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Applications of Fourier transform NMR spectroscopy : proton spin-lattice relaxation in organic molecules Preston, Caroline Margaret Callway 1975

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APPLICATIONS OF FOURIER TRANSFORM NMR SPECTROSCOPY: PROTON SPIN-LATTICE RELAXATION IN ORGANIC MOLECULES BY CAROLINE MARGARET CALLWAY PRESTON B.Sc. (Hon.), McMaster U n i v e r s i t y , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shal make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shal not be alowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date did $ /f1S ) ABSTRACT This t h e s i s describes a study of proton s p i n - l a t t i c e r e l a x a t i o n i n organic molecules i n s o l u t i o n , a study made possible l a r g e l y by the development of Fourier transform n.m.r. ( S p i n - l a t t i c e r e l a x a t i o n , which concerns the t r a n s f e r of energy between the spins and the molecular l a t t i c e , can occur by several d i f f e r e n t mechanisms, d i r e c t l y r e l a t e d to such l a t t i c e parameters as molecular geometry and motions.) A q u a l i t a t i v e survey of some mono-saccharides and t h e i r d e r i v a t i v e s revealed a s t e r e o s p e c i f i c dependence of the proton s p i n - l a t t i c e r e l a x a t i o n times (T values); further, the data strongly suggested that the dominant source of r e l a x a t i o n for the protons of an organic molecule i n d i l u t e degassed s o l u t i o n , i n a magnetically i n e r t , solvent, was the intramolecular dipole-dipole mechanism. The s t e r e o s p e c i f i c d i f f e r e n t i a l s , which had c l e a r diagnostic p o t e n t i a l , extended to d i - and polysaccharides, and an a l k a l o i d , vindolene, but not to several high-molecular-weight polysaccharides. Study of two s t r u c t u r a l l y - r e l a t e d groups of molecules a l l s i x -membered rings with known chair conformations i n s o l u t i o n , showed that the proton r e l a x a t i o n could be analyzed as a sum of independent intramolecular dipole-dipole i n t e r a c t i o n s between pai r s of protons; moreover, these pairwise i n t e r a c t i o n s were d i r e c t l y r e l a t e d to r a t i o s of interproton distances. This model was further probed by two methods, s e l e c t i v e deuteration and t a i l o r e d e x c i t a t i o n , and the r e s u l t s obtained by t a i l o r e d e x c i t a t i o n served to strengthen the assumption that the dominant source of r e l a x a t i o n was the intramolecular dipole-dipole mechanism. Other observations included the e f f e c t of changing temperature, concentration, and solvent, the extent of non-exponentiality i n the decay curves, and the much more strongly non-exponential decay of some methyl protons, p o s s i b l y due to c r o s s - c o r r e l a t i o n s . F i n a l l y , our new i n s i g h t into proton r e l a x a t i o n patterns, combined with Fourier transform technology provided some p r a c t i c a l methods f o r improving - i i i -the high-resolution n.m.r. of large organic molecules, i n c l u d i n g manipulation of the na t u r a l l y - o c c u r r i n g r e l a x a t i o n times by the addition of paramagnetic gadolinium ( i l l ) ' i o n s . This t h e s i s also describes the empirical methods adopted, i n the absence of a comprehensive theory, f o r determination of values i n coupled, multi-spin systems, and f i n a l l y , the preparation of some s e l e c t i v e l y deuterated sugar d e r i v a t i v e s . - i v -TABLE OF CONTENTS Page CHAPTER I. AN INTRODUCTION TO SOME ASPECTS OF NUCLEAR SPIN-LATTICE RELAXATION 1 CHAPTER II.. A GENERAL SURVEY OF PROTON SPIN-LATTICE RELAXATION TIMES IN MONO-SACCHARIDES AND SOME "LARGE" ORGANIC MOLECULES 33 CHAPTER I I I . PATTERNS OF PROTON SPIN-LATTICE RELAXATION IN A STX-MEMBERED . RING 75 CHAPTER IV. PHYSICAL AND•CHEMICAL APPROACHES TO THE QUANTITATIVE STUDY OF DIPOLE-DIPOLE PROTON RELAXATION IN ORGANIC MOLECULES 90 Introduction 90 The Six-membered Rings 93 The Five-membered Rings 107 Conclusions 119 CHAPTER V. SOME APPLICATIONS AND EXTENSIONS OF FOURIER TRANSFORM RELAXATION STUDIES 122 Introduction 122 Instrumental Methods 125 Chemical Methods..: 139 CHAPTER VI. EXPERIMENTAL METHODS 151 NMR Measurements 151 Errors . 152 Sources of Samples 159 Syntheses 160 REFERENCES l66 APPENDIX. AN NMR STUDY OF TWO BRANCHED-CHAIN SUGARS 172 -v-LIST OF TABLES Table Page CHAPTER I I . I I - l S p i n - l a t t i c e r e l a x a t i o n times f o r the anomeric protons of some free pyranose sugars 36 II - 2 S p i n - l a t t i c e r e l a x a t i o n times f o r the anomeric protons of some 2-acetamido-2-deoxy-g-hexopyranoses and some . methyl D-hexopyranosides 39 II - 3 Temperature dependence of the s p i n - l a t t i c e r e l a x a t i o n times of the anomeric protons of D-glucose k2 Il-k Anomeric s p i n - l a t t i c e r e l a x a t i o n times of furanose forms of several free hexoses and pentoses k6 II-5 Anomeric s p i n - l a t t i c e r e l a x a t i o n times of some peracetylated g-hexopyranoses... hQ II - 6 Ratios of s p i n - l a t t i c e r e l a x a t i o n times of penta-O-acetyl-3-g-galactopyranose and i t s (perdeuteromethyl) acetylated counterpart 50 II-T Non-exponential character of the decay of methyl resonances of vindolene 62 II - 8 Anomeric s p i n - l a t t i c e r e l a x a t i o n times f o r some disaccharides ; 6k II - 9 Anomeric s p i n - l a t t i c e r e l a x a t i o n times of some oligomers of D-glucose.... 67 11-10 Anomeric s p i n - l a t t i c e r e l a x a t i o n times of some f u l l y methylated polymers of g-glucose 69 I I - 11 Anomeric s p i n - l a t t i c e r e l a x a t i o n times for b a c t e r i a l capsular polysaccharide K l e b s i e l l a 2k .•. TO CHAPTER I I I I I I - l . Proton s p i n - l a t t i c e r e l a x a t i o n rates f o r four i n o s i t o l s 82 I I I - 2 . Pairwise intramolecular dipole-dipole-proton r e l a x a t i o n rates derived f o r t h e ' i n o s i t o l s 8k I I I - 3 Comparison of r a t i o s of interproton distances obtained from pairwise relaxation-rates and a- t h e o r e t i c a l model fo r the i n o s i t o l s 87 - v i -Table CHAPTER IV IV-1 IV-2 IV-3 TV-k IV-5 IV-6 IV-T IV-8 IV-9 IV-10 IV- 11 CHAPTER V V- l V-2 Page Comparison of s p i n - l a t t i c e r e l a x a t i o n rates of pyranoside compounds (51) and (52) i n two solvents 95 Contribution of H - 2 a x to the t o t a l r e l a x a t i o n rate of H-1, H - 2 e g, H-3 and H-^ i n compound (51) 98 Comparison of r a t i o s of interproton distances obtained from pairwise r e l a x a t i o n rates of (51) with two t h e o r e t i c a l models 99 S e l e c t i v e and n o n s e l e c t i v e r e l a x a t i o n rates f o r compound (51), together with r a t i o s and en-hancements 102 Relaxation rates obtained i n " t a i l o r e d e x c i t a t i o n " experiments on pyranoside compound (52) 10U Ratios of r e l a x a t i o n rates and enhancements from the t a i l o r e d e x c i t a t i o n experiments on ( 5 2 ) . . . 105 1 Relaxation rates f o r furanose d e r i v a t i v e s (^9) and (50), showing the contr i b u t i o n of H-3 to the rel a x a t i o n rate of the other protons 110 Relaxation rates obtained i n t a i l o r e d e x c i t a t i o n experiments on alio-furanose d e r i v a t i v e (^9).. 113 Ratios of re l a x a t i o n rates and enhancements from the t a i l o r e d e x c i t a t i o n experiments on ( U 9 ) . . . 11^ Relaxation rates of four s e l e c t i v e l y deuterated glueo-furanose d e r i v a t i v e s , showing the con-t r i b u t i o n of methyl protons to the re l a x a t i o n of the r i n g protons 117 Relaxation rates of allo-furanose compounds (^9) and (53) showing the contr i b u t i o n of the acetate protons to the re l a x a t i o n of the r i n g protons.. 118 E f f e c t of gadolinium (III) n i t r a t e on the anomeric s p i n - l a t t i c e r e l a x a t i o n rates of D-glucose..... ihl E f f e c t of gadolinium (III) n i t r a t e on the anomeric s p i n - l a t t i c e r e l a x a t i o n rates'of maltose. 1^ 2 1 - v i i -Table Page V-3 E f f e c t . o f gadolinium ( i l l ) n i t r a t e on the anomeric s p i n - l a t t i c e r e l a x a t i o n rates o f c e l l o b i o s e 1^3 V-k E f f e c t of gadolinium ( i l l ) n i t r a t e on the anomeric s p i n - l a t t i c e r e l a x a t i o n rates of maltotriose ihh - v i i i -Figure CHAPTER I 1-1 1-2 1-3 1-1+ I- 5 CHAPTER II I I - l II-2 H-3 11-h H-5 II-6 II-T II-8 II-9 LIST OF FIGURES Page Energy l e v e l s f o r a s p i n - % p a r t i c l e i n a magnetic f i e l d h Vector model showing the two-pulse sequence f o r determination of s p i n - l a t t i c e r e l a x a t i o n times... 6 Return of the magnetization to equilibrium following a l80-degree pulse. (A) Mt, (B) Mo-Mt T Energy l e v e l s and t r a n s i t i o n p r o b a b i l i t i e s f o r two s p i n - % p a r t i c l e i n a magnetic f i e l d IT Return to equilibrium i n a two-spin system following s e l e c t i v e and non-selective l80-degree pulses.... 22 P a r t i a l l y relaxed 100 MHz proton n.m.r. spectra of g-glucose, showing the anomeric region 35 Concentration dependence of the anomeric s p i n - l a t t i c e r e l a x a t i o n times of D-glucose hi S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of some peracetylated pyranoses h9 100 MHz proton n.m.r. spectra of penta-0_-acetyl-3-D-galactopyranose, and i t s (perdeuteromethyl) acetylated counterpart, showing the r e l a x a t i o n times of the i n d i v i d u a l t r a n s i t i o n s 52 S p i n - l a t t i c e r e l a x a t i o n times for the alkene protons of v i n y l acetate 55 S p i n - l a t t i c e r e l a x a t i o n times f o r the protons of the a l k a l o i d vindolene. 56 Photograph of a molecular model of vindolene, showing the o v e r a l l s p a t i a l d i s t r i b u t i o n of the substituents.. 58 Plot of ln(MQ-Mf.) vs delay time f o r the down-field t r a n s i t i o n of the E-lh doublet of vindolene. 6 l S p i n - l a t t i c e r e l a x a t i o n times for the anomeric protons of sucrose, r a f f i n o s e , and stachyose.. 68 - i x -Figure Page 11-10 P a r t i a l . 1 0 0 MHz proton n.m.r. spectrum of K l e b s i e l l a 2k polysaccharide at 95°C 71 I I - 11 Plots of ln(Mo-M-t) vs delay, time for the acetate peaks of K l e b s i e l l a 2k b a c t e r i a l polysaccharide 73 CHAPTER III. I I I - l Geometrical proton-proton r e l a t i o n s h i p s i n a s i x -membered r i n g i n chair form 76 III-2 S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of the halp-sugar d e r i v a t i v e s (39) to (k3) 1 77 I I I - 3 S t r u c t u r a l formulae of f i v e i n o s i t o l isomers studied, showing the normal s o l u t i o n conformations 8 l CHAPTER IV IV- 1 S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of pyranoside d e r i v a t i v e s ( 5 l ) and (52) i n deuterochloroform and deuterobenzene 9^ IV-2 P a r t i a l l y - r e l a x e d two-pulse 100 MHz proton n.m.r. spectra of compound (5 1 ) , obtained at Varian Associates, Palo A l t o , C a l i f o r n i a 101 IV-3 P a r t i a l 100 MHz proton n.m.r. spectra of allo-furanose d e r i v a t i v e (k9) and i t s counterpart deuterated at C-3 (50) 108 TV-k S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of allo-furanose compounds (k9), (50) and (53) 109 IV-5 P a r t i a l l y - r e l a x e d two-pulse 100 MHz proton n.m.r. spectra of allo-furanose (^9), obtained at Varian Associates, P a l o ; A l t o , C a l i f o r n i a 112 IV-6 S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of four gluco-furanose d e r i v a t i v e s (5k) to (57)» d i f f e r i n g only i n extent of deuteration I l 6 CHAPTER V V-1 P a r t i a l 100. MHz proton n.m.r. spectra of 1,2,3,^-tetra-0_-acetyl - B - P-ribopyranose ( A ) one FT t r a n s i e n t , (B) 100 t r a n s i e n t s . 126 - X -Figure V-2 . V-3 V-U V-5 v-6 V-T V-8 V-9 V-10 V - l l CHAPTER VI VI.-1 Page P a r t i a l 100 MHz proton n.m.r. spectra of 1 , 2 , 3 , U-tetra-0_-acetyl-3-D-ribopyranose, . showing the e f f e c t s of (B) time-averaging and (C) r e s o l u t i o n enhancement 128 P a r t i a l 100 MHz proton n.m.r. spectra of l , 2 , 3 , U - t e t r a -0-acetyl - 3-D-ribopyranose, showing the e f f e c t of re s o l u t i o n , enhancement on the H-5 resonances 130 P a r t i a l 100 MHz proton n.m.r. spectra of l , 2 , 3 , U - t e t r a -(D-acetyl-S-D-ribopyranose, showing the e f f e c t of progressively greater r e s o l u t i o n enhancement of the H-1 resonance 131 P a r t i a l l y - r e l a x e d 100 MHz proton n.m.r. spectra of g-glucose, i l l u s t r a t i n g the use of a two-pulse sequence to remove the r e s i d u a l HOD peak 133 P a r t i a l l y - r e l a x e d 100 MHz proton n.m.r. spectra of compound ( U l ) , i l l u s t r a t i n g the use of a two-pulse sequence to remove the si g n a l of a more slowly-relaxing proton, H-2 135 P a r t i a l l y - r e l a x e d 100 MHz proton n.m.r. spectra of compound ( U l ) , i l l u s t r a t i n g the use of a three-pulse sequence to remove\the signals of the f a s t e r - r e l a x i n g H-6 protons 136 Mutarotation o f a freshly-mixed s o l u t i o n o f a-D-allose: calcium chloride complex 138 Anomeric s p i n - l a t t i c e r e l a x a t i o n rates of D-allose as a function of the molar r a t i o of gadolinium T i l l ) n i t r a t e . lU6 100 MHz proton n.m.r. spectra of D-allose, showing changes i n the anomeric region from the pure s o l u t i o n (A) with progressive addition of gadolinium ( i l l ) n i t r a t e (B), •(C) 1U8 100 MHz proton n.nur. spectra of a fully-mutarotated s o l u t i o n of a-p-allose:calcium chloride complex, showing changes i n the anomeric region from the pure s o l u t i o n (A), with progressive a d d i t i o n of gadolinium ( i l l ) n i t r a t e '(B), (C), (D) lk9 Graph showing .variation of the apparent T i values obtained for the anomeric protons of D-glucose, as data from longer delay times are included i n the ca l c u l a t i o n s 156 1 - x i -Figure 1 Page VI - 2 . Ty p i c a l decay p l o t of ln(M 0-M^) vs delay time f o r the u p f i e l d t r a n s i t i o n of H-1 of D-glucose. 157 APPENDIX A-1 S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of two branched-chain sugars (59) and ( 6 0 ) . . 173 A - 2 P a r t i a l 220 MHz proton n.m.r. spectra of two branched-chain sugars (59) and (60)......• 175 - x i i -ACKNOWLEDGMENT It i s a pleasure to thank the many colleagues and friends who have contributed to the r e a l i z a t i o n of t h i s work. F i r s t and foremost, the guiding i n s i g h t and imagination, and constant encouragement o f Laurie H a l l w i l l long be remembered and appreciated. In the laboratory, help support and t e c h n i c a l advice were given by many, incl u d i n g Chris Grant and Liane Evelyn. The many discussions of physics with Jon Preston were also invaluable. Expert t e c h n i c a l support was provided by Roland Burton and P h y l l i s Watson i n the n.m.r. laboratory, by the members of the e l e c t r o n i c s shop, by Steve Rak and Joseph Molner who prepared innumerable sealable n.m.r. tubes, by Rasiya Mia who c a r r i e d out some of the T^ measurements, and by G l o r i a Dumoulin who expertly prepared the typed manuscript. Special thanks are due Ray Freeman, Howard H i l l and Bob Jones of Varian Associates at Palo A l t o , C a l i f o r n i a f or many h e l p f u l discussions and the unparalleled opportunity to carry out " t a i l o r e d e x c i t a t i o n " experiments on t h e i r newly-developed equipment. F i n a l l y , generous f i n a n c i a l support was provided by the National Research Council of Canada. We leave with many regrets for our time i n the "magic kingdom", with longing f o r now-distant f r i e n d s , a l i n g e r i n g i n t o x i c a t i o n with mountain and sea and a renewed outlook. - x i i i -PROLOG-UE fragments o f t r u t h , g l i m p s e d d a r k l y f i x e d i n a r t f u l image':' movements and shapes o f r e s t l e s s t u m b l i n g atoms, s c u l p t o r s m o u l d i n g our p a l e and f r a g i l e forms, we s e i z e ' u n c e r t a i n t y . CHAPTER I AN INTRODUCTION TO SOME ASPECTS OF NUCLEAR SPIN-LATTICE RELAXATION noctes v i g i l a r e serenas, quaerentem d i c t i s quibus et quo carmine demum c l a r a tuae possim praepandere lumina menti, res quibus occultas penitus convisere p o s s i s . Hunc i g i t u r terrorem animi tenebrasque necessest non r a d i i s o l i s neque l u c i d a t e l a d i e i d i s c u t i a n t sed Naturae species ratioque. (Lucretius, De Rerum Natura I, 1U2-8) leads me to stay awake through the quiet of the night, studying how by choice of words and the poet's art I can display before your mind a c l e a r l i g h t by which you can gaze i n t o the heart of hidden things. This dread and darkness of the mind cannot be d i s p e l l e d by the sunbeams, the shining shafts of day, but only by an understanding of the outward form and inner workings of nature. ( t r . R. Latham, Penguin Books Ltd., 1951). Organic chemists have long been f a m i l i a r with high r e s o l u t i o n proton nuclear magnetic resonance (n.m.r.) spectroscopy, and the information that can be obtained from the three n.m.r. parameters: integrated areas, chemical s h i f t s and coupling constants. Two further sources of information which are i n p r i n c i p l e , a v a i l a b l e from high-resolution studies, the spin-spin and s p i n - l a t t i c e r e l a x a t i o n times (H^ and ), have l a r g e l y been ignored by chemists. Of the many good reasons f o r t h i s neglect, the most cogent i s that, u n t i l r e c e n t l y , experimental methods for determining r e l a x a t i o n times have been completely incompatible with the complex spectra one obtains for a l l but the simplest organic molecules. Some progress was made with the development of the "audio-pulse" method"'", but a number of serious disadvantages precluded i t s routine a p p l i c a t i o n to organic systems. F i r s t , as only one t r a n s i t i o n at a time 1 could be studied, the experiments became p r o h i b i t i v e l y long. Second, the pulses were weak, and hence long, so that appreciable r e l a x a t i o n might occur during the pulse. However, the most serious handicap was the frequency condition, that t r a n s i t i o n s be separated by at l e a s t f i v e or s i x hertz, so that u s u a l l y only a few of the t r a n s i t i o n s i n a complex proton spectrum were amenable to study. Nonetheless, the few studies c a r r i e d out i n t h i s 2-k laboratory suggested considerable p o t e n t i a l for chemical a p p l i c a t i o n of these almost accessible parameters. Although has yet to be reduced to an "everyday" parameter, the p o s s i b i l i t y of easy and routine.study of the s p i n - l a t t i c e r e l a x a t i o n time, T , was suddenly r e a l i z e d with the development of Fourier transform (FT) 5 8 l6"a spectroscopy . Among i t s now well-known advantages are the following: a l l t r a n s i t i o n s can be studied simultaneously, so that the time required for a complete experiment i s reasonable; the pulses are extremely short, and r e l a x a t i o n during a pulse i s n e g l i g i b l e ; f i n a l l y , the instrumentation lends i t s e l f extremely w e l l to signal-averaging, which can be applied both to the reduction of experimental s c a t t e r , and to dramatic and routine improvement i n 88—90 s e n s i t i v i t y . Recent FT r e l a x a t i o n studies , e x p l o i t i n g t h i s increased s e n s i t i v i t y , have been concentrated on the carbon-13 nucleus; i t seemed worthwhile, however, to investigate also proton r e l a x a t i o n of organic molecules i n s o l u t i o n by the Fourier transform method. We hoped that an understanding of proton r e l a x a t i o n might complement the other three parameters i n s i m p l i f y i n g and i n t e r p r e t i n g high-resolution spectra, and also y i e l d information on molecular geometry, complementing and extending the carbon-13 r e s u l t s , which are a better probe of molecular motion. Spin r e l a x a t i o n i s e s s e n t i a l l y concerned with attainment of thermal 9-12 equilibrium i n the system of spins plus l a t t i c e , and r e l a x a t i o n times are the time constants for these exponential approaches to equilibrium. The processes of spin-spin (transverse) r e l a x a t i o n lead to thermal equilibrium 3 within the spin system, and a sing l e spin temperature. S p i n - l a t t i c e ( l o n g i t u d i n a l ) r e l a x a t i o n , on the other hand, i s concerned with thermal equilibrium between the spins and the l a t t i c e , and the approach to a common system temperature. To study s p i n - l a t t i c e r e l a x a t i o n therefore, the system of spins plus l a t t i c e i s perturbed i n some way, and i t s return to equilibrium monitored. Experimentally, t h i s i s often most conveniently achieved by having the spins absorb energy, and monitoring the flow of t h i s excess energy from the spin system to the l a t t i c e . As n.m.r. energies are very low, of the same order as t r a n s l a t i o n and r o t a t i o n , i t i s into these motions of the l a t t i c e that the energy i s diss i p a t e d . By " l a t t i c e " , therefore, we often mean just these degrees of freedom of the system; under, most conditions, the l a t t i c e heat capacity i s i n f i n i t e l y l a r g e r than that of the spin system. The spin system i s the assembly of spin-l/2 p a r t i c l e s , such as hydrogen n u c l e i , occupying the two energy states defined by the s t a t i c external f i e l d . ( F i g . I - l ) with a very small excess i n the lower (+.) state. AE i s the energy diffe r e n c e between the two spin states, k i s the Boltzmann constant, and T i s the spin temperature (and, at equilibrium, the l a t t i c e temperature). There i s a dynamic exchange of spins between the two l e v e l s , but at equilibrium the upward t r a n s i t i o n s (energy absorption) are balanced by the downward ones (energy l o s s ) . To measure the s p i n - l a t t i c e r e l a x a t i o n time, T^, the system i s i n i t i a l l y perturbed from equilibrium by adding enough energy to the spin system to inv e r t i t s population d i s t r i b u t i o n ; there i s a higher population i n the upper state, 9 and the spin temperature i s negative . As equilibrium i s restored, the rate of downward t r a n s i t i o n s exceeds that of upward t r a n s i t i o n s , and the energy i s At equilibrium, the population d i s t r i b u t i o n of the spins i s described by the Boltzmann condition: AE/kt (1) AE = XnH m = _1 2 N + Figure 1 - 1 . Energy levels for a spin-% particle in a magnetic.field H. m i s the component of spin angular momentum in the direction of the f i e l d , and N + and N_ are the populations of the two states. -W i s Planck's constant over 2TT, and y, the magnetogyric ratio, i s the ratio of the magnetic moment to the angular momentum of the particle. The ordering of the energy levels i s that for positive y, such as that of the hydrogen nucleus, and i s reversed for a negative y. 5 l o s t to the l a t t i c e (whose heat capacity i s so much l a r g e r , that i t s temperature i s unchanged). This process i s monitored at some delay time a f t e r the i n i t i a l perturbation, and the whole sequence i s repeated: perturb, wait and monitor, with varying delays, and from these data sampling population d i s t r i b u t i o n , the s p i n - l a t t i c e r e l a x a t i o n time i s determined. Of course, s u f f i c i e n t time i s allowed between sequences for the whole system to reach equilibrium. To describe the Fourier transform (and other pulsed) methods of 13-1U determining r e l a x a t i o n times , a vector model i s p a r t i c u l a r l y u s e f u l . F i g . 1-2 shows the standard co-ordinate system, and the experimental two-pulse sequence. The s t a t i c magnetic f i e l d i s represented by the vector H i n the +Z d i r e c t i o n , and instrumental transmission and detection occur i n the XY plane. At equilibrium, the l a r g e r spin population i n the lower energy state r e s u l t s i n a net macroscopic magnetization, Mq, i n the d i r e c t i o n of the external f i e l d . The i n i t i a l population inversion i s accomplished by a " l80-degree pulse", ->• which instantaneously rotates M , unchanged i n magnitude, into the -Z a x i s . The return to equilibrium corresponds to M shrinking i n the -Z d i r e c t i o n to zero, then i n c r e a s i n g i n the p o s i t i v e Z d i r e c t i o n to the e q u i l i b r i u m value of +Mq. The magnitude and d i r e c t i o n of M, at some time a f t e r the l80-degree pulse, i s measured by a "90-degree pulse", which rotates M into the XY plane. The pulse sequence used to measure T^ i s thus [.. .180°...delay.. .90°...], with i n c r e a s i n g delay times; each sequence can be time-averaged i f necessary to increase the signal-to-noise r a t i o . The experimental data, the signals recorded from the 90-degree pulses, are a ser i e s of n.m.r. spectra, whose peaks are negative at short delay times, decrease i n magnitude to zero, and increase i n the p o s i t i v e sense to the f u l l e quilibrium s i g n a l i n t e n s i t y . This progression i s shown gr a p h i c a l l y i n F i g . 1-3, and i t s mathematical form i s e a s i l y derived. The approach to equilibrium i s given by: 6 T W O - P U L S E SEQUENCE  FOR DETERMINATION  OF Tj - VALUES Hi 180 0 PULSE E Q U I L I B R I U M M A G N E T I S A T I O N D E L A Y ( T - S E C O N D S ) Figure 1-2. The co-ordinate system and vector model of the two-pulse determination of the s p i n - l a t t i c e r e l a x a t i o n time, . H represents the s t a t i c magnetic f i e l d . The l80-degree pulse perturbs the system; the sig n a l of the monitoring 90-degree pulse a f t e r a suitable delay time i s recorded. Figure 1-3. [A] Return of the magnetization to equilibrium following a l80-degree pulse. follows an exponential recovery curve from -MQ at t=0, through zero, to +MQ as " t " i s increased to i n f i n i t y . [B] i s a plot of (M 0 - M-£) which- decreases from an i n i t i a l value of +2MQ to zero at i n f i n i t e delay time. When the three-pulse sequence i s used, the data are obtained i n t h i s always-positive form, the peaks with the shorter Tj values decreasing more quickly. 8 d t = - T 7 ( M o - M t } ( 2 ) where i s the magnetization a f t e r a delay time " t " , and c l e a r l y i s the inverse of a rate constant. The s o l u t i o n , f o r the i n i t i a l condition of M = - M at t = 0 i s : t o -t/T M = M ( l - 2e ) (2a) U O As shown i n F i g . 1-3, t h i s function increases exponentially from an i n i t i a l value of -MQ, through zero, to +Mq at t=°°. Equation (2a) can also be written i n the form: -t/T (M - M J = 2M e (2b) o t o F i g . 1-3 also shows the function (M - M ) , which i s always p o s i t i v e , and O Xt decreases exponentially from an i n i t i a l value of +2M q to zero at i n f i n i t e delay time. T^ i s conveniently obtained from the slope of a semi-logarithmic p l o t of (M - M, ) v.s. t: o t l n (M - M,) = + In (2M ) (2c) O TJ 1^ o Experimentally, M q i s equivalent to the s i g n a l from a single 90-degree pulse applied at equilibrium, or the s i g n a l i n the two-pulse sequence at very long delay times. In FT experiments i t i s also possible to obtain the data d i r e c t l y i n the form (M - M ) , using a three-pulse sequence [.. .180°...delay. o "0 1 5 90°...DELAY...90°], with computer subtraction of the signals from the two 90-degree pulses. The "delay" i s var i e d as before, while the constant 9 "DELAY" i s several times longer than the longest to be measured, and allows the system to return to complete equilibrium. Both two- and three-pulse sequences are used experimentally; most o f these studies were c a r r i e d out using a three-pulse sequence. These models of r e l a x a t i o n , i n terms of population d i s t r i b u t i o n s and macroscopic properties have not explained how or why i t happens i n the f i r s t place. Spontaneous emission, an important r e l a x a t i o n mechanism i n higher energy forms of spectroscopy, i s completely n e g l i g i b l e at the very low frequencies of n.m.r.; r e l a x a t i o n proceeds by t r a n s f e r of energy to the l a t t i c e , i n the presence of f l u c t u a t i n g l o c a l f i e l d s generated within the l a t t i c e . As the coupling of the two systems i s not p a r t i c u l a r l y e f f e c t i v e , s p i n - l a t t i c e relaxation times are measured i n seconds, minutes and even hours; t h i s i s the basis of the saturation problems that often l i m i t n.m.r. s e n s i t i v i t y . Four mechanisms may cause r e l a x a t i o n of spin-l/2 n u c l e i " ^ : these are the d i p o l e - d i p o l e , s p i n - r o t a t i o n , chemical s h i f t anisotropy, and scalar coupling mechanisms, which have i n common the generation within the l a t t i c e of f l u c t u a t i n g f i e l d s , whose components at s u i t a b l e frequencies cause r e l a x a t i o n . In the dipole-dipole mechanism, the f i e l d s are produced by the random thermal motion of magnetic dipoles (spin-l/2 n u c l e i and unpaired e l e c t r o n s ) . F l u c t u a t i n g f i e l d s are also produced when c o l l i s i o n s cause changes i n the o v e r a l l angular v e l o c i t y of a molecule; r e l a x a t i o n occurs by d i r e c t t r a n s f e r of energy from the spin to the molecular r o t a t i o n a l motion, through the s p i n - r o t a t i o n i n t e r a c t i o n . I f the chemical s h i f t of a nucleus i s a n i s o t r o p i c , the e f f e c t i v e f i e l d at the nucleus, the sum of the external f i e l d and chemical s h i f t " s h i e l d i n g f a c t o r " , changes as the nucleus rotates i n space, and i n t h i s way.chemical s h i f t anisotropy generates a f l u c t u a t i n g f i e l d , and may contribute to r e l a x a t i o n . F i n a l l y , a spin undergoing f a s t r e l a x a t i o n or chemical exchange on a s u i t a b l e time scale may become a source of r e l a x a t i o n f o r the spin coupled to i t , as 10 f l u c t u a t i n g f i e l d s are produced by the changes i n the s c a l a r coupling i n t e r -action between the n u c l e i (the f a m i l i a r " J " s p l i t t i n g ^ ) . In p r i n c i p l e then, the experimentally determined r e l a x a t i o n rate ( l / T ^ value) may be a composite one, r e f l e c t i n g the simultaneous operation of more than one mechanism: \ = l ( V i <3) or i n terms of r e l a x a t i o n times: T r ? - = I T T p S ( 3a ) 1 i v l ; i where the subscript " i " r e f e r s to the d i f f e r e n t r e l a x a t i o n mechanisms. To i n t e r p r e t experimental r e l a x a t i o n measurements therefore, i t i s necessary to determine which r e l a x a t i o n mechanisms they represent, and the r e l a t i v e con-t r i b u t i o n of each. However, while a l l four r e l a x a t i o n mechanisms are p o s s i b l e , fortunately f or the chemist, some are much l e s s probable than others for the protons of an organic molecule i n s o l u t i o n , and indeed, r e l a x a t i o n rates are often dominated by one mechanism. Relaxation by chemical s h i f t anisotropy i s r a r e l y s i g n i f i c a n t , and for protons i t i s completely n e g l i g i b l e . U n t i l r e c e n t l y , i t had been c l e a r l y demonstrated only for the f l u o r i n e nucleus of CHFCl^ at low 17 temperatures , although i t has now been found for carbon-13 n u c l e i i n several 18-19 . 