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Applications of proton and Fluorine nuclear magnetic resonance spectroscopy to the study of large organic… Grant, Christopher William Maitland 1972

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APPLICATIONS OF PROTON AND FLUORINE NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY TO THE STUDY OF LARGE ORGANIC MOLECULES  BY CHRISTOPHER WILLIAM MAITLAND GRANT B.Sc.  (Hon.), McMaster U n i v e r s i t y ,  1968  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  March ,1972  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  (Lh4-t~\'i si~i  The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  Date  s h a l l not be  permission.  Department of  n  2. ?  ;  /f 7Z  Department or  I t i s understood t h a t copying or  of t h i s t h e s i s f o r f i n a n c i a l g a i n written  the Head of my  Columbia  publication  allowed without  my  - ii ABSTRACT This t h e s i s i s d i v i d e d i n t o three chapters; each i n v o l v i n g a 1 19 d i f f e r e n t approach to the use of both proton ( H) and f l u o r i n e ( F) nuclear magnetic resonance (n.m.r.) to study large organic molecules in solution.  To some extent the three chapters represent an e v o l u t i o n  i n technique of the s c i e n t i f i c world i n general and of our laboratory in particular. Previous students i n t h i s laboratory have used high r e s o l u t i o n 1 n.m.r. spectroscopy (of H,  31  19  P and  F n u c l e i ) combined w i t h  double i r r a d i a t i o n techniques to study p r o g r e s s i v e l y l a r g e r organic molecules.  In the f i r s t chapter t h i s work has been extended to s  "natural products" - molecules not p r e v i o u s l y very s u s c e p t i b l e , because of t h e i r s p e c t r a l complexity, t o d e t a i l e d n.m.r. s t u d i e s . In i particular  19 ti and  F n.m.r. have been employed i n conjunction with  heteronuclear noise modulated decoupling and *H-{ >H} INDOR.  A  s e r i e s of s t e r o i d s s u b s t i t u t e d w i t h f l u o r i n e i n the A, B or D rings have been examined as model systems: 6a- and 63-fluoro-cholest-4-en-3-one 33-ol-17-one.  2a-fluoro-cholestan-3-one, and 16,16-difluoro-androst-5-en-  In each case i t has been p o s s i b l e to obtain coupling  constants and chemical s h i f t s f o r the n u c l e i i n the area of the f l u o r i n e atom and hence to derive s t r u c t u r a l data from the n.m.r. spectra i n s p i t e of t h e i r complexity.  The H-{  INDOR technique alone  has been f u r t h e r applied to s e v e r a l problems posed by organic chemists involved w i t h n a t u r a l products. successful.  In each case t h i s approach has been  - iii  -  Recently very large molecules such as enzymes have been studied by n.m.r. v i a t h e i r e f f e c t s on the chemical s h i f t and l i n e width of smaller molecules with which they i n t e r a c t .  As part of a programme to  investigate the application of heteronuclear  n.m.r. to problems of  b i o l o g i c a l i n t e r e s t , we have used the above technique to study the interaction of various N-trifluoroacetylated monosaccharides with 19 the enzyme, lysozyme.  The  F chemical s h i f t s of N-trifluoroacetyl-D-  glucosamine and i t s methyl glycosides have been studied as a function of enzyme concentration.  The r e s u l t s suggest that the f l u o r i n e  substituents a f f e c t the binding process to some extent but that such effects can ba informative.  The p o s s i b i l i t y of using n.m.r. to study  the e f f e c t s of substituents on enzyme-inhibitor  interactions led to a  study of the monosaccharides, N-acetyl-D-glucosamine-a-methyl glycoside and i t s Cg-iodo and C^-methyl derivatives, a l l three of which gave s i m i l a r r e s u l t s .  This work i s treated i n Chapter II along  with a b r i e f discussion of the conformations of the i n h i b i t o r s involved. Very recently organic chemists and biochemists have begun employing pulsed n.m.r. equipment i n a variety of problems.  We have become  interested i n the applications of relaxation time measurements to 1 s t r u c t u r a l problems.  In Chapter I I I pulsed  involving several model systems are reported: trans isomers of 1,2-dichloro  H n.m.r. experiments mixtures of the c i s and  and 1,2-dibromoethylene and of the  ethyl esters of maleic and fumaric acids have been studied.  The  results of these experiments are encouraging, i n d i c a t i n g that, i n this case at least, relaxation times are s e n s i t i v e to structure and i n a consistent fashion.  substituent  We have experimented almost exclusively with  - iv-  s e l e c t i v e pulse techniques and have b u i l t and used a variety of equipment.  In the Appendix are described two audiofrequency-pulse  units which can be attached to a Varian HA-100 n.m.r. spectrometer and which were used f o r the experiments discussed i n Chapter I I I . The same audiofrequency-pulse techniques have been applied to the measurement of nuclear relaxation times of i n d i v i d u a l protons in.the a l k a l o i d , vindoline, and i n the sugar, 3,4,6-tri-0_-acetyl-l-0-benzoyl-2-bromo-2deoxy-g-D-glucopyranose and i t s 2-chloro analogue with less encouraging 19 results.  In addition we have reported the use of  calculate the rate constants, k  F pulsed n.m.r. to  and k^, f o r the association of the  a-anomer of N-trifluoroacetyl-D-glucosamine with  lysozyme.  - vTABLE OF CONTENTS Page CHAPTER I.  FLUORINE N.M.R. AND THE INDOR TECHNIQUE AS  PROBES OF NATURAL PRODUCT STRUCTURE  1  Introduction  1  Results and Discussion  8  A.  Studies of Fluorosteroids  B.  Application of H-{"hs> INDOR to Natural 1  Products Experimental  CHAPTER I I .  8  31 41  FLUORINE AND PROTON N.M.R. AS APPLIED TO CERTAIN  ASPECTS OF THE INTERACTION OF LYSOZYME WITH MONOSACCHARIDE INHIBITORS  42  Introduction  42  Results and Discussion  52  A.  Choice of a Suitable Chemical S h i f t Reference  B.  Experiments with N-Trifluoroacetyl-Dglucosamine  C.  52  53  Experiments with C^-Substituted N-Acetyl-Dglucosamine-a-methyl glycosides  D.  Conformation of Free Monosaccharide Inhibitors  E.  N.M.R. as a Probe for Conformation of Bound Monosaccharide Inhibitors  Experimental  71 83  92 98  - vi -  Page CHAPTER I I I .  APPLICATION OF THE AUDIOFREQUENCY PULSE  TECHNIQUE TO THE STUDY OF LARGE ORGANIC MOLECULES IN SOLUTION  105  Introduction  •• ••  Results and Discussion A.  A Case for the A p p l i c a b i l i t y of T^ and  113 to  Structural Organic Chemistry B.  ^05  113  Measurement of Nuclear Relaxation Times of an Alkaloid by the Audiofrequency Pulse Technique  C.  123  Measurement of Nuclear Relaxation Times of Carbohydrate Derivatives by the Audiofrequency Pulse Technique  D.  The Audiofrequency Pulse Technique and EnzymeInhibitor Rate Constants  Experimental APPENDIX A.  REFERENCES  138 145  PRINCIPLES OF PULSED N.M.R. SPECTROMETRY WITH  PARTICULAR REFERENCE TO HIGH RESOLUTION EXPERIMENTS ... APPENDIX B.  131  AUDIOFREQUENCY PULSE SPECTROMETERS EMPLOYED  148 153 162  - viiLIST OF TABLES Table  Page  CHAPTER I 1  N.M.R. Parameters f o r the Ring A Resonances of 2aFluorocholestan-3-one (1)  2  18  N.M.R. Parameters f o r Parts of Rings A and B of 6a- and 6g-Fluoro-cholest-4-en-3-one (3 and 4) ...  3  N.M.R. Parameters f o r the Ring D of 16,16-Difluoroandrost-5-en-3g-ol-17-one  4  23  (5)  2 9  N.M.R. Parameters f o r Compounds 6 (Cyano-aldehyde), 9, 10, and 11 (Decalones)  36  CHAPTER I I 19 1  F Chemical S h i f t Data f o r N - T r i f l u o r o a c e t y l - D glucosamine (1 and 2) and i t s Methyl Glycosides  63  (3 and 4) i n the Presence of Lysozyme 2  Linewidth Data f o r N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl Glycosides (3 and 4) i n  69  the Presence of Lysozyme 3  K^, Kg and A Values f o r Compounds 1 to 4; a l s o ^H Chemical S h i f t Data f o r 3 and 4 Competing w i t h NAcetyl-D-glucosamine-a-methyl-glycoside f o r  66  Lyzozyme 4  Chemical S h i f t Data f o r the N-Acetyl Protons of 7, 9, and 10 and f o r the C^-Methyl of 10 I n t e r a c t i n g x^ith Lysozyme  78  - viii Page Table 5  Kp, Kg and A Values for Compounds 7, 9, and 10; 1 also  H Chemical Shift of Glycoside Methyl on  Binding 6  79  N.M.R. Parameters for the 0-Acetate Derivatives  of  3*, 4' , 7' , 9' and 10' CHAPTER 1  90  III Relaxation Time Data for 3 , 4 , 6 - T r i - 0 - a c e t y l - l - 0 benzoyl-2-bromo-2-deoxy-g-D-glucopyranose  (1) and  i t s 2-Chloro analogue (2) 2  136  Nuclear Relaxation Time Data for the a- and (3Anomers of the Free Sugar, N - T r i f l u o r o a c e t y l - D glucosamine  142  - ix -  LIST OF FIGURES Figure  Page  CHAPTER I 1  The "^H n.m.r. spectrum (100 MHz) of 2a-fluorocholestan-3-one (1) i n CDCl^ solution.  A. The  normal spectrum; B. The spectrum measured with 19 simultaneous i r r a d i a t i o n at the  F resonance  frequency 2 3  9  The J H - {>H}INDOR experiments performed on the 19 F decoupled spectrum of 1 i n CDCl^ solution .... The >H-{ .H} INDOR  experiments performed on the  normal spectrum of 1 i n CDCl^ solution 4  11  ± x  13  P a r t i a l >H n.m.r. spectrum of the h i g h f i e l d region of 1 i n CDCl^ solution.  A.  The normal spectrum;  19 B.  the  F decoupled spectrum  14  19  5  The  F n.m.r. spectra (94.071 MHz) of 1 i n CDC1  3  solution  16  6  The normal ^H n.m.r. spectra of 3 (A) and 4 (B)  7  i n CDCl^ solution I9 The F n.m.r. spectra (94.071 MHz) of 3 (A), 4 (B) and 5 (C) i n CDC1 solution I I 19 3  8  The  H-{ H} INDOR experiments performed on the  27  The "*"H n.m.r. spectrum (100 MHz) of a steroid (6) reaction mixture i n CDCl^ solution  10  24  F  decoupled spectrum of 5 i n CDCl^ solution 9  20  32  The >H n.m.r. spectra of decalones 9 (A), 10 (B) , and 11 (C) at 100 MHz i n CDC1, solution  38  - x Figure  Page  CHAPTER I I 1  The section of a natural polysaccharide substrate which would occupy lysozyme's active s i t e during lysis  47  19 2  F n.m.r. spectra (94,071 MHz) of a mutarotating solution of N-trifluoroacetyl-D-glucosamine (aanomer, 1 ) i n pH 5 . 5 c i t r a t e buffer 19  3  55  F n.m.r. spectra recorded during a study of 1 and 2 (a- and g-anomers of N - t r i f l u o r o a c e t y l glucos57  amine) with lysozyme 19 4  A.  Graph of  F chemical s h i f t data from the study  of 1 with lysozyme B.  Increase i n 6 as a function of time during the  mutarotation of pure a-anomer ( 1 ) to an equilibrium 61  mixture i n the presence of lysozyme 5  ''"H n.m.r. spectra recorded during a study of C,.methyl-N-acetyl glucosamine-a-methyl  glycoside  ( 1 0 ) with lysozyme 6  74  Graphs of chemical s h i f t data for 1 0 , 7 and 9 i n t e r a c t i n g with lysozyme  7  A.  ^TI n.m.r. spectrum  glucosamine B.  77  ( 1 0 0 MHz) of N-acetyl  a-methyl glycoside (7) i n D2O  P a r t i a l hi n.m.r. spectrum  ( 1 0 0 MHz) of C,-  o iodo-N-acetyl glucosamine-a-methyl diacetate (9') i n C D C 1 „ solution  glycoside 84  -  xi  -  Figure 8  Page P a r t i a l >H n.m.r.  spectra (100 MHz) of A.  N-  t r i f l u o r o a c e t y l glucosamine-!*-methyl glycoside t r i a c e t a t e (3')  and B.  N-trifluoroacetyl  amine g-methyl glycoside triacetate (4')  glucosi n CDCl^  solution 9  86  Computer-simulated spectra of the H^ 'doublet' region of a hypothetical N-acetyl glucosamine-amethyl glycoside i n aqueous solution  CHAPTER 1  94  III The alkene proton regions of the n.m.r. (100 MHz, solvent CDC1 ) of A. 3  1,2-dlchloroethylene B.  spectra  1,2-dibromo- and  (cis and trans isomers) and  maleic and fumaric acid d i e t h y l esters (plus  cis-l,2-dichloroethylene) 2  Effect of a modified C a r r - P u r c e l l sequence on the magnetization vector, M  3  q  121  The H n.m.r. 1  spectrum (100 MHz) of the a l k a l o i d ,  v i n d o l i n e , i n CDCl^ solution 5  119  Effect of a T^ sequence on the magnetization vector, M o  4  115  A.  125  Scope photograph of a t y p i c a l C a r r - P u r c e l l  sequence on the C^-acetate resonance of v i n d o l i n e . B.  Scope photograph of a t y p i c a l T^ pulse sequence  on the same resonance  126  - xii  -  Figure 6  Page Relaxation time data obtained on the a l k a l o i d , vindoline,  7 .  i n CDC1 solution  129  3  P a r t i a l H n.m.r. spectra (100 MHz) of 3 , 4 , 6 - t r i 1  0-acetyl-l-0-benzoyl-2-bromo-2-deoxy-B-D-glucopyranose (A) and i t s 2-chloro analogue (B) i n C^D^ 8  A.  Plots of 1/T  and 1/T  2  vs.  [ E ] for the Nq  t r i f l u o r o a c e t y l group of the a-anomer. of (1/T--1/TJ 2 1  vs.  133  B.  Plot  [E ] for the a-anomer o  143  APPENDIX 1  Scope traces of various pulses on the alkene resonance of the d i e t h y l ester of maleic acid . . . .  2  Block diagram of the components used i n the "Mark I"  3  154  pulse unit  156  Block diagram of the components used i n the "Mark II"  5  audiofrequency-pulse spectrometer  Block diagram of the components used i n the "Mark I"  4  151  audiofrequency-pulse spectrometer  159  Block diagram of the components used i n the "Mark II"  pulse unit  I  60  - xiii -  ACKNOWLEDGEMENTS  F i r s t l y , I would l i k e to thank Dr. L.D. H a l l f o r his d i r e c t i o n i n this work - not only as a source of information and ideas, but also for his guidance i n developing a more mature s c i e n t i f i c philosophy. Secondly I would l i k e to acknowledge my numerous h e l p f u l discussions with Ben Malcolm and Dr. P. Steiner of this laboratory regarding my early work, with Dr. A.G. Marshall about t h e o r e t i c a l aspects and with Roland Burton concerning n.m.r. e l e c t r o n i c s .  CHAPTER I FLUORINE N.M.R. AND THE INDOR* TECHNIQUE AS PROBES OF NATURAL PRODUCT STRUCTURE Introduction The use of >H nuclear magnetic resonance  (n.m.r.) spectroscopy as  a method for studying small organic molecules i s well known. But with increasing molecular s i z e and complexity, the method becomes considerably less useful simply because the spectrum becomes so complex as to be not readily analysable; indeed i t often happens that a resonance of p a r t i c u l a r interest i s obscured by the overlapping transitions of other resonances, which i s the so-called resonance" problem.  "hidden-  A number of methods have evolved to overcome this  problem and these include the development of ever larger and more powerful magnets.  Also various double resonance techniques have  become available i n the l a s t decade and these can, i n many cases, help considerably i n the interpretation of complex n.m.r. spectra. Another method of obtaining n.m.r. data on molecules with complex  <H  n.m.r. spectra i s to look at the n.m.r. spectrum of some heteronuclear label ( i . e . some spin 1/2 nucleus other than protons) which has been  An acronym coined by Baker  f o r InterNuclear Double Resonance.  - 2 -  specifically incorporated for that purpose or of heteronuclei  31 present  in  the molecule (e.g.  already  13  P or  C).  This laboratory  has  investigated both of the above techniques and t h e i r application to  2 3  ever-larger molecules, e.g. sugars '  and c y c l i c phenyl phosphates.  4  Success i n such studies encouraged us to investigate the p o t e n t i a l of these methods with s t i l l larger and more complex molecules and "natural products" presented a next l o g i c a l step i n this d i r e c t i o n . Various >H n.m.r. methods have already been t r i e d i n this area but have met with limited success."'  In some areas researchers  have looked for  empirical chemical s h i f t r e l a t i o n s h i p s ; for example, i n the s t e r o i d area the relationship between the has  been studied.^'''  methyl group and ring geometry  More recently, however, s h i f t reagents have  8 been applied to natural products  and this approach promises to be  quite useful, although l i t t l e has appeared i n the l i t e r a t u r e on steroids as yet. "Natural products" chemists who  are of considerable importance, yet  organic  i s o l a t e and synthesize them often have considerable  d i f f i c u l t y i n learning their chemical i d e n t i t y or configuration.  The  n.m.r. problem i n this case i s often that t r a n s i t i o n s of i n t e r e s t are located i n the "methylene envelope" which generally extends from x to T 9.0.  7.5  Our f i r s t experiments involved the use of n.m.r. spectroscopy  i n an attempt to examine the configuration of the basic s t e r o i d 9 skeleton.  Sites of interest were l a b e l l e d with f l u o r i n e .  This  had  two major benefits: 19 1.  the electronegative  F nucleus tended to deshield one or more  neighbouring protons to low f i e l d of the methylene envelope where they  - 3could be used for ^H-{^H} 2.  INDOR. experiments,  since fluorine i s a 100% spin 1/2 atom, i t s own n.m.r. spectrum 1  19  could be readily examined and the Haugment the proton data.  F coupling constant data used to  Fluorine was also found (see text) to be useful  as a subject of spin decoupling experiments which made spectral assignments very easy. The information obtained by the above methods i s b a s i c a l l y a set of coupling constants and chemical s h i f t s f o r the groups of n u c l e i investigated.  Relative signs of coupling constants can i n many cases  also be obtained by the double resonance techniques used but were not investigated here.  Small differences i n chemical s h i f t  between different molecules provide a very limited s t r u c t u r a l probe because of the many un-predictable factors involved.  Coupling constants,  on the other hand, are a very useful s t r u c t u r a l probe. 1 and  Vicinal  ^H-"4l  19 H- F coupling constants are known to follow f a i r l y well-defined 3 13  relationships as a function of bond angle. ' The double resonance techniques employed i n this work have been quite thoroughly described i n the l i t e r a t u r e .  They a l l involve the  perturbing effect of a second radio-frequency f i e l d  ( i . e . , i n addition  to the extremely weak f i e l d being used to observe the spectrum).  This  effect depends on the strength of the perturbing f i e l d , yU^/?.^ Hz, (where y i s the nuclear gyromagnetic r a t i o and B.^ i s the perturbing r . f . f i e l d ) compared with i t s distance from a given l i n e .  Early use  of double resonance was confined to perturbing f i e l d s such that {yR^/l-n) > J where J i s the spin coupling constant which i s to be removed.  Such power levels lead to"spin decoupling" experiments.  '  -  4 -  In many situations however, the high perturbing f i e l d power levels necessary for spin decoupling are unsatisfactory because of the low  16 s e l e c t i v i t y of the resultant experiment.  Freeman and Anderson  have  discussed the theory and merits of using weak perturbing f i e l d s (yH^/^iT  of the order of the t r a n s i t i o n linewidth at half height).  1 17-1 These lower power techniques include "spin t i c k l i n g " and "INDOR". ' We have found that f o r ^H-{^"H} double resonance experiments, the lowest power (and hence most selective) form of INDOR i s convenient. This technique involves observing one sharp, well-resolved peak i n a spectrum while slowly scanning a weak perturbing f i e l d through other regions of the spectrum.  The peak being observed  t r a n s i t i o n between two nuclear energy 1  1  corresponds to a  levels:  a  (8 , . . ,. , . , . . population g The i n t e n s i t y of this peak i s related to the r a t i o , -—-—= ; . population a m  c  If the weak perturbing f i e l d reduces this r a t i o f o r the peak one i s observing, the peak w i l l become smaller,  and vice versa.  