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Selective chemical modification of polysaccharides 1980

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SELECTIVE CHEMICAL MODIFICATION OF POLYSACCHARIDES by MANSUR YALPANI B.Sc, Simon Fraser-University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1980 (c)Mansur Yalpani, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ZhfllgO ABSTRACT Various synthetic procedures were designed for selectively trans- forming carbohydrate polymers into versatile products with a wide range of applications using as substrates several abundant and industrially important polysaccharides (alginate, cellulose, chitin, chitosan, guaran, locust bean gum, xanthan gum). Thus, alkyl amide, amine, ester, and hydrazine derivatives of alginic acid as well as similar derivatives of cellulose and xanthan gum were prepared. Selective modification of chitosan afforded N-aryl derivatives which were found to be highly efficient metal-chelating agents. Attachment of carbo- hydrate moieties to the amino groups of chitosan using reductive alkyla- tion yielded a new class of branched, comb-like, 1-deoxyglucit-l-yl derivatives which exhibited a great diversity of useful properties in terms of solubility, gel-formation, compatibility, and interaction with other polysaccharides. The synthetic principles of this procedure were found to be amenable for adaption to other polysaccharide systems and branching types, as exemplified by a guaran derivative with chain- extended trisaccharide branches. Reductive alkylation of chitosan using ferrocenylaldehyde and sodium cyanoborohydride produced a new type of organometallic polysaccharide derivative. Combined enzymic and chemical modifications were found to be high-yielding for guaran and locust bean gum: specific oxidations using galactose oxidase afforded versatile C6' aldehyde intermediates which were reductively aminated to produce a wide variety of useful derivatives including i i synthetic glycoproteins and glycopeptide analogues. Various spectroscopic and other instrumental techniques were employed for the characterization of the polysaccharide derivatives and some native polymers in terms of their primary structure, three dimen- sional shape, and surface structure, as well as the molecular mobility of the hydrated polymer chains. Thus, nitroxide spin-labelling was utilized to monitor the course of many of the modification reactions, and provided evidence for a heterogeneous galactosyl-branch distribution for guaran and locust bean gum which was in agreement with a recently proposed structural model for these polymers. Esr experiments sug- gested heterogeneities in surface structure for chitosan and cellulose. The structural elucidation of several branched-chain chitosan deriva- tives and of guaran and locust bean gum was accomplished using 10C-nmr spectroscopy. Electron microscopy (SEM) of xerogels derived from branched-chain chitosan derivatives revealed a wide variety of ultra- structures ranging from smooth, non-porous to microporous. i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENT x i INTRODUCTION 1 Chapter I BACKGROUND 7 I-A. Polysaccharide Structure, O r i g i n and Use . . . 7 1. A l g i n i c Acid 7 2. Xanthan Gum 8 3. C e l l u l o s e 10 4. Galactomannans (Guar gum and Locust Bean gum) 13 5. Chitin/Chitosan 16 I-B. Solution Properties of Polysaccharides . . . . 19 1. S o l u b i l i t y 19 2. V i s c o s i t y 22 3. Gels 26 4. Applications 27 I-C. Polysaccharide M o d i f i c a t i o n 29 I- D. A n a l y t i c a l Methods 30 1. Magnetic Resonance 31 ( i ) Nmr 31 ( i i ) Esr 31 2. Elemental Microanalysis 47 II CHEMICAL MODIFICATION OF ALGINIC ACID, XANTHAN GUM, AND CELLULOSE 54 I I - A. Background 54 1. Introduction 54 2. Covalent Linkages 54 ( i ) C r i t e r i a used i n previous studies . . 55 ( i i ) C r i t e r i a used i n t h i s study 57 3. Selection C r i t e r i a 58 i v II-B. Chemical Modifications 63 1. A l g i n i c Acid 63 (i ) A l k y l ether 64 ( i i ) A l k y l ester 66 ( i i i ) Amides 68 (iv) Amines 71 (v) Hydrazines 75 (vi) s-Triazine derivatives 76 2. Xanthan Gum - 77 (i ) Amides 78 3. C e l l u l o s e 81 (i ) Amines 81 ( i i ) Urethanes 86 ( i i i ) Hydrazine 88 I I - C. Summary 89 II I BRANCHED-CHAIN CHITOSAN DERIVATIVES 94 I I I - A. Introduction 94 III-B. Synthesis and Properties of Branched-chain Chitosan Derivatives 95 1. Synthesis 95 2. Solute Interactions of Chitosan 99 3. G e l l i n g Processes 101 ( i ) Solute-mediated gelation 101 ( i i ) Gelation of branched-chain d e r i v a t i v e s 102 4. Other Properties 102 III-C. 1 3 C nmr 104 III-D. Spin-Labelling of C h i t i n and Chitosan . . . . 112 1. Introduction 112 2. Spin-Labelling of C h i t i n and Chitosan . . 113 3. Esr Spectra 116 4. S e l e c t i v e Broadening Experiments 122 I I I - E. Scanning Electron Microscopy 128 1. Introduction 128 2. SEM of Chitosan 130 3. SEM of Branched-Chain Chitosan Derivatives 130 IV COMBINED ENZYMIC AND CHEMICAL MODIFICATIONS 140 IV- A. Introduction 140 IV-B. Galactomannans 141 1. Modif i c a t i o n with Galactose Oxidase . . . 141 (i ) Guar gum 141 ( i i ) Locust bean gum 143 ( i i i ) Esr 144 (iv) Applications 150 (v) Nmr 152 (vi) V i s c o s i t y 157 ( v i i ) Compatibility 160 2. Periodate Oxidation 161 v V MODIFICATION OF THE METAL-CHELATING CAPACITY OF CHITIN AND CHITOSAN 167 V-A. Introduction 167 V-B. Mod i f i c a t i o n of Chelating Capacity and S o l u b i l i t y 168 1. Enhancement of Chelating Performance . . . 168 (i) Synthesis and copper complexes . . 168 ( i i ) Esr of copper complexes 174 2. Reduction of Chelating Capacity 179 3. S o l u b i l i t y M o d i f i c a t i o n 180 V-C. Organometallic Chitosan Derivative 182 VI SUMMARY 186 VII EXPERIMENTAL 194 VII-A. General Methods 194 1. Electron Spin Resonance 194 2. Nuclear Magnetic Resonance 195 3. Synthetic Methods 196 4. Materials 196 5. Product Nomenclature 197 VII-B. Chapter II 198 1. Materials 198 2. Synthesis 198 ( i ) Carbodiimide coupling 199 ( i i ) Via propylene g l y c o l esters . . . . 199 VII-C. Chapter I II 205 1. Scanning Electron Microscopy (SEM) . . . . 205 2. Materials 206 3. Preparation of 1- d e o x y g l y c i t - l - y l Chitosan Derivatives 206 (i) General procedure 206 ( i i ) Reactions of chitosan with reducing sugars 206 ( i i i ) Attempted chitosan d e r i v a t i v e s . . 210 (iv) Reactions of chitosan with non- reducing sugars 210 (v) [N-cyclohexane] chitosan [16] . . . 211 4. Oxidations with Galactose Oxidase . . . . 211 5. Interaction of [5] with Other Polysaccharides 212 6. Spin L a b e l l i n g of Chitosan and C h i t i n . . 212 VII-D. Chapter IV 215 1. V i s c o s i t y Measurements 215 2. Oxidation Procedures 215 3. Reductive Amination—General Procedure . . 217 4. Borodeuteride Reduction of C6-Aldehyde Derivatives 219 v i VII-E. Chapter V 220 1. Materials 220 2. Synthesis 220 3. Copper Complexation Reactions 224 APPENDIX 226 v i i LIST OF TABLES Table I - l . V i s c o s i t i e s of 1.0% Gum Solutions at 25°C 23 1-2. G e l l i n g Mechanism of Polysaccharides 27 1-3. Relationship Between Certain Properties and Applications 28 I I I - l . Reactions of Chitosan with Carbohydrates and Other Compounds 97 III-2. Chemical S h i f t Assignment of Chitosan and Some Derivatives 105 III-3. T Values for some Spin-labelled C h i t i n and c Chitosan Derivatives 120 III-4. U l t r a s t r u c t u r e C h a r a c t e r i s t i c s 136 IV-1. Compatibility of some Guaran Derivatives 161 V - l . Copper Chelation Performance of some C h i t i n and Chitosan Derivatives 170 v i i i LIST OF FIGURES Chapter I Figure I - l . S t r u c t u r e of a l g i n i c a c i d 8 1-2. S t r u c t u r e of xanthan gum 9 1-3. S t r u c t u r e of c e l l u l o s e 11 1-4. Proposed s t r u c t u r e s f o r the c e l l u l o s e m i c r o f i b r i l . . 12 1-5. Composition of guaran and l o c u s t bean gum 14 1-6. S t r u c t u r a l models proposed f o r guaran and l o c u s t bean gum 15 1-7. S t r u c t u r e of c h i t i n 16 1-8. Proposed s t r u c t u r e s of (a) a - c h i t i n , (b) g - c h i t i n . . 18 1-9. Representation of the space occupied by the g y r a t i o n of polysaccharides 21 1-10. R h e o l o g i c a l phenotypes 24 1-11. Rheogram of pseudoplastic f l u i d 25 1-12. Rheograms of polysaccharides 25 1-13. Generalized scheme f o r g e l l i n g of polysaccharides . . 26 1-14. Energy l e v e l diagram f o r a n i t r o x i d e 33 1-15. D i r e c t i o n a l dependence of Zeeman and h y p e r f i n e i n t e r a c t i o n s 34 1-16. Esr s p e c t r a of m a g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e showing g- and A - a n i s o t r o p i e s 36 1-17. Esr sp e c t r a of n i t r o x i d e s p i n - l a b e l i n aqueous g l y c e r o l s o l u t i o n s 37 1-18. Simulated s p e c t r a f o r i s o t r o p i c a l l y tumbling n i t r o x i d e s 41 1-19. Power spectrum showing the heights d i and d and the s p l i t t i n g 2A 42 1-20. Spin s t a t e energy l e v e l diagram f o r a copper ( I I ) nucleus 46 I - 21. Scheme of copper ( I I ) esr spectrum 46 Chapter I I I I - l . Esr s p e c t r a of [10] 65 I I - 2 . Esr s p e c t r a of [12] 67 I I - 3 . Esr sp e c t r a of [16A] 69 I I - 4 . Esr sp e c t r a of [16B] 70 I I - 5 . Esr s p e c t r a of [18A] 72 I I - 6 . 1 3 C nmr spectrum of p - f l u o r o a n i l i n e a l g i n a t e d e r i v a t i v e 74 I I - 7 . Esr spectrum of [21] 76 I I - 8 . Esr spectrum of [24] 77 I I - 9 . Esr spectrum of [26] 79 i x 11-10. Esr spectrum of [27] 80 11-11. Esr spe c t r a of [31] 82 11-12. Esr spe c t r a of [34], [35] 85 11-13. Esr spe c t r a of [37] 87 I I - 14. Esr spectrum of [40] 89 Chapter I I I I I I - l . I3c-nmr sp e c t r a of [1] v 106 I I I - 2 . l^c-nmr spectrum of [5] 107 I I I - 3 . 13c-nmr sp e c t r a of [7] and [6] 109 I I I - 4 . -^c-nmr spectrum of [16] 110 I I I - 5 . Esr spectrum of [25] 117 I I I - 6 . Esr s p e c t r a of [31] 118 I I I - 7 . Esr sp e c t r a of [34], [35] 119 I I I - 8 . Esr s p e c t r a of [33], [35] i n the presence of N i ( I I ) 123 I I I - 9. P l o t of c e n t r e - f i e l d l i n e w i d t h of [35] as a f u n c t i o n of added n i c k e l s u l f a t e 124 111-10. Esr s p e c t r a of [31], [34] i n the presence of N i ( I I ) 126 I I I - l l . Esr s p e c t r a of [29], [30] i n the presence of N i ( I I ) 127 111-12. P l o t of i n c r e a s e of c e n t r e - f i e l d l i n e w i d t h of [31], [35], [33] as a f u n c t i o n of added n i c k e l s u l f a t e 127A 111-13. SEM of [ 1 ] , [ 2 ] , [3] 131 111-14. SEM of [ 6 ] , [ 7 ] , [10] 132 Chapter IV IV-1. Esr sp e c t r a of [3] and [7] 145 IV-2. Proposed s t r u c t u r e s f o r guaran and l o c u s t bean gum 146 IV-3. Esr sp e c t r a of [3] 148 IV-4. D e r i v a t i v e s obtained from aldehyde intermediates [2] and [6] 151 IV-5. l aC-nmr spec t r a of [ 1 ] , [5] 153 IV-6. 1 3 C - s p e c t r a l r e g i o n of C l , C4, C5, C6 f o r [1] . . . 155 IV-7. Apparent v i s c o s i t i e s of guaran d e r i v a t i v e s as a f u n c t i o n of shear s t r e s s 158 IV-8. Logarithmic p l o t of apparent v i s c o s i t i e s of guaran d e r i v a t i v e s versus shear s t r e s s 159 IV-9. Rheogram of a 0.3% food-grade guar gum s o l u t i o n . . 160 IV- 10. Esr s p e c t r a of aqueous s o l u t i o n s of [21], [22] at 298K 162 IV-11. Esr spectra of [21] and [22] at 77K 162 Chapter V V-1. Esr s p e c t r a of Cu(II) complexes of [ 3 ] , [ 4 ] , [5] [10], [11], [12], [13] at ambient temperature . . 175 V-2. Esr spe c t r a of Cu(II) complexes of [ 3 ] , [ 4 ] , [ 5 ] , [10], [11], [12] at 77K 176 V-3. Esr spe c t r a of Cu(II) complexes of [1] and [7] . . . 177 V-4. Esr s p e c t r a of Cu(II) complex of [5] a f t e r v a r i o u s r e a c t i o n times 178 x ACKNOWLEDGEMENT I would like to express my deep appreciation to Dr. L. D. Hall for his interest and constant encouragement throughout the course of this work. It is a pleasure to thank a l l members and visitors of this lab, particularly Liane Evelyn, Mike Adam, Jeremy Sanders, Subramanian Sukumar, David Verrinder, John Waterton, and Kim Wong, for numerous helpful discussions and inspiration. I am also grateful to Mr. V. Bourne and Drs. D. Brooks, C. Culling, P. Reid, P. Salisbury, L. Srivastava, and I. Whyte for the generous loan of equipment, advice, and assistance, as well as to Dr. S. Ganapathy for obtaining solid state spectra of some derivatives prepared here. My special thanks are due to my wife, Talieh, without whose support this project could not have been completed, and to my brother, Nasser, whose efforts provided special 'insight' to some aspects of this work. xi INTRODUCTION Carbohydrates are incr e a s i n g l y being recognized f o r t h e i r immense 1 2 value i n numerous i n d u s t r i e s ranging from foods, pharmaceuticals, 3 4 t e x t i l e s , t e r t i a r y o i l recovery to the treatment of environmental p o l l u t a n t s . ^ They include the most abundant organic molecules on earth^ ( c e l l u l o s e and c h i t i n being produced at ca. 1 0 1 1 tonnes/year) and play a v i t a l r o l e i n the plant world, 7 where c e l l u l o s e i s the p r i n c i p a l g s t r u c t u r a l component and starch the main energy store, i n Crustacea and 9 i n s e c t s , where c h i t i n provides s k e l e t a l support, i n many b a c t e r i a , where e x o c e l l u l a r polysaccharides constitute a protective mechanism against a c c i d e n t a l d e h y d r a t i o n , ^ through to many facets of human l i f e where they are components of antibodies, enzymes, nuc l e i c acids, and c e l l surface g l y c o c o n j u g a t e s t h e y are of i n t e r e s t as a renewable 12 13 source of carbon, for energy storage, and as r e a d i l y manipulable 14 natural source of c h i r a l i t y . 15-17 Considerable attention has been directed i n recent years towards the development of an understanding of the properties of carbo- hydrate polymers i n aqueous s o l u t i o n where they can n a t u r a l l y occur i n a va r i e t y of sequences, as free e n t i t i e s as w e l l as i n d i f f e r e n t types of associations, and i n a corresponding multitude of conformational states. The o v e r a l l s t r u c t u r a l complexity of t h i s s i t u a t i o n i s frequently added to by s t r u c t u r a l inhomogeneities of the polymers both i n terms of d i s t r i b u t i o n s i n molecular weight or degree of polymerization (poly- 2 m o l e c u l a r i t y ) , or small d i f f e r e n c e s i n the sequence or s u b s t i t u t i o n pat- t e r n ( p o l y d i s p e r s i t y ) . Although these f a c t o r s have c o n t r i b u t e d t o the d i f f i c u l t y of e s t a b l i s h i n g the primary s t r u c t u r e and three-dimensional shape of many polysa c c h a r i d e systems i n general, new technology, i n the form of chemical and p h y s i c a l methodology, i s r a p i d l y being developed which i s p r o v i d i n g i n s i g h t i n t o the va r i o u s hydrated and condensed s t a t e s of many carbohydrates. Nevertheless, the p r e d i c t i v e understanding of the important r e l a t i o n s h i p between t h e i r s t r u c t u r e and f u n c t i o n i s s t i l l i n the s t a t e of infancy;'*'"' i t s extent can be summarized i n the d i s t i n c t i o n of v a r i o u s conformational f a m i l i e s of polysaccharides which are a s s o c i - ated w i t h c e r t a i n sequence f a m i l i e s , each of which may produce i n t r i n s i c p h y s i c a l p r o p e r t i e s . ^ ' C l e a r l y , new techniques f o r determining primary and secondary s t r u c t u r e and f o r studying t h e i r f u n c t i o n are re q u i r e d . Another challenge f a c i n g the polysaccharide chemist d e r i v e s from the growing demands f o r the sy n t h e s i s of c o n t r o l l e d - s o l u b i l i t y polymers 18 f o r s p e c i a l purposes such as blood plasma expanders, drug r e l e a s i n g 19 m a t r i c e s , or c o l l e c t i n g agents f o r precious or i n d u s t r i a l l y important 5 9 metals. ' With t h e i r r i c h abundance, low c o s t , and general v e r s a t i l i t y , p o lysaccharides c o n s t i t u t e i d e a l candidates f o r such a p p l i c a t i o n s . At the same time, the l i m i t a t i o n s of many of the procedures t r a d i t i o n a l l y 7 8 used ' f o r the d e r i v a t i z a t i o n of polysaccharides are i n c r e a s i n g l y becom- ing obvious. Although the s p e c i f i c d e r i v a t i z a t i o n of polysaccharide 20 f u n c t i o n a l i t i e s , such as f o r example c a r b o x y l a t e s , i s by no means a no v e l t y nowadays, the p r e v a i l i n g trend i n poly s a c c h a r i d e m o d i f i c a t i o n s c o u l d , u n t i l r e c e n t l y , be a p p r o p r i a t e l y termed as " s t a t i s t i c a l chemistry"; 3 the polysaccharides, often activated under strongly alkaline conditions, were subjected to treatment with unselective reagents, affording products whose exact composition could usually be determined only with great 21 d i f f i c u l t y , i f at a l l . Xanthates and carboxyalkyl derivatives typify such products. The intractability of many of the native polymers, such as cellulose and chitin, and the limited range of functional groups available for derivatization (mostly hydroxyl groups, some carboxylate, amine, or sulfate functions) has, of course, greatly contributed to this situtation. The resolution of the above problems associated with polysaccharide systems i s envisaged by the author to require a two-fold strategy incorporating, on the one hand, the design of specific and high-yielding reactions which, simultaneously, are of sufficient v e r s a t i l i t y to offer the opportunity for systematic modification of polysaccharide structure, and, on the other, the combination of various spectroscopic and analytical techniques with which the induced structural changes can be probed at both the macro- and molecular-levels. The work to be described in this thesis deals with various aspects of polysaccharide modification using several different naturally- occurring polysaccharides. Before proceeding to the discussion of the results of this investigation, i t s scope and the organization in this thesis need to be defined. It was the intention of this study to develop various procedures whereby polysaccharides can be transformed from intractable materials which, for the purposes of derivatization, offer few functionalities with only slightly different reactivity, into versatile polymers with a wide range of applications; i t was consequently deliberately decided at 4 the onset to seek a general overview of the subject area in terms of the strategy proposed above, in preference to the detailed and comprehensive pursuit of any one particular of i t s aspects. The investigations to be described followed the course outlined below: i n i t i a l l y (Chapter II),-a series of chemical reactions were evaluated as to their u t i l i t y for derivatizing several representative types of polysaccharides (alginate, cellulose, xanthan gum). The reductive amination reaction emerged as the most versatile procedure and i t was subsequently (Chapter III) applied to chitosan, which, with i t s prominent amine function, offered an ideal substrate for specific and efficient modifications affording a new class of branched-chain deriva- tives. Next (Chapter IV), the same reaction was combined with an enzymic modification procedure to prepare, again specifically and in high yield, various galactomannan derivatives. Lastly (Chapter V), an effort was made to direct the experience gained towards a specific application area, exemplified here by the synthesis of metal-chelating derivatives of chitosan. An intercomparison of the various results i s given i n the short summary (Chapter VI) preceding the Experimental data (Chapter VII). Chapter I provides some background information about the polysaccharide substrates and some of the instrumental techniques used. As w i l l be seen, these techniques have played an important role in helping with the development of the basic chemistry. 5 References 1. I. W. Cottrell and K. S. Rang, Development in Industrial Micro- biology, 19, 117 (1978); R. K. Robinson and P. Khan, Plant Foods Man, 2, 113 (1978). 2. H. H. Baer and J. L. Strominger, The Amino Sugars, Academic Press, New York, LA, 1969; L. B. Jaques, Science, 206, 528 (1979). 3. R. L. Whistler, Industrial Gums, Academic Press, New York, (1973); J. D. Reid and R. M. Reinhardt, in Modified Cellulosics, R. M. Rowell and R. A. Young, eds., Academic Press, New York, 11 (1978). 4. J. Mulderink, Erzmetall., 29., 560 (1976); C. T. Githens and J. W. Burnham, Soc. Petr. Eng. J., 17, 5 (1977); B. Sandiford, Energy Comm., j4, 53 (1978). 5. B. Tabushi, Y. Kobuke, and T. Nishiya, Nature, 280, 665 (1979); M. Takahashi, K. Shinoda, T. Mori, and T. Kikyo, Japan Pat., 7803982 (1978), Chem. Abstr., 89, 64708 (1978). 6. R. M. Rowell and R. A. Young (eds.), Modified Cellulosics, Academic Press, New York, (1978); D. A.. Rees, Polysaccharide Shapes, Chapman and Hall, London, (1977). 7. R. L. Whistler, ed., Methods in Carbohydrate Chemistry, III, (1963). 8. R. L. Whistler, ed., ib i d . , IV (1963). 9. R. A. A. Muzzarelli, Chitin, Pergamon Press, New York, (1977). 10. S. A. Barker and P. J. Somers, in The Carbohydrates, IIB, W. Pigman and D. Horton, eds., Academic Press, New York, 573 (1970). 11. M. I. Horowitz and W. Pigman, eds., The Glycoconjugates, Academic Press, New York, (1977); R. C. Hughes, Membrane Glycoproteins, Butterworths, London, (1976); E. G. Brunngraber, Neurochemistry of Aminosugars, C. C. Thomas, Springfield, (1979); N. Sharon, Complex Carbohydrates, Addison-Wesley, Reading, (1975). 12. T. A. Hsu, M. R. Ladish, and G. T. Tsau, Chem. Techn., 315 (1980); K. Selby, in Industrial Aspects of Biochemistry, L. Spencer, ed., 787 (1973). 13. D. J. Manners, Adv. Carbohydr. Chem., 12, 262 (1957). 14. H. Brunner, Angew. Chem. Internat. Edn., 10, 249 (1971). 15. D. A. Rees, Polysaccharide Shapes, Chapman and Hall, London, (1977). 6 16. E. M. Morris, i n Techniques and Applications of Fast Reactions i n Solution, W. J . Gettins and E. Wyn-Jones, eds., 379 (1979); D. A. Rees, i n MTP Intern. Rev. S c i . , Org. Chem. Ser. One, VII, G. 0. A s p i n a l l , ed., 251 (1973). 17. J . H. E l l i o t t , i n E x t r a c e l l u l a r M i c r o b i a l Polysaccharides, ACS Symp. Ser., 45, 144 (1977). 18. P. Calvert, Nature, 280, 108 (1979). " 19. H. Moldenhauer and H. J . Loh, Pharmazie, 33_, 216 (1978). 20. D. Horton and T. Usui, i n Carbohydrate Sulfates, R. G. Schweiger, ed., ACS. Symp. Ser., 77, 95 (1978). 21. Y. L. Meltzer, Water-soluble Resins and Polymers, Noyes Data Corp., London, (1976). CHAPTER I BACKGROUND I-A. Polysaccharide Structure, Origin, and Use Polysaccharides can be variously classified in terms of primary structure, overall charge (neutral or polyelectrolyte), origin, etc."'" For the purposes of chemical modification i t may be most appropriate to categorize the polysaccharides used in this study according to their most prominent functional group. Thus, alginic acid and xanthan gum are anionic species characterized to a large extent by their C-6 carboxy- late functions, chitosan, with i t s 2-deoxy-2-amino group, belongs to the less commonly encountered class of cationic polysaccharides, while cellulose, guar, and locust bean gum are representative examples of the large group of polysaccharides which offer only hydroxyl groups for derivatization. The following provides a brief summary of some of the most pertinent aspects of the above polysaccharides. 1. Alginic Acid Alginic acid has been known and util i z e d for almost a century, while the details of i t s chemical structure and properties have only recently been established. The principal sources of this watersoluble gum are various species of marine brown algae (Phaeophyceae).^ The thickening, suspending, stabilizing, gel- and film forming properties of alginic acid have found various applications in the food, cosmetic, coating, paint, pharmaceutical, and other industries. 7 7 8 Fig. I - l . Structure of alginic acid. Alginic acid i s a (1-4) linked, linear block polymer of fs-D-mannopyranosyluronic (M) and a-L-gulo-pyranosyluronic (G) acid residues, containing sequences of both types (poly M) and (poly G), g alternating with regions of mixed structure (poly MG). The M/G ratio varies depending on the source of the material. The molecular weight of alginic acid has been reported to be as high as 10 6, but is commonly around 2.0-2.5 x 10 5 with degrees of polymerization ranging between 9 180-930. The structure i s depicted in Figure I - l . 2. Xanthan Gum Xanthan gum typifies microbial polysaccharides which, with their unique physical and chemical properties, have found numerous successful applications i n food, textile, agricultural, paint, and petroleum industries in recent years.^ Xanthan gum, produced by some 30 species of the genus Xanthomonas, is a high molecular weight (> 106) poly- saccharide which contains D-glucose, D-mannose, and D-glucuronate as well as acetyl (4.7%) and pyruvate (3%) as depicted below (Fig. 1-2).^ The secondary and tertiary xanthan structures are s t i l l largely unknown. However, evidence obtained from various instrumental techniques 12 has revealed order-disorder transitions of the polymer i n solution. The native, probably double-stranded, polymer fibre can be denatured 9 into single strands by heat treatment. The denatured xanthan strands can in turn be renatured by addition of sodium chloride as schematically shown below.^ Native Oanalurtd Rtnatured 10 One of the most s i g n i f i c a n t functional properties of xanthan gum 14 i s i t s a b i l i t y to control aqueous f l u i d rheology; i t forms homogeneous aqueous dispersions and solutions exhibiting high v i s c o s i t y , as well as having characteristics of both pseudoplastic and p l a s t i c polymer systems; thus, a rapid decrease i n v i s c o s i t y i s observed with increasing shear rate and, upon the release of shear, t o t a l v i s c o s i t y recovery occurs almost instantaneously. 3. Cellulose Ubiquitous cellulose i s one of the most important natural products on e a r t h . ^ I t i s produced, consumed and destroyed i n nature i n t r e - mendous quantities (10 1 1 tonnes/year).^»^ Man h a s u t i l i z e d t h i s material for an almost unlimited number of purposes ranging from f i b r e s , t e x t i l e s , f i l m s , p l a s t i c s , coatings, pharmaceuticals, cosmetics, foods, to t e r t i a r y o i l recovery. ' More recent interests i n cel l u l o s e derive from i t s potential use as a renewable source of carbon 17 6 CH-OH After more than 150 years of study, the question of the formation and structure of cellulose s t i l l remains to be f u l l y resolved. C e l l u - lose i s a linea r polymer of 0(1-4) linked D-glucose with chains assuming 1 18 a f l a t ribbon conformation, similar to c h i t i n (Fig. 1-3). ' The ribbons, i n turn, are packed into sheets. The biosynthesis of cellulose i n plant c e l l walls produces amorphous regions and regions, constituting F i g . 1-3. Structure of c e l l u l o s e : (a)sequence; (b)chains resemble • f l a t ribbon; (c) , (d)proposed chain packing 12 »oX • ,1 "t's Fig. 1-4. Proposed structures for the cellulose microfibril: (a) cross-section of f i b r i l ; order increases towards a central core (from ref. 18); (b) amorphous regions and microcrystallites within a single micro f i b r i l ; (c) model incorporating elements of (a) and (b), three surface types are shown (A,B,C,). 13 19 20 50% or more, of high c r y s t a l l i n i t y ( m i c r o c r y s t a l l i n e ) ( F i g . 1-4). ' The l a t t e r can be c h a r a c t e r i z e d by c r y s t a l l o g r a p h y . The e f f i c i e n t chain packing and the numerous i n t e r c h a i n hydrogen bonds account f o r the i n s o l u b i l i t y of c e l l u l o s e i n conventional s o l v e n t s , and i t s s t a b i l i t y 21 and mechanical s t r e n g t h . The a c c e s s i b i l i t y and r e a c t i v i t y of the hydroxyl groups i n the d i f f e r e n t regions of c e l l u l o s e have been the subject of c o n s i d e r a b l e a t t e n t i o n . The amorphous regions are known to have greater a c c e s s i b i l - 16 22 i t y to solvent or s o l u t e p e n e t r a t i o n than the m i c r o c r y s t a l l i n e r e g i o n s . ' Such, and other f i n d i n g s , have l e d to the proposal of s e v e r a l models f o r 18 23 the three dimensional s t r u c t u r e f o r c e l l u l o s e . ' The f i r s t model envisages an ordered c r y s t a l l i n e core surrounded by a r e g i o n whose order decreases w i t h d i s t a n c e from the core ( F i g . I-4a), whereas i n the second model, c r y s t a l l i n e and amorphous regions a l t e r n a t e along the micro- f i b r i l l a r a x i s ( F i g . I-4b). Figure I-4c shows a model which accommo- dates features of both a and b, i n v o l v i n g three types of s u r f a c e s , v i z . , h i g h l y ordered ones (type a ) , type b which are l e s s ordered but p h y s i c - a l l y s t i l l c l o s e to c r y s t a l l i n e r e g i o n s , which are l i k e l y to c o n t a i n i n t e r - c h a i n hydrogen bonds. Type c surfaces are much l e s s ordered due to t w i s t and t i l t d i s t o r t i o n s of the f i b r i l . 4. Galactomannans (Guar gum and Locust bean gum) Galactomannans are assuming an ever i n c r e a s i n g r o l e i n v a r i o u s branches of i n d u s t r y , notably i n foods, pharmaceuticals, paper products, 24 cosmetics, p a i n t s , d r i l l i n g , and e x p l o s i v e s . Guar gum and l o c u s t bean gum are two of the more important galactomannan polysaccharides which are mainly derived from the seeds of leguminous p l a n t s or from m i c r o b i a l sources. Their primary p h y s i o l o g i c a l f u n c t i o n appears to be the HO 6 CH,OH 14 Fig. 1-5. Composition of guaran (x/y = 1.8) and locust bean gum (x/y = 4.3) used i n t h i s study. retention of water (by solvation), preventing the drying out of the seeds, and also t h e i r capacity as food reserves. In addition, galactomannans assume important roles i n the i n h i b i t i o n of viruses and i n interferon 25 induction. The synergistic interactions of these non-ionic polymers with other polysaccharides, e.g., with xanthan gum, and with themselves, has attracted considerable attention"'" and e f f o r t s directed at the elucidation of t h e i r primary structure have so far been only p a r t i a l l y successful. Both guar gum (MW 220,000) and locust bean gum (MW 310,000) con- t a i n a 3-D (1-4) linked mannan backbone which carries a-D-galactosyl residues at the C-6 positions and assumes a ribbon-like structure. The mannose (M) to galactose (G) r a t i o varies from 1.8:1 for guaran to 4:1 24 for locust bean gum. The d i s t r i b u t i o n of galactose bearing mannose units (MG) was u n t i l recently, subject to some controversy. Previously i t was thought that the structure of these polysaccharides was regular 26 consisting of either a homogeneous d i s t r i b u t i o n of galactose residues (Fig. I-6a) or of alternating sequences of (poly M) and (poly G) blocks 28—30 (Fig. I-6b); however, recent evidence has suggested otherwise as shown by the representative structures i n Figure I-6c. Whereas i n 27 ^ G, r ? 0 0 n [ M - M - M - M - J ^ [ M - M M - M M - M - M J ^ 0 0 0 . O Q o o o o o o o o o • M - M - M M - M <A-l^ft-M^-M-I^M^-M-^-M-M.M-M-M-M-M-M-liil-M-M-tt-ft-M. O O O O O O 9 9 I O O O O O O O 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 9 9 9 9 9 0 9 O O J 9 O G O O O O ' O O MMMMMMMMMMMMMW-tfM^M-MMMKMM-MWMMMIl 9 9 9 999 9 o 0 j 9 9 9 9 0 0 0 [ i i ] [ i ] Fig. 1-6. Three structural models proposed for guaran and locust bean gum: (a) regular galactosyl d i s t r i b u t i o n , 2 6 (b) s t r i c t l y alternating block sequence,27 (c) most recent model (Painter, Grasdalen, Gonzalez 2 8 - 3 0) for (i) guaran, ( i i ) locust bean gum. 16 guaran MG units occur i n groups of mostly two to four, alternating with blocks of two or three contiguous M un i t s , one finds i n locust bean gum, long blocks of contiguous M uni t s , together with long sequences of alternating M and MG unit s , as well as sections with two or three contiguous MG units. The rheological properties of aqueous solutions of both gums are of p a r t i c u l a r interest for many reasons: they behave as non-Newtonian solutions which, by themselves, form no gels unless borate or t r a n s i t i o n 24 metal ions are added. The gums are stable over a wide pH range and are compatible with many s a l t s . The v i s c o s i t y and shear s t a b i l i t y of guar and i t s derivatives have made these preferred g e l l i n g agents for 31 fracturing f l u i d s i n the o i l industry. 5. Chitin/Chitosan 32 C h i t i n was f i r s t isolated from mushrooms i n 1811 and from 33 34 insects i n 1823. Chitosan was discovered i n 1859. The development of c h i t i n chemistry has proceeded at an e r r a t i c pace. I t took almost a century before the chemical constitution of both polysaccharides was firmly established and most of the information presently available on these polymers has been obtained since 1950. The f i r s t descriptions of 35 chitosan as a metal-chelating agent appeared only i n 1969 but compar- a t i v e l y l i t t l e research has so far been carried out on these important polymers. Fig. 1-7. Structure of c h i t i n . 17 C h i t i n i s very w i d e l y d i s t r i b u t e d i n the p l a n t and, to an even greater extent, animal kingdom, where i t s main f u n c t i o n i s the p r o v i s i o n 36 of s t r u c t u r a l and s k e l e t a l support. C h i t i n i s found most abundantly i n f u n g i , w h i l e c h i t o s a n i s obtained mainly by N - d e a c e t y l a t i o n of c h i t i n , but a l s o occurs i n nature. The importance of c h i t i n i s emphasized by i t s n a t u r a l abundance, an estimated 10 ̂ - 1 0 1 1 tons annually,"*" which makes i t one of the most abundant organic m a t e r i a l s on earth. C h i t i n i s a l i n e a r polymer of 3-D (1-4) l i n k e d 2-acetamido-2-deoxy- D-glucopyranose u n i t s , of which a p r o p o r t i o n , t y p i c a l l y ^15%, i s 37 N-deacetylated (the f u l l y a c e t y l a t e d polymer i s c a l l e d c h i t a n ) . The molecular weight of c h i t i n has been estimated at 1.04 * 1 0 6 , w h i l e that 38 of c h i t o s a n ranges between 1.45-1.80 * 10 s. C h i t i n , l i k e c e l l u l o s e , has a r i b b o n - l i k e s t r u c t u r e ( F i g . 1-8). Three c r y s t a l l i n e forms, ot, 3, and Y - c h i t i n are d i s t i n g u i s h e d on the b a s i s of d i f f e r e n t c h a i n 36 arrangements and the presence of bound water ( F i g . 1-8). In c o n t r a s t to most other p o l y s a c c h a r i d e s , c h i t i n and c h i t o s a n have b a s i c c h a r a c t e r - 39 i s t i c s (pK of c h i t o s a n i s 6.3) which impart them w i t h unique prop- e r t i e s i n terms of s o l u b i l i t y , v i s c o s i t y , p o l y e l e c t r o l y t e behaviour, membrane forming a b i l i t y and metal c h e l a t i o n . Both these aminopolysaccharides are i n s o l u b l e i n common organic 36 s o l v e n t s , water, d i l u t e a c i d s , or c o l d a l k a l i s of any c o n c e n t r a t i o n . There are only a few s o l v e n t s or solvent systems which do not give r i s e to h y d r o l y s i s , degradation, or N - d e a c e t y l a t i o n . C h i t i n d i s s o l v e s , f o r example, i n 9N HC£, or >9N H2SO4 w i t h h y d r o l y s i s of the g l y c o s i d i c and amide l i n k a g e s . The solvent systems f o r c h i t i n which are more s a t i s f a c t o r y i n c l u d e h e x a f l u o r o i s o p r o p a n o l , hexafluoroacetone s e s q u i - hydrate, c e r t a i n c h l o r o a l c o h o l s , and hot concentrated s o l u t i o n s of 18 Fig. 1-8. Proposed structures of (a) c t-chitin, (b) g-chitin. 19 n e u t r a l s a l t s which are capable of high degrees of h y d r a t i o n , such as 40 saturated l i t h i u m thiocyanate s o l u t i o n at 95°C. Chitosan i s s o l u b l e i n a number of organic a c i d s , i n c l u d i n g formic and a c e t i c a c i d . Chitosan products have found a wide range of i n d u s t r i a l a p p l i c a t i o n s and 41 c e r t a i n d e r i v a t i v e s have been i m p l i c a t e d i n cancer therapy, wound h e a l - i n g , ̂  and other m e d i c i n a l uses."^ I-B. S o l u t i o n P r o p e r t i e s of Polysaccharides 1. S o l u b i l i t y The outstanding i n d u s t r i a l importance of polysaccharides a r i s e s from t h e i r u s e f u l i n t e r a c t i o n s w i t h a wide v a r i e t y of molecules ranging from food i n g r e d i e n t s and pharmaceuticals, to i n o r g a n i c p a r t i c l e s , c l a y s l i p s and o i l w e l l muds. These i n t e r a c t i o n s occur mostly i n aqueous media and the s o l u b i l i t y p r o p e r t i e s of polysaccharides are t h e r e f o r e of great i n t e r e s t . Their unique s o l u b i l i t y p r o p e r t i e s c o n s t i t u t e , at the same time, one of the major problems i n the development of polysaccharide chemistry. Although these p r o p e r t i e s are known to f o l l o w c e r t a i n trends, a systematic understanding of the r e l a t i o n of primary p o l y - saccharide s t r u c t u r e to p h y s i c a l behaviour i n aqueous s o l u t i o n , and of p o l y s a c c h a r i d e - s o l u t e i n t e r a c t i o n s , e t c . , remains to be e s t a b l i s h e d . In contrast to other s y n t h e t i c polymers, r e l a t i v e l y l i t t l e e f f o r t appears to have been c a r r i e d out i n t h i s area so f a r , which can presumably be a s c r i b e d , to some extent, to the i n t r a c t a b i l i t y of these m a t e r i a l s . The f o l l o w i n g i s a b r i e f d e s c r i p t i o n of some p e r t i n e n t aspects of 2 43 'hydrated' p o l y s a c c h a r i d e s . Their d i s s o l u t i o n i s envisaged ' to proceed through a process i n which, beginning i n amorphous r e g i o n s , i . e . , where i n t e r m o l e c u l a r i n t e r a c t i o n s are only p a r t i a l l y o p e r a t i v e due to 20 the disorganized s p a t i a l arrangement of molecules or chain segments, c o n t i n u a l h y d r a t i o n of the polysaccharide r e p l a c e s i n t e r m o l e c u l a r H- bonding. The competition of water molecules f o r a v a i l a b l e polymer s i t e s leads e v e n t u a l l y to an intermediate " g e l s t a t e " i n which l a r g e s e c t i o n s of the polysaccharide are f u l l y s o l v a t e d w h i l e some incompletely s o l v a t e d areas are s t i l l a s s o c i a t e d w i t h other polysaccharide chains. The completion of t h i s d i s s o l u t i o n process f a c i l i t a t e s , under approp- r i a t e c o n d i t i o n s , subsequent arrangement of the polysaccharides i n t o more or l e s s ordered s t r u c t u r e s such as g e l s . 