Material Science of Chitin and Chitosan

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Tadashi Uragami, Seiichi Tokura (Eds.)

Material Science of Chitin and Chitosan

Tadashi Uragami • Seiichi Tokura (Eds.)

Material Science of Chitin and Chitosan With 140 Figures and 42 Tables

^ g Kodansha

^

Spri ringer

Tadashi Uragami Professor Unit of Chemistry, Kansai University Suita, Osaka, Japan E-mail: [email protected] Seiichi Tokura Professor Chemical Laboratory Group, Kansai University Suita, Osaka, Japan E-mail: [email protected]

ISBN 4-06-212194-8 Kodansha Ltd., Tokyo ISBN-10 3-540-32813-0 Springer Berlin Heidelberg New York ISBN-13 13 978-3-540-32813-1 Springer Berlin Heidelberg New York

Library of Congress Control Number: 2006921555 This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springer.com © Kodansha Ltd. 2006 Printed in Japan The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. The instructions given for carrying out practical experiments do not absolve the reader from being responsible for safety precautions. Liability is not accepted by the authors. Coverdesign: design & production^ Heidelberg, Germany Printed on acid-free paper - 5 4 3 2 1 0

List of Contributors Numbers in parentheses refer to the chapters.

Abe, Koji (8)

Department of Functional Polymer Sciences, Shinshu University

Fukamizo, Tamo (4)

Department of Bioscience, Kinki University

Kurita, Keisuke (3)

Department of Materials and Life Science, Seikei University

Minami, Saburo (7)

Department of Clinical Medicine, Tottori University

Morimoto, Minoru (7)

Research Center for Bioscience and Technology, Tottori University

Nagahata, Misao (8)

National Institute of Health Sciences

Ogawa, Kozo (2)

Center for R&D of Bioresources, Osaka Prefecture University

Okamoto, Yoshiharu (7)

Department of Clinical Medicine, Tottori University

Ouchi, Tatsuro (6)

Department of Applied Chemistry, Kansai University

Saimoto, Hiroyuki (7)

Department of Materials Science, Tottori University

Shigemasa, Yoshihiro (7)

Department of Materials Science, Tottori University

Tamura, Hiroshi (9)

Unit of Chemistry, Kansai University

Teramoto, Akira (8)

Department of Functional Polymer Sciences, Shinshu University

Tokura, Seiichi(l, 10)

Chemical Laboratory Group, Kansai University

Uragami, Tadashi (5)

Unit of Chemistry, Kansai University

Yui, Toshifumi (2)

Department of Applied Science, Miyazaki University

Preface

Chitin and chitosan have been attracting attention as a final biomass all over the world. Interest in chitin and chitosan in fundamental science and applied research grows year by year. Chitin, chitosan and their derivatives have multifunctional properties with applications in the resource and energy sectors, as well as environmental problem-solving and practical life sciences. Thus, chitin and chitosan researchers continue to pursue further scientific and technological applications, particularly in the life sciences. This volume is a comprehensive introduction to the science and practical applications of chitin and chitosan and their related enzymes. It covers the methods of synthesis, structures, properties and applications of chitin, chitosan and related enzymes. One of our objectives is to review the basic physicochemical properties of chitin and chitosan and to present the fundamental preparative methods. We hope this will prove useful to those who are interested in the science and application of chitin and chitosan, as well as to those who wish to expand their knowledge in this area. Another objective of this monograph is to examine recent research and provide basic information on current topics. We have tried to present the new trends and to anticipate potential applications in the various fields of chitin, chitosan, and related enzymes. We have covered as wide a range of relevant topics as possible and discussed those areas where future work should be concentrated. However, the emphasis throughout this book is on those principles and topics which are based on the related subject matter. The present volume consists of several main sections containing recent topics reported by a number of prominent researchers in this field. Chapter 1 is an introduction to the fundamental properties of chitin and chitosan. A basic understanding of the outline, dissolution, viscoelastic properties and regeneration of chitin and chitosan is very important in the development of science and technology related to chitin and chitosan. In Chapter 2, the conformation of the chitin and chitosan molecule in the solid state, the inorganic salt, the metal chelate of chitosan and the conformation of the chitosan molecule in an acidic solution are discussed. The chemical modifications of chitin and chitosan discussed in Chapter 3 are important for the development of new materials. Direct modifications, modifications by reactive precursors and modifications based on protectiondeprotection techniques of chitin and chitosan are described. In Chapter 4, the

viii

Preface

enzymes responsible for the biosynthesis and decomposition of chitin and chitosan are reviewed, their mechanisms are discussed and their applications in a variety of fields reported. In Chapter 5, adsorbents, coagulants and membranes for the separation of materials from chitin, chitosan and their derivatives are reviewed. The mechanisms of this separation are described and their applications discussed. In Chapter 6, the immunological properties and drug delivery systems for the pharmaceutical applications of derivatives of chitin and chitosan are discussed from the viewpoint of the chemical and physical structure of their derivatives. In Chapter 7, the biocompatibility, biodegradability, biological activity and applications of biomaterials derived from chitin, chitosan and their derivatives are reviewed using various examples. In Chapter 8, the use of a polyelectrolyte complex composed of chitin and chitosan for tissue regeneration is introduced. The role of chitin, chitosan and their derivatives in the regeneration of tissues in vitro and in vivo is reported. In Chapter 9, the various applications to fibers, films, hydrogels, and the hybridization of chitin, chitosan and their derivatives are reviewed. The applications of chitinous materials such as cosmetics, food additives, agricultural materials and water cleaning are also introduced. Finally, in Chapter 10, the future prospects of chitin and chitosan are discussed from the viewpoint of their science and technology. Chitin and chitosan will become very important key materials in the life sciences in the 21st century. This volume on chitin and chitosan was planned while envisioning the science and technology of the 21st century. It is our hope that it will make a positive contribution to the future development of the scientific and technological applications for chitin, chitosan and related enzymes. In the preparation of this book, the most active scientists in this and related fields have been carefully selected as authors. We are grateful to all the contributors. Finally, we are indebted to Mr. Ippei Ohta of Kodansha Scientific Ltd. for his helpful suggestions and discussions concerning the organization of this book. March 2006 Tadashi Uragami Seiichi Tokura

