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UBC Theses and Dissertations

NMR studies of micelle forming model glycolipids Talagala, Sardha Lalith 1982

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Cl NMR STUDIES OF MICELLE FORMING MODEL GLYCOL IP IDS by SARDHA LALITH TALAGALA B.Sc. (Hons.), University of Peradem'ya, S r i Lanka, 1977 THESIS SUBMITTED IN PARTIAL FULFILLMENT .OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in 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 May 1982 © Sardha L a i i t h Talagala 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 available 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 his or her representatives. I t i s understood that copying or publication 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 C # £ W - g 7 # /  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date =1/ %~ «ft*n£ DE-6 n/sn i i ABSTRACT The work described herein fa l l s into three major categories: synthesis of model glycolipids, NMR studies on'model glycolipid'.micelles, and application of 2D-NMR'. spectroscopy in spectral assignment. Three synthetic routes, namely the glycosidation reaction, reductive amination reaction, and amide bond formation have been investigated in relation to their eff iciency and convenience in coupling carbohydrates with al iphatic chains. The reaction of amide bond formation was found to be a superior method over the others for the preparation of long alkyl chain derivatives. H^-NMR spectroscopy has been ut i l ized to study and detect the micelle formation by the model glycolipids. The studies described i l lustrate that the spin- latt ice relaxation rate (R-j) is well suited for the determination of c r i t i ca l micelle concentration providing i t is s u f f i -ciently high. The contrasting behaviour of R-j of the anomeric proton (H -1 ) of n-octyl 3-Q-glucoside in relation to that of H-2 and cu-CH^ upori micel l izat ion, has been tentatively attributed to the conformational changes 1 3 accompanying micelle formation. The observed upfield sh i f t of the C resonances of the alkyl chain has been explained as being due to the increased proportion of trans conformers in the micellar state. The question 1 3 of the downfield C sh i f t observed for the sugar resonances has been discussed. Study of N-alkyllactobionamides with H^-NMR proved to be d i f f i c u l t due to their extremely low c r i t i ca l micelle concentrations. Application of 2 D J-resolved spectroscopy and spin-echo correlated i i i spectroscopy (SECSY) i'n spectral assignment of unprotected sugar d e r i -vatives has b.een demonstrated. Using above techniques, complete assignment of the sugar region of n-octyl g-Q-glucoside and M-hexyllactobionamide has been achieved. i v TABLE OF CONTENTS Page No. CHAPTER I - GENERAL BACKGROUND 1 1.1 - Biochemical aspects 2 1.2 - Structures of natural g l y c o l i p i d s 3 1.3 - Behaviour of b i o l o g i c a l l i p i d s in water 7 1 .4 - Objective and format of the thesis 8 - References ^0 CHAPTER II - MICELLES 13 2.1 - Introduction 14 2.2 - Structure of micelles 16 2.3 - Physical properties of micelles 16 2.4 - Application of NMR for the study of amphiphile 23 aggregation. - References 27 CHAPTER III - SYNTHESIS OF MODEL GLYCOLIPIDS 31 3.1 - Previous work 32 3.2 - Method of glycoside formation 33 3.3 - Method of amide bond formation 37 3.4 - Via reductive amination 40 3.5 - Experimental 41 - References 47 V (Table of Contents continued) CHAPTER IV - NMR STUDIES OF MODEL GLYCOLIPIDS 49 1 4.1 - H NMR studies of n-octyl 6-Q-glucopyranoside 50 4.2 - C NMR studies of n-octyl B-Q-glucopyranoside 60 4.3 - H^ NMR studies of N-alkyllactobionamide series 65 4.4 - Comments 67 4.5 - Experimental 68 - References 69 CHAPTER V - TWO DIMENSIONAL FOURIER TRANSFORM NMR SPECTROSCOPY 71 5.1 - Introduction 72 5.2 - Description of homonuclear J-resolved and I'1 SECSY experiments 5.2.1 - Pulse sequence and data a c q u i s i t i o n 7> 5.2.2 - Data processing 5.3 - Application o f 2D spectroscopy i n spectral ^ assignment of unprotected sugars 5.3.1 - n-Octyl B-Q-glucopyranoside 8. 5.3.2 - N-Hexyllactobionamide 8 5 5.4 - Experimental 9-- References 9f vi CHAPTER III 3.1 CHAPTER IV 4.1 4.2 CHAPTER V 5.1 LIST OF TABLES Page No. Physical constants of N-al kyllactobionamide s e r i e s . 46 S p i n - l a t t i c e relaxation rates (R-|) of selected protons of octyl glucoside at d i f f e r e n t concen-trations in D^O. 1 3 C-NMR chemical s h i f t data f o r octyl glucoside with s h i f t changes ( A 6 ) on mice l l e formation. Proton chemical s h i f t and coupling constant data 86 of the sugar region of n-octyl 3-Q-glycopyranoside. Proton chemical s h i f t and coupling constant data of 94 the sugar resonances of N-hexyllactobionamide. v i i CHAPTER I 1 .1 1 .2 1.3 CHAPTER II 2.1 2.2 2.3 2.4 CHAPTER III 3.1 3.2 CHAPTER IV 4.1 4.2 LIST OF FIGURES Page No. Structures of some selected g l y c o g l y c e r o l i p i d s . 4 General structure of glycosphingolipids. 5 Structure of a ganglioside. 6 Schematic representation of the concentration 14 dependence of some physical properties for solutions of a'micelle forming amphiphile. Structures of common amphiphiles. 15 Mi c e l l ar models . 17 Relaxation between monomeric concentration and 18 total added concentration in true phase separation and micelle formation. H and C NMR spectra of n-octyl g-Q-glucoside. H and 1 3 C NMR spectra of N-hexyllactobionamide. 400 MHz 'H NMR spectra of octyl glucoside in n 2 0 51 at d i f f e r e n t concentrations. Variation of R-| with inverse total concentration for 55 o j-CH q, H-2 and H-l protons of octyl glucoside. ( L i s t of Figures continued) 4.3 Stereochemical view of the preferred conforma-tion about the C,-0., and C-Q, bonds of a I I a I glucoside. 4.4 Conformational projections involving C-|, , and C of a glycoside. a 13 4.5 100.6 MHz proton decoupled C NMR spectra of monomer and m i c e l l a r solutions of octyl glycoside. 4.6 400 MHz H^ NMR of N-dodecyllactobionamide in D ?0. CHAPTER V 5.1 Schematic representation, of d i f f e r e n t 2D-resolved NMR experiments. 5.2 I l l u s t r a t i o n of C0SY[A] and SECSY[B] spectra. 5.3 Pulse sequence f or 2D homonuclear J-resolved experiment. 5.4 Pulse sequences for SECSY[A] and C0SY[B] experiments. 5.5 Summary of the data manipulation procedure in 2D-experiments. 5.6 I l l u s t r a t i o n of various display modes and the t i l t routine used: in 20 spectra. 5.7 Part i a l 400 MHz ^H. ID and 2D NMR spectra of n-octyl g-D-glucopyranoside in D^O. 5.8 Individual J spectra of n-octyl g-Q-glucopyranoside 5.9 P a r t i a l 400 MHz 1H 1D and 2D NMR spectra of N-hexyl 1actobionamide in D o0. ( L i s t of Figures continued) 5.10. Part i a l 400 MHz 1 H' 2D NMR spectra of N-hexyl-1actobionamide in D2O. 5.11 Part i a l 400 MHz SECSY spectrum of N-hexyl-1actobionamide in d^O. 5.12 Pa r t i a l SECSY spectrum of N-hexyl1actobio-namide: an expansion of the dotted region of Fig. 5.11 . X ACKNOWLEDGEMENT I express my deepest gratitude to Dr. L.D. Hall for his guidance and constant encouragement throughout the course of this work. I am greatly indebted to Dr. S. Sukumar and Mr. M.A. Bernstein for many helpful discussions and c r i t i c i s m . The generous help of Messrs. N. Rajapakse, K.H. Holmes and Miss N. Partovi i n proof-reading the manuscript is g r a t e f u l l y acknowledged. Special thanks are also due to Mrs. R. Theeparajah for typing the manuscript and for her patience. De di-oation my late Father 1 CHAPTER I GENERAL BACKGROUND 2 1.1 - Biochemical aspects The surfaces of animal c e l l s play a c r i t i c a l role in inte r a c t i o n s between c e l l s and in the responses of c e l l s to t h e i r external environment which may contain b i o l o g i c a l l y a c t i v e substances such as drugs, hormones, antigens, etc. These int e r a c t i o n s are of prime importance in the growth, development and maintenance of m u l t i c e l l u l a r organisms. The simple fa c t that c e l l s are antigenic and induce an immune response when injected into unrelated animals suggests that c e l l s d i f f e r i n the structure of compo-nents located at t h e i r c e l l surfaces [1]. Since the c l a s s i c a l work on blood group substances [2], i t has long been known that complex carbo-hydrates play an important part in defining certain structural s p e c i f i c i -t i e s of c e l l surfaces [3,4,5], Extensive evidence [6] has accumulated sharing the view that the outer surface of mammalian c e l l s i s covered by a "layer" of carbohydrates consisting of oligosaccharides projecting into the external medium. These saccharides may be very complex. In addition to variations in the type and sequence of the constituent monomers, each of which may vary i n ring s i z e , the configuration of each g l y c o s i d i c bond and branching of the saccharide chain ensure a great v a r i a b i l i t y of struc-tures, meaning that these oligosaccharide structures can encode a r i c h biochemical language. This i s one of the reasons why c e l l surface carbohydrates are being considered as candidates responsible f o r some aspects of b i o l o g i c a l recognition [7,8]. It i s currently held that c e l l surface carbohydrates are possible p a r t i c i p a n t s in such events as receptor i n t e r a c t i o n s [9,10], permiablity change [11], c e l l u l a r adhesion and 3 recognition [12], which are fundamental processes governing various functions i n m u l t i c e l l u l a r organisms. Carbohydrates occur on mammalian c e l l surfaces p r imarily as components of g l y c o l i p i d s and glycoproteins. In the discussions which follow, attention w i l l be focussed mainly on g l y c o l i p i d s . G l y c o l i p i d s have been shown to be concentrated primarily in the plasma membrane [13] with some l o c a l i z a t i o n also occurring i n the endoplasmic reticulum [14]. They are generally minor l i p i d components of the membrane, accounting for 0.5 - 5% of the t o t a l membrane l i p i d . G l y c o l i p i d s are responsible f o r most of the serological s p e c i f i c i t y [15] exhibited by mammalian c e l l s and many organs have t h e i r s p e c i f i c types of g l y c o l i p i d [16]. The two best-characterised examples of membrane g l y c o l i p i d s serving s p e c i f i c b i o l o g i c a l functions are the ABO blood group antigens [17-19] and the ganglioside GM^  receptor f o r cholera toxin [20]; others have been proposed [21-24]. 1.2 - Structures of natural g l y c o l i p i d s As has already been implied, complex l i p i d s which contain a carbohydrate component are referred to as g l y c o l i p i d s . Based on the structure of the l i p i d component, g l y c o l i p i d s can be sub-divided into two cla s s e s , 1. g l y c o g l y c e r o l i p i d s 2. glycosphingolipids G l y c o g l y c e r o l i p i d s : These occur i n higher plants and algae (mainly i n the chloroplasts) and also in bacteria [25,26]. Glycosides of d i a c y l g l y c e r o l belong to t h i s c l a s s . The carbohydrate moiety can be a monosaccharide or a disaccharide 4 and structures containing galactose, mannose, and glucose residues have been characterized. Sulphated monosaccharide residues (glycosyl sulphates) have been found in plants and photosynthetic algae. The acyl chains are normally saturated and occur in varying lengths. Structures of some selected compounds in t h i s series are shown in F i g . 1.1. S-o I C H T — CH—CHo I I 0 o 1 I CO CO I I CH2 CH2 I I Long a l i p h a t i c chains Monogalactosyl d i a c y l g l y c e r o l ct-0-Gal actosyl (1 - 6) g-D- gal actosyl d i a c y l g l y c e r o l 6-Sul pho-a-Q-qui novosyl d i a c y l g l y c e r o l Figure 1.1: Structures of some selected g l y c o g l y c e r o l i p i d s 5 Glycosphingol i p i d s : These are e s p e c i a l l y abundant i n the membranes of brain and nerve c e l l s of higher organisms [27]. Those characterized from these sources are 1-0-glycosyl derivatives of ceramides (cerebrosides). The ceramides are N-(fatty acyl) sphingosines or derivatives thereof. ( F i g . 1.2). ! C H 2 —CH—CH(OH); ; NH CH ' 4 - " 1 II CH I C13H27 R Spingosine R = H, R' = H Ceramide R = long-chain a c y l , R' = H Cerebroside R = long-chain a c y l , R' = glycosyl Figure 1.2: General Structure of glycosphingolipids The glycosyl moiety can vary from a si n g l e galactose or glucose unit to complex oligosaccharides containing D-galactose, D-glucose, D-galactose-3-sulphate, L-fucose, 2-acylamido-2-deoxy-D-galactose (or D-glucose), and N-acetyl neuraminic acid ( S i a l i c a c i d ) . Cerebrosides often contain long - chain f a t t y acids, the most abundant of which are l i g n o c e r i c (24:0),cerebronic (q-hydroxylignoceric) and nervonic (24:1) acids. 6 More complex glycosphingolipids are usually s t r u c t u r a l l y derived from l a c t o s y l ceramide by extension of the carbohydrate head group, and have been c l a s s i f i e d according to the nature of the carbohydrate back bone. Thus the ganglio-series contains gangliotetrose, g-galactosyl (1-3) 3-N-acetylgalactosaminyl (1-4) l a c t o s e , the globo-series contains B-N-acetylgalactosaminyl (1-3) g-galactosyl (1-3 or 4) lactose, and the l a c t o - s e r i e s contains g-galactosyl (1-3 or 4) g-N-acetylgalactosaminyl-(1-3)1actose. Members of the ganglio-series are further substituted by s i a l i c acid residues while those of lactose series can be substituted by s i a l i c acid or fucose. Structure of a ganglioside i s given in Fig. 1.3. galactose N-acetyl-galactosami ne galactose glucose AcN ~CH2—CH CH(OH) (CH2)16 (CH2)12 CH3 CH3 CO NH CH CH HOCH HOCH HOCH2 HOCH2 s i a l i c acid s i a l i c acid Figure 1.3: Structure of a ganglioside 7 1.3 - Behaviour of bi o l o g i c a l l i p i d s in water Lipids in b i o l o g i c a l systems demonstrate a broad range of behaviour in water, from hydrocarbons which are i n s o l u b l e , to amphibilic molecules that possess potent detergent properties and interact with water rather dynamically. Most of the b i o l o g i c a l membrane l i p i d s including g l y c o l i p i d s possess amphipathic character due to the presence of polar and non polar regions in the same molecule. The surface and bulk properties of a given l i p i d depend upon the r e l a t i v e strength of the hydrophilic and l i p o p h i l i c portions of the molecule: i . e , the hydrophylic-1ipophiTic balance. Glyco-l i p i d s as well as other polar l i p i d s extracted from c e l l membranes and c e l l u l a r organelles can be c l a s s f i e d as "insoluble swelling amphiphi 1ic l i p i d s " on the basis of t h e i r behaviour in water and at an air-water i n t e r f a c e [28]. They spread to form stable monolayers at air-water int e r f a c e s [28] and although insoluble in bulk water, they swell in water to form l y o t r o p i c l i q u i d c r y s t a l s such as lamellar [28,29], reversed hexagonal or cubic phases [30]. Although the lamellar l i p i d b i l a y e r structure i s considered as the only compatible structure for the functioning of the membrane [31], the p o s s i b i l i t y of existence of other mesophases i s not t o t a l l y excluded [30]. Formation of d i f f e r e n t mesophases within the membrane during short time periods or in the proximity of integral membrane proteins has..