43 N a t u r a l p o l y s a c c h a r i d e s , of which some 300 are known to date, can be c l a s s e d i n t o d i f f e r e n t s o l u b i l i t y groups according to t h e i r s t r u c t u r e and conformation. Thus, l i n e a r polysaccharides w i t h a r e g u l a r , r i b b o n - l i k e s t r u c t u r e such as c e l l u l o s e and c h i t i n , form h i g h l y ordered, o f t e n c r y s t a l l i n e a r r a y s which are d i f f i c u l t to d i s s o l v e due to strong cohesive f o r c e s , whereas branching u s u a l l y leads to an enhancement i n s o l u b i l i t y and a r e d u c t i o n i n the i n t e r m o l e c u l a r i n t e r a c t i o n s . I t can a l s o be envisaged that f o r equal molecular weight p o l y s a c c h a r i d e s , a branched molecule w i l l r e q u i r e a smaller volume i n s o l u t i o n f o r g y r a t i o n than an extended l i n e a r one, as i n d i c a t e d i n Figure 1-9. Hig h l y branched p o l y - saccharides are almost always wa t e r s o l u b l e . S o l u b i l i t y can a l s o be a f f e c t e d by a s e r i e s of other f a c t o r s such as i o n i c charges, s t r u c t u r a l i r r e g u l a r i t i e s , g l y c o s i d i c l i n k a g e s which preclude ribbon s t r u c t u r e s , low molecular weight, and a wide molecular weight d i s t r i b u t i o n . The p o s s i b i l i t y of modifying polysaccharide s o l u b i l i t y by chemical d e r i v a t i z a t i o n i s a r e l a t i v e l y novel concept which i s ga i n i n g i n c r e a s i n g 44 importance f o r a wide v a r i e t y of m e d i c i n a l a p p l i c a t i o n s , i n c l u d i n g 45 polymer-mediated drug r e l e a s e . I t has long been known that the F i g . 1-9. Representation of the space occupied by the g y r a t i o n of (a) an extended l i n e a r p o l y s a c c h a r i d e ; (b) a branched polysaccharide of equal molecular weight (from r e f . 2). 22 i n t r o d u c t i o n of s u b s t i t u e n t s i n t o the l i n e a r c e l l u l o s e s t r u c t u r e can a f f o r d s o l u b l e d e r i v a t i v e s , w h i l e , conversely, the removal of branches, as i n the h y d r o l y s i s of the galactose side-chains of guaran, or the i n t r o d u c t i o n of hydrophobic groups or 3,6-anhydro r i n g s , as f o r s t a r c h 46 s u l f a t e s , leads to a r e d u c t i o n of polymer s o l u b i l i t y . Pfannemuller 47 et a l . have r e c e n t l y converted a s e r i e s of l i n e a r polysaccharides i n t o branched d e r i v a t i v e s , which r e s u l t e d , i n the case of amylose, i n d r a s t i c changes i n s o l u t i o n v i s c o s i t y . However, no precedent was known f o r applying t h i s concept f o r the purposes of s o l u b i l i z i n g an i n s o l u b l e p o l y s a c c h a r i d e . In t h i s study (TII-B) f a c i l e methods were explored f o r transforming an i n s o l u b l e , l i n e a r p olysaccharide (chitosan) i n t o s o l u b l e , branched d e r i v a t i v e s , which provides the opportunity f o r systematic s t u d i e s of the e f f e c t s of s t r u c t u r e on p h y s i c a l p r o p e r t i e s . One aspect of such s t u d i e s , the " t a i l o r i n g " of the h y d r o p h o b i c i t y / h y d r o p h i l i c i t y c h a r a c t e r i s t i c s of p olysaccharide d e r i v a t i v e s w i l l be demonstrated i n V-B. 2. V i s c o s i t y An important property of polysaccharides i s t h e i r a b i l i t y to impart 2 48 a wide range of v i s c o s i t i e s to aqueous s o l u t i o n s , ' as demonstrated by the r e p r e s e n t a t i v e s e l e c t i o n l i s t e d i n Table I - l ; (the comparable v i s c o s i t y of water i s ^1 cps at room temperature!). The d e f i n i t i o n of some b a s i c r h e o l o g i c a l terminology"''*" may be u s e f u l here. The r a t i o of shear s t r e s s , T (shearing f o r c e per area sheared) to shear r a t e , y (gradient of v e l o c i t y over clearance or f i l m t h i c k n e s s ) i s commonly defined as v i s c o s i t y , n.* The standard u n i t s f o r these are *or apparent v i s c o s i t y , u, f o r non-Newtonian f l u i d s 23 Table I - l . V i s c o s i t i e s of 1.0% Gum S o l u t i o n s at 25°C a Gum cps Gum a r a b i c (20% by wt.) 50 Locust bean gum - 100 M e t h y l c e l l u l o s e 150 Gum tragacanth 200 Carrageenan 300 Xanthan gumb 1000 H i g h - v i s c o s i t y sodium carboxymethyl c e l l u l o s e 1200 Gum karaya 1500 Sodium a l g i n a t e 2000 Chitosan (pH 4.1) C 2780 Guar gum 4200 adapted from r e f . 2 (except f o r ) b r e f . 49; c r e f . 50 -2 -1 dynes cm , sec , and poise or c e n t i p o i s e , r e s p e c t i v e l y . R h e o l o g i c a l f l u i d behaviour i s c l a s s i f i e d i n terms of c e r t a i n phenotypes which are diagrammatically shown i n Figure 1-10 as i d e a l i z e d curves. Of the var i o u s types, only three w i l l be o f concern to us, namely ( i ) Newtonian systems, which e x h i b i t a l i n e a r r e l a t i o n between shear s t r e s s and shear r a t e and which are u s u a l l y confined to low molecular l i q u i d s such as water; ( i i ) p s e u d o p l a s t i c ; and ( i i i ) d i l a t e n t systems. The l a t t e r two can be described by the r e l a t i o n n T = V where n.̂  i s the zero shear v i s c o s i t y ; f o r pseudoplastic or shear- t h i c k e n i n g systems n < 1, w h i l e n > 1 f o r d i l a t e n t o r shear t h i n n i n g systems. The most common type of non-Newtonian behaviour i s pseudo- p l a s t i c flow f o r which three d i s t i n c t regions are u s u a l l y encountered 24 thwr M i (torque) i F i g . 1-10. R h e o l o g i c a l phenotypes (from r e f . 49). as i l l u s t r a t e d i n the l o g a r i t h m i c p l o t of apparent v i s c o s i t y versus shear 48 r a t e ( F i g . 1-11). Most of the i n d u s t r i a l l y important polysaccharides f a l l i n t o the p s e u d o p l a s t i c category (e.g., xanthan, a l g i n a t e , guar gum, etc.) w h i l e 52 d i l a t e n c y has been observed f o r few (e.g., c e r t a i n s t a r c h d e r i v a t i v e s ) ; some t y p i c a l rheograms are shown i n Figure 1-12. 53 D i l a t e n c y can be envisaged to a r i s e from chain entanglement of polymers which, w i t h i n c r e a s i n g shear r a t e , leads t o v i s c o s i t y i n c r e a s e s , w h i l e s h e a r - t h i n n i n g can a r i s e from shear degradation of the polymer or from a p a r a l l e l alignment of the polymer chains i n the flow d i r e c t i o n r e s u l t i n g i n a r e d u c t i o n of the f l u i d ' s flow r e s i s t a n c e . The molecular o r i g i n of these processes, p a r t i c u l a r l y those g i v i n g r i s e to d i l a t e n t flow behaviour, i s , however, only incompletely understood. The c o n t r o l of r h e o l o g i c a l f l u i d behaviour has obviously important i m p l i c a t i o n s f o r the v a r i o u s a p p l i c a t i o n s of polysaccharides i n which they provide v i s c o s i t y , s o l u t i o n s t a b i l i t y , s u s p e n d a b i l i t y , e m u l s i f y i n g a c t i o n , g e l a t i o n , e t c . L i k e w i s e , the a b i l i t y to modify pol y s a c c h a r i d e c d Fig. 1-12. Rheograms of (a) 1% aqueous guar gum solution (from ref. 2); (b) xanthan gum (from ref. 49); (c) alginate (from ref. 54); (d) mixture of alginate and xanthan (from ref. 49). 26 2 rheology by chemical d e r i v a t i z a t i o n as f o r carboxymethylcellulose, or by i n t e r a c t i o n w i t h other polysaccharides as f o r the r e d u c t i o n of the p s e u d o p l a s t i c i t y of xanthan by a l g i n a t e ( F i g . I - l l e ) , w i l l c o n s t i t u t e an i n c r e a s i n g l y important concept i n the f u t u r e . 3. Gels Many polysaccharides have been shown''" to adapt ordered g e l s t r u c - tures by way of ( i ) a s s o c i a t i o n s i n t o double h e l i c e s as f o r e-carrageenan, ( i i ) bundles of double h e l i c e s as f o r agarose, ( i i i ) r i b b on-ribbon aggregations as f o r a l g i n a t e ; or ( i v ) s y n e r g i s t i c h e l i x - r i b b o n a s s o c i a - t i o n s as f o r mixed systems c o n t a i n i n g agarose and l o c u s t bean gum. Rees and coworkers^" have summarized these d i f f e r e n t c r o s s - l i n k e d networks of chains i n t o a g e n e r a l i z e d scheme ( F i g . 1-13). The common f e a t u r e of a l l G o n c r a l i M d j u n c t i o n t o n e s ' , w h i c h m a y • 4 M v o r a l t y p o s n a m e l y Fig. 1-13. Generalized scheme f o r g e l l i n g of polysaccharides (from r e f . 1 ) . these g e l l i n g mechanisms i s the cooperative a s s o c i a t i o n of two or more chain segments which i s o f t e n terminated by " i n t e r r u p t e d sequences," i . e . , by changes i n the sequence of sugar u n i t s , by ch a i n branching, or other a l t e r e d s u b s t i t u t i o n . Without going i n t o the d e t a i l s of the v a r i o u s i n d i v i d u a l g e l l i n g mechanisms, i t i s worthwhile t o p o i n t out the s e r i e s of d i s t i n g u i s h a b l e 27 Table I I - 2 . Polysaccharide gelling mechanism (from r e f . 5 5 ) Pictorial view of molecular states Name Significance for gel structure Corresponding range of gel properties Random coil Predominant in the sol; can exist as connecting lengths in gel structure and impart elasticity when they do so Double helix Provide cross- linking junctions in the gel " i f * Aggregate Add cross-linking to consolidate the gel structure, thus acting as super- junctions Sol ̂  Incipient gel ̂  Clear elastic gel ̂  Stiff gel «* Turbid rigid gel ̂  Phase separation; syneresed gel s t a g e s , s u m m a r i z e d i n Table 1-2 i n the p r o g r e s s i v e a s s o c i a t i o n of chains which leads to g e l networks, by way of , f o r example, h e l i c e s . In the s o l s t a t e , which can be c h a r a c t e r i z e d as a s t a t e of incomplete h y d r a t i o n of the polysaccharide chains due to aggregation phenomena, the chains assume the random c o i l conformation. The g e l , obtained when s u f f i c i e n t h e l i x has formed to provide c r o s s l i n k s , becomes i n c r e a s i n g l y more r i g i d w i t h f u r t h e r h e l i x a s s o c i a t i o n s a c t i n g as "s u p e r - j u n c t i o n s . " At a l a t e r stage, t h i s process may be accompanied by a l o s s of o p t i c a l transparency, and f i n a l l y , by synereses, i . e . , a c o n t r a c t i o n of the g e l network con- comitant w i t h the e x c l u s i o n of s o l v e n t . 4. A p p l i c a t i o n s I t i s apparent t h a t the aforementioned polysaccharide p r o p e r t i e s (and numerous others which could not be discussed here) a v a i l themselves to a v a r i e t y of a p p l i c a t i o n s and Table 1 - 3 (adapated from r e f . 5 6 ) 28 Table 1 - 3 . Relationship Between Certain Properties and Applications c 4-1 >> o c • H 4J • H « t H • H 4-1 a) cu • H r H e 3 4-1 >̂  o cu rQ • H r H r H • H o 4J a a cc) r O • H o > C •H n) C 4-1 CO C <4H CO • H cd O >> 4-1 « co - 4-t o M 4-1 t-l • H CO 4-1 CO • H 00 O cu 4-1 4-1 • H • H CO (U 4J C CO r H CO (3 r-l CO • H V4 c CO • H • H 0) o >> nJ • H 4-1 • H a) CO 3 o 6 r ^ r * V-l 4-1 4J O • H r Q cu 4J • H rJ C • H ft P, r H • H u cfl CO o • H • H c 4J CO o • H 4 - u C 4-1 r H r H • H C O T3 r O co 6 u 0) 0) CO CO cu cu O 3 r H 3 a. ct) & a. e CO CO 4-1 > ca CU CU r H e N QJ B CO r H o o o r H • H CO • H O o c r C 0) 3 • H V-i V-i VJ o > PH u w cn H CO c_> PH C/3 Tertiary o i l - recovery + + + + Oil-well d r i l l i n g + + + + + + + Paints + + + + Paper + + + + + + Textiles + + + + Explosives + + + Photography + + + + + Cosmetics + + + + + Toothpaste + + + Food + + + + + + + indicates some of the major relationships between polysaccharide prop- erties and various end uses. Table 1 - 3 shows that for a particular poly- saccharide to be of u t i l i t y to any one area of application, i t has to f u l l f i l in most cases more than a single requirement. It is with this consideration, that throughout this study potential applications of v a r i - ous polysaccharide derivatives are suggested on the basis of their observed characteristics. 29 I-C. Polysaccharide Modification In view of the immense u t i l i t y of polysaccharides in their native form, a substantial amount of work"^ has been directed at modifying them in order to obtain derivatives with altered chemical and physical properties. Such changes can frequently, particularly for neutral polysaccharides, be provoked by the introduction of only very small amounts of ionic substituents. However, in most of the derivatization procedures employed, the exact composition of the resulting products, in terms of the sequences of substituted and unsubstituted polymer functionality (e.g., hydroxyl groups), remains unknown due to the l i m i - tations of conventional analytical techniques, and one i s l e f t with the d i f f i c u l t y of characterizing the derivatives. A minimal description of the product can be given by the degree of substitution (d.s.), which i s generally defined as the average number of hydroxyl groups derivatized per monosaccharide unit. In view of the large numbers of, and differently positioned, hydroxyl groups which display very similar reactivities, a major d i f f i - culty encountered in polysaccharide modification arises from the fact that the reactions are often heterogeneous and do not proceed to completion. Although a few reactions employed in this study reflect some of these problems, the general conceptual approach chosen herein attempts to minimize such d i f f i c u l t i e s by exploring novel chemical and other routes, or combinations thereof, for preparing, in high yield, specifically derivatized polysaccharides. In this context, one reaction in particular, the reductive amination 58 reaction using sodium cyanoborohydride, deserves to be mentioned for i t s key role in achieving this goal. This versatile reaction, whose 30 mechanism is summarized below; is operative for a range of aqueous or organic solvents and pHs. Requiring only very mild conditions, the reductive amination reaction has been successfully applied to a wide variety of carbohydrates and other biological m a t e r i a l s . ^ ' ^ R-C ' 0 H+ » &C + H 2 N-R' R-C H o-NH-R' <- R-C -^N+- R' i i H H V H-B-OT H I-D. Analytical Methods The d i f f i c u l t i e s associated with the structural elucidation of polysaccharide derivatives were already alluded to; further complications arise when attempts are made to characterize the polymeric materials in terms of their molecular behaviour in the various solution (sol, gel) or solid states. Fortunately, the development of various modern instru- mental techniques in recent years has facilitated an immense progress in this area. 31 In this section i t is attempted to bri e f l y outline salient features of the magnetic resonance techniques employed without going into the details of several other methods (e.g., Scanning Electron Microscopy and Viscometry). Fuller descriptions of both the f o r m e r ^ a n d the l a t - t e r ~ ^ ' ^ techniques are found elsewhere. 1. Magnetic Resonance (i) Nmr Nuclear magnetic resonance (nmr) spectroscopy, particularly ^^C nmr, 60 i s a powerful analytical tool which i s routinely used for the elucida- tion of the primary structure and conformation of polysaccharides in solutions, gels, and more recently, in the solid state; no elaboration of the technique i s required here. However, the author would like to 63 direct attention to the u t i l i t y of nuclei other than carbon and proton which have not yet found extensive application in the analysis of poly- saccharide modification products. With the avai l a b i l i t y of modern instrumentation, nmr studies can be made of a practically unlimited range of magnetic nuclides which can be chemically incorporated into 63 polysaccharides. Thus, the wide chemical shift range of 1 9 F can be exploited to distinguish "products" from "unreacted reagent" as prelimin- ary experiments on alginate derivates have shown. Unfortunately, time did not permit the author to pursue these studies to any extent, ( i i ) Esr The following i s a very brief summary of some fundamental aspects of esr spectroscopy pertinent to the concepts and methods employed in this work; such treatment necessarily omits a vigorous quantitative derivatization (which the reader w i l l find elsewhere, e.g., in references 62, 64-68). 32 (a) Nitroxide esr Esr spectroscopy monitors the net absorption of energy by a para- magnetic molecule whose electron magnetic dipole i n t e r a c t s with an electromagnetic f i e l d Hg of frequency v. The magnetic moment of the elect r o n , characterized as the magnetic dipole moment u, can be simplis- t i c a l l y considered to derive from the spinning of the electron about an axis through i t s centre (spin magnetic dipole) with small contributions from the electron's motion about the nucleus of an atom ( o r b i t a l magnetic d i p o l e ) . Resonance occurs when t r a n s i t i o n s between the Zeeman l e v e l s , whose degeneracy may be l i f t e d by the a p p l i c a t i o n of a magnetic f i e l d Hg, are induced by an electromagnetic f i e l d , i . e . , when hv = ggH 0 (1) where h i s Planck's constant, f3 the Bohr magneton (enV2m) , m the electron mass, and g a dimensionless parameter rel a t e d to the e f f e c t i v e magnetic moment of the electron (u ) e w e = -ges (2) SE being the spin angular momentum vector. Differences i n the Zeeman energy between d i f f e r e n t molecules are described as changes i n g from i t s free electron spin-only value of 2.00232, as a r e s u l t of s p i n - o r b i t coupling. The g-value characterizes the p o s i t i o n of the resonance i n the frequency spectrum. Equation (1) i s s a t i s f i e d over a wide range of frequencies and magnetic f i e l d s ; most esr experiments including those described here, are conducted at X-band, i . e . , at about 9.5 GHz corres- ponding to an H value of -\-3.4 kG. The energy absorption i s monitored as the f i r s t d e r i v a t i v e of the absorption s i g n a l . F i g . 1-14. Energy l e v e l diagram f o r a n i t r o x i d e (s - i ) i n a magnetic f i e l d w i t h h y p e r f i n e c o u p l i n g to the s p i n 1 n i t r o g e n nucleus (from r e f . 67). 34 F i g . 1-15. D i r e c t i o n a l dependence of Zeeman and h y p e r f i n e i n t e r a c - t i o n s i n a n i t r o x i d e o r i e n t e d i n a diamagnetic host c r y s t a l , which was r o t a t e d i n the molecular xz plane (from r e f . 62). 67 For n i t r o x i d e f r e e r a d i c a l molecules such as 1 / V _ ,N= 0 - 1- 0 the p r i n c i p a l h y p e r f i n e i n t e r a c t i o n s occurs between the e l e c t r o n , shown i n the n i t r o g e n 2p z o r b i t a l and the n i t r o g e n nucleus ( 1 = 1 ) , l e a d i n g to the c h a r a c t e r i s t i c t h r e e - l i n e spectrum ( F i g . 1-14) i n which t r a n s i t i o n s obey the s e l e c t i o n r u l e s Am̂ . = 0, Am^ = ±1, (where m̂  and m̂. are values of the components of the e l e c t r o n and nuclear s p i n operators along the e x t e r n a l f i e l d d i r e c t i o n ) . The Zeeman and h y p e r f i n e i n t e r a c t i o n s are d i r e c t i o n dependent as demonstrated by the esr spectrum of a diamagnetic host c r y s t a l "doped" w i t h n i t r o x i d e ( F i g . 1-15). Both the p o s i t i o n (g) and the s p l i t t i n g of the l i n e s (A) are d i r e c t i o n dependent l e a d i n g to the p r i n c i p a l values g , g , g : A , A , A . The a x i a l symmetry of both tensors & x x & y y b z z ' xx' yy' zz J J a r i s i n g from the molecular symmetry, a f f o r d s 8 x x = Syy * 8 z z ° r 8 x x = g y y = ^ 3 n d g z z = 8 " (3) A = A ^ A or A = A = A, and A = A„ xx yy zz xx yy - zz A d i l u t e n i t r o x i d e s o l u t i o n a l s o produces a 3 - l i n e spectrum ( F i g . 1-16) but due to the averaging of the g and A a n i s o t r o p i e s (g„, g^ and A,,, A,) as a r e s u l t of molecular motion only the i s o t r o p i c s p l i t t i n g constant a 0 remains. Figure 1-16 a l s o shows the n i t r o x i d e spectrum i n a p o l y c r y s t a l l i n e s o l i d (e.g., at 77 K) i n which a l l p o s s i b l e n i t r o x i d e o r i e n t a t i o n s c o n t r i b u t e to the spectrum which i s simply the sum of resonances i n d i c a t e d i n F i gure 1-15. I t i s evident that w h i l e the c e n t r a l maximum contains c o n t r i b u t i o n s from a l l o r i e n t a t i o n s , the outer 36 F i g . 1-16. Esr sp e c t r a of m a g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e (a) i n a p o l y c r y s t a l l i n e s o l i d , (b) a v i s c o u s s o l u t i o n , and (c) a non-viscous s o l u t i o n showing g- and A - a n i s o t r o p i e s (from r e f . 67). 20 G F i g . 1-17. Esr spectra of n i t r o x i d e s p i n - l a b e l i n aqueous g l y c e r o l s o l u t i o n s ; v i s c o s i t y increases and temperature decreases from ( a ) - ( f ) (from r e f . 67). 3 8 extrema arise from radicals oriented with a molecular z-axis parallel to the external f i e l d . (b) Rotational correlation time, x c 6 2 It i s well known that nitroxide esr lineshapes are a sensitive - 1 1 - 7 function of molecular motion on a timescale of 1 0 to 1 0 sec. In order to affect the lineshape, such motion must partially average A or g tensor anisotropy (Fig. 1 - 1 6 ) . Reduced molecular motion leads to broadening of the high f i e l d line followed by the low f i e l d line and lastly the central line. Figure 1 - 1 7 demonstrates these effects for a system where the isotropic motion decreases and the correlation times (T ) increase. c The line shapes of the peaks in the spectra of isotropically tumbling molecules are assumed to be Lorentzian in which case the line width and the peak-to-peak height are a function of the transverse relax- ation time T2 given by tT 2(m I ) r 1 = T C { [ 3 1 ( 1 + 1 ) + 5 m 2 ] ^ + ^ ( A Y H Q ) 2 - j^AyK^} + X (4) where HQ is the applied f i e l d strength and b " 3* ( Azz " Axx> i n H z ( 5 ) ^ = ^ [ s 2 z - ^ x x + sy )] ( 6 ) X is a function which accounts for other contributions to the line width. This expression is only valid under the following conditions: ( 1 ) the hyperfine interaction is axially symmetric, i.e., A^ x = ( 2 ) the molecular motion is isotropic and sufficiently slow that W 2T 2 >> 1 (where co = p>eH0/h and b 2 T 2 « 1 ) , so that line widths are influenced; and ( 3 ) that T 2ag << 1 , which ensures that the three lines do not overlap. 39 Thus at X band the equations are applicable i n the range -1 -9 5 x 10 < t" c < 5 x 10 s. Usually f o r ni t r o x i d e s i n low v i s c o s i t y solvents these conditions are met so that i f we set 1 = 1 f o r nitrogen i n (4) the following expression i s obtained: T 2 ( 0 ) A " b 2 2 ^ - y = l - x c T 2 ( 0 ) [ I 5 b A Y H 0 m I - g m^ (7) The r a t i o T 2 (0) /T 2 (m̂ .) can be expressed i n terms of the r a t i o of the peak- to-peak heights by T 2(0) T 2 (m].) ho h m I (8) where h i s the peak-to-peak height i n a r b i t r a r y u n i t s . T 2(0) i s m l r e l a t e d to the l i n e widths by where Av(0) i s the l i n e width f or the c e n t r a l peak i n H z. I f we then i n s e r t (8) and (9) into (7) we obtain h 0 T h ~ = 1 " ^ 3 A v ( 0 ) [ C i m I + C 2 m i ] m I (10) where = -^bAyH 0 " (11) C 2 = b 2/8 (12) It i s now pos s i b l e to solve f o r x with mT = +1 and m_ = -1 i n terms of r c I I CJL or C 2 . Values of T c derived with C\ have been found to be very dependent on the microwave power^ so that i t i s best to avoid using unless the microwave dependence i s known. Therefore we solve (10) f o r T i n terms of c ^ 40 \ • i & + V->4 - 2 ' ' T I T 2 1 < 1 3 > c n^ n_^ Z C2 Because i t i s e a s i e r to measure the l i n e widths i n gauss than i n h e r t z we convert Av(0) to AH(0) and A - A from h e r t z to gauss by Eq. (1) Z Z X X w i t h g taken as the averaged g v a l u e . In gauss, Eq. (14) i s : x - + - 2] 9 / 3 h * H<°> (16) C n i -1 4TTgB (A -A )2 0 e zz xx For a n i s o t r o p i c r e o r i e n t a t i o n s of n i t r o x i d e s , a s i t u a t i o n more l i k e l y to p r e v a i l f o r a l a b e l l e d macromolecule, some a d d i t i o n a l f a c t o r s have to be noted. Many d i f f e r e n t and h i g h l y complex lineshapes can a r i s e from a n i s o t r o p i c r e o r i e n t a t i o n , and the best to q u a n t i t a t e the motion i s by s p e c t r a l s i m u l a t i o n . Programmes are a v a i l a b l e f o r a x i a l l y symmetric r e o r i e n t a t i o n i n which two c o r r e l a t i o n times, i n and TL are 69 d e r i v e d . In p r a c t i c e , however, at l e a s t where motional a n i s t r o p y i s -9 small and motions are r a p i d (T 'V. T m a* T, < 10 S ) , T has o f t e n been c a l - c u l ated using equation (16) or using s i m u l a t i o n methods which assume 62 i s o t r o p i c r e o r i e n t a t i o n . Some workers have even completely dispensed w i t h x u s i n g other parameters as e m p i r i c a l i n d i c e s of m o b i l i t y . ^ F i g ure 1-18 i l l u s t r a t e s lineshapes simulated^"*" on the assumption of i s o t r o p i c tumbling w i t h T values of 2.0 ns ( a ) , 3.2 ns ( b ) , and 5.0 ns ( c ) . Thus, the appearance of two s p e c t r a l 'components' at high f i e l d (as i n (c)) i s not, as might be i n i t i a l l y thought, n e c e s s a r i l y a s s o c i a t e d w i t h the e x i s t e n c e of two d i s t i n c t c o r r e l a t i o n times or w i t h the occurrence of motional anisotropy. In the present work, the exact F i g . 1-18. Simulated s p e c t r a f o r i s o t r o p i c a l l y tumbling n i t r o x i d e s w i t h x c (a) 2 ns; (b) 3.2 ns; (c) 5 ns (from r e f . 71). 42 2 A Fig. 1-19. Powder spectrum showing the heights di and d and the s p l i t t i n g 2A. quantitative evaluation of tumbling motions was, i n general, considered less important than a q u a l i t a t i v e comparison of motional rates of d i f f e r - ent polysaccharides or of a given system i n varying physical states, and, hence, computer simulations were not employed for x c determinations, (c) Distance measurements The esr s p i n - l a b e l l i n g technique provides, i n p r i n c i p a l , a number of independent methods for determining intermolecular distances. The method used here i s based on dipolar linebroadening. I f the r e l a t i v e reorientation of two spin-half radicals i s s u f f i c i e n t l y slow, the esr signal of each w i l l show a dipolar coupling with the other, whose magni- tude i s proportional to (3 cos 2 8 - l ) r where r i s the electron-electron distance and 8 i s the angle between the distance vector and the magnetic f i e l d . In a magnetically d i l u t e sample of radicals neither whose orientations nor whose positions are correlated, the esr signal should — 3 suffer a Laurentzian broadening AH^^ which i s proportional to r , where r i s the mean nearest-neighbour distance. For a random, three dimen- 43 sional spin distribution, r can be related to the spin label density, p (nm 3 ) , by r = ( ^ r p r 1 7 3 ! ^ ) (17) with r(n) = /Q e X x n - 1 d x , r ( j ) = 0.89261, and thus r = 0.55373 P~ 1 / 3 (18) The density i s related to the molar concentration by: p = 0.602 [C] (19) where [C] is the molar concentration of spin label. Substitution gives r = 0.656 [ C ] " 1 / 3 (20) The effective concentration of spin labels cannot be measured directly, since, however, AH, . is concentration dependent i t can be indirectly d i p obtained. The dj/d spectral parameter, f i r s t introduced by Kokorin et 72 a l . , i s a measure of AH^ , where and d can be derived from the powder spectrum, as defined in Figure 1-19. From calibration studies at 77 K, the concentration dependence of dj/d has been shown^ to be given by (T> " (T>-dii + 2' 0 4 t c ] ( 2 1 ) No dipolar broadening was found to exist for [C] < ^5 mM below which dj/d remains constant. This is the value of dj/d at i n f i n i t e dilution (d,/d) i.e., where no dipolar interactions occur between spins. This value varies somewhat from system to system and is a function of solvent polarity and the degree of residual motion at 77 K. Typical values are close 0.4. Combination of (21) and (19) yields d l d i _ _ o T " (T }»dii + °- 5 8 r ( 2 2 ) Eq. (22) can be used to measure distances in the regime of 1.0-2.4 nm.^ 44 (d) Spin-label-spin-probe experiments Interactions between spin labels, usually nitroxides, and spin probes, paramagnetic metal ions can provide valuable information about the topography of biological materials. ^ Two types of inter- actions may arise from the paramagnetic species: (i) dipole-dipole interactions which arise from induction of local magnetic fields by the magnetic dipole of paramagnetic group at the site of another; and ( i i ) Heisenberg exchange, which is caused by orbital overlap of unpaired electrons of proximal paramagnetic species. The interactions may be modulated by the spin-lattice relaxation times of the metal ions (Tj), by rotational diffusion of the metal-ion-free-radical assembly, or by translational diffusion of the metal ion with respect to the free radical. It i s essential to establish both the nature of the interactions and the significance of the modulation of the dominant interaction. No adequate theory exists to account for the range of possible situations, although 68 for transition metal complexes and nitroxides in low viscosity solu- 73 tions Heisenberg exchange has been found to be the dominant interaction. Likhtenshtein favours^"* the same mechanism for interactions between metal ions and nitroxide radicals covalently bound to proteins. Previous studies^"*" carried out in this laboratory have employed transition metal 2+ ions with very short Ti values, e.g., Ni for which Ti has been -̂ 2 —13 estimated at 'vLO - 1 0 S, in order to minimize dipolar contributions and maximize exchange interactions. The present study uses the same method. The spin-label-spin-probe method allows, in principal, for the resolution of complex esr spectra arising from a heterogeneous distribu- tion of radicals covalently attached to a macromolecule, since the esr 45 linewidth of the r a d i c a l AHQ i s re l a t e d to the rate constant for exchange r e l a x a t i o n (K) f o r encounters between the r a d i c a l and the paramagnetic metal ion by AH 0 = 6.5 x 10~8.K.C (23) . 65 where C i s the concentration of the spin probe. Hence, any hetero- geneities due to d i f f e r e n t a c c e s s i b i l i t i e s of lab e l s to the spin probe w i l l appear as d i s c o n t i n u i t i e s i n plot s of AH 0 versus C, because r e a d i l y a ccessible spin l a b e l s have higher K values than t h e i r l e s s accessible counterparts; the esr signals of the former should be broadened at lower probe concentrations. When rapid exchange occurs between the d i f f e r e n t l a b e l s i t e s , the experimental K values should become equal leading to the disappearance of the d i s c o n t i n u i t i e s i n the AHQ versus C pl o t s . 68 (e) Copper (II) esr For the d y configuration of copper(II) the e f f e c t i v e electron spin S = i and the spin angular momentum mg = ±% give r i s e to a double degenerate energy state. If the copper ion i s located i n a s i t e of lower symmetry than a perfect cubic c r y s t a l , the g and A values, as before, are o r i e n t a t i o n dependent (or anisotropic) as expressed i n equation (3), where the z-axis i s defined as coinciding with the highest- f o l d notation a x i s . When the unpaired Cu(II) electron couples with the nuclear spin (I = 3/2) the absorption s i g n a l i s s p l i t with 2 1 + 1 components, i n t h i s 74 case four l i n e s ( F ig. 1-20). In a p o l y c r y s t a l l i n e sample no averaging of the g and A anisotropies occurs. With the random d i s t r i b u t i o n of molecular symmetry axis, the observed resonance l i n e s represent the sum of the superimposed i n d i v i d u a l resonances. The spectrum shows a F i g . 1-21. Scheme of copper (II) esr spectrum showing both g„ and g A components. 47 weak set of l i n e s at g n , corresponding to molecules whose symmetry axis are p a r a l l e l to the applied f i e l d and a set of strong l i n e s at g x corresponding to perpendicular alignment as schematically shown i n Figure 1-21. Superhyperfine structure, r e s u l t i n g from hyperfine i n t e r a c t i o n s of spin 1 nitrogen nucleus and the electron spin, i s sometimes resolved. In the copper-complexes discussed i n Chapter V no such i n t e r a c t i o n s were observable. The A and g values can be abstracted d i r e c t l y , i f desired, from the f i e l d - c o r r e c t e d spectra. The A,, values are taken as the separation between the +i and -i l i n e s , and a 0 values are derived from the ~i, -3/2 separation. Both g„ and gg are obtained from the centre point between the +i and — i l i n e s . Equation (24) converts the values of these hyperfine constants (units of magnetic f i e l d ) i n t o frequency u n i t s . A(cm - 1) = A(gauss) x -S- x 9.3484 x l ( f 5 (24) 8 e 2. Elemental Microanalysis Elemental microanalysis i s a standard technique for studying poly- saccharide modifications since i t provides a f a c i l e determination of the, otherwise often d i f f i c u l t to e s t a b l i s h , degree of s u b s t i t u t i o n (d.s.). It i s , however, necessary to explain to the reader the nature of the "fudging" factor which accompanies most of the elemental analysis r e s u l t s i n the Experimental section. Most polysaccharides have a saturation vapour pressure of hydration roughly equivalent to that of phosphorous pentaoxide, and at normal 43 humidities they contain 8-10% water as water of hydration. It w i l l be consequently understood, why, despite even the greatest precautions and the most stringent specimen drying procedures, a c e r t a i n small percentage 48 of water of h y d r a t i o n i s u s u a l l y a s s o c i a t e d w i t h elemental a n a l y s i s data. To i l l u s t r a t e the p o i n t , the e f f e c t of water on the C, H, N percentages of N-c y c l o h e x y l c h i t o s a n (d.s. 0.5) i s l i s t e d below. Anal, f o r [C 8H!3 N O 5 ) 0. 02 (C 6Hi 1 N O 4 )„. k B ( C j 3 H 2 3 N 0 U ) 0 . 5 l n [MW] H 2 0 present C - H N C/N c a l c d . 209. 96 0 54.53 8.18 6.67 8.18 212. 05 0.12 (1%) 53.99 8.21 6.60 8.18 216. 25 0.35 (3%) 52.94 8.27 6.47 8.18 218. 35 0.47 (4%) 52.43 8.30 6.41 8.18 found 52.56 8.36 6.44 8.16 The C/N r a t i o s p r o v i d e , of course, another measure of the r e l i a b i l i t y of the a n a l y t i c a l r e s u l t s . Throughout t h i s work two s i t u a t i o n s were encountered where elemental a n a l y s i s d i d not provide u s e f u l i n f o r m a t i o n . The f i r s t was i n systems where the degree of s u b s t i t u t i o n was very low and m i c r o a n a l y s i s proved to be not s e n s i t i v e enough. The second case was found f o r a s e r i e s of a l g i n a t e d e r i v a t i v e s which gave u n e x p l i c a b l y low C, H, and, f o r s p i n - l a b e l l e d d e r i v a t i v e s , N percentages, d e s p i t e v a r i o u s e f f o r t s to remove any i m p u r i t i e s ; ( i t should be noted that a l g i n a t e and most other p o l y - saccharides c o n t a i n considerable q u a n t i t i e s of v a r i o u s t r a c e metals and were consequently p u r i f i e d p r i o r to use). In such s i t u a t i o n s , recourse was taken to esr double i n t e g r a t i o n and/or other s p e c t r o s c o p i c t e c h - niques, such as 1 3 C nmr. 49 References 1. D. A. Rees, Polysaccharide Shapes, Chapman and Hall, London, (1977). 2. R. L. Whistler (ed.), Industrial gums, Academic Press, New York, . 2nd edition, (1973). 3. G. 0. Aspinall, Polysaccharides, Pergamon, New York (1970). 4. G. 0. Aspinall (ed.), MTP Intern. Rev. Sci., Org. Chem., Series One, 1_ (1973). 5. F. A. Bettelheim, in Biological Polyelectrolytes, (A. Veis, ed.), Marcel Dekker, New York,. 131 (1970). 6. E. Percival and R. H. McDowell, Chemistry and Enzymology of Marine Algal Polysaccharides, Academic Press, New. York (1970). 7. K. B. Guiseley, 'Seaweed Colloids,' Kirk-Othmer Encycl. Them. Technol., 2nd Edition, 17, 763-784, Wiley, New York, (1968); W. H. McNeely and D. J. Pettit, in ref. 2, p. 49. 8. A. Haug, B. Larsen, and 0. Smidsryid, Acta Chem. Scand., 21, 691-704 (1967); A. Haug, B. Larsen, and 0. Smidsr^d, ib i d . , 20, 183-190 (1966); A. Penman, and G. R. Sanderson, Carbohydr. Res., 25, 273-282 (1972); J. Boyd, and J. R. Turvey, ib i d . , 66, 187-194 (1978). 9. F. G. Donnan and R. C. Rose, Can. J. Res., B-28, 105 (1950); A. Haug, Norwegian Inst. Seaweed Res., Trondheim, Norway, Rept. No. 30, (1964). 10. W. H. McNeely and K. S. Kang, in ref. 2, p. 486; R. Moorhouse, M. D. Walkinshaw, and S. Arnott, in Extracellular Microbial Poly- saccharides, (P. A. Sandford and A. Laskin, eds.), ACS Symp. Ser. 45, 90 (1977); A. Jeanes, J. Polym. Sci., Symp. No. 45, 209 (1974). 11. G. Holzwarth, Carbohydr. Res., 66, 173 (1978). 12. G. Holzwarth, Biochemistry, 15, 4333 (1976); E. R. Morris, D. A. Rees, G. Young, M. D. Walkinshaw, and A. Drake, J. Moi. Biol., 110, 1 (1977); C. S. H. Chen and E. W. Sheppard, J. Macromol. Sci. Chem., A13, 239 (1979). 13. G. Holzwarth and E. B. Prestridge, Science, 197, 757 (1977). 14. P. J. Whitcomb, B. J. Ek, and C. W. Macosko, ACS Symp. Ser., 45, 160 (1977). 15. F. Hoyle, A. H. Olaveson, and N. C. Wickramasinghe, Nature, 271, 229 (1978). 50 16. R. M. Rowell and R. A. Young (eds.), Modified Cellulosics, Academic Press, New York (1978). 17. T. A. Hsu, M. R. Ladisch, and G. T. Tsau, Chem. Techn., 315 (1980). 18. 0. H. Northcote, Essays Biochem., 8, 89 (1972). 19. R. H. Marchessault, and A. Sarko, Adv". Carbohydr. Chem., 22_, 421 (1967). 20. A. J. Stipanovic, and A. Sarko, Macromolecules, 9_, 851 (1976); A. Sarko, J. Southwick, and J. Hayashi, ibid., % 857 (1976); A. D. French, Carbohydr. Res., 61, 67 (1978). 21. A. H. Nissan, Macromolecules, 10, 660 (1977); A. F. Turbak, R. B. Hammer, R. E. Davies, and N. A. Portnoy, ACS Symp. Ser., _58, 12 (1977). 22. H. T. Kokhande, J. Appl. Polym. Sci., 20, 2313 (1976). 23. For a review, see, S. P. Rowland, and E. J. Roberts, J. Polym. Sci., A- l , 10, 2447 (1972). 24. A. M. Goldstein, E. N. Alter, and J. K. Seaman, in ref. 2, p. 303; R. Roi, ib i d . , p. 323. 25. P. M. Dey, Adv. Carbohydr. Chem. Biochem., 35, 341 (1978). 26. R. L. Whistler, Adv. Chem. Ser., 11, 45 (1954). 27. C. W. Baker and R. L. Whistler, Carbohydr. Res., 45, 237 (1975). 28. J. J. Gonzalez, Macromolecules, 11, 1074 (1978). 29. T. J. Painter, J. J. Gonzalez, and P. C. Hemmer, Carbohydr. Res., 69, 217 (1979). 30. H. Grasdalen and T. Painter, ib i d . , 81, 59 (1980). 31. C. J. Githens and J. W. Burnheim, Soc. Pet. Eng. J., 17, 5 (1977). 32. H. Braconnot, Ann. Chi. Phys., 79, 265 (1811). 33. A. Odier, Mem. Soc. Hist. Nat. Paris Jl, 29 (1823). 34. C. Rouget, Comp. Rend., 48, 792 (1859). 35. R. A. A. Muzzarelli and 0. Tubertini, Talanta, 16, 1571 (1969). 36. R. A. A. Muzzarelli, Chitin, Pergamon Press, New York (1977). 51 37. R. L. Whistler, in ref. 2, p. 465; J. N. BeMiller, in Methods in Carbohydr. Chemistry, _5, 103 (1965). 38. R. A. A. Muzzarelli, Natural Chelating Polymers, Pergamon Press, New York (1973). 39. L. A. Nud'ga, E. A. Plisko, and S. N. Danilov, Zhur. Obs. Khim., 43, 2752 (1973). 40. C. L. McCormick and D. K. Lichatowich, J. Polym. Sci., Polym. Lett. Edn., 17, 479 (1979). 41. A. E. Sirica and R. J. Woodman, J. Nat. Cancer Inst., 47, 377 (1971). 42. L. L. Balassa and J. F. Prudden, Proc. Fir s t Intern. Conf. Chitin/ Chitosan, (R. A. A. Muzzarelli and E. R. Panser, eds.), 296 (1978). 43. R. L. Whistler, in Carbohydrates in Solution, ACS Ser., 117, 242 (1973). 44. P. Calvert, Nature, 280, 108 (1979); C. Schuerch, Adv. Polym. Sci., 10, 173 (1972); L. B. Jaques, Science, 206, 528 (1979). 45. P. Ferruti, M. C. Tanzi, and F. Vaccaroni, Makromol. Chem., 180, 375 (1979); H. Moldenhauer and H. J. Loh, Pharmazie, 33, 216 (1978). 46. R. L. Whistler and S. J. Hirase, J. Org. Chem., ̂ 26, 4600 (1961). 47. B. Pfannemuller, G. Richter, and E. Husemann, Carbohydr. Res., 56, 139 (1977); H. Andresz, G. C. Richter, and B. Pfannemuller, Makromol. Chem., 179, 301 (1978). 48. J. H. E l l i o t t , in Extracellular Microbial Polysaccharides, ACS Symp. Ser., 45, 144 (1977). 49. Xanthan Gum, Kelco Co., Los Angeles, 1976. 50. L. J. Fi l a r and M. G. Wirick, in Proc. First Intern. Conf. Chitin/ Chitosan, 144 (1978). 