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Chitosan modification : toward the rational tailoring of properties Holme, Kevin R. 1986

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CHITOSAN MODIFICATION: TOWARD THE RATIONAL TAILORING OF PROPERTIES By Kevin R. Holme B.Sc, Carleton University, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 198 6 © Kevin R. Holme, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or pu b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ABSTRACT The main objective of t h i s work was to develop a method whereby chitosan could be modified to give synthetic anal-ogues of branched polysaccharides. To t h i s end, a v a r i e t y of a l l y l glycosides were prepared ( 99-102. 105. 108.109 and 111) and reductively ozonolyzed, to give the acetaldehydo glycosides 112-119. These aldehydes were then reductively alkylated to chitosan (1), to give branched chitosan deriva-t i v e s (120-127) of the general structure 157. Pendant residues of a and ^9-glucopyranose, a and /^-D-galactopyranose, 2-acetamido-2-deoxy-D- a and jQ-glucopyranose, glucopyranuronic acid and /j}-D-lactose were incorporated by t h i s method, at various l e v e l s of s u b s t i t u t i o n . Rheological evaluations of these derivatives by steady-shear viscometry demonstrated a r e l a t i o n s h i p between the degree of s u b s t i t u t i o n and rheological properties, as well as the e f f e c t of branch s i z e and f u n c t i o n a l i t y on aqueous solu-t i o n properties. Importantly, many of the trends seen i n t h i s study are s i m i l a r to established explanations f o r the aqueous so l u t i o n properties of seed galactomannans. I t was also shown that i n t r i n s i c v i s c o s i t i e s of the derivatives were supportive of observations based on concentrated s o l u t i o n properties. Also, i t was demonstrated that these water soluble chitosan d e r i v a t i v e s interacted, sometimes i n a s y n e r g i s t i c manner, with xanthan gum solutions. A s i m i l a r route, involving the synthesis of 10-undece-nyl - /3~D-glycopyranosides (134 -136) , reductive ozonolysis and reductive amination to chitosan, provided combined hydropho-bic / h y d r o p h i l i c branched chitosan derivatives (140-142) of the general structure 158. This methodology was demonstrated with the 10 -undecenyl ^Q-D-glycosides of glucopyranose, g a l -actopyranose and lactose. Compounds 140a and 141a. bearing glucose and galactose pendant residues, showed uncommon ther-mally induced gelation properties i n d i l u t e aqueous acid sol u t i o n . This property was studied by -^H-nmr relaxation measurements and 1 3C-nmr spectroscopy. I t was found that a high degree of sub s t i t u t i o n was necessary for gel formation, and that the pendant sugar was required, but excess hydroph-i l i c i t y (such as the disaccharide branch, lactose) precluded gelation. In addition, a derivative (151) was prepared, which contained a metal-chelating moiety and a hydrophilic spacer group. This compound had substantial copper (II) binding capacity, and useful ion-exchange a b i l i t y . F i n a l l y , a c h i t o -san d e r i v a t i v e (156) was synthesized, bearing a pendant l-thio - /3-D-glucopyranose moiety, and was shown to be useful for the a f f i n i t y chromatographic p u r i f i c a t i o n of the enzyme ^S-glucos idase. -v-TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES i v LIST OF FIGURES V ACKNOWLE DGEMENTS X ABBREVIATIONS x i CHAPTER 1: INTRODUCTION 1 1.1 Overview 1 1.2 Background 7 1.2.1 Introduction to Polysaccharide Modification. 7 1.2.2 Synthesis and Modification of Amino Polysaccharides * 10 1.2.3 NMR Spectroscopy of Polysaccharides 37 1.2.4 Polysaccharides i n Solution 59 CHAPTER 2: BRANCHED CHITOSAN DERIVATIVES 81 2.1 Introduction 81 2.2 N-[2 1-O-(glvcopvranosyl)ethyl1chitosan  Derivatives 87 2.2.1 Synthesis and Characterization 87 2.2.2 Viscometry 102 2.2.3 Synergistic Interactions 132 2 . 3 N- [ 10 ' -0- (/3-D-glycopyranosyl) decyl ~| chitosan Derivatives 137 2.3.1 Synthesis and Characterization 137 2.3.2..NMR Investigations 147 2.3.3 Mixed Branch Chitosan Derivatives 152 2.4 Conclusion 155 CHAPTER 3: METAL CHELATING AND AFFINITY CONJUGATES OF CHITOSAN 157 3.1 Introduction 157 3.1.1 Metal Chelating Chitosan Derivative 157 3.1.2 Potential A f f i n i t y Chromatography Support... 158 3.2 Metal Chelating Chitosan Derivative 159 3.2.1 Synthesis 159 3.2.2 Characterization 161 3.2.3 pH T i t r a t i o n 164 3.2.4 Copper(II) Chelation 164 3.2.5 Viscometry 165 3.3 A f f i n i t y Chromatography Derivative 167 3.3.1 Synthesis and Characterization 167 3.3.2 Enzyme Binding Studies 168 CHAPTER 4: EXPERIMENTAL 172 4.1 General 172 4.1.1 Methods 172 4.1.2 NMR Spectroscopy 174 4.1.3 Materials 174 4.2 Experimental for Chapter 2 175 4.2.1 General Synthetic Procedures 175 4.2.2 Synthesis of A l l y l Glycosides 179 4.2.3 Synthesis of 10'-Undecenyl /3-D-Glycosides... 190 4.2.4 Synthesis of Branched Chitosan Derivatives.. 194 - v i i -4.2.5 Viscometry 206 4.2.6 I n t r i n s i c V i s c o s i t y 209 4.3 Experimental f o r Chapter 3 210 4.3.1 General Procedures 210 4.3.2 Synthesis of Chelating Chitosan Derivatives. 211 4.3.3 Synthesis of Thio Glycoside A f f i n i t y Conjugate and Precursors 218 4.3.4 Enzyme Studies 222 BI BLIOGRAPHY 225 APPENDIX A. . 240 - v i i i -LIST OF TABLES Page Table 1. 1H and 1 3C-nmr data f o r carbohydrates and common sub s t i t i t u e n t s 41 Table 2. 1 3C-nmr data for the a l l y l glycosides 90 Table 3. Description of N-[2 1-O-(D-glycopyranosyl) ethyl]chitosan derivatives 94 Table 4 . 1 3C-nmr chemical s h i f t data f o r branch residues of N-[2'-0-(D-glycopyranosyl)ethyl] chitosan derivatives 96 Table 5. Power-law parameters f o r 2.0% aqueous solutions of derivatives 120-127 104 Table 6. Comparison of molar and percent concentra-t i o n f o r branched chitosans 126 Table 7. I n t r i n i s i c v i s c o s i t i e s f o r some branched chitosan derivatives 131 Table 8. Qu a l i t a t i v e description of s y n e r g i s t i c mixtures 132 Table 9. Power-law parameters f o r s y n e r g i s t i c solutions 133 Table 10. 1 3C-nmr chemical s h i f t data f o r Undecenyl /3-D-gly cos ides 140 Table 11. Description of N-ClO'-O-f^-D-glycopyranosyl)-decyl] chitosan products 143 Table 12. 1 3C-nmr chemical s h i f t data f o r N- [ 10' -O- (/3-D-glycopyranosyl) decyl ] chitosan products 145 Table 13. •'•H-nmr T^-relaxation times f o r 141a 147 Table 14. Power-law parameters f o r 2.0% aqueous solutions of 144 and 126c. at 30°and 50°C... 154 Table 15. Characterization and copper (II) uptake of derivatives 151a. 15lb and chitosan......... 163 Table 16. Description of a f f i n i t y conjugates 169 - i x -LIST OF FIGURES Page Figure 1. Angles 4> and defining the g l y c o s i d i c linkage 45 Figure 2. 1 3C-nmr spectrum of Dextran B 742 48 Figure 3. a) Anomeric region of ^ -nmr spectra of mannans 49 b) 1 3C-nmr spectrum of yeast mannan 49 Figure 4. ^H-nmr spectra of amylopectin and panose... 51 Figure 5. 1 3C-nmr spectra of amylopectin and panose.. 52 Figure 6. ^-3C-nmr spectral region showing C-4 resonances of various galactomannans 53 Figure 7 . 1 3C-nmr spectrum of a branched amylose, glycogen and methyl j3-D-glucopyranoside.... 55 Figure 8 . Interactions of hydroxyl groups i n starch.. 56 Figure 9. ^-3C-nmr spectrum of lentinan gel and a low molecular weight f r a c t i o n 58 Figure 10. Schematic representation of g l y c o s i d i c linkage and polysaccharide conformation.... 61 Figure 11. Depiction of the "Egg Box" model f o r C a + 2 induced gelation of alginate... 63 Figure 12. a) Proposed interactions i n galactomannan solutions 65 b) Proposed interactions i n galactomannan solutions 65 Figure 13. Plot of the e f f e c t of d e - e s t e r i f i c a t i o n of pectin on the CD response upon C a + 2 induced gelation 66 Figure 14. Interactions i n polygalacturonate gels 66 Figure 15. Rheograms of id e a l i z e d pseudoplastic flow.. 73 Figure 16. Idealized power-law rheograms 74 Figure 17. Rheograms of time dependent flow 76 Figure 18. Stress and s t r a i n waves fo r viscous, e l a s t i c and v i s c o e l a s t i c materials 78 -x-Figure 19. Dynamic flow behaviour i n gels, concentrated solutions, and d i l u t e solutions 80 Figure 20. 100.6 MHz 1 3C-nmr spectrum of 121a and 121b 97 Figure 21. 100.6 MHz 1 3C-nmr spectrum of 125a and 125c 98 Figure 22. 100.6 MHz 1 3C-nmr spectrum of 126b and 126d 99 Figure 23. Rheograms of derivatives 125a-d on a r i t h -metic coordinates 106 Figure 24. Rheograms of derivatives 125a-d on logarithmic coordinates 107 Figure 25. Rheograms of 126a-d on arithmetic coordin-ates 108 Figure 26. Rheograms of 121a-c on arithmetic coordin-ates 109 Figure 27. Rheograms of derivatives 121a-c and 126a-d on logarithmic coordinates 110 Figure 28. Rheograms of deriv a t i v e 120a on l i n e a r and logarithmic axes I l l Figure 29. Rheograms of 122a on l i n e a r and logar-ithmic axes 112 Figure 30. Rheograms of 124a and 127c-e on l i n e a r axes 113 Figure 31. Rheograms of 123a-d on arithmetic axes 114 Figure 32. Rheograms of 123a-d, 124 and 127c-e on logarithmic coordinates 115 Figure 33. Rheograms of xanthan gum solutions on arithmetic coordinates 116 Figure 34. Rheograms of hydroxyethylcellulose (HEC) and sodium alginate (NaALG) solutions on arithmetic coordinates 117 Figure 35. Rheograms of xanthan, hydroxyethyl c e l l u l o s e and sodium alginate solutions on logarithmic axes 118 Figure 36. Rheograms of xanthan, sodium alginate 121c and 126c on l i n e a r axes 119 Figure 37. Rheograms of xanthan, sodium alginate and - x i -125d on arithmetic coordinates 120 Figure 38. a)Rheograms of 2.0% aqueous solutions of xanthan, sodium alginate, 121c and 126c.. b)Rheograms of 1.0% aqueous solutions of xanthan, sodium alginate and 125d on logarithmic coordinates 121 Figure 39. Idealized " i n t e r d i g i t i z a t i o n " i n t e r a c t i o n of chitosan derivatives 123 Figure 40. Idealized association of branched chitosan chains 124 Figure 41. Graph of n vs d.s. f o r neutral monosacch-aride branched derivatives 125 Figure 42. Rheograms of sy n e r g i s t i c mixtures on l i n e a r axes 134 Figure 43. Rheograms of sy n e r g i s t i c mixtures on logarithmic axes 135 Figure 44. Stacked p l o t of inversion recovery T x-relaxation -^H-nmr spectra f o r 14la 148 Figure 45. 100.6 MHz 1 3C-nmr spectra of 141a at 30 and 50° C 150 Figure 46. 100.6 MHz 1 3C-nmr spectra of 144 at 30 and 50° C 153 Figure 47. Rheograms of 144 and 126c on logarithmic axes...... 155 Figure 48. Rheograms of solutions of 151a i n water, and 1.0 mM Cu(II)S0 4 s o l u t i o n 166 Figure 49. E l u t i o n of jS-glucosidase from a f f i n i t y column 171 - x i i -ACKNOWLEDGEMENTS I would l i k e to thank my supervisor, Dr. L.D. H a l l , f or providing support and encouragement throughout t h i s work, and for introducing me to the f i e l d of polysaccharide chemistry. I must also acknowledge the members of "the group" f o r t h e i r valuable discussions. I extend my gratitude to Dr. S.G. Withers f o r discussions and collaboration, to Dr. G.G.S. Dutton and h i s research group f o r including me i n t h e i r weekly seminars, and to numerous other f a c u l t y and s t a f f i n the Chemistry Department. Also, I thank NSERC f o r f i n a n c i a l support. I am extremely indebted to Dr. M.A. Tung, of the Food Science Department, f o r i n s t r u c t i o n i n rheology and generous access to instrumentation, and to R.A. Speers f o r discussions and tec h n i c a l advice on rheometry. As well, a general thanks goes to other members of the Food Science Department f o r t h e i r help. I would l i k e to acknowledge the contribution of two NSERC summer students, L. Ivany and K. Shklanka, with whom I had the pleasure of working. I would l i k e to extend my sp e c i a l thanks to Dr. G.L. Jung, f o r her invaluable assistance, patience and encourage-ment throughout the production of t h i s t h e s i s , and to my parents, f o r t h e i r guidance and support. * * J x i i i ABBREVIATIONS Ac = acetyl A C 2 O = ac e t i c anhydide b = broad bz = benzene DCC = dicyclohexylcarbodiimide DMF = dimethylformamide DMSO = dimethylsulfoxide EDC = l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide FT = Fourier transform Gal = D-galactose Glc = D-glucose g l c = gas l i q u i d chromatography GlcA = D-glucuronic ac i d GlcNAc <= 2-acetamido-2-deoxy-D-glucose G I C N H 2 = 2-amino-2-deoxy-D-glucose HEC = hydroxyethylcellulose HOAc = a c e t i c a c i d i r = i n f r a r e d Lact = D-lactose m = multipl e t Me = methyl ms = mass spectroscopy Ms = methanesulfonyl (mesyl) NaALG = sodium alginate nmr — nuclear magnetic resonance xiv PC = phenylcarbamoyl PF = para-formaldehyde Ph = phenyl i-PrOH = iso-propanol pyr = pyridine q = quartet RaNi = Raney n i c k e l s = s i n g l e t t t r i p l e t THF = tetrahydrofuran t i c = t h i n layer chromatography TMS = tetramethy1si1ane Ts = E-toluenesulfonyl (tosyl) Tr = t r i t y l Xan xanthan gum - 1 -CHAPTER 1  INTRODUCTION 1.1 OVERVIEW Polysaccharides are ubiquitous i n nature and p a r t i c i p a t e i n many v i t a l b i o l o g i c a l systems and processes. 1' 2 Their ro l e s f a l l i nto two general c l a s s i f i c a t i o n s : (1) Structural, as exemplified by c e l l u l o s e , the main s t r u c t u r a l polysaccharide i n the plant world, and by c h i t i n , which serves as the main s t r u c t u r a l material i n crustacean s h e l l s and insect exoskeletons; and (2) functional, as with starch, the main energy storage material i n plants, and glycogen, which serves analogously i n mammals. These are but a few of the better known polysaccharides which occur i n nature. Myriad others e x i s t , 3 ' 4 whether they be plant exudates (gums), substances i n seaweeds (algal polysaccharides), or the exocell u l a r polysaccharides of bact e r i a . I t has long been of i n t e r e s t to learn more about the various r o l e s of polysaccharides i n nature. However i n recent years a separate, a l b e i t related aspect i n the study of carbohydrate polymers has attracted increasing attention from both the academic and i n d u s t r i a l sectors, and has led to a d i v e r s i t y of i n d u s t r i a l applications. Polysaccharides, being extremely abundant and often possessing desirable p r o p e r t i e s , 5 are i d e a l candidates for - 2 -use i n a v a r i e t y of applied a r e a s . 6 ' 7 Some of the major i n d u s t r i a l uses of these materials occur i n food and food processing, a g r i c u l t u r a l products, cosmetics, pharmaceuti-c a l s , paints, adhesives, paper making, mining aids, waste water treatment, and enhanced o i l recovery. They serve as thickening or g e l l i n g agents, suspending agents, lubricants and metal sequestrants, amongst other purposes. 6 As well, polysaccharides have found growing biochemical a p p l i c a t i o n i n the form of s o l i d phase polymeric support m a t e r i a l s 1 ' 8 ' 9 for ion exhange and a f f i n i t y chromatography, and enzyme and c e l l immobilizations. While many polysaccharides are i s o l a t e d from natural sources i n a r e a d i l y u t i l i z a b l e form, many are insoluble, i n t r a c t a b l e materials unsuitable f o r many applications. The f a c t that the very abundant and inexpensive substances, c e l l u l o s e , starch and c h i t i n f a l l into the l a t t e r category has stimulated research into methods fo r t h e i r modifica-t i o n . 1 0 ' 1 1 Since the turn of the century i n d u s t r i a l polysac-charide derivatives of c e l l u l o s e and starch have been known. Various methods fo r the chemical modification of polysaccha-r i d e s have been employed, 1 2' 1 3 t y p i c a l l y involving d e r i v a t i -zation of the polysaccharide hydroxyl groups under strongly a l k a l i n e conditions with reactive organic compounds. While control of the o v e r a l l degree of su b s t i t u t i o n (d.s., number of substituents per monosaccharide residue) i s possible, these reactions usually a f f o r d a random or " s t a t i s t i c a l " d i s t r i b u t i o n of derivatized s i t e s on the monosaccharide -3-r e s i d u e . 1 0 ' - 1 - 1 ' 1 4 In other words, such reactions display l i t t l e s i t e - s e l e c t i v i t y , which can be a major drawback for some applications. As a r e s u l t , over the l a s t 10-15 years there have been greater e f f o r t s to develop new d e r i v a t i z a t i o n methods which o f f e r more control over a l l aspects of the reaction. This requires that a c c e s s i b i l i t y and r e a c t i v i t y d i f f i c u l t i e s of these l a r g e l y i n t r a c t a b l e polymers be over-come or circumvented. The current i n t e r e s t i n the modification of polysaccharides f o r the preparation of " t a i l o r e d d e r i v a t i v e s " has given t h i s f i e l d great impetus, and indeed, novel methodologies 1 5 are now emerging which have s i g n i f i c a n t p o t e n t i a l . However, there must be a concurrent e f f o r t d irected toward the characterization and evaluation of these complex materials, both s t r u c t u r a l l y and f u n c t i o n a l l y , leading to an understanding of the i n t e r - r e l a t i o n s h i p of structure and function. A large number of applications of polysaccharides make use of t h e i r properties i n aqueous so l u t i o n ( i . e . thickeners, g e l l i n g agents, suspending agents and l u b r i c a n t s ) , and not s u r p r i s i n g l y these properties have been subject to intensive s t u d y . 5 ' 1 6 Such studies require consideration of the d i v e r s i t y of possible sequences, of the multitude of conformational states available to a p a r t i c u l a r polymer sequence, as well as the various i n t e r - and intra-molecular associations which may o c c u r . 1 7 In l i g h t of other factors such as s t r u c t u r a l i r r e g u l a r i t i e s , i n the form of minor - 4 -differences i n sequences or s u b s t i t u t i o n pattern (polydispersity) or molecular weight inhomogeneity (polymolecularity), i t i s c l e a r that such investigations are inherently complex. In recent years there have been s i g n i f i c a n t advances made i n determining the primary structure of complex polysaccharide systems, l a r g e l y as a r e s u l t of new chemical and physical m e t h o d o l o g i e s . 1 ' 1 8 * 1 9 More importantly, modern techniques are providing a means to gain i n s i g h t into the hydrated and condensed states of these hydrocolloid polymers. 1 7 However, the r e l a t i o n s h i p between structure and properties, from a p r e d i c t i v e standpoint, remains elusive. To date, i t has been possible only to correlate the properties of polysaccharides that are related through e i t h e r sequence or conformation, both of which may produce i n t r i n s i c physical p r o p e r t i e s . 1 7 The main obstacles to understanding these re l a t i o n s h i p s have been the lack of a n a l y t i c a l methodology, the u n a v a i l a b i l i t y of systematically varied substrates f o r examination, and the o v e r a l l complexity of the systems of i n t e r e s t . This second point provides a locus of i n t e r e s t f o r chemists working i n polysaccharide modification, who could develop methods fo r preparing an array of related derivatives for structure/property investigations. The " s t a t i s t i c a l " methods avail a b l e f o r polysaccharide modification r e s u l t i n g i n complex, random substituent d i s t r i b u t i o n s are unsuitable f o r t h i s purpose. The development of s p e c i f i c modifications -5-to control product composition i s necessary f o r studies systematically r e l a t i n g structure to macroscopic properties. Some requirements for s i t e s e l e c t i v e control of the modification of polysaccharides are: (1) the presence of a s i t e of unique r e a c t i v i t y ; and (2) mild reaction conditions suitable f o r both polysaccharide and ligand. The primary objective of t h i s work was to incorporate chemical modification, spectroscopic characterisation and rheological evaluation into a concerted study of branched polysaccharides. As w i l l be seen, excellent examples of studies i n each of these areas e x i s t i n the current l i t e r a -ture; however, remarkably l i t t l e work has been addressed toward combined studies i n these areas. The f i r s t task was therefore to develop methodology whereby polysaccharides could be chemically modified to produce materials with s p e c i f i c , desired properties. We required that some degree of s e l e c t i v i t y be inherent i n t h i s methodology, such that a systematically varied "family" of branched polysaccharide derivatives could be obtained. We wished to prepare deriva-t i v e s s i m i l a r i n composition and structure to n a t u r a l l y occurring branched polysaccharides, which are already known to have i n t e r e s t i n g rheological p r o p e r t i e s . 5 ' 1 7 And f i n a l l y , we wished to investigate the r e l a t i o n s h i p between the struc-t u r a l features of the derivatives prepared and t h e i r macros-copic s o l u t i o n properties. In addition, while the potential immunological a p p l i c a t i o n s 1 ' 2 0 ' 2 1 stemming from t h i s methodology w i l l not be d i r e c t l y addressed i n t h i s work, i t -6-was intended that the strategy used would be s u f f i c i e n t l y general that i t could f i n d application i n that area. With these goals i n mind, we have .chosen the amino polysaccharide chitosan (1), as an exemplar. I t i s a l i n e a r (1+4)-linked homopolymer of 2-amino-2-deoxy-j3~D-glucopyranose, and i t i s derived from c h i t i n . C h i t i n (2), a (1+4)-linked 2-acetamido-2-deoxy-jS-D-glucopyranose polymer, i s a v a i l a b l e i n large quantities from Crustacea s h e l l wastes, and has found use i n numerous i n d u s t r i a l 2 2 * 2 3 and biomedi-c a l 2 4 " 2 6 applications. The free amine f u n c t i o n a l i t y on chitosan renders i t an i d e a l substrate f o r high y i e l d i n g s i t e - s e l e c t i v e reactions under mild, aqueous, a l k y l a t i o n and acylation c o n d i t i o n s . 2 7 This work w i l l describe the use of the reductive amination reaction to transform chitosan into metal chelating derivatives, a f f i n i t y chromatography sup-ports, v i s c o s i t y modifiers and g e l l i n g agents. I t was intended that the preparation of a s e r i e s of synthetic branched polysaccharides would provide some insight into the e f f e c t s of branching and branch composition on rheological properties. 1 2 - 7 -1 . 2 BACKGROUND 1 . 2 . 1 Introduction to Polysaccharide Modification The modification of polysaccharides has been employed for over a century and numerous monographs and review a r t i c l e s 7 ' 1 0 - 1 4 / 2 8 - 3 1 have appeared on t h i s t o p i c . T r a d i t i o n a l l y the well-known and abundant polysaccharides c e l l u l o s e 7 ' 1 0 " 1 4 ' 2 8 and s t a r c h , 7 ' 1 3 ' 2 9 ' 3 0 were the substrates of i n t e r e s t . Typical modification reactions have involved r e l a t i v e l y harsh treatment of the polymer with reagents, often under heterogeneous conditions, r e s u l t i n g i n derivatives with a " s t a t i s t i c a l " substituent d i s t r i b u t i o n . 1 0 ' 1 1 ' 1 4 Some of the well-known modifications f o r celluose and starch include e t h e r i f i c a t i o n , e s t e r i f i c a t i o n and oxidation. These methods have been used to prepare polysaccharides for various i n d u s t r i a l applications, and they have been described i n d e t a i l i n numerous other a r t i c l e s . 7 ' 1 0 - 1 4 > 2 8 - 3 1 some of the more common e t h e r i f i c a t i o n products include the hydrox-yethyl, hydroxypropyl, methyl and carboxymethyl ethers. These are produced by heterogeneous reaction of the polysaccharide (celluose or starch) under a l k a l i n e conditions with the respective a l k y l epoxides and a l k y l halides. These treatments give products with s t a t i s t i c a l substituent d i s t r i b u t i o n s , as dictated by the r e l a t i v e r e a c t i v i t i e s and a c c e s s i b i l i t i e s of the polymer hydroxyl g r o u p s . 1 0 ' 1 1 ' 1 4 A large amount of work has been done with c e l l u l o s e to determine su b s t i t u t i o n - 8 -patterns ob.tained with various reagents and s o l v e n t s , 1 0 ' 1 1 ' 1 4 and to explain these i n terms of i t s s o l i d state s t r u c t u r e . 3 2 Much of the d i f f i c u l t y i n achieving s e l e c t i v i t y with c e l l u -lose modifications has stemmed from the necessity of heter-ogeneous reaction conditions. While t h i s i s not the case for a l l polysaccharides, hydroxyl group a c c e s s i b i l i t y , of both primary and secondary positions, i s affected by the molecular associations of the polymer i n solution, often masking the inherent r e a c t i v i t y differences of the i n d i v i d u a l s i t e s . Hence, the r e s u l t i s l i m i t e d and unpredictable r e a c t i v i t y of the hydroxyl groups, s i m i l a r to the more extreme case of c e l l u l o s e . Well-known e s t e r i f i c a t i o n products are c e l l u l o s e acetate, n i t r a t e and xanthate. These c e l l u l o s e esters are used i n va r i e t y of ind u s t r i e s . The former two are used i n t e x t i l e manufacture, and c e l l u l o s e acetate i s also an important f i l m and packaging material. Cellulose n i t r a t e i s a commonly used explosive material. Preparing highly and f u l l y substituted esters i s l e s s d i f f i c u l t than f o r c e l l u l o s e ethers, but the sub s t i t u t i o n pattern f o r p a r t i a l l y e s t e r i f i e d derivatives remains s t a t i s t i c a l . The major oxidative treatments which have been applied to c e l l u l o s e 1 2 and/or s t a r c h 1 3 include hypochlorite, hypobromite, nitrogen dioxide, chromic acid, oxygen and periodate oxidations. These conditions/treatments give a va r i e t y of oxy-cellulose or oxy-starch derivatives respectively, containing aldehyde, ketone and carboxylate - 9 -groups. I t i s not within the scope of t h i s discussion to go into the h i s t o r i c a l development of these d e r i v a t i z a t i o n methods, which have already been well, reviewed i n the literature.12,13,31 The advancements i n polysaccharide modification are attri b u t e d to several factors. The a v a i l a b i l i t y of new reagents, c a t a l y s t s and solvents have had a profound, influence. Also, the greater v a r i e t y of natural polysaccharides employed as substrates has increased the array of reactions and possible products. However, the introduction of new strategies, which generally involve some degree of s e l e c t i v i t y , has created a much greater p o t e n t i a l fo r the generation of " t a i l o r e d " d e r i v a t i v e s . 1 5 Also, the many new areas i n which polysaccharides have found ap p l i c a t i o n ( i . e . biochemical p u r i f i c a t i o n s , s o l i d phase chromatography media, and biomolecule immobilizations), have provided a new range of target materials and increased m o t i v a t i o n . 1 * 8 ' 9 ' 2 8 ' 3 3 ' 3 4 S i t e - s p e c i f i c modification of polysaccharide carboxyl groups are well-known. 1 5 However, u n t i l the recent renaissance i n polysaccharide modification research, r e l a t i v e l y few methods for s i t e - s e l e c t i v e chemical d e r i v a t i z a t i o n of polysaccharides existed. In the l a s t 15 years, considerable attention has been directed toward amine-containing polysaccharides such as chitosan, a (1+4)-linked 2-amino-2-deoxy-^Q-D-glucopyranose homopolymer, as substrates for s e l e c t i v e m o d i f i c a t i o n s . 2 3 Such inter e s t has l e d to the development of new approaches f o r the modifi-- l o -cation of amine functions, and perhaps of greater importance, methods f o r s i t e - s e l e c t i v e preparations of synthetic amino polysaccharides. 1 5 A much larger choice of substrates bearing amino groups i s thus available f o r application i n " t a i l o r e d " modification strategies. In the following section, a review of the e x i s t i n g methods for the preparation and modification of amino polysaccharides w i l l be undertaken. 1 . 2 . 2 Synthesis and Modification of Amino Polysaccharides One of the most popular methods of introducing an amine function involves the intermediary formation of a polysaccha-r i d e bearing carbonyl functions through oxidation. Hence, methods f o r the s e l e c t i v e oxidation of polysaccharides are important i n amino polysaccharide synthesis, as well as f o r isotope l a b e l l i n g , epimerizations, and p o t e n t i a l l y f o r reaction with Grignard and Wit t i g reagents. Normally, keto groups are introduced at secondary positions while aldehydes or carboxylates r e s u l t at primary centers; however, over-oxi-dation can cause cleavage of the monosaccharide unit, and the formation of dialdehydes or d i c a r b o x y l a t e s . 3 5 In most instances t h i s i s undesirable as the i n t e g r i t y of the poly-saccharide has been decreased. Nevertheless, products of t h i s sort have found application as t e x t i l e and paper s i z e s . 1 3 Fortunately, while many oxidations work i n a non-selective or over-oxidative manner, mild reagents and strategies involving protecting groups have provided ways f o r overcoming some of the problems of polysaccharide oxidations. Scheme 1 -12-The oxidation of primary C-6 positions to an aldehyde occurs under many oxidative conditions. However, the aldehyde usually r a p i d l y oxidizes to the carboxylate under most aqueous conditions. Selective methods, t y p i c a l l y involving multistep procedures, have been developed f o r preparing 6-aldehydo-cellulose 9. 3 6' 3 7 For example, as shown by Scheme 1, the 6-0-p_-tolylsulfonyl d e r i v a t i v e 2 was converted into the azide 8, which was subsequently photolyzed to the aldehyde 9_.36 S i m i l a r l y , a "one-pot" preparation of a (a) i ) M s C l , DMF i i ) Na 2C0 3 > H 20 (b ) NaN^ ( c ) hV Scheme 2 -13-6-chloro-6-deoxy-cellulose d e r i v a t i v e H , followed by n u c l e o p h i l i c s u b s t i t u t i o n by azide ion and photolysis led to the 6-aldehydo product (Scheme 2) having d.s. values of 0.03-0.45, with greater s e l e c t i v i t y . 3 7 While none of the 6-aldehydo-cellulose derivatives have been converted to the corresponding 6-amino-celluloses, there i s c e r t a i n l y poten-t i a l f o r t h i s transformation. Scheme 3 outlines the analogous preparation of the 6-amino- and 6-aldehydo-cyclcodextrin compounds 16 and 12 from the s e l e c t i v e l y tosylated cyclcodextrin 2 4 . 3 8 » 3 9 Similar oxidation procedures have been applied to amylose. 4 0 Unfortunately, these procedures are i n v a r i a b l y accompanied by mild to appreciable polymer hydro-l y s i s . The enzyme galactose oxidase has been employed to generate s p e c i f i c a l l y the C-6 aldehyde moiety i n galactose residues of p o l y s a c c h a r i d e s . 4 1 ' 4 2 This enzymatic oxidation has been applied to a g a r o s e , 4 3 ' 4 4 locust bean gum 4 5 and guar gum.41,45-47 I n the l a t t e r case, the 6-aldehydo-guar 19.was reductively aminated to provide the amine-containing derivatives 20-25 (Scheme 4 ) 4 5 bearing a v a r i e t y of functional groups. The enzyme appears to be i n h i b i t e d by 4-0- and 3-0-substituted galactose residues, and by 2-amino-2-deoxy-D-galactose. 4 1' 4 6 The oxidative e f f i c i e n c y i s t y p i c a l l y quite high (60-90%). Treatment of the aldehyde with -16-aqueous bromine has provided 26,with a D-galacturonic acid branch residue on the mannan backbone . 4 5 The oxidation of secondary hydroxyl groups of polysaccharides, to keto- or glycosylulose residues, has been accomplished using the dimethyl su l f o x i d e / a c e t i c anhydride (DMSO/Ac20) reagent. 4 8 The t r i t y l ether i s usually employed as a C-6 protecting group, to prevent C-6 oxidation. For example, both 6-0-trityl-amylose 27, and 6 - 0 - t r i t y l - c e l l u l o s e 4, gave predominantly the respective 2-keto products upon treatment with DMSO/Ac20 (Scheme 5 ) . 4 9 - 5 1 Native c e l l u l o s e 3, under the same conditions gave mixtures of 2-oxy, 3-oxy and 2,3-dioxy r e s i d u e s . 5 2 The 3-oxy product 30 was obtained e x c l u s i v e l y i n 60-70% y i e l d when the reaction was performed using dimethyl sulfoxide/paraformaldehyde (DMSO/PF) as the solvent system (Scheme 6). 5 3 * 5 4 s i m i l a r oxidations of amylose (32.) gave predominantly the 3-oxy residue 33, with 10% of the 2-keto product 34.. Interestingly, i t was found that 6-O-trityl-amylose gave exclusively the C-2 oxidized - 1 7 -(a) DMSO/Ac 20 Scheme 5 product 23. i n the DMSO/Ac20/PF system, while 6 - 0 - t r i t y l -c e l l u l o s e yielded a mixture of 2-oxy 29 (56%) and 3-oxy 31. (36%) r e s i d u e s . 5 4 Analogously, 6-0-acetyl-amylose gave 56% and 30% of the 2- and 3-oxy monosaccharide residues respec-t i v e l y . Apparently, the bulkiness of the 6-0 substitutent influences the s i t e of oxidation, tending to promote C-2 oxidation. Selective oxidation of the C-3 p o s i t i o n of c e l l u -lose and amylose with DMSO/AC2O/PF, i s considered to be due -18--19-to r e v e r s i b l e formation of covalent hydroxymethyl and poly(oxymethylene)ol groups at 0-2 and 0-6. 5 3 Thus, when 6 - 0 - t r i t y l derivatives are treated with t h i s reagent, i n s i t u protection of 0-2 occurs, thereby s h i f t i n g s e l e c t i v i t y from 0-2 to 0-3. The Pfitzner-Moffat r e a g e n t 4 8 has been reported to achieve s i m i l a r oxidations and has been applied to c e l l u -l o s e . 5 2 The 2-oxy-amylose deriv a t i v e £8 has been further elaborated, v i a oximation, reduction and d e t r i t y l a t i o n (Scheme 7) to give 2-amino-amylose 3_6 with d.s. 0.8, and predominantly the D-gluco c o n f i g u r a t i o n . 4 9 Other workers have (a) DMS0/Ac20 (b) H0NH2C1 (c) LiAlH^, THF Scheme 7 -20-(a-) NaCNBH3, amine Scheme 8 -21-also applied t h i s oximation/reduction sequence i n 2-amino-polysaccharide s y n t h e s e s . 5 5 * 5 6 The 2- and 3-keto-cellulose derivatives 30 and 32, have been reductively aminated to provide the 2-amino- and 3-amino-cellulose derivatives 38-41 seen seen i n Scheme 8. 5 7 * 5 8 The reaction of glucosamine with 37 gave the (3-»2 •) -amine linked 4J., a unique branched polysaccharide. Dextran, a branched polysaccharide with a (1+6)-a-D-glucopyranose main chain with the 0-6 p o s i t i o n blocked, i s an i d e a l candidate f o r s e l e c t i v e oxidation of secondary centers. The DMSO/AC2O reagent, when applied to dextran (Eq. 1), gave mainly the 3-keto de r i v a t i v e 43..59 (a) DMS0/Ac20 Aqueous bromine 6 0 i s known to oxidize polysaccharides, and has been used to prepare dextran derivatives having 2-oxy (44) and 4-oxy (4J5) f u n c t i o n a l i t i e s i n a 0.85:1.00 r a t i o (Scheme 9 ) . 