Chiral Analysis: Advances in Spectroscopy, Chromatography and Emerging Methods 0444640274, 9780444640277

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Chiral Analysis

Advances in Spectroscopy, Chromatography and Emerging Methods Second Edition

Edited by

PRASAD L. POLAVARAPU Vanderbilt University, Nashville, TN, United States

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2018 Elsevier B.V. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). NOTICES Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-64027-7 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

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Chiral Analysis

Advances in Spectroscopy, Chromatography and Emerging Methods Second Edition

Edited by

PRASAD L. POLAVARAPU Vanderbilt University, Nashville, TN, United States

CONTENTS List of Contributors xiii Prefacexvii

Part One  Chirality in Nature

1

1. Chiral Asymmetry in Nature

3

Dilip Kondepudi Introduction3 Chirality: terminology and quantification issues 3 Chiral asymmetry in nature 10 Theory of spontaneous chiral symmetry breaking 15 Sensitivity of chiral symmetry breaking transitions to asymmetric interactions19 1.6 Examples of spontaneous chiral symmetry breaking 22 1.7 Concluding remarks 25 References25 1.1 1.2 1.3 1.4 1.5

2. Remote Sensing of Homochirality: A Proxy for the Detection of Extraterrestrial Life

29

C.H. Lucas Patty, Inge Loes ten Kate, William B. Sparks, Frans Snik 2.1 Introduction29 2.2 Homochirality30 2.3 Chemical and biological mechanisms for creating circular spectropolarimetric signals 35 2.4 Considerations for the remote sensing of homochirality in our solar system and beyond 45 2.5 Instrumentation50 2.6 Conclusion and outlook 60 References61

Part Two  Spectroscopic Methods and Analyses

71

3. Light Polarization and Signal Processing in Chiroptical Instrumentation73 Kenneth W. Busch, Marianna A. Busch 3.1 Introduction73 Part A—The polarization of light 73 3.2 Light as a wave 74 v

vi

Contents

3.3 Types of polarized light 76 3.4 Production of linearly polarized light 81 3.5 Production of circularly polarized light 98 3.6 Production of elliptically polarized light 101 3.7 Photoelastic modulators 101 Part B—Signal handling 105 3.8 Noise in electrical circuits 105 3.9 Noise reduction strategies 108 3.10 Application of phase-sensitive detection in chiroptical instrumentation 123 Appendix A basic optics 126 Appendix B signal handling 136 References150

4. Chiroptical Spectroscopic Studies on Soft Aggregates and Their Interactions

153

Vijay Raghavan, Prasad L. Polavarapu 4.1 Introduction153 4.2 OR studies 158 4.3 ECD studies 172 4.4 VCD studies 184 4.5 ROA studies 193 4.6 Summary195 References195

5. Vibrational Optical Activity in Chiral Analysis

201

Laurence A. Nafie, Rina K. Dukor 5.1 Introduction201 5.2 Definitions of VOA 204 5.3 Measurement of VOA 206 5.4 Theoretical basis of VOA 208 5.5 Calculation of VOA 214 5.6 Determination of AC 216 5.7 Determination of ee of multiple chiral species 226 5.8 VOA of solids and formulated products 235 5.9 Summary and conclusions 238 References239

6. Raman Optical Activity

249

Saeideh Ostovar pour, Laurence D. Barron, Shaun T. Mutter, Ewan W. Blanch 6.1 Fundamental principles 6.2 Experimental ROA studies

250 256

Contents

vii

6.3 Enhancement of ROA signals 270 6.4 Recent ROA instrumentation developments 273 6.5 Computational modeling of ROA spectra 274 6.6 Concluding remarks: future opportunities 282 References284

7. Chiral Molecular Tools Powerful for the Preparation of Enantiopure Compounds and Unambiguous Determination of Their Absolute Configurations by X-Ray crystallography and/or 1H NMR Diamagnetic Anisotropy

293

Nobuyuki Harada Introduction293 Methodologies for determining AC and their evaluations 294 Methodologies for chiral synthesis and their evaluations 297 Camphorsultam dichlorophthalic acid, CSDP acid (−)-1, and camphorsultam phthalic acid, CPS acid (−)-5, useful for enantioresolution of alcohols by HPLC and determination of their ACs by X-ray crystallography 299 7.5 A novel chiral molecular tool, 2-methoxy-2-(1-naphthyl)-propionic acid {MαNP acid (s)-(+)-2}, useful for enantioresolution of alcohols and determination of their ACs by the 1H nmr diamagnetic anisotropy method312 7.6 Complementary use of CSDP acid (−)-1 and MαNP acid (s)-(+)-2 for enantioresolution of alcohols and determination of their ACs by X-ray crystallographic and 1H nmr diamagnetic anisotropy methods325 7.7 Conclusions338 References339 7.1 7.2 7.3 7.4

8. Chiroptical Probes for Determination of Absolute Stereochemistry by Circular Dichroism Exciton Chirality Method345 Kuwahara Shunsuke, Ikeda Mari, Habata Yoichi 8.1 Introduction345 8.2 A chiroptical probe for chiral resolution and determination of the absolute configuration of aromatic alcohols 346 8.3 A chiroptical probe for determination of the absolute configuration of primary amines 352 8.4 A chiroptical probe for chirality transcription and amplification by the forming of [2]pseudorotaxanes 358 8.5 Conclusions363 References363

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Contents

9. Chiral Analysis by NMR Spectroscopy: Chiral Solvating Agents

367

Federica Balzano, Gloria Uccello-Barretta, Federica Aiello 9.1 Introduction367 9.2 Low-molecular-weight csas 371 9.3 CSAs involving ion pairing processes 383 9.4 Molecular tweezer csas 389 9.5 Synthetic macrocycle csas 391 9.6 Cyclodextrins401 9.7 Natural products 406 9.8 Lyotropic chiral liquid crystals 408 9.9 Chiral sensing 409 9.10 Configurational assignments 409 9.11 Conclusions411 References412

10. Chiroptical Spectroscopy of Biofluids

429

Vladimír Setnička, Lucie Habartová 10.1 Introduction429 10.2 Blood and blood-based derivatives 430 10.3 Hen egg white 446 10.4 Vitreous humor 447 10.5 Urine447 10.6 Chiroclinics—chiroptical methods as diagnostic tools 452 10.7 Concluding remarks 459 References460

Part Three  Chromatographic and Electromigration Methods

467

11. Chiral Gas Chromatography

469

Rahul A. Patil, Choyce A. Weatherly, Daniel W. Armstrong 11.1 Introduction469 11.2 Commercial stationary phases 471 11.3 Non-commercial stationary phases 490 11.4 Conclusions500 References500

12. Chiral Liquid Chromatography

507

Muhammad F. Wahab, Choyce A. Weatherly, Rahul A. Patil, Daniel W. Armstrong 12.1 Introduction507 12.2 Historical background of chiral liquid chromatography 508

Contents

ix

Interactions in chiral liquid chromatography 509 Chiral separations in liquid chromatography today 512 Salient features of chiral liquid chromatography 512 Major classes of modern chiral stationary phases (CSPs) for liquid chromatography: the “α” of the resolution equation 515 12.7 Packing process of high-efficiency chiral phases (“N” in the resolution equation)534 12.8 Method development in chiral LC (adjustment of “k” in the resolution equation)535 12.9 Special detectors in liquid chiral chromatography 536 12.10 Recent developments in enhancing chiral resolution using ultrahigh efficiency supports 539 12.11 Instrumental considerations for fast chiral LC 544 12.12 Future directions and developments in chiral liquid chromatography 558 References558 12.3 12.4 12.5 12.6

13. Enantioseparations by Capillary Electromigration Techniques

565

Bezhan Chankvetadze Introduction565 Separation principle in chiral CE: electrophoretic or chromatographic? 566 Enantioseparations with charged and uncharged chiral selectors 567 Enantioselective and nonselective phenomena in chiral CEKC567 Similarities and differences between enantioseparations by pressure-driven chromatography and CEKC569 13.6 Modes of enantioseparations in CEKC574 13.7 Chiral selectors 584 13.8 Selector–selectand interaction in chiral CE586 13.9 Mathematical models of CE enantioseparations 589 13.10 Enantioseparations in capillary electrochromatography 593 13.11 Future trends 600 References601 13.1 13.2 13.3 13.4 13.5

14. Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

607

Roberta Franzini, Alessia Ciogli, Francesco Gasparrini, Omar H. Ismail, Claudio Villani 14.1 Introduction607 14.2 Chiral stationary phases for SFC610 14.3 Analytical separations 612 14.4 Ultrafast high-efficient SFC separations 618 14.5 Conclusions626 References626

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Contents

15. Chiral Separation Strategies in Mass Spectrometry: Integration of Chromatography, Electrophoresis, and Gas-Phase Mobility

631

James N. Dodds, Jody C. May, John A. McLean 15.1 Introduction631 15.2 Chromatography and mass spectrometry 633 15.3 Liquid chromatography 635 15.4 Electrophoresis–mass spectrometry 637 15.5 Isomer separations by IM-MS 640 15.6 Conclusions643 References644

Part Four  Emerging Methods

647

16. Cavity-based Chiral Polarimetry

649

Dimitris Sofikitis, George E. Katsoprinakis, Alexandros K. Spiliotis, T. Peter Rakitzis 16.1 Introduction649 16.2 Optical activity 650 16.3 Single-pass polarimetry 652 16.4 Cavity ring-down polarimetry 654 16.5 Cavity ring-down polarimetry with signal reversals 660 16.6 Continuous-wave cavity-enhanced polarimetry with signal reversals 672 16.7 Future outlook and conclusions 675 References676

17. Quantitative Chiral Analysis by Molecular Rotational Spectroscopy679 Brooks H. Pate, Luca Evangelisti, Walther Caminati, Yunjie Xu, Javix Thomas, David Patterson, Cristobal Perez, Melanie Schnell 17.1 Introduction679 17.2 Basic principles of molecular rotational spectroscopy 682 17.3 Chiral tag rotational spectroscopy for enantiomer analysis 698 17.4 Three-wave mixing rotational spectroscopy for enantiomer analysis 714 17.5 Conclusions723 References725

18. Chiral Rotational Spectroscopy

731

Robert P. Cameron, Jörg B. Götte, Stephen M. Barnett 18.1 Introduction731 18.2 Chiral rotational spectroscopy 732

Contents

xi

18.3 Chiral rotational spectra 734 18.4 Chiral rotational spectrometer 743 References747

19. Chiral Analysis and Separation Using Molecular Rotation

753

Mirianas Chachisvilis 19.1 Introduction753 19.2 Chiral separations in SFs754 19.3 Chiral separations in REFs762 19.4 Summary776 References776 Index779

LIST OF CONTRIBUTORS Federica Aiello University of Pisa, Pisa, Italy Daniel W. Armstrong Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington,TX, United States Federica Balzano University of Pisa, Pisa, Italy Stephen M. Barnett University of Strathclyde, Glasgow, United Kingdom Laurence D. Barron University of Glasgow, Glasgow, United Kingdom Ewan W. Blanch RMIT University, Melbourne,Vic., Australia Kenneth W. Busch Baylor University, Waco, TX, United States Marianna A. Busch Baylor University, Waco, TX, United States Robert P. Cameron University of Strathclyde; University of Glasgow, Glasgow, United Kingdom; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany Walther Caminati Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy Mirianas Chachisvilis Solvexa LLC, Keswick, CT, San Diego, CA, United States Bezhan Chankvetadze Tbilisi State University, Tbilisi, Georgia Alessia Ciogli Sapienza University of Rome, Rome, Italy James N. Dodds Vanderbilt University, Nashville, TN, United States Rina K. Dukor BioTools, Inc., Jupiter, FL, United States Luca Evangelisti Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy

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List of Contributors

Roberta Franzini Sapienza University of Rome, Rome, Italy Francesco Gasparrini Sapienza University of Rome, Rome, Italy Jörg B. Götte University of Strathclyde; University of Glasgow, Glasgow, United Kingdom; Nanjing University, Nanjing, China Lucie Habartová University of Chemistry and Technology Prague, Prague, Czech Republic Nobuyuki Harada Tohoku University, Sendai, Japan Omar H. Ismail Sapienza University of Rome, Rome, Italy Inge Loes ten Kate Utrecht University, Utrecht, The Netherlands George E. Katsoprinakis IESL-FORTH; University of Crete, Heraklion-Crete, Greece Dilip Kondepudi Wake Forest University, Winston-Salem, NC, United States Ikeda Mari Department of Chemistry, Education Center, Faculty of Engineering, Chiba Institute of Technology, Chiba, Japan Jody C. May Vanderbilt University, Nashville, TN, United States John A. McLean Vanderbilt University, Nashville, TN, United States Shaun T. Mutter Cardiff University, Cardiff, United Kingdom Laurence A. Nafie Syracuse University, Syracuse, New York, NY; BioTools, Inc., Jupiter, FL, United States Saeideh Ostovar pour RMIT University, Melbourne,Vic., Australia Brooks H. Pate Department of Chemistry, University of Virginia, Charlottesville,VA, United States Rahul A. Patil Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington,TX, United States

List of Contributors

xv

David Patterson Department of Physics, University of California Santa Barbara, Santa Barbara, CA, United States C.H. Lucas Patty Vrije Universiteit Amsterdam, Amsterdam, The Netherlands Cristobal Perez Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Prasad L. Polavarapu Vanderbilt University, Nashville, TN, United States Vijay Raghavan Vanderbilt University, Nashville, TN, United States T. Peter Rakitzis IESL-FORTH; University of Crete, Heraklion-Crete, Greece Melanie Schnell Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Vladimír Setnička University of Chemistry and Technology Prague, Prague, Czech Republic Kuwahara Shunsuke Department of Chemistry, Faculty of Science, Toho University; Research Center for Materials with Integrated Properties, Toho University, Chiba, Japan Frans Snik Leiden University, Leiden, The Netherlands Dimitris Sofikitis IESL-FORTH; University of Crete, Heraklion-Crete, Greece William B. Sparks Space Telescope Science Institute, Baltimore, MD, United States Alexandros K. Spiliotis IESL-FORTH; University of Crete, Heraklion-Crete, Greece Javix Thomas Department of Chemistry, University of Alberta, Edmonton, AB, Canada Gloria Uccello-Barretta University of Pisa, Pisa, Italy Claudio Villani Sapienza University of Rome, Rome, Italy Muhammad F. Wahab Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington,TX, United States

xvi

List of Contributors

Choyce A. Weatherly Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington,TX, United States Yunjie Xu Department of Chemistry, University of Alberta, Edmonton, AB, Canada Habata Yoichi Department of Chemistry, Faculty of Science, Toho University; Research Center for Materials with Integrated Properties, Toho University, Chiba, Japan

PREFACE The subject of chirality spans different disciplines and has become a focal point for the discussion on its role in the origin of life.The ubiquitous presence of chirality that drives the sustenance of life on this planet raises the specter of the potential for its presence outside of this planet and for the existence of extraterrestrial life. These fundamental topics render chirality an important role for scientific curiosity and investigations. Modern drug development and pharmaceutical research are heavily influenced by the role of chirality in their respective goals. Separations of the enantiomers of chiral compounds, distinguishing among diastereomers, determining the three dimensional structures of chiral molecules are the topics that dominate natural sciences today and are broadly categorized under the banner of Chiral Analysis. The first edition of Chiral Analysis, edited by Kenneth Busch and Marianna Busch, appeared in 2006 describing the current-state-of research at that time. As fundamental science undergoes never-ending development, one finds 12 years later, that the then existing methods have undergone major refinements and several new methods have come into practice. For active researchers, it is important to stay informed of these developments. Therefore, the purpose of this second edition is to update the then existing methods as well as to describe the newly developed methods. This book is divided into four sections. The first section, Chirality in Nature, contains two chapters. The first chapter has been updated from the first edition. The second chapter is newly written and presents state of the art in remote sensing of homo chirality. The second section, Spectroscopic Methods and Analyses, presents eight chapters on different chiroptical spectroscopic methods. Among these, Chapters 3, 5, 6 and 8 are updated versions from the first edition, while the others, namely, Chapters 4, 7, 9 and 10 are newly written. The third section, Chromatographic and Electromigration Methods, provides state of the art in chromatographic chiral separations in five different chapters. All these five chapters are newly written. The fourth section, Emerging Methods, contains four new chapters that describe newly emerged methods in recent years, each describing a new independent method with novel applications.

xvii

xviii

Preface

This book is intended to serve as a guide for those interested in chiral analysis. Graduate students as well as scientific professionals pursuing chirality in physical chemistry, analytical chemistry, organic chemistry, biology and pharmaceutical research will find the relevant topics needed to pursue their independent research. In addition, researchers interested in chirality, as well as seasoned practitioners in this area, will find the chapters written by leading experts in their respective areas, a valuable resource. I would like to thank all of the authors of the chapters in this volume for their dedication and willingness in deciphering their scientific innovations to wider scientific community and for their cooperation throughout this project.Without assistance from these dedicated scientists this volume could not have been produced. Finally, I thank Kathryn Morrissey, Acquisitions Editor at Elsevier, for reaching out to me to undertake this book project. Prasad L. Polavarapu Vanderbilt University, Nashville, TN, United States

PART ONE

Chirality in Nature

CHAPTER 1

Chiral Asymmetry in Nature Dilip Kondepudi Wake Forest University, Winston-Salem, NC, United States

1.1  INTRODUCTION The wide-ranging chiral asymmetry in nature has given rise to a multidisciplinary interest in chirality. It is remarkable that nature does not exhibit left–right symmetry at any level: morphological, molecular, and even at the most fundamental level of elementary particle interactions [1–3]. At the astronomical level, circularly polarized light has been found in Orion Nebula [4], indicating the existence of large regions of space with asymmetry. Chiral asymmetry in biomolecules—the predominance of l-amino acids and d-sugars—has profound consequences for pharmaceutical and agricultural chemistry. It necessitates us to develop a theoretical framework in which we can quantitatively study chirality and study processes that generate chiral asymmetry. In this chapter, we will discuss the basic definitions and nomenclature used to describe chiral systems. This will be followed by a brief survey of known chiral asymmetries in nature and questions they raise.We will then present a general theory of spontaneous chiral symmetry breaking and discuss examples of spontaneous generation of asymmetry.

1.2  CHIRALITY: TERMINOLOGY AND QUANTIFICATION ISSUES When an object is not identical to its mirror image, it is said to be “chiral”. Lord Kelvin, who introduced this terminology, defined it thus: “I call any geometrical figure, or any group of points, chiral, and say it has chirality, if its image in a plane mirror, ideally realized, cannot be brought to coincide with itself ” [5]. A chiral object or a geometrical figure and its mirror image are thus distinguishable and they could be simply identified as “left-handed” or “right-handed”, a terminology that can readily be understood because we are asymmetric in the use of our hands. But there is a subtle issue here: how do you specify what we mean by a “left-handed Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00001-X Copyright © 2018 Elsevier B.V. All rights reserved.

3

4

Dilip Kondepudi

Figure 1.1  Examples of basic chiral units. All complex objects can be reduced to a set of basic chiral units. As shown in the lower part, every basic chiral unit can be associated with a directed circle and a vector which together define a right- or left-handed helix.

object” to the one who does not know what it is, say to a being on a distant planet? Could we send this information as a radio signal coded as a sequence of digits (“0” and “1”)? The answer to this question turns out to be negative; it is not possible to specify what one means by “left” or “right” in a linear sequence of digits. A three-dimensional object or a chirally asymmetric phenomenon in nature is needed to specify this information [6–8]. While Kelvin’s definition clearly identifies chiral object, we often have a need to distinguish between a simple chiral object, such as a tetrahedral molecule with four different atoms, and a complex chiral object, such as a protein. To describe chirality of assemblies of simple achiral building blocks, we may define a basic chiral unit as an object that cannot be divided into two or more chiral objects; any disassembly of such a unit will result in achiral subunits. Some examples of basic chiral units are shown in Fig. 1.1. According to this definition, a helix made of a continuous line segment is not a basic chiral unit; however, a helical assembly of sphere consisting of four spheres is a basic chiral unit. All basic chiral units can be associated with a direction of rotation and an arrow that together define a helix as shown in Fig. 1.1. (In certain situations, one might identify the direction of rotation with an axial vector, while the arrow is a vector. The combination of

Chiral Asymmetry in Nature

5

a vector and an axial vector defines chirality.) Through such association, we can specify the absolute configuration of all basic chiral units. The chirality of a large assembly of simple achiral units has to be described in terms of chirality of such basic chiral units. We will discuss more about this point later in this chapter.

1.2.1  Cahn–Ingold–Prelog classification of chiral molecules In the case of chiral molecules, a system of rules that identifies the absolute structure of the two enantiomers as R (Rectus, Latin word for “right”) and S (Sinister, Latin word for “left”) was proposed by Cahn et al. [9]. These rules have been adopted by IUPAC.The two molecular structures are called enantiomers of a chiral molecule. This designation is particularly suited for chiral organic molecules in which a carbon is bonded to four different atoms or groups, R1, R2, R3, and R4 as shown in Fig. 1.2. Each of the four groups bonded to the carbon are ranked by the atomic number of the atom directly bonded to the carbon, higher atomic number corresponding to a higher rank. If two or more atoms have the same atomic number, then other atoms in each group’s chain are compared until a difference is found. If all the atomic numbers are the same, then atomic masses are used instead

Figure 1.2  An example of R-enantiomer in accordance with Cahn–Ingold–Prelog rules. The four groups, R1–R4, attached to the central carbon are ranked, R1 > R2 > R3 > R4. Pointing the lowest ranked group away from the viewer, the clockwise rotation R1 → R2 → R3, defines the molecule as R-enantiomer. If the rotation is in the counterclockwise direction, then it is a S-enantiomer.

6

Dilip Kondepudi

for ranking the groups. Thus, a CH3 has a lower rank than CH2OH or CH2CH3; similarly, the isotope D has a higher ranking than H. In Fig. 1.2, the ranking is assumed to be R1 > R2 > R3 > R4. For such a molecule, the “arrow” is defined as that pointing from the central carbon to the group with the lowest rank, R4. The rotation direction is obtained from the three highest ranking groups by moving from the highest to the lowest ranking group, that is, R1 → R2 → R3. If this rotation and the arrow define a righthanded helix, the molecule is classified as R; if the helix is left-handed, the molecule is classified as S. It is possible that the two of the four groups, say R1 and R2, are stereoisomers. In that case, the Cahn–Ingold–Prelog rules specify a precise way of ranking the two groups. These rules may be found in Ref. [9]. Before the Cahn–Ingold–Prelog rules were formulated, the terminology for identifying the two enantiomers of a chiral molecule was based on optical rotation. Linearly polarized light of a particular wavelength undergoes a rotation as it propagates through a solution or a crystal of a chiral compound. A solution or solid that rotates linearly polarized light is said to be optically active. In fact, the discovery of optical activity by the physicist Biot in 1815 and Louis Pasteur’s insight that it was related to molecular asymmetry gave us a general means to identify enantiomers. Consequently, the handedness of a molecule was identified by its optical activity. If the light was rotated clockwise as it approaches the viewer (Fig. 1.3), the solution was designated “dextro-rotatory” or “+” and the enantiomer causing such a rotation was called d-enantiomer; if the light rotated counter-clockwise, the rotation was called “levo-rotatory” or “−” and the corresponding enantiomer is called l-enantiomer. This designation does not give us the absolute molecular structure, that is, one cannot say which one of the two mirror-image structures of the molecule gives rise to the observed optical rotation. In fact, the optical rotation for a given compound depends on the wavelength of the light and it can be dextro at one wavelength and levo at another.

Figure 1.3  Optical activity is the rotation of linearly polarized light as it passes through the sample. As shown in the figure, if the direction of polarization rotates clockwise as it approaches the viewer, the substance is dextro-rotatory and is designated “d” or “+”. If the rotation is in counter-clockwise direction, the substance is levo-rotatory or “l”. or “−”. Optical activity arises when the refractive indices of left- and right-circularly polarized light are unequal.

Chiral Asymmetry in Nature

7

Figure 1.4  The terminology “l- and d-amino acids” refers to the geometric structure as shown. Solutions of l-amino acids can be levo- or dextro-rotatory.

In addition, for a given wavelength, the direction of rotation can change with the solvent. Clearly the designation “R and S” specifies the molecular structure more precisely and it is preferred over the “l and d” designation. Since optical activity is readily measured and often used to analyze chiral products of a reaction, if the optical activity of enantiomer is known, it is specified by combining the R–S and l–d notation; thus R(–) indicates that the R-enantiomer is levo-rotatory. In addition to the R–S designation, another designation that is also used in the context of amino acids and sugars identifies the enantiomers as l and d, which in the case of sugars is tied to the designation given to glyceraldehyde. This designation refers to the absolute structure as shown in Fig. 1.4. This designation is independent of the side chain and hence all l-amino acids have a similar basic structure. However, depending on the side chain, an l-amino acid may be designated R or S, a point that needs to be noted when using the R/S designation for the biologically dominant l-amino acids.

1.2.2  Optical rotation The angle through which linearly polarized light is rotated, that is, the optical rotation, is usually denoted by α. Optical rotation is due to the difference in the refractive indices of right- and left-circularly polarized light. Linearly polarized light may be considered as a combination of the right- and leftcircularly polarized light, that is, linearly polarized light may be resolved into right- and left-circularly polarized components. If the two components travel at different speeds, the resultant linear polarization undergoes a rotation. If nR and nL are the refractive indices of right- and left-circularly

8

Dilip Kondepudi

polarized light, respectively, for light of wavelength λ, traversing a path in a chiral medium of length l, the optical rotation α is given by

α=

πl (n − nR ) λ L

(1.1)

In most practical situations, to a good approximation, optical activity of a solution is directly proportional to its concentration and the path length (length of the polarimeter tube). In addition, α also depends on the wavelength, the solvent, and the temperature. Consequently, specific rotation, [α], of light of wavelength λ, at a temperature T is defined as [α ]Tλ =



λ lc

(1.2)

in which c is the concentration in g/mL and l is the path length in decimeters.

1.2.3  Enantiomeric excess Optical activity of a solution is proportional to the difference in the amounts of the two enantiomers. This difference, which is a measure of the chiral asymmetry of the systems, is often quantified in terms of enantiomeric excess (EE), which is defined as

|[ R ] − [S ]| Enantiometric Excess= ([ R ] + [S ])

(1.3)

in which [R] and [S] are the concentrations of the two enantiomers. It is a measure of asymmetry relative to the total amount of the substance, not an absolute measure of the difference in the amount of R and S. As we shall see below, in formulating a general theory of spontaneous chiral symmetry breaking, the difference ([R] − [S]) is mathematically more convenient to use than EE.

1.2.4  Crystal enantiomeric excess In nucleation of chiral crystals, asymmetries can arise due to chirally autocatalytic processes. In this case, the asymmetry is due to the difference in the number of levo- and dextro-rotatory crystals. For this reason, one might define a crystal enantiomeric excess (CEE) as



CEE =

Nl − Nd Nl + Nd

(1.4)

Chiral Asymmetry in Nature

9

in which Nl and Nd are the number of levo- and dextro-rotatory crystals. This quantity measures the asymmetry in nucleation, not in the total number of molecules.

1.2.5  Chirality measures When we look at chiral objects, we often have a sense that one is “more chiral” than another. This notion could be intrinsic to the chiral objects themselves or in their interaction with other chiral objects. For example, when we compare two molecules that are in the shape of a twisted H, one could say the one with the larger twist angle is “more chiral”, which is a measure intrinsic to the object.The specific rotation for a given wavelength and other well-specified conditions, a measure not intrinsic to the object, could be used to compare the chirality of molecules, the molecules with larger rotation being “more chiral”. It is desirable to have the measure be such that: (i) the value of the chirality measure changes continuously with the shape of the object and is equal in magnitude but opposite in sign for enantiomers, (ii) the chirality measure is zero if and only if the object is achiral. It turns out that it is impossible to define a universal measure with these two properties! This is because enantiomers of certain chiral objects can be transformed from one to the other through a series of states which are all chiral—just as a left-hand glove can be transformed to a right-hand glove by turning it inside out one finger at a time. Examples of such transformations were noted by Mislow [10,11]. Molecules in which such a transformation can be realized are called “molecular gloves”. During such a transformation, the corresponding chirality measure has to change continuously from positive to negative (or vice versa) and hence must cross zero.The transformation, however, consists entirely of chiral states. Hence, there must be a chiral state whose chirality measure is zero, contrary to the requirement (ii). If an R-enantiomer of a molecule can be transformed into an S-enantiomer through intermediate states which are all chiral, it means there is no well-defined achiral state at which the change from R to S occurs; hence the point at which the change in designation is made is arbitrary: the notion of “left” and “right” cannot be made absolute. Avoiding the ambiguity that arises with the sign of a chirality measure, the intrinsic chirality of an object can be viewed, for example, in terms of how different the two enantiomers are from each other. In this regard, several measures have been proposed [12–17]. Buda et al. [13] define a degree of chirality as: “the value of a real-valued, continuous function that is zero if and only if the object is achiral. The degree of chirality is the measure of

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an absolute quantity and its value is therefore the same for both enantiomorphs of the measure object”. An example of the degree of chirality is the maximum amount of overlap between a chiral object and its enantiomer. Clearly, in this measure, the degree of all achiral objects is zero and all chiral objects is nonzero. Only chiral objects can distinguish between the enantiomers of a chiral molecule. Could we not define a measure of chirality on the basis of interaction of reference chiral object with all chiral molecules? For example, could we take optical rotation (at specified wavelength, temperature, and other conditions) as a measure of chirality? This approach too has difficulties because there are some chiral molecules [11] whose optical activity is for all practical purposes immeasurable and there is no simple, reliable, theoretical way to predict what it would be. Furthermore, a molecule that is classified as “left-handed” on the basis of optical rotation with one achiral solvent, may have to be classified as “right-handed” with another achiral solvent which indicates that this notion of “left” and “right”, derived from the interaction of the molecule with right- and left-circularly polarized photons of a particular wavelength, is also dependent on the environment and somewhat arbitrary. Though there is no universal measure of chirality, it must be noted that there are situations in which quantifying chirality could be extremely useful in organizing and understanding chiral interactions [18–20].

1.3  CHIRAL ASYMMETRY IN NATURE That nature does not treat “left” and “right” equally is not only remarkable, but also puzzling because the fundamental laws have a high degree of symmetry in many respects. All basic laws of physics are invariant under translations in space and time and rotations in space. Electromagnetic phenomenon is also invariant under mirror reflection, more precisely, parity (which is equivalent to mirror reflection and a rotation). But other fundamental interactions, at the nuclear level, are not invariant under parity. In this section, we will briefly survey the chiral asymmetries found at various levels of scale, from the nuclear level to the level of morphology of mammals.

1.3.1  Asymmetry in nuclear processes Chiral asymmetry in nuclear processes responsible for β-radioactivity was discovered in 1957 by Wu [21]. Some unstable nuclei “decay” to a lower

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state of energy by emitting electrons or positrons, also called β-particles. The isotope of cobalt, cobalt-60, for example, emits electrons. The nucleus of cobalt-60 has spin, but a stationary particle with a spin is not chiral; it does not possess handedness unless there is a direction associated with spin that could be used to define chirality. (In identifying the “north pole” using the right hand, we are imposing the chirality of the hand on the spinning sphere; a stationary spinning object is not chiral.) If there is an additional direction associated with a static spinning object, however, one could define left and right. In the case of radioactivity in cobalt-60, when the nuclei are placed in a magnetic field, their spin axes line up along the magnetic field. In cobalt-60, nuclei aligned in a magnetic field, more electrons were emitted from one of the two hemispheres, thus indicating the handedness of this phenomenon. If the two hemispheres of a spinning sphere are not identical, then that object is chiral because the lack of identity of the two hemispheres could be used to define a direction, which along with the rotation defines a helix. Further studies of β-radioactivity revealed that the emitted electrons themselves exhibit asymmetry. Since electrons have spin, one can associate left- or right-helicity to each electron by projecting its momentum vector along the spin axis. It was found that the electrons emitted by radioactive nuclei are predominantly left-helical. Chiral asymmetry in β-radioactivity appears in other ways as well.The emission of β-particles is accompanied by the emission of elusive particles called the neutrinos, which also have spin. All neutrinos generated in β-radioactivity are left-helical and antineutrinos are right-helical. Neither right-helical neutrinos nor left-helical antineutrinos have been detected; they are presumed not to exist. If there were equal number left-helical neutrinos and right-helical anti-neutrinos then we might say that left–right symmetry is restored, but this would require a balance between matter and antimatter. As far as we can tell, there is matter in the universe but not antimatter.The consequence is that present universe is filled exclusively with left-helical neutrinos.

1.3.2  Asymmetry in atoms We now move from the level of the nucleus to the level of the atom. Before the forces that held the particles in the atomic nucleus were discovered, physicists knew two fundamental forces in nature: gravity and electromagnetism. With the discovery of the nucleus, in which positively charged protons and uncharged neutrons were held together, new forces came to light. Soon after, studies in nuclear physics showed that there were

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Figure 1.5  Asymmetry in electro-weak interactions. The interaction between the electron and the nucleons depends on the electron helicity; the interaction energies of the left- and right-helical electrons are unequal. Due to electro-weak interaction, the ground-state energies of enantiomers are unequal.

basically two types of nuclear forces, which were termed, “strong” and “weak” forces. The weak forces were responsible for β-radioactivity, while the strong force was responsible for nuclear fission and fusion. Ever since James Clerk Maxwell realized that the laws of electricity and magnetism can be unified as “electromagnetic” laws, there has been a strong interest among physicists to unify different forces. Einstein, for example, tried very hard, but unsuccessfully, to unify gravity and electromagnetism. But the search for unification of fundamental forces continues and one of its great achievements is the unification of electromagnetic and weak forces, which necessitated the coining of the term “electro-weak” force. The formulators of the theory of electro-weak force, Steven Wienberg, Abdus Salam, and Sheldon Glashow were awarded the Nobel Prize for Physics in 1979. The exact nature of this unification is not important for us, but the chiral asymmetry this unification revealed is. One of the predictions of the electro-weak theory is that the electron interacts with the nucleons (particles in the nucleus, protons, and the neutrons) with a force that depends on its helicity. Left- and right-helical electrons are acted upon by different forces (Fig. 1.5). This fact brings the chiral asymmetry to the level of atoms, making them optically active. Atoms were thought to be spherically symmetric entities with no chiral attributes until the electro-weak force between the electron and the nucleons was discovered. A tube filled with vapors of the element bismuth, for example, is found to be levo-rotatory [22]. Due to electro-weak interactions we only find levo-bismuth in nature, its mirror-image twin does not exist. The optical activity of atoms, though very small, has been measured and found to be as predicted by the electro-weak theory, of the order of 10−7 radians, depending on the density of the vapor [22].

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1.3.3  Asymmetry in molecules The electro-weak interaction between the electron and the nucleons also brings chiral asymmetry to the level of molecules. In most chemistry texts it is stated that the ground-state energies of enantiomers are identical. This would be the case if the interaction between the electron and the nucleons were entirely governed by the laws of electromagnetism which are parity invariant. But since the electron–nucleon interaction has also a component of the parity violating weak-force, the ground-state energies of enantiomers must be different. This difference has been calculated by Mason and Tranter [23,24] using electro-weak theory and it is found to be of the order of 10−14 J/mol, too small to be detected by current experimental methods. Calculations done on amino acids, indicate that the ground-state energies of l-amino acids are lower than those of d-amino acids. Since then, many more accurate calculations have been performed by Schewerdtfeger’s group [25–30] and some recent theoretical calculations found that the effect in excited states could be larger by a factor of 103 [31,32].This raises hope that we might be able to measure electro-weak energy differences in enantiomers in the laboratory.

1.3.4  Biomolecular asymmetry Louis Pasteur’s discovery in 1857 that microbes metabolized only the dextro-rotatory sodium-ammonium-tartrate is a momentous one. It not only presented us with one of the most fundamental and universal aspects of the biological chemistry, it left us with a fundamental question regarding life: Is chiral symmetry essential for the evolution of life? With our present knowledge of biochemistry we know that, all of life is dominated by l-amino acids and d-riboses. In the words of Francis Crick, “The first great unifying principle biochemistry is that the key molecules have the same hand in all organisms” [33]. The food we eat, the medicines we take, and pesticides we use all have to conform to the chemistry dictated by this asymmetry.The evolutionary origins of this asymmetry remain an enigma. Though we have learned much about processes that can spontaneously generate chiral asymmetry in the last 15 years, we are still far from having plausible examples of processes that could have caused the dominance of l-amino acids and d-riboses.

1.3.5  Morphological asymmetry The asymmetry in the morphology of living organisms is all too wellknown: Most of the spiral seashells are right-helical; the organ placement in

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mammals is asymmetric—except for the rare one-in-ten-thousand inversion in humans (a condition called situs inversus). Chiral asymmetry of the brain is reflected in behavior: about 90% of humans in all societies and cultures are right-handed. A good compendium of asymmetries in the animal world was assembled by Neville in his Animal Asymmetries [34]. Morphological asymmetry is inherited. How does this happen? This question has a different significance compared to other inherited traits that are coded in a DNA sequence of A, T, G, and C. Handedness cannot be coded in a linear sequence of letters, that is, the information about the placement of the liver on the right, for example, could not be coded in a sequence such as ATTGCGGTAC … while placement on the left be coded in a different sequence GTTACCTGA…. To understand why this is so, we only have to look at the process that places the liver on the right in a mirror to realize that the sequence ATTGCGGTAC …could also carry the code to place the liver on the left.The information about right or left has to come from some existing asymmetry. Although asymmetry is easy to find at the molecular level of l-amino acids and d-riboses, we do not know how the molecular asymmetry gets translated to morphological asymmetry. The connection between the structure of amino acids and organs is similar to the connection between bricks and the shape of a building. Clearly the shape of a building is quite independent from the shape of a brick; yet morphological asymmetry seems to have its origins at the molecular level. Elucidating this link is one of the great challenges of developmental biology [35].

1.3.6  Astrophysical asymmetries On the astronomical scale, asymmetry was found in large regions of interstellar space in the form of circularly polarized light. Bailey et al. [4,36] reported the detection of infrared circularly polarized light in star-forming regions of Orion Nebula. Though this is not a global asymmetry, it nevertheless shows that chiral asymmetry can arise in vast regions in the universe and it may have important implications for the origin of molecular asymmetry in these regions. An obvious place to look for chiral asymmetry on the astrophysical scale is in spiral galaxies. With respect to an observer on the earth, all galaxies are receding and hence have a well-defined direction of motion. For spiral galaxies, if we combine the direction of the spiral with the recession velocity vector, we can define a helicity or handedness of spiral galaxies. The Carnegie Atlas of Galaxies [37] is a good resource to look at the statistics of spiral galaxies. A simple count of the left- and right-helical spiral galaxies

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shows an equal number, indicating no apparent asymmetry. However, if the statistics are extended to the different classes of spiral galaxies, an interesting trend seems to emerge [38]. Spiral galaxies are classified into ordinary spirals (S) and barred spirals (SB), a classification initiated by Edwin Hubble. The two classes of galaxies show opposite asymmetries that cancel each other. If we assign +1 to “Z-shaped” spirals and –1 to the “S-shaped” in a sample of 540 galaxies that consists of both S and SB spirals, the sum was found to be –4 (–0.172 standard deviations from zero), indicating that there is no observable asymmetry in the total sample. But in a subsample of 378 galaxies of class S, the sum was found to be –26 (–1.34 standard deviations from zero), while in the subsample of 162 galaxies of class SB the sum was +22 (+1.73 standard deviations from zero). The probability of observing these sums as a consequence of random fluctuations in a system that has no real asymmetry is 0.09 for class S and 0.04 for class SB. With such low probabilities for the observed statistics, we may consider the above data as an indication that there is an asymmetry in the distribution of the subclasses of ordinary spiral galaxies and barred spiral galaxies but of opposite sign. More details and references to previous work on this topic can be found in Ref. [38].This observation should be considered only an indication and not a definite conclusion that there is chiral asymmetry in subclasses of spiral galaxies; to be certain, analysis based on a larger dataset is essential.

1.4  THEORY OF SPONTANEOUS CHIRAL SYMMETRY BREAKING The chiral asymmetry at the biomolecular level lies in stark contrast to the paucity of chemical reactions that spontaneously generate asymmetry. When chiral molecules are synthesized from achiral reactants, we find equal amount of the two enantiomers. Since chemical reactions are governed by the electromagnetic laws that are mirror-symmetric, the rates of production of the two enantiomers are equal. The deviation from chiral symmetry due to the electro-weak component of the interaction is too small to influence chemical reactions on the laboratory volume and time scales. (As we shall see below, when very large scales of time and volume are involved, even very small asymmetries can have a large effect.) The mirror-symmetry of the electromagnetic laws implies that a process and its mirror image are equally realizable in nature; thus, if we ignore the electro-weak interactions, there can be no noticeable difference in chemical reaction rates of enantiomers. But, identical reaction rates need not always lead to equal amounts of the

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two enantiomers. In reactions that are autocatalytic, symmetric kinetic rate laws could produce asymmetric product.The products of a chemical process need not reflect the symmetries of the process that generated it. When this happens, the system is said to exhibit “spontaneous symmetry breaking”. In mathematical terms, the solutions of a system of differential equations need not have the same symmetries as the system of equations; the solutions could “break the symmetries” of the system. This phenomenon could be illustrated using a simple model that is an extension of a model suggested by Sir Charles Frederick Frank in 1953 [39]. The modification to Frank’s model was made to elucidate the thermodynamic nature of the model and to assess the impact of small external asymmetries on such models. This modified model is shown in Fig. 1.6. In

Figure 1.6  An autocatalytic reaction scheme in which XL and XD have identical kinetic rate laws. However, in an open system, this reaction gives rise to a state of broken symmetry in which the concentrations of XL and XD are unequal. Lower part of the figure shows a “bifurcation diagram” used in describing the transitions to asymmetric states. The parameter α = (XL − XD)/2 is a measure of the asymmetry. λ = [S][T], the product of the concentrations of S and T, is the bifurcation parameter. When the value of λ exceeds λC the system becomes unstable and makes a transition to an asymmetric state in which α is not zero.

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this model, achiral molecules, S and T react to form a chiral molecule XL or XD. The molecule X is chirally autocatalytic: XL catalyzes the production of XL and XD catalyzes the production of XD. In addition, XL and XD react to form an inactive product P. The system consists of a “reaction chamber” into which there is an inflow of reactants S and T and an outflow of P. For a given set of flows, the system reaches a stationary state in which the concentrations of all the species remain constant. The flows maintain the concentrations of S and T at a constant value. If the flows are reduced to zero, the system will eventually reach thermodynamic equilibrium in which the concentrations of XL and XD are equal. If the flows are slowly increased from zero, the concentrations of S, T as well as XL and XD increase but still the concentrations of XL and XD remain equal until the concentrations of S and T reach a critical value. Below the critical value, any difference in the concentrations of XL and XD that might arise through a random fluctuation will decay to zero; here the symmetric state of equal XL and XD is stable. Above the critical value, the system becomes “unstable” in the sense any small difference between the concentrations of XL and XD will grow; the system can no longer maintain the symmetry between XL and XD. If the concentrations of S and T surpass the critical value, the system will be forced to make a transition to an asymmetric state in which concentrations of XL and XD are not equal.Which one will be greater is entirely random, but one of the two enantiomers will dominate. This symmetry-breaking transition is summarized in the lower part of Fig. 1.6. In the terminology of nonequilibrium chemistry, the system undergoes a nonequilibrium transition to a symmetry breaking state. The thermodynamic state thus reached through a nonequilibrium transition is more ordered or has more structure. This structure is maintained by the “dissipative” or entropy-generating chemical processes; they were therefore called “dissipative structures” by Ilya Prigogine, who was awarded the 1977 Nobel Prize in Chemistry for his contributions to nonequilibrium thermodynamics. There are numerous other examples of dissipative structures that are a result of symmetry breaking transitions [40]. The above example also makes it clear that only a system far from thermodynamic equilibrium can generate and sustain asymmetry of the form we see in proteins and DNA.Though the racemization rates of amino acids are very small, a steady generation of l-amino acids is necessary to maintain the chiral purity of active proteins. The above model can be used to illustrate general features that all chiral symmetry breaking transitions will exhibit. As shown in Fig. 1.6, there is a quantity α = ([XL] − [XD])/2 which is a measure of the asymmetry; α = 0

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represents a symmetric state in which two enantiomers are present in equal amounts; α  ≠ 0 represents an asymmetric state in which one of the two enantiomers is in excess. α is more mathematically convenient than EE in analyzing symmetry-breaking transitions. The parameter λ = [S][T] is a measure of the system’s distance from thermodynamic equilibrium because, through appropriate inflow of S and T, their concentrations are maintained at a nonequilibrium value. It is also called the bifurcation parameter. If λ is such that it corresponds to equilibrium values of [S] and [T], the system will be in a symmetric state corresponding to α = 0. When λ reaches a critical value, indicated by λC, the systems begin to become unstable to small deviations of α from zero; if λ > λC, then random fluctuations in α will grow and drive it to either a positive or a negative value (a steady state) depending on the fluctuation. At λ = λC, two new asymmetric solutions “bifurcate” from the symmetric solution. In the vicinity of the critical point λC, it is possible to obtain an equation for the time evolution of α that will have the same general form for all systems that break chiral symmetry [41,42]. This equation is of the form

dα = − Aa 3 + B( λ − λC )α + ε f (t ) dt

(1.5)

In this equation, the coefficients A and B depend on the kinetic rate constants; the term ε f (t ) represents random fluctuations in which ε is the root-mean-square value of the fluctuations and f(t) a normalized random variable with zero mean. (As a first approximation, one may assume f(t) to be a Gaussian white noise.) The fluctuations make the evolution of α stochastic or random. Normally, random fluctuations in concentrations are quite unimportant for predicting the time-variation of concentrations during the reaction and the final product distribution. The kinetic rate equations enable us to predict these within the accuracy of the experiments. Not so in systems that spontaneously break chiral symmetry; the evolution of α is random, not deterministic. For example, if λ moves from a value below the critical point to a value above the critical point, α could evolve from zero to a positive or a negative value. Hence, we can only assign a probability, P+ or P–, that α will evolve to either value because final state of α depends on a random fluctuation. In the absence of a chiral influence that favors one of the enantiomers, we must have P+ = P– = 0.5. Eq. (1.5), which contains a term representing random fluctuation, is an example of a Langevin equation. For such equations, with appropriate specification of the property of the random variable f(t), it is possible to obtain an equation

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for the evolution of the probability distribution P(α,t), called the Fokker– Planck equation. We shall not discuss these mathematical details in this introductory chapter but focus on the general theoretical aspects. Note that the mirror symmetry of the chemical reactions is manifest in the general equation (1.5) in that both α and –α are its solutions. When we consider steady states (dα/dt = 0) if α = –α = 0, then the solution is symmetric; on the other hand, if α ≠ 0, the system has two possible solutions, α and –α, each representing a state of broken symmetry. For any chemical system that is capable of spontaneous chiral symmetry breaking, the general equation of the form (1.5) can be derived using group theory, without reference to particular details of the kinetic mechanism; in addition, general expressions relating the coefficients A and B to the kinetic rate constants can be derived [42]. This theory is similar to the Ginzberg–Landau theory of second-order phase transitions.

1.5  SENSITIVITY OF CHIRAL SYMMETRY BREAKING TRANSITIONS TO ASYMMETRIC INTERACTIONS The above general formalism can be used to analyze the effects of small asymmetric interaction on symmetry breaking transitions. For example, we can analyze how circularly polarized light might influence the symmetry breaking transition if it enhances the reaction rate of one of the two enantiomers. Due to the asymmetric interaction, the probabilities of transition, P+ and P–, to asymmetric states will be different from 0.5. Let us say, that due to the asymmetric interaction, the forward rate constant of the reaction S + T → XL is larger than that of S + T → XD by a factor g, that is, the rate constant kL = kD (1 + g), in which kL and kD are the rate constants of the two reactions, respectively. Given such a chiral influence, the task is to determine the probabilities P+ and P– = (1 − P+). Using the same general approach that leads to Eq. (1.5), the following modification of this equation can be obtained in the presence of the asymmetric interaction [41,42]:



dα = Aα 3 + B( λ − λC )α + Cg + ε f (t ) dt

(1.6)

in which C is a constant that depends on the kinetic rate constants. As before, for any given set of reactions, one can obtain C using general expressions [42]. The effect of the bias term Cg is countered by the random fluctuations whose strength, as measured by the root-mean-square value, is ε .

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Figure 1.7  In the presence of a chiral influence that favors one enantiomer (here corresponding to α > 0), the bifurcation diagram in Fig. 1.6 is modified to the one shown above. As λ increase from a value below λC to a value above λC, the system makes a transition to the favored upper branch with a higher probability. In this figure, a fluctuating trajectory of α shows its evolution to the favored branch in which α > 0. This process is very sensitive to small chiral influences. The probability of transition to the favored branch is given by the formula (1.7).

At first sight, it might seem that if Cg  ε then the effect of the chiral interaction on P+ would be negligible. But a careful analysis shows that the system could in fact be very sensitive to the chiral interaction even if Cg  ε [41]. Such sensitivity arises when the parameter λ sweeps through the critical value, λC, from a subcritical to a supercritical value. Let us assume that this sweep of λ happens at an average rate of γ, that is, λ = λ0 + γt in which λ0 2%) and is essentially

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Figure 2.2  Portrayal of the Frank model with autocatalysis and mutual antagonism. With every autocatalytic cycle the enantiomeric excess increases. Adapted from [47].

Figure 2.3  An electronic transition dipole, a magnetic transition dipole, and a possible nonzero dot product of the former two which can interact with the polarization of light.

the alkylation of an aldehyde to produce an alcohol. Even the Soai reaction, however, does not produce homochirality and is very much dependent on the reaction iteration or initial imbalance. Recent modifications of the Frank model show that homochirality might be a spontaneous consequence of symmetry breaking processes in closed noisy autocatalytic systems without the need for chiral inhibition [3]. Others have shown that chiral amplification might also be possible without chiral cross-inhibition and explicit enantioselective autocatalysis, but by using asymmetric synthesis and polymerization [50]. Both models, however, do await experimental validation. Other enantioselective mechanisms based around an autocatalytic feedback have been proposed and shown experimentally. One of these mechanisms is the Viedma ripening process [51,52]. Viedma ripening describes the solid state asymmetric amplification of initial enantiomeric imbalances. Through mechanical interaction, that is, grinding, a conglomerate of enantiomeric crystals spontaneously convert to one of the forms. The final excess depends on the initial enantiomeric excess, the differences in crystal size distribution, chiral impurities, and chiral additives [53]. Unlike the

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previously mentioned models, it has been shown that Viedma ripening can lead to pure chiral end products. It has additionally been proposed that the nature of life’s homochirality is ubiquitous. Parity-violating energy differences, an asymmetry (albeit very small) of physics, cause a difference in energy between molecules, and this in turn might have been at the basis of life’s homochirality [54–57]. It has been claimed that the parity violations might also lead to differences in amino acid solubility [58], although it has been shown that this also could be the consequence of impurities [59]. This prevalent homochirality, however, has not been without controversy and extensive arguments exist against this hypothesis [60,61]. Nonetheless, the influence of parity violations on homochirality cannot be ruled out completely at this point [62,63].

2.3  CHEMICAL AND BIOLOGICAL MECHANISMS FOR CREATING CIRCULAR SPECTROPOLARIMETRIC SIGNALS 2.3.1  The discovery of chirality and its relation to the polarization of light The molecular dissymmetry of chiral molecules has a specific response to electromagnetic radiation. Polarization is a fundamental property of electromagnetic radiation and can be created or modified by any system that is asymmetric, such as chiral molecules. It was also by this phenomenon that chirality was first discovered: molecular chirality was reported for the first time in 1815 by the French astronomer and physicist Jean Baptiste Biot. Biot noticed how some quartz crystals differed in optical rotatory dispersion; the rotation of the plane of linearly polarized light traveling through the crystals. He found that some crystals were dextrorotatory, rotating the plane clockwise, while others were levorotatory, rotating the plane counterclockwise. Soon he also found that various solutions of organic compounds share this phenomenon. It was a student of Biot: Louis Pasteur, the French biologist and chemist, who in 1848 first connected optical rotatory dispersion to the molecular chirality of life [64,65]. Pasteur was prompted by a paradoxical report by the German mineralogist Eilhard Mitscherlich, who observed that there was a difference in the optical activity by two otherwise chemically identical substances; tartaric acid and the so-called “paratartaric acid”. Tartaric acid is a common precipitate in wine fermentation (commonly known as “wine diamonds”) and paratartaric acid was a by-product

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in the industrial production of tartaric acid. But while dissolved tartaric acid displayed optical activity, paratartaric acid did not. Upon further investigation, Pasteur observed that the paratartarate crystals showed two forms with slightly different mineral faces; they were the mirror images of each other and thus not superimposable. He separated the two crystals and found that paratartaric acid was in fact a mixture of dextrorotatory and levorotatory tartaric acid; the two separated fractions showed left- and right-handed optical activity.

2.3.2  Optical rotatory dispersion, electronic circular dichroism, and circular polarization There exists a large discrepancy between measurements carried out in a laboratory and the measurements required for “passive” remote sensing. One of the most important differences is that in all laboratory measurements, be it for optical activity or circular dichroism (CD), both the intensity and the polarization state of the incoming light is controlled. In remote sensing, this is not possible. From an Earth point of view, sunlight coming directly from the sun is unpolarized. Sunlight does get polarized in the atmosphere by scattering, and as a result, the polarization of the sky can easily vary between ∼0% and 70% [66]. The Jones formalism is often used in relation to laboratory experiments, that is, due to its relatively easy applicability. However, the Jones formalism is not valid anymore in a remote sensing context in that it is dealing only with monochromatic light. It therefore also does not allow partially polarized or unpolarized light. It is therefore necessary to introduce the Stokes formalism; where the optical parameters of the Jones formalism are amplitudes and phases, the Stokes formalism deals with light intensities and thus also directionally measurable quantities. With the electric field vectors Ex in the x-direction (0°) and Ey in the y-direction (90°), the Stokes vector is S given by  I Q S= U V 

 〈E x E x∗ + E y E y∗ 〉   I 0º + I 90º    ∗ ∗   〈E x E x − E y E y 〉   I 0º − I 90º  =  〈E E ∗ − E E ∗ 〉  =  I − I x y y x   45º -45º    I RHC − I LHC   ∗ ∗   i 〈E x E y − E y E x 〉 

    

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Furthermore, in the Stokes formalism, any optical element can be described by the 4 × 4 Mueller matrix M:

Sout

  = MS in =    

M 11M 12 M 13 M 14 M 21M 22 M 23 M 24 M 31M 32 M 33 M 34 M 41M 42 M 43 M 44

 I   Q  U   V 

     in

Any set of optical elements in a system can be described by a total system matrix, the product of the multiplication of the individual elements: MT = MnMn−1…M2M1. The Stokes parameters I, Q, U, and V refer to intensities which relate to measurable quantities. The absolute intensity is given by Stokes I. Stokes Q and U denote the linear polarization, where Q is the difference between horizontal and vertical polarization and U gives the difference in linear polarization but with a 45° offset. Circular polarization is finally given by V, which gives the difference in intensity between left- and right-handed circularly polarized light. By division through the absolute intensity I, the polarization state can be completely described by Q/I, U/I, and V/I. Furthermore, I0°, I90°, I45°, and I−45° are the intensities oriented in the plane perpendicular to the propagation axis and ILHC and IRHC are, respectively, the intensities of left- and right-handed circularly polarized light. Let us consider a beam of light shining on a detector and a plane perpendicular to that beam for reference orientation. If we would insert an ideal linear polarizer into this beam with a transmission perpendicular to the beam and along the reference plane, the detector would measure the intensity I0°. If we would then rotate the polarizer by 90°, the detector would measure the intensity I90°. Similarly, from that position we can rotate the polarizer by −45° to obtain I45° or by +45° to obtain I−45°. In order to measure IRHC and ILHC we additionally need a quarter-wave plate (a retarder for which δ = π / 2 ). If we position the quarter-wave plate in front of the polarizer with a fast axis along the beam, we can rotate the polarizer to +45° to obtain IRHC and similarly we can rotate to −45° to obtain ILHC. Linearly polarized light can be described by the superposition of leftand right-handed circularly polarized light. Chiral molecules have a slight difference in refractive index for left- and right-handed circularly polarized

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light; when linearly polarized light thus interacts with chiral molecules this will result in a relative phase shift (∆) between those two polarization states [67]. Upon vector addition, this will thus result in linearly polarized light but with a rotated plane of polarization. If we have linearly polarized incident light, the angle of optical activity is then given by

α = 0.5∆ϕ =

π ( ∆n )z λ

where λ is the wavelength and ∆n is the difference in refractive index (circular birefringence). Both chemical and biological sciences also use the specific rotation, which is the optical activity corrected for concentration c and pathlength z : [α ] = α / zc , and the molar rotation, which is the amount of rotation per mole of substance: [φ] = [α] M × 10−2. Often, optical rotatory dispersion is expressed as the molar rotation per wavelength [φ]λ. Although optical activity is related to circular polarization and CD, and all the three under certain conditions can be interconverted, the principle of measurement is very different. Most importantly, in remote sensing again we have no control of the input polarization and we only know when it is safe to assume unpolarized incoming light. CD is the differential absorbance of left- and right-handed circularly polarized light and can additionally be described by Mueller matrix element m14. Circular polarization is the fractional polarization of incoming unpolarized light and can be described by Mueller matrix element m41. Traditionally, in chemical and biological sciences, CD is often expressed in degrees of ellipticity (θ), where the ratio of the minor to the major axis of the resultant polarization ellipse defines the tangent of the ellipticity [68]. Under the assumption that the amount of CD is small, this ellipticity is defined as tan(θ ) ≈ θ rad =

| ER | − | EL | = | ER | + | EL |

IR − IL IR + IL

where E refers to the magnitude of the electric field vectors for leftand right-handed circularly polarized light and I refers to the intensity of left- and right-handed circularly polarized light. Substituting I using Lambert–Beer law: I R − I L e ∆A ln10/4 − e −∆A ln10/4  ∆A ln10  ∆A ln10 = ∆A ln10/4 −∆A ln10/4 = tanh   ≈  +e 4 4 IR + IL e

Remote Sensing of Homochirality: A Proxy for the Detection of Extraterrestrial Life

θ deg =

39

180 ∆A ln10 4 π

where ∆A is the difference in absorption (∆A = AL  −  AR). Circular polarization is normally expressed in V/I. Under certain conditions the amount of CD is similar to the amount of fraction circular polarization (that is m41 = m14), that is, when the optical elements are isotropic. Circular polarization can be described as V IR − IL = . I IR + IL Substituting I using Lambert–Beer law: e ∆A ln10/2 − e −∆A ln10/2  ∆A ln10  ∆A ln10 = tanh  ≈ ∆A ln10/2 −∆A ln10/2  2  2 e +e Circular polarization (for unpolarized incoming light) and CD are then interchangeable by: V 2πθ deg ∆A ln(10) = = I 180 2 Additionally, CD and optical activity are interchangeable using a Kramers–Kronig transformation [69] (for a more detailed description of the numerical methods see [70]): [ϕ k ( λ )] =

2 π

∫

∞

0

[θ ( µ )]

µ cl180 ∞ µ dµ d µ = 2 ∫ [(V / I )( µ )] 2 2 0 λ −µ π λ − µ2 2

Notably, when CD is measured using equipment wherein the incident light is modulated, such as is the case using commercially available circular dichrographs, special care should be taken in the case of materials that are linearly anisotropic. The presence of linear dichroism can result in significant differences by apparent CD [71,72]. In general, this is caused by crosstalk issues with the modulation of the incoming light. As the processes in linear polarization often results in effects more than one order of magnitude higher than those in circular polarization, even a slight linear component can have a large effect on the outcome. In a similar fashion, cross-talk can

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have a large effect in circular polarization spectroscopy as we will further discuss in Section 2.5.

2.3.3  Electronic transitions and rotational strength Circular polarization, optical rotatory dispersion and CD, depending on the wavelength, for absorption phenomena all correspond to specific orbital transitions. This results from a change in charge distribution, from a ground to an excited state. Importantly, for every electronic transition both a magnetic and electric dipole transition can be specified. The electric dipole transition is caused by the interaction of the electric field component E with the electric operator of the molecules dipole moment, which can result in a change in charge distribution. While in ordinary spectroscopy the magnetic dipole transitions produce negligible effects, its component is essential when dealing with polarization as it correlates with charge rotation. The transition dipole vector thus describes both the strength (which is proportional to the strength of the absorption) and the ideal interaction polarization state. The dipole strength of these transitions can thus be different for left- and right-handed circularly polarized light, which is essentially CD. We will provide a small background in the essential molecular processes leading to CD. For a more extended theoretical background, see, for example [68,73–76]. If we look at molecular absorbance, the integral of a normal absorption band, the integrated intensity, is proportional to the strength of the electric dipole D: D=

3(2303)hc 8π 3 N 0

ε

∫ λ dλ

where h, c, N0, and ε are the Planck’s constant, the speed of light in vacuum, Avogadro’s number, and the extinction coefficient, respectively, and ε is the molar extinction coefficient. The strength of the electric dipole is related to the electric dipole transition moment (µ) by D = µ0m ⋅ µm 0 = µ02m , for the transition 0 → m . Often the dipole strength is also expressed by the oscillator strength f, which is proportional: f =

8π 2m vD 3he

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where m is the mass, e the charge of an electron, and v the wavelength of maximal absorbance. The integrated CD spectrum of the transition is then proportional to the rotational strength R: R=

3(2303)hc 32π 3 N 0

∫

∆ε dλ λ

Note that ∆ε is the difference between the molar extinction coefficients for left- and right-handed circularly polarized light, respectively, and the integral is taken over the entire band corresponding to a single transition, µ. The rotational strength of a transition is additionally defined as the imaginary part of the dot product of the electric (µ) and magnetic dipole (m): R = Im{µ0m ⋅ mm 0 } Furthermore, the electric dipole transition moment is defined by:

µ0m = ∫ Ψ ∗0 µΨ a dτ = e ∫ Ψ ∗0 ( ∑i ri ) Ψ a dτ where Ψ ∗0 and ψa are the complex conjugate of the ground state wave function and the excited state waveform, respectively, and ri is the location of electron i. Importantly, this also shows why for absorption phenomena there can be no CD in the absence of absorption (i.e. when µ0m, R = 0). Again, the rotational strength is thus also dependent on the magnetic dipole transition moment: mm 0 = ∫ ψ a∗µψ 0 dτ =

e ψ a∗ ( Σ i ri × pi )ψ 0 dτ ∫ 2mc

where pi is the linear momentum operator. Importantly, for achiral molecules, which thus have an inversion center or a mirror plane, respectively, either the dot product of the electric and or magnetic dipole moment is zero (i.e. if µ0m ≠ 0 then mm0 = 0 and the other way around) or the electric and magnetic dipole vectors are orthogonal. In the case of the mirror plane, either µ0m or mm0 may lie in the plane but the other must be normal to this plane, that is, they are orthogonal. More generally, if we assume that there can be no CD or circular polarization without absorption, then in achiral molecules we will observe that the magnetic charge rotation has no preferred direction.

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Figure 2.4  Main bonding and antibonding orbital transitions and the typical associated bonds and wavelengths in organic molecules.

This additionally also implies why molecules need to be three-dimensionally asymmetric in order to show any optical activity and cannot be planar. All these situations are resulting in a R = 0, and display no intrinsic CD and circular polarization. R can thus only be nonzero if µ0m and mm0 are parallel or antiparallel, allowing them both to be nonzero. Additionally, in the case of chiral molecules, it should be noted that upon evaluation of the mirror enantiomer, µ0m changes to µm0, while mm0 remains the same, thus R becomes −R. Lastly, it is important to remember the sum rule [77]: ∑m R0m = 0, that is, the integral of circular polarization over all transitions is zero. An overview of the main bonding transitions relevant to biological molecules is given in Fig. 2.4.

2.3.4  Exciton coupling While these rules are valid for small organic molecules, biologically produced organic molecules are often much more complex and can contain various groups that can absorb electromagnetic radiation or even be optically active. Many of these molecules can additionally be found in molecular complexes such as oligomers. Often, these oligomers exhibit large circular polarization signals, which are the result of interactions between the different transition dipoles leading to a larger R than that of the individual noninteraction transition dipoles. It is very well possible to have an oligomer exhibiting circular polarization while the individual molecules do not, for instance, when they are planar but also when completely achiral [68,78]. A good example of this is the circular polarization of chlorophyll. Chlorophyll has a very planar structure and its intrinsic circular polarization signal is thus very small. When multiple chlorophylls interact excitonically, this results in the addition of multiple absorption bands and circular polarization signal that is more than one

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Figure 2.5  Exciton splitting by two chromophores with an oblique orientation and the corresponding result on the circular polarization spectrum.

order of magnitude larger [79]. Additionally, when, for instance, a strong absorbing achiral molecule binds or complexes with a chiral molecule, the induced chirality might produce circular polarization around the absorption band of the first molecule [80]. Although there are multiple possible combinations of mixing between electric and magnetic dipole transitions, these interactions are the most significant when the transitions of two or more molecules allows coupling (exciton coupling) [81]. Generally, when the transition dipoles are positioned parallel or antiparallel to each other the transition moments will be coupled. This results in a higher energy state if the transitions are parallel (thus a blueshift in the absorbance band) and results in a lower energy state if the transitions are antiparallel (thus a redshift in the absorbance band). This is due to repelling or attracting interactions, respectively, and is similar when the transition dipoles are positioned collinear [82]. In most cases the dipoles will be positioned neither perfectly parallel nor perfectly collinear to each other, but are positioned in an oblique orientation. Such an orientation results in exciton band splitting; two new bands appear in normal absorbance and in circular polarization at lower and higher energy and thus wavelengths, see also Fig. 2.5. The dipole strength for these two bands can then be given by: D + = 21 ( µ0m( a ) − µ0m( b ) )2

and

D − = 21 ( µ0m( a ) + µ0m( b ) )2

We can additionally identify multiple contributing processes to the total rotational strength. Such as the contribution of the intrinsic rotational strengths or the coupling between the electric transition dipole of the first molecule with the magnetic transition dipole of the second. However, in electronically coupled transitions the exciton coupling is much larger. The rotational strength is then given by:

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Figure 2.6  The protein myoglobin, which comprise mostly α-helices and the corresponding transitions which together result in the final circular polarization spectrum. Data obtained from The Protein Circular Dichroism Data Bank (PCDDB) [84,85].

R=

π rab ⋅ ( µ0m( a ) × µ0m( b ) ) 2 λ0 m

where rab is the vector between the center of the two molecules. Exciton coupled circular polarization too is conservative; the sum rule still applies. It is important to note that if an environmental factor causes the transition energy to change, such as is the case of changing temperature, the circular polarization spectrum changes but the first moment of R remains unchanged [79]. As the coupling strongly depends on the position and orientation of the transitions, measurements on such systems have proven to be a very powerful probe in determining the secondary structure of molecules such as proteins. For instance, by observing conformational changes using CD, the left-handed double helical structure of Z-DNA was first discovered [83]. An example of a biological molecule, the corresponding secondary structure, and the final spectrum is shown in Fig. 2.6.

2.3.5  Large aggregates (PSI-type) A very interesting phenomenon arises when observing large and dense supramolecular systems, which has been labeled polymer and salt-induced (PSI) CD. Measuring the CD of these systems can result in anomalously large circular polarization signals with optical activity even outside of the absorbance bands [86–88]. These signals are attributed to the combined long range chiral structure of such aggregates and the occurrence of energetic interactions over the whole aggregate.Whereas in smaller aggregates, the entire aggregate is in the same wave phase upon interaction, this is not the case in larger aggregates (larger than 1/20th of the wavelength) where radiation coupling mechanisms by multiple internal scattering

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will become as significant as normal dipole coupling [89,90]. As a consequence, large signals have been observed which are additionally largely determined by the pitch and the handedness of the aggregates [79,91]. The amplitude of the CD signal of condensed DNA, for instance, can be 2 orders of magnitude higher than that of dispersed DNA [86]. PSI-type signals of isolated chloroplasts also show large anomalously shaped bands superimposed on the excitonic signals [79]. Signals of similar shape and magnitude have also been detected in situ on intact vegetation leaves [92–95]. While the photosystems of bacteria can display large circular polarization signals [96], these are often not PSI-type signals but result from excitonic interactions. In other cases, the size of the photosystems and the presence or absence of PSI-type signals is dependent on the physiological conditions. An example of this is the size dependency of the chlorosomes of green sulfur bacteria [97].

2.4  CONSIDERATIONS FOR THE REMOTE SENSING OF HOMOCHIRALITY IN OUR SOLAR SYSTEM AND BEYOND 2.4.1  Wavelength considerations In the far-UV (FUV), amino acid enantiomers typically display fractional circular polarization signals. Many amino acids show strong signals at wavelengths well below 190 nm [98], while larger amino acids or aromatic amino acids generally only display signals above 190 nm. Transitions below 190 nm are generally associated with σ → σ ∗ transitions (see also Fig. 2.4) but also with charge-transfer transitions displayed by proteins [99,100]. In the FUV above 190 nm, circular polarization arises when peptide bonds are located in a regular, folded environment. The transitions associated with these bonds generally absorb FUV light in the range 190–230 nm and the shape of the polarization spectrum can be used to extract conformational information about the protein backbone and its secondary structure. The main transitions in peptides are n → π ∗ at ∼220 nm and π → π ∗ at ∼190 nm, with a contribution from aromatic amino acid side chains. The α-helix (see also Fig. 2.5), β-sheet, and random structures give rise to characteristic shapes in the CD spectrum [68,101]. In the near-UV (250–350 nm), the circular polarization spectrum is sensitive to protein side chains and disulfide bonds. Many aromatic side chains only show induced circular polarization and are therefore indicative of the local molecular environment. Hence, throughout the entire UV, there

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is a wealth of structure in the polarization spectrum related to the hierarchy of chiral structures of fundamental biological material [68,102–104]. While some electronic transitions display circular polarization in Vis, often these are broad and weak. Generally, strong signals are displayed by ligands and conjugated systems. Good examples of a ligand are cyclic tetrapyrroles, such as the iron ligand heme (which colors our blood red) or the magnesium ligand chlorophyll a which is used in photosynthesis. Importantly, especially heme but also largely chlorophyll are planar molecules. Both molecules will thus only display circular polarization by induced chirality. Another important contribution to visible circular polarization is conjugated systems. Each additional bond in a conjugated π system will redshift the overall absorbance. Note that both heme and chlorophyll are also conjugated systems; the ring systems that allow complexation contain a lot of conjugating π transitions. Infrared (IR) CD spectroscopy, like UV-Vis CD spectroscopy, is used to provide chiral molecular conformational information in stereochemistry through rotational, vibrational, and Raman optical activity [105–107]. The near-IR has amide bands and stretching mode transitions in the range 2–3.5 µm, and the mid-IR reveals vibrational transitions, sensitive to the presence of amide bands associated with α-helix and β-pleated sheet structures in the range 5.8–6.8 µm [108,109]. While proven to be an excellent probe for molecular stereochemical investigations, the signals are often weak in nature [110]. For in situ detection of homochirality in the IR, however, vibrational Raman spectroscopy shows to be promising [111].

2.4.2  In situ observations While currently no instruments capable of detecting (vibrational) CD have been deployed in solar system missions, up to 2017 two other instruments capable of making chirality measurements have been deployed on extraterrestrial surfaces. The COSAC instrument onboard the Philae lander, part of the Rosetta mission, contained a multicolumn enantio-selective gas chromatograph (GC) coupled to a linear reflectron time-of-flight mass-spectrometer instrument to analyze organic compounds on the surface of comet 67P/Churyumov-Gerasimenko [112]. Unfortunately, due to the short lifetime of the lander on the comet’s surface no chirality data were obtained [113].The Sample Analysis at Mars instrument suite (SAM) onboard the Curiosity rover also is equipped with a chiral column to measure enantiomers of volatile organic compounds [114]. So far, no data have been reported on the enantiomers of the organic compounds detected [115,116]. It has been

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stated that the search for amino acid homochirality is one of the highest science goals of the ExoMars Rover mission [117]. The Urey Instrument suite developed for the ExoMars mission, but downselected, employed a combination of extraction, reaction with an agent, and detection with microcapillary electrophoresis to test for enantiomers [118]. The Mars Organic Molecule Analyzer (MOMA) onboard ExoMars 2020, an instrument suite combining pyrolysis GC and laser desorption with mass spectrometry, will carry one chiral column to enable the GC to separate enantiomers [119]. Near future missions will also include dedicated Raman spectrometers. These are the Raman Laser Spectrometer (RLS) on board the 2018 ExoMars Rover mission [120,121] and the Scanning Habitable Environments with Raman & Luminescence for Organics & Chemicals (SHERLOC) which will be deployed on the Mars 2020 rover [122,123]. The mentioned instruments are, however, incapable of detecting Raman optical activity. F ­ uture missions could potentially include such an instrument and/or a dedicated circular spectropolarimeter (see Section 2.5 for instrumental considerations) to indisputably show the possible presence of homochirality on Mars or other solar system bodies. The use of a full Stokes spectropolarimeter will allow greater characterization of the surface. Importantly, such in situ spectropolarimetry will aid in improving remote observations.

2.4.3  Solar system observations (remote) Circular polarization of scattered light from planetary surfaces is a common phenomenon observed throughout the solar system [124] and fractional circular polarization has been measured for Venus, Mercury, Moon, Mars, Jupiter, Saturn Uranus, and Neptune [125–128]. Different mechanisms are at work for gaseous surfaces (Jupiter, Saturn, Uranus, Neptune), including Venus, where the dense atmosphere obscures the surface, and solid surfaces like Mercury and the Moon. Circular polarization of light scattered from gaseous surfaces is attributed to the presence of spherical and nonspherical particles in the atmosphere [129–131]. Circular polarization of light scattered from solid, rough surfaces is a double reflection mechanism, where the first reflection from a crystal grain leads to linear polarization of the incident light, which subsequently hits a neighboring grain. Due to absorption by the second grain the linearly polarized incident light gets elliptically polarized upon this second reflection [132]. When combined with analog research and laboratory measurements, observations of the circular polarization of scattered light, therefore, can give a good remote insight into the composition of the atmosphere or the surface of these planets.

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As described earlier, CD, the differential absorbance of left- and righthanded circularly polarized light, is an excellent indicator of the chirality of organic compounds. In addition to chiral organic molecules, however, a range of morphologically chiral minerals exists, with d-(+)- and l-(−)quartz (SiO2) as a commonly occurring example [133].These chiral properties of minerals and their subsequent asymmetrical catalytic functions have been suggested as leading to the first enantioenrichments in chiral molecules [134–136]. Several instruments have been proposed for remote observations of circular polarization of planetary surfaces, including for Mercury [124] and Mars [137,138]. The instrument proposed for Mercury, the optical detector for enantiomorphism (ODE) is designed to detect enantiomorph crystals [124]. Spectral polarimetry has been proposed before in the case of Mars as a technique for the remote observation of homochirality [137,138]. For instruments focused on the detection of homochirality as biosignature care has to be taken to ensure an optimal distinction between homochirality of organic compounds and crystal chirality, to avoid false-positives. Laboratory measurements have consistently shown that mineral surfaces show a much weaker circular polarization signal that is also different in shape than those of biological nature [96,139]. Additionally, attesting to a general lack of false-positives, surface circular polarization imaging of Mars has so far not yielded any significant results [133]. The next targets within our solar system can be the liquid water containing moons of Jupiter and Saturns. Unlike Mars, however, such observations will likely need to be achieved through an orbiter rather than from the Earth.

2.4.4  Exoplanet observations To increase our chances of finding extraterrestrial life, we ultimately have to look outside our solar system. It has been estimated that every star out of the 100–400 billion stars in our galaxy contains at least one planet [140]. It has furthermore been estimated that the occurrence of rocky exoplanets in the habitable zone ranges from 2% to 20% per stellar system ([141] and references therein). While the majority of these stellar systems are very far away, which renders the detection of life on any orbiting exoplanet unlikely, already at least 80 systems can be found within 5 parsecs or 16.3 light-years [142]. Polarimetry in general has a lot of advantages in both the detection and characterization of exoplanets. Polarimetry allows for enhancing the contracts between the very bright light of a star (which light can be assumed

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to be unpolarized [143]) and the very dim light reflected of an exoplanet (which often is very strongly linearly polarized) [144,145]. As the linear polarization depends (beside other parameters) on both surface and atmospheric parameters, these observations will additionally allow to characterize potential atmospheric biosignatures, for example, O2 in combination with other gases out of thermodynamic equilibrium, such as CH4 [144,146]. Detection of neither of these gases is, however, free of false-positive scenarios [147–150]. While these means are very indicative of a planet’s habitability, they are thus not conclusive. Homochirality and its corresponding signals in circular polarization is a much more exclusive and unambiguous biosignature. An important consideration, however, is that the signal is relatively weak: typically, the circular polarization displayed by biological materials is less than 1% (see Section 2.5 for instrumentation considerations). In order to detect life on an exoplanet, it thus has to be relatively abundant at the surface. On Earth, this role is fulfilled by photosynthetic organisms. Photosynthesis is one of the most important hallmarks of life on Earth: the capacity to collect energy from the sun and to convert this into chemical energy. Photosynthesis is the major driving force of life on Earth and evolved very soon after the emergence of life itself [151–153]. The organic carbon production is more than 4 orders of magnitude higher than any other source [151]. Additionally, virtually all O2 on Earth derives from biological photosynthesis and if photosynthesis came to halt, the atmosphere would be depleted [154]. In terms of productivity and surface features, photosynthesis is thus the most likely target. Phototrophic organisms show a clear circular polarization signal around their maximum absorbance bands, in the blue but also mainly in the red. Typically, a sign change in V/I is observed around the maximum absorbance. See also Fig. 2.7 for an example of the circular polarization spectra produced by phototrophic organism. Similar to Earth, it is likely that the dominant phototrophic organism on an exoplanet evolves towards the optimal use of the light irradiated by its star [155]. Within our galaxy, M stars or “red dwarfs” are the most common stars and comprise nearly all of the stars that are close to Earth [156]. The incident radiation of red dwarfs is more red-shifted and it is thus likely that the pigments are redshifted as well [155]. As the photonic energy decreases with increasing wavelength, this might put constraints on the excited state redox potential. However, even water splitting oxygenic photosynthesis is, in theory, possible using light up to 2100 nm, although it might be difficult

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Figure 2.7  Circular polarization spectra of photosynthetic organisms. (A) Ivy leaves in transmission. (B) Ivy leaves in reflectance. A and B are taken with TreePol. (C) Cyanobacteria in transmission. (D) Cyanobacteria in reflectance. (C) and (D) are taken with the PEM-based polarimeter of Sparks et al. (A) is adapted from [92], (C) and (D) are adapted from [96].

to evolve the incredibly complex molecular machinery required for such reactions [157]. In any case, it will be most likely to observe circular polarization signals in the wavelength corresponding to the star’s maximum photon flux.

2.5  INSTRUMENTATION It is clear that any remotely observed circular polarization signal due to homochirality in living organisms will be tiny and that instrumentation to observe it has to be highly dedicated and optimized. Moreover, generally the incident light upon the “sample” under consideration cannot be controlled, and, in the case of direct sunlight or starlight, can be assumed unpolarized. Also, the scene or target under study can be quite dynamic,

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either because it is alive or otherwise moving around, or it is observed from an orbiting platform or, in the case of ground-based astronomical observations, through a turbulent atmosphere. Spectropolarimetric instrumentation for remote sensing of (homo)chiral molecules is therefore quite different from laboratory equipment that is used for chiral sensing. Here, we review polarimetric measurement approaches commensurate with the challenging task of remotely measuring circular polarization spectra (through spectropolarimetry or hyperspectral polarimetric imaging) at high sensitivity and accuracy. We confine ourselves to the “optical” spectral range from the UV to the mid-IR.

2.5.1  Polarization measurement approaches 2.5.1.1  Terminology: polarimetric sensitivity and accuracy The literature often suffers from conflicting or even erroneous definitions and conventions for describing the quality of polarimetric measurements. Therefore, we first lay some groundwork by stating some basic definitions (cf. [158]). The polarimetric sensitivity is defined to be the noise level in fractional polarization above which a real signal (in this case V/I(λ)) can be detected. Noise in spectropolarimetric observations can be due to photon noise, detector noise, etc., but can also come in the form of noise-like systematic effects like polarized spectral fringing. Fundamentally, the polarimetric sensitivity is limited by photon noise, as the standard deviation upon the differential intensity measurement to derive Stokes V is identical to that of the total intensity measurement (because the photon shot noise is governed by a completely random process). Hence cf. Poissonian statistics—the standard deviation of Stokes V is equal to the square root of the total number of collected photoelectrons, and NI V = σ  = I NI

1 NI

with NI the number of collected photoelectrons per spectral bin.Therefore, to reach a polarimetric sensitivity of better than 10−4, one needs to collect at least 108 photons per bins, and control all other noise-like systematic effects down to this level too. Once a real polarization signal has been detected well above the noise, the polarimetric accuracy describes to what extent the measurement after application of the polarization calibration agrees with the true input signal.

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Generally, for a complete Stokes vector measurement, the polarimetric accuracy is described by a 4 × 4 matrix. For a mere measurement of V/I(λ) in the absence of any linear polarization effects, the polarimetric accuracy has two components: the zero point (I → V component) and the scaling (V → V component) of the measurement. For the tiny signals that we are after for remote sensing of homochirality, it is mostly the inaccurate knowledge of the zero point that is particularly bothersome. Polarization calibration is typically accurate to 10−3 for the zero point and has a relative accuracy of ∼1% on the scaling. However, zero-point drifts are often spectrally relatively flat, such that a 25% of Stokes U (defined here at ±45° w.r.t. the mirror’s S and P axes) into Stokes V [158]. But also a totally rotationally symmetric refractive imaging system on-axis can easily induce cross-talk at the ∼1% level, due to stress-induced birefringence of any of the lenses.This stress can be intrinsic, as a residual from the annealing process of the glass, or externally applied by the mechanical mounting, or by a coating

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or other glass element that is applied/connected at a different temperature and has a different thermal expansion coefficient. It is therefore crucial to design an instrument such that such cross-talk effects are eliminated, or at least mitigated. One solution would be to locate the polarimetric optics in front of any optics [143]. Still, one will then need to calibrate the full-Stokes response of the polarization modulator at relative levels of 85

>85

>85

>85

20,000

200,000

200,000

10,000

10

8–20

8–13

10

20

6

15–30

20 (Continued)

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Kenneth W. Busch and Marianna A. Busch

Table 3.1  Comparison of Some Biréfringent Two-Prism Polarizersa (cont.) Material Wollaston Glan-Taylor Glan-ThompRochon MgF2 Calcite Calcite son Calcite

Optical path 18–28 17–29 length (mm) Applications Experiments Spectroscopy; where both laser applicapolarizations tions are needed

28–40

16–28

High-quality imaging

Experiments where both polarizations are needed

a Source of information: https://www.edmundoptics.com/resources/application-notes/optics/polarizer-selection-guide/.

Figure 3.21  The wire-grid polarizer: Ax and Ay are the electric vectors of the unpolarized light, where Ax = Ay.

Consider an unpolarized beam of light made up of two orthogonal components Ax and Ay as shown in Fig. 3.21. When the beam encounters the wire grid, the vertical electric vector Ay, which is parallel to the wires, will induce alternating currents in the wires that will lead to dissipation by Joule heating (I2R, where I is the current induced in the wire and R is its resistance). As a result, the vertical electric vector will be absorbed by the grid. By contrast, the horizontal electric vector Ax is not shorted out by the wire grid and is transmitted. Because the spacing between the wires must be small with respect to the wavelength of the incident light, wire polarizers are only feasible for long-wavelength radiation like the infrared.24 24

 ire grid polarization can easily be demonstrated with microwaves, where an ordinary W oven rack can be used.

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3.4.5  Polarization by dichroism Polarization by dichroism is similar, in some respects, to polarization with wire grids in that polarization by dichroism involves preferential absorption of one component of unpolarized light and transmission of the other. Dichroism is a property of certain materials that have the ability to absorb light vibrating in a particular plane more strongly than light vibrating in an orthogonal plane. The phenomenon of dichroism was discovered in 1815 by the French scientist Jean Biot while studying the optical properties of tourmaline [13]. Biot observed that the color of a tourmaline crystal varied when viewed through a slowly rotating linear polarizer. While certain single crystals, like tourmaline, exhibit dichroism, their use as polarizers is limited by the high cost and poor performance parameters. Perhaps the most serious limitation is the lack of availability of large, single crystals with which to make polarizers with sufficient apertures. In spite of efforts to grow single crystals of dichroic materials in the laboratory, the results achieved to date have been limited [14]. To circumvent the problem caused by a general lack of suitable, large, single crystals of dichroic materials, Edwin Land reasoned that it should be possible to duplicate the effect of a single crystal with a statistical array of microcrystals of the appropriate type [15]. If an array of microcrystals could, by some means, be suspended in a transparent matrix and aligned so that their absorption axes were roughly parallel, a sheet of this material would behave like a thin single crystal of dichroic material. Using crystals of quinine iodosulfate25 [16,17] suspended in cellulose acetate, Land was able to fabricate the first dichroic sheet polarizer (termed the J-sheet polarizer by the Polaroid Corporation). Today, the J-sheet microcrystalline polarizers have largely been ­superseded by molecular-sheet polarizers. Molecular-sheet polarizers are made by orienting the transition dipole moments of molecules in a plastic sheet so that they are all aligned in a particular direction. In this way, light waves with their electric vectors aligned with the transition dipole moments of the absorbing molecules will be absorbed. Light waves with their electric vectors orthogonal to the transition dipole moments of the aligned ­molecules will not be absorbed. In H-type sheet polarizers, sheets 25

 uinine iodosulfate (C80H194I6N8O20S3), a derivative of quinine from the bark of the Q cinchona tree, is a crystalline alkaloid material with a polarizing power five times that of tourmaline. It is sometimes called Herpathite after its discoverer William Herapath, an English physician.

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of ­polyvinyl alcohol (PVA) are stretched prior to being stained with molecular iodine. The procedure orients the iodine molecules so that they align parallel to the PVA polymer axis (which has been aligned in a particular direction by stretching). In K-type sheet polarizers, an oriented PVA film is heated in the presence of a catalyst (typically HCl), where it undergoes dehydration. After dehydration, the film becomes highly dichroic. K-type sheets have their transmission axis perpendicular to the direction of stretch. Because they are not doped (like H-sheet polarizers), K-sheet polarizers are more tolerant of higher temperatures. Sheet polarizers have the following advantages: • low cost, • large acceptance angle (∼30°), and • large linear aperture (5 cm or larger). While sheet polarizers are available in the near-UV region, their extinction ratios compared with prism polarizers are poor. In addition, most sheet polarizers that employ glass protective plates will not transmit light below 330 nm. Finally, sheet polarizers are not recommended for high-power applications because the unwanted polarization component is actually absorbed by the polarizing film where it is ultimately converted into heat, which must be dissipated.

3.5  PRODUCTION OF CIRCULARLY POLARIZED LIGHT Generation of circularly polarized light is important in chiroptical instruments like circular dichroism spectrometers. Production of circularly polarized light can be envisioned as a two-step process. First, unpolarized light is linearly polarized and then the linearly polarized beam is subsequently decomposed into circularly polarized light by means of a polarization-form converter known as a retarder.26 Retarders perform two basic functions: They resolve an incident linearly polarized beam into two orthogonal beams (a vertical component and a horizontal component) and then retard one of the beams relative to the other so that the two beams emerge from the retarder with a phase difference of ±γ. Fig. 3.22 illustrates the process to produce circularly polarized light. As shown in the figure, linearly polarized light strikes the calcite retarder so that there is a 45° angle between the vibration plane of the 26

Retarders are also known as phase shifters or wave plates. If a retarder produces a phase shift of ±90°, it is known as a quarter-wave plate.

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Figure 3.22  Production of circularly polarized light using a birefringent retarder plate: Ae and Ao are the electric vectors that make up the incident linearly polarized light whose vibration plane is at a 45° angle to the optical axis of the retarder plate. Because the vibration plane is at 45°, Ae = Ao.

polarized light and the optic axis (the fast axis27) of the retarder. Because the beam is at a 45° to the axis of the retarder, it can be ­resolved into two orthogonal components with equal magnitudes— an O-­component (Ao) in the horizontal plane and an E-component (Ae) in the vertical plane. If the retarder is made of a negative crystal like calcite, where no > ne , the E-ray, whose vibrations are parallel to the optic axis of the retarder, will travel faster than the O-ray. Both orthogonal beams traverse the retarder along the same ray path, but with different optical path lengths.28 For the O-ray, the optical path length is given by nod, where d is the thickness of the retarder plate. For the E-ray, the optical path length will be ned. Comparing both rays, the optical path length difference, ∆, between the rays when they emerge from the retarder will be (3.8) ∆ = (no − ne )d This optical path length difference, ∆, can be converted into a corresponding phase difference, γ (in radians), by multiplying by 2π/λ (see Eq. (3.2)) to give  or a negative crystal like calcite, the fast axis is parallel to the optic axis. For a positive F crystal, the reverse is true (i.e. the slow axis is parallel to the optic axis). 28 The optical path of a ray is the product of the refractive index for the ray times the distance the ray travels in the medium. 27

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2π γ =   (no − ne )d  λ  (3.9) Eq. (3.9) shows that the phase difference produced by the retarder for a given wavelength depends on the refractive index difference between the O- and E-rays and the thickness of the retarder plate. Since a phase-shift of 2π does not alter the waves, a multiple of 2π can be added to the left-hand side of Eq. (3.9) to give plate thicknesses that are realistic: 2π  m(2π ) + γ =  ( n − ne )d  λ  o (3.10) where m is an integer known as the order. When m = 0, Eq. (3.10) reduces to Eq. (3.9). Wave plates where m = 0 are known as zero-order plates, whereas those with a positive value for m are known as multiple-order retarders. Using calcite as an example, Eq. (3.10) can be used to get some idea of the plate thickness needed to produce circularly polarized light. For calcite at 589 nm, no is 1.658 and ne is 1.486. To produce circularly polarized light, a phase difference of 90° (π/2) between the O- and E-rays is needed.29 For a zero-order retarder, Eq. (3.9) gives a value of 0.856 µm for d. This is far too thin to be practical to fabricate and to use. By contrast, a multiple-order (m = 100, for example) quarter-wave plate can be made from calcite with a plate thickness of d = 0.343 mm. Such a plate must be ground to the precise thickness and optically polished. Since, in calcite, the E-ray travels faster than the O-ray, a quarter-wave plate of calcite will produce left-circularly polarized light. Since the zeroth-order wave plates have a larger acceptance angle than multiple-order wave plates, other materials besides calcite are often used as retarders. Mica, a birefringent mineral substance, offers a number of advantages as a retarder. At 589 nm, (no − ne ) is on the order of 0.005. Using this value in Eq. (3.9) gives a zeroth-order thickness for mica quarterwave plates of 0.059 mm. Since mica is made up of thin sheets, thicknesses of this magnitude can easily be achieved. Of considerable significance to its use as a retarder, the refractive indices for the O- and E-rays in mica 29

Since one wavelength corresponds to a phasor rotation of 360° or 2π, a phase shift of 90° corresponds to λ/4. As a result, a retarder that produces a 90° phase shift is referred to as a quarter-wave plate. By analogy, a half-wave plate would produce a phase shift of 180°.

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do not vary appreciably with wavelength. This means that mica can be used over a wide range of wavelengths (400–700 nm) with satisfactory performance.30

3.6  PRODUCTION OF ELLIPTICALLY POLARIZED LIGHT Elliptically polarized light is produced basically in the same manner as circularly polarized light with linearly polarized light incident on a retarder as shown previously in Fig. 3.22. Instead of orienting the vibration plane of the incident linearly polarized light at 45° with respect to the optic axis of the retarder (as was done to produce circularly polarized light), another angle is used. At this other angle, Ao ≠ Ae, and the result is elliptically polarized light.

3.7  PHOTOELASTIC MODULATORS Certain modern chiroptical instruments, like circular dichroism s­pectrometers, measure the absorbance difference of a sample for left- and right-circularly polarized light. Because this difference is quite small, AC measurements are used to extract the desired information (the small absorbance difference) from other larger potential instrumental variations like source drift, which would interfere with measurements on a longer time scale. To accomplish this goal, a high-speed alternating source of left- and right-circularly polarized light is needed. High-speed generation of an alternating source of left- and right-circularly polarized light can be accomplished with a device known as a photoelastic modulator (PEM). PEMs are based on the photoelastic effect whereby birefringence is induced in a transparent solid material like silica by application of stress.31 Fig. 3.23A shows a schematic diagram of a PEM, where a rectangular bar of a suitable material like silica is coupled to a piezoelectric transducer. PEMs can be thought of as variable retarders. When an electrical signal is applied to the piezoelectric transducer, the silica bar vibrates at a resonant frequency determined by the length of the bar.The piezoelectric transducer, which is driven by an electric control circuit that controls the amplitude of  y comparison, for quartz, only a 14-nm range can be tolerated for a 90 ± 1° phase shift. B This means that a quartz wave plate must be selected for the wavelength of anticipated use. 31 The birefringence induced in the optical element by the applied stress is proportional to the resulting strain produced. 30

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Figure 3.23  PEM. (A) Axis of the modulator; (B) Linearly polarized light incident at 45° to optical axis of modulator: Ex and Ey are the electric vectors that make up the incident linearly polarized light. Because the vibration plane is at a 45°, Ex = Ey.

the vibration, is tuned to the resonant frequency of the optical element (say 50 kHz). The birefringence induced in the rectangular bar by the ­vibration of the piezoelectric transducer varies with time and is a maximum in the center of the optical bar. Fig. 3.23B illustrates the effect of the PEM on a linearly polarized beam that is incident at 45° to the modulator axis (horizontal plane). This 45° incident beam can be considered to be composed of a vertical oscillating electric vector (Ey) and a horizontal oscillating electric vector (Ex) of equal magnitude. As the piezoelectric transducer applies stress to the

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optical bar, birefringence is induced, and the bar behaves like a variable retarder. The amount of retardation, ∆(t), produced is a function of time and is given by ∆(t ) = d[ nx (t ) − ny (t )] (3.11) where ∆(t) is the optical path difference as a function of time, d is the thickness of the optical bar, nx(t) is the instantaneous value of the ­refractive index in the horizontal plane, and ny(t) is the instantaneous value of the refractive index in the vertical plane. In terms of a phase difference, γ, Eq. (3.11) can be written as 2π (3.12) γ (t ) =   [ nx (t ) − ny (t )]d  λ  where λ is the wavelength of the linearly polarized incident beam. We are now in a position to consider the retardation produced over a complete cycle of the piezoelectric oscillator. When the optical bar is not stressed, no birefringence is produced, and nx = ny. At this point in the cycle, γ (0) = 0 , and the incident linearly polarized light at 45° passes through the modulator unaffected (see point a in Fig. 3.24A). When the optical bar is stretched by the piezoelectric transducer, the refractive index in the vertical plane, ny, decreases relative to that in the horizontal plane, nx. This means that electric vector of the vertical component will lead the electric vector of the horizontal component. According to Eq. (3.12), this will produce a positive phase shift that will increase until the difference between nx and ny reaches a maximum (when the optical bar is stretched to the maximum by the piezoelectric transducer—see point b in Fig. 3.24A). By adjusting the magnitude of the control signal applied to the piezoelectric modulator, the maximum positive phase shift can be adjusted so that it is equal to γ = +90° . In this condition, the PEM will behave as a quarter-wave plate, and right-circularly polarized light will emerge from the modulator. When the optical bar is compressed by the piezoelectric transducer, the refractive index of the horizontal component, nx, decreases relative to the refractive index in the vertical plane, ny. In this condition, the electric vector of the horizontal component will lead the electric vector of the vertical component. According to Eq. (3.12), this will produce a negative phase shift that will increase until the difference between nx and ny once again reaches

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Figure 3.24 (A) γ(t) versus time. (B) Polarization state of emerging light. (C) Condition of optical element as a function of time.

a maximum32 (when the optical bar is compressed to the maximum by the piezoelectric transducer— see point c in Fig. 3.24A). With the control ­voltage set to the proper level, the maximum phase shift in this condition will be γ = −90° . A phase shift of −90° will produce left-circularly polarized light. As shown in Fig. 3.24B, as the piezoelectric transducer goes through a complete cycle, the output of the modulator will cycle between linearly polarized light, elliptically polarized light, and circularly polarized light. At the maximum stretching condition of the cycle, right-circularly ­polarized light will emerge from the modulator. At the maximum ­compression ­condition of the piezoelectric cycle, left-circularly polarized light will be produced. 32

Since ny>nx, the difference will be negative.

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PART B—SIGNAL HANDLING  

3.8  NOISE IN ELECTRICAL CIRCUITS It is a fact that all electrical signals are contaminated to a greater or lesser extent by unwanted, extraneous signals that are termed noise [18,19]. Recovering weak signals that have been contaminated by large amounts of noise can be challenging and requires a basic knowledge of noise sources and how they behave. We will begin by discussing two major categories of noise based on their frequency dependence.

3.8.1  White noise There are two universal, fundamental sources of white noise in electrical circuits that arise because of the granular nature of charge carriers. The first of these is shot noise described in 1918 by W. Schottky [20]. Shot noise arises when charge carriers randomly cross an energy barrier. Schottky showed that the mean-squared current that arises from the combined effect of charge carriers crossing an energy barrier is given by i 2 = 2ei ∆f (3.13) where i 2 is the mean-squared shot noise current in amperes, e is the value of the electronic charge in coulombs (1.6 × 10−19 C), i is the average current in amperes, and ∆f is the frequency bandwidth of the measurement system in Hz. Eq. (3.13) reveals that shot noise is independent of frequency, and its magnitude depends on the average current and the bandwidth of the measurement system. If the mean-squared shot noise current flows through a resistor, the noise power produced will be given by (3.14) Pshot = i 2 R = 2eiR ∆f where Pshot is the shot-noise power in watts. Eq. (3.14) is useful because noise powers are additive. Since the magnitude of the shot-noise power does not vary with frequency, it is referred to as “white” noise (by analogy with white light being made up of all frequencies). If we take the square root of Eq. (3.13), the result is

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irms = i 2 = 2ei ∆f (3.15) where irms is the root-mean-square value of the shot-noise current. In preparing a plot of noise versus frequency, it is useful to express Eq. (3.13) in terms of the spectral density [21] given by

i2 ∆f

(3.16) S( f ) = = 2ei where S(  f  ) is the spectral noise density in A2 Hz−1 According to Eq. (3.16), for a signal with an average current of 10 nA, the spectral noise density arising from shot noise will be 3.2 × 10−9 nA2/Hz. Another source of white noise arises from the random fluctuation of electrons in resistive components. This type of white noise is known as thermal noise or Johnson [22] noise. Based on statistical thermodynamic arguments, Nyquist [23] showed that the thermal noise voltage in a resistor would be given by e 2 = 4 RkT ∆f (3.17) where e 2 is the mean-squared thermal noise voltage, R is the resistance in ohms, k is Boltzmann’s constant (1.37 × 10−23 W s K−1), T is the temperature in Kelvin, and ∆f is the frequency response bandwidth in Hz. Eq. (3.17) can be converted into a mean-squared current by dividing by R2, giving e2 4kT ∆f (3.18) i2 = 2 = R R Eq. (3.18) reveals that the thermal noise current can be reduced by ­cooling the resistor. Converting Eq. (3.18) into a spectral density gives

i2 4 kT (3.19) S( f ) = = ∆f R To get an idea about the magnitude of thermal noise, consider an amplifier with an input resistance of 10 MΩ at 300 K. From Eq. (3.19), the spectral noise density from thermal noise would be 1.6 × 10−9 nA2/Hz.

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3.8.2  Flicker noise In contrast to shot noise and thermal noise, both of which are independent of frequency, flicker noise or 1/f noise is a function of frequency [24]. Since the exact origin of flicker noise is not known, the usual expression given for it is an empirical one: Ki α ∆f (3.20) i2 = fβ where K is a constant, i is the current, α is a constant that is ∼2, β is a ­constant that is ∼1, ∆f is the frequency response bandwidth of the system in Hz, and f is the frequency in Hz. Expressing Eq. (3.20) in terms of spectral density gives i2 Ki 2 (3.21) S( f ) = = f ∆f If we take the log of Eq. (3.21), the result is (3.22) log S( f ) = log( Ki 2 ) − log f which is the equation for a straight line with a slope of −1 on a log–log plot. We will use Eq. (3.22) when we plot spectral noise density versus frequency. With this background in mind, we can represent the two major categories of noise in graphical form on a log–log plot. Fig. 3.25 shows a hypothetical plot of the log of noise spectral density in nA2/ per unit bandwidth in Hz versus the log of the frequency in Hz. The plot is hypothetical because, while we know the slope of the 1/fnoise line from Eq. (3.22), we do not know the value for the first term, and K must be determined empirically.33 In the lower frequency range of the plot, the noise is dominated by flicker noise or 1/f noise, so called because it follows an approximately 1/f distribution. At higher frequencies, white noise, which is independent of frequency, becomes dominant. At harmonics of the power line frequency (60 Hz), there are spikes in the spectral density plot due to interference. These interference spikes are not fundamental sources of noise and, in principle, can be reduced or eliminated by ­shielding 33

I n the plot, a corner frequency of about 10Hz was chosen arbitrarily, which is a reasonable choice for the onset of 1/f dominance.

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Figure 3.25  Plot of the logarithm of the noise spectral density (nA2 Hz−1) versus the ­logarithm of the frequency (Hz).

the experiment in a Faraday cage. In practice, it is best simply to avoid frequencies near 60 Hz and its odd multiples.

3.9  NOISE REDUCTION STRATEGIES The goal of all noise reduction strategies is to improve the signal-tonoise ratio (see Appendix B). For measurement systems dominated by white noise, signal-to-noise performance can be enhanced by bandwidth reduction, signal averaging, and integration. Eqs. (3.13) and (3.18) reveal that both shot noise and thermal noise depend on the frequency response bandwidth (∆f) of the measurement system. Reducing the bandwidth (∆f ) of a measurement system can be accomplished by increasing the time constant34 of a low-pass filter in the output of the measurement train. As with all forms of noise reduction, the trade-off for improved signal-to-noise ratio with bandwidth reduction is an increase in the time required to make a measurement (since the response time for the measurement is longer, the measurement system will respond more slowly to changes in signal level). Signal averaging is another means for improving signal-to-noise performance under white-noise limited conditions. With signal averaging, it can be shown (see Appendix B) that the signal-to-noise ratio increases as the square-root of the number of observations included in the average. Again

34

 he time constant for a simple RC low-pass filter is the product of the resistance in ohms T and the capacitance in farads, which has units of seconds. The time constant is the time required for current flowing through the resistor to charge the output capacitor to 63% of full charge.