51. J. H. Bradbury, in Physical Principles and Techniques of Protein Chemistry, JB, (S. J. Leach ed.), Academic Press (1970); R. S. Lenk, Polymer Rheology, Applied Science Publ., London, (1978); J. D. Ferry, Viscoelastic Properties of Polymers, Wiley, (1970). 52. A. R. Eastwood and H. A. Barnes, Rheol. Acta, 14_, 795 (1974). 53. R. Darby, Viscoelastic Fluids, Marcel Dekker. New York (1976). 54. Kelco Algin, Kelco Co., Los Angeles, 1976. 55. D. A. Rees, Chem. Ind., 630 (1972). 52 56. R. G. Schweiger, Carbohydr. Res., 70, 185 (1979). 57. J. F. Kennedy, Adv. Carbohydr. Chem. Biochem., 29_, 306 (1974), W. G. Overend, Chem. Ind., 61 (1976); J. F. Kennedy, Carbohydrate Chemistry, 11, 445 (1979). 58. R. F. Borch, M. D. Bernstein, and H. D. Durst, J. Am. Chem. Soc, 88, 1024 (1966); G. R. Gray, Methods Enzymology, 50, 155 (1978). 59. L. D. Hall and M. Yalpani, Carbohydr. Res., 78, C4 (1980); L. D. Hall and M. Yalpani, ibid., in press; M. A. Bernstein, L. D. Hall, and W. E. Hull, J. Amer. Chem. Soc, 101, 2744 (1979); M. Brownlee and A. Cerami, Science, 206, 1190 (1979); H. Wiegandt and W. Ziegler, Hoppe-Seyler's Z. Physiol. Chem., 355, 11 (1974). 60. A. S. Perlin, MTP Internat. Rev. Sci., Org. Chem. Ser. Two, ]_, 1 (1976) . 61. R. G. Kessel and C. Y. Shih, Scanning Electron Microscopy in Biology, Springer, New York (1974). 62. L. J. Berliner, ed., Spin Labelling, I, Academic Press, New York .(1976); II, (1979). 63. R. A. Dwek, Nuclear Magnetic Resonance in Biochemistry, Clarendon Press, Oxford, (1975). 64. E. G. Rozantsev, 'Free Nitroxyl Radicals,' Plenum, New York :(1970). 65. G. I. Likhtenshtein, 'Spin Labeling Methods in Molecular Biology,' Wiley, New York, (1976). 66. J. R. Bolton, D. Borg and H. Swartz, Biological Applications:df Electron Spin Resonance, Wiley-, New York, (1972). 67. J. E. Wertz, and J. R. Bolton, 'Electron Spin Resonance, Elementary Theory and Practical Applications,' McGraw-Hill, New York ;(1972). 68. C. Alleyne, M.Sc thesis, University of British Columbia, 1973; B. V. McGarvey, Transition Metal Chemistry, 3> 8 9 (1966); B. J. Hathaway and D. E. Bil l i n g s , Coordination Chem. Reviews, 4, 143 (1970); S. S. Eaton, and G. R. Eaton, Coord. Chem. Rev., 26. > 207 (1978). 69. J. H. Freed, in ref. 62, p. 53. 70. S. A. Goldman, G. V. Bruno, and J. H. Freed, J. Chem. Phys., _59, 3071 (1973); F. J. Sharom, and C. W. M. Grant, Biochem. Biophys. Res. Commn., 74_, 1039 (1977). 71. J. D. Aplin, Ph.D. thesis, University of British Columbia (1979). 53 72. A. I. Kokorin, K. I. Zamarayev, G. L. Grigoryan, V. P. Ivanov, and E. G. Rosantsev, Biofizika, 17, 34 (1972) (31 i n transl.). 73. J. Hyde, H. M. Swartz, and W. E. Antholine, in Spin Labelling II, (L. J. Berliner, ed.), 71 (1979). 74. M. J. Adam, Ph.D. thesis, University of British Columbia, (1980). CHAPTER II CHEMICAL MODIFICATION OF ALGINIC ACID, XANTHAN GUM, AND CELLULOSE II-A. Background 1. Introduction In light of the consideration discussed in the Introduction, the work described in this chapter was principally aimed at investigating the synthetic aspects of several derivatization procedures, some of them previously documented, using as substrates three important and represent- ative polysaccharides, alginic acid, xanthan gum, and cellulose. For these particular studies esr spectroscopy of the products obtained by reactions with reagents based on stable nitroxide spin labels was used to obtain insight to the general course of the reactions. As w i l l be seen, the particular merit of this approach is that i t enabled a faci l e distinc- tion to be made between material which had been covalently "bound" to the polysaccharide from that which was "unbound" or merely absorbed. Before discussing the chemistry undertaken i t is appropriate to provide some background information concerning covalent chemical linkages, the c r i t e r i a employed here for establishing their presence, and for selecting the various modification procedures. 2. Covalent Linkages It i s appropriate to begin with a discussion of the general nature of the reactions performed which in a l l cases afforded covalently linked 54 55 s p i n - l a b e l conjugates, as monitored by esr. To the organic chemist, unfamiliar with t h i s f i e l d , some of the chemical transformations presented here, f o r instance the amidation or e s t e r i f i c a t i o n of alginate deriva- t i v e s , w i l l appear to be rather s u r p r i s i n g , at least under the given experimental conditions. It i s necessary therefore, to b r i e f l y summarize the c r i t e r i a which were used i n t h i s study to determine the existence of covalent linkages as w e l l as evidence obtained from other sources. I t i s convenient to begin with the l a t t e r . ( i ) C r i t e r i a used i n previous studies 1 2 In two pre-1960 patents ' p e r t a i n i n g to the preparation and use of alginamides from esters of a l g i n i c acid under mild conditions the follow- ing evidence i s given i n support of the reactions: ( i ) the s o l u b i l i t y properties of the amide products depend on the hydrophilicity/hydrophobic- i t y of the s t a r t i n g amine, i . e . , the former y i e l d water-soluble compounds while the l a t t e r a f f ord water-insoluble compounds which are soluble i n organic solvents; when two d i f f e r e n t amines are used the r e s u l t i n g mixed amides have s o l u b i l i t y features intermediate to the above ones; ( i i ) the products can be characterized by nitrogen analysis and, more importantly, the t h e o r e t i c a l amounts of alcohol can be i s o l a t e d from the reaction of 1 3 a l g i n i c esters and ammonia. In another study, nitrogen analysis was also used to confirm the quantitative conversion of esters into amides. 1 2 In both of the above patents, ' the inventors consider i t probable that some of the a l g i n i c esters could be saponified to form the corresponding alginate s a l t s ; no evidence i s , however, c i t e d why the t r a n s - e s t e r i f i c a - t ions or amidations should not go to completion. In a more recent 4 survey, McDowell has demonstrated that the physicochemical properties of alginamides derived from a l g i n i c esters are consistent with the 56 formation of covalent products rather than salts. McDowell proposed the co-existence of several competing reactions, including cross-link formations, where the conditions for the breakdown of links (i.e., of alginic esters) are not markedly different from those for their formation (i.e., of products); thus, a narrow range of conditions has to be found for the formation rate to be greater than the destruction rate. Having dwelt on the formation of alginamides and esters, evidence for other reactions can next be considered. Incorporation of radio- active labels has been used to demonstrate the carbodiimide-mediated, quantitative esterification of polyuronides.^ A wealth of information about covalent linkages has been established for various industrially important processes such as the dyeing of textiles and cellulose deriv- atives.^ As w i l l be subsequently discussed, alkylations of cellulose using chloroacetic acid or other halogenated compounds, requires prior alkali-activation of the polymer for the reaction to proceed. Thus, i t has been demonstrated for chlorotriazine-dye-treated cellulose that the dye is removed by washing with water i f the reaction had been conducted in a neutral bath, whereas the dye cannot be washed off i f a l k a l i treat- ment precedes the reaction. Organic solvents do not remove the dye in the latter case, whereas they do i f cellulose i s treated with an unreac- tive vat- or azoic-dye under similar conditions. Dyed cellulose is insoluble in cuprammonium solutions whereas the undyed material is soluble. Furthermore, the dyed portion of treated materials can be chemically cleaved to produce a reactive moiety for further derivatiza- tions, e.g., diazotation, amine or phenol coupling with new dyes. In a recent Ph.D. thesis^ chlorotriazine-based nitroxide labels have been used in similar fashion to demonstrate covalent couplings to several poly- 57 saccharides. In addition, for many polysaccharide reactions the equiva- lent monosaccharide chemistry has been established. Numerous studies have used various combinations of chemical and spectroscopic techniques to characterize the products obtained from (often multi-step) d e r i v a t i z a t i o n s of polysaccharides. The above examples c l e a r l y reveal the p o s s i b i l i t y of demonstrating the covalent nature of various polysaccharide d e r i v a t i v e s although f o r t h i s proof to be unambiguous several d i r e c t and i n d i r e c t methods rather than a s i n g l e and f a c i l e spectroscopic one are required i n most cases. The advantages of the esr technique over other spectroscopic techniques f o r these purposes have already been alluded to. Before d e t a i l i n g the c r i t e r i a used i n t h i s study f o r e s t a b l i s h i n g covalent linkages, i t should be mentioned that s i m i l a r c r i t e r i a have been employed i n another recent Ph.D. t h e s i s , g which e x c l u s i v e l y deals with the s p i n - l a b e l l i n g of polysaccharides, ( i i ) C r i t e r i a used i n t h i s study The evidence derived i n t h i s study from esr spectroscopy was based on the following f a c t o r s : 1. The s p e c t r a l lineshapes and widths obtained from, i n most cases, aqueous s o l u t i o n are a good measure of the degree of motional immobilization of n i t r o x i d e moieties attached to the polymers. 2. The s p e c t r a l lineshapes obtained from s o l i d or frozen (77°K) samples allow f o r a f a c i l e d i s t i n c t i o n of "bound" and " f r e e " l a b e l s ( i n the former case) and they provide a d d i t i o n a l information about i n t e r a c - tions between proximate spin l a b e l s ( i n the l a t t e r case). 3. Control experiments i n which the unreactive spin l a b e l [1] was employed under i d e n t i c a l conditions as the rea c t i v e counterparts, demon- strated that very l i t t l e , i f any, non-covalent associations or other 58 phenomena such as t r a p p i n g , occur f o r the v a r i o u s polysaccharides used. A l t e r n a t i v e l y , the leakage of any " f r e e " s p i n l a b e l s from the polymeric matrices was monitored w i t h time, whenever such species were suspected t o c o e x i s t w i t h bound l a b e l s . - Q - 0 [1] Complimentary evidence was gathered from other s p e c t r o s c o p i c t e c h - niques and methods, wherever p o s s i b l e . In many cases i t would have been d e s i r a b l e to c h a r a c t e r i z e f u n c t i o n a l groups by i n f r a red ( i r ) s p e c t r o - scopy. This technique, however, i n my hand proved to be somewhat u n s a t i s f a c t o r y l a r g e l y due t o the many l i m i t a t i o n s imposed by the po l y - saccharide d e r i v a t i v e s themselves. In many cases, s p e c i f i c s p e c t r a l bands could not be detected due to overlapping bands, or the degree of s u b s t i t u t i o n was so sm a l l that p o s i t i v e i d e n t i f i c a t i o n was impossible w i t h the a v a i l a b l e i n s t r u m e n t a t i o n . S i m i l a r d i f f i c u l t i e s have presumably a l s o c o n t r i b u t e d to the l a c k of i r - e v i d e n c e i n any of the above c i t e d r e f e r - ences . From the aforementioned i t i s c l e a r that none of the c r i t e r i a by i t s e l f can unambiguously e s t a b l i s h covalent l i n k a g e s , but, i n t o t o , t h e i r combination together w i t h other a v a i l a b l e evidence from the l i t e r a t u r e should provide s u f f i c i e n t support f o r the presence of such bonds. 3 . S e l e c t i o n C r i t e r i a Turning now to the s t r a t e g y employed f o r the syntheses, previous 59 9 experience of t h i s laboratory had demonstrated that the reductive amination reaction promised the greatest general v e r s a t i l i t y for the purposes of t h i s work. As w i l l be seen, most of the modifications described herein were consequently designed to introduce appropriately e i t h e r carbonyl or amine f u n c t i o n a l i t i e s into the polysaccharides for subsequent d e r i v a t i z a t i o n s . The choice of synthetic procedures was further guided by the following main considerations: ( i ) to maintain the polysaccharide chain l a r g e l y , i f not completely i n t a c t and to avoid acid h y d r o l y s i s of the n i t r o x i d e spin l a b e l s ; and ( i i ) to form covalent linkages between the n i t r o x i d e l a b e l l e d reagents and the polymers i n the simplest and most d i r e c t way. As a consequence, strongly a c i d i c or basic conditions were not employed and the reactions were conducted at ambient temperature and pressure. Of the numerous a v a i l a b l e modification procedures two, periodate oxidation"^ and carbodiimide coupling,"'""'" were found to be p a r t i c u l a r l y u s eful and deserve further mention here. Dialdehyde derivatives of polysaccharides obtained by periodate oxidation are widely used; for example, dialdehyde dextran d e r i v a t i v e s 12 have been combined with various amine-bearing pharmaceuticals, and dialdehyde starch and c e l l u l o s e have been employed for graft polymeriza- tion,"'" 3 medical uses,"'"4 and enzyme immobilization."'"^ For a l g i n a t e , 16 17 Painter and coworkers ' have established c a r e f u l l y c o n t r o l l e d condi- tions for preparing periodate-oxidized alginate with degrees of oxidation ranging from 0 to 60%. These dialdehyde d e r i v a t i v e s constitute versa- t i l e intermediates since they can be further d e r i v a t i z e d , * say with *Another i n t e r e s t i n g variant has been demonstrated by the oxidation (NaC102) of the aldehyde groups to the corresponding carboxylic acid derivatives.12a 60 amines, to afford products which are associated with varying degrees of structural perturbation and substitution. It should be noted, however, that the periodate oxidation method can be complicated by a number of factors such as radical initiated-chain scission, and formation of inter- residue hemiacetals and hydrated species. Thus, Painter et a l . have shown that six-membered hemiacetal rings can be formed between aldehyde groups of hexuronic acid residues of alginate and the closest hydroxyl 18 groups on adjacent unoxidized residues [ 2 ] , while Nadzhimutdinov et a l . found dialdehyde cellulose to exist in two forms in aqueous solutions ( [ 3 ] , [A]). 61 Carbodiimides, p a r t i c u l a r l y N,N'-dicyclohexyl-carbodiimide (DCC) [5], have found widespread applications as condensing agents i n amide 19 20 and ester syntheses. ' More recently, watersoluble derivatives such as N-ethyl-N'-dimethyl aminopropyl carbodiimide hydrochloride (EDG) [6] have been developed which are wel l suited'for polysaccharide"* derivatiza- tions since they offer the additional advantage of watersolubility of [ 5 ] [6] CHgCHiN-C-NtCHj fe NvCH 5) 2 Cl" the N-acyl urea byproducts which may otherwise complicate the i s o l a t i o n of the desired products. The condensation of amines (R'Nr^) with carboxylic acids (R"C02H) i n the presence of DCC i s envisaged to proceed 21 v i a the activated ester intermediate [7]: 0 r OCR2 r&OoH + R-N = C = N-R [ R-W-C = NR [7] 1 a* o * 9 COR2 l̂ -NHC-F? + PW-CO-NUR WH-€-AR 62 The d e t a i l s of the reaction mechanism for EDC coupling have not yet 22 been f u l l y elaborated, but i t i s known that i n neutral aqueous solution a ring-chain tautomerism exists which predominantly (93%) favors the rin g tautomers as shown below: m-c^5 N-C2H5 L a s t l y , a comment i s required concerning the reaction yi e l d s obtained i n t h i s chapter. While some reactions proceeded i n es s e n t i a l l y quantitative fashion, for many others the conversion yi e l d s were r e l a t i v e l y low. However, no attempts were made to f u l l y optimize the reaction conditions i n such cases since some of these studies were designed to i n i t i a l l y prove the formation of covalent derivatives. I t would have been r e l a - t i v e l y easy i n many instances to substantially improve the r e s u l t s simply by varying the reaction conditions such as temperature, use of surfac- tants, and cat a l y s t s , etc. Reference can be made here to a recent patent which exclusively describes procedures for the preparation of 23 high d.s. polysaccharides. In view of the low d.s. obtained i n many reactions, esr double integration was used to determine the le v e l s of spin-label incorporation; microanalysis was performed, wherever possible on products not containing nitroxide moieties. The reactions to be described are organized into separate sections based on the polysaccharide substrates used. 63 II-B. Chemical Modifications 1. A l g i n i c Acid Of the three polysaccharides discussed here, a l g i n i c acid [8], A bo' J n [ 8 ] provided the greatest challenge for modifications as a result of i t s g " f r a g i l i t y " and low r e a c t i v i t y . Some previous attempts at spin- l a b e l l i n g of [8] had met with l i t t l e success and i t was found necessary to activate [8] p r i o r to several modification reactions. Various work- 24 ers had previously noted that the acetylation of a l g i n i c acid never pro- ceeded to completion, introducing no more than one acetyl group per uronic 25 acid residue. However, Schweiger discovered that a peracetylated product could be obtained with minimal polymer degradation i f a l g i n i c acid was i n i t i a l l y p a r t i a l l y (80-90%) dehydrated (complete dehydration leading to i n a c t i v a t i o n of [8]). Thus, following Schweiger's method p a r t i a l l y dehydrated a l g i n i c acid was obtained by treatment of [8] with cold, g l a c i a l acetic acid; t h i s procedure reduces the extensive hydrogen- bonding i n [8] thereby rendering the hydroxyl groups available for reac- t i o n . 64 ( i ) A l k y l ether Following the above procedure, stable acetamido ethers [10] of a l g i n i c acid [8] were prepared using 4-chloroacetamido-2,2,6,6-tetra- methylpiperidine-l-oxyl [9], i n either pyridine or aqueous acetone (65%) using s o l i d sodium bicarbonate as base. The mild conditions which were employed to avoid degradation of [8] led to very low d.s. (0.03-0.04). The esr spectrum of [10] i n aqueous solution (Fig. I l - l a ) reveals a very mobile nitroxide moiety. The r e l a t i v e l y high mobility can be ascribed to some extent to the ro t a t i o n a l freedom associated with the four-bond linkage between the polymer and probe. [ 8 ] [9] [10] The spectrum of s o l i d [10] (Fig. I l - l b ) resembles that of a poly- c r y s t a l l i n e sample and shows no presence of free l a b e l . 65 a b F i g . I I - l . Esr s p e c t r a of [10] (a) i n aqueous s o l u t i o n ; (at 298 K ) . (b) s o l i d ; 66 ( i i ) A l k y l e s t e r The e s t e r i f i c a t i o n of p a r t i a l l y dehydrated a l g i n i c a c i d [8] was accomplished u s i n g 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl [11] i n aqueous acetone a f f o r d i n g the e s t e r [12] (d.s. 0.05). [ - • C O f e H + H O ^ ^ O > | - 0 0 8 - ( ^ 0 [ 8 ] [11] [12] Evidence f o r the formation of [12] was derived from the esr spectrum of the s o l i d under methanol ( F i g . II-2b) which revealed a broad "bound" component together w i t h a superimposed s i g n a l a r i s i n g from " f r e e " l a b e l ( despite extensive d i a l y s i s of the produ c t ! ) . This " f r e e " l a b e l may be present as the s a l t which previous workers have a l l u d e d to (see II-B 2 ( i ) ) . The T c value of [12] i n aqueous s o l u t i o n was 20 ns ( F i g . I I - 2 a ) . A d d i t i o n a l evidence f o r the occurrence of e s t e r i f i c a t i o n was derived from a c o n t r o l experiment i n which the s p i n l a b e l [11] was replaced by the unre a c t i v e analogue [ 1 ] . Only a very s m a l l p r o p o r t i o n of [1] remained i n the sample a f t e r d i a l y s i s , as i n d i c a t e d i n Figure I I - 2 c . The above r e a c t i o n appears to provide a v i a b l e a l t e r n a t i v e to the known e s t e r i f i c a t i o n procedures f o r a l g i n i c a c i d which i n v o l v e the use 26 of a l k a l y n e oxides or the a c i d c h l o r i d e intermediate [14] proposed by Wypych.^ 67 Fig. II -2 . Esr spectra of [12] (a) in aqueous solution; (b) solid; (at 298K); (c) control experiment: [8] treated with [1] under identical conditions. 68 ( i i i ) Amides (a) v i a carbodiimide c o u p l i n g Amide d e r i v a t i v e s of a l g i n i c a c i d could be r e a d i l y prepared by carbodiimide-mediated c o u p l i n g i n organic or mixed aqueous-organic media. As i n the previous r e a c t i o n s , however, a l g i n i c a c i d i s only r e a c t i v e i n i t s p a r t i a l l y dehydrated form. Amidation can be achieved u s i n g DCC i n dimethylformamide (DMF), or w i t h EDC i n 65% aqueous acetone to a f f o r d the amide [16A] w i t h d.s. 0.10 and 1.00, r e s p e c t i v e l y . C l e a r l y , EDC i s the p r e f e r r e d reagent f o r high d.s. products. The esr s p e c t r a of [16A] pro- v i d e evidence f o r the covalent nature of the l i n k a g e ( F i g . I I - 3 ); i n the c o n t r o l experiment w i t h l a b e l [1] no s i g n a l was d e t e c t a b l e a f t e r i d e n t i c a l treatment ( F i g . I I - 3 c ) . The n i t r o x i d e moiety of [16A] i s moderately immobile i n aqueous s o l u t i o n , (T 15 ns) ( F i g . I I - 3 a ) . (b) v i a n u c l e o p h i l i c s u b s t i t u t i o n of a l g i n i c e s t e r s Propylene g l y c o l e s t e r s of a l g i n a t e [17] have been reported to 1-4 r e a c t w i t h amines to form amides i n high y i e l d s at ambient temperature. Commercial propylene g l y c o l a l g i n a t e (PGA) samples w i t h d i f f e r e n t degrees of e s t e r i f i c a t i o n (d.s. 0.80-0.85 and d.s. 0.50-0.60, r e s p e c t i v e l y ) were condensed w i t h amine s p i n l a b e l [15] i n DMF i n the presence of s m a l l amounts of water to y i e l d the amide [16B]. 69 Fig. II-3. Esr spectra of [16A] (a) in aqueous solution; (b) solid; (at 298K); (c) control experiment: [8] treated with [1] under identical conditions. 70 Fig. II-4. Esr spectra of [16B] (a) in aqueous solution; (b) solid; (at 298K); (c) frozen (77K); (d) control experiment: [17] treated with [1]. 71 j — CO2CH2CHOHCH3 + [15] [17] This r e a c t i o n i s of c o n s i d e r a b l e u t i l i t y s i n c e i t can be performed w i t h a wide range of amines such as primary or secondary a l i p h a t i c , c y c l o a l i p h a t i c , aromatic, and diamines.^" The r e s u l t i n g products are of i n t e r e s t f o r t h e i r gel-forming p r o p e r t i e s . The esr s p e c t r a of amides of the type [16B] i n aqueous s o l u t i o n ( F i g . II-4a) i n d i c a t e moderately immobile n i t r o x i d e moieties (T 33 ns) and the corresponding s p e c t r a of the s o l i d samples r e v e a l not only the covalent nature of the l i n k a g e s to the polymer but a l s o strong d i p o l a r i n t e r a c t i o n s ( F i g . I I - 4 b ) . S i m i l a r i n f o r m a t i o n can be derived from r a p i d l y f r o z e n (77K) samples ( F i g . I I - 4 c ) . The c o n t r o l experiment w i t h l a b e l [1] revealed no d e t e c t a b l e esr s i g n a l ( F i g . I I - 4 d ) . ( i v ) Amines F o l l o w i n g the methods of P a i n t e r and c o w o r k e r s , ^ dialdehyde a l g i n - ates w i t h v a r y i n g degrees of o x i d a t i o n (d.o.) were prepared and subse- quently converted to the corresponding amine d e r i v a t i v e s [18] using r e d u c t i v e amination and l a b e l [15]; t y p i c a l l y , amines w i t h d.s. 0.05 [18A] and 0.13 [18B] were obtained from dialdehyde precursors w i t h d.o. 0.10 and 0.44, r e s p e c t i v e l y . The esr s p e c t r a of [18A] and [18B] i n aqueous s o l u t i o n ( F i g . II-5a) revealed s u b s t a n t i a l l i n e broadening and values of 51 ns and 57 ns, r e s p e c t i v e l y . When these s o l u t i o n s were 72 Fig. II-5. Esr spectra of [18A] (left) and [18B] (right) (a) in aqueous solution; (b) solid; (at 2 9 8 K ) ; (c) frozen (77K). 73 CDoH C O C L \ [1*] f r o z e n , d i p o l a r broadening was again apparent ( F i g . I I - 5 c ) . The spectra derived from the s o l i d m a t e r i a l s under methanol show the presence of small p r o p o r t i o n s of f r e e l a b e l ( F i g . I I - 5 b ) . The above f i n d i n g s seem t o i m p l i c a t e the presence of two s p i n l a b e l s u n i t s per periodate-cleaved hexuronic a c i d residue of [18] s i n c e even at low d.s., d i p o l a r i n t e r a c - t i o n s are evident. P o s s i b l e c o n t r i b u t i o n s to the observed lineshapes from i n t e r a c t i o n s between n i t r o x i d e s attached to d i f f e r e n t chains or to neighbouring uronide r e s i d u e s , should be n e g l i g i b l e , at l e a s t f o r d e r i v a t i v e s of [18] w i t h low o v e r a l l d.s. I f the above i n f e r e n c e i s c o r r e c t , i t would be i n co n t r a s t to s e v e r a l observations of other d i a l - dehyde r e a c t i o n products f o r which i n c o r p o r a t i o n of only one amine- 28 bearing u n i t per dialdehyde has been found. The r e a c t i o n of equivalent dialdehyde c e l l u l o s e d e r i v a t i v e s w i l l be discussed i n I I - B 3. A sample of pe r i o d a t e o x i d i z e d a l g i n a t e (d.o. 0.10) was t r e a t e d w i t h p - f l u o r o a n i l i n e under i d e n t i c a l c o n d i t i o n s as above to produce a d e r i v a t i v e which could be c h a r a c t e r i z e d by 1 3 C nmr. Figure I I - 6 demon- s t r a t e s the u t i l i t y of 1 3 C nmr i n d e t e c t i n g even low (between 5-10%) l e v e l s of s u b s t i t u e n t i n c o r p o r a t i o n . S i m i l a r l y , p r e l i m i n a r y s t u d i e s of t h i s d e r i v a t e have shown that 1 9 F nmr i s w e l l s u i t e d f o r d i s t i n g u i s h i n g F i g . H-6. 100.6 MHz 1 3 C nmr spectrum of p-fluoroaniline alginate derivative, (4%) i n D2O at 308K, showing aromatic resonances at 117 ppm. 75 c o v a l e n t l y bound m a t e r i a l from r e a c t a n t ; when p - f l u o r o a n i l i n e was added to an aqueous s o l u t i o n of the f l u o r o a n i l i n e a l g i n a t e d e r i v a t i v e , the two species were found to be separated by ca 1 ppm i n chemical s h i f t . (v) Hydrazines 29 Pfannemuller et a l . have r e c e n t l y reported a s e r i e s of p o l y - saccharide hydrazine d e r i v a t i v e s which were employed f o r the s y n t h e s i s of branched-chain d e r i v a t i v e s . Thus, a l g i n i c a c i d hydrazine [19] was pre- pared, i n adaption of t h e i r method, from propylene g l y c o l a l g i n a t e [17] PGA + ttyff^lty) [17] [19] [21] and hydrazine hydrate. Reductive amination of [19], usi n g 4-oxy-2,2,6,6- t e t r a m e t h y l p i p e r i d i n e - l - o x y l [20] a f f o r d e d the s p i n l a b e l l e d d e r i v a t i v e [21] (d.s. 0.21), whose aqueous s o l u t i o n spectrum i s shown i n F i g u r e I I - 7 . This method i s p o t e n t i a l l y u s e f u l f o r the p r e p a r a t i o n of po l y s a c c h a r i d e intermediates s i n c e i t introduces a very short spacer group between the polymer and other molecules to be attached to i t . 76 Fig. II-7. Esr spectrum of [21] in aqueous solution (at 298K) (vi) s-Triazine derivatives 30 Lee and Maekawa have reported a method for incorporating the carboxyl groups of polyuronides into s-triazine rings using dimethyl- biguanidine hydrochloride [22]. Following their method [22] (d.s. 0.12) was prepared from propylene glycol alginate [17] and subsequently labelled using [20] to afford a product [24] (d.s. 0.02), whose aqueous solution [23] 77 spectrum ( F i g . II - 8 ) revealed a r e l a t i v e l y slow tumbling n i t r o x i d e moiety (x 31 n s ) . The s - t r i a z i n e type d e r i v a t i v e may be of i n t e r e s t f o r a p p l i c a t i o n s i n which a p a r t i a l masking of carboxylate groups of polyuronides i s d e s i r e d . F i g . I I - 8 . Esr spectrum of [24] i n aqueous s o l u t i o n (at 298K). 2. Xanthan Gum I n view of i t s recent disc o v e r y r e l a t i v e l y very few d e r i v a t i v e s of 31 xanthan gum are p r e s e n t l y known. However, s i g n i f i c a n t developments i n t h i s area can be expected c o n s i d e r i n g the wide range of i n d u s t r i a l a p p l i c a t i o n s which the n a t i v e polymer has already found. The c a r b o x y l f u n c t i o n s of the g l u c u r o n i c a c i d residues are the most prominent candi- dates f o r s p e c i f i c d e r i v a t i z a t i o n s of xanthan gum and should undergo the same r e a c t i o n s as t h e i r counterparts i n a l g i n a t e , as demonstrated here by 78 t h e c a r b o d i i m i d e - m e d i a t e d p r e p a r a t i o n o f t h e c a r b o x a m i d e d e r i v a t i v e . A m i d e s (a) v i a EDC c o u p l i n g P u r i f i e d x a n t h a n gum [25] was a c t i v a t e d v i a t h e c a r b o d i i m i d e p r o c e d u r e u s i n g EDC and s u b s e q u e n t l y condensed w i t h amine l a b e l [15] t o a f f o r d t h e amide [26] i n w h i c h 43% o f t h e c a r b o x y l g r o u p s had b e e n [ 2 5 ] R = 0 H [ 2 6 ] / [ 2 7 ] R«= [ 2 8 ] R = NH(CH 2 ) 3 CH 3 t r a n s f o r m e d . I n l i g h t o f t h e m i l d r e a c t i o n c o n d i t i o n s e m p l o y e d , one c o u l d a l s o e x p e c t d e r i v a t i z a t i o n o f t h e p y r u v i c a c i d a c e t a l g r o u p s t o o c c u r . The e s r s p e c t r u m o f [26] i n aqueous s o l u t i o n r e v e a l s a f a s t t u m b l i n g (T 6.1 x 10 1 1 s ) n i t r o x i d e m o i e t y w h i c h i s p r e s u m a b l y a t t r i b u t a b l e t o t h e m o t i o n a l f r e e d o m e n j o y e d by t h e 6ide - c h a i n s ( F i g . I I - 9 ) . 79 K r Fig. II-9. Esr spectrum of [26] i n aqueous solution (at 298K). ( b ) v i a direct condensation 32 A procedure has been described for the preparation of "amine" alginates which involves the condensation of amines with s o l i d alginate i n the presence of small amounts of water. I t i s unclear, however, from t h i s report what the term "amine alginates" actually implies i n terms of the structure of such derivatives, since no further information i s pro- vided; assuming the formation of covalent linkages between the amines and hexuronide carboxylates, such products should be more appropriately c l a s s i f i e d as amides. inter e s t i n g enough to warrant some preliminary investigations using xanthan gum as substrate. Thus, xanthan gum [25] was condensed with Nevertheless, the procedure appeared to be 80 amine l a b e l [15] f o l l o w i n g t h i s procedure to y i e l d a product [27] whose esr spectrum ( F i g . 11-10) revealed the presence of both "bound" and some " f r e e " l a b e l , a f t e r p u r i f i c a t i o n by d i a l y s i s . F i g . 11-10. Esr spectrum of s o l i d [27] under methanol (at 298K). Encouraged by t h i s evidence f o r the formation of a covalent d e r i v a - t i v e , the r e a c t i o n was performed us i n g a diamagnetic amine, n - o c t a d e c y l - amine [ C H 3 ( C H 2 ) 1 7 N H 2 ] . The d.s. of the r e s u l t i n g product [28] could not, however, be e x a c t l y e s t a b l i s h e d by m i c r o a n a l y s i s i n view of the presence of lower molecular weight f r a c t i o n s contained i n the amine (which was 90% t e c h n i c a l grade). The C/N r a t i o found corresponded roughly to a f u l l y s u b s t i t u t e d product and p r e l i m i n a r y 1 3 C nmr experiments of aqueous s o l u t i o n s of [28] (at 37°C) confirmed the presence of the amine s u b s t i t u e n t w i t h a broad, unresolved peak, centered at 32 ppm which can be assigned to the methylene carbons (see Appendix). 81 Pending f u r t h e r i n v e s t i g a t i o n s , t h i s low-cost m o d i f i c a t i o n procedure may be of s u b s t a n t i a l i n d u s t r i a l b e n e f i t . 3. C e l l u l o s e ( i ) Amines (a) v i a r e d u c t i v e amination" of dialdehyde c e l l u l o s e 33 F o l l o w i n g N e v e l l ' s procedure, c e l l u l o s e [29] was o x i d i z e d w i t h p eriodate to the dialdehyde d e r i v a t i v e [30] to o b t a i n products w i t h three d i f f e r e n t degrees of o x i d a t i o n (d.o.), 0.05, 0.19, and 0.34, r e s p e c t i v e l y . Subsequent r e d u c t i v e amination of these m a t e r i a l s produced the s p i n - l a b e l l e d d e r i v a t i v e s [31]. As i n the case of a l g i n a t e , the conversion i n t o s p i n - l a b e l l e d amines [31] d i d not seem to l i n e a r l y correspond to the i n c r e a s i n g d.o. of the p r e c u r s o r s . D.s. values of 0.01, 0.03, and 0.03 were obtained f o r [31] from the dialdehyde samples w i t h d.o. 0.05, 0.19, I: H2NSL ™ *T , CHO I L OH CHO NaCNBH3 2 [29] [30] [31] and 0.34, r e s p e c t i v e l y . The low o v e r a l l s p i n l a b e l i n c o r p o r a t i o n s can probably be a t t r i b u t e d to the f a c t that p e r i o d a t e s , i n c o n t r a s t to most other o x i d i z i n g agents, can penetrate both m i c r o c r y s t a l l i n e and amorphous 33 regions of c e l l u l o s e f i b r e s . The low y i e l d s may consequently be merely a r e f l e c t i o n of the i n a b i l i t y of the bulky n i t r o x i d e molecules to f u l l y penetrate the m i c r o c r y s t a l l i n e r e g i o n s . Such reasoning seems j u s t i f i a b l e i n l i g h t of the otherwise great e f f i c i e n c y of the r e d u c t i v e 81A CH2 CH2 SL-W fUSL [31A] C r L O H 0 [31B] F i g . 11-11. Esr s p e c t r a of [31] derived from dialdehyde c e l l u l o s e samples ([30]) w i t h d.o. 0.05 ( l e f t ) , 0.19 ( c e n t r e ) , 0.34 ( r i g h t ) , r e s p e c t i v e l y ; (a) aqueous suspension; (b) s o l i d ; (298K) oo 83 amination procedure, and of a recent esr study of the a c c e s s i b i l t y of 8 34 c e l l u l o s e which came to s i m i l a r c o n c l u s i o n s . ' I n t e r e s t i n g l y , the esr spe c t r a of [31] i n aqueous suspension ( F i g . I I - l l a ) , i n d i c a t e two d i s t i n c t n i t r o x i d e populations of which one enjoys a s u b s t a n t i a l l y greater freedom of motional r e o r i e n t a t i o n than the other; t h i s f i n d i n g gives f u r t h e r credence to the above " a c c e s s i b i l i t y " arguments. As f o r the corresponding a l g i n a t e d e r i v a t i v e s ( [ 1 8 ] ) , the esr spe c t r a ( F i g . I I - l l b ) of the c e l l u l o s e d e r i v a t i v e s [31] r e v e a l d i p o l e - d i p o l e i n t e r a c t i o n s between neighbouring spins from which i t may again be i n f e r r e d that s t r u c t u r e s c o n t a i n i n g two n i t r o x i d e s per p e r i o d a t e - cleaved glucose re s i d u e [31A] predominate over those bearing only one l a b e l , as ex e m p l i f i e d (from a s e r i e s of other p o s s i b i l i t i e s ) by [31B]. The strongest evidence f o r t h i s i n f e r e n c e d e r i v e s from the observation of d i p o l a r i n t e r a c t i o n s f o r c e l l u l o s e d e r i v a t i v e s w i t h low d.o. Assuming random o x i d a t i o n and subsequent l a b e l l i n g of the polymer and w i t h due c o n s i d e r a t i o n of the aforementioned " a c c e s s i b i l i t y " f a c t o r , i t i s reason- able to expect the sep a r a t i o n between s i n g l e n i t r o x i d e s attached to d i f f e r e n t hexose u n i t s to be too great f o r s i g n i f i c a n t c o n t r i b u t i o n s to the d i p o l a r i n t e r a c t i o n s to a r i s e from t h i s source. Thus, these f i n d i n g s are best accommodated by s t r u c t u r a l u n i t s of the type [31A]. (b) v i a r e d u c t i v e amination of C2- and C3-oxycellulose In the next round of experiments c e l l u l o s e was o x i d i z e d * without 35 any r i n g cleavage, using a c e t i c anhydride-DMSO, to a f f o r d C2- and C3- o x y c e l l u l o s e , [32] and [33], r e s p e c t i v e l y . The degrees of o x i d a t i o n of these d e r i v a t i v e s , as determined by m i c r o a n a l y s i s , were 0.85 and 0.80, *These samples were k i n d l y provided by Dr. J . Defaye, C.N.R.S., Grenoble, France. 84 r e s p e c t i v e l y . Reductive amination i n methanolic medium afforded the corresponding amine d e r i v a t i v e s [34] and [35] (d.s. 0.28 and 0.09, [33] X = C=0 I 3 5 ] X = C H - N H - S L [32] X = C=0 [34] X = C H - N H - S L r e s p e c t i v e l y ) . The lower d.s. value obtained f o r [34] may be due to s t e r i c hindrance by the bulky C 6 - t r i t y l group. The two products e x h i b i t e d i n t e r e s t i n g d i f f e r e n c e s i n t h e i r s o l u - b i l i t y p r o p e r t i e s ; the C 3 - l a b e l l e d [35] i s wat e r s o l u b l e but i n s o l u b l e i n o rganic s o l v e n t s , whereas [34] i s w a t e r - i n s o l u b l e but s l o w l y d i s s o l v e s i n chloroform (see I-B). The esr spec t r a of these m a t e r i a l s c l e a r l y demonstrate these s o l u b i l i t y d i f f e r e n c e s ( F i g . 11-12): the aqueous s o l u - t i o n spectrum of [35] r e v e a l s a slow-tumbling n i t r o x i d e moiety w h i l e [34] produces i n the same medium a " p o l y c r y s t a l l i n e " spectrum ( F i g . II-12a) (see I-C ( i ) ) ; on the other hand, the n i t r o x i d e moiety of [34] i n chloroform s o l u t i o n i s h i g h l y mobile i n comparison to th a t of [35] (F i g . I I - 1 2 b ) . The hy d r o p h o b i c i t y of the C 6 - t r i t y l of [34] i s mainly r e s p o n s i b l e F i g . 1 1 - 1 2 . Esr s p e c t r a of . [ 3 4 ] ( l e f t ) and [ 3 5 ] ( r i g h t ) , (a) i n water; (c) i n C H C 1 3 ; (at 298K); (c) f r o z e n (77K). 86 for the observed s o l u b i l i t y differences. I t i s quite obvious that these oxycellulose derivatives, i n conjunction with the reductive amination procedure, would be i d e a l l y suited for various studies some of which w i l l be discussed i n Chapter I I I . ( i i ) Urethanes Five-membered c y c l i c carbonate derivatives, obtained from the reac- t i o n of ethylchloroformate with v i c i n a l d i o l s , have been reported for a 36 number of carbohydrates including polysaccharides. These derivatives form stable urethane products upon reaction with amine nucleophiles. Commercial cellulose carbonate (d.s. 0.21) [36] was derivatized i n aqueous medium to afford the spin labelled derivative [37] (d.s. 0.08). °v f - O - C - N H - S L ^ - 0 + H 2 N - S L OH l > ] [37] I t i s interesting to note that (as for [31]) an aqueous suspension of [37] produced a complex spectrum consisting of a p a r t i a l l y resolved broad component and a more mobile (T 9.1 x 10 ̂ s ) one. Differences i n the morphology of [37] are therefore again indicated; indeed the properties of [37] varied with i t s h i s t o r y : when prepared from methanol, the product had a lower d.s. and i t s esr spectrum (Fig. II-13b) resembled that of a p o l y c r y s t a l l i n e sample, whereas the product obtained from reaction i n aqueous medium revealed strong dipolar interactions. I t can therefore be concluded that these differences are attributable to the greater a c c e s s i b i l i t y of [36] to aqueous solvent. Fig. 11-13. Esr spectra of [37] (a) aqueous suspension, ( 2 9 8 K ) ; (b) frozen (77K); (c) control experiment: [36] treated with [1] under i d e n t i c a l conditions. 