A weak  perturbing f i e l d can cause such population changes to occur when the nucleus being monitored i s spin-coupled to other n u c l e i . For instance, consider monitoring the peak corresponding to a t r a n s i t i o n A^ of the following two spin AX system:^  _ 5 -  aa  A, pa  X  The peak  A'  2  Ai  2  A-  Xi  corresponds to a t r a n s i t i o n between levels g a and act.  levels are also involved with the A£ l i n e , of the spectrum. f i e l d through X^ and  and  X  These  l i n e s , but not with the  Sweeping a weak perturbing radio-frequency  should cause i n t e n s i t y changes i n A^:  a  decrease as the a g - a a t r a n s i t i o n pumps spins into the act l e v e l , and an increase as the g g - get t r a n s i t i o n pumps spins into the g a l e v e l . A convenient way to run such experiments i s to arrange the n.m.r. equipment so that the recorder pen Y-axis monitors the i n t e n s i t y of a p a r t i c u l a r peak while the X-axis corresponds to the position of the weak perturbing f i e l d as i t i s swept through the spectrum. result of the above experiment would be^^:  I  AT  A  2  X,  X  2  The  2  - 6-  This model i s readily extended to a system of three or more spins.  '  From this then i t can be seen that one proton can be monitored and used to find the t r a n s i t i o n s of other protons with which i t i s spin coupled (e.g., i n the previous diagram, the X lines would have been detected even had they been e n t i r e l y hidden beneath the methylene envelope of a s t e r o i d ) .  The INDOR technique i s so s e l e c t i v e as to be  applicable as long as i n d i v i d u a l l i n e s can be i d e n t i f i e d , preferably on a f i r s t order basis.  The l i m i t a t i o n i s that some proton connected  with the s i t e of interest must be v i s i b l e for monitoring. We report here the use of the above approach to study the A-ring of 2a-fluoro-cholestan-3-one, the B-ring of 6a- and 6$-fluoro-cholest4-en-3-one and the D-ring of 16,16-difluoro-androst-5-en-3|3-ol-17-one. These steroids a l l have the commonly-occurring ring junctions.  trans 5-10 and 13-14  An additional interest i n these p a r t i c u l a r molecules  i s associated with the fluorine substituent i t s e l f since fluorine as a steroid " l a b e l " i s interesting from a b i o l o g i c a l viewpoint (a number 20 of fluorinated steroids have shown b i o l o g i c a l a c t i v i t y  - sometimes  21 enhanced  ). Partly for this reason, the l i t e r a t u r e on introduction of 22  such a l a b e l i s considerable. Nevertheless, i n terms of the general a p p l i c a b i l i t y of the n.m.r. method,one would hope to be able to routinely analyze natural product n.m.r. spectra without having to resort to spin l a b e l l i n g .  We have  found that i n many cases the natural product s i t e of i n t e r e s t , being the reaction s i t e , i s marked by a double bond, an oxygen-containing or some other electronegative species.  Such a group often  group,  serves to  deshield at least one neighbouring proton so that i t appears to low  - 7 -  f i e l d of the methylene envelope.  I t can then usually be employed i n  INDOR experiments to pick out the t r a n s i t i o n s of neighbouring protons which are s t i l l buried i n the methylene envelope.  This technique  was found to work consistently well i n i d e n t i f y i n g the products of several new reactions on natural products.  The results of some t y p i c a l  problems are included at the end of this chapter.  - 8Results and Discussion A.  Studies of Flubrostefoids 2a-Fluoro-choles tan-3-one (1) This compound w i l l be discussed i n some d e t a i l as i t represents  a " t y p i c a l " case; the other molecules w i l l be more b r i e f l y dealt with except where they posed s p e c i a l problems. The normal "^H n.m.r. spectrum of 1 (Fig. 1A) c l e a r l y shows the  2, R = Br  - 9 -  9 10 11 12  C-18  ' / // 5 6  2/3F|  J  1 2 3 Fig. 1.  4  8  6.6  The H n.m.r. spectra (100 MHz) in deuterochloroform solution. A. B.  7.6  8.6  of 2ct—f luoro—cholestan—3—one (1)  The normal spectrum The spectrum measured with simultaneous i r r a d i a t i o n at the resonance frequency (94, 076, 140.0 Hz). The assignment of the C-methyl resonances follows previously established c r i t e r i a . ^ CHCl^ was used for the field-frequency lock.  - 10 -  proton which has been deshielded from the methylene envelope by the electronegative fluorine atom.  I r r a d i a t i o n at the  19  F resonance  19 frequency removes the 48 Hz geminal  Y-Yl  coupling, collapsing  0  this l o w - f i e l d octet to a quartet (Fig. IB).  Changes also occur i n  the methylene envelope region during fluorine decoupling and these w i l l be discussed l a t e r . Two series of ^H-{"'"H} INDOR measurements were made while monitoring transitions of the c l e a r l y - d i s c e r n i b l e  proton.  The f i r s t of these  19 was performed on the of this experiment;  F decoupled spectrum.  F i g . 2 shows the results  thus, F i g . 2A i s the INDOR spectrum obtained by  scanning a weak radio-frequency f i e l d through the methylene envelope while monitoring i t s effect on t r a n s i t i o n 12 (using the numbering system of F i g . IB).  Sequential monitoring of transitions 11 and 9 gave  the responses shown i n F i g . 2B and C.  The frequency at which an INDOR  response occurs marks the position of a t r a n s i t i o n connected to the one being monitored.  The sense of the response  (either up or down)  contains information as to the r e l a t i v e signs of the coupling constants involved - but i n this work the signs were not of i n t e r e s t . e f f e c t i v e summation of a l l the INDOR responses  The  (shown diagrammatically  above the normal spectrum i n F i g . 2) picks out a l l transitions corresponding to the C^ protons.  The four low-field INDOR responses confirm  that the four low-field transitions of  the normal spectrum belong to a  single proton and indicate that i t i s one of the C^ protons.  Perhaps  more importantly, the h i g h e r - f i e l d set of INDOR responses now i d e n t i f y the transitions of the other C^ proton which are normally t o t a l l y obscured by the resonances of other methylene protons.  - 11 -  . 2.  The H-{ H} INDOR experiments performed on the F decoupled spectrum of 1 i n deuterochloroform solution. Using the t r a n s i t i o n numbering system of F i g . 1, the spectrum i n A corresponds to monitoring t r a n s i t i o n 12, B corresponds to monitoring t r a n s i t i o n 11, and C to t r a n s i t i o n 9. The composite assignment r e s u l t i n g from these spectra i s shown diagrammatically above the normal proton spectrum.CHCI3 was used as the internal-reference s i g n a l for the field-frequency lock.  - 12 -  The second series of INDOR measurements ( F i g . 3) was done without 19 F decoupling.  Five l i n e s of the H  resonance were monitored i n turn  zp 0  to pick out a l l t r a n s i t i o n s belonging to the  protons.  Because of  the decrease i n i n t e n s i t y of the t r a n s i t i o n s being monitored, the e f f e c t i v e signal-to-noise r a t i o f o r these responses i s poorer than that of the responses shown i n F i g . 2.  Nevertheless i t i s possible to make  a summation of the i n d i v i d u a l responses and obtain the complete set of t r a n s i t i o n frequencies f o r both of the  protons.  Due to the  fortuitous, near equality of the s p e c t r a l s p l i t t i n g s , there i s considerable degeneracy f o r the u p f i e l d  proton. 19  Inspection of the lower-field portion of the normal and  F  decoupled methylene region (Fig. 4) indicated that i n addition to the changes associated with the collapse of the known  t r a n s i t i o n s , two  further proton multiplets were subject to some decoupling; l i k e l y these were from the  protons.  Several of these t r a n s i t i o n s were c l e a r l y  resolved and a series of INDOR experiments  i n which these t r a n s i t i o n s  were monitored made possible a reasonably accurate assignment of a l l the resonances of both  protons.  Further responses were also observed  around x 8.4 and these were assigned to H,.. 1 19 work the a b i l i t y to remove or r e t a i n  H-  In this l a t t e r part of the  F couplings at w i l l was very  useful for i d e n t i f y i n g t r a n s i t i o n s . Close inspection of the INDOR spectra shown i n F i g . 2 and F i g . 3 reveals that some responses occur at frequencies corresponding to the C-methyl resonances.  These seem to be instrumental a r t i f a c t s a r i s i n g  from overloading of some amplifier stage i n the spectrometer. 19 The normal  F spectrum of 1 i s shown i n F i g . 5.  The comparatively  F i g . 3.  The H-{ H} INDOR experiments performed on the normal spectrum of 1 i n deuterochloroform containing enough CHCl^ for a lock s i g n a l . The transitions were monitored as follows: A t r a n s i t i o n 8; B t r a n s i t i o n 6; C t r a n s i t i o n 7; D t r a n s i t i o n 2; E t r a n s i t i o n 3. A diagrammatic summary of these responses i s given above the normal spectrum.  -  14  B  ^4(3 ^ 4 a 4/35oc  J  -  J4a5ot J4a4/3  Fig. 4 .  P a r t i a l H n.m.r. spectrum of the high f i e l d region of 1 i n deuterochloroform solution (CHCI3 lock). The normal spectrum i s shown i n A and the 1 9 F decoupled spectrum i n B . The f i r s t - o r d e r assignments of these spectra were based on the INDOR experiments discussed i n the text.  - 15 poor resolution of the undecoupled the  19  spectrum i s due to the fact that  F substituent i s part of a f a i r l y highly-coupled  arid to the large number of small, long-range couplings. to the large geminal coupling with H  proton  spectrum  In addition  (ca. 48 Hz) a number of smaller Zp  proton couplings are resolved (Fig. 5). The foregoing set of experiments  served to i d e n t i f y the  resonances  of a l l the protons associated with ring A of derivative 1. Comparison 19 of the t r a n s i t i o n s assigned to the 'normal' and F decoupled resonances of H. and H,„ provided an estimate of the magnitudes of the ^H-^H la IB 19 1, and F- H. couplings, which proved to be i n reasonable accord with the 19 estimates obtained by direct measurement of the F spectrum (Fig. 5). These data are included i n Table 1. H  i  and H  zp  0  Simulation of the spectra of the  resonances using the parameters involving these three  n u c l e i and the T?^ substituent, gave calculated t r a n s i t i o n energies which were i n close accord with the experimental values; on this basis i t was  concluded that a f u l l , i t e r a t i v e analysis was  Interestingly, the ^H-"'"H couplings f o r the H^ and  unnecessary. protons l i s t e d  i n Table 1 are i d e n t i c a l , within experimental error, with those 23 previously reported by A l l i n g e r et a l .  for 2a-fluoro-5a-androstane-  3,17-dione. Evaluation of the H^ and H^ resonances proved to be more d i f f i c u l t . However, i n spite of the small chemical s h i f t separation of the two C^ protons, a s a t i s f a c t o r y estimate of the inter-proton couplings 19 was  readily made by examination of the  F decoupled spectra and by  i t e r a t i v e , computer-based analysis involving the H^, resonances.  and H,.  Because of several fortuitous degeneracies, several  - 16 -  F  H -40  Fig. 5.  , HZ  1  . •I  .  I  .  1  .  1  .  I  ,  I ,, I 1 1  1  + 194.5 PPM. FROM CFCI3  19 The F n.m.r. spectra (94.071 MHz) of 1 i n deuterochloroform solution. The field-frequency lock i n this case was C F C I 3 . The normal spectrum i s shown i n the lower trace. The insert was recorded at the same gain but with simultaneous i r r a d i a t i o n of the entire . H spectrum using a noise-modulated radio-frequency centered at 99, 997, 945.0 Hz. A p a r t i a l f i r s t order assignment of the smaller couplings i s shown.  +40  HZ  - 17 19 attempts were required before a f u l l analysis which included the F couplings could be made. It should be noted that the presence of a spin 1/2 heteronucleus was  c r u c i a l for these assignments since i n i t s  absence i t would have been impossible to assign the  and  19 resonances.  The  F-H. coupling was assigned an absolutely p o s i t i v e 4-a , , . . 24 sign on the basis of previous studies. We turn now to consider the conformational significance of the coupling constants determined  for 1.  In a previous study, Abraham  25 and Holker  had r a t i o n a l i z e d the v i c i n a l H^-^  couplings of the 2a-  bromo analog (2) of the derivative we were studying i n terms of a s l i g h t l y distorted chair conformation for ring A.  Since the H^-H^  couplings of  1 are closely similar to those of 2 i t seems probable that both systems have similar conformations.  The remaining couplings determined  for 1  appear to support this contention and hence, i n d i r e c t l y , the v a l i d i t y of previous conclusions.  Thus the  ^ coupling of 14.4 Hz c l e a r l y  accords with a t r a n s - d i a x i a l relationship between these two protons. 19 1 Furthermore, the magnitudes of the v i c i n a l F- H couplings are only consistent with a gauche relationship between the F„ substituent and 2a the two protons at C^.  Although there i s a paucity of data concerning  4 J couplings, the values l i s t e d i n Table 1 for the couplings between 24 F„ , H. and H, appear to be i n reasonable accord with expectation. 2a 4 4g 1 h  a  One of the more interesting points which we wished to evaluate i n the course of this study was whether i t might be possible to use a fluorine substituent to l a b e l a s p e c i f i c s i t e of a steroid for subsequent n.m.r. studies.  molecule 19  It i s obvious from the normal  F  spectrum of 1 (Fig. 5) that, for this molecule at l e a s t , conventional  Table 1.  N.M.R. Parameters f o r the Ring A Resonances of 2a-Fluorocholestan-3-one (1)  Chemical s h i f t s (x-values, <J> -values)  la  H  13  7.52  8.52  2B  4a  43  5.01  7.79  7.65  H  H  F  5  <j> +194.5 c  8.47  Coupling constants (Hz)  H  la 13 H  -12.2  +  H  la 23 H  12.3  +  H  13 23 H  V  6.9*  48.0  H. F 4a  R. H. 4a 43  H. H 4a 5a  H. H 43 5a  -13.9*  2.1*  14.4*  c  V  c  +  7.3*  V 11.5*  4.5*  H. F 43  H F 5a  -2.0  c  §  ^0  Measured i n deuterochloroform solution containing CHCl^ or CFCl^ *  Estimated error  + 0.2 Hz  Estimated error  + 0.4 Hz  Estimated error  + 1.0 Hz  +  §  Note: The error l i m i t s indicated by t and § represent the l i m i t s between which the coupling constants were found to vary when the i n d i v i d u a l transitions were varied over the maximum possible limit.  19 F measurements have l i t t l e to recommend them. However, the detection of a F resonance while simultaneously i r r a d i a t i n g the proton spectrum 19 appears to be a more a t t r a c t i v e proposition.  In the case of 1, the  "*"H resonances were spread over ca. 4 . 5 p.p.m. and continuous-wave 19 1 F - { H ) decoupling experiments the proton couplings.  only resulted i n p a r t i a l collapse of 19 1 However, noise-modulated F- H decoupling  e f f e c t i v e l y removed a l l of the proton couplings and, as i s indicated 19 i n the insert of F i g . 5 , the  F resonance was then detected as a  reasonably sharp s i n g l e t . 6 a - and 6g-Fluofo-cholest-4-en-3-one The normal ''"H n.m.r. spectra of 63-fluoro-cholest-4-en-3-one (compound 3) and 6ct-fluoro-cholest-4-en-3-one (compound 4 ) i n deuterochloroform solution are shown f o r comparison i n F i g . 6.  A few drops  of chloroform was used f o r the i n t e r n a l field-frequency lock.  and H, appear to low f i e l d i n each case. b 19 1 geminal  Both H.  H, shows the c h a r a c t e r i s t i c o  F- H coupling of ca. 50 Hz.  An interesting difference between the two spectra i s the s p l i t t i n g of the C^Q methyl group into a doublet i n the 6g-fluoro steroid ( F i g . 6A) whereas i t i s a sharp singlet i n the 6«-fluoro analog (Fig. 6 B ) .  H,  4.0  Fig. 6.  H 6/5  7.0  10.0  The normal H n.m.r. spectra of 3 (A) and 4 (B)in deuterochloroform solution containing CHCl^ for a field-frequency lock. The inserts are expansions of the C^Q methyl group region showing the effect of i r r a d i a t i o n at the 19p resonance frequency (94, 078, 509.0 Hz for 3 and 94, 079, 100.0 Hz for 4).  - 21 This phenomenon has been investigated and discussed for a number of fluorinated steroids by Cross and L a n d i s . ^ > ^ > B o t h 2  a  c  and  C^  methyl groups have been observed to be s p l i t into doublets under certain 19 conditions i n steroids containing a  F substituent.  Cross and Landis 19 l r a t i o n a l i z e d the s p l i t t i n g s i n terms of a long-range F- a coupling. 19 This explanation has now been confirmed for the f i r s t time by F 19 decoupling (see expanded inserts with and without Fig.  6.  F decoupling) i n  Such s p l i t t i n g s could be quite readily understood i f the 19  f l u o r i n e were close to the methyl group i n question - but when the  F  i s f i v e or s i x a bonds away, the phenomenon seems worth remarking on. The actual transmission mechanism of the coupling (both 'through bond' and 'through space' have been argued) i s s t i l l q u e s t i o n a b l e ^ ' ^ but 0  26c a l l known cases have been found to follow the "converging-vector rule": "long range coupling between angular methyl protons and fluorine five or more a-bonds apart may occur only when a vector directed along the C-F bond, and originating at the carbon atom, converges upon and intersects a vector drawn along an angular methyl C-H bond i n the d i r e c t i o n of the proton, and originating at the methyl carbon." 19 In both 3 and 4, the  resonance could be readily examined by  decoupling to determine the FH^ s p l i t t i n g .  The H^H^  s p l i t t i n g was  c l e a r l y assignable during these decoupling experiments. anomers part of the highly coupled H^H^  F also  In both  proton system i s v i s i b l e at the  extreme low-field end of the methylene envelope, but unambiguous INDOR work was not possible with this system - i t was both second order and d i f f i c u l t to assign. The systems comprising H,, H,, H., , H,„, H„ and 4 6 7a 76' 8 r  19  F were close  - 22 /  enough to f i r s t order to enable INDOR analysis. set  was the l o g i c a l  of transitions to monitor during INDOR experiments  find the  protons and Hg.  Unfortunately the  designed to  lines were broadened  19 and poorly-resolved f o r both isomers. possible to monitor protons (Fig. 6).  With  F decoupling i t was  while running INDOR scans to find both However, the quality of the INDOR responses did not  allow an unambiguous determination of the p o s i t i o n of Hg.  For both  3 and 4 the 7a-proton was e n t i r e l y hidden i n the methylene envelope, but several t r a n s i t i o n s of the 7g-proton could be distinguished. 19 Without the lower.  F decoupling, the INDOR response quality was even  However, the g ~ F s p l i t t i n g s could be picked out f o r 3 and 4 H  7  by direct comparison between the decoupled and undecoupled spectra. 19 A good estimate of the H -F couplings was also obtained from the F /a  7  spectra. At this stage, the next step should have been to analyze the chemical s h i f t and s p l i t t i n g data (with the help of a computer program) to obtain 'true' chemical s h i f t s and coupling constants.  This was not  done here because a r e l i a b l e value for the H_ chemical s h i f t was not o  found through the INDOR experiments. Cg, and  Nevertheless, the n u c l e i at C^,  can be seen (Fig. 6) to form a v i r t u a l l y f i r s t order system -  and the H^ transitions found by INDOR seem to indicate that Hg i s not very close to either.  This means that one can f a i r l y safely take the  observed chemical s h i f t s and s p l i t t i n g s to represent true, f i r s t order values within experimental error (Table 2). 19 The normal  F n.m.r. spectrum of 3 i s shown i n F i g . 7A, and that  of 4 i n F i g . 7B. That of compound 3 i n p a r t i c u l a r i s broadened by  - 23 -  Table 2. N.M.R. Parameters f o r Farts of Rings A and B of 6 a - and 6g-Fluoro-cholest-4-en-3-one  (3 and 4)  F i r s t order chemical s h i f t s (x-values, (j^-values) Compound  H. 4  H, 6  F, 6  3 (63-F)  4.16  5.05  4 (6a-F)  3.97  4.94  H., 7a  H., 7g  $ + 165.61  8.81  7.84  $ + 183.63  8.79  7.74  C .