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the trade-off for increasing the signal-to-noise ratio is the increased time required to accumulate the observations in the average. A third means of improving the signal-to-noise ratio is by means of signal integration where the signal is integrated by a circuit (see Appendix B) over some time period. In this case, the signal will increase directly with time, while the random white-noise component (which is equally likely to be positive or negative) will tend to average to zero over time. While bandwidth reduction, signal averaging, and integration are all effective means of reducing white noise, they are all ineffective in reducing 1/f noise. One way to solve this dilemma is to avoid the frequency region where 1/f noise is dominant. If the signal of interest falls in the frequency range where 1/f noise is dominant, we can shift it to higher frequencies by a process known as amplitude modulation [25] as shown in Fig. 3.26. Fig. 3.26 shows a plot of noise power versus frequency. Superimposed on this plot is a small band of frequencies centered on ws that constitute the signal of interest. Notice that the signal of interest falls in a region of high 1/f-noise power. Also shown in the figure is a higher frequency signal wc, known as the carrier. By the process of amplitude modulation, the low-frequency signal band can be imposed on the amplitude of a high-frequency carrier signal, generating two side bands symmetrically displaced about the carrier frequency (see Appendix B). In this way, the information of interest can be shifted from the region of high 1/f noise power to a region where 1/f noise is minimal.

Figure 3.26  Noise power versus frequency showing the side bands superimposed on a 1/f-noise distribution, where ws is the signal of interest and wc is the higher frequency carrier signal.

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Before discussing the details of amplitude modulation, it is first important to discuss the notion of a transmission channel. To appreciate the process, it is necessary to understand exactly which noise sources will be reduced and which will not. Fig. 3.27 illustrates the broad features of the process. A low-frequency signal of interest is encoded on a high-frequency carrier by the process of amplitude modulation. This high-frequency carrier signal is then transmitted by some means to a detector, and the detector signal is down-shifted back to the original frequency range through the process of demodulation. If we look at the process in terms of a signal train in space as shown in Fig. 3.27, the region starting with the modulator and ending with the demodulator is the transmission channel. Only noise introduced in this transmission channel can be reduced through the process of modulation/ demodulation. Any noise (including 1/f noise) that exists on the signal prior to the modulator will be modulated along with the signal of interest and subsequently demodulated by the demodulator. Since this noise will be encoded on the carrier wave along with the signal of interest, the demodulator will not be able to distinguish it from the signal of interest. By contrast, any extraneous signals and noise introduced in the transmission channel will not be modulated and can therefore be blocked by the demodulator. Aside from ambient signals introduced in the transmission channel, noise introduced from the detector itself falls within the transmission channel. In the infrared region of the spectrum, in particular, spectrometers are often detector-noise limited by 1/f noise arising in the detector. In addition to detector noise, any amplifier drift will also be blocked. We are now in a position to look at amplitude modulation in more detail.

3.9.1  Amplitude modulation As discussed previously, amplitude modulation occurs when the amplitude of a high-frequency signal (the carrier) is varied or modulated by a lower

Figure 3.27  The modulation/demodulation process showing the transmission channel.

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frequency signal containing the information to be transmitted. Consider a high-frequency carrier signal given by (3.23) Carrier = Ac cos ω ct If this signal is multiplied by a low-frequency signal given by (3.24) Signal = As cos ω st where Ac > As and ω c  ω s , the result will be (3.25) {( As cos ω st )Ac }cos ω ct Notice now that the amplitude of the high-frequency carrier (indicated by the bar over the terms) is now a function of the low-frequency signal (i.e. the amplitude of the carrier has been modulated by the instantaneous value of As cos ω st ). Fig. 3.28 shows the effect of the process graphically. Notice that the modulated waveform given by Eq. (3.25) is a pure AC signal with no DC level. The information that was previously present in the low-frequency signal (Eq. (3.24)) is now encoded in the amplitude of the high-frequency carrier. As a consequence, the information has been shifted from a low-frequency domain (where 1/f noise is dominant) to a high-frequency domain that is less subject to 1/f noise fluctuations in the transmission channel. Fig. 3.29 shows the process graphically with a computer-generated plot of a modulated waveform obtained by multiplying a low-frequency cosine wave by a higher-frequency cosine wave (the carrier). It is clear from the figure that the information that was once present in the lower-frequency signal is now encoded in the variation of the amplitude of the carrier wave with time (i.e. the envelope of the carrier signal).

3.9.2 Demodulation To recover the original signal, the process must be reversed by demodulation. There are two basic ways to demodulate the signal. One way to do it is by envelope detection. Fig. 3.30 illustrates the use of envelope detection as a means of demodulation.

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Figure 3.28  Modulated waveform generated by multiplying the signal waveform by the carrier waveform.

Figure 3.29  Computer-generated plot obtained by multiplying a high-frequency cosine wave by a low-frequency cosine wave.

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Figure 3.30  Demodulation by envelope detection using a diode half-wave rectifier as a demodulator.

The process is similar to half-wave rectification. As shown in the figure, the modulated signal is passed through a diode, where the negative portion of the carrier wave is removed. The diode is then followed by a low-pass RC filter, which removes the high-frequency spikes, restoring the original low-frequency signal (i.e. the information).35 While envelope detection is a simple means of signal recovery, it is not particularly efficient because half of the signal (the negative portion of the carrier) is rejected. Furthermore, envelope detection is not as powerful as synchronous detection when it comes to noise rejection. The other way of demodulation is by synchronous demodulation. Synchronous detection or synchronous demodulation is analogous to full-wave rectification and is a powerful way to reduce extraneous signals and 1/f noise introduced in the transmission channel. In synchronous demodulation, the modulated waveform (Eq. (3.25)) is multiplied once again by the original carrier (Eq. (3.23)). This gives (3.26) {( As cos ω st )Ac cos ω ct}Ac cos ω ct = {As cos ω st}Ac2 cos 2 ω ct

35

The DC level in the demodulated signal can be removed by subsequent signal processing.

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Notice now that the demodulated signal is a function of cos 2 ω ct , which is now entirely positive. Moreover, the original information is still encoded in the amplitude of the cosine-squared waveform. The demodulated signal has been synchronously rectified and now has a DC level. If this high-frequency signal is passed through a low-pass filter, the high-frequency “spikes” will be filtered out, leaving the low-frequency variation (Eq. (3.24)) superimposed on a DC level. Fig. 3.31 shows the results obtained when the two waveforms are actually multiplied together by the computer. Notice that in this plot, all the spikes are positive, and the information is still contained in the envelope of the demodulated waveform (the l­ow-frequency cosine wave). When this high-frequency waveform is passed through a low-pass RC filter, the high-frequency spikes are removed, and the original signal is recovered. The horizontal line in Fig. 3.31 shows the DC level of the demodulated waveform, which can be removed by subsequent signal processing, thereby regenerating the original signal (Eq. (3.24)).

Figure 3.31 Computer-generated plot of the result obtained when the modulated waveform is multiplied by the carrier with the same frequency and phase. The horizontal line in the figure shows the positive DC level of the resulting demodulated signal.

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Fig. 3.32 shows a plot of the carrier signal and the modulated signal on the same time axis. In this plot, it is clear that both the frequency and the phase of the two waveforms are the same (they are “in-sync”). If we multiply these two waveforms together, the demodulated waveform that results will be entirely positive because positive portions of the carrier are always multiplied by positive portions of the modulated wave and negative peaks in the carrier are always multiplied by negative peaks in the modulated waveform. It should be clear from Fig. 3.32 that the phase relationship between the carrier and the signal being demodulated is an important consideration in the demodulation process. Consider now a low-frequency signal of interest is given by (3.27) Signal(t ) = As cos(ω st + φ )

Figure 3.32  Computer-generated plot showing the modulated waveform and the carrier waveform on the same time axis. Both waveforms are synchronized (same frequency and phase). In practice, the amplitude of the carrier would be much greater than shown.

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where As is the amplitude, ws is the frequency, and φ is an arbitrary fixed-phase angle. If this signal is modulated and then synchronously demodulated,36 it can be shown (see Appendix B) that the demodulated signal will be given by  A A2  (3.28) Demodulated signal =  s c cos φ  cos ω st  2  where Ac is the amplitude of the carrier signal. Eq. (3.28) shows that the amplitude of the demodulated signal is proportional to the fixed phase angle φ of the signal of interest in Eq. (3.27). Fig. 3.33 shows a computer-generated plot of the result obtained when the signal of interest (Eq. (3.27)) has a phase angle of 90° and the carrier has a phase angle of 0°. Comparing this plot to the one in Fig. 3.31, it is seen that a signal phase angle of 90° results in a product whose waveform has both positive and negative components.When this waveform is passed through a low-pass filter, the output will be zero, because there is no DC level associated with this waveform. As a result, in order for a modulated signal to produce an output from the synchronous demodulator, the demodulated signal must have a DC level. To produce the maximum output, the original signal of interest and the carrier must have the same frequency and a fixed phase relationship (for

Figure 3.33  Computer-generated plot of the result obtained if the modulated signal is shifted by 90° from the carrier waveform. Both signals still have the same frequency. The horizontal line in the figure shows that the demodulated signal in this case has a DC level of zero. The demodulated signal is entirely high-frequency AC components that are rejected by the low-pass RC filter. 36

By a carrier signal with a phase angle of 0°.

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maximum output, both the signal of interest and the carrier should have a phase angle of 0°). For this reason, synchronous demodulation is sometimes referred to as phase-sensitive detection. Consider now a noise signal with the same frequency as the signal of interest given by (3.29) Noise(t ) = AN cos(ω st + φ ) where AN is the amplitude of the noise signal, ws is the frequency of the noise signal (which happens to coincide with the signal of interest given by Eq. (3.24)), and φ is the phase angle of the noise signal. It can be shown (see Appendix B) that when this noise signal is amplitude modulated and synchronously demodulated with a carrier signal with a 0° phase angle, the demodulated result will be  A A2  (3.30) Demodulated noise =  N c cos φ(t ) cos ω st  2  Once again we see that the demodulated signal is proportional to cos φ . In contrast to Eq. (3.28), Eq. (3.30) gives a different result because, in the case of noise, φ(t) does not have a constant fixed value (like the signal of interest), but, instead, varies randomly with time, taking on all values from 0 to 2π over time. To illustrate graphically the random variation of cos φ(t ) (associated with noise) with time a hypothetical plot shown in Fig. 3.34 was constructed. A random number generator was used to create a sequence of 100 random phase angles, φ(n), varying randomly from 0 to 2π. Fig. 3.34 shows the cosine of these 100 random phase angles plotted versus their index (n) in the sequence. If we assume an arbitrary sampling rate of one sample per

Figure 3.34  Computer-generated plot of a random noise phase angle versus time.

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­ illisecond, the horizontal axis can be converted into an arbitrary time m scale for illustration purposes. It can be seen in the figure that the cosine of the noise phase angle varies approximately equally from positive to negative over the 100-ms interval. If the signal given by Eq. (3.30) were to be averaged over this time period, the average value would tend toward zero. Since the cos φ(t ) term is in the amplitude of Eq. (3.30), it means that even a noise signal at ws will be averaged to zero by an output low-pass filter with a sufficiently long time constant.

3.9.3  Implementing phase-sensitive detection We have seen that synchronous demodulation is essentially a m ­ ultiplication process whereby the amplitude-modulated signal is multiplied by the original carrier signal that was used to produce modulated signal. Fig. 3.35 shows a block diagram of a lock-in amplifier (LIA) for implementing ­phase-sensitive detection (synchronous demodulation). The heart of the system is the mixer, which performs the multiplication of the reference signal (i.e. the “carrier”) with the modulated input signal. Prior to the multiplication step, the modulated signal is amplified by an input amplifier and then passed through a bandpass filter (centered on the modulation frequency) to remove unwanted frequency components. The output of the mixer is then fed to a low-pass filter, which removes the unwanted high-frequency components from the mixer output (see Eqs. (B.36) and (B.39)). A phase-shifter is provided in the reference channel to assure that the modulated input signal and the reference signal are completely in phase for maximum output. While the concept of synchronous demodulation is straightforward enough, its practical implementation can be problematic. For instance, in the analog domain, a switching amplifier (whose gain switches between +G and –G) can be used to perform the multiplication step [26]. In this approach, the switching is performed by a square-wave reference signal that is

Figure 3.35  Block diagram of a lock-in amplifier.

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phase-locked with the original modulating signal. The switching amplifier is followed by a low-pass filter that removes the high-frequency components of the demodulated signal. One problem with using a square-wave reference is the presence of odd-harmonic components of the square wave that can be down-converted in the demodulation process (along with the fundamental) to DC, producing an offset error. In general, the analog approach is subject to • noisy input prefilters, • nonlinear demodulators, • limited rejection by output analog low-pass filters, and • drift from high-gain DC amplifiers. With the advent of sophisticated digital-signal processing (DSP) ­techniques [27], most modern LIAs use a digital approach. In the digital ­approach, the analog demodulator (mixer), analog low-pass filters, and ­analog DC amplifiers are all replaced by digital circuitry. In a digital LIA, the input signal is amplified and then immediately converted into the digital domain with a high-resolution, high-frequency analog-to-digital converter (ADC).37 The digitized sample is then fed to a digital signal processing (DSP) chip. This DSP chip then synthesizes a highresolution (24 bit) reference sine wave38 from the external reference signal supplied to the LIA. Next, this reference sine wave is multiplied by the digitized input signal using a high-speed digital multiplier. Finally, the demodulated digital signal is filtered with sophisticated digital low-pass filters that can have rolloffs of −20 dB/decade up to −80 dB/decade. Some of the advantages of using digital technology to implement phasesensitive detection are: • higher dynamic reserve,39 • lower drift with time and temperature changes, • lower distortion, and • high phase resolution.

3.9.4  Implementing modulation Fig. 3.36 shows an experiment employing modulation.  ypically, a 14–18 bit ADC operating at 256 kHz is used. T The use of a true sine wave eliminates the problems with harmonics caused by the use of square waves. 39 The dynamic reserve is the ratio (in dB) of the largest interfering signal to the full-scale input voltage at the given sensitivity. Typical values are >100 dB. 37 38

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Figure 3.36  Modulation of a light beam with an optical chopper.

In this example, fluorescence from a sample, induced by irradiation from a continuous laser, is to be determined.The continuous laser radiation passes through a rotating sectored disk (the “chopper”), where it is converted into a series of square-wave pulses, which irradiate the sample. The sample fluorescence is then detected by a photodiode. Along with the small chopped fluorescence signal, the detector is also exposed to ambient light from the room, whose signal overwhelms the small fluorescence signal of interest. In addition, the signal of interest is further contaminated with 1/f detector noise from the detector. The signal from the photodiode is fed into the signal channel of the LIA. As the sectored chopper rotates, it also simultaneously chops the light from a second reference light source that is detected by a reference photodiode. Since the laser radiation and the reference light source are both chopped by the same rotating sectored wheel, they both have exactly the same frequency and a constant phase relationship.The signal from the reference photodiode is fed into the reference channel of the lock-in amplifier. Any phase difference between the two signals can be adjusted by the phase shifter of the LIA.

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Note that in this procedure, both the signal of interest and the reference signal are square-wave modulated and both have a DC component that must be removed by the input filters of the LIA to convert them into true AC signals. Also note that the Fourier transform of a square wave is the sum of odd harmonics that make up the square wave (i.e. 3f, 5f, etc.) so that this modulation scheme employing square-wave modulation can potentially result in offset errors from these unwanted harmonics. Modulation can also be accomplished by applying a stimulating signal to some system and monitoring the response (sometimes referred to as derivative modulation). Suppose that we wish to modulate the magnetic field from an electromagnet. We could apply an alternating current with some amplitude to the coils of the electromagnet. This will produce the desired alternating magnetic field. The amplitude of the alternating magnetic field will not necessarily be equal to the amplitude of the alternating current. To determine that we will need to use a transfer function (in this case, a plot of magnetic field strength versus electromagnet current). The modulation of the output will depend on the shape of the transfer function and its slope. Fig. 3.37 shows a hypothetical transfer function for some output (y-axis) versus some input (x-axis).

Figure 3.37  Hypothetical transfer function showing the modulated output produced by a modulating stimulus.

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This particular transfer function is symmetrical about the y-axis. In the right-hand side of the figure, a stimulus is applied to the x-variable so that X(t) oscillates about some average value +X with an amplitude of s. A tangent to the transfer function at +X has a slope of −m. Tracing the oscillation about the x-axis on this tangent as a function of time generates a modulated output given by Y ± Amod , where Y is the average output for +X and Amod is the amplitude of the modulated output. The amplitude of the modulated output is determined by the amplitude of the modulating stimulus (s(t)) and the slope of the tangent to the transfer function at the average value of +X. Because the slope of the tangent in the right-hand side of the figure is –m, the phase of the modulated output is shifted by 180° from the modulating signal (the modulated output leads the stimulating signal by 180°). Now consider the effect of applying the same stimulus (s(t)) to an average value of –X as shown in the left-hand portion of Fig. 3.37. Once again we generate a modulated output of Y ± Amod . While the amplitude of the modulated output is the same as that obtained previously for +X, the phase of the modulated signal is unchanged from that of the modulating signal because the slope of the tangent is +m. So for –X, the modulated output and the stimulating signal have the same phase. Notice that as the average value of X approaches zero, the slope of the transfer function approaches zero. This means that for small values of |X|, the amplitude of the modulated signal approaches zero even if s(t) remains the same. So, at X = 0, the modulated output essentially disappears. The phase shift that occurs when the slope of the transfer function is –m can be used in conjunction with phase-sensitive detection to distinguish whether +X or –X is being modulated. This allows lock-in amplifiers to be used in instrument control in addition to simple signal recovery.

3.9.5  Benefits of modulation and synchronous demodulation The following lists some of the benefits of the amplitude modulation/synchronous demodulation process (i.e. phase-sensitive detection). • Any DC component associated with the modulated signal is blocked by the bandpass filter (centered at the modulation frequency) in the input of the LIA. • The modulation process shifts the signal of interest from a low-frequency region to a high-frequency region centered at the modulation frequency, thereby reducing interference from 1/f noise introduced in the transmission channel.

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• In order for the demodulated signal to have a DC level (and pass through the output low-pass filter), the modulated signal and the carrier must have the same frequency and a fixed phase relationship (ideally a phase angle of 0° for maximum output, but avoiding phase angles of 90° and 270° where cos φ = 0 ). • Any noise at the carrier frequency will result in a demodulated signal proportional to cos φ(t ) , where φ(t) is a random phase angle that varies with time. Since cos φ(t ) will vary randomly with time, it is equally likely to be positive or negative at any instant, and will be removed by the low-pass filter in the output of the LIA whose time constant can be set to a value commensurate with the response time needed for the measurement.40 As a caveat, it should be remembered that these benefits accrue only to noise reduction in the transmission channel. Modulating a noisy, unconditioned signal will not give the full benefit, since the noise will be modulated along with the signal of interest. As a result, the modulator should be placed as early as possible in the signal train (see Fig. 3.36). For example, in Fig. 3.36, if the modulator were placed after the photodiode in the signal channel, the ambient light signal would still be removed, but the 1/f detector noise would not.

3.10  APPLICATION OF PHASE-SENSITIVE DETECTION IN CHIROPTICAL INSTRUMENTATION Phase-sensitive detection is a powerful tool in reducing detector noise, and is, therefore, extremely important in infrared spectrometric measurements, where detector noise can be a problem. In a vibrational circular dichroism experiment, for example, if a PEM is modulated to sequentially produce left- and right-circularly polarized light (at 50 kHz), the control signal used to control the piezoelectric transducer can be used as a reference signal (i.e. the carrier) in synchronous demodulation. In this way, the left- and right-circularly polarized light will be generated in phase with the control signal. The DC output of the phase detector will be proportional to the intensity difference for left- and right-circularly polarized light. For samples that are not optically active, this difference will be zero. When this

40

 s a rule of thumb, it generally takes 5τ for a capacitor to reach full charge, so a time conA stant of 1 s would require ∼5 s for the signal to stabilize.

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occurs, no AC input waveform will be generated, and the output of the lock-in amplifier will be zero. Phase-sensitive detection can also be used for instrument control as in the following example. Fig. 3.38 shows a schematic diagram of an automatic polarimeter with no moving parts that uses the Faraday effect (see ­Appendix A). The instrument is like an ordinary polarimeter with a fixed polarizer and a fixed analyzer with their planes of polarization parallel in the vertical direction (along the y-axis).The parallel orientation of the polarizer and the analyzer allows the maximum amount of light from the source to strike the detector. This is the null condition. When an optically active sample is placed in the sample cell, it rotates the plane of polarization of the light emerging from the sample cell so that the new plane of polarization of the emerging light is no longer parallel with the vertical plane of polarization of the analyzer. This rotation alters the signal from the photodetector that is fed into a lock-in amplifier whose output generates an error voltage related to the magnitude of the sample rotation as well as the direction of the rotation. The error voltage is supplied to a variable, bi-polar DC power supply that supplies current to the ­compensator

Figure 3.38  Automatic polarimeter based on the Faraday effect.

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Faraday cell in order to rotate the plane of polarization back to the original null position. As the plane of polarization is returned to the original vertical direction (null condition) by applying the proper current to the coil of the compensator Faraday cell, the error voltage decreases to zero. When the plane of polarization is returned to the original vertical direction, the error voltage becomes zero, and the variable DC power supply continues to maintain the proper current through the coil. The direction and magnitude of the DC current (as measured by the current meter) required to reestablish the original null condition is proportional to the optical rotation produced by the sample. By calibrating the instrument with known samples of optically active materials (whose rotational magnitude and direction are known independently), the current applied to the compensator Faraday cell can be related to optical rotation of the sample. We are now in a position to understand how the error signal is generated by the lock-in amplifier. As shown in Fig. 3.38, the automatic polarimeter has two Faraday cells, each of which consists of an electromagnetic coil surrounding a glass rod. The first Faraday cell is the modulator cell that is driven by a sinusoidal AC current. The sinusoidal signal from the signal generator that supplies current to the modulator Faraday cell is also fed into the reference channel of the lock-in amplifier and acts as the reference signal. Due to the Faraday effect, the AC current supplied to the coil of the modulator Faraday cell causes the plane of polarization from the polarizer to oscillate back and forth about the original static vertical plane produced by the polarizer. Fig. A3.5 (see Appendix A) shows the transfer function for the modulation. In the absence of an optically active sample in the sample cell, the static plane of polarization will be unaltered from the vertical null position (0°). Since the slope of the transfer curve (Fig. A3.5) at 0° optical rotation is essentially zero, the modulation of the plane of polarization has virtually no effect on the intensity of the radiation passing through the analyzer when the instrument is in the null condition. As a result, the signal from the photodetector is unmodulated, and the output of the phase-sensitive detector is zero when the plane of polarization is in the vertical position (0°). Thus, when the static plane of polarization is 0°, there is no error signal from the lock-in amplifier and no current is supplied to the coil of the compensator Faraday cell. Now suppose that an optically active sample is placed in the sample cell that rotates the plane of polarization by +25°. For this angle of rotation, the slope of the transfer curve (Fig. A3.5) is negative and the

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­ odulated light intensity is 180° out of phase with the reference signal m from the signal generator. Since the photodetector signal is in phase with the light intensity, the photodetector signal is also 180° out of phase with the reference signal. According to Eq. (3.28), the demodulated DC output from the lock-in amplifier will be negative because the cosine of 180° is −1. This will result in the generation of a negative DC error signal from the lock-in amplifier. Now suppose that an optically active sample is placed in the sample cell that rotates the plane of polarization by −25°. For this angle of rotation, the slope of the transfer curve (Fig. A3.5) is positive, and the modulated light intensity is in phase with the reference signal from the signal generator. As a result, the demodulated DC output from the lock-in amplifier will be positive because the cosine of 0o is +1 (see Eq. (3.28)). So for a negative optical rotation, the error signal from the lock-in amplifier will be positive. So the magnitude of the error signal from the lock-in amplifier will be determined by the slope of the tangent to the transfer curve (Fig. A3.5) at the particular rotation caused by the optically active sample. The polarity of the error signal from the lock-in amplifier will be determined by the direction of the optical rotation. The error signal will cause the power supply to increase or decrease the current through coil of the compensator cell until the error voltage becomes zero, which point the power supply will maintain the proper current through the coil until a new sample with a different optical rotation is placed in the sample cell.

APPENDIX A BASIC OPTICS  

A1  REFRACTIVE INDEX When light propagates through a transparent dielectric medium41 like glass, the electric vector of the light wave interacts with the electron clouds around the atoms to induce oscillating dipoles in the medium that, in turn, reemit the radiation. This virtual absorption and reemission process retards the progress of the wave in the medium, thereby reducing its speed relative 41

 he dielectric medium is considered to be an array of atomic nuclei with their associated T bound electrons. Since the atomic nuclei are relatively massive compared with the mass of an electron, only the bound electrons are forced into oscillation by a high-frequency, periodic electromagnetic field.

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to the speed of light in a vacuum. The refractive index, n, of the medium is defined as 

n=

c v

(A.1)

where c is the speed of light in a vacuum and v is the speed of the light in the medium. In general, the denser the medium, the slower the light propagates and the larger the refractive index. The refractive index is also a function of the wavelength of the radiation, and the change in refractive index with wavelength is referred to as dispersion.

A2  SNELL’S LAW When light traveling in one medium strikes the interface of another medium at some angle of incidence, its direction of travel is altered by the phenomenon of refraction. Fig. A3.1 shows the refraction of light as it ­travels

Figure A3.1  Snell’s law: n1 and n2 are the refractive indices of medium 1 and 2, respectively; w is the hypotenuse of the two triangles shown in bold lines; i is the angle of incidence and φ is the angle of refraction.

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from a medium of lower density (medium 1) to one of higher density (medium 2). As shown in Fig. A3.1, when the wave fronts enter medium 2, they slow down and begin to stack up, causing the wavelength to decrease in the more-dense medium. At the same time, the direction of the wave fronts in medium 2 is altered. Referring to Fig. A3.1, consider a plane wave front AB, which strikes medium 2 at an angle of incidence i. The left-hand part of the wave front, indicated by point A, immediately enters medium 2. The right-hand part of the wavefront, indicated by point B, has to travel an additional distance X before it strikes medium 2. This additional distance X will be given by v1t , where v1 is the velocity of the ray in medium 1 and t is time. According to Eq. (A.1), v1 = c / n1 . From the figure, it can be seen that X = w sin i , so X will be given by c (A.2) X = t = w sin i n1 While the right-hand part of the wave front (point B in the figure) is traveling the distance X to reach medium 2, the left-hand part is already traveling in medium 2. In the time that it takes the right-hand portion of the wave front to traverse distance X, the left-hand portion of the wave front (point A in the figure) will have traveled into medium 2 by distance Y, given by Y = v 2t , where v2 is the velocity of the ray in medium 2 and t is time. As a result c (A.3) Y = t = w sin φ n2 In medium 2, in order for the left-hand portion of the wave front (point C in the figure) to be in phase with the right-hand portion of the wave front (point D in the figure), the time, t, in Eqs. (A.2) and (A.3) must be the same. Therefore, n n (A.4) t = 1 w sin i = 2 w sin φ c c which reduces to Snell’s law, n1 sin i = n2 sin φ (A.5) where n1 and n2 are the refractive indices, i is the angle of incidence, and φ is the angle of refraction.

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Fig. A3.2A shows the path of a light beam incident on a block of transparent dielectric material with parallel faces. At each interface, light is reflected and refracted. For a block of dielectric material with parallel faces, the ray emerging on the opposite face from the incident surface is undeflected from its original direction and is simply displaced from the dashed line by an amount dependent on Snell’s law and the thickness of the block. Fig. A3.2B shows the effect when light is incident on a trigonal prism. Light entering the prism will be refracted toward the normal to the first

Figure A3.2  Reflection and refraction (A) from a block of dielectric material with parallel faces where n1 and n2 are the refractive indices of the two media; i is the angle of incidence; r is the angle of reflection; and φ is the angle of refraction. (B) Total internal reflection from the interior surface of a prism when the critical angle is exceeded, where i2 is the angle of incidence at the second surface and φ2 is the angle of refraction for the emerging ray at the second surface.

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surface as shown in the figure because n2 > n1 . This refracted ray will strike the normal to the second surface at an angle i2.The ray incident on the second surface from the interior of the prism will be refracted away from the second-surface normal on exiting the prism because n1 < n2 . According to Snell’s law, the ray emerging from the prism at the second surface will be given by (A.6) n2 sin i2 = n1 sin φ where φ is the angle that the emerging ray makes with the normal to the second surface. As the angle i2 increases, the ray emerging from the prism begins to approach a value of φ = 90° with respect to the normal to the second surface. When this point is reached, the ray no longer emerges from the prism, but is reflected to the right as shown in Fig. A3.2B. The angle of incidence (i2) for which this occurs is known as the critical angle and is given by n (A.7) sin ic = 1 n2 where ic is the critical angle.

A3  LISSAJOUS FIGURES AND FORMS OF POLARIZATION As mentioned at the beginning of this chapter, light can be considered to be composed of an oscillating electric vector and an oscillating orthogonal magnetic vector. In a vacuum, the propagation of the magnetic and electric fields is in phase, but the amplitude of the electric field is larger by a factor of c, the speed of light in a vacuum.42 Because of this difference in amplitude, changes in the electric field are easier to measure. As a result, the phenomenon of polarization is generally studied considering only the electric component of light. From this point of view, the orientation of the electric field vector is taken as the orientation of the polarization of the radiation. An electric field vector of light, E, with any arbitrary orientation can be resolved into two orthogonal components as shown in Fig. A3.3A.

42

 he amplitude of the magnetic vector Bo will be given by Eo/c, where Eo is the amplitude T of the electric vector and c is the speed of light. If Eo has units of V m−1 and c is in m s−1, Bo will have units of V s m−2 or T.

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Figure A3.3  (A) An arbitrary electric vector E resolved into Ex and Ey components. (B) The orthogonal Ex and Ey vectors generate a Lissajous pattern in the xy-plane. The z-axis is the direction of propagation.

If the z-axis is taken as the direction of propagation, these two orthogonal electric vectors can be represented as Ex and Ey. The former can be visualized to lie in the plane perpendicular to the paper, and the latter can be visualized to be in the plane of the paper. Consider two orthogonal oscillating waveforms given by the following two parametric equations: (A.8) x(t ) = E x cos(ω t + φx ) y(t ) = E y cos(ω t + φy ) (A.9) where x is oscillating in the horizontal plane and y is oscillating in the vertical plane. If the x(t) waveform is applied to the x-input of an oscilloscope and the y(t) waveform is applied to the y-input, the combined effect of the vertical and horizontal oscillations will produce a two-dimensional figure, known as a Lissajous figure, on the screen as shown by the circle in the xyplane in Fig. A3.3B. The shape of the Lissajous figure produced depends on the amplitudes Ex and Ey, their relative frequencies, w, and their relative phase angles φx and φy. When the frequencies of both waveforms are the

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same, the two-dimensional pattern produced takes the general form of an ellipse. An ellipse is a conic section produced when a plane intersects with a right circular cone at some angle. The limiting forms of an ellipse are the circle and a line segment. In considering the polarization state of light, it is the phase relationship of φx with respect to φy that is important. This phase relationship can be expressed as a phase difference γ, and can be taken as the difference between φx and φy. As γ varies, different forms of polarization occur. Circular polarization and linear polarization, which are special limiting cases of elliptical polarization, occur when γ takes on specific values. As γ varies, the Lissajous figure produced can have the shapes shown in Fig. A3.4.

Figure A3.4  Lissajous patterns produced for various values of γ, the phase difference between two orthogonal oscillating waveforms.

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Fig. A3.4 shows that when γ is 0° or 180°, the light is linearly polarized. If γ is ±90°, the light is circularly polarized. For all other angles, the light is elliptically polarized. For two orthogonal waveforms given by Eqs. (A.8) and (A.9), having the same frequency w, it is possible to combine the two parametric equations so as to eliminate t [28]. When this is done, the resulting equation is x2 x y y2 (A.10) 2 cos − γ + = sin 2 γ 2 2 Ex Ex Ey Ey where x is the horizontal component of the Lissajous figure, y is the vertical component, and Ex and Ey are the amplitudes of the respective waveforms. Eq. (A.10) can be used to determine the polarization forms produced by different values of γ. A3.1  Linear polarization Consider the situation where Ex = Ey and γ is 0° or 180°. When γ is 0°, cos γ is 1 and sin γ is 0. When γ is 0° and E  = E , Eq. (A.10) reduces to x y (A.11) x 2 − 2xy + y 2 = 0 (A.12) ( x − y )2 = 0 (A.13) x−y=0 y=x (A.14) Eq. (A.14) is the equation for a straight line with a slope of +1. When γ is 180° and Ex = E y, cos γ is −1, sin γ is 0, and Eq. (A.10) reduces to (A.15) x 2 + 2xy + y 2 = 0 (A.16) ( x + y )2 = 0 (A.17) x+y=0 y = −x (A.18) Again, a linear relationship is observed, only this time the slope is −1.

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A3.2  Circularly polarized light If Ex = Ey and γ is equal to ±90°, then cos γ = 0 and sin γ = ±1 . In this case, Eq. (A.10) reduces to (A.19) x2 + y2 = E2 where E = Ex = Ey. Eq. (A.19) is the general equation for a circle with a radius of E. When γ = +90° , the Lissajous figure for this condition is a circle traced in a clockwise fashion (when looking toward the source), corresponding to right-circularly polarized light. When γ = −90°, the Lissajous figure for this condition is a circle traced in a counterclockwise fashion, ­corresponding to left-circularly polarized light.

A4  THE LAW OF MALUS Polarizers are frequently used in pairs where the first polarizer is followed by a second, known as the analyzer. Unpolarized light incident on the polarizer will be converted into plane polarized light vibrating in a particular plane. When the polarization plane of the analyzer is coincident with that of the polarizer (the polarizers are said to be in a parallel alignment), the maximum intensity will be passed by the pair. If the polarization plane of the analyzer is rotated by 90°, the two polarization planes will be orthogonal (or crossed), and an observer will notice that virtually no light will be transmitted by the pair of polarizers. If the analyzer is rotated through an additional 90° in the same direction, the polarization planes of both polarizers will again be parallel, and the maximum intensity will again be passed by the pair. Finally, as the analyzer is rotated again by 90° in the same direction (270° total), the polarizers will be crossed and the transmitted light will again be extinguished. Consider a polarizer/analyzer combination where the polarization plane of the polarizer is oriented in the vertical direction. Let the polarization plane of the analyzer be set at some arbitrary angle θ to that of the polarizer. Let A be the amplitude of the light beam transmitted by the polarizer. For any angle θ, A can be resolved into two components— one parallel to the plane of the analyzer (Ap) and the other orthogonal to the plane of the analyzer (Ao). Since the orthogonal component is rejected by the analyzer (its electric vector is crossed with the plane of the analyzer), only Ap will be transmitted by the analyzer. The magnitude of Ap is given by (A.20) Ap = Acos θ

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Since the intensity of a light beam is proportional to the square of the amplitude, the intensity of the light transmitted by the analyzer (assuming a perfect analyzer with no losses) will be given by (A.21) I = Ap2 = A 2 cos 2 θ = I o cos 2 θ where Io is the intensity of the light beam incident on the analyzer and I is the intensity of the light transmitted by the analyzer. The cosine-squared relationship between the transmitted intensity and the angle θ between the two polarization planes was discovered in 1808 by Etienne-Louis Malus, a French scientist, and is known as Malus’ law [29]. Fig. A3.5 shows a plot of the transmitted intensity of a pair of polarizers versus the angle θ between their polarization planes as predicted by Malus’ law.

A5  THE FARADAY EFFECT In 1845, Michael Faraday was studying the effect of applied magnetic fields on light passing through different materials [30].Working with a cylinder of lead glass, Faraday observed that when a magnetic field was applied along the axis of the cylinder, the plane of polarized light passing through the cylinder was rotated by an amount that was proportional to the strength of the magnetic field. The effect was not unique to glass, and other optically

Figure A3.5  (A) Relative intensity I/Io versus the angle between the analyzer and polarizer, where Io is the intensity of the light beam incident on the analyzer and I is the intensity of the light transmitted by the analyzer. (B) Plot of the slope of the curve shown in (A) versus the polarization angle between the polarizer and the analyzer.

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transparent dielectric materials produced a similar rotation to a greater or lesser amount. The rotation that Faraday observed was due to circular birefringence induced in the dielectric material by the magnetic field. The extent of the rotation can be calculated by  (A.22) θ = VBl where θ is the angle of rotation in radians, V is the Verdet constant  (a function of wavelength) in rad T−1 m−1, B is the magnetic induction (a vector quantity) in Tesla, and l is the length of the dielectric cylinder in meters. To get some idea of the magnitude of the effect, we will consider a flint glass rod 0.15 m long surrounded by a solenoid with 1000 turns oriented  along the axis of the cylindrical glass rod. The magnetic induction B produced by the solenoid will be given by  (A.23) B = µonI where µo is the permittivity of free space (4π × 10−7 T  m A−1), n is the number of turns of the solenoid coil per meter, and I is the current through the coil in amperes. For a current of 3.0 A, Eq. (A.23) gives a magnetic induction of 0.025 T. Using Eq. (A.22), we can calculate the extent of the rotation produced by 0.025 T. For SF-59 glass, the Verdet constant (at 650 nm) is 23 rad T−1 m−1. Assuming the glass rod is 0.15 m long, θ will be 0.086 rad or 4.9°. So, a 3 A current supplied to the solenoid will produce a rotation of a little less than 5° in the plane of a polarized light beam at 650 nm with SF-59 glass. If we reverse the direction of the current, the rotation of the plane of polarization will reverse.

APPENDIX B  SIGNAL HANDLING

B1  PHASOR BASICS Fig. B3.1 shows how a sine wave43 can be generated by a rotating vector, known as a phasor.

43

The term sine wave as used in this context refers to a generic sinusoidal wave.

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Figure B3.1  Generation of two sine waves by two counterclockwise rotating phasors with equal amplitudes and equal angular frequencies w and a phase difference of γ. Phasor 2 leads phasor 1 by γ. The figure also shows the angular shift (in radians) along the time axis due to γ.

In the figure, two phasors of equal amplitudes A are shown, each rotating in a counterclockwise direction with an angular frequency of w radians per second. As each phasor rotates, its y-projection generates a sine wave. The length of the phasor determines the amplitude of the sine wave. Since the phasors are rotating in a counterclockwise direction, phasor 2 is said to lead phasor 1 by γ radians. Since the tip of the phasor indicates the start of the sine wave, sine wave 2 is shifted along the time axis by γ as shown in the figure. The generalized equation for a sine wave generated by the rotating phasor is (B.1) y(t ) = A sin(ω t + φ ) where y(t) is the amplitude of the sine wave as a function of time, A is the amplitude of the sine wave determined by the length of the phasor, w is the angular frequency in radians per second, and φ is the phase angle in radians. If the phase angle is −90° (−π/2 rad), the result is

π (B.2) A sin  ω t −  = A cos ω t  2

B2  RC FILTER BASICS The impedance of a series combination of a resistor and a capacitor is the sum of the resistance of the resistor, which is independent of frequency, and the capacitive reactance of the capacitor, given by −1 (B.3) XC = ωC

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where XC is the capacitive reactance, w is the angular frequency in radians per second (i.e. 2πf where f is the frequency in Hz) of the applied AC signal, and C is the capacitance of the capacitor in farads. The series combination of a resistor and capacitor can be conveniently represented by a counterclockwise rotating phasor.The phasor rotates about the origin with an angular frequency of w radians per second. In a typical phasor diagram, the phasor in the complex plane is shown in a stationary position with the real axis as the positive x-axis and the imaginary axis as the y-axis. In phasor notation, the impedance of a series combination of a resistor and capacitor is given by  (B.4) Z = R + jX C  where Z is a phasor vector in the complex plane, R is the resistance of the resistor, and XC is the capacitive reactance given by Eq. (B.3), and j is the imaginary number equal to −1 .44 Fig. B3.2 shows a phasor diagram for a hypothetical series RC circuit. Notice that the impedance phasor is in the fourth quadrant because the capacitive reactance is a negative imaginary quantity (see Eq. (B.4)). The two angles shown are the phase angles. φ is the angle between the  impedance phasor and the real axis (i.e. the resistance). If we take Z as the direction of the applied signal, these two angles specify the directions of the current and voltage in the circuit. For example, the real axis (i.e. the resistance) shows the direction of the current through the resistor (and  also the voltage drop across the resistor, since they are in phase). Since Z is rotating counterclockwise, the entire frame in Fig. B3.2 is rotating as well. As a result, the current and voltage drop across the resistor are said to lead the applied signal by φ degrees. Likewise, the applied signal, indicated by Z , leads the voltage drop across the capacitor by θ degrees. To determine the magnitude of the impedance Z, we must determine the length of the phasor, which is given by Z = R 2 + X C2 (B.5) We are now in a position to discuss a simple, single-stage low-pass filter, which acts as an AC voltage divider, shown in Fig. B3.3A.

44

In electrical discussions, the imaginary number is generally represented as j instead of i to avoid confusion with current.

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Figure B3.2  Phasor diagram for a series RC circuit where Ris the resistance of the resistor, XC is the capacitive reactance of the capacitor, and Z is the resulting impedance represented as a phasor vector in the complex plane. Angle φ is the phase angle between the impedance phasor and the resistance, and θ is the phase angle between the impedance phasor and the capacitive reactance.

As shown in the figure, the input signal is applied across the series combination of the resistor and the capacitor, whereas the output is taken across the capacitor. By analogy with a simple voltage divider, the output of the circuit is related to the input by   XC (B.6) e out =  e 2 2  in  R + XC  where eout is the output voltage of the filter, ein is the input voltage to the filter, and |XC| is the magnitude of XC, which is simply the value of XC in Eq. (B.3) without the negative sign (i.e. X C = 1 / ωC ). Since the gain of any circuit is the output divided by the input, the gain of the low-pass RC filter is e 1 / ωC (B.7) G = out = 2 e in R + (1 / ωC )2 where G is the gain of the filter. By analogy, the gain of the high-pass RC filter shown in Fig. B3.3B is similarly given by R (B.8) G= 2 R + (1 / ωC )2 because the output of the high-pass filter is taken across the resistor.

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Figure B3.3  RC filters. (A) Low pass; (B) high pass, where eout is the output voltage of the filter, ein is the input voltage to the filter, R is the resistance, and C is the capacitance.

B3  BODE PLOTS While Eq. (B.7) describes the gain of a simple low-pass filter mathematically as a function of frequency, engineers often use a graphical approach to illustrate circuit performance. This approach is known as a Bode plot after Dutch engineer Hendrick Bode, who developed the plot in the 1930s. A Bode plot is a log–log graph where the gain in decibels is plotted against the logarithm of the angular frequency. The gain in decibels is given by e  (B.9) G (dB) = 20 log  out   e in  where G is the gain in decibels. To see how the plot is developed, we will divide the frequency range into two basic regions. Consider the frequency region where XC ≫ R.This will be the case for low frequencies.When XC ≫ R, the denominator in Eq. (B.6) reduces to XC and the gain given by Eq. (B.6) becomes 1. Since the RC filter does not have any amplifying ability, this is the largest gain that can be expected. If we express this gain in decibels, it becomes G (dB) = 0 for this frequency regime. Now consider the frequency range where R ≫ XC. This is the highfrequency region. In this frequency range, the gain given by Eq. (B.6) ­reduces to

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X 1 (B.10) G= C = R ω RC If we express this gain in decibels, we get (B.11) G (dB) = −20 log( RC ) − 20 log ω which is the equation for a straight line with a slope of −20 dB per log unit along the frequency axis. Finally, let us consider the case where R = |XC|. For this special case, the gain given by Eq. (B.6) will be XC 1 (B.12) G= = 2 2 2XC Converting the result into decibels gives G = −3.0. When R = Xc = 1/wC, the frequency where this condition is true is ω o = 1 / RC = 1 / τ , where τ is the RC time constant45 for the filter. (Note:The product of resistance in ohms times the capacitance in farads has units of seconds.) We are now in a position to draw a Bode plot for a simple RC filter.We start with a log–log plot where the gain in decibels is plotted on the vertical axis and the log of the angular frequency is plotted on the horizontal axis as shown in Fig. B3.4. Begin by drawing a horizontal line at 0 dB. Determine wo and locate log ω o on the horizontal line.Then draw a line with a slope of −20 dB per log unit through the point for log ω o on the horizontal line.The gain of the filter where the two line segments cross is −3 dB below the horizontal line. Connect the two line segments with a smooth curve that passes through the −3 dB point. Fig. B3.4 shows a Bode plot for a low-pass filter with a resistance of 10 kΩ and a capacitance of 1.25 µF. The Bode plot is a simple way to visualize the performance of RC filters. The high-frequency bandpass of the low-pass filter is taken to be the frequency of the 3 dB point. At frequencies higher than the 3 dB point, the gain of the filter decreases with a rolloff of −20 dB per log frequency unit. The Bode diagram for a high-pass filter can be constructed in an analogous manner to that for the low-pass filter. Again, start by drawing a horizontal line segment at 0 dB. Locate log ω o on the horizontal line. Then 45

 he time constant is the time it takes current flowing through the resistor to charge the T capacitor to 63.2% of being fully charged.

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Figure B3.4  Bode plot for a simple low-pass RC filter, where G (dB) is the gain in decibels and w is the angular frequency. For this Bode diagram, the resistance is taken as 10 kΩ, and the capacitance is taken as 1.25 µF.

draw a line with a slope of +20 dB per log unit through the log ω o point and connect the two line segments with a smooth curve that passes −3 dB below the horizontal line at the point where the two line segments cross.

B4  SIGNAL-TO-NOISE BASICS The signal-to-noise ratio is the essential figure-of-merit in optimizing instrument performance and can be expressed by Ssignal S (B.13) = N Noise rms where S/N is the signal-to-noise ratio, Ssignal is the average signal, and Noise rms is the root-mean-square value of the noise. Since the root-meansquare value of a quantity is associated with the standard deviation of the quantity, the signal-to-ratio can be considered to be the reciprocal of the relative standard deviation of the measurement equal to X / s , where s is the standard deviation of a series of measurements and X is the average value. If the output of an instrument is steady and in a digital format, the signal-to-ratio can be estimated conveniently by collecting a number of

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observations of the signal. From this sample of data, the average value can be computed by summing the observations and dividing by n, the number of samples collected. An estimate of the standard deviation can then be calculated from

∑ (X − X ) (B.14) s= N

1

2

i

n −1

where s is an estimate of the standard deviation, Xi is an individual observation, and X is the average of the observations. Fig. B3.5 shows how the signal-to-noise ratio can be estimated from a recorder tracing. We start by drawing a line through the center of the baseline noise and another line through the center of the signal noise envelope. The distance between the two lines is the average signal (in this case, 33 mm). Then two lines are drawn at the upper and lower bounds of the noise envelope to encompass about 99% of the pen excursions (in this case, 25 mm). Recall from statistics ±2.5 standard deviations from the mean includes about 99% of the area under a Gaussian distribution. Therefore, 1 standard deviation is estimated to be 1/5 of the noise envelope determined above.The signal-to-noise ratio is then 33 mm/5 mm or 6.6, which we can round off to 7, since this is only an estimate. The smallest signal that an instrument can determine reliably must be based on signal-to-noise considerations. The question to be answered is what signal-to-noise ratio is required to say that a signal is present in the presence of background noise. To answer this question, a standard statistical comparison of averages will be used, which is given by

Figure B3.5  Estimating the signal-to-noise ratio

S / N rms

from a recorder tracing.

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2 2   ssignal s background (B.15) ∆X = X signal − X background ≥ t  +   nsignal n background 

(

)

where ∆X is the difference between the average signal and the average background, t is the Student t value, ssignal is the standard deviation of the signal measurements, nsignal is the number of measurements of the signal taken, sbackground is the standard deviation of the background measurements, and nbackground is the number of background measurements taken. The value of t used will be determined by the confidence level and the number of degrees of freedom associated with the sample set (nsignal + nbackground – 2). Eq. (B.15) can be simplified by making some assumptions. Firstly, we can assume that, in the case of a very small signal, ssignal  ≈  sbackground. For convenience, we can assume that nsignal = nbackground = n. Substituting these assumptions into Eq. (B.15) and rearranging gives ∆X  S  2 (B.16) = ≥ t99  s n  N rms  detection limit where t99 is the value of t for 2n  − 2 degrees of freedom at the 99% ­confidence level. To convert this to a number, assume that five measurements are made of the signal and five measurements are made of the background (n = 5).The value for t at the 99% confidence level for eight degrees of freedom is 3.355. Substituting this into Eq. (B.16) gives 2.12, which means that the signal-to-noise ratio must be equal to or greater than 2.1 to be reliably detected on a statistical basis. Based on the above discussion, it is clear that the smallest discernible signal-to-noise ratio is not a fixed quantity, but, instead, depends on the confidence level selected and the number of measurements included in estimating the signal and the background.

B5  SIGNAL AVERAGING The effect of signal averaging on a steady signal S can be envisioned in the following manner. Consider a signal S with an associated noise variance of σ 2 . Suppose that the signal is sampled n times over some period of time. If we add these observations together, the result will be n

(B.17) ST = ∑Si i =1

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where ST is the total signal (i.e. the sum of the n signals) and Si is an individual observation. If the signal is relatively constant, each observation of the signal will be approximately constant, so (B.18) S1 ≈ S2 ≈  ≈ Sn As a result, Eq. (B.17) becomes (B.19) ST ≈ nS1 The total variance associated with the total signal will be the sum of the variances for the individual observations n

(B.20) σ T2 = ∑σ i2 i =1

where σ is the total variance associated with the total signal and σ i is the variance associated with an individual observation. Once again, if we assume that the individual variances are approximately equal, Eq. (B.20) reduces to 2 T

2

(B.21) σ T2 = nσ 12 Converting the total variance in Eq. (B.21) to a root-mean-square value gives (B.22) σ T = n σ1 The signal-to-noise ratio for the total signal will be given by S  ST nS1 (B.23) ≈ = n 1 n σ1 σT  σ1  Eq. (B.23) reveals that the signal-to-noise ratio associated with the total signal ST is improved by a factor of n compared with the signal-to-noise ratio associated with a single observation.

B6  SIGNAL INTEGRATION Fig. B3.6 shows a simple analog circuit for performing signal integration that can be used to illustrate the noise-reducing properties of the process. The figure shows an analog integrator, consisting of an operational amplifier [31], a resistor, and a capacitor. The output of this circuit is given by

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Figure B3.6  A simple analog integrator based on an operational amplifier where the input voltage Vin is a DC signal with a superimposed white-noise component, Vout is the integrated output of the circuit, and S&H is a sample-and-hold circuit that keeps the output constant until the reset switch is momentarily closed prior to another integration.

−1 t Vout = V in dt (B.24) RC ∫t =0 where Vin is a DC signal with a superimposed white-noise component, Vout is the integrated output of the circuit, R is the resistance, and C is the capacitance.The duration of the integration period, determined by the reset switch, is the time difference between when the switch is closed (the start of the integration period, t = 0) and when the switch is opened (the end of the integration period, t). The output of the operational amplifier is fed into a sample-and-hold circuit that keeps the output constant until the reset switch is momentarily closed prior to another integration. If the input to the circuit is essentially DC, the output will be given by −1  (B.25) Vout =  V t  RC  in integration where tintegration is the length of the integration period. Fig. B3.6 shows the polarity of the voltage across the capacitor. Since the input signal is DC, the direction of current through the capacitor due to the signal will always be in the same direction as shown in the figure by the signal arrow. As the integration period proceeds the signal voltage across the capacitor will increase with time. By contrast, any white-noise component superimposed on the DC signal of interest will fluctuate in a random manner about the DC signal. As a result, roughly half of the time the noise will charge the capacitor in the positive direction and

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roughly half of the time it will discharge the capacitor in the negative direction. As a result, the noise component will tend to average to zero during the integration period, improving the signal-to-noise of the output signal.

B7  MATHEMATICAL BASIS FOR AMPLITUDE MODULATION AND SYNCHRONOUS DEMODULATION Consider a signal containing the information of interest given by (B.26) Signal(t ) = As cos(ω st + φ ) (B.27) Signal(t ) = As [cos ω st cos φ − sin ω st sin φ ] where As is the amplitude of the signal, ws is the angular frequency of the signal in radians per second, t is the time, and φ is an arbitrary phase angle. Consider also a carrier signal given by Carrier(t ) = Ac cos ω ct (B.28) where Ac is the amplitude of the carrier and wc is the angular frequency of the carrier signal. We also assume that Ac > As and ω c  ω s . In the amplitude modulation process, the carrier signal is multiplied by the signal of interest, giving  Signal(t ) × Carrier(t ) = {As [cos(ω st )cos φ − sin(ω st )sin φ ]}Ac cos ω ct (B.29) Signal(t ) × Carrier(t ) = [( As cos ω st )( Ac cos ω ct )]cos φ − [( As sin ω st ) ( Ac cos ω ct )]sin φ

 (B.30) Eq. (B.30) consists of two terms that we can treat separately to keep the length of the equations manageable.46 Performing the multiplication on the first term in Eq. (B.30) gives

46

cos a cos b = 21 [cos(a + b ) + cos(a − b )] ; also sin( a + b ) = sin a cos b + cos a sin b and sin(a − b ) = sin a cos b − cos a sin b ; therefore, sin a cos b = 21 [sin(a + b ) + sin(a − b )] .

Recall that

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AA (B.31) First term =  s c  [cos(ω c − ω s )t + cos(ω c + ω s )t ]cos φ  2  AA (B.32) First term =  s c cos φ  [cos(ω c − ω s )t + cos(ω c + ω s )t ]  2  Performing the multiplication in the second term gives AA  Second term =  s c  [sin(ω c + ω s )t + sin(ω c − ω s )t ]sin φ  2  (B.33) AA (B.34) Second term =  s c sin φ  [sin(ω c + ω s )t + sin(ω c − ω s )t ]  2  The sum of Eqs. (B.32) and (B.34) is the modulated signal. Eqs. (B.32) and (B.34) reveal that the multiplication of the carrier signal by the signal of interest generates two new frequencies, a sum frequency (ω c + ω s ) and a difference frequency (ω c − ω s ) , that are symmetrically located about the original carrier signal frequency wc (see Fig. 3.26). The signal of interest has been shifted to higher frequency by the amplitude modulation process, thereby moving it out of the 1/f noise-dominated region in the transmission channel. To recover the signal of interest, the information must be down-shifted by the process of demodulation. To demodulate this signal, we must multiply both Eqs. (B.32) and (B.34) by the original carrier signal. Starting with the first term (Eq. (B.32)), we get  As Ac  (B.35) cos φ  [ cos(ω c − ω s )t + cos(ω c + ω s )t ] Ac cos ω ct    2  which, after evaluating and combining equivalent terms,47 yields  A A2   A A2   A A2  =  s c cos φ  cos ω st +  s c cos φ  cos(2ω c − ω s )t +  s c cos φ   2   4   4  ω ω cos(2 ) t + c s (B.36)

47

Recall that

cos ω st is an even function so that cos( −ω st ) = cos ω st .

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Multiplying the second term (Eq. (B.34)) by the carrier, evaluating, and combining equivalent terms48 yields  AA  −  s c sin φ  [sin(ω c + ω s )t + sin(ω c − ω s )t ] Ac cos ω ct   2   (B.37)  A A2  = −  s c sin φ  {(sin(ω c + ω s )t )(cos ω ct ) + (sin(ω c − ω s )t )(cos ω ct )}  2 

(B.38)  A A2  (B.39) = −  s c sin φ  {(sin(2ω c + ω s )t ) + (sin(2ω c − ω s )t )}  4  Examining Eqs. (B.36)and (B.39), we see that all the terms are at high frequency except the first term in Eq. (B.36). Passing the demodulated signal through an output low-pass filter can remove all the high-frequency components in Eqs. (B.36) and (B.39), leaving the demodulated signal given by  A A2  (B.40) Demodulated signal =  s c cos φ  cos ω st  2  Eq. (B.40) shows that the amplitude of the demodulated signal of interest at ws is proportional to the cosine of the phase angle, φ, in Eq. (B.26). If the signal of interest (Eq. (B.26)) and the carrier have the same phase ( φ = 0 ), the filtered demodulated signal given by Eq. (B.40) reduces to  A A2  (B.41) Demodulated signal =  s c  cos ω st  2  For mathematical simplicity, the signal of interest was taken as a single frequency. In practice, the information of interest would be contained in a small range of frequencies (i.e. a band). Since each frequency in the band would undergo the same modulation/demodulation sequence, the result would yield the same small range of frequencies. In the demodulation process, only the signal that has been modulated at the carrier frequency (Eq. (B.29)) will be successfully demodulated in the 48

Recall that

sin ω st is an odd function so that sin( −ω st ) = − sin ω st .

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synchronous demodulation step. Extraneous signals at other frequencies (i.e. noise) will be rejected. But what about noise that just happens to have the same frequency as the signal of interest? Consider a noise signal given by (B.42) Noise(t ) = AN cos(ω st + φ ) (B.43) Noise(t ) = AN [cos ω st cos φ − sin ω st sin φ ] where AN is the amplitude of the noise signal at ws. Following the same steps taken above, it can be shown that the demodulated noise signal will be given by  A A2  (B.44) Demodulated noise =  N c cos φ(t ) cos ω st  2  Once again the amplitude of the demodulated signal is proportional to cos φ . However, in the case of noise, φ(t) is not constant, but varies randomly with time and takes on all values from 0 to 2π over time. This means that, on average, cos φ(t ) will tend to be positive half of the time and negative half of the time. Summed over time by the capacitor in the output low-pass filter, the amplitude of the noise signal given by Eq. (B.44) will average to zero:   AN (n )Ac2  cos φ(n )   ∑   2  (B.45) lim  →0 n→∞ n     where n is the number of samples in the average. Note that the amplitude of the noise signal, AN(n), will also vary over time (in an uncorrelated manner with φ) from positive to negative. So even if a noise signal happens to have the same frequency as the signal of interest, it will be averaged out by the low-pass output filter.

REFERENCES [1] Jenkins, F. A.; White, H. E. Fundamentals of Optics, 4th ed.; McGraw-Hill: New York, 1976. [2] Shurcliff, W. A. Polarized Light Harvard University Press: Cambridge, MA, 1966. [3] Kliger, D. S.; Lewis, J. W.; Randall, C. E. Polarized Light in Optics and Spectroscopy Academic Press: Boston, MA, 1990.

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[4] Goldstein, D. Polarized Light, 2nd ed.; Marcel Dekker: New York, 2003. [5] Berova, N.; Polavarapu, P. L.; Nakanishi, K.; Woody, R. W. Comprehensive Chiroptical Spectroscopy, 1. Wiley: New York, 2014. [6] Polavarapu, P. L. Chiroptical Spectroscopy—Fundamentals and Applications CRC Press,Taylor & Francis Group: Boca Raton, FL, 2017. [7] D. Brewster, Phil. Trans. 1815, 105, 125. [8] Makas, A. S.; Shurcliff, W. A. J. Opt. Soc. Am. 1955, 45, 998. [9] Kliger, D. S.; Lewis, J. W.; Randall, C. E. Polarized Light in Optics and Spectroscopy Academic Press: Boston, MA, 1990 36. [10] Kliger, D. S.; Lewis, J. W.; Randall, C. E. Polarized Light in Optics and Spectroscopy Academic Press: Boston, MA, 1990 37. [11] Kristjánsson, L. Iceland Spar and its Influence on the Development of Science and Technology in the Period 1780–1930: Notes and References, 3rd ed.; Institute of Earth Sciences, Science Institute: University of Iceland, 2010 203, Report RH-20-2010. [12] Archard, J. F.; Taylor, A. M. J. Sci. Instrum. 1948, 25, 407. [13] Biot, J. R. Bull. Soc. Phil. Paris 1815, 6, 26. [14] Shurcliff, W. A. Polarized Light Harvard University Press: Cambridge, MA, 1966 59. [15] Land, E. H. J. Opt. Soc. Am. 1951, 41, 957. [16] Herapath, W. B. Phil. Mag. 1855, 3 (4), 161. [17] Herapath, W. B. Phil. Mag. 1855, 9, 366. [18] Davenport, W. B., Jr.; Root, W. L. An Introduction to the Theory of Random Signals and Noise McGraw-Hill: New York, 1958. [19] Ambrózy, A. Electronic Noise McGraw-Hill: New York, 1982. [20] Shottky, W. Ann. Phys. 1918, 57, 541–567. [21] Ambrózy, A. Electronic Noise McGraw-Hill: New York, 1982 18. [22] Johnson, J. Phys. Rev. 1928, 32, 97. [23] Nyquist, H. Phys. Rev. 1928, 32, 13. [24] Ambrózy, A. Electronic Noise McGraw-Hill: New York, 1982 113. [25] Goldman, S. Frequency Analysis, Modulation, and Noise McGraw-Hill: New York, 1948. [26] Ahsan, S. T.; McCann, H. Sci. Int. (Lahore) 2014, 26 (1), 65–73. [27] Lyons, R. G. Understanding Digital Signal Processing, 3rd ed.; Prentice-Hall: New York, 2011. [28] Jenkins, F. A.; White, H. E. Fundamentals of Optics, 4th ed.; McGraw-Hill: New York, 1976 253. [29] Malus, E. Mém. Soc. Arcueil 1808, 1, 113. [30] Martin, T., Ed. Faraday’s Diary; George Bell and Sons: London, 1933; pp. 263 paragraph 7498,Volume IV, Nov. 12, 1839—June 26, 1847. [31] Bruce, Carter; Ron, Mancini Op Amps for Everyone, 5th ed.; Newnes: Boston, MA, 2017.

CHAPTER 4

Chiroptical Spectroscopic Studies on Soft Aggregates and Their Interactions Vijay Raghavan, Prasad L. Polavarapu Vanderbilt University, Nashville, TN, United States

4.1  INTRODUCTION The word “aggregation” can encompass several situations ranging from two molecules coming together to form a dimeric structure [1] to several molecules coming together to form an organized assembly. Soft aggregates belong to the latter case where the varying number of molecules can come together to form a supramolecular assembly. This situation often occurs for surfactants (also popularly referred to as amphiphiles) [2], which contain both hydrophilic and hydrophobic groups in their chemical structures. Although there are a number of peptides that are known to aggregate, our focus in this chapter is limited to aggregates formed by surfactants. For a surfactant forming regular micellar structure in water, its molecules initially stay at the air–water interface because the hydrophobic part of surfactant molecules do not have enough affinity to mingle with water molecules. However, at a certain concentration, known as critical micelle concentration (CMC) or critical aggregation concentration (CAC), the air–water interface would have been filled up and surfactant molecules are forced to go into the water solvent. As a consequence, the surfactant molecules assemble together, inside the water solvent, in such a way that the hydrophobic portions of the surfactant molecules form their own interior surface facing away from water molecules. One classic example of these cases is sodium dodecyl sulfate (SDS), which when placed in water forms, above CMC, micellar structures with hydrophilic head groups pointing towards water and hydrophobic tails pointing away from water forming their own interior surface. A different example is Bis(2-ethylhexyl) sulfosuccinate sodium salt (also known as Aerosol-OT and abbreviated as AOT), which when dissolved in nonpolar solvents forms, above CMC, reverse micellar structures with hydrophobic tails pointing towards nonpolar solvent molecules and Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00004-5 Copyright © 2018 Elsevier B.V. All rights reserved.

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hydrophilic groups pointing away from the solvent forming their own interior surface. AOTs are known to encapsulate polar molecules, such as water, protecting them from unfavorable repulsive interactions with nonpolar solvent molecules. The assemblies, including micelles, reverse micelles, along with other possibilities such as lamellar, vesicles, ribbon, and rod-like structures [2] are referred to as soft aggregates. There are several achiral surfactants, such as tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), and 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), also in wide use, in addition to SDS and AOT mentioned above. The routine techniques for studying the aggregation properties of achiral surfactants include tensiometry, conductance, dynamic light scattering, fluorescence quenching, nuclear magnetic resonance (NMR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The focus in this chapter is on chiral surfactants, which have chiral centers in their hydrophilic head groups and/or in hydrophobic tails, and also on the interaction between achiral surfactants and chiral molecules. Although the literature studies on achiral surfactants and their interactions with achiral molecules are abundant, those on chiral surfactants and on interaction between achiral surfactants and chiral molecules are limited. Chiroptical spectroscopic methods [1,3,4] are best suited for studying the chirality-dependent properties. Thus, for studying the soft aggregates endowed with chirality, the techniques mentioned above for achiral surfactants are to be augmented with chiroptical spectroscopic methods. Four widely used independent techniques [1,3,4] that fall under the banner of chiroptical spectroscopy are optical rotatory dispersion (ORD), electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and vibrational Raman optical activity (ROA). These methods are briefly introduced below.

4.1.1  Optical rotatory dispersion [1,3] When linearly polarized light is passed through an isotropic sample of chiral molecules, the polarization axis of transmitted light is rotated. The direction and magnitude of rotation depend on the nature of chirality in the molecules investigated. The measured rotation, designated as optical rotation (OR), depends on the sample concentration and length of the light travel in the sample (referred to as the path length) and the wavelength of

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the light used. It can also depend on the temperature and solvent used for preparing the solution. OR is associated with ±sign (reflecting the direction of rotation) and its measurement method is often referred to as polarimetry. Commercial polarimeters are available for measuring OR. In the earlier literature, OR measurements using continuous scan dispersive instruments used to be reported.These expensive instruments are less commonly used in the current times. Instead, inexpensive discrete wavelength polarimeters are routinely used.These later instruments enable OR measurements at discrete wavelengths, facilitated by the use of electronically controlled appropriate narrow band light filters. Conventional polarimetric methods are not sensitive enough to measure OR for gas-phase samples. Nevertheless, the measurement of OR for gasphase samples is achieved in recent years using special techniques referred to as cavity ring down polarimetry [5] (see Chapter 14 in this volume). Since the measured OR depends on the sample concentration, and path length, a quantity normalized with these values, is commonly reported as a property that is considered to be the characteristic of the molecules comprising the sample. This normalized quantity is referred to as specific rotation (SR), and in more recent literature as specific optical rotation (SOR).

α [α ] = (4.1) c ×l In the above equation, [α] is SOR, α is OR, c is the concentration in g cc−1 (cc = cubic centimeter), and l is path length expressed in decimeters (dm). The units used for reporting [α] are deg cc g–1 dm–1. It is common practice to measure and report [α] at sodium D line wavelength (589 nm) and designate it as [α]D. When electronic transitions in the molecule occur at shorter wavelengths, Drude’s equation [6] (vide infra) indicates that the measured ORs will have larger magnitudes toward those shorter wavelengths and therefore it is advantageous to measure OR at several shorter wavelengths. The plot of [α] (or sometimes α itself) as a function of wavelength is referred to as the ORD curve. The measurement of SOR was limited in the older literature to one wavelength at 589 nm, but ORD, or wavelength resolved SOR, has become more common in recent years for two reasons: (a) The magnitude of SOR at shorter wavelengths is normally larger than that at longer wavelengths. This fact allows for measurements on samples that are available in limited quantities and (b) the ability to predict ORD accurately using quantum chemical (QC) methods, [1,3,7] for small-to-medium-range molecules,

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allowing for the interpretation of the experimentally measured values using corresponding predicted values.

4.1.2  Circular dichroism Differential absorption, ∆A, of left and right circularly polarized incident light by chiral molecules is referred to as circular dichroism (CD). ∆A = AL − AR (4.2) In the above equation, AL and AR are, respectively, the absorbances for left and right circularly polarized light. Absorbance, A, the logarithmic ratio of incident light intensity to transmitted light intensity, is related to the sample concentration (c) and pathlength (l) through molar extinction coefficient, ε. That is, A = ε × c × l). The CD phenomenon can originate from electronic transitions or vibrational transitions in the molecule. CD originating from electronic transitions is referred to as ECD and that from vibrational transitions is referred to as VCD. 4.1.2.1  Electronic circular dichroism [1,3] ECD is measured using commercial ECD spectrometers operating in the UV–Visible spectral region (∼190–600 nm). ECD spectra are normally presented as differential molar extinction, ∆ε, in units of M−1 cm−1, where M is molarity (mol L−1) as a function of wavelength. Most of the early literature studies used the ECD for monitoring the molecular structural changes by following the corresponding changes in the experimental ECD spectra. Recent advances in QC predictions of ECD are permitting the comparison of experimental spectra with those predicted using high-level QC predictions, for small-to-medium range molecules [1,3,8]. 4.1.2.2  Vibrational circular dichroism [1,3,4,9,10] VCD is measured in the infrared spectral region, ranging from 4000– 900 cm-1, using commercial Fourier transform (FT) VCD spectrometers or home-built dispersive VCD instruments. The nature of FT-VCD method requires the use of light filters restricting the measurements to either O–H/N–H/C–H stretching region (∼4000–2500 cm−1) or finger print region (∼2000–900 cm−1). Most of the reported VCD measurements are for the latter region. Routine VCD measurements below ∼900 cm−1 region are not yet feasible. VCD spectra are normally reported as differential molar extinction, ∆ε, in units of M−1 cm−1, as a function of the wavenumber (or cm−1). Most of the VCD spectra of

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biological molecules are used for monitoring the molecular structural changes by following the corresponding changes in experimental VCD spectra (see Chapter 5). Recent advances in QC predictions of VCD are permitting the comparison of experimental spectra with those predicted, using high-level QC predictions, for small-to-medium-range molecules [1,3,4,9–11].

4.1.3  Vibrational ROA [1,3,4,10,12] ROA is phenomenologically different from CD. It represents differential Raman scattering when chiral molecules are excited with right and left circularly polarized incident laser light or difference in intensities of Ramanshifted scattered right and left circularly polarized light components when chiral molecules are excited with linear/unpolarized incident laser light. ∆I = I αγ − I βδ (4.3) In the above equation, the subscripts and superscripts represent, respectively, the polarization state of scattered light components and incident laser radiation. A variety of combinations of polarizations for incident and scattered light components and scattering geometries can be used for experimental measurements.Three different scattering geometries can be used: (a) 90° scattering; (b) 180° back scattering and (c) 0° forward scattering. The most common measurements utilize the 180° backscattering geometry with unpolarized incident laser light and detecting right and left circularly polarized scattered light components. Unlike for VCD spectra, a larger width of vibrational region can be measured for ROA. Most common measurements are done for the ∼2000–200 cm−1 region. Moreover, water serves as an excellent solvent for Raman spectroscopy and hence biological molecules can be conveniently studied in their native water environment using ROA spectroscopy (see Chapter 6). Most of the ROA spectra of biological molecules are used for monitoring the molecular structural changes by following the corresponding changes in experimental ROA spectra. Recent advances in QC predictions of ROA are permitting the comparison of experimental spectra with those predicted, using high-level QC predictions, for small-tomedium-range molecules [1,3,13]. In this chapter, we provide a review of the applications of chiroptical spectroscopic methods for studying the soft aggregates derived from chiral surfactants, achiral surfactants, and achiral surfactants interacting with chiral molecules.

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4.2  OR STUDIES 4.2.1  Chiral surfactants The early studies on chiral surfactants using OR can be traced to Perrin et al. [14]. N-decyl-N,N-dimethylalanine hydrobromide (referred to as betaine) exhibited anomalous ORD curves for its enantiomers and the position of the ORD peak in the 225–230 nm region varied with both concentration and pH of the surfactant, as did the OR at a given wavelength. Perrin et al. noted that in the plot of OR at 232.5 nm as a function of concentration, for l-N-decyl-N,N-dimethylalanine hydrobromide, the slope changes very slightly at the CMC. This observation led them to suggest the possibility of detecting the micelle formation from OR studies. The authors stated that the change in OR and the concomitant shift of the ORD peak to higher wavelength on micelle formation, when compared with the shifts due to pH changes, are due to the change in the degree of ionization of the betaine on micelle formation. They further stated that the change in the behavior of OR on micelle formation is due to a change in the apparent dissociation constant of the weak acid. Mukerjee et al. [15] found that the observed deviations in OR, from that calculated as 0.587c (c being the concentration) at 320 nm as a function of concentration of β-d-octyl glucoside in water follow two different patterns. The deviation (a) is constant below CMC and (b) increases with concentration above CMC. Based on this observation, they concluded that OR measurements are capable of determining the CMC for some optically active surfactants. Using one term Drude’s equation [8], A [α ]λ = 2 2 (4.4) ( λ − λ0 ) in its rearranged form

λ02  1 1 (4.5) = − 1 [α ]λ λ 2 A  λ 2  1 the constants A and λ0 could be determined by plotting versus [α ]λ λ 2 1 .This analysis indicated that λ0 remained approximately the same below λ2 and above CMC. Therefore, the change in OR on micelle formation is considered to arise mainly from a change in the A term. The enhancement in the change in OR was thus considered to be due to the high concentration of glucoside head groups at the micelle surface rather than any significant conformational restraint at this surface.

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Chlorhexidine digluconate, where chirality is centered in the counterion rather than on the core of the micelle itself, also showed a deviation in OR upon micellation [16]. The plain ORD curves observed for chlorhexidine digluconate solution could not be fit to one term Drude equation, unlike the ORD curves for β–d-octyl glucoside [15]. This observation was interpreted as more than one electronic transition being responsible for the gluconate curves. The deviation in OR found after micelle formation was considered to be related to the change in the ionization of the gluconate on aggregation. OR and conductance studies were undertaken on morphine sulfate and related salts by Perrin et al. [17], which revealed that these compounds aggregate in aqueous solutions, and CMC can be identified from a break in the changes in ORs. Above CMC, SOR was found to increase for two salts and decrease for two other salts. However, the reasons for the changes in the SORs on micelle formation were considered to be difficult to interpret. Except for the studies reported above, studies on chiral surfactants specifically focusing on characterizing the SOR property appeared to have been unexplored until recent studies in our laboratory (vide infra). In the interim, however, numerous studies have reported the SOR as one of the physicochemical properties, but not with major focus on SOR itself. Shamsi et al. [18] synthesized three sulfated amino acid-based surfactants from l-leucinol, l-isoleucinol, and l-valinol (Fig. 4.1) for using them as effective surfactants for micellar electrokinetic chromatography (MEKC). These are N-undecenoxycarbonyl-l-leucine sulfate, N-undecenoxycarbonyl-l-isoleucine sulfate, and undecenoxycarbonyl-l-valine sulfate. Each of the synthesized surfactants had a polymerizable terminal double bond in the alkyl side chain (Fig. 4.1).

Fig. 4.1  N-undecenoxy carbonyl-l-amino acid-sulfated surfactants in monomeric form.

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The simple aggregates (at 100 mM) formed by these surfactants were converted into polymeric aggregates, polysodium N-undecenoxycarbonyl-l-leucine sulfate (poly-SUCLS), polysodium N-undecenoxycarbonyl-l-isoleucine sulfate (poly-l-SUCILS), and polysodium undecenoxycarbonyl-l-valine sulfate (poly-LSUCVS), by polymerizing the terminal double bond using [19] Co γ-irradiation. The ORs at 589 nm were reported for 10 mg/mL solutions of the simple aggregates and polymeric aggregates. Based on the magnitudes of the reported values, the reported ORs actually appear to be SORs, although that was not clearly mentioned, and we will assume them to be SORs in the following discussions. The aggregation numbers changed from 71 to 32, 66 to 42, and 74 to 36 upon polymerization, respectively, for leucine-, isoleucine-, and valine-based surfactants, while the corresponding SORs changed from −19.35 to −22.65, −14.10 to −18.10, and −16.20 to −19.80.The magnitude of SOR of polymeric aggregates was slightly higher than that of simple aggregates but the polymeric aggregation number was significantly lower than the monomeric aggregation number. Two other chiral surface-active polymerizable ionic liquids, undecenoxycarbonyl-l-pryrrolidinol bromide (l-UCPB) and undecenoxycarbonyll-leucinol bromide (l-UCLB) gave somewhat a different trend. In the case of l-UCPB, upon polymerization aggregation number decreased (from 95 to 34) and magnitude of SOR increased (from −2.35 to −7.84). In the case of l-UCLB, upon polymerization aggregation number decreased (from 97 to 25) and magnitude of SOR also decreased (from +21.67 to +17.45) [20]. Four alkenoxy leucine-based surfactants containing a terminal double bond, namely sodium N-octenoxy carbonyl-l-leucinate (l-SOcCL), sodium N-nonenoxy carbonyl-l-leucinate (l-SNoCL), sodium N-decenoxy carbonyl-l-leucinate (l-SDeCL), and sodium N-undecenoxycarbonyl-l-leucinate (l-SUCL) with C8, C9, C10, and C11 hydrocarbon tails, respectively, and one C11 chain surfactant with a terminal triple bond, namely sodium N-undecynoxy carbonyl-l-leucinate (l-SUCyL), were also characterized in monomeric and polymeric forms [21].The magnitude of the SOR values at 1% (w/v) for the alkenoxy leucine-based surfactants decreased (−16 to −9) with a decrease in the chain length (C11–C8), for both simple aggregates and polymeric aggregates. Interestingly, there was a proportional increase in the aggregation number (75–130 for monomeric surfactants and 37–46 for polymeric surfactants) with an increase in the chain length from 8 to 11 carbon atoms. The aggregates from the alkenoxy leucine-based surfactant also showed a decrease in the aggregation number (from 58 for a polymer to 18 for a monomer), but SOR did not change significantly.

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Similar results were reported for polysodium N-undecenoxy carbonyll-leucinate (poly-l-SUCL) and polysodium N-undecenoxy carbonyll-isoleucinate (poly-l-SUCIL) [22]. Dipeptide surfactants derived from the sodium salt of N- undecanoyl-l-Rleucinate where the R group was alanyl, valyl, seryl, or threonyl were studied by Shasmi et al. [23]. The monomer aggregates were also polymerized to make polymer aggregates. For the dipeptide polymer surfactant aggregates, the SOR and aggregate number were measured but only the SOR values were measured for dipeptide monomer surfactant. The SOR for the micelle polymers followed the same trend as that of the monomers, as the R group changed from alanine to threonine. However, for polymeric aggregates, there was no correlation between the aggregate number and SOR. As R changed from alanine, valine, serine to threonine, the corresponding aggregation numbers were 28, 32, 26 and 29, while SORs were ∼ −49, −37, −36, and −28. No effort was made in the above-mentioned studies to explore the connection between the changes in SOR above CMC and the sizes of aggregates. Recent studies in our laboratory addressed this connection (vide infra). In our laboratory, we undertook wavelength-resolved SOR studies on chiral surfactants and on achiral surfactants interacting with chiral molecules and supplemented them with those using various other techniques, importantly, tensiometry, NMR, XRD, and, wherever appropriate, TEM and SEM. The discovery that sodium salts of simple fluorenyl methyl oxy carbonyl (FMOC)-amino acids (AAs) exhibit surfactant properties led us to undertake detailed investigations on them. From tensiometry, it was found that sodium salts of FMOC-l-valine, FMOC-l-leucine, and FMOCl-isoleucine formed aggregates at ∼0.1 M [24]. No such aggregates were formed in methanol. Small-angle powder XRD studies revealed that the FMOC amino acids formed bilayers beyond 0.2 M. Interestingly, the labile FMOC group did not cleave from the amino acids probably because it was protected inside the bilayers. The SOR as a function of concentration of aqueous FMOC-AA-Na solutions changed significantly. At concentrations in the ∼0.07–0.3 g/mL range, SOR changed from −22.6 to −9.8 for FMOC-Val-Na, −20.6 to −6.9 for FMOC-Leu-Na, and −11.4 to −1.2 for FMOC-iLeu-Na (Fig. 4.2). Due to its gelation at these concentrations, the SOR of aqueous FMOC-nLeu-Na could not be measured. At concentrations above CMC, SOR is determined by aggregates in the bulk solution and is expected to be influenced by the intermolecular interactions within, and between, the aggregates. The experimentally observed

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Fig. 4.2  The SOR at 405 nm (A, C, E G) and surface tension (B, D, F, H) as a function of logarithmic concentration in water (black) and methanol (blue) for FMOC-Val-Na (A and B), FMOC-Leu-Na (C and D), FMOC-iLeu-Na (E and F), and FMOC-nLeu-Na (G and H). The structures of the FMOC-amino acids are displayed in I. The insets in A, C, and E display SOR as a function of concentration in the 50–300 mg/mL range. Data from Vijay and Polavarapu [24]. All amino acids used have l-configuration.

concentration dependence of SOR confirms this hypothesis. As the concentration increases, the number and size of the aggregates are expected to increase, which leads to varying intermolecular interactions and hence to varying SOR as a function of concentration. In the case of sodium salts of FMOC-AA in water, SOR changed steeply with concentrations above CMC. Small-angle XRD indicated the formation of bilayers at these concentrations.Therefore, steep changes in SOR beyond CMC were attributed to the formation of these bilayers. In a separate work, for two cationic surfactants, lauryl ester of l-tyrosine·HCl (LET) and lauryl ester of l-phenylalanine·HCl (LEP), we found direct correlation between the increase in the size of the aggregates and their SORs, with an increase in concentration (Fig. 4.3) [25]. The size of the aggregate was measured by two independent methods: time-resolved fluorescence quenching (TRFQ) and TEM. The sizes of the LET and LEP aggregates as a function concentration and temperature measured using TRFQ were already available in the literature [26]. We synthesized LET and LEP according to the procedure reported elsewhere [27] and estimated [25] the size of the aggregates at 50 and 200 mM for LEP and at 200 mM for LET using TEM. The increase in the size of the LEP aggregates with a concentration from TRFQ and TEM corroborated well. The aggregation number from TRFQ experiments, at 32 °C, varied from 80 to 160 for LEP and 80 to 100 for LET in the concentration range 50–200 mM. Interestingly, the SOR for LEP at 405 nm increased linearly from 37 to 56 deg cc g–1 dm–1 with increase in the aggregate size. A similar trend was observed for LEP at other wavelengths. LEP showed a significant decrease in the aggregation number with an increase in temperature at 50, 150 and 200 mM [26]. In the case of LET, contrary to LEP, the aggregation numbers increased both with concentration and temperature, but the increase was not substantial. The behavior of LET is opposite to that of LEP (even though both are cationic surfactants) and could be associated with the

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Fig. 4.3  (A) Correlation between SOR at 405 nm and the aggregation number of LEP and LET. (B) The structures of LEP (R = H) and LET (R = OH); Data from Vijay et al. [25].

p­ resence of Cl− bridge between OH and NH3+ groups in LET [26,27]. Note that in the case of LEP, the OH group is absent on the aromatic ring [25,27]. Although the aggregation number of LET did not change significantly with temperature, the SOR did change significantly, which may be associated with the disruption of hydrogen bonding in LET with increasing temperature and also with large vibrational contributions to SR expected from low frequency vibrational modes [28]. Nevertheless, good linear correlation between the aggregation number and SOR was evident in the 40−60 °C range (see Fig. 4.3). Synthesis and chiroptical spectroscopic studies on chiral surfactants derived from tartaric acid were initiated in our laboratory recently. The salt (T12M) formed between lauryl monoester of diacetyl-tartaric acid (T12OH) and α-methylbenzylamine (MBA) (see Fig. 4.4) [29] is one such chiral surfactant, which also had the attribute of being ionic liquid at room temperature. Extensive investigations on the chiroptical properties of the diastereomeric T12M salts formed from l-T12OH-(S)-MBA, d-T12OH-(S)-MBA, l-T12OH-(R)-MBA, and d-T12OH- (R)-MBA (designated, respectively, as LS, DS, LR, and DR) were undertaken [30]. In all these cases, an inflection point was found in the concentration-dependent SOR values at the CMC (∼0.3 mM). Thus, concentration-dependent SOR measurements can provide a convenient approach for the determination of CMCs. The magnitudes of SOR values increased beyond CMC indicating the growth of aggregates. As a control experiment, SOR was measured for a chiral tartaric acid-based nonaggregating analog and these SOR values did not

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Fig. 4.4  Chemical structure of the ionic liquid, T12M, formed from l-T12OH and (S)-MBA.

exhibit concentration dependence. This observation, along with that of oppositely signed SOR values for the enantiomers, suggested that the presence of aggregation size-mediated artifacts are not present in the abovementioned SOR measurements. In a separate work, we used SOR to follow the formation of small micelles and its subsequent transformation to large vesicles. It was reported that the aqueous solution of (1R,2S)-(–)-N-dodecyl-N-methylephedrinium bromide (DMEB; Fig. 4.5A) forms small micelles at 190 nm, of ECD spectra of LEP as a function of concentration (0.01–200 mM) (Fig. 4.9), despite the presence of chromophores from aromatic and ester groups [25]. LET also did not exhibit concentration-dependent ECD spectral changes, despite formation of aggregates.

Fig. 4.9  ECD (bottom panel) and absorbance (top panel) spectra of LEP at concentrations in the range 0.01–200 mM. Data from Vijay et al. [25].

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FMOC-AA-Na surfactants have CMC around 0.1 M (vide supra). Powder XRD studies on these surfactants indicated that bilayer structures and significant changes in SOR could be observed above the CMC. However, the ECD spectra of FMOC-AA-Na, specifically FMOC-Leu-Na, FMOCnLeu-Na in the concentration range 1–10 mM showed no ECD bands in the 190–300 nm region [24]. ECD measurements above this concentration range were not possible because of excessive absorbance. It is worth mentioning that the corresponding parent acids at 10 mM in CH3OH also did not reveal measurable ECD signals in the 205–300 nm region [24]. Since ECD and ORD are related through the Kramers–Kronig transform [47,48], the presence of concentration-dependent changes in SOR and their absence in ECD suggest that the changes in SOR in the visible spectral region could be originating from the electronic transitions that appear in the far-ultraviolet region (10 mM due to the change in CTAB aggregates from micelles to giant-worm like [38].

4.3.4  Interaction between surfactants and proteins/peptides Studies on surfactant–protein systems have received much attention because of their biochemical importance as well as their physicochemical interest. Many studies on the protein–surfactant interactions have been carried out for more than half a century. Some studies have used ECD to monitor the changes in the conformations of proteins upon interaction with surfactants. The mechanism of protein denaturation induced by surfactants was discussed from the perspective of kinetic data using ECD measurements [56]. The interaction of ethoxylate-based surfactants with bovine serum albumin (BSA) has been studied [57]. Specifically, mono-disperse tetra-(C12E4), hexa-(C12E6), and octa-ethyleneglycol mono-n-dodecyl ether (C12E8), and

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poly-disperse eicosa-ethyleneglycol mono-n-tetradecyl ether (C14EO20), with respective CMCs of 4.6 × 10−5, 7 × 10−5, 8 × 10−5, and 2 × 10−5 M, were employed for the interaction studies. ECD measurements were performed on BSA with and without various concentrations of C12En (n = 4, 6, 8) and C14EO20, at surfactant/protein molar ratios lower and higher than 1. The % α-helix content was determined from the ECD spectra using - ME  208 - 4000  × 100 % a − helix =  (4.7) [33,000 - 4000 ] where the numbers 4000 and 33,000 refer to ME208 for random coil and pure α-helix conformation, respectively. ME208 represents the mean residual ellipticity (MRE) at one of the characteristic minima for α-helices (208 nm), calculated using the following equation: observed ECD MRE = (4.8) C p × n × l × 10 where “observed ECD” means the ECD observed at 208 nm, Cp is the molar concentration of the protein, n the number of amino acids in the protein (583 for BSA), and l the path length (in cm). It was found that the secondary structure of BSA in the presence of ethoxylates increases the percent α-helix structure at the cost of decrease in the turns and unordered structures. The helical content of BSA in the presence of surfactant reached a maximum near the CMC of the surfactant. Note that the α-helix content is also higher for surfactant concentrations much higher than CMC due to protein–surfactant interaction. The interaction of BSA with three imidazolium-based gemini surfactants, with imidazolium head groups, dodecyl side chains, and different connectors between the head groups, was also studied using ECD. The connectors used were: (a) 2,5-dioxahexyl, (b) 2,15-dioxahexadecyl, or (c) butyl group. These gemini surfactants were, respectively, labeled as: oxyC2, oxyC12, and C4. The BSA concentration for the ECD experiments was kept constant at 0.15 mg/mL, and a 25 mM solution of sodium phosphate (pH 7.7) was used as a buffer. The surfactant concentration was varied from 1 µM to 1.05 mM. The results of these studies showed that the conformation of BSA has changed dramatically in the presence of all studied surfactants (see Table 4.1). The authors showed that the drop in the α-helix content versus surfactant concentration is more pronounced for oxyC2 and oxyC12 gemini surfactants. At a surfactant:BSA ratio of 491:1, the largest

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Table 4.1  Secondary Structures of BSA Obtained from ECD Spectraa α-Helix β-Sheet Surfactant csurf (mM) csurf:cBSA (%) (%) Turns (%)

Others (%)

oxyC2 oxyC2 oxyC2 oxyC2 oxyC12 oxyC12 C4 C4 C4

26 26 37 33 26 32 26 28 32

0 0.001 0.125 1.050 0 0.063 0 0.048 0.760

0:1 0.5:1 58:1 491:1 0:1 31:1 0:1 23:1 364:1

54 54 24 40 55 35 56 50 41

9 9 27 14 7 21 7 11 12

11 11 13 13 11 12 11 11 14

Concentrations of surfactant and BSA are denoted as csurf and cBSA, respectively. Representative data taken from Gospodarczyk et al. [58]. a

decrease in the α-helical content of BSA was observed for oxyC2 gemini surfactant. The C4 gemini surfactant required higher concentrations, than oxyC2 and oxyC12, for inducing the major conformational changes [58]. The interactions of BSA with ionic surfactants (SDS and CTAB) and β-cyclodextrin (β-CD) were also investigated by ECD measurements. The anionic surfactant (SDS) induced changes in protein conformation at a higher concentration compared to the cationic surfactant (CTAB). The effect of SDS on the ellipticity of BSA depended on the concentration of the surfactant, but not with a linear correlation. The ability of cyclodextrin to help recover the initial ellipticity of the protein depended on the molar ratio between the surfactant and β-CD [59]. When β-CD was added at 10–2 M concentration to the [SDS]/[BSA] = 103 or [CTAB]/[BSA] = 102 solutions, the recovery of the initial α-helical contribution to the secondary structure of the protein was almost complete. At concentrations below CMC, β-CD hindered the interaction between the employed surfactants and the protein. The interaction of BSA with two cationic bis-quaternary ammonium gemini surfactants, in buffer solutions (pH 7.0) also showed that they change the secondary structure of BSA [60]. In addition, it was interesting to note that the secondary structure of proteins was influenced significantly by a surfactant with longer hydrophobic chain than the shorter ones. In a similar experiment, unfolding of rabbit serum albumin (RSA) by cationic surfactants CTAB and TTAB was studied using ECD [19]. It was shown that the addition of surfactant to 1 wt% RSA in pH 7 phosphate buffer, the α-helix content progressively decreased up to 10 mM of CTAB

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or TTAB. At and above 20 mM, the % α-helix content was extremely low and % β-sheet content increased concomitantly, indicating a complete loss of secondary structure. Gelamo et al. performed spectroscopic studies on the interaction of BSA and human serum albumins with ionic surfactants, namely SDS, cationic cetyltrimethylammonium chloride, and zwitterionic N-hexadecyl-N,Ndimethyl-3-ammonium-1-propanesulfonate. The ECD data corroborated the partial loss of secondary structure upon addition of surfactant indicating high stability of serum albumins [61]. The effect of five surfactants, sodium dodecyl sarcosinate, dodecyltrimethylammonium bromide, SDS, sodium decyl sulfate, and sodium octyl sulfate on the secondary structure of bovine β-lactoglobulin B was studied, as a function of surfactant concentration, using ECD spectra [62]. In the absence of a surfactant, the secondary structure content of β-lactoglobulin B was determined to be ∼16% α-helix, 18% β-helix, and 66% random coil. Near the CMC of each surfactant, an increase in the amount of two structured forms and a 20%–25% decrease in the amount of unordered form was noted. However, the relative amounts of the two structured forms depended on the used surfactant and its concentration, probably reflecting the influence of ionic and nonionic forces that govern the secondary structure of β-lactoglobulin. The effect of a nonionic surfactant, dodecyl octaethyleneglycol monoether, on the conformation of a series of proteins (histones H2B, Hf2b, H4, and H1), oxidized ribonuclease, protamine phosphate, reduced and carboxymethylated ribonuclease, soybean trypsin inhibitor (3-casein, concanavalin A, Lens culinaris agglutinin, and leucoagglutinin) was studied. The surfactant did not influence the conformation of H1 but showed specificity in influencing the conformation of histones H2B, Hf2b, and H4. Also, the surfactant did not influence the conformation of several complex and open chain proteins that were thought to be sensitive. The results indicated that nonionic surfactant may serve as a weak perturbant whose induction of conformational transition requires a specific protein structure. On the other hand, the ionic surfactants such as SDS serve as strong perturbants leading to the enhancement of the helical content of the protein [63]. Overall, nonionic surfactants in general appeared to be much less effective as protein denaturants than ionic surfactants. The conformational characterization of bombolitins I and III peptides in the presence of SDS micelles was reported by measuring their ECD spectra as a function of the concentration of SDS varying from 0 to 23 mM. The ECD spectra of bombolitin I and III in the absence of SDS indicated the absence of a well-defined secondary structure. But the structures are not

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completely random, as revealed by a broad negative shoulder at 222 nm and a low intensity negative band below 200 nm. At concentrations below the CMC, the spectra indicated the presence of type β-turn as suggested by the appearance of ECD at 228 nm. At SDS concentrations above the CMC, the ECD spectra indicated that the peptides tend to fold into α-helical conformation. At a ratio of about 1:1, the characteristic bands in the ECD spectra indicated 60% α-helix content in bombolitin III and 70% in bombolitin I [64]. The influence of micelles of cationic CTAB, anionic SDS, zwitterionic lysophosphatidylcholine, and dodecylphosphorylcholine lipid on the structure of human β-endorphin, and its 12–26 fragment, was investigated using ECD. The effect of surfactants on human β-endorphin and its fragment, as determined by the ECD bands at 208 and 222 nm, is shown in Table 4.2. The polypeptide concentration was 0.1 mg/mL. It is worth noting that the surfactant’s concentration used in this work was greater than CMC. In the absence of a surfactant, the polypeptide showed an unordered structure.The surfactants induced helicity in the polypeptide.The highest α-helix content was found with the SDS surfactant [65]. It has been found that α-chymotrypsin also adopts higher helix content than that of native by the addition of SDS [66]. CD has been used to study the interaction of reverse micelles with proteins as well. Interactions of horse heart cytochrome c derivatives (zinc porphyrin cytochrome c and porphyrin cytochrome c) with surfactant interfaces in AOT and CTAB reverse micellar solutions were studied with ECD. These studies revealed that the secondary structure of protein was lost in AOT-reverse micelles. However, the secondary structure seems to be preserved in CTAB-reverse micelles [67].

4.3.5  Interaction between surfactants and DNA aggregates and related materials The chromophores of the nucleic acid base themselves do not show ECD as they have a plane of symmetry. The ECD signals shown by nucleosides and nucleotides are of low intensity as they derive signal from the interactions Table 4.2  Influence of Surfactants on % α-Helix Content of β-Endorphin Condition α-Helix (%)

Buffer SDS Lysophosphatidylcholine CTAB Representative data from Pasta et al. [65].

a

4 29 21 15

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of the base with the asymmetric sugar moiety. Enhanced ECD bands are found for polynucleotides and nucleic acids, due to macromolecular organization driven by base–base interactions [68]. Therefore, ECD is considered to be a valuable method to study the conformation of both polynucleotides and nucleic acids. The structure of DNA lipoplexes with hydroxyethylated alkylammonium gemini surfactants that showed high transfection activity was studied using ECD. It was shown that the transfection efficiency of HEK293T cells with the pEGFP_N1 circular plasmid was high only when they formed chiral supramolecular DNA–gemini surfactant complexes. The transfection efficiency was maximum for concentrations at which the spontaneous aggregation of components was observed [69]. The ECD of DNA (extracted from salmon sperm) complexed with cetyltrimethyl ammonium (CTMA) was studied. The DNA–CTMA complex formed self-assembled film probably due to the alignment of the alkyl chains of the cationic surfactant molecules and ordering of the rod-like DNA helices [70,71]. Aggregates formed from annealed samples of dilauroyl-phosphatidyladenosine (DLPA), dilauroylphosphatidyl-uridine (DLPU) (Fig. 4.11), and their 1:1 mixture were investigated using ECD. These two surfactants self-assemble in solution to form supramolecular structures that behave dissimilarly, due to the difference in the nucleoside at the phospholipid polar head group [72]. ECD spectra of DLPU do not show dependence on surfactant concentration. Moreover, they are identical to those of uridine monophosphate (UMP), except for a slight difference at shorter wavelengths in the negative band, due to the different substitution of the phosphate in the case of the surfactant (Fig. 4.12). In other words, ECD indicates a similar base conformation both in supramolecular aggre-

Fig. 4.11  Chemical structures of (a) dilauroyl-phosphatidyl-adenosine (DLPA) and (b) dilauroyl-phosphatidyl-uridine (DLPU).

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Fig. 4.12  Concentration dependence of ECD spectra for (A) DLPU and (B) DLPA micellar solutions in 0.1 M PBS pH 7.5. Corresponding nucleosidic monophosphate monomer spectra (pink, a), UMP and AMP (10 mM), are reported in the same medium. Surfactant concentrations: (black, b) 1 mM; (red, c) 4 mM; (blue, d) 10 mM; (cyan, e) 20 mM. Adapted (digitized and replotted) with permission from Bombelli et al. [72]. Copyright (2006) American Chemical Society.

gates and in monophosphate monomer due to the poor stacking of the uridine base (Fig. 4.12). On the other hand, ECD spectra of DLPA are strongly affected by surfactant concentration. They show significant changes in the molar ellipticities and also different absorption from that of AMP. These ECD observations can be correlated to the respective aggregate shapes. The uridine derivative forms long worm-like aggregates that do not change with the aging of the solution. The worm-like aggregates of the adenosine derivative however undergo, upon aging, a self-assembling process resulting in the formation of giant helicoidal aggregates that exist along with the smaller wormlike aggregates [72].

4.3.6  Induced ECD When dissolved in chiral solvents, electronic transitions of achiral molecules can exhibit ECD.This phenomenon is referred to as induced circular dichroism (IECD).Tachibana et al. reported that water-insoluble achiral dye (Sudan III) showed IECD when solubilized in the aqueous micellar solutions of a chiral surfactant, potassium d-12-hydroxystearate.The CMC of chiral surfactant is 7.9 mM in water and 1.4 mM in 0.1 N KOH solution. The ECD for 0.12 mM Sudan III was measured at 70 °C in 0.1 N KOH aqueous ­solution

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of chiral surfactant at 19.2 mM. Interestingly, in contrast to the absorption spectra, the molecular ellipticity [θ] associated with IECD increased with time indicating a process toward equilibration. No IECD was observed in equimolar solutions of the dye and the surfactant in a nonpolar solvent benzene, where aggregation does not occur, indicating IECD in aqueous solution was due to the presence of, and interaction with, chiral micelles. A watersoluble dye, pinacyanol chloride, showed no IECD in the micellar solution, whereas it exhibited a strong IECD spectrum below the CMC, because of formation of the cationic dye/anionic surfactant complex [73]. Achiral micelles can also influence the ECD associated with electronic transitions of chiral molecules, such as lutein (Fig. 4.13), a carotenoid. Although lutein is a chiral molecule, its sample dissolved in ethanol does not exhibit ECD in the visible region. Takagi et al. [74] reported that upon the addition of SDS, lutein exhibits ECD in the visible wavelength region with positive and negative extrema on either side of crossover at about 390 nm. The ECD signs were inverted when the amount of SDS was increased. Further addition of SDS led to the disappearance of ECD. These observations were associated with the formation of helical assembly of lutein molecules, inversion of helicity, and breakdown of the helical assembly followed by inclusion of leutin molecules into SDS micelles. In a following work, the latter phenomenon has been considered to be due to the chiral orientation of the lutein molecules in combination with the surfactant molecules bound to them [75]. Interestingly, DTAB, a cationic surfactant was found to influence the ECD of lutein below CMC in a different way compared to SDS [76]. The CMC of DTAB in 20 mM phosphate buffer solution, at pH 7 and 25 °C is 10 mM. When lutein was dispersed in a solution of DTAB in 20 mM phosphate buffer solution, pH 7, the absorption spectra showed significant changes that are dependent on concentrations of DTAB. (a) Below 5 mM DTAB, there was only one absorption peak at 378 nm. (b) At 8 and 10 mM DTAB, absorption band shifted to longer wavelength, with fine structure between 410 and 510 nm. (c). At 15 mM DTAB, the absorption spectrum showed one band at 452 nm with a shoulder on either side. This latter pattern is similar to that observed for lutein solution in an organic

Fig. 4.13  Chemical structure of lutein.

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Fig. 4.14  Structures of (P)-5HM and (R)-TAPA.

s­olvent. The corresponding ECD spectra of lutein in DTAB solution at its CMC (10 mM) showed a sharp increase in ECD intensities, but no sign reversal occurred unlike in SDS solution. Above CMC, the ECD intensity decreased, as also happened in SDS solution, and was considered to be due to the incorporation of lutein into micelles and weakened interactions by their dispersion in the micelles. The authors stated that lutein molecules seem to form polymeric aggregates below the CMC, oligomers at CMC and monomers above CMC of DTAB solution.

4.3.7  Interaction between surfactants and charge transfer complexes (R)-2-(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy)propionic acid (TAPA; Fig. 4.14) is insoluble in water, but can be dissolved in SDS micellar solution. TAPA exists as a dimer in chloroform, but as a monomer in SDS. As a result, the ECD spectra of (R)-TAPA in chloroform appear with opposite signs to those in SDS micellar solution. Racemic 2-hydroxymethylthieno[3,2-e:4,5-e8]di[1]benzothiophene (5HM; Fig. 4.14) is also insoluble in water. When 5HM is incorporated into aqueous SDS micelles containing TAPA, charge transfer (CT) complex between 5 HM and TAPA occurs. This CT complex exhibits intense IECD signal that reverses sign with time or sonication.This observation was attributed to alteration from (M)-helix to (P)-helix of 5HM accompanied by composition change of charge transfer complex from 1:2 to 1:1 [77].

