Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1: Protecting Groups [1] 9781774911525

Citation preview

AMINO ACIDS Insights and Roles in Heterocyclic Chemistry Volume 1 Protecting Groups

Amino Acids: Insights and Roles in Heterocyclic Chemistry, 4-volume set ISBN: 978-1-77491-150-1 (hbk) ISBN: 978-1-77491-151-8 (pbk) ISBN: 978-1-00333-019-6 (ebk) Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1: Protecting Groups ISBN: 978-1-77491-152-5 (hbk) ISBN: 978-1-77491-153-2 (pbk) ISBN: 978-1-00332-979-4 (ebk) Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 2: Hydantoins, Thiohydantoins, and 2,5-Diketopiperazines ISBN: 978-1-77491-154-9 (hbk) ISBN: 978-1-77491-155-6 (pbk) ISBN: 978-1-00332-983-1 (ebk) Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 3: N-Carboxyanhydrides, N-Thiocarboxyanhydrides, Sydnones, and Sydnonimines ISBN: 978-1-77491-156-3 (hbk) ISBN: 978-1-77491-157-0 (pbk) ISBN: 978-1-00332-987-9 (ebk) Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 4: Azlactones and Oxazolidin-5-ones ISBN: 978-1-77491-158-7 (hbk) ISBN: 978-1-77491-159-4 (pbk) ISBN: 978-1-00333-015-8 (ebk)

AMINO ACIDS Insights and Roles in Heterocyclic Chemistry Volume 1 Protecting Groups

Zerong Wang, PhD

First edition published 2023 Apple Academic Press Inc. 1265 Goldenrod Circle, NE, Palm Bay, FL 32905 USA

CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 USA

760 Laurentian Drive, Unit 19, Burlington, ON L7N 0A4, CANADA

4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN UK

© 2023 by Apple Academic Press, Inc. Apple Academic Press exclusively co-publishes with CRC Press, an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the authors, editors, and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors, editors, and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged, please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library and Archives Canada Cataloguing in Publication Title: Amino acids : insights and roles in heterocyclic chemistry / Zerong Wang, PhD. Names: Wang, Zerong (Daniel Zerong), author. Description: First edition. | Includes bibliographical references and indexes. | Contents: Volume 1: Protecting Groups. Identifiers: Canadiana (print) 20220276242 | Canadiana (ebook) 20220276269 | ISBN 9781774911501 (set) | ISBN 9781774911525 (v. 1 ; hardcover) | ISBN 9781774911532 (v. 1 ; softcover) | ISBN 9781003329794 (v. 1 ; ebook) Subjects: LCSH: Heterocyclic compounds. | LCSH: Amino acids. Classification: LCC QD400 .W36 2023 | DDC 547/.59—dc23 Library of Congress Cataloging-in-Publication Data

CIP data on file with US Library of C ​ ​ongress

ISBN: 978-1-77491-152-5 (hbk) ISBN: 978-1-77491-153-2 (pbk) ISBN: 978-1-00332-979-4 (ebk)

About the Author

Zerong Wang, PhD Professor of Chemistry, College of Science and Engineering, University of Houston–Clear Lake, Texas Zerong Wang, PhD, is a full Professor at the University of Houston-Clear Lake, Texas. Prior to that, he worked at the Institute for Biological Sciences of the National Research Council of Canada for several years. Through his career, the author has gained specific training and expertise in organic chemistry, particularly in physical organic chemistry and other subdisciplines that include photochemistry, spectroscopies, carbohydrate chemistry, sulfur chemistry, nucleosides and heterocycles, material science, reaction methodology, computational chemistry, among other. Dr. Wang has developed research projects relating to sulfur chemistry, computational chemistry, nucleoside analogs, heterocycle chemistry, materials science, and macromolecules (pillarene, calix[n]arene, and melamine-based dendrimers, etc.) and has received 22 research grants, including from NSF-MRI, NSFSTEM, Welch Research Grant, Welch Departmental Research Grant, and University of Houston-Clear Lake’s Faculty Research and Support Fund (FRSF) Grants. The author has developed two compendiums in organic chemistry: Comprehensive Organic Named Reactions, with Detailed Mechanism Discussions and Updated Experimental Procedures (3 volumes) (Wiley, 2009) and Encyclopedia of Physical Organic Chemistry (6 volumes) (Wiley, 2017), the PROSE Award winner in 2018. While conducting research activities, the author also teaches courses for both graduate and undergraduate students. To date, the author has taught courses on General Chemistry, General Chemistry Laboratory, Analytical Chemistry, Quantitative Chemical Analysis, Forensic Chemistry, Organic Chemistry, Organic Chemistry Laboratory, Advanced Organic Chemistry, Physical Organic Chemistry, Synthetic Organic Chemistry, Organometallic Chemistry, Biochemistry, Biochemistry Laboratory, Polymer Chemistry, Introduction to Chemical Engineering, Nutrition and Diet Chemistry, Green Chemistry, Introduction to NMR Spectroscopy, Chemistry Seminar, Graduate Research, and Chemistry for Non-Science Majors.

vi

About the Author

Dr. Wang earned his BS degree in Chemistry from Lanzhou University, PR China, and earned his MS and PhD degrees from the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences. He conducted his postdoctoral research at the Department of Chemistry, University of California Berkeley and York University (Canada).

Contents

Abbreviations............................................................................................................ix Acknowledgments....................................................................................................xiii Preface..................................................................................................................... xv 1.

Introduction to Amino Acids...........................................................................1

2.

The Dilemma of Working with α-Amino Acids...........................................29

3.

The Carboxyl Protecting Groups.................................................................41

4.

Amino Protecting Groups...........................................................................165

5.

Side Chain Protecting Groups....................................................................277

Index......................................................................................................................397

Abbreviations

1-Adom 1-adamantyloxymethyl 2-Adoc 2-adamantyloxycarbonyl 4-PriOCO 4-isopropyloxycarbonyl-oxybenzyloxy-carbonyl ABz azidomethoxybenzyl Acm acetamidomethyl ACP acyl carrier protein Adoc adamantyloxycarbonyl Adpoc 1-(1-adamantyl)-1-methylethoxycarbonyl Adpoc-F adpoc fluoride Aib 2-aminoisobutyric acid Alloc allyloxycarbonyl Anpe 2-(4-acetyl-2-nitrophenyl)ethyl BAL backbone amide linker BBTO bis(tributyltin) oxide BIMOC benz[f]inden-3-ylmethyloxycarbonyl Bn benzyl Boc tert-butoxycarbonyl Boc-Thr tert-butoxycarbonyl-L-threonine BOM benzyloxymethyl BOMCl benzyloxymethyl chloride BPFPPB 4-(10,19-bis(perfluorophenyl)-20H-porphyrin-5-yl)benzoyl BS2 bacillus subtilis BTFA boron tris(trifluoroacetate) Bum t-butoxymethyl CAL-A Candida antarctica CAN ceric ammonium nitrate Cbz benzyloxycarbonyl CLIMOC 2-chloro-3-indenylmethyloxycarbonyl DABCO 1,4-diazabicyclo[2.2.2]-octane DCC dicyclohexylcarbodiimide DCHA dicyclohexylammonium DCU dicyclohexylurea DEM diethyleneglycol-monomethylether DMA N,N-dimethylacetamide

x Abbreviations

DMAP 4-dimethylaminopyridine dimethylformamide DMF Dmp dimethylphosphinyl DMS dimethyl sulfide dimethyl sulfoxide DMSO DNBzh 2,2’-dinitrobenzhydryl Dnp 2,4-dinitrophenyl DNPS 2,4-dinitrophenylsulfenyl Dpm diphenylmethyl Dppe 2-(diphenylphosphino)ethyl DTBMS di-tert-butylmethylsilyl DTNP 2,2’-dithiobis(5-nitropyridine) DTP 2,2’-dithiodipyridine Dts N-dithiasuccinoyl DTT dithiothreitol ESR electron spin resonance Etoc ethoxycarbonyl Fm fluorenylmethyl Fmoc 9-fluorenylmethoxycarbonyl Foc furfuryloxycarbonyl GABA γ-aminobutyric acid HMFS N-[(9-hydroxymethyl)-2-fluorenyl] succinamic acid HOBt N-hydroxybenzotriazole HODhbt 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine Iboc isobornyloxycarbonyl Iboc-Cl isobornyloxycarbonyl chloride IUPAC union of pure and applied chemistry MBom 4-methoxy-benzyloxymethyl Mbs 4-methoxybenzenesulfonyl MBS p-methoxybenzenesulfonyl Meb 4-methylbenzyl MEE 2-[(2-methoxy)ethoxy]ethyl Menpoc α-methyl-nitropiperonyloxycarbonyl Menvoc α-methyl-nitroveratryloxycarbonyl MMT monomethoxytrityl MMT-Cl 4-monomethoxytrityl chloride Mob 4-methoxybenzyl Moc methoxycarbonyl MoEt 2-morpholinoethyl

Abbreviations xi

Moz-Ser (4-methoxybenzyloxy)carbonyl-L-serine methylsulfonylethyloxycarbonyl Msc Msc-Cl methylsulfonylethyloxycarbonyl chloride Msc-OSu methylsulfonylethyl succinimido carbonate p-methylsulfinylbenzyl Msib MsrA methionine sulfoxide reductase A Msz 4-methylsulfinylbenzyloxycarbonyl MTBE methyl tert-butyl ether MTM methylthiomethyl Mtt methyltrityl Nboc nitrobenzyloxycarbonyl NCAs N-carboxyanhydrides NCL native chemical ligation NHC N-heterocyclic carbene NMDA N-methyl-D-asparate Npoc nitropiperonyl-oxycarbonyl Nsc 2-(4-nitrophenyl) sulfonylethoxycarbonyl Ntc 2-(4-nitrophenylthio)-ethoxycarbonyl Nvoc 2-nitroveratryloxycarbonyl OAbc p-azobenzenecarboxamidomethyl – OH hydroxide OPEC 2-oxo-2-phenylethoxycarbonyl Pac phenacyl Pacm phenylacetamidomethyl pAF p-aminophenylalanine Paoc isopropylideneaminooxycarbonyl PCL poly(ε-caprolactone) PEG polyethylene glycol PEO polyethylene oxide Pet 2-pyridylethyl Pht phthaloyl Pms p-tolylmethylsulfonyl PMSF phenylmethylsulfonyl fluoride PNA protein nucleic acid p-NB p-nitrobenzyl POC phenoxycarbonyl POE polyoxyethylene PTMSE 2-phenyl-2-trimethylsilylethyl PTMSEL (2-phenyl-2-trimethylsilyl)ethyl

xii

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

PTSA p-toluenesulfonic acid 4-pyridyldiphenylmethyl PyBzh SEM β-(trimethylsilyl)ethoxymethyl 2-(trimethylsilyl)ethoxymethyl chloride SEM-Cl SPPS solid phase peptide synthesis Tacm trimethylacetamidomethyl t-Amoc tert-amyloxycarbonyl TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl TBDPS tri(isopropyl)silyl, tert-butyldiphenylsilyl Tbfmoc tetrabenzo-[a,c,g,i]fluorenyl-17-methyloxycarbonyl t-Bu tert-butyl t-Bumeoc 1-(3,5-di-tert-butylphenyl)-1-methylethoxycarbonyl TCP tetrachlorophthaloyl TES triethylsilane TFA trifluoraceate acid Tfa trifluoroacetyl TFMSA trifluoromethanesulfonic acid Thp tetrahydropyranyl TIS triisopropylsilane TMDBB 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl Tmob 2,4,6-trimethoxybenzyl TMS trimethylsilyl TMSBr trimethylsilyl bromide TMSE 2-trimethylsilylethyl TMSI trimethylsilyl iodide TOAC 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid Tos toluenesulfonyl TPC thiophene-2-carbonyl Trityl/Trt triphenylmethyl Tsc 2-(4-trifluoromethylphenyl)-sulfonylethoxycarbonyl Xan 9-xanthenyl

Acknowledgments

Writing this book series was harder than I expected, even though I had already been through this process for my three-volume book, Comprehensive Organic Named Reactions, with Detailed Mechanism Discussions and Updated Experimental Procedures (2009, ISBN: 978-0-471-70450-8), as well as in editing the six-volume set, Encyclopedia of Physical Organic Chemistry (2017, ISBN: 978-1-118-47045-9), winner of 2018 PROSE Award for Multivolume Reference/Science. I’m eternally grateful to my wife, Xi Liu, and my daughter, Izellah, who have taken care of me so that I could focus on this book series in my spare time. It would not have been possible to complete these five books without their long-time support. A very special thanks to our librarians in the Newman Library of the University of Houston-Clear Lake, who helped me locate necessary references in a timely manner. Finally, I thank my colleagues and friends who have provided me with endless guidance.

Preface

This book, Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1: Protecting Groups, is the first installment of a series regarding amino acids and their related simple heterocyclic derivatives. The subjects contained in this book are divided into five chapters: an introduction to amino acids, the dilemma of working with α-amino acids, the carboxyl protecting groups, the amino protecting groups, and α-amino acid side chain protecting groups. The following three volumes of this book series will describe some very simple heterocyclic compounds directly formed from amino acids. Hydantoin, thiohydantoins (including 2-thiohydantoins, 4-thiohydantoins, and 2,4-dithiohydantoins), and 2,5-diketopiperazines are organized in Volume 2; α-amino acid N-carboxyanhydrides (NCA), α-amino acid N-thiocarboxyanhydrides, and sydnones are collected in Volume 3; azlactones and oxazolidin-5-ones are assembled in Volume 4. a-amino acids are a group of organic molecules with very important biological roles, forming the essential constitutional units of proteins and enzymes. Amino acids are indispensable in peptide and protein syntheses. In addition, as the majority of amino acids have at least one chiral center, the naturally occurring amino acids are de novo synthons with high optical purity, which can be utilized in organic synthesis to make other types of target molecules besides the peptides and proteins. The two adjacent amino and carboxyl groups on α-amino acids not only synergistically strengthen the acidity of both carboxy and amino groups (the conjugate acid form) but also provide the opportunity for further functionalization of these two groups with largely different reactivities. However, the opposite nature of amino and carboxyl groups leads to the transformation of amino acids into inner salts after the transfer of protons from the carboxyl group to the amino group. In addition, the carboxyl can be ionized under basic conditions, whereas the amino group will be ionized under the acidic condition, rendering a net charge to the corresponding amino acid. As a result, the charged amino acid is not likely soluble in most organic solvents, and only with certain solubility in aqueous solutions. Due to the opposite reactivity between carboxyl and amino groups, they often interfere with each other in the transformation of amino acids

xvi Preface

into their derivatives, particularly for peptides and proteins. Therefore, the protection of one group and activation of the other is a common practice in protein chemistry. Moreover, most organic reactions are performed in organic solvents, many of which are undertaken under anhydrous conditions. Therefore, it is necessary to introduce the protecting group to either amino or carboxyl group in order to temporarily cap these groups and avoid the formation of ionic species so that the corresponding amino acid derivatives of enhanced solubility in organic solvents can be smoothly transformed into the expected products under conditions analogous to traditional organic synthesis. Furthermore, among the 20 amino acids, more than 10 amino acids have ionizable side chains, which demonstrate a wide variation in acidity, polarity, nucleophilicity, and the ability to be oxidized. These side chains may also interfere with the expected reactions of the α-amino and α-carboxyl groups. While there are many examples of practices using protected amino acids without the protection of their side chains, further protection of amino acid side chains has been proved beneficial in many circumstances. Overall, the application of protecting groups during organic synthesis involving amino acids as the starting material is nearly unavoidable, and the correct selection of protecting groups often impacts the outcome of the resulting transformation of amino acids. Due to the importance of protecting groups for amino acids, nearly 260 protecting groups have been collected and organized in this book, which is further classified into different categories according to their unique reactivities. Although some protecting groups can be applied to protect both carboxyl and side-chain functional groups, or both amino and side-chain protecting groups, some protecting groups are only suitable for one or a few functional groups. It should be pointed out that the choice of protecting group not only should consider the stability of the protecting group during storage and subsequent reaction conditions but also the ease of deprotection condition should it no longer be needed. For example, during the solid-phase synthesis of protein (or peptide), the amino protecting group must be stable under a basic condition so that the carboxylate end of amino acid can be mounted onto the solid support. Meanwhile, the condition to remove the amino protecting group, which allows the amino group to further react with subsequent amino acid residue, should not impact the carboxyl side linkage and trigger the cleavage of the amino acid from the solid support. Such orthogonality of the protecting groups for carboxyl and amino groups is often required during organic synthesis using amino acid derivatives as the starting materials or intermediates.



xvii

In order to provide readers with the most comprehensive set of information possible, I typically read through all collected references, create my own abstracts and summaries on the relevant literature, and then organize these collected contents in the designated sections. The many practical experimental procedures for the introduction of these protecting groups and the corresponding deprotection procedures provided in this book have been modified and improved from the original references; some are translated from German literature published more than 100 years ago. In addition, the structures of these protecting groups have been summarized in the appendix alongside the chemical reagents used during the protections and deprotections of amino acids. The author sincerely wishes that readers will find this book very useful for their research in the areas of heterocycles, physical organic chemistry, organic synthesis, protecting group, as well as protein/peptide synthesis.

CHAPTER 1

Introduction to Amino Acids

1.1 INTRODUCTION TO CARBOXYLIC ACIDS An amino acid is an organic compound containing an amino group and a carboxyl group. This definition fits a very large number of compounds, most of which are non-physiological. However, in the field of Biochemistry, the term “amino acid” generally refers to one of the 20 organic compounds most commonly used to construct proteins, although there are still many physiologically important amino acids, such as gamma-aminobutyric acid or γ-aminobutyric acid (GABA) [1], that are often omitted. According to the nomenclature rules of the International Union of Pure and Applied Chemistry (IUPAC), the carboxyl group (COOH or CO2H) has higher nomenclature priority than most of the remaining functional groups; therefore, organic compounds that contain a carboxyl group are generally called carboxylic acids, or simply acids. Despite the fact that molecules containing amino groups can also be called amines, the lower priority of the amino group would not lead to the names of carboxyl amines for organic compounds containing both carboxyl and amino groups. The IUPAC nomenclature for carboxylic acid uses the name of the alkane corresponding to the longest continuous chain of carbon atoms within the carboxylic acid, and the final -e in the alkane name is replaced by the suffix -oic acid, indicating that this type of molecule is distinctively acidic. The chain is numbered starting from the carboxyl carbon atom to show the positions of substituents along the main chain. Notwithstanding that the IUPAC nomenclature is precise and technically clear, “trivial” or historical names and shorthand notations are frequently used in the scientific literature for the long-chain aliphatic acids (also known as fatty acids for those acids with three carbon atoms and above) [2, 3], as they are derived from the hydrolysis of fats and oils. In a few cases, the trivial names also reflect the origins of these acids. For example, propionic acid is considered to be the first fatty

2

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

acid, with its name derived from the Greek protos pion, meaning “first fat.” Butyric acid results from the oxidation of butyraldehyde, the principal flavor of butter: butyrum in Latin. Capric acid is found in the skin secretions of goats, and capric in Latin indicates anything smelling like goats. Likewise, palmitic acid originates from the Palmaceae (palm oil) family, stearic acid results from the Greek stear ‘tallow,’ i.e., animal fat, and linoleic acid comes from Latin linum (‘flax’) and oleum (‘oil’). In the trivial nomenclature system, the position of substituent on the hydrocarbon chain of fatty acid is not labeled by number but a Greek symbol. For example, the “first” carbon of the carboxylic acid is the carboxyl carbon. The next carbon is called the α-carbon, because it is in the position α to the first carbon. The remaining carbon atoms along the hydrocarbon chain are given Greek letters in the order of β, γ, δ, ε, counting away from the α-carbon, and the last carbon along the main hydrocarbon chain is labeled as a “ω” which is the last letter in the Greek alphabet. Due to the presence of the polar carboxyl group, which can form a hydrogen bond with water, the carboxylic acid has higher solubility in water than the corresponding hydrocarbon of the same carbon numbers, and formic acid to butyric acid are all miscible with water. However, as the hydrocarbon chain increases, the solubility of respective fatty acids in water swiftly decreases, because the influence of the carboxyl group within the whole molecule reduces. Interestingly, the solubility of fatty acids in organic solvents, such as cyclohexane, also decreases as the hydrocarbon chain increase [4]. Some important physical properties of the common saturated fatty acids are shown in Table 1.1. Notably, the majority of natural fatty acids contain an even number of carbon atoms. As oxygen has a greater electronegativity than that of hydrogen (3.5 versus 2.1), the hydroxyl group, connecting to an electron-withdrawing carbonyl group in carboxylic acid, has a higher attendance to dissociate its proton than that in water, causing a generally stronger acidity of fatty acid with respect to water. Thus, fatty acids once dissolved in water consist of two types of carboxylic acids, the free carboxylic acid and the ionized carboxylate, as shown in Scheme 1.1. Ka is known as the acidity constant or dissociation constant, and the pKa value is defined as the pH value of the aqueous carboxylic acid solution at which 50% of hydrogen atoms are removed from the carboxyl group by the existing hydroxide (OH–) in the solution. This is easily approved with a series of equations (Eqs. (1)–(6)), which lead to the general Henderson-Hasselbalch equation (Eq. (7)) that describes the relationship between the pH scale, the ratio for the concentration of dissociated

Systematic Name

Trivial Name

Carbon Numbers

MP (°C)

Solubility in Water (g/100 g)b

Solubility in Cyclohexane (g/100 g) [4]

pKa

Propanoic acid

Propionic acid

3

–21

∞c

–

4.88 [5]

Butanoic acid

Butyric acid

4

–7.9 [6]

∞

–

4.82 [5]

Pentanoic acid

Valeric acid

5

–33.6 [7]

4.97

–

4.83 [8]

Hexanoic acid

Caproic acid

6

–3.5 [9]

1.019 [10]

–

4.85

Heptanoic acid

Enanthic acid

7

–7.5 [7]

0.24 [7]

–

–

Octanoic acid

Caprylic acid

8

16.2 [9]

0.079 [10]

∞

4.9 [11, 12]

Nonanoic acid

Pelargonic acid

9

12.4 [7]

0.032 [10]

∞

4.9 [13]

Decanoic acid

Capric acid

10

30.4 [9]

0.018 [10]

7,600

5.3

Dodecanoic acid

Lauric acid

12

44.2 [9]

0.0063 [10]

215

5.31 [14]

Tetradecanoic acid

Myristic acid

14

54.4 [9]

0.0024 [10]

72

palmitic > myristic > lauric acid [19]. To an extreme extent, the acidity of long fatty acids has been identified much weak, with a pKa value often greater than 8.0 [16]. When a carboxylic acid is converted into carboxylate, the sodium or potassium salt of carboxylic acid has a relatively higher solubility in water than the neutral form of carboxylic acid. This is because sodium or potassium carboxylate is much easier to dissociate than carboxylic acid, and the resulting carboxylate is hydrated with water. For example, the potassium salt of nonanoic acid has a minimal solubility of 18 g/100 mL in water at 90°C, which is even possibly miscible with water [20]; whereas the solubility of nonanoic acid in water was reported at 0.051 g per 100 grams of water at 60°C, and 0.032 g/100 g water at 30°C [10]. It is conceivable that the solubility of nonanoic acid will not be as large as 18 g/100 mL at 90°C, in comparison to potassium nonanate. Likewise, the corresponding sodium salt of nonanoic acid is reported to form a complex solution that contains the free ionic (C8H17COO–) and neutral molecule (nonanoic acid), as well as ionic and neutral micelles [20]. This is because sodium cation is smaller than potassium cation, so the columbic interaction between the carboxylate and sodium cation is stronger than that in the corresponding potassium salt, according to Eq. (8). In Eq. (8), Q1 and Q2 are the valent charges of cation and anion, r is the average distance between the cation and anion, and ε is the dielectric constant of solvent. Obviously, the potassium salt of fatty acid, with a larger diameter for potassium cation, has a smaller columbic force between the cation and anion, and is more likely to dissociate than the corresponding sodium salt. For an even longer fatty acid, the solubility of corresponding salt in water is still greater than the neutral carboxylic acid. For example, although arachidonic acid can be dissolved in either ethanol, dimethyl sulfoxide (DMSO), or dimethylformamide at a level of 10 g/100

Introduction to Amino Acids 7

mL solvent, or in chloroform and methanol with an upper limit of 5 g/100 mL solvent, arachidonic acid is insoluble in water [21]. However, sodium arachidonate has a solubility of around 0.25 g/100 mL, or 1 mg of arachidonic acid per 400 microliter of aqueous buffer at pH 8 [22].

F=

Q1Q2 (8) ε r2

Overall, for a particular carboxylic acid, the relative ratio of the free carboxylic acid and carboxylate in water depends on temperature, pKa value of the carboxylic group, pH of the solution, as well as other factors, such as ionic strength, the presence of surfactants, etc. For example, the solubility of fatty acids in water can be improved in the presence of surfactants [23]. In the presence of surfactant, it is found that the pKa of lauric acid increases in the order of cationic < neutral < anionic surfactant [24]. According to Scheme 1.1, for the aqueous solution of carboxylic acid, the addition of certain amount of base will shift the equilibrium to form more carboxylate which functions as the conjugate base, then a pH buffering system can form, if the ratio of carboxylate over free carboxylic acid is more than 0.1 or less than 10. In other words, the mixture of a weak acid and its conjugate base can form a pH buffer that resists the change of pH about 1 pH unit away from its pKa when additional acid or base is added to the buffering system, i.e., the working range of a pH buffer is: pKa –1 < pH < pKa +1. When acid is added to the pH buffered solution, carboxylate will couple with additionally added proton to form neutral carboxylic acid to counteract the variation of proton concentration. For comparison, when a base is added to such solution, the existing carboxylic acid will react with the added hydroxide to form more carboxylate to balance the proton concentration as well. Therefore, a pH buffer with the highest buffering capacity is when the concentration of acid equals that of the conjugate base. Otherwise, when acid concentration is more than 10 times the concentration of conjugate base, there will not be enough conjugate base to counteract with the added acid, or vice versa. Besides the saturated fatty acids listed in Table 1.1, there are also many unsaturated fatty acids, as listed in Table 1.2. For these unsaturated fatty acids, the extra-functional groups are the carbon-carbon double bonds, which in most cases are in cis-configurations among the natural fatty acids. As illustrated in Figure 1.1, the big difference between a saturated fatty acid and an unsaturated fatty acid with a cis-double bond is that the saturated fatty acid can be packed tightly in space, rendering a stronger intermolecular

8

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

force and a higher melting point than that of unsaturated fatty acids in cisconfiguration (case ‘a’ in Figure 1.1); whilst the unsaturated fatty acid, due to the spatial requirement of the cis-double bond, cannot be filled in the same space like the saturated one, resulting in a relatively lower melting point (case ‘b’ in Figure 1.1). Obviously, the unsaturated fatty acid having a trans-double bond would behave just like the saturated counterpart, with a relatively high melting point (similar to case ‘a’ in Figure 1.1). This is the reason when these fatty acids are esterified with glycerol to form triacylglycerides (fats and oils), fats have a higher percentage of long and saturated fatty acids, whereas oils have more content of unsaturated and short fatty acids. For example, olive oil containing approximately 72% oleic acid [25] has a melting point of –6°C, whereas beef fat with less content of unsaturated fatty acids forms solid or semi-solid at room temperature. In addition, to enhance the advantage of cis-double bond that lowers the melting point of unsaturated fatty acids, the additional cis-double bonds are usually not conjugated to the existing double bond, and often are isolated with a methylene group (CH2). This is because when all the cis-double bonds are conjugated together, the resulting hydrocarbon chains would be packed in a manner similar to that of saturated fatty acids or unsaturated fatty acids with a trans-double bond, as illustrated in case ‘c’ of Figure 1.1. It should be pointed out that the fatty acids with all conjugated trans-double bonds behave like the ones of all conjugated cis-double bonds. From Table 1.2, it is clear that the effect of lowering the melting points of fatty acids by a cis-double bond is more apparent when such double bond is located in the middle of the molecule. In contrast, when the cis-double bond is close to the terminus of fatty acid, the melting point will not be affected to the same level. For example, the melting point of stearic acid is 68.8°C, whereas the melting points for petroselinic acid and oleic acid are 30°C and 13.4°C, respectively. These three fatty acids all contain 18 carbon atoms. However, stearic acid is a saturated acid, petroselinic acid has a cisdouble bond more closer to the carboxyl group with a melting point lower by 38.8°C from stearic acid, but oleic acid containing a cis-double bond right in the middle has a melting point of only 13.4°C. Additionally, when more cis-double bonds are introduced into fatty acids, the effect introduced by additional cis-double bonds reduces gradually, as shown by the melting points of stearic acid (68.8°C), oleic acid (13.4°C), linoleic acid (–7°C) and α-linoleic acid (–11°C).

Introduction to Amino Acids 9

COOH COOH COOH

COOH

COOH

a

COOH

COOH

COOH

COOH

COOH

c

b

FIGURE 1.1 Possible structural effect on melting point of fatty acids.

TABLE 1.2 The General Information About Unsaturated Carboxylic Acidsa Systematic Name

Trivial Name

Designation

MP (°C)

Solubility pKa in Water (g/100 g)

cis-4-Decanoic acid

Obtusilic acid

10:1(n–6)

–

–

–

cis-9-Decenoic acid

Caproleic acid

10:1(n–1)

26.5 [7]

–

4.78

cis-5-Lauroleic acid

Lauroleic acid

12:1(n–7)

–

–

–

cis-4-Dodecenoic acid

Linderic acid

12:1(n–8)

–

–

–

cis-9-Tetradecenoic acid

Myristoleic acid

14:1(n–5)

–

–

–

cis-5-Tetradecenoic acid

Physeteric acid

14:1(n–9)

–

–

–

cis-4-Tetradecenoic acid

Tsuzuic acid

14:1(n–10)

–

–

–

cis-9-Hexadecenoic acid

Palmitoleic acid

16:1(n–7)

0.5 [7]

–

–

cis-6-Octadecenoic acid

Petroselinic acid

18:1(n–12)

30

–

–

10

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

TABLE 1.2 (Continued) Systematic Name

Trivial Name

Designation

MP (°C)

Solubility pKa in Water (g/100 g)

cis-9-Octadecenoic acid

Oleic acid

18:1(n–9)

13.4 [7]

–

5.02 [26] 9.85 [16]

trans-9-Octadecenoic acid

Elaidic acid

tr18:1(n–9)

44 [7]

–

9.95 [16]

trans-11-Octadecenoic acid

Vaccenic (asclepic) acid

tr18:1(n–7)

44 [7]

–

–

cis-9-Eicosenoic acid

Gadoleic acid

20:1(n–11)

25

–

–

cis-11-Eicosenoic acid

Gondoic acid

20:1(n–9)

–

–

–

cis-11-Docosenoic acid

Cetoleic acid

22:1(n–11)

–

–

–

cis-13-Docosenoic acid

Erucic acid

22:1(n–9)

34.7 [7]

–

–

trans-13-Docosenoic acid

Brassidic acid

tr22:1(n–9)

61.9 [7]

–

–

cis-15-Tetracosenoic acid

Nervonic acid

24:1(n–9)

43 [7]

–

–

9,12-Octadecadienoic acid

Linoleic acid

18:2(n–6)

–7 [7]

5

9.24 [16]

9,12,15-Octadecatrienoic acid

α-Linolenic acid

18:3(n–6)

–11 [7]

–

8.28 [16]

6,9,12-Octadecatrienoic acid

γ-Linolenic acid

18:3(n–6)

–

–

–

8,11,14-Eicosatrienoic acid

Dihomo-γlinolenic acid

20:3(n–6)

–

–

–

6,9,12,15Octadecatetraenoic acid

Stearidonic acid

18:4(n–3)

–57

–

–

5,8,11,14-Eicosatetraenoic acid

Arachidonic acid

20:4(n–6)

–49.5 [7]

–

4.752

a The melting points and pKa values of listed acids are obtained from commercial catalogs, TOXNET or Wikipedia if not cited.

1.2 INTRODUCTION TO ALIPHATIC AMINES In contrast to carboxylic acid, aliphatic amines are distinctively basic, due to their amino groups. Similar to hydroxyl group and carboxyl group, amino group can also form a hydrogen bond with water, so that aliphatic amines of

Introduction to Amino Acids 11

short hydrocarbon chains (up to 5 carbon atoms) are also miscible with water [27]. As the hydrocarbon chain increase further, the solubility of amines in water decreases dramatically, as shown by the representative amines listed in Table 1.3. For amines, once partially protonated, the mixture of ammonium and free amine can form a pH buffering system in water as well. TABLE 1.3 Some Properties of Common Amines [7] Name

Melting Point (°C)

Boiling Point (°C)

Density (g/mL)

Solubility in Water (g/100 mL)

pKa

Ethylamine

–80.5

16.5

0.677

∞

10.65

Propylamine

–84.75

47.22

0.7173

∞

10.54

Butylamine

–49.1

77.0

0.7414

∞

10.60

Pentylamine

–55

104.3

0.7544

∞

10.63

Hexylamine

–22.9

132.8

0.7660

0.97(30°C) [27]

10.56

Heptylamine

–18

156

0.7754

0.30(30°C) [27]

10.67

Pyrrolidine

–57.79

86.56

0.8586

∞

11.31

Imidazole

89.5

257

1.0303

66.3 6.8 (pH 10.5)

6.99

Note: Pyrrolidine and imidazole are included here for the purpose of comparison with amino acid proline and histidine later on.

1.3 INTRODUCTION TO GENERAL AMINO ACIDS When both amino and carboxyl groups coexist in a single molecule, many physical properties of the amino acid are different from the respective properties of either amine or carboxylic acid of the same hydrocarbon chain. This is because a proton can transfer from the carboxyl group to the nearby amino group to form an ionized carboxylate and ammonium functionality, so that the amino acid exists as a zwitterion or inner salt. In general, the amino group can function as a weak base, and the conjugate acid of amine, i.e., the ammonium salt is a weak acid; likewise, the carboxyl group can function as a weak acid, and the corresponding carboxylate is a weak base. Therefore, amino acids generally have two pH buffering ranges. In an acidic solution, the dissociation of the carboxyl group is depressed, and the amino group is protonated, so the cationic group (-NH3+) sitting close to the carboxyl group will have a strong inductive effect, resulting in an even stronger acidity for the carboxyl group within the amino acid than that of simple fatty acid. Likewise, the

12

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

carboxyl group is also generally considered as an electron-withdrawing group, rendering the nearby protonated amino group a lower pKa value in comparison to normal aliphatic amine in Table 1.3. However, the carboxylate group should have less inductive effect on the protonated amino group than that of the free carboxyl group, due to its negative charge. The general reaction pattern for amino acids with base is illustrated in Scheme 1.2. In addition, the magnitude of mutual influence on the acidity of both amino group and carboxyl group depends on the distance between the amino group and carboxyl group along the hydrocarbon chain. Such effect is most apparently displayed in α-amino acids, as the inductive effect is generally limited to up to three carbon atoms. Table 1.4 shows the pKa values of carboxyl group and amino group of amino acids with different distances between the amino and carboxyl groups, from which the inductive effect is noticeable for more than five carbon atoms away. For example, the pKa of acetic acid is 4.76, but the pKa of the carboxyl group in 7-amino-heptanoic acid is 4.502, a value slightly smaller than that of acetic acid. According to the pKa of fatty acids listed in Table 1.1, the pKa of the carboxyl group of 7-amino-heptanoic acid is predicted to be between 4.85 and 4.9, provided that there is no inductive effect from the distal amino group at all. This might be the reason that α-amino acids are more important than many other types of amino acids. The importance of α-amino acids also explains the fact that the amino acids that constitute proteins are all α-amino acids.

COOH H 3N H R Net Charge: +1

OH Ka1

COO H3 N H R 0

OH Ka2

COO H2 N H R -1

SCHEME 1.2 The titration pattern for a general amino acid. TABLE 1.4 The pKa Values Different Amino Acids Acid

Structure

pKa (COOH)

pKa (NH2)

Acetic acid

4.76 [28]

~

Glycine

2.34 [28]

9.6 [28]

β-Alanine

3.60 [28]

10.19 [28]

Introduction to Amino Acids 13

TABLE 1.4 (Continued) Acid

Structure

pKa (COOH)

pKa (NH2)

γ-Aminobutyric acid

4.031 [29] 10.556 4.23 [30] [29]

δ-Aminovaleric acid

4.27 [28]

6-NH2hexanoic acid

4.373 [29] 10.804 4.43 [31] [29]

7-NH2heptanoic acid

4.502 [29] –

10.43 [30] –

10.75 [31]

1.4 THE COMMON α-AMINO ACIDS THAT CONSTRUCT PROTEINS 1.4.1 THE COMMON α-AMINO ACIDS Even if only α-amino acids are considered, there would still be many types of amino acids. However, it had been generally accepted that only 20 α-amino acids are commonly used to construct proteins, for which the names were given by their discoverers and bear no relationship to their chemical structures [32]. All these 20 amino acids share a common ground that all have a direct connection with the genetic codes. Genetic codes are the sequence of nucleotides in nucleic acids. There are only four nucleotides in DNA (i.e., A, C, G, and T), as well as in RNA (i.e., A, C, G, U). During protein synthesis, the genetic code in DNA is kept in RNA via transcription, and specific amino acids will be brought into the ribosome by tRNA to construct protein, according to the genetic code on mRNA. If three consecutive nucleotide sequences in DNA can determine one particular amino acid, then there would be 64 (=43) different triplet combinations of the nucleotide sequences. However, excluding the three termination codons (TAA, TAG, and TGA), there are only 61 sense codons, for which several amino acids among the 20 α-amino acids have been connected to multiple codons, as summarized in Table 1.5. Recently, the riddle that has persisted for a while as to why only 20 amino acids were selected for ribosomal protein synthesis has been resolved as two more amino acids have been identified that share the common features of the 20 canonical amino acids. These two amino acids

14

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

are selenocysteine [33–37] and pyrrolysine [38–41]. For this triplet codon relationship, AUG codon has dual senses: initiation of protein synthesis and methionine insertion in the internal protein position. Likewise, TGA codon is used for the termination of protein synthesis as well as internal selenocysteine insertion; although TAG also indicates the termination of protein synthesis, it alternatively represents the internal site to insert pyrrolysine. Although it has not been generally accepted, p-aminophenylalanine (pAF) has also been claimed as the 21st amino acid for the reasons that it can be synthesized from simple carbon sources by bacteria, an aminoacyl-tRNA synthetase uniquely utilizes this amino acid and a tRNA efficiently delivers pAF into proteins in response to the amber codon, TAG [42]. It should be pointed out that besides these canonical amino acids, over 120 additional amino acids have been identified in natural proteins [43], which are incorporated into proteins by post-translational modification of the canonical amino acids of ribosomally made proteins [44]. The structures of the 22 α-amino acids (Table 1.6) are shown in Figure 1.2. TABLE 1.5 The Relationship between the Genetic Code and 22 α-Amino Acids Second Letter C

TTT TTC T

A

TCT Phe

TTA

TAT

TCC TCA

TAC Ser

A

Stop Pyl [39]

TGG

Trp

G

First Letter

CTT

CCT

CAT

CGT

T

His CCC Leu

CAC

CGC

Pro CCA

C Arg

CAA

CGA

A

CGG

G

Gln

CTG

CCG

CAG

ATT

ACT

AAT

AGT Asn

Ile

ATA ATG

C Stop Sec [34]

TAG

ATC

TGC

T Cys

TGA

TCG

CTC

A

TGT

Stop

TTG

CTA

Tyr

TAA

Leu

C

G

ACC ACA

Thr

AAC

AGC

AAA

ACG

AAG

C

AGA Lys

Met

T Ser A Arg

AGG

G

Third Letter

T

Introduction to Amino Acids 15

TABLE 1.5 (Continued) Second Letter C

GTT GTC G

GTA

A

GCT

G

GAT

GGT

T

Asp GCC

Val

GAC

GGC

C

Ala GCA

Gly GAA

GGA

A

GGG

G

Glu

GTG

GCG

GAG

Third Letter

First Letter

T

TABLE 1.6 General Properties of 22 Common α-Amino Acids [45] Amino Acid

Abbreviation

Symbol

pKa1

pKa2

pKaR

pIa

9.60

–

5.97

MP (°C) [7]

Solubility (g/100 g) [7]

290d

25.09

Amino Acids with Non-Polar Side Chains Glycine

Gly

G

2.34

b

Alanine

Ala

A

2.34

9.69

–

6.02

297d

16.50

Valine

Val

V

2.32

9.61

–

5.97

315

8.85

Leucine

Leu

L

2.36

9.60

–

5.98

293

2.20

Isoleucine

Ile

I

2.35

9.68

–

6.02

284d

3.42

Proline

Pro

P

1.99

10.96

–

6.48

221d

16.23

Methionine

Met

M

2.28

9.20

–

5.74

281d

5.6

8.80

–

5.41

235

2.51

Amino Acids with Polar Side Chains Asparagine

Asn

N

2.01

c

Cysteine

Cys

C

1.96

10.29

8.18

5.07

240d

–

Glutamine

Gln

Q

2.17

9.13

–

5.65

185d

4.2

Selenocysteine

Sec

U

–

–

5 . 2 [37] 5.47 [36]

–

–

39.2 [46]

Serine

Ser

S

2.21

9.15

13.60

5.68

228d

5.02

Threonine

Thr

T

2.11

9.62

13.60

5.87

256d

9.81

Amino Acids with Aromatic Side Chains Histidine

His

H

1.80

9.17

6.00

7.59

287d

4.35

Phenylalanine

Phe

F

1.83

9.12

–

5.48

283d

2.79

Tryptophan

Trp

W

2.38

9.39

–

5.89

289d

1.32

16

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

TABLE 1.6 (Continued) Amino Acid

Abbreviation

Symbol

pKa1

pKa2

pKaR

pIa

MP (°C) [7]

Solubility (g/100 g) [7]

Tyrosine

Tyr

Y

2.20

9.11

10.06

5.66

343d

0.046

Amino Acids with Positively Charged Side Chains Arginine

Arg

R

2.17

9.04

12.48

10.76

244d

18.26

Lysine

Lys

K

2.18

8.95

10.52

9.74

224d

0.58

Pyrrolysine

Pyl [41]

–

–

–

–

–

–

–

Amino Acids with Negatively Charged Side Chains Aspartic acid

Asp

D

1.89

9.60

3.65

2.77

270

0.495

Glutamic acid

Glu

E

2.19

9.67

4.25

3.22

160d

0.861

Calculated from pKa values. d stands for decomposition temperature of the specific amino acid. c pKa2 and pKaR are swapped from the Merck Index after verification with other databases. a

b

Amino Acids with Non-polar Side Chains O O O H 2N

OH OH

L-alanine

OH

O OH

OH NH L-proline

NH2

L-isoleucine

L-methionine

Amino Acids with Polar Side Chains O O HS

OH O

L-leucine

O S

NH2

H2N

NH2

L-valine

O

O

OH

OH NH2

NH2

glycine

O

OH

O

H2N

OH

NH2

NH2

L-asparagine

NH2

L-cysteine O

HSe

L-glutamine

O OH

NH2 L-selenocysteine

HO

OH O OH

NH2 L-serine

OH NH2 L-threonine

FIGURE 1.2 The 22 basic α-amino acids for the construction of proteins.

Introduction to Amino Acids 17

Amino Acids with Aromatic Side Chains H N O

O N

NH2

NH2 L-histidine

OH

OH

OH

HN

O

NH2

L-phenylalanine

O L-tryptophan

OH

NH2

HO

L-tyrosine

Amino Acids with Positively Charged Side Chains NH H2N

O

N H

O OH

H2 N

NH2

O OH

OH

N

NH2

L-arginine

O

L-lysine

HN

NH2

L-pyrrolysine

Amino Acids with Negatively Charged Side Chains O O O HO OH HO OH NH2 O NH2 L-aspartic acid

L-glutamic acid

FIGURE 1.2 (Continued).

1.4.2 STEREOCHEMISTRY OF α-AMINO ACIDS As shown in Figure 1.2, all the common α-amino acids except for glycine have at least one chiral center, as their “α-carbon” is bonded to four different groups: an amino group, a carboxyl group, hydrogen, and the side-chain. Regarding the stereochemistry of α-amino acids, the CahnIngold-Prelog’s R/S nomenclature commonly used in organic chemistry is not used, whilst the traditional D/L nomenclature dominates the current literature and everyday laboratory conversation. This system has been adopted from the one that was initially developed by Fischer and Rosanoff to describe the structure of carbohydrates, the so-called Fischer projection. In this system, glyceraldehyde was chosen as the reference molecule, for which (+)-glyceraldehyde was assigned to be in D-configuration because

18

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

the OH group attached to the C-2 of such molecule was initially assumed to be on the right-hand side of the hydrocarbon chain (dexter in Latin) in its correct Fischer projection, in which the CHO group appears at the top, and the hydroxymethyl group sits at the bottom. This arbitrary arrangement for the configuration of (+)-glyceraldehyde had been confirmed about 50 years later with X-ray crystallography. In comparison, the enantiomeric (-)-glyceraldehyde was defined in L-configuration because the OH group is on the left-hand side of the hydrocarbon chain (laevus in Latin). It should be pointed out that the vertical line in the Fischer projection is going back behind the plane of the page, whereas the horizontal line is coming out of the plane of the page.

FIGURE 1.3 The illustration of Fischer projection for the stereochemistry of α-amino acid.

When this notation is extended to α-amino acids, the carboxyl group will appear at the top, the side chain sits at the bottom, and the amino group and hydrogen reside at the horizontal positions. L-α-amino acids are those enantiomers in which the NH2 group is on the left-hand side of the Fischer projection. Conversely, the D-α-amino acids are those in which the NH2 group is on the right-hand side of the Fischer projection. The D/L-configuration of amino acid is illustrated in Figure 1.3. However, when the configuration of α-amino acid is unknown, then the Greek symbol of ξ is used in front of the amino acid name [47]. Interestingly, physiological amino acids are almost exclusively in L-configurations. It should be pointed out that the symbols D and L do not relate to the sign of rotation of an optically active molecule which is designated (+)- (or

Introduction to Amino Acids 19

d) and (-)- (or l), which indicates the direction in optical rotation (clockwise for +). 1.4.3 AMPHIPROTIC PROPERTIES OF α-AMINO ACIDS The amino and carboxyl functional groups found in α-amino acids allow the amino acids to have amphiprotic properties, for that the carboxyl groups (–CO2H) can be deprotonated to become carboxylates (–CO2–) with negative charges, and α-amino groups (NH2–) can be protonated to become positive α-ammonium groups (+NH3–). According to the pKa values in Table 1.4, the averaged pKa value for the carboxyl group is about 2.15 ± 0.15, and the amino group has an average of 9.47 ± 0.36 for pKa2. When the pH of the aqueous α-amino acid solution is less than 2.0, the neutral carboxyl group predominates and the amino group is fully protonated with a positive charge, according to the Henderson-Hasselbalch equation (eq. 7). Thus, the net charge of amino acid is +1. Conversely, when the pH of this solution is adjusted to above 9.5 with extra base, the carboxyl group will be completely deprotonated to carboxylate, and the neutral amino group will predominate, then the overall charge of α-amino acid will be –1. However, at pH between 2.2 and 9.5, the predominant form adopted by α-amino acid contains a negative carboxylate and a positive α-ammonium group, as shown by the middle structure of Scheme 1.2, though it coexists in dynamic equilibrium with small amounts of net negative and net positive ions. When the pH equals the average of pKa1 and pKa2 of α-amino acid that does not contain any ionizable side chain, this amino acid has net-zero charges, even though other forms of α-amino acid might still exist in small content which will cancel out the charges. This molecular state is known as a zwitterion, and the pH is known as the isoelectric point, pI. For those α-amino acids with a positively charged side chain, including arginine, histidine, lysine, and pyrrolysine, their pI values equal the average of pKa2 and pKaR, due to the contribution of charge from these side chains. Likewise, for those α-amino acids with negatively charged side chains (e.g., aspartic acid, glutamic acid, cysteine, and selenocysteine), their pIs are calculated by ½(pKa1 + pKaR). For the rest of amino acids with negatively charged side chains such as serine, threonine, and tyrosine, their pI values are calculated in a way similar to normal α-amino acids without ionizable side chains, as the pKaR of these α-amino acids are greater than that of pKa2 so that they will be deprotonated after ammonium group dissociates, so they do not contribute to pI values.

20

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

The isoelectric point is very important for an α-amino acid because α-amino acid has zero mobility in electrophoresis at this pH value, as well as minimum solubility in water. Thus, some amino acids (in particular, with non-polar side-chains) can be isolated by precipitation from water via adjusting the pH of the solution to the required isoelectric point. It should be pointed out that amino acids also exist as zwitterions in the solid phase. 1.4.4 ESSENTIAL AMINO ACIDS AND NONESSENTIAL AMINO ACIDS While all 20 common α-amino acids exist in the human body, their concentrations are different. Some amino acids can be synthesized de nono by the organism, or transformed from other molecules. These amino acids are known as nonessential amino acids. However, some other amino acids that either cannot be synthesized inside the human body due to the lack of synthetic pathway or cannot be synthesized at a sufficient rate to meet the metabolic need, they must be obtained by animals in the diet. These amino acids are called essential amino acids or indispensable amino acids. There are a total of nine amino acids that are essential for all vertebrates, including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine [48]. However, some vertebrates have extra essential amino acids. Also, some nonessential amino acids might become essential amino acids under certain conditions. These amino acids are also known as semi-essential or conditionally essential amino acids, such as arginine [49]. 1.4.5 UNNATURAL AMINO ACIDS It is often intriguing why nature only chooses 20 common amino acids in most cases, or possibly up to 22 amino acids as shown in Table 1.6 to form a variety type of proteins. What is the relationship between the genetic code and amino acids? In general, there are two theories that can reasonably explain such relationship, i.e., affinity matching [50] and code coevolution [51]. It is hypothesized in affinity matching theory that the relationship between genetic code and amino acids is established on the basis of stereochemical affinity matching between an amino acid and certain triplet sequences, for that the genetic code was developed in a way that was very closely connected to the development of amino acid repertoire. This close biochemical connection is fundamental to specific protein-nucleic acid interactions. In code coevolution theory, it is hypothesized that canonical genetic code evolved

Introduction to Amino Acids 21

from a simpler primordial form that only encoded fewer amino acids. Then the genetic code coevolved with the invention of biosynthetic pathways for new amino acids. After arranging a periodic table of codons and amino acids, it is found out that the central nucleotide in the triplet codons is important, for which purines control the charge while pyrimidines determine the polarity of the amino acids [52]. Still, it is hard to understand that the function of a protein can be fully demonstrated by these limited numbers of amino acids. As a matter of fact, many other amino acids have been identified in proteins, which are generally called unnatural or non-natural amino acids. Often these unnatural amino acids are found at a specific site within proteins. In order to study the structure and function of proteins, some unnatural amino acids have been purposely inserted into a specific site of proteins. As of today, many methods have been known to incorporate unnatural amino acids into proteins, including reassignment of sense (residue-specific incorporation) [53], non-sense suppression (site-specific incorporation) [54], and extended codons and frameshift suppression [55]. Although both the reassignment of sense and non-sense suppression methods have successfully altered the protein functionalities, and over 100 different unnatural residues have been introduced into proteins using the non-sense suppression method [56], these two methods are limited to incorporate a single unnatural amino acid into the recombinant protein [57]. This is because the genetic code only contains three stop codons, which limits the theoretical numbers of different unnatural amino acids that can be incorporated in a single protein. Conversely, in the extended codons and frameshift suppression approach, more than one unnatural amino acid can be inserted into proteins. In this approach, an mRNA containing an extended codon consisting of four or five bases is read by a modified aa-tRNA (acylated with unnatural amino acid and containing the corresponding extended anticodon) to obtain a full-length protein that contains an unnatural amino acid at the specific site. However, if the extended codon is read as a threebase codon by an endogenous tRNA, the reading frame will be shifted by one base. This will eventually result in a premature encounter with a stop codon and early termination of protein synthesis. As of today, several four-base codons (AGGU, CGGU, CCCU, CUCU, and GGGU) have been successfully read by the corresponding tRNAs [58], and up to 16 different mRNAs, each containing one of the five-base codons CGGN1N2 (N1 and N2 indicate one of the four bases, e.g., CGGUA, and CGGUG), have been decoded by aa-tRNAs with complementary five base anticodons to produce proteins, each containing an unnatural amino acid [59–61].

22

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Also, many unnatural amino acids found in proteins can also be formed via post-translational modification, which is one of the later steps in protein biosynthesis for many proteins. These post-translational processes include phosphorylation [62], acylation [63], thio-acylation [64], O-glycosylation, and N-glycosylation [65], oxidation [66], methylation [67], vitamin K-dependent carboxylation [68], sulfation [69], ADP-ribosylation [69], hydroxylation [69], prenylation [69], etc. 1.4.6 D-α-AMINO ACIDS Although the majority of α-amino acids belong to L-enantiomers, some D-α-amino acids do exist and also have important biological roles. The D-α-amino acid was first reported in 1939 by Kogl for D-glutamic acid that was isolated from the ovarian tumor [70]. The D-amino acids have a role in defense mechanisms, and have been detected in a variety of peptides synthesized by animal cells, such as the peptide of Tyr-Xaa-Phe, where Xaa could be D-alanine, D-methionine, or D-leucine in South American tree frog Phyllomedusa sauvagei [71]; Gly-D-Phe-Ala-Asp in the ganglia and atrium of African snail Achatina fulica [72]; occurrence of D-amino acids in acid hydrolysates of cells of Lactobacillus arabinosus, Bacillus brevis, Bacillus subtilis (BS2), Torulopsis utilis, and mycelia of Penicillium chrysogenum [73]. Also, D-amino acids occur in fungi [74]; microalgae and macroalgae [75]; higher plants [76]; marine invertebrates [77]; and mammals [78]. The D-amino acid-containing compounds have important applications in antibiotics, such as ampicillin, amoxicillin, cephalexin, cephadroxil, etc. [79]. KEYWORDS • • • •

amino acids unnatural amino acid D-amino acid Fischer Projection

• solubility

Introduction to Amino Acids 23

REFERENCES 1.

2. 3. 4. 5. 6.

7. 8.

9.

10. 11.

12. 13.

14. 15.

Olsen, R. W., (2002). GABA. In: Davis, K. L., Charney, D., Coyle, J. T., & Nemeroff, C., (eds.), Neuropsychopharmacology: The Fifth Generation of Progress (pp. 159–168). American College of Neuropsychopharmacology. Kalish, B. T., Fallon, E. M., & Puder, M., (2012). A tutorial on fatty acid biology. J Parenter Enteral Nutr., 5, 0148607112449650. Davidson, B. C., & Cantrill, R. C., (1985). Fatty acid nomenclature. South Afr. Med. J., 67, 633–643. Hoerr, C. W., & Ralston, A. W., (1944). The solubilities of the normal saturated fatty acids, II. J. Org. Chem., 9(4), 329–337. Harding, A. P., Wedge, D. C., & Popelier, P. L. A., (2009). pKa prediction from “quantum chemical topology” descriptors. J. Chem. Inf. Model., 49(8), 1914–1924. Dennis, S. C., Belcher, M., Dawson, T., Delaney, B., Fine, J., Flickinger, B., Friedman, P., et al., (2006). AOCS Resource Directory. Institute of Shortening and Edible Oils: New York. Lide, D. R., (2005). Properties of fatty acids. In: CRC Handbook of Chemistry and Physics. CRC Press LLC. Tao, L., Han, J., & Tao, F. M., (2008). Correlations and predictions of carboxylic acid pKa values using intermolecular structure and properties of hydrogen-bonded complexes. J. Phys. Chem. A, 112(4), 775–782. Privett, O. S., Breault, E., Covell, J. B., Norcia, L. N., & Lundberg, W. O., (1958). Solubilities of fatty acids and derivatives in acetone. J. Am. Oil Chemist Soc., 35, 366–370. Ralston, A. W., & Hoerr, C. W., (1942). The solubilities of the normal saturated fatty acids. J. Org. Chem., 7(6), 546–555. Lenne, P. F., Bonosi, F., Renault, A., Bellet-Amalric, E., Legrand, J. F., Petit, J. M., Rieutord, F., & Berge, B., (2000). Growth of two-dimensional solids in alcohol monolayers in the presence of soluble amphiphilic molecules. Langmuir, 16(5), 2306–2310. Ang, W. S., & Elimelech, M., (2008). Fatty acid fouling of reverse osmosis membranes: Implications for wastewater reclamation. Water Research, 42(16), 4393–4403. Nonanoic-Acid, Directive 98/8/EC Concerning the Placing Biocidal Products on the Market, (2004). Table 8.1: pKA values of organic materials in water at 25°C. in Section 8.49: Electrolytes, electromotive force, and chemical equilibrium. In: Gokel, & George W., (eds.), Dean’s Handbook of Organic Chemistry (2nd Edn., p. 851). McGraw-Hill, New York, ISBN: 0-07-137593-7. Nyren, V., & Back, E., (1958). The ionization constant, solubility product and solubility of lauric and myristic acid. Acta Chem. Scand., 12, 1305–1311. Patton, J. S., Stone, B., Papa, C., Abramowitz, R., & Yalkowsky, S. H., (1984). Solubility of fatty acids and other hydrophobic molecules in liquid trioleoylglycerol. Journal of Lipid Research, 25, 189–197.

24

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

16. Kanicky, J. R., & Shah, D. O., (2002). Effect of degree, type, and position of unsaturation on the pKa of long-chain fatty acids. Journal of Colloid and Interface Science, 256, 201–207. 17. Voelgyi, G., Baka, E., Box, K. J., Comer, J. E. A., & Takacs-Novak, K., (2010). Study of pH-dependent solubility of organic bases. Revisit of Henderson-Hasselbalch relationship Anal. Chim. Acta, 673(1), 40–46. 18. De Levie, R., (2003). The Henderson-Hasselbalch equation: Its history and limitations J. Chem. Edu., 80(2), 146–146. 19. Rahman, M. A., Ghosh, A. K., & Bose, R. N., (1979). Dissociation constants of longchain fatty acids in methanol-water and ethanol-water mixtures. Journal of Chemical Technology and Biotechnology, 29(3), 158–162. 20. Anderson, T., (1999). National Industrial Chemicals Notification and Assessment Scheme, Full Public Report: Nonanoic Acid, Potassium Salt. http://www.nicnas.gov.au/ publications/car/new/na/nafullr/na0100fr/na114fr.pdf (accessed on 21 February 2022). 21. Sigma-Aldrich(R) Safety Data Sheet. Version 6.5. Revision Date: 10/11/2021, Print Date: 02/26/2022. https://www.sigmaaldrich.com/US/en/sds/sigma/10931 (accessed on 2 March 2022). 22. MP Biomedicals. LLC, Technical Information, Catalog Number: 150384, 194625, https://www.mpbio.com/media/document/file/datasheet/dest/m/p/_/d/s/_/0/2/1/5/0/ MP_DS_02150384.pdf (accessed on 2 March 2022). 23. Tzocheva, S. S., Kralchevsky, P. A., Danov, K. D., Georgieva, G. S., Post, A. J., & Ananthapadmanabhan, K. P., (2012). Solubility limits and phase diagrams for fatty acids in anionic (SLES) and zwitterionic (CAPB) micellar surfactant solutions. Journal of Colloid and Interface Science, 369, 274–286. 24. Abraham, W., Harris, T. M., & Wilson, D. J., (1987). Electrical aspects of adsorbing colloid flotation. XX. NMR studies of lauric acid solubilization. Separation Science and Technology, 22(11), 2269–2280. 25. Waterman, E., & Lockwood, B., (2007). Active components and clinical applications of olive oil. Alternative Medicine Review, 12(4), 331–342. 26. Riddick, J. A., Bunger, W. B., & Sakano, T. K., (1986). Organic Solvents: Physical Properties and Methods of Purification. Wiley-Interscience. 27. Stephenson, R. M., (1993). Mutual solubility of water and aliphatic amines. J. Chem. Eng. Data, 38(4), 625–629. 28. Jencks, W. P., & Regenstein, J., (2010). Ionization constants of acids and bases. In: Lundblad, R. L., & Macdonald, F., (eds.), Handbook of Biochemistry and Molecular Biology. CRC Press. 29. Section 8. Electrolytes, Electromotive Force, and Chemical Equilibrium. (1998). In: Dean, J. A., (ed.), Lange’s Handbook of Chemistry. McGraw-Hill Professional. 30. Dawson, R. M. C., Elliott, D. C., & Elliott, W. H., (1989). Data for Biochemical Research. Oxford University Press. 31. Sigma Aldrich. 6-Aminohexanoic Acid. https://www.sigmaaldrich.com/deepweb/assets/ sigmaaldrich/product/documents/355/131/a7824pis.pdf (accessed on 2 March 2022).

Introduction to Amino Acids 25

32. Vickery, H. B., (1972). History of the discovery of the amino acids. II. Review of amino acids described since 1931 as components of native proteins. Advances in Protein Chemistry, 26, 81–171. 33. Castellano, S., Andrés, A. M., Bosch, E., Bayes, M., Guigó, R., & Clark, A. G., (2009). Low exchangeability of selenocysteine, the 21st amino acid, in vertebrate proteins. Mol. Biol. Evol., 26(9), 2031–2040. 34. Longtin, R., (2004). A forgotten debate: Is selenocysteine the 21st amino acid? Journal of the National Cancer Institute, 96(7), 504, 505. 35. Turanov, A. A., Xu, X. M., Carlson, B. A., Yoo, M. H., Gladyshev, V. N., & Hatfield, D. L., (2011). Biosynthesis of selenocysteine, the 21st amino acid in the genetic code, and a novel pathway for cysteine biosynthesis. Adv. Nutr., 2, 122–128. 36. Byun, B. J., & Kang, Y. K., (2011). Conformational preferences and pK(a) value of selenocysteine residue. Biopolymers, 95(5), 345–353. 37. Axley, M. J., Böck, A., & Stadtman, T. C., (1991). Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium. Proc. Nati. Acad. Sci. USA, 88(19), 8450–8454. 38. Atkins, J. F., & Gesteland, R., (2002). The 22nd amino acid. Science, 296, 1409, 1410. 39. Lukashenko, N. P., (2010). Expanding genetic code: Amino acids 21 and 22, selenocysteine and pyrrolysine. Russian Journal of Genetics, 46(8), 899–916. 40. Hao, B., Zhao, G., Kang, P. T., Soares, J. A., Ferguson, T. K., Gallucci, J., Krzycki, J. A., & Chan, M. K., (2004). Reactivity and chemical synthesis of L-pyrrolysine—the 22nd genetically encoded amino acid. Chemistry & Biology, 11, 1317–1324. 41. Quitterer, F., List, A., Beck, P., Bacher, A., & Groll, M., (2012). Biosynthesis of the 22nd genetically encoded amino acid pyrrolysine: Structure and reaction mechanism of PylC at 1.5 Å resolution. Journal of Molecular Biology, 424(5), 270–282. 42. Mehl, R. A., Anderson, J. C., Santoro, S. W., Wang, L., Martin, A. B., King, D. S., Horn, D. M., & Schultz, P. G., (2003). Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc., 125, 935–939. 43. Uy, R., & Wold, F., (1977). Posttranslational covalent modification of proteins. Science, 198, 890–896. 44. Ibba, M., Stathopoulos, C., & Söll, D., (2001). Protein synthesis: Twenty three amino acids and counting. Current Biology, 11, R563–R565. 45. Maryadele, J. O., Ann, S., Patricia, E. H., & Susan, B., (2001). The Merck Index (13th edn.). John Wiley & Sons, Inc. 46. Stephen, H., & Stephen, T., (1963). Solubilities of Inorganic and Organic Compounds. Pergamon Press: New York. 47. Dixon, H. B. F., Cornish-Bowden, A., Liebecq, C., Loening, K. L., Moss, G. P., Reedijk, J., Velick, S. F., & Vliegenthart, J. F. G., (1984). Nomenclature and symbolism for amino acids and peptides. Pure & Appl. Chem., 56(5), 595–624. 48. Fitzgerald, L. M., & Szmant, A. M., (1997). Biosynthesis of ‘essential’ amino acids by scleractinian corals. Biochem. J., 322, 213–221.

26

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

49. Appleton, J., (2002). Arginine: Clinical potential of a semi-essential amino acid. Alternative Medicine Review, 7(6), 512–522. 50. Woese, C. R., (1967). The Genetic Code: The Molecular Basis for Gene Expression (pp. 156–160). Harper & Row: New York. 51. Crick, F. H. C., (1968). The origin of the genetic code. J. Mol. Biol., 38, 367–379. 52. Biro, J. C., Benyó, B., Sansom, C., Szlávecz, Á., Fördös, G., Micsik, T., & Benyó, Z., (2003). A common periodic table of codons and amino acids. Biochemical and Biophysical Research Communications, 306, 408–415. 53. Minks, C., Alefelder, S., Moroder, L., Huber, R., & Budisa, N., (2000). Towards new protein engineering: In vivo building and folding of protein shuttles for drug delivery and targeting by the selective pressure incorporation (SPI) method. Tetrahedron, 56(48), 9431–9442. 54. Köhrer, C., Yoo, J. H., Bennett, M., Schaack, J., & RajBhandary, U. L., (2003). A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression. Chemistry & Biology, 10, 1095–1102. 55. Hohsaka, T., & Sisido, M., (2002). Incorporation of non-natural amino acids into proteins. Current Opinion in Chemical Biology, 6(6), 809–815. 56. England, P. M., (2004). Unnatural amino acid mutagenesis: A precise tool for probing protein structure and function. Biochemistry, 43(37), 11623–11629. 57. Ayyadurai, N., Deepankumar, K., Prabhu, N. S., Lee, S., & Yun, H., (2011). A facile and efficient method for the incorporation of multiple unnatural amino acids into a single protein. Chem. Commun., 47, 3430–3432. 58. Strømgaard, A., Jensen, A. A., & Strømgaard, K., (2004). Site-specific incorporation of unnatural amino acids into proteins. ChemBioChem, 5, 909–916. 59. Hohsaka, T., Ashizuka, Y., Murakami, H., & Sisido, M., (2001). Five-base codons for incorporation of non-natural amino acids into proteins. Nucleic Acids Research, 29(17), 3646–3651. 60. Hohsaka, T., & Sisido, M., (2000). Incorporation of non-natural amino acids into proteins by using five-base codon-anticodon pairs. Nucleic Acids Symposium Series, (44), 99–100. 61. Landweber, L. F., (2002). Custom codons come in threes, fours, and fives. Chemistry & Biology, 9(2), 143. 62. Papaleo, E., Casiraghi, N., Arrigoni, A., & Ranzani, V., (2012). Atomistic insights into the regulatory mechanisms mediated by post-translational modifications: Molecular dynamics investigations. Current Physical Chemistry, 2(4), 344–362. 63. Merrick, B. A., Dhungana, S., Williams, J. G., Aloor, J. J., Peddada, S., Tomer, K. B., & Fessler, M. B., (2011). Proteomic profiling of s-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3. Molecular and Cellular Proteomics, 10(10), M110.006007. 64. Barclay, E., O’Reilly, M., & Milligan, G., (2005). Activation of an α2A-adrenoceptor-Gαo1 fusion protein dynamically regulates the palmitoylation status of the G protein but not of the receptor. Biochemical Journal, 385(1), 197–206.

Introduction to Amino Acids 27

65. Berger, M., Kaup, M., & Blanchard, V., (2012). Protein glycosylation and its impact on biotechnology. Advances in Biochemical Engineering/Biotechnology, 127(Genomics and Systems Biology of Mammalian Cell Culture), 165–185. 66. Bourles, E., Alves De, S. R., Galardon, E., Giorgi, M., & Artaud, I., (2005). Direct synthesis of a thiolato-S and sulfinato-S CoIII complex related to the active site of nitrile hydratase: A pathway to the post-translational oxidation of the protein. Angewandte Chemie, International Edition, 44(38), 6162–6165. 67. Bothwell, I. R., Islam, K., Chen, Y., Zheng, W., Blum, G., Deng, H., & Luo, M., (2012). Se-adenosyl-L-selenomethionine cofactor analogue as a reporter of protein methylation. J. Am. Chem. Soc., 134(36), 14905–14912. 68. Gallaher, K. J., Wolpert, E. B., & Rannels, S. R., (1990). Gamma-carboxyglutamic acid excretion into rat amniotic fluid during late gestation. Journal of Developmental Physiology, 13(6), 327–332. 69. Stone, M. J., Chuang, S., Hou, X., Shoham, M., & Zhu, J. Z., (2009). Tyrosine sulfation: An increasingly recognized post-translational modification of secreted proteins. New Biotechnology, 25(5), 299–317. 70. Kogl, F., & Erxleben, H., (1939). Chemistry of tumors. Etiology of malignant tumors. Z. Physiol. Chem., 258, 57–95. 71. Kreil, G., (1997). D-amino acids in animal peptides. Annu. Rev. Biochem., 66, 337–345. 72. Fujimoto, K., Kubota, I., Yasuda-Kamatani, Y., Minakata, H., Nomoto, K., Yoshida, M., Harada, A., et al., (1991). Purification of achatin-I from the atria of the African giant snail, Achatina fulica, and its possible function. Biochemical and Biophysical Research Communications, 177(2), 847–853. 73. Stevens, C. M., Halpern, P. E., & Gigger, R. P., (1951). Occurrence of D-amino acids in some natural materials. J. Biol. Chem., 705–710. 74. Brueckner, H., Becker, D., Gams, W., & Degenkolb, T., (2009). Aib and Iva in the biosphere: Neither rare nor necessarily extraterrestrial. Chemistry & Biodiversity, 6(1), 38–56. 75. Yokoyama, T., Kan-no, N., Ogata, T., Kotaki, Y., Sato, M., & Nagahisa, E., (2003). Presence of free D-amino acids in microalgae. Bioscience, Biotechnology, and Biochemistry, 67(2), 388–392. 76. Brueckner, H., & Westhauser, T., (2003). Chromatographic determination of L- and D-amino acids in plants. Amino Acids, 24(1, 2), 43–55. 77. Abe, H., Yoshikawa, N., Sarower, M. G., & Okada, S., (2005). Physiological function and metabolism of free D-alanine in aquatic animals. Biological & Pharmaceutical Bulletin, 28(9), 1571–1577. 78. Hamase, K., Morikawa, A., & Zaitsu, K., (2002). D-amino acids in mammals and their diagnostic value. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences, 781(1, 2), 73–91. 79. Martínez-Rodríguez, S., Martínez-Gómez, A. I., Rodríguez-Vico, F., Clemente-Jiménez, J. M., & Heras-Vázquez, F. J. L., (2010). Natural occurrence and industrial applications of D-amino acids: An Overview. Chemistry & Biodiversity, 7, 1531–1548.

CHAPTER 2

The Dilemma of Working with α-Amino Acids

2.1 IMPACT OF SOLVENT ON THE SOLUBILITY OF α-AMINO ACIDS α-Amino acids are a class of important starting materials for organic synthesis, not only because they are the basic building blocks of proteins and peptides, but also can they be converted into a variety of heterocycles due to their multiple functional groups. However, it is always a challenge when α-amino acids are directly used as the starting materials in organic synthesis. The difficulty may be associated with α-amino acids’ insolubility in common organic solvents, high boiling points, the opposite reactivity between the amino and carboxyl groups, and possibly the instability and involvement of side chain of certain amino acids during the transformations of α-amino acids. α-Amino acids, due to the formation of inner salts, are generally insoluble in common organic solvents, especially for nonpolar solvents. Consequently, water becomes the ideal solvent for α-amino acids. Even in this case, α-amino acids with nonpolar side chains are still not very soluble in water, as indicated by the solubility at 25°C for glycine (25.09 g/100 g water), phenylalanine (2.79 g/100 g water) and tryptophan (1.32 g/100 g water), as listed in Table 1.6 of Chapter 1. The insolubility of α-amino acids in organic solvents can be demonstrated by the solubility change in water when organic solvents are mixed with an aqueous solution. It is reported that the absolute solubility is affected by the chemical potential of the solid phase and the solution thermodynamics, and the chemical potential, and the activity of the solute in a saturated solution is independent of the solvent composition [1]. Because of the existence of hydroxyl group, alcohols with small aliphatic moiety are generally miscible with water due to the formation of hydrogen bonding between alcohol and water. However, from methanol,

30

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

ethanol to propanol and butanol, the strength of hydrogen bond decreases as the hydrophobicity increases in this order, therefore, the dissolving power of water for α-amino acids would be less affected by methanol than that of butanol. Still, the solubility of α-amino acids with polar side chains in water is dramatically affected by the addition of methanol. The change of solubility in water is better illustrated by the change of relative solubility that is defined as the solubility in a mixed solvent system divided by the solubility of the same amino acid in water [1]. For comparison, the molar solubility of glycine and phenylalanine in different aqueous-organic mixed solutions are listed in Table 2.1. From Table 2.1, it is obvious for hydrophilic α-amino acids, as represented by glycine, the solubility in water decreases when organic solvents are added to the aqueous solution. Such change can be as large as more than three orders of magnitude [1]. The effect of propanol is more apparent than that of methanol, because methanol with a small methyl group is more miscible with water than isopropanol that contains three carbon atoms. For example, the molar solubility of glycine in water (3.3 M) decreases to 0.01 M in 100% methanol; however, it decreases to 0.00015 M in 100% of 2-propanol, corresponding to 22,000 times difference of the solubility change for 2-propanol. However, for α-amino acids with lipophilic side chains, such as phenylalanine, the molecular interactions between the nonpolar side chain of amino acid and the organic solvent(s) lead to a relative solubility in solutions with a small mole fraction of water (i.e., less water) that is higher than that for amino acids with a polar side chain. As a result, these alcohols are poorer crystallizing agents for amino acids with nonpolar side chains since there will be a higher yield loss in the solution after the crystallization is complete [1]. In fact, the data for phenylalanine illustrate that the interaction between the strongly nonpolar aromatic side chain and 1-propanol is so strong that the solubility in aqueous 1-propanol can be 25% larger than the solubility in water at the same temperature. For the case of tryptophan, the solubility even increases when the organic solvent is added to water, especially when the ratio of water to methanol is close to 1. It sounds that the solubility of tryptophan in water may not be decreased by the addition of methanol [8]. Interestingly, when polyol, such as sorbitol and inositol, is added to water, the solubility for amino acids only slightly decreases [9]. For example, the solubility of glycine only decreases from 25 g/100 mL in water to 19.31 and 17.42 g/100 mL in 2.0 M of xylitol and sorbitol solution, respectively. Such solubility only drops to 24.29 g/100 mL in 10% of inositol solution. Likewise, for amino acids with a non-polar side chain, such

Solvent Methanol WT% Gly [2] Phe [2]

Ethanol

1-Propanol Gly

2-Propanol

Phe

Gly

t-Butanol

Phe

Gly [3]

Dioxane

Gly

Phe 0.18 [2] 0.1689 [7] –

3.397 [4] –

0.1616 3.4103 [4] 0.1616 3.333 [4] [4] – 3.325 [2] 0.180 [2] –

0.18

3.33 [5]

–

–

–

2.7576 [4] –

–

2.8908 [4] –

–

–

–

–

2.825 [2] 0.157 [2] 2.625

–

0

3.325

0.18

–

–

–

0.047

–

–

3.325 [2] 3.3517 [7] –

0.048

–

–

–

–

0.08

3.175

0.169

0.10

–

–

3.100 [2] –

0.163 [2] –

0.103

–

–

–

–

–

0.1447 – [4] – –

0.149

–

–

–

–

–

–

0.153

–

–

–

–

0.16

3.025

0.08

–

–

1.7984 [4] –

0.163

–

–

–

–

0.205

–

–

0.0785 – [2] 0.1126 – [7] – –

–

0.20

2.875 [2] 1.5053 [7] –

0.251

–

–

–

–

–

–

1.150 [2] 0.064 [2] 1.0125

0.252

1.3625 0.072

1.275 [2]

0.067 [2]

–

–

–

Phe [3]

Gly

Acetonitrile

Phe

Gly

Phe 0.1695 [6] –

–

3.3517 [7] –

0.18 [5] 3.3304 [6] 0.1689 – [7] – –

–

–

–

–

–

0.155

–

–

–

–

–

–

–

–

–

–

–

–

2.32 [5]

–

–

1.6385 [4] –

–

–

–

0.149 [5] –

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

2.35

0.0745 –

–

–

–

–

–

–

–

–

0.1314 [4] –

2.675 [2] 0.0765 [2] 0.1435 – 0.1035 [4] [4] – – –

–

–

–

1.2229 0.1641 [6] [6] – –

0.063

1.452 [7] 0.1495 [7] 1.65 [5] 0.141 [5] – –

–

–

–

–

–

–

–

The Dilemma of Working with α-Amino Acids 31

TABLE 2.1 The Molar Solubility of Glycine and Phenylalanine in Mixed Aqueous-Organic Solvent System

32

TABLE 2.1 (Continued) Solvent Methanol WT% Gly [2] Phe [2] –

–

0.30

–

–

0.303

–

0.306

Gly –

1-Propanol

Phe

Gly

–

2-Propanol

Phe –

–

1.091 [4] 0.9099 0.0944 – [7] [7] – – –

–

–

–

–

0.343

1.225

0.032

0.40

–

–

0.407

–

–

1.150 [2] 0.5662 [7] –

0.439

–

–

0.447

0.95

0.449

Gly

Phe

Phe [3]

Gly

Acetonitrile

Phe

Gly

Phe

–

–

–

–

–

0.1495 – 0.0975 [4] [4] – 0.7673 [4] –

–

– –

0.1356 – [7] – –

–

–

0.8433 [7] –

–

–

–

–

–

1.11 [5]

–

–

0.028 – [2] 0.0896 – [7] – –

–

0.975 [2] 0.027 [2] 0.8875

0.026

–

0.134 [5] –

–

–

0.095 [4] –

–

–

–

–

0.4396 [7] 0.6 [5]

0.4929 0.158 [6] [6] – –

–

0.0215 –

–

–

–

0.7325 [2] 0.0225 [2] – –

0.65

0.029

0.9125 0.0235 – [2] [2] – – –

0.115 [7] 0.122 [5] –

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

0.452

–

–

–

–

–

0.3717 [4] –

–

–

–

–

–

–

0.50

–

–

–

–

–

–

–

–

–

0.1338 – [4] – –

–

0.508

0.3424 0.0835 – [7] [7] – – –

–

–

–

0.54

0.1575 0.026

–

–

0.125 [2] 0.018 [2] 0.105

0.017

0.60

–

– –

0.109 [4] –

0.1279 [4] 0.0592 [4] – –

–

–

0.2225 [4] –

–

0.607

0.1525 [2] 0.1865 [7] –

0.321 [5] 0.0933 – [5] – – –

–

–

0.019 [2] 0.0745 [7] –

0.1477 – [4] – –

–

Gly [3]

Dioxane

–

0.5795 [4] –

–

t-Butanol

–

0.0799 [4] –

0.0853 0.066 [7] [7] 0.167 [5] 0.074 [5]

–

–

0.0457 0.0386 [6] [6] – –

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

0.298

Ethanol

Solvent Methanol WT% Gly [2] Phe [2]

Ethanol Gly

1-Propanol

Phe

Gly

2-Propanol

Phe

Gly

t-Butanol

Phe

0.641

0.115

0.0092

–

–

–

–

–

0.646

–

–

0.0825 [2] 0.006 [2] 0.0575

–

–

0.0062 – [2] – –

–

0.70

0.100 [2] –

0.706

–

–

–

–

–

0.75

–

–

–

–

0.76

0.105

0.0082

–

–

0.805

–

–

0.005 [2] 0.0364 – [7] – –

–

0.80

0.0975 [2] 0.0324 [7] –

0.052 [4] –

0.875

–

–

0.879

0.04

0.0062

0.037 [2] –

0.0038 – [2] – –

0.90

–

–

0.903

–

–

1.0

0.0115 0.0018

–

–

–

Gly

Phe –

0.0056 –

–

–

–

–

–

–

–

–

–

–

–

–

–

0.042 [5] –

–

–

0.0375 [5] –

–

–

0.075 [2] 0.0048 [2] 0.0383 – 0.0171 [4] [4] – – –

0.0425

0.0044 –

–

–

–

–

–

–

–

–

0.004

0.016 [7] 0.019 [5] –

–

–

0.0083 [7] – 0.0213 [5] 0.0032 –

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

0.003 [7] 0.00573 [5] 0.007 [5] –

–

–

–

–

–

–

–

0.0226 [4] –

0.007 [2] 0.0034 [2] – –

0.0067 0.0148 0.0047 [7] [7] [4] – – –

0.0114 0.004 [4] 0.0045 [4] [4] – – –

0.0085 0.0011 – [2] [2] – – –

0.0011 0.000015 0.0004 [4] [2] [4] – – 0.0009 [2]

WT%: weight percentage of organic solvent in water.

Phe –

–

–

–

Gly –

0.0378 [4] –

–

Phe [3]

Acetonitrile

–

0.0745 – [4] – –

–

Gly [3]

Dioxane

0.00271 [5] 0.000013 0.00085 0.00195 [5] – – –

The Dilemma of Working with α-Amino Acids 33

TABLE 2.1 (Continued)

34

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

as phenylalanine, the same trend is noticeable, as indicated by the solution of phenylalanine in water (2.8 g/100 mL) and in 2.0 M xylitol solution (2.12 g/100 mL) [9]. The effect on solubility change by the addition of organic solvent to aqueous solution has important application as it can guide the purification of amino acid for a large change of solubility so that a high yield can be achieved for the purification of the amino acid via crystallization. For the case of tryptophan, the addition of methanol certainly does not help the purification of such amino acid. Therefore, for the alcohols added to aqueous solution, alcohols of larger hydrocarbon chains have a more significant effect than smaller alcohol, such as methanol. For the case of 1-butanol, the addition of hydrophilic amino acids, such as serine or glycine, increases the width of the miscibility gap in the water/1-butanol system. In contrast, hydrophobic amino acids with large nonpolar side chains, such as phenylalanine or tryptophan, increase the mutual solubility of the solvent components, resulting in a narrow miscibility gap [10]. However, it is reported that the solubility of amino acid in 2-propanol and t-butanol are almost the same, as indicated by the solubility of glycine (0.004 M) and phenylalanine (0.001 M) in both alcohols [11], although 2-propanol has a more profound effect on the solubility of phenylalanine than 1-propanol, as shown in Table 2.1.

FIGURE 2.1 The plot of log(Sw/So-w) versus the fraction of organic solvent in the mixed solvent system for glycine. Sw is the solubility of glycine in water, and So-w is the solubility of glycine in mixed solvent system (organic + water).

The Dilemma of Working with α-Amino Acids 35

It is suggested that the solubility change can be better shown by the change of relative solubility that is defined as the ratio of the solubility of amino acid at a specific composition of organic solvent and water over the solubility of the same amino acid in water [1]. The effect of the addition of organic solvents in water on the solubility of amino acids is represented by glycine and phenylalanine as illustrated in Figures 2.1 and 2.2. Obviously, for the hydrophilic amino acids, dioxane, and acetonitrile have a more profound effect on the relative solubility than alcoholic solvent, due to less hydrogen bond formed between water and these solvents. However, for the amino acids with hydrophobic side chains, the effect from acetonitrile or dioxane is much less than that from alcoholic solvent, as shown in Figure 2.2.

FIGURE 2.2 The plot of log(Sw/So-w) versus the fraction of organic solvent in the mixed solvent system for phenylalanine. Sw is the solubility of phenylalanine in water, and So-w is the solubility of phenylalanine in mixed solvent system (organic + water).

2.2 TEMPERATURE EFFECT ON THE SOLUBILITY OF α-AMINO ACIDS Besides the effect of solvent, temperature would be another factor to affect the solubility of amino acids. In general, the higher the temperature, the greater the solubility of amino acids, as energy at a higher temperature is provided to overcome the intermolecular interaction of solute molecules. However, in

36

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

an aqueous solution, the temperature cannot be changed dramatically, due to the boiling point of water. Also, when dealing with peptides or proteins, the high temperature often leads to the denaturation of proteins or peptides. For example, the solubility of glycine in water increases about 60% when the temperature increases from 25°C to 60°C, whilst the solubility increases about 130% for the same temperature range in 20% of EtOH solution. However, when propanol is mixed with water, the temperature effect on the relative solubility of DL-alanine is almost negligible [8]. 2.3 SALTING EFFECT AND pH EFFECT ON THE SOLUBILITY OF α-AMINO ACIDS Other factors that can affect the solubility of amino acids, peptides, and proteins are salting effect and pH effect. The salting effect is caused by the alteration of ionic strength of aqueous solution when salt is added, which can affect the solubility of amino acid as amino acid exists as an inner salt. In general, the presence of salts in the water could either have a saltingin effect or a salting-out effect on the solubilities of amino acids [12]. The salting-in effect increases the solubility of amino acids, whilst the salting-out effect decreases the corresponding solubility. The salting-in effect occurs when the added salt can positively interact with the ionic form of amino acids or proteins and facilitate further hydration of amino acids and proteins. In contrast, when the salting-out effect predominates, the added salts will compete with amino acids or proteins for hydration, which then decrease their solubility. Interestingly enough, the effect of pH on the solubility of amino acid in water parallels that in an organic solvent, such as alcohol. For example, the solubility of glycine in methanol is 11.5 mM (Table 2.1), when HCl is added to methanol at a concentration of 2.6 mM, the solubility of glycine decreases to 2.8 mM, however, when the HCl concentration is increased to 100.4 mM, the solubility of glycine in MeOH is increased to 76.5 mM, and at a concentration of 120 mM for HCl, the solubility of glycine in such methanol is found at 119.8 mM [13]. Finally, as amino acids are zwitterions in water, the ionic state of amino acids strongly depends on the pH of the aqueous solution. At the isoelectric point (pI), the net charge of amino acid is zero; however, at a pH lower than the pI value, the corresponding amino acid will carry a more positive charge than the negative charge and hold a net positive charge. Conversely, at pH

The Dilemma of Working with α-Amino Acids 37

greater than the pI value, amino acid will carry a more negative charge and the net charge will be negative. In either case, the ionic amino acid can be better hydrated by water, thus becoming more soluble in water. In the isoelectric solutions, the neutral dipolar amino acids are the predominant species. Because the thermodynamic solubility is only related to the activity of neutral dipolar amino acid species [12], the solubility of amino acid reaches the minimal number at its pI, and gradually increases when pH is moved away from the pI. For example, the pI of tyrosine is 5.66 (Table 1.6 in Chapter 1), the solubility of tyrosine stays low when pH is between 2.2 to 8.2 (less than 0.001 M), however, when pH is close to 1, the solubility increases to 0.003 M. Conversely, when pH increases to 10, the solubility increases to 0.006 M [12]. The solubility curve may be more flat at the bottom for a wider pH range for amino acids with a lower solubility in water, such as phenylalanine; whilst a narrower curve is found for the amino acid with high solubility in water, such as glycine [11]. In contrast, a deeper curve is found for the solubility of amino acid in methanol corresponding to the pH change [11]. Overall, due to the nature of inner salt for amino acids and the very low solubility of amino acids in organic solvents, especially in nonpolar solvents, the common solvents used for organic synthesis including hexane, acetone, ethyl acetate, methylene chloride, chloroform, ethanol, methanol, acetic acid, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), diethylene glycol, etc., cannot be used for the initial preparation of amino acid derivatives. Although the neat amino acids can be used in organic preparation, in the absence of solvent to avoid the solubility limitation, such as the direct preparation of thiohydantoins from amino acids and thiourea at temperatures around 220°C [14], the high reaction temperature may exclude many good reagents that have boiling points lower than the melting points of amino acids, or limit the number of amino acids or reagents that are not stable at such high temperature which may decompose and lead to unexpected side reactions. Reagents with boiling points lower than the melting points of amino acids may remain in the gas phase or can be easily distilled out before amino acids melt. In this case, amino acids are difficult to react because of the refluxing of the reagents with low boiling points. At the temperature around the melting points of amino acids, some amino acids may decompose, and result in many unexpected reactions, such as in the case of serine and threonine, which often dehydrate at this temperature.

38

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

2.4 MEASURES TO DEAL WITH AMINO ACID SOLUBILITY In order to solve the solubility problem of amino acids, the initial preparation from amino acids has been generally carried out in an aqueous solution. Once the derivatives are formed, which have relatively high solubility in organic solvents, the following transformations are performed in organic solvents. For example, to protect the amino group with t-butoxycarbonyl group, the standard protocol is to dissolve the amino acid in water and treat it with NaOH; once the amino acid is dissolved, t-butyl carbonate is added and the reaction is stirred for 3 days to allow the completion of the reaction. This protocol, although being repeated for many times, often affords the protected amino acids of not very high yields, due to the decomposition of t-butyl carbonate in the aqueous solution before it reacts with the amino acid [15]. The use of organic bases, such as Phosphazene bases (Schwesinger bases) instead of NaOH, allows the preparation of amino acid derivatives in the organic solvent, such as acetonitrile [15]. On the other hand, the carboxyl group is acidic and is often converted into ester, amide, or anhydride functional group in normal organic synthesis, whilst the amino group is basic and is often transformed into secondary amino, imino, enamino, or amido group. During these transformations, the carboxyl group may exist as carboxylate group in amino acid due to hydrogen transfer from the carboxyl group to the nearby amino group, and the resulting carboxylate group is much less reactive than the carboxyl group in the formation of ester and amide. Likewise, the amino group is often used as nucleophile in the formation of secondary amino, imino or other functional groups. The protonated ammonium ion is not nucleophilic enough and can only participate in a limited number of reactions. Moreover, some amino acids due to their labile side chains toward oxidizing reagents, or added acid/base, may not be used as the starting materials. Therefore, amino acids are often protected at either carboxyl group, amino group or both and then used for organic synthesis. Although amino acids have many drawbacks in organic synthesis as mentioned above, amino acids also have some advantages for being used as the starting materials in organic syntheses, if either carboxyl group or amino group is protected so that their reactivity has been changed or modified, because all naturally abundant amino acids are chiral molecules, in L-configuration. Therefore, amino acids can be converted into enantiomerically pure chiral starting materials, and the formed products may naturally be enantiomerically pure without further chiral resolution. This is because the chiral center in most cases

The Dilemma of Working with α-Amino Acids 39

is not involved in the transformation of functional group for amino acids, whereas the racemization at the chiral center would involve the breakage of at least one bond at the chiral center, which is seldom observed. Because the chirality of amino acids is so stable, the change of chirality of amino acids can be applied to measure the age of antique stuff. In contrast, in normal organic syntheses, many efforts have been developed in order to achieve high purity enantiomeric organic compounds from achiral starting materials, chiral resolution is often applied at the final stage of purification, including the formation of diastereomeric isomers and purification by crystallization or column chromatography, as well as using chiral HPLC separation. In order to solve many problems mentioned above for direct preparation from α-amino acids, all α-amino acids should be protected either at the carboxyl group, amino group or both so that the solubility of the resulting derivatives as well as their reactivity of the protected functional groups can be adjusted to meet the condition in order to run the expected reactions. The protection of the carboxyl group and amino group will be the focused on Chapters 3 and 4. KEYWORDS • • • • • •

α-amino acids glycine isoelectric point molar solubility phenylalanine salting effect

REFERENCES Orella, C. J., & Kirwan, D. J., (1989). The solubility of amino acids in mixtures of water and aliphatic alcohols. Biotechnology Progress, 5(3), 89–91. 2. Dey, B. P., & Lahiri, S. C., (1986). Solubilities of amino acids in different mixed solvents. Ind. J. Chem., 25A, 136–140. 3. Pal, A., Dey, B. P., & Lahiri, S. C., (1986). Studies on the dissociation constants & solubilities of amino acids in t-butanol + water mixtures. Ind. J. Chem., 25A, 322–329. 4. Orella, C. J., & Kirwan, D. J., (1991). Correlation of amino acid solubilities in aqueous aliphatic alcohol solutions. Ind. Eng. Chem. Res., 30, 1040–1045. 1.



5.

6.

7. 8. 9. 10.

11. 12. 13. 14. 15.

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Majumder, K., Majumder, K., & Lahiri, S. C., (2000). Solubilities of amino acids in dioxane + water mixtures and the determination of transfer free energies of interaction of amino acid from water to aquo-organic mixtures. Zeitschrift Phy. Chem., 214(3), 285–299. Gekko, K., Ohmae, E., Kameyama, K., & Takagi, T., (1998). Acetonitrile-protein interactions: Amino acid solubility and preferential solvation. Biochimica et Biophysica Acta, 1387, 195–205. Nozaki, Y., & Tanford, C., (1971). The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. J. Biol. Chem., 246(7), 2211–2217. Ferreira, L. A., Macedo, E. A., & Pinho, S. P., (2004). Solubility of amino acids and diglycine in aqueous–alkanol solutions. Chemical Engineering Science, 59, 3117–3124. Gekko, K., (1981). Mechanism of polyol-induced protein stabilization: Solubility of amino acids and diglycine in aqueous polyol solutions. J. Biochem., 90, 1633–1641. Gude, M. T., Meuwissen, H. H. J., Van, D. W. L. A. M., & Luyben, K. C. A. M., (1996). Partition coefficients and solubilities of α-amino acids in aqueous 1-butanol solutions. Ind. Eng. Chem. Res., 35, 4700–4712. Needham, T. E., Paruta, A. N., & Gerraughty, R. J., (1971). Solubility of amino acids in pure solvent systems. J. Pharmaceutical Sci., 60(4), 565–567. Chen, C. C., Zhu, Y., & Evans, L. B., (1989). Phase partitioning of biomolecules: Solubilities of amino acids. Biotechnology Progress, 5(3), 111–118. Dhar, S. K., & Chung, D. B., (1968). Solubility of amino acids in CH3OH-HCl medium, I. Solubility of glycine. Inorg. Nucl. Chem. Lett., 4, 701–704. Wang, Z. D., Sheikh, S. O., & Zhang, Y., (2006). A simple synthesis of 2-thiohydantoins. Molecules, 11, 739–750. Palomo, C., Palomo, A. L., Palomo, F., & Mielgo, A., (2002). Soluble α-amino acid salts in acetonitrile: Practical technology for the production of some dipeptides. Org. Lett., 4(23), 4005–4008.

CHAPTER 3

The Carboxyl Protecting Groups

3.1 INTRODUCTION TO CARBOXYL PROTECTING GROUPS In general, carboxylic acid can be converted into acid derivatives in order to protect the carboxyl group, and the acid derivatives can be changed back to carboxylic acid if needed. The acid derivatives include acid halide, anhydride, ester, amide, orthoester, as well as nitrile and oxazoline. Since acid halide and anhydride are more reactive than carboxylic acid, they are not used as the protecting groups for carboxyl; instead, the acid halide and anhydride are often applied as the activated carboxyl functional groups for chemical transformations. Therefore, the carboxyl group is often converted into ester, amide or orthoester to be protected. In general, the amide is less reactive than an ester, and is more difficult to be deprotected; as a result, only a few amido groups have been developed to protect the carboxyl group, whilst the majority of the protecting groups for carboxyl belong to esters. So far, several types of carboxyl protecting groups have been developed, and they are alkyl groups (e.g., alkyl, heteroatom substituted methyl group, ethyl group with a β-heteroatom, β-keto alcohol), unsaturated alkyl groups (e.g., allyl, propargyl, benzyl, trityl, benzhydryl), aryl groups, orthoester groups, silyl ester groups, amino groups and oxazoline, etc. In general, the more stable the protecting group formed, the less reactivity they bear for further transformation, and the more difficult they will be deprotected. Consequently, some special methods or reagents have to be applied for the deprotection of these stable protecting groups. For the ester protecting groups, they can be prepared from carboxyl in two general approaches: the Fischer esterification of carboxylic acid with the corresponding alcohol in the presence of an acid catalyst, and the deprotonation of the carboxyl group (with a base) followed by the esterification of carboxylate with an alkyl halide (or equivalent). All these protecting groups will be described with detailed experimental procedures in the following order: alkyl groups, heteroatom substituted methyl

42

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

groups, heteroatom substituted ethyl groups, β-keto alkyl, unsaturated alkyl groups (allyl, benzyl, benzhydryl, trityl, propargyl), aryl groups, orthoester groups, amino groups, silyl group and oxazoline group. 3.2 ALKYL PROTECTING GROUPS 3.2.1 METHYL PROTECTING GROUP Methyl ester is probably the simplest ester group for the protection of the carboxyl group, which can be easily formed by refluxing amino acid in methanol in the presence of an acid catalyst. The acid catalysts used include HCl [1], H2SO4 [2], p-toluenesulfonic acid (PTSA) [3], phosphoric acid [4], ion-exchange resin (e.g., AmberlystTM-15) [5], or reagents that can convert carboxyl group into acyl halide or generate acid when they are mixed with methanol, such as thionyl chloride [6], trimethylchlorosilane [7]. Alternatively, the amino acid methyl ester can be formed with another source of methyl group, such as methyl p-toluenesulfonate [8], dialkylsulfite [9], 2,2-dimethoxypropane [10], or diazomethane [11]. In contrast, the methyl group can be removed by means of the treatment of amino acid methyl esters with base (e.g., LiOH) [11, 12] or acid, (e.g., 45% HBr in acetic acid) [6], or under a mild condition, such as using enzyme [13] or AlCl3/N,N-dimethylaniline [14–18]. However, it is reported that the deprotection of amino acid methyl esters by saponification might be accompanied with racemization [19]. 3.2.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID METHYL ESTERS 3.2.1.1.1 Preparation of L-Phenylalanine Methyl Ester in the Presence of HCl [1] O

O OH + CH3OH

NH2

HCl 40 OC

OCH3 NH2

To 165.2 g of methanol containing 79 g of hydrogen chloride was added 165.2 g of L-phenylalanine, and the solution was then stirred at 40°C for 4 hours. HPLC confirmed that 99% or more of L-phenylalanine had been converted into L-phenylalanine methyl ester hydrochloride. The reaction

The Carboxyl Protecting Groups 43

solution was cooled to 3°C, and the precipitate was filtered to afford 258.6 g filter cake. According to analysis, the water and methanol contents in this cake were 3.6% and 13.0%, respectively. The filter cake was then dried by airflow at 50°C for 10 hours to obtain 197.6 g of L-phenylalanine methyl ester hydrochloride having a purity of 96.8% and containing 0.2% of water and 1.2% of L-phenylalanine, in a yield of 88.7%. (Note: gaseous HCl was passed into methanol, and the amount is difficult to control. However, according to the author’s experience, HCl in methanol can be quickly generated by means of the addition of acetyl chloride into cold anhydrous methanol, as the reaction between acetyl chloride and methanol would form methyl acetate and hydrogen chloride. Alternatively, thionyl chloride can be added to cold methanol. In either case, the amount of HCl is easily controlled). 3.2.1.1.2 Preparation of D-Phenylglycine Methyl Ester in the Presence of H2SO4 [2]

O OH NH2

+ CH3OH

H2SO4 O 73 C

O OCH3 NH2

To a suspension of 135 g D-phenylglycine in 252 mL (200 g) methanol was added 107 g (58 mL) concentrated sulfuric acid. The mixture was kept at reflux for 2 hours at approximately 73°C and concentrated at a reduced pressure using a vacuum pump. The pressure dropped from 1 atm to 20 mBar while at the same time the temperature of the reaction mixture was increased from 40 to 80°C. Then, the second portion of 126 mL (100 g) methanol was added, and the mixture was kept at reflux for 1 hour at approximately 81°C and again concentrated at a reduced pressure as described above. This procedure was repeated for another three times (adding methanol, refluxing, and concentrating). Finally, 126 mL (100 g) methanol was added and the solution was refluxed for another hour and cooled to ambient temperature. A total of 700 g methanol had been consumed in the end. Subsequently, 15 ml ammonia was added to the reaction mixture at a constant rate in 30 minutes until the pH was 2.3–2.4. Then, 75 mL water was added, and methanol was distilled off at a reduced pressure while the temperature was kept below 50°C. The pH of the final D-phenylglycine methyl ester solution was 2.0. (Note: A serious drawback of this process is that the yield is rather low, and a large amount of alcohol is consumed, up to 8 folds).

44

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.2.1.1.3 Preparation of L-Phenylalanine Methyl Ester in the Presence of Phosphoric Acid [4] O OH

+

O

+

NH2

O

O P OH + CH3OH HO OH

OCH3 NH2

A flask equipped with a magnetic stirrer and a reflux condenser was loaded with 330 mg L-phenylalanine (2 mmol) and 164 mg phosphorous acid (2 mmol). The solvent mixture of 2 mL water and 10 mL methanol was added and, after dissolution, 0.46 mL propylene oxide (6 mmol) was introduced. The reaction mixture was stirred for 1 hour at room temperature, then the second portion of propylene oxide (2 mmol) was added and the reaction mixture was stirred for 15 minutes at 40°C. Methanol was evaporated, and 1 mL 0.5 N HCl was added. Water was vacuum-evaporated, and the residue was washed with ether. The residue was then mixed with 5 mL methylene chloride and treated with 0.417 mL triethylamine, dissolved in 5 mL methylene chloride. The obtained solution was washed three times with 5 mL water. The organic phase was dried with MgSO4. Methylene chloride was evaporated, and the residue was dissolved in a mixture of chloroform/methanol (9:1) and was purified by column chromatography. From the solution, 0.222 g L-phenylalanine methyl ester was crystallized, in a yield of 62%. (Note: The formation of methyl ester is assumed to involve a cyclic ester in the presence of propylene oxide, as illustrated in Scheme 3.1). O OH NH2

+

O

+

O P OH HO OH

O O P NH3 CH OH HO 3 O Bn O

HO OH

SCHEME 3.1 Formation of amino acid methyl ester.

HO O OH O P O H 3N H Bn O O Bn O O P N HO H3 OH

O O

CH3

The Carboxyl Protecting Groups 45

3.2.1.1.4 General Procedure to Prepare Amino Acid Methyl Ester Hydrochlorides in the Presence of Trimethylsilyl (TMS) Chloride [7] To a round-bottomed flask was added 0.1 mol of amino acid. Then, under magnetic stirring, 0.2 mol of freshly distilled TMS chloride (0.1 mmol) was added slowly. After that, 100 mL methanol was added and the resulting solution or suspension was stirred at room temperature. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was concentrated on a rotary evaporator to give the amino acid methyl ester hydrochloride. (Note: this method has several advantages due to the following features: easy operation, mild reaction conditions, simple workup and good to excellent yields. For example, compared with other methods, the yields obtained with the TMSCl/MeOH system are in most cases comparable to or even higher than those obtained with the thionyl chloride/MeOH and HCl (H2SO4)/MeOH systems. In addition, the TMSCl/MeOH method is certainly more convenient from an operational point of view, as the thionyl chloride/MeOH system should be strictly maintained between –5~0°C and HCl gas must be continuously passed through the refluxing mixture in the MeOH/HCl method. In general, two equivalents of TMSCl are necessary per one carboxyl group, and the reaction time is around 12 hours). 3.2.1.1.5 General Procedure to Prepare Amino Acid Methyl Ester with 2,2-Dimethoxypropane [10] O H 2N

+

OH

HCl MeO

OMe

O H 2N

OCH3

One millimole of the amino acid (the hydrochloride in the case of L-lysine) was suspended in 10–15 mL of 2,2-dimethoxypropane (b.p. 79–81°C). To this suspension was added 1 mL concentrated HCl. The reaction mixture was allowed to stand at room temperature for 18 hrs. In the case of L-lysine and L-glutamic acid, because of their insolubility, the mixtures were supplemented with 3–4 mL methanol, heated to reflux for 2 and 5 hrs., respectively, and then allowed to stand for 18 hrs. at room temperature. After the reaction was completed, the reaction mixtures were concentrated under vacuum at 50–61°C and the residues were dissolved in a minimum amount of absolute methanol. Addition of about 25 mL absolute ethyl ether resulted in crystallization of the desired products.

46

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.2.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF METHYL ESTERS 3.2.1.2.1 Removal of Methyl Ester with AlCl3/N,N-dimethylaniline [17, 20] O

O

S

OCH3

Fmoc

NH

1) AlCl3/DMA (7:11)

S

2) H3O+

OH

Fmoc

98%

NH

To a suspension of 1 mmol N-Fmoc-methionine methyl ester and 11 mmol of N,N-dimethylaniline in dry dichloromethane was added a freshly grounded AlCl3 (7 mmol) at room temperature. The reaction was monitored by TLC. After the reaction was completed, the reaction was quenched by the addition of ice-cold water. The aqueous layer was extracted with ether and the combined organic layers were washed with 1 N HCl, and extracted with 1 N NaOH. The aqueous layer was acidified to pH 2 by addition of 1 N HCl and extracted with Et2O. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated to afford 98% of N-Fmoc-methionine. (Note: Excesses of reagents can also be removed via chromatographic purification, this procedure is summarized from two experimental procedures). 3.2.1.2.2 Removal of Methyl Ester with LiOH [11]

t-Boc H

H N Ph

CO2Me

LiOH (aq) MeOH

t-Boc H

H N

CO2H 97%

Ph

A mixture of 44 mg (1S,2R)-methyl 1-[N-(tert-butoxycarbonyl)amino]2-phenylcyclo-propane-1-carboxylate (0.15 mmol, 1.0 equiv), LiOH·H 2O (63 mg, 1.50 mmol, 10.0 equiv.), MeOH (4.0 mL), and H2O (2.0 mL) was refluxed for 2.5 hrs. The reaction mixture was then cooled to room temperature and acidified to pH ≈ 2.5, and the product was extracted with CH2Cl2 (3 × 5 mL). The organic extracts were combined, dried over Na2SO4, filtered. Evaporation of the solvent provided 40 mg of (1S,2R)-l-[N-(tert-butoxycarbonyl)amino]2-phenylcyclopropane-l-carboxylic acid as a clear oil, in a yield of 97%.

The Carboxyl Protecting Groups 47

3.2.1.2.3 General Method for Enzymatic Hydrolysis [13] O

H N

O O

3

N H

O

BS2 or CAL-A/pH 7.4 hexane/MeOH

O

O

H N

O O

N H

3

OH O

To a stirred solution of the substrate (0.15–0.20 mmol) in n-hexane (1 mL) and CH3OH (100 µL) was added a solution of the enzyme (either an esterase from Bacillus subtilis (BS2) or a lipase from Candida antarctica (CAL-A)) (50 mg) in 9 mL of phosphate buffer (50 mM pH 7.4). The reaction mixture was stirred for 24–72 h at 37°C. After acidification until pH 6 and extraction with EtOAc (3 × 5 mL), the organic layers were combined and washed with 5% NaHCO3 (3 × 5 mL). The aqueous layer was acidified until pH 6 and extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na2SO4, and the organic solvent was removed under reduced pressure to give the product. For the case of (S)-2-(N-(tert-butoxycarbonyl) hexanamido)acetic acid, the methyl ester was hydrolyzed for 48 hours with BS2 (87% yield) or 72 hours with CAL-A (74% yield). 3.2.2 ETHYL PROTECTING GROUP In theory, the protection of carboxyl with ethyl group would be very similar to the one with a methyl group, and the methods that have been applied to form the amino acid methyl esters will also be applicable for the formation of amino acid ethyl esters. 3.2.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID ETHYL ESTERS 3.2.2.1.1 Preparation of Amino Acid Ethyl Ester Hydrochloride with Thionyl Chloride [21] NH H 2N

N H

O

NH OH

O

NH OH

+ EtOH

SOCl2

H 2N

N H

O O O

NH OH

48

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To 2 mL absolute ethanol cooled with a mixture of salt and ice was added 0.16 mL thionyl chloride (2.2 mmol) dropwise. Then, 590 mg of salicyl arginine ((2-hydroxybenzoyl)-L-arginine, 2.0 mmol) was added and the mixture was refluxed for 5 hours. The reaction mixture was then cooled, and the solvent was evaporated under reduced pressure. The evaporation was repeated several times after the addition of a small amount of absolute ethanol to the resulting oil. The oily crude product was purified by chromatography on Sephadex LH-20 to afford 558 mg of salicyl arginine ethyl ester hydrochloride (ethyl (2-hydroxybenzoyl)-L-argininate hydrochloride) as a hygroscopic product, in a yield of 77.8%. 3.2.2.1.2 General Procedure for the Synthesis of Amino Acid Ethyl Ester in the Presence of HCl [22]

To a 100-mL round-bottomed flask, which was securely connected to a suction flask, were added 1.0 g of α-amino acid and 15 mL absolute ethanol. The HCI gas was generated by the controlled addition of concentrated hydrochloric acid to concentrated sulfuric acid in the suction flask. To do that, 50 milliliters of 18 M H2SO4 was added to a 1-L suction flask containing a Teflon-coated magnetic stirring bar. The sidearm of the suction flask was fitted with a piece of latex tubing to a 9-inch Pasteur pipet. Then, 20 mL of 12 M HCl was added to a long-stem 125-ml dropping funnel and attached to the top of the suction flask with a rubber stopper. The suction flask was securely clamped atop a magnetic stirrer and the dropping funnel was also, in turn, securely clamped. The round-bottomed flask containing the amino acid was clamped on a separate ring stand at a 45-angle, and the Pasteur pipet was secured so that the tip was just below the surface of the ethanol. The hydrochloric acid was added drop by drop to the stirred H2SO4, and the HCl gas generated was bubbled through the shaken suspension of the amino acid in ethanol. Note, care must be taken to prevent the accumulation of solids in the tip of the pipet. The dissolution of HCl gas in ethanol is quite exothermic, and the contents of the round-bottomed flask become quite warm in the process. It is found that the formation of HCl gas in this way is less cumbersome than the addition of H2SO4 to solid NaCl for routine use; also, it is not necessary to dry the generated HCl by passing it through

The Carboxyl Protecting Groups 49

a H2SO4 trap. After all the hydrochloric acid was added, the contents of the round-bottomed flask were refluxed for at least 0.5 hours or until a complete solution was obtained. Crystallization of the products was first attempted by placing the stoppered reaction flask in the freezer for one or two days. If no crystals were obtained, the reaction mixture was evaporated to remove the volatile solvent and excess HCl. 3.2.2.1.3 Formation of N-Norborn-2-ene-5-Carboxy-LPhenylalanine Ethyl Ester in the Presence of p-Toluenesulfonic Acid [23] O

O

H N H

COOH

+ EtOH

p-TsOH

H N H

CO2Et

A mixture of 0.578 g N-(norborn-2-ene-5-carbonyl)-L-phenylalanine (2 mmol) and 16 mg PTSA (0.084 mmol) in 30 mL of dry ethanol was refluxed for 6 hours. The ethanol was then evaporated, and the white residue was dissolved in diethyl ether. The ether solution was subsequently washed with water, aqueous sodium hydrogen carbonate solution, and water. The ether solution was concentrated and flash-chromatographed over silica gel 60 with diethyl ether: pentane (5:1) as the mobile phase, to afford 74% of N-norborn2-ene-5-carboxy-L-phenylalanine ethyl ester. 3.2.2.1.4 Preparation of Amino Acid with Orthoester Under Microwave Irradiation [24]

The protected amino acids (e.g., Fmoc-L-Ala-OH) and 2.5 to 5.0 equivalents of triethyl orthoformate were added to a heavy-walled Emrys process glass vial and sealed with an aluminum crimp cap fitted with a septum. The reaction mixture was subjected to microwave irradiation for 60 minutes at 200°C, to afford 85% of Fmoc-L-alanine ethyl ester.

50

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.2.2.1.5 Preparation of Amino Acid Ethyl Ester in the Presence of α-Chymotrypsin (CT) [25] CO2Et

CO2H N H

O

+ EtOH

N H

-Chymotrypsin

N H

0.1 M Phosphate buffer pH 6.8

O

N H

To a mixture of 0.05 g N-acetyl-L-tryptophan (0.203 mmol) and 20 mL ethanol was added 0.01 g α-chymotrypsin in 0.5 mL phosphate buffer (0.1 M, pH 6.8). The mixture was incubated at 30°C with constant shaking for 24 hours. In a two-phase method, the initial reaction mixture consisted of 1 M ethanol in chloroform (20 mL), 0.05 g of N-acetyl-L-tryptophan, and 0.01 g of α-chymotrypsin in 0.5 mL phosphate buffer (0.1 M, pH 6.8) was constantly shaken for 24 hours. After the reaction, the solvents were evaporated by a rotary evaporator, and the amount of N-acetyl-L-tryptophan and N-acetyl-L-tryptophan ethyl ester were determined by HPLC. 3.2.2.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF ETHYL ESTERS 3.2.2.2.1 Removal of Ethyl Group by Protease Trypsin [21] NH H 2N

N H HCl

NH

O H 2N

O O

Trypsin 0.1 M Phosphate buffer pH 7.5

NH O O

N H

O OH O

NH O O

To a solution of 300 mg aspirin-arginine ethyl ester hydrochloride (0.75 mmol) in 75 mL 0.1 M phosphate buffer (pH 7.5) was added 30 mg trypsin. The reaction mixture was incubated at 25°C for about 5 minutes. After checking the disappearance of aspirin-Arg-OEt⋅HCl by HPLC, the reaction mixture was frozen and subjected to freeze-drying. The lyophilizate was dissolved in 2 mL of MeOH, filtered, and the filtrate was purified on Sephadex LH-20 column to afford 168 mg of aspirin-arginine, in a yield of 66.8%.

The Carboxyl Protecting Groups 51

3.2.2.2.2 Removal of Ethyl Group with LiOH [26] NEt2

NEt2 OEt

N

O

LiOH THF/MeOH/H2O

OH N

O

To a solution of 0.200 g (R,S)-ethyl 2-(4-pyridyl)-2-(N,N-dimethylamino) acetate (0.960 mmol) in a 6 mL mixed solvent of THF-methanol-H2O (1:1:1) was added 0.120 g powdered LiOH (4.99 mmol) at room temperature. The solution was stirred for 3 hours and then acidified to pH 6 using 1 N HCl. The aqueous phase was washed with EtOAc and then lyophilized to give (R,S)-2-(4-pyridyl)-2-(N,N-dimethylamino)acetic acid dihydrochloride as a yellow solid (containing LiCl). 3.2.3 ISOPROPYL PROTECTING GROUP The isopropyl group is not as popular as the methyl, ethyl, or t-butyl group in terms of being used as the carboxyl protecting group. However, it has also been occasionally used. In general, amino acid isopropyl ester can be prepared and deprotected in a manner similar to that of the methyl or ethyl group. 3.2.3.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID ISOPROPYL ESTERS 3.2.3.1.1 Preparation of Isopropyl Phenylglycinate Hydrochloride O H 2N

OH Ph

+

OH

O HCl

H 2N

O Ph

Anhydrous HCl gas was bubbled slowly through a refluxing suspension of 12.0 g of racemic phenylglycine (80 mmol) in 250 mL of dry 2-propanol for 4.5 hours. Then the solvent was evaporated in vacuo, and the remaining powder was recrystallized from acetone-water to give 8.8 g isopropyl phenylglycinate hydrochloride as white crystals, in a yield of 48%, mp 225–228°C. The corresponding valine isopropyl ester hydrochloride was prepared in the same way by bubbling HCl gas for 6 hours to afford 52% of yield [27].

