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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.), U n i v e r s i t y o f 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 o f Chemistry)  We a c c e p t t h i s  t h e s i s as conforming  to the r e q u i r e d  standard  THE UNIVERSITY;-OF BRITISH COLUMBIA May 1982 ©  Sardha L a i i t h  Talagala  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head o f  representatives.  my  It i s  understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  C#£W-g7#/  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  n/sn  =1/ %~ «ft*n£  written  ii  ABSTRACT  The work described herein f a l l s synthesis  into three major categories:  of model g l y c o l i p i d s , NMR studies on'model g l y c o l i p i d ' . m i c e l l e s ,  and application of 2D-NMR'. spectroscopy in spectral assignment. Three synthetic routes, namely the glycosidation r e a c t i o n , reductive amination reaction, and amide bond formation have been investigated in relation to their e f f i c i e n c y and convenience in coupling carbohydrates with a l i p h a t i c 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 u t i l i z e d to study and detect the micelle formation by the model g l y c o l i p i d s . i l l u s t r a t e that the  The studies described  s p i n - l a t t i c e relaxation rate (R-j) is well suited for  the determination of c r i t i c a l micelle concentration providing i t is s u f f i c i e n t l y 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 m i c e l l i z a t i o n , has been tentatively attributed to the conformational changes accompanying micelle formation.  The observed upfield s h i f t of the  1 3  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 discussed.  C s h i f t observed for the sugar resonances has been  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 c a l micelle concentrations. Application of 2 D J-resolved spectroscopy and spin-echo correlated  i i i  s p e c t r o s c o p y (SECSY) i'n s p e c t r a l v a t i v e s has  b.een demonstrated.  o f the sugar r e g i o n o f n - o c t y l been a c h i e v e d .  assignment o f u n p r o t e c t e d sugar Using above t e c h n i q u e s , complete  deriassignment  g-Q-glucoside and M-hexyllactobionamide  has  iv TABLE OF CONTENTS Page No. CHAPTER I  -  GENERAL BACKGROUND  1  1.1  -  B i o c h e m i c a l aspects  2  1.2  -  Structures o f natural  1.3  -  Behaviour o f b i o l o g i c a l  1 .4  -  O b j e c t i v e and format o f the t h e s i s  -  References  ^0  -  MICELLES  13  2.1  -  Introduction  14  2.2  -  Structure of micelles  16  2.3  -  Physical  16  2.4  -  A p p l i c a t i o n o f NMR f o r the study o f amphiphile  CHAPTER II  glycolipids lipids  i n water  properties of micelles  3 7 8  23  aggregation. -  CHAPTER I I I -  References  27  SYNTHESIS OF MODEL GLYCOLIPIDS  31  3.1  -  P r e v i o u s work  32  3.2  -  Method o f g l y c o s i d e f o r m a t i o n  33  3.3  -  Method o f amide bond f o r m a t i o n  37  3.4  -  V i a r e d u c t i v e amination  40  3.5  -  Experimental  41  -  References  47  V (Table o f Contents CHAPTER IV  -  continued)  NMR STUDIES OF MODEL GLYCOLIPIDS  49  1 4.1  -  4.2  -  4.3  H NMR s t u d i e s o f n - o c t y l  6-Q-glucopyranoside  50  B-Q-glucopyranoside  60  -  ^H NMR s t u d i e s o f N - a l k y l l a c t o b i o n a m i d e s e r i e s  65  4.4  -  Comments  67  4.5  -  Experimental  68  -  References  69  CHAPTER V  -  TWO  5.1  -  Introduction  5.2  -  D e s c r i p t i o n o f homonuclear J - r e s o l v e d and  C NMR s t u d i e s o f n - o c t y l  DIMENSIONAL FOURIER TRANSFORM  NMR SPECTROSCOPY  71 72 I'  1  SECSY experiments 5.2.1  -  Pulse sequence and data a c q u i s i t i o n  5.2.2  -  Data p r o c e s s i n g  5.3  -  A p p l i c a t i o n o f 2D s p e c t r o s c o p y i n s p e c t r a l  7>  ^  assignment o f u n p r o t e c t e d sugars 5.3.1  -  n-Octyl B-Q-glucopyranoside  5.3.2  -  N-Hexyllactobionamide  8 5  5.4  -  Experimental  9-  -  References  9  8.  f  vi  LIST OF TABLES Page No. CHAPTER I I I 3.1  Physical  c o n s t a n t s o f N-al k y l l a c t o b i o n a m i d e s e r i e s .  46  CHAPTER IV 4.1  Spin-lattice relaxation protons o f o c t y l trations  4.2  r a t e s (R-|) o f s e l e c t e d  glucoside at different  concen-  i n D^O.  13 C-NMR chemical  shift  data f o r o c t y l  glucoside  w i t h s h i f t changes ( A 6 ) on m i c e l l e f o r m a t i o n . CHAPTER V 5.1  Proton chemical of  s h i f t and c o u p l i n g c o n s t a n t data  the sugar r e g i o n o f n - o c t y l  Proton chemical  3-Q-glycopyranoside.  s h i f t and c o u p l i n g c o n s t a n t data o f  the sugar resonances  86  o f N-hexyllactobionamide.  94  vii  LIST OF FIGURES  Page No. CHAPTER I 1 .1  Structures  o f some s e l e c t e d g l y c o g l y c e r o l i p i d s .  4  1 .2  General s t r u c t u r e o f g l y c o s p h i n g o l i p i d s .  5  1.3  Structure o f a ganglioside.  6  CHAPTER II 2.1  Schematic r e p r e s e n t a t i o n  o f the c o n c e n t r a t i o n  14  dependence o f some p h y s i c a l p r o p e r t i e s f o r solutions  of a'micelle  2.2  Structures  2.3  Mi c e l l a r models .  2.4  Relaxation total and  forming  amphiphile.  o f common a m p h i p h i l e s .  17  between monomeric  added c o n c e n t r a t i o n  micelle  15  concentration  and  18  i n true phase s e p a r a t i o n  formation.  CHAPTER I I I 3.1  H and  3.2  H and  C NMR s p e c t r a o f n - o c t y l 1 3  C  g-Q-glucoside.  NMR s p e c t r a o f N - h e x y l l a c t o b i o n a m i d e .  CHAPTER IV 4.1  400 MHz  'H NMR s p e c t r a o f o c t y l  at d i f f e r e n t 4.2  glucoside  n 2  0  51  concentrations.  V a r i a t i o n o f R-| with  inverse  total  o j - C H , H-2 and H-l protons o f o c t y l q  in  concentration glucoside.  for  55  ( L i s t o f Figures 4.3  continued)  S t e r e o c h e m i c a l view o f the p r e f e r r e d tion  about the C,-0., and C-Q,  I I  conforma-  bonds o f a  I  a  glucoside. 4.4  Conformational p r o j e c t i o n s C  i n v o l v i n g C-|,  , and  of a glycoside.  a 13 4.5  100.6 MHz proton decoupled  C NMR s p e c t r a o f  monomer and m i c e l l a r s o l u t i o n s o f o c t y l 4.6  glycoside.  400 MHz ^H NMR o f N-dodecyllactobionamide i n D 0. ?  CHAPTER V 5.1  Schematic NMR  representation, o f d i f f e r e n t  2D-resolved  experiments.  5.2  I l l u s t r a t i o n o f C0SY[A] and SECSY[B] s p e c t r a .  5.3  Pulse sequence f o r 2D homonuclear  J-resolved  experiment. 5.4  Pulse sequences  f o r SECSY[A] and C0SY[B]  experiments. 5.5  Summary o f the data m a n i p u l a t i o n procedure i n 2Dexperiments.  5.6  5.7  I l l u s t r a t i o n o f various  d i s p l a y modes and the t i l t  routine  used: i n 20 s p e c t r a .  Partial  400 MHz ^H. ID and 2D NMR s p e c t r a o f n - o c t y l  g-D-glucopyranoside 5.8  Individual  5.9  Partial  i n D^O.  J spectra o f n-octyl  400 MHz H  1actobionamide  1  g-Q-glucopyranoside  1D and 2D NMR s p e c t r a  i n D 0. o  o f N-hexyl  ( L i s t o f Figures 5.10.  continued)  Partial  400 MHz  1actobionamide 5.11  Partial  Partial  H' 2D NMR  s p e c t r a o f N-hexyl-  i n D2O.  400 MHz SECSY spectrum o f N-hexyl-  1actobionamide 5.12  1  i n d^O.  SECSY spectrum o f N - h e x y l 1 a c t o b i o -  namide: an expansion o f the dotted of  F i g . 5.11 .  region  X  ACKNOWLEDGEMENT  I express my deepest g r a t i t u d e to Dr. L.D. H a l l  f o r h i s guidance and  c o n s t a n t encouragement throughout the course o f t h i s work. I am g r e a t l y i n d e b t e d t o Dr. S. Sukumar and Mr. M.A. B e r n s t e i n f o r many h e l p f u l  d i s c u s s i o n s and c r i t i c i s m .  The generous  N. Rajapakse, K.H. Holmes and Miss N. P a r t o v i manuscript i s g r a t e f u l l y acknowledged.  h e l p o f Messrs.  i n p r o o f - r e a d i n g the  Special  thanks a r e a l s o due to  Mrs. R. Theeparajah f o r t y p i n g the manuscript and f o r her p a t i e n c e .  De  my  di-oation  late  Father  1  CHAPTER I  GENERAL BACKGROUND  2  1.1 -  Biochemical The  aspects  surfaces  o f animal  c e l l s play a c r i t i c a l  role in interactions  between c e l l s and i n t h e responses o f c e l l s t o t h e i r e x t e r n a l which may c o n t a i n antigens,  etc.  environment  b i o l o g i c a l l y a c t i v e substances such as drugs, hormones,  These i n t e r a c t i o n s are o f prime importance i n the growth,  development and maintenance o f m u l t i c e l l u l a r organisms.  The simple  fact  t h a t c e l l s a r e a n t i g e n i c and induce an immune response when i n j e c t e d i n t o unrelated  animals suggests t h a t c e l l s d i f f e r i n t h e s t r u c t u r e o f compo-  nents l o c a t e d a t t h e i r c e l l blood  surfaces  [1].  Since  the c l a s s i c a l  work on  group substances [ 2 ] , i t has long been known t h a t complex carbo-  hydrates play an important p a r t i n d e f i n i n g c e r t a i n s t r u c t u r a l s p e c i f i c i ties  of c e l l  sharing  surfaces  [3,4,5],  t h e view t h a t t h e outer  Extensive surface  evidence [ 6 ] has accumulated  o f mammalian c e l l s  " l a y e r " o f carbohydrates c o n s i s t i n g o f o l i g o s a c c h a r i d e s the e x t e r n a l  may be very complex.  i n t h e type and sequence o f t h e c o n s t i t u e n t i n r i n g s i z e , t h e c o n f i g u r a t i o n o f each  branching o f t h e s a c c h a r i d e  chain  language.  monomers, each o f which may g l y c o s i d i c bond and variability  considered  recognition  of s t r u c -  s t r u c t u r e s can encode a r i c h  This i s one o f the reasons why c e l l  c a r b o h y d r a t e s a r e being aspects of b i o l o g i c a l  In a d d i t i o n t o v a r i a t i o n s  ensure a great  t u r e s , meaning t h a t t h e s e o l i g o s a c c h a r i d e biochemical  projecting into  medium.  These s a c c h a r i d e s  vary  i s covered by a  surface  as c a n d i d a t e s r e s p o n s i b l e [7,8].  f o r some  It i s c u r r e n t l y held t h a t  cell  s u r f a c e c a r b o h y d r a t e s a r e p o s s i b l e p a r t i c i p a n t s i n such events as r e c e p t o r i n t e r a c t i o n s [9,10],  permiablity  change [ 1 1 ] , c e l l u l a r  adhesion and  3  recognition  [12], which a r e fundamental  functions i n m u l t i c e l l u l a r Carbohydrates occur  processes  on mammalian c e l l  be f o c u s s e d  have been shown t o be c o n c e n t r a t e d with  some l o c a l i z a t i o n  s u r f a c e s p r i m a r i l y as In the d i s c u s s i o n s which  mainly on g l y c o l i p i d s .  p r i m a r i l y i n the plasma membrane [13]  components o f the membrane, accounting f o r  membrane l i p i d .  most o f t h e s e r o l o g i c a l s p e c i f i c i t y  G l y c o l i p i d s are responsible f o r  [15] e x h i b i t e d by mammalian c e l l s and  many organs have t h e i r s p e c i f i c t y p e s o f g l y c o l i p i d c h a r a c t e r i s e d examples o f membrane g l y c o l i p i d s f u n c t i o n s a r e t h e ABO blood GM^ r e c e p t o r  1.2 -  group a n t i g e n s  of n a t u r a l  As has a l r e a d y  [ 1 6 ] . The two best-  serving s p e c i f i c  have been proposed  [21-24].  glycolipids  been i m p l i e d , complex l i p i d s which c o n t a i n a  c a r b o h y d r a t e component a r e r e f e r r e d t o as g l y c o l i p i d s . s t r u c t u r e of the l i p i d  biological  [17-19] and t h e g a n g l i o s i d e  f o r c h o l e r a t o x i n [ 2 0 ] ; others  Structures  Glycolipids  a l s o o c c u r r i n g i n t h e endoplasmic r e t i c u l u m [ 1 4 ] .  They a r e g e n e r a l l y minor l i p i d 0.5 - 5% o f t h e t o t a l  various  organisms.  components o f g l y c o l i p i d s and g l y c o p r o t e i n s . follow, attention w i l l  governing  Based on t h e  component, g l y c o l i p i d s can be s u b - d i v i d e d  i n t o two  classes, 1.  glycoglycerolipids  2.  glycosphingolipids  Glycoglycerolipids: These occur and  i n higher  a l s o i n b a c t e r i a [25,26].  class.  p l a n t s and algae Glycosides  (mainly  i n the chloroplasts)  o f d i a c y l g l y c e r o l belong t o t h i s  The c a r b o h y d r a t e moiety can be a monosaccharide o r a d i s a c c h a r i d e  4  and s t r u c t u r e s c o n t a i n i n g g a l a c t o s e , mannose, and glucose been c h a r a c t e r i z e d . sulphates) chains  Sulphated  have  monosaccharide r e s i d u e s ( g l y c o s y l  have been found i n p l a n t s and p h o t o s y n t h e t i c  are normally  residues  s a t u r a t e d and occur  i n varying  algae.  lengths.  The a c y l  S t r u c t u r e s of  some s e l e c t e d compounds i n t h i s s e r i e s a r e shown i n F i g . 1.1.  S-o I  C H T — CH—CHo  I  I  0  o  1  I  CO  CO  I  I  CH CH  I  Long  2  I  2  aliphatic chains  Monogalactosyl  diacylglycerol  ct-0-Gal a c t o s y l (1 - 6) g-D- gal a c t o s y l diacylglycerol  6-Sul pho-a-Q-qui novosyl  F i g u r e 1.1:  Structures  diacylglycerol  o f some s e l e c t e d 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 b r a i n and c e l l s of h i g h e r organisms [ 2 7 ] . are 1 - 0 - g l y c o s y l  Those c h a r a c t e r i z e d from these  d e r i v a t i v e s of ceramides ( c e r e b r o s i d e s ) .  are N - ( f a t t y a c y l ) s p h i n g o s i n e s  or d e r i v a t i v e s t h e r e o f .  The  nerve  sources ceramides  ( F i g . 1.2).  ! C H —CH—CH(OH); 2  ; NH CH ' 4 - " 1 II R CH I 13 27 C  Spingosine  R = H,  Ceramide  R = l o n g - c h a i n a c y l , R'  = H  Cerebroside  R = l o n g - c h a i n a c y l , R'  = glycosyl  F i g u r e 1.2:  The  H  glycosyl  General  R'  = H  Structure of glycosphingolipids  moiety can vary from a s i n g l e g a l a c t o s e or  u n i t t o complex o l i g o s a c c h a r i d e s c o n t a i n i n g D - g a l a c t o s e , D-galactose-3-sulphate, D-glucose),  and  N-acetyl  glucose  D-glucose,  L - f u c o s e , 2-acylamido-2-deoxy-D-galactose (or neuraminic  acid  (Sialic  acid).  Cerebrosides  o f t e n c o n t a i n long - c h a i n f a t t y a c i d s , the most abundant o f which a r e lignoceric acids.  (24:0),cerebronic  ( q - h y d r o x y l i g n o c e r i c ) and  nervonic  (24:1)  6  More complex g l y c o s p h i n g o l i p i d s are u s u a l l y s t r u c t u r a l l y from l a c t o s y l  ceramide by e x t e n s i o n  have been c l a s s i f i e d a c c o r d i n g bone.  Thus the  derived  of the c a r b o h y d r a t e head group,  t o the nature o f the c a r b o h y d r a t e back  ganglio-series contains  gangliotetrose, g-galactosyl  3-N-acetylgalactosaminyl  (1-4)  l a c t o s e , the  globo-series  B-N-acetylgalactosaminyl  (1-3)  g-galactosyl  (1-3 o r 4) l a c t o s e , and  l a c t o - s e r i e s contains (1-3)1actose.  and  g-galactosyl  (1-3  or 4)  (1-3)  contains the  g-N-acetylgalactosaminyl-  Members of the g a n g l i o - s e r i e s are f u r t h e r s u b s t i t u t e d  sialic  a c i d residues while  sialic  a c i d or f u c o s e .  galactose  those of l a c t o s e s e r i e s can  S t r u c t u r e of a g a n g l i o s i d e  be s u b s t i t u t e d by  i s given  in Fig.  1.3.  N-acetylgalactosami ne galactose  glucose  ~CH—CH  CH(OH)  2  NH CO  AcN  CH CH  (CH )  (CH )  CH  CH  2 16  HOCH HOCH2  sialic  HOCH HOCH  3  2  acid  sialic  Figure  by  1.3:  acid  Structure of a ganglioside  2  3  12  7  1.3 -  Behaviour of b i o l o g i c a l Lipids i n biological  lipids  i n water  systems demonstrate a broad range o f behaviour  i n water, from hydrocarbons which a r e i n s o l u b l e , t o a m p h i b i l i c molecules t h a t possess potent dynamically.  detergent  Most o f t h e b i o l o g i c a l  possess amphipathic c h a r a c t e r regions lipid  p r o p e r t i e s and i n t e r a c t with water membrane l i p i d s  rather  including glycolipids  due t o the presence o f p o l a r and non p o l a r  i n t h e same m o l e c u l e .  The s u r f a c e and bulk  p r o p e r t i e s o f a given  depend upon t h e r e l a t i v e s t r e n g t h o f the h y d r o p h i l i c and l i p o p h i l i c  p o r t i o n s o f the molecule: i . e , t h e h y d r o p h y l i c - 1 i p o p h i T i c lipids  as well  cellular  as other  polar l i p i d s extracted  from c e l l  balance.  Glyco-  membranes and  o r g a n e l l e s can be c l a s s f i e d as " i n s o l u b l e s w e l l i n g amphiphi 1 i c  l i p i d s " on t h e b a s i s o f t h e i r behaviour i n water and at an a i r - w a t e r i n t e r f a c e [28]. interfaces  They spread  [28] and although i n s o l u b l e i n bulk  t o form l y o t r o p i c l i q u i d hexagonal  t o form s t a b l e monolayers a t a i r - w a t e r water, they swell  c r y s t a l s such as l a m e l l a r [28,29],  i n water  reversed  o r c u b i c phases [ 3 0 ] .  Although t h e l a m e l l a r l i p i d  b i l a y e r structure i s considered  as t h e  only compatible s t r u c t u r e f o r the f u n c t i o n i n g o f the membrane [ 3 1 ] , t h e possibility  o f e x i s t e n c e o f other mesophases i s not t o t a l l y  Formation o f d i f f e r e n t mesophases w i t h i n the membrane d u r i n g periods  or i n the proximity  of i n t e g r a l  proposed t o be advantageous t o c e l l [32,33], exo- and e n d o - c y t o s i s , [34].  The u s e f u l n e s s  been p o i n t e d The  excluded  [30].  short time  membrane p r o t e i n s has..- been  functions  such as membrane f u s i o n  and t r a n s - b i l a y e r movements of l i p i d s  i n b i o l o g y o f such phase e q u i l i b r i u m systems has  out [ 3 5 ] .  