20-21 molecules e s p e c i a l l y at very high f i e l d s . In every case, i t accounted for only part of the t o t a l r e l a x a t i o n r a t e . Relaxation by s c a l a r coupling can be important, e s p e c i a l l y f o r n u c l e i of spin > l/2 (quadrupolar n u c l e i ) , but i s l 6 a unimportant for protons i n the absence of chemical exchange . For protons (and for carbon-13), the most important r e l a x a t i o n mechanism i s the dipole-dipole one, although spi n - r o t a t i o n may contribute to the r e l a x a t i o n of more 11 f r e e l y - r o t a t i n g methyl g r o u p s ^ 28,52,56^ ^ s proton r e l a x a t i o n rates found i n these studies appeared to "be almost completely dipole-dipole i n o r i g i n , only t h i s mechanism w i l l be discussed i n d e t a i l . The theory of dipole-dipole r e l a x a t i o n has been extensively 16 IT 22 developed and tested by experiment ' ' : we w i l l discuss only the r e s u l t s and i n p a r t i c u l a r , t h e i r p h y s i c a l s i g n i f i c a n c e , and relevance to organic chemical (proton) studies. As mentioned previously, the dipole-dipole mechanism involves spin r e l a x a t i o n i n the presence of components at the Larmor frequency i n the f l u c t u a t i n g f i e l d s generated wi t h i n the l a t t i c e by the random motions of magnetic dipoles. Two factors a f f e c t the rate of dipole-dipole r e l a x a t i o n between spins: the magnitude of the l o c a l f i e l d s , and the r e l a t i v e proportion of f l u c t u a t i o n s at the Larmor frequency. The f i e l d associated with a magnetic dipole increases with I t s magnetogyric r a t i o (magnetic moment), and decreases with distance while the s p e c t r a l d i s t r i b u t i o n of l o c a l f i e l d f l u c t u a t i o n s i s determined, i n l i q u i d s , by molecular c o l l i s i o n s . These parameters appear i n the equation f o r the r e l a x a t i o n rate of two i n t e r a c t i n g spin —1/2 p a r t i c l e s I and S : 2 2 1 Y I YS ( VIS = 7 T X T " 6 ~ T 0 0 1 IS r I S where -H" i s Planck's constant over' 2ir, Y T a n ^ Y a are the magnetogyric r a t i o s of I o the two spins, r i s the distance between I and S and x, the c o r r e l a t i o n time, l o characterizes the l a t t i c e motions producing the f i e l d f l u c t u a t i o n s . This equation i s v a l i d only i n the extreme narrowing l i m i t , the usual s i t u a t i o n i n l i q u i d s , where l/x i s much greater than the Larmor frequency. The dependence on y , r and x, each has s i g n i f i c a n t implications for the dipole-dipole r e l a x a t i o n of organic molecules (and of protons i n p a r t i c u l a r ) i n s o l u t i o n . F i r s t , the r e l a x a t i o n rate between two spins i s strongly dependent on t h e i r magnetogyric r a t i o s . In organic molecules, the only commonly occurring 12 nu c l e i of high y are hydrogen, and more oc c a s i o n a l l y , f l u o r i n e , while carbon-13 has both a low natural abundance, and a low y. Thus, f o r protons, the most important internuclear dipole-dipole i n t e r a c t i o n s are with other protons. However, the magnetic moment of the electron i s very much l a r g e r , arid i f unpaired electrons are present i n such paramagnetic species as molecular oxygen, organic r a d i c a l s , and some metal ions, even at low molar r a t i o s they w i l l dominate the re l a x a t i o n of protons, and other spin-l/2 n u c l e i . Experimentally, paramagnetic r e l a x a t i o n can be eliminated i f the sample i s free of paramagnetic impurities, and the dissolved oxygen has been removed by degassing. Second, dipole-dipole r e l a x a t i o n i s even more c r i t i c a l l y dependent on r , the internuclear distance, and hence i t may be an t i c i p a t e d that r e l a x a t i o n times of hydrogen (and f l u o r i n e ) n u c l e i i n r i g i d molecules w i l l have strong geometric dependencies, and thus be a possible probe of molecular geometry i n s o l u t i o n . F i n a l l y , the re l a x a t i o n rate i s l i n e a r l y dependent on the dipole-dipole c o r r e l a t i o n time, an average time between molecular c o l l i s i o n s , a number af f e c t e d , among other things by s o l u t i o n temperature and v i s c o s i t y . In the extreme-narrowing l i m i t 1/T i s of the order of l C T ^ - l O ^ sec much greater than the Larmor frequency; the rather low dipole-dipole r e l a x a t i o n rates decrease as the temperature (and frequency of c o l l i s i o n s ) increases. Dipole-dipole i n t e r a c t i o n s may be i n t e r - or intramolecular, among spins on the. same, or d i f f e r e n t molecules; however, the c o r r e l a t i o n times f o r i n t e r - and intramolecular dipole-dipole i n t e r a c t i o n s correspond to d i f f e r e n t degrees of freedom of molecular motions, and only the intramolecular con-t r i b u t i o n s to r e l a x a t i o n can be d i r e c t l y r e l a t e d to molecular structure through the r T C , f o r i n d i v i d u a l pairwise i n t e r a c t i o n s . The f l u c t u a t i n g f i e l d s which induce r e l a x a t i o n are produced by molecular t r a n s l a t i o n and r o t a t i o n ; to have e f f e c t , these must change ei t h e r the length, or the o r i e n t a t i o n with respect to the external f i e l d H, of the vector r j o i n i n g I and S. I f I and S are on the same molecule, the length of r can only be changed by molecular v i b r a t i o n s , 13 but these are, i n general so fast on the n.m.r. time scale as to have no e f f e c t on r e l a x a t i o n . Changes i n the o r i e n t a t i o n of r with respect to H are caused by molecular r o t a t i o n ; for intramolecular i n t e r a c t i o n s , therefore, T i s e s s e n t i a l l y a r o t a t i o n a l c o r r e l a t i o n time. As the average time between c o l l i s i o n s i n a l i q u i d i s much l e s s than the time required for a molecular r o t a t i o n , r o t a t i o n l6b occurs i n jumps caused by random c o l l i s i o n s . A simple p h y s i c a l model i s -> that of the molecule having a constant angular p o s i t i o n with respect to H f o r an average time x between the c o l l i s i o n s which cause i t to r e o r i e n t . In l i q u i d s with low v i s c o s i t y , at higher temperatures, r o t a t i o n may become l e s s hindered or g a s l i k e , e s p e c i a l l y for small molecules and methyl groups, and then a completely d i f f e r e n t r e l a x a t i o n mechanism, the spin-rotation i n t e r a c t i o n ' may become s i g n i f i c a n t . I f , on the other hand, I and S are on d i f f e r e n t molecules, t h e i r motions with respect to each other are completely random, and thus molecular v i b r a t i o n s , changes i n t r a n s l a t i o n a l v e l o c i t y and molecular r e -o r i e n t a t i o n can a l l produce f l u c t u a t i n g f i e l d s at I and S. The e f f e c t s of v i b r a t i o n are i n s i g n i f i c a n t , again due to the high frequencies involved, and as angular e f f e c t s are considered to be of l e s s s i g n i f i c a n c e than t r a n s l a t i o n a l ones, the former are l a r g e l y ignored i n the theory, and thus f o r intermolecular e f f e c t s , x i s a t r a n s l a t i o n a l c o r r e l a t i o n time. C l e a r l y , x i s a rather crude attempt to grasp, i n a single parameter, the o v e r a l l impact of l a t t i c e dynamics on r e l a x a t i o n . For intermolecular i n t e r -23 actions e s p e c i a l l y , the i n t e r p r e t a t i o n of x i s imprecise ; i n the intramolecular case, the use of a single c o r r e l a t i o n time i n equation (h) assumes i s o t r o p i c r o t a t i o n a l d i f f u s i o n . To make any progress at a l l , i t i s also common to assume that x i s the same for each i n t e r a c t i n g IS p a i r i n small r i g i d molecules with (assumed) i s o t r o p i c r o t a t i o n . I f a molecule i s rather large and non-spherical, which i n organic chemistry, i s more often than not the case, i t s motion may be strongly a n i s o t r o p i c ; furthermore, a s i n g l e c o r r e l a t i o n time i s inadequate to ik deal with " i n t e r n a l motion"; i . e . , the motions of methyl groups, side chains, and non-rigid carton structures. Not only departures from s p h e r i c a l shape, hut also the occurrence of weak p h y s i c a l i n t e r a c t i o n s between solute-solute, or solute-solvent p a i r s could also complicate the picture of molecular r o t a t i o n . This work, with a few exceptions, has studied proton r e l a x a t i o n i n small (on the organic scale) r i g i d molecules, where neither r o t a t i o n a l 2k 25 anisotropy ' or i n t e r n a l degrees of freedom should be s i g n i f i c a n t . The approach has also been to compare data from f a m i l i e s of s t r u c t u r a l l y s i m i l a r molecules under i d e n t i c a l experimental conditions, so that i t could be reasonably assumed that T, whatever i t s precise i n t e r p r e t a t i o n , would be the same f o r each molecule. To summarize, i n the absence of paramagnetic species, or f l u o r i n e , the dominant source of r e l a x a t i o n f o r the protons of an organic molecule i s the dipole-dipole (proton-proton) mechanism; sp i n - r o t a t i o n r e l a x a t i o n may be s i g n i f i c a n t f o r methyl groups and very small molecules at higher temperatures, i n solutions of low v i s c o s i t y . Where the dipole-dipole mechanism i s the only source of proton r e l a x a t i o n , the t o t a l r e l a x a t i o n rate of proton I can be expressed as a sum of the i n t e r - and intramolecular contributions: U l ' DD X l , . . V . DD ( i n t e r ) ( i n t r a ) where the intramolecular rate, i n turn, can be approximated as a sum of the independent pairwise i n t e r a c t i o n s of proton I with protons S^, ... , on the same molecule: (T, ) " f (T. ) I,. . , IS. (in t r a ) 1 I f these i n d i v i d u a l contributions to the intramolecular r e l a x a t i o n rate of 15 proton I can be determined, and i f t h e i r c o r r e l a t i o n times are approximately equal, then r a t i o s of interproton distances, can be obtained from the s i x t h root of r a t i o s of the i n d i v i d u a l T values: (T ) v Vis. J 1/6 '(VlS.' i 1/6 r IS. ,1 (T ) ^ 1 ;IS. 1 X J (VlS. 1 JJ ris. 1 (6) To put these r a t i o s of distances on an absolute b a s i s , an independent measure 29 of x i s necessary Ignoring the t h e o r e t i c a l l i m i t a t i o n s of such a s i m p l i s t i c a n a l y s i s , the most serious experimental problem i s to eliminate or determine the i n t e r -molecular contributions to r e l a x a t i o n . Intermolecular dipole-dipole r e l a x a t i o n may be minimized by using d i l u t e s o l u t i o n s , to reduce solute-solute i n t e r a c t i o n s , and also by choosing a non-magnetic solvent. Such solvents include carbon t e t r a c h l o r i d e and carbon d i s u l f i d e ; i t i s also common to use the deuterated analogues of such proton-containing solvents as benzene, water, and chloroform. The very much smaller magnetic moment of the spin-1 deuterium nucleus does not s i g n i f i c a n t l y add to the r e l a x a t i o n of solute hydrogen n u c l e i . The i n t e r -molecular r e l a x a t i o n rate can be determined by studying the e f f e c t of d i l u t i o n on T^ values. The changes i n v i s c o s i t y (and x) with d i l u t i o n can be minimized by re p l a c i n g an increasing proportion of the solute with i t s deuterated analogue, i f i t i s a v a i l a b l e . Deuterium s u b s t i t u t i o n also o f f e r s a way of e x t r a c t i n g i n d i v i d u a l pairwise r e l a x a t i o n rates from the t o t a l intramolecular r e l a x a t i o n rate of a proton. I f spin S^ i s replaced by a deuterium nucleus, then the contribution of the proton to the r e l a x a t i o n of proton I can be determined d i r e c t l y from a comparison of the intramolecular r e l a x a t i o n rate of spin I i n the normal and deuterated compound under the same experimental conditions. Again, there 1.6 w i l l be only a small error- due to dipole-dipole r e l a x a t i o n by the deuterium nucleus. Rather s u r p r i s i n g l y , i t was found that t h i s completely naive analysis accounted for many of the features of proton s p i n - l a t t i c e r e l a x a t i o n i n organic molecules. Even at high concentrations, the r e l a x a t i o n rates c l e a r l y r e f l e c t e d the stereochemical environment of the proton. The data obtained from d i l u t e degassed solutions implied strongly that proton s p i n -l a t t i c e r e l a x a t i o n was completely intramolecular dipole-dipole i n o r i g i n ; i n some cases i t was possible to c a l c u l a t e i n d i v i d u a l pairwise r a t e s , which appeared to add i n a simple way, and to give chemically reasonable r a t i o s of interproton distances. This p i c t u r e , however, has not accounted f o r a l l the observations, i n p a r t i c u l a r the many "non-exponential" recovery data. Some in s i g h t into dipole-dipole proton r e l a x a t i o n i n large organic molecules can be gained from consideration of the theory for a system of two non-identical spins. 30 31 This discussion w i l l follow the treatment by Solomon ' to a large extent, and again, for mathematical s i m p l i c i t y , w i l l be l i m i t e d to sp i n - % p a r t i c l e s , although s i m i l a r p r i n c i p l e s apply to higher spins. Consider a system of two s p i n - % p a r t i c l e s I and S. F i g . I-k shows the four energy l e v e l s ; the are the p r o b a b i l i t i e s per second f o r t r a n s i t i o n s between the states. The t r a n s i t i o n s described by W^ , W^ ,, Wg and Wgt, change the energy of only one spin, while i s the p r o b a b i l i t y of a double-quantum t r a n s i t i o n , i n which both I and S gain or lose energy. ¥ q i s the zero-quantum t r a n s i t i o n p r o b a b i l i t y f o r a mutual exchange of spin states, which preserves the t o t a l spin energy. In the absence of sca l a r coupling,. = W^ ,, and Wg = Wg,; t h i s i s a reasonable assumption also f o r f i r s t - o r d e r scalar coupling, where the chemical s h i f t d i f f e r e n c e of I and S i s much l a r g e r than t h e i r coupling constant. Under these conditions, changes i n the energy l e v e l populations, N , N , N and N can be described by the four k i n e t i c equations: 17 Figure l-h. T r a n s i t i o n p r o b a b i l i t i e s per second (Wj_) among the four states of a.system of two s p i n - % p a r t i c l e s I and S i n a s t a t i c magnetic f i e l d . ±%, as for the i s o l a t e d spin (Figure I - l ) . i s the component of spin angular momentum i n the d i r e c t i o n of the: f i e l d . The magnetogyric r a t i o s of I and S, and scalar coupling, i f present, determine the-precise ordering of the spin states. 18 d N+ + dt = -(WT + ¥ c + ¥ 0)N + WGN + WTN + ¥ 0 N + constant (7a) dW+_ — — = ¥ J , , - (¥ + ¥ T + ¥ rjN, + ¥ N + ¥ T N + constant (To) dt S ++ o I S +- o -+ I — dW_ — — = ¥ T N + ¥ N - (W- + W-, + W-,)N + ¥ 0 N + constant (7c) dt I ++ o +- o I S -+ S — dN = W01T L + ¥ J T + ¥ nN _,_ - (WT + ¥ 0 + w_)N + constant (7d) dt 2++ . "I"+- S -+ V " I S 2' — Again, i n the absence of scalar coupling, I and S are each r e -presented by a single n.m.r. s i g n a l ; the observable net macroscopic magnetizations I and S (corresponding to M i n the vector model for a single spin ( F i g . 1-2)), are proportional to the population differences between the two upper, and two lower states of each spin: I « (N • • +• I T ) - (N + N ) (8a) z ++ -+— —+ — S « N ) - (W + N ) (8b) z ++ —+ +— — Combining equations (7) and (8) gives: d l — £ = _(w + 2¥ T + ¥ Q)(I - I ) - (W„ - ¥ )(S - S ) (9a) dt o I 2 z o 2 o z o dS d T = " ( W o + 2 W S + W 2 ) ( S z - So> " ( W2 " W o ) ( l z " I o ) ( 9 b ) where S and I are the equilibrium magnetizations. I t i s convenient to o o define: 19 p T = W + 2¥ T + ¥ 0 p = ¥ + 2¥ Q + ¥ 0 a = w . - ¥ 2 o The general solutions of (9a,b) have the form of sums of two exponential terms: - X + t -X_t I - I = A + Be (10a) z o e -X+t -X_t S - S = Ae - Be (lOb) z o where the constants A and B are determined by the i n i t i a l conditions, and the exponents A and X are functions of p T , p c and o. ' — X o The exact form of the exponents depends on the values of the various t r a n s i t i o n p r o b a b i l i t i e s , which i n turn are determined by the mechanisms causing r e l a x a t i o n . The chemical s h i f t anisotropy, s p i n - r o t a t i o n , and ( f o r n u c l e i of spin > %) quadrupolar r e l a x a t i o n mechanisms contribute only to the one-spin t r a n s i t i o n p r o b a b i l i t i e s ¥ T , ¥ ,, ¥ and ¥ the " c r o s s - r e l a x a t i o n " terms, J. i D O ¥_ and W are zero. In t h i s case, the d i f f e r e n t i a l equations (9a,9t>) for I t— o z and S become: z d l — - = -2W,(I - I ) (9a') dt I z o dS — - = -2¥ q(S - S ) (913') dt S z o Thus, when spin r e l a x a t i o n occurs s o l e l y by these mechanisms, 1^ and return to t h e i r equilibrium values independently, and follow simple exponential functions: comparison with equation (2) for an i s o l a t e d spin shows that l/(T-. ) t = 2¥ T and l / ( T ) c = 2¥ . ( i f more than one r e l a x a t i o n mechanism i s X X X X o o occurring simultaneously, the t o t a l ¥^ or ¥ g i s the sum of the t r a n s i t i o n p r o b a b i l i t i e s f or each mechanism.) 20 For those r e l a x a t i o n mechanisms which require i n t e r a c t i o n of two spins, dipole-dipole and sc a l a r coupling, the cross-relaxation t r a n s i t i o n p r o b a b i l i t i e s are non-zero; these mechanisms contribute therefore to both p and a. The t r a n s i t i o n p r o b a b i l i t i e s f o r dipole-dipole r e l a x a t i o n between two s p i n - % p a r t i c l e s I and S, i n the absence of scalar coupling are given by: where, i n the extreme-narrowing l i m i t , l / ^ ^ D D ^ s a s previously defined i n equation {h): 2 2 ^ 2  1 Y l T S * 1>W r I g The approximation WT = Wc i s reasonable even when I and S are heteronuclei, i f the difference i n t h e i r magnetogyric r a t i o s i s small, as, for example, hydrogen 30 31 and f l u o r i n e ' . More complex expressions are required f o r the t r a n s i t i o n p r o b a b i l i t i e s i f I and S are strongly coupled. When I and S relax s o l e l y by t h e i r mutual dipole-dipole i n t e r -a c t i o n , and i n the l i m i t s described above where W-j. ~ W^ , ~ Wg » Wg, = W^ , and thus ~ pg = p, the exponents X+ and \_ i n the solutions (l0a,10b) for I and are simply (p+o) and (p-a). These functions w i l l be examined f or two i n i t i a l conditions o f experimental i n t e r e s t ; the inversion of both I and S by a non-selective 180-degree pulse (the ordinary Fourier transform z experiment), and the inversion of only one of the spin populations by a se l e c t i v e 180-degree pulse, which i s now also possible with FT methods. The behaviour of I and S i n t h i s very simple system provides valuable i n s i g h t , z z as the same p r i n c i p l e s govern dipole-dipole r e l a x a t i o n i n r e a l organic molecules, although the equations must be modified to include strong coupling, 21 multispin systems, and contribution of r e l a x a t i o n mechanisms other than the intramolecular (homonuclear) dipole-dipole one. For the non-selective, experiment, the i n i t i a l conditions are: I - I = -21 and S - S =' -2S z o o z o o which gives f o r the constants, A = (I + S ) and B = (I - S ) . However, f o r o o o o two s p i n - % n u c l e i with the same value of y•> the equilibrium magnetizations are equal, and B = 0. The solutions (l0a,10b) reduce again, to simple exponential functions, with a single rate constant, l / ( T )^ = (p+a): I = S = I (1 - 2 e " ( p + a ) t ) ' (10') z z o The recovery curves of I and S are, i n f a c t , i d e n t i c a l . z z I f the l80-degree pulse now a f f e c t s only one of the spins, the i n i t i a l conditions are: I - I = -21 and S - S = 0 z o o z o and A = B = -I . The solutions f o r I and S are now: o I = I [1 - ( e - ^ t + e - ( p " a ) t ) ] (I0a") S = I [1 - ( e - ( p + 0 ) t - e - ( p - 0 ) t ) ] (lOb") z o The perturbation of I causes a deviation of S from i t s equilibrium value, z z and two time constants are required to characterize the "non-exponential" return of I (and of S ) to equilibrium. F i g . 1-5 shows the functions I and z z z S vs. time, following a s e l e c t i v e i n v e r s i o n of I (l0a",10b") and also the z z fa s t e r common exponential recovery of both spins following a non-selective l80-degree pulse ( 1 0 ' ) . 22 Mo M t - 0 . 8 - ; - i o -Figure 1 - 5 . Return to equilibrium of the magnetization of spins I and. S, showing t h e i r common recovery path ( 1 0 ' ) , following a non-selective 180-degree pulse, and the: changes i n I z ( 1 0 a " ) and S z ( 1 0 b " ) , following a s e l e c t i v e inversion of I z . The l i m i t s f o r which these functions were derived have been explained i n the text. 2 3 While i t i s possible to f i t experimental data to functions of the type ( l 0 a " , 1 0 b " ) , the i n i t i a l rate of recovery immediately following a s e l e c t i v e l80-degree pulse can be approximated by a simple exponential function, with a si n g l e "T ". Looking again at the d i f f e r e n t i a l equations (9a,9t>), the population deviation (S^ - S Q) i s zero at the time of the pulse, and close to zero f o r a short time a f t e r i t . The k i n e t i c expressions for I and S are z z thus i n i t i a l l y : d l —2-= - p ( l - I ) (9a") dt z o dS and, f o r delay times which are short compared with l / a seconds, l / ( T )^ ~ p. I f (T^)-]- i s measured following both a non-selective and a s e l e c t i v e pulse, the r a t i o of the two time constants i s given by (using, f o r the s e l e c t i v e pulse, the i n i t i a l slope): W + 2W + W R 2 1 o R W„ - W o 2 o where R = l / ( T )^ following a non-selective pulse, and R q = l / ( T ^ ) I following a s e l e c t i v e l80-degree pulse on spin I. As the three t r a n s i t i o n p r o b a b i l i t i e s are r e l a t e d by W :W2:Wq•= 3:12:2, (p+a)/p =,,1.5-Thus, i f two s p i n - % p a r t i c l e s , i n the l i m i t s described, relax.only through a mutual dipole-dipole i n t e r a c t i o n , the r e l a x a t i o n rate measured following a non-selective pulse i s greater than the i n i t i a l rate following a s e l e c t i v e pulse on one of the spins, by a factor of 1.5. This r a t i o , and the perturbation of S following a s e l e c t i v e Inversion.of only I , are a dynamic z z 32 33 version of the nuclear Overhauser e f f e c t (WOE) ' , which occurs because, whenever two-spin t r a n s i t i o n s are allowed, the populations of I and S are 2k not independent. The " s t a t i c WOE" i s f a m i l i a r i n chemical a p p l i c a t i o n s of n.m.r.; i n t h i s experiment, (under steady-state conditions), an increase i n the s i g n a l of one spin i s observed upon saturation of the other"^ The two-spin theory has been generalized to l a r g e r systems"^ 3 , by simply adding the independent pairwise i n t e r a c t i o n s of each spin; the k i n e t i c equation f o r I i n a multispin system of I,S n,S„, ... S becomes: z ' 1' 2 n d l # • aT = " ( p I + I p I S . ) ( l z " V " ? °IS. ( S z . " S o . } ( 1 2 ) i i 1 1 1 1 n s i m i l a r equations describe the behaviour of the S spins. As before, p and l u . 1 0j.g describe dipole-dipole t r a n s i t i o n p r o b a b i l i t i e s between I and another 1 * spin S^. Pj i s the re l a x a t i o n rate of I due to such mechanisms as spi n -r o t a t i o n , and chemical s h i f t anisotropy, which do not contribute to two-spin t r a n s i t i o n p r o b a b i l i t i e s . For s i m p l i c i t y , i t w i l l be assumed that only d i p o l e -dipole r e l a x a t i o n contributes to p „ and a ; cross-relaxation by the scalar l b . l b . 1 1 coupling mechanism would be included i n a completely general d e s c r i p t i o n . The general solutions are sums of (n+l) exponential terms. We w i l l examine only the i n i t i a l rates of recovery of I f o r a few i n i t i a l conditions of experimental i n t e r e s t . I f the perturbation i s a s e l e c t i v e inversion of I , a l l of the z deviations (S - S ) are i n i t i a l l y zero, and the i n i t i a l change i n I can be z. o. z l l approximated by: d l z = - ( P T + I P I S )CF - I J (13) i i The s o l u t i o n i s , of course, a simple exponential, with: T T V = r 0 = ( p i + ? PIS . 5 1 I 1 1 I f , on the other hand, the i n i t i a l perturbation i s a non-selective 25 180-degree pulse, i n i t i a l l y , the population deviations are r e l a t e d by: YS. (I - I ) = — — (s - s ) z o y T z. o. I x 1 and equation ( 12) becomes d l dt Yl f H I S . Y T - IS.'S. / v z o' 1 1 1 I X X X I Where only dipole-dipole r e l a x a t i o n contributes to p ^ g and C g , i i p = 2 a , and while, i n a completely general treatment, a l l of the y Xb. Xb. b. I X X could be d i f f e r e n t , a more normal condition would be to set Yg ~ Yg> where i a l l of the S spins are the same nuclear species, and thus s i m p l i f y equation U M : Comparing i n i t i a l rates for the two experiments: R P / + ( 1 + 2 f " } I P I S . R _ I x x R ~ * M v P l + I P I S . x x where, as before, R and R q are the r e l a x a t i o n rates of spin I following, r e s p e c t i v e l y , a non-selective ( i n i t i a l rate) and s e l e c t i v e l 8 0 -degree pulse. I f the only r e l a x a t i o n mechanism of the I spins i s the dipole-dipole i n t e r -a ction with the S spins, p = 0 , and the r a t i o R/R = ( l + y„/2yT). I f X O o X y z y as, for example, a l l of the spins are hydrogen n u c l e i , R/R = 1 . 5 . o J_ O The enhancement over the s e l e c t i v e r e l a x a t i o n rate i s given by: R — R E I = — g - 2 - = 0 . 5 ( 16) 26 Thus, i n . a multispin system, measurement of the i n i t i a l r e l a x a t i o n rates of a spin a f t e r both s e l e c t i v e and non-selective pulses can give information about 3S 39 the type of re l a x a t i o n mechanisms operating ' . The enhancements decrease from the maximum of 0.5 as the contribution from "external" r e l a x a t i o n mechanisms, i . e . , , increases. Information about the r e l a t i v e magnitude of the d i f f e r e n t pairwise dipole-dipole i n t e r a c t i o n s can also be obtained, when i t i s instrumentally possible to perturb s e l e c t i v e l y d i f f e r e n t subgroups of the spin system^ >-^9 ^ while l e a v i n g other spins undisturbed. I f a l 8 0-degree pulse inverts 1^ and the subgroup S^jS^, ... S^, the population deviations of the S spins immediately following the pulse are approximately: YS. (S - S ) = — - (I - I ) 1 <. i <. m z. z y T z o 1 o I (S - S ) = 0 m < i <_ n z. o. I I I n i t i a l l y , I can be approximated by: (I - I ) (IT) = - R' (I - I ) z o I f p = 0 , y = y , and p I g_ = 2o : 1 i x Y R ' = ? P I S . + 2^1 .1 PIS. I I 'I i=l ,m. i and the enhancement over the s e l e c t i v e r e l a x a t i o n rate i s now given by: 27 Yg R' - R 2Y7 • I P I S . E T = -^—S. = 1 , 1 = 1 ^ m 3, ( 1 8 ) 1 xi o I P IS. I t i s convenient to set ][ p^g = p, and £ = Xp, where 0<X<1. I f Yg = Y j 5 i i i=l,m the enhancement "becomes simply: , R' - R o E x = — = 0 . 5 X ( 1 8 ' ) To study the relaxation of, for example, the chemically s h i f t e d protons of an organic molecule, two types of r e l a x a t i o n experiments, therefore, may be combined. F i r s t , the r e l a x a t i o n rates measured a f t e r s e l e c t i v e and non-s e l e c t i v e pulses may be compared; enhancements of 0 . 5 i n d i c a t e that the 38 r e l a x a t i o n i s completely dipole-dipole i n o r i g i n . Then, to determine r e l a t i v e magnitudes of the pairwise i n t e r a c t i o n s , groups of two or more spins may be s e l e c t i v e l y inverted. I f spin S^ i s inverted along with spin I, then the f r a c t i o n of the t o t a l dipole-dipole r e l a x a t i o n of I a r i s i n g from the i n t e r a c t i o n with S^ i s simply (E^- / 0 . 5 ) . These experiments can also provide information about molecular geometry. I f I, and then the p a i r s IS^ and IS^ are studied, the enhancements of the r e l a x a t i o n rate of I are proportional to p.j.g and i p g , which i n turn are r e l a t e d d i r e c t l y to the s i x t h power of the interproton 3 distances, r^g and r^g : i i ( s i ) . W I S ± l r i S j E I ( S . ) P I S : r I S i , (19) Our experimental r e s u l t s could, i n large part be adequately treated by these rather simple mathematics, although two phenomena, strong scalar 28 coupling and cr o s s - c o r r e l a t i o n s of the f l u c t u a t i n g l o c a l f i e l d s were not included i n the model. (Scalar coupling i s , of course, f a m i l i a r ; ^ "cross-U-5-51 c o r r e l a t i o n s " w i l l "be defined l a t e r . ) There i s , i n f a c t , no completely general theory to d e t a i l the r e l a x a t i o n behaviour of each t r a n s i t i o n i n a complex proton spectrum: t h e o r e t i c a l treatments have included the multispin system i n the l i m i t of f i r s t - o r d e r coupling, as out l i n e d i n the previous sec t i o n , the strongly-coupled two-spin system, and the e f f e c t s of cross-c o r r e l a t i o n s i n small groups of equivalent spins. Fortunately, except i n ce r t a i n l i m i t i n g cases, the e f f e c t s of scalar coupling or cro s s - c o r r e l a t i o n s i n our " r e a l " systems appeared to be s l i g h t , causing only small perturbations i n the re l a x a t i o n behaviour predicted by the simple multi-spin theory. We s h a l l therefore give only a b r i e f review at t h i s point of some relevant t h e o r e t i c a l studies, while observations of minor perturbations, and also some pathological ones, w i l l be noted i n l a t e r chapters. Wo attempts were made to tr e a t these q u a n t i t a t i v e l y . Interest i n the dipole-dipole r e l a x a t i o n of coupled spin systems was spurred by the i n i t i a l development of high-resolution n.m.r., which made possible the study of i n d i v i d u a l spins, and then of Individual t r a n s i t i o n s of 1+1 a m u l t i p l e t . Shimuzu and Fujiwara made a d e t a i l e d t h e o r e t i c a l study of re l a x a t i o n i n a coupled two-spin system. Theoretical and experimental aspects 37 were combined i n a study by Noggle using the saturation-recovery method, and 1+2 a more recent audio-pulse study by Freeman . Freeman has now re-examined the two-spin system with p a r t i c u l a r emphasis on Fourier transform experiments, i n c l u d i n g the l i m i t s of strong and weak coupling, several d i f f e r e n t i n i t i a l conditions, homo- and heteronuclear systems, and varying proportions of "external" r e l a x a t i o n (that i n ad d i t i o n to the mutual dipole-dipole i n t e r a c t i o n ) by other r e l a x a t i o n mechanisms, and by dipole-dipole i n t e r a c t i o n s with spins outside the system. These parameters a l l a f f e c t the degree to which r e l a x a t i o n (even a f t e r a non-selective pulse) can be non-exponential, the differences 29 among the i n d i v i d u a l t r a n s i t i o n s of a m u l t i p l e t , and the d e f i n i t i o n of the rate constants i n terms of t r a n s i t i o n p r o b a b i l i t i e s . While t h i s study dealt with groups o f only two coupled spins, many of the conclusions seemed consistent with our experience of multispin systems and with our e m p i r i c a l l y derived methods of data handling. The reader i s r e f e r r e d to the o r i g i n a l paper f o r 30 d e t a i l s ; the approach i s s i m i l a r to Solomon's for two non-coupled spins, but there are now four observables f o r which k i n e t i c equations (corresponding to eq. (9a,9b)) must be solved, using the f u l l expressions incorporating coupling strength for the dipole-dipole t r a n s i t i o n p r o b a b i l i t i e s , and i n c l u d i n g terms for external r e l a x a t i o n (the t r a n s i t i o n p r o b a b i l i t i e s p and p c ). Again, i t may be appropriate sometimes to measure i n i t i a l r a t e s , sometimes to f i t non-exponential data to t h e o r e t i c a l curves. U n t i l r e c e n t l y , experimental demonstrations of c r o s s - c o r r e l a t i o n kk i+5 e f f e c t s ' had been even rarer than those of r e l a x a t i o n by chemical s h i f t kG kl anisotropy, but i n t e r e s t i n t h i s phenomenon too, has now revived. ' . As previously discussed for the dipole-dipole r e l a x a t i o n mechanism, the f l u c t u a t i n g l o c a l f i e l d s can be considered to a r i s e from changes with respect to the external f i e l d i n the magnitude or o r i e n t a t i o n of a vector connecting two spins and the g e n e r a l i z a t i o n to multispin systems simply adds a l l possible pairwise i n t e r a c t i o n s to derive the t o t a l dipole-dipole r e l a x a t i o n rate of a spin. I m p l i c i t i n t h i s treatment i s the assumption that the: motions of the d i f f e r e n t r e l a x a t i o n vectors with respect to each other are completely random, that there i s no c o r r e l a t i o n between the dipole-dipole i n t e r a c t i o n s of the p a i r IS. and the IS.. This i s v a l i d f or intermolecular i n t e r a c t i o n s , where the 1 J r e l a t i v e motions of n u c l e i are indeed random f o r p r a c t i c a l purposes. However, i t can not be true f o r intramolecular i n t e r a c t i o n s , where n u c l e i are e i t h e r held i n a f i x e d geometric pattern, or" have only l i m i t e d i n t e r n a l degrees of freedom. The e f f e c t s of c r o s s - c o r r e l a t i o n s of the l o c a l f l u c t u a t i n g f i e l d s have been included i n several t h e o r e t i c a l studies of dipole-dipole r e l a x a t i o n 30 among three or four i d e n t i c a l s p i n - % p a r t i c l e s i n f i x e d arrangements. Early s t u d i e s ^ ^ of an e q u i l a t e r a l t r i a n g l e of three spins i n a l i q u i d , or under-going hindered r o t a t i o n about a f i x e d axis i n a s o l i d showed that r e l a x a t i o n was changed from a s i n g l e exponential to a sum of several exponential terms, and was always slower than i f c r o s s - c o r r e l a t i o n s were neglected. The perturbations, however, were very small, and seemed of l i t t l e p r a c t i c a l i n t e r e s t . Later c a l c u l a t i o n s for a three-spin group allowed to rotate about an axis f i x e d to a molecule i n s o l u t i o n " ^ showed that the r e l a x a t i o n might be very non-exponential i n c e r t a i n l i m i t s . For the p r a c t i c a l case of a methyl group undergoing hindered r o t a t i o n which i s r a p i d compared with the r o t a t i o n a l motion of the whole molecule ( i t s e l f i n the extreme-narrowing l i m i t ) , the r e l a x a t i o n i s the sum of two exponential terms: - s t - s t I - I = I {A e + A 0e } ( 2 0 ) z o . o 1 2 where Hubbard''"'" estimated that A^ ~ 1 / 6 , A^ " 5/6 and s^ << s^. Some examples of non-exponential methyl group r e l a x a t i o n , which may p o s s i b l y be due to cross-c o r r e l a t i o n s , are pointed out i n Chapter I I . And while the suggestion from theory c e r t a i n l y i s that c r o s s - c o r r e l a t i o n e f f e c t s should be n e g l i g i b l e for non-methyl protons, i t i s i n t e r e s t i n g to speculate whether some of t h e i r non-exponentiality following non-selective 180-degree pulses might not be caused not only by scalar coupling, which i n two-spin systems can cause both increases and decreases from the i n i t i a l rate , but also by c r o s s - c o r r e l a t i o n s . Interpretation of r e l a x a t i o n data f o r methyl protons i s complicated "by ye* another f a c t o r , the p o s s i b i l i t y of s p i n - r o t a t i o n r e l a x a t i o n , e s p e c i a l l y at higher temperatures. Several studies have shown the s i g n i f i c a n c e of s p i n -r o t a t i o n for methyl g r o u p s ^ ,5 2 , 5 6 ^ m o l e c u l e s as toluene at room temperature. Thus, to use the r e l a x a t i o n times, one must separate contributions of dipole-dipole and s p i n - r o t a t i o n r e l a x a t i o n . Further, any contributions by 31 s p i n - r o t a t i o n and intermolecular dipole-dipole r e l a x a t i o n , which both follow a simple exponential function, w i l l tend to obscure the small non-exponential component, due to c r o s s - c o r r e l a t i o n s , i n the intramolecular part. Even i f t h i s i s a l l sorted out, which can be done, geometrical information concerning interproton distances within.a methyl group i s of l i m i t e d chemical i n t e r e s t , and carbon-13 r e l a x a t i o n would be a more sensible probe of motional parameters. For a number of reasons then, these studies included only a few q u a l i t a t i v e observations of methyl proton r e l a x a t i o n . While proton r e l a x a t i o n i n s o l u t i o n had been studied for some small, highly symmetrical molecules 2^'^8,52 56 s u c l l a s • ) 3 e n Z i e n e 5 toluene and a c e t o n i t r i l e , and explained i n terms of i n t e r - and intramolecular dipole-dipole and s p i n -r o t a t i o n r e l a x a t i o n , nothing was known of proton r e l a x a t i o n i n the very complex high-resolution spectra of the l a r g e r molecules of i n t e r e s t to organic chemists. Our i n i t i a l study was, therefore, a general survey of proton r e l a x a t i o n i n some mono-saccharides and t h e i r d e r i v a t i v e s , which was then extended on the one hand to a few representative examples of other organic types, i n c l u d i n g one natural product, and on the other to d i - , o l i g o - and polysaccharides. The main points that emerged from t h i s were that the T^ values showed a wide and p o t e n t i a l l y u s e f u l range, and c l e a r l y r e f l e c t e d the stereochemical environment of non-methyl protons. The stereochemical dependencies, i n f a c t , strongly suggested the predominance of the intramolecular dipole-dipole mechanism. Chapter II describes t h i s e s s e n t i a l l y q u a l i t a t i v e work. Chapter I I I takes a chemical approach to t r a c i n g the pairwise intramolecular i n t e r a c t i o n s , by studying sets of organic molecules with very s i m i l a r structures and s o l u t i o n conformations. This study of two groups of six-membered rings implied even more strongly that proton r e l a x a t i o n i n d i l u t e degassed solutions occurs predominantly by the intramolecular dipole-dipole mechanism, and that the i n d i v i d u a l pairwise contributions do appear to be a d d i t i v e . 