52

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.2.4 n-HEPTYL PROTECTING GROUP The n-heptyl group has been rarely applied as the carboxyl protecting group in peptide synthesis. However, as the n-heptyl group is an enzyme-labile protecting group, the base-labile biologically relevant phosphopeptides can advantageously be synthesized under very mild conditions (pH 7, 37°C). Although most of the biocatalysts can saponify the n-heptyl ester, it is found that in the presence of lipase from Aspergillus niger, the C-terminal deprotection proceeded with the highest velocity, without attacking at the N-terminal blocking groups and the peptide bonds or the phosphoric acid esters [28]. The formation of amino acid n-heptyl ester should be performed in a manner similar to the preparation of methyl or ethyl ester, whilst the removal of the n-heptyl group will be performed in the presence of lipase from Aspergillus niger in a 95:5 ratio of 0.2 M phosphate buffer (pH = 7)/acetone at 37°C [28]. As this protecting group is seldom used, no experimental detail will be provided here. 3.2.5 tert-BUTYL PROTECTING GROUP As one of the alkyl-protecting groups for amino acids, the t-butyl group shares the common features as other alkyl groups, such as the methyl and ethyl groups mentioned above. However, as the t-butyl group is very bulky in comparison to methyl and ethyl groups, it provides additional advantages to the protected amino acids, especially the stability. For instance, samples of freshly prepared t-butyl glycinate and ethyl glycinate were placed in two sets of vials, of which one set was stored at 24°C and the other set was stored at –20°C to determine the storage stability characteristics over a period of time. Loss of stability was measured by the development of a precipitate of diketopiperazine and a significant change in the index of refraction of the liquid esters. It was found that at 24°C the ethyl glycinate showed some precipitate of diketopiperazine after only 1 day and the resulting mixture became completely solid after 4 days. In contrast, the t-butyl glycinate remained a perfectly clear liquid with an index of refraction value unchanged for 27 days, becoming only slightly hazy with a precipitate of diketopiperazine. For comparison, ethyl glycinate stored at –20°C remained a clear liquid for 4 days but showed a heavy precipitate of diketopiperazine after 14 days and was one-quarter solidified after 21 days. In contrast, the t-butyl glycinate remained a clear liquid with refraction value unchanged for at least 55 days. Therefore, the stable t-butyl esters of naturally occurring α-amino acids can be stored as reagents in peptide synthesis, thereby avoiding the use of HCl or

The Carboxyl Protecting Groups 53

HBr salts with accompanying racemization [29]. Also, t-butyl group can be removed under acidic conditions instead of using LiOH, thus the potential γ→α transpeptidation can be avoided involving glutamic acid residue [30]. In general, amino acid t-butyl esters can be prepared in three different ways, by means of peptide condensation reagent (e.g., DCC with t-butanol) [31], or in the presence of a base (KOBut [32], K2CO3 [31]) or acid. Among these choices of preparations, the reaction between amino acid and isobutylene in the presence of acid is the most popular method. [27, 29, 31, 33] Tert-butyl group can be easily removed by acid (e.g., HNO3 [31], CF3CO2H/Et3SiH [26], 85% H3PO4 [34], and ZnBr2 [35]). 3.2.5.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID t-BUTYL ESTERS 3.2.5.1.1 Preparation of t-Butyl Carbobenzoxyglycinate with Isobutylene [29] Ph

H N

O

O OH

+

p-TsOH MeC(O)-i-Pr

Ph

H N

O

O

O O

O

To a solution of 20.9 g ((benzyloxy)carbonyl)glycine in methyl isopropyl ketone was added 2.0 g of PTSA as a catalyst. Then 23 g of isobutylene was passed into the solution at room temperature, during which an exothermic reaction was observed. The solution was kept in a stoppered flask for a period of 18 hours. Isolation of the product was accomplished by adding the reaction product to a 4% NaOH solution. The amount of NaOH was such that a slight excess of alkali would remain after neutralization of unconverted ((benzyloxy)carbonyl)glycine. The ketone layer was separated, washed with distilled water, and dried. Then the ketone was completely removed by distillation at a very low pressure (0.35 mmHg). A light amber-colored oil remained which was tert-butyl ((benzyloxy)carbonyl)glycinate. 3.2.5.1.2 Preparation of t-Butyl Phenylglycinate Hydrochloride [27] O H 2N

O OH +

Ph

H2SO4 dioxane

H 2N

O Ph

54

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a pressure bottle were placed 5.0 g of racemic phenylglycine (33.3 mmol), 50 mL of purified dioxane and 5 mL of concentrated H2SO4. The reaction mixture turned yellow and became homogeneous. To the solution was added 50 mL of liquid isobutylene, and the bottle was then sealed and shaken for 20 hours. The precipitate that formed was collected and the filtrate was extracted with several portions of ether. The solid precipitate was combined with the extracts, and anhydrous HCl gas was bubbled through the solution. The precipitate that separated was recrystallized from acetone-water to give 1.85 g of pure t-butyl phenylglycinate hydrochloride, in a yield of 23%, mp 219–225°C. A similar procedure with isobutylene was performed in a highpressure flask in the presence of catalytic amount of H2SO4 (0.2 mL) for 30 mL of isobutylene, that afforded 33% of t-butyl 2(S)-2-[(benzyloxycarbonyl) amino]-3-(neopentyloxysulfonyl) propanoate [33]. 3.2.5.1.3 General Procedure for the Preparation of Amino Acid t-Butyl Ester with DCC [31] O H 2N

O OH + HO

R

DCC/DMAP CH2Cl2

H2N

O R

To an 8 mL vigorously stirred CH2Cl2 solution containing 20.0 mmol of amino acid, 40.0 mmol of t-butanol, and 3.2 mmol of 4-(dimethylamino) pyridine (DMAP) was added a solution of 20.8 mmol of DCC in 4.0 mL of CH2Cl2 dropwise at 0°C. After the addition was complete, the reaction mixture was allowed to reach room temperature and stirred overnight; the formed dicyclohexylurea (DCU) was filtered off, the solvent was evaporated and the residue was dissolved in 40 mL of Et2O. The obtained solution was additionally filtered, if necessary, washed with 0.5 M HCl (3 x 20 mL), 5% aqueous NaHCO3 (2 x 20 mL), 30% aqueous NaCl (20 mL), dried over Na2SO4. Upon removal of the solvent by evaporation, the residue was purified by column chromatography. 3.2.5.1.4 General Procedure to Prepare Amino Acid t-Butyl Ester under Basic Condition [31]

The Carboxyl Protecting Groups 55

To a 75 mL N,N-dimethylacetamide (DMA) solution containing 10.0 mmol of amino acid, and 10.0 mmol of benzyl triethylammonium chloride was added 260 mmol of the finely ground anhydrous K2CO3 in one lot, and the resulting suspension was kept under vigorous mechanical stirring. 2-Bromo-2-methylpropane (480 mmol) was added dropwise and the resulting mixture was stirred at 55°C for 24 hours. After that time, the reaction mixture was cooled to room temperature, poured under stirring into 1 liter of cold water. The oily residue which separated was extracted with EtOAc (2 × 200 mL). The combined organic phase was washed with 30% aqueous NaCl (2 × 200 mL), dried, and evaporated. The residue was purified by column chromatography. 3.2.5.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF t-BUTYL GROUP 3.2.5.2.1 Removal of t-Butyl Protecting Group by HNO3 [31]

A chilled solution of 100% HNO3 in CH2Cl2 (60.0 mmol in 8.0 mL) was added dropwise at 0°C and under stirring to a solution of 20.0 mmol of amino acid t-butyl ester in 12.0 mL of CH2Cl2, and the homogeneous mixture was stirred for 2 hours at 0°C with protection from the light. Then, the reaction mixture was diluted with 100 mL of CH2Cl2, washed with 30% aqueous NaCl (2 × 100 mL), dried over anhydrous Na2SO4. Upon evaporation of solvent, the residue was purified by crystallization or column chromatography. 3.2.5.2.2 Removal of t-Butyl Group by Trifluoroacetic Acid and Triethylsilane [26] O

O O

NH

MeO O

TFA/Et3SiH CH2Cl2

OH NH

MeO O

56

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Trifluoroacetic acid (343 mL, 4.62 mol) and Et3SiH (142 mL, 0.887 mol) were added sequentially to a solution of 82.0 g (S)-t-butyl 2-(methoxycarbonylamino)-3-methylbutanoate (0.355 mol) in 675 mL of CH2Cl2, and the mixture was stirred at ambient temperature for 4 hours. The volatile component was removed under diminished pressure and the resultant oil was triturated with 600 mL petroleum ether to afford a white solid, which was filtered and washed with hexane (500 mL) and petroleum ether (500 mL). Recrystallization from EtOAc/petroleum ether afford 54.8 g of (S)-2-(methoxycarbonylamino)3-methylbutanoic acid as white flaky crystals, in a yield of 88%, m.p. 108.5–109.5°C. 3.2.5.2.3 General Procedure for the Selective Removal of tert-Butyl Esters with ZnBr2 [35] Ph N

CO2But

ZnBr2 CH2Cl2

Ph N CO2H

A stirred solution of N-protected amino acid t-butyl ester (1 mmol) in 5 mL of dichloromethane was added 5 equivalents of ZnBr2 and the mixture was stirred at room temperature for 24 hours. After the addition of 20 mL of water, the mixture was stirred again for 2 hours. The organic phase was separated, and the aqueous layer was extracted twice with dichloromethane (20 mL). The combined organic portions were dried, filtered, and evaporated. The residue was chromatographed with EtOAc/hexane (1:1) containing 1% acetic acid as eluent to yield the corresponding acid. For the case of (2S)-N-9-(9-phenylfluorenyl) azetidine-2-carboxylic acid t-butyl ester, 80% of (2S)-N-9-(9-phenylfluorenyl) azetidine-2-carboxylic acid was obtained, m.p. 107.5–108.5°C. 3.2.6 ADAMANTYL PROTECTING GROUP Similar to the t-butyl group, adamantyl group has also been used as the carboxyl protecting group. However, due to the cost of the adamantyl alcohol, it is not as popular as the t-butyl group. Like the t-butyl group, adamantyl group can be introduced into amino acid in the presence of an acid catalyst [36]. In addition, an even more bulky alkyl group, such as 3,5-dimethyl-1-adamantyl group has been applied as the carboxyl protecting group [37]. The adamantyl group can be removed in the presence of trifluoroacetic acid and anisole.

The Carboxyl Protecting Groups 57

3.2.6.1 GENERAL PROCEDURE TO FORM AMINO ACID ADAMANTYL ESTER [36] O OH + NH2

O

+

HO

S O

O

O p-TsOH toluene,

O NH2

A mixture of 4 mmol amino acid 4-toluenesulfonic acid salt, 0.8 g of adamantyl alcohol (AdOH, 5.2 mmol), 440 µL of dimethyl sulfite ((CH3O)2SO, 4.8 mmol) and a catalytic amount of anhydrous TsOH (~ 20 mg, 0.12 mmol) in 4 mL toluene was refluxed under magnetic stirring. After the reaction was completed, the homogenous solution was diluted with 30 mL of CHCl3, washed with 5 mL H2O. The organic layer was dried (Na2SO4) and evaporated in vacuo. Et2O (30 mL) was added to the residue and the crystalline precipitate was filtered, washed with Et2O (2 × 20 mL) and recrystallized. When the amino acid is isoleucine, the yield of isoleucine adamantyl ester was 74%. 3.2.6.2 Representative Procedure to Remove Adamantyl Protecting Group [36] O

NH2

N H

H N O

O

O O

+

O

TFA

NH2

N H

H N

O OH

O

Leucyl-alanyl-valine 1-adamantyl ester hemioxalate salt ([(CO2H)2⋅HLeu-Ala-Val-OAd], 0.13 g, 0.25 mmol) was partitioned between Et2O and 10% aqueous Na2CO3, and the organic layer was dried over Na2SO4 and evaporated to dryness (This is to remove the oxalic acid). Then 580 mL of TFA (7.5 mmol) and 55 mL of anisole (0.5 mmol) were added to the residue and the resulting solution was left at room temperature for 1 hour. The solvent was removed under reduced pressure and the residue was partitioned between Et2O and water. The aqueous layer was extracted again with Et2O, and the combined organic layer was concentrated to dryness. The solid residue was dissolved in 5 mL of acetone, and a solution of Et3N in acetone was added dropwise until the solution appeared slightly acidic on moist indicator paper. The precipitated solid was filtered and washed well with acetone to afford 0.06 g of Leucyl-Alanyl-Valine, in a yield of 70%.

58

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.2.7 α-PHENYLETHYL PROTECTING GROUP α-Phenylethyl protecting group for carboxyl shares some feature characteristic of 9-fluorenylmethyl group as it can also be cleaved in the presence of acid (HCl). In general, the amino acid α-phenylethyl ester can be prepared by means of the esterification between amino acid and 1-phenyl ethanol. Alternatively, it can also be prepared via substitution between deprotonated amino acid and α-bromoethylbenzene. 3.2.7.1 SYNTHESIS OF N-PHTHALOYL-DL-PHENYLALANINE α-PHENYLETHYL ESTER [36] O

O O

N O

OH

Br + Et3N

+

O

, 10 hrs. EtOAc

N O

Ph

O Ph

The mixture of 2.95 g N-phthaloyl-DL-phenylalanine (10 mmol), 1.01 g of triethylamine (10 mmol) and 2.78 g of α-bromoethylbenzene (15 mmol) in 15 mL of EtOAc was refluxed for 10 hours. In the course of the reaction, triethylamine hydrobromide was precipitated and filtered off after the reaction had been completed. The filtrate was washed with 1 N HCl, saturated NaHCO3, water, and dried over anhydrous MgSO4. Upon removal of EtOAc under vacuum, the oily residue was dissolved in ether. The addition of petroleum ether afforded a crystalline substance which was further recrystallized from methanol to yield 2.55 g of N-phthaloyl-DL-phenylalanine α-phenylethyl ester, in a yield of 73%, m.p. 104–105°C. 3.2.7.2 REMOVAL OF THE α-PHENYLETHYL PROTECTING GROUP [36] O

O O

N O

O Ph

O

HCl Benzene

N O

OH Ph

Dry HCl gas was bubbled through a 4 mL dry benzene solution containing 1 mmol of N-phthaloyl-DL-phenylalanine α-phenylethyl ester at 0°C. The solution was either left for 24 hours at 0°C or the HCl and benzene were

The Carboxyl Protecting Groups 59

immediately removed under reduced pressure. Then 10 mL of EtOAc was added. The solution was washed with saturated NaHCO3 solution to a distinct alkaline reaction, followed by a small amount of water, and dried over anhydrous MgSO4. The organic solvent was removed under reduced pressure and the unreacted ester was dried to constant weight. The acid was precipitated from the alkaline aqueous layer with HCl and filtered by suction. 3.2.8 THE 2-(4-ACETYL-2-NITROPHENYL)ETHYL (ANPE) PROTECTING GROUP The 2-(4-acetyl-2-nitrophenyl)ethyl (Anpe) protecting group is a base-labile carboxyl protecting group, which can be easily removed by means of LiOH treatment. The amino acid ester of this protecting group can be prepared in the presence of DCC/DMAP. However, prior to the treatment of aspartic acid with Et3B in THF, the β-carboxyl group is protected with Anpe group instead of the α-carboxyl group, so that the two carboxyl groups within aspartic acid can be differentiated. 3.2.8.1 PREPARATION OF Nα-BOC-L-ASPARTIC ACID β-ANPE ESTERS, BOC-ASP(X)-OH [37] 3.2.8.1.1 Route A

1. Formation of N-Boc-Aspartic Acid Di-Anpe Ester: To a cooled suspension of N-Boc-Asp-OH and two equivalents of Anpe alcohol in CH2Cl2 in an ice bath were added three equivalents of DCC dissolved in CH2Cl2 dropwise for 2 hours, followed by 0.1 equivalent of 4-dimethylaminopyridine (DMAP). Then, the ice-water bath was removed and the reaction mixture was stirred overnight. The mixture

60

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

was chilled for 1 hour to promote the precipitation of DCU, which was separated by filtration. The filtrate was diluted with CH2Cl2 and washed twice with 5% aq. NaHCO3 and 1 M aq. KHSO4, dried over Na2SO4 and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with a gradient of MeOH (0–5%) in CH2Cl2 to afford 68% of N-Boc-Asp(Anpe)2 as a yellow foam. 2. Boc-Asp(X)-OH: To an ice-cooled solution of Boc-Asp(Anpe)2 in acetone was added a solution of LiOH⋅H2O (0.8 eq.) in water over a period of 1.5 hours. The reaction mixture was allowed to warm up to room temperature and the stirring was maintained for an additional 0.5 hour, after which time the acetone was removed. The aqueous solution was diluted and washed with EtOAc (2 × 25 mL) and after cooling in an ice bath, it was acidified to pH 2 with 10% aq. HCl. The desired product, Boc-Asp(X)-OH was then extracted with EtOAc (2 × 25 mL). The combined organic phase was dried over Na2SO4 and evaporated to dryness to afford 76% of N-Boc-Asp(Anpe)-OH as a yellow foam. 3.2.8.1.2 Route B Preparation of Aspartic Acid β-Anpe Ester Hydrochloride (HCl⋅HAsp(X)-OH) [37]. O

NH2

HO

OH Et3B THF

O

Et B Et O

O H2 N HO

1. Anpe-OH/DCC/CH2Cl2 2. HCl (g)/Et2O

O O

O NO2

O O

NH3+ClOH O

Boc2O/Na2CO3 dioxane/H2O

NO2

O O

NHBoc OH O

To the solution of aspartic acid 2,2-diethyl-5-oxotetrahydro-1,3,2-oxazaborole in CH2Cl2 was added 1.2 equivalent of Anpe alcohol and 1.2 equivalent of DCC. The mixture was stirred for 3 hours at room temperature. Then the suspension was cooled in an ice bath to promote the precipitation of DCU which was filtered. The filtrate was evaporated to dryness, re-dissolved in anhydrous Et2O, cooled in an ice bath and gaseous HCl was passed through the solution for about 10 minutes. The solid that separated was decanted,

The Carboxyl Protecting Groups 61

washed twice with Et2O and dried to afford 75% of aspartic acid β-Anpe ester hydrochloride as a yellow foam. The resulting ester can be further treated with t-butyl carbonate to form N-Boc-Asp(Anpe)-OH in route A. 3.2.9 FLUORENYLMETHYL (FM) PROTECTING GROUP 9-Fluorenylmethyl group (Fm) is still considered as an alkyl protecting group for the carboxyl group, as the carbon atom that will form ester functionality with the carboxyl group is still not directly connected to an aromatic ring. This protecting group, due to its orthogonality with protecting groups such as Boc and its facile deprotection with a secondary amine (e.g., piperidine) in DMF [38], has been applied in solid-phase peptide synthesis (SPPS) [39]. The amino acid Fm esters have been synthesized by reacting the N-protected amino acids with 9-fluorenylmethanol in the presence of dicyclohexylcarbodiimide (DCC)/DMAP [38, 40] or with diazofluorene [41]. Alternatively, the amino acid Fm ester can also be prepared via transesterification by means of either reacting the N-protected amino acid with 9-fluorenylmethylchloroformate in the presence of diisopropyl-ethylamine/DMAP [39], or reacting the N-protected amino acid p-nitrophenyl esters with 9-fluorenylmethyl alcohol [42] in the presence of imidazole, although the prolonged reaction time (overnight) was required for the second transformation, which may lead to the imidazole induced decomposition [39]. More preparation procedures are also available in the cited references. [43, 44] The base labile 9-fluorenylmethyl group can be easily removed in the presence of other ester-protecting functional groups with 10% piperidine in DMF at 0°C [38]. 3.2.9.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID FLUORENYLMETHYL ESTER 3.2.9.1.1 General Procedure for the Synthesis of Boc-Amino Acid Fluorenylmethyl Esters [45]

t-Bu-O O

OH

O

H N

OH + R

+

N C N

DMAP CH2Cl2

O

H N

t-Bu-O O

O R

62

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a dry CH2Cl2 solution containing 0.05 mol of the Boc-amino acid derivative, 4.7 g of fluorenylmethanol (0.024 mol) and 244 mg of DMAP (0.002 mol) cooled to 0–4°C, was added 3.72 mL of diisopropylcarbodiimide (0.024 mol). The resulting mixture was stirred for 16 hours at room temperature. The precipitated diisopropylurea was filtered off and the solvent was evaporated to an extent that the solution remained filterable. Once again, the urea was filtered off and the solution was evaporated to dryness, resulting in an oily residue. This residue was dissolved in EtOAc, and washed with a citric acid solution (three times), concentrated Na2CO3 solution (three times) and water, then dried over Na2SO4. Upon removal of the solvent, the oily residue was obtained. Further purification can be applied if necessary. 3.2.9.1.2 Preparation of Nα-Boc-Glycine α-Fluorenylmethyl Ester [40] H N

t-Bu-O

OH

O OH

+

+

DMAP CH2Cl2

N C N

O

H N

t-Bu-O

O

O

O

To a mixture of Boc-Gly-OH (10.0 g, 0.057 mol), 9-fluorenylmethanol (10.67 g, 0.0543 mol) and DMAP (66 mg, 0.543 mmol) in 250 mL CH2Cl2 cooled in ice-water bath, was added a solution of 11.77 g DCC (0.057 mol) in 50 mL CH2Cl2 dropwise over 20 minutes. The resulting mixture was stirred overnight and the solution was filtered to remove DCU. The filtrate was extracted with saturated NaHCO3 solution (3 times), 10% citric acid (2 times), water, and brine, and dried over anhydrous MgSO4. Upon evaporation of the solvent, the oily residue was purified by flash column chromatography (hexane/EtOAc 5:1) to afford 16.0 g of Nα-Boc-glycine α-fluorenylmethyl ester, in a yield of 79.3%, mp 80–82°C. 3.2.9.1.3 Preparation of N-Boc-Leucine 9-Fluorenylmethyl Ester via Transesterification [42] H N

t-Bu-O O

OH

O O

+

NO2

H N N toluene

H N

t-Bu-O O

O O

The Carboxyl Protecting Groups 63

To a suspension of 0.22 g 9-fluorenylmethyl alcohol (Fm-OH, 1.1 mmol) and 0.1 g of imidazole (1.5 mmol) in 2 mL dry toluene was added 0.35 g Boc-Leucine o-nitrophenyl ester (1 mmol). After being stirred overnight at room temperature, the reaction was complete as verified by TLC. The solvent was removed in vacuo, the residue was dissolved in EtOAc. The solution was washed sequentially with 1% citric acid, H2O, 0.5 N NaHCO3, H2O and dried over MgSO4. Removal of the solvent gave an oil which was chromatographed on a silica gel column (2 × 60 cm) with hexane (200 mL) followed by 100 mL each of 5%, 10%, 20%, 30% solution of EtOAc in hexane. Fractions containing the Fm ester were pooled and the solvent was removed in vacuo to afford 0.29 g of N-Boc-leucine 9-fluorenylmethyl ester, in a yield of 73%. This ester was soluble in EtOAc, hexane, ether, CHCl3, MeOH but crystallized on prolonged storage (m.p. 61–67°C). 3.2.9.2 REMOVAL OF THE 9-FLUORENYLMETHYL ESTER PROTECTING GROUP [42] O

H N

t-Bu-O O

O R

15% Piperidine or DEA DMF

O

H N

t-Bu-O O

OH R

To a 0.3 mL solution of DMF containing 15% of diethylamine or piperidine was added 5 mg of amino acid 9-fluorenylmethyl ester. The mixture was stirred and monitored by TLC. It was found that the deprotection was complete within 1 hour. For comparison, the deprotection in 15% of triethylamine in DMF required three hours to complete, and diisopropylethylamine took an even longer time, indicating the secondary amine is much better than tertiary amine. 3.2.10 2-PYRIDYLETHYL (PET) PROTECTING GROUP The 2-pyridylethyl protecting group (Pet) has a high UV absorption due to the pyridyl chromophore, which facilitates the detection of amino acid Pet ester during column chromatography [46]. In addition, the obvious advantage to form amino acid Pet ester is that such ester is equally well soluble in water and ether, and can be easily purified by flash chromatography or filtration over silica gel or Sephadex after removal of the unreacted N-protected amino acids [47]. The amino acid Pet ester can be prepared via

64

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

the esterification of amino acid and Pet alcohol in the presence of DCC and N-hydroxybenzotriazole (HOBt), or in the presence of DCC and DMAP [47]. The Pet group can be easily removed by sequential treatment with methyl iodide and diethylamine [47]. 3.2.10.1 GENERAL PROCEDURE FOR THE PREPARATION OF AMINO ACID 2-PYRIDYLETHYL ESTER [47] H N

t-Bu-O

O

N

OH

OH +

O

HOBt/DCC CH2Cl2 or DMF

H N

t-Bu-O

O O

N

O

To a stirred solution of 5 mmol N-Boc-amino acid, 169 mg of HOBt (1.25 mmol), 1.69 mL of freshly distilled Pet alcohol (15 mmol) in 10–30 mL of DMF (or CH2Cl2), was added 1.13 g of DCC (5.5 mmol) at 0°C. The reaction mixture was allowed to warm to about 21°C. After removal of precipitate DCU by filtration, the solvent was evaporated. The oily residue was re-dissolved in about 200 mL of EtOAc (and filtered again if necessary). The solution was washed with a small volume of saturated NaHCO3 and NaCl solution, and dried over Na2CO3. After removal of the solvent under vacuum, the oil was re-dissolved in methanol or DMF and purified through chromatography on a Sephadex LH20 column (7.8 cm, CH3OH or DMF respectively). Excess DCC was removed during chromatography. Alternatively, such preparation can also be carried out in the absence of HOBt, as illustrated below. To a CH2Cl2 solution containing 5 mmol of N-Boc-amino acid cooled at –20°C, were added sequentially 0.84 mL of Pet alcohol (7.5 mmol), 46 mg of DMAP (0.375 mmol) and 1.55 g of DCC (7.5 mmol). After stirring at this temperature for 15 minutes, the reaction mixture was allowed to warm to about 21°C and stirred for about 12 hours. The product can be worked up as described above. For the case of N-Boc-glycine, the yield of the corresponding ester was 87%. 3.2.10.2 REMOVAL OF THE 2-PYRIDYLETHYL GROUP [47] H N

t-Bu-O O

O O

N

1. CH3I 2. Et2NH DMF

H N

t-Bu-O O

O OH

The Carboxyl Protecting Groups 65

To an acetonitrile or DMF solution containing 0.5 mmol of amino acid Pet ester was added 94 µL of CH3I (1.5 mmol). The reaction mixture was stirred at about 21°C for 12 hours (the alkylation is time and solvent-dependent). Then the solvent was evaporated in vacuo. To remove the excess amount of CH3I, the reaction mixture was re-dissolved in acetonitrile and evaporated several times. After that, the residue was re-dissolved in CH2Cl2, acetonitrile or DMF, 414 µL of HNEt2 (4 mmol) was added and the solution was stirred at about 21°C for 12 hours. After evaporation of solvent and removal of excess diethylamine, the residue was distributed between EtOAc and 10% NaHCO3. The organic phase was re-extracted, the aqueous phase combined was cooled to 0°C and acidified with concentrated HCl. The product was extracted with EtOAc, dried over Na2CO3. Upon removal of EtOAc, the amino acid can be crystallized with an appropriate solvent system. 3.3 α-HETEROATOM SUBSTITUTED METHYL PROTECTING GROUPS Although alkyl groups and substituted alkyl groups have been widely applied as the carboxyl protecting groups, the relative difficulty in the removal of the above-mentioned alkyl protecting groups has promoted the development of more labile alkyl protecting groups, such as the introduction of a heteroatom substituted methyl group and ethyl group. The heteroatom substituted methyl groups, with structural similarity to acetal functionality, has relatively high reactivity than the simple methyl group. Up to now, several different protecting groups of this class have been developed, including methylthiomethyl (MTM), β-(trimethylsilyl)ethoxymethyl (SEM), acetyloxymethyl, benzyloxymethyl (BOM), and phthalimidomethyl groups, etc. 3.3.1 METHYLTHIOMETHYL (MTM) PROTECTING GROUP The amino acid methylthiomethyl (MTM) ester can be easily formed between N-protected amino acid and chloromethyl methyl sulfide in the presence of a base [48]. However, it is surprisingly interesting that almost quantitative yield of amino acid MTM ester can be prepared between amino acid, t-butyl bromide, and dimethylsulfoxide in the presence of NaHCO3 [49–51], as it is generally accepted that alkyl bromides are inert to dimethyl sulfoxide (DMSO) in the absence of metal ion assisting agents even at high temperature [52], especially for this case where t-butyl bromide is used. Under this

66

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

condition, the corresponding amino acid MTM ester can be obtained in a high yield without racemization [49]. The MTM group, on the other hand, is also stable enough so that NPS and trityl groups can be orthogonally removed by means of 2 N HCl in anhydrous Et2O with complete retention of the MTM group for a short time (a few minutes) [50]. However, the MTM group can be removed under the conditions where Boc and Z groups are removed [50]. Especially, the MTM group can be removed under mild condition by the treatment of AlCl3 and N,N-dimethylaniline in CH2Cl2 [18]. Under this condition, other carboxyl protecting groups, such as methyl, benzyl, methoxymethyl, methoxyethoxymethyl, and SEM can also be removed [18]. 3.3.1.1 GENERAL PROCEDURE FOR ESTERIFICATION OF N-BOC PROTECTED L-AMINO ACIDS WITH CHLOROMETHYL METHYL SULFIDE [48] NHBoc OH + Cl R O

S

CH3

(i-Pr)2NEt R

CHCl3, N2,

NHBoc O

S

CH3

O

To the refluxing solution of N-Boc protected amino acid in dry chloroform under static pressure of nitrogen, were added 3 equivalents of diisopropylethylamine and 1.2 equivalents of chloromethyl methyl sulfide sequentially. The reaction mixture was refluxed for 8 hours and then cooled to room temperature. The reaction mixture was diluted with chloroform and washed with distilled water, brine, and dried over anhydrous Na2SO4. Upon removal of the solvent under vacuum, the residue was chromatographed on a silica gel column using 1% triethylamine in chloroform as the eluent to afford pure N-Boc L-amino acid MTM ester. 3.3.1.2 GENERAL PROCEDURE FOR THE METHYLTHIOMETHYLATION OF N-PROTECTED AMINO ACIDS [50] NHBoc OH + R O

O S

+

NaHCO3 Br DMSO

R

NHBoc O

S

CH3

O

To a 5 mL of DMSO containing 1 mmol of the N-protected amino acid was added 10 mmol of solid NaHCO3. To this suspension, 10 mmol of

The Carboxyl Protecting Groups 67

t-butyl bromide in 5 mL of DMSO was added over a period of a few minutes. The reaction mixture was stirred at 30°C for 5 hours (Note: It is strongly suggested to monitor the reaction, as extended hours, e.g., 12 hours [51] or 16 hours [49] has been reported). After that, the solution was poured into water, extracted twice with EtOAc. The combined organic phase was dried and reduced to small bulk under vacuum. The residue was purified on a silica gel column (1.5 × 40 cm), by eluting with a mixture of hexane and EtOAc, with UV detection. The ester can be further purified by crystallization from hexane and EtOAc. 3.3.2 ACETYLOXYMETHYL PROTECTING GROUP The preparation of amino acid acetyloxymethyl ester consists of a two-step procedure, i.e., the preparation of bromomethyl acetate and the subsequent esterification with amino acid [53].   Step A: Preparation of bromomethyl acetate: O

+ (CH2O)n

Br

ZnCl2 CH2Cl2

O O

Br

To a solution of 23.45 g acetyl bromide (19 mmol) in 20 mL CH2Cl2, was added 200 mg of anhydrous ZnCl2 (1.67 mmol) at 25°C. Then the mixture was cooled by ice, and 6.0 g paraformaldehyde (200 mmol) was added to the ice-cooled mixture. The reaction mixture was stirred overnight at 25°C. Distillation of the mixture at 750 torr using a Vigreux headpiece (35 cm) gave first the unreacted acetyl bromide and CH2Cl2, followed by the crude acetyloxymethyl bromide boiling at 120–140°C (750 torr). Repeated distillations at 750 torr gave 23 g of acetyloxymethyl bromide (b.p. 132–134°C), in a yield of 75%. Step B: Preparation of acetoxymethyl N-[1-Oxy-2,2,5,5  tetramethylpyrroline-3-carbonyl]-glycinate: O

O N H

N O

OH O

O

(i-Pr)2NEt

+

O

Br

DMF

N H N O

O O

O O

To a solution of 0.085 g N-[1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl]glycine (0.35 mmol) in 10 mL of DMF at 10°C were added 0.5 mL of N,N-diisopropylethylamine (2.90 mmol) and then 0.23 mL of bromomethyl

68

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

acetate (2.47 mmol). The reaction mixture was stirred at 25°C for 48 hours. Upon removal of the solvent at 25°C (2 torr), the oily residue was purified by flash chromatography on silica gel using CH2Cl2/MeOH (95:5) as eluent to afford 50% of acetoxymethyl N-[1-oxy-2,2,5,5-tetramethylpyrroline3-carbonyl]glycinate as an oil. 3.3.3 THE β-(TRIMETHYLSILYL)ETHOXYMETHYL (SEM) PROTECTING GROUP The SEM has popularly been applied as the hydroxyl protecting group [54], due to easy formation of the corresponding ether, stability under a number of synthetic transformations and facile removal conditions (e.g., cleaved by 1.5% HCl in MeOH) [55]. When this group is extended to protect the carboxyl group, the corresponding amino acid 2-(trimethylsilyl)ethoxymethyl ester can be prepared in a manner similar to the formation of 2-(trimethylsilyl)ethoxymethyl ether. This protecting group often is a good choice for protecting the carboxyl group whenever the subsequent deprotection requires a mild, non-hydrolytic condition, which is usually removed under acidic conditions or with a fluoride ion source [56]. However, this protecting group may not be orthogonally applicable when other acid labile groups such as tert-butyldimethylsilyl (TBDMS) and tert-butoxycarbonyl (Boc) groups or fluoride sensitive N-9-fluorenylmethoxycarbonyl (Fmoc) group exist. Recently, it has been found that in the presence of MgBr2, the SEM group can be removed while Boc, Fmoc or Cbz group remains unaffected [56]. 3.3.3.1 GENERAL PROCEDURE FOR THE PREPARATION OF AMINO ACID SEM ESTER [56] R Boc

N H

R OH + O

Si

O

Cl

Li2CO3 DMF

Boc

N H

O

O

Si

O

The mixture of 0.25 M of the protected amino acid in DMF, 0.8 equivalent of 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl) and 1.1 equivalent of Li2CO3 was stirred at room temperature overnight. Then additional 0.4 equivalent of SEM-Cl was added, and the reaction mixture was stirred for another 4 hours. After that, the reaction mixtures were diluted with brine, and extracted with Et2O. Upon removal of the solvent under reduced pressure, the

The Carboxyl Protecting Groups 69

residue was purified by flash silica gel chromatography to afford 60–80% of amino acid SEM ester, usually as a pale-yellow oil or white solid. If there is not a base-labile group, a catalytic amount of 4-N,N-dimethylaminopyridine (DMAP) can be applied to increase the yield of ester. 3.3.3.2 GENERAL PROCEDURE TO REMOVE THE SEM GROUP FROM AMINO ACID ESTER [56] OMe

OMe

O

O O

N

N O

HN Z

OSEM NHBoc

O MgBr2 Et2O CH2Cl2

O O

N

N

O

O

HN

OH NHBoc

O

Z

To a solution of amino acid SEM ester in CH2Cl2 (0.0178 M) cooled at –20°C, was added three equivalents of MgBr2 etherate. After being stirred for half an hour, the reaction mixture was brought up to 0°C. Further warming to room temperature allowed the reactions to go to completion. The reaction mixture was then washed by dilute HCl and brine. Upon removal of solvent, the residue was purified by flash column chromatography to afford the deprotected amino acid. Alternatively, the reaction mixture was concentrated to remove CH2Cl2 solvent, and re-dissolved in saturated NaHCO3 solution. The aqueous solution was acidified with dilute HCl, and extracted with EtOAc. After removing EtOAc, the residue can be purified by column chromatography to afforded the deprotected amino acid. 3.3.4 PHTHALIMIDOMETHYL PROTECTING GROUP The phthalimidomethyl group should be a good carboxyl protecting group as it can be removed under either acidic or basic conditions [57]. The amino acid phthalimidomethyl ester can be formed in EtOAc, but will be formed at a much faster rate in a polar solvent such as DMF or DMSO with dicyclohexylamine as the base. Compared to the also acid-labile t-butyl group, although both phthalimidomethyl and t-butyl groups can be removed in the presence of dry HCl or HBr, the phthalimidomethyl ester of amino acid can survive

70

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

in the presence of PTSA [57]. Thus, the t-butyl group can be selectively removed in peptide synthesis with the coexistence of phthalimidomethyl group. Also, the phthalimidomethyl ester group can also be removed with zinc and acetic acid [58]. On the other hand, as phthalimidomethyl group can also be removed by a base, it is also possible to orthogonally deprotect the phthalimidomethyl for the compounds containing the t-butyl ester functionality in the presence of base reagents, such as with excess of Et2NH or hydrazine hydrate in alcohol (3 hours at 20°C), or NaOH in alcohol/water (< 1 hour at 20°C). It is found that the removal of phthalimidomethyl group under acidic conditions in an aprotic solvent requires longer reaction time (e.g., 16 to 18 hours for HCl in dioxane or EtOAc at 20°C), but a much shorter time in acidic protic solvent (e.g., 15 minutes for HBr in acetic acid). In addition, the phthalimidomethyl ester functionality can sustain under the catalytic hydrogenation condition [57]. 3.3.4.1 GENERAL PROCEDURE TO SYNTHESIZE PHTHALIMIDOMETHYL ESTERS OF N-SUBSTITUTED AMINO ACIDS [57]

Boc

N H

R

O

R OH + O

N Cl

Et2NH EtOAc

Boc

N H

O O O

O

N O

To a solution of dry EtOAc containing one equivalent of N-protected amino acid and one equivalent of diethylamine, was added one equivalent of N-chloromethylphthalimide. The resulting solution was stirred at 37–40°C overnight. Then the reaction mixture was washed twice with water and once with NaHCO3 solution. Upon removal of solvent, the amino acid phthalimidomethyl ester was obtained as either oil or crystal. The esterification would be run faster when it is carried out in a polar solvent such as DMF or DMSO with dicyclohexylamine as the base. 3.3.4.2 REMOVAL OF THE PHTHALIMIDOMETHYL GROUP As the phthalimidomethyl group can be removed under either acidic or basic conditions, different procedures can be followed in terms of deprotecting such carboxyl protecting group, as shown below.

The Carboxyl Protecting Groups 71

3.3.4.2.1 Removal of the Phthalimidomethyl Group under Acidic Condition [57] R Boc

O O

N H

N

O

R HCl dioxane or EtOAc

Boc

OH

N H

O

O

The amino acid phthalimidomethyl ester was dissolved in dioxane or EtOAc and the solution was saturated with dry HCl, or the ester was dissolved in a saturated solution of HBr in acetic acid. After the deprotection was complete, the solvent was evaporated and the residue was dissolved in NaHCO3 solution. The aqueous solution was washed with EtOAc and acidified to afford amino acid as an oil or crystalline compound. 3.3.4.2.2 Removal of the Phthalimidomethyl Group Under Basic Condition [57] R Boc

N H

O O O

N O

Et2NH or NH2NH2 MeOH

R Boc

N H

OH O

To a solution or suspension of one equivalent of amino acid phthalimidomethyl ester in methanol was added 2–4 equivalents of diethylamine or hydrazine hydrate. When diethylamine is used, the reaction mixture was stirred at room temperature for 24 hours, unless a large excess of diethylamine is used then the reaction can be worked up in a few hours. For this case, upon removal of solvent, the residue was worked up as in procedure 2.4.2.1. However, when hydrazine hydrate is applied, after several hours, acetic acid was added to destroy the salt giving a precipitate of 2,3-dihydrophthalazine-1,4-dione acetate which was removed by filtration. The filtrate was evaporated to dryness and the residue was worked up as in procedure 2.4.2.1. Alternatively, the amino acid phthalimidomethyl ester can be suspended in a 1:1 ratio of methanol/water with two equivalents of NaOH. The reaction mixture is then worked up similarly.

72

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.3.5 BENZYLOXYMETHYL (BOM) PROTECTING GROUP Although BOM has not been popularly applied as the carboxyl protecting group in amino acids, it has been used to protect regular carboxylic acid and should be extendable to the carboxyl group in amino acid. 3.3.5.1 PREPARATION OF BENZYLOXYMETHYL 2-IODOBENZOATE [59] I

I NaH OH DMF O

I ONa

O

Cl

O

Ph O

DMF

O

Ph

O

To a suspension of 0.3 g NaH (12 mmol) in 20 mL dry DMF was added a solution of 2.48 g 2-iodobenzoic acid (10 mmol) in 20 mL of DMF dropwise, and the mixture was stirred for 1 hour at ambient temperature then 12 mmol of benzyloxymethyl chloride (BOMCl) was added. The mixture was stirred at room temperature until the carboxylic acid was consumed, after which the mixture was mixed with 300 mL of water (300 mL) and 150 mL of tert-butyl methyl ether. The combined organic layer was washed with water (2 × 300 mL), brine, and dried over Na2SO4. Upon removal of the solvent under reduced pressure, the residue was purified by flash chromatography using tert-butyl methyl ether/hexanes as eluent to afford 2.67 g of BOM 2-iodobenzoate as a colorless oil, in a yield of 73%. 3.4 β-HETEROATOM SUBSTITUTED ETHYL PROTECTING GROUP Compared to the α-heteroatom substituted methyl protecting group, there have been more β-heteroatom substituted ethyl groups applied for the protection of the carboxyl group, presumably because of the relatively easy availability and better stability of the formed amino acid esters. The heteroatom at the β position can be nitrogen, phosphorus, oxygen, sulfur, and halogens. The developed β-heteroatom substituted ethyl groups include 2-[(2-methoxy)ethoxy]ethyl (MEE), diethylene glycol monomethyl ether, polyethyleneglycol (all with an oxygen atom at the β-positions); 2-(4-nitrophenylsulfonyl)ethyl and thiomethylethyl (a sulfur atom at the β-position); 2-morpholinoethyl (MoEt), 2-aminoethyl, 1-(2,5-dioxo-1-pyrrolidinyl)ethyl, and cyanomethyl (for a nitrogen atom at the β-position);

The Carboxyl Protecting Groups 73

2-trimethylsilylethyl (TMSE) and 2-phenyl-2-trimethylsilyl group (for a silicon atom at the β-position); and 2-(diphenylphosphino)ethyl (Dppe) group (when β-heteroatom is phosphorus). 3.4.1 2-HALOETHYL PROTECTING GROUP 2-Haloethyl protecting group normally include 2-chloro- and 2-bromoethyl groups (possibly 2-iodoethyl group), and the corresponding amino acid 2-haloethyl esters can be easily prepared via either esterification of amino acid with 2-haloethanol in the presence of DCC, or under conditions by prior converting amino acid into the corresponding acyl chloride with thionyl chloride which then reacts with 2-haloethanol [60]. This kind of hydrophobic esters can be smoothly coupled with Boc or Z-protected amino acids to form respective dipeptides. In addition, the amino acid 2-haloethyl esters are stable under moderate acidic (e.g., 40% HBr in acetic acid) or basic (e.g., 2 N NaOH) conditions [60]. On the other hand, 2-haloethyl groups can also be removed easily by three methods. The first method is the conversion of amino acid 2-haloethyl esters into amino acid 2-iodoethyl esters via the reaction of amino acid 2-haloethyl esters with KI and the subsequent treatment of amino acid 2-iodoethyl esters with Zn in DMF. This two-step process can be performed in a manner of one-pot procedure. Secondly, the amino acid 2-haloethyl esters can be treated with trimethylamine to form amino acid choline esters, which are very stable towards acids, but can easily be removed in the presence of 10–4 M hydroxide ion without racemization [60]. The third deprotecting method is to treat the amino acid 2-haloethyl esters with sodium sulfide [61]. 3.4.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID 2-HALOETHYL ESTERS 3.4.1.1.1 Preparation of Z-L-Alanine 2-Bromoethyl Ester in the Presence of DCC [60]

74

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a mixture of 10 g N-benzyloxycarbonyl-L-alanine (45 mmol) and 5.6 g of 2-bromoethanol (45 mmol) in 30 mL dry CH2Cl2 was added 11.0 g of DCC (60 mmol) at 4°C under stirring. After 36 hours, the precipitated DCU is filtered, the solution was washed with 0.5 N HCl, 0.5 N NaOH, and water, and then dried over Na2SO4. After evaporation of the solvent under vacuum, the residue was dissolved in acetone, filtered, and evaporated in vacuo to afford 10.2 g of N-benzyloxycarbonyl-L-alanine 2-bromoethyl ester as a colorless oil, in a yield of 69%. 3.4.1.1.2 General Procedure for the Preparation of Amino Acid 2-Chloro- or Bromoethyl Ester [60]

OH

H2N

+

HO

Cl

SOCl2 O

H 2N

Cl

O

O

To a suspension of 38 mmol isoleucine in 32 g (0.4 mol) chloroethanol cooled at –10°C was slowly added 6.0 g of thionyl chloride (50 mmol) dropwise. Then the mixture was warmed to room temperature and stirred until a clear solution of the amino acid is reached. Excess amount of alcohol and thionyl chloride was removed at 25–35°C via vacuum distillation, the residue was placed in water and the pH was adjusted to 9.8 with ammonia, and the solution was extracted with ether. The ether solution was washed with 1 N HCl and the aqueous layer was brought to pH 9.8 again and extracted with ether. The combined ether layer was dried and evaporated to afford 98% of isoleucine 2-chloroethyl ester. 3.4.1.1.3 Conversion of Amino Acid 2-Bromoethyl Ester to Corresponding 2-Iodoethyl Ester (Preparation of N-Benzyloxycarbonyl-L-Phenylalanyl-L-Leucine 2-Iodoethyl Ester) [60]

Ph

H N

O O

O N H Ph

O O

Br

+ NaI

CH3CN

Ph

H N

O O

O N H Ph

O

I

O

To a solution of 3.0 g of N-benzyloxycarbonyl-L-phenylalanyl-L-leucine 2-bromoethyl ester (5.8 mmol) in 10 mL of acetonitrile was added 1.3 g dry

The Carboxyl Protecting Groups 75

NaI and the mixture was refluxed for 12 hours. After being cooled to room temperature, 0.62 g of NaBr was filtered (99%). The filtrate was concentrated under vacuum to solvent-free, and the residue was dissolved in CH2Cl2 and filtered again. Upon removal of solvent, about 3.25 g N-benzyloxycarbonylL-phenylalanyl-L-leucine 2-iodoethyl ester was obtained as an almost colorless oil, in a yield of 99%. 3.4.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF 2-HALOETHYL GROUP 3.4.1.2.1 Removal of 2-Haloethyl Protecting Group (Preparation of N-Benzyloxycarbonyl-L-Phenylalanyl-L-Leucine) [60] Ph

H N

O O

O O

N H Ph

Zn I

Ph

O

O

H N

O

OH

N H Ph

O

O

To a solution of 3.4 g N-benzyloxycarbonyl-L-phenylalanyl-L-leucine 2-iodoethyl ester (6 mmol) was added 0.8 g zinc powder (12 mmol), and the mixture was refluxed for 24 hours. After the solution was cooled to room temperature, 50 mL ether and 50 mL ammonia-water solution (pH 9–10) were added. The zinc and zinc hydroxide were removed via filtration. The organic phase was separated and about 0.3 g of N-benzyloxycarbonyl-Lphenylalanyl-L-leucine 2-iodoethyl ester was recovered upon removal of ether. The aqueous solution was brought to pH 1 with 6 N HCl, and shaken with ether. The ethereal layer was dried over Na2SO4 and concentrated to afford 1.4 g of N-benzyloxycarbonyl-L-phenylalanyl-L-leucine, in a yield of 58%, m.p. 70°C. 3.4.1.2.2 One-Pot Procedure to Remove 2-Haloethyl Protecting Group [60]

Ph

H N

O O

O N H Ph

O O

1) NaI/DMF Br 2) Zn/DMF

Ph

H N

O O

O N H Ph

OH O

76

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

The mixture of 3.0 g of N-benzyloxycarbonyl-L-phenylalanyl-L-leucine 2-bromoethyl ester (5.8 mmol), 1.3 g of dry NaI (8.7 mmol) in 10 mL DMF was stirred at 80°C for 3 hours. After that, 1.0 g of zinc powder (15 mmol) was added in portions, and the mixture was continually stirred for 12 hours after the development of gas was completed. Then, the reaction was mixed with diluted Na2CO3 solution. The mixture was filtered, and the filtrate was extracted with ether. The aqueous layer was adjusted to pH 1 and extracted with ether again. The combined ether solution was washed with sodium thiosulfate and dried over Na2SO4. After removal of the solvent under vacuum, 1.8 g of N-benzyloxycarbonyl-L-phenylalanyl-L-leucine was obtained, in a yield of 75%. 3.4.1.2.3 Removal of 2-Chloroethyl Ester Group with Na2S [61]

To a 10 mL of aqueous acetonitrile (2:3) were added 1.47 g of N-Bocglycylglycyl 2-chloroethyl ester (0.005 mol) and 1.44 g of sodium sulfide nonahydrate (0.006 mol), and the mixture was refluxed for about 1 hour under stirring. The reaction mixture was cooled, and after addition with 30 mL water, was extracted with diethyl ether. The aqueous layer was mixed with EtOAc, titrated with aqueous 2 N HCl at pH 3. The aqueous phase was separated from the EtOAc layer and washed several times with EtOAc. The combined organic extracts were dried over MgSO4 and evaporated to dryness to yield the N-Boc-Gly-Gly-OH, which was further recrystallized from EtOAc/petroleum ether, in a yield of 50%, m.p. 135–136°C. 3.4.2 POLYETHYLENE GLYCOL (PEG) AND ETHYLENE GLYCOL RELATED PROTECTING GROUPS Polyethylene glycol (PEG), or more properly, poly(ethylene glycol), refers to a chemical compound composed of repeating ethylene glycol units. However, depending on how one chooses to define the constituent monomer or parent molecule (as ethylene glycol, ethylene oxide, or oxyethylene), PEG compounds are also known as PEO (polyethylene oxide) [62] and POE (polyoxyethylene) [63]. Often, PEG is followed by a number that denotes the averaged molecular weight of ethylene glycol

The Carboxyl Protecting Groups 77

oligomer mixtures in broadly or narrowly defined molecular weight range. Due to the high percentage of oxygen atoms in PEG, it is highly hydrophilic and water-soluble, and confers greater water solubility to proteins and peptides. Also, PEG molecules are soluble in some organic solvents, such as methylene dichloride. Such dual solubility of PEG in both aqueous solution as well as organic solvent renders peptide chemist flexibility while carrying out the preparations. In addition, PEG has been applied as a labeling tag and crosslinker in protein synthesis. Similar to PEG, some other ethylene glycol related molecules have also been applied as the carboxyl protecting groups, including 2-methoxyethyl, diethyleneglycol-monomethylether (DEM) [64] (also known as 2-[(2methoxy)ethoxy]ethyl (MEE)) [65], and ethylene glycol oligomers with a methyl group at one end. The amino acid PEG esters and amino acid ethylene glycol related esters can be easily prepared via the condensation of amino acid with the respective alcohol in the presence of DCC [66]. On the other hand, these protecting groups can also be removed under mild conditions with lipase [65]. 3.4.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID PEG ESTERS 3.4.2.1.1 Preparation of Dioleoylserinyldodecaethylene Glycol [67]

To a solution of 5.61 g methoxy dodecaethylene glycol (i.e., 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-ol, 0.01 mol) in 50 mL anhydrous CH2Cl2, were added 2.06 g of DCC (0.01 mol) and 6.34 g of dioleoyl-L-serine (0.01 mol) at 0°C. The resulting mixture was first stirred at 0°C for 2 hours, and then allowed to warm up to room temperature and stirred for additional 24 hours. When the reaction was completed, the white precipitate was filtered off over Celite. The residue was rinsed with a small amount of CH2Cl2 twice. The combined organic portions were washed with saturated NH4Cl, and dried over MgSO4. Upon evaporation of solvent, methoxy dodecaethylene glycol dioleoyl-L-serinate (i.e., 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-heptatriacontan-37-yl

78

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

dioleoyl-L-serinate) was obtained as pale yellowish oil. (Note: the structure in the original literature was wrong, and corrected here). 3.4.2.1.2 Preparation of Poly(Ethylene Glycol)-2000 Di(tertButyloxycarbonylglycinate) [68]

A mixture of 40.0 g poly(ethylene glycol)-2000 (20 mmol, with 33–34 ethylene glycol unit on average), 7.0 g of tert-butyloxycarbonyl glycine (40 mmol), and 4.9 g of DMAP (40 mmol) in 200 mL of CH2Cl2 was cooled to 0°C. After 10 minutes, 8.2 g of DCC (40 mmol) was added. The reaction mixture was stirred for 14 hours at room temperature. Then the precipitate of DCU was filtered, and the solution was concentrated and precipitated in Et2O. The product was filtered and dried in vacuo to yield 37.6 g of poly(ethyleneglycol)-2000 di(tert-butyloxycarbonylglycinate), in a yield of 81%. 3.4.2.1.3 Synthesis of Bis(Tyrosyl Hydrochloride) Poly(Ethylene Glycol)-6000 Diester (Bis-Tyr·HCl-PEG-6000) [66]

In a 100 mL conical flask, were added 6.0 g of PEG-6000 (1 mmol, 100 ethylene glycol unit in average), 0.435 g of tyrosine hydrochloride (2 mmol) and 10 mL DMF. The contents of the flask were gently heated to dissolve the solids to form a clear solution. To this solution was then added 0.412 g of DCC (2 mmol) in 5 mL DMF in a single portion. The reaction mixture was stirred at room temperature at 25°C for 24 hours. It was then filtered to separate out dicyclohexyl urea formed and the clear solution was poured in 200 mL Et2O to precipitate out white powdery product. The product was isolated and purified by reprecipitation from methanol into Et2O, in a yield of 78%, m.p. 58°C.

The Carboxyl Protecting Groups 79

3.4.2.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF ETHYLENE GLYCOL TYPE GROUP 3.4.2.2.1 Removal of 2-[(2-Methoxy)Ethoxy]Ethyl (MEE) Protecting Group [65]

In a 2 mL of 0.2 M aqueous sodium phosphate buffer (pH 7), containing 200 mg of lipase N or CE was added 3.5 mg of phenylmethylsulfonyl fluoride (PMSF), and the solution was stirred for 1 hour at 0°C to inhibit residual protease activity. After the addition of another 18 mL sodium phosphate buffer solution at 37 °C, the solution was stirred again for 1 hour. To a prepared solution of 200 mg lipase in 20 mL of 0.2 M sodium phosphate buffer (pH 7.0), a solution of 0.5 mmol of peptide ester (one of 2-(2-methoxyethoxy)ethyl (tert-butoxycarbonyl)-L-valyl-L-phenyl-alaninate, 2-(2-methoxyethoxy)ethyl (tert-butoxycarbonyl)-L-seryl-L-phenyl-alaninate, 2-(2-methoxyethoxy)ethyl ((2,2,2-trichloroethoxy)carbonyl)-L-alanyl-L-serinate or 2-(2-methoxyethoxy) ethyl ((benzyloxy)carbonyl)-L-alanyl-L-prolinate) in 2 mL of acetone was added dropwise. Then, the solution was vigorously stirred at 37°C for 24 hours. After saturation with NaCl, the solution was extracted with 25 mL EtOAc five times. The combined organic solutions were dried over MgSO4, and concentrated. The N-protected peptide was obtained by flash chromatography of the residue on 30 g silica gel with petroleum ether/EtOAc as eluent. The peptide was dissolved in Et2O and converted into their dicyclohexylammonium (DCHA) salt. 3.4.3 2-TRIMETHYLSILYLETHYL (TMSE) AND 2-PHENYL-2TRIMETHYLSILYLETHYL (PTMSE) PROTECTING GROUPS 2-Trimethylsilylethyl group (TMSE) can be conveniently introduced into the N-protected amino acid by means of the Steglich Esterification, i.e., treatment of the mixture of N-protected amino acid and trimethylsilylethanol with DCC in the presence of DMAP. Nα-Benzyloxycarbonyl-amino acid trimethylsilylethyl esters can also be prepared in a good yield in the presence of pyridine. The corresponding amino acid trimethylsilylethyl esters are stable under a wide variety of conditions used during coupling and work-up in peptide

80

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

synthesis [69–71]. On the other hand, 2-trimethylsilylethyl (TMSE) group can be readily cleaved by fluoride ion without racemization, preferably using a quaternary ammonium fluoride (e.g., tetrabutylammonium fluoride (TBAF)) in DMF or THF [69–71]. It is reported that the delocalization of electrons on the Si-C bond into the σ*-orbital of the vicinal C-O bond is crucial for the cleavage of TMSE esters [72]. On the basis of TMSE protecting group, and even better β-silicon substituted ethyl protecting group has been developed for carboxyl, i.e., 2-phenyl-2-trimethylsilylethyl (PTMSE) group. This group can be introduced into amino acid under conditions similar to that of TMSE, but it can be cleaved by treatment with TBAF much more rapidly than the TMSE group, leading to fewer side reactions [72]. For example, the cleavage of PTMSE group by treatment of all tested amino acid PTMSE esters in CH2Cl2 was completed within 3–5 minutes, leading to the formation of styrene, trimethylsilyl fluoride and tetra-n-butylammonium salt of carboxylic acid [72]. In addition, the amino acid PTMSE ester can also be prepared with a condensation agent, such as 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyNTP), in CH2Cl2 in the presence of pyridine [73]. Amino acid PTMSE ester is stable under a wide variety of conditions used in peptide synthesis, such as under the coupling conditions, or in the presence of amines (40% MeNH2 in water, morpholine, triethylamine), acetic acid, and to the condition of Pd/C catalyzed hydrogenolysis. However, it is unstable in the presence of trifluoroacetic acid (TFA). With a phenyl group, the amino acid PTMSE ester can be detected under UV light during purification with column and TLC. The 2-phenyl-2-trimethylsilyl ethanol can be prepared from trimethylvinylsilane in two steps, i.e., epoxidation of trimethylvinylsilane with mCPBA followed by the treatment of 2-(trimethylsilyl)oxirane with lithium diphenylcuprate in Et2O [72]. 3.4.3.1 GENERAL PROCEDURE FOR THE PREPARATION OF AMINO ACID PTMSE ESTER [72]

Si

O

O

Ph

DCC/DMAP OH CH2Cl2

OH + H2N R

H 2N

O R

Si Ph

To a solution of amino acid derivative (2 mmol), DMAP (0.2 mmol) and (2-phenyl-2-trimethylsilyl)ethanol (1.95 mmol) in 10 mL CH2Cl2 cooled at 0°C was added 2.2 mmol of DCC. The reaction mixture was initially stirred

The Carboxyl Protecting Groups 81

at 0°C for 30 minutes under an argon atmosphere, then at room temperature for 4 hours. After that, the reaction mixture was filtrated, and the filtrate was diluted with 20 mL CH2Cl2 and washed with saturated NaHCO3 and brine, dried over anhydrous MgSO4 and concentrated. The resulting residue was purified by column chromatography over silica gel (eluent: hexane/EtOAc) to afford amino acid PTMSE ester. 3.4.3.2 TYPICAL PROCEDURE FOR DEPROTECTION OF AMINO ACID PTMSE ESTER [72] O

O H2N

O R

Si Ph

Bu4NF 3H2O CH2Cl2

H 2N

OH R

To a solution of 3 mL CH2Cl2 containing 0.1 mmol of amino acid PTMSE ester, was added 0.3 mmol of TBAF·3H2O. The reaction mixture was stirred for 3–5 minutes at room temperature. After the reaction had been completed (TLC analysis), the reaction mixture was diluted with 10 mL EtOAc and 10 mL H2O. The organic layer was washed with brine, dried over anhydrous MgSO4 and concentrated. The resulting residue was purified by column chromatography over silica gel (eluent: CH2Cl2/MeOH/AcOH/H2O) to give deprotected amino acid derivative. 3.4.4 2-MORPHOLINO-ETHYL PROTECTING GROUP Amino acid 2-morpholino-ethyl ester is formed from the corresponding amino acid 2-haloethyl ester and morpholine, and the 2-morpholino-ethyl protecting group can be cleaved by lipase N in high yield. 3.4.4.1 GENERAL PROCEDURE FOR THE PRODUCTION OF AMINO ACID 2-MORPHOLINO-ETHYL ESTERS [64]

82

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

A solution of 5 mmol 2-bromoethyl (tert-butoxycarbonyl)-L-valylL-phenylalanine in 15 mL morpholine was stirred at 25°C for 1–4 hours. Then morpholine was distilled off under vacuum, the remaining material was taken up in ether and washed with ice-cold 0.5 N HCl solution. The combined aqueous phase was adjusted to pH 9 with sodium bicarbonate and extracted with methylene chloride. The organic phase containing amino acid morpholino-ethyl ester was dried over magnesium sulfate. Upon removal of the solvent under vacuum, the residue was crystallized in little ether to afford MoEt (tert-butoxycarbonyl)-L-valyl-L-phenylalanine. 3.4.5 2-(DIPHENYLPHOSPHINO)ETHYL PROTECTING GROUP The 2-(diphenylphosphino)ethyl group (Dppe) can be easily introduced into amino acid derivatives under the standard Steglich esterification condition, i.e., reaction of amino acid with 2-(diphenylphosphino)ethanol in the presence of DCC and DMAP. The Dppe ester is stable under standard conditions for peptide synthesis. The deprotection is carried out under mild conditions by quaternization of such amino acid ester with methyl iodide followed by a β-elimination induced by fluoride ion or potassium carbonate. 3.4.5.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID DPPE ESTER [74] Step 1: Preparation of 2-(diphenylphosphino)ethanol (DppeOH):   Ph

P Ph

Ph

Ph + Li

THF

P Ph

Li

Cl

HO THF

Ph

P Ph

OH

Under oxygen-free nitrogen atmosphere, dry THF (250 mL) was slowly added to a stirred mixture of 26.6 g triphenylphosphine (0.1 mol) and 3.5 g finely cut lithium wire (0.5 mol) kept at –10°C, and the mixture was stirred at room temperature for 2 hours. In order to eliminate the excess of lithium metal, the solution is transferred to another flask under nitrogen. The solution was cooled again to –10°C, and 0.1 mole of 2-chlorethanol (or 2-bromoethanol) was slowly added to this cooled solution. The mixture was then allowed to reach room temperature and stirred for additional 3 hours. After that, the solution was hydrolyzed, the organic layer was separated and the aqueous layer was extracted with THF, the combined organic layers were dried and evaporated in vacuum. The residue was laid on a 10 cm silica gel

The Carboxyl Protecting Groups 83

column and rapidly eluted with 250 mL of CHCl3-EtOAc mixture (70–30). The concentrated oil was then chromatographed on two silica-gel columns using CHCl3-EtOAc mixture (90–10) as eluent, to afford diphenylphosphine (10–12%) and 2-diphenylphos-phinoethanol (60–65%).   Step 2: Esterification of N-protected amino acids with DppeOH: O

H N

O

OH

+

O

Ph

P Ph

DCC (1.0 eq.) OH DMAP (0.05 eq.) CH2Cl2

Ph P Ph

O

H N

O

O

O

To a solution of 30 mL CH2Cl2 containing 10 mmol of DppeOH, 10 mmol of N-protected amino acid such as ((benzyloxy)carbonyl)-L-alanine and 0.5 mmol of DMAP cooled at 0°C was added 10 mmol of DCC. The mixture was stirred at 0°C for 15 minutes then at room temperature for additional 3 hours. The solution was cooled to 0°C again and repeatedly filtered until complete removal of DCU. The filtrate was washed three times with 10 mL of water, dried over sodium sulfate and concentrated under reduced pressure. The residual oil was rapidly run through a short column of silica (10 cm) to afford 85–95% of amino acid Dppe ester, e.g., 2-(diphenylphosphaneyl) ethyl ((benzyloxy)carbonyl)-L-alaninate (90% yield). Other Dppe esters are 2-(diphenylphosphaneyl)ethyl ((benzyloxy)carbonyl)glycinate (93%), 2-(diphenylphosphaneyl)ethyl (tert-butoxy-carbonyl)glycinate (90%), 2-(diphenylphosphaneyl)ethyl (tert-butoxycarbonyl)-L-alaninate (90%), 2-(diphenylphosphaneyl)ethyl ((benzyloxy)carbonyl)-L-phenyl-alanine (85%), 2-(diphenylphosphaneyl)ethyl (tert-butoxycarbonyl)-L-valinate (80%) and 2-(diphenylphosphaneyl)ethyl (tert-butoxycarbonyl)-L-leucine (85%). 3.4.5.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF DPPE GROUP [74] Step 1: Quaternization of Dppe esters:   H N

O O

O O

Ph P Ph

MeI (2.0 eq.) dry MeOH

H N

O O

Ph P Ph

O O I

To the amino acid Dppe ester, such as 2-(diphenylphosphaneyl)ethyl ((benzyloxy)carbonyl)-L-alaninate, in 5 to 10 times its volume of anhydrous methanol was added two equivalents of methyl iodide, the reaction mixture protected from the moisture was stirred for 8 to 10 hours at room temperature. The solvent was then removed under reduced pressure to give the methyl

84

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

diphenylphosphonium iodide (e.g., (2-((((benzyloxy)carbonyl)-L-alanyl) oxy)ethyl)-(methyl)diphenylphosphonium iodide) in quantitative yield as oil, which was difficult to crystallize. This compound can be obtained as a solid by treating the methanolic reaction mixture with a large volume of ether and stirring the solution continuously for several hours. Under this condition, the yield was about 50%.   Step 2: β-Elimination of Dppe with KF in acetone: H N

O

Ph P Ph

O O

I

O

KF acetone

H N

O

O OH

O

The methiodide of the Dppe ester was dissolved in 10 times its volume of anhydrous acetone and one equivalent of KF was added. The mixture was stirred for 5 or 6 hours at room temperature and the precipitated potassium salt of the amino acid (or peptide) was filtered and washed several times with acetone. It was then dissolved in water and acidified to pH 1 with hydrochloric acid. The solution was extracted several times with dichloromethane and the extract was then dried over Na2SO4. Upon removal of solvent, the residue was recrystallized according to conditions given in the literature, in a yield ranging from 80 to 85%.   Step 3: Alternative β-elimination with K2CO3 in acetonitrile: H N

O O

O O

I

Ph K2CO3 (1.5 eq.) P Ph H2O/CH3CN

H N

O

O OH

O

The methiodide of the Dppe ester was dissolved in 10 times its volume of acetonitrile and 1.5 equivalents of 10% K2CO3 aqueous solution was added. The mixture was stirred for 30 minutes at room temperature followed by solvent evaporation under reduced pressure. The residue was dissolved in water and extracted with dichloromethane to remove the hydroxyethyldiphenyl-methyl-phosphonium iodide. The aqueous layer was acidified to pH 1 and treated as described above. 3.4.6 β-METHYLTHIOETHYL PROTECTING GROUP This protecting group is compatible with the removal of N-o-nitrophenylsulfenyl group (HCl in ethanol), N-trityl (toluenesulfonic acid at 60°C in methanol), N-formyl (1 N or 1.5 N HCl in ethanol), and N-phthaloyl (refluxing with hydrazine hydrate in ethanol) group. The amino acid β-methylthioethyl

The Carboxyl Protecting Groups 85

esters can be formed from the reaction between amino acid and β-chloroethyl methyl sulfide in the presence of a base. The β-methylthioethyl group can be removed in a two-step process, i.e., the reaction of amino acid β-methylthioethyl ester with methyl iodide followed by treatment with a base. 3.4.6.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID β-METHYLTHIOETHYL ESTERS 3.4.6.1.1 Preparation of β-Methylthioethyl Esters of N-Protected Amino Acids without Solvent [75]

The N-protected amino acid such as N-trityl-DL-alanine (0.1 mole) was heated overnight at 65°C with β-chloroethyl methyl sulfide (0.15 mole) and anhydrous triethylamine (0.175 mole). The cooled product was dissolved in chloroform, ether or EtOAc and the solution was washed successively with water, saturated NaHCO3 and water, dried, and evaporated to dryness. The product 2-(methylthio)ethyl N-trityl-alaninate was then recrystallized from EtOH, the yield was 60%, m.p. 97–99°C. 3.4.6.1.2 Preparation of β-Methylthioethyl Esters of N-Protected Amino Acids in EtOAc [75] H N

O O

O + Cl OH

S

Et3N EtOAc, 18 hrs.

H N

O

O O

S

O

The mixture of N-protected amino acid such as ((benzyloxy)carbonyl) glycine (0.1 mole), β-chloroethyl methyl sulfide (0.2 mole), and anhydrous triethylamine (0.15 mole) in 25–50 mL anhydrous EtOAc was heated at 85°C under stirring. The product was cooled, diluted with EtOAc and filtered. The filtrate was worked up as described in the previous procedure. The N-benzyloxycarbonylglycine β-methylthioethyl ester was obtained in 90% yield, m.p. 34.5°C (from light petroleum).

86

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.4.6.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF β-METHYLTHIOETHYL GROUP 3.4.6.2.1 Removal of the β-Methylthioethyl Ester Group [75] H N

O O

O O

S

+ CH3I

H N

O O

O O

S I

  Step 1: Preparation of methiodide: The β-methylthioethyl ester, i.e., 2-(methylthio)ethyl ((benzyloxy)carbonyl)-glycinate (0.005 mole), was treated with an excess amount (5 equivalents or more) of methyl iodide. The methiodide was collected by filtration, after adding ether if necessary, and recrystallized. The β-methylthioethyl ester methiodide of N-benzyloxycarbonylglycine, i.e., 10-methyl-3,6dioxo-1-phenyl-2,7-dioxane-10-thia-4-azaundecan-10-ium iodide was obtained (no solvent, refluxed overnight) in 94% yield, after recrystallization from acetone-ether or ethanol, m.p. 74°C (decom).

  Step 2: Action of alkali on methiodide: The methiodide (0.005 mole) was dissolved in 15 mL of water or aqueous ethanol, and 1 N NaOH was titrated into the solution from a burette at such a rate as to keep the pH at 10–10.5 using a suitable internal indicator. When no more alkali was consumed (5–10 minutes), the solution was acidified and the N-protected amino acid was isolated by filtration or by extraction with a suitable solvent. For the case of N-benzyloxycarbonylglycine, the yield was 87%, m.p. 121°C. 3.4.6.2.2 Removal of β-Methylthioethyl Group without Isolation of Methiodide [75]

The β-methylthioethyl ester (0.01 mole), such as 2-(methylthio)ethyl ((benzyloxy)carbonyl)phenylalaninate, was heated under reflux for 5–6 hours

The Carboxyl Protecting Groups 87

with 10–20 mL of methyl iodide. The product was evaporated to dryness and the residue was triturated with ether. The ether-insoluble methiodide was dissolved in water and titrated with 1 N NaOH or 0.5 N Ba(OH)2 at pH 10–10.5 until no more alkali was consumed. The solution was acidified and the product was isolated by filtration or by extraction. If Ba(OH)2 was used, the equivalent amount of 1 N H2SO4 was added and the product was isolated by lyophilization after removal of BaSO4 by filtration or centrifugation. For the case of N-benzyloxycarbonyl-DL-phenylalanine, the yield was 76%, m.p. 102°C. 3.4.7 CYANOMETHYL PROTECTING GROUP The cyanomethyl group should be classified into α-substituted methyl protecting group, however, as the nitrogen atom in cyanomethyl is at the β-position, this group is considered as the β-heteroatom substituted ethyl group. 3.4.7.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID CYANOMETHYL ESTER Step 1: Preparation of cyanomethyl p-toluenesulfonate [76]:  

To a solution of 130 g of KCN (2 mol) in 250 mL of water-cooled in an ice-salt bath, were added a solution of 170 mL 37% formaldehyde solution (2.0 mol) and 130 mL water under stirring, at such a rate that the temperature never rises above 10°C (ca. 60 minutes). The mixture was cooled to –10°C and a solution of 380 g of tosyl chloride (2.0 moles) and 320 mL acetone (320 mL) were added under vigorous stirring, maintaining the internal temperature between –5 and –10°C (about 3 hours). Simultaneously, the pH of the reaction mixture must be maintained between 7 and 9 by adding a 1 N KOH solution. The flask is then removed from the cooling bath, and after standing overnight, 1,200 mL of water was added with stirring. After 30 minutes, the precipitate was filtered, washed thoroughly with cold water (5 × 50 mL) and dried at room temperature. The crude material melts at 44–47°C and weighs 400 g. It was purified by recrystallization from 600 mL ethanol using 20 g of decolorizing charcoal, under refluxing for 1 hour, filtering, and then cooling the solution in an ice-box. The product was filtered, washed with cold water

88

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

and dried. The recrystallized p-toluenesulfonyl cyanomethyl ester melts at 47–48°C, weighed 272 g. A second crop of 30 g was secured by working up the mother liquor, in a yield of yield 71.5%.   Step 2: Preparation of N-(2-ethoxycarbonyl-cyclopenten-1-yl)glycine cyanomethyl ester: O

O O N H

K O

O O S O

+

O CN

DMF/acetone

N H

O

O

CN

O 94%

A solution of 10 g potassium N-(2-ethoxycarbonyl cyclopentene-1-yl)glycinate (40 mmol) in 25 mL DMF and 50 mL acetone was added 7.0 g cyanomethyl p-toluenesulfonate (33 mmol). After the solution was refluxed for 3 hours, the solution was diluted with 1,200 mL water, and 7.8 g of N-(2-ethoxycarbonyl-cyclopentene-1-yl)-glycine cyanomethyl ester, i.e., ethyl 2-((2-(cyanomethoxy)-2-oxoethyl)amino)-cyclopent-1-ene-1-carboxylate, was separated, in a yield of 94%. 3.4.8 2-(4-NITROPHENYLSULFONYL)ETHYL PROTECTING GROUP There is only a limited application of this particular protecting group, as shown in the following example [77]. O

Ph OH + HO

H 2N O

S

Ph

O

TsOH Benzene, NO 2

NO 2 O

H 2N O

S O O

The glycine and L-phenylalanine 2-(4-nitrophenylsulfonyl)ethyl esters were prepared in good yields (89–93%) by direct azeotropic esterification with 2-(4-nitrophenylsulfonyl)-ethanol in benzene. This protecting group can be selectively removed by alkaline hydrolysis by the action of aqueous potassium hydroxide (pH 10 for 2–3 hours) in aqueous acetone at room temperature (46–85%), or alternatively, by the action of 1,5-diazabicyclo[4.3.0] non-5-ene in benzene (78–89%) to afford the N-protected dipeptides. 3.5 β-KETO ALCOHOL PROTECTING GROUPS Due to the relatively high stability of amino acid methyl ester, the methyl group has been modified by introducing an α-heteroatom on the methyl

The Carboxyl Protecting Groups 89

group as mentioned above. In addition, the methyl group can also be activated by introducing an acyl group linking to the methyl group, i.e., the β-keto alcohol that has been extensively applied to the protection of the carboxyl group in peptide synthesis. There are in general two kinds of β-keto alcohols, the acetol (when acetyl group, CH3CO is used as the β-keto group) and phenacyl (Pac, when benzoyl or substituted benzoyl group is applied as the acyl group). The amino acid ester of acetol is stable under hydrogenolysis (H2, Pd/C) and acidolysis (HCl/dioxane, TFA/CH2Cl2) conditions normally used in peptide synthesis, which can be easily formed under the Steglich esterification condition. However, within 5–45 minutes, the amino acid acetol esters can be deprotected with TBAF in THF [78]. Compared to the acetol group, phenacyl is more versatile as the stability of amino acid phenacyl ester can be further tuned by using phenacyl group with different substituents on the aromatic ring, such as p-methoxyphenacyl [79], α-methylphenacyl [79], p-bromophenacyl [80], and α-phenylphenacyl group [81]. As phenacyl protecting group is much more labile than the methyl group, the amino acid phenacyl ester with a free amino group in the presence of other amines will undergo one or all of the following reactions: (a) decomposition to give free amino acid and phenacyl alcohol, (b) cyclic dimerization to afford 2,5-diketopiperazine and phenacyl alcohol, and (c) the formation of Schiff base between the free amino group and the phenacyl keto functionality [82]. In addition, the electron-withdrawing acyl group in the phenacyl protecting group in some circumstances renders the corresponding amino acid phenacyl ester as acylating agents [80]. Moreover, it is found that the stability of amino acid phenacyl esters is affected by the length of the side-chain and the carbon-chain length between an amino group and a carboxyl group, for that the amino acid phenacyl esters of two to four methylene groups between the amino and carboxyl group are very unstable due to the intramolecular cyclization to form lactam [83]. Interestingly, amino acid phenacyl esters having a bulky side chain such as Leu, lle, Val, and Phe are extremely liable to degrade compared with those having a less bulky side chain such as Gly and Ala [84]. Also, racemization specific to proline and hydroxyproline has been observed when the corresponding amino acid phenacyl esters are used for coupling [85]. Therefore, amino acid phenacyl esters should not be used in excess in peptide synthesis, and often the freshly prepared salts of amino acid phenacyl esters (e.g., hydrochloride [82], trifluoroacetic acid salt [83, 84]) are applied in peptide synthesis. Nevertheless, the phenacyl group is a useful

90

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

carboxyl-protecting group in peptide synthesis because it is easily removable by treatment with zinc powder [82] or magnesium [86] in acetic acid without interfering with other protecting groups, such as Boc, Z, OMe, and OEt. In this case, zinc powder should be preliminarily activated by momentary treatment with 5–10% HCl and the following washing with water, alcohol, and ether. However, as not all peptide phenacyl esters are soluble in acetic acid, it is suggested to carry out this deprotection in DMF or trifluoroethanol in the presence of acetic acid [82]. Due to the potential deblocking of N-Boc group by acetic acid [82], other deprotection methods for phenacyl group have been developed, such as the action of thiophenoxide ion in non-aqueous media under mild conditions (e.g., sodium thiophenoxide in DMF) [80, 87], application of potassium xanthate [80], tetrabutylammonium fluoride (TBAF) [88], and the use of trimethyltin hydroxide (Me3SnOH) [89], bis(tributyltin) oxide (BBTO) in aprotic solvents [90]. It should be pointed out that even though it is necessary to remove the tin impurities from the final product completely, the deprotection of phenacyl group with BBTO does not cause racemization of amino acid and peptide products, and BBTO is a cheap reagent which does not require special handling techniques and equipment [90]. Other special deprotection methods have been developed for a specific type of phenacyl ester groups, such as the application of TBAF·xH2Othiol system for the deprotection of α-phenyl the phenacyl group [81], and photolytic cleavage of p-methoxphenacyl and α-methylphenacyl group which are photosensitive [79]. These two phenacyl groups have been applied to the esters of N-protected analine, glycine, phenylalanine, tryptophan, glycylglycine, and benzyl-aspartylserine [79]. 3.5.1 ACETOL PROTECTING GROUP 3.5.1.1 GENERAL PROCEDURE FOR PREPARATION OF AMINO ACID ACETOL ESTER [78] P

O

H N

OH + R

O OH

DCC/DMAP CH2Cl2

P

O

H N

O R

O

To a solution of suitably protected amino acid (1 mmol), 0.089 g of acetol (1.2 mmol) and a catalytic amount of DMAP (5 mg) in CH2Cl2 at 0°C was added 0.227 g of DCC (1.1 mmol). The reaction mixture was initially stirred

The Carboxyl Protecting Groups 91

at 0°C for 1 hour and then at room temperature overnight. DCU was filtered off and the filtrate was evaporated under reduced pressure. The residue was then taken up in EtOAc, washed successively with 5% aq. citric acid, water, 5% aq. NaHCO3 and finally with brine. The organic layer was dried over Na2SO4 and evaporated to dryness. The crude product was either crystallized from EtOAc/hexane or purified wherever necessary by silica gel column chromatography using EtOAc-hexane (1: 2) as an eluent. 3.5.1.2 GENERAL PROCEDURE FOR DEPROTECTION OF ACETOL [78]

P

O

H N

Bu4NF O

R

THF O

P

O

H N

OH R

A solution of peptide acetol ester in THF was treated with 1.0 M Bu4NF in THF (2–4 equivalents) at room temperature under stirring. After completion of the reaction as monitored by TLC, the reaction was quenched by adding cold water and concentrated under reduced pressure. The residue was then dissolved in EtOAc and washed with 5% aqueous KHSO4 several times and finally with brine. The organic layer was dried over Na2SO4 and evaporated to dryness to afford respective acid in excellent yield. 3.5.2 PHENACYL PROTECTING GROUP Only a few examples of using the phenacyl group as the carboxyl protecting group are available. Typical examples are provided below. 3.5.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID PHENACYL ESTERS 3.5.2.1.1 Synthesis of H-L-Hyp(Bzl)-OPac·HCl [85]

92

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a solution of 6.42 g Boc-L-Hyp(Bzl)-OH (i.e., (2S)-4-(benzyloxy)1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid, 20 mmol) and 4.18 g of phenacyl bromide (21 mmol) in 40 mL of DMF cooled at –10°C was added 2.94 mL of triethylamine (21 mmol) dropwise. Then, the solution was stirred at room temperature for 3 hours, and poured into a 5% NaHCO3 solution. The ester 1-(tert-butyl) 2-(2-oxo-2-phenylethyl) (2S)-4-(benzyloxy) pyrrolidine-1,2-dicarboxylate was extracted with ether, and washed with 1 N HCl and water, dried over MgSO4. Upon removal of the solvent in vacuo, the resulting oil was treated with 50 mL of TFA at –10°C for 10 minutes and then at room temperature for 40 minutes. The TFA solution was concentrated in vacuo to a residue, which was then dissolved in 4.2 mL of 5.7 N HCl in dioxane, and concentrated again. The residue was crystallized by trituration with diisopropyl ether, the crystals were collected by filtration and dried over NaOH pellets to afford 6.41 g of H-L-Hyp(Bzl)-OPac·HCl, i.e., 2-oxo2-phenylethyl (2S)-4-(benzyloxy)pyrrolidine-2-carboxylate hydrochloride, in a yield of 85.2%, m.p. 133–135°C. 3.5.2.1.2 Preparation of Benzyloxycarbonylamino Acid p-Bromophenacyl Esters

To a cooled solution of a benzyloxycarbonylamino acid such as ((benzyloxy)carbonyl)-L-proline (3 mmol) in 2.5 mL of DMF at 0°C was added 0.42 mL of triethylamine (3 mmol), followed by 0.834 g of p-bromophenacyl bromide (4.2 mmol) added. After being stirred at room temperature for 1–2 hours, the reaction mixture was diluted with 1 N NaHCO3, and kept at 0°C for some hours. The product, if solid, was collected, washed with water, dried in vacuo, and recrystallized. For the case of benzyloxycarbonyl-L-proline p-bromophenacyl ester, i.e., 1-benzyl 2-(2-(4-bromophenyl)-2-oxoethyl) (S)-pyrrolidine-1,2-dicarboxylate, was extracted with ethyl acetate, and concentrated in vacuo as an oil, which failed to crystallize on prolonged cooling.

The Carboxyl Protecting Groups 93

3.5.2.1.3 Preparation of N-(9-Fluorenylmethyloxycarbonyl)-LSerine Phenacyl Ester [91]

To a solution of 0.53 g L-serine (5.0 mmol) in a mixed solvent of 10 mL 10% Na2CO3 and 5 mL dioxane cooled in ice was added 1.30 g of 9-fluorenylmethyloxycarbonyl chloride (5.02 mmol) in 7.5 mL dioxane over a period of 1 hour. The reaction was kept at 0°C for one hour and then at room temperature overnight. After that, 150 mL ice water was added and the reaction mixture was washed with Et2O (2 × 40 mL). The aqueous layer was cooled in ice while 2 N HCl was added to adjust pH to 2. The aqueous layer was then extracted with EtOAc (2 × 30 mL). The combined organic phases were washed with 0.1 N HCl (30 mL), dried with MgSO4 and concentrated to an oil. This oily residue (((9H-fluoren9-yl)methoxy)carbonyl)-L-serine was then dissolved in 5 mL DMF and added to a prior prepared solution of 0.64 g KF (11 mmol) and 1.0 g of α-bromoacetophenone (5.0 mmol) in 5 mL DMF. The resulting mixture was stirred overnight, then diluted with 25 mL of Et2O, and washed with water (3 × 20 mL). The organic phase was dried over MgSO4 and concentrated. The residue was purified by column chromatography (toluene/EtOAc 7:3) to afford 1.52 g of N-(9-fluorenylmethyl-oxycarbonyl)-L-serine phenacyl ester, i.e., 2-oxo-2-phenylethyl (((9H-fluoren-9-yl)methoxy)carbonyl)-Lserinate (3.42 mmol), as a white crystalline solid, in a yield of 68%, m.p. 105–108°C. 3.5.2.1.4 General Synthetic Procedure for the Preparation of N-Nosyl-α-Amino Acid Phenacyl Esters [87] O

O 2N O

OH

S NH O

Cs 2 CO 3 EtOH

O

O 2N O

S NH O

O

O

OCs

Br

Ph DMF

O

O 2N O

O

Ph

S NH O

To a solution of 1 mmol N-((4-nitrophenyl)sulfonyl) (Nosyl)-α-amino acid such as ((4-nitrophenyl)sulfonyl)-L-isoleucine in ethanol cooled at 0°C

94

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

was slowly added an aqueous solution of 0.5 mmol Cs2CO3, and the mixture was stirred at room temperature for 1 hour. Then the solvent was evaporated under reduced pressure to afford cesium ((4-nitrophenyl)sulfonyl)-L-isoleucinate as yellow solid in quantitative yield. The solid was then dissolved in DMF and transferred to a DMF solution of phenacyl bromide (1 mmol). During the reaction a white solid of cesium bromide was formed. The reaction mixture was stirred for about 1 hour, monitored by TLC (diethyl ether/ petroleum ether 70:30 v/v). The white solid was then separated by filtration and the solvent was evaporated under reduced pressure. The residue was treated with a 9% aqueous solution of sodium carbonate and extracted with chloroform (3 × 10 mL). The combined organic extracts were washed with water and a saturated aqueous solution of NaCl, dried over Na2SO4. Upon removal of the solvent, the 2-oxo-2-phenylethyl ((4-nitrophenyl)sulfonyl)L-isoleucinate was obtained as a pale-yellow solid, in a yield of 95%, m.p. 191–192°C. 3.5.2.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF PHENACYL GROUP 3.5.2.2.1 Cleavage of p-Bromophenacyl by PhSNa [80] Br

O O Ph

NH

O O

O

O PhSNa DMF

OH Ph

NH

O O

To a solution of 421 mg of N-benzyloxycarbonyl-L-alanine p-bromophenacyl ester, i.e., 2-(4-bromophenyl)-2-oxoethyl ((benzyloxy)carbonyl)L-alaninate (0.001 mmol) in 1 mL DMF was added 0.264 g of sodium thiophenoxide (PhSNa, 0.002 mol). After the solution was stirred at room temperature for 30 minutes, the reaction mixture was diluted with 3 mL 1 N HCl, and extracted with ethyl acetate. The organic layer was washed with water, which was then extracted with 1 N NaHCO3. The combined aqueous layer, upon acidification, was filtered, and the solid was recrystallized from ether/heptane to afford 163 mg of pure N-benzyloxycarbonyl-L-alanine, in a yield of 73%, m.p. 86°C.

The Carboxyl Protecting Groups 95

3.5.2.2.2 General Procedure for Deprotection of N-Methyl-N-Nosylα-Amino Acid Phenacyl Esters [87]

To a stirred solution of 1 mmol N-methyl-N-Nosyl-α-amino acid phenacyl ester, such as 2-oxo-2-phenylethyl N-methyl-N-((4-nitrophenyl)sulfonyl)L-isoleucinate in DMF was added 5 mmol of sodium benzenethiolate with caution. The resulting mixture was maintained under an inert atmosphere (N2) and stirred at room temperature for 30 minutes while monitored by TLC (diethyl ether/petroleum ether 70:30 v/v). After evaporation of the solvent under reduced pressure, the obtained residue was treated with 1 N NaOH solution and extracted with chloroform (3 × 10 mL). The resulting aqueous basic solution was then acidified with a solution of 1 N HCl and then extracted with chloroform (3 × 10 mL). The combined organic extracts were washed with brine and dried over Na2SO4. Upon removal of solvent, 70% of N-methyl-N((4-nitrophenyl)sulfonyl)-L-isoleucine was obtained as a yellow oil. 3.5.2.2.3 General Procedure for the Deprotection of Phenacyl Esters in N-Protected Amino Acids and Dipeptides by Bis(Tributyltin) Oxide (BBTO) [90]

To a solution of 0.3 mL BBTO in 10 mL of toluene or benzene, was added 0.3 mmol of the N-protected amino acid or dipeptide phenacyl ester. The mixture was stirred at 70–100°C for 24–36 hours and monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure. The residue was pre-purified by C-18 reverse-phase silica gel column chromatography (CH3CN/H2O = 70:30) after which, the impure fraction was dissolved in 10 mL of EtOAc (10 mL) and extracted with 5% aq. NaHCO3 (3 × 5 mL). The aqueous phase was acidified to pH 4 with 10% aqueous KHSO4 and extracted with EtOAc (3 × 5 mL). The combined organic phase was washed with brine (2 × 5 mL), and dried over Na2SO4. Upon removal of solvent, the deprotected amino acid or peptide was obtained.

96

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.5.2.2.4 Deprotection of Phenacyl Group with Magnesium and Acetic Acid [86] H N

O O

O

O N H

O O

Mg/CH3CO2H CH3OH/DMF

H N

O O

O

O N H

O

OH CF3CO2H CH2Cl2

H 3N CF3CO2

N H

OH O

To a solution of 0.5 g phenacyl-(N-Boc-L-phenylalanine)-L-leucinate, i.e., 2-oxo-2-phenylethyl (tert-butoxycarbonyl)-L-phenylalanyl-L-leucinate (1 mmol) in 10 mL MeOH/DMF (8:2), were added 0.75 mL of acetic acid (12 mmol) and 160 mg of magnesium turnings (7 mmol). After being stirred at room temperature for 100 minutes, the reaction mixture was filtered. The filtrate was concentrated in vacuo and the residue was diluted with 10 mL 5% NaHCO3 and 10 mL ether-EtOAc (1:1). The organic layer was washed with 5% NaHCO3 (2 × 10 mL). The combined aqueous layers were acidified to pH 2–3 with saturated KHSO4 and extracted with EtOAc (2 × 10 mL). Then the combined organic layers were washed with brine (2 × 10 mL), dried over sodium sulfate and filtered. The filtrate was evaporated and the residue was dried over P2O5-KOH in vacuo for 24 hours. The solid material was dissolved in 0.5 mL dichloromethane and cooled to 0°C, then 0.5 mL of trifluoroacetic acid was added. The reaction mixture was stirred at 25°C for 1 hour and evaporated, the residue was dried over P2O5-KOH in vacuo for 24 hours to afford L-phenylalanine-L-leucine trifluoroacetic acid salt. 3.6 UNSATURATED PROTECTING GROUPS Different from the β-keto alcohol protecting group, the unsaturated protecting groups collected here have an unsaturated carbon-carbon double bond or triple bond attached to the methylene group that forms the ester bond with the carboxyl group of α-amino acids. These unsaturated protecting groups include the simple allyl groups, benzyl or substituted benzyl groups and propargyl group. Among these three kinds of protecting groups, benzyl groups are the most diversified, that consist of simple benzyl groups (including benzyl groups with further substituents on the aromatic ring), biphenylmethyl (or benzhydryl) groups, trityl

The Carboxyl Protecting Groups 97

groups, and fluorenyl groups. All these protecting groups, due to their unsaturated bonds at the β,γ-position, can be further modified or can be easily removed due to such functionality. Especially, all benzyl groups can be easily removed through hydrogenation in the presence of a transition metal catalyst. 3.6.1 ALLYL AND SUBSTITUTED ALLYL PROTECTING GROUPS The allyl protecting group can be introduced into amino acids under the Steglich Esterification condition, like the introduction of normal alkyl protecting groups. However, as the allylic halides are more reactive than alkyl halides in nucleophilic substitution, the amino acid alkyl esters can also be formed via nucleophilic substitution [92]. This protecting group, due to the presence of a double bond, has advantages over many saturated alkyl groups that have been used to protect the carboxyl group in α-amino acids and peptides, as the allyl group can be easily removed through catalytic hydrogenation, in the presence of Pd(PPh3)4 and a proton source [93]. Yet it can also be removed by enzymes, such as esterase from BS2 and its double mutant E188W/M193C that avoids side reactions [92]. In addition, the allyl carboxyl protecting group is compatible with Boc/Bn and Fmoc/tBu strategies [93], and is very useful in solid-phase supported peptide synthesis when amino acids are linked to a solid support via a backbone amide linker (BAL) anchor via reductive amination, with a variety of C-terminal functionalities [94]. The catalytic removal of the allyl group can be performed in nonaqueous solution (e.g., dichloromethane [95] or THF [96] in the presence of PhSiH3), or aqueous solution (e.g., DMSO/ THF/0.5 N HCl) [94]. In addition, the presence of an electrofuge moiety (e.g., trimethylsilyl) α to the allyl group will help the catalytic deprotection taking place under an even mild condition. For example, the 4-(trimethylsilyl)-2-buten-1-ol ester can be catalytically converted to TMS ester with Pd(PPh3)4 which is readily hydrolyzed with alcohol or water [92]. Therefore, a stable allyl ester can be converted to a very labile TMS ester under neutral conditions without the addition of any nucleophilic species. However, the presence of additional PPh3 or the use of solvent which can coordinate with palladium would noticeably decrease the conversion rate to the corresponding TMS ester.

98

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.6.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID ALLYL ESTERS 3.6.1.1.1 Preparation of (S)-1-Allyl 5-Ethyl 2-Benzyloxycarbonylamino-Pentanedioate [92]

To a mixture of 0.16 g Cs2CO3 (0.5 mmol) and 0.31 g of Z-Glu(OEt)-OH, i.e., (S)-2-(((benzyloxy)carbonyl)amino)-5-ethoxy-5-oxopentanoic acid (1.0 mmol) in 5 mL DMF was added a few drops of water. The mixture was distilled under reduced pressure to dryness and the residue was re-distilled twice from DMF (10 mL) until all the water has been removed. The solid cesium salt was stirred with 0.1 mL allyl bromide (1.15 mmol) in 0.6 mL of DMF overnight at room temperature. After removal of DMF, 20 mL of EtOAc was added, and the organic layer was washed consecutively with a saturated solution of NaHCO3, water, and brine, dried over Na2SO4. Upon removal of the solvent under reduced pressure, the residue was purified by column chromatography eluting with CHCl3 to afford 0.25 g of 1-allyl 5-ethyl ((benzyloxy)carbonyl)-Lglutamate, i.e., (S)-1-allyl 5-ethyl 2-benzyloxy-carbonylamino-pentanedioate, as a white solid, in a yield of 73%, m.p. 41–43°C. 3.6.1.1.2 Preparation of Glycine Allyl Ester, Trifluoroacetate Salt (H-Gly-OAllyl·TFA) [94]

  Method A: To a suspension of 8.8 g N-Boc-glycine (50 mmol) in 100 mL dioxane-H2O (4:1), was added a solution of 10.7 g CsHCO3 (55 mmol) in 20 mL H2O over 5 min. After 10 minutes, the suspension was concentrated in vacuo to give a foam, which was suspended in 80 mL of DMF. Then, 4.8 mL of allyl bromide (55 mmol) was added, and the solution was stirred at 25°C for 14 hours, and concentrated to dryness in vacuo. The resultant solid was suspended in 300 mL of EtOAc and washed with 10% aqueous NaHCO3 (3 × 150 mL). The combined aqueous phases were back-extracted with 100 mL EtOAc. The combined organic phases were concentrated in vacuo to an oil

The Carboxyl Protecting Groups 99

(10.6 g) which was purified by vacuum liquid chromatography over TLC-grade Silica Gel 60 G, using EtOAc-hexane (1:4) as the eluent. The resultant oil (9.3 g) was treated with TFA-CH2Cl2 (1:1, 50 mL) for 1 hour to remove the Boc group and then concentrated in vacuo to give an oil, which upon washing with Et2O (3 × 50 mL), provided 7.74 g of off-white crystals, in a yield of 68%, m.p. 95.5–96.5°C.

  Method B: To a solution of 3.0 g N-Boc-Gly-OH (17 mmol) in 25 mL CH3CN-allyl bromide (2:3), was added 5.8 mL diisopropylethylamine (DIPEA, 34 mmol). The mixture was refluxed (ca. 75–80°C) for 90 minutes, at which point TLC [CHCl3/MeOH/AcOH (95:5:3)] indicated complete esterification. The reaction mixture was diluted with 300 mL EtOAc, and washed with 0.1 N aqueous HCl (3 × 100 mL), 5% aqueous NaHCO3 (3 × 100 mL), and brine (3 × 100 mL), dried (MgSO4), and concentrated in vacuo. The resultant yellow oil was dissolved in 50 mL TFA-CH2Cl2 (1:1) and stirred for 1 hour, at which point TLC indicated complete Boc removal. The homogeneous reaction was concentrated in vacuo and chased with Et2O (3 × 50 mL, followed by re-concentration). The residual solid was once again suspended in 100 mL Et2O, filtered and washed with additional Et2O (50 mL) to give 3.3 g of NMR-pure glycine allyl ester trifluoroacetic salt as a white solid, m.p. 103–104°C. 3.6.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF ALLYL GROUP 3.6.1.2.1 Removal of the Allyl Group from Peptide Resin [95]

O O

H N

O

H N O

N H

O 1. PhSiH3/CH2Cl2

O

2. Pd(PPh3)4/CH2Cl2

O

O O

H N

O

H N O

N H

O

O OH

100

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

The allyl protected peptide loaded on resin was washed with dichloromethane (5 × 1 min), then argon was passed through the resin. Under the atmosphere of argon, a solution of PhSiH3 (10.0 equiv.) in dichloromethane was added and the resin was manually stirred. Then, a solution of Pd(PPh3)4 (0.10 equiv.) in dichloromethane was added and the reaction was shaken for 10 min. After that, the peptide resin was washed with CH2Cl2 (8 × 30 seconds), and the process was repeated once to assure completeness of the removal of the allyl group. 3.6.1.2.2 Catalytic Removal of Allyl Group to Form Boc-D-Leu-LLeu-L-threo-O-[Fmoc-L-HyAsn(Trt)-L-Ser(OtBu)]-HyPheL-Leu(OTBS)-L-Leu-D-Arg(Boc)2-L-Ile-L-aThr-Gly-OH [96]

To a solution of 1.3 mg Pd(PPh3)2Cl2 (1.9 μmol) in 237 μL of DMF was added 1.5 mg of triphenylphosphine (6 μmol), and the solution was stirred for 30 minutes. To this solution were added 174 mg of Boc-D-Leu-L-Leu-Lthreo-O-[Fmoc-LHyAsn(Trt)-L-Ser(O-tBu)]-HyPhe-L-Leu(OTBS)-L-LeuD-Arg(Boc)2-L-Ile-L-aThr-Gly-OAll (77 μmol) and 19 μL of phenylsilane (153 μmol) under argon, and the resulting mixture was stirred for 3 hours at room temperature. After that, the reaction was diluted with EtOAc and washed with a saturated solution of aqueous NH4Cl. The organic layer was separated, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel, eluting from 2%–20% methanol in chloroform. The concentration of the pure fractions provided 158 mg of Boc-D-Leu-L-Leu-L-threo-O-[Fmoc-L-HyAsn(Trt)-L-Ser(OtBu)]-HyPhe-L-Leu(OTBS)-L-Leu-D-Arg(Boc)2-L-Ile-L-aThr-Gly-OH as a clear solid, in a yield of 93%.

The Carboxyl Protecting Groups 101

3.6.2 PROPARGYL PROTECTING GROUP The propargyl group is unique when it is used as a carboxyl protecting group in peptide synthesis, as the amino acid propargyl ester is stable when such ester is treated with neat TFA or 20% piperidine in DMF [97]. Thus, the propargyl group is orthogonal to Fmoc and Boc protecting groups. On the other hand, although the propargyl group can be removed under the condition to remove allyl protecting group [with Pd(PPh3)4], the propargyl group can be selectively removed in the presence of an allyl group using the neutral reagent benzyltriethylammonium tetrathiomolybdate, which does not react with other commonly used protecting groups. The common esterification methods for the preparation of amino acid propargyl esters, such as the treatment of amino acid in the presence of an excess amount of propargyl alcohol with SOCl2 or PTSA, are unsuccessful for alanine. Instead, the reaction mixture turns dark, presumably due to the polymerization of propargyl alcohol. However, when alanine was treated with propargyl alcohol saturated with HCl, 72% of alanine propargyl ester was obtained. Other amino acids with β-carbon atoms, such as valine and isoleucine, do not react completely with propargyl alcohol, and the propargyl ester of 2-aminoisobutyric acid (Aib) cannot be prepared. When the amino acid propargyl ester cannot be formed in this way, these esters can be prepared from the Boc derivatives of these amino acids upon removal of Boc with TFA. The resulting trifluoroacetic acid salts of the amino propargyl esters obtained can directly be used for peptide synthesis. Alternatively, propargyl esters of a number of N-protected amino acids have been prepared by treating them with propargyl bromide (DMF, K2CO3, 0°C) in excellent yields. All the conditions for the introduction or deprotection of the propargyl esters do not result in the racemization of the amino acids. 3.6.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID PROPARGYL ESTER 3.6.2.1.1 General Procedure for the Synthesis of Propargyl Esters Under Basic Condition [97]

To a 10 mL solution of 5 mmol N-protected amino acid (Boc, Fmoc or Cbz) in anhydrous DMF cooled at –10°C, was added 0.69 g of anhydrous

102

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

K2CO3 (5 mmol). The mixture was stirred until a syrupy solution was formed. Propargyl bromide (0.55 mL of 80% solution in toluene, 5 mmol) was added dropwise to the reaction mixture, and the stirring was continued at –10°C for 1 hour. The reaction mixture was then allowed to attain room temperature, and DMF was removed under vacuum. The residue was transferred to a separating funnel with 50 mL of EtOAc, and washed subsequently with a saturated citric acid solution (2 × 25 mL), water (2 × 25 mL), and brine (25 mL). After being dried over anhydrous Na2SO4 and concentrated, the crude product was then purified by column chromatography (silica gel, 100–200 mesh) using a solution of ethyl acetate (10–30%) in petroleum ether as eluent. 3.6.2.1.2 General Procedure for the Synthesis of Propargyl Esters Under Acidic Condition [97]

Dry HCl was bubbled through propargyl alcohol (20 mL) at 0°C for 1 hour (Note: this may waste a lot of HCl, acetyl chloride can be added to propargyl alcohol to create HCl instead). Then 5 mmol of amino acid was added to this propargyl alcohol at 0°C, and the resulting mixture was stirred at room temperature for 12 hours. The excess of propargyl alcohol was removed under vacuum and the residue was washed with anhydrous diethyl ether (10 × 25 mL), dried under vacuum, and stored in a desiccator. The yield was from 65% to 90%. 3.6.2.2 GENERAL PROCEDURE FOR THE DEPROTECTION OF PROPARGYL ESTERS [97]

  Step 1: Preparation of benzyltriethylammonium tetrathiomolybdate: Ammonium molybdate (10 g) was dissolved in a mixture of ammonium hydroxide (60 mL) and water (20 mL), and the solution was filtered. Hydrogen sulfide was bubbled rapidly into the

The Carboxyl Protecting Groups 103

solution at room temperature until the solution was saturated with H2S. Then the temperature was raised to 60°C, while maintaining a slow stream of H2S. After 60 minutes, the mixture was cooled to 0°C and kept under refrigeration for 30 minutes. The granular product thus obtained was isolated by filtration. The crystalline solid was washed with isopropyl alcohol (2 × 25 mL), ether (4 × 25 mL), and dried under vacuum to afford 13.4 g of brick red crystals of ammonium tetrathiomolybdate, in a yield of 92%.   A solution of 23.31 g of benzyl triethylammonium chloride (102.5 mmol) in 60 mL distilled water was added in portions over 30 minutes to a well-stirred solution of ammonium tetrathiomolybdate (13 g, 50 mL) in 60 mL distilled water. The reaction mixture was vigorously stirred at room temperature for 2 hours, and the solid that separated was filtered, washed with isopropyl alcohol (2 × 40 mL), and ether (4 × 40 mL). The brick-red powder of benzyltriethylammonium tetrathiomolybdate was dried under vacuum and stored in a desiccator (24 g, 80%).   Step 2: Deprotection of propargyl esters: To a solution of 1 mmol amino acid propargyl ester in 5 mL of acetonitrile, was added 0.67 g of benzyltriethylammonium tetrathiomolybdate (1.1 mmol) at room temperature, and the reaction mixture was stirred for 2 hours. After that, acetonitrile was removed under vacuum, and the residue was mixed with a mixture of ethyl acetate and chloroform (9: 1). The crude product was purified by column chromatography (silica gel, 100–200 mesh) eluting with a solution of ethyl acetate in petroleum ether or methanol in chloroform. The yield in general was more than 80%. 3.6.3 BENZYL AND SUBSTITUTED BENZYL PROTECTING GROUPS 3.6.3.1 BENZYL PROTECTING GROUPS Benzyl (Bn) is one of a few widely used carboxyl protecting groups due to its relative stability towards acid and easy removal through catalytic hydrogenation [98]. However, employment of this protecting group commonly requires the use of acidic (e.g., PTSA [99–101]) or basic reaction media and/ or otherwise non-mild reaction conditions, which sometimes are not compatible with other functional groups in the molecule. Recently, a commercially available 2-benzyloxypyridine developed by Dudley that can be applied for the benzylation of any acid-sensitive substrate has been applied to prepare

104

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

amino acid benzyl esters. This reagent, known as the Dudley reagent, is trivial to prepare, and stable indefinitely to storage. This reagent can offer high yields of carboxylic acid benzyl esters in the presence of alcohols, phenols, protected amines, acetals, and other functionality [98, 102]. The pyridine byproduct can be easily removed as it is water-soluble. Usually, amino acids and two equivalents of the Dudley reagent and triethylamine (the optimal acid scavenger for the benzylation of carboxylic acid) in trifluoromethylbenzene (PhCF3) are stirred at 83°C for 24 hours to afford high yields of amino acid benzyl esters [102]. No evidence of competing benzyl ether formation or base-promoted elimination of the β-alcohol has been observed for such alternative benzylation. The mechanism for the benzylation with the Dudley reagent is illustrated in Scheme 3.2. It should be pointed out that another similar reagent, i.e., 2-((4-methoxybenzyl) oxy)-4-methyl quinoline, for the convenient formation of p-methoxybenzyl ether or ester is also known as the Dudley reagent [103]. Acidolysis study on synthetic peptide benzyl ester indicates a sharp changeover point in the mechanism from SN2 to SN1 in a ternary mixture of trifluoromethanesulfonic acid (TFMSA)-trifluoroacetic acid (TFA)-dimethyl sulfide (DMS), when the concentration of TFMSA in TFA is increased [104]. Therefore, peptides with nucleophilic side chains are strongly susceptible to react with the carbocations generated under the SN1 deprotection condition, including alkylation, arylation, and acylation side products of methionine, cysteine, tryptophan, tyrosine, aspartic acid, and glutamic acid. Due to this shortcoming, catalytic transfer hydrogenation is often applied for the removal of the benzyl group, using cyclohexene and 1,4-cyclohexadiene as the hydrogen donors [105]. Especially, 1,4-cyclohexadiene greatly facilitates the deprotection of peptide benzyl and benzyloxycarbonyl derivatives and allows for more rapid deprotection at a lower temperature [105]. Compared to cyclohexene and 1,4-cyclohexadiene, formic acid is a better hydrogen source as the nonpolar cyclohexene and 1,4-cyclohexadiene are immiscible with most peptides or peptide derivatives. For example, N-(benzyloxycarbonyl)lysine in the presence of an equal weight of palladium black in 88% formic acid is completely deprotected in 30 seconds at room temperature. Hydrogenation with lower concentrations of formic acid in methanol also leads to the rapid removal of benzyl and benzyloxycarbonyl protecting groups but reduces the possibility of removal of acid-labile protecting groups. Besides Pd/C, SiliaCat, a sol-gel organosilicate-based matrix loaded with entrapped palladium nanoparticles has also been tested as a good hydrogenation catalyst, which is easy to handle, non-pyrophoric, leach-proof, recoverable, and solvent-independent

The Carboxyl Protecting Groups 105

[106]. Like alkyl protecting groups, the benzyl protecting group can also be removed via enzyme hydrolysis, by using an esterase from BS2 and lipase from CAL-A under mild conditions [13].

SCHEME 3.2 Formation of amino acid benzyl ester involving the Dudley reagent.

Besides the simple benzyl group, many benzyl-related carboxyl protecting groups have been developed, due to the expandability of the benzyl group by introducing a variety of substituents on the aromatic ring. These benzyl groups can be divided into two groups, i.e., benzyls with either electron-donating or electron-withdrawing groups. The presence of these extra functional groups on the benzyl moiety regulates the stability of resulting amino acid benzyl esters and the removal of the substituted benzyl esters, as the ease of acidic cleavage of substituted benzyl esters is related to the stability of the corresponding carbonium ions [107]. Overall, the reported substituted benzyl groups with electron-donating groups include p-methylbenzyl [107], 2,4,6-trimethylbenzyl [107–112], pentamethylbenzyl [107], p-methoxybenzyl (anisyl) [100, 111, 113], azidomethoxybenzyl ester (ABz) [114], p-methylthiobenzyl [115, 116], and 4-((1-(4,4-dimethyl2,6-dioxocyclohexylidene)-3-methylbutyl)amino)benzyl (Dmab) [117–122]. The substituted benzyl groups with electron-withdrawing groups are p-chlorobenzyl [123], 4-nitrobenzyl [124], o-cyanobenzyl [125], p-methylsulfinylbenzyl (Msib) [115, 116], 4-sulfobenzyl [126], p-methoxycarbonylbenzyl [127], and p-(p-(dimethylaminophenylazo)benzyl [128]. Among these

106

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

substituted benzyl groups, the ones with electron-donating groups such as 2,4,6-trimethylbenzyl, 4-methoxybenzyl (Mob) and Dmab and the ones with electron-withdrawing groups such as 4-nitrobenzyl and 4-methylsulfinylbenzyl are especially common. 2,4,6-Trimethylbenzyl esters of amino acids often form good crystals during purification [112]. The three electron-donating methyl groups render 2,4,6-trimethylbenzyl a useful acid-labile carboxyl-protecting group which differs from the widely used benzyl ester system in that it can be readily removed along with benzyloxycarbonyl from the protected peptides by mild treatment with hydrogen bromide in acetic acid whilst leaves benzyl esters largely intact [108]. In addition, 2,4,6-trimethylbenzyl esters can also be cleaved selectively by cold trifluoroacetic acid without affecting benzyloxycarbonyl, formyl, or phthaloyl (Pht) amino-protecting groups present [109]. Like benzyl group, 2,4,6-trimethylbenzyl group is also subject to basic hydrolysis, but this option is rarely applied in peptide synthesis [112]. In addition, 2,4,6-trimethylbenzyl can be removed by catalytic hydrogenation as well, such as the hydrogenation over 10% palladium on charcoal in methanol [112]. Compared to 2,4,6-trimethylbenzyl group, Mob group with a strong electron-donating group at the para position of the aromatic ring, can also form a stable cation during acidic cleavage. The corresponding amino acid anisyl ester has been prepared via direct esterification with Mob alcohol in the presence of PTSA (azeotropic distillation) [100], or in the presence of a condensation reagent (DCC) [129]. However, due to the acid-labile nature of the anisyl group, the azeotropic distillation of amino acid with Mob alcohol requires a large excess of such alcohol while still offering the ester of very low yield [100]. The alternative imidazole-promoted condensation of the corresponding amino acid 4-nitrophenyl esters with Mob alcohol is limited to glycine and peptides bearing a glycine residue as C-terminal due to potential racemization. Therefore, better yields of amino acid Mob esters can be obtained by the indirect route, involving the interaction of Mob chloride or bromide with the silver (or ammonium) salts of N-(2-nitophenylthio)amino acids in chloroform [100] or with the sodium salt of the corresponding amino acid copper complex. In order to increase the yield of amino acid anisyl esters, two other methods have been applied. The first method involves the use of the pre-formed reagent N,N-diisopropyl-O-(4-methoxybenzyl)isourea under very mild conditions, allowing selective protection of carboxylic acids in the presence of other functionalities such as enolizable ketones and alcohol groups [113]. The second method consists of converting N-nitroso derivatives of amino acids into the oily p-methoxybenzyl esters by treatment

The Carboxyl Protecting Groups 107

of amino acids with p-methoxybenzyl chloride in dimethylformamide (DMF) in the presence of triethylamine at room temperature, followed by rapid cleavage of the nitroso group with HCl in dioxane [111]. Similar to the 2,4,6-trimethylbenzyl group, the Mob ester group can also be cleaved by brief action of cold trifluoroacetic acid without removal of benzyl group. Dmab, the abbreviation of 4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methylbutyl)amino)benzyl group [119], is compatible with Fmoc/tert-butyl strategy and will not be cleaved during the final TFA treatment [121]. This group possesses several advantages over the allyl group, including facile deprotection with hydrazine [117] and easy monitoring of the deprotection process [119]. As amino acid Dmab ester is readily cleaved using 2% v/v hydrazine monohydrate in DMF at room temperature within minutes, it negates the need for expensive palladium catalysts. In addition, such deprotection reaction generates a UV active indazole moiety, helping the track of the reaction progress spectrophotometrically [119]. The cleavage of the Dmab is suggested to involve a mechanism as shown in Scheme 3.3 [118, 119]. However, the hydrophobic nature of this protecting group renders the corresponding amino acid ester poor solubility in aqueous solution, which can be a drawback for achieving high concentrations required for the native chemical ligation (NCL) [121]. In addition, the Dmab group, when used for temporary protection of the glutamyl side-chain acid functionality, is prone to two deleterious side-reactions. These two drawbacks include the generation of truncated Nα-pyroglutamyl peptides via intramolecular cyclization either during Nα-Fmoc removal or subsequent coupling reactions, and the presence of the 4-aminobenzyl ester due to the slow 1,6-elimination reaction of the 4-aminobenzyl group derived from hydrazine treatment of Glu(ODmab), and the latter precludes activation of the glutamyl side-chain functionality [118]. O H N O

O

NH 2 NH 2 O

R spontaneous

N

H N

NH 2 + O

O

NH 2

O R

O

O R

SCHEME 3.3 Deprotection of the Dmab group via hydrazinolysis.

In contrast, the 4-nitrobenzyl group with a strong electron-withdrawing nitro group, causes the corresponding amino acid ester to form a stable

108

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

crystalline compound. As a result, the 4-nitrobenzyl group cannot be selectively cleaved by either hydrogenolysis or acidolysis in the presence of unsubstituted benzyl ester, benzyloxycarbonyl, and Boc groups [130]. In addition, the amino acid 4-nitrobenzyl esters can tolerate the treatment with hydrogen fluoride-anisole that is often used to remove other protecting groups other than β-phenacyl [124]. These esters can be prepared in 80–95% yields with little racemization by the addition of aqueous Na2S2O4-Na2CO3 to the ester in MeCN at 40°C. Although 4-nitrobenzyl esters are difficult to be cleaved via acidolysis, they can be removed by the treatment of Zn/HCl, Zn/ AcOH and Na2S [130]. The advantage of using 4-nitrobenzyl as the carboxyl protecting group probably is its easy cleavage under photo-irradiation [131]. It is found that the cleaving rate can be dramatically enhanced when there are two alkoxy substituents on the phenyl ring which parallel the increase in absorbance at 365 nm. A further increase in rate was observed by the introduction of a methyl group at the benzylic carbon [132]. Accordingly, different types of substituted nitrobenzyl groups have been developed, including nitrobenzyloxycarbonyl (Nboc), 2-nitroveratryloxycarbonyl (Nvoc), nitropiperonyl-oxycarbonyl (Npoc), α-methyl-nitroveratryloxycarbonyl (Menvoc) and α-methyl-nitropiperonyloxycarbonyl (Menpoc), as shown in Figure 3.1. Among these carboxyl protecting groups, the corresponding esters of amino acid with Menvoc and Menpoc offer both faster photo-kinetics and higher quantum yields than the known Nboc and Nvoc groups. The photolysis of the nitropiperonyloxycarbonyl (Npoc) group is particularly enhanced in acidic dioxane [132]. NO2 NO2 O

R

O

R

NO2 O

R

O

R

R MeO

MeO

O OMe

Nboc

NO2

NO2 O

O OMe

O Npoc

Nvoc

Menvoc

O Menpoc

O R=

N H

CO2Et

or COCH3

FIGURE 3.1  Representative 2-nitrobenyl protecting groups.