- been proposed to be advantageous to c e l l functions such as membrane fusion [32,33], exo- and endo-cytosis, and t r a n s - b i l a y e r movements of l i p i d s [34]. The usefulness in biology of such phase equilibrium systems has been pointed out [35]. The assertion [28], by one of the active researchers i n the f i e l d , that - "processes such as membrane budding and fusion may ultimately 8 be explained by the composition and states of the l i p i d s taking part i n these processes" - summarizes concisely the probable importance of polymorphism of l i p i d s in biology. 1.4 - Objective and format of the thesis The complexity of b i o l o g i c a l membranes, lar g e l y precludes an in-vivo study of the g l y c o l i p i d s in order to ascertain t h e i r s p e c i f i c r o l e s . This fa c t has promoted the synthesis and study of many 'model' g l y c o l i p i d s . Their synthesis has been necessitated because of the d i f f i c u l t i e s involved in i s o l a t i n g them in large quantities and in pure form from natural sources. Apart from helping to understand t h e i r n a t u r a l l y occuring counterparts, model g l y c o l i p i d s have found a p p l i c a t i o n as detergents for the s o l u b i l i z a t i o n of membrane components [36] and as acceptors for carbohydrate binding proteins, such as l e c t i n s [37,38], In s p i t e of many previous e f f o r t s , there continues to be a pressing need for the development of new synthetic methods and a n a l y t i c a l t o o l s to probe the behaviour of these "model" compounds in so l u t i o n . Realization of t h i s fact l a i d the foundation f o r the studies described i n t h i s t h e s i s and the objective of the work presented here has been two f o l d . F i r s t l y , to evaluate the e f f i c i e n c y of various synthetic schemes for the prepara-ti o n of model g l y c o l i p i d s so that large quantities could be obtained r a p i d l y , and in high y i e l d s . Secondly, to evaluate the advantages and l i m i t a t i o n s of Nuclear Magnetic Resonance (NMR) spectroscopy as a structural and an a n a l y t i c a l tool in studying the solution properties of these compounds. In t h i s context, i t was decided t o confine the attention to the class of compounds having a long alkyl chain as the hydrophobic portion 9 and a carbohydrate moiety as the hydrophilic group. These compounds f a l l into the class of "soluble amphiphiles" [28], which form micelles in d i l u t e aqueous s o l u t i o n s . This thesis describes the synthesis and NMR studies on micelle formation of the above d e r i v a t i v e s . H^-NMR was chosen 13 2 to s c r u t i n i z e micelle formation i n preference to ei t h e r C or H NMR because of the predictable need to work with very d i l u t e solutions. In the event t h i s proved to be a prudent decision since, as w i l l be seen, these derivatives have very low c r i t i c a l micelle concentrations and i t proved very d i f f i c u l t to study t h e i r m i c e l l a r properties even by ^ H-NMR. The format of t h i s thesis i s as follows: Chapter II introduces the reader to various aspects of micelles as currently understood and i s accompanied by a b r i e f survey of applications of NMR techniques in studying amphiphile aggregation. A discussion on the s y n t h e s i s o f 'model' g l y c o l i p i d s forms the basis for Chapter I I I . Chapter IV i s devoted to an evaluation of the experimental NMR resu l t s pertaining to micelle formation by these compounds. F i n a l l y , in Chapter V the reader i s exposed to a discussion on various 'two-dimensional 1 NMR experiments which have been u t i l i z e d i n spectral assignments. 10 References 1. Hood, L.E., Weissman, I.L., Wood, W.B., "Immunology", Benjamin/ Cummings Publishing Co., C a l i f o r n i a , 1978. 2. Gottschalk, A., "The Chemistry and Biology of S i a l i c acids and Related Substances-," Cambridge University Press, 1960. 3. Watkins, W.M., i n "Glycoproteins: Their Composition, Structure and Function", Part B; Gottschalk, A., Ed., E l s e v i e r , Amsterdam, 1972, p. 830-891. 4. Ginsburg, V., Kobata, A., in "Structure and Function of Bi o l o g i c a l Membranes", Rothfield, L.I. Ed., Academic Press, New York, 1971, p. 439. 5. Cook, G.M.W., Stoddart, R.W., "Surface Carbohydrates of Eukaryotic C e l l " , Academic Press, New York, 1973. 6. Parsons, D.F., Subjeck, J.R., Biochim. Biophys. Acta, (1972), 265, 85. 7. Hughes, C.L., Sharon, N., Trends Biochem. S c i . , (1978), 3, N275. 8. Marchesi, V.T., Ginsburg, V., Robbins, P.W., Fox, C.F., Eds., "Cell Surface Carbohydrates and B i o l o g i c a l Recognition", Alan R. Lis s Inc., New York, 1978. 9. Mullin, B.R., Fishman, P.H., Lee, G., A l o j , S.M., Ledley, F.D., Winand, R.J., Kohn, L.D., and Brady, R.O., Proc. Natl. Acad. S c i .  U.S.A., (1976), 73, 842. 10. Lee, G., A l o j , S.M., Brady, R.O., Kohn, L.D., Biochem. Biophys. Res.  Commun., (1976), 73_, 370. 11. G l i c k , J . C , Githen, S., Nature, (1965), ^08, 88. 12. Rosemann, S., Chem. Phys. L i p i d s , (1970), 5_, 270. 11 13. Weinstein, D.B., Marsh, J.B., G l i c k , M.C, Warren, L., J . B i o l .  Chem., (1970), 245, 3928. 14. C r i t c h l e y , D.R., Graham, J.M., Macpherson, I., FEBS L e t t . , (1973), 32, 37. 15. Talmadge, K.W., Burger, M.M., i n "Biochemistry of Carbohydrates", MTP International Review of Science, Biochemistry Series one, Vol. 5; Whelan, W.J., Ed., Butterworths, London, 1975, p. 59. 16. Carter, H.E., Johnson, P., Weber, E.J., Annu. Rev. Biochem., (1965), 34, 109. 17. Horowitz, M.I., in "The Glycoconjugates", Vol. I I , Horowitz M.I., Pigman, W., Eds., Academic Press, New York, 1978, p. 387. 18. Hughes, R.C, "Membrane Glycoproteins: A Review of Structure and Function", Butterworths, London, 1976, p. 114. 19. Sharon, N., "Complex Carbohydrates: Their Chemistry Biosynthesis and Function". Addison-Wesley, Reading, Massachusetts, 1975, p. 215. 20. Moss, J . , Vaughan, M., Annu. Rev. Biochem., (1979), 48, 581. 21. Jacques, L.W., Brown, E.B., Barret, J.M., Brey, W.S. J r . , Weltner, W. J r . , J . B i o l . Chem., (1977), 252^ , 4533. 22. Pappenheimer, A.M. J r . , Annu. Rev. Biochem., (1977), 46, 69. 23. Besanocon, F., Ankel, H., Nature, (1974), 252, 478. 24. Kohn, L.D. in "Receptor and Recognition",'Series A, Vol 5, Cuatrecas, P., Greaves, M.F., Eds., Chapman and H a l l , London, 1978, p. 133. 25. Shaw, N., Baddiley, J . , Nature, (1968), 217, 142. 26. Shaw, N., B a c t e r i o l . Rev., (1970), 34, 365. 27. Wiegandt, H., Adv. L i p i d Res., (1971), 9, 241. 28. Small, D.M., Pure and Appl. Chem., (1981), 5_3, 2095. 12 29. Rucco, M., Atkinson, D., Small, D.M., Skarjune, R., 0 1 d f i e l d , E., Shipley, G.G., Biochemistry, (1981), 20, 5957. 30. Wieslander, A., R i l f o r s , L., Johanssan, L.B., Lindblom, G., Biochemistry, (1981), 20, 730. 31. Singer, S.J., Nicolson, G.L., Science, (1972), 175, 720. 32. Lucy, J.A., in "Cell Membranes: Biochemistry, Cell Biology and Pathology", Weissman, G., Claiborne, R., Eds., H.P. Publishing Co., New York, 1975. 33. C u l l i s , P.R., de K r u i j f f , B., Biochim. Biophys. Acta, (1979), 559, 339. 34. C u l l i s , P.R., Farren, S.B., Hope, M.J., Can. J . Spectros., (1981), 26, 89. 35. Small, D.M., J . C o l l o i d Interface S c i . , (1977), 58, 581. 36. Stubbs, G.W., Smith, H.G. J r . , Litman, B.J., Biochem. Biophys. Acta, (1976) , 426, 46. 37. Read, B.O., Demel, R.A., Wiengandt, H., Van Deenen, L.L.M., i b i d . , (1977) , 470, 325. 38. Williams, T.J., Plessas, N.R., Goldstein, I . J ., Arch. Biochem.  Biophys., (1979), 195, 145. CHAPTER II MICELLES 14 2.1 Introduction The term "micelle" (from l a t . micella meaning "small b i t " ) was introduced by the pioneer in the f i e l d , J.W. McBain, in 1913 to describe the formation of aggregates of c o l l o i d a l dimensions by detergents and soaps i n aqueous s o l u t i o n . The idea evolved from the observation that amphiphilic molecules i . e . molecules possessing separate hydrophilic and hydrophobic portions, behave rather uniquely when dissolved in water above a c e r t a i n concentration [1]. Above t h i s concentration many physical properties of the solution (e.g. v i s c o s i t y , conductivity, o p t i c a l and spectroscopic properties) were found to show an abrupt change as i l l u s t r a t e d i n F i g . 2.1. Figure 2.1: Schematic representation of the concentration depen-dence of some physical properties for solutions of a micelle forming amphiphile (from r e f . [16]). This behaviour i s now attributed to the association of amphiphiles to form small aggregates - m i c e l l e s . The association of amphiphiles i n concentration 15 aqueous solution into m i c e l l a r aggregates i s ascribed to the hydrophobic e f f e c t [2] which in turn arises from the strong a t t r a c t i v e forces between water molecules [3], Such associations lead to a reduction in the t o t a l contact area of the hydrophobic groups with water. The concentration at which the association i s shown to occur i s c a l l e d the " c r i t i c a l micelle concentration" (cmc); i . e . i n d i l u t e solution (concentration <cmc) the amphiphiles e x i s t as monomers while at higher concentrations (»cmc) they spontaneously assemble to form stable m i c e l l a r aggregates. M i c e l l e - forming amphiphiles can be c l a s s i f i e d as c a t i o n i c , anionic, nonionic or z w i t t e r i o n i c according to the charge on the hydrophilic "head group". The hydrophobic " t a i l " commonly comprises a long hydrocarbon chain. Examples of a few common amphiphiles are given in Fig. 2.2. + Hexadecyltrimethyl ammonium bromide C H ^ - ( C H 2 } ^ ( C H ^ B r Sodium dodecyl sul phate CHg- (CH2)-| •] -S0^Na + f H3 N-Dodecyl-N,N-dimethyl glycine CH 3-(CH 2) 1 1-N-CH 2-C0 2 CH 3 Polyoxyethylene (6) hexadecanol CH 3-(CH 2) 1 5-0-(CH 2CH 20) 6H Figure 2.2: Structures of common amphiphiles 16 2.2 - Structure of micelles The structure of micelles has been a subject of controversy, l i k e many other aspects of m i c e l l e s , for a long time. Even at present there seems to be no universal agreement. The basic concept of a roughly spherical micelle in aqueous solution i s due to the pioneering work of G.S. Hartley [4]. The essential feature of t h i s Hartley model i s that a shell of hydrated polar head groups encases a hydrocarbon core (Fig. 2.3a). At present i t i s customary to consider micelles as s p h e r i c a l -e l l i p s o i d a l species (Fig. 2.3b), and the idea i s supported by sedimentation, d i f f u s i o n , and l i g h t s c a ttering data [5], Even though current discussions routinely depict the Hartley s p h e r i c a l - e l l i p s o i d a l m i c e l l e as a proven fa c t [6,7], the v a l i d i t y of the model has recently 13 been questioned [8]. According to Menger [8], neither the C-NMR data [9], ORD data [10], k i n e t i c data [11], nor the molecular model studies [12] support the Hartley concept, but strongly suggest a "porous m i c e l l e " with a rough surface and deep water f i l l e d c a v i t i e s . More recently, Fromherz [1,3] has proposed a surfactant-block (Fig. 2.3c) model for m i c e l l a r aggregates based on c r i t i c a l inspection of some experimental data. Since none of the newer models have yet gained wide acceptance, i n the following discussion the modfied Hartley model w i l l be regarded as the accepted structure of m i c e l l e s . 2.3 - Physical properties of micelles C r i t i c a l m icelle concentration (cmc) The c h a r a c t e r i s t i c cooperative nature of m i c e l l i z a t i o n makes i t often possible to describe the aggregation process using only a few 17 (a) Schematic cross-sectional representation of a Hartley spherical m i c e l l e . core E l l i p t i c a l cross- hydrocarbon section o f an id e a l i z e d anionic detergent micelle (from Ref. 7). Stern layer containing headgroups and 'bound' — — — _ counterions Gouy-Chapman diffuse double layer containing 'unbound' counterions (c) Surfactant-Block model 1. Orthogonal assembly of blocks with minimal headgroup contacts. 2. Modification of block-assembly to reduce the headgroup repulsion by the introduction of a gauche isomer near the head group. The stick-models represent the e f f e c t i v e volume of the molecules in a l i q u i d -c r y s t a l l i n e state (from Ref. 13). Figure 2.3: M i c e l l a r Models 1 8 parameters. It has, for example, proven to be most useful to ascribe a s p e c i f i c cmc to each micelle-forming amphiphile. In the "mass action law model" [14] of m i c e l l i z a t i o n , i t i s assumed that a sin g l e s i z e m i c e l l a r species i s in fast equilibrium with the monomers. This can be represented by, nA1 ^ An ; [An]/[A.,] n = K 2.1 where A-| and A n represent the monomer and the m i c e l l a r species of a fixed aggregation number, n, re s p e c t i v e l y . This equilibrium demands that, when n i s large, the concentration of micelles remains small up to a certain level of surfactant and increases rapidly t h e r e a f t e r . The l a r g e r the value of n, the sharper being the d i s c o n t i n u i t y . The presumption of single size micelles (monodispersity) i s not s t r i c t l y correct since in r e a l i t y micelles of d i f f e r e n t aggregation numbers can occur ( p o l y d i s p e r s i t y ) . X c r i , 0.5 1.0 1.5 2.0 Total added concentration Total amphiphite concentration Ca) (b) Figure 2.4: Relation between monomeric concentration i n solution and total added concentration in (a) true phase separation and (b) micelle formation. Figure 2.4(b) is based on calc u l a t i o n s and the dashed l i n e shows i d e a l i s e d behaviour and empirical procedures for determining the cmc (from Ref. [ 2 ] ) . 19 An important feature of micelle formation i s that when the t o t a l amphiphile concentration ( C t o t ) i s increased above the cmc, the concentration of free amphiphiles stays equal to the cmc over a wide range, while the concentration of amphiphiles present in m i c e l l a r form i s increased. Figure 2.4 shows the v a r i a t i o n in monomeric and m i c e l l a r concentrations with the t o t a l amphiphile concentration. In r e a l i t y the t r a n s i t i o n from monomeric to m i c e l l a r state i s not sharp, but occurs over a narrow range of concentrations. Thus dashed l i n e in Figure 2.4 shows only the i d e a l i s e d behaviour. The s i z e d i s t r i b u t i o n of micelles has been analyzed [15] using the "multiple equilibrium model" of m i c e l l i z a t i o n [16], This model can be formally represented in two equivalent ways; as a stepwise growth of micelles according to the equation 2.2, A, + A , *=? A ; n = 2,3 2.2 1 n-1 n or as a number of e q u i l i b r i a according to the equation 2.3, nA, A ; n = 2,3 .... . 2.3 1 n These two schemes are thermodynamically equivalent but they have d i f f e r e n t implications for the k i n e t i c behaviour of m i c e l l e s . At t h i s point the reader i s referred to the a r t i c l e by Aniansson [15] for a more elaborate discussion on the usefulness of t h i s model. The cmc has i t s most clear-cut i n t e r p r e t a t i o n within the (pseudo) "phase-separation model" of m i c e l l e formation [16]. According to t h i s model, the cmc i s regarded as the concentration at which the system enters a two phase region, the two pseudophases formed being the aqueous system and m i c e l l e s . This model i s p a r t i c u l a r l y useful for describing the amount 20 of mi cellized-amphiphile present, and how the molecular properties vary with the amphiphile concentration. The average of a quantity Q (which can be a d i f f u s i o n c o e f f i c i e n t , NMR chemical s h i f t , or relaxation time) i s determined by the fr a c t i o n s of amphiphile m i c e l l i z e d , p ™ l c , and free, p^ q such that for a t o t a l concentration C t Q t larger than the cmc, <Q> = pfc Q m i c + pjq Q a q = ( 1 - Q m i c + cmc 2 . 