-CH„ 10 J  n  ir  8.70 8.84  F i r s t order coupling constants (Hz) Compound 3  Compound 4 r  H.H, 4 oa  H. F 6a  H^ H_„ 6a 73  49.5  2.5±0.8  H-j H., 7a 73  H H_ 73 8  C -CH F 10 3  -13.5±1.0  3.0±0.8  1.9  in  H^ H^ 6a 7a 2.6±0.8  H^ F 7a 47±3  H^ H„ 7a o 13.0±1.0  H., F 73  ^,F 4  13.6  H.H, 4 66  H,.F 63  H,.H . 63 73  H,H._ 63 7a  H H 7a 8  1.8  47.8  5.8  11.8  11.4  H_. 7a 76  H_ H 76 8  C -CH_F 10 3  D  H  7  -10.8  Q  Q  2.5  7  in  0  Q  H.. F 7a 8.8  7  5.0  Q  H..F 73 4.7  H.F 4 0.7  Measured i n deuterochloroform solution containing CHCl^ or CFCl^ Unless otherwise indicated, the errors i n the above f i r s t order coupling constants are roughly 1 0.3 Hz.  B  100  F i g . 7.  i  q  100.5  HZ  t  100 p  HZ "1  109.6  The F n.m.r. spectra (94.071 MHz) of 3 (A), 4(B) and 5 (C) i n deuterochloroform solution. CFCl^ was used for the i n t e r n a l field-frequency lock.  -  25 1  unresolved s p l i t t i n g s - but i n both cases, the recognizable i n the fluorine spectra.  19  H-  F couplings are  In this series of experiments we have been able to find the resonances of nuclei associated with an appreciable portion of rings 4 A and B.  Wittstruck et a l . have studied several A -3-keto fluoro  steroids by n.m.r. spin-coupled to  They showed that the 6g-fluorine i s more strongly than i s a 6a-fluorine.  We have observed the same  phenomenon here. The INDOR technique i n this case supplied several useful points. The location of the  proton chemical s h i f t s permitted a check on the  f i r s t order nature of the observed H,-H_, s p l i t t i n g s and the summation o of the INDOR responses gave the H..-H  / splittings.  The  combination  19 of a  F probe and noise modulated  decoupling gave a set of v i c i n a l 1 1  H-F couplings as well as making the study possible. 1 and  Both  H- H  19  H-  F v i n c i n a l couplings i n compounds 3 and 4 are i n d i c a t i v e of  an approximate  chair conformation f o r ring B i n spite of the double  bond i n ring A. 19 With regard to the p o s s i b i l i t y of using normal F spectra to study a labelled steroid, the spectrum of compound 4 seems to be more 19 encouraging than that of 1 or 3.  Although the  F spectrum of 3 i s broad,  16,16-Difluoro-androst-5-en-3g-ol-17-one that of 4 could be useful. This compound provided, amongst other things, insight into the 6 7 D-ring conformation - a subject of some speculation. '  Fig. 8  19 displays the  F decoupled proton spectrum of 16,16-difluoro-androst-4-  en-3,17-dione (5) i n deuterochloroform solution:  chloroform was used for  - 26 -  the i n t e r n a l protons:  field-frequency  The i n s e r t  t h e i r normal spectrum on the l e f t  spectrum on the r i g h t . vector  lock.  shows the C^g and  19  and t h e i r  F decoupled  Here we have a n o t h e r example of the  r u l e " but t h i s time i t  "converging-  i s the C^^ m e t h y l group w h i c h i s  by a f i v e bond c o u p l i n g to the 3 - f l u o r i n e .  The a b i l i t y t o  split  decouple  19 the  F n u c l e u s a l l o w e d an a c c u r a t e measurement of  18  Once a g a i n ,  two s e t s of d a t a were a v a i l a b l e  d e c o u p l i n g the  1 D  O  19 of n o i s e  J  due t o the  1 F nuclei.  H transitions  associated with  the r e g i o n of i n t e r e s t were r e a d i l y found by s w i t c h i n g the power on and o f f , set  and comparing the s p e c t r a .  o f INDOR experiments  simplicity  (Fig.  decoupling  Then, a f i r s t ,  simple  8A, B, C, D) was run w h i l e i r r a d i a t i n g  19 b o t h of the  of b o t h H.. and 15a H - ^ g c o u l d be m o n i t o r e d w h i l e s c a n n i n g a weak r a d i o - f r e q u e n c y f i e l d through the methylene r e g i o n . showed up as a f a i r l y complex s e t 19 of t r a n s i t i o n s  F nuclei.  In t h i s case, t r a n s i t i o n s  because even w i t h o u t the  four other protons.  However, i t s  c  F n u c l e i i t i s coupled  into  c h e m i c a l s h i f t was r e a d i l y f o u n d .  Note t h a t m o n i t o r i n g l i n e 4 (a degenerate l i n e of H _ ) l e a d s  to  I 8.  .  I  ,  1  7.5  .  I  •  1  .  !  8.0  .  I  8.5  .  I  • . I  r  9.0  , . I  1 1 19 The H-{ H} INDOR experiments performed on the F decoupled spectrum of 5 i n d e u t e r o c h l o r o f o r m s o l u t i o n . H 1 5 t r a n s i t i o n s were m o n i t o r e d as f o l l o w s : A t r a n s i t i o n 1; B t r a n s i t i o n 2 ; C t r a n s i t i o n 4 ; D t r a n s i t i o n 3. The assignment r e s u l t i n g from t h e s e s p e c t r a i s shown d i a g r a m m a t i c a l l y above the n o r m a l p r o t o n s p e c t r u m . The i n s e r t shows the e f f e c t of - F d e c o u p l i n g on the C 1 0 and ( ^ 3 methyl groups. D e c o u p l i n g was by n o i s e modulated i r r a d i a t i o n at 94, 084, 210.0 Hz. L 9  - 28 -  responses for a l l four l i n e s of H.^.^ (Fig- 8C). The e f f e c t i v e summation of the INDOR responses i s shown above the spectrum and c l e a r l y gives a l l the information needed for analysis of the D-ring protons. 19 After switching off the  F decoupling  i t was possible to obtain  a second series of INDOR responses for H.. ' and H loot  1 C  „ which included  i j p  1 19 H- F s p l i t t i n g s . Note that the C-methyl resonances give r i s e to their usual a r t i f a c t responses i n the INDOR spectra. 19 The normal  F spectrum of 5 i s shown i n Fig. 7C. The spectrum  i s sharp and well resolved.  I t displays the t y p i c a l large (ca. 284.6 Hz)  . . 19_ 19_ , . geminal F- F coupling. The experiments described above provided enough data to completely analyze the D-ring n u c l e i of 5.  The data was analyzed with the help of  a computer program to obtain the true coupling constants  and chemical :  s h i f t s (Table 3). One would expect to be able to say something about the D-ring conformation at this stage.  I t i s well known that the Karplus-type  curves for v i c i n a l ^"H-^H and ^H-^F coupling constants must be empirically derived to a large extent and depend upon substituents and h y b r i d i z a t i o n amongst other things; furthermore, applications to five-membered rings appear to be p a r t i c u l a r l y d i f f i c u l t .  I t seems that the most sensible  approach when using such curves to determine ring geometry i s to consider the general trend of as large as possible a number of couplings around the ring. B  Thus J., . .. _ = 14.0 Hz would d e f i n i t e l y indicate a 14,153 r  trans-diaxial relationship of the two protons and J.. . .. 14,15a c  r  v  = 5.9 Hz  - 29 Table 3.  N.M.R. Parameters f o r the Ring D of 16,16-Difluoro-androst5-en-3g-ol-17-one (5)  Chemical s h i f t s (x-values, <j) -values)  H  14  H  8.55  15a  H  15g  7.60  F  8.04  16a  F  < j > + 109.57 c  16g  C  13~ 3 CH  a) + 100.49 c  8.95  Coupling constants (Hz)  H  14 15a H  H  14 15B  5.9*  H  15g 16B F  16.3*  H  14.0 H  +  F 15p*16a 22.6*  H  15a 15B  H  H  -14.2  CH -CH F 3  +  0.9  F  Estimated error ± 0.4 Hz.  F  19.6*  16g  13  3 16a  0  Measured i n deuterochloroform containing CHCl^ or CFC1 Estimated error ± 0 . 3 Hz.  ^15a 16a  -0.7*  +  13  15a 163  F  16a 16g F  284.6*  - 30 -  indicates roughly a 60° dihedral angle for this pair. resonances  are now  suggests a ^90° dihedral  lop  15a  the same geometry.  F  readily i d e n t i f i e d as 16a u p f i e l d and 16|3 downfield.  The -0.7 Hz coupling between H . a n d F., angle between them.  The  The remaining three H-j.5^16 couplings agree with It seems then pretty safe to draw the D-ring of 5 as:  where carbons 13, 17, 16 and 15 are i n a plane.  However, the geometry  3 may well be d i f f e r e n t when carbon 17 i s sp  hydridized.  In fact i t  has been suggested that carbons 14, 15, 16 and 17 are i n a plane for the 17-OH  and 176-acetyl  7 28 analogues.' 19  It seems that i n this compound at l e a s t , the useful "normal" n.m.r. spectra.  F labels have very  Nevertheless, they can be reduced to  sharp singlets by noise modulated decoupling of the entire proton 19 region. S i m i l a r l y , the F s p l i t t i n g s could be readily removed from 19 the proton spectrum by noise modulated decoupling at the F frequency. 19 1 It can be concluded from the above studies that 1 H-{  F-{ H} and  19 F} heteronuclear  decoupling experiments can considerably f a c i l i t a t e  n.m.r. studies of fluorinated s t e r o i d a l systems.  In p a r t i c u l a r , the  observation that noise-modulated i r r a d i a t i o n of the entire ^H spectrum 19 can e f f e c t i v e l y reduce a F resonance to a narrow singlet augurs well  - 31 for the future of fluorine as a probe for evaluating the i n t e r a c t i o n of s t e r o i d a l derivatives with other systems of biochemical significance such as membranes.  We also conclude that ^"H-{^H} INDOR measurements  should find widespread application i n a number of s t r u c t u r a l problems commonly encountered i n natural product chemistry.  For, although the  19 combination of technique,  F l a b e l l i n g and double resonance can be a powerful the presence of a fluorine substituent i s not a  necessary prerequisite for such measurements.  The next section w i l l  discuss t h i s . B.  Application of H-{ H} INDOR to Natural Products 1  1  The examples discussed i n the previous section of this thesis were chosen with a variety of d i f f e r e n t points i n mind; however they a l l indicated that "^-{"Si} INDOR measurements greatly f a c i l i t a t e d the assignment  of  n.m.r. spectra of "natural products".  It now remained  to show whether or not such measurements could be u s e f u l l y applied routinely to solve the type of s t r u c t u r a l problem which often occurs during laboratory syntheses of natural products or of related precursors. The following examples were chosen at random from several of the U.B.C. laboratories concerned with natural product chemistry i n order to investigate this point. (i)  A Steroidal Problem  As part of another programme, Weiler and Paisley (private communication) were attempting to perform the conversions indicated i n the flow sheet below.  The problem with which we', were presented was as  follows:- the workers had a crude reaction mixture whose n.m.r. spectrum i s shown i n F i g . 9.  The mixture contains starting material and at least  H •  2  i NJ  I  0.0 Fig. 9.  5.0  9.0  X  The h n.m.r. spectrum (100 MHz) of a steroid (6) reaction mixture i n deuterochloroform solution. The position of the C H C I 3 i n t e r n a l field-frequency lock i s shown. Chemical s h i f t positions of the protons of i n t e r e s t are shown.  - 33 -  one side product.  The only clear spectral features associated with  the molecular s i t e of interest are two aldehyde groups around x 0.2. Of the several possible alternative products, only the desired product should involve the aldehyde proton as part of an AMX system ( i . e . only 3 protons:  one at  and two at C^). Paisley believed  that the more intense aldehyde group represented the desired product while the other was a biproduct.  The peaks of the downfield  aldehyde  group were monitored i n turn while sweeping a perturbing radiofrequency f i e l d through the methylene envelope. was obtained by summing the INDOR responses:  A p a i r of quartets  one at x 7.93 and the  other at x 7.48 (Fig. 9 and Table 4). Both of these quartets were otherwise e n t i r e l y hidden i n the methylene envelope. In this case, the INDOR technique provided n.m.r. data which agreed with the proposed structure of the major product.  I.R. and  mass spectroscopy were of no use i n d i f f e r e n t i a t i n g amongst the various possibilities.  Chemical confirmation was doubtful because of the known  p o s s i b i l i t y of anomerization during reaction.  Several other possible  products could also have produced an aldehyde quartet e.g.  (8 could have a quartet f o r the aldehyde v i a second order coupling to one of the C, protons). 6  But neither of these compounds would have  produced the simple AMX system found by the INDOR experiments. one very useful feat  lire  Thus  cf the INDOR method i s that i t can be applied  to crude mixtures of compounds. (ii)  Some Decalone Problems  A reaction sequence involving the A-B ring junction was thought to have produced the following two decalones which were subsequently separated by G.L.C. (Piers and P h i l l i p s - private communication).  Proposed minor product  It  Proposed major product  was hoped that their n.m.r. spectra would provide a check of the above assignment. Fig.  The  n.m.r. spectrum of the minor product i s given i n  10A and that of the major product i n F i g . 10B.  dissolved i n deuterochloroform.  In each case the  Both samples were methyl group i s  readily i d e n t i f i e d by comparison with other compounds and by resonance experiments region.  double  (Fig. 10) as the downfield doublet i n the methyl  In the case of the minor product  (9), several protons are  partly resolved at the low f i e l d end of the methylene envelope.  In the  case of the major product (10), only one proton i s resolved. For both compounds i t was possible to perform INDOR experiments i n which the  methyl group l i n e s were monitored i n turn while  sweeping a weak radio-frequency f i e l d through the downfield methylene envelope region.  In each case a complex series of responses  was  obtained for H^ due to the degeneracy of the methyl group protons and the overlap of  transitions.  chemical s h i f t value f o r  The information so obtained was a  (Table 4) and the fact that i n both compounds  the H. t r a n s i t i o n s were spread over at least 32 Hz. 4  If the  H.-H_ 4 5  coupling were not a x i a l - a x i a l (e.g. axial-equatorial) the H^ transitions would be spread over only 22-25 Hz at most. the proposed  This result agrees with  structures given above.  The remaining v i s i b l e downfield t r a n s i t i o n s for 9 were assigned, on the basis of their chemical s h i f t and small number of couplings, to B.^  and  ^  monitoring these t r a n s i t i o n s i n turn while scanning  a weak perturbing f i e l d through the rest of the spectrum, i t was possible to pick out a l l of the  proton transitions and to see that  they formed a r e l a t i v e l y f i r s t order system (Fig. 10A and Table 4 ) . was not unambiguously located for 9 as only 8 mg of sample was  H^  available  -  Table 4.  36 -  N.M.R. Parameters f o r Compounds 6 (Cyano-aldehyde), and 11  (Decalones)  *  F i r s t order chemical s h i f t s Compound  6  %  7.48  (x-values)  l  H  H  7.93  C.-CH. 4 3  H  2  0.14  4  H  26  H  2a  H  l  9  8.97  7.33  7.52  7.86  -  10  8.99  7.88  7.93  7.66  8.50  C -CH 1  11  3  l  H  9.08  H  H  2  2  1 7.20  8.08  7.93  F i r s t order coupling constants (Hz) Compound H  6  9  6.4* C, — CH _H, 4 3 4  10  1  1.4* C.—CH„H, 4 3 4  H 2  H  H  2a 23 H  +  6.4*  12.6  7.2*  Measured i n deuterochr'oroform Estimated error + 0.4 Hz.  H  2B 13 H  +  3.9 H  2B la t 12.6 H  -  H  2a 16 H  13.0*  ^2a^la t 3.8 T  H H  H  14.5*  Estimated error I 0,2 Hz.  H 1 2  14.5+1.0  2a 2B t 13.0  H  1 11  H  2  4.0*  H  *  9, 10,  2 1.4*  2 t 6.2*  solution containing CHC1  - 37 and the s i g n a l - t o - n o i s e r a t i o was low. About three times as much of 10 was available so that i t was possible to locate a l l of the  proton transitions and a set of weaker  INDOR responses gave a chemical s h i f t value for H^.  In the case of 10,  only one proton was v i s i b l e at the l o w - f i e l d end of the methylene envelope and i t s transitions were monitored during INDOR experiments to f i n d the others (Fig.  10B and Table 4).  The s p l i t t i n g s quoted above are c e r t a i n l y close enough to f i r s t order values to be used for gross conformational a n a l y s i s . results are i n t e r e s t i n g . expect J  0  -  Za ,J-p  and J  Zp,lp  The  From the structure proposed for 9 one would <  6 Hz.  But the values in Table 4 suggest  that H^ i s a x i a l rather than e q u a t o r i a l .  Thus the n.m.r. data agree  with the structure proposed for 10 but not for 9. Partly as a result of this c o n f l i c t i n g n.m.r.  data, Piers  and P h i l l i p s were led to propose that the AB ring junction had formed  c i s instead of trans during the Birch reduction of a 4,5 double bond. This would avoid the s t e r i c s t r a i n caused by an a x i a l isopropyl group. It  should be noted that the n.m.r.  between 9 and 10.  It  INDOR data does not distinguish  only says that 9 i s not the compound i t was  8.0 10.  9.0  The % n.m.r. spectra of decalones 9 (A), 10 (B) and 11 (C) at 100 MHz' i n deuterochloroform containing enough C H C I 3 for a field-frequency lock. The f i r s t order proton assignments of interest are indicated diagrammatically.  - 39 -  o r i g i n a l l y thought to be.  But once again, INDOR provided important  s t r u c t u r a l information. Fig. IOC shows the ^"H n.m.r. spectrum of a decalone produced v i a Birch reduction i n a manner very s i m i l a r to 9 and 10.  It was  thought  to have one of the following structures:  The downfield quartet (Fig. 10C) was made the subject of a set of INDOR experiments.  Strong responses were obtained for a geminal partner,  and weaker responses for a v i c i n a l proton.  Both of these other protons  were hidden i n the methylene envelope and they f e l l close together (Table 4).  By monitoring the C^ methyl doublet while scanning a weak  radio-frequency f i e l d through the methylene envelope, the abovementioned v i c i n a l partner was  shown to be  H^.  The data shown i n Table 4 support structure (a). would be expected to have one of the H^-H^ Hz.  The  Structure (b)  s p l i t t i n g s greater than 10  protons have not been assigned as a or 3 i n the table.  However, i t seems quite l i k e l y that the downfield proton i s the upfield one i s H  . Za  an<  ^  The a x i a l C. methyl group might be expected 1  to d i s t o r t the A-ring i n such a fashion that the dihedral angle between H. and H„„ became less than 60° (.'. J a. 6.2 Hz) and that between H.. and 1 23 1 H  0  somewhat more than 60° (.'. J ^ 1.4 Hz).  - 40 -  From the above examples, i t seem reasonable to conclude that the *"H-{^H} INDOR technique should f i n d considerable application as an a n a l y t i c a l tool i n natural product chemistry.  -  41 -  Experimental General Methods (a)  A l l n.m.r. measurements were made with a modified Varian  HA-100 spectrometer operating i n the frequency-swept mode. (b)  The equipment required for the heteronuclear decoupling 29 30  experiments has been described elsewhere.  '  An account of the  modifications necessary for the measurement of has also been given (c)  H-{  INDOR spectra  previously."^  A l l n.m.r. measurements were made i n deuterochloroform  solution.  Since the spectrometer gain i s generally quite high for  INDOR experiments i t  i s often desirable to lock onto something as far  away from the region of interest as p o s s i b l e . CHCl^ was found to be u s e f u l .  For natural products  Proton chemical s h i f t s are reported i n  the T s c a l e . (d)  Where p o s s i b l e , analyses of the n.m.r.  with a modified version of the LA0C00N III  spectra were made  program and an I.B.M. 360-67  computer i n the U.B.C. Computer Centre. 2ot-Fluoro-cholestan-3-one  ( 1 ) was prepared as described i n the  32  literature. 6g- and 6q-Fluoro-cholest-4-en-3-one ( 3 and 4) were prepared 20 32 33  according to general procedures described i n the l i t e r a t u r e .  '  '  1 6 , 1 6 - D i f luoro-andros t-5-en-3-ol-17^-one ( 5 ) was prepared as 34  described i n the  literature.  The non-fluor'inated natural products studied were obtained from L. Weiler and K. Paisley (steroid) and E. Piers and N. P h i l l i p s (decalones) -  (private communication).  