6 1 ' 6 2 These p a r t i a l l y oxidized dextrans have been reductively aminated with alkylamines and albumin . Unfortu-nately, a c i d i c products a r i s i n g from r i n g cleavage made up 11% of the product mixture when 1.0 equivalent of oxidant per R a - N r U C H ^CHs b - N H ( C H 2 ) 6 N H 2 C —NH—Alb. (a) Br 2/H 20 (b) NaCNBH^» amine S c h e m e 9 - 2 3 -residue was used. The use of borate, a c i s - d i o l complexing agent, 6 3 i n conjunction with aqueous bromine oxidation has been employed i n the oxidation of T-40 d e x t r a n . 6 4 I t was found that i n the presence of borate the degree of oxidation increased to 65%, from 43% without borate, but with no change i n d i s t r i b u t i o n of ketone groups. Also the extent of over-oxidation was reduced from 11% to 3%. S i m i l a r l y , Sepharose™ (cross-linked agarose) has been treated with aqueous bromine to give residues having mainly the 4-keto f u n c t i o n a l i t y . This was then reductively aminated with 1-aminodecane, 1,6-diaminohexane and a l b u m i n . 6 1 ' 6 5 Cellulose treated under s i m i l a r conditions showed only a low degree of oxidation at the C-2 and C-3 p o s i t i o n s . 6 5 ' 6 6 The heparin analogue 52 has been prepared from p a r t i a l l y reduced (50%) a l g i n i c acid 49. as outlined i n Scheme 10, by s e l e c t i v e aqueous bromine oxidation, reductive amination with ammonium acetate, and s u l f a t i o n . 6 7 Substantial depolymerization of the product was noted. Oxidations have been performed on amine-containing polysaccharides i n order to generate carboxylates at C-6. For example, as depicted i n Scheme 11, 2-amino-6-0-trityl-amylose (53), was converted into the heparin analogue 55 having 46% carboxylates at C-6 . 5 6 S i m i l a r l y , chitosan (l) has been treated with p e r c h l o r i c acid, chromium t r i o x i d e and subsequently chlorosulfonic acid, to give the heparin anal-ogue 57 (Scheme 1 2 ) . 6 8 Interestingly, the perchlorate s a l t acted as a bulky group, s t e r i c a l l y l i m i t i n g oxidation at C-3. -24-Scheme 10 -25-TrO 27 n TrO 35 •n 55 R, = H,COCF s R 2 = SO s Na (a) i ) A c 2 0 , pyr i i ) HONH 2 > pyr (b) L i A l H ^ (c) i ) ( C F 3 C O ) 2 0 , pyr i i ) H C l , CHC1 3 (d) i ) 0 2 / P t i i ) H 3 0 + i i i ) OH" i v ) S 0 3 > pyr v) C1S0 3 H, pyr (e) HCl Scheme 11 57 (a) i ) HC10 A i i ) C r 0 3 (b) i ) C1S0 3H, pyr i i ) NaOH Scheme 12 C e l l u l o s e and amylose derivatives bearing 6-amino moieties have been prepared i n low y i e l d s by routes s i m i l a r to those f o r 6-aldehydo-cellulose. Scheme 13 outlines a route that has been applied to both c e l l u l o s e 6 9 and amylose, 7 0 i n which the 2,3-di-0-phenylcarbamoyl-6-0-p_-tolylsulfonyl de r i v a t i v e 58 was converted into the amine 61 v i a azide formation. Amino groups have been introduced at C-6 of - 2 7 -(a) NaN 3 , DMSO (b) L i A l H ^ , THF (c) NaOMe/MeOH Scheme 13 amylose by treatment with s u l f u r y l chloride to give the 6-chloro d e r i v a t i v e 62, followed by hydrazinolysis and reduction to give 63 (Scheme 14). 7 l The previous section i l l u s t r a t e d the many approaches that have been employed to prepare a d i v e r s i t y of synthetic amino polysaccharide derivatives. One of the main drawbacks to many of the procedures was concomitant depolymerization. The use of natu r a l l y occuring amino polysaccharides as 28-Scheme 14 substrates f o r mild chemical modification has thus become a favored method f o r preparing functionalized amino polysaccha-r i d e s without compromising molecular weight. Modified amino-polysaccharides have been prepared by N-alkylation and N-acylation of natural amine-containing polysaccharides. C h i t i n (2), a (1+4)-linked 2-acetami-do-2-deoxy-/3-D-glucopyranose polymer ( with 15% 2-ami-no-2-deoxy groups), i s the most abundant amino polysaccharide , and the second most abundant organic polymer, i n n a t u r e . 2 2 I t i s e a s i l y N-deacetylated to provide the 2-amino-2-deoxy-jS- (1+4) -D-glucopyranose homopolymer chitosan ( i ) , an i d e a l substrate for chemical modification. Both of these materials have attracted substantial i n t e r e s t i n the form of review a r t i c l e s and monographs which have focussed on t h e i r diverse and commercially useful p r o p e r t i e s . 2 2 - 2 6 The added advantage of f a c i l e chemical modification has stimu-l a t e d researchers to delve into " t a i l o r i n g " the properties of these r e l a t i v e l y i n t r a c t a b l e materials and expand the poten-t i a l i n d u s t r i a l u t i l i t y of these polysaccharides. As such, chitosan has been employed extensively as a substrate f o r N-acylation and N-alkylation. Insoluble i n water and most organic solvents, chitosan i s r e a d i l y s o l u b i l i z e d i n d i l u t e aqueous organic acids (e.g. acet i c , formic, o x a l i c , e t c . ) , i n which most of i t s reactions are conducted. These solvent systems give c l e a r , viscous chitosan solutions suitable f o r homogeneous chemical modification. Sel e c t i v e N-acylations have been performed on chitosan with a v a r i e t y of a r y l and a l k y l carboxylic a n h y d r i d e s . 7 2 " 8 0 These reactions were done (in 2-10% aqueous a c e t i c acid) with 2-3 molar equivalents of anhydride to give products 64a-c and 65a-n. with high d.s. (0.8-1.0) i n good y i e l d s (77-96%), as shown i n Eq.2. Succinylation of chitosan provided products -30-1 (a) H0Ac/Me0H/H 20, (RCO) 20 64,65 _B 6 5 a H b C H 3 C - k < C H 2 ) N C H 3 n= 1,2,4,6,8,10,12,14,16 ] C H 2 C I m C H 2 C H 2 C 0 2 H n u -31-with d.s. 0.2-0.60, with the higher d.s. samples being w a t e r - s o l u b l e . 7 4 ' 8 1 Cross-linking (0.5-1.0%) of these derivatives produced transparent gels su i t a b l e f o r enzyme immobilization matrices. An N-2'-acetoxybenzoyl (aspirin) d e r i v a t i v e 64b (d.s. 0.65), obtained by N-acylation of chitosan with 2-acetoxybenzoic anhydride has been evaluated as a p o t e n t i a l drug delivery and co n t r o l l e d release system. 8 2 The N-alkylation of chitosan has received much attention over the l a s t 10 years. These studies are generally categorized i n two ways: (1) involving reaction with a l k y l or a r y l haiides, and (2) by reductive amination with aldehydes or ketones. The former has been used to prepare quaternary alkylammonium derivatives from regenerated chitosan and a l k y l iodides i n the presence of pyridine or t r i e t h y l a m i n e . 8 3 The tri-N-alkylammonium iodide s a l t s of chitosan are water-soluble, with d.s. values ranging from 0.52-0.78. A N,N-dimethyl-N-hydroxyethyl-chitosan s a l t was obtained by reaction of chitosan with methyl iodide and ethylene o x i d e . 8 4 The reaction of amines with carbonyls i s well known. 8 5 The i n i t i a l equilibrium y i e l d i n g the imine or S c h i f f base, i s driven to completion upon addition of borohydride 7 6 or other reducing agents, which transform the imine i n t o the corresponding substituted amine. This reaction has found extensive a p p l i c a t i o n to chitosan, affording derivatives which vary i n f u n c t i o n a l i t y and properties. In some cases, i t i s of i n t e r e s t to i s o l a t e the S c h i f f base de r i v a t i v e rather than the reduced amine. N-Alkylidene -32-and N-arylidene derivatives of chitosan, 66a-q. have been reportedly derived from formaldehyde and a v a r i e t y of a l k y l 8 7 and a r y l 8 7 - 8 9 aldehydes, some examples of which are given i n Eq. 3. These derivatives have served as the protected forms of the chitosan amine moiety during O-alkylations and O-acetylations. T y p i c a l l y , these N-alkylidene and N-arylidene adducts are obtained as gels from reaction i n methanolic a c e t i c acid. These gels are insoluble i n water and organic (a) H0Ac/Me0H/H90, RCHO - 3 3 -solvents, although i n the l a t t e r , some N-arylidene products do s w e l l . 7 8 These materials are of i n t e r e s t f o r t h e i r membrane-forming a b i l i t y and f o r t h e i r porous ul t r a s t r u c t u r e , a desirable a t t r i b u t e for gel f i l t r a t i o n a p p l i c a t i o n s . 9 0 ' 9 1 Reductive a l k y l a t i o n s have the advantage of affording products of greater h y d r o l y t i c s t a b i l i t y than the corresponding imine, and are thus of greater a p p l i c a b i l i t y f o r many end uses. T y p i c a l l y , sodium cyanoborohydride i s the reducing agent of choice, due to i t s low r e a c t i v i t y toward aldehydes and ketones, and s t a b i l i t y i n aqueous reaction media. 8 6 However, hydrogen/Raney n i c k e l (H 2/RaNi) treatment i s also s u i t a b l e 8 5 and reacts more quickly. Sodium cyanoboro-hydride has been used to prepare a wide range of N-alkyl chitosan compounds, with v i r t u a l l y no depolymerization. 1 5 A v a r i e t y of a l k y l and a r y l aldehydes have been reacted using t h i s procedure. P a r t i c u l a r attention has been centered on the attachment of functionalized molecules to chitosan i n order to generate or enhance s p e c i f i c properties. For example, Eq. 4 shows that s a l i c y l a l d e h y d e , 9 2 o-phthalaldehyde, 9 3 g l y o x y l i c a c i d 9 4 and ascorbic a c i d 9 5 have been reductively alkylated to chitosan to produce, respectively, compounds 67-70. a l l of which demonstrated substantial metal-chelating capacity . A v a r i e t y of carbohydrate molecules have been attached to c h i t o s a n , 9 6 ' 9 7 giving the branched chain derivatives 71-76 (d.s. 0.10-0.97) amongst others. In the case of 7_6, i n t e r e s t -ing rheological properties were observed. 9 8 I t was demon-strated by N-acetylation of 76 to give 7_7, and by a l k y l a t i o n 7 0 -35-36-of 76. with propanal to y i e l d 78, that properties such as s o l u b i l i t y and hydrophobicity could be manipulated by prepar-ing "mixed" d e r i v a t i v e s . 9 6 In the same report, streptomycin s u l f a t e was s i m i l a r l y coupled to chitosan as was a selec-t i v e l y oxidized cyclodextrin, affording conjugates with p o t e n t i a l use i n drug delivery and drug c a r r i e r s . Branch copolymers of chitosan formed by reductive amination with T-10 dextran (d.s. 0.15) 9 6 and an aldehyde d e r i v a t i v e of polyethylene g l y c o l 9 3 (MW 8,000) have also been described. 76-78 R 76 H 77 78 O H -37-1 .2 .3 Kmr Spectroscopy of Polysaccharides A current trend associated with s t r u c t u r a l studies of polysaccharides revolves around the use of spectroscopic methods to probe both structure and molecular dynamics. 1 8 Spectroscopic methods can be used on both i n t a c t species or i n conjugation with wet chemical and chromatographic tech-niques. Ultimately, one would l i k e to determine the struc-ture, de novo, of i n t a c t polymers with minimal chemical manipulation. Some of the various spectroscopic techniques that have been used i n polysaccharide studies are i r , ORD/CD, •^ -H-nrnr and 1 3C-nmr spectroscopy. The l a t t e r two, proton and carbon-13 nuclear magnetic resonance spectroscopy, have become extremely valuable i n the l a s t ten years, as evidenced by the numerous recent reviews of t h e i r a p p l i c a t i o n to o l i g o 9 9 " 1 0 1 and p o l y s a c c h a r i d e s , 1 8 ' 1 0 2 " 1 0 4 i n both s t r u c t u r a l and physicochemical investigations. Because these two spec-troscopic t o o l s have become invaluable i n the area of poly-saccharide studies, a b r i e f overview i s i n order. Nuclear magnetic resonance spectroscopy has a number of inherent features which make i t p a r t i c u l a r l y indispensable i n polysaccharide investigations. The parameters of chemical s h i f t (S), coupling constants (J), and relaxation times (T^ and T 2) impart information about the chemical structure and i d e n t i t y of the carbohydrate residues present, as well as conformational and dynamic aspects of the system. There i s a large volume of l i t e r a t u r e dealing s p e c i f i c a l l y with these parameters as they apply to the study of carbohy--38-d r a t e s . 1 8 » 9 9 - 1 0 5 Nmr spectra are usually obtained on samples i n s o l u t i o n ; however, i n the l a s t decade i t has become possible to perform routine nmr experiments on samples i n the s o l i d s t a t e 1 0 5 using the 1 3 C - c r o s s polarization/magic angle spinning ( 1 3C CP/MAS) experiment. This advancement has greatly enhanced the p o t e n t i a l of nmr i n the study of i n t r a c t a b l e materials. These factors, coupled with technolog-i c a l advances i n the design of nmr instrumentation and computerization make nmr an invaluable spectroscopic t o o l i n the study of complex carbohydrate polymers. The discussion presented herein w i l l deal s o l e l y with s o l u t i o n state nmr spectroscopy. Before a discussion of and 1 3C-nmr spectroscopic a p p l i c a t i o n to polysaccharides i s undertaken, c e r t a i n factors which a f f e c t the a c q u i s i t i o n of high resolution spectra ( for 1H i n p a r t i c u l a r ) must be addressed. These factors are line-broadening of the signals and interference of exchange-able protons (N-H_, 0-H). T y p i c a l l y , solutions of polysaccha-rides are prepared i n deuterium oxide, as opposed to water, thereby removing the s i g n a l from 0-H and N-H protons. However, an HOD peak (S~4.8 ppm) a r i s e s from exchange, and may i n t e r f e r e with proton signals of the sample. Also, poly-saccharides often contain water of hydration, which further increases the HOD s i g n a l . To minimize t h i s interference i t i s prudent to subject the sample to deuterium exchange, i . e . d i s s o l u t i o n i n D 20 and l y o p h i l i z a t i o n , repeatedly. Generally, t h i s procedure diminishes the HOD signal s u f f i c i e n t l y to give -39-an unobscured spectrum of the sample of i n t e r e s t . Other methods e x i s t f o r minimizing the interference of the HOD signa l i n FT-nmr experiments. 1 0 7 For example, saturation decoupling takes advantage of the d i f f e r e n t i a l relaxation of solvent (Ti of H 20 > 2 s) and polymer (T± < 0.5 s) protons. Thus a suit a b l e pulse sequence can l a r g e l y remove the i n t e r -f e r i n g resonance, although spurious peaks may r e s u l t . Line-broadening i s a manifestation of the short spin-spin rela x a t i o n times (T 2) of the polymer protons C^i/2 a V T 2 ) • This problem can be p a r t i a l l y overcome by acquiring spectra at elevated temperatures. The use of h i g h - f i e l d spectrometers by which greater s i g n a l dispersion i s obtained, helps to counteract line-broadening e f f e c t s . Another way to obtain spectra with better resolution i s to use computer lineshape manipulation techniques, such as convolution difference p r o c e s s i n g . 1 0 3 This problem i s not as c r i t i c a l i n FT 1 3C-nmr because of the much greater s i g n a l dispersion, as w i l l be discussed shortly, which makes 1 3C-nmr extremely a t t r a c t i v e f o r studying polysaccharides. Carbon-13 nmr spectroscopy suffers from a drawback which does not apply to the 1H-nmr experiment. The inherent d i f f i c u l t y i n detection of 1 3 C l i e s i n the low natural abundance (1.1%) of t h i s n u c l e i , and the low r e l a t i v e sensi-t i v i t y (1.59 x 10~ 2), giving an o v e r a l l s e n s i t i v i t y decrease of -10~ 4, compared to that f o r Thus, longer time requirements become a major factor i n FT 1 3C-nmr experiments. The design of higher f i e l d instruments (300-500 MHz) has -40-helped to reduce these requirements, as have the technologi-c a l advancements i n modern FT nmr spectrometers and probes. However, the f a c t remains that 1 3C-nmr experiments require r e l a t i v e l y large amounts of machine time. •^H-nmr spectroscopy ^-nmr spectroscopy can provide s p e c i f i c information about a number of aspects of polysaccharides. The chemical s h i f t of the g l y c o s i d i c proton can give some i n d i c a t i o n of the i d e n t i t i e s of constitutent monosaccharides. However, due to l i m i t e d dispersion and broad resonances, absolute assignment on t h i s basis i s often d i f f i c u l t . The chemical s h i f t s of anomeric protons does d i s t i n g u i s h between the el-and j9-anomeric configurations, p a r t i c u l a r l y f o r gluco and galacto residues with H-l of ^-glycosides resonating at 4.5-5.0 ppm and that of the a-anomer occurring s l i g h t l y downfield at 5.0-5.5 ppm. This s h i f t difference r e s u l t s from the respective a x i a l and equatorial orientations of the C^-H^ b o n d . 1 0 9 The s p l i t t i n g or coupling constant of the anomeric signal can give a clue as to the r e l a t i v e configuration at C-2, and hence of sugar i d e n t i t y . However, broad resonances often preclude v i s u a l i z a t i o n of ^ ^ H coupling (Table 1) . One of the most important contributions of ^-nmr i s the ease of quantitating the respective anomeric resonances, thereby revealing r e l a t i v e proportions of constituent monosaccha-ri d e s . I t may be possible to determine the positions of -41-'H i> ippm) "C i> (ppm) CHjC - 1.5 CHjC -15 CHjCON CHjCO, 1.8-2.1 2.0-2.2 CHjCOH) CHjCO, j 20-23 CH(NH) 3.0-3.2 CHjC 38 C H , 0 3.3-3.5 CHjO 55-61 H-2 lo H-6' 3.5-4.5 CH(NH) 58-61 H-5 4.5-4.6 CHjOH 60-65 H-l (ax) 4.5-4.8 C-2 lo C-5 65-75 U-C(OH), 5.2 C—X' 80-87 HO 5.0-5.4 C-1 (ax-O, red) 90-95 H-i (eq) 5.3-5.8 C-1 (eq-O. red) 95-98 HCO, 5.9 C-1 (ax-O. glyc) C-1 (eq-O, glyc) C-1 (fur) COOH c=o 98-103 103-106 106-109 174- 175 175- 180 Substituent effects on x-'H and s-"Clppmr O-Alkyl O-Acyl O-Sulfate O-Phosphate 'H - 0.2-0.3 + 0.3-0.5 + 0.3-0.6 + 0.3-0.5 "C + 7-10 + <3 + 6-10 + 2-3 1 13 Table 1. Representative chemical shifts for H and C nuclei of common functional groups found on polysaccharides. a a. Abbreviations: ax, axial; eq, equatorial; red, reducing; glyc, glycosidic; fur, furanosyl. 13 b. Non-anomeric C involved i n glycosidic linkage, .c. Downfield, +; Upfield, -. g l y c o s i d i c attachment, at which the protons exhibit reso-nances s h i f t e d downfield by -0.2 ppm r e l a t i v e to the free hydroxyl equivalent. While i t i s usually d i f f i c u l t to assign other r i n g proton signals because of overlap, at high f i e l d s i s o l a t e d peaks can provide some i n d i c a t i o n of residue compo-s i t i o n . Proton nmr spectroscopy i s p a r t i c u l a r l y useful for est a b l i s h i n g the presence of substitutents which are often hydrolyzed or undetected by other a n a l y t i c a l methods. Typical substituents are acetates, methyl esters, methyl ethers and carboxyethylidene (pyruvate acetal) groups. The po s i t i o n of subs t i t u t i o n can often be assigned as well, due to downfield s h i f t s i n r i n g proton signals. For example, acetates can -42-s h i f t the corresponding r i n g protons into the anomeric chemical s h i f t r e g i o n . 1 1 0 The relaxation parameters, T j and T 2 f o r the various proton resonances can supply information about the motional dynamics of the p o l y m e r . 1 0 2 ' 1 1 1 ' 1 1 2 These parameters are e s p e c i a l l y useful for distinguishing between f r e e l y rotating and r i g i d groups (e.g. H-6 vs r i n g protons) and between less motion-restricted monosaccharide residues (e.g. branch residues y_s backbone residues) . The T 2 e f f e c t s are apparent i n s i g n a l linewidth ^ i / 2 <* 1/ T2)' a n a - c a n D e quantitated using spin echo pulse sequences. 1 1 3 T^ can be measured i n a v a r i e t y of w a y s 1 1 3 ' 1 1 4 including inversion recovery, saturation recovery, progressive saturation as well as various modifications of these pulse sequences. The nuclear Overhauser enhancement (n.O.e) 1 1 5 e f f e c t can be used to give conformational information which has been p a r t i c u l a r l y useful f o r determining g l y c o s i d i c linkage and geometry i n o l i g o s a c c h a r i d e s . 1 1 5 ' 1 1 6 The spectra of polysaccharides i n deuterated DMSO solu t i o n contain the hydroxyl proton resonances, the relaxation c h a r a c t e r i s t i c s of which can provide i n s i g h t into hydrogen-bonding interactions. S i m i l a r l y , hydrogen-bonding i s often r e f l e c t e d i n the chemical s h i f t of the hydroxyl group protons involved, again r e f l e c t i n g molecular conformation. 1 3c-Nmr Spectroscopy 1 3C-nmr spectroscopy i s undoubtedly a preferred method -43-f o r the study of polysaccharides, and indeed polymers i n general. The problem of solvent interference i s eliminated, concomitant with greater dispersion of resonances over a 200 ppm chemical s h i f t range as opposed to 10 ppm f o r that of 1H. While 1H- 1 3C coupling constants can be obtained, the f u l l y coupled spectra are often d i f f i c u l t to in t e r p r e t and require longer a c q u i s i t i o n times. Usually i t s u f f i c e s to obtain f u l l y decoupled spectra, which give a s i n g l e resonance per nonequivalent carbon. This gives increased si g n a l amplitude due to collapse of the multiplets and a proton-carbon n.O.e. e f f e c t which provides up to 3 times s i g n a l enhancement. P a r t i a l l y decoupled spectra from SSFORD (single frequency o f f resonance decoupled) experiments 1 1 3 may help i n determining the m u l t i p l i c i t y of the carbon reso-nances. The multiplets are s p l i t by, a much reduced amount over f u l l y coupled spectra, which have coupling constants of up to 200 Hz, thus reducing the d i f f i c u l t y i n v i s u a l i z i n g the mu l t i p l e t . This does not however, y i e l d the heteronuclear coupling constants. Complementary t e c h n i q u e s 1 1 8 are used to a i d i n assignments, as are the commonly used s p e c t r a l compar-isons to previously characterized model compounds such as methyl glycosides and oligosaccharides. The information content of a proton-decoupled 1 3C-nmr spectrum of a polysac-charide i s usually greater than that of a 1H spectrum. The chemical s h i f t of the g l y c o s i d i c carbon i s located downfield from those of the r i n g c a r b o n s , 1 1 9 - 1 2 1 and r e f l e c t s to a greater degree than 1H-nmr, the i d e n t i t y of the respective - 4 4 -sugar. The g l y c o s i d i c configuration can often be i n f e r r e d from 1 3 C chemical s h i f t data, with the a anomer t y p i c a l l y resonating at 98-103 ppm and the /3 at 103-106 ppm. The signals of r i n g carbons of most monosaccharides are resolved i n 1 3 C spectra at high f i e l d (50-100 MHz f o r 1 3 C n u c l e i ) , although linebroadening can cause overlap p a r t i c u l a r l y i n complex multiresidue polysaccharides. However, resonance separation i n spectra recorded at elevated temperatures i s usually s u f f i c i e n t to make assignment possible. The carbon involved i n g l y c o s i d i c attachment generally resonates 6-9 ppm downfield from the signals of the corresponding hydrox-y l i c c a r b o n , 1 1 9 - 1 2 1 and i s usually e a s i l y d i s c e r n i b l e . I f the linkage i s at a primary center, t h i s downfield s h i f t w i l l p o s i t i o n the resonance of the methylene carbon (C-6) i n the r i n g carbon region. The absence of a s i g n a l at 60-65 ppm i s an i n d i c a t i o n of a (1+6)-linked or 6-0-substituted polysac-charide. P a r t i a l l y decoupled spectra may also a i d i n t h i s assignment as the m u l t i p l i c i t y of the primary carbon ( t r i p -l e t ) amongst the r i n g carbon doublets w i l l be diagnostic. I t i s possible to d i s t i n g u i s h C-6 from other carbons using relaxation measurements, since the former has l e s s r e s t r i c t e d motion and w i l l undergo relaxation at a f a s t e r r a t e . 1 0 2 ' 1 1 1 * 1 1 2 There are methods, also based on relaxation phenomena, to d i s t i n g u i s h between primary, secondary, t e r -t i a r y and quaternary c e n t e r s . 9 9 ' 1 1 8 The presence of substitutents i s r e a d i l y established by 1 3 C chemical s h i f t s (see Table 1). To obtain meaningful 45-quantitative data from 1 3C-nmr spectra, i t i s c r u c i a l to take care i n s e t t i n g a q u i s i t i o n parameters 1 1 3 and i n sample p r e p a r a t i o n . 1 8 One must forego the nuclear Overhauser enhancement, and allow long relaxation delays (3-5 T^'s), thereby increasing the time requirement f o r the experiment. This i s also true f o r T^ and T 2 relaxation time determina-tions i n which various relaxation delays and pulse widths must be determined. 1 1 3 However, despite the time requirement, 1 3C-nmr relaxa t i o n measurements may provide useful informa-t i o n about the motional dynamics of the polymer. 2 7 The 1 3C-nmr spectral linewidths give a q u a l i t a t i v e i n d i c a t i o n of T 2 relaxation, which i n turn r e f l e c t s the motional c o r r e l a -t i o n t i m e s . 1 0 2 While most of t h i s discussion has been directed to proton-decoupled spectra, useful information can be obtained from the f u l l y coupled spectrum. In p a r t i c u l a r , the 3 J _ C H value can provide information about the angles d> and (Fig. 1 ) , representing the geometry about the g l y c o s i d i c bond, as formulated by the Karplus r e l a t i o n s h i p . 1 0 4 F igu re 1. Diagram showing g l y c o s i d i c con fo rmat ion , and the angles and Y which de f i ne i t . 46-2D-NMR Spectroscopy To date, two-dimensional nuclear magnetic resonance spectroscopy experiments 1 2 2 have found r e l a t i v e l y l i m i t e d a p p l i c a t i o n to p o l y s a c c h a r i d e s , 1 2 3 - 1 2 5 mainly due to complex signa l overlap as well as the added problem of l i n e -broadening. However, i t i s d e f i n i t e that 2D nmr methods w i l l f i n d increased a p p l i c a t i o n i n polysaccharide s t r u c t u r a l studies of both i n t a c t species and p a r t i c u l a r l y i n conjunc-t i o n with p a r t i a l l y hydrolyzed products, such as bacteriopha-ge-degraded oligosaccharides. The 2D-J experiment has been applied to some polysaccharide systems with s u c c e s s , 1 2 5 as a method to overcome the hidden resonance problems. This experiment provides a "homo-nuclear decoupled" ( i . e . a single l i n e per resonance) 1H -nmr spectrum i n one dimension, as well as coupling information i n the other dimension. The COSY and SECSY m e t h o d s 1 2 6 * 1 2 7 have found extensive applica-t i o n to o l i g o s a c c h a r i d e s , 1 2 8 - 1 3 0 and are being applied more frequently to polysaccharides having well dispersed •'-H-nmr s p e c t r a . 1 2 3 ' 1 2 4 These methods e s t a b l i s h connectivity between coupled 1H n u c l e i . There are 1 3 C 2D-nmr techniques which have been applied to polysaccharides. For example, proton-carbon c o r r e l a t i o n spectroscopy, which establishes the connectivity between carbon resonances and attached or coupled protons, has proven p o t e n t i a l i n oligosaccharide studies and limi t e d but expanding u t i l i t y i n polysaccharide structure investiga-t i o n s . 1 2 3 ' 1 2 4 These can be used to assign anomeric protons, based on the carbon resonance assignments which are often - 4 7 -easier. Another 1 3 C 2D-nmr experiment which has been used i n polysaccharide structure determination i s 2D 1 3C-nmr sp e c t r o s c o p y , 1 3 1 i n which carbon chemical s h i f t s are along one axis and 13C-^H coupling constants are along the other. At t h i s point, i t i s appropriate to i l l u s t r a t e with l i t e r a t u r e examples, the use of nmr spectroscopy i n the study of polysaccharides as outlined i n the previous section. Quite d e t a i l e d r e v i e w s 1 8 ' 1 0 2 " 1 0 4 on t h i s subject have appeared i n the l a s t f i v e years, so an exhaustive survey of t h i s material w i l l not be presented. Instead a few examples i l l u s t r a t i n g ' h e the important aspects of ^ -H and 1 3C-nmr spectroscopy i n the ' study of carbohydrate polymers, w i l l be b r i e f l y discussed. L i t e r a t u r e studies One family of polysaccharides extensively studied by nmr spectroscopy are the d e x t r a n s , 1 3 2 - 1 4 3 polymers of (l+6)-a-D-glucopyranose. The ^-nmr spectrum of d e x t r a n 1 3 3 (B-742) has resonances at S 4.9 and 5.2 ppm, due to the H-l of a-(1+6)-linked glucopyranose backbone and to those bearing a (1*3) or (1+4) branch, respectively. The r a t i o of these peaks provides a means f o r determining the degree of branching, which i s v e r i f i e d also by chemical methods. Comparison of C-l i n t e n s i t i e s i n 1 3C-nmr spectra of dextran B-742 (Fig. 2) affords a reasonable estimate of r e l a t i v e proportions of a -(1+6) (57%), a-(l+4) (9%) and a-(l-*3) (34%) l i n -k a g e s . 1 0 3 ' 1 3 9 " 1 4 3 I t was also found that r i n g resonances i n the 70-85 ppm region were diagnostic f o r a-(l-*-2), a-(i-»-3) and -48-a-(l+4) branches. Relative estimates from 1 3 C spectra agree to within 10% with methylation r e s u l t s , and are thus consid ered useful f o r analyses of branching i n dextrans. Yeast mannans have undergone s i m i l a r d e t a i l e d -^H and 1 3C-nmr investigations by Gorin and c o w o r k e r s . 1 4 4 - 1 5 1 These polysaccharides possess a (1+6)-a-D-mannopyranose backbone with mainly (1+2)-a- and some (1+3)-a-branches of varying length . ^ H-Nmr spectra of these polysaccha-r i d e s 1 4 4 " 1 4 6 have well resolved anomeric regions (Fig. 3a). Chemical s h i f t s of H-l vary with s u b s t i t u t i o n at 0-2, and with l o c a t i o n of the monosaccharide i n the branch , giving r i s e to a number of anomeric proton sign a l s . The anomeric -49-CO (D (0 = <o«>cng Pi H Pi A A I I to OS — CM CO ">r> • . co cx 8 10 03 M «-c-i-» F i g u r e 3 . a) The anomeric p ro ton s i g n a l s from a v a r i e t y of yeast mannans a t 100 MHz i n D ,0 , showing the d i f f e r e n t chemica l s h i f t s and r e l a t i v e i n t e n s i t i e s ; and b) the C-nmr spectrum of branched mannan from bakers yeas t i n D 2 0 , a t 70 6 C ( r e f . e x t e r n a l TMS). - 5 0 -Numbering e r r o r . Text not a v a i l a b l e . -51-J I ' » 5.4 J L J L 5.0 P P M 4.6 F i g u r e 4 . An expanded r e g i o n of the ^ - n m r s p e c t r a of a) panose, b) waxy-maize s t a r c h , and c) a ^degraded s t a r c h sample ob ta ined i n D 2 0 s o l u t i o n at 90 C. H O - 1 8 0 100 80 PPM 60 13, F i gu re 5 . C-nmr s p e c t r a of a) waxy-maize s t a r c h , b) panose, and c) degraded s t a r c h i n D 2 0 a t 90°C. Arrows i n a ) , and c) a r i s e from non- reduc ing t e r m i n a l r e s i d u e s , and reduc ing t e r m i n a l r e s i d u e s r e s p e c t i v e l y . The galactomannans of legume seeds, containing a ^3-(1+4) mannan backbone with sing l e residue side chains of (1+6)-linked-a-D-galactopyranose (31), d i f f e r from previous examples of branched polysaccharides i n the monomeric compo-Gal G a l 1 1 +a +<* fl 6 fl B fl 6 fl -Man- (1+4 ) -Man- (1+4 ) -Man- (1+4 ) -Man- (1+4 ) -Man- (1+4)-81 -53-s i t i o n of the b r a n c h . 1 5 5 Both 1H and 1 3C-nmr spectroscopy gave manno/galacto r a t i o s which agreed with chemical determi-nations. 1 5 6 » 1 5 7 An in t e r e s t i n g feature was the s p l i t t i n g of resonances i n the anomeric region of the 1 3C-spectrum (Fig. 6), r e f l e c t i n g the i d e n t i t y of the nearest neighbor. For example, three signals f o r the C-4 of mannose residues appeared, a r i s i n g from any two adjacent residues (diads) i n which (1) both were substituted, (2) one was substituted and (3) both were unsubstituted. I n t e n s i t i e s of these resonances gave r e l a t i v e amounts of the diad sequences. I l l 1 p p m F igure 6. C-nmr s p e c t r a l r e g i o n , a t 25 MHz, showing C-4 of the D-mannose r e s i d u e s i n a) l o c u s t bean gum; b) guaran; and c) the galactomannan from c l o v e r seeds . The d iad sequences cor respond ing to each peak, are shown a t the r i g h t , w i th the u n i t i n v o l v e d u n d e r l i n e d . -54-1 3C-nmr was applied f r u i t f u l l y to determine the structure of some s y n t h e t i c a l l y branched amylose d e r i v a t i v e s . 1 5 8 Addition of glucopyranosyl monomers to amylose produced a polysaccharide with unknown branch linkages. While a linkage was expected based on coupling methods, the 1 3 C s p e c t r a l data was necessary to confirm t h i s . The 1 3 C spectrum of the synthetic amylose was compared to those of methyl jS-D-glucopyranoside and the (1+6)- a branched (1+4)-a-linked polysaccharide g l y c o g e n , 1 0 3 * 1 4 3 as seen i n F i g . 7. The resonance at 8 104.4 ppm confirmed a /3-linkage f o r the branch, and a resonance at 8 80.3 ppm indicated the predom-inance of branching at the 0-6 p o s i t i o n of amylose. Other-wise, the spectrum of the synthetic compound looks l i k e a composite of the methyl jS-D-glucopyranoside and glycogen spectra. This example demonstrates the importance of nmr spectroscopy i n the study of branched polysaccharides, p a r t i c u l a r l y i n conjunction with polysaccharide modifica-t i o n s . The u t i l i t y of and 1 3C-nmr spectroscopy i s borne out by i t s increasing a p p l i c a t i o n to studies on complex and regular heteropolysaccharides. An excellent example i s provided by the work of Dutton and coworkers i n which nmr spectroscopy was employed i n conjunction with chemical methods i n s t r u c t u r a l investigations of the capsular polysaccharides of K l e b s i e l l a . 1 5 9 - 1 6 7 Nmr spectral analyses are e s p e c i a l l y applicable to oligosaccharides i s o l a t e d from the chemical degradation of these complex polysaccharides, as -55-Me j3 _ n _ Glucopyranoside c Glycogen b F i g u r e 7. C-nmr s p e c t r a of methy l /3 -D-g lucopyranos ide , a branched amylose d e r i v a t i v e , and g lycogen a t 90°C. -56-well as to repeating unit oligosaccharides obtained from bacteriophage degradations.166-169 Both 1H and 1 3C-nmr spectroscopy have found application i n the analysis of i n d u s t r i a l l y u t i l i z e d c e l l u l o s e deriva-t i v e s . Comprehensive a r t i c l e s on 2 - 0 - h y d r o x y p r o p y l , 1 7 0 - 1 7 2 2 - 0 - h y d r o x y e t h y l , 1 7 3 ' 1 7 4 m e t h y l 1 7 3 and c a r b o x y m e t h y l 1 7 3 ' 1 7 5 c e l l u l o s e s have appeared. These studies demonstrate the power of nmr spectroscopy as used fo r the detection of substitu-ents, since the derivatives of i n t e r e s t are complex and highly substituted. Nmr methods enable one to d i s t i n g u i s h the r e l a t i v e degree of s u b s t i t u t i o n at each p o s i t i o n due to the downfield chemical s h i f t s of substituted s i t e s . Besides i t s demonstrated u t i l i t y i n probing s t r u c t u r a l aspects of polysaccharides, nmr spectroscopy has also been used f o r conformational studies. The ^-nmr spectrum of amylose i n DMSO soluti o n exhibited signals f o r 0H-2 and 0H-3 at lower f i e l d (S>5) than anticipated, a phenomenon a t t r i b -uted to intramolecular hydrogen bonding a s s o c i a t i o n , 1 7 6 ' 1 7 7 as i l l u s t r a t e d i n F i g . 