4.3.8  Interaction between surfactants and β-cyclodextrin The surfactants Igepal CA720 (IplCA7) and its interaction with β-cyclodextrin (β-CDx) was studied using IECD [78]. The IECD of IplCA7 increased with increase in the concentration of cyclodextrin. The orientation of the phenyl moiety of the surfactant molecule inside

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the β-CDx cavity was determined from the sign of IECD using established procedures [63,79,80] and Kirkwood–Tinoco coupled oscillator model [81].

4.3.9  Interaction between surfactants and polymers The binding of surfactants tetradecylpyridinium chloride (TPC) and decylammonium chloride (DAC) and their mixtures (TPC: DAC = 1:3 TPC: DAC = 1:1) to sodium poly(l-glutamate) (P(Glu); average MW 36 000) at pH 7.9 was studied at surfactant concentrations of ∼0.01–5 mM. In the absence of surfactants, P(Glu) formed random coil structure. With increase in the surfactant concentration, which results in increase in the degree of binding of the DAC surfactant, the ECD spectra of P(Glu) showed the formation of double minimum at 208 and 222 nm, which is characteristic of α-helical structure. In the pure TPC or binary-surfactant solution, ECD assignable to ordered conformations was not found [82]. The interaction between sodium dodecylbenzenesulfonate (SDBS) and gelatin solutions was studied above the gelation temperature by viscosity and ECD measurements [83].The ECD spectra demonstrated that the addition of SDBS increased the negative minima, due to the swelling of gelatin and exposure of the hydrophobic residues to the electrostatic interactions.

4.4  VCD STUDIES VCD provides structure-specific information in the analysis of chiral compounds. It is well established for the study of peptide solution structure and has been used to study both proteins and peptides. However,VCD studies specifically focusing on peptide surfactants are not available.

4.4.1  Chiral surfactants The VCD spectra of chiral assemblies of LEP were measured at 200 and 170 mM in D2O (Fig. 4.15) [25]. Neither significant VCD bands nor differences therein were found at these two concentrations.VCD measurements at lower concentrations were not possible due to low sample absorbance and path length limitation imposed by strong D2O absorption. Overall, VCD spectra did not provide any significant information on the chiral assemblies of LEP in D2O. VCD spectra of chiral assemblies of sodium salts of FMOC-AA-Na, namely FMOC-Val-Na, FMOC-Leu-Na, and FMOC-iLeu-Na in D2O were measured at 200 mM and compared with the VCD spectra of the

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Fig. 4.15  VCD (bottom, b and c) and absorbance (top, a and d) spectra of LEP at (a, b) 170 mM and (c, d) 200 mM measured using 50 and 100 µm SL3 cell, respectively. In (b) and (c), red traces represent the noise level in the respective VCD spectra (black traces). Data from Vijay et al. [25].

corresponding FMOC-AA-Na in methanol, where they do not form chiral assemblies [24]. The VCD bands of FMOC-Val-Na, FMOC-Leu-Na, and FMOC-iLeu-Na in D2O were weak. A prominent but weak negative band associated with symmetric COO– stretching (at ∼1582 cm–1 for FMOCVal-Na, 1574 cm–1 for FMOC-Leu-Na, and 1578 cm–1 for FMOC-iLeuNa) was present for the FMOC-AA-Na in methanol. Overall, the weak bands of FMOC-AA-Na for the aggregates in D2O and the corresponding nonaggregate solution in CD3OD showed no significant differences in the VCD spectra [24]. It is worth mentioning that the VCD spectra of the corresponding parent FMOC-amino acids (FMOC-AA-H) were also measured in CD3OD and compared (Fig. 4.16) with the corresponding VCD of FMOC-AA-Na aggregates in D2O [24]. Strong VCD band intensities for acid solutions in CD3OD contrast the weak VCD band intensities of FMOC-AA-Na aggregates in D2O. This difference may originate from three different sources: (a) aggregate formation for sodium salts versus nonaggregate solution for acids; (b) different vibrational functional groups (C═O stretching vibration from COOH in the parent acid vs C–O stretching vibration from delocalized COO– in the salt); (c) differences in the solvent environment (CD3OD for

Fig. 4.16  VCD spectra (A1, A3, B1, B3, C1, and C3) and the corresponding absorbance spectra (A2, A4, B2, B4, C2, and C4) of (A) FMOC-Valine, (B) FMOC-Leucine and (C) FMOCNorleucine as acid (left vertical panels) and their comparison to the corresponding spectra of respective sodium salts (right vertical panels). Data from Vijay and Polavarapu [24].

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acids and D2O for salts). Nonmicelle forming disodium salts of hibiscus and garcinia acids, however, showed the reverse trend, where salts have larger VCD signals than parent acids [84]. VCD investigations were also conducted for the recently synthesized and characterized surfactant T12M (Fig. 4.4), which is also a room temperature anionic liquid [29]. The vibrational absorption (VA) spectra (Fig. 4.17) of four diastereomers of T12M aggregates in D2O at 200 mM showed a band at 1743 cm–1associated with the ester carbonyl group; a band at 1623 cm–1 associated with COO–group; at 1562, 1500, and 1458 cm–1 associated with C═C stretching vibrations of the methylbenzylamine; a band at 1384 cm–1 associated with CH bending vibrations. The VCD spectra of four T12M diastereomers showed a bisignate couplet corresponding to the C═O stretching vibrations at 1743 cm–1and a negative VCD band associated with stretching vibration of COO– group [29,49]. At the concentrations employed, the MBA portion of T12M did not exhibit significant intensities, and therefore no major differences are seen among the VA and VCD of T12M diastereomers. Abbate et al. [85] used VCD to study the aggregates of DMEB (see Fig. 4.5 for structure) dispersed in carbon tetrachloride. They followed the VCD associated with deuterated hydroxyl bond stretching motion of the self-assembled DMEB molecules and their interaction with D2O molecules. The DMEB aggregates showed a negative VCD feature associated with OH(OD) bonds. The trapped D2O in the aggregate showed a weak negative VCD. From the OD-stretching region of the IR and VCD spectra, Abbate et al. showed evidence for a specific interaction between DMEB-d1 and D2O. On the higher frequency side of the strong negative VCD band,

Fig. 4.17  VCD (left) and VA (right) spectra of four T12M diastereomers (see Fig. 4.4). DR: d-T12OH-(R)-MBA; DS: d-T12OH-(S)-MBA; LR: l-T12OH-(R)-MBA, and LS: l-T12OH-(S)-MBA. Data from Vijay and Polavarapu [49].

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a shoulder associated with the OD-stretching of D2O molecules indicated the chiral influence of DMEB on trapped D2O molecules [85].

4.4.2  Chiral surfactant films VCD measurements on the films of proteins, carbohydrates, and amino acids revealed certain advantages, in terms of enhanced signal-to-noise (S/N) ratio, over those for the aqueous solutions [86,87]. Taking cognizance of this previous work, we tried to enhance the weak VCD of chiral aggregates by forming films of aggregates, hoping to avoid solvent absorption interference and improve the S/N in VCD measurements. However, the VCD spectra of two different films prepared from identical CH3OH solutions at times yielded mirror image spectra (Fig. 4.18) [24].The optical micrographs of the films that yielded mirror image VCD showed crystalline features. However, the SEM images of these films appeared very different. The SEM images of one of the films that showed the mirror image VCD indicated that the film was composed of only one layer, but the other film showed the presence of multiple layers. From these results, we speculated that the mirror image VCD features must be due to the formation of multiple layers versus single layer and that one cannot take it for granted that VCD measurements can always be made for films.

Fig. 4.18  VA (middle top) and VCD (middle bottom) spectra, optical micrographs (left), and the SEM image (right) of the FMOC-iLeu-Na films prepared from CH3OH solutions. The color of the border of the optical and electron micrographs correspond to the color of the spectral traces. Data from Vijay et al. [24].

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Fig. 4.19  VCD (right) and VA (left) spectra for (R)-methyl lactate in (0.2 M) in CCl4 solution, in 0.164 M/ 0.082 M AOT micelles in CCl4 and in DMSO solution. Adapted (digitized and replotted) with permission from Abbate et al. [88]. Copyright (2012) Elsevier B.V.

It is also worth mentioning that the spectra of some of the films, both from aggregates in D2O and methanol solution, were at times time-dependent. The films showed broad absorption bands initially (suggesting amorphous nature of film) associated with no VCD and transforming in a few days into sharp absorption bands (suggesting the crystalline nature of the film) [24].

4.4.3  Interaction between chiral organic molecules and achiral surfactants Abbate et al. also investigated chiral methyl lactate in the presence of reverse micelles using VA and VCD [88]. The VA and VCD spectra were recorded for (S)- and -(R)-methyl lactate for neat samples and at different concentrations in DMSO and CCl4 solutions and compared them with those of methyl lactates in reverse micelles of AOT in CCl4 (Fig. 4.19). The tendency of methyl lactate to interact with AOT aggregates was inferred from these studies. [88] In a similar work, Abbate et al. studied the VCD of d- and l-dimethyl tartrate in lecithin reverse micelles dispersed in cyclohexane, where larger interactions were noted [89]. The state of d- and l-dimethyl tartrate trapped in dry AOT reverse micelles in CCl4 was

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also investigated using VCD. The experimental data were considered to be consistent with the hypothesis that both enantiomers of dimethyl tartrate are mainly entrapped in the reverse micelles and their location was in the proximity to the surfactant head-group region [37].

4.4.4  Interaction between chiral polypeptides and achiral surfactants VCD was used to study the interaction of polymers and surfactants in the C–H stretching vibration region and in the Amide I region both in solution and in mulls. The interaction of poly-l-lysine (PLL) and poly-l-arginine (PLAG) with SDS and the interaction of poly-l-glutamic acid (PLGA) and poly-l-aspartic acid (PLAA) with achiral TTAB surfactant has been studied using VCD [90]. It was observed that SDS changed the secondary structure of PLAG to α-helix and of PLL to β-sheet. TTAB disrupted the structures of both PLGA and PLAA. These results corroborated those obtained from ECD studies. Novotna et al. also used VCD to study the interaction between model membranes and poly-l-lysine and poly-l-arginine. For this study, micelles composed of SDS were used as a model for monolayer membrane and large unilamellar vesicles composed of phospholipids as a model for bilayer membrane. The presence of the liposomes in the solution generated special conditions for the formation of the α-helical structure of poly-l-arginine; the presence of SDS induced the formation of the β-structure of polyl-lysine [91].

4.4.5  Limitations on the use of VCD for studying soft aggregates In another work related to VCD of chiral surfactants, we identified a major drawback associated with VCD to study aggregating systems like surfactants and proteins [92]. We demonstrated that new structural transformations were induced in surfactant aggregates (and in peptides also) when they were confined in narrow spaces of the order of 50 µm between BaF2 plates (a standard cell used for VCD measurements). Two chiral surfactants with opposite charges viz a cationic surfactant LEP and an anionic surfactant diacetyl-l-tartaric acid mono lauryl ester sodium salt (T12O_COONa, Fig. 4.20) were investigated at 100 mM in D2O, in a sample cell equipped with BaF2 windows at 50 µm spacing. At 100 mM, both surfactants in D2O exist as chiral aggregates since the CMC of LEP and T12O_COONa are ∼0.1 and 1 mM, respectively.

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Fig. 4.20  Structure of diacetyl-l-tartaric acid mono lauryl ester sodium salt (T12O_COONa).

The confined aggregates showed time-dependent changes in the intensities of several VCD bands. Concomitant changes in the VA intensities of the corresponding bands were also observed. The evidence for structural transformations deduced from VCD experiments were corroborated using optical microscopy and TEM. The optical micrograph of the BaF2 cell with LEP solution aged in the same BaF2 cell showed deposition of material on the surface of the window, which is conceivably a surfactant film.

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The deposition of the material on the surface of the window appeared to be time-dependent, with no deposition of materials initially at least for a few hours. For TEM studies, the chiral surfactant solutions were deposited on carbon-coated TEM grids. The TEM image of the 4-day old control solution aged outside a BaF2 cell showed spherical micellar aggregates as small as ∼100 nm, whereas the TEM images of the LEP solution aged inside the 50 µm BaF2 cell showed different types of morphologies, viz., sheet like structures, larger spherical aggregates, and even micrometer size rods [92]. Similar observations were noted for some aggregating peptide solutions as well. The structural transformation of aggregating surfactants (and also peptides) due to their confinement between BaF2 windows in a narrow space can be explained as follows. In a BaF2 window every barium cation is surrounded by two fluoride anions. Therefore, the charge on the surfactant (or peptide) would be attracted toward oppositely charged ions on the BaF2 window surface. We speculate that the initial attraction to the surface of the window, due to either surface adsorption or electrostatic interactions, could direct the structural transformation in the rest of the surfactant (or peptide solution) inside the cell. The solvent is expected to resist these structural changes directed from the surface of the window. However, in view of limited space between the windows, the amount of solution present between the BaF2 windows, per unit area of the BaF2 surface is substantially less. The presence of limited amount of solvent between windows inhibits the ability of the solvent to reverse the effect induced by the BaF2 surfaces, back to solvent-mediated structure, leading to the domination of surfaceand space-induced changes [92]. For this reason, larger changes in the VCD spectra were seen in the smaller space (∼50 µm and less) BaF2 cell compared to those in the larger space (∼100  µm and greater) BaF2 cell, and the extent of the changes are inversely proportional to the path length of the BaF2 cell. In other words, the structural changes induced by surface and spatial confinement become more prominent for smaller path length cells. Overall, these results demonstrated that the time-dependent spectral changes observed with VA and VCD spectroscopies using narrow path length cells must now undertake control experiments to clarify if the observed changes are due to native molecular properties or spatial confinement [92]. A continuous flow sample arrangement could provide one viable option to avoid such space and surface induced transformations. The above-mentioned studies indicated that, using spatial confinement, small micelles

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can be converted into larger aggregates and peptide monomers can be converted into β-sheets, even in monomer-promoting 1,1,1,3,3,3-hexafluoro-2-propanol solvent [92]. The restrictions imposed by the requirement for high concentrations needed for VCD measurements limits the applications of VCD. Therefore, concentration-dependent VCD studies, covering the concentrations below and above CMC, are not possible for most chiral surfactants [24,34].

4.5  ROA STUDIES 4.5.1  ROA studies on surfactants ROA has not been used in the literature to study surfactants by themselves. ROA studies on tartaric acid-based chiral surfactants are currently ongoing in our laboratory.

4.5.2  ROA studies on protein–surfactant interactions ROA of BSA in the presence of anionic SDS, cationic DTAB, and neutral hexaethylene glycol monododecyl ether (C12E6) surfactants was investigated [93]. The concentrations of the surfactants for these studies varied from that at below CMC, slightly above CMC, and well above CMC. The Raman spectra of these surfactants by themselves demonstrated minimal or no interference with the main protein marker band at 1558 cm–1, also known as W3 (originating from the stretching vibration of the indole ring in the tryptophan side chains). Upon addition of SDS or DTAB to protein, the intensity of W3 band initially decreased and then disappeared at high concentrations of SDS or DTAB. Two bands belonging to the amide III modes were also influenced by the addition of charged surfactants: one related to α-helix at 1340 cm–1 decreased, while the second one related to random coil at 1323 cm–1 increased. The Raman spectra of the neutral surfactants showed minor changes than those of the ionic surfactants, the most prominent change being weakening of the above-mentioned W3 Raman band at 1558 cm–1. The following information could be inferred based on the ROA spectra of BSA–SDS system: (i) The amide I bisignate features, positive at 1666 and negative at 1639 cm–1, considered to be spectroscopic marker of the α-helix conformation did not change significantly indicating that SDS does not significantly alter the α-helix conformation of BSA. (ii) The weak negative band of BSA at 1450 cm–1due to bending vibrations of the CH2 and CH3 aliphatic groups upon addition of SDS become sharper and stronger

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with the concomitant appearance of two new weak negative ROA bands at 1246 and 1227 cm–1 indicating the formation of β-type structures. (iii) Upon the addition of SDS, the intensity of the amide III band at 1308 cm–1, considered to originate from nonhydrated α-helix, showed enhancement (relative to that at 1346 cm–1, which is considered to originate from hydrated α-helix) and its frequency shifted to 1296 cm–1. Overall, these data indicated that the nonhydrated (hydrophobic) sites in the protein were transformed by exposure to the SDS surfactant, while the hydrated (hydrophilic) domain did not change much with the addition of SDS. The ROA spectra of DTAB-BSA showed some differences from those in the BSA–SDS system: the negative band in the BSA–SDS system at 1450 cm–1, considered to originate from the bending vibrations of CH2/ CH3 groups, changes sign in the BSA-DTAB system and shifts toward higher energy by 10 cm–1; the invariant band in the BSA–SDS system at 1006 cm–1, which was considered to originate from the breathing modes of phenyl groups becomes negative in the BSA–DTAB system. These changes associated with hydrophobic groups are considered to suggest interaction of DTAB with hydrophobic portion of the protein. There were differences in the ROA spectra of the BSA–C12E6 solutions from those of ionic surfactants. The ROA bands of BSA at 1660 and 1300– 1350 cm–1 showed minimal changes upon the addition of neural surfactant. The interaction of neural surfactant with BSA leads to two new negative ROA bands at 1245 and 1212 cm–1. Additionally, the band corresponding to the phenyl breathing vibrations of the side chain at 1006 cm–1showed sign inversion similar to that observed after the BSA-DTAB system. In contrast to BSA-DTAB spectra, the sign of band at 1450 cm–1 (due to CH2/CH3 bending) remains the same as that in BSA–SDS spectra. All these observations were interpreted to suggest that hydrophobic interaction is common to all their surfactants, independent of the charge. ROA was also used to detect heat-induced molecular instability in an immunoglobulin G4 subclass therapeutic monoclonal antibody present in its formulation matrix containing excipients like surfactants [94]. In this study, the antibody was heated to 50 °C and ROA was measured as a function of time for 1 month. The results demonstrated that ROA was sensitive to detect heat-induced instability of the antibody and changes in the secondary and tertiary structure level. The interference from excipients like surfactants in drug product formulation were suppressed by data processing involving spectral subtraction and principal component analysis.

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4.6  SUMMARY The most widely used chiroptical spectroscopic technique for studying the interactions in, or with, soft aggregates has been ECD followed by ORD. VCD and ROA, on the other hand, have not yet found any broad applications in this area as these two are relatively new techniques. From our experience in using all four chiroptical spectroscopic techniques for studying surfactants, we can confidently state that ORD appears to be the most sensitive method for studying the aggregation phenomena, starting from lower premicellar concentrations to higher postmicellar concentrations. ORD is also the most inexpensive among all four chiroptical spectroscopic instruments. Nevertheless, it is important to apply as many different methods as possible for solving a given research problem. In this regard, all four chiroptical spectroscopic methods are expected to find future applications in the emerging research areas dealing with soft aggregates.

ACKNOWLEDGMENT The authors thank the National Science Foundation for financial support (CHE 1464874).

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Products: Avoiding the Aggregation Effects of Chiral Carboxylic Acids. J. Nat. Prod. 2012, 75 (8), 1441–1450. Abbate, S.; Passarello, M.; Lebon, F.; Longhi, G.; Ruggirello, A.;Turco Liveri,V.;Viani, F.; Castiglione, F.; Mendola, D.; Mele, A. Chiroptical Phenomena in Reverse Micelles:The Case of (1R,2S)-dodecyl (2-hydroxy-1-methyl-2-phenylethyl)dimethylammonium bromide (DMEB). Chirality 2014, 26 (9), 532–538. Shanmugam, G.; Polavarapu, P. L. Vibrational Circular Dichroism of Protein Films. J. Am. Chem. Soc. 2004, 126 (33), 10292–10295. Zhang, P.; Polavarapu, P. L. Vibrational Circular Dichroism of Matrix-assisted Amino Acid Films in the Mid-infrared Region. Appl. Spectrosc. 2006, 60 (4), 378–385. Abbate, S.; Gangemi, R.; Lebon, F.; Longhi, G.; Passarello, M.; Ruggirello, A.; Liveri,V. T. Investigations of Methyl Lactate in the Presence of Reverse Micelles by Vibrational Spectroscopy and Circular Dichroism. Vib. Spectrosc. 2012, 60, 54–62. Abbate, S.; Castiglione, F.; Lebon, F.; Longhi, G.; Longo, A.; Mele, A.; Panzeri, W.; Ruggirello, A.; Liveri, V. T. Spectroscopic and Structural Investigation of the Confinement of d and l Dimethyl Tartrate in Lecithin Reverse Micelles. J. Phys. Chem. B 2009, 113 (10), 3024–3033. Novotna, P.; Urbanova, M. A Solid Phase Vibrational Circular Dichroism Study of Polypeptide–Surfactant Interaction. Chirality 2015, 27 (12), 965–972. Novotna, P.; Urbanova, M.Vibrational Circular Dichroism Study of Polypeptide Model–Membrane Systems. Anal. Biochem. 2012, 427 (2), 211–218. Vijay, R.; Polavarapu, P. L. Molecular Structural Transformations Induced by Spatial Confinement in Barium Fluoride Cells. J. Phys. Chem. A 2013, 117 (51), 14086–14094. Nieto-Ortega, B.; Hierrezuelo, J. M.; Carnero Ruiz, C.; López Navarrete, J. T.; Casado, J.; Ramírez, F. J. Unfolding Pathway of a Globular Protein by Surfactants Monitored with Raman Optical Activity. J. Phys. Chem. Lett. 2014, 5 (1), 8–13. Thiagarajan, G.;Widjaja, E.; Heo, J. H.; Cheung, J. K.;Wabuyele, B.; Mou, X.; Shameem, M. Use of Raman and Raman Optical Activity for the Structural Characterization of a Therapeutic Monoclonal Antibody Formulation Subjected to Heat Stress. J. Raman Spectrosc. 2015, 46 (6), 531–536. Petrovic, A. G.; Bose, P. K.; Polavarapu, P. L. Vibrational Circular Dichroism of Car­ bohydrate Films Formed From Aqueous Solutions. Carbohydr. Res. 2004, 339 (16), 2713–2720.

CHAPTER 5

Vibrational Optical Activity in Chiral Analysis Laurence A. Nafie*,**, Rina K. Dukor**

* Syracuse University, Syracuse, New York, NY, United States ** BioTools, Inc., Jupiter, FL, United States

5.1  INTRODUCTION Chiral analysis encompasses all analytical techniques focused on quantitative measures of the chiral properties of molecules. The properties unique to chiral molecules are absolute configuration (AC) and enantiomeric excess (ee). As will be discussed below, vibrational optical activity (VOA) can provide information on samples of chiral molecules by direct spectroscopic investigation without modifying the molecules by reacting them, sometimes irreversibly, with other chiral molecules or substrates. From a molecular point of view, VOA chiral analysis may be regarded as noninvasive in that the molecule is not changed or altered from its natural solution or solid-state condition in order to carry out the analysis. An important property of VOA is its sensitivity to the conformational states of chiral molecules, both in solution and in the solid state. Apart from the determination of the absolute chirality of a molecule, VOA can be used to specify these states, in conjunction with quantum mechanical calculations, and determine populations of solution-state conformers that are interconverting faster than the nuclear magnetic resonance (NMR) timescale.VOA can also be used to probe the solid-state conformations of chiral molecules as well as their polymorphic crystal forms. Although this information is not strictly chiral in nature,VOA has extraordinary sensitivity to both the absolute structure and the conformation of chiral molecules and this aspect of chiral analysis by VOA should not be overlooked. The most important physical characteristic of any chiral molecule of a known primary structure is its AC. While this can often be achieved with optical rotation (OR) or electronic circular dichroism (ECD) by classical methods [1], the results are sometimes not definitive and may depend on rules or trends for which there are exceptions. When exceptions occur Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00005-7 Copyright © 2018 Elsevier B.V. All rights reserved.

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for the first time, a mistake in prediction usually occurs. Instead, standard analytical methodology has turned almost exclusively to anomalous singlecrystal X-ray diffraction for the a priori unambiguous determination of the AC of a chiral molecule [2]. In some cases, however, it is difficult to obtain single crystals with a heavy atom or single crystals of sufficient quality for X-ray analysis, including molecules that are liquids or oils in their pure states and can therefore never crystallize. Vibrational circular dichroism (VCD), together with Raman optical activity (ROA), offers an alternative route to the a priori unambiguous determination of AC [3–6].The method, as explained in more detail below, involves the comparison of measured and calculated VOA spectra across a range of frequencies for the molecule in question. This method has proved to be highly successful, and the ACs of literally hundreds of molecules have been solved in the last several years [6–14]. Most major pharmaceutical companies today use or are exploring the use of VCD to determine the AC of new chiral drug molecules or their precursors [15–20]. The second important characteristic of chiral molecules is the ee [20–23] of the constituent molecules. When new chiral molecules are brought to market as active pharmaceutical ingredients (APIs), the Food and Drug Administration (FDA) requires proof of the absence, usually at the level of a few tenths of a percent, that there are no significant amounts of the other enantiomer of the API present in the product. The other enantiomer can, in some cases, cause serious undesirable side effects. The standard method today for the determination of ee is chiral chromatography [20]. In this method, a separation of enantiomers is achieved with the use of a chiral stationary phase. Once the separation of the enantiomers is achieved for a racemic sample of the chiral molecule, the sensitivity of the method to the presence of the undesired enantiomer can be established. Sensitivities on the order of one-tenth of a percent are typical for chiral high-performance liquid chromatography (HPLC). Both VCD and ROA offer interesting alternatives to chiral HPLC for the determination of ee of chiral molecules. Since the intensity of the VCD spectrum relative to its parent infrared (IR) absorption spectrum varies linearly with ee, from zero for a racemic mixture to the maximum size for the pure enantiomer sample, once calibrated, VCD or ROA can be used to determine ee without the step of separating enantiomers. Furthermore, because the IR or Raman spectrum always accompanies the measurement of a VOA spectrum and because the VOA spectrum, obtained with a Fourier transform or a multichannel spectrometer, typically embraces

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hundreds of wave number frequencies and dozens of vibrational bands, structure determination is obtained simultaneously with ee determination. By contrast, bands eluting from an HPLC typically must be analyzed later by some other technique to verify the structural identity of the compound of each eluted band. As we shall demonstrate below, this simultaneous sensitivity to both structure and chirality across wide spectral ranges permits the use of VOA to monitor both the mole fraction and %ee of more than one chiral molecule undergoing reaction as a function of time [20–23].This kind of information is currently impossible to obtain in real time by any other technique and permits access to a deeper understanding of the course of the reactions of chiral molecules both for studying reaction dynamics and for optimizing the production of large batches of chiral molecules [24]. VCD and ROA can also be used to study the conformations of large biological molecules, such as peptides, proteins, carbohydrates, glycoproteins, and nucleic acids, and are starting to be used to study the conformational structures of protein pharmaceuticals in various stages of development and formulation [25–29]. The current spectroscopic range of VCD is being extended into the near-IR to be better able to routinely monitor the protein secondary structure and ensure the pharmaceutical viability of these new classes of drugs [30]. ROA can now be used to classify the folding family of newly isolated or engineered proteins using factor analysis routines and this methodology has been extended to the classification of the coat protein structure of viruses [31,32]. More recently, ROA and VCD have been shown to highly sensitive to higher-order structure in proteins, in general, and biopharmaceuticals, in particular [33–35]. A particularly important application ofVCD to biomolecules is its unusual sensitivity to the formation and development of amyloid fibrils [36]. It is well known that amyloid fibrils are implicated in many neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, and a number of prion diseases.Amyloid fibrils possess a dual-layer beta-sheet structure, called the cross-beta core, with axial extension perpendicular to the beta-sheet strands. Fibrils are highly stable and represent a thermodynamic state that is lower than that of natively folded proteins and hence, under appropriate conditions, any protein can fall into a fibril state. The unusual sensitivity of VCD fibrils is manifest in several ways, the most notable is the fact that the VCD of fibrils can be nearly two orders of magnitude larger than the VCD of the native structure of the fibril—from protein or peptide. Since the initial discovery of this enhanced sensitivity, a number of papers have been published [37–42] that explore various aspects of the s­upramolecular chiral

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structure of amyloid fibrils which, because they are very large and do not crystallize, cannot be studied directly by X-ray crystallography or NMR spectroscopy. VCD has recently been extended from solution-state studies to various forms of solids, including KBr pellets [41,43], mulls, films [44–47], and spraydried powders [48]. This greatly extends the utility of VCD to monitor broader classes of pharmaceutical products and opens the way for using VCD to study APIs in the presence of both achiral and chiral excipients in the solid phase and even in final formulated pills and tablets. In all areas of application, except the studies of conformations of biological molecules, ROA currently lags VCD in development and use. But as demonstrated below, instrumentation for measurement [49,50] and software for carrying out ab initio calculations are now available commercially for both VCD and ROA [51]. In principle, there is no reason why ROA will not catch up to VCD as a reliable method for chiral analysis, in the same way that neither IR nor Raman are regarded as more important than the other as achiral probes of the structure or physical state of molecular samples. Each has relative advantages and disadvantages, and strengths and weaknesses.What is clear is that VCD and ROA now provide very powerful supplements to IR and Raman, as well as the entire range of other analytical probes for monitoring and characterizing chiral molecules. It is important to keep in mind the power of vibrational spectroscopy in general, and VOA, in particular, to characterize the structure and stereospecific properties of chiral molecules.

5.2  DEFINITIONS OF VOA VOA consists of two complementary spectroscopic methods, VCD and vibrational ROA. For both VCD and ROA, one measures the differential response of a chiral molecule to left versus right circularly polarized radiation associated with a vibrational transition in a molecule. VCD consists of a single form derived from IR absorption. One could imagine the fluorescence forms of VCD, such as the fluorescence detected VCD or circularly polarized vibrational fluorescence, but fluorescence intensity in the IR is very weak and has not been observed. In addition, IR vibrational optical rotatory dispersion (VORD), also called vibrational circular birefringence (VCB), in the IR has been measured in two different ways [52,53], but this is technically not a new form of optical activity in the sense that CD and ORD are Kramers–Kronig transforms of

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one another and therefore contain redundant sets of information.VCD is more readily interpreted than VCB and hence is the preferred from of IR VOA. On the other hand, there are four independent forms of circularly polarized (CP) ROA and four forms of linearly polarized (LP) ROA derived from their associated ordinary Raman scattering. Further, ROA and Raman can be observed using a variety of scattering geometries and polarization modulation schemes. Other forms of VOA may well evolve in the future, but for the present, VCD and ROA are the two overall methods. The general definition of VOA is the differential interaction of a chiral molecule with left versus right circularly polarized radiation during vibrational excitation. In particular, there is one form of VCD and four forms of CP-ROA [54,55].The additional forms of CP-ROA arise due to the flexibility of modulating between left and right circular polarization states of the incident laser beam or the scattered Raman radiation, or both, either in-phase (right–right minus left–left) or out-of-phase (right– left minus left–right). These are called incident circular polarization (ICP)-ROA, scattered circularly polarization ROA (SCP-ROA), and two forms of dual circular polarization ROA (DCPI ROA and DCPII ROA), respectively. Definitions for these five forms of VOA are given below. Note that the convention for VCD is left minus right, whereas for ROA it is right minus left, a curiosity in the history of the development of these two forms of VOA [56,57]. VCD ICP-ROA SCP-ROA DCPI -ROA DCPII -ROA

∆A ( v ) = AL ( v ) − AR ( v ) ∆I α ( v ) = I αR ( v ) − I αL ( v ) ∆I α ( v ) = I Rα ( v ) − I Lα ( v ) ∆I I ( v ) = I RR ( v ) − I LL ( v ) ∆I II ( v ) = I LR ( v ) − I RL ( v ).

Close analogies exist between the ICP and SCP forms of ROA and two forms of electronic optical activity in fluorescence or luminescence. The background fluorescence associated with ICP-ROA corresponds to the fluorescence detected circular dichroism (FDCD) [58] since in this measurement the total fluorescence is measured while modulating the incident radiation between left and right circular polarization states. Alternatively, the fluorescence background associated with SCP-ROA is usually called circularly polarized emission (CPE) or circularly polarized luminescence (CPL) [59,60].

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While the one form of VCD has been measured, and in addition the redundant measurements of VCB/VORD as noted above, all four forms of ROA have also been measured and published. VCD was first measured in 1974 [61], and confirmed [62] and then extended [63] starting 1 year later using dispersive grating instrumentation. This was followed by the first demonstrations of Fourier transform VCD measurements from our Syracuse laboratory starting in 1979 [64,65]. ICP-ROA was the first form of ROA to be discovered and developed in the early to mid-1970s [66]. SCP-ROA was predicted to exist theoretically as a quantity originally called the degree of circularity (of the scattered radiation) [67], but it was not until 1988 that a practical method for the measurement of this form of ROA, now called SCP-ROA, was first devised and demonstrated in our laboratory [68]. Shortly thereafter, we predicted two new forms of ROA, the two forms of DCP-ROA, to exist as distinct from the ICP and SCP forms of ROA [69]. DCPI-ROA was first measured in backscattering in our laboratory in 1991 [70] and we succeeded to isolate the DCPII form of ROA, which only becomes nonzero as preresonance enhancement of Raman intensities becomes significant a few years later [71]. Linear polarization ROA (LP-ROA) has been predicted theoretically and can only exist close to resonance Raman conditions, and then only for right-angle scattering [72,73]. While ICP-ROA is the old known form of ROA, the first, and to date only, commercial ROA spectrometer, is designed to measure SCP-ROA. In the following section, we explain the fundamentals of the measurement and calculation of VOA spectra before going onto particular applications of chiral analysis.

5.3  MEASUREMENT OF VOA Because VCD and ROA intensities represent very small differences in the parent IR absorption or Raman scattering, respectively, neither can be measured without specialized instrumentation, which is why neither was discovered until the 1970s. VCD intensities are typically four to five orders of magnitude smaller than the parent IR spectra and similarly ROA intensities are three to four orders of magnitude smaller than the parent Raman-scattering spectra.The major obstacle to overcome in the measurement of VOA is to make sure that the spectra are free of offsets or distortions arising from optical components in the instrumentation that interfere with the true VOA spectrum. Various approaches to eliminate or reduce these spectral artifacts significantly have been devised and implemented

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for the commercially available instruments that measure VCD [74–76] and ROA [77–79]. While the measurement of VCD can be carried out on either dispersive grating or Fourier transform infrared (FT-IR) instruments, all commercially available VCD spectrometers are based on the Fourier transform design pioneered at Syracuse University [64,65]. The first and the only company to offer a dedicated, factory-aligned FT-IR VCD spectrometer, the Chiral IR, was BioTools, Inc., currently of Jupiter, Florida, in 1997. The FT-IR spectrometer base is manufactured for BioTools by ABB Bomem (now ABB Process Analytics), Inc. of Quebec, Canada. Since then, several other major manufacturers of FT-IR spectrometers, such as Bruker and Thermo/ Nicolet offer, a VCD accessory bench which requires on-site alignment after installation, although in the case of Thermo, they now offer a dual-PEM side-bench accessory, the Mantis, manufactured by BioTools. Jasco offers a single base single PEM FT-VCD spectrometer.The measurement procedure for VCD involves preparing a sample with an average transmission in the regions of interest roughly between 10% and 90%, with 40% optimum for signal-to-noise ratio, or an absorbance of approximately 0.4.This is achieved by adjusting the path length or concentration until the desired absorbance is obtained resulting in an IR spectrum of high quality. Using standard sampling accessories, a good VCD spectrum can typically be obtained from a few milligrams of sample. More recently, BioTools, Inc. has offered an upgrade for the ChiralIR, called the ChiralIR-2X, that consists of a dual polarization modulation accessory bench that automatically corrects VCD baseline offsets in real time [75].The wave number frequency range of the standard mid-IR VCD spectrometer from 800 to 2000 cm−1 has been extended in optimized stages to 4000 cm−1 covering the hydrogen-stretching region, where VCD was first discovered, to 7000 cm−1 covering the first combination and overtone region [80], and then onto 10,000 and even 14,000 cm−1 that actually connects to the long-wavelength end of the visible region of the spectrum where electronic transitions and ECD are measured [30]. Examples of VCD in all of these regions will be presented by example or application below. ROA spectra can now also be measured using a commercially available instrument. BioTools, Inc. announced the availability of the ChiralRAMAN SCP-ROA spectrometer in 2003, the ChiralRAMAN-2X in 2010, and has placed over 30 such instruments throughout the world. This instrument, based on the recent design of Werner Hug, features active real-time a­ rtifact

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suppression optics, fiber optic image transformation, and ­ simultaneous collection of left and right circularly polarized scattered Raman radiation on a back-thinned charge coupled device (CCD) detector [77–79]. The resulting Raman spectrometer is nearly an order of magnitude faster than all previous ROA spectrometers. As a side benefit, the ordinary Raman performance is so exceptional that Raman spectra of proteins in water at moderate concentrations have been obtained with signal-to-noise ratios in the range of 100 in less than 150 ms, the current minimum exposure time of the CCD camera electronics. Because of the ease of sampling of all classes of biological molecules, ROA holds great promise as a sensitive probe of the conformational state of these molecules. A number of applications of ROA to problems of biological interest, including unfolded proteins and viruses, have appeared in recent years [81–92]. Biological applications featuringVCD are also prominent in the recent literature, typically for smaller molecules, such as peptides and polypeptides [40,42,93–103].

5.4  THEORETICAL BASIS OF VOA An appreciation of the theoretical aspects of VOA is an important prerequisite to its full utilization for chiral analysis. This is important not only for the design and execution of experiments for particular applications, but also for the understanding of how to bring together measured and calculated VOA spectra for the elucidation of molecular properties such as AC and solution-state conformation. The development of theoretical methodology for VCD and ROA has followed different paths in the last 25 years.The formal theory of ICP-ROA was published prior to the first experimental observation [104], whereas a complete formal theory of VCD was not achieved until nearly a decade after the first experimental observation, due to the need to include nonBorn–Oppenheimer (BO) coupling effects into the expression for the magnetic dipole transition moment [105,106]. On the other hand, VCD was first calculated using ab initio quantum mechanical methods [107,108] before this was achieved for ROA [109]. Because VCD passed the key experimental and theoretical hurdles, such as commercial availability of instrumentation and software for calculations, at an earlier stage than for ROA and because it is inherently simpler both to measure and to calculate, there is a more extensive literature of VCD, compared to that of ROA. This ­literature for both VCD and ROA covers measurement techniques, theoretical calculations, and applications to molecules of biological and

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pharmaceutical ­interest.­Today, both VCD and ROA possess the advantage of a fully dedicated, ­commercially available instrument as well as commercially available software for the calculation of VCD and ROA intensities from the first principles [110].

5.4.1  Theory of VCD VCD is an extension of ECD from electronic to vibrational transitions [4,5,13,54,55]. The differential absorbance of left and right circularly polarized IR radiation by a chiral molecule during vibrational excitation is measured as ∆A = AL − AR for absorbance or ∆ε = ε L − ε R for molar absorptivity, where anisotropy ratios, g = ∆A / A = ∆ε / ε , are typically in the range from 10−3 to 10−6, a factor of 10–100 smaller than for electronic CD. The integrated IR absorption and VCD intensities are proportional to the dipole strength (D) and rotational strength (R), respectively, with g = 4R/D. IR intensities depend on the absolute square of the electric dipole transition moment of the molecule given by 2

Dra ( g 0 → g1) = Ψg1a µˆ r Ψg0a (5.1) and VCD intensity arises from the imaginary part of the scalar product of the electric and magnetic dipole transition moments of the molecule given by

(

)

R ra ( g 0 → g1) = Im Ψga0 µˆ r Ψg1a • Ψg1a mˆ Ψga0 (5.2) for a fundamental vibrational transition between the ground and the first excited vibrational states, Ψga0 and Ψg1a , of normal mode “a” in the ground electronic state “g”. The position-form electric dipole moment operator ( µˆ r ) and the magnetic dipole moment operator (mˆ ) consist of electronic and nuclear contributions for electrons j with position rj, velocity rj , mass m and charge −e, and nuclei J with position RJ, velocity R j , mass MJ, and charge ZJe, 

µˆ r = µˆ rE + µˆ rN = − ∑ er j + ∑ Z J e R J j

(5.3)

J

Z Je e mˆ = mˆ E + mˆ N = − ∑ r j × rj + ∑ R J × R J c c 2 2 j J (5.4) iehZ j ieh ∂ ∂ rj × −∑ RJ × =∑ ∂r j RJ j 2mc J 2M J c

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The theory of IR absorption for a vibrational transition within a given electronic state, usually the ground electronic state of the molecule, is straightforward. One invokes a separation of the electronic and vibrational parts of the wave functions Ψga0 and Ψg1a by implementing the BO approximation. At this level, one obtains the correlation between the positions of the nuclei and the electron probability density of the molecule. While this is sufficient for the position formulation of the dipole strength with the electric dipole moment operator given in Eq. (5.3), the magnetic dipole transition moment in Eq. (5.4), necessary for VCD, has nuclear and electronic velocity operators and the electronic contribution to the vibrational magnetic dipole transition moment vanishes within the BO approximation. To solve this unrealistic description, the lowest order correction to the BO approximation is necessary [105]. Further, it has been shown that this lowest order non-BO contribution to the magnetic dipole transition moment, and also the velocity formulation of the electric dipole transition moment, carries the exact correlation needed between nuclear velocities and vibrationally generated current density in molecules [106]. With these non-BO contributions in place, a complete vibronic coupling theory was available for implementation using quantum chemistry programs. The implementation of these basic theoretical expressions is a subject unto itself, and descriptions at various levels can be found in articles and reviews on the theoretical formulation and calculation of VCD. More recently, the vibronic theory of VCD has been developed in a nuclear velocity gauge formalism [111] and was extended to the case of VCD intensities in molecules with low-lying electronic states, but this theory has not yet been implemented for theoretical calculations [112].

5.4.2  Theory of ROA The theory of ROA is intrinsically more complex than that of VCD [4,5,113,114].The complexity arises from the fact that two photons instead of one are required to complete a Raman scattering event. The theory changes with variations in the relative geometry of the incident and scattered beams and the polarization modulation schemes of the two photons. As discussed in the introduction, there are four basic forms of circular polarization––ROA, ICP, SCP, and DCPI and DCPII ROA. In addition, for each of these forms there are three basic geometrical setups, right-angle scattering, backscattering, and forward scattering. Finally, as with ordinary Raman scattering, there are three basic cases: the general theory, appropriate for all circumstances, the far-from-resonance (FFR) limit, for transparent

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samples when using visible excitation, and the single-electronic-state (SES) resonance case when there is direct resonance with a single electronic state [115]. In addition to these forms of ROA, there are four forms of linear polarization ROA; these are more specialized forms of ROA that require near or direct resonance conditions to be observed [72]. In this section, we summarize and discuss the expression for the most common forms and limiting cases of ROA. For the sake of conciseness, we consider explicitly only expressions for unpolarized backscattering ICPu ROA and backscattering DCPI ROA. These account for nearly all of the ROA spectra measured in recent years. Other forms have been measured on occasion, but mainly for theoretical interest. The major expressions for all cases of circular and linear polarization ROA and associated Raman intensity have been published previously [4,114]. Here, we describe qualitatively only the general form of the theory and the interested reader may seek the mathematical expressions elsewhere if desired. In the general case, which includes the possibility of direct resonance interactions, the Raman tensor is not symmetric. This results in three Raman tensor invariants, the isotropic Raman invariant Ψga0 , the symmetric anisotropic Raman invariant Ψg1a , and the antisymmetric Raman invariant (α 2 ) , where the tilde above a symbol indicates a complex quantity. The last of these invariants is only active under resonance conditions and is responsible for the phenomena of inverse polarized Raman scattering. The three Raman invariants are appropriate linear combinations of products of Raman tensor elements. For ROA in the general case there are 10 ROA invariants, 5 in Roman font, conceptually associated with ­ ICP-ROA, and 5 in script font, conceptually associated with SCP-ROA, where the difference between these two is the order of the operators in contributing tensors. The five types of invariants are the isotropic ROA invariants, αG and αG, the symmetric, and antisymmetric magnetic dipole ROA invariants,

(

( )2 ) ,

βs G

(

βa

( G )2 ) ,

β s (G ) n

2

, and

(

βa

(G)2 ) , and the symmetric

and antisymmetric electric quadrupole ROA invariants, β

a

( A)2 , and

βs

β

a

( A)2 ,

βs

( A )2 ,

(A)2 . Analogously, the ROA invariants are appropriate linear

combinations of products of the ordinary Raman tensor elements with ROA tensor elements, where in the latter, one of the two electric dipole moment operators in the Raman tensor is substituted with a magnetic dipole moment operator or an electric quadrupole moment operator. This is analogous to the rotational strength invariant of VCD, which differs from

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the dipole strength by substitution of a magnetic dipole moment operator for one of the two electric dipole moment operators in the absolute square of the electric dipole transition moment. Ordinary Raman intensities are appropriate linear combinations of Raman tensor invariants and similarly ROA intensities are linear combinations of ROA invariants. The original theory of ROA was couched in theoretical expressions of the FFR approximation. In this limit, the Raman tensor becomes symmetric and the number of Raman invariants reduces from three to 2 2 two, β a (G ) and β s ( Ã ) , where the antisymmetric anisotropic Raman invariant vanishes and the remaining anisotropic invariant is symmetric and needs no subscript. More dramatically, in the FFR limit the number of ROA ­invariants reduces from 10 to 3. Half of these vanish when the difference between the script and bold ROA tensors vanishes owing to the FFR equivalence of the i­ncident and scattered radiation interactions. A further reduction ­ occurs due to the vanishing of the remaining antisymmetric invariants, leaving only β ( A)2, β a (Ã) 2, and β a (Ã) 2 . The prime on the magnetic dipole ­optical activity tensor symbol indicates an imaginary part, which produces a real observable quantity as in the expression for the rotational strength in Eq. (5.2). Theoretical expressions for these two Raman and three ROA invariants in the FFR approximation are given by  

1 α 2 = α αα α ββ 9 1 β (α )2 − (3α αβ α αβ − α αα α ββ ) 2

(5.5) (5.6)

1 αG ′ = α ααGββ ′ (5.7) 9 

1 β (G ′ )2 = (3α αβGαβ ′ − α ααG ′ − α ααGββ ′ ) 2

(5.8)

1 β ( A)2 = ω 0α α β ε αγδ Aγ ,δβ , (5.9) 2 where the polarizability and optical activity tensors are given by the following quantum mechanical expressions that, unlike IR and VCD, involve no contributions from the nuclear motion,

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α αβ =



ω jn 2 Re  n µˆ α j ∑ 2   j ≠n ω jn − ω 02



Gαβ ′ =



Aα ,βγ =

j µˆ β n  

−2 ω0 Im  n µˆ α j ∑ 2   j ≠n ω jn − ω 02 2 ω0 Re  n µˆ α j ∑ 2   j ≠n ω jn − ω 02

(5.10)

j mˆ β n  

(5.11)

ˆ n  jΘ βγ 

(5.12)

Here, repeated Greek subscripts are summed over all three Cartesian ˆ coordinates and the symbol Θ represents the electric quadrupole βγ operator. With these Raman and ROA invariants, we can write the following expressions for the Raman and ROA intensities associated with unpolarized ICP (ICPu) scattering and DCPI scattering [70]: 8K [12β (G ′ )2 + 4 β ( A)2 ] c



I uR (180°) − I uL (180°) =



I uR (180°) + I uL (180°) = 4 K [45α 2 + 7β (α )2 ]

(5.13) (5.14)

and  

I RR (180°) − I LL (180°) =

8K [12β (G ′ )2 + 4 β ( A)2 ] c

(5.15)

(5.16) I RR (180°) + I LL (180°) = 4 K [6β (α )2 ] where K is a constant associated with the scattering of radiation and c is the speed of light in vacuum. From these expressions, it can be seen that the ROA intensities for these two backscattering setups are the same and involve only the two anisotropic ROA invariants.These intensities are significantly larger than the depolarized ROA intensities associated with right-angle scattering, and hence the increased use of backscattering ROA in recent years. It is also clear that even though the ROA intensities of ICPu and DCPI are identical, their Raman intensities differ. The reason for this is that DCPI scattering includes an additional polarization discrimination of the scattering radiation before detection whereas in ICPu scattering, all the scattered light coming from the sample is measured. The additional Raman scattered radiation in ICPu is actually polarized DCPII-Raman backscattering which, in the FFR approximation carries zero ROA intensity. Hence no additional ROA intensity is present, but strongly polarized DCPII-Raman intensity is

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measured in ICPu ROA that is not measured for the same ROA spectrum using the DCPI setup. The additional Raman intensity for ICPu necessarily increases the noise in the measured ROA spectrum of a given magnitude. It is interesting to note that DCPI-Raman intensity corresponds to the classical depolarized Raman intensity, whereas ICPu-Raman intensity is the same as the corresponding polarized Raman intensity, and the ratio between these two intensities is the classical depolarization ratio for unpolarized incident radiation, yielding a value of 6/7 for depolarized bands. Aside from the theoretical noise advantage for depolarized bands, there is a significant advantage for DCPI ROA in the case of strongly polarized bands which can have substantially higher noise, and the potential for interfering artifacts, in the ICPu ROA measurement.

5.5  CALCULATION OF VOA For more than a decade following the discovery of ROA and VCD, the interpretation of VOA spectra was carried out on an empirical basis by correlation of spectra to structure or by means of model calculations which in turn were based on empirical concepts and parameters. With the appearance of ab initio quantum mechanical routines for the accurate calculation of VOA spectra, a new era emerged in the interpretation, analysis, and practical application of these spectra. The ab initio calculation of VCD was pioneered by Stephens, Rauk, and Nafie and Freedman [105,107,116,117]. A significant advance in the formulation of VCD occurred with the introduction of gauge-invariant atom orbitals (GIAOs) [111,118] also known as London atomic orbitals (LAOs) [119]. Commercial software for carrying out ab initio quantum mechanical calculations of VCD became available about the same time as VCD instrumentation with the release of Gaussian 98 by Gaussian Inc. currently located in New Haven, CT. Subsequent releases of Gaussian 03, 09 and 16 have added many more features for VCD calculations including full simulation of the spectrum using GaussView for the ease of comparison of the calculated VCD and IR spectra to the measured ones. Extensive testing has shown that the most efficient level of calculation for the simulation of VCD spectra of typical organic molecules is density functional theory (DFT) using GIAOs with hybrid functionals such as B3LYP and a basis set of 6–31G(d) or higher [7,8,10]. The ab initio calculation of ROA has been pioneered primarily by ­Polavarapu [109], Helgekar and coworkers [120], and more recently by

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Hug [121–123]. Gaussian 03 and subsequent programs (09 and 16) include subroutines for running ROA in the far-from-resonance approximation. With this program, it is possible to calculate ROA spectra for typical experimental setups as well as all Raman and ROA tensor quantities from which any form of ROA and any experimental measurement can be calculated. At this time, the ROA tensor routines are not yet programmed with analytical field derivatives and hence must be determined by finite difference calculations. In addition, it has been found that higher level basis sets, including diffuse functions, are important for the accurate calculation of ROA intensities [124–130]. As a result, calculated ROA intensities require a greater time investment compared to the corresponding VCD calculations for the same molecule, and consequently the upper size limit of a molecule for which ROA can be calculated is smaller than that of VCD. This is an intrinsic advantage of VCD calculations over those for the corresponding ROA case, and hence it is likely that VCD will always hold an advantage over ROA in terms of speed or accuracy for the VOA calculation of a particular molecule with a similar degree of accuracy, despite increases in the computational speed and efficiency in the future. With the advent of powerful software packages for the application of quantum mechanical calculations of molecular properties and the rapidly increasing performance of modern computer hardware, it is now possible to simulate the spectra of molecules from the first principles and compare these theoretical spectra with the traditional experimental ones. Although this can be carried out for electronic CD (and OR), as well as for VCD and ROA spectra, there are a number of advantages inherent in vibrational spectra that make this comparison of theory to experiment more informative and accurate than the corresponding comparisons for electronic spectra. First, vibrational spectra typically contain many more transitions through which the molecular stereochemistry can be probed. Each molecule of N atoms contains 3N − 6 degrees of vibrational freedom and most of these vibrational transitions can be observed with IR or Raman instrumentation. Thus,VCD and ROA spectra contain many vibrational bands representing all parts of the molecule. Second, VCD and nonresonant ROA sample the ground electronic state properties of molecules. The molecular transition involves only the modulation of the equilibrium ground state and hence it is easier to calculate from a quantum mechanical standpoint.To the extent, the excited electronic states enter the theoretical description of VCD and ROA intensities, they do so as sums over all allowed electronic states and the detailed description

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of individual excited electronic states is not a critical part of the calculation. On the other hand, accurate electronic CD calculations require an accurate description of both the ground electronic state of the molecule, the starting point, but also of each excited electronic state for which a CD band is to be calculated. Further, an accurate simulation of an ECD spectrum requires some description of the band shapes and the underlying vibronic substructure of the electronic state, which is currently beyond the available theoretical technology. Third, VCD and ROA spectra are not restricted to the presence in the molecule of a chromophore that will report on the molecular stereochemistry from a particular and sometimes remote vantage point. Virtually all chiral molecules exhibit VCD and ROA spectra that have bands in an expected intensity range. Molecules do not need to be modified in order to exhibit VCD or ROA spectra that are useful for analysis or comparison to theoretical calculations. Despite the many advantages of VCD and ROA, they, like all the methods in molecular spectroscopy, play a complementary role relative to other techniques such as electronic CD, NMR, X-ray crystallography, and fluorescence, in gaining a deeper understanding of the structure and conformation of molecules. The principal contribution of VCD and ROA is that they are structure-rich probes of the stereochemistry of molecules in solution and disordered states. They can resolve multiple conformational states that are interchanging on time scales in the sub-picosecond region and they show great promise for the accurate comparison of ab initio simulations to experimental measurements. It is clear now that, with further improvements and availability of instrumentation and software, VCD and ROA will emerge in the coming years as incisive, important tools for the precise determination of molecular stereochemistry in the solution and solid states.

5.6  DETERMINATION OF AC The determination of the AC of chiral molecules has a long history in the field of molecular stereochemistry. X-ray crystallography is the method used to obtain the most definitive information about the AC of a chiral molecule. Additionally, AC can often be deduced from the knowledge of reaction mechanisms in organic chemistry applied to well-characterized transformations from starting materials of known AC to the final products.

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OR and ECD can also be used to predict AC, usually based on rules for the sign of the rotation angle or ECD bands. NMR is blind to chirality, but ancillary methods for deducing AC using chiral shift reagents or chemical derivatives have been devised. However, whenever methods such as organic reaction mechanisms, OR, ECD, or NMR are relied upon fully over time, exceptions inevitably arise that result in erroneous predictions. Conversely, the Bijovet method of X-ray crystallography, with many technical improvements in X-ray technology in recent years, has become the recognized standard for the a priori determination of the AC of chiral molecules [2]. There is, however, one major drawback with X-ray crystallography. A pure single crystal of the enantiomer of the sample is required. In the case of liquids or oils, X-ray crystallography is precluded and other less definitive methods must be used if they possess the capability. In some other cases, crystals cannot be grown within reasonable periods of time, thereby either precluding this method or waiting for months to years for crystals. Another recommended requirement of the Bijovet method is the inclusion in the crystal of a heavy atom. When this is not the case, the determination of the phase required for the X-ray analysis is more difficult and sometimes mistakes are made, as was recently discovered by comparing the X-ray prediction to the results of a VCD determination of the AC [131]. Within the past decade, VCD has emerged as a powerful new method for the determination of the AC of chiral molecules. All molecules absorb radiation in the IR region, where their absorption pattern across the spectrum serves as a rich fingerprint of molecular structure and shape. In addition, all chiral molecules have a VCD spectrum that consists of an even more powerful fingerprint spectrum of the structure and shape of the molecule. The additional power afforded to VCD is due to its stereospecific sensitivity. Molecules with opposite AC, pairs of enantiomers, have the same IR spectrum, but opposite VCD spectra.This point is illustrated in Fig. 5.1, where the IR and VCD spectra of the (+)- and (−)-enantiomers of camphor in the mid-IR region of the spectrum are presented. Although the magnitude of the VCD spectrum is approximately 10,000 times smaller than that of the parent IR spectrum, with modern FT-VCD instrumentation, the measurement of a VCD spectrum is routine. Each absorbance band in the IR spectrum has a corresponding VCD band.There is no correlation between strong IR and strong VCD bands, but each band in the VCD spectrum reports on the structure of the molecule as well as its AC. Because VCD measurements can be carried out in the solution phase, and because the results are direct with rapid turnaround, the VCD method

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Figure 5.1  The VCD and IR spectra of (+)-R-camphor (upper structure) and (−)-S-camphor (lower structure). Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

of AC determination stands as an alternative to X-ray crystallography for the routine determination of AC in chiral molecules [15]. The AC of a chiral molecule is determined from VCD by comparing the results of an experimental measurement of the IR and VCD spectra of the chiral molecule with the quantum mechanical calculation of the same IR and VCD spectra for a particular choice of the AC of that molecule [3–6,132,133]. The sample is measured as a neat liquid or as a solution in a suitable solvent. Crystallization is not required, and the VCD measurement takes anywhere from 5 min to several hours depending on the quality of the spectrum desired and intensity of the VCD spectrum relative to the noise level. The calculation for a particular conformation typically takes on the order of a day of computer time depending on the size and complexity of the molecule and the speed and memory capacity of the computer. The ACs of molecules with up to 100 heavy atoms, where a heavy atom is any element

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beyond hydrogen, are now been determined by VCD. If the measured and calculated VCD spectra agree sign-for-sign for the major bands across the entire spectrum, and the relative magnitudes of those bands in both the VCD and IR spectra agree, then the AC is deduced without ambiguity or reference to any prior structure or calculation. If the bands across the VCD spectrum are opposite in sign, then the wrong enantiomer was chosen for the calculation. Calculation of the mirror-image structure then produces the desired agreement and again the AC is determined unambiguously. In the last four years, nearly every major pharmaceutical company in the world has either purchased a VCD spectrometer for AC determination or outsourced the determination of the AC of selected molecules by VCD through collaboration, feasibility study, or contract [11]. Some companies from which researchers have reported the results of VCD measurements at scientific meetings include Organon, GlaxoSmithKline, AstraZeneca, Johnson & Johnson Pharmaceutical Research & Development, Bristol Myers Squibb, Pfizer, Abbott, and Wyeth Laboratories.Through this activity, the ACs of hundreds of chiral molecules have been determined over the past two decades. Currently, confidentiality restricts much of this work from public disclosure, but a number of articles have been published after the results were approved for dissemination [15–20]. The simplest application of VCD to the determination of AC is for structures that are rigid to the degree that only one conformation is dominant in solution. In Fig. 5.2, we provide such an example where the AC of a newly synthesized chiral iminolactone (1,4-oxazin-2-one) was determined by VCD spectroscopy [134]. The stereo-specific line structures as well as its space filling optimized geometry from the DFT calculation are shown. The comparison of the measured (+)-enantiomer with the calculated R-enantiomer is also shown. The close agreement of all the measured features, together with the agreement of the intensity pattern of the IR spectrum, make available the unambiguous determination of the AC of this molecule. A more challenging example is the assignment of AC of the aryl axial chirality of the two atropisomers named dicurcuphenol B and C, isolated from the marine sponge Didiscus aceratus, the structures of which are shown in Fig. 5.3 [135]. The VCD and IR of these two isolates are also shown in this figure. The dicurcuphenols B and C are diasteriomers in that they possess a common chiral tail that breaks the mirror symmetry of the VCD of these two molecules. In addition, this tail contains a high degree of conformational

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Figure 5.2  Line structure and optimized geometry of a new chiral iminolactone with comparison of observed (+)-enantiomer VCD and IR to the calculated R-enantiomer VCD and IR as a proof of AC. Abbreviations: VCD, vibrational circular dichroism; IR, infrared; AC, absolute configuration.

freedom which is undesirable from a computational point of view. The strategy adopted was to subtract the VCD of the two dicurcuphenols, thereby i­solating the contributions of opposite VCD originating from the axial chirality. In Fig. 5.4, the comparison of the subtracted VCD to that calculated for the rigid chiral model compound (M)-10, which is the structure of dicurcuphenol minus its chiral tail, is shown. The excellent agreement allows the axial chirality of dicurcuphenol B to be assigned as M, therefore dicurcuphenol C has P axial chirality. In Fig. 5.5, results are presented for the determination of the AC of the molecule McN 5652-X, a high-affinity ligand for transport of seratonin in the brain, for which more than one conformer contributes to the observed VCD spectrum [19]. The theoretical analysis of this compound yielded two low-energy conformers, SR and SRb, shown in Fig. 5.5. Separate IR and VCD spectra were calculated for each conformer. Comparison of the calculated VCD for the two conformers with the experimental VCD spectra identifies bands in the experimental spectrum associated with each of the conformers. Many

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Figure 5.3  Structures, IR, and VCD of the two atropisomers dicurcuphenol B and C isolated from the marine sponge Didiscus aceratus. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

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Figure 5.4  Structures of the model phenol atropisomers (P)-10 and (M)-10 used for theoretical VCD calculations and comparison of the calculated IR and VCD spectra of (P)-10 with the difference in VCD spectrum of the atropisomers, curcuphenol B minus curcuphenol C shown divided by two, thereby establishing the absolute aryl axial chirality of the latter isolated natural products. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

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Figure 5.5  Comparison of the measured and calculated VCD and IR spectra of (+)-McN 5652-X. The calculated spectra are for two conformers, SRa and SRb. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

VCD bands for the two conformers are the same and are also seen in the experimental spectrum. From the agreement in sign of all the major VCD features and the relative intensities in the experimental and calculated spectra for both IR and VCD, the AC of (+)-McN 5652-X can be assigned to be 6S,10R, as shown in the structure in Fig. 5.5. This example illustrates that fromVCD analysis it is possible to determine the solution-state conformation of a molecule as well as its AC. In fact, agreement between the experimental and calculated IR and VCD spectra cannot be achieved unless the correct conformational states of the molecule are found and used as the basis for the calculations. Hence,VCD analysis provides not only the AC, but in addition, the solution-state conformation or conformational population. Within the past several years, a number of reviews have appeared for the determination of AC using VCD [12,14,94,133,136–139]. The extent and variety of these examples are testaments to the growing importance of the use of VCD for the determination of AC in chiral molecules.

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The methodology for the determination of AC using ROA is identical to that for VCD. Presently, the measurement of the ROA of chiral molecules in nonaqueous solution is problematic due to interference from strong solvent Raman bands that may interfere with ROA spectrum of the solute, particularly for dilute solutions. Much better for ROA analysis are compounds whose natural solvent is water, such as amino acids, sugars, and pharmaceutical molecules soluble in H2O at any pH. Water has a relatively weak Raman scattering spectrum and hence poses no serious problem to the measurement of the ROA spectrum. The procedure to determine the AC is essentially the same as for VCD, namely to start with an ab initio computational analysis of the Raman and ROA spectra of the preferred conformer or conformers of the molecule. Comparison of the experimental Raman and ROA spectra to the corresponding calculated spectra yields the AC. A few examples of the comparison of measured and ab initio calculated ROA spectra have appeared in the literature [121,122,124,125,140,141]. Results to date have demonstrated that a more extensive basis set using diffuse basis sets is necessary to capture an accurate Raman and ROA spectral response to the vibrational modes of the molecule. This, together with the limitations in the implementation of ROA subroutines, limits the size of the molecules that can currently be considered, and hence ROA is currently more limited in its range of application to AC than is VCD.This discrepancy is likely to continue in the future since it is intrinsic to the difference in complexity and computation demands of ROA compared to those of VCD. Although VCD and ROA are the most powerful and effective methods for the determination of AC in chiral molecule using optical activity, at least two other methods are currently under development and exploration. The first of these is the determination of AC by measured and ab initio calculated OR [142–150]. Although the experimental data are more widely available than experimental VCD spectra, the calculations require a higher level of quality for good reliability and only one datum is produced rather than an entire spectrum, so there is no way to be sure that good agreement between the experiment and calculation has been achieved.VOA, on the other hand, requires not only sign agreement, but also spectral confirmation through the corresponding relative intensities of the IR/Raman and VCD/ROA bands for which the correct conformation, or distribution of conformers, has been determined and which forms the basis of the comparison between the measured and calculated results.

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The other approach is the corresponding analysis for ECD [151–153]. For the comparison of experiment to theory, typically only a few ECD bands are available. Again, higher level calculations than those needed for VCD analysis are required for good reliability, but here, predicting ­correctly the relative signs for two or more transitions gives some confidence of ­having a valid analysis. The software for calculating OR and ECD is available in Gaussian 03 [32] and subsequent releases. Here again, calculation of the UV– visible absorption and ECD spectra for one-to-several excited electronic state transitions offers no definitive proof that the correct solution-state conformation, or distribution of conformations, is being used for the calculated ECD spectrum. Nevertheless, there is now a growing awareness of the value of using any two or all three of these ab initio methods,VCD, OR, and ECD, for the determination of AC from the experimentally determined data. In particular, there is value to include VCD or ROA in the determination to ensure that the conformational state or states of the molecule have been correctly identified. If the same prediction of AC, using the correct conformations, emerges from such a multiple analysis, an even higher level of confidence in prediction is achieved. Within the past few years, a new approach to enantiomeric detection of chiral molecules has emerged in the microwave region [154–164]. Here chirped pulse broad band microwave radiation is employed to interrogate rotational transitions in gas phase chiral molecules. It has been demonstrated that by simultaneously exciting two rotational states having orthogonal linear polarization (say x and z) in such a way that emission from the third orthogonal rotational (along y) can be coherently induced for which the phases of the microwave radiation of enantiomers of the molecule are opposite. If the signs of the three rotational electric dipole transition moments are known, and if all experimental instrumental phase shifts are known, the determination of the AC of a chiral molecule can be determined. To date such AC determination has not been demonstrated, but many other advantages have been identified. Specifically, the high resolution of the microwave rotational lines allows different conformers of a molecule to be distinguished by comparison to quantum chemistry calculations of the rotational constants of the conformers. Further, ee determination of a mixture of enantiomers can be directly determined without the need for separation or calibration. As technology advances in the future to make available even more efficient instrumentation for the measurement and ab initio computation of VCD, ROA, OR, ECD, and the new microwave spectra the determination

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of AC of chiral pharmaceuticals will be increasingly carried out by these methods because of their ease of sample preparation and time to final results.

5.7  DETERMINATION OF ee OF MULTIPLE CHIRAL SPECIES Another important property of a sample of optically active molecules is the ee of each chiral species present. Using conventional techniques, the fractional composition and the EEs of each species cannot be determined in the intact sample for reasons explained below. The traditional method for the determination of ee is chiral chromatography [20]. This is routinely accomplished within an accuracy of 0.1%, approximately an order of magnitude better than that demonstrated by VCD or ROA to date. On the other hand,VOA has demonstrated sensitivity in the range of one-to-several %ee. As such, VOA should not be considered as a replacement of chiral chromatography for the definitive measurement of the %ee of any sample. Nevertheless,VOA possesses several very important unique advantages over chiral chromatography as it is currently practiced.The most unique of these is the potential of VOA to be used as a real-time in situ monitor of the ee of multiple chiral species in a reacting medium [165].The second advantage of VOA is the determination of ee of chiral-active ingredients in various states or environments such as liquids, solutions, solids, and a variety of formulated forms in the presence of excipients [47,166,167] as tablets, powders, or liquids. All other methods of ee determination, currently devised or in use, require pure dissolved samples, and in the case of chiral chromatography require separation of enantiomers. The power of VOA to enable these in situ measurements relies on two important facts. The first is that the measurement of the VOA is always accompanied by a simultaneous measure of the parent IR or Raman spectrum. This provides an independent spectral measurement that contains information about the structural identity, composition, and state of the sample being measured. Second, and perhaps more important, the magnitude of the VCD or ROA is measured relative to the corresponding IR or Raman spectrum. Any changes in concentration, path length, density, or temperature that may affect the magnitude of the measured VOA spectrum can be automatically normalized out by corresponding changes in the parent vibrational spectrum. Absolute values of ee can be obtained in this way for any species under any conditions following a single calibration measurement of the VOA relative to the parent IR or Raman for a known value of ee.

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Recently, a flow-cell apparatus was constructed for the continuous measurement of VCD for a sample changing with time whereby the sample cell is never removed from the instrument [20]. This apparatus has been used in various ways to demonstrate the potential for VCD to monitor a reaction mixture that may be changing in time in both the composition and ee of each chiral species present. Initial studies focused just on following changes in ee of a single species, such as α-pinene or camphor in CCl4 solution. Here, a starting solution of ee was first measured and then in successive steps measured amounts of a solution of the opposite enantiomer at the same concentration of the opposite enantiomer were first mixed with the original solution, circulated through the flow cell, and then measured for the new ee. The resulting sets of VCD spectra, varying from 100% ee in 10 steps or so to a few %ee, could be analyzed using chemometric partial least squares (PLS) analysis to an accuracy of 1%–2% ee for measurement times of 10 or 20 min and a spectral resolution of 4 cm–1. Subsequently, we found that similar accuracy could be achieved for measurement times as short as 1 min if the resolution was lowered to 8 or 16 cm–1. A more challenging experiment is to use the FT-VCD-PLS method to follow changes in a two-component mixture, where both the mole fraction and the %ee of each species is changing as a function of time.The two chiral molecules chosen were (1S)-(–)-camphor and {(1S)-endo}-(−)-borneol, where the structures and AC of these molecules are shown in Scheme 1 [20]. It is clear that these molecules differ only in one functional group that is a carbonyl group in camphor and a hydroxyl group in borneol.We used the same mixing apparatus described above for these studies. Before carrying out the desired mixing experiment, a series of carefully designed calibration measurements were carried out to establish a training set with which the mixing experiments were to be analyzed using PLS. Mixtures of camphor and borneol, each at 100% ee were prepared across the full range of expected concentrations for the mixing experiments. This

Scheme 1  Simulated reaction of camphor to borneol.

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Figure 5.6  Comparison of the IR and VCD spectra of (1S)-(−)-camphor and [(1S)-endo](−)-borneol, where both are 1.000 M solutions in CC14. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

included the IR and VCD spectra of pure camphor and borneol, as shown in Fig. 5.6. From this figure, it can be seen that while the IR and VCD of these two molecules are distinct from one another, they do possess a high degree of similarity. This not only adds a challenge to the experiment as a whole, but also a degree of realism since any pair of molecules that are related as reactant and product are likely to have similar IR spectra, and quite possibly similar VCD spectra as well. The resulting set of training spectra were subjected to the cross-correlation regression analysis using PLS.

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Following the calibration measurements, a mixing experiment involving each species at 100% ee was set up in two parts, one starting from camphor and adding borneol and the other with the solutions reversed. By ­combining these two studies, a simulation of a complete reaction starting with pure camphor and ending with pure borneol was achieved. Since the solutions were all at 100% ee, the sets of both the IR and the VCD can be used independently to predict the mole fraction of the mixing experiments, where, because of higher signal quality, the errors in prediction for the IR were approximately an order of magnitude better than those for VCD. ­Taking the ratio of these two determinations for each mixing point allowed prediction of the %ee of each species, which was known by design to be 100% ee. E ­ rrors of prediction, based on the ratio of the IR and VCD determinations were between 2% and 3% ee for each species. With these calibrations and preliminary measurements completed, a more general mixing experiment was undertaken. The original solution was 9 mL 0.600 M at 100.00% ee (–)-borneol and 0.400 M at 100.00% ee (–)-camphor mixture in CCl4 solution. The add-ins were 0.400 M at 0.00% ee (–)-borneol and 0.600 M at 0.00% ee (–)-camphor in CCl4 solution. The volume of add-in was 1 mL for each injection. In total, 10 samples were prepared and measured by this flow-cell sampling method and the IR and VCD spectra generated by these experiments are displayed in Fig. 5.7. The final solution ended at mole fractions of camphor and borneol each at 0.500 and the %ee for camphor 40% and that of borneol 60%. For this system, to obtain the %ee of camphor and borneol at each step, both the IR and VCD spectra are required, since during the experiment, both the mole fractions and the EEs of the two components change. After these spectra were analyzed using PLS chemometric software, it was found that the EEs of both camphor and borneol could be determined at each mixing point with an accuracy of approximately 2%–3%. The general method for analyzing these spectra to determine mole fractions and %ee at each mixing stage is as follows: in principle, multiple ­species can be present, each with changing mole fraction and changing %ee. The IR spectra of each species at known concentration can be used to determine the mole fraction of each species present in the mixture.TheVCD spectra of each species at known %ee, say 100%, can be used to determine the apparent mole fractions of each of the chiral species present. These apparent mole fractions are less than the actual mole fractions, depressed by %ee values less than 100%. By normalizing the apparent mole fractions

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Figure 5.7  Ten correlated sets of VCD and IR spectra for flow-cell measurements of mixtures of (1S)-(−)-camphor and [(1S)-endo]-(−)-borneol in CCl4, where the IR ­spectral changes reflect only the changing the composition between camphor and borneol, whereas the VCD spectra change as the result of both changing composition and change ee of both species. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

by those obtained from the IR for each species, one is left with population coefficients between 0 and 1 that represent the ee for that species and after multiplying by 100, the %ee of each species is determined. The results presented here demonstrate that FT-VCD can be used to monitor simultaneously the %ee of multiple chiral molecules as a function of time. Although the intrinsic signal-to-noise ratio of VCD is not as high as other traditional monitors of the kinetics of chiral molecules, there are a number of advantages of FT-VCD that arise from its information content and from the multiplex nature of its measurement process. FT-VCD spectra typically contain many bands representing different vibrational modes from all portions of the molecule. For VCD, there is no concern about the concept of chromophore since all molecules have “vibrational

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chromophores” representing all structural locations in the molecule. Since different molecules have different vibrational bands and different vibrational frequencies for these bands, it is often possible to identify individual peaks in a mixture that belong to particular molecules. In the case of monitoring the %ee of a single species, the multiplex advantage of FT-VCD means that each point in the spectrum, of which there may be hundreds or thousands across the spectrum, represents an independent measure of the %ee. Combining these data points using PLS, with higher weighting accorded to regions of higher signal-to-noise ratio, the disadvantage of a relatively low signal-tonoise ratio at individual spectral locations is in large measure to overcome. These studies have been extended in two ways. One is a spectral extension of FT-VCD from the mid-IR into the near-IR region above 4000 cm–1. As an example, the mid-IR FT-VCD spectra are compared to various regions of the near-IR FT-VCD for a 1.0 M solution of (1R)-(–)-camphorquinone in CDCl3 solution [30]. The first spectral segment covers the typical region of the mid-IR from 800 to 1600 cm−1 using a liquid-nitrogen cooled HgCdTe at a path length of 100 µm. For the region of CH-stretching modes, a thermoelectrically cooled HgCdTe photovoltaic detector with a cut-off near 2000 cm−1 was employed and a sample path length of 50 µm. Beyond 3800 cm−1 much longer path lengths are needed and room-temperature detectors are now sufficient for optimum signal quality. Here, relatively weak combination and overtone bands from predominantly hydrogen-stretching modes occur. The first region from 3800 to 6200 cm−1, using an InGaAs detector and a path length of 2 mm, contains combination bands that represent one CH stretching and one CH bending mode below 5000 cm−1 and overtones of the CH stretching modes between 5500 and 6000 cm−1. In the next region between 6000 and 10,000 cm−1, a path length of 20 mm and a Ge detector is used. Here, the bands represent the second combination band region (2 CH stretches and one CH bend) and the second CH-stretching overtone region (3 CH stretches). We have noted certain similarities among some of these regions in terms of the relative intensities and sign patterns of the VCD. As can be seen in Fig. 5.8, the near-IR has the intrinsic advantage of longer sampling path lengths and, as a result, greater ease of sampling. The experiments described above for %ee determination of one- and twocomponent samples with a flow cell have been repeated for the near-IR [22]. In this region, it has been found that approximately nearly the same level of accuracy in the prediction of %ee can be achieved. Because detectors and VCD baselines are more sensitive to environmental perturbations in the

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Figure 5.8  VCD and IR absorption spectra of 1.0 M (1R)-(−)-camphorquinone in CDCl3 solution. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

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near-IR, a high level of chemometric analysis and more extensive calibration measurements are needed to achieve similar results, where we find errors in the 2%–3% range, rather than the 1%–2% range found in the mid-IR. Since the ­intrinsic bandwidths are broader in the near-IR, a resolution of 16 cm–1 was used instead of the 4 cm–1 used in the mid-IR region. Again, lowering the resolution further to 32 or 64 cm–1 provides the opportunity to achieve the same accuracy at shorter collection times approaching 1 min per measurement. Following the extension of our FT-VCD instrumentation into the near-IR region, flow-cell mixing measurements have been extended to the r­eaction of real molecules in a reaction vessel. Here, the reacting solution is circulated through a flow cell without the need to add in a secondary solution to achieve a change in mole fraction or %ee or both. One such experiment is the epimerization of 2,2-dimethyl-1,3-dioxolane4-methanol (DDM) carried out in the near-IR using an InGaAs detector with a spectral resolution of 32 cm−1. The structure and epimerization pathway of this molecule, together with a set of near-IR VCD spectra are shown in Fig. 5.9 [168]. The reaction intermediate is a protonated species which has longer lifetimes in solvents that give greater stability to this intermediate. Once the intermediate forms, it has two equally probable pathways back to either the original structure or its enantiomer. In Fig. 5.10, the reaction kinetics of %ee determined by the FT-VCD-PLS method is shown for three different solvents. Of these solvents, methylcyclohexane best stabilizes the intermediate and has the fastest loss of %ee. Toluene and carbon tetrachloride show nearly the same half-life, but have distinctly different kinetic detail that is not understood at present. In addition to this study, the reaction of 2-butanol to 2-chlorobutane by the addition of thionyl chloride has been monitored using the near-IR FT-VCD-PLS method described here [168]. It was possible to confirm that the reaction proceeded from reactant to product under the conditions tested, without loss of chirality at the 3% ee confidence level, while simultaneously monitoring the change in mole fraction from pure butanol to nearly pure chlorobutane.

Figure 5.9  NIR-VCD spectra for the calibration samples in toluene displayed in the range of 5300–4700 cm−1 at a resolution of 32 cm−1. Abbreviations: NIR-VCD, nearinfrared-vibrational circular dichroism.

Figure 5.10  %ee of (S)-DDM as a function of reaction time under the treatment of trifluoroacetic acid in toluene, carbon tetrachloride, and methylcyclohexane.

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5.8  VOA OF SOLIDS AND FORMULATED PRODUCTS Most VCD studies to date have focused on samples that are neat liquids or solutions. Several early investigations of solid-phase mulls and films have been published but without much success [43,169,170]. The main reason for not continuing these studies at the time was that, due to poor VCD artifact control, spectra proved difficult to reproduce because of small differences in birefringence between samples. More recently, with the ­improvements in artifact level and control made possible by the use of the dual photoelastic modulator (PEM) method [75], and greater care in sampling, solid-phase VCD studies have resumed. A number of years ago, we published a study of a spin-coated conducting organic polymer doped with a chiral camphor-based molecule [171]. The spin-coating process led to a sample film that had virtually no orientation dependence and as a result no linear birefringence artifacts. Several additional VCD studies of films have been published recently using a dual PEM setup that report success for a variety of samples including proteins and carbohydrates [44–47,172]. In most cases, the film VCD was very close to the VCD measured in solution. The main advantage of using films is that aqueous solvent absorption is avoided and roughly two orders of magnitude less sample is needed to obtain a high-quality VCD spectrum. Mulls and KBr pellets are alternatives to films for VCD studies. Mulls preserve the original crystal form of the solid, but as with IR absorption, scattering from the small crystal particles must be avoided. This is achieved for mulls by grinding the particles to a distribution with an average crystal size less than the wavelength of the light being used and then adding an index-matching fluid to decrease the contrast between the solid particles and their immediate optical environment.The tell-tale signature of the presence of a scattering contribution is the shape of the IR and VCD bands. Called the Christiansen effect [43], scattering produces IR bands that have a sharp rise on the high-frequency side of a peak, where intensity has been reduced and an extended tail to low frequency where intensity has been added. The corresponding effect on positive VCD bands is a scattering contribution that produces a negative VCD contribution to high frequency and a positive one to low frequency, superimposed on the normal VCD spectrum. The contributions are reversed in sign for negative VCD bands. As a result,VCD spectra with high scattering contributions can exhibit bisignate couplets wherein the absence of scattering only a monosignate band is observed.

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Figure 5.11  Solid-phase mull IR and VCD of propranolol as the hydrochloride chloride salt (grey, upper) and as the free base (black, lower). Abbreviations: VCD, vibrational ­circular dichroism.

An advantage of solid-phase sampling in mulls is that it permits different crystal forms of a molecule to be compared. As an example, in Fig. 5.11, the IR and VCD spectra of propranolol in two different solid forms are presented. The band shapes for both the IR spectra are symmetric indicating the absence of observable levels of scattering. The IR and VCD spectra show a high degree of similarity and at the same time some significant ­differences can be observed. For example, a band in the hydrochloride salt appears at 1320 cm–1 with significant VCD that is absent in the free base, and there is a very strong VCD couplet in the free base centered just above 1100 cm–1, which is only a small couplet in the hydrochloride salt even though the corresponding IR bands for these two couplets are almost the same. Detailed interpretation of these spectra must await the development of accurate ab initio descriptions of the unit cell of these solids with periodic boundary conditions describing the interactions of the cell with the neighboring unit cells. Several years ago, it was discovered that spray-dried films produce very large VCD intensities compared with mulls and KBr pellets for small

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Figure 5.12  Comparison of the VCD and IR of l-alanine as a mull and as a spray-dried film. Abbreviations: VCD, vibrational circular dichroism; IR, infrared.

molecules of biological significance such as amino acids, small peptides, and active pharmaceuticals [4]. The films are produced from aqueous solutions that are sprayed as a fine mist toward an IR window, such as BaF2, which has been heated to approximately 80 °C. The mist vaporizes rapidly to a crystalline film upon contact with the window. As an example, in Fig. 5.12, we show the spray-dried film of l-alanine compared with the corresponding mull. The spray-dried sample has VCD intensity that is nearly two orders of magnitude larger than that of the mull for nearly identical IR absorbance spectra. It appears that a different crystal form of l-alanine forms on the window under these conditions of rapid drying that is different from commercially available crystalline powders and used for the mull. The most intense VCD band has a ratio of the VCD to IR intensity of approximately

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0.02 that requires an extent of chiral order in the sample on the order of tens of alanine molecules. This illustrates how VCD can be used to probe different crystal structures of the same molecule and in this case the VCD spectrum reveals that spray-dried crystals of l-alanine, and the other amino acids, have a long range chiral order that extends well beyond that of the ordinary l-alanine crystals produced from precipitating solutions. With the ability to observe VCD in solids comes the opportunity to observe VCD in mixtures of APIs and excipients. Since some excipients are chiral, such as dextrose, mannitol, and cyclodextrins, interactions between APIs and excipients that change the state, AC, or ee of the API cannot automatically be ruled out. Thus, VCD offers the opportunity to investigate the crystal state and all chiral properties of APIs in situ as the final formulated products. Additionally, preliminary results show the potential of VCD to monitor not only the state of the API, but also any chiral precipitates that may be present. Research toward the goal of the complete chiral determination of solid-phase pharmaceutical products is currently in progress, and the prognosis for success is excellent as no serious obstacles are currently seen that prevent progress in this direction. The discussion of solid VOA to this point has focused on VCD. ROA spectra of solids have yet to be reported, however nothing a priori precludes this type of measurement since it is well known that Raman scattering can be successfully measured for solids. Scattering in the visible region is much more of a problem than it is in the IR region. On the other hand, scattering is virtually all Rayleigh scattering, and with proper optical filtering and avoiding fluorescent solids, ROA measurements in the solid phase should be reported in the future. Such a capability would add yet another probe of chirality to pharmaceutical products with chiral APIs and chiral excipients, if used.

5.9  SUMMARY AND CONCLUSIONS VOA spectra are rich in structural and stereo-specific information coming from vibrational chromophores extending over the entire molecular frame. The theory of VCD and ROA has advanced sufficiently in recent years to enable the accurate ab initio calculation of observed spectra for small-to-medium-sized molecules. This opens the way for the use of VOA to solve the stereochemical problems involving AC and solution conformation in a direct way without the need to modify the molecule, to calculate

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excited electronic state properties, or to crystallize the sample.The development of flow-cell techniques makes possible the monitoring of the reaction kinetics of chiral molecules in solution. Because both FT-VCD and CCD-ROA are multiplex techniques, it is possible to follow in real time the evolution of the mole fraction and ee of multiple species as a reaction proceeds. No other forms of optical activity currently possess this capability. Extension of VCD into the near-IR region further enhances the potential for VCD to be used routinely to monitor processes involving the synthesis, manufacture, and processing of pharmaceutical products containing chiral APIs. The commercialization of hardware and software for the measurement and calculation of VCD and ROA opens the way for these two forms of VOA to be broadly available for all those interested in solving problems involving molecular chirality and chiral analysis [11,173].

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CHAPTER 6

Raman Optical Activity Saeideh Ostovar pour*, Laurence D. Barron**, Shaun T. Mutter†, Ewan W. Blanch* *RMIT University, Melbourne,Vic., Australia **University of Glasgow, Glasgow, United Kingdom † Cardiff University, Cardiff, United Kingdom

Raman optical activity (ROA) has many advantages over conventional Raman spectroscopy for investigation of chiral molecules, in general, and biological molecules, in particular, on account of their homochirality. Since it is a manifestation of vibrational optical activity [1], being complementary to vibrational circular dichroism (VCD), ROA is more sensitive to the 3D structures of chiral molecules through its dependence on their absolute handedness, which has led to its development as an incisive probe of the structure and behavior of biomolecules in aqueous solution. For example, ROA spectra obtained from proteins can provide detailed information on the secondary and tertiary structures adopted by polypeptide backbones. Additionally, ROA can probe the tautomers of side chains. In common with other vibrational spectroscopies, there is no restriction on the size of molecules, or in their type, that can be investigated by ROA, broadening its application to unfolded proteins, viruses, and carbohydrates. More detailed accounts of the history of the use of ROA for studying small molecules and proteins can be found elsewhere [1–4]. These studies established the sensitivity of ROA to conformational subunits, particularly to the secondary structural motifs that are the building blocks of proteins, as well as to side-chain tautomers. This current review will focus on more recent applications of ROA to specific questions of protein behavior and to other biological systems such as carbohydrates, RNA, viruses, and blood plasma, as well as of resonance and other phenomena of growing scientific interest. From these examples, we hope to illuminate readers as to the potential of ROA to inform about diverse biological and chemical processes. Although many reviews of the theory of ROA scattering are available, we will begin with a summary of these principles in order to better understand how ROA spectroscopy is providing important new insights into diverse problems. Following is an account of some of the ROA research, both experimental and computational, from the past 10–15 years, as a guide to Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00006-9 Copyright © 2018 Elsevier B.V. All rights reserved.

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how this exquisitely sensitive chiroptical technique is revealing new insights into molecular behavior.

6.1  FUNDAMENTAL PRINCIPLES 6.1.1  The ROA observables In the fundamental scattering mechanism responsible for ROA discovered by Atkins and Barron [5], interference between light waves scattered via the molecular polarizability and optical activity tensors of a molecule yields a dependence of the scattered intensity on the degree of circular polarization of the incident light (known as incident circular polarization— ICP) and also to a circular component in the scattered light (scattered circular polarization—SCP). A subsequent and more definitive paper [6] introduced an appropriate dimensionless quantity that can be experimentally measured, the circular intensity difference (CID): ∆=

 R

(I R − I L ) (I R + I L )

(6.1)

L

where I and I are the scattered intensities in right- and left-circularly polarized incident light, respectively. ROA measurements can be performed using various different experimental configurations [3,7]. In particular, the scattering angle can be varied, with the backward direction (180° scattering) being the most important for studies of biomolecules in aqueous solution. In terms of the electric dipole–electric dipole molecular polarizability tensor, ααβ and the electric dipole–magnetic dipole and electric dipole–electric quadrupole optical activity tensors, G′αβ and Aαβγ, ICP CIDs associated with forward (0°) and backward (180°) scattering for an isotropic collection of chiral molecules with dimensions much smaller than the wavelength of the incident light are [3]: 4[45αG ′ + β (G ′)2 − β ( A)2 ] ∆(0°) = (6.2a) c[45α 2 + 7β (α )2 ]  ∆(180°) = 

24[ β (G ′)2 + (1/ 3)β ( A)2 ] c[45α 2 + 7β (α )2 ]

(6.2b)

These various tensor component products have been averaged over all orientations of the scattering molecule to generate collections of products that are invariant to axis rotations. Specifically, 

α = 13 α αα = 13 (α xx + α yy + α zz )

(6.3a)

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G ′ = 13 Gαα ′ = 13 (Gxx ′ + Gyy′ + Gzz ′ )

(6.3b)

are the isotropic invariants, and 

β (α )2 = 21 (3α αβ α αβ − α αα α ββ )

(6.3c)



β (G ′ )2 = 21 (3α αβGαβ ′ − α ααG ββ ′ )

(6.3d)



β ( A)2 = 21 ωα αβ ε αγδ Aγδβ

(6.3e)

are the anisotropic invariants in which the tensor components are defined with respect to molecule-fixed axes.These invariants are independent of the choice of the origin and each is measurable. In the above expressions, a Cartesian tensor notation is used in which a repeated Greek suffix denotes summation over the three components and εαβγ is the third-rank antisymmetric unit tensor. As was introduced above, as well as the case of ICP, ROA is also manifest as a small circularly polarized component in the scattered beam (SCP) [1,3,8–10]. Within the far-from-resonance approximation, measurement of this circular component (SCP ROA) as (IR − IL) − (IR + IL), where IR and IL denote the intensities of the right- and left-circularly polarized components, respectively, of the scattered light, provides equivalent information to the CID measurement (ICP ROA). It should be remembered that these relationships apply specifically to Rayleigh (elastic) scattering. For Raman (inelastic) scattering, the same basic CID expressions apply but with the molecular property tensors being replaced by the corresponding vibrational Raman transition tensors between the initial and final vibrational states, nv and jv. Therefore, ααβ, etc. are replaced by 〈 jv |ααβ (Q )| nv 〉, etc., where ααβ (Q), etc. are the effective polarizability and optical activity operators that depend parametrically on the normal vibrational coordinates, Q. Within the Placzek polarizability theory of the Raman effect [3,11], the ROA intensity depends on products ′ / ∂Q )0 and (∂ααβ / ∂Q )0 εαγδ (∂ Aγδβ / ∂Q )0, where such as (∂ααβ / ∂Q )0 (∂Gαβ the subscript 0 indicates the corresponding value for the case of the equilibrium structure. In the case of a molecule composed entirely of idealized axially symmetric achiral bonds, for which β(G′)2 = β(A)2 and αG′ = 0 [3,12], ROA is generated exclusively by anisotropic scattering, and the forward and backward CID expressions (6.2a) and (6.2b) reduce to [3] ∆(0°) = 0, (6.4a)

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∆(180°) = 

32β (G ′)2 c[45α 2 + 7β (α )2 ]

(6.4b)

The weak intensity of ROA scattering coupled with high background signals that can be encountered, particularly for biological or resonant samples, can complicate ROA measurements. Since conventional Raman intensities are the same in forward and backward scattering, the ROA signal in backscattering is increased relative to the background Raman intensity and so is the best experimental strategy for most ROA studies of biomolecules in aqueous solution [13,14].

6.1.2 Instrumentation As explained above, a backscattering geometry has proved essential for the routine measurement of ROA spectra of biomolecules in aqueous solution. Backscattered ROA spectra may be acquired using both the ICP and SCP measurement strategies, although the designs of the corresponding instruments are different. A backscattering ICP measurement strategy was used in Glasgow for some years and this strategy was used to successfully investigate the conformations of a large number of biomolecules. The final version of the Glasgow backscattering biomolecular ICP ROA instrument has been described in detail elsewhere [15]. A visible 514.5 nm argon ion laser beam was weakly focused into the sample solution contained in a small rectangular fused quartz cell. The cone of backscattered light was reflected off a 45° mirror, which had a small central hole drilled to allow passage of the incident laser beam, through an edge filter to remove the Rayleigh line and into the collection optics of a single-grating spectrograph containing a volume holographic transmission grating with a backthinned CCD detector, allowing the full spectral range to be measured in a single acquisition. To build up the signal-to-noise ratio of ROA signals, the spectral acquisition was synchronized with an electro-optic modulator used to switch the state of polarization of the incident laser beam between the right and left circular states at a suitable rate.The spectra were displayed in analog-to-digital converter units as a function of the Stokes–Raman wave number shift with respect to the exciting laser line. Typical laser power at the sample was ∼700 mW and sample concentrations of proteins, polypeptides, and nucleic acids were ∼30–100 mg/mL, while those of intact viruses were ∼5–30 mg/ mL. Under these conditions, ROA spectra over the range ∼600–1700 cm−1

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Fig. 6.1  The optical design of the final version of the Glasgow backscattering ICP ROA instrument. Adapted from Ref. [16]. Abbreviation: ICP ROA, incident circular polarization Raman optical activity.

were obtained in ∼5–24 h for proteins and nucleic acids and ∼1–4 days for intact viruses. Although ROA spectra may be recorded down to ∼200 cm−1 on favorable samples such as carbohydrates, spectra below ∼600 cm−1 can be unreliable from highly scattering samples like proteins due to offsets associated with the intense Rayleigh wing. A diagram of this instrument is shown in Fig. 6.1. Although this backscattering ICP ROA instrument served to establish ROA in biomolecular science and the design remains useful, routine ROA studies were often frustrated by the delicate nature of the associated measurements. This situation changed, thanks to a new design of ROA instrument by Werner Hug based on the use of the SCP strategy [9,16]. In particular, “flicker noise” arising from dust particles, density fluctuations, laser power fluctuations, etc. are eliminated since the intensity difference measurements required to extract the circularly polarized components of the scattered beam are taken between two orthogonal components of the scattered light measured during the same acquisition period.The flicker noise consequently cancels out, resulting in considerably improved signal-to-noise characteristics. The basic design of the SCP configuration is illustrated in Fig. 6.2. The incident visible laser beam at 532 nm from a frequency-doubled continuous Nd/YAG laser, the initial linear polarization of which is “scrambled” by a fast

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Fig. 6.2  Simplified optical design of the BioTools ChiralRAMAN backscattering SCP ROA instrument. Lenses are represented as double-headed arrows. Adapted from Ref. [43]. Abbreviation: SCP ROA, scattered circular polarization Raman optical activity.

rotation of the azimuth, is deflected into the sample cell using a small prism. The cone of backscattered light is collimated onto a liquid crystal retarder set to convert right- and left-circular polarization states into linear polarization states with azimuths perpendicular and parallel, respectively, to the plane of the instrument, followed by an edge filter to remove the intense Rayleigh line. A beam-splitting cube then diverts the perpendicular component at 90̊ to the propagation direction of the parallel component, which passes through undiverted. In this way, the right- and left-circularly polarized components of the backscattered light are separated and collected into the ends of two fiber optics. Each fiber-optic converts the cross-section from circular at the input end into a linear configuration at the output end that matches the entrance slit of a fast imaging spectrograph, thereby enabling separate Raman spectra for the right- and left-circularly polarized components of the scattered light to be dispersed simultaneously one above the other onto

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the chip of a backthinned CCD detector. Subtraction then reveals the SCP ROA spectrum corresponding to small circularly polarized components in the Raman bands. Small differences in the two detection channels are compensated for by interconverting their function through the switching of the liquid crystal retarder from the −λ/4 to the +λ/4 state. A commercial instrument based on the Hug design and incorporating a sophisticated artifact-suppression protocol, based on a “virtual enantiomers” approach which greatly facilitates the routine acquisition of reliable ROA spectra [9,16], was developed (the ChiralRAMAN from BioTools, Inc.). This instrument provides high-quality protein ROA spectra in ∼2–5 h, several times faster than the ICP ROA instruments, using a sample volume of ∼30 µL and ∼500 mW of focused laser power at the sample and extends protein ROA data acquisition routinely to the low-wave number region ∼200–600 cm−1. For neat liquid samples of small chiral molecules, the SCP ChiralRAMAN spectrometer measures high-quality ROA spectra in just a few minutes. On account of the tiny signals and the delicacy of the measurements, polarization artifacts have plagued ROA studies from the beginning. Indeed, the first reported ROA spectra were gross artifacts, with measurements of the first genuine ROA spectra, reported by Barron et al. in 1973 [17], only being achieved when the origins of the artifacts were properly understood and effective strategies implemented to suppress them. Thanks to Hug’s artifact suppression protocol incorporated into the ChiralRAMAN instrument, artifact-free ROA spectra may now be acquired routinely. Alas history keeps repeating itself with occasional reports of ROA data from home-built instruments that are artifacts of some sort. For example, a paper appeared claiming the first observation of surface-enhanced ROA [18]. Since the sample, adenine, is not chiral, the authors suggested that the signals resulted from surface-induced chirality. But because equal numbers of mirror-image chiral surface domains are expected on the silver nanoparticles employed in the bulk sample, resulting in zero ROA, the reported observations almost certainly originate in experimental artifacts. More recently, a combination of ellipsometry with Raman spectroscopy was reported which claimed to provide ROA spectra along with other polarization data [19]. However, among many other problems, the reported ROA spectrum of their model compound (S)-(−)-1-phenylethanol is several orders of magnitude too large and looks nothing like the negative of the definitive ROA spectrum of (R)-(+)1-phenylethanol shown in Fig. 6.7!

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6.2  EXPERIMENTAL ROA STUDIES A key advantage of ROA spectroscopy is that it is widely applicable to most types of biomolecules in aqueous solution, without restrictions due to size or conformational flexibility. Consequently, the majority of researchers now using ROA appear to be particularly interested in studying biological molecules. ROA spectroscopy provides more informative and, paradoxically, less complex spectra than conventional Raman since the most intense signals typically originate from vibrational coordinates that sample the most rigid and chiral parts of the structure. Usually, for proteins, these are located within the backbone and give rise to ROA band patterns characteristic of the local backbone conformation. Polypeptides and protein sequences in the standard conformations defined by the characteristic Ramachandran, φ,ψ angles found in secondary, loop, and turn structure are particularly favorable for ROA studies as their spectra are highly sensitive to secondary and tertiary structure. By comparison, the parent conventional Raman spectrum of a protein is dominated by bands arising from the amino acid side chains which often obscure the peptide backbone bands. Carbohydrate ROA spectra are similarly dominated by signals from skeletal vibrations, in this case mainly from the constituent sugar rings and the connecting glycosidic linkages. Although the parent Raman spectra of nucleic acids are dominated by bands from the intrinsic base vibrations, signals characteristic of the stereochemical arrangements of the bases, the sugar rings, and the sugar–phosphate backbone dominate their ROA spectra. The timescale of the Raman scattering event (∼3.3  × 10−14 s for a vibration with Stokes wave number shift ∼1000 cm−1 excited in the visible) is much shorter than that of the fastest conformational fluctuations of molecules. Therefore, an ROA spectrum is a superposition of “snapshot” spectra from all the distinct conformations present in the sample at equilibrium. This contrasts with the long timescale of NMR, so that NMR only senses the average structure of interconverting conformers and generally requires reliable molecular dynamics (MD) simulations to interpret the data. Furthermore, since ROA observables depend on absolute chirality, there is a cancellation of contributions from quasi-enantiomeric structures, which can arise as mobile structures exploring the range of accessible conformations. ROA thus exhibits an enhanced sensitivity to the dynamical aspects of biomolecular structure, making it a powerful probe of order–disorder and other structural transitions. Observables that are “blind” to chirality, such as

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conventional Raman band intensities, are generally additive and are therefore less sensitive to conformational mobility. The early years of ROA studies have been reviewed elsewhere [1–4,20,21]. Here, we will review more recent studies on proteins and other biomolecules.

6.2.1 Proteins For proteins, vibrations of the peptide backbone are generally associated with three main regions of the Raman spectrum [22,23]. The backbone skeletal stretch region ∼870–1150 cm−1 originates in Cα–C, Cα–Cβ, and Cα–N stretch coordinates; the extended amide III region ∼1230–1340 cm−1 arises from mainly the in-phase combination of the N–H in-plane deformation with the Cα–N stretch along with, as is now recognized, mixing between the N–H and Cα–H deformations [24–26]; and the amide I region ∼1630–1700 cm−1 originates mainly in the C=O stretch. In protein ROA spectra, the extended amide III region is of particular importance as coupling between the N–H and Cα–H deformations is sensitive to geometry which results in a rich and informative band structure. Amide II vibrations, which occur in the region ∼1510–1570 cm−1, and are assigned to the outof-phase combination of the in-plane N–H deformation with the C–N stretch can also be more prominent and informative in ROA spectra than in the corresponding conventional Raman spectra [27]. As discussed above, side-chain vibrations generate many characteristic Raman bands [22,23,28] but are typically less prominent in ROA spectra due to some conformational freedom averaging out the net chirality in certain situations. Despite this, some side-chain vibrations give rise to useful ROA signals as will be discussed later. Over a period of 12–15 years starting from the mid-1990s, a considerable number of protein ROA spectra were reported, mainly from the Barron group in Glasgow. These studies established a library of band assignments that were important for developing ROA as a protein structural technique, that the frequencies and signs of specific ROA bands correlated well with atomistic details from X-ray diffraction and NMR studies, and also illustrated how chemometrics can reveal the sensitivity of ROA bands to both secondary and tertiary structural motifs. While many more proteins are yet to be structurally characterized by ROA, the technique has matured to the point that in the past several years researchers are now making use of this information to address challenging problems in protein biology. We outline a number of these recent studies below.