a s s e r t i o n [ 2 8 ] , by one o f t h e a c t i v e r e s e a r c h e r s  t h a t - "processes  i n the f i e l d ,  such as membrane budding and f u s i o n may u l t i m a t e l y  8  be e x p l a i n e d by t h e composition  and s t a t e s o f t h e l i p i d s t a k i n g p a r t i n  t h e s e p r o c e s s e s " - summarizes c o n c i s e l y t h e probable importance o f polymorphism o f l i p i d s  1.4 -  in biology.  O b j e c t i v e and format The  complexity  o f the t h e s i s  of b i o l o g i c a l  i n - v i v o study o f t h e g l y c o l i p i d s roles.  i n order t o a s c e r t a i n t h e i r  specific  T h i s f a c t has promoted t h e s y n t h e s i s and study o f many 'model'  glycolipids. difficulties form  membranes, l a r g e l y p r e c l u d e s an  T h e i r s y n t h e s i s has been n e c e s s i t a t e d because o f t h e i n v o l v e d i n i s o l a t i n g them i n l a r g e q u a n t i t i e s and i n pure  from n a t u r a l s o u r c e s .  Apart  from  h e l p i n g t o understand  their  n a t u r a l l y o c c u r i n g c o u n t e r p a r t s , model g l y c o l i p i d s have found as d e t e r g e n t s  f o r the s o l u b i l i z a t i o n  a c c e p t o r s f o r carbohydrate  application  o f membrane components [ 3 6 ] and as  b i n d i n g p r o t e i n s , such as l e c t i n s  [37,38],  In s p i t e o f many p r e v i o u s e f f o r t s , t h e r e c o n t i n u e s t o be a p r e s s i n g need f o r t h e development o f new s y n t h e t i c methods and a n a l y t i c a l probe t h e behaviour of t h i s f a c t  o f these  "model" compounds i n s o l u t i o n .  tools to  Realization  l a i d the foundation f o r the studies described i n t h i s  and t h e o b j e c t i v e o f t h e work presented  here has been two f o l d .  thesis  Firstly,  t o e v a l u a t e t h e e f f i c i e n c y o f v a r i o u s s y n t h e t i c schemes f o r t h e preparat i o n o f model g l y c o l i p i d s so t h a t l a r g e q u a n t i t i e s c o u l d be o b t a i n e d r a p i d l y , and i n high y i e l d s .  Secondly, t o e v a l u a t e t h e advantages and  l i m i t a t i o n s o f Nuclear Magnetic structural  and an a n a l y t i c a l  Resonance  tool  (NMR) s p e c t r o s c o p y as a  i n studying the solution properties of  these compounds. In t h i s c o n t e x t , i t was d e c i d e d t o c o n f i n e t h e a t t e n t i o n t o t h e c l a s s o f compounds having a long a l k y l  c h a i n as the hydrophobic  portion  9  and  a c a r b o h y d r a t e moiety as t h e h y d r o p h i l i c group.  i n t o the  fall  c l a s s o f " s o l u b l e a m p h i p h i l e s " [ 2 8 ] , which form m i c e l l e s i n  d i l u t e aqueous s o l u t i o n s . studies  These compounds  This t h e s i s describes  on m i c e l l e f o r m a t i o n o f the  the  synthesis  above d e r i v a t i v e s .  and NMR  ^H-NMR was chosen 13  t o s c r u t i n i z e m i c e l l e formation i n preference t o e i t h e r because o f the  predictable  need t o work with very d i l u t e  2 C or  solutions.  the event t h i s proved t o be a prudent d e c i s i o n s i n c e , as w i l l t h e s e d e r i v a t i v e s have very low c r i t i c a l proved very d i f f i c u l t The  micelle concentrations  t o study t h e i r m i c e l l a r p r o p e r t i e s  format o f t h i s t h e s i s i s as f o l l o w s :  reader t o v a r i o u s  aspects o f m i c e l l e s  H NMR In  be seen, and i t  even by ^H-NMR.  Chapter II i n t r o d u c e s the  as c u r r e n t l y understood and i s  accompanied by a b r i e f survey o f a p p l i c a t i o n s o f NMR t e c h n i q u e s i n studying  amphiphile a g g r e g a t i o n .  g l y c o l i p i d s forms the evaluation  o f the  utilized  f o r Chapter I I I .  experimental  by t h e s e compounds. discussion  basis  on v a r i o u s  i n spectral  A d i s c u s s i o n on the  synthesisof  'model'  Chapter IV i s devoted t o an  NMR r e s u l t s p e r t a i n i n g t o m i c e l l e f o r m a t i o n  F i n a l l y , i n Chapter V the 'two-dimensional assignments.  1  reader i s exposed t o a  NMR experiments which have been  10  References  1.  Hood, L.E., Weissman, I.L., Wood, W.B.,  "Immunology",  Benjamin/  Cummings P u b l i s h i n g Co., C a l i f o r n i a , 1978. 2.  G o t t s c h a l k , A., "The Chemistry and B i o l o g y o f S i a l i c Substances-,"  3.  Watkins, W.M.,  Cambridge U n i v e r s i t y  a c i d s and Related  P r e s s , 1960.  i n " G l y c o p r o t e i n s : T h e i r Composition, S t r u c t u r e and  F u n c t i o n " , Part B;  G o t t s c h a l k , A., Ed., E l s e v i e r , Amsterdam, 1972,  p. 830-891. 4.  Ginsburg, V., Kobata, A., i n " S t r u c t u r e and F u n c t i o n o f B i o l o g i c a l Membranes", R o t h f i e l d , L . I .  Ed., Academic P r e s s , New York, 1971,  p. 439. 5.  Cook, G.M.W., S t o d d a r t , R.W., Cell",  6.  "Surface Carbohydrates o f E u k a r y o t i c  Academic P r e s s , New York, 1973.  Parsons, D.F., Subjeck, J.R.,  Biochim. B i o p h y s . A c t a , (1972), 265,  85. 7.  Hughes, C.L., Sharon, N.,  Trends Biochem. S c i . , (1978), 3, N275.  8.  M a r c h e s i , V.T., Ginsburg, V., Robbins, S u r f a c e Carbohydrates and B i o l o g i c a l  P.W.,  Fox, C.F.,  Recognition",  Eds.,  "Cell  Alan R. L i s s I n c . ,  New York, 1978. 9.  M u l l i n , B.R., Fishman,  P.H., Lee, G., A l o j , S.M., L e d l e y , F.D.,  Winand, R.J., Kohn, L.D., and Brady, R.O.,  P r o c . N a t l . 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 , G i t h e n , S., 12. Rosemann, S.,  Nature,  Chem. Phys. L i p i d s ,  (1965), ^08, 88. (1970), 5_, 270.  11  13. W e i n s t e i n , D.B., Marsh, J.B., G l i c k , M.C, Warren, L.,  J . Biol.  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., International  Burger, M.M.,  in  " B i o c h e m i s t r y o f Carbohydrates", MTP  Review o f S c i e n c e , B i o c h e m i s t r y S e r i e s one, V o l . 5;  Whelan, W.J., Ed., B u t t e r w o r t h s , London, 1975, p . 59. 16. C a r t e r , H.E., Johnson,  P., Weber, E . J . ,  Annu. Rev. Biochem., (1965),  34, 109. 17. Horowitz, M.I., i n "The G l y c o c o n j u g a t e s " , V o l . I I , Horowitz M.I., Pigman, W.,  Eds., Academic P r e s s , New York, 1978, p. 387.  18. Hughes, R . C , Function",  B u t t e r w o r t h s , London, 1976, p. 114.  19. Sharon, N., Function". 20. Moss, J . ,  "Membrane G l y c o p r o t e i n s : A Review o f S t r u c t u r e and  "Complex C a r b o h y d r a t e s : Addison-Wesley, Vaughan, M.,  21. Jacques, L.W., W. J r . ,  T h e i r Chemistry B i o s y n t h e s i s and  Reading, Massachusetts, 1975, p. 215.  Annu. Rev. Biochem., (1979), 48, 581.  Brown, E.B., B a r r e t , J.M., Brey, W.S. J r . , Weltner,  J . B i o l . Chem., (1977), 252^, 4533.  22. Pappenheimer, A.M. J r . ,  Annu. Rev. Biochem., (1977), 46, 69.  23. Besanocon, F., A n k e l , H., 24. Kohn, L.D. i n  Nature, (1974), 252, 478.  "Receptor and R e c o g n i t i o n " , ' S e r i e s A, Vol 5, C u a t r e c a s ,  P., Greaves, M.F., Eds., Chapman and H a l l , London, 1978, p. 133. 25. Shaw, N., B a d d i l e y , J . , Nature, 26. Shaw, N.,  (1968), 217, 142.  B a c t e r i o l . Rev., (1970), 34, 365.  27. Wiegandt, H., 28. S m a l l , D.M.,  Adv. L i p i d Res., (1971), 9, 241. Pure and A p p l . Chem., (1981), 5_3, 2095.  12  29. Rucco, M.,  A t k i n s o n , D.,  S h i p l e y , G.G.,  S m a l l , D.M.,  S k a r j u n e , R.,  B i o c h e m i s t r y , (1981), 20,  30. W i e s l a n d e r , A., R i l f o r s , L., Johanssan, Biochemistry,  (1981), 20,  Lucy, J.A.,  in  "Cell  New  York,  5957.  L.B.,  Lindblom,  S c i e n c e , (1972), 175,  Membranes:  Pathology", Weissman, G.,  E.,  G.,  730.  31. S i n g e r , S.J., N i c o l s o n , G.L., 32.  01dfield,  Biochemistry, Cell  C l a i b o r n e , R.,  Eds., H.P.  720. B i o l o g y and Publishing  Co.,  1975.  33. C u l l i s , P.R.,  de K r u i j f f , B.,  Biochim. Biophys. A c t a , (1979),  559,  339. 34. C u l l i s , P.R.,  F a r r e n , S.B.,  Hope, M.J.,  Can. J . S p e c t r o s . , (1981),  26,  89. 35. S m a l l , D.M.,  J. Colloid  36.  Smith, H.G.  Stubbs, G.W., (1976) , 426,  37. Read, B.O.,  J r . , Litman, B.J.,  Wiengandt, H.,  Biochem. Biophys. A c t a ,  Van Deenen, L.L.M.,  ibid.,  325.  38. W i l l i a m s , T . J . , P l e s s a s , N.R., Biophys.,  581.  46. Demel, R.A.,  (1977) , 470,  I n t e r f a c e S c i . , (1977), 58,  (1979), 195,  145.  Goldstein, I.J.,  A r c h . Biochem.  CHAPTER II  MICELLES  14  2.1  Introduction The  introduced the  term " m i c e l l e " (from l a t . m i c e l l a meaning "small by t h e pioneer  formation  i n the f i e l d ,  amphiphilic  J.W. McBain, i n 1913 t o d e s c r i b e  o f aggregates o f c o l l o i d a l  soaps i n aqueous s o l u t i o n .  dimensions by d e t e r g e n t s and  The idea evolved  from the o b s e r v a t i o n  molecules i . e . m o l e c u l e s p o s s e s s i n g  hydrophobic p o r t i o n s , behave r a t h e r uniquely a c e r t a i n concentration  [1].  b i t " ) was  that  s e p a r a t e h y d r o p h i l i c and  when d i s s o l v e d i n water above  Above t h i s c o n c e n t r a t i o n  many p h y s i c a l  p r o p e r t i e s o f t h e s o l u t i o n ( e . g . v i s c o s i t y , c o n d u c t i v i t y , o p t i c a l and spectroscopic illustrated  p r o p e r t i e s ) were found t o show an abrupt change as  i n F i g . 2.1.  concentration  Figure  2.1:  Schematic r e p r e s e n t a t i o n dence o f some p h y s i c a l  of the concentration  properties  of a m i c e l l e forming amphiphile  depen-  for solutions  (from r e f .  [16]).  This behaviour i s now a t t r i b u t e d t o the a s s o c i a t i o n o f amphiphiles t o form small  aggregates - m i c e l l e s .  The a s s o c i a t i o n o f amphiphiles i n  15  aqueous s o l u t i o n effect  i n t o m i c e l l a r aggregates  i s a s c r i b e d t o t h e hydrophobic  [ 2 ] which i n t u r n a r i s e s from t h e s t r o n g a t t r a c t i v e  water molecules  [3],  Such a s s o c i a t i o n s l e a d t o a r e d u c t i o n i n t h e t o t a l  c o n t a c t area o f t h e hydrophobic which t h e a s s o c i a t i o n concentration"  f o r c e s between  groups with water.  The c o n c e n t r a t i o n a t  i s shown t o occur i s c a l l e d t h e " c r i t i c a l  (cmc); i . e . i n d i l u t e s o l u t i o n  micelle  ( c o n c e n t r a t i o n <cmc) t h e  amphiphiles e x i s t as monomers w h i l e a t h i g h e r c o n c e n t r a t i o n s ( » c m c ) they spontaneously  assemble t o form s t a b l e 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 , a n i o n i c , n o n i o n i c o r z w i t t e r i o n i c a c c o r d i n g t o the charge hydrophilic  "head group".  long hydrocarbon given  chain.  The hydrophobic  "tail"  on t h e  commonly comprises  Examples o f a few common amphiphiles a r e  i n F i g . 2.2.  +  H e x a d e c y l t r i m e t h y l ammonium bromide  Sodium dodecyl sul phate  CH^-(CH }^(CH^Br 2  CHg- (CH )-| •] -S0^Na  +  2  f3 H  N-Dodecyl-N,N-dimethyl  glycine  CH -(CH ) -N-CH -C0 CH 3  2  11  2  2  3  Polyoxyethylene  (6) hexadecanol  F i g u r e 2.2:  CH -(CH ) -0-(CH CH 0) H 3  S t r u c t u r e s o f common  2  1 5  2  amphiphiles  2  6  a  16  2.2  -  Structure The  of m i c e l l e s  s t r u c t u r e of m i c e l l e s has  many o t h e r a s p e c t s  been a s u b j e c t  of m i c e l l e s , f o r a long time.  seems t o be no u n i v e r s a l agreement. spherical G.S.  [4].  The  of hydrated  2.3a).  there  pioneering  work of  e s s e n t i a l f e a t u r e of t h i s H a r t l e y model i s t h a t a (Fig.  i t i s customary t o c o n s i d e r m i c e l l e s as s p h e r i c a l -  species  sedimentation,  t o the  like  roughly  p o l a r head groups encases a hydrocarbon core  At p r e s e n t  ellipsoidal  Even at present  b a s i c concept of a  m i c e l l e i n aqueous s o l u t i o n i s due  Hartley  shell  The  of c o n t r o v e r s y ,  (Fig.  2.3b), and  d i f f u s i o n , and  current discussions  light  the  s c a t t e r i n g data  r o u t i n e l y d e p i c t the  m i c e l l e as a proven f a c t [ 6 , 7 ] , the  idea i s supported  Hartley  validity  [5],  by  Even though  spherical-ellipsoidal  of the model has  recently  13 been q u e s t i o n e d [ 9 ] , ORD  data  [12] support with  [8].  According  [ 1 0 ] , k i n e t i c data the  to Menger [ 8 ] , n e i t h e r the [ 1 1 ] , nor the molecular  H a r t l e y concept, but  a rough s u r f a c e and  Fromherz [1,3] has  model  studies  s t r o n g l y suggest a "porous m i c e l l e "  deep water f i l l e d  cavities.  proposed a s u r f a c t a n t - b l o c k  m i c e l l a r aggregates based on c r i t i c a l  C-NMR data  More r e c e n t l y ,  ( F i g . 2.3c)  i n s p e c t i o n of some  model  for  experimental  data. S i n c e none o f the newer models have y e t gained  wide a c c e p t a n c e , i n  the f o l l o w i n g d i s c u s s i o n the modfied H a r t l e y model w i l l accepted  2.3  -  regarded as  s t r u c t u r e of m i c e l l e s .  Physical  Critical  be  p r o p e r t i e s of m i c e l l e s  micelle concentration  The  (cmc)  c h a r a c t e r i s t i c cooperative  nature o f m i c e l l i z a t i o n makes i t  o f t e n p o s s i b l e t o d e s c r i b e the aggregation  process  u s i n g only a  few  the  17  (a)  Schematic c r o s s - s e c t i o n a l of a Hartley  Elliptical  cross-  hydrocarbon core  —  s e c t i o n o f an idealized  anionic  detergent  micelle  spherical  — — _  representation  micelle.  Stern layer containing headgroups and 'bound' counterions  (from Ref. 7 ) . Gouy-Chapman diffuse double layer containing 'unbound'  (c)  counterions  Surfactant-Block 1.  Orthogonal assembly o f b l o c k s with minimal  2.  model  headgroup  Modification  contacts.  o f block-assembly to  reduce the headgroup r e p u l s i o n the  introduction  by  o f a gauche isomer  near the head group. The s t i c k - m o d e l s r e p r e s e n t the e f f e c t i v e volume o f the molecules i n a l i q u i d c r y s t a l l i n e state  Figure  2.3:  M i c e l l a r Models  (from Ref. 1 3 ) .  18  parameters.  It has, f o r example, proven t o be most u s e f u l  s p e c i f i c cmc t o each m i c e l l e - f o r m i n g  to ascribe a  amphiphile.  In t h e "mass a c t i o n law model" [14] o f m i c e l l i z a t i o n , i t i s assumed that  a s i n g l e s i z e m i c e l l a r species  monomers.  i s i n f a s t e q u i l i b r i u m with the  T h i s can be r e p r e s e n t e d by, nA  where A-| and A  n  ^  1  represent  An ; [An]/[A.,]  =  n  K  2.1  the monomer and the m i c e l l a r s p e c i e s  of a f i x e d  a g g r e g a t i o n number, n, r e s p e c t i v e l y . This of m i c e l l e s increases  e q u i l i b r i u m demands t h a t , when n i s l a r g e , t h e remains small  up t o a c e r t a i n l e v e l  rapidly thereafter.  being the d i s c o n t i n u i t y .  concentration  of s u r f a c t a n t and  The l a r g e r t h e value o f n, t h e sharper  The presumption of s i n g l e s i z e m i c e l l e s  ( m o n o d i s p e r s i t y ) i s not s t r i c t l y  correct since i n r e a l i t y micelles of  d i f f e r e n t a g g r e g a t i o n numbers can occur ( p o l y d i s p e r s i t y ) .  X , Total added concentration cri  0.5  1.0 1.5 2.0 Total amphiphite concentration  Ca) Figure  2.4:  Relation  (b) between monomeric c o n c e n t r a t i o n  added c o n c e n t r a t i o n formation.  Figure  i n (a) true  i n s o l u t i o n and t o t a l  phase s e p a r a t i o n  2.4(b) i s based on c a l c 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 e m p i r i c a l d e t e r m i n i n g the cmc  and (b) m i c e l l e  (from Ref.  [2]).  procedures f o r  19  An  important  f e a t u r e of m i c e l l e f o r m a t i o n  amphiphile c o n c e n t r a t i o n concentration  (C  t o t  increased.  F i g u r e 2.4  concentrations transition  ) i s i n c r e a s e d above the cmc,  of f r e e amphiphiles  range, w h i l e the c o n c e n t r a t i o n  of amphiphiles  t o t h e cmc present  In r e a l i t y  represented  i n two  i n F i g u r e 2.4  been analyzed [16],  A, + A , 1 n-1  nA, 1  [15] u s i n g  *=?  A  n  ; n = 2,3  2.2  A  n  ; n = 2,3  2.3,  .... .  2.3  i m p l i c a t i o n s f o r the k i n e t i c behaviour of m i c e l l e s .  p o i n t the  reader  i s r e f e r r e d t o the a r t i c l e by  e l a b o r a t e d i s c u s s i o n on the u s e f u l n e s s  model, the cmc a two  be  schemes are thermodynamically e q u i v a l e n t but they  has  this  of t h i s model.  model" o f m i c e l l e formation  phase r e g i o n , the two  At  have  Aniansson [15] f o r a more  i t s most c l e a r - c u t i n t e r p r e t a t i o n w i t h i n the  i s regarded  the  growth of  different  "phase-separation  shows  2.2,  or as a number of e q u i l i b r i a a c c o r d i n g t o t h e equation  cmc  over  This model can  e q u i v a l e n t ways; as a stepwise  m i c e l l e s a c c o r d i n g t o the equation  The  the  behaviour.  s i z e d i s t r i b u t i o n of m i c e l l e s has  These two  over a wide  sharp, but occurs  Thus dashed l i n e  " m u l t i p l e e q u i l i b r i u m model" of m i c e l l i z a t i o n formally  the  i n monomeric and m i c e l l a r  from monomeric t o m i c e l l a r s t a t e i s not  only the i d e a l i s e d  total  i n m i c e l l a r form i s  amphiphile c o n c e n t r a t i o n .  a narrow range of c o n c e n t r a t i o n s .  The  s t a y s equal  shows t h e v a r i a t i o n  with the t o t a l  i s t h a t when the  [16].  