8 . More recently 1 3C-nmr spectroscopy Figure 8. A disaccharide u n i t of amylose showing the inter-residue i n t e r a c t i o n between the 2-OH and 3-OH groups. 57-has been used to investigate the conformation of h e l i c a l complexes of amylose and amylopectin i n s o l u t i o n . 1 5 4 In t h i s study, the random c o i l - t o - h e l i x t r a n s i t i o n was studied by adding DMSO, t r i i o d i d e or alcohols, to induce h e l i x forma-t i o n . In general, C-1 and C-4 showed marked downfield s h i f t s , a t t r i b u t e d to ro t a t i o n of the C-0 bonds of the g l y c o s i d i c linkage upon h e l i x f o r m a t i o n . 1 5 4 Molecular mobility of polysaccharides (as indicated by the c o r r e l a t i o n time constant, r c ) i s e a s i l y probed by nmr spectroscopy. Line broadening of 1 3 C resonances i n (1+3)-^ -D-glucans (curdlan) was observed upon g e l a t i o n . 1 7 8 - 1 8 1 Inferences regarding conformation can be made, depending on linewidths of both solutions and gels, as some h e l i c a l associations allow a greater degree of mobility. Downfield chemical s h i f t s of C-1 and C-3 of curdlan resonances, r e l a t i v e to those of degraded fr a c t i o n s , were explained as an e f f e c t of r e s t r i c t e d r o t a t i o n about the g l y c o s i d i c bond i n the h e l i x c o n f o r m a t i o n . 1 3 2 ' 1 7 9 Nmr spectroscopic studies on (1+3)-/3-D-glucans having (1+6)-/3-1 inked sidechains (lentinan) i l l u s t r a t e the mobility differences which can e x i s t between the main chain and branch residues. In the gel state, the (1+3)—yQ—1inked main chain exhibits no resonances due to an ordered structure and concomitant low mobility, while resonances f o r branch r e s i -dues are observed (Fig. 9 ) .179-181 T n e studies on t h i s family of gel-forming fungal glucans showed that, at 25.2 MHz, linewidths f o r main chain residue carbons were as large as - 5 8 -1000 Hz, corresponding to a motional c o r r e l a t i o n time of >10~6 s - 1 , while side chain 1 3 C resonances had c o r r e l a t i o n times of 10~ 8-10~ 9 s, as determined from linewidths, T^ values and nuclear Overhauser enhancements. 1 8 2 Similar 3) backbone 100 80 PPM 60 13, F i g u r e 9. * C-nmr s p e c t r a of a) l e n t i n a n g e l , and b) a lower mo lecu la r weight f r a c t i o n , i n D20. The d isappearance of s i g n a l s from the /3- ( l -» 3) l i n k e d main c h a i n i n the g e l s t a t e spectrum can be seen . although smaller differences i n mobility of backbone and side chain residues were observed i n some mannans, as indicated by 1 3 c _ T l measurements. 1 8 3 For example, T^ values f o r C-1 of the terminal side chain residue, an adjacent sidechain residue, and a main chain residue i n mannans were 0.20, 0.13 and 0.09 s, respectively. Dextran studies gave analogous r e s u l t s , -59-showing that mobility differences i n branched polysaccharides appear to be a general t r e n d 1 4 3 and that the r e l a t i o n s h i p between mobility, values and linewidths can be used as a t o o l to d i s t i n g u i s h branch residues from those of the main chain. 1.2.4 Polysaccharides i n Solution Much of the i n d u s t r i a l i n t e r e s t i n polysaccharides and t h e i r d e rivatives arises from the properties exhibited by aqueous solutions or dispersions of these m a t e r i a l s . 6 While some of the properties follow understandable and predictable trends, such as l i m i t e d s o l u b i l i t y , thickening or gelation and h y d r o p h i l i c i t y , the s p e c i f i c interactions at the molecu-l a r l e v e l are more d i f f i c u l t to comprehend. Although i t i s known that these molecules are both polymeric and hydrophilic i n nature, a complete understanding of the r e l a t i o n s h i p between primary polysaccharide structure and the physicochem-i c a l properties of the aqueous solutions has yet to be established. The molecular interactions of the polymer, including both i n t e r - and intra-molecular associations as well as interactions with other solutes and solvent mole-cules, are wide ranging and complex. Some systems o f f e r examples of s p e c i f i c interactions which can be correlated with the respective resultant properties, allowing some insi g h t to be gained. Unfortunately, the d i v e r s i t y i n poly-saccharide primary structure and accompanying int e r a c -t i o n s 1 7 ' 1 8 3 serves to i s o l a t e these examples without -60-permitting predictable trends to be established. And yet, i t i s t h i s general understanding which would be invaluable f o r the " t a i l o r e d " preparation of polysaccharide d e r i v a t i v e s . The following discussion w i l l provide an overview of polysaccha-rides i n solution, focussing on both the causes and evalua-t i o n of physicochemical properties. S o l u b i l i z a t i o n or hydration of polysaccharides i s thought to proceed i n i t i a l l y at amorphous regions where intermolecular interactions are l i m i t e d by the disorganized s p a t i a l arrangement of the chain r e s i d u e s . 3 ' 1 8 4 Further hydration subsequently replaces the intermolecular hydrogen-bonding to an extent which determines s o l u b i l i t y . L i t t l e or no hydration leaves the polymer undissolved, while l i m i t e d hydration r e s u l t s i n a gel-state sol u t i o n and extensive hydration y i e l d s apparent t o t a l d i s s o l u t i o n . The aqueous s o l u b i l i t y of natural polysaccharides i s dependent on s t r u c t u r a l and conformational features. For example, l i n e a r polysaccharides such as c e l l u l o s e and c h i t i n are known to adopt highly ordered ribbon-like structures, with substantial c r y s t a l l i n e character, r e s u l t i n g i n d i f f i c u l t s o l u b i l i z a t i o n . 3 ' 1 7 ' 1 8 4 Branched polysaccharides on the other hand, have a le s s ordered structure and t y p i c a l l y s o l u b i l i z e r e a d i l y . Some of the other factors which often enhance s o l u b i l i t y are g l y c o s i d i c arrangement, charged f u n c t i o n a l i t i e s , and s t r u c t u r a l i r r e g u l a r i t i e s . The g l y c o s i d i c linkages i n polysaccharides influence s o l u b i l i t y and molecular associations i n s o l u t i o n . 1 7 ' 1 8 3 As -61-mentioned, -linear homopolysaccharides such as c e l l u l o s e , c h i t i n and some mannans, having (1+4)- jQ-linkages, form f l a t ribbon sequences of semi-crystalline nature. Similar polysaccharides having the l e s s l i n e a r (1+4)-a-linkage (e.g. amylose) form le s s r i g i d c o i l e d springs or h e l i c e s , and are generally more water-soluble. In f a c t , an examination of the geometry of the various linkages, i . e . (1+6), (1+4), (1+3) and (1+2) i n both a and j3 forms, shows that s t e r i c e f f e c t s w i l l d i c t a t e the order and h e l i c a l parameters (pitch) of the p o l y m e r . 1 ' 1 7 * 1 8 3 F i g . 10 depicts i n s t i c k diagram form the e f f e c t of linkage p o s i t i o n and configuration on conformation. eg-Type A l-»3)-o>D-galactan l-»4)-a-D-galactan l-»3)-P-D-glucan l-»4)-0-D-glucan l-»3)-a-D-mannan l-+4)-0-D-mannan l-»-3)-a-D-xylan l-O)-0-D-xylan Type B eg- l-*4)-0-D-galactan l-*3)-0-D-galactan l-*4)-or-D-glucan l-OHJ-D-glucan l-»2)-o>D-mannan l-»4)-a-D-mannan l-*3)-0-D-mannan l-*4)-a-D-xylan l-»3)-0-D-xyIan TypeC l-»2)<»-D-galactan l-*2)-0-D-galactan I-*2)-or-D-glucan l-»2)-0-D-glucan l-*-2H*-D-mannan l-*2)-a-D-xylan l-*2)-0-D-xylan Type D e.g. all 1,6-disubstituted homoglycans F igu re 10. D e p i c t i o n s o f the r e l a t i o n s h i p between t e r t i a r y s t r u c t u r e and l i n k a g e . Type A , extended r i b b o n ; Type B, f l e x i b l e h e l i x ; Type C , crumpled r i b b o n ; and Type D, f l e x i b l e c o i l . -62-A (1*6)-linkage f o r example, having a greater degree of freedom, does not e x i s t i n a single preferred conformation and i s thus usually disordered i n solut i o n . This s i t u a t i o n i s exemplified by (1*6)-a-1inked D-glucopyranose polysaccharides of the dextran f a m i l y , 1 7 and evidenced by the r e l a t i v e l y low v i s c o s i t y displayed by these polymers. Charged or ionizable f u n c t i o n a l i t i e s on a polysaccharide can influence s o l u b i l i t y and solut i o n properties by providing l o c i f o r hydration, perhaps upon a pH change, or by providing s i t e s f o r i o n i c i n t e r a c t i o n with other charged solutes. An example of the former would be the use of aqueous organic ac i d solutions to dissolve water insoluble chitosan, l i k e l y by d i s r u p t i o n of intermolecular hydrogen bonding involving the amine upon formation of the ammonium ion. Interaction of an ionizable polysaccharide with charged solutes i s i l l u s t r a t e d by the action of C a + 2 and other divalent metal ions on sodium alginate s o l u t i o n s . 1 8 5 - 1 8 9 According to the "egg-box" model (Fig. 11), the calcium i s sequestered between chain segments of L-guluronate residues by means of i o n i c c r o s s l i n k i n g i n t e r a c t i o n s . 1 9 0 - 1 9 5 Noncrosslinked D-mannuronate and mixed L-guluronate/D-mannuronate chain segments provide s u f f i c i e n t areas of hydration such that the calcium chelate does not p r e c i p i t a t e , but forms a stable g e l . 1 9 3 Other polysaccharides with charged f u n c t i o n a l i t i e s include glycosaminoglycans such as hyaluronate and chon-d r o i t i n , which have uronic acid and acetamido groups; derma--63-o F i g u r e 11. The "Egg box" model f o r c a l c i u m i o n induced i n t e r a c t i o n of p o l y ( L - g u l u r o n a t e ) c h a i n s . tan s u l f a t e with s u l f a t e , uronate and acetamido containing residues; and heparins having s u l f a t e , uronate, N-sulfate and acetamido moieties. These f u n c t i o n a l i t i e s give r i s e to i o n i c interactions which determine the solution properties of these polysaccharides, and numerous s o l i d state and solu t i o n studies have been done to delineate important s t r u c t u r a l interactions and the solution conformation of these com--64-pounds. 1 9 6 As mentioned e a r l i e r , the branches on a l i n e a r polysaccharide can have a marked e f f e c t on the s o l u t i o n properties. There are r e l a t i v e l y few families of polysaccharides which are based on a l i n e a r backbone with varying degree of branching, but one such ser i e s are the seed galactomannans. 1 5 5 The parent ( 1 * 4 ) i n k e d l i n e a r mannan (ivory nut mannan), having a ribbon-like structure s i m i l a r to c e l l u l o s e , i s insoluble i n water, and i s a very r e s i l i e n t s o l i d material. However, when -20% of the residues are substituted with (1*6)-a-D-galactopyranose (locust bean gum), the material i s soluble i n hot water and gels upon cooling. Guar gum, having § 55% s u b s t i t u t i o n , i s l a r g e l y soluble i n water, giv i n g viscous solutions. I t i s c l e a r that d i s t r i b u -t i o n of substituents along the backbone i s an important factor i n the interactions of galactomannan polysaccharides. The s e l f - a s s o c i a t i o n of galactomannans, which were reported to contain substituted and unsubstituted b l o c k s , 1 9 7 has been att r i b u t e d to intermolecular interactions between unsubsti-tuted "smooth" regions, while branched "hairy" regions remain hydrated (Fig. 12a). Proponents of a more random alt e r n a t i n g s u b s t i t u t i o n pattern, as supported by recent studies on some g a l a c t o m a n n a n s , 1 9 8 - 2 0 1 postulate a two-fold conformation of the mannan backbone, 2 0 2 > 2 0 3 r e s u l t i n g i n "smooth" and "hairy" faces which could i n t e r a c t 2 0 1 * 2 0 4 as shown i n F i g . 12b. -65-F i g u r e 1 2 . I l l u s t r a t i o n o f g a l a c t o m a n n a n i n t e r a c t i o n s i n w h i c h a ) b l o c k s u b s t i t u t i o n f o r m s " h a i r y " s o l v a t e d r e g i o n s and " s m o o t h " r e g i o n s w h i c h i n t e r a c t ; o r b ) r a n d o m l y s u b s t i t u t e d c h a i n s h a v e c o n f o r m a t i o n a l l y i n d u c e d " h a i r y " f a c e s and s e l f a s s o c i a t i n g " s m o o t h " f a c e s . -66-Structural i r r e g u l a r i t y i s another feature known to a f f e c t hydration and solution properties of polysaccharides. The i r r e g u l a r i t y can involve a substituent positioned on selected residues of the polysaccharide, or conformational aberrations caused by the primary structure of the polymer. An excellent example of these e f f e c t s i s found i n pectins, a poly-(1+4) -a-D-galacturonate polymer having intermittent (1+2)-linked L-rhamnose units. This polymer i s found i n nature i n p a r t i a l l y e s t e r i f i e d forms, 3 and i s known to bind calcium and form gels i n a manner analogous to a l g i -n a t e s . 1 9 ' 1 9 4 ' 2 0 5 ' 2 0 6 I t has been established that sequences of -15 residues of u n e s t e r i f i e d galacturonate are required f o r calcium-induced chain a s s o c i a t i o n , 2 0 7 ' 2 0 8 and that the L-rhamnose uni t s occur at uniform i n t e r v a l s of - 25 galactu-ronate r e s i d u e s . 1 9 4 ' 2 0 8 F u l l y e s t e r i f i e d chains found i n nature have been subjected to blockwise enzymatic and random chemical d e - e s t e r i f i c a t i o n to give a range of samples useful fo r probing the e f f e c t of e s t e r i f i c a t i o n pattern on calcium-induced gelation. The calcium-binding c a p a c i t i e s of block and randomly e s t e r i f i e d chains, as monitored by c i r c u l a r dichroism are compared i n F i g . 1 3 . 1 9 1 ' 2 0 8 The randomly d e - e s t e r i f i e d samples show l i t t l e binding capacity u n t i l -50% of the residues are l i b e r a t e d . Upon further d e - e s t e r i f i c a t i o n a sharp increase i n binding i s observed, while chelation a b i l i t y increases almost l i n e a r l y upon blockwise removal of ester functions. These observations are r a t i o n a l i z e d by the requirement of long chain segments f o r cooperative calcium-F igu re 13. Ca lc ium b i n d i n g c a p a c i t y , as moni tored by CD, f o r p a r t i a l l y e s t e r i f i e d p e c t i n samples hav ing random ( • ) and b l o c k ( O ) s u b s t i t u t i o n p a t t e r n s . F i g u r e 14. The i n t e r a c t i o n s p resen t i n c a l c i u m pec ta te g e l s . -68-binding. The (1*2)-L-rhamnose units serve to disrupt the highly ordered chelate by causing kinks, thereby providing s u f f i c i e n t regions of hydration to maintain a stable gel rather than a p r e c i p i t a t e (Fig. 14). This study c l e a r l y i l l u s t r a t e s the importance of a systematic strategy of preparing modified polysaccharides, over and above those n a t u r a l l y a v a i l a b l e , i n order to probe structure/function int e r a c t i o n s and ultimately, to allow one to t a i l o r polysac-charide s o l u t i o n properties. Synergistic interactions, another i n t e r e s t i n g phenomena exhibited by polysaccharides i n solutions, have received considerable attention i n the recent l i t e r a t u r e . These involve quaternary association of unlike polysaccharides, with concomitant enhancement or a l t e r a t i o n of s o l u t i o n properties. The i n t e r a c t i o n of seed galactomannans with xanthan gum t y p i f i e s the s y n e r g i s t i c e f f e c t , 1 5 5 ' 2 0 4 ' 2 0 9 - 2 1 4 where viscous solutions or gels can be obtained at concentra-tions considerably lower than those required f o r e i t h e r s i n g l e component. The galactomannan polysaccharides also p a r t i c i p a t e i n mixed associations with carrageenan and a g a r . 1 5 5 ' 2 1 5 - 2 1 7 Generally, l e s s substituted galactomannans (e.g. locust bean gum, d.s.-0.2) ex h i b i t more extensive i n t e r a c t i o n than those with a higher degree of branch-i n g 2 0 9 ' 2 1 8 (e.g. guar gum, d.s.-0.5). Also, galactomannans having a regular a l t e r n a t i n g branch pattern show substan-t i a l l y stronger i n t e r a c t i o n with xanthan than do other galactomannans with equivalent o v e r a l l galactose con-- 6 9 -t e n t . 2 0 1 ' 2 0 9 These interactions are of substantial i n d u s t r i a l i n t e r e s t f o r producing viscous solutions and stable gels at low material cost. Introduction to Rheology Numerous experimental methods e x i s t f o r probing the s o l u t i o n interactions of polysaccharides, including chiroop-t i c a l techniques (ORD/CD), disorder-order t r a n s i t i o n kinet-i c s , l i g h t - s c a t t e r i n g measurements, d i f f e r e n t i a l scanning calorimetry, nuclear magnetic resonance spectroscopy and v iscometry. 1 7 The l a s t of these o f f e r s the a d d i t i o n a l feature of characterizing the flow behaviour or rheological proper-t i e s of the system, as well as providing information about molecular i n t e r a c t i o n s . Since the p o t e n t i a l a p p l i c a b i l i t y i s often based on the rheological properties of the material, rheometry i s an important method fo r characterizing polysac-charides i n s o l u t i o n . In f a c t , commercial polysaccharides are often i d e n t i f i e d by the s o l u t i o n v i s c o s i t y at a defined concentration. Certainly, viscometry i s indispensable f o r purposes of c o r r e l a t i n g the interactions and properties of polysaccharides. However, some caution i s required i n desig-ning and i n t e r p r e t i n g rheometric measurements because various experimental factors w i l l influence the molecular inte r a c t i o n s that are probed. I t i s intended that the follow-ing discussion w i l l provide the reader with some background on the types of viscometry and rheometry commonly employed, and the information that can be extracted from these measure--70-ments. Generally, the information procured from studies on d i l u t e and concentrated solutions i s d i f f e r e n t , and thus these concentration regimes are treated separately. Di l u t e Solutions The a b i l i t y of many polysaccharides to form viscous solutions at r e l a t i v e l y low concentrations i s well-known and has importance i n many i n d u s t r i a l and b i o l o g i c a l applications. This behaviour arises l a r g e l y from the c o i l dimensions of the hydrated polymer; however, depending on solvent conditions, contributions from interchain int e r a c t i o n s can be appreciable. An index which r e f l e c t s polymer c o i l dimensions i n soluti o n i s i n t r i n s i c v i s c o s i t y , 2 1 9 ([T?])* the f r a c t i o n a l increase i n v i s c o s i t y per uni t concentration (c) for i s o l a t e d chains ( i . e . c i i P o ) . I n t r i n s i c v i s c o s i t y increases with c o i l dimensions according to the Flory-Fox r e l a t i o n s h i p (Eq. 5 ) : [n] = £±i [5] where i s a constant, L i s the average end-to-end chain length and M r i s polymer molecular weight. The molecular weight dependence of i n t r i n s i c v i s c o s i t y i s given by the Mark-Houwink equation (Eq. 6 ) : [nl = K M a (6] 'K . r where K i s a constant and a i s a parameter r e l a t i n g to c o i l dimensions. Experimentally, i n t r i n s i c v i s c o s i t i e s are obtained using -71-the Kraemer re l a t i o n s h i p : Ln (n r e l = tn] + k" [ n ] 2 c c [7] or the Huggins equation: [8] where k' and k' • are constants and r e l a t i v e v i s c o s i t y (Tjrei) i s the r a t i o of s o l u t i o n v i s c o s i t y ( 7 7 ) to solvent v i s c o s i t y (77s), as given i n Eq. 9: r e l n n s 19] and s p e c i f i c v i s c o s i t y (n Sp)) i s obtained from r e l a t i v e v i s c o s i t y according to Eq. 10: . «.p - "..1-1 I 1 C Plots of T 7 S p / c against c, or of l n 7 7 r e i / c against c, f o r a s e r i e s of d i l u t e solutions, extrapolated to zero i n t r i n s i c v i s c o s i t i e s f o r a s e r i e s of r e l a t e d samples varying i n molecular weight i t i s possible to determine the constant K, and a, of the Mark-Houwink r e l a t i o n s h i p (Eq. 6). These parameters i n turn r e l a t e to the shape and conformation of the polymer. I t i s important to point out that because i n t r i n s i c v i s c o s i t y represents the e f f e c t of the material on the s o l u t i o n behavior at i n f i n i t e d i l u t i o n , they are v i r t u -a l l y independent of contribution from inter-chain interac-t i o n s . Thus intermolecular interactions are not probed by concentration, give i n t r i n s i c v i s c o s i t i e s . 2 1 9 By determining 72-i n t r i n s i c v i s c o s i t y . Concentrated Solutions As one considers more concentrated polymer solutions, a c r i t i c a l concentration, c*, i s reached, at which the volume occupied by the polymer equals the solu t i o n volume, and the presence of more polymer can be accommodated only by entanglement or i n t e r a c t i o n of chains. Above t h i s concentration polysaccharide solutions t y p i c a l l y have non-Newtonian flow b e h a v i o u r . 2 2 0 That i s , the apparent v i s c o s i t y (TJ) i s dependent on the shear rate ("Y ). Newtonian f l u i d s on the other hand, have the same v i s c o s i t y at a l l shear rates. For polysaccharides, shear thinning or pseudoplastic behaviour i s most common, with regions of Newtonian flow behaviour at low shear rates (represented by zero shear v i s c o s i t y , 7 7 0) and at high shear rates ( c a l l e d i n f i n i t e shear v i s c o s i t y , 7jm ) (Fig. 15). A measure of shear thinning of a polysaccharide s o l u t i o n i s obtained by expressing v i s c o s i t y as a f r a c t i o n of zero shear v i s c o s i t y . Another parameter i s ^o.l» the s h e a r rate at which the apparent v i s c o s i t y i s one tenth the magnitude of the i n f i n i t e shear rate v i s c o s i t y 2 2 0 (V)=T)Q/10) . Empirical models have been developed to represent flow behaviour curves from steady shear viscometric determinations over r e l a t i v e l y large shear rate r a n g e s . 2 2 1 For example, the power-law model: r, = my1"11 or [11J a = my11 [12] - 7 3 -< S h e a r r a t e ( l o g s c a l e ) F igu re 15. I d e a l i z e d rheograms of p s e u d o p l a s t i c f l ow p l o t t e d as a) shear s t r e s s vs shear r a t e on a r i t h m e t i c c o o r d i n a t e s ; and" b) apparent v i s c o s i t y vs shear r a t e on l o g a r i t h m i c c o o r d i n a t e s . -74-F igu re 16. I d e a l i z e d power- law model rheograms on l o g a r i t h m i c c o o r d i n a t e s , p l o t t e d as a) shear s t r e s s vs shear r a t e ; and b) apparent v i s c o s i t y vs shear r a t e . - 7 5 -where m i s the consistency c o e f f i c i e n t and n i s the flow behaviour index, has found extensive use i n characterizing non-Newtonian flow behaviour over intermediate shear rate ranges (Fig. 16).222-224 A v a r i e t y of c o n s t i t u t i v e equations e x i s t which can be used to model pseudoplastic flow over shear rate ranges including zero shear or zero and i n f i n i t e shear behaviour. Parameters obtained from empirical modelling are p a r t i c u l a r l y useful f o r comparison of flow properties of d i f f e r e n t polysaccharide solutions. Steady shear viscometric measurements are useful f o r examining the e f f e c t s of parame-te r s such as concentration, temperature, pH, and i o n i c strength on the flow properties of polysaccharide solutions. The cause of shear thinning i n polysaccharide solutions i s usually s p e c i f i c to the system; however, i n general i t occurs as the rate of externally imposed movement exceeds the rate of re-entanglement of the polymer chains. Thus, the "cros-s l i n k " network i s reduced r e l a t i v e to the entanglement-disentanglement equilibrium e x i s t i n g under s t a t i c or low shear rate conditions. Some highly ordered polymer solutions and gels have a component of s o l i d - l i k e or p l a s t i c behaviour when undis-turbed. These materials are often characterized by a y i e l d stress value, or a minimum shear stress value, above which flow w i l l occur. The power-law p l a s t i c model [Eq. 13] incor-porates the y i e l d stress parameter into the f a m i l i a r power-law model: a = o y + my11 [13] -76-where o~v i s the y i e l d stress. In considering the rheology of polysaccharide solutions, we have not yet attempted to d i s t i n g u i s h between time independent (which has been assumed to t h i s point) and time dependent flow behaviour. While t h i s feature w i l l not be addressed i n any depth here, discussions on t h i s aspect of rheology are a v a i l a b l e i n monographs on the s u b j e c t . 2 1 9 ' 2 2 1 Time dependent flow describes f l u i d s i n which decreasing or increasing e f f e c t s on apparent v i s c o s i t y (or shear stress) are evident, at a constant shear rate. Usually t h i s e f f e c t i s r e v e r s i b l e . Samples ex h i b i t i n g decreasing apparent v i s c o s i t y are termed t h i x o t r o p i c , while those with increasing v i s c o s i t y are rheopectic. I f an i r r e v e r s i b l e l o s s i n apparent v i s c o s i t y occurs, the f l u i d i s said to be rheodestructive. F i g . 17 i l l u s t r a t e s some of these features. F igu re 17. I d e a l i z e d rheograms of t ime dependent f l ow i n cont inuous upcurve and downcurve exper iments . -77-Th e organization and intermolecular networks of hydrogels can be probed by o s c i l l a t o r y shear or dynamic v i s c o m e t r y . 2 2 5 This involves the applic a t i o n of a small o s c i l l a t o r y , sinu-s o i d a l , deformation (Eq. 14), and measurement of the sample's Y = Y 0sin(cot) t 1 4 ^ resistance to deformation. For true s o l i d s the greatest deformation ( s t r a i n , 7) occurs when the applied stress (c r ) i s at a maximum (Eq. 15). That i s to say, stress and s t r a i n o = kYQsin(ut) [15] are i n phase. Liquids, on the other hand, show greatest resistance to flow (stress) when the rate of deformation (Eq. 16) i s greatest, and i s thus 90° out of phase with s t r a i n (Eq. 17) with the applied s t r a i n wave. F i g . 18 shows the | X = y = o)Y0cos(wt) [161 a = T I O J Y Q c o s ^ t ) [17] respective stress and s t r a i n waves for a s o l i d , a viscous f l u i d and a v i s c o e l a s t i c material. V i s c o e l a s t i c materials w i l l have a phase s h i f t of between 0 and 90° depending on the r e l a t i v e contributions of viscous and e l a s t i c behaviour. The stre s s function f o r a v i s c o e l a s t i c material i s given i n Eq. 18; a = YlG'sin(cot) + G"cos (cot) ] [18] - 7 8 -T i me F igu re 18. I d e a l i z e d dynamic response of e l a s t i c , v i s c o u s and v i s c o -e l a s t i c systems to s i n u s o i d a l o s c i l l a t o r y shear . -79-having both viscous and e l a s t i c components and the respective constants G', the dynamic storage modulus, and G" the loss modulus. The former i s a measure of the energy recovered per cycle of deformation, while the l a t t e r i s a measure of the energy l o s t as heat f o r a deformation cycle. A r a t i o of the l o s s and storage moduli (Eq. 19) gives the tangent of the phase s h i f t (8 ), and i s c a l l e d the l o s s tangent. This parameter i s s e n s i t i v e to changes i n v i s c o e l a s t i c behaviour Tan 6 = [19] f o r a material. The dynamic v i s c o s i t y , as seen i n Eq. 20, [20] describes the energy loss r e s u l t i n g from o s c i l l a t o r y s t r a i n . The dependence of 17*, or of the loss and storage modulus, on angular frequency (OJ) , can provide information about molecu-l a r assocations, p a r t i c u l a r l y the r e l a t i v e extent of cros-s l i n k i n g interactions and t h e i r strength. Figure 19 i l l u s -t r a t e s the dependence of the storage and loss moduli, and dynamic v i s c o s i t y on angular frequency, f o r hydrated systems corresponding to the gel state, concentrated s o l u t i o n and d i l u t e s o l u t i o n . 1 7 1.3 SUMMARY In the previous sections an attempt has been made to introduce the Reader to three areas of major importance i n polysaccharide chemistry. These were, (1) polysaccharide SO-10* • F i g u r e 19. T y p i c a l rheograms from o s c i l l a t o r y rheometry showing the s o l i d - l i k e and l i q u i d - l i k e behav iour as c h a r a c t e r i s e d by the s to rage ( ) and l o s s ( ) modu l i G ' and G " r e s p e c t i v e l y , and by dynamic v i s c o s i t y Tf . The samples shown are 2% a g a r , 5% K -ca r rageenan , and 5% d e x t r a n . modification (with emphasis on amino polysaccharides), ( 2 ) nmr spectroscopy of polysaccharides, and ( 3 ) solu t i o n proper-t i e s of polysaccharides. I t was intended at the outset of t h i s work that attention would not be directed at a single facet of polysaccharide chemistry, but rather, that the studies should r e f l e c t the diverse i n t e r e s t i n polysaccha-r i d e s . This philosophy i s r e f l e c t e d i n the introduction, and w i l l be equally apparent throughout the discussion. -81-CHAPTER 2 BRANCHED CHITOSAN DERIVATIVES 2 . 1 INTRODUCTION Some natural branched polysaccharides, such as x a n t h a n 2 2 6 and guar gum, 1 5 5' 2 2 7 are known to possess unique aqueous sol u t i o n p r o p e r t i e s . 1 6 ' 1 5 5 ' 2 2 6 I t has also been established that many branched e x o c e l l u l a r polysaccharides have immuno-genic a c t i v i t y , 1 ' 2 ' 2 0 ' 2 1 a f a c t which renders them poten-t i a l l y useful i n biomedical and pharmacological a p p l i c a -t i o n s . 2 2 8 These two factors have helped to d i r e c t attention toward the synthesis of polysaccharides bearing pendant carbohydrate moieties. The synthesis of branched derivatives of polysaccharides has been accomplished using a v a r i e t y of d i f f e r e n t a p p r o a c h e s . 1 5 ' 2 2 9 One notable method involves the reaction of acetobromo sugars under glycosidation conditions with amylose and c e l l u l o s e to produce branched d e r i v a t i v e s . 1 5 8 ' 2 3 0 " 2 3 4 Pfannemuller et a l . . have coupled the acetobromo derivatives of glucose (8_5) , maltose and maltodextrins (up to heptasac-charides) to 2,3-di-0-phenylcarbamoyl-6-0-trityl derivative (83) of the p o l y s a c c h a r i d e s 1 5 8 ' 2 3 0 (Scheme 15a), or to the 2,3-di-0-phenylcarbamoyl d e r i v a t i v e 2 3 0 84 (Scheme 15b) . The l a t t e r produced (1+6)-a-linkages p r e f e r e n t i a l l y with l i t t l e or no depolymerization, while the former gave (1+6) - j3-lin--82-Scheme 15 -83-kages and extensive depolymerization. When acetobromoglucose was employed, the branched amylose was produced i n 50-85% y i e l d s having degrees of substitution ranging from 0.21-0.44, while the maltodextrin derivative of amylose had d.s. 0.01-0.04. 2 3 4 When c e l l u l o s e was treated i n a s i m i l a r fashion with acetobromoglucose, lower d.s. values of 0.09-0.14 were obtained. Carbohydrate 1,2-orthoesters have found a p p l i c a t i o n i n coupling to amylose and c e l l u l o s e derivatives v i a the c y c l i c orthoester glycosidation m e t h o d . 2 2 9 ' 2 3 1 ' 2 3 5 Kochetkov et a l . have reported the reaction of 3,4,6-tri-O-acetyl-a-D-glucopyranose l , 2 - ( t - b u t y l orthoacetate) with randomly substituted c e l l u l o s e diacetate, giving a product substituted mainly at primary p o s i t i o n s . 2 3 5 Pfannemuller et a l . have used both l,2-.(t-butyl orthoacetate) and 1,2-(ethyl orthoacetate) (87) derivatives of 3,4,6-tri-O-acetyl-a-D-glucopyranose i n reactions with 2,3-di-O-phenylcarbamoylamylose (£4) and c e l l u l o s e derivatives (Scheme 1 6 ) . 2 3 1 The product 88 (d.s. 0.25-0.30) contained l a r g e l y (1*6)-^Q-branches with a small amount of (1*6)-a. When the l,2 - ( e t h y l orthoacetates) of maltose, maltotetrose and maltohexose were s i m i l a r l y treated, branched polysaccharides of d.s. 0.05-0.20 were obtained. Starch polysaccharides have been reacted with 3,4-dihydro-2H-pyrans, as i n Eq. 21, to give derivatives bearing the tetrahydropyran-2-yl a c e t a l . These compounds were water-soluble at low le v e l s of subs t i t u t i o n and organic-soluble at high l e v e l s . 2 3 6 While the pendant group i n t h i s -84-O H (a) i ) lu t i d i n i u m perchlorate, chlorobenzene i i ) NaOMe/MeOH Scheme 16 Starch-OH [21] -85-case i s not a carbohydrate, i t i s coupled " g l y c o s i d i c a l l y " and indicates the pot e n t i a l of using known carbohydrate g l y c a l s (such as the glucal derivative 90) to prepare derivatives with 2-deoxy-saccharide branches. 9 0 A v a r i e t y of l i n e a r and branched synthetic polysaccha-rides have been prepared by polymerization of 1,6-anhydro sugar d e r i v a t i v e s . 2 2 9 ' 2 3 7 * 2 3 8 For example, branched dextran analogues have been prepared from two su i t a b l y protected 1,6-anhydro-D-glucose d e r i v a t i v e s . 2 3 7 The methods described here have s i g n i f i c a n t p o t e n t i a l and r e f l e c t the f i r s t generation of synthetic methods f o r making branched polysaccharides. However, various disadvantages are evident i n most cases, such as (1) requirement f o r s p e c i f i c protection, (2) multistep synthetic procedures, (3) a c t i v a -t i o n of the carbohydrate moiety, (4) low coupling e f f i c i e n -c i e s , (5) poor s i t e - s e l e c t i v i t y , and (6) harsh, degradative reaction conditions. The reductive N-alkylation methods described i n the introduction (section 1.2.1) have been used to produce chitosan derivatives, by reaction with reducing mono- and disaccharides, having a c y c l i c carbohydrate branches. 9 6 - 8 6 -S i m i l a r l y , enzymatically oxidized guar was extended by reductive amination with aminosugars. 4 5 In both cases, the branches d i f f e r s u b s t a n t i a l l y from those on natural polysac-charides. The work described i n t h i s chapter constitutes a new method fo r c o n t r o l l e d s o l u b i l i z a t i o n of chitosan v i a the introduction of hydrophilic groups. Aldehydes obtained from reductive ozonolysis of a l l y l glycosides have been reduc-t i v e l y aminated to the 2-amino group of chitosan to produce pendant g l y c o s i d i c b r a n c h e s . 