4 \ t o t / t o t where Q m i c and Qac' are the values of Q in micella r and aqueous environments respectively. Below the cmc, <Q> = Q a q. Thus, by p l o t t i n g <Q> = Q ^  vs C t Q t ^ one should obtain two straight l i n e s which in t e r s e c t at the cmc. Equation 2.4 provides only approximate description of the experimental data. Here, a smooth t r a n s i t i o n i s observed in the region of the cmc instead of the predicted sharp change. This i s due to the fact that m i c e l l i z a t i o n i s not rigorously a phase separation s i t u a t i o n [17]. In p r a c t i c e , the determination of the cmc i s based on the change i n slope when an appropriate physical property that distinguishes between mic e l l a r and free amphiphile state i s plotted against t o t a l concentration. However, i t i s important to emphasize that no procedure can y i e l d a unique cmc because none in fact e x i s t s [17]. The cmc obtained l a r g e l y depends on the method employed and various methods of measurement and results obtained by t h e i r use, have been c r i t i c a l l y discussed [18]. The primary factor governing the magnitude of the cmc i s the size (length) of the hydrophobic part of the molecule. For many classes of single chain amphiphiles the dependence of cmc on the number of carbon atoms (n c) can be represented by the relationship [19], log cmc = a - bn 2 . 5 21 where a and b are constants. This implies that an increase in the chain length causes a decrease in the cmc. The presence of double bonds in the alkyl chain has been observed to cause an increase in the cmc by a factor of 3 - 4 in comparison with the corresponding n-alkyl compounds; chain-branching and introduction of -OH groups also cause substantial increases in the cmc value [16]. With regards to the polar head group, the main influence comes from i t s charge so that the cmc for a given alkyl chain length i s much lower for nonionic than f o r i o n i c amphiphiles [16]. The cmc values are found to decrease with increasing bulk of the head group [14]. Addition of simple e l e c t r o l y t e s has a large e f f e c t on the cmc of i o n i c amphiphiles, whereas for nonionic systems t h i s e f f e c t i s r e l a t i v e l y small. The decrease in cmc f o r i o n i c systems with added counterions usually corresponds to a l i n e a r r e l a t i o n s h i p between the logarithm of the cmc and the t o t a l counterion concentration [14]. The influence of added non-electrolytes varies widely, depending on whether the added compound i s p r e f e r e n t i a l l y located in the m i c e l l e , or i n the i n t e r - m i c e l l a r solution [14]. The dependence of cmc on temperature i s small [16] compared to that of most chemical association phenomena. The cmc may increase or decrease with increasing temperature, or i t may show a pronounced minimum. The pressure dependence of the cmc i s weak even up to high pressures [16]. M i c e l l a r s i z e and p o l y d i s p e r s i t y The aggregation number i s defined as the number of molecules contained i n a m i c e l l e . However, as pointed out e a r l i e r , in practice amphiphiles give a d i s t r i b u t i o n of micelle sizes (polydispersity) of which the aggregation number represents the most abundant of many micelle sizes 22 in equilibrium with each other. Studies indicate that the d i s t r i b u t i o n of micelle s i z e i s narrow and somewhat unsymmetrical around the average value [20]. In contrast to the cmc, the m i c e l l a r s i z e varies with a number of factors in a manner which i s complex and presently d i f f i c u l t to predict [20]. Intramicellar structure and dynamics A vast amount of experimental evidence has accumulated which suggests that the hydrocarbon chains located at the i n t e r i o r of micelles e s s e n t i a l l y have a l i q u i d - l i k e character [21-23], but less f l u i d than hydrocarbon solvents of s i m i l a r chain length [24]. However, c o n f l i c t i n g data [25,26] consistent with the idea of a s o l i d - l i k e micelle core, are also a v a i l a b l e . The analogy with a l i q u i d hydrocarbon i s not adequate since from the general structure of micelles i t i s clear that the polar head groups are more or less fixed to the m i c e l l a r surface. This f i x a t i o n imposes constraints on the motion of the attached alkyl chain. Related studies indicate that the motional freedom of the chain increases along the chain away from the polar head group [27] and that there i s an increased proportion of trans conformation of the chain upon m i c e l l i z a t i o n [28]. Since micelle formation i s associated with the elimination of unfavourable contact between hydrophobic groups and water, i t i s important to know exactly how much of the amphiphi 1e-water contact i s retained on m i c e l l e formation. Here again c o n f l i c t i n g ideas have been put forward; Menger and co-workers have shown [9] that water penetrates upto at least the f i r s t seven carbons, whilst Lindman and Wennerstrom argue [29] against any penetration of water into the i n t e r i o r of the" micelle except to hydrate 23 the head group. Sol ubi 1 i z ati on The a b i l i t y of m i c e l l a r solutions to dissolve substances that are insoluble (or sparingly soluble) in pure water, makes the m i c e l l i z a t i o n phenomenon most s i g n i f i c a n t from an i n d u s t r i a l and b i o l o g i c a l point of view. Spectroscopic observations [20,30,31] suggest that the s o l u b i l i z a t i o n of non-polar molecules in micelles resembles non-specific d i s s o l u t i o n in a non-aqueous phase rather than s p e c i f i c binding to a s i t e on or in the m i c e l l e . However, the nature of the solute determines i t s a f f i n i t y towards d i f f e r e n t parts of the m i c e l l e . Molecules with a polar group are s o l u b i l ized c l o s e to the. .mice! 1 e-water interface [32>]--,while a l i p h a t i c hydrocarbons are p r e f e r e n t i a l l y s o l u b i l i z e d in the i n t e r i o r of the m i c e l l e . Recent studies [26,33]'indicate that aromatic compounds are also s o l u b i l i z e d close to the micelle-water i n t e r f a c e . The preceding discussion was mainly intended to give only an overview of the subject. Many other important properties of m i c e l l e s , for example, counterion binding, m i c e l l a r c a t a l y s i s have been omitted, and these aspects are thoroughly dealt with in the review a r t i c l e s [6,7,16,34]. 2.4 - Application of NMR for the study of amphiphile aggregation Micelles continue to be scrutinized by an unusually wide variety of techniques including, among others, NMR, X-rays, ESR, fluorescence, l i g h t s c a t t e r i n g , and calorimetry. The discussion here i s r e s t r i c t e d to a p p l i c a t i o n of NMR because of the d i v e r s i t y of the methods employed and the reader i s referred to the reviews by Wennerstrom et a l . [34] and 24 Anacker [35] f o r an indepth coverage on other techniques. NMR has become the most generally applicable tool for the study Of amphiphile systems; the advent of Fourier transform techniques as well as high f i e l d superconducting magnets have made studies f e a s i b l e at sub-m i l l i m o l a r concentrations. The most frequently used "probe" nuclei are 1 1 3 19 2 31 H, C, F, H and P. A l l the common NMR parameters (chemical s h i f t s ; relaxation times, l i n e widths, and quadrupole s p l i t t i n g s ) have been u t i l i z e d to study d i f f e r e n t aspects of micelle structure. In most cases m i c e l l a r solutions are considered to be i s o t r o p i c from an NMR point of view [36] due to the rapid exchange of molecules between monomeric and m i c e l l a r states. Thus, a l l the observed NMR parameters represent the weighted average between the two states. U t i l i z a t i o n of NMR, even with i t s very high s e n s i t i v i t y i s hampered by the fact that the dispersion of chemical s h i f t s i s quite small. As a r e s u l t , i t i s d i f f i c u l t to resolve overlapping resonances from methylene groups in an alkyl chain and to detect t h e i r chemical s h i f t changes accompanying m i c e l l i z a t i o n [37]. The use of proton chemical s h i f t s i s very much easier in the presence of aromatic groups because t h e i r associated ring-current s h i f t s are se n s i t i v e to geometric features of molecular packing. This feature has been used to probe the formation of micelles from various co-phenylalkyltrimethylammonium bromides [38] and to detect the s o l u b i l i z a t i o n s i t e s of various aromatic compounds in cetyltrimethylammonium bromide micelles [33]. The spin l a t t i c e relaxation of alkyl chain protons has been shown to be more e f f i c i e n t in micelles than for free monomer molecules in aqueous solution [29], In another study [40], the v a r i a t i o n of spin l a t t i c e relaxation rate of methylene protons with concentration has been used to determine the cmc of 25 n-al kyl ammoni um chlorides in D2C1. The chemical s h i f t s [38] and relaxation rates [41,42] of water protons have been u t i l i z e d to investigate micelle hydration. The relaxation studies c l e a r l y i l l u s t r a t e reduced amphiphile-water contact on micelle formation while the i n t e r p r e t a t i o n of chemical s h i f t changes i s associated with d i f f i c u l t i e s . 13 13 The advantage of using C NMR i s that C resonances occur over a wider frequency range than resonances; t h i s makes i t possible to assign a large number of resonances to individual carbons. However, one major 13 d i f f i c u l t y i s the low NMR d e t e c t i o n - s e n s i t i v i t y of C. In practice 13 C chemical s h i f t studies are l i m i t e d to concentrations of the order of 10 mM or greater, while relaxation studies require an order of magnitude 1 "3 higher concentrations. The observed C chemical s h i f t changes upon micel1ization are not yet completely understood, but in general for alkyl chains they appear to be mainly determined by trans-gauche isomerization 13 equilibrium. Thus the observed downfield s h i f t of C .resonances have been interpreted as due to an increase in trans conformation of the a l k y l chain accompanying m i c e l l i z a t i o n [28,30]. The magnitudes of 1 3 C - s h i f t changes have also been useful in following the progressive aggregation of amphiphiles as well as for evaluating the aggregation 13 numbers and equilibrium constants [28,43], Relaxation studies using C provide a wealth of quantitative information on the surfactant mobility [27,44]. The results show a progressive increase in segmental mobility of the hydrocarbon chain away from the head group. An evaluation of the motional anisotropy [45] of surfactant molecules within a micelle and the 13 f l u i d i t y [45] of the m i c e l l a r i n t e r i o r have also been obtained through C relaxation studies. 26 NMR studies of deuterons.[45-48] of both water and amphiphiles have become common in recent years. Information on micelle hydration have been obtained by H relaxation studies of D 20. Relaxation studies of deuterons in an a l k y l chain have provided quantitative information"on chain 2 dynamics. The special feature of H-NMR i s that i t i s very useful in phase e q u i l i b r i a studies [49,50] with which the presence of one or more mesophases could be e a s i l y established. 19 F NMR has been used extensively to study f l u o r i n a t e d amphiphiles [51,52]. The p o s s i b i l i t i e s of NMR are numerous for amphiphilic systems and studies using other nuclei are very well discussed in the l i t e r a t u r e [34]. 27 References 1. McBain, J.W., Trans. Faraday Soc., (1913), _9, 99. 2. Tanford, C , "The Hydrophobic E f f e c t : Formation of Micelles and Bi o l o g i c a l Membranes", 2nd E d i t i o n , John Wiley & Sons, New York, 1980, p. 57. 3. Tanford, C , i b i d . , p. 2. 4. Hartley, G.S., Trans. Faraday Soc., (1935), 3_1, 31. 5. Backus, J.K., Scheraga, H.A., J . C o l l o i d S c i . , (1951), 6_, 508. 6. Fendler, J.H., Fendler, E.J., "Catalysis in M i c e l l a r and Macromolecular Systems", Academic Press, New York, 1975, p. 31. 7. Fisher, L.R., Oakenfull, D.G., Chem. Soc. Rev., (1974), 6_, 25. 8. Menger, F.M., Bonicamp, J.M., J . Am. Chem. Soc., (1981), 103, 2140. 9. Menger, F.M., Jerkunica, J.M., Johnston, J . C , J . Am. Chem. S o c , (1978), 100, 4676. 10. Menger, F.M., Boyer, B.J., J . Am. Chem. S o c , (1980), 102, 5936. 11. Menger, F.M., Yoshinaga, H., Venkatasubban, K.S., Das, A.R., J . Org.  Chem., (1981), 46, 415. 12. Menger, F.M., Acc. Chem. Res., (1970), 12, 111. 13. Fromherz, P., Ber. Bunsenges. Phys. Chem., (1981), 85, 891. 14. Menger, F.M., in "Bioorganic Chemistry, Vol. I l l , Macro and Multimolecular Systems", Van Tamelen E.E., Ed., Academic Press, New York, 1977, p. 139. 15. Aniansson, E.A.G., Ber. Bunsenges. Phys. Chem., (1978), 82_, 981, and references c i t e d t h e r e i n . 16. Lindman, B., Wennerstrom, H., in "Micelles", Topics i n Current Chemistry, Vol. 87, Springer-Verlag, New York, 1980, p. 30. 28 17. Reference 2, p. 64-65. 18. Mukerjee, P., Mysels, K.J., " C r i t i c a l M i c e l l e Concentrations of Aqueous Micelle Systems", NSRDS-NBS 36, U.S. Government Printing • O f f i c e , Washington D.C, 1971. 19. Shinoda, K., Nakagawa, T., Tamamushi, B.-T., Isemura, T., "C o l l o i d a l Surfactants. Some Physico-chemical Properties", Academic Press, New York, 1963. 20. Reference 16, p. 44-45. 21. Ribeiro, A.A., Denis, E.A., J . Phys. Chem., (1977), 81, 957. 22. Shinitzsky, M., Dianoux, A.C, Gi t l e l , C , Weber, G., Biochemi s t r y , (1971), 10, 2106. 23. Waggoner, A.S., G r i f f i t h , O.H., Christensen, G.R., Proc. Natl• Acad.  S c i . U.S., (1967), 57., 1198. 24. Ohnishi, S., Cyr, T.J.R., Lukushima, H., Bui 1. Chem. Soc. Jpn., (1970), 43, 673. 25. Dorrance, R.C, Hunter, T.F., J . Chem. Soc. Faraday I, (1972), 68, 1312. 26. Mukerjee, P., Cardinal, J.R., Desai , N.R., in " M i c e l l i z a t i o n , S o l u b i l i z a t i o n and Microemulsions", M i t t a l , K.L. Ed. Vol. I, Plenum Press, New York, 1977, p. 241. 27. Williams, E., Sears, B., Allerhand, A., Cordes, E.H., J . Am. Chem.  Soc., (1973), 95, 4871. 28. Persson, B.0., Drakenberg, T., Lindmann, B., J . Phys. Chem., (1976), 80, 2124. 29. Reference 16, p. 56. 30. Rosenholm, J.B., Drakenberg, T., Lindmann, B., J . C o l l o i d Interface  S c i . , (1978), 63, 538. 29 31. Waggoner, A.S., Keith, A.D., G r i f f i t h , O.H., J . Phys. Chem., (1968), 72, 4129. 32. Erikkson, J.C., Gilberg, G., Acta Chem. Scand., (1966), 20, 2019. 33. Ulmius, J . , Lindmann, B., Lindblom, G., Drakenberg, T., J . C o l l o i d  Interface S c i . , (1978), 65, 88. 34. Wennerstrom, H., Lindmann, B., Phys. Rep., (1979), 52_, 2. 