In comparison to 4-nitrobenzyl group, Msib group also with an electronwithdrawing group, renders the corresponding amino acid Msib ester exceptional stability towards acidolysis. For example, the Msib ester of phenylalanine is stable to TFA treatment, and only a trace amount of the cleaved product phenylalanine (3.3%) has been detected after two months

The Carboxyl Protecting Groups 109

[116]. In addition, the Msib ester is relatively stable to reduction. Peptide or amino acid Msib esters can be readily prepared from the corresponding alcohols or alkyl halides, or from the oxidation of amino acid p-methylthiobenzyl ester with m-chloroperbenzoic acid, which can also be prepared from amino acid and p-methylthiobenzyl alcohol or halide [115]. Although the amino acid Msib esters are quite stable to acidolysis, they can be easily deoxygenated into amino acid p-methylthiobenzyl (Mtb) esters with excess Me3SiCl/Ph3P, Me3SiCl/Me2S, or anhydrous hydrogen chloride, and the resulting esters can be rapidly solvolyzed in trifluoroacetic acid within 30 minutes [115]. Besides the above substituted benzyl groups, the acid-labile 4-(4-phenylazo) benzyloxybenzyl (Abz) esters of various amino acids are easily obtained and cleanly cleaved in very mild reductive conditions [114]. While the chromogenic p-(p-dimethylamino)phenylazobenzyl ester group readily withstands successive treatments with 25% trifluoroacetic acid in dichloromethane yet it is cleanly removed by catalytic hydrogenation [128]. Both Abz and p-(p-dimethylamino)phenylazobenzyl groups with intense color allow facile purification of the protected esters on silica gel or ion exchange column chromatography as well as TLC monitoring. The p-chlorobenzyl ester of glutamic acid was found to be significantly more stable to trifluoroacetic acid cleavage than the benzyl ester, and yet it could be removed without difficulty by liquid HF at 0°C [123]. Therefore, p-chlorobenzyl group is recommended for side-chain protection of aspartic acid and glutamic acid residues. Yet another substituted benzyl group with an electron-withdrawing group, i.e., 4-sulfobenzyl, combines the advantages of the solubilizing sulfo- and the protecting benzyl function [126]. This new protecting group is introduced into amino acids by means of sodium 4-(bromomethyl)benzene-sulfonate into cesium or DCHA salts of N-protected amino acids and peptides. After N-terminal deblocking, the amino acid 4-sulfobenzyl esters are isolated as zwitterions, which are stable to 2 M HBr in acetic acid as well as to a 10-fold excess of TFMSA in trifluoroacetic acid. The protecting group was removable by catalytic hydrogenation or saponification. 3.6.3.2 α-SUBSTITUTED BENZYL GROUPS As mentioned for the 4-nitrobenzyl, the introduction of an α-methyl group at the benzylic position can further enhance the photolytic rate of the corresponding amino acid ester. Benzoin and substituted benzoin with structures analogous to the benzyl group have been employed as photo-removable

110

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

protecting groups for carboxylic acids. Among them, the application of benzoin as a traceless linker for solid-phase synthesis of oligopeptides and the introduction of Fmoc-protected amino acids are especially impressive [133]. As an example, the release of Fmoc-β-alanine shown in Scheme 3.4 occurs upon photolysis at 350 nm with a maximal yield after 2 hours of photolysis.

SCHEME 3.4 The protection of carboxyl with an α-substituted benzyl group and its deprotection.

Besides benzoin as the α-substituted benzyl group, diphenylmethyl (also known as benzhydryl [36], Dpm [101]) is another popular protecting group for amino acids, which can form an even more stable carbocation during acidolysis. Several methods have been applied for the preparation of amino acid benzhydryl esters, including the Mitsunobu condensation of N-tritylamino acids with diphenyl-methanol using excess triphenylphosphine and diethyl azodicarboxylate [134], reaction of amino acid with diphenylmethyldiazomethane [135], treatment of amino acid silver salt with diphenylchloromethane, and refluxing a dichloromethane solution of N-protected amino acid and tri-diphenylmethyl phosphate under the catalytic effect of trifluoroacetic acid [135]. The deblocking of Dpm group can be easily achieved by the action of dilute solution of HCl or HBr in nitromethane [136], by trifluoroacetic acid [136], or by catalytic hydrogenolysis [36]. Similar to 4-nitrobenzyl group, the introduction of nitro group to benzhydryl moiety would allow the corresponding amino acid ester suitable for photolytic cleavage. For example, N-methyl-D-aspartic acid 2,2’-dinitrobenzhydryl (DNBzh) ester can release N-methyl-D-aspartate (NMDA) within 4.2 µs under a single 308 nm laser pulse irradiation, with a quantum efficiency of 0.18 [137]. It is found that the time constant for the release of NMDA is pH-dependent, occurring within 3.8 μs at pH 3.8 and 13.8 μs at pH 10.6.

The Carboxyl Protecting Groups 111

However, the poor aqueous solubility of DNB-NMDA requires addition of 20% DMSO to attain complete dissolution. 9-Fuorenyl, as a variant of Dpm group where two phenyl groups are directly linked together, can also be used as the carboxyl protecting group. The 9-fluorenyl esters of N-protected or N-free amino acids are easily obtained by the action of 9-diazofluorene on the amino acids in an appropriate organic solvent. The resulting esters can be cleanly cleaved by mild acidolysis or hydrogenolysis [41]. Other aromatically substituted benzyl groups include α-pyrenyl-benzyl [138] and porphyrin-methyl groups [139]. The amino acid α-pyrenylbenzyl ester can be prepared from amino acid and 1-(α-diazobenzyl)pyrene which is very stable and easy to handle. The resulting ester is stable in MeCN containing HCl for at least four hours without a significant amount of decomposition, but completely decomposed in 2 weeks [138]. Likewise, the amino acid porphyrin-methyl ester can be prepared in quantitative yield between amino acid and hydroxymethyl-porphyrin in the presence of coupling reagents, and the reaction can be easily monitored by TLC. The esters are highly soluble in most organic solvents, and readily cleaved by an acid to give hydroxymethyl-porphyrin and amino acids [139]. Finally, 4-picolyl (pyridin-4-ylmethyl) as a heteroatom-substituted benzyl group, has also been applied as the carboxyl protecting group [140]. 3.6.3.3 EXEMPLARY EXPERIMENTAL PROCEDURES FOR PREPARATION OF AMINO ACID BENZYL ESTER 3.6.3.3.1 General Preparation of Amino Acid Benzyl Ester Under Microwave Irradiation [99]

A mixture of 0.891 g alanine (10 mmol), 3 mL of benzyl alcohol and 1.89 g of PTSA (11 mmol) was placed in a 100 mL glass beaker and exposed to microwave irradiation operating at its 40% power. The reaction mixture, after the completion of the reaction, was cooled to room temperature and precipitated with 25 mL ether. The crystalline p-toluenesulfonate salt of

112

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

alanine benzyl ester was collected on a filter, washed with ether and dried in air to afford 95% of yield, m.p. 115–117°C. 3.6.3.3.2 Preparation of L-Serine Benzyl Ester Benzenesulfonate (L-Ser-OBn·PhSO3H) [141]

A mixture of L-serine (21.02 g, 200.0 mmol), technical-grade benzenesulfonic acid (45.0 g; 90%, 256 mmol), and benzyl alcohol (100 mL) in 250 mL of carbon tetrachloride was distilled gently until no more water formed (ca. 6–8 hours). Carbon tetrachloride was added periodically to maintain the solvent level. After removal of the remaining solvent by distillation under reduced pressure, 200 mL of diethyl ether was added to the reaction mixture with vigorous shaking. Storage of the resulting oil at 4°C for 24 hours gave a solid product, which was collected, washed with cold ether, and dried. Recrystallization from 2-propanol-anhydrous diethyl ether yielded 52.29 g of L-serine benzyl ester benzenesulfonate as a white powder (124.3 mmol), in a yield of 62.1%, m.p. 97–98°C. 3.6.3.3.3 Preparation of N-Boc-L-Serine Benzyl Ester with Dudley’s Reagent [102]

A mixture of 150 mg 2-benzyloxy-1-methyl pyridinium triflate (0.43 mmol), 460 µL of PhCF3, 65 µL of NEt3 (0.46 mmol), and 48 mg of N-BocL-serine (0.23 mmol) was heated at 83°C for 1 day. Then the reaction mixture was cooled to room temperature, diluted with 5 mL of H2O and extracted with EtOAc (2 × 10 mL). The combined organic phase was washed with H2O (10 mL) and brine (10 mL), dried over MgSO4, filtered, and concentrated under a vacuum. The residue was purified by flash chromatography on silica gel to afford 61 mg of N-Boc-L-serine benzyl ester as a colorless oil, in a yield of 91%.

The Carboxyl Protecting Groups 113

3.6.3.3.4 Preparation of L-Proline 2,4,6-Trimethylbenzyl Ester Hydrochloride [111]

A solution of 2.18 g N-nitroso-L-proline (15 mmol) and 2.12 mL of triethylamine (15 mmol) in 7.7 mL of DMF was treated with 2.56 g of chloromethylmesitylene (15 mmol). After being stirred at room temperature for 1–2 days, the mixture was diluted with water. The oily precipitate of N-nitroso-proline 2,4,6-trimethylbenzyl ester soon solidified to afford 3.89 g of product, in a yield of 92%. The crude nitroso compound was treated with 4 N hydrogen chloride in 35 mL of dioxane for 10 minutes, and most of the solvent was then removed under vacuum. Addition of ether gave the crystalline ester hydrochloride, which was recrystallized from ethanol-ether, m.p. 156.5–157°C. The overall yield from L-proline was 61%. 3.6.3.3.5 Preparation of N-Benzyloxycarbonyl-L-Alanine 4-Methoxybenzyl Ester [100]

To a cold solution of 2.23 g N-benzyloxycarbonyl-L-alanine (0.01 mol) in 10 mL of chloroform were added 1.4 mL of triethylamine (0.01 mol) and 2.01 g of Mob bromide (0.01 mol). The mixture was stirred at room temperature in the dark for 24 hours, then chloroform was added. The solution was washed with water, aqueous potassium hydrogen carbonate, and water again, dried and concentrated to dryness. Light petroleum ether was added to the syrupy residue, and crystallization was induced by cooling and scratching in EtOAc-light petroleum ether to afford 2.0 g of N-benzyloxycarbonyl-Lalanine Mob ester, in a yield of 59%. 3.6.3.3.6 Preparation of N-Benzyloxycarbonyl Phenylalanine p-Methylsulfinylbenzyl Ester (Z-Phe-OMsib) [115]   Step A: Preparation of N-benzyloxycarbonyl phenylalanine p-methylsulfinylbenzyl ester (Z-Phe-OMtb):

114

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

A solution of 299 mg N-benzyloxycarbonyl phenylalanine (1 mmol), 616 mg of p-methylthiobenzyl alcohol (4 mmol), 211 mg of 1-ethyl-3-[3(dimethylamino)-propyl]carbodiimide (EDCI, 1.1 mmol), and 168 mg of HOBt (1.1 mmol) in 20 mL of CHCl3 was stirred at 25°C for 2 days. The solution was then washed with 1 N HCl (2 × 100 mL), 10% Na2CO3 (2 × 100 mL), and H2O (50 mL). The aqueous solutions were extracted with fresh CHCl3 (50 mL). The combined CHCl3 solution was dried over anhydrous Na2SO4, and rotary evaporated to an oil. Crystallization in 50% hexane/Et2O afforded 339 mg of N-Z-phenylalanine p-methylthiobenzyl ester as a hard white powder, in a yield of 78%, m.p. 62–65°C.   Step B: Conversion of Z-Phe-OMtb into Z-Phe-OMsib:

To a solution of 0.710 g Z-Phe-OMtb in 50 mL of CH2Cl2 at 0°C was added dropwise a solution of 0.308 g mCPBA (1.79 mmol) in 10 mL of CH2Cl2. After 30 minutes, the solution was allowed to warm to 25°C. After 2 hours, 50 mL of water was added and CH2Cl2 was partially removed (up to 10 mL) by rotary evaporation. The mixture was extracted with 50 mL of Et2O. The ether solution was then washed with 30 mL of 10% Na2CO3 and 10 mL H2O, dried over anhydrous Na2SO4, and rotary evaporated to an oil. The oil was separated by a silica gel column packed in CHCl3 with an eluent of CHCl3/AcOH (95: 5) to afford 0.37 g of Z-Phe-OMsib as a colorless oil, in a yield of 50%. Also, this ester can be directly prepared from Z-phenylalanine and Msib alcohol in the presence of EDCI and HOBt, following the procedure described above to afford 98% of yield. 3.6.3.3.7 Preparation of N-Tritylglycine Diphenylmethyl Ester [136]

The Carboxyl Protecting Groups 115

To a solution of 10.2 g N-tritylglycine (0.032 mol) in 30 mL 1 N NaOH was added an aqueous solution of silver nitrate (5.6 g, 0.03 mole). The silver salt formed was filtered off, washed with cold water, and dried in a desiccator over P2O5. The total yield of salt was suspended in 100 mL of chloroform, and 6.1 g of Dpm chloride (0.03 mol) was added. The mixture was stirred at room temperature for about 20 hours and then refluxed for 1 hour. The silver chloride formed was removed by filtration through Celite, and the filtrate was evaporated to dryness. The crystalline residue was triturated with cold methanol and recrystallized from ethyl acetate, giving 7.2 g of N-tritylglycine Dpm ester, in a yield of 50%, m.p. 139–140°C. 3.6.3.4 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF BENZYL PROTECTING GROUPS 3.6.3.4.1 Removal of 4-Methylsulfinylbenzyl Group [115]   Step A:

To a solution of 1 mL freshly distilled anhydrous THF containing 0.04 mmol of TFA-(2,6-Cl2Bzl)Tyr-D-Ala-Gly-Phe-Met-OMsib and 117 µL of Me2S (1.6 mmol) was added 101 µL of Me3SiCl (0.80 mmol) under argon atmosphere while stirring. A white precipitate that initially formed slowly dissolved with stirring over 30 minutes. After 4 hours, 5 mL of EtOH was added and the solution was stirred for 10 minutes. After rotary evaporation, the resulting residue was dissolved in 5 mL of ethanol and rotary evaporated after stirring for 10 minutes. The resulting residue was dissolved in EtOH (5 mL) and CCl4 (5 mL) and rotary evaporated until a white precipitate formed. The precipitate was triturated with fresh CCl4 and dried to afford 42.5 mg of TFA-(2,6-Cl2Bzl)Tyr-D-Ala-Gly-Phe-Met-OMtb as a white powder, in a yield of > 100% (impure at this stage).

116

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

  Step B: Removal of the 4-methylthiobenzyl group:

To a mixed solution of 0.5 mL CHCl3 and 0.5 mL Me2S containing 0.04 mmol of TFA-(2,6-Cl2Bzl)Tyr-D-Ala-Gly-Phe-Met-OMtb was added 4 mL solution of 50% Me2S/TFA under stirring. After 1 hour, 4 mL of CHCl3 was added and the solution was washed with 40 mL of H2O. The water layer was back-extracted with CHCl3 (2 × 5 mL). The CHCl3 solutions were combined, and 10 mL of MeOH and CCl4 each were added, and the mixture was rotary evaporated to afford 95% of TFA-(2,6-Cl2Bzl)Tyr-D-Ala-Gly-Phe-Met-OH as a white powder. 3.6.3.4.2 Synthesis of N-Linked Glycopeptide-Ac-Asn(β-GlcNAc)-GlyAla-Tyr-Ser-OH [119]   Step A: Deprotection of Dmab:

A 3 mL solution of 2% hydrazine monohydrate in DMF (v/v) was added a protected pentapeptide [(γ-Dmab)-N-acetyl]-Asp-[N-(2,4-dimethylbenzyl)]Gly-Ala-(O-t-butyl)-Tyr-O-t-butyl-Ser) loaded to the Wang resin, and the mixture was shaken for 3 minutes. This procedure was repeated a total of five times. Removal of the ivDde moiety was determined by measuring the UV absorbance at 290 nm until no further indazole adduct could be detected. Following removal of ivDde, the resin was treated with a 5 mM solution of NaOH in H2O/methanol (3 mL, 1:1 v/v) for 3 hours. The resin

The Carboxyl Protecting Groups 117

was subsequently washed with methanol (5 × 3 mL), DMF (5 × 3 mL), dichloromethane (5 × 3 mL) and DMF (5 × 3 mL).   Step B: Aspartylation:

A 0.5 mL DMF solution containing 8.3 mg of 2-acetamido-2-deoxy-β-Dglucopyranosylamine (37.5 μmol), 19.5 mg of benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP, 37.5 μmol) and 8.3 μL of N-methylmorpholine (NMM, 75 μmol) was added to the above resin and the mixture was shaken for 16 hours. The resin was subsequently washed with DMF (10 × 3 mL) then dichloromethane (10 × 3 mL). A mixture of TFA/thioanisole/ triisopropylsilane (TIS)/water (17:1:1:1 v/v/v/v) was added to the resin and the mixture was shaken for 1.5 hours. The resin was then washed with TFA (3 × 3 mL), and the combined cleavage and washing solutions were concentrated in vacuo. The resulting residue was dissolved in 1.5 mL of DMSO, purified by preparative HPLC and lyophilized to afford 9.5 mg of the desired glycopeptide as a fluffy white solid, in a yield of 50%. 3.6.3.4.3 Deprotection of Diphenylmethyl Group by Trifluoroacetic Acid [136]

To a solution of 0.2 g freshly distilled phenol in 1 mL of trifluoroacetic acid was added 0.41 g of N-benzyloxycarbonyl-L-proline Dpm ester (0.001 mol). After reacting at 20°C for 30 minutes, the solution was concentrated at 20°C to dryness (within 5–7 minutes). The residue was taken up in ethyl acetate and the solution was worked up as usual. The product isolated from the alkaline extracts was washed many times with light petroleum to remove traces of phenol, affording 0.2 g of N-benzyloxycarbonyl-L-proline, in a yield of 82%, m.p. 73–74°C.

118

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.6.3.4.4 Deprotection of Diphenylmethyl Group by Hydrogen Bromide in Nitromethane [136]

L-Valine Dpm ester hydrobromide (0.36 g, 0.001 mol) was suspended in 20 mL 0.15 N HBr solution in nitromethane. Part of the starting material did not dissolve immediately, whereas crystals of the product started separating. After the mixture had been shaken for 15 minutes at 20°C, the crystalline material was filtered off and washed with nitromethane and ether, yielding 0.17 g of L-valine hydrobromide, in a yield of 90%, m.p. 208–210°C (decomp.) unchanged after recrystallization from ethanol-ether. 3.6.3.4.5 Removal of Diphenylmethyl Group via Hydrogenolysis [136]

A solution of 0.52 g benzhydryl ((benzyloxy)carbonyl)-L-valyl-L-valinate (0.001 mol) in 20 mL of methanol-tetrahydrofuran (3: 1) was hydrogenated over palladium-black catalyst. After 3 hours, the free peptide formed and the catalyst was filtered off and washed with a small amount of methanoltetrahydrofuran. The dipeptide was dissolved in hot water and the filtrate was concentrated to dryness to yield 0.18 g of L-valyl-L-valine, in a yield of 85%. 3.7 THE ARYL PROTECTING GROUPS The above protecting groups, including the alkyls, α-heteroatom substituted methyls, β-heteroatom substituted ethyls, phenacyls, allyls, propargyl, and benzyls, all share a common feature that the carbon atom which forms an ester with the carboxyl group of amino acids is in sp3 hybridization. However, these protecting groups may not be as useful as aryl protecting groups in peptide

The Carboxyl Protecting Groups 119

synthesis, as phenoxide is a better leaving group during amino acid coupling in comparison with the alkoxide group. Therefore, most amino acid aryl esters are considered activated esters, especially for those aryl groups with additional electron-withdrawing substituents. In peptide synthesis, the unequivocal coupling of Nα-urethane protected amino acid to Cα-protected one can be achieved only if a single electrophilic center is present in the acylating agent, leading to the acylation of the nucleophile, i.e., the amino component of the subsequent amino acid, in a unique way. In this case, the protected carboxyl groups are activated either during the coupling step using various coupling reagents or in a different step as activated esters. So far, 2-methoxyphenyl [142], 2-methoxy-5-nitrophenyl [143], o-nitrophenyl [144], p-nitrophenyl [76, 144–147], p-amidinomethylphenyl [148], m-amidinomethylphenyl [148], p-methylthiophenyl [149, 150], 2,4,5-trichlorophenyl [144, 151], pentafluorophenyl [144, 151–155], pentachlorophenyl [144, 151, 156], p-[N’,N”-di(Boc) guanidino]phenyl [157], 4-guanidino-phenyl [158, 159], p-(guanidinomethyl) phenyl [158], and 3-t-butyl-4-hydroxylphenyl [160], have been applied as the aryl protecting groups. Especially, the esters of N-Fmoc amino acids with aryl groups such as pentafluorophenyl (OPfp), pentachlorophenol (OPcp), trichlorophenyl (OTcp), nitrophenyl (ONp) have several merits in peptide synthesis. Some of them include formation of highly crystalline compounds, adequate solubility in DMF, commercial availability, resistance to hydrolysis, good stability in solution and on storage, cleanliness of the reaction, attendance to act efficiently with a catalyst, and free from racemization [151, 153]. Although the amino acid aryl esters can be prepared in methods similar to the ones for amino acid alkyl esters, the preparation of activated amino acid esters often involves the application of activating chloroformate or carbonate, such as the use of aryl chloroformate/pyridine/DMAP [161], mixed carbonates of t-butyl 2,4,5-trichlorophenyl carbonate [162], t-butyl p-nitrophenyl carbonate [163], pentafluorophenyl trifluoroacetate [164–169]. Alternatively, ONp, and OPfp esters of Boc-/Z-/Fmoc-amino acids have been prepared via the activation of the carboxyl group with EtOCOCl and NMM [170], or the combination of DCC/pyridine [147]. However, DCC has known limitations, such as the creation of insoluble DCU and potential skin irritation for some individuals [144, 151]. Therefore, it is a definite advantage over the existing strategies, if protection and activation of amino acids can be achieved in the same step [153]. For example, pentafluorophenyl trifluoroacetate has been applied to protect the amino group as a trifluoroacetamide and activate the carboxyl group as a pentafluorophenyl ester [165]. Likewise, propargyl pentafluorophenyl carbonate is proved to be feasible for the protection of amino group by propargylcarbonyl group and activation of carboxyl with

120

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

pentafluorophenyl group [153]. However, the application of six equivalents of 9-fluorenylmethyl-pentafluorophenyl carbonate for simultaneous protection (of the amino group) and activation (of carboxyl group) is only limited to anthranilic acid [165]. As many activated amino acid esters are used in peptide synthesis, the deprotection of the carboxyl group takes place spontaneously during the formation of the peptide bond, therefore, the deprotection of the carboxyl group is not an issue. However, for 3-t-butyl-4-hydroxylphenyl group, it is easily removed under relatively mild oxidative condition, and ceric ammonium nitrate (CAN) gives the best result among the tested oxidizing reagents, including lead tetraacetate, silver oxide, bromine, NBS, and thallic trifluoroacetate, etc. [160]. Similarly, 4-methylthiophenyl group is removed via oxidation after it is converted into activated 4-methylsulfonylphenyl ester [149]. In addition, m-guanidinophenyl, and m-(guanidino-methyl)phenyl esters derived from N-(tert-butyloxycarbonyl)-amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis [158]. It is found that p-amidinophenyl and p-guanidinophenyl esters behave as specific substrates for trypsin and trypsin-like enzymes, thus they are termed “inverse substrates” [148, 158, 171]. 3.7.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID ARYL ESTERS 3.7.1.1 PREPARATION OF N-(1-BENZOYL-PROPEN-2-YL)-LPHENYLALANINE p-NITROPHENYL ESTER [76]

To a solution of 4.5 g of N-(1-benzoyl-propen-2-yl)-L-phenylalanine (i.e., (E)-(4-oxo-4-phenylbut-2-en-2-yl)-L-phenylalanine, 15 mmol) and 2.3 g of p-nitrophenol (18 mmol) in 50 mL EtOAc, was added 4.0 g of N,N’dicyclohexyl-carbodiimide (15 mmol) at 0°C. After 30 minutes, the mixture was allowed to reach room temperature and was kept at this temperature for 1 hour. The N,N’-DCU was filtered off and washed with EtOAc. The combined EtOAc solution was washed and evaporated to dryness. The crystalline residue was recrystallized from hot ethanol to afford 3.6 g of

The Carboxyl Protecting Groups 121

4-nitrophenyl (E)-(4-oxo-4-phenylbut-2-en-2-yl)-L-phenylalaninate, in a yield of 55%, m.p. 115–116°C. 3.7.1.2 PREPARATION OF 2-METHOXYPHENYL GLYCINATE HYDROBROMIDE [142]

To a stirred mixture of 4.18 g Z-glycine (20.0 mmol), 2.19 mL of guaiacol (i.e., 2-methoxyphenyl, 20 mmol), 20 mL ethyl acetate, and 20 mL of THF at 0°C, was added slowly a solution of 4.54 g DCC (22.0 mmol) in 20 mL of ethyl acetate. After being stirred at 0°C for 3 hours, the reaction mixture was stirred at room temperature for an additional 24 hours. N,N’-DCU was removed by filtration. After evaporation of the solvent, the remaining liquid was mixed with 20 mL acetic acid, followed by HBr/AcOH (10.9 mL, 20 mmol of HBr) at room temperature. After 3 hours of stirring at room temperature, 200 mL of dried diethyl ether was poured into the reaction mixture to give a crude white precipitate, which was further purified by reprecipitation from methanol. The yield of 2-methoxyphenyl glycinate hydrobromide was almost quantitative. 3.7.1.3 GENERAL PROCEDURE FOR THE SYNTHESIS OF BPOC-XXXOPFP, WHERE XXX = ALA, VAL, LEU, ILE, PRO, MET, LYS(BOC), PHE, LYS(TFA), SER(T-BU), THR(T-BU), AND TRP [155]

O O

O N

O

OH

N

DMF, 55 C °

OH + NH2

O O

NH2 F

O OH O

NH O

F

F

F

OH

F DCC, EtOAc

F O

F

F

O O

NH

F F

O

The amino acid zwitterion (162 mmol) was dissolved in Triton B (162 mmol of a 40% solution in MeOH) and then concentrated on a high vacuum

122

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

rotary evaporator to remove any excess H2O and CH3OH. The white syrupy solid was mixed well with 40 mL of DMF and the suspension was concentrated again to a syrup on a high vacuum rotary evaporator. This step was repeated three times. The resulting heavy syrup was mixed with 200 mL of DMF, and 162 mmol of Bpoc-O-Ph was added, and the mixture was stirred at 55°C. After stirring for 3 hours, DMF was removed on a high vacuum rotary evaporator. The pasty solid or oil was diluted with 100 mL H2O (100 mL), then 2 g of Na2SO4 (helps to prevent emulsions in some cases) and 10 mL 5% NaHCO3 were added subsequently. The aqueous solution was extracted with ether (3 × 200 mL), and the combined ether layer was back-extracted with 20 mL 1% NaHCO3. The combined aqueous phase was combined, cooled in a 0°C ice bath and over-layered with 200 mL ether. Dropwise addition of 0.5 M citrate buffer at pH 3.5 to the biphasic mixture caused clouding in the aqueous layer that was cleared upon swirling. The addition was continued until pH 3.5 was reached in the mixture. The aqueous phase was extracted with ether (2 × 150 mL) and the ether combined was washed with 100 mL citrate buffer, water (3 × 200 mL), and brine (100 mL). Upon being dried over MgSO4, the solution was filtered and concentrated in vacuo to afford Bpoc-XXX-OH as an oily solid (yield 60–95%). The Bpoc-XXX-OH prepared above was dissolved in 400 mL of EtOAc (400 mL) and cooled to –10°C in an acetone ice bath. Pentafluorophenol (150 mmol) and DCC (150 mmol) were added sequentially in one portion and the reaction was allowed to stir for 6 hours at –10°C and then placed in a freezer overnight. The reaction mixture was filtered to remove DCU, and the EtOAc solution was concentrated in vacuo to a dry foam or solid, which was taken up by 200 mL ether. The ethereal solution was put in a –20°C freezer overnight, residual DCU was removed by filtration and the ether was removed in vacuo. The resulting foam or solid was crystallized from hexanes containing a small amount of a more polar co-solvent (Et2O or isopropanol) to afford pure Bpoc-XXX-OPpf. 3.7.1.4 PREPARATION OF 2-METHOXY-5-NITROPHENYL N-BUTYLOXYCARBONYLGLYCINE ESTER [143]

The Carboxyl Protecting Groups 123

To a mixture of 193 mg N-butyloxycarbonylglycine (1.1 mmol), 170 mg 2-methoxy-5-nitrophenol (1 mmol), 10 mg of HOBt (0.075 mmol), 10 mg of DMAP (0.08 mmol), and 230 mg of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (1.2 mmol) stirred at –78°C, was added 7 mL of methylene chloride via syringe. The reaction was continued overnight, and the temperature was allowed to rise to room temperature. The reaction mixture was washed with water (2 × 5 ml), 5% aqueous NaHCO3 (3 × 5 ml), water (2 × 5 ml), 10% aqueous citric acid (3 × 5 ml), and brine (1 × 3 ml). Upon being dried over anhydrous MgSO4, the solution was loaded to a preparative TLC plate and eluted with hexane and ether to afford 200 mg of 2-methoxy-5-nitrophenyl (tert-butoxycarbonyl)glycinate, in a yield of 75%, m.p. 121–122°C.

3.7.1.5 GENERAL PROCEDURE FOR THE SYNTHESIS OF 4-GUANIDINOPHENYL ESTERS OF Z-PROTECTED ALANINE DERIVATIVES [172] HO

O N H

O

OH

O

HOAt, DIC THF

O

O

O O

N H

O O

N N H

Boc N H

Boc

ultrasound CF3CO2H

O O

N N N

O O

Boc

Boc N N H H TBTU, DIPEA, DMF

N N H

N

N H

O O

CF3CO2 NH2 N H

NH2

Nα-Z-protected amino acids were first activated with 1-benzotriazolyl esters through the reaction of Z-(αTfm)Ala-OH, Z-(αDfm)Ala-OH, or Z-Aib-OH with TBTU (1 equiv.) and DIPEA (2.0 equiv.) in 20 mL of DMF for 30 minutes at room temperature. Then a solution of 4-[N’,N”-bis(Boc) guanidino]phenol (1 equiv.) in 10 mL of DMF was slowly added to the above reaction mixture. After being stirred overnight at room temperature, to this solution was added an excess amount of distilled water. After sedimentation, the precipitate was filtered, washed with water, and purified by flash column chromatography (eluent, petroleum ether/ethyl acetate). Final deprotection

124

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

of the Boc functionality of the bis(Boc)-protected compounds with trifluoroacetic acid and ultrasound resulted in the 4-guanidinophenyl esters. 3.7.1.6 GENERAL PROCEDURE FOR THE SYNTHESIS OF 4-GUANIDINOPHENYL ESTERS OF Z-PROTECTED LEUCINE AND PHENYLALANINE DERIVATIVES [172] O O

N H

OH

HOAt, DIC THF

O O

O

N N H

O O

N N N

O

HO

N N H

Boc N H

Boc

LiO n-BuLi THF

N N H

Boc N H

O

N H

O

N

O

N H

Boc

ultrasound CF3CO2H

O O

N H

O O

Boc N H

Boc

CF3CO2 NH2 N H

NH2

To the 20 mL THF solution containing N-protected α-fluoroalkyl amino acid derivatives Z-(αTfm)Phe-OH, Z-(αDfm)Phe-OH, Z-L-(αMe)Phe-OH, Z-D-(αPhe)Me-OH, Z-(αTfm)Leu-OH, or Z-(αDfm)Leu-OH was added 1,3-diisopropyl-carbodiimide (DIC, 1 equiv.) and HOAt (1 equiv.). The resulting mixture was stirred at room temperature for 30 minutes. On the other hand, a solution of 1.6 M n-BuLi (in hexane) was added dropwise over 5 minutes to a stirred solution of 4-[N’,N”-bis(Boc)guanidino] phenol in 20 mL dry tetrahydrofuran at room temperature under an argon atmosphere, and this reaction mixture was also stirred for 30 minutes. This 4-guanidinophenolate solution was added to the above solution of activated Nα-Z-protected amino acid derivative. The reaction mixture was stirred for another 2 hours at room temperature, and then 50 mL citric acid (5 g/100 mL) was added. The organic solvent was removed under vacuum, and the aqueous solution was extracted with ethyl acetate, dried over magnesium sulfate, and concentrated to give a yellow oil. The residue was purified by flash column chromatography on silica gel using petroleum ether/ethyl acetate as eluent. The 4-guanidinophenyl esters were obtained by deprotection of the Boc functionality with trifluoroacetic acid and ultrasound, as described above.

The Carboxyl Protecting Groups 125

3.7.1.7 PREPARATION OF FMOC-PHENYLALANINE PENTAFLUOROPHENYL ESTER [144]

A mixture of 3.87 g Fmoc-phenylalanine (10 mmol), 9-fluorenylmethyl chloroformate (Fmoc-Cl, 10 mmol) and 10 mmol of N-methylmorpholine was well stirred at –15°C for 5 minutes, then 10.5 mmol of pentafluorophenol in 10 mL of THF was added. The reaction was allowed to warm up to room temperature and the stirring was continued till the completion of the reaction. The solvent was evaporated and the residue was dissolved in CHCl3. The chloroform solution was washed with 5% NaHCO3 (2 × 5 mL), 5% HCl, water, and dried over anhydrous Na2SO4. Evaporation of CHCl3 under reduced pressure and recrystallization from EtOH: n-hexane (3:1) gave 5.2 g Fmoc-Phe-OPfp as crystalline solid, in a yield of 95%. 3.7.1.8 GENERAL PROCEDURE FOR THE SIMULTANEOUS PROTECTION AND ACTIVATION OF AMINO ACIDS USING PROPARGYL PENTAFLUOROPHENYL CARBONATE (POCOPFP) [152]   Step A: Preparation of propargyloxycarbonyl chloride (PocCl):

To a stirred solution of 2.23 g triphosgene (7.5 mmol) in 30 mL dry ether, was added 0.05 g activated charcoal and the mixture was stirred at room temperature (28°C) for 1 hour. The solution was then cooled to 0°C and 0.9 mL of propargyl alcohol (15 mmol) in 10 mL dry ether was added dropwise.

126

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

The resultant solution was stirred for 12 hours and filtered. The ether layer was concentrated under reduced pressure, and the remaining liquid was used for reactions without any further purification.   Step B: Preparation of propargyl pentafluorophenyl carbonate (PocOPfp):

To a stirred solution of 1.84 g pentafluorophenol (10 mmol) in 30 mL of dichloromethane at –10°C was added 1.2 g of propargyloxycarbonyl chloride (10 mmol). The solution was stirred for 10 minutes and 1.4 mL of triethylamine (10 mmol) was added dropwise over a period of 15 minutes. After 4 hours, the reaction mixture was diluted with 50 mL of dichloromethane and washed sequentially with 0.5 N HCl (20 mL), water (2 × 20 mL) and brine (20 mL). Upon drying over anhydrous Na2SO4, the solution was concentrated, and the residue was purified by silica gel (100–200 mesh) column chromatography, eluting with 3% solution of ethyl acetate in hexane to afford propargyl pentafluorophenyl carbonate as a white crystalline solid, in a yield of 98%, m.p. 65°C.   Step C: Simultaneous protection and activation of leucine with propargyl pentafluorocarbonate:

To a 2 mL DMF solution containing 0.131 g of leucine (1 mmol) and 0.559 g of propargyl pentafluorophenyl carbonate (2.1 mmol) cooled to –10°C, was added 0.177 mL of pyridine (2.2 mmol) dropwise. The reaction mixture was allowed to attain room temperature slowly and stirred for 3–5 hours. The reaction mixture was then diluted with 30 mL of dichloromethane and washed sequentially with 0.5 N HCl (10 mL), water (2 × 10 mL) and brine (10 mL). The resulting solution was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel (100–200 mesh) column chromatography eluting with ethyl acetate-hexane mixtures of appropriate concentration (5–20%) to afford 85% of N-propargyloxycarbonyl-leucine pentafluorophenyl ester as a gummy solid.

The Carboxyl Protecting Groups 127

3.8 ORTHOESTER PROTECTING GROUPS An orthoester (or ortho ester) is a type of organic molecule that contains an orthoester group with a general formula of RC(OR’)3. Thus, the orthoester group is a functionality containing three alkoxy groups attached to the same carbon atom and shares a stability characteristic similar to that of acetal and ketal that are essentially inert under neutral or alkaline conditions but can be readily hydrolyzed even in mildly acidic environments. The advantages of using orthoester as a carboxyl protecting group for amino acids include the provision of stability under basic conditions and the concurrent markup of the acidity of the α-proton by preventing the formation of enolate and thereby anion formation which leads to the racemization of the α-chiral center [173]. Especially, the cyclic orthoester is more stable than the acyclic one, and the increased stability makes the bicyclic orthoesters chromatographically stable compared to their acyclic counterparts [174]. In addition, orthoester group is one of the few protecting groups that protect the carbonyl group against nucleophilic attack by hydroxide or other strong nucleophiles such as Grignard reagents. However, the orthoester has not been popularly applied as the carboxyl protecting group, due to the low yield of orthoester formation from acids or nitriles [173]. In the early days, orthoesters had been obtained by the acid-catalyzed esterification of the carboxylic acids with 2-substituted-2-hydroxymethyl-1,3-propanediol for bicyclic orthoesters, or via the Pinner synthesis involving the acidic alkoxylation of nitrile to form acyclic orthoester which is then converted into cyclic orthoester with triol (Scheme 3.5) [175, 176]. Lately, Corey developed a procedure to form OBO ester (2,6,7-trioxabicyclo[2.2.2] octane ring system) [177] that involves a Lewis acid (BF3) catalyzed opening of the oxetane ester by carbonyl participation to form the 6-membered zwitterion, and the rearrangement of such intermediate generates the orthoester, as shown in Scheme 3.6 [178]. In this procedure, the release of ring strain of the oxetane presumably provides the overall thermodynamic driving force for such transformation. These oxetane esters can be conveniently prepared via DCC promoted condensation between amino acids and (3-methyloxetan-3-yl)methanol [179], or the alkylation of the N-protected amino carboxylates with the corresponding oxetane tosylate or bromide, where the (3-methyloxetan-3-yl)methyl tosylate is prepared using standard conditions as a stable crystalline material in 90–95% yield, and 3-(bromomethyl)-3-methyloxetane can be prepared in a number of ways from 3-methyl-3-(hydroxymethyl)oxetane with bromine/

128

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

triphenylphosphine [180], carbon tetrabromide/triphenylphosphine [181], or by displacement of the corresponding tosylate with sodium bromide in acetone. Although the best yields for the alkylation reactions were obtained between the cesium salt of amino acid and oxetane bromide (~85–90%), the preferred alkylation condition is from the corresponding oxetane tosylate in DMF in the presence of sodium iodide due to the slow decomposition of oxetane bromide upon standing [182]. This preparation has been modified to form 2,7,8-trioxabicyclo[3.2.1]octanes (ABO-esters) under condition of kinetic control via cationic zirconocene-catalyzed rearrangement of epoxy esters, involving the neighboring group-assisted opening and rearrangement of an acyloxy oxirane. Under this condition, in the presence of 1–5 mol% of silver(I) salts with non-coordinating counterions, the abstraction of chloride ions from Cp2ZrCl2 provides cationic metallocene with a high selectivity toward oxirane which functions as the Lewis base [183]. However, under the condition of thermodynamic control or in the presence of a strong Lewis acid, a tetrahydrofuran is formed instead. This new protocol works for both acid and base-sensitive α-amino acids and affords ABO-esters in high yield.

NC

Br

MeOH/HCl 40 hrs.

Br

OMe OMe OMe

OH OH OH BF 3 Et 2 O

Br O O

O

SCHEME 3.5 Formation of orthoester from the reaction of triol.

SCHEME 3.6 Corey’s procedure to form orthoester.

Regarding the deprotection strategy of the orthoester group, several methods have been developed, including the acid hydrolysis to open the cyclic orthoester ring followed by base hydrolysis (e.g., H2SO4/MeOH then 1 N NaOH/MeOH [184], DME:H2O at pH 3 with Na2SO4 then LiOH [185], catalytic pyridinium p-TSA in MeOH/H2O then LiOH [186]); transesterification of the ring-opened orthoester (usually to methyl ester, with NaOMe/ MeOH or K2CO3/MeOH) followed by base hydrolysis of the methyl ester;

The Carboxyl Protecting Groups 129

[187, 188] and treatment of the orthoester with trimethylsilyl iodide (TMSI), trifluoroacetic acid [179] or 6 N HCl [189]. 3.8.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID ORTHOESTERS 3.8.1.1 PREPARATION OF 1-[N-(9FLUORENYLMETHYLOXYCARBONYL)-(1S)-1-AMINO-2HYDROXYETHYL]-4-METHYL-2,6,7-TRIOXABICYCLO[2.2.2] OCTANE, FMOC-L-SER-OBO ESTER [179]   Step A: Formation of N-(9-Fluorenylmethyloxycarbonyl)-L-serine 3-Methyl-3-(hydromethyl)oxetane Ester (Fmoc-L-Ser-oxetane Ester): H N

O O

O OH OH

+ O

OH

DCC/DMAP CH 2 Cl 2

H N

O O

O O

O

OH

To a cooled mixture of 1.47 g DCC (7.11 mmol), 29.0 mg of DMAP (0.237 mmol), and 9.68 g 3-methyl-3-(hydroxymethyl)oxetane (94.8 mmol) at 0°C, was added a solution of the 1.97 g Fmoc-L-Ser (4.74 mmol) in 50 mL CH2Cl2 dropwise over a period of 1 hour under stirring. After that, the reaction mixture was stirred for additional three hours, and the precipitated DCU was filtered. The filtrate was then washed with 1% NH4Cl (2 × 125 mL) and 5% NaHCO3 (1 × 125 mL). Upon being dried over MgSO4, the solution was evaporated to dryness. This product was purified by silica gel flash chromatography (CHCl3: isopropylamine (40:l) or EtOAc: hexane (2: l), loaded in CH2Cl2) to afford 1.76 g of a white foam, corresponding to 90% yield of (3-methyloxetan-3-yl)methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-Lserinate. Further crystallization from EtOAc/hexane gave 1.42 g of colorless crystals, in a yield of 73%. The aqueous fractions were saved for recovery of oxetane alcohol, via evaporating the aqueous fractions to near dryness and then extracting them with EtOAc (3 × 50 mL). The organic fractions were combined, dried (MgSO4), and evaporated to a viscous liquid. The liquid was distilled under vacuum to yield 6.16 g of 3-methyl-3-(hydroxymethyl) oxetane, in a 67% of the theoretical recovery, m.p. 106–107°C. Step B: Formation of Fmoc-L-Ser-OBO ester:  

130

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

H N

O O

O O OH

O

BF 3 Et 2 O CH 2 Cl 2

H N

O O

O

O O

OH

Fmoc-L-Ser-oxetane ester (i.e., (3-methyloxetan-3-yl)methyl (((9Hfluoren-9-yl)methoxy)carbonyl)-L-serinate, 1.00 g, 2.43 mmol) was dissolved in 15 mL freshly distilled CH2Cl2 and cooled to 0°C under N2 atmosphere. Then a solution of BF3·Et 2O (40 µL of a 20% (v/v) solution in CH2Cl2, 0.065 mmol) was added, and the solution was stirred and allowed to warm to room temperature. After 8 hours, 100 µL of Et3N (0.72 mmol) was added and the solution was evaporated to dryness. The residue was purified by silica gel flash column chromatography (EtOAc: hexane (3: 1), loaded in CH2Cl2) to afford 0.855 g of Fmoc-L-Ser-OBO ester, i.e., (9H-fluoren-9-yl) methyl (S)-(2-hydroxy-1-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-octan-1-yl) ethyl)carbamate, as a white foam, in a yield of 85%. Further recrystallization from EtOAc/hexane gave 0.757 g of colorless crystals, in a yield of 76%, m.p. 146–147°C. 3.8.1.2 PREPARATION OF 1-[N-(BENZYLOXYCARBONYL)-(1S)-1AMINO-2-ETHANOL]-4-METHYL-2,6,7-TRIOXA-BICYCLO[2.2.2] OCTANE (CBZ-L-SER-OBO ESTER) [189] Step A: Preparation of 3-Methyl-3-(toluenesulfonyloxymethyl)   oxetane (Oxetane Tosylate):

A dry, 1 L round-bottomed flask was charged with 57.20 g of toluene4-sulfonyl chloride (0.3 mol), to which 250 mL of pyridine was added whilst stirring under nitrogen atmosphere. The reaction flask was placed inside a container filled with ice/water to cool the flask if the reaction became too exothermic. 3-Methyl-3-oxetanemethanol (20.4 g, 0.2 mol) was then added slowly and the mixture was stirred for 1.5 hours. The mixture was then slowly added to a vigorously stirring mixture of 700 mL de-ionized water and 700 g of crushed ice in a 2 L Erlenmeyer flask, and the resulting mixture was allowed to stir for an additional 0.5 hour. The white precipitate was then collected on #1 Whatman filter paper and washed with cold H2O. The

The Carboxyl Protecting Groups 131

product was dried under high vacuum and/or over P2O5 to afford 49.11 g of (3-methyloxetan-3-yl)methyl 4-methylbenzenesulfonate as a white powder, in a yield of 92%, m.p. 49.5–51°C.   Step B: Formation of N-(Benzyloxycarbonyl)-L-serine-3-methyl-3hydroxymethyl-oxetane Ester (Cbz-L-Ser-Oxetane Ester):

A mixture of 11.36 g Cbz-L-Ser (0.047 mol), 9.19 g of Cs2CO3 (0.028 mol) and 100 mL of water were stirred and freeze-dried for 12 hours to give a white foam. To this foam was added 12.65 g of oxetane tosylate (0.049 mol) and 1.41 g of NaI (9.8 mmol) which was then taken up in 400 mL DMF and then stirred under argon for 48 hours. DMF was then removed in vacuo and the resulting solid was dissolved in 600 mL of EtOAc. This solution was sequentially washed with H2O (200 mL), 10% NaHCO3 (2 × 100 mL), saturated NaCl (100 mL) and dried over MgSO4. The solvent was removed under reduced pressure to yield a yellow oil which was recrystallized from ethyl acetate and hexanes to yield 11.85 g of Cbz-L-Ser-oxetane ester, i.e., (3-methyloxetan-3-yl)methyl ((benzyloxy)carbonyl)-L-serinate, as a colorless rod-like crystal, in a yield of 78%, m.p. 70–70.5°C.   Step C: Formation 1-[N-(Benzyloxycarbonyl)-(1S)-1-amino-2ethanol]-4-methyl-2,6,7-trioxabicyclo[2.2.2]octane (Cbz-L-SerOBO Ester): OH

O O

N H

O O

O

BF 3 Et 2 O CH 2 Cl 2

OH

O O

N H

O

O O

To a solution of 15.0 g of Cbz-L-Ser oxetane ester (46.2 mmol) in 450 mL of dry CH2Cl2 was added 5 mL of CH2Cl2 solution containing 0.11 mL of BF3·Et 2O (0.93 mmol) at 0°C. Then the reaction mixture was stirred at room temperature for 6 hours. After addition of 1.29 mL of Et3N (9.25 mmol), the reaction mixture was stirred for an additional 30 minutes before being concentrated to a thick oil. The crude product was dissolved in 400 mL of EtOAc and washed with 3% NH4Cl (2 × 250 mL), 10% NaHCO3 (100 mL), saturated NaCl (250 mL), dried (MgSO4), and evaporated to dryness. The residue weighed 14.2 g, corresponding to 95% of yield, which was crystallized from EtOAc to give 13.6 g of Cbz-L-Ser-OBO ester as rod-like shiny crystals, in a yield of 93%, m.p. 103.5–105.0°C.

132

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.8.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF THE ORTHOESTER GROUP 3.8.2.1 PROCEDURE A. REMOVAL OF ORTHOESTER GROUP WITH TFA [178]

H N

O O

O

piperidine

O

CH 2 Cl 2

O

O H 2N

O O

CF 3 CO 2 H/H 2 O HO CH 2 Cl 2

OH

OH

O OH NH 2

Fmoc-L-Ser-OBO ester (0.517 g, 1.26 mmol) was added to 15 mL of 20% piperidine in CH2Cl2 and the mixture was stirred at room temperature for 40 minutes. Upon removal of the solvent under vacuum, the white residue was mixed with 15 mL CH2Cl2, 350 µL of TFA and 250 µL of water. After the solution was stirred at room temperature for 15 minutes, the solvent was again removed under vacuum. The oily residue was dissolved in 15 mL of MeOH, and 4 mL of water and 21 mL of 10% (w/v) Cs2CO3 solution (6.4 mmol, 5.1 equiv.) were added. After being stirred at room temperature for 18 hours, the solution was filtered through Celite and acidified with 2 N HCl to pH < 3. The solution was loaded on a cation exchange column, washed with 0.01 N HCl and water, and then eluted with 5% Et3N in water. The eluate was evaporated to dryness under vacuum to give 0.120 g of L-serine as a white solid, in a yield of 91%. Recrystallization afforded 0.109 g of white needles, in a yield of 82%, m.p. 215.5–216.5°C. 3.8.2.2 PROCEDURE B. REMOVAL OF ORTHOESTER GROUP WITH TMSI [178]

H N

O O

O

O O

+ Me 3 SiI

75 C° 24 hrs.

O HO

OH NH 2

OH

Fmoc-L-Ser-OBO ester (0.106 g, 0.258 mmol) was stirred with 500 µL of TMSI (3.5 mmol, 14.0 equiv.) at 75°C for 24 hours. After the solution was cooled, 3 mL of Et2O was carefully added followed by the dropwise addition of 5 mL 0.5 N NaOH. The organic layer was removed and washed with 0.5 N NaOH (2 × 4 mL). The aqueous fractions were combined, washed with Et2O

The Carboxyl Protecting Groups 133

(2 × 5 mL), and then acidified to pH < 3 with 2 N HCl. The sample was purified on a cation exchange column, affording 0.0249 g of L-serine as a white solid, in a yield of 92%. Recrystallization from H2O/acetone gave 0.0183 g of L-serine as fine needles, in a yield of 67%, m.p. 213–214°C. 3.9 p-AZOBENZENECARBOXAMIDOMETHYL (OABC) PROTECTING GROUP p-Azobenzenecarboxamidomethyl (OAbc) is a colored alkali labile carboxyl protecting group that can be removed with aqueous potassium carbonate in 15–20 minutes [190]. The corresponding esters of α-amino acids can be prepared in three steps, i.e., treatment of 4-(phenyldiazenyl)aniline with 2-bromoacetyl bromide to form p-(bromoacetamido)azobenzene, treatment of α-amino acids with ethyl acetoacetate and 15-crown-5 to transiently protect the amino group, and esterification of amino acid complex with p-(bromoacetamido)azobenzene in the presence of toluenesulfonic acid, as shown in Scheme 3.7.

SCHEME 3.7 Introduction of the p-azobenzenecarboxamidomethyl group.

3.9.1 GENERAL PROCEDURE FOR THE PREPARATION OF AMINO ACID OABC ESTER Reaction schemes for Steps A and B can be found in Scheme 3.7 [190].   Step A: Preparation of p-(Bromoacetamido)azobenzene: p-Aminoazobenzene (5.0 g, 25.3 mmol) was dissolved in 40 mL of chloroform, and then 40 mL of water was added. The resulting mixture was cooled to 10°C and under vigorous stirring 2.3 mL of bromoacetyl bromide (27 mmol) was added dropwise at 10°C. By slow addition of Na2CO3, the pH of the solution was kept at 9. The mixture was stirred for additional 30 minutes. Precipitated p-(bromoacetamido)

134

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

azobenzene was filtered, washed with cold chloroform, dried, and recrystallized from chloroform-methanol (4:1) to give 7.66 g of acetamide, in a yield of 95%, m.p. 166–168°C.   Step B: Preparation of amino acid OAbc ester salts: A mixture of an amino acid (10 mmol), DMF (10 mL), benzene (10 mL), 5 mL 2 M NaOMe in methanol, 15-crown-5 (2.2 g, 10 mmol) and 1.5 mL ethyl acetoacetate (11 mmol) was refluxed for 20 minutes using Dean-Stark adapter containing benzene. Then, 3.18 g of p-(bromoacetamido)azobenzene (10 mmol) was added, the resulting mixture was stirred at 50°C for 3 hours and diluted with 50 mL of EtOAc. The solution was washed with brine, dried over MgSO4 and acidified with 1.72 g of anhydrous PTSA (10 mmol). Precipitated orange crystals of salt were filtered and washed with ether. 3.9.2 REMOVAL OF THE OABC PROTECTING GROUP A typical example is provided below [190]:

A solution of 1.3 g K2CO3 (9.4 mmol) in 5 mL water was added to a suspension of Glp-Asn-OAbc, i.e., 2-Oxo-2-((4-((E)-phenyldiazenyl) phenyl)-amino)ethyl ((S)-5-oxopyrrolidine-2-carbonyl)-L-asparaginate (4.7 mmol) in 20 mL of DMF. The reaction mixture was stirred for 30 minutes, and after dilution with 20 mL water, the mixture was extracted with EtOAc (3 × 20 mL). The aqueous layer was adjusted to pH 4–5 and evaporated to dryness. The residue was extracted by hot anhydrous ethanol (2 × 30 mL). The solution was filtered and evaporated to afford 1.0 g of Glp-Asn-OH, i.e., ((S)-5-oxopyrrolidine-2-carbonyl)-L-asparagine, m.p. 262–264°C. 3.10 HYDRAZINE PROTECTING GROUP So far, all the examples of carboxyl protecting groups essentially convert the carboxyl group of α-amino acid into an ester group that does not ionize and affect the reactivity of the α-amino group anymore. In addition to the protection of the carboxyl group with an oxygen atom directly linked

The Carboxyl Protecting Groups 135

to the acyl group, other heteroatoms such as nitrogen, sulfur can also be applied to protect the carboxyl group. When a nitrogen atom is involved in the protection of the carboxyl group, primary amines, secondary amines, hydrazines, and substituted hydrazines can be used, in the format of amides and hydrazides. However, as amides have relatively lower reactivity than esters, the deprotection of amides is often problematic, as the peptide bonds between individual amino acids are also the amide bonds. Consequently, the conditions to deprotect the C-terminal amide functionalities may result in the degradation of peptide bonds. Thus, amines are usually not used as the carboxyl protecting groups in peptide and protein synthesis, although a few exceptions do exist where the formed amides can be easily cleaved. Hydrazine, however, is occasionally applied as the carboxyl-protecting group, as the resulting acid hydrazides can be converted to acid azides or diimides that are effective peptide coupling agents [191–193]. Diazotization of hydrazides with an Nα-Boc residue that is usually unstable under acidic conditions is also possible, as the diazotization is carried out at very low temperature (e.g., –30°C). When N,N’-di-isopropylhydrazine is applied as the carboxyl protecting group, it reacts with carboxylic acid derivatives (either acyl chlorides or mixed anhydrides) to give only monoacylhydrazides, and diacylhydrazides have not been detected under the normal range of acylation conditions (e.g., Schotten-Baumann reaction condition) [194]. In the case of 6β-phenylacetamidopenicillanic acid, prior conversion into its mixed anhydride with ethyl chloroformate, followed by reaction with N,N’-diisorpopylhydrazine afforded the corresponding hydrazide. Carboxyl group protected with N,N’-diisopropyl-hydrazine would be stable to both acidic and basic conditions and also inert towards acylating agents. The carboxyl group can be released by selective oxidation of hydrazides under mild conditions. For example, treatment of N-benzoyl-N,N’diisopropyl-hydrazine with lead tetraacetate and pyridine in dry benzene at room temperature, followed by extraction with aqueous sodium hydrogen carbonate, gave, after acidification, quantitative recovery of benzoic acid. Other oxidants for deprotection of hydrazides include sodium periodate, aqueous N-Bromo-succinimide, and chromium trioxide in acetic acid [194]. For the N-unsubstituted hydrazides, perchloric acid is also applied as the oxidant [195]. In comparison, removal of hydrazine with POCl3/H2O, HCl/ AcOH, or HBr/AcOH leads to no more than 94% completion of deprotection, while accompanying with partial or total racemization of the protected amino acids [195].

136

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3.10.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF AMINO ACID HYDRAZIDE 3.10.1.1 PREPARATION OF N-BENZOYL GLYCINE HYDRAZIDE [195] O

H N

OMe

+ NH2NH2 H2O

H N

MeOH, r.t.

O NHNH2

O

O

A solution of 1.93 g benzoyl-Gly-OMe (10 mmol) in 10 mL MeOH was treated in portions of 0.1 mL with a total of 1.14 mL hydrazine hydrate (24 mmol) and the reaction mixture was stirred at room temperature for 6 hours. After the addition of 10 mL ether, the crystals were collected and dried to afford 1.72 g of N-benzoyl glycine hydrazide, in a yield of 88%. After recrystallization from EtOH/ether, the product had an m.p. of 159.5–160.5°C. 3.10.1.2 PREPARATION OF (1S)-N-6βPHENYLACETAMIDOPENICILLANOYL-N,N’-DIISOPROPYLHYDRAZINE S-OXIDE [194] H HH N

Ph O

H HH N

O S

O 1. ClCO2Et/Et3N/CHCl3

N

2. i-PrNHNHPr-i

O O

OH

O S

N O N NH

O

Triethylamine (1 g) was added to a stirred solution of 3.5 g of (1S)-N-6βphenylacetamidopenicillanic acid S-oxide, i.e., (4R,5R,6R)-3,3-dimethyl-7oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4-oxide, in 50 mL dry chloroform containing 1.2 g of ethyl chloroformate (1.1 equiv.) at 0°C under nitrogen. After 2 hours the solution was cooled to –20°C and 1.8 g of freshly distilled N,N’-di-isopropylhydrazine was added. After 20 minutes the solution was washed with water, 20 mL 0.5 M tartaric acid, and finally water (50 mL portions). After drying and removal of solvent, the crude product was precipitated from light petroleum. Crystallization from benzene afforded 3.54 g of sulfoxide hydrazide, i.e., N-((4R,5R,6R)-2-(1,2-diisopropylhydrazine-1-carbonyl)-3,3-dimethyl-4-

The Carboxyl Protecting Groups 137

oxido-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-6-yl)-2-phenylacetamide, in a yield of 78%, m.p. 158–160°C. 3.10.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF HYDRAZINE PROTECTING GROUP 3.10.2.1 DEPROTECTION OF N-BENZOYL-L-PHENYLALANINE HYDRAZIDE [195]

N H

H N O

60% HClO 4 48 C, 24 hrs.

O OH

°

O

NH 2

N H

O

N-Benzoyl-L-phenylalanine hydrazide (283.6 mg, 1 mmol) was mixed in an ice bath with 5 mL of 60% HClO4 solution in 0.5 mL portions. After incubation at 48°C for 1 day, the mixture was cooled in an ice bath while a large excess of H2O was added. The solution was extracted with EtOAc to separate N-benzoyl-L-phenylalanine that was further purified by taking it up in 2 M KHCO3, acidifying, and again extracting with EtOAc. This organic phase was washed with H2O until neutral, dried (Na2SO4), and filtered. After evaporation of the solvent, the residue was crystallized from EtOH/H2O, giving 227 mg of analytically pure N-benzoyl-L-phenylalanine, in a yield of 84%, m.p. 134–134.5°C. 3.10.2.2 REMOVAL OF N,N’-DIISOPROPYLHYDRAZINE VIA OXIDATIONS WITH NBS [194]

To a solution of 0.2 g N-6β-phenylacetamidopenicillanoyl-N,N’diisopropylhydrazine in 10 mL THF and 3 mL water were added 0.06 g

138

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

pyridine and 0.10 g N-bromosuccinimide. After 10 minutes, the acid was extracted with ether, dried, and evaporated to afford 0.17 g of (1S)-N-6βphenylacetamido-penicillanic acid, in a yield of 90%. 3.11 5,6-DIHYDROPHENANTHRIDINE PROTECTING GROUP Although 5,6-dihydrophenanthridine has not been applied as the carboxyl protecting group in peptide synthesis, it has been proved a good protecting group for a carboxyl group. One of the prominent features of using 5,6-dihydrophenanthridine as a carboxyl protecting group is that the amide is stable enough under normal conditions for acetate saponification and even the Grignard reaction [196]. While the amide derived from 5,6-dihydrophenanthridine is readily obtained from a carboxylic acid and 5,6-dihydrophenanthridine by using 2-halopyridinium salt as the condensation reagent in acetonitrile, dichloromethane, or 1,2-dichloroethane, it is found that in the presence of a catalytic amount of DMAP, the amide derived from 5,6-dihydro-phenanthridine can be obtained in best yield by the use of 2-chloro-6-methyl-1,3-diphenylpyridinium tetrafluoroborate and proton sponge as the condensation reagent and acid captor, respectively. Alternatively, the amide can also be prepared from acyl chloride and 5,6-dihydrophenanthridine, where the acyl chloride can be prepared in situ by treating a carboxylic acid with oxalyl chloride. On the other hand, this amide can also be easily converted to the original carboxylic acid by oxidation, for which CAN outperforms other tested oxidants, such as DDQ, trityl cation, sodium persulfate, and mercuric chloride [196]. 3.11.1 PREPARATION OF 4-PHENYLBUTYRIC ACID 5,6-DIHYDROPHENANTHRIDINE AMIDE [196] Ph

OH

Ph O

HN +

N Cl Ph BF 4 proton sponge cat. DMAP CH 3 CN, r.t.

O

Me

N

Ph

To a solution of 220 mg 2-chloro-6-methyl-1,3-diphenylpyridinium tetrafluoroborate (0.60 mmol) in 2.5 mL acetonitrile containing a catalytic

The Carboxyl Protecting Groups 139

amount of DMAP was added a solution of 82 mg 4-phenylbutyric acid (0.50 mmol), 259 mg proton sponge (1.21 mmol), and 101 mg 5,6-dihydrophenanthridine (0.56 mmol) in 9 mL acetonitrile at room temperature under an argon atmosphere. The resulting mixture was stirred for 1.5 hours at room temperature and then refluxed for 5.5 hours. The reaction mixture was quenched with water, and the amide was extracted with ether. The ethereal extract was successively washed with aqueous sodium hydrogen carbonate solution, 1 N aqueous HCl solution, and brine, and dried over sodium sulfate. After removal of the solvent under reduced pressure, the residue was chromatographed on silica gel to give 160 mg of 4-phenylbutyric acid 5,6-dihydrophenanthridine amide, i.e., 1-(phenanthridin-5(6H)-yl)-4-phenylbutan-1-one, in a yield of 98%. 3.11.2 DEPROTECTION OF 5,6-DIHYDROPHENANTHRIDINE AMIDE [196] O N

Ph

CAN CH 3 CN/H 2 O (4:1)

OH

Ph O

CAN (904 mg, 1.65 mmol) was added at once to a solution of 245 mg 4-phenylbutyric acid 5,6-dihydrophenanthridine amide (0.45 mmol) in 7.5 mL of acetonitrile/water (4:1). After being stirred for 15 minutes, the reaction mixture was diluted with ether and separated from the aqueous layer. The organic layer was washed successively with 1 N aqueous HCl solution and brine, and dried over sodium sulfate. The solvent was removed under reduced pressure to give 119 mg of pure 4-phenylbutyric acid, in a yield of 97%. 3.12 OXAZOLINE, OXAZOLIDINE, 5,6-DIHYDRO-1,3-OXAZINE AND BOROXAZOLIDONE PROTECTING GROUPS Besides the conversion of the carboxyl group into ester and amide, the carboxyl group can also be converted into heterocyclic functionalities which may tolerate harsh reaction conditions, such as in the presence of Grignard reagent. Among these heterocyclic functionalities, oxazoline [197],

140

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

oxazolidine [198], 5,6-dihydro-1,3-oxazine [199], and boroxazolidone [200], are the representative protecting groups for carboxyl functionality. Oxazoline is a five-membered heterocyclic organic compound containing one atom of oxygen and nitrogen on the ring that are separated by one carbon atom. It exists between oxazole and oxazolidine in terms of saturation, where oxazole is fully unsaturated whereas oxazolidine is fully saturated. In comparison, oxazine is a six-membered heterocyclic compound containing both oxygen and nitrogen atoms that are also separated by one carbon atom. In general, oxazoline is formed between carboxylic acid and 2-aminoalcohol under dehydration conditions, or formed from acyl halide with 2-amino-alcohol [197]. In comparison, 5-oxazolidinone derivatives of α-amino acids, which can be prepared from N-protected α-amino acids and paraformaldehyde, have been employed as dual protection of the α-amino and α-carboxyl group in the synthesis of β-aspartyl and γ-glutamyl peptides. The 5-oxazolidinone derivatives of aspartic acid and glutamic acid can be formed in good yields from two procedures: treatment of N-benzyloxycarbonyl-L-aspartic or N-benzyloxycarbonyl-Lglutamic acid with paraformaldehyde in a mixture of acetic anhydride, acetic acid and a trace amount of thionyl chloride; or azeotropic distillation of N-benzyloxycarbonyl-L-aspartic or N-benzyloxycarbonyl-L-glutamic acid with paraformaldehyde and PTSA in benzene [201]. The protecting oxazolidinone ring can readily be removed by alkaline hydrolysis or by catalytic hydrogenolysis [202]. The 2-methyl-5,6-dihydro-1,3-oxazine derivative of the carboxylic acid is totally inert to various Grignard reagents so that substituted carboxylic acid can be prepared from this derivative after deprotection [199]. 3.12.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF PROTECTING GROUP 3.12.1.1 PREPARATION OF 4,5-DIHYDRO-4,4-DIMETHYL-2-(CIS-8HEPTADECENYL) OXAZOLE (OLEIC ACID OXAZOLINE) [197] N

O

+

OH

HO

NH 2

xylene

O

  Method A: A solution of 2.5 g 2-methyl-2-aminopropanol (0.028 mol) and 7.1 g oleic acid (0.025 mol) in 10 mL of xylene was refluxed

The Carboxyl Protecting Groups 141

for 18 hours under nitrogen, water being removed continuously. The temperature rose from 140 to 152°C and the solution became light yellow. The excess xylene was evaporated at reduced pressure and the resulting yellow liquid was filtered through neutral alumina (ether) and distilled in vacuo to yield 4.5 g of (Z)-2-(heptadec-8en-1-yl)-4,4-dimethyl-4,5-dihydrooxazole as a colorless liquid, in a yield of 54%, b.p., 168–173°C/0.3 mmHg. N

O Cl

+

HO

NH 2

1. CH 2 Cl 2 2. SOCl

2

O

  Method B: Oleyl chloride was prepared quantitatively from oleic acid and oxalyl chloride. To an ice-cooled solution of 8.8 g oleyl chloride (0.029 mol) in 15 mL of dry methylene chloride, 5.2 g of 2-methyl-2-aminopropanol (0.05 mol) in 135 mL of dry methylene chloride was added slowly (~ 1 hour) at 0°C with stirring. The reaction mixture was stirred for another hour at 0°C and then at room temperature overnight. The mixture was washed with water, dried over MgSO4 and evaporated. The resulting colorless oil (9.7 g, 94%) was dissolved in 125 mL dry ether, and 11.5 g of thionyl chloride (7.0 mL, 0.097 mol) was added dropwise with stirring at 0°C. After being stirred at 0°C for 5 minutes, the ether solution was added with stirring to 300 mL 2 M NaOH at 0°C. The phases were separated and the aqueous layer was extracted with ether. The combined ether solutions were dried over K2CO3 and evaporated to afford a light-yellow liquid in 8.5 g. Distillation in vacuo yielded 6.9 g of product as a colorless liquid, in a yield of 70%, b.p. 139–141°C/0.03 mmHg. 3.12.1.2 PREPARATION OF (S)-3-BENZYLOXYCARBONYL-5-OXO-4OXAZOLIDINEACETIC ACID [198]

A mixture of 5.35 g N-benzyloxycarbonyl-L-aspartic acid, 1.8 g paraformaldehyde, 4.0 g acetic anhydride and 0.3 mL thionyl chloride in 80 mL of

142

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

acetic acid was heated at 100°C for 4 hours. Evaporation of acetic acid under reduced pressure gave an oily residue, which was dissolved in EtOAc and extracted with 5% NaHCO3 solution. The aqueous layer was acidified with 6 N HCl on ice-cooling and extracted with EtOAc. The extract was washed with water and dried over MgSO4. Evaporation of the solvent afforded 4.4 g (S)-2-(3-((benzyloxy)carbonyl)-5-oxooxazolidin-4-yl)acetic acid, also known as (S)-3-benzyloxycarbonyl-5-oxo-4-oxazolidineacetic acid as a pale-yellow syrup, in a yield of 80%. 3.12.1.3 PREPARATION OF L-ASPARTIC ACID BOROXAZOLIDONE (GENERAL PROCEDURE) [200]

Finely ground L-aspartic acid (1.33 g, 10 mmol) was suspended in 5–10 mL THF, then 12 mL 1 M solution of triethylborane in THF (12 mmol) was added and the mixture was stirred until the aspartic acid had dissolved. The reaction time strongly depends on the effectiveness of the grinding. With finely powdered amino acid, the reaction is completed within 5–30 minutes. The solution was then filtered if necessary and concentrated to dryness. The residue was treated with either toluene or cyclohexane, the product was collected by filtration and washed with cyclohexane or diisopropyl ether. 3.12.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF PROTECTING GROUP 3.12.2.1 SAPONIFICATION OF (S)-3-BENZYLOXYCARBONYL-5-OXO-4OXAZOLIDINEPROPIONIC ACID [198]

The Carboxyl Protecting Groups 143

A solution of 1.5 g (S)-3-benzyloxycarbonyl-5-oxo-4-oxazolidinepropionic acid in 20 mL methanol was treated with 10.0 mL 1 N NaOH at room temperature for 4 hours. The reaction mixture was neutralized with 1 N HCl, and the methanol was removed under reduced pressure. The product dissolved in EtOAc was extracted with 5% NaHCO3 solution. The extract was washed once with fresh EtOAc and acidified with 6 N HCl. The acidified solution was extracted with EtOAc and the organic layer was washed with water and dried over MgSO4. Evaporation of solvent gave an oily residue, which was solidified on standing. Recrystallization from water gave 1.0 g of N-benzyloxycarbonyl-L-glutamic acid, in a yield of 71%, m.p. 120–121°C.

3.12.2.2 CATALYTIC REDUCTION OF (S)-3-BENZYLOXYCARBONYL-5OXO-4-OXAZOLIDINEPROPIONIC ACID [198] O

O O

OH N

O O

Pd black CH 3 CO 2 H MeOH/dioxane

O

O

HO

OH NH 2

A solution of 2.2 g (S)-3-benzyloxycarbonyl-5-oxo-4-oxazolidinepropionic acid in a mixture of 25 mL methanol, 4 mL acetic acid and 20 mL dioxane was hydrogenated in the presence of palladium black for 6 hours. The catalyst was removed by filtration, the filtrate was evaporated under reduced pressure and the residue was recrystallized from water to give 0.8 g of L-glutamic acid, in a yield of 73%, m.p. 199–200°C (decomp.).

3.13 TRANSIENT OR TEMPORARY PROTECTION OF CARBOXYL GROUP In peptide synthesis, it is necessary to protect the carboxyl group of one amino acid residue and the amino group of another amino acid residue to form the desired peptide bond. However, in some cases, the carboxyl group that should be protected can be temporarily protected and quickly

144

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

converted into other functionalities. In this case, not very stable carboxyl derivatives are formed. Reagents that have been used for temporary protection of carboxyl group include phase transfer reagent [203–205], 1-hydroxybenzotriazine [206], 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt) [207–212], and silyl esters [213, 214]. It is believed that a phase-transfer reagent can form a salt with C-terminal free amino acids or peptides, and this reagent can be easily removed during the extraction of reaction product so that the side reactions caused by acid- or base-catalyzed deprotection of carboxyl protecting group can be avoided [203]. Besides the presence of phase-transfer reagent, the most commonly used method for carboxyl protection as well as activation is by means of 1-hydroxybenzotriazine in the presence of a condensation agent. In this case, a peptide can be easily constructed between amino acids or an amino acid and a smaller peptide in the presence of 1-hydroxybenzotriazine and peptide condensation agent (e.g., DCC), of which one amino acid has the unprotected carboxyl group for the formation of the peptide bond [206]. Also, the application of HODhbt allows the formation of activated amino acid esters, that have been applied in a fully automated peptide synthesis system in which acylation and deprotection steps can be checked automatically for completion before proceeding to the next amino acid residues [209, 215]. When silyl esters are applied as the carboxyl protecting groups, TMS ester is often used for temporary carboxyl protections. The amino acid TMS esters can be easily prepared by treating amino acids with TMS chloride [214], hexamethyldisilazane [216], or ditrimethylsilylamine ((Me3Si)2NH) [217, 218], and the resulting amino acid TMS esters are quite stable in a nonaqueous solution but might be hydrolyzed instantaneously upon exposure to very mild acid or base, or even water. From the unprotected amino acids, N,O-bis-silylated amino acid derivatives can also form, whereas amino acids with reactive side chains, such as Ser, Thr, and Tyr, trisilylated products can form as well. With respect to the TMS group, TBDMS [219] and di-tert-butylmethylsilyl (DTBMS) [220] are relatively bulky groups, forming stable carboxyl derivatives that can withstand hydrolysis. Also, tri(tert-butoxy)silyl ester has been applied as the carboxyl protecting group [221], which can be introduced by the treatment of amino acid with (t-BuO)3SiCl/pyridine or SiCl4 in t-BuOH/ base [222].

The Carboxyl Protecting Groups 145

3.13.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR INTRODUCTION OF TRANSIENT CARBOXYL PROTECTING GROUP 3.13.1.1 GENERAL PROCEDURE FOR SALT FORMATION OF AMINO ACIDS WITH VARIOUS PHASE TRANSFER REAGENTS USING ALANINE AS AN EXAMPLE [203]

To a 500 mL round-bottomed flask, was added 4.45 g L-alanine (50 mmol), 200 mL water, and a phase transfer reagent (e.g., Bu4N+OH–, 50 mmol). The resulting mixture was stirred until complete dissolution (occasionally, more water or organic solvents such as methanol or ethanol were needed to effect the complete dissolution). The clear solution was lyophilized to afford a white powder, which was stored in a desiccator for subsequent use. 3.13.1.2 GENERAL PROCEDURE FOR C-TERMINAL FREE PEPTIDE SYNTHESIS USING BOC-PHE-ALA-OH AS EXAMPLE [203]

O O

N H

OH

OH DCC N N CH 2 Cl 2 /DMF (4:1) N

+

O

O N H

O

O O NH 2

O O

N N N

NBu 4

O O

N H

H N

O OH

O

Boc-Phe-OH (2.67 g, 10 mmol) was dissolved in a mixed solvent of 40 mL of dichloromethane and 10 mL of DMF. Then 1.37 g of 1-hydroxybenzotriazole (11 mmol) and 2.17 g dicyclohexylcarbodiimide (10.5 mmol) were added and the mixture was stirred for 2 hours. Then, the previously prepared salt of alanyl phase-transfer reagent (15 mmol) was added, the solution was stirred for 5 hours at room temperature until the Boc-Phe-OH was consumed as monitored by TLC. The insoluble by-products formed

146

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

were filtered off and the filtrate was diluted with 100 mL dichloromethane. The organic phase was washed with water (3 × 30 mL), 0.1 M HCl (4 × 20 mL), and water (3 × 30 mL), dried (anhydrous Na2SO4), and evaporated to give an oily residue which was redissolved in 100 mL EtOAc and the resulting solution was stored overnight in a refrigerator. Most DCU that dissolved with the product precipitated during the low-temperature storage. The precipitate was filtered off and the EtOAc was evaporated to yield a crude product that was crystallized after adding n-hexane. The product was recrystallized from EtOAc/n-hexane (1:3) to afford 2.49 g of Boc-Phe-AlaOH, i.e., (tert-butoxycarbonyl)-L-phenylalanyl-L-alanine, in a yield of 72%, m.p. 134–136°C. 3.13.1.3 PREPARATION OF N-TRITYL-VAL-ODHBT ESTER [212]

To a chilled solution of 0.6 g N-trityl-Val-OH and 0.51 g HODhbt in 10 mL THF was added 0.35 g of DCC. The mixture was kept at 0°C for 20 minutes and then at room temperature for 1 hour. The solvent was removed under reduced pressure and the remaining residue was taken up with EtOAc. The N,N’-DCU which separated out was filtered off and washed with EtOAc. The combined filtrates were washed with 5% K2CO3 solution, water, and dried over Na2SO4. The solvent was evaporated under vacuum and the residue crystallized upon addition of petroleum ether, affording 80% of N-trityl-Val-ODhbt, i.e., 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl trityl-Lvalinate, m.p. 140–141°C. 3.13.1.4 PREPARATION OF N-TRITYL-VAL-PHE-OBZ (BENZYL ESTERS) VIA THE DHBT ESTER OF N-TRITYL-VAL-OH) [212] O O Ph TsO

NH 3

Et 3 N OBn THF

O

O Ph

OBn NH 2

Ph 3 C

NH

N N

N

O THF

Ph 3 C

N H

H N O

O O Ph

Bn

The Carboxyl Protecting Groups 147

Phenylalanine benzyl ester p-toluenesulfonate (2 mmol) was dissolved in 7 mL of THF, and neutralized with triethylamine. Then 2.2 mmol of N-tritylVal-ODhbt ester was added. The mixture was kept at 0°C for 30 minutes and then at room temperature for 20 hours (Note: progress of the coupling reaction was followed by TLC and the ninhydrin test). The solvent was removed under reduced pressure and the remaining oily residue was taken up with 50 mL EtOAc, washed with 5% NaHCO3 solution (3 × 5 mL) and water and dried over Na2SO4. The extract with NaHCO3 became yellow at the beginning and turned white at the end of washings. The solvent was evaporated under vacuum and the residue crystallized upon addition of petroleum ether to afford 80% of N-trityl-Val-Phe-OBz, m.p. 153–156°C. 3.13.1.5 PREPARATION OF N-TRITYL-LEUCINE BY MEANS OF TRANSIENT PROTECTION OF CARBOXYL GROUP WITH TMS [214] O

O OH + (CH 3 ) 3 SiCl NH 2

CHCl 3 /MeCN (5:1)

O Cl

NH 3

O Si(CH 3 ) 3 1. NaOH O 2. AcOH HN 3. Et 2 NH CPh 3

Si(CH 3 ) 3 1. Et 3 N 2. Ph 3 CCl CHCl 3

O HN

O Et NH 2 2 CPh 3

To a magnetically stirred suspension of 1.31 g leucine (10 mmol) in 18 mL of CHCl3-MeCN (5:1) was added 1.27 mL Me3SiCl (10 mmol) at room temperature. The reaction mixture was heated under reflux for 2 hours and then allowed to attain room temperature. The addition of 2.79 mL Et3N (20 mmol) at a rate sufficient to maintain gentle reflux was followed by 2.79 g of trityl chloride in portions (10 mmol) that was dissolved in 10 mL of CHCl3. The resulting mixture was stirred for 1 hour at room temperature, and then an excess of MeOH (50 mmol) was added. Evaporation under reduced pressure left a residue, which was partitioned between 50 mL of Et2O (50 mL) and 50 mL precooled solution of citric acid (5%). The organic phase was collected and washed with 1 N NaOH (2 × 20 mL) and water (2 × 10 mL). The combined aqueous layers were washed with 20 mL of Et2O, cooled to 0°C, and neutralized with glacial AcOH. The precipitated product was extracted with Et2O (2 × 30 mL), and the combined organic layers were washed twice with water and dried (MgSO4). Evaporation of the solvent in vacuo gave 3.47 g of the desired product as a light-yellow foam, which upon dissolution in 20 mL of Et2O and

148

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

addition of Et2NH (1 mL, 10 mmol) afforded the corresponding crystalline diethylammonium trityl-L-leucinate salt, in a yield of 83%, m.p. 152–154°C. KEYWORDS • • • • • • • • • •

5,6-dihydrophenanthridine allyl protecting group aryl protecting group benzyl protecting group ethyl protecting group methyl protecting group orthoester phenacyl protecting group propargyl protecting group transient protection

REFERENCES 1.

2. 3. 4. 5. 6.

7. 8.

Ajioka, M., Higuchi, C., Oura, T., Katoh, T., & Yamaguchi, A., (1992). Preparation and Isolation of Mineral Acid Salt of An Amino Acid Methyl Ester. United States Patent, 5,113,009. Moody, H. M., (2008). Process for the Preparation of Amino Acid Methyl Esters. PCT Int. Appl., WO 2008110529 A1. Bodanszky, M., (1984). Alkyl esters of amino acids. Int. J. Peptide Protein Res., 23(1), 111–111. Devedjiev, I. T., Bairyamov, S. G., & Videva, V. S., (2008). Biomimetic synthesis of esters of natural amino acids. Heteroatom Chemistry, 19(3), 252–255. Ramesh, C. A., & Vimal, (1998). A mild and convenient procedure for the esterification of amino acids. Synth. Commun., 28, 1963–1965. Andersson, P. G., Guijarro, D., & Tanner, D., (1997). Preparation and use of aziridino alcohols as promoters for the enantioselective addition of dialkylzinc reagents to N-(diphenylphosphinoyl) imines. J. Org. Chem., 62(21), 7364–7375. Li, J., & Sha, Y., (2008). A convenient synthesis of amino acid methyl esters. Molecules, 13, 1111–1119. Ueda, K., Waki, M., & Izumiya, N., (1984). Facile synthesis of amino acid methyl ester p-toluene sulfonates with methyl p-toluenesonate. Memoirs of the Faculty of Science, Kyushu University, Ser. C., 14(2), 307–312.

The Carboxyl Protecting Groups 149

9.

10. 11. 12. 13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

Theobald, J. M., Williams, M. W., & Young, G. T., (1963). Amino acids and peptides. XVII. The preparation of the methyl and benzyl esters of amino acids by means of dialkyl sulfites. J. Chem. Soc., 1927–1930. Rachele, J. R., (1963). The methyl esterification of amino acids with 2,2-dimethoxypropane and aqueous hydrogen chloride. J. Org. Chem., 28(10), 2898. Williams, R. M., & Fegley, G. J., (1993). Asymmetric synthesis of (1S,2R)-(+)-2-Phenyl1-aminocyclopropane-1-carboxylic acid. J. Org. Chem., 58(24), 6933–6935. Wu, L. Y., & Berkman, C. E., (2005). Synthesis of N-phosphoryl amino acids using bis(9-fluorenylmethyl) phosphite. Tetrahedron Lett., 46(32), 5301–5303. Barbayianni, E., Fotakopoulou, I., Schmidt, M., Constantinou-Kokotou, V., Bornscheuer, U. T., & Kokotos, G., (2005). Enzymatic removal of carboxyl protecting groups. 2. Cleavage of the benzyl and methyl moieties. J. Org. Chem., 70(22), 8730–8733. Unden, A., (1995). Preparation of Acyclic aliphatic branched alkyl groups as protective groups for side chains of amino-acid moieties in preparation of peptide, polypeptide or protein structures. PCT Int. Appl. WO 9526975 A1. Di Gioia, M. L., Leggio, A., Le Pera, A., Liguori, A., Perri, F., & Siciliano, C., (2004). Alternative and chemoselective deprotection of the α-amino and carboxy functions of N-Fmoc-amino acid and N-Fmoc-dipeptide methyl esters by modulation of the molar ratio in the AlCl3/N,N-dimethylaniline reagent system. European Journal of Organic Chemistry, (21), 4437–4441. Di Gioia, M. L., Leggio, A., Le Pera, A., Siciliano, C., Sindona, G., & Liguori, A., (2004). An Efficient and highly selective deprotection of N-Fmoc-α-amino acid and lipophilic N-Fmoc-dipeptide methyl esters with aluminum trichloride and N,N-dimethylaniline. Journal of Peptide Research, 63(4), 383–387. Papaioannou, D., Athanassopoulos, C., Magafa, V., Karamanos, N., Stavropoulos, G., Napoli, A., Sindona, G., Aksnes, D. W., & Francis, G. W., (1994). Redox N-alkylation of tosyl protected amino acid and peptide esters. Acta Chemica Scandinavica, 48(4), 324–333. Akiyama, T., Hirofuji, H., Hirose, A., & Ozaki, S., (1994). Mild deprotection of methyl, benzyl, methoxymethyl, methylthiomethyl, methoxyethoxymethyl, and β-(Trimethylsilyl) ethoxymethyl esters with AlCl3-N,N-dimethylaniline. Synthetic Commun., 24(15), 2179–2185. Tsunematsu, H., Ishida, E., Yoshida, S., & Yamamot, M., (1991). Synthesis and enzymatic hydrolysis of aspirin-basic amino acid ethyl esters. Int. J. Pharmaceuticals, 68, 77–86. Borders, C. L., Blech, D. M., & McElvany, K. D., (1984). L-amino acid ethyl ester hydrochlorides derivatives for qualitative organic analysis. J. Chem. Edu., 61(9), 814–815. Buchmeiser, M. R., Sinner, F., Mupa, M., & Wurst, K., (2000). Ring-opening metathesis polymerization for the preparation of surface-grafted polymer supports. Macromolecules, 33, 32–39. Zhang, S., & Arvidsson, P. I., (2008). Facile synthesis of N-protected amino acid esters assisted by microwave irradiation. Int J. Pept Res. Ther., 14, 219–222.

150

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

23. Kise, H., & Shirato, H., (1985). Synthesis of aromatic amino acid ethyl esters by α-chymotrypsin in solutions of high ethanol concentrations. Tetrahedron Lett., 26(49), 6081–6084. 24. Romine, J. L., (2012). Preparation of 1H-benzimidazole derivatives end-capped with amino acid or peptide derivatives as hepatitis C virus inhibitors. PCT Int. Appl. WO 2012018325A1. 25. Kyba, E. P., Timko, J. M., Kaplan, L. J., De Jong, F., Gokel, G. W., & Cram, D. J., (1978). Host-guest complexation. 11. Survey of chiral recognition of amine and amino ester salts by dilocular bisdinaphthyl hosts. J. Am. Chem. Soc., 100(14), 4555–4568. 26. Sebastian, D., & Waldmann, H., (1997). Chemoenzymatic synthesis of a characteristic phosphopeptide fragment of the raf-1 kinase. Tetrahedron Lett., 38(17), 2927–2930. 27. Anderson, G. W., & Callahan, F. M., (1967). Tert-Butyl Group as a Carboxyl Protecting Group in Peptide Syntheses. United States Patent, US 3325466. 28. Pawelczak, K., Jones, T. R., Kempny, M., Jackman, A. L., Newell, D. R., Krzyzanowski, L., & Rzeszotarska, B., (1989). Quinazoline antifolates inhibiting thymidylate synthase: Synthesis of four oligo(L-γ-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid and their enzyme inhibition. J. Med. Chem., 32, 160–165. 29. Strazzolini, P., Scuccato, M., & Giumanini, A. G., (2000). Deprotection of t-butyl esters of amino acid derivatives by nitric acid in dichloromethane. Tetrahedron, 56, 3625–3633. 30. Sliedregt, K. M., Schouten, A., Kroon, J., & Liskamp, R. M. J., (1996). Reaction of N-trityl amino acids with BOP: Efficient synthesis of t-butyl esters as well as N-trityl serine- and threonine-β-lactones. Tetrahedron Lett., 37(24), 4237–4240. 31. Bischoff, L., David, C., Roques, B. P., & Fournié-Zaluski, M. C., (1999). Side-chainmodified sulfonic analogues of aspartic and glutamic acids: Synthesis, protection, and incorporation into peptides. J. Org. Chem., 64, 1420–1423. 32. Li, B., Berliner, M., Buzon, R., Chiu, C. K. F., Colgan, S. T., Kaneko, T., Keene, N., et al., (2006). Aqueous phosphoric acid as a mild reagent for deprotection of tert-butyl carbamates, esters, and ethers. J. Org. Chem., 74(24), 9045–9050. 33. Kaul, R., Brouillette, Y., Sajjadi, Z., Hansford, K. A., & Lubell, W. D., (2004). Selective tert-butyl ester deprotection in the presence of acid-labile protecting groups with use of ZnBr2. J. Org. Chem., 69, 6131–6133. 34. Iossifidou, S. M., & Froussios, C. C., (1996). Facile synthesis of 1-adamantyl esters of L-α-amino acids, a new class of carboxy protected derivatives. Synthesis, 1355–1358. 35. Matsuoto, A., Watanabe, H., & Otsu, T., (1992). Synthesis and radical polymerization of itaconates containing an adamantyl ester group. Bull. Chem. Soc. Jpn., 65(3), 846–852. 36. Błotny, G., & Taschner, E., (1966). New carboxyl-protecting groups applicable in peptide synthesis. Bulletin de l’Academie Polonaise des Sciences, Serie des Sciences Chimiques, 14(9), 615–619. 37. Robles, J., Pedroso, E., & Grandas, A., (1993). 2-(4-acetyl-2-nitrophenyl)ethyl: A new base-labile carboxyl protecting group. Synthesis, 1261–1266.

The Carboxyl Protecting Groups 151

38. DiMaio, J., Jaramillo, J., Wernic, D., Grenier, L., Welchner, E., & Adams, J., (1990). Synthesis and biological activity of atrial natriuretic factor analogues: Effect of modifications to the disulfide bridge. J. Med. Chem., 33, 661–667. 39. Mérette, S. A. M., Burd, A. P., & Deadman, J. J., (1999). Synthesis of 9-fluorenylmethyl esters using 9-fluorenylmethylchloroformate. Tetrahedron Lett., 40, 753, 754. 40. Zhao, Z., & Felix, A. M., (1994). Solid-phase synthesis of extended lactam ring systems: Preparation of amino acid α-fluorenylmethyl esters for the synthesis of reverse-extended lactams. Peptide Research, 7(4), 218–223. 41. Froussios, C., & Kolovos, M., (1989). Novel method for protection of carboxylic acids α-amines: 9-fluorenyl esters. Tetrahedron Lett., 30(26), 3413, 3414. 42. Bednarek, M. A., & Bodanszky, M., (1983). 9-fluorenylmethyl esters. Int. J. Peptide Protein Res., 21, 196–201. 43. Henklein, P., Halatsch, W. R., Franke, P., & Zoepfl, H. J., (1988). Preparation of 9-Fluorenylmethyl Amino Acid Esters. DD 261780 A1. 44. Kozhich, A., Ostrovskii, A., & Pozdnev, V. F., (1987). Application of fluorenylmethyl esters in the synthesis of viral protein fragments. Pept., Proc. Eur. Pept. Symp., 19, 75–78. 45. Henkel, B., Zhang, L., & Bayer, E., (1997). Investigations on solid-phase peptide synthesis in N-to-C direction (inverse synthesis). Liebigs Ann. Recueil, 2161–2168. 46. Kessler, H., Becker, G., Kogler, H., & Wolff, M., (1984). 2-(2-pyridyl-)ethyl esters, a new carboxyl protecting group in peptide synthesis. Tetrahedron Lett., 25(36), 3971–3974. 47. Kessler, H., Becker, G., Kogler, H., Friese, J., & Kerssebaum, R., (1986). 2-(2’-pyridyl)ethyl ester (pet ester) derivatives of polyfunctional amino acids. Int. J. Peptide Protein Res., 28, 342–346. 48. Zaidi, J. H., Arfan, M., Khan, K. M., Perveen, S., & Ambreen, N., (2006). Reactions of N-protected L-amino acids with alkyl chloromethyl ethers and chloromethyl methyl sulfide. Lett. Org. Chem., 3, 242, 243. 49. Dossena, A., Marchelli, R., & Casnati, G., (1979). New system for ‘activation’ of dimethyl sulphoxide in pummerer-like reactions. J. Chem. Soc., Chem. Commun., 370–371. 50. Dossena, A., Palla, G., Marchelli, R., & Lodi, T., (1984). Methylthiomethyl, a new carboxyl protecting group in peptide synthesis. Int. J. Peptide Protein Res., 23, 198–202. 51. Mora, N., Lacombe, J. M., & Pavia, A. A., (1993). Methylthiomethy (MTM) Methodology for the synthesis of phosphoserine, phosphothreonine and thiophosphoserine synthons. Peptides; Chemistry, Structure, and Biology; Proceedings of the 13 American Peptide Symposium (pp. 187–189). Edmonton, Alberta, Canada. 52. Ganem, B., & Boeckman, R. K., (1974). Silver-assisted dimethyl sulfoxide oxidations. Improved synthesis of aldehydes and ketones. Tetrahedron Lett., (11), 917–920. 53. Sosnovsky, G., Rao, N. U. M., Li, S. W., & Swartz, H. M., (1989). Synthesis of nitroxyl (aminoxyl) labeled probes for studies of intracellular environment by EPR and MRI. J. Org. Chem., 54, 3667–3674.

152

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

54. Lipshutz, B. H., & Pegram, J. J., (1980). β-(Trimethylsilyl)ethoxymethyl chloride. A new reagent for the protection of the hydroxyl group. Tetrahedron Lett., 21(35), 3343–3346. 55. Pinto, B. M., Buiting, M. M. W., & Reimer, K. B., (1990). Use of the [β-(trimethylsilyl) ethoxy]methyl (SEM) protecting group in carbohydrate chemistry. Fully functionalized rhamnose acceptors and donors for use in oligosaccharide synthesis. J. Org. Chem., 55(7), 2177–2181. 56. Chen, W. C., Vera, M. D., & Joullié, M. M., (1997). Mild, selective cleavage of amino acid and peptide β-(trimethylsilyl)ethoxymethyl (SEM) esters by magnesium bromide. Tetrahedron Lett., 38(23), 4025–4028. 57. Nefkens, G. H. L., Tesser, G. I., & Nivard, R. J. F., (1963). Usefulness of the phthalimido methyl group for the reversible protection of carboxyl functions. Recueil des Travaux Chimiques des Pays-Bas, 82(9, 10), 941–953. 58. Turner, D. L., & Baczynski, E., (1970). Removal of a protecting group. Chem. & Ind., (37), 1204. 59. Fürstner, A., & Davies, P. W., (2005). Heterocycles by PtCl2-catalyzed intramolecular carboalkoxylation or carboamination of alkynes. J. Am. Chem. Soc., 127(43), 15024, 15025. 60. Kunz, H., & Buchholz, M., (1979). The system 2-haloethyl ester/choline ester as a two-step protective group of the carboxylic function in peptide. Chem. Ber., 112, 2145–2157. 61. Amaral, T. M. J. S. A., & Barbedo, I. M. R. E., (1982). The 2-chloroethyl group for Carboxyl protection in peptide synthesis. In: Blaha, K., (ed.), Peptide 1982; Proceeding of the 17th European Peptide Symposium (pp. 133–135). Prague, Czechoslovakia. Malon, Petr. 62. Dilamian, M., Montazer, M., & Masoumi, J., (2013). Antimicrobial electrospun membranes of chitosan/poly(ethylene oxide) incorporating poly(hexamethylene biguanide) hydrochloride. Carbohydrate Polymers, 94(1), 364–371. 63. Ala-Kleme, T., Maki, A., Maki, R., Kopperoinen, A., Heikkinen, M., & Haapakka, K., (2013). Micelle-encapsulated fullerenes in aqueous electrolytes. Journal of Luminescence, 135, 221–226. 64. Kunz, H., Braum, G., & Braun, P., (1993). Carboxylic acid ester protecting groups, process to prepare them, their coupling to functional groups and their use. Eur. Pat. Appl. EP 536671 A2. 65. Kunz, H., Kowalczyk, D., Braun, P., & Braum, G., (1994). Enzymic hydrolysis of hydrophilic diethylene glycol and polyethylene glycol esters of peptides and glycopeptides by lipases. Angew. Chem., 106(3), 353–355. 66. Lele, B. S., Gore, M. A., & Kulkarni, M. G., (1999). Direct esterification of poly(ethylene glycol) with amino acid hydrochlorides. Synth. Commun., 29(10), 1727–1739. 67. Wu, N., & Keller, B. C., (2011). Amino acid linked PEG-lipid conjugates. PCT Int. Appl. WO 2011139343 A2. 68. Sauvagnat, B., Kulig, K., Lamaty, F., Lazaro, R., & Martinez, J., (2000). Soluble polymer-supported synthesis of α-amino acid derivatives. J. Comb. Chem., 2, 134–142.

The Carboxyl Protecting Groups 153

69. Sieber, V. P., (1977). The 2-trimethylsilylethyl residue, a selective cleavable carboxyl protecting group. Helvetica Chimica Acta, 60 Fasc., 8(264), 2711–2716. 70. Gerlach, V. H., (1977). 2-(trimethylsilyl)ethyl esters as carboxyl protecting group; application in the synthesis of (-)-(S)-curvularin. Helvetica Chimica Acta, 60, Fasc., 8(298), 3039–3044. 71. Marlowe, C. K., (1993). Peptide cyclization on TFA labile resin using the trimethylsilyl (TMSE) ester as an orthogonal protecting group. Bioorg. Med. Chem. Lett., 3(3), 437–440. 72. Wagner, M., & Kunz, H., (2000). (2-phenyl-2-trimethylsilyl)ethyl (PTMSE) esters – a novel carboxyl protecting group. Synlett, (3), 400–402. 73. Wada, T., Tsuneyama, T., & Saigo, K., (2001). Synthesis of a new type of artificial nucleic acid derived from optically active serine. Nucleic Acids Research Supplement No. I, 187, 188. 74. Chantreux, D., Game, J. P., Jacquier, R., & Verducci, J., (1984). The 2-(diphenylphosphino) ethyl group (Dppe) as a new carboxyl-protecting group in peptide chemistry. Tetrahedron, 40(16), 3087–3094. 75. Joaquina, M., Amaral, S. A., Barrett, G. C., Rydon, H. N., & Willett, J. E., (1966). Polypeptides. Part XIII. The use of β-methylthioethyl esters for the protection of carboxyl groups in peptides synthesis. J. Chem. Soc. (C), 807–813. 76. Balog, A., Vargha, E., Breazu, D., Beu, L., & Gönczy, F., (1970). The activation and protection of the carboxyl group in the β-dicarbonyl N-protected amino acid series. Revue Roumaine de Chimie, 15, 1391–1407. 77. Amaral, T. M. J. A., & Gomes, M. J. R., (1988). Direct use of the 2-(4-nitrophenylsulfonyl) ethyl ester group in peptide synthesis. In: Peptide 1988; Proceeding 20th European Peptide Symposium (pp. 82–84). Tubingen, Germany. 78. Kundu, B. J., (1992). Acetol: A useful new protecting group for peptide synthesis. Tetrahedron Lett., 33(22), 3193–3196. 79. Sheehan, J. C., & Umezawa, K., (1973). Phenacyl photosensitive blocking groups. J. Org. Chem., 38(21), 3771–3774. 80. Ledger, R., & Stewart, F. H. C., (1967). p-bromophenacyl esters in peptide synthesis. Aust. J. Chem., 20, 787–791. 81. Ueki, M., Aoki, H., & Katoh, T., (1993). Selective removal of phenacyl ester group with a TBAF·xH2O-thiol system from amino acid derivatives containing benzyl or 4-nitrobenzyl ester. Tetrahedron Lett., 34(17), 2783–2786. 82. Popova, O., Jung, R., & Mitin, Y., (1982). Some properties of amino acid and peptide phenacyl esters. In: Peptide 1982; Proceeding 17th European Peptide Symposium (pp. 137–140). Prague, Czechoslovakia. 83. Hashimoto, C., Suzuki, T., & Kodomari, M., (2004). An efficient method for the peptide synthesis using phenacyl ester. Peptide Science; Proceedings of the Japanese Peptide Symposium, 41, 587–590. 84. Hashimoto, C., Sugimoto, K., Takido, T., & Kodomari, M., (2002). Stability of amino acid phenacyl esters and an efficient synthetic method for peptides containing phenacyl

154

85.

86.

87.

88.

89.

90.

91.

92.

93. 94.

95. 96. 97.

98.

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

ester using Na2CO3 supported on Al2O3. Peptide Science; Proceedings of the Japanese Peptide Symposium, 39, 135–138. Kuroda, H., Kubo, S., Chino, N., Kimura, T., & Sakakibara, S., (1992). Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids. Int. J. Peptide Protein Res., 40(2), 114–118. Kokinaki, S., Leondiadis, L., & Ferderigos, N., (2005). A novel and efficient method for cleavage of phenacylesters by magnesium reduction with acetic acid. Org. Lett., 7(9), 1723–1724. Leggio, A., Belsito, E. L., De Marco, R., Liguori, A., Perri, F., & Viscomi, M. C., (2010). An efficient preparation of N-methyl-α-amino acids from N-nosyl-α-amino acid phenacyl esters. J. Org. Chem., 75, 1386–1392. Namikoshi, M., Kundu, B., & Rinehart, K. L., (1991). Use of tetrabutylammonium fluoride as a facile deprotecting reagent for 4-nitrobenzyl, 2,2,2-trichloroethyl, and phenacyl esters of amino acids. J. Org. Chem., 56, 5464–5466. Furlán, R. L. E., Mata, E. G., & Mascaretti, O. A., (1998). Efficient, non-acidolytic method for the selective cleavage of N-Boc amino acid and peptide phenacyl esters linked to a polystyrene resin. J. Chem. Soc. Perkin Trans. I, 355–358. Salomon, C. J., Mata, E. G., & Mascaretti, O. A., (1996). Selective deprotection of phenacyl, benzyl and methyl esters of N-protected amino acids and dipeptides and N-protected amino acids benzyl ester-linked to resins with bis(tributyltin) oxide. J. Chem. Soc., Perkin Trans. I, 995–999. Lüning, B., Norberg, T., & Tejbrant, J., (1989). Synthesis of mono- and disaccharide amino-acid derivatives for use in solid-phase peptide synthesis. Glycoconjugate J., 6, 5–19. Barbayianni, E., Kokotos, C. G., Bartsch, S., Drakou, C., Bornscheuer, U. T., & Kokotos, G., (2009). Bacillus subtilis esterase (BS2) and its double mutant have different selectivity in the removal of carboxyl protecting groups. Adv. Synth. Catal., 351, 2325–2332. Isidro-Llobet, A., Álvarez, M., & Albericio, F., (2009). Amino acid-protecting groups. Chem. Rev., 109, 2455–2504. Jensen, K. J., Alsina, J., Songster, M. F., Vágner, J., Albericio, F., & Barany, G., (1998). Backbone amide linker (BAL) strategy for solid-phase synthesis of C-terminal-modified and cyclic peptides. J. Am. Chem. Soc., 120, 5441–5452. Montero, A., Albericio, F., Royo, M., & Herradón, B., (2007). Synthesis of a 24-membered cyclic peptide-biphenyl hybrid. Eur. J. Org. Chem., 1301–1308. Guzman-Martinez, A., Lamer, R., & VanNieuwenhze, M. S., (2007). Total synthesis of lysobactin. J. Am. Chem. Soc., 129, 6017–6021. Ramapanicker, R., Gupta, R., Megha, R., & Chandrasekaran, S., (2011). Applications of propargyl esters of amino acids in solution-phase peptide synthesis. International Journal of Peptides, 854952(1–10). Lopez, S. S., & Dudley, G. B., (2008). Convenient method for preparing benzyl ethers and esters using 2-benzyloxypyridine. Beilstein Journal of Organic Chemistry, 4(44), doi: 10.3762/bjoc.4.44.

The Carboxyl Protecting Groups 155

99. Vasanthakumar, G. R., Patil, B. S., & Babu, V. V. S., (2002). Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts. Letters in Peptide Science, 9, 207–209. 100. Stelakatos, G. C., & Argyropoulos, N., (1970). Amino-acid 4-methoxybenzyl esters. J. Chem. Soc. (C), 964–967. 101. Froussios, C., & Kolovos, M., (1987). Preparation of diphenylmethyl esters and ethers of unprotected amino acids and β-hydroxy-α-amino acids. Synthesis, (12), 1106–1108. 102. Tummatorn, J., Albiniak, P. A., & Dudley, G. B., (2007). Synthesis of benzyl esters using 2-benzyloxy-1-methyl pyridinium triflate. J. Org. Chem., 72, 8962–8964. 103. Schaeckermann, J. N., & Lindel, T., (2018). Macrocyclic core of salarin C: Synthesis and oxidation. Organic Letters, 20(21), 6948–6951. 104. Tam, J. P., Heath, W. F., & Merrifield, R. B., (1986). Mechanisms for the removal of benzyl protecting groups in synthetic peptides by trifluoromethanesulfonic acidtrifluoroacetic acid-dimethyl sulfide. J. Am. Chem. Soc., 108, 5242–5251. 105. Amin, B. E., Anantharamaiah, G. M., Royer, G. P., & Means, G. E., (1979). Removal of benzyl-type protecting groups from peptides by catalytic transfer hydrogenation with formic acid. J. Org. Chem., 44(19), 3442–3444. 106. Pandarus, V., Beland, F., Ciriminna, R., & Pagliaro, M., (2011). Selective debenzylation of benzyl protected groups with SiliaCat Pd(0) under mild conditions. ChemCatChem, 3(7), 1146–1150. 107. Stewart, F. H. C., (1967). Comparative acidic cleavage experiments with methylsubstituted benzyl esters of amino acids. Aust. J. Chem., 20, 2243–2249. 108. Stewart, F. H. C., (1966). The use of 2,4,6-trimethylbenzyl esters in peptide synthesis. Aust. J. Chem., 19(6), 1067–1083. 109. Stewart, F. H. C., (1966). Selective acid hydrolysis of 2,4,6-trimethylbenzyl esters and its application in peptide synthesis. Aust. J. Chem., 19(8), 1511–1518. 110. Stewart, F. H. C., (1968). The synthesis of some peptide derivatives using the 2,4,6-trimethylbenzyl carboxyl-protecting group. Aust. J. Chem., 21(11), 2831–2834. 111. Stewart, F. H. C., (1971). p-methoxybenzyl and 2,4,6-trimethylbenzyl ester hydrochlorides of sarcosine, L-proline, and L-4-hydroxyproline. Aust. J. Chem., 24(8), 1749–1752. 112. Rivett, D. E., & Stewart, F. H. C., (1980). Use of 2,4,6-trimethylbenzyl esters in the synthesis of tryptophan peptides. Aust. J. Chem., 33(12), 2693–2697. 113. Wang, M. F., Golding, B. T., & Potter, G. A., (2000). A convenient preparation of p-methoxybenzyl esters. Synth. Commun., 30(23), 4197–4204. 114. Loubinoux, B., & Gerardin, P., (1991). Protection of acids with azidomethoxybenzyl esters (Abz): Application in peptide synthesis. Tetrahedron, 47(2), 239–248. 115. Samanen, J. M., & Brandeis, E., (1988). The p-(methylsulfonyl)benzyl group: A TFA-stable carboxyl-protecting group readily convertible to a TFA-labile group. J. Org. Chem., 53, 561–569.

156

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

116. Samanen, J. M., & Brandeis, E., (1985). The p-methylsulfinylbenzyl group, A selectively cleavable carboxyl protecting group. Peptides: Structure and Function; Proceedings of the Ninth American Peptide Symposium, 225–228. 117. Chhabra, S. R., Parekh, H., Khan, A. N., Bycroft, B. W., & Kellam, B., (2001). A Dde-based carboxyl linker for solid-phase synthesis. Tetrahedron Lett., 42, 2189–2192. 118. Johnson, T., Liley, M., Cheeseright, T. J., & Begum, F., (2000). Problems in the synthesis of cyclic peptides through use of the Dmab protecting group. J. Chem. Soc., Perkin Trans. I, (16), 2811–2820. 119. Conroy, T., Jolliffe, K. A., & Payne, R. J., (2009). Efficient use of the Dmab protecting group: Applications for the solid-phase synthesis of N-linked glycopeptides. Org. Biomol. Chem., 7, 2255–2258. 120. Chan, W. C., Bycroft, B. W., Evans, D. J., & White, P. D., (1995). A novel 4-aminobenzyl ester-based carboxy-protecting group for synthesis of A typical peptides by Fmoc-but solid-phase chemistry. J. Chem. Soc. Chem. Commun., 2209, 2210. 121. Briand, B., Kotzur, N., Hagen, V., & Beyermann, M., (2008). A new photolabile carboxyl protecting group for native chemical ligation. Tetrahedron Lett., 49, 85–87. 122. Ruczynski, J., Lewandowska, B., Mucha, P., & Rekowski, P., (2008). Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid. J. Pept. Sci., 14, 335–341. 123. Prestidge, R. L., Harding, D. R. K., & Hancock, W. S., (1976). Use of substituted benzyl esters as carboxyl-protecting groups in solid-phase peptide synthesis. J. Org. Chem., 41(15), 2579–2583. 124. Suzuki, K., Nitta, K., & Sasaki, Y., (1976). The β-phenacyl and β-p-nitrobenzyl esters to suppress side reactions during treatment of aspartyl peptides with hydrogen fluoride. Chem. Pharm. Bull., 24(12), 3025–3033. 125. Stewart, F. H. C., (1965). Condensation experiments with the o-cyanobenzyl esters of some amino acid and peptide derivatives. Aust. J. Chem., 18, 1877–1883. 126. Hubbuch, A., Bindewald, R., Föhles, J., Naithani, V. K., & Zahn, H., (1980). 4-sulfobenzyl, a new carboxy protecting group. Angew. Chem. Int. Ed. Eng., 19(5), 394–396. 127. Corbett, D. F., & Eglington, A. J., (1980). Conversion of the olivanic acids into antibiotics of the PS-5 type: Use of a new carboxy protecting group. J. Chem. Soc. Chem. Commun., 1083, 1084. 128. Reynolds, G. D., Harding, D. R. K., & Hancock, W. S., (1981). Use of the chromogenic p-(p-(Dimethylamino)phenylazo)benzyl (Az) ester in the synthesis of leu-enkephalin. Int. J. Peptide Protein Res., 17(2), 231–234. 129. Hiskey, R. G., & Adams, J. B., (1965). Sulfur-containing polypeptides. I. Use of the N-benzhydryloxycarbonyl group and the benzhydryl ester. J. Am. Chem. Soc., 87(17). 130. Guibe-Jampel, E., & Wakselman, M., (1982). Selective cleavage of p-nitrobenzyl esters with sodium dithionite. Synth. Commun., 12(3), 219–223. 131. Aujard, I., Benbrahim, C., Gouget, M., Ruel, O., Baudin, J. B., Neveu, P., & Jullien, L., (2006). O-nitrobenzyl photolabile protecting groups with red-shifted absorption:

The Carboxyl Protecting Groups 157

Syntheses and uncaging cross-sections for one- and two-photon excitation. Chem. Eur. J., 12, 6865–6879. 132. Holmes, C. P., & Kiangsoontra, B., (1994). Development of a new photo-removable protecting group for the amino and carboxyl groups of amino acids. Peptides; Chemistry, Structure, and Biology; Proceedings of the Thirteenth American Peptide Symposium (pp. 110–112). Edmonton, Alberta, Canada. 133. Routledge, A., Abell, C., & Balasubramanian, S., (1997). The use of a dithiane protected benzoin photolabile safety catch linker for solid-phase synthesis. Tetrahedron Lett., 38, 1227–1230. 134. Barlos, K., Kallitsis, J., Mamos, P., Patrianakou, S., & Stavropoulos, G., (1987). A novel preparation of amino acid diphenylmethyl esters and their application in peptide synthesis. Liebigs Ann. Chem., 633–635. 135. Lapatsanis, L., (1978). A new method for the preparation of diphenylmethyl esters by using tri-diphenylmethyl phosphate as alkylating agent. Tetrahedron Lett., (47), 4697, 4698. 136. Stelakatos, G. C., Paganou, A., & Zervas, L., (1966). New methods in peptide synthesis. Part III. Protection of carboxyl group. J. Chem. Soc. (C), 1191–1199. 137. Gee, K. R., Niu, L., Schaper, K., Jayaraman, V., & Hess, G. P., (1999). Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor. Biochemistry, 38, 3140–3147. 138. Iwamura, M., Hodota, C., & Ishibashi, M., (1991). 1-(α-diazobenzyl)pyrene: A reagent for photolabile and fluorescent protection of carboxyl groups of amino acids and peptides. Synlett, 35, 36. 139. Tamiaki, H., Kumon, K., & Shibata, R., (2007). Synthetic hydroxymethyl-porphyrins for protection of carboxy group. Journal of Porphyrins and Phthalocyanines, 11(5, 6), 434–441. 140. MacLaren, J. A., (1972). A convenient preparative method for esters of amino acids. Aust. J. Chem., 25, 1293–1299. 141. Jackson, R. F. W., Wishart, N., Wood, A., James, J., & Wythes, M. J., (1992). Preparation of enantiomerically pure protected 4-oxo-α-amino acids and 3-aryl-α-amino acids from serine. J. Org. Chem., 57, 3397–3404. 142. Nemoto, T., Ando, D., Naka, K., & Chujo, Y., (2006). Polycondensation of α-amino acid esters in the presence of yttrium triflate as a Lewis acid. J. Polymer Sci. Part A: Poly. Chem., 44, 4731–4735. 143. Ramesh, D., Wieboldt, R., Niu, L., Carpentert, B. K., & Hess, G. P., (1993). Photolysis of a protecting group for the carboxyl function of neurotransmitters within 3 μs and with product quantum yield of 0.2. Proc. Nati. Acad. Sci. USA, 90, 11074–11078. 144. Tantry, S. J., & Babu, V. V. S., (2003). 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a useful reagent for the synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenol, p-nitrophenyl, o-nitrophenyl and succinimidyl esters of Nα-urethane protected amino acids. Letters in Peptide Science, 10, 655–662.