2 3 9 ' 2 4 0 While the linkage i s not s t r i c t l y a g l y c o s i d i c branch, the saccharide residues are i n t a c t pyranosides and v a r i a t i o n of the pendant sugar func-t i o n a l i t y , i d e n t i t y , and linkage configuration are possible, by preparing the appropriate alkenyl glycoside precursors. Viscometric studies on aqueous solutions of t h i s family of branched chitosan derivatives have been undertaken as a means for probing structure/property r e l a t i o n s h i p s , which may f i n d more general a p p l i c a b i l i t y to other synthetic and natural branched polysaccharides. The use of 10-undecenyl ^3-D-glycosides i n analogous d e r i v a t i z a t i o n s w i l l demonstrate that v a r i a t i o n s i n the a l k y l chain length of these branched chitosans adds another dimen-sion i n the t a i l o r i n g of polysaccharide s o l u t i o n properties. This i s an extension of the concept of c o n t r o l l i n g s o l u b i l i t y properties of chitosan by co-reaction with hydrophobic and hydrophilic groups to give mixed branch d e r i v a t i v e s 1 9 (section 1.2.2). -87-2.2 N-[2'-O-(D-GLYCOPYRANOSYL)ETHYL]CHITOSAN DERIVATIVES 2.2.1 Synthesis and Characterization A wide v a r i e t y of a l l y l glycosides have been reported for use i n biochemical s t u d i e s 2 4 1 " 2 4 4 and as intermediates i n carbohydrate s y n t h e s e s . 2 4 5 ' 2 4 6 The a l l y l glycosides used i n t h i s work were prepared according to methods described by Lee and L e e . 2 4 2 The ^9-D-glycosides were prepared from the respective peracetylated a-D-glycopyranosyl halides. The acetobromo or acetochloro sugars 91-94 and 103 are well described i n the l i t e r a t u r e , and were prepared using standard methods. 2 4 7 Koenigs-Knorr g l y c o s i d a t i o n s 2 2 7 ' 2 4 8 of the acetobromo sugars 91-94 with a l l y l alcohol (Scheme 17a), provided the peracetylated a l l y l ^Q-D-glycosides 95-98. A p a r a l l e l route to the 2-acetamido-2-deoxy-/3-D-glucoside from acetochloroglucose (103), i s given i n Scheme 17b. Subsequent de-Q-acetylation gave the unprotected a l l y l ^Q-D-glyco-pyranosides 99-102 and 105. The a l l y l a-D-glycopyranosides 108 and 109 were prepared by acid-catalyzed glycosidation, as shown i n Eq. 22, where the respective free sugars, D-glucose (106) and, D-galactose(107) were refluxed i n a l l y l alcohol with Dowex 50x8, H +, ion-exchange r e s i n . The reported y i e l d s of a l l y l 2-acetamido-2-deoxy-a-D-gluco pyranoside using t h i s methods were very low ( 1 0 % ) , 2 4 2 so the preferred method, involving treatment with boron t r i f l u o r i d e - e t h e r a t e as ca t a l y s t , was used to prepare 111 (Eq. 23) from 110 i n 40% 99 OH OH H CH,OH 1 0 0 OH H OH CHjOH 101 OH OH H CO,H (a) al lyl alcohol, Hg(CN>2. Drlerite (b) NaOMe/MeOH Scheme I7a AcO-.OAc Aco>L«jrci NHAc 103 (a) al lyl alcohol, Hg(CN>2 (b) NaOMe/HeOH Scheme 17b AcO 0.0 AcO 104 NHAc H O - i NHAc 105 i CO CO I H.OH 108.109 122] (a) al lyl alcohol, Dowex S0X8, HT 106.108 OH H 107.109 H OH -89-[23] 110 vn (a) a l l y l a l c o h o l , BF^etherate y i e l d . Characterization data, such as melting points and o p t i c a l r o t a t i o n values, agreed with those reported i n the l i t e r a t u r e . 2 4 2 While published •'•H and 1 3C-nmr spectroscopic data f o r a l l y l glycosides were not ava i l a b l e i n many cases, the % and 1 3C-nmr spectra obtained agreed c l o s e l y with published chemical s h i f t and coupling constant values of the respective methyl g l y c o s i d e s . 9 9 " 1 0 1 Previously unreported a l l y l ^Q-D-glucopyranuronic acid 101. gave a 1 3C-nmr spectrum comparable to i t s respective methyl glycoside analogue, 2 8 and containing the characteris-t i c a l l y l group carbon resonances. Unfortunately, t h i s compound was not successfully c r y s t a l l i z e d and an a n a l y t i -c a l l y pure sample f o r s p e c i f i c r o t a t i o n and melting point determination was not obtained. The c r y s t a l l i n e precursor, methyl ( a l l y l 2,3,4-tri-0-acetyl - j3-D-glucopyranoside)uronate, 97, was f u l l y characterized. De-O-acetylation of 97 was performed under standard Zemplen conditions, with the t i c analysis of the reaction mixture showing a major component having an Rf value higher than expected f o r 101. Treatment with aqueous sodium hydroxide converted the component having Derivative Branch C-1 C-? > c-: J C-< 1 C-5 C-l C-] L« c-: > i c-: J * 99 j3-Glc 99 .6 71. .5 74. ,2a 68. > 0 b 74. 2 a 59. ,2 68. ,9*> 131. .8 117, .0 100 j6-Gal 100 .2 69. .1 71. .1 67. .0 73. 4 59. ,3 68. .9 131. .9 116. .9 101 jS-GlcA 99 .7 71. .1 73. .7 69. .2 72. 8 170. .4 69. .5 131. .7 117. .1 102 £-Lact (/3-Gal) 100 .2 69. .2 70, ,9 a 66. .8 73. 5 59. .3 (j3-Glc) 99 .4 71. , l a 72. .7* 76. .8 73. 0 b 58. .4 68. .9 131. .7 117. .0 105. #-GlcNAc 98 .8 54. .2 72. .5 68. .7 74. 5 59. .5 68. .9 132. .3 116. .5 108 a-Glc 95 .7 69. ,6 71. .5 68. .0 70. 2 59. .0 66. .8 132. .1 116. .5 109 a-Gal 96 .1 66. . 9 * 68. .0 67. .7 69. 3 59. ,6 66. ,7 a 132. .2 116. .5 111 a-GlcNAc 94 .5 52. .0 69, .4 68. .4 70. 3 59. .0 66. .8 132. .1 116. .2 Table 2. 100.6 MHz 1 3C-nmr chemical s h i f t data (ppm) f o r the a l l y l glycosides i n D 20 solution (ref. external TMS). a. Assignments may be reversed. b. II -91-the high Rf into a lower Rf material corresponding to the product. A complete l i s t i n g of the 1 3C-chemical s h i f t values for the a l l y l glycosides prepared i s given i n Table 2. The a l l y l glycoside precursors 99-102. 105. 108. 109 and 111 were reductively o z o n o l y z e d 2 4 9 to provide the respective acetaldehydo glycosides 112-119 (Scheme 1 8 ) . 2 4 0 ' 2 5 0 Aldehydes of t h i s type e x i s t i n a v a r i e t y of equilibrium states, including the gem-diol. the intramolecular c y c l i c hemiacetals and acetal oligomers. Hence d i r e c t characterizations were not attempted. Previous work done i n t h i s l a b o r a t o r y , 2 4 0 ' 2 5 0 i n which aldehydes of t h i s sort were reduced and acetylated for characterization purposes, established that the ozonolysis of a l l y l glycosides proceeds i n a v i r t u a l l y quantitative manner. Thus i n most cases, the aldehyde products were used d i r e c t l y i n the subsequent step. The aldehyde 114 was reduced and characterized by 1 3C-nmr spectroscopy i n order to e s t a b l i s h the s t a b i l i t y of uronosides to ozonolytic conditions. The sol u t i o n 1 3C-nmr spectrum of the product 128 established that the carbohydrate portion of the molecule was unaltered, and that the expected 2-hydroxyethyl glycoside was the product. Reductive amination 8 6 of chitosan with the aldehydes 112-119. was performed as outlined i n Scheme 18, to y i e l d -92-Scheme 18 Ri R2 R 3 R* 9 9 , 1 0 8 , 1 1 2 , 1 1 7 , 1 2 0 , 1 2 5 OH OH H CH 2 0H 1 0 0 , 1 0 9 , 1 1 3 , 1 1 8 , 1 _ 2 1 , 1 2 6 OH H OH CHjOH 101 ,114 ,122 OH OH H C 0 2 H 1 0 5 , 1 1 1 , 1 1 6 , 1 1 9 , 1 2 4 , 1 2 7 NHAc OH H CHjOH HO-HO 1 0 2 , 1 1 5 , 1 2 3 OH J H CH 2 OH d e r i v a t i v e s 120-127. The aldehydes were dissolved i n 10-15 mL of the reaction media (5% aqueous a c e t i c acid) and added to a viscous chitosan solution (@ 1 mmol/10 mL). Treatment with excess sodium cyanoborohydride resulted i n appreciable foaming which dissipated over time. A f t e r the reaction had been s t i r r e d f o r 24 hours, i t was f i l t e r e d to remove i n s o l -uble material. The only case where f i l t r a t i o n was not pos-s i b l e was i n the preparation of 122a. where the product p r e c i p i t a t e d during the reaction. Varying the molar r a t i o of aldehyde to chitosan (A/C) gave products with a range of degree of s u b s t i t u t i o n (d.s.) values, as shown i n Table 3. For example, a molar r a t i o of - 3 was used to give f u l l y or highly substituted derivatives (e.g. 121a-126a), while a r a t i o of 0.50 gave products with low d.s. (124d. 125d, 127e). The degree of s u b s t i t u t i o n values were determined from C, H and N elemental microanalyses (see appendix A). Of note i s the f a c t that f o r the ^9-D-lactosyl d e r i v a t i v e 123a. an A/C r a t i o of 3.0 resulted i n a d.s. of 0.90, i n d i c a t i n g that the s i z e of the substituent influenced the coupling e f f i c i e n c y , as would be expected. This i s further supported by the r e s u l t s of derivatives 127c and 127d. i n which A/C r a t i o s of 1.5 and 0.75 provided d.s. values 0.35 and 0.19 respectively, s i g n i f i c a n t l y lower than r e s u l t s for the 121. 123 and 125 s e r i e s of d e r i v a t i v e s . In the case of the l a c t o s y l deriva-t i v e s , the s i z e e f f e c t seemed to be manifested mainly at high d.s. values, hindering complete sub s t i t u t i o n , while f o r the acetamido derivatives r e l a t i v e l y lower d.s. products were Derivative Branch A/C d.s. Yieldf%) (±.05) 120a iQ-Glc 3.1 1.00 95 121a 0-Gal 2.7 1.00 60 b 1.3 0.70 85 _c 0.75 0.38 80 122a fl-GlcA 3.0 1.00 70 1.0 0.67 80 123a fl-Lact 3.1 0.90 95 h 1.5 0.76 85 c 0.75 0.35 95 0.50 0.32 95 0.35 0.24 87 124a /3-GlcNAc 3.0 1.00 85 125a a-Glc 3.0 1.00 95 _b 1.5 0.59 60 c 0.75 0.38 70 i i 0.5 0.26 80 126a a-Gal 3.1 1.00 60 b 2.0 0.86 55 c 1.0 0.48 95 _d 0.75 0.32 95 127a a-GlcNAc 3.1 1.00 90 b 3.1 1.00 85 1.5 0.35 , 95 _d 0.75 0.19 95 e 0.5 0.17 95 Table 3. Cha r a c t e r i s t i c s of N-[2-0-(D-glycopyranosyl)ethyl]-chitosan derivatives. obtained at a l l A/C r a t i o s less than 3.0. This could be an i n d i c a t i o n that the l a t t e r e f f e c t was not s t r i c t l y due to s i z e , but may r e l a t e to molecular associations or repulsions involving free amino f u n c t i o n a l i t i e s on the backbone and -95-acetamido groups on the branch. 1 3C-nmr spectra were recorded f o r a l l of the highly substituted derivatives for each sugar branch. These deriva-t i v e s were highly soluble and gave free flowing 5% (w/w) solutions i n D 20. The linewidths of the branch carbon resonances were r e l a t i v e l y narrow (5-10 Hz) i n comparison to the chitosan main chain resonances (100-200 Hz), as shown i n F i g . 20. 1 3 C chemical s h i f t assignments (Table 4 ) were e a s i l y accomplished by comparison to published values f o r methyl g l y c o s i d e s 9 9 * 1 0 0 or to values given i n Table 2 f o r the respective a l l y l glycosides. 1 3C-nmr spectra of derivatives 121b. 125c and 126c. having lower d.s. values, show su b s t a n t i a l l y broader signals f o r branch carbons than f o r the high d.s. analogues 121a, 125a. and 126b as shown i n Figs. 20, 21 and 22. This i s i n d i c a t i v e of i n t e r r e l a t i o n s h i p s between the degree of substitution, s o l u t i o n v i s c o s i t y and branch mobility, as manifested i n the c o r r e l a t i o n time ( r c ) dependence of T 2 and linewidth ( ^ i / 2 ) . 1 1 1 I t i s i n t e r e s t i n g to note the increased linewidth and reduced i n t e n s i t y of the C - l ' carbon of the branch, when compared to C-6 or other ri n g carbon resonances on the same deriva t i v e , i l l u s t r a t i n g the reduced mobility of positions c l o s e r to the main chain. Thus, not only does 1 3C-nmr spectroscopy provide proof of struc-t u r a l modification, i t also allows one to discern resonances on the basis of mobility and i t provides a q u a l i t a t i v e i n d i c a t i o n of r e l a t i v e v i s c o s i t i e s . Derivative Branch C-1 C-2 C-3 C-4 C-5 C-6 C-2' C-1' 120a /3-Glc 100.8 71.7 74.2 68.2 74.4 59.4 67.3 45.8 121a /3-Gal 101.4 67.2 71.2 69.3 73.6 59.5 67.5 45.8 122a /3-GlcA 100.7 71.6 74.1 70.4 74.4 174.1 67.4 45.8 123a /9-Lact 0-Gal) 101.4 69.4 71.1 67.0 73.8 59.4 0-Glc) 100.5 71.3 72.8 77.1 73.2 58.7 67.2 45.7 124c jS-GlcNAc 99.5 54.0 72.3 68.4 74.3 59.3 67.2 45.8 125a a-Glc 97.0 69.9 71.6 68.2 70.4 59.2 65.5 45.6 126b a-Gal 97.2 66.9 68.1 67.8 69.5 59.7 65.6 45.6 127a a-GlcNAc 95.6 52.1 69.5 68.5 70.5 59.1 65.4 45.6 129 a-GlcNH 2 97.5 53.9 69.1 69.0 71.2 59.8 66.2 45.9 130a (a-Glc) 96.9 69.8 70.3 68.0 71.5 59.0 65.0 45.5 (jQ-GlcNAc) * 99.7 53.8 — 77.5 76.0 60.9 Table 4. 100.6 MHz 1 3C-nmr data for the N-ethyl glycoside branched chitosan derivatives, showing chemical s h i f t values (ppm) for pendant sugar resonances (ref. external TMS). * GlcNAc of backbone, NAc resonances; CH 3 20.5, C=0 173.2. @56,000 t r a n s i e n t s I i 1 1 1 1 1 1 1 1 1 l 1 I C - 4 i r c - i C —I (backbone) / C - 6 @60,000 t r a n s i e n t s C - 1 T 1 1 1 1 1 I I I 120 T 1 1 r 8 0 P P M 4 0 13, F i gu re 20. 100.6 MHz C-nmr s p e c t r a l r e g i o n showing branch res idue resonances f o r a) 121a ( d . s . 1 .0 ) ; and b) 121b ( d . s . 0 . 7 0 ) , i n D 2 0 ( r e f . e x t e r n a l TMS). @96,000 t r a n s i e n t s 1 1 1 1 1 1 i r T 1 1 1 1 i r c - i C - l ' (backbone) \ C - 4 C - 3 C - 5 JJMJ C - 2 C - 2 ' C - 6 (§87,000 t r a n s i e n t s I 1 1 1 I 1 1 1 1 1 1 1 1 i 1 1 1 120 8 0 4 0 P P M 13 F i g u r e " 2 1 . An expanded r e g i o n of the 100.6 MHz C-nmr spec t r a of a)126b ( d . s . I.00) ; and b) 126d ( d . s . 0 . 3 2 ) , i n ELO, showing the branch r e s i d u e resonances ( r e f . e x t e r n a l TMS). F igu re 22. An expanded r e g i o n of the 100.6 MHz C-nmr spec t r a of a) 125a ( d . s . 1 .00) ; and b) 125c ( d . s . 0 . 3 8 ) , i n D 2 0 , showing the branch res idue s i g n a l s ( r e f . e x t e r n a l TMS). -100-The resonances due to the chitosan backbone are evident to some extent i n most of the derivatives' spectra. In general, the resonances of the anomeric carbon (C-l^) and C-4 D carbon are d i s c e r n i b l e . No attempt has been made here to assign a l l the chitosan resonances, p a r t i c u l a r l y i n interme-diate and low d.s. samples where the s p l i t t i n g of resonances, due to substituted and unsubstituted residues, complicated assignment, and broad l i n e s obscured the peaks. In a l l cases, i t was possible to assign the C-l* 5 s i g n a l to the 98-102 ppm region (depending on substitution) and C-4*3 to the 75-80 ppm range. The u t i l i t y of secondary modification of chitosan deriva-t i v e s has been alluded to. The derivatives described here having d.s. <1.0, are suitable candidates f o r homogeneous chemical reaction i n aqueous media. To demonstrate t h i s , three secondary modification sequences were undertaken. Two of these w i l l be mentioned here, and the t h i r d w i l l be described i n the context of a study to be discussed l a t e r i n t h i s chapter. One comparison we f e l t would be of value was to contrast the properties of the 2-acetamido-a-D-glucose d e r i v a t i v e (127a) with those of a deri v a t i v e bearing pendant 2-amino-a-D-glucose un i t s . As such, d e r i v a t i v e 127b was subjected to treatment with 40% aqueous NaOH at 100°C (Eq. 24). Both elemental analyses and 1 3C-nmr spectra v e r i f i e d the absence of N-acetate groups i n the desired product 129. Although i t remained to be seen whether depolymerization occurred, q u a l i t a t i v e observations of deri v a t i v e 129 were -101-encouraging. A second procedure, which o f f e r s a f a c i l e method fo r c o n t r o l l i n g or modifying the s o l u b i l i t y properties of the N-ethylglycosyl chitosan derivatives i s N-acetylation. Treatment of a solu t i o n of 125c (d.s. 0.38) i n aqueous methanol (1:1) with a c e t i c anhydride provided d e r i v a t i v e 130 (Eq. 25), which showed c h a r a c t e r i s t i c N-acetate peaks i n i t s 1 2 5 (a) Me0H/H20 (1:1), Ac20 130 -102-1 3C-nmr spectrum and analyzed for f u l l N-acetylation at a l l unsubstituted amines (d.s. of HNAc 0.62). Compound 130, having a high degree of N-acetylation, would q u a l i f y as a N-[2-0(a-D-glucopyranosyl)ethyl] c h i t i n d e r i v a t i v e . We now have i n hand a family of s t r u c t u r a l l y - r e l a t e d water-soluble derivatives bearing pendant carbohydrates with vari e d f u n c t i o n a l i t y , g l y c o s i d i c configuration, and s i z e at a number of degrees of substitution. This constitutes an i d e a l array of compounds for use i n studies r e l a t i n g s o l u t i o n properties to s t r u c t u r a l features. 2.2.2 Viscometry Steady shear rheometric determinations were performed on 2.0% (w/w) solutions (unless otherwise specified) of polysac-charide derivatives and commercial polysaccharides, i n d i s t i l l e d water at 20° ±0.5°C. Measurements were done using a r o t a t i o n a l viscometer with cone and plate geometry, from which shear stress (cr) values at shear rates (f) ranging from 1-2500 s" 1, were obtained. Apparent v i s c o s i t i e s (i?) were determined according to Eq. 26. n = I [26] Rheograms of apparent v i s c o s i t y against shear rate for solutions of derivatives 120-127 and f o r commercial samples of xanthan gum, hydroxyethyl c e l l u l o s e and sodium alginate are presented on l i n e a r and logarithmic coordinates i n Figs. 23-38. For purposes of comparison, i t was decided to employ -103 the power-law equation: o = my1 [12] where n i s the flow behaviour index, and m i s the consistency c o e f f i c i e n t , to model the flow behaviour of the solutions examined. Thus, regression of logarithm of shear stress against logarithm of shear rate provided the parameter n and the logarithm of m. The parameters, n and m, determined for a l l solutions, are presented i n Table 5. The rheograms on logarithmic axes therefore represent the power-law modelling, and i t i s immediately obvious that these p l o t s are consider-ably easier to analyze than those on l i n e a r axes. A f t e r c a r e f u l examination of the logarithmic rheograms, i t might be concluded that the power-law model i s not t o t a l l y appropriate for a l l solutions studied. I t would be more correct to say that, i n some cases, the experimental data extends past the shear rate range where the power-law model i s appropriate. There i s no doubt, however, that a l l of the derivatives prepared are adequately modelled by the power-law equation over a substantial portion of the experimental shear rate range. This i s supported by the high c o r r e l a t i o n c o e f f i c i e n t s (R 2) shown i n Table 5. The power-law has been frequently used to model the flow behaviour of polysaccharide solutions and d i s p e r s i o n s . 2 2 3 * 2 2 4 This method of analysis was f e l t to be superior to examination of apparent v i s c o s i t i e s as a f r a c t i o n of zero shear v i s c o s i t y (17 D) or at s p e c i f i c shear rate values representative of the experimental range (e.g. 10 -104-Derivative Branch d.s. n m R- #Points (±.05) ± 3% (mPa-s) ± 3% 120a /3-G1C 1.00 1.01 22. 4 .998 84 121a £-Gal 1.00 0.879 82. 7 .999 36 b 0.70 0.783 434 .997 33 c 0.38 0.588 2300 .995 50 122a /3-GlcA 1.00 0.970 45 .999 36 b 0.67 0.911 109 .997 32 123a /3-Lact 0.90 0.997 24. 9 .999 53 h 0.76 0.939 49. 4 .999 28 c 0.35 0.841 222 .997 30 d 0.32 0.891 159 .997 34 124a /3-GlcNAc 1.00 1.05 14. 3 .999 24 125a a-Glc 1.00 0.929 65. 1 .998 62 b 0.59 0.695 803 .999 34 c 0.38 0.677 1090 .998 33 d a 0.26 0.502 3270 .988 41 126a a-Gal 1.00 1.00 25. 3 .999 56 h 0.86 0.931 54. 1 .999 59 0.48 0.554 5180 .992 43 d 0.32 0.778 525 .998 60 127c a-GlcNAc 0.35 0.864 196 .997 29 d 0.19 0.822 456 .994 31 _e 0.17 0.810 456 .996 36 129 a-GlcNH 2 1.00 0.845 110 .999 37 Xan. 0.296 10200 .938 38 HEC 0.426 9230 .985 24 NaALG 0.717 3180 .988 34 Table 5. Power-law parameters f o r branched chitosan deriva-t i v e solutions (2.0% w/w i n water), xanthan gum (Xan), hydroxyethylcellulose (HEC), and sodium alginate (NaALG). a. Data given i s f o r a 1% solution. -105-and 1000 s - 1 ) . The parameter n i s a d i r e c t measure of the shear rate dependence or pseudoplasticity of the so l u t i o n while m corresponds to v i s c o s i t y at a shear rate of 1 s " 1 . For shear thinning f l u i d s , of which polysaccharide solutions are t y p i c a l examples, n can vary from 1.0, f o r solutions having Newtonian (no shear rate dependence) to 0.0-0.2, which describe extremely pseudoplastic f l u i d s . Comparison of m values provides an index of r e l a t i v e v i s c o s i t y (at a shear rate of 1 s" 1) f o r sample solutions. Thus, using these two parameters, n and m, we can begin to examine and contrast the flow behaviour of the solutions characterized i n Table 5. Discussion w i l l be l i m i t e d l a r g e l y to power-law parameters and the respective logarithmic rheograms. The l i n e a r rheo-grams are included f o r the readers reference, but they w i l l not be discussed d i r e c t l y . The f i r s t notable trend i n the rheological properties of the d e r i v a t i v e solutions i s that the f u l l y substituted derivatives 120a-122a and 124a-126a and the highly substituted l a c t o s y l compound 123a have Newtonian or close to Newtonian flow behaviour, as indicated by n ~ l . This i s probably i n d i c a t i v e of r e l a t i v e l y l i t t l e i nterchain interac-t i o n , and hence the disorder-order e q u i l i b r i a f o r these der i v a t i v e s would l i e f a r to the l e f t , and are not further disrupted by shearing forces. Derivatives 121a and 125a are s l i g h t l y anomalous i n that they have n values of -0.9, and thus have a small shear rate dependence. A possible explana-t i o n i s that these derivatives are capable.of stronger -106-(A O CL J E , >-H ; CO O O (/} > 3000 2500-500 1000 1500 2000 S H E A R RATE ( s - 1 ) 2500 F igu re 23 . Rheograms f o r 2.0% aqueous s o l u t i o n s of d e r i v a t i v e s a) 125a ( O ) , 125b ( • ) , 125c ( D ) ; a n d b) 125d ( O ) a t 2 0 ' C , on l i n e a r axes . -107-l O O O q > 10 ^^^^^^^ 1 10 100 1000 10000 SHEAR RATE (s_1) 10000^ ; 1 C O > 1 10 100 1000 10000 SHEAR RATE (s_1) F igu re 24. Rheograms f o r 2.0% aqueous s o l u t i o n s of p roducts a) 125a ( O ) , 125b ( • ) , 125c ( D ) ; a n d 5) 125d ( O ) on l o g a r i t h m i c a x e s . -108-5000 (A 4 0 0 0 -I 3000 : O H 0 -mJL 100 200 300 SHEAR RATE (s _ 1 ) 400 500 re 25 . Rheograms f o r 2.0% aqueous s o l u t i o n s of d e r i v a t i v e s a) 126a ( O ) , 126b ( • ) , 126d ( • ) ; and b) 126c ( • ) on l i n e a r a x e s . -109-0 I i i i i i i i i i i i i i i i . . . . , . • . . 0 500 1000 1500 2000 2500 SHEAR RATE (s _ 1) 2000-. O 1500-J , >" 1000-0-500 1000 SHEAR RATE (s"1) 1500 Figure 26. Rheograms f o r 2.0% aqueous solutions of der i v a t i v e s a) 121a ( • ) , 121b ( • ) ; and b) 121c ( O ) on l i n e a r axes. -110-10000 6 CL co o CJ CO > 1000-100-10- I 1 1 1 TTTT I I III 10 100 1000 SHEAR RATE (s"') i i i 111111 10000 10000^ o CL CO o o CO > 1000-100-10 100 1000 SHEAR RATE (s' 1) 10000 F igu re 27. Rheograms f o r 2.0% aqueous s o l u t i o n s of compounds a) 126a ( O ) , 126b ( • ) , 126c ( ! ) and 126d ( • ) ; and b) 121a ( • ) , 121b ( • ) and 121c ( O ) on l o g a r i t h m i c c o o r d i n a t e s . -111-100 o CL o  O o 0 0 > 500 1000 1500 2000 2500 S H E A R R A T E ( s - 1 ) 1000q o C L 0 0 O o 0 0 > 100T 10 100 S H E A R R A T E ( s _ 1 ) i 1000 10000 F igu re 28. rheograms f o r 2.0% aqueous s o l u t i o n s of d e r i v a t i v e s a) 120a ( O ) on l i n e a r a x e s ; and b) 120a ( O ) on l o g a r i t h m i c a x e s . -112-100-1 o CL J , 00 o o 00 > o + 0 "^Bi Ofci ft O ' I 1 ' ' 1—I ' 1—1—'—I—' ' ' '—I ' ' ' ' i 500 1000 1500 2000 2500 S H E A R R A T E ( s _ l ) D CL E 00 o CJ 00 > 1000^ 100-1 " i n 10 100 1000 S H E A R R A T E ( s " 1 ) 10000 F igu re 29. Rheograms f o r 2.0% aqueous s o l u t i o n s of a) 122a ( O ) , 122b ( • ) on l i n e a r a x e s ; and b) 122a ( O ) and 122b ( • ) on l o g a r i t h m i c c o o r d i n a t e s . -113-O ,00 5 0 0 1000 1500 2000 S H E A R R A T E ( s " 1 ) 2500 350 O 250 500 1000 1500 2000 S H E A R R A T E ( s - 1 ) 2500 F igu re 30. Rheograms f o r 2.0% aqueous s o l u t i o n s of d e r i v a t i v e s a) 127a ( • ) , 127b ( O ) , 127c ( • ) and 127d ( • ) ; and b) 124a ( A ) a t 20 C, on l i n e a r a x e s . -114-200 150-1 500 1000 1500 2000 2500 S H E A R R A T E ( s _ 1 ) F i g u r e 31 . Rheograms f o r a) 2.0% aqueous s o l u t i o n s of 123a ( O ) , 123b ( # ) , 123c ( • ) and 123d ( • ) ; and b) 2.7% aqueous s o l u t i o n of 123a, a t 20 C on l i n e a r a x e s . -115-1000zr O Q_ >-CO O CJ CO > 10 100 1000 S H E A R R A T E ( s _ 1 ) 11.11 10000 1000q CO 6 CL co o o co > 100t t 10 T i i i 111111 i i i 111111 10 100 S H E A R R A T E ( s - 1 ) i i i1111 1000 I I I 1111 10000 F i g u r e 32 . rheograms f o r 2.0% aqueous s o l u t i o n s of d e r i v a t i v e s a) 124a ( • ) , 127a ( O ) , 127b ( • ) , 127c ( • ) ; and b) 123a ( O ) , 123b ( O ) , 123c ( • ) , 123d and a 2.7% s o l u t i o n of 123a ( A ) on l o g a r i t h m i c c o o r d i n a t e s . Figure 33. Rheograms for aqueous xanthan dispersions having concentrations of 0.25% ( • ) , 0.5% ( • ) , 1.0% ( • ) , and 2.0% ( O ) on l i n e a r coordinates, at 20°C. -117-5000 -r i7> 4 0 0 0 -6 Q_ >-3 0 0 0 T o  2 0 0 0 O o o  S 1 0 0 0 500 1000 1500 2000 S H E A R R A T E ( s _ 1 ) 2500 2500 ^ 2 0 0 0 : , ~ — W 9 WW  I ' ' ' ' I ' ' • • I • ' 1 1 I 1 1 1 1 I 1 1 1 ' 0 500 1000 1500 2000 2500 S H E A R R A T E ( s _ 1 ) F igu re 34. Rheograms f o r a) h y d r o x y e t h y l - c e l l u l o s e s o l u t i o n s of 1.0% ( O ) and 2.0% ( • ) c o n c e n t r a t i o n ; and b) sodium a l g i n a t e s o l u t i o n s of 1.0% ( • ) and 2.0% ( O ) c o n c e n t r a t i o n , on a r i t h m e t i c a x e s . -118-10000^ co 6 Q_ E c/) o o CO > 1000-100-I I I I I 111 I I I I I 11II 10 100 S H E A R R A T E ( s - 1 ) i — i 111 • i • [ — i — i 111111 1000 10000 10000 CO 6 CL £ CO o CJ CO > 1000-100^ S H E A R R A T E ( s _ 1 ) I I I I I I | l I I I I i I I | 1000 10000 F i g u r e 35 . Rheograms on l o g a r i t h m i c axes f o r a) 0.25% ( • ) , 0.5% ( • ) , 1.0% ( • ) and 2.0% ( O ) xanthan gum d i s p e r s i o n s ; and b) 1.0% ( • ) arid 2.0% ( • ) h y d r o x y e t h y l - c e l l u l o s e and 1.0% ( • ) and 2.0% ( O ) sodium a l g i n a t e s o l u t i o n s . Figure 36. Rheograms comparing 2.0% (w/w) aqueous s o l u t i o n s of xanthan ( O ) , sodium a l g i n a t e ( • ) , 121c ( • ) , and 126c ( I ) on l i n e a r c o o r d i n a t e s . o SHEAR RATE (s"1) F igu re 37. Rheograms fo r 1.0% aqueous s o l u t i o n s of xanthan (O). sodium a l g i n a t e ( • ) and d e r i v a t i v e 125d ( • ) , on l i n e a r axes . -121-10000-3-o CL E 00 o o 00 > 1000 100 I I I M ! l | I I I I I I I 10 100 1000 S H E A R R A T E ( s _ 1 ) 10000 10000^ 6 CL E 00 o o 00 > 1000. 100-104 1 i i i i 1111 i i i i 111 10 100 1000 S H E A R R A T E ( s _ 1 ) 10000 F igu re 38. Rheograms on l o g a r i t h m i c coo rd i na tes f o r a) 1.0% aqueous s o l u t i o n s of xanthan gum ( O ) , sodium a l g i n a t e ( • ) and d e r i v a t i v e 125d ( • ) ; and b) 2.0% s o l u t i o n s of xanthan gum ( O ) , sodium a l g i n a t e ( • ) , 121c ( • ) and 126c ( • ) . -122-s e l f - a s s o c i a t i o n than the other highly substituted deriva-t i v e s . However, despite these minor v a r i a t i o n s i t i s obvious that a l l solutions of highly substituted derivatives exhibit very l i t t l e shear rate dependence. The m parameters fo r the high d.s. derivatives range from 14-80 mPa*sn, compared to , water with a v i s c o s i t y of 1.0 mPa*s A related observation i s the increase i n m and decrease i n n as the d.s. decreases f o r a se r i e s of derivatives contain-ing the same branch. For example, of the -galactosyl series 121a. 121b. and 121c (Fig. 27b), 121c having a d.s. 0.38 i s considerably more viscous (m 2300 mPa*sn) and pseudoplastic (n 0.588) than 121b (with m 434 mPa*sn and n 0.783). A sim i l a r trend i s seen i n the a-D-glucosyl (Fig. 24), y8-D-lactosyl (Fig. 32b), o-D-2-acetamido-2-deoxy-glucosyl (Fig. 32a) and at -D-galactosyl (Fig. 27a) s e r i e s . In the l a c t o s y l s e r i e s , 123a-d, the magnitude of the change i s considerably l e s s , both i n terms of v i s c o s i t y and pseudoplasticity. This i s undoubtedly a r e s u l t of the larg e r subsitutent being unable to form interchain interactions at r e l a t i v e l y lower l e v e l s of sub s t i t u t i o n than a monosaccharide branch. A l l of these -observations indicate that samples with low d.s. are more capable of interchain associations. Two possible mechanisms for t h i s are: (1) i n t e r d i g i t i z a t i o n of chains, as depicted i n Fig.39, and (2) occurrence of interchain interactions analo-gous to those present i n native c h i t o s a n . 2 3 For the l a t t e r case, a p a r t i c u l a r conformational state might provide "open" and "branched" faces, much l i k e those proposed i n seed -123-F igu re 39. I d e a l i z e d schemat ic of the i n t e r a c t i o n s of branched c h i t o s a n d e r i v a t i v e s i n aqueous s o l u t i o n . galactomannan s e l f - a s s o c i a t i o n s . 1 7 ' 2 0 1 The "open" faces could then i n t e r a c t , much as i n chitosan, with regions of disrup-t i o n imparting o v e r a l l s o l u b i l i t y (Fig. 40). The l a t t e r model may provide an explanation f o r the apparently anomalous r e s u l t f o r deriv a t i v e 126c. which has a d.s. of 0.48 and i s considerably more viscous than the analogous deriv a t i v e 126d, which has d.s. 0.32. Assuming that interactions occur as -124-F igu re 40 . I d e a l i z e d schemat ic of i n t e r a c t i o n s i n branched c h i t o s a n s e l f - a s s o c i a t i o n , i n which backbone-backbone i n t e r a c t i o n , between u n s u b s t i t u t e d or c o n f o r m a t i o n a l l y a c c e s s i b l e cha in segment, o c c u r s . depicted i n F i g . 40, i t i s reasonable that an optimum degree of s u b s t i t u t i o n , and sub s t i t u t i o n pattern, w i l l e x i s t for maximum interchain association. Indeed, recent studies have shown that both su b s t i t u t i o n pattern and degree of branching greatly influence galactomannan i n t e r a c t i o n s . 2 0 0 ' 2 0 9 Thus, perhaps f o r t h i s system a random su b s t i t u t i o n of -50% i s near optimum and viscous properties decrease on e i t h e r side of t h i s value. Of course, i t i s l i k e l y that v i s c o s i t y and -125-pseudoplasticity would increase again at quite low d.s. values, as larger blocks of unsubstituted backbone could adopt chitosan-like interactions, eventually r e s u l t i n g i n gels and i n s o l u b i l i z a t i o n . Derivatives 123e and 125d, having d.s. < 0.3 and giving a gel and a very viscous sol u t i o n respectively, provide some support f o r t h i s proposal. F i g . 41 shows the experimental data pl o t t e d as n vs d.s., f o r neutral monosaccharide branched derivatives, and the dotted l i n e n d . s F igu re 4 1 . The cons i s t ency c o e f f i c i e n t s (n) of the n e u t r a l monosaccharide branch d e r i v a t i v e s , 121a-c ( A ) , 125a-d ( • ) , and 126a-d ( • ) p l o t t e d aga ins t degree of s u b s t i t u t i o n ( d . s . ) , and a curve (—) r e p r e s e n t i n g the p o s t u l a t e d r e l a t i o n s h i p between n and d . s . -126-represents an i d e a l i z e d r e l a t i o n s h i p as j u s t put f o r t h . Much of the reasoning behind t h i s postulation has been used i n explaining the behaviour of galactomannans such as locust bean and guar gum, and i s thus not completely without prece-d e n t . 1 7 * 2 0 0 ' 2 0 1 ' 2 0 9 Also, i t might be expected that deriva-t i v e s of the sort prepared here would have behaviour most s i m i l a r to that of natural branched polysaccharides bearing s i n g l e pendant residues. I t must be remembered that, although 2.0% (w/w) solutions were used fo r a l l derivatives throughout these studies, the molar concentration of the solutions varied with the degree of s u b s t i t u t i o n of the sample. Table 6 contains some calcu-l a t e d average molecular weights fo r hypothetical derivatives bearing neutral monosaccharide branches at a v a r i e t y of d.s. values. I t can be seen that a f u l l y substituted derivative at d.s. MW molar r a t i o equimolar cone.(%) 0.2 202.2 0.4 243.4 0.6 284.6 0.8 325.8 1.0 367.0 Table 6. V a r i a t i o n of molar concentration of 2.0% solutions with d.s., for N-[2-0-(glycopyranosyl)ethyl]chitosan d e r i v a t i v e s . 2.0% concentration w i l l have fewer polymer chains (67%) than a der i v a t i v e having d.s. of 0.4. However, i t i s believed that 1.82 1.51 1.29 1.13 1.00 3.64 3.02 2.58 2.26 2.00 -127-these concentration discrepancies could not account fo r the changes i n rheology that were observed. Tests done on a 2.7% s o l u t i o n of the l a c t o s y l d e r i v a t i v e 123a having d.s. 0.9, showed that the 2.7% s o l u t i o n d i d not vary s u b s t a n t i a l l y i n rheology from i t s 2.0% solution, and d i d not match the 2.0% s o l u t i o n of 123b having a molar concentration -20% lower (Fig.32b). In order to investigate the r o l e of functional groups i n the interactions of the branched chitosan d e r i v a t i v e s described here, polymers bearing pendant a and ^Q-acetamido-D-glucose and yQ-D-glucuronate residues were prepared. The s o l u t i o n properties of 127c-e having acetamido-a-glucose branches, are somewhat anomalous from the r e s t of the mono-saccharide branched compounds. Even at low d.s. values (0.19 and 0.17 respectively f o r 127d and 127e) the solutions are not very pseudoplastic. I t was e a r l i e r mentioned that the coupling e f f i c i e n c i e s i n the preparation of these compounds were poor, y i e l d i n g low d.s. at r e l a t i v e l y high molar r a t i o s , probably due to s t e r i c or e l e c t r o s t a t i c forces. This may also be the reason f o r low pseudoplasticity, since as described for the l a c t o s y l derivatives, a b u l k i e r substituent would l i k e l y i n t e r f e r e i n the interchain association. The deriva-t i v e 124a. containing the j3-D-acetamido-glucose moiety also seems to f i t t h i s trend. However, i f the sugar s i z e i s influencing both the reaction e f f i c i e n c y and resultant s o l u t i o n properties, i t seems somewhat su r p r i s i n g that at a mole r a t i o of 3.0, f u l l y substituted derivatives 124a. 127a -128-and 127b were e a s i l y obtained. Another i n t e r e s t i n g r e s u l t was obtained f o r 127a i n the acetamido-glucose s e r i e s . Surpris-ingly, t h i s f u l l y substituted d e r i v a t i v e d i d not dissolve i n water or aqueous a c e t i c acid, but formed a c l e a r r i g i d g e l . This was anomalous when compared to 124a. the f u l l y s u b s t i -tuted jQ-analogue which was soluble i n water. The 1 3C-nmr spectrum showed however, that 127a a c t u a l l y contained both a and ^Q-2-acetamido-2-deoxy-D-glucose residues i n a 7:1 r a t i o . When der i v a t i v e 127b. having only a-acetamido-glucose branches was prepared, i t was also found to be soluble i n water. Thus, i n contrast to 127b and 124a containing pure and j3-acetamido-glucose substituents respectively, 127a gave a g e l , seemingly due to the presence of both the a and isomers as co-branches. The N-deacetylation of 127b having 2-acetami-do-2-deoxy-a-glucose branches with d.s. 1.0, was undertaken i n order to provide the free amino de r i v a t i v e 129 (Eq. 24). I t i s somewhat inappropriate to d i r e c t l y compare the solution propeties of 129 to the other derivatives because of the extra chemical, and p o t e n t i a l l y degradative, treatment required f o r i t s preparation. Since degradation would r e s u l t i n reduced v i s c o s i t y , the increased v i s c o s i t y of 129 i n r e l a t i o n to 124a and 127c indicates that the free amino group i s involved i n stronger interactions than i t s acetylated analogue. I f any depolymerization did occur, i t i s masked by the increased v i s c o s i t y due to s e l f - a s s o c i a t i o n . The derivatives 122a and 122b containing pendant -129-jQ-D-glucuronate residues, had some i n t e r e s t i n g features. As mentioned previously the a c i d i c forms of these derivatives were water-insoluble. The sodium s a l t s were r e a d i l y soluble and gave solutions having r e l a t i v e l y low v i s c o s i t y and l i t t l e pseudoplastic character. Derivatives of t h i s v a r i e t y have some p o t e n t i a l ; i t i s apparent that, i n the a c i d i c form, they have increased s o l u b i l i t y at low d.s., while f o r the sodium s a l t , v i s c o s i t y increased with lower d.s. I t was believed that derivatives bearing pendant uronic acid residues might be useful as metal chelates. I t was found with 122a and 122b that the addition of varying amounts of C a + 2 and C u + 2 ions had no apparent e f f e c t on s o l u t i o n properties. In hindsight however, i t seems possible that the analogous derivatives bearing the galacturonate residue, rather than glucuronate, might have more success i n t h i s regard. This postulation i s based on the proven a b i l i t y of galacturonate residues (in pectins) to chelate calcium. An attempt to prepare a deriva-t i v e having galacturonic acid branches was undertaken, based on the galactose oxidase oxidation followed by treatment with aqueous bromine, as described f o r the synthesis of 26. (section 1.2.2). The oxidation appeared to proceed as expected and afforded a s o l i d p r e c i p i t a t e . Unfortunately, a f t e r d i r e c t treatment with an aqueous bromine solution, and subsequent d i a l y s i s , very l i t t l e material was recovered and a 1 3C-nmr spectrum was inconclusive. Presumably, hydrolysis occurred due to improper buffering of the bromine reaction and the product dialysed out. Further attempts at t h i s reaction -130-were not undertaken, although i t i s s t i l l believed that t h i s sequence o f f e r s an a t t r a c t i v e means f o r generating galacturo-nosyl branches. As a f i n a l point, the rheograms of commercial samples of xanthan (Fig. 35a), hydroxyethylcellulose (Fig. 35b) and sodium alginate (Fig. 35b) are provided f o r contrast. F i g . 