32 Chapter.IV describes more quantitative attempts to determine the re l a x a t i o n mechanism, and to measure the size of pairwise i n t e r a c t i o n s , using a combination of phys i c a l and chemical methods. Two sets of sugar d e r i v a t i v e s were prepared, with a f i v e - and a six-membered r i n g , and varying degrees of deuterium s u b s t i t u t i o n on r i n g and substituent methyl protons. Relaxation rates a f t e r s e l e c t i v e and non-selective pulses were compared to determine the importance of dipole-dipole r e l a x a t i o n . Then, the magnitudes of some pairwise i n t e r a c t i o n s were measured by two methods: chemically, by comparing r e l a x a t i o n rates i n normal and deuterated compounds, and p h y s i c a l l y , by.comparing r e l a x a t i o n rates f o r s e l e c t i v e i n v e r s i o n and for inversion of pair s and groups of protons ( t a i l o r e d excitation^'"'"'^). The s e l e c t i v e and t a i l o r e d experiments were c a r r i e d out with the help o f H.D.W. H i l l and R.C. Jones (and the e a r l i e r help of R. Freeman) at Varian Associates, Palo A l t o , C a l i f o r n i a . F i n a l l y , our new i n s i g h t into proton r e l a x a t i o n patterns was combined with Fourier transform technology i n some very p r a c t i c a l methods for high-resolution proton n.m.r. These could be furthered by manipulation of the natu r a l l y - o c c u r r i n g r e l a x a t i o n times by addition of paramagnetic gadolinium ( i l l ) ions to the s o l u t i o n . Chapter V ou t l i n e s the p r a c t i c a l aspects of our studies. The Experimental Section includes sources of er r o r , and e m p i r i c a l l y -derived methods of ex t r a c t i n g values from the raw data one obtains from coupled multispin systems, along with some synthesis. F i n a l l y , the Appendix shows an attempt to apply high-technology n.m.r., i n the form of T^ measurements and 220 MHz spectra,.to a conformational problem i n a pa i r of branched-chain sugars. CHAPTER II A GENERAL SURVEY OF PROTON SPIN-LATTICE RELAXATION TIMES IN MONO-SACCHARIDES AND SOME "LARGE" ORGANIC MOLECULES quo referemus enim? quid nobis c e r t i u s i p s i s sensibus esse potest, qui vera ac f a l s a notemus? (Lucretius, De Rerum Natura I, 699-700) For what i s to be our standard of reference? What can be a surer guide to the d i s t i n c t i o n of true from f a l s e than our own senses? ( t r . R. Latham, Penguin Books Ltd., 1951) As the research background of t h i s laboratory included chemical and magnetic resonance studies of sugars, our i n i t i a l foray i n t o r e l a x a t i o n was a survey of proton T^ values of free sugars and mono-saccharide d e r i v a t i v e s . As the r e s u l t s seemed promising, work was extended to a few other representative organic molecules and to d i - and polysaccharides. Generally i t was found that the s p i n - l a t t i c e r e l a x a t i o n times showed a wide range of e a s i l y measurable values, which had c l e a r , a l b e i t q u a l i t a t i v e , s t e r e o s p e c i f i c dependencies. A " number of other phenomena were also observed, i n c l u d i n g temperature and con-centration dependence, the e f f e c t s of scalar coupling, c r o s s - r e l a x a t i o n , con-formational averaging and deuterium s u b s t i t u t i o n , and f i n a l l y , some p e c u l i a r i t i e s i n the r e l a x a t i o n of methyl protons. The s p i n - l a t t i c e r e l a x a t i o n times (non-selective) of a l l compounds were determined with a standard three-pulse sequence"'"''; the main advantage claimed for t h i s method over the more conventional two-pulse sequence i s that i t compensates for small changes i n r e s o l u t i o n , and hence i s very u s e f u l when time-averaging i s required. It i s also a very convenient form for complex spectra, where signals may be broad or poorly dispersed. D e t a i l s of the 33 3h experimental method, and the handling of data are given in the Experimental Section. The spectra shown in F ig . I I- l are typ ica l of the-sets of "par t i a l l y relaxed" spectra that constitute the experimental data. Table I I-l summarizes the f i r s t data obtained, for the anomeric protons of free hexoses and pentoses in aqueous (deuterium oxide) solution; their structural formulae are shown below. Clearly, there is a stereospecific dependence of the relaxation times. 35 H1( 0.2 u 1/ HOD 1.6 2.9 4.1 5.3 Figure I I - l . Par t ia l l y relaxed proton n.m.r. spectra (100 MHz) of a degassed solution (10$ w/v) of D-glucose in 99-96$ D 2 0 , at 51-5°C, showing the resonances of the anomeric protons and residual water peak. The delay time in seconds, between the 1 8 0 - and 90-degree pulses i s indicated to the l e f t of each f igure. It should be noted that the signal of H-1ft, the more rapidly, relaxing spin, decreases faster than the i n i t i a l l y much smaller signal of H-lct. 7.3 36 Table I I - l . S p i n - l a t t i c e r e l a x a t i o n times (seconds) f o r the H-1 resonances of some free pyranose sugars, measured f o r degassed 10% w/v s o l u t i o n i n 9 9 . 9 6 % deuterium oxide at k2°C. Sugar Relaxation time (seconds) Ratio H-la H-lg T /T la' 13 D-Glucose ( l ) k.k 2.3 1.9 D-Galactose ( 2 ) k.o 2.k 1 . 7 D-Mannose (3) 5 . 9 1.8 3.k 2-Deoxy-D-glucose (H) 3.8 1 . 7 2.2 D-Allose (5) 7-0 U.5 1 . 6 D-Altrose ( 6 ) a k.k 2 . 9 1.5 L-Rhamnose (T) 6.6 2.1 3 . 1 D-Talose (8) 7-6 2.3 3.3 D-Idose ( 9 ) 3 .7 2 . 9 1.3 g-Xylose (10) 5-7 3.3 1 . 7 D-Ribose ( l l ) k.6 6 . 6 0 . 7 1 D-Arabinose (12) k.O 6.2 0 . 6 5 D-Lyxose (13) 7.0 3.U 2.0 zQ% w/v concentration 37 These data may be interpreted at two l e v e l s , by comparison of the i n d i v i d u a l numbers, or of r a t i o s of T^ values, and the f i r s t approach i s v a l i d only i f a l l the sugars have the same r o t a t i o n a l c o r r e l a t i o n times. For D-glucose ( l ) and D-galactose (2), the a x i a l l y oriented H-1 of the g anomer has a s i g n i f i c a n t l y shorter r e l a x a t i o n time than the e q u a t o r i a l l y attached proton of the a anomer. The close s i m i l a r i t y of the data for these two sugars implies that the remote proton on carbon four has l i t t l e e f f e c t on the r e l a x a t i o n of the anomeric proton. Comparison of the data for D-glucose, on the one hand, with those of g-mannose (3), L-rhamnose (7) and D-talose (8) on the other, suggests that a gauche i n t e r a c t i o n (between H-1 and H-2) i s very e f f e c t i v e at causing r e l a x a t i o n . (The geometrical r e l a t i o n s h i p s between pai r s of protons on a s i x -membered r i n g i n chair form - vicinal-gauche, v i c i n a l - t r a n s , and 1,3-diaxial -are shown i n Figure I I I - l ) . There i s also the geminal r e l a t i o n s h i p , not shown, between two protons on the same carbon.) The r a t i o of T, /T. „ r i s e s from 1.9 l a 13 for ( l ) to approximately 3.3 f o r (3), (7) and (8). The short r e l a x a t i o n times for both anomers of 2-deoxy-D-arabino-hexose (2-deoxy-D-glucose (h)) confirm the importance of the gauche i n t e r a c t i o n . Furthermore, evidence that a 1,3-d i a x i a l r e l a t i o n s h i p also contributes to r e l a x a t i o n can be seen i n the data for D-allose (5). Removal of one 1,3-diaxial i n t e r a c t i o n , by in v e r s i o n of configuration at carbon three lowers the r a t i o of T ^ / T ^ from 1.9 to 1.6. A l l of these very q u a l i t a t i v e observations imply a range of us e f u l dependencies that might f a c i l i t a t e assignment of anomeric configurations of pyranose sugars; the t h r e e f o l d d i f f e r e n c e , f or example between the r e l a x a t i o n times of the anomers of D-mannose c l e a r l y provides a r a t i o n a l basis f or assigning t h e i r anomeric configuration. For a l l of the above mentioned sugars, i n which the a x i a l proton has a much shorter T than the equatorial one, both anomers have the C. conformation i n s o l u t i o n . The data for both D-idose (9) and g-altrose (8), by comparison, are a f f e c t e d by time averaging of the two chair conformations i n s o l u t i o n . Thus, the p o s s i b i l i t y of the existence of 38 more than one s o l u t i o n conformation must be considered i n applying data to the assignment of anomeric c o n f i g u r a t i o n . The T data f o r the pentapyranoses D-xylose (10), D-ribose ( l l ) , D-arabinose (12) and D-lyxose (13), are also a f f e c t e d by time-averaging; t h i s w i l l be discussed i n more d e t a i l a l i t t l e l a t e r . Continuing f o r the moment, with a q u a l i t a t i v e look at some other hexoses, whose s t r u c t u r a l formulae are shown below, i t i s evident from the data presented i n Table II-2 that 2-acetamido-2-deoxy-D-hexopyranoses (lU,15), and methyl D-hexopyranosides (l6,17,l8) also have pronounced d i f f e r e n t i a l s i n the values of t h e i r anomeric protons. Although replacement of a 2-hydroxyl group by a 2-acetamido group has l i t t l e e f f e c t on the r e l a x a t i o n time of H-1, the i n t r o d u c t i o n of an anomeric methoxyl group does cause a decrease of approximately 50$ i n the r e l a x a t i o n time of the anomeric proton. That t h i s 39 Table l i - 2 . S p i n - l a t t i c e r e l a x a t i o n times (seconds) f o r the anomeric protons of some 2-acetamido-2-deoxy-D-hexopyranoses and of some methyl D-hexopyranosides, measured f or degassed 10% w/v solutions i n 9 9 - 9 6 % DO, at i l 2°C. _ Compound Relaxation time (seconds) Ratio H-1 a H-13 T /T la' 13 2-Acetamido-2-deoxy-D-glucose (Ik) U.3 2 . 0 2 . 2 2-Acetamido-2-deoxy-D-mannose (15) k.6 1 . 2 3 . 9 Methyl D-glucopyranoside ( l 6 ) 2 . 7 1 . 6 1 . 7 Methyl (d^) D-glucopyranoside a (16a) h.T 2 . 2 2 . 1 Methyl D-mannopyranoside (lT) 3 . 3 1 . 2 * 2 . 8 Methyl D-xylopyranoside ( l 8 ) 3 . 9 1 . 7 2 . 3 Measured as a mixture, t o t a l concentration, 10% w/v. ^Concentration ^ 8 % , w/v. ko e f f e c t includes a contribution from a dipole-dipole i n t e r a c t i o n with the methoxyl protons i s c l e a r l y demonstrated by the longer r e l a x a t i o n times of the anomeric protons of the corresponding perdeuteromethyl glucopyranosides ( l 6 a ) , also given i n Table II-2. It had been hoped that, by working at constant concentration and temperature, v a r i a t i o n s i n intermolecular contributions to T values would be eliminated. However, the observation of some seemingly random v a r i a t i o n s i n the experimental data for protons which would be expected to have the same intramolecular environment f o r r e l a x a t i o n , suggested that t h i s approach had not been completely successful, and hence, a separate study was made of the con-centration and temperature dependencies of the T^ values of the anomeric protons of D-glucose. These r e s u l t s are summarized i n F i g . II-2 and Table II-3. The plot of concentration dependence indicates that, over the range of 0 . 0 6 to 1.2 molar, the T^ values change by z35%- The decrease i n T^ values with increasing concentration i s probably due both to increasing intermolecular dipole-dipole r e l a x a t i o n , and also to a change i n the c o r r e l a t i o n time with increase i n the v i s c o s i t y of the s o l u t i o n . Unfortunately, the a r b i t r a r i l y chosen concentration (10% w/v) at which these f i r s t series of measurements were made i s c l e a r l y high enough f o r some intermolecular contributions to remain; l a t e r studies were done at s i g n i f i c a n t l y lower con-centrations. The data i n Table II-3 indic a t e that change i n temperature can have a very s i g n i f i c a n t e f f e c t on the T^ value measured; for both of the anomeric protons, a temperature change of ~30° causes a greater than twofold change i n the T value. C l e a r l y then, i f data f o r d i f f e r e n t compounds are to be intercompared, i t i s necessary to keep the temperature and concentrations constant. I t i s i n t e r e s t i n g to note, : however, that f o r D-glucose at l e a s t , the r a t i o of the two anomeric T^ values appears to be e s s e n t i a l l y independent of the concentration or temperature at which the experiment has been conducted. r - 2.CH 0.5 moles/1 iter Figure H - 2 . V a r i a t i o n of the s p i n - l a t t i c e r e l a x a t i o n times of the anomeric protons of E-glucose with change i n concentration, f or degassed solutions i n 99.96% deuterium oxide, a l l at k2°C. Table I I - 3 - S p i n - l a t t i c e r e l a x a t i o n times of the anomeric protons of p-glucose as a function of temperature, f o r a degassed 10$ w/v s o l u t i o n i n 9 9 . 9 6 $ D 2 0 . Temperature (centigrade degrees) Relaxation time (seconds) Ratio H-la H-1B T /T l a IB 2 2 . 6 2 . 9 l.h 2 . 0 3 U . 5 U.6 2 . 3 2 . 0 U 2 . 0 a k.k 2 . 3 1 . 9 5 1 . 5 6 . 2 3 . 3 1 . 9 "A re-determination on the same sample gave H-la = k.9 s e c , H-1B = 2 . 5 s e c , r a t i o of T^/" 1^ = 1-9-^3 This i n v e s t i g a t i o n s t i l l did not explain the v a r i a t i o n i n the absolute values of the r e l a x a t i o n times. Why i s T of D-glucose k.k seconds, of D-mannose 5 . 9 seconds, of L-rhamnose 6 . 6 seconds, and of D-talose J.6 h seconds, when these sugars a l l have the C_ chair conformation i n s o l u t i o n , and the anomeric protons presumably have the same.environment f o r r e l a x a t i o n , the i n t e r a c t i o n with a neighbouring proton i n a~gauche r e l a t i o n s h i p ? C l e a r l y , small v a r i a t i o n s i n sample temperature or concentration can not account for such d i s p a r i t y , but there are several other possible causes. I t was thought f i r s t that at the high concentrations used, 10% w/v, there might be gross differences i n s o l u t i o n v i s c o s i t y f o r the d i f f e r e n t sugars, due to t h e i r d i f f e r e n t i n t e r a c t i o n s with the solvent D^O (which might indeed have been r e f l e c t e d i n the r e l a x a t i o n time of the r e s i d u a l HOD peak). I f t h i s were so, the differences i n c o r r e l a t i o n time should have been r e f l e c t e d i n d i f f e r e n t carbon-13 r e l a x a t i o n times. To t e s t t h i s , the carbon-13 r e l a x a t i o n times of two of the sugars were measured at the same temperature and concentration used for the proton measurements; these samples, however, were not degassed. For both g-glucose and L-rhamnose, the T^ values for carbons one to f i v e were the same within experimental error: the average T^ was 1 . 5 seconds f o r D-glucose, and 1 . 6 seconds for L-rhamnose, i n d i c a t i n g very s i m i l a r c o r r e l a t i o n times i n s o l u t i o n . Reducing the concentrations could not have removed a l l the discrepancies i n the proton data, as the concentration study on D-glucose showed that the increase i n T^ values with decreasing concentration " l e v e l l e d out" at approximately s i x seconds f o r H-la at a concentration of 0.2 molar. This value was s t i l l l e s s than T^ for the a anomers of D-allose, L-rhamnose and D-talose at 10% w/v (O .56 molar). A more l i k e l y source of the v a r i a t i o n i s the presence of small amounts of paramagnetic species, possibly r e s i d u a l oxygen from incomplete degassing, and also trace amounts of paramagnetic cations, such as C u ( l l ) , which could a r i s e from the D_o or the sugar samples, or have contaminated any kk of the glassware used i n preparation.. It might be.possible to.check f o r t h i s 57 by adding a small amount of some chel a t i n g agent , such as EDTA to the sample, which would e f f e c t i v e l y remove such cations from s o l u t i o n . As the carbon nu c l e i are shielded to some extent by the surrounding hydrogens from t h i s influence, t h e i r r e l a x a t i o n times would have been aff e c t e d l e s s by paramagnetic impurities, whether oxygen or m e t a l l i c species. The problem remains: whether i t i s some genuine r e f l e c t i o n of microscopic differences i n so l u t i o n behaviour, or a simple problem of contamination i s not e a s i l y answered. The bulk of t h i s work was done at lower concentrations, and i n organic solvents, and generally, the data were very consistent f o r s i m i l a r hydrogens i n d i f f e r e n t molecules. Evaluation of the T^ values f o r the pentopyranoses (10-13) given i n Table I I - l , i s complicated by the fact that i n s o l u t i o n , several of these molecules are conformationally inhomogeneous. For systems undergoing r a p i d h 1 interconversion between the C_ and conformers, the observed r e l a x a t i o n time, T should be given by the expression = x (1-x) 1_ T„ (21) (obs) where x i s the mole f r a c t i o n of molecules i n the conformation. Unfortunately, the T^ values of the c o n f i g u r a t i o n a l l y r e l a t e d hexoses can not be used as reference points, due to the unexplained v a r i a t i o n s which have just been discussed. Some i n t e r e s t i n g comparisons may s t i l l be made, however. The T^ values f o r both anomers of g-xylose (10) and for g-p-lyxose (13) are consistent with exclusive favouring'of the C_ conformer by these molecules, whereas the data f o r (3-D-ribose ( l l ) and 3-g-arabinose (12) imply an equally marked favouring of the " 4 ^ conformer; furthermore i t h5 appears that solutions of a-D-ribose and a-D-arabinose are populated by both h i 1 the and the conformers, the proportion of the conformer being somewhat higher for a-D-arabinose than for a-g-ribose. Although these findings agree well with the evaluations reported by previous workers'^ ^ , a s ignif icant disagreement appears to exist for a-g-lyxose (13). Lemieux and 58 59 Stevens and Rudrum and Shaw postulated that solutions of this sugar should 60 contain equal populations of the two chair forms, and Angyal and Pickles also anticipated some degree of time-averaging. However, the very long value It for a-g-lyxose i s more consistent with the predominant favouring of the C_^  conformation of the molecule. Clearly, more data and experience are needed before any def in i t ive comments can be made. During this survey of mono-saccharides, a few data were obtained for furanose forms of free sugars, shown below. (Table II-U). C H 2 0 H C H 2 0 H H 0 + -H.OH HO OH H.OH CH 2 0H H O - b CH 2 OH H.OH While the interpretation i s less clear than for the pyranose forms, differences are s t i l l apparent between some of the a and 3 forms, notably of g-ribose, and further^work in this area might lead to a method for assigning the anomeric configuration of ribo-nucleoside and -nucleotide systems, a problem that i s 1+6 Table II-1+. S p i n - l a t t i c e r e l a x a t i o n times of furanose forms of several hexoses and pentoses i n f u l l y mutarotated s o l u t i o n s . The s o l u t i o n con-centrations were 10% w/v i n 9 9 . 9 6 % deuterium oxide, and the temperature f o r a l l measurements was 1+2°C. Sugar Relaxation Time (seconds) Ratio T /T l a ' 13. H-1 a H-13 D-Allose ( 5 ) 10±1 9±1 =1 D-Altrose ( 6 ) a 6.k 5-5 1.2 D-Talose (8) 8.0 Q.h 0.95 D-Idose ( 9 ) 9.2 5 . 9 1 .6 D-Ribose ( l l ) 8 . 5 13.2 0.61+ =8% w/v concentration o 7^ notoriously d i f f i c u l t on the basis of conventional nmr parameters^. Following this preliminary work on free sugars in aqueous solution, six peracetylated hexoses, whose structural formulae can be seen in Fig. I I - 3 , were also studied; the values of their anomeric protons are given in Table II - 5 . As before, there is a clear distinction between the relaxation times of the two anomers, although the ratios are approximately the same as for the corresponding free sugar. For c l a r i t y , the T^ values of the other protons that were clearly resolved are shown in Fig. II - 3 . Study of the g-g-galacto derivative (20) provided particular insight into the variation in T^ values which can occur among the protons of a single compound. This molecule contains four axially oriented protons, whose relaxation times vary from 0.92 to h .k2 seconds. H-2 has the longest relaxation time, because i t has i t has no other protons optimally oriented for relaxation. The 1 , 3 , 5-tri-axial arrangement of H-1, H-3 and H-5, on the other hand, provides an efficient mutual relaxation network, with both H-3 and H-5 benefitting additionally from the gauche interaction with E-h. The H-5 resonance has a characteristically short T^ value, and this appears to be associated with the effective interactions with the pair of protons on carbon six. Comparison of the T^ values of derivative (20) with the corresponding values for the per(trideuteromethyl)acetylated derivative (21) proved interesting. (See Table II - 6 ) . The times for the deuterated compound are between five- and twenty-five per cent longer than those of the normal one, indicating that there is a significant contribution to the relaxation of the ring protons from the protons of the acetyl groups: evidently these con-tributions vary from one ring position to another. The nicely-resolved spectrum of B-g-galactose pentaacetate (20), and i t s deuterated counterpart (21) also provided an ideal model of some of U8 Table H-5. S p i n ^ l a t t i c e r e l a x a t i o n times of the anomeric protons of some peracetylated D-hexopyranoses i n deuterobenzene s o l u t i o n , 10% w/v, measured at k2°C. Relaxation Time Ratio Compound (seconds) T /T l a ' 13 H-la H-13 D-Glucose penta-acetate (19) 3.3 2.0 1.7 D-Galactose penta-acetate (20) 3.7 2.3 1.6 3-D-Galactose penta- (21) 9 £ (perdeutero)acetate £ 1 . 0 a-D-Idose penta-acetate (22) 3.8 — — Figure I I - 3 . S p i n - l a t t i c e r e l a x a t i o n times f o r the assignable protons of some peracetylated pyranoses i n 10$ deuterobenzene s o l u t i o n , measured at k2°C. 50 Table IT-6. Ratios of s p i n - l a t t i c e r e l a x a t i o n times f o r penta-O-acetyl-g-D-galactopyranose (20), and i t s (perdeuteromethyl)acetylated counterpart ( 2 l J . Proton Ratio deuterated . non-deuterated ,H-1 1.15 H-2 1.25 H-3 1.22 E-h 1.12 H-5 1.05 H-6 1.05 51 the perturbations which cro s s - r e l a x a t i o n can cause i n highly-coupled multi-spin systems. The data f o r the' i n d i v i d u a l t r a n s i t i o n s are most e a s i l y displayed on the o r i g i n a l spectra, ( F i g . I I - U ) , where i t can be seen that the r e l a x a t i o n times of the i n d i v i d u a l t r a n s i t i o n s of a spin m u l t i p l e t can vary widely, as i n H-1 and H-2, or be i d e n t i c a l within experimental erro r . The decays f o r t h i s p a i r of compounds also showed the usual amount of non-exponentiality; the apparent "T^" changing by up to twenty per cent as data from longer and longer delay times were included i n the c a l c u l a t i o n s . Some i n s i g h t i s provided by the U3 theory f o r the coupled two-spin system : perturbations from the behaviour predicted by simple theory are encouraged by three conditions - strong coupling, strong dipole-dipole i n t e r a c t i o n s , and asymmetric external r e l a x a t i o n . Inspection of F i g . II - U c e r t a i n l y indicates accord with the f i r s t condition; thus, the t r a n s i t i o n s of H-1 and H-2, which form a closely-coupled system (J/6 = 0.U2), have a l a r g e r T d i f f e r e n t i a l than those of H-3 and E-k (J/6 = O . l U ) . A n t i c i p a t i n g r e s u l t s from l a t e r chapters, i t seems that the t h i r d condition -asymmetric "external" r e l a x a t i o n - i s also f u l f i l l e d . (The "non-external" r e l a x a t i o n , i n t h i s case, w i l l be the mutual dipole-dipole i n t e r a c t i o n of H-1 and H-2, considering H-1 and H-2 as an i s o l a t e d two-spin system.). Thus, i n addition to t h e i r mutual dipole-dipole i n t e r a c t i o n , both spins can r e l a x by i n t e r a c t i o n s with the acetate protons ( i n 20), and by intermolecular i n t e r -actions. H-1 has i n a d d i t i o n , however, the very e f f i c i e n t 1,3-diaxial i n t e r a c t i o n s with H-3 and H-5, so that i t s o v e r a l l r e l a x a t i o n time i s much shorter than that of H-2. The t h i r d condition, however, i s l e s s completely met, as the v i c i n a l - t r a n s r e l a t i o n s h i p of H-1 and H-2 probably puts them too f a r apart f o r a strong dipole-dipole i n t e r a c t i o n . (The work i n Chapter I I I on comparable molecular structures suggests that vicinal-gauche and 1,3-diaxial i n t e r a c t i o n s are much more e f f e c t i v e i n causing relaxation.) While these complications do add to the d i f f i c u l t i e s of quantitative' i n t e r p r e t a t i o n however, i t must be emphasized that t h e y do not s e r i o u s l y prejudice q u a l i t a t i v e 52 Hi H. H. A A 1.7 I.6 I.6 Figure I l - h . P a r t i a l 100 MHz proton n.m.r. spectra of 10$ w/v solutions of _AJ penta-0_-acetyl-D-galacto-pyranose (20) and (B) i t s perdeutero-acetylated analog (21), i n d i c a t i n g the r e l a x a t i o n times (seconds) of the i n d i v i d u a l t r a n s i t i o n s . • . •. 53 applications, as these variations are less than the stereospecific dependencies of interest, and indeed, their effects can he minimized by adoption of the standard, i f rather arbitrary, methods of data-processing outlined in the Experimental Section. It was obvious from these studies that the proton spin- latt ice relaxation times of pyranoses showed a number of stereospecific dependencies, and that while explanation of these on a quantitative basis would be d i f f i c u l t , i t was apparent they were suf f ic ient ly clear-cut to provide a useful basis for configurational assignment, as well as a probe of solution conformational preference. More importantly, i t was possible to distinguish an isomeric pair of cyclohexane derivatives, as may be seen by comparing the values of the H-1 protons on C-1 of the isomeric U-tert-butylcyclohexanes, shown below. In addition, previous audio-pulse work had c lear ly differentiated pairs of Proton sp in- latt ice relaxation times for the C-1 protons of the isomeric U-t-butylcylohexanols, measured in deuterobenzene at U2°C as a mixture with cis:trans ratio of onerthree. Total concentration, 0.2M. 3 cis-trans isomeric alkenes , the protons of the cis isomer having much shorter relaxation times than those of the trans isomer. The Fourier transform method allowed this study to be extended to a three-spin alkene system with a more 5h complex, spin-coupled spectrum, v i n y l acetate. Again, the'T values obtained for the three protons, i n agreement with the assignment obtained from coupling constants, c l e a r l y r e f l e c t the stereochemical environment of each proton. As shown i n F i g . II-5, H-2, which i s c i s to H-1 has a much shorter r e l a x a t i o n time than H-3, which i s trans to H-1. It was clear that at l e a s t on a q u a l i t a t i v e l e v e l , the s p i n - l a t t i c e r e l a x a t i o n times of protons of "small" organic molecules r e f l e c t e d t h e i r stereochemical environment - t h e i r degree of crowding by other protons - and al s o , i n some cases the conformational preference of the molecule i n s o l u t i o n . To evaluate how f a r . t h i s diagnostic p o t e n t i a l extended to l a r g e r organic systems, the l a s t systems to be surveyed were an example of a natural product and a number of polymeric carbohydrate systems. The a l k a l o i d vindolene, whose spectrum at 100 MHz i s f u l l y 62 assignable , had been previously studied i n t h i s laboratory using the audio-2 pulse method . Only a very few r e l a x a t i o n times could be obtained, of w e l l -separated peaks w h i c h ' s a t i s f i e d the instrumental frequency l i m i t a t i o n s . Now using Fourier transform methods, i t was possible to determine the s p i n - l a t t i c e r e l a x a t i o n times of e s s e n t i a l l y a l l the protons - i n approximately the same experimental time - and these are shown together with the s t r u c t u r a l formula i n F i g . II-6. ° It i s i n t e r e s t i n g to note that the r e l a x a t i o n times for vindolene given i n F i g . II-6 range from 0.5 to approximately 3.0 seconds; t h i s s i x f o l d d i f f e r e n t i a l bodes well f o r future studies of large organic molecules. Inspection of molecular models of vindolene, and the photograph i n F i g . II-7 i l l u s t r a t e s more c l e a r l y the p o s s i b i l i t i e s f o r intramolecular dipole-dipole i n t e r a c t i o n s f o r the d i f f e r e n t protons. Dealing f i r s t with the C-H protons, i t i s noteworthy that those near the centre of the molecule, and hence nearest to other protons, have somewhat shorter r e l a x a t i o n times than those d i r e c t e d to the periphery. Of those on the periphery of the molecule, the ones with the 55 F i g . I I - 5 . 'Proton s p i n - l a t t i c e r e l a x a t i o n t i m e s f o r t h e a l k e n e p r o t o n s o f v i n y l a c e t a t e , measured i n deuterobenzene a t 0.1 M c o n c e n t r a t i o n , a t k2°C. 0.50 2.7 1.4 1.8 Figure IT.-6. Spin-^lattice r e l a x a t i o n times (.seconds) f o r trie protons of vindolene, f o r a 0.1 molar s o l u t i o n i n deuteroB.enzene, at h2 C. The values for trie metfiyT protons are Based on i n i t i a l slopes. F i g u r e II-7. A photograph o f a m o l e c u l a r model o f v i n d o l e n e , showing t h e o v e r a l l s p a t i a l d i s t r i b u t i o n o f t h e s u b s t i t u e n t s . Note t h a t a l l o f t h e methyl groups p r o j e c t towards t h e p e r i p h e r y o f t h e m o l e c u l e , and t h a t t h e y and o t h e r p e r i p h e r a l p r o t o n s g e n e r a l l y have t h e l o n g e s t r e l a x a t i o n t i m e s . 