as the c o n c e n t r a t i o n  According  (pseudo) to t h i s  at which the system  enters  pseudophases formed being the aqueous system  and m i c e l l e s . T h i s model i s p a r t i c u l a r l y  u s e f u l f o r d e s c r i b i n g the amount  20  o f mi c e l l i z e d - a m p h i p h i l e p r e s e n t , and how the m o l e c u l a r with t h e amphiphile c o n c e n t r a t i o n . be a d i f f u s i o n  properties  The average o f a q u a n t i t y Q (which can  c o e f f i c i e n t , NMR chemical  s h i f t , o r r e l a x a t i o n time) i s  determined by t h e f r a c t i o n s o f amphiphile m i c e l l i z e d , p ™ such t h a t f o r a t o t a l  <Q> = pf  c  Q  concentration  + pjq Q  m i c  a q  C  m i c  Q  2.4  + cmc  m i c  tot /  tot  ac  vs C  t Q t  ^  Below the cmc, <Q> = Q  one should  at t h e cmc.  Equation  experimental  data.  that m i c e l l i z a t i o n  a q  o b t a i n two s t r a i g h t  2.4 p r o v i d e s  .  Thus, by p l o t t i n g  l i n e s which  Here, a smooth t r a n s i t i o n  i s observed i n t h e region o f T h i s i s due t o t h e f a c t  i s not r i g o r o u s l y a phase s e p a r a t i o n  In p r a c t i c e , t h e d e t e r m i n a t i o n s l o p e when an a p p r o p r i a t e  situation  [17].  o f the cmc i s based on t h e change i n  p h y s i c a l property  t h a t d i s t i n g u i s h e s between  m i c e l l a r and f r e e amphiphile s t a t e i s p l o t t e d a g a i n s t t o t a l However, i t i s important  intersect  only approximate d e s c r i p t i o n o f t h e  the cmc i n s t e a d o f the p r e d i c t e d sharp change.  cmc  concentration.  t o emphasize t h a t no procedure can y i e l d a unique  because none i n f a c t e x i s t s [ 1 7 ] .  The cmc o b t a i n e d  l a r g e l y depends on  the method employed and v a r i o u s methods o f measurement and r e s u l t s obtained  by t h e i r use,  The  have been c r i t i c a l l y d i s c u s s e d  primary f a c t o r governing  s i n g l e chain amphiphiles c  [18].  the magnitude o f t h e cmc i s the s i z e  ( l e n g t h ) o f t h e hydrophobic p a r t o f the m o l e c u l e .  atoms ( n )  q  and Q ' are t h e values o f Q i n m i c e l l a r and aqueous  environments r e s p e c t i v e l y . <Q> = Q ^  , and f r e e , p ^  l c  l a r g e r than t h e cmc,  t Q t  = (1 \  where Q  vary  For many c l a s s e s o f  t h e dependence o f cmc on the number o f carbon  can be r e p r e s e n t e d  by the  r e l a t i o n s h i p [19],  l o g cmc = a - bn  2.5  21  where a and  b are c o n s t a n t s .  l e n g t h causes alkyl  a decrease  T h i s i m p l i e s t h a t an i n c r e a s e i n the c h a i n  i n the cmc.  c h a i n has been observed  The  presence  i n c r e a s e s i n the cmc  bonds i n the  t o cause an i n c r e a s e i n the cmc  o f 3 - 4 i n comparison with the c o r r e s p o n d i n g c h a i n - b r a n c h i n g and  of double  i n t r o d u c t i o n of -OH  n-alkyl  by a f a c t o r  compounds;  groups a l s o cause  substantial  value [16].  With regards t o the p o l a r head group, the main i n f l u e n c e comes from i t s charge  so t h a t the cmc  f o r a given a l k y l  f o r n o n i o n i c than f o r i o n i c amphiphiles  chain l e n g t h i s much  [16].  The  cmc  decrease with i n c r e a s i n g bulk of the head group [ 1 4 ] . e l e c t r o l y t e s has  a l a r g e e f f e c t on the cmc  f o r n o n i o n i c systems t h i s e f f e c t for ionic  values are found A d d i t i o n of  is relatively  r e l a t i o n s h i p between the l o g a r i t h m of the cmc [14].  The  to  simple  of i o n i c a m p h i p h i l e s , whereas small.  The decrease  systems with added c o u n t e r i o n s u s u a l l y corresponds  concentration  lower  and the t o t a l  in  cmc  to a linear counterion  i n f l u e n c e of added n o n - e l e c t r o l y t e s v a r i e s  w i d e l y , depending on whether the added compound i s p r e f e r e n t i a l l y l o c a t e d i n t h e m i c e l l e , or i n the i n t e r - m i c e l l a r s o l u t i o n The dependence of cmc o f most chemical  association  with i n c r e a s i n g temperature,  on temperature phenomena. or i t may  p r e s s u r e dependence of the cmc  M i c e l l a r s i z e and The  i s small  The cmc  may  [16] compared t o t h a t i n c r e a s e or  decrease  show a pronounced minimum.  The  i s weak even up t o high p r e s s u r e s [ 1 6 ] .  polydispersity  a g g r e g a t i o n number i s d e f i n e d as the number of  contained i n a m i c e l l e . amphiphiles  [14].  However, as p o i n t e d out e a r l i e r ,  give a d i s t r i b u t i o n  of m i c e l l e s i z e s  molecules in practice  ( p o l y d i s p e r s i t y ) of which  the a g g r e g a t i o n number r e p r e s e n t s the most abundant of many m i c e l l e s i z e s  22  i n e q u i l i b r i u m with each o t h e r . m i c e l l e s i z e i s narrow and [20].  Studies  i n d i c a t e t h a t the  somewhat unsymmetrical  In c o n t r a s t t o the cmc,  d i s t r i b u t i o n of  around the average  value  the m i c e l l a r s i z e v a r i e s with a number of  f a c t o r s i n a manner which i s complex and  presently  difficult  to predict  [20].  I n t r a m i c e l l a r s t r u c t u r e and  dynamics  A vast amount of experimental suggests t h a t the essentially  hydrocarbon chains  evidence has  l o c a t e d at the  have a l i q u i d - l i k e c h a r a c t e r  hydrocarbon s o l v e n t s  of s i m i l a r chain  data [25,26] c o n s i s t e n t with the  accumulated which i n t e r i o r of m i c e l l e s  [21-23], but  length  [24].  less f l u i d  than  However, c o n f l i c t i n g  idea of a s o l i d - l i k e m i c e l l e c o r e ,  are  also available. The the  general  analogy w i t h a l i q u i d  hydrocarbon i s not  s t r u c t u r e of m i c e l l e s i t i s c l e a r t h a t the  are more or l e s s f i x e d t o the m i c e l l a r s u r f a c e . c o n s t r a i n t s on the motion of the attached i n d i c a t e t h a t the motional away from the proportion  p o l a r head group [27] and  of t r a n s conformation of the  unfavourable contact  alkyl  freedom of the c h a i n  Since m i c e l l e formation  chain.  i s associated  much of the  m i c e l l e formation.  Here again  along  i s an  first  seven carbons, w h i l s t Lindman and  any  penetration  o f water i n t o the  studies the  chain  increased  with the e l i m i n a t i o n  ideas  of  water, i t i s important i s retained  have been put  co-workers have shown [9] t h a t water p e n e t r a t e s  the  imposes  upon m i c e l l i z a t i o n [28].  amphiphi 1e-water c o n t a c t conflicting  Related  increases  t h a t there chain  p o l a r head groups  This f i x a t i o n  between hydrophobic groups and  t o know e x a c t l y how  Menger and  adequate s i n c e from  on  forward;  upto at l e a s t  Wennerstrom argue [29]  against  i n t e r i o r of the" m i c e l l e except to hydrate  23  the  head group.  Sol ubi 1 i z a t i on The insoluble  ability  of m i c e l l a r s o l u t i o n s t o d i s s o l v e substances t h a t  (or s p a r i n g l y s o l u b l e )  phenomenon most s i g n i f i c a n t Spectroscopic  observations  are  i n pure water, makes the m i c e l l i z a t i o n  from an  industrial  and  biological  [20,30,31] suggest t h a t the  p o i n t o f view.  solubilization  of  non-polar molecules i n m i c e l l e s resembles n o n - s p e c i f i c d i s s o l u t i o n i n a non-aqueous phase r a t h e r than s p e c i f i c micelle.  However, the  nature of the  d i f f e r e n t p a r t s o f the m i c e l l e . solubil ized c l o s e hydrocarbons are micelle.  solute  or i n  the  determines i t s a f f i n i t y  Molecules with a p o l a r group  preferentially solubilized [26,33]'indicate  s o l u b i l i z e d c l o s e t o the m i c e l l e - w a t e r preceding  overview of the  t o a s i t e on  towards  are  to the. .mice! 1 e-water i n t e r f a c e [32>]--,while a l i p h a t i c  Recent s t u d i e s  The  binding  d i s c u s s i o n was  subject.  example, c o u n t e r i o n  Many other  i n the  i n t e r i o r of  the  t h a t aromatic compounds are  also  interface.  mainly intended  to give only  an  important p r o p e r t i e s of m i c e l l e s , f o r  b i n d i n g , m i c e l l a r c a t a l y s i s have been o m i t t e d ,  these a s p e c t s are t h o r o u g h l y d e a l t with i n the  review  and  articles  [6,7,16,34].  2.4  -  A p p l i c a t i o n of NMR  f o r the  M i c e l l e s c o n t i n u e t o be  study of amphiphile  s c r u t i n i z e d by an  t e c h n i q u e s i n c l u d i n g , among o t h e r s , s c a t t e r i n g , and application the  calorimetry.  of NMR  The  because of the  reader i s r e f e r r e d t o the  NMR,  unusually  X-rays, ESR,  discussion  aggregation wide v a r i e t y of  fluorescence,  here i s r e s t r i c t e d  to  d i v e r s i t y of the methods employed  reviews by Wennerstrom et a l . [34]  light  and  and  24  Anacker [35] f o r an i n d e p t h coverage NMR  has become the most g e n e r a l l y a p p l i c a b l e t o o l  amphiphile high  systems;  field  t h e advent  H,  13 C,  f o r the study  Of  o f F o u r i e r t r a n s f o r m t e c h n i q u e s as well  as  s u p e r c o n d u c t i n g magnets have made s t u d i e s f e a s i b l e at  millimolar concentrations. 1  on o t h e r t e c h n i q u e s .  19 F,  2 H and  31 P.  The most f r e q u e n t l y used A l l the common NMR  "probe"  parameters  r e l a x a t i o n t i m e s , l i n e w i d t h s , and quadrupole  view [36] due t o the r a p i d exchange of molecules  weighted  average  between the two  Utilization  of  NMR,  In most cases point of  between monomeric and  parameters  r e p r e s e n t the  states.  d i s p e r s i o n of  As a r e s u l t , i t i s d i f f i c u l t  from methylene groups i n an a l k y l changes accompanying m i c e l l i z a t i o n shifts  shifts;  even with i t s very high s e n s i t i v i t y i s  hampered by t h e f a c t t h a t the small.  NMR  are  s p l i t t i n g s ) have been  m i c e l l a r s o l u t i o n s are c o n s i d e r e d t o be i s o t r o p i c from an NMR  Thus, a l l the observed  nuclei  (chemical  u t i l i z e d t o study d i f f e r e n t a s p e c t s of m i c e l l e s t r u c t u r e .  micellar states.  sub-  chemical  shifts i s quite  to resolve overlapping  resonances  c h a i n and t o d e t e c t t h e i r chemical [37].  The  use of proton  shift  chemical  i s very much e a s i e r i n the presence of aromatic groups because  t h e i r a s s o c i a t e d r i n g - c u r r e n t s h i f t s are s e n s i t i v e t o geometric f e a t u r e s of molecular packing.  T h i s f e a t u r e has been used t o probe the f o r m a t i o n  o f m i c e l l e s from v a r i o u s co-phenylalkyltrimethylammonium t o d e t e c t the s o l u b i l i z a t i o n cetyltrimethylammonium of a l k y l  bromides [38]  and  s i t e s of v a r i o u s aromatic compounds i n  bromide m i c e l l e s [ 3 3 ] .  The  spin l a t t i c e  c h a i n protons has been shown t o be more e f f i c i e n t  than f o r f r e e monomer molecules  i n aqueous s o l u t i o n  study [ 4 0 ] , the v a r i a t i o n o f s p i n l a t t i c e  [29],  relaxation  protons with c o n c e n t r a t i o n has been used t o determine  relaxation  in micelles In another  r a t e of methylene the cmc  of  25  n-al kyl ammoni um The  chlorides  chemical  i n D2C . 1  shifts  [38]  and  relaxation rates  protons have been u t i l i z e d t o i n v e s t i g a t e m i c e l l e relaxation  studies c l e a r l y i l l u s t r a t e  m i c e l l e f o r m a t i o n w h i l e the associated  with  The  [41,42] of water  hydration.  The  reduced amphiphile-water c o n t a c t  i n t e r p r e t a t i o n of chemical  shift  on  changes i s  difficulties.  advantage of using  wider frequency range than  13  C NMR  i s that  13  C resonances occur over a  resonances; t h i s makes i t p o s s i b l e t o  a l a r g e number o f resonances t o i n d i v i d u a l c a r b o n s .  However, one  assign  major  13 difficulty  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 10 mM  shift  studies  are  l i m i t e d to concentrations  or g r e a t e r , w h i l e r e l a x a t i o n s t u d i e s  require  an  of the order  of  order of magnitude  1 "3 higher concentrations. micel1ization  are  The  not y e t  observed  C chemical  s h i f t changes upon  completely understood, but  i n general  for alkyl  c h a i n s they appear t o be mainly determined by trans-gauche i s o m e r i z a t i o n 13 equilibrium. Thus the observed d o w n f i e l d s h i f t of C .resonances have been i n t e r p r e t e d as due t o an i n c r e a s e i n t r a n s conformation o f the alkyl  chain  accompanying m i c e l l i z a t i o n  [28,30].  The  magnitudes of  13 C-shift  changes have a l s o been u s e f u l  a g g r e g a t i o n of amphiphiles as well  as  i n f o l l o w i n g the  for evaluating  the  progressive aggregation 13  numbers and  equilibrium  c o n s t a n t s [28,43],  p r o v i d e a wealth of q u a n t i t a t i v e  information  Relaxation on  the  studies using  surfactant  C  mobility  [27,44]. The r e s u l t s show a p r o g r e s s i v e i n c r e a s e i n segmental m o b i l i t y of the hydrocarbon c h a i n away from the head group. An e v a l u a t i o n of the motional a n i s o t r o p y [45] of s u r f a c t a n t molecules w i t h i n a m i c e l l e and the 13 fluidity  [45] of the m i c e l l a r i n t e r i o r  relaxation  studies.  have a l s o been o b t a i n e d through  C  26  NMR  s t u d i e s o f deuterons.[45-48] o f both water and  become common i n recent y e a r s . obtained  by  i n an a l k y l dynamics.  Information  H r e l a x a t i o n s t u d i e s of D 0. 2  c h a i n have p r o v i d e d The  special  phase e q u i l i b r i a  amphiphiles  on m i c e l l e h y d r a t i o n Relaxation  2  have been  s t u d i e s o f deuterons  q u a n t i t a t i v e information"on  f e a t u r e of  have  chain  H-NMR i s t h a t i t i s very u s e f u l i n  s t u d i e s [49,50] with which the presence of one  or more  mesophases c o u l d be e a s i l y e s t a b l i s h e d . 19 F NMR [51,52]. and  The  has  been used e x t e n s i v e l y t o study  p o s s i b i l i t i e s of NMR  s t u d i e s u s i n g other  [34].  fluorinated  amphiphiles  are numerous f o r a m p h i p h i l i c  n u c l e i are very well d i s c u s s e d  i n the  systems  literature  27  References  1.  McBain, J.W.,  2.  Tanford, C , Biological  T r a n s . Faraday Soc., "The Hydrophobic  (1913), _9, 99.  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Phys. Chem., (1981), 85, 891.  P.,  14. Menger, F.M.,  i n " B i o o r g a n i c Chemistry, V o l . I l l ,  Macro and  M u l t i m o l e c u l a r Systems", Van Tamelen E.E., Ed., Academic P r e s s , New York, 1977, p. 139. 15. Aniansson, E.A.G., references c i t e d  B e r . Bunsenges. Phys. Chem., (1978), 82_, 981, and  therein.  16. Lindman, B., Wennerstrom, H., i n " M i c e l l e s " , T o p i c s i n C u r r e n t Chemistry, V o l . 87, S p r i n g e r - V e r l a g , New York, 1980, p. 30.  28  17. Reference 2, p. 64-65. 18. Mukerjee,  P., Mysels, K.J.,  "Critical  M i c e l l e Concentrations of  Aqueous M i c e l l e 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, Surfactants.  T., " C o l l o i d a l  Some Physico-chemical P r o p e r t i e s " , Academic P r e s s , New  York, 1963. 20. Reference 16, p. 44-45. 21. R i b e i r o , A.A., D e n i s , E.A., 22. S h i n i t z s k y , M., Dianoux,  J . Phys. Chem., (1977), 81, 957.  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., C h r i s t e n s e n , G.R., Sci.  Proc. Natl•  Acad.  U.S., (1967), 57., 1198.  24. O h n i s h i , S., C y r , T.J.R., Lukushima, H.,  Bui 1. Chem. Soc. J p n . ,  (1970), 43, 673. 25. Dorrance, R . C , Hunter, T.F., J . Chem. S o c . Faraday  I, (1972), 68,  1312. 26. Mukerjee,  P., C a r d i n a l , J.R., Desai , N.R., i n " 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 M i c r o e m u l s i o n s " , M i t t a l ,  K.L. Ed. V o l . I, Plenum  P r e s s , New York, 1977, p. 241. 27. W i l l i a m s , E., S e a r s , B., A l l e r h a n d , A., Cordes, E.H., Soc.,  J . Am. Chem.  (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, Sci.,  (1978), 63, 538.  T., Lindmann, B.,  J . Colloid  Interface  29  31. Waggoner, A.S., K e i t h , A.D., G r i f f i t h , O.H.,  J . Phys. Chem., (1968),  72, 4129. 32. E r i k k s o n ,  J.C., G i l b e r g , G.,  Acta Chem. Scand.,  (1966), 20, 2019.  33. Ulmius, J . , Lindmann, B., Lindblom, G., Drakenberg,  T.,  J . Colloid  I n t e r f a c e S c i . , (1978), 65, 88. 34. Wennerstrom, H., Lindmann, B., 35. Anacker,  E.W.,  Phys. Rep., (1979), 52_, 2.  i n "Cationic Surfactants",  Jungermann, E. Ed., Marcel  Dekker, New York, 1970, p. 203. 36. Reference 16, p. 20. 37. Odberg, L., Svens, B., D a n i e l s o n , I . , J . C o l l o i d  Interface S c i . ,  (1972), 41, 298. 38. Nakagawa, T., Inoue, H., Jizomoto, H., H o r i u c h i , K.,  Kol1oid-Z.Z-  Polym., (1969), 229, 159. 39. C l i f f o r d ,  J . , T r a n s . Faraday S o c , (1965), 61_,  40. Nery, H., Marchal, J.P., Canet, D.,  J . Colloid.  1276. Interface S c i . ,  (1980), 77, 174. 41. Walker,  T.,  42. C l i f f o r d ,  J . Colloid  I n t e r f a c e S c i . , (1973), 4, 372.  J . , P e t h i c a , B.A.,  43. Persson, B.0., Drakenberg,  T r a n s . Faraday S o c , (1965), 61, 182.  T., Lindmann, B.,  J . Phys. Chem., (1979),  83, 3011. 44. Hendriksson, U., Odberg, L., 45. Menger, F.M., J e r k u n i c a ,  Colloid  J.M.,  Polym. S c i . , (1976), 254, 35.  J . Am. Chem. Soc., (1978),, 100, 688.  46. Wennerstrom, H., P e r s s o n , N.-0., Lindmann, B.,  ACS Symp. S e r . ,  (1975), 9, 253. 