38a d i r e c t l y contrasts 2.0% solutions of 121c and 126c with xanthan and sodium alginate, while F i g . 38b compares 1.0% solutions of 125d. xanthan and sodium alginate. I t can be seen that the derivatives described here e x h i b i t flow behav-iour s i m i l a r to xanthan and hydroxyethylcellulose, although they are somewhat l e s s viscous and l e s s pseudoplastic. The sodium alginate solutions however can be seen to be quite d i f f e r e n t i n t h e i r behaviour, being considerably l e s s pseudo-p l a s t i c over the shear rate range studied. I n t r i n s i c v i s c o s i t i e s for derivatives 126a, 126b. 126d. 125a. 120a and 122a were determined i n order to further probe the behaviour of these derivatives i n solution, and the r e s u l t s are presented i n Table 7. Looking at the i n t r i n s i c v i s c o s i t i e s f o r 126a.b and d, there i s again an increasing trend with decreasing d.s. This alludes to a r e l a t i o n s h i p between conformation and d.s. that i s consistent with the proposed mechanism fo r s e l f - a s s o c i a t i o n given i n F i g . 40. In other words, there i s an apparent conformationally related change i n the polysaccharide derivatives at lower d.s. values. I t then becomes possible, at c e r t a i n d.s. values, for a maximal inter-chain association to occur, causing increased -131-Derivative Branch d.s. f7 l R z #Points (±.05) (dL-g - 1) 120a j8-GlC 1.00 0.487 .968 8 122a )3-GlcA 1.00 2.63 .998 10 125a ot-Glc 1.00 1.46 .947 8 126a b d a-Gal 1.00 0.86 0.32 0.725 2.39 5.41 .833 .931 .944 8 8 10 Table 7. I n t r i n s i c v i s c o s i t i e s f o r selected derivatives, determined according to the Kraemer r e l a t i o n s h i p (Eq. 7). v i s c o s i t y and pseudo-plasticity. The i n t r i n s i c v i s c o s i t i e s of the f u l l y substituted derivatives 120a and 125a. bearing and ot-glucosyl sugars respectively, are -.5 and 1.5 (d L * g _ 1 ) . This discrepancy could be an i n d i c a t i o n of greater molecular order i n the case of 125a. The fa c t that 125a had lower n (0.929) and m values (65.1 mPa*sn) than 120a (n 1.01 and m 22.4) at 2.0% concentration supports t h i s observation. I t has already been mentioned that the highly substituted derivatives such as 120a and 125a have l i t t l e shear rate dependence and are s t e r i c a l l y r e s i s t a n t to s e l f -association. Therefore differences i n v i s c o s i t y , even at high (2.0%) concentrations, are probably a r e f l e c t i o n of the shape of the polymer molecule i t s e l f . The i n t r i n s i c v i s c o s i t y values f o r 120a and 125a are then the f i r s t clue to conformational differences induced by a l t e r i n g the gl y c o s i d i c -132-configuration of the branch. The e f f e c t on s o l u t i o n proper-t i e s i s not p a r t i c u l a r l y large i n magnitude, and the similar-i t y i n trends f o r lower d.s. samples fo r a l l s e r i e s indicate that other associative forces come into play at 2.0% concen-t r a t i o n which dominate the resultant properties. 2.2.3 Synergistic Interaction The increasingly apparent s i m i l a r i t y between the N-2'-(glycopyranosyl)ethyl chitosan derivatives and the well studied seed galactomannans, led us to consider using these derivatives to probe s y n e r g i s t i c i n t e r a c t i o n s . 2 1 3 ' 2 1 4 As such, mixtures of xanthan with derivatives 121a. 121b. 121c, 126c. 126d. 125c. and 123b were prepared, such that upon addition of water the resultant solutions contained 0.25% xanthan, and ,0.25% of the d e r i v a t i v e , with an o v e r a l l poly-saccharide concentration of 0.50%, as described i n Table 8. Derivative Branch d.s. Observations (±.05) 121a /3-Gal 1.00 S,V h 0.70 S,V C 0.38 G,B,X 123b /3-Lact 0.76 G,F,X 125c a-Glc 0.38 G,B,X 126c a-Gal 0.48 G,B,X d 0.32 . G,B,X Table 8. Q u a l i t a t i v e observations from s y n e r g i s t i c mixtures containing 0.25% xanthan and 0.25% of the respective d e r i v a t i v e . Code; B, beads; F, fibrous; G, g e l a t i -nous; S, solution; V, viscous; X, excluded solvent. -133-The interactions between xanthan and derivatives 121c. 126c, 126d and 125 resulted i n the formation of gelatinous globules which excluded solvent. With 123b, an opaque gelatinous p r e c i p i t a t e was obtained while 121a and 121b provided viscous solutions. The rheometric evaluations of these s y n e r g i s t i c solutions were compared to those of 0.25% and 0.50% (w/w) xanthan solutions and to a known s y n e r g i s t i c mixture contain-ing 0.25% xanthan and 0.25% locust bean gum (Fig. 42 and 43). The power-law parameters obtained f o r these f i v e solutions are given i n Table 9. Interestingly, the mixture of 121b and xanthan was more viscous (m 1780 mPa s) and s l i g h t l y l e s s pseudoplastic than the 0.50% s o l u t i o n of xanthan (m 1400 mPa s ) . The i n t e r a c t i o n of 121a with xanthan produced a l e s s viscous solution, which s t i l l had v i s c o s i t y and pseudoplas-t i c i t y greater than 0.25% xanthan. Thus, i n the f i r s t case, Sample n m R 2 #Points • 3% (mPa-s) + 3% xan + 121a 0.478 605 .974 30 xan + 121b 0.409 1780 .986 24 xan + LBG 0.402 1670 .965 23 xan(0.5%) 0.397 1400 .946 22 xan(0.25%) 0.518 378 .968 27 Table 9. Power-law parameters obtained from rheological tests on the s y n e r g i s t i c mixtures of 0.25% xanthan and 0.25% derivative, at 20°C. Parameters for a xan-than-locust bean gum mixture and 0.50 and 0.25% xan-than solutions are provided for comparison. F i g u r e 4 2 . R h e o g r a m s f o r s y n e r g i s t i c m i x t u r e s c o n t a i n i n g 0 . 2 5 % (w/w) x a n t h a n gum a n d 0 . 2 5 % 1 2 1 a ( • ) , 0 . 2 5 % 121b (O), o r 0 . 2 5 % l o c u s t b e a n gum ( • ) , o n l i n e a r a x e s . 10000 10 I i i i i i i I I i i i i i i i 111 i i i i i i 111 i i i i i i 11 1 10 100 1000 10000 SHEAR RATE (s"') F i g u r e 4 3 . Rheograms f o r s y n e r g i s t i c m i x t u r e s c o n t a i n i n g 0 .25% x a n t h a n gum and 0.25% 121a ( • ) , 0.25% 121b ( O ) , o r 0 .25% l o c u s t b e a n gum ( • ) , on l o g a r i t h m i c c o o r d i n a t e s . -136-an appreciable s y n e r g i s t i c e f f e c t i s seen, giv i n g properties greater than an equivalent concentration of xanthan. The l a t t e r case indicates that a small i n t e r a c t i o n i s taking place, r e s u l t i n g i n a lower ri and larger m, than f o r the 0.25% xanthan solu t i o n . The locust bean gum/xanthan synergis-t i c i n t e r a c t i o n gave a sol u t i o n having a s i m i l a r v i s c o s i t y and pseudoplasticity to the mixture of 121b and xanthan. I t i s important to point out that the i n t e r a c t i o n of the d e r i v a t i v e s prepared i n t h i s study with xanthan gum are not necessarily analogous to those between galactomannans and xanthan. Indeed, upon consideration of the primary structure of xanthan, i t seems l i k e l y that an i o n i c i n t e r a c t i o n between the carboxylates of xanthan and the amine groups on the chitosan derivatives would occur. Thus, derivatives having low d.s. permit access of the xanthan branch (bearing the carboxylate) to the free amino groups, giv i n g a s a l t p r e c i p i -t a t e . The higher d.s. samples s t e r i c a l l y r e s t r i c t the extent of i o n i c i n t e r a c t i o n and y i e l d viscous solutions. Thus, while we have not necessarily probed the s y n e r g i s t i c i n t e r a c t i o n between galactomannans and xanthan, the p o t e n t i a l f o r synerg-i s t i c i n t e r a c t i o n between chitosan derivatives and xanthan gum has been established. This observation i s e x c i t i n g , and opens up a whole range of p o s s i b l i t i e s f o r v i s c o s i t y modifi-cation. For example, i t can be envisioned that chitosan derivatives, such as those presented here, could be further modified i n order to l i m i t and control the s y n e r g i s t i c response upon mixing with xanthan gum. The simplest way of -137-doing t h i s i s probably by N-acetylation as described i n the preparation of the N-ethyl-^S-glucosyl c h i t i n d e r i v a t i v e 130. Furthermore, there i s no reason to l i m i t t h i s e f f e c t to xanthan mixtures, as s i m i l a r and perhaps more i n t e r e s t i n g observations could r e s u l t from admixture of chitosan deriva-t i v e s with other carboxylate-containing polymers, such as proteins. This observation also provides some in s i g h t into the behaviour exhibited by the a c i d i c and s a l t forms of the glucuronoside derivatives 122a and b_. I t i s reasonable to expect that i o n i c i n t e r a c t i o n caused p r e c i p i t a t i o n of high d.s. derivatives i n the acid form, but at lower d.s. reduced i n t e r a c t i o n could y i e l d solutions or gels. These i o n i c c r o s s l i n k i n g a t t r a c t i o n s are removed when the derivatives are i n the carboxylate form. 2.3 N- [10 • -Q- (jQ-D-GLYCOPYRANOSYL) DECYL] CHITOSAN DERIVATIVES 2.3.1 Synthesis and Characterization The 10•-undecenyl j 3~ D~glycosides of glucose (134). galactose (135), and lactose (136) were prepared by methods 2 4 2 s i m i l a r to those f o r the synthesis of a l l y l jQ-D-glycosides, as outlined i n Scheme 19. The acetobromo su-gars were reacted under Koenigs-Knorr glycosidation condi-tions with two molar equivalents of 10-undecenol i n chloro-form to give the intermediate peracetylated glycosides 131-133. The crude residue was d i r e c t l y de-O-acetylated to y i e l d the desired 10'-undecenyl jS-D-glycopyranosides 134-136. 138-AcO-i ° \ ? ^ ( C H 2 ) 9 ^ S 91,92,94 OAc OAc 131-133 Ri Ra 91,131 OAc H b 92,132 H OAc f HO - i AcO-j ° ^ ( C H 2 ) B ^ A c O ^ O 0 kOH 94,133 \?AC ./ H -r 1 OH 1 1 OAc 134-136 134 OH H 135 H OH (a) 10-undecenol, H g ( C N ) 2 > CHC13 HO - i (b) NaOMe/MeOH H O J - O O 136 H r—r OH Scheme 19 I t was noted by -^H-nmr that some a-D-glycoside impurity was present i n the jS-lactoside product. Liquid chromatography of the crude material, using methods reported f o r long chain a l k y l g l y c o s i d e s , 2 5 2 afforded the compounds 134, 135 and 136 as waxy s o l i d s . Characterization of the 10'-undecenyl j3~D-glycosides was best accomplished using 1H and 1 3C-nmr. Since s i m i l a r -139-molecules are known to behave as non-ionic surfactants and form m i c e l l e s , 2 5 2 " " 2 5 4 i n aqueous solution, and because t h e i r water s o l u b i l i t y was lim i t e d , the glycosides were dissolved i n methanol- d 4 . This served to reduce aggregation of the molecules and permit higher resolution spectra to be obtained. I t was found that spectra recorded at 50°C were better resolved than those determined at 20°C, which had broader sign a l s . Thus, assignment of *H and 1 3C-nmr spectra recorded at 400 MHz and 100.6 MHz respectively at 50°C was possible. The 1 3C-nmr chemical s h i f t data f o r the three glycosides i s given i n Table 10. Despite attempts at p u r i f i c a t i o n by l i q u i d chromatogra-phy, the jQ-lactoside product contained some a-anomer (-10%) impurity and was c a r r i e d through as such. This was l i k e l y a r e s u l t of performing the glycosidation at elevated tempera-tures. Obtaining a n a l y t i c a l l y pure samples f o r o p t i c a l r o t a t i o n determinations and elemental microanalyses was precluded because of the d i f f i c u l t y i n c r y s t a l l i z a t i o n and drying of the glycosides. However, f a s t atom bombardment (fab) mass spectrometry provided the expected parent peaks as proof of product molecular weights. Ozonolysis of the alkenyl /5-D-glycopyranosides 134 and 135 was performed at -78°C i n methanol. Somewhat s u r p r i s -ingly, i t was necessary to use a chloroform-methanol mixture (1:5) to s o l u b i l i z e the disaccharide 136. A f t e r workup, the aldehydes 137-139 were d i r e c t l y employed i n reactions with chitosan. I t was found that upon standing f o r over a day, the Sample! Sugar C-l C-2 C-3 C-4 C-5 C-6 C - l ' C-91 C-10' C - l l ' 134 jQ-Gal 102.5 73.3 76.0 70.0 76.3 61.1 69.1 32.9 138.3 112.8 135 /3-Glc 103.1 70.8 73.3 69.0 74.7 60.7 68.5 32.9 138.2 112.8 136 /3-Lact (/3-Gal) 103.0 70.8 72.8 a 69.7 75.1 60.7 (/3-Glc) 102.2 72.8 a 74.5 b 79.0 74.5 b 60.2 68.4 32.9 138.3 113.2 i Table 10. 100.6 MHz 1 3C-nmr chemical s h i f t data (ppm), for saccharide and some 1 aglycon resonances, of the 10'-undecenyl^Q-D-glycopyranosides i n CD3OD (ref. external TMS). a. Assignments may be reversed. O -141-aldehydes became insoluble i n methanol and water, but d i s -solved slowly i f small amounts of a c e t i c or hydrochloric acid were added. Apparently o l i g o - and poly-acetal compounds formed, as might be expected for these r e l a t i v e l y unhindered decyl-aldehydes. The reductive amination 8 6 of the decanalyl yQ-D-glycopyranosides 137 and 138 to chitosan (Scheme 20) were performed i n 5% aqueous a c e t i c acid-methanol (1:1) solvent. The methanol was necessary to s o l u b i l i z e the aldehydes 137 and 138; however, the aldehyde 139 was soluble i n the t o t a l l y aqueous system. Upon addition of sodium cyanoborohydride to the reaction solutions, a marked decrease i n v i s c o s i t y occurred. A f t e r the reactions were s t i r r e d f o r 24 hours, they were dialyzed, f i l t e r e d and l y o p h i l i z e d . The reactions were performed using two d i f f e r e n t aldehyde-to-chitosan r a t i o s , f o r each of the aldehydes 137-139. to give the derivatives 140-142a and b, which are l i s t e d i n Table 11. The degree of s u b s t i t u t i o n values, as determined from elemental microanaly-s i s , immediately t o l d us that the coupling e f f i c i e n c y of the long chain aldehydes was much greater than f o r those of the a l l y l glycoside route. Thus i n t h i s s e r i e s , d e rivatives 140a and 141a had d.s. values of 1.47 and 1.37 when 3 equivalents of aldehyde were employed, while previously a d.s. of 1.0 appeared to be maximal (Table 3). Derivatives with d.s. values lower than 1.0 were prepared by reducing the amount of aldehyde used i n the coupling reaction. Obviously, t h i s was a r e s u l t of the le s s hindered nature of the decanalyl aide-(a) 0 3, -78 C, DMS, MeOH (b) HOAc/MeOH/Ry) (1:10:10), NaCNBH^ Scheme 20 -143-Derivative Branch A/C d.s. (±.05) Yieldm 140a h 141a b 142a c 143a b 144 /3-Glc /3-Gal jS-Lact a-Gal 1 /3-Gal 3.0 2.1 3.0 1.5 2.9 1.6 2.0 1.0 2.9 1.47 0.81 1.37 0.22 1.10 0.50 1.73 1.00 0.32 1.04 80 85 65 85 70 70 76 82 63 Table 11. C h a r a c t e r i s t i c s for the N-[10 • -O- (/3-D-glycopyr-anosy1)decyl]chitosan derivatives. * The N-ethyl-(a-galactosyl) branch was present on 126d p r i o r to i t s modification to give the mixed deriv a t i v e 144. hydes, compared to acetaldehydes, which allowed a substantial amount of N,N-disubstitution. Since t h i s f a c t complicated molecular formula determinations, i t was assumed, f o r s i m p l i -c i f i c a t i o n , that i f the d.s. was greater than 1.0, no unsub-s t i t u t e d residues remained. For comparison purposes the derivatives 143a and 143b were prepared using standard [27] - T l C H 2 0 H 1 4 3 (a) Me0H/i-Pr0H/ H 20 (2:1:2), NaCNBH^, 10-hydroxydecanal -144-conditions involving the reaction of chitosan with 10-hydrox-ydecanal (Eq. 27) , which was obtained from ozonolysis of 10-undecenol. Derivative 143a p r e c i p i t a t e d from the reaction s o l u t i o n and was c o l l e c t e d by f i l t r a t i o n , and 143b was i s o l a t e d using standard workup procedures. Again high d.s. values were obtained at t y p i c a l A/C r a t i o s (Table 11). Disappointingly, none of the derivatives 140-143 were water soluble. They were however, a l l soluble i n d i l u t e organic or mineral acid solutions i n water (e.g. 1-2% aqueous a c e t i c a c i d ) . The high d.s. samples 140a. 141a and 142a. bearing pendant j3~D-glucose, jS-D-galactose, and jS-D-lactose residues respectively, gave t h i n , mobile solutions at 5.0% (w/w) polysaccharide concentration i n 1% aqueous a c e t i c acid, while the lower d.s. analogues 140b. 141b and 142b. gave s l i g h t l y more viscous solutions. Solution 1 3C-nmr spectra of 140a. 14la and 142a had e a s i l y d i s c e r n i b l e resonances fo r the pendant sugars and a l k y l group, but v i r t u a l l y no d i s t i n g u i s h -able signals from the chitosan backbone. This i s i n d i c a t i v e of f r e e l y r o t a t i n g pendant sugars, more so even than that seen f o r the N-ethyl glycosyl s e r i e s of d e r i v a t i v e s . I t must however be borne i n mind that, due to much larger average residue molecular weights, the decyl samples at 5% concentra-t i o n have a lower molar concentration than the N-ethyl series at 5% concentration. Despite t h i s factor, however, one would i n t u i t i v e l y expect greater mobility i n a group having a ten carbon vs a two carbon spacer arm. Total assignment of the branch and a l k y l 1 3 C resonances for derivatives 140a. 141a Derivative Branch C-l C-2 C-3 C-4 C-5 C-6 C-10' 140a /3-Glc 100.8 71.8 74.5 69.0 74.6 59.5 68.4 141a /9-Gal 101.3 67.0 71.4 69.2 73.4 59.2 68.8 142a /3-Lact (£-Gal) 102.5 70.5 72.2 70.0 75.8 60.5 (/3-Glc) 101.7 72.4 74.1 78.5 74.3 60.0 68.1 144 a-Gal 97.0 66.7 68.0 67.6 69.9 60.4 /3-Gal 101.2 67.0 71.3 69.1 73.3 59.2 68.8 Table 12. 100.6 MHz 1 3C-nmr chemical s h i f t (ppm) data f o r pendant residues of the N-decyl-^-D-glycopyranosides, i n 1.0% CD3COOD/D20 (ref. external TMS). -146-and 142a are presented i n Table 12, and again they compare well with the methyl glycoside a n a l o g u e s , 9 9 - 1 0 1 the 10-un-decenyl jQ-D-glycoside precursors (Table 10) and to the respective N-ethyl D-glycopyranosyl chitosans (Table 4). To our chagrin, i t was immediately apparent that these solutions had uninteresting rheology at ambient temperatures. Serendi-p i t o u s l y , i t was noted that upon heating the 5.0% solutions of 140a and 141a to 50-°C a s t i f f opaque gel formed, which dissolved r e v e r s i b l y upon cooling. Solutions of 2.0% concen-t r a t i o n d i d not e x h i b i t t h i s behaviour. This type of rever-s i b l e temperature induced g e l l i n g process was considered to be of some i n t e r e s t and deserving of further i n v e s t i g a t i o n . The non-reversible gelation of some proteins upon heating i s known, and i s l a r g e l y a ttributed to hydrophobic interac-t i o n s . 2 5 5 Long chain (C 8 and longer) a l k y l glycosides are non-ionic surfactants, which form micelles at c e r t a i n c r i t i -c a l c o n c e n t r a t i o n s . 2 5 2 - 2 5 4 ' 2 5 6 Such m i c e l l a r solutions are characterized by a "cloud point", which r e s u l t s from aggrega-t i o n of micelles upon h e a t i n g . 2 5 6 Interestingly, the gelation of 141a i s accompanied by increased s o l u t i o n opacity, and seems to r e l a t e to the cloud point phenomena that occurs at the monomeric l e v e l . Thus, i t appears that the necessary components fo r temperature dependent behaviour are present on the derivatives described here. That i s , the combination of hydrophobic character and polymeric structure appear to be conducive to temperature induced g e l l i n g . The r e v e r s i b i l i t y of the i n t e r a c t i o n indicates that reorientation of the -147-polymer chains accompanies temperature reduction. For pro-t e i n s t h i s i s not usually the case as i r r e v e r s i b l e denatura-t i o n accompanies gel formation. Another polysaccharide d e r i v a t i v e known to have s i m i l a r behaviour i s methylcellu-l o s e . 2 5 7 2.3.2 —H and ^ lc-NMR Investigations In order to follow gel formation and to perhaps gain i n s i g h t into the mechanism, XH and 1 3C-nmr experiments were undertaken. I t was f e l t that the mobility of the components of the de r i v a t i v e could be probed by observing the tempera-ture dependence of T^-relaxation of resonances i n the 1H-nmr spectrum of 141a. As such, T^-relaxation measurements of three resonances, representing the pendant sugar, the a l k y l chain, and solvent, i n the ^ -H-nmr spectrum were performed at 20°, 40°, 60° and 80° using the inversion recovery method 1 1 3 (Fig. 44). The T^ values obtained are given i n Table 13. The Temperature T^ of Resonances (s)  (°C) Sugar A l k v l HOD 20 0.34 0.25 3.4 40 0.42 0.30 1.6 60 0.47 0.32 1.4 80 0.80 0.54 1.2 Table 13. T^ values ,at 20, 40, 60, and 80° C, f o r the resonances indicated i n the 300 MHz -^ H-nmr spectrum of 141a (Figure 44), i n 1% CD3COOD/D2O solution. -148-6.0 4.0 2.0 PPM F igu re 44. Stacked p l o t s showing the i n v e r s i o n recovery of the resonances i n the spectrum of 141a, i n 1.0% CD3(X>2D/D20 s o l u t i o n at 20°C, a t 300 MHz. The peaks f o r which Tj^  v a l u e s were c a l c u l a t e d are i n d i c a t e d . -149-increase i n the T^-relaxation time of the sugar and a l k y l protons indicates that the c o r r e l a t i o n time (T c) of the polymer i s s u f f i c i e n t l y slow at 20°C that i t has passed the minima i n the vs r c curve. This i s expected since at 300 MHz a T c ~3xl0~ 9 s _ 1 would r e s u l t i n a T± minimum, while chitosan derivatives i n solut i o n have been shown to have c o r r e l a t i o n times of 10~ 9-10~ 8 s - 1 . 2 5 8 Thus, increased T^ values at 40°, 60° and 80 °C r e s u l t from reduced mobility of the respective groups i n the gel state. The decreasing T^ value of the solvent or HOD resonance, i s supportive of reduced solvent mobility upon gelation. In t h i s case, the water molecules having T Q - I O ' ^ - I O " " 1 1 s " 1 i n solution, are "trapped" i n the gel matrix and t h e i r reduced mobility causes a reduction i n T^, i n the d i r e c t i o n of the T^ minimum at f. c~ 3 x l 0 - 9 s _ 1 . Unfortunately, there were no obvious chemical s h i f t changes upon heating that could help illuminate the g e l l i n g mechanism. As expected, a general broadening of resonances occurred upon heating, due to the dependence of T 2 and linewidth, on c o r r e l a t i o n time. As i n the 1 3C-nmr spectrum, no -^ H-nmr resonances from the chitosan main chain were discernable. Gelation was also monitored by 1 3C-nmr spectroscopy. In F i g . 45, the 1 3C-nmr spectrum of 141a at 30° and 50°C i s given. Substantial line-broadening i s immediately apparent at 50°C, with linewidths f o r C - l being § 15 and 150 Hz respectively, for the 30° and 50°C spectra, r e f l e c t i n g the su b s t a n t i a l l y reduced mobility of the pendant galactose unit i n the g e l . Again, no chemical s h i f t changes occur upon -150-heating the sample. Thus, while nmr was useful f o r following the g e l a t i o n phenomena, the experiments performed were i n s e n s i t i v e to the interactions which cause i t . While i t i s d i f f i c u l t to speculate on the exact mechanism for the observed temperature induced gelation, s i m i l a r phenomena have been observed i n other systems. Phase separa--151-t i o n i s observed i n solut i o n of non-ionic surfactants when heated, and i s referred to as the "cloud point".259 Although some s t r u c t u r a l s i m i l a r i t i e s do e x i s t between the branch units described here, and carbohydrate derived non-ionic surfactants, i t i s d i f f i c u l t to v i s u a l i z e a m i c e l l a r l i k e i n t e r a c t i o n f o r these compounds. I t i s l i k e l y that, upon heating, the a l k y l chains are increasingly repulsed from i n t e r a c t i o n with water. The behavior seen here f o r these polysaccharide systems i s probably analogous to the tempera-ture dependence observed f o r polyethylene g l y c o l s o l u -t i o n s . 260 I t was i n t e r e s t i n g to note that the lower d.s. monosac-charide d e r i v a t i v e s 140b and 141b. and the lactose derivatives, 142a.b even at 7.5% concentration, d i d not gel at elevated temperatures. Thus, substantial hydrophobic character appears necessary, and a large pendant group precludes gel formation. The l a t t e r i s l i k e l y a r e s u l t of the increased h y d r o p h i l i c i t y of the disaccharide, counteracting or i n t e r f e r i n g i n the hydrophobic i n t e r a c t i o n s . I t has been r e p o r t e d 2 5 4 that a l k y l lactosides are not as prone to micelle formation, and behave poorly as surfactants. This i s l i k e l y a further manifestation of that property. Interestingly, of the deri v a t i v e s 143a and b, which lacked the pendant carbohy-drate, 143a was insoluble i n aqueous a c e t i c acid and 143b gave a highly viscous solution showing no observable change upon heating. This g r a t i f y i n g l y indicated that gel formation was dependent on the hydrophilic character of the pendant -152-moiety. Both 143a and b gave c l e a r s t i f f gels i n 1% a c e t i c acid-methanol, while the branched derivatives gave gels i n aqueous a c e t i c acid-methanol systems. • 2.3.3 Mixed Branch Chitosan Derivatives The concept of preparing co-branched chitosan derivatives i n order to control or enhance s o l u b i l i t y properties has been i n t r o d u c e d 9 6 ( s e c t i o n 1.2.2). In t h i s study, we f e l t that N-10'-C:-(j3-D-glycopyranosyl)decyl branches, and the unique temperature dependence they impart, could be used i n conjunc-t i o n with the c o n t r o l l e d s o l u b i l i t y properties of the N-2'-O-(D-glycopyranosyl)ethyl branched chitosan derivatives to give products having viscous properties that were stable or enhanced at elevated temperature. To t h i s end, derivative 126c was reductively alkylated with the decanalyl glycoside 138 to give 144 (Eq. 28), as described i n Table 11. - [28] (a) MeOH/H20 (1:1), NaCNBH^, 138 -153-5 0 ° C @96,000 t r a n s i e n t s ( f i i r 1 1 1 1 1 r i 1 1 1 1 1 40 120 80 PPM C-1 I a 3 0 ° C C - 4 . .C-10 C " 3 0 ^ C " 2 C-6 @48,000 t r a n s i e n t s i » i i r 120 T 1 I 1 1 1 1 1 1 1 80 40 PPM F igu re 46 . 100.6 MHz 1 3 C - n m r s p e c t r a l r e g i o n c o n t a i n i n g the branch res idue s i g n a l s f o r d e r i v a t i v e _144 a t 30° and 50° C , i n 1.0% CD^OOD/ D 2 0 ( r e f . e x t e r n a l TMS) -154-Disappointingly, i t was found that while 144 would swell i n water, i t would not give a true solution. Again, i t was found that a viscous s o l u t i o n was obtained i n 2.0% aqueous acetic ac i d . The 1 3C-nmr spectrum (Fig. 46) of 144 contains resonances f o r both branch residues; however, mobility differences r e s u l t i n considerable suppression of the q-gal-actosyl branch compared with the more extended ^0-D-galactosyl branch. Steady shear viscometry on 2.0% (w/w) solutions of 144 and 126c i n 2.0% aqueous ac e t i c acid, at 20°C and 50°C provided the data shown i n the logarithmic rheograms i n F i g . 47. The rheology of the s o l u t i o n of 144 at 20°C was i n t e r e s t i n g , i n that both pseudoplasticity and v i s c o s i t y were appreciable, and the solution was considerably more viscous than that of 126c. However, the rheograms (and power-law parameters, Table 14) show that the temperature dependence of Derivative Temperature n m R 2 #Points (°C) 144 20 50 126c 20 50 • 3% (mPa*s) * 3% 0.488 7330 .994 12 0.499 4920 .996 14 0.607 1880 .997 13 0.644 1060 .997 11 Table 14. Power-law parameters obtained from rheological evaluation of 126c and 144. at 20°and 50°C, i n 1.0% aqueous ac e t i c acid solution. -155-10000^ CO, 6 Q_ £ o o C O > 1000 100-10 100 1000 SHEAR RATE (s _ 1) 10000 Figure 47. Rheograms of d e r i v a t i v e s 144, at 20° ( O ) and 50°C ( • ) , and 126d, at 20° (•) and 50°C (•) on logarithmic coordinates. both 144 and 126c are s i m i l a r and r e s u l t i n a reduction of both v i s c o s i t y and, to a l e s s e r extent, pseudoplasticity. As an exploratory experiment, these r e s u l t s were rewarding and are c e r t a i n l y i n d i c a t i v e of further p o t e n t i a l f o r mixed de r i v a t i v e s . 2 . 4 CONCLUSION The work described i n t h i s chapter constitutes a s i g n i f i -cant contribution to e x i s t i n g knowledge on the r a t i o n a l d e r i v a t i z a t i o n of chitosan for c o n t r o l l e d s o l u b i l i t y applica-t i o n s . Analogues of natural branched polysaccharides have been prepared and t h e i r s i m i l a r i t y to the n a t u r a l l y occurring branched legume seed galactomannans has been discussed. In - 1 5 6 -addition, p o t e n t i a l f o r modifying and c o n t r o l l i n g chitosan s e l f - a s s o c i a t i o n s as well as s y n e r g i s t i c interactions has been demonstrated. The concept of u t i l i z i n g "mixed" branch der i v a t i v e s to access unique properties and interactions was further explored, and was extended by incorporating mixed f u n c t i o n a l i t y (namely hydrophobicity and h y d r o p h i l i c i t y ) on a s i n g l e carbohydrate derived branch, to provide chitosan d e r i v a t i v e s having unique g e l l i n g properties. -157-CHAPTER 3 METAL CHELATING AND AFFINITY CONJUGATES OF CHITOSAN 3.1 INTRODUCTION 3.1.1 Metal Chelating Chitosan Derivative As mentioned i n Chapter 1, chitosan has been modified i n order to prepare c o n t r o l l e d - s o l u b i l i t y derivatives, metal sequestrants, drug delivery matrices, drug c a r r i e r s and immobilization supports.22-26 Although our main e f f o r t s have been directed toward controlled s o l u b i l i t y compounds, 9 6' 2 3 7 we f e l t that our experience i n chitosan modification could be exploited i n the synthesis of metal chelating and a f f i n i t y ligand conjugates. Some metal chelating chitosan derivatives have been mentioned (67, 68, 69, and 70).92-95 our objective was to incorporate c o n t r o l l e d s o l u b i l i t y features i n conjunction with chelating properties to create a novel chitosan d e r i v a t i v e . This was f e l t to be best accomplished by incorporating a hydrophilic spacer arm between the chitosan backbone and the desired chelating group. Previous reports from t h i s lab i n which free sugars were coupled to chitosan to give a c y c l i c hydrophilic branches 9 6 provided an i d e a l route to the desired spacer group. Iminodiacetic acid, e s s e n t i a l l y one h a l f of the well-known chelate, ethylene diamine t e t r a a c e t i c acid (EDTA), was chosen as an appropriate -158-chelating f u n c t i o n a l i t y . The synthetic route to a precursor having the desired s t r u c t u r a l features, and i t s attachment to chitosan w i l l be discussed. 3.1.2 Potential A f f i n i t y Chromatography Support While chitosan has attracted s i g n i f i c a n t attention i n biochemical and biomedical a p p l i c a t i o n s , 2 2 - 2 6 there has been s u r p r i s i n g l y l i t t l e research into the use of chitosan i n a f f i n i t y chromatography. In collaboration with a group* interested i n the enzyme /3-glucosidase, we f e l t the p o t e n t i a l of using chitosan conjugates i n a f f i n i t y chromatography could be demonstrated. A number of well-known concepts have developed i n a f f i -n i t y chromatography, 8' 3 2' 3 3 including h y d r o l y t i c s t a b i l i t y of covalent linkages, choice of a suitable ligand, and the use of a spacer group. In our case, the use of reductive amina-t i o n procedures 8 6 eliminated any worry about hydrolysis of the amine linkage to the backbone. As a ligand, a pendant glucose having a g l y c o s i d i c linkage r e s i s t a n t to enzymatic hydrolysis was desired. 1-Thio-jS-D-glycosides, being known i n h i b i t o r s f o r glycosidase enzymes and generally immune to enzymatic hydrolysis, provided a l o g i c a l choice f o r the l i g a n d . 2 6 1 The spacer group required a degree of b i f u n c t i o n a l i t y such that the thio- /3-D-glucose could be attached, and a remaining s i t e was present f o r reaction with chitosan. The reagent l-allyloxy-2,3-epoxypropane ( a l l y l g l y c i d y l ether) * Dr. S.G. Withers and coworkers, The Department of Chemistry, The University of B r i t i s h Columbia. -159-provided both features, with an epoxide amenable to t h i o l a t e attack and a double bond which was convertible to an alde-hyde. Thus, the synthesis of the appropriate l-thio - jQ-D-glucopyranoside and i t s conjugation with chitosan w i l l be presented, and preliminary evaluations of / 3-glucosi-dase binding w i l l i l l u s t r a t e the p o t e n t i a l f o r a f f i n i t y chromatography applications. 