158

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

145. Sandrin, E., & Boissonnas, R. A., (1963). Synthesis of structural analogs of eledoisin. I. Preparation of intermediates. Helvetica Chimica Acta, 46(5), 1637–1669. 146. [a] Stewart, F. H. C., (1966). The preparation of some sequential polypeptides by the p-nitrophenyl ester method. Aust. J. Chem., 19, 1503–1509; [b] Stewart, F. H. C., (1969). Synthesis of polydepsipeptides with regularly repeating unit sequences. Aust. J. Chem., 22, 1291–1298. 147. Sekizaki, H., Itoh, K., Shibuya, A., Toyota, E., & Tanizawa, K., (2007). A facile synthesis of p- and m-(amidinomethyl)phenyl esters derived from amino acid and tryptic hydrolysis of these synthetic inverse substrates. Chemical & Pharmaceutical Bulletin, 55(10), 1514–1517. 148. Sekizaki, H., Itoh, K., Shibuya, A., Toyota, E., Kojoma, M., & Tanizawa, K., (2008). Trypsin-catalyzed synthesis of dipeptide containing α-aminoisobutyric acid using p- and m-(Amidinomethyl)phenyl esters as acyl donor. Chemical & Pharmaceutical Bulletin, 56(5), 688–691. 149. Johnson, B. J., & Trask, E. G., (1968). 4-(methylthio)phenyl and 4-(methylsulfonyl) phenyl esters in the preparation of peptides and polypeptides. II. Synthesis of the protected heptapeptide (A82-A88) of bovine chymotrypsinogen A. J. Org. Chem., 33(12), 4521–4524. 150. Babu, V. V. S., Ananda, K., & Mathad, R. I., (2000). Synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl and pentachlorophenol esters of fmoc-amino acids using Fmocamino acid chlorides as intermediates. Lett. Peptide Science, 7, 239–242. 151. Kisfaludy, L., & Schon, I., (1983). Preparation and applications of pentafluorophenyl esters of 9-fluorenylmethyloxycarbonyl amino acids for peptide synthesis. Synthesis, 4, 325–327. 152. Ramesh, R., Rajasekaran, S., Gupta, R., & Chandrasekaran, S., (2006). Simultaneous protection and activation of amino acids using propargyl pentafluorophenyl carbonate. Org. Lett., 8(9), 1933–1936. 153. Penke, B., Baláspiri, L., Pallai, P., & Kovács, K., (1974). Application of pentafluorophenyl esters of boc-amino acids in solid-phase peptide synthesis. Acta Physica et Chemica, 20(4), 471–476. 154. Carey, R. I., Bordas, L. W., Slaughter, R. A., Meadows, B. C., Wadsworth, J. L., Huang, H., Smith, J. J., & Furusjö, E., (1997). Preparation and properties of Nα-Bpoc-amino acid pentafluorophenyl esters. J. Peptide Res., 49, 570–581. 155. Bodanszky, A., Bodanszky, M., Chandramouli, N., Kwei, J. Z., Martinez, J., & Tolle, J. C., (1980). Active esters of 9-fluorenylmethyloxycarbonyl amino acids and their application in the stepwise lengthening of a peptide chain. J. Org. Chem., 45(1), 72–76. 156. De Beer, R. J. A. C., Zarzycka, B., Amatdjais-Groenen, H. I. V., Jans, S. C. B., Nuijens, T., Quaedflieg, P. J. L. M., et al., (2011). Papain-catalyzed peptide bond formation: Enzymespecific activation with guanidinophenyl esters. ChemBioChem, 12, 2201–2207.

The Carboxyl Protecting Groups 159

157. Sikizaki, H., Itoh, K., Toyota, E., & Tanizawa, K., (1998). Enzymatic peptide synthesis with p-guanidinophenyl and p-(guanidinomethyl)phenyl esters as acyl donors. Chem. Pharm. Bull., 46(5), 846–849. 158. Thust, S., Koksch, B., & Burger, K., (2000). Protease-catalyzed synthesis of α-fluoroalkyl substituted peptides. In: Martinez, J., & Jean-Alain, F., (eds.), Peptides 2000, Proceedings of the European Peptide Symposium (pp. 347–348). Montpellier, France-2001. 159. Takimoto, S., Kodera, Y., & Ohta, H., (1983). A Novel Carboxyl Protecting Group, the Monocarboxylic Esters Derived from t-Butylhydroquinone (Vol. 13, No. 2, pp. 103, 104). Fukuoka Univ. Sci. Reports. 160. Kim, S., Lee, J. I., & Kim, Y. C., (1985). A simple and mild esterification method for carboxylic acids using mixed carboxylic-carbonic anhydrides. J. Org. Chem., 50(5), 560–565. 161. Broadbent, W., Morley, J. S., & Stone, B. E., (1967). Polypeptides. V. The use of t-butyl 2,4,5-trichlorophenyl carbonate in the synthesis of N-t-butoxycarbonyl amino-acids and their 2,4,5-trichlorophenyl esters. J. Chem. Soc. Perkin Trans. I, 24, 2632–2636. 162. Wolman, Y., Ladkany, D., & Frankel, M., (1967). Synthesis of activated esters of n-protected amino acids. J. Chem. Soc. Perkin Trans. I, 8, 689–901. 163. Vigh, G., & Estrada, R. T., (2012). Fluorescence Labeling Reagents and Uses Thereof. PCT Int. Appl. WO 2012027717 A2. 164. Gayo, L. M., & Suto, M. J., (1996). Use of pentafluorophenyl esters for one-pot protection/activation of amino and thiol carboxylic acids. Tetrahedron Lett., 37(28), 4915–4918. 165. Mayer, S. C., Ramanjulu, J., Vera, M. D., Pfizenmayer, A. J., & Joullie, M. M., (1994). Synthesis of new didemnin B analogs for investigations of structure/biological activity relationships. J. Org. Chem., 59(18), 5192–5205. 166. Adamczyk, M., & Johnson, D., (1993). Synthesis of pentafluorophenyl 4-(N-Maleimidomethyl)cyclohexane-1-carboxylate (FMCC). Organic Preparations and Procedures International, 25(5), 592–594. 167. Veveris, A., Luse, I., & Markuns, M., (1991). Aniline as a reagent in determination by potentiometric acid-base titration. Zavodskaya Laboratoriya, 57(11), 7–11. 168. Green, M., & Berman, J., (1990). Preparation of pentafluorophenyl esters of Fmoc protected amino acids with pentafluorophenyl trifluoroacetate. Tetrahedron Lett., 31(41), 5851, 5852. 169. Benoiton, N. L., Lee, Y. C., & Chen, F. M. F., (1993). Preparation of activated esters of N-alkoxycarbonylamino and other acids by a modification of the mixed anhydride procedure. Int. J. Peptide Protein Res., 42(3), 278–283. 170. Sekizaki, H., Itoh, K., Toyota, E., & Tanizawa, K., (1999). Trypsin-catalyzed peptide synthesis with m-guanidinophenyl and m-(guanidinomethyl)phenyl esters as acyl donor component. Amino Acids, 17, 285–291. 171. Thust, S., & Koksch, B., (2003). Protease-catalyzed peptide synthesis for the site-specific incorporation of α-fluoroalkyl amino acids into peptides. J. Org. Chem., 68, 2290–2296.

160

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

172. Wipf, P., Tsuchimoto, T., & Takahashi, H., (1999). Synthetic applications of orthoesters. Pure & Appl. Chem., 71(3), 415–421. 173. Petroski, R. J., (2008). Improved preparation of halopropyl bridged carboxylic orthoesters. Org. Commun., 1(3), 48–53. 174. Skinner, K., & Lawrence, B., (2011). Soft Lewis acids present an improvement to pinner synthesis: A new route to esters. In: Abstracts of Papers, 241st ACS National Meeting & Exposition (Vol. 2011, p. ORGN-135). Anaheim, CA, United States. 175. Voss, G., & Gerlach, H., (1983). Ortho esters of carboxylic acids with 2,4,10-trioxaadamantane structure as carboxyl protecting group; applications in the synthesis of substituted carboxylic acids by Grignard reagents. Helvetica Chimica Acta, 66(7), 2294–2307. 176. Herdeis, C., & Kelm, B., (2003). A stereoselective synthesis of 3-substituted (S)-pyroglutamic and glutamic acids via OBO ester derivatives. Tetrahedron, 59, 217–229. 177. Corey, E. J., & Raju, N., (1983). A new general synthetic route to bridged carboxylic orthoesters. Tetrahedron Lett., 24(50), 5571–5574. 178. Blaskovich, M. A., & Lajoie, G. A., (1993). Synthesis of a chiral serine aldehyde equivalent and its conversion to chiral α-amino acid derivatives. J. Am. Chem. Soc., 115, 5021–5030. 179. Lan, A. J. Y., Heuckeroth, R. O., & Mariano, P. S., (1987). Electron-transfer-induced photocyclization reactions of arene-iminium salt systems. Effects of cation diradical deprotonation and desilylation on the nature and efficiencies of reaction pathways followed. J. Am. Chem. Soc., 109(9), 2738–2745. 180. Borch, R. F., Evans, A. J., & Wade, J. J., (1977). Synthesis of 8-epi-dendrobine. J. Am. Chem. Soc., 99(5), 1612–1619. 181. Wiley, A., Hershkowitz, R. L., Rein, B. M., & Chung, B. C., (1964). Studies in organophosphorus chemistry. I. Conversion of alcohols and phenols to halides by tertiary phosphine dihalides. J. Am. Chem. Soc., 86(5), 964, 965. 182. Blaskovich, M. A., Evindar, G., Rose, N. G. W., Wilkinson, S., Luo, Y., & Lajoie, G. A., (1998). Stereoselective synthesis of threo and erythro β-hydroxy and β-disubstituted-βhydroxy α-amino acids. J. Org. Chem., 63(11), 3631–3646. 183. Wipf, P., Xu, W. J., Kim, H. Y., & Takahashi, H., (1997). Zirconocene-catalyzed epoxy ester-ortho ester rearrangement: A new method for the protection of polyfunctionalized carboxylic acids and the asymmetric synthesis of orthoesters. Tetrahedron, 53(48), 16575–16596. 184. Corey, E. J., & Shimoji, K., (1983). Total synthesis of the major human urinary metabolite of prostaglandin d2, a key diagnostic indicator. J. Am. Chem. Soc., 105(6), 1662–1664. 185. Corey, E. J., D’Alarcao, M., & Kyler, K. S., (1985). Synthesis of new lipoxygenase inhibitors 13-thia- and 10-thiaarachidonic acids. Tetrahedron Lett., 26(33), 3919–3922. 186. Bannai, K., Tanaka, T., Okamura, N., Hazato, A., Sugiura, S., Manabe, K., Tomimori, K., & Kurozumi, S., (1986). Prostaglandin chemistry. XXXIII. Improved synthesis of isocarbacyclin using regioselective alkylation of allylic alcohols. Tetrahedron Lett., 27(52), 6353–6356.

The Carboxyl Protecting Groups 161

187. Kwok, P. Y., Muellner, F. W., Chen, C. K., & Fried, J., (1987). Total synthesis of 7,7-, 10,10-, and 13,13-difluoroarachidonic acids. J. Am. Chem. Soc., 109(12), 3684–3692. 188. Nicolaou, K. C., Theodorakis, E. A., Rutjes, F. P. J. T., Sato, M., Tiebes, J., Xiao, X. Y., Hwang, C. K., et al., (1995). Total synthesis of brevetoxin B. 2. Second generation strategies and construction of the dioxepane region [DEFG]. J. Am. Chem. Soc., 117(41), 10239–10251. 189. Rose, N. G. W., Blaskovich, M. A., Wong, A., & Lajoie, G. A., (2001). Synthesis of enantiomerically enriched β,γ-unsaturated-α-amino acids. Tetrahedron, 57(8), 1497–1507. 190. Zhuravlev, V. G., Mazurov, A. A., & Andronati, S. A., (1992). p-azobenzenecarboxamidomethyl esters–new colored hydrophobic carboxyl protecting groups in peptide synthesis. Coll. Czech. Chem. Commun., 57(7), 1495–1504. 191. Koziolkiewicz, W., Wasiak, T., & Janecka, A., (1985). Synthesis of four analogs of 6-11 substance P fragment of increased water solubility. Polish Journal of Chemistry, 59, 819–826. 192. Moutevelis-Minakakis, P., & Photaki, I., (1985). Some applications of the curtius rearrangement. Journal of the Chemical Society, Perkin Transactions 1, 2277–2281. 193. Teno, N., Tsuboi, S., Shimamura, T., Okada, Y., Yanagida, Y., Yoshinaga, M., Ohgi, K., & Irie, M., (1987). Amino acids and peptides. XIV. Synthesis and biological activity of three S-peptide analogs of bovine pancreatic ribonuclease A (RNase A). Chemical & Pharmaceutical Bulletin, 35, 468–478. 194. Barton, D. H. R., Girijavallabhan, M., & Sammes, P. G., (1973). Transformations of penicillin. Part II. N,N’-diisopropylhydrazine, a new reagent for protection of carboxylic acids. J. Chem. Soc., Perkin Trans. I, 929–932. 195. Schnyder, J., & Rottenberg, M., (1975). Hydrazide as a carboxyl protecting group, deprotection by acidolysis. Helvetica Chimica Acta, 58(60, 61), 521–523. 196. Uchimaru, T., Narasaka, K., & Mukaiyama, T., (1981). A novel carboxyl protecting group: The carboxamide derived from 5,6-dihydrophenanthridine. Chem. Lett., 1551–1554. 197. Svenson, R., & Gronowitz, S., (1982). On the hydrozirconation of some long-chain unsaturated fatty acid oxazolines. Chemica Scripta, 19, 149–153. 198. Itoh, M., (1969). Peptides I: Selective protection of α- or side-chain carboxyl groups of aspartic and glutamic acid. A facile synthesis of β-aspartyl and γ-glutamyl peptides. Chem. Pharm. Bull., 17(8), 1679–1686. 199. Meyers, A. I., Politzer, I. R., Bandlish, B. K., & Malone, G. R., (1969). Syntheses via dihydro-1,3-oxazines. VI. A carboxyl protecting group stable to the Grignard reagent. A new synthesis of carboxylic acids. J. Am. Chem. Soc., 91(21), 5886, 5887. 200. Nefkens, G. H. L., & Zwanenburg, B., (1983). Boroxazolidones as simultaneous protection of the amino and carboxyl group in α-amino acids. Tetrahedron, 39(18), 2995–2998. 201. Ben-Ishai, D., (1957). Reaction of acylamino acids with paraformaldehyde. J. Am. Chem. Soc., 79(21), 5736–5738.

162

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

202. Itoh, M., (1969). Peptides. I. Selective protection of α- or side-chain carboxyl groups of aspartic and glutamic acid. A facile synthesis of β-aspartyl and γ-glutamyl peptides. Chem. Pharm. Bull., 17(8), 1679–1686. 203. Chen, S. T., & Wang, K. T., (1991). Facile synthesis of biologically active peptides using phase transfer reagents as C-terminal protecting group. J. Chin. Chem. Soc., 38, 93–96. 204. Chen, S. T., Chang, C. H., & Wang, K. T., (1991). Facile synthesis of N-protected peptide fragments using polymer-bound 1-hydroxybenzotriazole as an active ester. J. Chem. Res. Synopses, (8), 206, 207. 205. Chen, S. T., & Wang, K. T., (1990). Phase-transfer reagents as C-terminal protecting groups; facile incorporation of free amino acids or peptides into peptide sequences. J. Chem. Soc., Chem. Commun., 1045–1047. 206. Adang, A. E. P., Duindam, A. J. G., Brussee, J., Mulder, G. J., & Van, D. G. A., (1988). Synthesis and nucleophilic reactivity of a series of glutathione analogs, modified at the γ-glutamyl moiety. Biochem. J., 255(2), 715–720. 207. Meldal, M., & Sheppard, R. C., (1987). Esters of Fmoc amino acids with 3,4-dihydro3-hydroxy-4-oxo-1,2,3-benzotriazine. A new class of self-indicating, activated intermediates for solid-phase synthesis. Pept., Proc. Eur. Pept. Symp., 19, 131–134. 208. Koenig, W., Knolle, J., & Breipohl, G., (1987). Preparation of 4-Oxo-3,4-dihydro-1,2,3benzotriazin-3-yl esters of amino acids. Ger. Offen. DE 3618218 A1. 209. Cameron, L. R., Holder, J. L., Meldal, M., & Sheppard, R. C., (1988). Peptide synthesis. Part 13. Feedback control in solid-phase synthesis. Use of fluorenylmethoxycarbonyl amino acid 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl esters in a fully automated system. Journal of the Chemical Society, Perkin Transactions 1, (10), 2895–2901. 210. Atherton, E., Holder, J. L., Meldal, M., Sheppard, R. C., & Valerio, R. M., (1988). Peptide synthesis. Part 12. 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl esters of fluorenylmethoxycarbonyl amino acids as self-indicting reagents for solid-phase peptide synthesis. Journal of the Chemical Society, Perkin Transactions 1, (10), 2887–2894. 211. Jakobsen, M. H., Buchardt, O., Engdahl, T., & Holm, A., (1991). A new facile one-pot preparation of pentafluorophenyl and 3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl esters of Fmoc amino acids. Tetrahedron Lett., 32(43), 6199–6202. 212. Cordopatis, P., Manessi-Zoupa, E., & Theodoropoulos, D., (1990). New trityl-activated esters for peptide synthesis. Coll. Czech. Chem. Commun., 55(10), 2575–2579. 213. Bolin, D. R., (1984). Protected amino acid derivatives. Eur. Pat. Appl. EP 129075 A2. 214. Barlos, K., Papaioannou, D., & Theodoropoulos, D., (1982). Efficient “one-pot” synthesis of N-tritylamino acids. J. Org. Chem., 47(7), 1324–1326. 215. Koenig, W., & Geiger, R., (1970). Peptides: Activation of the carboxyl group with dicyclohexylcarbodiimide and 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine. Chemische Berichte, 103(7), 2034–2040. 216. Michael, G., (1980). Gas chromatography of trifluoroacetylamino acid trimethylsilylester. III. A simple method for the preparation of N-trifluoroacetylamino acid trimethylsilylester. Journal of Chromatography, 196(1), 160–162.

The Carboxyl Protecting Groups 163

217. Schwarz, M., & Michael, G., (1976). Trifluoroacetylamino acid trimethylsilyl esters for gas chromatography. Journal of Chromatography, 118(1), 101–103. 218. Birkofer, L., Ritter, A., & Neuhausen, P., (1962). Silylated di- and tripeptides and their use in peptide syntheses. Justus Liebigs Annalen der Chemie, 659, 190–199. 219. Tantry, S. J., & Babu, V. V. S., (2002). 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides. Letters in Peptide Science, 9(1), 35–41. 220. Bhide, R. S., Levison, B. S., Sharma, R. B., Ghosh, S., & Salomon, R. G., (1986). Di-tert-butylmethylsilyl (DTBMS) trifluoromethanesulfonate. Preparation and synthetic applications of DTBMS esters and enol ethers. Tetrahedron Lett., 27(6), 671–674. 221. Mérette, S. A. M., Green, D., Patel, G., Scully, M. F., Kakkar, V. V., & Deadman, J. J., (1999). A general method for N to C terminal solid-phase peptide synthesis applied to bioactive peptidic boronates. In: Epton, R., (ed.), Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries, 1998: Peptides, Proteins and Nucleic Acids: Small Molecule Organic Chemical Diversity: Collected Papers, Fifth Intern. (pp. 349, 350). Mayflower Scientific: Kingswinford. 222. Gruszecki, W., Gruszecka, M., & Bradaczek, H., (1991). Stable tri-t-butoxysilyl esters of aspartic and glutamic acid as a new protection of their carboxyl groups. In: Giralt, E., & Andreu, D., (eds.), Pept. 1990: Proc. Eur. Pept. Symp., 21st (pp. 27, 28).

CHAPTER 4

Amino Protecting Groups

4.1 INTRODUCTION ON AMINO PROTECTING GROUPS When an amino acid is applied as the starting material in organic synthesis, the amino group of amino acid should be protected if a desired reaction at the carboxyl group is necessary, as in the case of peptide synthesis. Generally, the optimal requirements for protecting the amino group include the easy transformation of the amino group into a latent moiety during the reaction at the carboxyl group and convenient or mild deblocking condition to recover the amino group. Also, the presence of protecting group at the amino group should not interfere with the functionality of the carboxyl group or the activated carboxyl group. Often, the carboxyl group is protected or activated in a form of ester functionality, whereas the amino group is protected in a form of urethane. In peptide chemistry, two popular strategies have been developed to protect the amino and carboxyl groups: tert-butoxycarbonyl (t-Boc)/benzyl (Bn), and 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t-Bu) [1]. In the t-Boc/Bn strategy, the amino group is protected with t-Boc whereas the carboxyl is converted into the benzyl ester functionality. The advantage of this strategy is that both protecting groups can be removed by acidolysis. In the Fmoc/t-Bu strategy, the amino group is protected with Fmoc and the carboxyl group is transformed into t-butyl ester group. In this protocol, the Fmoc is usually removed by base (e.g., 20% piperidine) whereas the t-Bu can be easily deblocked by acid, thus these two mutually orthogonal protecting groups can be removed separately. Especially, the Fmoc/t-Bu protocol avoids the use of HF in the final cleavage step and hence is especially suitable for the construction of peptides containing acid-labile moieties. However, due to the hydrophobicity and bulkiness of the Fmoc, the low solubility in common solvents used in solid-phase peptide synthesis (SPPS) [1], incomplete cleavage of Fmoc [2], and low coupling yields are often associated with amino acids and peptides containing the Fmoc group.

166

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Another drawback of Fmoc group is its insufficient stability in weakly basic solvents or even neutral DMF [2]. All these pitfalls might limit the usage of Fmoc in peptide synthesis, especially the automated peptide synthesis. For the special case involving lysine, two different groups are required to protect the α-amino group and terminal (ε-) amino group. When the t-Boc is used to protect the α-amino group, the Nε-protecting group is required to be stable under the condition of removing the t-Boc group, or vice versa. Otherwise, partial removal of the Nε-protecting group during the deprotection of the Nα-protecting group would result in the formation of branched peptides during the coupling process [3]. As a result of special needs for the amino protecting group under a particular reaction condition, many types of amino protecting groups have been developed so far. They can be classified into a total of more than 12 groups, according to the conditions to cleave these protecting groups. These amino protecting groups are acid-labile, alcohol cleavable, alkali-labile, enzyme cleavable, hydrazine cleavable, hydrogenolysis cleavable, organometallic cleavable, oxidation cleavable, photolytic, reductive acidolytic, TBAF cleavable, and thiolytic protecting groups. Also, several protecting groups have been specifically developed for protecting the amino group within amino sugars that might have not yet been applied to the protection of the amino groups in amino acids and peptides are also included in this chapter. These N-protecting groups will be described individually. 4.2 ACID-LABILE AMINO PROTECTING GROUPS The most popular acid-labile amino protecting group is tert-butyloxycarbonyl group (t-Boc), for which the corresponding amino acid or peptide derivatives are converted into carbon dioxide, reactive isobutene and unprotected amino groups or peptides upon the treatment with an acid. On the basis of the successful application of t-Boc in peptide chemistry, several t-Boc-like amino protecting groups have been developed that can also be removed by the treatment with acid. These protecting groups include 1-(1-adamantyl)1-methylethoxycarbonyl (Adpoc), adamantyloxycarbonyl (Adoc), tert-amyloxycarbonyl (t-Amoc), 1-(3,5-di-tert-butylphenyl)-1-methylethoxycarbonyl (t-Bumeoc), 1,1-dimethyl-2-methacryl-methanamidoethoxycarbonyl-, 1,1-dimethyl-3-methacrylmethanamidobutoxy-carbonyl-, and 2-(p-biphenylyl)-2-propyloxycarbonyl. Besides these tertiary alcohols, some secondary alcohols and even primary alcohols have also been developed

Amino Protecting Groups 167

for the protection of the amino groups that can be cleaved under acidic conditions. The acid-labile amino protecting groups arising from secondary alcohols include 2-adamantyloxycarbonyl (2-Adoc), and isobornyloxycarbonyl (Iboc) groups, whereas the example of protecting groups originating from the primary alcohol are benzyloxycarbonyl (Cbz, Bz or Z) and furfuryloxycarbonyl (Foc). Also, triphenylmethyl (Trityl) and its derivatives including 4,4’-dimethoxytrityl, 4,4’,4”-trimethoxytrityl, monomethoxytrityl (MMT), due to the formation of the corresponding stable carbocations, can also be cleaved from the amino group. However, these groups have not been popularly used in amino acid and peptide manipulations yet, although they have been used for the protection of amino group already. Moreover, several sulfur-containing groups have also been applied as the acid-labile amino protecting groups, such as 4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), alkyldithiocarbonyl (e.g., cyclohexyldithiocarbonyl, c-Hexyl-S2CO), p-phenylazophenylsulfonylamino-carbonyl (Azo-Tac), p-xylyl-α-sulfonyl (also known as p-tolylmethylsulphonyl, Pms). Finally, it has been reported that dimethoxy-, diethoxy-, and di-n-butoxyphosphinyl groups are also good amino protecting groups that can be cleaved under mild conditions. The structures of these acid-labile amino protecting groups are displayed in Figure 4.1. The protection and deprotection conditions for individual protecting groups of this type will be described in detail below, with modified experimental procedures if available. Benzyloxycarbonyl group will be discussed in Section 4.1 (protection groups removable by hydrogenolysis). 4.2.1 tert-BUTOXYLCARBONYL (t-BOC) GROUP It is known that the tert-butyl ester withstands the attack of strong alkali and the reduction by sodium, and it also resists the catalytic hydrogenation [4]. Therefore, the t-Boc group, when applied as the protection group for the amino group, is the ideal partner of the benzyl group in peptide chemistry, which can be easily removed via catalytic hydrogenation. The t-Boc group can be mounted onto the amino group by means of tert-butyl chloroformate [5, 6], tert-butyl fluoroformate [7], tert-butyl azidoformate [8–10], and a variety of mixed tert-butyl carbonates [11, 12]. Among these reagents developed, the tert-butyl azidoformate is the most prominent one [12], which usually affords Boc-amino acids in moderate yields in the presence of MgO, Na2CO3 or NaOH [9]. The yield of Boc-amino acid from tert-butyl azidoformate can be improved by strict control of reaction pH with NaOH as monitored by

168

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

autotitrator [13], or simply in dioxane/water (1:1) in the presence of Et3N [8]. In comparison, tert-butyl chloroformate or tert-butoxycarbonyl chloride can be generated in situ from t-butyl alcohol and phosgene in the presence of pyridine at –74°C, which can introduce the t-Boc group to the hindered amino acid in high yield [5]. To deprotect the t-Boc group, trifluoroacetic acid (TFA) is often used as a convenient reagent [4], whether TFA is used alone or diluted with CH2Cl2 with the concentration of TFA varying from 20% to 50% [12]. Other acidic systems such as 4 N HCl-dioxane, 1 N HCl/ acetic acid [12], 10% H2SO4/dioxane [14], CF3SO3H (TFMSA)/TFA [15] have also been applied for the removal of the t-Boc group.

FIGURE 4.1 The structures of acid-labile amino protecting groups.

Amino Protecting Groups 169

4.2.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF N-BOC GROUP 4.2.1.1.1 Preparation of Nα-Boc-γ-(p-Bromobenzyl)Glutamic Acid [10]

To a stirred suspension of 18.5 g γ-p-bromobenzyl glutamate (i.e., (S)-2amino-5-((4-bromobenzyl)oxy)-5-oxopentanoic acid) in 250 ml of DMSO, 16.0 mL of triethylamine (117 mmol) and 13.5 mL of Boc-azide (i.e., tertbutyl carbonazidate, 87.8 mmol) were added. The mixture was stirred at 24°C for 23 hours. The reaction mixture was then diluted with 840 mL of water and washed with ether (2 × 150 mL). The aqueous phase was then cooled and acidified with 3 N HCl to pH 2. The product was extracted with EtOAc (2 × 200 mL, then 100 mL). The combined ethyl acetate layer was washed with water (3 × 50 mL) and dried over anhydrous MgSO4. Removal of the drying agent and solvent gave an oil which crystallized from ether-petroleum ether (b.p. 30–60°C) in a cold room (4°C) overnight. The product of (S)-5-((4bromobenzyl)oxy)-2-((tert-butoxycarbonyl)-amino)-5-oxopentanoic acid was filtered and washed with ether-petroleum ether (1: 4), and weighed 19.3 g, in a yield of 79%, m.p. 91–92°C. 4.2.1.1.2 Preparation of N-Boc Thiozolidine from tertButoxycarbonyl Chloroformate [5]

A 2,500 mL round-bottomed flask was fitted with a glass stirrer, low temperature thermometer, gas outlet (connected to a U-tube containing paraffin oil for checking the phosgene absorption and CaCl2 tube), a 100 mL addition funnel, and a gas inlet tube ending about 2 cm above the bottom of the flask, which was connected with a three-way stopcock to allow the

170

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

alternative introduction of dry N2 or phosgene. To this flask, were added 1,800 mL of purified CH2Cl2, 144 mL of anhydrous t-BuOH (1.54 mol), and 72 mL of anhydrous pyridine (0.895 mol). The mixture was cooled to –74°C with dry ice/acetone under N2, and phosgene (72 g, 0.729 mol) was introduced within 1 hour under vigorous stirring with a flow rate of ca. 0.3 L/min controlled by a flow-meter while maintaining a temperature between –71 and –74°C with no gas escaping during the introduction of phosgene, whereupon the color changed to light yellow and pyridine hydrochloride was precipitated. On disconnecting the phosgene cylinder for weighing, a slow stream of dry N2 was bubbled through the reaction mixture. The reaction was continually stirred for 15 hours at –74°C under a slow stream of dry N2. After the addition of 72 mL of pyridine (0.895 mL) to the above reaction mixture at –74°C, within half an hour, 57.6 g of powdered cysteine acetonide (0.36 mol) was added at once under a stream of N2 and the addition funnel was rinsed with 70 mL of pyridine. The reaction was kept at –74°C and 0°C each for 2.5 hours, then at room temperature for 1 hour. The reaction mixture was poured into a 10 L separatory funnel containing 2,700 mL of cold water and 180 g NaOH (4.5 mol), then, 3,400 mL of a 1:1 mixture of benzene and Et2O was added and another 200 mL of such solvent mixture was used to rinse the flask and transferred into the separatory funnel as well. The phases were separated and the organic phase was extracted with ice-cold aqueous 2 N NaOH (3 × 480 mL). The combined alkaline extracts were shaken with portions of CH2Cl2 (5 × 1,400 mL). From the Et2O-benzene and CH2Cl2 extracts, a neutral material (11.1 g) was obtained after drying and evaporation, which was (5aR,10aS)-3,3,8,8-tetramethyltetrahydro-3H,5H,8H,10Hdithiazolo[3,4-a:3’,4’-d]pyrazine-5,10-dione, m.p. 233–234°C. The alkaline solution was mixed with cold CH2Cl2 (1,800 mL), cooled with ice, and solid citric acid was added to the stirring mixture until pH 4 was reached. After the separation of the phases, the aqueous layer was again extracted with CH2Cl2 (3 × 1,800 mL). The combined organic extracts were dried (Na2SO4) and evaporated under reduced pressure. The resulting light oil was dried at 0.5 mmHg pressure for 1 hour to give 75.3 g (81%) of crude (R)-3-(tert-butoxycarbonyl)-2,2-dimethylthiazolidine-4-carboxylic acid. The crude product was dissolved in 1,000 mL warm pentane and concentrated to 300 mL, whereupon on standing for several hours at room temperature, the first crop of a pure product in an amount of 36.8 g was obtained, with an m.p. of 108–113°C. More product can be obtained from the crystallization of the mother liquid.

Amino Protecting Groups 171

4.2.1.1.3 Preparation of N-(tert-Butoxycarbonyl)-L-Alanine from Mixed Carbonate [11]

To a 60 mL of a 1:1 mixture of THF and water containing 8.4 mL of Et3N (60 mmol) was added 1.78 g of L-alanine (20 mmol), followed by 5.7 g of 1,2,2,2-tetrachloroethyl tert-butyl carbonate (20 mmol) and the solution was stirred at room temperature for 6 hours. Then 50 mL of water was added, and the resulting solution was washed with 50 mL of tert-butyl methyl ether. The aqueous phase was acidified to pH 2–3 with dilute HCl and extracted with ethyl acetate (3 × 40 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The residue was then crystallized from ethyl acetate-petroleum ether to give 3.4 g of (tertbutoxycarbonyl)-L-alanine as white crystals, in a yield of 90%, m.p. 81°C. 4.2.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF THE BOC GROUP 4.2.1.2.1 General Procedure for the Deprotection of the Boc Group [16] The respective peptide was dissolved in CH2Cl2 or a mixed solvent of CH2Cl2: CH3OH (9:1) (depending on the solubility) and cooled to 0°C. TFA was added and the solution was allowed to warm to room temperature. After being stirred at room temperature until the deprotection was completed as monitored by TLC, the solution was concentrated in vacuo. When the uncharged, neutralized peptide was the desired product, the solution was extracted with water, saturated aqueous NaHCO3 solution (in case of longer peptides, CH3OH was added to assure solubility of the peptide), water, and brine. The combined organic layers were dried over MgSO4, filtered, and evaporated in vacuo to yield the crude product in quantitative yield. In the case of the remaining protected peptide, the procedure was repeated. When the ammonium salt was the desired product, the reaction mixture was evaporated in vacuo and evaporated several times after adding CH2Cl2 to remove residual TFA to give the product in quantitative yield.

172

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.2.1.2.2 Deprotection of N-Boc-Leu-O-TAGc [17]

To a 20 mL 50% TFA in CH2Cl2 was added 97.1 mg of N-Boc-Leu-OTAGc (0.100 µmol), i.e., 3,5-bis(docosyloxy)benzyl (tert-butoxycarbonyl)L-leucinate. The reaction mixture was stirred at room temperature until the reaction was completed. Then CH3CN was added to precipitate H-Leu-OTAGc, affording the product of 3,5-bis(docosyloxy)benzyl L-leucinate, in a quantitative yield.

4.2.2 1-(1-ADAMANTYL)-1-METHYLETHOXYCARBONYL (ADPOC) GROUP Adpoc is the abbreviation of adamantylisopropoxycarbonyl [18] or Adpoc group that has the following properties: (a) resistance against alkali and hydrogenolysis, and (b) high lipophilicity leading to excellent solubility of higher peptides in organic solvents [19]. In addition, the Adpoc protected amino acids often form crystalline compounds and are stable when exposed to sunlight. Also, these compounds are stable for months at room temperature as well. The Adpoc residue can be easily incorporated into amino acids and peptides by means of the crystalline and stable Adpoc derivatives, such as Adpoc phenylcarbonate (Adpoc-OPh), Adpoc fluoride (Adpoc-F) and Adpoc-oxiimino-2-phenylacetonitrile (Adpoc-ON) [19]. Among these reagents, Adpoc-F, a very reactive and stable compound, allows the fast introduction of the Adpoc group into amino acids under mild conditions (–10°C, only a few minutes) in high yields. The reagent is stable at –30°C in solution for several months. Alternatively, amino acids can be acylated in an aqueous system (e.g., water/dioxane, equimolar addition of Et3N) with the crystalline [1-(1-adamantyl)-1-methylethoxycarbonyl-oxiimidno]2-phenylacetonitrile (Adpoc-ON) within one hour at room temperature in excellent yields. On the other hand, the Adpoc group can be removed in a mild acidic medium, preferably 3% TFA in CH2Cl2 [20], within 4–5 minutes at 0°C

Amino Protecting Groups 173

or 1–2 minutes at room temperature. In a mixture of acetic acid/83% formic acid/water (7:1:2), deprotection of the Adpoc group at 40°C is complete within 100 minutes [19]. Under these conditions, the Adpoc group is removed 1,000 times faster than the t-Boc group [21]. Consequently, it is possible to cleave the Adpoc group selectively in peptides containing other acid-labile protecting groups as well, such as the t-Boc, Bz, benzyl ether, and benzyl groups [19]. For the case of Nε-t-Boc-NαAdpoc-lysine, 0.1 N HCl/CF3CH2OH/CHCl3 (1:9:1) and 50% HCOOH/ CF3CH2OH/CHCl3 (1:9:1) show high selectivity among several acidolytic reagents [18]. Due to the easy removal of the Adpoc, incorporation of Adpoc protected amino acids in SPPS allows selective deprotection of this residue on the resin-bound peptide and avoids premature resin cleavage [22].

4.2.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PROTECTION OF AMINO ACIDS WITH THE ADPOC GROUP 4.2.2.1.1 Preparation of 2-((3R,5R,7R)-Adamantan-1-yl)Propan-2yl Carbonofluoridate [23]

During the course of 2–3 hours, dry, SO3-free carbonyl chloride fluoride (obtained from 60 g of 65% oleum and 25 mL of trichlorofluoromethane) was directly added to a solution of 19.4 g 2-((3R,5R,7R)-adamantan-1-yl) propan-2-ol (0.1 mol) in 150 mL of dry ether and 14 mL triethylamine at –40°C. During this step, triethylamine hydrochloride was precipitated. After the gas evolution had ceased, the mixture was allowed to stand overnight at –20°C; then the mixture was degassed at 200 mmHg and 10°C. After filtration, the residue was additionally washed with dry ether on the filter. The filtrate contained 22.9 g of 2-((3R,5R,7R)-adamantan-1-yl)propan-2-yl carbonofluoridate (Adpoc-F). After removal of the solvent under vacuum, this compound was obtained in crystalline form, which can be utilized directly or in an ether solution.

174

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.2.2.1.2 Preparation of Adpoc-Amino Acid (Glycine as an Example)

Glycine (0.75 g, 10 mmol) was dissolved under gentle heating in 4.5 mL of an approximately 40% solution of “Triton B” (benzyl trimethylammonium hydroxide) in methanol. The mixture was evaporated under vacuum, the residue was taken up in 5–10 mL of dimethylformamide (DMF), whereupon the mixture was again evaporated and the process was repeated. The mixture was then dissolved in 10 mL of DMF, cooled to –10°C, and combined at this temperature with 2.65 g of Adpoc-F (11 mmol) and 1.6 mL Et3N. The mixture was agitated for 3 hours at –5°C, for 2 hours at 0°C, allowed to reach room temperature, and the reaction solution was then poured into 20 mL of ice water. After extracting the mixture twice with, respectively, 20 mL of ether-petroleum ether (1:1), the aqueous phase was acidified with citric acid and repeatedly extracted with, in total, 80 mL of ether. The combined ether extracts were washed to neutral with water, the ether was distilled off under vacuum after a brief drying over Na2SO4. The residue was made to crystallize directly to afford 74% of (((2-((3R,5R,7R)-adamantan-1-yl)propan-2-yl) oxy)carbonyl)glycine, m.p. 135°C. Adpoc derivatives of other amino acids can be converted into the corresponding dicycylohexylammonium (DCHA) salt to form crystals. 4.2.2.1.3 Preparation of 2-((3R,5R,7R)-Adamantan-1-yl)Propan-2yl Phenyl Carbonate [23]

To a mixture of 19.4 g 2-((3R,5R,7R)-adamantan-1-yl)propan-2-ol (0.1 mol), 100 mL CH2Cl2 and 12.0 mL pyridine at –5°C was added a solution of 15.2 mL of phenyl chloroformate in 50 mL of CH2Cl2 within 30 minutes. The precipitate formed during the dropwise addition was re-dissolved after stirring the mixture overnight at 0°C. The reaction mixture was poured on ice, diluted with 150 mL of CH2Cl2, and the organic phase was separated. After being washed three times with water, the organic layer was dried over

Amino Protecting Groups 175

Na2SO4 and evaporated under vacuum at 30°C. By recrystallizing twice from benzene/n-hexane, 26.25 g of 2-((3R,5R,7R)-adamantan-1-yl)propan-2-yl phenyl carbonate (Adpoc-phenyl carbonate, Adpoc-OPh) was obtained, in a yield of 83.5%, m.p. 72°C. 4.2.2.1.4 Preparation of Adpoc-Amino Acid (Glycine as an Example) [23]

Glycine (0.75 g, 10 mmol) was dissolved under gentle heating in 4.6 mL of an approximately 40% solution of “Triton B” (benzyl trimethylammonium hydroxide) in methanol. After evaporation of the solvent, the residue was dried twice by azeotropic distillation with, respectively, 30 mL of DMF. The residue was taken up in 30 mL of DMF at 50°C, combined with 3.8 g of Adpoc-OPh (12 mmol), and the reaction mixture was agitated for 3 hours at 50°C. To work up the mixture, the latter was divided between water and diethyl ether-petroleum ether (1:1); the separated aqueous phase was acidified at 0°C, with 1 N citric acid solution (pH 2–3) and exhaustively extracted with diethyl ether. After the combined ether extracts were washed and dried in the usual manner, the mixture was evaporated to dryness under a vacuum. The remaining substance was made to crystallize after conversion into the DCHA salt, m.p. 158°C. 4.2.3 1-ADAMANTYLOXYCARBONYL (ADOC) GROUP 1-Adamantyloxycarbonyl, like the t-Boc group, can also form stable tertiary carbocation during acidolysis. The Nα-Adoc-amino acids of the non-polar side chain can be prepared by a straightforward procedure from hydrophobic amino acids and 1-adamantyl chloroformate in water-dioxane in up to 89% yields, whereas the reactions between hydrophilic amino acids and 1-adamantyl chloroformate give quite modest yields [24]. It is reported that the t-Adoc derivatives give strong ninhydrin-positive spots when the thinlayer plates are heated at 100°C for a short time [25], possibly caused by the decomposition of the Adoc group. This might provide false detection during the protection of amino acids with the Adoc group. When amino acid

176

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

benzyl esters are used to react with 1-adamantyl chloroformate, mono-, and bis-Adoc amino acid benzyl esters may form, which are difficult to obtain as crystals. However, if crystallization occurs, the resulting crystals have melting points less than 100°C; by contrast, the Adoc protected free acids readily crystallize with melting points up to 200°C [24]. 4.2.3.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PROTECTION OF AMINO ACIDS WITH THE ADOC GROUP 4.2.3.1.1 Preparation of 1-Adamantyl Chloroformate [24]

To an ice-cooled 20% solution of phosgene in toluene (150 mL, 30 g) was added dropwise a solution of 1-adamantanol (7.05 g, 46.3 mmol) and pyridine (7.30 g, 92.3 mmol) in 100 mL toluene under stirring. When the addition was complete, the reaction mixture was stirred for 1 hour at 0°C and then 4 hours at room temperature. The precipitate was filtered off and the filtrate was poured into ice-water. The toluene layer was collected, dried over Na2SO4. Upon evaporation of toluene in vacuo, 7.60 g of (3S,5S,7S)adamantan-1-yl carbonochloridate was obtained as a semisolid white residue, in a yield of 77%. 4.2.3.1.2 Preparation of 1-Adamantyloxycarbonyl Amino Acids (General Procedure) [25]

The amino acid (5 mmol) was suspended in ca. 20 mL of water in an ice bath. Under stirring was added 5 mL 1 N NaOH solution to dissolve the amino acid. Then, 0.80 g of sodium carbonate (7.5 mmol) was added to this solution. By the same time, a solution of adamantyl chloroformate (a little over 5 mmol) in benzene was dried on a flash evaporator at a bath temperature of about 30°C. Then dry petroleum ether was added to the

Amino Protecting Groups 177

residue which may be oily or semisolid and removed in vacuo. This process was repeated once more in order to remove traces of phosgene which may be left from the preparation of the chloroformate. The residue was dissolved in 5 mL of anhydrous dioxane and added in about four portions to the solution of the amino acid over a period of about 1 hour with continued stirring and cooling. Since some solid usually precipitated, 5 mL of ether was added after the first and last addition of chloroformate. The container of the chloroformate solution was washed twice with a little dioxane. After stirring in ice for about 2 more hours, the solution was extracted three times with ether, and the aqueous phase was cooled in ice, layered with ether or ethyl acetate (in the case of tryptophan, the derivative of which is not very soluble in ether). Under stirring and cooling, the aqueous mixture was acidified with 85% phosphoric acid or 10% sulfuric acid to a pH of about 2. About 2.5 ml of phosphoric acid was required. The precipitated Adoc amino acid was extracted into the organic layer (usually three times). The combined extracts including the organic layers extracted before the acidification were dried over sodium sulfate and the solvent was removed in vacuo. The residue was recrystallized from a suitable solvent (ether-petroleum ether, ethyl acetate, ethyl acetate-petroleum ether). This procedure is demonstrated by the preparation of Adoc-D-tryptophan below. 4.2.3.1.3 Preparation of Adamantyloxycarbonyl-D-Tryptophan [25]

D-Tryptophan (5.1 g, 25 mmol) was suspended in 80 mL water in an ice bath, and 25 mL 1 N NaOH was added followed by 4 g of sodium carbonate which resulted in a clear solution. Meanwhile, the solvent from a 150 mL 5% adamantyl chloroformate in benzene was removed in vacuo at a bath temperature of 30°C. Cyclohexane was added to the residue and removed in vacuo while this process was repeated twice. The residue was dissolved in 20 mL dry dioxane and added to the tryptophan solution in five portions over 90 minutes with continuous stirring in ice. After the second addition, 20 mL ether was added. The mixture was stirred in ice for another 3 hours and then extracted three times with ether. The aqueous phase was layered with

178

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

ethyl acetate and cooled in ice, and 12.5 mL of 85% phosphoric acid was added with stirring. The ethyl acetate layer was removed and the aqueous phase was extracted two more times with ethyl acetate (250 mL in total). The combined extracts were dried over sodium sulfate and the solvent was removed in vacuo. The solid residue was dissolved in 25 mL ethyl acetate and cooled in a refrigerator to yield 6.23 g of ((((3S,5S,7S)-adamantan-1-yl) oxy)-carbonyl)-D-tryptophan as crystals, m.p. 149–151°C (dec.). The mother liquor was concentrated in vacuo to dryness and the residue was dissolved in 15 mL of ethyl acetate. A second crop weighing 2.05 g and melting at 147–151°C was obtained. 4.2.3.1.4 General Procedure for the Preparation of Nα-Adoc-Amino Acid Benzyl Esters [24]

To the solution of L-tyrosine benzyl ester 4-methylbenzenesulfonate (1 equivalent) in dry pyridine (3 mL/equiv.) and CHCl3 (10 mL /equiv.) cooled at –5°C, was added dropwise over 1 hour with stirring a solution of Adoc-Cl (1.5 equiv.) in CHCl3 (10 mL/equiv.). The reaction was monitored with TLC (light petroleum ether/ Et2O, 2:1), which generally took 6 hours to complete. After attaining room temperature, the reaction mixture was washed several times with brine and dried over Na2SO4. Evaporation of the solvent gave oils which were purified by column chromatography (CH2Cl2/acetone, 97:3) on silica. For a typical example, as shown in the preparation of Adoc-Tyr-OBzl, 3.0 g of tyrosine benzyl ester (Tyr-OBzl, 11 mmol) and 3.60 g of Adoc-Cl (16.8 mmol) were used. After work-up and column chromatography, benzyl ((((3R,5R,7R)-adamantan-1-yl)oxy) carbonyl)-L-tyrosinate (Adoc-Tyr-OBzl) was obtained as an oil, which by treatment with ether could be converted into an amorphous solid (4.20 g, 84%). By the same time, 1.10 g of benzyl (S)-2-(((((3R,5R,7R)adamantan-1-yl)oxy)carbonyl)amino)-3-(4-(((((3R, 5R,7R)-adamantan1-yl)oxy)carbonyl)oxy)phenyl)propanoate (Adoc-Tyr(Adoc)-OBzl) was isolated, in a yield of 16%.

Amino Protecting Groups 179

4.2.3.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF THE ADOC GROUP 4.2.3.2.1 Partial Deprotection of Adoc2-Tyr(Adoc)-OBzl [24]

Compound Adoc2-Tyr(Adoc)-OBzl (15 mg, 18 mmol) was treated with an equivalent amount of TFA in 1 mL of CH2Cl2 and the reaction was monitored by TLC. After 48 hours, only traces of the starting material had been consumed. The addition of 0.5 equivalent of TFA, allowing a reaction time of 24 hours with TLC monitoring twice, followed by reaction for an additional 48 hours indicated only traces of the starting material remained. The solvent was evaporated off and the residue was distributed between 15 mL of EtOAc and 10 mL of 1 M NaHCO3. After chromatography on silica (CH2Cl2), 10 mg of pure benzyl (S)-2-(((((3R,5R,7R)-adamantan-1-yl)oxy)carbonyl)amino)3-(4-(((((3R,5R,7R)-adamantan-1-yl)oxy)-carbonyl)oxy)phenyl)propanoate, i.e., Adoc-Tyr(Adoc)-OBzl, was obtained, in a yield of 78%. 4.2.4 tert-AMYLOXYCARBONYL (t-AMOC) GROUP Tert-Amyloxycarbonyl (t-Amoc) is a higher homolog of the commonly used tert-butoxycarbonyl group (t-Boc), it can be used almost where t-Boc can be used. When it is mounted to an amino acid, it is found that the geometry of the urethane group adopts both cis and trans conformations in crystals, and the small differences in bond angles about the trigonal C in comparison to the amide bond of the peptide is caused by the altered interactions when a CαH group of a peptide unit is replaced by ester oxygen [26]. As t-Amoc chloride is fairly resistant to cold H2O [27], the introduction of such protecting group into amino acid and peptide can be easily carried out under the Schotten-Baumann condition, in the presence of water and an inert solvent [6]. This procedure is applicable to almost all amino acids or their derivatives. It is found that both yield and purity of protected amino acids and their derivatives are satisfactory except in the cases of asparagine, glutamine, and nitroarginine [6]. However, when Et3N

180

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

is applied as the base under the Schotten-Baumann condition, the urea-type byproduct of the respective amino acid ester also forms. It is found that the weaker the basicity of the amino acid ester used, the more byproduct was formed. Such a side reaction could be almost entirely suppressed by the use of Et2NCH2CO2Et in place of Et3N [28]. Besides the commonly used t-Amoc chloride [6, 28–31], tert-amyl azidoformate has also been used for introducing the t-Amoc group into free amino acids, amino acid esters, and peptides, although CHCl3 may inhibit the reaction of tert-amyl azidoformate with amino acid esters [29]. Alternatively, t-Amoc chloride has been converted into tert-amyl 8-quinolyl carbonate with the reaction of 8-hydroxyquinoline in tetrahydrofuran in the presence of Et3N, which is then applied to protect amino acid to afford > 86% of t-Amoc amino acid [32]. Also, the t-Amoc amino acid can also be prepared by the treatment of amino acid salts with di-tert-amyl pyrocarbonate in either PrOH or aqueous PrOH at 20–45°C [33, 34]. The higher homolog of t-Amoc, i.e., tert-pentyloxycarbonyl group, has also been developed as a protective group for amino acids [30]. The t-Amoc group can be cleaved under the same condition where t-Boc is cleaved, without the formation of cyclic diketopiperazines which are frequently obtained by other techniques [35]. 4.2.4.1 GENERAL PROCEDURE FOR THE INTRODUCTION OF N-TERTAMYLOXYCARBONYL GROUP 4.2.4.1.1 General Procedure for the Preparation of N-tertAmyloxycarbonyl Amino Acids [30]

To a solution of 0.1 mol amino acid ester HCl salt or tosylate in 200 mL of CHCl3 at –10 to –5°C were added alternately portions of solutions containing t-Amoc chloride and 0.1 mol of Et3N, where the addition of solutions containing t-Amoc chloride was continued until all the amino acid ester was consumed as monitored by TLC with ninhydrin detection. Then, the solution was washed with H2O, 0.5 N HCl, and 5% aqueous NaHCO3, dried, and concentrated to give a quantitative yield of oily N-t-Amoc amino acid

Amino Protecting Groups 181

ester. This oily amino acid ester was saponified in Me2CO or, if the benzyl ester, was hydrogenated in MeOH to form N-t-Amoc amino acid. 4.2.4.1.2 Preparation of N-tert-Amyloxycarbonyl Leucine [27]

The solution of 13.1 g L-leucine (0.1 mol) in a mixed solvent prepared from 150 mL of aqueous NaOH and 50 mL of THF was treated alternately at –3 to +3°C with 30 mL Et2O containing t-Amoc chloride and 50 ml 2 N NaOH to give 61% N-tert-amyloxycarbonyl L-leucine monohydrate, m.p. 66–68°C. 4.2.4.1.3 Preparation of N-tert-Amyloxycarbonyl Glycine [36]

To a 15 mL aqueous solution containing 0.75 g of glycine (0.01 mol) was added 0.80 g of MgO, followed by 15 mL dioxane solution containing 1.9 g of t-Amoc azide, and the mixture was stirred at 40°C overnight. After the pH was adjusted to 8 with 1 N HCl, the mixture was evaporated, and the residue was adjusted to pH 1–2 with 1 N HCl and extracted with EtOAc. Upon removal of solvent, 1.4 g of t-Amoc glycine was obtained, m.p. 82°C. 4.2.4.1.4 Preparation of N-tert-Amyloxycarbonyl Glycine [31]

To a solution of 46 g of 2-methylbutan-2-ol in 500 mL dry Et2O cooled below –40°C was added 100 g COCl2, then a solution of 41 g pyridine in 500 mL of Et2O was added dropwise at the same temperature. After a few

182

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

hours below –20°C, the mixture was filtered and the filtrate was concentrated in vacuo to 120 mL of about 60% pure t-Amoc chloride, also known as tert-pentyl carbono-chloridate. To a solution of 2.8 g EtO2CCH2NH2·HCl and 3 mL Me 3N in 40 mL CHCl3 at –10°C containing glycine was added 8 mL t-Amoc chloride mixed with 3 mL of Me3N in three portions. The reaction mixture was washed successively with H2O, 0.5 N HCl, and 5% Na2CO3, dried, and concentrated to a syrup which was saponified (or catalytically hydrolyzed) to give 82% t-Amoc glycine, m.p. 82.5–4.0°C. 4.2.5 [1-(3,5-DI-TERT-BUTYLPHENYL)-1-METHYLETHOXY] CARBONYL (T-BUMEOC) GROUP The amino protecting group of [1-(3,5-di-tert-butylphenyl)-1-methylethoxy]carbonyl, often known as t-Bumeoc, can be introduced with t-Bumeoc-F for temporary Nα-protection of amino acids [37, 38]. This group is stable against hydrogenolysis and alkaline treatment [37, 39], but can be smoothly cleaved under very mild acidic conditions [40], such as by 1% TFA in CH2Cl2 [38] or HCl in EtOAc at 40°C for 6 hours [39]. Under the condition of 1% TFA treatment in organic solvents, Nε-t-Boc, t-Bu ester and t-Bu ether groups will remain [41]. It is found that the cleavage of t-Bumeoc at Nα is about 4,000–8,000 times faster than the cleavage of Nα-t-Boc protecting group under the same acidolytic condition [42]. 4.2.5.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF T-BUMEOC GROUP 4.2.5.1.1 Preparation of 3,5-Di-tert-Butylbenzoic Acid [40]

To a three-necked flask equipped with a stirrer and refluxing condenser were added 103.0 g of 1,3-di-tert-butyl-5-methylbenzene (0.5 mol), 360.0 g of pyridine (4.5 mol), 180 g of water and 45 g of KOH. To this mixture at 95°C was added 201.0 g of potassium permanganate (1.26 mol) in portions over 3–4 hours. After the addition of KMnO4, the mixture was stirred for

Amino Protecting Groups 183

another 3–4 hours at 95°C. The manganese dioxide formed was drained and washed with 2 N KOH. The filtrate was concentrated in a rotary evaporator to about 300 mL, and mixed with EtOAc. The mixture was acidified with 4 N H2SO4, organic phase was separated and the aqueous phase was extracted again with EtOAc. After being concentrated, 3,5-di-tert-butylbenzoic acid crystallized in small, colorless needles, in an amount of 67.1 g (57% yield). If necessary, it could be recrystallized from ethanol, m.p. 170°C. 4.2.5.1.2 Preparation of 2-(3,5-di-tert-Butylphenyl)-2-propanol [40]

To 11.7 g (0.48 mol) of magnesium in 100 mL of absolute diethyl ether was added approximately 2 mL of methyl iodide to start the reaction. Then, the rest of 68.1 g of methyl iodide (0.48 mol) in 100 mL of absolute Et2O was added dropwise so that the reaction mixture boiled gently. After being heated or until almost all the magnesium has dissolved (ca. 1–2 hours), 30.0 g methyl 3,5-di-tert-butylbenzoate (0.12 mol) in 80 mL of ether was added dropwise so that the solution boiled steadily. The reaction mixture was stirred for about 15 hours at 35°C. It was then poured onto ice and acidified with solid citric acid to pH ≈ 4. The organic phase was separated and the aqueous phase was extracted with ether three times. The combined organic phases were alternately washed three times by saturated Na2CO3 solution and water and dried over Na2SO4. After distilling off the ether in vacuo, 27.8 g of 2-(3,5-di-tert-butylphenyl)-2-propanol was obtained as a yellow oil which crystallized rapidly. It was recrystallized from n-hexane to afford 25.6 g of pure product, in a yield of 85%, m.p. 57–58°C. 4.2.5.1.3 Preparation of [1-(3,5-di-tert-Butylphenyl)-1Methylethyl] Fluoroformate (t-Bumeoc-F) [40]

184

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a solution of 24.8 g of 2-(3,5-di-tert-butylphenyl)-2-propanol (0.1 mol) in 150 mL absolute Et2O and 14 mL of triethylamine was added dry, SO3-free carbonyl chloride fluoride [prepared from 120 g of 65% Oleum and 25 mL trichlorofluoromethane (Freon 11) (0.45 mol)] at –40°C slowly. Triethylammonium hydrochloride was precipitated. After gas evolution ceased, the mixture was allowed to warm to 20°C and stirred for about 1 hour. Then, the mixture was degassed at 200 torrs and 10°C. The mixture was filtered, the residual solid was washed with dry ether, and the filtrate was concentrated in vacuo to 100 mL at 10°C. This solution could be stored at –18°C for some time. When solvent was completed removed, 20.2 g of 2-(3,5-di-tert-butylphenyl)propan-2-yl carbono-fluoridate, also known as [1-(3,5-di-tert-butylphenyl)-1-methylethyl] fluoroformate was obtained, in a yield of 71%.

4.2.5.1.4 Preparation of [1-(3,5-di-tert-Butylphenyl)-1Methylethyl] Phenyl Carbonate (t-Bumeoc-OPh) [40]

To a mixture of 5.0 g of 2-(3,5-di-tert-butylphenyl)-2-propanol (20 mmol) in 35 mL of absolute CH2Cl2 and 2.36 g of pyridine (30 mmol) at –5°C was added 3.3 g of phenyl chloroformate (21 mmol) in 20 mL of CH2Cl2 dropwise within 1 hour. It was stirred for about 15 hours at 0°C, and a light-yellow precipitate was formed during the dropping of phenyl chloroformate. Thereafter, the reaction mixture was poured on ice, diluted with 50 mL of CH2Cl2, and the organic phase was separated. It was washed five times with water and dried over Na2SO4. The solvent was evaporated to afford a pale yellow, oily residue, which was recrystallized from ether/ petroleum ether. A second recrystallization from absolute MeOH gave 3.9 g of 2-(3,5-di-tert-butylphenyl)propan-2-yl phenyl carbonate, also known as [1-(3,5-di-tert-butylphenyl)-1-methylethyl] phenyl carbonate, in a yield of 53%, m.p. 79°C.

Amino Protecting Groups 185

4.2.5.1.5 General Procedure for the Preparation of t-BumeocAmino Acid [40]

Amino acid (10 mmol) was dissolved/suspended in 50 mL of dioxane/ water (1:1). Then 3.5 g of 2-(3,5-di-tert-butylphenyl)propan-2-yl carbonofluoridate (12 mmol) in 10 mL of absolute dioxane was added dropwise under ice-cooling (0°C) at constant pH (autotitrator, 4 N NaOH). After 5 mL of ether was added to the reaction solution, a precipitate was formed. The reaction mixture was allowed to warm to room temperature. Heating and stirring were continued for some time to complete the reaction as monitored by TLC. Then, the reaction mixture were extracted twice with ether, the combined ether extracts were washed with water three times. The combined aqueous phase was mixed with EtOAc at 0°C and acidified with 2 N citric acid to pH = 3.5. After phase separation, the aqueous phase was extracted twice with EtOAc. The combined organic phases were washed to neutral with water, dried over Na2SO4 and concentrated in vacuo. The oily residue was recrystallized from EtOAc/petroleum ether to afford pure (((2-(3,5-di-tertbutylphenyl)propan-2-yl)oxy)carbonyl)-L-amino acid. 4.2.6 2-ADAMANTYLOXYCARBONYL (2-ADOC) GROUP The general purpose of using 2-Adoc group is to increase the solubility of the protected components in organic solvent [43, 44] and to enhance the stability of the protected peptide fragments under the conditions in convergent solidphase peptide synthesis [44]. 2-Adoc is suitable for ε-amino protection of Lys [45] in convergent solid-phase peptide synthesis in combination with Nα-Fmoc protection and TFA-labile solid support [46]. Also, 2-Adoc has also been applied to the protection of tyrosine side chain [45, 47], and the imidazole function of histidine residue in peptide synthesis [48, 49]. It is stable to trifluoroacetic acid (TFA) [43, 47], tertiary amines [47–49] and 1-hydroxybenzotriazole (HOBt) [49], 5.0 N HCl/dioxane [47], and hydrogenation over a Pd catalyst [47]. On the other hand, this group can be easily removed by treatment of the protected amino acid derivative with 1 M trifluoromethanesulfonic acid (TFMSA)-thioanisole/TFA and HF [47]. Due

186

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

to its relatively high stability towards acidic condition, the racemization rate during the peptide coupling is reduced [48]. Besides its feasibility to protect the amino group, it can also be used to protect the hydroxyl group of tyrosine through the Schotten-Bauman reaction of 2-Adoc chloride with the copper complex of tyrosine [47]. 4.2.6.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF 2-ADOC GROUP 4.2.6.1.1 Preparation of N-(2-Adamantyloxycarbonyl)-2-[9(Methylsulfonyl)-β-Carbolin-3-yl]-Alanine [50]

To a stirred solution of 0.5 g 2-[9-(methylsulfonyl)-β-carbolin-3-yl]alanine methyl ester (1.4 mmol, also known as methyl (R)-2-amino-2-(9(methylsulfonyl)-9H-pyrido[3,4-b]indol-3-yl)propanoate) in 10 mL of THF were added 345 mg of 2-Adoc chloride (1.7 mmol) followed by a solution of 219 mg of diisopropylethylamine (1.7 mmol) in 5 mL of THF. The reaction mixture was stirred for 4 hours at room temperature and then evaporated in a vacuum. The residue was partitioned between 100 mL of EtOAc and 50 mL of water. The organic layer was washed successively each with 50 mL of 7.5% citric acid solution and 8% NaHCO3 solution, and dried over Na2SO4. Upon removal of the solvent in a vacuum, 0.73 g of N-(2-adamantyloxycarbonyl)2-[9-methylsulfonyl-β-carbolin-3-yl]-alanine methyl ester was obtained as a yellow oil which solidified upon drying, in a yield of 97.7%. To the flask containing this ester were added 10 mL of dioxane, 5 mL of water and 131 mg of lithium hydroxide (5.5 mmol). The mixture was stirred over 16 hours at room temperature. Then, dioxane was evaporated in a vacuum, the residue was diluted with 10 mL of water, and 7.5% citric acid solution was added to adjust pH to 6. The aqueous solution was then extracted with 50 mL of EtOAc, and the organic extract was washed with saturated salt solution and dried over Na2SO4. After removal of solvent in

Amino Protecting Groups 187

vacuum, 0.68 g of N-(2-adamantyloxycarbonyl)-2-[9-(methylsulfonyl)-βcarbolin-3-yl]-alanine was obtained as a yellow foam, in a yield of 95.5%. 4.2.6.1.2 Preparation of α-Methyl-N-[(Tricyclo[3.3.1.13,7]dec-2yloxy)Carbonyl]-(R)-Tryptophan Methyl Ester [50]

To a stirred solution of 912 mg of 2-adamantanol (5.99 mmol) in 15 mL of anhydrous CH2Cl2 were added 653 mg of bis(trichloromethyl) carbonate (2.20 mmol) and 474 mg of pyridine (5.99 mmol) in 10 mL of anhydrous CH2Cl2 at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 hours. The solvent was removed in vacuo at 30°C and 30 mL of EtOAc was added, and the solution was stirred for 10 minutes. The resulting precipitate was filtered off and the solvent was removed in vacuo at 30°C to give 1.29 g of 2-adamantyl-oxycarbonyl chloride as an oil which solidified upon standing, in quantitative yield. To a stirred solution of 965 mg of 2-Adoc chloride (4.5 mmol) in 10 mL of anhydrous THF was added a solution of 928 mg of α-methyl-(R)-tryptophan methyl ester (4.00 mmol) in 20 mL of anhydrous THF, followed by a solution of 808 mg of triethylamine (7.98 mmol) in 20 mL of anhydrous THF dropwise. After 15 minutes, the reaction mixture was filtered, the solvent was removed in vacuo, and the residue was column chromatographed using 2% MeOH in CH2Cl2 as eluant to yield 1.42 g of α-methyl-N-2-adamantyloxycarbonyl(R)-tryptophan methyl ester as a syrup, in a yield of 89%. 4.2.6.1.3 Preparation of H-Tyr(2-Adoc)-OH [47]

188

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Tyrosine-copper complex, prepared from 15.6 g of tyrosine (86 mmol) and CuSO4·5H 2O as usual, was suspended in a mixed solvent of dioxane and H2O (50 mL each), and 86.0 mL of 1 N NaOH was added. After the solution was cooled with ice, 21.9 g of 2-Adoc chloride (103.1 mmol) in 50 mL of dioxane was added. Upon the completion of the reaction, the reaction mixture was filtered and the solid was washed with a mixture of dioxane and H2O (1:1). The copper was removed with saturated EDTA solution as usual, and the remaining solid was washed with MeOH to yield 19.3 g of H-Tyr(2Adoc)-OH, i.e., (S)-3-(4-(((((1R,3S,5S,7S)-adamantan-2-yl)oxy)carbonyl) oxy)phenyl)-2-aminopropanoic acid, in a yield of 62.5%, m.p. 213–215°C. 4.2.6.1.4 Preparation of Z-His(2-Adoc)-OH [48]

To an ice-cooled solution of 8.7 g Z-His-OH (30 mmol) in 30 mL of 2 M NaOH and 20 mL of 1,4-dioxane were added portions of 8.4 g of 2-adamantyl chloroformate (39 mmol) in 10 mL of 1,4-dioxane. The reaction mixture was stirred with ice-cooling for 2 hours, after which it was acidified with 6 N HCl, and then extracted with EtOAc. The extract was washed with water, dried over Na2SO4, and evaporated. To an ice-cooled solution of the residual oil in 100 mL of diethyl ether was added 3.0 g of cyclohexylamine (30 mmol) in 100 mL of diethyl ether. The resulting precipitate was filtered off and washed with diethyl ether to afford 12.9 g of Z-His(2-Adoc)-OH·CHA, in a yield of 75.9%, m.p., 150–153°C. The pure Z-His(2-Adoc)-OH can be obtained from Z-His(2-Adoc)OH·CHA according to the following procedure. To an ice-cooled suspension of 1.0 g of Z-His(2-Adoc)-OH·CHA (1.8 mmol) in 100 mL of EtOAc was added 20 mL of 10% aqueous citric acid, and the resultant biphasic solution was vigorously stirred for 30 minutes. The EtOAc layer was separated, washed with water, dried over Na2SO4 and evaporated to give 690 mg of Z-His(2-Adoc)-OH, i.e., Nτ-((((1R,3S, 5S,7S)-adamantan-2-yl)oxy) carbonyl)-Nα-((benzyloxy)carbonyl)-L-histidine, as an amorphous powder, in a yield of 81.9%.

Amino Protecting Groups 189

4.2.6.1.5 Preparation of 2-(Adamantyloxycarbonyl)Amino-3-indol1-yl-2-Methyl Propionic Acid [51]

To a solution of 0.090 g of methyl 2-amino-3-(1H-indol-1-yl)-2-methyl propanoate in 5 mL of dry THF were added sequentially a solution of 2-adamantyl chloroformate (prepared from 0.118 g of 2-adamantanol) in 5 mL of dry THF and 0.0135 mL of Et3N in 5 mL of THF. The reaction mixture was stirred for 1 hour, then filtered and concentrated in vacuo to give an oil which was dissolved in 10 mL of EtOAc, washed with a saturated aqueous solution of NH4Cl (2 × 10 mL) and brine (2 × 10 mL). The organic phase was dried and concentrated in vacuo to give the crude ester. This crude ester was then mixed with 2 mL of water and 5 mL of 1,4-dioxane, and 0.033 g of LiOH was added and the mixture was stirred at 25°C for 15 hours. Upon removal of solvent, the residue was purified by flash chromatography using CH2Cl2/MeOH (9/1) as eluent to afford 0.150 g of 2-(adamantyloxycarbonyl)amino-3-indol-1-yl-2-methyl propionic acid as an oil, in a yield of 95%. 4.2.7 ISOBORNYLOXYCARBONYL (IBOC) GROUP Isobornyloxycarbonyl (Iboc) group, is another acid-labile amino protecting group that can be easily cleaved with CF3CO2H [52, 53] or HCl in HOAc [54, 55], but is rather stable under the treatment with 1–2 N HCl in organic solvents [53]. In addition, this protecting group is stable to alkali and catalytic hydrogenation [55]. This protecting group can be easily introduced into amino acid by means of Iboc chloride (Iboc-Cl), a satisfactorily stable oil prepared from isoborneol and phosgene [53]. The resulting Iboc amino acids, in most cases, could be obtained in crystalline form and in good yields [53]. The presence of Iboc group is known to enhance the solubility of amino acids and peptides [54]. Besides the protection of the amino group, Iboc has also been applied to protect the guanidino group of arginine [55].

190

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.2.7.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF IBOC GROUP 4.2.7.1.1 Preparation of N-Isobornyloxycarbonyl-D-pChlorophenylalanine Isopropyl Ester [56]

To a solution of 100 g phosgene in 350 mL of benzene at –5 to +5°C was added a solution of 28 g D- or L-isoborneol in 400 mL benzene and 25 mL pyridine. Upon usual treatment after the reaction, 39.4 g of Iboc chloride was obtained. Then, a mixture of 6.96 g D-p-chlorophenylalanine isopropyl ester hydrochloride (25 mmol) and 6.25 mL of Iboc chloride (30 mmol) in 10 mL of dioxane and 10 mL of water at room temperature was treated with a total of 12.8 mL of 4 N NaOH solution dropwise, to adjust the pH to a maximum of 10.0. After the reaction was completed, the mixture was neutralized with citric acid. The product was taken up by ethyl acetate, and the ethyl acetate solution was washed with water, dried over sodium sulfate and evaporated in a vacuum to afford 10.55 g of N-Iboc-D-p-chlorophenylalanine, in a quantitative yield. 4.2.7.1.2 Preparation of DL-Isobornyl Chloroformate [53]

To a solution of 200 g phosgene (2.0 mol) in 600 mL of anhydrous THF, was added a solution of 246.4 g of DL-isoborneol (1.6 mol) in 800 mL of anhydrous ether dropwise under stirring at about 5°C for over 60 minutes. The reaction mixture was allowed to stand at room temperature for

Amino Protecting Groups 191

additional 6 hours. Upon removal of solvent in vacuo, 323 g of DL-isobornyl chloroformate was obtained as a sticky oil. 4.2.7.1.3 General Procedure for the Preparation of Isobornyloxycarbonylamino Acids, NaOH Procedure [53]

An amino acid (50 mmol) was dissolved in 50 mL ice-cold 1 N NaOH solution. To this ice-cooled solution were added alternately 13.0 g of isobornyl chloroformate (60 mmol) in 40 mL of THF or dioxane and 50 mL 1 N NaOH in portions over a period of 60 minutes with stirring. After stirring at room temperature for another 2–4 hours, the reaction mixture was diluted with 120 mL of water, and the solution was extracted two times with ether. The aqueous phase was acidified with 1 N HCl under stirring and cooling. The precipitated product (solid or oil) was extracted with 120 mL of EtOAc, and the aqueous phase was extracted with two more portions of fresh EtOAc (80 mL). The combined organic layers were dried over anhydrous Na2SO4. Upon removal of the solvent in vacuo, the residue was crystallized from a suitable solvent such as petroleum ether, ether-petroleum ether, EtOAcpetroleum ether or EtOAc. 4.2.7.1.4 NaHCO3 Procedure to Prepare D-Isobornyloxycarbonyl-LAspargine [53]

To an ice-cooled suspension of 3.0 g L-asparagine monohydrate (20 mmol) in 50 mL water was added 4.0 g of NaHCO3 under stirring. Then, 5.2 g of D-isobornyl chloroformate (24 mmol) in 20 mL of dioxane was added in three portions over 60 minutes under stirring. The resulting mixture was stirred at room temperature for another 5 hours, then, it was extracted two

192

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

times with ether. After the aqueous phase was acidified with 0.5 N HCl, the product separated was extracted into three portions of EtOAc (40 mL each). The combined extracts were dried over anhydrous Na2SO4. Upon removal of EtOAc in vacuo, the residue was recrystallized from EtOAc to give 5.70 g of N-D-isobornyloxycarbonyl L-asparagine, i.e., ((((1R,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)carbonyl)-L-asparagine, as fine platelets, in a yield of 96.6%, m.p. 169–170°C (decomp.). 4.2.7.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF IBOC GROUP 4.2.7.2.1 Acidolysis of N-Isobornyloxycarbonyl Glycine with 20% HBr in Acetic Acid [53]

To a 10 mL 20% HBr in acetic acid, was added 5.11 g of D-isobornyloxycarbonyl-glycine (20 mmol) under stirring. After 2 minutes at room temperature, 100 mL of dry ether was added into the reaction mixture, and the resulting crystals were collected by filtration. The crystals were dissolved in 50 mL water, and the solution was passed through a column of Amberlite IRA-4B (AcO–). The eluate was evaporated in vacuo to dryness. The residue was recrystallized from water-EtOH to afford 1.38 g of glycine, in a yield of 92%. 4.2.7.2.2 Acidolysis of N-Isobornyloxycarbonyl Glycine with TFA [53]

To 10 mL of TFA was added 1.275 g of D-isobornyloxycarbonyl-glycine (5 mmol), and the solution was stirred at room temperature (22°C) for 20 minutes. The TFA was removed by evaporation, and the residue was washed well with anhydrous ether. The residue was dissolved in 10 mL of EtOH and neutralized with 0.75 mL of Et3N. The resulting crystal was collected by filtration and washed with cold EtOH, and recrystallized from aqueous EtOH to give 354 mg of free glycine, in a yield of 94%.

Amino Protecting Groups 193

4.2.8 TRIPHENYLMETHYL (TRT) GROUP AND ITS ANALOGOUS GROUPS Triphenylmethyl group, often known as trityl group or abbreviated as Trt [57, 58], is also a good protecting group for the amino group, which resists the deprotection under basic conditions but can be easily removed under acidic conditions [59], due to the formation of a stable tertiary carbocation, i.e., triphenylmethyl cation. The advantages of using trityl as the amino protecting group include the increased crystallinity [59], and lower chance of amino acid racemization during the coupling step in comparison to other N-protected amino acids [57]. However, the trityl group has been only occasionally applied in peptide synthesis because of the low yield encountered in the preparation of Nα-trityl amino acid and its failure to couple with other amino acids in acceptable yields [60]. When the trityl group is mounted onto amino acid ester, the hydrolysis of the ester group under mild conditions is very slow except for the corresponding N-trityl glycine and N-trityl peptide esters [61], due to the increased steric hindrance arising from the trityl group. A similar effect of the steric hindrance has also been observed during the coupling of higher N-trityl amino acids with esters of other amino acids, so that the N-trityl-L-phenylalanine, N-trityl-L-leucine, N-trityl-L-asparagine are unable to couple with amino acid esters; although such steric effect has not been observed in the coupling of N-trityldi- or polypeptides [61]. Due to the high reactivity of trityl chloride or trityl bromide, often the sulfhydryl group of cysteine will be tritylated as well [61]. While triphenylmethyl chloride is often used to form N-trityl amino acid derivatives, it is proved that the reaction between amino acid and triphenylmethyl bromide in dry CHCl3/DMF (2:1) is superior to that from trityl chloride due to the higher reactivity and reduced hygroscopic character of trityl bromide [62]. In addition, the preparation of N-trityl amino acid in high yield and purity has become possible by means of temporary tritylation of the corresponding amino acid TMS ester and subsequent mild hydrolysis [57]. Under this condition, hydroxyl amino acid is preferentially tritylated with Me3SiCl or Me2SiCl2 (or Ph2SiCl2) for temporary protection of both hydroxy and carboxyl groups. Removal of the trityl group occurs when the N-trityl-amino acid or peptide ester is heated briefly with alcohol-containing one equivalent of hydrogen chloride [61], treatment of N-trityl-amino acids or peptides with dilute acetic acid or one equivalent of hydrochloric acid, or catalytic hydrogenation [61]. In addition, a selective Nα-detritylation method of mono or di-tritylated amino acids with metallic catalysis in the aprotic anhydrous organic solvent

194

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

has been developed that leads to the formation of amino acids tritylated only on the functional groups in the lateral chain [63]. Besides the simple triphenylmethyl group, analogous methylphenyldiphenylmethyl (or methyltrityl (Mtt)) [64], MMT [64, 65], dimethoxytrityl [64, 66], and trimethoxytrityl [66] groups have also been developed and applied in the protection of amino groups. These groups can probably be removed under even milder acidic conditions. When Mtt is applied as the protecting group, it is found that Mtt is not as readily cleaved as expected if the syntheses are performed on hydrophilic resins such as Tentagel [64]. 4.2.8.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE TRITYLATION OF AMINO GROUP 4.2.8.1.1 Preparation of N-Trityl-Glycine Methyl Ester [61]

To a suspension of 1.25 g of glycine methyl ester hydrochloride (0.01 mol) in 15 mL of dry chloroform was added 2.2 g of triethylamine (0.022 mol) followed by 2.8 g of triphenylchloromethane (0.01 mol). The mixture was allowed to react for 6 hours at room temperature. The solution was washed twice with water and dried with sodium sulfate. The solvent was evaporated in vacuo, and complete removal of the chloroform was ensured by the addition of a few mL of methanol and subsequent evaporation in vacuo. The residue was recrystallized from methanol to afford 70% of N-trityl-glycine methyl ester, m.p. 106–107°C. 4.2.8.1.2 Preparation of N-Trityl-Threonine [57]   Procedure A:

A mixture of 3.57 g of threonine (30 mmol) and 3.62 mL of Me2SiCl2 (30 mmol) in 40 mL of CH2Cl2 was refluxed for 2 hours with stirring and then

Amino Protecting Groups 195

allowed to reach room temperature. Then 12.55 mL of Et3N (90 mmol) was added dropwise followed by a solution of 8.36 g of triphenylchloromethane (30 mmol) in 20 mL of CH2Cl2, and the resulting suspension was stirred for 5 hours at room temperature. Subsequently, excess of MeOH and 4.18 mL of Et3N (30 mmol) were added, and volatile components were removed by rotary evaporation. The residue was partitioned between 50 mL of EtOAc and 50 mL of 5% precooled solution of citric acid. The organic phase was collected and washed with 1 N NaOH (2 × 20 mL) and water (2 × 10 mL). The combined aqueous layers were washed with 20 mL of EtOAc, cooled to 0°C, and neutralized with glacial AcOH. The precipitated product was extracted with EtOAc (2 × 30 mL), and the combined organic layers were washed twice with water and dried over MgSO4. Upon concentration of the solution in vacuo to about 20 mL, 1 mL of diethylamine (10 mmol) was added to afford 58% of N-trityl-threonine diethylammonium salt, m.p. 165°C.   Procedure B:

A mixture of 3.57 g of threonine (30 mmol) and 6.23 mL of Ph2SiCl2 (30 mmol) in 40 mL of CH2Cl2 was refluxed for 2 hours and tritylated exactly as in procedure A to afford 92% of N-trityl-threonine diethylammonium salt.   Procedure C:

To a stirred suspension of 4.76 g of threonine (40 mmol) in 70 mL of CH2Cl2 was added 17.75 mL of Me3SiCl (140 mmol), and the mixture was refluxed for 20 minutes and then cooled to room temperature. After that, a solution of 19.51 mL Et3N (140 mmol) in 40 mL of CH2Cl2 was added, and the mixture was refluxed for 45 minutes. The reaction mixture, at 0°C, was treated dropwise with 2.43 mL of anhydrous methanol (60 mmol) in 10 mL of CH2Cl2 and allowed to attain room temperature. Then 5.58 mL of Et3N (40 mmol) was added followed by the addition of 11.25 g of triphenylchloromethane (40 mmol) in two portions over a 15-min period. The reaction

196

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

mixture was stirred for 5 hours and worked up as described in procedure A to afford 83% of N-trityl-threonine diethylammonium salt. 4.2.8.1.3 Preparation of N-Trityl-Amino Acid Through Metallic Pertritylation of Amino Acid [63]   Step A: Preparation of N-trityl-glycine trityl ester (Trt-Gly-OTrt):

To a mixture of 0.18 g of anhydrous zinc chloride (1.3 mmol) and 1.51 g of trityl chloride (5.4 mmol) in 8 mL of anhydrous acetonitrile was added 0.20 g of glycine (2.7 mmol), and the suspension was stirred at 30–40°C for 30 minutes. Then 0.76 mL of triethylamine was added slowly. At the end of the addition, abundant precipitation of a crystalline solid was obtained. The suspension was allowed to rest for 24 hours then it was filtered. The solid was washed in sequence with acetonitrile, a 1:1 mixture of water and MeCN and again with MeCN. After drying, 1.16 g of chromatographically pure Trt-Gly-OTrt was obtained, in a yield of 77.9%, m.p. 176–182°C.   Step B: Preparation of N-trityl-glycine (Trt-Gly-OH) by selective O-detritylation of Trt-Gly-OTrt:

To a solution of 0.20 g of Trt-Gly-OTrt (0.36 mmol) in 5 mL of CHCl3 was added slowly a solution of 0.04 g of copper acetate monohydrate (0.2 mmol) in 5 mL of MeOH. At the end of the addition, the mixture was set aside and monitored with TLC until the spot due to the substrate disappeared. Then, the organic solvents were evaporated under vacuum, Et2O was added to the residue and the suspension thus obtained was extracted with a diluted solution of citric acid, maintaining the pH between 3 and 4. This treatment at first led to the formation of a purple solid, which then disappeared. The ethereal, clear, and colorless solution was separated, and the aqueous layer was washed twice with Et2O. The combined organic solution was washed with water, and dried over sodium sulfate. The addition of diethylamine to

Amino Protecting Groups 197

the ethereal solution caused the precipitation of 0.098 g of N-trityl-glycine diethylammonium salt, in a yield of a 70.2%. 4.2.8.1.4 General Procedure to Prepare N-Trityl α-Amino Acids with Triphenylmethyl Bromide [62]

The finely powdered α-L-amino acid (10 mmol) was suspended in a solution of 7.11 g of triphenylmethyl bromide (22 mmol) in 50 mL of mixed solvent of CHCl3/DMF (2:1) in a dry flask and vigorously stirred at room temperature until a clear solution was reached (30 minutes up to one hour). Then, 5.6 mL of Et3N (40 mmol) in 10 mL of CHCl3/DMF (2:1) was dropped to the reaction mixture over a period of 20 minutes, and stirring was continued for another 30 minutes. After addition of 50 mL of MeOH, the reaction mixture was heated to 50°C for 20 minutes to 2.5 hours. The solvent was evaporated at reduced pressure, and the residue was taken up in Et2O (3 × 50 mL). The combined organic phase was washed with H2O (3 × 50 mL), and dried over Na2SO4. Then a solution of 1 mL diethylamine (10 mmol) in 10 mL of Et2O was dropped to the solution. The precipitate was filtered off and washed several times with cold Et2O, and dried in vacuo. The yield ranged from 80% to 86%. 4.2.8.1.5 Preparation of L-Nα-(4-Monomethoxytrityl)-β -[Thymin1-yl]Alanine [65]

To 2 mL of TFA was added 103 mg of N-Boc-β-[thymine-1-yl]alanine (329 µmol), and the solution was stirred at room temperature for 2 hours. TFA was removed in vacuo and the residue was repeatedly co-evaporated with toluene. After lyophilization from H2O, the residue was dissolved in 40

198

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

mL of DMF, and a solution of 133 mg of triethylamine (1.32 mmol) and 102 mg of 4-monomethoxytrityl chloride (MMT-Cl, 329 µmol) in 2 mL of DMF was added dropwise. The solution was stirred at room temperature for 1 hour, and concentrated to remove the solvent. The remaining oil was dissolved in CH2Cl2, and washed with water. The aqueous layer was extracted twice with CH2Cl2 with the total amount of CH2Cl2 about 140 mL. The combined organic phase was dried over Na2SO4 and concentrated in vacuo. Then, 3 mL of diethyl ether was added and the suspension was stirred at –28°C for 1 hour. After centrifugation at –10°C and separation from diethyl ether, a white solid was obtained. This procedure was repeated to give 65.6 mg of L-Nα-(4monomethoxytrityl)-β-[thymin-1-yl]alanine, in a yield of 41%. TLC, Rf = 0.70 (CH2Cl2:MeOH:NEt3 = 4:1:0.05). 4.2.8.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE DETRITYLATION OF AMINO GROUP 4.2.8.2.1 Detritylation of N-Trityl-Glycine Ethyl Ester [61]

A solution of 1.72 g of N-trityl-glycine ethyl ester (0.005 mol) in 5 mL of 1 N HCl in absolute ethanol, or 1.9 g of N-trityl-glycine ethyl ester hydrochloride in 5 mL of absolute ethanol was heated for 1–2 minutes in a water bath. Evaporation of ethanol in vacuo and trituration of the residue with ether resulted in crystallization of the glycine ethyl ester hydrochloride, in an amount of 0.7 g, in a yield of 95%, m.p. 144°C. 4.2.8.2.2 N-Detritylation of Tr-Gly-OH in Aprotic Solvent Catalyzed by Metallic Salts [63]

To a suspension of 0.04 g of copper chloride (II) (0.3 mmol) in 3 mL of MeCN was added 0.10 g of Trt-Gly-OH (0.3 mmol). Rapidly, under agitation at ambient temperature, the brown solid went into solution giving rise to precipitation of pale green solid. The suspension was left under stirring for 1 hour and

Amino Protecting Groups 199

filtered. After washing with MeCN, 0.05 g of glycine was obtained. The filtrate was poured in 100 mL of water to afford 0.08 g of triphenylmethanol after filtration and washing with water. (Note: according to the amount of starting material, only 0.02 g of glycine can be obtained, not 0.05 g). 4.2.9 MISCELLANEOUS ACID LABILE AMINO PROTECTING GROUPS Besides the above eight popular amino protecting groups that have been applied in syntheses involving amino acids and peptides, many other acidlabile amino protecting groups have been developed as well, although they are not as commonly used as the above-mentioned ones. These not so common acid-labile amino protecting groups include 1,1-dimethyl-2-methacrylmethanamido-ethoxycarbonyl [4, 67], 1,1-dimethyl-3-methacryl-methanamido-butoxycarbonyl [4, 67], 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) [68], alkyldithio-carbonyl [69], Foc [70], p-phenylazophenylsulfonylaminocarbonyl (Azo-Tac) [71], and p-tolylmethylsulfonyl (also known as p-xylyl-α-sulfonyl) (Pms) [3, 72]. Both 1,1-dimethyl-2-methacrylmethanamido-ethoxycarbonyl and 1,1dimethyl-3-methacrylmethan-amido-butoxy-carbonyl are polymerizable N-meth-acrylamino modified Boc (m-Boc) type protecting groups, derived from the corresponding tertiary alcohols. The monomeric and polymeric N-methacrylamino m-Boc amino acids and the corresponding methyl esters have been applied for peptide synthesis following the (N→C)-assembly method. These protecting groups can be rapidly cleaved in the presence of a strong acid (e.g., HBr/HOAc) [67]. In contrast to the classical t-Boc protecting group that may form t-butyl cation or isobutene during deprotection, the deprotection of these new groups leads to the formation of oxazole and oxazine derivatives. In addition, these two protecting groups have controllable solubility by variation of the N-acyl residue that can be adjusted by copolymerization [67]. The 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) group has been used to protect the ε-amino group of lysine, which can be readily removed with 0.15–0.3 M methanesulfonic acid in TFA-thioanisole (9:1). However, this group is completely resistant to hydrogenolysis or treatment with neat TFA and HCl [68]. In contrast to the introduction of many amino protecting groups into amino acids under the Schotten-Baumann conditions, it is impractical to form alkyldithiocarbonyl protected amino acid under this condition,

200

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

due to the pronounced lability of the carbamoyl disulfide moiety in aqueous alkaline media. However, both tert-butyldithiocarbonyl and cyclohexyldithiocarbonyl can be easily mounted to amino acids from the corresponding chlorides in direct reaction with amino acid tert-butyl ester followed by treatment with TFA. These dithiocarbonyl groups can be removed by the treatment of p-toluenesulfonic acid (PTSA) and triphenylphosphine [69]. The Foc group is introduced by the reaction of the appropriate N-carbonylamino acid ester with furfuryl alcohol. It can be removed acidolytically with 6.5% HBr/AcOH, HCl/AcOH, or CF3CO2H. Such cleavage is selective over benzyl ester, benzyl ether, and benzyloxycarbonyl groups [70]. The p-phenylazophenylsulfonylaminocarbonyl (Azo-Tac) can be introduced into amino acid by means of p-phenylazophenylsulfonyl isocyanate, and removed by warming with 95% ethanol or EtOAc containing 1% acetic acid [71]. The Pms or p-xylyl-α-sulfonyl protected amino acids can be prepared from p-MeC6H4CH2SO2Cl, which has excellent stability against trifluoroacetic acid or dilute hydrogen chloride. For example, the Pms-protected amino acids can tolerate the treatment of TFA at 20°C for 24 hours. However, this group could be removed cleanly by treatment with HF in the presence of anisole at –20°C for 60 min [3]. 4.2.9.1 PREPARATION OF N-(1,1-DIMETHYL-2METHACRYLMETHANAMIDO-ETHOXYCARBONYL)-LPHENYLALANINE METHYL ESTER [67] 4.2.9.1.1 Preparation of 1,1-Dimethyl-2-Methacrylmethanamido2-Ethyl-(4-Nitrophenyl) Carbonate

To a stirred solution of 1.57 g of N-(2-hydroxy-2-methyl-propyl)-methacrylamide (10 mmol) and 0.79 g of pyridine (10 mmol) in 30 mL of dichloromethane (30 mL) was added slowly 2.01 g of p-nitrophenyl chloroformate (10 mmol) at –15°C. The reaction mixture was stirred at room temperature for 3 hours while an initial precipitate dissolved. The solution was washed with portions of 1 N HCl (15 mL) until the organic layer turned colorless,

Amino Protecting Groups 201

followed by saturated sodium carbonate solution and water. After being dried over magnesium sulfate, the solution was evaporated nearly to dryness. The residue was mixed with 20 mL of ether/petroleum ether (2:1, v/v) to form colorless crystals at –10°C, to afford 2.40 g of the title compound, also known as 1-methacrylamido-2-methylpropan-2-yl (4-nitrophenyl) carbonate, in a yield of 77%, m.p. 87–88°C. 4.2.9.1.2 Preparation of N-(1,1-Dimethyl-2Methacrylmethanamido-Ethoxycarbonyl)-LPhenylalanine Methyl Ester

To a stirred solution of 840 mg of L-phenylalanine methyl ester hydrochloride (4 mmol) and 0.56 mL of triethylamine (4 mmol) in 10 mL of absolute chloroform was added 1.28 g of 1,1-dimethyl-2-methacrylmethanamido-ethyl-(4-nitrophenyl)-carbonate (4 mmol). The mixture was heated at 50°C for 24 hours. The yellow solution was washed with 1 mL portions of cold 1 N NaOH until the organic layer was nearly colorless. Then, the organic layer was treated with 1 mL of 1 N HCl and water (0–5°C), respectively. After being dried over magnesium sulfate, the solution was concentrated via evaporation. The residue was dissolved in a small amount of acetoacetic acid ethyl ester, covered with 10 mL of ether and cooled at –20°C to induce 1.15 g of methyl (((2-methyl-1-(3-methyl-2-oxobut-3-enamido)propan-2-yl)oxy) carbonyl)-L-phenylalaninate as light-yellow crystals, in a yield of 80%. The product was a waxy oil at room temperature. 4.2.9.2 PREPARATION OF CYCLOHEXYLDITHIOCARBONYL PROTECTED AMINO ACID AND ITS DEPROTECTION [69] 4.2.9.2.1 Preparation of Cyclohexyldithiocarbonyl Chloride

202

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a pre-cooled solution (0°C) of 57 mL chlorocarbonylsulfenyl chloride (freshly prepared from trichloromethane sulfenylchloride) (0.68 mol) in 100 mL pentane saturated with argon, was added 79.5 mL of cyclohexyl mercaptan (0.65 mol, density = 0.95 g/mL) dropwise under vigorous stirring in 30 minutes. The reaction mixture was stirred at 35–40°C for additional 60 minutes and then evaporated in vacuo. The residual oil was fractionally distilled to afford 76% of cyclohexyldithiocarbonyl chloride, also known as SS-cyclohexyl carbonochlorido(dithioperoxoate), as a yellow oil. 4.2.9.2.2 Preparation of Alkyldithiocarbonyl Amino Acids (Method A)

To a solution of 10 mmol amino acid tert-butyl ester hydrochloride and 10 mmol triethylamine in 50 mL ether or EtOAc, was added 10 mmol of alkyldithiocarbonyl chloride and 10 mmol Et3N dropwise at 0°C in 10 minutes under stirring. The reaction was allowed to proceed at room temperature for 2 hours, insoluble material (Et3N⋅HCl) was then filtered off and the filtrate was evaporated in vacuo. The residue was dissolved in 5 mL ice-cold TFA, the resulting solution was kept at room temperature for 60 minutes and then evaporated to dryness. The residue was distributed between ether (or EtOAc) and water; and the organic phase was then washed with saturated NaCl solution, dried over Na2SO4 and evaporated. The amino acid derivatives were obtained in chromatographically homogeneous form, partly as oils and partly as crystalline materials upon crystallization from suitable solvents. For the preparation of N-butyldithiocarbonyl-alanine (But-S2CO-Ala-OH), the yield was 83% as crystallized from diisopropyl ether/petroleum ether, m.p. 112–113°C. 4.2.9.2.3 Preparation of Alkyldithiocarbonyl Amino Acids (Method B)

To a suspension of 20 mmol amino acid in 30 mL of dioxane, were added 40 mmol trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (or 40 mmol trimethylchlorosilane and 40 mmol triethylamine). The reaction mixture was warmed up to 75°C for 60 minutes, whereby a clear solution was formed.

Amino Protecting Groups 203

Upon cooling to 0°C, 20 mmol of alkyldithiocarbonyl chloride was added, and the reaction was allowed to proceed at 0°C for 30 minutes and at room temperature for additional 60 minutes; then the mixture was poured into ice-cold water and the product was extracted with EtOAc. The combined organic phases were washed with water, dried over Na2SO4, and evaporated to dryness. The amino acid derivatives were obtained in chromatographically homogeneous form as oils or crystalline materials from suitable solvents. 4.2.9.3 REMOVAL OF THE CYCLOHEXYLDITHIOCARBONYL GROUP, PREPARATION OF L-PHENYLALANYL-L-LEUCINE [69]

To a solution of 1.36 g of cyclohexyldithiocarbonyl-Phe-Leu-OH, i.e., (cyclohexyldisulfannecarbonyl)-L-phenylalanyl-L-leucine (30 mmol) in 20 mL of THF, were added 0.57 g of PTSA monohydrate (3 mmol) and 1.73 g of triphenylphosphine (6.6 mmol). The mixture was stirred at room temperature for 60 minutes, and the solvent was then removed in vacuo. The residue was triturated with ether and dried. The solid was dissolved in 10 mL of water and the solution was neutralized with 2 M NaOH to pH 7. The product was filtered off, washed with water and ethanol, and dried to afford 0.78 g of L-phenylalanyl-L-leucine, in a yield of 87%, m.p. 262–264°C. 4.3 ALKALI-LABILE AMINO PROTECTING GROUPS Besides many acid-labile protecting groups as mentioned previously for the amino group, several alkali cleavable amino protecting groups have also been developed, which can be applied orthogonally with the carboxyl protecting groups. These base-labile amino protecting groups include (9H-fluoren-9-yl) methanesulfonyl (Fms), 2-(4-chlorophenyl)-sulfonylethoxycarbonyl (Cps), 2-(4-nitrophenyl)sulfonylethoxycarbonyl (Nsc), 2-(4-trifluoromethylphenyl)sulfonylethoxycarbonyl (Tsc), 2-(tert-butylsulfonyl)-2-propenyl-oxycarbonyl (Bspoc), 2-chloro-3-indenylmethyloxycarbonyl (CLIMOC), 2-methylsulfonyl-3-phenyl-1-prop-2-enyloxycarbonyl (Mspoc), 4-isopropyloxycarbonyloxybenzyloxy-carbonyl (4-PriOCO), benz[f]inden-3-ylmethyloxycarbonyl

204

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

(BIMOC), benzo-thiophenesulfone-2-methoxycarbonyl (Bsmoc), fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethyloxycarbonyl (Msc), and tetrabenzo-[a,c,g,i]fluorenyl-17-methyloxycarbonyl (Tbfmoc), etc. These alkali-labile protecting groups are listed in Figure 4.2.

FIGURE 4.2 The structure of alkali-labile amino protecting groups.

Amino Protecting Groups 205

4.3.1 FLUORENYLMETHOXYCARBONYL (FMOC) GROUP 9-Fluorenylmethyloxycarbonyl (Fmoc) was first introduced by Carpino et al in 1970 [73], which has become one of the two most commonly used amino protecting groups, i.e., t-Boc, and Fmoc. It has high stability toward trifluoroacetic acid, HCl, HBr in organic solvent, and tertiary amines [2, 74], as well as catalytic hydrogenolysis over palladium-carbon [73]. However, it can be conveniently deprotected simply by allowing a solution of the Fmoc protected sample in liquid ammonia to stand for several hours [73, 75], or standing in the solvents of DMF, DMA [76] in the presence of piperidine, morpholine, ethanolamine [73, 75], 1,4-diazabicyclo[2.2.2]-octane (DABCO) [77], or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) [78], often by the treatment with 5–20% of piperidine in DMF [2, 74, 79, 80], or even 25% of piperidine in DMF [81]. In addition, piperazino-, and piperidinofunctionalized polystyrenes [82, 83], and piperazino-functionalized silica gel have been used as the deblocking/ scavenging agents for the Fmoc group [82]. The Fmoc derivatives can also be cleaved by catalytic hydrogenation for 3–33 hours under various conditions [76]. Deprotection of the Fmoc group leads to the formation of monomeric or polymeric dibenzofulvene or an amine-dibenzofulvene adduct depending on the reaction conditions and the reagent used, which can be easily separated from the desired amino acid or peptide product [73, 75]. This protecting group can be readily introduced by treatment of the parent amine with 9-fluorenylmethyl chloroformate [73, 75], also known as 9-fluorenyl-methoxycarbonyl chloride in a weak alkaline solution [84], such as the aqueous dioxane in the presence of sodium carbonate or bicarbonate [73], in yields ranging from 88% to 97% [84]. Alternatively, it can also be introduced into amino acid from 9-fluorenylmethyl azidoformate [73, 75], 9-fluorenylmethoxycarbonyl-1H-benzotriazole [78, 85], or more commonly from N-(9-fluorenylmethoxycarbonyloxy) succinimide, also known as 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu) [77, 86–90]. The application of Fmoc chloride has the advantages of fast derivatization of the amines using a fully automated instrument, good separation of the derivatives on common C18 columns and sensitive detection owing to their fluorescence [91]. 9-Fluorenylmethyl chloroformate can be readily obtained from fluorenylmethanol by reacting with phosgene [75, 84]. It is a stable and crystalline solid that keeps well at low temperature in the absence of moisture. The use of Fmoc amino acids generally requires that the reactive side chains of amino acids would be protected by acid-labile groups [84].

206

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

On the other hand, the application of Fmoc as the amino protecting group also faces some “trivial” weaknesses, such as the insufficient stability of Fmoc-protected amino acids and peptides in weakly basic and even neutral DMF solution, and inefficient trapping of dibenzofulvene released during Fmoc cleavage by piperidine, resulting in alkene polymerization. Also, during SPPS, incomplete cleavage of Fmoc may occur, owing to the steric hindrance and unfavorable hydrophobic interactions of the bulky Fmoc moiety [2]. Furthermore, in the slow coupling reactions, undesired loss of Nα-Fmoc groups over time would cause a buildup of by-products due to the presence of an excess amount of activated Nα-Fmoc protected amino acids used in each cycle of the SPPS [92]. 4.3.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF FMOC REAGENTS 4.3.1.1.1 Preparation of 9-Fluorenylmethyl Chloroformate [75]

To a solution of 7.12 g of phosgene in 75 ml of CH2Cl2 cooled in an ice bath was added 12.8 g of 9-fluorenylmethanol slowly with stirring. The solution was stirred for 1 hour in the ice bath and then kept at ice-bath temperature for 4 hours. Removal of solvent and excess phosgene under reduced pressure gave an oil which crystallized after several hours to give 16 g of crude 9-fluorenylmethyl chloroformate, in a yield of 95%, m.p. 61.5–63°C. Recrystallization of this crude product from ether for two times gave 14.5 g of the 9-fluorenylmethyl chloroformate as colorless crystals, in a yield of 86%, m.p. 61.5–63°C. 4.3.1.1.2 Preparation of 9-Fluorenylmethyl Azidoformate [75]

To an ice-cold solution of 0.52 g of NaN3 in 2 mL of H2O was added slowly a solution of 1.35 g of 9-fluorenylmethyl chloroformate in 2.5 mL of acetone under stirring. The mixture was stirred in the ice bath for 2 hours

Amino Protecting Groups 207

and at room temperature for another 2 hours. The precipitated solid was filtered, washed with water, and recrystallized from acetone to give 1.13 g of 9-fluorenylmethyl azidoformate, also known as (9H-fluoren-9-yl)methyl carbonazidate, as colorless crystals, in a yield of 82%, m.p. 83–85°C. The analytical sample recrystallized from hexane had a m.p. of 89–90°C. 4.3.1.1.3 Preparation of N-Fmoc-1H-Benzotriazole [85]

A mixture of 1.0 g of (9H-fluoren-9-yl)methyl chloroformate (3.87 mmol) and 0.92 g of benzotriazole (7.74 mmol) in 20 mL of CH2Cl2 was stirred at 10°C for 3 hours. The precipitate formed was filtered off and discarded. The filtrate was evaporated under reduced pressure to give a crude product, which was washed with diethyl ether and recrystallized from CH2Cl2-hexanes to afford N-Fmoc-1H-benzotriazole, also known as (9H-fluoren-9-yl)methyl 1H-benzo[d][1,2,3]triazole-1-carboxylate, as white microcrystals, in a yield of 88%, m.p. 90–91°C. 4.3.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR INTRODUCTION OF THE FMOC GROUP 4.3.1.2.1 Preparation of 9-Fluorenylmethoxycarbonylglycine from Fmoc-Cl [75] O Cl

O O

+ H N 2

10% Na2CO3 OH dioxane/H2O

H N

O

O OH

O

To a solution of 0.57 g of glycine dissolved in 20.2 mL of 10% Na2CO3 was added with stirring and cooling in an ice bath a solution of 1.96 g of Fmoc-Cl in dioxane. The mixture was stirred at room temperature for 2 hours, then poured into 400 mL of H2O, and extracted twice with ether to remove small amounts of 9-fluorenylmethanol and the high-melting polymer

208

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

of dibenzofulvene. The aqueous layer was cooled in an ice bath and acidified with concentrated HCl to congo red paper. The white precipitate was extracted with EtOAc, and the combined organic layer was washed with water, dried over MgSO4. Upon evaporation of the solvent, 2.0 g of (((9H-fluoren-9-yl) methoxy)carbonyl)glycine was obtained as white solid, in a yield of 89%, m.p. 173–176°C. The product was further purified by recrystallization from MeNO2 to afford 1.98 g of crystal, in a yield of 88%, m.p. 174–175°C. 4.3.1.2.2 Synthesis of N-Fmoc-L-Cysteine [85]

To a solution of 0.121 g of L-cysteine (1 mmol) and 0.100 g of triethylamine (1 mmol) in 8 mL mixed solvent of CH3CN and H2O (3:1) was added 0.341 g of N-Fmoc-1H-benzotriazole (1 mmol). The mixture was stirred at room temperature for 2 hours and the solvent was then removed under reduced pressure. The residue was dissolved in 10 mL of EtOAc, and the organic layer was washed with 2 N HCl and brine. Evaporation of solvent followed by recrystallization from EtOAc/hexanes (3:1) gave 0.24 g of N-Fmoc-L-cysteine, i.e., (((9H-fluoren-9-yl)methoxy)carbonyl)-L-cysteine, as white microcrystals, in a yield of 70%, m.p. 134–136°C. 4.3.1.2.3 Preparation of (2S,3S)-2-N-(9H-Fluorenylmethoxy) Carbonylamino-4-(Benzyloxy)-3-Hydroxy-4-Oxobutanoic Acid [86]

To a solution of 0.1 g of (2S,3S)-2-amino-4-benzyloxy-3-hydroxy4-oxobutanoic acid (0.42 mmol) and 0.07 g of NaHCO3 (0.84 mmol) in 15 mL of water/acetone mixture (1:2) was added dropwise a solution of 0.141 g of Fmoc-OSu (i.e., (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl)

Amino Protecting Groups 209

carbonate, 0.42 mmol) in acetone. The reaction was completed within 2 hours, as determined by TLC. The reaction mixture was concentrated via rotary evaporation, and the residual water was acidified with 2 N HCl until pH 4 and extracted two times with EtOAc. EtOAc was evaporated, and the crude residue was purified by silica gel column chromatography (toluene: EtOAc: HOAc = 5: 5: 1) to afford 0.185 g of (2S,3S)-2-((((9H-fluoren-9-yl) methoxy)carbonyl)amino)-4-(benzyloxy)-3-hydroxy-4-oxo-butanoic acid as a white solid, in a yield of 96%. 4.3.1.2.4 Synthesis of 3,4;5,6-di-O-Isopropylidene-2-Deoxy-2(Fmoc-Amino)-D-Mannonic Acid [89]

In a dry 100 mL round bottom flask, were added 1.24 g of methyl 3,4;5,6-di-O-isopropylidene-2-amino-2-deoxy-D-mannate (i.e., methyl (S)-2-amino-2-((4S, 4’R,5R)-2,2,2’,2’-tetramethyl-[4,4’-bi(1,3-dioxolan)]5-yl)acetate, 4.29 mmol), 20 mL of THF and 5 mL of MeOH, followed by a solution of 215.8 mg of LiOH⋅H2O (5.14 mmol) in 5 mL of water. This light-yellow solution was stirred at room temperature for 2 hours. TLC (10% methanol in methylene chloride) showed that all of the starting material had been consumed and the product had been formed. THF/MeOH was removed under reduced pressure. The aqueous phase was re-suspended in 10 mL saturated NaHCO3 solution. To this suspension was added 1.74 g of Fmoc-OSu (5.14 mmol) in 10 mL of 1,4-dioxane. The resulting heterogeneous solution was stirred at room temperature for 18 hours. TLC (10% methanol in methylene chloride) showed that most of the starting material had been consumed and product had been formed. Dioxane was removed under reduced pressure. The aqueous layer was extracted with diethyl ether to remove the less polar impurities. Then the aqueous layer was acidified to pH 6 using 0.2 N HCl, and re-extracted with EtOAc. The EtOAc layer was washed with brine, dried over Na2SO4, and concentrated to yield 1.6 g of 3,4;5,6-di-O-isopropylidene-2-deoxy-2-(Fmoc-amino)-D-mannonic acid, i.e., (S)-2-((((9H-fluoren-9-yl)methoxy)-carbonyl)amino)-2-((4S,4’R,5R)2,2,2’,2’-tetramethyl-[4,4’-bi(1,3-dioxolan)]-5-yl)-acetic acid, in a yield of 76%.

210

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.3.1.3 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF THE FMOC GROUP 4.3.1.3.1 Liquid Ammonia Cleavage of 9-Fluorenylmethoxycarbonyl-L-Phenylalanine [75]

A solution of 1.55 g of Fmoc-L-Phe-OH in 250 mL of liquid ammonia was stirred for 10 hours and evaporated to dryness, then, 200 mL of ether was added to the residue. Dibenzofulvene could be isolated from the ether solution in 95% yield as the dibromide. The insoluble portion was filtered and dissolved in a minimum amount of H2O. Filtration removed a trace amount of dibenzofulvene polymer and evaporation of the filtrate gave 0.66 g of L-phenylalanine, in a yield of 100%. 4.3.1.3.2 Cleavage of Fmoc by DABCO to Prepare (3-Clicylamino-2Methylenepropanoyl)-L-Phenylalaninyl-L-Leucine Methyl Ester [77]

To a solution of 505 mg of [3-(Fmoc-glicylamino)-2-methylenepropanoyl]L-phenylalaninyl-L-leucine methyl ester (0.77 mmol) in 10 mL of dry CH2Cl2, was added 87 mg of DABCO (0.77 mmol) and the clear solution was refluxed for 6 hours and stirred for additional 12 hours at room temperature. After removal of the solvent under reduced pressure, the residue was purified by silica gel chromatography (EtOAc: MeOH = 9: 1) to give 0.32 g of methyl (2-((2-aminoacetamido)methyl)acryloyl)-L-phenylalanyl-L-leucinate, also known as (3-clicylamino-2-methylenepropanoyl)-L-phenylalaninyl-Lleucine methyl ester as a white solid, in a yield of 94%, m.p. 47–48°C.

Amino Protecting Groups 211

4.3.1.3.3 Deprotection of Fmoc with DBU for the Preparation of Peptide H-Gly-L-Leu-L-Cys-S-(Z-L-Ala)-OH [78]

A peptide of N-(((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-leucylS-(((benzyloxy)carbonyl)-L-alanyl)-L-cysteine was dissolved in dry THF at 0°C under argon. Then 2 equivalents of DBU was added dropwise. The solution was stirred for 15 minutes, and THF was evaporated. The sticky solid was dissolved in 2 N HCl, and the pH of the solution was adjusted to 5 using Na2HPO4. The solid formed was filtered off, washed with water, methanol, and diethyl ether, and dried to give 90% of S-(((benzyloxy)carbonyl)-Lalanyl)-N-glycyl-L-leucyl-L-cysteine as white microcrystals, in a yield of 90%, m.p. 200–202°C. 4.3.1.3.4 Deprotection of Fmoc-Gly-Pz(H)-OEt by 20% Piperidine in DMF [93]

To 2 mL of 20% piperidine in DMF was added 200 mg of ethyl 5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-1H-pyrazole-3-carboxylate (0.46 mmol), and the solution was stirred at ambient temperature for 15 minutes. The solvent was removed in vacuo and the resulting solid was absorbed onto silica gel. After chromatographic purification over silica gel eluting with CH2Cl2: MeOH: NH3 (80: 20: 1), 91 mg of ethyl 5-(2-aminoacetamido)-1H-pyrazole-3-carboxylate was obtained as a colorless solid, in a yield of 93%, Rf = 0.54, m.p. 188°C (dec.). 4.3.2 METHYLSULFONYLETHYLOXYCARBONYL (MSC) GROUP Methylsulfonylethyloxycarbonyl, also known as Msc, was introduced following its prototype 2-tosylethoxycarbonyl group (Tec) that was reported in 1964 [94]. Compared to its prototype Tec, the main characteristics of

212

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Msc include extreme acid stability, high base lability and a high polarity (or hydrophilicity) which enhances solubility in polar solvents including water [95]. Thus, Msc is favored when high solubility in aqueous media is desired. In addition, amino acids and peptides protected with Msc have good crystallizing properties and resist catalytic hydrogenolysis without affecting the catalytic efficiency of the catalyst during the removal of other protecting groups (e.g., Bz) [95]. Msc can be readily introduced into amino acids and peptides by means of mixed carbonates Msc-OSu or Msc-ONp, especially for compounds with low reactivity, e.g., S-trityl-cysteine or peptides having this residue in N-terminal position [95]. Also, Msc-azide is a good substrate for mounting Msc group to amino acids and peptides. The resulting Mscderivatives can be isolated via extraction with EtOAc and water. On the other hand, Msc can be easily deprotected with base, in only minutes in NaOH solution of pH 10–12 at 0°C in the presence of MeOH where MeOH traps the byproduct of vinyl sulfone [95], or even 15 seconds [96], or to an extreme extent within 5 seconds with 1.0 N NaOH or NaOCH3 [95]. It is reported that the most suited reagent for the complete and rapid removal of the Msc group from amino acid or peptide derivatives was the mixture of dioxane/ methanol/4 N NaOH in a ratio of 7.5/ 2.25/0.25 [95]. For a special case, Msc was specifically introduced into the NH2 groups of α1 glycine and β29 lysine of insulin, which has increased the solubility of insulin in polar solvents, facilitated the purification of Msc2-insulin by partition chromatography [97]. Other applications of Msc as the amino protecting group can be found elsewhere [98–102]. 4.3.2.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF MSC REAGENTS 4.3.2.1.1 Preparation of Methylsulfonylethyloxycarbonyl Chloride (Msc-Cl) [95]

To a solution of 12.4 g of 2-(methylsulfonyl)ethan-1-ol (100 mmol) in 25 mL of dry tetrahydrofuran cooled at –15°C, was added 12 mL of liquid phosgene quickly. The flask was closed with a ground joint bearing an open glass valve and the cooling bath was removed. The temperature to

Amino Protecting Groups 213

about 40°C due to an exothermic reaction. After about 2 hours, the outlet valve was connected to a water aspirator and the solution was evaporated to dryness. A pale yellow-colored oil remained, which was dissolved in 15 mL of tetrahydrofuran and cooled in ice. The product crystallized upon addition of 25 mL of dry ethyl ether giving 14.9 g of white, nearly odorless methylsulfonylethyloxycarbonyl chloride (Msc-Cl), in a yield of 80%, m.p. 49.0–49.5°C. 4.3.2.1.2 Preparation of Methylsulfonylethyl p-Nitrophenyl Carbonate (Msc-ONp) [95]

To a solution of 80.5 g of 2-(methylsulfonyl)ethanol (648 mmol) in 200 mL of absolute pyridine cooled at 0°C was added 118.9 g of p-nitrophenyl chloroformate (590 mmol) under stirring. The mixture was left for 5 hours at room temperature and then concentrated in vacuo. The thick syrup was poured into 1 L of 1.5 N hydrochloride acid. The product, which crystallized immediately, was collected by filtration and was washed with isopropyl alcohol (4 × 50 mL) and subsequently with diisopropyl ether (3 × 50 mL). A total of 128 g of 2-(methylsulfonyl)ethyl (4-nitrophenyl) carbonate was obtained, in a yield of 75%, m.p. 102°C. 4.3.2.1.3 Preparation of Methylsulfonylethyl Succinimido Carbonate (Msc-OSu) [95]

To a solution of 5.75 g of N-hydroxysuccinimide (50 mmol) in 50 mL of dry, freshly distilled acetonitrile, was added 7 mL of triethylamine (50 mmol), and the mixture was cooled to 0°C. A solution of 9.35 g of Msc chloride (50 mmol) in 50 mL of dry acetonitrile was added dropwise under stirring. A precipitate (Et3N·HCl) that gradually formed was filtered after about 15 minutes. The filtrate was evaporated and the residue was taken up in warm isopropyl alcohol (about 40°C). Filtration gave 11.4 g

214

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

of methylsulfonylethyl succinimido carbonate (also known as 2,5-dioxopyrrolidin-1-yl (2-(methylsulfonyl)ethyl) carbonate) as a white solid, in a yield of 86%, m.p. 112°C. The compound was crystallized from acetone by the addition of an equal volume of diisopropyl ether, or from acetonitrile, m.p. 114°C. 4.3.2.1.4 Preparation of Methylsulfonylethyloxycarbonyl Azide (Msc-N3) [95]

To a solution of 1.0 g of 2-(methylsulfonyl)ethyl chloroformate (5.36 mmol) in a mixture of EtOAc (2 mL) and ether (5 mL) was added 1.0 g of NaN3 (15.4 mmol) in 3 mL of water. The two-phase system was stirred for 5 hours and then separated. The aqueous layer was extracted with EtOAc and discarded. The combined organic phases were washed with water and dried over Na2SO4. After evaporation of the solvent, the residue solidified to afford 845 mg of methylsulfonylethyl-oxycarbonyl azide, in a yield of 82%. Crystallization from EtOAc (4 mL) and ether (5 mL) gave 705 mg of perfectly colorless crystals, m.p. 41.5°C. 4.3.2.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR INTRODUCTION OF MSC GROUP 4.3.2.2.1 General Procedure for the Preparation of Methylsulfonylethyloxy-Carbonyl Amino Acid [95]

To a suspension or solution of 20 mmol of an amino acid in 25 mL of an acetonitrile-water mixture (4:1), was added 5.78 g of solid methylsulfonylethyl p-nitrophenyl carbonate (20 mmol). The reaction was started by the addition of 2.8 mL of triethylamine (20 mmol) and the mixture was left for a time interval dependent upon the reactivity of the amino acid. Sometimes a small excess of the acylating agent was used. Nonhomogeneous systems were stirred. At the end of the acylation period,

Amino Protecting Groups 215

the yellow reaction mixture was filtered, if necessary, and concentrated in vacuo. For the removal of p-nitrophenol, the residue was dissolved in water, and the solution was acidified to pH 5. The solution was repeatedly extracted with ether and processed further, depending on the solubility of the product formed. 4.3.2.2.2 Preparation of Nε-Methylsulfonylethyloxycarbonyl-LLysine [95]

After the solution of 1.83 g of lysine monohydrochloride (10 mmol) in about 50 mL of water containing 1.25 g of copper sulfate (5 mmol) was cooled to 4°C, the pH of the blue solution was increased from 2.2 to 10.8 by careful addition of 5 mL of 4 N NaOH (20 mmol). Maintaining the pH in an auto-titrator loaded with 4 N NaOH, a solution of 2.24 g of Msc chloride (12 mmol) in 7.5 mL of dry THF was added dropwise to the mixture. NaOH was rapidly consumed and a precipitate formed. The acylation was complete in about 20 minutes. The suspension was centrifuged and the blue precipitate was washed with water and alcohol and dried with ether. The dry complex, obtained in 85.6% yield, was dissolved in 11 mL of 2 N HCl (22 mmol) and diluted with 30 mL of water. The resulting green solution was saturated with hydrogen sulfide. After about 2 hours the suspension was freed from H2S by the passage of a current of nitrogen and was then filtered. The acidic solution was filtered through a weakly basic ion-exchange resin (Merck II, OH-cycle) to remove HCl. Upon concentration of the neutral eluent, the product crystallized. On recrystallization from water, the compound was obtained as glistening blades. The lysine derivative was stable in 1 N HCl at room temperature in the pH-stat, whereas at pH 10.5, the Msc-group was slowly lost during the course of 24 hours.