47. S e e l i g ,  J . , N i e d e r b e r g e r , W . , J . Am. Chem. S o c , (1974), 96, 2069.  48. H e n r i k s s o n , U., Odbnerg, L., E r i k s s o n , J . C , (1975),  M o l . C r y s t . L i q . Cry S t . ,  30  49. Persson, N.-0., Colloid  F o n t e l l , K., Lindmann, B., T i d d y , G.J.T.,  J.  I n t e r f a c e S c i . , (1975), 53, 461.  50. Ulmius, J . , Lindblom, G., Wennerstrom, H., A r v i d s o n , G., Biochemistry,  (1977), 16, 5742.  51. M u l l e r , N., Simsohn, H.,  J . Phys. Chem., (1971), 75, 942.  52. M u l l e r , N., P e l l e r i n , J.H., Chen, W.W., 3012.  J . Phys. Chem., (1972), 76,  CHAPTER I I I  SYNTHESIS OF MODEL GLYCOLIPIDS  32  3.1  -  Previous The  and  work  chemical  synthesis of n a t u r a l l y occurring  g l y c o g l y c e r o l i p i d s has  been the  glycosphingolipids  s u b j e c t of an e x c e l l e n t review [ 1 ] ; a  b r i e f d i s c u s s i o n on the numerous methods used i n model s y n t h e s i s has The chain  a l s o appeared r e c e n t l y [ 2 ] .  simplest  type of model g l y c o l i p i d i s the one  i s j o i n e d d i r e c t l y onto a sugar r i n g .  two  moieties  The  g l y c o s i d a t i o n r e a c t i o n [3,4]  glycosyl  can  be achieved  conditions  been widely  publications  different  an a l c o h o l  b e t t e r p u r i t y and y i e l d with medium  8 - 14)  has  been the  s u b j e c t of  of c a r b o h y d r a t e s t o a l d o b i o n i c a c i d s w i t h  an amide l i n k .  carbohydrate m o i e t i e s  Reductive ami n a t i o n  reaction length  recent  and  been demonstrated by with  been u t i l i z e d  glycolipids  of v a r y i n g  40 ) has  lengths.  arm.  reacting maltotriose  sodium c y a n o b o r o h y d r i d e .  1-alkylamines of v a r y i n g c h a i n  by Hoagland et a l . [ 1 1 ] .  by  with  been a p p l i e d t o  a h y d r o p h i l i c spacer  Read et a l . [10] by  amination o f l a c t o s e with  chains  (Scheme I I I , p.  octadecylamine and  has  a s e r i e s of model alkyl  subsequent  1-alkylamines t o produce  T h i s scheme (p. 38)  s y n t h e s i s of model g l y c o l i p i d s w i t h  been reported  protected  M o d i f i c a t i o n of the o r i g i n a l  et a l . [8,9] t o s y n t h e s i z e  maltotetrose  between these  i n the presence of a s u i t a b l e  t o l a c t o n e s , enables r e a c t i o n with  g l y c o l i p i d s with Williams  alkyl  [5,6,7],  Oxidation conversion  (carbon  coupling  (Scheme I, p 34), where a  employed.  i n order t o o b t a i n  aliphatic alcohols  The  i n which an  v i a e t h e r , e s t e r , amine or amide l i n k a g e s .  h a l i d e i s r e a c t e d with  c a t a l y s t , has  glycolipid  This  the has  and Reductive  lengths  has  also  33  Model g l y c o l i p i d synthesized. cholesterol  substances  based  Coupling o f 2-aminoethyl  on c h o l e s t e r o l  have a l s o been  1-thio-g-D-galactopyranoside  c h l o r o f o r m a t e u s i n g 8-amino-3,6-dioxaoctanoic  spacer group has been r e p o r t e d by Slama et a l . [ 1 2 ] . approaches  but with d i f f e r e n t  Two  with  a c i d as the similar  spacer groups have a l s o been s t u d i e d  [13,14]. K i s o et a l . [15] have r e c o g n i z e d the u t i l i t y  of N-fatty a c y l a t e d  2-amino-2-deoxy-g-D-glycosides as key i n t e r m e d i a t e s f o r s y n t h e s i s of a v a r i e t y o f complex model g l y c o l i p i d s , and Flowers  [16] has r e p o r t e d  synthesis of a s e r i e s of acylamidoalkyl g l y c o s i d e s . Of t h e v a r i e t y o f methods a v a i l a b l e f o r making model it  was  glycolipids,  d e c i d e d t o study t h r e e methods, namely, the g l y c o s i d a t i o n  l i n k a g e through  an amide bond, and the r e d u c t i v e amination  the f o l l o w i n g d i s c u s s i o n each o f these methods w i l l  reaction,  reaction.  In  be d e s c r i b e d  separately.  3.2  -  Method of g l y c o s i d e f o r m a t i o n n-Octyl g - Q - g l u c o s i d e  ( I I I , Scheme 1) was  prepared i n high y i e l d  a d a p t i n g the s y n t h e t i c procedure  d e s c r i b e d i n Ref.  p e r - a c e t y l a t i o n o f D-glucose  c a r r i e d out with a c e t i c anhydride  was  sodium a c e t a t e a t 100°C.  The  tography  judged t o be complete  hours.  ( t . l . c . ) and was The  r e a c t i o n was  5.  monitored  i s o l a t e d product showed the presence  In the f i r s t  step, and  by t h i n - l a y e r chroma-  a f t e r one and a h a l f of only the g-anomer, as  confirmed by ^H-NMR. Subsequent r e a c t i o n of the p e r - a c e t y l a t e d g l u c o s e with 30% HBr i n glacial bromide.  a c e t i c a c i d produced  by  2,3,4,6-tetra-0_-acetyl-a-Q-glucopyranosyl  A f t e r 30 minutes o f r e a c t i o n , the t . l . c . showed o n l y t r a c e s of  35  the s t a r t i n g m a t e r i a l . prolonged  The r e a c t i o n was quenched a t t h i s p o i n t  r e a c t i o n times a r e known t o r e s u l t i n formation o f d e g r a d a t i o n  products o f the glucosyl immediately  showed l i t t l e The  The product i s o l a t e d was  o f t h i s compound s t o r e d  o r no i m p u r i t i e s  glycosidation  bromide and 1-octanol  this  bromide.  used  without f u r t h e r p u r i f i c a t i o n f o r subsequent r e a c t i o n .  ^H-NMR spectrum  and  because  over sodium hydroxide  i n vacuum,  even a f t e r a week.  r e a c t i o n was c a r r i e d out w i t h t h e g l u c o s y l  i n t h e presence o f f r e s h l y prepared  i o d i n e as c a t a l y s t .  The  silver  A study by Chiang e t a l . [ 1 7 ] has proved  i s t h e best c a t a l y s t found thus f a r f o r g l y c o s i d a t i o n  sugars, w h i l s t , with other c a t a l y s t s  carbonate that  o f simple  ( s i l v e r o x i d e , m e r c u r i c o x i d e and  h a l i d e s , and m e r c u r i c c y a n i d e ) h i g h e r p r o p o r t i o n s o f o r t h o e s t e r s are o b t a i ned. N e v e r t h e l e s s , s i 1ver t r i f 1 u o r o m e t h a n e s u l p h o n a t e c o n j u n c t i o n with an a p p r o p r i a t e proton a c c e p t o r  (triflate) in  (1,1,3,3-tetramethylurea)  i s a l s o now i n c r e a s i n g l y used as a c a t a l y s t i n g l y c o s i d a t i o n  reactions  [18,19]. The  condensation  r e a c t i o n was judged t o be complete a f t e r about 6  hours (by t . l . c . ) but was u s u a l l y allowed t o proceed protected  glycoside,  overnight.  The  i s o l a t e d as a c o n c e n t r a t e d s y r u p , was then  d e a c e t y l a t e d with m e t h a n o l / t r i e t h y l a m i n e / w a t e r  (2:1:1).  P u r i f i c a t i o n of  the product was a c h i e v e d by e l u t i o n through a column o f Dowex 1 (2%, OH", 200-400 mesh) r e s i n e q u i l i b r a t e d and e l u t e d with methanol. The  reaction  scheme employed and t h e adapted  be a convenient and an e f f i c i e n t method t o prepare glycoside  i n high p u r i t y .  procedure was found t o large  amounts of t h e  HO  '\»/  C H  2-  CH-,  -(CH )  ~<CH )f  2  2  HO-*  5  W-CH  q  Hm H«<  [A] (3-CH2  00112M  C  10 5(ppm)  20  3.0'  Z,0  2 C n 4  C  3,5  [B]  0.0562M  "~n—  100  ~80~  60  40  20  5(ppm) CO  cn  Figure 3 . 1 :  1 13 H and C-NMR s p e c t r a o f n - o c t y l  8-D-glucoside,  37  The prepared  400  MHz  H-NMR spectrum i n D2O  i s shown i n F i g 3.1  of 2D-J  r e s o l v e d NMR  Chapter  V.  o f n - o c t y l 3-D-glucoside  [A] along with the assignments.  spectroscopy  Also, additional  in spectral  IV.  The  ^C-NMR spectrum o f n - o c t y l 6-D-glucoside  3.3  -  Method of amide bond  Application  assignment i s d i s c u s s e d i n  f e a t u r e s of the spectrum due  f o r m a t i o n are d i s c u s s e d i n Chapter  so  100.6  MHz  to m i c e l l e  partially  i s shown i n F i g . 3.1  assigned [B].  formation  A s e r i e s of N - a l k y l l a c t o b i o n a m i d e d e r i v a t i v e s (V) were prepared a d a p t i n g the procedure formation was  of N - s u b s t i t u t e d aldobionamides  originally  imide  of Ref. 8, which i s o u t l i n e d i n Scheme I I .  (DCC).  c a r r i e d out i n the presence However, DCC  t i o n t o the condensation l a c t o n e [20],  reason  by  of  be r e a d i l y c o n v e r t e d  evaporation  U s u a l l y the product  precipitated  z a t i o n s u f f i c e d t o g i v e an a n a l y t i c a l l y  Q  o  correspond-  DCC. C.J  14  and  IV  16  on s t i r r i n g o v e r n i g h t at room temperature i n  c o u l d be i s o l a t e d d i r e c t l y i n h i g h p u r i t y  C ,  i n t o the  amines (C._, C.., 12  T h i s was  proceeds v i a the  from a non-aqueous s o l v e n t , and  the r e a c t i o n s were performed without  Reactions with long c h a i n a l k y l  methanol.  amines  N,N'-dicyclohexylcarbodi-  r e a c t i o n [ 8 ] , which most l i k e l y  repeated  proceeded t o completion  The  has been found t o make o n l y a minor c o n t r i b u -  A l d o n i c a c i d s can  i n g l a c t o n e [21] for this  from a l d o n i c a c i d s and  by  from the r e a c t i o n medium  (by t . l . c ) ;  one  and  recrystalli-  pure sample.  not the case with medium l e n g t h a l k y l  C , ) , where the r e a c t i o n s d i d not proceed I0 n  c h a i n amines (C ,  t o completion  even on  6  stirring  o v e r n i g h t , and the product  d i d not p r e c i p i t a t e from the r e a c t i o n  medium.  As a r e s u l t t h e s e  had to.be  the  reaction mixture.  The  products  i s o l a t e d by e v a p o r a t i o n  r e s u l t i n g crude m a t e r i a l c o n t a i n e d  of  significant  38  1.  KOH/I  2.  Amberlite  3.  Repeated e v a p o r a t i o n  2  IR-120 ( H ) +  from  t o l u e n e and 3-methoxyethanol  n=5.7.9.11.13.15  Scheme II  Co,  175  125  25  75  <5(ppm)  OK;I  30 Figure 3.2:  1  H and  13  ~?0~  C-NMR s p e c t r a o f N-hexyllactobionamide.  10  5(ppm)  40  amounts o f s t a r t i n g m a t e r i a l  and was p u r i f i e d by passage through a column  o f A m b e r l i t e CG 400 (HCO3) f o l l o w e d by r e c r y s t a l 1 i z a t i o n .  These  reac-  t i o n s c o u l d be d r i v e n f u r t h e r towards completion by working a t e l e v a t e d temperature obtained.  (under r e f l u x i n methanol), which Repeated  recrystal1ization  improved  the y i e l d s  o f the crude product u s u a l l y  a f f o r d e d a high degree o f p u r i t y .  1 The 400 MHz bionamide  13 H-NMR and 100.6 MHz  C-NMR s p e c t r a o f N-hexyl1acto-  i n D2O are shown i n F i g 3.2 with p a r t i a l  assignments;  further  assignments o f t h e ^H spectrum are d i s c u s s e d i n Chapter V.  3.4 -  V i a r e d u c t i v e ami n a t i o n Several  attempts were made t o r e d u c t i v e l y aminate  1-alkylamines u s i n g t h e procedure o u t l i n e d by Hoagland this  r e a c t i o n the r e d u c i n g end o f t h e sugar i n i t i a l l y  amine t o produce an imine (VI) which sodium  i s selectively  l a c t o s e with  et a l . [11].  r e a c t s with t h e  reduced i n s i t u by  cyanoborohydride (NaCNBH^) (Scheme I I I ) .  imine  V1 NaCNBH  Scheme I I I  In  3  41  A major drawback o f t h i s  r e a c t i o n i s t h e tendency  t o form  Amadori  rearrangement [22] products when the r e a c t i o n i s done under a c i d i c conditions.  As r e p o r t e d by Hoagland e t a l . [ l l ] t h i s c o u l d be suppressed  by u s i n g a weak o r g a n i c a c i d  ( p r o p i o n i c or benzoic a c i d ) as the proton  donor. Another p o s s i b l e c o m p l i c a t i o n t h a t c o u l d a r i s e i s t h e formation o f h i g h e r m o l e c u l a r weight substances aldehydo  sugar.  by f u r t h e r r e a c t i o n o f VII with the  Even though p r e c a u t i o n s a g a i n s t t h i s have not been taken  i n t h e r e f e r e n c e c i t e d , r e d u c t i v e aminations  with s m a l l e r molecules are  u s u a l l y c a r r i e d out u s i n g a f i v e - f o l d excess o f t h e amine [ 2 3 ] . E f f o r t s t o c a r r y out t h i s l i v e up t o our e x p e c t a t i o n s . c o n d t i o n s employed t h e product  r e a c t i o n with medium c h a i n amines d i d not  Even with t h e v a r i e t y o f d i f f e r e n t i n each case was contaminated  amounts o f rearrangement products o r h i g h e r m o l e c u l a r weight Attempts t o p u r i f y t h e product mixture chromatography were not s u c c e s s f u l . encountered,  reaction  with v a r y i n g substances.  by ion-exchange or gel f i l t r a t i o n  Because o f these  i t was decided not t o i n v e s t i g a t e t h i s  problems  r e a c t i o n any f u r t h e r  as a means of making model g l y c o l i p i d s . We b e l i e v e t h a t our e x p e r i e n c e c a s t s some doubts on the  3.5 -  r e p o r t e d by Hoagland et a l .  apparatus  m e l t i n g p o i n t s were recorded u s i n g a F i s h e r - J o h n s m e l t i n g p o i n t and a r e u n c o r r e c t e d .  rotary evaporator.  A l l s o l u t i o n s were evaporated  Thin l a y e r chromatography  on 7.5 x 2.5 cm B a k e r - f l e x (J.T. Baker Chemical gel  plates.  ethyl  [11].  Experimental All  Buchi  C shifts  The s o l v e n t systems, I - e t h y l  acetate/methanol  using a  ( t . l . c . ) was  performed  Co. N.J.) precoated  acetate/hexane  silica  (1:1 v / v ) , I I -  (4:1 v/v) and I I I - 1 - b u t a n o l / a c e t i c a c i d / d i e t h y l  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 e f f e c t e d by  s p r a y i n g with 30% s u l p h u r i c a c i d i n ethanol and h e a t i n g . the samples  M i c r o a n a l y s e s of  were c a r r i e d out by Mr. P. Borda, M i c r o a n a l y s i s L a b o r a t o r y ,  University of B r i t i s h  Columbia.  Nuclear magnetic  resonance  s p e c t r a were  recorded with a Bruker WH-400 h i g h r e s o l u t i o n s p e c t r o m e t e r o p e r a t i n g a t 400 MHz  and equipped with an Aspect 2000 computer. All  t h e s o l v e n t s used were of s p e c t r o or reagent grade and were  used without f u r t h e r t r e a t m e n t .  Hexyl, dodecyl and hexadecylamines  purchased from Eastman Kodak Co. whereas t h e o c t y l , decyl amines were purchased from A l d r i c h  Chemical  Co.  were  and t e t r a d e c y l -  Anhydrous  silver  carbonate was prepared u s i n g t h e procedure o f Wolfrom e t a l . [ 2 5 ] ,  P r e p a r a t i o n o f 1,2,3,4,6-penta-O-acetyl-B-D-glucopyranose  D-Glucose a c e t a t e i n 50 ml a bath a t 100°C.  (10 g, 0.055 mol) was added t o 5 g o f anhydrous  A f t e r a l l t h e g l u c o s e was d i s s o l v e d , t h e r e a c t i o n was  by t . l . c .  one spot w i t h an  i n s o l v e n t system  Progress o f the r e a c t i o n  co-migrated with a u t h e n t i c  The r e a c t i o n mixture was then poured  in a fine  stream i n t o 500 ml o f i c e - w a t e r and s t i r r e d  f o r 2 hours.  p r e c i p i t a t e was f i l t e r e d and washed s e v e r a l  times w i t h sodium  s o l u t i o n and then with i c e c o l d water  and d r i e d  under  m.p.  133-134°C ( l i t .  The r e s u l t a n t  suction.  product was then r e c r y s t a l 1 i z e d from 95% ethanol t o y i e l d i n 90% y i e l d ,  was  I and a f t e r 1 1/2 h r s r e v e a l e d o n l y  v a l u e o f 0.30, which  glucose pentaacetate.  material  sodium  (0.52 mol) o f a c e t i c anhydride p r e v i o u s l y e q u i l i b r a t e d i n  allowed t o proceed f o r a f u r t h e r h a l f hour. monitored  (I)  v a l u e 134°C).  carbonate The  a crystalline  43  Preparation of 2,3,4,6-tetra-O^acetyl-a-D-glucopyranosyl  To a s t i r r e d 5 g (0.013 mol)  s o l u t i o n o f 10 ml  o f I was  at t h i s  o n l y t r a c e s of s t a r t i n g m a t e r i a l .  5 min.  c h l o r o f o r m and  The  poured  o r g a n i c l a y e r was  dure was  i n t o 150 ml  of i c e - w a t e r and  then s e p a r a t e d and washed w i t h  syrup was  the decomposition  then evaporated t o a t h i c k  syrup  i n 78% y i e l d  and used  immediately  B-D-glucopyranoside  for further  d i s s o l v e d i n 60 ml  The  ml  s t o r e d o v e r 3-A° m o l e c u l a r s i e v e s , 1.8 c a r b o n a t e , 0.15  r e a c t i o n was  35°C). petroleum  and s u c t i o n  dried  The  II  product  was  reaction.  bromide ( I I ) (4 g,  9.7  stirring  (11 mmol) of 1-octanol t h a t has g of f r e s h l y  g o f i o d i n e and 4.0  allowed t o proceed  dried  overnight in  of c h l o r o f o r m and with e f f i c i e n t  f o l l o w i n g a d d i t i o n s were made: 1.7  proce-  (III)  2,3,4,6-Tetra-0_-acetyl-a-D-glucopyranosyl  The  of I I .  allowed t o c r y s t a l l i z e  The c r y s t a l s were r e c o v e r e d by f i l t r a t i o n  Preparation of n-octyl  silver  After a  d r i e d over c a l c i u m  (temperature  t a k i n g c a r e t o prevent e x c e s s i v e exposure t o m o i s t u r e .  mmol) was  for  ice-cold  d i s s o l v e d i n 15 ml o f anhydrous e t h e r and 25 ml o f  added and the product was  the f r e e z e r .  isolated  stirred  the aqueous l a y e r stayed b a s i c .  kept t o a minimum t o minimize  e t h e r was  diluted  The time i n v o l v e d d u r i n g the above s e p a r a t i o n and washing  o r g a n i c e x t r a c t was The  The  point i n s o l v e n t system I showed  washing with i c e - c o l d water the o r g a n i c l a y e r was  chloride.  HOAc (w/w),  The homogeneous s o l u t i o n was  s a t u r a t e d sodium b i c a r b o n a t e u n t i l final  in glacial  added, and s t i r r i n g c o n t i n u e d f o r 30 min.  t . l . c . o f the r e a c t i o n mixture  w i t h 20 ml  of 30% HBr  bromide ( I I )  g of 4-A  prepared and 9  molecular  the been  dried  sieves.  