CHAPTER II FLUORINE AND PROTON N.M.R. AS APPLIED TO CERTAIN ASPECTS OF THE INTERACTION OF LYSOZYME* WITH MONOSACCHARIDE INHIBITORS Introduction The applications of n.m.r. spectroscopy to the study of structure and mechanism i n b i o l o g i c a l systems are becoming increasingly widespread.  Perhaps this should not be surprising as i t i s one of  the only two methods capable of detecting i n d i v i d u a l atoms i n macromolecular  systems - the other method being X-ray d i f f r a c t i o n 35  studies of c r y s t a l s .  In cases where a very large molecule (for  instance a protein) can be i s o l a t e d i n a pure, c r y s t a l l i n e form, X-ray d i f f r a c t i o n can y i e l d uniquely valuable information concerning i t s fi 36a,b,c , . 37a,b , , ^ .38 geometry (lysozyme, carboxypeptidase A, and chymotrypsm being cases i n point).  N.m.r. i s a t t r a c t i v e because i t o f f e r s , at  least i n p r i n c i p l e , straightforward techniques for the detailed investigation of the structure of large molecules i n solution as w e l l as techniques for studying the dynamic aspects of molecular structure and i n t e r a c t i o n .  An enzyme found i n many animal tissues. A l l work described here was done on lysozyme derived from the whites of hens' eggs.  - 43 There are a number of very good review a r t i c l e s i n the recent l i t e r a t u r e dealing with the uses of n.m.r. spectroscopy i n b i o l o g i c a l 39-43 systems.  There i s no one,universal n.m.r. approach to such  problems.  A whole area has grown up around the use of paramagnetic 40  probes i n n.m.r. studies of enzyme-substrate complexes.  A more  obvious approach i s the use of normal high resolution n.m.r. spectra to study structure, and pH effects thereon, of 'small' b i o l o g i c a l building 42 blocks such as amino acids, peptides etc.  A recent, but very  important, area of endeavour i s the application of n.m.r. to the study of rate phenomena i n biochemical systems.  Chemists have been using 44 n.m.r. to study rate processes for a good ten years, but b i o l o g i c a l 45 applications have been longer i n appearing. Recent technological advances have opened up whole new areas to the n.m.r. spectroscopist 41 interested i n biochemical studies. Typical examples include 13 Fourier transform methods f o r measuring natural abundance  C n.m.r.  spectra, superconducting magnets f o r measuring "hi spectra at 220 or 300 MHz and also studies of "^N n.m.r. spectra. In addition to weak signals, a major d i f f i c u l t y i n applying n.m.r. to the study of biopolymers i s that the combination of large linewidths due to slow reorientation, and the presence of many closely spaced lines i n the spectrum, often produces featureless broad bands.  The  signal-to-noise problem can be overcome to some extent by time-averaging methods.  The other d i f f i c u l t i e s have inspired a variety of more or  less i n d i r e c t approaches.  Obviously spectrometers with higher magnetic  f i e l d s give better separation of spectral features.  Also one may hope  to leam about macromolecules by studying the simpler spectra of their  - 44 -  breakdown products or by looking at synthetic model systems (e.g., the use of n.m.r. i n c e l l membrane studies  46  ). In favourable cases,  the n.m.r. spectrum of a biopolymer may contain peaks or regions i d e n t i f i a b l e with some s i t e of interest and such spectral features provide probes of their surroundings (e.g., lysozyme, ^ > ^ r i b o n u c l e a s e ^ ) . a  In less favourable cases, s e l e c t i v e deuteration or heteronuclear l a b e l l i n g may be t r i e d . It i s also possible to observe the spectrum of a small molecule or ion which interacts with the biopolymer.  Such interactions may  produce observable changes i n l i n e positions and/or relaxation times which can be related to phenomena of i n t e r e s t .  The use of ions i n  such experiments generally involves relaxation time measurements - the results being t y p i c a l l y treated as outlined i n Stengle and Baldeschwieler's 49 ion-probe method.  The technique of comparing the n.m.r. spectrum  of a small molecule before and a f t e r addition of some biopolymer i s an i n d i r e c t but very promising one and i s the method considered i n this chapter. The object of any such experiment i s to get information about some facet of a large molecule's geometry or behaviour.  A small  molecule i s chosen which has an observable spectrum and which interacts with the larger molecule i n such a way as to r e f l e c t upon some s i t e of interest. case"^'"^  A good k i n e t i c model for many problems i s the "two s i t e The small molecule i s thought of as existing either  free i n solution ( s i t e A) or associated i n some way with the biopolymer Relaxation phenomena w i l l be discussed i n Chapter I I I .  -  molecule ( s i t e B).  Thus  45  -  f E = enzyme I = inhibitor  E  +  =  ^  EI  I  Kg = the binding constant  [EI] [E][I]  There may be considerable information on the rates of the exchange processes involved contained i n the relaxation phenomena associated with such a system (see Chapter III).  In general, the two s i t e s  available to a small molecule may be characterized by different resonance frequencies, to. (bound) and UL ( f r e e ) . A  This i s so because  B  i n general the magnetic properties of s i t e A w i l l d i f f e r from those of s i t e B.  This may be p a r t i c u l a r l y noticeable i f the bound molecule  i s proximal  to an aromatic system or the e l e c t r i c f i e l d of a polar  group or metal i o n .  Here i t s u f f i c e s to say that i f the rate of  exchange (k_^sec ^) i s very small compared to the difference i n frequency, and uv,.  If  (u>^-u)g)  f  then separate resonances w i l l occur at  the reverse i s true, then a single average resonance w i l l  D  appear between the two frequencies at a distance 6  = P^A  from  uj /2ir where P „ i s the f r a c t i o n of the small molecule which i s bound A B A  at a given time and A i s the chemical s h i f t and unbound species.  (io^-u>g) /2TT between bound  This l a t t e r s i t u a t i o n has been found to occur  regularly for reversible i n h i b i t o r s interacting with lysozyme.  The  chemical s h i f t of an observed i n h i b i t o r as a function of concentration, t  See Experimental Section for a more complete description.  - 46 -  pH or temperature y i e l d s information which can be used to calculate K^, A and thermodynamic  constants.  Lysozyme 54 Like a l l enzymes, lysozyme i s a globular protein  - a series of  amino acids bound together i n a chain by peptide bonds and folded into a complex three-dimensional structure.  Lysozyme consists of 129 amino  acid subunits of 20 d i f f e r e n t kinds and has a molecular 14,400."^  Thus i t i s a very small enzyme.  weight of about  It i s also exceptionally  stable i n aqueous solution and i s readily p u r i f i e d and c r y s t a l l i z e d . These properties contributed  to i t s being the f i r s t enzyme to have i t s  complete three dimensional structure determined (X-ray d i f f r a c 36a,b,c,56,57,. _ ., . _ , ,, tion ). Lysozyme provides then a very useful model enzyme system f o r solution study. Lysozyme's b i o l o g i c a l function i n animals (where i t i s found i n many tissues) i s to destroy  the c e l l walls of certain bacteria by  catalysing the hydrolysis of the carbohydrate component, s p e c i f i c a l l y a g-l,4-linked polysaccharide  with alternate N-acetyl  and N-acetyl muramic acid (NAM) residues.  This reaction proceeds with  cleavage of a C^-oxygen bond i n the substrate. section of such a polysaccharide during l y s i s . residues.  glucosamine (NAG)  F i g . 1 shows the  which would be bound to the enzyme  A, C and E are NAG residues, B, D and F are NAM  - 47 -  \ \  The enzyme's a b i l i t y to perform this c a t a l y t i c function does not depend so much on the chemical nature of i t s contituents as on i t s folded, three-dimensional structure.  Several generalizations concern-  ing the gross conformation of lysozyme have been noted i n the l i t e r a t u r e . P e p t i d e s with hydrophilic side chains (-acid or base) are found largely on the surface of the enzyme.  On the other hand,  most of the markedly hydrophobic side chains are shielded from the surrounding aqueous medium by more polar parts of the molecule.  The  o v e r a l l folding of the lysozyme polypeptide chain has led to a structure with a deep c l e f t running up one side.  It i s this c l e f t which contains  the 'active s i t e ' . It i s not possible to perform X-ray studies of lysozyme interacting with a b a c t e r i a l c e l l w a l l .  But i t has been possible to grow crystals  of the enzyme Interacting with small " i n h i b i t o r " sugar molecules. These are species which can i n h i b i t the action of lysozyme by themselves binding to the active s i t e i n a manner s i m i l a r to that of the true substrate but which themselves react only slowly i f at a l l . Lysozyme's action i s known  to be i n h i b i t e d by such N-acetate-  containing polysaccharide breakdown products as the previously mentioned NAG  and NAM  and also by the C^-iodo derivative of NAG,  the disaccharide,  58 chitobiose (di-NAG) and the trisaccharide, c h i t o t r i o s e (tri-NAG). 36c It was  primarily X-ray studies of lysozyme i n t e r a c t i n g with  that led to the binding array shown i n F i g . 1.  tri-NAG  This array places a  monosaccharide subunit i n each of 6 subsites (A to F) i n the c l e f t of lysozyme.  The bond cleaved i s shown by a dotted l i n e .  consisting of NAG  Polysaccharides  up to the tetrasaccharide are known to bind with  58 59 their reducing end i n subsite C  '  and consecutive sugar rings i n  subsites B and then A (hence the monosaccharide NAG C).  occupies subsite  From the proximity of the sugar residues to polypeptide side  chains i n the c l e f t  (as indicated by X-ray s t u d i e s ) , modes of bonding  have been postulated"^'"^° f o r a l l s i x subsites.  Workers have been  quite s p e c i f i c about l i k e l y hydrogen bonding interactions but less s p e c i f i c about nonpolar ones.  Nevertheless nonpolar binding seems  l i k e l y to play i n important part i n view of the " o i l droplet with a 36c polar coat" nature of lysozyme. More recent solution studies have 60 tended to bear out the X-ray postulates.  The proposed mechanism  for l y s i s " ^ ' " ^ involves the c a t a l y t i c e f f e c t of the proximate peptide 0  side chains of residue 35 (glutamic acid) and 52 (aspartic acid) on the C-0 bond i n question (Fig. 1).  It has been suggested  that  of  -  49 -  ring D passes through a carbonium ion intermediate and that binding of the substrate d i s t o r t s ring D.  X-ray data on lysozyme has formed a  basis for a l l the more recent solution work.  Lysozyme and the Chemical S h i f t Technique Previous studies from this laboratory have shown^^ '^ that ~^F 3  n.m.r..parameters are more sensitive to changes i n chemical environment than are  n.m.r. parameters.  Because of this we have been interested  i n the p o s s i b i l i t y of using fluorine substituents as s t r u c t u r a l probes for investigating a variety of chemical problems.  From the n.m.r.  viewpoint any such heteronuclesr probe has a number of obvious advantages and this i s p a r t i c u l a r l y so i n the study of biomolecular associations where i t i s often d i f f i c u l t  to i d e n t i f y unequivocally a p a r t i c u l a r  proton resonance. In view of the strong stereoelectronic preference of the carbon62 63 fluorine bond,  '  i t seems l i k e l y that i n some cases the introduction  19 of a  F substituent may seriously perturb  the system of  interest.  However, such a perturbation might i t s e l f y i e l d valuable information as we s h a l l show.  Lysozyme seemed to be an i d e a l model for studying  this phenomenon because of the detailed X-ray and n . m . r . ' ^ ' ^ data which are already a v a i l a b l e . acetyl derivatives  ^  For these studies four N - t r i f l u o r o -  (1-4) of D-glucosamine  were synthesized  S t r i c t l y these are derivatives of 2-deoxy-2-trifluoroacetamido-Dglucopyranose.  - 50 -  CH OH 2  which correspond to four known i n h i b i t o r s (5-8)  of lysozyme.  These l a t t e r compounds (5-8) have been extensively by Raftery et a l . ^ ^  and by Sykes et a l . ^ ' ^  the N-acetate peaks).  Values of  studied v i a n.m.r.  (both of whom monitored  and A obtained for such compounds  necessarily have a high experimental error ( t y p i c a l l y + 10-20%). one must be c a r e f u l not to place undue emphasis on small  Hence  differences  i n measured values between different compounds. Our  studies of the t r i f l u o r o derivatives (in which we have monitored  the N-trifluoroacetate peaks) subsequently led us to investigate other problems:  two  (a)  The e f f e c t of substituents at C,  (b)  The conformation of i n h i b i t o r s .  The former problem was approached by synthesizing and studying the following series of compounds:  CH R 2  NHCOCH3  Inhibitor  conformations were studied by analysis of the ^Ti n.m.r.  spectra of t h e i r O-acetates.  We have also b r i e f l y considered the  p o s s i b i l i t y of studying the conformation of bound i n h i b i t o r s .  - 52 -  Results and Discussion A.  Choice of a Suitable Chemical S h i f t Reference The most accurate way  spectroscopy  to measure small chemical s h i f t s i n n.m.r.  i s by comparison with some reference compound.  When  using organic solvents i t i s common p r a c t i c e to employ tetramethylsilane (TMS)  as an i n t e r n a l reference for measuring proton chemical  and freon 11 (CFCl^)  for f l u o r i n e work.  are carried out i n aqueous s o l u t i o n . c a p i l l a r y of TMS  shifts,  However, enzyme studies  T h e o r e t i c a l l y one could use a  or freon 11 held concentric with the n.m.r. tube 69  (see Experimental  Section).  But i t has been shown  s h i f t s measured r e l a t i v e to such external standards  that  chemical  are very prone to  bulk magnetic s u s c e p t i b i l i t y e f f e c t s - e s p e c i a l l y when the temperature i s varied between samples. For proton work i t i s convenient frequency dependent.  lock;  to use the water peak for a f i e l d -  but this peak i s notoriously temperature and  pH  Hence a small quantity of some other species must be added  for use as an i n t e r n a l standard.  For lysozyme work, both a c e t o n e ^  ^  and t_-butanol^ have been employed. 19 For a was  used.  F field-frequency lock a c a p i l l a r y of t r i f l u o r o a c e t i c acid This was  found to be very suitable for work with a t r i f l u o r o -  acetyl label as extensive f i e l d offsets'were not required.  We have  considered the p o s s i b i l i t y of using sodium t r i f l u o r o a c e t a t e (NaOCOCF^) as an i n t e r n a l reference. Unfortunately, i t was to s h i f t and broaden.  I t i s very soluble and quite i n e r t .  found to interact with lysozyme i n such a way  as  Other i n t e r n a l references t r i e d were t r i f l u o r o -  ethanol (CFoCH„0H) and hexafluoroacetone  sesquihydrate.  The former  - 53 -  I  appears as a very sharp t r i p l e t quite close to the region of the fluoroacetate l a b e l (Fig. 2).  tri-  I t shows no sign of broadening or  s h i f t i n g on addition of lysozyme (Table 2) and was  used i n a l l f l u o r i n e  chemical s h i f t studies. Trifluoroethanol i s somewhat a c i d i c . made to see that i t was  For this reason a check was  not overloading the 0.1 M pH 5.5  buffer used for this work.  citrate  Twice the concentration of t r i f l u o r o e t h a n o l  actually employed during enzyme runs was  seen to have no noticeable  e f f e c t on the buffer pH, hence no problem i s to be expected.  B.  Experiments with  N-Trifluoroacetyl-D-glucosamine  The monosaccharide, N-acetyl-D-glucosamine (NAG-anomers 5 and are known"^'"^'^ from X-ray studies to bind s p e c i f i c a l l y  6)  and  reversibly at subsite C on the enzyme surface (although the a-anomer i s a s p e c i a l case).  The methyl glycosides (7 and 8) seem l i k e l y " ^ ' ^  to behave i n a s i m i l a r fashion to 6 with respect to binding to l y s o zyme.  60,58 In a l l four cases, the  resonance of the N-acetyl group shows  a d e f i n i t e u p f i e l d s h i f t ^ ' ^ and broadening on binding of the sugar to lysozyme.  This s h i f t has been a t t r i b u t e d to the proximity of the  N-acetyl methyl protons i n the bound sugar to the aromatic portion of a tryptophan  residue (residue 108) i n l y s o z y m e . ' ^  of the free sugar, NAG,  In the case  the a- and B-anomers have been observed to  58 59 show'a degenerate N-acetyl resonance when no lysozyme i s present  '  but this singlet gradually becomes a doublet as enzyme i s added.  This  58 59 68 degeneracy has considerably hampered  '  '  accurate chemical  shift  - 54 -  measurements on the system and no doubt p a r t l y explains why Raftery et a l . and A .  and Sykes et a l . get somewhat d i f f e r e n t values for K D  We have synthesized (see Experimental section) the following series of compounds i n which the normal N-acetate group of N-acetylD-glucosamine has been replaced by an N - t r i f l u o r o a c e t y l group.  I t was  expected that the fluorine resonances of 1 and 2 might not be degenerate CH OH 2  as i n the  H case.  Moreover, i t was hoped that fluorine chemical  s h i f t s on association with- the enzyme would be greater than proton  19 shifts.  At the same time we expected that the  F n u c l e i might w e l l  have some perturbing effect on the i n t e r a c t i o n . When a mixture of the a- and g-anomers of the free sugar was allowed to c r y s t a l l i z e slowly from water, the a-anomer was obtained as a pure, c r y s t a l l i n e hydrate.  The mutarotation of this species to  19 its  equilibrium mixture could be followed by  F n.m.r. ( F i g . 2)  and was observed to take about 45 minutes i n pH 5.5 c i t r a t e buffer at 31.5°C.  The f i n a l r a t i o of a to g was 46% a and 54% g.  The fact  that i t was indeed the a-anomer which was obtained pure could be readily proven from the proton n.m.r. spectrum i n which the H -H„ 1  - 55 -  Time  (min.)  .a  r m  12  300 19 Fig.  2  Hz. From TFA  200  F n.m.r. spectra (94.071 MHz) of a mutarotating solution of Ntrifluoroacetyl-D-glucosamine (a-anomer, 1) i n pH 5.5 c i t r a t e buffer (0.1 M). ~The i n t e r n a l standard, t r i f l u o r o e t h a n o l , i s seen as a sharp t r i p l e t some 100 Hz to higher f i e l d . A c a p i l l a r y of TFA was used f o r the field-frequency lock. The time after dissolving the pure a-anomer i s shown to the right of the spectra.  -  56 -  s p l i t t i n g i s c l e a r l y v i s i b l e i n the downfield C^-proton. be noted from F i g . 2 that, as was hoped, the  It  should  N-trifluoroacetyl  groups of 1 and 2 are non-degenerate (separation ca. 27.0 Hz), and that they f a l l i n a convenient p o s i t i o n r e l a t i v e to the i n t e r n a l ence compound, CF^Cr^OH. a-anomer i s s u f f i c i e n t l y  Unfortunately  refer-  the mutarotation of the pure  fast to result i n an appreciable amount of  the g-anomer (2) only several minutes a f t e r d i s s o l u t i o n .  The excellent  19 separation of the  F peaks of the two anomers permitted accurate  integration of the i n t e n s i t i e s . 19 F i g . 3 shows the e f f e c t of lysozyme on the F n.m.r. spectra of 1 and 2. The experiment was performed by varying the sugar concentration from 0.10 M to 0.02 M while holding the concentration of lysozyme constant _3 at 3 x 10  M (see experimental s e c t i o n ) .  