35. Anacker, E.W., in "Cationic Surfactants", Jungermann, E. Ed., Marcel Dekker, New York, 1970, p. 203. 36. Reference 16, p. 20. 37. Odberg, L., Svens, B., Danielson, I., J . C o l l o i d Interface S c i . , (1972), 41, 298. 38. Nakagawa, T., Inoue, H., Jizomoto, H., Horiuchi, K., Kol1oid-Z.Z-Polym., (1969), 229, 159. 39. C l i f f o r d , J . , Trans. Faraday S o c , (1965), 61_, 1276. 40. Nery, H., Marchal, J.P., Canet, D., J . C o l l o i d . Interface S c i . , (1980), 77, 174. 41. Walker, T., J . C o l l o i d Interface S c i . , (1973), 4, 372. 42. C l i f f o r d , J . , Pethica, B.A., Trans. Faraday S o c , (1965), 61, 182. 43. Persson, B.0., Drakenberg, T., Lindmann, B., J . Phys. Chem., (1979), 83, 3011. 44. Hendriksson, U., Odberg, L., C o l l o i d Polym. S c i . , (1976), 254, 35. 45. Menger, F.M., Jerkunica, J.M., J . Am. Chem. Soc., (1978),, 100, 688. 46. Wennerstrom, H., Persson, N.-0., Lindmann, B., ACS Symp. Ser., (1975), 9, 253. 47. Seelig, J . , Niederberger, W . , J . Am. Chem. S o c , (1974), 96, 2069. 48. Henriksson, U., Odbnerg, L., Eriksson, J . C , Mol. Cryst. L i q . Cry St., (1975), 30 49. Persson, N.-0., F o n t e l l , K., Lindmann, B., Tiddy, G.J.T., J .  C o l l o i d Interface S c i . , (1975), 53, 461. 50. Ulmius, J . , Lindblom, G., Wennerstrom, H., Arvidson, G., Biochemistry, (1977), 16, 5742. 51. Muller, N., Simsohn, H., J . Phys. Chem., (1971), 75, 942. 52. Muller, N., P e l l e r i n , J.H., Chen, W.W., J . Phys. Chem., (1972), 76, 3012. CHAPTER III SYNTHESIS OF MODEL GLYCOLIPIDS 32 3.1 - Previous work The chemical synthesis of n a t u r a l l y occurring glycosphingolipids and g l y c o g l y c e r o l i p i d s has been the subject of an excellent review [1]; a b r i e f discussion on the numerous methods used in model g l y c o l i p i d synthesis has also appeared recently [2]. The simplest type of model g l y c o l i p i d i s the one in which an a l k y l chain i s joined d i r e c t l y onto a sugar r i n g . The coupling between these two moieties can be achieved via ether, ester, amine or amide linkages. The glycosidation reaction [3,4] (Scheme I, p 34), where a protected glycosyl halide i s reacted with an alcohol in the presence of a s u i t a b l e c a t a l y s t , has been widely employed. Modification of the o r i g i n a l reaction conditions in order to obtain better purity and y i e l d with medium length a l i p h a t i c alcohols (carbon 8 - 14) has been the subject of recent publications [5,6,7], Oxidation of carbohydrates to aldobionic acids with subsequent conversion to lactones, enables reaction with 1-alkylamines to produce g l y c o l i p i d s with an amide l i n k . This scheme (p. 38) has been u t i l i z e d by Williams et a l . [8,9] to synthesize a series of model g l y c o l i p i d s with d i f f e r e n t carbohydrate moieties and alkyl chains of varying lengths. Reductive ami nation (Scheme I I I , p. 40 ) has been applied to the synthesis of model g l y c o l i p i d s with a hydrophilic spacer arm. This has been demonstrated by Read et a l . [10] by reacting maltotriose and maltotetrose with octadecylamine and sodium cyanoborohydride. Reductive amination of lactose with 1-alkylamines of varying chain lengths has also been reported by Hoagland et a l . [11]. 33 Model g l y c o l i p i d substances based on cholesterol have also been synthesized. Coupling of 2-aminoethyl 1-thio -g-D-galactopyranoside with cholesterol chloroformate using 8-amino-3,6-dioxaoctanoic acid as the spacer group has been reported by Slama et a l . [12]. Two s i m i l a r approaches but with d i f f e r e n t spacer groups have also been studied [13,14]. Kiso et a l . [15] have recognized the u t i l i t y of N-fatty acylated 2-amino-2-deoxy-g-D-glycosides as key intermediates for synthesis of a variety of complex model g l y c o l i p i d s , and Flowers [16] has reported synthesis of a series of acylamidoalkyl glycosides. Of the variety of methods a v a i l a b l e f o r making model g l y c o l i p i d s , i t was decided to study three methods, namely, the glycosidation reaction, linkage through an amide bond, and the reductive amination reaction. In the following discussion each of these methods w i l l be described separately. 3.2 - Method of glycoside formation n-Octyl g-Q-glucoside ( I I I , Scheme 1) was prepared in high y i e l d by adapting the synthetic procedure described in Ref. 5. In the f i r s t step, per-acetylation of D-glucose was c a r r i e d out with a c e t i c anhydride and sodium acetate at 100°C. The reaction was monitored by t h i n - l a y e r chroma-tography ( t . l . c . ) and was judged to be complete a f t e r one and a h a l f hours. The i s o l a t e d product showed the presence of only the g-anomer, as confirmed by H^-NMR. Subsequent reaction of the per-acetylated glucose with 30% HBr in g l a c i a l a c e t i c acid produced 2,3,4,6-tetra-0_-acetyl-a-Q-glucopyranosyl bromide. After 30 minutes of reaction, the t . l . c . showed only traces of 35 the s t a r t i n g material. The reaction was quenched at t h i s point because prolonged reaction times are known to result in formation of degradation products of the glucosyl bromide. The product i s o l a t e d was used immediately without further p u r i f i c a t i o n for subsequent reaction. The H^-NMR spectrum of t h i s compound stored over sodium hydroxide in vacuum, showed l i t t l e or no impurities even a f t e r a week. The glycosidation reaction was c a r r i e d out with the glucosyl bromide and 1-octanol i n the presence of fr e s h l y prepared s i l v e r carbonate and iodine as c a t a l y s t . A study by Chiang et a l . [17] has proved that t h i s i s the best c a t a l y s t found thus far for glycosidation of simple sugars, w h i l s t , with other c a t a l y s t s ( s i l v e r oxide, mercuric oxide and halides, and mercuric cyanide) higher proportions of ortho esters are obtai ned. Nevertheless, si 1ver trif1uoromethanesulphonate ( t r i f l a t e ) i n conjunction with an appropriate proton acceptor (1,1,3,3-tetramethylurea) i s also now increasingly used as a c a t a l y s t i n glycosidation reactions [18,19]. The condensation reaction was judged to be complete a f t e r about 6 hours (by t . l . c . ) but was usually allowed to proceed overnight. The protected glycoside, i s o l a t e d as a concentrated syrup, was then deacetylated with methanol/triethylamine/water (2:1:1). P u r i f i c a t i o n of the product was achieved by e l u t i o n through a column of Dowex 1 (2%, OH", 200-400 mesh) resin e q u i l i b r a t e d and eluted with methanol. The reaction scheme employed and the adapted procedure was found to be a convenient and an e f f i c i e n t method to prepare large amounts of the glycoside in high p u r i t y . [A] HO ' \ » / C H 2 -HO-* Hm H«< ~<CH2)f CH-, -(CH 2) 5 0 0 1 1 2 M (3-CH2 W-CHq Z,0 3.0' 20 10 5(ppm) [B] C3,5 C2 C 4n 0.0562M "~n— 100 ~80~ 60 40 20 5(ppm) 1 13 Figure 3 . 1 : H and C-NMR spec t ra of n -oc ty l 8 -D -g l ucos ide , CO cn 37 The 400 MHz H-NMR spectrum in D2O of n-octyl 3-D-glucoside so prepared i s shown i n Fig 3.1 [A] along with the assignments. Application of 2D-J resolved NMR spectroscopy in spectral assignment i s discussed in Chapter V. Also, additional features of the spectrum due to micelle formation are discussed in Chapter IV. The 100.6 MHz p a r t i a l l y assigned ^C-NMR spectrum of n-octyl 6-D-glucoside i s shown in F i g . 3.1 [B]. 3.3 - Method of amide bond formation A series of N-alkyllactobionamide derivatives (V) were prepared by adapting the procedure of Ref. 8, which i s outlined in Scheme II. The formation of N-substituted aldobionamides from aldonic acids and amines was o r i g i n a l l y c a r r i e d out in the presence of N,N'-dicyclohexylcarbodi-imide (DCC). However, DCC has been found to make only a minor contribu-t i o n to the condensation reaction [8], which most l i k e l y proceeds via the lactone [20], Aldonic acids can be re a d i l y converted into the correspond-ing lactone [21] by repeated evaporation from a non-aqueous solvent, and for t h i s reason the reactions were performed without DCC. Reactions with long chain alkyl amines (C._, C.., C . J and IV 12 14 16 proceeded to completion on s t i r r i n g overnight at room temperature i n methanol. Usually the product pre c i p i t a t e d from the reaction medium and could be i s o l a t e d d i r e c t l y i n high purity (by t . l . c ) ; one r e c r y s t a l l i -zation s u f f i c e d to give an a n a l y t i c a l l y pure sample. This was not the case with medium length alkyl chain amines (C , 6 C Q, C , n ) , where the reactions did not proceed to completion even on o I 0 s t i r r i n g overnight, and the product did not p r e c i p i t a t e from the reaction medium. As a r e s u l t these products had to.be i s o l a t e d by evaporation of the reaction mixture. The r e s u l t i n g crude material contained s i g n i f i c a n t 38 1. KOH/I2 2. Amberlite IR-120 (H +) 3. Repeated evaporation from toluene and 3-methoxyethanol n=5.7.9.11.13.15 Scheme II Co, 175 125 75 25 <5(ppm) 30 ~?0~ 1 13 Figure 3.2: H and C-NMR spectra of N-hexyllactobionamide. OK;I 10 5(ppm) 40 amounts of s t a r t i n g material and was p u r i f i e d by passage through a column of Amberlite CG 400 (HCO3) followed by r e c r y s t a l 1 i z a t i o n . These reac-tions could be driven further towards completion by working at elevated temperature (under reflux in methanol), which improved the y i e l d s obtained. Repeated r e c r y s t a l 1 i z a t i o n of the crude product usually afforded a high degree of pu r i t y . 1 13 The 400 MHz H-NMR and 100.6 MHz C-NMR spectra of N-hexyl1acto-bionamide in D2O are shown in Fig 3.2 with p a r t i a l assignments; further assignments of the ^ H spectrum are discussed in Chapter V. 3.4 - Via reductive ami nation Several attempts were made to reductively aminate lactose with 1-alkylamines using the procedure outlined by Hoagland et a l . [11]. In t h i s reaction the reducing end of the sugar i n i t i a l l y reacts with the amine to produce an imine (VI) which i s s e l e c t i v e l y reduced in s i t u by sodium cyanoborohydride (NaCNBH^) (Scheme I I I ) . imine V1 NaCNBH3 Scheme III 41 A major drawback of t h i s reaction i s the tendency to form Amadori rearrangement [22] products when the reaction i s done under a c i d i c conditions. As reported by Hoagland et a l . [ l l ] t h i s could be suppressed by using a weak organic acid (propionic or benzoic acid) as the proton donor. Another possible complication that could a r i s e i s the formation of higher molecular weight substances by further reaction of VII with the aldehydo sugar. Even though precautions against t h i s have not been taken in the reference c i t e d , reductive aminations with smaller molecules are usually c a r r i e d out using a f i v e - f o l d excess of the amine [23]. Eff o r t s to carry out t h i s reaction with medium chain amines did not l i v e up to our expectations. Even with the variety of d i f f e r e n t reaction condtions employed the product in each case was contaminated with varying amounts of rearrangement products or higher molecular weight substances. Attempts to purify the product mixture by ion-exchange or gel f i l t r a t i o n chromatography were not successful. Because of these problems encountered, i t was decided not to investigate t h i s reaction any further as a means of making model g l y c o l i p i d s . We believe that our experience casts some doubts on the C s h i f t s reported by Hoagland et a l . [11]. 3.5 - Experimental A l l melting points were recorded using a Fisher-Johns melting point apparatus and are uncorrected. A l l solutions were evaporated using a Buchi rotary evaporator. Thin layer chromatography ( t . l . c . ) was performed on 7.5 x 2.5 cm Baker-flex (J.T. Baker Chemical Co. N.J.) precoated s i l i c a gel p l a t e s . The solvent systems, I-ethyl acetate/hexane (1:1 v/v), I I -ethyl acetate/methanol (4:1 v/v) and III - 1-butanol/acetic acid/diethyl 42 ether/water (9:6:3:1 v/v) were used. V i s u a l i z a t i o n was effected by spraying with 30% sulphuric acid i n ethanol and heating. Microanalyses of the samples were c a r r i e d out by Mr. P. Borda, Microanalysis Laboratory, University of B r i t i s h Columbia. Nuclear magnetic resonance spectra were recorded with a Bruker WH-400 high resolution spectrometer operating at 400 MHz and equipped with an Aspect 2000 computer. A l l the solvents used were of spectro or reagent grade and were used without further treatment. Hexyl, dodecyl and hexadecylamines were purchased from Eastman Kodak Co. whereas the o c t y l , decyl and tet r a d e c y l -amines were purchased from Aldrich Chemical Co. Anhydrous s i l v e r carbonate was prepared using the procedure of Wolfrom et a l . [25], Preparation of 1,2,3,4,6-penta-O-acetyl-B-D-glucopyranose (I) D-Glucose (10 g, 0.055 mol) was added to 5 g of anhydrous sodium acetate i n 50 ml (0.52 mol) of acetic anhydride previously equ i l i b r a t e d in a bath at 100°C. After a l l the glucose was dissolved, the reaction was allowed to proceed f or a further half hour. Progress of the reaction was monitored by t . l . c . in solvent system I and a f t e r 1 1/2 hrs revealed only one spot with an value of 0.30, which co-migrated with authentic glucose pentaacetate. The reaction mixture was then poured i n a f i n e stream i n t o 500 ml of ice-water and s t i r r e d f o r 2 hours. The resultant p r e c i p i t a t e was f i l t e r e d and washed several times with sodium carbonate solution and then with i c e cold water and dried under suction. The product was then re c r y s t a l 1 i z e d from 95% ethanol to y i e l d a c r y s t a l l i n e material i n 90% y i e l d , m.p. 133-134°C ( l i t . value 134°C). 43 Preparation of 2,3,4,6-tetra-O^acetyl -a-D-glucopyranosyl bromide (II) To a s t i r r e d solution of 10 ml of 30% HBr in g l a c i a l HOAc (w/w), 5 g (0.013 mol) of I was added, and s t i r r i n g continued f o r 30 min. The t . l . c . of the reaction mixture at t h i s point in solvent system I showed only traces of s t a r t i n g material. The homogeneous solution was d i l u t e d with 20 ml chloroform and poured into 150 ml of ice-water and s t i r r e d for 5 min. The organic layer was then separated and washed with i c e - c o l d saturated sodium bicarbonate u n t i l the aqueous layer stayed basic. After a f i n a l washing with i c e - c o l d water the organic layer was dried over calcium c h l o r i d e . The time involved during the above separation and washing proce-dure was kept to a minimum to minimize the decomposition of I I . The dried organic extract was then evaporated to a thick syrup (temperature 35°C). The syrup was dissolved in 15 ml of anhydrous ether and 25 ml of petroleum ether was added and the product was allowed to c r y s t a l l i z e overnight in the freezer. The c r y s t a l s were recovered by f i l t r a t i o n and suction dried taking care to prevent excessive exposure to moisture. The product II was i s o l a t e d in 78% y i e l d and used immediately f o r further reaction. Preparation of n-octyl B-D-glucopyranoside (III) 2,3,4,6-Tetra-0_-acetyl-a-D-glucopyranosyl bromide (II) (4 g, 9.7 mmol) was dissolved i n 60 ml of chloroform and with e f f i c i e n t s t i r r i n g the following additions were made: 1.7 ml (11 mmol) of 1-octanol that has been stored over 3-A° molecular sieves, 1.8 g of fres h l y prepared and dried s i l v e r carbonate, 0.15 g of iodine and 4.0 g of 4-A9 molecular sieves. The reaction was allowed to proceed overnight even though the t . l . c . in 44 solvent system I showed completion of the reaction a f t e r 6 hrs (R^ of octyl glucoside per-acetate = 0.52). The suspension was then f i l t e r e d through a c e l i t e pad and washed with 50 ml chloroform and the f i l t r a t e concentrated to a syrup. Deacetylation was achieved by di s s o l v i n g the syrup in 20 ml of methanol/triethyl amine/water (2:1:1 v/v) and allowing the solution to stand 10 hrs at room temperature. Deacetylation was monitored.by -t.1 . e . ~ (solvent system I I ) , and af t e r 10 hrs the reaction mixture showed two spots corresponding to n-octyl B-Q-glucopyranoside (R f = 0.52) as the major product and small amounts of glucose (R f = 0.18). The reaction mix-ture was then concentrated to a syrup, simultaneously removing any unreac-ted 1-octanol. The crude material was then dissolved in a minimum amount of methanol and passed through a column of Dowex 1 (2% cross-linked; 0H~ form; 200-400 mesh) previously e q u i l i b r a t e d with methanol. P u r i f i e d n-octyl B-Q-glucopyranoside was then recrystal1 ized from 95% EtOH to pro-duce flaky c r y s t a l s in 76% y i e l d , m.p. 58-60°C; [ a]p 2-24.3° :(C 0.. 