216

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.3.2.2.3 Preparation of Methylsulfonylethyloxycarbonyl (Msc)Octadeuterophenylalanine [99]

Octadeuterophenylalanine (100 mg) was slowly dissolved in 3 mL of dimethylsulfoxide (DMSO), and 100 µL of N-ethylmorpholine were added to obtain a pH of 8.8. Msc-N-hydroxysuccinimide ester (350 mg, 2 equivalents) was added and the solution was stirred at room temperature. The reaction was followed by electrophoresis on paper at pH 6.5, and found to be quantitative after 1 hour. Then, 100 µL of acetic acid and 50 mL 0.1% trifluoroacetic acid (TFA) (w/v) were added to the reaction mixture and the product was purified on Sep-Pak-C18 cartridges (Waters Associates, Milford, MA, USA). After washing the cartridge with 10 mL of methanol and then 10 mL of 0.1% TFA, 8 mL portions of the reaction mixture were loaded, 10 mL 0.1% TFA was used to wash the cartridge and the product was eluted in 4 mL 50% acetonitrile containing 0.1% TFA. After washing with 10 mL of 80% acetonitrile and re-equilibration of the cartridge with 10 mL of 0.1% TFA, the loading and purification process was repeated seven times, and the fractions of the purified product were pooled, evaporated under a stream of nitrogen and freeze-dried. A white powder was obtained and stored at –20°C. 4.3.2.3 EXEMPLARY EXPERIMENTAL PROCEDURES FOR REMOVAL OF MSC GROUP 4.3.2.3.1 Deprotection of Msc-Protected Amines, Solvent Composition [95] The Msc group is quickly lost in a dioxane-methanol mixture containing sufficient aqueous NaOH to give a 0.1 or 0.2 N solution. The amount of water is critical and should be limited to concentration up to 5% for maximum reaction rate. Methanol acts as a scavenger for methylsulfonyl-ethylene and the concentration of this constituent appears to influence the reaction rate profoundly. The best ratio is dioxane/methanol/(2 or 4 N) NaOH = 14: 5: 1, corresponding to a final base (CH3O– and OH–) concentration of 0.1 or 0.2 N.

Amino Protecting Groups 217

4.3.2.3.2 Deprotection of Methylsulfonylethyloxycarbonyl-LPhenylalanyl-L-Arginyl-L-Tryptophyl-Glycine Methyl Ester Hydrochloride [95]

Dioxane (dry, peroxide-free) and absolute methanol were mixed in a proportion of 14: 5 (v/v). Crude methylsulfonylethyloxycarbonyl-Lphenylalanyl-L-arginyl-L-tryptophyl-glycine methyl ester hydrochloride (766 mg, 1 mmol) was dissolved in about 10 mL of the above solvent mixture with vigorous stirring. Then 1 mL of 4 N NaOH (4 equiv.) in 10 mL of the same solvent mixture was immediately added. After 45 seconds, the reaction mixture was acidified to pH < 4 by the addition of HCl and the solution was concentrated in vacuo. The product was precipitated by careful addition of NaOH to pH 11. After filtration and drying, 508 mg of the crude free base were isolated, in a yield of 90%. The monohydrochloride was obtained, after the dissolution of the base in a small amount of a water-acetonitrile mixture (1:1) containing one equivalent of hydrochloric acid, by slow addition of acetonitrile and cooling, to afford 419 mg of L-phenylalanyl-L-arginyl-L-tryptophylglycine hydrochloride, in a yield of 69.7%.

4.3.3 2-CHLORO-3-INDENYLMETHYLOXYCARBONYL (CLIMOC) AND BENZ[F]INDEN-3-YLMETHYLOXY-CARBONYL (BIMOC) PROTECTING GROUPS Both 2-chloro-3-indenylmethyloxycarbonyl (CLIMOC) and BIMOC are base labile amino protecting groups that are more sensitive to the base than the Fmoc group [103]. For example, they can be deblocked about 10 times faster than the Fmoc analogs, and the scavenging reactions are about 250 times as fast as that in the treatment of Fmoc derivatives. Although these two protecting groups are stable to acids, they are not as stable as

218

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

the Fmoc derivatives. Like other amino protecting groups, both CLIMOC and BIMOC can be mounted to amino acids or peptides by means of the corresponding chloroformate, azidoformate, or mixed carbonate such as the succinimido carbonate. The CLIMOC derivatives are better used in methylene dichloride but not in DMF, whereas the BIMOC protected amino acids and peptides can be used in any common solvents including DMF. Unfortunately, except for the glycine derivative, most of the CLIMOC- and BIMOC-amino acid derivatives synthesized are oils that are difficult or impossible to crystallize [103]. 4.3.3.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF CLIMOC REAGENTS 4.3.3.1.1 Preparation of (2-Chloroinden-3-yl)Methyl Chloroformate [103] O

Cl

OH O Cl

O

+ Cl

Cl

THF Cl

To a solution of 18.0 g of (2-chloro-1H-inden-3-yl)methanol (100 mmol) in 100 mL of dry THF cooled at 0°C, was added 30.0 g of phosgene (303 mmol), and the resulting mixture was stirred at 0°C for 3 hours. Excess phosgene and solvent were removed under water pump vacuum at 0°C, and the residue was recrystallized from hexane to give 22.0 g of (2-chloroinden3-yl)methyl chloroformate, in a yield of 91%, m.p. 52–53°C. 4.3.3.1.2 Preparation of (2-Chloroinden-3-y1)Methyl Succinimido Carbonate [103]

Amino Protecting Groups 219

To a solution of 18.4 g of (2-chloroinden-3-yl)methyl chloroformate (75.6 mmol) in 200 mL of CHCl3, was added 22.4 g of the dicyclohexylamine salt of N-hydroxysuccinimide (75.6 mmol) portion-wise under stirring over a half-hour period. The mixture was stirred overnight at room temperature and filtered and the filtrate was washed twice each with 10% citric acid, 10% NaHCO3, and water. Removal of the solvent from the dried solution (MgSO4) gave a gold-colored oily residue, which was recrystallized from CHCl3-Et2O to give 24.1 g of (2-chloroinden-3-yl)methyl succinimido carbonate, in a yield of 80%, m.p. 152–154°C. 4.3.3.1.3 Preparation of Benz[f]Inden-3-Ylmethyl Chloroformate [103]

To an ice-cold, stirred solution of 7 mL of phosgene in 10 mL of dry THF was added dropwise a solution of 2.5 g of benz[f]indene-3-methanol (12.7 mmol) in 25 mL of dry THF within 1 hour. The reaction mixture was allowed to stand at 0°C for another 2.5 hours. Excess amounts of phosgene and solvent were removed from a water bath (25°C) with a water aspirator (10 mmHg). In order to remove traces of phosgene, portions of 25 mL pentane were added, and the solution was evaporated three times. Eventually, 3.1 g of crude benz[f]inden-3-ylmethyl chloroformate was obtained as a yellow solid, in a yield of 94%. Recrystallization twice from ether gave 2.0 g of the pure chloroformate as colorless crystals, in a yield of 61%, m.p. 56–57°C. 4.3.3.1.4 Preparation of Benz[f]Inden-3-Ylmethyl Azidoformate [103]

220

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To an ice-cold, stirred solution of 0.16 g of NaN3 (2.5 mmol) in 1.1 mL of H2O was added slowly a solution of 0.42 g of benz[f]inden-3-ylmethyl chloroformate (0.77 mmol) in 0.9 mL of acetone. The mixture was stirred in an ice bath for 2 hours and at room temperature for 2 hours. The reaction mixture was extracted twice with 20-mL portions of ether. The combined ether extracts were washed three times with 10-mL portions of H2O and dried over MgSO4. Removal of the solvent from a water bath (30°C) with a rotary evaporator (7 mmHg) gave 0.46 g of benz[f]inden-3-ylmethyl azidoformate, m.p. 49–53°C. Recrystallization twice from hexane gave 0.33 g of the azide as colorless crystals, in a yield of 81%, m.p. 58–59°C. 4.3.3.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PROTECTION OF AMINO ACID WITH CLIMOC OR BIMOC GROUP 4.3.3.2.1 Preparation of N-(2-Chloroinden-3Ylmethyloxycarbonyl)-Phenylalanine [103]

To a 150 mL 0.6 N Na2CO3 solution cooled to 10°C was added 8.25 g of phenylalanine (50 mmol), followed by a cold solution of 13 g of (2-chloroinden-3-yl)methyl chloroformate (57.6 mmol) in 150 mL of dioxane. The resulting mixture was stirred in an ice-water bath for 15 minutes. The sodium salt which separated was filtered, washed with several portions of ether, suspended in water, and acidified with concentrated HCl to give 17.0 g of the free acid, in a yield of 92%. Because it was difficult to remove a persistent impurity, presumably CLIMOC-Phe-Phe-OH, a sample (2.5 g) of the crude product was purified on a Waters Prep-500 HPLC instrument using a C18 silica gel cartridge. Isocratic elution via MeOH-H2O-HOAc (70:29:1) gave the pure amino acid, which was collected in a volume of 2 L (flow rate 100 mL/min). Rotary evaporation gave 2.38 g (95% recovery) of the acid, which after recrystallization from ethanol was obtained as the ethanol complex, m.p. 113–135°C (softening at 75°C).

Amino Protecting Groups 221

4.3.3.2.2 Preparation of N-(Benz[f]Inden-3-Ylmethyloxycarbonyl) Phenylalanine [103]

To an ice-cold, stirred solution of 0.200 g of benz[f]inden-3-ylmethyl azidoformate (0.75 mmol) in 7.5 mL of THF was added very slowly an ice-cold solution of 0.105 g phenylalanine (0.64 mmol) in 3 mL of 10% aqueous Na2CO3 solution. The reaction mixture was stirred at 0°C for 96 hours and then treated with 20 mL of water. The aqueous solution was extracted three times with 10-mL portions of ether, cooled in an ice bath, and acidified with 10% aqueous HCl solution to Congo red. The resulting white precipitate was suction filtered to afford 220 mg of N-(benz[f]inden3-ylmethylo-xycarbonyl)phenylalanine, also known as (((1H-cyclopenta[b] naphthalen-3-yl)methoxy)carbonyl)-L-phenylalanine, in a yield of 76%, m.p. 176–177°C. Recrystallization from MeOH-H2O (3:1) gave 205 mg of pale yellow crystals as a methanol complex of the protected acid, in a yield of 70.2%, m.p. 191.5–192.5°C. 4.3.4 BENZOTHIOPHENESULFONE-2-METHYLOXYCARBONYL (BSMOC), 2-TERT-BUTYLSULFONYL-2-PROPENOXYCARBONYL (BSPOC) AND 2-METHYLSULFONYL-3-PHENYL-1-PROP-2ENYLOXY-CARBONYL (MSPOC) PROTECTING GROUPS These three related amino protecting groups, i.e., benzothiophenesulfone-2-methyloxycarbonyl (Bsmoc) [104], 2-tert-butylsulfonyl2-propenoxycarbonyl (Bspoc) [105], and 2-methylsulfonyl-3-phenyl1-prop-2-enyloxycarbonyl (Mspoc) [105], share a common structural feature that a Michael acceptor is incorporated into the structural scaffold so that treatment of the protected amino acids and peptides with these groups by a nucleophile will trigger the deprotection [106]. The common structural moiety among these protecting groups is the α,β-unsaturated sulfonyl group. It is reported that the reactivity order of the Michael addition decreases among the Michael acceptors containing the following electron-withdrawing groups, i.e., C6H5SO2 > Me3CSO2 > COOEt > C6H5SO > C6H4NO2-p [106]. However, as there is no additional group at the β position of Bspoc, and it

222

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

is known that the β-substituents in the Michael acceptor causes significant retardation of the Michael addition, amino acids and peptides protected with Bspoc are often suffered from the premature deprotection by the liberated amino group during the deprotecting process [105]. Thus, for both Msmoc and Mspoc with additional substituent at the β position, the corresponding derivatives yield clean free amine products during deprotection. However, they differ from each other regarding the byproducts formed upon piperidine induced deblocking. In the case of Bsmoc, the initial Michael-like adduct quickly isomerizes completely to the final stable adduct, whereas in the case of Mspoc, an equilibrium mixture of the two adducts is formed [105]. On the other hand, the reactivity of the nucleophile (e.g., primary, and secondary aliphatic amines) follows an order related to both intrinsic basicity and steric effects [106], as indicated in a dramatic difference in deblocking rate upon substitution of pyrrolidine for piperidine [105]. Bsmoc and Mspoc, being easily removed with base, are advantageous over Msc and Fmoc that involve β-elimination in the deblocking process, such as lower concentrations of piperidine or weaker bases (e.g., morpholine) required with minimal level of base-catalyzed side reactions, and suitability for rapid continuous solution synthesis of peptides [105]. Especially, the Bsmoc amino protecting group offers a significant improvement over the corresponding Fmoc-based method for rapid solution synthesis due to the opportunity to use water or saturated sodium chloride solution rather than an acidic phosphate buffer to remove all byproducts, with consequent cleaner phase separation and higher yields of the growing peptide [104]. For this case, the deprotecting process can involve the use of insoluble piperazino silica as well as the polyamine tris(2-aminoethyl)amine (TAEA, also known as N1,N1-bis(2-aminoethyl) ethane-1,2-diamine) which simplifies aqueous separation of the growing, but non-isolated peptide product, from excess acylating agent and other side products formed in the deblocking process [104]. 4.3.4.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF BSMOC, BSPOC, AND MSPOC REAGENTS 4.3.4.1.1 Preparation of Benzo[b]Thiophen-2-Methanol [104]

Amino Protecting Groups 223

While the solution of 45.5 g of benzo[b]thiophene (0.34 mol) in 225 mL of dry THF was cooled to –30°C under an atmosphere of N2, 330 mL of n-BuLi (1.6 M in hexane, 1.5 equiv.) was added dropwise over 2 hours with mechanical stirring. The dark blue solution was allowed to warm to 0°C and then re-cooled to –78°C. Then, 72.0 g of paraformaldehyde (2.4 mol) was added in small portions over 20 minutes, and the mixture was allowed to warm to room temperature overnight. Caution! A considerable amount of gas was evolved on warming, especially when the temperature reached 10–20°C. The apparatus should be well ventilated and stirring was maintained, otherwise, the mixture may boil uncontrollably. The mixture was acidified to pH ≈ 3 with 3 N HCl, the layers were separated, and the aqueous phase was extracted twice with 100 mL portions of ether. The solid remaining in the flask was washed with three 150 mL portions of ether, all ether extracts were combined and dried over MgSO4, and the solvent was removed by rotary evaporation. The residual oil was dissolved in 600 mL of CH2Cl2, and the solution was washed with saturated NaHCO3 (2 × 100 mL), saturated NaCl (100 mL), and dried over MgSO4. The solution was filtered, and the solvent was removed to give an oil that was recrystallized from CH2Cl2/hexane (4:1) to give 42.4 g of benzo[b]thiophen-2-methanol as a white solid, in a yield of 70%, m.p. 68–70°C. 4.3.4.1.2 Preparation of Benzothiophenesulfone-2-Methanol [104]

To a stirred solution of 20.36 g of benzo[b]thiophen-2-methanol (0.124 mol) in 500 mL of acetic acid held at 45–50°C was added 95.4 g of sodium perborate tetrahydrate (0.62 mol) portion-wise over a period of 20 minutes. Stirring was continued at this temperature until the completion of the oxidation (TLC). The acetic acid was removed by evaporation under reduced pressure, and the residue was stirred with 150 mL of water. The precipitated sulfone alcohol was collected by filtration and recrystallized from chloroform/hexane or dichloromethane/hexane to give 2-(hydroxymethyl)benzo[b] thiophene 1,1-dioxide in about 75% yield. The alcohol was obtained after recrystallization from CHCl3/hexane (4:1) in the form of white crystals, m.p. 113–114°C.

224

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.3.4.1.3 Preparation of Benzothiophenesulfone-2-Methyl Chloroformate (Bsmoc-Cl) [104]

To a stirred solution of 30.8 g of 2-(hydroxymethyl)benzo[b]thiophene 1,1-dioxide (0.16 mol) in 400 mL of dry THF cooled to –30°C in a dry iceacetone bath was added 104 g of phosgene (80 mL, 1.05 mmol) in one portion which had been condensed into a graduated cylinder. The solution was allowed to warm to room temperature overnight. Excess phosgene and solvent were removed with the aid of a water aspirator, the process was repeated using additional solvent: 200 mL of THF, 200 mL of THF/ether (1:1), and 200 mL of ether. Recrystallization of the residue from dry ether gave 36.1 g of benzothiophenesulfone-2-methyl chloroformate as white crystals, m.p. 76–77°C. 4.3.4.1.4 Preparation of N-(Benzothiophenesulfone-2-Methyl)-NSuccinimidyl Carbonate (Bsmoc-OSu) [104]

To a solution of 8.98 g Bsmoc-Cl (34.8 mmol) in 100 mL of dry CH2Cl2 that was protected from moisture (CaSO4), was added 10.3 g of dicyclohexylamine salt of N-hydroxysuccinimide (34.8 mmol) under stirring. After stirring for 20 hours, the solution was filtered, and the amine salt was washed with two 75-mL portions of CH2Cl2. The combined organic filtrates were washed with saturated NaHCO3 (2 × 100 mL) and saturated NaCl (2 × 100 mL), dried over MgSO4, filtered, and treated with decolorizing carbon. Filtration and evaporation of solvent gave 9.6 g of crude N-(benzothiophenesulfone-2-methyl)-Nsuccinimidyl carbonate (also known as (1,1-dioxidobenzo[b]thiophen-2-yl) methyl (2,5-dioxopyrrolidin-1-yl) carbonate), in a yield of 81%. The product was further recrystallized from CH2Cl2/hexane or EtOAc/hexane to afford 8.6 g of the carbonate as white crystals (74%), m.p. 170–172°C.

Amino Protecting Groups 225

4.3.4.1.5 Preparation of (E)-3-(Methylsulfonyl)-3-Phenyl-2Propenyl Alcohol [105]

The mixture of 25.6 g of (E)-(3-bromo-2-(methylsulfonyl)prop-1-en-1-yl) benzene (93.7 mmol) (prepared from the copper(II)-catalyzed addition of methanesulfonyl chloride to 1-phenylpropene followed by dehydrochlorination of the adduct with N-methylpiperidine in benzene to give (E)-(2-(methylsulfonyl) prop-1-en-1-yl)benzene and its subsequent radical bromination with NBS in CCl4) [276] and 15.82 g of sodium formate (0.23 mol) in 300 mL of methanol were refluxed for 6 hours. When the starting material had disappeared (TLC), the mixture was allowed to cool and concentrate with the aid of a water aspirator. The residue was diluted with water and extracted several times with 100 mL portions of CH2Cl2. The organic layer was dried over MgSO4, the solvent was removed in vacuo, and the crude alcohol was recrystallized from CHCl3hexane to give 15.8 g of the pure (E)-2-(methylsulfonyl)-3-phenylprop-2-en1-ol as white crystals, in a yield of 80.2%, m.p. 74–76°C. 4.3.4.1.6 Preparation of (E)-2-(Methylsulfonyl)-3-Phenyl-2Propenyl Chloroformate (Mspoc-Cl) [105]

To a stirred solution of 8.5 g (E)-2-(methylsulfonyl)-3-phenyl-2-propenyl alcohol (0.04 mol) in 50 mL of THF at 0°C was added in one portion of 37.5 mL phosgene. The reaction mixture was stirred overnight at room temperature, and excess phosgene and solvent were removed under reduced pressure with the aid of a water aspirator. The crude material was recrystallized from

226

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

CH2Cl2/hexane to give 9.5 g of (E)-2-(methylsulfonyl)-3-phenyl-2-propenyl chloroformate as colorless crystals, in a yield of 86.4%, m.p. 118–120°C. 4.3.4.1.7 Preparation of N-[(E)-2-(Methylsulfonyl)-3-Phenyl-2Propenyloxy-Carbonyloxy] Succinimide (Mspoc-OSu) [105]

To a stirred solution of 13.74 g of Mspoc-Cl (50 mmol) in 150 mL of CH2Cl2 was added 14.8 g of the dicyclohexylamine salt of N-hydroxysuccinimide (50 mmol) portion-wise. The reaction mixture was stirred overnight and filtered, and the precipitate was washed with CH2Cl2. The filtrate was washed with 10% citric acid (100 mL), 10% NaHCO3 (100 mL), and water (100 mL). The organic layer was dried (MgSO4), and the solvent was removed in vacuo to give the crude N-[(E)-2-(methylsulfonyl)-3-phenyl-2-propenyloxycarbonyloxy] succinimide, also known as (E)-2,5-dioxopyrrolidin-1-yl (2-(methylsulfonyl)-3-phenylallyl) carbonate, which was recrystallized from CH2Cl2/hexane to give 13.24 g of Mspoc-OSu as white crystals, in a yield of 75%, m.p. 152–154°C. 4.3.4.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PROTECTION OF AMINO ACIDS 4.3.4.2.1 General Procedures for the Preparation of Benzothiophenesulfone-2-Methoxycarbonyl Amino Acids [104]

Amino Protecting Groups 227

To a suspension of 3.87 mmol of an amino acid in 20 mL of CH2Cl2 was added in one portion 0.98 mL of chlorotrimethylsilane (7.73 mmol). The mixture was then refluxed for 1 hour and cooled in an ice bath. Diisopropylethylamine (1.3 mL, 7.31 mmol) was added slowly followed by 1 g of benzothiophenesulfone-2-methyl chloroformate (3.87 mmol). The reaction mixture was allowed to stand at 0°C for 20 minutes and then for 1–1.5 hours at room temperature. The solvent was removed in vacuo and the resulting oil was distributed between 40 mL of ether and 80 mL of 2.5% NaHCO3 solution. The combined aqueous layers were acidified to pH 2 with concentrated HCl and extracted with three 30-mL portions of EtOAc. The extracts were combined and washed with 30 mL of saturated NaCl and 30 mL of water, dried over MgSO4, and filtered. Upon evaporation of solvent, the white residue or oil was recrystallized from the appropriate solvent or solvent mixture to give the corresponding Bsmoc-protected amino acids. 4.3.4.2.2 Preparation of Bsmoc-Protected Amino Acids via Bsmoc-Osu in Acetonitrile-Water in the Presence of Triethylamine [104]

To a suspension of 2.96 mmol of the amino acid in 20 mL of acetonitrile/water (1/1) was added triethylamine to give an apparent pH of 9.0. The suspension became clear. After the addition of 1 g of Bsmoc-OSu (2.96 mmol), the pH was kept at 8.5–9.0 by adding triethylamine. The uptake of base ceased after about 15 minutes. The reaction mixture was stirred at room temperature for 40–45 minutes, acidified to pH 5 with 0.1 N HCl or 10% KHSO4, and concentrated in vacuo. The mixture was diluted with about 5 mL of water and acidified to pH 2 with 0.1 N HCl. The mixture was extracted with four 25-mL portions of ethyl acetate. The combined ethyl acetate extracts were washed several times with saturated NaCl solution, dried over MgSO4, filtered, and the solvent was removed in vacuo. The residue was recrystallized from the appropriate solvent or solvent mixture to give the corresponding Bsmoc-protected amino acids.

228

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.3.4.2.3 Preparation of Bsmoc-Protected Amino Acids via BsmocOSu in Acetone-Water in the Presence of NaHCO3 [104]

To a solution of 1.48 mmol of amino acid and 0.248 g of NaHCO3 (2.95 mmol) in 10 mL of water was added a solution of benzothiophenesulfone2-methyl N-succinimidyl carbonate (1.48 mmol) in 10 mL of acetone. The reaction mixture was stirred overnight at room temperature, diluted with water, and extracted twice with CH2Cl2 to remove a small amount of benzothiophenesulfone-2-methanol and unreacted Bsmoc-OSu. The aqueous layer was cooled in an ice bath and acidified with concentrated HCl to pH 2. The resulting white precipitate or oil was extracted with three 25-mL portions of EtOAc. The combined organic layer was washed with 30 mL brine and then 30 mL of water and dried over MgSO4. Upon filtration, the solvent was removed in vacuo. The residue was recrystallized from the appropriate solvent or solvent mixture to give the corresponding Bsmoc-protected amino acids. 4.3.4.2.4 Preparation of Mspoc-Phenylalanine from Mspoc-OSu [105]

To a stirred solution of 1.65 g of phenylalanine (10 mmol) and 2.10 g of NaHCO3 (25 mmol) in 40 mL of water, was added a solution of 3.53 g of Mspoc-OSu (10 mmol) in 50 mL of acetone. After stirring for 3–5 hours or overnight (TLC), the reaction mixture was extracted with CH2Cl2 to remove unreacted Mspoc-OSu. The acetone was removed by means of a

Amino Protecting Groups 229

water aspirator. After cooling, the reaction mixture was acidified with 10% HCl to Congo red to give a white solid, which was filtered and washed with water several times to give the crude Mspoc-Phe-OH. Recrystallization from EtOAc/hexane gave the pure acid, m.p. 166–168°C. 4.3.4.2.5 Preparation of tert-Butyl N-(2-(tert-Butylsulfonyl)-2Propenyl)Phenylalaninate [106]

A solution of 0.494 g of 2-(tert-butylsulfonyl)-2-propenyl chloroformate (Bspoc-Cl, 2.05 mmol) and 0.623 g of tert-butyl phenylalaninate hydrophosphite (2.05 mmol) in 20 mL of CH2Cl2 was stirred with 25 mL of 5% NaHCO3 at room temperature for 3 hours. The aqueous phase was separated, and the organic phase was washed with two 25 mL portions of 5% HCl. The organic layer was dried over MgSO4 and filtered, and the solvent was removed in vacuo from a water bath at 45°C to give 0.87 g of pure tert-butyl (((2-(tert-butylsulfonyl)allyl)oxy)-carbonyl)-L-phenylalaninate as an oil, in a yield of 100%. To the oil was added 80 mg of tert-butyl phenylalaninate (0.36 mmol, 0.18 equiv.). After standing at room temperature for 48 hours, 20 mL of Skelly F (petroleum ether, 30–60°C) was added, and the solvent was removed in vacuo to give a white solid. Recrystallization from 50% Skelly B (n-hexane)/Skelly F gave 0.56 g of tert-butyl (((2-(tert-butylsulfonyl)allyl) oxy)carbonyl)-L-phenyl-alaninate as a colorless solid, in a yield of 72%, m.p. 58.5–59.0°C. 4.3.5 (9H-FLUOREN-9-YL)METHANESULFONYL GROUP (9H-Fluoren-9-yl)methanesulfonyl (Fms) has been developed to overcome the potential problem associated with the Fmoc group where the Fmoc protected amino acids or amino phosphonic acids might be cleaved under certain conditions by the nucleophilic attack of the oxygen atom on the carbamate moiety onto the free carboxyl or phosphonyl group to give an oxazoline-like derivatives [107]. This potential problem becomes more significant in the synthesis of α-amino phosphonic acid containing peptides,

230

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

probably due to the high affinity of oxygen for phosphorus that greatly facilitates such cyclization [74]. Fms can be mounted into amino groups by means of Fms-Cl, which can be stored in a general vial at room temperature for at least six months without significant decomposition. It can be applied to protect various amino groups, either from free amines, α-amino carboxylic esters or α-amino phosphonic esters. Smooth and quantitative removal of the Fms group can be attained under conditions similar to the removal of Fmoc. The advantages of Fms over Fmoc have been demonstrated in the dehydrative condensation between an N-Fms-protected α-amino phosphoalanine monoester and an α-substituted α-amino carboxylic ester and amide [74]. 4.3.5.1 PREPARATION OF (9H-FLUOREN-9-YL)METHANESULFONYL CHLORIDE (FMS-CL) [74]   Step A: Chlorination of (9H-Fluoren-9-yl)methanol:

(9H-Fluoren-9-yl)methanol (35.0 g, 0.178 mmol) was added to 500 mL of SOCl2 and the solution was heated at 80°C for 4 hours, then the remaining SOCl2 was removed by distillation, and 30.6 g of (9H-fluoren-9-yl)methyl chloride, also known as 9-(chloromethyl)-9H-fluorene, was obtained by vacuum distillation, in a yield of 80%. Step B: Preparation of (9H-fluoren-9-yl)methanesulfonic acid via   sulfonylation:

To a 500-mL round bottom flask equipped with reflux condenser were added a Teflon-coated magnetic stirring bar, 30.0 g of (9H-fluoren-9-yl) methyl chloride (0.140 mol), 300 mL of acetone, and 300 mL of saturated aqueous Na2SO3 solution. The mixture was stirred vigorously at 90°C for 8 hours, and then cooled to ambient temperature. After rough removal of acetone at 30°C and 80 mmHg, 100 mL of 6 M HCl was added in an ice bath until pH of the solution became ca. 1, resulting in precipitation of white solid. This was separated by filtration and washed with 200 mL of ether.

Amino Protecting Groups 231

The solid was dried at 40°C and 10 mmHg to afford 35.6 g of nearly pure (9H-fluoren-9-yl)methanesulfonic acid as a white solid, in a yield of 98.2%.   Step C: Preparation of (9H-fluoren-9-yl)methanesulfonyl chloride (Fms-Cl):

To a 500-mL round bottom flask equipped with a Teflon-coated magnetic stirring bar, were added 35.0 g of (9H-fluoren-9-yl)methanesulfonic acid (0.135 mol), 57.6 g of PCl5 (0.277 mol) and 500 mL of POCl3. The whole mixture was stirred at 25°C for 18 hours under a gentle argon stream, the outlet of which was connected to a trap containing KOH. After removal of POCl3 at 80°C and 10 mmHg followed by cooling to 0°C by an ice bath, 300 mL of saturated aqueous NaHCO3 was carefully added. The mixture was extracted by ethyl acetate (500 mL × 3). The combined organic layers were washed sequentially with 200 mL of water and brine, dried over 100 g of Na2SO4. Filtration and concentration under reduced pressure gave 33.2 g of (9H-fluoren-9-yl)methanesulfonyl chloride as a white solid, in a yield of 88.2%yield. The overall yield for the three consecutive steps for the preparation of (9H-fluoren-9-yl)methanesulfonyl chloride was 69.0%. 4.3.5.2 PREPARATION OF FMS-ALA-O-T-BU [74]

A 20-mL Young’s type Schlenk flask containing a Teflon-coated magnetic stirring bar was charged with 182 mg of H-Ala-O-t-Bu·HCl (1.00 mmol) and 5.0 mL of CH2Cl2. The whole mixture was cooled to 0°C by an ice bath, and 490 μL of diisopropylethylamine (3.00 mmol), and 418 mg of Fms-Cl (1.50 mmol) were introduced. The ice bath was removed and the flask was then immersed into a 30°C water bath. After 3 hours of stirring for the colorless solution, 2 mL of water was added. The organic layer was separated, and the aqueous layer was extracted by CH2Cl2 (2.0 mL × 2). The combined organic layers were washed with 2 mL of brine and dried over Na2SO4. Upon filtration

232

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

and subsequent evaporation under reduced pressure, the oily residue was purified by column chromatography with 25 g of CH2Cl2/hexane mixture (4:1) to afford 322 mg of tert-butyl (((9H-fluoren-9-yl)methyl)sulfonyl)-Lalaninate, i.e., Fms-Ala-O-t-Bu, as a white solid, in a yield of 83%. 4.3.6 2-(4-NITROPHENYL)SULFONYLETHOXYCARBONYL (NSC) AND 2-(4-NITROPHENYLTHIO)-ETHOXYCARBONYL (NTC) GROUPS These two amino protecting groups are base-labile amino protecting groups alternative to Fmoc for amino acids [92]. Compared to the Fmoc, Nsc has additional features that include moderate absorption at 380 nm which is excellent for real-time monitoring of the deprotection process, good stability of the protected peptides in DMF-based solvent systems (more than 24 hours) [92], and decreased rearrangement of X-Asp, which can be a serious problem in SPPS. The Nsc-protected amino acids are easily synthesized and commercially available crystalline compounds that are prerequisites for SPPS applications. While both Nsc and Ntc are base labile groups, Ntc is more stable than Nsc under basic conditions [108]. 4.3.6.1 PREPARATION OF 2-(4-NITROPHENYLTHIO) ETHOXYCARBONYL-L-SERINE METHYL ESTER [108]

HCl·H-Ser-OMe (77.8 g, 0.5 mol) prepared from 52.5 g of L-serine (0.5 mol), 43.8 mL of thionyl chloride (0.6 mol) and 1.5 L of methanol was dissolved in 500 mL of EtOH/water mixture (9: 1). To this solution was added 146 mL of triethylamine, and the resulting mixture was cooled in ice bath for 30 minutes. Then a solution of 130.2 g of Ntc-Cl (0.5 mol) in 300 mL of dioxane was added dropwise with vigorous stirring and kept for an additional 1 hour. The reaction mixture was quenched by 5 mL of AcOH, and concentrated. The residual oil was diluted with 300 mL of water, and extracted with EtOAc (500 mL × 2). The combined organic layer was washed with water (400 mL × 3) and brine (400 mL × 2), dried over anhydrous Na2SO4, and concentrated. The residue was crystallized from Et2O and

Amino Protecting Groups 233

hexane to afford 154 g of 2-(4-nitrophenylthio)-ethoxycarbonyl-L-serine methyl ester as a light yellow powder, in a yield of 90%, m.p. 98–99°C. 4.3.6.2 PREPARATION OF 2-(4-NITROPHENYLTHIO) ETHOXYCARBONYL-O-T-BUTYL-L-SERINE [108]

A mixture of 172.2 g of 2-(4-nitrophenylthio)-ethoxycarbonyl-L-serine methyl ester (0.5 mol), 20 mL BF3·Et 2O and 600 mL of CHCl3 was stirred under isobutylene gas (2.5 psi) at room temperature for 5 hours. Then, the reaction was quenched by 25 mL of 1 N NaOH, and evaporated to an oil. The residue was dissolved in 500 mL of MeOH, followed by portion-wise addition of 550 mL 1 N NaOH (1.1 eq.). After standing at room temperature for 2 hours, the mixture was concentrated to dryness. To the residue was added 400 mL of Et2O and 400 mL of water. The aqueous layer was washed with Et2O (300 mL × 2), acidified to pH 2 with 1 N HCl, and extracted with EtOAc (500 mL × 2). The organic layer was washed with water (400 mL × 3) and brine (400 mL × 2), dried over anhydrous MgSO4, and concentrated to an oil. The residue was crystallized from Et2O and petroleum ether to give 172.6 g of methyl O-(tert-butyl)-N-((2-((4-nitrophenyl)thio)ethoxy) carbonyl)-L-serinate as white powder, in a yield of 90%, m.p. 108–109°C. 4.3.6.3 PREPARATION OF 2-(4-NITROPHENYSULFONYL) ETHOXYCARBONYL-O-T-BUTYL-L-SERINE [108]

To a solution of 193.2 g of 2-(4-nitrophenylthio)ethoxycarbonyl-Ot-butyl-L-serine (0.5 mol) in 500 mL of acetone were added 200 mL of 0.3 M Na2MoO4 and 200 mL of 35% H2O2, and the reaction mixture was kept at room temperature for 5 hours. Then, the mixture was concentrated and distributed between 600 mL of EtOAc and 300 mL of water. The organic layer was washed with 1% NaHCO3 (300 mL × 2), water (400 mL × 3),

234

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

and 200 mL of brine, dried over anhydrous MgSO4. Upon concentration to dryness, the residue was crystallized from Et2O and hexane to give 197.6 g of methyl O-(tert-butyl)-N-((2-((4-nitrophenyl)sulfonyl)-ethoxy)-carbonyl)L-serinate as a white powder, in a yield of 95%, m.p. 109–111°C. 4.3.7 MISCELLANEOUS BASE-LABILE AMINO PROTECTING GROUPS Besides the above-mentioned base-labile amino protecting groups, there are still some base-labile amino protecting groups, although they are not as popular as Fmoc, t-Boc, Msc, etc. These amino protecting groups include 2,2-bis(4-nitrophenyl) ethan-1-oxycarbonyl (Bnpeoc) [109], 2-(4-chlorophenyl)sulfonyl-ethoxycarbonyl (Cps) [110], 4-isopropyloxycarbonyloxybenzyloxy-carbonyl (4-PriOCO) [111], 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc) [112], 2-(4-trifluoromethyl-phenylsulfonyl)ethoxycarbonyl (Tsc) [113], and Tbfmoc [114]. Among these not so commonly used amino protecting groups, 4-PriOCO can be removed in a slightly alkaline medium via a 1,6-elimination, by hydrogenolysis, or by HBr in acetic acid, which however can sustain under the treatment of TFA in CH2Cl2. For example, 4-PriOCO can be quickly removed completely in 0.1 N NaOH. Although its removal is slow in 5% K2CO3, 4-PriOCO can be deprotected up to 98% within 2 hours in the presence of 1 equivalent of hydrazine [111]. While Nsc-protected amino acids have greater chemical and thermal stability than the Fmoc-protected amino acids when Nsc is installed on the exocyclic amino group of the heteroaromatic pyrrole amino acid, the general utility of the Nsc group in the solid-phase synthesis of polyamides is hampered by the poor solubility of the Nsc-protected imidazole amino acid in organic solvents [113]. By contrast, Tsc is superior in solubility to Nsc and in stability to Fmoc for the protection of exocyclic (hetero)aromatic amines [113]. 4.4 PROTECTING GROUPS REMOVABLE BY HYDROGENOLYSIS The first amino protecting group that can be removed via hydrogenolysis is benzyloxycarbonyl (Cbz or Z) group, introduced by Bergmann and Zervas in 1932 [115]. The introduction of this protecting group has not only led to a new epoch in the history of peptide synthesis, but also introduced a new perspective to the conception of protecting group chemistry in organic synthesis as a whole. This protecting group has been widely employed for Nα protection in peptide synthesis, preferably for solution-phase synthesis,

Amino Protecting Groups 235

due to its stability, facile introduction and removal conditions and minimum side reactions of the Z-protected amino acid derivatives. While catalyst poisoning may prevent its application in the preparation of peptides that contain cysteine, methionine, or other residues bearing divalent sulfur groups, this protecting group can also be removed by means of treatment with acid, such as HBr in acetic acid [116], TFA at high temperatures [117], TFA-thioanisole [118], 50% TFA in CH2Cl2 [119], liquid HF [120], and BBr3 [121]. Due to the side chain branching for the case of lysine, other groups that can be removed by hydrogenolysis have been developed, including p-nitrobenzyloxycarbonyl (pNZ) [122, 123], 2-chlorobenzyloxycarbonyl (2-ClZ) [124], 2,6-dichlorobenzyloxycarbonyl (2,6-Cl2Bzl), 2-bromobenzyloxycarbonyl (2-BrZ) [124, 125], 4,5-diaryl-4-oxazolin-2-one (e.g., 4,5-bis(p-methoxyphenyl)- and 4,5-bis(p-chlorophenyl)-4-oxazolin-2-one) [126], β-phenylethyloxycarbonyl group (“homobenzyloxycarbonyl” or “homocarbobenzoxy,” hZ) [127], isonicotinyloxycarbonyl [128], p-trimethylammonium chloridobenzyloxycarbonyl [129], isopropylideneaminooxycarbonyl (Paoc) [130], and 3-(3’,6’-dioxo-2’,4’,5’-trimethylcyclohexa-1’,4’-diene)-3,3-dimethylpropionyl (Q) [131]. These amino protecting groups are listed in Figure 4.3.

FIGURE 4.3 Amino protecting groups that can be removed by hydrogenolysis.

While benzyloxycarbonyl can be easily removed by the treatment of HBr in acetic acid, β-phenylethyloxycarbonyl is stable under this condition for

236

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

over a period of 24 hours at room temperature. Also, under normal hydrogenation conditions, benzyloxycarbonyl will be removed at atmospheric pressure over a palladium-carbon catalyst (10%), whilst β-phenylethyloxycarbonyl retains; both groups can be removed by means of catalytic transfer hydrogenolysis in the presence of ammonium formate [127]. For hydrogenolysis of benzyloxycarbonyl group with in situ generated molecular hydrogen by addition of triethylsilane (TES) to the palladium-charcoal catalyst, the benzyloxycarbonyl group can be removed in 5–10 minutes [132]. Likewise, 2-chlorobenzyloxycarbonyl (2-ClZ), 2,6-dichlorobenzyl-oxycarbonyl (2,6Cl2Bzl), and 2-bromobenzyloxycarbonyl (2-BrZ) are preferably removed by means of transfer hydrogenation, in methanol or DMF containing 10% palladium on carbon (1/10 to 1/5 weight of peptide) and 2 to 4 equivalents of ammonium formate at room temperature [124]. 4.4.1 BENZYLOXYCARBONYL (CBZ, BZ OR Z) GROUP Benzyloxycarbonyl (Cbz or Z) protecting group has been used by Bergmann and Zervas during the early days of rational peptide synthesis and is a milestone in modern peptide chemistry [133, 134]. This urethane-type protecting group is introduced into amino acids and related derivatives mainly under the Schotten-Baumann conditions using benzyl carbonochloridate [133] (also known as benzyl chloroformate [135], and benzyloxycarbonyl chloride [136]) as the acylating agent, which is readily accessible via reaction of benzyl alcohol with phosgene [133]. However, due to its low stability, thermal decomposition of benzyl chloroformate to benzyl chloride and carbonic anhydride can take place even at low temperature of storage, and a trace amount of hydrogen chloride can catalytically enhance the decomposition rates. In the worst case, it is found that when benzyl chloroformate was stored even for a short period of time and at low temperature, contents of up to 20% benzyl chloride had been detected [133]. Consequently, the preparation of highly pure benzyl chloroformate requires various precautions, such as the use of freshly distilled, benzaldehyde-, and peroxide-free benzyl alcohol, the quantitative removal of the hydrogen chloride formed in the reaction, and the exclusion of heavy metal ions even in a trace amount. Even under this condition, a small amount of benzyl chloride can still be detectable from the prepared benzyl chloroformate by NMR [133]. Thus, the preparation of Cbz-protected amino acid or peptide derivatives from Cbz-Cl is more or less contaminated with benzyl

Amino Protecting Groups 237

chloride, and is also accompanied by benzyloxycarbonyl-dipeptides to various extents. To overcome the intrinsic problem with benzyl chloroformate, a variety of alternative reagents have been developed to form benzyloxycarbonyl protected amino acid derivatives, including mixed benzyl carbonates with various phenols, with 1-hydroxypiperidine and N-hydroxysuccinimide, benzyloxycarbonyl-thiosulfate sodium salt, 1-benzyloxycarbonyl-3-methylimidazium chloride, 3-benzyloxycarbonyl-2-oxazolone, N-benzyloxycarbonyloxy-5-norbornene-2,3-dicarboximide, 3-benzyloxy-carbonylbenzo-xazoline-2-thione, 3-benzyloxy-carbonylbenzothiazoline-2-thione, 3-benzyloxycarbonylbenzimidazoline-2-thione, and 2-benzyloxycarbonyl-thiopyridine. However, none of these tested reagents is an efficient substitute for the benzyl chloroformate, partly because of the laborious or costly preparation of these reagents, lower acylating power or the formation of by-products [133]. It should be pointed out that dibenzyl dicarbonate [133] or dibenzylpyrocarbonate [137] is stable on storage and is well suited for the preparation of benzyloxycarbonyl amino acid derivatives, with a limited amount or no benzyloxycarbonyl dipeptide formed under special conditions [137]. From dibenzyl dicarbonate, equimolar amounts of amino acids can be converted into benzyloxycarbonyl amino acids under the Schotten-Baumann conditions, usually within 1 hour. Upon usual workup procedures, benzyloxycarbonyl amino acids or peptide derivatives are obtained in yields and homogeneity comparable with those only obtainable from the freshly prepared benzyl chloroformate of the best quality [133]. Also, benzyloxycarbonyl amino acid derivatives of good qualities can be prepared with 1-benzyloxycarbonyl-benzotriazole that is readily accessible from benzotriazole and benzyl carbonochloridate, although this reagent is less reactive and the corresponding reaction usually takes 24 hours [133]. Recently, it is found that in the presence of a catalytic amount of β-cyclodextrin (10%), good quality of N-benzyloxycarbonyl amino acid can be obtained in either aqueous or carbonate buffer (pH = 8) in reaction with benzyl chloroformate [138]. The benzyloxycarbonyl group is often removed via catalytic hydrogenation [139], which is also removable by acidic hydrolysis or alkaline hydrolysis [111]. It has been reported that the catalyst poisoning during hydrogenation can be greatly diminished when liquid ammonia is used as a solvent for palladium-catalyzed hydrogenation. This solvent enables quantitative cleavage of Nα-benzyloxy-carbonyl groups on many protected peptides bearing S-protected cysteine residues [140].

238

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.4.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PREPARATION OF CBZ REAGENTS 4.4.1.1.1 Preparation of Dibenzyl Dicarbonate [133]

Benzyl chloroformate (510 g, 3 mol) is reacted with 240 g of sodium benzylcarbonate (1.52 mol) at room temperature. After being stirred for 4–8 hours, ice water was added. The organic phase was separated and excess benzyl chloroformate was removed by distillation. The oily residue crystallized in the cold from diisopropyl ether to afford 230 g of dibenzyl dicarbonate, in a yield of 53%, m.p. 25–30°C. 4.4.1.1.2 Preparation of Dibenzyl Dicarbonate [137]

To a solution of 21.6 g of benzyl alcohol (0.2 mol) in 200 mL of THF was added 4.8 g of sodium hydride (0.2 mol), and the solution was refluxed for 2 hours. The solution was then cooled to room temperature and CO2 was bubbled in while maintaining efficient stirring and external cooling. After one hour, 28.5 mL of benzyl chloroformate (0.2 mol) was slowly added and the mixture was stirred for three hours at room temperature. The precipitate was centrifuged and the supernatant was evaporated to dryness under vacuum. The resulting oil slowly crystallized in the refrigerator. Crystals were then triturated with cold hexane, filtered, and dried under vacuum to afford 45.8 g of dibenzyl dicarbonate, in a yield of 79%, m.p. 28°C. 4.4.1.1.3 Preparation of 1-Benzyloxycarbonyl Benzotriazole [133]

Amino Protecting Groups 239

To a chilled solution of 357 g of benzotriazole (3 mol) and 511.5 g of freshly prepared benzyl chloroformate (3 mol) in 4.5 L of THF, was added 417 mL of triethylamine (3 mol) dropwise under stirring. The precipitate was filtered, and the solution was concentrated in vacuo to an oily residue. The product was crystallized twice from EtOAc to afford 682 g of 1-benzyloxycarbonyl benzotriazole, also known as benzyl 1H-benzo[d][1,2,3]triazole1-carboxylate, in a yield of 90%, m.p. 108–110°C. 4.4.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE INTRODUCTION OF THE CBZ GROUP 4.4.1.2.1 Preparation of N-Benzyloxycarbonyl-L-Threonine [141]

A 2-liter, 5-necked reaction flask equipped with a thermometer, a mechanical agitator, a pH electrode, and two constant-volume addition funnels was charged with 119.2 g of L-threonine (98% purity) and 600 g of water. Then, 80 grams of 50% aqueous NaOH solution was added to convert L-threonine to the corresponding sodium salt, with a pH of about 11.3. The addition of 174.0 g of benzyl chloroformate (99% purity), 80 g of 50% aqueous NaOH solution and concentrated HCl as separate streams were accomplished sequentially. The addition of NaOH started 5 minutes after the addition of benzyl chloroformate started, which took 70 minutes; and the addition of NaOH took 1.5 hours, so that pH stabilized at 11.0. Around 20 minutes after the completion of the addition of NaOH, the addition of HCl started, and during the hydrochloric acid addition, a precipitate suddenly began to form at about pH 4.0, which froze into a near-solid mixture to stop the agitator. About 600 mL of EtOAc was added to the flask and the agitator was manually turned until it could move slowly under its own power. As the reaction mixture was stirred, the precipitate dissolved and permitted the agitator to move freely. After the pH had stabilized at 1.7 (took about 40 minutes to adjust pH to 1.7), stirring was continued for about 15 minutes and then discontinued. The reaction mixture was then charged into a 2 L separation funnel and the

240

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

phases were allowed to separate. The bottom aqueous phase was drained off and re-extracted with an additional 300 mL of EtOAc. The combined organic phase was stored overnight over anhydrous MgSO4. The solid was then removed by filtration and the filtrate was stripped under vacuum in a Buchi rotary evaporator at about 40°C. The solids were dried for 4 hours in a vacuum at about 45°C and absolute pressure in the range from about 22 to about 127 mmHg, and then placed in a vacuum desiccator over P2O5 for 16 hours at ambient temperature and absolute pressure of about 0.1 to 0.2 mmHg. The resulting N-benzyloxycarbonyl-L-threonine weighed 210.20 g and melted at 93–95°C. The yield was 83% with a purity of 99.6%. 4.4.1.2.2 Preparation of N-Benzyloxycarbonyl Tryptophan with Dibenzyl Dicarbonate [133]

To a solution of 10.21 g of L-tryptophan (50 mmol) in a mixture of 50 mL 1 N NaOH and 50 mL of dioxane, was added 14.32 g of dibenzyl dicarbonate (50 mmol) in 50 mL of dioxane dropwise under stirring. After 60 minutes at room temperature, the bulk of the dioxane was evaporated, and the resulting aqueous solution was acidified with 1 N H2SO4 to pH 2 and extracted with EtOAc or methyl t-butyl ether (3 ×). The combined extracts were washed with water, dried over Na2SO4 and evaporated. The residue was crystallized from EtOAc/petroleum ether to afford 16.1 g of N-benzyloxycarbonyl-Ltryptophan, in a yield of 95%, m.p. 124–125°C. 4.4.1.2.3 Preparation of N-Benzyloxycarbonyl-L-Alanine with 1-Benzyloxycarbonyl-Benzotriazole [133]

Amino Protecting Groups 241

To a chilled solution of 4.45 g L-alanine (50 mmol) in a mixture of 50 mL 1 N NaOH and 50 mL of dioxane, was added 12.66 g of 1-benzyloxycarbonyl-benzotriazole (50 mmol) in 50 mL of dioxane dropwise under stirring. After 24 hours at room temperature, the dioxane was evaporated, and the resulting aqueous solution was diluted with water and extracted with EtOAc or methyl t-butyl ether (3 ×). The aqueous phase was acidified to pH 2 with 1 N H2SO4 and again extracted with EtOAc or methyl t-butyl ether (3 ×). The combined latter extracts were washed sulfate-free with water, dried over Na2SO4 and evaporated. The residue was crystallized from ether/petroleum ether to afford 9.6 g of N-benzyloxycarbonyl-L-alanine, in a yield of 86%, m.p. 84–85°C.

4.4.1.2.4 Catalytic Preparation of N-BenzyloxycarbonylTryptophan [138]

To 10 mL of water or 0.1 M carbonate buffer (pH = 8) was added 0.114 g of β-cyclodextrin (0.1 mmol) at room temperature, followed by 0.204 g of tryptophan (1 mmol). After the solution was stirred for 5 minutes, 0.171 g of Cbz-Cl (1 mmol) was added, and the resulting mixture was stirred at room temperature until the reaction was complete (ca. 7 minutes). The reaction mixture was extracted with EtOAc (the aqueous phase was neutralized with 10% Na2CO3 in the case of water-based reactions). The filtrate was cooled to 5°C and the precipitated β-CD was removed by filtration. The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under vacuum. The residue was purified by column chromatography to afford 0.328 g of N-benzyloxycarbonyl-tryptophan, in a yield of 97%.

242

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.4.1.3 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE REMOVAL OF THE CBZ GROUP 4.4.1.3.1 Synthesis of Poly(ε-Caprolactone) (PCL)-(CH2)6-NH2 [139]

To a suspension of 2.0 g of poly(ε-caprolactone)-(CH2)6-NH-Z in 40 mL of EtOAc/MeOH (v/v, 2:1) was added 1.0 g of 10% Pd supported on activated carbon. The resulting mixture was subjected to hydrogenation under an H2 blanket at room temperature for 24 hours. After filtration over Celite, the solution was concentrated and precipitated into cold methanol. The obtained polymer was re-precipitated from cold methanol by dissolving in chloroform to afford 70% of PCL-(CH2)6-NH2. 4.4.1.3.2 Deprotection of Cbz Group via Hydrogenation [142]

A 2.5-L Parr bottle was charged with 16.0 g of 5% Pd/C (50% wet) under a nitrogen atmosphere. A solution of 75 g of 4-((R)-benzyloxycarbonylamino-carboxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (190 mmol) in 1.5 L of EtOH/H2O (2:1) was added under a nitrogen purge. The mixture was hydrogenated under 50 psi hydrogen gas at room temperature for 16 hours. The mixture was then flushed with nitrogen and filtered. The catalyst cake was rinsed with 400 mL of EtOH. The organic solution was concentrated under vacuum until a gray solid was obtained (49 g, 100%). The gray solid was suspended in 750 mL of MeOH, stirred at 60°C for 2 hours, and further stirred at 0°C for an additional 1 hour. The solid was collected by filtration and rinsed with 200 mL cold MeOH (5°C). The solid was dried at 60°C under vacuum (2 mm Hg) to obtain 40 g of

Amino Protecting Groups 243

4-((R)-aminocarboxymethyl)-piperidine-1-carboxylic acid tert-butyl ester, also known as (R)-2-amino-2-(1-(tert-butoxycarbonyl)piperidin-4-yl)acetic acid, as an off-white solid, in a yield of 82%.

4.4.1.3.3 Deprotection of Cbz Group via Hydrogenation Over Pd(OH)2 [143]

To a solution of 339 mg of N-benzyloxycarbonyl-(2S,3aR,5S,6S,7aS)5,6-bis(methoxymethoxy)octahydro-1H-indole-2-carboxylic acid methyl ester (0.865 mmol) in 50 mL of MeOH was added 117 mg of Pd(OH)2. A hydrogen balloon was attached and the solution was stirred for 3 hours at room temperature. CH2Cl2 was added, and the mixture was filtered and concentrated to afford 95% of (2S,3aR,5S,6S,7aS)-5,6-bis(methoxymethoxy)octahydro1H-indole-2-carboxylic acid methyl ester.

4.4.1.3.4 Deprotection of Cbz-Protected Methionine [140]

A dry 1-L three-necked, round-bottomed flask is equipped with a dry ice reflux condenser, a gas-inlet tube, and a magnetic stirring bar, which is immersed in an acetone-dry ice bath. A total of 300 mL of ammonia is passed through a drying tower containing potassium hydroxide pellets and collected in the flask. A solution of 0.708 g (2.5 mmol) of N-benzyloxycarbonyl-Lmethionine in 10 mL of N,N-dimethylacetamide (DMA), 1.02 g of triethylamine (1.40 mL, 0.0101 mol), and 1.25 g of freshly prepared palladium black are added. The acetone-dry ice bath is removed to permit the reaction

244

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

to proceed at the boiling point of ammonia (–33°C), and a gentle stream of dry nitrogen is bubbled into the flask. The nitrogen stream is discontinued and replaced by a stream of hydrogen that has been passed through a concentrated sulfuric acid scrubber. The mixture is stirred under reflux for 5.5 hours to effect hydrogenolysis. The hydrogen stream is then discontinued, a flow of nitrogen is resumed, and the dry ice is removed from the reflux condenser, permitting rapid evaporation of ammonia. After all the ammonia has escaped from the flask, the flask is attached to a rotary evaporator, and the mixture is evaporated to dryness under reduced pressure. The residue is dissolved in water and filtered through a sintered funnel of medium porosity to remove the catalyst. The filtrate is evaporated to dryness, and the residue (354 mg, 95%) is crystallized from water-ethanol. The white crystalline product, after drying under reduced pressure at 25°C, weighs 272–305 mg, in a yield of 73–82%, m.p. 280–282°C (dec.).

4.4.2 p-NITROBENZYLOXYCARBONYL (PNZ) GROUP The pNZ group was first introduced by Carpenter and Gish as an amino protecting group alternative to the benzyloxycarbonyl group [144]. It has been used for the protection of the ε-amino group of Lys to prevent the potential side-chain branching associated by the Cbz group [145, 146]. The pNZ group can be mounted to amino acid by means of in situ generated pNZ-N3, i.e., pNZ-Cl is allowed to react with NaN3, and the resulting pNZ-N3 is directly treated with free amino acids to afford the pNZ-amino acids, in relatively high yields (71–94%) and purity. The resulting pNZ-amino acids have superior solubility in DMF to their corresponding Fmoc derivatives [122]. Besides the catalytic hydrogenation, the pNZ group can also be removed by other nitro reducing methods. When the p-nitro group is reduced to the p-amino group, the resulting p-aminobenzyloxycarbonyl derivative of amino acid suffers from a spontaneous collapse by 1,6-electron pair shift to afford the quinonimine methide and the carbamic acid; and the carbamic acid decomposes to the corresponding free amino acid. It is found that SnCl2 is one of the best nitro reducing group at a concentration of 6 M, especially in the presence of a catalytic amount of acid (e.g., 1.6 mM HCl) [122].

Amino Protecting Groups 245

4.4.2.1 PREPARATION OF P-NITROBENZYLOXYCARBONYL GLYCINE [122]

A solution of 0.624 g of sodium azide (9.6 mmol) in 2.5 mL of H2O was added to a solution of 1.73 g of p-nitrobenzyl chloroformate (8 mmol) in 3.5 mL of 1,4-dioxane. The resulting emulsion was stirred for 2 hours and the formation of the 4-nitrobenzyl carbonazidate was monitored by TLC (CH2Cl2). A 10 mL mixture of 1,4-dioxane/2% aqueous Na2CO3 (1:1) containing 0.60 g of glycine (8 mmol) was then added dropwise, and the resulting white suspension was stirred for 24 hours while keeping the pH between 9 and 10 by adding 10% aqueous Na2CO3. At this point, TLC (CH2Cl2) showed that there was no remaining azide, H2O (75 mL) was added and the suspension was washed with methyl tert-butyl ether (MTBE) (3 × 40 mL). The aqueous portion was acidified to pH = 2–3 with 3 N HCl, and a white precipitate appeared, which was filtered off and dried to yield 1.44 g of N-p-nitrobenzyloxycarbonyl glycine as a white solid, in a yield of 71%. 4.4.2.2 PREPARATION OF N-P-NITROBENZYLOXYCARBONYL-DGLUTAMIC ACID [123]

D-Glutamic acid (10.0 g, 0.07 mol) was dissolved in 75 mL of 2 M NaOH solution (0.14 mol), and 30 mL of 1,4-dioxane was added. The resulting solution was cooled to 0°C with vigorous stirring while a solution of 22.5 g of p-nitrobenzyl chloroformate (0.1 mol) in 115 mL of 1,4-dioxane and 75 mL of 2 M NaOH were added dropwise from two separate dropping funnels over 30 minutes. The resulting mixture was stirred at room temperature for 30 minutes. The precipitate was filtered and washed with 225 mL of water. The filtrate and washing were combined and extracted with 500 mL of ethyl acetate. The aqueous layer was acidified to pH 3 and extracted with 300 mL of ethyl acetate. The combined ethyl acetate extract was washed with saturated NaCl, dried (MgSO4) and concentrated in vacuo leaving (((4-nitrobenzyl)

246

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

oxy)carbonyl)-D-glutamic acid as a yellow oil, which was crystallized from ethyl acetate/light petroleum (b.p. 35–60°C) as yellow crystals, in 14.28 g (64.4% yield); m.p. 154–155°C. 4.4.3 4,5-DIARYL-4-OXAZOIN-2-ONE DERIVATIVE OF AMINO ACIDS 4,5-Diaryl-4-oxazolin-2-one derivatives of amino acids are prepared by condensation of 4,5-diaryl-1,3-dioxol-2-one with amino acids, in the presence of 1.0 equivalent tetramethylammonium hydroxide to furnish hydroxyoxazolidinone as an intermediate. This intermediate is directly converted into the oxazolone derivatives of amino acid by dehydration with TFA without isolation. The 4,5-diaryl-1,3-dioxol-2-ones are prepared by condensation of anisoin or 4,4’-dichlorobenzoin with phosgene or trichloromethyl chloroformate in the presence of 2.0 equivalents of N,Ndimethylaniline in toluene [126]. These oxazolone derivatives of amino acids are stable to NaOH-aqueous EtOH, liquid hydrogen fluoride and hydrobromic acid/acetic acid at room temperature, but are decomposed by hydrogenolysis over Pd/carbon, or under reductive or oxidative conditions. 4.4.3.1 PREPARATION OF 4,5-DIPHENYL-1,3-DIOXOL-2-ONE USING PHOSGENE [126]

To a stirred solution of 21.2 g of anisoin (i.e., 2-hydroxy-1,2-diphenylethan-1-one, 0.1 mol) in 400 mL of toluene containing 11.0 g of phosgene (0.11 mol) was added dropwise 24.2 g of N,N-dimethylaniline (0.2 mol) within 1 hour at 0°C. After stirring at room temperature for 12 hours, the resulting precipitate was collected by filtration, washed with toluene (30 mL), and dissolved in CHCl3. The solution was washed with H2O, dried, and then evaporated. The residue was recrystallized from toluene to give an 16.0 g of 4,5-diphenyl-1,3-dioxol-2-one, m.p. 175–176°C. The above toluene filtrate and washings were combined and warmed on a water bath at

Amino Protecting Groups 247

80–90°C for 3 hours. After cooling, the solution was washed with 0.5 N HCl and H2O, dried, and evaporated. The residue was recrystallized from toluene to give additional 4.0 g of 4,5-diphenyl-1,3-dioxol-2-one, m.p. 175–176°C. The total yield was 20.0 g (67%). 4.4.3.2 PREPARATION OF 4,5-DIPHENYL-1,3-DIOXOL-2-ONE USING TRICHLOROMETHYL CHLOROFORMATE [126]

To a stirred suspension of 13.6 g of anisoin (0.05 mol) in 150 mL of toluene containing trichloromethyl chloroformate was added dropwise 12.1 g of N,N-dimethylaniline (0.1 mol) within 1 hour at 0°C. The same workup procedure led to 12.2 g of 4,5-diphenyl-1,3-dioxol-2-one, in a yield of 82%, m.p. 175–176°C. 4.4.3.3 PREPARATION OF 4,5-DIARYL-4-OXAZOIN-2-ONE DERIVATIVE OF ALANINE [126]

To a solution of 5 mmol alanine and 10% methanolic tetramethylammonium hydroxide (4.5 g, 5 mmol) in 30 mL of dioxane or DMF, was added 5 mmol of 4,5-bis(4-methoxyphenyl)-1,3-dioxol-2-one, and the resulting solution was stirred at room temperature for 24–36 hours. The reaction mixture was then acidified with 1 N HCl and extracted with EtOAc. The extract was washed with H2O, dried, and evaporated. The residue was dissolved in 5 mL

248

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

of TFA and allowed to stand for 2 hours at room temperature. Removal of TFA in vacuo afforded a pale-yellow oil which solidified by trituration with petroleum ether/benzene and recrystallized from EtOAc-petroleum ether/ benzene to give (S)-2-(4,5-bis(4-methoxyphenyl)-2-oxooxazol-3(2H)-yl) propanoic acid. 4.4.3.4 CATALYTIC HYDROGENOLYSIS OF (S)-2-(4,5-BIS(4METHOXYPHENYL)-2-OXOOXAZOL-3(2H)-YL)PROPANOIC ACID [126]

A 100 milligrams of (S)-2-(4,5-bis(4-methoxyphenyl)-2-oxooxazol-3(2H)-yl)propanoic acid in a mixture of 50 mL MeOH and 2 mL of 1 N HCl was hydrogenated over 25 mg of 10% Pd/C until no fluorescent spot was noted on TLC. The mixture was filtered off and the filtrate was evaporated. The residue was recrystallized from EtOH-ether to afford 29 mg of alanine hydrochloride. 4.4.4 ISONICOTINYLOXYCARBONYL (INOC) PROTECTING GROUP The isonicotinyloxycarbonyl protected lysine does not have the potential problem of side-chain branching at the ε-position of lysine as that in the case of benzyloxycarbonyl group. This protecting group is stable to most of the conditions commonly employed in peptide synthesis, including the strongly acidic conditions often employed for the removal of peptides from resin supports in solid-phase peptide syntheses. However, it can be cleanly removed under mild conditions, by the action of either zinc dust in aqueous acetic acid or by catalytic hydrogenation using 5% Pd/C [128].

Amino Protecting Groups 249

4.4.4.1 PREPARATION OF ISONICOTINYL P-NITROPHENYL CARBONATE [128] 4.4.4.1.1 Method 1

To a solution of 152 g of bis(p-nitrophenyl) carbonate (0.67 mol) in 1,600 mL of methylene chloride was added dropwise a solution of 63.5 g of 4-pyridylcarbinol (0.58 mol) (azeotropically dried using benzene) in 500 mL of methylene chloride over 30 minutes with stirring, followed by a solution of 50 g of N-methylmorpholine (0.49 mol) in 150 mL of methylene chloride. The solution was allowed to stir for 2.5 days. TLC (silica gel, CHCl3) showed that this length of time is required for a complete reaction. The resulting solution was washed sequentially with H2O (2 × 1 L), 0.1 N H2SO4 (2 L), saturated NaHCO3 (4 × 2 L), and saturated NaCl (2 L). The organic phase was dried over Na2SO4, filtered, and evaporated to dryness in vacuo. The solid residue was dissolved in a minimum of warm ethyl acetate (30°C). The free base of isonicotinyl p-nitrophenyl carbonate crystallized on standing overnight, in an amount of 53 g (33%), m.p. 104–106°C. 4.4.4.1.2 Method 2

4-Pyridylcarbinol (13.12 g, 0.12 mol), which was azeotropically dried with benzene, and 27.2 g of bis(p-nitrophenyl) carbonate (0.12 mol) were dissolved in 200 mL of CH2Cl2 and the mixture was stirred for 3 days at 20°C. The mixture was filtered to remove a small amount of insoluble material and the solution was evaporated to dryness. The foamy solid was dissolved in 500 mL of ethyl acetate and crystallized by the addition of about 1,100 mL of hexane to give 26.2 g of isonicotinyl p-nitrophenyl carbonate, in a yield of 80%, m.p. 108–111°C.

250

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

4.4.4.1.3 Preparation of ε-iNoc-Lys [128]

A solution of 7.44 g of CuCl2·2H 2O (0.044 mol) in 160 mL of H2O was added to a solution of 14.56 g of L-lysine hydrochloride (0.08 mol) in 160 mL of H2O, and the solution was adjusted to pH 9.5 by the addition of 50% aqueous NaOH. Isonicotinyl p-nitrophenyl carbonate (22 g, 0.08 mol) was added over a period of 20 minutes with stirring. The reaction mixture was vigorously stirred at 20°C for 26 hours (heavy precipitate), after which acetic acid was added to adjust pH to 7.2. The copper complex of ε-isonicotinyloxycarbonyl lysine was isolated by filtration and washed with H2O (slow filtration). This material was dissolved in 900 mL of 10% aqueous acetic acid and treated with H2S to precipitate CuS. The mixture was filtered through Celite and the filtrate was evaporated to dryness in vacuo. The residue in the amount of 22 g contains small amounts of free lysine and p-nitrophenol. This material (20 g) was dissolved in 150 mL of water and sufficient Dowex 1 × 2 (OH–) was added to obtain pH 6.6, thus adsorbing residual p-nitrophenol. The solution was applied to a 1,400-mL column of IRC-50 (NH4+) and eluted with 20% ethanol in 0.1 N NH4OAc. The first four ninhydrin positive fractions (500 mL each) contained a single component as indicated by TLC (silica gel, CHCl3-MeOH-H2O/ 5:4:l). These were combined and evaporated to dryness in vacuo, and the residue was crystallized from water/ethanol to yield 13.3 g of ε-iNoc-Lys, i.e., N6-((pyridin-4-ylmethoxy)carbonyl)-L-lysine, in a yield of 47%, m.p. 235–236°C. Note: If the copper complex of ε-iNoc-Lys is thoroughly washed with water, clean product is obtained by crystallization of the free ε-iNoc-Lys without ion-exchange chromatography. Thorough washing is made difficult, however, by the physical nature of the complex.

Amino Protecting Groups 251

4.4.4.1.4 Removal of iNoc by Hydrogenation [128]

N-α-Boc-ε-iNoc-Lys (10 mg), i.e., N2-(tert-butoxycarbonyl)-N6((pyridin-4-ylmethoxy)carbonyl)-L-lysine, was dissolved in 1 mL of 5% aqueous acetic acid, then, 10 mg of Pd/C (10%) was added. The suspension was purged with nitrogen, and hydrogen was bubbled through the solution for 10 minutes. Analysis by TLC showed that the iNoc protecting group has been removed completely. 4.4.5 p-TRIMETHYLAMMONIUM CHLORIDOBENZYLOXYCARBONYL PROTECTING GROUP The purpose of introducing the p-trimethylammonium chloridobenzyloxycarbonyl group is to increase the solubility of the protected amino acid in a polar solvent, such as water, so that the formation of peptide linkage in aqueous media becomes possible. However, the resulting protected amino acid esters are difficult to crystalize due to their high hygroscopicity. 4.4.5.1 PREPARATION OF p-TRIMETHYLAMMONIUM CHLORIDOBENZYLOXYCARBONYL CHLORIDE [129]

Phosgene was passed slowly into a mixture of 6.0 g finely powdered 4-(hydroxymethyl)-N,N,N-trimethylbenzeneammonium chloride (0.03 mol) and 10 mL of dry toluene in a round-bottom flask protected from moisture, which was cooled in an ice-salt mixture with occasional shaking. The reaction took about 5 hours and the volume of the mixture became nearly doubled. The flask was kept in an ice-salt mixture overnight. The reactant first hardened and then changed into finely dispersed crystalline needles. Phosgene was removed by warming to 30°C, and toluene was decanted. The remaining crystalline mass was further dried with a water aspirator to remove residual phosgene and

252

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

toluene. p-Trimethylammonium chloridobenzyloxycarbonyl chloride, also known as 4-(((chlorocarbonyl)oxy)-methyl)-N,N,N-trimethylbenzenaminium chloride, gradually changed into a mass of colorless crystalline particles that should be kept from moisture because it was highly hygroscopic. 4.4.5.2 FORMATION OF p-TRIMETHYLAMMONIUM CHLORIDOBENZYLOXYCARBONYLGLYCINE METHYL ESTER [129]

To an ice-bath cooled aqueous solution of 1.0 g of glycine methyl ester hydrochloride (8 mmol) with pH adjusted to 8–9 with 4 N NaOH was added a solution of 2.5 g of p-trimethylammonium chloridobenzyloxycarbonyl chloride (approx. 9 mmol) in 10 mL of methanol with stirring. NaOH was added intermittently to maintain the reaction mixture at approximately pH 8–9. Stirring was continued for 2–3 hours and HCl was then added to adjust the mixture to pH 7. NaCl was filtered, and the filtrate was evaporated to dryness. Addition of absolute ethanol, filtration, and evaporation were repeated twice to remove NaCl. The filtrate was again concentrated, and the residue was recrystallized from absolute ethanol-EtOAc to afford 2.0 g of p-trimethylammonium chloridobenzyloxycarbonylglycine methyl ester, also known as 4-((((2-methoxy-2-oxoethyl)carbamoyl)oxy)methyl)-N,N,Ntrimethylbenzenaminium chloride, in a yield of 80%, m.p. 172–173°C (dec.). 4.4.5.3 HYDROGENOLYSIS OF p-TRIMETHYLAMMONIUM CHLORIDOBENZYLOXYCARBONYL-GLYCYL GLYCINE [129]

Amino Protecting Groups 253

The solution of about 1.0 g of p-trimethylammonium chloridobenzyloxycarbonyl glycylglycine (2.8 mmol) in 20 mL of ethanol was hydrogenated with 0.1 g of 10% Pd/C catalyst at ambient temperature and pressure. Ether was added and it was stood in the refrigerator to yield 0.1 g of crystalline glycylglycine. The filtrate was evaporated, then water and the alcohol solution of picric acid were added. p-Tolyl trimethylammonium picrate (0.95 g, 90%) was thus removed and recrystallized from ethanol, m.p. 197–198°C. The filtrate was concentrated to give another crop of glycylglycine (0.21 g, 58%), m.p. 209–214°C (dec.). 4.5 THIOLYSIS CLEAVABLE AMINO PROTECTING GROUP 4.5.1 N-DITHIASUCCINOYL (DTS) AND (ALKYLDITHIO)CARBONYL GROUPS The N-dithiasuccinoyl (Dts) group was initially developed by Barany and Merrifield in 1977 for the protection of the amino group [147]. The corresponding Dts-amino acid derivative is prepared in general from the treatment of N-ethyloxythiocarbonyl amino acid derivative with chlorocarbonylsulfenyl chloride in anhydrous solution, optionally in the presence of a tertiary amine, to form an initial adduct that undergoes ring closure followed by loss of ethyl chloride to yield the Dts-derivative, as illustrated in Scheme 4.1. It is found that this reaction proceeds exceedingly rapid at 0 to 45°C, often in good yields accompanyed by the major byproduct of isocyanate [147]. Besides its application in the protection of the primary amino group in α-amino acid derivatives and peptides, Dts has been applied to protect the primary amino group in aminosugars [148]. However, the free Dts-amino acids could not be prepared directly from the N-ethoxythiocarbonyl amino acids containing a free carboxyl group. Instead, the corresponding methyl, tert-butyl, or trimethylsilyl (TMS) esters of N-ethoxythiocarbonyl amino acids are used to react with the chlorocarbonylsulfenyl chloride to afford the Dts-protected amino acid derivatives that are then hydrolyzed in 12 N HCl/acetic acid (1:4), or anhydrous HBr in acetic acid at 25°C to yield the Dts-protected amino acids. Obviously, the Dts moiety is entirely resistant to the strongly acidic and mildly basic reagents [147], whereas treatment of the Dts-amino acid derivatives with aliphatic amines or strong aqueous alkali leads to the formation of mixed urea or deprotected amino acids. In addition, Dts-amines, and Dts-amino acid derivatives can tolerate photolytic treatment

254

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

with light of wavelength > 330 nm [147], rendering the orthogonal cleavage of acid-stable o-nitrobenzyl and α-methylphenacyl esters [149]. On the other hand, the Dts moiety can be rapidly and specifically cleaved under mild conditions by thiolysis [150]. Thiolytic cleavage of the heterocyclic disulfide under mild conditions due to the facile susceptibility of disulfide bridges to reductive cleavage gives, in an irreversible process via a carbamoyl disulfide intermediate, the parent amine, two carbonyl disulfide molecules and the disulfide corresponding to the reducing thiol [151]. Thus, the Dts-amino acids and Dts-amines can be used as mild oxidation reagents that promote the formation of intramolecular disulfide bridges within peptides and proteins. It is found that the thiolysis of Dts proceeds cleanly under a variety of conditions, which undergoes markedly fast in the presence of a tertiary amine. For example, the thiolytic deprotection of Dts generally completes within 5 minutes at 25°C with an excess amount of 0.2 M β-mercaptoethanol in 0.5 M Et3N in CH2Cl2 [147]. Besides the reductive thiolysis, Dts function can also be rapidly and quantitatively removed through mild and specific reductive cleavage with borohydrides or trialkylphosphines [149].

SCHEME 4.1 Formation of the Dts functionality.

Under comparable conditions, it is found that the rates of reductive thiolysis vary over three to four orders of magnitude, with the fastest rates in polar aprotic media of high dielectric constants. In addition, a synergistic effect has been observed for polar, auto-protolyzable solvents when a tertiary amine exists [149]. For example, the deprotection rates in pyridine-DMF mixtures are much faster than the rates in either neat pyridine or in neat DMF. The relative rates are estimated in the order of “buffered” DMF > DMF-pyridine (9:1) > pyridine ~ DMF-benzene (1:1) > pyridine-benzene (1:1) ~ DMF-acetic acid (9:1), in an approximate ratio of 400:150:20:10:1:1. Such synergism has also been observed in other solvents, such as N-methylpyrrolidone, DMA, and DMSO [149]. According to the two-step reaction mechanism involved in the thiolytic cleavage of the Dts amino-protecting group, open-chain (alkyldithio)carbonyl derivatives are formed as short-life intermediates. Thus, the (alkyldithio)

Amino Protecting Groups 255

carbonyl group can also be applied as the amino protecting group that can be removed under thiolysis condition [69], especially for the (cyclohexyldithio) carbonyl function. It is found that the N-(cyclohexyldithio)carbonyl-amino acid derivatives can be obtained by relatively simple procedures which are sufficiently stable under normal conditions of peptide synthesis. However, the (alkyldithio)carbonyl amino acids cannot be prepared directly under the Schotten-Baumann conditions owing to the pronounced lability of the carbamoyl disulfide function in aqueous alkaline media. The corresponding amino acid esters react smoothly with both (tert-butyldithio)carbonyl or (cyclohexyldithio)carbonyl chloride to yield the corresponding Nα-protected derivatives. Reductive cleavage with thiols or phosphines results in quantitative removal of this amino protecting group [69]. 4.5.1.1 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE PROTECTION OF AMINO ACIDS WITH DTS OR (ALKYLDITHIO) CARBONYL GROUPS 4.5.1.1.1 Preparation of N-[2-(N-Ethoxythiocarbonyl)Aminoethyl]-N[[6-N-(Benzyloxycarbonyl)-Adenin-9-yl]Acetyl]Glycine [152]

A solution of 409 mg of N-[2-(N-t-butoxycarbonyl)aminoethyl]-N-[[6N-(benzyloxy-carbonyl)adenin-9-yl]acetyl]glycine (0.78 mmol) in 16 mL of TFA-H2O (19:1) was stirred for 60 minutes. Concentration in vacuo (1 mmHg) gave an oil that was re-dissolved in 15 mL of EtOH-H2O (2:1). To this solution was then added 246 mg of bis(ethoxythiocarbonyl)sulfide (1.17 mmol, 1.5 equiv) and the resulting suspension was brought to a pH of 9–10 by the addition of 2.0 mL 2.5 M aqueous NaOH. An additional 0.6 mL of such NaOH solution was added dropwise over a period of 4 hours in order to maintain the pH. At this point, all of bis(ethoxythiocarbonyl)-sulfide

256

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

was dissolved and the pH remained constant overnight. The solution was diluted with 18 mL of H2O, acidified to pH 2 with 12 N HCl, and extracted with EtOAc (5 × 20 mL). The organic layers were combined, dried over MgSO4, and evaporated in vacuo to provide a colorless oil. Trituration of the oil with Et2O yielded N-[2-(N-ethoxythiocarbonyl) aminoethyl]-N-[[6-N-(benzyloxycarbonyl)-adenin-9-yl]acetyl]glycine, also known as N-(2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)N-(2-((ethoxycarbonothioyl)-amino)ethyl)glycine in a white solid, which was further dried over P2O5, in the amount of 385 mg (95% yield), m.p. 118–120°C. 4.5.1.1.2 Formation of N-[2-(N-Dithiasuccinoyl)Aminoethyl]-N-[[6N-(Benzyloxycarbonyl)Adenin-9-yl]Acetyl]GLycine [152] S S O S O

O OH

N H

N

N

Si N

N O

N

N O CH3CN

Si

Cl

S

O

Cl N

O

N N

N

NHCbz CbzHN

N

O

N

OH

O

To a suspension of 81 mg of freshly prepared N-[2-(N-ethoxythiocarbonyl)aminoethyl]-N-[[6-N-(benzyloxycarbonyl)adenin-9-yl]acetyl]glycine (0.16 mmol) in 9 mL of CH3CN was added 59 µL of TMS (E)-N-(trimethylsilyl) acetimidate (0.24 mmol, 1.5 equiv.), and the mixture was stirred for 30 minutes. Then, 14 µL of chlorocarbonylsulfenyl chloride [Cl(C=O)SCl, 0.18 mmol, 1.1 equiv.] was added, resulting in a yellow solution. The reaction was complete after 20 minutes as indicated by HPLC. The suspension was distributed between EtOAc (10 mL) and H2O (15 mL), and the aqueous phase was extracted with EtOAc (5 × 15 mL). The organic layers were combined, dried over MgSO4, and concentrated to a small volume that upon cooling to –20°C gave 70 mg of N-[2-(N-dithiasuccinoyl)-aminoethyl]N-[[6-N-(benzyloxycarbonyl)adenin-9-yl]acetyl]glycine, also known as N-(2-(6-(((benzyloxy)carbonyl)amino)-9H-purin-9-yl)acetyl)-N-(2-(3,5dioxo-1,2,4-dithiazolidin-4-yl)ethyl)glycine, in an off-white solid, in a yield of 80%, m.p. 138–140°C.

Amino Protecting Groups 257

4.5.1.1.3 Preparation of 1,3,4,6-Tetra-O-Acetyl-2-Deoxy2-Dithiasuccinimido-α-D-Glucopyranose and 1,3,4,6-Tetra-O-Acetyl-2-Deoxy-2-Dithiasuccinimido-β-DGlucopyranose [148]

To a solution of 5.0 g of (3R,4R,5S,6R)-6-(acetoxymethyl)-3-((ethoxycarbonothioyl)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate, also known as 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(ethoxythiocarbonylamino)-α,β-Dglucopyranose (11.6 mmol) in 50 mL of dry CH2Cl2, was added 1 mL of chlorocarbonylsulfenyl chloride (11.8 mmol) at 0°C. Development of the reaction was monitored by TLC (toluene: EtOAc = 2: 1). After 30 minutes, the reaction mixture was concentrated, re-dissolved in 100 mL of CHCl3, washed successively with 1 N HCl (3 × 100 mL) and water (2 × 100 mL). The organic layer was then dried over MgSO4 and concentrated to provide the crude product. Purification of the crude product by vacuum liquid chromatography (toluene: EtOAc = 7: 1) afforded 2.46 g of 1,3,4,6-tetra-O-acetyl2-deoxy-2-dithiasuccinimido-α-D-glucopyranose (45.6%, also known as (2R,3R,4R,5S,6R)-6-(acetoxymethyl)-3-(3,5-dioxo-1,2,4-dithiazolidin-4-yl) tetrahydro-2H-pyran-2,4,5-triyl triacetate) and 1.58 g of 1,3,4,6-tetra-Oacetyl-2-deoxy-2-dithiasuccinimido-β-D-glucopyranose (29%, also known as (2S,3R,4R,5S,6R)-6-(acetoxymethyl)-3-(3,5-dioxo-1,2,4-dithiazolidin4-yl)tetrahydro-2H-pyran-2,4,5-triyl triacetate), and 450 mg of the mixed fraction (8.3%). 4.5.1.1.4 Preparation of (Butyldithio)Carbonyl-Alanine [69]

258

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

To a solution of 1.82 g of alanine tert-butyl ester hydrochloride (10 mmol) and 1.39 mL of triethylamine (10 mmol) in 50 mL of ether or EtOAc, were added 1.84 g of (butyldithio)carbonyl chloride (10 mmol) and 1.39 mL of Et3N (10 mmol) dropwise at 0°C in 10 minutes under stirring. The reaction was allowed to proceed at room temperature for 2 hours, insoluble material (Et3N⋅HCl) was then filtered off and the filtrate was evaporated in vacuo. The residue was dissolved in a 5 mL ice-cold TFA. After being left at room temperature for 60 minutes, the resulting solution was evaporated to dryness. The residue was dissolved in ether (or EtOAc), washed with water, and then with saturated NaCl solution. Upon drying over Na2SO4, the filtrated solution was evaporated to dryness. The residue was purified by column chromatography and further crystallized from diisopropyl ether/petroleum ether to afford 1.97 g of N-(butyldithio)carbonyl-alanine, also known as (butyldisulfanne-carbonyl)-L-alanine, in a yield of 83%, m.p. 112–113°C. 4.5.1.2 EXEMPLARY EXPERIMENTAL PROCEDURES FOR THE DTS GROUP 4.5.1.2.1 Thiolysis of Dts-Glycine to β-Hydroxyethyldithiocarbonyl glycine [149]

To a solution of 35 mg of Dts-glycine (0.18 mmol, i.e., 2-(3,5-dioxo1,2,4-dithiazolidin-4-yl)acetic acid) in 7 mL of peroxide-free dioxane were added 100 µL of triethylamine (0.7 mmol) and 50 µL of β-mercaptoethanol (0.7 mmol, i.e., 2-mercaptoethan-1-ol). After being stirred for 10 minutes, the reaction mixture was diluted into a mixture of EtOAc and 1 N HCl (50 mL each). Amino acid analysis showed that at this point the reaction had proceeded to 65% conversion to ((2-hydroxyethyl)disulfannecarbonyl) glycine, and the ratio of Dts: Carb was 1:6. The aqueous phase removed all of the product glycine, but also about half of the carbamoyl disulfide. The organic phase was dried over magnesium sulfate, filtered, concentrated by rotary evaporation at 10 mmHg and 45°C, and then dissolved in 2 mL of CHCl3. In the course of the rotary evaporation, some carbamoyl disulfide was destroyed, so that the final ratio of Dts: Carb was 1.0: 1.5.

Amino Protecting Groups 259

4.5.1.3 PREPARATION OF 2-ACETAMIDO-3,4,6-TRI-O-ACETYL-2-DEOXYβ-D-GLUCOPYRANOSYL AZIDE [148]

To 4 mL of dry CH2Cl2-MeOH (1:1) was added 50 mg of 3,4,6-tri-Oacetyl-2-deoxy-2-dithiasuccinimido-β-D-glucopyranosyl azide (111.5 µmol) followed by 4.2 mg of sodium borohydride (NaBH4, 111.5 µmol). After 10 minutes, TLC (toluene/EtOAc = 2:1) showed complete disappearance of the starting material. The solution was then concentrated and the residue was dissolved in a mixture of acetic anhydride (1 mL)/pyridine (2 mL). After being kept at room temperature for 2 hours, the solution was concentrated, the residue was dissolved in 20 mL of CHCl3, and the solution was washed with water, dried over MgSO4 and concentrated. The product was crystallized from EtOAc-light petroleum to give 29.8 mg of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide, also known as (2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-azidotetrahydro-2H-pyran-3,4-diyl diacetate, in a yield of 71.8%, m.p. 162–163°C. 4.5.1.4 REMOVAL OF THE (CYCLOHEXYLDITHIO)CARBONYL GROUP FOR PREPARATION OF L-PHENYLALANYL-L-LEUCINE [69]

To an ice-cold solution of 4.1 mL of Et3N (31.5 mmol) and 2.5 mL of ethanedithiol (31.5 mmol) in 30 mL of THF, was added 2.85 g of cHxS2CO-Phe-Leu-OH (6.3 mmol, i.e., (cyclohexyldisulfannecarbonyl)-Lphenylalanyl-L-leucine). The resulting solution was stirred for 60 minutes at room temperature, the solvent was then removed in vacuo and the residue was dissolved in 10 mL of water and 3.5 mL of 2 M NaOH was added. The aqueous solution was extracted three times with ether, acidified to pH 2 with 1 M HCl and re-extracted with ether/petroleum ether (1:1, v/v) and finally neutralized with 4 M NaOH to pH 7. The crystalline product was filtered off,

260

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

washed with water and ethanol and dried to afford 1.54 g of L-phenylalanylL-leucine, in a yield of 88%. 4.5.2 O-NITROPHENYLSULFENYL GROUP o-Nitrophenylsulfenyl group, also known as NPS or Nps, is a base stable amino protecting group that can be applied to overcome the drawback of acyl migration from the internal backbone to the terminal amino group during the solid-phase synthesis of protein-nucleic acid (PNA) associated with the Fmoc strategy [153]. Due to the stability under basic conditions, the NPS protected amino acids or peptides can be prepared from the reaction between the corresponding amino acid or peptide esters (e.g., p-nitrophenyl ester) and o-nitrophenylsulfenyl chloride, and the resulting NPS protected amino acid or peptide esters are then saponified with an equivalent of alkali [154]. The resulting NPS derivatives of amino acids and peptides are yellow substances that crystallize easily [155]. Also, the NPS derivatives of amino acids have been prepared and isolated in a pure state as their dicyclohexylammonium (DCHA) salts [155]. In addition, o-nitrophenylsulfenyl chloride reacts almost quantitatively with amino acid N-carboxyanhydrides (NCAs) to give the N-NPS-NCAs that can be easily applied to peptide synthesis [156]. The NPS protecting group can be removed easily with acids, even with acetic acid, in aqueous alcohol (or acetone, dioxane, etc.), solutions [155]. More conveniently, the NPS group can be removed by two equivalents of HCl or HBr in alcoholic solutions or nonpolar solutions, such as in ether, EtOAc [155], or with anhydrous HCl [157]. When other acid-labile protecting groups exist in amino acid or peptide derivatives, the NPS group can be removed under thiolytic conditions, via the treatment of the NPS protected amino acid or peptide derivatives with 2-mercaptopyridine [154] or under reductive mild acidic conditions (3% dichloroacetic acid in 1 M thioacetamide) [153]. Under the condition of acidic reductive cleavage of NPS, the free amino group is naturally protonated, thus avoiding possible trans-acylation process. 4.5.2.1 PREPARATION OF O-NITROPHENYLSULFENYLAMINO ACIDS (GENERAL PROCEDURE) [155]

Amino Protecting Groups 261

To a solution or suspension of an amino acid (0.02 mol) in a mixed solvent of 10 mL 2 N NaOH and 25 mL dioxane, was added 0.022 mol of o-nitrophenylsulfenyl chloride in 10 equal portions as 12 mL of 2 N NaOH was added dropwise within 15 minutes with vigorous shaking. The solution was diluted with 200 mL of water, filtered, and acidified with 1 N H2SO4. The syrup precipitate usually crystallized on scratching and cooling. The product was filtered off, washed with water, dried, and dissolved in ethyl acetate or ether, precipitated again by the addition of petroleum ether. Alternatively, after the acidification with sulfuric acid, the o-nitrophenylsulfenyl derivatives were extracted with EtOAc or ether, or with a mixture of both of these solvents (1:1). The extract was repeatedly washed with water until the aqueous layer became neutral to congo red paper and then dried with Na2SO4. Upon addition of 4 mL of dicyclohexylamine, the corresponding salt separated out in most cases in the form of needles. 4.5.2.2 GENERAL PROCEDURE FOR THE SYNTHESIS OF NPS-NCAS [156]

To a solution of 0.1 mol of amino acid NCA in 300 mL of THF cooled to an ice bath, was added 19.0 g of o-nitrophenylsulfenyl chloride (0.1 mol) under stirring. Then, 14 mL of triethylamine was slowly dropped with vigorous stirring into the solution. After the addition of triethylamine, the system was stirred at 0°C for 15 minutes. The resulting crystals of triethylamine hydrochloride were removed by filtration. The filtrate was concentrated under reduced pressure at 35°C. The residual oil was crystallized by the addition of n-hexane. The crystals of NPS-NCA were dissolved in a small amount of EtOAc and the insoluble material was removed by filtration. The addition of n-hexane to the solution and cooling in a refrigerator gave the crystalline pale-yellow product. Recrystallization of the product from EtOAc gave a pure Nps-NCA. The product was collected by filtration and dried over P2O5. For a special amino acid NCA, such as L-leucine, the corresponding NPS-L-leucine NCA that could not be crystallized from EtOAc was dissolved in Et2O, and gradually diluted with diisopropyl ether and n-hexane (1:1) until the system

262

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

became cloudy. This system was cooled at –20°C in a refrigerator for 2 days to form crystals. 4.5.2.3 DEPROTECTION OF N-NPS-L-ISOLEUCYLGLYCINE [155]

To a solution of 1.7 g of o-nitrophenylsulfenyl-L-isoleucylglycine (0.005 mol) in 20 mL of EtOAc, was added EtOAc containing hydrogen chloride. Immediately, the dipeptide hydrochloride precipitated. The mixture was shaken for 10 minutes and then evaporated to dryness. The removal of the excess hydrogen chloride was achieved by repeated addition of ethyl acetate and evaporation to dryness. Finally, the residue after the addition of ethyl acetate was extracted several times with water. The combined aqueous extracts (25 mL) were extracted with ethyl acetate, then with ether, and were passed through a column of Amberlite IR 4B (OH form). Upon evaporation of the HCI-free eluate and addition of alcohol, 0.83 g of L-isoluecylglycine was obtained in the form of long needles, in a yield of 92%, m.p. > 240°C (dec.). 4.5.2.4 REMOVAL OF THE O-NITROPHENYLSULFENYL GROUP [154]

S NO2

H N

N

O

SH O

OH CH2Cl2

OH NH2

A solution of 93.1 mg of o-nitrophenylsulfenlphenylalanine (0.292 mmol) in 2 mL of CH2Cl2 was combined with a solution of 38.2 mg of 2-mercaptopyridine (0.34 mmol) in 1.5 mL of CH2Cl2. The initially clear homogeneous mixture turned turbid within a few seconds, and thin-layer chromatography revealed that such thiolysis was completed in less than a minute. The solid was collected by filtration, washed with CH2Cl2 and dried to afford 43.5 mg of phenylalanine, in a yield of 90%.

Amino Protecting Groups 263

4.6 HYDRAZINE LABILE AMINO PROTECTING GROUP N-Tetrachlorophthaloyl amino protecting group [158], also known as TCP, originating from the Gabriel primary amine synthesis with a phthalic acid anhydride, is compatible with both Fmoc and Boc protecting groups in peptide synthesis, which is stable under both acidic and basic conditions. Compared to the Fmoc group, the TCP is not affected by the treatment of piperidine or N,N-diisopropylethylamine. By contrast, the TCP group can be conveniently removed by hydrazine or ethylenediamine in DMF. For example, the TCP group can be completely removed with hydrazine/DMF (3: 17) at 35°C for 30 minutes or with ethylenediamine/DMF (1: 200) at 50°C for 30 minutes. However, treatment of TCP-protected amines with methylamine or with diamines does not lead to the deprotected amines, but the corresponding N,N’-disubstituted tetrachlorophthal-amides. This observation has been harnessed to prepare linear and macrocyclic peptide-arene hybrids with 1,3-diaminopropane/DMF (1:49) at 25°C for 5 minutes. Due to the four electron-withdrawing chlorine atoms at the TCP, the removal of such group is much milder than its analogous phthaloyl (Pht) that is commonly used in the preparation of the primary amines. 4.6.1 PREPARATION OF C(DAP-TCP-GLN) [158]

Upon removal of the Fmoc group from Fmoc-PAL-PEG-PS resin (100 mg, 0.16 mmol/g), 21.0 mg of TCP-Glu-OAl (3 equiv., i.e.,

264

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

(S)-5-(allyloxy)-5-oxo-4-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-yl) pentanoic acid) was introduced using 22 μL of N,N’-diisopropylcarbodiimide (3 equiv.) and 6 mg of 1-hydroxy-7-azabenzotriazole (HOAt, 3 equiv.) in 200 μL of DMF for 4 hours, followed by washing with DMF (5 × 1 min). The resin was next treated with 1,3-diaminopropane/DMF (1: 49) at 25°C for 5 minutes, and washed with DMF (6 × 1 min). The C-terminal allyl ester was cleaved by treatment with 92 mg of Pd(PPh3)4 (5 equiv.) in 1.5 mL of CHCl3/HOAc/NMM (37:2:1) under nitrogen at 25°C for 3 hours, and the resin was washed with THF (3 × 2 min), DMF (3 × 2 min), DIEA/ CH2Cl2 (1:19, 3 × 2 min), sodium N,N-diethyldithiocarbamate (0.03 M in DMF, 3 × 15 min), DMF (10 × 1 min), CH2Cl2 (3 × 2 min), and DMF (3 × 1 min). An aliquot of the resultant H-DAP-TCP-Glu-(PAL-PEG-PS)-OH resin (10 mg) was cleaved with TFA/H2O (19:1) for 2 hours. The filtrate from the cleavage reaction was collected and dried to give H-DAP-TCP-Gln-OH, which was characterized by analytical HPLC (tR = 13.2 min, 95% purity, 88% yield; condition A), and ESI-MS (C16H18Cl4N4O5, m/z calcd. for 486.5; found 487.3 [M + H]+). The bulk of the resin was subjected to cyclization by treatment with 37 mg of PyAOP (5 equiv.), 10 mg of HOAt (5 equiv.), and 25 μL of DIEA (10 equiv.) in NMP. After 6 hours at 25°C, the resin was negative to the Kaiser ninhydrin test. Treatment with TFA/H2O (19:1) for 2 hours gave the cyclic peptide-arene hybrid, which was characterized by analytical HPLC (tR = 14.6 min, 99% purity, 91% yield). KEYWORDS • • • • • • •

acid-labile protecting group base-labile protecting group Fmoc group hydrazine hydrogenolysis t-Boc thiolysis

REFERENCES 1.

Carreño, C., Méndez, M. E., Kim, Y. D., Kim, H. J., Kates, S. A., Andreu, D., & Albericio, F., (2000). Nsc and Fmoc Nα-amino protection for solid-phase peptide synthesis: A parallel study. J. Peptide Res., 56, 63–69.

Amino Protecting Groups 265

2.

3. 4.

5. 6.

7.

8. 9. 10.

11.

12. 13. 14.

15. 16.

17.

Sabirov, A. N., Kim, Y. D., Kim, H. J., & Samukov, V. V., (1998). Fmoc- and Nsc-groups as a base labile N(α)-amino protection: A comparative study in the automated SPPS. Protein and Peptide Letters, 5(2), 57–62. Fukuda, T., Kitada, C., & Fujino, M., (1978). p-tolylmethylsulphonyl: A new aminoprotecting group in peptide synthesis. J. Chem. Soc., Chem. Commun., 220–221. Gormanns, M., & Ritter, H., (1994). Polymeric protecting groups. 6. Synthesis of a novel N-ethenoxyamino-modified tert-butoxycarbonyl-type amino protecting group. Macromolecules, 27, 5221–5228. Vorbruggen, H., (2008). tert-butoxycarbonyl chloride for the introduction of N-Boc groups into amino acids. Synthesis, (23), 3739–3741. Sakakisbara, S., Honda, I., Takada, K., Miyoshi, M., Ohnishi, T., & Okumura, K., (1969). tert-amyloxycarbonyl as a new protecting group in peptide synthesis. V. Direct synthesis of tert-amyloxycarbonyl and tert-butyloxycarbonyl amino acids from the respective tertalkyl chloroformates. Bull. Chem. Soc. Jpn., 42(3), 809–811. Loffet, A., Galeotti, N., Jouin, P., & Castro, B., (1989). Tert-butyl esters of N-protected amino acids with tert-butyl fluorocarbonate (Boc-F). Tetrahedron Lett., 30(49), 6859–6860. Grzonka, Z., & Lammek, B., (1974). Simple method of preparation of tert-butoxycarbonyl amino acids. Synthesis, (9), 661–662. Schwyzer, R., Sieber, P., & Kappeler, H., (1959). N-tert-butyloxycarbonylamino acids. Helvetica Chimica Acta, 42, 2622–2624. Yamashiro, D., Noble, R. L., & Li, C. H., (1973). Human pituitary growth hormone. 36. Solid-phase synthesis of the carboxyl-terminal cyclic dodecapeptide. J. Org. Chem., 38(20), 3561–3565. Barcelo, G., Senet, J. P., & Sennyey, G., (1985). 1,2,2,2-tetrachloroethyl tert-butyl carbonate: A simple and efficient reagent for the tert-butoxycarbonylation of amines and amino acids. J. Org. Chem., 50(20), 3951–3953. Carpino, L. A., (1973). New amino-protecting groups in organic synthesis. Acc. Chem. Res., 6, 191–198. Schnabel, E., (1967). Improved synthesis of tert-butoxycarbonyl amino acids by a constant pH reaction. Liebigs Ann. Chem., 702, 188–196. Houghten, R. A., Beckman, A., & Ostresh, J. M., (1986). Use of 10% Sulfuric acid/ dioxane for removal of N-α-tertiary-butyloxycarbonyl group during solid-phase peptide synthesis. Int. J. Peptide Protein Res., 27(6), 653–658. Gharahdaghi, F., Mathers, J., & Mische, S. M., (1992). In: Angeletti, R. H., (ed.), TFMSA/TFA Cleavage in t-Boc Peptide Synthesis (pp. 199–208). Tech. Protein Chem. 3. Hartwig, S., Nguyen, M. M., & Hecht, S., (2010). Exponential growth of functional poly(glutamic acid) dendrimers with variable stereochemistry. Polymer Chemistry, 1(1), 69–71. Tana, G., Kitada, S., Fujita, S., Okada, Y., Kim, S., & Chiba, K., (2010). A practical solution-phase synthesis of an antagonistic peptide of TNF-α based on hydrophobic tag strategy. Chem. Commun., 46(43), 8219–8221.

266

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

18. Voelter, W., & Kalbacher, H., (1993). Investigations with selective deblocking reagents for Adpoc-protected amino acids and peptides. Liebigs Ann. Chem., (2), 131–136. 19. Kalbacher, H., & Voelter, W., (1981). 1-(1-adamantyl)-1-methylethoxycarbonyl (Adpoc): A new useful amino protecting group. Structure and Activity of Natural Peptides, 383–396. 20. Shao, J., Krauss, S., Shekhani, M. S., & Voelter, W., (1990). In: Giralt, E., & Andreu, D., (eds.), A Test Case for the 1-(1-Adamantyl)-1-Methylethoxycarbonyl (Adpoc) Group: Solid-Phase Synthesis of LH-RH Using the Adpoc Strategy with an Acid-Labile Amide Linkage, Pept. (pp. 29, 30). 21. Voelter, W., (1980). Advances in and comparison of protective groups for peptide synthesis. Miles International Symposium Series, 12(Polypept. Horm.), 135–147. 22. Marburg, S., Neckers, A. C., & Griffin, P. R., (1996). Introduction of the maleimide function onto resin-bound peptides: A simple, high-yield process useful for discriminating among several lysines. Bioconjugate Chemistry, 7(5), 612–616. 23. Kalbacher, H., & Voelter, W., (1984). Substituted Carbonic Acid Esters. United States Patent, US 4440692 A. 24. Nyasse, B., & Ragnarsson, U., (1993). Synthesis and application of N,N-bis-(1adamantyloxycarbonyl) amino acids. Acta Chem. Scand., 47, 374–379. 25. Haas, W. L., Krumkalns, E. V., & Gerzon, K., (1966). Adamantyloxycarbonyl, a new blocking group. Preparation of 1-adamantyl chloroformate. J. Am. Chem. Soc., 88(9), 1988–1992. 26. Benedetti, E., Pedone, C., Toniolo, C., Nemethy, G., Pottle, M. S., & Scheraga, H. A., (1980). Preferred conformation of the tert-butoxycarbonylamino group in peptides. Int. J. Peptide Protein Res., 16(2), 156–172. 27. Miyoshi, M., & Onishi, T., (1970). tert-amyloxycarbonylation of amino acids. Jpn. Tokkyo Koho. JP 45024766 B4. 28. Sakakibara, S., & Ito, M., (1967). Tert-Amyloxycarbonyl as a new protecting group in peptide synthesis. III. Unexpected side reaction during the synthesis of tertamyloxycarbonylamino acids. Bull. Chem. Soc. Jpn., 40, 646–649. 29. Honda, I., Shimonishi, Y., & Sakakibara, S., (1967). Tert-amyloxycarbonyl as a new protecting group in peptide synthesis. IV. Synthesis and use of tert-amyl azidoformate. Bull. Chem. Soc. Jpn., 40(10), 2415–2418. 30. Sakakibara, S., Shin, M., Fujino, M., Shimonishi, Y., Inoue, S., & Inukai, N., (1965). Tert-amyloxycarbonyl as a new protecting group in peptide synthesis. I. Synthesis and properties of N-tert-amyloxycarbonylamino acids and related compounds. Bull. Chem. Soc. Jpn., 38(9), 1522–1525. 31. Ajinomoto Co., Inc., (1967). Tert-Amyloxycarbonyl Group for Protecting the Amino or Imino Group in Amino Acids during Peptide Synthesis. French Patent Office, FR 1447532. 32. Rzeszotarska, B., & Wiejak, S., (1968). Synthesis of N-tert-butyloxycarbonyl- and N-tert-amyloxycarbonyl amino acids. Angew. Chem., Int. Ed. Engl., 7(5), 379, 380.

Amino Protecting Groups 267

33. Pozdnev, V. F., (1978). Synthesis of tert-amyloxycarbonyl derivatives of amino acids using di-tert-amyl pyrocarbonate. Bioorganicheskaya Khimiya, 4(9), 1273, 1274. 34. Pozdnev, V. F., (1979). N-tert-Amyloxycarbonyl Derivatives of Amino Compounds. U.S.S.R., SU 696010 A1. 35. Bailey, W. J., & Griffith, J. R., (1964). Thermal removal of N-tert-alkoxycarbonyl blocking groups of amino acids and peptides. Polymer Preprints (American Chemical Society, Division of Polymer Chemistry), 5(1), 279–290. 36. Sakakibara, S., (1968). Tertiary-amyloxy carbonyl amino acids. Jpn. Tokkyo Koho, JP 43006931 B. 37. Mueller, J., & Voelter, W., (1984). In 2-(3,5-Di-tert-butylphenyl)propyloxycarbonyl (t-Bumeoc) and 3,5-di-tert-butylbenzyloxycarbonyl (Dbz) amino acids and their use for syntheses of peptide hormones. In: Voelter, W., & Daves, D. G., (eds.), Biol. Act. Princ. Nat. Prod. (pp. 176–186). 38. Voelter, W., Breipohl, G., Tzougraki, C., & Jungfleisch-Turgut, E., (1992). Solid phase synthesis of a somatostatin amide analog using acid labile tert-bumeoc protection and an acid labile anchor group. Coll. Czech. Chem. Commun., 57(8), 1707–1718. 39. Voelter, W., Mueller, J., Heinzel, W., & Beni, C. T., (1983). In 2-(3,5-di-tert-butylphenyl) propyloxycarbonyl (t-Bumeoc), 3,5-di-tert-butylbenzyloxycarbonyl (Dbz), and 2-adamantylpropyloxycarbonyl (Adpoc) amino acids and their use for peptide syntheses. In: Hruby, V. J., & Rich, D. H., (eds.), Pept.: Struct. Funct., Proc. Am. Pept. Symp. 8th (pp. 131–134). 40. Voelter, W., & Mueller, J., (1983). The 1-(3,5-di-tert-butylphenyl)-1-methylethoxycarbonyl (T-Bumeoc) residue, a novel extremely acid-labile amino protecting group for peptide syntheses. Liebigs Ann. Chem., (2), 248–260. 41. Jungfleisch, E., Kalbacher, H., Voelter, W., & Tzougraki, C., (1989). In synthesis of a somatostatin analog with the acid-labile T-bumeoc amino protecting group. Pept., Proc. Eur. Pept. Symp., (pp. 73–75). 42. Voelter, W., Mueller, J., Beni, C., Heinzel, W., Kalbacher, H., Morel, A., & Wollmann, H., (1983). In the application of recently developed amino protecting groups in peptide synthesis. Petr, Pept., Proc. Eur. Pept. Symp., 17th (pp. 125–128). 43. Nishiyama, Y., & Okada, Y., (1994). Temporary Nα-deprotection/protection procedure to facilitate the purification of protected peptide fragments for use in convergent solidphase peptide synthesis. Tetrahedron Lett., 35(40), 7409–7412. 44. Mukherjee, A. K., & Agosta, W. C., (1994). New amino-protecting group, 2-adamantyloxycarbonyl (2-Adoc) and its application to the synthesis of protected peptides. Chemtracts-Organic Chemistry, 7, 415, 416. 45. Okada, Y., Joshi, S., Shintomi, N., Kondo, Y., Tsuda, Y., Ohgi, K., & Irie, M., (1999). Amino acids and peptides. LIV. Application of 2-adamantyl derivatives as protecting groups in the synthesis of peptide fragments related to sulfolobus solfataricus ribonuclease. I. Chem. Pharm. Bull., 47(8), 1089–1096.

268

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

46. Nishiyama, Y., Shintomi, N., Kondo, Y., & Okada, Y., (1994). Amino Acids and peptides, part 38. Development of a new amino-protecting group, 2-adamantyloxycarbonyl, and its application to peptide synthesis. J. Chem. Soc., Perkin Trans. 1, 3201–3207. 47. Okada, Y., Shintomi, N., Kondo, Y., Yokoi, T., Joshi, S., & Li, W., (1997). Amino acids and peptides, LI. Application of the 2-adamantyloxycarbonyl (2-Adoc) group to the protection of the hydroxyl function of tyrosine in peptide synthesis. Chem. Pharm. Bull., 45(11), 1860–1864. 48. Nishiyama, Y., Shintomi, N., Kondo, Y., Izumi, T., & Okada, Y., (1995). Amino acids and peptides. Part 42. Application of the 2-adamantyloxycarbonyl (2-Adoc) group to the protection of the imidazole function of histidine in peptide synthesis. J. Chem. Soc., Perkin Trans. 1, (18), 2309–2313. 49. Nishiyama, Y., Shintomi, N., Kondo, Y., & Okada, Y., (1994). Application of the 2-adamantyloxycarbonyl (2-Adoc) group to the protection of the imidazole function of histidine in peptide synthesis. J. Chem. Soc., Chem. Commun., (21), 2515, 2516. 50. Horwell, D. C., Aranda, J., Augelli-Szafran, C. E., Betche, H. J., Holmes, A., Mullican, M. D., Pritchard, M. C., et al., (1992). Amino acid analogs as CCK antagonists. PCT Int. Appl., WO 9204025 A1. 51. Araldi, G., Donati, D., Oliosi, B., Ursini, A., Van, A. F., Natalini, B., Pellicciari, R., & Tarzia, G., (1996). Synthesis and receptor binding affinity of cholecystokinin receptor ligands: 2- and 1-indolyl derivatives of PD134308. Farmaco, 51(7), 471–476. 52. Jäger, G., & Geiger, R., (1971). Isobornyloxycarbonylamino acids and their preparation methods. Ger. Offen., DE 2013033 A. 53. Fujino, M., Shinagawa, S., Nishimura, O., & Fukuda, T., (1972). Isobornyloxycarbonyl function, a new convenient amino-protecting group in peptide synthesis. I. Synthesis and properties of isobornyloxycarbonylamino acids. Chem. Pharm. Bull., 20(5), 1017–1020. 54. Jäger, G., & Geiger, R., (1973). Isobornyloxycarbonyl residue as a new protecting group for the amino function. Justus Liebigs Annalen der Chemie, 9, 1535–1544. 55. Jäger, G., & Geiger, R., (1973). In isobornyloxycarbonyl group as new amino- and guanidino-protecting groups. In: Nesvadba, H., (ed.), Pept., Proc. Eur. Pept. Symp., 11th (pp 78–81). 56. Jäger, G., & Geiger, R., (1971). Isobornyloxycarbonylamino Acids and Their Preparation Methods. German Patent, DE 2013033A1. 57. Barlos, K., Papaioannou, D., & Theodoropoulos, D., (1982). Efficient “one-pot” synthesis of N-trityl amino acids. J. Org. Chem., 47, 1324–1326. 58. De La Torte, B. G., Marcos, M. A., Eritja, R., & Albericio, F., (2002). Solid-phase peptide synthesis using N-trityl-amino acids. Letters in Peptide Science, 8, 331–338. 59. Megumi, K., Arif, F. N. B. M., Matsumoto, S., & Akazome, M., (2012). Design and evaluation of salts between N‑trityl amino acid and tert-butylamine as inclusion crystals of alcohols. Cryst. Growth Des., 12, 5680–5685. 60. Barlos, K., Papaioannou, D., & Theodoropoulos, D., (1984). Preparation and properties of Nα-trityl amino acid 1-hydroxybenzotriazole esters. Int. J. Peptide Protein Res., 23, 300–305.

Amino Protecting Groups 269

61. Zervas, L., & Theodoropoulos, D. M., (1956). N-tritylamino acids and peptides. A new method of peptide synthesis. J. Am. Chem. Soc., 78, 1359–1363. 62. Mutter, M., & Hersperger, R., (1989). Efficient synthesis of N-triphenylmethyl α-amino acids. Synthesis, (3), 198–200. 63. Maltese, M., (2007). Tritylation reactions based on metallic catalysts. PCT Int. Appl. WO 2007009944 A1. 64. Matysiak, S., Böldicke, T., Tegge, W., & Frank, R., (1998). Evaluation of monomethoxytrityl and dimethoxytrityl as orthogonal amino protecting groups in Fmoc solid-phase peptide synthesis. Tetrahedron Lett., 39, 1733, 1734. 65. Roviello, G. N., Gröschel, S., Pedone, C., & Diederichsen, U., (2010). Synthesis of novel MMT/Acyl-protected Nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras. Amino Acids, 38, 1301–1309. 66. Henderson, A. P., Riseborough, J., Bleasdale, C., Clegg, W., Elsegood, M. R. J., & Golding, B. T., (1997). 4,4’-dimethoxytrityl and 4,4’,4”-trimethoxytrityl as protecting groups for amino functions; Selectivity for primary amino groups and application in 15N-labelling. J. Chem. Soc., Perkin Trans. 1, 3407–3413. 67. Gormanns, M., & Ritter, H., (1995). Modified tert-butoxycarbonyl (m-BOC) derivatives as monomeric and polymeric amino protecting groups – VII. Tetrahedron, 51(12), 3525–3534. 68. Fujino, M., Wakimasu, M., & Kitada, C., (1982). 4-methoxy-2,3,6trimethylbenzenesulfonyl (Mtr): A new amino-protecting group in peptide synthesis. J. Chem. Soc., Chem. Commun., 445–446. 69. Wünsch, E., Moroder, L., Nyfeler, R., & Jaeger, E., (1982). (Alkyldithio)carbonyl groups for protection of amino functions in peptide synthesis. Hoppe-Seyler’s Z. Physiol. Chem., 363, 197–202. 70. Jeschkeit, H., Losse, G., & Neubert, K., (1966). Peptide synthesis with the furfuryloxycarbonyl group as an amino-protecting group. Chemische Berichte, 99(9), 2803–2812. 71. Weinstein, B., & Steiner, P. A., (1979). In: Gross, E., & Meienhofer, J., (eds.), A New Amino Protecting Group, the Azo-Tac Unit, Pept., Struct. Biol. Funct., Proc. Am. Pept. Symp. 6th, (pp. 329–331). 72. Fukuda, T., & Fujino, M., (1978). p-xylyl-α-sulfonyl: A new amino-protecting group in peptide synthesis. Peptide Chemistry, 15, 19–22. 73. Carpino, L. A., & Han, G. Y., (1970). The 9-fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc., 92(19), 5748, 5749. 74. Ishibashi, Y., Miyata, K., & Kitamura, M., (2010). (9H-fluoren-9-yl)methanesulfonyl (Fms): An amino protecting group complementary to Fmoc. Eur. J. Org. Chem., 4201–4204. 75. Carpino, L. A., & Han, G. Y., (1972). The 9-fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem., 37(22), 3404–3409.