In F i g . 3A no enzyme has  been added and the s h i f t s of a - and g-anomers are shown r e l a t i v e  to  the centre l i n e of the t r i f l u o r o e t h a n o l t r i p l e t (204.5 Hz downfield from the t r i f l u o r o a c e t i c acid c a p i l l a r y lock s i g n a l ) . concentration was 0.05 M.  Internal standard  F i g . 3B displays the spectrum obtained when _3  a sample of sugar at the same concentration (0.10 M) i s made 3 x 10 i n lysozyme.  M  The a-anomer (1) i s seen to broaden considerably and to s h i f t  u p f i e l d toward the i n t e r n a l reference s i g n a l . appreciable change.  The g-anomer (2) shows no  When the sugar concentration i s lowered to 0.04 M (Fig.  3C) the compound 1 shows even greater changes i n chemical s h i f t and l i n e width , whereas the spectrum of 2 reamins r e l a t i v e l y unchanged. s h i f t results of this experiment are l i s t e d i n Table 1.  The chemical  The data were  treated, as described i n the experimental s e c t i o n , according to the 66 method of Dahlquist and Raftery i n order to obtain the d i s s o c i a t i o n  - 57 -  0.10 M.  0.10 M. + LYS.  0.04 M. + LYS.  H Z . 19  Fig. 3  F R O M  T F A  F n.m.r. spectra recorded during a study of 1 and 2 (a- and 3 anomers of N - t r i f l u o r o a c e t y l glucosamine) with lysozyme i n pH 5.5 c i t r a t e buffer (0.1 M). A. Sugar concentration 0.10 M, no enzyme. -3 B. Sugar concentration 0.10 M, 3 x 10 M enzyme -3 C. Sugar concentration 0.04 M, 3 x 10 M enzyme In each case the center l i n e of the t r i f l u o r o e t h a n o l standard t r i p l e t i s shown to h i g h f i e l d .  - 58 -  constant, K  = ^— , and the bound chemical s h i f t , A. For this purpose B i n i t i a l l y assumed that because the g-anomer does not s h i f t D  i t was  K  or broaden appreciably, that therefore i t does not bind to lysozyme. Making this assumption (Fig. 4A, f i l l e d c i r c l e s ) leads to calculated values of Kg = 96.7 M  1  and A  = 0.947 ppm  for 1.  At this stage i t  seems appropriate to remark on the changes i n linewidth observed 19 for the  F resonances of 1 and 2 and for the centre l i n e of the  CF^CrL^OH t r i p l e t .  Such changes w i l l be discussed at length i n  Chapter I I I , but i t should be noted here that although the resonance of 1 broadens considerably (Table 2), that corresponding  to 2  broadens n e g l i g i b l y  and the reference remains as sharp as i t was  before lysozyme was  added.  However, lack of a detectable chemical s h i f t or broadening i n the spectrum of an i n h i b i t o r i n contact with an enzyme does not the p o s s i b i l i t y of i t s binding.  preclude  For one thing, the f r a c t i o n of  bound i n h i b i t o r i s low at any one time.  Also there are several  mechanisms for broadening and, as w i l l be shown i n Chapter I I I , that operating i n the case of the a-anomer (1) i s primarily "exchange 67 67 68 broadening." Sykes and Sykes and Parravano have shown that this i s also the primary broadening mechanism for the "*"H n.m.r. Nacetate resonances of the proton analogues of compounds 1 to 4. Exchange i s  a mechanism for broadening i f i t occurs between s i t e s  of d i f f e r e n t chemical s h i f t .  In other words, the observed lack of  19 change i n the  F spectrum of 2 could be explained by i t s binding to  lysozyme i n such a way  that the N - t r i f l u o r o a c e t y l  experience a very great change i n magnetic f i e l d .  i  group does not It might i n fact be  - 59 a stronger i n h i b i t o r of lysozyme than 1 and yet appear not to bind at a l l from chemical s h i f t and linewidth data.  66 Dahlquist and Raftery calculate  have derived equations permitting one to  = 1/Kg and A for two interconverting anomers, both of  which can be seen to s h i f t on binding.  They had l i m i t e d success i n  applying these equations to the compounds 5 and 6 because of the small  CH OH 2  5r  R  1 = H  R  2  = OH  1 = OH  R  2  = H  R  R  2  = OCH3  R  2  = H  R  HO\OH  Is  R  1  =  H  1 = OCH  3  s h i f t of the 0-anomer and the near degeneracy of the N-acetyl peaks. In our case, although there was no problem of degeneracy, the g-anomer gave no information which would permit solution of the equations. A p a r t i a l answer to this impasse was  to make use of the pure  a-anomer (1) by dissolving weighed amounts i n thermostated  solutions  19 just p r i o r to recording their section).  It was  anomerization.  F n.m.r. spectra (see experimental  then possible to monitor spectral changes during  This was  tions of sugar ( [ a ] Q  =  done repeatedly for several i n i t i a l concentra-  0.10 M and  [O. ] = 0.06 M) and the results were Q  compared with the results at mutarotation equilibrium to decide whether or not the g-anomer (2) was binding e f f e c t i v e l y . this i s best i l l u s t r a t e d by example.  Perhaps  Suppose an i n i t i a l concentration, -3  0.10 M, of pure a-anomer with 3 x 10 s h i f t 6 = x Hz.  M lysozyme shows a chemical  As mutarotation occurs, the concentration of a-anomer  - 6b (1) w i l l drop to 0.046 M and hence the bound f r a c t i o n , P „ , w i l l 13  increase and 6 w i l l increase ( i . e . , as shown i n F i g . 4A, smaller sugar concentrations give larger s h i f t s ) .  But the g-anomer concentra-  tion increases from 0 to 0.054 M as that of the a-anomer decreases. If  this g-anomer (2) binds to lysozyme as strongly as the a-anomer,  i t w i l l begin to compete for s i t e s on the enzyme and at anomeric equilibrium the number of available enzyme s i t e s w i l l have been decreased by a quantity  [Eg]  (the concentration of bound g-anomer).  The result w i l l be that although [a] w i l l have decreased by roughly 50%, so w i l l [Ea] and hence the observed chemical s h i f t  6 = x Hz  w i l l remain v i r t u a l l y the same (note that at these sugar concentrations nearly a l l of the sugar  i s i n the unbound form).  F i g . 4B shows the results of these mutarotation experiments. The data so obtained suffered from the fact that a f i n i t e time (3-5 minutes) was required to produce an i n i t i a l spectrum and from the fact that even once the sample was i n the probe mutarotation took place rapidly r e l a t i v e to the time required to make careful measurements.  Nevertheless, the data show  c l e a r l y that the a-anomer  (1) does undergo an u p f l e l d s h i f t during mutarotation (note that, conversely, a downfield s h i f t would indicate that the g-anomer was bound more strongly than the mutarotation s h i f t  a ) . Moreover the magnitude of this  (^2.7 Hz when [a ] = 0.06 M and o  ^ 2 . 1 Hz when [a ] = o  0.10 M) i s roughly within experimental error of the s h i f t  that  would be expected i f the g-anomer did not bind at a l l (see Table 1). It  seems safe then to say that for 2.  for 1 i s considerably larger than  But from these experiments i t i s impossible to  realistically  - 61 -  t  (min.)  19 Fig. 4. A. Graph of " F chemical s h i f t data from the study of 1 with lysozyme: f i l l e d c i r c l e s - mutarotated mixture; X's - maximum error from the study of pure a-anomer. The Y-axis i s i n units of molarity of 1. B. Increase i n 6" (5') as a function of time during the mutarotation of pure a-anomer (1) to an equilibrium mixture i n the presence of lysozyme: f i l l e d c i r c l e s - [a ] = 0.10 M, X's - [ a j = 0.06 M. T  - 62 -  estimate a  for 2.  I t ij; possible to use the results of the muta-  rotation studies to estimate a maximum correction to the o r i g i n a l graph for the a-anomer.  This i s done i n F i g . 4A:  the l i n e through the  f i l l e d c i r c l e s representing equilibrium data, and the l i n e through the "X's"  i n d i c a t i n g an approximate maximum correction to the pure  oranomer data.  As shown i n F i g . 4A, taking the g-anomer into consider-  ation may change the intercept on the sugar concentration axis by as much as 0.002 M and t h i s w i l l change roughly  0.012  M.  for 1 from 0.0103 M to  However, i n view of the error l i m i t s involved i n  the mutarotation studies (and indeed i n the technique i t s e l f ) such a correction i s not warranted. It should be emphasized here that there remains the p o s s i b i l i t y that the g-anomer i s binding to the enzyme i n such a way  that i t  neither s h i f t s on binding nor i n t e r f e r e s with the binding of the a-anomer or that i t binds non-competitively  Lysozyme and the Methyl Glycosides of  i n the slow exchange l i m i t .  N-Trifluoroacetyl-D-glucosamine  As mentioned previously, the methyl glycosides made as part of the series of fluorinated compounds. p u r i f i e d and separated  (3 and 4) were These were  by column chromatography on s i l i c a gel (see  CH OH 2  O,  NHCOCF3  3/  R  4/  R-, = O C H  1 =  H  R 3  =  2  R  2  OCH  =H  3  Table 1.  19 F Chemical Shift Data for N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl _3 Glycosides (3 and 4) i n the Presence of Lysozyme (3 x 10 M)  Total Sugar Concentration [io3 (M)  Chemical Shift from Trifluoroethanol (Hz)  102.96 103.21 103.41 103.47 103.57 103.60  0.10 (no lys.) 0.10 0.08 0.06 0.04 0.02  129.85 125.41 124.72 123.45 121.55 117.88  Sugar Concentration [ I ] (M)  Chemical Shift from Trifluoroethanol (Hz)  Q  0.10 (no lys.) 0.10 0.08 0.06 0.04 0.02  Changes i n S h i f t for 1 6 (Hz)  1/6 (Hz  0 4.57 5.27 6.53 8.43 12.11  0.219 0.190 0.153 0.119 0.0826  Sugar Concentration [ I ] 00 Q  130.95 131.43 131.40 131.46 131.65 131.75  0.10 (no lys.) 0.10 0.08 0.06 0.04 0.02  )  Chemical S h i f t from Trifluoroethanol (Hz) 4 99.82 100.20 100.68 100.28 100.45 100.35  - 64 Table 2.  Linewidth Data for the N-Trifluoroacetate resonances of NTrifluoroacetyl-D-glucosamine  (1 and 2) and i t s Methyl _3  Glycosides (3 and 4) i n the Presence of Lysozyme (3 x 10 Total Sugar Concentration [I ] (M) o 0.10 0.10 0.08 0.06 0.04 0.02  (no lys.)  Sugar Concentration [ I  0.10 Q.10 0.08 0.06 0.04 0.02  o  ]  ( M )  (no lys.)  Transition Linewidth at Half Height 1  2  0.70 1.10 1.15 1.28 1.50 2.00  0.70 0.75 0.76 0.80 0.80 0.79  Transition Linewidth at Half Height 3  Standard  0.74 0.77 0.78 0.79 0.77 0.84  0.48 0.48 0.47 0.48 0.48 0.47  4 0.10 0.10 0.08 0,0.6 0.04 0.02  (no lys.)  0.69 0.74 0.77 0.75 0.75 0.75  Standard 0.48 0.49 0.49 0.50 0.49 0.50  Standard 0.60 0.60 0.61 0.59 0.61 0.60  M)  - 65 experimental  section).  Hence they could be studied separately i n  interaction with lysozyme.  This was  done (once again using the 19  method of Pahlquist and Raftery) by monitoring  the  F resonances and  measuring their chemical s h i f t r e l a t i v e to the i n t e r n a l  standard,  t r i f l u o r o e t h a n o l , while varying the concentration of sugar from to 0.02  0.10  M) i n the presence of a constant concentration of enzyme (3 x  ^3 10  M).  The chemical s h i f t results are tabulated (Table 1) with  those for the free sugar.  The chemical s h i f t data for 3 and 4 i s  very similar to that for 2. (~Q.5  That i s , there i s only a very s l i g h t  Hz) downfield s h i f t as compared to the 12.0 Hz u p f i e l d s h i f t of  the a-anomer, 1,  This s i m i l a r i t y amongst 2, 3 and 4 also shows up  i n the l i n e broadening data:  i n each case a very s l i g h t broadening  (ca. 0.1 Hz) i s apparent over the t o t a l range of concentration data (Table 2) compared to a broadening of w e l l over 1.0 Hz for 1. The standard  (trifluoroethanol) can be seen not to broaden detectably.  In the case of the glycosides i t i s possible to measure their binding strength (or lack thereof) against that of some other sugar of known strength.  This was not possible with the free sugar,2,because  a l l effects would be swamped by those of the strong i n h i b i t o r , 1 . The known i n h i b i t o r , 2-deoxy-2-acetamido~a-methyl-glycoside (7), was chosen for comparison.  We have measured K  for 7 (as w i l l be described  l a t e r i n this chapter) and found i t to be 22.9 M  (K^ = 0.0437 M) .  _1  Th,is i n h i b i t o r i s assumed to bind at subsite C."^ performed i n each case invplved the same approach.  ^  The experiment The s h i f t , 6 ,  of the N-acetate group of 7 at a given concentration of enzyme and sugar was  compared to t;hat of another sample containing the same  - 66 -  Table 3.  Dissociation Constants (Kp), Binding Constants (Kg) and Bound Chemical S h i f t s (A) for Compounds 1 to 4  K  Compound  D  (M)  K  B  , -1 (M )  1  0.0103  2  >>0.0103  <<96.7  3  >0.0437  <22.9  4  >0.0437  <22.9  . A(ppm)  96.7  0.947  ^0 '  Competition of the N-Trifluoroacetyl-a-  ^0  and g-methyl  Glycosides (3 and 4) f o rSubsite C as Indicated by the N-Acetyl Proton Chemical S h i f t of 7  Concentration of 7 (M) 0.49  [E ] (M)  Other Sugar Added  0.003  none  u  TI  II  II  II  II  Cpd. 3 M).49 M none Cpd. 4 <v0.49 M  Observed Shift ( 6 ) for 7 (Hz) 2.2 1.7 2.2 1.5  - 67 -  concentration of the sugar, 7, and enzyme plus an equal of e i t h e r 3 or 4.  concentration  In each case the observed s h i f t , 6 , was  decrease by roughly  1/4  (Table 3).  seen to  This indicates that 3 and 4 bind  to subsite C (or at least i n h i b i t binding at subsite C) less strongly than N-acetyl-glucosamine-a-methy1 glycoside for which we have calculated  = 22.9  M  ( l i t e r a t u r e values ^ 20 M ^ ^ ) .  We have at several points mentioned the problem that i t i s d i f f i c u l t to separate the phenomenon of binding at one  specific  s i t e from that of a multiple equilibrium with several s i t e s . data cannot resolve this d i f f i c u l t y . and Sykes and Parravano^ ^  Our  Both Dahlquist and Raftery  59 64—66 '  have been quick to r e a l i z e the problem.  The best information on the subject i s that provided by the X-ray work 57 58 of Blake et a l . ' which indicates that monosaccharide NAG at subsite C (although  binds  a-NAG binds i n a different orientation from  58 p-NAG  ).  It i s certainly possible to state from our results that  the fluorinated analogues 1, 3 and 4 compete with normal NAG  for  s i t e s on the enzyme surface - but this i s not concrete proof of subsite C  occupation.  It seems appropriate the ILght of present  at this point to consider our results i n  theories regarding binding of i n h i b i t o r s and  substrates to lysozyme. Since the o r i g i n a l X-ray studies of Blake ^ .. 36,57,58 , ... j . 60 * , „ , ^ 35 et a l . a number of solution studies and further X-ray data e  have appeared which have to a large extent borne out the i n i t i a l  claims.  However, there i s s t i l l uncertainty as to the actual importance of the various possible binding modes.  Workers i n this area have  generally assumed that contributions from each of the s i x subsites  can  - 68 -  be added to obtain values for the unitary free energy of association (AF^ = -RT  In K ) ^  of an oligosaccharide.  In our case, we  are  dealing with monosaccharides which seem most l i k e l y to bind at subsite C.  A considerable number of i n h i b i t o r s have been studied  to date and intercomparisons permit several conclusions.  It i s  generally accepted that the mpst important contribution to binding at subsite C arises from interactions of the acetamido group with the enzyme.  In p a r t i c u l a r i t has been postulated  between i t s NH and CO groups and  to form hydrogen bonds  the main chain CO and NH groups of 58  amino acid residues  107 and 59 respectively.  Also i t has been  35 suggested  that there i s a strong nonpolar association of the  acetamido methyl group with the aromatic indole ring of tryptophan 108.  It i s of course the proximity  to this l a t t e r side chain which  i s proposed to account for the large value of A for acetamido methyl protons of sacpharides bound at subsite C.  There are i n fact a t o t a l o •  of 30 proposed Van  der Waals contacts  sugar residue at subsite C.  (< 4 A) with the enzyme for a 58  Blake et a l ,  have reported a s l i g h t l y  d i f f e r e n t bound orientation for the a- and 3-anomers of NAG.  They  suggest that the g-anomer occupies subsite C i n a manner t y p i c a l of the actual substrates, being subject to the above-mentioned interactions plus hydrogen bonds between i t s 0(6) and 0(3) atoms and of the tryptophan side chains 62 and 63 respectively. on the other hand i s thought to be subject  groups  The a-anomer  to the above mentioned  interactions except those involving 0(6) and 0(3) a way  the NH  as to achieve a hydrogen bond between 0(1)  and i s bound i n such (which points down  into the enzyme) and the main chain NH of residue 109.  On the other hand  -  69 -  the ct-methyl glycoside 9 has been shown not to achieve this hydrogen 58 bond v i a 0(1)  but rather to bind as the 3~anomer of the free 58  sugar - Blake et a l .  suggest that this i s due to the glycoside methyl  group's i n t e r f e r i n g s t e r i c a l l y with the formation of such a bond. Certainly the q u a l i t a t i v e trend of our results  (that 1 binds  d i f f e r e n t l y than do 2, 3 and 4 and that 2, 3 and 4 behave s i m i l a r l y to one another) i s i n agreement with what would be predicted from known f a c t s .  However, there are several discrepancies.  Perhaps the  most glaring i s that substitution of NHC0CF for NHC0CH has greatly 3  3  enhanced the differences between the a-anomer of the free sugar and p-anomer and glycosides.  Literature values of  for 5 and 6 show D  60 considerable v a r i a t i o n as remarked by Chipman and Sharon - nevertheless -1 -1 they f a l l between 20 and 50 M  whereas we arrive at 96.7 M  and something considerably less than 96.7 M ^ for 2, 3 and 4.  for 1 Average  l i t e r a t u r e values -quoted by Chipman and Sharon for 7 and 8 are K = D  20 and 27 M ^ respectively.  Although we have not measured a value  for the g-glycoside, 8, our value of  = 22.9 M  for the a-glycoside  (see next section) i s within experimental error of the data from other workers. Our observations for the N-trifluoroacetates  2, 3 and 4 can be  explained by postulating that replacement of the normal N-acetate by 19 its  F analogue has disrupted (or even destroyed)  certain modes of  binding v i a this group ( i . e .  the two hydrogen bonding s i t e s and the  Van der Waals i n t e r a c t i o n s ) .  Our previously mentioned observation  that the NH proton i s more readily exchanged for deuterium i n the trifluoroacetate derivatives would support such a postulate.  It  is  - 70 -  already widely accepted NAG  that binding v i a the acetamido group of  derivatives accounts for most of the free energy of association  at subsite C.  This same lack of association of the N - t r i f l u o r o a c e t y l  group with tryptophan  108 would explain the lack of chemical  shift  on binding ( p a r t i c u l a r l y i n the case of 3 and 4 which seem to bind x^eakly at l e a s t ) . However, such a simple argument does not explain the r e l a t i v e l y strong binding (for monosaccharide i n h i b i t o r s ) of the a-anomer, 1. If replacement of the N-acetyl group of 5 by an N - t r i f l u o r o a c e t y l group served only to weaken bonding v i a this group we would c e r t a i n l y not expect to observe K_, = 96.7  M  An observation which may  a clue i s that the t o t a l bound chemical s h i f t for 1 was  found to be  A = 95 Hz which i s not much more than the proton s h i f t s (A = 70 observed for monosaccharides binding to subsite C.  provide  Hz)  This apparent  19 lack of reflect  F chemical s h i f t s e n s i t i v i t y to environment may  simply  (as with 2, 3 and 4) a lack of binding v i a the t r i f l u o r o -  acetamido group.  The fact that a s h i f t i s seen here at a l l may  result from a strong 0(1) hydrogen bond to the NH of residue 109 which holds the i n h i b i t o r i n p o s i t i o n .  