59 shorter r e l a x a t i o n times are also the ones having close s t e r i c i n t e r a c t i o n s with other protons. For example, E-lh i s quite close to the protons on C-10 and C - l l , whereas H-15 and'H-17 can only r e l a x v i a the protons of the methyl groups located at p o s i t i o n s 1 and l 6 , which are further away. A s i m i l a r r a t i o n a l e can he o f f e r e d f o r the shorter T^ value of H-6 compared with H - 7 ; the former i s rather close to the methyl protons of the C-5 e t h y l moiety. The low values of the methylene protons at p o s i t i o n s 8 , 10 and 11 are also consistent with the f a c t that they are a l l i n close array. Like the penta-acetates ( 2 0 ) and ( 2 1 ) , vindolene provided some i n t e r e s t i n g examples of disturbances i n the r e l a x a t i o n of a m u l t i - s p i n , coupled system. For many of the protons, e s p e c i a l l y those with short r e l a x a t i o n times, the T^ values of the t r a n s i t i o n s of a m u l t i p l e t were i d e n t i c a l within experimental e r r o r , and the recovery curves deviated only s l i g h t l y from simple exponentials. This was not the case, however, for the three protons of the aromatic r i n g , H-IH-, H-15 and H - 1 7 , which form a closely-coupled ABC system. Examination again, of the molecular model of t h i s compound ( F i g . I I - 7 ) shows that several external dipole-dipole i n t e r a c t i o n s are possible for these three spins which are coupled only to each other. Thus, H-lU may i n t e r a c t with the methylene protons at C - l l , and H-15 with the methoxyl group at C - l 6 , while for H - 1 7 , the only s i g n i f i c a n t dipole-dipole i n t e r a c t i o n s possible are the external ones with the C - l 6 methoxyl and N-l methyl protons. This three-spin system i s s i m i l a r i n some ways to the H - 1 , H - 2 .group of g-D-galactose penta-acetate, i n that the two conditions of strong coupling and asymmetric external r e l a x a t i o n are met, but the t h i r d condition, of strong i n t e r n a l dipole-dipole i n t e r a c t i o n s i s not, as the only i n t e r n a l dipole-dipole i n t e r a c t i o n of s i g n i f i c a n c e would be that between H-lU and H - 1 5 . The r e l a x a t i o n of t h i s system was examined i n some d e t a i l , and a large number of data points were taken to define the recovery curves more exactly. F i r s t , the values of the i n d i v i d u a l t r a n s i t i o n s were, from low to high f i e l d (seconds): the E-lh doublet 1 . 5 5 , l.&T; the H-15 quartet, 2 . 5 5 , 2 . 5 8 , 3 . 1 7 , 3 . 1 6 ; and the H-17 doublet 2 . 6 8 and 2 . 7 5 . I n t e r e s t i n g l y , H-17, with the smallest r a t i o of J / 6 , also showed the. smallest d i f f e r e n c e s . Second, as data from longer and longer delay times were included, most of the apparent values increased, which was the usual s i t u a t i o n i n a l l of t h i s work, but for the two downfield t r a n s i t i o n s of H-15 , t h e y showed a steady decrease; divergence of the re l a x a t i o n rates within a spin m u l t i p l e t i s , i n ^3 f a c t , a p r e d i c t i o n of the theory f o r a coupled two-spin system. F i n a l l y , many of the semi-logarithmic p l o t s of (M -M ) a c t u a l l y showed an o s c i l l a t o r y O "0 type of decay, superimposed on the o v e r a l l increase or decrease from i n i t i a l slope. F i g . I I - 8 shows one of the decay p l o t s , f o r the l o w - f i e l d t r a n s i t i o n of the E-lh doublet, where a s l i g h t tendency f o r o s c i l l a t i o n of the value of the instantaneous slope i s just apparent over the experimental scatter. The ef f e c t s were rather more obvious i n the plot s f o r H-15; t h i s p a r t i c u l a r decay was chosen as an example of how l i n e a r the decay may be, i n sp i t e of a l l . Once again, these perturbations do not appear to prejudice q u a l i t a t i v e i n t e r p r e t a t i o n of the T^ values. The methyl protons showed a d i f f e r e n t type of disturbance, and t h e i r r e l a x a t i o n times can not be corr e l a t e d with molecular geometry i n a simple way. The resonances of the W-methyl and the acetate-methyl (C-h) had 62 previously been assigned , but. no d i s t i n c t i o n had been made between the methoxyl ( C - l 6 ) , and the carboxymethyl (C -3) resonances. One of these un-assigned resonances gave e s s e n t i a l l y an exponential decay, while the' semi-logarithmic p l o t s of the other three were s t r i k i n g l y non-exponential (see Table I I - 7 ) , so that even an i n i t i a l slope could not e a s i l y be assigned.* *To see whether t h i s d i f f e r e n c e might lead to an assignment of the two methoxy resonances, a so l u t i o n approximately 0 . 1 molar i n each of the four model compounds methyl benzoate, ani s o l e , phenyl acetate and acetone was studied; a l l of the methyl decays were quite exponential, and the T i values obtained were, r e s p e c t i v e l y , 1 0 . 9 , 1 0 . 5 , 1 0 . 7 and 2 0 . 6 seconds. 2.0 time (sec.) 62 Table I I - 7 . Variation-of the "T]/' obtained from different, decay lengths for the methyl resonances of vindolene, for a 0.1 molar degassed solution i n deuterobenzene at 1+2°C. Note that three of the four are strongly non-exponential. Apparent Tj-values obtained from semi-log plots for the time periods indicated below. Resonance l i m " 1 0 .8 1.3 2.0 2.1+ 3.8 - 0CH3 1 .8 1 • 3U 1.57 - 1.83 2.11 - 0CH3 1 .8 1 .68 1.69 1.76 1.80 1.89 - NCH3 1 .k 1 .28 1.28 1.38 1.1+5 1.63 - 0C0CH3 1 .7 1 .1+5 1.U5 - I . 6 9 2.03 63 It certainly seemed reasonable to:assume that spin-rotation i s not contributing 127 . to the methyl group relaxations . It may well be that the non-exponential behaviour is being caused by cross-correlation effects, and that for this molecule, the correlation times for the methyl and overall molecular rotation do f a l l into the limits which Hubbard suggested would cause significant non-exponential decay''''". The rotation of the single methyl group whose relaxation is closer to a simple exponential is presumably much less hindered, or is relaxed more by interactions with other spins outside the methyl group. It i s perhaps worth mentioning here that strikingly non-exponential behaviour, which w i l l not be discussed in any detail, was also observed for the methyl protons of the N-acetyl groups of 2-acetamido-2-deoxy-D-glucose (l^) and the corresponding mannose isomer ( 1 5 ) , and of the acetate groups of the a and 3 glucose and galactose penta-acetates ( 19 ) and (20), and the group of five halo-sugar'derivatives (39) to (k3), which are the subject of the next chapter. The study of vindolene proved interesting, therefore, in a number of ways. It showed that i t was feasable, using Fourier transform methods,.to measure proton relaxation times in even very complex spin-coupled spectra, and that the experimental relaxation times, in spite of theoretical limitations, showed a wide and potentially useful range which, except for methyl protons, clearly reflected stereochemical environments. Vindolene also seems an interesting candidate in which to investigate further both cross-correlation effects, and the detailed relaxation behaviour of a three-spin system, namely the group of H - lU, H-15 and H-17. As the synthesis of vindolene has been extensively studied, i t may even be possible to vary some relaxation parameters chemically by deuteration of, for example, the N-methyl group. Turning f i n a l l y to polymeric carbohydrate systems, data for the anomeric protons of a few representative disaccharides, (23) to ( 2 7 ) , whose structural formulae are shown below, are summarized In Table I I - 8 , and Table I I - 8 . Proton s p i n - l a t t i c e r e l a x a t i o n times (seconds) f o r the anomeric protons of disaccharides as 5% w/v solutions i n 9 9 . 9 6 % D^O at k2°C. O b t a i n e d by i n t e r p o l a t i o n i n the concentration study shown i n F i g . -H-2. b T l c / T l c t * ° r T1B / T13*' a S a P P r ° P r i a t e -Compound Reducing proton Non-reducing proton Acetone T1 T r a t i o T /T l a ' 13 T l a * T13* r a t i ° b D-glucose a ( l ) 5.3 2 . 6 2 . 0 Maltose (23) (M - - 0-a-D-glucopyranosyl-D-glucopyranose) 2 . 5 1 . 1 2 . 2 0 . 8 6 2 . 9 16.3 Cellobiose (2k) (k-0-3-D-gluc opyranosyl-D-glucopyranose) 2 . 1 1 . 1 1 . 9 0 . 5 2 2 . 1 1 7 . 2 Lactose (25) (U-C_-3-I>-galactopyranosyl-D-glucopyranose) 2 . 1 1 . 1 1 . 8 0 . 5 U 2 . 1 1 6 . 9 Gentiobiose ( 2 6 ) ( 6 - 0-3-D-glucopyranosyl-D-glucopyranose) 2.3 1 . 2 1 . 9 0 . 5 9 2 . 0 1 5 . 9 Melibiose (27) ( 6 - 0-a-D-galactopyranosyl-D-glucopyranose) 2 . 6 1.3 1 . 9 1 . 1 2.3 1 6 . 1 65 H OH OH -Melibiose t27) reve a l a number of i n t e r e s t i n g dependencies. At the reducing end, while the r e l a x a t i o n times are, i n general, shortened from those of corresponding monosaccharides, there i s s t i l l a l a r g e systematic d i f f e r e n t i a l between the a and the 3 protons. Going to the non-reducing protons, there i s a furth e r s u b s t a n t i a l reduction of r e l a x a t i o n times from those of the corresponding reducing proton. The precise reason for t h i s decrease i s not yet known. I t may be due to a di f f e r e n c e i n the c o r r e l a t i o n times at the two s i t e s o f the disaccharide, or the p o s s i b i l i t y o f a d d i t i o n a l "intramolecular" r e l a x a t i o n o f the non-reducing anomeric proton by r i n g protons of the other monomer u n i t ; f o r the present, i t i s noteworthy that the d i f f e r e n t i a l provides a means of d i s t i n g u i s h i n g the reducing and non-reducing resonances of a disaccharide. Even more i n t e r e s t i n g , i t appears that the systematic d i f f e r e n t i a l between a and 3 protons i s maintained at the non-reducing end, although there are only a few data, and the r a t i o s can only be obtained by intercomparing two d i f f e r e n t molecules. For the l 4 l i n k e d disaccharides, the T value of the a non-reducing proton of maltose i s approximately 1 . 6 times that of the 3 non-reducing proton 66 of c e l l o b i o s e and la c t o s e . The s i t u a t i o n f o r the 1-+6 linked.disaccharides i s s i m i l a r . At the non-reducing end, the r a t i o "between the T^ values of the a proton of melibiose and the 3 proton of gentiobiose i s 1.9. Further i n d i c a t i o n of the enhancement of r e l a x a t i o n with increase i n the length of the oligo-saccharide chain comes from comparison of the r e l a x a t i o n times of glucose, with those of maltose, maltotriose (28) and the a and 3-Schardinger dextrins (29 and 30 r e s p e c t i v e l y ) , a l l a ±-*k l i n k e d oligomers of glucose (Table II-9). This i s almost c e r t a i n l y associated with the increase i n motional c o r r e l a t i o n time T , corresponding to slower molecular tumbling as molecular si z e increases. (It i s important to note that the near-constancy of the T^ of the acetone added as an i n t e r n a l reference precludes the p o s s i b i l i t y that the observed e f f e c t s simply r e f l e c t changes i n bulk v i s c o s i t y . ) The series sucrose (31), r a f f i n o s e (32) and stachyose (33), which have only non-reducing anomeric protons, shows a s i m i l a r progressive enhancement of r e l a x a t i o n with increasing molecular weight. ( F i g . II-9). There i s also a d i f f e r e n t i a l of f i f t e e n per cent between the T^ values of the two chemically d i s t i n c t non-reducing protons, which i s maintained as the number of sugar units increases from three to four. Intrigued by the above demonstrations that s p e c i f i c intramolecular i n t e r a c t i o n s s t i l l l e ad to T^ differences i n short oligosaccharides, we were prompted.to see i f the same were true for polysaccharides. Table 11-10 l i s t s anomeric s p i n - l a t t i c e r e l a x a t i o n times for three f u l l y methylated polymers of glucose, whose s t r u c t u r a l repeating units are shown below; the T^ values are i d e n t i c a l within experimental error (although the bulk v i s c o s i t y of the c e l l u l o s e polymer (36) was much greater). We also studied a b a c t e r i a l capsular polysaccharide, K l e b s i e l l a 2k, of molecular weight (5-9) x 10^., whose structure 63 i s the five-sugar repeating unit shown i n Table 11-11. F i g . 11-10 shows the Table I 1 - 9 . Proton s p i n - l a t t i c e r e laxation times (seconds) for the anomeric protons of some oligomers of D-glucose, i n deuterium oxide solution at U2°C. Sugar Concentration % w/v Reducing proton Non-reducing proton Acetone H-la H-13 Ratio H-la H-la H-13 Ratio ^red H-13 H non-red Si D-glucose ( l ) 5 5-3 2 . 6 2 . 0 - - - -Maltose ( 2 3 ) 5 . 2 . 5 .1.1 . . .2.2 0 . 8 6 - 2 . 9 1 6 . 3 Maltotriose ( 2 8 ) 5 1 .7 0 . 7 3 2 . 3 0 . 5 3 - 3 . 2 1 6 . 3 a-Schardinger Dextrin (29) 1 - - 0 . 2 7 — — 1 6 . 8 B-Schardinger .' .Dextrin . . ( 3 0 ) 1 - - - 0 . 2 3 — _ 1 9 . 8 'obtained by i n t e r p o l a t i o n of concentration study (Fig. I I - 2 ) 68 Sucrose C3l) 1_0_ -D-glucopyranosyl- -D-fructofuranoside DH 0 CH2 \ k H / i — °' A H HO . OH II CH20H CH20H 0.86 OH OH H Raffinose (.32} -galactosyl sucrose Stachyose C33) F i g . II-9. Proton s p i n - l a t t i c e r e l a x a t i o n times (sec.) f o r the anomeric protons of three oligosaccharides as 5$ w/v solutions i n 99-96$ deuterium oxide, measured at U2°C. 69 Table 1 1 - 1 0 . Proton s p i n - l a t t i c e . r e l a x a t i o n times for.the anomeric protons of three f u l l y methylated polysaccharides as 1% w/v solutions i n deutero-chloroform at h2°C Compound Relaxation time (sec . ) Anomeric Proton Acetone 2 , 3, 1+-trimethyl dextran (3h) O .26 1 9 . 2 2 , 3 , 6 - t r i m e t h y l amylose ( 3 5 ) 0 . 2 2 2 1 . 1 2 , 3 , 6 - t r i m e t h y l c e l l u l o s e ( 3 6 ) 0 . 2 5 1 8 .T Table 11-11. S p i n - l a t t i c e r e l a x a t i o n times f o r b a c t e r i a l capsular poly-saccharide K e b s i e T l a 2h, undegassed, i n D_0 at 95°C. St r u c t u r a l u n i t : 6 3 GlcA k a 3 1 - Man -a 2 1 Man a 3 Man Peak number (see F i g . 11-10) assignment3 -seconds 1 a-GlcA - — - Man 1 3 a-Man Glc 0.23 2 a-Man - — — Man 0.31 3 1 It B-Man - — - GlcA 3-Glc - — - GlcA 0.22 1+ 0_-acetyl 0.55* 5 free acetate 2.U 'See F i g . II-10 Based on i n i t i a l slope; see discussion. — H O D Figure 11-10. P a r t i a l 100 MHz proton n.m.r. spectrum of K l e b s i e l l a 2k polysaccharide at 95°C i n D 2 0. Peaks 1,2, and 3 are the anomeric protons; t h e i r assignments are given i n Table 11-11. Peaks k and 5 are methyl groups, h representing f r e e , and 5, bound acetate groups. 72 '(36) 100 MHz n.m.r. spectrum of K2k in D^ O at 95°C, while the relaxation times are given in Table 11-11. Again, the results lack diagnostic potential; the small differences between the relaxation times of the anomeric protons are less than in mono- or di-saccharides. Whilst the relaxation data for the anomeric protons of the poly-saccharides were disappointing, the data for the acetate peaks of K2h showed some interesting features. The structural determination of K2k showed that there was one 0_-acetyl group (i t s precise location i s unknown) to seven or eight 63 sugar units . This group i s represented by peak four in the n.m.r. spectrum of ¥2h, while peak five is free acetate, resulting from decomposition in the sample, which had been held at 95°C several times. There i s a fourfold difference i n their relaxation times, which are 2.k sec. for the free acetate methyl, and approximately 0.55 sec. (based on i n i t i a l slope) for the bonded acetate methyl. Typical decay plots for the two peaks are shown in Fig. 11-11, and i t can be seen that the decay of the bonded acetate i s decidedly non-exponential. This behaviour, similar to, and more pronounced than, the non-exponentiality observed for three of the four methyl groups of vindolene, may Figure 11-11. Plots of l n ^ - M , - ) , vs delay time " t " f o r the acetate peaks i n the 100. MHz n.m.r. spectrum of b a c t e r i a l capsular polysaccharide K l e b s i e l l a 2h i n D 20 at 95°C . peak k free acetate; peak 5 bound acetate. again be due t o . c r o s s - c o r r e l a t i o n .effects,.which depend on the degree of hindrance of the methyl group r o t a t i o n , • the degree of anisotropy of the o v e r a l l motion of the large polymer, and f i n a l l y , the difference i n the k6 kl 51 c o r r e l a t i o n times for the two motions ' ' . I t i s also possible that at l e a s t part of the non-exponentiality may be due to the d i f f e r e n t r e l a x a t i o n times of O-acetyl groups i n two or more s i t e s i n the molecule, but with the same chemical s h i f t . U n t i l the p o s i t i o n ( s ) of the O-acetyl group have been c l a r i f i e d , t h i s point can not be s e t t l e d . From the rather small number of d i - and polysaccharides surveyed, i t i s d i f f i c u l t to draw f i n a l conclusions. For disaccharides at l e a s t , values seem to have the same sort of s t e r e o s p e c i f i c i t y and diagnostic p o t e n t i a l as for monosaccharides, but the s i t u a t i o n for higher polymers i s l e s s c l e a r . Obviously, the concepts of " c o r r e l a t i o n time" and "intramolecular" become more complex. In addition to the now anisotropic motion of the whole molecule, there may be f a s t e r l o c a l motions of short segments of the main chain, and of branching sugar u n i t s . It can also be expected that "intramolecular" dipole-dipole r e l a x a t i o n may occur between protons of adjacent monomer u n i t s , but these would lack the f i x e d geometrical r e l a t i o n s h i p s of protons of a single sugar u n i t . Both of these problems would make s i m p l i s t i c i n t e r p r e t a t i o n s of data meaningless. Experimentally, f o r high-resolution n.m.r. of polysaccharides, there are serious problems with increasing r e l a t i v e i n t e n s i t y of the r e s i d u a l HOD peak, and poor dispersion of the anomeric region. (The anomeric region of K2U i s exceptionally well-resolved; even then, peaks one and three each represent two d i f f e r e n t anomeric protons.) However, some of the instrumental and chemical methods described i n Chapter V could probably be applied i n t h i s area. F i n a l l y , systematic i n v e s t i g a t i o n s • o f both.proton and carbon-13 r e l a x a t i o n data, of the polymers and the solvent, should provide u s e f u l i n s i g h t into the microdynamics of polysaccharides i n s o l u t i o n , a problem of considerable i n t e r e s t i n b i o l o g i c a l studies. CHAPTER I I I PATTERNS OF PROTON SPIN-LATTICE RELAXATION IN A SIX-MEMBERED RING The r e s u l t s of the q u a l i t a t i v e survey i n Chapter II had strongly implied that the r e l a x a t i o n of protons of organic molecules i n d i l u t e degassed solution i n magnetically i n e r t solvents was predominantly intramolecular dipole-dipole i n o r i g i n , and thus c r i t i c a l l y dependent on the stereochemical environment of each proton. In p a r t i c u l a r , c h a r a c t e r i s t i c differences were observed i n the r e l a x a t i o n times of protons with equatorial or a x i a l o r i e n t a t i o n s on a six-membered r i n g . While there are, i n p r i n c i p l e , a rather large number of pairwise i n t e r a c t i o n s possible among the protons of a s i x -membered r i n g , the inverse sixth-power dependence of the intramolecular d i p o l e -dipole r e l a x a t i o n mechanism e f f e c t i v e l y reduces t h i s number, and for a s i x -membered r i n g i n the chair conformation, i t i s s u f f i c i e n t to consider the three stereochemically d i s t i n c t i n t e r a c t i o n s , vicinal-gauche (vg), v i c i n a l - t r a n s (vt) and 1,3-diaxial (aa), (see F i g . I I I - l ) . The geminal i n t e r a c t i o n must be added i f there are carbon atoms with two d i r e c t l y bonded protons. To understand the pattern of dipole-dipole proton r e l a x a t i o n , we returned therefore, to the protean six-membered r i n g , and f i r s t studied the f i v e pyranose derivatives whose s t r u c t u r a l formulae are shown below. 3,k,6-Tri-O-acetyl- l-0-benzoyl-2-C-bromo-2-B-D-glucopyranose (39), the corresponding a anomer (Uo), the corresponding 3 and a 2-C-chloro d e r i v a t i v e s (Ul) and (k2), and the 2-C_-a-D-mannopyranose d e r i v a t i v e (U3), had been previously prepared i n 6k k t h i s laboratory , and were known to have approximately the same C_ chair conformation i n s o l u t i o n and well-resolved n.m.r. spectra, so that t h e i r r e l a x a t i o n times could be extensively surveyed. F i g . III-2 summarizes the' T 75 Figure I I I - l . The three stereochemically dist inct proton-proton relationships in a six-membered ring in chair form, with no geminal protons: vicinal-gauche (vg), v ic inal-trans (vt) and 1,3-diaxial (aa). (39) (40) (43) Figure III-2. S p i n - l a t t i c e , r e l a x a t i o n times (seconds) f o r the assignable protons of the halo-sugar d e r i v a t i v e s (39) to (k3), measured f o r degassed 0.1 molar solutions i n deuterobenzene at H2°C. The acetate methyl protons had r e l a x a t i o n times between 2.1 and 2.U seconds (based on i n i t i a l slopes; the decays were very non-exponential). 7 8 AcO \OAc ( 3 9 ) Rj_=OBz, R2=H (hO) R =H, R2=0Bz AcO {hi) R^OBz, R2H (h2) Hjj-E, R2=0Bz ( U 3 ) values obtained from 0 . 1 molar* solutions in deuterobenzene, using again, a standard three-pulse sequence. For {ho), (hi) and {h2), the concentration was 0 . 1 M; for ( 3 9 ) approximately 0 . 0 7 M due to lack of material, and for ( U 3 ) s l i ght ly less than 0 . 1 M as some material fa i led to dissolve. These differences in concentration are probably the cause of some minor inconsistencies in the data, for example, the s l i ght ly higher values of ( 3 9 ) . There are several clear-cut dependencies which deserve comment. F i r s t , the close s imi lar i ty between the individual relaxation times of the configurationally ident ica l pairs of compounds ( 3 9 ) , {hi) and {ho), (h2) shows that the C-2 halogen substituents do not contribute appreciably to the proton relaxation, and implies ident ical rotational correlation times in solution. Second, the data show a number of marked configurational dependencies, which i t i s tempting to correlate with the dipole-dipole interactions of pairs of protons. For example, the increase in the T^ value of H-1 in going from the axia l orientation (as in ( 3 9 ) and (Hi)), to the equatorial orientation (as in {ho) and ( U 2 ) ) suggests a strong 1 ,3 - d i a x i a l dipole-dipole interaction•between H-la, and H-3a and H -5a . Supporting evidence for this comes from the mutual increase in 79 the T-|_ values of the H-3a resonances from approximately 2.1* to. 3.1* seconds that accompanies the removal o f the i n t e r a c t i o n with H-la. (Compare ( 3 9 ) with (1+0) and (1*1) with (1+2)). The same holds true for the H-1* resonances of (1*0) and (1*3) where inversion of the bromine at C-2 from the equatorial o r i e n t a t i o n (as i n (1*0)) , to the a x i a l o r i e n t a t i o n (as i n (1*3)) causes a s i m i l a r increase i n the T^ value of H-1*. Intercomparison of the T^ values also implies a mutual r e l a x a t i o n of vicinal-gauche protons. Compare, f o r example, the r e l a x a t i o n times of the H-2 resonances of the a,g p a i r s ( 3 9 ) , (^0) and (1*1), (1*2). In both instances, the change from a v i c i n a l - t r a n s to a vicinal-gauche i n t e r a c t i o n with H-1 r e s u l t s i n a s u b s t a n t i a l (ca. twofold) reduction i n the T^ value of H-2. A s i m i l a r dependence i s found f o r the H-3 resonance of (1*0) and (1*3), where the i n t r o d u c t i o n of the gauche i n t e r a c t i o n with H-2 again reduces the T^ value from 3.1* to 1 . 9 seconds. A l l of these distance dependencies strongly imply the dominance of the intramolecular dipole-dipole mechanism. I n t e r e s t i n g l y , the introduction of the gauche i n t e r a c t i o n between H-2 and H - 3 , as i n going from (1*0) to (1+3), causes a much smaller change i n the r e l a x a t i o n time of H-2 than of H - 3 . Evidently here the i n t r o d u c t i o n of the gauche (H-2 t c H-3) i n t e r a c t i o n c l o s e l y compensates f o r the l o s s of the 1 , 3 - d i a x i a l (H-2 to H-1*) i n t e r a c t i o n . And, as was found for the sugar penta-acetates described i n Chapter I I , the r e l a x a t i o n time of H-5 i s further shortened by the proximity of the two H-6's, which due to t h e i r a d d i t i o n a l geminal i n t e r a c t i o n , have the shortest (most e f f i c i e n t ) r e l a x a t i o n times i n the molecules. These data showed several c l e a r - c u t s t e r e o s p e c i f i c dependencies f o r the s p i n - l a t t i c e r e l a x a t i o n times of protons on a six-membered r i n g , and implied that both 1 , 3 - d i a x i a l and vicinal-gauche i n t e r a c t i o n s make a s i g n i f i c a n t contribution to the r e l a x a t i o n of an a x i a l l y oriented proton, while f o r an e q u a t o r i a l l y oriented proton, only vicinal-gauche i n t e r a c t i o n s are important. Both the s t e r e o s p e c i f i c dependence and the close agreement of data for equivalent protons i n d i f f e r e n t molecules encouraged a more quantitative 8 0 study, to.check more r i g o r o u s l y the assumption that r e l a x a t i o n was occurring p r i m a r i l y through the intramolecular dipole-dipole mechanism. ..It was hoped that the r e l a x a t i o n times obtained from a group of r e l a t e d molecules of known solut i o n conformation could be used to c a l c u l a t e i n d i v i d u a l contributions to r e l a x a t i o n of vicinal-gauche, v i c i n a l - t r a n s , and 1 , 3 - d i a x i a l i n t e r a c t i o n s , which i n turn could be used to predict r e l a x a t i o n rates ( l / T ^ values) i n another member of the set. While the halo-sugars had provided a large amount of data, and many q u a l i t a t i v e trends, the data were not s u i t a b l e f o r t h i s sort of c a l c u l a t i o n f o r several reasons. F i r s t , the i d e a l chair conformation i s d i s t o r t e d by the r i n g oxygen; second, protons i n the substituent groups contribute to the r e l a x a t i o n of the r i n g protons; f i n a l l y , the simple r e l a t i o n s h i p s are complicated by the geminal grouping of carbon s i x . What one wants, i n f a c t , i s a set of molecules, with known, undistorted chair conformations, and the same r o t a t i o n a l c o r r e l a t i o n time i n s o l u t i o n ; these c r i t e r i a appear to be met by the i n o s i t o l s ^ ( l , 2 , 3 , U ,5 ,6-hexahydroxycyclohexanes), e s p e c i a l l y as the substituent, the OH group, lends i t s e l f so well to deuteration. Of the nine isomers, n e o - i n o s i t o l i s v i r t u a l l y i n s o l u b l e i n water, the p a i r of stereoisomers are i d e n t i c a l for n.m.r. purposes, and two others, a l i o - and muco-inositol, which are time-averaging between the two chair forms i n s o l u t i o n , have complex spectra. Of the f i v e remaining for a r e l a x a t i o n study, c i s - i n o s i t o l (hQ) i s time-averaging between two i d e n t i c a l chair forms i n s o l u t i o n , each o f which has three a x i a l and three equatorial protons, and i t s n.m.r. spectrum i s a s i n g l e l i n e . Thus, four i n o s i t o l s can be used as a basis set to c a l c u l a t e the three i n d i v i d u a l contributions to r e l a x a t i o n , and from these a value f o r the r e l a x a t i o n time of the s i n g l e time-averaged.transition of c i s - i n o s i t o l can be estimated. F i g . I I I - 3 shows the normal s o l u t i o n conformations of these f i v e stereoisomers; the r e l a x a t i o n rates that could be measured for the basis set, e p i - ( U 5 ) , myo - ( U 6 ) , L-chiro - ( U 7), and s c y l l o - i n o s i t o l (hh), are given i n Table I I I - l , grouped 81 Scy l lo - inos i to l (Jth) Epxinositol (V?) Myoinositol {k6\ L-Chiroinositol (>7) C is inos i to l {_hQ\ Figure I I I - 3 . S t r u c t u r a l formulae of the f i v e i n o s i t o l s studied, showing the normal s o l u t i o n conformations. 82 Table I I I - l . Relaxation rates ( s e c . - 1 ) f o r four i n o s i t o l s , measured for 1% w/v degassed solutions i n 9 9 - 9 6 $ D 2 0 at 1+2°C. The data are grouped i n columns according to the number and type of proton-proton i n t e r a c t i o n s that can cause re l a x a t i o n . The values i n brackets are from poorly-dispersed regions of the spectra, and are considered l e s s r e l i a b l e . I n o s i t o l no 1 , 3 - d i a x i a l 1 1 , 3 - d i a x i a l 2 1 , 3 - d i a x i a l (1 ) 2 vg ( 2 ) 2 vt ( 3 ) 1 vg 1 vt ( M 2 vg ( 5 ) 2 vt ( 6 ) 1 vg 1 vt ( 7 ) 2 vg ( 8 ) 2 vt ( 9 ) 1 vg 1 vt s c y l l o - (1+1+) . 2 8 8 e p i - (U5) . 2 6 2 .101+ .1+58 . 3 9 1 myo- (1+6) .316 ( . 2 6 7 ) ( . 2 7 9 ) ( . 3 6 U ) L-chiro- (1+7) . 2 9 8 ( . 2 3 0 ) ( . 2 9 1 ) average . 2 9 2 .101+ . U 5 8 . 2 8 8 . 3 9 1 (average using a l l data) (.2U9) ( . 2 9 1 ) (.281+) ( . 3 7 8 ) 83 according-to:.the'.number'and type of pairwise intramolecular dipole-dipole i n t e r a c t i o n s possible for each, stereochemically d i s t i n c t type of proton. The values i n brackets are from regions of the spectrum that were strongly coupled or poorly dispersed, and are considered to be l e s s r e l i a b l e . As outlined i n the Introduction, the composite T^ value of a proton i s given by the sum of contributions from d i f f e r e n t r e l a x a t i o n mechanisms (eq. ( 3 a ) ) , or i f the experimentally measured T^ i s due s o l e l y to the intramolecular dipole-dipole mechanism, by the sum of contributions to the t o t a l rate from each pairwise proton-proton i n t e r a c t i o n (eq. ( 5 a ) ) . Thus, from column ( l ) of Table I I I - l , the average value of l / T ^ f o r a proton which relaxes only by i n t e r a c t i o n s with.two gauche neighbours (!) i s 0.292 seconds Hence: 2 = 2R = 0.292 sec. 1 (22) T vg vg and R = 0.1U6 sec. 1 . S i m i l a r l y from column ( 2 ) , the rate due to i n t e r a c t i o n s with two trans neighbours i s 0.101+ sec. \ and R _^ = 0.052 sec. vt Using these values for v i c i n a l i n t e r a c t i o n s , one can c a l c u l a t e the rate due to one 1 , 3 - d i a x i a l i n t e r a c t i o n , R . For example, using the data i n £13. column (T): 2R + 2R = 0.1+58 sec. 1 vg aa _ i (23) R = 0.083 sec. aa Data i n columns (8) and (9) gave 0.092 and 0.097 sec . " 1 f o r the 1 , 3 - d i a x i a l contribution; the average from the three sets of data i s 0.091 — 0.008 sec. where the range of approximately ± 10%. i s quite.reasonable. Table III-2 summarizes the r e l a x a t i o n rates and'corresponding r e l a x a t i o n times derived for the three types of pairwise i n t e r a c t i o n . To t e s t the v a l i d i t y of t h i s additive approach, an attempt was 8U T a b l e I I I - 2 . R e l a x a t i o n r a t e s and c o r r e s p o n d i n g r e l a x a t i o n t i m e s d e r i v e d f o r t h e t h r e e g e o m e t r i c a l l y d i s t i n c t p a i r w i s e i n t e r a c t i o n s o f a p r o t o n i n a six-membered r i n g i n c h a i r form. t y p e o f i n t e r a c t i o n R l 1 seconds 1 seconds v i c i n a l - g a u c h e 0 . 1 U 6 6 . 8 5 v i c i n a l - t r a n s 0 . 0 5 2 1 9 . 2 1 , 3 - d i a x i a l 0 . 0 9 1 1 1 . 0 85 made to estimate the r e l a x a t i o n rate of the single t r a n s i t i o n of c i s - i n o s i t o l , which i n s o l u t i o n i s an equal mixture of two. ( i d e n t i c a l ) ' c h a i r forms. In both forms, the three equatorial protons r e l a x through two vicinal-gauche i n t e r -actions, and the three a x i a l ones through two vicinal-gauche plus two 1 , 3 - d i a x i a l i n t e r a c t i o n s . The r e l a x a t i o n rates for the two s i t e s are: R = 2R = 0 . 2 9 2 sec eq vg - 1 R = 2R + 2R = O.hlh sec ax vg aa - 1 ( 2 3 a ) ( 2 3 b ) The r e l a x a t i o n rate for a proton undergoing time-averaging between two s i t e s i s given by the weighted average of the rates at the two s i t e s (eq. (21) i n Chapter I I ) ; i n t h i s case the populations of the two s i t e s are equal. The r e l a x a t i o n rate f o r a r i n g proton of c i s - i n o s i t o l i s therefore: R . = 0 . 5 (R + R ) = 0 . 3 8 3 sec c i s ax eq Tn = 1/R . = 2 . 6 l sec. 1 . C I S C I S -1 (210 ( 2 V ) The c a l c u l a t e d T^ for c i s - i n o s i t o l , 2 . 6 l seconds, i s i n reasonable agreement, within experimental e r r o r , with the observed value of 2 . 8 8 seconds. (A c a l c u l a t i o n i n c l u d i n g the l e s s r e l i a b l e data gave 2 . 5 5 seconds.) With t h i s s a t i s f a c t o r y i n t e r n a l consistency established, we then decided to see i f chemically reasonable r a t i o s of interproton distances could be derived from the pairwise rates i n Table I I I - 2 . As o u t l i n e d i n Chapter I, for intramolecular dipole-dipole r e l a x a t i o n , the s i x t h root of the r a t i o of two pairwise dipole-dipole r e l a x a t i o n rates gives the r a t i o of the two i n t e r -proton distances: (6) (T ) 1 v V l S . l 1/6. •• ^ i s . 1 ,i 1/6 . ris. i (T ) v V i s . ( V i s . 1 0 r i s . j 86 The t h e o r e t i c a l values for distances were c a l c u l a t e d f o r an i d e a l c hair using standard values for the C-C and C-H bond distances, and the t e t r a h e d r a l angle of 1 0 9 . 5 °• Table 111-3 shows a comparison of the c a l c u l a t e d and experimental r a t i o s of interproton distances, and the values are i n s u b s t a n t i a l agreement. Both the i n t e r n a l consistency of the data, and the chemically reasonable r a t i o s of interproton distances that can be derived support the assumption that the experimental T^ values were almost completely intramolecular dipole-dipole i n o r i g i n , and so, add i n a simple way, and r e f l e c t the precise stereochemical environment of each r i n g proton. It must be pointed out that these conclusions r e s t eventually on an assumption that a l l of the r e l a x a t i o n was intramolecular d i p o l e - d i p o l e . Apart from the usual experimental precautions of removing dissolved oxygen, and using pure samples and deuterated solvents of high i s o t o p i c p u r i t y , the T^ values were measured at a low enough concentration, 1% w/v, that i t was hoped that r e l a x a t i o n due to solute-solute i n t e r a c t i o n would be n e g l i g i b l e . As we had found i n a concentration study of D-glucose that no increase i n T^ values occurred below 0 . 