Contents

List of Contributors Preface 1 Chitin and Chitosan 1.1 Introduction 1.2 Identification of Chitin and Chitosan 1.3 Chemical Structures of Chitin and Chitosan 1.4 Significant Participation of Chitin and Chitosan in Biological Functions 2 Three-dimensional Assembly of Chitin and Chitosan 2.1 Introduction 2.2 Chitin Structures 2.2.1 Crystal Structures of Chitin 2.2.2 Computer Modeling of Chitin Crystals 2.2.3 Proposed Models for the Biosynthesis Mechanism of Chitin Microfibrils 2.2.4 Complexes with p-Chitin 2.3 Chitosan Structures 2.3.1 Crystal Structures of Chitosan 2.3.2 Acid Complexes 2.3.3 Transition Metal Complexes 2.3.4 Chitosan Conformation in Acidic Solutions 2.4 Spontaneous Transformation of Chitosan Conformation 2.4.1 At Room Atmosphere 2.4.2 In an Alcohol-Water Mixture 2.5 Conformational Diversity of the Chitosan Molecule

v vii 1 1 2 3 7 21 21 21 21 24 27 30 33 34 37 42 43 44 44 46 47

X

Contents

3 Introduction of Biologically Active Branches through Controlled Modification Reactions of Chitin and Chitosan 3.1 Introduction 3.2 O-Glycosyl-branched Derivatives 3.2.1 Synthesis Based on Phthaloyl-chitosan A. Phthaloylation B. Branching C. Properties 3.2.2 Synthesis Based on Trimethylsilyl-chitosan A. Silylation B. Branching 3.3 A^-Branched Derivatives 3.3.1 Reductive A^-Alkylation A. With Reducing Sugars B. With Formyl-bearing Sugars 3.3.2 A^-Acylation 3.4 Cyclodextrin-branched Derivatives 3.4.1 Reductive Alkylation 3.4.2 Other Reactions 3.5 Peptide-branched Derivatives 3.5.1 A^-Acylation 3.5.2 Other Reactions 3.6 Other Branched Derivatives 3.7 Conclusion

51 51 52 53 53 54 57 59 59 60 61 61 61 64 66 67 67 69 70 70 74 74 77

4 Enzymes Involved in Chitin and Chitosan Decomposition and Synthesis 4.1 Introduction 4.2 Chitinases 4.2.1 Chitinase Structure 4.2.2 Enzyme Mechanism 4.2.3 Chitin and Oligosaccharide Binding 4.3 Chitosanases 4.3.1 Chitosanase Structure and Mechanism 4.3.2 Hydrolytic Specificity....; 4.3.3 Chitosan and Oligosaccharide Binding 4.4 Chitin Synthase 4.4.1 Enzymatic Mechanism of Chitin Biosynthesis 4.4.2 Regulation of Enzyme Activity

81 81 81 82 86 89 92 92 95 97 98 98 99

Contents

4.4.3 Structure of Chitin Synthases 4.4.4 Chitosan Biosynthesis and Chitin Deacetylase 4.5 Applications 4.5.1 Oligosaccharide Production 4.5.2 Plant Protection 4.5.3 Chitinases as Targets for Blocking Host-Parasite Interaction 4.6 Conclusion 5 Separation Materials Derived from Chitin and Chitosan 5.1 Introduction 5.2 Structural Design of Separation Materials 5.2.1 Chemical Design of Separation Materials 5.2.2 Physical Construction of Separation Materials 5.3 Characteristics of Chitin and Chitosan 5.4 Preparation Methods for Separation Materials 5.4.1 Methods for Membrane Preparation 5.4.2 Membrane Structures 5.4.3 Preparation of Granular Porous Materials 5.5 Principles of Membrane Permeation 5.5.1 Dialysis 5.5.2 Reverse Osmosis 5.5.3 Ultrafiltration 5.5.4 Pervaporation 5.5.5 Evapomeation 5.5.6 Carrier Transport 5.5.7 Transport with Catalytic Function 5.5.8 Gas Permeation 5.6 Chitinous Materials for Separation 5.6.1 Membranes A. Dialysis Membranes B. Reverse Osmosis Membranes C. Ultrafiltration Membranes D. Pervaporation Membranes E. Evapomeation Membranes F. Carrier Transport Membranes G. Catalytic Membranes H. Gas Permeation Membranes 5.6.2 Adsorbents 5.6.3 Coagulants

xi

101 103 105 105 107 108 108 113 113 114 114 115 115 116 116 117 118 118 118 119 120 120 121 121 122 123 123 123 123 126 127 128 137 145 149 150 150 155

xii

Contents

5.6.4 Immobilization Supports 5.7 Superiority as Separation Materials

156 159

6 Pharmaceutical Applications of Chitin and Chitosan 6.1 Introduction 6.2 Immuno-enhancing Effects of A^-Acetylchitohexaose 6.3 Possibility of Application of Chitin and Chitosan as Drug Delivery Tools 6.3.1 Design of Macromolecular Prodrugs of Antitumor Agents Using Chitin and Chitosan as Carriers 6.3.2 Preparation of Chitosan Gel Microspheres as Antitumor Drug Vehicles 6.3.3 Celluar Gene Delivery Using Quaternary Chitosan Conjugates Having Antennary Galactose Residues 6.3.4 Preparation of PEG-grafted Chitosan Nano-aggregates as Polypeptide Drug Vehicles 6.4 Conclusion

165 165 165

185 189

7 Biomedical Materials from Chitin and Chitosan 7.1 Introduction 7.2 Biocompatibility 7.2.1 Toxicity 7.2.2 Biodegradability 7.3 Biological Activity 7.3.1 Polymorphonuclear Cells A. Migration B. Reactive Oxygen Species Release C. Cytokines 7.3.2 Macrophages A. Migration B. Cytokines 7.3.3 Fibroblasts and Vascular Endothelial Cells 7.3.4 Platelets 7.3.5 Complements 7.3.6 Extracellular Matrix A. Collagen B. Glycosaminoglycan and Proteoglycan C. A^-Acetyl-D-Glucosamine and D-Glucosamine 7.3.7 Analgesia

191 191 192 192 193 195 195 196 197 198 199 200 200 201 201 201 203 203 203 204 204

168 168 175 180

Contents

7.3.8 Antimicrobial Activity 7.3.9 Wound Healing Acceleration 7.4 Applications of Biomedical Materials 7.4.1 Preparation of Biomedical Materials A. ChitipackS B. ChitipackP C. ChitopackC D. Other Materials 7.4.2 Applications for Wound Healing A. Chitin Materials B. Chitosan Materials C. D-Glucosamine—Application in Injured Cartilage

xiii

205 206 207 208 208 208 209 209 209 209 211 212

8 Regeneration of Tissue in the Living Body 8.1 Polyelectrolyte Complexes 8.2 Scaffold for Tissue Culture of Various Cells 8.2.1 Extracellular Matrix 8.2.2 Biomedical Materials 8.3 Hard Tissue Regeneration In Vitro and In Vivo 8.3.1 Bone Regeneration 8.3.2 Cartilage Regeneration 8.3.3 Application in Dental Surgery

219 219 222 222 224 227 227 235 238

9 Miscellaneous Applications of Chitinous Materials 9.1 Introduction 9.2 New Solvent System for a-Chitin 9.3 Fabrication of Chitinous Material 9.3.1 Preparation of Fibers A. Chitosan-based Fibers B. Chitin-based Fibers 9.3.2 Preparation of Hydrogel 9.3.3 Preparation of Membrane A. Chitin Sheet B. Chitosan-based Membrane 9.4 Chitinous Materials in Application 9.4.1 Cosmetics 9.4.2 Food Additives 9.4.3 Agricultural Materials 9.4.4 Water Purification