270

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

76. Atherton, E., Bury, C., Sheppard, R. C., & Williams, B. J., (1979). Stability of fluorenylmethoxycarbonylamino groups in peptide synthesis. Cleavage by hydrogenolysis and by dipolar aprotic solvents. Tetrahedron Lett., (32), 3041, 3042. 77. Galeazzi, R., Martelli, G., Marcucci, E., Orena, M., Rinaldi, S., Lattanzi, R., & Negri, L., (2010). Analogues of both leu- and met-enkephalin containing a constrained dipeptide isostere prepared from a Baylis-hillman adduct. Amino Acids, 38, 1057–1065. 78. Katritzky, A. R., Tala, S. R., Abo-Dya, N. E., Ibrahim, T. S., El-Feky, S. A., Gyanda, K., & Pandya, K. M., (2011). Chemical ligation of S-acylated cysteine peptides to form native peptides via 5-, 11-, and 14-membered cyclic transition states. J. Org. Chem., 76, 85–96. 79. Eerland, M. F., & Hedberg, C., (2012). Design and synthesis of an Fmoc-SPPScompatible amino acid building block mimicking the transition state of phosphohistidine phosphatase. J. Org. Chem., 77, 2047–2052. 80. Zinieris, N., Leondiadis, L., & Ferderigos, N., (2005). Nα-Fmoc removal from resinbound amino acids by 5% piperidine solution. J. Comb. Chem., 7, 4–6. 81. Hwang, S. H., Lehman, A., Cong, X., Olmstead, M. M., Lam, K. S., Lebrilla, C. B., & Kurth, M. J., (2004). OBOC small-molecule combinatorial library encoded by halogenated mass-tags. Org. Lett., 6(21), 3829–3832. 82. Carpino, L. A., Mansour, E. M. E., & Knapczyk, J., (1983). Piperazine-functionalized silica gel as a deblocking-scavenging agent for the 9-fluorenylmethyloxycarbonyl amino-protecting group. J. Org. Chem., 48, 666–669. 83. Carpino, L. A., Mansour, E. M. E., Cheng, C. H., Williams, J. R., MacDonald, R., Knapczyk, J., Carman, M., & Lopusinski, A., (1983). Polystyrene-based deblockingscavenging agents for the 9-fluorenylmethyloxycarbonyl amino-protecting group. J. Org. Chem., 48, 661–665. 84. Atherton, E., & Sheppard, R. C., (1987). The fluorenylmethoxycarbonyl amino protecting group. The Peptides, 9, 1–38. 85. Ibrahim, T. S., Tala, S. R., El-Feky, S. A., Abdel-Samii, Z. K., & Katritzky, A. R., (2012). Cysteinoyl- and cysteine-containing dipeptidoylbenzotriazoles with free sulfhydryl groups: Easy access to N-terminal and internal cysteine peptides. Chem Biol Drug Des, 80(2), 194–202. 86. Bionda, N., Cudic, M., Barisic, L., Stawikowski, M., Stawikowska, R., Binetti, D., & Cudic, P., (2012). A practical synthesis of Nα-Fmoc protected L-threo-β-hydroxyaspartic acid derivatives for coupling via α- or β-carboxylic group. Amino Acids, 42, 285–293. 87. Boll, E., Dheur, J., Drobecq, H., & Melnyk, O., (2012). Access to cyclic or branched peptides using bis(2-sulfanylethyl)amido side-chain derivatives of Asp and Glu. Org. Lett., 14, 2222–2225. 88. Shu, L., & Wang, P., (2008). Synthesis of enantiopure Fmoc-α-methylvaline. Org. Proc. Res. Dev., 12, 298–300. 89. Vlahov, I. R., Santhapuram, H. K. R., You, F., Wang, Y., Kleindl, P. J., Hahn, S. J., Vaughn, J. F., et al., (2010). Carbohydrate-based synthetic approach to control toxicity profiles of folate-drug conjugates. J. Org. Chem., 75, 3685–3691.

Amino Protecting Groups 271

90. Gupta, S., & Schafmeister, C. E., (2009). Synthesis of a carboxylate functionalized bis-amino acid monomer. J. Org. Chem., 74, 3652–3658. 91. Brückner, H., Flassig, S., & Kirschbaum, J., (2012). Determination of biogenic amines in infusions of tea (camellia sinensis) by HPLC after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Amino Acids, 42, 877–885. 92. Ramage, R., Jiang, L., Kim, Y. D., Shaw, K., Park, J. L., & Kim, H. J., (1999). Comparative studies of Nsc and Fmoc as Nα-protecting groups for SPPS. J. Peptide Sci., 5, 195–200. 93. Rzepecki, P., Gallmeier, H., Geib, N., Cernovska, K., König, B., & Schrader, T., (2004). New heterocyclic β-sheet ligands with peptidic recognition elements. J. Org. Chem., 69(16), 5168–5178. 94. Kader, A. T., & Stirling, C. J. M., (1964). Elimination-addition. III. New procedures for the protection of amino groups. J. Chem. Soc., 258–266. 95. Tesser, G. I., & Balvert-Geers, I. C., (1975). The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function. Int. J. Peptide Protein Res., 7, 295–305. 96. Boon, P. J., Tesser, G. I., & Nivard, R. J. F., (1979). Semisynthetic horse heart [65-homoserine] cytochrome C from three fragments. Proc. Nati. Acad. Sci. USA, 76(1), 61–65. 97. Geiger, R., Obermeier, R., & Tesser, G. I., (1975). Methylsulfonylethyloxycarbonyl residue as a reversible amino protecting group for insulin. Chemische Berichte, 108(8), 2758–2763. 98. Filippov, D., Van, D. M. G. A., Kuyl-Yeheskiely, E., & Van, B. J. H., (1994). Methylsulfonylethyloxycarbonyl group as a protection for the guanidino function in arginine. Synlett, 922, 923. 99. Stöcklin, R., Rose, K., Green, B. N., & Offord, R. E., (1994). The semisynthesis of [octadeutero-PheB1-octadeutero-ValB2]-porcine insulin and its characterization by mass spectrometry. Protein Engineering, 7(2), 285–289. 100. Büllesbach, E. E., Schmitt, E. W., & Gattner, H. G., (1982). Preparation of partially protected Des-alanineB30-insulin-B-chain-disulphide and its use in semisynthesis. Int. J. Peptide Protein Res., 20, 207–217. 101. Smeets, P., Granger, M., Van, N. J. W., Bloemendal, H., & Tesser, G. I., (1977). Des-NαIacetyl-α-melanotropin, a synthetic substrate for specific N-terminal directed enzymatic acetylation. Int. J. Peptide Protein Res., 9, 52–56. 102. Ten, K. P. B. W., Adams, P. J. H. M., & Tesser, G. I., (1985). Semisynthesis of horse heart cytochrome c analogues from two or three fragments. Proc. Nati. Acad. Sci. USA, 82(24), 8279–8283. 103. Carpino, L. A., Cohen, B. J., Lin, Y. Z., Stephens, K. E., & Triolo, S. A., (1990). The 2-chloro-3-indenylmethyloxycarbonyl and Benz[f]inden-3-ylmethyloxycarbonyl basesensitive amino-protecting groups, application to an inverse Merrifield approach to peptide synthesis. J. Org. Chem., 55, 251–259. 104. Carpino, L. A., Ismail, M., Truran, G. A., Mansour, E. M. E., Iguchi, S., Ionescu, D., El-Faham, A., et al., (1999). The 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc) amino-protecting group. J. Org. Chem., 64, 4324–4338.

272

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

105. Carpino, L. A., & Mansour, E. M. E., (1999). The 2-methylsulfonyl-3-phenyl-1-prop-2enyloxycarbonyl (Mspoc)amino-protecting group. J. Org. Chem., 64, 8399–8401. 106. Carpino, L. A., & Philbin, M., (1999). Amino-protecting groups subject to deblocking under conditions of nucleophilic addition to a Michael acceptor. Structure-reactivity studies and use of the 2-(tert-butylsulfonyl)-2-propenyloxycarbonyl (Bspoc) group. J. Org. Chem., 64, 4315–4323. 107. Carpino, L. A., Chao, H. G., Beyermann, M., & Bienert, M., (1991). [(9-fluorenylmethyl) oxy]carbonyl (Fmoc) amino acid chlorides in solid-phase peptide synthesis. J. Org. Chem., 56(8), 2635–2642. 108. Park, J. I., Lee, S. H., Lee, Y. S., & Kim, H. J., (1999). Facile synthesis of 2-(4-Nitrophenylsulfonyl)ethoxycarbonyl-O-t-butyl-L-serine, threonine, and tyrosine. Bull. Korean Chem. Soc., 20(2), 244–246. 109. Ramage, R., Blake, A. J., Florence, M. R., Gray, T., Raphy, G., & Roach, P., (1991). A Base Labile protecting group for peptide synthesis: 2,2-bis(4-nitrophenyl)ethan-1oxycarbonyl urethanes. Tetrahedron, 47, 8001–8024. 110. Sabirov, A. N., Ofitserov, V. I., Shval’e, A. F., & Samukov, V. V., (1987). 2-(4-chlorophenyl) sulfonylethoxycarbonyl (Cps) as a new amino protecting group in liquid phase peptide synthesis: A synthesis of [Leu]enkephalin and Its [D-Ala2] analog. Bioorganicheskaya Khimiya, 13(6), 754–759. 111. Wakselman, M., & Guibe-Jampel, E., (1973). Alkali-labile substituted benzyloxycarbonyl amino-protecting group. J. Chem. Soc., Chem. Commun., (16), 593, 594. 112. Choi, J. S., Lee, H. S., Lee, Y., Jeong, N., Kim, H. J., Kim, Y. D., & Han, H., (2002). Nsc-mediated solid-phase synthesis of polyamides containing pyrrole amino acid. Tetrahedron Lett., 43(24), 4295–4299. 113. Choi, J. S., Lee, Y., Kim, E., Jeong, N., Yu, H., & Han, H., (2003). The 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc) amino-protecting group: Use in the solid-phase synthesis of pyrrole-imidazole polyamides. Tetrahedron Lett., 44, 1607–1610. 114. Ramage, R., & Raphy, G., (1992). Design of an affinity-based Nα-amino protecting group for peptide synthesis: Tetrabenzo[a,c,g,i]fluorenyl-17-methyl urethanes (Tbfmoc). Tetrahedron Lett., 33(3), 385–388. 115. Bergmann, M., & Zervas, L., (1932). A general process for the synthesis of peptides. Reports of the German Chemical Society (Berichte der Deutschen Chemischen Gesellschaft), 65B, 1192–1201. 116. Ben-Ishai, D., & Berger, A., (1952). Cleavage of N-carbobenzoxy groups by dry hydrogen bromide and hydrogen chloride. J. Org. Chem., 17(12), 1564–1570. 117. Mitchell, A. R., & Merrifield, R. B., (1976). Occurrence of N-alkylation during the acidolytic cleavage of urethane protecting groups. J. Org. Chem., 41(11), 2015–2019. 118. Kiso, Y., Ukawa, K., & Akita, T., (1980). Efficient removal of n-benzyloxycarbonyl group by A ‘push-pull’ mechanism using thioanisole-trifluoroacetic acid, exemplified by A synthesis of met-enkephalin. J. Chem. Soc., Chem. Commun., (3), 101–102. 119. Ragnarsson, U., Karlsson, S., & Lindeberg, G., (1970). Amino group protection in solidphase peptide synthesis. Acta Chem. Scand., 24, 2821–2825.

Amino Protecting Groups 273

120. Sakakibara, S., Shimonishi, Y., Kishida, Y., Okada, M., & Sugihara, H., (1967). Use of anhydrous hydrogen fluoride in peptide synthesis. I. Behavior of various protective groups in anhydrous hydrogen fluoride. Bull. Chem. Soc. Jpn., 40(9), 2164–2167. 121. Felix, A. M., (1974). Cleavage of protecting groups with boron tribromide. J. Org. Chem., 39(10), 1427–1429. 122. Isidro-Llobet, A., Guasch-Camell, J., Álvarez, M., & Albericio, F., (2005). p-nitrobenzyloxycarbonyl (pNZ) as a temporary Nα-protecting group in orthogonal solid-phase peptide synthesis – avoiding diketopiperazine and aspartimide formation. Eur. J. Org. Chem., 3031–3039. 123. Baldwin, J. E., Herchent, S. R., & Singh, P. D., (1980). Syntheses of penicillin N, [6a-3H] penicillin N and [10-14C,6a-3H]penicillin N. Biochem. J., 186, 881–887. 124. Gowda, D. C., (2002). Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation with formic acid and 10% palladium on carbon. Indian J. Chem., 41B, 1064–1067. 125. Gowda, D. C., Rajesh, B., & Gowda, S., (2000). Ammonium formate catalytic transfer hydrogenation: A convenient method for removal of halogenated benzyloxycarbonyl and benzyl protecting groups in peptide synthesis. Indian J. Chem., 39B, 504–508. 126. Kanaoka, M., & Kurata, Y., (1978). Chemical studies on amino acids and peptides. III. 4,5-diaryl-4-oxazolin-2-one derivatives as an amino protecting group. Chem. Pharm. Bull., 26(2), 660–664. 127. Carpino, L. A., & Tunga, A., (1986). The ((β-phenylethy1)oxy)carbonyl (“homobenzyloxycarbonyl”, hZ) amino-protecting group. J. Org. Chem., 51, 1930–1932. 128. Veber, D. F., Paleveda, W. J., Lee, Y. C., & Hirschmann, R., (1977). Isonicotinyloxycarbonyl, a novel amino protecting group for peptide synthesis. J. Org. Chem., 42(20), 3286–3288. 129. Zhang, Y., Wang, X., Li, L., & Zhang, P., (1986). p-trimethylammonium chloridobenzyloxycarbonyl as a water-soluble amino protecting group. Scientia Sinica (Series B), 29(10), 1009–1017. 130. Gillessen, D., & Trzeciak, A., (1981). Application of a new amino protecting group in a synthesis of lysine-vasopressin. In: Schlesinger, D. H., (ed.), Neurohypophyseal Peptide Hormones and Other Biologically Active Peptides (Vol. 13, pp. 37–47). 131. Carpino, L. A., & Nowshad, F., (1993). The redox-sensitive, colored N-3-(3’,6’-dioxo2’,4’,5’-trimethylcycloa-1’,4’-diene)-3,3-dimethylpropionyl (Q) amino-protecting group. Tetrahedron Lett., 34(44), 7009–7012. 132. Mandal, P. K., & McMurray, J. S., (2007). Pd-C-induced catalytic transfer hydrogenation with triethylsilane. J. Org. Chem., 72, 6599–6601. 133. Wünsch, E., Graf, W., Keller, O., Keller, W., & Wersin, G., (1986). On the synthesis of benzyloxycarbonyl amino acids. Synthesis, (11), 958–960. 134. Carpino, L. A., Mansour, E. S. M. E., & Sadat-Aalsee, D., (1991). Tert-butyloxycarbonyl and benzyloxycarbonyl amino acid fluorides. New, stable rapid-acting acylating agents for peptide synthesis. J. Org. Chem., 56, 2611–2614.

274

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

135. Kawahata, N., Weisberg, M., & Goodman, M., (1999). Synthesis of β,β-dimethylated amino acid building blocks utilizing the 9-phenylfluorenyl protecting group. J. Org. Chem., 64, 4362–4369. 136. Taylor-Papadimitriou, J., Yovanidis, C., Paganou, A., & Zervas, L., (1967). New methods in peptide synthesis. Part Vl. On α- and γ-diphenylmethyl and phenacyl esters of L-glutamic acid. J. Chem. Soc. (C), 1830–1836. 137. Sennyey, G., Barcelo, G., & Senet, J. P., (1986). Synthesis and use of dibenzylpyrocarbonate: Preparation of dipeptide free N-benzyloxycarbonyl glycine. Tetrahedron Lett., 27(44), 5375, 5376. 138. Kumar, V. P., Reddy, M. S., Narender, M., Surendra, K., Nageswar, Y. V. D., & Rao, K. R., (2006). Aqueous phase mono-protection of amines and amino acids as N-benzyloxycarbonyl derivatives in the presence of β-cyclodextrin. Tetrahedron Lett., 47, 6393–6396. 139. Lu, F. Z., Xiong, X. Y., Li, Z. C., Du, F. S., Zhang, B. Y., & Li, F. M., (2002). A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(ε-caprolactone). Bioconjugate Chemistry, 13, 1159–1162. 140. Felix, A. M., Jimenz, M. H., & Meienhofer, J., (1979). Removal of Nα-benzyloxycarbonyl groups from sulfur-containing peptides by catalytic hydrogenation in liquid ammonia: O-tert-butyl-L-seryl-S-tert-butyl-L-cysteine Tert-butyl ester. Organic Syntheses, 59, 159–169. 141. Krogh, J. A., (1985). N-Benzyloxycarbonyl Amino Acids Containing Additional Functionality. United State Patent, US 4500726A. 142. Shieh, W. C., Xue, S., Reel, N., Wu, R., Fitt, J., & Repič, O., (2001). An enantioselective synthesis of (R)-4-piperidinylglycine. Tetrahedron: Asymmetry, 12, 2421–2425. 143. Hanessian, S., Margarita, R., Hall, A., Johnstone, S., Tremblay, M., & Parlanti, L., (2002). Total synthesis and structural confirmation of the marine natural product dysinosin a: A novel inhibitor of thrombin and factor VIIa. J. Am. Chem. Soc., 124, 13342, 13343. 144. Carpenter, F. H., & Gish, D. T., (1952). The application of p-nitrobenzyl chloroformate to peptide Synthesis. J. Am. Chem. Soc., 74(15), 3818–3821. 145. Peluso, S., Dumy, P., Nkubana, C., Yokokawa, Y., & Mutter, M., (1999). Solid-phase strategies for the assembly of template-based protein mimetics. J. Org. Chem., 64(19), 7114–7120. 146. Shields, J. E., & Carpenter, F. H., (1961). Synthesis of a heptapeptide sequence derived from bovine insulin. J. Am. Chem. Soc., 83(14), 3066–3070. 147. Barany, G., & Merrifield, R. B., (1977). A new amino protecting group removable by reduction. Chemistry of the dithiasuccinoyl (Dts) function. J. Am. Chem. Soc., 99(22), 7363–7365. 148. Meinjohanns, E., Meldal, M., Paulsen, H., & Bock, K., (1995). Dithiasuccinoyl (Dts) amino-protecting group used in syntheses of 1,2-trans-amino sugar glycosides. J. Chem. Soc., Perkin Trans., 1, 405–415.

Amino Protecting Groups 275

149. Barany, G., & Merrifield, R. B., (1980). Kinetics and mechanism of the thiolytic removal of the dithiasuccinoyl (Dts) amino protecting group. J. Am. Chem. Soc., 102(9), 3084–3095. 150. Barany, G., & Albericio, F., (1985). A three-dimensional orthogonal protection scheme for solid-phase peptide synthesis under mild conditions. J. Am. Chem. Soc., 107, 4936–4942. 151. Chen, L., & Barany, G., (1996). N-dithiasuccinoyl (Dts)-glycine: A novel oxidation reagent for the formation of intramolecular disulfide bridges under mild conditions. Letters in Peptide Science, 3, 283–292. 152. Planas, M., Bardají, E., Jensen, K. J., & Barany, G., (1999). Use of the dithiasuccinoyl (Dts) amino protecting group for solid-phase synthesis of protected peptide nucleic acid (PNA) oligomers. J. Org. Chem., 64(20), 7281–7289. 153. Wexselblatt, E., Yavin, E., Katzhendler, J., Vaksman, O., & Reich, R., (2008). A new method for the synthesis of peptide nucleic acids. Collection Symposium Series: Chemistry of Nucleic Acid Components, 10, 471, 472. 154. Stern, M., Warshawsky, A., & Fridkin, M., (1979). Facile thiolytic removal of the o-nitrophenylsulfenyl amino-protecting group. Int. J. Peptide Protein Res., 13, 315–319. 155. Zervas, L., Borovas, D., & Gazis, E., (1963). New methods in peptide synthesis. I. Tritylsulfenyl and o-nitrophenylsulfenyl groups as N-protecting groups. J. Am. Chem. Soc., 85, 3660–3666. 156. Katakai, R., (1975). Peptide synthesis using o-nitrophenylsulfenyl n-carboxy α-amino acid anhydrides. J. Org. Chem., 40(19), 2697–2702. 157. Kawashiro, K., Yoshida, H., & Morimoto, S., (1977). The synthesis of α-aminonitriles starting from the corresponding amino acids. I. Use of o-nitrophenylsulfenyl as an N-protecting group. Bull. Chem. Soc. Jpn., 50(11), 2956–2960. 158. Cros, E., Planas, M., Barany, G., & Bardají, E., (2004). N-tetrachlorophthaloyl (TCP) protection for solid-phase peptide synthesis. Eur. J. Org. Chem., 3633–3642.

CHAPTER 5

Side Chain Protecting Groups

5.1 INTRODUCTION From the synthetic chemistry point of view, all α-amino acids with nonpolar side chains can be easily handled in the preparation/synthesis of peptides/proteins, by protecting either their carboxyl groups or amino groups. The protection of the carboxyl group has been covered in Chapter 3 and the protection of the amino group has been detailed in Chapter 4 of this book. Although so many protecting groups have been developed for the protection of the amino group, only two protecting groups are commonly used for the α-amino group, which are tert-butoxycarbonyl (t-Boc) and (((9H-fluoren-9-yl)methoxy)carbonyl) (Fmoc) protecting groups, corresponding to the two popular synthetic strategies of peptides [1]. These two strategies are t-Boc/Bn and Fmoc/t-Bu combinations, i.e., protection of the amino group with t-Boc and the carboxyl group with benzyl group in the former strategy where both protecting groups can be cleaved in the presence of acid or the benzyl group can be selectively removed by hydrogenolysis. In contrast, the amino group is protected with Fmoc and the carboxyl group is protected by the t-butyl group in the Fmoc/t-Bu strategy, where Fmoc can be removed in the presence of a base (e.g., 20% piperidine) whereas the t-Bu can be easily deblocked by acid, so that these two mutually orthogonal protecting groups can be removed separately. However, only a few amino acids are of non-polar side chains that include glycine, alanine, valine, leucine, isoleucine, methionine, proline, and phenylalanine. Other amino acids bearing additional functional groups can potentially complicate the synthetic outcomes, even though the conditions for peptide formation without the protection of amino acid side chains have been developed. Generally, the side chains of these amino acids can be classified as acidic, basic, and neutral but polar groups. The amino acids with acidic side chains include aspartic

278

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

acid (COOH, pKa = 4.25), glutamic acid (COOH, pKa = 3.65), cysteine (SH, pKa = 8.18) and tyrosine (phenol, pKa = 10.06). The amino acids with basic side chains are arginine (guanidino, pKa = 12.48), lysine (NH2, pKa = 10.52), and histidine (imidazole, pKa = 6.0), where the pKa values are the acidity constants of the corresponding conjugate acids of these functional groups. The side chain of these amino acids may participate in the reactions involving either amino group or carboxyl group during the formation of peptide bonds, or the deprotection of existing protecting group as well as cleavage of the peptides from the solid support, leading to the formation of byproducts. The amino acids with neutral polar side chains, such as serine (OH), threonine (OH), tryptophan (indole), asparagine (CONH2), glutamine (CONH2), may participate in other types of side reactions, such as acidic dehydration of threonine [2]. Similarly, the side chain of methionine can be easily oxidized to methylsulfonyl or methylsulfonyl group in the presence of oxidizing agents [3]. Therefore, many of these side chains should be protected prior to the formation of peptides involving these amino acids. In theory, the carboxyl side chain of aspartic acid and glutamic acids can be protected with any carboxyl protecting groups outlined in Chapter 3. However, in order to differentiate the reactivity between the α-carboxyl group and the side-chain carboxyl group, protecting group of different stability should be applied, depending on the actual synthetic route. Similarly, the side chain of lysine and arginine can be protected with any protecting groups suitable for the amino groups as collected in Chapter 4; protecting groups of different stability should be chosen for these side chains so that they can be retained while the α-amino protecting group is removed. Nevertheless, regardless of what protecting groups applied to the side chain, the conditions to remove these side-chain protecting groups should not lead to the cleavage of resulting peptides/proteins. Regarding the protection of functional groups in solution synthesis of large peptides, two different philosophies have arisen in the literature, i.e., minimal protection strategy and global protection strategy [4]. The minimal protection strategy has been applied when there is a difficulty in complete removal of side-chain protecting groups, and the peptides can potentially be damaged under the conditions of removing these side-chain protecting groups [5, 6]. This strategy is particularly useful in the segment condensation approach where the purification of products is relatively simple. In comparison, all side-chain functional groups will be protected throughout the synthesis in the global protection strategy to avoid any possible side

Side Chain Protecting Groups 279

reactions caused by the side chain functional groups [7]. This strategy is often applied in stepwise condensation protocols where the by-products arising from the side chain reactions may be very difficult to separate by ordinary purification methods. This strategy is mostly applied in solidphase peptide synthesis, where an excess amount of acylating amino acid derivatives is used to ensure the complete formation of peptide bonds at every step. As of the diversity of amino acid side chain functionalities, a variety of protecting groups have been developed, as summarized in further sections. 5.2 HYDROXYL PROTECTING GROUPS FOR SERINE, THREONINE, TYROSINE, AND HYDROXYPROLINE Besides the dehydration of threonine, the dehydration of serine has also been well documented. For example, when a series of serine-containing dipeptides and tripeptides, including Z-Gly-Ser-OEt (Z = PhCH2O2C), Z-X-Ser-OMe (X = Val, Gly-Gly), Z-Ser-X-OEt (X = Gly, Ala) and Z-GlySer-Gly-OEt were treated with carbodiimide (i.e., Me2N(CH2)3N:C:NHEt/ CuCl) in CHCl3 or DMF in the dark at room temperature for 5-7 hours, the corresponding dehydroalanine-containing peptides were formed in 75-84% yields [8]. The carbodiimide is a common condensation reagent for peptide synthesis. Similarly, N-protected β-hydroxy-α-amino esters have been converted into didehydroamino esters in the presence of a mild dehydrating agent (i.e., PhN(SO2CF3)2/Et3N) [9]. Likewise, dehydroalanine (ΔAla) peptides (e.g., Bz-Ala-ΔAla-OMe) has been obtained in 58% yield from Z-Ala-Ser-OMe when the peptide was treated with oxalyl chloride and triethylamine in methylene chloride at 0°C [10]. More examples about the dehydration of serine or threonine-containing peptides have been reported elsewhere [11]. In addition, saponification of urethane-type N-protected serine and threonine derivatives, as well as the N-urethane protected peptides of either serine or threonine as the N-terminal amino acid with free hydroxyl group afford benzyl alcohol and 2-oxazolidone derivatives, as illustrated in Scheme 5.1 [12]. Under this condition, saponification of (benzyloxy) carbonyl-L-threonyl-L-leucine (Z-Thr-Leu), methyl (4-methoxybenzyloxy)carbonyl-L-seryl-L-leucinate (Moz-Ser-Leu-OMe), (4-methoxybenzyloxy)carbonyl-L-serine (Moz-Ser), benzyloxycarbonyl-L-serine (Z-Ser), tert-butoxycarbonyl-L-threonine (Boc-Thr), and 4-methoxybenzyl

280

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)carbamate (Moz-Thr-NH2) also leads to the formation of the corresponding oxazolidone derivatives. All these potential reactions involving the unprotected hydroxyl group within serine/threonine demonstrate the necessity of side-chain protection. Nevertheless, there are still examples of peptide syntheses without the protection of the hydroxyl group of serine and threonine [13], such as in the synthesis of Histone H1 C-terminal segment 152–184 [14], and Me3SiCN mediated peptide synthesis where Ser, Thr, Tyr, and Cys have not been protected [15].

SCHEME 5.1 Formation of 2-oxazolidone ring during the hydrolysis of N-urethane protected serine.

Regarding the general protection of the hydroxyl group, it can be protected in the form of either ester or ether (including silyl ether) [16]. However, considering the compatibility and orthogonality with other protecting groups for amino acids, only a few protecting groups have been applied for protection of the hydroxyl group in serine, threonine, and 4-hydroxyproline. 5.2.1 PROTECTION OF HYDROXY GROUP BY ETHERS 5.2.1.1 TERT-BUTYL PROTECTING GROUP The t-butyl protecting group can be introduced into serine/threonine as well as tyrosine in reaction with isobutene in the presence of a strong anhydrous acid catalyst. For example, after threonine was converted to its N-benzyloxycarbonyl derivative, further esterification with p-O2NC6H4CH2Br at 80°C afforded 97% of Z-Thr-OCH2C6H4NO2-p (Z = PhCH2O2C). Subsequent treatment of this compound with isobutene at 0°C gave 81% of Z-Thr(CMe3)OCH2C6H4NO2-p. Hydrogenation of the resulting ester with Pd-C gave 85% of threonine tert-butyl ether, whereas alkaline hydrolysis of the ester yielded 80% of Z-Thr(CMe3)-OH [17]. Similarly, t-butyl group has been applied to

Side Chain Protecting Groups 281

make Fmoc-Ser(CMe3)-OH [18], and to protect the hydroxyl group of serine, threonine, and tyrosine in the synthesis of glucagon segment 7–29 [15] It is suggested that the t-butyl ether as the hydroxyl protecting group has stability similar to that of N-tert-butyloxycarbonyl group (t-Boc), indicating that the application of tert-butyl protecting group for the hydroxyl functionality is incompatible with the N-t-Boc for the amino group, as both can be removed by the application of trifluoroacetic acid [1]. However, the tert-butyl group is compatible with N-benzyloxycarbonyl (removed by hydrogenolysis), (((2-([1,1’-biphenyl]-4-yl)propan-2-yl)oxy)carbonyl) (Bpoc, removed by very dilute TFA), and N-o-nitrophenylsulfenyl (removed by an equivalent of HCl or thiolysis) [2]. It should be pointed out that triphenylmethyl (trityl) group has often been applied to protect regular hydroxyl group, but is rarely used for the protection of hydroxyl group of serine or threonine [3], as it is more labile than tert-butyl group due to the formation of more stable triphenylmethyl cation. 5.2.1.1.1 Preparation of tert-Butyl N-Benzyloxycarbonyl-O-(tertButyl)Serinate [4]

Isobutylene was passed into a suspension of 12.0 g of N-benzyloxycarbonyl-serine in 500 mL CH2Cl2, and 2 mL of concentrated sulfuric acid was added slowly. After 3 hours of passing the isobutylene into the suspension, the flask was stoppered and kept overnight to give 12.2 g of tert-butyl N-benzyloxycarbonyl-O-(tert-butyl)serinate as a cloudy oil. The alcoholic solution of this compound was mixed with 10 mL 2 N NaOH to afford N-benzyloxycarbonyl-O-(tert-butyl)serine. Similar procedure has been applied to make tert-butyl (S)-2(benzyloxycarbonyl-amino)-3-(4-(tert-butoxy)phenyl)propanoate (i.e., tert-butyl O-tert-butyl-N-benzyloxycarbonyl-L-tyrosinate and tert-butyl (S)-2-amino-3-(4-(tert-butoxy)phenyl)propanoate (i.e., tert-butyl O-tertbutyl-L-tyrosinate) after hydrogenation in EtOH in the presence of 10% Pd-C, as well as tert-butyl O-tert-butyl-N-benzyloxycarbonyl-L-threoninate [4].

282

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.2.1.2 BENZYL PROTECTING GROUP Benzyl has been commonly applied as a hydroxyl protecting group due to its easy introduction and relative stability as well as unique deblocking condition (e.g., hydrogenolysis or combination of oxidation and hydrolysis). The protection of serine side chain with benzyl group can be found in the synthesis of actinomycin analogs involving O-benzyl-L-serine methyl ester and O-benzyl-L-serine [5]. Likewise, N-Boc-O-benzyl-serine and N-Boc-O-benzyl-threonine have been applied in the synthesis of Barnacle Model Proteins [6], and O-benzyl-serine and O-benzyl-threonine have been used to form polymers via NCA polymerization [7]. N-Boc-Obenzyl-serine can be prepared by treatment of N-Boc-serine with sodium hydride (NaH) in DMF followed by the reaction with benzyl bromide. Removal of the Boc group would give O-benzyl-serine [8]. The corresponding benzyl protected threonine can be formed in similar condition. Alternatively, N-Boc-O-benzyl-serine, and N-Boc-O-benzyl-threonine can be prepared by benzylating N-Boc-serine and N-Boc-threonine with benzyl bromide in DMF in the presence of NaOH at –10°C to 0°C [9]. The relative stability of benzyl protected compounds have been tested in a 50:50 (V/V) mixture of TFA and CH2Cl2 at 20°C, with the increasing stability of O-benzyl-L-tyrosine < Nε-benzyloxycarbonyl-L-lysine < O-benzyloxycarbonyl-L-tyrosine ∼ S-(4-methoxybenzyl)-L-cysteine 95% pure by 1H NMR and was used without further purification.

Side Chain Protecting Groups 303

5.3.1.3.2 Decomposition of the 9-BBN-L-Lysine Complex (Method A)

The 9-BBN-L-lysine complex was dissolved in MeOH (2 mL/ mmol) and to this solution was added conc. HCl (1 mL/mmol). After the exothermic reaction ceased, the solution was stirred at room temperature for 10 minutes and then concentrated in vacuo (rotary evaporator). The residue was digested with THF, filtered, and the precipitate was washed with THF to remove the 9-BBN residue. The resultant solid was exposed to a high vacuum for 1 hour (< 0.1 mm Hg) to give NMR pure amino acid HCl salt, which could be converted to the free amino acid by warming with excess propylene oxide in aqueous methanol for several minutes. The yield of L-lysine was 78%. 5.3.1.3.3 Decomposition of the 9-BBN-L-Lysine Complex (Method B)

To a 50 mL Erlenmeyer flask were added 20 mL THF, 10 mmol of 9-BBN-L-lysine complex, and 3.0 mL of ethylenediamine (45 mmol). The mixture was heated with a heat gun to just below boiling for 1 minute. The mixture was then cooled and filtered (vacuum filtration). The precipitate was washed with THF (3 × 50 mL) and dried in vacuo to give 91% of pure L-lysine by NMR.

304

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.3.1.4 PREPARATION OF Nε-FMOC-L-LYSINE [71]

5.3.1.4.1 Preparation of Nε-Fmoc-L-Lysine Cu(II) Complex L-Lysine hydrochloride (5 g, 27.4 mmol) was dissolved in 40 mL of H2O, then 5 g of basic CuCO3 (45.2 mmol) was added. The mixture was refluxed for 30 minutes, and then the hot suspension was filtered and washed with H2O. After cooling to 25°C, the solution was basified with 1.5 g of MgO, and a solution of 10.6 g Fmoc-azide (40 mmol) dissolved in 75 mL dioxane was added. After stirring at 45°C for 16 hours, a bulky blue precipitate was formed. The whole mixture was stirred with 25 mL 2 N aqueous AcOH for 1 hour and then filtered. The residue was washed with H2O (3 × 50 mL), dioxane (2 × 25 mL), and CHCl3 (2 × 25 mL) in order to eliminate the excess of azide, and dried to give 6.98 g of Nε-fluorenylmethyloxy-carbonyl-Llysine Cu(II) complex, in a yield of 64%, m.p. 211–213°C (dec). 5.3.1.4.2 Preparation of Nε-Fmoc-L-Lysine To a solution of 1.42 g NaHCO3 in 20 mL water was added 2.53 g of EDTA disodium salt (8.67 mmol) in portions to form a supersaturated EDTA disodium salt solution. Then 5.19 g of finely powdered Nε-fluorenylmethyloxycarbonylL-lysine Cu(II) complex (6.5 mmol) was added. The suspension was vigorously shaken at 25°C until the initial blue complex was decomposed, and a white solid separated for approximately 1 hour. After filtering and washing with water, 4.6 g of Nε-Fmoc-L-lysine was obtained, in a yield of 96%, m.p. 209–211°C (dec).

Side Chain Protecting Groups 305

5.4 PROTECTING GROUPS FOR ARGININE While both arginine and lysine carry amino groups on their side chains, the overall chemical behavior of their side chains is different. For example, the side chain of arginine is a guanidino group with three nitrogen atoms, whereas the side chain of lysine is a simple primary amino group with only one nitrogen atom. Furthermore, the basicity of the arginine side chain is higher than that of lysine, possibly due to the effect of proton sponge, like the one in 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) and 1,5-diazabicyclo(4.3.0) non-5-ene (DBN). In addition, some ornithine can be formed during the stepwise peptide chain elongation of arginine-containing peptides or during the cleavage of α-amino protecting groups under strongly basic conditions while the guanidino was protected by adamantyloxycarbonyl (Adoc) or Me3CO2C (Boc) groups [94]. What is worse is the formation of δ-lactam during the synthesis of arginine-containing dipeptides from Z-Arg(Tos)-OH, BocArg(Tos)-OH, Fmoc-Arg(Boc)2-OH or Fmoc-Arg(Pmc)-OH, as a significant amount of δ-lactam was formed during the synthesis of Z-Arg(Ts)-OMe and Z-Arg(Ts)-NH2, as well as of Boc-Arg(Ts)-chloromethyl ketone. Particularly, mixed anhydride coupling procedure for arginine leads to the formation of more δ-lactam than other coupling methods, and the protection of the guanidino group with di-Boc also induces more δ-lactam formation than any other NG-protection method [95]. As a result, more activated arginine might be consumed or des-Arg peptides might be formed [96]. On the other hand, arginine is unavoidable during the peptide synthesis of arginine-containing peptides (or proteins), such as abaloparatide [97], angiotensin II [98], bremelanotide [99], desmopressin [100], leuprolide [101], and semaglutide [102], just to name a few. Due to the basicity of the guanidino group, it can be simply protonated to reduce its reactivity during the formation of the peptide bond, such as the protonation with HBr [103]. However, it is still necessary to protect this group either to reduce the potential side reactions mentioned above or to adjust its solubility in the common solvents used in SPPS [96]. Regarding the protection of guanidino group, some protecting groups used for the lysine side chain such as benzyloxycarbonyl (Z) [104], Boc [105–107], and 5,5,5-trifluoro-4-oxopent-2-en-2-yl [85], have also been applied to protect this particular group. The most frequently used arginine side chain protecting groups are based on electron-rich aromatic sulfonyl moiety, in order to be compatible with the TFA cleavage [108]. So far, the guanidino protecting groups of this type include toluenesulfonyl (Tos) [72,

306

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

92, 109, 110], 4-methoxy-benzenesulfonyl (also known as p-methoxybenzenesulfonyl) (Mbs) [111–113], 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds) [111, 112, 114], 2,4,6-trimethylbenzenesulfonyl (also known as mesitylene-sulfonyl [115], mesitylene-2-sulfonyl [116], or mesityl2-sulfonyl [96, 112]) (Mts), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) [111, 112, 115, 117, 118], 2,3,4,5,6-penta-methylbenzenesulfonyl (Pme) [111, 112], 2-methoxy-4,6-dimethylbenzenesulfonyl (iMds) [112], 2,4,6-trimethoxybenzenesulfonyl (Mtb) [112], 4-methoxy-2,3,5,6-tetramethylbenzenesulfonyl (Mte) [112], and bicyclic 1,2-dimethylindole-3-sulfonyl (MIS) [108], 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf) [108, 115, 119], and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) [108, 115, 120–123]. Also these sulfonyl type guanidino protecting groups are listed in Figure 5.2.

FIGURE 5.2 Sulfonyl type protecting groups for arginine side chain.

Besides these many aromatic sulfonyl guanidino protecting groups, TMS [104], 1-Adoc [124], methylsulfonylethyloxy-carbonyl (Msc) [125], and even nitro group (NO2) [96, 106, 107, 115, 126, 127], have been applied as the arginine side-chain protecting groups. Also, the linker or handle on solid-phase support to mount the N-terminal amino acid has also been

Side Chain Protecting Groups 307

applied as the temporary arginine side chain protecting group, such as (3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)acetyl group [128]. Regarding the removal of these guanidino protecting groups, Boc can be removed by TFA-H2O (95:5) at room temperature [96], toluenesulfonyl (Tos) group can be removed by anhydrous HF, trifluoromethanesulfonic acid (TFMSA)/thioanisole or Na/NH3 [109]. This group is the most used protecting group in the Boc/Bn solid-phase strategy [110]. The removal of Mtr is somewhat challenging, which requires a neat trifluoroacetic acid (TFA) in the presence of scavengers over a prolonged period and is often incomplete, especially when more than one Mtr-protected arginine residues exist in the peptide [96]. This is probably the reason for so many aromatic sulfonyl groups developed for protecting the arginine side chain which are more acid-labile. For example, the acidic stability of several such groups has been found in the following order: Mds > Mtr > Pmc > Pbf > MIS, and MIS is claimed to be the most acid-labile sulfonyl-type protecting group for arginine [108]. Similarly, the acidic stability of benzenesulfonyl protecting groups is in the order of iMds > Mts ~ Mbs > Mte > Pme > Mtb ~ Mds > Mtr [112]. Pmc can be removed by 50% of TFA, rather than a high percentage of TFA for Mtr [120], whereas removal of Mbs and 1-adamantyloxycarbonyl groups requires excessively vigorous reaction conditions (MeSO3H) [111]. In contrast, Msc is an extremely acid-stable group that could be rapidly eliminated with sodium hydroxide in dioxanemethanol (complete after 2.5 min at 20°C with NaOH) [125]. Interestingly, the sulfonyl type protecting group demonstrates different acidic stability when situated at different position, with higher stability at the α-position than that at the γ-position. For example, tosyl group when situated at the α-position is stable to liquid HF but can be readily deblocked by HF while it is at the side chain of arginine; similarly, Pmc could not be removed from the α-position by means of neat TFA at room temperature over a period of several hours [119]. Therefore, it is suggested that all sulfonyltype protecting groups require special care upon cleavage in order to suppress byproduct formation [123]. One possible byproduct form from the migration of Pmc to the position 2 of indole upon deprotection of a peptide containing both Trp and Pmc-protected Arg in anhydrous TFA; alternatively, the Pmc could migrate to the phenolic hydroxyl of tyrosine, or form sulfate esters with Tyr, Ser, and Thr [123]. The nitro protecting group can be removed by catalytic reduction with a wide range of catalysts, such as SnCl2 [127], and TiCl3 [126]. However, catalytic hydrogenation of peptides containing multiple nitro group protected

308

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

arginine residues may result in long reaction time and the concomitant degradation of the peptides. To mitigate this problem, SnCl2 is suggested as the reducing agent in a green solvent, such as 2-methyl tetrahydrofuran, using aqueous HCl as an acid rectifier [96]. 5.4.1 PROTECTION OF ARGININE WITH NITRO GROUP AND ITS DEPROTECTION [127] 5.4.1.1 PREPARATION OF N-NITRO-L-ARGININE

To 200 mL concentrated sulfuric acid at room temperature was added 100 g of L-arginine hydrochloride in portions under stirring. The hydrogen chloride thus produced was removed by a water pump until the gas bubbles disappeared. To this clear solution was added 50 g of powdered ammonium nitrate also in portions under stirring, and after 15 minutes, the gas bubbles were removed in vacuo again. The mixture was poured onto cracked ice with stirring. The resulting solution was brought to pH 6.8 with concentrated ammonium hydroxide under cooling, and finally allowed to stand at 0°C for several hours. The L-nitroarginine was filtered over suction, washed with cold water, recrystallized from hot water, filtered, then washed sequentially with water, ethanol, and ether, and dried. Please be noted that there is little difference for the solubility of L-nitro-arginine in cold or hot water, recrystallization from water was partially carried out repeatedly using the mother liquor which was used prior to the recrystallization. A total amount of 90 g of N-nitro-L-arginine was obtained, in a yield of 86.5%, m.p. 255°C (decomp.). 5.4.1.2 REMOVAL OF THE NITRO GROUP FROM N-NITRO-L-ARGININE

Side Chain Protecting Groups 309

5.4.1.2.1 Reduction of N-Nitro-L-Arginine in Boiling Water To a solution of 1.0 g N-nitro-L-arginine in 30 mL 60% formic acid was added 8.3 g of stannous chloride, and the solution was stirred in a boiling water bath for 3 hours. The precipitate was filtered, the filtrate was diluted with 30 mL of water, and then hydrogen sulfide was added until the precipitation of stannous sulfide ceased. The sulfide was then filtered and extracted with hot water. The combined solution of the filtrate and the extract was treated with active carbon. The clear solution was concentrated to dryness, and the residue was dried in a vacuum desiccator. To the syrup dissolved in 30 mL absolute ethanol was added 0.63 mL of triethylamine, the mixture was allowed to stand for 3 hours in a cold room. The crystalline formed was filtered, washed with ethanol, and dried. The crude product was 0.90 g (93.7%), which was recrystallized from water and ethanol to afford 0.67 g of L-arginine hydrochloride, in a yield of 69.7%, m.p. 215°C (decomp.). 5.4.1.2.2 Reduction of N-Nitro-L-Arginine in Warm Water To a solution of 1.0 g N-nitro-L-arginine in 30 mL 60% formic acid was added 8.3 g of stannous chloride, and the solution was stirred at 50°C for 25 hours. The reaction mixture was treated in the way as described in 4.1.2.1. The amount of crude product was 0.90 g (93.7%), which was recrystallized from water and ethanol to afford 0.65 g of L-arginine hydrochloride, in a yield of 67.8%. 5.4.2 PREPARATION OF Nω-2,4,6-TRIMETHOXYBENZENESULFONYLL-ARGININE (H-ARG(MTB)-OH) [112]

310

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.4.2.1 PREPARATION OF 2,4,6-TRIMETHOXYBENZENESULFONYL CHLORIDE (MTB-CL) To a solution of 5.05 g of 1,3,5-trimethoxybenzene in 500 mL CH2Cl2 cooled to –5 ~ –10°C was added slowly a solution of 6.0 mL chlorosulfonic acid in 400 mL CH2Cl2, and the resulting solution was stirred for 3 hours. The reaction mixture was then poured onto crushed ice mixed with 300 mL 5% NaHCO3. The organic layer was washed with water, dried over anhydrous MgSO4, and concentrated. The residue was crystallized from CCl4 to afford 0.61 g of 2,4,6-trimethoxybenzenesulfonyl chloride, in a yield of 7.6%, m.p. 130–133°C. 5.4.2.2 PREPARATION OF Z-ARG(MTB)-OH Nα-benzyloxycarbonyl-L-arginine (0.77 g) was dissolved in a mixture of 2.5 mL 4 N NaOH and 10 mL acetone cooled with ice. To this solution was added a solution of 1.0 g of Mtb-Cl in 4 mL acetone, and the mixture was stirred for 2 hours. After acidification with 10% citric acid, the solvent was evaporated off and the material was extracted with EtOAc. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated. The residue was triturated with ether to give a precipitate, which was collected and further purified by silica gel column chromatography (CHCl3-MeOH-AcOH = 9: 0.7: 0.35). The collected fractions were concentrated, and the residue was triturated with ether to give a 0.50 g of Z-Arg(Mtb)-OH, in a yield of 37.1%, m.p. 89–93°C. 5.4.2.3 PREPARATION OF H-ARG(MTB)-OH A solution of 0.15 g Nα-benzyloxycarbonyl-L-arginine(Mtb)-OH in 30 mL of MeOH was hydrogenated over Pd black. After the usual work-up, the material was triturated with ether to give 0.1 g of H-Arg(Mtb)-OH, in a yield of 88.8%, m.p. 115–120°C. 5.4.3 PREPARATION OF H-ARG(MIS)-OH [108]

Side Chain Protecting Groups 311

5.4.3.1 PREPARATION OF 1,2-DIMETHYL-1H-INDOLE-3-SULFONYL CHLORIDE (MIS-CL) To a round-bottomed flask filled with argon atmosphere were added 14.5 g of 1,2-dimethylindole (99.8 mmol), 19.1 g of sulfur trioxide pyridine complex (119.8 mmol) and 70 mL pyridine. The reaction mixture was refluxed for 1 hour and cooled to 60°C when the reaction was completed. The reaction mixture was concentrated under vacuum to give a solid, which was suspended in 200 mL dry CH2Cl2 under argon atmosphere. The suspension was then cooled in an ice bath and 13.5 mL of oxalyl chloride (20.0 g, 158 mmol) was slowly added. After that, 0.5 mL of DMF was slowly and carefully added, and vigorous effervescence was observed. The reaction mixture was stirred in an ice bath for an additional 30 minutes until the effervescence ceased and was then stirred at room temperature for 2 hours. CH2Cl2 (200 mL) was added to the reaction mixture, and after the mixture was cooled to below 5°C, 150 mL of cold water (2–8°C) was added, and the resulting mixture was stirred for 10 minutes. The organic phase was separated and washed with cold water (2–8°C, 2 × 150 mL), and dried over 15 g of anhydrous MgSO4. Upon filtration, the filtrate was concentrated to ca. 40 mL. The solid was filtered off and washed with 60 mL of CH2Cl2/n-hexane mixture (1: 1). The solid was dried under vacuum to give 19.6 g of 1,2-dimethyl-1H-indole-3-sulfonyl chloride as light pink solid, in a yield of 80.4%, m.p. 67.7–73.5°C. 5.4.3.2 PREPARATION OF Z-L-ARG(MIS)-OH To a suspension of 2.05 g of Nα-benzyloxycarbonyl-L-arginine (6.7 mmol) in 6.7 mL 3 N aqueous NaOH (20 mmol) was added 13.3 mL acetone to dissolve the amino acid. After the solution was cooled in an ice bath, an additional 6.7 mL 3 N aqueous NaOH and a solution of 3.69 g 1,2-dimethyl-1H-indole3-sulfonyl chloride (14.7 mmol) in 13.3 mL acetone were simultaneously added over 10 minutes. The reaction mixture was stirred at 0°C for 2 hours and an additional 2 hours at room temperature. Once no 1,2-dimethyl1H-indole-3-sulfonyl chloride was detected by TLC (hexane/EtOAc = 1: 1), 100 mL of H2O was added and the suspension was washed with diethyl ether (3 × 80 mL). The aqueous phase was acidified to pH 2~3 by the addition of 1 N HCl, the resulting precipitate was filtered, washed with acidic water (pH 2~3) and dried in vacuo. The crude product obtained was purified by column chromatography. Upon removal of solvent from the collected pure fractions

312

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

in vacuo, an oil was yielded. This process was repeated. Hexane and CH2Cl2 were then sequentially added and a precipitate appeared on scratching. The solvent was decanted and the solid was washed 4 times with CH2Cl2/hexane (enough hexane to precipitate all the product) to remove HOAc and give 0.70 g of N2-benzyloxycarbonyl-Nw-((1,2-dimethyl-1H-indol-3-yl)sulfonyl)-Larginine (Z-L-Arg(MIS)-OH), in a yield of 20.4%, m.p. 155.5–159.1°C. 5.4.3.3 PREPARATION OF H-L-ARG(MIS)-OH A mixture of 486 mg Z-L-Arg(MIS)-OH (0.94 mmol) and 110 mg of 10% Pd/C in 60 mL MeOH was hydrogenated overnight at atmospheric pressure. After this time, TLC (CH2Cl2-MeOH-HOAc, 90: 9: 1) still showed some starting material. Then additional 100 mg of 10% Pd/C was added and the mixture was hydrogenated for a further 24 hours, after which TLC showed the absence of starting material. The reaction mixture was filtered over Celite and evaporated to dryness to yield 352 mg of Nw-((1,2-dimethyl-1H-indol3-yl)sulfonyl)-L-arginine (H-L-Arg(MIS)-OH), in a yield of 98% yield, m.p. = 153.2–155.0°C. 5.5 PROTECTING GROUPS FOR HISTIDINE Histidine, as another amino acid with a basic side chain, has demonstrated some unique properties that are quite different from those of lysine and arginine. For example, although there are two nitrogen atoms within the imidazole functional group, only one such atom will behave as Lewis base, and its basicity is much less than that of the amino group or guanidino group in lysine and arginine, respectively. The two nitrogen atoms have been labeled as position π and τ, respectively, as illustrated in Figure 5.3. The lone electron pair on the τ-nitrogen is situated perpendicularly with respect to the imidazole ring and is conjugated to the two π bonds to form the aromatic system, with a total of 6 π electrons to meet the Hückel’s 4n+2 π electrons [129]. Therefore, the lone electron pair on the τ-nitrogen cannot be donated to Lewis acid, and this nitrogen atom is not basic either. In contrast, the lone electron pair on the π-nitrogen is parallel to the imidazole ring, without participating in conjugation with the two π bonds, so that it is readily available to bind with Lewis acid. Unfortunately, as this π-nitrogen is so close to the hydrogen atom on the chiral center within histidine, it can easily abstract the hydrogen atom from the chiral center once the histidine is activated

Side Chain Protecting Groups 313

during the formation of the peptide bond, leading to the racemization of the chiral center. (Note: protection of the α-amino group with Fmoc would increase the acidity of the hydrogen at the chiral center, enhancing the probability of racemization, particularly when the carboxyl group is activated with an electron-withdrawing group). This is the reason that among the 20 naturally occurring amino acids, Nα protected histidine when activated is especially prone to racemization [130, 131], as shown by the path ‘a’ in Scheme 5.4. Alternatively, the π-nitrogen might attack the activated carbonyl group, leading to a cyclized product as illustrated in path ‘b’ in Scheme 5.4, for the creation of (9H-fluoren-9-yl)methyl (S)-(5-oxo-6,7-dihydro-5Hpyrrolo[1,2-c]imidazol-6-yl)carbamate when the α-amino is protected with a Fmoc group [132]. Furthermore, the hydrogen atom on the τ-nitrogen may undergo tautomerization to have the hydrogen situating at the π-nitrogen, as shown in Scheme 5.5. This N-protonation and deprotonation on the imidazole ring of histidine may be crucial in optimum performance of several catalytic enzymes, as illustrated by the possible proton transfer via histidine between the aspartate and serine residues of the catalytic triad of serine proteinases [133]. It has been clearly evidenced that the suitable situation of histidine confirmation is essential for the formation of taxonomic substates in carbonmonoxy myoglobin [134]. a total of 43 optimized conformers for both tautomers of N-formyl-L-histidinamide at RHF/6-31G* level have been located, and the stabilization effect was found to be larger for the L-type backbone conformers than those of the D-type backbone conformers [135].

FIGURE 5.3 Labelling of nitrogen atoms on histidine side chain.

Based on the facts mentioned above, the necessity to protect the histidine side chain is obvious in order to avoid the potential racemization. While it has been stated that most protecting groups for histidine actually block the τ-nitrogen, and the only commercially available histidine derivative is Fmoc-His(Bum) (Bum = t-butoxymethylhistidine) [136], several histidine derivatives with protecting groups at the π-nitrogen are also known in the literature. Many of these histidine side-chain protecting groups are the alkyl type groups. For example, triphenylmethyl (trityl) [136–141], diphenylmethyl (Dpm) [139], diphenyl-4-pyridylmethyl [142], and carboxymethyl

314

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

[143], have been applied to protect the τ-nitrogen of histidine side chain. Also, histidine has been mounted to trityl [144], and 2-chloro-trityl [145] containing resins for solid-phase peptide synthesis. Some acyl protecting groups such as toluenesulfonyl (Tos) [137, 138], benzyloxycarbonyl (Z) [139] are also used to protect the histidine τ-nitrogen. For comparison, histidine derivatives with the π-nitrogen protected by t-butoxymethyl (Bum) [146], alloxymethyl [147], 1-adamantyloxymethyl (1-Adom) [148], allyl [149], phenacyl [141, 149], benzyloxymethyl (BOM) [149], 4-bromobenzyloxymethyl [150], 4-methoxy-benzyloxymethyl (MBom) [151], have been reported in the literatures. Likewise, Boc [136] and 2,6-dimethoxybenzoyl [140] have been applied to protect the histidine π-nitrogen.

SCHEME 5.4 Potential racemization and side reaction of carboxyl activated histidine.

SCHEME 5.5 Tautomerization of histidine side chain.

In addition to the above literature with clear protection of either the π- or τ-nitrogen on the histidine side chain, several papers also have protections on the imidazole rings, but the actual nitrogen position has not been specified, such as in the cases for the preparation of Fmoc-His(Bum), Trt-His(Trt), Fmoc-His(Trt); [152] Bpoc-His(2,6-Dmbz)-OH; [153] Boc-His(X)-OH (I,

Side Chain Protecting Groups 315

X=CPh3, CHPh2, 2,4,6-(O2N)3C6H2, piperidinocarbonyl) [154]. Sometimes, the symbol of “Nim” or “im-” in front of the protecting group indicates the side-chain protecting group, without a clear sign of at position of the π- or τ-nitrogen, possibly due to the potential tautomerization as shown in Scheme 5.5, such as im-benzyl, im-Dnp, im-toluenesulphonyl, and im-t-butoxycarbonyl [150], im-2,4-dinitrophenyl (Dnp) [150] or Nim-tritylhistidine [155], Nim-(2-cyanoethyl)-L-histidine [143], and Nim-Bzl-His [156]. Nevertheless, while many groups have been developed for the protection of the histidine side chain, it is still not quite clear which protecting group is superior to the others. For example, although the trityl group on the τ-nitrogen reduces the basicity of the imidazole through electronic effects and prevents the hydrogen abstraction from the chiral center due to its bulkiness, it does not circumvent the problem completely [136]. It is suggested that in order to completely block the potential racemization, protection at the π-nitrogen is indispensable [147, 150]. For this reason, it is possible to use the allyl group as a temporary protecting group for the regioselective introduction of other groups at the π-nitrogen as shown in the preparation of Nα-Boc-Nπ-phenacyl-L-histidine and Nα-Boc-Nπ-benzyloxymethyl-Lhistidine methyl esters [149]. On the other hand, some unexpected problems may arise. For example, the solubility of side-chain protected histidine derivatives might be an issue, particularly when it is protected with benzyl or Dnp [150]. Also, histidine protected with Tos, Dnp, and Boc might be vulnerable to nucleophilic attack [130]. Regarding the cleavage of these histidine side-chain protecting groups, the trityl group at the τ-nitrogen can tolerate the treatment of HCl in acetic acid but can be promptly removed with trifluoroacetic acid [130]. The histidine protected with BOM or 4-bromobenzyloxymethyl can sustain in the presence of an excess amount of nucleophilic and basic reagents for many hours at room temperature, as well as trifluoroacetic acid [150]. These two groups can be slowly cleaved by 6 N HBr in acetic acid but can be cleaved quantitatively in saturated HBr in TFA at room temperature within 1.5 hours, as well as catalytic hydrogenolysis [150]. Similarly, MBom is completely stable during the repetitive deprotection of Fmoc with 20% piperidine/DMF but is readily removable by TFA [151]. It should be pointed out that protection of histidine side chain with methyl and isopropyl groups at the π- and τ-nitrogen respectively allows the preparation of histidine derived N-heterocyclic carbene (NHC) ligand to form active ruthenium catalyst [157]. Similarly, temporary protection of the π-nitrogen with an acyl group provides another way of ligation to form peptides [158].

316

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.5.1 EXEMPLARY PROCEDURES TO PROTECT HISTIDINE SIDE CHAIN 5.5.1.1 PREPARATION OF FMOC-HIS(τ-2,6-DMBZ)-OH [140]

To a solution of 1.89 g Fmoc-His-OH (5.0 mmol) in 10 mL anhydrous DMF was added 1.9 mL of diisopropylethylamine (11 mmol). After the mixture was cooled in an ice bath, a solution of 1.10 g 2,6-dimethoxybenzoyl chloride (5.5 mmol) in 2.5 mL anhydrous DMF was added dropwise over a period of 30 minutes. The mixture was stirred for 4 hours at room temperature, and DMF was then evaporated in vacuo, and the resulting thick yellow oil was poured into a mixture of 30 mL EtOAc and 30 mL ice-chilled 10% citric acid. The aqueous phase was separated and extracted with EtOAc (2 × 20 mL). The combined organic phases were washed with water (2 × 20 mL), brine (20 mL), and dried over Na2SO4. After filtration and concentration under reduced pressure, the residual solution was slowly poured into n-hexane under vigorous stirring, which caused the precipitation of crude compound, which was collected by filtration and crystallized from EtOAc/n-hexane to afford 2.22 g of Fmoc-His(τ-2,6-Dmbz)-OH as a white solid, in a yield of 82%. 5.5.1.2 PREPARATION OF FMOC-HIS(π-2,6-DMBZ)-OH [140]

To an ice-chilled solution of 1.89 g Fmoc-His-OH (5.0 mmol) and 2.0 mL diisopropylethylamine (12 mmol) in 5.0 mL anhydrous CH2Cl2 was slowly added 1.35 mL of chlorotrimethylsilane (10.5 mmol), the resulting clear solution was stirred at room temperature for 15 minutes. Additional 1.0 mL

Side Chain Protecting Groups 317

of diisopropylethylamine (6.0 mmol) was added, and the mixture was cooled again with an ice bath, then a solution of 1.10 g of 2,6-dimethoxybenzoyl chloride (5.5 mmol) in 5.0 mL dry CH2Cl2 was slowly added under stirring over a period of 30 minutes. Then the reaction mixture was stirred at room temperature for 6 hours. After the addition of 20 mL CH2Cl2, the reaction mixture was washed with 40 mL ice-chilled 10% citric acid. The resulting precipitate was filtered off and set aside. The organic phase was further washed with water (3 × 30 mL), dried over Na2SO4, filtered, and evaporated. Trituration of the resulting yellowish residue with EtOAc/n-hexane afforded 1.88 g of a white powder. NMR spectroscopic analysis showed that this compound was Fmoc-His(τ-2,6-Dmbz)-OH. The precipitate from the acidic extraction (0.95 g) was suspended in boiling EtOAc and the slurry was filtered while hot. The insoluble residue mostly contained Fmoc-His-OH. A white residue precipitated upon cooling of the warm filtrate was identified as Fmoc-His(π-2,6-Dmbz)-OH, which weighed 0.35 g, in a yield of 13%. 5.5.1.3 PREPARATION OF Nα-T-BUTYLOXYCARBONYL-Nτ-2,4DINITROPHENYL­L-HISTIDINE [159]

The starting material of Nα-Boc-L-His-OMe was prepared from L-histidine dihydrochloride, which was first converted into L-histidine methyl ester dihydrochloride (95% yield, m.p. 204–205°C) and then treated with t-butoxycarbonyl chloride. Then, 2.7 g of Boc-His-OMe (10 mmol) was dissolved in 10 mL dioxane, and 10 mL 1 N NaOH solution was added, and the mixture was allowed to stand for 45 minutes at 20°C. The mixture was then adjusted to pH 8.0 and maintained there with a pH-stat (Radiometer) while 2.0 g of 2,4-dinitrofluorobenzene (11 mmol) in 10 mL dioxane was added dropwise over 1 hour. The reaction mixture was further stirred at pH 8 for 12 hours in the absence of light, which was then extracted with diethyl ether (3 × 50 mL). After the aqueous phase was adjusted to pH 3.5 at 0°C with 1 N HCl, it was extracted with ethyl acetate (3 × 50 mL). The ethyl acetate phases were combined, dried over anhydrous Na2SO4, and concentrated to afford an orange foam in vacuo at 40°C. The foam was dissolved in hot

318

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

2-propanol and cooled down to afford 88% of Nα-t-butyloxycarbonyl-Nτ-2,4dinitrophenyl-L­histidine, m.p. 92–93°C. 5.5.1.4 PREPARATION OF NIM-TRITYL-L-HISTIDINE [155]

To a stirred suspension of 1.55 g L-histidine (10 mmol) in 15 mL of CH2Cl2 was added 1.21 mL of Me2SiCl2 (10 mmol), and the mixture was refluxed for 4 hours. Then, 2.79 mL of Et3N (20 mmol) was added, the solution was further refluxed for 15 minutes. When the solution was cooled down to room temperature, 1.39 mL of Et3N (10 mmol) followed by a solution of 2.79 g of trityl chloride (Trt-Cl, 10 mmol) in 10 mL of CH2Cl2 was added under stirring. After 2 hours, an excess of MeOH was added and the solvent was evaporated in vacuo. Water was added to the residue, and the pH was adjusted to 8–8.5 by dropwise addition of Et3N. The resulting slurry was shaken well with CHCl3, and the insoluble material was filtered off with suction. Further washing with water and Et2O provided 3.85 g of Nim-tritylL-histidine, in a yield of 97%, m.p. 218–220°C. 5.5.1.5 PREPARATION OF Nα,NIM-DITRITYL-L-HISTIDINE [155]

A suspension of 0.77 g of L-histidine (5 mmol) and 0.63 mL of Me3SiCl (5 mmol) in 15 mL of CH2Cl2 was refluxed for 2 hours under stirring and cooled. Then, 0.65 mL of Et3N (5 mmol) was added, and the mixture was refluxed for 5 minutes. After being cooled to room temperature, 1.39 mL of Et3N (10 mmol) and a solution of 2.79 g trityl chloride (10 mmol) in 15 mL of CH2Cl2 were added slowly. The resulting mixture was stirred for 3 hours at room temperature, then an excess of MeOH was added. Upon the

Side Chain Protecting Groups 319

concentration of the reaction mixture in vacuo, the resulting oily residue was partitioned between CHCl3 and 5% citric acid solution. The chloroform layer was washed with brine and dried over MgSO4. When the majority of solvent was evaporated, n-hexane was added, and the mixture was allowed to stand for 4 days at room temperature to give 2.75 g of crystalline Nα,Nim-ditrityl-Lhistidine, in a yield of 86%, m.p. 198°C. 5.5.1.6 PREPARATION OF BPOC-HIS(2,6-DMBZ)-OH [153]

To a suspension of 985 mg of Bpoc-His-OH (2.5 mmol) in 5 mL anhydrous DMF was added 0.9 mL of diisopropylethylamine (5.3 mmol). The slurry was cooled on an ice bath while a solution of 525 mg of 2,6-Dmbz-Cl (2.6 mmol) in 2 mL of DMF was added dropwise over 15 minutes. The mixture was stirred at room temperature for 2 hours. The bulk of the solvent was evaporated under reduced pressure, and the resulting yellow paste was dissolved in 30 mL EtOAc. The organic phase was washed with 30 mL 0.5 M citrate buffer (pH 3.5), water (2 × 30 mL), and brine (30 mL). After being dried over Na2SO4 and filtered, the organic phase was evaporated to dryness, yielding a white powder, which was triturated with EtOAc/n-hexane and then recrystallized from MeCN/Et2O to afford 988 mg of Bpoc-His(2,6-Dmbz)-OH as a white solid, in a yield of 71%, Rf = 0.38 (CHCl3/MeOH/HOAc, 90:8:2, v/v/v). 5.5.1.7 PREPARATION OF Nα,Nτ-DITRITYL-L-HISTIDINE METHYL ESTER [138]

To a stirred suspension of 15.0 g of L-histidine methyl ester dihydrochloride (61.98 mmol) in 600 mL of CH3CN (600 mL) at 0°C were sequentially

320

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

added slowly 43.31 mL of Et3N (309.92 mmol) and 43.07 g of trityl chloride (154.96 mmol) in portions. The reaction mixture was allowed to stir at room temperature overnight. The CH3CN was concentrated under reduced pressure and the residue was dissolved in 500 mL of CH2Cl2, washed with an aqueous saturated solution of NaHCO3 (100 mL), water (100 mL), and brine (100 mL). Upon being dried over MgSO4 and concentrated under a high vacuum, 40.47 g of Nα,Nτ-ditrityl-L-histidine methyl ester was obtained as a yellow solid, in quantitative yield. 5.6 PROTECTING GROUPS FOR ASPARAGINE AND GLUTAMINE The functional group on the side chain of asparagine and glutamine is a primary amido group, which is very similar to the secondary amido group of peptide and protein, in terms of polarity and stability. As a result, the synthesis of peptides containing asparagine and/or glutamine has been achieved using asparagine and/or glutamine with their side-chain unprotected. For example, asparagine and glutamine as well as serine, threonine, and tyrosine have been coupled directly to amino acid esters in 82~95% yields using the combination of N,N,N’,N’,N,”N”-hexamethylphosphanetriamine or tris(4-methylpiperazin-1-yl)phosphane with hexachloroethane as condensation agent, where racemization was suppressed in the presence of 1-hydroxybenzotriazole [160]. When water-soluble coupling reagent 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) is used instead of DCC, N-9-fluorenylmethyloxycarbonyl-asparaginepentafluorophenyl ester can be directly prepared from N-Fmoc-Asn-OH and pentafluorophenol [161]. Likewise, diphenyl phosphorazidate is suitable for the formation of peptides involving serine, threonine, valine, glutamine, asparagine, methionine, histidine, and tryptophan without prior protection of their side chains [162]. Although most peptide syntheses are still performed with asparagine and glutamine of unprotected side-chain, an increased tendency to protect asparagine and glutamine side chain has been noticed in recent years [163], owing to the following reasons. First of all, different from the peptide bond that is secondary amido group, the primary amido group at the asparagine or glutamine side chain can potentially undergo dehydration to give cyano group in the presence of acid or carboxyl activating agent, resulting in β-cyano-L-alanine and γ-cyano-α-Laminobutyric acid [164]. Secondly, intramolecular reaction of the asparagine side chain and the activated α-carboxyl group leads to the formation of succinimide derivatives, causing slow or incomplete coupling with the asparagine

Side Chain Protecting Groups 321

[163, 165]. In addition, under alkaline conditions, asparagine-peptides may be deamidated, resulting in aspartic-acid derivatives [163]. Finally, peptides with asparagine or glutamine usually are polar than the ones with side-chain protected, forming hydrogen bonds and leading to low solubility [166, 167]. Under a special condition to convert the α-amino acids into the corresponding α-amino aldehydes for the purpose of making reduced amide pseudopeptides, the amino aldehydes cannot be prepared easily from asparagine and glutamine when their side chains are unprotected, due to the potential reaction of the side chain amido group with the newly generated aldehyde group. For this reason, the side chain amido group is protected with 9-xanthenyl, and the α-carboxyl group is converted into the Weinreb amide followed by reduction with LiAlH4 to afford the ideal aldehydes in good yields [168]. So far, the most common protecting groups for the asparagine and glutamine side chains are benzyl and substituted benzyl groups, such as trityl (triphenylmethyl) [123, 163, 169], and 4-Mtt (Figure 5.1) [170]. Other substituted benzyl groups are 4­methoxybenzyl (4-MeO-Bn) [171], 2,4-dimethoxybenzyl (Dmb) [172], 2,4,6-trimethoxybenzyl (Tmob) [173], 4-methoxy-2-methylbenzyl (4-MeO-2-Me-Bn) [166], 4-methoxy-2,5-dimethylbenzyl (4-MeO-2,5-Me2Bn) [174, 175], 2,3,4-trimethoxybenzyl (2,3,4-(MeO)3-Bn) [174], 2,4,5-trimethylbenzyl (2,4,5-Me3Bn) [174], 2,4,6-trimethylbenzyl (2,4,6-Me3Bn) [174, 175], 2,2,5,7,8-pentamethylchroman-6-methyl (Pmcm) [169], 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-methyl (Pbfm) [169], 1-(3,4-dimethylphenyl) ethyl (1-(3,4-Me2Ph)Et) [175], 1-(4-methoxyphenyl)ethyl (1-(4-MeO-Ph)Et) [175], and 1-tetralinyl (Ttyl) [176], as shown in Figure 5.4. In addition, Dpm (also known as benzhydryl) [175, 176], 4,4’-dimethoxybenzhydryl (Mbh) [123, 163, 177], 2,2’,4,4’-tetramethoxy-benzhydryl (Tbh) [163, 178], 9-xanthenyl (Xan) [163, 179], 2-methoxy-9H-xanthen-9-yl (2-Moxan) [179], 3-methoxy9H-xanthen-9-yl (3-Moxan) [179], can also be considered as substituted benzyl protecting groups, which are also shown in Figure 5.4. Furthermore, 1-naphthylmethyl (1-Naph-Me) [174], 2-methoxyl-1-naphthylmethyl (2-MeO1-Naph-Me) [174, 175], 4-methoxy-1-naphthylmethyl (4-MeO-1-Naph-Me) [174, 175], and cyclopropyldimethylcarbinyl (Cpd) [180] as well as urethanetype protecting groups such as t-Boc, 4-nitrobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, and 2,2,2-trichloroethoxycarbonyl [181], have also been applied to protect the amido group of asparagine and glutamine side chains. Interestingly, 4-chloro-3-nitro-benzaldehyde has been applied to react with Asn-OBut to form tetrahydropyrimidinone which protects both amino and amido group of asparagine [165].

322

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

FIGURE 5.4 Protecting groups for the side chain of asparagine and glutamine.

Regarding the protecting group for the asparagine and glutamine side chain, i.e., the amido group, it is generally required that the protecting group should be stable under the conditions to remove the amino protecting group (i.e., Boc), such as the application of TFA/dichloromethane [182], but can be

Side Chain Protecting Groups 323

readily removed with strong cleavage reagents like boron tris(trifluoroacetate) (BTFA) [183] and HF [184] which are used for complete removal of the protecting groups at the end of peptide synthesis. Among these protecting groups, only benzhydryl group has been used in the preparation of long peptides [166], whereas Asn and Gln with trityl group at the side chain are all crystalline and have much better solubilities in organic solvents than their non-tritylated counterparts, and the trityl group is completely stable to bases and nucleophiles [123]. The protecting groups of Pbfm and Pmcm should be less hydrophobic than the Trt group, and more easily removed during workup, where Pbfm is even more labile than the Pmcm group [169]. As far as the 1-tetralinyl group is concerned, its stability can be further tuned by the introduction of additional substituents on the phenyl ring. For example, when an electron-donating group like methyl or methoxy group is attached to positions 5, 6, 7, or 8 of the 1-tetralinyl group, the resulting tetralinyl group is more labile than the unsubstituted 1-tetralinyl group, particularly at positions 6 and 8 [166]. Trityl group is more labile than Tmob in TFA solution [163]. For the introduction of these protecting groups to the side chain of asparagine and glutamine, there are two strategies, one involves the reaction between the side-chain carboxyl group of the aspartic acid/glutamic acid with the corresponding amines of the relevant protecting groups [166, 174], and the other route is the acid-catalyzed reactions of the appropriate alcohols of the relevant protecting groups with Fmoc-Asn-OH and Fmoc-Gln-OH [123, 163, 169]. While the latter approach is not applicable for the direct introduction of the Trt group on Fmoc-Asn/Gln-OH [169], it could become feasible when the reaction temperature is raised to 50 to 60°C in the presence of acetic anhydride [163]. Regarding the deprotection of these protecting groups, the benzyl type protecting groups can be cleaved with BTFA in trifluoracetic acid (TFA) and in acetic acid solutions [166, 175]. Specifically, the Pbfm group can be removed by TFA/CH2Cl2 (90:5) containing 5% of either triethylsilane (TES) or TIS as scavenger, and TES is more efficient than TIS as a push-pull additive for enhancing the cleavage/deprotection reaction [169]. The tetralinyl group can be removed with TFA/CH2Cl2 in combination with anisole, or BTFA in trifluoroacetic acid [166]. The labile trityl group can be rapidly cleaved by TFA at room temperature, particularly for the deprotection of glutamine side chain which is faster than that of asparagine [123].

324

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.6.1 EXEMPLARY PROCEDURES TO PROTECT ASPARAGINE AND GLUTAMINE SIDE CHAINS 5.6.1.1 PREPARATION OF BENZYL N2-(TERT-BUTOXYCARBONYL)-N5(1,2,3,4-TETRAHYDRONAPHTHALEN-1-YL)-L-GLUTAMINATE [166]

A stirred mixture of 0.5 g of benzyl (tert-butoxycarbonyl)-L-glutaminate (1.5 mmol) and 0.26 g of N-hydroxysuccinimide (2.25 mmol) in 3 mL of CH2Cl2 was cooled to –5°C. To this mixture was added a solution of 0.34 g DCC (1.65 mmol) in 5 mL CH2Cl2, and the resulting mixture was stirred at –5°C for 50 minutes. A solution of 0.24 g of 1-aminotetralin (1.65 mmol) in CH2Cl2 was added and the mixture was stirred for 50 minutes at –5°C and an additional 24 hours at room temperature. Then, 0.075 mL of acetic acid was added, the mixture was stirred for 15 minutes, and the precipitate dicyclohexylurea (DCU) was filtered off and washed with CH2Cl2 (3 × 3 mL). The solvents were removed on a rotary evaporator in vacuo and the residue was dissolved in 5 mL CH2Cl2. Some insoluble crystals were filtered off. Chloroform (7 mL) was added to the filtrate, the solution was washed with 5% aqueous citric acid (3 × 9 mL), 5% aqueous sodium bicarbonate (3 × 12 mL), and de-ionized water (5 × 15 mL). The organic layer was dried over anhydrous Na2SO4, and the solvents were removed on a rotary evaporator in vacuo. The semi-solid was dissolved in 6 mL of hot EtOAc, cooled to room temperature and filtered off. To the filtrate, a solution of 24 mL of petroleum ether (b.p. 40–60°C) was added and the mixture was kept at 0–5°C overnight. The precipitate was filtered, washed with petroleum ether (40–60°C)­EtOAc (4:1) (3 × 5 mL) and dried in vacuo to give 0.31 g of benzyl N2-(tert-butoxycarbonyl)-N5-(1,2,3,4-tetrahydro-naphthalen-1-yl)L-glutaminate, in a yield of 45%, m.p. 82–83°C.

Side Chain Protecting Groups 325

5.6.1.2 PREPARATION OF BENZYL N4,N4-BIS(2,4-DIMETHOXYBENZYL)N2-((4-METHOXYBENZYLOXY)CARBONYL)-L-ASPARAGINATE [185]

After the mixture of 6.25 g of (S)-4-(benzyloxy)-3-(((4-methoxybenzyloxy)carbonyl)amino)-4-oxobutanoic acid (16.2 mmol) and 4.90 g of bis(2,4dimethoxybenzyl)amine (15.5 mmol) in 50 mL THF was stirred for 1 hour, a solution of 1.89 g of diethylisopropylamine (DEIPA, 17.0 mmol) in 15 mL THF was added dropwise. After the reaction, the solvent was removed by evaporation under vacuum, and the residue was taken up in ethyl acetate. This solution was washed twice each with dilute citric acid solution, sodium hydrogen carbonate solution and water. Upon being dried over Na2SO4 and removal of EtOAc, the residue was washed well with petroleum ether, and the residue solidified to afford 9.1 g of benzyl N4,N4-bis(2,4-dimethoxybenzyl)-N2-((4methoxybenzyloxy)carbonyl)-L-asparaginate, in a yield of 85%. 5.6.1.3 PREPARATION OF N2-(((9H-FLUOREN-9-YL)METHOXY) CARBONYL)-N4-((2,2,4,6,7-PENTAMETHYL-2,3DIHYDROBENZOFURAN-5-YL)METHYL)-L-ASPARAGINE [169]

326

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.6.1.3.1 Preparation of Benzyl N2-(Benzyloxycarbonyl)-N4((2,2,4,6,7-Pentamethyl-2,3-Dihydrobenzofuran-5-yl) Methyl)-L-Asparaginate A solution of 0.5 g of benzyl (benzyloxycarbonyl)-L-asparaginate (Z-AsnOBn, 1.4 mmol) and 0.236 g of 1H-benzo[d][1,2,3]triazol-1-ol (HOBt, 1.54 mmol) in 10 mL mixture of DMF and CH2Cl2 (1:1) was stirred at 0°C for 5 minutes. Then, 0.317 g of DCC (1.54 mmol) was added. The mixture was stirred at 0°C and then room temperature for 1 hour each. This solution was then transferred to a solution of 0.429 g of (2,2,4,6,7-pentamethyl2,3-dihydrobenzofuran-5-yl)methanamine (1.54 mmol) and 279 μL of Et3N (2 mmol) in 4 mL of DMF and CH2Cl2 (1:1). The resulting reaction mixture was further stirred at room temperature for 4 hours and then cooled to 0°C and filtered. Solids were washed with CH2Cl2, solvents were removed under vacuum, and the crude product was dissolved in 20 mL EtOAc. The resulting solution was washed with aqueous 1 N HCl solution (2 × 15 mL), saturated aqueous NaHCO3 solution, and brine (15 mL), and dried over anhydrous MgSO4. Upon filtration, the solvent was removed under vacuum. An oily solid was recovered, which was crystallized in Et2O/hexanes to afford 0.547 g of benzyl N2-(benzyloxycarbonyl)-N4-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)methyl)-L-asparaginate as a white solid, in a yield of 70%, m.p. 108–112°C. 5.6.1.3.2 Preparation of Nω-(2,2,4,6,7-Pentamethyl-2,3Dihydrobenzofuran-5-yl)Asparagine A mixture of 0.5 g of benzyl N2-(benzyloxycarbonyl)-N4-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)methyl)-L-asparaginate (0.9 mmol) and 0.1 g of 10% Pd/C (0.1 g) in 15 mL mixture of AcOH and H2O (4:1) was blown with a stream of hydrogen gas at room temperature for 2 hours under vigorous stirring. The mixture was filtered through Celite. The Celite pad was then washed with MeOH, and the volatiles was removed under vacuum. The resulting crude product was crushed and washed with hot methyl tertbutyl ether (MTBE) to afford 0.355 g of Nω-(2,2,4,6,7-pentamethyl-2,3dihydrobenzofuran-5-yl)asparagine as a white solid, in a yield of 87%, m.p. 158–161°C.

Side Chain Protecting Groups 327

5.6.1.3.3 Preparation of Nα-(9-Fluorenylmethoxycarbonyl)-Nω(2,2,4,6,7-Pentamethyl-2,3-Dihydrobenzofuran-5-yl) Asparagine To a solution of 0.15 g of (S)-1-carboxy-3-oxo-3-(((2,2,4,6,7-pentamethyl2,3-dihydrobenzofuran-5-yl)methyl)amino)propan-1-aminium acetate (0.38 mmol) in 15 mL mixed solvent of 6% aqueous Na2CO3 solution and CH3CN (2:1) cooled at 0°C, was added a solution of 0.141 g of Fmoc-OSu (0.42 mmol) in 2 mL CH3CN over 20 minutes. The resulting mixture was stirred at room temperature for 5 hours and 20 mL water was added. After the mixture was cooled to 0°C, a 1 N aqueous HCl solution was added to reach pH 2–3, and the mixture was washed with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous MgSO4. Filtration and evaporation of the solvent under vacuum afforded an oily solid, which was crystallized in EtOAc/hexanes to yield 0.112 g of Nα-(9-fluorenylmethoxycarbonyl)-Nω-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl) asparagine as a white solid, in a yield of 53%, m.p. 135–138°C. 5.6.1.4 PREPARATION OF METHYL N2-(BENZYLOXYCARBONYL)-N4(2,4-DIMETHOXYBENZYL)-L-ASPARAGINATE [172]

To a stirred solution of 6.6 g of (S)-3-((benzyloxycarbonyl)amino)4-methoxy-4-oxobutanoic acid (23.5 mmol) and 3.92 g of 2,4-dimethoxybenzylamine (23.5 mmol) in 100 mL CH2Cl2 at 5–10°C was added a solution of 2.6 g freshly distilled 1-diethylamino-1-propyne (23.5 mmol) in 25 mL CH2Cl2 dropwise during 30 minutes. The resulting mixture was stirred at room temperature for 30 minutes and the solvent was evaporated. Crystallization of the residue from ethyl acetate gave 7.4 g of methyl N2-((benzyloxy) carbonyl)-N4-(2,4-dimethoxybenzyl)-L-asparaginate, in a yield of 74%, m.p. 162–163°C.

328

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.7 PROTECTING GROUPS FOR ASPARTIC ACID AND GLUTAMIC ACID Both aspartic acid and glutamic acid have a carboxyl group on their side chain, which often interferes with the formation of peptide bonds if not protected. In theory, the protecting group suitable for protection of the α-carboxyl group should be able to protect the side-chain carboxyl group as well. However, if both α-carboxyl group and side-chain carboxyl group (for convenience here, ω-carboxyl group will be used for both amino acid) are blocked with the same protecting group, selectivity would be an issue, resulting in β-peptide or γ-peptide. As a result, these two carboxyl groups must be protected with different functional groups. Still, there are some challenges in making aspartic acid- and glutamic acid-containing peptides, particularly for the case of aspartic acid-containing peptides. The biggest issue associated with the aspartic acid-containing peptides is the formation of aspartimide [19, 186–194], (also known as aminosuccinmide derivative) [195–198] during the cleavage of protecting group from the synthesized peptides that is formed via nucleophilic attack by the peptide bond nitrogen atom of the subsequent amino acid residue on the carboxyl side of aspartic acid, as illustrated in Scheme 5.6 [186, 199]. The formation of such imide has been known to occur in the presence of acid [19] or base [200], or even under neutral conditions in water [193]. The formation of aspartimide in the presence of HF has been claimed to follow a AAC2 (acidic, acyl, bimolecular) mechanism [19]. Subsequent hydrolysis of the five-membered imide ring leads to either α-peptide (the normal peptide) and β-peptide, where the β-peptide is often the major product of hydrolysis which is difficult to separate it from the regular peptide [194, 201]. Similar situations may occur to glutamic acid-containing peptide to form a six-membered imide (known as glutamide or pyroglutamide [202]) although less frequently, and the hydrolysis of this imide results in the formation of α-peptide and γ-peptide [201, 203]. The formation of epimerized aspartimide and the subsequent ring-opening may lead to seven different by-products, including a mixture of D/L-α- and β-piperidides and undesired D/L-β-aspartyl peptide in a ratio of 3:1 to the α-aspartyl peptide, where the separation of the D/L-β-aspartyl peptides and epimerized D-α-aspartyl peptide is almost impossible [186]. As a result, the potential formation of aspartimide might be a serious problem for making multiple aspartic acid-containing peptides. For the aspartic acid-containing peptides, it is found that the tendency to form the aspartimide is strongly dependent on the nature of the i+1 amino

Side Chain Protecting Groups 329

acid and the aspartic acid side chain protecting group, where the aspartimide can be easily formed when the side chain of aspartic acid is activated [193]. For example, when the i+1 amino acid is glycine, serine or threonine, the corresponding aspartimide can form most rapidly in the presence of a base [194], particularly for the case of Asp-Gly [191]. Peptide with an Asp-Asn motif is also prone to the formation of aspartimide [186]. For the Fmoc-based SPPS synthesis of NH2-Gly-Yaa-Asp(OBn)-Xaa-OH, in addition to form aspartimide peptides and piperidide derivatives, 1,4-diazepane-2,5-dionecontaining peptides are also formed when Xaa is glycine. In this case, the formation of aspartimide and piperidide derivatives is influenced not only by the nature of the amino acid following the aspartic acid residue but also the bulkiness of the side chain of the amino acid preceding the aspartic acid (Yaa). Under certain conditions, the 1,4-diazepane-2,5-dione peptide derivatives may even be the predominant byproducts [199].

SCHEME 5.6 Potential side reaction of aspartic acid-containing peptides.

The effect of the side-chain protecting group can be viewed during the deprotection of the Fmoc group of the Rink amide linked peptides for which the side chain of aspartic acid is protected with N-[1-(4,4-dimethyl-2,6dioxocyclohexylidene)-3-methylbutyl]aminobenzyl (Dmab), where 11% of aspartyl peptide, as well as 72% of aspartimide and 17% of aspartyl methyl ester were detected under the condition of 20% piperidine in DMF; and 52% of aspartimide and 48% of aspartyl methy ester were detected when 2% DBU in DMF was applied for the deprotection. In contrast, peptides with aspartic acid protected with t-butyl at the side chain afforded 100% of

330

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

aspartyl peptides [189]. Likewise, the formation of pyroglutamide is more likely to happen during the elongation peptide sequence with Dmab-protected Glu, which does not occur with t-butyl protected Glu [204]. Even when the side chain of aspartic acid is protected with t-butyl group, the formation of aspartimide cannot be completely prevented [205], as the sequence and conformation of peptide may play a role as well [206]. In order to avoid the formation of aspartimide, different approaches have been developed, including: (a) the peptoid methodologies by protection of the aspartyl amide bond with N-(2-hydroxy-4-methoxybenzyl) (Hmb) group [191, 205, 207–209], or other protecting groups, such as 2,4-dimethoxybenzyl, Tmob, or 2-nitrobenzyl [210]; (b) use of resin with base-labile linker (handle), such as co(polyethyleneglycol-acrylamide) with a bifunctional linker of N-[(9-hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) in combination with morpholine as the cleavage reagent [211], and fluoride cleavable (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL) linker [212]; (c) use of different deprotection agents, such as acidolysis of β-benzyl aspartate peptides with HBr in TFA/p-cresol [197], and 1-hydroxypiperidine [213]; (d) addition of additives, such as the addition of phenol with electronwithdrawing group (e.g., 2,4-dinitrophenol, HOBt/pentachlorophenol or HOBt/2,4-dinitrophenol for the deprotection of Boc-L-Asp(Bn)-L-Asn-LTyr(OBn)-OCH3 and Boc-L-Thr(OBn)-L-Asp(Bn)-L-Asn-Tyr(OBn)-OCH3) [214]; and (e) use of different protecting groups. Regarding the protection of aspartic acid and glutamic acid side chains, benzyl [197, 200, 202, 215–220], and t-butyl [186, 191, 201, 220–225], are the two commonly used protecting groups, similar to their usages for the protection of the α-carboxyl groups of amino acids. Particularly, the benzyl group is often used during the synthesis of aspartic acid- or glutamic acid-containing polymers [226–230]. In addition, allyl has often been used as the side chain carboxyl protecting group [188, 190, 196, 205, 231–233]. Besides these three groups, there are many other protecting groups that have been occasionally used for protecting the side chain of aspartic acid and/or glutamic acid. These protecting groups include methyl [205, 234, 235], ethyl [195, 205], 3-methylpent-3-yl (Mpe) [191, 205], 2,4-dimethyl-3-pentyl [194] (or 2,4-dimethylpent-3-yl, Dmp) [192], cyclopentyl (c-Pt) [205, 236, 237], cyclohexyl (c-Hx) [205, 238, 239], cycloheptyl (c-Hp) [205, 240, 241], cyclododecyl (c-Ddc) [242], menthyl [241, 243, 244], and arylmethyl groups, such as 4-bromobenzyl (p-BrBn) [15, 245], p-chlorobenzyl (p-ClBn) [245], p-nitrobenzyl (p-NB) [245], 2-nitro-4,5-dimethoxybenzyl (nitroveratryl) [234], 2-phenylisopropyl

Side Chain Protecting Groups 331

(2-PhiPr, should be 2-phenylpropan-2-yl) [205, 246], 3-picolyl (3-pyridylmethyl) [247], 4-picolyl (4-pyridylmethyl) [247–249], 4-pyridyldiphenylmethyl (PyBzh) [205], 2,2’-dinitrobenzhydryl (DNBzh) [250], 9-phenylfluoren-9-yl (9-PhFl) [205], fluorenylmethyl (Fm) [193, 251], and N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]aminobenzyl (Dmab) [189, 205]. Furthermore, pentafluorophenyl or succinimide [198], 1-(2-(dicyanomethylene)-7-(dimethylamino)-2H-chromen-4-yl) ethyl (Dcmdmace) [187], and (2-(dicyanomethylene)-7-(dimethylamino)2H-chromen-4-yl)methyl (Dcmdmacm) [187] have been applied as the side chain protecting groups for aspartic acid and/or glutamic acid. Some of these protecting groups are listed in Figure 5.5. It should be pointed out that these protecting groups will form esters with the side chain carboxyl group of aspartic acid and glutamic acid. There are examples of using amine as the carboxyl protecting group as well, such as in the case of using tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate for glutamic acid [252]. While benzyl and t-butyl groups have been applied to protect the sidechain carboxyl groups of aspartic acid and glutamic acid, they often cause problems, particularly in the case of aspartic acid due to the formation of aspartimide as mentioned above. Therefore, the benzyl protected Asp and Glu are suitable for the preparation of peptides of moderate size, but not good for long peptide chains and small acidic proteins such as the acyl carrier protein (ACP) from E. coli [245]. The tendency to form pyroglutamyl moiety can be applied to form 9- or 10-membered cyclic dipeptides (bislactams) via aminoacyl incorporation reaction [198]. On the other hand, the benzyl group can be converted into other functional groups such as amide, carbonyl hydrazide, carbonyl azide, and thioester for the purpose of peptide side-chain ligation [219]. Alternatively, side-chain protecting group with a C-C triple bond or double bond moiety allows the Cu(I)-catalyzed alkyne-azide and thiol-ene “click” chemistry [253], as well as olefin metathesis [254]. Compared with the simple benzyl group, the ones with an electron-withdrawing group on the aromatic ring have demonstrated enhanced stability so that they can be used in the synthesis of longer peptides. For example, p-chlorobenzyl is about twice as stable as the benzyl group towards the hydrolysis with TFA-CH2Cl2 (1:1) at 45°C, which can be deprotected with liquid HF at 0°C; similarly, 4-bromobenzyl on glutamic acid side chain is about four times as stable as the benzyl counterpart, and 4-nitrobenzyl is more stable during the cleavage with HBr [245].

332

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

FIGURE 5.5 Protecting groups for the side chain of glutamic acid and aspartic acid.

In addition, the contrast between the polar nature of the peptide backbone and the hydrophobicity of some amino acid side chains (e.g., Phe, Leu, etc.), and protecting groups (e.g., benzyl, tert-butyl) of trifunctional amino acids may reduce the solubility of the relevant peptide segments.

Side Chain Protecting Groups 333

Incorporation of polar group (e.g., 3-picolyl and 4-picolyl groups) to the side chain of aspartic acid and glutamic acid results in a substantial increase in solubility of the protected peptide segments [247]. The 4-picolyl group is useful in the synthesis of highly acidic peptide sequences, which sustains during the acidiolytic cleavage of the peptide from the resin, whereas it can be quantitatively removed by hydrogenation using 5% Pd/C in ethyl acetate [248]. The aggregating segment potentially due to intramolecular hydrogen bonds may also cause the low solubility of peptide segment, as shown by the electron spin resonance (ESR) spectroscopy study of stable free radical 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) attaching to the ING fragment (65–74) of the ACP and polyalanine AAAA sequence. The result shows that the type of single-protecting group can significantly affect the overall peptide chain mobility during the peptide synthesis, and the side-chain protecting groups have a pronounced influence on the overall solvation of the peptide resin [255]. It has been shown that cyclohexyl group can induce greater chain immobilization than the benzyl group in Boc chemistry and the t-butyl group in Fmoc chemistry [255], and can markedly reduce the formation of aspartimide with respect to the benzyl group [19, 238]. This might be the reason that cyclohexyl has been commonly used in the protection of aspartic acid side chains in SPPS. However, it is more stable in anhydrous acid than most other protecting groups used in the Boc chemistry, so Dmp has been adopted, which is superior to the cyclohexyl group and is also compatible with the Fmoc chemistry [194]. The allyl group can be easily mounted to the carboxyl side chains, and can be removed under mild conditions (being base labile), which is also compatible with most N-protecting groups [196]. 2,2’-dinitrobenzhydryl (DNBzh) group is photo-cleavable at 308 nm [250]. As short-wavelength light might be limited in vivo application due to poor capacity of penetration as well as photocytotoxicity, both 1-(2-(dicyanomethylene)-7-(dimethylamino)-2Hchromen-4-yl)ethyl and (2-(dicyano-methylene)-7-(dimethylamino)-2Hchromen-4-yl)methyl have been developed which can be cleaved with green light (λ > 500 nm) [187]. Finally, it should be pointed out that Dmab cannot be used for the protection of the aspartic acid side chain without the protection of the amide nitrogen of the peptide backbone [189]. For the introduction of protecting group onto the side chain of aspartic acid or glutamic acid, the α-carboxyl group is generally protected first. However, as both α-carboxyl and ω-carboxyl groups are similar in reactivity, the selective introduction of protecting group to the side chain is somewhat

334

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

challenging. To achieve this goal, three general strategies can be applied. The first one is to protect both α-amino and carboxyl group by means of a copper(II) complex, as seen in the case of lysine and arginine. However, this method has demonstrated a shortage when protecting the aspartic acid side chain with fluorenylmethyl, due to the poor solubility of the salts of copper(II) complexes, such as cesium, pyridinium, dimethylaminopyridinium, N,N-diisopropylneopentylammonium salts in DMF and similar solvents. Although the corresponding DCHA salt was soluble in DMF (at the level of 1 g/10 mL), its reaction with fluorenylmethanol in the presence of N,N-diisopropylethylamine gave exclusively dibenzofulvene [71]. The second approach takes the advantage of stable boroxazolidone that is formed from both α-amino and carboxyl groups with boron reagent, leaving the free side chain for further reactions [256–258]. This boroxazolidone has also been known as mixed anhydride of amino acid and alkyl (or aryl) borinates [90], or borinic acid [259]. The third strategy uses the hexafluoroacetone to temporarily protect the α-amino and carboxyl group by forming (5-oxo2,2-bis(trifluoromethyl)oxazolidin-4-yl) ring with either aspartic acid or glutamic acid, allowing the further functionalization of the side carboxyl group [260–262]. It should be pointed out that the cyclohexyl group might be selectively introduced to the side-chain carboxyl group without prior protection of the α-carboxyl group under a special condition, with precise control of reaction temperature and reaction time (e.g., 70°C, in Et2O with H2SO4. Note, the boiling point of Et2O is much less than 70°C) [239]. As far as the deprotection of aspartic acid and glutamic acid side chain is concerned, the benzyl group can be removed by a saturated solution of HBr in acetic acid [229], t-butyl group can be deblocked during the final cleavage of the peptides from the resin by TFA or other protic acids, such as HCl, H2SO4, HNO3, as well as Lewis acids, such as TiCl4, TMSOTf, ZnBr2 and CeCl3 [202]. Pyridylmethyl (either 3-picolyl or 4-picolyl) is readily removed from peptides in high yield by hydrogenation [248]. Dmab is removable with 2% hydrazine within a second, but the removal of the remaining aminobenzyl ester part will be very slow [202]. p-Nitrobenzyl group can be mildly removed using a solution of SnCl2 in DMF/phenol and acetic acid [263], and the allyl group could be deprotected with tetrakis(triphenylphosphine) palladium [Pd(PPh3)4] and phenylsilane (PhSiH3) [232]. The removal of the rest protecting groups mentioned above can be located from the relevant references.

Side Chain Protecting Groups 335

5.7.1 EXEMPLARY PROCEDURES TO PROTECT ASPARTIC ACID AND GLUTAMIC ACID SIDE CHAINS 5.7.1.1 PREPARATION OF N4-CYCLOHEXYL-L-ASPARAGINE [239]

To 250 mL cooled Et2O was added 25 mL H2SO4 (500 mmol) under stirring, then 250 mL of cyclohexyl alcohol (2,500 mmol) and 33.0 g of L-aspartic acid (250 mmol) were added. The resulting suspension was heated in a rotary evaporator at 70°C for 2 hours under vacuum, during which the bulk of the solvent was removed. The resulting oil was partitioned between 250 mL EtOAc and 300 mL 5% aqueous KHCO3. The pH was adjusted to 7.0 with 4 N aqueous NaOH. The aqueous layer was concentrated under a vacuum until precipitation occurred. The resulting suspension was chilled overnight and filtered to give 32 g of L-aspartic acid β-cyclohexyl ester, in a yield of 60% with a purity of 93%, along with 7% of Na2SO4. 5.7.1.2 PREPARATION OF δ-CYCLOHEPTYL GLUTAMATE

5.7.1.2.1 Preparation of N-Benzyloxycarbonyl-L-Glutamic Acid [264] In an ice-cooled suspension of 44 g L-glutamic acid and 50 g sodium hydrogen carbonate in 230 mL water, was added 48 mL benzyloxycarbonyl chloride

336

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

under vigorous stirring, and at the same time, 150 mL 2 N NaOH solution was gradually dripped in within 1 and half hours to allow the pH remained almost exactly between 8 and 9. The reaction mixture was stirred for a further 2 hours without external cooling and then filtered. The solution was made Congo red with 18% HCl, and the oil layer was separated. The aqueous layer was extracted with EtOAc (3 × 100 mL), and the combined organic layers were mixed with the oil, and then washed with 50 mL 18% HCl. After being dried over sodium sulfate and filtration, EtOAc was removed under vacuum. The remaining thick colorless oil was taken up with a little hot EtOAc, and petroleum ether was added until it began to cloud. Crystallization started soon on rubbing, and an additional 200 mL of petroleum ether was added and the mixture was stirred until the mass has become completely powdery. After standing in the refrigerator, 55–67 g of N-benzyloxycarbonyl-L-glutamic acid was obtained after filtration and drying, corresponding to a yield of 65–80%. Recrystallization in water afford completely the pure compound as fibrous crystals, m.p. 120–121°C. 5.7.1.2.2 Preparation of Benzyl N-Benzyloxycarbonyl-L-Glutamate [264] The reaction between 60 g of N-benzyloxycarbonyl-L-glutamic acid and 150 mL of acetic anhydride at room temperature afford 48–49.5 g of N-benzyloxycarbonyl-L-glutamic acid anhydride, corresponding to a yield of 86–90%, m.p. 94°C. Then 35 g of this anhydride was allowed to react with benzyl alcohol. After removing the excess alcohol under vacuum, the remaining residue was mixed with ice water, acidified with 18% HCl to Congo red, and then extracted with ether. The combined ether extracts were successively washed with water 4 times, and then with aqueous sodium carbonate solution (each containing 1.5 g of sodium carbonate) until no more carbon dioxide evolved. This solution was then acidified with dilute hydrochloric acid to yield a colorless oil that soon solidified. From fractions 1 and 2 of sodium carbonate abstraction, only non-crystallizing oils were initially obtained by acidification, which on repeated fractionation with appropriate amounts of sodium carbonate solution, yielded a further small amount of the desired ester. N-Benzyloxycarbonyl-L-glutamic acid could be recovered from the non-crystallized portions and the residue of the ether solution by gentle saponification, which can be used again for the reactions.

Side Chain Protecting Groups 337

5.7.1.2.3 Preparation of Benzyl N-Benzyloxycarbonyl-ωCycloheptyl-Glutamate [240] To a solution of benzyl N-benzyloxycarbonyl-L-glutamate (18.1 mmol) in 100 mL THF were added 3.26 mL of cycloheptyl alcohol (1.5 eq.), 0.22 g of 4-N,N-dimethylaminopyridine (DMAP, 0.1 eq.) and 4.85 g of dicyclohexylcarbodiimide (DCC, 1.3 eq.). The resulting mixture was stirred overnight, filtered, and concentrated. The residue was taken up by EtOAc, and then washed with 5% citric acid, 5% NaHCO3, brine, and dried over Na2SO4. Upon filtration and concentration again, the residue was recrystallized from EtOAc and n-hexane to afford 6.35 g of benzyl N-benzyloxycarbonyl-ωcycloheptyl-glutamate, in a yield of 75%, m.p. 38–40°C. 5.7.1.2.4 Preparation of ω-Cycloheptyl-L-Glutamate [240] A mixture of 6.0 g benzyl N-benzyloxycarbonyl-ω-cycloheptyl-glutamate (12.83 mmol), 50 mL MeOH and a few drops of acetic acid was hydrogenated over a Pd catalyst for 24 hours. Upon filtration of the catalyst, the filtrate was concentrated and the residue was recrystallized from MeOH and EtOAc to afford 2.37 g of ω-cycloheptyl-L-glutamate, in a yield of 76%, m.p. 184–186°C. 5.8 PROTECTING GROUPS FOR CYSTEINE AND SELENOCYSTEINE Among the 20 amino acids, cysteine is a unique amino acid due to its sulfhydryl side chain, which demonstrates plenty of reactivities, such as acidity (pKa = 8.18), nucleophilicity (to be alkylated and acylated) and readiness to be oxidized to disulfide, as well as sulfonic acid [265]. Particularly, the formation of a disulfide linkage between two cysteines (known as cystine) within proteins is one of the several contributors to maintain the correct protein conformation and functions, in addition to hydrogen bond, electrostatic interaction and van der Waals interaction. Disulfide linkage is stronger than the above weak interactions, so it plays an important role in protein folding and structural stabilization for a functional conformation [266]. Considering the occurrence of cysteine in mammals (2.26%), cysteine is unavoidable for most proteins. In all organisms studied except plants, two cysteines are often separated apart by two other amino acids, in a motif of (Cys-AA-AA-Cys); even for Archea, more than 21% of cysteines have been

338

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

found on this particular motif [267]. As the number of cysteine in a protein increases, the possible combination to form cystine moiety within the protein increases dramatically. For example, the number of possible regioisomers a polypeptide chain can form due to the formation of cystine are 1, 3, 15, and 105, when the corresponding number of cysteines within the polypeptide are 2, 4, 6, and 8, respectively [268]. However, only the proteins, as well as peptide hormones and some messenger molecules with a specific disulfide linkage will exert their proper functions. Therefore, it is necessary to build the disulfide bonds sequentially while the proteins are synthesized [269]. To achieve this goal, all cysteines must be protected by orthogonal groups of different stabilities so that they can be sequentially deprotected under specific conditions to ensure the formation of correct disulfide linkages [269, 270]. Examples of applying delicate strategies for the selection of distinct sulfhydryl protecting groups can be found in the syntheses of linaclotide that consists of three disulfide linkages [265]; orexin-A, a neuropeptide with two intramolecular disulfide bonds [271]; and α-conotoxin SI, a tridecapeptide from marine cone snail venom that contains two “interlocking” disulfide linkages to connect Cys2-Cys7 and Cys3-Cys13, which binds selectively to the muscle subtype of nicotinic acetylcholine receptors [268]. Another feature for the protection of cysteine side chain has been demonstrated in regioselective ligation to form long-chain peptides by means of thioesters [272]. However, there are several side reactions involving the cysteine side chains. One of such side reactions is the racemization during the process of cysteine residue coupling [273], or coupling of a segments containing cysteine residue [274], or at the C-terminal cysteine [275, 276]. The easiness of cysteine racemization can be viewed by the formation of 4-thiazolidinecarboxylic acid derivatives during its reaction with aldehydes or ketones, such as the formation of 2,2-dimethyl-4-thiazolidinecarboxylic acid in reaction with acetone [277]. Another side reaction is the β-elimination of sulfhydryl and its protecting group during the deprotection of Fmoc, as demonstrated in the case of C-terminal cysteine with its side chain protected with acetamidomethyl (Acm) group when treated with piperidine, that forms dehydroalanine derivative which then reacts with piperidine. This β-elimination is governed by the quality of the leaving group and the acidity of the proton at the α-carbon, where the acidity of such proton is influenced by the nature of three nearby protecting groups on: a) the amino group, b) the carboxyl group (anchoring the C-terminal cysteine residue to the resin), and c) the protected sulfhydryl group [278]. It is plausible that the racemization of cysteine may involve the β-elimination mechanism.

Side Chain Protecting Groups 339

When the amino group of cysteine is protected with Fmoc, the N-Fmoc→S-Fm trans-protection has been noticed, possibly involving the E1cb elimination to evolve carbon dioxide and generation of dibenzofulvene. It is suggested that the addition of thiolate generated by the deprotonation of thiol with morpholine to dibenzofulvene yields the S-Fm protected product, as illustrated in the top portion of Scheme 5.7 [279]. As the hydrogen atom at 9-position of the fluorene ring is not acidic enough, with a pK of around 22 if one compares it with the pKa of 9-methylfluorene (pKa = 21.85 ~ 22.3) [279(a)]; on the other hand, the pKa of thiol is 8.5 [279(b)], likely this N-Fmoc→S-Fm trans-protection involves the deprotonation of thiol and subsequent intramolecular substitution, as illustrated in the bottom portion of Scheme 5.7. Similar phenomenon has been noticed during the protection of sulfhydryl group with the Fmoc group when methyl (tert-butoxycarbonyl)L-cysteinate was treated with (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin1-yl) carbonate (Fmoc-OSu) in the presence of Et3N, affording 91% of methyl S-((9H-fluoren-9-yl)methyl)-N-(tert-butoxycarbonyl)-L-cysteinate, rather than the expected methyl S-((9H-fluoren-9-yl)methoxycarbonyl)-N-(tertbutoxycarbonyl)-L-cysteinate. The latter can be exclusively formed when methyl (tert-butoxycarbonyl)-L-cysteinate was treated with (9H-fluoren9-yl)methyl carbonochloridate (i.e., (9H-fluoren-9-yl)methyloxycarbonyl chloride, Fmoc-Cl) in the presence of Et3N. The reactivity difference between these two Fmoc-reagents can be justified by the relative acidity of the conjugate acid of leaving group, where HCl is about 11 orders of magnitude more acidic than the N-hydroxysuccinimide, so that the β-elimination, decarboxylation, and the generation of thiolate and the dibenzofulvene specifically occur in the case of Fmoc-OSu [280].

SCHEME 5.7 The suggested mechanism for the N-Fmoc → S-Fm trans-protection.

It is suggested that the amounts of undesired side products during SPPS involving cysteine were dependent on the polymeric support, the side­chain protecting group, the linker incorporated to anchor cysteine to the resin, and the state of the amino group during chain elongation (either amino or

340

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

amide), as illustrated by the formation of 3-(1-piperidinyl)­alanine adduct on polystyrene resin and with Acm protection [278].

SCHEME 5.8 N-Fmoc → S-Fm trans-protection via intramolecular substitution.