2 molar, i t seemed that the O.O56 molar concentration chosen for the i n o s i t o l s would be "safe" i n t h i s respect. What would the e f f e c t be, of a small intermolecular c o n t r i b u t i o n to relaxation? It would reduce the s t e r e o s p e c i f i c dependencies by adding a small extra term to the r e l a x a t i o n rate of each proton. As i t was assumed that the experimentally measured values were purely intramolecular, the r e l a x a t i o n rates derived for i n d i v i d u a l pairwise i n t e r a c t i o n s would then a l l be a l i t t l e too high. I f intermolecular e f f e c t s were both small, and roughly equal from one proton to another, they would not have greatly a f f e c t e d e i t h e r the " i n t e r n a l " check (the estimation of the r e l a x a t i o n rate of c i s - i n o s i t o l ) , nor the "external" one, as the s i x t h root of a r a t i o (A+6):(B+6) would not d i f f e r s i g n i f i c a n t l y from that of A:B. 87 Table III-3. Comparison of r a t i o s of interproton distances obtained from (a) s i x t h root of r a t i o s of i n d i v i d u a l T i values (Table III-2) and (b) distances calculated f o r an i d e a l six-membered r i n g i n chair form. The bond distances used i n the c a l c u l a t i o n s were: C-C, 1.5^ and C-H, 1.09 and the r e s u l t i n g distances were: vicinal-gauche, 2.^9 X, v i c i n a l - t r a n s , 3.06 & and 1,3-diaxial 2.52 K. i n t e r a c t i o n s compared r a t i o s of T i values r a t i o s of interproton distances from s i x t h root of Ti r a t i o s from c a l c u l a t i o n v i c i n a l - t r a n s vie inal-gauche 2.81 1.19 1.23 1 , 3 - d i a x i a l vie inal-gauche 1 . 6 l 1.08 1.01 v i c i n a l - t r a n s 1 , 3 - d i a x i a l 1.75 1.10 1.22 88 A second assumption was that the f i v e i n o s i t o l s studied a l l had the same c o r r e l a t i o n times under the experimental conditions used. To summarize, i n the work on the i n o s i t o l s , two assumptions were made, which appear to have been j u s t i f i e d i n the r e s u l t s , and also are consistent with a l l of the work on other molecules. While experiments could have been done to check t h e i r v a l i d i t y , i t was f e l t at the time that other studies were more worthwhile. The p o t e n t i a l f o r chemical a p p l i c a t i o n i s c l e a r , although a few words of caution are appropriate here. While r e l a x a t i o n rates can be accurately and reproducibly measured, the precise meaning of the rates of a set of s p i n -coupled mu l t i p l e t s i s not always c l e a r . Even when the only r e l a x a t i o n mechanism i s the intramolecular dipole-dipole (proton-proton) one, the presence of cross-r e l a x a t i o n can lead to errors of up to twenty per cent, by causing the recovery to deviate from purely exponential behaviour, and the rates to d i f f e r from one t r a n s i t i o n of a m u l t i p l e t to another. How t h i s problem has been dealt with i n an empirical way to standardize our data, and make our r e s u l t s consistent and reproducible i s discussed i n the Experimental se c t i o n . Further, the r e s u l t s obtained from one set of structures can only be t r a n s f e r r e d i n a most general, q u a l i t a t i v e way to another. For example, when one uses the i n d i v i d u a l rates i n Table I I I - 2 to predict the r a t i o s of (T /T ) for glucose and mannose, one lex l p obtains r a t i o s of 1 . 6 and 2 . 2 , at considerable variance with the experimental r a t i o s of 1 . 9 and 3 . 3 (Chapter I I ) . However, even t h i s sort of naive c a l c u l a t i o n c o r r e c t l y predicts that f o r both sugars, T ^ i s much longer than T.^, a n ( i that the d i f f e r e n t i a l increases i n going from the gluco to the manno configuration. 89 P o s t s c r i p t : 38 Recent work by Freeman , on the same sealed sample of one of the halo-sugars, the 2-C_-chloro-3-gluco d e r i v a t i v e ( U l ) , using a combination of se l e c t i v e and non-selective Fourier transform measurements, together with Overhauser enhancements, has confirmed that f o r H-1, 100$ of the r e l a x a t i o n i s d i p o l e - d i p o l e , and that a minimum of 90$ i s intramolecular i n o r i g i n . CHAPTER IV PHYSICAL AND CHEMICAL APPROACHES TO THE QUANTITATIVE STUDY OF DIPOLE-DIPOLE PROTON RELAXATION IN ORGANIC MOLECULES Multaque praeterea t i b i possum commemorando argumenta fidem d i c t i s conradere n o s t r i s . verum animo s a t i s haec v e s t i g i a parva sagaci sunt, per quae possis cognoscere cetera t u t e . namque canes ut montivagae persaepe f e r a i naribus inveniunt intectas fronde quietes, cum semel i n s t i t e r u n t v e s t i g i a c e r t a v i a i , s i c a l i d ex a l i o per te tute ipse videre t a l i b u s i n rebus, p o t e r i s caecasque latebras insinuare omnis et verum protrahere inde. (Lucretius, De Rerum Natura I, U 0 0 - U 0 9 ) There, are many other proofs I could add to the p i l e i n order to strengthen c o n v i c t i o n ; but for an acute i n t e l l i -gence these small clues should s u f f i c e to enable you to discover the rest f o r y o u r s e l f . As hounds that range the h i l l s often smell out the l a i r s of w i l d beasts screened i n t h i c k e t s , when once they have got on to the r i g h t t r a i l , so i n such questions one thing w i l l lead on to another, t i l l you can succeed by y o u r s e l f i n tracking down the t r u t h to i t s lurking-places and dragging i t f o r t h . ( t r . R.E. Latham Penguin Books, 1951) Introduction This chapter describes work on nine compounds, the two pyranoside and seven furanose d e r i v a t i v e s whose structures are shown below. A l l were f i r s t surveyed i n the same manner as the molecules i n the e a r l i e r work; that i s , the non-selective r e l a x a t i o n times of degassed, sealed, 0 . 1 molar solutions were measured using the three-pulse sequence and the data-handling procedures o u t l i n e d i n the Experimental Section. The ambient probe temperature had, however, been lowered from 1+2 to 33°C. These experiments w i l l be r e f e r r e d to as "UBC", "conventional", or "three-pulse". In a d d i t i o n , three compounds, ( 5 1 ) 5 ( 5 2 ) and ( 5 3 ) , were studied by the " t a i l o r e d e x c i t a t i o n " method ' , i n the laboratory of Varian Associates, Palo A l t o , C a l i f o r n i a , using the same sealed samples. This instrumental development allows the Fourier transform 90 91 ALLO-PYRANOSIDES (51) R = H \ (52) R = D OCH ALLO-FURANOSES R = CH3 GS) R' = OCOCD3, R" = H (50) R' = OCOCD3, R" = D (53) R' = OCOCH3, R" = H GLUCO-FURANOSES (3\) R = CH3, R' = OCOCH3 (55) R = CH3, R' - OCOCD3 (56) R = CD3, R' = OCOCH3 (57) R = CD3, R' = OCOCD3 92 pulses to be applied to any region(s) of the spectrum; thus the experiments include inversion of one m u l t i p l e t ( s e l e c t i v e ) , of two or three m u l t i p l e t s simultaneously, and of the whole spectrum (non-selective). The experimental procedures were s l i g h t l y d i f f e r e n t from those of UBC experiments. The probe temperature was 2 8 ° , a l l experiments were c a r r i e d out using a two-pulse sequence with a strong s e n s i t i v i t y enhancement fa c t o r (see Chapter V) to reduce noise, and fewer data points were taken, u s u a l l y ten, o c c a s i o n a l l y f i f t e e n or twenty delay times when time permitted. Slopes were determined by the usual computer-fit using a l l the data points, unless otherwise mentioned, without regard as to how many T^ periods the decay length represented; i n most cases, the maximum delay time was l e s s than "T^". The r e l a x a t i o n times of i n d i v i d u a l t r a n s i t i o n s of a m u l t i p l e t were not determined, only the average rel a x a t i o n time of each chemically s h i f t e d proton, by p l o t t i n g the peak height sum of the i n d i v i d u a l t r a n s i t i o n s . These experiments w i l l be described as "Varian", " t a i l o r e d e x c i t a t i o n " , or "two-pulse" experiments. Note that a l l UBC experiments, i n t h i s and other chapters, measure non-selective T^'s, while the Varian experiments include both s e l e c t i v e and non-selective ones. There i s some redundancy i n reporting experimental r e s u l t s i n t h i s chapter. The UBC data, which are of a survey nature, are shown as T^ values, superimposed on s t r u c t u r a l formulae to emphasize the s t e r e o s p e c i f i c dependence. For purposes o f c a l c u l a t i o n however, many of the same numbers may be found i n t a b l e s , as r e l a x a t i o n rates. The Varian r e s u l t s are always given as ra t e s , i n tabular form, The f i r s t part of t h i s chapter discusses the six-membered r i n g pyranoside compounds ( 5 l ) and ( 5 2 ) , i n c l u d i n g the e f f e c t of changing the solvent from deuterochloroform to deuterobenzene. The second part concerns the five-membered r i n g furanose compounds (h9), ( 5 0 ) and ( 5 3 ) to ( 5 7 ) , i n c l u d i n g an i n v e s t i g a t i o n of the importance of the substituent methyl groups for the r e l a x a t i o n of the r i n g protons. 93 As there i s so much data i n t h i s chapter, i t i s perhaps worthwhile to review here our reasons f o r studying these nine molecules. Our main i n t e r e s t was i n the r e l a x a t i o n of the r i n g protons - to see i f t h e i r r e l a x a t i o n were completely dipole-dipole i n o r i g i n , by comparison of s e l e c t i v e and non-selective r e l a x a t i o n times, and also to determine i n d i v i d u a l pairwise r e l a x a t i o n rates by two independent methods, s e l e c t i v e deuteration and t a i l o r e d e x c i t a t i o n , and compare the r e s u l t s from the two methods. Two other projects were added, however, i n v e s t i g a t i o n of solvent e f f e c t s f o r ( 5 l ) and ( 5 2 ) , and of the importance of substituent methyl protons i n the r e l a x a t i o n of r i n g protons, by study of the. four.gluco-furanoses (5*0 to ( 5 7 ) . The Six-Membered Rings The non-selective proton r e l a x a t i o n times of methyl 3 - 0_-(trideutero-methyl)acetyl-U ,6-0-benzylidene -2-deoxy-a-E)-ribo-hexopyranoside ( 5 1 ) , and of i t s 2-C-deutero counterpart ( 5 2 ) were measured i n both deuterochloroform and deuterobenzene; the r e s u l t s are shown i n F i g . IV - 1 . As the s t e r e o s p e c i f i c dependencies are consistent with those discussed e a r l i e r , and with data f o r the s t r u c t u r a l l y - r e l a t e d p a i r of nitro-sugars ( 5 8 , 59) i n the Appendix, these q u a l i t a t i v e aspects w i l l not be discussed further. There i s a marked solvent e f f e c t , with the r e l a x a t i o n rates generally being greater i n deuterochloroform. Table IV - 1 shows the solvent e f f e c t s more qu a n t i t a t i v e l y , as r e l a x a t i o n rates, and differences i n r a t e s . (AR^ = RQ-QQ-^ R_ n ). The solvent differences vary from e s s e n t i a l l y zero - l e s s than C 6 D 6 experimental error - for and the two H-6's, to a small increment of ten to twenty per cent f o r the other protons. For H -5 , however, the differ e n c e i s a s t r i k i n g one hundred per cent. The close agreement of the AR^ values obtained from ( 5 l ) and ( 5 2 ) for H-1 , H-2 , H - 3 , H-U and H-5 i s remarkable, ^q e s p e c i a l l y as, for some protons, the degree of coupling and s i g n a l overlap were quite d i f f e r e n t i n the two solvents. Figure IV-1. Spin- latt ice relaxation times of methyl 3-Oj-(.trideuteromethyl)acetyl-k,6-0_-benzylidene-2-deoxy-a-D-ribo-hexopyranoside (.51) and i t s 2-C-deutero counterpart (52). in both deuterochloroform and deuterobenzene (0.1 molar) at,33 C, using a conventional three-pulse method. 95 Table IV -1 . Comparison of relaxation rates in two different solvents for hexopyranoside compounds (51) and ( 5 2 ) , for 0 . 1 molar solutions at 33°C. t and * are a rather subjective indication of the degree of spectral dispersion; * referring to strongly coupled and overlapping signals, and t to pairs where the overlap i s less serious. A l l other data are from well-dispersed parts of the spectra. A partial 100 MHz n.m.r. spectrum of the nondeuterated compound (51) in deuterochloroform i s shown in Fig. IV-2 of a Varian experiment. (Data are those shown as relaxation times in Fig. IV-1) Relaxation rate (seconds 1) (51) ( 5 2 ) Proton Hi CDCI3 Rl ARX Ri ' CDC13 Ri ARx H-1 0.32k 0 . 2 9 1 * 0.033 0 . 2 1 7 0 . 1 8 U 0 . 0 3 3 H-2 ax 1 . 0 0 1.01 (-0.01) H-2 eq O .896 0 . 8 3 2 • 0.061+ 0 . 2 8 5 0 . 2 1 7 0 . 0 6 8 H-3 0 . 3 5 3 0.291+ 0 . 0 5 9 0 . 2 7 0 0.212 0 . 0 5 8 H-H 0 . 7 2 8 f 0 . 5 8 7 0.11+1 0 . 5 6 8 t 0.1+1+2 0 . 1 2 6 H-5 0.1+8)4* . * 0.21+9 0 . 2 3 5 0.1+1+9* * 0 . 1 9 2 0 . 2 5 7 H-6 ax 0.9lkf 0 . 8 1 2 0.102 0 . 9 3 2 + 0 . 9 ^ 8 ( - 0 . 0 1 6 ) H-6 eq * O.I+93 0 . 6 0 9 * ( - 0 . 1 1 6 ) * 0 . 5 8 0 * 0 . 5 5 3 0 . 0 2 7 H-7 0.1+3U 0 . 3 9 2 0.01+2 0.1+55 0 . 3 7 8 0 . 0 7 7 96 Why these solvent differences e x i s t i s not known. The agreement of the r e s u l t s from two compounds suggests (hut by no means proves) that the higher r e l a x a t i o n rates i n deuterochloroform were not s o l e l y due to higher concentrations of paramagnetic impurities or diss o l v e d oxygen. Furthermore, the close agreement of most of the i n d i v i d u a l pairwise r e l a x a t i o n rates i n the two solvents (discussed i n the next section; see Table IV-2) e s s e n t i a l l y r u l e s out an explanation i n terms of d i f f e r e n t c o r r e l a t i o n times for the o v e r a l l molecular r e - o r i e n t a t i o n . The degree of coupling and s i g n a l overlap may have had some influence on the r e l a x a t i o n r a t e s , but generally, t h i s has not been found to have a large e f f e c t on our standard experimental T^ values. Another possible reason would be d i f f e r e n t arrangements and rotamers of the substituent groups i n the two solvents. A small change i n the d i s p o s i t i o n of the C-1 methoxyl group could have s i g n i f i c a n t e f f e c t s on the r e l a x a t i o n of H -1 , H-2 eq and H -5 . (The acetate group at C-3 was of course, deuterated i n both ( 5 l ) and ( 5 2 ) . The non-deuterated version, methyl k,6-0_-benzylidene-3-0_-acetyl-2-deoxy-q-D-ribo-hexopyranoside (D) was a c t u a l l y prepared as a standard during the synthesis of (51) and ( 5 2 ) , but i t s r e l a x a t i o n times were not measured; study of (D) might now be useful i n explaining the solvent e f f e c t s ) . The purpose of t h i s comparison was simply to assess the possible magnitude of solvent e f f e c t s , and a large number of experiments would be necessary for a complete explanation. C l e a r l y , solvent e f f e c t s can be s i g n i f i c a n t , and vary from one proton to another. Fortunately, except i n one case, the o v e r a l l q u a l i t a t i v e patterns were not upset, and as w i l l be shown i n the next section, the pairwise r e l a x a t i o n rates determined i n the two solvents were i d e n t i c a l w ithin experimental e r r o r . For the present, these r e s u l t s emphasize the need for i d e n t i c a l experimental conditions - of temperature, concentration, p u r i t y and solvent - i f data are to be used q u a n t i t a t i v e l y , or for diagnostic purposes. These e f f e c t s also support the r a t i o n a l e of working with sets of 97 s t r u c t u r a l l y - r e l a t e d molecules under standard conditions, rather than using data from one molecule on an absolute b a s i s , as the e f f e c t s of solvent, c r o s s -r e l a x a t i o n and s p e c t r a l dispersion are at l e a s t reduced to a reasonably constant background. This has been a di g r e s s i o n ; the o r i g i n a l purpose i n studying these molecules was to t r y and determine the contribution that H -2 makes to the ax r e l a x a t i o n of other protons by the two approaches of deuterium s u b s t i t u t i o n and t a i l o r e d e x c i t a t i o n , and to compare the r e s u l t s . The f i r s t r e s u l t may be found from the differences i n the r e l a x a t i o n rates i n the normal (51) and deuterated compound ( 5 2 ) , shown i n Table IV-2. The changes caused by deuterium s u b s t i t u t i o n of H -2 i n the two solvents.agree quite w e l l , and there are large differences i n the pairwise r e l a x a t i o n r a t e s , r e f l e c t i n g the d i f f e r e n t geometrical r e l a t i o n s h i p s . (Although the pyranose r i n g departs from the i d e a l chair form, because of the heteroatom i n the r i n g , the interproton r e l a t i o n s h i p s can be c l a s s i f i e d i n the same way,'as vicinal-gauche, v i e i n a l - t r a n s , 1 , 3 - d i a x i a l , and geminal.) Again, the only probe of chemical r e a l i t y , the only t e s t of whether these i n d i v i d u a l r e l a x a t i o n rates are measures of independent pairwise i n t r a -molecular dipole-dipole i n t e r a c t i o n s with a common c o r r e l a t i o n time, i s to see i f they give reasonable r a t i o s of interproton distances. These r a t i o s , c a l c u l a t e d from the s i x t h root of r a t i o s of the i n d i v i d u a l pairwise r e l a x a t i o n rates are given i n Table rV-3, and t h i s time, unfortunately, there i s no obvious standard against which they may be compared, an X-ray structure, for example. However, r a t i o s are shown f o r an i d e a l six-membered r i n g (those c a l c u l a t e d for the i n o s i t o l study i n Chapter III), and also f o r a pyranose r i n g , obtained by measuring a molecular model of an oxygen-containing six-membered r i n g . The substantial agreement of most of the r a t i o s suggests that the r e l a x a t i o n rates c a l c u l a t e d i n Table IV-2 do r e f l e c t pairwise intramolecular dipole-dipole i n t e r a c t i o n s , and hence may be a u s e f u l probe of molecular geometry. There i s 98 Table IV - 2 . C a l c u l a t i o n of the cont r i b u t i o n of H - 2 a x to the t o t a l r e l a x a t i o n rate of H -1 , H - 2 eg, H-3 and H-1* i n methyl 3 - 0_-(trideuteromethyl )acetyl-l+, 6-0-benzylidene -2-deoxy-a-D-ribo-hexopyranoside ( 5 1 ) , by the differences i n the rel a x a t i o n rates of (51) and i t s 2-C-deutero counterpart ( 5 2 ) . The data are those given i n Table I V - 1 , for 0 . 1 molar solutions i n deuterochloroform and deuterobenzene at 33°C, from conventional Tj determinations. solvent prot on ( 5 1 ) E l ( 5 2 ) Ri A Ei type of i n t e r a c t i o n CDC13 H-1 0 . 3 2 U 0 . 2 1 7 0 . 1 0 7 v i e i n a l -gauche H-2 eq 0 . 8 9 6 0 . 2 8 5 0 . 6 1 1 geminal H-3 0 . 3 5 3 0 . 2 7 0 0 . 0 8 3 v i c i n a l -gauche E-h 0 . 7 2 8 0 . 5 6 8 0 . 1 6 0 1 , 3 - d i a x i a l C6D6 H-1 0 . 2 9 1 0.18I* 0 . 1 0 7 v i c i n a l -gauche H-2 eq 0 . 8 3 2 0 . 2 1 7 0 . 6 1 5 geminal H-3 0 . 2 9 1 * 0 . 2 1 2 0 . 0 8 2 v i c i n a l -gauche H-1* 0 . 5 8 7 0.1*1*2 0.11*5 1 , 3 - d i a x i a l 99 Table IV-3. Comparison of r a t i o s of interproton distances obtained from the pairwise rates derived i n Table IV-2, with r a t i o s of distances f o r i d e a l r i n g systems. The values f o r the pyranose r i n g were obtained by measurement of a molecular model. The values i n the l a s t column are for a six-carbon cyclohexane r i n g i n chair form, and were ca l c u l a t e d using standard bond distances, and the t e t r a h e d r a l angle. (These are the same c a l c u l a t i o n s used i n Chapter III f o r the i n o s i t o l s . ) i n t e r a c t i o n s compared rat i o of T i values r a t i o of interproton distances f r o m T ! r a t i o s (6th root) from model of pyranose r i n g from ca l c u l a t e d cyclohexane r i n g H - H vicinal-gauche .^a o H2a " H2e g e m i n a l IM 1.1+0 1.1+2 1.1+0 H_ - H vicinal-gauche H2a ~ H2e § e m i n a l 5.73 i.3h 1.1+2 1.1+0 - 1,3-diaxial H 2 a - H 2 e geminal k.Oh 1.26 1.5k 1.1+2 H 0 - H vicinal-gauche H 2 a - vicinal-gauche 1.30 1.01+ 1.00 1.00. 100 considerable disagreement, however, when the data for the 1,3-diaxial i n t e r -a c t i o n - the H-2 to E-h distance - are included, suggesting a considerable ax ' shortening of t h i s distance. Any a p p l i c a t i o n of these data to conformational study at t h i s point would be pure conjecture; however, there may be a t w i s t i n g d i s t o r t i o n of the r i n g , which would increase the separation of the a x i a l substituents at positions one and three. This completes discussion of the conventional experiments on the pyranoside d e r i v a t i v e s , and the next section i s devoted to the Varian experiments. The o r i g i n a l plan had been, of course, to do the exact counterpart of the UBC study; that i s , to determine the cont r i b u t i o n of H-2 to the rel a x a t i o n of the other protons by s e l e c t i v e inversion experiments on the non-deuterated compound ( 5 l ) . However, the chemical s h i f t d i f f e r e n c e of the two H-2 protons was too small to inv e r t one without s e r i o u s l y perturbing the other. Therefore, only one experiment was c a r r i e d out on ( 5 l ) , a determination of the extent of dipole-dipole r e l a x a t i o n of H-1, but several experiments were instead c a r r i e d out on (52). (The Varian experiments were on the chloroform solutions only.) F i g . IV-2 shows p a r t i a l l y relaxed two-pulse spectra of (5l)» t y p i c a l of those obtained from the Varian experiments. Due to the a p p l i c a t i o n of a s e n s i t i v i t y enhancement factor to the free induction decay (see Chapter V), the signal-to-noise r a t i o i s high, at the expense of r e s o l u t i o n . The re l a x a t i o n rates obtained from s e l e c t i v e and non-selective inversion of H-1 are given i n Table TV-k, along with the r a t i o s and enhancements. Some i n d i c a t i o n i s also given of the non-exponential character of both decays. C e r t a i n l y , i n t h i s type of study, c a r e f u l determination of i n i t i a l slopes i s important, as the systematic error caused by non-exponential decay increases as data from longer and longer delay times are included i n the c a l c u l a t i o n s . The enhancement of 0.k3 or 0.U2 obtained for H-1 agrees within experimental error with the t h e o r e t i c a l maximum of 0.5, i n d i c a t i n g that i t s r e l a x a t i o n i s almost completely H 6 0 . H 4 U A Z Z ^ Y T - O C H Figure IV - 2 . P a r t i a l l y relaxed 100 MHz n.m.r. spectra of methyl 3 - 0 -(trideuteromethyl)acetyl - k ,6-0-benzylidene -2-deoxy-a-g-ribo-hexopyranoside ( 5 l ) , obtained f o r a n o n - s e l e c t i v e two-pulse T i experiment, on a degassed 0.1 molar s o l u t i o n i n deuterochloroform at 28°C. A s e n s i t i v i t y enhancement fa c t o r has been applied to the free induction decay i n order to increase signal-to-noise at the expense of r e s o l u t i o n . This experiment was c a r r i e d out i n the n.m.r. laboratory of Varian Associates at Palo A l t o , C a l i f o r n i a , i n August, 197^ 102 Table IV-k. V a r i a t i o n of r e l a x a t i o n rates and enhancements with decay length for H-1 of methyl 3 - 0-(trideuteromethyl)acetyl-U , 6 - 0-benzylidene - 2-a-D-ribo-hexopyranoside ( 5 1 ) . Data are f o r a 0 . 1 molar so l u t i o n i n CDCI3 at 28°C. Experiments c a r r i e d out at Varian Associates, Palo A l t o . a i n i t i a l slope ^average of maximum and minimum slopes. " t " Max. (no. of data points) 0 . 8 ( 8 ) 1 . 0 ( 1 0 ) 2 . 0 (15) 3 . 0 ( 2 0 ) "best" v i s u a l f i t or i n i t i a l slope s e l e c t i v e rate R ( s e c . - 1 ) 0 0 . 2 8 1 0 . 2 7 3 0 . 2 7 3 0 . 2 6 6 0 . 2 7 7 a non-s e l e c t i v e rate R ( s e c . - 1 ) 0 . 3 8 5 0 . 3 9 1 0 . 3 5 6 0 . 3 3 5 0 . 3 9 5 b r a t i o R/R 0 1 . 3 7 1 . U 3 1 . 3 0 1 . 2 6 1 . U 2 enhance-ment 0 . 3 7 0.k3 0.30 0 . 2 6 0.1*2 103. dipole-dipole i n o r i g i n . Without more data and experience of " t a i l o r e d e x c i t a t i o n " , i t i s impossible to t e l l from one experiment vhether an enhancement of 0 . U 3 simply r e f l e c t s scatter about a true value of 0 . 5 , or a genuine con-t r i b u t i o n to r e l a x a t i o n from some other mechanism. Several t a i l o r e d e x c i t a t i o n experiments were then c a r r i e d out on ( 5 2 ) , the deuterated pyranoside. The regions excited and the r e l a x a t i o n rates are given i n Table I V - 5 ; note that the s e l e c t i v e rates f o r H-2 and H-T were eq obtained i n one double i r r a d i a t i o n , as i t . seemed extremely u n l i k e l y that there could be any i n t e r a c t i o n between these two well-separated spins. Table IV - 6 shows the r a t i o s and enhancements of r e l a x a t i o n rates. The non-selective enhancements i n column ( l ) again i n d i c a t e the degree of dipole-dipole r e l a x a t i o n . The maximum of 0 . 5 was found f or H-7 , although the values were l e s s f o r the other spins; again, at t h i s stage i t i s not possible to t e l l whether the depression i s genuine, or a r e s u l t of random and systematic e r r o r s . A l l of the values for are suspect, because the peak was broadened by deuterium coupling, and a small and r a p i d l y - r e l a x i n g impurity on one side caused d i s -t o r t i o n of the baseline. C e r t a i n l y , f o r a l l protons, the dominant source of r e l a x a t i o n i s the dipole-dipole mechanism. The double and t r i p l e inversion experiments show that the r e l a x a t i o n rate of H-2 i s enhanced to the same extent by simultaneous inversion of e i t h e r eq H-1 or H-3; and conversely, i n v e r s i o n of H-2 causes equal enhancement of H-1 ' ' eq and H - 3 . Inversion of a l l three spins then increases the enhancement of the rate of H-2 to that obtained by non-selective i n v e r s i o n , but causes no further eq . ' increase i n the rates of H-1 and H - 3 , confirming the i n t u i t i v e assumption that molecular geometry prevents any s i g n i f i c a n t dipole-dipole i n t e r a c t i o n between these two spins. As' o u t l i n e d i n the Introduction, the f r a c t i o n a l contribution to the t o t a l r e l a x a t i o n rate of one spin by another (or others) i s given by the f r a c t i o n of the non-selective enhancement obtained i n the p a r t i a l enhancement. 10k Table IV-5. Relaxation rates ( i n seconds 1 ) for several protons of methyl 3_0-(trideuteromethyl)acetyl -H , 6 - 0-benzylidene - 2-deoxy - 2-C--deutero-a-Ii-altro-pyranoside ( 5 2 ) . The experiments were c a r r i e d out at Varian Associates, using a 0 . 1 molar sample i n C D C I 3 , at 28°C. Ten data points with delay times of ( 0 . 2 , O.k . . . 2 . 0 ) seconds were obtained f o r each experiment, and the rates were obtained by the standard computer least-squares f i t to a l l ten points. Relaxat 7 , 197^ ion rate (seco ads x ) •<—August 13-lk, 1 OYli ) < August y f *+ * proton non-s e l e c t i v e H " 2 eq a H-7 H " 2 e q H-1 H " 2 e q H-3 H -2eq + H-1 + H-3 non-sel e c t i v e 1 3 H-1 H-3 H-1 0 . 2 U 8 0.203 0 . 2 0 1 0 . 2 ^ 8 0 . 1 7 9 H-2 eq 0 . 2 9 0 0 . 2 1 7 0 . 2 3 U 0 . 2 3 1 0.300 0.300 H-3 0 . 2 9 5 0 . 2 U 6 0.2H-8 0 . 3 0 U 0 . 2 2 0 H-7 0.510 0 . 3 H 1 0.k9k 'Equivalent to two separate s e l e c t i v e inversions of H-7 and H-2 Average of two runs. 105 Table IV-6 . Ratios of r e l a x a t i o n rates and enhancements f o r methyl 3 - 0_-(trideuteromethyl)acetyl-U ,6-0_-benzylidene -2-deoxy -2-C-deutero-a-D-altropyranoside ( 5 2 ) , based on the data i n Table IV - 5 . The errors are" a c t u a l l y of the order of ± 0 . 1 , with systematic errors tending t o drive the values down. Proton Ratio of r e l a x a t i o n rates (enhancement) non-sel. H 1 + H 2 eq H 2 + H eq H l + H 2 + H 3 eq s e l e c t i v e s e l e c t i v e s e l e c t i v e s e l e c t i v e H-1 1 . 3 8 ( 0 . 3 8 ) 1 . 1 3 ( 0 . 1 3 ) 1 . 1 2 ( 0 . 1 2 ) H-2 eq 1 . 3 U (0.3*0 1 . 0 8 ( 0 . 0 8 ) 1 . 0 6 ( 0 . 0 6 ) 1 . 3 8 ( 0 . 3 8 ) H-3 1 . 3 8 ( 0 . 3 8 ) 1 . 12 ( 0 . 1 2 ) 1 . 1 3 ( 0 . 1 3 ) H-T 1 . U 9 ( 0 . U 9 ) 106 Thus a p a r t i a l enhancement of, f o r example 0.13,.indicates .that 26% of the r e l a x a t i o n a r i s e s from dipole-dipole i n t e r a c t i o n s with the spin(s) simultaneously-inverted (provided the non-selective enhancement i s 0 . 5 ) . For some protons studied, the experimental non-selective enhancements were l e s s (and i n one case more) than 0 . 5 ; at t h i s stage, as the balance of experimental error and r e l a x a t i o n by other mechanisms has not yet been assessed, i t seems reasonable to estimate the r e l a t i v e contribution of spin(s) to the t o t a l dipole-dipole r e l a x a t i o n rate of spin I as f r a c t i o n s of I's experimental non-selective enhancement, whether more or l e s s than 0 . 5 . Thus, the data i n Table IV - 6 suggest that i n t e r a c t i o n s with E-2^^ account f o r approximately 3k% ( i . e . , 13/38) of the r e l a x a t i o n of H-1 , and 32$ of that of H -3 . Unfortunately, i t i s not possible to compare d i r e c t l y the r e s u l t s of the deuteration study, which measured i n t e r a c t i o n s with the a x i a l H -2 . (The i n d i v i d u a l rates were 0 . 1 0 7 - 1 - 1 sec. for H-1 and 0 . 0 8 3 sec. f o r H - 3 ) . A rough comparison can be made, however, by using the range 0 . 0 8 3 - 0 . 1 0 7 sec. 1 to approximate the v i c i n a l -gauche int e r a c t i o n s of H-1 and .H-3 with H-2 a l s o ; the r e s u l t i s shown below: eq contribution of H-2 to t o t a l r e l a x a t i o n rate eq from deuteration from t a i l o r e d (estimated from H-2 data) e x c i t a t i o n ax H-1 38-1+9 % 3h% H-3 31-h0% 32% (The t o t a l r e l a x a t i o n rates of H-1 and H-3 i n (52) (UBC experiments) were 0 . 2 1 7 and 0 . 2 7 0 s e c . - 1 r e s p e c t i v e l y ) . Thus the p a r t i a l intramolecular dipole-dipole r e l a x a t i o n "budgets" obtained from ei t h e r method show the same pattern; s u b s t a n t i a l portions, however, remain to be accounted f o r . Other possible i n t e r a c t i o n s are, presumably, with the methoxyl protons for H-1 ( c f . the data f o r l 6 , l 6 a i n Chapter I I ) , and for H - 3 , the vicinal-gauche i n t e r a c t i o n with H-1+. Using the same estimate f o r 107 the H-3,H-1+ i n t e r a c t i o n would now account f o r 6 l - 7 9 $ of the r e l a x a t i o n of H -3 . C l e a r l y , much remains to be learned about the d e t a i l e d sources of proton r e l a x a t i o n i n a "large" molecule; using a combination of selective-deuteration and s u f f i c i e n t t a i l o r e d e x c i t a t i o n experiments, i t should be possible to account q u a n t i t a t i v e l y f or the relaxation, from each source. These studies of the two pyranoside d e r i v a t i v e s suggested a number of further experiments; however, discussion of these w i l l be deferred, and the work on the seven furanose derivatives.discussed next. The Five-membered Rings Similar studies were then c a r r i e d out on the p a i r of furanose d e r i v a t i v e s , 1 , 2 : 5 , 6-di - 0_-isopropylidene - 3 - 0-(trideuteromethyl) acetyl-cx-B-allofuranose (1+9) and i t s 3-C_-deuterated counterpart ( 5 0 ) . ( A l l of the furanose derivatives were studied i n deuterobenzene s o l u t i o n only.) P a r t i a l 100 MHz n.m.r. spectra of the r i n g protons of (1+9) and ( 5 0 ) are shown i n F i g . I V - 3 , i l l u s t r a t i n g the coupling pattern, and the extent of deuteration of H -3 . In spectrum IV - 3 B , the s i g n a l of H-3 has been completely removed from the spectrum, and the quartets of H-2 and H-l+.have been s i m p l i f i e d to doublets. The t r a n s i t i o n s are broadened s l i g h t l y , due to the unresolved coupling with the deuterium nucleus. Conventional three-pulse measurement of the r e l a x a t i o n times gave the values shown i n F i g . IV-1+; these show the expected s t e r e o s p e c i f i e i t y , and the q u a l i t a t i v e aspects, again, w i l l not be discussed. The differences i n r e l a x a t i o n rates caused by the deuterium s u b s t i t u t i o n at C - 3 , which give the co n t r i b u t i o n that dipole-dipole i n t e r a c t i o n with H-3 makes to the r e l a x a t i o n of the other protons are shown i n Table I I - 7 . Within experimental e r r o r , H-3 has no e f f e c t on the r e l a x a t i o n of H-1 or the two H-6's, as might be expected from inspection of a molecular model. The con t r i b u t i o n to the r e l a x a t i o n of H-1+ and H-5 i s small, but probably s i g n i f i c a n t , and to H-2 i s very l a r g e , nearly h a l f of the t o t a l r e l a x a t i o n r a t e . 108 F i g . IV-3. P a r t i a l 100 MHz proton n.m.r. spectra of 1,2:5,6-di-0-isopropylidene 3-0-(trideuteromethyl)acetyl-a - g-allofuranose (h9) QD , and of i t s counterpart deuterated at C-3 (50), (B}. Note the extent of deuteration, the s i m p l i f i c a t i o n of the m u l t i p l e t s of H-2 and E-h, and also the s l i g h t broadening of the H-2 and E-h signals i n (B), due to unresolved deuterium coupling. 