245 245 245 247 247 247 251 254 254 254 257 258 258 260 261 262

xiv

Contents

9.4.5 Drug Delivery System 10 Prospects for the Application of Chitin and Chitosan

264 271 Index 277

Chitin and Chitosan

1.1

Introduction

Chitin, a polysaccharide abundant in nature, is considered to be the most prominent natural polymer due to multifunctions such as biodegradability and low toxicity in animal body and acceleration of skin recovery. However, few applications have been reported due to its low solubility in general solvents. Chitosan, on the other hand, has been studied more for various practical applications because of functions such as biodegradability, low toxicity and acceleration of fibroblast formation in animal body, acceleration blood clotting, antimicrobial activity and high solubihty in water following the formation of salts with various organic acids and hydrochloric acid. As a result, numerous reports on chitosan have been published, including chemical modifications and biomedical applications.

Chitin (poly-A^-acetyl-glucosamine)

/OH

/OH

O. O

HO^^'^'^X NH2

/OH

O.

O"

O

HO^^^^^X

HO

NH2

NH2

Chitosan (poly-glucosamine) Fig. 1.1 Chemical structures of chitin and chitosan.

Although chitin, a naturally abundant muco-polysaccharide, is hardly found in plant support systems, it is present in animal support or tissue of lower evolutionary organisms such as crustaceans, insects and mushrooms. The yield of chitin in nature is said to be second only to that of cellulose (Fig. 1.1). Chitin is also known to be biodegradable in nature and in animal body due to the close chemical structure of sialic acid, which is a component of cell wall-supporting materials. Since chitinases contain an animal self-defense system to kill microbials by breaking down the cell wall, chitin is hydrolyzed easily in animal body. The hydrolysate.

2

I

Chitin and Chitosan

A^-acetylglucosamine (GlcNAc) joins the nutrient pathway without any of the toxicity of chitin hydrolysate. The main function of chitin is acceleration of skin recovery with little immunological response. The main target of recent research on chitin is regeneration under mild conditions for application in biomedical materials using this function, even though it is difficult to find a good solvent for chitin. On the other hand, chitosan, a deacetylated derivative of chitin, exists in small amounts in several kinds of mushrooms and is mainly used as a chemical product of chitin. The predominant functions of chitosan seem to be its antimicrobial activity and inhibitory function toward chitinases in addition to the acceleration of fibroblast formation in animal body. These predominant functions have been applied to biomedical purposes in addition to food preservation due to the high solubility of chitosan in aqueous organic acid solutions and hydrochloric acid aqueous solution.

1.2

Identification of Chitin and Chitosan

The identification of chitin required more than a century of study. The general recognition of the chitin chemical structure and crystalline structure was achieved first in the 1960's. The first report on chitin was made by Odier in 1823 regarding the similarity between the supporting substance of insects and that of plants, i.e., cellulose.'^ It took almost one and half centuries until chitin was recognized as one of nature's abundant polysaccharides. According to a description by Muzzarelli,^^ there were many investigations proving the chemical structure of chitin separate from that of cellulose. Lassaigne^^ isolated the chitinous exoskeleton of the silkworm Bombyx mori by warm potassium hydroxide and demonstrated the presence of nitrogen in chitinous exoskeleton to form potassium cyanide following the treatment of the exoskeleton with potassium hydroxide and potassium hipochloride. Finally, Ledderhose"^^ found chitin to be composed of glucosamine and acetic acid, a fact which was later confirmed by Gilson.^^ Reviews on chitin were published by Von Wieseligh,^^ Wester,^^ von Westein,^^ Leven and Lopez-Suarez,^^ and Von Franciis^^^ in the first stage of chitin research. They attempted to confirm the natural occurrence of chitin in living organisms, biological degradation, the chemical structure and, to a somewhat lesser degree, the technology of practical applications. The presence of chitin in fungi and Crustacea was confirmed by Rammelberg^^^ through its biological hydrolyzates. The presence of chitin-susceptible bacteria was reported in various marine sediments and animals by Zobell.^^^ In 1956, Purchase and Braun concluded that chitin was a polysaccharide consisting mainly of glucosamine residues following several hydrolytic reactions.'^> Regarding the research on chitosan after its discovery by Rouget,^"^^ dissolution of chitosan was studied by Von Furth and Russo^^^ following the formation of salts with organic acids and precipitation of chitosan by removing acids using alkaline reagents. Lowy found the most insoluble acid salt of chitosan to be sulfate salt.'^'

1.3

Chemical Structures of Chitin and Chitosan

3

As for the crystallography of chitin, the a structure has been proposed for the chitin from crab and shrimp shells and the p structure for the chitin from Loligo pen/^^ This was followed by a clear crystallographic analysis of the a structure for chitin by Blackwell in 1967/^^ Both chitins have been described as quite different figures not only in crystalline structure but also in the absorption profiles of solvents including water. Machine-made and hand-made nonwoven fabrics have been reported from aqueous gel of (J-chitin prepared by the mechanical agitation of p-chitin aqueous suspension. ^^^ However, a similar method did not work for achitin, probably due to its tight crystalline structure. Successful dissolution procedures were reported in the preparation of nonwoven fabrics from both types of chitin through hydrogel formation.^^^ However, the p-chitin crystalline structure was maintained in nonwoven fabrics prepared by mechanical agitation of P-chitin. A similar observation has also been reported regarding the maintenance of the p structure by applying ethylene-diamine as a solvent for chitin suspension by mechanical agitation.^^^ Few chemical modifications have been reported for P-chitin in contrast to the large number of reports on a-chitin.

1.3

Chemical Structures of Chitin and Chitosan

As noted above, the chemical structure of chitin was proposed by Purchase and Braun based on that of cellulose.^^^ Richard in 1951 noted chitin as a component of the integument of arthropods.^^^ The detection procedures and quantitative analysis for chitin were reviewed by Tracey in 1955.^^^ Jeuniaux reviewed the chitinous structure and the meaning of chitin in the biochemical evolution of insects in 1971,^"^^ following several reviews on the zoological participation of the chitin-protein complex.^^'^^^ The biochemistry of chitin in plant was described by Goodwin and Hercer in 1972,^^^ followed by the detection of chitin in bacterial cell surface.^^^ The participation of chitin in plant cell wall was mentioned by Preston in 1974^^^ and in cuticle of insect by Rockstein in the same year.^^^ Jeuniaux continued the study of the chitin-protein complex in insects and pubHshed the results in 1975.^^^ Although chitin is sparingly soluble in general mild solvents due to rigid crystalline structure through hydrogen bonding, chitosan becomes water soluble following the formation of salts with organic acids such as formic acid, acetic acid, acidic amino acids and ascorbic acid. Among the mineral acids, only hydrochloric acid is useful to make chitosan water soluble. Chitin was reported to dissolve in strong solvents such as concentrated hydrochloric acid, concentrated sulfuric acid, phosphoric acid (7 8%-97%) and methane sulfonic acid,^"^^ although the molecular weight of chitin starts to decrease immediately after dissolution. Dinitrogen tetroxide-A^,A^-dimethylformamide (DMF),^^^ lithium chloride-A^,A^-dimethylacetamide (DMAc)^^^ and hexafluoroisopropanol and hexafluoroacetone sesquihydrate were reported to be a mild solvent for chitin.^^^ Concentrated formic acid has been reported to give viscous chitin solution^^^ with spinnability following repeated freezing and thawing, if the entire procedure proceeds under temperatures below