63%, • MeOH). Microanalysis, ca l c u l a t e d : C, 57.51; H, 9.65; found: C, 57.23; H, 9.36%. Preparation of N-alkyllactobionamides (V) g-D-Lactose was f i r s t oxidized to potassium lactobionate with KOH/I2, by adapting the procedure by Moore et al [24]. To a s t i r r e d solution of 5.7 g of I^ and 80 ml of MeOH previously e q u i l i b r a t e d at 40°C, a warm concentrated solution of a-D-lactose (4 g, 0.012 mol) i n methanol was added. Then to the s t i r rVd mixture, 65 ml of 4% K0H in methanol was added dropwise (15 - 20 min) and allowed to s t i r f o r 10 min. A further 50 mis of 4% KOH/MeOH solution was added dropwise at t h i s point and the f i n a l color of the reaction mixture ( l i g h t straw-yellow) indicated the 45 removal of nearly a l l the free iodine. A temperature of 40°C was maintained throughout the reaction. The reaction mixture was s t i r r e d for a f i n a l 10 min and cooled to room temperature. At t h i s point potassium lactobionate s e t t l e d to the bottom of the flask and the solution was f i l t e r e d and the p r e c i p i t a t e washed with MeOH and ether. The crude product was then re c r y s t a l 1 i z e d from water and methanol to achieve a y i e l d of 94%. Pure potassium lactobionate was then dissolved i n water, passed through a Amberlite IR-120 (H +, 20-50 mesh) column and the aqueous eluant was evaporated and dried at 40°'C under vacuum to obtain l a c t o b i o n i c a c i d . The free acid was then converted to lactobiono-l,5-lactone (IV) by repeated evaporation from 2-methoxyethanol and toluene. Compound IV was obtained i n 92.5% y i e l d from the s t a r t i n g lactose. Reaction with the 1-alkylamines was then achieved by s t i r r i n g 1 g (2.9 mmol) of lactone IV with 3.5 mmol of 1-alkylamine overnight i n 25 ml of methanol. With hexyl (Cg), octyl (Cg) and decyl (C-|0) amines, the reaction was c a r r i e d out in b o i l i n g methanol, whereas with dodecyl (C-jpJ, tetradecyl ( C ^ ) , and hexadecyl (C-|g) amines the reaction proceeded smoothly at room temperature. The Cg, Cg, and C-jQ N-alkyllactobionamides were i s o l a t e d by evaporation of the reaction mixture and subsequent washing (ether) and drying. The C-^, C-^, and C-|g N-alkyllactobionamides usually p r e c i p i t a t e d from the reaction mixture and were iso l a t e d by f i l t r a t i followed by washing (ether) and drying. A ll lactobionamides were checked for t h e i r purity by t . l . c . i n solvent system I I I . Final p u r i f i c a t i o n was achieved by r e c r y s t a l l i z a t i o n e i t h e r from absolute ethanol or methanol and the y i e l d ranged from 90-92% based on the lactone. Physical constants of the compounds prepared are given below. 46 Tahle 3,1 - Physical constants of N-alkyllactonionamide series Compound N-Hexyl N-Octyl N-Decyl N-Dodecyl Microanalysis Mp t°C) % cal cul ated C H N % found C H [ a ] n 2 ° (MeOH) 142-143 48.97 7.99 3.17 48.96 8.18 3.09 + 31 .8 (C 0.82%) 146-147 51.16 8.37 2.98 51.25 8.48 2.97 + 25.4 (C 0.078%.) 145-146 53.10 8.71 2.81 52.84 8.76 2.85 + 28.8 (C 0.51%) 143_144 54.85 9.02 2.67 54.68 9.06 2.67 + 26.5 [C 0.30%) N-Tetradecyl 145-146 56.30 9.28 2.53 56.07 9.26 2.55 + 23.2 (C 0.30%) N-Hexadecyl 140-141 57.80 9.53 2.40 57.71 9.35 2.46 n.d. n.d. -' :ndt' determined 47 References 1. Gigg, R., Chem. Phys. L i p i d s , ( 1 9 8 0 ) , 26, 287. 2. Aplin, J.D., Wriston, J.C., C r i t i c a l Rev. Biochem., ( 1 9 8 1 ) , 1_, ( 4 ) , 289. 3. Whistler, R.L., Wolfrom, M.L., Eds., Methods in Carbohydrate Chemistry, Volume I I , Academic Press, NY, 1963. 4. Igarashi, K. Adv. Carbohydr. Chem. Biochem. Vol. 34., Tipson, R.S., Horton, D. Eds., Academic Press, NY, 1977, p. 243. 5. Rosevear, P., VanAken, T., Baxter, J . , Ferguson-Miller, S., Biochemistry, ( 1 9 8 0 ) , J_9; 4108. 6. De Grip, W.J., Bovee-Geurts, P.H.M., Chem. Phys. L i p i d s , ( 1 9 7 9 ) , 23_, 321 . 7. Keana, J.F.W., Roman, R.B., Membr. Biochem., ( 1 9 7 8 ) , _1, 323. 8. Williams, T.J., Plessas, N.R., Goldstein, I.J., Carbohydr. Res., (1 9 7 8 ) , 67, CI. 9. Williams, T.J., Plessas, N.R., Goldstein, I . J . , Arch. Biochem.  Biophys., ( 1 9 7 9 ) , 195_, 145. 10. Read, B.D., Demel, R.A., Wiegandt, H., Van Deenen, L.L.M., Biochem.  Biophys. Acta, ( 1 9 7 7 ) , 470, 325. 11. Hoagland, P.D., P f e f f e r , P.E., Valentine, K.M., Carbohydr. Res., (197 9 ) , 74, 135. 12. Slama, J . , Rando, R.R., Carbohydr. Res., ( 1 9 8 1 ) , 88, 213. 13. Orr, G.A., Rando, R.R., Baugerter, F.W., J . B i o l . Chem., ( 1 9 7 9 ) , 254, 4 7 21. 14. Chabala, J.C., Shen, T.Y., Carbohydr. Res., ( 1 9 7 8 ) , 67_, 55. 48 15. Kiso, M., Nishiguchi, H., Hasegawa, A., Carbohydr. Res., (1980), 81_, C13. 16. Flowers, H.M., Carbohydr. Res., (1976), 46, 133. 17. Chiang, CK., McAndrew, M., Barker, R., Carbohydr. Res., (1979), 70, 93. 18. Banoub, J . , Bundle, D.R., Can. J . Chem., (1979), 57, 2091. 19. Hanessian, S., Banoub, J., Carbohydr. Res., (1977), 53_, C13. 20. Fieser, M., Fies e r , L.F., Toromanoff, E., Hi rata, Y., Heymann, H., T e f f t , M., Bhattacharya, S., J . Am. Chem. S o c , (1956), 78, 2825. 21. I s b e l l , H.S., Flush, H.L., Methods in Carbohydrate Chemistry, Volume I I , Whistler R.L. and Wolfrom M.L. Eds. Academic Press, NY, 1963 p. 17. 22. Hodge, J.E., Fisher, B.E., i b i d , p. 99 23. Borch, R.F., Bernstein, M.D., Durst, H.D., J . Am. Chem. Soc., (1971), 93, 2897. 24. Moore, S., Link, K.P., J . B i o l . Chem., (1940), 133, 293. 25. Wolfrom, M.L., Lineback, D.R., Methods in Carbohydrate Chemistry, Whistler, R.L., Wolfrom, M.L., Eds., Academic Press, New York, Volume II , 1963, p. 342. CHAPTER IV NMR STUDIES OF MODEL GLYCOLIPIDS 50 4.1 - 'H-NMR Studies of n-octyl B-Q-glucopyranoside n-Octyl B-Q-glucopyranoside* i s well known to form mi c e l l e s , and the c r i t i c a l micelle concentration (cmc) has been determined by using fluorescent probes [1] and by surface tension measurements [2]. The use of octyl glucoside as a mild, d i a l y s a b l e , nonionic detergent i n s o l u b i l i z a t i o n of membrane proteins i s increasing [3], The 400 MHz ^ H-NMR spectra of octyl glucoside in D^ O recorded at di f f e r e n t concentrations of the amphiphile are shown in Fig. 4.1; the spectral assignment i s discussed in Chapter V. Each solution of d i f f e r e n t concentration was prepared both separately and by stepwise d i l u t i o n of the most concentrated s o l u t i o n . The spectra obtained from both sets of solutions were found to be i d e n t i c a l . The most s t r i k i n g changes in the spectra occur for the resonances between 3.0 - 4.0 6 ; these include those of the sugar protons as well as the a-CH2 of the alkyl chain. There i s a marked change i n chemical s h i f t of H-4, H-5, H-6.B; and H-aB with d i l u t i o n . The H-l and H-2 protons also show a minor but gradual, u p f i e l d and downfield s h i f t s respectively with increase in concentration. Moreover, a closer look at the methylene envelope at 1.4 <5 also shows a downfield s h i f t . Another feature which i s evident from the spectra i s the increase i n spectral line-width with increase in concentration. This e f f e c t i s most e a s i l y noticeable with H-2 and 0-CH2 resonances. It seems pla u s i b l e to a t t r i b u t e t h i s dependence of both chemical s h i f t and line-width on concentration of the amphiphile, to the formation * Hereafter referred to as octyl glucoside. 5 1 T I • i i i i i r 40 3.0 2-0 1-0 S/ppm Figure 4.1: 400 MHz H^ NMR spectra of octyl glucoside in d^O at d i f f e r e n t concentrations. Experimental parameters: number of scans = 12, a c q u i s i t i o n time = 1.8 s, sweep-width = 2202.6 Hz, temperature = 298°C. 52 of micelles at higher solute concentration [4], The sugar moieties of the molecules form the outer layer of the micelles, while the a l i p h a t i c chains occupy the i n t e r i o r . Mi eel 1ization brings the individual molecules closer together and hence can mutually influence the local as well as the e l e c t r o n i c environment of the atoms. Appreciable changes in e l e c t r o n i c environment could only be caused (through space) by the presence of polar or unsaturated (e.g. aromatic) groups i n the near proximity of the atoms concerned. The e f f e c t due to the -OH groups w i l l be mostly f e l t by the sugar protons and the a l i p h a t i c protons are not expected to experience i t to any s i g n i f i c a n t extent. On the contrary, the alkyl chains experience a major change i n local environment upon micelle formation since they are now imbedded in the hydrophobic i n t e r i o r of the micelle [5], whilst the sugar head groups s t i l l remain in a hydrophilic environment created by the water of hydration [6]. The above mentioned differences in environment are re f l e c t e d as changes in chemical s h i f t s i n the H^-NMR spectrum. The process of molecular aggregation i s associated with an increase in the rotational c o r r e l a t i o n time (xc) [7] of the molecule, and hence a decrease in the spin-spin relaxation time (T 2) of the nuclei [8]. Since the NMR spectral line-widths are inversely proportional to the spin-spin relaxation time [9], the spectral l i n e s become increasingly broader on formation of micell a r aggregates. S p i n - l a t t i c e relaxation studies: The continuous change in the NMR spectrum of octyl glucoside with increase i n concentration makes i t impossible to determine the cmc by d i r e c t inspection. Thus, i t was contemplated that an in v e s t i g a t i o n of s p i n - l a t t i c e relaxation rates (R-,) of protons would be more rewarding. 53 The values f o r R-| of the w-CHg, H-2 and H-l protons of octyl glucoside determined at d i f f e r e n t concentrations are given in Table 4.1, while the plots of R-j versus inverse t o t a l concentrations (1/C) are shown i n F i g . 4.2. The plots f o r H-2 and w-CH3 show a progressive t r a n s i t i o n around the cmc instead of the sharp d i s c o n t i n u i t y predicted by the phase separation model (Chapter I I , p 19). Nevertheless, the cmc of the system can be determined by extrapolation of the l i n e a r regions of these plots and t h e i r points of in t e r s e c t i o n taken as the cmc. The cmc values so obtained from the data for a>-CH3 and H-2 are 0.023 ± 0.001 M and 0.022 ± 0.002 M; these, within experimental e r r o r , agree well with the values quoted in the l i t e r a t u r e (0.023 M, Ref. 1). As can be seen from F i g . 4.2 , upon micel 1 i z a t i o n the H-2 and co-CHg resonances both show sharp increases in t h e i r R-j values. In contrast, H-l appears to relax at a constant rate ( i . e . unaffected by the phase t r a n s i t i o n ) ; c l e a r l y t h i s merits comment. For the protons of a small molecule undergoing i s o t r o p i c motion i n a d i l u t e solution i n a magnetically i n e r t solvent, there i s substantial experimental evidence to show that the dominant contribution to relaxation arises via the intramolecular dipole-dipole mechanism [10,11], Consequent-l y , the rate of relaxation (R-| ) i s given by 2 2 r i j where, ,YJ• and -,Y • are the gyromagnetic ratios of the two nuclei undergoing rel a x a t i o n , r . . i s the internuclear distance, and T i s the rotational i.J c c o r r e l a t i o n time of the vector between the nuclei i and j . 54 Table 4.1 - S p i n - l a t t i c e relaxation rates, (R-j) of selected protons of octyl glucoside at d i f f e r e n t concentrations in D2O. . .. R, Csee" 1) Concentration . 11.  moles/1 oj-CH? H,, 0.0068 0.36 0.40 1 .33 0.0112 0.36 0.42 1 .39 0.0165 0.35 0.40 1 .34 0.0225 0.41 0.46 1 .35 0.0274 0.49 0.46 1 .31 0.0337 0.61 0.53 1 .47 0.0449 0 i 71 0.58 1 ,42 0.0562 0.76 0.59 1 .35 0.1089 0.92 0.71 1 .42 o o • « • -\ a LU • CO a | o ' v<4 is CH %t A ^a)-CH 3 o I — — £ j B _e_ o o CMC H 1 I I I 0.0 40.0 BO.O 120.0 160.0 200 1 / C L / M 0 L E Figure 4.2: Variation of R1 with inverse total concentration. for w-CHo, H-2 and H-l protons of octyl glucoside. 56 Assuming that intramolecular dipole-dipole relaxation i s exclusive-ly dominant for the solutions studied here, the increase in the value of H-2 and U-CH 3 can be explained by an increase i n rotational c o r r e l a t i o n time (x ) upon m i c e l l i z a t i o n (equation 4.1). It follows from t h i s , that the r a t i o of ^1(micelle) c a n b g t a k e n a s a m e a s u r e 0 f the motional R](monomer) immobilization experienced by the groups in the molecule on formation of m i c e l l e s . The values of t h i s parameter calc u l a t e d , f o r H-2 and oj-CHg are 1.77 and 2.55 res p e c t i v e l y . This c a l c u l a t i o n i s based on the assumption that solutions with concentrations 0.0068 M and 0.1089 M t r u l y represent the motion of a monomer and a m i c e l l a r solution respectively. From those values i t can be in f e r r e d that upon m i c e l l i z a t i o n , the terminal methyl group undergoes immobilization to a greater degree than the sugar head group. The obvious question outstanding at t h i s juncture i s why H-l does not show the same signs of immobilization -upon m i c e l l i z a t i o n as does H-2; given that the sugar ring i s r i g i d , any hindrance to the motion of the sugar moiety should be experienced equally by a l l the sugar protons. This anomalous behaviour of R-| of H-l could be r a t i o n a l i z e d by the changes i n conformation which accompany micelle formation in order to f a c i l i t a t e the close-packing of molecules. If t h i s conformational change was such that the distance between H-l and other protons which contribute to i t s relaxa-t i o n i s increased ( i . e . an increase i n r-^ in equation 4.1), then t h i s would decrease R-|. Moreover, since R-j i s inversely proportional to the si x t h power of the inter-proton distance, a small change i n r would cause a substantial difference i n the relaxation rate (R-|). In t h i s way the ef f e c t of increase i n c o r r e l a t i o n time upon m i c e l l i z a t i o n , on the relaxa-t i o n rate of H-l, would be n u l l i f i e d by the accompanying conformational 57 change. It should also be noted that i t i s not necessary to postulate that a l l molecules within a micelle have undergone conformational change. It i s worthwhile pursuing t h i s explanation further because i t has been demonstrated that for methyl glycosides, the aglycon methyl group protons can contribute as much as 50% to the relaxation of the anomeric (H-l) proton [10]. Therefore the conformation about the two bonds from the g l y c o s i d i c oxygen (C, - 0, and C - 0 J can be of prime importance i n I I a I e s t a b l i s h i n g the relaxation pathways of H-l (Fig. 4.3). Figure 4.3: Stereochemical view of the preferred conformation about the C|-0.j and C -0.