2.3 2.3 Figure IV-k. Spin-lattice, relaxation times (seconds) for the protons of l,2:5,6-di-0-isopropylidene-3-0-acetyl-a-D-allofuranose (53), and two p a r t i a l l y deuterated analogues (1+9) and (50). Data are from conventional three-pulse experiments on 0.1 molar solutions i n CgDg at 33°C. Table TV-7. Relaxation rates f o r 1,2 :5 , 6-di - 0_-isopropylide - r 3 - 0 -(trideuteromethyl)acetyl-a-D-allofuranose (1+9) and i t s 3-C-deuterated counterpart ( 5 0 ) , f o r 0 .1 molar solutions i n deuterobenzene at 33°C, showing the contribution that dipole-dipole i n t e r a c t i o n with H-3 makes to the t o t a l r e l a x a t i o n rate of the other r i n g protons. (Data are f o r non-selective UBC experiments and are those shown as r e l a x a t i o n times i n F i g . IV-k) proton r e l a x a t i o n rate s e c . - 1 AR " R U 9 " R 5 0 per cent of rate due t o H-3 (h9) (50) H-1 0.127 0.121 0.006 5$ H-2 0.275 0.1U7 0.128 kl% H-3 0.26k — H-U 0.217 0.18k 0.033 15$ H-5 0.2U5 0.221 0 0.02k 10$ H - 6 d 0.U39 0.UU3 (-0.00U) ~0 H-6 0.510 0.56H (-0.05U) ~0 I l l A number.of tailored, excitation experiments were then carried out, as previously described, on the same sealed sample of (h9), this time including the exact counterpart of the UBC measurement, the contribution of H-3 to the relaxation rate of H-2. Fig. IV-5 shows some typical tailored excitation experiments on (h9) • f i r s t a non-selective inversion (IV-5A), then selective inversions of H-1 and H-2 (IV-5B and IV-5C respectively). The relaxation rates found in this series of experiments are given in Table IV-8, and the ratios and enhancements in Table IV-9. Within experimental error, the enhancement obtained by non-selective inversion is the theoretical maximum for each of the three protons, indicating that their relaxation is completely dipole-dipole in origin. The data from the pairwise experiments suggest that dipole-dipole interaction with H-2 causes nearly 75% of the relaxation of H-1 (i.e., 32/^3), and about h5% of the relaxation of H-3, while H^l and H-3 each cause about 30% of the relaxation of H-2. Dipole-dipole relaxation could also arise from interactions with the substituent methyl groups, and, for H-3, with E-h t H-5 and the two H-6's, depending on the configuration about the C-h to C-5 bond. (The deuteration study suggested a significant interaction of H-3 only with H-5). Of course, the original purpose was to compare the contribution of H-3 to the relaxation of H-2, as obtained by two independent methods. Comparison of the two results was, unfortunately, rather disappointing: U7% from selective deuteration and 30% by tailored excitation. Although such a difference is perhaps within the limits of experimental error, i t has not usually been necessary to invoke such generous ranges of. uncertainty.* *This is perhaps one place to indulge in a l i t t l e chemical rationalization of the discrepancy: the value of hl% from deuteration seems to be too high. As w i l l be discussed next, study of the four gluco-furanose derivatives suggests that something of the order of 20% of the relaxation rate of the ring protons is caused by dipole-dipole interactions with the isopropylidene methyl protons, and i t does not seem unreasonable to suggest that the situation might be similar for the allose derivatives. The enhancement study, and molecular models, suggest that dipole-dipole interactions of H-2 with H-1 and H-3 should be about equal. If the figure of U7% is correct, these interactions would then account for 9h% of the relaxation, of H-2, leaving only 6% for substituent methyl protons, and residual intermolecular effects. 112 Figure IV^5. Two-pulse Ti experiments on (H9), on a degassed 0.1 molar s o l u t i o n i n deuterobenzene at 28°C, carried.out at Varian Associates i n August. 197^. A strong s e n s i t i v i t y enhancement has been incorporated to reduce noise. A shows a non-selective i n v e r s i o n , B a s e l e c t i v e inversion of H-1, and C a s e l e c t i v e inversion of H-2. 112a 113 Table IV - 8 . Relaxation rates f o r 1 , 2 ; 5 , 6 - d i - 0 _ - i s o p r o p y l i d e n e - 3 - 0 -(trideuteromethyl)acetyl-a-D-allofuranose (h-9) obtained by the t a i l o r e d e x c i t a t i o n method at Varian Associates. Data are f o r a 0 . 1 molar s o l u t i o n i n deuterobenzene at 28°C. A l l rates were obtained by a least-squares computer f i t to ten data points, with delay times ( 0 . 2 , 0 . 6 . . . 3 . 8 ) seconds. proton r e l a x a t i o n rates (sec. non-select ive s e l e c t i v e H-1 + H-2 H-2 + H-3 H-1 0 . 1 U 7 0 . 1 0 3 0 . 1 3 6 H-2 0 . 2 9 6 0 . 2 0 2 0 . 2 3 1 0 . 2 3 3 H-3 0 . 2 8 3 0 . 1 6 8 0 . 2 1 8 I l l * Table IV-9. Ratios of relaxation rates, and enhancements for 1,2 :5 ,6-di-0_-isopropylidene-3-0_-(trideuteromethyl)acetyl-a-g-allofuranose (k9), obtained from the data in Table TV-8. proton Ratio of relaxation (enhancement) rates non-selective H-1 + H-2 H-2 + H-3 selective selective selective H-1 1.1*3 ( 0 . U 3 ) 1.32 (0.32) H-2 1.1*7 (0.U7) l.lk (o.ii*) 1.15 (0.15) H-3 1.68 (0.68) 1.30 (0.30) 115 Although our main concern was with the r e l a x a t i o n o f the r i n g protons, i t was obvious that the methyl groups of the acetoxy and isopropylidene moieties were also making a co n t r i b u t i o n , and to evaluate these, we studied the non-deuterated analogue of (1+9) and ( 5 0 ) ; i . e . , 1 , 2 ; 5 , 6-di - 0_-isopropylidene - 3 -O-acetyl-a-n-allofuranose ( 5 3 ) , and four forms of 1 , 2 ; 5 , 6 - d i - 0 - i s o p r o p y l i d e n e -3-0_-acetyl-a-g-glucofuranose, (5V) to ( 5 7 ) d i f f e r i n g only i n the extent of deuterium s u b s t i t u t i o n of the methyl protons. The T values of (53) to (57) were obtained by conventional experiments, and for c l a r i t y are shown with the s t r u c t u r a l formulae i n F i g . IV - 6 . (The non-selective r e l a x a t i o n times of (1+9) and (53) were shown i n F i g . IV-1 ;) . The differences i n the r e l a x a t i o n rates due to the acetate and isopropylidene methyl protons are c a l c u l a t e d i n Table IV - 1 0 for the glucose derivatives (5I+) to ( 5 7 ) , and i n Table IV -11 f o r the a l l o s e ones (1+9) and ( 5 3 ) . The data i n Table IV - 1 0 appear much more r e l i a b l e , as the four glucose derivatives provide two independent measurements of the contribution of the acetate and isopropylidene protons. Where the increments are small, and/or the agreement between the two sets of data i s poor, i t i s probably safe to regard the e f f e c t s as i n s i g n i f i c a n t . From Table IV - 1 0 for the glucose structures, i t i s apparent that the protons of the acetate group make only a small contribution to the r e l a x a t i o n of the r i n g protons; the only s i g n i f i c a n t contributions being to H - 3 , H-5 and po s s i b l y (Their e f f e c t on the r e l a x a t i o n of i f genuine, i s quite large.) The contribution to r i n g proton r e l a x a t i o n by the isopropylidene methyl groups i s l a r g e r , and the agreement between the two sets of data, except f o r H-6^, i s e x c e l l e n t . The isopropylidene methyl protons account for approximately twenty per cent of the r e l a x a t i o n of H-1 , H-2 and H-1+ (based on the non-deuterated compound ( 5 3 ) ) . The l a s t columns show-the contribution by a l l f i f t e e n methyl protons, which 1amounts'to something of the order of twenty per cent of the re l a x a t i o n of the r i n g protons. This type of study seems to have p o t e n t i a l as a probe of so l u t i o n conformation, although the r e s u l t s must be applied with 116 Figure IV-6. S p i n - l a t t i c e r e l a x a t i o n times (seconds) f o r the protons of l,2;5,6-di-0^-isopropylidene-3-0^-acetyl-a-g- glucofuranose (5*0 and three p a r t i a l l y deuterated analogues (55), (56) and (57), obtained from conventional three-pulse measurements on 0.1 molar solutions i n CgDg at 33°C. Table IV-10. Relaxation rates of the protons of 1,2;5,6-di-0_-isopropylidene-3-0-acetyl-a-g-glucofuranose (5I+) and some s e l e c t i v e l y deuterated forms (55), (56) and (57), and c a l c u l a t i o n of the contribution of substituent methyl protons to the relaxation of r i n g protons. Data are from conventional three-pulse experiments on 0.1 molar solutions i n deuterobenzene at 33°C, and are those shown as T values i n F i g . IV-6. rel a x a t i o n rates (sec. "'") contribution of acetat relaxation of r i n g pro e to tons. con t r i b u t i o n of i s o -propylidene methyls to rel a x a t i o n of r i n g protons. t o t a l c o ntribution of s u b s t i t -uent s. proton (5k) D0 (55) D3 (56) D12 (57) D15 (5k)-(55) (56)-(57) average % of t o t a l (5k)-(56) (55)-(57) average % of t o t a l (5k)-(57) % of t o t a l H-1 0.130 0.127 0.103 0.101+ 0.003 -0.001 0.001 2% 0.027 0.023 0.025 19% 0.026 20% H-2 0.221 0.209 0.168 0.166 0.012 0.002 0.007 3% • 0.053 0.01+3 0.01+8 22% 0.055 25% H-3 0.221 0.207 0.20U 0.191 0.011+ 0.013 0.013 6% 0.017 0.016 0.017 1% 0.030 lk% E-k 0.306 0.300 0.232 0.230 0.006 0.002 0.001+ 1% O.07I+ 0.070 0.072 2k% 0.076 25% H-5 0.278 0.25k 0.250 0.230 0.021+ 0.020 0.022 Q% 0.028 0.021+ 0.026 9% 0.01+8 11% H-6, d 0.533 0.k59 O.I+89 0.1+27 0.01k 0.062 0.068 13% 0.01+1+ 0.032 0.038 1% 0.106 20% H-6 u 0.61+7 0.559 O.56U 0.5kQ 0.088 0.016 0.052 8% 0.083 0.011 0.01+7 1% 0.099 15% H-6 av 0.590 0.509 0.527 0.1+88 0.081 0.039 0.060 10% 0.063 0.021 0.01+2 1% 0.102 11% 118 Table IV-11. Relaxation rates of the protons of 1,2 ;5 ,6-di-0_-isopropylidene-3-0_-acetyl-a-B-allofuranose (53) and i t s 3-0_-(trideuteromethyl) acetyl counter-part (1+9)» and calculation of the contribution of the acetate methyl protons to the relaxation of the ring protons. Data are for conventional three-pulse measurements of 0.1 molar solutions in deuterobenzene at 33°C, and are those shown as Tn values in Fig. TV-k. proton . relaxation rates (sec. - 1) contribution of acetate (53) D0 (k9) D 3 (53)-(U9) per cent of t o t a l H-1 0.139 0.127 0.012 9% H-2 0.272 0.275 (-0.003) 0% H-3 0.21k 0.26U 0.010 k% H-U 0.228 0.217 0.011 5% H-5 0.262 0.21+5 0.017 1% H-6 d 0.k62 0.1+39 0.023 5% H-6 u 0.600 0V510 0.090 15% H-6 av 0.531 0.1+75 0.056 10% 119 caution, as they probably represent an average of rotamers. Very l i t t l e data were a v a i l a b l e f o r allofuranose d e r i v a t i v e s , but Table IV -11 shows an attempt to c a l c u l a t e the contribution of the acetate methyl protons, by the differences i n the r e l a x a t i o n rates of (h9) and (53). The r e s u l t s , unfortunately, appear to represent experimental e r r o r , rather than r e a l e f f e c t s . In general, the increments seem too l a r g e , when compared with those for the glucofuranoses (5*0 to (57) - i t i s d i f f i c u l t to see how the s u b s t i t u t i o n could a f f e c t the r e l a x a t i o n rate of H -1 , f o r example, and while the e f f e c t on the r e l a x a t i o n of H-2 may be small, i t can hardly be negative. I show t h i s to emphasize how large experimental uncertainty can sometimes be, even when experiments are c a r r i e d out i n the standard way with the usual precautions and to emphasize the value of p a r a l l e l , independent data, where pos s i b l e . Conclusions The work i n t h i s chapter demonstrated, f i r s t , that the r e l a x a t i o n of protons of organic molecules i n d i l u t e degassed s o l u t i o n s , was predominantly dipole-dipole i n o r i g i n , a r e s u l t consistent with the conclusions suggested by e a r l i e r , more q u a l i t a t i v e work. Second, two independent methods f o r determining independent pairwise intramolecular dipole-dipole i n t e r a c t i o n s were compared, s e l e c t i v e deuteration, and t a i l o r e d e x c i t a t i o n . The r e s u l t s , although l a r g e l y compatible, were not completely s a t i s f a c t o r y , as the spectrum of (5l) was not suitable f o r carrying out the exact counterpart of the deuteration study, while, for the furanoses, the agreement, while p o s s i b l y within experimental e r r o r , was poor. -Two reasons were suggested for the problems encountered i n the t a i l o r e d e x c i t a t i o n studies, experimental e r r o r , and con-t r i b u t i o n to r e l a x a t i o n by meahanisms other than the dipole-dipole one; to these should be added a t h i r d , that r e l a x a t i o n i n these r i g i d molecules may w e l l have been aff e c t e d by c r o s s - c o r r e l a t i o n s of the l o c a l f l u c t u a t i n g f i e l d s , and thus 120 . the mathematical analysis may have been based on an inadequate model.. The t h i r d facet of t h i s chapter was an exploration of the e f f e c t of a change i n solvent, and the fourth, a determination of the extent to which i n t e r a c t i o n s with substituent methyl groups a f f e c t the r e l a x a t i o n of r i n g protons. These " f i n a l " studies, f a r from concluding the e a r l i e r i n v e s t i g a t i o n s , suggested a great deal of further work. For the pyranosides, several approaches could be taken to account more f u l l y f o r the r e l a x a t i o n of H-1 , the two H-2's, and H -3 ; the other protons, except for H-7 , are not amenable to study by. t a i l o r e d e x c i t a t i o n . While i t was not possible to perturb only one of the H-2's, an obvious experiment ( i n retrospect) would have been s e l e c t i v e inversion of the p a i r , with H-1 and with H -3 . An a l t e r n a t i v e approach would be preparation of the compound with deuteration of the e q u a t o r i a l H - 2 , so that H-2 could now ax be s e l e c t i v e l y perturbed. In a d d i t i o n , both deuteration and t a i l o r e d e x c i t a t i o n could be used to study the influence of the methoxyl protons on the r e l a x a t i o n of H-1 . For the furanoses, the errors associated with, the three a l l o s e d e r i v a t i v e s , ( M - 9 ) , ( 5 0 ) . and ( 5 3 ) , appeared high, judging from the c a l c u l a t i o n s concerning both H-3 and the acetate methyl protons, and i t might be worthwhile to repeat the UBC measurements, possibly .with d i f f e r e n t preparations. Due to the l i m i t e d quantity of the intermediate, 1 , 2 : 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - a - g - r i b o -hexofuranose - 3-ulose, a v a i l a b l e at the time, compounds (k9) and ( 5 0 ) were prepared on a very small scale, and were d i f f i c u l t to p u r i f y and c r y s t a l l i z e . More generally - and these, to some extent, form some of the general conclusions of t h i s thesis - monitoring the e f f e c t of methyl groups on the r e l a x a t i o n of the r i n g protons, as opposed to study of the methyl protons themselves, appears a.promising avenue for i n v e s t i g a t i o n , both f o r accounting for the r e l a x a t i o n of r i n g protons, and for studying s o l u t i o n conformations. The d i s p o s i t i o n of rotamers, for example, could be a l t e r e d by a small change i n temperature, choice of solvent, concentration, and even degree of deuteration. 121 Relaxation studies, using s e l e c t i v e deuteration, t a i l o r e d , e x c i t a t i o n , or the s t a t i c nuclear Overhauser e f f e c t , could provide a s e n s i t i v e probe of the e f f e c t of each parameter on the o v e r a l l energy balance. Further, carbon-13 studies should be included i n in v e s t i g a t i o n s of. proton r e l a x a t i o n , as these add a s e n s i t i v e probe of molecular motion to the probe of geometry and conformation which proton r e l a x a t i o n provides. On a more pragmatic l e v e l , the experimentor who: wishes to use t a i l o r e d e x c i t a t i o n must become f a m i l i a r with a l l sources of experimental e r r o r , i n p a r t i c u l a r , those associated with non-exponential decay, and the s u f f i c i e n t determination of i n i t i a l slopes. When a series of s t r u c t u r a l l y s i m i l a r molecules i s to be studied, a preliminary concentration study should be c a r r i e d out, so that , i n the main i n v e s t i g a t i o n , one may be confident that intermolecular'interactions have been reduced to.a n e g l i g i b l e l e v e l . In t h i s regard, the routine use of s e n s i t i v i t y enhancement should reduce the minimum concentration l e v e l s that can be e a s i l y studied without time-averaging. F i n a l l y , the wide choice of i n i t i a l conditions, inc l u d i n g a r b i t r a r y pulse strength, which t a i l o r e d e x c i t a t i o n o f f e r s , should provide a much:fuller experimental base f o r those intere s t e d i n the d e t a i l e d r e l a x a t i o n behaviour of the i n d i v i d u a l t r a n s i t i o n s i n a coupled m u l t i s p i n system, and thus stimulate the development of more comprehensive theory. CHAPTER V SOME APPLICATIONS AM) EXTENSIONS OF. FOURIER TRANSFORM RELAXATION STUDIES Introduction The development'of Fourier transform methods has made possible a whole new range of n.m.r.' experiments. At the simplest l e v e l , the f a c t that spectra can he obtained so r a p i d l y has greatly f a c i l i t a t e d the observation of very small amounts of m a t e r i a l , and of systems that are changing with time. Fourier transform technology has also provided the f i r s t reasonable method for measuring the r e l a x a t i o n times associated with the complex spectra one obtains for organic molecules i n s o l u t i o n . At one l e v e l , t h i s provides a very u s e f u l probe df both molecular geometry and molecular motion i n s o l u t i o n , and at a simpler l e v e l suggests many novel' means, some of which are already becoming routine, for s i m p l i f y i n g and extracting more information from n.m.r. spectra. In a continuous-wave (cw) n.m.r. experiment, a weak observing r a d i o -frequency f i e l d i s scanned slowly through the appropriate frequency range, e x c i t i n g each t r a n s i t i o n i n turn. For routine operation with a 100 MHz instrument, one scan takes of the order of f i v e hundred seconds, and a concentration of about 0.1 molar i s required for adequate signal-to-noise; even then, compromises must be made among the various instrumental s e t t i n g s . The signal-to-noise r a t i o can be improved by f i l t e r i n g out some of the noise, but t h i s causes some loss of r e s o l u t i o n ; the best r e s o l u t i o n i s obtained by scanning very slowly, but then the resonances saturate r e a d i l y (a property of t h e i r rather long r e l a x a t i o n times), and the observing f i e l d must be reduced, again reducing the s i g n a l - t o -noise. While i t i s possible to observe strong peaks, such as methyl resonances, down to concentrations of 0.01. M with.one scan, e s t a b l i s h i n g the best combination of parameters, e s p e c i a l l y f o r linewidth measurements, i s tedious, and the r e s u l t s poor. It i s also worth noting, that unless further compromises can be 122 123 accepted, i f one wishes to.time-average a number .of.scans i n the cw mode, each scan requires the f u l l f i v e hundred seconds or so established f or the best sing l e scan. In the FT mode, a l l of the resonances are excited at once, and the t o t a l time needed to acquire one scan i s the pulse time ( n e g l i g i b l e ) plus the a c q u i s i t i o n time, the length of time that the free induction decay (F.I.D.) i s recorded. Like the cw experiment, the FT experiment necessitates a number of decisions which sometimes c o n f l i c t . In theory, the F.I.D. i s an exponential decay extending to i n f i n i t y ; i n pr a c t i c e the a c q u i s i t i o n time (AT) i s t y p i c a l l y one to four seconds. In p r i n c i p l e then, one can time-average by pulsing every second or two, and the immense improvement in' s i g n a l - t o - n o i s e that can be obtained by pulsing one thousand times i n as many seconds, over scanning once i n cw mode for one thousand seconds has been documented^ , 2-®°. However, the spins have a f i n i t e r e l a x a t i o n time, and just as saturation occurs i n a cw scan at too high a power l e v e l , i n the FT.mode, s u f f i c i e n t r e l a x a t i o n time must be allowed between pulses to achieve the maximum undistorted s i g n a l height.for a l l the t r a n s i t i o n s . Two approaches are possible here: the pulse can be l e s s than ninety degrees, the spins are l e s s removed from equilibrium, and the pulse i n t e r v a l can be shortened.. However, the s i g n a l f o r each t r a n s i e n t w i l l be smaller as the maximum response i s obtained from a 9 0 - (or 2 7 0 ) degree pulse. A l t e r n a t i v e l y , a f u l l 90-degree pulse can be used and a waiting period (i n a d d i t i o n to the a c q u i s i t i o n time) introduced between pulses. Each spectrum w i l l then be i t s f u l l height, and fewer transients w i l l be needed. In p r a c t i c e , one often pulses rather r a p i d l y , allowing some saturation of the slower-relaxing spins, while achieving maximum signal-to-noise, f o r the f a s t e r - r e l a x i n g ones. The re l a x a t i o n times of protons of large organic molecules i n so l u t i o n are usua l l y l e s s than two or three seconds, so that except.for very d i l u t e solutions i t i s possible to gain good signal to noise i n a reasonable time without saturating any signals (except perhaps the so l v e n t ) . The problem i s much more 12k serious i n carbon-13 work, where the natural abundance and s e n s i t i v i t y are so low, and, while the r e l a x a t i o n t i m e s ^ ' of those carbons with d i r e c t l y bonded hydrogens are short (of the order of one second), the r e l a x a t i o n times of t e r t i a r y carbons, such as i n carbonyl functions can be many times greater, even i n large molecules. Rapid pulsing w i l l r e s u l t i n the signals f or these carbons (which also lack the extra i n t e n s i t y gained from the Overhauser e f f e c t i n noise-decoupled spectra) having a very reduced i n t e n s i t y , or disappearing altogether. A number of approaches have been t r i e d to overcome t h i s problem: 90 l e t t i n g them have reduced i n t e n s i t y , o ften a u s e f u l a i d i n s p e c t r a l assignment , the use of a pulse sequence to a r t i f i c i a l l y restore the magnetization to i t s 9 1 - 9 3 equilibrium value , and chemically, the use of a paramagnetic r e l a x a t i o n 9 U - 9 6 reagent to shorten r e l a x a t i o n times. I t i s the l a s t method that seems the most generally appli c a b l e . Having decided on the number of transients to acquire, the pulse strength, and the i n t e r v a l between pulses, one must choose the a c q u i s i t i o n time, which has a c r i t i c a l bearing on the r e s o l u t i o n or s e n s i t i v i t y of the f i n a l transformed spectrum. As the free induction decay proceeds i t s i n t e n s i t y becomes lower, while the noise l e v e l remains constant.- Thus,.the e a r l y part of the F..I.D. contributes a great deal to the signal-to-noise r a t i o , while the information c a r r i e d i n the l a t e r stages i s necessary to achieve the best r e s o l u t i o n . A longer a c q u i s i t i o n time r e s u l t s i n better and better r e s o l u t i o n , u n t i l increasing noise n u l l i f i e s the improvement. However, with a given a c q u i s i t i o n time, i t i s possible to mathematically weight the f i r s t or l a s t stages of the F.I.D. p r i o r to Fourier transformation i n order to enhance e i t h e r the s e n s i t i v i t y or'the r e s o l u t i o n of the f i n a l spectrum*. Enhancement of s e n s i t i v i t y . w i t h . l o s s of r e s o l u t i o n may prove useful f or very d i l u t e samples, but we have.found i t more convenient to increase the number of t r a n s i e n t s . * 9 7 - 1 0 1 There are a v a r i e t y of other methods^ f o r mathematical manipulation, which w i l l not be discussed here. 125 When re s o l u t i o n enhancement i s used, there i s a large increase i n the noise l e v e l , so that t h i s i s usually applied a f t e r the a c q u i s i t i o n of very many tr a n s i e n t s . F i n a l l y , the gated and computer-controlled nature of the FT mode of f e r s many p o s s i b i l i t i e s for s p e c t r a l manipulation, and t h i s , combined with a general understanding, of s o l u t i o n r e l a x a t i o n phenomena, becomes a powerful t o o l for organic n.m.r., and further suggests the' chemical modification of natural r e l a x a t i o n times to amplify the power of the instrumental methods. While many of the points discussed i n t h i s chapter have been applied to carbon-13 studies, t h e i r a p p l i c a t i o n f o r proton magnetic resonance has lagged behind. In the course of these studies on r e l a x a t i o n , we found many of the same methods to be useful for.proton n.m.r.; t h i s chapter documents some of the improvement that can r o u t i n e l y be obtained. Instrumental Methods Probably the best-known a p p l i c a t i o n of Fourier transform n.m.r. i s the routine improvement possible i n s e n s i t i v i t y , i l l u s t r a t e d i n F i g . V - l for a sample of l , 2,3,H-tetra - 0-acetyl - 3-D-ribopyranose. Spectrum A i s the r e s u l t of one FT scan, equivalent to the best cw si n g l e scan, and B shows the summation of one hundred FT t r a n s i e n t s , with a t o t a l accumulation time of only f i v e hundred seconds. The point here i s that'the t o t a l amount. of' t h i s r e l a t i v e l y high molecular weight compound i n a 5 mm n.m.r. tube was only one milligram, and c l e a r l y , the spectrum of even a smaller, amount of material could have been obtained i n the same time with a m i c r o c e l l . F i g . V-1B also i l l u s t r a t e s a l i m i t a t i o n * i n the time, averaging of proton spectra: the broad hump across the _ This f i g u r e , and several others i n t h i s chapter i l l u s t r a t e further what i s given and taken away i n FT spectroscopy. For t h i s complex spectrum, the presence.of foldover peaks l i m i t e d the smallest sweep width to f i v e hundred hertz, but the transformed spectra can be plotted.on any l a r g e r (or smaller) h o r i z o n t a l scale, so that coupling constants may be more e a s i l y measured. For F i g . V - l , the expansion factor was 2 . 0 ; the sweep width was 500 hz, but the spectra were'plotted as 250 hz. The l i m i t here i s that as the expansion V D O J A i • • • i i , , i • i 1 1 1 , 1 1 1 1 1 , i 1111111. • • i , 11,., i 11111 Figure V-1. P a r t i a l 100 MHz proton n.m.r. spectra of l,2,3 ,U-tetra-0_-acetyl-S-2-ribopyranose i n acetone-dg at 33°C QD shows the r e s u l t of a sing l e t r a n s i e n t ; \JS) shows that of the Fourier transform summation of 100 t r a n s i e n t s , r e q u i r i n g 500 seconds. >-> 127 factor increases beyond a c e r t a i n point,:the number of data points becomes i n s u f f i c i e n t to.' c l e a r l y . define the peak shape, and no further advantage i s gained. We have - used expansion factors of.up t o . f i v e , however. I t i s also worth pointing out that one can have many, spectra f o r the p r i c e of. one time-accumulation.; the o r i g i n a l F.I.D. i s stored on tape, and then f o r each type of spectrum, the computer can make a copy of the F.I.D., transform i t with or without s e n s i t i v i t y or r e s o l u t i o n enhancement f a c t o r s , and trace a v a r i e t y of p l o t s from each transformed F.I.D. A l a s t advantage of the FT mode i s that any sweep-width can be used, and one i s not r e s t r i c t e d to the standard settings found i n cw operation. baseline a r i s e s from the'material from which the probe i s constructed. Several approaches to t h i s problem would be to concentrate the same amount of material i n a m i c r o c e l l , or one of the recently developed 1 mm sample tubes 102 which contain 5 y l of s o l u t i o n , and so reduce the number of t r a n s i e n t s ; use the computer to subtract the spectrum of a reference (probably a tube of the solvent), s i m i l a r to the operation of a double-beam spectrometer; use a probe with a lower background; f i n a l l y , i f the of the background protons i s much 99 shorter than those o f the sample, as i s quite l i k e l y the case, delayed FT spectroscopy might be a simple instrumental s o l u t i o n , a l b e i t introducing some lo s s of s i g n a l height. F i g . V-2 i l l u s t r a t e s the improvement that can be obtained by r e s o l u t i o n enhancement (along with'time-averaging), again for a sample of 1,2,3 ,U-tetra-0_-acetyl-3 - g-ribopyranose. Spectrum A shows the r e s u l t of one cw scan for a 0.1M s o l u t i o n . The Fourier transform spectrum shown i n F i g . V-2B has a r e s o l u t i o n 103 comparable with that of a spectrum published previously , while that i n V-2C shows a remarkable increase i n r e s o l u t i o n , and was obtained by using the r e s o l u t i o n enhancement subroutine of a standard Varian FT programme. An intermediate a c q u i s i t i o n time of 3.0 seconds was used, and. the r e s o l u t i o n enhancement factor v a r i e d to f i n d the optimum value, with the o r i g i n a l F.I.D. stored on tape. C l e a r l y v i s i b l e i n t h i s spectrum are a d d i t i o n a l small s p l i t t i n g s , i n a l l resonances but H-2 and H-5a, a r i s i n g from long-range couplings. For example, each of the p r i n c i p l e components of the H-1 doublet.is now v i s i b l e as a quartet, which indicates that H-1 i s coupled with H-2,. H-3, H-U and H-5e. Most of the r i n g 128 Figure V-2. P a r t i a l 100 MHz proton n.m.r. spectra of l,2,3,^-tetra-0-acetyl-3 -g-ribopyranose i n acetone-d.5 (0.1M) at k2°. [A} , Shows the r e s u l t of a single continuous-wave scan, with a sweep-width of 250 Hz and a t o t a l scan time of 1000 seconds. The spectrum i n CH3 shows the Fourier transform summation of 100 t r a n s i e n t s , each with an a c q u i s i t i o n time of 3-0 seconds and a delay time between successive transients of 12 seconds; the t o t a l time used to obtain t h i s spectrum was 1500 seconds. The spectrum given i n © was derived from the same free induction decay s i g n a l as [B) but a resolution-enhancement weighting f a c t o r of 1.0 units was applied immediately p r i o r to the Fourier Transform. The marked increase i n r e s o l u t i o n and i n the noise l e v e l should be noted. 129 protons, i n f a c t , are extensively coupled, but H-5a shows only a simple geminal and v i c i n a l coupling. A plo t of the resonances of H-5a and H-5e, made at the same sweep-width and pl o t t e d with a f i v e - f o l d expansion f a c t o r i n F i g . V-3 provides a cl e a r e r p i c t u r e of the r e s o l u t i o n obtained i n t h i s way: the long-range couplings i n the H-5e resonance are only *0.5 hz. There i s a l i m i t to the increase i n r e s o l u t i o n p o s s i b l e , and t h i s i s r e l a t e d to the a c q u i s i t i o n time. As mentioned previously, as eit h e r the a c q u i s i t i o n time i t s e l f , or the weighting given to the l a t e r stages of the F.I.D. i s increased, there i s an increase i n the amount of noise introduced into the transformed spectrum. The e f f e c t of varying the weighting factor i s i l l u s t r a t e d i n the set of resonances shown i n F i g . V - U . As the weighting f a c t o r i s increased, there i s a steady improvement i n the r e s o l u t i o n from spectrum A to D, accompanied, as always, by an increase i n the n o i s e - l e v e l . The spectrum i n F i g . V-l+E shows the e f f e c t of applying too sharp a weighting function; only a small change causes the t r a n s i t i o n between an acceptable n o i s e - l e v e l and complete l o s s of s i g n a l . Another u s e f u l a p p l i c a t i o n of FT n.m.r. i s to sim p l i f y spectra, by combining two- or three-pulse sequences with a knowledge of re l a x a t i o n times. In the T^ experiment, as has been discussed i n Chapter I the magnetization of a l l the n u c l e i i s inverted by a 180-degree pulse and at one point during the recovery to equilibrium, the magnetization i n the Z-direction i s zero, (the " n u l l " point) and the a p p l i c a t i o n of a 90-degree pulse at t h i s point produces no s i g n a l . As the rel a x a t i o n times of protons and other n u c l e i vary widely, t h e i r n u l l point i s reached at d i f f e r e n t times, and t h i s becomes the basis of a powerful method to s i m p l i f y spectra. One of the problems of proton n.m.r. i s the presence of r e s i d u a l peaks of deuterated solvents; t h i s i s e s p e c i a l l y troublesome f o r d i l u t e solutions, and f o r aqueous solutions where i t i s often d i f f i c u l t to e f f i c i e n t l y replace the exchangeable protons of the sample beforehand. Apart from obscuring 130 Figure V-3. Both of these traces are expansions of the H-5e and H-5a resonances of l,2,3 ,U-tetra-0_-acetyl-B - r j-rihopyranose given previously i n F i g . V-2. [A] corresponds to the trace i n F i g . V-2B, and [Bj to that of F i g . V-2C. The very s i g n i f i c a n t improvement i n the r e s o l u t i o n of a long-range coupling (s0.5 hz) i n the H-5e resonance i s apparent. The fa c t that the half-height width of the H-5e t r a n s i t i o n s i n LB} i s l e s s than that of the H-5a t r a n s i t i o n s implies the presence of a small, but unresolved coupling into H-5a. Figure Y-k. The H-1 resonance o f l , 2 , 3 , U - t e t r a - 0 - a c e t y l - 3 - 2 - r i b o p y r a n o s e i n acetone-d 6 s o l u t i o n (0.1 M) at h2°. A l l the s p e c t r a were based on the f r e e i n d u c t i o n decay s i g n a l r e s u l t i n g from 100 t r a n s i e n t s w i t h an a c q u i s i t i o n time of 3.0 seconds and a pu l s e - d e l a y time o f 12 seconds ( t o t a l time 1500 seconds). {T| i s the r e s u l t of d i r e c t F o u r i e r Transformation, w i t h no r e s o l u t i o n enhancement. (B} was obtained by a p p l y i n g a r e s o l u t i o n enhancement f a c t o r of 1.5; © a f a c t o r of 1.0; Cfi) a f a c t o r of 0.8; and d3 a f a c t o r o f 0.7. The pro g r e s s i v e in c r e a s e i n both r e s o l u t i o n and n o i s e - l e v e l should be noted. In (D] the H-1 resonance i s c l e a r l y v i s i b l e as a doubletted-quartet w i t h a long-range c o u p l i n g o f *0.5 Hz. The spectrum shown i n [JD i l l u s t r a t e s the e f f e c t of a p p l y i n g too sharp a weighting f u n c t i o n . 