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ROA spectra can be used to determine a protein’s fold when more conventional techniques have been unable to do so, as was shown for α1-acid glycoprotein (AGP) [29,30]. This is a much studied glycoprotein but no X-ray crystal or NMR structure of the intact glycosylated protein had been reported. An X-ray structure, PDB 3KQ0, is now available but this is for a recombinant form, so is presumably deglycosylated! The polypeptide moiety consists of a single chain of 183 amino acids with two disulfide bridges. The carbohydrate content makes up 45% of the molecular weight of AGP and contains five or six highly sialylated oligosaccharide chains, N-glycosylated to asparagine residues. The polypeptide chain is thought to adopt a lipocalin-type fold based on an eight-stranded antiparallel β-sheet closed back on itself to form a continuously hydrogen bonded barrel with strands linked by β-hairpins and a long loop [31].The lipocalin fold is also found in β-lactoglobulin, which has been well studied by ROA. Many of the protein ROA bands of AGP were similar to those observed for β-lactoglobulin, indicating that their folds are also similar. ROA to studies of protein misfolding were first conducted two decades ago and work continues into this fascinating problem. Despite there being a large amount of information regarding the kinetics of the fibrillation process and the structure of mature fibrils, relatively little is known about the structures of the soluble prefibrillar toxic aggregates [1,2,32]. Early events in fibrillogenesis feature a dynamic and heterogeneously populated ensemble of oligomeric states. Because of their inherently transient nature, their exact characterization proves difficult using conventional methods. Previously, ROA was able to characterize the molten globule formed in the intermediate state of human lysozyme [33] that precedes fibril formation. More recently, ROA spectra were reported for a reversible prefibrillar intermediate state of insulin [34], though at that time analysis was limited to empirical band assignments. In a subsequent paper, further studies [35,36] were able to confirm and better interpret the spectral details using density functional theory (DFT) calculations and the CCT fragmentation approach. Interestingly, they also found that the spectral details were sensitive to local conformation rather than through space noncovalent interactions between structural components, that ROA bands appear to be sensitive to twisting of the fibrils, and that ROA spectra are likely to be complementary to VCD spectra of protein fibrils. As the authors stated, ROA spectra are difficult to obtain for fibrils due to sample inhomogeneities and generation of birefringent artifacts, but a combination of techniques such as ROA and VCD spectroscopies offers a potential route to the future structural characterization of prefibrillar and fibrillar proteins.

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Similarly, the highly conformationally flexible protein α-synuclein has been studied using ROA by Johannessen and coworkers [37]. This natively unfolded, or intrinsically disordered, protein plays a key role in the progression of Parkinson’s disease. Understanding the conformational stability of such disordered proteins is important for the future development of therapies for Parkinson’s and similar neuropathologies, a major challenge for medicine as Western populations age and the various forms of dementia place an increasing burden on society. ROA has previously been able to characterize the conformations of fibril intermediate states of human lysozyme [33] and insulin [34–36], providing insights into molecular mechanisms underlying protein misfolding diseases. In this more recent work, Mensch and colleagues investigated the induction of α-helical and β-sheet structures in different variants and mutants of α-synuclein. The combination of ROA’s sensitivity to conformation with its broad applicability to different experimental conditions raises the possibility of its use for the verification of the stability of biologics under the full range of conditions experienced during bioformulation. Protein-, nucleic acid-, and carbohydrate-based compounds now dominate the global pharmaceuticals market and their complexity over small organic pharmaceuticals compounds increases the problem of drug stability. During production and formulation, biologics experience widely ranging conditions (e.g. temperature, pH, concentration, ionic strength, etc.) and characterizing their structures and stability in solution under such stresses can be challenging. Recently, a research team from Merck Sharp and Dohme used ROA to monitor conformational changes induced in an immunoglobulin G4 (IgG4) therapeutic monoclonal antibody in its formulation matrix [38], establishing that even subtle changes in secondary and tertiary structures due to thermal stress could be identified. This illustration of how ROA can provide important information about conformational stability under bioformulation conditions suggests many future opportunities in the pharmaceutical sector. Although most ROA studies have been reported for pure samples of proteins measured in vitro, the potential to analyze protein structure under more physiologically relevant conditions has attracted interest. To date, such examples are few but Setnicˇka and coworkers [39] have recently shown that it is possible to measure ROA spectra of blood plasma proteins in human blood plasma. By adding high concentrations of a fluorescence quencher, namely sodium iodide, followed by photobleaching, these researchers were able to dramatically decrease the fluorescent background by 90% and increase the signal-to-noise ratio by a factor of 3.3, though

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baseline distortions remained the same. Nevertheless, good quality spectra were obtained from the biofluid. Although the application of ROA to more complex biomaterials will face other challenges, this demonstration that biofluids can now be investigated greatly broadens potential applications of ROA as a biomedical technology. The ability of ROA to investigate small structural changes within complexes has been demonstrated by the Unno group in Saga, Japan. Although resonance and preresonance effects are a subject of current interest, see other examples in this review, they can complicate the analysis of protein spectra. Unno and colleagues have used their near-infrared excited ROA spectrometer to probe the structural changes in the retinal Schiff-base chromophore of the light-driven proton pump bacteriorhodopsin. They found that ROA spectra, in combination with DFT calculations, were able to easily differentiate between the all-trans, 15-anti and 13-cis, and 15-syn isomers [40]. A recent example of the current power of quantum-chemical calculations in studies of peptide and protein conformation was the calculation of ROA spectra for ∼3000 model oligopeptide conformations over populated regions of the Ramachandran φ,ψ potential energy surface [37]. The results provided a database of calculated ROA spectra of model peptide structures which was used to make assignments in unprecedented detail of experimental ROA patterns to the allowed conformational elements, associated with very specific regions of the Ramachandran surface, of the peptides in solution. The stunning agreement between the observed and calculated ROA spectra of, for example, model oligopeptides known to take up the α-helical and poly(l-proline II) (PPII) conformations in solution demonstrated the power of the database for the assignment of ROA band patterns of peptides and proteins. The spectral trends contained in the database were used to refine previous interpretations of experimental ROA patterns of a number of different proteins including, inter alia, a reassessment of the previously assigned positive ROA bands at ∼1300 and 1340 cm−1 to unhydrated and hydrated α-helix, respectively, [41] with the opposite being more likely!

6.2.2  Nucleic acids Ribonucleic acids (RNAs) also support a broad range of secondary and tertiary structures that generate characteristic features in both Raman and ROA spectra. While relatively few ROA spectra have been reported to date for RNAs compared to proteins, a similar level of sensitivity to structure and dynamics has been shown. ROA spectra are sensitive to three different

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sources of chirality in nucleic acids: the chiral base-stacking arrangement of intrinsically achiral base rings, the chiral disposition of the base and sugar rings with respect to the C–N glycosidic link, and the inherent chirality associated with the asymmetric centers of the sugar rings. As RNA secondary structure motifs are generally stable in isolation, unlike the case for proteins, it is expected that distinctive structural fingerprints should be discernible from ROA spectra. Hobro et al. [42] reported ROA spectra for common RNA secondary structural motifs found in a 37 nucleotide fragment from Domain I of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) RNA. These spectra were able to identify differences in local conformations of the phosphodiester backbone corresponding to the GNRA tetraloop with stem in comparison with those of the same parent structure with either a U.C base mismatch or a pyrimidine-rich asymmetric bulge. While the limited availability of RNA samples in milligram quantities has restricted the number of studies that have been reported, ROA has also been shown to be sensitive to phosphorylation patterns in ribonucleotides [43]. The general features of the ROA spectra of a series of adenosine ribonucleotides varied significantly, thus providing a fingerprint sensitive to both the number and position of phosphate groups on the ribose. While the corresponding Raman and surface-enhanced Raman spectra (SERS) could distinguish between many of the adenosine ribonucleotides, these two techniques were unable to differentiate several of these compounds as these two techniques are blind to chirality. In contrast, the ROA spectra for even adenosine monophosphate (AMP), diphosphate (ADP), and triphosphate (ATP) were unique. It was also observed that distinct ROA bands arose from cyclization of the phosphate group in adenosine-(2,3)monophosphate and adenosine-(3,5)-monophosphate, presumably due to the increase in rigidity induced by cyclization. The signals associated with the phosphorus groups in these two cyclic ribonucleotides were opposite in sign, reflecting the opposite stereochemistries of the environments of the phosphorus atom in each case (Fig. 6.3).

6.2.3 Viruses Knowledge of the structure of viruses at the molecular level is essential for understanding their modus operandi. However, key structural biology techniques such as X-ray crystal or fiber diffraction, or cryo-electron microscopy are not always applicable. Conventional Raman is valuable in studies

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Fig. 6.3  ROA spectra of adenosine, adenosine 5′-monophosphate (AMP), adenos­ ine 5′-diphosphate (ADP), adenosine 5′-triphosphate (ATP), A(2)MP (pH 3.13), adenosine 2′,3′-cyclic monophosphate (A(2,3)MP), adenosine 3′-monophosphate (A(3)MP), and adenosine 3′,5′-cyclic monophosphate (A(3,5)MP) in solution. The concentration for each sample was 100 mg/mL and laser power at the sample was 0.6 W. Figure adapted from Ref. [43]. Abbreviation: ROA, Raman optical activity.

of intact viruses at the molecular level as it is able to simultaneously probe both the protein and nucleic acid constituents [44]. The additional incisiveness of ROA further enhances the value of Raman spectroscopy in structural virology. Remarkably, ROA spectra may be obtained for most types of intact viruses in aqueous solution, including filamentous, cylindrical, and icosahedral [45,46]. Even more remarkable is that valuable information such as the folds of the major coat proteins can simply be “read off ”! For

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example, the ROA band pattern for filamentous bacteriophages such as fd is highly characteristic of an extended helix and looks very similar to that of α-helical polypeptides [32,45,47–49]; that of tobacco mosaic virus is characteristic of proteins with a helix bundle fold; those of satellite tobacco virus (STMV) and the MS2 virus capsid are characteristic of up-down antiparallel β-sheet [50], with that of STMV containing additional bands characteristic of a significant amount of disordered structure from the many long strands. The importance of intrinsic disorder in viral coat proteins, especially to facilitate rapid changes at almost all stages of their life cycle, is being increasingly recognized with many functions attributed to disordered regions [51]. A remarkable discovery that tryptophan conformation and absolute stereochemistry could be determined from the signs of the tryptophan W3 ROA bands was made from ROA data on filamentous bacteriophages [52], further insights into this assignment being provided by later DFT calculations [53]. Finally, the special power of ROA in structural virology was illustrated by a study on cowpea mosaic virus CPMV [46]. CPMV is a member of the comovirus group of plant viruses and is a bipartite virus, that is, its genome consists of two different RNA molecules (RNA-1 and RNA-2) separately encapsidated in identical icosahedral capsids.The protein capsid is constructed from 60 copies of an asymmetric unit made up of three different protein domains each of which supports a “jelly-roll β-sandwich” fold. However, X-ray diffraction did not resolve the structures of RNA-1 and RNA-2. Preparations of CPMV were separated into empty protein capsids, capsids containing RNA-1, and capsids containing RNA-2. The top panel of Fig. 6.4 shows the Raman and ROA spectra of the empty protein capsid, with the spectral band patterns being characteristic of the jelly-roll β-sandwich fold of the capsid proteins. The middle panel shows the spectra of the capsid containing RNA-2, with bands from the nucleic acid now evident along with those from the protein. The Raman and ROA spectra for the capsid with RNA-1 was very similar. The bottom panel shows the spectra obtained by subtracting the top from the middle spectra, leading to Raman and ROA spectra for the encapsidated RNA-2 along with possible changes in protein bands due to protein–RNA interactions. The difference ROA spectrum reflects the single-stranded A-type helical conformation of the encapsidated RNA-2, which was not previously known. The ROA spectrum of RNA-1 was extracted using the same procedure and was found to be very similar to that of RNA-2, despite their having quite different lengths and low sequence homology.

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Fig. 6.4  Backscattered ICP Raman (IR + IL) and ROA (IR  −  IL) spectra measured for (A) the empty cowpea mosaic virus (CPMV) protein capsid (top pair), (B) the intact capsid containing RNA-2 (middle pair), and (C) the difference spectra obtained by subtracting the top from the middle spectra to reveal the spectra of the viral RNA-2 (bottom pair). Abbreviations: ICP, incident circular polarization; ROA, Raman optical activity.

6.2.4 Carbohydrates High-resolution structural techniques (X-ray crystallography and NMR) have revolutionized biology through their ability to reveal detailed information on proteins and nucleic acids, but they are less applicable to carbohydrates. Most glycoproteins and polysaccharides do not form diffractable

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crystals while their typical combination of large size and low sequence complexity often prevent the acquisition of well-resolved NMR spectra. Other biophysical techniques are required to improve our understanding of how sugars, glycans, and polysaccharides behave, and all sugars are chiral, making ROA an attractive technique for investigating their solvated conformations. The Barron group in Glasgow first showed that detailed and informative spectra could be measured on simple sugars [54–56], can determine the absolute configuration about each chiral center of a carbohydrate, and differentiate between α- and β-anomeric linkages, epimers, homomorphic sugars, and infer the relative conformations of CH2OH groups, which are important for intramolecular hydrogen bonding and for stabilizing backbone conformations. Further studies on cyclodextrins [57,58] established that ROA can provide detailed information about the conformational dynamics of oligosaccharides. The first ROA spectra of glycoproteins were also reported in Glasgow [30,59], as well as the first studies of the protein and glycan components of a glycoprotein [60]. ROA spectroscopies have since been used to characterize the conformations and behavior of complex polysaccharides. An example is the study by Yaffe et al. [61] on hyaluronan (HA), a nonsulfated glycosaminoglycan (GAG) composed entirely of repeating disaccharides of glucuronic acid (GlcA) and N-acetyl-glucosamine (GlcNAc) linked by alternating β-1,3 and β-1,4 glycosidic bonds. HA is found in all vertebrate tissues as a highmolecular-mass polysaccharide and performs a wide range of biological functions, leading to its widespread use in medicine, tissue engineering, and cosmetics, yet, we understand relatively little about the structural parameters regulating HA organization and function. Yaffe et al. [62] monitored characteristic intersaccharide interactions in a short HA subunit (the HA4 tetramer) but found no signs of extensive interchain interactions being formed in a much longer HA chain (see Fig. 6.5). This finding shows that there was no extensive tertiary structure formation in the HA polymer, in agreement with NMR studies. Rudd et al. [63] have also reported distinctive ROA spectra for several GAGs, further emphasizing the potential of the technique in glycobiology. Recently, Ashton et al. [64] have used ROA to investigate the interactions between mucin glycoproteins. Mucins are highly glycosylated, highmolecular-weight proteins which perform diverse roles in the formation of mucosal gels in metazoans, which function as mesh-like, size, and charge exclusion barriers. Most mucins have a block copolymer structure with nonglycosylated domains at both termini that are separated by an extended

Fig. 6.5  Raman (IR + IL) and ROA (IR − IL) spectra, respectively, of a 1:1 stoichiometric mix of GlcA and GlcNAc (A and B), HA4 tetramer (C and D), and the HApol polymer (E and F). Secondary structure marker bands are shown with arrows. The large differences between the ROA spectra shown in B and D reflect the formation of secondary structure–type interactions between saccharides, while the strong similarity between the ROA spectra for HA4 and HApol (D and F) indicate that no significant tertiary interactions are formed in the polymer. Figure is taken from Ref. [61]. Abbreviation: ROA, Raman optical activity.

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glycosylated domain rich in serine, threonine, and proline residues. O-linked glycosylation occurs through conjugation of N-acetylgalactosamine (GalNAc) sugars to the hydroxyl moieties of the serines and threonine side chains, giving rise to the so-called “hinge region” thought to be important for molecular control of the formation of the mucosal mesh. Ashton et al. determined the order of conformational changes occurring over concentration ranges relevant to formation of the gastric mucosal layer.They found that at mucin concentrations from 20 to 40 mg/mL, these GalNAc moieties underwent conformational changes, with other saccharides changing conformation above 40 mg/mL, together with other structural transitions observed in the protein core, notably the formation of β-structure. In this way, ROA was able to help monitor the formation of transient entanglements formed by what are thought to be brush–brush interactions between the oligosaccharide combs of mucin molecules.Though highly informative, this study was performed on a commercial mucin sample that was probably not intact. A following study on a functional human MUC5B mucin and several of its subdomains using a combination of ROA and other spectroscopies revealed further insights into mucin structure and glycosylation [65]. A challenge for glycobiology is to map and understand the expression patterns of component sugars in the glycan components of glycoproteins. With most techniques, it is difficult to determine linkage types and between oligosaccharides with similar compositions. Johannessen et al. [60] examined yeast invertase from Saccharomyces cerevisiae, a high mannose glycoprotein that is widely used in the sugar industry as a biocatalyst and a number of its component oligosaccharides. Significant differences were observed between their corresponding ROA spectra, arising from the different glycosidic linkages and the resulting effects on conformation and dynamics of the component sugars. Spectra for two mannose trisaccharides revealed ROA band patterns similar to those found in the ROA spectrum of the glycoprotein yeast invertase. The authors concluded that this band pattern originated from the α  − (1 → 2)- and α  − (1 → 3)-glycosidic linkages, possibly with some minor contributions from α − (1 → 6)-linkages. This close similarity in ROA band patterns indicated that the conformations around these glycosidic links in the glycoprotein are similar to those of the free mannose trisaccharides in aqueous solution. Recently, this group extended their study on glycoproteins to ribonuclease B (RNase B). RNase B has the same protein sequence as RNase A but with a single N-linked glycan containing two GlcNAc and a variable number of mannose residues.The ROA spectral

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signatures for this glycan component were reconstructed by subtracting the protein spectral signatures measured for the nonglycosylated RNase A [66]. The section on ROA calculations describes several further recent developments on carbohydrate studies with ROA (see below). ROA appears well positioned to make a major impact in the glycosciences.

6.2.5  Natural products Monoterpenes were widely studied during the early years of ROA and were important test cases for enabling both experimental and computational developments over the years. Although ROA is still less widely known than either VCD or electronic circular dichroism (ECD) in the world of natural products chemistry, there have been an increasing number of studies that suggest that ROA will be more important in this area in the future. The absolute configuration of the monoterpene (+)-(E)-junionone was determined by Hug and colleagues as the R-conformer [67]. Similarly, a new ROA group in Japan determined the absolute configurations of (+)-(R)and (−)-(S)-limonene and (+)-(E)-alpha-santalol [68]. Aeroplysinin-1 is an antiangiogenic drug extracted from the marine sponge Aplysina cavernicola. It has two stereogenic centers, and their absolute configurations, along with the conformational properties of the molecule in aqueous solution, were determined using experimental ROA measurements and DFT (B3LYP/aug-cc-pVDZ) calculations [69]. These calculations were performed for the four stereoisomers possible, resulting in the assignment as 1S,6R. The absolute configuration of the more flexible marine antibiotic (+)-synoxazolidinone A, isolated from Synoicum pulmonaria and with three stereogenic centers and eight possible conformers, was assigned as 6Z,10S,13R using a combination of ECD,VCD, and ROA spectroscopies with DFT calculations [70]. ROA was important for the reliable determination of the configuration at the double bond in the molecule.The ability to structurally investigate natural products using ROA even in plant extracts was shown in a study on borneol [71]. The active component in pichtae essential oil (from the Siberian fir needle pine, Abies sibirica), which is sold as an essential oil and home remedy for various ailments in Eastern Europe, is (−)-bornyl acetate. As bornyl acetate, like the pichtae essential oil, is an oil, it is interesting to note that gas phase DFT calculations were sufficiently accurate to interpret the experimental spectra and establish the conformation of (−)-bornyl acetate in situ. As shown in Fig. 6.6, it should be emphasized that in most other cases either implicit or explicit solvation is required for a reliable DFT calculation of ROA spectra.

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Fig. 6.6  The experimental ROA spectra of four different samples of pichtae oil (a–d) and ROA spectra of (−)-bornyl acetate and (+)-bornyl acetate. The signs of ROA bands from the pichtae oil samples clearly identify the presence of the (−)-conformer. Abbreviation: ROA, Raman optical activity.

There have also been a number of recent studies by Batista Jr. and colleagues on natural products extracted from Brazillian cerrado plant species. In one case [72], while a detailed NMR study was able to structurally characterize a new ent-halimane diterpenoid, 18-hydroxy-enthalima-1(10),13-(E)dien-15-oic acid, from ethanolic extracts from the flowers and leaves of Hymenaea stigonocarpa, ROA measurements and DFT calculations established the absolute configuration. In another example, a cyclic octapeptide, or orbitide, called ribifolin was isolated from Jatropha ribifolia and characterized using a battery of techniques, including ROA measurements and calculations [73]. A very good level of agreement between the DFT calculations and experimental ROA spectra was observed, allowing assignment of the classic and inverse γ-turn conformations supported by this cyclic peptide. This strategy was also successfully applied to a flavone C-diglycoside, where explicit solvation was again shown to be important [74]. A combination of ROA and VCD was shown to be more reliable than ECD for characterizing

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a two chiral-center homoisoflavonone [75] and revealed the different optical purities of this chiral natural product when produced by three different Polygonum (the knotweed and buckwheat family of flowering plants) species.

6.3  ENHANCEMENT OF ROA SIGNALS ROA is far more structurally sensitive technique than conventional Raman spectroscopy. However, it suffers from its inherent weakness in scattering efficiency, leading to long data collection times and high sample concentration requirements. Dramatic increases in ROA intensity can be obtained through mixing of electronic transitions with the vibrational transitions generating Raman and ROA spectra. We briefly discuss how observations of such enhancements are providing a new perspective on molecular behavior.

6.3.1  Surface-enhanced ROA A potential solution to this limitation in ROA scattering intensities is provided by application of the surface plasmon resonances that give rise to surface-enhanced Raman scattering (SERS), in order to enhance the corresponding ROA signal. However, reliable measurements of surface-enhanced Raman optical activity (SEROA) has generally proven to be difficult. A recent review by Ostovar pour and Blanch [76] discussed the challenges involved in attempts to measure reliable SEROA. In summary, controlling the spectral artifacts routinely observed during SEROA experiments was very challenging since the reflection of electromagnetic waves from metal surfaces can modify circularly polarized light and so create elliptically polarized light, which in ROA measurements lead to spectral artifacts. Abdali et al. reported SEROA spectra of two resonant proteins, cytochrome c and myoglobin, as well as of a nonresonant peptide, Met-enkephalin [77–79]. While reasonable signal-to-noise levels were obtained by the authors, these molecules did not have enantiomers to verify the authenticity of the reported spectra. For any chiroptical technique, the standard proof that a signal is real and not an artifact is the observation of mirror image band responses for the two enantiomers. As no enantiomers were available for these proteins or the peptide, it is difficult to verify these spectral features as being from the SEROA effect. As no other SEROA spectra have yet been reported for peptides or proteins, presently we have no other reference with which to compare these spectra.

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Later, Osinska et al. reported the measurement of mirror image SEROA spectra of l- and d-cysteine using an electrochemically roughened solid silver-based system [80]. Although this was an interesting observation, the authors reported that SERS spectra of l- and d-cysteine for the same experiment could not be observed. Since any SEROA measurement must logically be weaker than the corresponding SERS measurement, it is possible that the authors measured solution-phase ROA, as sample concentrations were very high in their study, complicated by large reflection-based signals from the metal surface. Owing to the complexity of the SERS enhancement process, it is important to control the experimental conditions such as colloid type, analyte concentration, aggregating agents, and pH as they have a direct effect on obtaining not only reliable SERS but also any associated SEROA spectra [81]. The time-dependent nature of the aggregation process also has a significant effect on the measured SEROA spectra. Control of these factors led to the first definitive confirmation of SEROA by Ostovar pour et al. [82]. This was achieved by using a hydrophilic polyacrylic acid “polycarbopol” polymer as a stabilizing medium to control nanoparticle aggregation since this polymer has small Raman and SER cross-sections, minimizing interference from background signals. The feasibility of obtaining resonant SEROA, or SERROA, has been recently confirmed through the use of single plasmonic nanomaterials. As plasmonic substrates offer huge electromagnetic fields for SERS measurements, fluorescent dye-labeled silver colloids were investigated to determine if they could provide the same level of enhancement for chiroptical spectroscopy. Ostovar pour et al. [83] synthesized silver silica nanotags with achiral plasmonic nanostructures. Chirality was induced into the achiral plasmonic surface of the substrate by binding to either l- or d-enantiomeric analytes. Interestingly, a chiral influence on the SERRS spectrum of the achiral benzotriazole dye was observed. This observation of chiroptical behavior monitored by dye-labeled nanotags was suggested due to long range radiative electromagnetic coupling between the surface plasmons generated by these nanotags and the surrounding chiral molecules.The chiral perturbation existent in the presence of achiral chromophores induced optical activity to the metal nanoparticles and the dye, both of which act as agents to enhance the Raman signal, the former via surface plasmons and the latter via electronic resonance. The experimental results were verified by computational modeling of the chiral response of the nanoplasmonic

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s­ystems once a chiral analyte was introduced. This study verified that quantum mechanical modeling is likely to be important for further developing our understanding the principles and parameters responsible for SEROA. The propagation of plasmons in metallic nanostructures has also been employed to enhance ROA for selected vibrational modes. Sun et al. [84] have reported enhancing ROA using propagating surface plasmons in chiral Ag nanowires.They used chiral fmoc-glycyl-glycine-OH molecules as analytes to study the local surface plasmon-enhanced ROA. The corresponding enhanced ROA spectra was very different to normal ROA and since the analytes they used did not have enantiomers it is currently difficult to confirm that the signal is from the molecules rather than the chiral Agnanowires. A comparison of experimental SEROA results with calculations was performed using matrix polarization theory where a model of the SEROA spectra of ribose and cysteine molecules was generated [85]. These findings showed a strong dependence of enhancement on the distance between molecules and the metal surface along with dependence of the CID ratios on distance and rotational averaging. Results from this study confirmed that maximum enhancement can be obtained by colloidal aggregates rather than from the asymmetry of individual particles and validated the experimental observation of SEROA from l- and d-ribose reported previously [83]. The importance of controlling the colloid–sugar distance was emphasized, which was also important in the experimental set up for the SEROA measurements on ribose. Recently, Chuntonov and Haran [86] have shown that the combination of a nanoparticle trimer with a single molecule can lead to maximizing of the SEROA signal. These researchers have presented a simple model that can be used to aid future design of devices to generate light with well-defined polarization once it couples with plasmons from metal nanoparticles.

6.3.2  Enhancement of ROA by other approaches Aggregation-induced resonance Raman optical activity (AIRROA) is a new phenomenon which has developed very recently to enhance ROA signals using the resonance resulting from supermolecular aggregation [87–89]. Observation of strong ROA intensity has been reported for J-aggregates of astaxanthin (AXT) [88]. ROA enhancement using this method is potentially very useful as a general approach since it led to resonance ROA for molecules that generally display low intrinsic ROA signals.

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Another approach to enhance the ROA effect is to use metal complex systems. Several studies investigated the effect of both chiral and nonchiral metal complexes on chiral structures and how this gives rise to resonance effects in ROA spectra [89–92]. These studies show that ROA experiments on transition-metal containing systems are a very promising approach for the probing of optical activity, the manifold of chiralities, and electronic and nuclear structures of metal complexes.

6.4  RECENT ROA INSTRUMENTATION DEVELOPMENTS Recent advances in ROA instrumentation have enabled studies of biological molecules which was not previously feasible. Unno et al. [40] have developed a near-infrared excitation ROA spectrometer and reported the first measurement of near infra-red ROA spectra for a light-driven photon pump, bacteriorhodopsin. This instrument demonstrated the potential of near-infrared ROA measurements for studying cofactor molecules in enzymes and photoreceptors [93,94].The same group has recently reported ROA measurements for orange carotenoid protein that provided information on the molecular mechanism of photoactivation [95]. An alternative approach taken by Hiramatsu et al. [96,97] to improve ROA signals was to employ a nonlinear Raman process, namely coherent anti-Stokes Raman scattering (CARS). ROA measurements with CARS had been previously proposed by Bjarnason et al. [98] and Oudar et al. [99]. CARS–ROA has potential advantages over spontaneous ROA due to its applicability to fluorescent samples. However, the chiral CARS–ROA signals measured in this first study were around six orders of magnitude less than typical nonchiral CARS signals. Despite the low intensity observed, this was the first report of CARS–ROA, as well as the first ROA observation with a pulsed laser source. The authors were able to make use of the phase sensitivity of CARS spectral interferometry (iCROA) to minimize experimental artifacts for beta-pinene measured with excitation wavelengths of 532 and 1064 nm [97]. A backscattering ICP ROA instrument using deep UV excitation at 244 nm from a frequency-doubled continuous argon ion laser has been developed by Kapitan et al. [100] and is starting to provide data on both small chiral organic molecules and biomolecules. The extension of ROA into the UV region promises the long-sought capability of studying biomolecules, particularly proteins and nucleic acids, at low concentration.

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6.5  COMPUTATIONAL MODELING OF ROA SPECTRA The calculation of ROA spectra is an important aspect of the technique since successful simulations can provide the complete solution structure (including conformation, absolute configuration, conformational populations) and conformational dynamics of chiral molecules. Calculations of the ROA observables, which are usually based on the Placzek approximation, can proceed by several different approaches. Modeling of ROA spectra can proceed using approaches such as the bond-polarizability (valence-optical) model, in which the molecule is decomposed into bonds or groups supporting local internal vibrational coordinates [3,101]. However, the approximations that are inherent to these models limit the ability of these calculations to reproduce experimental data accurately. Such models can, however, provide valuable physical insights into the generation of ROA bands [3], which is often not obvious from the computationally superior ab initio approach, now the method of choice for analyzing the details of ROA spectra [1,2,102]. An ab initio quantum-chemical method, based on calculations of ααβ, G′αβ , and Aαβγ in a static approximation, and the dependence of these property tensors on the normal vibrational coordinates, was first developed by Prasad Polavarapu in the late 1980s [20,103]. Although the first such calculations of ROA spectra did not reach the high levels of accuracy now attainable, they nonetheless proved valuable. For example, the absolute configuration of the archetypal chiral molecule CHFClBr, unknown for over a century, was reliably determined from a comparison of the experimental and ab initio theoretical ROA spectra [104]. Subsequent approaches produced significant improvements in the quality of ab initio ROA calculations. By including basis sets containing moderately diffuse p-type orbitals on hydrogen atoms, Zuber and Hug [105] demonstrated that ab initio ROA calculations of a similar high level of quality to those of VCD may be achieved. This is significant because, in many systems including small chiral organic molecules but especially in proteins (vide infra), it is found that vibrations with large contributions from C–H and N–H deformations often generate large ROA signals. Interest in ROA calculations among theoreticians is growing, with a plethora of publications providing further refinements and applications to ever-larger systems. Currently, ROA calculations may be performed using the DALTON [106] and Gaussian [107] software packages: the 09 version of Gaussian implements an analytic time-dependent protocol for the calculation of the ROA property-tensor derivatives, both Hartree–Fock (HF) and DFT, resulting in

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an order-of-magnitude increase in speed of the calculations. Further details, including many more references, may be found in some recent reviews of ROA computations [2,108–112]. As an example of what may routinely be achieved, Fig. 6.7 presents experimental and simulated Raman and ROA spectra of 1-phenylethanol.

Fig. 6.7  Experimental and calculated backscattered SCP Raman and ROA spectra of (+)-(R)1-phenylethanol (adapted from Ref. [113]). The absolute intensities are arbitrary, but the dimensionless ratios (IR + IL)/(IR  −  IL) are meaningful and may be compared between experimental and simulated spectra. Abbreviations: SCP, scattered circular polarization; ROA, Raman optical activity.

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This was taken from a study which revisited the samples used for the first observations of ROA, namely both enantiomers of 1-phenylethanol and 1-phenylethylamine, using state-of-the-art instrumentation and calculations [113]. The experimental backscattered SCP Raman and ROA spectra of both enantiomers of 1-phenylethanol are displayed in Fig. 6.7, together with the corresponding simulated Raman and ROA spectra of the (+)-(R)enantiomer, which closely reproduces the experimental spectra. It is often necessary to allow some degree of conformational freedom in order to simulate the observed Raman and ROA bandshapes, something especially important for many biomolecules [114,115]. In the present case, a Boltzmann average over several hundred rotameric conformations of the phenyl and −OH groups was taken. Clearly, assignments of absolute configurations are completely secure from ROA results of this quality. While there have been many other calculations of ROA spectra reported for small- and medium-sized molecules, we will limit the following discussion to a brief history of progress in the last decade and some examples of how computational modeling can reveal insights into experimental results on biomolecules. A significant advancement in the computation of ROA spectra came from the work of Liégeois et al. in 2007 [116]. Early ROA computations were hampered by the numerical derivative method for the calculation of the electric dipole–magnetic dipole polarizability tensor, which they addressed with the introduction of an analytical time-dependant HF algorithm that calculated derivatives of this tensor with respect to Cartesian coordinates. The introduction of this algorithm allowed for the first time the fully analytical evaluation of the necessary frequency dependant invariants for ROA calculations.

6.5.1  Standard computational approaches today DFT is now widely used in ROA computations. Ruud et al. [117] were the first to report ROA spectra calculated using DFT, with the popular B3LYP functional. Results were compared to those calculated using HF and a sum over states expansion, with DFT offering significant improvement in the calculation of harmonic vibrational frequencies. DFT also resulted in improvements in the calculation of the ROA CID, although to a lesser degree. Despite many advances, calculations can still be very expensive due to the calculation of the polarizability and optical activity tensors. Calculations are particularly time-consuming for larger molecules and for very large

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systems full evaluation is not practical at all. As a result of this, several groups have aimed to apply approximations to allow for more efficient calculation of spectra for large molecules. Bour et al. [118] proposed one of the most widely used approximations in the Cartesian coordinate tensor transfer (CTT) technique. CTT constructs the Hessian matrix and ROA intensities for a large molecule by calculating property tensors for a set of smaller overlapping fragments. This technique has been used to study molecules beyond the scope of a full quantum mechanical simulation, such as for the protein insulin [36] and the cyclic dodecadepsipeptide valinomycin [119]. The choice of level of theory for performing ROA calculations is of great importance with several studies addressing this. The choice of basis set is of particular importance, the strong basis set dependence was noted early on by Helgaker et al. [120], as such this is addressed foremost. The choice of method has also been studied, with the early work of Ruud et al. [117], noting that DFT outperforms HF. Reiher et al. [121] compared three different DFT functionals, representing local density approximation, generalized gradient approximation, and hybrid functionals, and reported that the hybrid functionals were superior. Danecek et al. [122] also explored method dependence by comparing a selection of common functionals and reported seven that gave the best results when compared to experimental data, BHandHLYP, MPW1PW91, B3P86, B3PW91, PBE0, P98, and B3LYP. Today, the most widely used functional in modern calculations is B3LYP, which has been shown to be effective in calculating ROA spectra of a variety of different biological and chemical systems, as well being considered one of the best options in terms of accuracy versus computational expense. Zuber and Hug [105] observed the importance of diffuse functions in basis sets used for the calculation of ROA scattering tensors and as such proposed a new basis set, rDPS. This basis set was formed from the relatively small 3-21++G, with semidiffuse p functions, with an exponent of 0.2 on all hydrogens. In benchmark studies, rDPS performed remarkably well against much larger basis sets, including aug-cc-pVDZ. Zuber and Hug noted that while rDPS offers very good qualitative and quantitative results for ROA tensor computation it is not suitable for geometry optimization or harmonic frequency calculations, as such force field calculations should be undertaken at a different and more appropriate level of theory. This problem was considered by Cheeseman and Frisch, who explored the basis set dependence for the optimization and force field calculations from the Raman and ROA tensor invariants [123]. This study was the first

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carried out after the advancement to fully analytical derivatives for the ROA tensor invariant and as such this paper explored the two fully analytical approaches available, known as the onestep and the twostep. The two-step procedure calculates the optimization and force field at a different level of theory to the ROA calculation, whereas the one-step procedure calculates everything at the same level of theory.The authors showed that the basis set requirements differ for force field and ROA calculation. Basis sets for ROA calculations require diffuse functions, as established in the other studies referenced above, while the force field and optimization calculations generally do not. A general set of rules were established, which are recommended to be followed by the reader, and the authors recommended that the twostep procedure should be used for greatest efficiency. It was also recommended for small system sizes that the basis sets cc-pVTZ and aug-cc-pVDZ be used for the force field and ROA calculations, respectively. When dealing with larger system sizes, the basis sets 6-31G* and rDPS were recommended for force field and ROA calculations, respectively. The choice of level of theory is of great significance when calculating ROA spectra but another aspect which must also be considered is the modeling of solvent. It is often essential to include the solvent in some form in ROA calculations, this is especially prevalent when studying biological systems as their vibrational behavior and conformation is often significantly influenced by the solvent environment, leading to drastic differences in certain ROA bands [124,125]. Solvent can be included in ab initio calculations in two ways, by using implicit models or by adding explicit solvent molecules. Implicit solvent models represent the solvent as a uniform polarizable medium with a homogenous dielectric constant and with the solute placed within a cavity. This approach is widely used in computational chemistry with the commonly used polarizable continuum model (PCM) and COnductor-like Screening MOdel (COSMO) models and adds relatively little computational cost compared to gas-phase calculations. PCM has, in the past, been shown to be somewhat effective for ROA computations on small peptides, but has also been shown to perform poorly for systems with high conformational flexibility and those with strong solvent interactions [111,126].

6.5.2  Insights gained from ROA calculations The development of explicit solvation models for ROA computation has progressed greatly in recent years with a greater number of publications. One subject that has witnessed great success from the use of explicit solvent

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models is the study of carbohydrates, where until recently there was a gap in the literature. Gas-phase and implicit solvent calculations have been shown to give very poor experimental agreement for a range of monosaccharides. Early studies on 1,6-anhydro-β-d-glucopyranose by Luber and Reiher showed that even a few explicit water molecules led to marked changes in the calculated spectrum [127]. Cheeseman et al. [126] used a combination of explicitly solvated MD simulations with quantum mechanics/molecular mechanics simulations (QM/MM) to compute the ROA spectrum of β-d-methylglucose to a high level of agreement with experiment. This work set the basis for further iterations on this MD QM/MM protocol to progress ROA computations of monosaccharides to near perfect agreement with experiment [128,129]. Improvements made when utilizing explicit solvation can also be obtained when calculating ROA spectra for small organic molecules [130] and small peptides [131,132]. Recently, Gorecki [133] used ROA, along with the three other chiroptical methods of ECD, VCD, and optical rotatory dispersion (ORD), to carry out a configurational and conformational study on the influenza drug Tamiflu. The use of multiple, optically active spectroscopic methods along with computational validation gave results that were in very good agreement with the reported details of a single crystal X-ray structure. It was, however, noted that it was important to consider the effect of solvent as many diverse conformers were noted to be stable in several solvents and X-ray studies on very flexible molecules may not be suitable to explore all possible conformers and methods such as ROA, which can provide information on conformational dynamics, can be excellent alternatives. One of the most important publications for the ROA field was the determination of the absolute configuration of chirally deuterated neopentane [134]. Hug and coworkers assigned the absolute configuration of this molecule which is chiral as a result of a dissymmetric mass distribution, an achievement of which no other structural technique had proven successful, which exhibits the incredible sensitivity possible with ROA. There have been many computational ROA studies on amino acids and in the last decade peptides and proteins have attracted considerable interest. These larger systems are much more computationally expensive and as a result other approximations, such as the CCT transfer, need to be incorporated. Bour and coworkers have published several important studies on peptide and protein computational ROA. They successfully modeled spectra of the cyclic peptide valinomycin containing 12 amino acids [119] and insulin [35], to date the largest system studied by this technique, containing

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51 amino acids. For a more in-depth assessment of ROA calculations on amino acids and peptides, the reader is directed to Refs. [110,135,136,137]. The study of carbohydrates by computational ROA has seen a burst in recent years. As mentioned above, gas-phase and implicit solvation models have proved ineffective for calculating the ROA spectra of even simple monosaccharides.The work of Cheeseman et al. [126] was the first to provide a methodology that accurately re-creates experimental spectra for monosaccharides, with methyl-β-d-glucose as a test case. The importance of the explicit solvation models was outlined above but another vital facet that was also covered by this protocol is the modeling of conformational dynamics. When carrying out calculations to reproduce experimental ROA ­spectra, the ratios of conformers in solution needs to be considered. Calculations in the gas phase and with implicit solvation models include multiple conformers by Bolztmann weighting spectra calculated for the individual conformers, based on their calculated free energies. These individual conformers are found by using software that performs conformational searches and gives the most likely low energy conformers based on fast molecular mechanics methods or by manual rotation around dihedral angles.This method has disadvantages as the energies used for the spectral weightings are susceptible to the errors induced when explicit solvent is not taken into account and as a result the conformer ratios may not be accurate representations of the molecule’s behavior in solution. To overcome this problem, Cheeseman et al. [126] used MD simulations to account for the conformational behavior in solution. In this test case, the conformer ratios of the important w dihedral angle were well established from other extensive studies on glycopyranisides [138] and as such constrained simulations were carried out on the two main conformers to fix that dihedral angle. A selection of snapshots was taken from the two MD trajectories to sample the conformational flexibility of the remaining hydroxyl groups and the final spectrum was generated from weighting the averaged spectra for each of the two main conformers, to give very good agreement with experiment, as shown in Fig. 6.8. Mutter et al. [128] used a similar method as above to model d-glucuronic acid and N-acetyl-d-glucosamine but took a different approach for modeling the conformational dynamics of the systems. The two monosaccharides in question lacked definitive conformational data and as such the authors used the conformer ratios obtained from the MD simulations to weight individual snapshot spectra. This approach proved very successful for reproducing the experimental spectra of the two molecules and had the added advantage that a minimum number of snapshots were used.

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Fig. 6.8  ROA spectra of methyl-β-d-glucose. (A) Experimental, (B) calculated spectrum in the gas phase, (C) calculated spectrum using PCM, (D) calculated spectrum using combined QM/MM and MD simulations. Adapted with permission from Ref. [126]. Abbreviations: ROA, Raman optical activity; PCM, polarizable continuum model.

The conformational analysis carried out resulted in only statistically relevant snapshots being extracted from the MD trajectories. The calculation for d-glucuronic acid gave excellent agreement with experiment, from a spectrum formed from only six MD snapshots, whereas other studies have reported needing more than a hundred snapshots when performing random sampling [40,130]. More recently, Zielinski et al. [129] used the above protocol to study β-d-xylose. This study further refined the conformational analysis and resulted in near perfect agreement between the experimental and theoretical spectra. The size dependence of the explicit solvation shell was also benchmarked in this study and found that adding water molecules beyond an approximate boundary of 10 Å from the center of β-d-xylose had little significance on the calculated ROA spectrum.This result not only allows for

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increased efficiency in future calculations, but also can be used to provide greater insights into conformational dynamics and potential new avenues of research into areas such as molecular crowding and the greater effect of water molecules in enzyme pockets. A subject that has attracted less interest in the literature, but is of significance for guiding future developments, is the study of chiral transition metal complexes. Luber and Reiher [89,90] have presented studies on octahedral cobalt and rhodium complexes, exhibiting axial chirality.The authors showed that ROA can be used to distinguish not only the local chirality of ligands, but also the axial chirality from ∆/Λ configurations, although their work did not explore experimental studies on chiral transition complexes. More recently, Humbert-Droz et al. [139] reported a combined experimental and theoretical study on the chiral transition metal complex tris(ethylenediamine)-rhodium(III). Very good experimental agreement was found for particular conformers, although these conformers did not correspond with the most energetically favored ones from the calculations and the origin of the observed bands was explored and vibrational modes assigned. Explicit solvation models were also examined in this study using ab initio MD and these were shown to further improve the agreement between experiment and theory, emphasizing the importance of conformational dynamics for nonbiological systems. Wu et al. [140] in a recent study used ROA, along with VCD and ECD, to study a chiral bipyridine–europium(III) complex.This work was the first to take ROA into the lanthanide elements, although the calculation aspect focused on the free ligands, due to the computational problems arising from relativistic effects when high atomic number elements are used. The authors noted that ROA provided the most sensitive probe of complex chirality for this molecule and that short measurement times and very low concentrations were sufficient.

6.6  CONCLUDING REMARKS: FUTURE OPPORTUNITIES A sufficient body of experimental data has now been accumulated and analyzed to demonstrate that ROA can provide new and incisive information about a vast range of chiral molecular systems that is highly complementary to that obtained from other techniques. The many applications of ROA to biomolecular science that are reviewed in this article provide the mere glimpse of what is now possible. Some promising future opportunities are outlined below.

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ROA should be particularly valuable for the determination of protein structure and function in the postgenomic era, especially for the many proteins specified by a genome, be they folded, unfolded, or partially unfolded, which are inaccessible to X-ray and NMR methods. ROA will be useful even for those proteins that do crystallize since it provides fold information, albeit not at atomic resolution, and could be valuable for solving X-ray crystal structures by molecular replacement methods because ROA data will identify those proteins with the most structural similarity to the proteins under study. The apparent lack of an upper size limit to the structures that may be studied with ROA, together with its short timescale which facilitates studies of conformational dynamics in aqueous solution, offers significant advantages over NMR for some applications. Furthermore, since many of the thousands of known viruses are likely to be inaccessible to the key techniques of structural biology, structural virology is a particularly promising area for ROA on account of the ease with which the folds of the major coat proteins may be “read off ” from their ROA spectra and differences of detail identified between coat proteins of different viruses having the same basic fold and also due to its ability to provide information about the nucleic acid core and protein−nucleic acid interactions. It may also be possible to obtain information about the carbohydrate structure and dynamics of viral envelope glycoproteins. In addition to the determination of the absolute configuration of c­ hiral drugs, the pharmaceutical industry is becoming increasingly aware of the value of ROA for characterizing and monitoring glycoprotein biopharmaceuticals since it is uniquely sensitive to the structural and conformational details of both the polypeptide backbones and carbohydrate chains of the intact biomolecules in aqueous solution. Now that patent protection is expiring on the first tranche of biopharmaceuticals, companies are working to produce “biosimilars” which reproduce the overall structure, function, and clinical characteristics of the original product. Biological drugs are produced in living cells, not chemical plants, so copies can at best only be highly similar to the original. Since the hurdles imposed by regulatory authorities will be much higher for biosimilars than for generic versions of small-molecule drugs, ROA is expected to be valuable for their characterization and validation on account of its unique ability to detect subtle structural and conformational differences compared with the original.

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Two-dimensional spectroscopic correlation methods, based on linear relationships between spectral data obtained under a perturbing influence such as change of temperature or pH, have the potential to increase the amount of information that may be extracted from ROA spectra. Spectral resolution is enhanced by spreading bands over a second dimension which can provide, inter alia, unambiguous band assignments. Studies of the α-helix to β-sheet transition of poly(l-lysine) as a function of temperature [101] and of the α-helix to disordered transition in poly(l-glutamic acid) as a function of pH [102] have demonstrated the value of two-dimensional ROA measurements in biomolecular science. The further development of computational simulations of ROA spectra will be especially important, in particular, the dramatic influence of water solvation, since this will be essential in order to exploit fully the wealth of information they can provide. A recent review of the progress that has been made in recent years in achieving reliable and accurate simulations also highlights how the remarkable sensitivity of ROA to molecular structure and dynamics can be exploited in force field design and vibrational analysis [58]. Despite the exciting new types of ROA experiments outlined earlier, “conventional” ROA measurements with transparent samples using visible laser excitation is expected to remain the simplest and most informative strategy for most routine studies of biomolecules in aqueous solution. Further reductions in measurement times, sample concentrations, etc. remain highly desirable to widen the realm of applications. From the first instruments in the early 1970s, ROA instrument development has fed voraciously on new optical−electronic technology, especially with regard to detectors for use in optical astronomy and this trend is expected to continue from advances in photonics for astronomical applications [41]. With apologies to the motto of the Royal Air Force, namely per ardua ad astra, we might finish with the exhortation: per astra ad ROA!

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CHAPTER 7

Chiral Molecular Tools Powerful for the Preparation of Enantiopure Compounds and Unambiguous Determination of Their Absolute Configurations by X-Ray Crystallography and/or 1H NMR Diamagnetic Anisotropy Nobuyuki Harada Tohoku University, Sendai, Japan

7.1 INTRODUCTION It is well recognized that molecular chirality is essential for life processes and that most biologically active compounds controlling physiological functions of living organisms are chiral. Hence, in the structural study of biologically active compounds, including natural products, determination of absolute configuration (AC) becomes the first major issue.The second issue is the chiral synthesis of natural products and biologically active compounds that become pharmaceutical targets and how efficiently desired enantiomers can be synthesized with 100% enantiopurity or enantiomeric excess (% ee). Furthermore, studies on chiral functional molecules and molecular machines, such as the light-powered chiral molecular motor developed in our laboratory, have been rapidly progressing in recent years. Therefore, the unambiguous determination of the AC of chiral compounds as well as their chiral syntheses are of vital importance in the field of material science. We have recently developed chiral carboxylic acids as novel molecular tools proved to be powerful for enantioresolution and simultaneous determination of AC of various alcohols. These chiral molecular tools are very useful for the facile synthesis of enantiomers with 100% ee and also for the AC determination by X-ray crystallography and 1H NMR diamagnetic anisotropy. The methods using these chiral molecular tools have been Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00007-0 Copyright © 2018 Elsevier B.V. All rights reserved.

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s­uccessfully applied to various compounds and their methodologies and applications are explained throughout this chapter.

7.2  METHODOLOGIES FOR DETERMINING AC AND THEIR EVALUATIONS The methodologies for determining the ACs of chiral compounds are classified into the following two categories.

7.2.1  Nonempirical methods for determining ACs of chiral compounds As methods of this category, there are the Bijvoet method of X-ray crystallography [1] and circular dichroism (CD) exciton chirality method [2]. These powerful methods provide nonempirical determination of a target molecule’s configuration without the knowledge of the AC of reference compounds. Namely AC of each compound can be independently determined. In X-ray crystallography, since the anomalous scattering effect of heavy atoms can be measured very accurately under proper conditions, the absolute stereostructure obtained is unambiguous and reliable. In addition, the relative configuration of the molecule can be projected as a three-dimensional structure and therefore the method has been employed extensively. However, the X-ray method needs single crystals of suitable size and quality good for X-ray diffraction experiments and so the problem is how to obtain such single crystals. So, in some cases, it needs time and efforts to obtain such single crystals. Recently, the Flack parameter has been employed as an extension of the Bijvoet method [3]. The method using the Flack parameter is convenient for determining the ACs of chiral compounds, but it is important to check whether the crystal used in the X-ray analysis is enantiopure or not. The CD exciton chirality method [2] is also useful because the AC can be determined in a nonempirical manner and it does not require crystallization. Furthermore, chiral chemical and biological reactions are traceable by CD and hence even the ACs and conformations of unstable compounds can be obtained by this method. When applying this CD exciton chirality method, it is important to understand the theory and mechanism of this method and also to select chromophores [2].

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7.2.2  Relative methods for determining AC using an internal reference with known AC AC can be obtained by determining the relative configuration at the position of interest against a reference compound or substituent with known AC. So, this methodology is called the relative method. A typical example is the X-ray crystallography taken after the introduction of a chiral auxiliary with known AC. For example, we have used (1S,2R,4R)-(−)- camphorsultam as a chiral auxiliary for enantioresolving racemic carboxylic acids and also for determining their ACs by X-ray crystallography [4–7]. As the extension of this methodology, we have first developed camphorsultam dichlorophthalic acid (CSDP acid) (1S,2R,4R)-(−)-1 and 2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid) (S)-(+)-2 as shown in Fig. 7.1.These chiral acids are very powerful for enantioresolution of racemic alcohols and simultaneous determination of their AC by Xray crystallography and/or 1H NMR diamagnetic anisotropy. The principle and applications of the method using these chiral acids will be discussed throughout this chapter. The principle of this method is simple. For example, alcohol is esterified with chiral acid (−)-1 yielding ester and if the ester is obtained as single crystals, the stereostructure can be determined by X-ray crystallography.

Figure 7.1  Enantioresolution and determination of absolute configuration of alcohols using chiral carboxylic acids. (Adapted with permission from Ref. [8].)

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In this case, the AC of the alcohol part can automatically be determined by using the acid part as an internal reference of AC. Consequently, the samples do not need to contain heavy atoms for anomalous scattering effect.The result obtained is very clear, even when the final R-value is not small enough due to the small size or poor quality of the single crystal.The AC can be determined with certainty, even if only the relative configuration is obtained. A variety of methods to link an internal reference to the target molecule have been developed. For example, there are ionic bonding such as conventional acid–base salts, covalent bonding such as esters or amides, and the use of recently developed inclusion complexes [9]. These relative X-ray methods are expected to find widespread application. Recently, the proton nuclear magnetic resonance (1H NMR) diamagnetic anisotropy method has often been employed as the relative method and is useful for the study of the AC of natural products. In particular, to determine the ACs of secondary alcohols, the so-called Mosher acid, α-methoxy-α-(trifluoromethyl)phenylacetic acid, MTPA acid (S)-(−)-3, was developed (Fig. 7.2) [10]. Similarly, α-methoxyphenylacetic acid, MPA acid (S)-(+)-4 was useful as studied by Trost and coworkers [11]. Later, the MTPA acid method was generalized as the advanced Mosher method by Kusumi et al. [12,13], which has been widely applied to various natural products. In these methods, the ACs of chiral auxiliaries, such as MTPA acid and MPA acid, have been already determined and the preferred conformation of the esters formed from chiral secondary alcohols and MTPA or MPA acid is rationalized. In addition, the aromatic substituent (phenyl group) generates a diamagnetic anisotropy effect due to the ring current effect induced under the external magnetic field and hence the proton NMR signals of the alcohol moiety facing the phenyl group in the preferred conformation are moved to a higher magnetic field (high field shift). By observing such 1H NMR diamagnetic anisotropy effect, that is, ∆δ values, the AC of alcohol part can be determined. One problem of this method is

Figure 7.2  Chiral acids with 1H NMR diamagnetic anisotropy effect.

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that it is based on the assumption of preferred conformation of molecules in solution. Namely, this is an empirical rule and hence it is important to check carefully the obtained data and also to compare the result with similar application results. The AC can be determined by chemical correlation or comparison of optical rotation, [α]D, and/or CD spectrum with that of reference compounds with known AC. Although the latter CD method has been frequently employed, a careful selection of reference compounds is necessary for reliable AC determination, because there were some examples where the final AC assignment was wrong due to the selection of unsuitable reference compounds of CD spectra.

7.3  METHODOLOGIES FOR CHIRAL SYNTHESIS AND THEIR EVALUATIONS The task after determination of AC is the synthesis of chiral compounds. The practical methods to synthesize chiral compounds are roughly divided into two categories, each of which is further divided and has advantages and disadvantages as described below. In this chapter, “chiral synthesis” includes not only the so-called asymmetric synthesis, but also enantioresolution. In addition, the method in which covalently bonded diastereomers are formed using a chiral auxiliary followed by HPLC separation and recovery of the target compound is also defined as enantioresolution.

7.3.1  Enantioresolution of racemates Type (a): In this method, a chiral auxiliary is ionically bonded to racemates as seen in the conventional cases of acid–base combination and a mixture of diastereomers formed is subjected to fractional recrystallization to obtain enantiopure compounds. This method is also applicable to the inclusion complexes formed by hydrogen bonding [9]. The critical point is whether the diastereomer can be obtained with 100% enantiopurity through fractional recrystallization or not. It should be noted that recrystallization does not always afford 100% enantiopure diastereomer. However, if this method is successful, it is suitable for the mass preparation of chiral compounds. Type (b): In this method, a chiral auxiliary is covalently bonded to racemates to produce a diastereomeric mixture, which is separated by conventional HPLC on silica gel or other methods to enantiopure diastereomers, and then the chiral auxiliary is cleaved off (Fig. 7.1). Hence, this method

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can yield an enantiopure compound. The point is whether diastereomers can clearly be separated by HPLC or not. If a clear separation is achieved, each diastereomer obtained is enantiopure, and the target compound after cleavage of the chiral auxiliary is also 100% enantiopure. It is advisable to use a chiral auxiliary that can be cleaved off easily. Type (c): This is an excellent method where racemates are directly enantioseparated by HPLC or GC using columns made of chiral stationary phases and a number of reports have been published [14,15]. The question is again whether racemates are clearly separated into two enantiomers or not. If a clear separation is achieved, 100% pure enantiomers are obtained by this method as well.The method is convenient and suitable for analytical separation, as it does not require derivatization. In general, chiral columns are expensive and are, therefore, mostly used for analytical purposes. However, in some cases, mass separation is conducted on an industrial scale to obtain chiral compounds such as pharmaceutical materials. It should be noted that AC determination by the elution order is difficult, as there are many exceptions. Type (d): This is a unique method where racemates undergo an enzymatic or asymmetric reaction to yield enantiomers by the kinetic resolution effect. In particular, high stereoselectivity of the enzymatic reaction leads to high enantiopurity [16]. However, care should be taken, since the method does not always yield products of 100% enantiopurity.

7.3.2  Asymmetric syntheses Type (a): This is a highly efficient and powerful method to obtain chiral products by the action of a chiral reagent or chiral catalyst on achiral compounds. Being a well-known method, many eminent reviews have been published for these asymmetric syntheses and so no further explanation is required here. The problem with this method is that the products obtained are not always enantiopure. Furthermore, it is generally difficult to determine the AC of the products based on the reaction mechanism. Accordingly, an independent AC determination by the methods described in this chapter is suggested. Type (b): There is also another method to obtain chiral compounds such as by enzymatic reaction on achiral or meso compounds. The asymmetric reaction of a meso compound by an enzyme is particularly interesting and is defined as the desymmetrization reaction. In this case too, the enantiopurity is not always 100% and the AC must be determined separately.

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7.4 CAMPHORSULTAM DICHLOROPHTHALIC ACID, CSDP ACID (−)-1, AND CAMPHORSULTAM PHTHALIC ACID, CSP ACID (−)-5, USEFUL FOR ENANTIORESOLUTION OF ALCOHOLS BY HPLC AND DETERMINATION OF THEIR ACS BY X-RAY CRYSTALLOGRAPHY The author considers that the most reliable and powerful method for determining the AC is the X-ray crystallography of compounds containing a chiral auxiliary with known AC as the internal reference, as described above. Namely the AC of the point in question can be unambiguously determined from the X-ray ORTEP drawing showing a relative stereochemistry, because the AC of the chiral auxiliary is already known.Therefore, it is rather easy to determine the AC. The author also considers that the highly efficient method for preparing an appropriate amount of various chiral compounds with 100% enantiopurity in a laboratory scale is the enantioresolution of type (b) in Section 7.3.1, as illustrated in Figs. 7.1 and 7.3. In this method, a chiral auxiliary is covalently bonded to racemates and the obtained diastereomeric mixture can be separated by conventional HPLC on silica gel. If the chromatogram shows a base-line separation, the diastereomers obtained are enantiopure. This method is characterized by a clear and efficient separation even with a small amount of sample, compared to the fractional recrystallization method described in type (a) in Section 7.3.1. As chiral auxiliaries satisfying these two requirements, the author and coworkers have designed and prepared the chiral molecular tools, camphorsultam dichlorophthalic acid, CSDP acid (−)-1, and camphorsultam phthalic acid, CSP acid (−)-5, connecting (1S,2R,4R)-2,10-­ camphorsultam with 4,5-dichlorophthalic or phthalic acid and have applied those chiral tools to various compounds (Fig. 7.3) [17–19,21–24,47,51].

Figure 7.3  Design of chiral molecular tools, CSDP and CSP acids containing 2,10-camphorsultam moiety.

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The 2,10-­camphor-sultam was selected because of its good affinity with silica gel used in HPLC, allowing good separation of two diastereomers. In addition, the sultam amide moiety is effective for providing prismatic single crystals suitable for X-ray diffraction experiment. Furthermore, the (1S,2R,4R) absolute stereochemistry of 2,10-camphorsultam established is useful as the internal reference of AC. In addition, the sulfur atom can be used as a heavy atom for the X-ray Bijvoet method. To connect the alcohols, an ester bond was chosen, because it could readily be formed and cleaved off. Accordingly, phthalic acids, especially 4,5-dichlorophthalic acid, were selected as a linker [17,18]. In telephthalic acid and succinic acid esters, the two chiral moieties are remote to each other in space. On the other hand, in phthalic acid ester, they are close enough to result in a stronger interaction. So, we have expected its diastereomeric recognition would be effective in HPLC (Fig. 7.3). The desired molecular tool, CSDP acid (−)-1, was synthesized by reacting (1S,2R,4R)- (−)-2,10-camphorsultam anion with 4,5-dichlorophthalic anhydride: acid (−)-1, mp 221 °C from EtOH; [α ]D20 −101.1 (c 1.375, MeOH). In a similar way, CSP acid (−)-5, was also prepared from (1S,2R,4R)-(−)-2,10-camphorsultam and phthalic anhydride: acid (−)-5, mp 184–187 °C from CHCl3; [α ]D20 −134.7 (c 2.218, MeOH). Compounds 1 and 5 should be formally called phthalic acid amides. However, here we adopt the common names, CSDP and CSP acids. These carboxylic acids were condensed with alcohol in the presence of 1,3-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) [17,18].

7.4.1  Application of the CSP acid method to various alcohols The following exemplifies a general procedure of this method. The CSP acid (−)-5 was allowed to react with (±)-(4-methoxyphenyl)phenylmethanol 12 using DCC and DMAP in CH2Cl2 yielding diastereomeric esters, which were separated by HPLC on silica gel: hexane/ EtOAc = 4:1; separation factor α = 1.1, resolution factor Rs = 1.13 (Fig. 7.4 and Table 7.1) [23]. The first-eluted ester 17a obtained was recrystallized from EtOAc giving large colorless prisms: mp, 172 °C. A single crystal of 17a was subjected to X-ray analysis affording the ORTEP drawing as shown in Fig. 7.4, from which the AC of the alcohol part was determined as S based on the AC of camphorsultam moiety used as an internal reference. The S AC of 17a was also determined by the heavy atom effect of a sulfur atom

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Figure 7.4  (A) Enantioresolution of alcohol 12 using CSP acid (−)-5 and determination of absolute configuration by X-ray crystallography. (B) ORTEP drawing of ester (S)-17a. (Reprinted with permission from Ref. [23].)

contained. Thus, the AC of the alcohol part was doubly established. The reduction of the first-eluted ester (S)-17a with LiAlH4 yielded enantiopure alcohol (S)-(−)-12. Other examples are listed in Table 7.1 and their structures are displayed below the table. The successful enantioresolution of various alcohols, determination of their ACs by X-ray analysis, and recovery of enantiopure alcohols listed in the table proved the effectiveness of this method. Compound 10 is an interesting example of atropisomeric and chiral substance studied by Toyota et al. In general, it is very difficult to determine the AC of such chiral compounds of atropisomerism based on steric hindrance. However, Toyota et al. [20] successfully solved this problem by application of the CSP acid method. In cyanohydrin 14 and amine 15, the diastereomeric separation and determination of their ACs were possible; however, there remains a problem how to recover enantiopure compounds 14 and 15, because the amide bond is not easily hydrolyzed in general.Amine 15 could be enantioresolved as the salt of (2R,3R)-(+)-tartaric acid [24], and its AC was established as (S)-(−) by this method. For compound 16, its AC was ­unambiguously ­determined

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Table 7.1  Enantioresolution of Alcohols and Amine by HPLC on Silica Gel Using (1S,2R,4R)-(−)-CSP Acid, and Determination of Their ACs by X-ray Crystallography Alcohol Solventa Rs X-ray AC of First Fr. Ref. α

6 7 8 9 10 11 12 13 14 15 16

H/EA = 3:1 H/EA = 5:1 H/EA = 4:1 H/EA = 7:1 DM/EA = 50:1 H/EA = 2:1 H/EA = 4:1 H/EA = 5:1 H/EA = 3:1 H/EA = 2:1 H/EA = 3:1

1.1 1.1 1.05 1.1 – 1.2 1.1 1.1 1.3 1.1 1.1

1.3 1.3 0.73 0.8 – 1.3 1.3 1.6 2.8 1.0 1.6

2nd Fr. 1st Fr. – – 1st Fr. – 1st Fr. 1st Fr. 1st Fr. 1st Fr. 2nd Fr.

R R R 3R,4R Msc aR,aR S R R S R

[17] [17] [5] [18,19] [20] [21,22] [23] [23] [24] [24] [17]

H, n-hexane; EA, ethyl acetate; DM, dichloromethane. Source: Reprinted with permission from Ref. [25]. a

by this internal reference method, in spite of the large R-value in X-ray analysis, owing to its poor crystallinity [17].

7.4.2  Application of the CSDP acid method to various alcohols We have then found that another chiral molecular tool, CSDP acid (−)-1, is much more powerful for enantioresolution of alcohols by HPLC on silica gel and also for providing prismatic single crystals suitable for X-ray analysis.

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As in the case of CSP acid (−)-5, this CSDP acid (−)-1 is also useful as an internal reference in determining AC by X-ray analysis. Moreover, CSDP acid (−)-1 contains two chlorine atoms as heavy atoms in addition to a sulfur atom, which leads to more efficient determination of AC by the anomalous scattering effect of heavy atoms. A typical example is illustrated in Fig. 7.5. Alcohol (±)-9 was condensed with CSDP acid (−)-1 in the presence of DCC and DMAP. The diastereomeric mixture of the two esters obtained was base-line separated by HPLC on silica gel: hexane/EtOAc = 7:1, α = 1.18, Rs = 1.06.The first-eluted ester 41a was obtained as silky and fine needle-like crystals when recrystallized from MeOH, but those were unsuitable for X-ray analysis. On the other hand, the second-eluted fraction 41b gave larger prisms good for X-ray analysis when recrystallized from EtOAc. The AC of ester 41b was unambiguously determined to be (3S,4S) using the 2,10-camphorsultam part as an internal reference and also the heavy atom effect as shown in Fig. 7.5 [18,19]. Ester 41b was reduced with LiAlH4 to remove the chiral auxiliary, yielding enantiopure alcohol (3S,4S)-(−)-9 [18,19].This AC was consistent with the result obtained by the CD exciton chirality method applied to

Figure 7.5  (A) Enantioresolution of alcohol (±)-9 using CSDP acid (−)-1 and determination of its absolute configuration by X-ray crystallography. (B) ORTEP drawing of ester (3S,4S)-41b. (Reprinted with permission from Ref. [19].)

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the corresponding 4-bromobenzoate of alcohol 9 [18]. Recently, we have found that the solvolysis with K2CO3/MeOH was very effective to recover enantiopure alcohols from CSDP esters in a high yield. It should be emphasized that starting from enantiopure alcohol (3S,4S)(−)-9, we have first invented a light-powered chiral molecular motor, in which the direction of motor rotation is controlled by the molecular chirality [26]. Therefore, it is important to determine the AC of molecular motor and to synthesize the motor in an enantiopure form (100% ee). Table 7.2 lists other application examples and their structures are displayed below the table. In relation to the above chiral molecular motor, Table 7.2  Enantioresolution of Alcohols by HPLC on Silica Gel Using (1S,2R,4R)-(−)CSDP Acid, and Determination of Their ACs by X-Ray Crystallography Alcohol Solventa Rs X-ray AC of First Fr. Ref. α

9 18

H/EA = 7:1 H/EA = 7:1

1.18 1.23

1.06 1.27

19 20 12 13 21 22 23 24 25 27 29 30 32 33 34 35 36 37 38 39 11 40

H/EA = 10:1 H/EA = 10:1 H/EA = 4:1 H/EA = 5:1 H/EA = 8:1 H/EA = 6:1 H/EA = 7:1 H/EA = 8:1 H/EA = 4:1 H/EA = 8:1 H/EA = 4:1 H/EA = 5:1 H/EA = 4:1 H/EA = 10:1 H/EA = 6:1 H/EA = 6:1 H/EA = 5:1 H/EA = 5:1 H/EA = 2:1 H/EA = 2:1 H/EA = 3:1 H/EA = 4:1

1.30 1.17 1.20 1.26 1.17 1.17 1.00 1.18 1.13 1.21 1.27 1.12 1.14 1.26 1.26 1.25 1.16 1.12 1.11 1.38 1.18 1.27

1.74 1.79 0.91 1.37 0.80 0.95 – 0.83 1.0 1.07 1.20 1.01 0.91 1.03 1.29 1.94 1.11 0.87 0.88 1.19 – 1.14

H, n-hexane; EA, ethyl acetate. X-ray analysis of camphanate ester. c X-ray analysis of 4-bromobenzoate. Source: Adapted with permission from Ref. [25]. a

b

2nd Fr. 1st Fr. 2nd Fr. – 2nd Fr. – – Yesb – – 1st Fr. – Yesb Yesb 1st Fr. 2nd Fr. – – 1st Fr. 1st Fr. 1st Fr. – 1st Fr. 2nd Fr. Yesc

3R,4R 1R,2S

[18,19] [27]

1S,4R 1R,2R S R R R – R R S S S R R R R S S R R aR,aR S

[18] [28,29] [23] [23] [30] [31] [23] [23] [31] [30] [32] [33] [33,34] [33] [34] [35] [36] [36] [36] [36] [21,22] [37]

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enantiopure alcohols (1R,2S)-(+)-18 and (1S,4R)-19 were prepared by the CSDP acid method and their ACs were determined by X-ray crystallography. Alcohol (1S,2S)-(+)-20 is a synthetic precursor of the lightpowered chiral molecular motor, which rotates much faster than the previous motor made from alcohol (3R,4R)-(+)-9 [28,29]. Thus, the CSDP acid method was useful for the synthesis of light-powered chiral molecular motors. CSDP acid esters of para-substituted diphenylmethanols 12, 13, 21, 22, 24, and 25 were clearly separated by HPLC on silica gel, although the para-substituents governing the chirality of the molecule are apart from the chiral center, that is, the carbon atom with hydroxyl group [23,30,31]. These results indicate that the CSDP acid recognizes the molecular chirality well, that is, the difference between a hydrogen atom and a para-substituted functional group. It was impossible to separate the diastereomeric CSDP acid esters of (4-methylphenyl) phenylmethanol 23 by HPLC on silica gel (Table 7.2). Namely, the chirality recognition of alcohol 23 as CSDP acid ester was difficult. The difference between hydrogen and methyl group constituting the molecular chirality of 23 is small and so it is very hard to recognize such a trivial difference [23]. Therefore, the following strategy was adopted. First, (4-bromophenyl)(4′-methylphenyl)methanol 24 was selected as a precursor, which was well enantioresolved as CSDP acid esters and their ACs were determined by X-ray crystallography. The reduction of the bromine atom led to the desired and enantiopure alcohol (S)-(−)-23 [23]. This strategy was also useful for the synthesis and determination of the AC of isotopesubstituted chiral diphenylmethanols as described below. The molecular chirality can also be generated by the substitution with isotopes as shown in 2H-substituted diphenylmethanol 26 and 13C-substituted diphenylmethanol 28 [30,32]. It is almost impossible to directly enantioresolve these isotope-substituted chiral compounds by chiral HPLC with chiral stationary phase or by usual HPLC on silica gel using chiral auxiliary. In such a case, the next strategy was adopted: (a) selection of (±)-(4-bromophenyl)(phenyl- 2,3,4,5,6-d5)methanol 27 as a precursor; (b) enantioresolution as CSDP acid esters; (c) determination of its AC; and (d) subsequent reduction of the bromine atom providing the desired and enantiopure (phenyl-2,3,4,5,6-d5)phenylmethanol 26 [30]. In fact, the preparation of chiral (phenyl-2,3,4,5,6-d5)phenylmethanol 26 was carried out as follows. Racemic alcohol (±)-27 was condensed with CSDP acid and then the esters formed were separated by HPLC on

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silica gel: α = 1.21. Both diastereomeric esters separated gave only fine ­needle-like crystals even after a series of recrystallizations. Accordingly, after recovering the enantiopure alcohol (−)-27 from the first-eluted fraction 42a, a part of (−)-27 was converted to ester 43 using (−)-camphanic acid chloride (Fig. 7.6). Ester 43 showed good crystallinity, providing prismatic crystals suitable for X-ray analysis and the AC of the alcohol moiety was determined as S, using the AC of (−)-camphanic acid part as an internal reference. Subsequently, alcohol (S)-(−)-27 was reduced with H2NNH2/H2O in the presence of Pd-C to yield the isotope-substituted and enantiopure (phenyl-2,3,4,5,6-d5)phenylmethanol [CD(−)270.4]-(S)-26, making possible the unambiguous determination of the chirality generated by the substitution with isotopes. The specific rotation [α]D measured at the wavelength of the sodium D-line (589 nm) is usually used to distinguish enantiomers. However, it is difficult to measure [α]D value of compounds with isotopesubstitution chirality. We have proposed the new definition method of ­enantiomers by the use of CD data, because CD is not only more sensitive than [α]D, but also accurately measurable even with a small amount of samples. For example, [CD(−)270.4]-(S)-26 stands for the enantiomer with negative CD at 270.4 nm and S AC [30]. It would be even more advantageous, if (−)-camphanic acid could be used from the beginning as the chiral auxiliary for enantioresolution. However, the enantioresolution power of (−)-camphanic acid is generally poor. For example, camphanic acid esters 43 prepared from racemic alcohol 27 could not be separated by HPLC on silica gel. Thus, it is sometimes necessary to select two chiral auxiliaries depending on the situation. A similar scheme could be applied to synthesize 13C-substituted chiral diphenylmethanol 28 [32]. Namely, (4-bromophenyl)(phenyl-1,2,3,4,5,6-13C)

Figure 7.6  Preparation of camphanate ester (S)-43, whose absolute configuration was determined by X-ray crystallography.

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methanol (±)-29 was chosen and then enantioresolution and determination of AC were carried out in a similar way as described above. Subsequently, the bromine atom was reduced to yield the 13C-substituted chiral (phenyl1,2,3,4,5,6-13C)phenylmethanol [CD(−)270]-(S)-28. Despite the very small molecular chirality due to the slight difference between isotopes 12C and 13 C, the CD spectrum of 13C-substituted chiral diphenylmethanol (S)-28 is clearly observable [32]. Interesting results were also obtained in the case of ortho-substituted diphenylmethanols. Using the method described above, enantiopure (2-methoxyphenyl)phenylmethanol (−)-30 was prepared and its AC was determined as S by X-ray crystallography [33]. On the other hand, chiral (2-methylphenyl)phenylmethanol 31 had previously been synthesized by asymmetric catalytic reaction and its AC had been estimated based on the chiral reaction mechanism. So, it gave a ­problem whether the absolute configurational assignment based on the reaction mechanism is reliable or not. The author thought that the independent and unambiguous determination of the AC was necessary. To solve this problem, we have carried out the following experiments. The direct enantioresolution of alcohol 31 as CSDP acid esters by HPLC was unsuccessful, as in the case of para-substituted alcohol 23. Namely, it was difficult to discriminate hydrogen atom and the methyl group in the orthoposition. We then ­attempted the enantioresolution of (4-bromophenyl) (2′-methylphenyl)methanol 44, because para-methyl analog 24 was separable as described above. However, the HPLC analysis exhibited only a single peak. Next, we adopted the following indirect method [33,34]. The strategy consisted of enantioresolution of (2-hydroxymethylphenyl)phenylmethanol 32 as CSDP acid ester, determination of AC by X-ray analysis, and conversion of the enantiopure ester obtained to the desired alcohol 31. CSDP acid (−)-1 (1 equiv.) was allowed to react with diol (±)-32 to yield a diastereomeric mixture of esters, in which the primary alcohol group was selectively esterified. In this case, the chiral auxiliary group bonding to the primary alcohol moiety was remote from the stereogenic center of 32, but the diastereomeric esters were clearly separated: α = 1.14. Single crystals were obtained from the second-eluted fraction (−)-45b, leading to the ­determination of its AC as S by X-ray analysis.The first-eluted fraction (R)– (−)-45a was converted to the desired enantiopure alcohol (R)–(−)-31 via several ­reaction steps.The above result indicated that the AC of 31 previously ­assigned on the basis of an asymmetric reaction mechanism was wrong [34].

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Since the absolute configurational assignment based on the reaction mechanism sometimes may result in an error, it is advisable to determine the AC of products independently. The CSDP acid method was applied also to alcohol 33, where the primary hydroxyl group was protected as silyl ether. In the CSDP esters of alcohol 33, the chiral acid group is close to the chiral center in the alcohol moiety.Therefore, a stronger interaction between two chiral moieties would lead to a larger separation in HPLC. In fact, diastereomeric CSDP esters of alcohol 33 were well separated: α = 1.26. Their ACs were determined by the conversion to alcohol (R)-(+)-32. Similarly, alcohol 34 was enantioresolved and its AC was determined. It was surprising to find the fact that the CSDP acid method was ­directly applicable to (2,6-dimethylphenyl)phenylmethanol 35, although ­(2-methylphenyl)phenylmethanol 31 could not be enantioresolved as ­described above. Racemic alcohol was esterified with CSDP acid (−)-1 yielding diastereomeric esters, which were separated well by HPLC on silica gel: α = 1.25. The first-eluted ester (−)-46a was obtained as single crystals, and hence its AC was determined to be R by X-ray crystallography. From ester (−)-46a, enantiopure alcohol (R)-(+)-35 was obtained [35]. The CSDP acid method is also effective for the preparation of a variety of benzyl alcohols 36–39 [36], which would be useful as chiral synthons for the total synthesis of natural products because of their unambiguous ACs and 100% enantiopurity. Atropisomer 11 is a unique chiral compound containing three naphthalene chromophores. Its enantioresolution and determination of AC were carried out by the following method [21,22]. Racemic diol, (±)-1,1′:4′,1″-ternaphthalene2,2″-dimethanol 11, was combined with CSDP acid (−)-1 forming diesters, which were separated by HPLC on silica gel: hexane/EtOAc = 3:1, α = 1.18. While the first-eluted fraction (−)-47a yielded fine needle-like crystals, the second-eluted fraction (+)-47b yielded large crystals by recrystallization from hexane/EtOAc 2:1. In general, single crystals suitable for X-ray analysis have prismatic or columnar forms or thick plate-like forms with definite surfaces and edges. The second-eluted ester (+)-47b gave odd crystals resembling airplanes with triangular wings upon recrystallization and did not look like single crystals. However, after removal of the wings, the body part was subjected to X-ray analysis, which revealed that it was a single crystal. Interestingly, the formula weight of an asymmetric unit estimated from the preliminary lattice constant did not agree with the molecular weight of (+)-47b. So, we

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had initially thought that molecular structure might be incorrect, assuming that the asymmetric unit of 47b contained one molecule because of the asymmetric structure of CSDP acid moiety. Careful investigation of the data obtained, however, revealed that one asymmetric unit contained a half of the molecule 47b. Namely, ester (+)-47b has a C2 symmetric structure even in crystals, despite the fact that the molecule contains complex CSDP acid moieties. The AC, that is, torsion among three naphthalene chromophores, was unambiguously determined as (aS,aS) on the basis of internal reference. The chiral auxiliaries were removed from ester (aS,aS)-(+)-47b to yield enantiopure diol (aS,aS)-(+)-11. The AC obtained from this X-ray analysis was consistent with that obtained by the application of the CD exciton chirality method to (+)-11 [21,22]. The compound, 2-(1-naphthyl)propane-1,2-diol 40, was isolated as a chiral metabolite of 1-isopropylnaphthalene in rabbits (Fig. 7.7). The metabolite, however, was not enantiopure and its AC had been only empirically estimated based on the reaction mechanism. To obtain the enantiopure diol 40 and to determine its AC in an unambiguous way, the method of CSDP acid was applied to (±)-40 [37]. In this case, only the primary alcohol part was esterified and a diastereomeric mixture obtained was clearly separated by HPLC on silica gel: hexane/EtOAc = 4:1, α = 1.27, Rs = 1.14. In this HPLC, the presence of free tertiary hydroxyl group is important, because the protection of the tertiary alcohol group led to poor separation.

Figure 7.7 (A) Enantioresolution and determination of the absolute configuration of 2-(1-naphthyl)propane-1,2-diol 40. (B) X-ray ORTEP drawing of 4-bromobenzoate (S)-(−)-49. Reprinted with permission from Ref. [37].

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Despite the repeated recrystallizations, both diastereomers were obtained only as amorphous solids. Therefore, the first-eluted fraction (−)-48a was reduced with LiAlH4 to yield enantiopure glycol (−)-40, which was further converted to 4-bromobenzoate (−)-49 (Fig. 7.7). By recrystallization from EtOH, ester (−)-49 gave good single crystals suitable for X-ray analysis and consequently its AC was explicitly determined as S by the Bijvoet pair measurement of the anomalous scattering effect of the bromine atom contained (Table 7.3) [37]. As listed in Table 7.3, the ratio of structural factors |Fc(h k l)|/|Fc(h k −l)| calculated for the S AC agreed with those obtained from the observed data, establishing the S configuration of ester (−)-49. It should be noted that in this crystal form, (−h −k −l) and (h k −l) reflections are equivalent to each other.

Table 7.3  The Bijvoet Pairs of (S)-(−)-2-(1-Naphthyl)Propane-1,2-Diol 4-Bromobenzoate 49: Observed and Calculated Absolute Values of the Structural Factors for (h k l) and (h k −l) Reflections and Their Ratiosa,b |Fo(h k l)| |Fo(h k −l)| |Fo(h k l)|/|Fo(h k −l)| h

k

l

[|Fc(h k l)|]

[|Fc(h k −l)|]

[|Fc(h k l)|/|Fc(h k −l)|]

1 1 2 4 5 2 4 5 1 2 2 2 3 5 2 2 7 4

4 5 8 1 5 1 4 6 3 1 3 5 7 4 1 10 5 4

1 1 1 1 1 2 2 2 3 3 3 3 3 3 4 3 3 4

39.4 [35.4] 39.3 [37.7] 78.4 [74.1] 102.6 [91.1] 10.1 [11.3] 162.2 [154.3] 83.0 [81.0] 71.0 [68.1] 76.0 [74.9] 75.8 [72.8] 89.7 [86.5] 80.9 [77.3] 66.8 [63.6] 40.0 [40.1] 104.6 [99.5] 49.4 [49.7] 42.2 [40.9] 80.9 [75.5]

32.1 [27.9] 46.2 [42.2] 73.8 [68.4] 92.8 [84.6] 20.2 [19.0] 149.3 [143.6] 90.7 [87.6] 66.4 [62.6] 83.6 [79.6] 69.5 [66.6] 99.6 [94.5] 73.8 [69.4] 73.2 [69.1] 46.4 [45.6] 98.0 [92.6] 45.0 [43.7] 36.3 [35.3] 87.0 [80.7]

1.23 [1.26] 0.85 [0.89] 1.06 [1.08] 1.11 [1.08] 0.50 [0.59] 1.09 [1.07] 0.92 [0.92] 1.07 [1.09] 0.91 [0.94] 1.09 [1.09] 0.90 [0.90] 1.10 [1.11] 0.91 [0.92] 0.86 [0.88] 1.07 [1.07] 1.10 [1.07] 1.16 [1.16] 0.93 [0.94]

In this crystal, ǀFo(−h −k −l)ǀ = ǀFo(h k −l)ǀ. Reflections satisfying ǀǀFo(h k l)ǀ−ǀFo(h k −l)ǀǀ > 10 σ(Fo) were selected, where 2 2 0.5 σ ( Fo ) = {σ count + (0.007 | Fo |) } . Source: Reprinted with permission from Ref. [37]. a

b

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Figure 7.8  Synthesis of a novel chiral molecular tool, (S)-(+)-2-methoxy-2-(1-naphthyl) propionic acid (MαNP acid) 2 from glycol (S)-(−)-40 and determination of its absolute configuration.

Furthermore, we have obtained enantiopure 2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid) (S)-(+)-2 via several reactions from diol (S)-(−)-40 (Fig. 7.8) [37]. We have discovered that this novel carboxylic acid, MαNP acid 2, was very powerful for enantioresolution and simultaneous determination of AC of various secondary alcohols by the 1H NMR anisotropy method [38–50]. The results obtained by the 1H NMR anisotropy method are of course consistent with those by the X-ray method. Therefore, the methods of CSDP and MαNP acids are useful as complementary molecular tools.

7.5 A NOVEL CHIRAL MOLECULAR TOOL, 2-METHOXY2-(1-NAPHTHYL)-PROPIONIC ACID {MαNP ACID (S)-(+)-2}, USEFUL FOR ENANTIORESOLUTION OF ALCOHOLS AND DETERMINATION OF THEIR ACS BY THE 1H NMR DIAMAGNETIC ANISOTROPY METHOD We have discussed the design and applications of CSP and CSDP acids useful for both the synthesis of enantiopure compounds and the unambiguous determination of their ACs by X-ray analysis. The X-ray crystallographic method using the internal reference of AC thus leads to the unambiguous and reliable determination of AC. However, the drawback of X-ray crystallography is that the method needs single crystals and therefore it is not applicable to noncrystalline materials. However, in routine experiments, prismatic single crystals suitable for X-ray analysis are not always obtainable. So, is there any other method applicable to noncrystalline materials? In addition, most of the applications listed in Tables 7.1 and 7.2 are limited to aromatic compounds. So, a powerful method applicable to aliphatic compounds is also required. We have recently discovered that 2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid 2, Fig. 7.9) is remarkably effective for the enantioresolution of aliphatic alcohols, especially acyclic aliphatic alcohols [38–50]. In the 1 H NMR spectra of the esters formed from MαNP acid 2 and alcohols, the chemical shifts of the protons in the alcohol moiety are strongly affected by

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Figure 7.9  Novel chiral MαNP acids with powerful ability to enantioresolve alcohols and strong 1H NMR diamagnetic anisotropy effect.

the diamagnetic anisotropy effect induced by the naphthyl group.Therefore, this MαNP acid 2 can be used as the chiral auxiliary useful for determining the AC of secondary alcohols. In this sense, MαNP acid 2 is similar to MTPA and MPA acids as described in Section 7.2.2 [10–13]. Another advantage of the MαNP acid 2 is that it does not racemize, because the α-position of acid 2 is fully substituted and therefore it is easy to prepare the enantiopure acid 2. As will be discussed below, MαNP acid 2 is a very powerful chiral derivatizing agent (CDA), which simultaneously enables both enantioresolution of secondary alcohols and determination of their ACs. Namely, the diastereomeric MαNP esters formed from racemic alcohol are easily separable by HPLC on silica gel and their ACs can be determined by the 1H NMR diamagnetic anisotropy effect. Therefore, this MαNP acid method is very useful for the preparation of natural products and biologically active synthetic chiral compounds, for example, chiral drugs, and also for simultaneous determination of their ACs. In this sense, the chiral MαNP acid 2 is superior to the conventional chiral acids, Mosher’s MTPA acid [10], MPA acid studied by Trost’s group [11], 1- and 2-NMA acids developed by Seco [13] and Ohtani [12] groups. The following sections describe in detail the principle and applications of this chiral MαNP acid method: (a) synthesis of chiral MαNP acid 2, (b) determination of its AC by X-ray analysis and chemical correlation, (c) enantioresolution of racemic acid 2 with chiral alcohols, (d) absolute configurational and conformational analyses of MαNP acid esters by NMR spectroscopy, (e) enantioresolution of racemic alcohols and determination of their ACs using chiral MαNP acid 2, and (f) recovery of chiral alcohols with 100% enantiopurity from the separated diastereomeric esters.

7.5.1  Facile synthesis of MαNP acid 2 and its extraordinary enantioresolution with natural (−)-menthol To synthesize a large amount of enantiopure chiral MαNP acid 2, the ­facile synthesis and enantioresolution of racemic acid 2 were carried out as shown in Fig. 7.10. In general, chiral synthetic amines or alkaloids have been used for enantioresolution of carboxylic acids. However, we have adopted the

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Figure 7.10  Facile synthesis and enantioresolution of novel chiral MαNP acid.

following novel strategy to use chiral alcohols. In this method, chiral alcohols are condensed with racemic acid 2 and the diastereomeric esters formed are largely separated by HPLC on silica gel.The separated esters are then hydrolyzed to yield both enantiomers of the desired carboxylic acids [37,39,49]. As a chiral alcohol, naturally occurring (−)-menthol was selected and esterified with racemic acid 2. It was much surprising that the diastereomeric esters 52a and 52b formed were very largely separated by HPLC on silica gel (hexane/EtOAc = 10:1) as illustrated in Fig. 7.11A.The separation and resolution factors were extraordinarily high (α = 1.83, Rs = 4.55), indicating that acid 2 has a great ability to recognize the chirality of the alcohols. The efficiency in separation enabled the HPLC of a large scale of sample; esters 52a/52b (1.0–1.8 g) were separable in one run using a glass column of silica gel (25 φ × 400 mm) as shown in Fig. 7.11B.

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Figure 7.11  HPLC separation of MαNP acid menthol esters: (A) 100 mg sample and (B) 1.0 g sample. (Reprinted with permission from Ref. [49].)

The first-eluted ester 52a was subjected to solvolysis to yield chiral acid (+)-2, while the second-eluted ester 52b gave acid (−)-2.To determine the ACs of chiral acids 2 obtained, those were converted to methyl esters, the CD spectra of which were measured. By comparison of those CD spectra with that of the authentic sample with known AC established by X-ray analysis and chemical correlation as shown in Figs. 7.7 and 7.8, the ACs of chiral acids 2 were determined as (S)-(+) and (R)-(−), respectively, leading to the assignment of (S)-(−)-52a and (R)-(−)-52b (Fig. 7.10).

7.5.2 The 1H NMR diamagnetic anisotropy method for determining the AC of secondary alcohols: the sector rule and applications As described above, the 1H NMR diamagnetic anisotropy method has been frequently used as a relative and empirical method for determining the ACs of chiral organic compounds [10–13]. In particular, the advanced Mosher method for chiral secondary alcohols has been successfully employed in the field of natural products [12]. In the cases of MTPA and MPA acids, the phenyl group exhibits the diamagnetic anisotropy effect induced by the aromatic ring current, affecting the chemical shift (δ) of protons in the alcohol part. Therefore, the AC of chiral alcohol can be determined by the difference (∆δ) of the chemical shifts of esters formed with (R) and (S) carboxylic acids: ∆δ = δ(R)−δ(S) or ∆δ = δ(S)−δ(R).We have found that MαNP acid 2 is superior to MTPA and MPA acids, because the diamagnetic anisotropy effect of naphthyl group is much stronger than that of the phenyl group and therefore larger ∆δ values are obtained [39,46]. So, the AC of chiral alcohols

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can be unambiguously determined, when using MαNP acid 2 as a chiral NMR anisotropy reagent. Moreover, MαNP acid has another advantage that it does not racemize, because the α-position of acid 2 is fully substituted. From these reasons, it is advisable to use MαNP acid 2, rather than other conventional chiral acids, for determining the AC of chiral alcohols including natural products. All 1H NMR peaks of diastereomeric menthol MαNP esters 52a and 52b were fully assigned by various methods including two-dimensional ones (1H, 1H-1H COSY, 13C, 1H-13C COSY, HMBC) as shown in Fig. 7.12A [39]. The protons of the isopropyl group in ester 52b appeared at much higher fields than in ester 52a. On the other hand, the protons in the 2-position in 52a appeared at higher fields than in ester 52b. Those high-field shifts are obviously due to the diamagnetic anisotropy effect induced by the naphthyl group of MαNP acid moiety. To determine the AC from the NMR anisotropy effect, it is required to determine the preferred conformation of each diastereomer. In esters 52a and 52b, the ACs of MαNP acid and menthol moieties are established as described above and so the following stable conformations are proposed to satisfy the anisotropy effects observed in the NMR spectra (Figs. 7.12 and 7.13). Namely, the two oxygen atoms of the methoxyl and ester carbonyl groups are synperiplanar (syn) to each other in their stable conformations (Fig. 7.13) [49]. In addition, the ester carbonyl oxygen atom is also

Figure 7.12  1H NMR chemical shift data (ppm) of menthol MαNP and HαNP esters. ­(Reprinted with permission from Ref. [39].)

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syn to the alcohol methine proton. Therefore, the methoxyl group, ester group, and alcohol methine proton lie in the same plane, which is called the MαNP plane (Fig. 7.13). These syn conformations are similar to those proposed for MPA esters. In addition, the methyl group of propionic acid moiety is close to the naphthyl proton at the position 2; namely these are in syn relation to each other, forming the Me/naphthyl plane as shown in Fig. 7.13. It should be noted that in both esters, the MαNP plane and Me/ naphthyl plane are perpendicular to each other. In the preferred conformation of ester 52a, the naphthyl group and H-2 protons of menthol moiety are on the same front side of the MαNP plane and the H-2 protons are located above the naphthyl plane. Therefore, the H-2 protons feel the diamagnetic anisotropy effect and hence they appear at higher-field. On the other hand, in ester 52b, the isopropyl group is ­located over the naphthyl group and hence the very large high-field shifts of isopropyl protons are observable as shown in Fig. 7.12A [39]. The predominance of the syn conformations in the MαNP plane of esters 52a and 52b are supported by the comparison of the NMR data with those of 2-hydroxy-2-(1-naphthyl)- propionic acid (HαNP) menthol esters 53a and 53b as shown in Fig. 7.12B [39]. From the NMR chemical shift and IR data, it is obvious that the tertiary hydroxyl group of HαNP esters is intramolecularly hydrogen bonded to the oxygen atom of the ester carbonyl group. Namely, the hydroxyl group and the ester carbonyl oxygen atom take a syn conformation. We have found a very interesting fact that the NMR chemical shift data of MαNP acid menthol ester (S;1R,3R,4S)-(−)-52a, especially those of the menthol part, are very similar to those of HαNP acid menthol ester (S;1R,3R,4S)-(−)-53a as shown in Fig. 7.12A and B.The same is true for the pairs of other diastereomers, (R;1R,3R,4S)-(−)-52b and HαNP acid menthol ester (R;1R,3R,4S)-(+)-53b (Fig. 7.12). These facts indicate that MαNP acid menthol esters take the syn conformation, as HαNP acid menthol esters usually do.This fully explains the observed diamagnetic anisotropy effects. From these data and many other MαNP ester cases, the sector rule for determining the AC of secondary alcohols could be deduced as shown in Fig. 7.13B.The basic procedure of this method is as follows; (R)-MαNP and (S)-MαNP acids are separately allowed to react with a chiral alcohol, the AC of which is defined as X. So, the ester prepared from (R)-MαNP acid has the (R,X) AC, while the other ester from (S)-MαNP acid has the (S,X) AC. All NMR proton signals of (R,X)- and (S,X)-esters are fully assigned by careful analysis. If necessary, the use of two-dimensional spectra is suggested.

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Figure 7.13  (A) The preferred conformation of menthol MαNP esters. (B) The sector rule for determining the absolute configuration of chiral alcohols by use of NMR ∆δ values. (Adapted with permission from Ref. [52].)

The parameter ∆δ expressing the diamagnetic anisotropy effect is defined as follows. ∆δ = δ ( R , X ) − δ ( S , X )

From the 1H NMR data of esters (R,X) and (S,X), ∆δ values are calculated for all protons in the alcohol moiety. For example, in the case of menthol MαNP esters 52a and 52b, ∆δ is expressed as follows. ∆ δ = δ (R , X) − 52b − δ (S , X) − 52a

The calculated ∆δ values are shown in Fig. 7.12A. Fig. 7.13B shows the sector rule for MαNP ester, where the MαNP-Ogroup is placed on the down and front side, while the methine proton of the secondary alcohol is on the down and rear side. The group R2 showing positive ∆δ values is placed on the right side, while the group R1 showing negative ∆δ values on the left side. From this projection, the AC X of chiral alcohol can be determined [49]. In the case of menthol MαNP esters 52a and 52b, it should be noted that the ∆δ values of all protons obey the sector rule. From these results,

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the AC of alcohol part was unambiguously determined to be R, which of course agreed with the AC of natural (−)-menthol. To confirm the above AC determination, we next attempted X-ray crystallography. It was difficult to obtain the single crystals of MαNP ester 52a or 52b, but finally we could obtain single crystals of ester 52b by recrystallization from diethyl ether/MeOH. As shown in Fig. 7.14, the AC of ester 52b determined by X-ray crystallography agreed with that assigned by the 1H NMR/MαNP acid method [51]. Thus, the empirical 1H NMR diamagnetic anisotropy method was supported by X-ray crystallography. The diamagnetic anisotropy effect of the chiral MαNP acid is much stronger than that of the conventional chiral carboxylic acid (Fig. 7.15) [39]. For instance, the ∆δ values of menthol MαNP ester are ca. four times larger than those of Mosher’s MTPA ester [10] (Fig. 7.15B); twice for MPA ester studied by Trost and coworkers [11] (Fig. 7.15C); comparable to 1-NMA and 2-NMA esters reported by Seco [13] and Ohtani [12] et al. MαNP acid is thus effective for determining the AC of natural products. Some application examples of this MαNP acid method to chiral alcohols are shown in Fig. 7.16.

Figure 7.14  X-ray ORTEP drawing of menthol MαNP ester (R;1R,3R,4S)-(−)-52b. (Reprinted with permission from Ref. [51].)

Figure 7.15  Comparison of the NMR ∆δ values of menthol esters formed with chiral carboxylic acids. (Reprinted with permission from Ref. [39].)

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Figure 7.16  The NMR ∆δ values and absolute configurations determined by the MαNP acid method. (Reprinted with permission from Ref. [39].)

7.5.3  Enantioresolution of various alcohols using MαNP acid and simultaneous determination of their ACs Another extraordinary quality of MαNP acid is its excellent ability in chiral recognition. For example, as discussed above, racemic MαNP acid could be successfully enantioresolved as the esters of natural (−)-­menthol; the diastereomeric esters formed were largely separated by HPLC on silica gel. MαNP acid could also be enantioresolved with other ­chiral alcohols as shown in Fig. 7.16. These facts logically indicate that if ­enantiopure MαNP acid is used, racemic alcohols can be e­ nantioresolved. In fact, we have succeeded in enantioresolution of various racemic alcohols using enantiopure MαNP acid (S)-(+)-2 as exemplified in Fig. 7.17 [41]. This novel chiral MαNP acid (S)-(+)-2 has thus a remarkable enantioresolving power for alcohols, especially for aliphatic alcohols. For instance, in the case of 2-butanol, the diastereomeric esters 54a/54b can be baseline separated with the separation factor α = 1.15 and resolution factor Rs = 1.18. In this case, it is obvious that the chiral carboxylic acid 2 recognizes well the slight difference between the methyl and the ethyl groups. This is an excellent and practical method since the chiral acid 2 exhibits a high resolving power to aliphatic alcohols, to which in general asymmetric syntheses are hardly applicable. The next question is then how the AC of alcohol moiety is determined. The ACs of separated diastereomers can be determined by applying the 1H NMR diamagnetic anisotropy method using chiral MαNP acid as described above. So, we have proposed a new general scheme as illustrated in Fig. 7.18 [41,49,52]. Racemic alcohol is esterified with MαNP acid (S)-(+)-2 yielding a mixture of diastereomeric esters, which is separated by HPLC on silica gel. The AC of the first-eluted ester is defined as (S,X), where S denotes the AC of MαNP acid part, while

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Figure 7.17  HPLC separation of diastereomeric esters formed from racemic aliphatic alcohols and (S)-(+)-MαNP acid (silica gel, 22 φ  × 300 mm, hexane/EtOAc = 20:1). (­Reprinted with permission from Ref. [41].)

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Figure 7.18  Enantioresolution of racemic alcohol as (S)-MαNP esters, and determination of the absolute configuration of the first-eluted fraction by the NMR anisotropy method. (Reprinted with permission from Ref. [52].)

X denotes that of the alcohol part to be determined. So, the AC of the second-eluted ester is expressed as (S,−X), where −X indicates the ­ ­opposite AC of X. The original definition of ∆δ value is defined as follows (Fig. 7.19): ∆δ = δ ( R , X ) − δ ( S , X )

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Figure 7.19  Determination of the absolute configurations of the alcoholic part of the first-eluted esters by the NMR diamagnetic anisotropy method using (S)-(+)-MαNP acid, and the observed ∆δ values. (Reprinted with permission from Ref. [41].)

So the value of δ(R,X) is required to calculate the ∆δ value. However, the enantiomer (R,X) does not exist in this scheme and so the original equation of ∆δ cannot be used here. To solve the above problem, the following conversion of the equation was performed. Since the ester (S,−X) is the enantiomer of ester (R,X), their NMR data should be identical with each other, that is, δ(R,X) = δ(S,−X). Therefore, ∆ δ = δ (R , X) − δ (S , X) = δ (R , X) − δ (R , − X) = δ (1st fr.) − δ (2nd fr.)

So, the AC X of the first-eluted fraction can be determined from the ∆δ value which is obtained by subtracting the chemical shift of the first-eluted fraction from that of the second-eluted fraction (Fig. 7.18). This method has been applied to the esters shown in Fig. 7.16, giving ∆δ values as shown in Fig. 7.19, from which the ACs of the first-eluted esters were determined. The ∆δ values are reasonably distributed; namely, positive values at the right and negative values at the left. The AC of the first-eluted ester can thus be determined and the opposite AC is of course assigned to the second-eluted ester.

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It should be noted that when MαNP acid (R)-(−)-2 is used, the ∆δ value is defined as follows. ∆ δ = δ (R , X) − δ (S , X) = δ (R , X) − δ (R , − X) = δ (1st fr.) − δ (2nd fr.)

The next step is the recovery of enantiopure alcohol and chiral MαNP acid 2. As exemplified in Fig. 7.20, both enantiopure alcohols were readily obtained by the solvolysis of the separated esters [45]. The chiral MαNP acid (S)-(+)-2 was also recovered and could be recycled. How good is the enantiopurity of the recovered alcohols? In our method, both diastereomeric esters obtained are enantiopure, if MαNP acid 2 used is enantiopure, because they are fully separated in HPLC. The MαNP acid 2 was enantioresolved with natural (−)-menthol, the enantiopurity of which was confirmed as 100% by the gas chromatography using the chiral stationary phase [49]. As described above, MαNP acid has excellent enantioresolving power regardless of its simple molecular structure and the absence of hetero atoms. Besides, the chiral acid 2 is superior to the Mosher’s MTPA and MPA acids in the diamagnetic anisotropy effect and therefore further development is expected.

Figure 7.20  Recovery of enantiopure alcohols and (S)-(+)-MαNP acid. (Reprinted with permission from Ref. [44].)

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7.6  COMPLEMENTARY USE OF CSDP ACID (−)-1 AND MαNP ACID (S)-(+)-2 FOR ENANTIORESOLUTION OF ALCOHOLS AND DETERMINATION OF THEIR ACS BY X-RAY CRYSTALLOGRAPHIC AND 1H NMR DIAMAGNETIC ANISOTROPY METHODS 7.6.1  Application to various alcohols including diphenylmethanols As discussed above, the method of MαNP acid is very useful for the preparation of enantiopure secondary alcohols and the simultaneous determination of their ACs. However, those ACs were determined by the empirical sector rule, which is based on the diamagnetic anisotropy of naphthalene ring and the preferred conformation of MαNP ester.Therefore, the ACs assigned by this method possess the empirical nature. How reliable are those ACs? To evaluate the reliability of the 1H NMR/MαNP acid method, we have compared the results by the MαNP acid method with those by the X-ray crystallographic analysis, as summarized in Table 7.4 [46,50]: see the structures shown in the figure attached to the table. Recently, much attention has been focused on chiral fluorinated organic compounds, since some chiral synthetic drugs consist of fluorinated aromatic moieties. To prepare enantiopure fluorinated diphenylmethanols and to determine their ACs by X-ray crystallography, we have applied the CSDP acid and MαNP acid methods [50]. For example, racemic (4-trifluoromethylphenyl)phenylmethanol 64 was esterified with CSDP acid (−)-1 yielding a diastereomeric mixture of esters, which was clearly separated by HPLC on silica gel: separation factor α = 1.34 (Table 7.4). The first-eluted ester 83a was recrystallized from EtOH giving prisms, one of which was subjected to X-ray crystallography affording the X-ray ORTEP drawing as shown in Fig. 7.21. Although the trifluoromethyl moiety takes a disordered structure, the AC of the first-eluted ester (−)-83a was unambiguously determined to be R by the internal reference method using the (1S,2R,4R) AC of the camphor part and also by the heavy atom effect. Thus, the AC of ester 83a was doubly determined [50]. Enantiopure alcohol (R)-(−)-64 was easily recovered by treating the first-eluted CSDP ester 83a with K2CO3 in MeOH. Although the AC of (+)-64 had previously been estimated as S by the Horeau’s method, the abnormality of its application had been pointed out [55].Therefore, the direct

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Table 7.4  Silica Gel–HPLC Separation of Diastereomeric Esters Formed From Racemic Alcohols With CSDP Acid (−)-1 and/or MαNP Acid (+)-2, Determination of Their Absolute Configurations by X-Ray Crystallography and/or by the 1H NMR Diamagnetic Anisotropy Method 1 AlcoH NMR Alcohol Acid hol Solventa α X-ray ∆δ b from 1st Fr. Ref.