o v e r n i g h t even though the t . l . c . i n  44  s o l v e n t system  I showed completion o f t h e r e a c t i o n a f t e r 6 h r s (R^ o f o c t y l  g l u c o s i d e p e r - a c e t a t e = 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 w i t h 50 ml c h l o r o f o r m and t h e f i l t r a t e c o n c e n t r a t e d to a syrup.  D e a c e t y l a t i o n was a c h i e v e d by d i s s o l v i n g t h e syrup i n 20 ml  o f m e t h a n o l / t r i e t h y l amine/water (2:1:1 s t a n d 10 h r s a t room temperature. (solvent  system  v/v) and a l l o w i n g t h e s o l u t i o n t o  D e a c e t y l a t i o n was monitored.by  I I ) , and a f t e r 10 h r s t h e r e a c t i o n mixture showed two  spots c o r r e s p o n d i n g t o n - o c t y l  B-Q-glucopyranoside  ( R = 0.52) as t h e f  major product and small amounts o f g l u c o s e ( R = 0.18).  The r e a c t i o n mix-  f  t u r e was then c o n c e n t r a t e d t o a syrup, s i m u l t a n e o u s l y removing ted 1-octanol. of methanol  -t.1 . e . ~  any unreac-  i n a minimum amount  The crude m a t e r i a l was then d i s s o l v e d  and passed through a column o f Dowex 1 (2% c r o s s - l i n k e d ; 0H~  form; 200-400 mesh) p r e v i o u s l y e q u i l i b r a t e d w i t h methanol. n - o c t y l B-Q-glucopyranoside was then  recrystal1ized  duce f l a k y c r y s t a l s i n 76% y i e l d , m.p.  58-60°C;  Purified  from 95% EtOH t o pro-  [ ] p - 2 4 . 3 ° ( C 0.. 63%, • MeOH). 2  :  a  M i c r o a n a l y s i s , c a l c u l a t e d : C, 57.51; H, 9.65; found:  C, 57.23; H, 9.36%.  P r e p a r a t i o n o f N - a l k y l l a c t o b i o n a m i d e s (V)  g-D-Lactose  was f i r s t  o x i d i z e d t o potassium l a c t o b i o n a t e w i t h  KOH/I2, by a d a p t i n g the procedure by Moore e t a l [ 2 4 ] .  To a s t i r r e d  s o l u t i o n o f 5.7 g o f I ^ and 80 ml o f MeOH p r e v i o u s l y e q u i l i b r a t e d a t 40°C, a warm c o n c e n t r a t e d s o l u t i o n o f a - D - l a c t o s e (4 g, 0.012 mol) was  added.  Then t o t h e s t i r r V d  added dropwise  (15 - 20 min)  mixture, 65 ml o f 4% K0H i n methanol was  and allowed t o s t i r f o r 10 min.  50 mis o f 4% KOH/MeOH s o l u t i o n was added dropwise final  i n methanol  c o l o r o f t h e r e a c t i o n mixture  (light  at t h i s  A further  p o i n t and t h e  straw-yellow) i n d i c a t e d t h e  45  removal  o f n e a r l y a l l the f r e e i o d i n e .  m a i n t a i n e d throughout t h e r e a c t i o n . a final  A temperature  The r e a c t i o n mixture was s t i r r e d f o r  10 min and c o o l e d t o room temperature.  l a c t o b i o n a t e s e t t l e d t o t h e bottom filtered  o f 40°C was  At t h i s p o i n t  of the f l a s k  and the s o l u t i o n was  and t h e p r e c i p i t a t e washed with MeOH and e t h e r .  product was then  recrystal1ized  from water  potassium  and methanol  The crude  t o achieve a y i e l d  of 94%. Pure potassium l a c t o b i o n a t e was then d i s s o l v e d  i n water,  passed  through a A m b e r l i t e IR-120 ( H , 20-50 mesh) column and t h e aqueous e l u a n t +  was  evaporated and d r i e d a t 40°'C under  vacuum t o o b t a i n l a c t o b i o n i c  The f r e e a c i d was then c o n v e r t e d t o l a c t o b i o n o - l , 5 - l a c t o n e repeated e v a p o r a t i o n from 2-methoxyethanol o b t a i n e d i n 92.5% y i e l d  from t h e s t a r t i n g  and t o l u e n e .  acid.  (IV) by  Compound IV was  lactose.  R e a c t i o n w i t h t h e 1-alkylamines was then a c h i e v e d by s t i r r i n g (2.9 mmol) o f l a c t o n e of methanol.  IV with 3.5 mmol o f 1-alkylamine o v e r n i g h t i n 25 ml  With hexyl  (Cg), o c t y l  (Cg) and decyl  r e a c t i o n was c a r r i e d out i n b o i l i n g methanol, tetradecyl smoothly  1 g  ( C ^ ) , and hexadecyl  a t room temperature.  (C-| ) amines, t h e 0  whereas with dodecyl  (C-|g) amines t h e r e a c t i o n  (C-jpJ,  proceeded  The Cg, Cg, and C-jQ N - a l k y l l a c t o b i o n a m i d e s  were i s o l a t e d by e v a p o r a t i o n o f t h e r e a c t i o n m i x t u r e and subsequent washing  ( e t h e r ) and d r y i n g .  usually  p r e c i p i t a t e d from  f o l l o w e d by washing for their  The C-^, C-^, and C-|g N - a l k y l l a c t o b i o n a m i d e s  the r e a c t i o n mixture and were i s o l a t e d  ( e t h e r ) and d r y i n g .  p u r i t y by t . l . c .  i n solvent  a c h i e v e d by r e c r y s t a l l i z a t i o n the y i e l d  ranged  either  by f i l t r a t i  A l l l a c t o b i o n a m i d e s were checked  system  III.  Final  p u r i f i c a t i o n was  from a b s o l u t e ethanol or methanol and  from 90-92% based on t h e l a c t o n e .  the compounds prepared a r e given below.  Physical  constants of  46  Tahle  3,1 - P h y s i c a l  constants  o f N-alkyllactonionamide  series  Microanalysis  Compound  %  found  C  H  Mp  % cal cul ated  t°C)  C  H  N-Hexyl  142-143  48.97  7.99  3.17  48.96  8.18  3.09  + 31 .8 (C 0.82%)  N-Octyl  146-147  51.16  8.37  2.98  51.25  8.48  2.97  + 25.4 (C 0.078%.)  N-Decyl  145-146  53.10  8.71  2.81  52.84  8.76  2.85  + 28.8 (C 0.51%)  N-Dodecyl  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  n.d.  [ a ]  2 n  °  (MeOH)  n.d.  -' :ndt' determined  47  References  1.  Gigg, R.,  Chem. Phys. L i p i d s ,  (1980), 26,  287.  2.  A p l i n , J.D., W r i s t o n , J.C., C r i t i c a l Rev. Biochem., ( 1 9 8 1 ) ,  1_,  (4),  289. 3.  W h i s t l e r , R.L., Wolfrom, M.L., Chemistry, Volume  4.  I g a r a s h i , K.  Eds., Methods i n Carbohydrate  I I , Academic Press, NY, 1 9 6 3 .  Adv. Carbohydr. Chem. Biochem. V o l . 3 4 . , T i p s o n , R.S.,  Horton, D. Eds., Academic P r e s s , NY, 1 9 7 7 , p. 2 4 3 . 5.  Rosevear, P., VanAken, T., B a x t e r , J . , F e r g u s o n - M i l l e r , S., Biochemistry,  6.  ( 1 9 8 0 ) , J_9; 4 1 0 8 .  De G r i p , 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.  W i l l i a m s , T . J . , P l e s s a s , N.R., G o l d s t e i n , I . J . , Carbohydr. Res., ( 1 9 7 8 ) , 6 7 , CI.  9.  W i l l i a m s , T . J . , P l e s s a s , N.R., G o l d s t e i n , I . J . , A r c h . Biochem. Biophys.,  10.  ( 1 9 7 9 ) , 195_, 1 4 5 .  Read, B.D., Demel, R.A., Wiegandt, H., Van Deenen, L.L.M., Biochem. Biophys. Acta,  11.  (1977), 470, 325.  Hoagland, P.D., P f e f f e r , P.E., V a l e n t i n e , K.M., Carbohydr. Res., (1979) , 74, 1 3 5 .  12.  Slama, J . , Rando, R.R.,  Carbohydr. Res., ( 1 9 8 1 ) , 8 8 ,  13.  O r r , G.A., Rando, R.R.,  Baugerter, F.W.,  213.  J . B i o l . Chem., ( 1 9 7 9 ) ,  254, 4 7 2 1 . 14.  Chabala, J.C., Shen, T.Y., Carbohydr. Res., ( 1 9 7 8 ) , 67_,  55.  48  15.  K i s o , M.,  N i s h i g u c h i , H., Hasegawa, A.,  Carbohydr. Res., (1980), 81_,  C13. 16.  Flowers, H.M.,  17.  Chiang, C K . ,  Carbohydr. Res., (1976), 46, 133. McAndrew, M.,  Barker, R.,  Carbohydr. Res., (1979), 70,  93. 18.  Banoub, J . , Bundle, D.R.,  19.  Hanessian, S., Banoub, J . ,  20.  F i e s e r , M., T e f f t , M.,  21.  Isbell,  Can. J . Chem., (1979), 57, 2091. Carbohydr. Res., (1977), 53_, C13.  F i e s e r , L.F., Toromanoff, E., Hi r a t a , Y., Heymann, H., B h a t t a c h a r y a , S.,  H.S.,  II, Whistler  F l u s h , H.L.,  J . Am. Chem. S o c , (1956), 78, 2825. Methods i n Carbohydrate Chemistry, Volume  R.L. and Wolfrom M.L.  Eds. Academic Press,  NY, 1963 p.  17. 22.  Hodge, J . E . , F i s h e r , B.E., i b i d , p. 99  23.  Borch, R.F., B e r n s t e i n , M.D., 93,  Durst, H.D.,  J . Am. Chem. Soc., (1971),  2897.  24.  Moore, S., L i n k , K.P., J . B i o l . Chem., (1940), 133, 293.  25.  Wolfrom, M.L., Whistler,  Lineback, D.R.,  R.L., Wolfrom, M.L.,  I I , 1963, p. 342.  Methods i n Carbohydrate  Chemistry,  Eds., Academic P r e s s , New York,  Volume  CHAPTER IV  NMR  STUDIES OF MODEL GLYCOLIPIDS  50  4.1  -  'H-NMR S t u d i e s n-Octyl  the c r i t i c a l fluorescent octyl  of n - o c t y l B-Q-glucopyranoside  B-Q-glucopyranoside* i s well  micelle concentration  (cmc)  known t o form m i c e l l e s , and  has been determined by u s i n g  probes [1] and by s u r f a c e t e n s i o n measurements [ 2 ] .  g l u c o s i d e as a m i l d , d i a l y s a b l e , n o n i o n i c detergent  The use o f  in solubilization  of membrane p r o t e i n s i s i n c r e a s i n g [ 3 ] , The  400 MHz ^H-NMR s p e c t r a o f o c t y l  d i f f e r e n t concentrations spectral  o f the amphiphile are shown i n F i g . 4.1; t h e  assignment i s d i s c u s s e d  concentration  was prepared  most c o n c e n t r a t e d  g l u c o s i d e i n D^O recorded a t  i n Chapter V.  Each s o l u t i o n o f d i f f e r e n t  both s e p a r a t e l y and by stepwise  solution.  The s p e c t r a o b t a i n e d  dilution  o f the  from both s e t s o f  s o l u t i o n s were found t o be i d e n t i c a l . The  most s t r i k i n g changes i n the s p e c t r a occur  between 3.0 - 4.0 6 ; these the a-CH2 of the a l k y l  i n c l u d e those  chain.  dilution.  Another f e a t u r e which i s evident  e a s i l y n o t i c e a b l e with  *  shift. from the s p e c t r a i s t h e i n c r e a s e  increase i n concentration.  This e f f e c t  i s most  H-2 and 0-CH2 resonances.  and l i n e - w i d t h on c o n c e n t r a t i o n  Hereafter  also  s h i f t s r e s p e c t i v e l y with  It seems p l a u s i b l e t o a t t r i b u t e t h i s dependence o f both shift  shift  Moreover, a c l o s e r look a t the methylene  envelope a t 1.4 <5 a l s o shows a d o w n f i e l d  s p e c t r a l l i n e - w i d t h with  as well as  The H-l and H-2 protons  but gradual, u p f i e l d and d o w n f i e l d  increase i n concentration.  in  o f the sugar protons  resonances  There i s a marked change i n chemical  of H-4, H-5, H-6.B; and H-aB with show a minor  f o r the  r e f e r r e d t o as o c t y l  o f the amphiphile,  glucoside.  t o the  chemical formation  51  T  I  •  i  40  i  i  i  3.0  i  r  1-0  2-0  S/ppm Figure 4.1:  400 MHz ^H NMR different  spectra of octyl  concentrations.  glucoside  Experimental  number o f scans = 12, a c q u i s i t i o n width = 2202.6 Hz, temperature  i n d^O a t  parameters:  time = 1.8 s, sweep-  = 298°C.  52  of m i c e l l e s a t h i g h e r s o l u t e c o n c e n t r a t i o n [ 4 ] ,  The sugar m o i e t i e s o f t h e  molecules form t h e o u t e r l a y e r o f t h e m i c e l l e s , w h i l e t h e a l i p h a t i c c h a i n s occupy t h e i n t e r i o r .  Mi eel 1 i z a t i o n  brings the individual  t o g e t h e r and hence can m u t u a l l y i n f l u e n c e the l o c a l e l e c t r o n i c environment environment  concerned.  as well  closer  as t h e  o f t h e atoms. A p p r e c i a b l e changes i n e l e c t r o n i c  c o u l d o n l y be caused  or u n s a t u r a t e d  molecules  (through space) by the presence o f p o l a r  (e.g. aromatic) groups  i n t h e near p r o x i m i t y o f t h e atoms  The e f f e c t due t o t h e -OH groups w i l l  be mostly f e l t  by t h e  sugar protons and t h e a l i p h a t i c protons a r e not expected t o e x p e r i e n c e i t t o any s i g n i f i c a n t e x t e n t . major change i n l o c a l  On t h e c o n t r a r y , the a l k y l  environment  chains experience a  upon m i c e l l e f o r m a t i o n s i n c e they a r e now  imbedded i n t h e hydrophobic i n t e r i o r o f the m i c e l l e [ 5 ] , w h i l s t t h e sugar head groups s t i l l  remain  of h y d r a t i o n [ 6 ] .  i n a h y d r o p h i l i c environment  The above mentioned  r e f l e c t e d as changes i n chemical  c r e a t e d by t h e water  d i f f e r e n c e s i n environment are  s h i f t s i n t h e ^H-NMR spectrum.  The p r o c e s s o f m o l e c u l a r a g g r e g a t i o n i s a s s o c i a t e d with an i n c r e a s e in the r o t a t i o n a l  c o r r e l a t i o n time  decrease i n t h e s p i n - s p i n the NMR  spectral  c  r e l a x a t i o n time  ( T ) o f the n u c l e i 2  [ 8 ] . Since  line-widths are i n v e r s e l y proportional t o the spin-spin  r e l a x a t i o n time [ 9 ] , t h e s p e c t r a l formation of m i c e l l a r  Spin-lattice  (x ) [ 7 ] o f t h e m o l e c u l e , and hence a  l i n e s become i n c r e a s i n g l y broader on  aggregates.  relaxation  studies:  The continuous change i n t h e NMR  spectrum o f o c t y l  glucoside with  i n c r e a s e i n c o n c e n t r a t i o n makes i t i m p o s s i b l e t o determine t h e cmc by direct  inspection.  spin-lattice  Thus, i t was contemplated t h a t an i n v e s t i g a t i o n o f  relaxation  rates  (R-,) o f protons would be more rewarding.  53  The  values f o r R-| o f t h e w-CHg, H-2 and H - l protons  determined  of octyl  glucoside  a t d i f f e r e n t c o n c e n t r a t i o n s are given i n Table 4.1, while the  p l o t s o f R-j versus  inverse total  c o n c e n t r a t i o n s (1/C) a r e shown i n F i g .  4.2. The  p l o t s f o r H-2 and w-CH  the cmc i n s t e a d o f t h e sharp s e p a r a t i o n model  (Chapter  can be determined  show a p r o g r e s s i v e t r a n s i t i o n  3  discontinuity  I I , p 19).  p r e d i c t e d by t h e phase  N e v e r t h e l e s s , t h e cmc o f t h e system  by e x t r a p o l a t i o n o f the l i n e a r  t h e i r p o i n t s o f i n t e r s e c t i o n taken  around  as t h e cmc.  r e g i o n s o f these  p l o t s and  The cmc v a l u e s so o b t a i n e d  from t h e data f o r a>-CH and H-2 a r e 0.023 ± 0.001 M and 0.022 ± 0.002 M; 3  t h e s e , w i t h i n experimental literature  e r r o r , agree w e l l with t h e v a l u e s quoted i n t h 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 t h e H-2 and co-CHg resonances  both  show sharp  i n c r e a s e s i n t h e i r R-j v a l u e s .  appears t o r e l a x a t a c o n s t a n t  r a t e ( i . e . u n a f f e c t e d by t h e phase  t r a n s i t i o n ) ; c l e a r l y t h i s merits For t h e protons a dilute solution experimental  comment.  o f a small molecule  i n a magnetically inert  evidence  In c o n t r a s t , H-l  undergoing  i s o t r o p i c motion i n  solvent, there i s substantial  t o show t h a t t h e dominant c o n t r i b u t i o n t o r e l a x a t i o n  a r i s e s v i a t h e i n t r a m o l e c u l a r d i p o l e - d i p o l e mechanism [10,11], l y , the rate of r e l a x a t i o n  Consequent-  (R-| ) i s given by 2  r  2  i j  where, ,YJ• and -,Y • a r e t h e gyromagnetic r a t i o s o f the two n u c l e i r e l a x a t i o n , r . . i s t h e i n t e r n u c l e a r d i s t a n c e , and T i.J  c o r r e l a t i o n time o f t h e v e c t o r between the n u c l e i  undergoing  i s the rotational c  i and j .  54  T a b l e 4.1 - S p i n - l a t t i c e octyl  .  r e l a x a t i o n r a t e s , (R-j) o f s e l e c t e d protons o f  g l u c o s i d e a t d i f f e r e n t c o n c e n t r a t i o n s i n D2O.  R, Csee" )  ..  1  . 11.  Concentration moles/1  oj-CH  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  ?  H,,  o  o •«•-  \  a  LU • CO a  |  o ' v<4  is  CH  o  I  %t  A  — — £ j  _e_  B  ^a)-CH  3  CMC o o  1  H 0.0  40.0  I  BO.O  1/C  F i g u r e 4.2:  Variation of R f o r w-CHo, H-2  1  160.0  L/M0LE  with and  I  120.0  inverse total  H-l  protons  concentration.  of o c t y l  glucoside.  I  200  56  Assuming t h a t i n t r a m o l e c u l a r d i p o l e - d i p o l e r e l a x a t i o n i s e x c l u s i v e l y dominant f o r t h e s o l u t i o n s s t u d i e d here, the i n c r e a s e i n t h e H-2 and - C H  can be e x p l a i n e d  U  3  time (x ) upon m i c e l l i z a t i o n the  r a t i o of  ^1(micelle) R](monomer)  immobilization micelles.  c  experienced  a  by an i n c r e a s e i n r o t a t i o n a l (equation  n  b  g  t  a  k  e  n  4.1). a  s  a  m  e  a  s  correlation  It f o l l o w s from t h i s , t h a t u  r  e  0  f t h e motional  by t h e groups i n the molecule on formation o f  The values o f t h i s parameter c a l c u l a t e d , f o r H-2 and oj-CHg are  1.77 and 2.55 r e s p e c t i v e l y .  This c a l c u l a t i o n  t h a t s o l u t i o n s with c o n c e n t r a t i o n s  i s based on the assumption  0.0068 M and 0.1089 M t r u l y  represent  the motion o f a monomer and a m i c e l l a r s o l u t i o n r e s p e c t i v e l y . values  value o f  From  i t can be i n f e r r e d t h a t upon m i c e l l i z a t i o n , t h e t e r m i n a l  group undergoes i m m o b i l i z a t i o n  those  methyl  t o a g r e a t e r degree than the sugar head  group. The  obvious q u e s t i o n  outstanding  a t t h i s j u n c t u r e i s why H - l does  not show t h e same s i g n s o f i m m o b i l i z a t i o n  -upon m i c e l l i z a t i o n  given t h a t t h e sugar r i n g i s r i g i d , any hindrance sugar moiety should  be e x p e r i e n c e d  as does H-2;  t o t h e motion o f t h e  e q u a l l y by a l l the sugar p r o t o n s .  This  anomalous behaviour o f R-| of H - l c o u l d be r a t i o n a l i z e d by t h e changes i n conformation close-packing  which accompany m i c e l l e formation of molecules.  If t h i s conformational  the d i s t a n c e between H-l and o t h e r tion  i s increased  would decrease R-|.  i n order t o f a c i l i t a t e t h e  protons  ( i . e . an i n c r e a s e i n r-^  change was such t h a t  which c o n t r i b u t e t o i t s r e l a x a i n equation  change i n r  a s u b s t a n t i a l d i f f e r e n c e i n t h e r e l a x a t i o n r a t e (R-|).  tion  this  Moreover, s i n c e R-j i s i n v e r s e l y p r o p o r t i o n a l t o t h e  s i x t h power of the i n t e r - p r o t o n d i s t a n c e , a small  effect  4.1), then  would cause  In t h i s way t h e  of i n c r e a s e 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 t h e r e l a x a -  r a t e o f H - l , would be n u l l i f i e d  by the accompanying  conformational  57  change. all  It should a l s o be noted t h a t i t i s not necessary t o p o s t u l a t e t h a t  molecules  w i t h i n a m i c e l l e have undergone c o n f o r m a t i o n a l  It i s worthwhile  change.  