132 regions of i n t e r e s t , the presence of one very much l a r g e r peak (usually HOD) causes problems with dynamic range; i . e . , i n order to accommodate the large peak, the computer tends to scale down the smaller peaks as more scans are accumulated. There are also problems i n phasing the f i n a l spectrum. The HOD peak can be s h i f t e d chemically, by the addition of t r i f l u o r o a c e t i c a c i d , or by changing the temperature, but both are perturbations of the sample, and fl u c t u a t i o n s of the operating temperature of a large spectrometer lead to poor s t a b i l i t y and r e s o l u t i o n . A number of double resonance and pulse techniques have been developed to overcome t h i s p r o b l e m 1 ^ 1 0 9 , 1 2 8 ^ ^ e s i m p ] _ e s t being the a p p l i c a t i o n of the two-pulse s e q u e n c e 1 ^ described above, u t i l i z i n g the large dif f e r e n c e i n re l a x a t i o n times between the solvent and the organic solute. The protons of the HOD molecules i n a D^O s o l u t i o n have a much longer r e l a x a t i o n time than the protons of a large organic molecule, such as a sugar. The l a t t e r have a much shorter c o r r e l a t i o n time, and many more dipole-dipole i n t e r a c t i o n s . In an undegassed s o l u t i o n the HOD peak commonly has a r e l a x a t i o n time of ten to f i f t e e n seconds, compared to two or three seconds f o r the solute peaks, and i t i s a simple matter to choose a delay time such that the HOD peak i s at i t s n u l l point, while the sample peaks have recovered f u l l i n t e n s i t y . This i s i l l u s t r a t e d , f o r f a i r l y r e a l i s t i c conditions i n F i g . V-5 for a 1% w/v s o l u t i o n of g-glucose i n 99'1% DgO; ^ e B-glucose was not previously exchanged with B^O. Eight transients were time-averaged i n order to increase the signal-to-noise, and the t o t a l accumulation time was f i f t e e n minutes. A few other points about t h i s procedure are worth mentioning. F i r s t , i n t e g r a t i o n can be c a r r i e d out on the f i n a l spectrum, and t h i s w i l l be accurate i f the n u l l i s accurate, and i f a l l the solute protons have recovered f u l l i n t e n s i t y (or recovered to the same degree). I f some with a longer r e l a x a t i o n time have not recovered f u l l y , then t h e i r i n t e n s i t i e s w i l l be reduced. Second, the rather long time needed f o r t h i s pulse sequence can be reduced by using 107 somewhat l e s s than a 180-degree pulse i n i t i a l l y , and f i n a l l y , there need not 1 3 3 Figure V-5. P a r t i a l l y relaxed spectra of a so l u t i o n {l% w/v) of D-glucose i n D 20 (99-7$) at h2°; the g-glucose was not l y o p h i l i z e d with D 20 p r i o r to t h i s experiment. (Xj shows a single-pulse, Fourier-transform spectrum. The spectra (B) and [Cj were the r e s u l t of eight a c q u i s i t i o n s each, using a two-pulse sequence with pulse delays set at 12.8 and 13.0 sec, r e s p e c t i v e l y . These two spectra were d e l i b e r a t e l y chosen to show how f i n e l y the delay time has to be selected i f complete elimination of the water peak i s to be achieved. 13U be a very.large d i f f e r e n t i a l i n the values to extract a good spectrum of the solute. It has been pointed out that even a diffe r e n c e factor of l.h should 107 s u f f i c e , and time-averaging can be used to b u i l d up the reduced i n t e n s i t y of the peaks of i n t e r e s t . Problems of poor i n t e g r a t i o n are more than compensated by the vast improvement i n the o v e r a l l q u a l i t y of the spectrum. In p r i n c i p l e , there i s no reason why the d i f f e r e n t i a l s i n the r e l a x a t i o n times o f the protons i n the same molecule can not also be used to s i m p l i f y spectra. We have observed values ranging over a f a c t o r of ten i n large ( i . e . molecular weight 3 0 0 - U 0 0 ) organic molecules. In the case of mono-saccharide d e r i v a t i v e s , analysis of the ~*"H n.m.r. spectra i s often complicated by the overlap of the H-5. and H-6 resonances (which have quite short r e l a x a t i o n times) with other r i n g protons. F i g . V -6 i l l u s t r a t e s the use of a two-pulse sequence to s i m p l i f y the spectrum of 3,h , 6-tri - 0_-acetyl-l - 0_-benzoyl - 2-C-chloro-2-deoxy-3-D-glucopyranose ( U l ) , one of the molecules discussed i n Chapter I I I . Because of the f o u r f o l d d i f f e r e n t i a l between the T values of H-2 and H -6g a delay could be chosen such that the H-2 resonances were at t h e i r n u l l point, while the H-6 resonances had recovered f u l l i n t e n s i t y . Another v a r i a t i o n of t h i s method allows detection of more slowly-r e l a x i n g resonances. In the three-pulse sequence, the spectra are displayed as (M -M ) , where M i s the equilibrium magnetization and M i s the magnetization O "t o t at time " t " a f t e r the l 8 0-degree pulse. As M i s a constant, while M varies O "C from -M (at zero delay time) to +M (at equilibrium), the value of (M -M ) O O O "C decreases from an i n i t i a l value of + 2 M q to zero. In t h i s mode, the more qui c k l y - r e l a x i n g resonances decay r a p i d l y , and "disappear" while the more slowly-relaxing ones s t i l l have a large i n t e n s i t y . The spectra shown i n F i g . V -7 for the same molecule i l l u s t r a t e an a p p l i c a t i o n of the three-pulse sequence to observe the H-2 resonances now, while the H-6 resonances have decayed to zero i n t e n s i t y . Note that i n the l a s t spectrum, the i n t e n s i t i e s of H-1, H -3 and K-k are also reduced, and H-5 has disappeared. Figure V-6. Par t ia l ly relaxed part ia l 100 MHz proton n.m.r. spectra of 3,H,6-tri-0_-acetyl-l-0-benzoyl-2-chloro-2-deoxy-6-g-glucopyranose (Ul), for a degassed 0.1 molar solution in deuterobenzene at~H2°C, using a two-pulse sequence. [SJ shows the normal Fourier transform spectrum, and [BJ the result of a two-pulse sequence with a delay-time of 3.0 seconds between the 180- and 90-degree pulses„ 136 He 2 H 2 Figure V-7. P a r t i a l l y relaxed p a r t i a l proton n.m.r. spectra of 3,H,5-tri-0-acetyl-l-0_-benzoyl-2-C_-chloro-2-deoxj r-B-D-glucopyranose ( U l ) , f o r a degassed 0.1 molar s o l u t i o n i n deuterobenzene at using a three-pulse sequence. (A) The spectrum obtained with the delay-time " t " = 0.1 sec; QD with t = 0.5 s e c ; [iD with 5=1-0 s e c ; Q)j with 5 = 3.0 sec. The o v e r a l l i n t e n s i t y o f a l l t r a n s i t i o n s decreases through the series [A) to CJTJ . Note however, that H-5, H - 6 i and H - 6 2 , which have the shortest r e l a x a t i o n times, have e f f e c t i v e l y decayed to zero i n t e n s i t y i n [Dj , so that the four t r a n s i t i o n s of H-2 are now c l e a r l y resolved. 137 One f i n a l a p p l i c a t i o n of the FT spectrometer i s the greatly improved study of systems that are changing with time11'"'. The main advantage here i s that a l l the peaks are monitored instantaneously, and provided the requirements f o r r e l a x a t i o n between pulses are met, scans can be repeated very r a p i d l y . I t i s also possible to pulse at short i n t e r v a l s , store a large number of F.I.D.'s on magnetic tape, so that the Fourier transformation and p l o t t i n g can be c a r r i e d out l a t e r (coming soon to UBC). The example i l l u s t r a t e d i n F i g . V - 8 , an experiment c a r r i e d out under manual c o n t r o l , i s the mutarotation of a f r e s h l y mixed s o l u t i o n of the calcium chloride complex of a - p - a r l o s e 1 1 1 . F i g . V-8A shows the f a l l i n i n t e n s i t y with time of the H-la t r a n s i t i o n s , and V - 8 B the corresponding r i s e i n the H-13 t r a n s i t i o n s . This p a r t i c u l a r experiment i n -corporated another FT bonus, i n that the data were obtained by using a two-pulse sequence to p a r t i a l l y n u l l the large r e s i d u a l HOD peak. This peak was large because the concentration of the sample, which had not been previously l y o p h i l i z e d was only 0 . 1 5 molar, and, while at the temperature of t h i s experiment, i t d i d not overlap the anomeric peaks, i t s spinning sidebands, and the d i s t o r t i o n introduced i n the baseline completely obscured the peaks of H-13 and d i s t o r t e d those o f H-la. P a r t i a l n u l l i n g o f the water peak allowed both t r a n s i t i o n s to be monitored cleanly: the scatter remaining i n the plots i s the r e s u l t of the i n e v i t a b l y changing r e s o l u t i o n of a f r e s h l y mixed sample. The scatt e r i s l a r g e r f or the H-13 resonances, which have a narrower linewidth, and could be reduced by p l o t t i n g i n t e g r a l s rather than peak heights. <• However, at the time of t h i s work, the i n t e g r a l s obtained i n the FT mode were not r e l i a b l e , and manual evaluation of peak areas i s extremely time-consuming. Another approach would be to include a s e n s i t i v i t y enhancement f a c t o r before Fourier transformation which has the e f f e c t of broadening the l i n e s , and reducing t h e i r s e n s i t i v i t y to changes i n r e s o l u t i o n . (The r e l a x a t i o n experiments c a r r i e d out at Varian Associates, described i n Chapter IV, incorporated a strong s e n s i t i v i t y enhancement f a c t o r , which c l e a r l y improved the q u a l i t y of the data). With 138 Figure V - 8. Mutarotation of a f r e s h l y mixed s o l u t i o n of 0 . 1 5 M a-g-allose: calcium chloride i n 9 9 - 9 6 % D 2 0 at 3 3 ° C The i n t e n s i t i e s were obtained by adding the peak heights of the two t r a n s i t i o n s of each anomeric proton. 139 c a r e f u l c o n t r o l of parameters, i t should he possible to monitor the mutarotation even at temperatures where the peaks coincide with the HOD peak.* Chemical Methods The f i r s t part of t h i s chapter i l l u s t r a t e d some ways to extend Fourier transform methods by using r e l a x a t i o n phenomena; one now asks i f there i s further p o t e n t i a l i n the chemical manipulation of proton r e l a x a t i o n times. Williams"*""^ and co-workers and LaMar and F a l l e r " ^ 9 have pointed out** that the _ — ———— There i s a large body of work on the e f f e c t s of other paramagnetic ions; f o r a general review and discussion, see reference 7 7 ' association of a gadolinium (III) ion with an organic molecule should lead to systematic changes i n the s p i n - l a t t i c e r e l a x a t i o n times of that molecule: these changes should have the form A(T 1) a r where " r " i s the distance between the gadolinium nucleus and the proton under l l 8 i n v e s t i g a t i o n . Although t h i s r e l a t i o n s h i p i s apparently quite simple f o r organic substrates having a single locus for as s o c i a t i o n with the gadolinium * I t i s worth noting here that t h i s method would be suitable f or a r e -i n v e s t i g a t i o n of the c l a s s i c problem of m u t a r o t a t i o n 1 1 4 i n sugars. The process i s slow enough that manual con t r o l of timing and p l o t t i n g i s s u f f i c i e n t , and i f necessary, the HOD peak can be cancelled. The only perturbation i s that the process w i l l occur i n D 20 rather than H 20. It should be possible to f i t a l l four rate constants for the case of simple mutarotation, o^acyclic^p 1. For some Sugars, such as glucose, two complementary experiments can be c a r r i e d out, as both pyranose forms can be obtained i n pure c r y s t a l l i n e form, and two of the four rate constants can be obtained from i n i t i a l rates alone. There are of course many other ways of studying t h i s phenomenon, such as p o l a r i m e t r y 1 1 1 * and p o l a r o g r a p h y 1 1 5 , but FT n.m.r. seems to o f f e r the maximum p o s s i b i l i t y of determining a l l the rate constants with minimum perturbation. I n t e r e s t i n g l y , the mathematics 1 1 7 f o r the k i n e t i c s i t u a t i o n X£Y£Z were worked out i n 1910 for the express p u r p o s e : 1 1 6 "The i n q u i r y , of which the r e s u l t s are now described was begun i n 1903... It was hoped that the study of the equations•for the action XtYtZ might throw l i g h t on the question of the existence i n aqueous solutions of dextrose of a substance intermediate between a- and g-glucose." iho i o n , i t s p o t e n t i a l f or f a c i l i t a t i n g studies of molecules with several binding s i t e s i s much l e s s obvious. Prompted by the e a r l i e r i n t e r e s t of t h i s laboratory i n the a s s o c i a t i o n T8—80 of lanthanide ions with carbohydrate d e r i v a t i v e s as a t o o l both to s i m p l i f y spectra and to probe s o l u t i o n geometry, and also by my own e f f o r t s to harness s p i n - l a t t i c e r e l a x a t i o n phenomena, I studied the e f f e c t s of very small quantities ( t y p i c a l l y 10 ^ to 10 ^ molar equivalents) of gadolinium ( i l l ) ions on the proton values of some sugars i n aqueous s o l u t i o n . One object of these experiments was to increase the d i f f e r e n t i a l between the T^-values of i n d i v i d u a l protons of a sugar, and thus extend the effectiveness of the p a r t i a l r e l a x a t i o n approach to s p e c t r a l assignments. The f i r s t measurements were made with sugars which were not a n t i c i p a t e d to have any s p e c i f i c locus f o r a s s o c i a t i o n with gadolinium, and constitute a sort of c o n t r o l study. Table V - l summarizes the s p i n - l a t t i c e r e l a x a t i o n rates of the anomeric protons of a 0 . 2 M s o l u t i o n of g-glucose. In the absence of gadolinium, there i s a systematic d i f f e r e n t i a l between the r e l a x a t i o n rates of the two anomeric protons, which r a p i d l y decreases with in c r e a s i n g molar pro--3 portions of gadolinium. The rates become i d e n t i c a l at about 1 . 5 x 10 molar equivalents of added gadolinium, and continue to increase as the gadolinium concentration i s increased s t i l l f u r t h e r , i n d i c a t i n g that the r e l a x a t i o n of the anomeric protons i s now dominated by the paramagnetic contribution from the . gadolinium ions which are a s s o c i a t i n g i n an essentially•random fashion with the two sugar species. Turning to a p a i r of simple disaccharides, the non-reducing anomeric proton of both maltose and c e l l o b i o s e has a f a s t e r r e l a x a t i o n rate than the anomeric protons at the reducing end. (Tables V - 2 , V-3). As before, addition of gadolinium causes a l l of the proton r e l a x a t i o n rates to increase: the e f f e c t s on the reducing protons, however, are more marked. A s i m i l a r e f f e c t was found f o r maltotriose (Table V-h) and i t i s again apparent that non-specific binding ll+i Table V - l . Experimental data showing the e f f e c t of added gadolinium (III) n i t r a t e on the s p i n - l a t t i c e r e l a x a t i o n rates of the anomeric forms of g-glucose. The s o l u t i o n of g-glucose was 0.2 molar i n D 20, and the experiments were c a r r i e d out at 1+2°C. Concentration (Molarity) Relaxation Rates (sec 1 ) Ratio l a IB [ C d I ] : i ] x l O U [ G d I 3 I I ] x l 0 3 [D-glucose] H-la H-16 0 0 0.22 0.37 0.60 0.20 0.10 0.21+ 0.1+0 0.60 o.Ho 0.20 0.33 0.50 0.66 '0.60 0.30 0.1+2 0.57 O.7U 1.0 0.50 0.57 0.70 0.81 1.6 0.80 0.79 0.83 0.95 2.6 1.3 1.2 1.1 1.1 3.0 1.8 l.U. 1.1+ 1.1 10.0 5.0 3.9 3.9 1.0 ± 1 + 2 Table V - 2 . The e f f e c t of added gadolinium (III), n i t r a t e on the s p i n - l a t t i c e relaxation.rates of the. anomeric protons of a s o l u t i o n of maltose. The maltose s o l u t i o n was 0 . 2 molar i n D 2 0 , and was not degassed. The ambient temperature was 1+2°C. H-la and H-13. are the reducing anomeric protons, and H-l*a i s the non-reducing anomeric proton. Concentration (Molarity) Relaxation Rate (sec 1 ) Ratio R n / R 1 * l a l * a [ G d I ] C I ] x l O U [ G dI I 3 : ] x i o 3 [Maltose] H-la H-13 H-1* a 0 0 0.1+3 0 . 8 1 1 . 2 0 . 3 7 0 . 2 0 . 1 0.1+9 0 . 9 U 1 . 2 0 . 3 9 0 . 5 0 . 2 5 0 . 5 8 1 . 1 1 . 3 0.1+1+ 0 . 8 0.1+ 0 . 7 0 1 . 0 1 . 3 0 . 5 3 1 .5 0 . 7 5 1 . 3 1 . 7 1 . 7 0 . 7 8 2 . 2 1 . 1 1 . 8 , 2 . 1 1 . 9 0 . 9 6 3 - 0 1 . 5 2 . 3 2.U 2 . 2 . 1 . 1 8 . 0 1+.0 5 . 0 5 . 8 + 1 . 5 1+.6 1 . 1 H+3 Table V-3. The e f f e c t of added gadolinium ( i l l ) n i t r a t e on the s p i n - l a t t i c e r e l a x a t i o n rates of the anomeric protons of c e l l o b i o s e , f o r a 0.2 molar s o l u t i o n i n D 20 at 1+2°C. The s o l u t i o n was not degassed. H-l*3 r e f e r s to the non-reducing anomeric proton. Concentration Relaxation Rate (Molarity) (sec 1 ) Ratio [ G d I I I ] x l 0 1 + [ G d ] x i 03 H-la H-13 H-l* 3 ~R 0/R, x.0 [Cellobiose] 13 1*3 0 0 0.52 1.1 2.1 0.50 0.2 0.10 0.53 1.0 2.2 0.U6 0.5 0.25 0.56 0.92 2.1 0.1+3 1.0 . 0.50 0.65 1.1 2.1 0.51 2.0 1.0 1.1 1.5 2.1 0.70 2.7 1.1+ 1.9 2.0 2.1+ 0.81+ 1+.0 2.0 2.9 2.9 2.6 1.1 10 5.0 7.1 8±3 1+±1 2.2 ihh Table Y-k. The e f f e c t of gadolinium ( H i ) n i t r a t e on the s p i n - l a t t i c e r e -l a x a t i o n rates of the anomeric protons of maltotriose, for a O.lh molar so l u t i o n i n D 2 0 at k2°C. The maltotriose s o l u t i o n was not degassed. H-l*a r e f e r s to the non-reducing anomeric protons. Concentration (Molarity) LMaltotrioseJ Relaxation Rate ( s e c - 1 ) H-la H-10 H-l*a Ratio l * a l a 0 0 o .6o 1 . 3 1 . 8 o.h 0 . 3 o . 6 i 1 . 3 1 . 8 1 . 6 1 . 2 1 . 8 2.k 2 . 3 3 . 0 2 . 2 2 . 9 3 . 6 2 . 9 6 . 0 k.k k.6 k.6 k.O 10 l.k , 7 . 1 1 1 . 0 5 . 6 0 . 3 3 0.3k 0 . 8 0 1 . 0 1 . 1 1 . 3 1U5 of gadolinium ions by. a sugar produces.substantial increases i n the proton r e l a x a t i o n rates of that sugar. These changes, however, do not seem to have any diagnostic p o t e n t i a l ; indeed the i n t r i n s i c R - d i f f e r e n t i a l s associated with intramolecular.dipole-dipole r e l a x a t i o n are destroyed i n these experiments. In some experiments, addition of large amounts of gadolinium appeared to again cause a divergence i n the r e l a x a t i o n r a t e s , and indeed t h i s i s apparent f o r the highest concentration experiments i n Tables V-2, V-3 and V-h. The problem i s that at these, and even higher gadolinium concentrations, the r e s i d u a l water peak becomes so broadened that i t precludes accurate measurements of the i n t e n s i t i e s of the anomeric proton resonances, and t h e i r own broadening of course reduces the signal'^to-noise.* _ - — There are a few other i n t e r e s t i n g points and p o s s i b i l i t i e s for future work associated with these, experiments. F i r s t , the HOD peak must be dealt with. While a rather sophisticated pulse sequence has been developed to combine s o l v e n t - n u l l i n g with the standard two-pulse T^-experiment 1 0 5, i t was not a v a i l a b l e at the time at UBC, and i t also requires a d i f f e r e n t i a l between the r e l a x a t i o n rates of the solvent and solute protons. However, as water binds, more strongly to gadolinium than to these sugars, the r e l a x a t i o n rate of the HOD peak also increases as the concentration of gadolinium i s increased, and soon exceeds the rate f o r the anomeric'protons of the sugar. In f a c t , f o r some of the experiments the anomeric resonance was measured a f t e r most of the i n t e n s i t y of the HOD peak had decayed (using the three-pulse sequence). Simple a l t e r n a t i v e s would be the addition of t r i f l u o r o a c e t i c a c i d to s h i f t the HOD peak downfield, or a large increase i n temperature, which would have the advantage of making a l l the r e l a x a t i o n times longer and thus increasing the accuracy of the measurements. Second., when the data i n the Tables were p l o t t e d as Rj vs. G d ( l l l ) concentration, there were c l e a r differences i n the general appearances of the p l o t s , suggesting perhaps differences i n the way the various sugars i n t e r a c t with both water and gadolinium ion. Studies measuring the changes i n the re l a x a t i o n rates of both anomeric and HOD protons might provide i n t e r e s t i n g , a l b e i t q u a l i t a t i v e i n s i g h t into the behaviour of these sugars i n aqueous s o l u t i o n . In view of recent studies u t i l i z i n g proton r e l a x a t i o n rates of water i n large b i o l o g i c a l s y s t e m s 8 1 - 8 5 model studies of very simple systems might be appropriate. Attention was next d i r e c t e d to g - a l l o s e . Angyal"'""1"'" had observed a highly s p e c i f i c a s s o c i a t i o n of cx-D-allose with europium and other metal ions, and we an t i c i p a t e d that gadolinium should have a marked e f f e c t on the proton r e l a x a t i o n rates of that species, and much l e s s e f f e c t on the 3-D-allopyranose. The data p l o t t e d i n F i g . V-9 bear t h i s out i n a most convincing manner: Figure V-9. V a r i a t i o n of the r e l a x a t i o n rates of the anomeric protons of D-allose i n B^OT" as a function of the molar r a t i o of added gadolinium ( i l l ) n i t r a t e . The a l l o s e concentration was 0.2 molar, and the temperature was h2°C. -3 addition of 1.5 x 10 molar equivalents of gadolinium enhances the r e l a x a t i o n of the a anomeric proton more than f i f t y - f o l d , whereas that of the 6 anomeric proton i s changed l e s s than t h r e e - f o l d . This change i s accompanied by a substantial change i n the line-width of the H-la resonance, which eventually becomes so broadened that i t i s removed from the spectrum. ( F i g . V-10). A s i m i l a r e f f e c t i s observed for the furanose forms (Figs. V-9 and V-10) and again the a form shows p r e f e r e n t i a l complexing with the gadolinium. These studies imply that attempts to use gadolinium ions to increase the d i f f e r e n t i a l between the s p i n - l a t t i c e r e l a x a t i o n times of i n d i v i d u a l protons o f sugars i n aqueous sol u t i o n are only l i k e l y to be f r u i t f u l i f there i s some s p e c i f i c locus of a s s o c i a t i o n between the sugar and the 111-113 gadolinium. The extensive studies of Angyal and others have shown that two suitable binding s i t e s are an a x i a l - e q u a t o r i a l - a x i a l arrangement of three hydroxyl groups on a six-membered (chair-form) r i n g , and also a c i s - c i s arrangement of three hydroxyl groups on a five-membered r i n g , the arrangements found i n the pyranose and furanose forms, r e s p e c t i v e l y , of a-g-allose. This type of geometry i s l i k e l y to occur i n a number of carbohydrate systems, and indeed the associations can be so strong as to cause r e v e r s a l of normal 113 sol u t i o n conformational preference , or change the outcome of a re a c t i o n from one anomeric form to another"*""'""'". Indeed, i n the same way that "broadening" and " s h i f t i n g " lanthanide reagents can be combined i n one experiment"'""'"^' ^ , one could combine a high concentration of a non-paramagnetic "binding" cati o n , such as calcium ( I I ) , to produce a large chemical perturbation, with a very small amount of a f r e e l y exchanging competitive "broadening" c a t i o n , such as G d ( l l l ) , to e f f e c t i v e l y remove one set of resonances from the spectrum. This type o f experiment i s i l l u s t r a t e d i n F i g . V - l l again f o r the calcium chloride complex of a-g-allose. F i g . V - l l A i s a spectrum of the equilibrium mixture, using a two-pulse sequence to almost completely n u l l the HOD peak, and sixteen transients to increase the signal-to-noise. B shows the e f f e c t of the addition Figure V - 1 0 . Proton 100 MHz n.m.r. signals of the anomeric protons of g-allose, for a 0 . 2 molar, so l u t i o n i n D 2 0 ( 9 9 . 9 6 $ ) at k2°C. TjD i s the spectrum for the ,pure s o l u t i o n . \B) was measured a f t e r the addition of i x l O - 3 molar equivalents of gadolinium ( i l l ) n i t r a t e ; [Cj a f t e r addition of 2><10 - 3 molar equivalents of gadolinium n i t r a t e . (This i s not the same.sample for which the data i n F i g . V - 9 were obtained; the broadening i n t h i s sample was greater, for the same molar r a t i o of added gadolinium ( i l l ) , than the broadening observed i n the o r i g i n a l s o l u t i o n . The type of broadening i s the same, however.) 149 Figure V - l l . 100 MHz 1H n.m.r. spectra of f u l l y mutarotated a - g - a l l o s e : calcium c h l o r i d e , 0.2.M i n 99-96% D 2 0 at 33°C. The peak assignments are o-furanose 1, B-furanose 2 , a-pyranose 3, B-pyranose h and HOD 5. QQ shows the normal spectrum, obtained by averaging l 6 t r a n s i e n t s , and incorporating a two-pulse sequence with a delay-time of 9.3 seconds to n u l l the r e s i d u a l HOD peak. In B , 0.5 x 10~ 3 molar equivalents of gadolinium (III) have been added, and the spectrum i s the sum of 2k t r a n s i e n t s . In [C] , the G d ( l l l ) concentration has been doubled to 1.0 x 1 0 - 3 molar equivalents, and 50 scans were used. In both [Bj and [J5) , the HOD peak has i t s f u l l i n t e n s i t y . For spectrum [D) , f i f t y transients were again used, but the HOD peak was reduced i n i n t e n s i t y by using a three-pulse sequence, with a delay-time of 0.3 seconds. 150 of 0 . 5 x 10 molar equivalents of G d ( l l l ) , and again the spectrum has been time-averaged. In t h i s case, the r e l a x a t i o n rate of water, which also binds to gadolinium, was comparable to that of the sugar protons, so that i t was not possible to n u l l the r e s i d u a l HOD peak. The H-1 doublet of the a-pyranose form has broadened much more than that of the B-pyranose form, and a small peak v i s i b l e as a shoulder on the downfield side of H-la i n spectrum A i s _3 emerging. In spectrum C, the G d ( l l l ) concentration was doubled, to 1 . 0 x 10 molar equivalents, and f i f t y t ransients were time-averaged. The s i g n a l due to H-1 of the a-pyranose has broadened so that i t has been completely removed from the spectrum, while a peak with a smaller unresolved s p l i t t i n g s , probably H-1 of the B-furanose can e a s i l y be distinguished. At t h i s gadolinium concentration, the r e l a x a t i o n rate of the HOD peak was f a s t e r than that of the sugar protons, so an attempt was made to remove i t from the spectrum, t h i s time using the three-pulse sequence. However, the d i f f e r e n t i a l was not l a r g e , and a great deal of noise and baseline d i s t o r t i o n was introduced. I t was possible to at l e a s t reduce the r e l a t i v e i n t e n s i t y of t h i s peak by using a short delay of 0 . 3 seconds i n the three-pulse sequence. The r e s u l t of t h i s i s shown i n spectrum D, which was made using the same settings and number of transients as C. While the signal-to-noise of the sugar peaks i s somewhat lower, the o v e r a l l q u a l i t y of the spectrum i s much be t t e r , and i t i s now possible to just resolve the s p l i t t i n g i n the B-furanose peak. In p r i n c i p l e , i t should even be possible to observe only the spectrum of the anomer which binds to gadolinium, the a form, by subtracting the broadened spectrum from the unbroadened one"^^. CHAPTER VI EXPERIMENTAL METHODS Nmr Measurements These studies were c a r r i e d out on a Varian XL - 1 0 0 . ( l 5 ) spectrometer, equipped with a Varian model 6 2 0 L - 1 6 K Fourier transform accessory, and also a magnetic tape u n i t (Line Tape, model C 0 6 0 0 ) , so that sets of p a r t i a l l y - r e l a x e d spectra could he accumulated, stored on tape, and p l o t t e d l a t e r . This was p a r t i c u l a r l y u s e f u l when time-averaging was required; the tape f a c i l i t y was also used for resolution-enhanced spectra, as a copy of the o r i g i n a l free induction decay could he stored, and then d i f f e r e n t r e s o l u t i o n enhancement factors applied to f i n d the optimum r e s o l u t i o n obtainable. The lock s i g n a l for field/frequency control was provided by the deuterium resonance of the deuterated solvent, and unless otherwise i n d i c a t e d , a l l experiments were at the normal operating temperature of the probe. This was U2°C for most of the work (Chapters I I , I I I and V), but i n A p r i l , 1 9 7 ^ , the probe temperature was lowered to 33°C, so that the l a s t s e r i e s of compounds (Chapter IV) were studied at t h i s new temperature. Relaxation measurements were made using the l6*K programmes provided by Varian Associates; i . e . , 8K of programme and 8K of data storage. The three-pulse sequence 1 5 [.. . 1 8 0 ° ...delay.. . 9 0 ° ...DELAY...9 0 ° ...] which could be time-averaged i f necessary, was u s u a l l y used. The long "DELAY" was s u f f i c i e n t to allow the magnetization o f a l l the spins to return to equilibrium; r e l a x a t i o n times were determined by varying the "delay" from a small f r a c t i o n of "T^" to several times T^. As o u t l i n e d i n Chapter I, t h i s three-pulse sequence produces spectra i n the form (MQ-M^), where M q i s the equilibrium magnetization, and M^ the magnetization a f t e r a delay time t ; as the delay time t i s increased, (M -M,) decreases from an i n i t i a l value of +2M to zero. The r e l a x a t i o n time v o t o 151 152 T i s obtained from the slope (-1/T ) of a plo t of ln(M -M ) vs t . This was -L _|_ O "C done by using a computer programme to c a l c u l a t e a simple f i r s t - o r d e r l e a s t -squares f i t of the data, and also to p l o t the f i t t e d l i n e together with the data points on the l i n e p r i n t e r , so that the amount of scatte r or non-exponential decay could be assessed. (These c a l c u l a t i o n s were c a r r i e d out on an IBM 36O-67 computer situated i n the UBC Computer Centre). For the i n i t i a l survey (Chapter I I ) , the sample concentration was 10% w/v for the monosaccharides and monosaccharide d e r i v a t i v e s , and 5% w/v f o r the d i - and oligosaccharides. Most l a t e r work was done at lower concentrations, u s u a l l y 0.1 molar, or 1%> w/v. Water-soluble samples were l y o p h i l i z e d and dissolved i n 99-96% E^O (Norell Chemical Co.), while the other samples were run i n deuterated chloroform, acetone or benzene. The prepared solutions were f i l t e r e d into 5 mm n.m.r. tubes f i t t e d with BIO ground-glass j o i n t s , degassed by s i x freeze-pump-thaw cycles, and sealed under vacuum. Only the samples i n the gadolinium study (Chapter V), the two carbon-13 samples (Chapter I I ) , and the K l e b s i e l l a 2h polysaccharide sample were not degassed. For the gadolinium study, sample concentration was 0.2 molar, except f o r maltotriose, which was O.lU molar f o r reasons of s o l u b i l i t y ; aliquots of gadolinium (one to ten m i c r o l i t e r s ) were added from stock solutions of 0.1, 0.01 and 0.001 molar gadolinium n i t r a t e i n D 20. Errors Some experimental guidelines were evolved to reduce the many possible contributions to experimental error i n these r e l a x a t i o n studies. As the presence of even small amounts of dissolved oxygen or other paramagnetic species i n the sample can shorten proton r e l a x a t i o n times considerably, t h i s was eliminated as f a r as possible by degassing and sea l i n g the samples, and using reasonably pure compounds. To minimize v a r i a t i o n s i n concentration, which d i r e c t l y a f f e c t r e l a x a t i o n times, samples were prepared accurately i n 153 volumetric flasks;some.solvent,.however, was probably l o s t during degassing cycle s . This would have been more serious f o r the non-aqueous samples, but i t was a small e f f e c t , and could be expected to be reasonably constant from one sample t o another. To reduce errors from the spectrometer, i t was only used f o r r e l a x a t i o n measurements when i n a very stable condition, so that r e s o l u t i o n (and probe temperature) could be maintained constant throughout the experiment. This point i s extremely important - to compare peak heights from one spectrum to another, the l e v e l of r e s o l u t i o n must have been the same f o r each. The three-pulse sequence developed at V a r i a n 1 ^ does help to reduce the e f f e c t of minor f l u c t u a t i o n s i n r e s o l u t i o n , e s p e c i a l l y when time-averaging i s involved; i n a d d i t i o n , the order of delay-times was u s u a l l y randomized. The sets of s t r u c t u r a l l y - r e l a t e d molecules were u s u a l l y run with s i m i l a r o f f s e t s , sweep-widths, and sets of delay times, and the length of the 360° pulse i n the frequency range of i n t e r e s t was checked f o r each T^ experiment. For proton experiments, the-sweep-width was u s u a l l y f i v e hundred hertz or l e s s , so that there was no appreciable decrease i n pulse strength away from the c e n t r a l frequency. For the p a r t i c u l a r Fourier transform accessory being used t h i s d i d become noticeable as the sweep-width was increased beyond one thousand hertz. For the carbon-13 measurements, where the sweep-widths were twelve to f i f t e e n hundred h e r t z , i t was necessary to use a compromise pulse length which was a l i t t l e too long for the u p f i e l d peaks, and a l i t t l e too short for the downfield ones. F i n a l l y , the long "DELAY" of the three-pulse sequence was u s u a l l y generous, t y p i c a l l y at l e a s t f i v e or s i x times the longest T^ of i n t e r e s t . This d i d seem necessary because, although a delay of two or three times T^ should be s u f f i c i e n t to restore equilibrium for purely exponential recovery, proton r e l a x a t i o n i s often non-exponential, the deviation most often being i n the d i r e c t i o n of a "slowing down" from purely exponential decay as the recovery proceeds. 