(−)-1 (±)-64 H/EA = 5:1 1.34 1st Fr. (+)-2 (±)-64 H/EA = 8:1 1.39 – (−)-1 (±)-65 H/EA = 5:1 1.16 1st Fr. 2nd Fr. (+)-2 (±)-65 H/EA = 10:1 1.07 – (−)-1 (±)-66 H/EA = 4:1 1.00 – (+)-2 (±)-66 H/EA = 15:1 1.08 – (−)-1 (±)-67 H/EA = 5:1 1.11 – (+)-2 (±)-67 H/EA = 10:1 1.18 – (−)-1 (±)-68 H/EA = 5:1 1.05 – (+)-2 (±)-68 H/EA = 10:1 1.07 – (−)-1 (±)-69 H/EA = 4:1 1.00 – (+)-2 (±)-69 H/EA = 7:1 1.00 – (−)-1 (±)-70 H/EA = 4:1 1.21 1st Fr. (+)-2 (±)-70 H/EA = 8:1 1.08 – (+)-2 (±)-31 H/EA = 15:1 1.12 1st Fr. 2nd Fr. (+)-2 (±)-71 H/EA = 15:1 1.10 1st Fr. 2nd Fr. (−)-1 (±)-72 H/EA = 4:1 1.15 1st Fr. (−)-1 (±)-73 H/EA = 5:1 1.16 1st Fr. (+)-2 (±)-74 H/EA = 20:1 1.21 1st Fr. (+)-2 (±)-75 H/EA = 20:1 1.88 1st Fr. (+)-2 (±)-76 H/EA = 2:1 1.35 – (+)-2 (±)-77 H/EA = 10:1 1.80 1st Fr.

– Yesc –

(R)-(−)-64 (R)-(−)-64 (R)-(−)-65

[50] [50] [50]

Yesc – Yes – Yes – Yes – – – Yesc Yesc

(R)-(−)-65 66d (R)-(+)-66 67d (R)-(−)-67 68d (R)-(−)-68 69d 69d (S)-(−)-70 (S)-(−)-70 (R)-(−)-31

[50] [50] [50] [50] [50] [50] [50] [50] [50] [50] [50] [49,51]

Yesc

(R)-(+)-71

[49,51]

– – Yesc Yesc Yes Yesc

(S)-(−)-72 (S)-(+)-73 (1R,2R)-(−)-74 (1R,2S)-(−)-75 (1R,2R)-(−)-76 (1R,6R,8aR)(−)-77

[46] [46] [49,51] [49,51] [49] [51,53]

(+)-2 (+)-2 (+)-2 (+)-2 (+)-2

Yes Yesc Yes Yes Yesc

(1S,6S,8aS)-(+)-78 (S)-(+)-79 (3R,4S)-(+)-80 (S)-(−)-81 (S)-(−)-82

[53] [49] [49] [49] [54]

(±)-78 (±)-79 (±)-80 (±)-81 (±)-82

H/EA = 1:1 H/EA = 10:1 H/EA = 15:1 H/EA = 10:1 H/EA = 50:1

1.27 1.22 1.46 1.30 1.78

– 1st Fr. – – 2nd Fr.

H, n-hexane; EA, ethyl acetate. ∆δ, 1H NMR diamagnetic anisotropy effect. Absolute configuration determined by 1H NMR diamagnetic anisotropy method agreed with that by X-ray crystallography. d Diastereomers were not separated. Source: Adapted with permission from Refs. [25,50]. a

b c

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and unambiguous determination of its AC has been desired for a long time. The AC of (4-trifluoromethylphenyl)phenylmethanol (R)-(−)-64 was thus first determined by X-ray analysis. To evaluate the reliability of the 1H NMR/MαNP acid method, next, racemic alcohol 64 was esterified with MαNP acid (S)-(+)-2, giving a diastereomeric mixture of esters. The mixture was separated well by HPLC on silica gel (hexane/EtOAc 8:1): separation factor α = 1.39 (Table 7.4). To determine the AC by the 1H NMR anisotropy method, all NMR signals were fully assigned by the 1H, 1H-1H COSY, 13C, HMQC, and HMBC methods. In the case of fluorinated diphenylmethanols, the 1H–19F coupling

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Figure 7.21  X-ray ORTEP drawing of CSDP ester (R)-(−)-83a. (Reprinted with permission from Ref. [50].)

was also helpful for assigning 1H NMR signals. From the observed data, the ∆δ values of 1H NMR anisotropy effect of (4-trifluoromethylphenyl) phenylmethanol MαNP esters 84a/84b were calculated; the phenyl group showed large positive ∆δ values (+0.11 ∼ +0.43 ppm), while the 4-trifluoromethylphenyl group large negative ∆δ values (−0.58 ∼ −0.37 ppm) as shown in Fig. 7.22A. So, the R AC was assigned to the first-eluted MαNP ester 84a [50]. Chiral fluorinated diphenylmethanols were recovered by reduction with LiAlH4 from the corresponding diastereomeric MαNP esters. So, the firsteluted MαNP ester 84a of (4-trifluoromethylphenyl)phenylmethanol was treated with LiAlH4, yielding enantiopure alcohol (R)-(−)-64, which was identical with the authentic sample recovered from the first-eluted CSDP ester (R)-(−)-83a. The R AC of (4-trifluoromethyl- phenyl)phenylmethanol (−)-64 determined by the 1H NMR anisotropy method was thus confirmed by X-ray crystallography (Table 7.4). Racemic (3-trifluoromethylphenyl)phenylmethanol 65 was similarly converted to diastereomeric CSDP esters, which were base-line separated by HPLC on silica gel: α = 1.16. It should be emphasized that both esters 85a and 85b were obtained as single crystals, which were subjected to X-ray analysis. In the case of ester 85a, the final X-ray R-value remained higher because of low crystallinity. However, even in such a case, its AC was unambiguously determined, based on the internal reference of AC of the camphor moiety. The second eluted CSDP ester 85b crystallized as prisms, allowing the unambiguous determination of AC by X-ray crystallography as listed in Table 7.4 [50].

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Figure 7.22 Observed ∆δ values of MαNP esters prepared from alcohols 64 and 73. (Reprinted with permission from Refs. [46,50].)

Diastereomeric MαNP esters were prepared from racemic alcohol 65. Although it was not easy to separate them by HPLC on silica gel, ­separation factor α = 1.07, we could separate them by repeating HPLC. From the NMR data, ∆δ values were calculated allowing the AC determination of the first-eluted ester. It should be noted that the AC obtained by the 1H NMR/MαNP acid method was again proved by X-ray crystallography (Table 7.4). The CSDP esters of (2-trifluoromethylphenyl)phenylmethanol 66 ­appeared as a single peak in HPLC on silica gel, indicating no separation at all. On the other hand, MαNP esters of alcohol 66 could be separated by HPLC on silica gel despite small α-value: α = 1.08. Based on the ∆δ values and the result of reduction, the AC of alcohol recovered from the firsteluted ester was determined as listed in Table 7.4 [50]. In the case of (4-fluorophenyl)phenylmethanol 67, diastereomeric CSDP esters was separated well with α values of more than 1.1. However, no single crystals suitable for X-ray analysis were obtained from both CSDP esters and therefore their ACs could not be determined by X-ray crystallography. On the other hand, MαNP esters of alcohol 67 were separated well, α = 1.18 and so the AC of alcohol (R)-(−)-67 was determined by the 1 H NMR diamagnetic anisotropy method (Table 7.4) [50]. Similar things happened in the case of (3-fluorophenyl)phenylmethanol 68; it was difficult to separate its CSDP esters, α = 1.05, and single crystals suitable for X-ray analysis could not be obtained. MαNP esters of alcohol 68 could be separated and from the ∆δ values, the AC of the alcohol ­recovered from the first-eluted ester was determined to be (R)-(−)-68. As seen above, when the CSDP acid method is not applicable, the MαNP acid method becomes useful [50]. Unfortunately, both CSDP acid and MαNP acid methods were useless for (2-fluorophenyl)-phenylmethanol 69; their diastereomeric esters appeared as

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single peaks in HPLC on silica gel, indicating no separation at all. Therefore, their ACs have remained undetermined (Table 7.4) [50]. On the other hand, both methods were applicable to (2,6-difluorophenyl)phenylmethanol 70. Namely, CSDP esters of alcohol 70 were separated well with α = 1.21.The first-eluted CSDP ester 87a was recrystallized from EtOH giving prisms, the X-ray analysis of which led to the unambiguous determination of an S AC to alcohol (−)-70. The same AC of alcohol (S)(−)-70 was obtained by the 1H NMR/MαNP acid method (Table 7.4) [50]. As discussed above, the CSDP acid method and/or MαNP acid method are useful for the preparation of enantiopure fluorinated diphenylmethanols and simultaneous determination of their ACs except the case of (2-fluorophenyl)phenylmethanol 69. In some cases, the AC determined by the 1 H NMR/MαNP acid method was confirmed by X-ray analysis of CSDP ester.Thus, the complementary use of both methods is useful and suggested. It was much surprising to find the fact that diastereomeric MαNP esters of (2-methyl- phenyl)phenylmethanol 31 could be directly separated and its AC was unambiguously determined by both 1H NMR and X-ray methods [49,51]. Remember that alcohol 31 could not be enantioresolved by the CSDP acid method, and so, we had to select the indirect route as discussed in Section 7.4. Here, racemic alcohol 31 was esterified with MαNP acid (+)-2 yielding diastereomeric esters 88a/88b, which were separated by HPLC on silica gel: α = 1.12. So, the ACs of esters were determined from the obtained ∆δ values. In addition, it was lucky that both MαNP esters 88a and 88b were obtained as a single crystal, and hence their ACs were unambiguously determined by X-ray crystallography. It is natural that both methods led to the same AC. Hydrolysis of ester 88a yielded enantiopure alcohol (R)-(−)-31 (Table 7.4). In a similar way, the MαNP acid method was applied to (2-chlorophenyl)phenylmethanol 71, which afforded enantiopure alcohol (R)-(+)-71 with AC established by 1H NMR and X-ray methods [49,51]. The CSDP acid method was applicable to meta-methoxy-substituted ­diphenylmethanols 72 and 73 (Table 7.4). For example, racemic (3,5-­dimethoxyphenyl)phenylmethanol 73 was esterified with CSDP acid (−)-1 yielding a diastereomeric mixture of esters, which was separated well by HPLC on silica gel: α = 1.16. The first-eluted ester (−)-89a was obtained as single crystals, when recrystallized from EtOH. The X-ray crystallography of ester 89a led to the unambiguous determination of its AC as S (Fig. 7.23) [46].

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Figure 7.23  X-ray ORTEP drawing of CSDP ester (S)-(−)-89a. (Reprinted with permission from Ref. [46].)

Ester (S)-(−)-89a was treated with K2CO3 in MeOH giving enantiopure alcohol (S)-(+)-73. The AC of (3,5-dimethoxyphenyl)phenylmethanol 73 was thus unambiguously determined for the first time. The MαNP acid method was next applied to chiral alcohol (S)-(+)-73, to confirm the reliability of the 1H NMR anisotropy method. Namely, alcohol (+)-73 was esterified with MαNP acids (R)-(−)-2 and (S)-(+)-2, respectively, yielding esters (R,X) and (S,X), where X denotes the AC of alcohol moiety. From the 1H NMR data of both esters, the ∆δ values {∆δ = δ(R,X) − δ(S,X)} were calculated as shown in Fig. 7.22B. Since the 3,5-dimethoxyphenyl ring showed positive ∆δ values and the remaining phenyl group negative ∆δ values, the S AC was assigned to alcohol (+)-73.This result, of course, agrees with the assignment by X-ray crystallography [46]. Similarly, the AC of (3-methoxyphenyl)phenylmethanol 72 was determined to be (S)-(−) by X-ray crystallography (Table 7.4) [46]. The MαNP acid method was next applied to cyclic secondary alcohols as listed in Table 7.4. Racemic trans-2-methylcyclohexanol 74 was esterified with MαNP acid (S)-(+)-2 affording diastereomeric esters 90a/90b, which were separated well by HPLC on silica gel: α = 1.21. From the 1H NMR ∆δ values, the AC of the first-eluted ester 90a was determined to be (S;1R,2R). So, the hydrolysis of the first-eluted ester 90a yielded enantiopure alcohol (1R,2R)-(−)-74 [49]. It was lucky that the first-eluted MαNP ester 90a was obtained as single crystals upon recrystallization, one of which was subjected to X-ray analysis. The AC of the alcohol part was determined to be (1R,2R) as shown in Fig. 7.24A, which agreed with the AC assigned by the 1H NMR/MαNP acid method [51].

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Figure 7.24 X-ray stereostructure of MαNP esters. (Reprinted with permission from Ref. [51].)

Similarly, diastereomeric MαNP esters 91a/91b prepared from racemic trans-2-isopropyl-cyclohexanol 75 were largely separated by HPLC on silica gel: α = 1.88; compare with the α-value of esters 90a/90b. Thus, the HPLC separation is sensitive to the difference of the bulkiness of the adjacent alkyl group. Namely, the isopropyl group is larger than the methyl group, and hence the α-value of 91a/91b is larger than that of 90a/90b. The first-eluted ester 91a was also obtained as single crystals and its AC was determined by X-ray analysis as shown in Fig. 7.24B. Of course, it agreed with the AC determined by the 1H NMR method [51]. Thus, the AC determination by the empirical 1H NMR/MαNP acid method was established by X-ray analysis of ester 91a itself. Therefore, it is unnecessary to apply the CSDP acid method to alcohol 75 for establishing its AC by X-ray analysis. It was interesting that the MαNP acid method was applicable to trans-1,2-cyclohexanediol 76. From the racemic glycol 76, mono-MαNP esters were prepared and separated by HPLC on silica gel despite the existence of a hydroxyl group. From the ∆δ values, the AC was determined; the result was consistent with the already established AC of glycol (1R,2R)(−)-76 [49]. The Wieland–Miescher (W–M) ketone is useful as a chiral synthetic precursor of the synthesis of various natural products. However, it is still difficult to prepare enantiopure W–M ketone. Therefore, we have applied

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the MαNP acid method to W–M ketone derivative (±)-77. Diastereomeric MαNP esters 92a/92b were largely separated by HPLC on silica gel: α = 1.80 (Table 7.4). Therefore, it is possible to separate in a preparative scale. The removal of TBDMS group yielded esters 93a/93b having a free hydroxyl group and it is interesting that HPLC separation of esters 93a/93b became less effective: α = 1.27 [53]. The AC of the first-eluted ester 92a was determined from the data of ∆δ values to be (S;1R,6R,8aR), which was confirmed by X-ray crystallography of ester 92a, where the MαNP acid part was used as the internal reference of AC. The 1H NMR/MαNP acid method was thus supported here again by X-ray analysis. It should be noted that the AC of ester 93a was opposite to that of ester 92a. Namely, the HPLC elution order was reversed [51,53]. We have recently succeeded in the invention of light-powered chiral molecular motors [26]. In relation to the studies, the MαNP acid method was applied to alcohols 79 and 80; MαNP esters 94a/94b formed from racemic alcohol 79 were separated well by HPLC on silica gel: α = 1.22. Furthermore, the first-eluted ester 94a was obtained as single crystals, one of which was analyzed by X-ray crystallography. In this case, the 1H NMR/MαNP acid and X-ray methods are again consistent with each other. Alcohol 80 was similarly enantioresolved with α = 1.46, and the AC of the first-eluted ester was determined from the ∆δ values as shown (Table 7.4) [49]. At last, the 1H NMR/MαNP acid method was applied to acetylene alcohols 81 and 82 (Table 7.4). Diastereomeric MαNP esters prepared from alcohol 81 were separated well by HPLC on silica gel: α = 1.30. From the ∆δ values obtained from 1H NMR spectra, its AC was determined to be (S)-(−)-81, which was consistent with the AC assigned by the CD exciton chirality method [49,56]. It was interesting that the MαNP acid method was applicable to acetylene alcohol 82 with two long side chains. Racemic alcohol 82 was esterified with MαNP acid (S)-(+)-2 yielding diastereomeric esters 95a/95b, which were largely separated by HPLC on silica gel: α = 1.78. From the 1H NMR ∆δ values, the AC of the first-eluted ester 95a was determined to be (S,19S). It was lucky that the second-eluted ester 95b was obtained as single crystals, when recrystallized from iso-PrOH. The crystals were very thin plates with 5 µm thickness and hence the X-ray experiment was carried out by using the strong X-ray of synchrotron radiation at the SPring-8 in Hyogo, Japan. Despite the large final X-ray R-value = 0.0814, the AC of ester 95b was unambiguously determined to be (S,19R). Thus, the AC assigned

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by 1H NMR/MαNP acid method was confirmed by X-ray crystallography. Hydrolysis of ester 95a yielded enantiopure (S)-(−)-17-octatriacontyn19-ol 82 [54]. As described above, the ACs assigned by the 1H NMR/MαNP acid method were confirmed by X-ray crystallography in many cases and hence the MαNP acid method for determining AC became much more reliable. Thus, the MαNP acid is powerful as a chiral molecular tool for preparation of enantiopure compounds and simultaneous determination of their ACs.

7.6.2  Synthesis of enantiopure cryptochiral hydrocarbon, (R)-(+)[VCD(−)984]-4-ethyl-4-methyloctane and determination of its AC 4-Ethyl-4-methyloctane 96 is a simple chiral hydrocarbon, where methyl, ethyl, propyl, and butyl groups are bonded to a quaternary carbon of ­chiral center (Fig. 7.25).The four alkyl groups are similar to each other, and hence its optical rotation value would be very small. Therefore, hydrocarbon 96 is one of cryptochiral compounds. As will be discussed below, we have succeeded in the synthesis of enantiopure hydrocarbon 96 and its unambiguous determination of AC by applying the CSDP acid and MαNP acid methods [57,58]. In addition, its AC was also determined by the ab initio molecular orbital (MO) calculation of vibrational circular dichroism (VCD) spectrum [59]. Before our synthesis, Wynberg and coworkers reported the synthesis of chiral hydrocarbon 96, but its ee was ca. 85%–95% ee, and its AC had remained undetermined [60]. Later, Lardicci and coworkers reported the synthesis of chiral hydrocarbon 96 and assigned its AC to be (R)-(+) by applying the CD exciton chirality method to a synthetic precursor [61]. However, the observed CD Cotton effect was very weak, ∆ε = +0.8, and hence it was difficult to say that the AC of compound 96 was unambiguously determined. We adopted the synthetic route shown in Fig. 7.26, where alcohol cis-97 was designed as the synthetic precursor of hydrocarbon 96. Racemic alcohol cis-97 was esterified with CSDP acid (−)-1 yielding diastereomeric esters, which were separated well by HPLC on silica gel as shown in Fig. 7.27A. The second eluted ester (+)-cis-98b was recrystallized from EtOH/CH2Cl2 affording single crystals, one of which was subjected to X-ray analysis giv-

Figure 7.25  Cryptochiral hydrocarbon, (S)-(−)-[VCD(+)984]-4-ethyl-4-methyloctane.

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Figure 7.26  Synthesis of cryptochiral hydrocarbon 96 by the CSDP acid method. (Reprinted with permission from Ref. [58].)

Figure 7.27  HPLC separation on silica gel: (A) CSDP esters 98a/98b, hexane/EtOAc 6:1; (B) MαNP esters 99a/99b, hexane/EtOAc 15:1. (Reprinted with permission from Ref. [58].)

ing the ORTEP drawing as shown in Fig. 7.26. The AC of the alcohol part was unambiguously determined by both X-ray internal reference and heavy atom effect methods to be (1R,2R) [58]. From the second eluted ester (+)-cis-98b, enantiopure alcohol (1R,2R)-(−)-cis-97 was easily recovered. The alcohol was then converted

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to enantiopure cryptochiral hydrocarbon (S)-(−)-96 via several reaction steps, where the four groups around the chiral center were always connected to the quaternary carbon. Therefore, no ambiguity is seen in the chemical correlation. Thus, the synthesis of enantiopure cryptochiral hydrocarbon 96 and the unambiguous determination of its AC were first achieved in an unambiguous manner [58]. The MαNP acid method was next applied for the synthesis of chiral hydrocarbon 96 and for the determination of its AC as shown in Fig. 7.28. Racemic alcohol cis-97 was esterified with MαNP acid (S)-(+)-2 ­affording diastereomeric esters, which were largely separated by HPLC on silica gel as shown in Fig. 7.27B: separation factor α = 1.81, resolution factor Rs = 5.97. It was thus surprising to find the result that MαNP esters 99a/99b are much more easily separated than CSDP esters 98a/98b, which indicates that the MαNP acid method is more practical for HPLC separation of larger amount of sample [57,58]. To determine the AC of the first-eluted ester cis-99a, the 1H NMR spectra of esters 99a and 99b were analyzed in detail using two-dimensional NMR spectra. The diamagnetic anisotropy ∆δ values were obtained as

Figure 7.28 Synthesis of cryptochiral hydrocarbon 96 by the MαNP acid method. ­(Reprinted with permission from Refs. [57,58].)

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shown in Fig. 7.28B, where aromatic group shows positive ∆δ values, while aliphatic side chains show negative values.Therefore, the AC of the first eluted ester (−)-cis-99a was unambiguously determined to be (S;1S,2S) [57]. It was lucky that the second-eluted MαNP ester (−)-cis-99b was obtained as single crystals, when recrystallized from hexane/EtOAc. A single crystal was subjected to X-ray crystallography, by which the AC of the alcohol part was determined to be (1R,2R) as illustrated in Fig. 7.28C, where the S AC of MαNP acid part was used as the internal reference of AC. The AC assigned by the 1H NMR method was thus corroborated by X-ray crystallography. Starting from the first-eluted ester (S;1S,2S)-(−)-cis-99a, enantiopure cryptochiral hydrocarbon (R)-(+)-96 was similarly synthesized as shown in Fig. 7.26D. Thus, we could synthesize both enantiomers (100% ee) of hydrocarbon 96 [57]. Recently, it became possible to calculate the VCD (i.e. CD in infra-red region) spectra based on the ab initio MO calculation, which enabled one to determine the AC by comparing the observed and theoretically calculated VCD spectra [62]. To study if the VCD spectral method is applicable to cryptochiral hydrocarbon 96, the VCD spectra of both enantiomers (S)(−)-96 and (R)-(+)-96 were measured as shown in Fig. 7.29A. In the studies of VCD spectra, it is advised to measure the VCD spectra of both enantiomers because of very weak signals; see the ∆ε value of VCD in Fig. 7.29A. The observed VCD spectra showed about ten VCD Cotton effects in the region of 1150–900 cm−1. It should be noted that in this region, two VCD spectral curves are mirror images indicating that these VCD signals are almost artifact-free. Furthermore, observed VCD of (S)-(−)-96 shows a strong positive Cotton effect at 984 cm−1 and hence the enantiomer can be specified by VCD as follows; (S)-[VCD(+)984]-96 indicates that the enantiomer 96 showing a positive VCD Cotton effect at 984 cm−1 has (S) AC [59]. The VCD spectrum curve of hydrocarbon (S)-96 was computed by the DFT (density functional theory) MO method as shown in Fig. 7.29B. It was surprising to find that the DFT calculation gave a VCD curve (solid line), which reasonably agreed with the observed VCD curve of (−)-[VCD(+)984]-96 (solid line), although the calculated VCD Cotton effects are shifted to higher wave-number, and the calculated VCD intensity is weaker than the observed one. Namely, the Cotton effects numbered shows good one-to-one correspondence between observed and calculated VCD bands. This result led to the unambiguous determination of AC, that is, (S)-(−)-[VCD(+)984]-96 [59].

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Figure 7.29  VCD spectra of cryptochiral hydrocarbon 96: (A) solid line, observed VCD of (S)-(−)-[VCD(+)984]-96; dotted line, observed VCD of (R)-(+)-[VCD(−)984]-96. (B) Solid line, calculated VCD of (S)-96; dotted line, calculated VCD of (R)-96. The numbers indicated on the solid line specify VCD Cotton effects and show the one-to-one correspondence between experimental and theoretical VCD bands. (Reprinted with permission from Ref. [59].)

A similar result was also obtained for opposite enantiomer (R)-(+)[VCD(−)984]-96, when dotted lines in Fig. 7.29A and B were compared. Of course, these ACs obtained from VCD spectra are consistent with those determined by X-ray crystallography and 1H NMR anisotropy method. It should be emphasized that the AC determination by the theoretical calculation of VCD spectra has thus been proved in an experimental manner.

7.7  CONCLUSIONS We have developed several novel chiral molecular tools, in particular, chiral carboxylic acids, and successfully applied those CDAs to the enantioresolution of alcohols by HPLC separation and also to the simultaneous determination of AC by X-ray crystallography and/or by 1H NMR

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anisotropy method. The X-ray crystallographic method using an internal reference is, of course, the best for determining AC. However, ideal single crystals are not always obtained. In such a case, the 1H NMR method using MαNP acid, which requires no crystallization, is effective. In enantioresolution, chiral CSDP acid and MαNP acid are thus useful as the complementary molecular tools. If the resolution with one CDA is u ­ nsuccessful, the use of the other is suggested. The methods described above are thus very powerful for the preparation of enantiomeric alcohols with 100% enantiopurity and also for the simultaneous determination of their ACs.

ACKNOWLEDGMENTS The author thanks Dr. George A. Ellestad, Department of Chemistry, Columbia University, for his valuable suggestions. The researches described in this chapter have been carried out in collaboration with many coworkers, whose names are listed in the references cited. The author sincerely thanks them for their cooperation and efforts.

REFERENCES [1] Bijvoet, J. M.; Peerdeman, A. F.;Van Bommel, A. J. Determination of the Absolute Configuration of Optically Active Compounds by Means of X-Rays. Nature 1951, 168, 271–272. [2] (a) Harada, N.; Nakanishi, K. A Method for Determining the Chiralities of Optically Active Glycols. J. Am. Chem. Soc. 1969, 91, 3989–3991. (b) Harada, N.; Nakanishi, K. The Exciton Chirality Method and Its Application to Configurational and Conformational Studies of Natural Products. Acc. Chem. Res. 1972, 5, 257–263. (c) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy—Exciton Coupling in Organic Stereochemistry University Science Books and Oxford University Press: Mill Valley, CA and ­Oxford, 1983. (d) Harada, N.; Nakanishi, K.; Berova, N. Electronic CD Exciton Chirality Method: Principles and Applications. Comprehensive Chiroptical Spectroscopy, 2, Berova, N., ­Polavarapu, P., Nakanishi, K.,Woody, R.W., Eds.;Wiley, 2012; pp. 115–166 Chapter Four. [3] (a) Flack, H. D. On Enantiomorph-Polarity Estimation. Acta Crystallogr. A 1983, 39, 876–881. (b) Flack, H. D.; Bernardinelli, D. The Use of X-Ray Crystallography to Determine Absolute Configuration. Chirality 2008, 20, 681–690. [4] Harada, N.; Soutome,T.; Murai, S.; Uda, H. A Chiral Probe Useful for Optical Resolution and X-Ray Crystallographic Determination of the Absolute Stereochemistry of Carboxylic Acids. Tetrahedron: Asymmetry 1993, 4, 1755–1758. [5] Harada, N.; Soutome, T.; Nehira, T.; Uda, H.; Oi, S.; Okamura, A.; Miyano, S. Revision of the Absolute Configurations of [8]Paracyclophane-10-Carboxylic and 15-Methyl[10]Paracyclophane-12-Carboxylic Acids. J. Am. Chem. Soc. 1993, 115, 7547–7548. [6] Harada, N.; Hattori,T.; Suzuki,T.; Okamura, A.; Ono, H.; Miyano, S.; Uda, H. Absolute Stereochemistry of 1-(9-Phenanthryl)-2-Naphthoic Acid as Determined by CD and X-Ray Methods. Tetrahedron: Asymmetry 1993, 4, 1789–1792. [7] Hattori, T.; Harada, N.; Oi, S.; Abe, H.; Miyano, S. 1,12-Dioxa[12](1,4)Naphthalenophane-14-Carboxylic Acid: Practical Synthesis, Resolution and Absolute Configuration of The Enantiomers. Tetrahedron: Asymmetry 1995, 6, 1043–1046. [8] Kasai, Y.; Naito, J.; Kuwahara, S.; Watanabe, M.; Ichikawa, A.; Harada, N. Novel Chiral Molecular Tools for Preparation of Enantiopure Alcohols by Resolution and Simultaneous Determination of Their Absolute Configurations by The 1H NMR Anisotropy Method. J. Synth. Org. Chem. 2004, 62, 1114–1127.

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[9] (a) Toda, F. Isolation and Optical Resolution of Materials Utilizing Inclusion Crystallization. Top. Curr. Chem. 1987, 140, 43–69. (b) Toda, F. Studies of Host-Guest Chemistry. Fundamentals and Applications of Molecular Recognition and Their Development to New Organic Solid State Chemistry. J. Synth. Org. Chem. Jpn. 1990, 47, 1118–1131. (c) Toda, F.; Tanaka, K.; Watanabe, M.; Abe, T.; Harada, N. E ­ nantiomer Resolution by Crystallization With Chiral Hosts: Application to Monoterpenes, Verbenone and Apoverbenone. Tetrahedron: Asymmetry 1995, 6, 1495–1498. [10] Dale, J. A.; Mosher, H. S. Nuclear Magnetic Resonance Enantiomer Reagents. ­Configurational Correlations Via Nuclear Magnetic Resonance Chemical Shifts of Diastereomeric Mandelate, O-Methylmandelate, and α-Methoxy-α-TrifluoromethylPhenylacetate (MTPA) Esters. J. Am. Chem. Soc. 1973, 95, 512–519. [11] Trost, B. M.; Belletire, J. L.; Godleski, S.; McDougal, P. G.; Balkovec, J. M.; Baldwin, J. J.; Christy, M. E.; Ponticello, G. S.;Varga, S. L.; Springer, J. P. On the Use of the O-Methylmandelate Ester for Establishment of Absolute Configuration of Secondary Alcohols. J. Org. Chem. 1986, 51, 2370–2374. [12] (a) Ohtani, I.; Kusumi, T.; Kashman,Y.; Kakisawa, H. High-Field FT NMR Application of Mosher’s Method. The Absolute Configurations of Marine Terpenoids. J. Am. Chem. Soc. 1991, 113, 4092–4096. (b) Yamase, H.; Ooi, T.; Kusumi, T. Determination of the Absolute Configuration of Linear Secondary Alcohols Adopting One Enantiomer of the Chiral Anisotropic Reagents, Methoxy-(1-and 2-Naphthyl)Acetic Acids. Tetrahedron Lett. 1998, 39, 8113–8116. (c) Arita, S.; Yabuuchi, T.; Kusumi, T. Resolution of 1- and 2-Naphthylmethoxyacetic Acids, NMR Reagents for Absolute Configuration Determination, by Use of l-Phenylalaninol. Chirality 2003, 15, 609–614. [13] (a) Seco, J. M.; Latypov, S. K.; Quinoa, E.; Riguera, R. New Chirality Recognizing Reagents for the Determination of Absolute Stereochemistry and Enantiomeric Purity by NMR. Tetrahedron Lett. 1994, 35, 2921–2924. (b) Seco, J. M.; Quinoa, E.; Riguera, R.The assignment of absolute configuration by NMR. Chem. Rev. 2004, 104, 17–117. [14] (a) Okamoto, Y.; Suzuki, K.; Ohta, K.; Hatada, K.; Yuki, H. Optically Active Poly(Triphenylmethyl Methacrylate) With One-Handed Helical Conformation. J. Am. Chem. Soc. 1979, 101, 4763–4765. (b) Okamoto, Y.; Honda, S.; Okamoto, I.; Yuki, H.; Murata, S.; Noyori, R.; Takaya, H. Novel Packing Material for Optical Resolution: (+)-Poly(Triphenylmethyl Methacrylate) Coated on Macroporous Silica Gel. J. Am. Chem. Soc. 1981, 103, 6971–6973. [15] Review articles. J. Chromatogr. A 2001, 906, 1–482. [16] Patel, R. N., Ed. Stereoselective Biocatalysis; Marcel Dekker: New York, NY, 2000. [17] Harada, N.; Nehira, T.; Soutome, T.; Hiyoshi, N.; Kido, F. Chiral Phthalic Acid Amide, A Chiral Auxiliary Useful for Enantiomer Resolution and X-Ray Crystallographic Determination of The Absolute Stereochemistry of Alcohols. Enantiomer 1996, 1, 35–39. [18] Harada, N.; Koumura, N.; Robillard, M. Chiral Dichlorophthalic Acid Amide, An Improved Chiral Auxiliary Useful for Enantioresolution and X-ray Crystallographic Determination of Absolute Stereochemistry. Enantiomer 1997, 2, 303–309. [19] Harada, N.; Koumura, N.; Feringa, B. L. Chemistry of Unique Chiral Olefins. 3. ­Synthesis and Absolute Stereochemistry of Trans- and Cis-1,1′,2,2′,3,3′,4,4′-Octahydro3,3′-Dimethyl-4,4′-Biphenanthrylidenes. J. Am. Chem. Soc. 1997, 119, 7256–7264. [20] Toyota, S.; Yasutomi, A.; Kojima, H.; Igarashi, Y.; Asakura, M.; Oki, M. Absolute Conformation and Chiroptical Properties. VI. 2,2′,3,3′-Tetramethoxy-9,9′-Bitriptycyl: A Stereochemical Analog of 1,2-Disubstituted Ethane With Identical Substituents. Bull. Chem. Soc. Jpn. 1998, 71, 2715–2720. [21] Harada, N.; Vassilev, V. P.; Hiyoshi, N. Absolute Configuration and Conformation of 1,1′:4′,1″-Ternaphthalene Compounds as Determined by X-Ray Crystallography. ­Enantiomer 1997, 2, 123–126.

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[22] Harada, N.; Hiyoshi, N.; Vassilev, V. P.; Hayashi, T. Synthesis, Circular Dichroism, and Absolute Stereochemistry of 1,1′:4′,1″-Ternaphthalene Compounds. Chirality 1997, 9, 623–625. [23] Harada, N.; Fujita, K.; Watanabe, M. Enantioresolution and Absolute Stereochemistry of Diarylmethanols. Enantiomer 1997, 2, 359–366. [24] Nehira, T.; Harada, N. unpublished data. [25] Harada, N.; Watanabe, M.; Kuwahara, S. Novel Chiral Derivatizing Agents Powerful for Enantioresolution and Determination of Absolute Stereochemistry by X-Ray Crystallographic and 1H NMR Anisotropy Methods. In Chiral Analysis; Busch, K. W., Busch, M. A., Eds.; Elsevier Science Publishers: Amsterdam,The Netherlands, 2006; pp. 661–691 Chapter Eighteen. [26] Koumura, N.; Zijlstra, R. W. J.; van Delden, R. A.; Harada, N.; Feringa, B. L. LightDriven Monodirectional Molecular Rotor. Nature 1999, 401, 152–155. [27] Koumura, N.; Harada, N. unpublished data. [28] Fujita, T.; Kuwahara, S.; Harada, N. A New Model of Light Powered Chiral Molecular Motor With Higher Speed of Rotation (1). Synthesis and Absolute Stereostructure. Eur. J. Org. Chem. 2005, 4533–4543. [29] Kuwahara, S.; Fujita, T.; Harada, N. A New Model of Light Powered Chiral Molecular Motor With Higher Speed of Rotation (2). Dynamics of Motor Rotation. Eur. J. Org. Chem. 2005, 4544–4556. [30] Harada, N.; Fujita, K.;Watanabe, M. Molecular Chirality by Isotopic Substitution. Synthesis, Absolute Configuration, and CD Spectra of Chiral Diphenylmethanols. Enantiomer 1998, 3, 64–70. [31] Fujita, K.; Harada, N. unpublished data. [32] Harada, N.; Fujita, K.;Watanabe, M. Molecular Chirality by Isotopic Substitution. Synthesis, Absolute Configuration, and CD Spectra of 13C Substituted Chiral Diphenylmethanol. J. Phys. Org. Chem. 2000, 13, 422–425. [33] Kuwahara, S.; Watanabe, M.; Harada, N.; Koizumi, M.; Ohkuma, T. Enantioresolution and Absolute Stereochemistry of o-Substituted Diphenylmethanols. Enantiomer 2000, 5, 109–114. [34] Watanabe, M.; Kuwahara, S.; Harada, N.; Koizumi, M.; Ohkuma, T. Enantioresolution by the Chiral Phthalic Acid Method: Absolute Configurations of (2-Methylphenyl)Phenylmethanol and Related Compounds. Tetrahedron: Asymmetry 1999, 10, 2075–2078. [35] Kosaka, M.; Kuwahara, S.;Watanabe, M.; Harada, N.; Job, G. E.; Pirkle, W. H. Comparison of CD Spectra of (2-Methylphenyl)- and (2,6-Dimethylphenyl)-Phenylmethanols Leads to Erroneous Absolute Configurations. Enantiomer 2002, 7, 213–217. [36] Kosaka, M.; Watanabe, M.; Harada, N. Enantioresolution by the Chiral Phthalic Acid Method: Absolute Configurations of Substituted Benzylic Alcohols. Chirality 2000, 12, 362–365. [37] Kuwahara, S.; Fujita, K.;Watanabe, M.; Harada, N.; Ishida,T. Enantioresolution and Absolute Stereochemistry of 2-(1-Naphthyl)Propane-1,2-Diol and Related Compounds. Enantiomer 1999, 4, 141–145. [38] Ichikawa, A.; Hiradate, S.; Sugio, A.; Kuwahara, S.; Watanabe, M.; Harada, N. Absolute Stereochemistry of 2-Hydroxy-2-(1-Naphthyl)Propionic Acid as Determined by the 1 H NMR Anisotropy Method. Tetrahedron: Asymmetry 1999, 10, 4075–4078. [39] Harada, N.; Watanabe, M.; Kuwahara, S.; Sugio, A.; Kasai,Y.; Ichikawa, A. 2-Methoxy2-(1-Naphthyl)Propionic Acid, A Powerful Chiral Auxiliary for Enantioresolution of Alcohols and Determination of Their Absolute Configurations by the 1H NMR Anisotropy Method. Tetrahedron: Asymmetry 2000, 11, 1249–1253. [40] Ichikawa, A.; Hiradate, S.; Sugio, A.; Kuwahara, S.; Watanabe, M.; Harada, N. Absolute Configuration of 2-Methoxy-2-(2-Naphthyl)Propionic Acid, as Determined by the 1 H NMR Anisotropy Method. Tetrahedron: Asymmetry 2000, 11, 2669–2675.

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[41] Taji, H.; Kasai,Y.; Sugio, A.; Kuwahara, S.;Watanabe, M.; Harada, N.; Ichikawa, A. Practical Enantioresolution of Alcohols With 2-Methoxy-2-(1-Naphthyl)Propionic Acid and Determination of Their Absolute Configurations by the 1H NMR Anisotropy Method. Chirality 2002, 14, 81–84. [42] Fujita, T.; Kuwahara, S.; Watanabe, M.; Harada, N. Crystalline State Conformation of 2-Methoxy-2-(1-Naphthyl)Propionic Acid Ester. Enantiomer 2002, 7, 219–224. [43] Ichikawa, A.; Ono, H.; Hiradate, S.; Watanabe, M.; Harada, N. Absolute Configuration of 2-Methoxy-2-(1-Naphthyl)Propionic and 2-Methoxy-2-(2-Naphthyl)Propionic Acids as Determined by the Phenylglycine Methyl Ester (PGME) Method. Tetrahedron: Asymmetry 2002, 13, 1167–1172. [44] Taji, H.; Watanabe, M.; Harada, N.; Naoki, N.; Ueda,Y. Diastereomer Method for Determining % ee by 1H NMR and/or MS Spectrometry With Complete Removal of the Kinetic Resolution Effect. Org. Lett. 2002, 4, 2699–2702. [45] Kasai, Y.; Watanabe, M.; Harada, N. Convenient Method for Determining the Absolute Configuration of Chiral Alcohols With Racemic 1H NMR Anisotropy Reagent, MαNP Acid. Use of HPLC-CD Detector. Chirality 2003, 15, 295–299. [46] Kosaka, M.; Sugito, T.; Kasai, Y.; Kuwahara, S.; Watanabe, M.; Harada, N.; Job, G. E.; Shvet, A.; Pirkle,W. H. Enantioresolution and Absolute Configurations of Chiral MetaSubstituted Diphenylmethanols as Determined by the X-Ray Crystallographic and 1H NMR Anisotropy Methods. Chirality 2003, 15, 324–328. [47] Ichikawa, A.; Ono, H.; Harada, N. Synthesis and Analytical Properties of (S)-(+)-2-Methoxy-2-(9-Phenanthryl)Propionic Acid. Tetrahedron: Asymmetry 2003, 14, 1593–1597. [48] Nishimura, T.; Taji, H.; Harada, N. Absolute Configuration of the Thyroid Hormone Analogue KAT-2003 as Determined by the 1H NMR Anisotropy Method With a Novel Chiral Auxiliary, MαNP Acid. Chirality 2004, 16, 13–21. [49] Kasai,Y.; Taji, H.; Fujita, T.;Yamamoto,Y.; Akagi, M.; Sugio, A.; Kuwahara, S.; Watanabe, M.; Harada, N.; Ichikawa, A.; Schurig, V. MαNP Acid, A Powerful Chiral Molecular Tool for Preparation of Enantiopure Alcohols by Resolution and Determination of Their Absolute Configurations by the 1H NMR Anisotropy Method. Chirality 2004, 16, 569–585. [50] Naito, J.; Kosaka, M.; Sugito, T.; Watanabe, M.; Harada, N.; Pirkle, W. H. Enantioresolution of Fluorinated Diphenylmethanols and Determination of Their Absolute Configurations by X-Ray Crystallographic and 1H NMR Anisotropy Methods. Chirality 2004, 16, 22–35. [51] Kuwahara, S.; Naito, J.;Yamamoto,Y.; Kasai,Y.; Fujita,T.; Noro, K.; Shimanuki, K.; ­Akagi, M.; Watanabe, M.; Matsumoto, T.; Watanabe, M.; Ichikawa, A.; Harada, N. C ­ rystalline State Conformational Analysis of MαNP Esters, Powerful Resolution and Chiral 1H NMR Anisotropy Tools. Eur. J. Org. Chem. 2007, 1827–1840. [52] Kasai,Y.; Sugio, A.; Sekiguchi, S.; Kuwahara, S.; Matsumoto,T.;Watanabe, M.; Ichikawa, A.; Harada, N. Conformational Analysis of MαNP Esters, Powerful Chiral Resolution and 1H NMR Anisotropy Tools. Aromatic Geometry and Solvent Effects on the ∆δ Values. Eur. J. Org. Chem. 2007, 1811–1826. [53] Kasai, Y.; Shimanuki, K.; Kuwahara, S.; Watanabe, M.; Harada, N. Preparation of Enantiopure Wieland–Miescher Ketone and Derivatives by the MαNP Acid Method. Substituent Effect on the HPLC Separation. Chirality 2006, 18, 177–187. [54] Sekiguchi, S.; Akagi, M.; Naito, J.; Yamamoto, Y.; Taji, H.; Kuwahara, S.; Watanabe, M.; Ozawa, Y.; Toriumi, K.; Harada, N. Synthesis of Enantiopure Aliphatic Acetylene ­Alcohols and Determination of Their Absolute Configurations by 1H NMR Anisotropy and/or X-Ray Crystallography. Eur. J. Org. Chem. 2008, 2313–2324. [55] Wu, B.; Mosher, H. S. Configuration of Some Para-Substituted Benzhydrols. J. Org. Chem. 1986, 51, 1904–1906.

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[56] Naito, J.;Yamamoto,Y.; Akagi, M.; Sekiguchi, S.; Watanabe, M.; Harada, N. Unambiguous Determination of the Absolute Configurations of Acetylene Alcohols by Combination of the Sonogashira Reaction and the CD Exciton Chirality Method—Exciton Coupling Between Phenylacetylene and Benzoate Chromophores. Monatsh. Chem. 2005, 136, 411–445. [57] Fujita, F.; Obata, K.; Kuwahara, S.; Miura, N.; Nakahashi, A.; Monde, K.; Decatur, J.; Harada, N. (R)-(+)-[VCD(+)945]-4-Ethyl-4-Methyloctane, The Simplest Chiral Saturated Hydrocarbon With a Quaternary Stereogenic Center. Tetrahedron Lett. 2007, 48, 4219–4222. [58] Fujita, T.; Obata, K.; Kuwahara, S.; Nakahashi, A.; Monde, K.; Decatur, J.; Harada, N. (R)-(+)-[VCD(−)984]-4-Ethyl-4-Methyloctane, a Cryptochiral Hydrocarbon With a Quaternary Chirality Center. (1) Synthesis of Enantiopure Compound and Unambiguous Determination of Absolute Configuration. Eur. J. Org. Chem. 2010, 6372–6384. [59] Kuwahara, S.; Obata, K.; Fujita, T.; Miura, N.; Nakahashi, A.; Monde, K.; Harada, N. (R)-(+)-[VCD(−)984]-4-Ethyl-4-Methyloctane, A Cryptochiral Hydrocarbon With a Quaternary Chirality Center. (2) Vibrational CD Spectra of Both Enantiomers and Absolute Configurational Assignment. Eur. J. Org. Chem. 2010, 6385–6392. [60] (a) Hoeve, W. T.; Wynberg, H. Chiral Tetraalkylmethanes. Two syntheses of Optically Active Butylethylmethylpropylmethane of Known and High Optical Purity. J. Org. Chem. 1980, 45, 2754–2763. (b) Wynberg, H.; Hekkert, G. L.; Houbier, J. P. M.; Bosch, H. W. The Optical Activity of Butylethylhexylpropylmethane. J. Am. Chem. Soc. 1965, 87, 2635–2639. [61] Caporusso, A. M.; Consoloni, C.; Lardicci, L. Stereoselective Construction of Quaternary Carbon Centers Via Reaction of Heterocuprates With Chiral Allenic Bromides: A Facile Synthesis of Butylethylmethylpropylmethane in High Enantiomeric Purity. Gazz. Chim. Ital. 1988, 118, 857–859. [62] Berova, N., Polavarapu, P., Nakanishi, K.,Woody, R.W., Eds. Comprehensive Chiroptical Spectroscopy, 1–2; Wiley: Hoboken, NJ, USA, 2012.

CHAPTER 8

Chiroptical Probes for Determination of Absolute Stereochemistry by Circular Dichroism Exciton Chirality Method Kuwahara Shunsuke*,**, Ikeda Mari†, Habata Yoichi*,**

*Department of Chemistry, Faculty of Science, Toho University, Chiba, Japan **Research Center for Materials with Integrated Properties, Toho University, Chiba, Japan † Department of Chemistry, Education Center, Faculty of Engineering, Chiba Institute of Technology, Chiba, Japan

8.1  INTRODUCTION The development of a practical approach to determine the absolute configuration of natural and artificial molecules is a challenge in the life and material sciences fields [1-3]. Although X-ray crystallography is a reliable method for the determination of absolute configuration, it can often be difficult to obtain good crystals suitable for X-ray analysis. Recently, the 1H nuclear magnetic resonance (NMR) anisotropy method has been employed as an empirical method to determine the absolute configurations of chiral compounds [4-6]. In particular, the modified Mosher method using the ring current effect of the aryl moiety has been widely used to determine the absolute configurations of chiral alcohols. Owing to the complexity of the conformational distribution, application of the method to chiral amines has been limited [7-10]. Circular dichroism (CD) is a powerful tool for determination of the stereochemistry of chiral molecules on the microgram scale [1-3,11]. Especially, exciton-coupled CD (ECCD) is a potentially sensitive method for chiral detection [12,13]. The relationship between the sign of the CD Cotton effect and the twist of the two electric transition moments of molecules has been established in a nonempirical manner. A variety of chromophores has been used in ECCD, including p-substituted benzoates, 2-naphthoates, 2-anthroates, and naphthalimide derivatives [3,12,13].

Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00008-2 Copyright © 2018 Elsevier B.V. All rights reserved.

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Figure 8.1  The design of chiroptical probes based on biphenyl chromophore.

A biphenyl chromophore exhibits intense absorptions at 252 nm (ε 19,000) due to the π–π* transition arising from polarization along the long axes in the biphenyl chromophore. A simple 4-biphenyl carboxylic acid has been used as the chromophore in CD [3,13,14] and fluorescencedetected CD (FDCD) [15,16]. Rosini, Toniolo, and coworkers have reported 2,2′-disubstituted biphenyls to detect the chirality of diols, carboxylic acids, and amino acids in induced CD (ICD) at A band [17-26]. We have recently designed new chiroptical probes based on biphenyl chromophores (Fig. 8.1). A biphenyl framework could be easily attached to a variety of functional groups such as a chiral recognition site, a binding site, and an auxochrome. When the binding site is coupled to the substrate molecules, the information on the chirality transfer to the recognition site in the ortho position. The absorbance of the biphenyl chromophore can be red-shifted introducing an auxochrome. Here, we report the development of chiroptical probes based on biphenyl chromophores and their application for determination of absolute stereochemistry by ECCD.

8.2  A CHIROPTICAL PROBE FOR CHIRAL RESOLUTION AND DETERMINATION OF THE ABSOLUTE CONFIGURATION OF AROMATIC ALCOHOLS Chiral aromatic and allylic alcohols are important building blocks for the preparation of chiral pharmaceuticals, agrochemicals, and functional materials [2]. In general, chiral compounds are prepared by techniques such as asymmetric synthesis, enzymatic kinetic resolution, separation by chiral high-performance liquid chromatography (HPLC) and recrystallization of diastereomeric salts [2]. However, these methods do not always provide the desired enantiomer with 100% enantiopurity. Determination of the absolute configuration of the prepared chiral compounds presents another challenge for researchers. Recently, (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid

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Figure 8.2  Conceptual diagram of the chiral resolution and determination of the absolute configuration by the formation of MBC esters. Abbreviation: MBC, minimum bactericidal concentration.

(MαNP acid) has been found to be useful not only for the chiral resolution of secondary alcohols but also for determination of their absolute configurations by the 1H NMR anisotropy method [27-34]. To develop a versatile reagent for chiral resolution and determination of the absolute configuration of aromatic and allylic alcohols, we designed a new chiral auxiliary, (−)-3-menthoxybiphenyl-4-carboxylic acid [(−)-1, MBC acid] [35]. The (1R,2S,5R)-(−)-menthoxy group has been used previously for the chiral resolution of carboxylic acids, amino acids, phosphinates, and silanes by recrystallization or chromatography [29,36-38]. In MBC acid, the (1R,2S,5R)-(−)-menthoxy group is introduced to the biphenyl chromophore in the ortho position of carboxylic acid. Carboxylic acid acts as a linker to the alcohols and amines (Fig. 8.2). It is expected that the diastereomeric MBC esters obtained when MBC acid (−)-1 covalently bonded to racemic aromatic or allylic alcohols can be separated by chromatography. Their absolute configurations could then be determined by ECCD between the biphenyl chromophore of the MBC part of the molecule and the chromophore of the alcohol. The advantage of ECCD is that it can be used to determine absolute configurations in a nonempirical manner. The chiral alcohols would then finally be recovered by hydrolysis of the MBC esters. MBC acid (−)-1 was synthesized from commercially available 4-bromo-2-fluorobenzoic acid via four steps with an overall yield of 55% [35].

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Scheme 1  Chiral resolution of MBC esters (–)-3a and (+)-3b. Abbreviation: MBC, minimum bactericidal concentration.

MBC acid (−)-1 was condensed with racemic aromatic alcohol (±)-trans-2, which is an intermediate used in the preparation of DNA-binding molecular motors [39], to yield MBC esters (−)-3a and (+)-3b (Scheme 1). (−)-3a and (+)-3b were separated by HPLC using ODS column (Fig. 8.3). The separation and resolution factors were high (α = 1.12; Rs = 1.86), indicating that (−)-1 has an ability to recognize the chirality of aromatic alcohols. The CD and UV spectra of (−)-3a and (+)-3b are illustrated in Fig. 8.4. The UV spectrum of (−)-3a exhibits intense absorptions at λmax = 269.2 nm (ε 23,600) and 233.4 nm (ε 86,000) due to the π–π* transition arising from polarization along the long axes in the biphenyl and naphthyl groups, respectively. These two transitions couple with each other in the CD spectrum of (−)-3a to generate exciton coupling Cotton effects: λext = 258.4 nm (∆ε1 = +28.4) and λext = 219.8 nm (∆ε2 = –36.6).The positive first and negative second Cotton effects indicate the positive exciton chirality between the two chromophores.This means that the two long axes of the chromophores constitute a clockwise screw sense. The preferred conformation of (−)-3a is illustrated in Fig. 8.4C. Although there may be some free rotation around the benzoate C–O bond, it is well known that the benzoate C–O bond is s-trans and the carbonyl oxygen atom of the benzoate is synperiplanar to the alcoholic methine proton [12,13,30,31]. The long axis of the biphenyl chromophore is nearly

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Figure 8.3  HPLC separation of MBC esters 3a/3b. ODS column (YMC-Pack Pro C18 RS (250 × 10 mm)), eluent: acetonitrile, flow rate: 9.0 mL/min. Separation factor α = (t2 − t0)/ (t1  −  t0), where t1 and t2 are the retention times of the first- and second-eluted fractions, respectively, and t0 is the retention time of an unretained compound. Resolution factor Rs = 2(t2 − t1)/(W1 + W2), where W1 and W2 are the bandwidths of the first- and second-eluted fractions at the baseline level, respectively. Abbreviations: HPLC, highperformance liquid chromatography; MBC, minimum bactericidal concentration; ODS, octadecylsilyl. Redrawn with permission from [35], copyright 2017, Elsevier.

Figure 8.4  CD spectra (A) and UV spectra (B) of (1S,2R)-(−)-3a (red line, 0.20 mM in EtOH, 293 K) and (1R,2S)-(+)-3b (blue line, 0.22 mM in EtOH, 293 K). Preferred conformations of (1S,2R)-(−)-3a and (1R,2S)-(+)-3b (C). Abbreviations: CD, circular dichroism; UV, ultraviolet. Redrawn with permission from [35], copyright 2017, Elsevier.

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Figure 8.5  ORTEP drawing of (1S,2R)-(−)-3a. Abbreviation: ORTEP, Oak Ridge Thermal Ellipsoid Plot. Redrawn with permission from [35], copyright 2017, Elsevier.

parallel to the alcoholic C–O bond. Therefore, the clockwise screw sense of the chromophores of (−)-3a leads to an S absolute configuration of the alcohol part of the molecule, resulting in the (1S,2R) absolute configuration of (−)-3a. (+)-3b exhibits an almost mirror image of the Cotton effects of (−)-3a in the CD spectrum [λext = 259.8 nm (∆ε1 = –24.6) and λext = 220.4 nm (∆ε2 = +38.8)], indicating that the arrangements of the chromophores in (−)-3a and (+)-3b are symmetrical. The negative first and positive second Cotton effects indicate that (+)-7b has the (1R,2S) absolute configuration. To confirm the configurational assignment by X-ray crystallography, (1S,2R)-(−)-3a was recrystallized from EtOAc to give single crystals. The absolute configuration of the alcohol part of the molecule was determined to be S using the known absolute configuration of the (1R,2S,5R)-(−)menthol part as an internal reference. As shown in Fig. 8.5, the long axes in the biphenyl and naphthyl groups constitute a clockwise screw sense. The dihedral angles φ(C9–O1–C1–H1) and φ(O2–C9–O1–C1) are –47.2° and 3.5°, respectively, which indicate that the carbonyl oxygen atom of the benzoate and the alcoholic methine proton are arranged in a nearly synperiplanar conformation in the crystalline state. Optimized structures of (1S,2R)-(−)-3a and (1R,2S)-(+)-3b determined by DFT at the B3LYP/6-31G* level of theory [40] also support the conformations described above. The absolute twist of the long axes in the biphenyl and naphthyl groups constitute a clockwise and counterclockwise

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Figure 8.6  Optimized structures of (1S,2R)-(−)-3a and (1R,2S)-(+)-3b at B3LYP/6-31G* level. Redrawn with permission from [35], copyright 2017, Elsevier.

screw sense in (1S,2R)-(−)-3a and (1R,2S)-(+)-3b, respectively (Fig. 8.6). The carbonyl oxygen atom of the benzoate and the alcoholic methine proton of (1S,2R)-(−)-3a and (1R,2S)-(+)-3b take the synperiplanar conformation. Enantiopure alcohols, (1S,2R)-(+)-2 and (1R,2S)-(−)-2, were obtained from the corresponding esters (1S,2R)-(−)-3a and (1R,2S)-(+)-3b, respectively, by hydrolysis with KOH in EtOH/H2O (Scheme 1). Applications of this technique to other aromatic alcohols (±)-4–7, yielding esters 8–11, are summarized in Table 8.1. Each diastereomeric mixture of MBC esters was separated by HPLC using an ODS column (α = 1.05–1.06).The diastereopurities of the MBC esters were determined to be >99% by 1H NMR.The amplitude of the CD signals of the MBC esters of 6-methoxy-1-tetralol (S)-9a and (R)-9b was low and their CD spectra did not show a mirror image.The difference CD curve was therefore calculated to detect the genuine ECCD [41]: ∆∆ε = ∆ε ((S)-9a)–∆ε ((−)-1) and ∆∆ε = ∆ε ((R)-9b)–∆ε ((−)-1). The difference CD curve showed an almost mirror image of the typical ECCD pattern. The MBC esters of acyclic alcohols (±)-6 and 7 also exhibited the typical ECCD pattern. Even if the naphthyl or styryl chromophore rotates around the C–C single bond connected to the center chiral carbon, the absolute twist between the biphenyl group and the chromophore is unchanged. Aromatic alcohol (S)-(−)-7, a key intermediate in the synthesis of anthracyclin antibiotics [42,43], has previously been prepared in 94%ee and the absolute configuration was assigned by considering the mechanism

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Table 8.1  Application of the technique to aromatic alcohols (±)-4–7 Alcohols Separated esters α λmax/nm (∆ε)

1.06 (1S,2S)-8a (1st eluted) (1R,2R)-8b (2nd eluted)

256.6 (+31.0), 219.6 (−7.7) 259.6 (−23.7), 220.6 (+30.8)

1.05 (S)-9a (1st eluted) 278.2 (+2.7), 233.2 (−5.7)a (R)-9b (2nd 284.4 (−1.1, sh), 233.8 eluted) (+5.1)b

1.05 (S)-10a (1st eluted) (R)-10b (2nd eluted)

264.4 (+11.2), 215.0 (−34.7)

1.05 (S)-11a (1st eluted) (R)-11b (2nd eluted)

284.0 (+3.7), 257.8 (−6.3)

265.0 (−7.4), 216.6 (+22.0)

296.8 (−4.1, sh), 259.0 (+7.8)

Difference CD curve: ∆∆ε = ∆ε ((S)-9a)−∆ε ((−)-1) and ∆∆ε = ∆ε ((R)-9b) − ∆ε ((−)-1). Difference CD curve: ∆∆ε = ∆ε ((S)-9a)−∆ε ((−)-1) and ∆∆ε = ∆ε ((R)-9b) − ∆ε ((−)-1). Adapted with permission from [35], copyright 2017, Elsevier.

a

b

of the asymmetric synthesis. We have prepared enantiopure (S)-(−)-11 and (R)-(+)-11 and unambiguously determined their absolute configurations by ECCD. Further application of this technique to allyl alcohols and amines is now in progress.

8.3  A CHIROPTICAL PROBE FOR DETERMINATION OF THE ABSOLUTE CONFIGURATION OF PRIMARY AMINES As described above, MBC acid (−)-1 is useful for chiral resolution and determination of the absolute configuration of aromatic alcohols. However, the application of this method cannot be extended to alcohols without a chromophore, because ECCD is observed only for the interaction between two chromophores [12,13]. This has been a week point of the ECCD method. Recently, researchers have reported effective probes to determine

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Figure 8.7  Conceptual diagram of the chirality detection by 2′,2″-quaterphenyl probe 12.

the absolute configurations of chiral mono-functional compounds without chromophores by ECCD [44-58]. We have designed a new chiroptical probe (12) with a 2′,2″-quaterphenyl group to determine the absolute configurations of primary amines [59]. Two biphenyl chromophores have been connected in the ortho position of bromo methyl groups (Fig. 8.7). We expected that (i) the probe 12 would couple readily with chiral primary amines yielding 12-chiral amine conjugates with a 7-membered ring, (ii) information on the absolute configurations of the amines would be transcribed into a spatial arrangement of two biphenyl units in the 2′,2″-quaterphenyl group which is detected directly by the ECCD method, and (iii) the conformers of the 12-chiral amine conjugates would be predicted by the analysis of conformational distributions using theoretical calculations. The chiroptical probe 12 was synthesized from 4-bromobenzoic acid via six steps with an overall yield of 20% [59]. 12-Amine conjugates, (S)-13a– (S)-19a, were prepared by the reaction of 12 with chiral primary amines in the presence of K2CO3 in acetonitrile (Scheme 2). Fig. 8.8 shows the UV and CD spectra of the 12-primary amine conjugates, (S)-13a, (S)-14a, (S)-17a and (S)-18a, in hexane. The UV spectrum of (S)-13a exhibits an intense absorption at 257 nm due to the π–π* transition arising from polarization along the long axes in the methoxybiphenyl chromophores. The CD spectrum of (S)-13a exhibits significant CD Cotton effects due to the exciton coupling between the two methoxybiphenyl chromophores; λext = 278.8 nm (∆ε1 = –9.2) and λext = 256.2 nm (∆ε2 = +8.5). The amplitude of ECCD (ACD value) [12,13], which is defined as ACD = ((1 (first Cotton effect) – ((2 (second Cotton effect), is −17.7. The negative exciton chirality indicates that the two long axes in

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Scheme 2  Coupling reaction of 12 and chiral primary amines.

Figure 8.8  CD spectra (A) and UV spectra (B) of (S)-13a, (S)-14a, (S)-17a, and (S)-18a (0.2 mM in hexane, 293 K). Abbreviations: CD, circular dichroism; UV, ultraviolet. Redrawn with permission from [59], copyright 2013, the American Chemical Society.

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the methoxy-biphenyl chromophores constitute an M twist. On the other hand, (R)-13a exhibited the mirror image of the CD spectrum of (S)-13a. The CD spectra of the 1-aliphatic amine conjugates, (S)-14a–(S)-17a, had the same sign for the Cotton effects as (S)-13a. It is important to note that even the differences in steric bulkiness for methyl and ethyl groups in (S)-17a can be discriminated. The CD spectra of 1-aromatic amine conjugates, (S)18a and (S)-19a, exhibited the opposite sign for the Cotton effects compared with (S)-13a–(S)-17a.This inversion of Cotton effects cannot be explained by the steric features of the substituents using the Charton steric parameters [60]. To clarify the mechanism of the inversion of the CD, theoretical calculations were carried out using a methoxy-omitted model. To obtain the relative amounts of M and P conformers of (S)-13b, preliminary conformational searches were carried out using an MMFF model and then all local minimum conformers were optimized with DFT using the B3LYP/6-31G* model [40]. Three conformers with lowest energies in (S)-13b were found within 3.0 kcal/mol (Fig. 8.9). The two long axes of the biphenyl chromophores of the conformers A and B constitute an M twist and those of the conformer C constitutes a P twist. Using Boltzmann’s equation (T = 298 K), the relative amounts of each conformer in (S)-13b were determined as 75:25 (M/P). In the 1-aliphatic amine conjugates, (S)-13b–(S)-15b and (S)-17b, the amounts of the M conformers were greater than those of the P conformers

Figure 8.9  Three major conformers of (S)-13b based on B3LYP/6-31G*. Dihedral angles (C6–C1–C1′–C6′) are −42.8° (A), −42.2° (B), and 43.5° (C). Redrawn with permission from [59], copyright 2013, the American Chemical Society.

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(Table 8.2). On the other hand, for the 1-aromatic amine conjugates, (S)18b and (S)-19b, the amounts of the P conformers were greater than those of the M conformers. These results were consistent regarding the sign of the observed CD Cotton effect. In addition, the ACD values were directly estimated by the relative amounts of the M and P conformers because the C6–C1–C1′–C6′ dihedral angle in all quaterphenyl units in the theoretical models was approximately constant (plus or minus c. 42°). A linear relationship between the ACD values and the calculated excess of M conformers was obtained with R2 = 0.975 (Fig. 8.10). By comparing the observed and calculated signs and the amplitude of the ACD values, the absolute configurations of chiral primary amines were determined. To evaluate the practical utility of the method for the determination of the ee of chiral primary amines, a calibration graph [52,57] was prepared using (S)- and (R)-13a for varying ee (−100, −80, −60, −40, −20, 0, +20, +40, +60, +80, +100 %ee of (S)-13a). The values for %ee were plotted versus the ACD values. The calibration graph was linear, with R2 = 0.999 (Fig. 8.11). This result indicated that the method is applicable to the quantitative determination of the ee of primary amines. Table 8.2  Comparison of the excess of M conformer and observed CD amplitude at B3LYP/6-31G* level

Entry

Compound

Calculated ratio (M/P)

Excess of M conformer, %a

Observed CD amplitude (ACD value)b

1 2 3 4 5 6

(S)-13b (S)-14b (S)-15b (S)-17b (S)-18b (S)-19b

75.2:24.8 56.0:44.0 57.6:42.4 52.8:47.2 31.0:69.0 22.7:77.3

50.4 12.0 15.2 5.6 −38.0 −54.6

−17.7 ((S)-14a) −7.5 ((S)-14a) −5.9 ((S)-15a) −1.8 ((S)-17a) +20.4 ((S)-18a) +22.2 ((S)-19a)

Excess of M conformer (%) = ((M conformer − P conformer)/((M conformer + P conformer)) × 100. ACD value: ACD = ((1 − ((2, where ((1 and ((2 are intensities of first and second Cotton effects, respectively. Adapted with permission from [59], copyright 2013, the American Chemical Society. a

b

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Figure 8.10  The relationship between the ACD values and excess of M conformer. Excess of M conformer (%) = ([M] − [P])/([M] + [P]) × 100, where [M] and [P] are the amounts of M and P conformers calculated by B3LYP/6-31G*, respectively. Redrawn with permission from [59], copyright 2013, the American Chemical Society.

Figure 8.11  Calibration graph for ACD values based on (S)-13a and (R)-13a with varying %ee values. Redrawn with permission from [59], copyright 2013, the American Chemical Society.

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The combination of the new chiroptical probe 12 and theoretical calculations represents an effective method to determine both the absolute configuration and the ee of chiral primary amines in a nonempirical manner. Further application of the method to amino alcohol and the amino acid is now in progress.

8.4  A CHIROPTICAL PROBE FOR CHIRALITY TRANSCRIPTION AND AMPLIFICATION BY THE FORMING OF [2]PSEUDOROTAXANES Chirality recognition in the supramolecular system plays an important role in many biological processes. Recently various artificial and biomimetic supramolecular systems for chirality recognition have been extensively studied [61–67]. A typical example of chirality recognition in supramolecular interaction is the complex formation by crown ethers. Crown ethers having chiral centers are widely used for chirality recognition toward chiral amines, ammonium salts, and amino acids by the difference of stabilities between diastereomeric complexes such as (R)-host–(R)-guest and (R)-host– (S)-guest complexes [68–70]. When we looked at the recognition system from a different angle, we came up with an idea for a supramolecular system. That is, an achiral crown ether having a functional group which can invert the chirality of an atropisomer depending on the chirality of guests could achieve chirality transcription to the crown ether from the guest. We designed a new benzo-2′,2″-quaterphenyl-26-crown-8 ether probe (20) [71]. Crown ether has been connected to 2′,2″-quaterphenyl chromophore. The 2′,2″-quaterphenyl group can flip such as a coaxis rotor blade and the macrocyclic ring can form [2]pseudorotaxanes [72] with cationic organic guests such as sec-ammonium salts. We expected that information on the chirality of the ammonium salts is transcribed into the 2′,2″-quaterphenyl unit of 20 (Fig. 8.12). Concretely, information on the absolute configurations of sec-ammonium ions would be transcribed into the spatial arrangement of two biphenyl chromophores in the 2′,2″-quaterphenyl group by ECCD. The new benzo-2′,2″-quaterphenyl-26-crown-8 ether probe (20) was prepared from biphenyl-2,2′-diol via five steps with an overall yield of 19% [71]. Crown ether probe (20) was treated with equimolar amounts of (R)21a-H·PF6, (R)-21b-H·PF6, (S)-21a-H·PF6, and (S)-21b-H·PF6 to give complexes [20·(R)-21a-H][PF6], [20·(R)-21b-H][PF6], [20·(S)-21a-H] [PF6], and [20·(S)-21b-H][PF6] in quantitative yield, respectively (Scheme 3).

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Figure 8.12  Conceptual diagram of the chiral transcription by the formation of [2]pseudorotaxanes.

Scheme 3  Complex formation of crown ether probe (20) with chiral sec-ammonium salts.

The structure of [20·(R)-21a-H][PF6] was confirmed by 1H NMR, 13C NMR, and nuclear Overhauser effect spectroscopy (NOESY). When equimolar amounts of 20 and (R)-21a-H·PF6 were mixed in CDCl3, the ethyleneoxy protons of 20 and the methyl protons of (R)-21a-H·PF6 are broadened in the 1H NMR spectrum, indicating the slow exchange complexation on the 1H NMR timescale [73,74]. In addition, NOESY spectrum of the mixture exhibits an NOE cross-peak between the ethyleneoxy protons of 20 and the methyl protons of (R)-21a-H+. From these results, it was confirmed that the combination of the crown ether probe (20) and the chiral sec-ammonium salts form the [2]pseudorotaxanes. The association constant Ka of [20·(R)-21a-H][PF6] was not determined because of the peak broadening

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in the 1H NMR spectrum.The Ka value of [20·(R)-21b-H][PF6] is obtained using 1H NMR titration experiments at 273 K to be 370 M−1. In the cold spray ionization-MS (CSI-MS) of [20·(R)-21a-H][PF6] and [20·(R)-21b-H][PF6], fragment ion peaks arising from [20·(R)-21aH]+ and [20·(R)-21b-H]+ were observed at m/z = 888 and 841, respectively. The CSI-MS spectral data also supports the formation of the [2] pseudorotaxanes. To investigate the chirality transcription and amplification of the [2] pseudorotaxanes, the UV–Vis and CD spectra of the crown ether probe (20), chiral sec-ammonium salts, and [2]pseudorotaxanes were measured (Fig. 8.13). Crown ether probe (20) has a λmax arising from π–π* transition

Figure 8.13  CD spectra (CHCl3, 273 K) (A) and UV spectra (CHCl3, 293 K) (B) of 20 (red), (R)-21a-H·PF6 (black), [20·(R)-21a-H][PF6] (pink), [20·(S)-21a-H][PF6] (purple), [20·(R)-21bH][PF6] (green), and [20·(S)-2b-H][PF6] (blue). Abbreviations: CD, circular dichroism; UV, ultraviolet. Redrawn with permission from [71], copyright 2013, the Royal Society of Chemistry.

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around 260 nm in the UV–Vis spectrum. This absorption is due to the polarization along the long axis of the biphenyl chromophore moieties in the 2′,2″-quaterphenyl group. Since 20 is an achiral molecule having a flexible 2′,2″-quaterphenyl group, no Cotton effect was observed. Although the chiral ammonium salt (R)-21a-H·PF6 shows CD Cotton effects around 260 nm (aromatic 1Lb band), the amplitude is very weak. On the other hand, the [2]pseudorotaxane [20·(R)-21a-H][PF6] exhibits significant CD Cotton effects due to the exciton coupling between the two biphenyl chromophores in the 2′,2″-quaterphenyl group; λext = 263.3 nm (∆ε1 = –3.6), λext = 242.6 nm (∆ε2 = +6.6). The negative first and positive second Cotton effects indicate the negative exciton chirality between the two chromophores. Therefore, the two long axes of the two chromophores of [20·(R)-21a-H][PF6] constitute a counterclockwise screw sense. In contrast, [2]pseudorotaxane [20·(S)-21a-H][PF6] shows the mirror image of the Cotton effects of [20·(R)-21a-H][PF6] in the CD spectrum (λext = 261.6 nm (∆ε1 = +3.9), λext = 242.3 nm (∆ε2 = –7.4)). The CD spectra of [20·(R)-21b-H][PF6] and [20·(S)-21b-H][PF6] show the same sign of Cotton effects as those of [20·(R)-21a-H][PF6] and [20·(S)-21a-H] [PF6], respectively. This means that the [2]rotaxanes containing the chiral sec-ammonium salts with the same absolute configuration show the same Cotton effects in CD spectra. Because good crystals of the [2]pseudorotaxanes suitable for X-ray crystallography were not obtained, a conformer search using molecular mechanics and then optimization using semi-empirical AM1 calculations followed by ab initio B3LYP/6-31G* calculations of [20·(R)-21a-H]+ were carried out (Fig. 8.14). The optimized structure shows that the sec-ammonium salt is incorporated in the crown ether ring and the two biphenyl moieties constitute a counterclockwise screw sense with the dihedral angle (C6–C1–C1’–C6’) of −84°. The modeling strongly supports the result of CD measurements. To evaluate the effectiveness of the [2]pseudorotaxane system for quantitative enantiomeric excess (ee) determination of chiral sec-ammonium ions, a calibration curve [52,57] was created using (R)- and (S)-21a-H·PF6 in varying ee (−100, −80, −60, −40, −20, 0, +20, +40, +60, +80, +100 %ee of (R)-21a-H·PF6). The CD amplitudes at 261.6 nm were plotted versus %ee (Fig. 8.15). The calibration curve shows a linear relationship with R2 = 0.998. The result indicates that the system is applicable to quantitative ee determination of chiral sec-ammonium ions. In this system, the crown ether probe (20) forms complexes with chiral sec-ammonium ions to make [2]pseudorotaxanes, and the chirality of the

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Figure 8.14  Preferred conformation of [20·(R)-21a-H][PF6] (A). Optimized structure of [21·(R)-21a-H]+ by ab initio B3LYP/6-31G* (B). Redrawn with permission from [71], copyright 2013, the Royal Society of Chemistry.

Figure 8.15  CD signals at 261.6 nm of [20·(R)-21a-H][PF6] and [20·(S)-21a-H][PF6] with varying %ee values. Abbreviation: CD, circular dichroism. Redrawn with permission from [71], copyright 2013, the Royal Society of Chemistry.

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sec-ammonium ion is transcribed into the crown ether. Since two biphenyl parts in the 2′,2″-quaterphenyl group rotate like a coaxial rotor, the chirality of the atropisomers of the crown ether can be inverted depending on the chirality of the guest ions. When the crown ether probe (20) formed the chiral [2]pseudorotaxane with (R)-sec-ammonium ions, the negative first and positive second Cotton effects indicating a counterclockwise screw sense were observed in the CD spectrum. The chirality transcription and amplification by forming of the pseudorotaxane would be a practical method to determine the absolute configuration and ee of chiral sec-amines.

8.5  CONCLUSIONS Many chiroptical probes have been reported in the literature with limited applications for chiral sensing and determination of absolute stereochemistry by ECCD.We have developed chiroptical probes based on biphenyl chromophore for determination of absolute stereochemistry by ECCD. In particular, the ECCD detection of biphenyl units in 2′,2″-quaterphenyl has the advantage to determine the absolute twist in a nonempirical manner. Furthermore, the absolute twist could be supported by theoretical calculations. The combination of ECCD detection of chiroptical probes and theoretical calculations would open a new way to determine the absolute configurations. Further application of the chiroptical probes is now in progress.

ACKNOWLEDGMENTS The authors sincerely thank the coworkers of this research for their contributions. This research was supported by Grant-in-Aid for Scientific Research (11011761, 25410100, 17K05845) and Supported Program for the Strategic Research Foundation at Private Universities (2012–2016) from MEXT (Japan). A grant from Futaba Electronics Memorial Foundation is also acknowledged.

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[33] Yamamoto, Y.; Akagi, M.; Shimanuki, K.; Kuwahara, S.; Watanabe, M.; Harada, N. Tetrahedron 2014, 25, 1456. [34] Akagi, M.; Sekiguchi, S.; Taji, H.; Kasai, Y.; Kuwahara, S.; Watanabe, M.; Harada, N. Tetrahedron 2014, 25, 1466. [35] Kuwahara, S.; Tasaki, N.; Suzuki, Y.; Nakagawa, M.; Ikeda, M.; Habata, N. Tetrahedron 2017, 28, 945. [36] Iwamoto, K.; Shimizu, H.; Araki, K.; Shinkai, S. J. Am. Chem. Soc. 1993, 115, 3997. [37] Vineyard, B. D.; Knowles, W. S.; Sabacky, M. J.; Bachman, G. L.; Weinkauff, D. J. J. Am. Chem. Soc. 1977, 99, 5946. [38] Xu, L. W.; Li, L.; Lai, G. Q.; Jiang, J. X. Chem. Soc. Rev. 2011, 40, 1777. [39] Nagatsugi, F.; Takahashi,Y.; Kobayashi, M.; Kuwahara, S.; Kusano, S.; Chikuni, T.; Hagihara, S.; Harada, N. Mol. BioSyst. 2013, 9, 969. [40] Spartan’10, Wavefunction, Inc.: Irvine, CA, 2010. [41] Ohmori, K.; Mori, K.; Ishikawa, Y.; Tsuruta, H.; Kuwahara, S.; Harada, N.; Suzuki, K. Angew. Chem. Int. Ed. Engl. 2004, 43, 3167. [42] Terashima, S.; Tanno, N.; Koga, K. Tetrahedron Lett. 1980, 21, 2753. [43] Ohkuma, T.; Ooka, H.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc. 1995, 117, 10417. [44] Huang, X. F.; Borhan, B.; Rickman, B. H.; Nakanishi, K.; Berova, N. Chem. Eur. J. 2000, 6, 216. [45] Borovkov,V.V.; Lintuluoto, J. M.; Inoue,Y. Org. Lett. 2000, 2, 1565. [46] Kurtan,T.; Nesnas, N.; Li,Y. Q.; Huang, X. F.; Nakanishi, K.; Berova, N. J. Am. Chem. Soc. 2001, 123, 5962. [47] Kurtan,T.; Nesnas, N.; Koehn, F. E.; Li,Y. Q.; Nakanishi, K.; Berova, N. J. Am. Chem. Soc. 2001, 123, 5974. [48] Borovkov,V.V.; Lintuluoto, J. M.; Inoue,Y. J. Am. Chem. Soc. 2001, 123, 2979. [49] Zhang, J.; Holmes, A. E.; Sharma, A.; Brooks, N. R.; Rarig, R. S.; Zubieta, J.; Canary, J. W. Chirality 2003, 15, 180. [50] Hosoi, S.; Serata, J.; Sakushima, A.; Kiuchi, F.; Takahashi, I.; Ohta, T. Lett. Org. Chem. 2006, 3, 58. [51] Nieto, S.; Dragna, J. M.; Anslyn, E.V. Chem. Eur. J. 2010, 16, 227. [52] Iwaniuk, D. P.; Wolf, C. J. Am. Chem. Soc. 2011, 133, 2414. [53] Iwaniuk, D. P.; Wolf, C. Org. Lett. 2011, 13, 2602. [54] Ghosn, M. W.; Wolf, C. Tetrahedron 2011, 67, 6799. [55] Fujiwara, T.; Taniguchi, Y.; Katsumoto, Y.; Tanaka, T.; Node, M.; Ozeki, M.; Yamashita, M.; Hosoi, S. Tetrahedron 2012, 23, 981. [56] Dragna, J. M.; Pescitelli, G.; Tran, L.; Lynch,V. M.; Anslyn, E.V.; Di Bari, L. J. Am. Chem. Soc. 2012, 134, 4398. [57] You, L.; Pescitelli, G.; Anslyn, E.V.; Di Bari, L. J. Am. Chem. Soc. 2012, 134, 7117–7125. [58] Hayashi, S.;Yotsukura, M.; Noji, M.; Takanami, T. Chem. Commun. 2015, 51, 11068. [59] Kuwahara, S.; Nakamura, M.; Yamaguchi, A.; Ikeda, M.; Habata, Y. Org. Lett. 2013, 15, 5738. [60] Charton, M. J. Am. Chem. Soc. 1975, 97, 1552. [61] Philp, D.; Stoddart, J. F. Angew. Chem. Int. Ed. Engl. 1996, 35, 1155. [62] Amabilino, D. B.; Stoddart, J. F. Chem. Rev. 1995, 95, 2725. [63] Cornelissen, J.; Rowan, A. E.; Nolte, R. J. M.; Sommerdijk, N. Chem. Rev. 2001, 101, 4039. [64] Green, M. M.; Cheon, K. S.;Yang, S.Y.; Park, J. W.; Swansburg, S.; Liu, W. H. Acc. Chem. Res. 2001, 34, 672. [65] Pu, L. Chem. Rev. 2004, 104, 1687. [66] Mateos-Timoneda, M. A.; Crego-Calama, M.; Reinhoudt, D. N. Chem. Soc. Rev. 2004, 33, 363. [67] Hembury, G. A.; Borovkov,V.V.; Inoue,Y. Chem. Rev. 2008, 108, 1.

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CHAPTER 9

Chiral Analysis by NMR Spectroscopy: Chiral Solvating Agents Federica Balzano, Gloria Uccello-Barretta, Federica Aiello University of Pisa, Pisa, Italy

9.1  INTRODUCTION Growing awareness of the role of chirality with respect to the therapeutic and regulatory effects of pharmaceutical products has made more and more demanding and urgent the need of reliable methods for monitoring and quantifying stereoisomeric products, among which direct and noninvasive methods of detection are privileged. In the area of spectroscopic methods, nuclear magnetic resonance (NMR) spectroscopy plays a leading role since it provides several measurable parameters for each of the observable nuclei of the stereoisomeric products, such as chemical shifts (δ ), coupling constants (J), and relaxation rates (R), which depend on the chemical environment or dynamic properties of the nuclei, even though chemical shift measurements are privileged in terms of operative convenience and also responsivity. Importantly, the integrated areas of NMR signals are the parameters which depend exclusively on the amount of substance and, hence, allow to perform accurate quantitative determinations. Intrinsic isochrony of enantiotopic nuclei of enantiomeric substrates is the counterpart to this copiousness of potentially available resources: enantiomers are in the same chemical environment and, hence, indistinguishable by NMR. On the other hand, enantiomer isochrony can be removed, at least in principle, by transferring enantiomers into a diastereomeric environment by exploiting suitable enantiomerically pure chiral auxiliaries. Three main kinds of chiral auxiliaries for NMR spectroscopy have been developed: chiral derivatizing agents (CDAs), chiral solvating agents (CSAs), and chiral lanthanide shift reagents (CLSRs). CDAs are enantiopure chiral compounds which are reacted with the enantiomeric substrates in order to form diastereomeric derivatives by formation of true covalent bonds. High enantiopurity Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00009-4 Copyright © 2018 Elsevier B.V. All rights reserved.

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of CDAs and the absence of phenomena of kinetic resolution are prerequisites for the reliability of enantiomers’ quantification. CSAs are simply mixed to the two enantiomers to be analyzed and form with them diastereomeric solvates which are stabilized by hydrogen bonds, dipole–dipole, or π–π interactions. CLSRs are similar to CSAs with regard to the absence of covalent chemical bond connections with the enantiomeric substrates, but they include a paramagnetic center which can potentially enhance chemical shift differentiation. Once enantiomers are transformed into diastereomers by covalent linking to CDAs or by noncovalent interactions with CSAs or CLSRs, distinct resonances can be detected for the two enantiomers, the magnitude of differentiation of which constitutes the nonequivalence (∆δ, absolute value of the difference between the chemical shifts of diastereotopic nuclei).The integration of the resonances of corresponding nuclei of the two enantiomers in the diastereomeric derivatives or complexes affords the enantiomeric purity, whereas their relative positions (sense of nonequivalence) depends on the absolute configuration. The enantioresolution quotient (E) [1,2], which is defined as the ratio between the nonequivalence and the overall width of the signal (singlet or multiplet) before adding the chiral auxiliary, represents another fundamental parameter. Enantioresolution quotient, nonequivalence, enantiomeric ratio, and overall experimental time (including sample preparation) all together give a real indication about the quality of the enantiodiscrimination experiments. The enormous interest toward chiral auxiliaries for NMR spectroscopy is witnessed by the huge literature on this topic. Several reviews and chapter books have been published over the last 10 years [3–14] in order to describe and critically compare peculiarities of every category of chiral auxiliaries for NMR. Focusing now on CSAs needs at least a concise historical introduction: the first experiments involving CSAs are due to Pirkle [15], who showed that the fluorine resonance of racemic 2,2,2-trifluoro-1-phenylethanol (Fig. 9.1), which appeared as a doublet due to H–F scalar coupling in the pure

Figure 9.1  α-Phenylethylamine (1) and 2,2,2-trifluoro-1-phenylethanol.

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compound, underwent doubling in optically active α-phenylethylamine (1, Fig. 9.1). In the case of enantiomerically enriched alcohol, the integrated areas of the two doublets corresponded to the enantiomeric excesses (ees) of the alcohol. Some peculiarities of the CSAs were pointed out by means of these first experiments [16]: very high enantiomeric purity of CSAs is not as fundamental as for CDAs. CSAs’ enantiopurity only affects the magnitude of differentiation of enantiomers, but not quantification reliability. The diastereomeric products do not need to be in a well-defined stoichiometry. CSA resonances of the two diastereomeric solvates are not differentiated and, hence, the nucleus to be selected as probe for the enantiomeric purity determinations must belong to the enantiomeric substrates. All of these properties can be rationalized on considering that, in the fast exchange conditions, which in general hold in equilibria involving CSAs, the measured chemical shifts (δobs) of each enantiomer are the weighted average of the corresponding chemical shifts in the free (δf) and bound (δb) states: 

δobs = x f δf + x bδb

(9.1)

where xf and xb are the molar fractions of the solute in the free and bound states, respectively. Therefore, the differentiation of enantiomers inside the diastereomeric solvates stems from two different sources: stability constants of two diastereomeric solvates, producing different xb values of the two enantiomers, and differences in their stereochemical environment (δb values). Interestingly, Bourˇ et al. explored by molecular dynamics simulations and density functional computations the origin of chemical shift changes due to the presence of the chiral auxiliaries, revealing that perturbed conformational equilibria are mainly responsible for chemical shift differentiations [17]. Dealing with complexation equilibria means that enantiomer differentiation can be controlled to some extent by using solvents which do not compete with the CSA for the interaction with the enantiomers, by lowering the temperature in order to affect the magnitude of the stability constants of the diastereomeric solvates and/or produce a conformational restriction, and, finally, by increasing the total concentration or CSA to substrate molar ratios. To this last respect, it must bear in mind that adding further equivalents of CSA bring about a saturation point beyond which any further increase in the separation between the signals of the two enantiomers is not observed [16].

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In the analysis of single enantiomers having very high enantiomeric purity without having access to the racemate, an important issue is either the sample is really enantiomerically pure or the analytical method is ineffective. In these cases, both enantiomers of the CSA can be employed, as suggested by Pirkle already in the late 1982 [16]. This method, which has been recently developed by Lewis et al. [18], is based on the fact that the signals of the analyte A in the (R)-CSA/(R)-A and (S)-CSA/(R)-A mixtures are, respectively, at the same positions of (S)-CSA/(S)-A and (R)-CSA/(S)-A mixtures. Each sample must be prepared accurately with regard to the total concentration and analyte to CSA molar ratio. Furthermore, the enantiomers of the CSA must be of similar enantiomeric purity in order to provide reliable results. Care has also to be taken to phenomena of competitive binding arising from high differences in the association constants of the two diastereomeric solvates: when the enantiomeric composition is changed, the enantiomers may be complexed to different extents, leading to changes in the chemical shifts, which could make difficult the identification of the minor isomer. In general, the better way is increasing the CSA to analyte ratio and to perform titrations at different ratios of CSAs. In principle, any NMR-active nucleus can be selected for the detection of the enantiomeric substrates in the NMR-based chiral recognition, but dipolar nuclei giving sharp signals are preferred. 1H nucleus is always privileged due to its high sensitivity and also responsivity to change of chemical environment due to the presence of the chiral auxiliary. Intrinsic problems in 1H observation entail the poor spectral dispersion and multiplicity, which, in some cases, cause severe signals overlapping, made aggravated by the copresence of the chiral auxiliary. Signals overlapping can be partially resolved by using special techniques [19–23] for the removal of scalar couplings, such as in the case of frequency-selective 1D 1H NMR experiments, in which fully homodecoupled 1H NMR resonances appear as resolved 1D singlets without their typical J(HH) coupling constant multiplet structures. The comparison of the integrated area of the signals of the minor isomer to the 13C satellite signal of the major isomer has been proposed for the assessment of stereoiomeric excesses to ≥98.9 [24,25]. 1 H NMR nonequivalences are, in general, higher than those of 13C; however, low-sensitivity 13C nuclei give better enantioresolution ratios due to their very low linewidth, and errors associated to enantiomeric purities’ determinations are consequently lower [2]. The unique problem in using 13 C observation is the intrinsic low sensitivity of this nucleus, but nowadays the use of a cold probe with direct detection for 13C allows to enhance

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considerably the sensitivity and reduce the experimental time needed. The value of 13C NMR spectroscopy in alternative to proton NMR has been extensively recognized for NMR metabolomic investigation [26]. The use of dissolution dynamic nuclear polarization (d-DNP) [27] could boost the use of 13C NMR spectroscopy for the detection of enantiomeric signals. d-DNP is based on the initial polarization of solely the chiral analyte, by insertion of the sample into the dynamic nuclear polarizer and hyperpolarization of the sample at low temperature, followed by a quick mix with the solvent containing the CSA; subsequently, the sample is transferred into the NMR magnet and 13C NMR spectrum acquired. In this way, the signals of the CSA would be minimized or not detected in the resulting spectrum.

9.2  LOW-MOLECULAR-WEIGHT CSAS Since its earliest applications, Pirkle’s alcohol [16] (S)-(+)-1-(9anthryl)-2,2,2-trifluoroethanol (TFAE, 2, Fig. 9.2) has represented one of the most popular low-molecular-weight CSAs and stood out for versatility and effectiveness. In the last decade, several applications of Pirkle’s CSAs have been proposed, as in the 13C NMR discrimination of all eight stereoisomers of α-tocopherol (Fig. 9.2) [28], at low temperature (250 K) and using a high concentration of α-tocopherol in the presence of 4–5-fold molar excess of TFAE, or in the enantiodifferentiation of β-hydroxylamides (Fig. 9.2),

Figure 9.2  Pirkle’s alcohol (TFAE, 2) and selected enantiodifferentiated compounds.

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aimed to evaluate the occurrence of chiral recognition during the enzymatic hydrolysis of N-acylaziridines [29]. TFAE has also been applied to the analysis of the stereogenic properties of cyclophosphazene derivatives like the ones of Fig. 9.2 [30–42], the 31P NMR signals of which underwent relevant doublings in the mixtures with the CSA in the case of the racemic chiral forms, together with remarkable shifts of complexed achiral/meso forms. Care should be taken of doublings caused by spin system changes rather than enantiodifferentiation. However, Yesilot demonstrated that chiral chromatography is more reliable than using 31P NMR spectroscopy under the addition of a CSA for characterizing the stereogenic properties of cyclotriphosphazene derivatives containing one [43] or two equivalent centers of chirality [44], trans (racemic) and cis (meso) respectively, as for di-spiro derivatives of the reaction of gem-disubstituted cyclotriphosphazenes, N3P3Cl4X2 (X = Ph, PhS, PhNH, PhO) with 3-amino-1-propanol. The enantiomers of monospirocyclotriphosphazenes (Fig. 9.2) have been distinguished by 31P NMR spectroscopy on the addition of (S)-2 in CDCl3 at a 30:1 CSA to substrate molar ratio [45]. Chiral and achiral forms of the product of octabromination of p-tertbutylcalix[8]arene octamethyl ether (Fig. 9.2) were distinguished on the basis of the splitting pattern of the methoxy signals of the macrocyclic system in a chiral medium (TFAE) [46]. The number of split signals is different in the case of methoxy groups which are enantiotopic by external comparison in the chiral form and enantiotopic by internal comparison in the achiral form. Trifluoromethyl carbinols, mainly TFAE, produced satisfactory enantiodiscrimination of proton signals of chiral ∆2-thiazolines-1,3 (Fig. 9.2) and also of 19F NMR resonances of fluorinated derivatives, with a good correlation between sense of nonequivalence and absolute configuration [47]. The same CSA (TFAE) guidedVirgili et al. in the development of (R,R)α,α′-bis(trifluoromethyl)-9,10-anthracenedimethanol [48] (3) and (R,R)-α, α′-bis(trifluoromethyl)-1,8-anthracenedimethanol [49] (4) (Fig. 9.3), as chiral complexing agents for a wide range of polar compounds with aryl groups, mainly amine and/or alcohol-based substrates. Their use has been successfully extended to more complex, highly functionalized structures, such as pharmaceutical agents carvedilol [50], fluoxetine [48,50], or colchicine [51] (Fig. 9.3). Interestingly, (S,S)-3 and its D8-deuterated form 5 (Fig. 9.3) have also been reported as CSAs for lamotrigine (Fig. 9.3), which presents enantiomerism due to axial chirality in addition to isomeric and tautomeric forms [52].

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Figure 9.3  CSAs 3–5 and some enantiodicriminated substrates.

Among phenylacetic acid analogs, (R)-(−)-(2-naphthyloxy)phenylacetic acid (6) (Fig. 9.4) has been used for the ee determination of hydroxymethylmexiletine (Fig. 9.4), one of the main metabolites of mexiletine, a chiral therapeutically relevant compound [53]. In a recent application to the discrimination of zolmitriptan enantiomers [54], four different CSAs were compared, that is, (R)-(−)-α-methoxyphenyl acetic acid (7, Fig. 9.4), (R)-(+)-α-methoxy-α-trifluoromethylphenyl acetic acid (8, Fig. 9.4), (S)-2 (Fig. 9.2), and (S)-(−)-1,1′-(2-naphthol) (BINOL), among which (R)-7 gave the best enantiodiscriminating effects in the 1H NMR spectra. An application of (S)-7 and (S)-(9-anthryl)methoxyacetic acid (9, Fig. 9.4) as CSAs has also been reported in the stereochemical analysis of desaturase-mediated sulfoxidation [55,56]. The use of chiral reference standards was required in order to properly assign the absolute configuration of analytes by 19F NMR. This is because fluorine nuclei are highly sensitive to subtle differences in the structures of the sulfoxide-solvating agent complex and hence are highly dependent on the nature of the chiral NMR solvating agent. Use of CSAs with larger aromatic rings such as 9 (Fig. 9.4) allows to extend the range at which the fluorine tag can function [56]. Enantiodiscriminating efficiency of phenylacetic acids can be enhanced by O-arylation or O-heteroarylation, which requires the availability of suitable synthetic procedure: Cavalluzzi and co-workers [57] developed a one-step copper-promoted arylation of hydroxyl groups that applied to the coupling of mandelic acid with several halobenzenes and haloheteroarenes. The corresponding derivatives 10 (Fig. 9.4) were efficient CSAs for the direct 1H NMR determination of ees of several clinically and pharmacologically relevant amines [57].

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Figure 9.4  CSAs 6–15 and selected applications.

Gil and Virgili [58] envisaged first the intrinsic advantages of having perdeuterated CSAs for ee determinations and realized perdeuterio-1-(9anthryl)-2,2,2-trifluoroethanol, which does not interfere with resonances of enantiomeric signals. Analogously, Cavalluzzi et al. [59] proposed (R)-(−)-2-(2,3,4,5,6-pentafluorophenoxy)-2-(phenyl-d5)acetic acid (11, Fig. 9.4) as a CSA.The compound, which was synthesized as racemate from (±)-mandelic acid-d5 and then resolved with (R)-1-phenylethylamine, allowed the differentiation of enantiomers of the three quinoline-containing antimalarial agents chloroquine, hydroxychloroquine, and mefloquine (Fig. 9.4) [59]. d-Camphorsulfonic acid (12, Fig. 9.4) represents another low-molecularweight CSA, which, as an example, allowed to confirm as trans the stereochemistry of the dispirocyclic cyclotriphosphazene shown in Fig. 9.4 on the basis of the fact that its 31P NMR resonances underwent doubling in the presence of the CSA [60].

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By reacting natural amino acids with 1,8-naphthalic anhydride, the two CSAs 13 and 14 (Fig. 9.4) were easily synthesized [61], which are effective in the enantiodifferentiation of chiral α-arylalkylamines. The majority of proton signals of the amines were split in the presence of the CSA, thus allowing accurate ee’s determinations.The molecular basis of enantiodiscrimination was also investigated by analyzing the complexation stoichiometries of the diastereomeric complexes, determining the association constants, and analyzing the stereochemical features by nuclear overhauser effect (NOE) methods. The simple structure of (S)-(−)-ethyl lactate (15, Fig. 9.4), which is an inexpensive reagent, lends the chiral reagent to potentially broad applications. It has been used to induce differentiation of 1H, 13C, and 15N NMR signals of phytoalexins, spirobrassinin, and 1-methoxyspirobrassinin (Fig. 9.4), lipophilic substances possessing antimicrobial properties [62]. Interestingly, the phenomenon of self-induced diastereomeric anisochronism (SIDA) has been observed for spirobrassinin (Fig. 9.4), where differentiation of enantiomeric signals was observed in the absence of chiral auxiliaries, due to the formation of dimeric self-aggregates [62], by noncovalent interactions. SIDA, which was firstly described by Uskokovic in 1969 for dihydroquinine (Fig. 9.5) [63], is very attractive but limited in its applications since significant signal splitting is often only observed in highly concentrated solutions, very low temperatures, and nonpolar solvents. In order to widen

Figure 9.5  Self-induced diastereomeric anisochronism.

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the applicability of SIDA in chiral analysis, a different approach has been recently proposed [64], involving the substrate modification by introduction of N-3,5-dinitrobenzoyl group which strongly promotes noncovalent self-aggregation phenomena. In the case of 3,5-DNB-Ala-OMe (Fig. 9.5), self-induced enantiodifferentiations up to 0.4 ppm were measured in C6D6. An alternative and elegant way to promote the formation of homo- and heterodimers of alcohol enantiomers for NMR self-discrimination involves the fast and selective cleavage of one of the Al-bonded pyridine groups of the nonchiral [EtAl(6-Me-2-py)3Li] reagent by chiral alcohols under mild conditions, to produce heteroleptic aluminum complexes (Fig. 9.5) [65]. The dimers are easily distinguished by 1H and 7Li NMR spectroscopy in toluene-d8, allowing the fast evaluation of ees [65]. From the observation that, in the absence of chiral auxiliaries, diketopiperazine (S)-1-benzyl-3-[(Z)-(dimethylamino)methylidene]-6-methylpiperazine-2,5-dione (DKP, Fig. 9.5) undergoes concentration-dependent dimerization processes which lead to self-discrimination phenomena and, hence, splitting of enantiomers NMR signals in the absence of an external CSA, the precursor of DKP, (S)-1-benzyl-6-methylpiperazine-2,5-dione (16, Fig. 9.5), was exploited as a CSA for DKP and also for cyclic and acyclic amides [66]. The relevant feature of this CSA is the presence of vicinal hydrogen-bond-accepting (C=O) and hydrogen-bond-donating (NH) groups. This stereoelectronic feature favored the differentiation of enantiomers of tryprostatin B analogues (Fig. 9.5) [67]. The relevance of the nature of N-substituents of diketopiperazines on their properties as CSAs was demonstrated by comparing (S)-16 (Fig. 9.5), its regioisomer (S)-1-benzyl-3-methylpiperazine-2,5-dione, (S)-17, and its close analogue (S)-1-(pentafluorobenzyl)-6-methylpiperazine-2,5-dione, (S)-18 (Fig. 9.5), in NMR enantiodiscrimination experiments of N-acylamino acid esters: CSA (S)-16 exhibited the strongest chiral solvating properties for racemic α-amino acid derivatives [68]. In view of the relevance of chiral carboxylic acids and their derivatives in a wide variety of biological processes, several CSAs for their enantiodiscrimination by NMR have been proposed, including chiral amines [69,70] and amino alcohols [71]. For instance, enantiodifferentiating efficiency of αphenylethylamine toward chiral carboxylic acids was remarkably improved by incorporation of two anisotropic groups as in 19 (Fig. 9.6) [69]. Significant splittings of the resonances of several aromatic and nonaromatic acids as well as N-derivatives of some amino acids and dicarboxylic acids were detected. Amines 20 and 21 (Fig. 9.6) produced very high differentiation of

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Figure 9.6  CSAs 19–38.