88 A control experiment i n which [36] was treated with the unreactive label [1], proved that no adsorption phenomena were present in this set of experiments (Fig. II-13c). Finally, i t should be noted that [36] in methanol may possibly 37 undergo side-reactions (formation of O-methoxycarbonyl derivatives [38]) which could be partially responsible for the lower d.s. of [37] obtained from this medium. [36] fEOH 9 0-C-0-CH3 r-OH [38] ( i i i ) Hydrazine Cellulose hydrazine derivatives have found various applications as 29 38 reactive intermediates, for enzyme immobilization, or for metal- 39 collection. Their synthesis can be achieved via the C6-chlorodeoxy 39 derivative or, more conveniently, via commercially available carboxy- methyl c e l l u l o s e . 2 9 ' ^ 0 Using the latter method, cellulose hydrazine [39] r - C H , C O N H N H 2 — ^ > l-CH2CONHNH - Q - O I NaCNBH, 1 [39] [40] (d.s. 0.5) was prepared and subsequently reductively aminated, using [20], to yield the hydrazine [40] (d.s. 0.003) whose aqueous solution spectrum i s shown in Figure 11-14. The nitroxide moiety of [40] dis played a relatively slow motional reorientation time (T 17 ns). 89 I | Fig. 11-14. Esr spectrum of [40] i n aqueous solution (298K). II-C. Summary It i s evident from the preceding section that a number of reactions s a t i s f i e d the requirements for s p e c i f i c and medium to high e f f i c i e n t conversion of polysaccharides. The carbodiimide-mediated amidaticn f a l l s into t h i s category, as does the formation of reactive intermediates v i a hydrazidation or s p e c i f i c oxidation of suitably-blocked c e l l u l o s e . The modification of v i c i n a l d i o l s v i a periodate-oxidation or c y c l i c carbonate formation i s less s p e c i f i c ; but the former procedure may, i f performed under controlled conditions, i n some cases be useful (see, e.g., V-B 2). Among the other methods examined, the e s t e r i f i c a t i o n of a l g i n i c acid with alcohols seems to be promising and should be investigated for some other polysaccharide systems. The same holds true for the amida- ti o n reactions of a l g i n i c esters. It is obvious that the use of sodium cyanoborohydride is mainly responsible for the u t i l i t y and v e r s a t i l i t y of almost a l l of the above modification procedures. 91 References 1. R. Kohler and W. D i e r i c h s , U.S. Pat., 2,881,161 (1959); Chem. Ab s t r . , 54, 7926d (1960). 2. B r i t . P at., 768,309 (1957), ( c i t e d i n r e f . 4 ) . 3. K. W. K i r b y , Ph.D. t h e s i s , Purdue U n i v e r s i t y , (1958). 4. R. H. McDowell, J . Soc. Cosmet. Chem.,21, 441 (1970). 5. R. L. Taylor and H. E. Conrad, Biochemistry, 11, 1383 (1972); see a l s o I . Danishefsky and E. S i s k o v i c , Carbohydr. Res., JL6_, 199 (1971). 6. R. L. M. A l l e n , Color Chemistry, Appleton-Century-Crofts, New York, 1971. 7. M. J . Adam, Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1980). 8. J . D. A p l i n , Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia, (1979). 9. J . D. A p l i n , M. A. B e r n s t e i n , C. F. A. C u l l i n g , L. D. H a l l , and P. E. Reid, Carbohydr. Res., 70, C9 (1979); L. D. H a l l and J . C. Waterton, J . Am. Chem. Soc., 101, 3697 (1979); L. D. H a l l and M. Y a l p a n i , Carbohydr. Res., J78, C4 (1980). 10. G. 0. A s p i n a l l , i n MTP I n t e r n . Rev. S c i . , Org. Chem. Ser. One, 295 (1973). 11. H. G. Khorana, Chem. Rev., 53, 145 (1953). 12. (a) K. P. Khomyakov, M. A. Penenzhik, A. D. V i r n i k , and Z. A. Rogovin, Vysokomol. Soyed., 1_, 1030, 1035 (1965), (1140, 1145 i n t r a n s l . ) ; (b) A. Gabert, F. Kretzschmann, W. D. Wiezorek, and P. Wolf, B r i t . Pat., 1,069,820 (1967). 13. A. Takahashi, T. Omata, and S. Takahashi, Sen-I G a k k a i s h i , 15, 70 (1973); Chem. A b s t r . , 81, 39195 (1974). 14. Neth. Pat. Ap p l . , 77,11034 (1977); Chem. A b s t r . , 91, 63007 (1980). 15. L. G o l d s t e i n , M. Pecht, S. Blumenberg, D. A t l a s , and Y. L e v i n , Biochemistry, 9_, 2322 (1970); T. J a r a s l a v a , V. J i r i , and V. Drahomira, C o l l e c t . Czech. Chem. Commun.,44, 3411 (1979). 16. T. P a i n t e r and B. Larsen, Acta Chem. Scand., _24, 813 (1970). 17. I . L. Andresen, T. P a i n t e r , and 0. Smidsr^d, Carbohydr. Res., 59, 563 (1977); and references t h e r e i n . 92 18. S. Nadzhimutdinov, A. A. Sarymsakov, and K. U. Usmanov, Dokl. Akad. Nauk SSSR, 219, 1371 (1974); (892 in transl.). 19. G. E. Means and R. E. Feeney, Chemical Modification of Proteins, Holden-Day Inc., San Francisco, (1971). 20. K. Holmberg and B. Hansen, Acta Chem. Scand., B33, 410 (1979). 21. A. F. Hegarty, M. T. McCormack, G. Ferguson, P. J. Roberts, J. Amer. Chem. Soc, 99, 2015 (1977). 22. T. Tenforde, R. A. Fawwaz, and N. K. Freeman, J. Org. Chem., 37, 3372 (1972). 23. C. P. Iovine and D. K. Ray-Chaudhuri, U.S. Pat., 4,129,722 (1978). 24. A. Wassermann, Nature, 158, 271 (1946); N. H. Chamberlain, G. E. Cunningham, and J. B. Speakman, ibid., 158, 553 (1946). 25. R. G. Schweiger, J. Org. Chem.,27, 1786 (1962). 26. A. B. Steiner and W. H. McNeely, Ind. Eng. Chem., 43, 2073 (1951). 27. Z. Wypych, Roczniki Chemii, 43, 1616 (1969). 28. See for example, J. C. Lee, H. L. Weith, and P. T. Gilham, Bio- chemistry, % 113 (1970); Z. I. Kuznetsova, E. G. Ivanova, and A. I. Usov, Izvest. Akad. Mauk SSSR, Ser. Khim., 1858 (1973). 29. H. Andresz, G. C. Richter, and B. Pfannemiiller, Makromol. Chem., 179, 301 (1978). 30. C. S. Lee and K. Maekawa, Agr. Biol. Chem., 40, 785 (1976). 31. W. H. McNeely and K. S. Kang, in Industrial Gums, R. L. Whistler ed., 473 (1973); G. Holzwarth, Carbohydr. Res., 66, 173 (1978). 32. Kelco Algin, (second edn.), Kelco Co., San Diego, California, 41 (1976). 33. P. T. Nevell, Methods Carbohydr. Chem., 3, 164 (1963). 34. L. D. Hall and J. D. Aplin, J. Amer. Chem. Soc, in press. 35. N. Pravdic and H. G. Fletcher, Carbohydr. Res., 19, 353 (1971). 36. W. M. Doane, B. S. Shasha, E. I. Stout, C. R. Russell, and C. E. Rist, i b i d . , 11, 321 (1969); J. F. Kennedy and H. C. Tun, Anti- microb. Ag. Chemother., 3_, 575 (1975). 37. E. I. Stout, W. M. Doane, B. S. Shasha, C. R. Russell, and C. E. Rist, Tetrahedr. Lett., 45, 4481 (1967). 93 38. E. Junowicz and S. E. Charm, Biochim. Biophys. Acta, 428, 157 (1976). 39. S. Machida and Y. Sueyoshi, Angew. Makromol. Chem.,49, 171 (1976). 40. H. L. Weith, J. L. Wiebers, and P. T. Gilham, Biochemistry, 9_, 4396 (1970). 41. H. Grasdalen, B. Larsen, and 0. Smidsr^d, Carbohydr. Res., _56_, CH (1977). CHAPTER III BRANCHED-CHAIN CHITOSAN DERIVATIVES III-A. Introduction The unique properties and importance of chitin and chitosan have already been briefly indicated in Chapter I. Despite the serious limitations imposed by their insolubility, various derivatives of both polymers have been successfully prepared in analogy to equivalent cellulose derivatives.''" More recently, the primary amine functions of 2 chitosan have been derivatized with a number of anhydrides, and common 3 4 aliphatic and aromatic aldehydes, ' invariably affording insoluble products. Although certain watersoluble ether and salt derivatives of chitosan are known,"'" no attempts had previously been made to affect the solubilization of chitosan by introducing suitable hydrophilic moieties into the polymer. The main impetus for the work described in this chapter derived from the goal to solubilize these rather intractable materials using the latter method. We were also interested i n studying the molecular properties of the branched-chain derivatives obtained by covalent attachment of various carbohydrate moieties to these polymers. Considerable efforts have been directed at the conversion of linear polysaccharides into branched-chain analogues which are of interest for a variety of reasons, ~* including the investigation of lectin-carbohydrate reactions,^ the preparation of model compounds in the fields of allergy,^ 8 9 enzymology, and immunology, and the study of the physical properties of 94 95 branched-chain derivatives."*"^ Previous workers have a p p l i e d v a r i o u s s y n t h e t i c routes"*"^" such as c o p o l y m e r i z a t i o n , ^ o r t h o e s t e r s a c e t o b r o m o - 13 , . 14 . . 1 5 ,., , sugars, hydrazones, or enzymic g l y c o l y s a t i o n s to c e l l u l o s e , amy- l o s e , a l g i n i c a c i d , and other p o l y s a c c h a r i d e s . These procedures, how- ever s u f f e r from v a r i o u s l i m i t a t i o n s s i n c e they r e q u i r e ( i ) s p e c i f i c p r o t e c t i o n of the l i n e a r p o l y s a c c h a r i d e , such as i n the r e a c t i o n of 1,2-orthoacetate sugars w i t h 2,3-di-0-phenylcarbomoyl d e r i v a t i v e s of amylose and c e l l u l o s e ; ( i i ) a c t i v a t i o n of the sugar which i s to form the s i d e chain; or ( i i i ) r e a c t i o n c o n d i t i o n s which lead to p a r t i a l or extensive p o l y s a c c h a r i d e degradation, e.g., u s i n g hydrazinehydrate. Most of the r e a c t i o n s are a l s o l a b o r i o u s and l o w - y i e l d i n g , a l l reasons which m i t i g a t e against r o u t i n e or l a r g e - s c a l e adaption. The work described i n t h i s chapter demonstrates the u t i l i t y of the r e d u c t i v e amination r e a c t i o n f o r the s y n t h e s i s of comb-like c h i t o s a n d e r i v a t i v e s , whose s o l u t i o n and molecular p r o p e r t i e s were examined. Although the study performed h e r e i n c o n s t i t u t e s only a beginning, i t , n e v e r t h e l e s s , c l e a r l y i n d i c a t e s the immense v a r i e t y of unique and u s e f u l p r o p e r t i e s which these d e r i v a t i v e s e x h i b i t and t h e i r s i g n i f i c a n c e f o r the p o t e n t i a l a p p l i c a t i o n i n v a r i o u s areas. I I I - B . Synthesis and P r o p e r t i e s of Branched-chain Chitosan D e r i v a t i v e s 1. Synthesis Using c h i t o s a n as an exemplar, a method was devised which i s s u i t a b l e f o r the tr a n s f o r m a t i o n of l i n e a r , amine-containing polysac- charides i n t o s t a b l e , branched-chain d e r i v a t i v e s . The r e a c t i o n i t s e l f , summarized i n scheme 1, represents a f u r t h e r example of the r e d u c t i v e amination procedure which i s compatible w i t h e s s e n t i a l l y any aldehydo 96 scheme 1 sugar. Under t y p i c a l conditions, chitosan [1], dissolved i n a mixture (1:1) of d i l u t e (1%) aqueous acetic acid and methanol, was reductively alkylated using a solution of the carbonyl-containing sugar [1.1-3.2 molar equivalents per hexosamine residue (mol/GlcN)] at room temperature. The reactions of chitosan with various aldehyde-, keto-, lactone, and non- reducing sugars and cyclohexanone are summarized i n Table I I I - l . Table I I I - l reveals the following important features. The reac- tions of chitosan with aldehydo sugars proceed, i n general, smoothly y i e l d i n g products with mostly high degrees of substitution (d.s.) i n almost a l l cases, these reactions were accompanied by the formation of soft to very r i g i d , transparent or milky-white gels with, i n the l a t t e r case, attendant synereses. Monosaccharides produced gels at almost twice the rate of disaccharides and the d.s. of the chitosan products increased with increasing amounts of aldehydo sugar used (with one exception for lactose). I t may, i n passing, be mentioned that the 97 Table I I I - l . Reactions of Chitosan w i t h Carbohydrates and Other Compounds Conditions Product Compound added (mol/GlcN) Time (hr) G e l a [code] d.s. glucose 1.33 8 " 6 [3] n.d. 3.00 8 1,5 0.9 galactose 2.03 4 1,3 [2] 0.7 2.22 4 1,3 1.0 glucosamine 1.3-1.6 8 4,5 [18] 0 2.7 72 4,5 0 galactosamine 1.17-2.73 72 6 [19] 0 N-acetylglucosamine 1.6 12 1,3 [4] n.d. 1.6 24 1,5 1.0 c e l l o b i o s e 1.17 12 7 [5] n.d. 2.2 36 7 0.3 l a c t o s e 1.17 10 6 [6] 0.25 1.5 30 6 0.14 1.54 144 1,5 0.8 2.92-2.94 24 1,5 [7] "2.0"d maltose 1.7 12 7 [9] n.d. 1.7 30 1,5 0.6 mel i b i o s e 1.11 4 1,4 [10] n.d. 1.11 18 1,5 0.6 m a l t o t r i o s e 1.3 12 6 [20] 0 f r u c t o s e 2.2-3.2 30 7 [13] 0 a-glucoheptonic l a c t o n e 3.2 72 6 [23] 0 m e l i z i t o s e 1.05 24 7 [12] n.d. 1.05 240 2 0 t r e h a l o s e 1.3 72 6 [11] 0 cyclohexanone 4.5 24 7 [16] 0.5 a ( l - 5 ) : g e l s formed; (1) r i g i d , (2) ropy, (3) transparent, (4) very s o f t , (5) white; (6) g e l not formed, (7) very v i s c o u s s o l u t i o n H c obtained from m i c r o a n a l y s i s not determined see t e x t see Experimental s e c t i o n 98 amounts of aldehydo sugars (as w e l l as aromatic aldehydes, i n Chapter V) re q u i r e d f o r the formation of f u l l y (or h i g h l y ) s u b s t i t u t e d c h i t o s a n d e r i v a t i v e s were found to be f a r l e s s (by a f a c t o r of 2-40) than those 3 reported by Hirano et a l . f o r t h e i r S c h i f f ' s base d e r i v a t i v e s . A r a t h e r unusual s i t u a t i o n was encountered f o r the r e a c t i o n of r e l a t i v e l y l a r g e amounts of l a c t o s e (2.9 mol/GlcN) which gave products w i t h d.s. corresponding to 2.0 as determined by m i c r o a n a l y s i s . The question as to whether t h i s value arose from two l a c t o s e residues c o v a l e n t l y l i n k e d to each glucosamine u n i t or from a mixture of bound and " f r e e " l a c t o s e u n i t could be r e a d i l y solved by both 1 3C-nmr and e l e c t r o n microscopy i n i n favour of the l a t t e r case as w i l l be subsequently discussed ( I I I - C , E ) . In t h i s s e r i e s of experiments, no products were obtained f o r the r e a c t i o n s w i t h glucosamine and galactosamine d e s p i t e the f a c t that very s o f t white gels were produced w i t h glucosamine. Coulombic r e p u l s i o n between the protonated amine groups of c h i t o s a n (pH -4.5) and those at C2 of the r e s p e c t i v e hexosamines seems to be p r i m a r i l y r e s p o n s i b l e f o r the l a c k of product formation i n the l a t t e r two cases s i n c e a f u l l y s u b s t i t u t e d c h i t o s a n d e r i v a t i v e [4] was obtained us i n g N-acetglucosamine. The amine f u n c t i o n at C2 of the s i d e - c h a i n of [4] p r o v i d e s , upon N- d e a c e t y l a t i o n , a convenient locus f o r f u r t h e r r e d u c t i v e a l k y l a t i o n r e a c t i o n s which could a f f o r d branched, t r e e - l i k e d e r i v a t i v e s . The f a c i l i t y w i t h which c h i t o s a n can be s e l e c t i v e l y modified i s a l s o demonstrated by i t s r e a c t i o n w i t h cyclohexanone to y i e l d [16] (d.s. 0.5). The v e r s a t i l i t y of t h i s procedure was f u r t h e r e x e m p l i f i e d by the s p e c i f i c a l l y o x i d i z e d C6'-aldehydo d e r i v a t i v e s [14] and [15] which were de r i v e d from the corresponding l a c t i t y l - and m e l o b i i t y l d e r i v a t i v e s [6] and [10] using galactose oxidase (see IV-B 1 ) . D e r i v a t i v e s [14] 9 9 and [15] are useful intermediates as demonstrated by t h e i r conversion into the spin-labelled derivatives [34] and [35], respectively (see III-D). [14] and [15] could s i m i l a r l y be used, i f desired, to prepare chitosan derivatives with even longer side-chains v i a reductive amination. The apparent f a i l u r e of the reactions with maltotriose, fructose, and a-glucoheptonic lactone i s considered to be spurious i n view of the ease with which the previous reactions proceeded. These reactions were, however, not further investigated. Noteworthy i s , nevertheless, the fact that the reaction of fructose afforded very viscous solutions. A more detailed account of the observed g e l l i n g processes w i l l be given i n III-B 3. 2. Solute Interactions of Chitosan Previous studies had shown that a c i d i f i e d blends of chitosan and polyols such as s o r b i t o l and gl y c e r o l , produce high v i s c o s i t y solutions,^' while aqueous oxa l i c acid solutions of chitosan afford gels."^ This, i n 100 c o n j u n c t i o n w i t h our observation that both f r u c t o s e and glucosamine s i g n i f i c a n t l y a l t e r e d the s o l u t i o n v i s c o s i t y of c h i t o s a n , appeared to i n d i c a t e that i n the r e a c t i o n s s t u d i e d here, processes other than the chemical r e a c t i o n between c h i t o s a n and sugar s u b s t r a t e alone, were, at l e a s t i n these s p e c i f i c cases, r e s p o n s i b l e f o r the s o l u t i o n p r o p e r t i e s of the r e a c t i o n mixtures. In order to t e s t t h i s hypothesis, two non- reducing saccharides, m e l i z i t o s e and t r e h a l o s e , were added to c h i t o s a n s o l u t i o n s and i t was found t h a t , i n the former case, the v i s c o s i t y g r a d u a l l y increased l e a d i n g e v e n t u a l l y (10 days) to the formation of a ropy g e l (see Table I I I - l ) . Next, an aqueous s o l u t i o n of sodium cyanoborohydride (700 mg, 11.1 mM, 15 ml) was added to a 1% s o l u t i o n of c h i t o s a n (30 ml) and a sharp increase i n s o l u t i o n v i s c o s i t y was observed w i t h i n 5 minutes; no g e l was obtained, however, w i t h i n 30 days.* When, on the other hand, the r e a c t i o n of c h i t o s a n w i t h l a c t o s e (3.9 mol/GlcN) was performed under omission of sodium cyanoborohydride, no g e l s were formed and a product [8] w i t h very low d.s. (0.1) was obtained. These f a c t s would seem to i n d i c a t e a s p e c i f i c i n t e r a c t i o n of c h i t o s a n and the reducing agent which s i g n i f i c a n t l y c o n t r i b u t e s to the formation of g e l s . In c o n t r o l experiments w i t h s o l u t i o n s of c h i t o s a n i t s e l f or of mixtures (1:2 w/w) of c h i t o s a n and a s e r i e s of other s o l u t e s , i n c l u d i n g a l k a l i e a r t h , l a n t h a n i d e , and t r a n s i t i o n metal s a l t s , no v i s c o s i t y i ncreases or g e l formations were observed over periods of up to 30 days. *Gel formation could a l s o not be induced by c o o l i n g (5°C). 3. G e l l i n g Processes Prom the r e s u l t s discussed above i t i s obvious that a complex s e r i e s of phenomena c o n t r i b u t e to the observed g e l l i n g processes, and we w i l l now attempt to i d e n t i f y some of these. ( i ) Solute-mediated g e l a t i o n Although the i n t e r a c t i o n s of s o l u t e s w i t h some a c i d i c polysacchar- 18 ides have been i n v e s t i g a t e d no equivalent work e x i s t s f o r c a t i o n i c p o l y s a c c h a r i d e s , w i t h the exception of s e v e r a l r e p o r t s on the formation 19 of p o l y e l e c t r o l y t e complexes of c h i t o s a n and a n i o n i c p o l y s a c c h a r i d e s . Assuming that the processes i n v o l v e d f o r both types of i o n i c p o l y - saccharides are f a i r l y s i m i l a r i n nature, two types of s o l u t e e f f e c t s can be considered: (a) E l e c t r o l y t e s have been i m p l i c a t e d i n the g e l a t i o n of 20 v a r i o u s p o l y s a c c h a r i d e s , most notably i n those of a l g i n a t e and 21 carrageenan, where they mediate h e l i x aggregations by way of s p e c i f i c bonding i n t e r a c t i o n s w i t h c a r b o x y l and hydroxyl groups. In general however, the presence of metal ions has un p r e d i c t a b l e e f f e c t s on p o l y - saccharides i n s o l u t i o n , which cannot be simply r a t i o n a l i z e d i n terms 22 of coulombic a t t r a c t i o n s . Thus, some e l e c t r o l y t e s decrease the v i s c o s i t y of a l g i n a t e and guaran but have the opposite e f f e c t f o r 22 23 carrageenan and hypnean. ' The a c t i o n of sodium cyanoborohydride (and o x a l i c acid) on c h i t o - san s o l u t i o n s may t h e r e f o r e be described as s p e c i f i c i n t e r a c t i o n s of these r e l a t i v e l y l a r g e anions w i t h c h i t o s a n . (b) N o n - e l e c t r o l y t e s , such as sugars and a l c o h o l s are 23 24 known ' to induce g e l a t i o n of hypnean d i s p e r s i o n s , w i t h only s m a l l q u a n t i t i e s of sugar being r e q u i r e d f o r the formation of very strong gels. In such systems sugars are considered*'"'"'*'"'' to act, on the one hand, as competing agents for solvent thereby leading to a decreased solvation of the polysaccharide chains and, on the other, as promoters of inter-chain hydrogen bonding; both modes of action resulting in gel formation. Similar phenomena must be also operative for the case of the viscosity increase or gelation of chitosan solutions in the presence of various carbohydrates, such as fructose, glucosamine, and melizitose, which did not form chitosan derivatives. ( i i ) Gelation of branched-chain derivatives The gelation of the branched-chain chitosan derivatives probably involves very similar factors as discussed above. Noteworthy is the generally greater gel strength of these derivatives in comparison with that of the solute-mediated products. It can be invoked that the covalently-linked saccharides allow for a more proximate interchain- association than in the latter case. In addition to these factors, the extent and regularity of the substitutions on chitosan appear to be of importance for gelation, since a l l derivatives with high d.s. formed ri g i d gels, whereas for derivatives with d.s. <0.3 no gels (or only viscosity increases) were observed, e.g., for cellobiose and lactose. 26 In a related study, Schweiger has attributed the unusual compatibility of cellulose sulfate derivatives to the uniformity of substitution. 4. Other Properties The u t i l i t y of the branched-chain chitosan derivatives i s further illustrated by the properties which they exhibit after isolation from the reaction mixtures. In contrast to other known derivatives, e.g., the arylidene chitosan derivatives discussed in Chapter V, a l l of these 103 samples r e t a i n e d t h e i r g e l l i n g p r o p e r t i e s when the dry powders were r e c o n s t i t u t e d i n e i t h e r n e u t r a l aqueous or s l i g h t l y a c i d i c media. Thus, the d e r i v a t i v e s [ 3 ] , [ 6 ] , [ 7 ] , and [ 9 ] , at concentrations above 3-5% (some even at much lower c o n c e n t r a t i o n s ) , formed r i g i d gels i n aqueous s o l u t i o n s , w h i l e [2] and [A] g e l l e d i n s l i g h t l y a c i d i c (pH 5-6) s o l u - t i o n s ; [10] was s o l u b l e i n the l a t t e r medium but produced g e l s on a d d i t i o n of base. Gels obtained from [2] thinned out w i t h time (10- 12 h ) . D e r i v a t i v e s [5] and [7] e x h i b i t e d s t a b i l i t y to a l k a l i n e media, and [5] and [10] were a l s o compatible w i t h 50% aqueous et h a n o l , the l a t t e r forming very v i s c o u s s o l u t i o n s . Aqueous s o l u t i o n s of [6] or [7] d i d not g e l or p r e c i p i t a t e when mixed w i t h calcium c h l o r i d e , chromium c h l o r i d e , t i n c h l o r i d e , potassium chromate, b o r i c a c i d , or s e v e r a l combinations of these. I n t e r e s t i n g l y , the d e r i v a t i v e [ 5 ] , which by i t s e l f d i d not g e l , was found t o form r i g i d white g e l s (which contracted a f t e r a few h o u r s ) , when mixed w i t h a l g i n a t e , and very v i s c o u s s o l u t i o n s , when mixed w i t h e i t h e r guaran or l o c u s t bean gum. Our i n i t i a l e v a l u a t i o n has demonstrated the great f a c i l i t y w i t h which an i n t r a c t a b l e m a t e r i a l , such as c h i t o s a n , can be converted, us i n g t h i s m o d i f i c a t i o n procedure, i n t o products which, even at r e l a t i v e l y very low d.s. (e.g., f o r [6] d.s. 0.1A), are s o l u b l e i n aqueous media. The wide range of s o l u b i l i t y , g e l l i n g , and c o m p a t i b i l i t y p r o p e r t i e s of these branched-chain d e r i v a t i v e s appears to be ra t h e r unique f o r p o l y - saccharide products and promises to be of p o t e n t i a l u t i l i t y f o r a v a r i e t y of a p p l i c a t i o n s , c o n s i d e r i n g the low cost of combining two surp l u s carbo- hydrates such as, f o r example, l a c t o s e and c h i t o s a n . III-C. 1 3C nmr * 3C nmr was employed for the spectral assignment and structural elucidation of chitosan and a selected number of the branched-chain derivatives; the spectra are shown in Figures III-1-4, and the proposed chemical shift assignments are recorded in Table III-2. The spectral assignment of chitosan [ 1 ] i t s e l f (Fig. I l l - l a ) was readily accomplished by comparison with previous data reported for the 27 28 monomeric aminoglycosides. ' The anomeric signals of both the aminoglucose and acetamidoglucose residues of [ 1 ] were clearly resolved; the signal at lower f i e l d was attributed to the acetamido derivative while that of the amino-derivative appeared 3 ppm upfield. The signals of the other ring carbons of the two types of hexosamine residues were indistinguishable at this pH (pD 4.0) and were assigned, based on the relative proportions of acetamidoglucose and glucosamine residues in [ 1 ] , to the latter (Table III-2). On lowering the pH (to pD 1.5) the resonances of [ 1 ] experienced an upfield shift of 2-3 ppm (Fig. I l l - l b ) , which was accompanied by the appearance of several additional signals. No previous 1 3C-data for equivalent monomeric aminoglycosides (at pD 1.5) were available to confirm the proposed assignments (Table III-2) of these additional resonances.* The degradation of [1] under strongly acidic conditions (pD 1.5) could be observed only after prolonged periods of time (3-4 weeks) at 298K with the appearance of additional resonances in the anomeric region and between 60-77 ppm (C3,C4,C6). TABLE I I I - 2 . C h e m i c a l S h i f t A s s i g n m e n t o f C h i t o s a n and some D e r i v a t i v e s a C h e m i c a l s h i f t , ppm Compound ( d . s •) b C l C2 C3 C4 C5 C6 C l ' C2' C3' C4' C5' C 6 ' [ l J b p D 4 . 0 1 _ j- B l , 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y l ) B l , 4 - ( 2 - d e o x y - 2 - a c e t a m i d o - g l u c o p y r a n o s y l ) 1 0 1 . 8 9 8 . 6 5 6 . 6 7 5 . 3 7 1 . 1 7 7 . 8 6 0 . 9 p D 1 . 5 B l , 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y l ) B l , 4 - ( 2 - d e o x y - 2 - a c e t a m i d o - g l u c o p y r a n o s y l ) 9 8 . 3 9 4 . 9 5 3 . 1 5 2 . 9 7 2 . 0 7 1 . 6 6 8 . 4 6 7 . 8 7 4 . 3 5 7 . 5 [51 ( 0 . 3 ) 6 1 , 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y l ) 8 - g l u c o p y r a n o s y l 1 0 2 . 0 1 0 3 . 0 5 6 . 9 7 4 . 1 7 5 . 2 7 6 . 3 7 1 . 5 n . r . 7 7 . 6 7 6 . 5 6 1 . 3 6 1 . 3 1 - d e o x y g l u c i t - l - y l A 5 1 . 3 7 3 . 9 7 5 . 5 8 0 . 6 7 1 . 5 6 2 . 6 [6J ( 0 . 9 ) B l , 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y 1 ) 1 0 2 . 8 5 6 . 8 7 5 . 7 C 7 2 . 0 7 7 . 7 6 1 . 8 B - g a l a c t o p y r a n o s y l 1 0 4 . 0 7 2 . 0 7 3 . 5 6 9 . 0 75 .7  C n . r . A A 1 - d e o x y g l u c i t - l - y l 5 1 . 3 7 2 . 0 7 2 . 0 7 9 . 0 n . r . 6 2 . 9 l a c t o s e n . r . n . r . 7 3 . 5 6 9 . 6 7 6 . 1 n . r . 9 2 . 6 9 6 . 5 i n . r . n . r . 7 9 . 0 e 7 5 . 7 c n . r . [6J ( 0 . 2 5 ) 8 1 - 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y 1 ) 1 0 2 . 7 5 7 . 0 7 4 . 9 n . r . 7 7 . 5 6 1 . 9 B - g a l a c t o p y r a n o s y l 1 0 4 . 0 7 2 . 0 7 2 . 8 6 9 . 1  f 7 6 . 0 n . r . c 1 - d e o x y g l u c i t - 1 - y l 5 1 . 1 7 2 . 0 7 2 . 0 7 9 . 0 6 9 . 1 I 6 3 . 0 [16J ( 0 . 5 ) b B - l , 4 - ( 2 - d e o x y - 2 - a m i n o - g l u c o p y r a n o s y l ) 1 0 2 . 1 9 8 . 5 5 6 . 6 7 5 . 3 7 1 . 0 7 9 . 0 6 1 . 9 6 0 . 6 J p D 4 . 0 c y c l o h e x y l 5 9 . 8 3 0 . 5 2 5 . 0 g 3 0 . 0 2 4 . 8 8 3 0 . 5 a i n D 0 r e l a t i v e t o I n t e r n a l p - d i o x a n e ( 6 7 . 4 0 ppm) a t 315 K ; b s o l v e n t D O A c - d , / D . O ; C c o i n c i d i n g r e s o n a n c e s ; g a s s i g n m e n t may be r e v e r s e d ; h ^ i k d e g r e e o f s u b s t i t u t i o n ; a , B r e s p e c t i v e l y ; r e s o n a n c e s c o i n c i d e e x c e p t as i n d i c a t e d ; n . r . - n o t r e s o l v e d . O 106 Fig. I I I - l . 100.6 MHz C-nmr spectra of [1] (4% in DOAc-d /D 0) at 315K (a) at pD 4; (b) at pD. 1.5; ( sweep width 26,000, pulse width 13 ysec, delay 0.1 sec, 50 000 scans).  1 0 8 T h e r e s o n a n c e s o f t h e p o l y s a c c h a r i d e b a c k b o n e w e r e , u p o n s u b s t i t u - t i o n , f o u n d t o b e d i s p l a c e d b y s m a l l i n c r e m e n t s o n l y . F o r e x a m p l e , t h e h e x o s a m i n e s i g n a l s o f C l , C 2 , C 4 , a n d C6 o f d e r i v a t i v e [ 5 ] w e r e s h i f t e d d o w n f i e l d b y a p p r o x i m a t e l y 0 . 3 p p m , w h i l e t h o s e o f C3 a n d C5 w e r e s h i f t e d u p f i e l d b y 0 . 2 ppm ( F i g . I I I - 2 ) . * - T h e r e s o n a n c e s o f t h e p e n d i n g c e l l o b i i t y l r e s i d u e s c o u l d b e i d e n t i f i e d b y c o m p a r i s o n w i t h t h e c h e m i c a l 29 s h i f t s r e p o r t e d f o r c e l l o b i o s e a n d 4 - [ ( l - D e o x y c e l l o b i i t - l - y l ) - a m i n o ] b e n z o p h e n o n e ( p r e p a r e d a n a l o g o u s l y b y r e d u c t i v e a m i n a t i o n o f c e l l o b i o s e , s e e r e f e r e n c e 3 0 ) . T h e c h e m i c a l s h i f t o f t h e d e o x y g l u c i t y l C l 1 w a s s h i f t e d u p f i e l d b y m o r e t h a n 4 0 ppm f r o m t h a t o f t h e p a r e n t c e l l o b i o s e . T h e a s s i g n m e n t s o f t h e t e r m i n a l g l u c o p y r a n o s y l r e s i d u e o f [ 5 ] w e r e f o u n d t o b e i n e x c e l l e n t a g r e e m e n t w i t h t h e p r e v i o u s r e s u l t s o f s e v e r a l 29 w o r k e r s . T h e r e d u c t i v e a l k y l a t i o n o f c h i t o s a n w i t h e x c e s s l a c t o s e ( 3 m o i / G l c N r e s i d u e ) p r o d u c e d , a s m e n t i o n e d e a r l i e r , a d e r i v a t i v e [ 7 ] w h o s e e l e m e n t a l a n a l y s i s i n d i c a t e d a d . s . o f " 2 . 0 " . T h e 1 3 C nmr s p e c t r u m o f [ 7 ] ( F i g . I I I - 3 a ) , h o w e v e r , r e v e a l e d q u i t e c l e a r l y t h a t t h i s d . s . v a l u e d e r i v e d f r o m o n e e q u i v a l e n t o f " f r e e " l a c t o s e p e r h e x o s a m i n e e q u i v a l e n t , a s e v i d e n c e d b y t h e p r e s e n c e o f t h e t w o a n o m e r i c g l u c o p y r a n o s e s i g n a l s ( b o t h a a n d 6) i n t h e r e g i o n b e t w e e n 9 0 - 1 0 0 p p m . T h e s p e c t r u m o f [ 7 ] w a s d o m i n a t e d b y t h e s h a r p e r s i g n a l s o f t h e t r a p p e d l a c t o s e a n d w a s n o t f u r t h e r a s s i g n e d . T h e l a c t o s e c o m p l e x [ 7 ] w a s s u b s e q u e n t l y d i a l y z e d f o r f i v e d a y s i n o r d e r t o r e m o v e t h e u n b o u n d l a c t o s e b u t t h e r e s u l t i n g p r o d u c t [ 6 ] w a s f o u n d t o s t i l l c o n t a i n r e s i d u a l q u a n t i t i e s o f t h e t r a p p e d l a c t o s e ( c a . 50%) a s s h o w n b y t h e a n o m e r i c C l ' s i g n a l s a t 9 2 . 6 ppm a n d * N o t e , h o w e v e r , p H d i f f e r e n c e s b e t w e e n t h e t w o s a m p l e s [ 1 ] a n d [ 5 ] ; t h e s p e c t r u m o f t h e l a t t e r w a s r e c o r d e d a t pD 7 . F i g . I I I - 3 . 1 0 0 . 6 MHz C-nmr s p e c t r a o f (a) [7] and (b) [6] ( d . s . 0 . 9 ) o b t a i n e d f r o m [7] (a) a f t e r 5d d i a l y s i s ; ( c o n t d . )  110 D 120 100 80 60 40 20 ppm F i g . I I I - 4 . 100.6 MHz C-nmr spectrum of [16] i n DOAc-d^/D^ at pD 4.0 and 315K, 60,000 scans. I l l 96.5 ppm, r e s p e c t i v e l y ( F i g . I l l - 3 b ) . These f i n d i n g s , consequently, provide evidence f o r the formation of a strong complex between 1-deoxy- l a c t i t - l - y l c h i t o s a n and l a c t o s e from which the " f r e e " l a c t o s e i s only incompletely r e l e a s e d upon extensive d i a l y s i s . From 'this i t may be p o s t u l a t e d that [7] c o n s t i t u t e s an i n c l u s i o n complex i n which l a c t o s e may r e s i d e i n the i n t e r s t i c e s of m u l t i s t r a n d e d h e l i c e s of [ 6 ] , somewhat s i m i l a r to the well-known iodine-amylose i n c l u s i o n complex. I t appears that t h i s type of a s s o c i a t i o n i s s p e c i f i c to [ 6 ] , s i n c e no s i m i l a r phenomena have so f a r been observed f o r any of the other branched-chain c h i t o s a n d e r i v a t i v e s . The chemical s h i f t s of the hexosamine residues of [6] followed the same trend as were e s t a b l i s h e d f o r those of [5] and the s i g n a l s of the c o v a l e n t l y - l i n k e d l a c t i t y l r e sidues could be r e a d i l y i d e n t i f i e d by 29 comparison w i t h previous assignments of l a c t o s e , as w e l l as of a s e r i e s of r e l a t e d a l k y l - d e o x y l a c t i t y l a m i n e d e r i v a t i v e s reported by 31 31 P f e f f e r et a l . and of two f l u o r e s c e n t d e o x y l a c t i t y l a m i n e d e r i v a t i v e s . The C l ' s i g n a l of the 1 - d e o x y g l u c i t - l - y l residues of [6] appeared at 51.3 ppm and was i n l i n e w i t h the corresponding s i g n a l s of the a l k y l 31 d e r i v a t i v e s of P f e f f e r et a l . Good agreement was a l s o obtained between the other chemical s h i f t assignments of [6] and the r e s u l t s . . 29,30 reported elsewhere. F i g u r e I I I - 3 c shows the spectrum of a sample of [6] w i t h lower d.s. (0.25) which contained no trapped l a c t o s e . The observed chemical s h i f t s of t h i s product agreed w i t h the r e s u l t s of the previous sample, r e v e a l i n g o n l y minor displacements of some resonances. The c y c l o h e x y l d e r i v a t i v e [16] produced a w e l l - d i s p e r s e d spectrum (at pD 4.0) i n which the methylenic r i n g carbons were observed i n the 112 r e g i o n between 24 to 31 ppm i n c l o s e agreement w i t h the corresponding 32 s i g n a l s of N-methylcyclohexylamine ( F i g . I I I - 4 ) ; the c y c l o h e x y l C l ' resonance appeared at 59.8 ppm, 1.2 ppm downfield from that of the corresponding N-methyl d e r i v a t i v e . The chemical s h i f t s of the carbons of the polymer backbone were found to be again i n good agreement w i t h those of the previous d e r i v a t i v e s w i t h the exception of the C5 s i g n a l which was d i s p l a c e d downfield by 1.2 ppm w i t h respect to that of [ 1 ] . The hexosamine resonances i n the s p e c t r a l r e g i o n between 70 to 80 ppm were f u r t h e r c h a r a c t e r i z e d by the presence of incompletely r e s o l v e d s i g n a l s at lower f i e l d which can presumably be assigned to the unbranched hexosamine residues s i n c e the branched residues produced, i n g e n e r a l , sharper s i g n a l s (compare the 1 3 C nmr s p e c t r a of galactomannans, discussed i n Chapter I V ) . The C6 resonances of both the glucosamine and acetamidoglucose residues were c l e a r l y r e s o l v e d and could be assigned on the b a s i s of the corresponding monomeric aminoglycosides. 2^ The carbonyl and methyl resonances of the N-acetate groups were observed at 175 ppm (±0.6) and at 22 ppm (±0.9), r e s p e c t i v e l y f o r a l l d e r i v a t i v e s discussed here. I I I - D . S p i n - L a b e l l i n g of C h i t i n and Chitosan 1. I n t r o d u c t i o n Concomitant w i t h the advent of novel c h i t i n based products, the need has a r i s e n f o r the development of s p e c t r o s c o p i c l a b e l l i n g methods that w i l l f a c i l i t a t e d e t a i l e d a n a l y s i s of the molecular parameters r e s p o n s i b l e f o r the s o l u t i o n and g e l behaviour of both the n a t i v e p o l y - mers and t h e i r d e r i v a t i v e s . The i n t r a c t a b i l i t y of the aminopoly- 113 saccharides i s mainly r e s p o n s i b l e f o r the p a u c i t y of such i n f o r m a t i o n i n the l i t e r a t u r e ; only r e c e n t l y have spe c t r o s c o p i c s t u d i e s begun to shed some l i g h t on the shape and molecular p r o p e r t i e s of these m a t e r i a l s . 33 B u f f i n g t o n and Stevens have concluded from c i r c u l a r d i c h r o i s m data that i n t e r - , r a t h e r than i n t r a m o l e c u l a r i n t e r a c t i o n s between amide groups play an important f a c t o r i n the g e l formation of c h i t a n and c h i t i n . 3 Hirano et a l . have observed pronounced changes i n s p e c i f i c r o t a t i o n of c h i t o s a n during S c h i f f s base formation and the attendant g e l a t i o n , but d i d not draw any s t r u c t u r a l c o n c l u s i o n from these. As an extension of our i n v e s t i g a t i o n s of the g e l l i n g and metal c h e l a t i n g p r o p e r t i e s and u l t r a s t r u c t u r e of c h i t i n and c h i t o s a n d e r i v a t i v e s i t was e s s e n t i a l to evaluate procedures f o r preparing n i t r o x i d e s p i n - l a b e l l e d d e r i v a t i v e s . The work described i n t h i s s e c t i o n covers three aspects: ( i ) the s y n t h e s i s of a number of s p i n - l a b e l l e d c h i t i n and c h i t o s a n d e r i v a t i v e s ; ( i i ) the assessment of s e v e r a l s t r u c t u r a l parameters of these m a t e r i a l s , such as the m o b i l i t y of the l a b e l m o i e t i e s and, by i n f e r e n c e , of the polymers; and ( i i i ) the e x p l o r a t i o n of i n t e r a c t i o n s between paramag- n e t i c ions and s p i n - l a b e l s bound to the p o l y s a c c h a r i d e backbone. 2. S p i n - L a b e l l i n g of G h i t i n and Chitosan The s p i n - l a b e l l i n g proved to be a somewhat greater challenge than other r e a c t i o n s of these m a t e r i a l s due to the a c i d l a b i l i t y of the n i t r o x i d e probes. Several s t r a t e g i e s were explored to r e s o l v e t h i s problem. F i r s t l y , c h i t i n [21],* p r e t r e a t e d w i t h methyl s u l f o x i d e and aqueous sodium hydroxide, reacted w i t h 4-chloroacetamido-2,2,6,6-tetramethyl- *Derived from crab s h e l l and c o n t a i n i n g 10% f r e e NH2. 114 p i p e r i d i n e - l - o x y l [24] to produce the spin-labelled c h i t i n [25] (d.s. L - + CLCH,C0NH [21] [24] Rj=CH2C0NHSL [25] 0.04). The derivative [25] swelled to some extent when suspended i n methanol or aqueous solution. Alternative attempts to s p e c i f i c a l l y l a b e l c h i t i n i n solution 34 (using the LiCl/DMAc solvent system) by reductive a l k y l a t i o n of the free amine groups with 4-oxy-2,2,6,6-tetramethylpiperidine-l-oxyl, [26] were unsuccessful. The expected product [27] was obtained i n such low y i e l d s * (from several attempts) that t h i s route was not further pursued. NaCNBH, CM-OH CM,0H [21] R = SL [25] Spin-labelling of chitosan [1] was conducted s i m i l a r l y using the labels [24] and [26]. Since, however, ac i d i c media are required for the d i s s o l u t i o n of [1], a number of routes were investigated to avoid or minimize acid-degradation of the nitroxide labe l s . The f i r s t approach consisted of converting chitosan into the N-sulfate derivative *As judged by i t s esr spectrum. 115 [28] which i s moderately w a t e r s o l u b l e at elevated temperatures, and subsequently l a b e l l i n g [28] under m i l d c o n d i t i o n s u s i n g [26] or [24] to a f f o r d the products [30], (d.s. 0.45) and [29] (d.s. 0.5), r e s p e c t i v e l y . Thus, the d e r i v a t i v e [28] i s a v e r s a t i l e intermediate and has r e c e n t l y 35 a l s o been employed f o r enzyme i m m o b i l i z a t i o n s . Both d e r i v a t i v e s [29] and [30], which were p a r t i a l l y N - s u l f a t e d , formed gels i n aqueous s o l u t i o n s . In a d i f f e r e n t approach, c h i t o s a n was next d i s s o l v e d i n a mixture of methanol and very d i l u t e (0.4%) aqueous a c e t i c a c i d and r e d u c t i v e l y a l k y l a t e d u s i n g [26] to a f f o r d a product [31] which g e l l e d c o n s i d e r a b l y i n methanol or aqueous s o l u t i o n . T h i s f e a t u r e combined w i t h the f o r t u i - t o u s l y low s u b s t i t u t i o n (d.s. 0.1) (which ensured a minimal p e r t u r b a t i o n of the polymer s t r u c t u r e ) made [31] of greater u t i l i t y than [29] or [30].