15U The signal-to-noise r a t i o was not u s u a l l y a problem, as an adequate l e v e l could be obtained for most 0.1 molar samples with only one or two t r a n s i e n t s , and time-averaging could be used r o u t i n e l y when necessary. Serious compromises between signal-to-noise and the amount of time a v a i l a b l e were necessary, however, f o r the two carbon-13 T measurements (Chapter I I ) , and the measurements of some very small proton s i g n a l s , i n p a r t i c u l a r the H-1 resonances of the a forms of g - a l l o s e . Some experimental s c a t t e r , of course, always remains; to compensate, one may acquire many t r a n s i e n t s , or use the same amount of time to acquire more data points, and both approaches were used. Calculations of "T " t y p i c a l l y included data from twenty to t h i r t y values of the delay time t and very o c c a s i o n a l l y a l a r g e r number. Another problem occurred when the peaks of i n t e r e s t were close to another large s i g n a l , such as a methyl or HOD resonance. This was e s p e c i a l l y troublesome for the studies of sugars with gadolinium, where the signals of the anomeric protons could become barely d i s c e r n i b l e shoulders of the HOD peak, and the l o c a t i o n of the baseline was doubtful. Where to draw the baseline was a r e a l problem i n many experiments, and a source of e r r o r ; t h i s was o c c a s i o n a l l y resolved by p l o t t i n g data obtained with both a " s t r a i g h t " baseline, and a baseline drawn along the r i s e of the l a r g e r peak, and then using the "best" value, or, i f both plots were equally bad, the average. (This was only necessary for the most concentrated of the gadolinium samples). In a d d i t i o n to these more or l e s s random e r r o r s , there are systematic and l e s s t r a c t a b l e errors inherent i n the thing being measured - the recovery of a set of spin-coupled m u l t i p l e t s to equilibrium a f t e r a non-selective 180-degree pulse. F i r s t , the T^ may be a composite value, i n c l u d i n g con-t r i b u t i o n s to r e l a x a t i o n other than the intramolecular dipole-dipole (proton-proton) one. For samples free of d i s s o l v e d oxygen or other paramagnetic species, i n d i l u t e s o l u t i o n i n a magnetically i n e r t solvent, where hydrogen i s 155 the only nucleus with an appreciable magnetic moment, and i n the absence of chemical exchange, the dominant source of proton r e l a x a t i o n appears to be the intramolecular dipole-dipole mechanism. However,- there may s t i l l be small intermolecular contributions to r e l a x a t i o n from other solute molecules, and even from the deuterated solvent. (As the magnetogyric r a t i o of deuterium i s only one-sixth that of hydrogen, i t s e f f e c t on proton r e l a x a t i o n i s very greatly-reduced.) In a d d i t i o n , the s p i n - r o t a t i o n mechanism may contribute to the r e l a x a t i o n of methyl protons. Even when t h i s i s overcome j there remains the problem of cross-relaxation i n spin-coupled systems, which can cause the T^ values o f the i n d i v i d u a l t r a n s i t i o n s of a m u l t i p l e t to d i f f e r considerably, and t h e i r recovery to deviate progressively from a simple exponential as the delay time increases. F i n a l l y , whether c r o s s - c o r r e l a t i o n s of the f l u c t u a t i n g l o c a l f i e l d s may have any e f f e c t on the dipole-dipole r e l a x a t i o n among the protons of a r i g i d molecule remains unknown. While the values w i t h i n a m u l t i p l e t could be i d e n t i c a l within experimental e r r o r , there were often large v a r i a t i o n s ; f o r example, the T^ values for the two t r a n s i t i o n s of 3-D-galactose penta-acetate (±0% w/v i n deuterobenzene at h2°C) were 1 . 8 5 and 2 . 7 1 seconds, a di f f e r e n c e of f i f t y per cent. The v a r i a t i o n due to non-exponential behaviour, on the other hand, seemed to be up to twenty per cent f o r non-methyl protons; f o r methyl protons, the deviation could be much greater, so that even an i n i t i a l slope was d i f f i c u l t to define. To examine t h i s e f f e c t , p l o t s from a si n g l e non-selective T^ experiment were truncated a f t e r i ncreasing delay times " t " , and the apparent slopes compared. F i g . VI - 1 shows t y p i c a l v a r i a t i o n s i n the values obtained when data from successively longer delays are included i n the c a l c u l a t i o n s . In t h i s example, the H-la t r a n s i t i o n s of a ten per cent s o l u t i o n of D-glucose, i t may be noted that a f t e r an i n i t i a l decrease, there i s a steady increase i n the values obtained as the plot i s c a r r i e d out to nine seconds (approximately twice T ), and the t o t a l v a r i a t i o n i s about twenty per cent. F i g . VT-2 shows 156 T, i-apparent (seconds) Figure VI-1. V a r i a t i o n of apparent values obtained as data from longer and longer delay.times are included i n the c a l c u l a t i o n , f o r the H-1 resonances of a 10% w/v solution of g-glucose i n 99.96% D20 at k2°C. Upper trace: average value for the two H-la t r a n s i t i o n s . Lower trace : values for the downfield t r a n s i t i o n of H-1P. (The u p f i e l d t r a n s i t i o n was obscured by the HOD peak.) 157 Figure VI - 2 . Typical decay p l o t of ln(Mo-Mt) versus t f o r the u p f i e l d t r a n s i t i o n of H-la of a solu t i o n of g-glucose (ioi w/v) i n D 2 0 (99-96$) measured at h2°C. Note the "slowing down" from purely exponential decay at the longer delay times of approximately one T j - period and greater. 158 the a ctual p l o t of a l l the data for one of these H-la t r a n s i t i o n s ; the deviation from the i n i t i a l slope becomes apparent at delay times longer than "T^" seconds. In general, apparent values increase as longer times are included, but t h i s i s by no means always true, and i n t h i s p a r t i c u l a r example, the r e s u l t s f o r the H-18 t r a n s i t i o n show only a steady decrease. Similar d e t a i l e d c a l c u l a t i o n s were made for the H-la resonances of a ten per cent so l u t i o n of D-mannose, and with the pl o t length v a r i e d from 1 . 6 to 1 2 . 0 seconds, the apparent increased from 5-5 to 6 . 3 seconds, again, a range of about twenty per cent. It i s important to note that within experimental error the r a t i o of T_^ to T appears to be independent of the time period of the p l o t . In the l i g h t of t h i s sort of v a r i a t i o n (which was found for many of the compounds studied), and i n the absence of any comprehensive theory for the d e t a i l e d r e l a x a t i o n behaviour of a simultaneously perturbed, say, seven-spin system, a standard procedure was adopted to at l e a s t increase the consistency of the r e s u l t s . * For each t r a n s i t i o n , a T^ was calculated using as much data _ This i s not a completely a r b i t r a r y choice, as the deviation from exponential behaviour becomes more pronounced as r e l a x a t i o n p r o c e e d s 3 9 , and also the signal-to-noise becomes progressively worse at very long delay times. as p o s s i b l e , often i n c l u d i n g delay times much la r g e r than "T ". This apparent T^ was noted, and then the data were r e p l o t t e d , truncated at a maximum delay time of ( l . l T ), although for some sets of data, values of (l.O T ) or ( 1 . 2 T ) were chosen. The r e s u l t from t h i s truncated pl o t i s the experimental T or that has been reported throughout, and where the r e s u l t s d i f f e r e d from one t r a n s i t i o n of a spin-coupled m u l t i p l e t to another the average of the i n d i v i d u a l rates has been reported. When the measurements were made and the data processed i n t h i s way, the consistency and r e p r o d u c i b i l i t y of the r e s u l t s were high, and the maximum normal experimental error appeared to be of the order of ±5%. This was the range, f o r example, observed for the 10% w/v so l u t i o n of D-glucose; the i n i t i a l 159 determination gave T = U .36 sec. and T = 2 . 2 6 sec. while the corresponding J-0t 1 P values from a re-determination on the same sealed sample were U .89 and 2 . 5 3 s e c , a diff e r e n c e of f i v e per cent. The errors were often, i n f a c t , l e s s . As another example, r e l a x a t i o n times f o r the anomeric, protons of D-altrose, measured on two per cent solutions of both a c r y s t a l l i n e sample, and of a syrup prepared i n the laboratory from methyl h,6-0_-benzylidene-a-g-altropyranoside, d i f f e r e d by only three per cent. Again, r e l a x a t i o n times determined for protons with the same stereochemical environment i n s t r u c t u r a l l y r e l a t e d molecules often agreed to within 0 . 1 - 0 . 2 seconds. This was the case, f o r example, for the f i v e halo-sugars ( 3 9 - ^ 3 ) discussed i n Chapter I I I , and f o r the p a i r of branched-chain nitro-sugars (58 and 59) i n the Appendix. Sources of Samples Deuterated solvents were purchased from several chemical companies, and were used without further p u r i f i c a t i o n . Many of the compounds were obtained from commercial sources, or were laboratory samples; these were p u r i f i e d by r e c r y s t a l l i z a t i o n i f necessary. The following samples were g i f t s from others: D-allose (K.N. Slessor and J.D. Stevens), g-talose (L.D. Hayward), a-D-allose: calcium chloride complex (J.D. Stevens); methyl 3-D-mannopyranoside (G.M. Bebault), a-D-idose penta-acetate (H. Paulsen), D-galactose penta-acetate as the a and 3 anomers and the 3-penta-(deuteromethyl) acetate (L. Evelyn), methyl U , 6 - 0 -benzylidene - 2-deoxy - 3-nitromethyl-a - g-ribo-hexopyranoside, and i t s x y l o -counterpart (A.R. Rosenthal), K l e b s i e l l a 2h b a c t e r i a l polysaccharide (G.G.S. Dutton), the seven i n o s i t o l s (S.J. Angyal), c i s - and trans - H-t-butyl-cyclo-hexanol (Z.M. Akhtar), and vindolene (J.P. Kutney). A d d i t i o n a l D-talose was purchased from Fluka, and a d d i t i o n a l D-allose from Calbiochem. Many of the d i - and oligosaccharides were purchased from P f a n s t i e h l Laboratories, Inc. The samples of 3,h , 6-tri - 0^-acetyl-l - 0_-benzoyl - 2-C_-bromo - 2-deoxy - 3 -p-glucopyranose ( 3 9 ) , the corresponding a anomer (Ho), the 2-C-chloro i 6 o derivatives (kl) and (k2)and the 2-C-bromo-a-manno d e r i v a t i v e (k-3) were 6k prepared by J . Manville i n t h i s laboratory . The melting points of these halogenated d e r i v a t i v e s were ( 3 9 ) l 6 l - l 6 2 ° , (kO) 112-113°, (hi) l 6 0 - l 6 l ° , (k2) 1 5 3 - 1 5 U 0 , and (1+3) l 6 8 - l 6 9 ° C . 3 - 0-Acetyl-l , 2 : 5 , 6-di - 0-isopropylidene-a-D-glucofuranose (5*+), and the corresponding D-allose compound ( 5 3 ) were laboratory samples. The melting points were: for (5V) 5 6 - 5 8 ° , lit T° 5 9 - 6 0 ° , and for (53) 7 ^ . 5 - 7 6 ° , l i t 7 0 7 U - 7 H . 5°C. Syntheses Many reactions were monitored by t h i n - l a y e r chromatography, using glass plates coated with " S i l i c a Gel H f o r TLC" (E. Merck AG, Darmstadt, Germany). The plates were run i n f i v e per cent methanol i n chloroform or two to one ether/toluene, and developed by s u l f u r i c a c i d spray and heat. Some products were p u r i f i e d on s i l i c a columns, using Mallinckrodt S i l i c a r CC-7 "Special f or Column Chromatography". A l l evaporations were c a r r i e d out under reduced pressure, and unless otherwise in d i c a t e d , " l i g h t petroleum" r e f e r s to the f r a c t i o n with b o i l i n g range 6 5 - 1 1 0°C. g-Idose ( 9 ) D-Idose was prepared from 0-D-glucose penta-acetate v i a the 67 acyloxonium ion by the method of Paulsen . Nine grams of f r e s h l y d i s t i l l e d antimony pentachloride i n dichloromethane were added to a s t i r r e d s o l u t i o n of ten grams o f 3-D-glucose penta-acetate i n dichloromethane, cooled i n an i c e - s a l t bath. Moisture was excluded from the r e a c t i o n . A f t e r the addition, the c o o l i n g bath was removed, and within f i f t e e n minutes, a white s a l t p r e c i p i t a t e d . The reaction was s t i r r e d f o r another t h i r t y minutes, and then f i l t e r e d , and the s o l i d washed with dichloromethane and dry ether. This product, the hexachloro-antimonate o f the D-ido acetoxonium ion was immediately hydrolyzed i n sodium 161 acetate and water. A f t e r twenty minutes, the so l u t i o n was extracted with chloroform, and the extract washed with water, drie d over calcium c h l o r i d e , and evaporated to-a syrup, which was a mixture of 1 , 2 , 3 , 6 - and l , 2 , 3 , U - t e t r a -0_-acetyl-a-D-idopyranose. . This was acetylated with ten ml of pyridine and ten ml a c e t i c anhydride at f i f t y degrees for t h i r t y minutes. The solvents were co-evaporated with ethanol, and the a-D-idose-penta-acetate c r y s t a l l i z e d from ethanol. Total y i e l d of penta-acetate, 1 . 2 grams, mp 9 3 - 9 ^ . 5 ° ( l i t . 9U-95°C). The penta-acetate was de-acetylated by a standard procedure^ using methanol/sodium methoxide, and the syrupy D-idose obtained was treated with amberlite IR -120 r e s i n to remove traces of antimony p r i o r to the T^ measurement. D-Altrose ( 6 ) This sugar was prepared from methyl k , 6 - 0_-benzylidene-a-D-altro-68 pyranoside (Paul S t e i n e r ) , following a procedure i n the l i t e r a t u r e . It was f i r s t hydrolyzed to methyl a-D-altropyranoside using s u l f u r i c acid/water, peracetylated with s u l f u r i c a c i d / a c e t i c anhydride, and deacetylated with sodium methoxide/methanol. The a l t r o s e did not c r y s t a l l i z e . The T^ values of a two per cent so l u t i o n of the preparation agreed to within three per cent with the values'obtained from a s i m i l a r s o l u t i o n of a c r y s t a l l i n e sample of D-altrose. (Trideutero)methyl-D-glucoside (a plus 3) ( l 6 a ) A mixture of the a and 3 anomers i n a r a t i o of approximately 69 o 5 : 3 5 was prepared according to a method m the l i t e r a t u r e , using methanol-d^ and amberlite IR -120 ion-exchange r e s i n . The mixture was treated with decolourising carbon i n ethanol, and evaporated to give a white s o l i d , which was used without further p u r i f i c a t i o n f o r the n.m.r. experiment. 162 3 - 0 j-(Trideuteromethyl)acety^ (55) TO This was prepared by a method analogous to a l i t e r a t u r e method for ( 5 * 0 . One gram of 1 , 2 : 5 , 6-di - 0_-isopropylidene-a-D-glucofuranose (diacetone glucose) was s t i r r e d overnight at room temperature with one ml (perdeuteromethyl) acetic anhydride and three ml p y r i d i n e . The solvent was repeatedly co-evaporated with toluene, and the r e s u l t i n g s l i g h t l y orange syrup was dissolved i n chloroform, treated with charcoal, d r i e d and evaporated. Light petroleum was added, and a seed c r y s t a l of ( 5 * 0 . The product c r y s t a l l i z e d i n large square 70 p l a t e s , mp.5 7 . 5 - 5 9 . 5 ° , l i t e r a t u r e . v a l u e ' f o r (5*0 5 9 - 6 0 ° . 3 - 0-(Trideuteromethyl)acetyl-l , 2 : 5 , 6-di - 0_-isopropylidene-a-D-allofuranose (U9) This was made i n the same way as i t s glucose counterpart ( 5 5 ) using 250 mg of 1 , 2 : 5 , 6-di - 0_-isopropylidene-a-g-allofuranose (diacetone a l l o s e ) , prepared by D. M i l l e r . It was r e c r y s t a l l i z e d from l i g h t petroleum, and formed TO long needle-like c r y s t a l s , melting at 7 ^ - 7 5 - 5 ° , l i t e r a t u r e value f o r ( 5 3 ) 7 U - 7 5 0 . 1 , 2 : 5 , 6-di - 0 _ - (Perdeuteromethyl )isopropylidene-a-D-glucofuranose (A) _ A l i t e r a t u r e preparation f o r diacetone glucose was used, with glucose that had previously been exchanged with D^O, (perdeuteromethyl)acetone, and phosphoric a c i d prepared from phosphorous pentoxide and D^O. The product was r e c r y s t a l l i z e d from l i g h t petroleum, and formed f i n e needle-like c r y s t a l s , 71 melting at 1 0 8 - 1 0 8 . 5 ° , l i t e r a t u r e value f o r diacetone glucose , 1 1 0 - 1 1 1 ° . 3 - 0-Acetyl-l , 2 : 5 , 6-di - 0 -(perdeuteromethyl)isapropylidene-a-p-glucofuranose (56) This was made i n the same way as ( 5 5 ) , by a c e t y l a t i o n of ikO mg of (A), and p u r i f i e d by e l u t i o n from a s i l i c a column with toluene:ether ( 1 : 2 ) . The c o l l e c t e d f r a c t i o n s were f i l t e r e d , evaporated, and dissolved i n l i g h t petroleum, and c r y s t a l l i z a t i o n was ef f e c t e d by a seed of ( 5 7 ) . This compound 163 formed square f l a t p l a t e s , which were s l i g h t l y discoloured, and melted at TO 5 6 - 5 7 ° , l i t e r a t u r e value 1 for (5*0, 59-60°. 3-0-(Trideuteromethyl)acetyl-l,2:5,6-di-0-(perdeuteromethyl)isopropylidene-a-D-glucofuranose (57) 150 mg of (A) was acetylated as previously described, using (perdeuteromethyl)acetic anhydride. The product, which i n i t i a l l y c r y s t a l l i z e d i n large square f l a t plates was r e c r y s t a l l i z e d from l i g h t petroleum, and formed one c r y s t a l mass made up of joined square p l a t e s , weighing 106 mg. mp 57.5-59°, l i t e r a t u r e 7 0 mp for (5U), 59-60°. 3-0-(Trideuteromethyl)acetyl-3-C-deutero-l,2:5,6-di-0-isopropylidene-a-D-allofuranose (50) 250 mg of l,2:5,6-di-0_-isopropylidene-a-rJ-ribo-hexofuranose-3-ulose 72 (D. M i l l e r ) was reduced with 230 mg of sodium borodeuteride (Stohler Isotope Chemical) by s t i r r i n g overnight at room temperature i n 7 0 % ethanol/water. The cloudy s o l u t i o n was added to water, and extracted seven times with e t h y l acetate, with sodium chloride added to the aqueous l a y e r to f a c i l i t a t e separation. The ethy l acetate s o l u t i o n was drie d over sodium s u l f a t e , and evaporated to give a white s o l i d (mp 6 3 - 6 9 ° ) . This crude product was drie d and acetylated as before i n a small amount of pyridine and (perdeuteromethyl)acetic anhydride. The product s o l i d i f i e d , but was contaminated with at l e a s t two impurities, probably the intermediates, the keto-sugar, and diacetone a l l o s e . It was p u r i f i e d by e l u t i o n from a s i l i c a column using f i v e per cent methanol i n chloroform. The product was then r e c r y s t a l l i z e d from l i g h t petroleum and, a f t e r seeding with (53) gave 137 mg of very large needle-like c r y s t a l s , mp 7 U - 7 U . 5 0 , . l i t e r a t u r e value T° f o r (53), 1^-75°. Methyl h ,6-0-benzylidene-2-deoxy-a-P_-ribo-hexopyranoside .(B) This compound was prepared from methyl 2,3-anhydro-U ,6-0-16k benzylidene-a-D-allopyranoside (Paul Steiner) by reduction with l i t h i u m 73 aluminum hydride i n anhydrous tetrahydrofuran . The s t a r t i n g material had been r e c r y s t a l l i z e d previously from acetone, and the tetrahydrofuran was d i s t i l l e d from l i t h i u m aluminum hydride immediately p r i o r to use. The product c r y s t a l l i z e d very r e a d i l y , and was r e c r y s t a l l i z e d from ethanol. mp 1 2 2 . 5 - 1 2 U . 5 ° , l i t e r a t u r e . v a l u e , 1 2 4 - 1 2 6 ° T 3 . Methyl h,6-0-benzylidene-2-deoxy-2-C_-deutero-a-g-altropyranoside (C) This was prepared i n the same way as the preceding compound, using l i t h i u m aluminum deuteride (Stohler Isotope Chemicals), mp 1 2 5 - 1 2 6 ° . Methyl h,6^0-benzylidene-3-0-acetyl-2-deoxy-a-g-ribo-hexopyranoside (D) A laboratory sample, which was an equal mixture of (B) and (D) with some minor impurities, was re-acetylated. S t i r r i n g overnight at room temperature with a c e t i c anhydride and pyridine produced no increase i n the proportion of acetate, and i t was necessary to heat the mixture at 1 0 0 - 1 1 5 ° f o r approximately s i x hours f o r the a c e t y l a t i o n to proceed e s s e n t i a l l y to completion. The re a c t i o n mixture, which became very dark during heating, was worked up by co-evaporating twice with toluene, and d i s s o l v i n g the dark syrup i n chloroform. The chloroform s o l u t i o n was washed twice with water, d r i e d over sodium s u l f a t e and evaporated, and the dark orange syrup dissolved i n ethanol and treated with charcoal. N.m.r. of the product at t h i s stage confirmed the presence of the acetate (D), which was then p u r i f i e d by e l u t i o n from a s i l i c a column with two:one ether:toluene to remove minor amounts of s t a r t i n g material, and a faster-running byproduct. The c o l l e c t e d f r a c t i o n s were dissolved i n a large volume of l i g h t petroleum, and the product formed large discoloured i r r e g u l a r c r y s t a l s a f t e r the solvent had evaporated to dryness at room temperature. Total y i e l d , 6 7 . 5 mg, mp. 7 ^ - 8 0 ° . U8 mg were r e c r y s t a l l i z e d from l i g h t petroleum and y i e l d e d hO mg 165 of i r r e g u l a r c r y s t a l s , which melted at 76.8-78°. Analysis. Calculated f o r C l 6 H 2 0 ° 6 : C ' 6 2 ' 3 3 ; H ' 6-^- Found: C, 62.39; H, 6.59. Methyl k ,6-0-"benzylidene -3-0-(trideuteromethyl)acetyl -2-deoxy-a-P_-ribo-hexo-pyranoside (51) 250 mg. of (B) heated at 100-120° with a small amount of (perdeuteromethyl)acetic anhydride and pyr i d i n e u n t i l a c e t y l a t i o n was e s s e n t i a l l y complete as shown "by TLC. The workup and p u r i f i c a t i o n were e s s e n t i a l l y as described f o r the corresponding (D). The f r a c t i o n s from the column were separated into two batches, the f i r s t cut (less pure) of lh9 mg, and a purer cut, also of 1^ 9 mg. Both batches c r y s t a l l i z e d from l i g h t petroleum, i n i r r e g u l a r c r y s t a l s , and were not r e c r y s t a l l i z e d . Batch one, y i e l d , 98.6 mg, mp 75.8-76.5°. Batch two, y i e l d , 100.5 mg, mp 75.5-76.5°. 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G i l l e n , Biochimica et Biophysica Acta 2 8 6 , 1 0 - 1 5 ( 1 9 7 2 ) . 8 U . K. H. Mil d , T. L. James and K. T. G i l l e n , Journal of C e l l u l a r Physiology 8 0 , 1 5 5 - 8 ( 1 9 7 2 ) . 8 5 . R. E. Block, FEBS L e t t . 3h_, 1 0 9 - 1 2 ( 1 9 7 3 ) . 8 6 . J . W. F a l l e r , M. A. Adams and G. N. LaMar, .Tetrahedron L e t t . , 6 9 9 - 7 0 2 (197*0. 8 7 . H. D. W. H i l l and R. Freeman, Introduction to Fourier Transform NMR, Varian Associates A n a l y t i c a l . Instrument D i v i s i o n , Palo A l t o , C a l i f o r n i a , 1 9 7 0 . 8 8 . A. Allerhand,. D. Doddrell and R. Komoroski, J . Chem. Phys. 55., 1 8 9 - 1 9 8 (1971) . 8 9 . G. C. Levy, Accounts.of Chemical Research 6 , 16I-I69 ( 1 9 7 3 ) . 9 0 . F. W. Wehrli, Chem. Commun., 3 7 9 - 8 0 ( 1 9 7 3 ) . 9 1 . R. L. Streever and H. Y. Carr, Phys. Rev. 1 2 1 , 2 0 - 2 1 ( l 9 6 l ) . 9 2 . E. D. Becker, J . A. F e r r e t t i and T. C. Farrar, JACS 91., 7 7 8 U - 5 ( 1 9 6 9 ) . 9 3 . R. R. Shoup, E. D. Becker and T. C. Far r a r , J . Mag. Res. 8_, 2 9 8 - 3 1 0 . ( 1972) . 9h. G. N. LaMar, JACS 93., lOUO-Ul ( 1 9 7 1 ) . .170 9 5 . R. Freeman, K. G. R. Pachler and G. N. LaMar, J . Chem. Phys. 55., 4 5 8 6 - 9 3 ( 1 9 7 1 ) . 9 6 . G. C. Levy and J . D. C a r g i o l i , J . Mag. Res. 10_, 2 3 1 - 4 ( 1 9 7 3 ) . 9 7 . R. R. Ernst, R. Freeman, B. Gestblom and T. R. Lusebrink, Mol. Phys. 13_, 2 8 3 - 6 ( 1 9 6 7 ) . 9 8 . N. L. R. King and J . H. Bradbury, Nature 2 2 9 , 404-6 ( l 9 7 l ) -9 9 . C. H. A.. S e i t e r , G. W. Feigenson, S. I. Chan and Ming-chu Hsu, JACS 9jt» 2 5 3 5 - 3 7 ( 1 9 7 2 ) . 1 0 0 . I. D. Campbell, C. M. Dobson, R. J . P. Williams and A. V. Xavier, J . Mag. Res. 1 1 , 1 7 2 - 8 1 ( 1 9 7 3 ) . 1 0 1 . W. B. Moniz, C. F . P o r a n s k i , J r . , and S. A. Sojka, J . Mag. Res. 13, 110-118 (197U). 1 0 2 . J. N. Schoolery, Varian Instrument Applications 8_, 1 0 - 1 1 ( 1 9 7 4 ) . 1 0 3 . L. D. H a l l and J . F. Manville, i n Deoxy Sugars, Advances i n Chemistry Series, Number 7 4 , American Chemical Society, 1 9 6 8 , pp. 2 2 8 - 5 3 . 1 0 4 . J . Feeney and G. C. K. Roberts, Chem. Commun., 2 0 5 - 6 ( l 9 7 l ) . 1 0 5 . A. G. Re d f i e l d and R. K. Gupta, J . Chem. Phys. 54., l 4 l 8 - 1 9 ( l 9 7 l ) . 1 0 6 . S. L. Patt and B. D. Sykes, J.. Chem. Phys. 56., 3 1 8 2 - 4 ( 1 9 7 2 ) . 1 0 7 . F. W. Benz, J . Feeney and G. C. K. Roberts, J . Mag. Res. 8 , 1 1 4 - 1 2 1 ( 1 9 7 2 ) . 1 0 8 . J . P. Jesson, P. Meakin and G. Kn e i s s e l , JACS 95., 6 l 8 - 2 0 ( 1 9 7 2 ) . 1 0 9 . B. L. Tomlinson and H. D. W. H i l l , J . Chem. Phys. 59., 1775-1784 ( 1 9 7 3 ) . 1 1 0 . J . Grimaldi, J . Baldo, C. McMurray and B. D. Sykes, JACS 94_, 7 6 4 1 - 4 5 ( 1 9 7 2 ) . 1 1 1 . M. E. Evans and S. J . Angyal, Carbohyd. Res. 25_, 43-48 ( 1 9 7 2 ) . 1 1 2 . S. J . Angyal, Aust. J. Chem. 25., 1 9 5 7 - 6 6 ( 1 9 7 2 ) . 113. S. J . Angyal, Pure and App. Chem. 35_, 131-146 ( 1 9 7 3 ) . 1 1 4 . C. S. Hudson, JACS 3 2 , 8 8 9 - 8 9 4 ( 1 9 1 0 ) . 115. T. Ikeda and M. Senda, Bull.. Chem. Soc. Japan 46, 2 1 0 7 - 1 1 ( 1 9 7 3 ) . 1 1 6 . T. M..Lowry and.W. T. John,.J. Chem. Soc. 9 7 , 2 6 3 4 - 4 5 ( 1 9 1 0 ) . 1 1 7 . A. A. Frost and R.. G. Pearson, K i n e t i c s and Mechanism, second e d i t i o n , John Wiley.and Sons Inc., 1 9 6 l . 1 1 8 . C. D. Barry, A. C. T. North, J . A. G l a s e l , R. J . P. Williams and A. V. Xavier, Nature 232, 2 3 6 - 4 5 ( l 9 7 l ) -171 119.. G. N. LaMar and J. W. F a l l e r , JACS 95., 3817-18 (1973)'. 120. C. M. Preston and L. D. H a l l , Carbohyd. Res. 37, 267-82 (197M. 121. L. D. H a l l and C. M. Preston, Can. J . Chem. 52, 829-32 (±97*0 . 122. C. W. M. Grant, L. D. H a l l and C. M. Preston, JACS 95., 77U2-H7 (1973). 123. L. D. H a l l and C. M. Preston, Varian Instrument. Applications 8_, 8-11 (1973). 12h. L. D. H a l l and C. M. Preston, Chem. Comm., 1319 (±972). 125. L. D. H a l l and C. M. Preston, Carbohyd. Res. 2J_, 286-88 (1973). 126. L. D. H a l l and C. M. Preston, Carbohyd. Res. 29, 522-2H (±973). 127. W. M. M. J . Bovee and J . Smidt, Mol. Phys. 26, 1133-36 (1973). 128. I. D. Campbell, C. M. Dobson, G. Jeminet and R. J . P. Williams, FEBS Let t . U_9_, 115-119 (197M. APPENDIX AN NMR STUDY OF .TWO BRANCHED-CHAIN SUGARS From there to here, from here to there, funny things are everywhere. (Dr. Seuss, One f i s h two f i s h red f i s h blue f i s h , Beginner Books, Random House, Inc., I 9 6 0 ) During the course of t h i s work, we studied a p a i r of branched-chain sugars d i f f e r i n g only i n t h e i r configuration at C - 3 . 4 ,6-0_-Benzylidene-2-deoxy-3-C-nitromethyl-a-g-ribo-hexopyranoside ( 5 8 ) and the arabino isomer ( 5 9 ) are both produced from the r e a c t i o n of the corresponding 3-keto sugar, 4 5 6 - 0 _ -benzylidene - 2-deoxy-a-D-erythro-hexopyranosid - 3-ulose, with nitromethane and sodium hydroxide, and are r e a d i l y separated by f r a c t i o n a l c r y s t a l l i z a t i o n . The isomers have been distinguished by a series of chemical conversions, and by c i r c u l a r dichroism s p e c t r a 7 ^ As the nitro-sugars are intermediates i n the preparation of amino-sugars, and eventually, of new nucleosides with modified b i o l o g i c a l a c t i v i t y , and as l i t t l e use has been made of n.m.r. because there i s no hydrogen at C - 3 , and the spectra seem complex, we decided to explore the p o t e n t i a l of modern n.m.r. techniques for these types of compounds. The proton r e l a x a t i o n times of ( 5 8 ) and ( 5 9 ) were obtained i n d i l u t e (0.1M) degassed CDCl^ s o l u t i o n ; these data are displayed on the s t r u c t u r a l formulae i n F i g . A-1. For the rib o isomer ( 5 8 ) , the 100 MHz spectrum was f u l l y assignable, and the T^ values of e s s e n t i a l l y a l l of the protons were measured. k They show a wide range, and as the sugar r i n g i s locked i n the C_ conformation by the benzylidene substituent, can be e a s i l y c o r r e l a t e d with the degree of i n t e r a c t i o n with other protons. For example, the a x i a l and equatorial H-6's are e a s i l y distinguished. The r e l a x a t i o n times that could be measured f o r the arabino isomer show a s i m i l a r pattern, and f o r protons i n the same stereochemical 172 173 2 . 4 1 1 . 1 6 1 . 4 2 Figure A-1. T x values (seconds) f o r the assignable protons of h,6-0-benzylidene-2-deoxy -3-C-nitromethyl-a -S-rTbo-hexopyranoside ( 5 8 ) , and the arabino isomer ( 5 9 ) , measured f o r 0.1 molar solutions i n CDC13 at U2°C. environment agree remarkably to within 0 . 1 0 seconds. However, without data f o r E-h and H-5 of ( 5 9 ) , the r e l a x a t i o n times can not be used to assign unequivocally the configuration of the two isomers. The hydroxyl proton i s the only other "probe" which i s accessible i n both molecules, and the possible e f f e c t s of chemical exchange and hydrogen bonding must be considered i n the i n t e r p r e t a t i o n of i t s T^ values. As normal precautions were taken to exclude moisture from the samples, and the OH peaks were sharp, i t i s l i k e l y that chemical exchange was absent, or too slow to a f f e c t the r e l a x a t i o n . Hydrogen-bonding of the OH to the methoxyl oxygen i n the r i b o isomer i s a d e f i n i t e p o s s i b i l i t y , and would cause a shortening of the r e l a x a t i o n time. However, hydrogen-bonding, by reducing the strength of the OH bond, would also cause a decrease i n the OH s t r e t c h i n g frequency. The i n f r a - r e d data, measured i n d i l u t e s o l u t i o n have been reported as 3600 cm f o r ( 5 8 ) , the r i b o form, and 3500 cm ^ for ( 5 9 ) , the arabino form; the difference i s small, and opposite to that expected i f hydrogen-bonding were occurring. With these cautions then, i t i s possible that the shorter T^ value of the a x i a l l y oriented OH may indeed r e f l e c t , i n a simple way, i t s greater degree of i n t e r a c t i o n with other protons i n the molecule. Although the r e l a x a t i o n study f a i l e d to d i s t i n g u i s h the isomers because of the complexity of the spectra, the spectra themselves were of s u f f i c i e n t i n t e r e s t that 220 MHz spectra were also obtained, and these, together with the coupling patterns, are shown i n F i g . A - 2 . The spectrum of ( 5 8 ) i s now f i r s t - o r d e r , and can be assigned by inspection, but unfortunately, the complex regions of the spectrum of ( 5 9 ) are v i r t u a l l y unchanged from t h e i r appearance at 100 MHz and are s t i l l unassignable. . There i s a small long-range coupling i n H-2a and one of the nitromethyl protons, which i s not present i n the spectrum 74 of ( 5 8 ) . This feature, which was pointed out i n the 60 MHz spectrum , i s a most valuable a i d to assigning the configuration at C - 3 , as i t i s only possible between the a x i a l H-2 and a proton of the a x i a l nitromethyl group, which can Figure A - 2 . High-resolution 220 MHz proton n.m.r. spectra of k,6-0--benzylidene-2-deoxy-3-C - n i t rome thy 1- a -D- r i b o-hexopy r ano s i de ( 5 8 ) A and B , i t s arabino counterpart (.59) 176 form a "planar W" arrangement. I t was f e l t from t h i s study that a greater use of high-resolution spectra, even at 100 MHz, could be of considerable a i d i n the study of complex carbohydrate d e r i v a t i v e s , e s p e c i a l l y now that high-quality spectra can be ro u t i n e l y obtained from minute quantities of material. I t has also been shown that f o r even large organic molecules, the rel a x a t i o n times of "equivalent" protons i n s i m i l a r structures are v i r t u a l l y i d e n t i c a l under the same experimental conditions and show a strong dependence on the stereochemical environment of the proton. Thus, the study of s p i n - l a t t i c e r e l a x a t i o n times has a d e f i n i t e p o t e n t i a l i n s t r u c t u r a l problems, and should prove a supplement to the t r a d i t i o n a l n.m.r. parameters. C. M. Preston and L. D. H a l l , Carbohyd. Res. 37_, 267-82 (1974). A general survey of the proton, s p i n - l a t t i c e r e l a x a t i o n times of monosaccharide de r i v a t i v e s L. D. H a l l and C. M. Preston, Carbohyd. Res. 27_, 286-88 (1973). Two, novel applications of Fourier transform spectroscopy. C. W. M. Grant, L. D. H a l l and C. M. Preston, JACS 95, 7742-7 (1973). S e l e c t i v e measurement of nuclear r e l a x a t i o n times of carbohydrate d e r i v a t i v e s . L. D. H a l l and C. M. Preston, Carbohyd. Res. 29, 522-4 (1973). Longitudinal r e l a x a t i o n times of the anomeric protons of o l i g o -saccharides. L. D. H a l l and C. M. Preston, Can. J . Chem. 52, 829-32 (1974). A s e l e c t i v e determination of the nuclear r e l a x a t i o n times of the a l k a l o i d vindolene: an extension v i a Fourier transform measurements. L. D. H a l l and C. M. Preston, Varian Instrument Applications 8, 8-11 (1973). S p i n - l a t t i c e r e l a x a t i o n times of protons. L. D. H a l l , C. M. Preston and J . D. Stevens, Carbohyd. Res. 41_, 41-52 (1975). Applications of Fourier transform proton N.M.R. spectroscopy to studies of carbohydrate d e r i v a t i v e s . L. D. H a l l and C. M. Preston, Carbohyd. Res. 41_, 53-61 (1975). The eff e c t s of gadolinium ions on the proton s p i n - l a t t i c e r e l a x a t i o n rates of sugars i n aqueous s o l u t i o n . 

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