4

1

Chitin and Chitosan

15°C to prevent suppression of molecular weight.^^^ In 1995, calcium chloride dihydrate saturated methanol or ethanol, one of the solvents of Nylon 6,6 including formic acid, was reported to be a good solvent for chitin."^^^ The solubility of chitin has been extensively investigated applying various calcium halide hydrates, and a higher degree of A^-acetylation of chitin was found to result in higher solubility in calcium chloride dihydrate saturated methanol or ethanol, as shown in Fig. 1.2. • Solubility of chitin in sat. CaCb • 2H20/MeOH

I

^^ 0.9 B 0.8

8

• y T

\

I

\I — — — Ti —L ^• r*i—rn—I

0.7

«-jd

"Bx) 0.6

L

! M W = 12000 •MW= 160000 MW = 40000

F L1

>. 0.5 ;3 0.4 0.3 =3 O 0? CO 0.1 0

I \m\ H*\J #

—\—m————I —

^

—{h

I*—

M^ I T—————h #

MM

MM

MM

IM,

MM

MM

MM

MM

0 10 20 30 40 50 60 70 80 90100 Degree of A^-acetylation (%)

Solubility Chitosan

Chitin

Fig. 1.2 Dependence of chitin solubility on the degree of A^-acetylation applying calcium chloride dihydrate saturated methanol as solvent.

Chitin solution made the determination of molecular weight of chitin possible by viscosity measurement applying A^-acetylated molecular weight standard chitosan to set up an arbitrary viscosity equation, as shown in Scheme 1.1. The theoretical viscosity equation was almost impossible to set up because optical and diffusion measurements were hard to apply due to the high salt concentration of solvent. A homogeneous chitin hydrogel has also been reported to be prepared by the addition of large excess of water to the chitin solution (in calcium chloride dihydratesaturated methanol or ethanol). This hydrogel was able to be chemically modified under much milder conditions than those required for chitin powder."^'^ Since the rigid hydrogen bonds were broken down during the dissolution of chitin into the calcium chloride solvent, preparation of nonwoven chitin fabrics was also reported without using any glue, probably due to the regeneration of hydrogen bonds. Moreover, adsorption of anionic compounds was shown in nonwoven chitin fabrics, suggesting that it may become an important drug carrier with biodegradability."^^^ Regarding dissolution of chitosan, hydrochloric acid and various organic acids were applied to form water-soluble salts with chitosan. However, the viscosity of these chitosan salt aqueous solutions tend to become unstable at room temperature.

1.3

Chemical Structures of Chitin and Chitosan

Chitosan

Chitin Deacetylation A^-acetylation

0.2 M AcOH-0.1 M NaCl-4 M urea Aqueous solution

sat. CaCb • 2H20/MeOH

,

Rotatory viscometer [77] Intrinsic viscosity of chitin

Calculation of molecular weight of chitin

[rj] vs. M

IL

,

Ubbelohde viscometer [7]] = 8.93X10 " M " ' ^

Molecular weight of chitosan

100 [T]] = KM" K = 2.54X10" a=0.45

J^ 10

10^

10'

10'

10^

MW Scheme 1.1 A schematic route to set up a tentative viscosity equation for chitin solution and working curve to estimate molecular weight of chitin directly by viscosity in sat. CaCl2/2H20methanol.

Acetic acid was shown to reduce the viscosity of chitosan aqueous solution more than other acids, including hydrochloric acid, as shown in Fig. 1.3. A neutral chitosan hydrogel has also been prepared to maintain the molecular weight constant for long standing at room temperature in addition to giving a clear chitosan solution by the addition of a minimum amount of acid, e.g., hydrochloric acid and acetic acid, as shown in Fig. 1.4."^^^

1

Chitin and Chitosan

Preparation of chitosan solution from chitosan powder Chitosan powder 1.53 g

Chitosan powder 3.06 g 4%CH3COOH100ml (0.64 M)

0.1MHC158ml

Chitosan solution (2.64 g/100 ml)

Chitosan solution (3.06 g/100 ml)

(pH = 3.8)

(pH = 3.8)

Viscosity measurements

r7tsp •••r]sp at r hours r]osp"77spatOhour

Chitosan solution (HCl)

Chitosan solution (CH3COOH)

30 20 10 0 20

40

60

80 100 Time (h)

120

140

160

180

Fig. 1.3 Depenence of stability of chitosan solution on the acid aqueous solution. (Prepared from chitosan powder)

1.4

Significant Participation of Chitin and Chitosan in Biological Functions Preparation of chitosan solution from chitosan hydrogel

Chitosan gel 10 g + H20 30 ml (Water content ...93.89%)

Chitosan gel 10 g + HiO 30 ml (Water content ...93.89%)

O.lMHCl 16 ml T Transparent chitosan solution (1.09g/100ml)

O.IMCH3COC 18 ml T Transparent chitosan solution (1.05 g/100 ml)

+ H2O

+ H2O

Chitosan solution (0.55 g/100 ml)

T Chitosan solution (0.53 g/100 ml)

>

pH = 5.37

>

pH = 5.36 T

Viscosity measurements

Fig. 1.4 Dependence of stability of chitosan solution on the acid aqueous solution. (Prepared from neutral chitosan hydrogel)

1.4

Significant Participation of Chitin and Chitosan in Biological Functions

The biological functions of chitin are quite different from those of chitosan due to a slight change in the functional group at the C-2 position of the glucose residue

8

1

Chitin and Chitosan

which becomes a main factor for inducing the conformational difference in crystaUine structure between them. Alhough two crystalline forms were proposed for chitin by Rudall in 1967, it is difficult to fmd the differences in biological properties. In general research on chitin is limited because it is not easily soluble. Exceptions are its acceleration of the recovery of epidermal cells in animal physiology suggested by Muzzarelli'^'^^ and pharmaceutical application reported by Miyazaki et al.''^ Chitin was classified as an inert polysaccharide chemically and biologically until a mild solvent was found that made it easy to modify by chemical reaction. Carboxymethyl-chitin (CM-chitin), a water-soluble and biodegradable chitin derivative, has been appHed in the biomedical field because of its close biological properties with hyaluronic acid. The induction of macrophage activation for short periods applying CM-chitin was reported by Nishimura et al. through in vivo study, as shown in Tables 1.1 and 1.2."^^^ Weak mitogenic activity on normal Table 1.1 Effect of 6-0-substituted derivatives of chitin or chitosan on macrophage activation Treatment^

Does (iUg)

Chitin CM-chitin P-chitin S-chitin Acetyl-chitin HE-chitin DHP-chitin CM-chitosan DHP-chitosan 70% DA-chitosan Pyran copolymer Control

500 500 500 500 500 500 500 500 500 500 500

-

% Lysis (mean ± s.e.)