* 0 [31] The s p i n - l a b e l l e d d e r i v a t i v e s [34] and [35] were obtained by r e d u c t i v e amination of the corresponding C6'-aldehydo, branched chitosans [14] and [15] (which were described i n I I I - B ) u s i n g 4-amino-2,2,6,6- t r e t r a m e t h y l p i p e r i d i n e - l - o x y l [32]. Both ]34] and [35] were s o l u b l e i n water, forming v i s c o u s s o l u t i o n s . • Several other c h i t o s a n d e r i v a t i v e s , obtained from r e a c t i o n s per- formed i n solvent systems such as aqueous p y r i d i n e or aqueous a c e t i c a c i d , were produced i n very low y i e l d s and were consequently considered to be unworthy of f u r t h e r a t t e n t i o n . 116 [34] l y x , R^H [35] Rj»H, Rj-X X- 3. Esr Spectra The esr s p e c t r a of the d e r i v a t i v e s described above are shown i n Figures I I I - 5 - 7 and the corresponding motional c o r r e l a t i o n times are l i s t e d i n Table I I I - 3 ; the T £ values represent approximate v a l u e s * s i n c e they were derived w i t h the assumption of i s o t r o p i c motion. Neverthe- l e s s , they do provide a measure of the r e l a t i v e d i f f e r e n c e s i n the degrees of motional freedom between the d e r i v a t i v e s . From Table I I I - 3 i t i s evident that the c h i t i n d e r i v a t i v e enjoys a smaller freedom of r o t a t i o n a l r e o r i e n t a t i o n than the c h i t o s a n d e r i v a t i v e s . I n t u i t i v e l y , t h i s i s not unreasonable s i n c e the former only swelled i n s o l u t i o n , w h i l e the l a t t e r , p a r t i c u l a r l y [31], formed voluminous gels which c o n s i s t e d of • P a r t i c u l a r l y f o r the complex s p e c t r a of [30] and [31]; an accurate q u a n t i t a t i o n of T C'S would r e q u i r e use of s p e c t r a l s i m u l a t i o n . Fig. III-5. Esr spectrum of [25] in aqueous solution (298K). 118 F i g . I I I - 6 . Esr sp e c t r a of [31] (a) g e l i n water, (b) gel i n methanol, (c) g e l (b) t r a n s f e r r e d i n t o water ( a f t e r 2 weeks e q u i l i b r i z a t i o n ) ; (at 298K). 119 Fig. II-I -7 . Esr spectra of (a) [34], (b) [35], in aqueous solution (at 2 9 8 K ) . 120 Table I I I - 3 . T Values f o r some S p i n - l a b e l l e d c C h i t i n and Chitosan D e r i v a t i v e s Compound [25] [30] [31] [34] [35] 35 61-400 a 68 43 V a l u e s a r i s e from d i f f e r e n t components, see text l a r g e proportions of s o l v e n t (>95%). The solvent pockets provided a m a t r i x i n which the n i t r o x i d e had a g r e a t e r m o b i l i t y than i n [25]. I t i s a l s o i n t e r e s t i n g to note the s u b s t a n t i a l d i f f e r e n c e s , both i n l i n e - shape and T , between [30] and [31], seemingly s i m i l a r d e r i v a t i v e s , d i f f e r i n g mainly i n t h e i r degree of s u b s t i t u t i o n . W i t h i n the s e r i e s of c h i t o s a n d e r i v a t i v e s , [34] and [35] revealed a somewhat greater m o b i l i t y than [30] which i s presumably a t t r i b u t a b l e to the f a c t that ( i ) [34] and [35] were w a t e r s o l u b l e , w h i l e [30] formed g e l s ; and ( i i ) the n i t r o x i d e m o i e t i e s of [34] and [35] are separated from the polymer backbone by a spacer group of two sugars, w h i l e i n [30] the n i t r o x i d e s are separated from the polymer by a four bond l i n k a g e only. I t might be expected that the l a t t e r f a c t o r should lead to a much greater n i t r o x i d e m o b i l i t y i n [34] and [35]. That t h i s e f f e c t i s apparently m i t i g a t e d can be p o s s i b l y a s c r i b e d to the entanglement of p o l y s a c c h a r i d e chains and/or i n t r a - c h a i n i n t e r a c t i o n s . This argument i s a l s o supported by the s u b s t a n t i a l (50%) d i f f e r e n c e s i n of [34] and [35], which i n a l l l i k e l i h o o d , can o n ly a r i s e from the d i f f e r e n t types 121 of l i n k a g e (l->4 and l->6, r e s p e c t i v e l y ) between the pendant sugar m o i e t i e s . The more extended chain of [35] allows f o r a more ordered, l e s s mobile, s o l u t i o n s t r u c t u r e , whereas the l-*4 l i n k a g e of [34] leads to l e s s ordered conformation i n which the n i t r o x i d e moiety enjoys a greater m o b i l i t y . The esr sp e c t r a of g e l l e d [31] ( F i g s . I I I - 6 a,b), i n e i t h e r methanol or aqueous s o l u t i o n , r e v e a l a very i n t e r e s t i n g lineshape which i s s t r o n g l y i n d i c a t i v e of s e v e r a l p a r t i a l l y r e s o l v e d s p e c t r a l components w i t h v a s t l y (̂  order of magnitude) d i f f e r e n t x c values and h y p e r f i n e l i n e w i d t h s . I t should be noted here that the "sharp" s i g n a l could not be removed d e s p i t e extensive washing of the m a t e r i a l and may o r i g i n a t e from a bound but very mobile n i t r o x i d e p o p u l a t i o n , although the p o s s i b i l - i t y f o r i t to d e r i v e from a s m a l l q u a n t i t y of adsorbed l a b e l cannot be t o t a l l y excluded. The esr sp e c t r a of [31] seem to i n d i c a t e the presence of n i t r o x i d e populations r e s i d i n g i n more or l e s s a c c e s s i b l e s i t e s . The l i k e l i h o o d that the observed lineshapes simply a r i s e from an i s o t r o p i c - a l l y tumbling, homogeneous p o p u l a t i o n of s p i n l a b e l s can be r u l e d out on the f o l l o w i n g grounds: ( i ) s i m i l a r lineshapes (with approximately equal p r o p o r t i o n s of s p i n populations i n the r e s p e c t i v e s i t e s ) are obtained f o r [31] i n two d i f f e r e n t s o l v e n t s ; and ( i i ) data obtained from experiments i n v o l v i n g paramagnetic ions show a heterogeneous d i s t r i b u t i o n of spins over the polysaccharide m a t r i x (discussed below). Precedence f o r the proposed e x i s t e n c e on c h i t o s a n of s e v e r a l types of s i t e s w i t h v a r y i n g degrees of a c c e s s i b i l i t y e x i s t s i n the l i t e r a t u r e s i m i l a r phenomena 36 37,38 having p r e v i o u s l y been observed f o r c e l l u l o s e and agarose using nmr and esr methods. The heterogeneity of s p i n - l a b e l d i s t r i b u t i o n i n [31] was a l s o demonstrated by an experiment i n which one solvent 122 (methanol) i n the i n t e r i o r of the g e l l e d [31] was replaced by another one (water) r e s u l t i n g , a f t e r s u f f i c i e n t time f o r e q u i l i b r i z a t i o n (2 weeks), i n changes i n the r e l a t i v e proportions of the s p e c t r a l compon- ents ( F i g . I I I - 6 c ) . Figure I I I - 6 c r e v e a l s a predominance of a very mobile n i t r o x i d e p o p u l a t i o n over a l e s s mobile, and b a r e l y d e t e c t a b l e , one. Although t h i s observation seems to c o n f l i c t at f i r s t s i g h t w i t h the s i m i l a r i t y i n lineshape d i s p l a y e d by f r e s h l y prepared gel s i n each of the two s o l v e n t s ( F i g s . I I I - 6 a,b), i t can be considered to a r i s e from a slow s t r u c t u r a l r e o r g a n i z a t i o n of the g e l which r e s u l t s i n an o v e r a l l l e s s ordered, more mobile assembly. Support f o r such " s o l v e n t - mediated" g e l expansion derives from the observation that the macroscopic g e l volume increased s i g n i f i c a n t l y (̂  2x) w i t h time (weeks) when methanol was replaced by water. This volume increase was not, however, accom- panied by a l o s s i n o v e r a l l g e l r i g i d i t y over extended periods of time ( s e v e r a l months) e l i m i n a t i n g the p o s s i b i l i t y f o r the increased m o b i l i t y to a r i s e from a slow d i s s o l u t i o n of the g e l . 4. S e l e c t i v e Broadening Experiments The " m u l t i - s i t e " model proposed above was next t e s t e d u s i n g the s p i n - p r o b e - s p i n - l a b e l method. Previous workers"^ ^ have shown that paramagnetic "probe" i o n s , such as t r a n s i t i o n metal i o n s , i n s o l u t i o n lead to exchange broadening of the esr l i n e s of s p i n l a b e l s both i n s o l u t i o n and attached to macromolecules. The l i n e width of the n i t r o x i d e esr s i g n a l of [33] increased r a p i d l y w i t h i n c r e a s i n g probe i o n c o n c e n t r a t i o n , as shown i n Figure I I I - 8 a , no s i g n a l being d e t e c t a b l e 2+ at N i c o n c e n t r a t i o n above 3 mM. In c o n t r a s t , f o r the d e r i v a t i v e [35] one f i n d s ( F i g . II I - 8 b ) that again the s i g n a l decreases w i t h i n c r e a s i n g c o n c e n t r a t i o n of probe i o n . However, a r e s i d u a l s i g n a l was a a F i g . I I I - 8 . Esr s p e c t r a of [33] ( l e f t ) and [35] ( r i g h t ) i n the presence of N i ( I I ) ; N i ( I I ) concentrations were (mM): [33] (a) 0,(b) 0.12,(c) 0.23,(d) 0.45, (e) 1.12; [35] (a) 0,(b) o.22,(c) 0.45,(d) 2.90,(e) 5.14,(f) 9.61,(g) 11.84,(h) 20.2,(i) 30.0. S 3 0 0 124 Fig. I I I - 9 . Plot of centre-field linewidth of [35] as a function of added nickel sulfate. 125 observable even a f t e r a d d i t i o n of 30 mM Ni*"' , although reduced i n i n t e n s i t y by approximately two orders of magnitude i n comparison to the s i g n a l obtained i n the absence of metal i o n . The p l o t of the l i n e w i d t h , (ACOQ(C)) (peak-to-peak) against c o n c e n t r a t i o n of added n i c k e l r e v e a l s d i s - 40 c o n t i n u i t i e s which i n d i c a t e the existence of s e v e r a l d i f f e r e n t s i t e s occupied by l a b e l s ( F i g . I I I - 9 ) . At concentrations above 12 mM, n i c k e l seems to exert l i t t l e or no i n f l u e n c e on AWQ(C). (A s i m i l a r o v e r a l l 2+ Aoo 0(c) dependency on (Ni ) was observed f o r the d i r e c t l y l a b e l l e d c h i t o s a n [31].) The l i n e broadening of the s p i n - l a b e l l e d d e r i v a t i v e s i n the presence of paramagnetic ions ( F i g s . 111-10, 11) can a l s o be expressed i n terms of the dependency of the increase i n peak-to-peak width of the centre l i n e on metal c o n c e n t r a t i o n as i l l u s t r a t e d i n Figure 111-12 f o r [31], [33], and [35]. For both [31] and [35] r a p i d broadening was observed at low 2+ (Ni ), whereas at higher concentrations t h i s e f f e c t was reduced ( f o r [ 3 1 ] ) * or v i r t u a l l y absent ( f o r [35]); 7 mM concentrations of metal, on the other hand, broadened the s i g n a l of [34] e s s e n t i a l l y beyond d e t e c t i o n . * S i m i l a r behaviour was e x h i b i t e d by [29] which showed a r e s i d u a l s i g n a l even at c o n c e n t r a t i o n of 26 mM N i l I ( F i g . I I I - l l ) ; peak o v e r l a p , however, precluded l i n e width measurements. Fig. 111-10. Esr spectra of [31] (left) and [34] (right) in the presence of Ni(II); Ni(II) concentrations were (mM): [31] (a) 0,(b) 2.23, (c) 4.47,(d) 6.70,(e) 8.94,(f) 13.40,(g) 31.28; [34] (a) 0,(b) 2.23,(c) 4.47,(d) 6.70. K> ON Esr spectra of [30] (left) and [29] (right) in the presence of Ni(II); Ni(II) concentrations were (mM): [30] (a) 0,(b) 1.12, (c) 2.23,(d) 3.35 , ( e ) 5.59,(f) 16.76,(g) 25.69; [29] (a)0,(b) 2.23,(c) 3.35,(d) 4.47. 12 7 A F i g . 111-12. P l o t of increase of c e n t r e - f i e l d l i n e w i d t h of (a) [31], (b) [35], (c) [33] as a f u n c t i o n of added n i c k e l s u l f a t e . 128 C l e a r l y , each of the former two systems seemed to c o n t a i n n i t r o x - ides i n s e v e r a l d i f f e r e n t s i t e s which were a c c e s s i b l e to d i f f e r e n t extents to metal i o n s . For the case of [31], other evidence f o r t h i s phenomenon was already c i t e d . In c o n c l u s i o n then, i t can be s a i d that there i s s u f f i c i e n t e v i - dence to i n d i c a t e the presence of s e v e r a l d i f f e r e n t s i t e s on c h i t o s a n and some of i t s d e r i v a t i v e s i n which the randomly d i s t r i b u t e d l a b e l s r e s i d e . A p h y s i c a l d e s c r i p t i o n which would accommodate such evidence can be sought i n the existence of e i t h e r pores or pockets w i t h i n the i n d i v i d u a l p o l y s a c c h a r i d e chains or j u n c t i o n zones ( i n ge l s ) between the chains c o n t a i n i n g n i t r o x i d e l a b e l s which are more or l e s s a c c e s s i b l e to so l v e n t s or s o l u t e s . E l e c t r o n microphotographs of xerogels derived from a s e r i e s of c h i t o s a n d e r i v a t i v e s bearing sugar-side chains e x h i b i t e d (see III-D) polyphasic microporous u l t r a s t r u c t u r e s , i n l i g h t of which the pro- posed " m u l t i - s i t e " model f o r the s p i n - l a b e l l e d d e r i v a t i v e s would seem to be even more probable. U n f o r t u n a t e l y , there was no opportunity to o b t a i n SEM microphotographs of these m a t e r i a l s . I I I - E . Scanning E l e c t r o n Microscopy 1. I n t r o d u c t i o n Scanning e l e c t r o n microscopy (SEM) i s a new and powerful technique which has a l r e a d y found widespread a p p l i c a t i o n s i n such areas as m a t e r i a l s c i e n c e , b i o l o g y , and medicine. More r e c e n t l y , i t has f a c i l i - t a t e d c o r r e l a t i o n s between s t r u c t u r a l o r g a n i z a t i o n and f u n c t i o n a l s i g n i f - i cance of s y n t h e t i c m a t e r i a l s such as polymers. SEM s t u d i e s have e s t a b l i s h e d c r u c i a l l i n k s between macromolecular polymer p r o p e r t i e s , i n c l u d i n g mechanical s t r e n g t h , solvent a d s o r p t i o n , s o l u t e s e p a r a t i o n , 42 e t c . , and u l t r a s t r u c t u r e of v a r i o u s m a t e r i a l s , e.g., polyacrylamides, 43 44 45 s i l i c a g e l , g e l a t i n , and p o l y s t y r e n e . Polysaccharides such as 46 , . 47 . 48 , , . , . , . c u r d l a n , a l g i n a t e , and c h i t i n have a l s o been examined i n t h e i r n a t i v e forms, w h i l e only r e l a t i v e l y few p o l y s a c c h a r i d e d e r i v a t i v e s , w i t h the notable exception of c e l l u l o s e products, have so f a r been 49 m o r p h o l o g i c a l l y c h a r a c t e r i z e d . Thus, M a s r i and Jones reported micro- graphs of commercial chitosans derived from crab s h e l l s which revealed l a m e l l a r phases w i t h i n t e r c o n n e c t i v e transverse f i b r i l s . Hirano and 48 coworkers have noted the s i m i l a r i t y between c e l l u l o s i c and N-acetylated c h i t o s a n g e l s both of which featured microporous polyphases;* a s i m i l a r morphology was found f o r x e r o g e l s * * of N-methylene c h i t o s a n . These s t u d i e s have, however, provided l i t t l e i n f o r m a t i o n about the g e l forma- t i o n of the c h i t o s a n products. The work presented i n t h i s s e c t i o n * * * p o r t r a y s some i n i t i a l attempts to c h a r a c t e r i z e the a r c h i t e c t u r e of s e v e r a l c h i t o s a n d e r i v a - t i v e s described i n the previous s e c t i o n s and to e s t a b l i s h c o r r e l a t i o n s w i t h t h e i r g e l l i n g p r o p e r t i e s . I t can be a n t i c i p a t e d that the SEM r e s u l t s w i l l a l s o f a c i l i t a t e a b e t t e r understanding of other aspects of the molecular p r o p e r t i e s of these m a t e r i a l s , such as m e t a l - c h e l a t i n g e f f i c a c i e s . The f o l l o w i n g d i s c u s s i o n w i l l focus on some nove l f e a t u r e s of c h i t o s a n i t s e l f as w e l l as on four branched-chain, gel-forming d e r i v a t i v e s thereof (see I I I - B ) . * I . e . , c o n s i s t e n t of s e v e r a l s t r u c t u r a l types, i n t h i s case, a mixture of membraneous and microporous reg i o n s . * * I . e . , d r i e d g e l s which r e t a i n most of t h e i r three dimensional morphology. ***SEM s t u d i e s were performed by Mr. Nasser Y a l p a n i . 130 2. SEM of Chitosan 49 M a s r i and Jones have, as already mentioned, c h a r a c t e r i z e d com- m e r c i a l , crab s h e l l - d e r i v e d c h i t o s a n . We examined c h i t o s a n d e r i v e d from shrimp s h e l l and found many s i m i l a r i t i e s between these two m a t e r i a l s as w e l l as some novel f e a t u r e s , t y p i c a l aspects of which are shown i n the photomicrographs ( F i g . 111-13 a, b ) . Chitosan f l a k e s ( F i g . III-13a) e x h i b i t e d f l a t l a m e l l a r phases w i t h some la m e l l a e extending at an angle of between ca. 45-90° w i t h respect to the outer s u r f a c e of the f l a k e s . A l a r g e number of p r o t r u d i n g m i c r o f i b r i l s , ca. 20-40 nm i n diameter and up to 0.2 um l o n g , are evident on these l a m e l l a e ; these m i c r o f i b r i l s are o r i e n t e d e i t h e r p a r a l l e l or perpendicular to the l a m e l l a r plane. 49 M a s r i and Jones concluded from s i m i l a r f i n d i n g s that the former assemble to form i n d i v i d u a l l a m e l l a e w h i l e the l a t t e r act as support and l i n k between the l a m e l l a r l a y e r s . Another i n t e r e s t i n g view of c h i t o s a n i s i l l u s t r a t e d i n Figure III-13b where r e l a t i v e l y l a r g e (ca. 10 um h e i g h t , 10-50 ym w i d t h , 5-10 um depth) dome-shaped o r i f i c e s are seen to be incorporated i n t o the membraneous s u r f a c e . M i c r o f i b r i l s , 20 nm i n diameter and up to 10 ym i n l e n g t h , are again evident as are some c r y s t a l l i t e s presumably from m i n e r a l sources. 3. SEM of Branched-Chain Chitosan D e r i v a t i v e s The xerogels of the branched-chain c h i t o s a n d e r i v a t i v e s synthe- s i z e d i n t h i s work d i s p l a y e d a vast range of u l t r a s t r u c t u r e s . Galac- t i t y l c h i t o s a n [2] xerogels have non-porous, smooth membraneous phases ( F i g . 111-13 e,f) of 2-4 ym t h i c k n e s s . The enlargement i n F i g u r e I I I - 1 3 f d i s c l o s e s a h i g h l y - o r d e r e d m i c r o f i b r i l l a r s u b s t r u c t u r e . The non-porous surface s t r u c t u r e of [2] i s r a t h e r unique among the c h i t o s a n d e r i v a t i v e s s t u d i e d and c o r r e l a t e s w e l l w i t h the observation that no m e t a l - c h e l a t i o n could be accomplished w i t h t h i s d e r i v a t i v e (see V-B 2 ) . F i g . 111-13. SEM of [1] ( a ) , (b); [3] ( c ) , ( d ) ; [2] ( e ) , ( f ) ; bars under the micrographs i n d i c a t e 10 um (a-d,f) and 100 um ( e ) .  J31.A [10] F i g . I l l - 1 4 . SEM of [7] ( a ) - ( c ) ; [10] (d); [6] (e), ( f ) ; bars under the micrographs indi c a t e 10 ym.  133 In c o n t r a s t to the above, the g l u e i t y 1 c h i t o s a n [3] e x h i b i t s a p o l y - p h a s i c , microporous u l t r a s t r u c t u r e ( F i g . 111-13 c,d). The pore dimensions are non-uniform ranging from 20-35 x 40-70 ym ( F i g . I I I - 1 3 d ) , w i t h very t h i n membrane w a l l s . The u l t r a s t r u c t u r e of [3] has c e r t a i n s i m i l a r i t i e s 4 to that of the N-methylene c h i t o s a n reported by Hirano et a l . , the pore dimensions of the l a t t e r being somewhat l a r g e r (see Table I I I - 4 ) . Photomicrographs of xerogels derived from the r e a c t i o n products [7] of c h i t o s a n w i t h three molar e q u i v a l e n t s of l a c t o s e per glucosamine r e s i - due r e v e a l a r a t h e r unusual polyphasic topography. F i g u r e I I I - 1 4 a shows 134 contiguous membraneous s u r f a c e s , s t r u c t u r e d i n i r r e g u l a r honeycomb- f a s h i o n , which are covered w i t h c r y s t a l l i t e s . The honeycomb regions c o n s i s t mainly of hexagonal, sometimes t r i a n g u l a r , formations w i t h t y p i c a l s i z e s of ca. 2-5 x 2-7 ym. The c r y s t a l l i t e s ( F i g s . 111-14 a,c) vary s u b s t a n t i a l l y i n s i z e (ca. 0.4-7 x 13 ym) and shape and appear to c o n s i s t of a m i c r o f i b r i l l a r s u b s t r u c t u r e . When the above l a c t i t y l c h i t o s a n gels were p u r i f i e d by extensive d i a l y s i s i n s t e a d of by washings onl y , the r e s u l t i n g product [6] d i s p l a y e d a d r a s t i c a l l y a l t e r e d micro- a r c h i t e c t u r e as shown i n Figures 111-14 e and f. A p o l y p h a s i c , micro- porous s t r u c t u r e i s obtained c o n t a i n i n g non-uniformly s i z e d pores (9-17 x 10-28 ym) w i t h t h i n w a l l s ; some i n d i v i d u a l m i c r o f i b r i l s , ca. 20-140 nm i n diameter, are a l s o evident ( F i g . I I I - 1 4 f ) . These f i n d i n g s confirm e a r l i e r conclusions (see I I I - B ) based on 1 3 C nmr spectroscopy that l a c t i t y l c h i t o s a n , i n the presence of excess l a c t o s e , produces gels which " t r a p " t h i s reagent ( i n a 1:1 r a t i o of the l a t t e r per l a c t i t y l glucosamine residue) to a f f o r d m a t e r i a l s w i t h " c l o s e d " ( i . e . , non-porous) u l t r a s t r u c t u r e ; upon removal of the "trapped" reagent by d i a l y s i s "open" (microporous) m a t e r i a l s are obtained. These p a r t i c u l a r observations have so f a r only been made f o r the r e a c t i o n of c h i t o s a n w i t h l a c t o s e . Xerogels d e r i v e d from m e l i b i i t y l c h i t o s a n [10] d i s p l a y e d a h i g h l y ordered m i c r o f i b r i l l a r u l t r a s t r u c t u r e . The edge view shown i n Figure III-14d r e v e a l s a p a r a l l e l array of m i c r o f i b r i l s which have diameters of ca. 150 nm. I t w i l l be r e c a l l e d from the previous s e c t i o n t h a t , i n e x p l a n a t i o n of the x c value observed f o r the C6'-spin l a b e l l e d d e r i v a - t i v e [35], an extended conformation of the 1-6 l i n k e d s i d e chains had been invoked. Assuming that the s o l u t i o n s t r u c t u r e s of [10] and [35] 135 are not very d i s s i m i l a r , one i s tempted to c o r r e l a t e the esr r e s u l t s w i t h the g e l s t r u c t u r e described here. The dimensions of the v a r i o u s xerogels are assembled i n Table I I I - 4 together w i t h the data obtained f o r some other m a t e r i a l s . The microporous, branched-chain c h i t o s a n d e r i v a t i v e s reported here have pore s i z e s intermediate between N-acetyl c h i t o s a n and N-methylene c h i t o s a n and the other s y n t h e t i c polymers l i s t e d i n Table I I I - 4 . The SEM r e s u l t s obtained so f a r do not i n d i c a t e c o n s i s t e n t trends i n the r e l a t i o n of the m i c r o - a r c h i t e c t u r e and the chemical s t r u c t u r e of the s i d e - c h a i n s of the c h i t o s a n d e r i v a t i v e s . The a-1,6 l i n k a g e s of the side - c h a i n s of [10] are presumably p a r t i a l l y r e s p o n s i b l e f o r the observed h i g h l y r e g u l a r m i c r o f i b r i l l a r u l t r a s t r u c t u r e , which, i n comparison w i t h the 3-1,4 l i n k a g e s of [ 6 ] , would a l l o w f o r a more extended and ordered assembly of the s i d e - c h a i n s . I t i s , however, unclear why the sm a l l conformational d i f f e r e n c e between the sid e - c h a i n s of [2] and [3] should r e s u l t i n such d r a s t i c changes i n u l t r a s t r u c t u r e as evidenced here. A more l i k e l y , l e s s s i m p l i s t i c e x p l a n a t i o n f o r the v a r i e t y i n the micro- a r c h i t e c t u r e of the c h i t o s a n d e r i v a t i v e s must consequently be sought i n f a c t o r s other than the d i f f e r e n c e s i n chemical s t r u c t u r e of the s i d e - chains alone. A combination of f a c t o r s , such as those l e a d i n g to g e l formation, i . e . , H-bonding and a v a r i e t y of a s s o c i a t i v e phenomena of polysaccharide chains i n s o l u t i o n , and the extent and u n i f o r m i t y of s u b s t i t u t i o n , e t c . , (see I I I - B ) presumably c o n t r i b u t e s to the observed topographies of the xe r o g e l s . 136 Table I I I - 4 . U l t r a s t r u c t u r e C h a r a c t e r i s t i c s Pore S t r u c t u r e W a l l M a t e r i a l Main Feature Dimensions(um) Thickness(ym) Reference [3] p o l y p h a s i c , microporous 20-35 x 40-70 very t h i n [2] p o l y p h a s i c , non-porous — — ™~ [6] polyphasic microporous 9-17 x 10-28 0.2-1.9 -~ [7] p o l y p h a s i c , non-porous — — "~ [10] p o l y p h a s i c , m i c r o f i b r i l l a r — . — — N-methylene- c h i t o s a n p o l y p h a s i c , microporous 40-70 x 50-100 very t h i n 4 N - a c e t y l - c h i t o s a n polyphasic microporous 30-50 x 80-300 very t h i n 48 polyacrylamide microporous 2 x 10-15 0.3-1.0 up to 200 i n sec- t i o n s 42 p o l y v i n y l a l c o h o l p o l y p h a s i c , microporous 0. 1-0.3 very t h i c k 42 polyethylene oxide p o l y p h a s i c , microporous 3-20 1.5-2.0 42 137 References 1. See R. A. A. M u z z a r e l l i , C h i t i n , Pergamon Press, New York, (1977). 2. S. Hirano, Y. Ohe, and H. Ono, Carbohydr. Res., 47, 315 (1976). 3. S. Hirano, N. Matsuda, 0. Miura, and il. Iwaki, i b i d . , 71, 339 (1979). 4. S. Hirano, N. Matsuda, 0. Miura, and T. Tanaka, i b i d . , 7_1, 344 (1979). 5. See, e.g., W. W. Graessley, Acc. Chem. Res., 10, 332 (1977); H. Grisebach and R. Schmid, Angew. Chem. I n t e r n . Ed., '11, 159 (1972). 6. I. J . G o l d s t e i n , R. D. P o r e t z , L. L. So, and Y. Yang, Arch. Biochem. Biophys., 127, 787 (1968). 7. S. F. Grappel, E x p e r i e n t i a , _27, 329 (1971). 8. E. T. Reese and F. W. P a r r i s h , Biopolymers, 4_, 1043 (1966). 9. J . C i s a r , E. A. Rabat, M. M. Dorner, and J . L i a o , J . Exp. Med., 142, 435 (1975). 10. B. Pfannemuller, G. C. R i c h t e r , and E. Husemann, Carbohydr. Res., 56, 139 (1977). 11. For reviews, see A. F. Bochkov and G. E. Zaikov, Chemistry of the 0- G l y c o s i d i c Bond, Pergamon P r e s s , New York, (1979); W. A. Szarek, i n MTP I n t e r n . Rev. S c i . , Org. Chem. Ser. One, ]_, 80 (1973). 12. H. I t o and C. Schuerch, J . Amer. Chem. S o c , 101, 5797 (1979). 13. B. Pfannemuller, G. R i c h t e r , and E. Husemann, Carbohydr. Res., 47, 63 (1976). 14. H. Andresz, G. C. R i c h t e r , and B. Pfannemuller, Makromol. Chem., 179, 301 (1978). 15. W. B. Neely, U.S. Pat., 3,133,856 (1964); Chem. A b s t r . , 61, 5899h (1964). 16. L. J . F i l a r and M. G. W i r i c k , Proc. F i r s t I n t e r n . Confer. C h i t i n / Chitosan, R. A. A. M u z z a r e l l i and E. R. P a r i s e r , eds., 169 (1978). 17. E. R. Hayes and D. H. Davies, i b i d . , 193 (1978). 18. F. A. Bettelheim, i n B i o l o g i c a l P o l y e l e c t r o l y t e s , A. V e i s , ed., Marcel Dekker, New York, 131 (1970). 138 19. Y. Kikuchi and H. Fukudu, Makromol. Chem., 175, 3593 (1974). 20. G. T. Grant, E. R. Morris, D. A. Rees, P. J. C. Smith, and D. Thorn, FEBS Lett., 32, 195 (1973). 21. G. Robinson, E. R. Morris, and D. A. Rees, J.C.S. Chem. Comm., 152 (1980). 22. R. J. Dobbins, in Industrial Gums, R." L. Whistler, ed., Academic Press, New York, 19 (1973). 23. R. E. Sand and M. Glicksman, i b i d . , 147 (1973). 24. H. J. Humm and L. G. Williams, Amer. J. Bot., 35, 287 (1948). 25. E. Pippen.T. H. Schultz, and H. Owens, J. Colloid Sci., 8, 97 (1953); H. Harvey, Chem. Ind., ]_, 29 (1960). 26. R. G. Schweiger, Carbohydr. Res., 70, 185 (1979). 27. N. Yamaoka, T. Usui, H. Sugiyama, and S. Seto, Chem. Pharm. Bull., 22, 2196 (1974). 28. S. J. Perkins, L. N. Johnson, D. C. Ph i l l i p s , and R. A. Dwek, Carbohdr. Res., 59, 19 (1977). 29. L. D. Hall, G. A. Morris, and S. Sukumar, J. Amer. Chem. Soc, 102, 1745 (1980); P. E. Pfeffer, K. M. Valentine, and F. W. Parrish, i b i d . , 101, 1265 (1979). 30. L. D. Hall and M. Yalpani, Carbohydr. Res., 78^, C4 (1980); L. D. Hall and M. Yalpani, in preparation. 31. P. D. Hoagland, P. E. Pfeffer, and K. M. Valentine, Carbohydr. Res., 74, 135 (1979). 32. L. F. Johnson and W. C. Jankowski, Carbon-13 NMR Spectra, Wiley, New York (1972). 33. L. A. Buffington and E. S. Stevens, J. Amer. Chem. Soc. , 101, 5159 (1979). 34. C. L. McCormick and D. K. Lichatowich, J. Polym. Sci., Polym. Lett. Ed., 17, 479 (1979). 35. M. S. Masri, V. G. Randall, and W. L. Stanley, i n Proc. Fir s t Intern. Conf. Chitin/Chitosan, R. A. A. Muzzurelli and E. R. Pariser, eds., 364 (1978). 36. M. F. Froix and R. Nelson, Macromolecules, 8, 726 (1975). 139 37. L. D. Hall and J. D. Aplin, J. Amer. Chem. Soc, 100, 1934 (1978); L. D. Hall and J. D. Aplin, i b i d . , in press. 38. J. D. Aplin, Ph.D. thesis, University of British Columbia, (1979). 39. E. Odgaard, T. B. Melo, and T. Henriksen, J. Magn. Res., 18, 436 (1975); G. I. Likhtenstein, Adv. Moi. Relax. Int. P r o c , 10, 47 (1977). 40. G. I. Likhtenstein, Spin Labels in Molecular Biology, Nauka, Moscow, (1974). 41. J. S. Hyde, H. M. Swartz, and W. E. Antholine, in Spin Labeling II, L. J. Berliner, ed., 71 (1979). 42. Z. Blank and A. C. Reimschussel, J. Mater. Sci., 9_, 1815 (1974). 43. J. C. Dennis, J. Colloid and Interf. Sci., 28, 32 (1968). 44. E. S. Halberstadt, H. K. Henisch, J. Nickl, and E. W. White, ibid., 29, 469 (1969). 45. P. Bajaj and S. K. Varshney, Polymer, _21, 201 (1980). 46. A. Koreeda, T. Harada, K. Ogawa, S. Sato, and N. Kasai, Carbohydr. Res., 33, 396 (1974). 47. E. S. Obolonkova, E. M. Belavtseva, E. E. Braudo, and V. B. Tostoguzo, Colloid. Polymer Sci., 2_52, 526 (1974). 48. S. Hirano, R. Yamaguchi, and N. Matsuda, Biopolymers, 16, 1987 (1977). 49. M. S. Masri and F. T. Jones, in Proc. First Intern. Conf. Chitin/ Chitosan, R. A. A. Muzzarelli and P. E. R. Pariser, eds., 483 (1978). CHAPTER IV COMBINED ENZYMIC AND CHEMICAL MODIFICATIONS IV-A. I n t r o d u c t i o n The work described i n the previous chapters i n v o l v e d p o l y s a c c h a r i d m o d i f i c a t i o n s by chemical means and the reader w i l l be now be f a m i l i a r w i t h some of the problems encountered i n accomplishing the task of s p e c i f i c f u n c t i o n a l transformations. Enzymes provide i n many cases a f a c i l e a l t e r n a t i v e f o r p o l y s a c c h a r i d e m o d i f i c a t i o n s . 1 2 From the numerous enzyme systems a v a i l a b l e , galactose oxidase (D-galactose : oxygen 6-oxidoreductase, EC 1.1.3.9) seemed p a r t i c u l a r l y a t t r a c t i v e f o r i t s s p e c i f i c i t y and o v e r a l l s i m p l i c i t y of r e a c t i o n (eqn. This enzyme e x i s t s as a s i n g l e polypeptide chain (MW 68,000) w i t h a s i n g l e metal i o n (copper) as i t s s o l e c o f a c t o r and i s produced by a 3 4 fungus. The enzyme has been employed f o r a range of mono, o l i g o - and polymeric** galactose s u b s t r a t e s ; i n a c t i v i t y i s apparently only observed f o r C4 s u b s t i t u t i o n of galactose or g a l a c t o s i d e s and f o r C3 140 141 2 5 7 s u b s t i t u t i o n i n the case of 2-deoxy-2-aminogalactose d e r i v a t i v e s . ' ' The enzyme d i s p l a y s a much greater a f f i n i t y f o r polymeric than f o r 2 monomeric substrates but i t s metabolic f u n c t i o n s remain obscure. A f u r t h e r u s e f u l aspect of t h i s enzyme c o n s i s t s i n i t s s t e r e o s p e c i f i c o x i d a - t i o n of the C6 primary a l c o h o l group i n the course of which the (pro-S)- hydrogen i s a b s t r a c t e d . These features of galactose oxidase have been s u c c e s s f u l l y e x p l o i t e d f o r a p p l i c a t i o n s to a number of p o l y s a c c h a r i d e s , ^ ^ •, A- 2,5,14 , 9 . t „ , . _ . 10,11 i n c l u d i n g guaran and agarose, and to c e l l surface g l y c o p r o t e i n s ; 11 . • 10,12 v a r i o u s s p e c t r o s c o p i c and other probes, such as deuterium, t r i t i u m , 13 and n i t r o x i d e s p i n - l a b e l s , have been incorp o r a t e d i n t o b i o l o g i c a l m a t e r i a l s of d i f f e r e n t complexity u s i n g t h i s procedure. The s u b s t a n t i a l u t i l i t y of t h i s enzymic m o d i f i c a t i o n can be f u l l y appreciated i f one con- s i d e r s equivalent chemical methods f o r the i n t r o d u c t i o n of C6 aldehyde f u n c t i o n s i n t o polysaccharides.'*'"' The work presented i n t h i s hapter was aimed at demonstrating the p o t e n t i a l of enzymic treatment of D-galactose c o n t a i n i n g p o l y s a c c h a r i d e s , e x e m p l i f i e d here by guaran and l o c u s t bean gum, two r e p r e s e n t a t i v e and important galactomannans f o r a f f e c t i n g s p e c i f i c and h i g h - y i e l d i n g t r a n s - formations . IV-B. Galactomannans 1. M o d i f i c a t i o n w i t h Galactose Oxidase ( i ) Guar gum 2 F o l l o w i n g the p r e s c i e n t suggestion of Avigad et a l . and of S c h l e g e l et a l . , " * galactose oxidase was used to introduce an aldehyde group at C6 of the pendant galactose u n i t s of guar gum [ 1 ] , r e d u c t i v e amination of which, using any primary or secondary amine and sodium 142 cyanoborohydride a f f o r d s a polymer bearing a s u b s t i t u e n t of choice at C6.- The general sequence of r e a c t i o n s i s summarized i n scheme 1, and the s p i n - l a b e l l i n g of [1] t y p i f i e s the c o n d i t i o n s used; p u r e ^ guar gum (60 mg, 0.12 mM e q u i v a l e n t s galactose) i n phosphate b u f f e r (pH 7, 25 mM, 15 ml) reacted w i t h galactose oxidase (90 u n i t s ) i n the presence of c a t a - l a s e (E.C. 1.11.1.6, 10500 u n i t s ) f o r 24 hours, a f f o r d i n g a very v i s c o u s , ropy m a t e r i a l . Omission of c a t a l a s e l e d to an approximately f o u r - f o l d r e d u c t i o n i n the y i e l d s of [3]*.^"' Reductive amination could be per- formed i n s i t u by the a d d i t i o n of aqueous s o l u t i o n s of 4-amino- 2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n e - l - o x y l ( [ 4 ] , 92 mg, 0.54 mM) and sodium cyanoborohydride (300 mg, 4.4 mM) over 36 hours; a l t e r n a t i v e l y the a l d e - hyde d e r i v a t i v e [2] could be i s o l a t e d by ethanol p r e c i p i t a t i o n , followed SCHEME 1 *by consuming i n h i b i t i n g H 2 0 2 i n the r e a c t i o n mixture. 143 by c e n t r i f u g a t i o n . P u r i f i c a t i o n of the product [3] could be achieved e i t h e r by d i a l y s i s (4 days) or by ethanol p r e c i p i t a t i o n * followed by c a r e f u l washing. Although no attempts were made to a s c e r t a i n complete removal of the s m a l l amounts of enzyme, no i n t e r f e r e n c e from t h i s source could be detected by nmr, or even p r i o r to p u r i f i c a t i o n , by e s r ; were t h i s to be a problem i t could be i d e a l l y r e s o l v e d by an immobilized 18 enzyme system. Although only some p r e l i m i n a r y attempts** have so f a r been made to f u l l y optimize the r e a c t i o n c o n d i t i o n s , the y i e l d s (based on galactose content) obtained are r a t h e r encouraging, t y p i c a l l y v a r y i n g between 60-70% as determined by elemental m i c r o a n a l y s i s , 1 3 C nmr, or by esr double i n t e g r a t i o n i n the case of [3 ] . ( i i ) Locust bean gum Fo l l o w i n g the above procedures l o c u s t bean gum [5] was o x i d i z e d to the corresponding C6 aldehyde [ 6 ] . The o x i d a t i o n i n t h i s case pro- ceeded w i t h a greater e f f i c i e n c y as judged by elemental a n a l y s i s and esr double i n t e g r a t i o n of the s p i n - l a b e l l e d d e r i v a t i v e [ 7 ] , t y p i c a l y i e l d s ranging from 70-90%. I t i s u n c e r t a i n at t h i s p o i n t , whether the higher y i e l d s obtained f o r l o c u s t bean gum have any s i g n i f i c a n c e i n terms of the s t r u c t u r a l d i f f e r e n c e s of the two gums a f f e c t i n g enzyme a c t i v i t y . How- ever, the high degrees of o x i d a t i o n obtained i n both cases are i n l i n e w i t h p r e v i o u s , l e s s accurate f i n d i n g s of Schlegel et a l . " ' who, using c o l o r i m e t r i c and other a n a l y t i c a l procedures, reported degrees of 2 o x i d a t i o n f o r guaran ranging from 3% to 76% and 95%. Avigad et a l . *Resultedu3ually i n i n s o l u b i z a t i o n of the product. **See Experimental s e c t i o n . 144 found for guaran an enzyme substrate a f f i n i t y constant (Km) value of -4 3.1 * 10 M which is three orders of magnitude smaller than that for galactose (Km = 2.4 x 10 Similarly, the relative rate of guaran 2 oxidation was 50% higher than for galactose i t s e l f , both factors clearly indicating the greater enzyme af f i n i t y for the high molecular weight sub- strate. No corresponding data were available for locust bean gum. The application of galactose oxidase to some synthetic chitosan derivatives was already discussed i n the previous chapter (II-D). ( i i i ) Esr The esr spectra of the spin-labelled guaran [3], and locust bean gum [7] i n aqueous solution were very similar, revealing relatively mobile nitroxide moieties with T values of 7.70 x 10 sec and c 7.61 x i o sec, respectively (Fig. IV-la). It is noteworthy that the corresponding spin-labelled gums prepared via periodate oxidation of the native polymers displayed similar spectral lineshapes as [3] and [7] (see Fig. IV-10). Esr spectroscopy of [3] and [7] at 77K provided novel information concerning the distribution of the nitroxide groups and, by inference, that of the pendant galactose units of the gums themselves whose struc- tures are illustrated in Figure IV-2. Thus, from calculation of the mean nearest-neighbour distance between spins, r, derived from the spectral parameter d^/d at 77K( see I-C), a value of 1.36 nm (±5%) was obtained for [3]. This value is most consistent with a structure in which the pendant galactose units are arranged in "blocks" as recently 19-21 proposed by Painter, Gonzalez and coworkers (Fig. IV-2); this conclusion i s based on the following arguments. From molecular model building studies, a rough distance estimate  146 GGGG GGG GGGG •M-M-M-M-M-MM-M-M-M-M MM-M-M-M-M^-M-M-M-MM-M-M-MM-M-M-M O G G G G G G G . M M M M M M-M*-M-M-M-M-M-M-M^-M-MH*-M-M^-M^-M-M4<4it-M-M b I G G G G G G G GGG 9 9 9 9 9 9 9 G GGGGOG G G G G G GGG G M-M-M-M-M-M-M-M-M-M-MM^^-M-M-M-M-M-M-M-M-M-M-M-M-pVl-M-M-M 9 e '• r+M-M-M-MM-MMM^-M-M-MM-M-M-M-M-M-M-MM-MM-MM-M-MM c a G GGGG G G •M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-MM-M-M-M-M-M-M-M-M-M-M-M-M- GG GGG GGG 0 GG 99 GG m.M .. J. Ja n fc* ».'* rf- tl|||'« I ' I I'j UH|I l'l ii il t'l ill tl U il il M I I lUt fci PfffŴ WWwWWWl'rW ^ n n n n rH W Fl Ml R W MlMMl Ml Ml Ml 06 GGG GGGG GGGG 00 00 Fig. IV-2. Proposed structures for guaran (a) and locust bean gum (b) (from ref. 20). of 1.1-1.7 nm was obtained for the separation of the nitroxide moieties of [3] within blocks of between two to four contiguous, branched mannose h - I I - H K NN " " 71' f If *| *M I I I N N N N I I I I 1 I I I I I G G G G G E) Co] W M (MG) residues [A]. The nearest-neighbour distance of nitroxides of adjacent MG blocks which are separated by one unbranched mannose (M) unit [B], was found to be -1.8-2.3 nm by the same method, whereas a value of ~1.5-2.0nmwas obtained for a regular M-MG sequence, i . e . , for a uniform galactose d i s t r i b u t i o n [C]. 