| bonds of a glycoside. According to Lemieux and co-workers [12,13], the preferred orientation of the Ca-0^,: bond i s determined by the so c a l l e d exo-anomeric e f f e c t ; the favoured conformation being that which has the aglycon carbon gauche to H-l and the ring oxygen (O5) as shown in the projection I, in Fig. 4.4. As depicted in the projection I I , ( F i g . 4.4) the most favoured conformation about the C'-0, bond would be the one with C, and C in the a 1 1 £ trans arrangement. When a l l t h i s i s combined we have the s i t u a t i o n where one of the protons (H-aA) on the aglycon carbon, i s closest to the anomeric proton (H-l) whilst the other (H-aB) i s in close proximity to the ring 58 oxygen (Og) ( F i g . 4 . 3 ) . It seems reasonable to suggest that t h i s would be the most preferred conformation of octyl glycoside in the monomeric state. This i s indeed supported by the fa c t that H-aA and H-aB have very d i f f e r e n t chemical s h i f t s ( F ig. 4 . 1 [ A ] ) , where H-aB i s more shielded by the i n t e r -action with the lone pairs of electrons of the ring oxygen (Or). Projection along Projection along 0,-C, bond C - 0 , bond 1 1 a 1 Figure 4 . 4 : Conformational projections involving C^  , 0-| and C^ of a glycoside. In the m i c e l l a r state, the conformation of the molecules has to allow for the maximum extent of hydrophobic i n t e r a c t i o n between the mole-cules. This requirement could be expected to override the normal conforma-t i o n a l forces, e s p e c i a l l y when the polar head-group i s rather bulky. In the case of octyl glucoside with the bulky sugar head-group, the molecule might favour a more pianar or a bent conformation; t h i s could be e a s i l y achieved by an increase in the dihedral angle, <l> (Fig. 4 . 3 ) . If t h i s happens, the distance between H - l and H-aA would be increased, causing a decrease in the relaxation contribution to H-l from H - a A . Further, an increase in the dihedral angle would force H-aB to move closer to the ring 59 oxygen thus causing i t to experience a greater s h i e l d i n g e f f e c t from the electron lone p a i r s . This i s r e f l e c t e d in the NMR spectra (Fig. 4.1 [A]-[D]) as an increase in chemical s h i f t of H-aB. The basis for the above argument depends on the assumption that at least the portion of the sugar ring involving H-2 i s r i g i d , so that interproton distances ( r-jj) to H-2 are constant and the change in i t s relaxation rate i s primarily due to the change i n c o r r e l a t i o n time ( T C ) . This could be v e r i f i e d by comparing the coupling constant (J) values for each proton i n the sugar ri n g i n monomer and m i c e l l a r s t a t e s . The change in J values (AJ) measured with the aid of 2D J-resolved NMR spectroscopy (Chapter V) for spectra [A] and [C] ( F i g . 4.1) are given below: J l , 2 J2,3 J3,4 J4,5 AJ 0.2 0.1 0.7 0.9 These rather small changes in J values may not be considered to indicate any s i g n i f i c a n t amount of ring puckering upon mic e l 1 i z a t i o n . An accurate estimate of t h i s would require a careful evaluation of other factors which can influence the J values. Even though the r a t i o n a l i z a t i o n discussed in t h i s section i s not absolutely conclusive i t i l l u s t r a t e s the important deformations in a molecule which can accompany micelle formation; and also the e f f e c t s these can have on spectral parameters. Temperature e f f e c t s : The temperature at which nonionic m i c e l l a r solutions show a sudden increase in t u r b i d i t y i s c a l l e d the cloud point. The size of the 60 surfactant aggregates has been shown to increase rapidly as the temperature i s raised towards t h i s point [14,15], This observation has been interpreted as i n d i c a t i v e of formation of larger micelles or s e l f -aggregation of smaller micelles [15], In an attempt to observe t h i s e f f e c t , 1 H-NMR spectra of mi c e l l a r octyl glucoside solutions were recorded at several temperatures in range the 25-85°'C. None of these spectra showed any substantial difference i n the line-widths or chemical s h i f t s with temperature. The same type of behaviour has been observed by Staples and Tiddy [16] with polyethylene oxide surfactants. Moreover, i t was not possible to observe any changes i n the H^-NMR spectrum of octyl glucoside solutions s l i g h t l y above or below the cmc within the same temperature range. This shows the weak temperature dependence of the cmc of octyl glucoside. 1 3 4.2 - C-NMR studies of octyl glucoside The 100.6 MHz 13C-NMR spectra of octyl glucoside at 0.0112 M and 0.0562 M concentrations corresponding to monomer and mic e l l a r solutions respectively are shown i n F i g . 4.5; the indicated spectral assignments of the sugar moiety are according to that given by Pfeffer et a l . [17]. The change i n chemical s h i f t s (A6) for each resonance between the two spectra are tabulated in Table 4.2 and :a downfield s h i f t in- the : m i c e l l a r spectrum compared to the monomer spectrum i s considered p o s i t i v e . 1 The figu r e accompanying Table 4.2 shows the r e l a t i v e signs of the s h i f t s observed for each carbon. The i n t e r e s t i n g observation i s the opposing trend of the s h i f t d ifferences observed f o r sugar (upfield) and alkyl chain (downfield) portions of the molecule. Precedent, f o r t h i s type of behaviour with d i f f e r e n t head groups can be found in the l i t e r a t u r e [18]. [ B ] °v / C H 2 ^ CH-i X C H , (^CH2>r <5(ppm) Figure 4.5: 1 100.6 MHz proton decoupled ' L NMR spectra in D 20 of [A] monomer (.0.0112 M) and [B] m i c e l l a r (0.0562 M) solutions of octyl glucoside. Experimental parameters: sweep width 20,000 Hz; Acquisition time = 0.8192 s; number of scans = 20,000, temperature = 300°C. 62 1 r> Table 4 . 2 - C chemical s h i f t data for octyl glucoside with s h i f t changes (AS) on micelle formation. Resonance fi(monomer) A<5 (micelle-monomer) C 1 1 0 2 . 9 3 0 . 1 5 C 3 7 6 . 6 2 ± 0 . 0 C c 7 6 . 6 2 - 0 . 1 3 b C 2 7 3 . 9 3 - 0 . 1 4 ( 7 1 . 3 4 - 0 . 3 5 1 4 ( 7 0 . 5 3 - 0 . 2 7 Cc 6 1 . 6 4 - 0 . 1 1 6 C' 3 1 . 7 1 . 0 . 3 7 2 9 . 4 5 0 . 3 0 2 9 . 0 7 : 0 . 5 3 c 1 c 1 c 1 c 3 ' 4 ' 5 ' 6 J 2 8 . 9 6 0 . 5 3 2 5 . 7 2 0 . 3 6 C y 2 2 . 5 9 0 . 2 7 r 1 1 4 . 0 0 0 . 1 4 63 Two p r i n c i p l e mechanisms can be v i s u a l i z e d to cause chemical s h i f t changes of the alkyl chain on micelle formation; "medium e f f e c t s " - i . e . d i r e c t e f f e c t s of the change i n p o l a r i t y of the environment, and "conformational e f f e c t s " - i . e . s h i f t changes caused as a d i r e c t result of a conformational change i n the molecule [19]. Since the m i c e l l a r i n t e r i o r i s hydrophobic [20], the alkyl chain i s expected to undergo a change in environment upon m i c e l l i z a t i o n . But according to Persson et a l . [19] medium e f f e c t s do not cause a sizeable 13 v a r i a t i o n in the C chemical s h i f t s of methylene and methyl carbons i n an alkyl chain. They also have demonstrated that observed s h i f t changes are quite d i f f e r e n t on micelle formation and on t r a n s f e r to an organic solvent. 1 3 It has also been observed separately, that C s h i f t s of carbonyl groups vary strongly with the solvent, whilst the solvent e f f e c t s are small f o r al k y l groups [20], 13 The observed downfield C s h i f t of carbons i n the alkyl chain can be r a t i o n a l i z e d on the basis of possible conformational changes that accompany m i c e l l i z a t i o n . It has been shown that, the t r a n s i t i o n from a gauche to trans conformation of an alkyl chain i s accompanied by a downfield s h i f t of the carbon resonances. This due to release of non-bonded i n t e r a c t i o n s operating between the y-carbons in the gauche conformation ( y e f f e c t ) [21,22]. gauche trans — • downfield s h i f t u p f i e l d s h i f t " ^ 64 A study by Batchelor et a l . [23] provides d i r e c t support for the predominance of conformational e f f e c t s . In th i s study with l e c i t h i n the 13 C s h i f t s of methylene carbons appear 0.3 ppm downfield in v e s i c l e s , i n comparison with l e c i t h i n i n chloroform s o l u t i o n . This result i s interpreted as being due to an increased proportion of alk y l chains with trans conformation when l e c i t h i n i s incorporated into v e s i c l e s . In d i l u t e aqueous solutions the alkyl chain conformation can be expected to a t t a i n a p a r t i a l l y c o i l e d state because of the tendency to reduce the unfavourable hydrocarbon-water contact, while the entropy e f f e c t s should also favour c o i l e d conformations in pure hydrocarbons [25]. Due to geometric constraints operating in a micell e , some alk y l chains should favour a more extended trans conformation [24]. From the data presented i t can be in f e r r e d that micelle formation i s accompanied by an increased proportion of trans conformers in the alkyl chain. The larger s h i f t s observed for carbons i n the middle of the chain suggests that conformational e f f e c t s are more pronounced in the centre of the chain than at either end. The issue of the u p f i e l d 1X s h i f t of the -C0 2 head group has been discussed i n the l i t e r a t u r e [25] as being dominated by the medium e f f e c t s . But the medium e f f e c t s strongly depend on the nature (structure) of the group that i s being observed [20]. In the case of sugar head groups, in d i l u t e solution these are strongly H-bonded in water and any increase or decrease in the H-bonding upon m i c e l l i z a t i o n would be accompanied by a corresponding downfield or an u p f i e l d s h i f t of the carbon resonances. In essence, the degree of H-bonding in the mi c e l l a r state compared to the monomer would determine the s h i f t caused. Another possible r a t i o n a l i z a -1 3 ti o n f o r the observed u p f i e l d C s h i f t s of the sugar resonances, i s the 65 shi e l d i n g e f f e c t created by the neighbouring -OH groups which would be operative i n a mi c e l l e . The p o s s i b i l i t y of the sugar ring undergoing a conformational change during m i c e l l i z a t i o n i s highly u n l i k e l y because of the high energy involved in such a change. In conclusion, the observed 13 u p f i e l d C s h i f t of sugar resonances can originate due to a variety of factors but a d e f i n i t e i n t e r p r e t a t i o n cannot be made at t h i s juncture. 4.3 - H^-NMR Studies of N-alkyllactobionamide series NMR studies with t h i s series of compounds proved to be somewhat of a challenge because of t h e i r low s o l u b i l i t y i n water and e s p e c i a l l y low c r i t i c a l micelle concentrations [26]. P a r t i a l 400 MHz ^ H-NMR spectra; of N-dodecyllactobionamide in 0^0 at 5.0 mM and 0.20 mM concentrations are shown i n Fi g . 4.6 . The spectra were assigned by comparison with the f u l l y assigned spectrum of N-hexyl1actobionamide (Chapter V). The considerable increase i n spectral line-width at higher concentration i s evident. This can be e a s i l y a t t r i b u t e d to the presence of micelles as discussed i n Section 4.1. The cmc of the same compound determined in a previous study [26] (0.28 mM) f a l l s within t h i s concentration range. A s p i n - l a t t i c e relaxation study of t h i s molecule was not undertaken due to the long experimental times involved. Investigation of the spectra of other derivatives in the series showed the following d i f f e r e n c e s . N-Hexyl and N-Octyl derivatives f a i l e d to show any s i g n i f i c a n t l i n e broadening with increase i n concentration upto t h e i r s o l u b i l i t y l i m i t s i n d i c a t i n g t h e i r i n a b i l i t y to assemble into m i c e l l e s . N-Decyl de r i v a t i v e produced some l i n e broadening with increase in concentration, while with N-tetradecyl and N-hexadecyl derivatives the monomer spectra (narrow l i n e s ) could not be obtained even at very low 1 1 I I I I I I U A.O 3.6 3.2 6(ppm) Figure 4.6: 400 MHz ^H NMR spectra of N-dodecyll actobionami de in D,,0. Experimental parameters: sweep width = 2202.6 Hz; Acquisi-tion time = 1.8596 s; number of scans = 8412 (monomer spec-trum), 124 (micelle spectrum), temperature = 290°C, HOD peak presaturated (not shown). 67 concentrations. This indicates that the l a t t e r two derivatives have extremely low cmc values which are not possible to detect even with ]H-NMR. 4.4 - Comments This chapter r e f l e c t s mainly the advantages and l i m i t a t i o n s of using H^-NMR in studying m i c e l l a r solutions. The main advantage of H^-NMR, as pointed out in Chapter II , i s i t s high signal/noise (S/N) s e n s i t i v i t y which makes i t f e a s i b l e to study solutions of sub-mi 11imolar concentration. Nevertheless, i t should be mentioned that with some derivatives encountered i n t h i s study, the work was act u a l l y performed only with l i m i t i n g s e n s i t i v i t y . This study also demonstrates the d i f f e r e n t parameters (chemical s h i f t , coupling constant, line-width and relaxation time), which could be used to provide an ins i g h t to the mole-x u l a r processes involved" in micelle formation. The use of the above para-meters with H^-NMR i s much more convenient than with any other n u c l e i , even though a study of other nuclei can provide additional information. Disadvantages of using H^-NMR have already been discussed in Section 2 . The novel aspect of t h i s study i s that i t has been possible to probe the behaviour of the head group of a class of micelle forming nonionic amphiphiles by ^ H-NMR. The common nonionic detergents that are being studied involve polyethylene oxide surfactants, and a study of t h e i r head groups i s e s p e c i a l l y d i f f i c u l t because of the unresolvable resonances produced by the head group. In t h i s respect, the studies described i n t h i s chapter with a carbohydrate moiety as the head group should a t t r a c t considerable attention. 