% Statis (mean ± s.e.)

H2O2 Release (nmol/mg protein)

2.5±3.0 53.0±4.0'= 3.4±4.2 2.8±6.6 0.3±2.6 22.2±4.8' 15.3±3.2 3.9±3.7 18.6±7.2 55.9±4.3 66.4±3.0' 1.0±5.6

58.2±1.3'^ 58.3±2.0' 55.6±2.9' 43.1±4.1 N.D. 44.1±2.8 66.9±1.5' 45.2±2.l' 49.2±2.l' 54.7±3.3' 97.5±0.5'^ 38.9±3.9

33.6±4.2'= 51.8±2.5' 33.0±5.3' 44.1 ±2.4' N.D. 43.8±3.3'^ 78.7±2.9' 59.6±3.0'= 59.6±3.0'^ 98.7±3.9'^ 62.4±6.4' 18.5±1.4

^ Mice were injected i.p. with each polysaccharide 3 days before harvest of macrophages. '' Significant difference from the control by Student's test (p 2. Affinity for solvents The glycosylation products 6, 10, 14 and 18 were soluble in common low-boiling organic solvents owing to the protecting groups and sugar branches. Deprotected chitosan derivatives 7, 11,15 and 19, and chitin derivatives 8, 12, 16 and 20 were readily soluble in neutral water and swelled considerably in organic solvents. As implied by the water-solubility of the deprotected branched chitosans and chitins thus prepared, they were characterized by a high capacity for moisture absorption and retention. For instance, 12, 16 and 20 with ds 0.4-0.5 showed 3240% weight increase due to moisture absorption at 93% relative humidity, much

58

3

Biologically Active Branches to Chitin and Chitosan

higher than that of the original chitin, 18%. The branched chitins were then kept at 32% relative humidity to evaluate the moisture retention ability. The weights decreased gradually to level off at around 20% weight increase based on the weights of the dried samples, which were again higher than 7% for chitin. The effect of the structures of branches was not so significant, but disaccharide maltose improved the hydrophilicity better than monosaccharide galactose. Of the three, A^-acetylglucosamine appeared to be the most effective.^^'^^^ 3. Biodegradability Polysaccharides chitin and chitosan are important as biodegradable polymeric materials, and the influence of the branches on biodegradability of chitins was elucidated in terms of the susceptibility to lysozyme in a phosphate buffer solution of pH 4.5. The reducing ends formed by the enzymatic hydrolysis were determined by oxidation-reduction titration with ferricyanide, and decreases in the amount of ferricyanide were measured by UV spectroscopy. Compared to the original chitin, branched chitins degraded rapidly probably because the hydrolysis of these watersoluble branched chitins took place in homogeneous solution, whereas hydrolysis of linear chitin proceeded under heterogeneous conditions. Typical examples of the degradation behavior are shown in Fig. 3.1. Of the branched chitins with similar ds values, monosaccharide-branched chitins 12 and 20 had almost the same biodegradability as evidenced in this figure. Disaccharide-branched chitin 16, however, degraded much more slowly, indicating the steric hindrance by the bulky branches.'^'^''^'^ The influence of the extent of branching on biodegradation was also examined with A^-acetylglucosamine-branched chitins 20 having various ds values, and u.ou 20 (ds 0.13) 0.50^ 0.40 H o

20 (ds 0.37)

i 1 0.3020 (ds 0.45) 0.20-

*12(ds0.40)

0.10-

20 (ds 0.63) 16 (ds 0.42) ^ Chitin ^

0.00 i

\/^^r^ •

_

• 1

1

1

10

15

20

25

Time (h) Fig. 3.1 Enzymatic degradation of chitin and branched chitins with lysozyme (medium, pH 4.5 acetate buffer; temperature, 36°C; AAbsorbance, decrease in the absorbance of ferricyanide used to determine the resulting reducing ends): • , chitin; A, 12 (P-galactose-chitin) with ds 0.40; • , 16 (p-maltose-chitin) with ds 0.42; # , 20 (A^-acetyl-p-glucosamine-chitins) with ds 0.13, 0.37, 0.45 and 0.63.

3.2

(9-Glycosyl-branched Derivatives

59

the results are included in Fig. 3.1. The degradation rate of 20 with ds 0.13 was the highest. The rate decreased with increasing ds, and that of 20 with ds 0.63 was fairly low, which was again interpreted as steric hindrance by the large number of branches. These results suggest that the biodegradability can be regulated by introducing sugar branches, and that a low extent of branching enough to solubilize would be desirable to efficiently promote biodegradation.^^^ 4. Antimicrobial activity Antimicrobial activity is one of the most attractive biological properties of chitosan from the standpoint of applications in the fields of food and medicine. Branched chitosans were expected to have considerable activity due to the presence of free amino groups and considered of interest as potential water-soluble antimicrobial agents. Some typical results of the activities of branched chitosans having similar ds values are summarized in Table 3.1. Although all the branched chitosans and the original chitosan showed antimicrobial activity, 15 showed the lowest activity among these chitosans, implying lowered accessibility to the amino group due to the bulky disaccharide branches. Both 15 and 7 were less effective than chitosan. Glucosamine-branched chitosan 19, on the other hand, exhibited a somewhat higher antimicrobial activity than chitosan and may be a useful watersoluble antimicrobial agent with low toxicity. The activity has thus proved to be considerably dependent on the chemical structures of the branches and most likely on the density of free amino groups in these poly saccharides. ^"^'^^'^^^ Table 3.1 Antimicrobial activity of chitosan and branched chitosans Suppression of growth (%)^

Bacillus suhtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Streptococcus mutans Candida albicans

7

15

19

Chitosan

26 17 n.d. 93 n.d. n.d.

20 n.d. n.d. 16 n.d. n.d.

96 93 24 84 56 96

62 81 21 83 51 72

^ Percentage of the colony forming units decreased by the treatment of microbe precultures with chitosans in aqueous lactic acid (c 5 ppm): 7 (a-mannose-chitosan) with ds 0.42; 15 (j8-maltose-chitosan) with ds 0.40; 19 (j3-glucosamine-chitosan) with ds 0.45; n.d., not determined.