This must be regarded  as a rather  unsatisfactory explanation though as i t does not explain the large value of K for 1. That this i s not a unique phenomenon i s B suggested i n a recent note by Kent and Dwek^ who  report high binding  constants for several halogenated monosaccharide i n h i b i t o r s . Since we completed our work a communication has appeared which reports the i n h i b i t i o n of lysozyme interacting with an actual substrate 71 by the free sugar, N-trifluoroacetyl-D-glucosamine.  These authors  - 71 -  have also looked at the  F n.m.r. spectra of the species, 1, 2,  and 4 interacting with lysozyme and report that and  A = 78 Hz  (at 100 MHz).  3,  for 1 i s 0.0091 M  Both of these values agree within  experimental error with our own.  These workers also report no n.m.r.  chemical s h i f t for 2, 3, and 4 i n t e r a c t i n g with the enzyme. 72 Although there are few examples i n the l i t e r a t u r e  73 '  of the  19 use of  F as a probe for investigating enzyme-inhibitor interactions,  the technique does seem to have p o t e n t i a l benefits. the a- and  For  g-anomers (1,2) were well separated from one  instance, another  i n t h e i r spectra i n contrast to t h e i r proton analogues (5,6). 19 also seems l i k e l y that the f a i l u r e of the  It  F probe to display a  large bound s h i f t , A, i s a p e c u l i a r i t y of this system as mentioned previously.  Certainly the e f f e c t s observed point out dramatically  that  i n b i o l o g i c a l systems a CF bond can have s p e c i f i c effects which are d i f f e r e n t from those of a CH bond, and these e f f e c t s may  throw l i g h t  on the mechanisms involved. C. Experiments with C^-Substituted N-Acetyl-D-glucosamine-a-methyl glycosides 19 In the previous section we  reported  studies v i a  F n.m.r. of  the i n t e r a c t i o n of lysozyme with various monosaccharide i n h i b i t o r s . These studies were carried out p a r t l y to study the a p p l i c a b i l i t y of fluorine labels to such systems and partly i n an e f f o r t to learn more about the nature of the i n t e r a c t i o n . came to was  One  of the conclusions  we  that the replacement of CH^ by CF^ i n the N-acetyl group  seemed to have modified (probably lessened) i t s binding  interactions  with the enzyme.  Nevertheless,  the N-trifluoroacetylated methyl  glycosides s t i l l showed evidence of weak binding.  As mentioned before,  saccharide units occupying subsite C i n the normal fashion have been postulated to have 30 Van der Waals contacts 'and 4 hydrogen bonding s i t e s (2 of the l a t t e r involving the N-acetyl group).  But of a l l the  interactions, those involving the N-acetyl group are known^ to be the most powerful.  We report i n this section an attempt to guage  58 the importance of the hydrogen bonding contact postulated 0(6) and the NH group of tryptophan side chain 62 using  between n.m.r.  Our approach has been to synthesize a series of C^-substituted mono-  58 saccharide i n h i b i t o r s , one of which has been previously studied  by  X-ray d i f f r a c t i o n and i s known to bind i n the ' t y p i c a l ' way at subsite C (compound 9), another which i s assumed to bind at subsite C i n the same way  (compound 7), and a t h i r d which i s a previously unknown  i n h i b i t o r of lysozyme (compound 10). shown below:  CH R 2  NHCOCH3  These three i n h i b i t o r s are  - 73 -  The proton spectra of each of these compounds contained sharp peaks corresponding to the N-acetyl and methoxyl groups - these were suitable f o r studies with lysozyme.  The new i n h i b i t o r , 10, also  displayed a sharp doublet corresponding to the  protons and this  proved to be useful as a further probe of the bound environment. was hoped that the changes at  It  i n going from 7 to 9 to 10 would be  s p e c i f i c i n their effects as measured by K  and  A.  Compound 10 The study of this compound's i n t e r a c t i o n with lysozyme may be taken as t y p i c a l of the proton work done here.  The compound i t s e l f  was produced by c a t a l y t i c hydrogenation at atmospheric pressure of the C,-iodo derivative 9, as described i n the experimental section, o As with the fluoro-derivatives of the previous section,  and A were  calculated from the results of a study of the chemical s h i f t of sharp i n h i b i t o r peaks as a function of sugar concentration (0.10 to 0.02 M) —3 in the presence of a constant enzyme concentration (3 x 10  M) i n  pH 5.5 c i t r a t e buffer (0.1 M). Sample spectra from the experiment with 10 are shown i n F i g . 5. Fig. 5A displays the condition, 6 = 0 , i n which no enzyme i s present. Spectral features shown are the N-acetate peak, the C^-methyl doublet and the internal reference compound, 0.025 M t e r t i a r y butanol. large water peak was used f o r a field-frequency lock.  The  Note that  _3 when 3 x 10 M lysozyme i s added (Fig. 5B) both the N-acetyl peak and the doublet due to the C, protons experience an upfield s h i f t o (and broadening).  When the sugar concentration i s decreased to 0.04  M, the value of 6 i s increased as i l l u s t r a t e d i n F i g . 5C.  Note that  - 74 -  NAc  C  5  CH  3  . Std. 0.10  M  0.10  M.  +  LYS.  0.04  C  +  LYS.  5 Hz i  269.9 HZ. Fig. 5  - ' F R O M  M  r  340.4 349.0 H O D  H n.m.r. spectra recorded during a study of C^-methyl-N-acetyl glucosamine-a-methyl glycoside (10) with lysozyme i n pH 5.5 c i t r a t e buffer (0.1 M). A. Sugar concentration 0.10 M, no enzyme -3 B. Sugar concentration 0.10 M, 3 x 10 M enzyme .-3 Sugar concentration 0.04 M, 3 x 10 "" M enzyme C. The acetate resonance, C^-methyl resonance and the t e r t i a r y butanol i n t e r n a l standard are shown.  -  75 -  the Cg protons s h i f t less than the acetyl protons. The s h i f t s ( r e l a t i v e to the i n t e r n a l standard) of both the Nacetate peak and the C^-methyl doublet were large enough to be readily measured during the course of the experiment.  In F i g . 6A,  1/6 i s plotted v s . the i n i t i a l sugar concentration for both of these s p e c t r a l features.  It  i s an encouraging check on the technique that  both the graph for the N-acetate peak ( f i l l l e d c i r c l e s ) and that for the C^-methyl peak (X's)  intersect at very nearly the same point.  Naturally this i s as should be because the i n t e r s e c t i o n p o i n t , -(Kp + [ E ] ) , should be the same for a l l peaks i n the same compound as q  described i n the experimental s e c t i o n .  The value of  as determined  from following the N-acetate peaks i f 27.0 M ^ and that determined -1 from the C^-methyl peaks i s 26.4 M  .  The former i s probably more  accurate i n view of the larger 6 values involved.  The bound  chemical s h i f t , A, for the N-acetate peak was found to be 0.728 ppm and that for the C^-methyl peak 0.254 ppm (see Tables 4 and 5). Compound 7 This compound, the a-methyl glycoside of NAG, has been studied i n solution with lysozyme p r e v i o u s l y ^ with the result that i t s values i  for Kg and A are already i n the l i t e r a t u r e . We have repeated the study to make aire that our values do have some generality and that comparison of data amongst 7, 9 and 10 i s as meaningful as p o s s i b l e .  Data  -3 obtained from the study with 3 x 10  M lysozyme over a sugar concentra-  tion range of 0.10-0.02 M are shown plotted i n F i g . 6B and are l i s t e d i n Table 4.  S h i f t values were again measured r e l a t i v e to the i n t e r n a l  standard, t e r t i a r y butanol.  The s h i f t s given i n the table and plotted  -  76 -  i n F i g . 6B are those observed for the N-acetyl peak. calculated for Kg and  The values  A (Table 5) from the intercept and slope  respectively are Kg = 22.9 M ^ and  A = 0.734 ppm.  The C^-protons  of 7 were not suitable for chemical s h i f t studies because of the complexity of t h e i r spectrum. Compound 9 58 Although X-ray studies have been made  of the compound, 9,  interacting with lysozyme, no solution data seem  60  to have been reported.  The compound was synthesized from the a-methyl glycoside of NAG (7) v i a the C^-tosylate by displacement with Nal as described i n the experimental section. most d i f f i c u l t  This p a r t i c u l a r compound was p h y s i c a l l y the  to work with because of i t s low s o l u b i l i t y  c i t r a t e bufer used.  i n the  Partly for this reason i t was considered a good  system on which to test the r e p e a t a b i l i t y of measurements.  The results  of two separate studies are shown graphically i n F i g . 6C and the data i s l i s t e d i n Tables 4 and 5. was used to calculate Kg.  As with 7, the N-acetate peak alone  The runs were performed i n a fashion  exactly analogous to that for 7 and 10 ( i . e . , by varying the sugar _3 concentration, [I ], while holding [E ] constant at 3 x 10 M). o o The same i n t e r n a l reference, t e r t i a r y butanol, was employed and the water peak was used for a fieId-frequency  lock.  A l l runs were made  on the same spectrometer whose probe temperature was found to be 31.5°C. The results calculated from the two separate runs are Kg 35.0 M  _ 1  and A A == 0.583 ppm ( f i l l e d c i r c l e s )  0.631 ppm (X's),  and Kg = 31.8 M  1  and  A=  1 Fig. 6  (Hz ) 1  Graphs of chemical s h i f t data for the N-acetate protons of 10, 7, and 9 ( f i l l e d c i r c l e s - A, B, and C respectively) interacting with lysozyme. In A the X's represent data for the C^-methyl resonance. In C the X's represent a run done one week l a t e r .  - 78 -  Table 4.  Chemical Shift Data f o r the N-Acetyl Protons of Compounds 7, 9 and 10 and f o r the C^-Methyl of 10 Interacting with -3 Lysozyme (3 x 10 M)  Compound 7 [ I ] (M)  6  Q  .10 .08 .06 .04 .02  (Hz)  1/6 (Hz ) l  1.44 1.70 2.01 2.45 3.17  0.694 0.588 0.498 0.408 0.316  Compound 9 (data from two runs) [I ] (K) o  -  6  (Hz)  1/6 (Hz X )  .10 .08 .06 .04 .02  1.29 1.58 1.90 2.46 3.21  .775 .633 .526 .406 .312  .10 .08 .06 .04 .02  1.34 1.57 1.95 2.43 3.25  . 749 .637 .513 .412 .308  .  Compound 10 [ I  o  ]  ( M )  -NHC0CH  6  3  "(Hz) .10 .08 .06 .04 .02  1.51 1.74 2.17 2.62 3.50  1/6  -NHC0CH  6 3  (Hz" ) 1  0.662 0.575 0.461 0.382 0.286  -CH  1 / 6 3  -CH  3  (Hz)  (Hz" )  0.53 0.60 0.74 0.87 1.26  1.888 1.670 1.351 1.149 0.794  1  - 79 -  Table 5.  Dissociation Constants (K_), Binding Constants (K ) and D  D  D  Bound Chemical S h i f t s (A) f o r Compounds 7, 9 and 10 Compound  K  (M)  IC, (M )  D  7  A(ppm)  _1  B  0.0437  22.9  0.734  9 (data from two runs)  0.0314 0.0286  31.8 35.0  0.631 0.583  10 NHC0CH  0.0370  27.0  0.728  0.0378  26.4  0.254  3  C -CH r  3  S h i f t of Glycoside Methyl on Binding (+ = u p f i e l d , - = downfield).  Compound  7  9  10  [ 1 ^ (M)  [ E ] (M) q  0.10  0  0.05  0.003  0.10  0  0.05  0.003  0.10  0  0.05  0.003  «_ HC0CH N  3  6  ~OCH  3  0  0  + 2.2  - 0.2  0  0  + 2.2  + 0.2  0  0  + 2.4  - 0.1  - 80 -  A further technique-checking experiment performed with this sugar was one designed to test f o r a sugar concentration dependence in the s h i f t between the standard and the acetate peak.  In this  experiment no enzyme was added but the sugar concentration was over i t s f u l l range.  varied  Without lysozyme being present the chemical  s h i f t between the N-acetate peak and the i n t e r n a l standard ( t e r t i a r y butanol) was  found to be invariant within less than 0.1  Hz.  It was mentioned at the beginning of this section that the C^methoxyl groups of 7, 9, and 10 were p o t e n t i a l l y suitable for chemical s h i f t measurements ( i . e . that they were sharp and readily v i s i b l e ) . However, Raftery et a l . ^  have reported that the methoxyl peak of 7  does not s h i f t appreciably on binding to lysozyme.  Indeed we  found  this to be true for a l l three compounds, 7, 9 and 10 (see Table 5). It i s encouraging that the NHCOCH^ 6 values found here f a l l exactly on graphs of data taken several months e a r l i e r . Since our measurements indicate that 7, 9 and 10 bind with approximately equal a f f i n i t y to lysozyme, then i f they are occupying the same subsite (presumably  subsite C) they should be approximately  equally affected by competitive i n h i b i t o r s . used the N-deuteroacetyl analogue of NAG  Raftery et a l . ^  have  (see experimental section) for  - 81 s i m i l a r problems because, although not a strong i n h i b i t o r , i t has the advantage of not displaying an i n t e r f e r i n g It was  N-acetate resonance.  indeed found that this compound affected 7, 9 and 10 to  roughly the same extent i n the presence of lysozyme. The results of measuring Kg and A for the three compounds, 7, 9 and 10 are that the values are quite similar (Table 5).  The  values  of A for 7 and 10 are i d e n t i c a l and the value for 9 i s not very d i f f e r e n t (perhaps not s i g n i f i c a n t l y d i f f e r e n t ) - however, i t may indicate that the bound orientation of 9 i s very s l i g h t l y d i f f e r e n t from that of 7 and  10.  The Kg values for the three compounds are also too similar to allow any clear d i s t i n c t i o n s .  Nevertheless  i t may  be s i g n i f i c a n t  that the order of r e l a t i v e magnitudes, IH (compound 9) > a 10) > K  K  (compound a  (compound 7), i s the same as the order of l i p i p h y l i c i t y of  the compounds (e.g. during T.L.C. on s i l i c a gel using MeOH/CHCl^ as eluent, 9 ran fastest, then 10 and then 7).  Certainly though,  i t seems safe to say that the reverse order of binding strength (as measured by K ) does not exist - that i s , K than K  for 10 and 9.  for 7 i s not larger  This suggests that e i t h e r :  D  (a)  hydrogen-bonding v i a the C^-oxygen i s unimportant or  (b)  there i s also some p o t e n t i a l for Van der Waals interactions  involving the Cg region and that these make up for the loss of a hydrogen-bonding s i t e . This l a t t e r p o s s i b i l i t y i s a t t r a c t i v e i n view of the appreciable 3 bound s h i f t seen for the C^-methyl group of 10. We have already mentioned that an appreciable Van der Waals i n t e r a c t i o n i s postulated  - 82 -  between the N-acetate methyl of NAG and the indole ring of tryptophan 108 and that i t i s the aromatic f i e l d of this ring which causes the large bound s h i f t of the N-acetate peak.  There are i n fact two  tryptophan residue indole rings which are close enough to interact to some extent with the C^-methyl group of 10:  tryptophan 62, which  60 i s postulated  to be involved i n hydrogen-bonding to 0(6) of  sugars i n subsite C, and tryptophan 63 which i s postulated*^ to be involved i n hydrogen-bonding to 0(3) of sugars i n subsite C. The imidazole ring contains an NH group which i s the proposed hydrogenbonding moiety; Van der Waals interactions v i a the same ring are less ... 60 specific. It seems from our studies that the n.m.r. technique does indeed have certain advantages over other solution methods of studying b i o l o g i c a l systems.  These have been pointed out i n several review  papers mentioned at the beginning of this chapter.  One r e a l l y  noteworthy advantage i s the a b i l i t y to investigate i n d i v i d u a l parts of complex systems (e.g. i n d i v i d u a l anomers i n mixtures and i n d i v i d u a l protons i n the same molecule - examples of both of which have been included i n this chapter).  A noteworthy drawback to  n.m.r. i n b i o l o g i c a l systems i s the low signal-to-noise r a t i o of the technique.  The recent introduction of Fourier transform n.m.r.  spectroscopy represents a quantum 5 P i um  t n  n  e  solution of this  l a t t e r problem, but i t requires expensive equipment. 19 n.m.r. probes such as  The search f o r  F which p o t e n t i a l l y display a large bound  chemical s h i f t , A , i s sensible because a large A permits the use of very d i l u t e enzyme solutions i n studies such as those performed here.  - 83 D.  Conformation of Free Monosaccharide I n h i b i t o r s The binding of an i n h i b i t o r or substrate to an enzyme a c t i v e  s i t e i s often h i g h l y s p e c i f i c and depends, not only on the substituent groups,but also on the c o n f i g u r a t i o n and conformation of the molecule 74 75 involved.  '  Also mechanisms f o r enzyme a c t i o n have postulated  both enzyme and substrate d i s t o r t i o n .  In these respects, the  conformations  of monosaccharide subunits of lysozyme substrates are  of i n t e r e s t .  The conformations  i n s o l u t i o n of the 2-deoxy-2-amino  sugars studied here have not been previously reported. apart from determining the absolute conformations  A l s o , quite  of NAG d e r i v a t i v e s ,  we wanted to e i t h e r confirm or r u l e out the p o s s i b i l i t y of conformational d i f f e r e n c e s being the cause of v a r i a t i o n i n The n.m.r. spectra  and A between i n h i b i t o r s .  of sugars i n aqueous s o l u t i o n are generally  unsuitable f o r conformational s t u d i e s . fact that the bulk of the r i n g protons  This i s a r e s u l t of the (often a l l but H^) f a l l  close together i n the region about x 6.0 t o x 6.5 (e.g. F i g . 7A). A standard method of g e t t i n g around t h i s problem i s to make the completely ()-acetylated d e r i v a t i v e of the sugar i n question.  This  serves the dual function of increasing i t s s o l u b i l i t y i n organic solvents and tending to spread the r i n g proton spectrum over a wider energy range.  I t i s then often p o s s i b l e to f i n d a solvent i n which the n.m.r.  spectrum i s p a r t l y or wholly analysable.  The assumption i s made  that the data so derived can be reasonably extrapolated to the nonacetylated species i n water.  For the molecules studied here t h i s  assumption could be checked v i a the coupling J.. ~. We report here l, z  the r e s u l t s of such a study on the completely O-acetylated d e r i v a t i v e s of  - 84 -  OMe  NAc  8.o  6.0  4.0  r  OMe  Ho  H  4 H  5.0  7  A. B.  6.0  6  H  6  7.0  T  H n . m . r . spectrum (100 MHz) of N - a c e t y l glucosamine a - m e t h y l g l y c o s i d e (7) i n D^O u s i n g a TMS c a p i l l a r y f o r f i e l d - f r e q u e n c y l o c k . 1 P a r t i a l H n . m . r . spectrum (100 MHz) of C g - i o d o - N - a c e t y l g l u c o s a m i n e - a - m e t h y l g l y c o s i d e d i a c e t a t e ( 9 ' ) i n CDCl^-TMS s o l u t i o n . The f i r s t o r d e r assignment i s shown. Note the remnant of the NH c o u p l i n g i n t o .  - 85 compounds 3, 4, 7, 9, and 10. These derivatives w i l l be labelled after t h e i r parent compounds as follows:  CH OAc 2  NHCOCF3  CH R 2  NHCOCH3  The  H n.m.r. spectra (100 MHz) of the ring proton regions of  3' and 4' are shown i n F i g . 8A and B respectively. In each case the solvent i s deuterochloroform-TMS.  N-trifluoroacetyl-a-methoxy-  3,4,6-pyranose triacetate (3') and i t s (3-methoxy counterpart (4') both displayed couplings into  due to the proton on the C2~nitrogen.  These couplings have been removed i n the spectra shown i n F i g . 8 by exchanging the nitrogen proton with deuterium. by adding several drops of  This was  accomplished  to the n.m.r. tube containing the  sample dissolved i n deuterochloroform-TMS and to this adding a tiny drop  - 86 -  Hi  5.0 Fig. 8  5.5  6.0  6.5  T  P a r t i a l H n.m.r. spectra (100 MHz) of A. N - t r i f l u o r o a c e t y l glucosaminea-methyl glycoside t r i a c e t a t e (3') and B. N - t r i f l u o r o a c e t y l glucosamineg-methyl glycoside t r i a c e t a t e (4'), both i n CDC1 s o l u t i o n using TMS f o r a field-frequency lock. 3  of triethylamine.  87 -  For the N - t r l f l u o r o a c e t y l  compounds this exchange  occurred very r e a d i l y , presumably due to the a c i d i t y of the -NH group brought about by the electronegative fluorine atoms. Both 3' and 4  1  show a w e l l resolved doublet corresponding to H^  at T 5.17 and T 5.33 respectively.  