3.2 METAL CHELATING CHITOSAN DERIVATIVE 3.2.1 Synthesis By design, i t was decided to prepare a compound which contained both an iminodiacetic acid f u n c t i o n a l i t y f o r metal chelation, and an aldehyde f o r coupling to chitosan. We chose to incorporate a carbohydrate-derived, hydrophilic spacer arm to enhance the s o l u b i l i t y c h a r a c t e r i s t i c s of the f i n a l product, and to act as a spacer arm. Scheme 21 outlines the synthesis of the coupling precursor 149. The epoxide 145 was prepared from l,2:3,4-di-0-iso propylidene galactose (146), by reaction with epichlorohydrin i n the presence of sodium hydride. Subsequent reaction of the epoxide with dimethyliminodiacetate (147) gave a 4:1 mixture of the dimethyliminodiacetate 148a. and the morpholone 148b. Although c y c l i z a t i o n s s i m i l a r to that producing 148b have been observed i n reactions of epoxides with amino acid e s t e r s , 2 6 2 i t appears to be more favoured f o r these dies t e r compounds. Total conversion of the mixture to the morpholone -161-148b was e a s i l y achieved by d i s t i l l a t i o n . Liquid chromatogra-phy on s i l i c a gel offered some separation of the mixture, but i t was determined that further c y c l i z a t i o n occurred on the column. Base hydrolysis of the 148 mixture, and subsequent treatment with acid at 50°C, gave the free sugar deri v a t i v e 149. Compound 149 was coupled to chitosan v i a reductive amination, with sodium cyanoborohydride i n 5% aqueous acetic acid, to give the derivatives 151a and 151b (Eq. 2 9 ) * Control of the degree of su b s t i t u t i o n was achieved by varying the r a t i o of aldehyde to chitosan (A/C) as shown i n Table 15. Derivative 151a was water-soluble, giving a c l e a r viscous solut i o n at 5.0% (w/w) i n d i s t i l l e d water. Compound 151b. on the other hand, gave an opaque gel at 5.0 % (w/w) concentra-t i o n and at 1.0% retained much of i t s gelatinous nature. 3.2.2 Characterization The s o l u t i o n state 1 3C-nmr spectrum of 151a was obtained and resonances f o r the hydroxypropyl and iminodiacetate f u n c t i o n a l i t i e s were e a s i l y d i s c e r n i b l e . Most of the carbohydrate resonances a r i s i n g from the galactosyl branch were assignable. The chitosan resonances, however, were very broad and assignment was d i f f i c u l t . I n terestingly, the linewidth f o r C - l of the chitosan backbone i s of the order of 90 Hz, while the carbon resonances of C-6' of the galactosyl group had a linewidth of 40 Hz. This r e f l e c t s the r e l a t i v e m o b i l i t i e s of these portions of the molecule. -162--163-Sample d.s. Cu(II) Uptake 5  (±.05) mitiol/g % T h e o r e t i c a l " 151a 1.00 2.97 152 151a c 1.00 0.33 16.9 151b 0.50 2.45 87.5 Chitosan 0- 2.40 39.0 Chitosan 0'^ 1.15 18.5 Chitosan e 0.06 2.0 Chitosan^ 3.12 51.0 Table 15. Cu(II) chelating capacity f o r derivatives 15la and 151b contrasted with values obtained i n p a r a l l e l experiments f o r chitosan, and with l i t e r a t u r e values f o r chitosan. a. A 200 mg sample was dissolved or dispersed i n @ 15 mL of d i s t i l l e d water and treated with a 0.5 M Cu(II) acetate solu t i o n (15 mL), exhuastively dialysed and freeze-dried. b. Based on the number of copper ions per GlcN residue. c. A f t e r saturation with Cu(II) these samples were dialysed for 3 d (3x250 mL) against water, 2 d against 0.1 M imi-nodiacetic acid solution, and f i n a l l y , exhaustively with water and freeze-dried. d. Precipitated, l y o p h i l i z e d chitosan. e. Chitosan flakes, r e f . 92 f. Chitosan, r e f . 263 -164-3.2.3 PH T i t r a t i o n The r e s u l t s f o r pH-titrations of derivatives 151a and 151b gave i n f l e c t i o n points corresponding to values of pK a! 2.1, pK a 2 5.9 and p^ 3.9. These values are s i m i l a r to those reported f o r N-carboxymethylchitosan, 9 4 which contains some s i m i l a r f u n c t i o n a l i t i e s . Some of the i n f l e c t i o n s are i l l - d e f i n e d , probably because of the number of t i t r a t a b l e groups present, and the i n s o l u b i l i z a t i o n at pH values of approximately 10 and higher. 3.2.4 Copper(II) Chelation Table 15 summarizes the evaluation of copper(II) uptake of the derivatives 151a and 151bf and contrasts these with native l y o p h i l i z e d chitosan. The d i f f e r e n t i a l uptake of 151a and 151b r e f l e c t s the higher d.s. value of 151a. The chelating capacity of native chitosan i t s e l f , i s 2.4 mmol/g and the percent uptake, i n terms of copper ions per residue, corresponds to an average of 0.39. For the derivatives 151a and b_, more than one ion binds per der i v a t i z e d residue ( i . e . uptake>100%), which indicates that additional binding to the backbone, over and above one equivalent binding per chelating branch, occurs i n the derivative. Indeed, the difference between the t h e o r e t i c a l maximum (100%) expected f o r binding to the iminodiacetate moieties, and the values determined for the derivatives i s s i m i l a r to the percent uptake of chitosan. This point was further examined by saturating both 151a and chitosan with copper(II) ions, and then d i a l y z i n g the r e s u l t --165-ing chelates against 0.1M iminodiacetic acid i n d i s t i l l e d water f o r 48 hours. The copper content of chitosan decreased by only h a l f to 1.15 g/mmol or 18.5%, while most of the copper (89%) was removed from 151a. Interestingly, the f i n a l % copper content of both 151a and chitosan a f t e r d i a l y s i s were very close. The major l i m i t a t i o n i n making comparisons o f . t h i s s o r t a r i s e s from the f a c t that the physical form of the polymer can be very important. Table 16 shows two very d i f f e r e n t values f o r copper(II) uptake fo r chitosan f l a k e s 9 2 and l y o p h i l i z e d c h i t o s a n . l t must also be noted that i n t h i s study, the derivatives were dissolved p r i o r to the addition of copper(II) acetate solution, while the l y o p h i l i z e d c h i t o -san remained as a hydrated, p a r t i c u l a t e dispersion. 3.2.5 Viscometry Since compound 151a formed viscous 1.0% (w/w) solutions, we f e l t i t would be of i n t e r e s t to evaluate i t s rheological properties. We also f e l t viscometry could o f f e r some insight into the i n t e r a c t i o n of the polymer with copper(II) ions. The steady shear viscometric determinations are shown i n F i g . 49 f o r 1.0% (w/w) solutions of 151a i n d i s t i l l e d water and i n 1.0 mM aqueous copper(II) s u l f a t e . Interestingly, the visco-s i t y of the s o l u t i o n containing copper ions i s l e s s than that for the d i s t i l l e d water solution. This was contrary to the known behaviour of other metal chelating polysaccharides, such as sodium alginate which increases i n v i s c o s i t y or gels -166-100.0-1 0.0 T 0.0 500 .0 1000.0 1500.0 2000.0 2500 .0 SHEAR RATE (s _ 1) Figure 48. Rheograms on l i n e a r coordinates f o r de r i v a t i v e 151a as a 1.0% s o l u t i o n i n water ( • ) , and i n 1.0 mM Cu(II)S0 (• ) s o l u t i o n . upon the addition of divalent metal ions such as calcium and c o p p e r 1 8 5 " 1 8 9 . This behaviour r e s u l t s from the c r o s s l i n k i n g e f f e c t of the metal ions, something which i s apparently not occurring i n the samples described here. This leads us to the conclusion that each branch or residue i s a s e l f -contained chelate for one copper ion. Thus to s a t i s f y the metal ion's ligand requirements, c r o s s l i n k i n g i s not neces-sary. The fa c t that the chelating r e s u l t s indicate >100% -167-binding supports the conclusion that each branch chelates one ion. 3.2.6 Conclusions We are g r a t i f i e d by the range of properties exhibited by t h i s chitosan derivative as they r e f l e c t the success of a r a t i o n a l approach to t a i l o r i n g the properties of polysaccha-r i d e s . Thus, i n accord with previous experience, the presence of the high l e v e l of N-substitution by a hydrophilic side-chain resulted i n a f r e e l y soluble d e r i v a t i v e . Functional groups, i n t h i s case the chelating iminodiacetate, modifying the side-chain o f f e r access to additional properties. This encourages further research into the r a t i o n a l t a i l o r i n g of chitosan f o r structure/property studies. 3.3 AFFINITY CHROMATOGRAPHY DERIVATIVE 3.3.1 Synthesis and Characterization The synthesis of the desired diastereomeric 1-thio-jQ-D-glucopyranoside 154. was accomplished from 2,3,4,6-tetra-0-acetyl-l-thio - /3-D-glucopyranoside (152) and l-allyloxy-2,3-epoxypropane, as outlined i n Scheme 22. Reductive ozonolysis of 154 provided the respective aldehyde 155,. which was d i r e c t l y employed fo r coupling to chitosan. Concern over possible oxidation of the s u l f u r during ozonoly-s i s prompted the use of a large excess of dimethyl s u l f i d e during workup. The aldehyde 155 was then reductively aminated -168-Scheroe 22 -169-to chitosan according to standard methods, to a f f o r d the respective derivatives 156a-d, with c h a r a c t e r i s t i c s l i s t e d i n Table 16. The 1 3C-spectrum of 156a showed the expected Derivative A/C d.s. Crosslink (±.05) (%) 156a 2.1 0.90 156b 2.1 0.90 5 156c 0.57 0.25 156d 0.57 0.25 15 Table 16. C h a r a c t e r i s t i c s of derivatives 156a-d. resonances i n d i c a t i n g attachment of the thio -^Q-D-gluco-pyranoside moiety. As a prelude to t e s t i n g of these materials as a f f i n i t y supports i t was necessary to determine whether the t h i o g l y -c o s i d i c linkage i s indeed r e s i s t a n t to enzymic hydrolysis. The monomer 154 was u t i l i s e d f o r these t e s t s , thus avoiding problems associated with the handling of polymeric materials, yet s t i l l providing a stringent t e s t of l a b i l i t y . An assay for glucose based upon the coupling of hexokinase and glucose-6-phosphate dehydrogenase was u t i l i s e d to detect enzyme catalysed turnover, since glycosidase catalysed hydrolysis of thioglycosides produces glucose and the free aglycone t h i o l . Incubation of 154 (3.0 mM) with ^9-glucosidase (~5 units) i n the presence of coupling enzymes and cofactors -170-overnight resulted i n no s i g n i f i c a n t increase i n absorbance; thus no s i g n i f i c a n t hydrolysis had occurred. The v i a b i l i t y of the coupling enzymes at t h i s stage was demonstrated by addition of a known amount of glucose and measurement of the expected response. Binding of the thioglucoside 154 to the enzyme was then investigated k i n e t i c a l l y using jS-glucosidases from almond and from Alcaliaenes f a e c a l i s . I n h i b i t i o n of hydrolysis of E-nitrophenyl jQ-D-glucopyranoside by 154 was measured and values of 35 mM and 1.5 mM determined f o r the jQ-glucosidases from almond and Alcaliaenes f a e c a l i s r espectively. The K m values f o r p_-nitrophenyl jS-D-glucopyranoside f o r these two enzymes are 3 mM263 and 0.08 mM 2 6 4 respectively. I t therefore appears that both enzymes w i l l bind the thioglucoside but since the jQ-glucosidase from Alcaligenes has the greater a f f i n i t y i t was used i n assaying the a f f i n i t y support for binding of enzyme. A preliminary t e s t of the a b i l i t y of the de r i v a t i s e d polymer to bind ^3-glucosidase was performed using the cross-linked preparation, 156d. No s p e c i f i c attempts were made to optimise the flow properties of the polymer. E f f l u e n t from the column was monitored by assaying the enzyme a c t i v i t y released using p_-nitrophenyl /9-D-glucopyranoside, as shown i n Figure 49. C l e a r l y jS-glucosidase i s bound to the column i n i t i a l l y and eluted at high s a l t concentrations, thus i n d i c a t i n g the pot e n t i a l of t h i s material as an a f f i n i t y support. 8 Fraction No. Figure 49. P r o f i l e of j3-glucosidase a c t i v i t y i n fract i o n s eluted from a column 156a. -172-CHAPTER 4  EXPERIMENTAL 4.1 GENERAL 4.1.1 Methods A l l evaporations were performed under diminished pressure on a Buchi rotary evaporator. Melting points were determined using a Fisher-Johns melting point apparatus and are uncor- • rected. Infrared (ir) spectra were recorded on a Perkin-Elmer model 710B i n f r a r e d spectrophotometer, and were ca l i b r a t e d using the 1601 cm"1 band of polystyrene f i l m . Optical rotations were obtained using a Perkin-Elmer 141 polarimeter. Low r e s o l u t i o n mass spectra (ms) were recorded on a Var-ian/MAT CH4B or Kratos/AEI MS 50 mass spectrometer. A n a l y t i -c a l g a s - l i q u i d chromatography was performed on a Hewlett-Packard 5832A gas chromatograph with a 6 f t x 0.125 i n s t a i n l e s s s t e e l column packed with 5% 0V-17 on 80-100 mesh Chromosorb W (HP). Carbon, hydrogen and nitrogen elemental microanalysis were performed by Mr. P. Borda, Microanalytical Laboratory, University of B r i t i s h Columbia. Copper microana-lyses were done by Canadian Microanalytical Ltd., Vancouver, Canada. A n a l y t i c a l thin-layer chromatography (t i c ) was done with 0.20 mm pre-coated aluminum back sheets of S i l i c a Gel 60 F254 (E* Merck,Darmstadt, Germany). Solvent systems employed f o r t i c analyses were; A) ethyl acetate-hexane -173-(3:2), B) ethyl acetate- 2-propanol-water (9:4:2), C) chloro-form-methanol (15:1), and D) chloroform-methanol (4:1). For detection of components, t i c sheets were sprayed with: a) 30% s u l f u r i c acid i n 95% ethanol followed by heating on a hot plate (for carbohydrates), b) 2.0% ammonium molybdate i n 10% s u l f u r i c acid-ethanol solvent, and subsequent heating on a hot plate, c) 2% ninhydrin i n acetone and heating on a hot plate (for amino groups), and d) 1% neutral, aqueous potas-sium permanganate so l u t i o n (for unsaturated moieties). Flash column chromatography was performed using 230-400 mesh s i l i c a gel (Kieselgel 60, E. Merck, Darmstadt, Germany) according to the procedure of S t i l l et a l r 2 6 6 Workup and p u r i f i c a t i o n of reactions involving polysac-charides generally involved exhaustive d i a l y s i s (Spectrapor, membrane tubing, M.W. cutoff 6,000-8,000) against d i s t i l l e d water, followed by freeze-drying. Polysaccharide samples were dried f o r 48 hours at 70°.C i n vacuo (0.05 mm Hg) and stored i n Schlenk tubes under nitrogen atmosphere p r i o r to elemental microanalysis. Ozonolyses were performed at -78°C, using a Welsbach Ozonator (90V, 2 p s i input 0 2 pressure) ozone source. The ozone was bubbled into the cooled sol u t i o n v i a a sintered glass bubbling tube u n t i l the reaction mixture turned pale blue. The ozone source was turned o f f and the soluti o n purged with 0 2 (g) u n t i l colourless. Two equivalents of dimethyl s u l f i d e were added to the reaction mixture, which was allowed to warm to room temperature with s t i r r i n g f o r 2 hours. -174-4.1.2 Nuclear Magnetic Resonance Spectroscopy •^ H-nmr: Proton nmr spectra were t y p i c a l l y measured at 270 MHz using a home-built unit comprised of an Oxford Instru-ments 63.4 KG superconducting solenoid, a Nicolet Model 1180 computer (32K) and a Bruker WP-60 console. Where indicated, 400 MHz spectra were recorded on a Bruker WH-400 spectrome-t e r , and 300 MHz spectra on a Varian XL-300 spectrometer. Samples dissolved i n deuterated chloroform were referenced r e l a t i v e to i n t e r n a l tetramethylsilane (TMS), those dissolved i n deuterium oxide r e l a t i v e to i n t e r n a l sodium 3-trimethylsi-lylproprionate-2,2,3,3-d 4 (TSP). 1 3C-nmr: Proton-decoupled carbon-13 nmr spectra were recorded at 100.6 MHz on a Bruker WH-400 spectrometer, or at 75.5 MHz with a Varian XL-300 spectrometer. Spectra were t y p i c a l l y obtained at temperatures of 305-310 K, unless otherwise s p e c i f i e d . Polysaccharide samples were prepared d i r e c t l y i n the 10 mm nmr tube to avoid handling of the viscous or gelatinous materials. Concentrations were t y p i -c a l l y 5% (w/w), unless further d i l u t i o n was necessary f o r d i s s o l u t i o n , i n which case the concentrations are s p e c i f i e d . 4.1.3 Materials Chemicals and reagents were purchased from suppliers as follows. l-Allyloxy-2,3-epoxypropane, boron t r i f l u o r i d e -methanol (50%) complex, dimethylsulfide, Dowex 50x8, H + (100-200 mesh) ion-exchange r e s i n , hydrogen bromide i n acetic -175-acid (30% w/w), iminodiacetic acid, sodium cyanoborohydride, sodium hydride (60% dispersion i n o i l ) and 10-undecenyl alcohol were obtained from A l d r i c h Chemical Co. D-Glucosamine hydrochloride was purchased from Sigma Chemical Co. A l l y l alcohol and epichlorohydrin were supplied by MC/B Chemical Co. Mercuric cyanide was from ICN Pharmaceuticals Inc., D-lactose from Eastman Kodak Co., and D-glucouronolactone from Eastman Organic Chemicals Ltd. N-Acetyl-D-glucosamine and l,2:3,4-di-0-isopropylidene-D-galactose were obtained from Koch-Light Laboratories, D-glucose from Fischer Scien-t i f i c Co., and D-galactose from Merck and Co. BDH supplied the pyridine and Mallinckrodt, Inc. supplied the a c e t i c anhydride. P u r i f i c a t i o n and d i s t i l l a t i o n of reagents were performed according to standard p r o c e d u r e s . 2 6 7 Chitosan (from crab s h e l l , N-acetyl <5%) was purchased from Sigma Chemical Co. 2-Hydroxyethylcellulose (medium v i s c o s i t y , 4500-6500 cPs, 2.0% solution) was obtained from Polysciences, Inc. Xanthan gum (Keltrol) and sodium alginate (Keltone) and locust bean gum were obtained from Kelco Co. 4.2 EXPERIMENTAL FOR CHAPTER 2 4.2.1 General Synthetic Procedures Synthesis of Acetobromo Sugar Precursors (91-94) The sugar was s t i r r e d i n a 3:2 mixture of a c e t i c anhy-dride/pyridine (10 mL/g sugar) at room temperature overnight -176-under a drying tube. The bulk of the solvent was then removed, ethanol was added and allowed to react f o r 15 min to remove re s i d u a l a c e t i c anhydride. The solvent was removed and the ethanol treatment was repeated. The r e s u l t i n g syrup was c r y s t a l l i z e d from ethanol to give a mixture of a- and ^-per-acetylated sugars i n y i e l d s of 80-95%. The sugar peracetates, e i t h e r the crude syrup or the c r y s t a l l i n e compound, of e i t h e r anomer, were then treated with 30% (w/w) hydrogen bromide i n a c e t i c acid (@ 4 mL/g of peracetate) f o r 45 min at room temperature under a drying tube. The reaction mixture was poured into ice-water and extracted twice with equal volumes of chloroform. The chloro-form layer was washed with saturated sodium bicarbonate to n e u t r a l i t y , then dried over magnesium s u l f a t e , f i l t e r e d , and the solvent removed. The resultant syrup was generally e a s i l y c r y s t a l l i z e d with e i t h e r anhydrous ether or with anhydrous ether/petroleum ether mixtures. These compounds were stored with minimum decomposition f o r months i n a desiccator over sodium hydroxide p e l l e t s . Typical y i e l d s were 85-90%. Methyl (l,2,3,4-tetra-0-acetyl)-D-glucuronate, was prepared by a l i t e r a t u r e method from D-glucurofuranose-3 , 6 - l a c t o n e , 2 7 2 g i v i n g mixtures of the o and £ acetates (1:2) i n 70-75% y i e l d s . Methyl (2,3,4-tri-0-acetyl-l-bromo)-tt-D-glucuronate (9_4) was prepared from the peracetate mixture, using the standard hydrogen bromide-acetic acid (30%) treatment, i n 61% y i e l d . 2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-a-D-glucopyranosyl -177-chloride (103) was prepared v i a a standard preparation i n which 2-acetamido-2-deoxy-l,3,4,6-tetra-O-acetyl -a-D-glucopyranose was treated with hydrogen chloride satu-rated a c e t i c a n h y d r i d e . 2 6 8 Workup and storage of the product was s i m i l a r to that of the acetobromosugars. Y i e l d a f t e r c r y s t a l l i z a t i o n was 79%, m.p. 126-127°C ( l i t . m.p. 126-127°C). Synthesis of Formylmethyl Glycosides (112-1191 12-116 11.7-119 R2 R 3 R4 112,117 OH OH H CH2OH 113,118 OH H OH CH 20H 114 OH OH H C0 2 H 116,119 NHAc OH H CH 20H H0-H0 115 OH |( Cf H CH 20H i f OH -178-A s o l u t i o n of the a l l y l D-glycoside i n methanol (with the exception of allyl -yQ-D-lactose which required methanol-water, 2:1), (@ 5-10 mL/mmol) was cooled to -78°C and saturated with ozone. The pale blue solu t i o n was then purged with oxygen u n t i l colourless and treated with excess (2-4 equiv.) of dimethylsulfide. The s t i r r e d reaction mixture was warmed to ambient temperature over 2 h and concentrated. The syrupy residue was taken up i n ethanol, p r e c i p i t a t e d with ether and decanted. This procedure was repeated, and the resultant gummy p r e c i p i t a t e was dried i n vacuo (0.05 mm Hg) to give a foamy s o l i d which gave a streak by t i c analysis at Rf (solvent B) lower than s t a r t i n g material. Recovery of mate-r i a l was t y p i c a l l y 85-95%, and the product was used d i r e c t l y i n the subsequent reaction without further characterization. Synthesis of 10• -Formylnonyl^Q-D-glycosides (137-139) H Ri R2 The s t a r t i n g 10•-undecenyl ^5-D-glycoside was dissolved i n methanol (§ 5-10 mL/mmol), (methanol-chloroform, 4:1 f o r undecenyl ^S-D-lactose), cooled to -78°C and saturated with -179-ozone* ( u n t i l the solu t i o n was pale blue). The s t i r r e d s o l u t i o n was then purged with oxygen u n t i l colourless, treated with excess dimethyl s u l f i d e and warmed to room temperature over 2 h. The solvent was removed and the residue was repeatedly taken up i n a minimum of ethanol and p r e c i p i -tated with ether. The gummy residue was dried i n vacuo to give a waxy s o l i d , usually i n 85-95% y i e l d s . I t was found that the aldehyde was best used immediately (within 24 h) because a methanol-insoluble residue formed when the aldehyde was l e f t standing at room temperature. This residue dissolved slowly on addition of a c e t i c or d i l u t e hydrochloric acid. 4.2.2 Synthesis of A l l y l Glycosides A l l y l 2,3,4,6-tetra-O-acetyl-^-D-glucopyranoside (95) A mixture of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (9_1, 18.0 g, 43.8 mmol), mercuric cyanide (12.8 g, 50.4 mmol) and D r i e r i t e (14.0 g) i n a l l y l alcohol (90 mL), was s t i r r e d at ambient temperature f o r 24 h. Excess a l l y l alcohol was evaporated, the residue taken up i n chloroform (250 mL) and f i l t e r e d . The f i l t r a t e was washed with saturated brine s o l u t i o n (2x100 mL), dried over magnesium s u l f a t e , f i l t e r e d and concentrated. The residue was dissolved i n methanol, f i l t e r e d and allowed to c r y s t a l l i z e to give 14.5 g (85%) of 95, as a white s o l i d , having a s i n g l e spot by t i c (solvent A), m.p. 87-88°C ( l i t . m.p. 86°, 88°C) ; -^H-nmr (270 MHz): 8 (CDC1 3), 5.84 (m, 1 H, H-2•), 5.29 (br d, 1 H, J 17.0 -180-Ri 95-98 ,104 R4 99-102,105 Ri R 2 R 3 . R4 95 OAc OAc H CH 20Ac 96 OAc H OAc CH 2OAc 97 OAc OAc H C0 2 C H 3 104 NHAc AcO-i AcO/ OAc — o o H CH 2OAc 98 0 A c f s ^ y H CH 20Ac 99 ,108 OH OAc OH H CH 20H 100 .109 OH H OH CH 20H 101 OH OH H C0 2 H 105,111 NHAc OH H CH 2OH 102 HO-n HO/ OH • H CH 2OH -181-Hz, H-3'a), 5.22 (br d, 1 H, J 12.0 Hz, H-3'b), 5.21 (t, 1 H, J 9.5, 9.5 HZ, H-4), 5.11 (t, 1 H, J 9.5, 9.5 Hz, H-3), 5.04 (dd, 1 H, J 9.5, 8.0 Hz, H-2), 4.48 (d, 1 H, J 8.0 Hz, H-l), 4.35 (dd, 1 H, J 14.0, 5.0 Hz, H-l'a), 4.28 (dd, 1 H, J 13.0, 2.0 Hz, H-6) , 4.14 (dd, 1 H, J . 13.0, 4.5 Hz, H-6a) , 4.11 (dd, 1 H, J 14.0, 6.5 Hz, H-l'b), 3.71 (m, 1 H, H-5), 2.10, 2.06, 2.04, 2.02 (4s, 4x3 H, 4 OAc). A l l y l j 3 ~ D ~ 9 l u c o P y r a n o s i d e (21) A l l y l 2,3,4,6-tetra-0_-acetyl-/3-D-glucopyranoside (95, 12.0 g, 31.0 mmol) was dissolved i n dry methanol (75 mL) and the resultant s t i r r e d solution was treated with 0.5N metha-nol i c sodium methoxide (10 mL). The reaction was monitored by t i c (solvent B). When the reaction was complete, Dowex 50x8 (H +, 100-200 mesh) and water (25 mL) were added to neutralize the reaction mixture, which was then f i l t e r e d and concen-trated. The residue was r e c r y s t a l l i z e d from ethanol to give 5.25 g (77%) Of 99, m.p. 99-100°C, [ a ] D 2 5 -39.0°(c 1.10, water) ( l i t . m.p. 100-101°C, [ a ] D 2 5 -40.0°in water) ; ^ -H-nmr (270 MHz): S ( D 2 0 ) , 5.85 (m, 1 H, H-2')/ 5.39 (d, 1 H, J 17.0 HZ, H-3'a), 5.30 (d, 1 H, J_ 10.0 Hz, H-3'a), 4.51 (d, 1 H, J 8.0 Hz, H-l), 4.39 (dd, 1 H, J 13.0, 5.0 Hz, H-l'a), 4.21 (dd, 1 H, J 13.0, 6.5 Hz, H-l'b), 3.93 (br d, 1 H, J 12.0, 2.0 Hz, H-6), 3.71 (dd, 1 H, J 12.0, 6.0 Hz, H-6a), 3.51 (t, 1 H, J 9.0, 9.0 HZ, H-3), 3.45 (dd, IH, J 9.0, 6.0 Hz, H-5), 3.37 (t, 1 H, J 9.0, 9.0 Hz, H-4), 3.28 (t, 1 H, J 9.0, 8.0 HZ, H-2); 1 3C-nmr (100.6 MHz): S(D 20), 131.8 (C-2'), 117.0 (C-3»), 99.6 (C-1), 74.21 (C-5), 74.17 (C-3), 71.5 (C-2), -182-68.0 (C-4 or 1')/ 68.9 ( C - l ' or 4), 59.2 (C-6). A l l y l 2 /3,4,6-tetra-0-acetyl - )Q-D-galactopyranoside (96) 2,3,4,6-Tetra-O-acetyl-ct-D-galactopyranosyl bromide (92. 10.00 g, 24.3 mmol) was s t i r r e d i n anhydrous a l l y l alcohol (50 mL) with mercuric cyanide (6.76 g, 26.6 mmol) and Drier-i t e (7.0 g) under a drying tube, at ambient temperature for 24 h. Ether (50 mL) was added, the suspension was f i l t e r e d and the solvent was removed. The residue was taken up i n chloroform (100 mL), and the organic layer was washed with saturated brine (2x100 mL), dried over magnesium su l f a t e , f i l t e r e d and concentrated. Flash column chromatography of the resultant residue gave 8.01 g (85%) of 96 as a syrup, which was homogeneous by t i c (solvent A) ; -^H-nmr (270 MHz) : S(CDC1 3), 5.85 (m, 1 H, H-2'), 5.40 (d, 1 H, J 3.5 Hz, H-4), 5.34-5.16 (m, 3 H, H-2,3 • a, 3 'b) , 5.04 (dd, 1 H, J_ 10.0, 3.5 Hz, H-3), 4.53 (d, 1 H, J 8.0 Hz, H-l), 4.36 (dd, 1 H, J 13.0, 4.5 Hz, H-l»a), 4.23-4.05 (m, 3 H, H-l'b,6,6a), 3.91 (t, 1 H, J 7.0, 7.0 HZ, H-5), 2.16, 2.07, 2.00 (3s, 12 H, 4 OAC) . A l l y l jS-D-galactopyranoside (100) To a s t i r r e d s o l u t i o n of a l l y l 2,3,4,6-tetra-O- ace-tyl -^S-D-galactopyranoside (9_6, 7.90 g, 20.4 mmol) i n anhydrous methanol (50 mL), was added a sol u t i o n of 0.5N sodium methoxide/methanol (5 mL). When the reaction was complete ( t i c ) , the mixture was neutralized with Dowex 50x8 (H +, 100-200 mesh) ion-exchange r e s i n , f i l t e r e d and concen-trated. The residue was c r y s t a l l i z e d from ethanol-ether to -183-give 4.30 g (96%) of 100 as a white s o l i d , which was homogeneous by t i c (solvent A), m.p. 100-101°C, [0,3 D 2 5 -11.0° (c 1.02, water ) ( l i t . m.p. 102-103°C, [ Q ! ] D 2 5 -10.9°in water); ^-nmr (270 MHz): 8(D 20), 5.97 (m, 1 H, H-2 1), 5.39 (d, 1 H, J 16.0 Hz, H-3'a), 5.28 (dd, 1 H, J 10.0, H-3'b), 4.43 (d, 1 H, J 8.0 Hz, H-l), 4.40 (dd, 1 H, J 13.0, 6.0 Hz, H-l'a), 4.22 (dd, 1 H, J 13.0, 7.0 Hz, H-l'b), 3.92 (d, 1 H, J 3.0 Hz, H-4), 3.77 (dd, 1 H, J 11.0, 7.0 Hz, H-6), 3.75 (dd, 1 H, J_ 11.0, 4.0 Hz, H-6a) , 3.67 (dd, 1 H, J 4.0, 7.0 Hz, H-5), 3.64 (dd, 1 H, J_ 10.0, 4.0 Hz, H-3), 3.53 (dd, 1 H, J 10.0, 8.0 Hz, H-2); 1 3C-nmr (100.6 MHz): 8(D 20), 131.9 (C-2'), 116.9 (C-3'), 100.2 (C-1), 73.4 (C-5), 71.2 (C-3), 69.1 (C-2), 68.9 (C-1'), 67.0 (C-4), 59.3 (C-6). Methyl ( a l l y l 2,3,4-tri-0-acetyl-j8-D-glucopyranosid)uronate (97) Methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl bro-mide) uronate (93, 16.0 g, 40.0 mmol) was s t i r r e d under anhydrous conditions with mercuric cyanide (12.5 g, 49.0 mmol) and D r i e r i t e (13.0 g) i n dry a l l y l alcohol (100 mL) for 19 h. The reaction mixture was f i l t e r e d and concentrated, and the resultant syrupy residue was dissolved i n chloroform (150 mL). The chloroform sol u t i o n was washed with saturated brine (2x100 mL), dried over magnesium su l f a t e , f i l t e r e d and concentrated. The s o l i d residue was r e c r y s t a l l i z e d from methanol to give c r y s t a l l i n e 97 (13.8 g, 92%), m.p. 137-138°C, [ a ] D 2 5 -33.3° (c 1.14, water); -^H-nmr (270 MHz): 8(CDC1 3), 5.74 (m, 1 H, H-2'), 5.32-5.16 (m, 4 H, -184-H-3,4,3'a,3'b), 5.04 (t, 1 H, J 9.0, 8.0 Hz, H-2), 4.60 (d, 1 H, J 8.0 Hz, H-l), 4.36 (dd, 1 H, J 13.5, 5.0 Hz, H-l'a), 4.09 (dd, 1 H, J 13.5, 6.5 Hz, H-l'b), 4.03 (d, 1 H, J 9.0 HZ, H-5), 3.76 (s, 3 H, C0 2CH 3), 2.06, 2.01 (2s, 9 H, 3 OAc) ; Anal. Calcd. f o r C 1 6H 2 2O 1 0:  c i 51.34; H, 5.92. Found: C, 51.39; H, 5.90. A l l y l jQ-D-glucopyranuronic acid (101) Methyl ( a l l y l 2,3,4-tri-O-acetyl -^Q-D-glucopyranosid )uronate (97, 8.5 g, 25.5 mmol) was dissolved i n dry methanol (100 mL) and treated with 0.5N sodium methoxide i n methanol (5.0 mL). A f t e r one hour, a l l s t a r t i n g material was consumed, as shown by t i c (solvent B), and two new spots were evident (Rf 0.62, major and Rf 0.43, minor). A f t e r the addition of 2N sodium hydroxide (aq) solut i o n (15 mL, 30 mmol) and the resultant reaction mixture was s t i r r e d f o r 1 h, a single spot (Rf 0.43) was observed by t i c . Dowex 50x8 (H +, 100-200 mesh) ion-exchange r e s i n was added to a c i d i f y the reaction mixture, which was then f i l t e r e d , decolourized and concentrated. Attempts to c r y s t a l l i z e the residue were unsuccessful, and a f t e r drying i n vacuo. 5.2 g (87%) of a discoloured, foamy s o l i d was obtained, 1 3C-nmr (100.6 MHz): S(D 20), 170.4 (C-6), 131.7 (C-2 1), 117.1 (C-3'), 99.7 ( C - l ) , 73.7 (C-3), 72.8 (C-5), 71.1 (C-2), 69.6 ( C - l 1 or C-4), 69.2 (C-4 or C - l ' ) . A l l y l 2•,3«,6'-tri-O-acetyl-4»-O-(2,3,4,6-tetra-O-acetyl ^S-D-galactopyranosyl) -jS-D-glucopyranoside, ( a l l y l 2,2',3,3'4,6,6' -hepta-0-acetyl - j 9-D-lactoside) (98) 2 •, 3 ', 6»-Tri-O-acetyl-4-O- (2,3,4,6-tetra-O-acetyl- /3-D-- 185 -galactopyranosyl)-a-D-glucopyranosiyl bromide (94, 1.60, 2.3 mmol) was s t i r r e d i n the dark with mercuric cyanide (0.64 g, 2.5 mmol) and D r i e r i t e (1.0 g) i n dry a l l y l alcohol (20 mL) for 24 h. Excess alcohol was removed and the residue taken up i n dichloromethane chloride (50 mL), washed with saturated brine (2x25 mL), dried over magnesium s u l f a t e , f i l t e r e d and concentrated, to y i e l d 1.50 g of a crude syrup. T i c (solvent A) showed a major spot (Rf 0.70) and some minor lower Rf spots. Flash column chromatography gave 1.12 g (72%) of 98 as a foamy s o l i d , which consisted of a si n g l e component by t i c . Large scale preparations with 10 g of "acetobromolactose" gave equally good or better y i e l d s of 98; •'-H-nmr (270 MHz): S(CDC13), 5.94 (m, 1 H, H-2"), 5.35 (d, 1 H, J 3.0 Hz, H-4), 5.32-5.16 (m, 3 H, H-3•,3"a,3"b), 5.12 (dd, 1 H, J 10.0, 8.0 Hz, H-2), 4.97 (dd, 1 H, J 10.0, 3.0 Hz, H-3), 4.94 (t, 1 H, J 9.0, 8.0 Hz, H-2 1), 4.53 (d, 1 H, J 8.0 Hz, H-l or l 1 ) , 4.50 (d, 1 H, J 8.0 Hz, H-l' or 1), 4.47 (dd, 1 H, J 12.0, 2.0 Hz, H-6'), 4.31 (dd, 1 H, J 13.0, 5.0 Hz, H-l"a), 4.15-4.04 (m, 4 H, H-6,6a, 6'a, l " b ) , 3.90 (t, 1 H, J 7.0, 7.0 HZ, H-5), 3.82 (t, 1 H, J 9.0, 9.0 Hz, H-4'), 3.61 (m, 1 H, H-5'), 2.16, 2.13, 2.07, 2.05, 1.96 (5s, 21 H, 7 OAc). A l l y l 4»-0 - ( £ - D-galactopyranosyl ) - j 3 - D-glucopyranoside, ( A l l y l j3 -D-lactoside) (102) A l l y l 2,2' ,3,3' ,4,6,6'-hepta-0-acetyl-/?-D-lactoside (98, 11.0 g, 16.0 mmol) was dissolved i n anhydrous methanol (50 mL) and the resultant s t i r r e d s o l u t i o n was treated with 0.5N sodium methoxide/methanol solution (5.0 mL). When the reac--186-t i o n was complete ( t i c , solvent B), water (25 mL) and Dowex 50x8 (H +, 100-200 mesh) were added u n t i l the s o l u t i o n was neutral. The suspension was f i l t e r e d , the f i l t r a t e was concentrated and the residue was r e c y r s t a l l i z e d from ethanol-water to give 4.9 g (80%) of a white s o l i d , which was homoge-neous by t i c (solvent B); m.p. 169-170°C ( l i t . m.p. 170-171°C) ; ^-nmr (270 MHz): 8(D 20), 5.94 (m, 1 H, H-2"), 5.33 (br d, 1 H, J_ 17.0 Hz, H-3'a), 5.24 (br d, 1 H, J 10.0 Hz, H-3'b), 4.49 ( d l H, 2 8.0 Hz, H-l or 1'), 4.40 (d, 1 H, J 7.5 Hz, H-l' or 1), 4.35 (dd, 1 H, J 12.5, 5.5 Hz, H-l"a), 4.19 (dd, 1 H, J_ 12.5, 6.0 Hz, H-l"b) , 3.98-3.48 (m, 11 H, H-2,2',3,3 l,4,4 ,,5 ,,6,6a,6 ,,6 la), 3.30 (br t , 1 H, J 7.0, 6.0 Hz, H-5); 1 3C-nmr (100.6 MHz): 8(D 20), 131.8 (C-2"), 117.0 (C-3"), 101.2 (C-1), 99.4 (C-1'), 76.8 (C-4'), 73.6 (C-5), 73.0 (C-5' or 3«), 72.7 (C-3' or 5'), 71.1 (C-2' or 3), 70.9 (C-3 or 2«), 69.2 (C-2), 68.9 (C-1"), 66.8 (C-4), 59.5 (C-6'), 59.3 (C-6). A l l y l 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl - /3-D-glucopyranoside (104) 2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-a-D-glucopyranosyl chloride (103, 14.0 g, 38.3 mmol) was s t i r r e d with mercuric cyanide (10.8 g, 42.5 mmol) and D r i e r i t e (10 g) i n dry a l l y l alcohol (75 mL) f o r 16 h under anhydrous conditions. The reaction mixture was d i l u t e d with chloroform (50 mL), f i l -tered and concentrated to a t h i c k syrup. The syrup was taken up i n chloroform (150 mL), f i l t e r e d and the organic phase was washed with saturated brine s o l u t i o n (2x100 mL), dried over -187-magnesium su l f a t e , f i l t e r e d and concentrated. The residue was c r y s t a l l i z e d from ethyl acetate/petroleum ether to give 11.17 g (75%) of a white c r y s t a l l i n e s o l i d , which consisted of one component by t i c analysis (solvent A); m.p. 161-162°C ( l i t . m.p. 160°, 162-163°C); 1H-nmr (270 MHz): 8(CDC1 3), 5.84 (m, 1 H, H-2«), 5.76 (d, 1 H, J 8.5 Hz, NHAc), 5.31 (t, 1 H, J 10.0, 10.0 Hz, H-4), 5.28 (br d, 1 H, J 18.0 Hz, H-3'a), 5.21 (br d, 1 H, J 11.0 HZ, H-3'b), 5.08 (t, 1 H, J 10.0, 10.0 Hz, H-3), 4.73 (d, 1 H, J_ 8.0 Hz, H - l ) , 4.35 (dd, 1 H, J 13.0, 5.0 Hz, H-l'a), 4.28 (dd, 1 H, J_ 13.0, 5.0 Hz, H-6), 4.15 (dd, 1 H, £ 13.0, 2.0 Hz, H-6a), 4.10 (dd, 1 H, J 13.0, 6.0 Hz, H-l'b), 3.90 (dt, 1 H, J 10.0, 8.5, 8.0 Hz, H-2), 3.72 (m, 1 H, H-5), 2.10, 2.04, 2.03 (3s, 3 H each, 3 OAc), 1.95 ( S , 3 H, NHAC). A l l y l 2-acetamido-2-deoxy-^3-D-glucopyranoside (105) De-O-acetylation of a l l y l 2-acetamido-3,4,6-tri-Q-acetyl-2-deoxy- jQ-D-glucopyranoside (104 f 10.0 g, 25.8 mmol) was done i n methanol with 0.5N methanolic sodium methoxide (5.0 mL). When the reaction was complete ( t i c ) , the mixture was neutralized with Dowex 50x8 (H +, 100-200 mesh) ion-exchange r e s i n , f i l t e r e d and concentrated. The residue was r e c r y s t a l l i z e d from methanol to give 4.96 g (74%) of 105 as a s o l i d , which consisted of one component by t i c (solvent B), m.p. 170-171°C, [ a ] D 2 5 -31.9° (c 0.58, water), ( l i t . m.p. 171-172°C, [O ; ] D 2 5 -33.9° i n water); ^-nmr (270 MHz): 8(D 20), 5.90 (m, 1 H, H-2'), 5.31 (d, 1 H, J 17.0 Hz, H-3'a), 5.26 (d, 1 H, J 10.0 Hz, H-3'b), 4.56 (d, 1 H, J 8.0 Hz, H-l), -188-4.34 (dd, 1 H, J 13.0, 5.0 Hz, H-l'a), 4.16 (dd, 1 H, J 13.0, 6.0 Hz, H-l'b), 3.93 (d, 1 H, J 12.0 Hz, H-6), 3.74 (dd, 1 H, J 12.0, 5.0 HZ, H-6a), 3.72 (br t, 1 H, J 9.0, 8.0 Hz, H-2), 3.43-3.58 (m, 3 H, H-3,4,5); 1 3C-nmr (100.6 MHz): 8(D 20), 173.0 (COCH3), 132.3 (C-2'), 116.5 (C-3 1), 98.8 (C-1), 74.5 (C-5), 72.5 (C-3), 68.9 (C-1' or 4), 68.7 (C-4 or 1'), 59.5 (C-6), 54.2 (C-2), 20.8 (CO_CH3). A l l y l a-D-glucopyranoside (108) Anhydrous D-glucose (106. 