3.2.2 Synthesis Based on Trimethylsilyl-chitosan A. Silylation Although the above-mentioned synthetic procedure based on phthaloyl-chitosan has assured fully regioselective introduction of branches into chitin and chitosan and is superb for the synthesis of branched products with well-defined structures, it consists of nine reaction steps from chitin to branched chitins. To facilitate practical use, a simpler method of synthesis is desirable. Direct glycosylation of

60

3

Biologically Active Branches to Chitin and Chitosan

chitin with oxazoline 17 was attempted with the expectation that the reaction would take place almost exclusively at the C-6 position because of the bulkiness of the branches, but this was unsuccessful with a- and P-chitins. This is attributable to the low solubility and hence the low reactivity of chitin due to strong intermolecular forces. Protection of the hydroxy groups of chitin by silylation would interfere with intermolecular hydrogen bonding and consequently enhance affinity for organic solvents. Unlike phthaloylation of the amino group, silylation of the hydroxy groups will possibly leave the groups available for modification reactions. As has been reported, trimethylsilylation of polysaccharides such as cellulose,^^'^^^ amylose^^^ and dextran^^^ improved affinity for organic solvents. Trimethylsilylation of chitin was also reported but resulted in partial substitution (ds 0.6) with hexamethyldisilazane in formamide at 70°C under the conditions where cellulose gave a fully substituted product (ds 3.0),^^^ indicating the poor reactivity of chitin. The reaction behavior was thus investigated under various conditions in pyridine with a mixture of hexamethyldisilazane and chlorotrimethylsilane on shrimp a-chitin and squid p-chitin to establish a procedure for full substitution. Although both a-chitin and p-chitin gave fully silylated chitin (ds 2.0) (21), p-chitin was more suitable for facile substitution (Scheme 3.9).^"^^ The 0-Si linkages of 21 were fairly stable in neutral aqueous solution, but readily hydrolyzed with dilute acid, supporting easy protection and deprotection.

NHAc

(Me3Si)2NH MeaSiCl

OSiMe3 MesSiO NHAc 21

HsO^ Scheme 3.9

The resulting silylated chitin was soluble in acetone and pyridine. Moreover, the silylated hydroxy groups showed considerable reactivity in organic solvents such as pyridine, indicating that silylated chitin 21 has high potential as a convenient organosoluble precursor for modification reactions.^^^ B. Branching Glycosylation of 21 with oxazoline 17 was conducted in 1,2-dichloroethane with camphorsulfonic acid as the catalyst to give a branched product (22) (Scheme 3.10). Hydrolytic deprotection of 22 with aqueous sodium hydroxide gave the corresponding branched chitosan (19), while transesterification of 22 with methoxide afforded the branched chitin (20). In this manner, 20 could be synthesized from chitin in three steps, making this procedure superior to the above method based on phthaloyl-chitosan in simplicity and overall yield.^"^^

3.3

A^-Branched Derivatives

61

.OH NH2

\^o _

NaOHaq OAc AcO—r^O .

NH2

19

NHA 21

17 CSA

^ 0

Ha3

NHAc . n 22

OH HO--c^O ( HO-^^^"^-" NHA NaOCH3

Ha3 NHAc _ 20

Scheme 3.10

3.3

A^-Branched Derivatives

3.3.1 Reductive A^-Alky lation A. With Reducing Sugars The amino group of chitosan is suitable for efficient substitution reactions provided appropriate reaction conditions are apphed, and sugar branches can be introduced. The resulting derivatives are structurally different from naturally occurring branched polysaccharides where branches are present as 0-glycosyls, but they are important from the viewpoint of easy structural diversification of chitosan. Reductive alkylation is a simple and convenient way to modify the amino group selectively. In the reaction, the amino group is condensed with an aldehyde or a ketone to give an imine (Schiff base), which is then reduced to form a C-N bond with borohydrides. Reducing sugars are regarded as aldehydes or ketones and used for reductive alkylation. Chitosan was thus subjected to the reaction with lactose followed by reduction with sodium cyanoborohydride in aqueous acetic acid to give A^-substituted product (23) (Scheme 3.11). This is the first example of synthetic sugar-branched chitosans.^^^ The xerogels formed after the reaction were examined by scanning electron micrography, and a wide variety of ultrastructures were observed, ranging from nonporous to microporous structures.^^^ Besides lactose, various reducing sugars were used in this reaction; they include glucose, A^-acetylglucosamine, glucosamine, galactose, cellobiose, maltose, melibiose, maltotriose, fructose and dextran.^^^ Streptomycin sulfate was similarly incorporated to give a chitosan/streptomycin conjugate of biomedical interest. A branched product (24) from A^-acetylglucosamine is shown in Scheme 3.12.

62

3

Biologically Active Branches to Chitin and Chitosan HO ^ O H 1) UO~^^T^^^ OH

OH HO-V^^^^-0NH2

^-^^^O

OH

CQJ^

HOA-^-^^^ONH

2) NaCNBHB HO ^ O H HO

OH C^,, OH

OH

23 Scheme 3.11

OH

HO

OH O NH2

HO 1) HO

OH NHAc

2) NaCNBHs

HO

OH O

o-

NH AcHN HO HO'^-^T^oi OH OH 24

Scheme 3.12

The resulting branched derivatives were soluble in water or aqueous acetic acid. Some derivatives are interesting as water-soluble polysaccharides with unique solution properties. For instance, an aqueous solution of 23 prepared from chitosan and lactose showed unusual non-newtonian features such as low-shear newtonian behavior, a medium shear viscosity increase (dilatancy) and a highshear viscosity drop (pseudoplasticity).^^^ Nitroxide spin labels could be attached to the C-6 position of the terminal galactose unit of the lactose residue to reveal the gel characteristics of the soluble chitosan derivatives. The spin-probe — spinlabel method was used to demonstrate structural heterogeneities for the chitosan derivatives."^^^ Dielectric relaxation of an aqueous solution of 23 was measured in the frequency range from 1 MHz to 1 GHz, and pronounced dielectric dispersions were observed. The high-frequency dielectric dispersion was attributed to the motion of the branches.^'^ Similarly, fructose-modified chitosan was prepared and may be useful as a scaffold for hepatocyte attachment.^^^ Chitosan derivatives obtained from various mono- and disaccharides were used to discuss the solubility and rheological behavior."^^^ A galactose derivative having an iminodiacetic acid unit at the C-6 position (25) was used for reductive alkylation to prepare a chitosan derivative having metal chelating groups (26) with the galactose moiety as a hydrophilic spacer arm

3.3

iV-Branched Derivatives

63

(Scheme 3.13). Although the Cu(II) binding capacity of 26 was comparable to that of chitosan, 26 had better ion-exchange ability. Derivative 26 was water soluble, and the solution viscosity decreased on addition of Cu(II) ions.'^'^^