On the other hand, selenocysteine (Sec, U) although is not one of the 20 standard amino acids, it has been considered as the “21st” proteinogenic amino acid because it shares three key properties with the other 20 amino acids, i.e., it has its own codon (UGA) and its own unique tRNA, and can be co-translationally inserted at the ribosome (Note: UAG for the codon of 22nd amino acid, pyrrolysine) [281]. The selenocysteine-containing proteins, known as selenoproteins, have been found in all three kingdoms (e.g., bacteria, archaea, and eukaryotes) of life which play essential roles in human health and development [282]. The unique element within selenocysteine is selenium, belonging to the same chalcogen group as oxygen and sulfur. Therefore, selenocysteine shares many common properties of cysteine [283], and even carries the name of cysteine. For example, cysteine has often been involved in the oxidation-reduction to form cystine, and selenocysteine is located in the active sites of enzymes (e.g., glutathione peroxidase, thioredoxin reductase, and iodothyronine deiodinase) that participate in oxidation-reduction reactions [284–286], and has been found in more than 25 human selenoproteins [283]. Due to the low pKa of its side chain (5.2, [285] see Chapter 1), high reactivity, and redox property, selenocysteine has been more commonly adopted in SPPS, and many protecting groups developed for the sulfhydryl group have been applied in the protection of selenocysteine side chain. Due to the unique properties of the sulfhydryl group as mentioned above, the most diversified distribution of protecting groups has been developed for this group [270]. The first group of sulfhydryl protecting groups are the benzyl and substituted benzyl groups. The application of simple benzyl group can be found in the preparation of mesotocin (a nonapeptide amide) [287], lysine-vasopressin (a nonapeptide) [288], arginine vasopressin [289],

Side Chain Protecting Groups 341

isotocin (a nonapeptide amide) [290], and β-chain of insulin [156], just to name a few. The substituted benzyl groups include 4-methylbenzyl (Meb) [291], p-methoxybenzyl (Mob, Figure 5.4) [270, 292–295], p-nitrobenzyl (pNB, Figure 5.5) [263, 266, 296], 2,4,6-trimethoxybenzyl (Tmob, Figure 5.4) [297–299], Dpm (benzhydryl, Figure 5.4) [300–302], 4-methoxytrityl (also known as monomethoxytrityl (MMT)) [303, 304], 4,4’,4”-trimethoxyphenylmethyl (TMTr) [298, 305], xanthenyl (Xan, Figure 5.4) [306, 307], monomethoxyxanthenyl (Figure 5.4) [307], 9-phenylxanthen-9-yl (Pixyl) [308], 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-methyl (Pbfm, Figure 5.4) [169], (4,5,6-trimethoxy-2,2-dimethyl-2,3-dihydrobenzofuran7-yl)methyl (Tmbm) [169], 2,2,5,7,8-pentamethyl-3,4-dihydro-6-benzopyranylmethyl (Pmbp, also abbreviated as Pmcm, Figure 5.4) [309], 2,2,4,6,7-pentamethyl-2,3-dihydro-5-benzofuranylmethyl (Pmbf, also abbreviated as Pbfm, Figure 5.4) [169, 309], 4,5,6-trimethoxy-2,3-dihydro7-benzofuranylmethyl (Tmbf) [309], 10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-yl (5H-dibenzo-suberyl, Sub), 5H-dibenzo[a,d]cyclohepten5-yl (5H-dibenzosuberenyl, Dbs) [310], 5-phenyl-10,11-dihydro-5Hdibenzo[a,d][7]annulen-5-yl (5-PhSub) and 5-phenyl-5H-dibenzo[a,d][7] annulen-5-yl (5-PhDbs) [311]. In addition to this group of sulfhydryl protecting groups, the second group of protecting groups for the sulfhydryl functionality are the substituted methyl groups, such as Acm [266, 268, 270, 271, 278, 292, 312], benzamidomethyl (Bam, Bzm) [313–315], 4-methoxybenzyloxymethyl (MBom) [316], phenylacetamidomethyl (Pacm, Phacm) [298, 317–319], trimethylacetamidomethyl (Tacm) [267, 320–322], phenacyl (Pac) [272, 323–325], and N-methyl-N-(2-oxo-2-phenylethoxycarbonyl)aminomethyl (also known as N-methyl-phenacyloxycarbamidomethyl, Pocam) [77]. Another type of protecting groups for the sulfhydryl group of cysteine are alkyl groups, including t-butyl [267, 291, 326–328], 2,3-dihydroxypropyl [329], 2-(2,4-dinitrophenyl)ethyl (Dnpe) [267, 298, 330], Thp [267, 331, 332], 4-picolyl (Figure 5.5) [142, 333], diphenyl-4-pyridylmethyl (PyBzh, Figure 5.5) [142, 334, 335], 3-amino-2-hydroxy-3-oxopropyl (often known as 2-carbamoyl-2-hydroxyethyl) [336, 337], allyl [338], propyl, and ethyl [338]. It should be pointed out that S-allyl protected cysteine is a natural product commonly found in garlic (Allium sativum), which has demonstrated enormous amount of biological activities, such as against the development of a proinflammatory status [339], skeletal muscle atrophy [340], and reduction of acute liver dysfunction induced by lipopolysaccharide/D-galactosamine

342

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

[341], just to name a few. Interested readers can easily find hundreds of literature about other biological functions of this compound. The fourth type of protecting groups for the sulfhydryl functionality are the carbamoyl or alkoxycarbonyl groups, such as the famous (9H-fluoren9-yl)methoxycarbonyl (Fmoc, Figure 5.1) [280], and less often used ethylcarbamoyl [267, 342–345], (2-chloroethyl)carbamoyl [344], and 2-nitrobenzyloxycarbonyl (Nboc) [346]. The fifth group of sulfhydryl protecting groups are sulfenyl thiocarbonate and carbamoylsulfenyl groups, such as (methoxycarbonyl)sulfenyl (Scm), (methoxycarbonyl)disulfanyl (Sscm), (N-methyl-N-phenylcarbamoyl) sulfenyl (Snm), and (N-methyl-N-phenylcarbamoyl)disulfanyl (Ssnm) [299]. Due to the readiness of forming disulfide linkage between cysteine, several alkylthio-groups have also been applied to protect the cysteine sulfhydryl group, that include tert-butylsulfanyl [347] or tert-butylsulfenyl (S-tBu) [348], 2,3-dimethylbutan-2-ylthio (also known as sec-isoamylthio, SIT) [269], 2-methyltetrahydrofuran-3-ylthio (also known as 2-methyloxolane3-thio, MOT) [269], 2,4,6-trimethoxyphenylthio (S-Tmp) [349], 2,6-dimethoxyphenylthio (S-Dmp) [349], 3-nitro-2-pyridinesulfenyl (3-Npys) [350], tritylsulfanyl (S-Trt) [351, 352], and (5-nitropyridin-2-yl)thio (also known as 2-thio(5-nitropyridyl), 5-Npys) [353, 354]. For the protection of the selenocysteine side chain, all protecting groups outlined above for cysteine’s side chain are theoretically feasible for selenocysteine. In fact, some of these groups have already been applied for selenocysteine, such as methyl [284], propyl [284], allyl [282–284], benzyl [355, 356], Meb [356–358], 4-methoxybenzyl (Mob) [312, 356, 359–363], p-nitrobenzyl (pNB) [266], Acm [266], 9-xanthenyl (Xan) [364]. In addition, phenyl [361], 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl (Nbde) [283], and nitroveratryl (Figure 5.5) [283] groups have also been applied to protect the selenocysteine side chain. Some of these protecting groups are shown in Figure 5.6. The disulfide bond is not so stable under basic conditions, as shown in the weak base treatment of EGF-like motif of neuregulin 1-β1 with three disulfide linkages in a single 71-residue chain [365], and alkaline cleavage of the disulfide bonds in black-eyed pea (Vigna unguiculata) trypsin and chymotrypsin inhibitor (BTCI) [366]. Likewise, the t-butylthio and 2,6-dimethoxybenzylthio groups are not very stable under basic conditions and have been deprotected to a certain degree after repeated treatments with 20% piperidine. The secondary thiol protecting groups, such as SIT and MOT with a branched point at the β-position, particularly for the MOT, are fully compatible with Fmoc​/t-Bu strategy and are more susceptible to be removed in the presence of piperidine

Side Chain Protecting Groups 343

[269]. On the other hand, the disulfide bond can be cleaved upon oxidation or reduction. For example, the relative stability of disulfide bond has been applied to the preparation of μO-Conotoxin MfVIA, where the Cys-Cys (3–6) linkage was formed by removal of S-Trt groups on Cys3 and Cys6 with iodine in a mixture of hexafluoroisopropanol and CH2Cl2, and additional S-S linkage between Cys2 and Cys5 was created after further oxidation with iodine in DMF [367]. In contrast, although 3-Npys is stable to the treatment of TFA, or HF/anisole (9:1) at 0°C for 45 minutes, it can be removed by thiolysis with either 2-mercaptopyridine and 2-mercaptomethylimidazole or with 3-mercaptoacetic acid and 2-mercaptoethanol [368]. While S-benzamidomethyl is unstable during HF cleavage and alkaline treatment, S-Tacm is stable under these conditions, which can be removed with iodine or Hg(II) treatment [320]. Upon the treatment with TFA, the relative stability has been found in the following order: Bn > p-methylbenzyl >> S-t-butyl group > t-butyl group, where the benzyl group is inert [369]. However, the stability of Meb and Acm progressively decreases in DMSO at elevated temperatures [291]. For the introduction of the sulfhydryl protecting group, the tetrahydropyran protected cysteine can be prepared in Et2O from the reaction of cysteine and dihydropyran in the presence of BF3·Et 2O at room temperature, or in CH2Cl2 in the presence of PTSA [331]. The protecting groups of Pmcm and Pbfm can be introduced to cysteine from the reaction of the corresponding alcohols with cysteine hydrochloride in the presence of anhydrous TFA [169]. Similarly, the pixyl protected cysteine is formed from the condensation of 9-hydroxy9-phenylxanthene with cysteine in AcOH in the presence of BF3·Et 2O [308]. The preparation of S-fluorenylmethylcysteine involves the reaction of cysteine with fluorenylmethyl chloride or (9H-fluoren-9-yl)methyl 4-methylbenzenesulfonate in the presence of N,N-diisopropylethylamine, where the protecting group is stable towards the treatment with anhydrous HF, iodine in DMF, and 2-mercapto-1-ethanol in DMF [71]. The Dnpe is a very useful alternative to the S-Fm group, as the Dnpe prepared cysteine can be easily prepared from the reaction of cysteine with 2-(2,4-dinitrophenyl)ethyl bromide and can be safely removed by treatment with piperidine, and the combination of Dnpe and Acm and/or Meb can facilitate the orthogonal synthesis of target peptides containing two or more disulfide bridges. The Dnpe group is base labile and compatible with Boc/Bn strategy [298], and can be introduced to cysteine from the reaction of cysteine and 2,4-dinitrophenethyl 4-methylbenzenesulfonate [330]. The protocols for the introduction of other protecting groups can be found in the relevant literature.

344

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

FIGURE 5.6 Protecting groups for the side chain of cysteine and selenocysteine.

Side Chain Protecting Groups 345

Regarding the removal of cysteine protecting groups, the t-butyl group can be removed with TFA and Hg(OAc)2 at pH 4 [326], or by TMSBr in TFA with a sufficient amount of ethane-1,2-dithiol (EDT) as a scavenger [328]. 4-Methoxytrityl (Mmt) is more acid-labile than trityl, it can be easily removed by 1% TFA in CH2Cl2 in the presence of t-butyl and trityl group, whereas both trityl and Mmt can be deblocked together with acidolytic mixture (TFA/EDT/H2O/TIS: 94/2.5/2.5/ 1; v/v/v/v) [265]. Tetrahydropyran (Thp) can be removed by acid, including H2SO4, aqueous acetic acid, PTSA in alcohol, 10% TFA in the presence of carbocation scavengers (e.g., water or TIS), as well as Lewis acids [331]. While Acm has been reported to be stable towards acid and will be deblocked with iodine or thallium salt oxidation [278], Acm and Tacm are also reported to be unstable in the presence of TFA and HF, particularly at elevated temperatures, whereas p-nitrobenzyl remains under this condition [266]. The strong Lewis acids such as TMSBr and TMS trifluoromethanesulfonate (TMSOTf) have also been used to remove benzyl and Mob groups from the side-chains of cysteine and selenocysteine; however, these reagents are extremely harsh and have limited solubility in ether, in addition, they can trigger side reactions with peptide or precipitate with the target peptides, causing peptides to become oily and difficult to handle upon lyophilization [312]. In contrast, Lewis acid Tl(CF3CO2)3 has been applied to deprotect t-butyl, benzyl, Mob, Meb, Acm, as well as Dbs and adamantyl groups, facilitating the formation of a disulfide linkage between two adjacent cysteine residues [370]. A similar report has pointed out that oxidative deprotection with iodine or thallium salt leads to the formation of an intramolecular disulfide linkage [297]. Similarly, Hg(OAc)2 in TFA has been applied to remove t-butyl, adamantyl, and Mob groups from cysteine, but failed to deblock benzyl group [371]. Deprotection of Acm with silver trifluoromethanesulfonates avoids the oxidization of methionines [372]. AgBF4 in TFA has been used to deblock both Acm and Tacm without affecting other protecting groups [373]. In addition, 2,2’-dithiobis(5nitropyridine) (DTNP) in TFA in the presence of 2% thioanisole has been applied for the deprotection of Mob and Acm from cysteine and selenocysteine, where DTNP or 2,2’-dithiodipyridine (DTP) is a much milder electrophile than the iodonium cation, which is a solid with good solubility in TFA [374]. The commonly used 2,4,6-trimethoxybenzyl (Tmob) group can be deprotected under the following conditions, 30% of TFA in CH2Cl2 along with

346

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5% each of PhOH, PhSMe, and water; 6% of TFA in CH2Cl2 in the presence of 0.5% of TES or TIS, affording the free sulfhydryl group. However, the cleavage of Tmob and transfer of Tmob to other carbocation scavengers is a reversible process, the choice of scavenger is very critical, only silane is effective to irreversibly convert the Tmob to 2,4,6-trimethoxytoluene. In the presence of co-solvent such as DMF or methanol, Tmob is partially deprotected at a variety of acid concentrations [297]. For comparison, Meb group must be removed under high acidity. For example, only 79% of cysteine has been recovered with 90% HF treatment along with conventional scavenger anisole, whereas substitution of anisole with 5% of p-cresol and 5% of p-thiocresol leads to complete removal of methylbenzyl group at 0°C in 1 hour [375]. The reductive deprotection of sulfhydryl protecting groups generally applies to the removal of disulfide protecting groups. In this case, thiols such as β-mercaptoethanol [330, 350], 2-mercaptopyridine, 2-mercaptomethylimidazole, 3-mercaptoacetic acid [350], and dithiothreitol (DTT) have been applied as the reducing agents, such as the removal of fluorenylmethyl group with 2-mercaptoethanol or DTT [71]. It should be pointed out that the thiolysis of S-Snm or S-Scm may produce intramolecular disulfide linkage, and potential homologation with S-Ssnm and S-Sscm could result in the formation of trisulfide and/or tetrasulfide [299]. The application of these thio-protecting groups and their deprotection to form trisulfide linkage may provide a unique way to form peptides with necessary trisulfide connection, as it has already been reported that as much as 10% of recombinant human growth hormone produced in Escherichia coli contains such trisulfide linkage [299]. For example, an extra sulfur has been identified between the Cys182 and Cys189 of a biosynthetic human growth hormone. [376, 377]. Also, several trisulfide-containing antibodies have been identified, such as mAb-Y antibody variant [378], and recombinant monoclonal antibody [379]. The reductive cleavages of sulfhydryl protecting groups also include the removals of phenacyl (Pac) [272], and N-methyl-N-(2-oxo2-phenylethoxycarbonyl)aminomethyl (Pocam) [77] with zinc in acetic acid. Finally, some protecting groups can be easily removed under basic conditions, such as ethylcarbamoyl group [342], whereas a few individual groups might be deblocked under special conditions, such as the Pacm group that is removed by penicillin amidohydrolase [298].

Side Chain Protecting Groups 347

5.8.1 EXEMPLARY PROCEDURES TO PROTECT CYSTEINE AND SELENOCYSTEINE SIDE CHAINS 5.8.1.1 PREPARATION OF S-BENZHYDRYL-L-CYSTEINE [302]

To a solution of 157.62 g of L-cysteine hydrochloride (1.00 mol) in 1 liter of acetic acid at 60°C, was added 184.23 g of benzhydrol (1.00 mol), followed by 140 mL of BF3·Et 2O under stirring. Then 1.5 L of 95% ethanol, 500 mL of water, and 300 g of sodium acetate were added sequentially. Upon cooling, 258.4 g of crude S-benzhydryl-L-cysteine was filtered. This compound can be purified by dissolving 15 g of S-benzhydryl-L-cysteine in 150 mL hot ethanol and 10 mL of 5 N HCl, followed by the addition of 8 mL pyridine. Filtration and washing afford pure S-benzhydryl-L-cysteine, m.p. 206–207°C (decomp.). A similar procedure has been applied to make N-Fmoc-Cys(Mmt)-OH [304], and N-(9-fluorenylmethoxycarbonyl)-S-(2,2,5,7,8-pentamethylchroman-6-yl)-L-cysteine [169], and S-(Tmob)-cysteine trifluoroacetate salt [297], for the direct reaction of cysteine or cysteine hydrochloride with the corresponding alcohols in the presence of acid. 5.8.1.2 PREPARATION OF (R)-2-AMINO-3-((4-METHOXYBENZYL) SELANYL)PROPANOIC ACID (Se-p-METHOXYBENZYL-LSELENOCYSTEINE)

5.8.1.2.1 Preparation of (2R,2’R)-3,3’-Diselanediylbis(2Aminopropanoic Acid) (Bis-L-Selenocysteine) [380] To 60 mL 1 M disodium diselenide solution was added 3.2 g of β-chloro-Lalanine (26 mmol) in 50 mL of H2O (pH 9.0) dropwise over 2 hours. The

348

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

resulting solution was stirred at 37°C under nitrogen atmosphere for 16 hours. After adjustment of pH to 2 with 6 N HCl, 6 mmol of hydroxyamine (NH2OH) was added to reduce contaminating elemental selenium to H2Se. This solution was flushed with N2 gas, and the exhaust gas (N2 and H2Se) was passed through two successive saturated solutions of lead acetate to trap H2Se. Then, the pH was adjusted between 6.0 and 6.5 with NaOH. After standing overnight at 4°C, bis-selenocysteine was obtained as yellow crystal, which was further recrystallized from water to afford 2.71 g of bisL-selenocysteine, in a yield of 62%, m.p. 184–185°C (decomp.). 5.8.1.2.2 Preparation of Se-p-Methoxybenzyl-L-Selenocysteine [362] To an ice-cooled solution of 1.90 g of bis-L-selenocysteine (5.69 mmol) in 5 mL 0.5 N NaOH was added 1.80 g of NaBH4 (44.9 mmol) in portions under stirring, and the reaction mixture was stirred at room temperature until the disappearance of yellow color. The solution was then cooled in an ice bath and 15 mL of 2 N NaOH was added, followed by the addition of 4.06 mL p-methoxybenzyl chloride (29.9 mmol) dropwise. After vigorous stirring at 4°C for 4 hours, the reaction mixture was acidified with concentrated HCI to form a precipitate of crude product. Filtration, washing with ether, and recrystallization from hot water afforded 2.44 g of Se-p-methoxybenzyl-Lselenocysteine, in a yield of 74%, m.p. 173–176°C (decomp.). 5.8.1.3 PREPARATION OF N-(((9H-FLUOREN-9-YL)METHOXY) CARBONYL)-S-((METHYL((2-OXO-2-PHENYLETHOXY) CARBONYL)AMINO)METHYL)-L-CYSTEINE [77]

The starting material of (2-phenyl-1,3-dioxolan-2-yl)methyl methylcarbamate was prepared from 2-hydroxy-1-phenylethan-1-one which was first protected with ethylene glycol in the form of (2-phenyl-1,3-dioxolan2-yl)methanol and then coupled with disuccinimidyl carbonate, and methylamine in DMSO in the presence of DMAP and DIPEA. This starting

Side Chain Protecting Groups 349

material in an amount of 260 mg (1.1 mmol) was dissolved in 1.5 mL of dimethoxyethane, and 0.5 mL of 36% aqueous formaldehyde solution and 120 mg of Na2CO3 (1.1 mmol) were added. After being stirred at room temperature for 2 hours, the reaction mixture was mixed with EtOAc, washed with H2O and brine, and dried over Na2SO4. Upon filtration and concentration, (2-phenyl-1,3-dioxolan-2-yl)methyl (hydroxymethyl) (methyl)-carbamate was obtained, which was mixed directly with 350 mg of cysteine hydrochloride (2.0 mmol) in 2 mL of trifluoroacetic acid without further purification. The reaction mixture was stirred at room temperature for 30 minutes, and TFA was removed under reduced pressure. The residue was chromatographed on silica gel with CHCl3/CH3OH/ AcOH (90/10/1) to give 370 mg of S-((methyl(((2-phenyl-1,3-dioxolan2-yl)methoxy)carbonyl)amino)methyl)-L-cysteine and S-((methyl((2-oxo2-phenylethoxy)carbonyl)amino)methyl)-L-cysteine as an inseparable mixture by column. To this mixture, was then added a solution of 370 mg (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (FmocOSu, 1.1 mmol) in 5 mL DMF which also contained 170 µL DIPEA (1.1 mmol). The resulting solution was stirred at room temperature for 2 hours and then mixed with EtOAc, washed with 1 M aqueous HCl and brine, and dried over Na2SO4. After filtration and concentration in vacuo, the residue was chromatographed on silica gel with toluene/EtOAc/AcOH (50/50/1) to afford 440 mg of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-((methyl((2oxo-2-phenylethoxy)carbonyl)amino)methyl)-L-cysteine as a colorless solid, with an overall yield of 73% for the three steps, Rf = 0.18 (Toluene/ EtOAc/AcOH, 50/50/1). 5.9 PROTECTING GROUPS FOR TRYPTOPHAN Among the 20 traditional proteogenic amino acids, tryptophan is the largest amino acid with an indole moiety at the side chain. It has demonstrated many important biological activities, such as the precursor of neurohormone serotonin [381]. However, due to the extended aromatic system, the indole ring is very reactive, rendering alkylation and acylation products at various positions. In addition, the indole ring can be oxidized or reduced [382, 383]. As a result, tryptophan is one of the most challenging residues during peptide synthesis [384]. One common problem associated with tryptophan is t-butylation during the acidolytic deprotection of the t-Boc protecting group or t-butyl esters on other amino acid residues with TFA or HF, arising from the capture of

350

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

t-butyl carbocation as of the enriched electron density on the indole ring [385]. In this case, the t-butyl group can be translocated to the nitrogen atom of the indole ring (Nin), as shown in the preparation of 1-(tert-butyl)L-tryptophan (or Nin-tert-butyltryptophan) from the reaction of benzyl (benzyloxycarbonyl)-L-tryptophanate and tert-butyl acetate in the presence of TFA, followed by hydrogenation, or alternatively from the reaction between methyl (benzyloxycarbonyl)-L-tryptophanate and isobutene in the presence of H2SO4, followed by saponification and hydrogenation [386]. More complicatedly, other C- and N-butylated tryptophan derivatives have also been detected with t-butyl acetate in the presence of TFA, such as 5-t-butyl, 1,5-di-t-butyl tryptophan derivatives [387]. During the acidolytic cleavage of the Boc group from methyl (tert-butoxycarbonyl)-L-tryptophylglycinate in the presence of TFA, TFA/anisole or HCl/dioxane, as well as t-butylation of tryptophan within Ac-Trp-Gly-OMe and Z-Trp-Gly-OH with Me3COH/CF3CO2H or H-Ala-OCMe3/CF3CO2H, it is clear that the reaction rate and specificity of the tert-butylation is highly dependent on the nature of the cleaving agents and on the environment of the tryptophan residue in the peptide chain [388]. When tryptophan was left in excess amount of t-butanol and TFA at room temperature for 48 hours, as many as 11 products have been identified, including two tri(t-butyl)tryptophan and one tetra(tbutyl)tryptophan, where 2,5,7-tri-t-butyltryptophan was obtained in 34.5% yield [389]. Many other examples of t-butylation of tryptophan have been summarized already [390]. Similarly, benzylation of tryptophan has been observed during the removal of benzyl protecting groups. For example, during the acidolysis of 4-methoxybenzyloxycarbonyl-L-tryptophan (p-MeOC6H4CH2O2CTrp-OH) by CF3CO2H/anisole, several tryptophan derivatives with p-methoxybenzyl group at various positions have been identified, although they can be suppressed by the use of thioanisole or Me2S containing 2% ethanedithiol-skatole as cation scavengers [391]. Likewise, in solutionphase synthesis of an anti-human immunodeficiency virus peptide (T22), a significant amount of p-methoxybenzyl has been translocated from cysteine to tryptophan during thioanisole-mediated deprotection with 1 M trimethylsilyl trifluoromethanesulfonate (TMSOTf) in trifluoroacetic acid [392]. The alkylation of tryptophan with Tmob group has been found during cleavage of the synthesized peptide from the solid support under acidic conditions, where the Tmob was used to protect glutamine and asparagine [173].

Side Chain Protecting Groups 351

Besides the translocation of t-butyl and substituted benzyl groups, some acyl groups have been translocated to tryptophan moiety during deprotection, such as the migration of 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) from arginine to tryptophan [393, 394], and the transfer of sulfenyl on nitrogen to indole ring of tryptophan, where sulfenyl group is 2-nitrophenylsulfenyl (NPS) or 2,4-dinitrophenylsulfenyl (DNPS) [395]. For the transfer of Pmc from arginine to tryptophan, it is found that when these two amino acids are separated by hydrophilic residues, the migration of Pmc is much more pronounced than the case when they are separated by hydrophobic residues. Particularly, when arginine and tryptophan are separated by one [394] or two [393] amino acid residues, such migration is most pronounced and cannot be suppressed by any scavenger mixture. The extent of migration depends on the amino acid that separated arginine and tryptophan as well as the position of tryptophan within the peptide chain and the type of solid-phase carrier [394]. It is found that minimization of Trp alkylation can be achieved by the inclusion of side-chain protecting group “scavengers” during the TFA treatment [384]. On the other hand, when the indole ring is protected with an electronwithdrawing group, the overall affinity of the indole moiety towards carbocation will be reduced, and the potential side reaction of tryptophan can be suppressed. Therefore, most of side chain protecting groups for tryptophan are acyl groups, that are mounted to position 1 of indole ring (Nin-position). These protecting groups are classified into four groups, i.e., formyl group [327], urethane groups, arylsulfonyl groups, and diphenylphosphinothioyl [396] groups. More than seven urethane groups have been applied to protect indole moiety of tryptophan, including benzyloxycarbonyl (Z) [397], 4-nitrobenzyloxycarbonyl (Z(NO2)) [398], 2,4-dichloro-benzyloxcarbonyl [399], t-butoxycarbonyl (Boc) [384, 400], 2,2,2-trichloroethoxycarbonyl (Troc) [401, 402], methoxycarbonyl (Moc) [403], ethoxycarbonyl (Etoc) [390], cyclohexyloxy-carbonyl (Hoc) [404], and 1-adamantyloxycarbonyl (Adc) [390], etc. The arylsulfonyl protecting groups include tosyl (Tos) [398], 4-methoxybenzenesulfonyl (Mbs) [405], 2,4-dimethoxybenzenesulfonyl (Dmb) [406], 2,4,6-trimethoxybenzenesulfonyl (Mtb) [406], 4-methoxy2,3,6-trimethylbenzenesulfonyl (Mtr) [406], 2,4,6-trimethylbenzenesulfonyl (mesitylenesulfonyl, Mts) [407, 408], and 2,4,6-triisopropylbenzenesulfonyl (Tip) [390]. Besides these many acyl protecting groups, some alkyl groups have been applied to protect the nitrogen of indole moiety, such as methyl [409],

352

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

3-pentyl [404], and carboxylmethyl, where the carboxymethyl was then connected with amino acid derivative [409]. Due to the instability of the formyl group under various experimental conditions, it is suggested that Nin-formyl group is only of limited use in peptide synthesis [385]. Similarly, Boc-protected tryptophan has unexpected low stability during purification on silica gel chromatography [410]. Regarding the removal of these tryptophan side-chain protecting groups, formyl group can be removed by base treatment (NaOH/DMF, pH 13, 10 min) [411], whereas tosyl and benzoyl groups can be deprotected under mild conditions either by controlled-potential electrolysis or chemical reduction using either magnesium in anhydrous methanol or mercury-activated aluminum [398]. However, there are a few situations that the side chain of tryptophan is not necessarily protected, such as when possible acidic reagents are not used, as demonstrated in the solid-phase synthesis of [15-leucine]little gastrin on a polar polyamide resin support [412]. Also, no side chain protection has been applied for tryptophan during the solid-phase synthesis of ribonuclease T1 [413]. When the peptide remains attached to the solid support throughout the deprotection reaction, the t-butylation of tryptophan is reduced during acidic cleavage of the tert-butyl protecting groups in the absence of thiol-scavenging reagents [414]. During the study of structure and stability of molten globule state of guinea pig α-lactalbumin, the advantage of Nin-formyl protection versus no protection is not so obvious, thus it is suggested that the tryptophan side chain protection is unnecessary in this case [415]. 5.9.1 EXEMPLARY PROCEDURES TO PROTECT TRYPTOPHAN SIDE CHAIN 5.9.1.1 PREPARATION OF 1-FORMYL-DL-TRYPTOPHAN HYDROCHLORIDE [416]

Side Chain Protecting Groups 353

A solution of 0.50 g of DL-tryptophan in 10 mL 98–100% formic acid was saturated with gaseous HCl with vigorous agitation and maintained in an HCl atmosphere at room temperature for 60–90 minutes. After removal of the solvent, the residue was crystallized from ethanol/ethyl ether, collected by filtration, washed with ethyl ether, and dried. 5.9.1.2 PREPARATION OF 1-TOSYL-L-TRYPTOPHAN [406]

5.9.1.2.1 Preparation of Benzyl 1-Tosyl-Nα-Trityl-Tryptophanate To a solution of 2.15 g of benzyl Nα-trityl-L-tryptophanate (4 mmol) in 10 mL DMF was added 240 mg of 50% NaH in mineral oil (5 mmol) under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 hours. Then, 950 mg of p-toluenesulfonyl chloride (5 mmol) was added, and the mixture was stirred for 15 hours. After the addition of 50 mL EtOAc, the mixture was stirred for an additional hour, then mixed with 30 mL water. The isolated organic layer was washed with 5% NaHCO3 and water and dried over Na2SO4. Upon removal of solvent, the residue was purified by column chromatography on silica gel using toluene as eluent, to afford 1.70 g of benzyl 1-tosyl-Nα-trityl-tryptophanate, in a yield of 61.5%, m.p. 101–103°C. 5.9.1.2.2 Preparation of 1-Tosyl-L-Tryptophan To 20 mL 80% acetic acid, was added 1.3 g of benzyl 1-tosyl-Nα-trityltryptophanate (1.88 mmol), the resulting suspension was stirred at room temperature. After a while, triphenylmethanol precipitated out. Upon filtration of triphenylmethanol, the filtrate was subjected to catalytic hydrogenolysis at room temperature for 4 hours in the presence of palladium black. The reaction mixture was filtered and the filtrate was evaporated to dryness. The residue was recrystallized from water to afford 585 mg of 1-tosyl-Ltryptophan, in a yield of 86.9%, m.p. 227°C (dec.).

354

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

5.9.1.3 PREPARATION OF 1-MESITYLENESULFONYL-L-TRYPTOPHAN [407]

5.9.1.3.1 Preparation of N-(4-Methoxybenzyloxycarbonyl)-NinMesitylenesulfonyl-L-Tryptophanate (Z(OMe)-Trp(Mts)-OH) To an ice-NaCl chilled mixture of 27.51 g benzyl (4-methoxybenzyloxycarbonyl)-L-tryptophanate (Z(OMe)-Trp-OBzl, 60 mmol), 6.0 g of NaOH powder (150 mmol) and 0.19 g of cetyltrimethylammonium chloride (0.6 mmol) in 300 mL CH2Cl2, was added 32.85 g of mesitylenesulfonyl chloride (Mts-Cl, 150 mmol) in 300 mL CH2Cl2 dropwise. After being stirred in an ice bath for 16 hours, the mixture was acidified with 1 N HCl. The organic phase was washed with brine, 5% NaHCO3 and H2O, then dried over Na2SO4 and concentrated. The residue was dissolved in 600 mL EtOH and treated with 90 mL 1 N NaOH in an ice bath for 2.5 hours. Then, the solvent was evaporated off in vacuo and the residue was dissolved in 400 mL H2O. The aqueous phase, after being washed with ether, was acidified with 1 N HCl and the resulting oily precipitate was extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated. Treatment of the residue with n-hexane afforded 32.4 g of N-(4-methoxybenzyloxycarbonyl)-Nin-mesitylenesulfonyl-L-tryptophanate as a powder, in a yield of 98%. 5.9.1.3.2 Preparation of Nin-Mesitylenesulfonyl-L-Tryptophan (H-Trp(Mts)-OH) N-(4-Methoxybenzyloxycarbonyl)-Nin-mesitylenesulfonyl-L-tryptophanate (0.55 g, 1.0 mmol) was treated with a mixture of 1.5 mL TFA and 0.3 mL of anisole in an ice-bath for 60 minutes, then TFA was removed by evaporation. Treatment of the residue with ether afforded a powder, which was recrystallized from MeOH and ether in the presence of a few drops of Et3N

Side Chain Protecting Groups 355

to afford 0.34 g of Nin-mesitylenesulfonyl-L-tryptophan, in a yield of 87%, m.p. 211–213°C. 5.10 PROTECTING GROUPS FOR METHIONINE Methionine is a very important amino acid within a protein, often because of its rule as the initial amino acid during protein synthesis [417]. In addition, the smooth conversion of methionine to methionine sulfoxide with oxidizing agent and reduction of methionine sulfoxide back to methionine by ubiquitous methionine sulfoxide reductases render methionine an antioxidant activity [418]. Also, methionine is important for cancer cell growth and metabolism [419]. Due to so many biological activities of methionine, there have been more than 400 review articles about amino acid methionine. On the other hand, regarding the peptide synthesis involving methionine, the side chain of methionine theoretically should not be an issue during peptide synthesis, as the thioether group is much less reactive than other acidic or basic functionality. This might be the reason that there in fact are not many specific reports about the protection and deprotection of the methionine side chain [420]. However, there are obvious differences in reactivity between thioether and ether, including the higher nucleophilicity of thioether versus ether and instability of thioether in exposure to oxidizing agents. As sulfur is more polarizable than oxygen, and sulfur atom within methionine is more nucleophilic than the oxygen in ether, it is quite common for the reaction of thioether, also known as sulfide, with alkylating agents to form sulfonium salts [421, 422]. The resulting sulfonium salt is much more polar than the thioether itself, resulting in an enhanced solubility in an aqueous solution. For example, alkylation of methionine side chain has been applied to modulate the lower critical solubility temperature (LCST) of temperature-responsive recombinant elastin-like polypeptides [423]. Likewise, chemoselective alkylation of methionine residues on methioninecontaining polymers allows the “methionine click” functionalization to form multifunctional and multi-reactive polypeptides [424]. The good nucleophilicity of methionine side chain over other nucleophilic side chains, such as hydroxyl group in serine, phenoxy in tyrosine, the amino group in lysine and guanidino group in arginine has been demonstrated in the treatment of 1 mmol of amino acid in 10 mL water in the presence of 21 mmol NaHCO3 with 20 mmol of triethyloxonium fluoroborate to afford nearly pure

356

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

amino acid ethyl ester along with the alkylation of methionine and histidine side chains, whereas no alkylations occurring on the above mentioned OH, NH2 and guanidino groups [425]. The nucleophilicity of methionine side chain has been widely applied for the nonenzymatic cleavage of methionyl peptide bond with cyanogen bromide during protein sequencing under mild conditions, where the intermediate cyanosulfonium bromide of methionine is isolated, leading to the formation of iminolactone of homoserine, which is unstable in the aqueous acidic reaction medium and is readily hydrolyzed to homoserine with the liberation of the amino acid or the aminoacyl peptide that followed methionine [426]. Furthermore, allyl, but-2-yn-1-yl groups and 2-oxo-2-phenylethyl have been attached to methionine and cysteine side chains, as well as histidine and lysine by means of allyl bromide, 1-bromo-2butyne and 2-bromoacetophenone for further modifications or introduction of additional functional group, such as the “click chemistry” for the C-C triple bond [427]. Furthermore, the high nucleophilicity of thioether side chain of methionine is not only demonstrated in many side-chain alkylations to form sulfonium salts but also seen in conjugate addition to α,β-unsaturated ketones, such as the addition of methionine to ortho-benzoquinone (i.e., cyclohexa-3,5diene-1,2-dione) to form (3,4-dihydroxyphenyl)sulfonium salt [428]. In addition to converting the hydrophobic side chain of methionine into hydrophilic by means of alkylation, oxidation of thioether into sulfoxide will also increase the polarity of the side chain. When 19 out of 20 amino acids (cysteine excluded) are treated with peroxymonosulfate without explicit activation, a strong oxidizing agent with EH0 = 1.82 V at environmental pH values, the reaction half-life ranges from milliseconds to hours, with four most reactive amino acids of decreasing rate in the order of methionine > tryptophan > tyrosine > histidine. This result indicates that methionine and tryptophan are likely the initial targets of oxidation in peptides and proteins [429]. The Boc-derivatives of Met, Cys, and Trp can be effectively oxidized with methyltrioxorhenium/H2O2 system; even when they are embedded in peptides, they can be regioselectively oxidized with such a system [430]. Due to the susceptibility of methionine toward the oxidizing agents, biological oxidation of methionine has affected the functions of many proteins, for which one of the key enzymes, the methionine sulfoxide reductase A (MsrA) is implicated in a number of diseases [431, 432]. Also, oxidation of methionine may affect the stability of proteins, as shown in the development of β-amyloid-​dependent pathogenesis of Alzheimer’s disease [433], or even

Side Chain Protecting Groups 357

lead to loss of biological activity of proteins, such as the oxidation of methionine in calmodulin [434]. For example, once the side chain of methionine in collagen is made hydrophilic by either alkylation with iodoacetate or oxidation with H2O2, the collagen forms fibrils only very slowly at neutral pH and 37°C whereas unmodified collagen forms fibrils easily, because nucleation of fibrils is retarded by promoting stabilization of the bound water which must be expelled for proper alignment of the collagen molecule [435]. Similarly, the oxidation of methionine is one of the major chemical degradations of biotherapeutic drugs [436]. In a particular example involving the oxidation of methionine, the oxidation of human parathyroid hormone (1–34) in the presence of ferrous EDTA with oxygen result in the conversion of methionine[8] to methionine sulfoxide and histidine[9] to 2-oxo-hisitidne, and subsequent hydrolytic cleavage between Met[8] and His[9] [437]. Based on the information provided above regarding methionine, there is a potential difficulty during the synthesis of peptides containing methionine or tryptophan residue during the TFA based acidic cleavage [438]. In order to minimize the potential oxidation of methionine for the cleavage of methionine-containing peptides prepared by Fmoc-based solid-phase synthesis, a mixture of reagents containing 81% of trifluoroacetic acid, 5% of phenol, 5% of thioanisole, 2.5% of 1,2-ethanedithiol, 3% of water, 2% of dimethylsulfide and 1.5% of ammonium iodide (all in w/w) has been designed, which works very well for the model pentadecapeptide from the active site of DsbC, a periplasmic protein involved in protein disulfide bond formation. When 1.5% of NH4I was added to other cleavage reagent cocktails, it is still effective to reduce the amount of methionine sulfoxide [439]. Nevertheless, there are only a few practical examples for protecting and deprotection of the methionine side chain during peptide synthesis. The conversion of methylsulfide into methyl sulfoxide in methionine side chain can greatly avoid the potential alkylation to form sulfonium salt, although the resulting sulfonium salt would enhance the solubility of peptides [420]. In addition to common oxidizing agents such as H2O2, NaIO4 has been used as well [440, 441]. The resulting sulfoxide can then be converted back to methylsulfide at the end of peptide synthesis. For example, in the process of making N-benzyloxycarbonyl-N-methyl methionine, the intermediate of benzyl (R)-4-(2-(methylthio)ethyl)-5-oxooxazolidine3-carboxylate was oxidized with mCPBA in CH2Cl2 to give benzyl (4R)-4-(2-(methylsulfonyl)ethyl)-5-oxooxazolidine-3-carboxylate, which was then treated with TES in combination with TFA in CHCl3 to afford (2R)-2-(((benzyloxy)carbonyl)-(methyl)amino)-4-(methylsulfonyl)

358

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

butanoic acid. Subsequent treatment with ammonium iodide and dimethylsulfide yields the final product. In this way, the potential reductive cleavage of the methionine side chain is avoided during the reduction of oxazolidinone in the prior step by means of sulfoxide [107]. The last step applies a general reaction for the conversion of sulfoxide into sulfide with iodide [442]. Likely, the sulfoxide can also be converted into sulfide with dimethylsulfide and trifluoroacetic anhydride [443], or a combination of oxalyl chloride and iodide salt [444]. 5.10.1 EXEMPLARY PROCEDURES TO PROTECT METHIONINE SIDE CHAIN 5.10.1.1  OXIDATION OF (4-METHOXYBENZYLOXY)CARBONYL-LMETHIONINE [440]

A mixture of 15.67 g (4-methoxybenzyloxy)carbonyl-L-methionine (Z(OMe)-Met-OH, 0.050 mol) in 100 mL EtOAc and 13.9 g of NaIO4 (0.065 mol) in 80 mL water was stirred efficiently at room temperature for 7 hours until the starting material disappeared by thin-layer chromatography analysis. After acidification with citric acid, the organic phase was separated, washed with brine and dried over Na2SO4. Upon filtration and condensation in vacuo, the residue was triturated with EtOAc and recrystallized twice from MeOH and EtOAc to afford 11.82 g of (2S)-2-((4-methoxybenzyloxycarbonyl) amino)-4-(methylsulfonyl)butanoic acid, in a yield of 71.8%, m.p. 97–99°C. 5.10.1.2  ALLYLATION OF N-ACETYL-DL­METHIONINE [427]

A mixture of 1.21 g allyl bromide (10 mmol) and 0.96 g of N-acetylDL­methionine (5 mmol) in 50 mL 50% aqueous ethanol was stirred at

Side Chain Protecting Groups 359

25°C for 24 hours. After addition of 40 mL water, the aqueous mixture was washed with chloroform (4 × 50 mL). The aqueous solution was then evaporated at 40°C. The residual gum was dried in vacuo over P2O5 to give 1.62 g of N-acetyl-S-allyl-DL-methionine sulfonium bromide (also known as (3-acetamido-3-carboxypropyl)(allyl)(methyl)sulfonium bromide) as a non-crystallizable, deliquescent glass. 5.10.1.3  PREPARATION OF POLY(S-METHYL-L-METHIONINE SULFONIUM CHLORIDE) [424]

Poly(L-methionine) was suspended in either DMF, water, or 0.2 M aqueous formic acid (10 mg/mL). Then methyl iodide was added (3 equivalent MeI/Met residue). The reaction mixture was covered with foil and stirred at room temperature for 48 hours. If the extended reaction time is necessary, an additional 1.1 equivalent MeI/Met can be added with an increased reaction time of 72 hours. After the completion of the reaction, the solution was diluted with twice the amount of water in volume and transferred to a dialysis bag of molecular weight cut-off (MWCO) at 2,000 Daltons. The solution was dialyzed against 0.10 M NaCl for 24 hours, followed by DI water for 48 hours with water changes twice per day. Dialysis against NaCl serves to exchange counterions so that only chloride is present. The contents of the dialysis bag were then lyophilized to dryness to give the product as a white solid. KEYWORDS • hydroxy protecting group • lysine • arginine • histidine • asparagine

360

• • • • • • •

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

glutamine aspartic acid glutamic acid cysteine selenocysteine tryptophan methionine

REFERENCES 1.

2.

3.

4. 5.

6.

7.

8. 9.

Vadolas, D., Germann, H. P., Thakur, S., Keller, W., & Heidemann, E., (1985). Protection of the hydroxyl function of hydroxyproline and serine in peptide synthesis by the 2-methoxyethoxymethyl (mem) group. International Journal of Peptide & Protein Research, 25(5), 554–559. doi: 10.1111/j.1399-3011.1985.tb02210.x. Stewart, J. M., (1981). Chapter 4. Protection of the hydroxyl group in peptide synthesis. In: Gross, E., & Meienhofer, J., (eds.), The Peptides: Analysis, Synthesis, Biology (Vol. 3 (Protection of Functional Groups in Peptide Synthesis), pp. 169–201). Academic Press: New York. Barlos, K., Gatos, D., Koutsogianni, S., Schafer, W., Stavropoulos, G., & Wenqing, Y., (1991). Preparation and application of N-Fmoc-O-trt-hydroxy amino acids for solid-phase synthesis of peptides. Tetrahedron Letters, 32(4), 471–474. doi: 10.1016/ S0040-4039(00)79471–8. Callahan, F. M., & Zimmerman, J. E., (1967). Hydroxy Amino Acid Derivatives. United States Patent, US 3340274. Takahayashi, I. N., Mio, Y., Oka, T., Shima, T., Shimahara, N., & Iki, M., (1962). Synthesis of actinomycin analogs. Chemical & Pharmaceutical Bulletin, 10, 147–151. https://doi.org/10.1248/cpb.10.147. Yamamoto, H., Nagai, A., Okada, T., & Nishida, A., (1989). Synthesis and Adhesive studies of barnacle model proteins. Marine Chemistry, 26(4), 331–338. doi: 10.1016/0304-4203(89)90038–8. Habraken, G. J. M., Wilsens, K. H. R. M., Koning, C. E., & Heise, A., (2011). Optimization of N-carboxyanhydride (NCA) polymerization by variation of reaction temperature and pressure. Polymer Chemistry, 2(6), 1322–1330. doi: 10.1039/c1py00079a. Sugano, H., & Miyoshi, M., (1976). A convenient synthesis of N-tert-butyloxycarbonylO-benzyl-L-serine. Journal of Organic Chemistry, 41(13), 2352, 2353. Hever, I., & Palmai, G., (1980). Optically active N-tert-butyloxycarbonyl-O-benzyl serine and threonine. Hung. Teljes, HU 17887 A2.

Side Chain Protecting Groups 361

10. Erickson, B. W., & Merrifield, R. B., (1973). Acid stability of several benzylic protecting groups used in solid-phase peptide synthesis. Rearrangement of O-benzyltyrosine to 3-benzyltyrosine. Journal of the American Chemical Society, 95(11), 3750–3756. 11. Gowda, D. C., (2002). Removal of some commonly used protecting groups in peptide syntheses by catalytic transfer hydrogenation with formic acid and 10% palladium on carbon. Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 41B (5), 1064–1067. http://nopr.niscair.res.in/handle/123456789/21919 (accessed on 4 March 2022). 12. Futaki, S., Taike, T., Akita, T., & Kitagawa, K., (1990). A new approach for the synthesis of tyrosine sulfate containing peptides: Use of the p-(methylsulfonyl)benzyl group as a key protecting group of serine. Journal of the Chemical Society, Chemical Communications (7), 523–524. 13. Chauhan, V. S., Ratcliffe, S. J., & Young, G. T., (1980). Hydroxy- and amino-protection based on the 4-dimethylcarbamoylbenzyl group. International Journal of Peptide & Protein Research, 15(2), 96–101. doi: 10.1111/j.1399-3011.1980.tb02555.x. 14. Engelhard, M., & Merrifield, R. B., (1978). Tyrosine protecting groups: Minimization of rearrangement to 3-alkyltyrosine during acidolysis. Journal of the American Chemical Society, 100(11), 3559–3563. 15. Yamashiro, D., (1977). Protection of aspartic acid, serine, and threonine in solidphase peptide synthesis. Journal of Organic Chemistry, 42(3), 523–525. doi: 10.1021/ jo00423a027. 16. Nishiyama, Y., Shikama, S., Morita, K. I., & Kurita, K., (2000). Cyclohexyl ether as a new hydroxy-protecting group for serine and threonine in peptide synthesis. Journal of the Chemical Society, Perkin Transactions 1, (12), 1949–1954. doi: 10.1039/b001261k. 17. Weygand, F., Steglich, W., Fraunberger, F., Pietta, P. G., & Schmid, J., (1968). 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl radical as a protective group for the hydroxyl groups of serine and threonine in peptide syntheses. Chemische Berichte, 101(3), 923–934. 18. Polzhofer, K. P., (1969). Application of the 2,2,2-trifluoro-1-(benzyloxycarbonylamino) ethyl group as a hydroxyl-protecting group in the Merrifield synthesis. Tetrahedron, 25(17), 4127–4132. 19. Tam, J. P., Riemen, M. W., & Merrifield, R. B., (1988). Mechanisms of aspartimide formation: The effects of protecting groups, acid, base, temperature and time. Peptide Research, 1(1), 6–18. 20. Hughes, L. J., Andreatta, R. H., & Scheraga, H. A., (1972). Helix-coil stability constants for the naturally occurring amino acids in water. V. Serine parameters from random poly(hydroxybutylglutamine-co-L-serine). Macromolecules, 5(2), 187–197. 21. Hecht, M. H., Zweifel, B. O., & Scheraga, H. A., (1978). Helix-coil stability constants for the naturally occurring amino acids in water. 17. Threonine parameters from random poly(hydroxybutylglutamine-co-L-threonine). Macromolecules, 11(3), 545–551.

362

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

22. Fischer, P. M., (1992). Application of tert-butyldimethylsilyl ethers of serine, threonine and tyrosine in peptide synthesis. Tetrahedron Letters, 33(49), 7605–7608. doi: 10.1016/ S0040-4039(00)60836-5. 23. Otake, Y., Nakamura, H., & Fuse, S., (2018). Rapid and mild synthesis of amino acid N-carboxy anhydrides: Basic-to-acidic flash switching in a microflow reactor. Angewandte Chemie, International Edition, 57(35), 11389–11393. doi: 10.1002/ anie.201803549. 24. Yang, X., & Yu, B., (2013). Total synthesis of jadomycins B, S, T, and ILEVS1080. Chemistry: A European Journal, 19(26), 8431–8434. doi: 10.1002/chem.201301297. 25. Sarnowski, M. P., Pedretty, K. P., Giddings, N., Woodcock, H. L., & Del, V. J. R., (2018). Synthesis and β-sheet propensity of constrained N-amino peptides. Bioorganic & Medicinal Chemistry, 26(6), 1162–1166. doi: 10.1016/j.bmc.2017.08.017. 26. Shimogaki, M., Takeshima, A., Kano, T., & Maruoka, K., (2020). Enantioselective synthesis of monosaccharide analogues by two-step sequential enamine catalysis: Benzoyloxylation and Aldol reaction. European Journal of Organic Chemistry, (14), 2028–2032. doi.org/10.1002/ejoc.202000073. 27. Xu, Y. N., Zhu, M. Z., & Tian, S. K., (2019). Chiral α-amino acid/palladium-catalyzed asymmetric allylation of α-branched β-ketoesters with allylic amines: Highly enantioselective construction of all-carbon quaternary stereocenters. Journal of Organic Chemistry, 84(22), 14936–14942. doi: 10.1021/acs.joc.9b02282. 28. Yoshida, M., Masaki, E., Terumine, T., & Hara, S., (2014). Asymmetric α-allylation of α-branched aldehydes with allyl alcohols by synergistic catalysis using an achiral palladium complex and a chiral primary amino acid. Synthesis, 46(10), 1367–1373. doi: 10.1055/s-0033-1340901. 29. Yoshida, M., Terumine, T., Masaki, E., & Hara, S., (2013). Direct asymmetric α-allylation of α-branched aldehydes by two catalytic systems with an achiral Pd complex and a chiral primary α-amino acid. Journal of Organic Chemistry, 78(21), 10853–10859. dx.doi.org/10.1021/jo4018414. 30. Cheng, L., Han, X., Huang, H., Wong, M. W., & Lu, Y., (2007). Highly diastereoselective and enantioselective direct organocatalytic anti-selective mannich reactions employing N-tosylimines. Chemical Communications (Cambridge, United Kingdom), 40, 4143– 4145. doi: 10.1039/B706793C. 31. Cheng, L., Wu, X., & Lu, Y., (2007). Direct asymmetric three-component organocatalytic anti-selective mannich reactions in a purely aqueous system. Organic & Biomolecular Chemistry, 5(7), 1018–1020. doi: 10.1039/b701579h. 32. Wu, X., Jiang, Z., Shen, H. M., & Lu, Y., (2007). Highly efficient threonine-derived organocatalysts for direct asymmetric aldol reactions in water. Advanced Synthesis & Catalysis, 349(6), 812–816. doi: 10.1002/adsc.200600564. 33. Yong, F. F., & Teo, Y. C., (2011). Recyclable siloxy serine organocatalyst for the direct asymmetric mannich reactions in ionic liquids. Synthetic Communications, 41(9), 1293–1300. doi: 10.1080/00397911.2010.481750.

Side Chain Protecting Groups 363

34. Yoshida, M., Ukigai, H., Shibatomi, K., & Hara, S., (2015). Organocatalytic asymmetric Michael addition of α-branched aldehydes to vinyl ketones: Synthesis of 5-ketoaldehydes possessing a stereo-controlled all-carbon quaternary stereogenic center. Tetrahedron Letters, 56(25), 3890–3893. doi: 10.1016/j.tetlet.2015.04.107. 35. Popik, O., Zambron, B., & Mlynarski, J., (2013). Biomimetic syn-aldol reaction of dihydroxyacetone promoted by water-compatible catalysts. European Journal of Organic Chemistry, (33), 7484–7487. doi: 10.1002/ejoc.201301436. 36. Robertson, J., & Stafford, P. M., (2003). Selective hydroxyl protection and deprotection. In: Osborn, H. M. I., (ed.), Carbohydrates (pp. 9–68). 37. Sharma, A., Ramos-Tomillero, I., El-Faham, A., Rodriguez, H., De La Torre, B. G., & Albericio, F., (2017). Tetrahydropyranyl: A non-aromatic, mild-acid-labile group for hydroxyl protection in solid-phase peptide synthesis. ChemistryOpen, 6(2), 206–210. doi: 10.1002/open.201600157. 38. Rastetter, W. H., Erickson, T. J., & Venuti, M. C., (1980). Syntheses of enterobactin and enantioenterobactin. Journal of Organic Chemistry, 45(24), 5011–5012. 39. Rastetter, W. H., Erickson, T. J., & Venuti, M. C., (1981). Synthesis of iron chelators. enterobactin, enantioenterobactin, and a chiral analog. Journal of Organic Chemistry, 46(18), 3579–3590. 40. Olsen, R. K., Ramasamy, K., Bhat, K. L., Low, C. M. L., & Waring, M. J., (1986). Synthesis and DNA-binding studies of [lac2,lac6]TANDEM, an analog of des-Ntetramethyltriostin A (TANDEM) having L-lactic acid substituted for each L-alanine residue. Journal of the American Chemical Society, 108(19), 6032–6036. 41. Yu, X., Dai, Y., Yang, T., Gagne, M. R., & Gong, H., (2011). Facile synthesis of salmochelin S1, S2, MGE, DGE, and TGE. Tetrahedron, 67(1), 144–151. 42. Tan, L., & Ma, D., (2008). Total synthesis of safinamide A: A potent anti-inflammatory bicyclic depsipeptide. Angewandte Chemie, International Edition, 47(19), 3614–3617. 43. Krishnamoorthy, R., Vazquez-Serrano, L. D., Turk, J. A., Kowalski, J. A., Benson, A. G., Breaux, N. T., & Lipton, M. A., (2006). Solid-phase total synthesis and structure proof of callipeltin B. Journal of the American Chemical Society, 128(48), 15392, 15393. 44. Iijima, Y., Kimata, O., Decharin, S., Masui, H., Hirose, Y., & Takahashi, T., (2014). Total synthesis of (+)-antimycin A3b on solid supports. European Journal of Organic Chemistry, (22), 4725–4732. 45. Scheule, R. K., Cardinaux, F., Taylor, G. T., & Scheraga, H. A., (1976). Helix-coil stability constants for the naturally occurring amino acids in water. X. Tyrosine parameters from random poly(Hydroxypropylglutamine-co-L-tyrosine). Macromolecules, 9(1), 23–33. 46. Ozawa, T., Ikota, N., & Nakagawa, H., (2000). Preparation of dithiocarbamates as spin trapping agents for nitrogen monoxide scavengers. Jpn. Kokai Tokkyo Koho. JP 2000128893 A. 47. Smyth, D. G., Stein, W. H., & Moore, S., (1963). The sequence of amino acid residues in bovine pancreatic ribonuclease: Revisions and confirmations. Journal of Biological Chemistry, 238, 227–234.

364

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

48. Jain, J. C., Sharma, I. K., Sahni, M. K., Gupta, K. C., & Mathur, N. K., (1977). Selective O-acetylation of hydroxyamino acids in presence of sulfonic acid resin. Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 15B(12), 1149. 49. Jain, J. C., Sahni, M. K., Gupta, K. C., & Narang, C. K., (1978). Some resin-catalyzed synthetic reactions of amino acids and peptides. In: Gadre, G. T., (ed.), Proc. Ion-Exch. Symp. (pp. 233–235). 50. Arakawa, K., Smeby, R. R., & Bumpus, F. M., (1962). Synthesis of Succinyl1-isoleucyl5angiotensin II and N-(poly-O-acetylseryl)-isoleucyl5-angiotensin II. Journal of the American Chemical Society, 84, 1424–1426. 51. Sheehan, J. C., Goodman, M., & Hess, G. P., (1956). Peptide derivatives containing hydroxy amino acids. Journal of the American Chemical Society, 78, 1367–1369. 52. Kristensen, T. E., (2015). Chemoselective O-acylation of hydroxyamino acids and amino alcohols under acidic reaction conditions: History, scope and applications. Beilstein Journal of Organic Chemistry, 11, 446–468. doi: 10.3762/bjoc.11.51. 53. Kurano, Y., Kimura, T., & Sakakibara, S., (1987). Total synthesis of porcine cholecystokinin-33 by the classical solution procedure. Peptide Chemistry, 24, 151–156. 54. Penke, B., Zarandi, M., Toth, G., Varga, J., Zsigo, J., & Kovacs, K., (1983). Application of some non-proteinogen amino acids in the synthesis of peptide hormone analogs. Wissenschaftliche Beitraege-Martin-Luther-Universitaet Halle-Wittenberg, 52(Enzymes Horm. Med. Res. Diagn.), 104–112. 55. Penke, B., & Rivier, J., (1983). New synthetic approach to peptide sulfate esters. Synthesis and biological activities of CCK analogs. In: Hruby, V. J., & Rich, D. H., (eds.), Pept.: Struct. Funct., Proc. Am. Pept. Symp., 8th (pp. 119–122). 56. Ueki, M., Saito, M., & Oyamada, H., (1988). Protection of side-chain alcoholic hydroxyl groups of serine and threonine by the dimethylphosphinyl (Dmp) group. In: Marshall, G. R., (ed.), Pept.: Chem. Biol. Proc. Am. Pept. Symp. 10th (pp. 282, 283). 57. Ueki, M., Kobayashi, H., Karasawa, M., & Shinozaki, K., (1984). Peptide synthesis by use of dimethylphosphinothioyl chloride (Mpt-Cl) - reactivity toward side-chain hydroxyl groups. Peptide Chemistry, 21, 67–70. 58. Wu, C., Long, X., Li, S., & Fu, X., (2012). Simple and inexpensive threonine-based organocatalysts as highly active and recoverable catalysts for large-scale asymmetric direct stoichiometric aldol reactions on water. Tetrahedron: Asymmetry, 23(5), 315–328. doi: 10.1016/j.tetasy.2012.02.023. 59. Wu, C., Fu, X., & Li, S., (2011). Simple and inexpensive threonine-based organocatalysts for the highly diastereo- and enantioselective direct large-scale Syn-aldol and Antimannich reactions of α-hydroxyacetone. Tetrahedron: Asymmetry, 22(10), 1063–1073. doi: 10.1016/j.tetasy.2011.06.022. 60. Kiso, Y., Kimura, T., Yoshida, M., Shimokura, M., Akaji, K., & Mimoto, T., (1989). A new class of amino protecting group removable by reductive acidolysis: The 4-methylsulfinylbenzyloxycarbonyl (Msz) group. Journal of the Chemical Society, Chemical Communications, (20), 1511–1513. doi: 10.1039/c39890001511.

Side Chain Protecting Groups 365

61. Kiso, Y., Tanaka, S., Kimura, T., Itoh, H., & Akaji, K., (1991). New hydroxyl protecting groups of a safety-catch type removable by reductive acidolysis. Chemical & Pharmaceutical Bulletin, 39(11), 3097–3099. 62. Overell, B. G., & Petrow, V., (1955). Polymers of some basic and acidic α-amino acids. Journal of the Chemical Society, 232–236. doi: 10.1039/JR9550000232. 63. Balboni, G., Cocco, M. T., Salvadori, S., Romagnoli, R., Sasaki, Y., Okada, Y., Bryant, S. D., et al., (2005). From the potent and selective μ opioid receptor agonist H-DmtD-Arg-Phe-Lys-NH2 to the potent δ antagonist H-Dmt-Tic-Phe-Lys(Z)-OH. Journal of Medicinal Chemistry, 48(17), 5608–5611. doi: 10.1021/jm0504959. 64. Kimura, K., & Fukushima, Y., (2014). Fluorescence Studies on self-association of biodegradable pyrene-labeled poly(L-lysine) grafted with poly(caprolactone). Journal of Photopolymer Science and Technology, 27(6), 703–709. doi: 10.2494/ photopolymer.27.703. 65. Li, W., Zhang, X., Wang, J., Qiao, X., Liu, K., & Zhang, A., (2012). Peptidic molecular brushes with enhanced chirality. Journal of Polymer Science, Part A: Polymer Chemistry, 50(19), 4063–4072, S4063/1-S4063/16. doi: 10.1002/pola.26208. 66. Luebbert, A., Castelletto, V., Hamley, I. W., Nuhn, H., Scholl, M., Bourdillon, L., Wandrey, C., & Klok, H. A., (2005). Nonspherical assemblies generated from polystyrene-bpoly(L-lysine) polyelectrolyte block copolymers. Langmuir, 21(14), 6582–6589. doi: 10.1021/la0502600. 67. Rodriguez-Hernandez, J., Gatti, M., & Klok, H. A., (2003). Highly branched poly(Llysine). Biomacromolecules, 4(2), 249–258. doi: 10.1021/bm020096k. 68. Sulistio, A., Blencowe, A., Widjaya, A., Zhang, X., & Qiao, G., (2012). Development of functional amino acid-based star polymers. Polymer Chemistry, 3(1), 224–234. doi: 10.1039/C1PY00436K. 69. Salem, E. M., & Schou, O., (1980). New side-chain protecting groups for lysine and tyrosine suitable for solid-phase peptide synthesis. Indian Journal of Chemistry, Section B: Organic Chemistry Including Medicinal Chemistry, 19B(1), 62–64. 70. Hernandez, J. R., & Klok, H. A., (2003). Synthesis and ring-opening (Co)polymerization of L-lysine N-carboxyanhydrides containing labile side chain protective groups. Journal of Polymer Science, Part A: Polymer Chemistry, 41(9), 1167–1187. doi: 10.1002/ pola.10660. 71. Albericio, F., Nicolas, E., Rizo, J., Ruiz-Gayo, M., Pedroso, E., & Giralt, E., (1990). Convenient syntheses of fluorenylmethyl-based side-chain derivatives of glutamic and aspartic acids, lysine, and cysteine. Synthesis, (2), 119–122. doi: 10.1055/s-1990-26804. 72. Bouzide, A., Sauve, G., & Yelle, J., (2005). Lysine derivatives as potent HIV protease inhibitors. Discovery, synthesis and structure-activity relationship studies. Bioorganic & Medicinal Chemistry Letters, 15(5), 1509–1513. doi: 10.1016/j.bmcl.2004.12.068. 73. Dose, C., & Seitz, O., (2008). Single nucleotide specific detection of DNA by native chemical ligation of fluorescence-labeled PNA-probes. Bioorganic & Medicinal Chemistry, 16(1), 65–77. doi: 10.1016/j.bmc.2007.04.059.

366

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

74. Rijkers, D. T. S., Den, H. J. A. J., & Liskamp, R. M. J., (2002). An optimized solid-phase synthesis strategy - including on-resin lactamization - of Astressin, its retro-, inverso-, and retro-Inverso isomers as corticotropin-releasing factor antagonists. Biopolymers, 63(2), 141–149. doi: 10.1002/bip.10052. 75. Sitnikov, N. S., Malysheva, Y. B., Fedorov, A. Y., & Schmalz, H. G., (2019). Design and synthesis of new protease-triggered co-releasing peptide-metal-complex conjugates. European Journal of Organic Chemistry, (40), 6830–6837. doi: 10.1002/ejoc.201901206. 76. Akbulut, H., Ando, S., Yamada, S., & Endo, T., (2018). Synthesis of poly(Nεphenoxycarbonyl-L-lysine) by polycondensation of activated urethane derivative and its application for selective modification of side chain with amines. Journal of Polymer Science, Part A: Polymer Chemistry, 56(22), 2522–2530. doi: 10.1002/pola.29230. 77. Katayama, H., Nakahara, Y., & Hojo, H., (2011). N-methyl-phenacyloxycarbamidomethyl (Pocam) group: A novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions. Organic & Biomolecular Chemistry, 9(12), 4653–4661. doi: 10.1039/c1ob05253e. 78. Mulvey, J. J., Feinberg, E. N., Alidori, S., McDevitt, M. R., Heller, D. A., & Scheinberg, D. A., (2014). Synthesis, pharmacokinetics, and biological use of lysine-modified single-walled carbon nanotubes. International Journal of Nanomedicine, 9, 4245–4255. DOI:10.2147/IJN.S66050. 79. Monroc, S., Feliu, L., Serra, J., Planas, M., & Bardaji, E., (2006). Synthesis and solidphase applications of N-tetrachlorophthaloyl (TCP) side-chain-protected amino acids. Synlett, (17), 2743–2746. doi: 10.1055/s-2006-950255. 80. Rizo, J., Albericio, F., Giralt, E., & Pedroso, E., (1992). Reversible protection of lysine to facilitate the purification of protected peptide segments. Tetrahedron Letters, 33(3), 397–400. doi: 10.1016/S0040-4039(00)74141-4. 81. Lescrinier, T., Pannecouque, C., Rozenski, J., Van, A. A., & Herdewijn, P., (1995). Amino acids derived from ornithine. Letters in Peptide Science, 2(3, 4), 206–208. doi: 10.1007/BF00119154. 82. Katayama, H., Utsumi, T., Ozawa, C., Nakahara, Y., Hojo, H., & Nakahara, Y., (2009). Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction. Tetrahedron Letters, 50(7), 818–821. doi: 10.1016/j. tetlet.2008.12.005. 83. Morin, C., & Thimon, C., (2004). Synthesis and evaluation of boronated lysine and bis(carboranylated) γ-amino acids as monomers for peptide assembly. European Journal of Organic Chemistry, (18), 3828–3832. doi: 10.1002/ejoc.200400342. 84. Oelmann, J., Miller, R. G., Baabe, D., Metzler-Nolte, N., & Broering, M., (2020). Biometal corrole active esters and their amino acid and peptide conjugates. European Journal of Inorganic Chemistry, (32), 3059–3069. doi:10.1002/ejic.202000472. 85. Zanatta, N., Squizani, A. M. C., Fantinel, L., Nachtigall, F. M., Bonacorso, H. G., & Martins, M. A. P., (2002). Application of 4-alkoxy-1,1,1-trifluoro[chloro]alk-3-en2-ones as selective protecting groups of amino acids. Synthesis, (16), 2409–2415. doi:10.1055/s-2002-35218.

Side Chain Protecting Groups 367

86. Connolly, P. J., Beers, K. N., Wetter, S. K., & Murray, W. V., (2000). Solid-phase synthesis of Nα-benzyl-Nα-cinnamyl lysine and glutamic acid derivatives. Tetrahedron Letters, 41(27), 5187–5191. doi: 10.1016/S0040-4039(00)00834-0. 87. Aletras, A., Barlos, K., Gatos, D., Koutsogianni, S., & Mamos, P., (1995). Application of Fmoc-Lys(Mtt)-OH in the preparation of MAPs and MAP-libraries. Epitheorese Klinikes Farmakologias kai Farmakokinetikes, International Edition, 9(2, 3), 129–133. 88. Gesquiere, J. C., Najib, J., Letailleur, T., Maes, P., & Tartar, A., (1993). Formation of Nε-Dnp lysine during deprotection of Nε-Fmoc lysine in Nim-Dnp-histidine-containing peptides. Tetrahedron Letters, 34(12), 1921–1924. doi: 10.1016/S0040-4039(00)91963-4. 89. Balajthy, Z., (1991). An improved preparation of N-benzyloxycarbonyl-L-lysine methyl ester hydrochloride. Organic Preparations and Procedures International, 23(3), 375, 376. doi: 10.1080/00304949109458213. 90. Skoog, I. H., (1964). Reaction of alkyl borinates with α-amino acids. Journal of Organic Chemistry, 29(2), 492, 493. doi: 10.1021/jo01025a509. 91. Dent, W. H. III., Erickson, W. R., Fields, S. C., Parker, M. H., & Tromiczak, E. G., (2002). 9-BBN: An amino acid protecting group for functionalization of amino acid side chains in organic solvents. Organic Letters, 4(8), 1249–1251. doi: 10.1021/ol017241b. 92. Sanchez, A., Calderon, E., & Vazquez, A., (2013). Using the 9-BBN group as a transient protective group for the functionalization of reactive chains of α-amino acids. Synthesis, 45(10), 1364–1372. doi: 10.1055/s-0032-1316848. 93. Cudic, M., Mari, F., & Fields, G. B., (2007). Synthesis and solid-phase application of suitably protected γ-hydroxyvaline building blocks. Journal of Organic Chemistry, 72(15), 5581–5586. doi: 10.1021/jo070436y. 94. Rink, H., Sieber, P., & Raschdorf, F., (1984). Conversion of NG-urethane protected arginine to ornithine in peptide solid-phase synthesis. Tetrahedron Letters, 25(6), 621–624. doi: 10.1016/S0040-4039(00)99954-4. 95. Cezari, M. H. S., & Juliano, L., (1996). Studies on lactam formation during coupling procedures of Nα-Nω-protected arginine derivatives. Peptide Research, 9(2), 88–91. 96. Alhassan, M., Kumar, A., Lopez, J., Albericio, F., & De La Torre, B. G., (2020). Revisiting NO2 as protecting group of arginine in solid-phase peptide synthesis. International Journal of Molecular Sciences, 21(12), 4464/1–4464/13. doi: 10.3390/ijms21124464. 97. Stefano, G., & Carla, C., (2016). Abaloparatide. Clinical Cases in Mineral and Bone Metabolism: The Official Journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases, 13(2), 106–109. 98. Galaktionov, S. G., Nikiforovich, G. V., Shenderovich, M. D., Cipens, G. I., & Vegners, R., (1976). Three-dimensional structure of angiotensin II. In: Loffet, A., (ed.), Pept., Proc. Eur. Pept. Symp., 14th (pp. 617–624). 99. Durek, T., Cromm, P. M., White, A. M., Schroeder, C. I., Kaas, Q., Weidmann, J., Ahmad, F. A., et al., (2018). Development of novel melanocortin receptor agonists based on the cyclic peptide framework of sunflower trypsin inhibitor‑1 Journal of Medicinal Chemistry, 61(8), 3674–3684. doi: 10.1021/acs.jmedchem.8b00170.

368

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

100. Walse, B., Kihlberg, J., & Drakenberg, T., (1998). Confirmation of desmopressin, an analog of the peptide hormone vasopressin, in aqueous solution as determined by NMR spectroscopy. European Journal of Biochemistry, 252(3), 428–440. 101. Iino, Y., & Mori, H., (1995). Structure and characterization of gonadotropin-releasing hormone (GnRH) analogs. Hormone Frontier in Gynecology, 2(1), 15–21. 102. Lau, J., Bloch, P., Schaffer, L., Pettersson, I., Spetzler, J., Kofoed, J., Madsen, K., et al., (2015). Discovery of the once-weekly glucagon-like peptide-1(GLP-1) analogue semaglutide. Journal of Medicinal Chemistry, 58(18), 7370–7380. doi: 10.1021/acs. jmedchem.5b00726. 103. Barbier, B., Caille, A., & Brack, A., (1984). Synthesis and β-conformation of sequential polypeptides containing arginine, histidine, and leucine. Biopolymers, 23(11, Pt. 1), 2299–2310. doi: 10.1002/bip.360231112. 104. Jetten, M., Peters, C. A. M., Van, N. J. W. F. M., & Ottenheijm, H. C. J., (1991). A one-pot N-protection of L-arginine. Tetrahedron Letters, 32(42), 6025–6028. doi: 10.1016/S0040-4039(00)79455-X. 105. Zhang, Y., & Kennan, A. J., (2001). Efficient introduction of protected guanidines in BOC solid-phase peptide synthesis. Organic Letters, 3(15), 2341–2344. doi: 10.1021/ ol016139b. 106. Ding, X., Miller, P. G., Hwang, M. P., Fu, J., & Wang, Y., (2019). Scale-up synthesis of a polymer designed for protein therapy. European Polymer Journal, 117, 353–362. doi: 10.1016/j.eurpolymj.2019.05.032. 107. Aurelio, L., Box, J. S., Brownlee, R. T. C., Hughes, A. B., & Sleebs, M. M., (2003). An efficient synthesis of N-methyl amino acids by way of intermediate 5-oxazolidinones. Journal of Organic Chemistry, 68(7), 2652–2667. doi: 10.1021/jo026722l. 108. Isidro, A., Latassa, D., Giraud, M., Alvarez, M., & Albericio, F., (2009). 1,2-dimethylindole-3-sulfonyl (MIS) as protecting group for the side chain of arginine. Organic & Biomolecular Chemistry, 7(12), 2565–2569. doi: 10.1039/b904836g. 109. Kiso, Y., Satomi, M., Ukawa, K., & Akita, T., (1980). Efficient deprotection of NG-tosylarginine with a thioanisole-trifluoromethanesulfonic acid system. Journal of the Chemical Society, Chemical Communications, (22), 1063, 1064. doi: 10.1039/ C39800001063. 110. Ramachandran, J., & Li, C. H., (1962). Preparation of crystalline NG-tosylarginine derivatives. Journal of Organic Chemistry, 27(11), 4006–4009. 111. Atherton, E., Sheppard, R. C., & Wade, J. D., (1983). Side chain protected nα-fluorenylmethoxycarbonyl amino acids for solid phase peptide synthesis. Nα-fluorenylmethoxycarbonyl-NG-4-methoxy-2,3,6-trimethylbenzenesulfonyl-Larginine. Journal of the Chemical Society, Chemical Communications, (19), 1060–1062. doi: 10.1039/C39830001060. 112. Fujino, M., Wakimasu, M., & Kitada, C., (1981). Further studies on the use of multisubstituted benzenesulfonyl groups for protection of the guanidino function of arginine. Chemical & Pharmaceutical Bulletin, 29(10), 2825–2831. doi: 10.1248/cpb.29.2825.

Side Chain Protecting Groups 369

113. Atherton, E., Woolley, V., & Sheppard, R. C., (1980). Internal association in solid phase peptide synthesis. Synthesis of cytochrome C residues 66–104 on polyamide supports. Journal of the Chemical Society, Chemical Communications, (20), 970, 971. doi: 10.1039/C39800000970. 114. Fujino, M., Nishimura, O., Wakimasu, M., & Kitada, C., (1980). 4-methoxy-2,6dimethylbenzenesulfonyl (Mds): A new protecting group of the guanidino function in peptide synthesis. Journal of the Chemical Society, Chemical Communications, (14), 668, 669. doi: 10.1039/c39800000668. 115. De Luca, S., Ulhaq, S., Dixon, M. J., Essex, J., & Bradley, M., (2003). Solid-phase synthesis of a focused library of trypanothione reductase inhibitors. Tetrahedron Letters, 44(15), 3195–3197. doi: 10.1016/S0040-4039(03)00438-6. 116. Yajima, H., Takeyama, M., Kanaki, J., & Mitani, K., (1978). The mesitylene-2sulfonyl group, a acidolytically removable NG-protecting group for arginine. Journal of the Chemical Society, Chemical Communications, (11), 482, 483. doi: 10.1039/ c39780000482. 117. Penke, B., & Rivier, J., (1987). Solid-phase synthesis of peptide amides on a polystyrene support using fluorenylmethoxycarbonyl protecting groups. Journal of Organic Chemistry, 52(7), 1197–1200. doi: 10.1021/jo00383a004. 118. Schlagel, L. J., Bors, L., Mitchell, G. W., King, J. L., Cao, L., Kirk, M., & Whitaker, J. N., (1997). Immunological effects of an arginine side chain contaminating synthetically prepared peptides. Molecular Immunology, 34(2), 185–194. doi: 10.1016/ S0161-5890(97)00013-8. 119. Carpino, L. A., Shroff, H., Triolo, S. A., Mansour, E. S. M. E., Wenschuh, H., & Albericio, F., (1993). The 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf) as arginine side chain protectant. Tetrahedron Letters, 34(49), 7829–7832. doi: 10.1016/S0040-4039(00)61487-9. 120. Stephenson, R. J., Plieger, P. G., & Harding, D. R. K., (2011). Improved Fmoc synthesis of bradykinin. Protein & Peptide Letters, 18(9), 952–955. 121. Peytou, V., Condom, R., Patino, N., Guedj, R., Aubertin, A. M., Gelus, N., Bailly, C., et al., (1999). Synthesis and antiviral activity of ethidium-arginine conjugates directed against the TAR RNA of HIV-1. Journal of Medicinal Chemistry, 42(20), 4042–4053. doi: 10.1021/jm980728e. 122. Susaki, H., Suzuki, K., Ikeda, M., Yamada, H., & Watanabe, H. K., (1994). Synthesis of artificial glycoconjugates of arginine-vasopressin and their antidiuretic activities. Chemical & Pharmaceutical Bulletin, 42(10), 2090–2096. doi: 10.1248/cpb.42.2090. 123. Riniker, B., Floersheimer, A., Fretz, H., Sieber, P., & Kamber, B., (1993). A general strategy for the synthesis of large peptides: The combined solid-phase and solution approach. Tetrahedron, 49(41), 9307–9320. doi: 10.1016/0040-4020(93)80017-N. 124. Jaeger, G., & Geiger, R., (1970). 1-Adamantyloxycarbonyl residue as blocking group for the guanidino function of arginine. Chemische Berichte, 103(6), 1727–1747. doi: 10.1002/cber.19701030608.

370

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

125. Filippov, D., Van, D. M. G. A., Kuyl-Yeheskiely, E., & Van, B. J. H., (1994). Methylsulfonylethyloxycarbonyl group as a protection for the guanidino function in arginine. Synlett, (11), 922–924. doi: 10.1055/s-1994-23049. 126. Freidinger, R. M., Hirschmann, R., & Veber, D. F., (1978). Titanium(III) as a selective reducing agent for nitroarginyl peptides: Synthesis of arginine vasotocin. Journal of Organic Chemistry, 43(25), 4800–4803. 127. Hayakawa, T., Fujiwara, Y., & Noguchi, J., (1967). A new method of reducing nitroarginine-peptide into arginine-peptide, with reference to the synthesis of poly(Larginine hydrochloride). Bulletin of the Chemical Society of Japan, 40(5), 1205–1208. doi: 10.1246/bcsj.40.1205. 128. Garcia, O., Nicolas, E., & Albericio, F., (2003). Solid-phase synthesis: A linker for side-chain anchoring of arginine. Tetrahedron Letters, 44(28), 5319–5321. doi: 10.1016/ S0040-4039(03)01203-6. 129. Martin, B. G., (1997). The preparation and properties of polyfluoro aromatic and heteroaromatic compounds. Journal of Fluorine Chemistry, 86(1), 1–76. doi: 10.1016/ S0022-1139(97)00006-7. 130. Sieber, P., & Riniker, B., (1987). Protection of histidine in peptide synthesis: A reassessment of the trityl group. Tetrahedron Letters, 28(48), 6031–6034. doi: 10.1016/ S0040-4039(00)96856-4. 131. Terada, S., Kawabata, A., Mitsuyasu, N., Aoyagi, H., & Izumiya, N., (1978). Racemization during the synthesis of histidine-containing peptides. Bulletin of the Chemical Society of Japan, 51(11), 3409, 3410. doi: 10.1246/bcsj.51.3409. 132. Sheehan, J. C., Hasspacher, K., & Yeh, Y. L., (1959). Activated cyclic derivatives of amino acids. Journal of the American Chemical Society, 81(22), 6086. doi: 10.1021/ ja01531a062. 133. Ash, E. L., Sudmeier, J. L., Day, R. M., Vincent, M., Torchilin, E. V., Haddad, K. C., Bradshaw, E. M., et al., (2000). Unusual 1H NMR chemical shifts support (His) Cε1-H···O=C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis. Proceedings of the National Academy of Sciences of the United States of America, 97(19), 10371–10376. doi: 10.1073/pnas.97.19.10371. 134. Muller, J. D., McMahon, B. H., Chien, E. Y. T., Sligar, S. G., & Nienhaus, G. U., (1999). Connection between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin. Biophysical Journal, 77(2), 1036–1051. 135. Hudáky, P., Beke, T., & Perczel, A., (2002). Peptide models XXXIV. Side-chain conformational potential energy surfaces associated with all major backbone folds of neutral tautomers of N- and C-protected L-histidine. An Ab initio study on ethylimidazole and N-formyl-L-histidinamide. Journal of Molecular Structure: THEOCHEM, 583, 117–135. doi: 10.1016/S0166-1280(01)00804-1. 136. Robertson, N., Jiang, L., & Ramage, R., (1999). Racemization studies of a novel coupling reagent for solid phase peptide synthesis. Tetrahedron, 55(9), 2713–2720. doi: 10.1016/ S0040-4020(99)00043-5.

Side Chain Protecting Groups 371

137. Lelais, G., Micuch, P., Josien-Lefebvre, D., Rossi, F., & Seebach, D., (2004). Preparation of protected β2- and β3-homocysteine, β2- and β3-homohistidine, and β2-homoserine for solid phase syntheses. Helvetica Chimica Acta, 87(12), 3131–3159. doi: 10.1002/ hlca.200490280. 138. Nelli, Y. R., Douat-Casassus, C., Claudon, P., Kauffmann, B., Didierjean, C., & Guichard, G., (2012). An Activated building block for the introduction of the histidine side chain in aliphatic oligourea foldamers. Tetrahedron, 68(23), 4492–4500. doi: 10.1016/j. tet.2011.11.066. 139. Losse, G., & Krychowski, U., (1970). New histidine derivatives for solid-phase peptide synthesis. Journal fuer Praktische Chemie (Leipzig), 312(6), 1097–1104. doi: 10.1002/ prac.19703120616. 140. Zaramella, S., Strömberg, R., & Yeheskiely, E., (2003). Application of Nim-2,6dimethoxybenzoyl histidine in solid-phase peptide synthesis. European Journal of Organic Chemistry, (13), 2454–2461. doi: 10.1002/ejoc.200200668. 141. Jones, J. H., & Ramage, W. I., (1978). An approach to the prevention of racemization in the synthesis of histidine-containing peptides. Journal of the Chemical Society, Chemical Communications, (11), 472, 473. doi: 10.1039/c39780000472. 142. Young, G. T., (1981). Protection during peptide synthesis: Some new directions. Biopolymers, 20(9), 1805–1809. doi: 10.1002/bip.1981.360200905. 143. Climie, I. J. G., & Evans, D. A., (1982). Carbon-13 nuclear magnetic resonance spectroscopy as a probe of enzyme environment. II. Effect of solvent and pH on carbon-13 chemical shifts in derivatized amino acid models. Tetrahedron, 38(5), 697–711. doi: 10.1016/0040-4020(82)80213-5. 144. Sabatino, G., Chelli, M., Mazzucco, S., Ginanneschi, M., & Papini, A. M., (1999). Cyclization of histidine containing peptides in the solid-phase by anchoring the imidazole ring to trityl resins. Tetrahedron Letters, 40(4), 809–812. doi: 10.1016/ S0040-4039(98)02459-9. 145. Dixon, M. J., Andersen, O. A., Van, A. D. M. F., & Eggleston, I. M., (2006). First synthesis of argadin: A nanomolar inhibitor of family-18 chitinases. European Journal of Organic Chemistry, (22), 5002–5006. doi: 10.1002/ejoc.200600599. 146. Colombo, R., Colombo, F., & Jones, J. H., (1984). Acid-labile histidine side-chain protection: The N(π)-tert-butoxymethyl group. Journal of the Chemical Society, Chemical Communications, (5), 292, 293. 147. Harding, S. J., & Jones, J. H., (1999). π-allyloxymethyl protection of histidine. Journal of Peptide Science, 5(9), 399–402. doi: 10.1002/(SICI) 1099-1387(199909)5:93.0.CO;2-7. 148. Okada, Y., Wang, J., Yamamoto, T., Mu, Y., & Yokoi, T., (1996). Amino acids and peptides. Part 45. Development of a new Nπ-protecting group of histidine, Nπ-(1adamantyloxymethyl)histidine, and its evaluation for peptide synthesis. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, (17), 2139–2143.

372

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

149. Kimbonguila, A. M., Boucida, S., Guibe, F., & Loffet, A., (1997). Allyl protection of the imidazole ring of histidine. Tetrahedron, 53(37), 12525–12538. 150. Brown, T., & Jones, J. H., (1981). Protection of histidine side-chains with π-benzyloxymethyl or π-bromobenzyloxymethyl groups. Journal of the Chemical Society, Chemical Communications, (13), 648, 649. doi: 10.1039/C39810000648. 151. Hibino, H., & Nishiuchi, Y., (2011). 4-methoxybenzyloxymethyl group as an N p-protecting group for histidine to eliminate side-chain-induced racemization in the fmoc strategy. Tetrahedron Letters, 52(38), 4947–4949. doi: 10.1016/j.tetlet.2011.07.065. 152. Sieber, P., (1987). An improved method for anchoring of 9-fluorenylmethoxycarbonyl amino acids to 4-alkoxybenzyl alcohol resins. Tetrahedron Letters, 28(49), 6147–6150. doi: 10.1016/S0040-4039(00)61832-4. 153. Zaramella, S., Yeheskiely, E., & Strömberg, R., (2004). A method for solid-phase synthesis of oligonucleotide 5’-peptide-conjugates using acid-labile α-amino protections. Journal of the American Chemical Society, 126(43), 14029–14035. doi: 10.1021/ja046945o. 154. Losse, G., & Krychowski, U., (1971). New histidine derivatives for Merrifield peptide synthesis. Tetrahedron Letters, (44), 4121–4124. 155. Barlos, K., Papaioannou, D., & Theodoropoulos, D., (1982). Efficient “one-pot” synthesis of N-tritylamino acids. Journal of Organic Chemistry, 47(7), 1324–1326. 156. Marglin, A., & Merrifield, R. B., (1966). The synthesis of bovine insulin by the solid phase method. Journal of the American Chemical Society, 88(21), 5051, 5052. DOI:10.1021/ja00973a068. 157. Monney, A., Venkatachalam, G., & Albrecht, M., (2011). Synthesis and catalytic activity of histidine-based NHC ruthenium complexes. Dalton Transactions, 40(12), 2716–2719. doi: 10.1039/c0dt01768j. 158. Huang, K. J., Huang, Y. C., & Lin, Y. A., (2018). Synthesis of histidine-containing oligopeptides via histidine-promoted peptide ligation. Chemistry - An Asian Journal, 13(4), 400–403. doi: 10.1002/asia.201701802. 159. Bauce, L. G., & Goren, H. J., (1979). Synthesis of a series of residue 1 (pyroglutamic acid) analogs of thyrotrophin releasing hormone. International Journal of Peptide and Protein Research, 14(3), 216–226. doi: 10.1111/j.1399-3011.1979.tb01928.x. 160. Appel, R., & Willms, L., (1979). Organophosphorus peptide-coupling reagents. Part 7. Tertiary phosphine/hexachloroethane as a condensing reagent in peptide synthesis. Chemische Berichte, 112(3), 1057–1063. 161. Gyi, J. I., Kinsman, R. G., & Rees, A. R., (1995). Preparation of N-9-fluorenylmethyloxycarbonyl-asparagine-pentafluorophenyl ester from the free acid. Synlett, (2), 205, 206. doi: 10.1055/s-1995-4897. 162. Shioiri, T., & Yamada, S., (1974). Amino acids and peptides. XI. Phosphorus in organic synthesis. VI. Application of diphenyl phosphorazidate to the synthesis of peptides containing various functions. Chemical & Pharmaceutical Bulletin, 22(4), 859–863. 163. Sieber, P., & Riniker, B., (1991). Protection of carboxamide functions by the trityl residue. Application to peptide synthesis. Tetrahedron Letters, 32(6), 739–742.

Side Chain Protecting Groups 373

164. Ressler, C., & Ratzkin, H., (1961). Synthesis of β-cyano-L-alanine and γ-cyano-α-Laminobutyric acid, dehydration products of L-asparagine and L-glutamine; a new synthesis of amino acid nitriles. Journal of Organic Chemistry, 26(9), 3356–3360. doi: 10.1021/jo01067a080. 165. Konopelski, J. P., Filonova, L. K., & Olmstead, M. M., (1997). Dipeptide surrogates containing asparagine-derived tetrahydropyrimidinones: Preparation, structure, and use in solid phase synthesis. Journal of the American Chemical Society, 119(18), 4305, 4306. doi: 10.1021/ja9639271. 166. Gitu, P. M., Yusuf, A. O., & Bhatt, B. M., (1998). Application of tetralinyls as carboxamide protecting groups in peptide synthesis. Bulletin of the Chemical Society of Ethiopia, 12(1), 35–43. doi: 10.4314/bcse.v12i1.21032. 167. Sieber, P., & Riniker, B., (1990). Side-chain protection of asparagine and glutamine ty trityl. Application to solid-phase peptide synthesis. In: Epton, R., (ed.), Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 1st (pp. 577–583). 168. Carreno, L. F., Alba, M. P., Varela, Y., Patarroyo, M. E., & Lozano, J. M., (2011). A new approach to obtain Nα-t-Boc-amino acid aldehydes from asparagine and glutamine for reduced amide pseudopeptide solid-phase synthesis. Chemical Biology & Drug Design, 78(4), 603–611. 169. Garcia, O., Bofill, J. M., Nicolas, E., & Albericio, F., (2010). 2,2,4,6,7-pentamethyl-2,3dihydrobenzofuran-5-methyl (Pbfm) as an alternative to the trityl group for the sidechain protection of cysteine and asparagine/glutamine. European Journal of Organic Chemistry, (19), 3631–3640. doi: 10.1002/ejoc.201000201. 170. Sax, B., Dick, F., Tanner, R., & Gosteli, J., (1992). 4-methyltrityl (Mtt): A new protecting group for the side chain protection of Asn and Gln in solid-phase peptide synthesis. Peptide Research, 5(4), 245–246. 171. Pietta, P. G., & Marshall, G. R., (1970). Amide protection and amide supports in solid-phase peptide synthesis. Journal of the Chemical Society [Section] D: Chemical Communications, (11), 650, 651. doi: 10.1039/c29700000650. 172. Marshall, G. R., Pietta, P. G., & Cavallo, P., (1971). 2,4-dimethoxybenzyl as a protecting group for glutamine and asparagine in peptide synthesis. Journal of Organic Chemistry, 36(25), 3966–3970. doi: 10.1021/jo00824a025. 173. Shah, D., Schneider, A., Babler, S., Gandhi, R., Van, N. E., & Chess, E., (1992). Alkylation of tryptophan during deprotection of Tmob-protected carboxamide side chains. Peptide Research, 5(4), 241–244. 174. Gitu, P. M., Bhatt, B. M., Nyarang’o, D., & Hruby, V. J., (1994). Synthesis of carboxamide protected asparagine and glutamine derivatives. International Journal of BioChemiPhysics, 3(1&2), 42–46. 175. Gitu, P. M., Bhatt, B. M., Nyarango, D., & Hruby, V. J., (1993). Solvolysis of carboxamide protected asparagine and glutamine derivatives with boron tris(trifluoroacetate) in trifluoroacetic acid and in acetic acid solutions. International Journal of BioChemiPhysics, 2(1, 2), 161, 162.

374

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

176. Gitu, P. M., Yusuf, A. O., Ogutu, V. O., & Bhatt, B. M., (1998). Application of tetralinyl group in solid-phase peptide synthesis. International Journal of BioChemiPhysics, 6, 7(1, 2), 7–10. 177. Koenig, W., & Geiger, R., (1970). New amide protecting group. Chemische Berichte, 103(7), 2041–2051. 178. Juhasz, A., & Bajusz, S., (1979). A novel amide-protecting group. Acta Chimica Academiae Scientiarum Hungaricae, 102(3), 289–296. 179. Han, Y., Sole, N. A., Tejbrant, J., & Barany, G., (1996). Novel NO-xanthenylprotecting groups for asparagine and glutamine, and applications to Nα-9fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. Peptide Research, 9(4), 166–173. 180. Carpino, L. A., Shroff, H. N., Chao, H. G., Mansour, E. M. E., & Albericio, F., (1995). Cyclopropyldimethylcarbinyl: A new acid-labile side-chain protecting group for asparagine and glutamine. In: Maia, H. L. S., (ed.), Peptides 1994: Proceedings of the European Peptide Symposium, 23rd (pp. 155, 156). Braga, Portugal; Braga, Portugal. 181. Sakura, N., Hirose, K., Nishijima, M., Uchida, Y., & Hashimoto, T., (1985). Synthesis of L-α-amino acid derivatives with ω-amide acylated by urethane type protecting groups and the application to peptide chemistry. Peptide Chemistry, 85–90. 182. Gutte, B., & Merrifield, R. B., (1971). Synthesis of ribonuclease A. Journal of Biological Chemistry, 246(6), 1922–1941. 183. Pless, J., & Bauer, W., (1973). Boron tris(trifluoroacetate) for removal of protecting groups in peptide chemistry. Angewandte Chemie (International ed. in English), 12(2), 147, 148. doi: 10.1002/anie.197301471. 184. Sakakibara, S., Shimonishi, Y., Kishida, Y., Okada, M., & Sugihara, H., (1967). Use of anhydrous hydrogen fluoride in peptide synthesis. I. Behavior of various protective groups in anhydrous hydrogen fluoride. Bulletin of the Chemical Society of Japan, 40(9), 2164–2167. doi: 10.1246/bcsj.40.2164. 185. Weygand, F., Steglich, W., & Bjarnason, J., (1968). Easily cleavable protective groups for acid amide groups. III. Derivatives of asparagine and glutamine with 2,4-dimethoxybenzyl- and 2,4,6-trimethoxybenzyl-protected amide groups. Chemische Berichte, 101(10), 3642–3648. 186. Ferreira, V. F. C., Correia, J. D. G., Farinha, C. M., & Mendes, F., (2020). Improved Fmoc-solid-phase peptide synthesis of an extracellular loop of CFTR for antibody selection by the phage display technology. Journal of Peptide Science, 26(7), e3253/1– e3253/9. doi: 10.1002/psc.3253. 187. Gandioso, A., Cano, M., Massaguer, A., & Marchán, V., (2016). A green light-triggerable RGD peptide for photo controlled targeted drug delivery: Synthesis and photolysis studies. Journal of Organic Chemistry, 81(23), 11556–11564. doi: 10.1021/acs. joc.6b02415. 188. Conroy, T., Jolliffe, K. A., & Payne, R. J., (2010). Synthesis of N-linked glycopeptides via solid-phase aspartylation. Organic & Biomolecular Chemistry, 8(16), 3723–3733. doi: 10.1039/c003673k.

Side Chain Protecting Groups 375

189. Ruczynski, J., Lewandowska, B., Mucha, P., & Rekowski, P., (2008). Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid. Journal of Peptide Science, 14(3), 335–341. doi: 10.1002/psc.941. 190. Flora, D., Mo, H., Mayer, J. P., Khan, M. A., & Yan, L. Z., (2005). Detection and control of aspartimide formation in the synthesis of cyclic peptides. Bioorganic & Medicinal Chemistry Letters, 15(4), 1065–1068. doi: 10.1016/j.bmcl.2004.12.025. 191. Mergler, M., Dick, F., Sax, B., Weiler, P., & Vorherr, T., (2001). Systematic investigation of the aspartimide problem. In: Lebl, M., & Houghten, R. A., (eds.), Peptides: The Wave of the Future, Proceedings of the Second International and the Seventeenth American Peptide Symposium (pp. 63, 64). San Diego, CA, United States, San Diego, CA, United States. 192. Karlstrom, A., Rosenthal, K., & Unden, A., (2000). Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry. Journal of Peptide Research, 55(1), 36–40. doi: 10.1034/j.1399-3011.2000.00147.x. 193. Meutermans, W. D. F., Alewood, P. F., Bourne, G. T., Hawkins, B., & Smythe, M. L., (1997). Synthesis of α-aspartyl-containing cyclic peptides. Letters in Peptide Science, 4(2), 79–84. doi: 10.1007/BF02443518. 194. Karlstroem, A. H., & Unden, A. E., (1995). The β-2,4-dimethyl-3-pentyl ester as a new protecting group for aspartic acid that prevents base-catalyzed aspartimide formation in solid phase peptide synthesis. Tetrahedron Letters, 36(22), 3909–3912. doi: 10.1016/0040-4039(95)00639-T. 195. Conde, S., & Lopez-Serrano, P., (2002). Regioselective lipase-catalyzed amidation of N-blocked L- and D-aspartic acid diesters. European Journal of Organic Chemistry, (5), 922–929. doi: 10.1002/1099-0690(200203)2002:53.0.CO;2-E. 196. Xaus, N., Clapes, P., Bardaji, E., Torres, J. L., Jorba, X., Mata, J., & Valencia, G., (1989). New enzymic approach to the synthesis of convenient aspartic acid intermediates in peptide chemistry. Synthesis of N-(benzylocarbonyl)-L-aspartic acid β-allyl ester. Tetrahedron, 45(23), 7421–7426. doi: 10.1016/S0040-4020(01)89204-8. 197. Bodanszky, M., Tolle, J. C., Deshmane, S. S., & Bodanszky, A., (1978). side reactions in peptide synthesis. VI. A reexamination of the benzyl group in the protection of the side chains of tyrosine and aspartic acid. International Journal of Peptide & Protein Research, 12(2), 57–68. doi: 10.1111/j.1399-3011.1978.tb02868.x. 198. Chulin, A. N., Rodionov, I. L., & Ivanov, V. T., (2005). Synthesis of 10- and 9-membered dilactams derived from aspartic and glutamic acids. Journal of Peptide Research, 65(2), 292–297. doi: 10.1111/j.1399-3011.2005.00233.x. 199. Süli-Vargha, H., Schlosser, G., & Ilaš, J., (2007). 1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid β-benzyl ester. Journal of Peptide Science, 13(11), 742–748. doi: 10.1002/psc.885. 200. Bodanszky, M., & Kwei, J. Z., (1978). Side reactions in peptide synthesis. VII. Sequence dependence in the formation of aminosuccinyl derivatives from β-benzyl-aspartyl

376

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

peptides. International Journal of Peptide & Protein Research, 12(2), 69–74. doi: 10.1111/j.1399-3011.1978.tb02869.x. 201. Kates, S. A., & Albericio, F., (1995). Rearrangement of Glu(OtBu)- and Asp(OtBu)containing peptides upon fluoride treatment in solid-phase synthesis. Letters in Peptide Science, 1(5), 213–220. doi: 10.1007/BF00127267. 202. Giri, R. S., Manne, S. R., Dolai, G., Paul, A., Kalita, T., & Mandal, B., (2017). FeCl3mediated side chain modification of aspartic acid- and glutamic acid-containing peptides on a solid support. ACS Omega, 2(10), 6586–6597. doi: 10.1021/acsomega.7b01143. 203. Capasso, S., Mazzarella, L., Sica, F., & Zagari, A., (1991). First evidence of spontaneous deamidation of a glutamine residue via a cyclic imide to an α- and γ-glutamic residue under physiological conditions. Journal of the Chemical Society, Chemical Communications, (23), 1667, 1668. doi: 10.1039/C39910001667. 204. Johnson, T., Liley, M., Cheeseright, T. J., & Begum, F., (2000). Problems in the synthesis of cyclic peptides through use of the Dmab protecting group. Perkin 1: An International Journal of Organic and Bio-Organic Chemistry, (16), 2811–2820. 205. Mergler, M., Dick, F., Sax, B., Weiler, P., & Vorherr, T., (2003). The aspartimide problem in fmoc-based SPPS. Part I. Journal of Peptide Science, 9(1), 36–46. doi: 10.1002/ psc.430. 206. Dölling, R., Beyermann, M., Haenel, J., Kernchen, F., Krause, E., Franke, P., Brudel, M., & Bienert, M., (1994). Piperidine-mediated side product formation for Asp(OBut)containing peptides. Journal of the Chemical Society, Chemical Communications, (7), 853, 854. doi: 10.1039/C39940000853. 207. Quibell, M., Owen, D., Peckman, L. C., & Johnson, T., (1994). Suppression of piperidinemediated side product formation for Asp(OBut)-containing peptides by the use of N-(2hydroxy-4-methoxybenzyl) (Hmb) backbone amide protection. Journal of the Chemical Society, Chemical Communications, (20), 2343, 2344. doi: 10.1039/c39940002343. 208. Packman, L. C., (1995). N-2-hydroxy-4-methoxybenxyl (Hmb) backbone protection strategy prevents double aspartimide formation in a ‘difficult’ peptide sequence. Tetrahedron Letters, 36(41), 7523–7526. doi: 10.1016/0040-4039(95)01522-1. 209. Nicolas, E., Pujades, M., Bacardit, J., Giralt, E., & Albericio, F., (1997). A new approach to Hmb-backbone protection of peptides: Synthesis and reactivity of Nα-Fmoc-Nα-(Hmb)amino acids. Tetrahedron Letters, 38(13), 2317–2320. doi: 10.1016/ S0040-4039(97)00303-1. 210. Zahariev, S., Guarnaccia, C., Pongor, C. I., Quaroni, L., Cemazar, M., & Pongor, S., (2006). Synthesis of ‘difficult’ peptides free of aspartimide and related products, using peptoid methodology. Tetrahedron Letters, 47(25), 4121–4124. doi: 10.1016/j. tetlet.2006.04.074. 211. Rabanal, F., Pastor, J. J., Nicolas, E., Albericio, F., & Giralt, E., (2000). Synthesis of aspartimide-free protected peptides on base-labile functionalized resins. Tetrahedron Letters, 41(42), 8093–8096. doi: 10.1016/S0040-4039(00)01398-8. 212. Wagner, M., Dziadek, S., & Kunz, H., (2003). The (2-phenyl-2-trimethylsilyl)ethyl(PTMSEL)-linker in the synthesis of glycopeptide partial structures of complex cell

Side Chain Protecting Groups 377

surface glycoproteins. Chemistry - A European Journal, 9(24), 6018–6030. doi: 10.1002/ chem.200305304. 213. Wade, J. D., Mathieu, M. N., Macris, M., & Tregear, G. W., (2000). Base-induced side reactions in Fmoc-solid phase peptide synthesis: Minimization by use of piperazine as Nα-deprotection reagent. Letters in Peptide Science, 7(2), 107–112. doi: 10.1007/ BF02443569. 214. Martinez, J., & Bodanszky, M., (1978). Side reactions in peptide synthesis. IX. Suppression of the formation of aminosuccinyl peptides with additives. International Journal of Peptide & Protein Research, 12(5), 277–283. doi: 10.1111/j.1399-3011.1978. tb02898.x. 215. Ajayaghosh, A., & Pillai, V. N. R., (1990). Solid-phase synthesis of N-methyl- and N-ethylamides of peptides using photolytically detachable [(3-nitro-4-[(alkylamino) methyl]benzamido]methyl]polystyrene resin. Journal of Organic Chemistry, 55(9), 2826–2829. doi: 10.1021/jo00296a049. 216. Nozaki, S., & Muramatsu, I., (1988). Convenient synthesis of N-protected amino acid amides. Bulletin of the Chemical Society of Japan, 61(7), 2647, 2648. doi: 10.1246/ bcsj.61.2647. 217. Stewart, F. H. C., (1971). Experiments on the acid-stability of γ-benzyl ester protecting groups in the synthesis of l-glutamyl peptides. Australian Journal of Chemistry, 24(12), 3739–3743. 218. Tamamura, H., Nakamura, J., Noguchi, K., Funakoshi, S., & Fujii, N., (1993). Acceleration of the Nα-deprotection rate by the addition of m-cresol to diluted methanesulfonic acid and its application to the Z(Ome)-based solid-phase syntheses of human pancreastatin-29 and magainin 1. Chemical & Pharmaceutical Bulletin, 41(5), 954–957. doi: 10.1248/ cpb.41.954. 219. Tian, X., Yu, P., Tang, Y., Le, Z., & Huang, W., (2017). Aspartic acid side-chain benzyl ester as a multifunctionalization precursor for synthesis of branched and cyclic arginylglycyl-aspartic acid peptides. Synlett, 28(15), 1966–1970. doi: 10.1055/s-0036-1588870. 220. Kaneshiro, C. M., & Michael, K., (2006). A convergent synthesis of N-glycopeptides. Angewandte Chemie, International Edition, 45(7), 1077–1081. doi: 10.1002/ anie.200502687. 221. Clapes, P., Adlercreutz, P., & Mattiasson, B., (1990). Enzymic peptide synthesis in organic media: Nucleophile specificity and medium engineering in α-chymotrypsincatalyzed reactions. Biotechnology and Applied Biochemistry, 12(4), 376–386. 222. McDermott, J. R., & Benoiton, N. L., (1973). N-methylamino acids in peptide synthesis. II. New synthesis of n-benzyloxycarbonyl-N-methylamino acids. Canadian Journal of Chemistry, 51(12), 1915–1919. doi: 10.1139/v73-286. 223. Moore, A. T., Rydon, H. N., & Smithers, M. J., (1966). Polypeptides. XIV. The synthesis of some oligopeptides containing lysine and glutamic acid residues. Journal of the Chemical Society [Section] C: Organic, (24), 2349–2359. doi: 10.1039/j39660002349.

378

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

224. Wang, C. T., Kulesha, I. D., Stefko, P. L., & Wang, S. S., (1974). Solid phase synthesis of Penta gastrin and other peptide amides by a modified technique. International Journal of Peptide & Protein Research, 6(2), 59–64. doi: 10.1111/j.1399–3011.1974.tb02361.x. 225. Yanaihara, N., Hashimoto, T., Yanaihara, C., & Sakura, N., (1970). Synthesis of proinsulin. I. Synthesis of partially protected tritriacontapeptide related to the connecting peptide fragment of porcine proinsulin. Chemical & Pharmaceutical Bulletin, 18(2), 417–420. doi: 10.1248/cpb.18.417. 226. Koga, T., Nagaoka, A., & Higashi, N., (2006). Fabrication of a switchable nanosurface composed of acidic and basic block-polypeptides. Colloids and Surfaces, A: Physicochemical and Engineering Aspects, 284, 285, 521–527. doi: 10.1016/j. colsurfa.2005.10.013. 227. Ponnusamy, E., (2011). A new and greener method to manufacture copolymer-1. WIT Transactions on Ecology and the Environment, 154(Sustainable Chemistry), 33–38. doi: 10.2495/CHEM110041. 228. Subra, G., Mehdi, A., Enjalbal, C., Amblard, M., Brunel, L., Corriu, R., & Martinez, J., (2011). Functionalized mesoporous silica: A good opportunity for controlled peptide oligomerization. Journal of Materials Chemistry, 21(17), 6321–6326. doi: 10.1039/ c0jm04492j. 229. Trudelle, Y., (1973). Sequential polypeptides. synthesis of poly(L-tyrosyl-L-glutamylL-tyrosyl-L-glutamyl), poly(L-glutamyl-L-tyrosyl-L-glutamyl), and poly(L-glutamylL-glutamyl-L-tyrosyl-L-glutamyl) by use of catechol esters. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972–1999), (10), 1001–1005. 230. Yang, Q., Wang, L., Lin, W., Ma, G., Yuan, J., & Chen, S., (2014). Development of nonfouling polypeptides with uniform alternating charges by polycondensation of the covalently bonded dimer of glutamic acid and lysine. Journal of Materials Chemistry B: Materials for Biology and Medicine, 2(5), 577–584. doi: 10.1039/C3TB21333A. 231. Handa, B. K., & Keech, E., (1992). Fmoc solid phase synthesis of an endothelin converting enzyme substrate. use of allyl ester as the third orthogonal protecting group. International Journal of Peptide & Protein Research, 40(1), 66–71. doi: 10.1111/j.13993011.1992.tb00106.x. 232. Ning, X., Liu, D., Liu, S., Zhang, M., & Shen, H., (2018). Modification of oligopeptides on aspartic acid or lysine residues by solid-phase synthesis through on-resin side-chain conjugation. Synlett, 29(19), 2588–2594. doi: 10.1055/s-0037-1611060. 233. Patel, G., Husman, W., Jehanli, A. M., Deadman, J. J., Green, D., Kakkar, V. V., & Brennand, D. M., (1999). A cyclic peptide analog of the loop III region of plateletderived growth factor-BB is a synthetic antigen for the native protein. Journal of Peptide Research, 53(1), 68–74. doi: 10.1111/j.1399-3011.1999.tb01618.x. 234. Lodder, M., Crasto, C. F., Laikhter, A. L., An, H., Arslan, T., Karginov, V. A., Short, G. F. III., & Hecht, S. M., (2000). Synthesis of aspartic acid derivatives useful for the preparation of misacylated transfer RNAs. Canadian Journal of Chemistry, 78(6), 884–891.

Side Chain Protecting Groups 379

235. Wood, H. W., (1958). Synthetic polypeptide substitutes for gelatin in photographic emulsions and observations on gelation. Nature (London, United Kingdom), 182, 106, 107. 236. Izdebski, J., Yamashiro, D., Li, C. H., & Viti, G., (1982). Synthesis and properties of human γ-lipotropin. International Journal of Peptide & Protein Research, 20(1), 87–92. doi: 10.1111/j.1399-3011.1982.tb02657.x. 237. Blake, J., (1979). Use of cyclopentyl ester protection for aspartic acid to reduce base catalyzed succinimide formation in solid-phase peptide synthesis. International Journal of Peptide & Protein Research, 13(4), 418–425. doi: 10.1111/j.1399-3011.1979. tb01901.x. 238. Plaue, S., (1990). Synthesis of cyclic peptides on solid support. Application to analogs of hemagglutinin of influenza virus. International Journal of Peptide & Protein Research, 35(6), 510–517. doi: 10.1111/j.1399-3011.1990.tb00255.x. 239. Toth, G. K., & Penke, B., (1992). An improved method for the preparation of ω-cyclohexyl esters of aspartic and glutamic acid. Synthesis, (4), 361, 362. doi: 10.1055/s-1992-26108. 240. Fujii, N., Sakurai, M., Akaji, K., Nomizu, M., Yajima, H., Mizuta, K., Aono, M., et al., (1986). Studies on peptides. CXXXIX. Solution synthesis of a 42-residue peptide corresponding to the entire amino acid sequence of human glucose-dependent insulinotropic polypeptide (GIP). Chemical & Pharmaceutical Bulletin, 34(6), 2397– 2410. doi: 10.1248/cpb.34.2397. 241. Yajima, H., Funakoshi, S., & Akaji, K., (1986). Several methodological improvements for the synthesis of biologically active polypeptides. Biopolymers, 25(Suppl.), S39–S46. 242. Kawasaki, K., Murakami, T., Koshino, K., Namikawa, M., Maeda, M., Hama, T., & Mayumi, T., (1994). Amino acids and peptides. XX. Preparation of β-cyclododecyl aspartate and its application to synthesis of fibronectin- and laminin-related peptides. Chemical & Pharmaceutical Bulletin, 42(4), 792–795. doi: 10.1248/cpb.42.792. 243. Yajima, H., Futaki, S., Otaka, A., Yamashita, T., Funakoshi, S., Bessho, K., Fujii, N., & Akaji, K., (1986). Studies on peptides. CXLIII. Evaluation of β-menthyl aspartate for peptide synthesis. Chemical & Pharmaceutical Bulletin, 34(10), 4356–4361. doi: 10.1248/cpb.34.4356. 244. Hayakawa, T., & Yamamoto, H., (1972). Synthesis and conformational studies of polyacidic amino acids containing optically active side chains. In: Rohlfing, D. L., (ed.), Mol. Evol. (pp. 247–260). 245. Prestidge, R. L., Harding, D. R. K., & Hancock, W. S., (1976). Use of substituted benzyl esters as carboxyl-protecting groups in solid-phase peptide synthesis. Journal of Organic Chemistry, 41(15), 2579–2583. doi: 10.1021/jo00877a015. 246. Chen, R., Pawlicki, M. A., & Tolbert, T. J., (2014). Versatile on-resin synthesis of high mannose glycosylated asparagine with functional handles. Carbohydrate Research, 383, 69–75. doi: 10.1016/j.carres.2013.11.002. 247. Rizo, J., Albericio, F., Romero, G., Garcia-Echeverria, C., Claret, J., Muller, C., Giralt, E., & Pedroso, E., (1988). Use of polar picolyl protecting groups in peptide synthesis. Journal of Organic Chemistry, 53(22), 5386–5389. doi: 10.1021/jo00257a044.

380

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

248. Garapati, S., & Burns, C. S., (2014). The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: Flipping peptide charge states for characterization by positive ion mode ESI-MS. Journal of Peptide Science, 20(3), 191–195. doi: 10.1002/ psc.2598. 249. Young, G. T., & Garner, R., (1971). Amino acids and peptides. XXXIII. Synthesis of val5-angiotensin-II by the picolyl ester method. Journal of the Chemical Society [Section] C: Organic, (1), 50–53. 250. Gee, K. R., Niu, L., Schaper, K., Jayaraman, V., & Hess, G. P., (1999). Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor. Biochemistry, 38(10), 3140–3147. doi: 10.1021/ bi9826557. 251. Al-Obeidi, F., Sanderson, D. G., & Hruby, V. J., (1990). Synthesis of β- or γ-fluorenylmethyl esters of respectively Nα-Boc-L-aspartic acid and Nα-Boc-L-glutamic acid. International Journal of Peptide & Protein Research, 35(3), 215–218. doi: 10.1111/j.1399-3011.1990. tb00940.x. 252. Miltschitzky, S., & Koenig, B., (2004). Synthesis of an amino acid with protected cyclen side chain functionality. Synthetic Communications, 34(11), 2077–2084. doi: 10.1081/ SCC-120037922. 253. Sleczkowski, M. L., Segers, I., Liu, Y., & Palmans, A. R. A., (2020). Sequence-defined L-glutamamide oligomers with pendant supramolecular motifs via iterative synthesis and orthogonal post-functionalization. Polymer Chemistry, 11(46), 7393–7401. doi: 10.1039/d0py01157f. 254. Aihara, K., Shigenaga, A., Takahashi, D., & Otaka, A., (2013). New approach for synthesis of lactam bridged peptides using olefin metathesis on AJIPHASE. Peptide Science (Proceedings of the Japanese Peptide Symposium), 50, 143, 144. 255. Cilli, E. M., Vicente, E. F., Crusca, E., & Nakaie, C. R., (2007). EPR investigation of the influence of side chain protecting groups on peptide-resin solvation of the Asx and Glx model containing peptides. Tetrahedron Letters, 48(31), 5521–5524. doi: 10.1016/j. tetlet.2007.05.164. 256. Nefkens, G. H. L., & Zwanenburg, B., (1983). Boroxazolidones as simultaneous protection of the amino and carboxyl group in α-amino acids. Tetrahedron, 39(18), 2995–2998. doi: 10.1016/S0040–4020(01)92162–3. 257. Fernandez-Vargas, J., Farfan, N., Castillo, D., Santillan, R., Carabez, A., & LotinaHennsen, B., (1995). OEC complex enzyme and photophosphorylation inhibition behavior of 1,1-diphenylboroxazolidone derivatives of α-amino acids. In: Mathis, P., (ed.), Photosynthesis: From Light to Biosphere, Proceedings of the International Photosynthesis Congress, 10th (pp. 839–842). Montpellier, Fr. 258. González, A. G., Zavala, L. C., Moreno, A. P. A., San, J. E. R., Ferrara, J. G. T., Espinosa, L. R., & Jiménez, G. M., (2011). Pharmacokinetics of diphenylboroxazolidones of L-α-amino acids with activity on the CNS: Quantification in rat DBS by UPLC-MS/MS. Bioanalysis, 3(4), 439–448. doi: 10.4155/bio.10.208.

Side Chain Protecting Groups 381

259. Baum, G., (1970). Carbon-boron bond cleavage by amino acids: An improved route to mixed anhydrides of diphenylborinic and amino acids. Journal of Organometallic Chemistry, 22(2), 269–271. doi: 10.1016/S0022-328X(00)86042-8. 260. Ruehl, T., Boettcher, C., Pumpor, K., Hennig, L., Sieler, J., & Burger, K., (2004). Hexafluoroacetone as protecting and activating reagent: Site-selective functionalization of α-amino alkanedioic acids. Synthesis, (18), 3065–3069. doi: 10.1055/s-2004-834918. 261. Boettcher, C., Spengler, J., Essawy, S. A., & Burger, K., (2004). Hexafluoroacetone as protecting and activating reagent. A new approach to N-glycosides. Monatshefte für Chemie, 135(7), 853–863. doi: 10.1007/s00706-004-0183-9. 262. Burger, K., Gold, M., Neuhauser, H., & Rudolph, M., (1991). Regiospecific reactions with ω-carboxy-α-amino acids. Part III. Aspartic acid. Chemiker-Zeitung, 115(3), 77–82. 263. Hocker, M. D., Caldwell, C. G., Macsata, R. W., & Lyttle, M. H., (1995). p-nitrobenzyl side-chain protection for solid-phase synthesis. Peptide Research, 8(6), 310–315. 264. Goldschmidt, S., & Jutz, C., (1953). Über peptid-synthesen, III. Mitteil.: Eine neue synthese des glutathions. Chemische Berichte, 86(9), 1116–1121. doi: 10.1002/ cber.19530860914. 265. Stathopoulos, P., Papas, S., Sakka, M., Tzakos, A. G., & Tsikaris, V., (2014). A rapid and efficient method for the synthesis of selectively S-Trt or S-Mmt protected Cys-containing peptides. Amino Acids, 46(5), 1367–1376. doi: 10.1007/s00726-014-1696-0. 266. Muttenthaler, M., Ramos, Y. G., Feytens, D., De Araujo, A. D., & Alewood, P. F., (2010). p-Nitrobenzyl protection for cysteine and selenocysteine: A more stable alternative to the acetamidomethyl group. Biopolymers, 94(4), 423–432. doi: 10.1002/bip.21502. 267. Miseta, A., & Csutora, P., (2000). Relationship between the occurrence of cysteine in proteins and the complexity of organisms. Molecular Biology and Evolution, 17(8), 1232–1239. doi: 10.1093/oxfordjournals.molbev.a026406. 268. Hargittai, B., & Barany, G., (1999). Controlled syntheses of natural and disulfidemispaired regioisomers of α-conotoxin SI. Journal of Peptide Research, 54(6), 468–479. doi: 10.1034/j.1399-3011.1999.00127.x. 269. Chakraborty, A., Sharma, A., Albericio, F., & De La Torre, B. G., (2020). Disulfidebased protecting groups for the cysteine side chain. Organic Letters, 22(24), 9644–9647. doi: 10.1021/acs.orglett.0c03705. 270. Schroll, A. L., Hondal, R. J., & Flemer, S. Jr., (2012). 2,2’-dithiobis(5-nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups. Journal of Peptide Science, 18(1), 1–9. doi: 10.1002/psc.1403. 271. Soll, R., & Beck-Sickinger, A. G., (2000). On the synthesis of orexin a: A novel one-step procedure to obtain peptides with two intramolecular disulfide bonds. Journal of Peptide Science, 6(8), 387–397. 272. Katayama, H., & Hojo, H., (2013). The phenacyl group as an efficient thiol protecting group in a peptide condensation reaction by the thioester method. Organic & Biomolecular Chemistry, 11(26), 4405–4413. doi: 10.1039/c3ob40644j.