147 I t should be noted here t h a t , f o r t u i t o u s l y , the maximal d i s t a n c e s of the two l a t t e r estimates approach the upper d e t e c t i o n l i m i t (-2.2 nm) f o r which the d^/d parameter provides r e l i a b l e d i s t a n c e i n f o r m a t i o n (see I-C). Consequently, c o n t r i b u t i o n s of d i p o l a r i n t e r a c t i o n s from di s t a n c e s greater than the above l i m i t can be e f f e c t i v e l y ignored i n l i g h t of the r ^ dependency of d i p o l a r c o u p l i n g s ; . d i p o l a r c o n t r i b u - t i o n s a r i s i n g from i n t e r a c t i o n s c l o s e to the d e t e c t i o n l i m i t should be s m a l l i n comparison w i t h short-range i n t e r a c t i o n s . Thus, i n view of the above, a r e g u l a r guaran s t r u c t u r e appears u n l i k e l y . However, l a c k - i n g knowledge of the exact conformations of e i t h e r of the polymer d e r i v a - t i v e s [3] and [7] i n s o l u t i o n or i n the s o l i d s t a t e , the above d i s t a n c e estimates were derived by assuming a range o f , what was deemed, p r e f e r r e d conformations of a s i n g l e polymer chain w i t h a twofold conformation of the mannose backbone; n i t r o x i d e i n t e r a c t i o n s due to c h a i n - f o l d i n g or other i n t e r - and i n t r a - m o l e c u l a r sources were a l s o not taken i n t o account. I t i s t h e r e f o r e obvious that no f i r m conclusions could be reached, based s o l e l y on these estimates. Further supporting evidence was, however, obtained from a d d i t i o n a l experiments. In the f i r s t set of experiments the extent of s p i n - l a b e l l i n g of guar gum was continuously reduced f o r a comparison of the esr s p e c t r a of the r e s u l t i n g products. Figure IV-3 i l l u s t r a t e s the f a c t that the pronounced l i n e broadening, observed f o r d e r i v a t i v e s w i t h h i g h degrees of s u b s t i t u t i o n (d.s. 0.71, F i g . IV-3a), i s l a r g e l y r e t a i n e d f o r samples bearing one h a l f (d.s. 0.34, F i g , IV-3b) or only one quarter (d.s. 0.14, F i g . IV-3c) the number of n i t r o x i d e r e s i d u e s . From these r e s u l t s , assuming random l a b e l l i n g of the p o l y s a c c h a r i d e , a non-regular n i t r o x i d e d i s t r i b u t i o n can be i n f e r r e d , s i n c e the n i t r o x i d e s of low d.s. d e r i v a t i v e : 148 F i g . IV-3. Esr s p e c t r a of [3] (a) d.s. 0.71, (b) d.s. 0.34, (c) d.s. 0.14; at 77K. of [3] would be too f a r apart i n a s t r i c t l y r e g u l a r s t r u c t u r e f o r the above i n t e r a c t i o n s to occur, even i f i n t e r m o l e c u l a r i n t e r a c t i o n s between chains were o p e r a t i v e . These r e s u l t s alone do not, however, r u l e out 22 another proposed s t r u c t u r e which i s based on a l t e r n a t i n g long b l o c k s of contiguous MG and M u n i t s ; but f u r t h e r r e s u l t s obtained from l o c u s t 21 bean gum and those most r e c e n t l y reported by Grasdalen and P a i n t e r , exclude t h i s p o s s i b i l i t y as w e l l . Thus, s i g n i f i c a n t l y , f o r [7] a value of r = 1.75 nm (±5%) was found i n s i m i l a r f a s h i o n ( F i g . I V - l b ) ; t h i s value i s ~30% greater than that of [3 ] , c l e a r l y r e f l e c t i n g the proposed s t r u c t u r a l d i f f e r e n c e s between guaran and l o c u s t bean gum as expressed i n Figure IV-2. According to 20 P a i n t e r et a l . l o c u s t bean gum has, i n comparison to guaran, only one f i f t h and one quarter the number of doublet and t r i p l e t frequencies f o r two and three consecutive MG u n i t s , r e s p e c t i v e l y , w h i l e i t s t r i p l e t frequency f o r a l t e r n a t i n g M and MG u n i t s , i . e . , M-MG-M or MG-M-MG, i s greater than that of guaran by a f a c t o r of 4.5. In other words, the observed g r e a t e r r value of [7] a r i s e s from a r e l a t i v e l y s m a l l e r c o n t r i - b u t i o n from consecutive MG blocks and a l a r g e r c o n t r i b u t i o n from a l t e r - n a t i n g sequences. The r value of [ 7 ] , which i s a l s o i n agreement w i t h the molecular model c a l c u l a t i o n s discussed e a r l i e r , r u l e s out the 150 p o s s i b i l i t y of s t r u c t u r e s w i t h r e g u l a r g a l a c t o s y l d i s t r i b u t i o n [D] s i n c e the d i s t a n c e s i n v o l v e d would f a l l o u t s i d e the d e t e c t i o n range of the esr method, as discussed e a r l i e r . Thus, i n c o n c l u s i o n , i t can be s a i d that the esr data are i n good q u a l i t a t i v e agreement w i t h the galactomannan s t r u c t u r e s of P a i n t e r and 19-20 coworkers, although, s t r i c t l y speaking, separations between n i t r o x i d e s r a t h e r than galactose u n i t s were measured here w i t h the assumption that the conformations of [3] and [7] are not very d i s s i m i l a r to those of t h e i r r e s p e c t i v e n a t i v e polymers. ( i v ) A p p l i c a t i o n s The extreme v e r s a t i l i t y of the aldehyde intermediates [2] and [6] could be demonstrated by performing the r e a c t i o n s , summarized i n Figure IV-4, which range from r e d u c t i v e amination to o x i d a t i o n and r e d u c t i o n . The r e d u c t i v e amination r e a c t i o n , e x e m p l i f i e d here by a v a r i e t y of products, i n c l u d i n g [ 3 ] , [ 7 ] - [ l l ] , has obv i o u s l y many i n t e r e s t i n g v a r i - a nts, such as the p o s s i b i l i t y to introduce amine f u n c t i o n s i n t o the p o l y - saccharide by use of ammonium acetate a f f o r d i n g [12], a c a t i o n i c key intermediate. Conversely, a n i o n i c d e r i v a t i v e s can be prepared by o x i d a - t i o n of the aldehyde f u n c t i o n using bromine water at pH 6, to y i e l d the c a r b o x y l i c a c i d [14]. Reduction of the aldehyde intermediate w i t h sodium borohydride or i t s deuterated analogue a f f o r d s the o r i g i n a l polymer which i n the l a t t e r case has incorporated a probe w i t h minimal s t r u c t u r a l p e r t u r b a t i o n s , [15],[16]. Of the products obtained by r e d u c t i v e amination the BSA-derivative [11] c o n s t i t u t e s a novel type of p o l y s a c c h a r i d e - p r o t e i n conjugate which t y p i f i e s a wide range of p o t e n t i a l l y u s e f u l d e r i v a t i v e s , i n c l u d i n g 151 Fig. I V - A . Derivatives obtained from aldehyde intermediates [ 2 ] and [ 6 ] . 152 immobilized enzymes. Similarly, reaction of [2] with glycine to yield [9], another anionic species, illustrates the preparation of a new class of glycopeptides. The hydroxypropylamine guar derivative [8] (d.s. -0.8) is of interest for i t s similarity to hydroxyethyl and hydroxypropyl derivatives of guar, prepared from the respective alkylene oxides, which 23 have applications as efficient fracturing fluids for o i l well stimula- 24 tion. In contrast to the latter, [8] i s specifically substituted at C6 of the galactose units which seems to result in substantial d i f f e r - ences in the rheological properties (see v i ) . The imidazole guaran [10] 25 could serve as a model for medicinal derivatives. Lastly, the u t i l i t y of the intermediates derived from [2] and [6] is exemplified here by the reductive alkylation of the amino guaran deriva- tive [12] using lactose to afford the branch-extended guaran derivative [13] i n acceptable yield (d.s. 0.4). H ? CH2NHR1 (v) Nmr High resolution 1 3C nmr was employed in an effort to elucidate the structures-of the native galactomannans and their derivatives. Figure IV-5 shows the proton-decoupled, natural abundance 100.6 MHz 1 3C nmr Fig. IV-5. 100.6 MHz C-nmr spectra of (a) [1] and (b) [5] in D,0; ( sweep width 23,000 Hz, pulse width 14 ys, delay 0.1"s, 50,000 scans). 154 s p e c t r a o f g u a r a n a n d l o c u s t b e a n gum a t a p r o b e t e m p e r a t u r e o f 3 0 ° C . F r o m t h e k n o w n m a n n o s e t o g a l a c t o s e r a t i o s o f t h e t w o p o l y m e r s , a n i n i t i a l c o m p a r i s o n o f t h e s e s p e c t r a a l l o w s f o r f a c i l e i d e n t i f i c a t i o n o f s e v e r a l r e s o n a n c e s : f o r g u a r a n ( v a l u e s o f [ 5 ] a r e g i v e n i n p a r e n t h e s i s ) t h e a n o m e r i c s i g n a l s a t 1 0 0 . 1 ppm ( 1 0 0 . 6 ppm) a n d 9 9 . 4 ppm ( 9 9 . 6 ppm) c a n b e a s s i g n e d t o C l o f t h e 0 - D - m a n n o s e a n d a - D - g a l a c t o s e r e s i d u e s , r e s p e c - t i v e l y , a n d t h e m e t h y l e n e r e s o n a n c e s a t 6 1 . 1 ppm ( 6 1 . 1 ppm) a n d 6 0 . 6 ppm ( 6 0 . 7 ppm) a r e a t t r i b u t a b l e t o C6 o f t h e a - D - g a l a c t o s e a n d g - D - m a n n o s e r e s i d u e s . T h e s e v a l u e s a g r e e w i t h t h o s e r e p o r t e d f o r t h e a - D - g a l a c t o - 26 p y r a n o s y l u n i t o f r a f f i n o s e a n d t h e 1 , 4 - l i n k e d 3 - D - m a n n o p y r a n o s e r e s i - 27 d u e s o f a n u n b r a n c h e d m a n n a n ( w h i c h w i l l h e n c e f o r t h b e r e f e r r e d t o a s a - D - g a l a c t o s y l u n i t a n d 1 , 4 - m a n n a n , r e s p e c t i v e l y ) . T h e r e s o n a n c e s a t 6 3 . 3 ppm ( 6 9 . 5 p p m ) , 6 9 . 9 ppm ( 6 9 . 9 p p m ) , a n d 7 1 . 3 ppm ( 7 1 . 4 ppm) c a n b e a s s i g n e d t o C 2 , C 3 , a n d C 5 , r e s p e c t i v e l y o f t h e g a l a c t o s y l u n i t s b a s e d o n t h e c l o s e a g r e e m e n t w i t h t h e v a l u e s o f t h e m o n o - m e r i c e q u i v a l e n t ( 6 9 . 3 0 p p m , 7 0 . 2 5 p p m , 7 1 . 8 3 p p m ) . T h e C4 r e s o n a n c e o f t h e g a l a c t o s e r e s i d u e s e x p e c t e d a t 7 0 . 0 3 ppm i s u n r e s o l v e d i n t h e g u a r a n s p e c t r u m , b u t p a r t i a l l y r e s o l v e d i n t h e l o c u s t b e a n gum s p e c t r u m a t 7 0 . 6 p p m . T h e r e s o n a n c e s a t 7 2 . 9 ppm ( u n r e s o l v e d i n t h e s p e c t r u m o f [ 5 ] ) a n d 7 3 . 4 ppm ( 7 3 . 5 ppm) c a n b e r e a d i l y a s s i g n e d t o C2 a n d C3 o f t h e m a n n o s e u n i t s , r e s p e c t i v e l y , f r o m c o m p a r i s o n w i t h t h e v a l u e s f o r t h e 1 , 4 - m a n n a n ( 7 2 . 0 p p m , 7 3 . 2 p p m ) . T h e d e d u c t i o n o f t h e r e m a i n i n g m a n n o s e s i g n a l s i n F i g u r e I V - 5 i s 27 l e s s t r i v i a l i n v i e w o f t h e e x p e c t e d c o m p l e x i t y a r i s i n g f r o m b r a n c h i n g a t C6 o f a p p r o x i m a t e l y h a l f t h e m a n n o s e u n i t s . T h u s , t h e m u l t i p l e t c e n t r e d a t 7 6 . 7 ppm ( 7 6 . 6 ppm) i s s h i f t e d u p f i e l d b y 1 . 6 ppm ( 1 . 7 ppm) 155 r e l a t i v e to the p o s i t i o n of the corresponding C4 resonance of 1,4-mannan. The resonance at 74.2 ppm (74.4 ppm), s h i f t e d u p f i e l d by 2.5 ppm (2.3 ppm), i s more r e a d i l y i d e n t i f i e d as o r i g i n a t i n g from C5 of the unbranched man- nose re s i d u e s i n l i g h t of i t s greater r e l a t i v e i n t e n s i t y i n the spectrum of l o c u s t bean gum compared to that of [ 1 ] . From t h i s i t can be i m p l i e d that the resonance at 73.4 ppm (73.5 ppm) a r i s e s from C5 of the branched mannose u n i t s ; i n both s p e c t r a ( F i g . IV-5) the r a t i o of the r e l a t i v e i n t e n s i t i e s of the l a t t e r two s i g n a l s i s almost i d e n t i c a l to that of the r e s p e c t i v e anomeric s i g n a l s confirming thereby the above i n f e r e n c e . The 1 3 C nmr s p e c t r a , at l e a s t i n the case of guaran, appear to con- t a i n i n f o r m a t i o n concerning the sequences of branched and unbranched man- nose u n i t s . The p o s i t i o n of the carbon resonances of the unbranched man- nose u n i t s i s seemingly s e n s i t i v e to branching at the neighbouring mannose re s i d u e as i n d i c a t e d by the s p l i t t i n g of the resonances of C l , C4, C5, and C6 (Fig. IV-6). M4 T — — i 1 1 I I I 1 r — — i — i i f (, ppm) F i g . IV-6. i 3 C - s p e c t r a l region of (a) C l , (b) C4, C5, (c) C6 f o r [ 1 ] . 156 21 A f t e r completion of these assignments, Grasdalen and Painter reported recently on 1 3 C nmr studies of p a r t i a l l y degraded guaran and locust bean gum. The authors obtained a complete s p e c t r a l assign- ment, recording t h e i r spectra at lower magnetic f i e l d (25 MHz) and higher temperature (90°C). Their data are i n good q u a l i t a t i v e agreement with those described here, from which they d i f f e r e d only i n two aspects: (i) the C6 resonance of the branched mannose units was resolved i n t h e i r spectra (69.7 ppm); and ( i i ) differences i n chemical s h i f t (downfield s h i f t s of up to 2 ppm) of some of the corresponding resonances were observed, p a r t i c u l a r l y f o r those of the galactose residues. The l a t t e r observation derives presumably from the combined e f f e c t of various f a c - t o r s , such as differences i n probe temperature and sample i n t e g r i t y . * Some of the postulates advanced here were confirmed by Grasdalen and Painter who, from the s p l i t t i n g pattern of the C4 resonance of the mannose residues, derived nearest-neighbour p r o b a b i l i t i e s f o r both polymers which 19 20 were i n good agreement with t h e i r previous fi n d i n g s . ' In contrast to the guaran spectra discussed here, these authors did not, however, observe peak s p l i t t i n g f o r any other carbon of the unbranched mannose u n i t s . Thus, i n conclusion, the experiments performed here have demonstrated that high r e s o l u t i o n 1 3 C spectra of high-molecular weight polysaccharides can be obtained, using admittedly h i g h - f i e l d spectrometers, at r e l a t i v e l y low temperatures and without extensive degradation of the materials. Although such i n v e s t i g a t i o n can be associated with the disadvantage of s l i g h t l y lower r e s o l u t i o n , obvious advantages accrue from the study of the native, undegraded polysaccharides. *Chemical s h i f t displacements, a l b e i t to higher f i e l d , were observed i n t h i s study when the experiments were conducted at higher temperatures (55°C). 157 ( v i ) V i s c o s i t y The v a r i e t y of chemical m o d i f i c a t i o n s of the gums can induce a correspondingly l a r g e range of i n t e r e s t i n g r h e o l o g i c a l p r o p e r t i e s of the r e s u l t i n g d e r i v a t i v e s , as demonstrated by a s e l e c t i o n of the guar d e r i v a - t i v e s discussed above. Some of the observed r h e o l o g i c a l f e a t u r e s are s i m i l a r i n nature to those of the n a t i v e polymer, w h i l e others appear to be novel and unique. Thus, the p l o t of apparent v i s c o s i t y versus shear r a t e ( F i g . IV-7) of 1% aqueous s o l u t i o n s of the aldehyde d e r i v a t i v e [2] shows the same c h a r a c t e r i s t i c s of p s e u d o p l a s t i c i t y as guar gum i t s e l f , a l b e i t at f a r lower v i s c o s i t i e s (see I-B). In marked c o n t r a s t , the c a r b o x y l i c a c i d d e r i v a t i v e [14] and both the hydroxy propylamine [8] and the s p i n - l a b e l l e d ([3]) d e r i v a t i v e s seem to e x h i b i t d i l a t e n t behaviour which i s l e s s pronounced at higher shear r a t e s (>10 sec "*") . A b e t t e r understanding of the l a t t e r behaviour i s gained when the flow curves are p l o t t e d on a l o g a r i t h m i c s c a l e ( F i g . IV-8) si n c e (as i l l u s t r a t e d i n I-B 2) p s e u d o p l a s t i c i t y and d i l a t e n c y can be expressed ( w i t h i n a l i m i t e d shear s t r e s s range) i n terms of a power law. Comparison of Figure s IV-7 and 8 rev e a l s a new f e a t u r e . D e r i v a t i v e s [ 3 ] , [ 8 ] , and [14] e x h i b i t two d i s - t i n c t r h e o l o g i c a l r e g i o n s : shear r a t e s below 10-15 sec produce d i l a t e n t flow, w h i l e at higher shear r a t e s no v i s c o s i t y changes are apparent (Newtonian r e g i o n ) . D e r i v a t i v e [ 2 ] , on the other hand, r e v e a l s the expected fe a t u r e s of pse u d o p l a s t i c flow (see I-B). The nature of the v i s c o s i t y p l o t s of e i t h e r of the above d e r i v a t i v e s was not a f f e c t e d by changes i n co n c e n t r a t i o n between 0.1-1.0%. The apparent d i l a t e n t f l o w behaviour observed here i s uncommon f o r guar or i t s d e r i v a t i v e s ; the shear s t a b i l i t y of the l a t t e r has, however, 29 found many i n d u s t r i a l a p p l i c a t i o n s . G o l d s t e i n et a l . found a reduced 158 o [14] R = COOH j 0 10 20 30 4 0 50 SHEAR RATE (sec" 1 ) Fig. IV-7. Apparent viscosities of 1% aqueous solutions of guaran derivatives as a function of shear stress. 159 0- • [8] R = CH2NH(CH2)30H A £3] R = CH 2 NH-{Jj-0 = CH0 ^ . • L21 R o [141 R = C00H 10 io2 SHEAR RATE (seer 1 ) F i g , I V - 8 . Logarithmic p l o t of apparent v i s c o s i t i e s of guaran d e r i v a t i v e s versus shear s t r e s s . 160 pseudoplastic f l o w response f o r d i l u t e (0.3%) aqueous s o l u t i o n s of food- grade guar gum at shear r a t e s between 40-60 rpm ( F i g . I V - 9 ) , w h i l e 23 Holocomb and Smith reported a reduced s e n s i t i v i t y to changes i n shear r a t e f o r c e r t a i n c r o s s - l i n k e d hydroxy a l k y l guar d e r i v a t i v e s . GUAR GUM I S O V) l i t 5 o. P z U l B O O O 0 1 0 2 0 M 4 0 B O tO RPM F i g . IV - 9 . Rheogram of a 0.3% food-grade guar gum s o l u t i o n at 25°C (from r e f . 29). The i n t e r e s t i n g changes i n the r h e o l o g i c a l p r o p e r t i e s which were induced here coupled w i t h the ease w i t h which f u r t h e r chemical v a r i a t i o n s can be made, suggests a number of p o t e n t i a l l y important a p p l i c a t i o n s f o r these and s i m i l a r guaran d e r i v a t i v e s . This w i l l be f u r t h e r i l l u s t r a t e d by the experiments discussed i n the subsequent s e c t i o n . I t should, how- ever, be pointed out t h a t , f o r the d e r i v a t i v e s described here to be of use i n s p e c i f i c areas, a f u l l e r r h e o l o g i c a l e v a l u a t i o n of the r e s p e c t i v e d e r i v a t i v e would, of course, be r e q u i r e d , ( v i i ) C o m p a t i b i l i t y Some of the above guar d e r i v a t i v e s were t e s t e d f o r t h e i r c o m p a t a b i l i t y w i t h aqueous s a l t s o l u t i o n s , b o r i c a c i d as w e l l as w i t h absolute ethanol 161 Table IV-1. C o m p a t i b i l i t y of some guaran d e r i v a t i v e s S a l t Compound C a C l 2 S n C l 2 K2CrOi+ C r C l 3 H3BO3 Ethanol [1] + + + - - c [2] + _ b + + + + b c [3] + + + + [8] + _ b + + + + [13] + _ b + + + _ c a l % aqueous s o l u t i o n s of polysaccharide w i t h s a t u r a t e d , aqueous s o l u t i o n s of metal s a l t s ; + i n d i c a t e s compati- b i l i t y , - i n c o m p a t i b i l i t y ; b g e l formed; C p r e c i p i t a t e . and the r e s u l t s are compared w i t h the n a t i v e guaran [1] i n Table IV-1. The guaran d e r i v a t i v e s show good c o m p a t i b i l i t y w i t h metal i o n s , none being g e l l e d i n c o n t r a s t to [1] by e i t h e r chromium c h l o r i d e or b o r i c a c i d . U n l i k e the n a t i v e guaran, however, they are g e l l e d by t i n c h l o r - i d e . Noteworthy, a l s o i s the s t a b i l i t y of the aldehyde and hydroxy- propylamine d e r i v a t i v e s [2] and [8] towards ethanol. These complementary c o m p a t i b i l i t y p r o p e r t i e s found here could serve to g r e a t l y extend the range of u s e f u l a p p l i c a t i o n s , p a r t i c u l a r l y i n the o i l i n d u s t r i e s , of guar gum. 2. P e r i o d a t e O x i d a t i o n The foregoing o x i d a t i o n procedures u s i n g galactose oxidase were shown to be h i g h - y i e l d i n g and s p e c i f i c , but may, nevertheless be not d i r e c t l y compatible w i t h i n d u s t r i a l a p p l i c a t i o n s i n view of the costs i n v o l v e d . Enzyme i m m o b i l i z a t i o n and c o n t r o l l e d periodate o x i d a t i o n can be suggested as two a l t e r n a t i v e , l e s s expensive r o u t e s , of which the f e a s i b i l i t y of the l a t t e r was i n v e s t i g a t e d to some extent here. F i g . IV-10. Esr s p e c t r a of aqueous s o l u t i o n s of (a) [ 2 1 ] , (b) [22] at 298K. F i g . IV-11. Esr s p e c t r a of [ 2 1 ] ( l e f t ) and [ 2 2 ] ( r i g h t ) (a) s o l i d (at 2 9 8 K ) , (b) frozen at 77K. 163 The p r e f e r e n t i a l o x i d a t i o n of the g a l a c t o p y r a n o s y l groups of guar gum and l o c u s t bean gum by l i m i t e d q u a n t i t i e s of sodium metaperiodate had been p r e v i o u s l y reported to leave the mannose backbone of these polymers 28 28 l a r g e l y i n t a c t . Thus, Opie and Keen, using 0.25 mole NalO^ per mole of guaran hexose u n i t , found 77% of the o x i d i z e d hexose u n i t s of the product to d e r i v e from the galactose r e s i d u e s . F o l l o w i n g t h e i r method, guar gum and l o c u s t bean gum were o x i d i z e d u s i n g even smaller amounts of periodate (0.19 mole and 0.14 mole NalO^/mole of hexose, r e s p e c t i v e l y ) which correspond to 14% and 12%, r e s p e c t i v e l y , of the t h e o r e t i c a l r e q u i r e - ment. I f , as assumed, periodate consumption i s confined to the galactose u n i t s i n each case, the expected degrees of o x i d a t i o n (d.o.) of these u n i t s would be 0.53 and 0.70, r e s p e c t i v e l y , i . e . , the polymers would be incompletely o x i d i z e d at these periodate l e v e l s . Another l i m i t a t i o n of t h i s method becomes apparent when one pro- ceeds to examine the s t r u c t u r e of the dialdehyde products. Although i t i s known that the r e a c t i v i t y of the g a l a c t o s y l c i s - d i o l (C3 and C4) i s much greater than that of the t r a n s - d i o l (C2 and C3), and t h a t , hence, the product should predominantly be [17] r a t h e r than [19], complications a r i s e from the v a r i e t y of p o s s i b l e i n t r a - r e s i d u e c y c l i c hemiacetal 19 s t r u c t u r e s ( i n t e r - r e s i d u e hemiacetal formation i s not favoured) and the s m a l l ( i n view of the very low l e v e l s of periodate used) but f i n i t e p r o b a b i l i t y f o r a monooxidized galactose r e s i d u e to consume a second mole 20 of p e r i o d a t e a f f o r d i n g [20]. Thus, upon r e d u c t i v e amination of the dialdehyde products derived from guaran ([17]) and l o c u s t bean gum ( [ 1 8 ] ) , using s p i n - l a b e l [ 4 ] , a s e r i e s of s p i n - l a b e l l e d product s t r u c t u r e s can be envisaged to e x i s t f o r each of the polymers some of which are i n d i c a t e d i n scheme 2. HO [21] SCHEME 2 164 In view of the above, extensive esr s t u d i e s of the s p i n l a b e l l e d guar gum [21] and l o c u s t bean gum [22] d e r i v a t i v e s were considered to be not worthwhile. S u f f i c e i t to say that the esr spec t r a of [21] and [22] i n aqueous s o l u t i o n ( F i g . IV-10) resembled i n lineshape those of the corresponding galactose oxidase-derived samples ( F i g . IV-1) and had s i m i l a r apparent values ( f o r [21], = 6.36 x 10 sec and f o r [22], = 5.31 x 10 ^" s e c ) . The esr spec t r a of the s o l i d d e r i v a t i v e s r e v e a l two components of which one appears to a r i s e from a more mobile l a b e l p o p u l a t i o n ( F i g . I V - l l a ) . This i n t e r p r e t a t i o n i s not unreasonable i n view of the p o s s i b i l i t y of d i f f e r e n t o x i d a t i o n products and the f a c t that the products were p u r i f i e d by extensive (5 days) d i a l y s i s . No d i p o l a r i n t e r a c t i o n s are evident i n e i t h e r the above spe c t r a or i n those obtained at 77K ( F i g . I V - l l b ) , i n d i c a t i n g a l a r g e r mean separation between spins than i n the corresponding enzyme-treated m a t e r i a l s . This can presumably be a s c r i b e d to a more extended, s t e r i c a l l y l e s s crowded con- formation of the side-chains i n the r i n g - c l e a v e d d e r i v a t i v e s i n compari- son to the enzyme-derived samples. 165 References 1. See, for example, J. F. Kennedy,ih Carbohydrate Chemistry, Specialist Periodical Reports, 11, 371 (1979). 2. G. Avigad, D. Amaral, C. Asensio, and B. L. Horecker, J. Bio l . Chem., 237, 2736 (1962). 3. D. Amaral, L. Bernstein, D. Morse, and B. L. Horecker, i b i d . , 238 (1963); D. J. Kosman, M. J. Ettinger, R. E. Weiner, and E. J. Massaro, Arch. Biochem. Biophys., 165, 456 (1974). 4. J. M. Sempere, C. Gancedo, and C. Asensio, Anal. Biochem., 12_, 509 (1965); J. N. C. Whyte and J. R. Englar, Carbohydr. Res.,57, 273 (1977). 5. R. A. Schlegel, C. M. Gerbeck, and R. Montgomery, Carbohydr. Res., I, 193 (1968). 6. S. M. Rosen, M. J. Osborn, and B. L. Horecker, J. Biol. Chem., 239, 3196 (1964); W. Jack and R. J. Sturgeon, Carbohydr. Res., 49, 335 (1976). 7. A. Maradufu and A. S. Perlin, ibid., 32, 43 (1974). 8. A. Maradufu, G. M. Cree, and A. S. Perlin, Can. J. Chem.,50, 768 (1971); 49, 3429 (1971). 9. M. W. C. Hatton and E. Regoeczi, Biochim. Biophys. Acta, 438, 339 (1976). 10. K. Y. Chen, R. H. Kramer, and E. S. Canellakis, i b i d . , 507, 107 (1978). 11. M. A. Bernstein, L. D. Hall, and W. E. Hull, J. Amer. Chem. Soc, 101, 2744 (1979). 12. G. Avigad, Carbohydr. Res., _3> 430 (1967). 13. J. D. Aplin, M. A. Bernstein, C. F. Culling, L. D. Hall, and P. E. Reid, ibid., 70, C9 (1979). 14. L.D. Hall and M. Yalpani, ib i d . , 81, C10 (1980). 15. See, for example, D. M. Clode and D. Horton, i b i d . , 19., 329 (1971). 16. R. L. Whistler and J. W. Marx, Methods Carbohydr. Chem., 5, 143 (1965). 17. G. A. Hamilton, P. K. Adolf, J. de Jersey, G. C. Du Bois, G. R. Drykacz, and R. D. Libby, J. Amer. Chem. Soc, 100, 1899 (1978). 166 18. See, for example, W. Hartmeier and G. Tegge, Starch, 31, 348 (1979). 19. J. J. Gonzalez, Macromolecules, 11, 1074 (1978). 20. T. J. Painter, J. J. Gonzalez, and P. C. Hemmer, Carbohydr. Res., 69, 217 (1979). 21. H. Grasdalen and T. Painter, ibid., 81_, 59 (1980). 22. C. W. Baker and R. L. Whistler, ibid., 45, 237 (1975). 23. D. L. Holcomb and M. 0. Smith, Southw. Petrol. Short Course, 22, 129 (1975). 24. C. J. Githens and J. W. Burnham, Soc. Pet. Eng. J., 17_, 5 (1977). 25. H. Guglielmi and A. Jung, Hoppe-Seyler's Z. Physiol. Chem., 358, 1462 (1977); J. A. Hickman and D. H. Melzack, Biochem. Pharmacol., 25, 2489 (1976). 26. G. A. Morris and L. D. Hall, submitted for publication. 27. P. A. J. Gorin, Carbohydr. Res., 39, 3 (1975). 28. J. W. Opie and J. L. Keen, Germ. Pat., 1,262,756 (1968); Chem. Abstr., 68, 88358K (1968). 29. A. M. Goldstein, E. N. Alter, and J. K. Seaman, in Industrial Gums, R. L. Whistler (ed.), Academic Press, New York, 303 (1973). CHAPTER V MODIFICATION OF THE METAL-CHELATING CAPACITY OF CHITIN AND CHITOSAN V-A. Introduction The application of polymers as support matrices for chelation,''" c l i n - 2 3 i c a l use, catalysis, as well as for synthesis has grown rapidly since their use i n peptide synthesis was demonstrated by Merrifield. One facet of this effort has been directed at incorporating metal ions or metal com- plexes into polymers using a variety of chelating groups. Some problems encountered in many of these studies derive from the often complex and costly synthesis, from inefficient metal chelation, and from the leaching into solution of the metal complex from the polymer. The search for new and efficient chelating polymers constitutes therefore a major area of research. Numerous other reasons exist for interest in metal-polymer con- A jugates including the study of metal complexes in biological systems, metal-based a f f i n i t y chromatography,~* and the treatment of environmental - , , 6 pollutants. The work described in this chapter represents an effort to direct some of the previously gained experience towards some specific application. Chitin and chitosan appeared to be well-suited for advancing the development of metal chelation since both materials are inexpensive, abundant 'natural' chelating agents^ and since an extensive knowledge of the chelating prop- g erties of their monomeric constituent has been established for many years. We found, surprisingly, that, in spite of a number of studies of metal 7 9 chelation by the native polymers, ' only a few very recent attempts to 167 168 improve t h e i r c h e l a t i n g c a p a c i t y by means of chemical d e r i v a t i z a t i o n have been described i n the l i t e r a t u r e . ^ Thus, as an extension of a l o n g - standing i n t e r e s t i n t h i s l a b o r a t o r y i n "metal-conjugation" by monosac- 11 12 cha r i d e s , i n c l u d i n g amino-sugars, methods were f i r s t evaluated whereby the m e t a l - c h e l a t i n g performance of both c h i t i n and c h i t o s a n can be enhanced or modified. The i n c o r p o r a t i o n of a r e l a t i v e l y novel concept, namely the " t a i l o r i n g " of the s o l u b i l i t y p r o p e r t i e s of the polymeric d e r i v a t i v e s , was demonstrated f o r these procedures. L a s t l y , a method was i n v e s t i g a t e d f o r the p r e p a r a t i o n of a new type of organometallic p o l y s a c c h a r i d e d e r i v a t i v e . V-B. M o d i f i c a t i o n of C h e l a t i n g Capacity and S o l u b i l i t y 1. Enhancement of C h e l a t i n g Performance ( i ) Synthesis and copper complexes Chitosan [1] was condensed w i t h s a l i c y l a l d e h y d e [2] f o l l o w i n g the 12 13 methods of Nudga et a l . and Hirano et a l . to a f f o r d the S c h i f f s base d e r i v a t i v e [3] (scheme 1 ) : CHgOH C H 0 CHgOH ^ " "I C U R = R f 122 H C3D N = C H HO C 6 J - C 0 ^ H C4J N H - C H j ^ 3 HO scheme! H 6 C O 2 H 169 I n t e r e s t i n g l y , r e d u c t i o n of the a c i d - l a b i l e * azomethine f u n c t i o n of s a l i c y l i d e n e - c h i t o s a n [ 3 ] , w i t h sodium cyanoborohydride simultaneous w i t h i t s formation produced a very s o f t , i v o r y coloured g e l , which a f t e r d i a l y s i s and l y o p h i l i z a t i o n , gave the amine [4] (d.s. 0.6) as a f l u f f y , o f f - w h i t e m a t e r i a l . (Attempts to c a r r y out the r e d u c t i o n consecutive to the formation of [3] were only p a r t i a l l y s u c c e s s f u l as i n d i c a t e d by the r e t e n t i o n of most of the y e l l o w colour and r i g i d i t y of the g e l i n i t i a l l y produced.) The S c h i f f ' s base d e r i v a t i v e [5] (d.s. 1.0) was produced i n s i m i l a r f a s h i o n from 3-formyl-2-hydroxy benzoic a c i d [ 6 ] . * * The s a l i c y l i d e n e chitosans [ 3 ] , [ 4 ] , and [ 5 ] , l i k e c h i t i n [7] and ch i t o s a n [ 1 ] , r e a d i l y reacted w i t h copper(II) acetate i n e i t h e r aqueous or methanolic*** s o l u t i o n to produce, by analogy w i t h the monosaccharide 14 e q u i v a l e n t s [8] and [ 9 ] , coloured complexes (see Table V-1), which could be c h a r a c t e r i z e d by esr spectroscopy. C 8 ] C 9 1 13 *The d e r i v a t i v e i s , however, s t a b l e to base. * * K i n d l y provided by Dr. M. J . Adam. ***No s u b s t a n t i a l d i f f e r e n c e s i n Cu-chelation c a p a c i t y between these media were observed. Table V-1. Copper C h e l a t i o n Performance of some C h i t i n and Chitosan D e r i v a t i v e s 3 Cmpd. Time (hr) Copper i • mmol g / content I of theory Colour parent polymer Cu(II) complex [3] 1 0.54 23 deep y e l l o w l i g h t green 12 0.62 26 dark green [A] 1 2.19 72 white green 12 3.03 100 dark green [5] 1 0.02 1 deep y e l l o w deep ye l l o w 12 0.06 2 deep y e l l o w 20 - - green y e l l o w [10] 1 0.42 13 white l i g h t blue 12 2.64 80 tu r q u o i s e [11] 1 0.26 7 l i g h t y e l l o w l i g h t green 12 0.40 11 turqu o i s e [1] 12 0.06 1 white blue [7] 12 0.18 4 ye l l o w l i g h t blue [12] 12 0.07 1 yel l o w l i g h t green [13] 12 0.01 0.3 l i g h t y e l l o w l i g h t y e l l o w a i n methanol at 25°C contact time c m i l l i m o l e per gram polymer 171 Atomic abs o r p t i o n spectroscopy was used to q u a n t i t a t i v e l y determine the amounts of Cu(II) incorporated i n t o the d e r i v a t i v e s . Table V - l shows that the copper c h e l a t i o n c a p a c i t y of the amine [ 4 ] , sampled a f t e r 12 hr r e a c t i o n time, was enhanced by a f a c t o r of four over that of [ 3 ] , and a 50 f a c t o r over that of [5] or c h i t o s a n . This increased c h e l a t i n g c a p a c i t y of [4] over [3] i s i n l i n e w i t h the observed s t a b i l i t y constants of the copper(II) complexes of r e l a t e d l i g a n d systems ( f o r a comparison, see r e f . 16). Furthermore, the 7a greater p o r o s i t y of [ 4 ] , a f l u f f y , w a t e r - i n s o l u b l e m a t e r i a l which i n con t r a s t to i t s s o l i d analogue [3] and the other d e r i v a t i v e s , swelled c o n s i d e r a b l y i n aqueous or a l c o h o l i c s o l u t i o n , i s presumably a l s o p a r t l y r e s p o n s i b l e f o r t h i s o b servation. More i n f o r m a t i o n was gained from experiments i n which attempts were made to e l u t e the copper ions from the complexes of [3] and [ 4 ] , us i n g 0.1M EDTA s o l u t i o n at p H 8 , t h i s proceeded s u c c e s s f u l l y f o r the former case, whereas ^30-40% of the cop- per was r e t a i n e d by the l a t t e r complex, which was, however, completely "demetallated" by treatment w i t h aqueous a c i d (pH. 2). These f i n d i n g s t e s t i f y again to the greater c h e l a t i n g a b i l i t y of [ 4 ] . I t i s i n t e r e s t i n g to note (see Table V - l ) , that the amine [ 4 ] , i t s analogue [ 3 ] , and c h i t o s a n * chelated a r e l a t i v e l y l a r g e p r o p o r t i o n (72%, 87%, and 86%, r e s p e c t i v e l y ) of t h e i r t o t a l uptake w i t h i n a short p e r i o d (1 h r ) , whereas [5] complexed a r e l a t i v e l y s m a l l e r amount (30%) of copper. The hi g h c h e l a t i n g r a t e of [4] was a l s o r e f l e c t e d i n the almost i n s t a n - taneous c o l o u r a t i o n (green) when t h i s m a t e r i a l was added to a s o l u t i o n of c u p r i c or n i c k e l o u s i o n s . *From refe r e n c e 7a (obtained under equivalent c o n d i t i o n s ) 172 CH20R CH2OR NHR C113 R=CH2C02H,H CK)3 R=CH2CC2H,H A number of studies" 1' have suggested the use of c h i t o s a n f o r metal- based chromatography and from the foregoing d i s c u s s i o n i t i s apparent that the s a l i c y l d i m i n e type c h i t o s a n d e r i v a t i v e s may be of i n t e r e s t f o r s i m i l a r a p p l i c a t i o n s , two aspects of which are suggested here: 1. Metal ions can be c o l l e c t e d on columns packed w i t h these m a t e r i a l s and subsequently e l u t e d therefrom using complexing agents, such as EDTA. The metal ions can be a l t e r n a t i v e l y removed w i t h a c i d i c s o l u t i o n s (pH2) as the s a l i c y l a l d e h y d e complex and recovered a f t e r degradation of the complex. O v e r a l l , i t i s t h e r e f o r e p o s s i b l e to regenerate both the c h i t o s a n column and the s a l i c y l a l d e h y d e d e r i v - a t i v e . This l a t t e r procedure i s of importance because i t could be u t i l i z e d f o r the l a r g e - s c a l e p u r i f i c a t i o n of s a l i c y l a d e h y d e d e r i v - 18 a t i v e s i n analogy to e s t a b l i s h e d methods. 2. Reduction of the S c h i f f ' s base d e r i v a t i v e s a f f o r d s v e r s a t i l e d e r i v a t i v e s which are h y d r o l y t i c a l l y s t a b l e to a c i d i c and b a s i c c o n d i t i o n s . We have a l s o evaluated a number of other d e r i v a t i v e s of c h i t i n and c h i t o s a n which may be p r e f e r a b l e complexing agents f o r l a r g e s c a l e a p p l i c a t i o n s s i n c e they are l e s s expensive to prepare. The carboxy- methyl d e r i v a t i v e s of c h i t i n [10] (d.s. 1.0) and c h i t o s a n [11] (d.s. 1.2) 173 19 both prepared* by a procedure s i m i l a r to that of T r u j i l l o , formed turquoise copper(II) complexes. Both d e r i v a t i v e s had a greater c h e l a - t i o n e f f i c a c y than t h e i r u n d e r i v a t i z e d precursors [1] and [7] (20 times and 11 times, r e s p e c t i v e l y ) (see Table V-1). The carboxymethyl c h i t o - san d e r i v a t i v e [11] i s t h e r e f o r e an e x c e l l e n t candidate f o r commercial metal complexation. Another c h i t o s a n d e r i v a t i v e t e s t e d was the N-methylene compound 20 [12] which was r e c e n t l y described as m a t e r i a l f o r g e l chromatography. Reaction of [12] w i t h copper(II) acetate produced a blue g e l . The c h e l a t i n g c a p a c i t y of [12] was, however, found to be not s i g n i f i c a n t l y greater than t h a t of c h i t o s a n (Table V-1). CH20H N = C H 2 C12J I t should be noted that due to the i n t r i n s i c v a r i a t i o n s i n p o r o s i t y and g r a i n s i z e (e.g., c h i t o s a n f l a k e s were employed) of the m a t e r i a l s s t u d i e d , absolute l e v e l s of metal c h e l a t i o n are not as i n f o r m a t i v e as the r e l a t i v e (or enhanced) va l u e s . These m a t e r i a l s should a l s o not be evaluated s o l e l y on the b a s i s of t h e i r absolute c h e l a t i o n c a p a c i t y s i n c e , under s u i t a b l e c o n d i t i o n s , v a r i o u s s y n t h e t i c polymers may a l s o e x h i b i t high metal complexing e f f i c a c i e s . Other f a c t o r s , i n a d d i t i o n to the ones *The degrees of s u b s t i t u t i o n of both products were found to be q u i t e s e n s i t i v e to experimental c o n d i t i o n s , i . e . , r e a c t i o n time w i t h a l k a l i and c h l o r o a c i d — s e e Experimental. 174 p r e v i o u s l y a l l u d e d t o , such as the s t a b i l i t y and r a t e of formation of the polymer bound metal complex, i t s s e l e c t i v i t y to and general s e n s i - t i v i t y towards v a r i o u s c l a s s e s of metal ions (e.g., a l k a l i e a r t h , and t r a n s i t i o n m e t a l s ) , and the v e r s a t i l i t y of the m a t e r i a l s , should be taken i n t o c o n s i d e r a t i o n . Although time d i d not permit the author to pursue some of the above aspects, the f i n d i n g s described here c l e a r l y i n d i c a t e the u t i l i t y of these aminopolysaccharides as h y d r o p h i l i c sup- port matrices f o r metal c h e l a t i n g agents; we suggest that systematic s t u d i e s might be rewarding. ( i i ) Esr of copper complexes The esr sp e c t r a of the copper complexes of the n a t i v e polymers and of the d e r i v a t i v e s unambiguously e s t a b l i s h e d the presence of polymer- bound copper complexes ( F i g s . V - l - 4 ) . The spec t r a of a l l the samples r e v e a l c l o s e l y r e l a t e d f e a t u r e s : the n i t r o g e n h y p e r f i n e couplings are unresolved but an a x i a l symmetry about the copper atom i s d i s p l a y e d . The spectrum of [5] i s d i s t i n g u i s h e d by an a d d i t i o n a l g^ band ( F i g . V-4). Attempts to i d e n t i f y the c o o r d i n a t i o n geometry of these copper complexes were undertaken, but were i n c o n c l u s i v e due to the complexity and v a r i e t y of p o s s i b l e c h e l a t i o n s i t e s ( i . e . , -OH, -NH2, -NHAc, -CO2H, etc.) of these m a t e r i a l s . Thus, an impression that the predom- inance of one or more c o o r d i n a t i o n geometries could be dependent on metal i o n co n c e n t r a t i o n (very vaguely apparent i n the case of the chitos a n - C u ( I I ) complex, F i g . V-3) could not be confirmed when the d e r i v a t i v e [5] was exposed to i n c r e a s i n g amounts of metal i o n s ; the induced esr s p e c t r a l changes were unrevealing ( F i g . V-4). The esr spe c t r a of the fr o z e n (77K) Cu(II)-complexes d i d l i k e w i s e not provide 175 F i g . V-1. Esr s p e c t r a of Cu(II) complexes of (a) [ 3 ] , (b) [ 4 ] , (c) [ 5 ] , (d) [10], (e) [11], ( f ) [12], (g) [13] at ambient temperature. 