HO

OH O NH2

L^o 1)

HO-^^^^T"^OH OH 2) NaCNBHs

25

26 Scheme 3.13

Chitosan was reductively A^-alkylated with a trisaccharide having a reactive 2,5-anhydromannofuranose unit (27)/^^ which was prepared from partially deacetylated chitin, to give derivatives (28) with ds 0.07-0.40 (Scheme 3.14). A derivative with ds 0.40 was soluble in water. The apparent pATa values of the primary amine (unsubstituted) and secondary amine (substituted) were determined to be 6.5-6.9 and 5.0-5.2, respectively. Size exclusion chromatography indicated that the incorporation of such sugar branches did not affect the molar hydrodynamic volume of chitosan."^^^ The reductive alkylation of chitosan with carbonyl compounds is usually conducted in aqueous acetic acid or a mixture with methanol, but supercritical carbon

OH HO

o oNH2

HO^ 28 Scheme 3.14

64

3

Biologically Active Branches to Chitin and Chitosan

dioxide could also be used as a medium. The reaction with glucose and maltooligosaccharides in CO2 was more facile and complete than in conventional media, resulting in water-soluble imine derivatives with high ds values."^^^ B. With Formyl-bearing Sugars The method described above is simple for introducing sugar groups at the amino group of chitosan, but as a result of reductive alkylation using a reducing end, the sugar moieties are linked to chitosan in open-chain forms. When a sugar derivative having aldehyde functionality in the aglycone unit is applied, it will enable sugar groups to be incorporated without opening the ring. Actually, as shown in Scheme 3.15, glucose and galactose were introduced with a spacer arm of Cio. The products (29 and 30) were soluble in aqueous acetic acid to give solutions that formed gels at 50°C, but the gels reverted to solutions on cooling to room temperature.^^^ ^OH HO ,(CH2)9-CHO O^^^^T-^O 1) HO OH 2) NaCNBHs

OH HOA^-^^^^^ONH

^OH H O - ^Vr ^^ Q O ^ .(CH2),( H HO-W^-^OOH 29

^OH NH2

HO OH t ^ O (CH2)9-CHO 1) H O - V - - r ^ ^ OH 2) NaCNBHs

OH HO^^^^^^O NH

HO

OH

I

OH 30 Scheme 3.15

Ozonolysis of allyl glycosides gave sugar derivatives having an aldehyde group (31), formylmethyl glycosides, which allowed the introduction of sugar moieties with a shorter C2 spacer arm (Scheme 3.16). The ds was controlled by the amount of glycosides. In this way, chitosan derivatives containing galactose (32) were prepared, and the rheology of aqueous solutions of the derivatives was dependent on the ds."^^^ Other sugars such as glucose, lactose and A^-acetylglucosamine were also introduced by the same method, and the resulting derivatives with ds above 0.3 were soluble in water. Rheological behavior of aqueous solutions of these derivatives having glycosyloxyethyl groups was examined, and the solution viscosity and pseudoplasticity were found to increase with decrease in

3.3

A^-Branched Derivatives

65

ds.^^'^^^ Mannose, lactose, fucose and A^-acetylglucosamine were introduced in a similar manner to give derivatives whose influences on the aggregation of lectins and bacteria and on the active oxygen generation by canine polymorphonuclear leukocyte cells were elucidated.^^^ The water-soluble derivatives appeared to sensitize the cells by a priming mechanism.^^^ HO

OH

HO^OH "*

HO

£5 HO

31

H

OH

NaCNBHs

HO^V^-^-^-^^^ONH OH HO^S^-^^^^^0NH2

HO

OH

lA^O

/(CH2)2

32 Scheme 3.16

A chitosan derivative bearing pendant 1-thio-P-glucose groups via a spacer arm was prepared by reductive alkylation and crosslinked with glutaraldehyde. The product exhibited a specific interaction with p-glucosidase and was suggested to be useful for affinity chromatography.^'*^ Reductive alkylation with pformylphenyl melibioside gave a disaccharide-branched chitosan that had a lectinbinding capacity ascribable to the presence of a-galactosyl groups.^^^ A formyl derivative of sialic acid, which has important biological functions, was used to introduce sialic acid into chitosan. /7-Formylphenyl a-sialoside (33) was convenient for reductive alkylation (Scheme 3.17), and the derivative (34) with ds 0.53 was soluble in water, but those with ds below 0.44 were not soluble.^^'^^^ They were treated with succinic anhydride to improve water solubility, and the products aggregated wheat germ agglutinin and Grijfonia simplicifolia lectin due to the sialic acid moieties. Using branched polyamines as spacers, the sialic acid group could be introduced in a dendritic form, although the ds was rather low, and C02Na

HO

OH O

o-

NH2

34 Scheme 3.17

66

3

Biologically Active Branches to Chitin and Chitosan

the products are expected to exhibit some biological activities characteristic of sialic acid.^^'^^^ Starting from/?-isothiocyanatophenyl a-sialoside, sialodendrimers containing an aldehyde group (35) were synthesized, and they were introduced into chitosan similarly to obtain derivatives (36) having the sialic acid groups as exemplified by the trivalent dendrimer in Scheme 3.18. After deprotection with aqueous alkali, the resulting derivatives were only slightly soluble in neutral water and thus treated with succinic anhydride to modify the remaining free amino groups to enhance solubility.^^^

2) NaCNBH3 3) NaOHaq

3.3.2

A^-Acylation

Compared to the easy and simple method of reductive alkylation using aldehydes or ketones, A^-acylation of chitosan with carboxylic acids has not been explored much to form chitosan/sugar conjugates. Acylation of chitosan with a-glucoheptanoic acid y-lactone at room temperature gave the corresponding A^-substituted product.^^^ In an attempt to produce a hepatocyte-targeting DNA carrier, lactobionic acid having a galactose group was coupled with chitosan by a water-soluble carbodiimide, and some of the remaining free amino groups were reductively A^-alkylated with dextran to increase hydrophilicity.^^^ Acylation of chitosan with gluconic acid in the presence of a watersoluble carbodiimide was also attempted, but the ds was low.^"^^

3.4

3.4

Cyclodextrin-branched Derivatives

67

Cyclodextrin-branched Derivatives

Hydrophobic organic substances form host-guest inclusion complexes with cyclodextrins, which are cyclic amylose oligomers composed of a-(1^4) linked glucopyranose units. The affinity depends on the size of both organic substances as guests and cyclodextrins as hosts. Two kinds of cyclodextrins, a- and p-cyclodextrins (hexamer and heptamer), have been used for introduction into chitosan, and the specific interactions of cyclodextrins on chitosan with some organic substances were studied.