p u r s u i n g t h i s e x p l a n a t i o n f u r t h e r because i t has  been demonstrated t h a t f o r methyl  g l y c o s i d e s , the aglycon methyl  group  protons can c o n t r i b u t e as much as 50% t o the r e l a x a t i o n o f the anomeric (H-l) proton  [10].  Therefore the conformation  g l y c o s i d i c oxygen (C, - 0, and C  I I  - 0J  about the two  bonds from  can be of prime importance  the  in  I  a  e s t a b l i s h i n g the r e l a x a t i o n pathways of H-l ( F i g . 4.3).  Figure 4.3:  Stereochemical  view o f the p r e f e r r e d conformation  about  the C|-0.j and C -0.| bonds o f a g l y c o s i d e . A c c o r d i n g t o Lemieux and co-workers [12,13], the p r e f e r r e d o r i e n t a t i o n of the C -0^, bond i s determined a  :  by the so c a l l e d  exo-anomeric  e f f e c t ; the favoured conformation  being t h a t which has the aglycon  gauche t o H-l and the r i n g oxygen  (O5)  Fig.  I, i n  4.4. As d e p i c t e d i n the p r o j e c t i o n  conformation  about the C'-0,  a t r a n s arrangement. one of the protons proton  as shown i n the p r o j e c t i o n  carbon  1  I I , ( F i g . 4.4)  the most favoured  bond would be the one with C, and C  When a l l t h i s i s combined we  1  £  i n the  have the s i t u a t i o n where  (H-aA) on the aglycon carbon, i s c l o s e s t t o the anomeric  (H-l) w h i l s t the o t h e r (H-aB) i s i n c l o s e p r o x i m i t y t o the  ring  58  oxygen  (Og)  (Fig. 4 . 3 ) .  It seems reasonable t o suggest t h a t t h i s would  the most p r e f e r r e d conformation  of o c t y l  g l y c o s i d e i n the monomeric  T h i s i s indeed supported by the f a c t t h a t H-aA and chemical  shifts  (Fig. 4 . 1 [ A ] ) ,  where H-aB  H-aB  inter-  (Or).  a c t i o n with the lone p a i r s o f e l e c t r o n s o f the r i n g oxygen  P r o j e c t i o n along C - 0 , bond  1 1  Figure 4 . 4 :  state.  have very d i f f e r e n t  i s more s h i e l d e d by the  P r o j e c t i o n along 0,-C, bond  be  a  Conformational  projections  involving  1  C^ , 0-| and  C^  of a glycoside. In the m i c e l l a r s t a t e , the conformation a l l o w f o r t h e maximum e x t e n t o f hydrophobic cules. tional  This requirement  of the molecules  has t o  i n t e r a c t i o n between the mole-  c o u l d be expected t o o v e r r i d e the normal  conforma-  f o r c e s , e s p e c i a l l y when the p o l a r head-group i s r a t h e r b u l k y .  the case o f o c t y l  g l u c o s i d e with the bulky sugar head-group, the  might f a v o u r a more p i a n a r or a bent c o n f o r m a t i o n ; t h i s c o u l d be a c h i e v e d by an i n c r e a s e i n the d i h e d r a l happens, the d i s t a n c e between H - l and  a n g l e , <>l ( F i g . 4 . 3 ) .  molecule easily  If t h i s  H-aA would be i n c r e a s e d , c a u s i n g a  decrease i n the r e l a x a t i o n c o n t r i b u t i o n t o H - l from H - a A . i n c r e a s e i n the d i h e d r a l  In  F u r t h e r , an  angle would f o r c e H-aB t o move c l o s e r t o the  ring  59  oxygen thus c a u s i n g  i t to experience  a g r e a t e r s h i e l d i n g e f f e c t from  e l e c t r o n lone p a i r s . This i s r e f l e c t e d i n the NMR [ A ] - [ D ] ) as an i n c r e a s e i n chemical The  s h i f t of  spectra  l e a s t the p o r t i o n of the sugar r i n g i n v o l v i n g H-2  relaxation  ( - j j ) t o H-2 r  r a t e i s p r i m a r i l y due  are constant  the assumption t h a t a t  i s r i g i d , so t h a t  and  the change i n i t s  t o the change i n c o r r e l a t i o n time ( T ) . C  This c o u l d be v e r i f i e d by comparing the c o u p l i n g constant  (J) values  each proton  i n the sugar r i n g i n monomer and m i c e l l a r s t a t e s .  i n J values  (AJ)  measured with the a i d o f 2D J - r e s o l v e d  (Chapter V) f o r s p e c t r a  J  AJ  [A] and  l,2  J  0.2  i n d i c a t e any accurate  [C] ( F i g . 4.1)  2,3  0.1  These r a t h e r small  J  f a c t o r s which can  NMR  are given  3,4  J  The  spectroscopy  below:  not  be c o n s i d e r e d  upon m i c e l 1 i z a t i o n .  of t h i s would r e q u i r e a c a r e f u l e v a l u a t i o n of  Even though the  change  0.9  changes i n J values may  i n f l u e n c e the J  for  4,5  0.7  s i g n i f i c a n t amount of r i n g puckering  estimate  4.1  H-aB.  b a s i s f o r the above argument depends on  interproton distances  (Fig.  the  to An  other  values.  r a t i o n a l i z a t i o n discussed  i n t h i s s e c t i o n i s not  a b s o l u t e l y c o n c l u s i v e i t i l l u s t r a t e s the important deformations i n a molecule which can can  have on  Temperature The  accompany m i c e l l e f o r m a t i o n ;  and  a l s o the e f f e c t s these  s p e c t r a l parameters.  effects: temperature a t which n o n i o n i c m i c e l l a r s o l u t i o n s show a sudden  increase in t u r b i d i t y  i s c a l l e d the c l o u d p o i n t .  The  s i z e of  the  60  s u r f a c t a n t aggregates has been shown t o i n c r e a s e r a p i d l y as t h e temperature is  r a i s e d towards  t h i s point  [14,15],  T h i s o b s e r v a t i o n has been  i n t e r p r e t e d as i n d i c a t i v e o f f o r m a t i o n o f l a r g e r m i c e l l e s o r  self-  aggregation o f smaller m i c e l l e s [15], In an attempt t o observe t h i s e f f e c t , octyl  1  H-NMR s p e c t r a o f m i c e l l a r  g l u c o s i d e s o l u t i o n s were recorded a t s e v e r a l  25-85°'C.  temperatures  None o f these s p e c t r a showed any s u b s t a n t i a l  l i n e - w i d t h s o r chemical s h i f t s with temperature.  i n range the  difference i n the  The same type o f  behaviour has been observed by S t a p l e s and Tiddy [ 1 6 ] w i t h p o l y e t h y l e n e oxide s u r f a c t a n t s .  Moreover,  the ^H-NMR spectrum o f o c t y l  i t was not p o s s i b l e t o observe any changes i n g l u c o s i d e s o l u t i o n s s l i g h t l y above o r below  the cmc w i t h i n t h e same temperature dependence o f the cmc o f o c t y l  4.2 -  13  100.6 MHz C-NMR 13  This shows the weak  temperature  glucoside.  C-NMR s t u d i e s o f o c t y l  The  range.  glucoside  spectra of octyl  g l u c o s i d e a t 0.0112 M and  0.0562 M c o n c e n t r a t i o n s c o r r e s p o n d i n g t o monomer and m i c e l l a r r e s p e c t i v e l y a r e shown i n F i g . 4.5; t h e i n d i c a t e d s p e c t r a l  solutions assignments o f  the sugar moiety a r e a c c o r d i n g t o t h a t given by P f e f f e r e t a l . [ 1 7 ] . The change i n chemical s h i f t s  ( A 6 ) f o r each resonance between t h e two s p e c t r a are  t a b u l a t e d i n Table 4.2 and a d o w n f i e l d s h i f t in- the m i c e l l a r spectrum :  :  t o t h e monomer spectrum  i s considered p o s i t i v e .  1  The f i g u r e  compared  accompanying  T a b l e 4.2 shows t h e r e l a t i v e s i g n s o f t h e s h i f t s observed f o r each  carbon.  The i n t e r e s t i n g o b s e r v a t i o n i s t h e opposing t r e n d o f t h e s h i f t d i f f e r e n c e s observed f o r sugar portions of the molecule. different  head  ( u p f i e l d ) and a l k y l  chain (downfield)  Precedent, f o r t h i s type o f behaviour w i t h  groups can be found i n t h e l i t e r a t u r e [ 1 8 ] .  [ B ]  ° vX  CH,  /  C  H  2^  ^(CH>r  CH-i  2  <5(ppm) Figure 4.5:  100.6  MHz proton decoupled  (0.0562 M) s o l u t i o n s Acquisition  1 ' L NMR s p e c t r a  of octyl  glucoside.  i n D 0 o f [A] monomer (.0.0112 M) and [B] m i c e l l a r 2  Experimental parameters:  time = 0.8192 s; number o f scans = 20,000, temperature  sweep width 20,000 Hz; = 300°C.  62  Table 4 . 2  1  -  r>  C chemical  changes  shift  data  (AS) on m i c e l l e  Resonance  for octyl  g l u c o s i d e with  shift  formation.  fi(monomer)  A<5 (micelle-monomer)  C  1  102.93  0.15  C  3  76.62  ±0.0  C  c  76.62  -0.1 3  73.93  -0.14  (71.34  -0.35  ( 70.53  -0.27  61.64  -0.11  31.71.  0.37  29.45  0.30  29.07  :0.53  J 28.96 25.72  0.53 0.36  22.59  0.27  b C  2  1  C  4  c  6  C'  c  c cc  1  1  '  3  Cy r  1  4  1  '  5  '  6  14.00  0.14  63  Two  p r i n c i p l e mechanisms can  changes of the a l k y l  be v i s u a l i z e d t o cause chemical  c h a i n on m i c e l l e f o r m a t i o n ;  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 "conformational  effects"  a conformational  - i.e. shift  "medium e f f e c t s " - i . e .  of t h e environment,  and  changes caused as a d i r e c t  result  of  change i n t h e molecule [ 1 9 ] .  Since the m i c e l l a r i n t e r i o r  i s hydrophobic [ 2 0 ] , the a l k y l  expected t o undergo a change i n environment upon m i c e l l i z a t i o n . according  shift  chain i s But  t o Persson e t a l . [19] medium e f f e c t s do not cause a s i z e a b l e 13  variation alkyl  i n the  chain.  C chemical  s h i f t s of methylene and methyl carbons i n an  They a l s o have demonstrated t h a t observed s h i f t  q u i t e d i f f e r e n t on m i c e l l e formation  It has vary alkyl  and  changes are  on t r a n s f e r t o an o r g a n i c 13  a l s o been observed s e p a r a t e l y , t h a t  C s h i f t s of carbonyl  solvent.  groups  s t r o n g l y w i t h t h e s o l v e n t , w h i l s t the s o l v e n t e f f e c t s are small groups  for  [20], 13  The  observed d o w n f i e l d  C shift  of carbons i n the a l k y l  be r a t i o n a l i z e d on t h e b a s i s of p o s s i b l e conformational accompany m i c e l l i z a t i o n .  It has  gauche t o t r a n s conformation downfield  changes t h a t  been shown t h a t , t h e t r a n s i t i o n from a  of an a l k y l  s h i f t of the carbon resonances.  chain i s accompanied by a T h i s due  t o r e l e a s e of  non-bonded i n t e r a c t i o n s o p e r a t i n g between the y - c a r b o n s i n the conformation  ( y e f f e c t ) [21,22].  gauche  trans — •  upfield  chain  shift"^  downfield  shift  gauche  can  64  A study by B a t c h e l o r e t a l . [ 2 3 ] p r o v i d e s d i r e c t predominance o f c o n f o r m a t i o n a l e f f e c t s .  In t h i s  support f o r t h e  study with l e c i t h i n t h e  13 C shifts comparison  o f methylene carbons appear 0.3 ppm d o w n f i e l d i n v e s i c l e s , i n with l e c i t h i n  i n chloroform s o l u t i o n .  This r e s u l t i s  i n t e r p r e t e d as being due t o an i n c r e a s e d p r o p o r t i o n o f a l k y l t r a n s conformation when l e c i t h i n  i s incorporated into  In d i l u t e aqueous s o l u t i o n s t h e a l k y l expected t o a t t a i n a p a r t i a l l y  coiled  vesicles.  c h a i n conformation can be  s t a t e because  reduce the u n f a v o u r a b l e hydrocarbon-water  chains with  o f the tendency t o  c o n t a c t , w h i l e t h e entropy  e f f e c t s s h o u l d a l s o f a v o u r c o i l e d conformations i n pure hydrocarbons [ 2 5 ] . Due t o geometric c o n s t r a i n t s o p e r a t i n g i n a m i c e l l e , some a l k y l should f a v o u r a more extended  t r a n s conformation  [24].  chains  From the data  p r e s e n t e d i t can be i n f e r r e d t h a t m i c e l l e f o r m a t i o n i s accompanied by an i n c r e a s e d p r o p o r t i o n o f t r a n s conformers  i n the a l k y l  chain.  The l a r g e r  s h i f t s observed f o r carbons i n t h e middle o f t h e c h a i n suggests t h a t conformational  e f f e c t s a r e more pronounced  i n the c e n t r e o f the c h a i n than  at e i t h e r end. The i s s u e o f t h e u p f i e l d discussed i n the l i t e r a t u r e  1  X  shift  of the -C0  [25] as being dominated  But t h e medium e f f e c t s s t r o n g l y depend on t h e nature group t h a t i s being observed  [20].  2  head group  has been  by the medium e f f e c t s . (structure) of the  In the case o f sugar head groups, i n  d i l u t e s o l u t i o n t h e s e a r e s t r o n g l y H-bonded i n water and any i n c r e a s e or decrease i n t h e H-bonding upon m i c e l l i z a t i o n would be accompanied by a c o r r e s p o n d i n g d o w n f i e l d o r an u p f i e l d  shift  o f t h e carbon  resonances.  In  essence, t h e degree o f H-bonding i n the m i c e l l a r s t a t e compared t o t h e monomer would determine t h e s h i f t caused. Another p o s s i b l e r a t i o n a l i z a 1 3 t i o n f o r t h e observed u p f i e l d C s h i f t s o f the sugar resonances, i s t h e  65  s h i e l d i n g e f f e c t c r e a t e d by t h e n e i g h b o u r i n g operative i n a m i c e l l e . conformational  The p o s s i b i l i t y  -OH groups which would be  o f the sugar r i n g undergoing a  change d u r i n g m i c e l l i z a t i o n  i s h i g h l y u n l i k e l y because o f  the high energy i n v o l v e d i n such a change.  In c o n c l u s i o n , the observed  13 upfield  C s h i f t o f sugar resonances can o r i g i n a t e due t o a v a r i e t y o f  f a c t o r s 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 a t t h i s  4.3 -  ^H-NMR S t u d i e s  of N-alkyllactobionamide  NMR s t u d i e s with t h i s  series  s e r i e s o f compounds proved t o be somewhat o f  a c h a l l e n g e because o f t h e i r low s o l u b i l i t y critical  juncture.  micelle concentrations  [26].  i n water and e s p e c i a l l y low  Partial  400 MHz ^H-NMR spectra; o f  N-dodecyllactobionamide i n 0^0 a t 5.0 mM and 0.20 mM c o n c e n t r a t i o n s a r e shown i n F i g . 4.6 . fully  assigned  considerable  The s p e c t r a were assigned  by comparison with t h e  spectrum o f N-hexyl1actobionamide (Chapter  V ) . The  i n c r e a s e i n s p e c t r a l l i n e - w i d t h a t higher c o n c e n t r a t i o n i s  evident.  T h i s can be e a s i l y a t t r i b u t e d t o t h e presence o f m i c e l l e s as  discussed  i n S e c t i o n 4.1.  previous  study  spin-lattice  The cmc o f the same compound determined i n a  [ 2 6 ] (0.28 mM) f a l l s w i t h i n t h i s c o n c e n t r a t i o n  r e l a x a t i o n study  t h e long experimental  range.  A  o f t h i s molecule was not undertaken due t o  times i n v o l v e d .  I n v e s t i g a t i o n o f the spectra o f other d e r i v a t i v e s i n the s e r i e s showed t h e f o l l o w i n g d i f f e r e n c e s . to  show any s i g n i f i c a n t  upto t h e i r s o l u b i l i t y micelles. in  N-Hexyl and N-Octyl  l i n e broadening with  derivatives failed  increase i n concentration  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 t o assemble i n t o  N-Decyl d e r i v a t i v e produced some l i n e broadening w i t h  c o n c e n t r a t i o n , w h i l e with  monomer s p e c t r a  N-tetradecyl  and N-hexadecyl  (narrow l i n e s ) c o u l d not be o b t a i n e d  increase  derivatives the  even a t very low  1  1  I  U  I  I  I  I  3.6  A.O  I  3.2  6(ppm) F i g u r e 4.6:  400 MHz ^H NMR s p e c t r a o f N-dodecyll actobionami de i n D,,0. Experimental tion  parameters:  sweep width  = 2202.6 Hz; A c q u i s i -  time = 1.8596 s; number o f scans  = 8412 (monomer spec-  trum), 124 ( m i c e l l e spectrum), temperature peak p r e s a t u r a t e d ( n o t shown).  = 290°C, HOD  67  concentrations.  T h i s i n d i c a t e s t h a t t h e l a t t e r two d e r i v a t i v e s have  extremely low cmc values which are not p o s s i b l e t o d e t e c t even  with  H-NMR.  ]  4.4 - Comments T h i s chapter using  r e f l e c t s mainly t h e advantages and l i m i t a t i o n s o f  ^H-NMR i n s t u d y i n g m i c e l l a r s o l u t i o n s .  The main advantage o f  ^H-NMR, as p o i n t e d out i n Chapter I I , i s i t s high s i g n a l / n o i s e (S/N) s o l u t i o n s o f sub-mi 11imolar  s e n s i t i v i t y which makes i t f e a s i b l e t o study concentration.  Nevertheless,  i t should  d e r i v a t i v e s encountered i n t h i s only w i t h  limiting  study,  sensitivity.  d i f f e r e n t parameters (chemical  be mentioned t h a t w i t h some the work was a c t u a l l y performed  This study  a l s o demonstrates t h e  s h i f t , coupling constant,  l i n e - w i d t h and  r e l a x a t i o n t i m e ) , which c o u l d be used t o p r o v i d e an i n s i g h t t o t h e molexular  processes  meters w i t h  involved" i n m i c e l l e f o r m a t i o n .  ^H-NMR i s much more convenient  even though a study  novel  than with  o f o t h e r n u c l e i can provide  Disadvantages o f u s i n g The  The use o f the above  ^H-NMR have a l r e a d y  aspect  o f t h i s study  any o t h e r  being  by ^H-NMR.  been d i s c u s s e d  i n Section 2 .  i s t h a t i t has been p o s s i b l e t o  head groups i s e s p e c i a l l y d i f f i c u l t  oxide  should  that are  s u r f a c t a n t s , and a study  of their  because o f the u n r e s o l v a b l e  resonances produced by t h e head group. d e s c r i b e d i n t h i s chapter with  forming  The common n o n i o n i c detergents  studied involve polyethylene  nuclei,  additional information.  probe t h e behaviour o f t h e head group o f a c l a s s o f m i c e l l e n o n i o n i c amphiphiles  para-  In t h i s  a carbohydrate  attract considerable attention.  respect, the studies  moiety as t h e head group  68  4.5 -  Experimental Samples f o r NMR s p e c t r a were prepared  d i s s o l v i n g i n 99.7% D 0 2  s p e c t r a were run with All  by a c c u r a t e l y weighing and  (Merck Sharp and Dohme Canada Ltd.) and t h e  r e f e r e n c e t o e x t e r n a l t e t r a m e t h y l s i 1ane (TMS).  