20.0 g, 110 mmol) was s t i r r e d with Dowex 50x8 (H +, 100-200 mesh) i n dry a l l y l alcohol (200 mL) at r e f l u x temperature f o r 90 min. The reaction mixture was f i l t e r e d and concentrated to af f o r d a syrupy residue. C r y s t a l l i z a t i o n of t h i s material from absolute ethanol gave 4.1 g (17%) of 108. Decolourization and reprocessing of the mother l i q u o r provided an additional 4.4 g (18%), gi v i n g an o v e r a l l 8.5 g (35%) of a product, which exhibited one spot by t i c analysis (Rf 0.486, solvent B); m.p. 97-98°C, [ a ] D 2 5 +136.4° (c, 1.06, water), ( l i t . m.p. 95-97°C; [a ] o 2 5 +133.8° i n water); ^-nmr (270 MHz): 8(D 20), 5.99 (m, 1 H, H-2'), 5.38 (br d, 1 H, J_ 17.0 Hz, H-3'a), 5.27 (br d, 1 H, J_ 10.0 HZ, H-3'b), 4.98 (d, 1 H, J_ 3.5 Hz, H-l), 4.24 (dd, 1 H, J 12.0, 5.0 HZ, H-l'a), 4.07 (dd, 1 H, J 12.0, 6.0 Hz, H-l'b), 3.89-3.66 (m, 4 H, H-3,5,6,6a), 3.57 (dd, 1 H, J 10.0, 3.5 Hz, H-2), 3.42 (t, 1 H, J 10.0, 10.0 Hz, H-4); 1 3C-nmr (100.6 MHz): 8(D 20), 132.1 (C-2'), 116.5 (C-3'), 95.7 (C-1), 71.5 (C-3), 70.2 (C-5), 69.6 (C-2), 68.0 (C-4), 66.8 (C-1'), 59.0 (C-6). -189-A l l y l a-D-galactopyranoside (109) D-Galactose (107. 20.0 g, 110 mmol) was suspended i n dry a l l y l alcohol (200 mL) and s t i r r e d with Dowex 50x8 (H +, 100-200 mesh, 12.0 g) at r e f l u x temperature f o r 90 min. The reaction mixture was f i l t e r e d and concentrated, and the resultant syrupy residue was c r y s t a l l i z e d from absolute ethanol to give 10.2 g (42%) of a white c r y s t a l l i n e s o l i d . Further r e c r y s t a l l i z a t i o n from absolute ethanol gave material homogeneous by t i c analysis (solvent B); m.p. 146-147°C; [ a ] D 2 5 +178.0° (c 0.99, water); ( l i t . m.p. 143-145°C, [ a ] D 2 5 +181.3°, i n water); -^H-nmr (270 MHz): 8(D 20), 5.99 (m, 1 H, H-2'), 5.39 (d, 1 H, J 17.0 Hz, H-3'a), 5.28 (d, 1 H, J 11.0 Hz, H-3'b), 4.89 (d, 1 H, J 2.0 Hz, H-l), 4.25 (dd, 1 H, J 13.0, 5.0 Hz, H-l'a), 4.08 (dd, 1 H, J 13.0, 6.0, H-l'b), 3.98 (br s, 1 H, I 1.0 HZ, H-4), 3.96 (d, 1 H, J 8.0 Hz, H-5), 3.89-3.80 (m, 2 H, H-2,3), 3.77-3.63 (m, 2 H, H-6,6a); 1 3C-nmr (100.6 MHz): 8(D 20), 132.2 (C-2 1), 116.5 (C-3'), 96.1 ( C - l ) , 69.3 (C-5), 68.0 (C-3), 67.7 (C-4), 66.9 (C-2 or 1')/ 66.7 ( C - l 1 or 2), 59.6 (C-6). A l l y l 2-acetamido-2-deoxy-ot-D-glucopyranoside (111) 2-Acetamido-2-deoxy-glucose (110, 25 g, 113 mmol) was suspended i n dry a l l y l alcohol (350 mL) and the resultant mixture was treated with boron t r i f l u o r i d e etherate complex (2.5 mL). The reaction mixture was refluxed f o r 2 h and allowed to stand at room temperature f o r 24 h. Removal of solvent l e f t a c r y s t a l l i n e residue which was r e c r y s t a l l i z e d from ethanol/ether to give 13.9 g (47%) of a white c r y s t a l --190-l i n e s o l i d , which consisted of a major spot (Rf 0.417, solvent B) and a minor spot (Rf 0.33) by t i c analysis. The less e r component was shown by 1H-nmr to be the jQ-isomer ( a l l y l 2-acetamido-2-deoxy-jQ-D-glucopyranoside) , which was present i n -1:8 r a t i o with the a-isomer. Subsequent recrys-t a l l i z a t i o n s provided 111 as a single component by t i c analysis (Rf 0.417, solvent B); m.p. 172-173°C; [a]r>25 +147.6° (c 1.12, water); ( l i t . m.p. 172-174°C, [ a ] D 2 5 +148.8° i n water); ^-nmr (270 MHz): 8(D 20), 5.94 (m, 1 H, H-2'), 5.36 (d, 1 H, J_ 16.5 Hz, H-3'a), 5.27 (d, 1 H, 1 11.0 Hz, H-3'b), 4.93 (d, I H , J 3.0 Hz, H-l), 4.23 (dd, 1 H, J_ 13.0, 5.5 HZ, H-l'a), 4.03 (dd, 1 H, J_ 13.0, 6.5 Hz, H-l'b), 3.95-3.63 (m, 5 H, H-2,3,5,6,6a), 3.49 (t, 1 H, H-4); 1 3C-nmr (100.6 MHZ) S ( D 2 0 ) , 172.7 (COCH3), 132.1 (C-2'), 116.2 (C-3'), 94.5 ( C - l ) , 70.3 (C-5), 69.4 (C-3), 68.5 (C-4), 66.8 ( C - l ' ) f 59.9 (C-6), 52.0 (C-2), 20.3 ( C O C H 3 ) . 4.2.3 Synthesis of lO'-Undecenyl ff-D-Glycosides 10'-Undecenyl j9-D-glucopyranoside (134) A suspension of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (91, 20.0 g, 48.7 mmol), 10-undecenol (16.58 g, 97.3 mmol), mercuric cyanide (13.53 g, 53.6 mmol) and D r i e r i t e (14 g) were s t i r r e d i n chloroform (125 mL) at r e f l u x f o r 10 h. The reaction mixture was f i l t e r e d , washed with saturated brine sol u t i o n (2x75 mL), dried over magnesium s u l f a t e , f i l t e r e d and concentrated. The residue was taken up i n -191-anhydrous methanol and treated with 0.5N methanolic sodium methoxide (10.0 mL). When the reaction was complete ( t i c , solvent D), Dowex 50x8 (H +, 100-200 mesh) ion-exchange r e s i n was added to neutralize the solution, which was then f i l -tered. The f i l t r a t e was concentrated and the resultant residue was subjected to l i q u i d chromatography (chloroform-methanol, 5:1) to af f o r d 12.9 g (80%) of a waxy s o l i d , 134. •^H-nmr (270 MHz): 8(CD3OD) , 5.79 (m, 1 H, H-10'), 4.96 (br d, 1 H, J_ 17.0, 1.5 Hz, H - l l ' a ) , 4.90 (d, 1 H, J 10.0 Hz, H - l l ' b ) , 4.26 (d, 1 H, J 8.0 Hz, H-l), 3.86 (dd, 1 H, J 11.5, 2.4 Hz, H-6a), 3.69 (dd, 1 H, J 11.5, 5.2 Hz, H-6b), 3.55 (m, 2 H, H-l«a,b), 3.38 (t, 1 H, J 9.0, 8.5 Hz, H-3), 3.32 (t, 1 H, J 9.0, 8.5 HZ, H-4), 3.27 (m, 1 H> H-5), 3.19 (t, 1 H, J 8.5, 8.0 Hz, H-2), 2.04 (m, 2 H, H-9'a,b), 1.63 (m, 2 H, H-2'a,b), 1.43-1.29 (m, 12 H, a l k y l protons); 1 3C-nmr (100.6 -192-MHz): 8(CD3OD), 138.3 (C-10'), 112.8 ( C - l l ' ) , 102.5 (C-1), 76.3 (C-5), 76.0 (C-3), 73.3 (C-2), 70.0 (C-4), 69.1 (C-1 1), 61.1 (C-6), 32.9 (C-9 1), 29.0-28.2 and 25.4 (C-2' to 8'); ms (fab), m/z: 333 (M+H)+. 10'-Undecenylj9-D~galactopyranoside (135) A mixture of 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (92, 17.9 g, 43.6 mmol), 10-undecenal (14.8 g, 87.6 mmol), mercuric cyanide (12.1 g, 48.0 mmol) and D r i e r i t e (18 g) i n chloroform (125 mL) was s t i r r e d at r e f l u x f o r 9 h under anhydrous conditions. The reaction mixture was cooled, d i l u t e d with ether (60 mL), f i l t e r e d and concentrated. The residue was dissolved i n chloroform (200 mL), and the chloro-form layer was washed with saturated brine (2x100 mL), dried over magnesium s u l f a t e and f i l t e r e d . A f t e r the solvent was removed, the residue was dissolved i n anhydrous methanol (80 mL) and deacetylated with 0.5N sodium methoxide i n methanol (10.0 mL). When the reaction was complete ( t i c , solvent C), Dowex 50x8 (H +, 100-200 mesh) was added and the mixture was f i l t e r e d and concentrated. The crude product (16.0 g) was chromatographed (chloroform-methanol, 5:1) to give 12.2 (84%) of a waxy s o l i d having a single spot by t i c analysis (solvent D), -^H-nmr (270 MHz): 8(CD3OD), 5.83 (m, 1 H, H-10'), 4.99 (br d, 1 H, J 17.0 Hz, H - l l ' a ) , 4.93 (br d, 1 H, J 17.0 Hz, H - l l ' b ) , 4.24 (d, 1 H, J 7.2 Hz, H-l), 3.90 (m, 3 H, H-l«a f 1^,5) , 3.77 (d, 1 H, J 6.2 Hz, H-4), 3.63-3.45 (m, 4 H, H-6,6b,2,3), 2.04 (m, 2 H, H-9'a,b), 1.63 (m, 2 H, H-2'a,b), 1.43-1.30 (br s, 12 H, a l k y l protons); 1 3C-nmr -193-(100.6 MHz): S(CD3OD) , 138.2 (C-10'), 112.8 ( O i l ' ) , 103.1 ( C - l ) , 74.65 (C-5), 73.3 (C-3), 70.8 (C-2), 69.0 (C-4 or 68.5 ( C - l 1 or 4), 60.7 (C-6), 32.9 (C-9'), 29.0-28.2 and 25.2 (C-2' to 8'); ms (fab), m/z: 333 (M+H)+. 10"-Undecenyl 4'-0-t)9-D-galactopyranosyl)-^3-D-glucopyr anoside, (10"-Undecenylj3""D-lactoside) (136) "Acetobromolactose" (SA, 25.0 g, 35.8 mmol), 10-undecenol (12.2 g, 72.0 mmol), mercuric cyanide (9.94 g, 39.4 mmol) and D r i e r i t e were s t i r r e d together i n chloroform (100 mL) at r e f l u x f o r 11 h. The reaction mixture was cooled and f i l -tered, and the f i l t r a t e was washed with saturated brine s o l u t i o n (2x75 mL), dried over magnesium s u l f a t e and concen-trated. The residue was dissolved i n anhydrous methanol and the resultant s t i r r e d solution was treated with 0.5N sodium methoxide i n methanol (10.0 mL). When the reaction was complete ( t i c , solvent D), the reaction was neutralized with Dowex 50x8 (H +, 100-200 mesh) ion-exchange r e s i n and f i l t e r e d . The solvent was removed and the residue was p r e c i -p i t a t e d from methanol to y i e l d 14.9 g (84%) of 136 as a waxy s o l i d , -^H-nmr (270 MHz): S(CD3OD), 5.80 (m, 1 H, H-10") , 4.98 (br d, 1 H, J_ 17.0 Hz, H - l l " a ) , 4.93 (d, 1 H, J 10.5 Hz, H - l l " b ) , 4.42 (br d, 1 H, J 7.5 Hz, H-l' ) , 4.28 (d, 1 H, J 8.0 Hz, H-l), 3.94-3.83 (m, 3 H, H-6a,6b,4), 3.80 (br d, 1 H, J 11.5 Hz, H-6«a), 3.74 (dd, 1 H, J 11.5, 4.5 Hz, H-6«b), 3.70-3.49 (m, 5 H, H-2,3,3•,4•,5), 3.46 (m, 1 H, H-5'), 3.30 (t, 1 H, J 9.0, 8.0 Hz, H-2')* 2.04 (m, 2 H, CH 2-9"), 1.63 (m, 2 H, CH 2-2"), 1.42-1.29 (br s, 12 H, a l k y l protons); -194-1 3C-nmr (100.6MHz): 8(00300), 138.3 (C-10"), 113.2 ( C - l l " ) , 103.0 (C-1), 102.2 (C-l«), 79.0 (C-4'), 75.1 (C-5 or 5' or 3 1 ) , 74.5 (C-5' and 3 1 or 5), 72.8 (C-2' or 3), 72.78 (C-3 or 2'), 70.8 (C-2), 69.7 (C-4), 68.4 (C-1"), 60.7 (C-6), 60.2 (C-6 1), 32.9 (C-9 1), 28.9-28.2 and 25.1 (C-2" to 8"); ms (fab), m/z: 495 (M+H)+, 333. 4.2.3 Synthesis of Branched Chitosan Derivatives General Procedure f o r the Preparation of N-[2'-o-(D-glycopyr anosyl)ethyl]chitosans Chitosan flakes were dissolved i n 5.0% aqueous a c e t i c a c i d (§ 10 mL/1.0 mmol) with s t i r r i n g . A so l u t i o n of the aldehyde (0.5-3.0 molar equivalents) i n 5.0% aqueous ac e t i c a c i d (10-15 mL) was added, followed by treatment with sodium cyanoborohydride (§ 4.0 molar equivalents) f o r 24 h. The reaction mixture was then dialyzed against d i s t i l l e d water (6x2 L) f o r 6 days, f i l t e r e d through a medium pore glass f r i t f i l t e r and freeze dried. Yields varied between 55 and 95%. N-[2'-o-( jQ-D-glucopyranosyl)ethyl]chitosan (120) a) Chitosan (0.50 g, 3.11 mmol) reacted with formylmethyl j8-D-glucopyranoside (112. 2.10 g, 9.50 mmol) to give 1.08 g, (95%) of compound 120a. Anal. Calcd. f o r [(Ci 4H 25N0 1 0)i.on]*0.51H 2O: C, 44.66; H, 6.91; N, 3.72. Found: C, 44.67; H, 6.92; N, 3.62. N- [2 1 -O- tj8-D-galactopyranosyl) ethyl] chitosan (121) a) Chitosan (0.65 g, 4.04 mmol) was treated with the -195--196-acetaldehydo-glycoside 113 (2.7 equiv) to give 0.86 g (58%) of der i v a t i v e 121a. Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 . 0 4 (C 1 4H 2 5NO 1 0) 0 > 9 6 ] *2.3 H 20: C, 41.12; H, 7.25; N, 3.43. Found: C, 41.12: H/6.94: N, 3.51. b) When chitosan (0.55 g, 3.42 mmol) was reacted with the aldehyde 113 (1.00 g, 4.50 mmol), 0.86 g (83%) of l y o p h i l i z e d product 121b was obtained. Anal. Calcd. f o r [ ( 0 ^ ^ 0 4 ) 0 . 3 3 ( c14 H25 N 0 lo) 0.67] * 0 - 5 0 H 20: C, 43.34; H, 7.12; N, 4.45. Found: C, 43.35; H, 7.11; N, 4.46. c) Chitosan (0.65 g, 4.04 mmol) reacted with aldehydo sugar 113 (0.67 g, 3.00 mmol) to y i e l d 0.82 g (81%) of compound 121c. Anal. Calcd. f o r [ (Cgl^NC^) 0 # 6 2 (C 1 4H 2 5NO 1 0) 0.38 3 *0.63 H 20: C, 43.31; H, 7.03; N, 5.59. Found: C, 43.31; H, 6.86; N, 5.59. N- [2'-o- ty3-D-glucuronopyranosyl)ethyl]chitosan (122) a) Coupling of the aldehyde 114 (1.75 g, 7.5 mmol) to chitosan (0.40 g, 2.5 mmol) gave a p r e c i p i t a t e d product a f t e r 15 min. The sol u t i o n was made basic (pH-8), dialyzed as usual and freeze-dried. The l y o p h i l i z e d product was dissolved i n 0.5N sodium hydroxide (10 mL), pr e c i p i t a t e d with ethanol, f i l t e r e d and washed with methanol to give, a f t e r drying, 0.67 g (67%) of 122a. Anal. Calcd. f o r [(C 1 4H 2 2N0 1 1Na)]•0.32H 2 0 : C, 41.40; H, 5.40; N, 3.45. Found: C, 41.40; H, 5.40; N, 3.18. -197-b) Chitosan (0.40 g, 2.5 mmol), when treated with the aldehydo-sugar 114 (0.60 g, 2.6 mmol), produced a viscous solu t i o n a f t e r 24 h. The solution was dialyzed and freeze-dried to y i e l d 0.67 g (81%) of deriva t i v e 122b. Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 . 3 3 (C 1 4H 2 3N0 1 1) 0 . 673 * 1-* 3 H 20: C, 41.61; H, 6.47; N, 4.26. Found: C, 41.61; H, 6.43; N, 4.30. N- (2"-0- [4' -0- (j9-D-galactopyranosyl) -^3-D-glucopyranosyl] ethyl)chitosan (123) a) Chitosan (0.40 g, 2.5 mmol) was treated with acetalde-hydo j9-D-lactoside 115 (3.0 g, 7.8 mmol) to give 1.30 g (95%) of d e r i v a t i v e 123a. Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 > 1 X ( C 2 0 H 3 6 N O 1 5 ) 0 m 8 9 ] • 0.22H20 : C, 44.90; H, 6.83; N, 2.84. Found: C, 44.89; H, 6.84; N, 2.83. b) When chitosan (0.65 g, 4.0 mmol) was treated with compound 115 (2.30 g, 6.00 mmol), 1.52 g (86%) of derivative 123b was obtained. Anal. Calcd. f o r [ ( C 6 H 1 3 N 0 4 ) 0 . 2 4 ( C 2 0 H 3 6 N 0 1 5 ) 0 . 7 6 ] > 0 - 6 6 H 20: C, 44.12; H, 6.92; N, 3.09. Found: C, 44.12; H, 7.17; N, 3.09. c) When chitosan (0.65 g, 4.04 mmol) was reacted with the aldehyde 115 (1.15 g, 3.00 mmol), 1.21 g (97%) of compound 123c was i s o l a t e d . Anal. Calcd. f o r [ (C 6H nN0 4) 0.65( c20 H36 N O15) 0.35) ' 0 • 7 4 H 20: C, 43.15; H, 7.00; N, 4.62. Found: C, 43.15; H, 7.19; N, 4.63. -198-d) Chitosan (0.80 g, 5.00 mmol) and 115 (1.00 g, 2.60 mmol) were reacted to give 1.36 g (93%) of der i v a t i v e 123d. Anal. Calcd. f o r [ (C 6H 1 : LN04) 0 > 6 7 (C 2 oH3 6N0 1 5) 0.33]*0.44H2O: C, 43.84; H, 6.92; N, 4.82. Found: C, 43.84; H, 7.27; N, 4.84. e) Chitosan (0.60 g, 3.70 mmol) and 115 (0.50 g, 1.30 mmol) were reacted to give 0.80 g (87%) of 123e. Anal. Calcd f o r [ (C 6H 1 : LN04) 0 . 7 6 (C 2 0H 3 6NO 1 5) 0 . 2 4 ] • 0.49H20: C, 43.47; H, 6.97; N, 5.41. Found: C, 43.46; H, 7.29; N, 5.37. N- [2' -O- (2-acetamido-2-deoxy-j@-D-glucopyranosyl) ethyl]chitosan (124) a) Reaction of chitosan (0.30 g, 1.9 mmol) with the acetaldehydo-$-D-glycoside 116 (1.50 g, 5.6 mmol) gave 0.67 g (85%) of the der i v a t i v e 124a. Anal. Calcd. f o r [(C 1 6H 2 8N 2O 1 0)]•0.85H 20: C, 45.36; H, 7.02; N, 6.61. Found: C, 45.36; H, 7.33; N, 6.43. N-(2'-Qr(a-D-glucopyranosyl)ethyl]chitosan (125) a) Chitosan (0.65 g, 4.04 mmol) when treated with ace-taldehydo a-D-glucopyranoside 117 (2.65 g, 11.9 mmol) gave 1.42 g (96%) of derivative 125a. Anal. Calcd. f o r [(C 1 4H 25NO 1 0) l t 0 0]•0.61H 20: C, 44.44; H, 6.94; N, 3.70. Found: C, 44.45; H, 6.85; N, 3.40. b) Chitosan (0.60 g, 3.73 mmol) was coupled with the aldehyde 117 (1.24 g, 5.59 mmol) to give 0.66 g (61%) of 125b. Anal. Calcd. f o r [ (C 6H 1 ; LN04) 0 < 4 1 (C 1 4H 2 5NO 1 0) 0 . 59] • 0. 87 H 20: C, 43.14; H, 7.04; N, 4.69. Found: C, 43.14; H, 7.10; N, -199-4.69. c) Chitosan (0.60 g, 3.73 mmol) was reacted with the aldehyde 117 (0.62 g, 2.80 mmol) to give 0.67 g (72%) of compound 125c. Anal. Calcd. f o r [ ( C 6 H i : L N 0 4 ) 0 . 6 2 (C 1 4H 2 5NO 1 0) 0 . 3 8 ] • 0.62 H 20: C, 43.30; H, 7.00; N, 5.59. Found: C, 43.30; H, 7.01; N, 5.60. d) Chitosan (0.80 g, 5.0 mmol) was treated with aldehyde 117 (1.11 g, 5.00 mmol) to y i e l d 0.91 g (80%) of 125d. Anal. Calcd, f o r [ ( ^ ^ ^ 0 4 ) 0 > 7 4 (C 1 4H 2 5NO 1 0) 0 # 2 6 ] • 0.66H20 : C, 42.82; H, 7.05; N, 6.18. Found: C, 42.82; H, 7.15; N, 6.17. N -[2» - O - ( a - D-galactopyranosyl)ethyl]chitosan (126) a) Reaction of chitosan (0.45 g, 2.8 mmol) and the aldehyde (118. 1.90 g, 8.64 mmol) gave 0.60 g (57%) of l y o p h i l i z e d product. Anal. Calcd. f o r [(C 1 4H 2 5NO 1 0)]•0.63H 20: C, 44.40; H, 6.94; N, 3.70. Found: C, 44.41; H, 7.01; N, 3.74. b) Reaction of the aldehyde (118, 1.67 g, 7.52 mmol) and chitosan (0.60 g, 3.73 mmol) afforded 0.68 g (54%) of a white f l u f f y product 126b. Anal. Calcd. f o r [ ( 0 5 ^ ^ 0 4 ) 0 < 1 4 (C 1 4H 2 5NO 1 0) 0 > 8 6 ] • 1.06H2O: C,43.27; H, 7.03; N, 3.92. Found: C, 43.27; H, 7.12; N, 3.91. c) When chitosan (0.80 g, 5.0 mmol) was reacted with the aldehyde 118 (1.11 g, 5.00 mmol), 1.34 g (96%) of product 126c was obtained. -200-Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 . 5 2 (C 1 4H 2 5NO 1 0) 0 > 4 8 ] -0.49 H 20: C, 43.95; H, 6.97; N, 5.21. Found: C, 43.95; H, 7.19; N, 5.20. d) When chitosan (0.80 g, 5.0 mmol) was treated with the aldehyde 118 (0.83 g, 3.74 mmol), 1.10 g (95%) of product 126d was obtained. Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 < 6 8 (C 1 4H 2 5N0 1 0) 0 . 3 2 ] • 0.41H20 : C, 43.85; H, 6.96; N, 5.98. Found: C, 43.85; H, 7.30; N, 5.98. K-[2"-0-(2-acetamido-2-deoxy-a-D-glucopyranosyl) ethyl]chitosan (127) a) Chitosan (0.60 g, 3.7 mmol) was treated with the aldehydo sugar 119 (3.05 g, 11.5 mmol) to give 1.35 g (92%) of l y o p h i l i z e d product 127a. Anal. Calcd. f o r [ (C 1 6 H 2 8 N 2 O 1 0 ) ' 0 . 3 6 ^ 0 ] : C, 46.32: H, 6.92; N, 6.76. Found: C, 46.32; H, 6.61; N, 6.78. b) Reaction of chitosan (0.60 g, 3.7 mmol) with the acetaldehydo-glycoside 119 (3.00 g, 11.4 mmol) gave 1.25 g (85%) of product a f t e r freeze-drying. Anal. Calcd. f o r [(C 1 6H 2 8NO 1 0)•1.53H 20]: C, 44.08; H, 7.12; N, 6.43. Found: C, 44.08; H, 7.05; N, 6.23. c) Coupling of the aldehyde 119 (1.60 g, 6.0 mmol) to chitosan (0.65 g, 4.0 mmol) yielded 1.0 g (95%) of derivative 127c. Anal. Calcd. f o r [ (C 6H 1 : LN0 4) 0 . 6 3 (C 1 6H 2 8N 2O 1 0) 0 . 3 7 ] •0.52H2O: C, 44.47; H, 7.01; N, 7.33. Found: C, 44.47; H, 6.86; N, 7.31. -201-d) Treatment of chitosan (0.65 g, 4.0 mmol) with the aldehydo-glucoside 119 (0.79 g, 3.0 mmol) gave 0.80 g (93%) of compound 127d. Anal. Calcd. for t(C 6H 1 1NO 4) 0. 83(C 1 6H2 8N 2O 1 0) 0. 1 7]-0.58 H 20: C, 43.30; H, 7.05; N, 7.68. Found: C, 43.30; H, 7.10; N, 7.71. e) Chitosan (0.80 g, 5.0 mmol) was reacted with 119 (0.70 g, 2.5 mmol) to produce 1.03 g (95%) of product 127e. Anal. Calcd. f o r [ (C6HnN04) o.8l( t'16^28 N2 0lo) 0.193 '0.46 H 20: C, 43.84; H, 7.00; N, 7.70. Found: C, 43.83; H, 7.18; N, 7.70. Synthesis of N-[2 • -0- (2-amino-2-deoxy-^3-D-glucopyranosyl) ethyl]chitosan (129) Derivative 127b (1.25 g, 3.10 mmol) was suspended i n 40% aqueous sodium hydroxide (60 mL) and heated at 100 C under a nitrogen atmosphere for 6 h. The reaction mixture was then cooled, neutralized, dialysed and freeze dried to give 1.1 g (97%) of 129. Anal. Calcd. f o r [ ( C 1 4 H 2 6 N 2 0 9 ) 1 # 0 ] • 3 . 0 H 2 O : C, 40.00; H, 7.63; N, 6.67. Found: C, 40.00; H, 7.97; N, 6.76. Synthesis of N-[2»-0-(0-D-glucopyranosyl)ethyl]chitin (130) Derivative 125c (160 mg, 0.67 mmol) was dissolved i n a water-methanol mixture (1:1, 13 mL) and s t i r r e d with acetic anhydride (70 mg, 65 mL, 0.67 mmol) for 24 h. Exhaustive d i a l y s i s against d i s t i l l e d water gave d e r i v a t i v e 130 (95%). Anal. Calcd. for [ ( C 8 H 1 3 N 0 5 ) 0 . 6 2 ( C 1 4 H 2 5 N O 1 0 ) 0 . 3 8 ] • 0 . 8 3 H 20: C,44.03; H, 6.86; N, 5.00. Found: C, 44.03; H, 6.84; N, -202-4.80. General Procedure f o r the Preparation of N-[10' -O- (jg-D-glycopyranosyl) decyl] chitosan Derivatives Ri R2 140 OH H 141 H OH H O - i H 0 > - 0 0 142 KQH J H 140-142 OH CH20H 143 144 -203-A s t i r r e d solution of chitosan flakes i n a mixture of 5% aqueous a c e t i c acid-methanol (1:1, @ 15 mL/mmol) was treated with a solution of the 10-decanalyl jS-D-glycoside (1.5-3.0 equiv.) i n the reaction media (10-15 mL) and subsequently with sodium cyanoborohydride (§ 4.0 molar equiv). The r e s u l -tant mixture was s t i r r e d for 24 h and then dialyzed for 3 days against methanol-water, 1:1 (3x1 L) and 3 days against d i s t i l l e d water (3x1 L). The solution was f i l t e r e d through a medium pore sintered glass f i l t e r and freeze-dried. Yields were 50-90%. N-[10'-O-( jS-D-glucopyranosidyl)decyl]chitosan (140) a) Chitosan (0.65 g, 4.0 mmol) was treated with the aldehyde 137 (2,8 g, 8.4 mmol) to give, a f t e r workup, 0.93 g (54%) of deriva t i v e 140a. Anal. Calcd. f o r [ (C 6H^N0 4) 0 < 3 ^ (C 2 2H 4 1N) 1 0 ) 0 # 8 1 ] • 0.83 H 20: C, 52.49; H, 8.53: N, 3.23. Found: C, 52.49; H, 8.20; N, 3.23. b) When chitosan (0.45 g, 2.8 mmol) was reacted with the decanalyl jS-D-glycoside 137 (2.85 g, 8.5 mmol), 1.64 g (91%) of product 140b was obtained. Anal. Calcd. f o r [ ( C 2 2 H 4 1 N O 1 0 ) 0 . 5 3 ( C 3 8 H 7 1 N 0 1 6 ) 0 < 4 7 ) ] - 1 . 0 H 20: C, 54.80; H, 8.83; N, 2.17. Found: C, 54.80; H, 8.60; N, 2.17. N- [10' -o- (jS-D-galactopyranosyl) decyl] chitosan (141) a) Treatment of chitosan (0.65 g, 4.0 mmol) with the glycoside 138 (4.0 g, 12.0 mmol) provided 1.43 g (58%) of 141a. -204-Anal. Calcd. for [ ( C 2 2 H 4 ] N O 1 0 ) 0 . 6 3 ( C 3 8 H 7 1 N 0 1 6 ) 0 . 3 7 ] 0 . 8 2 H 20: C, 54.79; H, 8.79; N, 2.29. Found: C, 54.79; H, 8.89; N, 2.29. b) Chitosan (0.65 g, 4.0 mmol) was reacted with the aldehyde 138 (2.0 g, 6.0 mmol) to y i e l d 0.89 g (90%) of deri v a t i v e 141b. Anal. Calcd. for [ (CgHijNG^) o.78 (^22H4^NO) ] • 1.55H20: C, 44.13; H, 8.00; N, 5.41. Found: C, 44.13; H, 7.83; N, 5.46. N-[10"-0- (^3-D-galactopyranosyl) -£-D-glucopyranosyl) decyl]chitosan or N-[10"-0-(£-D-lactosyl)decyl]chitosan (142) a) Reaction of chitosan (0.65 g, 4.0 mmol) with the aldehyde 139 (5.8 g, 11.7 mmol) provided 1.95 g (67%) of product 142a. Anal. Calcd. for [ ( C 2 8 H 5 1 N 0 1 5 ) 0 > 9 0 ( C 5 0 H 9 1 N O 2 6 ) 0 . x ] - 2 . 0 4 H 20: C, 49.94; H, 8.14; N, 1.93. Found: C, 49.94; H, 8.11; N, 1.93. b) Treatment of chitosan (0.65 g, 4.0 mmol) with the aldehyde 139 (3.10 g, 6.3 mmol) yielded 1.18 g (69%) of l y o p h i l i z e d 142b. Anal. Calcd. f o r [ ( C 6 H 1 3 N 0 4 ) 0 # 5 0 ( C 2 8 H 5 1 N 0 1 5 ) 0 > 5 0 ] • 1 . 4 5 H 20: C, 47.77; H, 7.94; N, 3.28. Found: C, 47.77; H, 8.05; N, 3.26. N-[10-hydroxydecyl]chitosan (143) a) A solution of chitosan (0.65 g, 4.0 mmol) i n 5% aqueous ac e t i c acid-methanol-i-propanol (5:4:1, 60 mL) was treated with 10-hydroxydecanal (2.1 g, 12.0 mmol) and subsequently with sodium cyanoborohydride (1.0 g, 16.0 mmol) -205-for 24 h. A flocculent p r e c i p i t a t e formed rapidly, which upon completion of reaction, was co l l e c t e d by f i l t r a t i o n and washed with water to give 1.30 g (76%) of derivative 143a. Anal. Calcd. f o r [ ( C 1 0 H 3 1 N O 5 ) 0 . 2 7 ( C 2 6 H 5 1 N 0 6 ) 0 . 7 3 ] * 0 . 2 3 H 20: C, 64.26; H, 10.59; N, 3.22. Found: C, 64.26; H, 10.62; N, 3.22. b) Treatment of chitosan (0.65 g, 4.0 mmol) as i n part (a), with 10-hydroxydecanal (0.70 g, 4.0 mmol) and sodium cyanoborohydride (1.0 g, 16.0 mmol) fo r 24 h, provided a solution which a f t e r d i a l y s i s against methanol-water (2:1) for 2 days (2x1 L), methanol-water (1:1) f o r 2 days (2x1 L) and f i n a l l y water f o r 2 days (2x2 L), gave 1.04 g (82%) of freeze-dried product 143b. Anal. Calcd. f o r [(C 1 6H 3 1N0 5)]•0.42H 2O: C, 59.17; H, 9.81; N, 4.31. Found: C, 59.17; H, 9.65; N, 4.52. Synthesis of mixed N-ethyl and N-decyl-D-glycopyranosyl chitosan derivative Derivative 126d (150 mg, 0.68 mmol) i n 5% aqueous acetic acid-methanol (1:1) was s t i r r e d with the aldehyde 138 (0.65 g, 1.95 mmol) and sodium cyanoborohydride (0.30 g, 4.80 mmol) for 24 h. The reaction mixture was transfered to a d i a l y s i s sack and dialysed against water-methanol (1:1) f o r 3 days (3x1 L), against d i s t i l l e d water f o r 3 days (3x1 L), and freeze dried to give 220 mg (63%) of 144. Anal. Calcd. f o r [ ( C 1 4 H 2 5 N 0 1 0 ) 0 # 4 8 ( C 3 8 H 7 1 N 0 1 6 ) 0 > 5 2 ] •1.28H20: C, 51.79; H, 8.39; N, 2.28. Found: C, 51.78; H, 8.56; N, 2.29. -206-4.2.4 ViscometrY Polysaccharide solutions f o r viscometry were prepared by d i s s o l v i n g the sample i n d i s t i l l e d water (5.0 mL) containing 50 ppm sodium benzoate as s t a b i l i z e r . T y p i c a l l y 2.0% (w/w) solutions of a l l synthetic derivatives were prepared, except where otherwise indicated. Commercial polysaccharide deriva-t i v e s were prepared i n a s i m i l a r manner, at concentrations of 1.0 and 2.0%. A l l samples were given at l e a s t 24 hours to disperse, with intermittent mixing on a Vortex-Genie™ t e s t tube mixer. Any samples which contained entrapped a i r bubbles were centrifuged on a bench-top serum centrifuge f o r 30 min. The steady shear viscometric measurements were performed on a r o t a t i o n a l viscometer ( V i s c o - E l a s t i c Analyzer, Sangamo Transducers, W. Sussex, England) with truncated cone and plate f i x t u r e s (d 50.0 mm, a 2.5°, gap 90 fim). A co n t r o l l e d temperature g l y c o l bath under the plate provided temperature co n t r o l . A l l measurements were recorded at 20.0° ( ± 0.2°C). The gap s e t t i n g was zeroed with no sample present by lowering the cone f i x t u r e u n t i l contact was j u s t made with the plate. The cone f i x t u r e was then raised ~ 2 mm to allow sample loading. A sample solution (-1.5 mL) was loaded into a syringe with an 18 gauge needle or, for very viscous samples, a 7" Pasteur pipette. The sample was then discharged onto the center of the plate, taking care to avoid the formation of -207-a i r bubbles. The cone f i x t u r e was lowered onto the sample to the desired 90 ^.m gap set t i n g . The sample was given f i v e minutes to equlibrate to 20.0 (+0.5) C, as v e r i f i e d by measurement with a thermocouple. The sample was then stepped through a series of increasing torque settings, applied to the rotating cone f i x t u r e , t y p i c a l l y from 0.1 to 60 G*cm. For each torque set t i n g , the resultant angular v e l o c i t y ( radians*s _ 1) was recorded on a s t r i p chart, allowing the equilibrium value to be reached (usually within 30 s fo r the majority of samples). When eithe r the maximum torque (60 G*cm) or maximum angular v e l o c i t y (100 r a d i a n s * s _ 1 ) was approached, the torque was stepped through a s i m i l a r decreas-ing s e r i e s , and angular v e l o c i t y was recorded. Duplicate measurements were performed on a l l samples. Three Newtonian standard o i l samples were subjected to i d e n t i c a l measurement, at a l l measured torque values, i n order to c a l i b r a t e the torque settings over the f u l l range of observed v i s c o s i t i e s . Shear stress (cr) i s related to torque (T) according to equation 30 : 3T a = [30] 2irr where T i s torque i n dynes*cm and r i s radius; and shear rate (?) to angular v e l o c i t y (co) according to Eq. 31: •• * - t = T T 1 3 1 1 Substitution of values from cone geometry can provide shear -208-stress and shear rate factors as seen i n equations 32 and 33. o = 2996.7T (mPa) [32] y = 22.920) ( s - 1 ) [33] Apparent v i s c o s i t y w i l l then be given by equation 34: n = | = 130.76(|) (mPa-s) [34] Corrected torque values ( T c o r r ) were calculated from standard o i l measurements by rearranging equation 34, to give: ri • 1 co P = 0 1 1 c o r r 130.76 [35] where ^ o i i i s the known standard o i l v i s c o s i t y , and to was the measured angular v e l o c i t y . Corrected torque values and determined angular v e l o c i t i e s provided shear stress, shear rates and apparent v i s c o s i t i e s from equations 32, 33 and 34. Logarithm of shear stress, shear rate and apparent v i s c o s i t y were calculated. Linear regression of logarithm of shear stress against logarithm of shear rate, gave power-law parameters according to equation 36: l o g (a) = log(m) + nlog ( -y) [36] Rheograms of steady shear viscometric data were plotted on both l i n e a r and logarithmic coordinates as apparent v i s c o s i t y vs shear rate. Rheometric evaluations reported i n section 2.3.3 were done using a d i f f e r e n t rheometer, which was also a controlled -209-stress instrument, capable of elevated temperature measur-ments (Controlled Stress Rheometer, Carri-Med Ltd. , Dorking, Surrey, England). The data was obtained i n the same way as described previously, gi v i n g torque (dyne*cm) and angular v e l o c i t y (w) values, and converted to shear stress and shear rate using Eq.s 30 and 31. The cone-plate dimensions were r 20.0 mm, and a 2.0°. Data treatment followed exactly the procedure already outlined. 4.2.5 I n t r i n s i c V i s c o s i t y Solutions of 0.075, 0.050, 0.025 and 0.010% (w/w) f o r i n t r i n s i c v i s c o s i t y determinations were prepared by d i l u t i o n of 0.10% w/w stock solutions (100 mg/100 mL). The solutions were loaded into the Canon-Fenske c a p i l l a r y viscometer, according to standard pr ocedures, 2 1 9 and the viscometer was placed i n a temperature-controlled jacket and allowed to e q u i l i b r a t e to 25 (± 0.5)°C fo r 10 min. Duplicate determina-tions of e f f l u x time were recorded f o r each solution. Canon-Fenske #50 or #100 viscometers were used accordingly i n order to keep e f f l u x times i n the optimal 200-800 s range. . The viscometers were cal i b r a t e d with water and standard o i l ( 7.798 mPa*s at 25°C ) to give the constant k according to equation 37; n = k ^ p [37] where 7 7 i s v i s c o s i t y (mPa*s), t i s e f f l u x time and p i s -210-density. From equation 37, v i s c o s i t i e s f o r a l l sample solu-tions were determined and used to cal c u l a t e r e l a t i v e v i s c o s i t y ( i 7 r e l ) according to Eq. 38; r e l n s w here^ s i s the solvent v i s c o s i t y . I n t r i n s i c v i s c o s i t i e s [n] were obtained according to the Kraemer [Eq. 7] re l a t i o n s h i p : L n ( n r e l ) 2 r ^ - = [ n l +k ' [ n ] c [7] where c i s the concentration (g/100 mL) and k]/ i s a constant. Linear regression of l n i 7 r e i / c against concentra-t i o n provided, i n t r i n s i c v i s c o s i t i e s f o r the sample solu-t i o n s . 4.3 EXPERIMENTAL FOR CHAPTER 3 4.3.1 General Procedures pH T i t r a t i o n curves were determined on solutions of -0.25 g of polysaccharide derivative i n 0.1N HCl (aq) sol u t i o n (25.0 mL) with a pH meter. Copper chelation samples were prepared by d i s s o l v i n g the derivative (-200 mg) i n d i s t i l l e d water (15 mL) and t r e a t i n g with 2.0M copper(II) acetate (aq) solution (15 mL). A f t e r s t i r r i n g f o r 24 h, the suspensions were dialyzed against 1) d i s t i l l e d water exhaustively (6x1 L) -211-or 2) d i s t i l l e d water f o r 3 days (3x1 L), 0.1N iminodiacetic acid (250 mL) fo r 48 h and then exhaustively with d i s t i l l e d water (6x1 L). The samples were freeze-dried and then dried i n vacuo (70°C, 0.05 mm Hg). For viscometry, sample solutions of 1.0% (w/w) concentration were prepared i n d i s t i l l e d water (containing 5 ppm sodium benzoate as s t a b i l i z e r ) or i n O.lmM copper(II) s u l f a t e (aq) solution. Dissolution was aided by periodic mixing (Vortex-Genie) over a 24 h period. Entrapped a i r bubbles were removed by centrifugation on a bench-top centrifuge for 30 min. Viscometric measurements were performed with a r o t a t i o n a l , controlled stress rheometer (Vis c o - E l a s t i c Analyzer) as described i n section 4.2.4. 4.3.2 Synthesis of Chelating Chitosan Derivatives Dimethyl iminodiacetate (Iminodiacetic acid dimethylester), (147) Iminodiacetic acid (5.15 g, 38.7 mmol) was s t i r r e d i n anhydrous methanol (25 mL) to which was added 50% boron trifluoride-methanol complex (10 mL). The reaction mixture was refluxed f o r 9 h, cooled to room temperature, and poured into 150 mL of chloroform and 75 mL saturated sodium bicarbonate solution. Additional s o l i d sodium bicarbonate was added u n t i l the aqueous layer was s l i g h t l y basic (pH paper). The chloroform layer was separated, the aqueous layer was extracted twice more with chloroform (2x150 mL), and the -213-chloroform extracts were combined. The chloroform solution was then dried over magnesium su l f a t e and f i l t e r e d . Concentration gave 5.96 g (96%) of a l i q u i d , which corresponded to the dieste r 147. This material was one component by t i c analysis (Rf 0.27, solvent A); i r (fi l m ) : *max 1 7 5 0 cm"1; ^-nmr (270 MHz): S(CDC13), 2.07 (s, 1 H, NH), 3.49 (S, 4 H, -CH 2-), 3.74 (s, 6 H, C0 2CH 3); ms, m/z ( r e l . i n t e n s i t y ) : 161 (15), 129 (6), 103 (7), 102 (94), 74 (37). 6-0-(2,3-Epoxypropyl)-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (146) 1,2:3,4-Di-O-isopropylidene-a-D-galactopyranose, 145 (10.00 g, 38.4 mmol) and epichlorohydrin (3.55 g, 38.4 mmol) were dissolved i n anhydrous tetrahydrofuran (THF, 100 mL). Sodium hydride (1.84 g, 60% o i l dispersion, 46.1 mmol) was washed with three portions of hexane (10 mL) and added to the THF so l u t i o n . The reaction mixture was refluxed f o r 9 h, and a f t e r d i l u t i o n with methanol (2 mL), was cooled and poured int o water (50 mL). The aqueous layer was extracted with three portions of ether (100 mL) and the combined ethereal extracts were washed twice with saturated brine solution (50 mL). The ether layer was dried with magnesium s u l f a t e , f i l t e r e d and concentrated to give 11.40 g of a crude syrup. T i c analysis (solvent A) of t h i s material showed a major component at Rf 0.55 and minor components at lower Rf values. Flash l i q u i d chromatography (2:1 ether-petroleum ether) afforded 6.88 (57%) of a white c r y s t a l l i n e s o l i d -214-corresponding to diastereomeric 146; Further processing of mixed fractions gave additional product. I r (KBr): vmSLX 1450 (CH 3 deform), 1380 [-C(CH 3) 2 sym deform], 1250 cm"1 (C-0 epoxide); -^H-nmr (270 MHz): S(CDC1 3), 1.34 (s, 6 H, CH.3) , 1.45 (s, 3 H, CH 3), 1.54 (s, 3 H, CH 3), 2.63 (m, 1 H, J 5.5, 2.5 HZ, O-CH.) , 2.78 (t, 1 H, J_ 5.5, 5.5 Hz, O-CH) , 3.17 (m, 1 H, -CH-C), 3.41, 3.49 (2dd, 1 H, J_ 1.0, 5.5 Hz, H-l'aR,S), 3.59-3.83 (m, 3 H, H-6a,6b, H-lbR,s), 3.98 (br t , 1 H, J 5.5, 5.5 HZ, H-5), 4.24 (ddd, 1 H, J_ 8.0, 2.0, 2.0 Hz, H-4), 4.30 (dd, 1 H, 2 5.5, 3.0 Hz, H-2), 4.57 (dd, 1 H, J_ 8.0, 3.0 Hz, H-3), 5.52 (d, 1 H, J_ 5.5 Hz, H - l ) ; ms, m/e ( r e l . i n t e n s i t y ) : 316 (0.1), 301 (66), 258 (3), 243 (3), 229 (9); Anal. Calcd. f o r C 1 5H240 7: C, 56.95; H, 7.65. Found: C, 56.95; H, 7.78. 6 -0 -[2-Hydroxy-3-(methyliminodiacetate)propyl]-1,2:3,4-di-O-isopropylidene -ct-D-galactopyranose (148a)and the morpholin-3-one 148b 6-0- (2,3-epoxypropyl) -1,2:3,4-di-0_-isopropylidene-a-D galactopyranose (146. 2.00 g, 6.33 mmol) and dimethylimino-diaacetate (147. 1.00 g, 6.2 mmol) were dissolved i n ethanol (50 mL) and refluxed for 8 h. The ethanol was removed and the resultant syrup dissolved i n chloroform (50 mL). The chloro-form layer was washed with two portions (50 mL) of saturated brine, dried over magnesium sul f a t e , f i l t e r e d and concen-trated to give 2.95 g of a syrup. T i c analysis confirmed the absence of s t a r t i n g material and showed two major components -215-(R f 0.41, 0^34, solvent A). Glc analysis of the product showed one major peak (>95% peak area) . The -^H-nmr spectrum of the syrup indicates a mixture of products 148a and 148b i n a r a t i o of 2:1 respectively. A f t e r f l a s h l i q u i d chromatogra-phy p u r i f i c a t i o n , 2.45 g of a 1:4 mixture of 148a, 148b was obtained (86%). Subsequent short path d i s t i l l a t i o n (200 CC, 0.05 mm Hg) gave 1.85 g of pure 148b by ^-nmr spectroscopy. Glc analysis of t h i s component gave one peak at the same retention time observed f o r the product mixture. 148a: i r ( f i l m ) : v m a x 1755 (C=0 ester), 3500 cm"1 (OH); -^H-nmr (270 MHz): 8(CDCl 3), 1.33 (s, 6 H, 2CH 3), 1.45 (s, 3 H, CH_3) , 1.54 (s, 3 H, CH 3), 2.64 (ddd, 1 H, J 12.5, 10.0, 2.0 Hz, -CH-N), 3.00 (dd, 1 H, J 12.5, 2.0 Hz, - C H-N), 3.39-3.79 (m, 8 H, H-6, H-6», -CH20, 2 -CH2C02Me), 3.72 (s, 6 H, 2 C0 2CH 3), 3.83 (m, 1 H, CHOH), 3.97 (br s, 1 H, H-5), 4.25 (dd, 1 H, J 8.0, 1.0 Hz, H-4), 4.33 (dd, 1 H, J 5.0, 2.0 Hz, H-2), 4.61 (dd, 1 H, J 8.0, 2.0 Hz, H-3), 5.54 (d, 1 H, J 5.0 Hz, H - l ) . 