3.4.1 Reductive Alkylation For the reductive alkylation of chitosan with cyclodextrins, an aldehyde group should be introduced into cyclodextrins beforehand. An aldehyde-bearing cyclodextrin, Cf-aldehydo-cyclohcpiamylost, was synthesized by controlled oxidation of (J-cyclodextrin and allowed to react with chitosan in the presence of sodium cyanoborohydride. The product could possibly be used for catalysis, metal complexation and pharmaceutical preparations.^^^ a-Cyclodextrin and p-cyclodextrin were transformed into formylmethylated cyclodextrins (37) by allylation followed by ozonolysis, and the products were then subjected to reductive alkylation of chitosan (Scheme 3.19). The ds could be raised to 0.59. The derivatives (38) having a- and p-cyclodextrins included /7-nitrophenoxide in aqueous solution, and the dissociation constants of the host-guest complexes were calculated to be comparable to those of the free cyclodextrins.^^'^^^ Chitosan beads having a-cyclodextrin or p-cyclodextrin branches were prepared by crosslinking chitosan beads with hexamethylene diisocyanate followed by reductive alkylation with formylmethylated a-cyclodextrin or P-cyclodextrin. They were suggested for use in affinity chromatography, controlled release of drugs and removal of endocrine disrupting chemicals.^^'^^^ A P-cyclodextrin bearing an amino group, 6-amino-6-deoxy-P-cyclodextrin (39), was allowed to react with uronic acid to give A^-acylated derivative (40), and CHO ^O OH HO

o

NH2

o-

37 NaCNBHa

^OH HO^Vi^^^^O NH

O 38 Scheme 3.19

68

3

Biologically Active Branches to Chitin and Chitosan

the resulting reducing part was used for reductive alkylation of chitosan (Scheme 3.20). The affinity of the chitosan derivative (41) for organic substances was evaluated with ^butylbenzoic acid and catechin as model guests. The inclusion properties of the grafted cyclodextrin were confirmed to be similar to those of free cyclodextrin in terms of complex geometry and complexation ability. The derivatives may thus be useful for encapsulation and delivery of biologically active 69) species OH

NH.

H O ^ ; ^ O H

^ ^ ^

HO CO2H 40

39 OH HOA^^^^^ONH2

NaCNBHs

Scheme 3.20

Supramolecular assemblies based on chitosan/cyclodextrin derivative 41 were observed in solution. As a hydrophobically modified polymer, chitosan having adamantyl groups (42) was prepared by reductive alkylation with an aldehydecontaining adamantane (Scheme 3.21). Polyethylene glycol and its diamino derivative were modified with a carboxy-containing adamantane through esterification and amidation to give diadamantyl products (43 and 44). Specific interactions of 41 with 42, 43 and 44 were elucidated in solution by rheological measurements, and the behavior was dependent on the guest macromolecules. A network structure formed as a result of physical crosslinking with multiadamantane-grafted chitosan 42, whereas a marked increase in the viscosity was observed with endcapped polyethylene glycols 43 and 44.^°^

3.4

Cyclodextrin-branched Derivatives

69

,CHO

HO

OH O NH2

NaCNBHa

/CO2H

HO-fCH2CH20-fCH2CH20H

C-0-fCH2CH204-CH2CH20-C

Carbodiimide

Jm 43

/CO2H

H2N CH2CH2O

CH2CH2NH2

-NH

Carbodiimide

CH2CH2O

CH2CH2NH-

44 Scheme 3.21

3.4.2

Other Reactions

Low molecular weight chitosan prepared by partial degradation was acylated with carboxymethylated P-cyclodextrin (45) with a water-soluble carbodiimide in aqueous solution, giving a derivative (46) with ds 0.25 (Scheme 3.22). The inclusion behavior was discussed using 6-(/7-toluidino)-2-naphthalene-6-sulfonate as the guest molecule. Both 46 and free cyclodextrin form 1:1 complexes, but the former showed a lower equilibrium constant than the latter.^^^ CO2H

p ^OH NH2

45 Water-soluble carbodiimide

^OH HO^i^^^^^O NH

46 Scheme 3.22

70

3

Biologically Active Branches to Chitin and Chitosan

6-Amino-6-deoxy-P-cyclodextrin 39 could be used for amidation of A^-carboxymethyl-chitosan by a water-soluble carbodiimide (Scheme 3.23). The product (47), covalently linked to macroporous silica gels, exhibited chiral recognition ability to separate some DL-amino acids.^^^ p-Cyclodextrin was introduced into chitosan by treatment with a monochlorotriazinyl derivative of p-cyclodextrin. The extent of substitution was apparently low, but the product decontaminated water containing textile dyes.^^^ Oxidation of P-cyclodextrin with periodate gave a cyclic polyaldehyde (although the structure may not be simple) which crosslinked chitosan by Schiff base formation. The product loaded with Cu(II) worked as a catalyst for the oxidation of adrenaline.^"^^ OH HO

o

NH2

OCHCO2H NaCNBH3

HO

OH O

O"

NH CH2

COOH 39 Water-soluble carbodiimide

HO

OH O NH I PH2

o=cV[H 47 Scheme 3.23

3.5

Peptide-branched Derivatives

In addition to the presence of free amino groups, chitosan has notable biological features including biocompatibility, biodegradability and low toxicity. These characteristics have made chitosan quite attractive as a carrier for pharmaceutically active peptides. The conjugation makes possible control of the release rates and enzymatic degradation of the peptides.

3.5.1 N-Acylation Peptide side chains could be introduced by ring-opening graft copolymerization of a-amino acid A^-carboxy anhydrides (NCAs). Free amino groups of chitosan may work as initiators, but attempts in organic solvents resulted in poor extent of grafting on account of the limited swelling. However, because of the water solubility, randomly 50% deacetylated chitin (48)^^"^^^ showed high reactivity in aqueous solution. The graft copolymerization of y-methyl L-glutamate NCA was accomplished by treating an aqueous solution of 48 with an ethyl acetate solution of the NCA at

3.5

Peptide-branched Derivatives

71

0°C to give chitin/polypeptide conjugates (chitin-gra/]t-poly(Y-methyl L-glutamate)s) (49), which were subsequently converted to water-soluble derivatives (50) by alkaline hydrolysis (Scheme 3.24)/^^ Even though NCAs are susceptible to hydrolysis, the grafting efficiency was as high as 91% in aqueous media under these conditions/^^ Graft copolymerization of NCAs onto partially deacetylated chitins was also possible in DMSO, but the grafting efficiency was moderate owing to the heterogeneous reaction conditions.^^^ The polypeptide chains introduced were useful as spacer arms having a terminal free amino group, and the polyalanine chains of chitin-gra/r-poly(L-alanine) were available to immobilize the dihydronicotinamide group.^^^ COOMe (CH2)2

^OH

1

HO-V^^^^-O^ NHAc \p[

r

H N ^

^OH HO-V^^^^O NH2

^OH

1

r

HO^V^^^^'O^ NHAc jp

^OH HO^V^-^^:^0 NH

I

C-CH-NH^H II

Water-soluble chitin (/?=