It  i s thus immediately possible  to distinguish between a - and 6-anomers by the smaller H^ s p l i t t i n g of the a-compound.  B.^ was readily assigned i n each case by INDOR  experiments performed while monitoring H^.  Subsequent INDOR  experiments permitted a l l transitions to be assigned as shown diagrammatically i n F i g . 8A and B. group occurs at highest f i e l d . system to l o w f i e l d .  The  In each case the C^-methoxyl  H^ and H^ form a s l i g h t l y coupled  protons form a f a i r l y highly coupled  system at h i g h f i e l d and H,. can be seen at x 6.02 (3')  and T 6.18 (4 ) 1  as an octet. The l i n e positions for compounds 3' and 4' were used to calculate coupling constants and chemical s h i f t s by means of computer-based analyses. spin systems.  iterative,  Both compounds could be treated as seven  The data so obtained are l i s t e d i n Table 6.  The completely O-acetylated derivatives of 7, 9 and 10 ( 7 ' , 9  1  and 10') were treated i n the same way as 3' and 4 ' .  For example,  the ring proton spectrum of 9' i s shown i n F i g . 7B.  Once again the  c h a r a c t e r i s t i c H^ resonance can be i d e n t i f i e d (at x 5.24).  ^H-{"^H}  INDOR experiments run while monitoring these transitions enabled one to pick out the B^ t r a n s i t i o n s .  The B^ t r a n s i t i o n s were then made  the subject of INDOR experiments to locate H_ and H..  The replacement  - 88 -  of the C^-O-acetate by iodine has caused the C^-protons to s h i f t to highfield.  7' and 10' were treated i n exactly the same fashion.  As with the N - t r i f l u o r o a c e t y l compounds i t was found  convenient  to simplify the spectrum somewhat by exchanging the C 2 n i t r o g e n _  proton f o r deuterium as described e a r l i e r .  This was considerably  more d i f f i c u l t when the adjacent acetate group was a normal acetate as opposed to a t r i f l u o r o - s u b s t i t u t e d group as pointed out previously. In fact a remnant of the R^-NH coupling can be seen i n F i g . 7B as weak s a t e l l i t e s of  since the exchange was not 100%.  In general  the C2~nitrogen proton appeared to lowfield when not replaced by deuterium (e.g. f o r 10' -NH occurred at T 3.99 and the I^-NH s p l i t t i n g was 9.5 Hz). There i s no point i n discussing each of these three compounds i n detail.  In each case, t r a n s i t i o n s were assigned and used as the  basis of an i t e r a t i v e c a l c u l a t i o n (by computer) of the true coupling constants and chemical s h i f t s (see Table 6). The proton spectra of a l l compounds 3', 4', 7', 9' and 10' were very s i m i l a r with two exceptions: (a)  4' has a larger H-^ ^ -  c o u  pli g n  ( a x i a l - a x i a l as opposed to  equatorial-axial) . (b)  the Cg-protons of 10' appear as a doublet rather than 8  lines as they are equivalent. The spectra obtained f o r the acetates should be compared to that shown i n F i g . 7A.  I t i s the H"*" n.m.r. spectrum of 7 i n D 2 O - obviously  very l i t t l e information i s available from such a spectrum.  The only  c l e a r l y i d e n t i f i a b l e features are H. (which suffers a degeneracy with  -  the HOD  89  -  peak) at x 4.5 and the methoxyl and N-acetate peaks to h i g h f i e l d .  The bulk of the ring protons form an extremely highly coupled and degenerate region from x 6.0 to x 7.0.  This spectrum was run using  a c a p i l l a r y of TMS held concentric with the n.m.r. tube as a f i e l d frequency lock. The conformations of the 2-deoxy-2-amino sugars studied here are reflected i n the coupling constants  ^,  ^,  ^, and  ^.  The  conformations suggested by the data i n Table 6 are shown below - one very important feature being that, regardless of their absolute conformation, each of the f i v e sugars studied seems to have the same conformation as indicated by the close s i m i l a r i t y of the values. In each case (except that of 4') J  „ would suggest an equatorial-  a x i a l coupling with a dihedral angle i n the neighbourhood  of 60°.  In  the case of 4', the H -H„ relationship seems to approach a x i a l - a x i a l .  0 Me  - 90 -  Table 6.  N.M.R. Parameters f o r the O-Acetate Derivatives 3', 4', 7', 9' and 10'  Chemical Shifts (x-values)  H  l  H  2  H  3  H  4  H  1  fi 2  H  5  6  6  3'  5.17  5.67  4.70  4.86  6.02  5.72  5.87  4'  5.33  5.98  4.59  4.90  6.18  5.69  5.82  7'  5.25  5.66  4.78  4.90  6.06  5.74  5.89  9'  5.24  5.66  4.80  5.09  6.22  6.69  6.85  10'  5.33  5.68  4.83  5.15  6.17  8.81  -  4 5  5 6  t Coupling Constants (Hz) H  1 2 H  H  2 3 H  H  3 4 H  H  H  6  1  1 2 6  3'  3.7  11.3  9.4  10.5  4.7  2.2  -12.4  4'  8.6  10.8  9.6  9.7  5.2  1.9  -13.0  7'  3.6  10.9  9.3  9.7  2.4  4.7  -12.2  9'  3.7  10.5  9.4  9.6  2.2  8.9  -10.7  10'  3.6  10.4  9.6  9.8  6.2  -  * t  Measured i n deuterochloroform solution containing TMS. The errors i n these coupling constants are +0.2 Hz  -  - 91 -  The values of the ^-H^,  H-j-H^  a n d  H  4 ~ 5 couplings ( a l l - 10 Hz) are H  i n close accord with a t r a n s - d i a x i a l relationship i n each case ( i . e . the 180° v i c i n a l angle indicated i n the sugar diagrams). As mentioned  previously, i t i s not immediately obvious that the  conformations arrived at for the above compounds may be meaningfully extrapolated to those of their un-acetylated parent compounds i n water. However i t i s possible to check the observed s p l i t t i n g of H^ f o r a sugar dissolved i n water (e.g. F i g . 7A) against the H^-H^  coupling  constant of the O-acetylated system; for instance the compounds 3, 7, 9, and 10 were seen to have H^ s p l i t t i n g s i n the neighbourhood 3.1 Hz.  of  At f i r s t sight this would suggest that their conformations  are s l i g h t l y different from those of their O-acetylated derivatives i n deuterochloroform.  However, as w i l l be discussed i n more d e t a i l i n  the rext section,  and H^ f a l l quite close together i n the parent  sugars (Fig. 7A) and are strongly coupled (J„ „ = 10-11 Hz ^ t h e i r chemical s h i f t separation). H^ spectrum assuming  ^  =  In fact a t h e o r e t i c a l c a l c u l a t i o n of the 3.6 Hz but taking into account the close  coupling of H^ and H^ gives a s p l i t t i n g for H^ of 3.1 Hz (see F i g . 8) as observed.  This observation would argue favourably for the  extrapolation from O-acetylated sugar conformations to those of their unacetylated precursors. The conformations arrived at for the amino sugars studied are those to be expected on s t e r i c grounds and on the basis of previous n.m.r. 2 3 studies of non-amino sugars. '  - 92 -  E.  N.M.R. as a Probe f o r Conformation o f Bound Monosaccharide Inhibitors There has  conformational enzymes.  been c o n s i d e r a b l e s p e c u l a t i o n c o n c e r n i n g changes on b i n d i n g of s u b s t r a t e s  (or i n h i b i t o r s )  Perhaps the b e s t known proponent of enzyme  changes on b i n d i n g i s K o s h l a n d ^ whose "induced put  forward  as a mechanism of enzyme a c t i o n .  a r e l a t i v e l y small, r i g i d  enzyme - X-ray work has  d i s t o r t i o n of the enzyme occurs  on b i n d i n g .  been  case of lysozyme -  shown t h a t v e r y  little  36  '  the same workers have p o s t u l a t e d t h a t d u r i n g  to  conformational  f i t t h e o r y " has  In the  35  the r o l e of  On  cleavage  the o t h e r hand, of o l i g o s a c c h a r i d e s  the sugar u n i t bound i n s u b s i t e D of the a c t i v e s i t e i s d i s t o r t e d toward a h a l f - c h a i r conformation. molecular  models.  No  T h i s c o n c l u s i o n was  concrete proposals  drawn from  have been made r e g a r d i n g 60  d i s t o r t i o n of sugars at other  subsites although  i t has been suggested  that such d i s t o r t i o n s are p o s s i b l e . In p r i n c i p l e n.m.r. chemical be  s h i f t s and  used to measure d i s t o r t i o n on b i n d i n g .  w i l l be  considered  S i n c e , i n the  coupling constants  The  latter  f a s t exchange l i m i t , the i n h i b i t o r  unbound  observed  to those of the t o t a l l y bound s p e c i e s .  has been emphasized p r e v i o u s l y (Chapter taken as  resonances  average of the bound and  s p e c i e s , i t should be p o s s i b l e to e x t r a p o l a t e from the  only be  possibility  here.  represent a population-weighted  coupling constants  could  true c o u p l i n g c o n s t a n t s  However, as  I ) , observed s p l i t t i n g s i n very s p e c i a l  circumstances  (the b a s i c c o n d i t i o n  f o r t h i s assumption to be v a l i d b e i n g  n u c l e i be  shifts  s e p r a t e d by  can  that a l l  l a r g e w i t h r e s p e c t to the c o u p l i n g  constants).  - 93 -  In none of the i n h i b i t o r s examined here i s this true. the  For instance,  n.m.r. spectrum of 7 dissolved i n D2O.is shown i n F i g . 7A.  H^  can be seen to lowfield (T 5.25) and the s p l i t t i n g due to coupling with E^ i s c l e a r l y v i s i b l e .  and  are known to f a l l generally  i n the lowfield region of the ring proton envelope f o r free sugars dissolved ±1 aqueous media. monitoring  Indeed INDOR experiments run while  can be used to pick out part of the E^ spectrum at the  lowfield end of the envelope (Fig. 7A).  The coupling between E^ and  i s known from the l a s t section to be 10.9 Hz.  By inspection i t i s  possible to guess a rough chemical s h i f t position for H^. Armed with this data i t i s a simple matter to calculate t h e o r e t i c a l spectra for the system formed by H^, E^  a n c  * H^'  This should give a  f a i r l y r e l i a b l e representation of H^ to be compared with that observable experimentally.  A computer plot programme was used which  actually drew out the calculated H^ spectrum.  F i g . 9A shows the  result of aich a computer p r i n t out for H^ when  ^  =  3.6 Hz and  J„ „ = 10.9 Hz ( i . e . , the coupling constants calculated i n the previous section for 7'), and when the separation between H^ and H^ i s taken as 12.0 Hz (see Table i n F i g . 9). spectrum i s found to be 3.1 Hz.  The s p l i t t i n g seen i n the simulated  F i g . 9B shows the result of allowing  H^ to s h i f t u p f i e l d toward H^ by 1.7 Hz: H^ has now become 3.0 Hz.  the observed s p l i t t i n g i n  In F i g . 9C, H^ has been allowed to s h i f t  another 1.0 Hz toward H^ with the result that the H 2.9 Hz.  s p l i t t i n g becomes  The point we have t r i e d to make here i s that rather small  changes i n the chemical s h i f t s of H^ or H^ (changes which are on the scale of those expected from binding to the enzyme) can cause appreciable  10 H z  D  B C H E M I C A L SHIFT H1  5.253  5.253  5.253  5.253  H;  6.103  6.120  6.130  6.183  H,  6.223  6.223  6.223  6.223  Hz  3.1  3.0  2.9  Hz  3.6  3.6  3.6  APPARENT  J  ACTUAL  '12  Fig. 9.  T  1 2  3.6  Computer-simulated spectra of the 'doublet' region of a hypothetical N-acetyl glucosamine-a-methyl glycoside i n aqueous solution. The resonance has been generated from a consideration of H^, and by assuming ^ 3.6 Hz, ^ = 0 Hz, ^= =  10.9 Hz and the chemical s h i f t values shown above. only the chemical s h i f t of Hj i s varied.  Note that  - 95 changes i n the s p l i t t i n g observed i n H^. by showing what happens to the  F i g . 9D emphasizes the point  'doublet' when E^ has s h i f t e d 8.0 Hz  toward H^ - obviously there i s now no simple interpretation of the H^ s p l i t t i n g i n terms of J, „.  In fact the s p l i t t i n g observed experi-  mentally (Fig. 7A) i n H^ was found to be 3.1 Hz which agrees w e l l with the proposed arrangement of  H^, E^ and H^ i n F i g . 9A.  In spite of what has been said above, i t would be good evidence i  !  for i n h i b i t o r d i s t o r t i o n on binding 'to see a large change i n the observed s p l i t t i n g of H^.  We have calculated an order of magnitude  for errors l i k e l y to be introduced  by chemical s h i f t changes i n E.  and H^ (this error being roughly + 0.2-0.3 Hz).  I t i s now a simple  matter to calculate the changes to be expected i n the H^ s p l i t t i n g of an i n h i b i t o r with K  - 50 M  and  A -  0.70 ppm.  J3  K  -  h  ~  [ E I ]  [E][I]  -  50 M"  1  5  0  M  where [EI] = concentration of bound i n h i b i t o r  if  [E]  =  concentration of free enzyme  [I]  =  concentration of free i n h i b i t o r  [I] - [I ] = 0.04 M o  and [E] =  t  h  6  n  (where [I ] i s the i n i t i a l i n h i b i t o r concentration) o  0.003 - [EI]  [EI] (0.003-[EI])(0.04)  5  U  - 96 -  /.  and  [EI] =  0.002 M  Fraction of i n h i b i t o r bound = I  =  1/20  = I  =  19/20  Fraction of i n h i b i t o r free  Now i n the fast exchange l i m i t the observed s p l i t t i n g , J ^  g  » i s a sum  of two terms:  J  where  ^  obs.  T  B obs.B J  +  r  F obs.F J  g  -  s p l i t t i n g i n t o t a l l y bound case  J', „ obs.F  =  s p l i t t i n g i n t o t a l l y free case. °  If we assume quite a large d i s t o r t i o n on binding so that  J' _ obs.B  Then J '  =  6 Hz  and J' _ obs.F  =  (1/20) 6  =  0.3  =  3.25 Hz  +  =  3.1 Hz  (19/20) 3.1  ODS .  +  2.95  It should be obvious then that to use spectral s p l i t t i n g changes to measure i n h i b i t o r d i s t o r t i o n on binding w i l l require careful spectral analysis.  Nevertheless, the technique should be viable  provided the following steps are followed:  (a)  O b t a i n a h i g h f r a c t i o n o f bound i n h i b i t o r - t h i s w i l l i n v o l v e d i l u t e s o l u t i o n s and broad  (b)  Use a time a v e r a g i n g  lines.  technique  (esp. F o u r i e r transform) t o  a c h i e v e good s i g n a l - t o - n o i s e . Cc)  Choose an i n h i b i t o r which shows l i t t l e  exchange  broadening  (and/or work a t h i g h e r temperatures) - t h i s w i l l g r e a t l y i n c r e a s e l i n e sharpness (d)  and accuracy,  Choose a system whose spectrum i s e i t h e r f i r s t analysed  to a v o i d a m b i g u i t i e s due t o h i g h o r d e r  Another p o t e n t i a l technique  o r d e r o r can be effects.  f o r the i n v e s t i g a t i o n o f c o n f o r m a t i o n a l  changes on b i n d i n g i s one which t h i s l a b o r a t o r y has been working  19 toward f o r some y e a r s . chemical  I t i n v o l v e s the extreme s e n s i t i v i t y o f F 76 s h i f t s to i n t r a m o l e c u l a r e f f e c t s (such as c o n f o r m a t i o n o r  2 c o n f i g u r a t i o n changes ) .  F o r i n s t a n c e , i n the system s t u d i e d here  19 one would observe " n o i s e decouple" project.  a  F s u b s t i t u e n t on the r i n g .  The a b i l i t y t o  t h e r i n g p r o t o n s would be v e r y u s e f u l i n any such  - 98 Experimental General Methods (a)  A l l *H n.m.r. spectra were measured with a Varian HA-100  spectrometer operating i n the frequency sweep mode.  For enzyme studies  the water resonance was used f o r the field-frequency lock.  Sugar  solutions i n CDCl^ were run locked on tetramethylsilane (TMS).  All  ^"H chemical s h i f t s are reported on the x scale. 19 (b)  All  F spectra were measured at 94.071 MHz using a Varian  HA-100 spectrometer operating i n the frequency sweep mode. A c a p i l l a r y of t r i f l u o r o a c e t i c acid (TFA) held concentric with the n.m.r. tube was used for the field-frequency lock. (c)  Melting points were performed  on a Thomas-Hoover c a p i l l a r y  -m.p. apparatus and are corrected for thermometer error. (d)  The HA-100 probe temperature  was 31.5°C f o r a l l enzyme  experiments. (e)  Two different i n t e r n a l reference compounds x^ere used f o r  the measurement of chemical s h i f t s (<5) i n enzyme studies: f o r proton work, t e r t i a r y butanol and f o r fluorine work, t r i f l u o r o e t h a n o l (Aldrich). (f)  A l l pH measurements were performed on an Instrumentation  Lab. Inc. pH meter with expandable scale. (g)  A l l enzyme measurements were made i n 0.1 M c i t r a t e buffer  at pH 5.5 (Documenta G e i g y ^ ) . (h)  The technique of measuring 6 r e l a t i v e to the i n t e r n a l  standards i n enzyme runs was as follows.  The spectrum of the standard  was recorded and calibrated followed by that of the sugar of interest.  resonance  Then the standard spectrum was run again and the results  - 99 -  were averaged. (i)  This was repeated several times for each sample.  A l l enzyme measurements were made with the same batch of hen  egg white lysozyme (Worthington Biochemical - s a l t - f r e e , twice recrystallized). use.  Solutions were made up freshly immediately p r i o r to  Enzyme concentrations were calculated by removing a 25 y£  aliquot and d i l u t i n g to 25.00 ml with buffer.  The absorbance was  then read at 280 my on a double beam Bausch a Lomb Spectronic 600 and used to calculate [E ] from the known extinction c o e f f i c i e n t . o  78  Organic Synthesis 2-Amino-2-deoxy-N-trifluoroacetyl-D-glucose (1 and 2)  was the  precursor of a l l fluoro derivatives studied here and was prepared by the action of S-ethyl t r i f l u o r o t h i o l a c e t a t e (Pierce Chemical Co.) on D79 glucosamine in methanol as described i n the l i t e r a t u r e .  D-glucos-  amine i t s e l f was produced i n the reaction vessel from glucosamine hydrochloride (Aldrich) and sodium methoxide p r i o r to adding the trifluorothiolacetate.  The a-anomer (1) c r y s t a l l i z e d s e l e c t i v e l y  from an aqueous solution of the mutarotated pair as solvent was allowed to evaporate slowly.  1 was obtained as large, clear, colourless  crystals which when carefully dried became white and powdery:  m.p.  188-188.5°C (with decomposition). 2-Amino-2-deoxy-N-trifluoroacetyl-D-glucose-a (and g)-methyl glycoside (3 and 4) were prepared from pure, dry N - t r i f l u o r o a c e t y l glucosamine v i a the general method of acid c a t a l y s i s i n methanol using a r e s i n (Dowex-Ag 50 W X8, Bio Rad) as catalyst.  The resin i n i t s acid  - 100 -  form was washed thoroughly with methanol over a period of several days before use.  Sample conditions were:  4 g of s t a r t i n g material  was dissolved i n about 50 ml of dry methanol.  To t h i s x^as added 30 ml  of resin and the mixture was refluxed with s t i r r i n g f o r 3 hours. The mixture was then f i l t e r e d and the resin washed thoroughly. Stripping of the solvent from the f i l t r a t e produced a syrupy mixture of a - and g-glycosides (in about a 2:3 r a t i o ) .  Column chromatography  on s i l i c a gel (Mallinckrodt "SilicAR" CC-7), using f i r s t  10%  MeOH/CHCl and then 20% MeOH/CHCl gave the pure anomers i n roughly 3  60% y i e l d .  3  M.p.  was 216.0-217.0°C  of 3 was 196.5-197.5°C (with decomp.  (with decomp.), m.p.  of 4  Both were white s o l i d s .  2-Amino-2-deoxy-N-acetyl-D-glucose-a-methyl  glycoside (7)  was  prepared from N-acetyl glucosamine (Pfanstiel Lab.) as described i n 80 the l i t e r a t u r e  using Dowex resin i n the acid form and MeOH.  P u r i f i c a t i o n was achieved by means of column chromatography on s i l i c a gel using 10% MeOH/CHCl as eluent. 3  s o l i d was  The m.p.  of the white  192-193°C.  2-Amino-2-deoxy-N-acetyl(d )-D-glucose was prepared by selective 3  N-acylation of glucosamine with acetic anhydride-d^ (Merck, Sharp o 80 and Dohme) as described i n the l i t e r a t u r e .  2-Amino-2-deoxy-N-acetyl-Cg-0-p-tolylsulfonyl-D-glucose-a-methylglycoside was produced i n a fashion similar to that described i n the  i  - 101 -  l i t e r a t u r e f o r i t s glucose analogue.  3 . 9 g of 7 was dissolved i n  dry pyridine (55 ml) i n a flask equipped with drying tube.  1.1 mole  equivalents of p - t o l y l s u l f o n y l chloride (Eastman) was dissolved i n 8 ml of dry pyridine. ice bath.  