68 4.5 - Experimental Samples f o r NMR spectra were prepared by accurately weighing and di s s o l v i n g i n 99.7% D 20 (Merck Sharp and Dohme Canada Ltd.) and the spectra were run with reference to external tetramethylsi 1ane (TMS). Al l spectra were recorded at room temperature on a Bruker WH-400 (9.4T) high resolution spectrometer equipped with an Aspect 2000 computer, operating at 400 MHz for and 100.6 MHz for 1 3C at the Department of Chemistry, University of B r i t i s h Columbia. Data were accumulated i n the quadrature detection mode and stored in a disk for subsequent processing. A l l the data were m u l t i p l i e d by an exponential l i n e broadening factor (0.1 1 "I ? Hz for H and 5.0 Hz for l oC) and z e r o - f i l l e d p r i or to Fourier transfor-mation. S p i n - l a t t i c e relaxation times (T|) of protons were determined [27] by using a (180°-T-90°-Acquisition-RD) pulse sequence, where T i s the variable delay between the two pulses and RD, the relaxation delay. The experiments were performed with phase a l t e r a t i o n of the 180° pulse to reduce errors due to any imperfection i n the pulse length and RD was set to ~5T^ or higher. The data f o r , at least eight T- values were c o l l e c t e d f o r each T-| measurement, and the relaxation time was calculated by f i t t i n g the data to the T13IR data reduction routine included in the Nicolet NTCFTB (NMR) program. Given the data f o r the i n t e n s i t y of the signal and time, the TI31R program calculates the T-| without assuming a perfect 180° pulse and indicates how close t h i s inversion pulse was to i t s correct value [28]. For a l l the T-| values calculated the accuracy of the 180° was found to be within the acceptable range. In the case of a mul t i p l e t , the t o t a l i n t e n s i t y of the multiplet was plotted against time. From the T-| values, the relaxation rate (R-j) was calculated using the rel a t i o n s h i p R-| = VT, and the values given are reproducible to within better than ±5%. 69 References 1. De Grip, W.J., Bovee-Geurts, P.H.M., Chem. Phys. L i p i d s , (1979), 23, 321. 2. Shinoda, K., Yamaguchi, T., Hori, R., Bui 1. Chem. Soc. Jpn., (1961), 34, 239. 3. Helenius, A., Simons, K., Biochim. Biophys. Acta., (1975) 415, 29. 4. Wennerstrom, H., Lindman, B., Phys. Rep., (1979), 5_2, 33. 5. Wennerstrom, H., Lindman, B., i b i d . , p. 60. 6. Wennerstrom, H., Lindman, B., i b i d . , p. 69. 7. Shaw, D., "Fourier Transform N.M.R. Spectroscopy", E l s e v i e r , Amsterdam, 1976, p. 300. 8. Shaw, D., i b i d . , p. 306. 9. Shaw, D., i b i d . , p. 9. 10. Preston, CM., H a l l , L.D., Carbohydr. Res., (1974), 37_> 267. 11. H a l l , L.D., Chem. in Canada, (1976), 28, 19. 12. Lemieux, R.U., Pavia, A.A., Martin, J.L., Watanabe, K.A., Can. J .  Chem., (1969), 47, 4427. 13. Lemieux, R.U., Koto, S., Tetrahedron, (1974), 30., 1933. 14. Tanford, C , Nozaki , Y.U., Rohde, M.F., J . Phys. Chem., (1977), 81, 1555. 15. Atwood, D., J . Phys. Chem. , (1968), 7_2, 339. 16. Staples, E.J., Tiddy, G.J.T., J . Chem. Soc. Faraday I, (1978), 74, 2530. 17. P f e f f e r , P.E., Valentine, K.M., Pa r r i s h , F.W., J . Am. Chem. S o c , (1979), 101, 1265. 70 18. Persson, B., Drakenberg, T., Lindman, B., J . Phys. Chem., (1979), 83, 3011. 19. Persson, B., Drakenberg, T., Lindman, B., J . Phys. Chem., (1976), 80, 2124. 20. Stothers, J . , "Carbon-13 NMR Spectroscopy" i n "Organic Chemistry, A Series of Monographs", Vol. 24, Academic Press, New York, 1972. 21. Cheney, B.V., Grant, D.M., J . Am. Chem. S o c , (1967), 89, 5319. 22. Cheney, B.V., Grant, D.M., i b i d . , p. 5315. 23. Batchelor, J.G., Prestegard, J.H., Cushley, R.J., Lipsky, S.R., Biochem. Biophys. Res. Commun., (1972), 4_8, 70. 24. Lindman, B., Wennerstrom, H., in "Topics i n Current Chemistry", Vol. 87, Micelles: Springer-Verlag, New York, 1980, p. 49. 25. Rosenholm, J.B., Drakenberg, T., Lindman, B., J . C o l l o i d Interface  S c i . (1978), 63, 538. 26. Williams, T.J., Plessas, N.R., Goldstein, I.J., Arch. Biochem.  Biophys., (1977), 470, 325. 27. Martin, M.L., Martin, G.J., Delpuech, J . - J . , " P r a c t i c a l NMR Spectros-copy", Heyden and Sons Ltd., 1980, p. 244. 28. Levy, G., Peat, I., J . Magn. Reson., (1975), 18, 500. CHAPTER V TWO DIMENSIONAL FOURIER TRANSFORM NMR SPECTROSCOPY 72 5.1 - Introduction The concept of two-dimensional Fourier transformation was f i r s t proposed by Jeener [1] i n 1971, but i t ' s widespread s i g n i f i c a n c e was not re a l i z e d u n t i l several years l a t e r . The f i r s t NMR experiments using t h i s technique were published i n 1975 [2,3], to be followed l a t e r by a de t a i l e d t h e o r e t i c a l analysis [4], which l a i d the foundation f o r the subsequent development i n t h i s area. Comprehensive reviews [5,6] on two-dimensional NMR (2D-NMR) spectroscopy have been published, and a more recent a r t i c l e [7] deals with i t s b i o l o g i c a l a p p l i c a t i o n s . It w i l l be r e c a l l e d that i n a conventional Fourier transform NMR experiment, the nuclear spins are excited by application of a single radio frequency pulse, and t h e i r responses are measured during a sing l e time period ( t ^ ) , the a c q u i s i t i o n time. Subsequent Fourier transformation of t h i s time domain data set, s(^)» produces the frequency domain NMR spectrum, S(F_2) [8]. Insofar that t h i s displays a l l the frequency dependent parameters along a single frequency axis, t h i s can be regarded as a one-dimensional NMR (1D-NMR) experiment. B r i e f l y , the 2D-NMR technique involves, c o l l e c t i o n of a data matrix s(t_-|, t ^ ) , as a function of two independent time domains (;t-| and t_ 2 ) , followed by double Fourier transformation. The r e s u l t i n g 2D spectrum S(F_-|> contains one i n t e n s i t y axis and two frequency axes (F^ and £_,,). A large variety of experiments -is possible depending on which perturba-t i o n s (frequency or phase of the RF ra d i a t i o n , decoupler l e v e l , etc.) are applied to the nuclear spins during the time i n t e r v a l s t^ and t ^ . Thus i t i s important to recognize that d i f f e r e n t types of information can be derived from d i f f e r e n t types of 2D experiments. At present, the 2D experiments which are of p r i n c i p l e importance 73 to the p r a c t i c i n g chemist can be b a s i c a l l y divided i n t o two categories [8], 1. 2D-Resolved NMR Spectroscopy: homonuclear or heteronuclear J-resolved NMR [9-12], chemical s h i f t resolved NMR [13-15], dipolar resolved NMR in s o l i d s [16,17]. 2. 2D-Correlated NMR Spectroscopy: autocorrelation through J-coupling [4,7], autocorrelation through dynamic processes such as chemical exchange or Overhauser enhancement [18,19]. 2D-Resolved NMR, s i m p l i f i e s complex spectra by spreading the t r a n s i t i o n s of a conventional ID spectrum into a second dimension. Examples of spreading parameters are scalar coupling constants (J_-resolved NMR), chemical s h i f t s (chemical s h i f t resolved NMR) or dipolar coupling constants (dipolar resolved NMR). In J_-resolved and dipolar resolved NMR, one of the frequency axes, ( F ^ ) , in the 2D spectrum corresponds to chemical s h i f t s while the other, (F_y), consists of scalar coupling (Jj or dip o l a r coupling constants. In chemical s h i f t resolved NMR, both axes and F_2 represent chemical s h i f t s but of two d i f f e r e n t nuclei (e.g. and 1 3 C). This i s summarized in F i g . 5.1. \H J_-Resolved technique has become one of the most powerful and commonly used 2D-NMR experiments since i t often provides unprecedented dispersion of H^-NMR spectra and helps to resolve overlapping resonances. This leads to a wealth of information which can only be obtained with extreme d i f f i c u l t y from a normal ID spectrum of a complex molecule. It has already been proved to be valuable in the analysis of H^-NMR spectra of oligosaccharides [13,20], steroids [21,22], and peptides [23,24]. 74, [A] [B] [C] [D] J - A * D Figure 5.1; Schematic representation of d i f f e r e n t 2D resolved NMR experiments. [A] - Homonuclear J-resolved NMR [B] - Heteronuclear J-resolved NMR [C] - Dipolar resolved NMR [D] - Chemical s h i f t resolved NMR 5 - chemical s h i f t ; J - s c a l a r coupling constant; D - d i p o l a r coupling constant. A and X represent d i f f e r e n t nuclides. 75 Heteronuclear J_-resolved spectroscopy has been used to determine C- H 13 13 coupling constants [12,25], C- C coupling constants [26] as well as 1 31 that of H- P [27,28]. Chemical s h i f t resolved ( s h i f t c o r r e l a t i o n ) 13 1 spectroscopy has been applied to obtain the co r r e l a t i o n between C and H resonances [13,29]. This enables one to i d e n t i f y the protons attached to a p a r t i c u l a r carbon and to make additional assignments in individual spectra. The two common c o r r e l a t i o n experiments now in use are,2D-Correl a-t i o n Spectroscopy (COSY) [30-32] and Spin-Echo Correlated Spectroscopy (SECSY) [24,33,34]. In both experiments the" c o r r e l a t i o n i s obtained through ^-coup-l i n g and thus provide i d e n t i c a l information [33]. These techniques are extremely useful i n determining the connectivity of the resonances in complex spectra, and t h e i r application to oligosaccharides and peptides have already been demonstrated. In a COSY spectrum, both F_-j and F_2 frequency axes represent chemical s h i f t s of the same nuclei and the normal spectrum i s displayed along the diagonal of the spectrum [ F i g . 5.2(A)]. Moreover, the spin-coupled multiplets give r i s e to off-diagonal responses (correlated peaks) at the points corresponding to the chemical s h i f t s of coupled n u c l e i . In • a SECSY spectrum, the F_^  axis corresponds to one half of the difference i n chemical s h i f t s (A6/2 ) of the spin coupled n u c l e i , whereas F_2 corresponds to the normal chemical s h i f t a x i s . Here, the normal spectrum i s displayed at the zero frequency of the F_^  axis [ F i g . 5.2(B)], and the c o n n e c t i v i t i e s of spin coupled multiplets. are indicated by.the responses which appear on ei t h e r side of the spectrum. The l i n e s showing the c o n n e c t i v i t i e s are aligned at an angle of 135° to the F_2 a x 1 s « The general area of 2D-NMR, also includes multiple quantum 76 Figure 5.2: I l l u s t r a t i o n of COSY(A) and SECSY(B) s p e c t r a . 77 t r a n s i t i o n detection [4,35,36], zeumatography [37] and heteronuclear cross-relaxation [38] experiments, to name just a few. This chapter i l l u s t r a t e s the a p p l i c a t i o n of ^H J-resolved spectroscopy and SECSY in the analysis of 1 H NMR spectra of two model g l y c o l i p i d s , syntheses of which were described i n Chapter I I . 5.2 - Description of homonuclear J-resolved and SECSY experiments 5.2.1 - Pulse sequences and data a c q u i s i t i o n Homonuclear J-resolved spectroscopy i s a variant of c l a s s i c a l Carr-Purcell spin echo experiment [39], in which the 90° pulse used to perturb the nuclear spins i s followed by a 180° pulse, a f t e r a certain delay time (t,, ). The pulse sequence for the J-resolved experiment can be 2 represented by {90° - t. - 180° - t, - A c q u i s i t i o n ( t ') - Relaxation delay (RD)} where n i s the number of a c q u i s i t i o n s ; t h i s can be diagramatically represented as shown below in Fig. 5.3. 90°pulse 180° pulse spin-echo Defocussing Refocussing Acquisition interval interval tj 2 2 Evolution period Detection period t2 Figure 5.3: Pulse sequence for 2D homonuclear J-resolved experiment. 78 In a J_-resolved experiment a seri e s of Carr-Purcell pulse sequences is performed, in which the evolution time (t^) i s incremented by a constant value (At^), so that, l l = k.At,; where k = 0, 1, (N -1) and = number of experiments performed. In each case only the refocussed signal i s acquired ( Fig. 5.3) as a function of time ( t ^ ) . This half-echo can be treated as i f i t were a conventional free induction decay and the parameters for the corresponding frequency domain (F_2, which contains chemical s h i f t information) are set in the normal manner. The increment in (At-]) determines the spectral width (SW2) in the corresponding frequency domain (F^,, which contains coupling constant information), according to the equation, SW-j = 2{yt~ ' T^e n u m ' 3 e r °^ experiments performed, N-j, corresponds to the number of points in £_-[, r e s u l t i n g in a d i g i t a l resolution of 1/At.^N1 or in the F^ domain. In practice the data are accumulated and stored by a computer as a s t r i n g of consecutive f i l e s on a disk, f or subsequent data processing. The pulse sequence for the SECSY experiment follows the same pattern as that for the J-resolved experiment except that the second pulse i s a 90°' pulse as indicated below and in Fig. 5.4(A). {90° - t i - 90° - t] - Acquisition ( t 9 ) - Relaxation delay} 2 2 L n The data a c q u i s i t i o n f o r the SECSY experiment involves the same procedure as used in the J-resolved experiment. Figure 5.4 also depicts the pulse sequence for the COSY experiment where the a c q u i s i t i o n begins immediately a f t e r the second 90° pulse. 9 0 ° pulse 9 0 ° pulse 79 [A] I tl 2 2 Acquisit ion (t, 90° pulse 9 0 ° pulse [fi] Acquisit ion (t2 Figure 5.4: Pulse sequences for SECSY[A] and COSY[B] experiments. 80 5.2.2 - Data processing [20,40,41] The data processing procedures f o r J_-resolved and SECSY experi-ments follow the same scheme which i s summarized in Fig. 5.5, except that the ' t i l t ' routine at the f i n a l step i s omitted i n the SECSY experiment. The i n i t i a l 2D time domain data matrix s(t_-|, t_2) consists of N-| number of free induction decays ( f . i . d ) . Each of these f . i . d ' s i s f i r s t subjected to Fourier transformation with respect to t_2 to produce a data matrix s(t_^, F_2); this..consists of a set of phase modulated spectra, the phase of which depends on the t_^  value. For weakly coupled spin systems the phase v a r i a t i o n with t^ for a p a r t i c u l a r t r a n s i t i o n depends not on the chemical s h i f t , but only on the multiplet structure corresponding to that p a r t i c u l a r t r a n s i t i o n . This fa c t i s u t i l i z e d to d i f f e r e n t i a t e between resonance l i n e s with d i f f e r e n t multiplet s p l i t t i n g s by means of a second Fourier transformation. To accomplish t h i s , the s(t_ n, F_2) data matrix i s f i r s t transposed to produce the s(F_ 2 > t^) matrix, and then Fourier transformed with respect to jb to produce the frequency domain data set S(F_ 2 > F^). Transposition of S(F_2, F^) y i e l d s the S(F_^, F_2) data matrix which corresponds to the (untilted) J-resolved spectrum, or the SECSY spectrum. The peaks i n the J_-resolved spectrum at t h i s stage are aligned at angle of 45° providing that the scales of the F_ and F_2 axes are the same. Projection of t h i s (untilted) spectrum onto the £ 2 axis y i e l d s the normal spectrum and the projection at an angle of 45° gives the equivalent of a "homonuclear broad-band decoupled spectrum", with a l l the coupling constant information suppressed ( F i g . 5.6). A more useful display of the J-resolved spectrum i s obtained a f t e r i t has been " t i l t e d " by an angle of 45° so that a l l the t r a n s i t i o n s of a p a r t i c u l a r multiplet l i e s p a r a l l e l to the _F_1 axis (Fig. 5.6). This t i l t routine eliminates the p o s s i b i l i t y of 81 2D TIME SIGNAL ,t 2 ) N ] f . i . d ' s s t 1 FT 2D SPECTRUM (TO BE TRANSPOSED) (Real) |( Imaginary) 1 . 2 n d FT F l N , l 2N 1 1. Absolute Va!ue 2. Transposition 2D SPECTRUM (UNTILTED) SCF-, ,F 2) TILT by 45° PHASE MODULATED SPECTRA s ( t j , f 2 ) N, SPECTRA WITH I • PHASE MODULATIONS DEPENDING ON t, (Real) •(Imaginary) N2'-2 Transposition 2 Sffg.ty) N 2 INTERFEROORAMS .. 