148b: i r ( f i l m ) : v m a x 1755 cm"1 (C=0 ester, morpholone); ^-nmr (270 MHz): 8(CDC13), 1.33 (s, 6 H, 2 C H 3 ) , 1.44 (s, 3 H, CH 3), I. 54 (s, 3 H, CH 3), 2.86 (ddd, 1 H, J 12.5, 10.0, 2.0 Hz, CH-N), 3.08 (dd, 1 H, I 12.5, 2.0 Hz, -CH-N), 3.39 (s, 2 H, -CH2C02Me), 3.40-3.79 (m, 6 H, H-6, H-6', -CH2C02Me, -CH 20), 3.73 (s, 3 H, CH 3), 3.97 (br t, 1 H, J 5.0, 1.0 Hz, H-5), 4.25 (d, 1 H, J 8.0, 1.0 Hz, H-4), 4.34 (dd, 1 H, £ 5.0, 2.0 HZ, H-2), 4.63 (dd, 1 H, J 8.0, 2.0 Hz, H-3), 4.68 (m, 1 H, CHOR), 5.54 (d, 1 H, J 5.0 Hz, H - l ) . 148a. 148b mixture: ms, m/z ( r e l . i n t e n s i t y ) : 477 (5), 462 (10), 445 (2), 430 (6), -216-418 (30), 387 (12), 386 (14). 6-0-(2-Hydroxy-3-iminodiaceto-propyl)-D-galactose (149) Compound 148 (2:1 mixture of 148a and 148b, 10.0 g, 21.4 mmol) was dissolved i n 2N sodium hydroxide i n 9:1 methanol-water (100 mL). The reaction mixture was refluxed for 4 h and cooled i n an ice bath. 6N hydrochloric acid (60 mL) was added to give a IN solution, which was then refluxed for 5 h. Af t e r cooling the solution was f i l t e r e d and the f i l t r a t e was concentrated to a volume of 10 mL. Ethanol (20 mL) was added and the mixture was cooled i n i c e , f i l t e r e d and concentrated. The residue was taken up i n ethanol and p r e c i p i t a t i o n was achieved by the addition of acetone. The gummy p r e c i p i t a t e was taken up i n ethanol and treated with decolourizing carbon, and the f i l t r a t e was treated with acetone to give a gummy p r e c i p i t a t e . This material was a foamy hydroscopic s o l i d (7.15 g, 86%) a f t e r drying i n vacuo (25 C, 0.05 mm Hg) and consisted of a mixture of a and j8 6-0-D-galactose isomers 149. 1 3C-nmr (100.6 MHz, main isomer): S(D 20), 166.7 (COOH), 98 ( C - l ) , 71.8, 71.2 (C-5,2), 69.3, 69.2 (CHOH), 67.9, 67.2 (C-3,4), 66.7 (C-6), 63.4 (CH 20), 57.1, 57.2 (-CH2NH), 54.3 (NHCH2C02H). 6-0-[2-hydroxy-3-(iminodiacetic acid hydrochloride) propyl]-D-galactitol (150) Compound 149 (0.90 g, 2.3 mmol) was dissolved i n 95% ethanol (10 mL) to which was added an aqueous solut i o n of saturated sodium bicarbonate (2 mL). To t h i s mixture, sodium borohydride (0.50 g, 13.2 mmol) was added and allowed to s t i r -217-fo r 2 h at room temperature. The reaction was then a c i d i f i e d to pH 2 (pH paper) by dropwise addition of 6N hydrochloric acid. The s o l u t i o n was f i l t e r e d and the solvent removed. The residue was taken up i n methanol and concentrated t h r i c e more to remove borate s a l t s . The residue was then dissolved i n ethanol-methanol (1:1), and the resultant s o l u t i o n was f i l -tered and p r e c i p i t a t e d with acetone to give 0.75 g (83%) of a foamy hygroscopic s o l i d . Attempts to c r y s t a l l i z e the diaste-reomic mixture were unsuccessful. 1 3C-nmr (100.6 KHz): 8(D 20), 167.3 (COOH), 71.7, 71.3, 69.1 (C-5,4,3,2), 68.8, 68.6 (CHOH), 67.3 (C-6), 63.3, 63.0 (CH 20-), 62.0 ( C - l ) , 56.37, 56.07 (CH2NH), 54.1, 53.9 (NHCH2C02H). Chelating Chitosan Derivative (151) a) Chitosan (0.55 g, 3.4 mmol) was dissolved i n 5% aqueous ac e t i c acid (50 mL) and the galactose d e r i v a t i v e 149 (2.0 g, 4.9 mmol) i n 5% aqueous a c e t i c acid (10 mL) was added to the resultant s t i r r e d solution. A f t e r 15 min, sodium cyanoborohydride (1.25 g, 20.0 mmol) i n 5 mL of reaction solvent was added. After s t i r r i n g f o r 24 h, the mixture was poured into a d i a l y s i s sac and dialyzed f o r 6 days (6x2 L) against d i s t i l l e d water and subsequently freeze-dried to give 1.62 g (84%) of a white f l u f f y s o l i d ; 1 3C-nmr (100.6 MHz): S(D 20), 168.6 (COOH), 98.4 ( C - l 1 ) , 75.9 (C-4'), 73.2 (C-5«), 71.2, 71.1 (C-5,2), 68.9 (=CH0H), 67.9, 67.4 (C-3,2), 66.7 (C-6), 63.2 (-CH20-), 58.7 (-CH2NH), 56.3 (NHCH2C02H), 54.7 ( C - l ) . Anal.Calcd f o r (C 19H 3 4N 20 14)'1.56H 20: C,42.06; H, 6.85; -218-N, 5.16.Found: C, 42.06; H,7.10; N, 4.81. b) The procedure used for the preparation of 151b was as described for the preparation of 151a. with the exception that 1.00 g (2.45 mmol) of the galactose d e r i v a t i v e 149 was used. The resultant y i e l d was 1.16 g (63%) of a f l u f f y white s o l i d . Anal. Calcd. f o r [ (C 6H 1 : LN0 4) o.50( c19 H34 N2°14) 0.50^ H 20: C, 41.98; H, 6.91; N, 5.88. Found: C, 41.98; H, 7.17; N, 5.85. 4.3.3 Synthesis of Thio Glycoside A f f i n i t y Conjugate and  Precursors (3 • - O - A l l y l - 2 • -hydroxypropyl) 2 ,3,4,6-tetra -o-acetyl-l-thio - j S " D-glucopyranoside (153) 2,3,4,6-Tetra-O-acetyl-l-thio-jS-D-glucopyranose (152. , 10.0 g, 27.5 mmol), l-allyloxy-2,3-epoxypropane (3.14 g, 27.5 mmol) and sodium bicarbonate (2.31 g, 27.5 mmol) were s t i r r e d together i n ethanol (60 mL) at re f l u x temperature f o r 4 h. The solu t i o n was cooled, poured into chloroform (200 mL), washed with saturated sodium bicarbonate aqueous solution (100 mL), saturated aqueous brine (2x100 mL), dried over magnesium sul f a t e , f i l t e r e d and concentrated. Liquid chromatography afforded 7.56 g (58%) of diastereomeric 153. l-H-nmr (270 MHz): S(CDC13) , 5.87 (m, 1 H, H-2"), 5.32-4.96 (m, 5 H, H-2,3,4,3»a,3"b), 4.55 (d, 1 H, J 10.0 Hz, H-l), 4.30-4.05 (m, 2 H, H-6a,b), 4.00 (d, 2 H, J 5.0 Hz, H-3'), -219--220-3.93 (m, 1 H, H-2'), 3.73 (m, 1 H, H-5), 3.47 (m, 2 H, H-l'a,b), 3.15-2.58 (m, 2 H, H-l"a,b), 2.10, 2.06, 2.04, 2.00 (4s, 3 H each, 4 OAc); ms, m/z 478 (M+), 418, 358. (3• -O-Allyl-2 • -hydroxypropyl) l-thio - /3-D-glucopyranoside (154) A solution of compound 153 (7.3 g, 15.3 mmol) i n anhydrous methanol (75 mL) was treated with 0.5N sodium methoxide i n methanol (5 mL) u n t i l the reaction was complete ( t i c , solvent B). The solut i o n was neutralized with Dowex 50x8 (H +-form, 100-200 mesh) ion-exchange r e s i n , f i l t e r e d , decolourized and concentrated. Attempts to c r y s t a l l i z e the syrupy residue were unsuccessful. Drying i n vacuo (0.05 mm Hg) gave 4.5 g (95%) of the diastereomeric mixture 154 as a foamy s o l i d , ^-nmr (270 MHz): 8 (D20) , 5.89 (m, 1 H, H-2"), 5.28 (br d, 1 H, J_ 17.0 Hz, H-3"a), 5.20 (br d, 1 H, £ 12.0 Hz, H-3"b), 4.49, 4.47 (2d, 1 H, 1 7.0 Hz, H-l), 4.02 (d, 2 H, J 6.0 Hz, H-3'), 3.99 (m, 1 H, H-2')/ 3.83 (dd, 1 H, J_ 12.0, 1.0 Hz, H-6a) , 3.64 (dd, 1 H, J_ 12.0, 3.0 Hz, H-6b) , 3.60-3.31 (m, 3 H, H-3,4,5), 3.26 (t, 1 H, J_ 9.0, 7.0 Hz, H-2), 2.92 (dd, 1 H, J_ 14.0, 5.0 Hz, H-l"a) , 2.82 (m, 2 H, H-l')/ 2.70 (dd, 1 H, J 14.0, 8.0 Hz, H-l"b). Ozonolysis of 154 The 1-thio-^S-D-glucopyranoside 154 (3.42 g, 11.0 mmol) was dissolved i n methanol (50 mL), cooled to -78 C and satu-rated with ozone. Dimethylsuifide (3.43 g, 4.1 mL, 55.0 mmol) was added, and the reaction was allowed to warm to ambient temperature f o r 2 h with s t i r r i n g . Excess solvent was removed -221-and the syrupy residue dissolved i n ethanol and preci p i t a t e d by the addition of ether. A f t e r p r e c i p i t a t i n g twice and dry-ing i n vacuo. 3.30 g (96%) of crude 155 was obtained. l-Thio-^3-D-glucopyranoside A f f i n i t y Conjugate (156) a) A solution of chitosan (0.85 g, 5.3 mmol) i n 5% aqueous ac e t i c acid (50 mL) was treated with a solut i o n of the thio-glucosealdehyde 155 (3.4 g, 10.9 mmol) i n the reaction solvent (10 mL) and sodium cyanoborohydride (1.25 g, 20.0 mmol) f o r 24 h. The solut i o n was dialyzed f o r 6 days against d i s t i l l e d water (6x1 L), f i l t e r e d and freeze-dried to give 2.25 g (95%) of the deriv a t i v e 156a; 1 3C-nmr (100.6 MHz): 8(D 20), 99.5 (C-1*), 87.0 (C-1), 78.5 (C-5), 75.5 (C-3), 72.3,71.8 (C-2 and C-4), 67.6,67.5,66.5 (C - l ' , 2 1 , and 2"), 59.1 (C-6), 49.0 (C-3«), 45.5 (C-1"). Anal. Calcd f o r [ ( C g H n N O ^ 0 . 1 0 (C^HajNO-QS) 0.90] * 1 ' 9 H 2 0 : C,41.21; H, 7.13; N, 3.02; S, 6.22. Found: C, 41.21; H, 7.00; N, 3.46; S, 6.83.) b) A s t i r r e d dispersion of deriv a t i v e 156a (0.50 g, 1.3 mmol) i n 2.0% aqueous ac e t i c acid was treated with glutaraldehyde (25% aqueous solution, 0.10 mL, 0.26 mmol) and sodium cyanoborohydride (0.25 g, 4.0 mmol) for 24 h. The suspension was f i l t e r e d and the p r e c i p i t a t e was washed with water. Drying i n vacuo (0.05 mm Hg) provided 0.40 g (69%) of 156b. Anal. Calcd. for [ ( C 1 7 H 3 1 N 0 1 1 S ) o . 9 o ( c 8 . 5 H 1 6 N 0 4 ) O . i o l ' ) - 6 5 H 20: C, 45.68; H, 6.67; N, 3.16; S, 6.51. Found: C, 45.68; H, 6.92; N, 3.05; S, 6.40. * denotes carbons on the chitosan backbone. -222-c) A s t i r r e d solution of chitosan (0.70 g, 4.35 mmol) i n 5% aqueous acetic acid (50 mL) was treated with the thio-glycoside 155 (0.8 g, 2.56 mmol) and sodium cyanoborohydride (1.0 g, 16.0 mmol) fo r 24 h. The solution was dialyzed f o r 6 days (6x1 L) against d i s t i l l e d water and freeze-dried to give 0.67 g (61%) of 156c. Anal. Calcd. f o r [ (C6Hi;LNC-4) 0 . 7 5 ( C 1 7 H 3 1 N 0 1 1 S ) 0 . 2 5 3 * 0.88 H 20: C, 42.06; H, 7.11; N, 5.61; S, 3.20. Found: C, 42.06; H, 6.85; N, 5.80; S, 3.29. d) A s t i r r e d s o l u t i o n of chitosan (0.70 g, 4.35 mmol) i n 5% aqueous a c e t i c acid (50 mL) was treated with the thio-sugar 155 (0.80 g, 2.56 mmol) and sodium cyanoborohydride (1.0 g, 16.0 mmol) fo r 24 h. 25% Aqueous glutaraldehyde (0.40 mL, 1.0 mmol) and sodium cyanoborohydride (0.25 g, 4.0 mmol) were then added and allowed to s t i r f o r 24 h, affording a s t i f f , c l e a r foamy g e l . Af t e r d i a l y s i s 0.70 g (64%) of 156d was obtained. Anal. Calcd. f o r [ (C 6H 1 : LN04) 0 . 4 5 (C 1 7H 3 1N0 1 : LS) 0.25 ( c8.5 H16 N O4)0.3]'°.43H 20: C, 45.13; H, 7.27; N, 5.54; S, 3.17. Found: C, 45.14; H, 7.22; N, 4.96; S, 3.04. 4.3.4 Enzyme Studies A l l buffer chemicals, substrates, cofactors and coupling enzymes were obtained from Sigma Chemical Co. Assays of glucose l i b e r a t i o n were performed e s s e n t i a l l y as described p r e v i o u s l y 2 6 5 * 2 6 9 and standardized against a glucose standard -223-solution. Quantities of coupling enzymes employed were s u f f i c i e n t that a l l glucose had been consumed within 5 minutes. I n h i b i t i o n constants for the thioglucoside (154) with ^5-glucosidases from almond emulsin and A. f a e c a l i s were determined using a range of i n h i b i t o r concentrations (0.2-2.0 times Kj^ ) at a fixed concentration (5 mM and 0.1 mM respectively) of p_-nitrophenyl glycoside i n 50 mM sodium phosphate buffer pH 6.8 at 25° C. Data were analyzed by means of a p l o t of ( v u n i r ^ i b i t e d ) / ( v i n h i b i t e d ) versus i n h i b i t o r concentration. The slope of such a p l o t equals K m/[Ki(S+K m)] from which the K^ values can be determined. The effectiveness of the a f f i n i t y support was evaluated as follows. A small column (5x35 mm) was packed with the cross-linked polymer (156d), equilibrated with buffer (5 mM sodium phosphate, pH 6.8) and a small sample of A. f a e c a l i s ^S-glucosidase loaded on. Af t e r washing with the same buffer (5 mL), e l u t i o n was effected using a buffer containing sodium chloride (0.5 M) and sodium phosphate (5 mM), pH 6 . 8 . jS-Glucosidase a c t i v i t y eluted from the a f f i n i t y column was estimated spectrophotometrically by measuring the rate of hydrolysis of p_-nitrophenyl glucoside (0.25 mM) i n 50 mM sodium phosphate buffer, pH 6.8 upon addition of a fixed aliquot (10 fiL) of column e f f l u e n t . -224-BIBLIOGRAPHY 1. J.F. Kennedy and C.A. White (eds.), "Bioactive Carbo-hydrates", John Wiley and Sons, New York (1983) 2. D.J. Candy, ed., " B i o l o g i c a l Function of Carbohydrates", John Wiley and Sons, New York (1980) 3. G.O. A s p i n a l l (ed.), "Polysaccharides", Pergamon, Oxford, (1970) 4. G.O. A s p i n a l l (ed.), "The Polysaccharides", Vol. 2, Academic Press, New York (1983) 5. R.L. Whistler (ed.), "In d u s t r i a l Gums", Academic Press, New York (1973) 6. P.A. Sandford and J . Baird, "The Polysaccharides", G.O. A s p i n a l l (ed.), Vol. 2, p. 411-490, Academic Press, New York (1983) 7. Y.L. Meltzer, "Water Soluble Polymers, Developments Since 1978", Noyes Data Corporation, Park Ridge, New Jersey (1981) 8. I. Parikh and P. Guatrecasas, Chem. Eng. News, Aug. 26 (1985), p. 17 9. B.P. Sharma, L.F. Bailey and R.A. Messing, Angew. Chem. Int. Ed. Engl., 21, 837 (1982) 10. E. Ott and H.M. Spurlin, eds. "High Polymers", Vol. V, Parts I-III, Interscience (1954) 11. N.M. Bikales and L. Segal, eds. "High Polymers", Vol. V, Parts IV and V, Interscience, New York (1971) 12. R.L. Whistler (ed.), Methods Carbohydr. Chem., Vol. I l l . p. 193 (1963) 13. R.L. Whistler (ed.), Methods Carbohydr. Chem., Vol. IV, p. 279 (1964) 14. R.M. Rowell and R.A. Young (eds.), "Modified C e l l u -loses", Academic Press, New York (1978) 15. M. Yalpani, Tetrahedron, 41(15), 2957 (1985) 16. D.A. Brant (ed.), "Solution Properties of Polysacchar-ides", Vol. 150, ACS Symp. Ser. (1981) -225-17. D.A. Rees, E.R. Morris, D. Thorn and J.K. Madden, "The Polysaccharides", G.O. A s p i n a l l (ed.), Vol. Xf P« 195, Academic Press, New York (1982) 18. A.S. P e r l i n and B. Casu, "The Polysaccharides", G.O. As p i n a l l (ed.), Vol. X , p. 133, Academic Press, New York (1982) 19. G.O. A s p i n a l l (ed.), "The Polysaccharides", Vol. X , p. 36-131, Academic Press, New York (1982) 20. C T . Bishop and H.J. Jennings, "The Polysaccharides", G.O. A s p i n a l l (ed.), Vol. X»P«292, Academic Press, New York, (1982), p.292 21. H.J. Jennings, Adv. Carbohydr. Chem. Biochem., 41, 155 (1983) 22. R.A.A. Muzzarelli (ed.), " C h i t i n " , Pergamon Press, New York (1977) 23. R.A.A. Muzzarelli, "The Polysaccharides", G.O. A s p i n a l l (ed.), Vol. 3_, p. 417, Academic Press, New York (1985) 24. J.P. Zikadis (ed.), " C h i t i n , Chitosan and Related Enzymes", Academic Press, New York (1984) 25. R.A.A. Muzzarelli and E.R. Pariser (eds.), "Proceedings of the F i r s t International Conference on C h i t i n and Chitosan", Cambridge, Massachusetts (1978) 26. S. Hirano and S. Tokura (eds.), "Proceedings of the Second International Conference on C h i t i n and Chitosan", Japan Soc. C h i t i n , T o t t o r i , Japan (1982) 27. M. Yalpani and L.D. H a l l , Macromolecules, 17, 272 (1984) 28. J.F. Kennedy, Adv. Carbohydr. Chem. Biochem. 2JJ, 306 (1974) 29. J.A. Radley (ed.), "Starch and I t s Derivatives", Chapman and H a l l , London (1968) 30. R.W. James (ed.), " I n d u s t r i a l Starches", Noyes Data Corp., Park Ridge, New Jersey (1974) 31. R.L. Whistler (ed.), Methods Carbohydr. Chem., Vol. V, p. 395-411 (1965) 32. R.H. Marchessault and P.R. Sundararagan, "The Polysacch-arides", G.O. A s p i n a l l (ed.), Vol. 2, p. 12, Academic Press, New York (1983) -226-33. P.D.G. Dean, W.S. Johnson and F.A. Middle (eds.), " A f f i n i t y Chromatography, A P r a c t i c a l Approach", IRL Press, Oxford (1985) 34. J.H. Pazur, Adv. Carbohydr. Chem. Biochem., 39, 405 (1981) 35. L. Kuinak, B. Alince, V. Masura and J . A l f o l d i , Svensk Paperstidn., 72, 205 (1969) 36. D.M. Clode and D. Horton, Carbohydr. Res., 19, 329 (1971) 37. D. Horton, A.E. Luetzow and O. Theander, Carbohydr. Res., 2_6, 1 (1973) 38. A.R. Gibson, L. D. Melton and K.N. Slessor, Can. J . Chem., 52, 3905 (1974) 39. L.D. Melton and K.N. Slessor, Carbohydr. Res., 18., 29 (1971) 40. D.M. Clode and D. Horton, Carbohydr. Res., 17, 365 (1971) 41. G. Avigad, D.M. Amaral, C. Asensio and B.L. Horecker, J . B i o l . Chem., 237, 2736 (1962) 42. A. Maradufu, G.M. Cree and A.S. P e r l i n , Can. J . Chem., 50, 768 (1971) 43. W. Jack and R.J. Sturgeon, Carbohydr. Res., 49, 335 (1976) 44. M.W.C. Halton and E. Regoeczi, Biochem. Biophys. Acta, 438. 339 (1976) 45. M. Yalpani and L.D. H a l l , J . Polym. S c i . , Polym. Chem. Edn., 20, 339 (1976) 46. J.K. Rogers and N.S. Thompson, Carbohydr. Res., 7, 665 (1968) 47. R.A. Schlegel, CM. Gerbeck and R. Montgomery, Carbo-hydr. Res., 7, 193 (1968) 48. See J . March (ed.), "Advanced Organic Chemistry", 3rd ed., Wiley, New York (1985), p. 1081-1083, and r e f e r -ences c i t e d therein 49. M.L. Wolfrom and P.Y. Wang, Carbohydr. Res., 12, 104 (1970) -227-50. J . Defaye, H. Driguez and A. Gadelle, Appl. Polym. Symp., 28, 955 (1976) 51. J . Defaye and A. Gadelle, Carbohydr. Res., 5.6, 411 (1977) 52. K. Bredereck, Tetrahedron Lett., 695 (1967) 53. C. Bosso, J . Defaye, A. Gadelle, C.C. Wong and C. Pedersen, J . Chem. Soc. Perkin Trans., X , 1579 (1982) 54. J . Defaye and A. Gadelle, Pulp Paper Canada, 75, 50 (1974) 55. D. Horton and T. Usui, "Carbohydrate Sulfates", R.G. Schweiger (ed.), Vol 77, p. 95, ACS Symp. Ser. (1978) 56. D. Horton and E.K. Just, Carbohydr. Res., 30, 349 (1973) 57. M. Yalpani, L.D. H a l l , J . Defaye and A. Gadelle, Can. J . Chem., 62, 260 (1984) 58. T. Teshirogi, H. Yamamoto, M. Sakamoto and H. Tonami, Sen-I Gakkaishi, 36, T501 (1980) 59. A.N. de Belder, B. Lindberg and S. Svensson, Acta. Chem. Scand., 22, 949 (1968) 60. see r e f . 40, p. 1057-1060 and references therein 61. O. Larm and E. Scholander, Carbohydr. Res., 58, 249 (1977) 62. O. Larm, K. Larsson, E. Scholander, B. Meyer and J . Thiem, Carbohydr. Res., £1, 13 (1981) 63. J . Boeseken, Adv. Carbohydr. Chem., 4., 189-210 64. B. Augustinsson and E. Scholander, Carbohydr. Res., 126. 162 (1984) 65. M. Einarsson, B. Forsberg, O. Larm, M.E. Rigneline and E. Scholander, J . Chromatogr., 214., 45 (1981) 66. L.O. Andersson, J . Hoffman, E. Hohner, O. Larm, K. Larsson and G. Soderstrom, Thromb. Res., 2J3, 741 (1982) 67. J . Hoffman, O. Larm, K. Larsson, L.O. Andersson, E. Holmer and G. Soderstroem, Carbohydr. Polym., 2, 115 (1982) 68. D. Horton and K. Just, Carbohydr. Res., £9, 173 (1973) -228-69. T. Teshirogi, H. Yamamoto, M. Sakamoto and H. Tonami, Sen-I Gakkaishi, 34* T510 (1978) 70. T. Teshirogi, H. Yamamoto, M. Sakamoto and H. Tonami, Sen-I Gakkaishi, 35, T479 (1979) 71. M.L. Wolfrom, K.C. Gupta, K.K. De, A.K. Chatterjee, T. Kinoshita and P.Y. Wong, Staerke, 2_1, 39 (1969) 72. S. Hirano and R. Yamaguchi, Biopolymers, 15, 1685 (1976) 73. S. Hirano, Y. Ohe and H. Ono, Carbohydr. Res., 47, 315 (1976) 74. R. Yamaguchi, Y. Ar a i , T. Itoh and S. Hirano, Carbohydr. Res., 88; 172 (1981) 75. S. Hirano and T. Moriyasu, Carbohydr. Res., £2., 323 (1981) 76. S. Hirano and Y. Kondo, J . Chem. Soc. Jpn., 1622 (1982) 77. S. Hirano, Agric. B i o l . Chem., ±2, 1939 (1978) 78. S. Hirano, N. Matsuda, O. Miura and H. Iwaki, Carbohydr. Res., 21, 339 (1979) 79. S. Hirano, N. Matsuda, 0. Miura and T. Tanaka, Carbo-hydr. Res., 71, 334 (1979) 80. K. Kurita, H. Ichikawa, S. I s h i z e k i , H. F u j i s a k i and Y. Iwakura, Makromol. Chem., 183. 1161 (1982) 81. R. Yamaguchi, Y. A r a i , T. Kaneko and T. Itoh, Biotech-nol. Bioeng., 24* 1081 (1982) 82. S. Hirano and Y. Ohe, Carbohydr. Polym., 4, 15 (1984) 83. L.A. Nud'ga, E.A. Plisko and S.N. Danilov, Zh. Obshch. Khim., 43, 2756 (1973) (p. 2733 i n Transl.) 84. S. Okimasu, B u l l . Agr. Chem. Soc. Jpn., 20_, 29 (1956) 85. see r e f . 40, p. 798-800 and references c i t e d therein 86. R.F. Borch, M.D. Bernstein and H.D. Durst, J . Amer. Chem. S o c , 93, 2897 (1971) 87. R.A.A. Muzzarelli, Carbohydr. Polym., 3, 53 (1983) 88. L.A. Nud'ga, E.A. Plisko and S.N. Danilov, Zh. Obshch. Khim., 43, 2752 (1973) (p. 2729 i n Transl.) -229-89. S. Hirano and T. Osaka, Agric. B i o l . Chem., 47, 1389 (1983) 90. S. Hirano, R. Yamaguchi and N. Matsuda, Biopolymers, 16. 2752 (1977) 91. L.D. H a l l , M. Yalpani and N. Yalpani, Biopolymers, 20. 1413 (1981) 92. R.A.A. Muzzarelli, F. Tanfani, S. M a r i o t t i and M. Emanuelli, Carbohydr. Polym., 2_, 145 (1982) 93. L.D. H a l l and M. Yalpani, Carbohydr. Res., 8_3, C5 (1980) 94. R.A.A. Muzzarelli, F. Tanfani, M. Emanuelli and S. Ma r i o t t i , Carbohydr. Res., 102, 199 (1982) 95. R.A.A. Muzzarelli, F. Tanfani and M. Emanuelli, Carbo-hydr. Polym., 1, 137 (1984) 96. M. Yalpani and L.D. H a l l , Macromolecules, 17 272 (1984) 97. L.D. H a l l and M. Yalpani, J . Chem. S o c , Chem. Commun., 1153 (1980) 98. M. Yalpani, L.D. H a l l , M.A. Tung and D.E. Brooks, Nature (London) , 302., 812 (1983) 99. K. Bock and H. Thogersen, Annu. Rep. NMR Spectrosc., 13. 1 (1982) 100. K. Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem., 41, 27 (1983) 101. K. Bock, C. Pedersen and H. Pedersen, Adv. Carbohydr. Chem. Biochem., 42., 193 (1984) 102. P.A.J. Gorin, Adv. Carbohydr. Chem. Biochem., 38, 13 (1981) 103. F.R. Seymour, "Carbon-13 NMR i n Polymer Science", W.M. Pasika (ed.), ACS Symp. Ser. 103. p. 27, Wash., DC (1979) 104. A.S. P e r l i n and G.K. Hamer, "Carbon-13 NMR i n Polymer Science", W.M. Pasika (ed.), ACS Symp. Ser. 103. p. 123, Wash. DC (1979) 105. L.D. H a l l , "The Carbohydrates", W. Pigman and D. Horton (eds.), Vol. IB, p. 1300, Academic Press, New York (1980) 106. P.E. Pf e f f e r , J . Carbohydr. Chem., 3(4), 613 (1984) -230-107. A.G. Redfield, Methods i n Enzymology, Vol. 4£, p. 243 (1978) 108. D. Shaw (ed.), "Fourier Transform N.M.R. Spectroscopy", El s e v i e r , New York (1984), p. 207 109. P. Soher (ed.), "Nuclear Magnetic Resonance Spectro-scopy", Vol. I, CRC Press, Boca Raton (1983), p. 34 and Vol. I I , p. 27 110. G. Annison and G.G.S. Dutton, private communication 111. R.A. Dwek, "Nuclear Magnetic Resonance i n Biochemistry", Oxford Univ. Press (London), London and New York (1973) 112. J.L. James (ed.), "Nuclear Magnetic Resonance i n Biochemistry", Academic Press, New York (1975) 113. M.L. Martin, J . J . Delpuech and G.J. Martin (eds.), " P r a c t i c a l NMR Spectroscopy", Heyden, London (1980) 114. G.C. Levy and I.R. Peat, J . Magn. Reson., 75, 500 (1970) 115. J.H. Noggle and R.E. Schirmer (eds.), "The Nuclear Overhauser E f f e c t " , Academic Press, New York (1971) 116. H. Thogersen, R.U. Lemieux, K. Bock and B. Meyer, Can. J. Chem., 60, 44 (1982) 117. K. Bock, D. Bundle and S. Josephson, J . Chem. Soc. Per-kin I I , 59 (1982) 118. D.M. Doddrell and D.T. Pegg, J . Am. Chem. S o c , 102, 6388 (1980) 119. D.E. Dorman and J.D. Roberts, J . Am. Chem. S o c , 92., 1355 (1970) 120. I.CP. Smith, Acc. Chem. Res., 8, 131 (1975) 121. A.S. P e r l i n , MTP Int. Rev. S c i . : Org. Chem., Ser. Two, 7, 1 (1976) 122. A. Bax (ed.), "Two Dimensional Nuclear Magnetic Resonance i n Liquids", D. Reidel Publishing Co., Hintham, Mass. (1982) 123. F. Michon, J.R. Brisson, R. Roy, F.E. Ashton and H.J. Jennings, Biochem., 24., 5592 (1985). 124. F. Michon, J.R. Brisson, R. Roy, H.J. Jennings and F.E. -231-Ashton, Can. J . Chem., 63., 2781 (1985) 125. D.Y. Gagnaire, F.R. Taravel and M.R. Vignon, Macro-molecules, 15, 126 (1982) 126. W.P. Ave, E. Bartholdi and R.R. Ernst, J . Chem. Phys., 64/ 2229 (1976) 127. A. Bax, R. Freeman and G.A. Morris, J . Magn. Reson., 44, 542 (1981) 128. L.D. H a l l and S. Sukumar, J . Am. Chem. S o c , 101. 3120 (1979) 129. M.A. Bernstein and L.D. H a l l , J . Am. Chem. S o c , 104. 5553 (1982) 130. J . Dabrowski, H. Egge and U. Dabrowski, Carbohydr. Res., 114, 1 (1983) 131. L.D. H a l l and G.A. Morris, Carbohydr. Res., 82., 175 (1980) 132. P. Colson, H.J. Jennings and I.CP. Smith, J . Am. Chem. S o c , £6, 8081 (1974) 133. W.M. Pasika and L.H. Cragg, Can. J . Chem., 4_1, 777 (1963) 134. R.L. Sidebotham, L. Weigel and W.H. Bowen, Carbohydr. Res., 19, 151 (1971) 135. E.J. Bourne, R.L. Sidebotham and L. Weigel, Carbohydr. Res., 22, 13 (1972) 136. T. Usui, M. Yokoyama, N. Yamaoka, K. Matsuda, K. Tuzimura, H. Sugiyama and S. Seto, Carbohydr. Res., 33, 105 (1974) 137. D. Gagnaire and M. Vignon, Makromol. Chem., 178. 2321-(1977) 138. F.R. Seymour, R.D. Knapp and S.H. Bishop, Carbohydr. Res., 74, 77 (1979) 139. F.R. Seymour, R.D. Knapp and S.H. Bishop, Carbohydr. Res., 51, 179 (1976) 140. F.R. Seymour, R.D. Knapp, S.H. Bishop and A. Jeanes, Carbohydr. Res., 68, 123 (1979) 141. F.R. Seymour, R.D. Knapp, E.C.M. Chen, A. Jeanes and S.H. Bishop, Carbohydr. Res., 7_1, 231 (1979) -232-142. F.R. Seymour, R.D. Knapp, E.C.M. Chen, S.H. Bishop and A. Jeanes, Carbohydr. Res., 74, 41 (1979) 143. F.R. Seymour and R.D. Knapp, Carbohydr. Res., 8 1 , 67 (1980) 144. P.A.J. Gorin and J.F.T. Spencer, Can. J . Chem., 46, 2305 (1968) 145. P.A.J. Gorin, J.F.T. Spencer and S.S. Battacharjee, Can. J. Chem., 47, 1499 (1969) 146. P.A.J. Gorin, J.F.T. Spencer and R.J. Magus, Can. J . Chem., 47, 3569 (1969) 147. P.A.J. Gorin, Can. J . Chem., 5.1, 2105 (1973) 148. P.A.J. Gorin, R.H. Haskins, L.R. Travassos and L. Mendonca-Previato, Carbohydr. Res., 55, 21 (1977) 149. L. Mendonca-Previato, P.A.J. Gorin and J.O. Previato, Biochemistry, 1 8 , 149 (1979) 150. P.A.J. Gorin, L. Mendonca-Previato, J.P. Previato and L.R. Travassos, J . Protozool., 26, 473 (1979) 151. E.M. Barreto-Bergter, L.R. Travassos and P.A.J. Gorin, Carbohydr. Res., B6, 272 (1980) 152. P. Dais and A.S. P e r l i n , Carbohydr. Res., 100, 103 (1982) 153. M.J. Gidley, Carbohydr. Res., 139, 85 (1985) 154. J . Jane, J.F. Robyt and D.H. Huang, Carbohydr. Res., 140. 21 (1985) 155. I.CM. Dea and A. Morrison, Adv. Carbohydr. Chem. Bio-chem., Vol. 31, 241 (1975) 156. A.E. Manzi, A.S. Cerezo and J.N. Schoolery, Carbohydr. Res., 148, 189 (1986) 157. H. Grasdalen and T. Painter, Carbohydr. Res., 81, 59 (1980) 158. B. Pfannemuller, G.C Richter and E. Husemann, Carbo-hydr. Res., 43, 151 (1975) 159. Y.M. Choy and G.G.S. Dutton, Can. J . Chem., 51. 3021 (1973) -233-160. J.M. Berry, G.G.S. Dutton, L.D. H a l l and K.L. Mackie, Carbohydr. Res., C8 (1977) 161. G.G.S. Dutton and A.V. Savage, Carbohydr. Res., 83/ 351-(1980) 162. C.-C. Cheng, S.-L. Wang and Y.M. Choy, Carbohydr. Res., 73, 169 (1979) 163. G.G.S. Dutton and T.E. Falkman, Carbohydr. Res., 8, 147 (1980) 164. G.G.S. Dutton, K.L. Mackie, A.V. Savage, D. Reiger Hug and S. Stirm, Carbohydr. Res., 84, 161 (1980) 165. K. Okutani and G.G.S. Dutton, Carbohydr. Res., 86. 259 (1980) 166. G.G.S. Dutton and D.N. Karunaratne, Carbohydr. Res., 138. 277 (1985) 167. J.L. Fabio, D.N. Karunaratne and G.G.S. Dutton, Carbo-hydr. Res., M l , 251 (1985) 168. G.G.S. Dutton and A.V.S. Lim, Carbohydr. Res., 144, 263 (1985) 169. G.G.S. Dutton and A.V.S. Lim, Carbohydr. Res., 145. 67 (1985) 170. D.-S. Lee and A.S. P e r l i n , Carbohydr. Res., 106, 1 (1982) 171. K. Kimura, T. Shigemura, M. Kubo and Y. Maru, Makromol. Chem., I86_r 61 (1985) 172. F.F.-L. Ho, R.R. Kohler and G.A. Ward, Anal. Chem., 44, 178 (1972) 173. A. Parfondry and A.S. P e r l i n , Carbohydr. Res., 57, 39 (1977) 174. J.R. DeMember, L.D. Taylor, S. Trummer, L.E. Rubin and C.K. C h i k l i s , J . Appl. Polym. S c i . , 21, 621 (1977) 175. F.F.-L. Ho and D.W. Klosiewicz, Anal. Chem., 52., 913 (1980) 176. B. Casu, M. Regiani, G.G. Gallo and A. Vigevani, Tetra-hedron, 22, 3061 (1966) 177. M. St. Jacques, P.R. Sundararajan, K.J. Taylor and R.M. Marchessault, J . Am. Chem. S o c , 9_8, 4386 (1976) -234-178. H. Saito, T. Ohki, Y. Yoshioka and F. Fukuoka, FEBS Lett.," 68, 15 (1976) 179. H. Saito, T. Ohki, N. Takasuka and T. Sasaki, Carbohydr. Res., 58, 293 (1977) 180. H. Saito, T. Ohki and T. Sasaki, Biochemistry, 1 6 , 908 (1977) 181. H. Saito, E. Miyata and T. Sasaki, Macromolecules, 11, 1244 (1978) 182. R.T. Boere and R.G. Kidd, Ann. Rep. NMR Spectros., Vol. 1 3 , P. 319 (1982) 183. P.A.J. Gorin and M. Mazurek, Carbohydr. Res., 72, C l (1979) 184. R.L. Whistler (ed.), "Carbohydrates i n Solution", ACS Symp. Ser., 117, 242 (1973) 185. E. H i r s t and D.A. Rees, J . Chem. S o c , p. 1182 (1965) 186. D.A. Rees and J.W.B. Samuel, J . Chem. Soc. C, p. 2295 (1967) 187. A. Haug, B. Larsen and O. Smidsrod, Acta. Chem. Scand., 20, 183 (1966) 188. A. Haug, B. Larsen and 0. Smidsrod, Acta. Chem. Scand., 21, 691 (1967) 189. W. Mackie,^Biochem. J . , 125, 89P (1971) 190. E.R. Morris, D.A. Rees and D.Thom, J . Chem. S o c , Com-mun., p. 245 (1973) 191. G.T. Grant, E.R. Morris, D.A. Rees, P.J.C. Smith and D. Thorn, FEBS Lett., 32., !95 (1973) 192. T.A. Bryce, A.A. McKinnon, E.R. Morris, D.A. Rees and D. Thorn, Faraday Discuss. Chem. S o c , 57, 221 (1974) 193. E.R. Morris, D.A. Rees, D. Thom and J . Boyd, Carbohydr. Res., 66, 145 (1978) 194. M.J. Gidley, E.R. Morris, E.J. Murray, D.A. Powell and D.A. Rees, J . Chem. S o c , Chem. Commun., 990 (1979) 195. D. Thom, G.T. Grant, E.R. Morris and D.A. Rees, Carbo-hydr. Res., 100, 29 (1982) -235-196. For a detailed discussion of glycosaminoglycans, see Ref. 17, p. 248 197. J.E. Courtois and P. Le Dizet, Carbohydr. Res., 3_, 141 (1966) 198. B.V. McCleary, E. Nurthen, F.R. Taravel and J.-P. Joseleau, Carbohydr. Res., 118. 91 (1983) 199. B.V. McCleary and N.K. Matheson, Carbohydr. Res., 119. 191 (1983) 200. B.V. McCleary, A.H. Clark, I.CM. Dea and D.A. Rees, Carbohydr. Res., 139, 237 (1985) 201. B.V. McCleary, Carbohydr. Res., 2 1 , 205 (1979) 202. E. F r e i and R.D. Preston, Proc. R. Soc. London, Ser. B, 169. 127 (1968) 203. P. Zugenmaier, Biopolymers, 1 3 , 1127 (1974) 204. I.CM. Dea, E.R. Morris, D.A. Rees, E.J. Welsh, A.H. Barnes and J . Price, Carbohydr. Res., 52, 249 (1977) 205. E.R. Morris, D.A. Powell, M.J. Gidley and D.A. ;ees, J . Mol. B i o l . , 155, 507 (1982) 206. C. S t e r l i n g , Biochem. Biophys. Acta., 26, 186 (1957) 207. D.A. Powell, E.R. Morris, M.J. Gidley and D.A. Rees, J . Mol. B i o l . , 155, 517 (1982) 208. R. Kohn, Pure Appl. Chem., 42., 371 (1975) 209. I.CM. Dea, A.H. Clark and B.V. McCleary, Carbohydr. Res., 142, 275 (1986) 210. B.V. McCleary, I.CM. Dea, J . Windustand, D. Cooke, Carbohydr. Polym., 4, 253 (1984) 211. D.A. Rees, Biochem. J . , 126, 257 (1972) 212. I.CM. Dea and E.R. Morris, ACS Symp. Ser., 45, 174 (1977) 213. M. Tako and s. Nakamura, Carbohydr. Res., 138. 207 (1985) 214. M. Tako and S. Nakamura, Agr. B i o l . Chem., 48/ 2987 (1984) -236-215. A. Darke, E.R. Morris, D.A. Rees and E.J. Welsh, Carbo-hydr. Res., 66, 133 (1978) 216. E.R. Morris, D.A. Rees, M.D. Walkinshaw and A. Darke, J . Mol. B i o l . , HO, 1 (1977) 217. S. Arnott, A. Fulmer, W.E. Scott, I.CM. Dea, R. Moorhouse and D.A. Rees, J . Mol. B i o l . , £P_, 269 (1974) 218. B.V. McCleary, A.H. Clark, I.CM. Dea and D.A. Rees, Carbohydr. Res., 139, 237 (1985) 219. J.R. van Wazer, J.W. Lyons, K.Y. L i n and R.E. Colwell, "Viscosity and Flow Measurement: A Laboratory Handbook of Rheology", Wiley, New York (1963) 220. E.R. Morris, A.N. Cutler, S.B. Ross-Murphy, D.A. Rees and J . Price, Carbohydr. Polym., 1, 15 (1981) 221. M.A. Tung, "Concepts i n Rheology", The University of B r i t i s h Columbia, Vancouver, and references therein 222. J.H. E l l i o t , ACS Symp. Ser., 45, 144 (1981) 223. H.-T. Chang and D.F. O l l i s , Biotech. Bioeng., Vol. XXIV, 2309 (1982) 224. R.A. Speers and M.A. Tung, J . Food S c i . , 5_1(1), 96 (1986) 225. J.D. Ferry, " V i s c o e l a s t i c Properties of Polymers", Wiley, New York (1980) 226. P.A. Sanford and A. Laskin (eds.), " E x t r a c e l l u l a r Micro-b i a l Polysaccharides", Vol. A5_, ACS Symp. Ser. (1977) 227. R.L. Whistler and C.L. Smart, "Polysaccharide Chemis-t r y " , Academic Press, New York, 1953, p. 291 228. G. Franz, Adv. Polym. S c i . , Vol. 7_6, 3 (1986) 229. A.F. Bochkov and G.E. Zaikov, "Chemistry of the 0-Glyco-s i d i c Bond", Pergamon Press, New York (1979) 230. B. Pfannemuller, G.C Richter and E. Husemann, Carbo-hydr. Res., 47, 63 (1976) 231. B. Pfannemuller, G.C. Richter and E. Husemann, Carbo-hydr. Res., 56, 139 (1977) 232. B. Pfannemuller, G.C. Richter and E. Husemann, Carbo-hydr. Res., 5J6, 147 (1977) -237-233. W.N. Emmerling and B. Pfannemuller, Makromol. Chem., 179. 1627 (1978) 234. B. Pfannemuller and A. Berg, Makromol. Chem., 180, 1183 (1979) 235. N.K. Kochetkov, A.F. Bochkov and T.A. Sokolowskaya, Carbo-hydr. Res., 19, 1 (1971) 236. See ref . 28, p. 339 and references c i t e d therein 237. H. Ito and C. Schuerch, J . Amer. Chem. S o c , 101. 5797 (1979) 238. V. Masura and C. Schuerch, Carbohydr. Res., 15, 65 (1970) 239. L.D. H a l l and K.R. Holme, J . Chem. S o c , Chem. Commun., 3, 217 (1986) 240. M.A. Bernstein and L.D. H a l l , Carbohydr. Res., 78i, C l (1980) 241. E. Falent-Kwast, P. Kovac, A. Bax and C.P.J. Glaudemans, Carbohydr. Res., 145, 332 (1986) 242. R.T. Lee and Y.C. Lee, Carbohydr. Res., 37, 193 (1974) 243. E.M. Bessell and J.H. Westwood, Carbohydr. Res., 25. 11 (1972) 244. E.W. Thomas, Carbohydr. Res., 13, 225 (1970) 245. J . Gigg, R. Gigg, S. Payne and R. Conant, Carbohydr. Res., 141, 91 (1985) 246. R. Roy and H.J. Jennings, Carbohydr. Res., 112. 63 (1983) 247. R.U. Lemieux, Methods i n Carbohydr. Chem., Vol. I I , p. 221 (1963) 248. K. I g a r i s h i , Adv. Carbohydr. Chem. Biochem., 34., 243 (1977) 249. P.S. Bailey, Chem. Rev., 58, 925 (1958) 250. J . Balatoni, B.Sc Thesis, University of B r i t i s h Columbia, 1981 251. P.E. Jansson, L. Keene and B. Lindberg, Carbohydr. Res., 45 275 (1975) -238-252. W.J. DeGrip and P.H.M. Bovee-Geurts, Chem. Phys. of Lipi d s , 23, 321 (1979) 253. J.F.W. Keana and R.B. Roman, Membrane Biochemistry, 1, 323 (1978) 254. P. Rosevar, T. VanAken, J . Baxter and S. Ferguson-Miller, Biochemistry, 19, 4108 (1980) 255. A.T. Paulson, Ph.D. Thesis, Department of Food Science, The University of B r i t i s h Columbia, Vancouver, B.C., 1986. 256. B. Lindman and H. Wennerstrom, Topics i n Current Chemis-t r y , Vol 87, 27 (1980) 257. G.K. Greminger, J r . and A.B. Savage, "I n d u s t r i a l Gums", R.L. Whistler (ed.), Academic Press, New York (1973) 258. M. Yalpani and L.D. H a l l , Can. J . Chem., 62, 975 (1984) 259. C. Tanford (ed.), "The Hydrophobic E f f e c t " , John Wiley and Sons, New York (1980) 260. H. Morawetz (ed.), "Macromolecules i n Solution", John Wiley and Sons, New York (1975) 261. C-H. Kuo and W.W. Wells, J . B i o l . Chem., 253, 3550 (1978) 262. K. Jankowski and B. Casimir, Can. J . Chem., 45, 2865 (1967) 263. See re f . 22, p.149 264. M.P. Dale, H.E.Ensley, K. Kern, K.A.R. Sastry and L.D. Byers, Biochemistry, 24/ 3530 (1985) 265. A.G. Day and S.G. Withers, Biochem. C e l l . B i o l . , i n press 266. W.C. S t i l l , M. Kahn and A. Mitra, J . Org. Chem., 43., 2923 (1978) 267. D.D. Perrin, W.L.F. Armarego and D.R. Perrin (eds.), " P u r i f i c a t i o n of Laboratory Chemicals", Pergamon Press, New York (1980) 268. G.N. Bollenback, J.W. Long, D.G. Benjamin and J.A. Lindquist, J . Chem. S o c , Vol. 7_7, 3310 (1955) 269 . Y Hsuanyu and K.J. La i d l e r , Can. J . Biochem. C e l l B i o l . 63, 167 (1985) -239-APPENDIX A Calculation of d.s. from C and N elemental microanalysis The C/N r a t i o of the polymer product can be generally expressed as a l i n e a r combination, as shown i n Eq. 39: respectively, C^ and N^ are the number of carbon and nitrogen atoms i n the i t h residue, and x^ i s the degree of subs t i t u -t i o n ( or molar ratio) of the i t h residue. When only two d i f f e r e n t monomer units are to be considered, Eq. 39 i s expanded to give Eq. 40. In cases where both units contain only a single nitrogen atom, the re l a t i o n s h i p i s s i m p l i f i e d to give Eq. 41. [39] where M c and M n are the atomic weights of carbon and nitrogen C N 12(x 1C 1+(l-x 1)C 2) [40] 14(x 1N ]+(l-x 1)N 2) C N 12(x 1C 1+(l-x 1)C 2) [41] 14 

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