176 F i g . V-2. Esr spectra of Cu(II) complexes of (a) [3], (b) [A], (c) [5], (d) [10], (e) [11], (f) [12] at 77K. 200 G F i g . V-3. Esr spectra of Cu(II) complexes of (a) [1] a f t e r 8 hr reaction time (298K), (b) [1] af t e r 20 hr reaction time (298K), (c) [7] a f t e r 8 hr reaction time, (d) sample (b) at 77K, (e) sample (c) at 77K (inset: 5* a m p l i f i c a t i o n ) . 178 C 200 G F i g . V-4. Ambient temperature esr spe c t r a of Cu(II) complex of [5] a f t e r (a) 8 h r , (b) 16 h r , (c) 28 hr r e a c t i o n time. 179 any a d d i t i o n a l i n f o r m a t i o n ( F i g . V-2). I t i s most l i k e l y t hat a mixture of geometries r a t h e r than one pure c o o r d i n a t i o n geometry p r e v a i l s . I t can nevertheless be noted t h a t the corresponding mono- and o l i g o s a c c h a r i d e copper conjugates (of glucosamine) r e p o r t e d l y form b i n u c l e a r complexes^"* and species c o n s i s t i n g of one Cu(II) i o n and four moles of glucosamine 22 u n i t s , r e s p e c t i v e l y . For the n a t i v e c h i t o s a n i n aqueous s o l u t i o n , 23 I n a k i et a l . have r e c e n t l y proposed an e q u i l i b r i u m between Cu(II) complexes which, depending on the pH of the medium, i n v o l v e s two or three glucosamine u n i t s per metal atom and i s accompanied by conformation changes of the polymer as shown below. PH 7-9 2. Reduction of C h e l a t i n g Capacity I n s o f a r as a l l the above d e r i v a t i v e s possessed enhanced c h e l a t i n g c a p a c i t i e s , i t i s i n t e r e s t i n g to a l s o observe the converse phenomenon, i . e . , r e d u c t i o n or complete i n h i b i t i o n of metal complexation. For example, c h i t o s a n r e d u c t i v e l y aminated w i t h galactose a f f o r d s a branched- c h a i n d e r i v a t i v e [13] (see I I I - B ) which, on exposure to Cu(II) ions does not a l t e r i t s c o l o u r nor does i t produce a d e t e c t a b l e esr s i g n a l ( F i g . V-19). This c h e l a t i n g " i n h i b i t i o n , " a l s o determined by atomic absorp- t i o n spectroscopy (Table V - l ) , i s presumably due, to a l a r g e extent, to CH2OH 180 ( \ NH-CH2 h O H HO- HO- hOH C13D CH20H the molecular architecture of this material as evidenced by scanning electron microscopy (see III-E). The smooth, non-porous surface struc- ture of [13] appears to be impervious to metal ions. In contrast, the amine [4], along with most other aminopolysaccharide derivatives, i s a porous material. 3. Solubility Modification The versatile combination of Schiff's base-formation and reductive amination provides a convenient route for attaching and/or controllably releasing a wide range of medicinally or otherwise important molecules to chitosan, which i t s e l f i s a biodegradable material. Medicinal 7b 2 A application, ' in particular, would clearly benefit from the addi- tional options of selectively either solubilizing the polymer backbone, (an example of which w i l l be described subsequently) or conversely, reducing i t s solubility by reaction of chitosan [1] with another poly- meric species. As an example of the latter effect, the polyanhydride [14] (Gulf PA-18)* was reacted to afford the white insoluble derivative [15] (d.s. 1.0): *A sample of which was provided by Gulf Specialty Chemicals. 181 C H 2 O H C H - C H 2 - C H - C H I i 1 + C U C * H 3 2 Q^^O in LM C = 0 . ^ 3 3 C0 2 H _ C H — C H ^ - C H - C H n Reactions of t h i s type have been used to i n s o l u b i l i z e enzymes u s i n g On the other hand, i t i s f e a s i b l e to prepare polymeric compounds der i v e d from [1] which have enhanced s o l u b i l i t y . The amine [ 4 ] , as already mentioned, was not w a t e r s o l u b l e , whereas r e d u c t i v e amination of c h i t o s a n w i t h 1.2 molar e q u i v a l e n t s of s a l i c y l a l d e h y d e [2] and 0.3 molai e q u i v a l e n t s of l a c t o s e produced a s l i g h t l y w a t e r s o l u b l e product [16] (d.s. 1.0) which contained a s a l i c y l d i m i n e to sugar r a t i o of 3:1. This product, i n view of i t s s o l u b i l i t y p r o p e r t i e s and s t r u c t u r a l s i m i l a r i t y 24 to c e r t a i n p o l y ( v i n y l s a l i c y l i c a cid) d e r i v a t i v e s of the type [17], was s e l e c t e d f o r some p r e l i m i n a r y t e s t s of a n t i b a c t e r i a l a c t i v i t i e s . copolymers of ethylene and maleic anhydride. 25 OH 182 24 D e r i v a t i v e s of the type [17] have been reported to have a n t i b a c t e r i a l a c t i v i t i e s against gram-positive and gram-negative b a c t e r i a . The d e r i v a t i v e [16] was t e s t e d * f o r a n t i b a c t e r i a l a c t i v i t y a g a i n s t B a c i l i u s mycoides, Staphylococcus aureus and Pseudomonas p u t i d a by the a g a r-plate t e s t . The t e s t c o n s i s t s of a p p l y i n g the sample on small f i l t e r d i s c s to the r e s p e c t i v e c u l t u r e d b a c t e r i a ; the development and s i z e of a zone of growth i n h i b i t i o n can then be used as a q u a l i t a t i v e measure of the a n t i b a c t e r i a l potency of the sample. Although the t e s t r e s u l t s i n d i c a t e d no d e t e c t a b l e a c t i v i t y of d e r i v a t i v e [16] a gainst e i t h e r of the gram-positive B. Mycoides and S. aureus or the gram-negative P. p u t i d a , the p r e l i m i n a r y nature of these t e s t s , e.g., only one d e r i v a t i v e was i n v e s t i g a t e d , should be noted. In view of the potency and s p e c i f i c i t y of the p o l y - ( v i n y l - s a l i c y l i c a c i d ) d e r i v a t i v e s , f u r t h e r d e t a i l e d s t u d i e s , i n v o l v i n g , f o r i n s t a n c e , a v a r i e t y of s t r u c t u r a l l y r e l a t e d d e r i v a t i v e s , could w e l l prove more s u c c e s s f u l . OH C17D *With the k i n d a s s i s t a n c e of Dr. P. J . S a l i s b u r y . **A sample of which was k i n d l y provided by Dr. M. J . Adam. 183 V-C. Organometallic Chitosan D e r i v a t i v e Complimentary to the foregoing procedures are methods f o r c o v a l e n t l y a t t a c h i n g metals t o p o l y s a c c h a r i d e s . This can be e x e m p l i f i e d by the r e d u c t i v e a l k y l a t i o n of c h i t o s a n u s i n g ferrocene-aldehyde** [18] which produces the brown organometallic d e r i v a t i v e [19] (d.s. 0.455). Compound [19] c o n s t i t u t e s , to our knowledge, the f i r s t such organometallic p o l y - saccharide d e r i v a t i v e . Polymer attached metallocenes are p o t e n t i a l l y of 26 i n t e r e s t f o r a v a r i e t y of reasons i n c l u d i n g c a t a l y s i s , and medical , . . 27 a p p l i c a t i o n s . 184 References 1. See for example, H. Rapoport, Acc. Chem. Res., 9_y 135 (1976); R. S. Card and D. C. Neckers, J. Am. Chem. Soc.,'99_, 7734 (1977); L. D. Rollman, ib i d . , 97_, 2132 (1975); R. S. Drago, J. Gaul, A. Zombeck, and D. K. Straub, i b i d . , 102, 1033 (1980). 2. R. S. Ramirez and J. D. Andrade, in Polymer Grafts in Biochemistry, (H. F. Hixson and E. P. Goldberg, eds.), Marcel Dekker, New York, 309 (1976). 3. F. R. Hartley and P. N. Vezey, Adv. Organometallic Chem., 15, 189 (1977); E. Kalalova and J. Kalal,F. Svec, Angew. Makromol. Chem., 54, 141 (1976). 4. J. F. Kennedy, Chem. Soc. Rev., j}, 221 (1979); R. B. Martin,:.in Metal Ions in Biological Systems, (H. Sigel, ed.), Marcel Dekker, New York, Vol..8, (1979). 5. B. Lonnerdal, J. Carlsson, and J. Porath, FEBS Letters, 75, 89 (1977). 6. I. Tabushi, Y. Kobuke, and T. Nishiya, Nature, 280, 665 (1979). 7a. R. A. A. Muzzarelli, Natural Chelating Polymers, Pergamon Press, New York, (1973). 7b. R. A. A. Muzzarelli, Chitin, Pergamon Press. New York, (1977). 8. J. C. Irvine and J. C. Earl, J. Chem. Soc, 121, 2376 (1922); D. Horton, in The Amino Sugars (R. W. Jeanloz, ed.), Academic Press, New York, IA, 70 (1969). 9. F. Yaku, and T. Koshijima, in Proceedings of the First International Conference on Chitin/Chitosan, (R. A. A. Muzzarelli and E. R. Pariser, eds.), 386 (1978). 10. M. Takahashi, K. Shinoda, T. Mori, and T. Kikyo, Japan Pat., 78 03982 (1978), [Chem. Abstr., 89 (1978) 64708g]; T. Sakaguchi, A. Nakajima, and T. Horikoshi, Nippon Nogei Kagaku Kaishi, 53, 149 (1979), [Chem. Abstr., 91 (1970) 126598y]. 11. V. G. Gibb and L. D. Hall, Carbohydr. Res., 63, Cl (1978); L. D. Hall, P. R. Steiner, and D. C. Miller, Can. J. Chem., 57_, 38 (1979); P. R. Steiner, Ph.D. thesis, University of British Columbia (1971); A. M. Slee, M.Sc thesis, University of British Columbia (1973); D. C. Miller, M.Sc thesis, University of British Columbia (1977). 12. M. J. Adam and L. D. Hall, Chem. Comm., 234 (1979). 185 13. L. A. Nud'ga, E. A. Pllsko, and S. N. Danilov, Zh. Obshch. Khim., 43, 2752 (2729 i n transl.) (1973). 14. S. Hirano, N. Matsuda, 0. Miura, and H. Iwaki, Carbohydr. Res., 71, 339 (1979). 15. M. J. Adam, Ph.D. thesis, University of British Columbia (1980). 16. Stability Constants, Supplement No. 1, The Chemical Society London, Specialist Publication 24, 521; 551 (1971). 17. See R. A. A. Muzzarelli, in Proceedings of the First International Conference on Chitin/Chitosan (R. A. A. Muzzarelli and E. R. Pariser, eds.), 335 (1978); and references cited therein. 18. A. I. Vogel, Practical Organic Chemistry, Longmans, Greens and Co., London, 704 (1956). 19. R. T r u j i l l o , Carbohydr. Res., 483 (1968). 20. S. Hirano, N. Matsuda, 0. Miura, and T. Tanaka, ibi d . , 71, 344 (1979). 21. B. J. Hathaway and D. E. B i l l i n g , Coordin. Chem. Rev., _5, 143 (1970); H. Yokoi, Bull. Chem. Soc. Japan, 47, 3037 (1974); R. S. Drago, J. Gaul, A. Zombeck, and D. K. Straub, J. Amer. Chem. Soc, 102, 1033 (1980). 22. F. Yaku, E. Muraki, K. Tsuchiya, Y. Shibata, and T. Koshijima, Cell . Chem. Technol., 11, 421 (1977). 23. Y. Inaki, M. Otsuru, and K. Takemoto, J. Macromol. Sci. Chem., A12, 953 (1978). 24. 0. Vogl and D. T i r r e l l , i b i d . , A13, 415 (1979). 25. M. S. Masri, V. G. Randall, and A. G. Pittman, in Proceedings of the First International Conference on Chitin/Chitosan (R. A. A. Muzzarelli and E. R. Pariser, eds.), 306 (1978). 26. H. Brunner, Angew. Chem. Internat. Edn., 10, 249 (1971). 27. E. I. Edwards, R. Epton, and G. Marr, J. Organometallic Chem., 107, 351 (1976); J. C. Johnson, Metallocene Technology, Noyes Data Corp., New York, 230 (1973) and references therein. CHAPTER VI SUMMARY It w i l l be recalled that the objectives for this thesis were to develop versatile synthetic methods for selective and efficient modifica- tions of carbohydrate polymers for a wide variety of different applica- tions using modern analytical tools for the characterization of the products. Since this study was intended only to provide an overview i n preparation for subsequent more detailed investigations i t is important in this chapter to draw together the experiences gained for comparison. It was demonstrated in the different chapters that the task of transforming abundant and industrially important carbohydrate polymers (alginate, cellulose, chitin, chitosan, guaran, locust bean gum, xanthan gum) into a great variety of readily manipulable products can be success- f u l l y accomplished using, in many cases, inexpensive synthetic methods. From the synthetic aspect, i t was demonstrated in Chapter II that a range of representative polysaccharides can be readily modified in a selective and efficient manner from which i t may be inferred that the methods used are applicable to other polysaccharides as well. The derivatization of carboxylate groups was accomplished in several ways affording amide, ester, hydrazine, and t r i a z i n y l derivatives. Of these, the amidation methods were found to be high-yielding where amines were either directly coupled, as for the xanthan amides, or through carbo- diimide mediation, as for both alginate and xanthan gum, or via alkyl 186 187 e s t e r s , such as the propylene g l y c o l a l g i n a t e s . The s i m p l i c i t y of the e s t e r i f i c a t i o n of the c a r b o x y l i c a c i d m o i e t i e s of a l g i n i c a c i d w i t h a l c o h o l s appeared to be r a t h e r promising d e s p i t e the low y i e l d s p r e s e n t l y obtained. Hydrazide d e r i v a t i v e s of po l y s a c c h a r i d e s , e x e m p l i f i e d here f o r a l g i n a t e and c e l l u l o s e , c o n s t i t u t e u s e f u l intermediates f o r the attachment of c a r b o n y l - c o n t a i n i n g molecules v i a r e d u c t i v e a l k y l a t i o n . Carbonyl f u n c t i o n s can, conversely, be introduced i n t o polysaccharides by s e v e r a l e q u a l l y f a c i l e routes f o r subsequent d e r i v a t i z a t i o n s as ex e m p l i f i e d by the o x y c e l l u l o s e d e r i v a t i v e s and the v a r i o u s p e r i o d a t e - cleaved m a t e r i a l s . The syntheses performed i n Chapter I I I , transformed an i n t r a c t a b l e p o l y s a c c h a r i d e , c h i t o s a n , i n t o s o l u b l e products which comprise a novel c l a s s of branched-chain d e r i v a t i v e s and e x h i b i t e d a wide range of u s e f u l p r o p e r t i e s i n terms of gel - f o r m a t i o n , s o l u b i l i t y , c o m p a t i b i l i t y , and i n t e r a c t i o n s w i t h other p o l y s a c c h a r i d e s . The s p i n - l a b e l l i n g of c h i t i n and c h i t o s a n i l l u s t r a t e d s e v e r a l a l t e r n a t i v e methods of d e r i v a t i z a t i o n u s i n g comparatively m i l d e r c o n d i t i o n s . I t may be pointed out that the s y n t h e t i c p r i n c i p l e s employed f o r the p r e p a r a t i o n of the branched-chain c h i t o s a n d e r i v a t i v e s are amenable to adaption f o r the systematic syntheses of branched polysaccharides w i t h both comb-like and t r e e - l i k e s t r u c t u r e s . This method i s a l s o more f a c i l e than any of the known procedures f o r the p r e p a r a t i o n of branched 1 2 d e r i v a t i v e s ; ' although i t s u t i l i t y was demonstrated here only by the attachment of mono- and o l i g o s a c c h a r i d e s to c h i t o s a n , one can envisage the combination of d i f f e r e n t polysaccharides or poly s a c c h a r i d e fragments to produce another novel c l a s s of polysaccharides u s i n g s i m i l a r proce- dures. The key f o r such r e a c t i o n s i s the i n t r o d u c t i o n , i f not already 188 present, of suitable prominent functional groups into the polymeric materials, a task which can be usually achieved in a variety of ways. This was nicely born out by the galactomannan modifications where galactose oxidase treatment in conjunction with the reductive amination reaction afforded a variety of novel polysaccharide conjugates ranging from chain-extended guaran, to synthetic glycoproteins and glycopeptides. Several of the polysaccharide derivatives displayed an interesting range of rheological and compatibility properties. Although this enzymic procedure i s , i n i t s present form, incompatible with large-scale appli- cations, i t s optimization, using existing enzyme reactor technology, 3 should not pose too d i f f i c u l t a task. The controlled periodate oxida- tion may, for some applications, also provide a viable alternative. In Chapter V the advantages of selective modification were i l l u s - trated in terms of one specific polysaccharide application and i t was found that salicylidene chitosan derivatives constituted highly e f f i c i - ent metal chelating agents, the hydrolytic s t a b i l i t y of which could be controlled using sodium cyanoborohydride. Procedures were designed to tai l o r the solubility of these derivatives in analogy with the branched- chain chitosan derivatives. The reductive amination reaction was also employed for the preparation of new organometallic polysaccharide derivatives. Various spectroscopic and other instrumental techniques were employed throughout this study for the characterization of the poly- saccharide derivatives and some native polymers in terms of their primary structure, three dimensional shape, and surface structure, as well as their molecular mobility i n solution. The nitroxide spin-labelling method was successfully u t i l i z e d for 189 determining various r e a c t i o n parameters, such as y i e l d and p u r i t y of product, as w e l l as extracting information about motional c o r r e l a t i o n times and, to some extent, structures of the products obtained. The distance measurements performed on guaran and locust bean gum were found to be i n good q u a l i t a t i v e agreement with the s t r u c t u r a l models proposed 4 5 f o r these polysaccharides by Painter and coworkers. ' For the s p i n - l a b e l l e d amine de r i v a t i v e s of alginate and c e l l u l o s e , derived from the periodate-oxidized dialdehyde precursors, the esr r e s u l t s suggested the presence of two n i t r o x i d e residues per cleaved hexose u n i t . Indications of the heterogeneity i n surface structure and a c c e s s i b i l i t y of c e l l u l o s e and chitosan could be obtained with the aid of esr methods, which also provided evidence f o r the formation of stable copper(II) complexes of several chitosan d e r i v a t i v e s . I t should be noted that esr spectroscopy and most of the esr techniques used i n t h i s study have only recently found a p p l i c a t i o n to polysaccharides^ and can be expected to provide a wealth of information for such systems i n future. High r e s o l u t i o n 1 3 C nmr was employed i n e f f o r t s to elucidate the structure of several branched-chain chitosan d e r i v a t i v e s and of guaran and locust bean gum. Using a 100.6 MHz (for 1 3C) spectrometer, i t could be demonstrated that the 1 3 C nmr spectra of high molecular weight polysaccharides are adequately resolved at r e l a t i v e l y low probe tempera- tures without the need f o r extensive p r i o r depolymerization as i s 5 , o commonly practiced at lower f i e l d s . For guaran, the 1SC nmr spectra appeared to contain information concerning the sequences of branched and unbranched mannose u n i t s , which was independently confirmed by the work of Painter and coworkers f o r p a r t i a l l y depolymerized guaran samples."* The nmr studies performed i n t h i s study are representative of the 190 enormous potential of this method as applied to polysaccharides. The multitude of observable nuclei in conjunction with new rimr techniques,^ 8 of which some have only recently been developed in this lab, ensure an increasingly important role to nmr spectroscopy for studies in this area. This is exemplified by the preliminary results obtained by 1 3C magic angle spinning-cross polarization and 1 3C spin-echo nmr experiments of polymers which cannot be analyzed by conventional techniques (see Appendix). It is regrettable that I could not pursue these studies to any great extent, particularly since many of the polysaccharide deriva- tives which were prepared were well-suited for model studies. SEM provided evidence for a wide variety of ultra-structures for several of the branched-chain chitosan derivatives and revealed interest- ing aspects of shrimp shell-derived chitosan i t s e l f . The microarchitec- ture of the chitosan derivatives ranged from smooth non-porous to micro- porous with evidence, in most cases, for microfibrillar substructures. 1 3 SEM studies proved valuable in establishing, together with C nmr, the formation of the lactose inclusion complex of 1-deoxy-lactit-l-yl chito- san. Although the results obtained so far do not allow for s t r i c t correlations between ultrastructure and the conformation of the poly- saccharide side-chains, certain relationships seemed to be indicated for 1-deoxymelibiit-l-yl chitosan whose al,6-linked side-chain gave rise to a highly ordered parallel array of microfibrils. The non-porous surface structure of 1-deoxy-galactit-l-yl chitosan appeared to correlate with the observed lack of metal-chelating capacity. During the course of this study several underlying concepts were apparent, the significance of which extends beyond the merely synthetic aspects discussed so far. Perhaps the most important, and at the same 191 9 time generally least documented so far, idea i s that of inducing system- atic changes in polysaccharide structure and physical properties. The d i f f i c u l t i e s encountered in this area originate mainly from the lack of facile chemical methodology for the preparation of suitable model com- pounds the structures of which can be systematically varied in terms of, for example, branching. The synthesis of the branched-chain chitosan derivatives, as already alluded to, seems to provide access to such model polysaccharides, particularly in view of the possibility of reduc- ing the size of this high molecular weight polymer to the level of oligo- saccharides. The ease with which branching can be affected for poly- saccharides other than chitosan was demonstrated for guaran and should be equally f a c i l e for xanthan gum, cellulose, and alginate in analogy with the various non-saccharidic derivatives prepared (e.g., octadecyl xanthan amide, C2 and C3 spin-labelled cellulose derivatives, and spin- labelled algin amides or esters). The concept of solubilizing intractable, but abundant polysacchar- ides by way of reactions with other surplus carbohydrates, such as lac- tose, i s certain to find applications to cellulose, chitin, and other important polymers. It is equally certain that the a b i l i t y to modify the solubility, chelating efficacy, viscosity, and surface structure of polysaccharide derivatives w i l l , i f properly understood, be of great u t i l i t y to industry and other areas. The relevance of the above ideas i s , of course, not confined to the presently known polysaccharides. Other types of polysaccharides, particularly bacterial v a r i e t i e s , ^ could be screened and developed with the aid of such concepts to drastically expand the scope of carbo- hydrate polymer applications. Such developments, together with the obvious extension of many of the chemical reactions which are presented here in preliminary form only, should in due course, serve to place the present studies into context. 193 References 1. For reviews, see A. F. Bochkov and G. E. Zaikov, Chemistry of the 0- Glycosidic Bond, Pergamon Press, New York, (1979); W. A. Szarek, in MTP Intern. Rev. Sci., Org. Chem. Ser. One, 7, 80 (1973). 2 . H. Ito and G. Schuerch, J. Amer. Chem. Soc, 101, 5797 (1979); B. Pfannemuller and A. Berg, Makromol. Chem., 180, 1201 (1979); W. B. Neely, U.S. Pat., 3,133,856 (1964), Chem. Abstr., 61, 5899h (1964). 3. W. Hartmeier and G. Tegge, Starch, 31, 348 (1979). 4. J. J. Gonzalez, Macromolecules, 11, 1074 (1978); T. J. Painter, J. J. Gonzalez, and P. C. Hemmer, Carbohydr. Res., 217 (1979). 5. H. Grasdalen and T. J. Painter, ib i d . , 81, 59 (1980). 6. J. D. Aplin, Ph.D. thesis, University of British Columbia, (1979); J. D. Aplin and L. D. Hall, J. Amer. Chem. Soc, in press; L. D. Hall and J. D. Aplin, i b i d . , 100, 1934 (1978); D. Gagnaire and L. Odier, Bull. Soc. Chim. Fr., 2325 (1974). 7. W. L. Earl and D. L. VanderHart, J. Amer. Chem. Soc, 102, 3251 (1980). 8. L. D. Hall and S. Sukumar, ib i d . , 102, 1745 (1980). 9. D. A. Rees, Polysaccharide Shapes, Chapman and Hall, (1977). 10. P. A. Sandford and A. Laskin, Extracellular Microbial Polysaccharides, ACS Symp. Ser., 45, (1977). CHAPTER V I I EXPERIMENTAL VII-A. General Methods 1. E l e c t r o n Spin Resonance Esr s p e c t r a were recorded at X-band using a V a r i a n E-3 instrument i n the d e r i v a t i v e a b s o r p t i o n mode and i n t e g r a t e d using a P a c i f i c P r e c i - s i o n Co. MO-1012A i n t e g r a t o r . The second i n t e g r a t i o n was performed by peak c u t t i n g and weighing and comparison w i t h f r e s h l y - p r e p a r e d standard s o l u t i o n s of 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl. Spectro- meter s e t t i n g s — m o d u l a t i o n amplitude, f i l t e r time constant and scan r a t e — w e r e chosen i n each case to avoid d i s t o r t i o n of the s p e c t r a l l i n e s , and power l e v e l s were non-saturating. The ambient temperature was always 25°C + 1, and the f i e l d always increased from l e f t to r i g h t . L i n e widths were measured using the sm a l l e s t p o s s i b l e scan range ( g e n e r a l l y not shown i n the diagrams) and at l e a s t two measurements were made usin g d i f - f e r e n t scans i n each case. The f i e l d was c a l i b r a t e d using a proton nmr magnetometer and the X-band microwave frequency was monitored on a Hew- l e t t - P a c k a r d 5245-L e l e c t r o n i c counter equipped w i t h a 8-18 GHz frequency converter. Spectra at 77K were obtained u s i n g a Dewar i n s e r t c o n t a i n i n g l i q u i d n i t r o g e n , at 0.16 mW microwave power, the lowest a v a i l a b l e . Oxygen was prevented from condensing i n the sample tube by s e a l i n g the top w i t h a rubber septum cap. A l l low temperature sp e c t r a were recorded i n 3 mm 194 195 i.d. quartz tubes when non polar organic solvents were used and in 1 mm i.d. Pyrex tubes for aqueous solutions. At room temperature, both these types of tubes were used along with a f l a t high-quality quartz c e l l , capacity 73 yL, with ground glass joints at both ends (J. Scanlon Co.). For aqueous suspensions and gels of polysaccharides a teflon insert designed by Dr. F. G. Herring was used. This consisted of a cylinder of diameter 10 mm with a half-cylinder section 30 mm long cut away in the center, forming a fl a t surface 10 x 30 mm upon which the wet poly- saccharide was placed beneath a glass cover s l i p , the latter retained by surface tension. Solution of nitroxides, whose spectra were to be used for correlation time ( T ) measurements, were deoxygenated by bubbling nitrogen through for several minutes; in aqueous solutions this was found to be unnecessary. * 2. Nuclear Magnetic Resonance *H nmr. Proton nmr spectra were measured at 270 MHz with a prototype of a home-built spectrometer based on a Bruker WP-60 console, a Nicolet 1180 computer (32K), a Nicolet 293A pulse controller unit, a Diablo Disk, and an Oxford Instruments Superconducting solenoid, or at AOO MHz on a Bruker WH-A00 spectrometer. 1 3C nmr. Proton-decoupled carbon nmr spectra were recorded at 20 MHz with a Varian CFT-20 spectrometer or with a Bruker WP-80 * Spectra of solid polysaccharide derivatives were recorded under under deoxgygenated chloroform unless otherwise indicated. 196 instrument, or at 100.6 MHz with a Bruker WH-400 spectrometer; the latter two instruments being equipped with a variable temperature control unit. Polysaccharide samples were dissolved in D20 directly in the 10 mm nmr tubes to avoid handling of the viscous or gelling materials. Final sample concentrations ranged between 3-8% (w/v). Unless otherwise indicated, spectra were obtained at 305-310°K and were referenced to internal dioxane. 1 9 F . Fluorine nmr spectra were recorded at 94.08 MHz on a Varian XL-100 spectrometer and were referenced to external trifluoroacetic acid. 3. Synthetic Methods A l l reactions involving polysaccharides were conducted at ambient temperature. A l l concentrations were performed on a Buchi rotary evap- orator. Soluble polysaccharide reaction products were purified by dialysis using Spectropore #1 or #2 tubing from Spectrum Medical Inc. (Los Angeles, California), against 0.01M EDTA / 0.01M NaCl solutions or d i s t i l l e d water for 4-6 days, and subsequently lyophilized. A l l samples were stored refrigerated with desiccation. C, H, N microanalyses were carried out by Mr. P. Borda of this department; Cu and Fe microanalysis was performed by Canadian Micro- analytical Service Ltd. (Vancouver) using n i t r i c acid digestion of the polysaccharides and atomic absorption. 4. Materials The following alginate samples were a gif t from Kelco Co., San Diego, California: sodium alginate (Keltone), alginic acid (Kelacid), and propylene glycol alginate esterified 50-60% (Kelcoloid-HVF) and 80-85% (Kelcoloid-0). Sodium alginate was purified following Schweiger's method. Cellulose carbonate was purchased from Sigma Chemical Co.; 197 Cellulose powder (Whatman CF11) from W. & R. Balston Ltd., U.K. Samples of 2- and 3-oxycellulose were gifts from Dr. J. Defaye, Centre de Recherches sur les Macromolecules Veg^tales, Grenoble. Carboxymethyl- cellulose (12M31P) was a gift from Hercules Inc., Willington, Delaware. Xanthan gum samples were gifts from Kelco Co. (Keltrol), and Tate & Lyle Ltd., London (77A3). The former samples were purified according to the 2 method of Holzwarth while the latter were supplied in purified form. Guar gum and locust bean gum were gif t s from Kelco Co. and were purified 3 according to Whistler's method with slight modifications. Chitosan (from shrimp shell) and chitin (from crab shell) were purchased from Sigma and used without further purification. Sodium cyanoborohydride was from Aldrich Chemical Co. 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 4-oxo-2,2,6,6-tetra- methylpiperidine-l-oxyl, and the spin label analogue 4-amino-2,2,6,6- tetramethylpiperidine-l-oxyl were purchased from Alrich Chemical Co. The other spin labels, 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl, and 4-chloroacetamido-2,2,6,6-tetramethylpiperidine-l-oxyl were synthe- 4 sized by L. Evelyn of this lab using the method of McConnell. 5. Product Nomenclature The application of systematic nomenclature to most of the poly- saccharide derivatives reported here i s not feasible due to the hetero- geneity i n the structure of the original polymers or in the substitution pattern of the reaction products; in a number of cases, such as for the products derived from periodate-oxidized materials, the exact structure of the product i s unknown (see text). The nomenclature system adapted here attempts to indicate the substituent(s) introduced followed by the name of the native polysaccharide. 198 VII-B. Chapter II 1. Materials l-Ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HC1 (EDC) was pur- chased from Sigma Chemical Co., and dicyclohexylcarbodiimide (DCC) from Eastman Chemical Co. Dimethylbiguanidine HC1 was from Aldrich Chemical Co., sodium periodate was from Fisher Scientific Co. Hydrazine hydrate was purchased from Mallinckrodt Co. 2. Synthesis [A-(Acetamido-2,2,6,6-tetramethylpiperidine-l-oxyl)] alginic acid [10] 5 Alginic acid was partially dehydrated following Schweiger's method with some modifications. Alginic acid (1 g) was swelled in glacial acetic acid (5 ml) and then centrifuged in a disk-top centrifuge. The supernatent was decanted, and the procedure repeated several times. The wet material thus obtained was used for subsequent reaction. A solution of chloroacetamide label [9] i n dry pyridine (130 mg, 5.3 mM, A ml) was added to partially dehydrated alginic acid (AO mg, 0.23 mM) and the pH of the dispersion was adjusted with solid NaHC03 to -7.5. The reaction mixture was gently shaken for 18 hr. The resulting product [10] had d.s. 0.0A. When 65% aqueous acetone was used instead of pyridine the d.s. of [10] was lowered by one fourth. [A-(2,2,6,6-tetramethylpiperidine-l-oxyl)] alginate [12] Alginic acid (150 mg, 0.85 mM) was partially dehydrated and sus- pended in 65% aqueous acetone (5 ml). After addition of hydroxy label [11] (300 mg, 1.7 mM) the pH was adjusted to 6 using NaHC03 and the mix- ture was shaken for 18 hr. The product [12] had d.s. 0.05. 199 [4-(2,2,6,6-tetramethylpiperidine-l-oxyl)] alginamide [16] (i) Carbodiimide coupling (a) EDC coupling. To partially dehydrated alginic acid (150 mg, 0.85 mM) was added a 65% aqueous acetone solution of EDC (240 mg, 1.25 mM, 5 ml), and after adjustment of the pH to 6.5 using N a 2 C 0 3 , a solution of amine label [15] (270 mg, 1.6 mM, 2 ml). The mixture was shaken for 18 hr and the resulting product [16A] had d.s. 1.00. (b) DCC coupling. The reaction conditions were the same as i n the EDC coupling procedure with the exception of using DCC (100 mg, 0.48 mM), and DMF (3 ml) as solvent. This reaction, however, proceeded in lower (~10x) yields than the EDC coupling. ( i i ) Via propylene glycol esters A solution of amine label [15] (276 mg, 1.6 mM) in DMF (2 ml) was added to a suspension of propylene glycol alginate (PGA), with a degree of esterification 0.80-0.85 (193 mg, 0.86 mM) in DMF (2 ml). The mix- ture was shaken for 14 hr to yield a product [16B] with d.s. -0.15 (18% conversion of available esters groups). For PGA with a lower degree of esterification (0.5-0.6) a product with d.s. -0.10 (18% conversion) was obtained. [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl)] algin amine [18] Sodium alginate was periodate-oxidized according to the procedure of Painter et a l . ^ In a typical experiment, a 0.78% aqueous solution of sodium alginate was oxidized with 0.25M NaI0i+ in the presence of 1-pro- panol at 4°C in the dark for 24 hr. The oxidation was terminated by addition of ethylene glycol. Reductive amination was performed by addition of an aqueous solution of amine spin-label [15] (3-5 fold molar 200 excess) and sodium cyanoborohydride (7-10 fold molar excess). The reac- tion mixture was shaken for 24 hr. For alginate samples oxidized 10%, products with d.s. 0.05 (52% conversion of available aldehyde groups) were obtained, while for samples oxidized 44% the products [18] had d.s. 0.13 (30% conversion). [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl)] algin hydrazine [21] (a) The method of Andresz et a l . ^ was modified to prepare hydrazine derivatives. Samples of propylene glycol alginate (Kelcoloid- 0, 0.5 g, 2.2 mM), were mixed with hydrazine hydrate, (10 ml, 0.2 M), in a 50 ml Erlenmeyer flask. The reaction mixture was gently shaken over- night at 22°C, affording a viscous solution which was diluted with water (25 ml). The product was precipitated i n methanol (300 ml), the precip- itate was collected after centrifugation (9000 rpm, 50 min.), and dialyzed against 0.01 M EDTA for 2d, before lyophilization. Elemental analysis showed a product [19] with d.s. 0.76, i.e., 92% conversion of the ester. Anal, for [ (C 6H 80 6) 0 i 1 ? ( C 9 H l l t O 7 ) 0 > Q ? ( C ^ o ^ O g ) ^ ? g]-0.88 H20; calcd. C 36.25, H 5.74, N 10.35, (C/N3.53); found C 36.01, H 5.37, N 10.36, (C/N 3.48). Similarly, for PGA with lower degrees of esterification, products [19] with d.s. -0.45 (i.e., -80% conversion) were obtained (C/N 5.71). These d.s. obtained here correspond well with those of Andresz et a l . ^ (d.s. 0.67). (b) Reductive alkylation of hydrazine alginate [19] (d.s. 0.76) was carried out by dissolving i t (27 mg, 0.13 mM) in water (10 ml) and adding a solution (7 ml) of keto spin label [20] (102 mg, 0.58 mM) and NaCNBH3 (140 mg, 2.2 mM). The reaction mixture was l e f t on a shaker overnight. The hydrazine product [21] had d.s. 0.21. 201 [(2-amino-4-N-dimethylamino)-s-triazin-6-yl] alginate [23] and [4-(N-dimethylamino)-2-(4-N-amino-2,2,6,6-tetramethylpiperine-l-oxyl)- s-triazin-6-yl] alginate [25] (a) The s-triazinyl derivate was prepared according to 8 Lee and Maekawa using propylene glycol alginate, (Kelcoloid-0) instead of the methyl ester of alginate. Alginate (1 g, 5.44 mM) was suspended in MeOH (100 ml) at 0°C and dimethylbiguanidine hydrochloride [22] (1 g, 6.1 mM) in THF/MeOH (1:1) (60 ml) was added with sti r r i n g to the suspen- sion over a 20 min period. After one hour, the reaction mixture was allowed to warm to room temperature and le f t s t i r r i n g for four days. The mixture was reduced to dryness, and d i s t i l l e d water (20 ml) was added affording a gel, which was purified. The product had d.s. -0.12 (i.e., 15% conversion of esters) as determined by microanalysis. Anal, for [ (C 6H 8O 6) 0 > 2 (CgHj l f 0 7 ) Q > 6 g ( C j ̂  5 ^ ) ^ u]-2.01 H20; calcd. C 38.88, N 3.20, (C/N 12.2); found C 38.59, N 3.05 (C/N 12.7) (b) The s-triazinyl alginate [23] was labelled by dis- solving 40 mg (-0.18 mM) in water (10 ml) and adding a solution of keto label [20] (80 mg, 0.47 mM) and NaCNBH3 (100 mg, 1.6 mM) to the mixture, which was shaken for 13 hr. The product [24] had d.s. 0.02. [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl)] xanthan amide [26] To xanthan gum (150 mg, 0.16 mM) dissolved in water (10 ml) was added EDC (100 mg, 0.78 mM) and the reaction mixture (pH 5) was cooled to 5°C for ten minutes before the amine label [15] (80 mg, 0.47 mM) was added with s t i r r i n g . The reaction was continued for twelve hours, yielding a product [26] with d.s. 0.43 (based on carboxyl groups) after dialysis (4 d). 202 [4-N-(2,2,6,6-tetramethylpiperidine-1-oxyl)] xanthan amide' [27] 9 Following a method reported for the synthesis of "amine alginates," xanthan gum (50 mg, 0.05 mM) was mixed in a 25 ml Erlenmeyer flask with solid amine spin label [15] (57 mg, 0.33 mM) using a spatula. Three drops of water were added and mixed in to'produce a heavy paste, which was shaken for 11 hr, and then dialysed (4 d). The product [27] had d.s. 0.05 (based on carboxyl groups). [18-N-(n-octadecyl)] xanthan amide [28] Following the above procedure, octadecylamine (60 mg, 0.22 mM) was admixed with xanthan gum (75 mg, 0.08 mM) to afford, after dialysis (3 d), [28]. The microanalytical results (C 48.04, H 7.99, N 2.27) could not be matched with a corresponding molecular formula, presumably because of the presence of lower molecular weight fractions in the starting amine (90%, technical grade—Aldrich Chemical Co.), but the C/N ratio corre- sponds roughly to a d.s. 1.0. [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl)] cellulose amine [31] (a) The periodate oxidation method of N e v e l l ^ was used. Three samples (500 mg, 3.1 mM) of cellulose powder were each reacted with NaI04 (0.01M, 0.05M, and 0.1M) in d i s t i l l e d water (25 ml) in the dark for 60 hours at 20°C in a constant temperature shaker to afford products which had a degree of oxidation (d.o.) of 5%, 19%, and 34%, respectively. The reaction was quenched by dumping the dispersions into d i s t i l l e d water (250 ml) and decanting the supernatent. This process was repeated 6-7 times. The samples were l e f t in d i s t i l l e d water overnight, then washed with water (100 ml) and subsequently dried in vacuo at 56°C. 203 (b) Periodate-oxidized cellulose samples (1 mM) were labelled in MeOH (7 ml) by adding 2.6 equivalents of amine spin label [15] and 6.4 equivalents Na.CNBH3 and a few drops of water. After gentle shaking (28 hr) the samples were successively washed on a f r i t t e d glass f i l t e r with 100 ml of water, ethanol, acetone, and ether, and subsequently dried under vacuum for 24 hr at 56°C. The following d.s. were obtained (d.o.): (A) d.s. 0.03 (5%); (B) d.s. 0.063 (19%); (C) d.s. 0.065 (34%). Anal, for (A) [ ( C ^ 0O5)Q^ G5(C&R805)Q^ ^ C C j 3H 2 7 N 20 5) 0 > 0 3 1 * ° - 1 3 H2°5 calcd. C 44.30, H 6.43, N 0.50; found C 44.01, H 6.38, N 0.51. Anal.for (B) [(C 6H 1 0O 5) o > 8 1(C 6H 8O 5) o > 1 2 7(C 1 3H 2 7N2O5) o_ O 6 3]-0.14 H20; calcd. C 44.81, H 6.49, N 1.02; found C 44.70, H 6.49, N 1.03. Anal, for (C) [ ( C 6 H 1 0 O 5 ) ( ) < 6 6 ( C 6 H 8 O 5 ) 0 > 2 7 5 ( C 1 3 H 2 7 N 2 0 5 ) 0 > 0 6 5 ] • 0. 