s p e c t r a were recorded  a t room temperature on a Bruker WH-400  (9.4T) high r e s o l u t i o n spectrometer o p e r a t i n g a t 400 MHz f o r  equipped with an Aspect  2000 computer,  and 100.6 MHz f o r C at t h e Department o f 1 3  Chemistry,  University of B r i t i s h  quadrature  d e t e c t i o n mode and s t o r e d i n a d i s k f o r subsequent  All  t h e data were m u l t i p l i e d  Data were accumulated i n t h e  by an e x p o n e n t i a l  l i n e broadening  processing. f a c t o r (0.1  "I ?  1 Hz f o r H and  Columbia.  5.0 Hz f o r C ) and z e r o - f i l l e d l o  prior to Fourier transfor-  mation. Spin-lattice  r e l a x a t i o n times (T|) o f protons  by u s i n g a (180°-T-90°-Acquisition-RD) v a r i a b l e delay  were determined [ 2 7 ]  pulse sequence, where T i s t h e  between t h e two p u l s e s and RD, t h e r e l a x a t i o n d e l a y .  experiments were performed with  The  phase a l t e r a t i o n o f t h e 180° pulse t o  reduce e r r o r s due t o any i m p e r f e c t i o n i n t h e p u l s e l e n g t h and RD was s e t t o ~5T^ o r h i g h e r .  The data  f o r , a t l e a s t e i g h t T- values were  collected  f o r each T-| measurement, and t h e r e l a x a t i o n time was c a l c u l a t e d by f i t t i n g the data t o t h e T13IR data NTCFTB (NMR) program.  reduction routine included i n the N i c o l e t  Given  t h e data f o r t h e i n t e n s i t y o f t h e s i g n a l and  time, t h e TI31R program c a l c u l a t e s t h e T-| without  assuming a p e r f e c t 180°  p u l s e and i n d i c a t e s how c l o s e t h i s  i n v e r s i o n p u l s e was t o i t s c o r r e c t  value  [ 2 8 ] . For a l l t h e T-| values  c a l c u l a t e d t h e accuracy  found  t o be w i t h i n t h e a c c e p t a b l e  total  i n t e n s i t y o f t h e m u l t i p l e t was p l o t t e d a g a i n s t time.  range.  In t h e case  o f t h e 180° was  of a m u l t i p l e t , t h e From t h e T-|  v a l u e s , t h e r e l a x a t i o n r a t e (R-j) was c a l c u l a t e d u s i n g t h e r e l a t i o n s h i p R-| = VT,  and t h e values  given a r e r e p r o d u c i b l e t o w i t h i n b e t t e r than ±5%.  69  References  1.  De G r i p , W.J., Bovee-Geurts,  P.H.M.,  Chem. Phys. L i p i d s ,  (1979), 23,  321. 2.  Shinoda, K., Yamaguchi, T., H o r i , R.,  Bui 1. Chem. Soc. Jpn., (1961),  34, 239. 3.  H e l e n i u s , A., Simons, K.,  Biochim. B i o p h y s . A c t a . , (1975) 415,  4.  Wennerstrom, H., Lindman, B.,  5.  Wennerstrom, H., Lindman, B., i b i d . ,  6.  Wennerstrom, H., Lindman, B.,  7.  Shaw, D.,  29.  Phys. Rep., (1979), 5_2, 33.  ibid.,  p. 60. p. 69.  " F o u r i e r Transform N.M.R. Spectroscopy", E l s e v i e r ,  Amsterdam, 1976, p. 300. 8.  Shaw, D., i b i d . ,  9.  Shaw, D.,  ibid.,  p. 306. p. 9.  10.  P r e s t o n , C M . , H a l l , L.D., Carbohydr. Res., (1974), 37_>  267.  11.  H a l l , L.D., Chem. i n Canada, (1976), 28, 19.  12.  Lemieux, R.U., P a v i a , A.A., M a r t i n , J . L . , Watanabe, K.A., Can. J . Chem., (1969), 47, 4427.  13.  Lemieux, R.U., Koto, S.,  T e t r a h e d r o n , (1974), 30.,  1933.  14.  T a n f o r d , C , Nozaki , Y.U., Rohde, M.F., J . Phys. Chem., (1977), 81, 1555.  15.  Atwood, D.,  J . Phys. Chem. , (1968), 7_2,  16.  S t a p l e s , E . J . , Tiddy, G.J.T.,  339.  J . Chem. Soc. Faraday  I , (1978), 74,  2530. 17.  P f e f f e r , P.E., V a l e n t i n e , K.M., P a r r i s h , F.W., (1979), 101, 1265.  J . Am. Chem. S o c ,  70  18.  Persson, B., Drakenberg,  T., Lindman, B.,  J . Phys. Chem., (1979), 83,  T., Lindman, B.,  J . Phys. Chem., (1976), 80,  3011. 19.  Persson, B., Drakenberg, 2124.  20.  Stothers, J . ,  "Carbon-13 NMR Spectroscopy" i n "Organic Chemistry, A  S e r i e s o f Monographs", V o l . 24, Academic P r e s s , 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.  B a t c h e l o r , J.G., P r e s t e g a r d , J.H., C u s h l e y , R.J., L i p s k y , S.R., Biochem. Biophys. Res. Commun., (1972), 4_8, 70.  24.  Lindman, B., Wennerstrom, H., i n 87, M i c e l l e s :  25.  26.  T., Lindman, B.,  J . Colloid  W i l l i a m s , T . J . , P l e s s a s , N.R., G o l d s t e i n , I . J . ,  A r c h . Biochem.  (1977), 470, 325.  M a r t i n , M.L., M a r t i n , G.J., Delpuech, J . - J . ,  "Practical  copy", Heyden and Sons L t d . , 1980, p. 244. 28.  Interface  (1978), 63, 538.  Biophys., 27.  S p r i n g e r - V e r l a g , New York, 1980, p. 49.  Rosenholm, J.B., Drakenberg, Sci.  "Topics i n C u r r e n t Chemistry", V o l .  Levy, G., Peat, I . , J . Magn. Reson., (1975), 18, 500.  NMR S p e c t r o s -  CHAPTER V  TWO DIMENSIONAL FOURIER TRANSFORM  NMR SPECTROSCOPY  72  5.1  -  Introduction The concept o f two-dimensional  F o u r i e r t r a n s f o r m a t i o n was f i r s t  proposed by Jeener [ 1 ] i n 1971, but i t ' s realized until  several years l a t e r .  widespread s i g n i f i c a n c e was not  The f i r s t  NMR experiments u s i n g  this  t e c h n i q u e were p u b l i s h e d i n 1975 [ 2 , 3 ] , t o be f o l l o w e d l a t e r by a d e t a i l e d theoretical  a n a l y s i s [ 4 ] , which  development  i n t h i s area.  l a i d t h e f o u n d a t i o n f o r t h e subsequent  Comprehensive  reviews [5,6] on two-dimensional  NMR  (2D-NMR) s p e c t r o s c o p y have been p u b l i s h e d , and a more recent  [7]  d e a l s with i t s b i o l o g i c a l It w i l l  article  applications.  be r e c a l l e d t h a t i n a c o n v e n t i o n a l F o u r i e r t r a n s f o r m NMR  experiment, t h e n u c l e a r s p i n s a r e e x c i t e d by a p p l i c a t i o n o f a s i n g l e r a d i o frequency p u l s e , and t h e i r time p e r i o d of  (t^),  responses a r e measured d u r i n g a s i n g l e  the a c q u i s i t i o n time.  Subsequent  Fourier transformation  t h i s time domain data s e t , s ( ^ ) » produces t h e frequency domain NMR  spectrum, S(F_2) [ 8 ] . dependent  I n s o f a r t h a t t h i s d i s p l a y s a l l t h e frequency  parameters along a s i n g l e frequency a x i s , t h i s can be regarded  as a one-dimensional  NMR  (1D-NMR) experiment.  B r i e f l y , t h e 2D-NMR t e c h n i q u e i n v o l v e s , c o l l e c t i o n o f a data m a t r i x s(t_-|, t ^ ) , as a f u n c t i o n o f two independent time domains (;t-| and t_ ), 2  f o l l o w e d by double F o u r i e r t r a n s f o r m a t i o n .  S(F_-|>  c o n t a i n s one i n t e n s i t y a x i s and two frequency axes  A l a r g e v a r i e t y o f experiments tions  The r e s u l t i n g 2D spectrum (F^ and £_,,).  -is p o s s i b l e depending on which p e r t u r b a -  (frequency o r phase o f t h e RF r a d i a t i o n , d e c o u p l e r l e v e l , e t c . ) a r e  a p p l i e d t o t h e n u c l e a r s p i n s d u r i n g t h e time i n t e r v a l s t ^ and t ^ . is  Thus i t  important t o r e c o g n i z e t h a t d i f f e r e n t types o f i n f o r m a t i o n can be  d e r i v e d from d i f f e r e n t types o f 2D experiments. At  p r e s e n t , t h e 2D experiments which a r e o f p r i n c i p l e  importance  73  t o the p r a c t i c i n g chemist can be b a s i c a l l y d i v i d e d i n t o two c a t e g o r i e s [8], 1.  2D-Resolved  NMR  Spectroscopy:  homonuclear or h e t e r o n u c l e a r J - r e s o l v e d NMR chemical NMR 2.  shift  in solids  r e s o l v e d NMR  [9-12],  [13-15], d i p o l a r  resolved  [16,17].  2 D - C o r r e l a t e d NMR  Spectroscopy:  a u t o c o r r e l a t i o n through J - c o u p l i n g [ 4 , 7 ] , a u t o c o r r e l a t i o n through dynamic p r o c e s s e s such as chemical exchange or Overhauser 2D-Resolved  NMR,  enhancement  s i m p l i f i e s complex s p e c t r a by s p r e a d i n g the  t r a n s i t i o n s o f a c o n v e n t i o n a l ID spectrum Examples o f s p r e a d i n g parameters NMR),  chemical  shifts  one o f the frequency axes,  i n t o a second  dimension.  are s c a l a r c o u p l i n g c o n s t a n t s  (chemical s h i f t  c o n s t a n t s ( d i p o l a r r e s o l v e d NMR).  chemical  [18,19].  r e s o l v e d NMR)  (J_-resolved  or d i p o l a r c o u p l i n g  In J_-resolved and d i p o l a r r e s o l v e d  ( F ^ ) , i n the 2D spectrum  NMR,  corresponds t o  s h i f t s w h i l e the o t h e r , (F_y), c o n s i s t s of s c a l a r c o u p l i n g ( J j o r  dipolar coupling constants. and F_2 r e p r e s e n t chemical  In chemical  shift  s h i f t s but o f two  r e s o l v e d NMR,  different  nuclei  both axes (e.g.  and  13 C).  This i s summarized i n F i g . 5.1. \H  J_-Resolved t e c h n i q u e has become one o f the most powerful  commonly used 2D-NMR experiments  since i t often provides  and  unprecedented  d i s p e r s i o n of ^H-NMR s p e c t r a and h e l p s t o r e s o l v e o v e r l a p p i n g resonances. T h i s l e a d s t o a wealth of i n f o r m a t i o n extreme d i f f i c u l t y  from a normal  which can only be o b t a i n e d with  ID spectrum o f a complex m o l e c u l e .  It  has a l r e a d y been proved t o be v a l u a b l e i n the a n a l y s i s of ^H-NMR s p e c t r a of o l i g o s a c c h a r i d e s [13,20], s t e r o i d s [21,22], and p e p t i d e s [23,24].  74,  [A]  [B]  [C]  J  D  -A*  Figure  5.1;  [D]  Schematic r e p r e s e n t a t i o n o f d i f f e r e n t 2D r e s o l v e d  NMR  experiments. [A] - Homonuclear J - r e s o l v e d [B] - H e t e r o n u c l e a r  NMR  J-resolved  [C] - D i p o l a r r e s o l v e d  NMR  [D] - Chemical s h i f t r e s o l v e d 5 - chemical coupling  shift;  NMR  NMR  J - s c a l a r coupling constant;  constant.  A and X r e p r e s e n t  different  nuclides.  D - dipolar  75  H e t e r o n u c l e a r J_-resolved s p e c t r o s c o p y has been used t o determine 13 coupling constants that o f  [12,25],  1 31 H- P [27,28].  spectroscopy resonances  a p a r t i c u l a r carbon  H  13 C-  C c o u p l i n g c o n s t a n t s [26] as well as  Chemical  shift  resolved ( s h i f t  has been a p p l i e d t o o b t a i n t h e c o r r e l a t i o n  [13,29].  C-  correlation) 13 between  1  C and  H  T h i s enables one t o i d e n t i f y t h e protons a t t a c h e d t o and t o make a d d i t i o n a l  assignments i n i n d i v i d u a l  spectra. The two common c o r r e l a t i o n experiments tion  Spectroscopy  [24,33,34].  (COSY) [30-32] and Spin-Echo  In both experiments  l i n g and thus p r o v i d e extremely  useful  now i n use are,2D-Correl aC o r r e l a t e d Spectroscopy  the" c o r r e l a t i o n i s o b t a i n e d through  identical  information [33].  (SECSY)  ^-coup-  These t e c h n i q u e s a r e  i n determining t h e c o n n e c t i v i t y o f t h e resonances i n  complex s p e c t r a , and t h e i r a p p l i c a t i o n t o o l i g o s a c c h a r i d e s and p e p t i d e s have a l r e a d y been demonstrated. In a COSY spectrum, both F_-j and F_ frequency 2  chemical  s h i f t s o f t h e same n u c l e i  and the normal  a l o n g t h e diagonal o f t h e spectrum  [ F i g . 5.2(A)].  axes r e p r e s e n t  spectrum  Moreover, t h e s p i n -  coupled m u l t i p l e t s g i v e r i s e t o o f f - d i a g o n a l responses at t h e p o i n t s c o r r e s p o n d i n g t o t h e chemical a SECSY spectrum, t h e F_^ a x i s corresponds chemical  shifts  (A6/2  t o t h e normal chemical  i s displayed  (correlated  s h i f t s o f coupled n u c l e i .  2  axis.  In •  t o one h a l f o f the d i f f e r e n c e i n  ) o f t h e s p i n coupled n u c l e i , whereas F_ shift  peaks)  Here, the normal  spectrum  corresponds  i s displayed  at t h e z e r o frequency o f t h e F_^ a x i s [ F i g . 5.2(B)], and t h e c o n n e c t i v i t i e s of  s p i n coupled m u l t i p l e t s . are i n d i c a t e d by.the  on e i t h e r s i d e o f t h e spectrum.  responses  which appear  The l i n e s showing t h e c o n n e c t i v i t i e s a r e  a l i g n e d a t an angle o f 135° t o t h e F_ « The general area o f 2D-NMR, a l s o i n c l u d e s m u l t i p l e quantum a x 1 s  2  76  F i g u r e 5.2:  I l l u s t r a t i o n o f COSY(A) and SECSY(B) s p e c t r a .  77  t r a n s i t i o n d e t e c t i o n [4,35,36], zeumatography [37] and cross-relaxation  [38] experiments, to name j u s t a  T h i s chapter spectroscopy  and  few.  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  SECSY i n the a n a l y s i s of  1  H NMR  heteronuclear  J-resolved  s p e c t r a of two  model  g l y c o l i p i d s , syntheses o f which were d e s c r i b e d i n Chapter I I .  5.2  -  5.2.1  D e s c r i p t i o n of homonuclear J - r e s o l v e d and SECSY experiments - Pulse  sequences and  data  acquisition  Homonuclear J - r e s o l v e d spectroscopy Carr-Purcell  i s a v a r i a n t of  s p i n echo experiment [ 3 9 ] , i n which the 90° p u l s e used t o  p e r t u r b the n u c l e a r s p i n s i s f o l l o w e d by a 180° d e l a y time ( t , , ). 2 represented {90°  The  pulse sequence f o r the J - r e s o l v e d experiment can  - t, - A c q u i s i t i o n ( t ') - R e l a x a t i o n  where n i s the number of a c q u i s i t i o n s ; t h i s can as shown below i n F i g .  90°pulse  2  Evolution period  delay  (RD)}  be d i a g r a m a t i c a l l y  5.3.  180° pulse  Defocussing interval  F i g u r e 5.3:  pulse, a f t e r a c e r t a i n  by  - t . - 180°  represented  classical  spin-echo  Refocussing interval tj  Acquisition  2  Detection period  t  2  Pulse sequence f o r 2D homonuclear J - r e s o l v e d experiment.  be  78  In is  a J_-resolved experiment a s e r i e s o f C a r r - P u r c e l l  performed, i n which the e v o l u t i o n time  p u l s e sequences  ( t ^ ) i s incremented by a  c o n s t a n t v a l u e ( A t ^ ) , so t h a t , l l  = k.At,; where k = 0, 1, and  In  (N  = number of experiments  each case only the r e f o c u s s e d s i g n a l  a f u n c t i o n o f time conventional  (t^).  ( F i g . 5.3)  as  T h i s h a l f - e c h o can be t r e a t e d as i f i t were a  f r e e i n d u c t i o n decay and the parameters f o r the c o r r e s p o n d i n g 2  i n the normal  the  performed.  i s acquired  frequency domain (F_ , which c o n t a i n s chemical s h i f t  The  -1)  i n f o r m a t i o n ) are set  manner.  increment i n  (At-]) determines the s p e c t r a l width  c o r r e s p o n d i n g frequency domain  (SW ) 2  in  (F^,, which c o n t a i n s c o u p l i n g c o n s t a n t  information), according to the equation,  SW-j  =  2{yt~ ' ^ T  e  n u m  '  3 e r  °^  experiments performed, N-j, corresponds t o the number of p o i n t s i n £_-[, resulting  in a digital  r e s o l u t i o n o f /At.^N 1  1  In p r a c t i c e the data are accumulated  or  i n the F^  and s t o r e d by a computer as  a s t r i n g o f c o n s e c u t i v e f i l e s on a d i s k , f o r subsequent The p u l s e sequence  domain.  f o r the SECSY experiment  data p r o c e s s i n g .  f o l l o w s the same  p a t t e r n as t h a t f o r the J - r e s o l v e d experiment except t h a t the second p u l s e is  a 90°' p u l s e as i n d i c a t e d below and i n F i g . 5.4(A).  { 9 0 ° - t i - 90° - t] - A c q u i s i t i o n 2 2  ( t ) - Relaxation 9  L  n  The data a c q u i s i t i o n f o r the SECSY experiment procedure as used i n the J - r e s o l v e d experiment. the  p u l s e sequence  delay}  i n v o l v e s the same  F i g u r e 5.4 a l s o  f o r t h e COSY experiment where the a c q u i s i t i o n  immediately a f t e r the second 90°  pulse.  depicts begins  79  9 0 ° pulse  9 0 ° pulse  I  [A] tl 2  2 Acquisition (t,  9 0 ° pulse  9 0 ° pulse  [fi]  Acquisition (t  2  F i g u r e 5.4:  Pulse sequences f o r SECSY[A] and COSY[B] experiments.  80  5.2.2  - Data The  p r o c e s s i n g [20,40,41] data p r o c e s s i n g procedures  f o r J_-resolved and  SECSY e x p e r i -  ments f o l l o w the same scheme which i s summarized i n F i g . 5.5, the  'tilt'  routine at the f i n a l  The  initial  s t e p i s o m i t t e d i n t h e SECSY  2D time domain data m a t r i x  number of f r e e i n d u c t i o n decays  (f.i.d).  except  experiment.  s(t_-|, t_ ) c o n s i s t s o f N-| 2  Each of these f . i . d ' s i s f i r s t  s u b j e c t e d t o F o u r i e r t r a n s f o r m a t i o n with r e s p e c t t o t_ t o produce  a data  2  m a t r i x s(t_^, F_ ); 2  that  this..consists o f a s e t of phase modulated s p e c t r a , the  phase o f which depends on t h e t_^ v a l u e .  For weakly coupled s p i n  systems  the phase v a r i a t i o n with t ^ f o r a p a r t i c u l a r t r a n s i t i o n depends not on chemical  s h i f t , but o n l y on the m u l t i p l e t  particular transition. resonance  This f a c t  first  n  t r a n s p o s e d t o produce  F^).  2>  at  the s(F_  2>  2  t ^ ) m a t r i x , and then  t o produce  2  ( u n t i l t e d ) J - r e s o l v e d spectrum,  The peaks i n t h e J_-resolved spectrum  P r o j e c t i o n of t h i s  normal  spectrum  ( u n t i l t e d ) spectrum  or the SECSY  at t h i s stage are a l i g n e d  angle of 45° p r o v i d i n g t h a t t h e s c a l e s o f the F_  and F_ axes are the  onto the £  2  2  c o n s t a n t i n f o r m a t i o n suppressed J - r e s o l v e d spectrum  spectrum",  the  ( F i g . 5.6).  i s obtained a f t e r  with a l l the c o u p l i n g  A more u s e f u l  ( F i g . 5.6).  This t i l t  display of the  i t has been " t i l t e d " by an angle o f  4 5 ° so t h a t a l l the t r a n s i t i o n s o f a p a r t i c u l a r m u l t i p l e t 1  axis y i e l d s  and the p r o j e c t i o n a t an angle of 45° g i v e s the e q u i v a l e n t  a "homonuclear broad-band decoupled  the _F_ a x i s  Fourier  the frequency domain data s e t  2  same.  of  second  T r a n s p o s i t i o n o f S(F_ , F^) y i e l d s the S(F_^, F_ ) data m a t r i x  which corresponds t o the spectrum.  