P NMR signals of enantiomers of BINOL and H8-BINOL-based chiral phosphoric acids (Fig. 9.6), as the consequence of the formation of diastereomeric ammonium phosphates [70]. Chiral amino alcohols 22–26 (Fig. 9.6), which incorporate anisotropic aromatic groups and multiple chiral centers, were applied as CSAs to the analysis of chiral carboxylic acids, including Ts-derivatives of some α-amino acids, α-hydroxyl acids, and dibenzoyl-tartaric acid [71]. 1-(α-Aminobenzyl)-2-naphthol (27, Fig. 9.6) represents a chiral substrate which can be readily prepared in enantiomerically pure form and can also be easily modified to tune its enantiodiscriminating properties on the basis of structural requirements of enantiomeric substrates [72]. For this reason, a series of its enantiomerically pure derivatives 28–30 (Fig. 9.6) has been probed in the NMR differentiation of mandelic acid enantiomers. CSA 30 with a free NH group and the benzyl group produced the best nonequivalence of the methine proton of mandelic acid in the 1H NMR spectrum. 31

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Among the variety of commercially available amino alcohol, prolinederived diphenylprolinols have been widely employed as chiral ligands in asymmetric synthesis and catalysis and also selected as CSAs for chiral acids [73]. In particular, (S)-diphenyl(pyrrolidin-2-yl)methanol (31, Fig. 9.6) with free NH and OH groups was effective in the enantiodiscrimination of several classes of carboxylic acids [73]. Roof-shaped molecules were firstly introduced by Weber et al. [74,75] as geometrically designed molecules, the basic unit of which resembles a roof of a house with functional groups protruding like antenna and a bulky skeleton similar to its foundation (32–34, Fig. 9.6). After probing enantiodiscriminating efficiency of optically pure roof-shaped amines, like 35 (Fig. 9.6), toward mandelic acid as the standard substrate, amines were screened in the differentiation by the 1H, 13C, 19F, and 31P NMR of enantiomers of several α-functionalized acids [76]. Roof-shaped secondary amines 36–38 (Fig. 9.6) gave enantiodiscrimination not only of carboxylic acids, but also of weakly acidic BINOL and its derivatives [77]. Chiral amides, for the first time proposed by Deshmukh et al. [78] (39, Fig. 9.7), represent a versatile family of CSAs extensively employed for the enantiodiscrimination of several classes of compounds.Their effectiveness is

Figure 9.7  CSAs 39–54.

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mainly due to their ability to act simultaneously as hydrogen bond donors and acceptors, with the aromatic moiety favoring π–π interactions. Derivatives like (R)-40 (Fig. 9.7) with perpendicularly arranged dinitrobenzoyl and naphthalene rings generate chiral cleft structures inside which a good fit can be obtained only for one enantiomer [79,80]. The enantioselective complexation yields significant nonequivalences of the diastereotopic protons of different kinds of multifunctional tertiary alcohols [79] and protected amines [80] even in the presence of substoichiometric amounts of CSAs. Kagan’s amides (S)-39 and (S)-40 (Fig. 9.7), which were employed in the 31P NMR enantiodifferentiation of phopholene oxides [81], constituted poor enantiodiscriminating agents for chiral nonracemic atropoisomeric bis-phosphine dioxides, but represented the best choice for monophosphine oxides (Fig. 9.7), superior to (S)-naproxen, (S)-mandelic acid, dibenzoyltartaric acid, and its mono-dimethylamide derivative [82]. The introduction of a strong electron-withdrawing 1–3,5-bis(trifluoromethyl)phenyl group [83] as in (S)-41 (Fig. 9.7), in which a quite open arrangement of the aromatic rings is present, allowed the hydrogen attached to the nitrogen of the amide group of CSAs to form stronger hydrogen bonding with chiral amides, sulfoxides, α-substituted acids, α-hydroxy ketone, keto epoxides, and N-protected amino acids. Chiral amino alcohol 42 (Fig. 9.7) and chiral amino alcohol imides 43–46 (Fig. 9.7), which were synthesized by regioselective ring opening reaction of (R)-N-(2,3-epoxypropyl)phthalimide with appropriate chiral amines, were probed as CSAs for the NMR enantiodiscrimination of carboxylic acids [84]. Derivatives of optically active trans-1,2-diaminocyclohexane are very popular building blocks for new CSAs, in which aromatic functional groups can be introduced with the purpose of enhancing enantiomer differentiation by magnetic anisotropic effects. By following this rationale, a new class of imide CSAs 47–49 (Fig. 9.7) was obtained [85], by reacting (1R,2R)-1,2-diaminocyclohexane with 1,8-naphthalic anhydrides, which showed better enantiodiscriminating efficiency toward several classes of carboxylic acids, compared to α-phenylethylamine. Remarkable nonequivalences were measured, but only in the cases of lipophilic systems, and the enantioselective discrimination required the use of less polar solvents. New amphiphilic CSAs 50 and 51 (Fig. 9.7) were versatile for a wide range of chiral carboxylic acids including both lipophilic and hydrophilic carboxylic acids, such as chiral hydroxylated acid in protic polar solvent [86].

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The observation that the (1S,2S)-1,2-diaminocyclohexane-derived chiral selector for HPLC, endowed with an ureide tethering linkage and devoid of aromatic moieties, could be successfully employed for the resolution of N-(3,5-dinitrobenzoyl)amino acids [87] suggested to obtain an analogous CSA 52 (Fig. 9.7) by reacting diamine with ethyl isocyanate. The chiral auxiliary was applied to the 1H NMR differentiation of enantiomers of the same class of amino acids derivatives [88]. As an attempt to obtain a chiral auxiliary which serves as a single probe for the enantiodiscrimination of chiral molecules containing different kinds of functional groups, (1S,2S)-N,N′-dihydroxy-N,N′-bis(diphenylacetyl)-1, 2-cyclohexanediamine (53, Fig. 9.7), a C2 symmetric chiral hydroxamic acid, has been proposed [89]. This CSA was remarkably versatile, probably due to the presence of multiple hydrogen bonding sites. The discrimination of a large number of molecules with different functional groups was achieved, such as acids, hydroxy acids, diacids, primary, secondary and tertiary amines, alcohols, amino alcohols, oxazolidones, oxathiones, carbonates, diols, epoxides, phosphoric acids, cyanohydrins, piperidine, piperazine, hydroxy esters, sulfoxides, prochiral amines, prochiral acids, prochiral alcohols, and prochiral amino alcohols. Differentiation of enantiomeric signals was detected also in 13 C, 19F, and 31P spectra and in a wide range of deuterated solvents, satisfying solubility requirements of the enantiomeric substrates. In the cases of remarkably overcrowded spectral regions, due to multiplicity pattern and overlapping of peaks from both (R)- and (S)-enantiomers, pure shift NMR approach [20,21] was exploited for decoupling of all the coupled protons and obtaining single peak at the chemical shift positions of both the enantiomers, enabling the visualization of discrimination. By reacting 2 equiv. of (S)-1-phenylethylamine or (S)-1-(1-naphthyl)ethylamine with 1 equiv. of thiophosgene, chiral thioureas (S,S)-54 (Fig. 9.7) were obtained [90], which acted as CSAs for a broad variety of ammonium salts of chiral acids, including α-amino acids, α-hydroxy acids, α-halo acids, and naproxen. A reliable correlation between the relative positions of α-proton of enantiomers and absolute configuration was found, which suggested the possibility of using the CSAs for the assignment of the absolute configuration of some classes of acids. Among low-molecular-weight CSAs, great attention has been focused on 1,1′-binaphthyl-2,2′-diol (55, Fig. 9.8) [91]: its hydroxy groups can form hydrogen bonds, and the naphthyl rings cause shielding effects that give a strong contribution to the differentiation of diastereomeric pairs. By means of this chiral auxiliary, several classes of organic compounds have been differentiated

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Figure 9.8  CSAs 55–63 and selected applications.

including sulfinimines [92], phytoalexins [93], alkaloids [94], and some active pharmaceutical ingredients including promethazine [95], omeprazole and its analogues [96], and natural isoflavanones [97]. In this last case, both enantiomeric purity and absolute configuration could be defined by 1H NMR analysis [97]. Enantiopure 55 differentiated the 1H NMR resonances of chiral phytoalexins, spirobrassinin, 1-methoxyspirobrassinin (Fig. 9.4), and cisand trans-1-methoxyspirobrassinol methyl ethers (Fig. 9.8) [93]. In this last application, Klika also described an interesting experiment of “TLC in an NMR tube”: by adding silica gel to NMR samples containing the analyte and the CSA in C6D6, the enantiomeric composition of the sample remaining in solution was perturbed and changes of between 0.8% and 2.9% were observed, revealing that one enantiomer is more stabilized in its complex with the CSA under non-polar conditions relative to a polar environment in comparison with the opposite enantiomer in its complex with the CSA [93]. 3-(2″-Pyrrolidinyl)-BINOL (56, Fig. 9.8) has a pyrrolidinyl group acting as the binding site for carboxylic acids [98]. The commercially available derivative of BINOL, (S)-3,3′-dibromo-1,1′-bi-2-naphthol (57, Fig. 9.8), has been used as a CSA for flavanones [99]. Sulfonimide derivative of binaphthol 58 (Fig. 9.8) induced relevant differentiation of selected proton signals of lactides, four-, six- and seven-membered lactones, O-carboxy-anhydrides, cyclic carbonates, and epoxides [100]. Interestingly, neither the disulfonamides (RCHNHSO2CF3)2 nor the binaphthol induced noticeable differentiation between d- and l-lactides [100].

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Urea derivatives of (R)-1,10-binaphthalene-2,2′-diamine (59–62, Fig. 9.8) were very simply obtained by reaction with the appropriate isocyanates and then employed for the enantiodifferentiation of several chiral aromatic and aliphatic sulfoxides [101]. In some cases, doublings of the enantiomeric signals were also detected in the presence only of 0.05 equiv. of CSA in the presence of 59 (Fig. 9.8). Electron-withdrawing substituents on the CSA favored the interactions between the NH groups of the urea CSA, as well as a positive effect was observed in the case of ureas bearing aryl groups in comparison to aliphatic derivatives. Chiral phosphoric acid 63 (Fig. 9.8) differentiated enantiomers of indoloquinazoline alkaloid-type tertiary alcohols (Fig. 9.8), pyrazine-type tertiary alcohols (Fig. 9.8), cyclic amino alcohols, and diamines with high efficiency. Interestingly, (R)-55 induced no differentiation of the enantiomers of alkaloid-type tertiary alcohols [102]. The use of (S)- or (R)-tert-butylphenylphosphinothioic acid ((R)-64, Fig. 9.9) and tert-butylphenylphosphinoselenoic acid ((R)-65, Fig. 9.9) as CSAs has been exhaustively reviewed by Drabowicz et al. [103–106]. The two CSAs showed uncommon versatility, being applied to NMR analyses of many classes of chiral organic compounds including racemic phosphinate esters, phosphinothioates, phosphinic amides, alcohols, diols, thiols, mercaptoalcohols, amines, amino alcohols, and hydroxyacids.The same author described the use of 64 and 65 (Fig. 9.9) for the determination of enantiomeric excesses

Figure 9.9  CSAs 64–68 and applications.

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of alkaloids such as crispine A [107], harmicine [107], and trypargine [108]. Very recently, chiral atropisomers obtained from 3,5-dibromo-2,4,6-collidine and bromo derivatives of 2,6- and 2,4-lutidine (Fig. 9.9) [109], as well as sterically demanding, stable at room temperature, atropisomeric derivatives of penta-(ortho-substitutedphenyl)pyridines (Fig. 9.9) [110], were distinguished in the presence of the phosphorated CSAs by 1H NMR analyses. In some cases, the selenium analogue 65 (Fig. 9.9) was more effective [107]. Enantiomers of alkaloids (+)-salsolidine and (−)-norlaudanosine (Fig. 9.9) were also differentiated by proton and carbon spectroscopy in the presence of (R)-64 [111]. The same CSA was effective toward the enantiomers of trans-1,10(cyclohexane-1,2-diyl)bis(imidazole) N-oxides [112] (Fig. 9.9) and allowed to determine by 31P NMR the enantiomeric excesses of the adducts obtained by Michael addition of sulfur and nitrogen nucleophiles to a chiral nonracemic 2-phosphono-2,3-didehydrothiolane S-oxide [113]. Enantiopure O-ethyl phenylphosphonothioic acid (66, Fig. 9.9), obtained by resolution with brucine, was successfully applied to the 1H NMR chiral analysis of chiral amines, amino alcohols, and chiral alcohols. Nonequivalences were detected not only for protons at the stereogenic center, but also at its α- and β-positions [114]. Simple commercially available amino acid derivatives, Fmoc-Trp(Boc)OH (67, Fig. 9.9), in particular, have been proposed [115] as a practical alternative to chiral phosphinothioic acids for the NMR discrimination of enantiomers of chiral phosphorus compounds including phosphonates, phosphinates, phosphates, phosphine oxides, and phosphonamidates. Even though the majority of organophosphorus compounds derived from chiral C2-symmetric diamines have been used as chiral-derivatizating reagents for NMR [116,117], P-stereogenic heteroatom-substituted secondary phosphine oxides like 68 (Fig. 9.9) have been explored as CSAs [118]. Both 19F and 1H NMR splittings of signals of α-(trifluoromethyl) benzyl alcohol and mandelic acid were detected in toluene-d8 at room temperature, encouraging further applications.

9.3  CSAS INVOLVING ION PAIRING PROCESSES Enantiodiscrimination can be efficiently achieved by ion-pairing interaction mechanisms, as in the case of hexacoordinated phosphates anions 69 (BINPHAT) and 70 (TRISPHAT) (Fig. 9.10) [119,120], which tightly associate in diastereomeric ion pairs with chiral organic, organometallic, or metalloorganic cations, leading to NMR differentiations. Substrates with

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Figure 9.10  CSAs 69–75.

central, axial, planar, or helical chirality can be differentiated. Different kinds of nuclei can be observed (1H, 13C, 15N, 19F, and 31P nuclei). TRISPHAT is overall more efficient with cationic metalloorganic and organometallic substrates, whereas BINPHAT is frequently more efficient toward organic cations. Although the efficient NMR enantiodifferentiation by such a kind of CSAs usually requires low-polarity halogenated solvents, in the case of quaternary N-alkyl derivatives of Tröger base a distinction of the enantiomers was observed even in polar solvent as acetonitrile [121]. Same CSAs led to differentiation of M and P enantiomers of the azaphosphatrane-hemicryptophane cages [122] and octanuclear metalla-boxes [123]. In this last application, NMR differentiation was remarkable also in CD3CN and in the presence of very low amounts (5–10 mol%) of BINPHAT. BINPHAT, which contains a strained seven-membered ring, is sensitive to acidic conditions, and decomposes over time when associated with Brøndsted or Lewis acidic cations. Therefore, their mannose derivatives 71–73 (Fig. 9.10) were proposed, which showed even superior performances in the case of M and P stereoisomer of organic monomethinium cations (Fig. 9.10) [124].

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Negatively charged Al(III) complexes 74–75 (Fig. 9.10) with stable chirality at the metal center can be obtained by means of newly designed hexadentate N2O4 ligands prepared from 1,2-diaminocyclohexane [125]. In the case of analyte peak overlapping with the cyclohexyl region of Al(III) complex, 1,2-diphenylethylenediamine can be used for the preparation of the CSA. These compounds are remarkably versatile CSAs both for positively and negatively charged chiral molecules in polar or nonpolar solvents also at substoichiometric amounts and with stereogenic centers at the α- to δposition of the charged functional groups. For the enantiodiscrimination of chiral amines, the Al(III) complex, initially prepared as sodium salt (74a–75a, Fig. 9.10), was converted into the acidic form (74b–75b, Fig. 9.10) by addition of an equimolecular amount of trifluoroacetic acid [125]. Imidazolinium salts with bulky silyl groups 76–82 (Fig. 9.11), with applications in the palladium-catalyzed reactions, were probed as CSAs for potassium Mosher’s carboxylate: the salt 78 with one hydroxy group protected with triisopropylsilyl (TIPS) showed the maximum splitting in the 19 F NMR spectrum, probably due to the formation of a hydrogen bond between the hydroxy group of 78 and Mosher’s carboxylate [126].

Figure 9.11  CSAs 76–95.

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Chiral zwitterionic phosphorus-containing heterocycles 83–84 (Fig. 9.11) are highly polar molecules, combining a concentration of electron density on the PO−2 moiety, with a highly diffused cation within the bisimidazoline framework. These features make them good candidates as CSAs for chiral acids, as confirmed by the enantiodiscrimination experiments carried out on an α-hydroxy acid, a diacid, and a sulfonic acid [127]. Mixing triphenoxyborane and a chiral amine, and subsequently adding C2-symmetric chiral BINOL (55, Fig. 9.8) represents the simple mix and shake strategy for the formation of a stereoisomeric chiral ammonium borate salt, directly into the NMR tube. The final optimized concentration of the mixture that yielded better discrimination for all the molecules is: 1 equiv. of triphenoxyborane, 4 equiv. of chiral amine, and 4 equiv. of (R)-BINOL. In this way, differentiation of amine signals is detected by 1H NMR spectroscopy [128]. The use of the high-resolution two-dimensional pureshift zCOSY NMR method with homonuclear band-selective decoupling in both dimensions led to precise quantification of enantiomers in chiral mixtures with strong 1H–1H scalar (J) coupling network or high enantiomeric excess, as demonstrated in the case of ternary mixtures containing 1-aminoindan, triphenoxyborane, and (R)-BINOL [129]. The use of ternary ion-pair complexes [130–132] constitutes an interesting approach to the optimization of CSA performances and relies on pairing effects. The mixture of BINOL (55, Fig. 9.8) and an achiral organic base, such as 4-(dimethylamino)pyridine (DMAP, Fig. 9.11) or 1,4-diazabicyclo[2.2.2]octane (DABCO, Fig. 9.11), produces better differentiating effects of chiral carboxylic acid in comparison with BINOL, alone [130]. The acid and the base ion-pair behaves as a single moiety and stabilize the formation of the ternary ion-pair complex with BINOL, by accepting a proton from BINOL. The application of the same approach to the chiral analysis of hydroxy acids suggested the possibility to establish simultaneously the ee and the absolute configuration [130]. The addition of 1 equiv. of DMAP to (R)-mandelic acid allowed to resolve enantiomeric signals of 3-butyn-2-ol, which are isochronous without the amine; the same enantiodifferentiation enhancement is obtained for other chiral secondary alcohols as well as for the 19F NMR signals of trifluoromethyl benzyl alcohol [131]; in these cases, the role of DMAP was very significant. The efficacy and widespread applicability of this approach has been clearly demonstrated also in the NMR differentiation by (S)-1-phenylethanol as CSAs, which failed to discriminate carboxylic acid without DMAP, whereas differentiation of carboxylic acid signals was obtained in the presence of the amine [131].

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Ion-pair interaction between optically active mandelic acid and DMAP was the basis of the assignment of enantiomeric purity and absolute configuration also of cyanohydrins, via formation of one to one ternary complexes with strongly differentiated association constants [132]. The same approach was followed for enhancing enantodifferentiation by 1,10-binaphthyl-2,20-diyl hydrogenphosphate (85, Fig. 9.11) in its ternary mixture containing DMAP and a chiral analyte in CDCl3 [133]. In this way, well-dispersed peaks for each enantiomer of amino alcohols, secondary alcohols, cyanohydrins, oxazolidones, diols, thiones, and epoxides were obtained in the 1H NMR spectrum and not only for the proton at the chiral center, but also for remote proton sites, allowing precise measurement of the ees. As another example, (R)-2′-amino-[1,1′-binaphthalene]-2-ol (86, Fig. 9.11) failed to serve as a CSA for acids, hydroxy acids, and their derivatives, but efficient enantiodiscrimination of the same kind of chiral substrates was achieved by using p-toluenesulfonic acid as a linker, which induces hydrogen bonding interactions [134]. The enantioselective interaction between a simple C2-symmetric chiral bisthiourea CSA 87 (Fig. 9.11) and enantiomers of chiral carboxylic acids is promoted by DMAP, by formation of geometrically different diastereomeric ternary complexes between the chiral bisthiourea and carboxylate-DMAPH+ ion pair [135]. An interaction mechanism was proposed involving the interaction of each enantiomer of racemic carboxylic acids with DMAP to form a carboxylate-DMAPH+ ion-pair through a NH···O bond, which strengthens the interaction between the carboxylic acid and chiral CSA 87 to further form a ternary complex via multiple intermolecular H-bonding interactions between the carboxylate group of the ion pair and the two thiourea groups of CSAs. CSA 87 can be easily synthesized from commercially available chemical reagents (S,S)-1,2diphenylethane-1,2-diamine and 3,5-bis(trifluoromethyl)phenyl isothiocyanate and represents a highly efficient chemical solvating agent for varied α-carboxylic acids. In the ternary system containing the same CSA, the amine DABCO and mandelic acid, large enough nonequivalences of methine proton of mandelic acid and various α-hydroxyl acids and their derivatives were detected, which were exploited for configurational assignments since a reliable correlation between the shift induced by each enantiomer of the CSA in the methine protons of the acids and the absolute configuration was found [136]. The use of 87 as CSA has been extended to several other classes of chiral compounds [137].

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Chiral isobornyl amine, which is a readily available material, has been condensed with various aromatic acid chlorides in order to obtain a new class of amide CSAs 88–95 (Fig. 9.11) [138], in which the rigid bicyclic framework of isobornyl amine with three stereogenic centers provides the steric bulk for chiral molecular recognition during the NMR analysis. Derivatives with strong electron-withdrawing nitro groups attached on the acid component of the amides allowed to differentiate isomers of 1,10-binaphthyl-2,2′-diyl hydrogen phosphates by 31P NMR analysis. An external base was needed for enantiodiscrimination to assist the abstraction of the acidic proton of the phosphorilated compounds for the subsequent ionic interactions in the three-component mixture CSA/enantiomeric mixture/base. Among the bases, DMAP showed the best effect, due to its basicity [139] and the presence of the aromatic ring for π–π interactions. Ionic liquids (ILs) are a group of organic salts that are liquid at room temperature and are obtained by the combination of organic cations and a variety of anions. They are stable, have high solubilizing power, and virtually no vapor pressure. These favorable properties make them a valuable alternative to commonly used volatile solvents. Chiral ILs (CILs) may have central, axial, or planar chirality either in the cation or in the anion or in both of them [140–141]. The major advantage of CILs is tunability of their structures and/or functionalities through judicious selection of their ions. The first spectroscopic application of CILs as CSAs was reported in 2002 by Wasserscheid et al. [142], who described the use of CIL 96 (Fig. 9.12), derived from ephedrine, in the differentiation of 19F resonances of enantiomers

Figure 9.12  CSAs 96–103.

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of Mosher’s acid: the amount of water present in the solvent (CD2Cl2) constituted the factor mainly affecting the magnitude of the splitting [142]. Due to the remarkable structural variability of chiral ILs (CILs), only few examples will be described. Among imidazolium-based chiral ionic liquids, CILs derived from amino acids [143,144] as 97 (Fig. 9.12) have been applied to the enantiodiscrimination of racemic chiral carboxylic acids; d-xylose derivatives 98 (Fig. 9.12) showed good efficiency in the differentiation of enantiomers of racemic Mosher’s acid salt in CD3CN [145]. Stereopure imidazolium salts (99–102, Fig. 9.12) with cyclophane-type planar chirality, in which the length of the bridge unit was systematically varied, were probed as CSAs for the 19F and 31P NMR enantiodiscrimination, respectively, of α-methoxyα-(trifluoromethyl)phenylacetate anion and O-ethyl phenylphosphonothioate anion [146].The magnitude of nonequivalence was more affected by the nature of the side chain on the bridge rather than the bridge length. Zwitterion CILs incorporating imidazolinium and alkylsulfonate or sulfamate groups as 103 (Fig. 9.12) showed remarkable versatility, leading to very high nonequivalence in the enantiodifferentiation of Mosher’s acid, alcohols, cyanohydrins, amino alcohols, nitro alcohols, thiols, and carboxylic acids [147]. In order to rationalize the effect of structural changes either in the cation or anion on enantiodiscriminating properties,Wasserscheid et al. compared the interactions between CILs and racemic Mosher’s salt in deuterated solvent by using 19F NMR spectroscopy [142]. These interactions were quantified using the chemical shift difference or peak splitting of the CF3 group of racemic Mosher’s salt in the two diastereomeric associates. Just in consideration of the high structural variability of this class of CSAs, a combinatorial approach is more suited for ascertaining the effect of structural modifications on the enantiodiscriminating efficiency. By following this way, a library of novel CILmodified silanes was synthesized [148], using three chiral tertiary amines, Pro, Eph, and Peu (Fig. 9.12), which include hydroxyl, phenyl, pyridyl, urea, and ester functional groups, in order to synthesize chiral cations by quaternization reactions. 19F NMR was used as a quick method for the comparison of CILs’ enantiodiscriminating efficiency. The effect of the nature of chiral cations, anions, and linker chain lengths (Fig. 9.12) was investigated.

9.4  MOLECULAR TWEEZER CSAS Molecular tweezer CSAs include U-shaped chiral compounds with two aromatic groups separated by a semirigid spacer.Their ability to differentiate several classes of enantiomeric substrates relies on their conformational

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adaptability and flexibility, which leads to the formation of the most favorable diastereomeric complexes. Virgili proposed some CSAs having such a kind of features, like compound 104 (Fig. 9.13), which has been obtained by the condensation of (R,R)-α,α′-(bistrifluoromethyl)-9,10anthracenedimethanol with muconic acid [149]. Muconic acid allowed to set a distance between the pincers of the tweezer, which is ideal for favoring the inclusion of a single aromatic guest containing a hydrogen bond acceptor or donor group. Other examples by the same author include di-(R,R)1-[10-(1-hydroxy-2,2,2-trifluoroethyl)-9-anthryl]-2,2,2-trifluoroethyl phthalate 105 (Fig. 9.13), isophthalate 106 (Fig. 9.13), and terephthalate 107 (Fig. 9.13), which were obtained by esterification of (R,R)-α,α′(bistrifluoromethyl)-9,10-anthracenedimethanol with phthalic, isophthalic, and terephthalic acid dichlorides and employed for the analysis of monoand polyfunctional chiral compounds, such as diols or amines [150]. CSAs 105–107 change their conformation in the presence of the enantiomeric

Figure 9.13  CSAs 104–119.

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substrates and, in this way, the most favorable association complexes can be formed. The pyridine-2,6-biscarboxamide moiety has also been selected for imposing the required U-shaped geometry which is stabilized by means of the two H-bonds formed between the NH groups of trans-cyclopentane-1,2-diamine or trans-cyclohexane-1,2-diamine as in the compounds 108–109 (Fig. 9.13), which have been addressed to the enantiodiscrimination of carboxylic acids [151,152]. The comparison between several structurally correlated receptors 108–112 (Fig. 9.13) pointed out the importance of the pyridine-2,6-bis-carboxamide moiety in the enantiodiscrimination processes. In the presence of CSA 112 (Fig. 9.13), nonequivalences are remarkably reduced, confirming the relevance of the cooperative effects of the two side arms of the molecular tweezer in the stabilization and differentiation of the diastereomeric solvates. Both enantiomers of a trans-2-aminocyclopentanol and a trans-N,N-dialkylcyclopentano-1,2-diamine bearer of an anthracene unit were obtained by chemoenzymatic methods and connected with pyridine-2,6-dicarbonyl dichloride in the pincer-like structure of the CSA 113 (Fig. 9.13) that showed a great efficiency in the enantiodiscrimination of mandelic-type acids [153]. Salen ligand-based molecular tweezers 114–117 (Fig. 9.13) were proposed as very efficient CSAs for chiral carboxylic acids, mainly at the 3:1 CSA/substrate molar ratio, corresponding to the complexation stoichiometry [154]. The phenolic OH group was fundamental, since analogous structures devoid of such a kind of functional group did not differentiate the resonances of the enantiomeric substrates. The simultaneous presence of amide and amino groups in the bis(amino amide) ligands 118 (Fig. 9.13), which was obtained from natural amino acids, favored the enantioselective interaction with carboxylic acids [155]. The enantiodiscriminating efficiency depended on the length of the spacer, being maximum for 3–5 carbon atoms. Also, the presence of an aromatic moiety in the side chain favors the differentiation of the enantiomeric substrates [155]. Usnic acid units can be connected by trans-1,2-diaminocyclohexane (119, Fig. 9.13) for the generation of the U-shaped geometry, which fits with various chiral esters containing an electron-poor aromatic moiety [156].

9.5  SYNTHETIC MACROCYCLE CSAS Macrocycle chiral auxiliaries for NMR spectroscopy provide highly preorganized structures, which are able to surround the enantiomeric substrates by inclusion processes as well as peripheral functional groups at their

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external surface may be exploited for the stabilization of diastereomeric complexes [157]. In general, the rationale in the design of synthetic macrocyclic CSAs is the generation of a host cavity endowed with hydrogen bond donor and acceptor sites connected to an anisotropic ring-current moiety. On this basis, Ema et al. [158–160] designed several kinds of polyfunctional hosts, selecting 2,6-diacylaminopyridine as a binding unit, which has both hydrogen-bond donor and acceptor sites in the compact structures 120– 122 (Fig. 9.14) with BINOL as source of anisotropic ring-current effects. The bifunctional macrocyclic hosts showed very good efficiency and versatility in the enantiodiscrimination of a variety of chiral compounds, such as carboxylic acid, oxazolidinone, lactone, alcohol, sulfoxide, sulfoximine, isocyanate, and epoxide compounds. Nonequivalences up to 0.55 ppm were

Figure 9.14  CSAs 120–134.

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measured, with remarkably differentiated association constants of the diastereomeric pairs. Open chain diamide 123 (Fig. 9.14) was selected as a model compound to evaluate the role of the macrocyclic structure [159]. Nitro group in 120 and 122 (Fig. 9.14) enhanced not only the binding capacity, but also the degree of enantioselectivity, leading to efficient differentiation also at very low CSA/substrate molar ratios. The V-shaped disposition of the two 2,6-diacylaminopyridine moieties of 120 (Fig. 9.14) enhanced the effectiveness of binding with respect to the parallel alignment of the two binding moieties in 124 (Fig. 9.14) [159]. The size and the shape of the cavity can be tuned acting on the binaphthyl like in 122 (Fig. 9.14) [160], where the 3,5-bis(trifluoromethyl)phenyl group allows to obtain a more compact structure suitable for the efficient enantiodiscrimination of small molecules such as 2-chloropropionic acid and methyl lactate. In virtue of the presence of four protonable amino groups and highly anisotropic environments due to pyridine moieties, the symmetrical polyazamacrocycle 125 (Fig. 9.14) represented a good CSA for the enantiodifferentiation of carboxylic acids, with the possibility of recovering both components (CSA and substrate) by simple acid–base extraction procedures. Doublings of enantiomeric substrates were dependent on the CSA to acid molar ratio, with splitting at low ratios and loss of differentiation at high ratios, to indicate the formation of multimolecular complexes [161]. With the idea that a complex based on a salt bridge is more stable than that via an ordinary hydrogen bond, Zhang et al. proposed several macrocyclic hosts for acids. Novel chiral macrocyclic compounds 126–127 (Fig. 9.14) from a C2-symmetric aminonaphthol with (S)-α-(2-naphthyl)ethylamine and (S)-α-phenylethylamine in proximity of the chiral centers constituted excellent CSAs for chiral carboxylic acids, phosphinic, phosphonic, and phosphoric acids [162–164]. 1H NMR chemical shift nonequivalences up to 0.80 ppm were measured for mandelic acid derivatives. The change in the anisotropic group from phenyl to naphthyl did not improve the chiral discrimination. Macrocyclic compounds 126–133 (Fig. 9.14) were compared in the enantiodiscrimination of carboxylic acids, with the acyclic compound 134 (Fig. 9.14) designed to investigate the importance of macrocyclic structures [164]. Although the structures of the CSAs 126–134 (Fig. 9.14) are similar, their chiral discrimination abilities toward mandelic acid were remarkably different.The change in the pyridine ring (CSAs 126 and 132) to benzene (CSAs 129 and 133) caused a significant loss of chiral discrimination ability; macrocyclic compounds 132 and 133 derived from p-benzenedialdehydes showed less chiral discriminating

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ability than those (126 and 129) derived from m-benzenedialdehydes, and the acyclic compound 134 was ineffective. The large anisochrony detected in racemic mandelic acid protons in the presence of 126 inspired the authors to explore different α-chiral carboxylic acids, such as α-halo acids, α-alkyl acids, and some derivatives of α-amino acids [164]. Chiral macrocycles 135 and 136 (Fig. 9.15) have been synthesized starting from d-phenylalanine and have been proved to be effective CSAs for chiral carboxylic acids and α-amino acid derivatives [165]. It is interesting to note that the introduction of multiple amino, amide, and phenolic hydroxyl groups as in 137–144 (Fig. 9.15) in an open structure had led to highly effective and practical CSAs for the discrimination of enantiomers of α-hydroxy acids and N-Ts-α-amino acids by 1H NMR spectroscopy [166]. Enantiomeric discrimination of chiral compounds with two or more chiral centers, like dipeptides, represents a difficult task and a contribution in this area came from the tetraazamacrocyclic CSAs 145–148 (Fig. 9.15); their C2 symmetry and the presence of a well-defined cavity enable to stabilize strong hydrogen bond interactions with the stereoisomeric mixtures in a 1-to-1 association complexes [167]. The same CSAs were subsequently employed for the enantiodiscrimination of α-hydroxy acids and N-Ts-α-amino acids [168]. The macrocyclic compounds 149–151 (Fig. 9.16) derived from l-phenylalanine methyl ester with pyridine-2,6-biscarboxamide moiety as a binding unit and both hydrogen-bond donor and acceptor sites, which

Figure 9.15  CSAs 135–148.

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Figure 9.16  CSAs 149–160.

made the compound an useful and efficient CSA for a wide range of carboxylic acids [169]. Macrocyclic amines with D3 symmetry, like 152 (Fig. 9.16), obtained from enantiomerically pure 1,2-diaminocyclohexane, provided a highly asymmetric environment for chiral guest molecules through various interactions such as hydrogen bonding, π–π stacking, and CH–π interactions and represent efficient CSAs for the NMR differentiation of enantiomers of secondary alcohols, cyanohydrins, and propargylic alcohols [170]. However, the same CSA failed to double the signals of carboxylic acids. Better results for the determination of the enantiomeric purity of carboxylic acids were obtained by using small amounts (0.25–0.50 equiv.) of the host 153 (Fig. 9.16), having phenolic hydrogen bond donor groups [171]. Periasamy et al. reported [172] that also hosts 152 and 154–155 (Fig. 9.16) were efficient in the enantiodiscrimination of chiral acids, but, in virtue of their polytopic nature, low CSA-to-substrate ratios were required.

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l-/d-Tartaric acids constituted alternative building blocks endowed with two chiral units, the two hydroxyl groups of which could be easily modified in the design of the chiral macrocyclic polyamides 156–158 (Fig. 9.16), which led to differentiation of enantiomers of mandelic acid and its derivatives or dibenzoyltartaric acid [173]. Two diastereomers of optically active N,O-containing new macrocycles with chiral backbone and chiral pendent groups (S,S,S)-159 and (R,R,S)-159 (Fig. 9.16) were proposed. The different accessibility of the cavities of (S,S,S)-159 and (R,R,S)-159 is responsible for their well-differentiated enantiodiscriminating efficiency toward organo phosphoric and phosphonic acid derivatives [174]. In order to obtain larger cavities simultaneously avoiding the collapse of large-volume structures, the backbone was made rigid by aromatic components. The chiral macrocycle 160 (Fig. 9.16), proposed by PalominoSchaetzlein et al. [175], satisfies these requisites since it consists of six units of terephthalic acid and of α,α′-(bistrifluoromethyl)-9,10-anthracendimethanol. Molecular dynamics simulations demonstrated a completely folded conformation in vacuum, favored by intramolecular hydrogen bonding and π-stacking interactions, whereas unfolded conformations, leaving an open cavity for host–guest interactions, were found in a nonprotic solvent [175]. Calixarenes and calix[4]resorcinarenes (161–162, Fig. 9.17) are different kinds of host compounds, the applications of which as CSAs has been exhaustively reviewed by Wenzel in 2014 [176]. The same author gave several contributions in the field of the development of water-soluble calix[4] resorcinarenes by introduction of polar ionic groups, as in the case of tetrasulfonated calix[4]resorcinarenes 163–166 (Fig. 9.17) containing proline moieties or pipecolinic acid groups, which gave efficient enantiodiscrimination of water-soluble or cationic and anionic derivatives of several classes of organic compounds, allowing to realize a wide scheme of chiral analyses in aqueous medium [177–185]. α-Methyl-l-prolinylmethyl pendants (164, Fig. 9.17) at the aromatic moieties of calix[4]resorcinarene exalted enantiodifferentiation in comparison to analogous reagents with proline (163, Fig. 9.17) and hydroxyproline (166, Fig. 9.17) moieties [183,184]. The best performances were given by the system with appended pipecolinic groups [182]. Wenzel proposed the use of water-soluble calix[4]resorcinarenes 163–165 (Fig. 9.17) also for the differentiation of proton resonances of enantiomers of simple naphthyl amide derivatives of amine and amino alcohols. The derivatization scheme (Fig. 9.17) for the substrates involved the use of naphtho[2,3-c]furan-1,3-dione and the protons of newly introduced

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Figure 9.17  CSAs 161–174.

naphthyl moiety represented the probe for the determination of ees. The relative positions of 1H NMR signals of the two enantiomers in the presence of the CSA showed a well-defined trend which resulted useful for absolute configuration determinations [185]. Several other kinds of functional groups were appended at the rims of calix[4]arene in order to modulate their enantiodiscriminating properties: this is the case of chiral mono and diamide derivatives of calix[4]arenes 167–169 (Fig. 9.17), the complexation properties of which toward racemic 1-phenylethylamine were described [186]. Amine or amino alcohol-armed calix[4]arenes 170–172 (Fig. 9.17) formed 1:2 instantaneous complexes with acidic guests [187]. Chiral calix[4]arenes with aminonaphthol units at the lower rim 173–174 (Fig. 9.17) were proposed as chiral NMR solvating agents to determine the enantiomeric purity of mandelic acid at room temperature [188].

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The role of the increasing molecular complexity due to the macrocyclic structure has been investigated by comparing the enantiodiscriminating ability of chiral polyamide selectors, deriving from (R,R)-1,2-diaminocyclohexane and (S,S)-1,2-diphenylethylendiamine in molecular tweezertype systems 175 and 176 (Fig. 9.18) and connected to resorcin[4]arene

Figure 9.18  CSAs 175–180.

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structures 177 and 178 (Fig. 9.18), by using mandelic acid as probe compound [189]. The comparison of 177 and 178 with resorcin[4]arene 179 (Fig. 9.18) pointed out the relevance of the lateral polyamide cycle. The NMR experiments demonstrated that the presence of amide moieties in chiral selectors 175–179 (Fig. 9.18) is fundamental for the thermodynamic stabilization of the diastereoisomeric solvates formed with the two enantiomers of mandelic acid. However, only the cooperation of the abovesaid polar groups with the resorcin[4]arene core makes possible an efficient enantiodiscrimination. A more challenging approach in the design of chiral macrocycles is to have “inherently” chiral hosts as in the case of 180 (Fig. 9.18), with proximal amino and hydroxy groups in a fixed cone conformation. The formation of a 1:1 complex with the two enantiomers of mandelic acid was responsible for the relevant differentiation, which were produced in the proton nuclei of racemic acid. Interestingly, nonequivalences were not responsive to the molar ratio CSA/substrate since the addition of more than an equimolar amount of CSAs showed no further change in the chemical shifts of the mandelic acids [190]. Heteroatom-bridged calixaromatics are a new generation of macrocyclic host molecules, which can adopt different conformations and give tunable cavity sizes, depending on the nature of the heteroatoms on the bridging units. In particular, calix[2]arene[2]triazines can exhibit π–π interactions of aromatic rings, and hydrogen bonding interactions on the triazine nitrogen atoms as chiral host molecules. Oxo-bridged calix[2] arene[2]triazine derivatives 181–182 (Fig. 9.19), which were synthesized from (1S,2R)-1-amino-2-indanol or (1S,2R)-2-amino-1,2-diphenylethanol, differentiated the proton spectra of carboxylic acids and α-hydroxy

Figure 9.19  CSAs 181–182.

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acids, especially those containing aromatic groups such as mandelic acid and α-methoxyphenylacetic acid [191]. Among CSAs endowed with cavities, the chiral crown ether with an 18-crown-6 unit and peripheral carboxyl groups (18-crown-6)-2,3,11,12tetracarboxylic acid (183, Fig. 9.20) is well known for its ability to form three hydrogen bonds with protonated primary amino groups. In virtue of this peculiarity, it is suitable for the enantiodiscrimination of primary amines, including diamines, α-amino acids, β-amino acids, and isoxazoline-fused β-amino acid derivatives, as shown by Wenzel in several papers dedicated to this topic [185,192–194]. The same CSA was effective for determining enantiomeric purities of chiral pyrrolidines [195]: by mixing the neutral amine with the CSA, a neutralization reaction occurs, producing the corresponding ammonium and carboxylate ions. CSA 183 (Fig. 9.20) showed enantiodiscriminating ability toward cyclic β-amino acids with cyclopentane, cyclohexane, cycloheptane, cyclopentene, cyclohexene, bicyclo[2.2.1] heptane, and bicyclo[2.2.1]heptene rings [196]. Not only enantiomeric discriminations of the methine hydrogen atoms α to the amine and carboxylic acid moieties were observed for all substrates, but also a consistent correlation between the relative positions of enantiomer signals and the absolute configuration was found for some proton nuclei. In spite of the fact that symmetrical macrocyclic CSAs are, in general, preferred, the introduction of bulky substituent in C1-symmetry selectors

Figure 9.20  CSAs 183–188.

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could make a more rigid CSA structure and originate better structure complementarity, which could be beneficial in enantiorecognition processes. For these reasons, C1-symmetric chiral crown ethers including 28-crown-8, 20-crown-6 (like 184–186, Fig. 9.20), 17-crown-5, and 14-crown-3 were obtained from maleopimaric acid and proposed for the enantiodiscrimination of protonated primary amines and amino acid methyl ester salts by 1H NMR spectroscopy [197]. By incorporating a chiral azacrown ether moiety as a binding site into the molecular framework of a calix[4]arene, the chiral calix[4]azacrown ethers 187 and 188 (Fig. 9.20), in which a furfuryl group is attached on the nitrogen atom, were synthesized for the enantioselective recognition of carboxylic acids [198]. The basicity of the nitrogen atom in the azacrown loop played a major role in the enantioselective recognition of carboxylic acids.

9.6  CYCLODEXTRINS The enormous popularity of cyclodextrins (CDs) (189–191, Fig. 9.21) relies on their unique structural features: six (α-CD), seven (β-CD), or eight (γ-CD) glucopyranose units assembled in a symmetrical truncated cone shape, with an apolar cavity inside which apolar moieties or apolar molecular portions can be accommodated, and an external surface made hydrophilic by the presence of the hydroxyl groups bound to the secondary and primary sites on the wide and narrow rims, respectively.Therefore, CDs are soluble in aqueous medium and able to complex both polar and apolar substrates. In the virtue of chirality of glucopyranose moieties, binding of chiral substrates can lead to enantiodiscrimination. Furthermore, solubility, complexing, and enantiodiscriminating properties of CDs can be modulated by means of derivatization reactions of the hydroxyl groups, which are endowed with well-differentiated reactivities. Casy and Greatbanks published some of the earliest papers on the use of CDs as CSAs for the NMR differentiation of pharmacologically relevant chiral compounds in aqueous medium [199–200]. From then on literature on this topic flourished to a huge amount [3,5–9,13,14]. The detection of (R,S)- and (S,R)-enantiomers of emtricitabine (Fig. 9.21), an analog of cytidine, by 19F NMR spectroscopy [201], using native CDs as chiral recognition agents in aqueous medium, and the differentiation of aminophosphonic acids enantiomers [202] represent recent examples. In the first case [201], among all CDs, α-CD showed the largest separation due to complementarity in the fit between the guest and cavity size of host molecules.

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Figure 9.21  CSAs 189–205 and selected applications.

The magnitude of 31P nonequivalence of aminophosphonic acids [202] was strongly pH-dependent, being optimal at alkaline pH. Native CDs have also found applications as water-soluble CSAs for the direct analysis of chiral metabolites in intact biological fluids, remarkably widening their applicative perspectives: the analysis of samples does not require any pretreatment, apart from the addition of a small aliquot of deuterated water for lock and shimming, which, however, may be alternatively put into a coaxial tube in order to avoid any contact with the biological sample or H–D exchange processes. In this way, the direct 1H NMR enantiodifferentiation in urine of ibuprofen (Fig. 9.21), and one of its major metabolites, the glucuronidated (Gluc) carboxylate derivative 2-[4-(2-carboxy-2-methylpropyl)phenyl]propionic acid (IBUD, Fig. 9.21), has been

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achieved by direct addition of the CSA β-CD. A limiting factor in this kind of applications is the remarkable spectral complexity of the samples and the difficulty to control the molar ratio between the chiral metabolites and the chiral auxiliary, since the amount of metabolite cannot be exactly predetermined [203]. Poor solubility in aqueous medium represents the major drawback of β-CD, which is less expensive of the three native CDs. Therefore, several water-soluble CD derivatives are now commercially available as an alternative to the parent CDs, which allows to select CDs not only based on the differences in the size of their inner cavities, but also on the nature of substituents on their rims, charged or not. As an example, in the chiral recognition of the racemic organophosphorus pesticide fenamiphos [204], water-soluble CDs were compared for affording a rapid, selective, and accurate quantitative 31P NMR spectroscopy analytical method. Among six neutral CDs—α-CD (189), β-CD (190), methyl-β-CD (Me-β-CD, 192), hydroxyethyl-β-CD (HE-β-CD, 193), hydroxypropyl-β-CD (HP-β-CD, 194), and hydroxypropyl-γ-CD (HP-γ-CD, 195)—and two anionic CDs— carboxymethyl-β-CD (CM-β-CD, 196) and carboxyethyl-β-CD (CE-βCD, 197)—β-CD and HP-β-CD were the best CSAs for the enantiomeric discrimination of fenamiphos (Fig. 9.21). Rudzinska et al. [205] showed that CDs constitute the most general and efficient agents for the 31P NMR enantiodiscrimination of various N-protected aminophosphonates derivatives (Fig. 9.21), by exploiting the different sizes of the cavities of α-CD, β-CD, γ-CD, and HP-γ-CD for enhancing the affinity of the chiral substrates toward the chiral selectors. The development of CDs as CSAs for NMR has been enormously stimulated by enantioselective chiral chromatography [206,207], as in the case of capillary electrophoresis, the robust correlation of which with NMR spectroscopy has been extensively pointed out by Chankvetadze et al. [208–215]. Lipophilic groups on the CD rims break the ordered network of hydrogen bonds among the secondary hydroxyl groups of adjacent rings of underivatized CDs and perturb the truncated cone shape of CDs, strongly affecting the enantiodifferentiating properties.The development of lipophilic CDs as CSAs for ee determinations in organic solvents by NMR mainly builds on the activities in the field of enantioselective gas chromatography by Koehler et al. [216] and Schurig [217–222]. As an example, the very high gas-chromatographic enantioseparation factor produced by heptakis(2,3di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (AcSiCD7, 198, Fig. 9.21) for 1,1,1,3,3-pentafluoro-2-(fluoromethoxy)-3-methoxypropane

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(compound B) (Fig. 9.21), a chiral degradation product of the inhalation anesthetic sevoflurane [223], was mimicked by NMR [224]. In this case, the relevance of superficial Si–F attractive interaction was demonstrated by NMR. 198 was an efficient CSA also for N-trifluoroacetylated derivatives of amino acids (Fig. 9.21) with free carboxylic functions: both silyl and acetyl groups in the CSA resulted in the stabilization of diastereomeric complexes [225]. A regular correlation was found between the relative positions of enantiomers’ amide resonances and absolute configuration, thus suggesting the possibility of employing the CD derivative for configurational assignments of the same class of α-amino acid derivatives [225]. Importantly, the strong effect of trace amounts of water in the enantiodiscriminating processes by lipophilic CDs in apolar solvents has been clearly evidenced by NMR experiments involving octakis(3-O-butanoyl2,6-di-O-pentyl)-γ-cyclodextrin (199, Fig. 9.21), a well-known chiral selector for gas chromatography (Lipodex E), and the two enantiomers of methyl-2-chloropropionate (MCP, Fig. 9.21): water was deeply included into the CD and favored the formation of the inclusion complex with (S)MCP, whereas (R)-MCP was only slightly affected, thus causing a significant increase of NMR differentiation [226].The observed effects encourage the use of small achiral modifiers in the development of new effective chiral auxiliaries for enantiomeric purities determination by NMR spectroscopy. Relevance of impurities or additives on enantiodiscrimination processes has also been stressed by Waibel et al. [227] inside an accurate NMR investigation dealing with the role of urea, which is commonly employed as solubility enhancer for the poorly water-soluble β-CD in capillary electrophoresis, in the enantiodiscrimination of dipeptide enantiomers. The additive can originate hydrogen bond interactions with dipeptide enantiomers, thus affecting enantioselective complexation phenomena. In general, anionic and cationic CDs are more effective chiral NMR solvating agents, respectively, for cationic and anionic substrates than neutral native CDs. Ion-pairing interactions, the extension of the CD cavity due to the presence of the substituents and the enhancement of the hydrophilicity of the external surface, all contribute to enhance their enantiodifferentiating ability in comparison to native CDs. In these applications, the degree of substitution of the hydroxyl groups can critically affect the enantiodifferentiation in the NMR spectra. In 2006, Dignam et al. described [228] the use of randomly substituted carboxymethylated CD and selectively substituted at the 6 and 2 sites of the CDs as CSAs for cationic substrates. In the enantiodiscrimination of pheniramine, chlorpheniramine, and brompheniramine

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(Fig. 9.21), the indiscriminately substituted α-, β-, and γ-CDs were more effective than native CDs or the derivatives with carboxymethyl groups at the primary or secondary positions: derivatives at C-2 sites were more similar to randomly substituted CDs and derivatives at the primary sites more similar to native CDs. Incorporation of the carboxymethyl groups on the primary face did not appear to alter the association of pheniramine-type substrates with the CDs. At the light of these results, randomly carboxymethylated CDs were used [229,230] as CSAs for cationic aromatic amines and aromatic hydroxy amines, with the β-CD derivative, in general, more efficient with respect to the others. Adding praseodymium(III)nitrate or ytterbium(III) nitrate to mixtures of the carboxymethylated CD and substrate constituted an interesting strategy for enhancing the differentiation of enantiomeric signals, taking care of the line broadening which is produced by the lanthanide. A series of compounds with a variety of aromatic functionalities including phenyl, pyridyl, indoline, and naphthyl rings were considered. The reaction with glycidyltrimethylammonium chloride was exploited to obtain cationic CDs 200 (Fig. 9.21) with different degrees of substitution [231]. These CDs are positively charged independent of pH. A wide range of anionic substrates, including indole, dihydroindole, phenyl, imidazole, lactam, pyrrolidine, and piperidine rings, were explored in order to assess the enantiodiscriminating efficiency and versatility of CSAs. The effectiveness of randomly substituted cationic trialkylammoniumderivatives of CDs in the enantiodiscrimination of 13 different anionic substrates was evaluated by 1H NMR spectroscopy [232]. CDs with a single stereochemistry at the hydroxy group on the (2-hydroxypropyl)trialkylammonium chloride substituent were often but not always more effective than the corresponding CD in which the C-2 position was racemic, with the bulkier triethyl or tri-n-propyl derivatives more effective than the corresponding trimethyl derivatives. Very recently, the use of phosphated α-, β- and γ-CDs (201, Fig. 9.21) has been recommended for protonated cationic enantiomeric substrates spanning from simple amines to amino alcohols or amino acids and their derivatives [233]. In some cases, phosphated CDs, which are commercially available with low and high degrees of substitutions, showed better enantiodiscriminating versatility in comparison with carboxymethylated CDs. The addition of a paramagnetic lanthanide ion positively affected enantiomer differentiation [233].

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Armstrong et al. showed an interesting case of NMR differentiation of ∆ and Λ enantiomers of octahedral tris-chelate metal complexes, such as ruthenium(II) trisdiimine complexes (Fig. 9.21), by anionic sulfated, anionic sulfoalkylated, and neutral aromatic modified CDs (202–205, Fig. 9.21), showing that aromatic and anionic derivatizing groups were beneficial for chiral recognition [234].

9.7  NATURAL PRODUCTS Natural products represent a precious source of versatile multifunctional chiral reagents, the enantiodiscrimination properties of which can be tuned by suitable chemical modifications. A noticeable example is represented by quinine (206, Fig. 9.22), a natural alkaloid firstly proposed in 1988 for the enantiodiscrimination of binaphthyl compounds and alkylarylcarbinols [235] and subsequently successfully employed both in its native form (together with its quinidine stereoisomer 207, Fig. 9.22) or as derivatives

Figure 9.22  CSAs 206–220 and selected applications.

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for the enantiodiscrimination of a variety of mono-functional and polyfunctional organic compounds. In addition to carbamoylated derivatives at C-9 (208, Fig. 9.22) with enantiodiscriminating properties complementing native alkaloid, C-10 derivatives (209, Fig. 9.22) have also been proposed [236–238]. Applications of native alkaloids include the use of (−)-cinchonidine (210, Fig. 9.22) in the enantiodiscrimination of montelukast (Fig. 9.22), a selective leukotriene receptor antagonist indicated for the treatment of asthma and seasonal allergic rhinitis [239], or the use of quinine for determining a tentative absolute configuration at the phosphorus atom of hydroxyphosphinates (Fig. 9.22) with two stereogenic centers (at the phosphorus and α-carbon atoms) [240]. Quinidine derivatives with pyridazine (211, Fig. 9.22) or anthraquinone core at C-9 (212, Fig. 9.22) gave the opportunity to monitor enantioselectivity in the asymmetric alcoholysis of meso-anhydrides carried out by using the same quinidine derivatives as catalysts [241]. Quinine and quinidine (206–207, Fig. 9.22) as well as their derivatives 9-O-tert-butylcarbamoylquinine (213, Fig. 9.22) and 9-O-tert-butylcarbamoylquinidine (214, Fig. 9.22) were also studied as CSAs for the enantiodifferentiation of N-benzyloxycarbonyl derivatives of 1-aminoalkanephosphonic and phosphinic acids by means of 31P NMR spectroscopy [242]. Phosphorylation at the C-9 hydroxyl group (215–216, Fig. 9.22) produced nonequivalences up to 0.1–0.2 ppm for 3,5-dinitrobenzoylsubstituted amino acids with superior efficiency with respect to the parent alkaloid [243]. These promising results also suggested to exploit the phosphate moiety at C-9 for producing zwitterionic quinine derivatives 217–219 (Fig. 9.22), with a negatively charged phosphate group and a positively charged quinuclidine tertiary amine fragment, which were effective with N-(3,5-dinitrobenzoyl)-protected amino acids and their methyl esters and secondary amines [244]. Quinine O,O-diethyl-phosphorodithioate 220 (Fig. 9.22), an organocatalyst obtained by conjugating the alkaloid with the dithiophosphoric acid moiety, was also tested as a CSA in the enantiodiscrimination of NBoc-phenylglycine by 31P and 1H NMR. Any differentiation was detected in the phosphorous spectra, whereas the α-methine proton underwent doubling [245]. Recently, the readily available softwood resin derivative (+)-dehydroabietylamine (221, Fig. 9.23) offered the opportunity to develop novel CSAs 222–223 (Fig. 9.23) for the enantiomeric resolution of racemic carboxylic

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Figure 9.23  CSAs 221–224.

acids and their n-Bu4N salts [246,247]. In this last application, a modification of RES-TOCSY NMR pulse sequence [23] was exploited for the enhancement of enantiomeric discrimination, when the resolution of multiplets was insufficient. (−)-Epigallocatechin gallate (224, Fig. 9.23), a polyphenolic bioflavonoid abundantly present in green tea, represents another example of CSAs from natural sources, which allowed the enantiodifferentiation of a large number of α-amino acids in a polar solvent (DMSO) [248]. The same CSA differentiated racemic propranolol in D2O [249].

9.8  LYOTROPIC CHIRAL LIQUID CRYSTALS Several reviews in this last decade focused on CSAs based on lyotropic chiral liquid crystals (CLCs) [250–255], therefore here only a very short outline of their features will be presented. CLCs are based on cholesteric lyopolymer such as polypeptide, polynucleotide, or polysaccharide, dissolved in organic liquids or water. At well-defined composition and temperature ranges, they form chiral nematic mesophases exhibiting alignment with respect to the external magnetic field. Especially convenient CLCs are based on optically active poly-γ-benzyl-l-glutamate (PBLG) or polyγ-benzyl-d-glutamate (PBDG) in suitable organic cosolvents. In spite of small alignment of solutes in these solvents, anisotropic interactions remain measurable and enantioselective ordering of chiral solute occurs giving rise

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to differentiation of their NMR spectra. Their use does not require the presence of specific functional groups of the enantiomeric substrates and all magnetically active nuclei can be observed for detecting enantiodiscrimination, even in the case of very low abundant isotopes.

9.9  CHIRAL SENSING The determination of enantiomeric excesses by using non-CSAs represents an intriguing issue and it is based on the use of nonchiral receptors, which are called prochiral solvating agent (pro-CSA) since they operate under conditions of fast exchange between a free form and bound to chiral analyte similarly to standard CSAs. Pro-chirality of the reagent means that upon formation of noncovalent host–guest complexes, the chiral information is transferred from chiral guest to the originally achiral host, which becomes chiral and, hence, responsive to the enantiomeric excess of the chiral substrates to be analyzed. The magnitude of the signals’ separation is linearly correlated to the ees of the chiral substrate. The main inconveniences of nonchiral–chiral sensing systems are that a calibration curve must be built and low-temperature measurements are required. The main advantage in these applications is the accuracy of ee determinations increases approaching the enantiopure state, opposite to the accuracy trend of traditional CSAs [256,257]. Labuta et al. found that in solutions of tetraphenylporphine (225, Fig. 9.24) with (R)-2-phenoxypropionic acid at −32.5 °C, the resonances corresponding to the ortho-phenyl protons and the pyrrole protons of 225 were split into two sets of symmetrical signals, the separation of which depended on the ee of the chiral carboxylic acid [258]. Same authors demonstrated that 5,10,15,20-tetrakis(3,5-di-tert-butyl-4-oxocyclohexa-2,5dienylidene)porphyrinogen (226, Fig. 9.24) constitutes a sensor of chirality due to the presence of a site for molecular recognition. Guest species interact with 226 through hydrogen bonding at the pyrrolic NH groups. Mandelic acid chirality causes splitting of the resonances of 226 resonances, the magnitude of which was confirmed to be linearly dependent on the ees of mandelic acid [257].

9.10  CONFIGURATIONAL ASSIGNMENTS Absolute configurations are more commonly determined by NMR on the basis of the use of CDAs [4,11,12]: diastereomeric derivatives involving covalent binding between the chiral auxiliary and the enantiomeric

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Figure 9.24  Chiral sensing systems 225–226.

substrates adopt a preferred conformation which can be predicted on the basis of the differential shielding that is caused by an aromatic ring incorporated into the chiral discriminating reagent. In the case of CSAs, the mechanism of discrimination is not always understood; however, a regular trend can be found in the relative magnitude of the changes of chemical shifts for the two enantiomers in the presence of the CSA. When this trend is consistently followed by several components of a specific class of compounds, then the absolute stereochemistry of an unknown compound of the same class can be reliably assigned. Alternatively, an approach which is similar to the one employed for configurational assignments by CDAs can be exploited, involving the comparison of the chemical shifts of one enantiomer of the chiral substrate in the presence of each enantiomer of the CSA [4,11,12]. The chemical shifts differences are interpreted on the basis of suitable interaction models, which can be obtained by computational method or, experimentally, by NOE measurements. As an example, the configurational assignment of bavachinin was based on the relative positions of the signals of proton H-3a of bavachinin in mixtures containing (S)-bavachinin or (R)-bavachinin (Fig. 9.24) with (S)-57 and (R)-57 (Fig. 9.8) [99]. Another recent example of configurational assignment by means of CSAs has been described by Chaudhari and Suryaprakash in the case of the ternary ion-pair complexation of BINOL, mandelic acid, and the organic base DMAP [130].The signs of chemical shift differences ∆δR,S between the diastereomers formed by each enantiomer of mandelic acid and both enantiomers of BINOL were interpreted as due to the location of α proton, the phenyl ring, and the OH of mandelic acid with respect to the shielding/ deshielding cone of BINOL. In the ion-pair complex, the (R)-mandelic acid/(R)-BINOL diastereomer, the alpha proton of mandelic acid is located

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in the deshielding region of the napthyl ring of BINOL, resulting in the high-frequency shift of its resonance, whereas in the case of (R)-mandelic acid/(S)-BINOL diastereomer, it resides in the shielding region of the naphthyl ring. Thus, the differences in chemical shifts of diastereomeric signals (∆δR,S) for α proton show positive value for (R)-mandelic acid, while in the case of (S)-mandelic acid show reverse behavior, resulting in the negative value of ∆δR,S.The possibility that shielding and deshielding effects could arise from the aromatic ring of DMAP was ruled out in experiments carried out by using DABCO instead of DMAP. A punctual analysis of all cases of configurational assignments involving CSAs is out of the scope of this chapter.

9.11  CONCLUSIONS Advantages of CSAs over other classes of chiral auxiliaries for NMR spectroscopy are remarkable in terms of simplicity of use, as witnessed by the great efforts which are devoted to the development of more and more efficient and versatile systems. Research activities regarding low-to-medium-molecularweight CSAs seem to be focused more to methodological aspects in order to make the chiral analysis more robust in terms of reliability, reproducibility, and accuracy, also exploiting several powerful NMR techniques of spectral simplification, which could have great impact in the validation of NMR methods for the assessment of enantiomeric purities. The major contribution on the design of new CSAs comes from synthetic macrocyclic systems which are specifically targeted to increment the interaction enantioselectivity inside a wide range of chiral substrates. Natural products are greatly attractive since they are, in general, multifunctional and their enantiodiscriminating properties can be modulated by simple modification reactions.Water-soluble CSAs, mainly CDs, have remarkable potentialities in the field of the noninvasive analysis of chiral metabolites directly into the biological fluids. The counterpart to the intrinsic advantages of CSAs in the determination of enantiomeric purities is the lack of criteria for the selection of the most effective CSA and the lack of data comparing the effectiveness of different CSAs. It must also be taken into account that several CSAs, although efficient, are not commercially available. All of this makes the choice of the optimal CSA for a given analyte a challenging task.This issue has been faced by proposing [259] automated high-throughput combinatorial experimental approaches which allow to compare enantiodiscriminating efficiency and versatility of CSA libraries.

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CHAPTER 10

Chiroptical Spectroscopy of Biofluids Vladimír Setnicˇka, Lucie Habartová

University of Chemistry and Technology Prague, Prague, Czech Republic

10.1  INTRODUCTION Chirality, an omnipresent unique feature, is regarded as the signature of life. Starting with DNA double helix and l-amino acids as essential building blocks of the human body, over β-sheet structures in neurodegenerative amyloid plaques, to rare left-handed snail shells and the noncoincident left and right human hands, chiral elements shape up the world. Although chiroptical techniques have revealed a large amount of significant information about many biomolecules [1–12], the analyses have been focused primarily on pure substances or molecular systems in model environments and performed under controlled conditions. The determination of absolute configuration became a gold standard in pharmaceutical research [13–17]; the structure of essential biomolecules has also been resolved [12,18,19]. Detailed studies on interactions between human serum albumin (HSA) and other proteins with bile pigments [20,21], drugs [22], cell membranes [23,24], etc. have been conducted, and protein folding/ misfolding under various experimental conditions has been thoroughly investigated [25–27]. However, the behavior and structure of a molecule may be significantly affected by its natural environment and surrounding moieties present therein.Thus, the analysis of essential biomolecules in biological fluids holds great potential for the understanding of many biological processes.The so far conducted chiroptical studies have been limited to single molecules or simple mixtures, so that the researchers would not have to overcome the obstacles presented by the complexity of biofluids. Despite low concentrations of clinically significant molecules, high level of fluorescence and various undesirable interferences overlapping the signals of interest, numerous efforts have been made to introduce chiroptical methods into biofluid analysis [28–36]. The key studies clarifying the abilities and limitations of chiroptical spectroscopy as a probe for real biological samples are discussed in this chapter. Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00010-2 Copyright © 2018 Elsevier B.V. All rights reserved.

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10.2  BLOOD AND BLOOD-BASED DERIVATIVES Blood, an indispensable biofluid comprising more than 4000 components of diverse features and functions, is formed as a suspension of blood cells in liquid blood plasma [37]. Blood plasma accounts for ∼55% of total blood volume and its primary function is the maintenance of homeostasis provided by the transport of blood gasses, various nutrients, metabolites, hormones, and waste products throughout the body. This yellow opalescent aqueous solution of proteins, inorganic salts, and small organic molecules is obtained from anticoagulant-treated blood via sedimentation or, more effectively, centrifugation of the cellular part of the blood represented by red and white blood cells and platelets. If aiming for serum, no anticoagulants are used when the blood is drawn from the patient, allowing it to coagulate naturally and, subsequently, the clot formed is removed. On top of lacking blood cells and platelets as in plasma, the resulting serum is free from fibrinogen and other coagulation factors [37,38]. Blood plasma contains about 500 different proteins, whose biological function is determined by their structural arrangement, that is, conformation and/or folding [38]. It was proved that some diseases may induce changes in both the concentration and the structure of plasmatic proteins and that some structural forms of a protein are even disease-specific. For example, in the case of neurodegenerative protein misfolding diseases, plaques comprising fibrils of amyloid-β protein or neurofibrillary tangles consisting of helical tau-protein filaments are formed in the brain [39,40] and a portion thereof passes the blood–brain barrier into the blood stream [41]. Unfortunately, the levels of many commonly used protein markers are rather low in blood. Thus, for a simplified monitoring facilitating the diagnostic process, it would be convenient to identify new molecules, whose concentration would be higher and structural changes more specific.

10.2.1  Electronic circular dichroism The first chiroptical analyses of blood-based samples were performed as early as 1977, when Jung et al. [28] monitored serum levels of O-(βhydroxyethyl)-rutosides in patients administering a flavone glycoside-based vascular therapeutic agent Venoruton. As their molecules contain chromophores, the flavone glycosides are optically active and, thus, detectable by electronic circular dichroism (ECD) [42]. The interaction of the aglycone part with the covalently attached sugar moiety results in different extinction coefficients, which may be useful in investigating not only the conformation,

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but also the composition of such compounds. Moreover, ECD is suitable also for the kinetic studies of flavone glycosides, specifically hydroxyethylated rutosides. The metabolic cleavage of the rutoside molecule usually results in the complete disappearance of the ECD signal, whereas the interaction with blood biomolecules (proteins) increases chiroptical properties. As hydroxyethyl-rutosides bind to serum albumin and globulins [43], it was essential to measure serum samples without any preprocessing or modification, such as protein precipitation, which might have caused partial removal of the studied compounds. In addition to eliminating conformational changes and sample degradation, the authors recommended analyses at a strictly controlled temperature (4 °C). It was found that the ECD signals of serum or plasma had positive amplitudes, whereas the bands located at the same wavelength (∼345 nm) showed negative values for the active ingredients (tri- and tetrahydroxyethyl-rutoside) (Fig. 10.1). Although the ECD spectra of hydroxyethyl-rutosides contain multiple bands, these were not considered for the study because they are either located in the range of a fluorescence band (390 nm), less intense (312, 292, and 269 nm), or may be influenced by protein interference ( 250,000 plates/m by chiral chromatography. Mass transfer kinetics are very different for chiral versus achiral on the same column. Like achiral chromatography, the goal of any chiral separation is to have desired resolution in the shortest possible time. Eq. (12.1) can be used to evaluate and understand the factors affecting resolutions in isocratic chiral LC. For the sake of discussion, with average plates N, selectivity α and average retention factor k values, one can arrive at the following resolution Eq. (12.1) [31]. This equation clearly shows the three top factors, which affect resolution, which we have written in the order of importance. Selectivity is the most important variable, followed by efficiency and then comes the retention factor, which is especially true for subsecond separations.  α − 1 N  k  (12.1) Rs =  α + 1  2  k + 1 

Note that the other commonly used resolution equations use the efficiency of the second peak, which can be markedly different from the first eluting enantiomer [32]. The selectivity is solely controlled by the chemistry of the stationary phase, mobile phase, and the temperature, that is, it is purely a thermodynamic variable. The efficiency N is another critical variable, which is dependent on the nature of the packed bed, particle

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Figure 12.3  Effect of particle sizes, particle morphologies, and column geometries on chiral separation of 5-methyl-5-phenylhydantoin on teicoplanin bonded silica. Mobile phase is 100% MeOH at a flow rate of 1.0 mL/min for 0.46 cm i.d. columns and 0.425 mL/ min for the 0.30 cm i.d. to maintain the same linear velocity. Note the changes in efficiency. UV detection at 220 nm (Original work).

size/morphology, and kinetics of sorption-desorption. The efficiency has been dramatically improved by using modern particle morphologies such as FPP and SPP. The driving force behind using such small particles is to allow the use of shorter columns 1 cm to 5 cm for faster separations with the same resolution as obtained in traditional 5 µm and 25 cm long column format. This concept is illustrated in Fig. 12.3. The same chiral separation of 5-methyl-5-phenylhydantoin is demonstrated on four different column geometries (25 × 0.46 cm i.d., 10 × 0.46 cm i.d., 5 × 0.46 cm i.d, and 2 × 0.30 cm i.d.). The 25 to 5 cm columns are packed with 5 µm teicoplanin bonded columns, whereas the 2 cm column is packed with 2.7 µm SPP in a 0.3 cm i.d. column. A significant reduction of time can be achieved (from 4.6 min to 0.34 min) simply by using small particles.

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12.6  MAJOR CLASSES OF MODERN CHIRAL STATIONARY PHASES (CSPs) FOR LIQUID CHROMATOGRAPHY: THE “α” OF THE RESOLUTION EQUATION As stated earlier, selectivity can be altered by developing new surface chemistries or adding chiral additives to the mobile phase. To provide an anisotropic environment for the enantiomers the most straightforward approach is to add a chiral selector in the mobile phase and use an achiral column such as C18 bonded silica or to derivatize a chiral molecule so that it becomes a diastereomer. In general, adding additives to the mobile phase can lead to the formation of so-called system peaks, which results from the disturbance of the mobile phase-stationary phase equilibria. The system peaks become visible if the chiral selector has some absorbance, or in other cases, they can be detected by refractive index detectors. To avoid these problems and long equilibration times, chiral separations, coated or bonded stationary phases are preferred. Newer chiral column chemistries are developed based on three criteria (1) broader selectivity than all existing chemistries, (2) superior functional group specificity (e.g., primary amines, etc.) separations, or (3) resolution of those enantiomers, which have not been resolved before. The chiral selectors can be coated onto silica, polymers, or other inorganic oxides (ZrO2, etc.). The historical evolution of chiral LC phases has been discussed elsewhere [33, 34]; herein our focus will be on currently employed phases. Two terms in chiral LC are currently used in the literature for solvent stable chiral phases namely “immobilized” versus “bonded” phases. An immobilized chiral phase is rather an ambiguous term, which typically implies a situation where a chiral selector has been crosslinked in and out of the particle. This term is popularly associated with polysaccharide phases as used earlier. A chiral selector (e.g., derivatized cellulose of appropriate molecule weight) is first dissolved in a solvent such as tetrahydrofuran, the particles are equilibrated with a solution, and the solvent is slowly evaporated in the presence of particles with a rotary evaporator [35]. Although coated phases are relatively simple to make, they have limited solvent compatibilities. For example, dichloromethane, tetrahydrofuran, and ethyl acetate mobile phases dissolve and remove the chiral selectors. Interestingly, bonded and coated chiral selectors show different retention times and similar or lower selectivity compared to coated ones [36]. In general, coated phases show higher selectivity than their “immobilized” counterparts.

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A bonded CSP refers to a covalent bond between the support particle and the chiral selector; organosilane chemistry is typically used for this purpose. The bonding procedures for chiral selectors on silica, typically utilize trichloro or trialkoxysilanes with an alkyl spacer. The alkyl spacer end has a reactive functional group such as glycidyl, amine, isocyanate, isothiocyanate, mercapto (thiol), or Si-H group. This terminal unit on the silane is then coupled with a reactive site on the chiral selector. For example, the isocyanate and glycidyl are very active toward amines/alcohols, which are abundantly available on polysaccharides or macrocyclic glycopeptides. The mercapto units can also be activated in the presence of free radical initiators such as azobisisobutyronitrile, and Si-H can be coupled to terminal alkenes on a chiral selector, for example, in quinine. Alternatively, double bonds on chiral selectors can be coupled via Pt catalyst in hydrosilylation reactions. For example, quinine molecule has a vinylic side chain. Using a triethoxysilane, with one hydride group, one can couple the quinine’s side chain and the ─Si-H group in the presence of Pt (“Speier’s catalyst,” H2PtCl6), catalyst under anhydrous conditions. Presence of a hydroxyl group can be used to react isocyanates to form carbamate linkages. With amino groups, the isocyanate containing silanes form ureas.The functional group coupling the silane and the chiral selector should be resistant to hydrolysis. In general, the resistance to hydrolysis is as follows: urea linkage > carbamate linkage > ester linkage. The latter is very prone to hydrolysis by acids and bases.The former linkages are sufficiently stable toward acid or base hydrolysis. By optimizing the molar ratio of coupling silane with the chiral selector, the selectivity of the stationary phase can be optimized to maximize the value of α. The effect of alkyl spacer length and type on the column selectivity has also been studied in detail by bonding silanes of different length and a terminal epoxy group with cyclodextrin phases [37]. π-Complex Stationary Phases. These synthetic chiral phases are functionalized aromatic hydrocarbons to provide π-π interactions to the analytes. In older literature, the term charge transfer stationary phases were also used to describe them. These π-complex phases participate in donor– acceptor interactions such as H-bonding, face-to-face, face-to-edge, and dipole-dipole stacking. Additional bulky nonpolar groups can provide steric repulsion to the chiral analyte. The idea behind synthetically introducing many discrete interactions was to increase the probability of chiral recognition. The logic behind the design of these CSPs was summarized as “In almost every case, successful CSPs contain some aromatic or extended π-functionality, and this is not accidental. Because of the sterically demanding nature of π-π interactions (face-to-face or face-to-edge), they are often

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the controlling factor in enantiomer separations on these CSPs. π-π interactions are inherently multipoint” [38]. Thus, π-complex CSPs such as (R, R)-N-3,5-dinitrobenzoyl-1,2-diphenyl-1,2-diamine [39] have found applications in separating various classes of compounds such as amides, alcohols, esters, ketones, acids, sulfoxides, phosphine oxides, phosphonates, thiophosphineoxide, β-lactams, organometallics, atropisomers, and heterocycles. Often the analyte is first derivatized with a dinitrophenyl group or another large aromatic group to provide interactions with the chiral selector [40]. Most of these π-complex stationary phases have been utilized using hexane/heptane mixtures with alcohols and basic or acidic additives. The main advantage of using a purely synthetic chiral selector is to control the elution order of enantiomers. By individually bonding a (+) or a (−) version of the derivatized aromatic hydrocarbon onto silica, opposite elution orders can be obtained. For example, Mikes [15] noted that the optical sign of the adsorbed chiral selector determines which enantiomer is retained more selectively on the column. This approach is beneficial for specific analysis when one of the enantiomers is an undesired trace impurity. It is desirable to elute the impurity first to avoid the tail of the primary component. Among all π-complex phases, Whelk-O1 chemistry, which is 1-(3,5-dinitrobenzamido)-1,2,3,4-tetrahydrophenanthrene bonded silica, is used for diverse applications (Fig. 12.4). It is available in both enantiomeric forms (S, S) and (R, R). The electron withdrawing nitro groups on the aromatic rings are essential for chiral recognition via π-π stacking interactions and amide/aromatic n-π interactions. Recent studies have bonded Whelk-O1 on sub-2 µm fully porous supports. The latest applications will be shown in the applications section. Macrocyclic Bonded Stationary Phases. Macrocyclic stationary phases are supports, which have a bonded molecule of moderate molecular

Figure 12.4  Structural characteristics of π-complex stationary phases. Example: S,S Whelk-O1.

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mass. The three useful classes of such chiral phases are (1) crown ether phases by Cram et al. [16, 17]), (2) bonded cyclic oligosaccharides such as cyclodextrins and cyclofructans [18, 41, 42], and (3) macrocyclic antibiotics, which are glycopeptides [43, 44]. Armstrong group pioneered the last two classes. The latter group of macrocyclics is utilized in a variety of chiral separations as detailed in later sections. 1. Crown Ethers. Crown ether CSPs consist of cyclic oligomers of ethylene oxide where the ethyleneoxy, that is, ─CH2CH2O─ is the repeating unit. Ions, the size of K+ and NH4+, tend to form inclusion complexes with [18] crown-6 polyethers. Primary amines, when protonated in acidic mobile phases, also participate in hydrogen bonding via three H atoms of the protonated amine and three oxygen atoms of 18-crown-6. As a result, these crown ether phases work only for primary amines but not for secondary amines and amino acids (e.g., proline) or other such molecules. This type of “adduct” formation is termed as a host–guest relationship (inclusion complexes) as the protonated amine sits in the cavity of the crown ether. The earliest crown ethers phases comprised of crosslinked polystyrene supports [16]. Crown ether columns consisting of bis-(1,1’ binaphthyl)-22crown-6 moiety or bonded (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid are popular CSP chemistries. Fig. 12.5 shows the schematic of the latter and its interactions with a α-amino acid. Specialty columns exist to separate primary amines using dilute (ca. 10 mM) perchloric acid (HClO4). The acid is required to protonate the amines. The use of other acids greatly decreases or negate the separation on this column. Such separations can be challenging on preparative scales

Figure 12.5  (A) Tetracarboxylate crown ether. (B) Formation of an inclusion complex between crown ether oxygens and a quaternary ammonium group.

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because perchloric acid can explode upon concentrating in the presence of organic compounds. Coated versions of crown ether chemistries also exist. Crown ethers based CSPs have severe limitations on solvent compatibility. For example, adding more than 15% methanol in perchloric acid on a Crown-Pak column, the column degrades rapidly. Similarly, the presence of K+ in the sample will alter the chiral selectivity. A detailed review in 2016 covers the status of crown ether phases and their applications [45]. 2. Cyclic Oligosaccharides. Bonded cyclic oligosaccharides are one of the most useful and versatile chiral phases which can be used in normal phase, reversed phases, polar organic mode, and polar ionic mode ­interchangeably. Very few CSPs have this capability to handle such a wide variety of solvents. In this section, two important classes of cyclic oligosaccharides will be discussed namely, cyclodextrins and cyclofructans. In the former, the macrocycle consists of α-(1→4) linked D-glucopyranose units, whereas the latter consists of β-(2→1) linked D-fructofuranose units. Both oligosaccharides are prepared by bacterial action upon starch and inulin which results in cyclodextrins and cyclofructans. Cyclic units of 6, 7, and 8 glucopyranose units are termed as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin respectively, whereas 6, 7, and 8 membered cycloinuloligosaccharides are called as cyclofructan-6 (CF-6), 7(CF-7), and 8(CF-8) respectively. Fig. 12.6 shows the structure of native β-cyclodextrin and cyclofructan-6. The primary and secondary hydroxyl groups on the sugars provide a very versatile functionalizing group, which is amenable to functionalization with several reactive organic functional groups such as isocyanates with ─OHs to form carbamate linked alkyl chains or aromatic rings. Another successful bonding functionalization of the hydroxyl groups is epoxide chemistry. The

Figure 12.6  Structures of (A) β-cyclodextrin and (B) Cyclofructan (CF6).

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remaining hydroxyl groups can also be employed for binding a tetraethoxysilane, which allows silica bonding chemistry. There is a significant difference between cyclofructans and cyclodextrins, which can be a source of confusion. Cyclodextrins have a ∼ 5.5 to 8 Å diameter hydrophobic cavities (depending on which cyclodextrin is being considered) whereas cyclofructans have a small crown ether core surrounded by hydrophilic moieties (Fig. 12.6). The lipophilic toroidal cavities of the cyclodextrins can bind neutral molecules and ions such as NH4+ or Ba2+. A variety of water-soluble and insoluble molecules can fit in the toroidal cavities forming inclusion complexes.When using aqueous-organic mobile phases, retention is mainly by inclusion (or host guest mechanism). Because molecules of different chemistry, size, shape, and spatial geometry form different strength inclusion complexes with cyclodextrins, chiral or other shape selective separations are readily achieved. Organic solvents such as ethanol or acetonitrile compete for inclusion in the cyclodextrin cavity. One prominent chiral phase is HP-RSP cyclodextrin, which is a high performance (R, S)-hydroxypropyl modified β-cyclodextrin “bonded” to silica. This modified cyclodextrin column provides the additional steric and H-bonding capability, leading to broad chiral selectivity for basic, acidic, and neutral compounds in all chromatographic modes [46]. Cyclofructans offer a unique selectivity to chromatographers. Three hydroxyls in the structure completely cap the upper side of the ring. This side of the macrocycle is relatively hydrophilic as the result of the directionality of all its hydroxyl groups. The lower side of the molecule is lipophilic due to methylene group of gauche ─O─C─CH2─O─ units [47]. Thus inclusion complexation by hydrophobic interaction which is present in cyclodextrins is absent in cyclofructans. Attempts at resolving enantiomers using native cyclofructan have been less than marginally successful. Consequently, cyclofructans need to be derivatized to open like a flower bloom and allow chiral interactions. Cyclofructan 6 modified with isopropyl carbamate (Larihc-P column) is a unique stationary phase for primary amines [48] and works with even supercritical fluids. Cyclofructan-7 modified with dimethylphenylcarbamate (CF7-DMP) groups acts as more like a π-complex column with the additional benefits of the unique features of cyclofructan. Similarly, R-naphthyl methyl-carbamate cyclofructan-6 (CF6-RN) provides enantioselectivity toward a broad range of compounds, including chiral acids, amines, metal complexes, and neutral compounds [49]. Interestingly both column chemistries (CF6 Larihc-P and CF6-RN) show complementary selectivity, that is, a separation that is not possible on the first one, is typically resolved on the other column. 3. Macrocyclic glycopeptides. Most chiral stationary phases (Table 12.2) are from natural products rather than totally synthetic routes.

Table 12.2  List of chiral stationary phases and their applications CSP class CSP

π-complex phases

Analytes

Mobile phase

1-(3,5-Dinitrobenzamido)-1,2,3,4,-tetrahydrophenanthrene (Whelk-O1 and Whelko-O2)

Amides, epoxides, esters, urea, carbamates, ethers, aziri- In reversed-phase modes, mobile dines, phosphonates, aldehydes, ketones, carboxylic phases for these CSPs can acids, alcohols, and nonsteroidal antiinflammatory consist of any polarity but drugs (NSAIDs). must remain within a pH 3,5-dinitrobenzoyl leucine This π-electron acceptor phase demonstrates enhanced range of 2.5 to 7.5. Operations enantioselectivities for several classes of compounds, in normal or reversed-phase including benzodiazepines. mobile phases are possible. 3,5-dinitrobenzoyl phenyl glycine Compounds which contain π-basic groups: aryl Mobile phases can consist of substituted cyclic sulfoxides, bi-β-naphthol and its IPA, hexane, heptane, ethyl analogs, α-indanol and α-tetralol analogs, and aryl acetate, ethanol, water, acetosubstituted hydantoins. nitrile, methanol, 1,2-dichlodimethyl N-3,5-dinitro-benzoyl-α-aminoβ-blockers without chemical derivatization. It also romethane and THF. Common 2,2-dimethyl- 4-pentenyl phosphonate resolves the enantiomers of many compounds sepamobile phase additives include (α-Burke 2) rated on π-electron acceptor type chiral stationary acetic acid, TFA, triethylamine, phases. diethylamine, and ammonium N-3,5-dinitrobenzoyl-3-amino-3-phenylAnilide derivatives of a wide variety of chiral carboxacetate. 2-(1,1-dimethylethyl)-propanoate (β-Gem ylic acids, including nonsteroidal antiinflammatory 1) agents Bonded β-lactam (Pirkle 1-J) Underivatized β-blocker enantiomers, and can also be used for the separation of the enantiomers of aryl propionic acid NSAIDs as well as other drugs. 3,5-dinitrobenzoyl derivative of 1,2-diamino- A broad range of racemate classes including amides, cyclohexane (DACH-DNB) alcohols, esters, ketones, acids, sulfoxides, phosphine oxides, selenoxides, phosphonates, thiophosphineoxide, phosphine selenide, phosphine-borane, beta-lactams, organometallics, atropisomers, and heterocycles. 3,5-dintrobenzoyl derivative of phenyl ethyl- General ability to separate the enantiomers of many enediamine (ULMO) racemate classes, and is particularly good at separating the enantiomers of aryl carbinols (Continued)

Table 12.2  List of chiral chemistry classes and their applications (cont.) CSP class CSP Analytes

Crown ethers

(18-Crown-6)-tetracarboxylic acid (Chirosil RCA (+) and SCA (−) ) Amino acids and primary amines

Crown ether type binaphthyl derivatives (CrownPack-CR-I(+), CrownPak-CRI(−)

Mobile phase

This CSP operates in acidic RP mode. Typical pH range of the mobile phase is around pH 2.5. Lower pH will result in a good resolution but a shorter column life. Enantioselectivity differs from the type of acid used. It is recommended that you find the proper acid by screening. Acids such as acetic acid, perchloric acid, sulfuric acid, phosphoric acid, and trifluoroacetic acid are used.

CSP class

CSP

Analytes

Mobile phase

Cyclic oligosaccha- Native β-cyclodextrin (Cyclobond I 2000) rides (cyclodextrin-based phases) Native γ-cyclodextrin (Cyclobond II)

Small analytes of general interest in the pharmaceuti- There are three distinct ways cal, chemical and environmental areas preferred to develop separations on inclusion functional groups: iodide>bromide>chl cyclodextrin-based phases. oride>fluoride; nitrate, sulfate, phosphate hydroxyl polar organic phase mode (as a function of pH). Preferred hydrogen bonding (acetonitrile/methanol/triethgroups carboxyl, carbonyl amines, primary, secondylamine/acetic Acid) Typical ary, tertiary, free or in ring structures composition: 95/5/0.1/0.1. β-cyclodextrin with dinitrophenyl group Small molecules, pharmaceuticals, π basic analytes. Normal Phase Mode (Hex(Cyclobond I 2000 DNP) ane/IPA) Typical composition: Methylated β –cyclodextrin at 2,3 hydroxyl Separates a variety of structural and geometric isomers 90/10. Reversed Phase Mode groups (Cyclobond I 2000 DM) as well as enantiomers not resolved on α and β na(Acetonitrile/Buffer) Typitive cyclodextrin. cal composition: 20/80 (pH Modified β –cyclodextrin using propylene Marginal separations obtained on native cyclodextrins 3.0–7.5). Choice of condioxide (Cyclobond I 2000 SP, RSP) are often enhanced on this CSP in the reversed tions varies with solubility and phase mode with significantly long-term stability. analyte structure. Cyclodextrin This CSP separates nonaromatic racemates. It has phases are stable in all known been one of the best methods for t-boc amino acids solvents. However, halogenated and cyclic hydrocarbons. solvents form strong inclu(R,S)-Hydroxypropyl modified - β cyclodexsion complexes and should be trin (Cyclobond I 2000 HP-RSP) A modified version of Cyclobond I 2000 RSP. avoided both as a mobile phase (R,S)-Hydroxypropyl modified - β cycloImproved selective for most analytes compared to solvent and solubilizing solvent dextrin bonded to SPP particles (CDShellCyclobond I 2000 RSP. for analytes. RSP) Per acetylated β cyclodextrin (Cyclobond I This CSP is beneficial when the includable aromatic 2000 AC) portion of an analyte has a stereogenic center with a hydroxyl or amine group in the alpha or beta position. This CSP will enhance the complex formation Per acetylated γ-cyclodextrin (Cyclobond II leading to higher selectivity and shorter retention AC) than experienced with the native cyclodextrin phases. 3,5-dimethylphenylisocyanate modified β – Small molecules, pharmaceuticals, π acidic or basic cyclodextrin (Cyclobond I 2000 DMP) analytes. (Continued)

Table 12.2  List of chiral chemistry classes and their applications (cont.) CSP class CSP Analytes

Cyclic oligosaccha- Cyclofructan-6 modified with isopropyl ride (Cyclofructan carbamate (LarihcShell-P) based phases)

Macrocyclic glycopeptides

Mobile phase

Primary amines, such as amino alcohols, amino esters, This phase can be operated NP and amino amides and POM. However, better resolution can be obtained in the POM mode. Unlike crown ether CSP, this CSP works more effectively with organic solvents. Cyclofructan-6 modified with R-naphthyl Secondary and tertiary amines, alcohols, and many This phase can be operated RP, ethyl carbamate (Larihc CF6-RN) neutral compounds NP, and POM. However, better resolution can be obtained in the NP mode. Cyclofructan-7 modified with 3,5-dimethyl- A broad variety of compounds. LARIHC CF7-DMP Most effective in the normal phenyl carbamate (Larihc CF7-DMP) CSP demonstrates complementary enantioselecphase tivity when compared to the LARIHC CF6-RN phase Vancomycin (Chirobiotic V, Chirobiotic V2, Neutral molecules, amides, acids, esters, and amines These CSPs can be used in and VancoShell) (specifically secondary and tertiary amines) PIM, POM, RP, and NP Teicoplanin (Chirobiotic T and Chirobiotic Underivatized α, β, γ, or cyclic amino acids, N(for neutral molecules). Any T2, and TeicoShell) derivatized amino acids (FMOC, CBZ, t-BOC), organic solvent can be used. α hydroxy-carboxylic acids, acidic compounds Screening Mobile Phases for including carboxylic acids and phenols, small pepPIM: 100/0.1 wt% MeOH/ tides, neutral aromatic analytes and cyclic aromatic NH4TFA or 100/0.1/0.1 and aliphatic amines. beta-blockers (amino alcoMeOH/acetic acid (AA)/trihols), and dihydrocoumarins methylamine (TEA), for POM: Teicoplanin aglycone (Chirobiotic TAG) Sulfur containing molecules (sulfoxides) including 60/40/0.3/0.2 ACN/MeOH/ amino acids, methionine, histidine and cysteine, AA/TEA, for RP: 30/70 amino alcohols, and neutral molecules like the MeOH/20 mM NH4OAc oxazolidinones, hydantoins, and diazepines (pH 4) buffer, NP: 30/70 Chemically modified macrocyclic glycopep- Similarly selectivity of vancomycin based phase, with EtOH/Hexane (or Heptane) tide for nicotine isomers (NicoShell) increased chiral selectivity to nicotine and nicotine analogs

CSP class

CSP

Amylose based coated phases

tris(3,5-dimethylphenylcarbamate) modified amylose (AD)

Amylose based immobilized phases

Analytes

A wide range of basic, neutral, and acidic analytes that contain structural components of carboxylic acids, alcohols, secondary and tertiary amines, carboxylic ester, sulfur, amides, halogens, ketones, cyclic, aromatics, and ether tris ((S)-α-methylbenzyl carbamate) modified A wide range of basic, neutral and some acidic anaamylose (AS) lytes that contain structural components of carboxylic acids, alcohols, tertiary amines, sulfur, amides, halogens, cyclic, aromatic, and ether tris(5-chloro-2-methylphenylcarbamate) A wide range of basic, neutral and some acidic analytmodified amylose (AY) es that contain structural components of carboxylic acids, alcohols, secondary and tertiary amines, esters, halogens, keto, cyclic, aromatic, and ether tris(3-chloro-4-methylphenylcarbamate) A wide range of acidic, neutral and few basic analytes modified amylose (AZ and Trefoil AMY1) that contain structural components of carboxylic acids, alcohols, halogens, keto, cyclic, and aromatic. tris(3,5-dimethylphenylcarbamate) amylose A wide range of basic, neutral and few acidic analytes (IA) that contain structural components of carboxylic acids, alcohols, esters, sulfur, amide, halogens, keto, cyclic, aromatic, and ether tris(3-chlorophenylcarbamate) amylose poly- Basic and neutral compounds separate from these mer (ID) CSPs. Analytes that contain alcohols, amines, carboxylic esters, sulfur, amides, halogens, cyclic, aromatic, nitro, and ester group are separated on these CSPs.

Mobile phase

All modes are used with the following success POM and PIM = RP>NP

All modes are used with the following success RP>POM and PIM>NP NP, POM, and PIM are used with the following success NP>POM and PIM NP, POM, and PIM are used with the following success NP>POM and PIM NP is mostly used with limited success in POM and PIM

NP is mostly used with limited success in RP

(Continued)

Table 12.2  List of chiral chemistry classes and their applications (cont.) CSP class CSP Analytes

tris(3,5-dichlorophenylcarbamate) amylose polymer (IE)

tris(3-chloro-4-methylphenylcarbamate) modified amylose (IF)

tris(3-chloro-5-methylphenylcarbamate) modified amylose (IG)

Cellulose-based coated phases

Cellulose tris(3,5-dimethylphenylcarbamate) (OD and Trefoil Cel1)

Cellulose tris(4-methylbenzoate) (OJ)

Mainly basic compounds with limited success with acid and neutral compounds separate on this CSP. Amino acid derivatives and compounds that contain carboxylic acid, alcohol, amines, carboxylic ester, sulfur, amide, halogen, cyclic, aromatic, and ether groups are separated on this CSP This CSP tends to separate basic compounds well. Some acid and neutral compounds can be separated on the CSP. Amino acid and their derivatives canbe separated. Analytes that contain carboxylic acid, alcohol, amines, carboxylic esters, sulfur, amides, halogens, keto, cyclic, aromatic, and ether groups can be separated on this CSP. As a new CSP, some separations on a variety of classes of compounds have been shown. Some include anti-inflammatory drugs, amino acids, flavonoids, antiarrhythmic drugs, catecholamines, and antifungal drugs. This CSP separates acids, bases, and neutral compounds. Compounds that contain carboxylic acids, alcohols, amines, carboxylic ester, halogen, cyclic, aromatic, and ether groups are separatedin this phase. This CSP separates mostly basic and neutral compounds. Few acid compounds can be separated. Compounds that contain carboxylic acid, alcohols, amines, carboxylic esters, sulfur, halogens, keto, cyclic, aromatics, and ether groups can separate on this CSP.

Mobile phase

NP

NP is used most often. However, few separations have been performed in RP, POM, and PIM.

This CSP has been shown to separate compound in mostly NP, but RP separations are possible on this CSP. All modes are used with the following success NP>RP>POM and PIM

NP, POM, and PIM are commonly used. RP can be used but has shown limited separations.

CSP class

Cellulose-based immobilized phases

Specialty Chemistries: Proteins, Ion-Exchangers, and Zwitterions

CSP

Analytes

Cellulose tris(3-chloro-4-methylphenylcarba- This CSP separates mostly basic and neutral commate) (OZ and Trefoil Cel2) pounds. Few acid compounds can be separated. Compounds that contain carboxylic acid, alcohols, amines, sulfur, halogens, keto, cyclic, aromatics, and ether groups can separate on this CSP. Cellulose tris (3,5-dimethylphenylcarbamate) This CSP separates acidic, basic, and neutral com(IB) pounds. Some amino acids and their derivatives can be separated. Analytes that contain carboxylic acid, alcohol, amines, amides, halogen, cyclic, aromatic, and ether groups have shown chiral separations. Cellulose tris (3,5-dichlorophenylcarbamate) This CSP separates acidic, basic, and neutral com(IC) pounds. Some amino acids and their derivatives can be separated. Analytes that contain carboxylic acid, alcohol, amines, sulfur, amides, halogen, cyclic, aromatic, and ether groups have shown chiral separations. Immobilized α1-acid glycoprotein (AGP) This CSP works well at separating basic compounds and few acidic and neutral compounds. Compounds that contain carboxylic acid, alcohol, amines, carboxylic ester, sulfur, halogens, keto, cyclic, aromatic and ether groups have shown chiral separations. Immobilized cellobiohydrolase (CBH) The CSP works well with basic compounds. Compounds that contain alcohols, amines, keto, aromatic Immobilized human serum albumin (HSA). This CSP separates acidic compounds and a few neutral compounds. Analytes that contain carboxylic acid, alcohol, amide carbamate, halogen, ketone, cyclic and aromatic groups separate on this CSP, Nderivatized amino acids are separated on this phase.

Mobile phase

NP is most frequently used, followed by POM and PIM.

NP is most utilized. Some separation can be obtained by RP

NP is most utilized. Some separation can be obtained by RP, POM, and PIM

RP mode is used with the CSP

(Continued)

Table 12.2  List of chiral chemistry classes and their applications (cont.) CSP class CSP Analytes

Sulfonated (ChiralpakZWIX(+) & (−))

O-9-(tert-butyl carbamoyl) quinine (Chiralpak-QN AX)

O-9-(tert-butyl carbamoyl) quinidine (Chiralpak-QD-AX)

Mobile phase

This CSP is utilized for amphoteric compounds such A type of PO mode which uses as native amino acids and peptides. This CSP has a few percentage of water. also been shown to separate N-derivatized amino An example moble phase acids. Compounds that contain carboxylic acid, alconsist of 50 mM formic cohol, amine, secondary amide and aromatic groups acid + 25 mM diethylamine have been shown to separate on this CSP. in methanol/acetonitrile/water = 49/49/2 These CSP separate acidic compounds and N-deriva- PO and RP mode are utilized. tized amino acids. Analytes that contain carboxylic acid, alcohol, carboxylic ester, amide, ketone, aromatic and ether groups have shown to separate on these phases.

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Figure 12.7  Structures of (A) Vancomycin and (B) Teicoplanin selectors.

Attempts to synthesize these antibiotics takes about 20 to 40 steps [50, 51], and hence commercial stationary phases (Chirobiotic Series or the newly introduced VancoShell and Teicoshell) are made by fermented processes. Fig. 12.7 shows the structure vancomycin and teicoplanin chiral selectors. For example, vancomycin, ristocetin are fermentation products of Streptomyces orientalis (a bacterium found in Borneo soil), Amycolatopsis lurida (a pathogenic bacterium), and Antinoplanes teichomyceticus (soil bacterium). All macrocyclic glycopeptides are relatively large molecules with molecular weights in the range of 600 to 2200. There are hundreds of such macrocyclic antibiotics. Four such compounds have become versatile commercial chiral stationary phases namely teicoplanin, teicoplanin aglycone (same as teicoplanin but sugar units removed), vancomycin, and risotecin.These molecules share several common properties by having a multitude of stereogenic centers , for example, vancomycin (18), teicoplanin (23), and risotecin (38).The polyfunctional molecules contain hydroxyls, peptide groups, carboxylic acids, amines, aromatic, groups, methyl esters, amido groups, and several sugar units (although not all listed functional groups are present in one macrocyclic glycopeptide).The structure of these molecules show that the interactions defined for protein phases (see section on Specialty Columns) are present along with interactions present in modified cellulose/amylose chiral selectors. As a result, strong π-π complexation, hydrogen bonding, dipole stacking, strong anionic or cationic bonding is present along with weak inclusion complex. Further, the fused macrocyclic rings give these compounds additional rigidity and fixed spatial configurations that can be beneficial for chiral recognition.This multitude of functional groups allow the columns to be used in reversed phase mode, normal phase

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mode, polar organic mode, and polar ionic mode interchangeably. These chiral stationary phases contain peptide bonds.The macrocyclic glycopeptides used for bonding can be chemically pure (e.g., vancomycin) or they may consist of structurally similar compounds differing in the number of carbons chain attached with one of its sugars like teicoplanin. An interesting study probed the mechanism of chiral recognition by vancomycin selector. Electrophoretic separations were performed with and without Cu2+ in the buffer. The buffer containing copper, completely lost the enantiomeric selectivity [52]. It was shown that complexation of vancomycin with copper involved coordination of copper with the secondary amine present on its aglycone basket, while the primary amine on the disaccharide side chain remained free. It was concluded based on X-ray crystallography that the complexation of vancomycin with copper through the secondary amine plays a significant role in chiral selectivity in liquid chromatography. Vancomycin- and teicoplanin-based CSPs are commercially available. The primary difference is how the macrocyclic glycopeptide is bonded to the silica surface. Commercial Chirobiotic V2 and T2 columns have a six-carbon chain spacer, between the amino-silane and the macrocyclic glycopeptide.Vancomycin-based chiral columns work best for acidic, basic, and neutral compounds in reversed phase. Neutral molecules can be separated by normal phase, reversed phase, and polar organic mobile phases. The same applies to teicoplanin columns. One of the interesting features of macrocyclic glycopeptides columns is the “principle of complementary separations” for macrocyclic glycopeptide columns. This implies that if a partial enantiomeric separation is found on one column, then it is very likely to obtain baseline resolution on the other with a similar mobile phase. This observation originates from structural similarities of macrocyclic glycopeptide along with their subtle differences in stereoselective binding sites [53].