21 H20: calcd. C 44.61, H 6.37, N 1.05; found C 44.33, H 6.25, N 1.04. [2-deoxy-2-(4-N-amino-2,2,6,6-tetramethylpiperidine-l-oxyl)] cellulose [34] and [3-deoxy-3-(4-N-amino-2,2,6,6-tetramethylpiperidine-l-oxyl)-6- t r i t y l ] cellulose [35] (a) 2-oxycellulose [32] and 3-bxy-6-trityl cellulose [33] were prepared (Dr. J. Defaye)^ by oxidation of cellulose using acetic anhydride in DMS0/Ac20 and subsequent precipitation from water. Detritylation was accomplished by sti r r i n g the product in acetone (100 ml) containing cone. HC1 (4 ml) for 16 hr to obtain a product [32] with d ,p. 150, d.s. 0.8. Anal, for [32] [(C 6H 8O 5) 0 g(C 6H 1 0O 5) Q_ 2]•0.18 H20; calcd. C 44.00, H 5.39; found C 43.78, H 5.20. Anal, for [33] (D.P. 150, d.s. 0.85) [ ( C 6 H 1 0 O 5 ) 0 a 5 ( C 2 5 H 2 3 O 5 ) o > 8 5 ] . 204 0.2 H20; calcd. C 71.71, H 5.83; found C 71.94, H 5.63. (b) Spin-labelling of 2-oxycellulose [32] was achieved by reacting i t (378 mg, 2.36 mM) in methanol (7 ml) with the amine spin label [15] (412 mg, 2.41 mM) and NaCNBH3 (400 mg, 6.4 mM) and a few drops of water for 33 hr on a shaker at 20°C. The products were f i l - tered, and successively washed with water (100 ml), ethanol (150 ml), acetone (50 ml), diethylether (50 ml), and subsequently dried in vacuo at 50°C. The product was found to have d.s. -0.28. Anal, for [ ( C ^ 0 O 5 ) Q # 2(C 6H 8O 5) 0_ ̂ CCj 5 H 2 7 N 2 0 5 ) Q 2 Q ] • 1.59 H20; calcd. C 43.93, N 3.37, (C/N 13.04); found C 43.73, N 3.37, (C/N 12.98). (Esr double integration gave d.s. 0.12.) 3-oxy-6-trityl cellulose [33] was labelled under the same conditions yielding a product with d.s. -0.09. Anal, for [ ( C 6 H 1 0 O 5 ) 0 > 1 5 ( C 2 5 H 2 3 O 5 ) 0 > 7 6 4 ( C 3 J t H 1 | 2 N 2 O 5 ) 0 i 0 8 6 ] - 0 . 3 H20: calcd. C 71.33, H 6.08, N 0.62; found C 71.22, H 5.54, N 0.61. (Esr double integration gave d.s. 0.04.) [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl] cellulose urethane [37] A solution of the amine label [15] (366 mg, 2.1 mM) in phosphate buffer (0.5M, pH 7, 2 ml) was added to a suspension of cellulose carbon- ate (d.s. 0.21, 80 mg, 0.48 mM) in the buffer (10 ml). The reaction mixture was stirred overnight at 5°C, and then dialyzed (5 d) at the same temperature to afford a product [37] with d.s. 0.08. Anal, for [(C 6H 1 0O 5) 0 i 7 4(C 7H 8O 6) 0 > 1 8(C 1 6H 2 7N 2O 6) 0 < ( ) 8].0.75 H20; calcd. C 43.22, H 6.50, N 1.16; found C 43.08, H 6.54, N 1.10. When the reaction was carried out i n methanol a product with lower d.s. (0.05) was obtained. 205 Anal, for [ ( C ^ O 0 5 ) 0 > g ( C 7 H 8 0 6 ) 0 > 1 5 ( C j BH 2 7N 20 6) 0 > Q 5]-0.34 H20; calcd. C 44.06, H 6.25, N 0.77; found C 43.86, H 6.16, N 0.75. [4-(2,2,6,6-tetramethylpiperidine-l-oxyl)] cellulose hydrazine [40] (a) The cellulose hydrazine derivative [39] was prepared following the method of Andresz et a l . , 7 using sodium carboxymethyl- cellulose (d.3. 1.2-1.4) as starting material. The product had a d.s. value of -0.5 (0.68 l i t . ) 7 as calculated from elemental analysis. Anal, for [ ( C 8 H 1 2 0 7 ) n r (C 8H l i fN 20 6) ]'2.9 H20: calcd. C 34.37, N 5.01, (C/N 6.86); found C 34.20, N 4.90, (C/N 6.98). (b) Cellulose hydrazine (40 mg, 0.16 mM) was labelled i n phosphate buffer (0.5M, pH 7, 10 ml) using keto spin label [20] (97 mg, 0.6 mM) and NaCNBH3 (100 mg, 1.6 mM). The mixture was shaken for 15 hr and then dialyzed (4 d), to afford [40], d.s. 0.003 (from esr double integration). VII-C. Chapter III 1. Scanning Electron Microscopy (SEM)* 12 The methods of Hirano et a l . were followed for the preparation of the gel samples. Portions of the chitosan derived gels were cut, washed, and rapidly frozen (ca -60°C), before lyophilization. The xerogels were dried at 80°C under reduced pressure (0.01 mm) for 12 hr prior to examination. Specimens were mounted on metal holders with double-coated sticky cellophane tape and silver cement and coated with -4 gold at a vacuum of <10 mm. An Etec Autoscan electron microscope was used, operated at 20 kV. Unless otherwise indicated, only those *These experiments were performed by Mr. Nasser Yalpani. 206 portions of the specimens were viewed which were not damaged by handling. 2. Materials The carbohydrates used in this section were purchased from the following suppliers: maltose, cellobiose, maltotriose, galactosamine hydrochloride, glucose (Aldrich Chemical Co.); melizitose, trehalose, a-glucoheptonic lactone (Pfanstiehl Lab.); lactose, melibiose (Eastman Chemicals); fructose (BDH Chemicals); galactose (Merck); glucosamine hydrochloride (Sigma Chemical Co.). 3. Preparation of 1-deoxyglycit-l-yl Chitosan Derivatives (i) General procedure Chitosan (500 mg, 3 mM) was dissolved with s t i r r i n g i n a mixture (1:1) of methanol and 1% aqueous acetic acid (solvent A) or in the 13 latter medium (solvent B) (30 ml). To the resulting viscous solution was added with vigorous sti r r i n g a solution (10-20 ml) of the carbonyl- containing compound (3.3-10 mM) and sodium cyanoborohydride (20 mM). The reaction mixture was l e f t s t i r r i n g at room temperature for 3-18 hr unti l a gel had formed. The solvent excluded by the gel was decanted, the gel was broken up, repeatedly washed with methanol (150 ml) and f i n a l l y with diethyl ether (150 ml). The solid products thus obtained were f i r s t air-dried for several hours, then dried in vacuo at 56°C for 12-18 hr , and f i n a l l y crushed into a fine powder. In the cases where no gel was formed, the reaction mixture was dialyzed i n dialysis bags against d i s t i l l e d water for periods of 4-6 d with frequent (-15-20) changes of water to obtain, after lyophilization, mostly white materials. ( i i ) Reactions of chitosan with reducing sugars [1-deoxy-l-galactit-l-yl] chitosan [2] Addition of galactose (1.20 g, 6.7 mM) led to the formation of a 207 s t i f f , glassy gel within 1-2 hr. The product (d.s. 0.9) was ivory coloured. Anal, for [(C 8H 1 3N0 5) ( C 1 2 H 2 3 N 0 9 ) J ' 1 . 9 H 20: calcd. C 40.07, H 7.49, N 4.03; found C 40.09, H 7.58, N 3.97. When a smaller amount of galactose ( 1 . 1 0 g, 6.1 mM) was used, the resulting product had a lower d.s. ( 0 . 7 ) . Anal, for [(CgH^ JNO^OQ 3 ( C 1 2 H 2 3 N O G ) 0 ? ] ' 0 . 6 1 H 20; calcd. C 42.65, H 7.24, N 4.88; found C 42.59, H 7.20, N 4.97. [1-deoxy-l-glucit-l-yl] chitosan [ 3 ] Addition of glucose (0.72 g, 4 mM) in aqueous methanol produced no gel. Further addition of 0.9 g ( 5 mM) afforded a firm white gel which was washed and dried. The solid obtained (0.86 g) appeared to be inhomogeneous and was dialyzed for 4 days. After work-up the product had d.s. 0.9 (0.64 g, 7 1 % ) . Anal, for [ ( C 8 H 1 3 N O 5 ) 0 > ( ) 2 ( C 6 H 1 1 N 0 4 ) 0 0 8 ( C 1 2 H 2 3 N 0 9 ) 0 > G ] • 0. 51 H 20; calcd. C 43.05, H 7.22, N 4.39; found C 42.80, H 7.10, N 4.60. [l-deoxy-l-(2-deoxy-2-N-acetylamino),glucit-l-yl] chitosan [ 4 ] Addition of N-acetylglucosamine (1.07 g, 4.84 mM) produced an elastic clear gel after standing overnight, which hardened and turned white after 24 hr. The product had d.s. 0.97. Anal, for [ ( C ^ A N 0 5 ) 0 > 4 H 2 5 N 2 0 9 ) 0 > Q J ] • 2.9 H 20: calcd. C 40.20, H 7.44, N 6.69;. found C 39.99, H 6.90, N 6.55. [1-deoxy-l-cellobiit-l-yl] chitosan [ 5 ] Addition of cellobiose (3.5-6.6 mM) produced no gel after 2 d. The product had d.s. 0.3. 208 Anal, for [(C 8Hi3N0 5) 0 > 0 5(C 6HiiNOu) Q > 6 5(Ci8 H 3 3 N O 1 K)]-0.72 H20; calcd. C 42.47, H 7.04, N 5.11; found C 42.38, H 7.06, N 5.15. [l-deoxy-l-lactit-l-yl] chitosan [6] Addition of lactose (1.2 g, 3.5 mM) produced a milky solution but no gel when the reaction mixture was le f t s t i r r i n g for 10 hr. This product (A) had d.s. 0.23. Similarly, no gel was formed when the lac- tose to glucosamine (L/G) ratio was increased to 1.5 (1.5 g, 4.5 mM lactose); the product isolated after 30 hr had d.s. 0.12 (B), while the same L/G ratio produced a white gel when the reaction mixture was le f t undisturbed for 144 hr (with occasional addition of reducing agent). This product (C) had d.s. 0.78 after dialysis. When the L/G ratio was increased to 2.94 (3.0 g lactose) a white soft gel was formed within 24 hr, which, after nine washes with methanol (150 ml) and ether (150 ml), produced a material (D) whose elemental analysis indicated a f u l l y sub- stituted (d.s. 0.94) product [7] containing one equivalent of unreacted lactose per repeating unit. Subsequent extensive dialysis (5 d) of (D) produced a clear sol (E) with d.s. 0.94. When the reaction was carried out in the absense of sodium cyano- borohydride, using an L/G ratio of 3.90, no gel formed after 28 hr and the resulting product (F), [8] had d.s. 0.1. 209 Anal, f o r [(C 8H 13N0 5) m(C 6H 1 3N0 l t) n(C i eH 23N0 l l t) i 3] * H 20 Product Formula C H N (d.s.) m n p x calcd. found calcd. found c a l c d . found A (0.23) 0.07 0.7 0.23 - 44.01 44.29 6.99 7.27 5.85 5.96 B (0.12) 0.07 0.81 0.12 0.79 41.85 41.69 7.12 7.03 6.44 6.60 C (0.78) 0.07 0.15 0.78 2.9 39.52 39.29 7.30 6.88 2.98 3.00 F (0.1) 0.03 0.87 0.1 0.92 41.22 41.01 7.13 7.01 6.63 6.71 Anal, f o r (E) [ ( C 8 H i 3 N O 5 ) 0 > 0 1 ( C 1 8 H 3 3 N O l l t ) 0 > 9 4 + 0.05 ( C ^ H ^ O n ) - 1.62 H 20; c a l c d . C 41.72, H 7.06, N 2.63; found C 41.44, H 7.06, N 2.81. Anal, f o r (D) [ (CeHj 3 N O 5 ) 0 _ 0 1 ( C 1 8 H 3 3 N O 1 1 + ) 0 > 9 4 + C 1 2H 220iiM.9 H 20; calcd. C 41.80, H 6.86, N 1.59; found C 41.49, H 6.96, N 1.55. [1 - d e o x y - l - m a l t i t - l - y l ] chitosan [9] Addition of maltose (1.74 g, 5.09 mM) produced a r e l a t i v e l y viscous s o l u t i o n a f t e r standing of the re a c t i o n mixture f o r 12 hr, and a s t i f f , white gel was formed within 24 hr. The product had d.s. 0.6. Anal, f o r [ (C 8Hi 3 N O 5 ) 0 > 0 1 ( C 6 H 1 1 N O i t ) 0 _ 3 9 ( C i 8 H 3 3 N 0 1 i t ) ( ) > fi]-1.98 H 20: calcd. C 40.38, H 7.23, N 3.56; found C 40.14, H 6.72, N 3.67. [ 1 - d e o x y - l - m e l i b i i t - l - y l ] chitosan [10] Addition of melibiose (1.20 g, 3.5 mM) led to the formation of an i n i t i a l l y (4 hr) so f t gel which hardened on standing (12 h r ) . The product had d.s. 0.6. Anal, f o r [ ( C 8 H i 3 N O 5 ) 0 _ 0 1 ( C 6 H i i N O 1 + ) 0 > 3 9 ( C 1 8 H 3 3 N O i l t ) 0 ^ 3 * 0 . 5 9 H 20; calcd. C 43.12, H 6.96, N 3.81; found C 43.05, H 6.87, N 3.80. 210 ( i i i ) Attempted chitosan derivatizations Reaction with glucosamine [18] Addition of glucosamine hydrochloride (0.86 g, A mM) in a 1:1 mix- ture of methanol and aqueous NaHC03 (10 ml) produced a viscous solution but no gel after 8 hr. Upon further addition of glucosamine (0.86 g) the reaction was stirred for 0.5 hr, and then l e f t standing for 1 d. A very soft gel formed, to which methanol (150 ml) was added, leading to a hardening of the white gel. This gel, which constituted only -1/3 of the reaction mixture, was fil t e r e d and worked up as before to yield 0.20 g of a white product and the f i l t r a t e was dialyzed (3 d) to afford a flu f f y material. Microanalysis revealed that no product was formed in either of the above cases; similarly, no reaction occurred when smaller quantities (1.06 g, A.93 mM) of glucosamine were used. No firm gel was obtained i n i t i a l l y after overnight s t i r r i n g , but when the reaction mix- ture was le f t undisturbed for 2 weeks, a white, soft gel was formed. Reaction with galactosamine [19] No reaction occurred when galactosamine hydrochloride (3.5-8.2 mM) was used under the above conditions. Reaction with maltotriose [20] To chitosan (170 mg, 1 mM) dissolved i n solvent A was added a solu- tion of maltotriose (670 mg, 1.3 mM) and sodium cyanoborohydride (5 mM). No gel was formed and no reaction occurred after 8 hr as indicated by elemental analysis. (iv) Reactions of chitosan with non-reducing sugars Reaction with trehalose [11] Trehalose (1.5 g, A mM) dissolved in solvent A (10 ml), was added to chitosan (3.0 mM) and mixed for 12 hr. No gel was formed. 211 Reaction with melizitose [12] A yellow, viscous solution was obtained when melizitose (1.70 g, 3.2 mM) was mixed with chitosan in solvent A. After standing for 11 d a ropy gel was produced from the reaction mixture. Reaction with fructose [13] No gel was obtained on addition of a solution of fructose (9.6 mM) in solvent A (10 ml) after 24 hr. Reaction with a-glucoheptonic lactone [23] Addition of a-glucoheptonic lactone (9.6 mM) produced no derivative after 3 d . (v) [N-cyclohexane] chitosan [16] The milky-white solution obtained on addition of cyclohexanone (4.8-13.5 mM) produced no gel. The product [16] was isolated after dialysis (10 d) (d.s. 0.5). Anal, for [ (C 8Hi 3 N 0 5 ) 0 _ Q 2 ( C 6 H j i N 0 1 | ) 0 < 4 g(C 1 3H 2 3NOu) 0 > 5] • 0.41 H20; calcd. C 52.68, H 8.28, N6.44; found C 52.56, H 8.36, N 6.44. 4. Oxidations with Galactose Oxidase [l-deoxy-6'-aldehydo - l a c t i t - l - y l ] chitosan [14] Compound [6]. (103 mg, 0.13 mequiv. galactose) was dispersed in phosphate buffer (25 mM, pH 7, 10 ml) and formed a soft glassy gel which was purged with 0 2 for 1 minute. Catalase (14400 units) and galactose oxidase (90 units) solutions were added and a viscous, ropy material formed after a few hours. The polysaccharide was diluted with water (10 ml) after 2 d and then poured into absolute ethanol (150 ml). The precipitate was collected by centrifugation (9000 rpm, 40 min) and dried, yielding 93 mg of the oxidized product [14]. 212 [1-deoxy-'6'-aldehydo -meli b i i t - l - y l ] chitosan [15] Compound [10] (100 mg, 0.13 mequiv. galactose) was dissolved in dilute acetic acid and the pH was raised to 4.5 by addition of aqueous NaHC03 solution yielding a gel which was treated as above. 95 mg of the oxidized product [15] were isolated. 5. Interaction of [5] with Other Polysaccharides To each of three portions of the [ c e l l o b i i t - l - y l ] chitosan deriva- tive [5] dissolved in d i s t i l l e d water (10 ml) was added a solution (0.2 g, 25 ml) of (i) sodium alginate, ( i i ) guar gum, and ( i i i ) locust bean gum. The mixtures were vigorously stirred and diluted to 40 ml. A white gel formed immediately for alginate; the gel volume decreased considerably (~15x) over a 12 hr period, the surrounding solution being very viscous. No gels were produced in the other cases, but the guar gum mixture devel- oped a considerable viscosity. 6. Spin Labelling of Chitosan and Chitin [4-N-(2,2,6,6-tetramethylpiperidine-l-oxyl)] chitosans [30], [31] Method A. Chitosan (500 mg, 3 mM) was dissolved in a mixture (1:1) of methanol and 0.4% aqueous acetic acid (50 ml) to which was added, with sti r r i n g , a solution of 4-oxy-2,2,6,6-tetramethylpiperidine-l-oxyl [26] (1.0 g, 5.8 mM) and NaCNBH3 (0.9 g, 15 mM) in methanol (10 ml). The solution was stirred for 12 hr at which point a soft white gel had formed. The product .[31] had a d.s. 0.1. Anal, for [ (C8H! 3NO 5) 0_ Q 2(C 6Hi 1N0 1 +) Q_ g 8(C x 5H 2 7N 20 7) ]-0.49 H20; calcd. C 44.72, H 7.38, N 8.27; found C44.47, H 7.10, N 8.15. 14 Method B. Via chitosan N-sulfate [28]. Chitosan (250 mg, 1.5 mM) was dissolved i n 10% aqueous acetic acid (50 ml) and precipitated by addition of an aqueous ammonium sulfate solution. The precipitate was 213 collected by centrifugation, and then suspended in phosphate buffer (20 ml, pH 7). After adjusting the pH to 6.5, the solution was heated to 50° to dissolve the sulfate salt upon which keto spin label [26] (340 mg, 2.0 mM), and NaNCBH3 (0.44 g, 7.2 mM) were added with st i r r i n g . The reaction mixture was kept at 50°C for . 5 hr, then aqueous sodium hydroxide solution was added to precipitate the product at slightly alkaline pH. The product, [30] had d.s. 0.45 (d.s. 0.1 from esr double integration). Anal, for [ ( C ^ 3 ^ 5 ) ^ 1(C 6H 1 2N0 7S) Q > ̂  {C15H27N205)0< 4 5 ]' 0 • 6 Hz0; calcd. C 43.63, H 7.17, N 7.20; found C 43.55, H 6.99, N 7.44. The spin labelling was also attempted by reacting chitosan with spin label in 2M aqueous pyridine and 1% aqueous acetic acid resulting, however, in a much lower yield. [4-acetamido-2,2,6,6-tetramethylpiperidine-l-oxyl] chitosan [29] The labelling was carried out via the chitosan sulfate intermediate (200 mg, 0.8 mM) described above, which was dissolved i n hot water (20 ml, pH 6.5). A solution of 4-chloroacetamido-2,2,6,6-tetramethylpiperidine- l-oxyl [24], (176 mg, 1 mM) in 65% aqueous acetone (2 ml) was added with s t i r r i n g and the reaction mixture was kept at 40-50°C for 6 hr at which time gelation occurred. The gel was separated, washed consecutively with water, ethanol, acetone, and ether and then dried; (d.s. 0.25). Anal, for [(C 8Hi3N0 5) 0 > 1(C 6H 1 2N0 7S) 0 > 6 5(C 1 7H 3 0N 3O 6) o > 2 5]•1.2 H20; calcd. C 36.73, H 6.55, N 7.18; found C 36.60, H 6.49, N 7.14. The same reaction carried out in the presence of 4-dimethylamino- pyridine (DMAP) catalyst (100 mg, 0.8 mM) gave a product with greater d.s. (0.5). 214 Anal, for [(CgHi 3NO 5) 0^ 1(C 6H 1 2NO 7S) 0 > 4(Ci 7H 3 0N 3O 5) o > 5]-1.35 H20; calcd. C 42.87, H 7.32, N 8.55; found C 42.64, H 7.28, N 8.88. Reductive Amination of C-6 Aldehyde Chitosan Derivatives (a) The C6'' aldehydo l a c t i t y l chitosan [14], (43 mg, 0.06 mequiv. galactose) was suspended in aqueous methanol (15 ml) to which was added a solution of amine spin label [32] (150 mg, 0.9 mM) and NaCNBH3 (0.1 g, 2 mM) in the same solvent (5 ml). The amination was carried out for 12 hr and the product was purified by dialysis (3 d); esr double integration gave a d.s. ~0.7 (the microanalytical results could not be exactly matched with a molecular formula; found C 40.82, H 6.53, N 3.87). (b) The C6' aldehydo melibiityl chitosan [15] (58 mg, 0.08 mequiv. galactose) was treated as above yielding a product [35] with d.s. ~0.15 (from esr); (found C 41.47, H 6.77, N 3.92). [4-acetamido-2,2,6,6-tetramethylpiperidine-l-oxyl] chitin [25] Chitin (180 mg, 0.85 mM) was soaked i n DMSO (15 ml) for 48 hr, then fil t e r e d and suspended in aqueous (65%) NaOH solution (40 ml) for 1 hr, after which the a l k a l i c h i t i n was f i l t e r e d , pressed, and resuspended in iso-propanol (25 ml). A solution of the label [24] (250 mg, 0.92 mM) was added with st i r r i n g . After 2 hr the product was fi l t e r e d , washed (water 100 ml, methanol 150 ml), and dried. The product had d.s. 0.04. Anal, for [(CgR^ 3NO 5) 0 > 9 6(C 1 9H 3 2N 3O 7) 0 > 0 4]-0.47 H20; calcd. C 45.77, H 6.70, N 6.83; found C 45.58, H 6.45, N 6.78. Attempted Reductive Amination Chitin (1 g, 4.72 mM) was dissolved in a mixture containing 3.6% (w/v) LiCl and dimethylacetamide (DMAC) (40 ml) and the undissolved 215 material was separated by f i l t r a t i o n through glass wool. To the f i l t r a t e was added a solution of keto spin label [26] (3.6 g, 21 mM) in DMAC and NaCNBH3 (20 g, 33 mM). The reaction mixture was warmed (50°C) for 24 hr, after which aqueous methanol (70 ml) was added to separate the product out as a gel. The gel was fi l t e r e d , washed (methanol 200 ml), and dried, and was found to contain no bound label. VII-D. Chapter IV 1. Viscosity Measurements Viscosity determinations were performed on a Contravese Low Shear 2/Rheomat-30 viscometer using a Fisher 5000 chart recorder and a Haake constant temperature bath. The instrument was calibrated using a Brook- f i e l d viscosity standard (10.3 cps). Measurements were conducted for three polysaccharide concentrations (0.01, 0.1, and 1.0%) at 25.00°C (± 0.05), a minimum of three readings were recorded for each data point. 2. Oxidation Procedures (a) Galactose oxidase. The galactomannan (70-200 mg) was dissolved in phosphate buffer (25 mM, pH 7, 20 ml) by shaking for several hours. The resulting solution was purged with oxygen for several minutes before adding catalase (90000 units/0.1 mM galactose equivalents), and galactose oxidase (100 units/0.1 mM galactose equivalents). The samples were kept at 24°C on a constant temperature shaker for 24-36 hr. The viscosity of the reaction mixture increase sharply during the course of the oxidation affording a ropy gel for both polysaccharides after a few hours. The gel formation could be avoided by performing the reac- tion at greater dilutions (-50 ml solution volume). The aldehyde products were isolated by (i) diluting the sample with an equal volume of 216 phosphate buffer prior to ethanol precipitation (250 ml). The precipitate was then collected by centrifugation (7000 rpm, 40 min); or ( i i ) exten- sive (5 d) dialysis and lyophilization. Efforts to avoid possible coprecipitation of the small amounts (few mg) of enzyme by fractional precipitation were not very successful, although no interference in either the esr or nmr spectra could be detected. Several attempts to optimize the oxidation conditions, such as vari - ations in temperature (23-31°C), time (22-36 hr), or enzyme to substrate ratio (100-150 units/0.1 mM galactose equivalents) produced no s i g n i f i - cant changes i n the degree of oxidation of [2] as judged by analysis of the d.s. of i t s spin-labelled derivative [3]. (b) Periodate oxidations. These were carried out, with slight modification, according to the method of Opie and Keen.''""' The polysaccharide (0.3 mM galactose equivalents) was dissolved in phosphate buffer (20 ml) as before and 1-propanol (1 ml) was added followed by aqueous solutions (2 ml) of sodium metaperiodate (0.23 mM /moi. hexose unit for guaran and 0.17 mM/mol. hexose unit for locust bean gum). The oxidation was conducted at 5°C in the dark for 15 hr after which i t was stopped by addition of ethylene glycol (1 ml). When the oxidation was carried out in smaller solution volumes (~8 ml) the same ropy gels were obtained as in the case of galactose oxidase treatments. The dialde- hyde products were isolated after dialysis (3 d). (c) Bromine oxidation of [2]. The bromine oxidation of the C6 aldehyde guaran derivative [2] was similar to that of Avigad et a l . ^ An aqueous solution of [2] (75 mg, 0.15 mM, 7 ml) was added to bromine water (0.38 mM, 2 ml), the pH was adjusted with barium benzoate to -6 and the reaction mixture was kept at 26°C in a constant temperature 217 shaker for 14 hr. The galacturopyranosyl product [14] was precipitated with ethanol (50 ml), and collected after centrifugation and dialysis (3 d). 3. Reductive Amination—General Procedure The reductive aminations of the C6 aldehyde derivatives [2] and [6] were carried out in situ after oxidation or by dissolving the isolated aldehyde products in aqueous solution followed by treatment with the amine (4-8 moi./galactose equivalent) and sodium cyanoborohydride (20-40 moi./galactose equivalent) at ambient temperature for 24-36 hr. The products were isolated by dialysis (4-6 d) and lyophilization. 6-[N-4(-amino-2,2,6,6-tetramethylpiperidine-l-oxyl)] guaran derivative [3] The yields of the spin-labelled derivative [3] ranged between 60- 70% as determined by esr double integration and microanalysis (the latter being generally more reliable). A typical sample (d.s. 0.61) gave the following analytical results. Anal, for t ( C 6 H 1 0 O 5 ) 0 i 6 4 ( C 6 H 1 0 O 5 ) 0 > l 4 ( C 1 5 H 2 8 N 2 O 6 ) o > 2 2 ] ' 0 . 7 8 H20; calcd. C 44.86, H 7.32, N 2.89; found C 44.83, H 7.15, N 2.71. 6-[N-3 -amino-1-propanol ] guaran derivative [8] Reaction of [2] with 3-amino-1-propanol (7.5 mM/aldohexose equiv- alent) afforded [8]. The d.s. was estimated from 13C-nmr to be ca. 0.8. Microanalysis gave a C/N ratio of 17.33; (calcd. for d.s. 1.0; C/N 16.86). 6-(N-glycine) guaran derivative [9] Reaction of [2] with glycine (4.0 mM/aldohexose equivalent) afforded [9], d.s. 0.2. 218 Anal, for [(C 6H 1 0O5) o > 6 4(C 6H 9O 6) 0 > 1 6(C 8H 1 3N0 7) 0 > 2]-0.89 H20; calcd. C 39.35, H 6.31, N 1.43; found C 39.14, H 6.16, N 1.37. 6-[N-(4-amino)-5-imidazolecarboxamide] guaran derivative [10] Reaction of [2] with 4-amino-5-imidazolecarboxamide HC1 (Chemical Dynamics Corp.) (4.3 mM/aldohexose equivalent) afforded [10], d.s. 0.05. Anal, for [(CgHj 0O 5) 0 i 6 4(C 6H 90 6) Q > 3 1(C x 0YL X5N4O5) Q Q 5]-1.05 H20; calcd. C 38.94, H 6.35, N 1.48; found C 38.83, H 6.28, N 1.46. 6-[N-4-(amino-2,2,6,6-tetramethylpiperidine-l-oxyl)] locust bean gum derivative [7] Spin-labelled derivatives of locust bean gum with d.s. ranging between 0.7-0.8 were obtained, as before; a sample with d.s. 0.7 had the following data. Anal, for [ ( C ^ 0 O 5 ) 0 > 8 1 ( C 6 H 1 O 0 6 ) 0 > Q ( J ( C L 5H 2 8N 20 6) 0_ 13]-0.56 H20; calcd. C 44.33, H 6.99, N 1.91; found C 44.07, H 6.76, N 1.79. Spin-labelled guaran [21] obtained from periodate oxidized guaran, [17]. The product [21] had d.s. 0.85 (based on d.o. = 0.53 for [17]) according to elemental analysis (calculated with one spin-label per dialdehyde unit). Anal, for [(C 5H 1o05) 0 > 6 4(C 6H 90 6) 0 > 2 0(C 1 5H 2 8N 20 6) 0 ^1-1.34 H20; calcd. C 41.32, H 7.16, N 2.10; found C 41.08, H 6.88, N 2.03. Spin-labelled locust bean gum [22], obtained from periodate oxidized locust bean gum, [18]. The product [22] had d.s. 1.0 (based on d.o. 0.70 for [18]) as for [21]. 219 Anal, for t(C 6H 1 0O 5) O f g l(C 6H 9O 6) 0 > D 6(C 15H 2 8N 2O 6) 0 > l 3]-0.93 H20; calcd. C 42.70, H 7.02, N 1.84; found C 42.50, H 6.94, N 1.97. 6-[N-amino] guaran derivative [12] Reductive amination of [2] with ammonium acetate (6 mol/aldohexose unit) for 3 d yielded [12], d.s. 0.55. Anal, for [ (C6Hj O 0 5 ) 0 _ ^ ( C ^ O 0 5 ) 0 > ^ ( C ^ 2 N 0 5 ) 0 > 2 Q]-0.56 H20; calcd. C 40.48, H 6.52, N 1.56; found C 40.27, H 6.46, N 1.54. 6-[N-l-deoxy-l-lactit-l-yl amine] guaran derivative [13] C6-N-amino guaran [12] (55 mg, 0.33 mM) was reductively alkylated with lactose (400 mg, 1.17 mM) to afford, after 2 d, [13], d.s. 0.4. (based on [12]). Anal, for [ ( C ^ 0 O 5 ) Q > ̂ (C^ O 0 6 ) 0 > 1 6 ( C 6 H 1 2 N 0 5 ) Q > u ( C A 5 ) Q > o g - 1.82 H20; calcd. C 38.94, H 6.74, N 1.41; found C 38.65, H 6.49, N 1.29. Bovum Serum Albumin—conjugate of guaran [11] To a solution of the aldehyde [2] i n d i s t i l l e d water (119 mg, 0.71 mM, 20 ml) was added an aqueous solution of BSA (96.1 mg, 1.4 uM) and sodium cyanoborohydride (250 mg, 4 mM, 5 ml). After stirring the reaction mixture for 2 d, the product [11] was isolated after dialysis. 4. Borodeuteride Reduction of C6-aldehyde Derivatives Sodium borodeuteride (0.1 g, 2.4 mM) was added to the dissolved C6 aldehyde derivatives [2] and [6], respectively (0.3 mM aldohexose equivalent, 15 ml) and the aqueous reaction mixture was stirred for 1 d, after which excess reducing agent was destroyed by addition JO f 2N HC1 (0.5ml). The monodeuterated products [15] and [16] (respectively), wer< subsequently dialyzed (3 d) and lyophilized. 220 VII-E. Chapter V 1. Materials The chemicals were purchased from the following suppliers: salicylaldehyde was vacuum d i s t i l l e d before use (Fisher); s a l i c y l i c acid (Malinckrodt); cupric acetate (Fisher); PA-18 polyanhydride was a g i f t of Gulf Specialty Chemicals; ferrocene aldehyde [18] was kindly provided by Dr. M. J. Adam. 2. Synthesis [N-Salicylidene] chitosan [3] 13 17 The methods of Hirano et a l . and Nud'ga et a l . were employed with some modifications: to chitosan (500 mg, 3.0 mM), dissolved in a mixture (1:1, 25 ml) of methanol and 1% aqueous acetic acid, was added dropwise and with vigorous st i r r i n g , salicylaldehyde [2] (0.35 ml, 3.5 mM). The resulting yellow, i n i t i a l l y very viscous solution turned into a thick gel within minutes. A saturated aqueous solution of NaHC03 was added (2 ml) to prevent acid hydrolysis of the S c h i f f s base. A further portion of [2] (0.35 ml, 3.5 mM) was added dropwise resulting in a further stiffening of the gel, to which was then added methanol (80 ml) and NaHC03 solution (1 ml). After a few minutes the solvents were decanted and the wash was repeated twice, the pH of the f i n a l wash being neutral. The gel was l e f t standing in methanol (100 ml) for 4 hr, then f i l t e r e d on a sintered glass funnel, washed with methanol and diethyl ether (100 ml each), air-dried for several hours and f i n a l l y dried in vacuo at 56°C. Yield 0.75 g, d.s. 0.97. Anal, for [ (C 8Hi 3NO 5) ( ) > 0 2(C 1 3Hj 5 ^ 5 ) ^ 9 Q]-0.65 H20; calcd. C 56.20, H 5.94, N 5.08; found C 56.36, H 5.84, N 5.09. Addition of a total 4.2 mM [2] resulted in a product with lower 221 d.s. (0.7). Anal, for [(C 8H! 3NO 5) 0 > 0 3(C 6HiiNO^) Q > 2 ?(C xgHj 5N0 5) Q_ ?]-0.72 H20; calcd. C 53.02, H 6.21, N 5.64; found C 52.84, H 5.97, N 5.67. [N-(2-cresol)]-chitosan [4] The same procedure as for [3] was employed using NaCNBH3 (0.2 g, mM) concommitant with the addition of [2]. Addition of the reducing agent caused the yellow colour of the product to fade and no gel was formed i n i t i a l l y . The solution was l e f t s t i r r i n g overnight resulting in a soft gel. Further addition of the same quantities of [2] and NaCNBH3 afforded a soft, ivory coloured gel which was dialyzed (3 d) against d i s t i l l e d water, and lyophilized to give a f l u f f y white material (0.44 g), d.s. 0.7. Anal, for [ (CeH! 3NO 5) ( ) ! 0 3(C eH 1 1NO„) ( ) > 2 7(C 1 3H 1 7NO 5) 0 > 7]-0.76 H20; calcd. C 52.55, H 6.76, N 5.59; found C 52.30, H 6.50, N 5.55. When the reduction was carried out after the S c h i f f s base gel had formed, the latter retained i t s r i g i d i t y and, to a large extent, i t s yellow colour. After the usual workup, a yellow product with d.s. 0.85 was obtained (0.70 g). Anal, for [ (C8H! 3NO 5) 0 > ( ) 2 (C^ 1N0i t) Q u (Cj 3H 1 7NO 5) 0 > g 5 ] - 0 . 63 H20; calcd. C 54.63, H 6.66, N 5.32; found C 54.42, H 6.34, N 5.49. [N-(3-carboxy-)salicylidene] chitosan [5] As before, chitosan (0.33 g, 2.0 mM) was condensed with 3-formyl-2- hydroxy-benzoic acid [6] (0.39 g, 2.35 mM) dissolved in methanol (10 ml) to produce a bright yellow and very rig i d gel within 3 min. The product was yellow and odourless and had a d.s. 1.0 (0.53 g). 222 Anal, for [(C 8H l aNO5) 0 > 0 2(CmH 1 5NO 7) 0 > 9 8]-1.01 H20; calcd. C 51.24, H 5.26, N 4.31; found C 51.26, H 5.20, N 4.17. Carboxymethyl derivatives of chitosan [10] and chitin [11] 18 The method of T r u j i l l o was adopted with slight modifications. Chitin was suspended in DMSO (15 ml) for 1 d prior to the treatment which was used for the preparation of both [10] and [11]. The polysaccharides (0.5 g) were suspended i n an aqueous (65%) NaOH solution (50 ml) for 0.5 hr to produce the a l k a l i derivatives which were f i l t e r e d , pressed, and then added to a solution of monochloroacetic acid (2.6 g) in i-propanol (50 ml). The suspensions were stirred for 1 hr, f i l t e r e d , resuspended in water (100 ml) and the solution pH was raised (from 3.5-4.0) with cone. NaOH solution to neutral. The chitosan derivative [10] formed a gel at this stage, which was lyophilized. The solid carboxymethyl chitin [11] was f i l t e r e d , pressed and dried. Anal, for [10] d.s. 1.2 [ (Ci 2H 1 7N0! 0 ) O - 1 ( C 8 H 1 3 m O Q , g]-1.02 H20; calcd. C 40.46, H 6.25, N 5.62; found C 40.29, H 6.67, N 5.56. Anal, for [11] d.s. 1.0 [(C 8H 1 3NO b) 0 1(C 1QHJ 5NO 7) 0 g ] ; calcd. C 45.78, H 5.81, N 5.45; found C 45.89, H 6.86, N 5.46. N-Methylene Chitosan [12] This derivative was prepared following the procedure of Hirano et 19 a l . Chitosan (500 mg, 3.0 mM) was dissolved in 2% aqueous acetic acid (25 ml). Dropwise addition of 37% formaldehyde solution (2 ml, 24 mM) produced a colourless gel in less than 3 hr, which was broken up and suspended in methanol (100 ml) for I d , and then f i l t e r e d . This washing procedure was repeated twice, before the gel was f i n a l l y sus- pended in diethyl ether (100 ml) for 1 d. Fi l t r a t i o n and drying yielded , 223 the product [12]. Cross-linked polyanhydride chitosan [15] To chitosan (500 mg, 3 mM), dissolved in a mixture (1:1, 45 ml) of methanol and 2% aqueous acetic acid, was added polyanhydride [14] (Gulf PA-18)(1.15 g, 3.3 mM) dissolved in benzene (8 ml). The white viscous solution formed a very soft gel after standing overnight which was sus- pended in methanol (100 ml) for 1 d. After two such washes with meth- anol, and with diethyl ether, the product was fi l t e r e d and dried yielding 0.08 g of a white powder (A). Alternatively, chitosan dissolved in 10% aqueous acetic acid (15 ml) was mixed with a solution of the polyan- hydride (5.05 g, 14.4 mM) in ethyl acetate (30 ml) to produce a milky gel within seconds, which was worked up as before. 0.25 g white powder was obtained (B) (d.s. -1.0). Anal, for [C 5 0H 8 7NO 1 2]•1.49 H20; calcd. C 65.18, H 9.85, N 1.52; found C 65.20, H 9.89, N 1.53. Solubilized salicylidene chitosan [16] To chitosan (0.50 g, 3.0 mM), dissolved in a mixture (1:1) of methanol and 1% aqueous HOAc, was added lactose (0.30 g, 0.9 mM) in MeOH (4 ml) and subsequently, salicylaldehyde [2].(0,. B5 ml, 3.5 mM) and NaCNBH3 (0.3 g, 4.8 mM) dissolved in water (4 ml). The vigorously stirred mix- ture lost i t s yellow colour after a short time and produced a soft, faintly yellow gel. The product had an overall d.s. 1.0 with 25% sugar substitution. Anal, for [ (CQ^ 3 N 0 5 ) 0 > Q 5 ( C : ̂  7N0 5) Q^ N (Cj 8H33 N Om> 0. 24 ] ; c a l c d - C 52.85, H 6.57, N 4.42; found C 52.51, H 5.95, N 4.20. 224 N-Ferrocenyl chitosan [19] To chitosan (0.20 g, 1.2 mM), dissolved in a mixture of methanol and 1% aqueous acetic acid (1:1, 50 ml) was carefully added, with s t i r - ring, a solution of ferrocenealdehyde [18] (0.30 g, 1.4 mM) and NaCNBH3 (0.9 g, 14.4 mM) i n methanol (10 ml). The i n i t i a l l y red reaction mix- ture was l e f t s t i r r i n g overnight yielding a fine brown precipitate which was f i l t e r e d , washed (methanol), and dried. 0.38 g of the brown product (d.s. 0.445) was isolated. Anal, for [ (C 8Hi 3 N O 5 ) Q > Q 2 ( C 6 H i i N O O Q > ^ ( C 1 7 H 2 i F e N 0 l t ) Q > ^ ] ; calcd. C 48.83, H 5.83, N 5.26, Fe 9.37; found C48.60,H 5.90, N 5.32, Fe 10.06. 3. Copper Complexation Reactions The polysaccharides were complexed by dispersing portions (0.1 g) in methanol (20 ml) with vigorous magnetic st i r r i n g . Aqueous solutions were not used due to the water-solubility of some derivatives and i n order to keep reaction conditions constant. (However, no substantial differences were observed when the complexation was carried out i n water.) After the desired times, the polysaccharides were fil t e r e d and washed (150 ml) before being dried in vacuo at 80°C for 24 hr. The samples were kept desiccated before elemental analysis. The copper determinations were obtained by n i t r i c acid digestion of the polysaccharide samples followed by atomic absorption measurements. 225 References 1. R. G. Schweiger, J. Org. Chem., Al, 90 (1976). 2. G. Holzwarth, Biochemistry, 19, A333 (1976). 3. R. L. Whistler and J. W. Marx, Methods Carbohydr. Chem., JS, 1A3 (1965). A. W. G. Struve and H. M. McConnell, Biochem. Biophys. Res. Commun., A9, 1631 (1972), H. W. Wien, J. D. Morrisett, and H. M. McConnell, Biochemistry, 11, 3707 (1972). 5. R. G. Schweiger, J. Org. Chem., 27_, 1786 (1962). 6. I. L. Andresen, T. J. Painter, and 0. Smidsr^d, Carbohydr. Res., 59, 563 (1977); and references cited therein. 7. H. Andresz, G. C. Richter, and B. Pfannemiiller, Makromol. Chem., 179, 301 (1978). 8. C. S. Lee and K. Maekawa, Agr. Biol. Chem., AO, 785 (1976). 9. Kelco Algin (Second Edn.), Kelco Co., San Diego, California, Al (1976). 10. P. T.Nevell, Methods Carbohydr. Chem., 3_, 16A (1973). 11. Dr. J. Defaye, private communication to Dr. L. D. Hall. 12. S. Hirano, R. Yamaguchi, and N. Matsuda, Biopolymers, 16, 1987 (1977); Int. J. Biochem., % 501 (1978). 13. S. Hirano, N. Matsuda, 0. Miura, and H. Iwaki, Carbohydr. Res., 71, 339 (1979). 1A. M. S. Masri, V. G. Randall, and W. L. Stanley, in Proc. First Intern. Conf. Chitin/Chitosan, (R. A. A. Muzzuralli and E. R. Pariser, eds.), 36A (1978). 15. J. W. Opie and J. L. Keen, Germ. Pat., 1, 262, 756 (1968); Chem. Abstr., 68, 88, 358k (1968). 16. G. Avigad, D. Amaral, C. Asensio, and B. L. Horecker, J. Biol. Chem., 237, 2736 (1962). 17. L. A. Nud'ga, E. A. Plisko, and S. N. Danilov, Zh. Obshch. Khim., A3, 2752 (1973), (2729 in transl.). 18. R. T r u j i l l o , Carbohydr. Res., A83 (1968). 19. S. Hirano, N. Matsuda, 0. Miura, and T. Tanaka, ibi d . , 71_, 3AA (1979). 226 APPENDIX 13 C nmr spectroscopy of natural polysaccharides has estblished i t s e l f as a very powerful tool in recent years. However, with the excep- tion of permethylated derivatives, modified polysaccharides have not yet been widely studied. Both native and modified derivatives are, unfortunately, not always amenable to analysis by conventional nmr techniques, e.g. for 6 1 13 xanthan gum (MW 10 ) no C nmr spectra have so far been observed due to 2 i t s extremely high solution viscosities. This Appendix contains the p r e l i - 13 minary results from C nmr experiments which were designed to remedy such situations. 13 In the f i r s t set of experiments we foud that a C nmr spectrum of xanthan gum in aqueous solution could be obtained using conditions (pulse vidth 32 us, ca. 144°- delay 2 s) which were essentially those for the spin- 3 echo technique. Figure A-1 illustrates the complex ( yet unassigned ) spectrum of xanthan gum which presumably derives only from the pendant, more mobile trisaccharide side-chains. 13 Another, very powerful recent development is C Magic Angle Spinning-Cross Polarization (MAS-CP) nmr , which allows for the analysis of solid materials. Figure A-2 demonstrates the enormous u t i l i t y of this high resolution technique for both native xanthan gum (unpurified Keltrol) and the octadecyl xanthan amide derivative whose sythesis was discussed in Chapter II-B. The spectrum of xanthan gum (Fig. A-2A) shows the carbonyl signals at 173 ppm, the anomeric signals (partially resolved) at 102 ppm, the overlapping signals from C2 to C5 of the various monosaccharide residues at 73 ppm, and the pyruvate methyl resonance 2 at 21 ppm. The spectrum of the amide (Fig. A-2B) reveals an \  228 0 2 0 0 ppm 100 13 Fig. A-2. C-MAS-CP nmr spectra of (A) xanthan gum (unpurified Keltrol), 10,000 scans, 2 s repetition, 0.5 ms contact time, 2 KHz spinning 10 G iH decoupling; (B) octadecyl xanthan amide, 100.mg sample, same conditions. Spectra were obtained on a Bruker CXP-200 spectrometer ( by Dr. S.Ganapathy). 229 additional resonance at 33 ppm which can be assigned to the methylene carbons of the hydrophobic side-chains. The spectrum required a total of three hours experimental time providing considerably more detail than the corresponding l 3C solution experiment in an equivalent period (using conventional nmr techniques). The MAS-CP method i s , of course, of particular importance for substances which are not soluble in common solvent systems, as exempli- fied here by the 1 3C nmr spectra of cellulose powder (Whatman CF11) and [N-(3-fluoro)-benzyl] chitosan (prepared analogously to the s a l i c y l - dimine chitosan derivatives in Chapter V) (Fig. A-3). The spectrum of the former (Fig. A-3a) appears to be intermediate to those of purely microcrystalline and amorphous cellulose samples"' providing support for the esr results (see II-B 3) which were indicative of heterogeneities in accessibility for this material. The aromatic signals of the chitosan derivative (Fig. A-3b) are.observed between 120 to 145 ppm and the N-methylene signal appears at 23 ppm. 230 200 100 0 ppm Fie A-3. 13C-MAS-CP nmr spectra of (A) cellulose (Whatman CF11), 10,000 scans, 2 s repetition, 0.5 ms conact time, 2 KHz spinning, 10,G lH decoupling; (B) [ N-(3-fluoro)-benzyl] chitosan, 20,000 scans, same conditions. 231 References 1. G. Holzwarth, Carbohydr. Res., 66, 173 (1978). 2. E. R. Morris, D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke, J. Moi. Biol., 110, 1 (1977); P. J. Garegg, P. E. Jannson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78, 127 (1980). 3. L. D. Hall and S. Sukumar, J. Magn. Res., .38, 559 (1980); and references therein. 4. J. Schaefer and E. 0. Stejskal,in Topics in Carbon-13 NMR Spectroscopy, G. Levy, ed., 283 (1979); W. E. Earl and D. L. Hart, Macromole- cules, 12, 762 (1979). 5. R. H. Atalla, J. C. Gast, D. W. Sindorf, V. J. Bartuska, and G. E. Maciel, J. Amer. Chem. Soc, 102, 3249 (1980).

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