382

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

273. Park, J. H., Carlin, K. P., Wu, G., Ilyin, V. I., & Kyle, D. J., (2012). Cysteine racemization during the Fmoc solid phase peptide synthesis of the nav1.7-selective peptide - protoxin II. Journal of Peptide Science, 18(7), 442–448. doi: 10.1002/psc.2407. 274. Loidl, G., Dick, F., Mergler, M., & Schoenleber, R. O., (2009). Optimized coupling protocols for the incorporation of Cys derivatives during Fmoc-SPPS. Advances in Experimental Medicine and Biology, 611(Peptides for Youth), 163, 164. doi: 10.1007/978-0-387-73657-0_74. 275. Low, M., Rill, A., & Kisfaludy, L., (1984). Racemization of the C-terminal Cys(Bzl)group during ammonolysis of peptide esters and a novel method for amidation of carboxylic acids. In: Ragnarsson, U., (ed.), Pept., Proc. Eur. Pept. Symp., 18th (pp. 267–270). 276. Atherton, E., Hardy, P. M., Harris, D. E., & Matthews, B. H., (1991). Racemization of C-terminal cysteine during peptide assembly. In: Giralt, E., & Andreu, D., (eds.), Pept. 1990: Proc. Eur. Pept. Symp., 21st (pp. 243, 244). 277. Shiraiwa, T., Kataoka, K., Sakata, S., & Kurokawa, H., (1988). Racemization of optically active cysteine via formation of 2,2-dimethyl-4-thiazolidinecarboxylic acid. Bulletin of the Chemical Society of Japan, 61(11), 4158–4160. doi: 10.1246/bcsj.61.4158. 278. Lukszo, J., Patterson, D., Albericio, F., & Kates, S. A., (1996). 3-(1-piperidinyl)alanine formation during the preparation of C-terminal cysteine peptides with the Fmoc/t-Bu strategy. Letters in Peptide Science, 3(3), 157–166. doi: 10.1007/BF00132978. 279. Wehner, J. W., & Lindhorst, T. K., (2012). S-fluorenylmethyl protection of the cysteine side chain upon Nα-Fmoc deprotection. Beilstein Journal of Organic Chemistry, 8, 2149–2155. doi: 10.3762/bjoc.8.242. (a) Bors, D. A., Kaufman, M. J., & Streitwieser, A., (1985). Carbon acidity. 67. The indicator scale of cesium ion pairs in tetrahydrofuran. Journal of the American Chemical Society, 107(24), 6975–6982. doi: 10.1021/ ja00310a038. (b) Hofer, F., Kraml, J., Kahler, U., Kamenik, A. S., & Liedl, K. R., (2020). Catalytic site pKa values of aspartic, cysteine, and serine proteases: Constant pH MD simulations. Journal of Chemical Information and Modeling, 60(6), 3030–3042. doi: 10.1021/acs.jcim.0c00190. 280. West, C. W., Estiarte, M. A., & Rich, D. H., (2001). New methods for side-chain protection of cysteine. Organic Letters, 3(8), 1205–1208. doi: 10.1021/ol015678d. 281. Lukashenko, N. P., (2010). Expanding genetic code: Amino acids 21 and 22, selenocysteine and pyrrolysine. Russian Journal of Genetics, 46(8), 899–916. doi: 10.1134/S1022795410080016. 282. Liu, J., Zheng, F., Cheng, R., Li, S., Rozovsky, S., Wang, Q., & Wang, L., (2018). Sitespecific incorporation of selenocysteine using an expanded genetic code and palladiummediated chemical deprotection. Journal of the American Chemical Society, 140(28), 8807–8816. doi: 10.1021/jacs.8b04603. 283. Peeler, J. C., & Weerapana, E., (2019). Chemical biology approaches to interrogate the selenoproteome. Accounts of Chemical Research, 52(10), 2832–2840. doi: 10.1021/acs. accounts.9b00379.

Side Chain Protecting Groups 383

284. Soriano-García, M., (2004). Organoselenium compounds as potential therapeutic and chemopreventive agents: A review. Current Medicinal Chemistry, 11(12), 1657–1669. doi: 10.2174/0929867043365053. 285. Ren, X., Zou, L., Lu, J., & Holmgren, A., (2018). Selenocysteine in mammalian thioredoxin reductase and application of ebselen as a therapeutic. Free Radical Biology & Medicine, 127, 238–247. doi: 10.1016/j.freeradbiomed.2018.05.081. 286. Low, S. C., Harney, J. W., & Berry, M. J., (1995). Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis. Journal of Biological Chemistry, 270(37), 21659–21664. doi: 10.1074/jbc.270.37.21659. 287. Yusuf, A. O., Bhatt, B. M., & Gitu, P. M., (2004). Tetralin group for asparagine sidechain protection and application to BOC-solid-phase peptide synthesis of mesotocin. Journal of the Kenya Chemical Society, 2(1), 14–17. 288. Yusuf, A. O., Bhatt, B. M., & Gitu, P. M., (2002). Protection of the amide side-chain of asparagine with the 1-tetralinyl group in the solid-phase peptide synthesis of lysinevasopressin. South African Journal of Chemistry [Online Computer File], 55, 87–96. 289. Gish, D. T., & Du Vigneaud, V., (1957). Synthesis of peptides of arginine related to arginine vasopressin. Journal of the American Chemical Society, 79(13), 3579–3581. doi: 10.1021/ja01570a074. 290. Yusuf, A. O., Bhatt, B. M., & Gitu, P. M., (2001). Solid-phase peptide synthesis of isotocin with amide of asparagine protected with 1-tetralinyl. Trifluoromethanesulphonic acid (TFMSA) Deprotection, cleavage and air oxidation of mercapto groups to disulphide. Bulletin of the Chemical Society of Ethiopia, 15(2), 143–149. 291. Cuthbertson, A., & Indrevoll, B., (2000). A method for the one-pot regioselective formation of the two disulfide bonds of α-conotoxin SI. Tetrahedron Letters, 41(19), 3661–3663. doi: 10.1016/S0040-4039(00)00437-8. 292. Schroll, A. L., & Hondal, R. J., (2009). Further development of new deprotection chemistry for cysteine and selenocysteine side chain protecting groups. Advances in Experimental Medicine and Biology, 611(Peptides for Youth), 135–136. doi: 10.1007/978-0-387-73657-0_60. 293. Akaji, K., Fujii, N., Yajima, H., Hayashi, K., Mizuta, K., Aono, M., & Moriga, M., (1985). Studies on peptides. CXXVII. Synthesis of a tripentacontapeptide with epidermal growth factor activity. Chemical & Pharmaceutical Bulletin, 33(1), 184–201. doi: 10.1248/cpb.33.184. 294. Sakakibara, S., Honda, I., Naruse, M., & Kanaoka, M., (1969). Direct synthesis of p-methoxybenzyl carbazate and p-methoxybenzyloxycarbonylamino acids using p-methoxybenzyl chloroformate as reagent. Experientia, 25(6), 576–578. doi: 10.1007/ BF01896518. 295. Ohkawa, H., & Eto, M., (1969). Alkylation of mercaptans and inhibition of “SH enzymes” by saligenin cyclic phosphate and phosphorothiolate esters. Agricultural and Biological Chemistry, 33(3), 443–451. doi: 10.1080/00021369.1969.10859328.

384

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

296. Berse, C., Boucher, R., & Piché, L., (1957). Preparation of L-cystinyl and L-cysteinyl peptides through catalytic hydrogenation of intermediates. Journal of Organic Chemistry, 22(7), 805–808. doi: 10.1021/jo01358a024. 297. Munson, M. C., Garcia-Echeverria, C., Albericio, F., & Barany, G., (1992). S-2,4,6-trimethoxybenzyl (Tmob): A novel cysteine protecting group for the Nα-(9fluorenylmethoxycarbonyl) (Fmoc) strategy of peptide synthesis. Journal of Organic Chemistry, 57(11), 3013–3018. doi: 10.1021/jo00037a013. 298. Royo, M., Garcia-Echeverria, C., Munson, M. C., Slomczynska, U., Eritja, R., Giralt, E., Barnay, G., & Albericio, F., (1992). Cysteine protection in solid phase peptide synthesis. In: Epton, R., (ed.), Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 2nd (pp. 461–465). 299. Chen, L., Zoulikova, I., Slaninova, J., & Barany, G., (1997). Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: Directed methods for the construction of disulfide and trisulfide bridges in peptides. Journal of Medicinal Chemistry, 40(6), 864–876. doi: 10.1021/jm9607156. 300. Kaufmann, K. D., Schoenherr, C., Jeschke, C., Bauschke, S., Kietzer, M., & Dölling, R., (1979). Synthesis of an insulin α-chain fragment A14–21 with the diphenylmethyl protecting group for cysteine. Journal fuer Praktische Chemie (Leipzig), 321(4), 613–618. doi: 10.1002/prac.19793210414. 301. Schreier, E., & Hermann, P., (1976). Synthesis of a pentapeptide derivative of the insulin sequence A17-A21. Journal fuer Praktische Chemie (Leipzig), 318(3), 502–506. doi: 10.1002/prac.19763180322. 302. Hiskey, R. G., & Adams, J. B. Jr., (1968). S-benzhydryl-L-cysteine. Biochemical Preparations, 12, 92–94. 303. Ogo, N., Oishi, S., Matsuno, K., Sawada, J. I., Fujii, N., & Asai, A., (2007). Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorganic & Medicinal Chemistry Letters, 17(14), 3921–3924. doi: 10.1016/j. bmcl.2007.04.101. 304. Barlos, K., Gatos, D., Hatzi, O., Koch, N., & Koutsogianni, S., (1996). Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis. International Journal of Peptide & Protein Research, 47(3), 148–153. doi: 10.1111/j.1399-3011.1996.tb01338.x. 305. Munson, M. C., Garcia-Echeverria, C., Albericio, F., & Barany, G., (1992). Novel cysteine protecting groups for the Nα-9-fluorenylmethyloxycarbonyl (Fmoc) strategy of peptide synthesis. In: Smith, J. A., & Rivier, J. E., (eds.), Pept.: Chem. Biol., Proc. Am. Pept. Symp., 12th (pp. 605, 606). 306. Hargittai, B., Han, Y., Kates, S. A., & Barany, G., (1999). S-xanthenyl side-chain anchoring for solid-phase synthesis of cysteine-containing peptides. In: Tam, J. P., & Kaumaya, P. T. P., (eds.), Peptides: Frontiers of Peptide Science, Proceedings of the American Peptide Symposium, 15th (pp. 273, 274). Nashville. 307. Sole, N. A., Han, Y., Vagner, J., Gross, C. M., Tejbrant, J., & Barany, G., (1996). Novel N- and S-xanthenyl protecting groups for side-chains of asparagine, glutamine and

Side Chain Protecting Groups 385

cysteine and their applications for Fmoc solid phase peptide synthesis. In: Kaumaya, P. T. P., & Hodges, R. S., (eds.), Peptides: Chemistry, Structure and Biology, Proceedings of the American Peptide Symposium, 14th (pp. 113, 114) Columbus, Ohio. 308. Echner, H., & Voelter, W., (1992). 9-phenylxanthen-9-yl- (Pixyl): A new thiol protecting group and its use in solid phase peptide chemistry. In: Epton, R., (ed.), Innovation Perspect. Solid Phase Synth. Collect. Pap., Int. Symp., 2nd (pp. 371–375). 309. Garcia, O., Nicolas, E., & Albericio, F., (2001). New dihydrobenzopyran and dihydrobenzofuran based cysteine protecting groups for Fmoc chemistry. In: Epton, R., (ed.), Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids--Small Molecule Organic Chemistry Diversity, Collected Papers, International Symposium, 6th (pp. 289, 290). York, United Kingdom. 310. Echner, H., & Voelter, W., (1995). Application of the 10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-yl (5H-dibenzo-suberyl, Sub) and 5H-dibenzo[a,d]cyclohepten-5-yl (5H-dibenzosuberenyl, Dbs) Groups for thiol protection in Fmoc solid phase peptide chemistry. In: Maia, H. L. S., (ed.), Peptides 1994, Proceedings of the European Peptide Symposium, 23rd (pp. 157, 158). Braga, Portugal. 311. Ogo, N., Ishikawa, Y., Sawada, J. I., Matsuno, K., Hashimoto, A., & Asai, A., (2015). Structure-guided design of novel L-cysteine derivatives as potent KSP inhibitors. ACS Medicinal Chemistry Letters, 6(9), 1004–1009. doi: 10.1021/acsmedchemlett.5b00221. 312. Harris, K. M., Flemer, S. Jr., & Hondal, R. J., (2007). Studies on deprotection of cysteine and selenocysteine side-chain protecting groups. Journal of Peptide Science, 13(2), 81–93. doi: 10.1002/psc.795. 313. Chakravarty, P. K., & Olsen, R. K., (1978). Benzamidomethyl Group as a thiol protecting group for cysteine, N-methylcysteine, and corresponding N-tert-butyloxycarbonyl derivatives. Journal of Organic Chemistry, 43(6), 1270, 1271. doi: 10.1021/jo00400a063. 314. Fujii, N., Otaka, A., Watanabe, T., Okamachi, A., Tamamura, H., Yajima, H., Inagaki, Y., et al., (1989). Silver trifluoromethanesulfonate as an S-deprotecting reagent for the synthesis of cystine peptides. Journal of the Chemical Society, Chemical Communications, (5), 283, 284. doi: 10.1039/c39890000283. 315. Papsuevich, O. S., Miksta, S., Cipens, G., Straujuma, R., & Kalejs, U., (1985). Synthesis of deaminooxytocin using s-benzamidomethyl protecting group for cysteine and β-mercaptopropionic acid. Latvijas PSR Zinatnu Akademijas Vestis, Kimijas Serija, (6), 749–755. 316. Hibino, H., & Nishiuchi, Y., (2012). 4-methoxybenzyloxymethyl group, a racemizationresistant protecting group for cysteine in Fmoc solid phase peptide synthesis. Organic Letters, 14(7), 1926–1929. doi: 10.1021/ol300592w. 317. Gongora-Benitez, M., Basso, A., Bruckdorfer, T., Royo, M., Tulla-Puche, J., & Albericio, F., (2012). Eco-friendly combination of the immobilized PGA enzyme and the S-phacm protecting group for the synthesis of Cys-containing peptides. Chemistry - A European Journal, 18(50), 16166–16176. doi: 10.1002/chem.201201370. 318. Royo, M., Alsina, J., Giralt, E., Slomcyznska, U., & Albericio, F., (1995). S-phenylacetamidomethyl (Phacm): An orthogonal cysteine protecting group for Boc

386

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

and Fmoc solid-phase peptide synthesis strategies. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, (9), 1095–1102. 319. Royo, M., Alsina, J., Giralt, E., Slomczynska, U., & Albericio, F., (1994). S-Phenylacetamidomethyl (Phacm). A versatile cysteine protecting group for Boc and Fmoc solid-phase synthesis strategies. In: Hodges, R. S., & Smith, J. A., (eds.), Pept.: Chem., Struct. Biol., Proc. Am. Pept. Symp., 13th (pp. 116–118). 320. Kiso, Y., Yoshida, M., Kimura, T., Fujiwara, Y., & Shimokura, M., (1989). A new thiol protecting trimethylacetamidomethyl group. Synthesis of a new porcine brain natriuretic peptide using the S-trimethylacetamidomethyl-cysteine. Tetrahedron Letters, 30(15), 1979–1982. doi: 10.1016/S0040-4039(00)99629-1. 321. Kiso, Y., Yoshida, M., Fujiwara, Y., Kimura, T., Akaji, K., & Yajima, H., (1994). Syntheses of natriuretic peptides using a new S-protecting group, S-trimethylacetamidomethyl (Tacm) group. In: Basava, C., & Anantharamaiah, G. N., (eds.), Peptides (pp. 27–37). 322. Yoshida, M., Akaji, K., Tatsumi, T., Fujiwara, Y., Kimura, T., & Kiso, Y., (1990). Deprotection of S-trimethylacetamidomethyl group using silver tetrafluoroborate. In: Peptide Chemistry (pp. 33–38). 323. Mukherjee, S., Matveenko, M., & Becker, C. F. W., (2020). Highly precise protein semisynthesis through ligation-desulfurization chemistry in combination with phenacyl protection of native cysteines. Methods in Molecular Biology (New York, NY, United States), 2133 (Expressed Protein Ligation), 343–358. doi: 10.1007/978-1-0716-0434-2_17. 324. Tang, G., Ji, T., Hu, A. F., & Zhao, Y. F., (2008). Novel N,S-phenacyl protecting group and its application for peptide synthesis. Synlett, (12), 1907–1909. doi: 10.1055/s-2008-1077887. 325. Katayama, H., & Hojo, H., (2013). Phenacyl Group is an efficient protecting group of the Cys side chain in peptide condensation reaction by the thioester method. Peptide Science (Proceedings of the Japanese Peptide Symposium), 49, 171, 172. 326. Felix, A. M., Jimenez, M. H., Mowles, T., & Meienhofer, J., (1978). Reactions in liquid ammonia. VII. Catalytic hydrogenolysis in liquid ammonia. Cleavage of Nα-benzyloxycarbonyl groups from cysteine-containing peptides with tert-butyl side chain protection. Application to a stepwise synthesis of somatostatin. International Journal of Peptide & Protein Research, 11(5), 329–339. doi: 10.1111/j.1399-3011.1978. tb02857.x. 327. Pavo, I., Penke, B., Varga, J. R., Vecsei, L., & Kovacs, K., (1986). A new synthesis of somatostatin and some potential metabolites. Acta Chimica Hungarica, 122(3, 4), 261–272. 328. Wang, H., Miao, Z., Lai, L., & Xu, X., (2000). An efficient procedure for cleavage of t-butyl protected cysteine in solid phase peptide synthesis. Synthetic Communications, 30(4), 727–735. doi: 10.1080/00397910008087375. 329. Metzger, J. W., Wiesmueller, K. H., & Jung, G., (1991). Synthesis of Nα-Fmoc protected derivatives of S-(2,3-dihydroxypropyl)-cysteine and their application in peptide synthesis. International Journal of Peptide & Protein Research, 38(6), 545–554. doi: 10.1111/j.1399-3011.1991.tb01538.x.

Side Chain Protecting Groups 387

330. Royo, M., García-Echeverría, C., Giralt, E., Eritja, R., & Albericio, F., (1992). S-2-(2,4dinitrophenyl)ethyl-L-cysteine: A new derivative for solid-phase peptide synthesis. Tetrahedron Letters, 33(17), 2391–2394. doi: 10.1016/S0040-4039(00)74220-1. 331. Sharma, A., Ramos-Tomillero, I., El-Faham, A., Nicolas, E., Rodriguez, H., De La Torre, B. G., & Albericio, F., (2017). Understanding tetrahydropyranyl as a protecting group in peptide chemistry. ChemistryOpen, 6(2), 168–177. doi: 10.1002/open.201600156. 332. Ramos-Tomillero, I., Rodriguez, H., & Albericio, F., (2015). Tetrahydropyranyl, a nonaromatic acid-labile Cys protecting group for Fmoc peptide chemistry. Organic Letters, 17(7), 1680–1683. doi: 10.1021/acs.orglett.5b00444. 333. Ruegg, U. T., Jarvis, D., & Rudinger, J., (1979). 4-pyridylmethyl; a thiol-protecting group suitable for the partial synthesis of proteins. Biochemical Journal, 179(1), 119–126. doi: 10.1042/bj1790119. 334. Coyle, S., Hallett, A., Munns, M. S., & Young, G. T., (1981). Amino acids and peptides. Part 45. The protection of the thiol function of cysteine and the imidazole-N of histidine by the diphenyl-4-pyridylmethyl group. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, (2), 522–528. 335. Coyle, S., & Young, G. T., (1976). Protection of cysteine and histidine by the diphenyl4-pyridylmethyl group during peptide synthesis. Journal of the Chemical Society, Chemical Communications, (23), 980, 981. 336. Mojska, H., Gielecinska, I., Jasinska-Melon, E., Winiarek, J., & Sawicki, W., (2020). Are AAMA and GAMA levels in urine after childbirth a suitable marker to assess exposure to acrylamide from passive smoking during pregnancy? -a pilot study. International Journal of Environmental Research and Public Health, 17(20), 7391/1–7391/15. doi: 10.3390/ijerph17207391. 337. Chung, C. J., Hsu, H. T., Chang, C. H., Li, S. W., Liu, C. S., Chung, M. C., Wu, G. W., et al., (2020). Relationships among cigarette smoking, urinary biomarkers, and urothelial carcinoma risk: A case-control study. Environmental Science and Pollution Research, 27(34), 43177–43185. doi: 10.1007/s11356-020-10196-2. 338. Kos, Y., (2020). Neuroprotective mechanisms of S-allyl-L-cysteine in neurological disease (review). Experimental and Therapeutic Medicine, 19(2), 1565–1569. doi: 10.3892/etm.2019.839. 339. Ayeleso, A. O., Lembede, B. W., Nyakudya, T. T., Adepoju, A. E., Chegou, N. N., & Mukwevho, E., (2020). Administration of S-allyl cysteine to neonatal rats modulates inflammatory biomarkers in high-fructose-fed rats in adulthood. Tropical Journal of Pharmaceutical Research, 19(5), 1053–1058. doi: 10.4314/tjpr.v19i5.21. 340. Gupta, P., Dutt, V., Kaur, N., Kalra, P., Gupta, S., Dua, A., Dabur, R., Saini, V., & Mittal, A., (2020). S-allyl cysteine: A potential compound against skeletal muscle atrophy. Biochimica et Biophysica Acta, General Subjects, 1864(10), 129676/1–129676/14. doi: 10.1016/j.bbagen.2020.129676. 341. Rousta, A. M., Mirahmadi, S. M. S., Shahmohammadi, A., Ramzi, S., Baluchnejadmojarad, T., & Roghani, M., (2020). S-allyl cysteine, an active ingredient of garlic, attenuates acute liver dysfunction induced by lipopolysaccharide/D-galactosamine in mouse:

388

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Underlying mechanisms. Journal of Biochemical and Molecular Toxicology, 34(9), e22518/1–e22518/10. doi: 10.1002/jbt.22518. 342. Blake, J., Woodworth, B. A., Litzi-Davis, L., & Cosand, W. L., (1992). Ethylcarbamoyl protection for cysteine in the preparation of peptide-conjugate immunogens. International Journal of Peptide & Protein Research, 40(1), 62–65. doi: 10.1111/j.1399-3011.1992. tb00105.x. 343. Smith, C. W., Skala, G., & Walter, R., (1977). Synthesis and some pharmacological properties of [4-β-(2-thienyl)-L-alanine]oxytocin. Journal of Medicinal Chemistry, 21(1), 115–117. doi: 10.1021/jm00199a022. 344. Németh, L., Somfai-Relle, S., Kellner, B., Sugár, J., Bognar, R., Farkas, J., Bálint, J., et al., (1978). Study of the antitumoral activity of S-carbamoyl-L-cysteine derivatives in animal experiments. Arzneimittel-Forschung, 28(7), 1119–1123. 345. Storey, H. T., Beacham, J., Cernosek, S. F., Finn, F. M., Yanaihara, C., & Hofmann, K., (1972). Polypeptides. LI. Application of S-ethylcarbamoylcysteine to the synthesis of a protected heptatetracontapeptide related to the primary sequence of ribonuclease T1. Journal of the American Chemical Society, 94(17), 6170–6178. doi: 10.1021/ ja00772a040. 346. Shimizu, M., Yumoto, N., & Tatsu, Y., (2006). Preparation of caged compounds using an antibody against the photocleavable protecting group. Analytical Biochemistry, 348(2), 318–320. doi: 10.1016/j.ab.2005.09.029. 347. Rijkers, D. T. S., Kruijtzer, J. A. W., Killian, J. A., & Liskamp, R. M. J., (2005). A Convenient solid phase synthesis of s-palmitoyl transmembrane peptides. Tetrahedron Letters, 46(19), 3341–3345. doi: 10.1016/j.tetlet.2005.03.079. 348. Eritja, R., Ziehler-Martin, J. P., Walker, P. A., Lee, T. D., Legesse, K., Albericio, F., & Kaplan, B. E., (1987). Use of the S-tert-butylsulfenyl group for protection of cysteine in solid-phase peptide synthesis using Fmoc amino acids. Tetrahedron, 43(12), 2675–2680. doi: 10.1016/S0040-4020(01)86872-1. 349. Postma, T. M., Giraud, M., & Albericio, F., (2012). Trimethoxyphenylthio as a highly labile replacement for tert-butylthio cysteine protection in Fmoc solid phase synthesis. Organic Letters, 14(21), 5468–5471. doi: 10.1021/ol3025499. 350. Rosen, O., Rubinraut, S., & Fridkin, M., (1990). Thiolysis of the 3-nitro-2-pyridinesulfenyl (Npys) protecting group. An approach towards a general deprotection scheme in peptide synthesis. International Journal of Peptide & Protein Research, 35(6), 545–549. doi: 10.1111/j.1399-3011.1990.tb00260.x. 351. Rietman, B. H., Peters, R. F. R., & Tesser, G. I., (1995). On the stability of S-(alkylsulfanyl) cysteine derivatives during solid-phase synthesis. Recueil des Travaux Chimiques des Pays-Bas, 114(1), 1–5. 352. Rietman, B. H., Peters, R. F. R., & Tesser, G. I., (1994). A facile method for the preparation of S-(alkylsulfenyl)cysteines. Synthetic Communications, 24(9), 1323–1332. doi: 10.1080/00397919408011734. 353. Ste Marie, E., Ruggles, E., & Hondal, R., (2016). Removal of benzyl groups from cysteine and selenocysteine using 2,2’-dithiobis-5-nitropyridine and ascorbolysis.

Side Chain Protecting Groups 389

In: Abstracts of Papers, 252nd ACS National Meeting & Exposition (pp. ORGN-530). Philadelphia, PA, United States. 354. Bucks, M. E., & Savinov, S. N., (2010). Direct evaluation of cellular internalization rates using chromogenic disulfides. Molecular BioSystems, 6(7), 1176–1179. doi: 10.1039/ c003969a. 355. Theodoropoulos, D., Schwartz, I. L., & Walter, R., (1967). Synthesis of seleniumcontaining peptides. Biochemistry, 6(12), 3927–3932. doi: 10.1021/bi00864a039. 356. Schroll, A. L., Hondal, R. J., & Flemer, S. Jr., (2012). The use of 2,2’-dithiobis(5nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine-containing peptides. Journal of Peptide Science, 18(3), 155–162. doi: 10.1002/psc.1430. 357. Metanis, N., Keinan, E., & Dawson, P. E., (2006). Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency. Journal of the American Chemical Society, 128(51), 16684–16691. doi: 10.1021/ ja0661414. 358. Oikawa, T., Esaki, N., Tanaka, H., & Soda, K., (1991). Metalloselenonein, the selenium analog of metallothionein: Synthesis and characterization of its complex with copper ions. Proceedings of the National Academy of Sciences of the United States of America, 88(8), 3057–3059. doi: 10.1073/pnas.88.8.3057. 359. Casi, G., Roelfes, G., & Hilvert, D., (2008). Selenoglutaredoxin as a Glutathione peroxidase mimic. ChemBioChem, 9(10), 1623–1631. doi: 10.1002/cbic.200700745. 360. Flogel, O., Casi, G., Hilvert, D., & Seebach, D., (2007). Preparation of the β3-homoselenocysteine derivatives Fmoc-β3hSec(PMB)-OH and Boc-β3hSec(PMB)-OH for solution and solid-phase-peptide synthesis and selenoligation. Helvetica Chimica Acta, 90(9), 1651–1667. doi: 10.1002/hlca.200790171. 361. Gieselman, M. D., Xie, L., & Van, D. D. W. A., (2001). Synthesis of a selenocysteinecontaining peptide by native chemical ligation. Organic Letters, 3(9), 1331–1334. doi: 10.1021/ol015712o. 362. Koide, T., Itoh, H., Otaka, A., Yasui, H., Kuroda, M., Esaki, N., Soda, K., & Fujii, N., (1993). Synthetic study on selenocystine-containing peptides. Chemical & Pharmaceutical Bulletin, 41(3), 502–506. doi: 10.1248/cpb.41.502. 363. Tamura, T., Oikawa, T., Ohtaka, A., Fujii, N., Esaki, N., & Soda, K., (1993). Synthesis and characterization of the selenium analog of glutathione disulfide. Analytical Biochemistry, 208(1), 151–154. doi: 10.1006/abio.1993.1021. 364. Gokula, R. P., Patel, K., Maurya, S. K., & Singh, H. B., (2019). Facile synthesis of stable selenocystine peptides and their solution state NMR studies. Organic & Biomolecular Chemistry, 17(37), 8533–8536. doi: 10.1039/c9ob01910c. 365. Saito, K., Yasuo, I., Uchimura, H., Koide-Yoshida, S., Mizuguchi, T., & Kiso, Y., (2010). Quantitative proteolytic 18O-labeling analysis of protein disulfide bond rearrangement. In: Peptide Science (pp. 393, 394).

390

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

366. Ikemoto, H., Mizuta, K., & Mateus, V. M., (1985). Alkaline Cleavage of disulfide bonds in the black-eyed pea trypsin and chymotrypsin inhibitor. Anais da Academia Brasileira de Ciencias [Annals of the Brazilian Academy of Sciences], 57(1), 87–93. 367. Vetter, I., Dekan, Z., Knapp, O., Adams, D. J., Alewood, P. F., & Lewis, R. J., (2012). Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels. Biochemical Pharmacology, 84(4), 540–548. doi: 10.1016/j.bcp.2012.05.008. 368. Ridge, R. J., Matsueda, G. R., Haber, E., & Matsueda, R., (1982). Sulfur protection with the 3-nitro-2-pyridinesulfenyl group in solid-phase peptide synthesis. Synthesis of lysine8-vasopressin. International Journal of Peptide & Protein Research, 19(5), 490–498. doi: 10.1111/j.1399-3011.1982.tb02634.x. 369. Schroll, A., & Hondal, R., (2008). Advances in 1) the development of new deprotection chemistry for cysteine and selenocysteine side chain protecting groups and 2) the synthesis of a new selenocysteine derivative that have applications in peptide synthesis. In: Abstracts, 35th Northeast Regional Meeting of the American Chemical Society (p. NERM-301). Burlington, VT, United States. 370. Fujii, N., Otaka, A., Funakoshi, S., Bessho, K., & Yajima, H., (1987). new procedure for the synthesis of cystine peptides by oxidation of S-substituted cysteine peptides with thallium(III) trifluoroacetate. Journal of the Chemical Society, Chemical Communications, (3), 163, 164. doi: 10.1039/c39870000163. 371. Nishimura, O., Kitada, C., & Fujino, M., (1978). New method for removing the S-p-methoxybenzyl and S-tert-butyl groups of cysteine residues with mercuric trifluoroacetate. Chemical & Pharmaceutical Bulletin, 26(5), 1576–1585. doi: 10.1248/ cpb.26.1576. 372. Hunter, M. J., & Komives, E. A., (1995). Deprotection of S-acetamidomethyl cysteinecontaining peptides by silver trifluoromethanesulfonates avoids the oxidization of methionines. Analytical Biochemistry, 228(1), 173–177. doi: 10.1006/abio.1995.1333. 373. Yoshida, M., Akaji, K., Tatsumi, T., Iinuma, S., Fujiwara, Y., Kimura, T., & Kiso, Y., (1990). Synthesis of porcine brain natriuretic peptide-32 using silver tetrafluoroborate as a new deprotecting reagent of the S-trimethylacetamidomethyl group. Chemical & Pharmaceutical Bulletin, 38(1), 273–275. doi: 10.1248/cpb.38.273. 374. Flemer, S. Jr., (2011). Selenol protecting groups in organic chemistry: Special emphasis on selenocysteine se-protection in solid phase peptide synthesis. Molecules, 16, 3232–3251. doi: 10.3390/molecules16043232. 375. Heath, W. F., Tam, J. P., & Merrifield, R. B., (1986). Improved deprotection of cysteinecontaining peptides in hydrofluoric acid. International Journal of Peptide & Protein Research, 28(5), 498–507. doi: 10.1111/j.1399-3011.1986.tb03284.x. 376. Jespersen, A. M., Christensen, T., Klausen, N. K., Nielsen, P. F., & Soerensen, H. H., (1994). Characterization of a trisulfide derivative of biosynthetic human growth hormone produced in Escherichia coli. European Journal of Biochemistry, 219(1, 2), 365–373. doi: 10.1111/j.1432-1033.1994.tb19948.x.

Side Chain Protecting Groups 391

377. Andersson, C., Edlund, P. O., Gellerfors, P., Hansson, Y., Holmberg, E., Hult, C., Johansson, S., et al., (1996). Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli. International Journal of Peptide & Protein Research, 47(4), 311–321. doi: 10.1111/ j.1399-3011.1996.tb01360.x. 378. Li, W., Yang, B., Zhou, D., Xu, J., Ke, Z., & Suen, W. C., (2016). Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1025, 57–67. doi: 10.1016/j.jchromb.2016.05.004. 379. Kita, A., Ponniah, G., Nowak, C., & Liu, H., (2016). Characterization of cysteinylation and trisulfide bonds in a recombinant monoclonal antibody. Analytical Chemistry (Washington, DC, United States), 88(10), 5430–5437. doi: 10.1021/acs. analchem.6b00822. 380. Chocat, P., Esaki, N., Tanaka, H., & Soda, K., (1985). Synthesis of L-selenodjenkolate and its degradation with methionine γ-lyase. Analytical Biochemistry, 148(2), 485–489. doi: 10.1016/0003-2697(85)90256-8. 381. Daly, J. W., Mauger, A. B., Yonemitsu, O., Antonov, V. K., Takase, K., & Witkop, B., (1967). Synthesis and metabolism of 2,3-dihydro-L-tryptophan and 2,3-dihydro-5hydroxy-DL-tryptophan. Biochemistry, 6(3), 648–654. doi: 10.1021/bi00855a002. 382. Semenov, A. N., Lomonosova, I. V., Berezin, V. I., & Titov, M. I., (1993). Peroxidase and laccase as catalysts for removal of the phenylhydrazide protecting group under mild conditions. Biotechnology and Bioengineering, 42(10), 1137–1141. 383. Kikugawa, Y., (1978). Chemistry of amine-boranes. Part 3. Reduction of tryptophan derivatives with pyridine-borane. Journal of Chemical Research, Synopses, (5), 184, 185. 384. Fields, C. G., & Fields, G. B., (1993). Minimization of tryptophan alkylation following 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Tetrahedron Letters, 34(42), 6661–6664. doi: 10.1016/S0040-4039(00)61669-6. 385. Low, M., & Kisfaludy, L., (1979). Studies on the Nin-formyl group as possible protecting group against tryptophan indole side chain tert-butylation. Hoppe-Seyler’s Zeitschrift fur Physiologische Chemie [Hoppe-Seyler’s Journal of Physiological Chemistry], 360(1), 13–18. doi: 10.1515/bchm2.1979.360.1.13. 386. Jaeger, E., Thamm, P., Knof, S., Wuensch, E., Low, M., & Kisfaludy, L., (1978). Sidereactions in peptide synthesis, III: Synthesis and characterization of Nin-tert-butylated tryptophan derivatives. Hoppe-Seyler’s Zeitschrift fuer Physiologische Chemie [HoppeSeyler’s Journal of Physiological Chemistry], 359(12), 1617–1628. 387. Jaeger, E., Thamm, P., Knof, S., & Wuensch, E., (1978). Side-reactions in peptide synthesis, IV. Characterization of C- and C,N-tert-butylated tryptophan derivatives. Hoppe-Seyler’s Zeitschrift fuer Physiologische Chemie, 359(12), 1629–1636.

392

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

388. Low, M., Kisfaludy, L., & Sohar, P., (1978). Tertiary butylation of the tryptophan indole ring during the removal of the Tert-butoxycarbonyl group in peptide synthesis. HoppeSeyler’s Zeitschrift fuer Physiologische Chemie, 359(12), 1643–1651. 389. Low, M., Kisfaludy, L., Jaeger, E., Thamm, P., Knof, S., & Wuensch, E., (1978). Direct tertiary butylation of tryptophan. Preparation of 2,5,7-Tri-tert-butyltryptophan. HoppeSeyler’s Zeitschrift fuer Physiologische Chemie, 359(12), 1637–1642. 390. Rzeszotarska, B., & Masiukiewicz, E., (1990). Arginine, histidine and tryptophan in peptide synthesis. The indole function of tryptophan. Organic Preparations and Procedures International: The New Journal for Organic Synthesis, 22(6), 655–706. doi: 10.1080/00304949009457901. 391. Ogawa, H., Sasaki, T., Irie, H., & Yajima, H., (1978). Studies on peptides. LXXIX. By-products derived from Nα-protected tryptophan by acids. Chemical & Pharmaceutical Bulletin, 26(10), 3144–3149. 392. Tamamura, H., Otaka, A., Takada, W., Terakawa, Y., Yoshizawa, H., Masuda, M., Ibuka, T., et al., (1995). Solution-phase synthesis of an anti-human immunodeficiency virus peptide, T22([Tyr5,12, Lys7]-polyphemusin II), and the modification of Trp by the p-methoxybenzyl group of Cys during trimethylsilyl trifluoromethanesulfonate deprotection. Chemical & Pharmaceutical Bulletin, 43(1), 12–18. doi: 10.1248/ cpb.43.12. 393. Lebl, M., Pires, J., Poncar, P., & Pokorny, V., (1999). Evaluation of gaseous hydrogen fluoride as a convenient reagent for parallel cleavage from the solid support. Journal of Combinatorial Chemistry, 1(6), 474–479. doi: 10.1021/cc9900302. 394. Stierandova, A., Sepetov, N. F., Nikiforovich, G. V., & Lebl, M., (1994). Sequencedependent modification of Trp by the Pmc protecting group of Arg during TFA deprotection. International Journal of Peptide & Protein Research, 43(1), 31–38. doi: 10.1111/j.1399-3011.1994.tb00373.x. 395. Fontana, A., Marchiori, F., Rocchi, R., & Pajetta, P., (1966). Amino group protection by sulfenyl residues in peptide synthesis. I. Reaction of tryptophane with sulfenyl chlorides. Gazzetta Chimica Italiana, 96(10), 1301–1312. 396. Kiso, Y., Kimura, T., Shimokura, M., & Narukami, T., (1988). Nin-diphenylphosphinothioyltryptophan, a Useful derivative for peptide synthesis by the methanesulfonic acid-thioanisole system and fluoride ion deprotection methods. Journal of the Chemical Society, Chemical Communications, (4), 287–289. doi: 10.1039/ C39880000287. 397. Chorev, M., & Klausner, Y. S., (1976). Protection of tryptophan in peptide synthesis. The use of crown ethers. Journal of the Chemical Society, Chemical Communications, (15), 596. doi: 10.1039/c3976000596a. 398. Maia, H. L. S., Monteiro, L. S., & Sebastiao, J., (2001). Mild reductive cleavage of tryptophan and histidine side-chain protecting groups. European Journal of Organic Chemistry, (10), 1967–1970. doi: 10.1002/1099-0690(200105)2001:103.0.CO;2-X.

Side Chain Protecting Groups 393

399. Klausner, Y. S., & Chorev, M., (1977). Crown ethers as catalysts of fluorideanion-mediated reactions in peptide synthesis. Part 1. Protection of tryptophan by benzyloxycarbonyl and 2,4-dichlorobenzyloxycarbonyl groups. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972–1999), (6), 627–631. 400. Franzen, H., Grehn, L., & Ragnarsson, U., (1984). Synthesis, properties, and use of Nin-Boc-tryptophan derivatives. Journal of the Chemical Society, Chemical Communications, (24), 1699–1700. doi: 10.1039/C39840001699. 401. Kiso, Y., Inai, M., Kitagawa, K., & Akita, T., (1983). Protection of the tryptophan indole ring in peptide synthesis. Use of a new derivative, Nin-2,2,2-trichloroethoxycarbonyltryp tophan. Chemistry Letters, (5), 739–742. doi: 10.1246/cl.1983.739. 402. He, F., Foxman, B. M., & Snider, B. B., (1998). Total syntheses of (-)-asperlicin and (-)-asperlicin C. Journal of the American Chemical Society, 120(25), 6417, 6418. doi: 10.1021/ja9809408. 403. Anthoni, U., Christophersen, C., Obel, A., & Halfdan, N. P., (1994). Cyclization of tryptophans. III. The crystal structure of a product derived from trifluoroacetylation of Nβ-methoxycarbonyl-L-tryptophan methyl ester in pyridine. Acta Chemica Scandinavica, 48(4), 334–339. doi: 10.3891/acta.chem.scand.48-0334. 404. Nishiuchi, Y., Nishio, H., Inui, T., Bodi, J., & Kimura, T., (2001). Evaluation of new base-resistant side chain protecting groups for tryptophan and tyrosine during convergent synthesis with Boc chemistry. In: Epton, R., (ed.), Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids--Small Molecule Organic Chemistry Diversity, Collected Papers, International Symposium, 6th (pp. 331, 332). York, United Kingdom, York, United Kingdom. 405. Crich, D., & Huang, X., (1999). On the reaction of tryptophan derivatives with N-phenylselenyl phthalimide: The nature of the kinetic and thermodynamic hexahydropyrrolo[2,3-b]indole products. Alkylation of tryptophan with inversion of configuration. Journal of Organic Chemistry, 64(19), 7218–7223. doi: 10.1021/ jo991093+. 406. Fukuda, T., Wakimasu, M., Kobayashi, S., & Fujino, M., (1982). New protecting groups for the indole ring of tryptophan in peptide synthesis: 2,4,6-trimethoxybenzenesulfonyl and 4-methoxy-2,3,6-trimethylbenzenesulfonyl groups. Chemical & Pharmaceutical Bulletin, 30(8), 2825–2835. doi: 10.1248/cpb.30.2825. 407. Fujii, N., Futaki, S., Yasumura, K., & Yajima, H., (1984). Studies on peptides. CXXI. Nin-mesitylenesulfonyltryptophan, a new derivative for peptide synthesis. Chemical & Pharmaceutical Bulletin, 32(7), 2660–2665. doi: 10.1248/cpb.32.2660. 408. Yajima, H., Fujii, N., Akaji, K., Sakurai, M., Nomizu, M., Mizuta, K., Aono, M., et al., (1985). Synthesis of a 42 residue peptide corresponding to the entire amino acid sequence of human GIP. Chemical & Pharmaceutical Bulletin, 33(8), 3578–3581. doi: 10.1248/cpb.33.3578. 409. Abdel-Rahman, A. A. H., Abdel-Megied, A. E. S., Abdel-Bary, H. M., Abdel-Aleem, A. A. H., Morcy, E. M. I., & Shabaan, M. T., (2009). Amino acid derivatives, X [1]:

394

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

Synthesis and antimicrobial evaluation of α-amino acid esters bearing a N1-protected tryptophan side chain. Monatshefte für Chemie [Monthly Bulletins for Chemistry], 140(5), 559–564. doi: 10.1007/s00706-008-0089-z. 410. Nadvornik, M., Langer, V., Jirasko, R., Holcapek, M., Weidlich, T., Lycka, A., & Popkov, A., (2008). Syntheses, X-ray, MSn, NMR and CD structure determination of nickel(II) complexes of Schiff bases of (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine2-carboxamide and aromatic α-amino acids. Polyhedron, 27(17), 3477–3483. doi: 10.1016/j.poly.2008.08.009. 411. Meloen, R. H., Turkstra, J. A., Lankhof, H., Puijk, W. C., Schaaper, W. M. M., Dijkstra, G., Wensing, C. J. G., & Oonk, R. B., (1994). Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide. Vaccine, 12(8), 741–746. doi: 10.1016/0264-410X(94)90226-7. 412. Brown, E., Sheppard, R. C., & Williams, B. J., (1983). Peptide synthesis. Part 5. Solidphase synthesis of [15-leucine]little gastrin. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972–1999), (6), 1161–1167. 413. Izumiya, N., Waki, M., Kato, T., Ohno, M., Aoyagi, H., & Mitsuyasu, N., (1972). Solidphase synthesis of ribonuclease T1. In: Meienhofer, J., (ed.), Chem. Biol. Pept., Proc. Am. Pept. Symp., 3rd (pp. 269–279). 414. Johnson, T., & Sheppard, R. C., (1991). Resin effects in solid-phase peptide synthesis. Enhanced purity of tryptophan-containing peptides through two-step cleavage of side chain protecting groups and peptide-resin linkage. Journal of the Chemical Society, Chemical Communications, (22), 1653–1655. doi: 10.1039/C39910001653. 415. Chyan, C. L., Wormald, C., Dobson, C. M., Evans, P. A., & Baum, J., (1993). Structure and stability of the molten globule state of guinea Pig α-lactalbumin: A hydrogen exchange study. Biochemistry, 32(21), 5681–5691. doi: 10.1021/bi00072a025. 416. Previero, A., Coletti-Previero, M. A., & Cavadore, J. C., (1967). Reversible chemical modification of the tryptophan residue. Biochimica et Biophysica Acta, Protein Structure, 147(3), 453–461. doi: 10.1016/0005-2795(67)90005-0. 417. Lim, J. M., Kim, G., & Levine, R. L., (2019). Methionine in proteins: It’s not just for protein initiation anymore. Neurochemical Research, 44(1), 247–257. doi: 10.1007/ s11064-017-2460-0. 418. Jaune-Pons, E., & Vasseur, S., (2020). Role of amino acids in regulation of ROS balance in cancer. Archives of Biochemistry and Biophysics, 689, 108438/1–108438/8. doi: 10.1016/j.abb.2020.108438. 419. Wanders, D., Hobson, K., & Ji, X., (2020). Methionine restriction and cancer biology. Nutrients, 12(3), 684/1–684/14. doi: 10.3390/nu12030684. 420. Bodanszky, M., & Bednarek, M. A., (1982). Experiments on the Protection of the thioether in methionine. International Journal of Peptide & Protein Research, 20(5), 408–413. doi: 10.1111/j.1399-3011.1982.tb03060.x. 421. Lopez-Gonzalez, R., Gnecco, D., Juarez, J. R., Orea, M. L., Gomez-Calvario, V., Bernes, S., Aparicio, D. M., & Teran, J. L., (2020). Synthesis of (+)- and (-)-geissman-waiss

Side Chain Protecting Groups 395

lactone from chiral sulfonium salts. Tetrahedron Letters, 61(13), 151697/1–151697/4. doi: 10.1016/j.tetlet.2020.151697. 422. Capps, P. A., & Jones, A. R., (1974). Sulfonium salt formation from the reaction of methionine with aziridine alkylating agents. Journal of the Chemical Society, Chemical Communications, (9), 320, 321. doi: 10.1039/c39740000320. 423. Kramer, J. R., Petitdemange, R., Bataille, L., Bathany, K., Wirotius, A. L., Garbay, B., Deming, T. J., et al., (2015). Quantitative side-chain modifications of methioninecontaining elastin-like polypeptides as a versatile tool to tune their properties. ACS Macro Letters, 4(11), 1283–1286. doi: 10.1021/acsmacrolett.5b00651. 424. Kramer, J. R., & Deming, T. J., (2012). Preparation of multifunctional and multireactive polypeptides via methionine alkylation. Biomacromolecules, 13(6), 1719–1723. doi: 10.1021/bm300807b. 425. Yonemitsu, O., Hamada, T., & Kanaoka, Y., (1969). Esterification of peptides in aqueous solution. Tetrahedron Letters, (23), 1819–1820. doi: 10.1016/S0040-4039(01)88021-7. 426. Gross, E., (1967). The cyanogen bromide reaction. Methods in Enzymology, 11, 238–255. doi: 10.1016/S0076-6879(67)11029-X. 427. Jones, J. B., & Hysert, D. W., (1971). Alkylations of the side-chain nucleophiles of cysteine, methionine, histidine, and lysine derivatives with allyl bromide, 1-bromo-2butyne, and 2-bromoacetophenone. Canadian Journal of Chemistry, 49(18), 3012–3019. doi: 10.1139/v71-502. 428. Vithayathil, P. J., & Murthy, G. S., (1972). New reaction of O-benzoquinone at the thioether group of methionine. Nature (London), New Biology, 236(65), 101–103. doi: 10.1038/newbio236101b0. 429. Ruiz, M., Yang, Y., Lochbaum, C. A., Delafield, D. G., Pignatello, J. J., Li, L., & Pedersen, J. A., (2019). Peroxymonosulfate oxidizes amino acids in water without activation. Environmental Science & Technology, 53(18), 10845–10854. doi: 10.1021/ acs.est.9b01322. 430. Lazzaro, F., Crucianelli, M., De Angelis, F., Neri, V., & Saladino, R., (2004). A novel oxidative side-chain transformation of α-amino acids and peptides by methyltrioxorhenium/H2O2 system. Tetrahedron Letters, 45(50), 9237–9240. doi: 10.1016/j.tetlet.2004.10.074. 431. Makukhin, N., Nosek, V., & Misek, J., (2018). Development of a ratiometric fluorescent probe with two reactive sulfoxides for monitoring the activity of methionine sulfoxide reductase A. Synthesis, 50(4), 772–777. doi: 10.1055/s-0036-1591888. 432. Oien, D. B., & Moskovitz, J., (2008). Substrates of the methionine sulfoxide reductase system and their physiological relevance. Current Topics in Developmental Biology, 80, 93–133. doi: 10.1016/S0070-2153(07)80003-2. 433. Mozziconacci, O., Bhagavathy, G. V., Yamamoto, T., Wilson, G. S., Glass, R. S., & Schoneich, C., (2016). Neighboring amide participation in the Fenton oxidation of a sulfide to sulfoxide, vinyl sulfide and ketone relevant to oxidation of methionine thioether side chains in peptides. Tetrahedron, 72(48), 7770–7789. doi: 10.1016/j.tet.2016.08.075.

396

Amino Acids: Insights and Roles in Heterocyclic Chemistry, Volume 1

434. Jas, G. S., & Kuczera, K., (2002). Free-energy simulations of the oxidation of C-terminal methionines in calmodulin. Proteins, 48(2), 257–268. doi: 10.1002/prot.10133. 435. Joseph, K. T., Venkatasubramanian, K., & Kuehn, K., (1975). Effect of modification of methionine side chains on the interaction properties of collagen. Indian Journal of Biochemistry & Biophysics, 12(2), 158–162. 436. Mulinacci, F., Poirier, E., Capelle, M. A. H., Gurny, R., & Arvinte, T., (2013). Influence of methionine oxidation on the aggregation of recombinant human growth hormone. European Journal of Pharmaceutics and Biopharmaceutics, 85(1), 42–52. doi: 10.1016/j. ejpb.2013.03.015. 437. Mozziconacci, O., Ji, J. A., Wang, Y. J., & Schoneich, C., (2013). Metal-catalyzed oxidation of protein methionine residues in human parathyroid hormone (1–34): Formation of homocysteine and a novel methionine-dependent hydrolysis reaction. Molecular Pharmaceutics, 10(2), 739–755. doi: 10.1021/mp300563m. 438. Lee, H. J., Chweh, W., Kim, Y. D., Lee, S. S., & Kim, H. J., (2002). Synthesis of Met& Trp-containing peptides using Nα-Nsc-O-(Trt)-hydroxy amino acids; Nsc-Ser(trt), Thr(trt), Tyr(trt)-OH. In: Benedetti, E., & Pedone, C., (eds.), Peptides 2002, Proceedings of the European Peptide Symposium, 27th (pp. 184, 185). Sorrento, Italy. 439. Huang, H., & Rabenstein, D. L., (1999). A cleavage cocktail for methionine-containing peptides. The Journal of Peptide Research: Official Journal of the American Peptide Society, 53(5), 548–553. doi: 10.1034/j.1399-3011.1999.00059.x. 440. Fujii, N., Sasaki, T., Funakoshi, S., Irie, H., & Yajima, H., (1978). Studies on peptides. LXXIV. Convenient procedure for the preparation of methionine sulfoxide derivatives. Chemical & Pharmaceutical Bulletin, 26(2), 650–653. doi: 10.1248/cpb.26.650. 441. Okamoto, K., Yasumura, K., Shimamura, S., Nakanishi, S., Numa, S., Imura, H., Tanaka, A., et al., (1980). Synthesis of the dodecapeptide designated as bovine γ-melanotropin (γ-MSH). Chemical & Pharmaceutical Bulletin, 28(9), 2839–2843. doi: 10.1248/ cpb.28.2839. 442. Nicolas, E., Vilaseca, M., & Giralt, E., (1995). A study of the use of NH4I for the reduction of methionine sulfoxide in peptides containing cysteine and cystine. Tetrahedron, 51(19), 5701–5710. doi: 10.1016/0040-4020(95)00234-Y. 443. Tanikaga, R., Nakayama, K., Tanaka, K., & Kaji, A., (1977). Rapid reaction between sulfonium ion and sulfide. Preparative reduction of sulfoxide to sulfide. Chemistry Letters, (4), 395, 396. doi: 10.1246/cl.1977.395. 444. Babu, J. R., Brink, A. E., Konas, M., & Riffle, J. S., (1994). High performance polymer particles for composite matrixes: Poly(arylene ether-ether sulfide)s. Polymer, 35(23), 4949–4955. doi: 10.1016/0032-3861(94)90649-1.

Index

1 1-(1,2,3,4,5-pentamethylcyclopenta2,4-dien-1-yl)ethyl (c-Ppe), 285 1-(1-adamantyl)-1-methylethoxycarbonyl (ADPOC), 172, 173 fluoride (Adpoc-F), 172–174 oxiimino-2-phenylacetonitrile (AdpocON), 172 phenylcarbonate (Adpoc-OPh), 172, 175 1-(2-(dicyanomethylene)7-(dimethylamino)-2H- chromen-4-yl) ethyl, 333 1-(3,5-di-tert-butylphenyl)-1-methylethoxycarbonyl (t-Bumeoc), 166, 182–185 1-(4,4-dimethyl- 2,6-dioxocyclohexylidene) ethyl (Dde), 297, 300 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl (Nbde), 342 1,1-dimethyl-2-methacrylmethanamido ethoxycarbonyl, 199 ethyl-(4-nitrophenyl)-carbonate, 201 1,2,2,2-tetrachloroethyl tert-butyl carbonate, 171 1,2-dimethyl-1H-indole-3-sulfonyl chloride, 311 1,2-dimethylindole, 306, 311 3-sulfonyl (MIS), 306 1,3,5-trimethoxybenzene, 310 1,3-di-tert-butyl-5-methylbenzene, 182 1,4-cyclohexadiene, 104 1,4-diazabicyclo[2.2.2]-octane (DABCO), 205, 210 1,5-diazabicyclo(4.3.0) non-5-ene (DBN), 305 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 205, 211, 289, 290, 305, 329 15-crown-5, 133, 134 1-adamantanol, 176 1-adamantyloxycarbonyl (Adc), 351 1-adamantyloxymethyl (1-Adom), 314 1-aminotetralin, 324

1-benzyloxycarbonyl-benzotriazole, 237, 240, 241 1-diethylamino-1-propyne, 327 1-hydroxybenzotriazine, 144 1-hydroxybenzotriazole, 145, 185, 320 1-hydroxypiperidine, 237, 330 1-naphthylmethyl (1-Naph-Me), 321 1-tetralinyl (Ttyl), 321

2 2-(2,4-dinitrophenyl)ethyl (Dnpe), 341 2-(3,5-di-tert-butylphenyl)propan-2-yl carbonofluoridate, 184, 185 2-(4-acetyl-2-nitrophenyl)ethyl (Anpe), 59–61 2-(4-chlorophenyl)sulfonyl-ethoxycarbonyl (Cps), 203, 234 2-(4-nitrophenyl)sulfonylethoxycarbonyl (NSC), 232 2-(4-nitrophenylsulfonyl)ethyl, 72, 88 2-(4-nitrophenylthio)-ethoxycarbonyl (NTC), 232 L-serine methyl ester, 233 2-(4-trifluoromethyl-phenylsulfonyl)ethoxycarbonyl (Tsc), 203, 234 2-(bromomethyl)anthracene-9,10-dione, 290 2-(diphenylphosphaneyl)ethyl benzyloxy-carbonyl-L-alaninate, 83 tert-butoxy-carbonyl-glycinate, 83 2-(diphenylphosphino)ethanol, 82 2-(diphenylphosphino)ethyl (Dppe), 73, 82–84 2-(hydroxymethyl)benzo[b]thiophene 1,1-dioxide, 224 2-(methylsulfonyl)ethan-1-ol, 212 2-(methylsulfonyl)ethyl 4-nitrophenyl-carbonate, 213 chloroformate, 214 2-(tert-butylsulfonyl)-2-propenyl-oxycarbonyl (Bspoc), 203, 221, 222, 229 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl), 68

398

2-(trimethylsilyl)oxirane, 80 2,2,2-trichloroethoxycarbonyl (Troc), 351 2,2,4,6,7-pentamethyldihydrobenzofurane5-sulfonyl (Pbf), 306 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 305–307, 351 2,2,6,6-tetramethylpiperidine-1-oxyl4-amino-4-carboxylic acid (TOAC), 333 2,2-dinitrobenzhydryl (DNBzh), 110, 331, 333 2,2-dithiodipyridine, 345 2,2-dithiobis(5-nitropyridine) (DTNP), 345 2,2-bis(4-nitrophenyl) ethan-1-oxycarbonyl (Bnpeoc), 234 2,2-dimethoxypropane, 42, 45 2,2-dimethyl-4-thiazolidinecarboxylic acid, 338 2,3,4-trimethoxybenzyl (2,3,4-(MeO)3-Bn), 321 2,3-dihydroxypropyl, 341 2,4,5-trichlorophenyl, 119 2,4,6-triisopropylbenzenesulfonyl (Tip), 351 2,4,6-trimethoxybenzenesulfonyl (Mtb), 306, 307, 310, 351 2,4,6-trimethoxybenzyl (Tmob), 321, 341, 345 2,4-dimethoxybenzenesulfonyl (Dmb), 351 2,4-dimethoxybenzyl (Dmb), 321, 325, 327, 330 2,4-dinitrofluorobenzene, 317 2,4-dinitrophenethyl 4-methylbenzenesulfonate, 343 2,4-dinitrophenyl (Dnp), 298, 315, 318, 343, 351 2,4-dinitrophenylsulfenyl (DNPS), 351 2,5-diketopiperazine, 89 2,6-dichlorobenzyl, 235, 236, 282, 285 group, 282, 285 2,6-dichlorobenzyloxycarbonyl (2,6Cl2Bzl), 235 2,6-dimethoxybenzoyl, 314, 316, 317 2,6-dimethoxyphenylthio (S-Dmp), 342 2.2.2-trifluoro-1-(benzyloxycarbonylamino) ethyl (Z-TF), 283 2-[(2-methoxy)ethoxy]ethyl (MEE), 72, 77, 79 2-acetamido-2-deoxy-β-D- glucopyranosylamine, 117

Index

2-adamantanol, 187, 189 2-adamantyloxycarbonyl (2-Adoc), 167, 185–187 2-amino acetic diphenylborinic anhydride, 299 2-aminoethyl, 72, 222 2-aminoisobutyric acid (Aib), 101, 123 2-benzyloxypyridine, 103 2-bromo-2-methylpropane, 55 2-bromobenzyloxycarbonyl (2-BrZ), 235, 236 2-chlorethanol, 82 2-chloro-3-indenylmethyloxycarbonyl (CLIMOC), 203, 217, 218, 220 2-chloro-2-bromoethyl groups, 73 2-chlorobenzyloxycarbonyl (2-ClCbz), 296 2-diphenylphos-phinoethanol, 83 2-hydroxy-1-phenylethan-1-one, 348 2-iodobenzoic acid, 72 2-mercaptoethanol, 343, 346 2-mercaptopyridine, 260, 262, 343, 346 2-methoxy 4,6-dimethylbenzenesulfonyl (iMds), 306 5-nitrophenyl, 119, 123 9H-xanthen-9-yl (2-Moxan), 321 2-methoxyethoxymethyl (Mem), 290 2-methoxyethyl, 77 2-methoxyphenyl, 119, 121 2-methyl-2-aminopropanol, 140, 141 2-methylbutan-2-ol, 181 2-methylsulfonyl-3-phenyl-1-prop-2-enyloxycarbonyl (Mspoc), 203 2-morpholinoethyl (MoEt), 72, 82 2-nitro-4,5-dimethoxybenzyl (nitroveratryl), 330 2-nitrobenzyl, 330, 342 2-nitrobenzyloxycarbonyl (Nboc), 108, 342 2-nitrophenylsulfenyl (NPS), 66, 260–262, 351 2-nitroveratryloxycarbonyl (Nvoc), 108 2-oxo-2-phenylethoxycarbonyl, 296, 341 2-oxo-hisitidne, 357 2-phenyl-2-trimethylsilylethyl (PTMSE), 80, 81 2-propanol, 30, 34, 51, 112, 183, 184, 318 anhydrous diethyl ether, 112 2-pyridylethyl (Pet), 63–65 2-trimethylsilylethyl (TMSE), 73, 79, 80

Index 399

3 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), 144, 146 3,5-bis(docosyloxy)benzyl (tertbutoxycarbonyl)-L-leucinate, 172 3-bromocyclohex-1-ene, 285, 287 3-mercaptoacetic acid, 343, 346 3-methyl-3-(hydroxymethyl) oxetane, 129 3-methylpent-3-yl (Mpe), 330 3-nitro-2-pyridinesulfenyl (3-Npys), 342 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin1-yl) phosphonium hexafluorophosphate (PyNTP), 80 3-pentyl, 330 3-t-butyl-4-hydroxylphenyl, 119, 120

4 4-(10,19-bis(perfluorophenyl)-20H-porphyrin-5-yl)benzoyl (BPFPPB), 297 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)benzoyl (TMDBB), 297 4-(dimethylamino) pyridine (DMAP), 54 4-(hydroxymethyl)-N,N,N-trimethylbenzeneammonium chloride, 251 4,4-dimethoxytrityl, 167 4,5-dimethoxy-2-nitrobenzyloxycarbonyl (NVOC), 296 4,5,6-trimethoxy-2,2-dimethyl-2,3-dihydrobenzofuran-7-yl-methyl (Tmbm), 341 4-aminobenzyl group, 107 4-bromobenzyl, 169, 282, 314, 315, 330, 331 4-bromobenzyloxymethyl, 314, 315 4-chlorobenzyl, 282, 321 4-chlorobenzyloxycarbonyl, 321 4-dimethylaminopyridine, 59 4-dimethylcarbamoylbenzyl, 282 4-guanidinophenolate, 124 4-guanidinophenyl, 124 4-isopropyloxycarbonyloxybenzyloxycarbonyl (4-PriOCO), 203, 234 4-methoxy 1-naphthylmethyl (4-MeO-1-Naph-Me), 321 2,3,6-trimethyl-benzenesulfonyl (Mtr), 167, 199, 306, 307, 351 2,6-dimethylbenzenesulfonyl (Mds), 306, 307 4-methoxybenzenesulfonyl, 351

4-methoxybenzyl, 104, 106, 279, 282, 325, 330, 341, 342, 350, 354, 358 4-methoxybenzyloxycarbonyl-L-tryptophan, 350 4-methoxybenzyloxymethyl (MBom), 314, 341 4-methylbenzyl, 282, 341 4-methylsulfinylbenzyloxycarbonyl (Msz), 295 4-methylsulfonylphenyl ester, 120 4-methylthiophenyl group, 120 4-monomethoxytrityl chloride, 198 4-nitrobenzyl group, 107, 108, 110 4-nitrobenzyloxycarbonyl (Z(NO2)), 351 4-nitrophenyl chloroformate, 295 4-N,N-dimethylaminopyridine (DMAP), 59, 61, 62, 64, 69, 78–80, 82, 83, 90, 119, 123, 129, 138, 139, 337, 348 4-phenylbutyric acid, 138, 139 4-pyridylcarbinol, 249 4-sulfobenzyl, 105, 109

5 5,6-dihydrophenanthridine, 138, 139, 148 amide, 139 protecting group, 138

7 7-amino-heptanoic acid, 12

9 9-borabicyclononane (9-BBN), 299, 301–303 9-diazofluorene, 111 9-fluorenylmethoxycarbonyl (Fmoc), 68, 165, 205, 327, 347 1H-benzotriazole, 205 L-phenylalanine, 210 9-fluorenylmethyl azidoformate, 205–207 group, 58, 61 pentafluorophenyl carbonate, 120 9-fluorenylmethylchloroformate, 61 9H-fluoren-9-yl-methanesulfonyl group, 229 9H-fluoren-9-yl-methoxy-carbonyl (Fmoc), 46, 49, 68, 97, 100, 101, 107, 110, 119, 125, 129, 130, 132, 165, 166, 185, 204–211, 217, 218, 222, 229, 230, 232,

400

Index

234, 244, 260, 263, 264, 277, 281, 285, 292, 296–299, 304, 305, 313–317, 320, 323, 327, 329, 333, 338–340, 342, 347, 349, 357 group, 165, 166, 205, 206, 217, 229, 263, 264, 313, 329, 339 L-alanine ethyl ester, 49 L-ser-OBO ester, 129, 130, 132 9-phenylfluoren-9-yl (9-PhFl), 331 9-phenylxanthen-9-yl (Pixyl), 341 9-xanthenyl (Xan), 321, 342

A Acidic acyl bimolecular mechanism (AAC2), 328 Abaloparatide, 305 Acetal, 65, 127, 290 Acetamidomethyl, 338, 341 Acetic anhydride, 140, 141, 259, 293, 323, 336 Acetol protecting group, 90 Acetonitrile, 35, 38, 65, 74, 76, 84, 103, 138, 139, 196, 213, 214, 216, 217, 227, 289 Acetyl bromide, 67 chloride, 43, 102, 293 Acetyloxymethyl, 65, 67 bromide, 67 Achatina fulica, 22 Acid catalyst, 41, 42, 56, 280 halide, 41 labile amino protecting groups, 166 protecting group, 264 Acidic basic functionality, 355 catalyst, 290 Acidity constant, 2, 278 Actinomycin, 282 Acyl carrier protein (ACP), 331, 333 rearrangement, 292 Acylation, 22, 104, 119, 135, 144, 214, 215, 260, 293, 349 Adamantyl groups, 345 oxycarbonyl (Adoc), 166, 175–179, 185–189, 305–307, 351

Affinity matching, 20 Aldol reaction, 288 Aliphatic moiety, 29 Alkyl protecting groups, 42, 52 2-(4-acetyl-2-nitrophenyl)ethyl (ANPE) protecting group, 59 nα-boc-l-aspartic acid β-ANPE esters boc-asp(x)-oh, 59 2-pyridylethyl (PET) protecting group, 63 preparation of amino acid 2-pyridylethyl ester, 64 removal of 2-pyridylethyl group, 64 adamantyl protecting group, 56 amino acid adamantyl ester, 57 ethyl protecting group, 47 preparation of amino acid ethyl esters, 47 removal of ethyl esters, 50 fluorenylmethyl (FM) protecting group, 61 preparation of amino acid fluorenylmethyl ester, 61 removal (9-fluorenylmethyl ester protecting group), 63 isopropyl protecting group, 51 preparation of amino acid isopropyl esters, 51 methyl protecting group, 42 preparation of amino acid methyl esters, 42 removal of methyl esters, 46 n-heptyl protecting group, 52 tert-butyl protecting group, 52 preparation of amino acid t-butyl esters, 53 removal of t-butyl group, 55 α-phenylethyl protecting group, 58 removal of α-phenylethyl protecting group, 58 synthesis of n-phthaloyl-dl-phenylalanine α-phenylethyl ester, 58 Alkylation, 65, 104, 127, 128, 349–351, 355–357 Alkyldithiocarbonyl chloride, 202, 203 Allium sativum, 341 Alloxymethyl, 314 Allyl bromide, 98, 99, 356, 358 oxycarbonyl (Alloc), 296 protecting group, 97, 101, 148 Aluminum crimp cap, 49

Index 401

Amide, 38, 41, 135, 138, 139, 179, 230, 297, 299, 321, 330, 331, 333, 340, 341 Amine, 1, 10, 11, 61, 63, 80, 89, 104, 135, 185, 205, 222, 224, 230, 234, 253, 254, 263, 288, 299, 325, 331, 323 Amino acid, 1, 11–14, 16–22, 29, 30, 34–39, 42, 44, 45, 47–49, 51–59, 61–74, 77, 79–86, 88–90, 93, 95–97, 101–111, 118–121, 123, 124, 127, 128, 133–135, 140, 142–144, 165–168, 172–177, 179–182, 185, 189, 191, 193, 194, 197, 199, 200, 202, 203, 205, 206, 212, 214, 218, 220–222, 227–229, 232, 234–237, 244, 246, 251, 253–255, 260, 261, 277–280, 293, 296, 299, 303, 306, 311–313, 320, 321, 328–330, 332, 334, 337, 340, 349, 351, 352, 355, 356 ester, 59, 72, 82, 89, 107, 109, 110, 119, 120, 144, 180, 181, 193, 251, 255, 320 ethyl esters, 47 methyl ester, 42, 44, 45, 47, 88 N-carboxyanhydrides (NCAs), 260 phenacyl esters, 89 porphyrin-methyl ester, 111 trimethylsilylethyl esters, 79 propargyl esters, 101 protecting groups (lysine), 296 decomposition (9-bbn-l-lysine complex), 302 nε-benzyloxycarbonyl-l-lysine via 9-bbn complex, 301 nε-benzyloxycarbonyl-l-lysine via copper complex, 300 nε-fmoc-l-lysine, 304 protecting lysine side chain, 300 Ammonia, 43, 74, 75, 205, 210, 237, 243, 244 Ammonium formate, 236 Amoxicillin, 22 Ampicillin, 22 Angiotensin II, 285, 305 Anhydride, 38, 41, 135, 236, 293, 305, 334, 336, 358 Anhydrous HCl gas, 54 Anisoin, 246, 247 Anisole, 56, 57, 108, 200, 323, 343, 346, 350, 354

Antibiotics, 22 Antioxidant activity, 355 Aqueous acidic reaction medium, 356 carboxylic acid, 2 organic mixed solutions, 30 sodium hydrogen carbonate solution, 49, 139 Arachidonic acid, 6, 7 Arginine, 19, 20, 48, 50, 189, 278, 299, 305–312, 334, 340, 351, 355, 359 Aromatic sulfonyl groups, 307 Aryl groups, 41, 42, 119 protecting group, 148 Arylation, 104 Arylsulfonyl groups, 351 protecting groups, 351 Asparagine, 134, 179, 191–193, 278, 320–323, 326, 327, 350, 359 peptides, 321 Aspartic acid, 19, 59–61, 104, 109, 110, 140–142, 278, 282, 299, 323, 328–335, 360 Aspartimide, 328–331, 333 Aspergillus niger, 52 Aspirin-arginine ethyl ester hydrochloride, 50 Azeotropic distillation, 106, 140, 175 Azidoformate, 167, 180, 218 Azidomethoxybenzyl, 105

B Bacillus, 22, 47 brevis, 22 subtilis (BS2), 22, 47, 97, 105 Backbone amide linker (BAL), 97 Barnacle model protein, 282 Base catalyzed deprotection, 144 side reactions, 222 labile protecting group, 264 Benz[f]inden 3-methanol, 219 3-ylmethyl azidoformate, 220, 221 chloroformate, 219, 220

402

ylmethyloxycarbonyl (BIMOC), 203, 204, 217, 218, 220 Benzamidomethyl (Bam), 341 Benzenesulfonic acid, 112 Benzhydrol, 347 Benzo[b]thiophene, 223 Benzoin, 109, 110 Benzothiophenesulfone 2-methanol, 223 2-methoxycarbonyl (Bsmoc), 204, 221, 222, 224, 227, 228 2-methyl chloroformate, 224, 227 N-succinimidyl carbonate, 228 oxycarbonyl, 221 Benzotriazole, 207, 208, 237–239 Benzoyl chloride, 293, 294 groups, 352 Benzyl (Bn), 41, 42, 55, 66, 90, 92, 96, 97, 103–107, 109–112, 165, 167, 172–176, 178, 179, 181, 200, 236–239, 277, 279, 282–285, 291, 292, 297, 299–302, 307, 315, 321, 323–326, 330–334, 336, 337, 340–343, 345, 350, 351, 353, 354, 357 1-chloro-2,2,2-trifluoroethyl-carbamate, 283, 284 alcohol, 111, 112, 236, 238, 279, 336 bromide, 282, 283 chloroformate, 236–239, 300, 301 group, 96, 97, 104–107, 109–111, 167, 173, 277, 282, 283, 299, 321, 330, 331, 333, 334, 340, 341, 343, 345, 351 protecting group, 105, 148, 282, 290 triethylammonium chloride, 55, 103 trimethylammonium hydroxide, 174, 175 carboxyl protecting groups, 105 Benzylation, 103, 104, 350 Benzyloxycarbonyl, 54, 74, 75, 79, 87, 92, 94, 104, 106, 108, 113, 114, 117, 131, 140–143, 167, 200, 234–241, 243, 244, 248, 255, 256, 279–283, 285, 290, 292, 296, 301, 302, 305, 310–312, 314, 326, 327, 335–337, 350, 351, 357 dipeptide, 237 group, 167, 200, 236, 237, 244, 248, 285

Index

Benzyloxymethyl (BOM), 65, 72, 314, 315 chloride, 72 Benzyltriethylammonium tetrathiomolybdate, 101–103 Biochemical connection, 20 Biometal corrole active ester, 297 Biosynthetic human growth hormone, 346 Biotherapeutic drugs, 357 Bis(ethoxythiocarbonyl)-sulfide, 255 Bis(p-nitrophenyl) carbonate, 249 Bis(tributyltin) oxide (BBTO), 90, 95 Bis(trichloromethyl) carbonate, 187 Boron tris(trifluoroacetate) (BTFA), 323 Boroxazolidone, 140, 334 Bremelanotide, 305 Bromine, 120, 127 Bromoacetyl bromide, 133 Bromomethyl acetate, 67 Buffering capacity, 7 system, 7, 11 Butoxycarbonyl (Boc), 59–64, 66, 68, 73, 76, 90, 92, 96–101, 108, 112, 119, 123, 124, 135, 145, 146, 165–169, 171–173, 179, 182, 197, 199, 251, 263, 277, 279, 281, 282, 284, 287, 295–297, 299, 305, 307, 314, 315, 317, 322, 330, 333, 343, 350–352, 356 Butyraldehyde, 2 Butyric acid, 2

C Calmodulin, 357 Candida antarctica, 47 Canonical amino acids, 13, 14 genetic code, 20 Carbocations, 104, 167 Carbon carbon double bonds, 7 tetrachloride, 112 Carbonyl carbon, 1, 2 chloride fluoride, 173, 184 derivatives, 144 disulfide molecules, 254 functionality, 140 group, 1, 2, 5, 8, 10–12, 17–19, 29, 38, 39, 41, 42, 45, 59, 61, 68, 72, 89, 96,

Index 403

97, 118–120, 134, 135, 138–140, 143, 144, 165, 193, 253, 277, 278, 291, 299, 313, 320, 321, 323, 328, 330, 331, 333, 334, 338 hydrazide, 331 protecting groups, 41, 65, 66, 77, 103, 108, 134, 135, 144, 203, 278 side chain, 278, 333 Carboxylate, 2, 5–7, 11, 12, 19, 38, 41, 46, 88, 92, 207, 211, 239, 357 Carboxylic acid, 1, 2, 5–7, 10, 11, 41, 46, 56, 72, 80, 92, 104, 106, 110, 127, 135, 136, 138, 140, 170, 242, 243 Carboxylmethyl, 352 Catalytic hydrogenation, 70, 97, 103, 106, 109, 167, 189, 193, 205, 237, 244, 248, 282, 284, 307 condition, 70 hydrogenolysis, 110, 140, 205, 212, 315, 353 Cation exchange column, 132, 133 Celite, 290 Cephadroxil, 22 Cephalexin, 22 Ceric ammonium nitrate (CAN), 120, 138, 139 Cetyltrimethylammonium chloride, 354 Chemical transformations, 41 Chlolecystokinin-33 (CCK-33), 293 Chlorocarbonylsulfenyl chloride, 202, 253, 256, 257 Chloroethanol, 74 Chloroform, 7, 37, 44, 50, 66, 85, 94, 95, 100, 103, 106, 113, 115, 125, 133, 134, 136, 194, 201, 223, 242, 292, 319, 359 Chloroformate, 119, 125, 167, 168, 175–177, 188, 189, 191, 205–207, 218–220, 226, 229, 236–239, 246, 247, 296 Chloromethyl mesitylene, 113 methyl sulfide, 65, 66 Chlorosulfonic acid, 310 Chromatographically, 48, 63, 64, 68, 69, 72, 79, 99, 100, 112, 129, 178, 179, 189, 210, 212, 250, 257, 262, 286, 290, 352, 358 homogeneous, 202, 203 Chromium trioxide, 135

Chymotrypsin, 50, 342 Cis-configurations, 7, 8 Cis-double bond, 7, 8 Code coevolution, 20 Collagen, 357 Column chromatography, 39, 44, 54, 55, 60, 62, 63, 69, 81, 91, 93, 98, 102, 103, 109, 123, 124, 126, 130, 178, 209, 232, 241, 258, 286, 287, 290, 292, 310, 311, 353 Common α-amino acids, 13 amphiprotic properties (α-amino acids), 19 common α-amino acids, 13 D-α-amino acids, 22 essential amino acids-nonessential amino acids, 20 stereochemistry (α-amino acids), 17 unnatural amino acids, 20 Concentrated hydrochloric acid, 48, 296 sulfuric acid, 43, 48, 244, 281, 308 Conjugated cis-double bonds, 8 trans-double bonds, 8 Crystalline ester hydrochloride, 113 Crystallization, 30, 34, 39, 45, 55, 67, 113, 129, 170, 176, 198, 202, 250 agents, 30 properties, 212 C-terminal cysteine, 338 deprotection, 52 Cyanomethyl, 72, 87, 88 Cyclic diketopiperazines, 180 dimerization, 89 Cyclododecyl (c-Ddc), 330 Cycloheptyl (c-Hp), 330 alcohol, 337 Cyclohexane, 2, 104, 142, 285, 286 Cyclohexyl alcohol, 335 aspartate, 285 group, 285, 286, 333, 334 mercaptan, 202 Cyclohexylamine, 188, 283, 287, 288 Cyclohexyldithiocarbonyl, 167, 200, 202, 203

404

Index

Cyclopentyl (c-Pt), 330 Cyclopropyldimethylcarbinyl (Cpd), 321 Cysteine, 19, 104, 170, 193, 208, 211, 235, 237, 278, 282, 299, 337–347, 349, 350, 356, 360 hydrochloride, 343, 347, 349 racemization, 338 Cystine, 337, 338, 340 moiety, 338

D Dean-stark adapter, 134 Decomposition rates, 236 Defense mechanisms, 22 Dehydration, 140, 246, 278, 279, 320 Dehydroalanine-containing peptides, 279 Denaturation, 36 Deprotection methods, 90 Deprotonation, 41, 313, 339 Desiccator, 102, 103, 115, 145, 240, 309 Desmopressin, 305 Diacylhydrazides, 135 Diazomethane, 42 Diazotization, 135 Dibenzofulvene, 205, 206, 208, 210, 334, 339 Dibenzyl dicarbonate, 237, 238, 240 pyrocarbonate, 237 Dichloromethane, 46, 56, 84, 96, 97, 100, 109, 110, 117, 126, 138, 145, 146, 200, 223, 290, 322 Dicyclohexyl amine, 69, 70, 219, 224, 226, 261, 284 ammonium (DCHA), 79, 109, 174, 175, 260, 284, 334 carbodiimide (DCC), 53, 54, 59–62, 64, 73, 74, 77–80, 82, 83, 90, 106, 119, 121, 122, 127, 129, 144–146, 320, 324, 326, 337 urea (DCU), 54, 60, 62, 64, 74, 78, 83, 91, 119–122, 129, 146, 324 Diethyl azodicarboxylate, 110 ether, 49, 76, 94, 95, 102, 112, 121, 175, 183, 188, 198, 207, 209, 211, 287, 288, 302, 311, 317 Diethylamine, 63–65, 70, 71, 195–197

Diethyleneglycol-monomethylether (DEM), 77 Diethylisopropylamine, 325 Dihydroxyacetone, 289 Diisopropylcarbodiimide, 62, 264 Diisopropylethylamine, 63, 66, 67, 99, 186, 227, 231, 263, 316, 317, 319, 334, 343 Diketopiperazine, 52 Dimethoxyethane, 349 Dimethyl sulfide (DMS), 104 sulfite, 57 sulfoxide, 6, 65 Dimethylaminopyridinium, 334 Dimethylformamide, 6, 37, 107, 174 Dimethylphosphinothioyl (Mpt), 293 Dimethylphosphinyl (Dmp), 293 Dimethylsulfoxide (DMSO), 6, 37, 65–67, 69, 70, 97, 111, 117, 169, 216, 254, 293, 299, 343, 348 Di-n-butoxyphosphinyl, 167 Dioxane, 35, 54, 70, 71, 86, 89, 92, 93, 98, 107, 108, 113, 143, 168, 172, 175, 177, 181, 185, 186, 188–191, 202, 205, 207, 209, 212, 216, 220, 232, 240, 241, 245, 247, 258, 260, 261, 284, 304, 317, 350 methanol mixture, 216 Diphenylmethyl, 110, 313 diazomethane, 110 Diphenylphosphine, 83 D-isobornyloxycarbonyl-L-aspargine, 191 Disulfide, 200, 254, 255, 258, 337, 338, 342, 343, 345, 346, 357 bonds, 338, 342 linkage, 337 Di-tert-butylmethylsilyl (DTBMS), 144 Dithiothreitol (DTT), 346 D-phenylglycine, 43 Dudley reagent, 104, 105

E E1cb elimination, 339 Electron donating group, 6, 105, 106, 323 spin resonance (ESR), 333 withdrawing acyl group, 89

Index 405

carbonyl group, 2 group, 5, 12, 105, 106, 108, 109, 221, 313, 330, 331, 351 Electronegativity, 2 Electrophilic center, 119 Enantiomeric, 18, 22 (-)-glyceraldehyde, 18 Enolate, 127 Enterobactin, 290 Environmental pH values, 356 Enzyme cleavable, 166 Escherichia coli, 346 Essential amino acid, 20 Ester, 38, 41–44, 46–52, 55–59, 61–71, 74–76, 79–86, 88–97, 99, 101, 103–114, 118, 119, 126–128, 130, 131, 134, 139, 144, 147, 165, 178–182, 186, 187, 189, 190, 193, 194, 196, 198, 200, 201, 210, 216, 217, 230, 243, 252, 260, 264, 280, 282, 292, 299, 317, 319, 320, 329, 334–336, 356 Esterase, 47, 97, 105 Ethane-1,2-dithiol (EDT), 345 Ethanolamine, 205, 302 Ethoxycarbonyl (Etoc), 88, 233, 234, 351 Ethyl, 37, 41, 42, 45–52, 65, 72, 77, 79–88, 92, 94, 98, 102, 103, 114, 115, 117, 121, 123, 124, 126, 130, 131, 133–136, 148, 169, 171, 177, 178, 190, 198, 201, 211, 213, 214, 227, 231, 245, 246, 249, 253, 256, 261, 262, 283, 284, 290, 292, 317, 320, 321, 325, 327, 330, 331, 333, 341, 343, 353, 356, 357 acetate, 37, 92, 94, 102, 103, 115, 117, 121, 123, 124, 126, 131, 169, 171, 177, 178, 190, 227, 231, 245, 246, 249, 261, 262, 283, 284, 290, 292, 317, 325, 327, 333 acetoacetate, 133, 134 chloroformate, 135, 136 glycinate, 52 protecting group, 80, 81, 148, 283 Ethylcarbamoyl, 342, 346 Ethylenediamine, 263, 299, 300, 303 EtOAc-petroleum ether, 56, 76, 185, 240 Exothermic, 48, 53, 130, 213, 303 Extra-functional groups, 7

F Fischer esterification, 41 projection, 17, 18, 22 Fluorenylmethoxycarbonyl (Fmoc), 204, 205 Fluorenylmethyl, 61–63, 93, 125, 205, 206, 331, 334, 343, 346 Formaldehyde, 87, 349 Formic acid, 2, 104, 173, 282, 309, 353, 359 Formyl, 84, 106, 351, 352 group, 351, 352 Fractionation, 336 Frameshift suppression approach, 21 Free carboxylic acid, 2, 5, 7 Functional groups, 1, 19, 29, 38, 39, 41, 61, 103, 105, 194, 277–279, 328, 331 Furfuryl alcohol, 200 oxycarbonyl (Foc), 167, 199, 200

G Gabriel primary amine synthesis, 263 Gamma-aminobutyric acid (GABA), 1 Genetic code, 13, 20, 21 Global protection strategy, 278, 293 Glutamic acid, 17, 19, 22, 45, 53, 104, 109, 140, 143, 246, 278, 282, 323, 328, 330–336, 360 Glutamide, 328 Glutamine, 179, 278, 320–323, 350, 360 Glutathione peroxidase, 340 Glyceraldehyde, 17, 18, 289 Glycine, 12, 15, 17, 29–31, 34–37, 39, 53, 62, 64, 67, 78, 85, 88, 90, 98, 99, 106, 121, 136, 174, 175, 181, 182, 192–194, 196–199, 207, 208, 212, 217, 218, 245, 252, 255, 256, 258, 277, 299, 329 Glycylglycine, 90, 253 Grignard reaction, 138 reagent, 127, 139, 140 Guaiacol, 121

H Henderson-Hasselbalch equation, 2, 19 Heteroaromatic pyrrole amino acid, 234 Heterocyclic functionalities, 139

406

Index

organic compound, 140 Histidine, 11, 19, 20, 185, 188, 278, 298, 312–315, 317–320, 356, 357, 359 Homogeneous reaction, 99 Homoserine, 356 Hydrazides, 135, 282 Hydrazine, 70, 71, 84, 107, 116, 135, 136, 166, 234, 263, 264, 282, 300, 334 hydrate, 70, 71, 84, 136 labile amino protecting group, 263 preparation of c(DAP-TCP-GLN), 263 protecting group, 134, 137 Hydrocarbon chain, 2, 6, 8, 11, 12, 18, 34 Hydrochloric acid, 48, 49, 84, 193, 217, 239, 336 Hydrogen chloride, 42, 43, 109, 113, 193, 200, 236, 262, 294, 308 sulfide, 215, 309 Hydrogenation, 97, 104, 106, 109, 167, 185, 236, 237, 242, 281, 285, 333, 334, 350 Hydrogenolysis, 80, 89, 108, 111, 166, 167, 172, 182, 199, 234–236, 244, 246, 264, 277, 281–283 Hydrolysis, 1, 88, 105, 106, 119, 128, 140, 144, 193, 237, 280, 282, 328, 331 Hydrophilic, 212 amino acids, 34, 35, 175 Hydrophobic, 30, 165, 332 side chains, 35 Hydroxide (OH–), 2, 7, 73, 75, 102, 127, 145, 186, 216, 246, 247, 250, 307, 308 Hydroxy amine (NH2OH), 348 ethyldiphenyl-methyl-phosphonium iodide, 84 methyl group, 18 porphyrin, 111 oxazolidinone, 246 proline, 89, 280, 291, 293 protecting group, 359

I Imidazole functional group, 312 ring, 312–314 Indispensable amino acid, 20

Inductive effect, 5, 6, 11, 12 Insulin, 212, 341 Internal selenocysteine insertion, 14 International Union of Pure Applied Chemistry (IUPAC), 1 Intramolecular disulfide linkage, 345, 346 Inverse substrate, 120 Iodothyronine deiodinase, 340 Ion-exchange resin, 42, 215 Ionophore, 290 Isoborneol, 189, 190 Isobornyl (i-Bor), 285 Isobornyloxycarbonyl (Iboc), 167, 189, 190, 192 Isobutene, 166, 199, 280, 350 Isobutylene, 53, 54, 233, 281 Isoelectric point (pI), 19, 20, 36, 37, 39 solutions, 37 Isoleucine 2-chloroethyl ester, 74 adamantyl ester, 57 Isonicotinyl oxycarbonyl, 235, 248, 250 p-nitrophenyl carbonate, 249, 250 Isopropyl, 51, 285 dimethylchlorosilane, 289 phenylglycinate hydrochloride, 51 Isopropylideneaminooxycarbonyl (Paoc), 235 Isotocin, 341

J Jadomycin S, 288

K Ketal, 127, 290

L Lactam, 89, 305 Lactobacillus arabinosus, 22 L-arginine hydrochloride, 308, 309 Laser pulse irradiation, 110 Lauric acid, 6, 7 Leucyl-alanyl-valine, 57 Leuprolide, 305 Linaclotide, 338 Linoleic acid, 2, 8

Index 407

Lipase, 47, 52, 77, 79, 81, 105 Lipophilicity, 172 L-isoluecylglycine, 262 L-leucine, 75, 83, 96, 181, 193, 203, 210, 259–261, 279 Long-chain aliphatic acids, 1 Lower critical solubility temperature (LCST), 355 L-phenylalanine methyl ester hydrochloride, 42, 43, 201 Lyophilization, 87, 197, 345 Lysine, 19, 20, 45, 166, 173, 199, 212, 215, 235, 248, 250, 251, 278, 282, 296–305, 312, 334, 340, 355, 356, 359

M Magnesium, 82, 90, 96, 124, 171, 183, 201, 258, 283, 286, 352 sulfate, 82, 124, 171, 201, 258, 283, 286 M-amidinomethylphenyl, 119 Mannich reaction, 288 M-chloroperbenzoic acid, 109 Menthyl, 330 Mercuric chloride, 138 Merrifield synthesis, 284 Mesotocin, 340 Methanol, 6, 7, 29, 30, 34, 36, 37, 42–45, 51, 58, 64, 71, 78, 83, 84, 100, 103, 104, 106, 110, 115–118, 121, 127, 134, 143, 145, 174, 175, 194, 195, 209, 211, 212, 216–218, 221, 223, 225, 228, 230, 232, 236, 242, 252, 282, 290, 299, 300,301, 303, 346, 348, 352 tetrahydrofuran, 118 Methionine, 14, 20, 22, 46, 104, 235, 243, 277, 278, 320, 355–360 click, 355 containing peptides, 357 polymers, 355 sulfoxide, 355–357 reductase A (MsrA), 356 Methionyl peptide bond, 356 Methoxy carbonyl (Moc), 205, 291, 339, 342, 351 dodecaethylene glycol, 77 dioleoyl-L-serinate, 77 ethoxymethyl, 66, 291

Methyl 4-methoxybenzyloxy-carbonyl-L-seryl-Lleucinate, 279 benzyloxycarbonyl-L-tryptophanate, 350 ester, 42 group, 30, 41, 42, 47, 65, 77, 88, 89, 106, 108, 109, 290, 341 N-benzyloxycarbonyl-L-serinate, 284 O-(tert-butyl)-N-((2-((4-nitrophenyl)thio) ethoxy) carbonyl)-L-serinate, 233 p-toluenesulfonate, 42 protecting group, 72, 87, 148 tert-butoxycarbonyl-L-tryptophylglycinate, 350 tert-butyl ether (MTBE), 245, 326 Methylene chloride, 37, 44, 82, 123, 141, 209, 249, 279 dichloride, 77, 218 group, 8, 89, 96 Methylsulfonyl ethylene, 216 group, 278 Methylsulfonylethyl oxycarbonyl azide, 214 p-nitrophenyl carbonate, 214 succinimido carbonate, 213, 214 Methylsulfonylethyloxycarbonyl (MSC), 204, 211–217, 222, 234, 306, 307 chloride (Msc-Cl), 212, 213 Methylthiomethyl (MTM), 65, 66 Methyltrityl (Mtt), 194, 297, 321 Michael acceptors, 221 addition, 221, 222, 288 Minimal protection strategy, 278 Mitsunobu condensation, 110 Mixed solvent system, 6, 30, 34, 35 Molar solubility, 30, 39 Molecular weight cut-off (MWCO), 359 Monoacylhydrazides, 135 Monohydrochloride, 215, 217 Monomethoxytrityl (MMT), 167, 194, 198, 341 Morpholine, 80–82, 205, 222, 330, 339

N N-(benz[f]inden- 3-ylmethylo-xycarbonyl) phenylalanine, 221

408

N-(benzothiophenesulfone-2-methyl)-Nsuccinimidyl carbonate, 224 N-(benzyloxycarbonyl)lysine, 104 N-(cyclohexyldithio)carbonyl-amino acid derivatives, 255 N-(tert-butyloxycarbonyl)-amino acid, 120 N,N-diisopropyl-hydrazine, 135, 137 N,N-dimethylacetamide (DMA), 55, 243 N,N-dimethylaniline, 42, 46, 66, 246, 247 N,N-dimethylformamide (DMF), 37, 61, 63–65, 67–70, 72, 73, 76, 78, 80, 88, 90, 92–96, 98, 100–102, 107, 113, 116, 117, 119, 122, 123, 126, 128, 131, 134, 145, 166, 174, 175, 193, 197, 198, 205, 206, 211, 218, 232, 236, 244, 247, 254, 263, 264, 279, 282, 283, 285, 287, 289, 295, 299, 300, 311, 315, 316, 319, 326, 329, 334, 343, 346, 349, 352, 353, 359 N-[(9-hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS), 330 N-acetyl-S-allyl-DL-methionine sulfonium bromide, 359 N-acyl peptide derivatives, 283 Narrow miscibility gap, 34 Native chemical ligation (NCL), 107 N-benzoyl-L-phenylalanine, 137 N-benzyloxycarbonylglycine, 85, 86 N-benzyloxycarbonyl L-alanine, 74, 94, 113, 241 2-bromoethyl ester, 74 L-phenylalanyl-L-leucine, 74–76 2-iodoethyl ester, 75 L-proline, 117 Dpm ester, 117 L-serine, 289, 290 oxy-5-norbornene-2,3-dicarboximide, 237 N-Boc glycylglycyl 2-chloroethyl ester, 76 leucine 9-fluorenylmethyl ester, 63 O-benzyl-serine, 282 threonine, 282 N-butyldithiocarbonyl-alanine, 202 N-carboxyanhydrides (NCAs), 260 N-chloromethylphthalimide, 70 N-dithiasuccinoyl (DTS), 253, 255, 256, 258 N-ethylmorpholine, 216 Neutral carboxylic acid, 6, 7

Index

Neutralization, 53, 293 N-Fmoc-methionine, 46 N-formyl-L-histidinamide, 313 N-heterocyclic carbene (NHC), 315 N-hydroxybenzotriazole (HOBt), 64, 114, 123, 185, 326, 330 N-hydroxysuccinimide, 213, 216, 219, 224, 226, 237, 324, 339 N-Iboc-D-p-chlorophenylalanine, 190 Ninhydrin-positive spots, 175 N-Isobornyloxycarbonyl-D-p-chlorophenylalanine isopropyl ester, 190 Nitrile, 41, 127 Nitroarginine, 179, 308 Nitrobenzyloxycarbonyl, 108, 321 Nitromethane, 110, 118 Nitrophenyl (ONp), 119, 212, 213, 295 Nitropiperonyloxycarbonyl (Npoc), 108 Nitroveratryl, 342 N-methyl-D-aspartate (NMDA), 110, 111 N-nitroso-proline 2,4,6-trimethylbenzyl ester, 113 Nonanoic acid, 6 Nonessential amino acid, 20 N-o-nitrophenylsulfenyl group, 84 Nonpolar side chains, 29, 30, 34, 277 Non-pyrophoric, 104 N-protecting groups, 166, 333 N-trityl peptide esters, 193 threonine diethylammonium salt, 195, 196 N-tritylglycine Dpm ester, 115 Nucleic acid, 13, 20 Nucleophilic, 38, 97, 104, 127, 229, 285, 293, 315, 328, 355 species, 97 substitution, 97, 285 Nucleotide sequences, 13 N-urethane, 279, 280 N-Z-phenylalanine p-methylthiobenzyl ester, 114

O O-(cyclohexyl)-L-tyrosine, 286 O-(tert-butyldiphenylsilyl)-L-serine, 290 O-cyanobenzyl, 105 O-cyclohexyl tyrosine, 285

Index 409

Olefin metathesis, 331 Oleic acid, 8, 140, 141 Oleum, 2, 173 Oleyl chloride, 141 Olive oil, 8 O-nitrophenyl, 63, 119, 260–262, 281 O-nitrophenylsulfenyl chloride, 260, 261 derivatives, 261 group, 260, 262 Optical rotation, 19 Organic compounds, 1, 39 preparation, 37 solvents, 2, 29, 30, 35, 37, 38, 77, 111, 145, 172, 182, 189, 196, 234, 323 syntheses, 38, 39 Organometallic cleavable, 166 Orthoester, 41, 42, 127–129, 148 group, 41, 42, 127, 128 protecting groups, 127 removal of orthoester group, 132 preparation of amino acid orthoesters, 129 1-[n-(9- fluorenylmethyloxycarbonyl)(1s)-1-amino-2- hydroxyethyl]4-methyl-2,6,7-trioxabicyclo[2.2.2] octane fmoc-l-ser-obo este), 129 1-[n-(benzyloxycarbonyl)-(1s)-1amino-2-ethanol]-4-methyl2,6,7-trioxa-bicyclo[2.2.2] octane (cbz-l-ser-obo ester), 130 removal of TFA, 132 removal of TMSI, 132 Oxalyl chloride, 138, 141, 279, 311, 358 Oxazaborolidinone, 302 Oxazine, 140, 199 Oxazole, 140, 199 Oxazolidine, 140 Oxazolidone, 279, 280 Oxazoline, 41, 42, 139, 140, 229 oxazolidine 5,6-dihydro-1,3-oxazineboroxazolidone protecting groups, 139 (s)-3-benzyloxycarbonyl-5-oxo-4oxazolidineacetic acid, 141 4,5-dihydro-4,4-dimethyl-2-(cis-8heptadecenyl) oxazole, 140

catalytic reduction ((s)-3-benzyloxycarbonyl-5-oxo-4-oxazolidinepropionic acid), 143 introduction of protecting group, 140 l-aspartic acid boroxazolidone, 142 Oxidation, 2, 22, 109, 120, 138, 166, 223, 254, 282, 295, 340, 343, 345, 356, 357 removal of protecting group, 142 saponification of (s)-3-benzyloxycarbonyl-5-oxo-4-oxazolidinepropionic acid, 142 reduction, 340 Oxidizing agents, 278, 355–357

P P-(bromoacetamido)azobenzene, 133, 134 P-(p-(dimethylaminophenylazo)benzyl, 105 Palladium, 97, 104, 106, 107, 118, 143, 205, 236, 237, 243, 334, 353 catalyzed hydrogenation, 237 Palmaceae, 2 Palmitic acid, 2 P-amidinomethylphenyl, 119 P-aminobenzyloxycarbonyl derivative, 244 P-aminophenylalanine (PAF), 14 Paraformaldehyde, 67, 140, 141, 223 Pasteur pipet, 48 P-azobenzenecarboxamidomethyl (OAbc), 133, 134 P-bromophenacyl, 89, 92, 94 bromide, 92 P-chlorobenzyl, 105, 109, 330, 331 Penicillium chrysogenum, 22 Pentachlorophenol (OPcp), 119 Pentachlorophenyl, 119 Pentafluorophenyl (OPfp), 119, 120, 122, 125, 126, 320, 331 trifluoroacetate, 119 Pentamethylbenzyl, 105 Peptide, 22, 52, 53, 70, 77, 79, 80, 82, 84, 89–91, 95, 97, 100, 101, 104, 106, 118–120, 135, 138, 143, 144, 165–167, 171, 173, 179, 185, 186, 193, 199, 205, 211, 212, 222, 234, 236, 237, 248, 251, 255, 260, 263, 264, 277–280, 285, 293, 296, 297, 299, 305, 307, 313, 314, 320, 323, 328–333, 338, 345, 349–352, 355–357

410

bonds, 52, 135, 278, 279, 328 Peptoid, 330 Perchloric acid, 135 Peroxymonosulfate, 356 Petroselinic acid, 8 pH effect, 36 Phase transfer reagent, 144, 145 Phenacyl, 89–96, 108, 314, 315, 341, 346 keto functionality, 89 protecting group, 89, 91, 148 Phenoxide, 119 Phenoxycarbonyl (POC), 296 Phenyl, 58, 73, 79, 80, 83, 86, 90, 108, 111, 119, 120, 133, 134, 174, 175, 178, 179, 182, 184, 188, 221, 225, 226, 229, 281, 323, 330, 341, 342, 348, 349 acetamidomethyl (Pacm), 341 alanine, 20, 29, 30, 34, 35, 37, 39, 42–44, 49, 58, 82, 87, 88, 90, 96, 108, 113, 114, 120, 125, 137, 193, 210, 220, 221, 228, 262, 277 benzyl ester p-toluenesulfonate, 147 chloroformate, 174, 184 methylsulfonyl fluoride (PMSF), 79 silane, 100, 334 Phosphopeptides, 52 Phosphoric acid, 42, 52, 177, 178 Photo-irradiation, 108 Photo-kinetics, 108 Photolytic cleavage, 90, 110 Photosensitive, 90 Phthalic acid anhydride, 263 Phthalimidomethyl ester, 69–71 group, 65, 69, 70 Phthaloyl (Pht), 58, 84, 106, 263 Phyllomedusa sauvagei, 22 Pinner synthesis, 127 Piperidine, 61, 63, 101, 132, 165, 205, 206, 211, 222, 242, 243, 263, 277, 285, 298, 299, 315, 329, 338, 342, 343 Piperidinofunctionalized polystyrenes, 205 P-methoxybenzenesulfonyl (Mbs), 306, 307, 351 P-methoxybenzyl chloride, 107, 348 ether, 104 P-methoxycarbonylbenzyl, 105

Index

P-methoxyphenacyl, 89 P-methylbenzyl, 105, 343 P-methylthiobenzyl (Mtb), 109 alcohol, 109, 114 ester, 109 P-methylthiophenyl, 119 P-nitrobenzyl, 235, 245, 330, 341, 342, 345 chloroformate, 245 oxycarbonyl (pNZ), 235 P-nitrophenyl, 61, 119, 200, 213, 260 chloroformate, 200, 213 Polar co-solvent, 122 impurities, 209 Poly(hydroxybutylglutamineco-L-serine), 288 Polyethylene glycol (PEG), 72, 76–78, 263, 264, 330 oxide (PEO), 76 Polyoxyethylene, 76 Porphyrin-methyl, 111 Post-translational modification, 14, 22 processes, 22 Potassium carboxylate, 6 hydroxide, 88, 243 nonanate, 6 salt, 6, 84 P-phenylazophenylsulfonyl aminocarbonyl (Azo-Tac), 167, 199, 200 isocyanate, 200 Propargyl alcohol, 101, 102, 125 bromide, 101, 102 oxycarbonyl chloride, 125, 126 pentafluorophenyl carbonate (PocOPfp), 119, 125, 126 protecting group, 148 Propionic acid, 1, 189 Propyl, 114, 200, 341, 342 Propylene oxide, 44, 303 Protecting groups (histidine), 312 exemplary procedures (protect histidine side chain), 316 BPOC-HIS(2,6-dmbz)-oh, 319 FMOC-HIS(π-2,6-dmbz)-oh, 316 FMOC-HIS(τ-2,6-dmbz)-oh, 316 nim-trityl-l-histidine, 318

Index 411

nα-nim-ditrityl-l-histidine, 318 nα-nτ-ditrityl-l-histidine methyl ester, 319 nα-t-butyloxycarbonyl-nτ-2,4- dinitrophenyll-histidine, 317 Protecting groups (methionine), 355 exemplary procedures (protect methionine side chain), 358 allylation (n-acetyl-dlmethionine), 358 oxidation of 4-methoxybenzyloxycarbonyl-l-methionine, 358 preparation of poly(s-methyl-l-methionine sulfonium chloride), 359 Protecting groups (tryptophan), 349 exemplary procedures, 352 1-formyl-dl-tryptophan hydrochloride, 352 1-mesitylenesulfonyl-l-tryptophan, 354 1-tosyl-l-tryptophan, 353 Protecting groups (removable hydrogenolysis), 234 4,5-diaryl-4-oxazoin-2-one derivative of amino acids, 246 catalytic hydrogenolysis of (s)-2-(4,5bis(methoxyphenyl)-2-oxooxazol-3(2H)-yl propanoic acid, 248 4,5-diaryl-4-oxazoin-2-one derivative of alanine, 247 4,5-diphenyl-1,3-dioxol-2-one using trichloromethyl chloroformate, 247 4,5-diphenyl-1,3-dioxol-2-one-phosgene, 246 benzyloxycarbonyl (cbz-bz-z) group, 236 introduction of CBZ group, 239 preparation of CBZ reagents, 238 removal of CBZ group, 242 isonicotinyloxycarbonyl (INOC) protecting group, 248 isonicotinyl p-nitrophenyl carbonate, 249 p-nitrobenzyloxycarbonyl (PNZ) group, 244 n-p-nitrobenzyloxycarbonyl-d-glutamic acid, 245 p-nitrobenzyloxycarbonyl glycine, 245 p-trimethylammonium chloridobenzyloxycarbonyl protecting group, 251 formation (p-trimethylammonium chloridobenzyloxycarbonylglycine methyl ester), 252

hydrogenolysis (p-trimethylammonium chloridobenzyloxycarbonyl-glycyl glycine), 252 preparation (p-trimethylammonium chloridobenzyloxycarbonyl chloride), 251 Protein-nucleic acid (PNA), 260 Proton sponge, 138, 139, 305 P-toluenesulfonic acid (PTSA), 42, 49, 53, 70, 101, 103, 106, 111, 134, 140, 200, 203, 291, 292, 343, 345 P-toluenesulfonyl chloride, 353 P-tolylmethylsulfonyl, 199 P-trimethylammonium chloridobenzyloxycarbonyl chloride, 252 glycylglycine, 253 group, 251 Pyridine hydrochloride, 170 Pyroglutamide, 330 Pyroglutamyl moiety, 331 Pyrrolysine, 14, 19, 340

Q Quinoline-8-sulfonyl (Q-SO2), 297

R Racemization, 39, 42, 53, 66, 73, 80, 89, 90, 101, 106, 108, 119, 127, 135, 186, 193, 313–315, 320, 338 Recrystallization, 86, 87, 118, 125, 130, 136, 184, 208, 215, 220, 223, 283, 308, 348 p-toluenesulfonyl cyanomethyl ester, 88 Reduction, 109, 167, 307, 321, 340, 341, 343, 352, 355, 358 Regioselective ligation, 338 Relative solubility, 30, 35, 36 Rhamnulose aldolases, 289 Ribosomal, 13, 340 Ring-opening polymerization, 296, 297 Rink amide, 329 Rotary evaporation, 114, 115, 195, 209, 220, 223, 258 evaporator, 45, 50, 122, 183, 220, 240, 244, 302, 303, 324, 335

412

Index

Round-bottomed flask, 45, 48, 49, 130, 145, 169, 243, 294, 311

S S-(((benzyloxy)carbonyl)-L- alanyl)-Nglycyl-L-leucyl-L-cysteine, 211 S-(Tmob)-cysteine trifluoroacetate salt, 347 Salting effect, 36, 39 Saponification, 42, 109, 138, 279, 336, 350 Saturated acid, 8 fatty acid, 2, 7, 8 S-benzhydryl-L-cysteine, 347 Schiff base, 89 Schotten-Bauman condition, 179, 180, 199, 236, 237, 255 reaction, 135, 186 Schwesinger base, 38 Segment condensation approach, 278 Selective oxidation, 135 Selenocysteine, 14, 19, 340, 342, 344, 345, 348, 360 Selenoproteins, 340 Semaglutide, 305 Sense codon, 13 Sephadex LH-20, 48, 50 Se-p-methoxybenzyl-L- selenocysteine, 348 Serine, 19, 34, 37, 77, 93, 112, 129, 131–133, 232, 233, 278–294, 299, 313, 320, 329, 355 Serotonin, 349 Side-chain alkylations, 356 protecting groups, 278, 298, 306, 313, 315, 333, 352 Siderophore, 290 Silver oxide, 120 Sodium 4-(bromomethyl)benzene-sulfonate, 109 acetate, 347 arachidonate, 7 benzenethiolate, 95 bromite trihydrate, 295 carbonate, 94, 176, 177, 201, 205, 336 perborate tetrahydrate, 223 sulfate, 83, 96, 139, 177, 178, 190, 194, 196, 284, 336 sulfide, 73, 76

thiophenoxide (PhSNa), 90, 94 thiosulfate, 76 Solid-phase peptide synthesis (SPPS), 61, 165, 173, 185, 206, 232, 279, 283, 297, 305, 329, 333, 339, 340 Solubility, 2, 6, 7, 11, 20, 22, 29, 30, 34–39, 77, 107, 111, 119, 165, 171, 172, 185, 189, 199, 212, 215, 234, 244, 251, 290, 297, 299, 305, 308, 315, 321, 332–334, 345, 355, 357 Solution-phase synthesis, 234 Stable tertiary carbocation, 175, 193 triphenylmethyl cation, 281 Standard steglich esterification condition, 82 Stearic acid, 2, 8 Steglich esterification, 79, 97 condition, 89 Steric effect, 193, 222 Stoppered reaction flask, 49 S-trityl-cysteine, 212 Styrene, 80 Succinimide derivatives, 320 Sulfhydryl protecting groups, 338, 340–342, 346

T Tautomerization, 313, 315 T-butoxymethyl (Bum), 313, 314 T-butyl acetate, 350 bromide, 65, 67 carbonate, 38, 61 phenylglycinate hydrochloride, 54 T-butylation, 349, 350, 352 Teflon-coated magnetic stirring bar, 48, 230, 231 Tentagel, 194 Termination codons, 13 Tert-amyloxycarbonyl (t-Amoc), 166, 179–182 Tert-butoxylcarbonyl (t-BOC), 46, 47, 68, 79, 82, 83, 92, 96, 123, 146, 165–170, 173, 175, 179, 180, 182, 199, 205, 234, 243, 251, 264, 277, 279, 281, 285, 287, 288, 291, 299, 321, 324, 339, 349 L-threonine, 279

Index 413

Tert-butyl, 53, 68, 72, 78, 92, 107, 165–168, 171, 182–184, 200, 202, 221, 229, 232, 234, 242, 243, 253, 255, 258, 280, 281, 283, 288–290, 331, 332, 342, 350, 352 azidoformate, 167 carbonate, 167 carbonazidate, 169 ester, 167, 200, 202, 242, 243, 258 fluoroformate, 167 N-benzyloxycarbonyl-O-(tert-butyl) serinate, 281 phenylalaninate, 229 protecting group, 280 Tert-butyldimethylsilyl (TBDMS), 68, 144, 288, 299 Tert-butyldiphenylsilyl (TBDPS), 288, 289 Tert-butylsulfanyl, 342 Tetra(t-butyl)tryptophan, 350 Tetrabenzo-[a,c,g,i]fluorenyl-17-methyloxycarbonyl (Tbfmoc), 204, 234 Tetrabutylammonium fluoride (TBAF), 80, 81, 89, 90, 166 Tetrachlorophthaloyl (TCP), 263, 264, 297, 300 Tetrahydrofuran, 124, 128, 180, 212, 213, 284, 308 Tetrahydropyranyl (Thp), 290, 292 Thallic trifluoroacetate, 120 Thermodynamics, 29 Thioanisole, 117, 199, 235, 285, 307, 345, 350, 357 Thioester, 331 Thiohydantoins, 37 Thiolysis, 254, 255, 262, 264, 281, 343, 346 Thiomethylethyl, 72 Thionyl chloride, 42, 43, 45, 48, 73, 74, 140, 141, 232 MeOH system, 45 Thiophene-2-carbonyl (TPC), 297 Thioredoxin reductase, 340 Threonine, 19, 20, 37, 194, 195, 239, 240, 278–283, 285, 288–294, 320, 329 Toluenesulfonyl (Tos), 305, 307, 314, 315, 351 Torulopsis utilis, 22 Transcription, 13 Trans-double bond, 8 Transesterification, 61, 128

Transient protection, 148 Transpeptidation, 53 Triisopropylsilane (TIS), 117, 291, 323, 345, 346 Tri(isopropyl)silyl, 288 Tri(t-butyl)tryptophan, 350 Triacylglycerides, 8 Tributylamine, 293 Trichlorofluoromethane (Freon 11), 184 Trichlorophenyl (OTcp), 119 Tri-diphenylmethyl phosphate, 110 Triethylamine, 44, 58, 63, 66, 80, 85, 92, 104, 107, 113, 126, 147, 169, 173, 184, 187, 194, 196, 198, 201, 202, 208, 213, 214, 227, 232, 239, 243, 258, 261, 279, 284, 294, 309 hydrochloride, 173, 261 Triethyloxonium fluoroborate, 355 Triethylsilane (TES), 236, 323, 346, 357 Trifluoroacetamide, 119 Trifluoroacetic acid (TFA), 56, 57, 80, 89, 92, 96, 98, 99, 101, 104, 107–109, 115–117, 121, 124, 129, 132, 168, 171, 172, 179, 182, 185, 192, 197, 199, 200, 202, 205, 216, 234, 235, 246, 248, 255, 258, 264, 281, 282, 285, 291, 299, 305, 307, 315, 322, 323, 330, 331, 334, 343, 345, 346, 349–351, 354, 357 Trifluoroacetyl (Tfa), 286, 297, 300 Trifluoromethanesulfonate (TMSOTf), 345 Trifluoromethanesulfonic acid (TFMSA), 104, 109, 168, 185, 285, 307 Triisopropylsilane, 117, 291 Triisopropylsilyl, 288 Trimethoxytrityl, 167, 194 Trimethylchlorosilane, 42, 202 Trimethylsilyl (TMS), 45, 65, 68, 73, 80, 97, 129, 144, 147, 193, 202, 253, 256, 285, 288, 306, 330, 345, 350 (E)-N-(trimethylsilyl)acetimidate, 202 bromide (TMSBr), 285, 345 fluoride, 80 iodide (TMSI), 129, 132 trifluoromethanesulfonate (TMSOTf), 350 Trimethylsilylethanol, 79 Trimethyltin hydroxide (Me3SnOH), 90 Trimethylvinylsilane, 80 Triphenylchloromethane, 194, 195

414

Index

Triphenylmethanol, 199, 353 Triphenylmethyl (TRT), 167, 193, 194, 197, 281, 313, 321 bromide, 193, 197 Triphenylphosphine, 82, 100, 110, 128, 200, 203, 334 Triphosgene, 125 Tris(2-aminoethyl)amine (TAEA), 222 Tris(4-methylpiperazin-1-yl)phosphane, 320 Trityl, 167, 194, 196–198, 323, 353 chloride, 147, 193, 196, 318, 320 Tritylsulfanyl (S-Trt), 342 Trivial nomenclature system, 2 Trypsin, 50, 120, 342 catalyzed peptide, 120 Tryptophan, 20, 29, 30, 34, 50, 90, 104, 177, 178, 187, 240, 241, 278, 320, 349–353, 355–357, 360 Tyrosine, 19, 37, 78, 104, 178, 185, 186, 188, 278, 280–282, 285, 288, 291–293, 295, 296, 299, 307, 320, 355, 356

U Unnatural amino acid, 21, 22 Unsaturated carbon-carbon double bond, 96 fatty acid, 7, 8 protecting groups, 96 allyl group, 99 allyl substituted allyl protecting groups, 97 amino acid allyl esters, 98

amino acid benzyl ester, 111 amino acid propargyl ester, 101 benzyl protecting groups, 103 benzyl protecting groups, 115 benzyl substituted benzyl protecting groups, 103 propargyl esters, 102 propargyl protecting group, 101 α-substituted benzyl groups, 109 Unsubstituted benzyl ester, 108 Urethane, 119, 165, 179, 236, 279, 351 UV absorbance, 116

V Vacuum rotary evaporator, 122 Vasopressin, 340

W Wang resin, 116, 297 Water aspirator, 213, 219, 224, 225, 229, 251 Weinreb amide, 299, 321 Williamson ether synthesis, 285

X X-ray crystallography, 18

Z Zinc, 70, 75, 76, 90, 196, 248, 346 Zwitterion, 11, 19, 20, 36, 109, 121, 127