12.6.1  Polysaccharide CSPs Among a large number of CSPs so far developed, derivatives of cellulose and amylose exhibit broad applicability to a wide range of enantiomeric compounds. While cellulose triacetate was an adequate initial CSP [14, 54], Okamoto and coworkers further developed the coated polysaccharide stationary phases in the mid-1980s by derivatizing cellulose with tribenzoate and trisphenylcarbamate [35] and then derivatizing both cellulose and amylose with tris(3,5-dimethylphenylcarbamate) [55, 56]. Today, multiple manufacturers make and sell polysaccharide-based CSPs (Fig. 12.8). It is

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Figure 12.8  Structures of cellulose and amylose phases.

estimated 70 to 80% of liquid chiral separations are performed on polysaccharide-based CSPs [57]. The ubiquity of polysaccharides-based CSPs in separation science relies on its unique structure and the degree of various derivatization that has been performed. Fig. 12.8 shows the most common derivatives of cellulose and amylose [58]. The chiral recognition of polysaccharide-based CSPs depends on the secondary structure of the polymer and not the individual monomer. Derivatives of cellulose and amylose show much higher chiral recognition than nonderivatized forms. Phenylcarbamates and their various substituents, as shown in Fig. 12.8, provided reasonably broad enantioselectivity when coated on silica [35, 55, 59]. The success of various derivatives of phenylcarbamates of cellulose and amylose arises due to the electron donating or withdrawing effects of the phenyl substituents and their effect on the secondary structure [59]. Despite the chiral selectivity afforded by cellulose and amylose derivatives, which were coated onto silica supports, there are drawbacks. These phases were conventionally prepared by physically coating the

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stationary phase without any covalent bonding to the support [55, 60]. Due to the preparation of these coated phases, solvents such as tetrahydrofuran, chloroform, dichloromethane, toluene, ethyl acetate, and acetone cannot be used. These solvents cause problems such as swelling and dissolution of the stationary phase. Additionally, the problem of leakage of the chiral selector with the products of preparative separations. Eluents that are acceptable to use include alkanes, alcohols, acetonitrile, and water. Due to the solvent limitations of the coated polysaccharide phases, immobilization became a requirement for the versatility of these CSPs. Many immobilization approaches have been established since the inception and commercialization of cellulose and amylose CSPs [57, 61–65]. Okamoto and coworkers immobilized cellulose and amylose derivatives as early as 1987 by using 3-aminopropyl-functionalized silica and a diisocyanate crosslinker [66]. These first generation immobilized CSPs exhibited little chiral recognition due to an excessive amount of crosslinkage needed to immobilize the stationary phase. To overcome this obstacle, it was shown that covalent bonding to the silica support at the reducing terminal end of an amylose chain defeated the excessive linkage strategy and improved chiral recognition [67, 68]. The limitation of this technique is its complicated preparation process and selectivity for amylose only. Immobilization method utilizing triethoxysilyl groups on derivatized polysaccharides show more promise than other immobilization techniques with respect to processing, immobilization efficiency, chiral recognition, and applicability to different polysaccharide derivatives [69, 70]. During the immobilization process, the higher polymeric structure of the polysaccharide is suggested to be less distorted. It is thought to be due to small amounts of triethoxysilyl groups needed for cross-linkage and immobilization. Francotte et al. patented [71] photochemical and free radical crosslinking approaches for immobilizing polysaccharides onto silica. Many commercial immobilized phases use this technology to create stable chiral phases, which are marketed as immobilized phases. These types of immobilized polysaccharide CSPs displayed similar chiral selectivity to traditional coated derivatized polysaccharide analogs. There are several commercially available immobilized polysaccharide phases available (Table 12.2). These phases have been shown to be excellent and robust analytical and preparative CSPs in comparison to their coated counterparts. With the plethora of information on these phases regarding chiral separations, the reader is referred to other literature whose sole focus is

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Figure 12.9  Structures of (A) Quinine and (B) tert-carboamylated quinine bonded to silica.

on polysaccharide-based CSPs and not in a general survey of chiral liquid chromatography techniques [72–77]. Specialty Columns. There are several commercial specialty chiral columns which perform exceptionally well for certain classes of compounds. Among them, chiral ion-exchangers, zwitterions, and protein-based phases deserve a special mention. Cinchona alkaloids such as quinine and quinidine can be converted into chiral anion-exchange-type stationary phases (Fig. 12.9). The quinine-based columns developed by Lindner et al. show good enantioselectivity toward chiral acids [78]. The nitrogen group of these alkaloids is positively charged. By incorporating a sulfonic acid group in the molecule, these alkaloids can also be converted into zwitterions. The zwitterionic phases have somewhat broader selectivity especially with chiral acids, chiral amines, chiral amino acids, and peptides [79]. Recently, Linder et al. showed a fascinating insight, which confirmed the initial studies by Mikes. Using modified quinine selector, the authors showed the enantiomeric purity of bonded chiral selector is critical. Small concentrations of the opposite enantiomer of a selector caused a significant decrease in selectivity. For instance, bonding just a 0.05 mole fraction of opposite enantiomer adamantyl, neopentyl derivative of quinine on silica resulted in a drop in α value of 2.4 to 1.9 of (R, S)-N-FMOC-phenylalanine [80]. Many proteins have the potential to discriminate between the separate forms of a chiral compound. Proteins are also chiral in nature regarding both their primary structure (i.e., being made of L-amino acids) and their secondary or higher order structures. Among the bonded proteins, α1-acid glycoprotein bonded packing material was found to be particularly useful for early pharmaceutical applications, particularly for cyclic and secondary aromatic amine compounds. The large size of the protein has a drawback regarding capacity. They have the least capacity among chiral stationary phases. Complications can arise from denaturation; these

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protein-based columns are the least stable/robust of all the chiral stationary phases. Consequently, despite their historical importance, they are rarely used today. Among other proteins, bovine serum albumin, human serum albumin, ovamucoid, and cellobiohydrolase have also been bonded on silica supports for separating aromatic acids, primary amines, and anionic compounds [81]. More sophisticated synthetic approaches exist for binding proteins and glycopeptides onto silica. The hydroxyl or free amine groups are utilized for bonding. High temperatures cannot be used as the proteins readily denature. Some of these techniques are the Schiff base method (i.e., reductive amination), the N-hydroxysuccinimide (NHS) method, the carbonyl diimidazole (CDI) method, the epoxy method, the ethyl dimethyl aminopropyl carbodiimide (EDC) method, and the tresyl chloride process, as well as others [82]. A number of protein-based CSPs have been prepared using the Schiff base method. This is often done by taking diol-containing support, oxidizing this support to form aldehyde groups, and then allowing these aldehyde groups to combine with free amine groups on a protein and followed by sodium cyanoborohydride treatment [82]. As is evident from the above discussions, synthetic approaches for the chiral phase are far more complicated than most achiral phases. Similarly, chiral bonded polymeric phases have been introduced based on tartaric acid derivatives bonded to silica such as O,O’-bis (3,5-dimethylbenzoyl)-N, N’-diallyl-L-tartar diamide, and O,O’-bis (4-tertbutylbenzoyl)-N, N’-diallyl-L-tartar diamide. These phases are stable in all solvents, albeit using trifluoroacetic acid can cause some hydrolysis. Other polymeric phases such as poly(trans-1,2-cyclohexanediyl-bis-acrylamide) and poly(diphenylethylenediamine-bis-acryloyl) also exist for enantiomeric separations.

12.7  PACKING PROCESS OF HIGH-EFFICIENCY CHIRAL PHASES (“N” IN THE RESOLUTION EQUATION) After bonding the chiral selector to the stationary phase (fully porous or superficially porous), the material must be packed into chromatographic columns to perform separations. Besides optimized synthesis and mass loading, the column packing procedure governs the peak width and the shape of chiral separations. Commercial packing methods are usually trade secrets. All the engineering details have been addressed in a recent perspective [83]; however, a brief practical overview is given for current sub 2 µm to 5 µm particles for readers desiring to pack new chiral stationary phases provided

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column packing hardware is available. Chiral phases in 0.5, 2, 3, 5, 10, and 15 cm columns (with 2.1, 3.0, and 4.6 mm i.d.) have been successfully packed with the procedures outlined later. A high-pressure pneumatically driven pump, which can generate pressures up to 10,000 psi or 30,000 psi, is required. To successfully pack the particles, a solvent system, which can disperse the particles must be chosen. The solvent system typically consists of binary solvents such as chlorinated solvents/alcohols, and alcohols/cyclohexanols. The suspension should be observed under an optical microscope for agglomeration of silica particles.There have been numerous studies with chiral fully porous and superficially porous particles which show that solvents which aggregate particles lead to poor column efficiency, whereas dispersive suspensions have yielded reduced plates height (plate height/particle diameter)  1.5), defining the “column cutting” parameter as a speed that can be obtained through an actual reduction in column length, or by changes in mobile phase composition, flow rate, or column temperature. Using this approach, a large number of baseline enantioseparations in the sub-minute time scale were obtained on short columns packed with 3-micron CSPs based on amylose or cellulose derivatives and with 5-micron Whelk-O1 CSP, all of them using CO2 based eluents [29].

14.3.2  Pirkle-type CSPs Originally introduced and later developed by Pirkle, these CSPs are characterized by low-molecular-size selectors whose molecules form an ordered layer (brush-type) on the inert matrix surface. These selectors typically show the presence of polar sites capable of dipole–dipole, H-bonding, and aromatic–aromatic interactions with complementary sites on the analyte molecules. The widely used Whelk-O1 selector [32] bears a π–acidic 3,5-dinitrobenzamide and a π–basic naphthyl fragments close to the stereogenic center, held together in a rigid fashion and forming a cleft where aromatic portion of the analyte molecules can be accommodated while simultaneously establishing H-bond, face-to-face, and face-to-edge aromatic interactions. Spectroscopic data taken both in solution and in the solid state support this picture of enantioselective recognition.This simple 1:1 association mode and the ordered brush-type organization of the chiral selector on the silica surface have direct relevance not only on the large enantioselectivity usually observed, but also on the kinetic side of the process and ultimately on the chromatographic efficiency. The excellent kinetic performances of columns packed with CSPs incorporating brush-type selectors are peculiar of the high speed–high efficiency results obtained on the newly introduced sub-2-micron materials (see further). Working preferentially in the normal-phase mode, an easy transfer to SFC mode can be obtained with Pirkle-type CSPs. The effect of cosolvent nature was investigated by Szczerba and Wrezel [33] in chiral SFC method development on a Whelk-O1 column. Focusing the results only on the three alcohols, the authors found out that selectivity and retention decrease as polarity of alcohol decrease

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and that isopropanol ensures highest selectivity even if compared with nonalcoholic modifier (tetrahydrofuran and acetonitrile). Byrne et al. [34] employed Whelk-O1 CSP together with polysaccharide-based CSP in the evaluation of 2,2,2-trifluoroethanol as organic modifier in CO2. Alcoholsensitive chiral compounds in addition to the other standard chiral ones were analyzed, and results were compared with those obtained with typical cosolvents (methanol, ethanol, isopropanol, and acetonitrile). It was shown that trifluoroethanol is a useful alternative to standard cosolvents and it represents a solution for separation of alcohol-sensitive compounds. Recently, in a fundamental SFC investigation, columns packed with the Whelk-O1 CSP have been included in a group of 10 chiral columns to investigate the effect of column backpressure on retention and selectivity [35].

14.3.3  Macrocyclic glycopeptide antibiotic CSPs Macrocyclic glycopeptide antibiotics are a class of medium-sized molecular size whose structure contains an aglycone cyclopeptide “basket” surrounded by carbohydrate moieties. Teicoplanin (T), teicoplanin aglycone (TAG), ristocetin (R), and vancomycin (V) are used in the preparation of a family of CSPs that have common features like a broad spectrum of application even for strongly polar and ionic compounds and the ability to work in a variety of eluting conditions including reversed-phase, polar organic, and normal-phase modes. However, only a few examples of SFC separation, in comparison to their use in liquid chromatography, have been recently reported in the literature [36,37]. Lavison and Thiébaut [36] employed a ristocetin stationary phase in SFC for the resolution of a series of structurally diverse samples. Perhaps the strong retention showed in normal elution conditions (20% of organic modifier) have restricted the development of analytical methods in SFC. However, the renewed interest about these CSPs in supercritical mode is very recent and overlaps with the introduction of a new generation of silica particles, developed for ultrafast separations (see following section).

14.3.4  Ion exchange CSPs The tert-butyl carbamates of quinine and quinidine immobilized on silica turned out to be the most useful structure variations in the design of weak-anion exchange chiral selector [38]. These selectors showed good enantioselectivity for the same compound classes of acidic chiral compounds also under SFC conditions. Pell and Lindner [39] showed in 2012

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that anion-exchange, the primary interaction governing retention during HPLC elution with polar organic or aqueous mobile phases, is preserved under SFC elution with carbon dioxide containing methanol and additives. The effects of different types and amounts of additives (acids, bases, water) and of temperature on chromatographic performance were evaluated. The results indicated that retention, but not enantioselectivity, can be modulated by the amounts and types of co- and counterions (salts) in the modifier. In addition, the authors studied the effect of the “inherent acidity” of CO2–methanol mobile phases on chromatographic parameters. As counterpart of anion exchange CSPs, a strong cation exchange CSP based on the syringic acid amide derivative of trans-(R,R)-2-aminocyclohexanesulfonic acid was prepared and employed in SFC to resolve the enantiomers of chiral amines [40]. A systematic study of retention and selectivity as a function of the nature and concentration of additives (formic acid, ammonium formate, ammonia, tertiary amines) demonstrated also in this case that retention under SFC conditions is predominantly based on an ion exchange mechanism.

14.3.5  Cyclodextrin CSPs Cyclodextrins (CDs) are cyclic oligosaccharides consisting of several (usually 6, 7, or 8) glucose units connected by α-1,4-linkages. They possess a three-dimensional shape resembling a hollow torus. The primary 6-hydroxyl groups are located on the narrow rim of the torus, while the 2and 3-hydroxyl secondary groups are located on the wider rim, generating a hydrophilic outer surface and a hydrophobic internal cavity.The latter can accommodate nonpolar portions of the analytes and can form 1:1 inclusion complexes in water-rich media.The hydroxyl groups are amenable of selective modification, yielding a variety of derivatized cyclodextrins with extended interaction and recognition abilities.An overview about the synthesis and applications of cyclodextrins, in native or derivatized form, has been reported few years ago [41]. The use of CDs in SFC with packed columns is not widespread, probably because the low polarity of the mobile phases, even in the presence of polar modifiers, does not favor the formation of inclusion complexes with the analytes, thus limiting their interactions with the external surface of the CDs. On the other hand, cationic functionalized CDs immobilized onto vinyl silica via radical copolymerization, demonstrated good enantioselectivity in the separation of analytes containing ionizable moieties (anions) [42,43].These works highlighted the importance of

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electrostatic forces between the ionizable group of the enantiomers and the cationic sites of CDs.

14.4  ULTRAFAST HIGH-EFFICIENT SFC SEPARATIONS The favorable fluidic properties of carbon dioxide-based eluents allow ultrafast enantiomeric separations to be performed at extremely high flow rates, with reduced drawbacks due to high column back-pressure or degraded efficiency compared to the same separations obtained with classical eluents. While some examples of fast, sub-minute SFC separations have been reported in the past [6,9,10], only in recent years the field has been deeply investigated leading to high efficient processes that are routinely completed on the seconds time scale. A number of factors have contributed to this gain in analysis speed, including improved chromatographic particle technology and dedicated instrumentation [44–46]. Particle technology is important because, to a first approximation, the column efficiency (expressed as the number of theoretical plates, N) is inversely proportional to the average size of the CSP particles that fill the column. Thus, with smaller particles, the analyte molecules can travel a shorter pathway and improve their mass transfer kinetics, which is one of the factors contributing to H, the height equivalent to a theoretical plate (H = L/N, smaller H corresponds to higher column efficiency). A compact equation accounting for the whole set of factors that contribute to H is H = Hl + He + Hm + Hf where the subscripts indicate longitudinal diffusion (l), eddy dispersion (e), liquid–solid mass transfer resistances due to slow diffusion rate across the particles (m), and frictional heating generated at elevated flow rates (f  ). Van Deemter plots correlate plate height H with mobile-phase velocity (u) according to H = A+

B + Cu u

where A, B, and C represent eddy dispersion, longitudinal diffusion, and mass transfer, respectively. Plots of experimentally measured H versus flow rate velocity are commonly used to explore the column kinetic performance and locate the optimal flow rate.

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The decrease in particle size has a direct impact on the pressure required to pump the mobile phase through the column, according to the Darcy’s equation: ∆P =

Φu0 Lη d p2

where η is the viscosity of the mobile phase, L the column length, u0 the linear mobile-phase velocity (u0 = L/t0, with t0 being the hold-up time), Φ is the column resistance factor, and dp is the particle diameter. Therefore, low-viscosity mobile phases like those based on carbon dioxide are ideal when small particles (e.g. sub-2-micron fully porous particles), or long columns, are used in combination with high eluent flow rates. High speed and high efficiency enantioseparations can be realized under SFC conditions using short chiral columns packed with small particles and delivering the eluent at high flow rates: baseline resolutions that are completed in the seconds time domain have been demonstrated recently possible for a large variety of chiral solute structures. Asymmetric synthesis, enzymatic reactions, and resolution by crystallization are research areas where quick analytical methods for optical purity determinations are highly desirable. Fast enantiomeric excess measurements of a large number of chiral products can accelerate the screening of experimental conditions and the overall optimization process. SFC on CSPs is amenable to high-throughput screening of enantiomeric excess (hundreds of samples per day) and can compete with optical or sensor-based methods whose analysis cycle time is smaller than 60 s. Distinct additional advantages of high-throughput screening methods based on SFC on CSPs are the broad applicability (also for mixtures) and the inherent quantitation ability [47–49]. In a seminal paper, Gasparrini and coworkers [50] realized a proof-ofconcept study demonstrating the feasibility of high-throughput screening of large compound libraries by enantioselective ultra-high-performance SFC. Van Deemter analysis of columns packed with standard 5.0-micron and with 1.7-micron Whelk-O1 particles clearly showed the superior kinetic performance of the latter CSP when used in conjunction with carbon dioxide-based eluents (Fig. 14.5). In the high flow rate region of the plots, the efficiency loss of the columns was always less pronounced under SFC elution conditions, thus enabling short analysis times and high efficiency.

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Figure 14.5  Comparison of van Deemter plots under LC and SFC conditions of the first eluted enantiomer of trans-stilbene oxide on 1.7-micron Whelk-O1 CSP. Adapted from [50]. Abbreviations: LC, liquid chromatography; SFC, supercritical fluid chromatography; CSP, chiral stationary phase.

A 5-cm long column packed with 1.7-micron totally porous WhelkO1 particles was used in conjunction with fast CO2–methanol gradient elution, to screen for enantioselectivity a library composed by 129 chiral compounds featuring ample structural variation. A high success rate was observed for polar neutral, and both acidic and basic compounds and transfer of the gradient conditions to unoptimized isocratic elution led to very fast, high-efficient separations with sub-minute run times as shown in Fig. 14.6.

Figure 14.6  Fast and high-efficient separations obtained on 1.7-micron Whelk-O1 CSP. Adapted from [50]. Abbreviation: CSP, chiral stationary phase.

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Figure 14.7  Ultrafast, high-efficiency enantioseparations using CSPs based on 1.7-micron Whelk-O1 CSP, CO2/MeOH 80/20, 4.0 mL/min flow rate, Tcol = 35 °C, 12.4 MPa back pressure, UV detection at 220 nm. Top trace 50 × 4.6 mm column, bottom trace 10 × 4.6 mm column. Abbreviations: CSP, chiral stationary phase; UV, ultraviolet.

Ultrafast enantioseparations accompanied by high efficiency were achieved using a 1-cm long column, as shown in Fig. 14.7.Taking advantage of the large enantioselectivity (α = 2.5) and efficiency (N/m ∼ 250,000 on the second peak) offered by the 5 cm column packed with sub-2-micron Whelk-O1 CSP, the excess resolution available (Rs = 15) can be traded for speed of analysis by reducing the column length to 1 cm (Fig. 14.8). An extremely fast and complete separation, lasting only 10 s, was obtained on the short column (α = 2.5; N/m ∼ 205,000; Rs = 5). It should be noted that ultrafast, high-efficiency SFC enantioseparations realized on short columns, or on columns having small void volumes, pose severe hardware limitations

Figure 14.8  Preparative SFC large-scale resolution on immobilized cellulose tris(3,5-dichlorophenylcarbamate) 5-micron CSP packed on a 3 × 25 cm column, 10% methanol in CO2 as eluent delivered at 200 mL/min, T = 45 °C, 10 MPa back pressure. Abbreviations: SFC, supercritical fluid chromatography; CSP, chiral stationary phase.

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in terms of extremely small extra-column volumes and to high detector sampling rates [49]. Ultrafast separations under SFC conditions have been reported for a variety of chiral samples on short columns packed with fully porous sub2-micron CSPs based on immobilized amylose derivative [51], Whelk-O1 [52], teicoplanin [53,54] selectors. Quinine immobilized on sub-3-micron superficially porous particle has been reported as an effective CSP capable to afford SFC separation with run times in the seconds time scale [55].

14.4.1 Preparative separations Chiral drugs are administered as single enantiomers or as racemic mixture, and the properties of every enantiomer should be studied independently as required by the regulatory authorities, so it is of high importance in pharmaceutical R&D to develop a method that allows to obtain the single isomers. Development of an asymmetric synthesis can be time consuming; on the contrary, the preparation of a racemic mixture and its resolution by chromatography shortens significantly the discovery times. Preparativescale-SFC using carbon dioxide-based eluents is nowadays extensively adopted in both academic and industrial environments to obtain enantiomers with high enantiomeric purity, bringing numerous advantages including high speed, low pressure drop, reduced solvent consumption and costs, greater safety concerning flammability and toxicity, reduced environmental impact, and fast solvent removal [56]. In chiral pSFC, the most common CSPs are those based on polysaccharide-derived selector adsorbed or immobilized on silica or Pirkle-type phases, because of the good selectivity and loadability, allowing increased amounts of racemate to be applied to a column and still achieve resolution. Other CSPs suitable for analytical SFC, like those based on cyclodextrins or on glycopeptide antibiotics, are not used on a preparative scale. 14.4.1.1  pSFC Versus pHPLC The principal difference between SFC and HPLC emerge from the properties of the mobile phase: supercritical fluid or liquid. In a preparative HPLC, hexane or heptane are used in mixture with solvents like isopropanol, methanol, acetonitrile, and methylene chloride that control both sample solubility and retention. In a preparative SFC, carbon dioxide constitutes the principal component of the mobile phase, generally above 60%, with the addition of the same polar solvents used in LC. The low viscosity and high diffusivity of the CO2-based mobile phase allows high flow rates,

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thus resulting in greater chromatographic efficiencies and resolutions with shorter run times; in fact, SFC is 3–5 times faster than HPLC. Increasing the flow rates often results in higher productivity and this is of particular importance to achieve purified products for pharmaceutical testing in good yield and short times. Owing to the low viscosity of the supercritical CO2, columns packed with small-particle-size CSPs can be used, maintaining high efficiencies even at high flow rates [57].The high throughput in a preparative SFC permits the use of smaller size column compared to HPLC, thus reducing the cost of CSP material and column hardware. Another advantage of the preparative SFC versus preparative HPLC is the lower organic solvent usage. The lower solvent usage in the preparative SFC is achieved by replacing a majority of the mobile phase with CO2. Most of the mobile phase (60%–95%) is removed after chromatography by decreasing pressure, leaving only the modifier that consists usually of methanol, ethanol, or acetonitrile. The result is a reduction of the time required for post purification solvent removal and product isolation. Additionally, most of the solvents used in the preparative scale HPLC, especially in normal phase where alkanes are usually employed, are flammable. The consequence is that the preparative HPLC process must be performed in appropriate bunkers where the fire risk in minimized. Greater amounts of solvents are consumed in the preparative HPLC process compared to SFC, and usually the volume of organic solvents that can be stored in a standard laboratory are restrictive. The replacement of organic solvent with CO2 is desirable because it is nonflammable and nontoxic, so it offers less concern about safety and it is a renewable ecofriendly source, as it is generally recovered as a byproduct of manufacturing processes or condensed from the atmosphere, thus resulting in no net increase in CO2. Overall, organic solvent volumes for the preparative SFC are 2–10 times less than those used in the preparative HPLC allowing to reduce running costs considerably. However, a complete cost comparison between SFC and HPLC should consider additional aspects like initial equipment cost, maintenance time, cost of the operator, and elimination of the solvent after purification. Additionally, SFC is a new technology compared to HPLC and it still requires development because some parameters like pressure and temperature play a fundamental role in a preparative method development. Furthermore, the scale up from analytical to preparative scale that is straightforward in HPLC and is based on general rules, such as adjusting the volumetric flow and injection volume to the square of the ratio of the columns diameters, is more complicated in SFC. Indeed, an altered pressure drop along

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the column, because of the high compressibility of the CO2-based mobile phase, will generate a density change which eventually will affect retention of the analytes and their separation. Some additional strategies, like control of the backpressure to hold up the same average density along the column, should be carried out to maintain the same chromatographic resolution and selectivity when transferring a method from the analytical to preparative scale in SFC compared to LC [58,59].

14.4.2 Application and strategy Early-stage toxicological studies require from 100 g up to few kilograms of the enantiomerically pure product, and SFC is a technique of choice for the purification in the pharmaceutical field, spacing in a range from small preparative up to larger enantioseparation scale. In the past years, successful separations of different amounts of chiral compounds have been presented in many publications. In 2008, Miller [60] reported the approach in separation of a racemate that were proprietary pharmaceutical compounds belonging to and synthesized at Amgen (Cambridge, MA, USA). In a recent publication, Leek et al. [15] reported a strategy to obtain a 5 kg separation of a compound using four CSP screening (three polysaccharide-based and Whelk-O1). When performing a large-scale separation, different aspects must be taken under consideration and one of the most important is the choice of the organic modifier in the mobile phase. The solvent is usually chosen on the basis of cost, availability, volatility, and environmental impact, but the most considerable feature is the solubility of the analyte. This is sometimes the limiting factor for preparative SFC, reducing the amount of racemate that can be injected. A method set up to afford greater separations may result unsuccessful and even causing precipitation of the compound in the column or eventually system shutdown. The solubility of the compound could change substantially in the CO2/modifier mixture compared to that in the modifier alone [16]. Eventually, the racemate must be dissolved in a solvent different from the pure modifier and depending on the injection technique, solubility issues can have undesirable effect on the chromatographic separation. Another useful strategy for increasing the productivity of the process is the “stacked injection” technique that is often applied during a preparativescale SFC [56].

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With this technique, a second injection is performed before the elution of the second enantiomer of the compound is complete. The time between two subsequent injections is based on the time calculated from the beginning of the first peak and the end of the second eluted peak. Several examples of pSFC resolutions of drugs or drug-like molecules have been described by Francotte. The stacked injection technique was used to resolve a 50 g sample of chiral N-benzyl-3-methyl-piperydin-4-one on a 30 × 250 mm column packed with an adsorbed polysaccharide CSP. Using 5% ethanol in CO2 delivered at 120 mL/min and a cycle time of 1.8 min, the two enantiomers were obtained with greater than 99% enantiomeric excess, and the overall process had a productivity of 2.33 kg of racemate per day per kg of CSP [57]. Recently, an interesting paper reported a large-scale chiral SFC applications with fast run times and high loadings by using a chlorinated polysaccharide-CSP in both coated and immobilized versions [61]. In particular, the bonded version of the CSP permits the use of dichloromethane or acetonitrile as cosolvents gaining in sample solubility and loading ability for each injection and allowing the development of robust methods to resolve poorly alcohol-soluble racemates. Fig. 14.8 shows a chromatographic trace of a large-scale SFC separation of a racemate in short time (less than 6 min) and with high throughput (8.5 g/h). Additional examples highlighting the advantages of pSFC in a pharmaceutical research and development context are reported by Leek and Andersson [62]. With the availability of the immobilized version of the polysaccharides CSPs, the wider choice of solvent additives greatly improves the solute solubility in the CO2-based eluents, giving clear advantages in terms of productivity. In one example, the authors report a preparative isolation of the desired enantiomer of a chiral carboxylic acid on a 250 × 50 mm column packed with cellulose tris(3,5dichlorophenylcarbamate) immobilized on 5-micron silica particles, using 20% ethanol and 0.5% formic acid in CO2 as eluent at 12 MPa and 40 °C. Since the whole separation was completed in about 3 min, a total of 1.1 kg of the desired enantiomer was obtained in short time, with high yield and 99% enantiomeric excess. The pSFC process showed a chromatographic throughput of 5.7 kg racemate/kg CSP/day, with a solvent consumption of only 0.07 m3/kg racemate and the additional benefit of efficient solute recovery by evaporation of the mobile phase. Preparative separation of enantiomers by SFC has nowadays become the method of choice for milligrams to kilogram scale. Distinct advantages of pSFC are the high productivity,

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the reduced consumption of organic solvent, and the easy recovery of the desired enantiomers [63].

14.5  CONCLUSIONS Enantiomer separation by SFC on packed columns is now a mature technique, thanks to the advancement in the theory behind the mechanism of retention and selectivity and to the development of improved CSPs. Parallel improvement in instrumentation quality and robustness has contributed to the acceptance of SFC as a reliable technique in the chiral separation field, complementary to HPLC and in some cases with superior performance. The unique properties of carbon dioxide-based eluents render chiral SFC the method of choice for ultrafast high-efficient separations on modern sub-2-micron CSPs and for preparative separations on a large scale.

REFERENCES [1] Darr, J. A.; Poliakoff, M. New Directions in Inorganic and Metal-Organic Coordination Chemistry in Supercritical Fluids. Chem. Rev. 1999, 99, 495–541. [2] Klesper, E.; Corwin, A. H.; Turner, D. A. High Pressure Gas Chromatography Above Critical Temperatures. J. Org. Chem. 1962, 27, 700–701. [3] Lesellier, E. Retention Mechanisms in Super/Subcritical Fluid Chromatography on Packed Columns. J. Chromatogr. A 2009, 1216, 1881–1890. [4] Perrenoud, A. G. G.; Veuthey, J. L.; Guillarme, D. Comparison of Ultra-high Performance Supercritical Fluid Chromatography and Ultra-high Performance Liquid Chromatography for the Analysis of Pharmaceutical Compounds. J. Chromatogr. A 2012, 1266, 158–167. [5] Mourier, P. A.; Eliot, E.; Caude, M. H.; Rosset, R. H.; Tambuté, A. G. Supercritical and Subcritical Fluid Chromatography on a Chiral Stationary Phase for the Resolution of Phosphine Oxide Enantiomers. Anal. Chem. 1985, 57, 2819–2823. [6] Gasparrini, F.; Misiti, D.; Villani, C. Direct Resolution in Sub- and Supercritical Fluid Chromatography on Packed Columns Containing Trans-1,2-diaminocyclohexane Derivatives as Selectors. Trends Anal. Chem. 1993, 12, 137–144. [7] Saito, M. History of Supercritical Fluid Chromatography: Instrumental Development. J. Biosci. Bioeng. 2013, 115, 590–599. [8] Taylor, L. T. Supercritical Fluid Chromatography for the 21st Century. J. Supercrit. Fluids 2009, 47, 566–573. [9] Mangelings, D.; Vander Heyden, Y. Chiral Separations in Sub- and Supercritical Fluid Chromatography. J. Sep. Sci. 2008, 31, 1252–1273. [10] Farrell, W. P.; Aurigemma, C. M.; Masters-Moore, D. F. Advances in High Throughput Supercritical Fluid Chromatography. J. Liq. Chrom. Relat. Tech. 2009, 32 (11–12), 1689–1710. [11] Lesellier, E.; West, C. The Many Faces of Packed Column Supercritical Fluid Chromatography—A Critical Review. J. Chromatogr. A 2015, 1382, 2–46. [12] Kalíková, K.; Šlechtová, T.;Vozka, J.; Tesarˇová, E. Supercritical Fluid Chromatography as a Tool for Enantioselective Separation: A Review. Anal. Chim. Acta 2014, 821, 1–33.

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[13] Płotka, J. M.; Biziuk, M.; Morrison, C.; Namieśnik, J. Pharmaceutical and Forensic Drug Applications of Chiral Supercritical Fluid Chromatography. Trends Anal. Chem. 2014, 56, 74–89. [14] Desfontaine,V.; Guillarme, D.; Francotte, E.; Novákovác, L. Supercritical Fluid Chromatography in Pharmaceutical Analysis. J. Pharm. Biomed. Anal. 2015, 113, 56–71. [15] Leek, H.; Thunberg, L.; Jonson, A. C.; Öhleń, K.; Klarqvist, M. Strategy for Large-Scale Isolation of Enantiomers in Drug Discovery. Drug Discov.Today 2017, 22 (1), 133–139. [16] Miller, L. Preparative Enantioseparations Using Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 250–255. [17] Guiochon, G.; Tarafder, A. Fundamental Challenges and opportunities for preparative supercritical fluid chromatography. J. Chromatogr. A 2011, 1218, 1037–1114. [18] Rajendran, A. Design of Preparative–Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 227–249. [19] Vajda, P.; Guiochon, G. The Modeling of Overloaded Elution Band Profiles in Supercritical Fluid Chromatography. J. Chromatogr. A 2014, 1333, 116–123. [20] Shibala, T.; Mori, K.; Okamob, Y. Polysaccharide Phases. In Chiral Separation by HPLC; Krstulovic, A. M., Ed.; Ellis Horwood: New York, 1989; pp. 336–398. [21] Francotte, E. R. Enantioselective Chromatography as a Powerful Alternative for the Preparation of Drug Enantiomers. J. Chromatogr. A 2001, 906, 379–397. [22] Francotte, E.; Huynh, D. Immobilized Halogenophenylcarbamate Derivatives of Cellulose as Novel Stationary Phases for Enantioselective Drug Analysis. J. Pharm. Biomed. Anal. 2002, 27, 421–429. [23] Chankvetadze, B. Recent Developments on Polysaccharide-based Chiral Stationary Phases for Liquid-phase Separation of Enantiomers. J. Chromatogr. A 2012, 1269, 26–51. [24] West, C.; Guenegou, G.; Zhang, Y.; Morin-Allory, L. Insights into Chiral Recognition Mechanisms in Supercritical Fluid Chromatography. II. Factors Contributing to Enantiomer Separation on Tris-(3,5-dimethylphenylcarbamate) of Amylose and Cellulose Stationary Phases. J. Chromatogr. A 2011, 1218, 2033–2057. [25] Felix, G.; Berthod, A.; Piras, P.; Roussel, C. Part III Supercritical Fluid Separations. Sep. Purif. Rev. 2008, 37, 229–301. [26] West, C.; Zhang,Y.; Morin-Allory, L. Insights into Chiral Recognition Mechanisms in Supercritical Fluid Chromatography. I. Non-enantiospecific Interactions Contributing to the Retention on Tris-(3,5-dimethylphenylcarbamate) Amylose and Cellulose Stationary Phases. J. Chromatogr. A 2011, 1218, 2019–2032. [27] Geryk, R.; Kalíkova, K.; Schmid, M. G.; Tesarˇová, E. Enantioselective Separation of Biologically Active Basic Compounds in Ultra-performance Supercritical Fluid Chromatography. Anal. Chim. Acta 2016, 932, 98–105. [28] Khater, S.; Lozac’h, M. -A.; Adam, I.; Francotte, E.; West, C. Comparison of Liquid and Supercritical Fluid Chromatography Mobile Phases for Enantioselective Separations on Polysaccharide Stationary Phases. J. Chromatogr. A 2016, 1467, 463–472. [29] Regalado, E. L.; Welch, C. J. Pushing the Speed Limit in Enantioselective Supercritical Fluid Chromatography. J. Sep. Sci. 2015, 38, 2826–2832. [30] Regalado, E. L.; Helmy, R.; Green, M. D.; Welch, C. J. Chromatographic Resolution of Closely Related Species: Drug Metabolites and Analogs. J. Sep. Sci. 2014, 37, 1094–1102. [31] Schafer, W.; Chandrasekaran, T.; Pirzada, Z.; Zhang, C.; Gong, X.; Biba, M.; Regalado, E. L.;Welch, C. J. Improved Chiral SFC Screening for Analytical Method Development. Chirality 2013, 25, 799–804. [32] Pirkle, W. H.; Welch, C. J. An Improved Chiral Stationary Phase for the Chromatographic Separation of Underivatized Naproxen Enantiomers. J. Liq. Chromatogr. 1992, 15, 1947–1955. [33] Szczerba, T.; Wrezel, P. Effect of Varying Co-solvents in SFC Method Development on a Whelk-O1 Chiral Stationary phase. LCGC N. Am. 2008, 21.

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[34] Byrne, N.; Hayes-Larson, E.; Liao, W. W.; Kraml, C. M. Analysis and Purification of Alcohol-Sensitive Chiral Compounds Using 2,2,2-Trifluoro Ethanol as a Modifier in Supercritical Fluid Chromatography. J. Chromatogr. B 2008, 875, 237–242. [35] Wang, C. L.; Zhang,Y. R. Effects of Column Backpressure on Supercritical Fluid Chromatography Separations of Enantiomers Using Binary Mobile Phases on 10 Chiral Stationary Phases. J. Chromatogr. A 2013, 1281, 127–134. [36] Lavison, G.; Thiébaut, D. Evaluation of a Ristocetin Bonded Stationary Phase for Subcritical Fluid Chromatography of Enantiomers. Chirality 2003, 15, 630–636. [37] Liu, Y.; Berthod, A.; Mitchell, C. R.; Xiao, T. L.; Zhang, B.; Armstrong, D. W. Super/ Subcritical Fluid Chromatography Chiral Separations with Macrocyclic Glycopeptide Stationary Phases. J. Chromatogr. A 2002, 978, 185–204. [38] Maier, N. M.; Nicoletti, L.; Lämmerhofer, M.; Lindner, W. Enantioselective Anion Exchangers Based on Cinchona Alkaloid-Derived Carbamates: Influence of C8/C9 Stereochemistry on Chiral Recognition. Chirality 1999, 11, 522–528. [39] Pell, R.; Lindner, W. Potential of Chiral Anion-Exchangers Operated in Various Subcritical Fluid Chromatography Modes for Resolution of Chiral Acids. J. Chromatogr. A 2012, 1245, 175–182. [40] Wolrab, D.; Kohout, M.; Boras, M.; Lindner, W. Strong Cation Exchange-type Chiral Stationary Phase for Enantioseparation of Chiral Amines in Subcritical Fluid Chromatography. J. Chromatogr. A 2013, 1289, 94–104. [41] Xiao,Y.; Ng, S.; Tan, T. T.Y.; Wang,Y. Recent Development of Cyclodextrin Chiral Stationary Phases and Their Applications in Chromatography. J. Chromatogr. A 2012, 1269, 52–69. [42] Wang, R. Q.; Ong, T. T.; Ng, S. C. Chemically Bonded Cationic β-Cyclodextrin Derivatives as Chiral Stationary Phases for Enantioseparation Applications. Tetrahedron Lett. 2012, 53, 2312–2315. [43] Wang, R. Q.; Ong, T. T.; Ng, S. C. Chemically Bonded Cationic (–)Cyclodextrin Derivatives and Their Application in Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1224, 97–103. [44] Guillarme, D.; Bonvin, G.; Badoud, F.; Schappler, J.; Rudaz, S.;Veuthey, J. -L. Fast Chiral Separation of Drugs Using Columns Packed with Sub-2 µm Particles and Ultra-high Pressure. Chirality 2010, 22, 320–330. [45] Kotoni, D.; Ciogli, A.; D’Acquarica, I.; Kocergin, J.; Szczerba, T.; Ritchie, H.; Villani, C.; Gasparrini, F. Enantioselective Ultra-high Performance Liquid Chromatography: A Comparative Study of Columns Based on the Whelk-O1 Selector. J. Chromatogr. A 2012, 1269, 226–241. [46] Grand-Guillaume Perrenoud, A.; Veuthey, J. -L.; Guillarme, D. The Use of Columns Packed with Sub-2 µm Particles in Supercritical Fluid Chromatography. Trends Anal. Chem. 2014, 63, 44–54. [47] Hamman, C.;Wong, M.; Aliagas, I.; Ortwine, D. F.; Pease, J.; Schmidt, D. E., Jr.;Victorino, J. The Evaluation of 25 Chiral Stationary Phases and the Utilization of Sub-2.0 µm Coated Polysaccharide Chiral Stationary Phases via Supercritical Fluid Chromatography. J. Chromatogr. A 2013, 1305, 310–319. [48] Hamman, C.; Wong, M.; Hayes, M.; Gibbons, P. A High Throughput Approach to Purifying Chiral Molecules Using 3-µm Analytical Chiral Stationary Phases via Supercritical Fluid Chromatography. J. Chromatogr. A 2011, 1218, 3529–3536. [49] Barhate, C. L.; Joyce, L. A.; Makarov, A. A.; Zawatzky, K.; Bernardoni, F.; Schafer, W. A.; Armstrong, D. W.; Welch, C. J.; Regalado, E. L. Ultrafast Chiral Separations for High Throughput Enantiopurity Analysis. Chem. Commun. 2017, 53, 509–512. [50] Sciascera, L.; Ismail, O.; Ciogli, A.; Kotoni, D.; Cavazzini, A.; Botta, L.; Szczerba, T.; Kocergin, J.;Villani, C.; Gasparrini, F. Expanding the Potential of Chiral Chromatography for High-throughput Screening of Large Compound Libraries by Means of Sub-2 µm

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Whelk-O1 Stationary Phase in Supercritical Fluid Conditions. J. Chromatogr. A 2015, 1383, 160–168. [51] Berger, T. A. Preliminary Kinetic Evaluation of an Immobilized Polysaccharide Sub2µm Column Using a Low Dispersion Supercritical Fluid Chromatograph. J. Chromatogr. A 2017, 1510, 82–88. [52] Berger, T. A. Kinetic Performance of a 50 mm Long 1.8 µm Chiral Column in Supercritical Fluid Chromatography. J. Chromatogr. A 2016, 1459, 136–144. [53] Barhate, C. L.; Farooq Wahab, M.;Tognarelli, D. J.; Berger,T. A.; Armstrong, D.W. Instrumental Idiosyncrasies Affecting the Performance of Ultrafast Chiral and Achiral Sub/ Supercritical Fluid Chromatography. Anal. Chem. 2016, 88, 8664–8672. [54] Ismail, O. H.; Ciogli, A.;Villani, C.; De Martino, M.; Pierini, M.; Cavazzini, A.; Bell, D. S.; Gasparrini, F. Ultra-fast High-efficiency Enantioseparations by Means of a Teicoplaninbased Chiral Stationary Phase Made on Sub-2 µm Totally Porous Silica Particles of Narrow Size Distribution. J. Chromatogr. A 2016, 1427, 55–68. [55] Patel, D. C.; Breitbach, Z. S.;Yu, J. J.; Nguyen, K. A.; Armstrong, D. W. Quinine Bonded to Superficially Porous Particles for High-efficiency and Ultrafast Liquid and Supercritical Fluid Chromatography. Anal. Chim. Acta 2017, 963, 164–174. [56] Speybrouck, D. Preparative Supercritical Fluid Chromatography: A Powerful Tool for Chiral Separations. J. Chromatogr. A 2016, 1467, 33–55. [57] Francotte, E. Practical Advances in SFC for the Purification of Pharmaceutical Molecules. LC–GC Europe April 2016. [58] Tarafder, A. A Scaling Rule in Supercritical Fluid Chromatography. I. Theory for Isocratic Systems. J. Chromatogr. A 2014, 1362, 278–293. [59] Åsberg, D.; Enmark, M.; Samuelsson, J.; Fornstedt, T. Evaluation of Co-solvent Fraction, Pressure and Temperature Effects in Analytical and Preparative Supercritical Fluid Chromatography. J. Chromatogr. A 2014, 1374, 254–260. [60] Miller, L. Preparative Enantioseparations Using Supercritical Fluid Chromatography. J. Chromatogr. A 2012, 1250, 250–255. [61] Wu, D. -R.; Yip, S. H.; Li, P.; Sun, D.; Mathur, A. From Analytical Methods to Largescale Chiral Supercritical Fluid Chromatography Using Chlorinated Chiral Stationary Phases. J. Chromatogr. A 2016, 1432, 122–131. [62] Leek, H.; Andersson, S. Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development. Molecules 2017, 22, 158–166. [63] Welch, C. J. The Use of Preparative Chiral Chromatography for Accessing Enantiopurity in Pharmaceutical Discovery and Development. Comprehensive Org. Synth. 2014, 9, 143–159 Second Edition.

CHAPTER 15

Chiral Separation Strategies in Mass Spectrometry: Integration of Chromatography, Electrophoresis, and Gas-Phase Mobility James N. Dodds, Jody C. May, John A. McLean Vanderbilt University, Nashville, TN, United States

15.1  INTRODUCTION As chiral molecules, by definition, are characterized by rotation of plane polarized light, it may initially seem odd that many analytical studies have utilized mass spectrometry (MS), a nonspectroscopic technique, to analyze chiral systems. As the biological function of compounds is derived from the molecular structure, many MS studies specifically focus on what structural forms of the analyte contribute to an observed phenotype [1]. Modern mass spectrometers are highly selective, often able to identify small molecule analytes (99% (97%ee) >99% (97%ee) >99% (97%ee) >99% (97%ee) >99% >99% >99% (97%ee) >98% >99%

355 633 355 355 633 355 633 355 355 355

+191.2 ± 2.8 +48.1± 1.9 –188.8 ± 2.8 +70.5 ± 2.0 –12.1 ± 1.9 –63.0 ± 5.9 +5.2 ± 1.1 +304.2 ± 11.0 –180.3 ± 9.3 +10.2 ± 2.9

+165.2 +45.5 –165.2 +21.8 –17.0 –88.4 +19.0 +416.9 –157.8 –26.4

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with a cavity, in which the chiral signals are proportional to the number of cavity passes N (and hence the effective pathlength, which was of order 1 km), and therefore can be enhanced by about N  ≈ 1000. The second breakthrough is the ability to suppress birefringent backgrounds with the large intracavity circular birefringence; this is an important breakthrough, as birefringent backgrounds are one of the factors limiting polarimetry measurements reaching the shot-noise limit. In Section 16.5, we describe one final breakthrough, the ability to measure absolute chiral signals, without needing to remove the chiral sample.

16.5  CAVITY RING-DOWN POLARIMETRY WITH SIGNAL REVERSALS In the previous section, we described a method of chiral measurement, with sensitivity enhanced by an optical cavity. However, despite this, the obtained sensitivity was below the single-pass shot-noise limit. An important limiting factor, for both single-pass and cavity-enhanced cases, is the ability to measure the null sample, needed as a reference for the signal of the chiral sample.The act of removing the sample often introduces errors to the measurement system, as there will be small changes to the alignment, positioning, background birefringence, etc. In this section, we describe a method that allow the absolute measurement of the chiral signals, without needing to remove the sample. In contrast to the linear cavity shown in Fig. 16.3, we now consider a ring cavity shown in Fig. 16.7; the discussion in this chapter will be based on the results in Refs. [35,36]. A ring cavity is different from a linear cavity, in that two directions of propagation are supported (which we label as clockwise CW, and counterclockwise CCW), and the output from these two directions can be separated. For a linear cavity, the two directions of the light through the sample cannot be easily separated spatially (though they can, in principle, be separated temporally, if each pulse through the cavity can be resolved). For the linear cavity in Fig. 16.3, the large intracavity birefringence was produced by two quarter-wave plates with optical axes offset by angle α, producing a chiral rotation of angle 2α per roundtrip. The angle is chiral, because the angle has the same sign (in the light-propagation frame) for both CW and CCW; therefore, if a chiral sample is added with rotation angle , the total single-roundtrip rotation angle will be (2α + ) for both CW and CCW directions. At this point, we note that there is another form of circular birefringence that has different symmetry: Faraday rotation.

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Figure 16.7  Experimental setup for signal-reversing CRDP, with a bowtie cavity. Abbreviation: CRDP, cavity ring-down polarimetry.

There exist transparent crystals with a large Faraday Effect, known as magneto-optic crystals, which have a large Verdet constant, such as TGG [37]. TGG absorbs less than 10−4 cm−1 from about 500–1400 nm, can be antireflection coated so that it is compatible with a low-loss cavity, and has a Verdet constant of 134 rad T−1 m−1 at 632 nm. Therefore, a TGG crystal with length 1 cm, with an applied magnetic field of 400 G, will produce a Faraday rotation of about 3°, which is of similar magnitude to that produced using the quarter-wave plates. Such magnetic fields can be produced with solenoids and can be reversed in sign from B to –B, at will. Therefore, we see that the intracavity circular birefringence can be produced magneto-optically. The sign of the Faraday rotation angle θF is determined by the magnetic field direction B and not by the direction of propagation of the light (as is the case for chiral optical rotation). This difference in symmetry, between Faraday rotation θF and chiral rotation c, leads to a very interesting result when both are present in a ring cavity: the total single-pass rotation angle for CW and CCW, ΘCW and ΘCCW, are different (breaking the symmetry between CW and CCW), as in one case the two angles add, and in the other case the two angles subtract: ΘCW = θ F + ϕ c (16.17a) (16.17b) ΘCCW = θ F − ϕ c

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In analogy to Eq. (16.15), the different single-pass optical rotation of ΘCW and ΘCCW produce different polarization beating frequencies: (16.18a) ω CW = (θ F + ϕ c ) tr = (θ F + ϕ c ) c L

ω CCW = (θ F − ϕ c ) tr = (θ F − ϕ c ) c L (16.18b) where tr = L/c is the ring cavity round-trip time and L is the ring cavity round-trip length. The sign of the Faraday rotation angle θF can be controlled by the sign of the applied magnetic field B, so that the polarization beating frequencies can be written as

ω CW ( ±B ) = ( ±θ F + ϕ c ) c L + ω offset (t ) (16.19a) ω CCW ( ±B ) = ( ±θ F − ϕ c ) c L + ω offset (t ) (16.19b) where we have added the term ω offset (t ), to include all nonideal, timedependent offset and noise terms, such as from poor cavity alignment and from residual birefringence. Notice that the experimental polarization beating signals, as described by Eq. (16.13), are only sensitive to the absolute value of the polarization beating frequency w. Therefore, the difference in the polarization beating frequencies ∆ω ( ± B ), using Eq. (16.19) and assuming that θ F is the largest term, is given by (16.20) ∆ω ( ± B ) = ω CW ( ±B ) − ω CCW ( ± B ) = ± 2ϕ c ( c L ) Notice that the Faraday rotation θ F cancels, as does the noise ω offset (t ) (assuming that both CW and CCW have common noise), leaving only the desired term proportional to c, and with a sign controlled by the sign of the B field. Therefore, the additional subtraction can be performed: ∆ω ( + B ) − ∆ω ( − B ) = 4ϕ c ( c L ) (16.21) Eqs. (16.20) and (16.21) demonstrate the strength of signal reversals, where two experimental signals, in principle, have everything in common except that the contribution from the desired signal differs in sign; subsequently, subtraction of the signals cancels all undesired components, while the desired signal is doubled, which gives the factor of 2 in Eq. (16.20). The

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application of the second signal reversal gives a second factor of 2 (and a total factor of 4) in Eq. (16.21). These signal reversals allow the determination of c without needing to remove the chiral sample, but simply by comparing the polarization beating frequencies of CW and CCW (i.e. one direction provides a background reference for the other), and an additional check is given by reversing the applied magnetic field. This ability to measure chiral samples without needing a null measurement opens the way for measuring samples that cannot be done otherwise (such as samples at surfaces which cannot be easily or noninvasively removed, or in open-air samples under ambient pressure where clearly the atmosphere cannot be pumped out). Following below, we describe the application of signal-reversing CRDP to samples in (a) gas phase, (b) open air, (c) solution, and (d) evanescent waves, using an 800 nm laser pulse. The experimental setup for a signal-reversing CRDP bowtie cavity is shown in Fig. 16.7. The bowtie ring cavity is chosen because the angle of incidence (AOI) at the mirrors is small (less than 5°), which allows the birefringence at each reflection to be small (less than 0.1°) and allows 0° AOI mirrors to be used (so that one can use the same mirrors for linear or bowtie cavities). In contrast, conventional ring cavities, with 45° AOI, have several disadvantages: the birefringence at each reflection can be very large (in the 10° range or larger); the cavity lifetime is typically very different for s and p modes (by about a factor of 10), and the circular birefringence from one of the four mirrors being out of plane is much bigger than that for a bowtie cavity. A gas cell with antireflection-coated windows was inserted in the CRDP cavity, shown in Fig. 16.7 (inset). Vapor of α-pinene, of either (+) and (–) enantiomeric form, was introduced with pressures ranging up to 4 mbar, whereas the magnetic field was alternated between states +B and –B (where the magnitude of B was 0.3 T, and produced with an electromagnet). The four experimental ring-down traces, corresponding to the four states (CW, +B), (CW, –B), (CCW, +B), and (CCW, –B), are shown in Fig. 16.8. Notice that the four traces are virtually indistinguishable and that to observe small differences, the traces (at long times of a few microseconds) need to be blown up twice (see inset of Fig. 16.8). Here, notice that, as described by Eq. (16.19), compared to the polarization beating frequency for the empty cavity w0, two of the frequencies increase slightly by the same amount, ω CW ( + B ) and ω CCW ( − B ), whereas two of the frequencies decrease slightly by the same amount, ω CW ( − B ) and ω CCW ( + B ). The differences ∆ω ( + B ) and ∆ω ( − B ) from Eq. (16.20) are shown in

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Figure 16.8  Experimental signals for signal-reversing CRDP for 4 mbar of (+)-α-pinene vapor, showing the polarization beating for the four channels corresponding to the combinations of CW and CCW propagation directions, and +B and –B magnetic fields, from [35]. Abbreviations: CRDP, cavity ring-down polarimetry; CW, clockwise; CCW, counterclockwise.

Figure 16.9  Signal-reversing CRDP measurements of optical rotation versus pressure for (+)-α-pinene and (–)-α-pinene vapor, for +B and –B magnetic fields, showing the highly symmetric results and demonstrating the signal reversals for each case, from [35]. Abbreviation: CRDP, cavity ring-down polarimetry.

Fig. 16.9, as a function of (+)-α-pinene and (–)-α-pinene vapor pressure. Notice the resulting highly symmetric graph, in which ∆ω ( + B ) and ∆ω ( − B ) traces mirror each other through the x-axis, whereas the traces associated with (+)-α-pinene and (–)-α-pinene mirror each other through the y-axis. This high degree of symmetry, predicted by Eqs. (16.20) and

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(16.21), is readily observed because the system studied has very little systematic noise, and mainly the signals are visible. Now that this has been demonstrated, we can use this high degree of symmetry to isolate chiral signals in high-noise environments (with noises that do not demonstrate the symmetry to B field exchange and can thus be subtracted). An example of where the advantages of signal-reversing CRDP are well demonstrated is in a high-noise environment where no background subtraction is possible for conventional single-pass polarimetry. The measurement of vapor of α-pinene in an open-air setting at ambient pressure is such an example: the evaporation of the α-pinene produces large timedependent and spatial variations in the refractive index of the air in the cavity, causing various instabilities and large time-dependent backgrounds. The setup where an open tray of α-pinene is allowed to evaporate into one arm of the CRDP cavity is shown in the inset of Fig. 16.7; the tray can be removed manually, to demonstrate a null measurement. In Fig. 16.10A, all four polarization beating frequencies are shown, for the tray IN and the tray OUT. Eq. (16.19) predicts that, compared to tray OUT, ω CW ( + B ) and ω CCW ( − B ) should increase, whereas ω CW ( − B ) and ω CCW ( +B ) should decrease. In contrast, we see that for the tray IN, all four frequencies decrease; the reason for this is that the dominant component of the frequency change is not a chiral signal, but a nonchiral background due to the vapor refractive index (likely causing a slight misalignment of the cavity). Therefore, if any one of the four channels was used to measure the chirality of the gas, all four would yield an incorrect value, and two of the channels would even yield the incorrect sign of the chirality. However, when the two subtraction procedures from Eqs. (16.20) and (16.21) are applied to the four frequencies to yield the absolute optical rotation c, we see in Fig. 16.10B, a much more clear picture: when the tray is OUT, the optical rotation is zero (within experimental error), for both enantiomers of α-pinene; when the tray is IN for (+)-α-pinene, the optical rotation is positive, and when the tray in IN for (–)-α-pinene, the optical rotation is negative (and the magnitude of the optical rotation agrees with the optical rotation of 4 mbar vapor pressure of α-pinene).We note that the increased noise for the tray IN occurs because the density of α-pinene is actually varying; whereas for tray OUT, conditions are more stable, and the error bars are smaller.These measurements demonstrate the ability of signalreversing CRDP to isolate chiral signals in larger time-varying backgrounds. Next, we demonstrate measurement in solution phase using a syringepumped flow cell. The fused-silica (FS) windows of the flow cell are

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Figure 16.10  Open-air signal-reversing CRDP measurements of optical rotation for (+)-α-pinene and (–)-α-pinene vapor, showing (a) the signals of the four channels and (b) the chiral signal after two signal-reversal subtractions, from [35]. Abbreviation: CRDP, cavity ring-down polarimetry.

antireflection coated on the outer surface, for air/FS, and on the inner surface for FS/water (where the approximate refractive indices at 800 nm for air, water, and FS are 1.00, 1.33, and 1.45, respectively). Sucrose solutions of 0%, 1%, 2%, and 3% were passed in succession and the optical rotation was measured, and shown in Figure 16.11. The limiting factor in measurement

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Figure 16.11  Signal-reversing CRDP measurements of optical rotation for sucrose solutions for concentrations from 0% to 3%, from [36]. Abbreviation: CRDP, cavity ring-down polarimetry.

precision in this case seems to be the quality of the flow cell; we believe that much more precise measurements can be obtained with the more uniform flow and better compound separation obtained through high-performance liquid chromatography (HPLC). Finally, we present measurements of chiral samples in the evanescent wave (EW) of the probe light formed from total internal reflection (TIR) at the surfaces of an FS dove prism. We note that optical rotation measurements in an EW had never been measured before. The reasons for this are several: (a) the effective pathlength in the EW is very small (a few micrometers), so that the signals are necessarily very small, (b) there are two main sources of birefringence that are difficult to compensate, the bulk birefringence of the prism, and the birefringence from the different penetration depths of the s and p polarizations at TIR, and (c) the act of removing the sample at the surface causes changes in surface birefringence and temperature (unless there is excellent temperature stabilization), which cause effects larger than the small chiral optical rotation. However, signalreversing CRDP is uniquely adapted to suppress the birefringence and isolate the chiral signals. The experimental setup is shown in the inset of Fig. 16.7, where the light beam refracts at the first surface of the antireflection-coated prism, then performs TIR with an AOI of approximately 84°, and finally refracts at the last prism surface so that the light beam propagates along the original propagation direction (before entering the prism). An antireflection-coated

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MgF2 plate is used as a birefringence compensator, to ensure that the total single-pass birefringence is much less than the induced Faraday rotation θF. Before describing the measurements, we first give a theoretical background for the optical rotation in EWs. The penetration depth of the EW is described by an exponential decay from the distances from the prism z: I ( z ) = I 0e − z d , where d is given by (see bottom of Fig. 16.12):

λ (16.22) d= 4π n p sin 2 θ − N 2 where λ is the wavelength of the light, np is the refractive index of the prism (here np = 1.45), θ is AOI at the TIR surface (here θ = 84°), and N = n/np (where n is the refractive index of the sample). Similarly, the Goos–Hänchen

Figure 16.12  EW signal-reversing CRDP measurements of optical rotation for maltodextrin, fructose, and glycerin solutions, showing (A) the signals of the four channels, (B) the chiral signal after two signal-reversal subtractions where the chiral signals have been isolated from the noise, (C) a plot of the optical rotation versus refractive index, and (D) a schematic of the size of the EW at the prism surface, controlled by the refractive index; from [35]. Abbreviations: EW, evanescent wave; CRDP, cavity ring-down polarimetry.

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shift LGH (see bottom of Fig. 16.12) which is the distance between the point where the light impinges at the TIR surfaces and the point where the light leaves the TIR surface, and is given by

λ tan θ (16.23) LGH = π n p sin 2 θ − N 2 For illustration, we give some typical values of d and LGH. For λ = 800 nm, θ = 84°, np = 1.45, n = 1 (meaning that only air is in the EW), and N = 1/1.45 = 0.690, then d = 61 nm and LGH = 2.3 µm. For n = 1.44 (for a solution close to the refractive index of the prism), N = 1.44/1.45 = 0.9931, and d = 0.83 µm and LGH = 31.4 µm. Therefore, we see that the size of the LGH of the EW can be varied from about 2 to 30 µm, by varying the refractive index in the EW. The optical rotation in the EW, according to the Drude–Condon model for Maxwell’s equations in isotropic optically active media [38–40], is well approximated by [36]: 2 cos θ ∆n N  π   ∆n   cos θ  L ϕ EW ≈ = (16.24) 2 2     λ   1 − N   sin θ  GH n 1− N sin 2 θ − N 2

where ∆n = ( n+ − n− ), n = ( n+ + n− ) 2, and n+ and n− are the refractive indices for left and right circularly polarized light, respectively. We also express EW in terms of LGH, so that we can compare EW to conventional transmission optical rotation, with T = (π∆n/λ)Leff, where Leff is the pathlength for transmission optical rotation. Setting EW = T, we obtain the effective pathlength for the optical rotation in the EW in terms of the Goos–Hänchen shift LGH:  cos 2 θ 1  (16.25) L eff =  LGH  sin θ 1 − N 2  This equation gives some intuition as to the expected magnitude of the EW optical rotation, compared to the geometrical pathlength LGH. For example, there is a regime where the factor in the parentheses is approximately equal to 1, specifically for grazing incidence (θ ≈ 90°) and for index matching (N ≈ 1); in this case, sin θ ≈ N ≈ 1, and therefore cos 2 θ (1 − N 2 ) ≈ 1, so that the entire prefactor is approximately equal to 1, yielding L eff ≈ LGH . This simple result shows that in this limiting case (of grazing incidence and index matching), the effective optical rotation pathlength is simply the

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geometric optical path in the EW parallel to the prism surface (whereas the optical path perpendicular to the prism surface, going out and coming back, cancels by the symmetry of chiral optical rotation); we can interpret the optical rotation as behaving entirely classically in this regime. For other conditions, Leff can be slightly larger than LGH, or can be much smaller than LGH. For example, at the critical value of TIR, where sin θ = N, and cos2  θ = 1 −  N2, then Leff = LGH/sin  θ; therefore, for a typical value of θ = 45°, then L eff = 2LGH , and the optical rotation is somewhat larger than that for normal transmission measurements. In contrast, close to grazing incidence, but far from index matching makes Leff very small; for example, for θ = 85°, and N = 0.5, then L eff ≈ (0.01)LGH , and the optical rotation is suppressed by about a factor of 100 compared to equivalent transmission measurements. In an attempt to make EW as large as possible, notice that it can be maximized in three separate ways: (a) choosing a sample with a large ∆n (which we have done with the choice of concentrated solutions of maltodextrin and fructose, which have ∆n up to about 3 × 10−6, for solutions with concentration of about 50%); (b) choosing N to be nearly 1 (index matching), for example, N = 1.442/1.453 = 0.9924, as term 1/(1 − N2) becomes large, in this case 1/(1 −  N2)  ≈ 66.3; and (c) choosing θ close to 90° (known −1 2 as grazing incidence), for example, θ = 84°, as the term ( sin 2 θ − N 2 ) 2 2 −1 2 becomes large, in this case ( sin θ − N ) ≈ 15. Notice that the last two enhancement factors give a total enhancement of 66.3 × 15 ≈ 1000, compared to values of N and θ that are not favorable. This large enhancement factor was used to help observe optical rotation in the EW for the first time (in addition to the advantages offered by signal reversing CRDP). Solutions of maltodextrin, fructose, and glycerol are passed over the TIR surface using a flow cell. The solutions were chosen because maltodextrin solutions can produce large positive optical rotations, fructose solutions can produce large negative optical rotations, and glycerol solutions produce no optical rotation. All three solutions can easily be used to vary the refractive index between 1.41 and 1.45, by varying the concentrations; varying the refractive index in this range allowed the size of the EW to be varied over about an order of magnitude, for an effective pathlength of about 2–30 µm, as discussed above. For a fixed refractive index, all three solutions of maltodextrin, fructose, and glycerol are alternated through the EW with the flow cell, and all four polarization beating frequencies, ω CW ( + B ), ω CCW ( − B ), ω CW ( − B ), and ω CCW ( + B ) , are measured and shown in Fig. 16.12A, for refractive

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index n = 1.442. Notice that all four frequencies show common noise, as they all drift up and down together. In contrast, the chiral signal is not obvious to the eye, as it is not clear that the pairs ω CW ( + B ) and ω CCW ( − B ), and ω CW ( −B ) and ω CCW ( +B ) have a relative splitting when the three solutions are alternated. Again, no single channel does give the correct answer, and the chiral signal cannot be identified. However, when the two subtraction procedures are performed (Eqs. (16.20) and (16.21)), it now becomes clear that maltodextrin gives a positive optical rotation, and fructose gives a negative optical rotation (see Fig. 16.12B). Notice that the optical rotation signals have small systematic drifts that are not random noise. We assume that these drifts are caused by variations in the concentration and refractive index of the solutions in the EW, which will cause variations in the size of the EW and, therefore, to the magnitude of the optical rotation signal. Finally, we plot the optical rotation of maltodextrin and fructose solutions versus refractive index n (with n ranging from about 1.41 to 1.45), while also comparing to the exact theoretical predictions; the agreement is very good (see Fig. 16.12C). These results represent the first demonstration of chiral optical rotation measurements in an EW. Despite the successes of the novel open-air and EW chiral optical rotation measurements, it is important to note that the sensitivity of this chiral polarimeter (with an absolute optical rotation sensitivity of about 100 microdegrees Hz−1/2 per pass, which is better than most commercial polarimeters) is approximately five orders of magnitude worse than the shot-noise limit, calculated using the energy of the input light to the cavity. Specifically, our input power was ∼100 mW, giving a total shot noise of ∼100 nanodegrees Hz−1/2. However, we have at least 100 cavity passes, therefore the single-pass shot noise is the total divided by the number of passes, or ∼1 nanodegree Hz−1/2. We will discuss the origin of the loss of sensitivity, to see what can be done to recover some of these five orders of magnitude. In the previous paragraph, a shot noise of ∼1 nanodegree Hz−1/2 was calculated, assuming a cavity transmission of 100%. However, for cavity ring-down experiments, the input mirror rejects a very large fraction of light, equal to the mirrors reflectivity, typically ranging from 0.99 to 0.9999; therefore, about 2–4 orders of magnitude of light is lost at the first mirror, resulting in a loss in sensitivity of 1–2 orders of magnitude. A resonant cavity using cw light can have a very high transmission, close to 100%, so that the shot noise can be improved significantly compared to a CRDP cavity.

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Another source of noise in CRDP is the fact that a very large number of cavity modes are excited, each of which has different noise dependence, such as time-dependent cavity misalignment. Additionally, variations in the laser profile will cause a variation in the excitation of modes, also leading to noise. A solution to this problem is again the use of a resonant cavity using narrow-bandwidth cw light, so that a single cavity mode can be excited. In the next section, we discuss such a solution, CW-CRDP with signal reversals.

16.6  CONTINUOUS-WAVE CAVITY-ENHANCED POLARIMETRY WITH SIGNAL REVERSALS Continuous-wave (cw) cavity-enhanced spectroscopies (CES) have been used to measure absorption and birefringence with extremely high sensitivities. For example, linear birefringence has been measured with a sensitivity of about 2 × 10−11 degrees, or 20 picodegrees [41], after extensive averaging. The Cotton–Mouton effect has been measured in neon gas, at 1064 nm and by applying a 1 T transverse magnetic field, yielding the specific birefringence value of ∆nu = (5.9 ± 0.2) × 10−16 at atmospheric pressure [42]. Such sensitivities are 5–6 orders of magnitude better than those reported using CRDP, due to the advantages of cwCES, allowing the shot-noise limit to be reached. Two main advantages are the high transmission of the light through the cavity and the ability to modulate the signal rapidly (e.g. modulating the magnetic field in the Cotton–Mouton measurements), which can then be measured using lock-in detection. An important goal of ours is to combine the advantages of cw-CES with chirality detection, to produce cw-CEP with signal reversals.We present the experimental setup for cw-CEP in Fig. 16.13. The cavity is essentially the same, compared to signal-reversing CRDP, the main differences relating to the light input and output optics. First we discuss the cavity mode structure, to understand these differences and how cw-CEP works. The cavity frequency mode structure is shown in Fig. 16.14. First, in the absence of any cavity, linear or circular birefringence (i.e. θF = 0 and c = 0), a cavity resonance is shown in the center (defined as the center, with zero detuning), in Fig. 16.14A; the next cavity resonance is far off scale, being approximately 100 to 10,000 cavity linewidths away, known as the free spectral range, corresponding to a cavity finesse between 100 and 10,000. The transmission is very low for pulsed-laser excitation, due to lack

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Figure 16.13  The experimental setup for cw-CEP, showing the optics for the input light and the output recombination optics (insets). Abbreviation: cw-CEP, continuous-wave cavity-enhanced polarimetry.

of resonant excitation in the cavity. In contrast, for a CW laser with a laser bandwidth similar to or narrower than the cavity bandwidth, ideally up to 100% of the light can be transmitted through the cavity. Subsequently, when an intracavity Faraday effect is introduced (with θF nonzero, e.g. θF ≈ 3°), the cavity resonance is split symmetrically into

Figure 16.14  The mode structure of the (bowtie) CEP setup. Abbreviation: CEP, cavityenhanced polarimetry.

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two peaks at frequency splittings ω F = ± θ F ( c L ), one resonant for right circularly polarized light and the other for left circularly polarized light (see Fig. 16.14A); in contract, the central unsplit resonance transmits any light polarization. We note that the splitting is the same for both CW and CCW propagation directions: ∆ω CW = ∆ω CCW = 2ω F . Finally, a chiral sample is introduced in one arm of the cavity (i.e. c is nonzero). In this case, the splitting between the two circularly polarized modes is now different between the CW and CCW propagation directions (see Fig. 16.14B): ∆ω CW = 2 (ω F + ω c ) and ∆ω CCW = 2 (ω F − ω c ), where ω c = ϕ c ( c L ). Therefore, ϕ c can be determined from measuring the frequency splitting ω c . In signal-reversing CRDP, the measured polarization beating frequencies for CW and CCW (achieved by inputting linearly polarized light, with a laser linewidth much broader than the polarization splitting, so that both circularly polarized modes are coherently excited) were given by ∆ω CW and ∆ω CCW , so that ω c = ( ∆ω CW − ∆ω CCW ) 4 (and the sign can be reversed by inverting the magnetic field). For cw-CEP, ω c is measured in a somewhat different fashion. The splitting ω c is very small, typically much smaller than the cavity linewidth, so that the two modes for CW and CCW shown in Fig. 16.14B are nearly degenerate (for clarity the splitting is shown to be comparable to the cavity linewidth); therefore, these two modes cannot beat with each other, as the beating frequency will be very slow and cannot be resolved (as it is washed out by the larger cavity linewidth). To avoid this problem, we can pass the cavity output through an acousto-optic modulator (AOM), which produces a beam that is shifted by the AOM frequency ω AOM (where ≈ 80 MHz), as well as keeping the original unshifted beam. Therefore, passing both the CW and CCW outputs through the AOM, four beams are produced cw0, ccw0, cw1, and ccw1, with four frequen0 0 1 1 cies: ω CW , ω CCW , ω CW , and ω CCW , respectively, where the superscript “0” denotes an unshifted frequency, and the superscript “1” denotes a fre1 0 1 0 quency shifted by ω AOM : ω CW  = ω CW  + ω AOM , and ω CCW  = ω CCW  + ω AOM 1 0 . Subsequently, beams cw and ccw are recombined at detector 1, producing a beating signal proportional to sin(w1t + φ1); similarly beams cw0 and ccw1 are recombined at detector 2, producing a beating signal proportional to sin(w2t + φ2). Note that (16.26a) ω 1 = ω AOM + 2ω c (16.26b) ω 2 = ω AOM − 2ω c

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Figure 16.15  The experimental signals of frequency beating for the CW and CCW propagation directions. Differences are proportional to chirality, see Eq. (16.27). Abbreviations: CW, clockwise; CCW, counterclockwise.

Experimental signals demonstrating the beating frequencies ω 1 and ω 2 from detectors 1 and 2 are shown in Fig. 16.15; these frequencies can be measured with extremely high precision (e.g. 12 significant figures) using a frequency counter. Finally, ω c can determined by subtracting Eqs. (16.26a) and (16.26b):

ω c = (ω 1 − ω 2 ) 4 (16.27) Eq. (16.27) gives the desired single-pass chiral optical rotation angle ϕ c , from ϕ c = ω c ( L c ). Two signal reversals can be applied, to help isolate the chiral signals from noise: (a) reversing the input light polarization from left to right circularly polarized (this can be performed rapidly, up to the kHz timescale, using devices such as electro-optic or photo-electric modulators), and (b) by inverting the sign of the magnetic field applied to the intracavity magnetic-optic crystal (this reversal can also be performed relatively quickly, up to about 100 Hz). Current work is in progress, using the procedure described above, to measure ϕ c with very high sensitivity, well below the microdegree range.

16.7  FUTURE OUTLOOK AND CONCLUSIONS We have given a historical description of the development of CRDP, and signal-reversing CRDP, which have solved many of the problems facing measurement of chirality with cavities, and have opened the way for further development. The successful demonstration of cw-CEP in the near future should measure chiral optical rotation with record sensitivity, and open new

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fields of application. Two such new directions include the use of intracavity gain and the use of microresonators. Intra-cavity laser absorption spectroscopy (ICLAS) has been shown to achieve effective pathlengths of about 100,000 km and cavity linewidths of ∼1 Hz [43]; the combination of ICLAS with cw-CEP could achieve a further reduction in the detection sensitivity of a few orders of magnitude, by increasing the effective number of cavity passes from about 103 (for a normal passive cavity) to about 107 (for a cavity with gain). Microresonators, with cavity sizes of about 100 µm, have been used to detect single molecules, by measuring the shift in the cavity modes from the presence of the molecule [44]. Such microresonators support CW and CCW modes, and an intracavity magnetic field (which is parallel or antiparallel to the CW and CCW propagation directions) can be applied by a wire carrying a current perpendicular to the cavity plane. Therefore, the development of cw-CEP for a microresonator can be achieved in principle. The goal of the measurement of the chirality of a single molecule seems to be realizable goal for the future; it represents the ultimate challenge, in the long line of improvement in the optical methods for measurement of chirality.

ACKNOWLEDGMENTS The authors gratefully acknowledge Patrick H.Vaccaro for providing high-resolution images of Figs. 16.3, 16.5, and 16.6. This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Reinforcement of Postdoctoral Researchers” (MIS-5001552), implemented by the State Scholarships Foundation (ΙΚΥ). We also acknowledge partial financial support from the GSRT under the ERA.NET co-fund action (EPOCHSE, grant no. Τ3ΕΡΑ-00043), and the European Commission Horizon 2020, ULTRACHIRAL Project (grant no. FETOPEN-737071).

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[31] Caricato, M.; Vaccaro, P. H.; Crawford, T. D.; Wiberg, K. B.; Lahiri, P. Insights on the Origin of the Unusually Large Specific Rotation of (1S,4S)-Norbornenone. J. Phys. Chem. A 2014, 118, 4863. [32] Bougas, B.; Katsoprinakis, G. E.; von Klitzing, W.; Sapirstein, J.; Rakitzis, T. P. CavityEnhanced Parity-Nonconserving Optical Rotation in Metastable Xe and Hg. Phys. Rev. Lett. 2012, 108, 210801. [33] Jacob, D.; Bretenaker, F.; Pourcelot, P.; Rio, P.; Dumont, M.; Dore, A. Pulsed Measurement of High-Reflectivity Mirror Phase Retardances. Appl. Opt. 1994, 33, 3175. [34] Bougas, L.; Katsoprinakis, G. E.; von Klitzing,W.; Rakitzis,T. P. Fundamentals of CavityEnhanced Polarimetry for Parity-Nonconserving Optical Rotation Measurements: Application to Xe, Hg, and I. Phys. Rev. A 2014, 89, 052127. [35] Sofikitis, D.; Bougas, L.; Katsoprinakis, G. E.; Spiliotis, A. K.; Loppinet, B.; Rakitzis, T. P. Evanescent-Wave and Ambient Chiral Sensing by Signal-Reversing Cavity Ringdown Polarimetry. Nature 2014, 514, 76. [36] Bougas, L.; Sofikitis, D.; Katsoprinakis, G. E.; Spiliotis, A. K.; Tzallas, P.; Loppinet, B.; Peter Rakitzis, T. Chiral Cavity Ring Down Polarimetry: Chirality and Magnetometry Measurements Using Signal Reversals. J. Chem. Phys. 2015, 143, 104202. [37] Balbin Villaverde, A.; Donatti, D. A.; Bozinis, D. G.Terbium Gallium Garnet Verdet Constant Measurements with Pulsed Magnetic Field. J. Phys. C 1978, 11, L495–L498. [38] Condon, E. U.Theories of Optical Rotatory Power. Rev. Mod. Phys. B 1937, 9, 432–457. [39] Silverman, M. P. Reflection and Refraction at the Surface of a Chiral Medium: Comparison of Gyrotropic Constitutive Relations Invariant or Noninvariant Under a Duality Transformation. J. Opt. Soc. Am. A 1986, 3, 830–837. [40] Lekner, J. Optical Properties of Isotropic Chiral Media. Pure Appl. Opt. 1996, 5, 417–443. [41] Durand, M.; Morville, J.; Romanini, D. Shot-Noise-Limited Measurement of Subparts-per-trillion Birefringence Phase Shift in a High-Finesse Cavity. Phys. Rev. A 2010, 82, 031803. [42] Bregant, M.; Cantatore, G.; Carusotto, S.; Cimino, R.; Della Valle, F.; Di Domenico, G.; Gastaldi, U.; Karuza, M.; Lozza, V.; Polacco, E.; Ruoso, G.; Zavattini, E.; Zavattini, G. A Precise Measurement of the Cotton–Mouton Effect in Neon. Chem. Phys. Lett. 2005, 410, 288–292. [43] Baev,V. M.; Latz, T.; Toschek, P. E. Laser Intracavity Absorption Spectroscopy. Appl. Phys. B 1999, 69, 171–202. [44] Baaske, M. D.; Foreman, M. R.; Vollmer, F. Single-Molecule Nucleic Acid Interactions Monitored on a Label-Free Microcavity Biosensor Platform. Nature Nanotechnol. 2014, 9, 933–939.

CHAPTER 17

Quantitative Chiral Analysis by Molecular Rotational Spectroscopy Brooks H. Pate*, Luca Evangelisti**, Walther Caminati**, Yunjie Xu†, Javix Thomas†, David Patterson‡, Cristobal Perez§ , Melanie Schnell§ *Department of Chemistry, University of Virginia, Charlottesville,VA, United States **Department of Chemistry “Giacomo Ciamician”,University of Bologna, Bologna, Italy † Department of Chemistry, University of Alberta, Edmonton, AB, Canada ‡ Department of Physics, University of California Santa Barbara, Santa Barbara, CA, United States § Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany

17.1  INTRODUCTION This chapter presents early results from the emerging field of quantitative chiral analysis by molecular rotational spectroscopy. The focus is on the development of measurement techniques to solve the challenging analytical chemistry problem of determining the ratios of all stereoisomers for a chiral molecule. This analysis becomes particularly challenging as the number of chiral centers in the molecule increases. Furthermore, this area of spectroscopy has the goal of creating measurement techniques that can be used directly on complex chemical mixtures to perform chiral analysis without the need of chemical separation by chromatography. Examples of chemical samples that fall into this category include natural products like essential oils from plants that are a rich mixture of volatile species and reaction flask samples where stereospecific chemical reactions are performed and which contain unreacted reagents, desired and undesired reaction products, and solvents in the mixture.

17.1.1  Challenges in quantitative chiral analysis An illustration of the analysis challenges is the synthesis of isopulegol from citronellal in the commercial production of menthol shown in Fig. 17.1 [1–3]. Two new stereocenters are produced in the cyclization reaction that generates isopulegol. Subsequent hydrogenation of the olefin produces the final menthol product. The goal in menthol production is to generate only the isopulegol diastereomer because the other isomers have bad taste. The Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00019-7 Copyright © 2018 Elsevier B.V. All rights reserved.

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Figure 17.1  The cyclization of citronellal is shown as an illustration of the challenges for chiral analysis of molecules with multiple stereocenters. Chiral catalysts are designed to optimize the production of isopulegol. The cyclization sets two new chiral centers producing a product with 23 = 8 possible stereoisomers. If the reaction proceeds without racemization, then the stereochemistry at one site is fixed by the citronellal reagent. In this case, the reaction can produce the four diastereomer products shown on the right. Racemization, or a starting material with low enantiopurity, can produce the enantiomers of each of these products with all eight stereoisomers possibly present in the reaction mixture.

development of asymmetric catalysts for this process by Noyori was recognized with the 2001 Nobel Prize in Chemistry [4]. The reaction intermediate has three chiral centers and can exist in 23 = 8 stereoisomers. Four of these stereoisomers are diastereomers and have distinct molecular geometries. Traditional spectroscopy methods can distinguish these isomers. Each diastereomer exists in two nonsuperimposable mirror image forms—the enantiomers—to complete the full set of eight stereoisomers. The measurement challenge is to determine the fractional composition of stereoisomers produced in the reaction.This includes both the analysis of the diastereomer ratio and the enantiomer ratio for each diastereomer. In practical applications, there are two additional challenges: (1) The analysis should be possible without the use of any reference samples. For example, if the molecule is newly created in the lab, there will be no sample of known absolute configuration (e.g., useful to determine the order of elution in chromatography) and no sample of known enantiomeric excess (EE) (to

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calibrate measurements such as optical rotation or vibrational circular dichroism (VCD)). (2) The technique needs to have a large dynamic range so that minor stereoisomer impurities—or even chiral impurities produced by unexpected side reactions [5]—can be detected. The ability to perform the analysis without the need to develop a chromatographic chiral separation protocol would be a significant strength.

17.1.2  Rotational spectroscopy for chemical analysis Rotational spectroscopy measures a high-resolution spectrum where the spectral pattern is determined by the three-dimensional structure of the molecule [6]. The quantized energy levels for the spectroscopy come from the overall rotational motion of the molecule. The rotational kinetic energy is determined by the three moments-of-inertia in the principal axis system. Any changes in the mass distribution will produce a different energy level structure and spectroscopic transition frequencies. Therefore, structural isomers have distinct rotational spectra but enantiomers, which have the same set of bond lengths and bond angles, have identical rotational spectra. In order for the rotational motion of the molecule to couple with light, it is necessary for the molecule to have a permanent dipole moment. Although there are special cases of chiral molecules with elements of molecular symmetry, they generally have C1-symmetry and are polar. The important feature of molecular rotational spectroscopy for chemical analysis is that the resolution of the spectral transitions is sufficiently high that the spectra of isomers with only small changes in mass distribution can be measured without spectral overlap. For example, the 13C- isotopologues of molecules, isomers created when a single carbon atom is isotopically substituted, are routinely resolved in the instruments used for rotational spectroscopy [7].The extreme sensitivity to changes in the mass distribution make rotational spectroscopy well-suited to distinguishing diastereomers of molecules with multiple chiral centers. A second advantage of the high-resolution of the spectrometers is that complex sample mixtures can be analyzed directly without spectral overlap. An example of the rotational spectrum from the vapor head space of an essential oil will be presented later to illustrate this capability.

17.1.3  Bringing enantiomer-specific measurement capabilities to rotational spectroscopy Until recently, there has been limited application of molecular rotational spectroscopy to the study of chiral molecules. One impediment to the use of the technique was the inability to differentiate enantiomers with high sensitivity. The common spectroscopic approach to enantiomer-specific

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spectroscopy is to use circular dichroism. However, circular dichroism becomes weaker as the wavelength of the resonant radiation gets longer [8– 10]. The rotational circular dichroism properties of propylene oxide have been calculated and show that the fractional differential absorption of left and right circularly polarized light is more than an order-of-magnitude weaker than for VCD and is probably below the sensitivity of spectrometers used in the field [9,10]. The two approaches described here adapt other strategies for enantiomer analysis that have been used for vibrational and nuclear magnetic resonance (NMR) spectroscopy. The major breakthrough in rotational spectroscopy applications in chiral analysis occurred in 2013 with the demonstration of enantiomer-specific detection using three-wave mixing methods [11,12]. These methods are based on the realization that a gas sample with an EE lacks a center of symmetry and, therefore, can support sum and difference frequency generation [13].Three-wave mixing can be observed using pulses of light with orthogonal polarizations and in these experiments the phase of the coherent emission signal contains information about the absolute configuration. This concept had previously been demonstrated using infrared laser spectroscopy [14–16]. The advantages of performing rotational spectroscopy three-wave mixing measurements for quantitative chiral analysis will be described in this chapter [17]. The second approach to enantiomer analysis is to convert the enantiomers into spectroscopically distinct species using a chiral resolving agent. This method has been developed extensively for NMR spectroscopy using both chemical derivatization, with reagents such as Mosher's ester, and molecular complexation [18–21]. The rotational spectroscopy version of this enantiomers-to-diastereomers strategy uses noncovalent interactions to form a weakly bound complex between the molecule of interest and a small, enantiopure tag molecule in a pulsed jet expansion. This measurement approach is an extension of pioneering studies of the structures of complexes of small chiral molecules from the field of rotational spectroscopy [22–26].

17.2  BASIC PRINCIPLES OF MOLECULAR ROTATIONAL SPECTROSCOPY 17.2.1  Molecular rotational spectroscopy The theory for molecular rotational spectroscopy is highly developed and there are several excellent texts [6,27–29] on the topic with the work by Gordy and Cook [6] being the most frequently cited reference. In this

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s­ection, the theoretical principles of rotation spectroscopy are presented at minimum level required to understand the spectral observations. The fundamental challenges for performing rotational spectroscopy on large molecules are also discussed. The first consideration is the energy levels for molecular rotation. These energy levels come from the kinetic energy of rotation for the free molecule. As a result, molecular rotational spectroscopy requires the molecule to be in the gas phase and at low enough pressure so that molecular collisions do not produce significant line broadening. Because angular momentum is quantized, the allowed energies for molecular rotation are also quantized. They are obtained from the eigenvalues of the Hamiltonian operator for rotational kinetic energy. For a rigid rotor where the geometry is fixed as a function of the rotational energy, the Hamiltonian is: 

Hrot = A Pa2 + BPb2 + C Pc2

(17.1)

In this expression, Pa, Pb, and Pc are the angular momentum operators for rotational motion about the three principal axes of rotation. There are three molecular parameters in the Hamiltonian (A, B, C), and these are called the rotational constants. The constants are ordered in terms of their magnitudes: A > B > C. For a spherical top, all three constants are identical. For a symmetric top, two of the constants are equal (giving either a prolate symmetric top with (A, B = C) or an oblate top symmetric top with (A = B, C)). Except for a few special cases, chiral molecules have C1symmetry and are asymmetric tops with three distinct rotational constants. The rotational constants are inversely related to the principal moments-ofinertia (Ia, Ib, and Ic) that characterize the three-dimensional mass distribution of the molecule: 

A=  2 2Ia

(17.2)

with Ia the moment-of-inertia for the a-principal axis and with similar expressions for B and C. It is common in rotational spectroscopy to work with the Hamiltonian in frequency instead of energy units (through the relation ∆E = hν) and the rotational constants are commonly reported in MHz units. The rigid rotor Hamiltonian involves only angular momentum operators and can, therefore, be calculated exactly using computers. The resulting energy levels are labeled with three quantum numbers as JKaKc. Here, J is the usual quantum number related to the square of the length of the angular momentum vector. For a closed shell molecule, J is quantized and takes

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integer values starting from zero. The other two quantum numbers, Ka and Kc, describe the orientation of the angular momentum vector with respect to the molecular principal axis system. They are actually pseudo quantum numbers and give the projection of the angular momentum vector on the rotational axes in the two symmetric top limits: the one associated with the smallest moment-of-inertia (Ka) for prolate tops and the one associated with the largest moment-of-inertia (Kc) for oblate tops. Since these are projections, they must take integer values between –J..0..J.They are reported as unsigned quantities using the magnitude of the projection. For any value of J, there are (2J + 1) possible orientations of the angular momentum in the molecular principal axis system. As an example, for total rotational angular momentum quantum number J = 1, the labelling for the three energy levels in JKaKc notation is: 111, 101, and 110. Because these correspond to different amounts of angular momentum about the stable rotational axes, they will correspond to different allowed rotational kinetic energies for an asymmetric top. There is an additional (2J + 1)-degeneracy of the rotational energy levels that comes from the different projections of the total angular momentum in a space-fixed axis (the quantum number is MJ = −J..0..J). This orientation does not affect the rotational kinetic energy giving rise to the (2J + 1)-spatial degeneracy for each energy level. Although this degeneracy affects the transition intensities (since a single transition frequency has a series of exactly overlapping transitions in field-free space), it is not important to the interpretation of molecular rotational spectra in the absence of an external electric field. For large, rigid molecules at the low temperatures of a pulsed jet expansion it is often possible to obtain a quantitative fit of the experimental spectrum using just the rigid rotor Hamiltonian of Eq. (17.1). In rotational spectroscopy, a spectrum “fit” generally reproduces the transition frequencies to the experimental accuracy. For example, the root-mean-squared frequency error in an analysis is often on the order of 10 kHz and is significantly less than the experimental line width of the transitions at about 70 kHz in broadband rotational spectrometers. To achieve this level of accuracy, it is often necessary to include additional terms in the rotational kinetic energy Hamiltonian that account for distortion of the geometry as a function of the rotational kinetic energy. In this case, the Hamiltonian for rotational spectroscopy is the Watson Hamiltonian [30] and with the first correction for distortable rotation contains five experimentally determinable quartic centrifugal distortion terms:

685

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(

)

∆



(

4 2 2 4 2 2 2 J P − ∆ JK P Pa − ∆ K Pa − 2 δ J P Pb − Pc  − δ K Pa2 Pb2 − Pc2 + Pb2 − Pc2 Pa2

2 2 2 H A-reduction = A Pa + B Pb + C Pc + 

( (

) (

) )

) 

(17.3)

where P2 is the square of the total angular momentum: P  = Pa2 + Pb2 + Pc2 [6]. In most cases, the Hamiltonian uses the so-called A-reduction that is given above. In the case where the molecule is very close to a symmetric top, there is an alternative form of the Hamiltonian known as the S-reduction that gives a more suitable treatment of centrifugal distortion [6]. The Watson Hamiltonian still uses only angular momentum operators and parameters that are constants and, therefore, can be solved exactly. There are two other additions to the molecular rotational Hamiltonian that are encountered in the field of molecular rotational spectroscopy. For molecules that contain atoms with nuclear quadrupole moments (e.g., 14N with nuclear spin quantum number I = 1), the nuclear quadrupole hyperfine coupling must be included [6]. This Hamiltonian also includes only angular momentum operators and can be solved exactly. The second effect, that is, common comes from internal rotation of “tops” attached to the molecule—like methyl groups. The theory of internal rotation effects on molecular rotational spectra is also a well-developed topic and methods to include these effects are known [6,31]. However, for larger molecules the observation of “splitting” of the rotational transitions due to an internally rotating functional group is rare. The prediction of the rotational spectrum requires the calculation of the intensities for the allowed transitions between quantized energy levels. The coupling of light to the molecular rotation occurs through the permanent dipole moment of the molecule (and, as a result, rotational spectroscopy requires polar molecules). The torque on the molecule through the interaction of the electric field of the light source and electric dipole moment can change the kinetic energy of rotation around any of the three principal axes. As a result, there are three different rotational spectra that come from the interaction of light with the dipole moment vector components in the principal axis system where µ = (µa, µb, µc). Each of these three spectra, known as the a-type, b-type, and c-type spectra, have their own selection rules. The relative intensities of the three spectra are proportional to the squares of the dipole moment components. In this way, the relative intensities of the a-, b-, and c-type spectra give information about the direction of 2

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the dipole moment in the principal axis system. The theory for calculating the transition intensities in the rotational spectrum is known and described in the texts for this field of spectroscopy [6].

17.2.2  Challenges for large molecule rotational spectroscopy As the size of the molecule, as characterized by the principal moments-ofinertia, increases, it becomes more challenging to measure the spectrum at high sensitivity.The main issue is the size of the rotational partition function: Q rot ( T ) ≈ ( kT )

32



12

( ABC )

(17.4)

where A, B, and C are the rotational constants for the molecule.The sensitivity also depends on the population difference between energy levels of the rotational transition and the Boltzmann distribution adds an additional linear dependence bringing the overall temperature dependence of the signals to T−5/2. There are two important points in these results. First, for any measurement the sensitivity is improved by lowering the temperature of the gas sample. As a result, molecular rotational spectroscopy is most commonly performed on gas samples cooled in a pulsed molecular beam—an approach pioneered in the Balle-Flygare cavity-enhanced Fourier transform microwave spectrometer [32]. By seeding the molecule of interest in an inert gas, the rotational temperature of the sample in the pulsed jet expansion is typically 1–10 K. The pulsed jet expansion also effectively cools the vibrational excitation of the molecule. For larger molecules like those presented in this work, the population of vibrational excited states is generally  B > C associated with rotations about the molecule-fixed, principal axes of inertia X, Y, and Z as illustrated in Fig. 18.1. Suppose now that the molecule is illuminated for a short time by weak, far off-resonance circularly polarized light [41] to which it has been intro­ duced adiabatically, in the visible or near infrared, say. The illuminating light drives oscillations in the charge and current distributions of the molecule, affecting the rotation of the molecule while shifting the energy of the mol­ ecule in an orientationally and chirally sensitive manner: the leading-order perturbative contributions to this energy shift take the form [29–31]

 1 α XX a  ε 0 c   2

−

I

 

+ σ k B XX + b

1 α  2 YY

+ σ k BYY

 + c 1 α   2 ZZ

+ σ k BZZ

 (18.1) 

where I is the intensity of the light; k is the wavevector of the light; σ is the polarization parameter of the light, equal to ±1 for left- or right-handed cir­ cular polarization in the optics convention [42]; a, b, and c with a + b + c = 1 are constants particular to the rotational state of the molecule; αXX , αYY , and αZZ are molecule-fixed components of the molecule’s polarizability tensor, which are each identical for enantiomers; BXX , BYY , and BZZ are moleculefixed components of the molecule’s optical activity pseudotensor [43,44], which are each of equal magnitude but opposite sign for enantiomers.

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When the rotational spectrum [45] of such molecules is measured, typically using microwaves, it will appear distorted: the light-shifted resonance frequen­ cy for a given rotational transition and enantiomer follows from Eq. (18.1) as 1  1  ∆a α XX + σ k B XX  + ∆b  α YY + σ k BYY        I 2 2 ∆f = ∆f 0 −   (18.2) 1 2π ε 0 h-     + ∆c α ZZ + σ k BZZ 2    

where ∆f0 is the unperturbed transition frequency and ∆a, ∆b, and ∆c are differences in the values of a, b, and c for the rotational states involved in the transition. In Section 18.3, we show how such distortions can be exploited in chiral rotational spectroscopy. It is interesting to note that Eq. (18.1) is the a.c. Stark shift, calculated to higher order than usual [46]. Such shifts also govern the refraction of light propagating through a medium [46], with circular birefringence due to BXX , BYY , and BZZ giving rise to natural optical rotation [46]. Spatial gradients in such shifts give rise, moreover, to forces, including the dipole optical force used to trap atoms in optical lattices [47] and the discriminatory optical force [30,46,48–60]; a viable manifestation of chirality in the translational degrees of freedom of chiral molecules and, indeed, a means by which to spatially separate enantiomers.

18.3  CHIRAL ROTATIONAL SPECTRA In the present section, our goal is to illustrate, simply, some of the features that might be seen in chiral rotational spectra for various types of sample. To produce Fig. 18.2 and Figs. 18.4–18.6, we plotted Lorentzians cen­ tered at the relevant rotational transition frequencies, calculated from Eq. (18.2) as described in [29]. Each Lorentzian was ascribed a frequency fullwidth at ­half-maximum of 1.0 × 103 s−1 (1 s−1 = 1 Hz) and taken to be pro­ portional in amplitude to the number of contributing molecules. Rotation­ al transitions are labeled as J τ′ ′,m′ ← J τ ,m , with J ∈ {0,1, …} determining the magnitude of the rotor angular momentum, τ ∈ {0, … , ± J } labeling the rotor energy, and m ∈ {0, … , ± J } determining the z-component of the rotor angular momentum (with the light propagating in the +z-direction) for a particular rotational state, as usual [32,37].

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Figure 18.2  A rotational line for an enantiopure sample of the lowest energy conformer of (S)-propylene glycol in the absence of light (A), illuminated by left-handed light (B) and illuminated by right-handed light (C). The separation between rotational lines (B) and (C) reveals orientationally and chirally sensitive information about the response of the molecules to the light. Adapted from [29].

The same features persist when corrections to Eq. (18.2) are included and for larger rotational linewidths: it is acceptable to have rotational lines overlap significantly if their centers, say, can still be distinguished with suf­ ficient resolution.The forms of the rotational lines seen in a real chiral rota­ tional spectrum will depend, of course, upon the nature and functionality of

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Figure 18.3  Singly deuterated chlorofluoromethane derives chirality from the arrangement of its neutrons. These molecules should exist in small quantities in some refrigerators. Adapted from [29].

Figure 18.4  A rotational line for a 60:40 mixture (A), a 50:50 mixture (B) and a 40:60 mixture (C) of enantiomers of isotopically chiral housane in the presence of lefthanded light. The chiral splitting is apparent even for the racemate while the relative heights of the constituent lines reveal the enantiomeric excesses of the samples. Adapted from [29].

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Figure 18.5  A rotational line for a sample of the three different stereoisomers of tartaric acid as it might appear in standard rotational spectroscopy (A) and in chiral rotational spectroscopy using left-handed light (B). The standard rotational spectrum fails to distinguish between the enantiomers whilst the chiral rotational spectrum instead distinguishes between all three molecular forms. Here ∆ = A2 + B 2 + C 2 − AB − AC − BC .Adapted from [29].

the chiral rotational spectrometer used to obtain the spectrum, but should nevertheless offer the same information. The reader will observe the high precision with which I and 2π/|k| are quoted. In principle, this represents no difficulty and ensures that Fig. 18.2 and Figs. 18.4–18.6 are drawn accurately to a frequency resolution of 102 s−1. In practice, it should be possible in many cases to reduce strin­ gent requirements on the uniformity and stability of the intensity of the light by exploiting certain, “magic” rotational transitions, as discussed in Section 18.3.5.

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Figure 18.6  A chiral splitting induced in a rotational line of a racemate of ibuprofen using a relatively low intensity of left-handed light. Adapted from [29].

18.3.1  Oriented chiroptical information The basic property of a chiral molecule that is probed in typical optical rotation experiments using fluid samples is the isotropic sum [14,36,61–63]



1 (B XX + BYY + BZZ ) 3

(18.3)

These experiments yield no information about BXX, BYY , or BZZ indi­ vidually. Other well-established chiroptical techniques such as circular dichroism [14,63–68] and Raman optical activity [14,63,67–71] yield other chirally sensitive molecular properties but the fact remains that it is the isotropically averaged forms of these that are usually observed in practice. The ability to determine oriented rather than isotropically averaged chiroptical information, in particular, the individual, molecule-fixed com­ ponents BXX, BYY , and BZZ is highly attractive, as these offer a wealth of in­ formation about molecular chirality that is only partially embodied by the isotropic sum seen in Eq. (18.3). At present, such information can only be obtained, however, using an oriented sample as in a crystalline phase [72,73]. The preparation of such samples is not always feasible and even when it can be achieved, signatures of chirality are usually very difficult to distinguish from other effects, in particular, those due to linear birefringence. Indeed, it was noted in 2012 that “we have a shockingly small amount of data on the

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chiroptical responses of orientated molecules, a vast chasm in the science of molecular chirality” [73]. Consider first an enantiopure sample of the lowest energy conformer of (S)-propylene glycol. Racemic propylene glycol is employed as an antifreeze and is a key ingredient in electronic cigarettes. Depicted in Fig. 18.2 is: (A) the 2−1 ← 1−1 rotational line in the absence of light; (B) the 2−1,0 ← 1−1,0 rotational line in the presence of light with I = 2.0000 × 1012 kg s−3 (1 kg s−3 = 10−4 W  cm−2), 2π/|k| = 5.320 × 10−7 m, and σ = 1; (C) the same as in (B) but with σ = −1. The separation between rotational line (A) and the centroid of rotational lines (B) and (C) yields a certain combina­ tion of αXX   , αYY  , and αZZ while that between rotational lines (B) and (C) provides a particular combination of BXX  , BYY , and BZZ. BXX  , BYY , and BZZ can be determined individually by recording such energies for two distinct rotor transitions and both circular polarizations of the light and making use of the measured value of the isotropic sum seen in Eq. (18.3) [29,31]. Knowledge of BXX, BYY , and BZZ might assist in the assignment of abso­ lute configuration, as the measured signs of these should be easier to corre­ late with those predicted by quantum chemical calculations than in the case of the isotropic sum seen in Eq. (18.3), which is often somewhat smaller in magnitude than its constituents BXX/3, BYY/3, and BZZ/3 [74]. BXX, BYY , and BZZ might also serve as probes of isotopic molecular chirality and crypto­ chirality in general, as explained in Section 18.3.2. Although our focus here is upon the chirality of individual molecules, we observe that knowledge of BXX, BYY , and BZZ might, in some cases, facilitate the exploration and exploitation of the myriad contributions to the optical properties of crystals [72,73] comprised wholly or in part of such molecules.We recognize more­ over that our proposed technique offers αXX, αYY , and αZZ (and potentially even the distortion of such quantities by static fields) as byproducts, which is in itself an attractive feature that could see our proposed technique find use even for achiral molecules [29,31].

18.3.2  Isotopic molecular chirality Chirality is more widespread at the molecular level than is sometimes ap­ preciated, for even a molecule with an achiral arrangement of atoms may in fact be chiral by virtue of its isotopic constitution, as illustrated in Fig. 18.3. Isotopically chiral molecules might have been among the very first chiral molecules, formed perhaps in primordial molecular clouds [75].They might even have given rise to biological homochirality, by triggering dissymmetric

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autocatalysis reactions [75–79]. At a more fundamental level still, isotopi­ cally chiral molecules have been put forward [80] as promising candidates for the measurement of minuscule differences believed to exist between the energies of enantiomers [14,37,39,81–84]. It is well established that isotopic substitution in certain achiral molecules can significantly modify their in­ teraction with living things. Heavy water can change the phase and period of circadian oscillations [85], for example. In spite of this, there “have been very few studies on isotope-generated chirality in biochemistry” [78]. Isotopic molecular chirality can already be probed using various tech­ niques, in particular, vibrational circular dichroism and Raman optical ac­ tivity, which are inherently sensitive to chiral mass distributions [66,86–90]. A difficulty, however, is that enantiopurified samples of isotopically chiral molecules can often only be synthesized in small quantities [87] while reso­ lution of racemates is extremely challenging [91]. Chiral rotational spec­ troscopy may prove particularly useful here as it is, like vibrational circular dichroism and Raman optical activity, inherently sensitive to isotopic mo­ lecular chirality and, in addition, gives an incisive signal even for a racemate, thus negating the need for dissymmetric synthesis or resolution. We find in electronic calculations within the Born–Oppenheimer ap­ proximation for a rigid nuclear skeleton [34,37] that the isotropic sum seen in Eq. (18.3) vanishes for an isotopically chiral molecule, as it is rotationally invariant and the electronic charge and current distributions of the mol­ ecule are achiral. Chirally sensitive vibrational corrections to this picture do exist but are usually small at visible or near-infrared frequencies, as con­ sidered here. The individual components BXX, BYY , and BZZ, and therefore chiral splittings in chiral rotational spectroscopy, can nevertheless attain ap­ preciable magnitudes for an isotopically chiral molecule as each of these is dependent upon the orientation of the principal axes of inertia relative to the molecule and is, therefore, sensitive to the distribution of mass through­ out the molecule, which is where the molecule’s chirality resides. Chiral rotational spectroscopy might be similarly useful for other molecules ex­ hibiting cryptochirality [92], where the isotropic sum seen in Eq. (18.3) is essentially zero while two or three of its constituents BXX, BYY , and BZZ are instead of appreciable magnitude. Consider next then a nonenantiopure sample of housane with the usual C atom at either the bottom-left or bottom-right of the “house” substi­ tuted with a 13C atom to give the enantiomers of an isotopically chiral molecule. Depicted in Fig. 18.4 is the 1−1,0 ← 00,0 rotational line for light with I = 4.0000 × 1012 kg s−3, 2π/|k| = 5.320 × 10−7 m, and σ = 1

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illuminating a sample comprised of: (A) a 60:40 mixture of enantiomers; (B) a 50:50 mixture; (C) a 40:60 mixture. In all three cases, the chiral split­ ting is apparent while the relative heights of the constituent lines reveal the enantiomeric excess of the sample and so enable its determination. Let us highlight the significance of panel (B) in particular. We have here an obvi­ ous and revealing signature of chirality from a racemate of isotopically chiral molecules, as claimed.The chirality of each of these molecules derives solely from the placement of a single neutron, which constitutes but 1% of the total mass of the molecule. In contrast,Techniques such as electronic optical rotation and electronic circular dichroism, are nearly double blind under such circumstances, in that they are insensitive to isotopic molecular chiral­ ity and, moreover, chirality at the molecular level in racemic samples. Even vibrational circular dichroism, Raman optical activity, and chiral microwave three wave mixing, in spite of their inherent sensitivity to chirality and mass distribution, would yield vanishing signals here, again because of their in­ sensitivity to chirality at the molecular level in racemic samples.