s p l i t t i n g s by means o f a  To accomplish t h i s , t h e s ( t _ , F_ ) data m a t r i x i s  t r a n s f o r m e d with r e s p e c t t o jb S(F_  structure corresponding to that  i s u t i l i z e d t o d i f f e r e n t i a t e between  l i n e s with d i f f e r e n t m u l t i p l e t  Fourier transformation.  the  lies parallel  r o u t i n e e l i m i n a t e s the p o s s i b i l i t y  of  to  81 PHASE MODULATED SPECTRA s(tj ,f )  2D TIME SIGNAL ,t )  2  2  N, SPECTRA WITH N  1  f.i.d's  ]  st  I  FT  •  PHASE MODULATIONS DEPENDING ON t, (Real)  •(Imaginary) N2'2  Transposition  Sffg.ty) 2  2D SPECTRUM 1. 2  (TO BE TRANSPOSED)  (Real) F  l  n d  FT  2N  ,l  2  INTERFEROORAMS ..  2  ;  (Real)  |( Imaginary) N  N  |(Imaginary) 2N,  "1  1  1. A b s o l u t e Va!ue 2. T r a n s p o s i t i o n 2D SPECTRUM (TILTED) S(F £ ')  2D SPECTRUM (UNTILTED) SCF-, ,F )  r  2  2  TILT by 45°  F ' ?  L  Figure 5.5;  Summary o f the data m a n i p u l a t i o n  procedure  (Chemical shift)  i n 2D-experiments  82  f (S,J)  f'(S)  2  Figure  5.6:  Illustration  2  o f various  used i n proton  2D J - r e s o l v e d s p e c t r o s c o p y .  J - r e s o l v e d spectrum the normal  d i s p l a y modes and the t i l t  A 45° p r o j e c t i o n gives  decoupled-proton spectrum ( b ) . 2D spectrum quartet. triplets  [B] onto  2  axis y i e l d s  a proton-  P r o j e c t i o n o f a s e c t i o n o f the  axis.gives  the J^ spectrum o f the  S i m i l a r l y , p r o j e c t i o n o f [C] y i e l d s a t r a c e with the superimposed  (d).  Tilting  by 45° y i e l d s the S ( 6 , J) matrix a t the r e s p e c t i v e chemical triplets  The ( u n t i l t e d )  [A] when p r o j e c t e d onto the £  spectrum ( a ) .  routine  (from  Ref. 2 0 ) .  shifts  the J - r e s o l v e d  spectrum  [ D ] . Cross s e c t i o n s o f [D] give  the J s p e c t r a o f the  83  o v e r l a p p i n g between t h e t r a n s i t i o n s o f c h e m i c a l l y s h i f t e d resonances, when the  p r o j e c t i o n s onto £-| are t a k e n .  The p r o j e c t i o n of the t i l t e d  onto ¥_2 produces the "decoupled spectrum" and the m u l t i p l e t each resonance chemical  p r o j e c t i n g i t onto F_^. centre.  5.3  -  5.3.1  t o F i a c r o s s the whole F_-| dimension, and The J_ s p e c t r a so o b t a i n e d are symmetrical  This i s i l l u s t r a t e d  o f 2D-spectroscopy i n s p e c t r a l  unprotected  sugars  - n-Octyl  about  i n F i g . 5.6.  Application  assignment  of  g-D-glucopyranoside (I)  The p a r t i a l D^O  structure f o r  (J s p e c t r a ) can be o b t a i n e d by t a k i n g the t r a c e at the  shift parallel  the  spectrum  400 MHz  i s shown i n F i g . 5.7(A).  ^H spectrum o f n-octyl  3-D-g1ucopyranoside i n  The o v e r l a p p i n g of m u l t i p l e t s o f d i f f e r e n t  protons p r e c l u d e s unequivocal s p e c t r a l  assignment  by c o n v e n t i o n a l  t e c h n i q u e s such as d e c o u p l i n g and matching of s p e c t r a l  splittings.  Furthermore, i t makes t h e d e t e r m i n a t i o n o f chemical s h i f t s and c o n s t a n t s o f the protons i n v o l v e d almost an i m p o s s i b l e t a s k . problems can be e a s i l y overcome through  1  coupling These  H J_-resol ved s p e c t r o s c o p y .  proton-decoupled-proton spectrum o b t a i n e d by the p r o j e c t i o n o f the  The tilted  2D J - r e s o l v e d spectrum onto ^ ( c h e m i c a l s h i f t ) a x i s i s shown i n F i g . 5.7(B).  As can be seen, the spectrum  i s greatly  simplified  by the  s u p r e s s i o n o f the c o u p l i n g c o n s t a n t i n f o r m a t i o n and each peak i n the spectrum appear a t the c o r r e s p o n d i n g chemical s h i f t determination a t r i v i a l  v a l u e thus making i t s  matter.  The c o u p l i n g i n f o r m a t i o n i s e x t r a c t e d by o b t a i n i n g a c r o s s the t i l t e d  2D J - r e s o l v e d spectrum  cross-sections  (J s p e c t r a ) at the chemical  shift  v a l u e s i n d i c a t e d i n t h e proton-decoupled-proton spectrum; t h e s e are shown  -T  1  »  44 Figure 5.7:  Partial  »  r—  1  40  r  6 (ppm)  3-6  400 MHz 'H ID and 2D NMR s p e c t r a o f n - o c t y l 6-D-glucopyranoside  [A] - The normal Relaxation  0.0112 M i n D 0. 2  spectrum. Sweep width 1000.0 Hz, A c q u i s i t i o n time = 2.04 s, Data s i z e = 4K,  delay = 0.5 s , Number o f scans = 30.  o f the same r e g i o n . +SW  2  filling),  3-2  = 1000.0 Hz,  Total a c q u i s i t i o n  time =5.6  SW  ]  [B] - The proton-decoupled proton spectrum  =15.6 Hz, N  hours.  2  = 4K and N  ]  =128 ( a f t e r  zero-  [H-6AXA  10 Hz  6A,B  I - I  H-1  5,6 A  H-6B  H- ocfi  u  Ju  hK  H-5  H-3  U  F i g u r e 5.8:  Individual spectrum.  H-2  JUUL, J spectra  of  n-octyl  -D-gl ucopyranoside  obtained  JUUL, f r o m 2D  J-resolved  86  T a b l e 5,1.:  Proton chemical  shift  region o f n-octyl  and c o u p l i n g c o n s t a n t data o f the sugar  g-D-glucopyranoside,  Chemical  shifts,  Coupling c o n s t a n t s , Hz  ppm Glucose  ring: = 8.1  H-l  4.44  J  H-2  3.25  J  H-3  3.47  J  3,4  =  H-4  3.37  J  4,5  =  H-5  3.45  J  5,6A  H-6A  3.91  J  5,6B  H-6B  3.71  J  6A,6B  H-aA  3.91  J  H-aB  3.68  2 j 3  =9.6  9  9  -°  -  =  8  2  J  - -° 6  =  n  -  6  A l i p h a t i c chain: n D aA,aB  =  9.9  87  i n F i g . 5.8.  With t h e chemical s h i f t and c o u p l i n g i n f o r m a t i o n i n hand,  the assignment o f t h e resonances c o u l d be accomplished by matching o f individual  coupling constants.  were c o n f i r m e d by c a r e f u l  The i n d i v i d u a l  assignments o f H-3  d e c o u p l i n g o f the H-2  resonance.  i n Table  H-4  The'  assignments are i n d i c a t e d i n F i g . 5.7(B) and t h e chemical s h i f t c o u p l i n g c o n s t a n t v a l u e s are t a b u l a t e d  and  and  5.1.  At t h i s j u n c t u r e , i t i s a p p r o p r i a t e t o comment on some f e a t u r e s d i s p l a y e d i n the s p e c t r a . limitation  Strong c o u p l i n g e f f e c t s present a s e r i o u s  i n 2D-spectroscopy [4,33,41] as i n l D - s p e c t r o s c o p y .  spectroscopy, strong coupling gives r i s e to additional  In  2D-  l i n e s of low  inten-  s i t y which appear inbetween t h e chemical s h i f t s o f t h e s t r o n g l y c o u p l e d peaks. ling  The  s p e c t r a o f the a d d i t i o n a l  show an unsymmetrical  identification.  For t h a t m a t t e r , a l l the a r t i f a c t s  i n a proton-decoupled-  i n a s i m i l a r manner.  constants.  The a r t i f a c t s  H-4  and  In t h e normal  of the coupling  i n t h e 2D p r o j e c t i o n due t o s t r o n g  e f f e c t s are i n d i c a t e d w i t h an a s t e r i s k  i n F i g . 5.7  spectrum the m u l t i p l e t  coupling  (B). 6 due t o H-aA  at 3.92  and  appear as a s i n g l e resonance i n t h e proton-decoupled-proton spectrum,  i n d i c a t i n g t h a t t h e s e two the c r o s s - s e c t i o n let  The H-3,  resonances form a s t r o n g l y c o u p l e d system i n which the d i f f e r e n c e i n  t h e i r chemical s h i f t s a r e i n t h e same o r d e r o f magnitude  H-6A  coup-  p a t t e r n a l o n g F_^, which h e l p s i n t h e i r  proton spectrum can be i d e n t i f i e d H-5  peaks a r i s i n g due t o s t r o n g  protons have i d e n t i c a l  chemical s h i f t s .  (J_ spectrum) a t t h i s chemical s h i f t  s t r u c t u r e s o f both protons which can be e a s i l y  shown by t h e s t i c k The not e a s i l y  Hence,  includes the multip-  unscrambled  as  diagram.  spectrum o f H-5  i n d i c a t e s i t s complex  seen i n t h e ID spectrum.  Also the H-4  m u l t i p l i c i t y , which i s  resonance appears as a  88  four l i n e pattern  (double d o u b l e t ) i n t h e J_ spectrum whereas only t h r e e  l i n e s c o u l d be seen i n t h e normal  spectrum.  This i s due t o t h e i n h e r e n t  enhanced r e s o l u t i o n i n t h e J_ dimension (F_j a x i s ) o b t a i n e d i n . J - r e s o l v e d s p e c t r o s c o p y s i n c e , t h e observed l i n e - w i d t h i n t h i s dimension has no contribution  from t h e inhomogeneities o f magnetic  field.  5 . 3 . 2 - N-Hexyllactobionamide ( I I ) Assignment  o f t h e sugar r e g i o n i n t h e ^ H NMR  spectrum o f t h e  d i s a c c h a r i d e d e r i v a t i v e was a c h i e v e d by a combination o f 2D methods: J - r e s o l v e d s p e c t r o s c o p y and s p i n - e c h o c o r r e l a t e d s p e c t r o s c o p y (SECSY). The normal  and t h e proton-decoupled-proton spectrum o f the sugar r e g i o n o f  N-hexyllactobionamide The  'tilted'  p l o t t e d as  ( I I ) i s shown i n F i g . 5.9 along w i t h t h e J_-traces.  J - r e s o l v e d s p e c t r a and the SECSY s p e c t r a o f the same r e g i o n contour diagrams, where t h e spectrum i s viewed down t h e  i n t e n s i t y a x i s , a r e shown i n F i g . 5.10 and 5.11 r e s p e c t i v e l y .  An expan-  s i o n o f t h e d o t t e d region o f t h e SECSY spectrum i s shown i n F i g . 5.12.  The molecule I I c o n t a i n s 13 sugar protons and t h e i r numbering i s as i n d i c a t e d above. H-l  By i n s p e c t i o n o f SECSY s p e c t r a , t h e p o s i t i o n s o f t h e  through H-4 protons i n t h e g a l a c t o s e ''residue as well  as t h a t o f a l l the  protons i n t h e g l u c o s e (open c h a i n ) r e s i d u e can be determined.  The  89 H-6A  T  t  1  1  1  t  4-4 Figure 5.9:  Partial  1  r  I  I  I  40 400 MHz H ID and 2D NMR s p e c t r a  nionamide  ]  0.025 M i n D^O.  spectrum.  del ay = 1 s , Number o f scans = 24.  SW-j = 23.4 Hz, N  z e r o f i l l ing).  Total  g  [B] - The  = 4K and N., = 256 ( a f t e r  a c q u i s i t i o n time = 8.7 hours.  Upper t r a c e - J s p e c t r a  Sweep  time 1.86 s , Data s i z e = 8K,  proton-decoupled-proton spectrum o f t h e same r e g i o n . 750.7 Hz,  .  o f N-hexyl1acto-  [A] - The normal  width = 2202.6 Hz, A c q u i s i t i o n Relaxation  "  3-6 6(ppm)  of individual  multiplets.  ±SU^  =  90  [B]  U-U  LO  3.6 S/ppm  Figure 5.10:  Partial  400 MHz  0.025 M i n D 0. 2  resolved  H 2D NMR s p e c t r a  [A] - A contour diagram o f the 2D 3-  spectrum.  [B] - The proton-decoupled-proton  spectrum (same as i n F i g . 5.9). mental  o f N-hexyl1actobionamide  parameters.  See F i g . 5.9 f o r e x p e r i -  91  H-6A£J3'B 5  ' 4-5  » 4.0  > 3-5  S/ppm Figure 5.11:  Partial  400 MHz SECSY spectrum o f N-hexyl1actobionamide  0.025 M i n D 0. 2  The l i n e s  show the c o n n e c t i v i t i e s . N  2  = 4096 and  shows the normal  joining SW  2  the c o r r e l a t e d  peaks  = 750,7 Hz, iSW-, = 250.0. Hz,  = 256 [ a f t e r z e r o f i l l i n g ) . spectrum o f the same r e g i o n .  Upper t r a c e  F i g u r e 5.12:  Partial  SECSY spectrum  o f N-hexyllactobionamide:  expansion o f the dotted r e g i o n o f F i g . 5,11.  an  93  c o n n e c t i v i t i e s o f these s p i n coupled protons a r e shown i n F i g . 5.11 and 5.12.  The c o n n e c t i v i t i e s o f H-5, H-6A and H-6B o f the g a l a c t o s e r e s i d u e  cannot  be seen  i n t h e SECSY s i n c e they appear  i n t h e crowded r e g i o n  <5 and form a very s t r o n g l y coupled s p i n system.  between 3.71 - 3.85  can be seen t h a t H-6'B  It  a l s o appears a t t h e l o w f i e l d end o f t h e above  crowded r e g i o n . The  proton-decoupl ed-proton  spectrum  m a j o r i t y o f protons and hence t h e i r chemical can be d e r i v e d without d i f f i c u l t y .  shows w e l l  s h i f t s and c o u p l i n g c o n s t a n t s  The m u l t i p l e t due t o H-4 and H-5'  appear as a s i n g l e peak i n t h e "decoupled" spectrum identical  chemical  shifts.  r e s o l v e d peaks f o r  showing t h a t they have  The H-5 resonance appears as a low i n t e n s i t y  peak amidst t h e second o r d e r a r t i f a c t s , but c o u l d 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 p a t t e r n i n t h e J_ spectrum. In t h e proton decoupled proton spectrum appear  the four H-6 protons  a t 3.87, 3.80, 3.78 and 3.76 £ , and t h e resonances  a t 3.87 and 3.76 6  c o u l d be a s s i g n e d t o H-6'A and H-6'B on t h e b a s i s o f the c o n n e c t i v i t i e s shown i n t h e SECSY spectrum. f o r t h e H-6A and H-6B.  T h i s l e a v e s t h e resonances a t 3.80 and 3.78 <5  O b v i o u s l y , these protons are s t r o n g l y coupled and  accounts f o r t h e complex J-spectrum  observed f o r H-6B.  Thus a l l chemical s h i f t s and c o u p l i n g c o n s t a n t s f o r i n d i v i d u a l sugar protons can be determined  5.4 -  and t h e data a r e t a b u l a t e d i n T a b l e 5.2.  Experimental The  NMR  s p e c t r a were recorded with r e f e r e n c e t o e x t e r n a l  m e t h y l s i l a n e , on a Bruker WH-400 (9.4 T) high r e s o l u t i o n equipped with an Aspect 2000 computer.  tetra-  spectrometer  The Bruker FTNMR-2D software  program ( v e r s i o n # 810515) was used t o a c q u i r e t h e 2D d a t a .  94  Table 5 , 2 -  Proton  chemical  resonances  s h i f t and c o u p l i n g c o n s t a n t data o f the sugar  o f N-h.exyll actobionami de.  Chemical  shifts,  Coupling  ppm  Galactose  constants, Hz  ring:  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  H-6A  3.80  J  5 j 6 B ;  H-6B  3.78  Glucose  J  j  6  = 8.0  A  = 5.2  6A,6B  -  = 1 1  7  open c h a i n :  H-2'  4.39  J , , = 2.8  H-3'  4.17  J , , = 4.2  H-4'  3.97  J ,  H-5'  3.94  J  H-6A'  3.87  J , ,  H-6'B  3.76  2  3  4  4  = 6.5  g l  5',6'A= 3  5  J  3  5  B  6'A,6'B  2  = 6.57  =  ] 1  '  7  95  The the s p e c t r a l block  sweep width  (SW^) i n t h e  r e g i o n d e s i r e d (~1000 Hz) and t h e data were a c q u i r e d on a  s i z e , t y p i c a l l y , o f 4K r e s u l t i n g  Hz/PT.  The number o f p o i n t s a c q u i r e d  corresponded  dimension was s e t t o i n c l u d e only  t o 64 o r 128.  in a digital i n the  The sweep width  r e s o l u t i o n o f 0.48  dimension (SW ) 2  (N-j) u s u a l l y  i n t h e F_-j dimension f o r  J_-resolved experiments was s e t t o a value so as t o i n c l u d e the widest multiplet. corresponded  The d i g i t a l  r e s o l u t i o n obtained  t o 0.28 Hz/PT.  the d i f f e r e n c e i n chemical nuclei.  i n t h i s dimension  typically  In SECSY, the SW^ was set so as t o cover s h i f t s o f t h e f a r t h e s t apart s p i n  The time r e q u i r e d f o r data  half  coupled  a c q u i s i t i o n u s u a l l y amounted t o 4 ' -  8  hours depending on t h e c o n c e n t r a t i o n o f t h e s o l u t i o n . All used.  t h e 2D data p r o c e s s i n g stages  The data were z e r o - f i l l e d  enhancement f u n c t i o n b e f o r e  are automated i n t h e program  and m u l t i p l i e d by s i n e r e s o l u t i o n  Fourier transformation  data p r o c e s s i n g and subsequent p l o t t i n g t y p i c a l l y  i n each dimension. r e q u i r e d about 4  The  hours.  96  References  1.  Jeener, J . , Yugoslavia,  Ampere I n t e r n a t i o n a l Summer School,  Basko P o l j e ,  A p r i l , 1971.  2.  E r n s t , R., C h i m i , (1975), 29, 179.  3.  Muller,  4.  Aue, W. P., B a r t h o l d i , E., E r n s t , R.R.,  L., Kumar, A., E r n s t , R.R.,  J . Chem. Phys., (1975), 63, 5490. J . Chem. Phys., (1976), 64,  2229. 5.  Freeman, R., M o r r i s , G.A., Bui 1. Mag. Reson., (1979), 1_, 5.  6.  Freeman, R.,  7.  Nagayama, K.,  8.  Shaw, D., F o u r i e r Transform N.M.R. Spectroscopy, E l s e v i e r ,  P r o c . R. Soc. Lond. A,  (1980), 373, 149.  Adv. B i o p h y s . , (1981), 14, 139. Amsterdam,  1976. 9. 10.  Aue, W.P.,  Karhan, J . , E r n s t , R.R.,  J . Chem. Phys., (1976), 64, 4226.  Nagayama, K., Wuthrich, K., Bachmann, P., E r n s t , R.R.,  Biochem. .  B i o p h y s . Res. Commun., (1977), 78, 99. 11.  H a l l , L.D., Sukumar, S.,  12.  H a l l , L.D., M o r r i s , G.A., references  13.  J . Am. Chem. S o c , (1979), H U , 3120. Carbohydr. Res., (1980), 82, 175 and  cited therein.  H a l l , L.D., M o r r i s , G.A., Sukumar, S.,  J . Am. Chem. S o c , (1980),  102, 1745. 14.  M u l l e r , L., Kumar, A., E r n s t , R.R.,  15.  Bolton, and  16.  P.H., Bodenhausen, G.,  references  J . Chem. Phys., (1975), 63, 5490.  J . Am. Chem. S o c , (1979), 101, 1080  cited therein.  S t o l l , M.E., Vega, A . J . , Vaughan, R.W.,  J . Chem. Phys., (1976), 65_,  4093. 17.  O p e l l a , S.J., Waugh, J.S., J . Chem. Phys., (1977), 66, 4919.  97  18.  Meier, B.H., E r n s t , R.R.,  J . Am. Chem. S o c , (1979), 101, 6441.  19.  J e e n e r , J . , M e i e r , B.H., Bachmann, P., E r n s t , R.R.,  J . Chem. Phys.,  (1979) , ] ± , 4546. 20.  Sukumar, S.,  Ph.D. T h e s i s ,  U n i v e r s i t y of B r i t i s h  Columbia,  Vancouver, 1979. 21.  H a l l , L.D., Sanders, J.K.M.,  J . Am. Chem. S o c , (1980), 1_02, 5703.  22.  Hall,  J . Org. Chem.,  23.  Nagayama, K., Bachmann, P., Wuthrich, K., E r n s t , R.R.,  L.D., Sanders, J.K.M.,  (1981), 46, 1132. J . Magn.  Reson., (1978), 31, 133. 24.  Nagayama, K., Wuthrich, K.,  E u r . J . Biochem., (1981), 114, 365.  25.  Turner, D.L., Freeman, R.,  26.  Niedermeyer, R., Freeman, R.,  27.  Bodenhausen, G.,  28.  B o l t o n , P.H.,  29.  Bodenhausen, G., Freeman, R.,  30.  Wagner, G., Kumar, A., Wuthrich, K.,  J . Magn. Reson., (1978), ^ 9 , 587. J . Magn. Reson., (1978), 617, 30.  J . Magn. Reson., (1980), 39, 175.  J . Magn, Reson., (1981), 45, 239. J . Am. Chem. Soc., (1978), 100, 320. E u r . J . Biochem., (1981), 114,  375. 31.  Bax, A., Freeman, R.,  J . 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