2 ; (Real) |(Imaginary) "1 2N, 2D SPECTRUM (TILTED) S ( F r £ 2 ' ) F ?' (Chemical L s h i f t ) Figure 5.5; Summary of the data manipulation procedure in 2D-experiments 82 f2(S,J) f2'(S) Figure 5.6: I l l u s t r a t i o n of various display modes and the t i l t routine used in proton 2D J-resolved spectroscopy. The ( u n t i l t e d ) J-resolved spectrum [A] when projected onto the £ 2 axis y i e l d s the normal spectrum (a). A 45° projection gives a proton-decoupled-proton spectrum (b). Projection of a section of the 2D spectrum [B] onto axis.gives the J^  spectrum of the quartet. S i m i l a r l y , projection of [C] y i e l d s a trace with the t r i p l e t s superimposed (d). T i l t i n g the J-resolved spectrum by 45° y i e l d s the S ( 6 , J) matrix [D]. Cross sections of [D] at the respective chemical s h i f t s give the J spectra of the t r i p l e t s (from Ref. 20). 83 overlapping between the t r a n s i t i o n s of chemically s h i f t e d resonances, when the projections onto £-| are taken. The projection of the t i l t e d spectrum onto ¥_2 produces the "decoupled spectrum" and the multiplet structure f o r each resonance (J spectra) can be obtained by taking the trace at the chemical s h i f t p a r a l l e l to F i across the whole F_-| dimension, and projecting i t onto F_^ . The J_ spectra so obtained are symmetrical about the centre. This i s i l l u s t r a t e d i n F i g . 5.6. 5.3 - Application of 2D-spectroscopy in spectral assignment of unprotected sugars 5.3.1 - n-Octyl g-D-glucopyranoside (I) The p a r t i a l 400 MHz H^ spectrum of n-octyl 3-D-g1ucopyranoside in D^ O i s shown in Fig. 5.7(A). The overlapping of multiplets of d i f f e r e n t protons precludes unequivocal spectral assignment by conventional techniques such as decoupling and matching of spectral s p l i t t i n g s . Furthermore, i t makes the determination of chemical s h i f t s and coupling constants of the protons involved almost an impossible task. These problems can be e a s i l y overcome through 1 H J_-resol ved spectroscopy. The proton-decoupled-proton spectrum obtained by the projection of the t i l t e d 2D J-resolved spectrum onto ^ ( c h e m i c a l s h i f t ) axis i s shown i n F i g . 5.7(B). As can be seen, the spectrum i s greatly s i m p l i f i e d by the supression of the coupling constant information and each peak in the spectrum appear at the corresponding chemical s h i f t value thus making i t s determination a t r i v i a l matter. The coupling information i s extracted by obtaining cross-sections across the t i l t e d 2D J-resolved spectrum (J spectra) at the chemical s h i f t values indicated i n the proton-decoupled-proton spectrum; these are shown -T 1 » » 1 r — r 4 4 40 3-6 6 (ppm) 3-2 Figure 5.7: Partial 400 MHz 'H ID and 2D NMR spectra of n-octyl 6-D-glucopyranoside 0.0112 M in D20. [A] - The normal spectrum. Sweep width 1000.0 Hz, Acq u i s i t i o n time = 2.04 s, Data s i z e = 4K, Relaxation delay = 0.5 s, Number of scans = 30. [B] - The proton-decoupled proton spectrum of the same region. +SW2 = 1000.0 Hz, SW] =15.6 Hz, N 2 = 4K and N] =128 (a f t e r zero-f i l l i n g ) , Total acqu i s i t i o n time =5.6 hours. [ H - 6 A X A H-1 u 6A,B 5,6 A H-3 H-5 J u U F i g u r e 5.8: I n d i v i d u a l J s p e c t r a o f n - o c t y l s p e c t r u m . 10 Hz I - I H-6B H- ocfi h K H-2 JUUL, JUUL, - D - g l u c o p y r a n o s i d e o b t a i n e d f rom 2D J - r e s o l v e d 86 Table 5,1.: Proton chemical s h i f t and coupling constant data of the sugar region of n-octyl g-D-glucopyranoside, Chemical s h i f t s , ppm Coupling constants, Hz Glucose ring: H-l H-2 H-3 H-4 H-5 H-6A H-6B 4.44 3.25 3.47 3.37 3.45 3.91 3.71 J = 8.1 J 2 j 3 = 9 . 6 J3,4 = 9 - ° J4,5 = 9 - 8 J5,6A = 2 J J5,6B - 6 - ° J6A,6B = n - 6 A l i p h a t i c chain: H-aA H-aB 3.91 3.68 J n D = 9.9 aA,aB 87 in F i g . 5.8. With the chemical s h i f t and coupling information i n hand, the assignment of the resonances could be accomplished by matching of individual coupling constants. The individual assignments of H-3 and H-4 were confirmed by careful decoupling of the H-2 resonance. The' assignments are indicated i n F i g . 5.7(B) and the chemical s h i f t and coupling constant values are tabulated in Table 5.1. At t h i s juncture, i t i s appropriate to comment on some features displayed in the spectra. Strong coupling e f f e c t s present a serious l i m i t a t i o n in 2D-spectroscopy [4,33,41] as in lD-spectroscopy. In 2D-spectroscopy, strong coupling gives r i s e to additional l i n e s of low inten-s i t y which appear inbetween the chemical s h i f t s of the strongly coupled peaks. The spectra of the additional peaks a r i s i n g due to strong coup-l i n g show an unsymmetrical pattern along F_^ , which helps in t h e i r i d e n t i f i c a t i o n . For that matter, a l l the a r t i f a c t s in a proton-decoupled-proton spectrum can be i d e n t i f i e d i n a s i m i l a r manner. The H-3, H-4 and H-5 resonances form a strongly coupled system in which the difference i n t h e i r chemical s h i f t s are i n the same order of magnitude of the coupling constants. The a r t i f a c t s i n the 2D projection due to strong coupling e f f e c t s are indicated with an asterisk in F i g . 5.7 (B). In the normal spectrum the multiplet at 3.92 6 due to H-aA and H-6A appear as a single resonance in the proton-decoupled-proton spectrum, i n d i c a t i n g that these two protons have i d e n t i c a l chemical s h i f t s . Hence, the cross-section (J_ spectrum) at t h i s chemical s h i f t includes the multip-l e t structures of both protons which can be e a s i l y unscrambled as shown by the s t i c k diagram. The spectrum of H-5 indicates i t s complex m u l t i p l i c i t y , which i s not e a s i l y seen in the ID spectrum. Also the H-4 resonance appears as a 88 four l i n e pattern (double doublet) i n the J_ spectrum whereas only three l i n e s could be seen in the normal spectrum. This i s due to the inherent enhanced resolution i n the J_ dimension (F_j axis) obtained i n .J-resolved spectroscopy since, the observed line-width in t h i s dimension has no contribution from the inhomogeneities of magnetic f i e l d . 5.3.2- N-Hexyllactobionamide (II) Assignment of the sugar region i n t he^ H NMR spectrum of the disaccharide d e r i v a t i v e was achieved by a combination of 2D methods: J-resolved spectroscopy and spin-echo correlated spectroscopy (SECSY). The normal and the proton-decoupled-proton spectrum of the sugar region of N-hexyllactobionamide (II) i s shown in F i g . 5.9 along with the J_-traces. The ' t i l t e d ' J-resolved spectra and the SECSY spectra of the same region p l o t t e d as contour diagrams, where the spectrum i s viewed down the i n t e n s i t y axis, are shown in Fig. 5.10 and 5.11 respectively. An expan-sion of the dotted region of the SECSY spectrum i s shown in F i g . 5.12. The molecule II contains 13 sugar protons and t h e i r numbering i s as indicated above. By inspection of SECSY spectra, the positions of the H-l through H-4 protons in the galactose ''residue as well as that of a l l the protons i n the glucose (open chain) residue can be determined. The 89 H-6A T 1 1 1 r t 1 t I I I " . 4-4 4 0 3-6 6(ppm) Figure 5.9: P a r t i a l 400 MHz ]H ID and 2D NMR spectra of N-hexyl1acto-nionamide 0.025 M in D^ O. [A] - The normal spectrum. Sweep width = 2202.6 Hz, Acquisition time 1.86 s, Data size = 8K, Relaxation del ay = 1 s, Number of scans = 24. [B] - The proton-decoupled-proton spectrum of the same region. ±SU^ = 750.7 Hz, SW-j = 23.4 Hz, N g = 4K and N., = 256 ( a f t e r z e r o f i l l ing). Total a c q u i s i t i o n time = 8.7 hours. Upper trace - J spectra of individual m u l t i p l e t s . 90 [B] U-U LO 3.6 S/ppm Figure 5.10: P a r t i a l 400 MHz H 2D NMR spectra of N-hexyl1actobionamide 0.025 M in D20. [A] - A contour diagram of the 2D 3-resolved spectrum. [B] - The proton-decoupled-proton spectrum (same as in F i g . 5.9). See Fig. 5.9 for experi-mental parameters. 91 H-6A£J3'B 5 ' » > 4-5 4.0 3-5 S/ppm Figure 5.11: P a r t i a l 400 MHz SECSY spectrum of N-hexyl1actobionamide 0.025 M in D20. The lines j o i n i n g the correlated peaks show the c o n n e c t i v i t i e s . SW2 = 750,7 Hz, iSW-, = 250.0. Hz, N 2 = 4096 and = 256 [ a f t e r z e r o f i l l i n g ) . Upper trace shows the normal spectrum of the same region. Figure 5.12: P a r t i a l SECSY spectrum of N-hexyllactobionamide: an expansion of the dotted region of Fig. 5,11. 93 c o n n e c t i v i t i e s of these spin coupled protons are shown in F i g . 5.11 and 5.12. The c o n n e c t i v i t i e s of H-5, H-6A and H-6B of the galactose residue cannot be seen in the SECSY since they appear i n the crowded region between 3.71 - 3.85 <5 and form a very strongly coupled spin system. It can be seen that H-6'B also appears at the l o w f i e l d end of the above crowded region. The proton-decoupl ed-proton spectrum shows well resolved peaks f or majority of protons and hence t h e i r chemical s h i f t s and coupling constants can be derived without d i f f i c u l t y . The multiplet due to H-4 and H-5' appear as a sing l e peak in the "decoupled" spectrum showing that they have i d e n t i c a l chemical s h i f t s . The H-5 resonance appears as a low i n t e n s i t y peak amidst the second order a r t i f a c t s , but could be i d e n t i f i e d by i t s c h a r a c t e r i s t i c 8 l i n e pattern i n the J_ spectrum. In the proton decoupled proton spectrum the four H-6 protons appear at 3.87, 3.80, 3.78 and 3.76 £ , and the resonances at 3.87 and 3.76 6 could be assigned to H-6'A and H-6'B on the basis of the c o n n e c t i v i t i e s shown i n the SECSY spectrum. This leaves the resonances at 3.80 and 3.78 <5 for the H-6A and H-6B. Obviously, these protons are strongly coupled and accounts f o r the complex J-spectrum observed f o r H-6B. Thus a l l chemical s h i f t s and coupling constants for ind i v i d u a l sugar protons can be determined and the data are tabulated i n Table 5.2. 5.4 - Experimental The NMR spectra were recorded with reference to external t e t r a -methylsi lane, on a Bruker WH-400 (9.4 T) high resolution spectrometer equipped with an Aspect 2000 computer. The Bruker FTNMR-2D software program (version # 810515) was used to acquire the 2D data. 94 Table 5,2- Proton chemical s h i f t and coupling constant data of the sugar resonances of N-h.exyll actobionami de. Chemical s h i f t s , Coupling constants, ppm Hz Galactose r i n g : H-l 4.57 J 1 2 = 7.7 H-2 3.57 J 2 3 = 9.8 H-3 3.67 J 3 4 = 3.3 H-4 3.94 J 4 5 = 1 .2 H-5 3.72 J 5 j 6 A = 8.0 H-6A 3.80 J 5 j 6 B ; = 5.2 H-6B 3.78 J 6 A , 6 B = 1 1 - 7 Glucose open chain: H-2' 4.39 J 2 , 3 , = 2.8 H-3' 4.17 J 3 , 4 , = 4.2 H-4' 3.97 J 4 , g l = 6.5 H-5' 3.94 J 5 ' , 6 ' A = 3 - 2 H-6A' 3.87 J 5 , 5 , B = 6.57 H-6'B 3.76 J6'A,6'B = ] 1 ' 7 95 The sweep width (SW^) i n the dimension was set to include only the spectral region desired (~1000 Hz) and the data were acquired on a block s i z e , t y p i c a l l y , of 4K r e s u l t i n g in a d i g i t a l resolution of 0.48 Hz/PT. The number of points acquired in the dimension (N-j) usually corresponded to 64 or 128. The sweep width (SW2) in the F_-j dimension f or J_-resolved experiments was set to a value so as to include the widest m u l t i p l e t . The d i g i t a l resolution obtained i n t h i s dimension t y p i c a l l y corresponded to 0.28 Hz/PT. In SECSY, the SW^  was set so as to cover half the difference in chemical s h i f t s of the farthest apart spin coupled n u c l e i . The time required for data a c q u i s i t i o n usually amounted to 4 ' - 8 hours depending on the concentration of the s o l u t i o n . A l l the 2D data processing stages are automated in the program used. The data were z e r o - f i l l e d and m u l t i p l i e d by sine resolution enhancement function before Fourier transformation i n each dimension. The data processing and subsequent p l o t t i n g t y p i c a l l y required about 4 hours. 96 References 1. Jeener, J . , Ampere International Summer School, Basko P o l j e , Yugoslavia, A p r i l , 1971. 2. Ernst, R., Chimi, (1975), 29, 179. 3. Muller, L., Kumar, A., Ernst, R.R., J . Chem. Phys., (1975), 63, 5490. 4. Aue, W. P., Bartholdi, E., Ernst, R.R., J . Chem. Phys., (1976), 64, 2229. 5. Freeman, R., Morris, G.A., Bui 1. Mag. Reson., (1979), 1_, 5. 6. Freeman, R., Proc. R. Soc. Lond. A, (1980), 373, 149. 7. Nagayama, K., Adv. Biophys., (1981), 14, 139. 8. Shaw, D., Fourier Transform N.M.R. Spectroscopy, E l s e v i e r , Amsterdam, 1976. 9. Aue, W.P., Karhan, J . , Ernst, R.R., J . Chem. Phys., (1976), 64, 4226. 10. Nagayama, K., Wuthrich, K., Bachmann, P., Ernst, R.R., Biochem. . Biophys. Res. Commun., (1977), 78, 99. 11. H a l l , L.D., Sukumar, S., J . Am. Chem. S o c , (1979), HU, 3120. 12. H a l l , L.D., Morris, G.A., Carbohydr. Res., (1980), 82, 175 and references c i t e d t h e r e i n . 13. H a l l , L.D., Morris, G.A., Sukumar, S., J . Am. Chem. S o c , (1980), 102, 1745. 14. Muller, L., Kumar, A., Ernst, R.R., J . Chem. Phys., (1975), 63, 5490. 15. Bolton, P.H., Bodenhausen, G., J . Am. Chem. S o c , (1979), 101, 1080 and references c i t e d t h e r e i n . 16. S t o l l , M.E., Vega, A.J., Vaughan, R.W., J . Chem. Phys., (1976), 65_, 4093. 17. Opella, S.J., Waugh, J.S., J . Chem. Phys., (1977), 66, 4919. 97 18. Meier, B.H., Ernst, R.R., J . Am. Chem. S o c , (1979), 101, 6441. 19. Jeener, J . , Meier, B.H., Bachmann, P., Ernst, R.R., J . Chem. Phys., (1979) , ]±, 4546. 20. Sukumar, S., Ph.D. Thesis, University of B r i t i s h Columbia, Vancouver, 1979. 21. H a l l , L.D., Sanders, J.K.M., J . Am. Chem. S o c , (1980), 1_02, 5703. 22. H a l l , L.D., Sanders, J.K.M., J . Org. Chem., (1981), 46, 1132. 23. Nagayama, K., Bachmann, P., Wuthrich, K., Ernst, R.R., J . Magn.  Reson., (1978), 31, 133. 24. Nagayama, K., Wuthrich, K., Eur. J . Biochem., (1981), 114, 365. 25. Turner, D.L., Freeman, R., J . Magn. Reson., (1978), ^9, 587. 26. Niedermeyer, R., Freeman, R., J . Magn. Reson., (1978), 617, 30. 27. Bodenhausen, G., J . Magn. Reson., (1980), 39, 175. 28. Bolton, P.H., J . Magn, Reson., (1981), 45, 239. 29. Bodenhausen, G., Freeman, R., J . Am. Chem. Soc., (1978), 100, 320. 30. Wagner, G., Kumar, A., Wuthrich, K., Eur. J . Biochem., (1981), 114, 375. 31. Bax, A., Freeman, R., J . Magn. Reson., (1981), 44, 542. 32. Bernstein, M.A., H a l l , L.D., Sukumar, S., Carbohydr. Res., in press. 33. Nagayama, K., Kumar, A., Wuthrich, K., Ernst, R.R., J . Magn. Reson., (1980) , 40, 321. 34. Nagayama, K., Wuthrich, K., Ernst, R.R., Biochem. Biophys. Res.  Commun., (1979), 90, 305. 35. Bodenhausen, G., Void, R.L., Void, R.R., J . Magn. Reson., (1980), 37_, 93. 36. Pouzard, G., Sukumar, S., H a l l , L.D., J . Am. Chem. S o c , (1981), 103, 4209. 98 37. Kumar, A., Welti, D., Ernst, R.R., J . Magn. Reson., (1975), 18, 69. 38. Bodenhausen, G., Freeman, R., J . Am. Chem. S o c , (1978), 100, 320. 38. Carr, H.Y., P u r c e l l , E.M., Phys. Rev., (1954), 94_, 630. 39. Bodenhausen, G., Freeman, R., Niedermeyer, R., Turner, D.L., J . Magn.  Reson., (1977), 26, 133. 40. Nagayama, K., Bachmann, P., Wuthrich, K., Ernst, R.R., J . Magn.  Reson., (1978), 31, 133. 41. Bodenhausen, G., Freeman, R., Morris, G.A., Turner, O.L., J . Magn.  Reson., (1978), 31, 75. 

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