18.3.3  Molecules with multiple chiral centers Standard rotational spectroscopy can often distinguish well between dif­ ferent isomers, provided they are not enantiomers [93]. Chiral rotational spectroscopy can distinguish well between different isomers including en­ antiomers. It may find particular use, therefore, in the analysis of molecules with multiple chiral centers, which permit an exponentially large number of different stereoisomers, many of which are enantiomers. This could see chiral rotational spectroscopy find particular use in the food and pharma­ ceutical industries, where different isomers must be individually justified [94] and molecules with multiple chiral centers are recognized as being “challenging” [95]. Consider a sample of tartaric acid. The N = 2 chiral centers permit 2N = 4 stereoisomers, two of which are equivalent, leaving three distinct stereoisomers. One of these, mesotartaric acid, is achiral, whereas the oth­ er two, l-tartaric acid and d-tartaric acid, are enantiomers. Depicted in Fig. 18.5(A) is the 2−2 ← 1−1 rotational line for a 50:n:(50 − n) mixture of mesotartaric acid, l-tartaric acid, and d-tartaric acid in the absence of light, where n is any number between 0 and 50 inclusive and we have assumed equal line strengths for each molecule.The contribution due to mesotartaric acid appears well separated from that due to l-tartaric acid and d-tartaric acid. The spectrum gives no information, however, about the relative abun­ dances of l-tartaric acid and d-tartaric acid (determined by n), only their

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combination (n + (50 − n) = 50, independent of n). Depicted in Fig. 18.5(B) is the 2−2,0 ← 1−1,±1 rotational line for a 50:20:30 mixture in the presence of light with I = 1.0000 × 1012 kg s−3, 2π/|k| = 5.320 × 10−7 m, and σ = 1. Contributions due to all three stereoisomers now appear well distinguished while yielding a wealth of new information, as claimed. Rotational spectra are sufficiently sparse that the analysis of molecules with significantly more chiral centers in this way should not be met with any fundamental difficulties. This ability to distinguish well and in a chi­ rally sensitive manner between subtly different molecular forms persists moreover for more general mixtures containing multiple types of molecule. The chirally sensitive analysis of complicated mixtures using traditional techniques represents a serious challenge. Indeed, it was suggested in 2014 that “only one mixture analysis (based upon circular dichroism, vibrational circular dichroism or Raman optical activity) was reported so far” [19], although the use of chiral microwave three wave mixing to analyze various mixtures has now been well demonstrated [19,20,23,24].

18.3.4 Scaling Polarizabilities tend to increase with the size of a molecule.The light intensity required to induce observable shifts in a rotational spectrum therefore tends to decrease with the size of a molecule, as is evident in the examples above. This favorable scaling is ultimately counteracted in that larger molecules are usually more difficult to sample appropriately, tend to exhibit lower rotational transition frequencies, are often more likely to absorb light, and might require higher levels of theory to accurately describe. It seems then that there should be a certain molecular size range for which chiral rotational spectroscopy is particularly well suited. To illustrate these ideas, let us consider a racemate of a particular conformer of ibuprofen, which is somewhat more massive than the molecules considered in the other examples above. Such a sample would yield no information about the chirality of the molecules when analyzed using traditional techniques. Depicted in Fig. 18.6 is the chiral splitting of the 1−1,0 ← 00,0 rotational line due to light with I = 2.0000 × 1011kg s−3, 2π/|k| = 5.320 × 10−7 m, and σ = 1. The presence and chiral character of the two enantiomeric forms is revealed, with a light intensity considerably lower than in the other examples above, as claimed. The rotational transition frequencies seen here are also considerably lower than in the other examples above, although it should be noted that these are among the very lowest rotational transition frequencies available for these molecules and that signifi­ cantly higher rotational transition frequencies do exist.

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The chiral splittings seen in Fig. 18.2 and Figs. 18.4–18.6 are neither the smallest nor the largest to be found in the chiral rotational spectra of these molecules.

18.3.5  Practical considerations Requirements on the monochromaticity and stability of the wavelength of the light are stringent but are eased somewhat by the fact that the αAB vary slowly with wavelength far off-resonance [14]. For most rotational transitions, requirements on the uniformity and stability of the intensity of the light are very stringent, as small variations in the intensity can eas­ ily overwhelm chiral splittings. In many cases, rotational transitions can be found, however, for which the chirally insensitive piece of the rotational transition frequency shift due to the light is considerably smaller than is typical while the chirality-sensitive piece remains appreciable. These magic rotational transitions should be particularly well suited to chiral rotational spectroscopy as they reduce requirements on the uniformity and stability of the intensity of the light. It should be possible moreover to significantly refine some magic transitions by fine-tuning the polarization properties of the light or even the strength and direction of an applied static field.

18.4  CHIRAL ROTATIONAL SPECTROMETER In the present section, we discuss a basic design for a chiral rotational spectrometer [28,29]. This represents but one of many conceivable pos­ sibilities for the implementation of chiral rotational spectroscopy: the ideas introduced in Sections 18.2 and 18.3 have a generality reaching beyond this particular design. The design certainly has its limitations, but should nevertheless permit high-precision measurements based upon αXX, αYY , and αZZ for many types of molecule, be they chiral or achiral, as well as mea­ surements based upon BXX, BYY , and BZZ for some types of chiral molecule under favorable circumstances. The key components of the spectrometer are depicted in Fig. 18.7(A) with an expanded view of the active region in Fig. 18.7(B). We summarize their functionality as follows and refer for a quantitative model of the spec­ trometer to Ref. [29]: (i) A pulsed supersonic expansion nozzle together with a collima­ tion stage is employed to generate narrow pulses of internally cold chiral molecules with unimpeded rotational degrees of freedom. A piezoelectric nozzle permits a high rate of measurement [96].

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Figure 18.7  Key components of a chiral rotational spectrometer. Drawn approximately to scale but with portions of the Helmholtz coils and the vacuum chamber removed, for the sake of clarity. Adapted from [29].

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The collimation stage might include a skimmer augmented by an aperture [97–99]. (ii) An optical cavity houses far off-resonance visible or perhaps nearinfrared circularly polarized light of moderate intensity, to shift the rotational energies of the molecules in a chirally sensitive manner. Fine-tuning the polarization properties of the light in the active re­ gion enables the refinement of magic transitions, to help overcome stringent requirements on the intensity of the light and thus ob­ tain a clean chiral rotational spectrum. The optical cavity might be of the skew-square ring variety, comprised of low-loss, ultra-highreflectivity, low-anisotropy mirrors [100,101] while the light might originate from an external cavity diode laser, the output of which is fiber-amplified and mode matched into the ring with stability actively enforced [102,103].We envisage the light to be continuous wave here, with a central intensity of at least 1011 kg s−3 [102,104]. Each molecule takes some 10−3 s to traverse the light, a time interval large enough to facilitate a microwave frequency linewidth of around 104 s−1. Variants of our design that use pulsed light rather than continuous wave light are also conceivable and might prove easier to implement. (iii) A microwave cavity and associated components generate and detect microwaves as in the well-established technique of cavity-enhanced Fourier transform microwave spectroscopy [35,40,93,105–112] but here with the aim of measuring chirally sensitive distortions of the rotational spectrum of the molecules due to the light. The microwave cavity might be of the Fabry–Pérot variety, comprised of spherical mirrors with microwaves coupled in and out of the microwave cavity via waveguide or perhaps via antennas. (iv) An evacuated chamber encompasses the key components described above to eliminate atmospheric interference with the molecular puls­ es and facilitate the removal of molecules between measurements.The absence of air, dust, and other such influences should assist, moreover, in maintaining the stability of the optical cavity [103]. (v) A static magnetic field of moderate strength and high uniformity de­ fines a quantization axis parallel to that defined by the direction of propagation of the light in the active region while enabling addi­ tional refinement of magic transitions if necessary.The static magnetic field might be produced by a pair of superconducting Helmholtz coils [113] or perhaps even an appropriate arrangement of permanent mag­ nets with some degree of tunability. Note that the static magnetic field

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plays no direct role in probing the chirality of the molecules. Its influ­ ence is discussed in more detail in [29]. A chiral rotational spectrum is recorded as the average of many measure­ ments, each of which proceeds as follows.The nozzle is opened at some ini­ tial time, allowing a molecular pulse to begin expanding toward the active region. In the initial stage of this expansion, the internal temperature of the molecules decreases dramatically, as collisions convert enthalpy into directed translational energy. The molecules thus occupy their electronic and vibra­ tional ground states and a small collection of rotational and nuclear spin states, with their internal angular momenta preferentially quantized parallel to the static magnetic field. Following this initial stage, the molecules pro­ ceed largely collision-free. A subset of the molecules selected by the colli­ mation stage eventually permeate the light in the active region, which shifts their rotational energies in a chirally sensitive manner. When the overlap between the molecules, the light, and the microwave mode is optimum, a microwave pulse permeates the microwave cavity and induces coherence in those (light-shifted) rotational transitions that lie near the chosen micro­ wave cavity frequency and within the microwave cavity frequency band­ width. The molecules then radiate back into the microwave cavity over a longer time, with the signal diminishing primarily as a result of residual collisions. This free induction decay signal is monitored and the real part of its Fourier transform, say, calculated and regarded as the measurement [114]. We have estimated the signal-to-noise ratio [29] and find that an entirely acceptable chiral rotational spectrum could be obtained for a recording time of a few hours under favorable operating conditions. This is approaching the time usually taken to record a complete standard rotational spectrum [93], but here with the effort focused entirely upon a single rotational line. This is acceptable as four spectra spread over two lines for opposite circular polarizations might already permit the extraction of all of the chirally sensi­ tive information on offer here for a particular enantiomer.We are reminded of early Raman optical activity spectrometers, which demanded recording times of several hours [71]. Even now, “traditional chiroptical spectroscopy techniques take minutes to hours” [16]. Chiral microwave three wave mix­ ing in contrast exhibits an excellent signal-to-noise ratio, with measurement times “as fast as tens of seconds” having been claimed in one of the earliest publications [16]. A linearly polarized standing wave of light with a significantly lower in­ tensity, housed simply in a two-mirror optical cavity perhaps, might already suffice if measurements based upon αXX, αYY , and αZZ, for either chiral or

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achiral molecules, are all that is sought. Measurements of individual, ori­ entated components of αAB using a combination of optically induced a.c. Stark shifts and microwaves have recently been reported for heteronuclear molecules [115]. This work, performed independently of ours, confirms the validity of the basic theory first presented by us [29] for chiral rotational spectroscopy.

ACKNOWLEDGMENTS This work was supported by the Engineering and Physical Sciences Research Council grants EP/M004694/1, EP/101245/1, and EP/M01326X/1; the alumnus programme of the Newton International Fellowship; the Max Planck Institute for the Physics of Complex Systems; the National Key Research and Development Programme of China under con­ tract number 2017YFA0303700 and a Royal Society Research Professorship RP150122.We thank Laurence D. Barron and Fiona C. Speirits for helpful correspondences.

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CHAPTER 19

Chiral Analysis and Separation Using Molecular Rotation Mirianas Chachisvilis Solvexa LLC, Keswick, CT, San Diego, CA, United States

19.1  INTRODUCTION Separation and analysis of chiral molecules also plays an important role in the pharmaceutical industry [1]. Most of the new small-molecule drugs reaching the market today are single enantiomers, rather than the racemic mixtures. Therefore, it is expected that separation and analysis of chiral molecules will continue to play an increasingly important role in the pharmaceutical industry [1,2]. More generally, the relevance of chirality in nature is well established [3]. A variety of mechanisms have been proposed to explain symmetry-breaking interactions and the origins of enantiomeric homogeneity in biological systems, such as circularly polarized light [4], gravitational fields and vortex motion, parity violation, time-dependent optical and magnetic fields, or photochemistry [5]. These mechanisms mostly lead to very small enantiomeric excess and thus require additional amplification to reach enantiopure state. Current separation methods typically rely on interactions with various chiral selectors, for example, chiral chromatography or recrystallization and related Viedma ripening [6,7], whereas determination of absolute configuration (AbCon) relies on X-ray crystallography and chiroptical spectroscopy [8], which encompasses a range of spectroscopic techniques, including vibrational circular dichroism [9], and typically requires ab initio simulations and/or large amount of sample and suffers from low fidelity. All these methods are time consuming and they do not lend themselves to a priori predictions of performance for newly synthesized molecules (e.g. chiral high-performance liquid chromatography requires a method development step such as selection of appropriate chiral column, solvent, and separation conditions). Other recently proposed chiral separation/analysis methods include chiral gratings [10] and nuclear magnetic resonance in the presence of static electric field [11,12]. Very recently, AbCon has been determined using microwaves [13] and Coulomb explosion imaging [14]; however, Chiral Analysis. http://dx.doi.org/10.1016/B978-0-444-64027-7.00019-5 Copyright © 2018 Elsevier B.V. All rights reserved.

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these new methods are applicable to molecules that can be sampled in the gas phase and have not been demonstrated for larger molecules with high degree of conformational freedom. In this chapter, we will describe and review two different chiral separation and analysis approaches that rely on molecular propeller effect (MPE). The macroscopic propeller effect is a well-known hydrodynamic phenomenon which manifests itself through rotational–translational coupling in left–right dissymmetrical bodies such as helical filaments [15–17]. Already Pasteur [18] claimed that dissymmetry generated by a rotation coupled with linear motion is similar to spatial dissymmetry (chirality) as encountered in chemical structures [3].Thus, chiral molecules can be envisaged as tiny propellers with their “handedness” and propulsion direction being determined by the AbCon of the molecule. It has long been hypothesized that the MPE may lead to separation of enantiomers exposed to radio-frequency electric fields of rotating polarization, however due to deficiencies in the theoretical derivations, the MPE was predicted to be small, which may have discouraged experimental verification of such an effect [19]. Recently, it has been experimentally demonstrated that the MPE is real and can be used to separate enantiomers of small molecules in solution using rotating electric field (REF) [20]; moreover, it was shown that the MPE can be used for AbCon determination. Rotating the molecules with electric fields requires a high electric dipole moment of the molecule, strong electric field, and nonpolar solvents [20]; such requirements may pose challenges for achieving commercial acceptance in the near future. Alternative approach is to use other possible means of imposing rotation on the molecules such as shear flows (SFs). Both approaches have their advantages and disadvantages as will be described in this chapter.

19.2  CHIRAL SEPARATIONS IN SFs There have been multiple theoretical [21–28] but only a few experimental studies [29–32] on separation of macroscopic chiral objects (such as helical colloidal particles, model chiral helices, helical-shaped bacteria, >1  µm) in helical flows, vortices, microfluidic SFs, or rotating magnetic fields; one of the more recent studies [30] points out that despite many theoretical approaches proposed so far, there is still no agreement as on the magnitude and even the direction of motion of chiral particles in SFs. Most of the theoretical studies presented in the above papers are phenomenological and

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rely on numerical simulations arriving at sometimes conflicting conclusions, although all agree that separation of chiral objects in SFs should be possible, at least on the microscale.The most detailed theoretical study by Makino and Doi [22] suggested that chiral separation for small molecules in SFs is possible but only at high shear rates because the separation effect is a third-order effect in the shear rate; the authors postulate that the effect must be zero at low shear rates (i.e. in the linear regime) due to random orientations of small molecules in solution (i.e. isotropic system). The magnitude of the shear rate . g relative to the rotational diffusion rate can be represented by dimension. less Péclet number, Pe, defined as: Pe = γ /Dr , where Dr = kBT / πηd 3 is rotational diffusion constant (η is the viscosity and d the size of the molecule). For small molecules discussed in this proposal, Pe vesc, veff is inversely dependent on the field rotation frequency and proportional to the dipole–field interaction torque squared. The velocity of a molecule due to propeller motion can be expressed as 

v = L rev × v eff ( E ) × Acor ( E ) × F ( E )

(19.14)

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where veff is an effective molecular rotation frequency and Acor is the angular correction factor. Excluding the Acor(E) × F(E) factor, Eq. (19.14) simply means that linear propulsion velocity is due to rotational–translational coupling. As indicated above, F(E) accounts for the fact that only a small fraction of molecules follow the REF, whereas the factor Acor accounts for the random orientation of the propeller axis. For weak electric fields, the dipoles diffuse out of plane perpendicular to the axis of the separation chamber. The electric field torque still causes these molecules to rotate around x axis, however the molecular propeller axis with highest rotational-translational coupling is not always aligned along chamber axis, hence the propulsion efficiency is reduced by a certain factor which we call Acor; Acor ≈ 0.5 for parameters used in this analysis (see Ref. [20] for details on calculation of Acor). Fig. 19.5G shows that for weak electric fields, propulsion velocity is linearly proportional to the rotation frequency and field magnitude (only in the case if field rotates slower than vesc). Also note that the linear dependence of propeller velocity on REF frequency Eq. (19.14) suggests that increasing frequency can more than compensate for the low F(E), enabling achievement of high-propulsion velocities and separation and/or analysis in a short time period. However, in Eq. (19.14), veff is equal to the rotation frequency of the REF only in the case when the molecular population is able to respond to a change in the orientation of the external electric field sufficiently fast, that is, it is able to follow the REF. The responding fraction of the molecules will be able to rotate at the frequency of the external REF if the maximal torque imposed by the dipole–field interaction is larger than the rotational drag torque due to the rotational friction experienced by the molecule in solution at the REF frequency. Above a certain REF frequency, the molecules are not able to follow the REF (see Fig. 19.6); the value of this “escape” frequency νesc is determined by the rotational friction, the dipole moment, and electric field strength. More specifically, νesc is a frequency at which the torque due to rotational drag is equal to the maximal torque imposed by the electric field on the molecular dipole: v esc = µ E / 2πξ r where ξ r = kBT / Dr is the rotational friction coefficient. vesc is ∼0.51 and ∼2.1 MHz for molecules III and II, respectively. At REF frequencies above νesc, the molecules “slip”, rotating at greatly reduced effective frequency veff (Fig. 19.6B). For example, for molecule I exposed to the REF of 0.9 MHz, veff is only156 kHz; in this case, Eq. (19.13) yields a velocity value of 25 nm s−1 at which molecule III is propelled in solution due to the propeller effect. The propulsion velocity of molecule II is larger

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at ∼45 nm s−1, mostly due to its much higher rotational escape frequency since Lrev for molecule II is significantly lower. Eq. (19.14) is only strictly valid when vesc is significantly higher than the frequency of the REF (i.e. when the assumption of equilibrium distribution Eq. (19.9) applies); nonetheless, our approximation based on introduction of veff is supported by experiment (see below). Note that when the field rotation frequency is much higher than vesc, veff ∼ E2 (see Eq. (19.13)); it then follows from Eq. (19.14) that the overall propeller velocity is proportional to the cube of the electric field magnitude. For this reason, our setup was designed to operate in the regime when the dependence on the electric field is nearly linear, that is, at higher voltage rather than higher frequency.

19.3.2  Experimental demonstration of MPE To experimentally confirm the MPE, we have performed experiments on solutions of molecules III and II using the experimental apparatus depicted in Fig. 19.7A. The REF inside the microfluidic chamber is generated by applying π/2 phase-shifted voltages to the four pairs of electrodes surrounding the chamber (Fig. 19.7B and C). A small amount of a racemic solution of molecule III was injected into the center of the separation chamber and exposed to the REF. Data in Fig. 19.8A show that the material collected from the leading and trailing sides of the exposed sample (which was split into two halves at the center of the absorption chromatogram) have finite and opposite signs of circular dichroism (CD) signal. If the rotation direction of the REF is inverted, the CD signals from the leading and trailing fractions are inverted too. Furthermore, when the experiment is performed on a pure enantiomer sample, no inversion of the CD signal for the leading and trailing fractions occurs; these results unequivocally prove that exposure to the REF leads to enantiomeric separation of binaphthyl molecules. Importantly, the experimentally detected direction of propulsion of (S)- and (R)-enantiomers is the same as predicted by MD simulations (compare signs of Lrev in Fig. 19.5C and D and CD signals in Fig. 19.8A and Ref. [20]; e.g. the leading fraction is enriched with the (S)-enantiomer for the clockwise REF, see Fig. 19.8A, middle panel). This offers a new approach for determining the absolute configuration of a chiral molecule. To quantify chiral separation efficiency, we use enantiomeric excess, ee (ee = 0% for racemic sample and ee = 100% for pure enantiomer) defined above. Based on the experimentally determined translational diffusion

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Figure 19.7  Experimental setup. (A) A three-dimensional slice of the separation chamber showing the four electrodes, A–D, surrounding the microfluidic capillary. (B) Cross-sectional schematic showing how the electric field rotates within the separation chamber at four selected time points during a single cycle, t1–t4. At each time point, two electrodes are in the high-voltage state (+), and the opposite two electrodes are in a zero-voltage state (0). This results in a 90° rotation of the orientation of the electric field (E) within the separation chamber between each time point. (C) The voltage waveforms on each of the four electrode pairs during one full cycle of the electric field rotation. These square-like waveforms (1100 V, 900 kHz) show the π/2 phase shift between electrodes A–D. The four time points t1–t4 from Fig. 2B are also shown. (D) Expected directions of motion of the (S)- and (R)-enantiomers of molecule III for the indicated direction of rotation of the REF (clockwise, curved black arrow). α is the relative angle between the electric dipole moment and the electric field. The electric field rotates around the x-axis in the plane zy. The gray arrows show the (opposite) directions of motion for the (S)- and (R)-enantiomers of molecule III. The structure of molecule III ((S)-enantiomer) is also shown with the dipole moment direction indicated.

Figure 19.8  Experimental results for molecule III. (A) Absorbance and CD chromatograms (obtained simultaneously) for samples of molecule III after being exposed to the REF for 83 h and subsequently collected. All process conditions were identical, except where noted. The first (left) peak of each chromatogram represents the leading half of the slug and the second (right) peak represents the trailing half of the slug. Upper: racemic molecule III after exposure to counter clockwise (CCW) REF. Middle: racemic molecule III after clockwise (CW) REF. Lower: pure (S)-enantiomer of molecule III after CW REF. (B) Absorbance chromatogram from the in-line detector of a slug of racemic molecule III after exposure to CW REF for 45 h. The sample collected from the shaded left-hand side of the chromatogram had enantiomeric excess (ee) of 26% of the (S)-enantiomer of molecule III, whereas the right shaded section of the chromatogram had an ee of 61% of the (R)-enantiomer of molecule III. This is consistent with the edge of the sample slug being more enantiomerically enriched than in the center. The boundaries of the active area of the separation chamber are also shown after conversion to the elution time scale.

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coefficient (molecule III: D = 8.3 × 10−6 ± 3 × 10−7 cm2 s−1; molecule II: D = 1.4 × 10−5 ± 5 × 10−7 cm2 s−1) and a drift term of 25 nm s−1, Eq. (19.7) predicts the ee value of 19% which is reasonably close to the experimental value of ∼26% (Fig. 19.8B) considering uncertainties in many parameters used to calculate v. Moreover, Fig. 19.8B indicates that enantiomeric enrichment is higher if the sample is collected from an off-center location of the chromatographic absorption profile, as compared to the ee value from one-full half of the sample (ee = 61%, corresponding to 80.5% enrichment level); this is expected for a diffusive spreading process with the presence of a drift term. We further tested if higher separation of enantiomers could be achieved using molecule II (Fig. 19.9A) which has a higher dipole moment (10.9 Da). CD chromatograms in Fig. 19.9B clearly show that after exposure to the REF, the leading and trailing fractions of the initially racemic sample become enantiomerically enriched. In contrast, when the experiment is performed on a pure enantiomer sample, no inversion of the CD signal from the leading and trailing fractions occurs. Moreover, and similar to molecule III, the sign of the CD signature depends on the direction of rotation of the REF, and the direction of translational motion is correctly predicted by MD simulation for (S)- and (R)-enantiomers, for example, the leading fraction is enriched in the (S)-enantiomer after exposure to the clockwise REF (compare CD signs in Fig. 19.9B, upper panel, and Supplementary Fig. 19.3 in Ref. [20]). Fig. 19.9C shows the concentration profiles after various exposure times to the REF, whereas Fig. 19.9D summarizes enantiomeric excess values as a function of separation time. These data indicate that enantiomeric excess is already detectable after 1 h of exposure to the REF. The enrichment curve in Fig. 19.9D exhibits a square root time dependence, as predicted by Eq. (19.7). This confirms that the underlying separation mechanism is linear in time, as expected for the propeller motion. A fit of Eq. (19.7) to the ee data in Fig. 19.9D using the experimentally determined value of D results in a propulsion velocity of ∼50 ± 5 nm s−1, which is very close to the theoretically predicted value (45 nm s−1, see above). The theoretical enrichment estimate for molecule II is 27% after 46 h which is also very close to the experimentally observed value of ∼32%. At higher exposure times, separation efficiency can deviate from the theoretical value primarily due to the finite length of the active area of the separation chamber (10 cm). The material that diffuses outside the chamber boundaries is not covered by the electrodes and therefore is not exposed to the REF.

Figure 19.9  Experimental results for molecule II. (A) Binaphthyl molecule II ((S)-enantiomer). Dipole moment, µ = 10.9 Debye. (B) Absorption and CD chromatograms (obtained simultaneously) for racemic molecule II, after being exposed to the REF for 21 h and subsequently collected. All experimental conditions were identical, except where noted. The first (left) peak of each chromatogram represents the signal from the leading half of the slug and the second (right) peak represents the trailing half of the slug. Upper panel: racemic molecule II after exposure to CW REF. Middle panel: racemic molecule II after exposure to CCW REF. Lower panel: pure (S)-enantiomer of molecule II after exposure to CW REF. (C) Three overlaid and normalized absorbance chromatograms from the in-line detector of racemic molecule II after exposure to CW REF for 8 min (blue), 6 h (red), and 60 h (black). The separation chamber boundaries, converted into the elution time scale, are also shown. The black chromatogram shows shape distortions due to effects from the chamber boundaries. (D) Enantiomeric excess (ee) versus CW REF exposure time for racemic molecule II. Each sample of molecule II was collected by splitting the sample at the center of the in-line chromatographic absorption profile into the leading and trailing halves, with the leading half being enriched in the (S)-enantiomer and the trailing half being enriched in the (R)-enantiomer. Each data point is an average of at least six samples (both (S) and (R) ee values averaged together), with standard error bars shown. The blue curve is the least-squares fit of Eq. (19.7) to the data.

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Our theoretical findings on the MPE are fundamentally different from an earlier theoretical study by Baranova and Zeldovich [19] which predicted a quadratic dependence on the electric field magnitude based on a phenomenological assumption that the propeller velocity should be proportional to the radio-frequency field intensity (∼E2). Moreover, they have assumed that a rate at which the molecules settle in response to the field change is faster than 100 MHz which as we show above is not correct for typical small molecules (i.e. they did not account for the fact that molecules would not be able to rotate at radio frequencies). Using the field magnitude of 3 × 105 V  m−1 and radio field rotation frequency of 100 MHz proposed in their study [19], veff would be only 308 Hz for molecule III, making experimental observation of the effect not practically feasible. In contrast, our derivation of Eq. (19.13) is based on the solution of rotational diffusion equation with two degrees of freedom, is valid for any electric field magnitude, and predicts that the propeller effect at practical electric field magnitudes will be multiple orders of magnitude stronger than those predicted by Baranova and Zeldovich, making experimental verification and potential applications feasible. For example, if we use the theory proposed in the Baranova and Zeldovich’s paper to calculate the propulsion velocity for molecule III in our experimental setup, we obtain a value of only ∼0.3 nm s−1 which is about two orders of magnitude lower than that predicted by our approach. As illustration if a solution of enantiomers is placed into a closed container of length L and exposed to an REF, over time an exponential concentration distribution will develop: vL (19.15) e( v / D ) x C ( x ) = C Ave D(e vL /D − 1) where CAve is the average concentration of enantiomers and D is the translation diffusion coefficient. The distribution profile is inverted for opposite enantiomers. Eq. (19.15) follows directly from the lateral diffusion equation with a drift term due to the propeller motion. Quantity D/v has a dimension of length and characterizes the balance between the diffusion and drift motion due to the propeller effect. A smaller characteristic length indicates that the propeller effect is stronger than diffusion, allowing a more complete separation of enantiomers within a shorter time period. Since the values of D/v are 3.4 and 3.0 cm for molecules III and II, respectively, it is expected that separation efficiency will be higher for molecule II.

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19.4  SUMMARY Theoretical and experimental evidence discussed above show that the strength of the MPE is underappreciated and can indeed enable separation of chiral molecules into enantiomerically enriched states within hours when exposed to REFs or SFs. The fields of drug discovery, development, and manufacturing increasingly require molecules that are enantiopure. In this chapter, we have described a novel approach for analysis and separation of chiral small molecules that utilizes MPE to enable simultaneous separation and absolute configuration analysis in a single step, without any need for chiral selectors. The technology may enable significant cost and time savings for chiral chemistry research and industry: (1) by eliminating the need for expensive chiral stationary phases, (2) by significantly shortening separation method development, and (3) through the use of predictive software for performance prediction and stereochemistry determination.

ACKNOWLEDGMENTS The work presented in this chapter was supported in part by the NIH grant R43GM119431 to Solvexa, LLC.

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INDEX A N-Aacetylgalactosamine (GalNAc) sugars, 265 1-(α-Aminobenzyl)-2-naphthol, 377 AbCon, 753 Ab initio molecular orbital (MO) calculation, 334 Absolute configuration (AbCon), 201, 293, 753 determination and evaluations, methodologies for, 294–297 nonempirical methods, 294 relative methods using an internal reference with known AC, 295–297 determination of, 224 Absolute configuration determinations, 700, 701, 713 via M3WM, 721 Accurate quantum chemistry methods, 692 Acetonitrile, 434, 642 N-Acetyl-glucosamine (GlcNAc), 265 N-Acetyl-D-glucosamine, 280 Achiral benzotriazole dye, 271 Achiral bonds, 251 Achiral micelles, 182 Achiral surfactants, 166 chiral probes in, 167–170, 175 and polypeptides, interaction between, 170–171 Acid-base combination, 297 Acid-base salts, 296 Active pharmaceutical ingredients (APIs), 202 AC voltage divider, 138 Acyclic alcohols, MBC esters, 351 Adenosine diphosphate (ADP), ROA spectra, 262 Adenosine-(2, 3)- monophosphate, 261 Adenosine-(3, 5)-monophosphate, 261 Adenosine monophosphate (AMP), 261 ROA spectra of, 262 Adenosine triphosphate (ATP), 261 ROA spectra, 262 Aeroplysinin-1, 268

Aerosol-OT, 153 Aggregation, 153 Aggregation-induced resonance Raman optical activity (AIRROA), 272 Ahrens polarizer, 89 Air-water interface, 153 Al-bonded pyridine groups, 376 Albumin, 431 Alcohol enantioresolution, 301 Alcohol methine proton, 316 Alcohols enantioresolution and determination of absolute configuration, using chiral carboxylic acids, 295 Al(III) complexes, 385 Aliphatic amine conjugates, 355 Alkaloids, 380 Allain–Trinder reaction scheme, 432 Amines enantioseparations, by SFC, 613 primary, 518 secondary, 518 Amino acid, 236 α-amino acids, Ts-derivatives of, 377 9-amino acid system, 641 formation, 32 Amino alcohols, 378, 382 3-Amino-1-propanol, 372 (R)-sec-Ammonium ions, 361 Amphetamine, 612 Amplitude-modulated signal, 118 Amplitude modulation mathematical basis for, 147–150 Amyloid-β protein, 430 Amyloid fibrils cross-beta core, 203 Amylose tris(3, 5-dimethylphenylcarbamate), 612 Analog-to-digital converter (ADC), 119 Analytical separations, 612 cyclodextrin CSPs, 617 ion exchange CSPs, 616 macrocyclic glycopeptide antibiotic CSPs, 616 Pirkle-type CSPs, 615 polysaccharide CSPs, 612–615 779

780 Analytic time-dependent protocol, 274 Angle of incidence (AOI), at mirrors, 663 Angle of optical activity, 37 1, 6-Anhydro-β-D-glucopyranose, 278 Anisotropic invariants, 250 Anthracyclin antibiotics, 351 (S)-(+)-1-(9- Anthryl)-2, 2, 2-trifluoroethanol, 371 Antinoplanes teichomyceticus, 520 Anti-Stokes lines lines, 731 APIs chiral properties, 238 Aplysina cavernicola, 268 Apparent mole fractions, 229 Aqueous background electrolyte (BGE), 581 Arbitrary electric vector, 131 A-reduction, 684 Arg 8 produces, 641 Aromatic alcohols, 351 application, 352 Artifact-suppression protocol, 255 Astaxanthin (AXT), j-aggregates, 272 Astrophysical asymmetries, 14 Asymmetric interaction on symmetry breaking transitions, 19 Asymmetric photochemistry, 33 Asymmetric photolysis, 33 Asymmetric reaction, 298 Asymmetric synthesis, 297 Atomic resolution, 283 Atoms chiral asymmetry, 11 Atropisomer, 309 AUTOFIT program, 694

B Backscattering ICP measurement strategy, 252 BaF2 window surface, 192 Balle-Flygare cavity, 686 Balle-Flygare design, 689 Basic chiral units, 4 absolute configuration of, 4 definition, 4 Benzo-2′, 2′′-quaterphenyl-26-crown-8 ether probe, 358

Index

Benzyl alcohols, 309 (S)-1-Benzyl-3-methylpiperazine-2, 5-dione, 376 Bijvoet method, 294 Binaphthyl diamine, 758 (S)-(–)-1, 1′-Binaphthyl-2, 2′-diamine, 756 1, 1′-Binaphthyl-2, 2′-diol, 380 1, 10-Binaphthyl-2, 20-diyl hydrogenphosphate, 387 1, 10-Binaphthyl-2, 2′-diyl hydrogen phosphates, 388 Binaphthyl molecule III, 763 Biological molecules, 203 Biomolecular asymmetry, 13 Biophysical techniques, 264 Biot, Jean, 97 BioTools, Inc., 207 Biphenyl chromophore, 346, 348 Birefringent materials, 88 Biréfringent two-prism polarizers, 95 Bis(2-ethylhexyl) sulfosuccinate sodium salt, 153 trans-1, 10- (cyclohexane-1, 2-diyl) bis(imidazole) N-oxides, 382 1-3, 5-Bis(trifluoromethyl)phenyl group, 378 3, 5-Bis(trifluoromethyl)phenyl isothiocyanate, 387 Block copolymer structure, 265 Blood plasma detection and quantitation, of proteins, 457 electronic circular dichroism, 430–435 fluorescence-detected circular dichroism, 438 Raman optical activity (ROA), 439–443 vibrational circular dichroism (VCD), 436–437 BO approximation, 210 Bode diagram for high-pass filter, 141 for simple low-pass RC filter, 142 Boltzmann constant, 765 Boltzmann distribution, 686, 765 Boltzmann’s constant, 106 Bombolitins I and III peptides, 178

Index

Bond-polarizability (valence-optical) model, 274 Born-Oppenheimer approximation, 687 rigid nuclear skeleton, 740 Born-Oppenheimer (BO) coupling effects, 208 (-)-Bornyl acetate, 268, 269 Bovine serum albumin, 533 B2PLYP method, 692 Brewster’s angle, 84–87 calcite crystal, 88 Broadband circular polarization spectroscopy, 32 4-Bromobenzoate (-), 311 (4-Bromophenyl)(4′-methylphenyl) methanol, 306 (±)-(4-Bromophenyl)(phenyl- 2, 3, 4, 5, 6-d5)methanol, 306 Brøndsted acidic cations, 383 Brownian diffusion, 757 Brush-brush interactions, 265 BSA-DTAB system, 194 BSA-SDS system, 193 Buchu oil (betulina), 718 GC-MS analysis, 718 menthone, 720 absolute configuration of higherabundance enantiomer, 722 three-wave mixing measurements, 719, 720 (S)-2-Butanol, 641 9-O-tert-Butylcarbamoylquinine, 407 (S)-(-)-2, 2’-(1, 4- Butylenedioxy)-6, 6’-dinitro-1, 1’-binaphthalene separation simulation of counterpart enantiomers, 761 (S)- or (R)-tert-Butylphenylphosphinothioic acid, 382

C Cahn-Ingold-Prelog classification of chiral molecules, 5 Calibration measurements, 227, 229 Calixarenes, 396 Calix[4]resorcinarenes, 396 Camphanate ester (S) preparation of, 307

781 (-)-Camphanic acid, 307 D-Camphorsulfonic acid, 374 2, 10-Camphor-sultam, 299, 303 Camphorsultam dichlorophthalic acid (CSDP), 295, 299–312 acid method, 309, 330 acid moiety asymmetric structure of, 309 Camphorsultam phthalic acid, 299–312 Camphor to borneol, simulated reaction of, 227 Canada balsam, 90, 93 Capillary electrochromatography dependence, of EOF velocity, 600 enantioseparations in, 593 capillaries packed with achiral stationary phases, 596–597 capillaries packed with CSPs, 597–598 EOF velocity, 600 plate height for, 601 wall-coated open tubular (WCOT) capillaries, 594–596 Capillary electrochromatography (CEC), 565 Capillary electrokinetic chromatography (CEKC), 565 effect, of increasing counterpressure, 570 enantioselective and nonselective phenomena, 567 modes of enantioseparations, 574 counterbalancing driving force, 576 flow counterbalanced capillary electrophoresis (FCCE), 575 partial filling technique, 575 self-electrophoretic mobility, 575 pressure-driven chromatography and, 569–574 Capillary electrophoresis (CE), 565, 632 combination, of chiral selectors, 578 continuous separation, of (±)-chlorpheniramine, 577 enantioseparations, with charged and uncharged chiral selectors, 567 mathematical models, 589 advantage, 589 chiral charged resolving agent migration model (CHARM), 591

782 Capillary electrophoresis (CE) (cont.) concentration, of chiral selector, 591 dual chiral separation systems, 592 duoselective chiral separation model, 591 microfabricated electrophoretic devices, 583 nonaqueous enantioseparations, 580 reversal, of EMO on, 572 schematic representation, flowcounterbalanced separation principle, 571 selector–selectand interaction, 586 binding energies and component contributions, 590 electropherograms, of CPT mixtures, 588 geometry-optimized snapshots, from simulated MD trajectories of, 589 nuclear magnetic resonance (NMR) spectroscopy, 586 schematic of β-CD:CPT and HDA-β-CD:CPT, 589 separation principle, 566 simultaneous carrier mode separation, 580 Capillary gel electrophoresis (CGE), 566 Capillary isoelectric focusing (CIEF), 566 Capillary isotachophoresis (CITP), 582 Capillary liquid chromatography (CLC) applications, 548 baseline, between two enantiomeric separations, 554 odd van Deemter shapes, 549 separation of epimers/ diastereomers, 553 subsecond chiral chromatography, 548 tunable peak shapes, 555 two dimensional chiral LC, 550 chiral separations, 512 chiral stationary phases (CSPS) for, 515 π-complex stationary phases, 516 detectors, choice of, 538 development method, 535 efect of particle sizes, particle morphologies, and column geometries, 514 enantiomeric separations, 554

Index

features of, 512 isocratic mode, 512 theoretical plates, 513 future perspective, 558 historical background, 508 instrumental considerations for, 544 interactions in, 509 anisotropic selectors, 509 Cahn-Ingold-Prelog rules, 510 diastereoisomeric complexes, 509 nature, of electronic interactions, 511 three-point interaction model, of chiral stationary phase, 510 macrocyclic bonded stationary phases, 517 Odd van Deemter shapes, 549 resolution equation, 515 separation, of epimers/diastereomers, 553 special detectors, 536 circular dichroism detector (CD), 537–539 polarimetric and optical rotation detectors (ORD), 537 tobacco-specific racemic nitrosamine, direct separation, 554 tunable peak shapes, 555 Van Deemter coefficients for, 599 tert-Carboamylated quinine, structure, 533 Carbohydrates, 264–268 based compounds, 259 Carbonyl diimidazole (CDI) method, 533 O-Carboxy-anhydrides, 381 Carboxylate-DMAPH+ ion pair, 387 Carboxylic acid signals, 386 2-[4-(2-carboxy-2-methylpropyl)phenyl] propionic acid, 402 Carnegie Atlas of Galaxies, 14 Carrier waveform, modulated waveform, 112 CARS spectral interferometry (iCROA) phase sensitivity of, 273 Cartesian coordinate tensor transfer (CTT) technique, 276 Cathinone, 612 Cavity-based chiral polarimetry, 649 limitations, 649 optical cavity, advantages, 649–650

Index

Cavity circular birefringence linear polarization, protection of, 657 Cavity-enhanced spectrometers chirped-pulse, 690 Cavity-enhanced spectroscopies (CES), 654, 672 Cavity frequency mode structure, 672 Cavity ring-down cavity, 655 Cavity ring-down polarimetry (CRDP), 650, 654 bowtie cavity signal-reversing, 663 cavity antireflection-coated windows, 663 experimental setup for two-mirror, 656 experimental signals for, 658 optical cavities, 654 polarization rotation frequency, 656 with signal reversals, 660–672 signal-reversing advantages of, 665 EW, 668 measurements, 665 open-air, 666 optical rotation measurements, 667 optical rotation versus pressure, 664 signal-reversing, experimental setup for, 661 signal-reversing, experimental signals for, 664 source of noise, 672 Cavity ring-down spectroscopy (CRDS), 654 CCD detector, 252 CCT fragmentation approach, 258 CCT transfer, 279 CD. See also Circular dichroism detector (CD) Cefotaxime, 434 ECD spectra of, 434 Cefoxitin, 434 ECD spectra of, 434 Cefuroxime, 434 ECD spectra of, 434 Cellobiohydrolase, 533 CEP setup, mode structure, 673 Cetyltrimethyl ammonium (CTMA), 180

783 Cetyltrimethylammonium bromide (CTAB), 154 Cetyltrimethylammonium chloride, 178 Charge coupled device (CCD) detector, 207 Charged resolving agent migration model (CHARM), 591 CHARMM general force field (CGenFF), 764 Chemical correlation, 315 Chemical shifts, 367 diastereotopic nuclei, 368 differentiations, 369 in the free and bound states, 369 Chemometric analysis, 231 Chiral α-arylalkylamines, 375 Chiral acids, 324 with 1H NMR diamagnetic anisotropy effect, 296 Chiral alcohol, 314, 315, 317 Chiral amides, 378 Chiral amino acid derivatives, 471 Chirasil-Val, structure, 471 Chiral amino alcohols, 377, 379 Chiral analysis, 201 Chiral asymmetry, 3, 10, 25 astrophysical, 14 in atoms, 11 biomolecular, 13 in biomolecules, 3 in molecules, 13 morphological, 13 in nuclear processes, 10 for radioactivity, 10 Chiral autocatalysis, 23, 33 Chiral auxiliaries, 297, 299, 309 Chiral base-stacking arrangement, 260 Chiral bipyridine-europium(III) complex, 282 Chiral bisthiourea, 387 Chiral camphor-based molecule, 235 Chiral carboxylic acids, 293 Chiral-center homoisoflavonone, 269 Chiral chemistry classes and applications, 521 Chiral chromatography, 372 Chiral compounds, 299 Chiral derivatizing agents (CDAs), 313, 367 Chiral ∆2-thiazolines-1, 3, 372

784 Chiral fluorinated diphenylmethanols, 328 Chiral fmoc-glycyl-glycine-OH molecules, 272 Chiral GC stationary phases, 469 application of, 470, 496 CC3, 497 CC9 and CC10, 497 classification, 470 as GC stationary phases, 499 Chiral ILs (CILs), 388 ChiralIR-2X, 207 Chiral isobornyl amine, 388 Chirality, 3, 367 Cahn-Ingold-Prelog classification of, 5 crystal enantiomeric excess, 8 definition, 3 discovery of, 35 enantiomeric excess, 8 measures, 9 ambiguity with sign of, 9 on basis of interaction of reference chiral object, 10 changes in, 9 degree of chirality, 9 optical rotation, 7 relation to polarization of light, 35 and relation to polarization of light, 35 terminology issues, 3 Chirality concept, with nonsuperimposable mirror images, 508 Chirality recognition, 358 Chirality transcription, 360 Chiral lanthanide shift reagents (CLSRs), 367 Chirally autocatalytic reactions, 23 Chirally sensitive vibrational corrections, 740 Chiral macrocycles, 394, 396 Chiral MαNP acid, 320 Chiral mobile phase additive (CMPA), 569 Chiral molecular motor, 304 Chiral molecular systems, 282 Chiral molecular tools, 299, 302, 338 synthesis of, 312 Chiral molecules, 29, 32 Cahn-Ingold-Prelog classification of, 5 characteristic of, 202 enantiomeric detection of, 225

Index

enantiomers of, 5 molecular dissymmetry of, 35 Chiral N-acylamino acid surfactants (NAASs), 173 Chiral nonracemic atropoisomeric bis-phosphine dioxides, 378 Chiral organic molecules, 274 and achiral surfactants, interaction between, 189 Chiralpak AD-H (amylose tris(3, 5-dimethylphenylcarbamate), 613 Chiralpak IC (cellulose tris(3, 5-dichlorophenylcarbamate), 613 Chiral (2-methylphenyl) phenylmethanol, 308 Chiral phosphoric acid, 382 Chiral polypeptides and achiral surfactants, interaction between, 190 Chiral primary amines, 356 coupling reaction of, 354 ChiralRAMAN instrument, 255 ChiralRAMAN SCP-ROA spectrometer, 207 Chiral resolution, ecent developments, 539 enantiomeric separations, of BINAM, 542 enantioseparations, in different elution modes, 540 particle morphology, 539 separation, of TSO enantiomers, 544 ultrafast enantiomeric separations, 541 Chiral rotational spectra, 734 Chiral rotational spectrometer, 743 evacuated chamber encompasses, 745 key components, 743, 744 microwave cavity, and associated components, 745 moderate strength, static magnetic field of, 745 optical cavity houses far off-resonance, 745 pulsed supersonic expansion nozzle, 743 Chiral rotational spectroscopy, 731, 732, 740 energy shift, 733 far off-resonance circularly polarized light, 733 intensity of light, 733 isotopic molecular chirality, 739

Index

monochromaticity, 743 multiple chiral centers, molecules with, 741 oriented chiroptical information, 738 practical considerations, 743 scaling, 742 un-ionized L-α-alanine molecule, 733 Chiral samples gas- and solution-phase specific rotation measurements, 659 Chiral selectors, 584–585 Chiral sensing, 409 Chiral separation, intermolecular interactions, 636 Chiral solvating agents (CSAs), 367 amphiphilic, 379 6-15 and selected applications, 374 3-5 and some enantiodicriminated substrates, 373 involving ion pairing processes, 383–389 low-molecular-weight, 371 molecular tweezer, 389–391 Pirkle’s, 371 synthetic macrocycle, 391–401 Chiral splittings, 743 Chiral stationary phases (CSPs), 469, 507, 610, 635 benefits, 610 structures, of chiral selectors, 611 Chiral surfactants molecular dynamics simulations, 171 quantum chemical predictions, 171 Chiral symmetry breaking at different levels, 20 spontaneous. See Spontaneous chiral symmetry breaking transitions to asymmetric interactions, 19, 25 Chiral synthesis, 297 evaluations, methodologies for, 297–298 asymmetric syntheses, 298 racemates, enantioresolution of, 297–298 Chiral tag rotational spectroscopy, 707 confidence method for absolute configuration of molecule, 708 intensity of rotational transitions, 709

785 Chiral tag rotational spectroscopy, for enantiomer analysis, 698 absolute configuration, determination of, 700 challenges of, 713 EE, in fenchyl alcohol, 710 EE, measurement of, 707 3-methylcyclohexanone, absolute configuration of, 701 Chiral transition metal complex, 282 Chiral zwitterionic phosphorus-containing heterocycles, 386 Chirasil-Dex, 483 Chirasil-Val stationary phase, 471 structure of, 471 Chiroclinics—chiroptical methods, 452 cancer and degenerative diseases, 453 albumin-to-total-protein ratio, 453 in Alzheimer’s disease, 456 ECD spectra, 453 leave-one-out cross-validation (LOOCV), 455 linear discriminant analysis (LDA), 455 metabolic disorders, 456–458 Chiroptical instrumentation, 73 light as wave, 74 counterclockwise rotating phasors, 75 transverse electromagnetic wave, 74 light polarization, 73, 78 linearly production, 81 by birefringence, 87–94 by dichroism, 97–98 by reflection/refraction, 83–87 by scattering, 82–83 by wire grids, 94–96 time-invariant exponential, 76 types of, 76 circularly polarized light, 79 elliptically polarized light, 80 linearly polarized light, 78 s- and p-polarizations, diagrammatic representation, 77 phase-sensitive detection, application of, 123–126 signal processing, 73 Chiroptical probes, 353, 358, 363 Chiroptical probes design based on biphenyl chromophore, 346

786 Chiroptical spectroscopic methods, 154 Chiroptical techniques, 429 Chlorophyll circular polarization, 42 Chlorophyll pigments, in green plants, 633 2-Chloropropionic acid, 391 Chloroquine, 374 Cholesterol, 433 Chondrites, 32 Christiansen effect, 235 Chromatography methods, 633 integration of, 631 precolumn derivatization, 634 stereoisomer separations, 634 Chromophores, 43, 179 exciton splitting by, 43 Chugaev reagent, 433 CID ratios, 272 Cinchona alkaloids, 533 Circular Couette flow rotational direction and expected propeller motion, 758 Circular dichroism (CD), 36, 38, 156, 345 Cotton effects, 334, 345, 355, 360 detector, 537–539 exciton chirality method, 294 absolute configuration of primary amines determination chiroptical probe for, 352–358 chiral resolution and determination, chiroptical probe for, 346–352 chiroptical probes for determination of absolute stereochemistry, 345–363 overview, 345–346 [2]pseudorotaxanes forming, chirality transcription and amplification, 358–363 polymer and salt-induced (PSI), 44 signals, 351, 770 Circular intensity difference (CID), 250 Circularly polarized emission (CPE), 205 Circularly polarized light, 134, 655 production of, 98–100 Circularly polarized luminescence (CPL), 205 Circularly polarized scattered Raman radiation, 207

Index

Circular polarization, 36, 39, 43, 44, 47, 50, 210 of chlorophyll, 42 of light, 33 by synchrotron radiation, 33 oligomer exhibiting, 42 spectra of photosynthetic organisms, 50 Circular spectropolarimetric signals, 35, 53 chirality, 35 circular dichroism (CD), 36, 38 circular polarization, 39 electronic transitions and rotational strength, 40 exciton coupling, 42 large aggregates (PSI-type), 44 linear dichroism, 39 linearly polarized light, 37, 38 Stokes formalism, 36, 37 13 C isotope, 687 Citronellal cyclization of, 680 isopulegol, synthesis of, 679 13 C NMR spectroscopy, 370 Cobalt-60, 10 Coherent anti-Stokes Raman scattering (CARS), 273 Cold spray ionization-MS (CSI-MS), 360 Collision cross-section (CCS), 637 Colloid-sugar distance, 272 Colon cancer patients ECD spectra, 454 ROA spectra, 454 Commercial stationary phases, 471 chiral amino acid derivatives, 471 cyclodextrins, 471 Competitive binding, 370 Complex analytical mixtures separation of, 634 Complex chiral object, 4 π-Complex stationary phases, 516 advantage, 517 structural characteristics of, 517 Concentration dependence, of ECD spectra for, 181 Conductor-like screening model (COSMO), 278 Configurational assignments, 411

787

Index

Continuous-wave (cw), 672 cw-CEP, experimental setup, 673 frequency beating, experimental signals of, 675 Continuous-wave cavity-enhanced polarimetry (cw-CEP), 650, 673 Conventional chiral acids, 315 Conventional polarimetric methods, 155 Cosine wave, low-frequency computer-generated plot, 112 Cotton effects, 350, 353 Cotton-Mouton effect, 672 Cotton-Mouton measurements, 672 Coulomb explosion imaging, 753 Coupling constants, 367 Coupling Cotton effects, 348 Cowpea mosaic virus (CPMV) protein, 264 Crispine A, 382 Critical aggregation concentration (CAC), 153 Critical micelle concentration (CMC), 153 Cross-correlation regression analysis, 227 Crown ether probe, 361 complex formation of, 359 Crown ethers, 518 Cryo-electron microscopy, 261 Cryptochiral hydrocarbon synthesis of, 335, 336 Cryptochiral hydrocarbon (S)-(−)[VCD(+)984]-4-ethyl-4methyloctane, 334 Crystal enantiomeric excess (CEE), 8 Crystal particles, 235 13 C satellite signal, 370 CSDP acid (-)-1, 299–312 application to various alcohols, 302–312 including diphenylmethanols, 325–334 enantiopure cryptochiral hydrocarbon synthesis, (R)-(+)- [VCD(-)984]4-ethyl-4-methyloctane, 334–338 for enantioresolution of alcohols and determination of their AC, complementary use of, 325–338 CSDP ester (S)-(-) X-ray ORTEP drawing of, 331 CSP acid (−)-5, 299–312 application to various alcohols, 300–302

C-substituted chiral (phenyl- 1, 2, 3, 4, 5, 6-13C)phenylmethanol [CD(-) 270]-(S), 307 Cyanohydrins, 301, 380, 386 Cyclic carbonates, 381 Cyclic oligosaccharides, 519 β-Cyclodextrin (β-CDx), 183 Cyclodextrins (CD), 401–406, 471, 617 applications, 485–489 cone structure, 474 phases and applications, 475 preparation, of cyclodextrin-based columns, 472 diluted selectors, 474 grafted chiral selectors, 483 undiluted selectors, 472 separation, mechanism, 484 inclusion interactions, 484 substituents, effect of, 485 surface interactions, 484 structures of, 472, 473 β-Cyclodextrin, structure, 519 Cyclofructans, 490 applications, 491 mechanism of separation, 491 structure, 490, 519 Cycloheptane, 400 Cyclohexane, 400 Cyclohexene, 400 Cyclopentene, 400 Cyclophosphazene derivatives, 372 Cyclotriphosphazenes, 372 L-/D-Cysteine SEROA spectra of, 271 13

D D-amino acids, 7 Decylammonium chloride (DAC), 184 def2TZVP basis set, 692 (+)-Dehydroabietylamine, 407 Demodulated noise, 117 Demodulated signal, 115, 149 Demodulation waveform, 113 DC level, 114 synchronous detection/synchronous demodulation, 113 D-enantiomer, 6

788 Density functional (DFT) methods, 692 Grimme’s dispersion, 696 Density functional theory (DFT), 214 calculations, 258 Deoxyribonucleic acid (DNA), 29 Deproteinization agent, 434 Desmopressin, 642 diastereomers, experimental separation of, 642 Desymmetrization reaction, 298 DFT derivatives, 274 Diabetes diagnostics and monitoring, 457 ECD spectra, of urine, 458 Diacetyl-L-tartaric acid mono lauryl ester sodium salt (T12O_COONa), 191 2, 6-Diacylaminopyridine, 391 Diamagnetic anisotropy effect, 296, 317–319 1, 2-Diaminocyclohexane, 395 trans-1, 2-Diaminocyclohexane, 379 Diastereoisomer, 507 Diastereomeric esters, 330 Diastereomeric MBC esters, 347 Diastereomer ratio, quantitative determination of, 694 Diastereotopic protons, 378 4, 5-Dichlorophthalic acid, 299 4, 5-Dichlorophthalic/phthalic acid, 299 Dicurcuphenol B axial chirality of, 220 1, 3-Dicyclohexylcarbodiimide (DCC), 300 Didiscus aceratus, 219 Dielectric material block reflection and refraction, 129 Digital-signal processing (DSP), 119 Digital technology, advantages of, 119 Dihedral angles, 350 Diketopiperazine (S)-1-benzyl-3-[(Z)(dimethylamino)methylidene]-6methylpiperazine-2, 5-dione, 376 Dilauroyl-phosphatidyl-adenosine (DLPA), 180 Dilauroyl-phosphatidyl-uridine (DLPU), 180 Diluted selectors, 474 Dimeric structure, 153

Index

Dimethylacetamide (DMA), 580 4-Dimethylaminopyridine (DMAP), 300, 386 (S)-2, 2-Dimethyl-1, 3-dioxolane4-methanol (DDM), 234 2, 2-Dimethyl-1, 3-dioxolane- 4-methanol (DDM), 233 N, N-Dimethylformamide (DMF), 580 (2, 6-Dimethylphenyl)phenylmethanol, 309 Dimethylsiloxane and (2-carboxypropyl) methylsiloxane, 471 (S)-Diphenyl(pyrrolidin-2-yl)methanol, 378 Dipole-dipole interaction, 367 Dipole strength, 43 Di-spiro derivatives, 372 Dissipative structures, 17 Dissymmetric mass distribution, 279 Dodecyl octaethyleneglycol monoether, 178 Dodecyltrimethylammonium bromide (DTAB), 154, 178 Doppler broadening, 690 D-riboses, 13 Drift tube ion mobility spectrometer, 638 Drude-Condon model, 669 Drude’s equation, 158 Drug product formulation, 194 Dual-beam implementation, 55 Dye-labeled nanotags, 271 Dynamic nuclear polarization (d-DNP), 370

E ECCD detection of biphenyl units, 363 chiroptical probes, 363 ECD. See Electronic circular dichroism (ECD) ECD spectra of SDLV in water, 174 EE determination, 710 Electric dipole-electric dipole molecular polarizability tensor, 250 Electric dipole-electric quadrupole optical activity tensors, 250 Electric dipole moment operator, 209 Electric dipole strength, 40 electric dipole transition moment, 41 Electric field magnitude, 766

Index

Electric field rotational frequency, 766 Electric field torque, 766 Electric field vector, 765 Electric quadrupole moment operator, 211 Electric vectors, 77 unpolarized light, 77 Electrochemical sensing, 632 Electromagnetic and weak forces unification of, 11 Electronic circular dichroism (ECD), 36, 154, 156, 201, 268, 430 achiral surfactants, 174 calculations, 215 calibration curves, for quantitation of Venoruton, 432 chiral surfactants, 172–174 derivatization, of cholesterol, 433 ECD spectra, of human blood plasma and hen egg white, 436 endogenous interference, advantage, 435 hen egg white, 446 human serum, 431 hydroxyethyl-rutosides, 431 induced, 181–183 relative content, secondary structures proteins, 437 serum levels of, O-(β- hydroxyethyl)rutosides, 430 spectra , urine of diabetic patients, 458 studies of, flavone glycosides, 430 urinalysis, 449 vitreous humor, 447, 448 Electronic transition dipole, 34 Electronic transitions, 40 magnetic and electric dipole transition, 40 and rotational strength, 40–42 Electron-nucleon interaction, 13 Electroosmotic flow (EOF), 566 Electrophoresis, integration of, 631 Electrophoresis-mass spectrometry, 637 gas-phase ion, 637 Electrophoretic separations, 520 Electrospray ionization (ESI), 635 Electro-weak forces, 11 Electro-weak interactions asymmetry in, 12 Electro-weak parity violation, 21

789 Elliptically polarized light, production of, 101 Elliptical polarization, 33 Ellipticity, 38 Emerging light, polarization state of, 104 Enantiodiscriminating efficiency, 373, 378, 383 Enantiomeric excess, 636 amplification of, 33 Enantiomeric excess (EE), 8, 32, 201, 680 on meteorites, 32 Enantiomeric purity of CSAs, 369 Enantiomeric ratio, 368 Enantiomers, 5, 73, 507 ground-state energies of, 13 polarized light and symmetry breaking transitions, 19, 20 Enantiomer-sensitive three-wave measurement, 721 Enantiomer-specific molecular rotational spectroscopy basic principle of, 715 (−)-menthone measurement, 715 Enantiomers-to-diastereomers strategy, 682 Enantiopure alcohol, 306, 324, 325, 351 recovery of, 324 Enantiopure cryptochiral hydrocarbon, 335 Enantioresolution quotient, 368 Enantioselective complexation, 378 Enantioselective mechanisms, 34 Enantioseparations, 567 Enantiotopic nuclei, 367 Encephalomyocarditis virus (EMCV), 261 Enhanced fluidity chromatography, 608 Ephedrine, 388 (−)-Epigallocatechin gallate, 408 Epoxides, 381 E-ray, 99 Ester, 303 carbonyl oxygen atom, 316 Ethyl dimethyl aminopropyl carbodiimide (EDC) method, 533 (S)-(−)-Ethyl lactate, 375 4-Ethyl-4-methyloctane, 334 Evanescent wave (EW), 667 CRDP signal-reversing, 668 optical rotation, 669 Exciton-coupled CD (ECCD), 345

790 Exciton coupling, 42–44 Exciton splitting by chromophores, 43 Experimental polarization beating signals, 662 Explicit solvation models, 282

F F and 31P NMR enantiodiscrimination, 388 Faraday effect, 125, 135–136, 661, 673 automatic polarimeter, 124 Faraday rotation, 653, 660, 662 angle, 653, 662 sign of, 661 Far-from-resonance (FFR) limit, 210 Fast imaging spectrograph, 253 Fenchyl alcohol, 695, 710 diastereomer identification, 694 enantiomeric excess (EE) determinations, 713 with propylene oxide tag, 712 relaxed potential energy surface for hydroxyl internal rotation, 696 two diastereomers of, 695 Fenchyl alcohol, spectrum of, 697 FFR limit, 212 FID signal, 721 Field magnitude, 775 First-eluted ester, 332 Five wave mixing process, 731 Flack parameter, 294 Flow-cell apparatus, 227 Fluorenyl methyl oxy carbonyl (FMOC)amino acids (AAs), 161 Fluorescence-detected circular dichroism (FDCD), 205, 346 of human blood plasma, 438 Fluorescence quencher acrylamide, 440 sodium iodide, 440 Fluorescent dye-labeled silver colloids, 271 (2-Fluorophenyl)-phenylmethanol, 329 (4-Fluorophenyl)phenylmethanol, 329 Flurbiprofen, analysis time reduction for enantioseparation of, 614 FMOC-AA-Na surfactants, 173 19

Index

FMOC amino acids representative chromatograms of, 552 Food and Drug Administration (FDA), 202 Fourier transform, 202, 746 Fourier transform infrared (FT-IR) instruments, 207 Fourier transform microwave spectrometer, 686, 718 chirped-pulse, 690 low-frequency, schematic diagram of, 691 Fourier transform rotational spectroscopy chirped-pulse, 690 Fractional recrystallization, 297 Fragmentation techniques, 632 angle between analyzer and polarizer, 135 circular birefringence, 136 Frank model with autocatalysis, and mutual antagonism, 34 Frequency-doubled continuous argon ion laser, 273 Frequency-selective 1D 1H NMR experiments, 370 Fresnel’s equations, 84 FT-VCD instrumentation, 217 advantages of, 230 extension of, 233 spectral extension of, 231 FT-VCD-PLS method, 227, 233 Fused-silica (FS) windows, 665

G Gas chromatography (GC), 632 separation, 469 Gas-liquid chromatography (GLC), 508 Gas-phase mobility integration of, 631 Gauge-invariant atom orbitals (GIAOs), 214 Gaussian concentration profiles, 760 Gaussian software packages, 274 Gedamine, 93 General Valve Series 9 solenoid nozzles, 695 Geometric isomer dyads, 507 Glan-Foucault configuration, 92 Glan polarizers, 92

Index

Glan-Taylor arrangement, 94 Glan-Taylor polarizer, 95 Glan-Thompson polarizer, 92 Glasgow backscattering ICP ROA instrument version of, 253 Glazebrook polarizer, 90, 91 Globulins, 431 Glucuronic acid (GlcA), 265 Glycidyltrimethylammonium chloride, 405 Glycoprotein, 258 Glycoprotein yeast invertase ROA spectrum of, 267 Glycosaminoglycan (GAG), 265 Goos-Hänchen shift, 668, 669 Grafted chiral selector advantages, 484 structure of, 483 Grafted chiral selectors, 483

H Hamiltonian operator, 683 rigid rotor, 683 rotational spectroscopy, 684 Hamiltonian spectral parameters rigid rotor, 692 1 H and Li NMR spectroscopy, 376 Harmicine, 382 Hartley model of micelles, 167 Hartree-Fock (HF) derivatives, 274 HA subunit, 265 α-Helical polypeptides, 261 Hen egg white, 446 components, 446 ECD spectra of, 446 Raman spectra, 446 VCD analyses of, 446 Hepatitis C protease inhibitor chiral×chiral 2D-LC method, 553 Herriot cell, 655 Heteroatom-bridged calixaromatics, 399 Heteroleptic aluminum complexes, 376 Hexacoordinated phosphates anions, 383 H-F scalar coupling, 368 HgCdTe photovoltaic detector pectral extension of, 231

791 High-affinity ligand, 220 High-density lipoprotein (HDL), 433 High-efficiency chiral phases, packing process, 534 High-performance liquid chromatography (HPLC), 202, 346, 608, 665 High-pressure liquid chromatography (HPLC), 508 High-quality protein ROA spectra, 255 High-resolution structural techniques NMR, 264 X-ray crystallography, 264 Hinge region, 265 Homochirality, 29–32, 45 of life, 30 racemic nucleotide mixture, 30 ribozymes, 30 theories about, 30 origin of, 32 amplification of enantiomeric excess, 33–35 initial imbalance, 32–33 Homogenous dielectric constant, 278 H-2 protons, 317 H-type sheet polarizers, 97 Human serum albumin (HSA), 429, 533 Huntington’s disease, 203 Hyaluronan (HA), 265 Hybrid solvents, 607 Hydrated (hydrophilic) domain, 193 Hydrocarbon (S) VCD spectrum curve, 337 Hydrogen bonds, 367 Hydrophilic interaction chromatography (HILIC), 608 Hydroxyacids, 382 Hydroxychloroquine, 374 Hydroxy esters, 380 Hydroxyethyl-rutosides, 431 β-Hydroxylamides, 371 (2-Hydroxymethylphenyl)phenylmethanol, 308 2-Hydroxymethylthieno[3, 2-e:4, 5-e8] di[1]benzothiophene, 183 2-Hydroxy-2-(1-naphthyl)- propionic acid (HαNP) menthol esters, 317 Hymenaea stigonocarpa, 269 Hyperglycemia, 457

792

I Ibuprofen, chiral splitting induced in rotational line, 739 ICP ROA instrument, 251, 253 IM drift times, 637 Imidazolinium salts, 385 Imidazolium-based chiral ionic liquids, 388 Imidazolium-based gemini surfactants, 176 IM-mass spectrometer Agilent 6560, instrument schematic of, 638 isomer separations, 640 Immunoglobulin G4 (IgG4) therapeutic monoclonal antibody, 259 IM separations, 640 Incident circular polarization (ICP), 250 Inclusion interactions, 484 Induced CD (ICD), 346 Induced circular dichroism (IECD), 181 Infrared (IR) absorption spectrum, 202 Infrared (IR) CD spectroscopy, 46 InGaAs detector, 231 Inorganic cage complexes, 171 Instrumentation, 50 current and future instrument concepts, 57–60 mitigating linear polarization cross-talk, 56–57 polarization measurement approaches, 51 snapshot modulation, 55–56 temporal modulation, 52–55 terminology, 51–52 Interfaced to separate, 638 inherent properties, 639 Internal ribosome entry site (IRES) RNA, 261 Intramolecular hydrogen bonding, 264 Intrinsic problems in 1H observation, 370 Ion exchange CSPs, 616 Ionic liquids (ILs), 388, 497 applications of, 497 dissolution of chiral selectors, 498 Ionic permethyl ß-cyclodextrins, 498 Ion mobility (IM), 632 Ion mobility spectrometry (IMS), 637 Ion-pairing interactions, 404 Ion-pair interaction, 386 IR absorption theory, 210

Index

Isoflavanones, 380 Isoleucine 11 isomers, skeletal structures, 641 Isopropyl carbamate, 519 Isotopic molecular chirality, 739, 740 Isotropic invariants, 250

J Jatropha ribifolia, 269 Jefferey’s orbits, 756 Jones formalism, 36 J-sheet microcrystalline polarizers, 97 J-sheet polarizer, 97

K Kisiel’s Programs for Rotational Spectroscopy (PROSPE) website, 693 Kraitchman analysis, 689 Kramers-Kronig transformation, 39 Kramers-Kronig transforms, 204 Kuhn’s g-factor, 539

L Labeled polymer and salt-induced (PSI) CD, 44 β-Lactam antibiotics chiroptical analysis of, 433 deproteinization, 434 ECD spectra of, 433 Lambert-Beer law, 38 L-Amino acids, 7, 13 Lamotrigine, 372 Lansoprazole, analysis time reduction for enantioseparation of, 614 Large aggregates (PSI-type), 44 Lartaric acid, rotational line, 737 LC-IM-QTOF experiment timescale of analytical separations, 639 Leave-one-out cross-validation (LOOCV), 455 Le Bel-van’t Hoff rule, 512 L-Enantiomer, 6 Leucine 11 isomers , skeletal structures, 641 Leukotriene receptor antagonist, 407 Lewis acidic cations, 383 Library free, 692 Life homochirality, 30

793

Index

Light as wave, 74 counterclockwise rotating phasors, 75 transverse electromagnetic wave, 74 Light, electric field vector, 130 Light polarization, 73, 78 beam of light scattered by small particles in liquid medium, 83 in chiroptical instrumentation, 73 elliptically, 80, 81 left-circularly polarized light, 104 forms helix rotating counterclockwise, 80 linearly production, 81 by birefringence, 87–94 by dichroism, 97–98 by reflection/refraction, 83–87 by scattering, 82–83 by wire grids, 94–96 reflection and refraction when light strikes, 85 right-circularly polarized light, production of, 80 stacked-plate, 86, 87 time-invariant exponential, 76 types of, 76 circularly polarized light, 79 elliptically polarized light, 80 linearly polarized light, 78 s- and p-polarizations, diagrammatic representation, 77 Light polarization, chirality relation, 35 Light wave, electric vector, 76 Linear discriminant analysis model, 455 graphical representation of, 455, 457 spectral bands for, 456 Linearly polarized light, 7 Linear polarization, 7, 57, 133 Lipophilic systems, 379 Liquid chromatography (LC), 508, 632 L- and D-amino acids, 634 Lissajous figures, 127–129, 131, 132, 134 Living organisms, morphological asymmetry, 13 Local density approximation, 277 Lock-in amplifier, block diagram, 118 Low-molecular-weight CSAS, 371 Lunar Observatory for Unresolved Polarimetry of Earth (LOUPE), 59

Lutein, 182 L-Valine t-butylamide chiral selector, 471 Lyotropic chiral liquid crystals, 408

M Macrocyclic amines with D3 symmetry, 395 Macrocyclic bonded stationary phases, 517 crown ether, 518 cyclic oligosaccharides, 519 Macrocyclic glycopeptides, 520 antibiotic CSPs, 616 Macrocyclic system, 372 Macroscopic ellipsoidal particles, 756 Magnetic dipole optical activity tensor symbol, 212, 213 magnetic transition dipole, 34 Main bonding and antibonding orbital transitions, 42 Malus’ law, 134, 653 Mandelic acid, 378, 398 MαNP acid (S)-(+)-2 application to various alcohols including diphenylmethanols, 325–334 enantiopure cryptochiral hydrocarbon synthesis, (R)-(+)- [VCD(-)984]4-ethyl-4-methyloctane, 334–338 for enantioresolution of alcohols and determination of their AC, complementary use of, 325–338 MαNP esters, x-ray stereostructure of, 332 Mason-Schamp relationship, 641 equation, 637 Mass spectrometry (MS), 631, 633 mass-to-charge ratio, 631 Maxwell’s equations, 669 MBC acid, 347, 352 MBC esters chiral resolution of, 348 diastereomeric mixture of, 351 HPLC separation of, 349 Mean residual ellipticity (MRE), 175 Mefloquine, 374 Menthol MαNP esters conformation of, 318 sector rule for, 318 x-ray ORTEP drawing of, 319 Menthone buchu oil, 720

794 (−)-3-Menthoxybiphenyl-4-carboxylic acid, 347 Metabolic disorders, 456 characteristics, 456 diabetes, 457 diagnostics, of microalbuminuria, 457 hyperglycemia causes, 456 Metal-organic frameworks (MOFs), 492 applications of, 494 mechanism of separation, 492 view of 1-D left-handed helix channels in, 493 Methoxy-biphenyl chromophores, 353 (S)-(+)-2-Methoxy-2-(1-naphthyl) propionic acid, 346 2-Methoxy-2-(1-naphthyl)propionic acid (MαNP acid), 295, 312 advantage of, 313 configurational and conformational analyses, 313 facile synthesis and enantioresolution of, 314 menthol esters, HPLC separation of, 315 principle and applications, 313 2-Methoxy-2-(1-naphthyl)propionic acid (MαNP acid) (S)-(+), 312 enantioresolution of various alcohols, 320–324 facile synthesis and its extraordinary enantioresolution with natural (-)-menthol, 313–315 1 H NMR diamagnetic anisotropy method for determining AC, 315–319 novel chiral molecular tool, 312–325 Methoxy-omitted model, 355 α Methoxyphenylacetic acid, 399 (±)-(4-Methoxyphenyl)phenylmethanol, 300 1-Methoxyspirobrassinin, 375, 380 trans-1-Methoxyspirobrassinol methyl ethers, 380 α−Methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA acid), 296 per-O-Methylated cycloinuloheptose (PM-CF7), 491 per-O-Methylated cycloinulohexaose (PMCF6), 491

Index

α-Methylbenzylamine (MBA), 164 (R)-(+)-3-Methylcyclohexanone, 701, 702 (S)-(+)-3-Methylcyclohexanone, 702 3-Methylcyclohexanone with 3-butyn-2-ol chiral tag experimental and theoretical rotational constants, 706 experimental and theoretical rotational constants, 703 (R)-3-Methylcyclohexanone commercial sample with enantiopure (R)-3-butyn-2-ol, 705 Methyl-β-D–glucose, 280 ROA spectra of, 281 Methyl lactate, 391 (2-Methylphenyl)phenylmethanol, 309 (4-Methylphenyl) phenylmethanol CSDP acid esters of, 306 MgF2 plate, 667 Micellar electrokinetic chromatography (MEKC), 159 Microwaves, rotational spectrum, 734 Microwave three-wave mixing (M3WM), 714 Mirrors reflectivity, 671 MMFF model, 355 Mobile-phase composition, 634 Modulated waveform computer-generated plot, 114, 115 Modulation, 121 hypothetical transfer function, 121 Moffitt-Yang plots, 170 Molar ratios, 369 Molecular chirality, 293, 306 Molecular dynamics (MD) simulations, 171, 256, 280, 758–760 diamine binaphthyl molecule, 759 molecular gloves, 9 Molecular propeller effect (MPE), 754 magnitude of, 762 Molecular rotation chiral analysis/separation, 753 absolute configuration (AbCon), 753 experimental setup, 771, 772, 774 molecular propeller effect (MPE), 754 in MPE, experimental demonstration, 770–775

Index

in REFs, 762, 764–769 in SFs, 754–762 theoretical findings, 775 X-ray crystallography, 753 spectroscopy, 682 basic principles of, 682 bringing enantiomer-specific measurement capabilities, 681–682 energy levels, 683 feature of, 681 Molecular stereochemistry, 215 determination of, 216 Molecules, chiral asymmetry in, 13 Mole fraction, 229 Moment-of-inertia, 683 Monomer-promoting 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol solvent, 192 Monophosphine oxides, 378 Monosaccharides, ROA computations of, 278 Monospirocyclotriphosphazenes, 372 Monoterpenes, 268 Morphological asymmetry, 13 inheritance, 14 living organisms, 13 Mosher acid, 296, 388 salt in CD3CN, 388 Mosher’s carboxylate, 385 Mosher’s MTPA ester, 319 MS2 virus capsid, 261 Mueller matrix, 37 Multichannel spectrometer, 202 Multiple chiral centers, molecules, 741 Multiple chiral species, determination of ee, 226–233 Multiple stereocenters, challenges for chiral analysis, 680 M3WM method, 721 Myoglobin, circular polarization spectrum, 44

N N-Acylamino acid esters, 376 N-Acylaziridines, 371 Naphthalene ring diamagnetic anisotropy of, 325

795 1, 8-Naphthalic anhydride, 375, 379 Naphtho[2, 3-c]furan-1, 3-dione, 396 2-(1-Naphthyl)propane-1, 2-diol, 310 Naproxen, 636 R-naproxen, 636 Natural alkaloid, 406 Natural products, 268–270, 406–408 N-3, 5-Dinitrobenzoyl group, 375 amino acids, 380 Near-infrared excitation ROA spectrometer, 273 FT-VCD-PLS method, 233 region, 238 Negative ROA bands, 193 Neurofibrillary tangles, 430 Neutral hexaethylene glycol monododecyl ether (C12E6) surfactants, 193 Newtonian fluids, 756 in narrow gap Couette flows, 760 N-Hexadecyl-N, N-dimethyl-3ammonium-1-propanesulfonate, 178 N-Hydroxysuccinimide (NHS) method, 533 N-Methylformamide (NMF), 580 NMR-active nucleus, 370 NMR anisotropy effect, 316 NMR chemical shift, 317 NMR spectroscopy, 368, 682 trans-N, N-dialkylcyclopentano-1, 2-diamine, 391 Noise low-pass filter, 108 random noise phase angle vs. time, 117 Noise, in electrical circuits, 105 flicker noise, 107 white noise, 105–106 Noise power versus frequency, 109 Noise reduction strategies, 108 amplitude modulation, 110–111 demodulation, 111–118 implementing modulation, 119–122 implementing phase-sensitive detection, 118–119 modulation/synchronous demodulation, benefits of, 122–123 Noise spectral density logarithm of frequency, 108

796 Non-commercial stationary phases, 490 chiral ILs, 497 cyclofructans, 490 metal-organic frameworks, 492 porous organic cages, 494 Nuclear magnetic resonance (NMR), 201 1 H nuclear magnetic resonance (NMR) anisotropy method, 312, 338, 345, 346 determination, 373 diamagnetic anisotropy, effect, 296, 315 MαNP acid method, 333 spectroscopy, 367, 586, 682 Nuclear Overhauser effect (NOE), 375 Nuclear Overhauser effect spectroscopy (NOESY), 359 Nuclear processes, chiral asymmetry in, 10 Nucleic acids, 260–261 Raman spectra of, 256 NWChem, 764

O O-Arylation, 373 Octakis(3-O-butanoyl- 2, 6-di-O-pentyl)γ-cyclodextrin, 404 O-Heteroarylation, 373 Olefin produces, hydrogenation of, 679 Oligomer, exhibiting circular polarization, 42 Omeprazole, 380 Optical activity, 6 of solution, 8 Optical cavities cavity ring-down polarimetry, 654 chiral measurement method, 660 polarization rotation, 657 Optical cavity, 650 advantages, 649–650 asymmetry factor, 651 circularly polarized light, 650 dispersion, 652 electric dipole, 650 transition, 651 transition probability, 651 Optical chopper, light beam modulation, 120 Optical detector for enantiomorphism (ODE), 48 Optical-electronic technology, 284

Index

Optically active molecules, sample of, 226 solution/solid, 6 Optical rotation (OR), 6, 7, 154, 201 achiral surfactants, 166–167 causes of, 7 chiral, 655 single-pass, 657 chiral surfactants, 158–166 formula for, 7 as measure of chirality, 10 optical rotation detectors (ORD), 36, 154, 279, 537 O-rays, 90, 99 birefringence, 90 experiences, 92 ORD. See Optical rotation detectors (ORD) Organophosphorus compounds, 383 Orthogonal linear polarization, 225 Oscillator strength, 40 Ovamucoid, 533 Oxazepam detector data sampling rate and response time, effect, 547 subsecond separation of, 548 Oxazolidones, 387 Oxo-bridged calix[2] arene[2]triazine derivatives, 399

P Paratartaric acid, 35 Partial least squares (PLS) analysis, 227 PEM FT-VCD spectrometer, 207 Penta-(ortho-substitutedphenyl)pyridines, 382 Pe numbers, 754 Peptides, 236 backbone, 257 bands, 256 Perchloric acid, 518 PGOPHER graphical spectrum analysis, 694 Phase-sensitive detection, 116, 123, 124 Phase-shift, 100 Phasor amplitude, 76 counterclockwise rotating, 137 diagram for series RC circuit, 139

Index

Phenol atropisomers model, structures of, 222 Phenylacetic acids, 373 analogs, 373 (+)-(R)-1-Phenylethanol, 275 (S)-(-)-1-Phenylethanol, 255 1-Phenylethanol enantiomers of, 275 (S)-1-Phenylethylamine, 380 α-Phenylethylamine, 368, 376 Phopholene oxides, 378 Phosphinic amides, 382 Phosphinothioates, 382 Phosphinothioic acids, 383 2-Phosphono-2, 3-didehydrothiolane S-oxide, 382 Phosphoric acids, 380 Phosphorylation at the C-9 hydroxyl group, 407 Photoelastic modulator (PEM), 101 axis of modulator, 102 effect of, 102 method, 235 Photoelastic modulators, 101–104 Photosynthesis, 49 Phytoalexins, 375, 380 Piezoelectric transducer, 102, 103 α−Pinene, 658 optical rotation versus pressure, 659 Piperazine, 380 Piperidine, 380 Pirkle’s alcohol, 371 Pirkle’s Rule, 632 Pirkle-type CSPs, 615, 636 Placzek approximation, 274 Plasma, 430 Plasmons, propagation of, 272 POCs. See Porous organic cages (POCs) Polarimetric, 48, 59 accuracy, 51 detectors, 537 optical rotation detectors (ORD), 537 sensitivity, 51 Polar ionic mode (PIM), 536 Polarizabilities, 742 Polarizable continuum model (PCM), 278 Polarization forms of, 127–129

797 of light, 73 polarized light and symmetry breaking transitions, 19, 20 Polarizers, 56, 81 Biréfringent two-prism, 95 characterized by, 82 H-sheet, 98 K-type sheet, 98 wire-grid, 96 Polar organic mode (POM), 536 Poly-DL-alanine (PDLA), 170 Poly-γ-benzyl-l-glutamate (PBLG), 408 Poly(L-glutamic acid), 284 Poly-L-lysine (PLL), 190 Poly(L-proline II) (PPII) conformations, 260 Poly-N5-2- (2′-hydroxyethoxy)-ethyl-Lglutamine (PEEG), 170 Poly-N5-(2- hydroxyethyl)-L-glutamine (PHEG), 170 Poly-N5-(3-hydroxypropyl)-L-glutamine (PHPG), 170 Polysaccharide CSPs, 530–532, 612 amylose, 612 cellulose, 612 column cutting parameter, 615 separation mechanisms, 613 Polyvinyl alcohol (PVA), 97 Polyvinylidenedifluoride (PVDF), 435 Porous organic cages (POCs), 494 list of chiral metal organic frameworks, 494, 496 mechanism of separation, 494 Porphyrins, 24 Praseodymium(III)nitrate, 405 Prochiral acids, 380 Prochiral alcohols, 380 Prochiral amines, 380 Prochiral amino alcohols, 380 L-Proline, 634 Proline-derived diphenylprolinols, 378 Promethazine, 380 Propranolol, peak shape tunability, 557 Propranolol,VCD spectra of, 236 (S)-Propylene glycol rotational line for enantiopure sample, 735 Protein-nucleic acid interactions, 283

798 Protein-RNA interactions, 263 Proteins, 257–260 homochirality, 29 misfolding, 258 diseases, 259 molecule higher-level chiral asymmetries of, 21 Proteinuria, 451 Proton nuclear magnetic resonance (1H NMR) diamagnetic anisotropy method, 296 Protons, chemical shifts of, 312 [2]Pseudorotaxanes, 360, 361 formation, chiral transcription, 359 Pulsed jet expansion, 709 Pulsed-laser excitation, 672 Pyridine-2, 6-biscarboxamide moiety, 391 3-(2′′-Pyrrolidinyl)-BINOL, 381

Q Quantitative chiral analysis challenges, 679 by molecular rotational spectroscopy, 679 Quantum chemical (QC) calculations, 260 methods, 155 predictions, 171 Quantum mechanical calculations, application of, 215 Quantum mechanics/molecular mechanics simulations (QM/MM), 278 2′, 2′′-Quaterphenyl group, 361 2′, 2′-Quaterphenyl probe, chirality detection by, 353 Quinidine derivatives with pyridazine, 407 Quinine iodosulfate, 97 structure, 533 Quinine O, O-diethyl-phosphorodithioate, 407

R Racemic acid synthesis/enantioresolution of, 313 Racemic alcohol, 306, 330, 336 enantioresolution of, 322 Racemic (3, 5-dimethoxyphenyl) phenylmethanol, 330

Index

Racemic (3-trifluoromethylphenyl) phenylmethanol, 328 Racemic (4-trifluoromethylphenyl) phenylmethanol, 325 Racemic propylene glycol, 739 β-Radioactivity chiral asymmetry in, 10 Radiofrequency (RF) electrodes, 718 Raman intensity, 211 Raman laser spectrometer (RLS), 47 Raman optical activity (ROA), 201, 249–284, 439, 738, 740 ability of, 260 ab initio calculation of, 214 analysis, 224 applications of, 249 assignments, of characteristic Raman/ ROA bands, 444 calculation, 277 ChiralRAMAN-2X, 440 circularly polarized (CP), 204 computational modeling of spectra, 274–282 explicit solvation models for, 278 insights gained from ROA calculations, 278–282 standard computational approaches today, 276–278 dual circular polarization, 204 experimental studies, 256–270 carbohydrates, 264–268 natural products, 268–270 nucleic acids, 260–261 proteins, 257–260 viruses, 261–264 experiments, types of, 284 fluorescence of, 440 future opportunities, 282–284 hematoporphyrin, 439 hen egg white, 446 illumination period, 440 incident circular polarization (ICP), 204, 206 intensities, 213 kinetic quenchers, on fluorescence reduction, 440, 442 linearly polarized (LP), 204 original theory of, 212

Index

photobleaching, 440 physical/chemical reduction, 439 potential of, 249 principles, 250–255 instrumentation, 252–255 observables, 250–252 scattering circular polarization, 206 weak intensity of, 252 sensitivity of, 249 signals enhancement of, 270–273 recent instrumentation developments, 273 signal-to-noise ratio of, 252 surface-enhanced ROA, 270–272 spectra of, 441, 443 calculation, 274, 276 spectrometers, 738, 746 spectroscopy, advantage of, 256 studies on surfactants, 193 protein-surfactant interactions, 193–194 tensor invariants, 277 tensor quantities, 214 theory, 210–214 vs. Raman spectroscopy, 249 Raman scattering, 206 event, 210 spectrum, 224 Raman signal, 271 Raman tensor, 211 elements, 211 invariants, 211 (R)-(+)-α-methoxy-αtrifluoromethylphenyl acetic acid, 373 (R)-(+)-α-methoxyphenyl acetic acid, 373 (R)-2′-amino-[1, 1′-binaphthalene]-2-ol, 387 Rayleigh scattering, 83, 238 (R)-1, 10-binaphthalene-2, 2′-diamine, 382 RC circuit, hypothetical series, 138 Recognition system, 358 Recrystallizations, 311 Rectangular fused quartz cell, 252 Refractive index, 126

799 maltodextrin/fructose solutions, optical rotation of, 671 Relative intensity, angle between analyzer and polarizer, 135 Relaxation rates, 367 Remote sensing, 45 of homochirality, 45 solar system and, 45 exoplanet observations, 48–50 in situ observations, 46–47 solar system observations (remote), 47–48 wavelength considerations, 45–46 R-Enantiomer, 219 Resolution equation, 534 Resonant cavity u, sing narrow-bandwidth cw light, 672 Resonant proteins SEROA spectra of, 270 Ribonuclease B (RNase B), 267 Ribonucleic acid (RNA), 29, 260 Ribozymes, 30 Ring cavity clockwise (CW), 660 counterclockwise (CCW), 660 Risotecin, 520 Ristocetin, 616 (R)-mandelic acid, 386 (R)-(−)-(2-naphthyloxy)phenylacetic acid, 373 (R)-N-(2, 3-epoxypropyl)phthalimide, 379 ROA. See Raman optical activity (ROA) Rochon polarizer, 92, 93 Roof-shaped secondary amines, 378 Rotating electric field (REF), 754 frequencies, 767, 769 phase-shifted voltages, 770 Rotational constants, 225 Rotational frequency, effective, 768 Rotational kinetic energy, Hamiltonian operator, 683 Rotational line, for mixtures, 736 Rotational partition function, large molecule challenges, 686 Rotational Raman optical activity, 731 Rotational spectra complicated mixtures, chirally sensitive analysis of, 742

800 Rotational spectroscopy advantages of, 682 chemical analysis, 681, 689 molecular structure, 687 Rotational strength, 41 Rotational transitions, 685, 734 frequencies, 742 translational coupling, 756 (R)-(−)-2-(2, 3, 4, 5, 6-pentafluorophenoxy)-2(phenyl-d5)acetic acid, 374 (R, R)- α, α′-bis(trifluoromethyl)-9, 10-anthracenedimethanol, 372, 389 (1R, 2R)-1, 2-diaminocyclohexane, 379 (1R, 2S)-(−)-N-dodecyl-Nmethylephedrinium bromide (DMEB), 165 (R)-2-(2, 4, 5, 7-tetranitrofluoren-9ylideneaminooxy)propionic acid, 183

S Saccharomyces cerevisiae, 267 Sample Analysis at Mars instrument suite (SAM) onboard, 46 Satellite tobacco virus (STMV), 261 Scanning electron microscopy (SEM), 154 Scattered circular polarization (SCP), 250, 251 Scattering mechanism, 250 Secondary structures of BSA obtained from ECD spectra, 177 Self-induced diastereomeric anisochronism (SIDA), 375 Separation factor, 300, 327 Sevoflurane, 404 SFC. See Supercritical fluid chromatography (SFC) Shear flows (SFs), 754 Newtonian fluid, 756 Shear rate, magnitude of, 754 Shot noise, 105 Signal amplitude-modulated, 118 averaging, 108, 144–145 carrier waveform, 116

Index

enhancement, 270–273 other approaches, 272–273 recent instrumentation developments, 273 surface-enhanced ROA, 270–272 handling, 136 Bode plots, 140–141 phasor basics, 136 RC filter basics, 137–139 sine waves, generation of, 137 high-frequency carrier, 110 integration, 145–146 low-frequency, 110, 111 processing in chiroptical instrumentation, 73 recovering weak signals, 105 reversals, 675 Signal-to-noise basics, 142–144 characteristics, 253 levels, 270 ratio, 108, 109, 143, 207, 230, 259 Signal waveform, modulated waveform, 112 Simple chiral object, 4 Sine waves, generation of, 75 Single-crystal X-ray diffraction, 201 Single-electronic-state (SES) resonance, 210 Single-pass optical rotation, 662 Single-pass polarimetry, 652 error limit optical rotation, 654 extinction polarimeter, 653 linear polarization, 652 rotation angle, determination of, 653 Singly deuterated chlorofluoromethane derives, 736 Sinusoidal wave motion, 75 Small-molecule drugs, 283 Small-to-medium-range molecules, 155 (S)-(−)-1, 1′-(2-naphthol) (BINOL), 373 (S)-(-)-2-(1-Naphthyl)propane-1, 2-diol 4-bromobenzoate Bijvoet pairs of, 311 Snell’s law, 84, 85, 127–129 Soai reaction, 33 Sodium-ammonium-tartrate, 13 Sodium dodecyl sarcosinate, 178 Sodium dodecyl sulfate (SDS), 153

Index

Sodium N-(4-dodecyloxybenzoyl)Lvalinate, 173 Sodium N-undecynoxy carbonyl-Lleucinate, 160 Software development, for spectrum analysis, 693 Solid-phase pharmaceutical products, 238 Solid-phase sampling, advantage of, 236 Solubility, 403 Solution-state conformation, 220 Solvation models, 280 SPCAT, 693 Specific optical rotation (SOR), 155 Specific rotation (SR), 155 Spectral modulation, 55 Spectrum simulations, for molecules, 688 (S)-1-(Pentafluorobenzyl)-6methylpiperazine-2, 5-dione, 376 SPFIT spectral fitting engine, 693 SPHERE-ZIMPOL instrument, 59 Spiral galaxies, astrophysical asymmetries of, 14, 15 Spirobrassinin, 375 Spontaneous chiral symmetry breaking in Langmuir monolayers, 24 in molecular aggregation, 24 in stirred crystallization of NaClO3, 22–24 theory of, 15 consequence of, 21 Frank’s model, 16 general features, 17, 18 in mathematical terms, 15 nonequilibrium transition, 17 Spray-dried films, 236 Spray-dried sample, 237 S-reduction, 684 (S, S)-1, 2- Diphenylethane-1, 2-diamine, 387 (1S, 2S)-N, N′-Dihydroxy-N, N′bis(diphenylacetyl)-1, 2-cyclohexanediamine, 380 (1S, 2S, 4R)-1, 3, 3-Trimethylbicyclo[2.2.1]heptan-2-ol (fenchyl alcohol) enantiomeric excess measurement, 711 Standard rotational spectroscopy, 741 Stark effect, 687

801 Stark shift, 734 Stationary-phase composition, 634 Stereoisomer, 507 for chiral molecule, 679 products, 367 Stokes formalism, 37 Stokes lines, 731 Stokes-Raman wave number shift, 252 Stokes vector, 36 Structural isomer complexity, illustration of, 632 Styryl chromophore, 351 Subsecond chiral chromatography, 548 N-Substituents of diketopiperazines, 376 Ortho-Substituted diphenylmethanols, 308 Para-Substituted diphenylmethanols CSDP acid esters of, 306 Sulfinimines, 380 Sulfonimide derivative of binaphthol, 381 Sulfoxides, 380 Supercritical fluid chromatography (SFC), 607, 632 advantage of, 609 applications, 608 chiral stationary phases for, 610 comparison, of physical properties of gases, supercritical fluids, and liquids, 608 milestones, in SFC evolution, 610 Supramolecular vesicles, 174 Surface-enhanced Raman optical activity (SEROA), 270 Surface-enhanced Raman scattering (SERS), 261, 270 enhancement process complexity of, 271 measurements, 271 Surface interactions, 484 Surface tension, 170 Surfactants, 178 β-cyclodextrin, interaction between, 183 charge transfer complexes, Interaction between, 183 DNA aggregates and related materials, Interaction between, 179–181 polymers, interaction between, 184 proteins/peptides, Interaction between, 175–179

802 Symmetry-breaking bias, 30 Synchronous cyclic capillary electrophoresis (SCCE), 576 Synchronous demodulation, mathematical basis for, 147–150 Synchrotron radiation, x-ray of, 333 Synoicum pulmonaria, 268

T Tartaric acid, 35, 741 (2R, 3R)-(+)-Tartaric acid, 301 Tartaric acids, 396 chiral surfactants, 193 Tau-protein, 430 Teicoplanin aglycone, 616 Teicoplanin, structure, 529 Temporal modulation, 52 Terbium gallium garnet (TGG), 653 absorbs, 661 Ternary ion-pair complexes, 386 Terrestrial biochemistry, 29 Tertiary alcohols, 378 Tetracarboxylate crown ether, 518 Tetracycline, 449 Tetradecylpyridinium chloride (TPC), 184 Tetradecyltrimethylammonium bromide (TTAB), 154 5, 10, 15, 20-Tetrakis(3, 5-di-tert-butyl-4oxocyclohexa-2, 5- dienylidene) porphyrinogen, 409 4-(1, 1, 3, 3-Tetramethylbutyl)phenylpolyethylene glycol (Triton X-100), 154 Thermal expansion coefficient, 56 Thermal noise voltage, 106 Three-point model, 632 stereoselection with the stationary phase in condensed-phase chromatography, 633 Three-wave mixing measurements buchu oil (betulina), 719, 720 menthone, 720 on menthone, 716 rotational spectroscopy absolute configuration of higherabundance enantiomer, 722

Index

challenges and future, 721 for enantiomer analysis, 714 buchu oil as sample, 719, 720 example measurement, 716 menthone in buchu oil, predominant enantiomer of, 718 microwave three-wave mixing (M3WM), 714 molecular rotational spectroscopy, basic principle of, 715 Threonine, 161 Time-dependant HF algorithm, 276 Time-resolved fluorescence quenching (TRFQ), 161 Titrations at different ratios of CSAs, 370 α-Tocopherol, 371 Toluene calibration samples, NIR-VCD spectra for, 234 p-Toluenesulfonic acid, 387 Total internal reflection (TIR) angle of incidence (AOI), 668 antireflection-coated prism, 667 evanescent wave (EW), 667 surfaces light impinges, 668 maltodextrin, fructose, and glycerol, 670 Traditional spectroscopy methods, 679 Trans-1, 2-cyclohexanediol, 332 Trans-2-methylcyclohexanol, 331 Transmission channel, modulation/ demodulation process, 110 Transmission electron microscopy (TEM), 154 Transverse electromagnetic wave light, 74 Trifluoroacetic acid, 385 Trifluoromethyl carbinols, 372 Trifluoromethyl moiety, 325 (2-Trifluoromethylphenyl)phenylmethanol, 329 (4-Trifluoromethyl- phenyl) phenylmethanol (-), 328 (4-Trifluoromethylphenyl)phenylmethanol (R), 325 (4-Trifluoromethylphenyl) phenylmethanol MαNP esters, 327

Index

2, 2, 2-Trifluoro-1-phenylethanol, 368 Trihydroxyethyl-rutosides, 431 Triisopropylsilyl (TIPS), 385 Triphenoxyborane, 386 Tris(ethylenediamine)-rhodium(III), 282 Tröger base, 383 Trypargine, 382 Tryptophan, 438 conformation, 263 Twist excitation, low-frequency pulse, use of, 717 Two dimensional chiral LC, 550 Two-dimensional spectroscopic correlation methods, 284

U Ultrafast high-efficient SFC separations, 618 application and strategy, 624–625 comparison, of van Deemter plots under LC and SFC conditions, 620 Darcy’s equation, 619 fast and high-efficient separations, 620 preparative separations, 622 pSFC Versus pHPLC, 622–623 SFC large-scale resolution on, 621 Ultra-high-performance liquid chromatography (UHPLC), 609 Ultra-high-pressure gas chromatography, 608 N-Undecenoxy carbonyl-L-amino acidsulfated surfactants, 159 Undiluted selectors, 472 Unpolarized light beam, 83 Uridine monophosphate (UMP), 180 Urinary albumin, 457 Urine, 447 composition, of human urine, 447 ECD spectrum of, 449–452 urinary concentration of, 448 UV absorption spectra of, 449 UV region, 98 UV-Vis spectrum, 360

V Vancomycin, 616 structure, 529 van Deemter plots, van Deemter plots, 550

803 VCD. See Vibrational circular dichroism (VCD) Venoruton, 430 Very large telescope (VLT), 59 FORS observations, 59 Vibrational chromophores, 230 Vibrational circular birefringence (VCB), 204 Vibrational circular dichroism (VCD), 154, 156, 201, 249, 334, 436, 680 advantages of, 216 application of, 203, 219 bands, 220, 235 baselines, 231 centrifugation, 437 chiral surfactants, 184–188 films, 188–189 current spectroscopic range of, 203 extensive literature of, 208 hen egg white, 446 intensity, 237 IR absorption spectra of, 232 measurement of, 207 mechanical calculations of, 214 mirror symmetry of, 219 order-of-magnitude weaker, 681 principal contribution, 216 spectroscopy, 700, 723 spectrum, 217, 218 for studying soft aggregates, limitations on, 190–193 theory, 209–210 Vibrational cooling, 686 Vibrational optical activity (VOA) AC, determination of, 216–226 calculation of, 214–216 definitions of, 204–206 measurement of, 206–208 multiple chiral species, determination of ee, 226–233 overview, 201–204 potential of, 226 property of, 201 of solids and formulated products, 235–238 theoretical basis of, 208–214 ROA theory, 210–214 VCD theory, 209–210 use of, 202

804 Vibrational optical rotatory dispersion (VORD), 204 Vibrational Raman optical activity (ROA), 154, 157 Vibrational spectroscopy, 204 Viedma ripening process, 34 “Virtual enantiomers” approach, 255 Viruses, 261–264 Vitreous humor, 447 ECD spectra of, 447, 448

W Wall-coated open tubular (WCOT) capillaries, 594 Warfarin (WF), 578 analysis time reduction for the enantioseparation of, 614 enantioseparation of, 579 (R)-(-)-warfarin, 762 Waveform, cosine-squared, 114 Wave functions, 210 W3 band, 193 weak forces, 11 Whelk-O1 columns CSP, 636 geometrical characteristics of, 543 overlapped van deemter curves, 545 Wieland-Miescher (W-M) ketone, 332

Index

Wire grid polarization, 96 Wollaston polarizer, 90, 91

X X-ray analysis, 294, 300, 301, 303, 308, 329 crystallography, 203, 216, 217, 293–295, 299, 309, 319, 330, 331, 334, 337, 338, 345, 361 analysis, 312, 325 Bijovet method of, 217 configurational assignment by, 350 X-ray Bijvoet method, 299 X-ray diffraction (XRD), 154, 257, 263, 701 experiment, 294, 299 X-ray prediction, 217 X-ray structure, 279 X-ray technology, 217 β-D-Xylose, 281

Y Ytterbium(III) nitrate, 405

Z Zeroth-order wave plates, 100 Zolmitriptan enantiomers, 373 Zwitterion CILs, 389 Zwitterionic phase, 533