The solution of 7 was cooled to 0°C i n a s a l t -  The p - t o l y l s u l f o n y l chloride solution was then added  dropwise with s t i r r i n g over a period of 20 min while maintaining the temperature at or below 0°C. warm up to room temperature.  The mixture was then l e f t to  After 48 hours the solvent was  stripped o f f . The resulting o i l was dissolved i n CHCl^ and washed with an aqueous solution of KHCO^.  The y i e l d was 50-60% of a l i g h t  tan o i l .  2-Amino-2-deoxy-N-acetyl-Cg-iodo-D-glucose-a-methyl  glycoside ( 9 )  was prepared from the C^—tosylate by a method analogous to that  81 described i n the l i t e r a t u r e f o r i t s glucose analogue.  The o i l  obtained as described above (4 g) was dissolved i n dry acetone (40 ml).  3 mole equivalents of dry Nal ( A l l i e d Chemical) was then  added and the solution was refluxed with s t i r r i n g for 3 hours after which the platey crystals of sodium p-tolylsulfonate were f i l t e r e d off.  Workup afforded a s l i g h t l y tacky powder.  achieved by column chromatography CHCly  P u r i f i c a t i o n was  on s i l i c a gel eluted with 5% MeOH/  The result was a good y i e l d of a white s o l i d , m.p. 162-164°C.  2-Amino-2-deoxy-N-acetyl-C^-methyl-D-glucose-a-methyl  glycoside (10)  was prepared by c a t a l y t i c hydrogenation of 9 at atmospheric pressure. The general technique for hydrogenation of a l k y l halides i s discussed  - 102 -  elsewhere.  82  2.0 g of 9 was dissolved In 25 ml of MeOH i n a 50 ml  Erlenmeyer flask.  3-4 equivalents (1 g) of s o l i d KOH was added and  300 mg of palladium on powdered charcoal (Matheson, Coleman and B e l l 10% c a t a l y s t ) .  The mixture was hydrogenated at room temperature and  atmospheric pressure for 3 hours.  Workup afforded a clear, colourless  glass (yield > 90%).  O-Acetylated derivatives of sugars were found to be extremely useful for product i d e n t i f i c a t i o n v i a their  n.m.r.  They were  produced by reaction with excess acetic anhydride (Baker Chemical  Co.)  i n dry pyridine.  Treatment of Data The correlation of chemical s h i f t ( 6 ) and i n h i b i t o r concentration ([I ]) with bound chemical s h i f t (A) and d i s s o c i a t i o n constant o 1 66 (K = ——) has been described by Dahlquist and Raftery i n some B D  K  detail.  The basic treatment i s shown below for an i n h i b i t o r interacting  reversibly with an enzyme.  E  K  +  D  I  =  y  [hi J  EI  (1)  [E]  =  enzyme concentration  [I]  =  i n h i b i t o r concentration  [EI] =  concentration of enzyme-inhibitor complex  In the fast exchange l i m i t we have:  - 103 -  6  =  P_ A B  where 6 i s the observed s h i f t  (2)  of a p a r t i c u l a r sugar peak from i t s unbound position and P i s D  the f r a c t i o n of t o t a l i n h i b i t o r bound at a given time from (2)  •  6  =  jy^j- A o  [EI] =  [I ] | o  but  [E] =  [ E l - [EI] = o  [E ] - [I ] | o o A  and  [I] =  [ l ] - [EI] =  [I ] - [I ] |  o  o  o  where the subscript 'o' indicates i n i t i a l concentration. Substitution into equation (1) gives  D  5  o  o  o  Making the following approximations that:  and  (a)  6 <<  (b)  K  Q  A  i s of the order of [I ]  we may drop the term  — [ I ] to get: Q  [E ]A  U  O  O  [E J A or  [ I J  |  ~  O  ~  [  V  o  A  - 104 -  Hence a plot of [I ] vs. 1/6 should y i e l d a straight l i n e of slope [E ]A and intercept -(K o  + [E ] ) . D o  CHAPTER I I I APPLICATION OF THE AUDIOFREQUENCY PULSE TECHNIQUE TO THE  STUDY OF LARGE ORGANIC MOLECULES IN SOLUTION  Introduction Several techniques involving the use of n.m.r. spectroscopy to study large organic molecules i n solution have already been discussed. Magnetic resonance methods (both e.s.r. and n.m.r.) involve r e l a t i v e l y low energy spectroscopic processes and, hence, r e f l e c t i n their spectra phenomena of great i n t e r e s t to the chemist.  Previously i n  this thesis we have made use of the well known s p e c t r a l features: chemical s h i f t , scalar coupling and integrated i n t e n s i t y .  A fourth  feature, the relaxation times associated with a given resonance, w i l l now  be considered.  U n t i l quite recently this l a s t feature has been  used primarily by physicists and physical chemists i n problems involving small molecules. The term relaxation here refers to the transfer of spin  populations  between nuclear energy l e v e l s as a r e s u l t of their energy d i f f e r e n t i a l in a magnetic f i e l d .  Obviously  i f there i s some mechanism for this  transfer there w i l l be a tendency to reach a Boltzmann d i s t r i b u t i o n such that a resultant macroscopic magnetic moment vector w i l l e x i s t along the magnetic f i e l d axis.  At temperatures greater than a few degrees  - 106 Kelvin, the r a t i o  N. _± N  =  of lower to upper state population can be written  !  +  2yH o kT  T i s the Boltzmann spin temperature:  the spins are immersed i n a  thermal bath (the bulk solution) at roughly room temperature.  However,  there i s only a very weak thermal coupling between the nuclear spin system and the bath so that the Boltzmann spin temperature can become very high during the course of some perturbation such as i r r a d i a t i o n at the resonance frequency.  This weak coupling arises from time-  dependent l o c a l magnetic f i e l d s (the time dependence being derived from molecular motion).  These fluctuating l o c a l magnetic f i e l d s to which  a molecule i n solution i s exposed are responsible for a l l nuclear relaxation processes but there are some half dozen major mechanisms f o r 51 83 84 production aid i n t e r a c t i o n of such f i e l d s .  '  '  Moreover there are  two macroscopically observable features of relaxation processes: ease of saturation of a t r a n s i t i o n (related to the above  the  spin-temperature  discussion) and the linewidth of the resonance corresponding  to the  t r a n s i t i o n (related to the nuclear spin state l i f e t i m e ) . In dealing with nuclear relaxation, i t i s generally convenient to consider the above-mentioned macroscopic magnetic moment which i s experimentally observable and which at equilibrium l i e s along the Z-axis (H - a x i s ) .  The behaviour  of this vector can be described by c l a s s i c a l  53 mechanics and basic relaxation phenomena are readily dealt with using , , , , • • •, 50,83-85, such a model (more complete discussions have been given elsewhere )  - 107 -  With any t r a n s i t i o n i n a n.m.r. spectrum there i s associated such a vector.  When this vector i s disturbed from i t s equilibrium p o s i t i o n  (say by i r r a d i a t i o n at i t s resonance frequency, oi) i t recovers exponentially The  at a c h a r a c t e r i s t i c rate upon removal of the perturbation.  time constant for i t s recovery p a r a l l e l to the Z-axis i s c a l l e d T^  and that for i t s collapse i n the XY-plane i s c a l l e d T^. Experimentally techniques. according  both T^ and 1^ can be measured by a variety of  i s r e f l e c t e d i n the linewidth of a single t r a n s i t i o n  to the relationship T^ = 1/TTAV  at h a l f height).  Unfortunately,  (where A v i s the linewidth  for most common organic molecules i n  solution the 'true' value of A v i s obscured by f i e l d inhomogeneity 86 87 and/or small, unresolved s p l i t t i n g s .  Spin-echo methods  '  and the  88 89 ' are more generally applicable for measurement of T„. lp z The spin-echo technique was chosen for use i n this work, although both 89 other methods have been t r i e d . T, can be measured by T, , adiabatic 1 lp 90 91a rapid passage, saturation recovery and progressive  T  1  technique  91b saturation  techniques.  In this work T^ was measured by a very  simple pulse technique i n which a single pulse i s used to invert the magnetization along the Z-axis (see Appendix A) and i t s return to equilibrium i s monitored at i n t e r v a l s . B i o l o g i c a l Applications Because T^ and T^ are determined by time-dependent i n t e r - and intramolecular  r e l a t i o n s h i p s , they contain information  about r e l a t i v e  rates of motion, orientations and distances i n the molecules involved. Although the b i o l o g i c a l applications of relaxation studies are not as  - 108 -  numerous to date as those of chemical s h i f t and coupling constant they are very important and becoming more so.  data,  A very general review 41  of the area has been included i n the a r t i c l e by Allerhand and T r u l l . More s p e c i f i c review a r t i c l e s e x i s t on various aspects of the use of nuclear relaxation rates to study macromolecules.  In p a r t i c u l a r ,  Mildred Cohn has described i n d e t a i l the use of paramagnetic 40 probes.  For instance considerable work has been done by introducing  paramagnetic species at the active s i t e of an enzyme f o r enzymei n h i b i t o r i n t e r a c t i o n studies.  The e f f e c t of the unpaired  electron  spins on the relaxation rates of nuclear spins i s a function of the distance between the two i n t e r a c t i n g species and of the motional freedom i n the region of the paramagnetic species  (the reason f o r  using a paramagnetic probe i n the f i r s t place i s that unpaired  electrons  are extremely e f f e c t i v e i n causing rapid relaxation of neighbouring nuclei).  Thus the exploitation of relaxation rate changes effected  by paramagnetic probes at the active s i t e provides  the p o s s i b i l i t y  of estimating interatomic distances and of mapping the substrates at  the active s i t e i n solution as well as characterizing the molecular  motion of highly l o c a l i z e d regions of the active s i t e .  The same approach  may permit c a l c u l a t i o n of the rate of chemical exchange between the 'enzyme-bound' and 'unbound' substrate conditions.  This rate i s  related to the rate of the f i r s t elementary step i n the reaction sequence of enzyme c a t a l y s i s .  A well known example of the paramagnetic  probe technique i s the study of creatine kinase using paramagnetic 40 manganous ions and a stable nitroxide free r a d i c a l .  - 109 45 Sykes and Scott have produced an excellent review on the use of n.m.r. to study dynamic aspects of molecular structure and interaction i n b i o l o g i c a l systems exclusive of paramagnetic methods.  probe  They point out that there are two approaches whereby  b i o l o g i c a l systems have been studied for k i n e t i c data. involve solution of the phenomenological  Both approaches  Bloch equations f o r the  92 nuclear magnetization,  as modified by McConnell to include the effects  of chemical exchange, for a spin system where a nucleus i s transferred back and forth between two d i s t i n c t environments, order l i f e t i m e s , x^ and i .  A and B, with f i r s t  Perhaps the most obvious technique i s  that applicable when both s i t e s are approximately equally populated.  44 45 51 It i s then possible to derive equations describing the lineshape for  ' '  a variety of exchange l i f e t i m e s and to compare these to the  observed spectrum.  With this approach i t i s desirable to be able to A B vary the exchange l i f e t i m e , x = ( — — — ) , over a f a i r l y wide range A B r e l a t i v e to the difference i n resonance frequencies (u> -uO i f this A B is non-zero or r e l a t i v e to the relaxation rates of the s i t e s i f to. = A T  T  T  T  V In  the system (lysozyme and i n h i b i t o r ) studied i n this chapter,  the condition of equal s i t e populations was not f u l f i l l e d .  However,  an interesting area where the lineshape approach has been applied (with somewhat ambiguous results) i s the study of h e l i x - c o i l transitions  45 94 i n certain synthetic polypeptides.  '  In these studies the resonances  corresponding to peptide bond NH groups and a-CH groups were monitored. The other approach described by Sykes and Scott f o r calculation of  forward and reverse exchange rate constants i s applicable when  - 110  -  the population of one s i t e i s much greater than that of the other This i s a common s i t u a t i o n i n enzyme-substrate and studies where the concentration  site.  enzyme-inhibitor  of free substrate or i n h i b i t o r i s much  larger than the concentration of bound species.  In fact i n these cases  only one resonance i s observed even i n the slow exchange l i m i t and  rate  constants must be calculated from a study of T^, 1^ and to for this •resonance as a function of some variable such as temperature, spectrometer frequency or r e l a t i v e population of s i t e s A and B.  In our case  we have used the l a t t e r approach by varying the enzyme concentration. 45 Sykes and Scott  point out that for the case of Population A >> 95  Population B , S w i f t and Connick the modified Bloch equation exchange rates.  have derived a general s o l u t i o n to  for T^ and CJ which i s v a l i d for a l l  A corresponding  equation  for T^ has been derived by  Luz and Meiboom^ and by O'Reilly and Poole. 97a 97b Sykes and Sykes and Parravano have manipulated these equations a  into forms suitable for dealing (via n.m.r.) with the case of an enzyme-inhibitor  system i n which the bound magnetic environment of  some portion of the i n h i b i t o r d i f f e r s from that of i t s unbound counterpart.  Naturally these equations are most sensitive to exchange  rates which are "on the n.m.r. time scale" - to date, a l l rate  constant  data reported for monosaccharide i n h i b i t o r s of lysozyme seems to be 97a 97b i n this intermediate range. Sykes and Sykes and Parravano have applied t h e i r equations to the i n t e r a c t i o n of lysozyme with four N-acety1-D-glucosamine compounds (5-8) of the previous  chapter by  98 making use of the N-acetyl resonances, and Sykes  has reported a  s i m i l a r measurement on trifluoroacetyl-D-phenylalanine i n t e r a c t i n g  - Ill with a-chymotrypsin. In this chapter, amongst other uses of the audiofrequency technique,  99  pulse  we report the results of rate constant calculations on  the system of lysozyme and N-trifluoroacetyl-a-D-glucosamine as 19 measured by pulsed  F n.m.r.  This data was  of p a r t i c u l a r interest 19  to us because we were curious as to the e f f e c t s of  F labels with  regard to the work of Chapter I I . Applications to Large Organic Although  Molecules  the phenomenon of nuclear relaxation i s of fundamental  importance to the measurement of n.m.r. spectra, and has as such received a great deal of attention from p h y s i c i s t s , i t has been largely neglected by organic chemists.  There are several good  reasons for this lack of i n t e r e s t , of which the most cogent i s that u n t i l recently, instrumentation suitable for the routine and s e l e c t i v e measurement of the nuclear relaxation times of anything other than very simple systems has been unavailable. s e l e c t i v e , audiofrequency-pulse  However, the development of the  (n.m.r.) technique by Freeman and  99 Wxttekoek  has made possible, at least i n p r i n c i p l e , relaxation  studies of complex organic systems. about which very l i t t l e i s known.  This i s an extremely  new  area  As w i l l be seen, 'large' organic  molecules such as sugars and steroids have quite different relaxation times from simple molecules  such as benzene or chloroform and there are  considerable problems to be overcome i n dealing with them.  The work  reported here (and indeed elsewhere i n the l i t e r a t u r e ) i s only a scratch on the surface of the f i e l d of s e l e c t i v e relaxation studies of large  - 112 -  organic molecules; eventually i t may be possible to relate the r e s u l t s of such studies to intramolecular phenomena of i n t e r e s t .  In this  regard we describe here some preliminary considerations of the problem of whether  and  are sensitive to intramolecular phenomena  by measuring the relaxation times of a series of c l o s e l y related simple compounds (the c i s and trans isomers of  1,2-dichloroethylene,  1,2-dibromoethylene and the e t h y l esters of maleic and fumaric a c i d s ) . As w i l l be shown, the results of these experiments are encouraging. We have also gone on to apply s i m i l a r methods to more p r a c t i c a l examples:  the measurement of proton relaxation times of various n u c l e i  i n an alkaloid and several sugars.  - 113 -  Results and Discussion A.  A Case for the A p p l i c a b i l i t y of  and  to S t r u c t u r a l Organic  Chemistry Physicists and physical chemists have measured nuclear relaxation times of small molecules  i n attempts to elucidate the mechanisms of  relaxation. ^ ' ^ ' " ^ 53,100  ^ t y p i c a l approach i s to calculate  and  for a simple molecule by considering the various possible mechanisms and comparing the calculated values to those observed  experimentally.  In such cases measurements are usually made i n a solvent possessing no hydrogen m c l e i or i n a deuterated solvent and the data extrapolated to i n f i n i t e d i l u t i o n to rule out intermolecular e f f e c t s .  As chemists  dealing with the structure of large organic molecules, we were curious as to whether"relaxation time measurements are sensitive to molecular parameters of interest to us - and we were w i l l i n g to s e t t l e for empirical relationships.  For instance, are  and  sensitive to changes i n  substituent groups or orientation and proximity of substituents? I f not, a number of p o t e n t i a l uses of pulsed n.m.r. would be closed to us.  * As a f i r s t step i n this d i r e c t i o n we have measured the relaxation times of a series of substituted c i s and trans isomers of ethylene:  The apparatus used for pulse experiments described i n this chapter (with the exception of those i n Section B) was the "Mark I I " pulse spectrometer (see Appendix B) designed and constructed i n collaboration with Mr. Roland Burton of this Department.  c\ R = CI  1,2-dichloroethylene  (M.W.  = 96.96)  R =. Br  1,2-dibromoethylene  (M.W.  = 185.88)  R = COOEt  maleic (cis) and fumaric (trans) acid d i e t h y l esters (M.W. = 172.20)  Since we were interested primarily i n changes i n  and  brought  about by intramolecular chemical differences rather than i n absolute values, we wished to maintain a l l other factors as constant as possible.  A very simple method of ensuring this i s to dissolve a l l  compounds i n the same sample - a method which i s b e a u t i f u l l y with selective pulse techniques. the ~^H n.m.r. spectrum (100 MHz)  As a t y p i c a l example F i g . 1A shows of a mixture of the c i s and trans  isomers of 1,2-dichloro- and 1,2-dibromoethylenes. deuterochloroform with a few drops of TMS  The solvent used was  for a field-frequency lock.  Although the bromo-compounds were purchased  as a mixture of isomers,  the concentrations of the four compounds were approximately (= 0.5 M).  The sample was  compatible  equal  c a r e f u l l y degassed (6 freeze-pump-thaw  cycles) before being sealed off to exclude (paramagnetic)  oxygen.  Each  compound gives a single, symmetric, very sharp resonance whose linewidth  - 115 -  Br  \  /  /  Br  \  V  ^COOEt Cl  EtOOC  EtOOC  - / "\  COOEt  V  / "  "\  7 i  B  3.0  as Line'  1 2 3 4  5 6 7 1.  T-\  58.5 95.4 78.4 114.9 28.8 12.5  T  2  r  (sec.)  15.7 28.0 6.7 11.9 20.1 7.5 9.3  The alkene proton regions of the n.m.r. spectra (100 MHz) of degassed samples (solvent deuterochloroform) of A. 1,2-dibromoand 1,2-dichloroethylene (cis and trans isomers) and B. maleic and fumaric acid diethyl esters (plus c i s 1,2-dichloroethylene as a reference). TMS was used for the i n t e r n a l field-frequency lock. In each case the resonances are numbered and the compounds to which they correspond are indicated. The nuclear relaxation data for each resonance i s tabulated below the spectra.  - 116 -  i s determined mainly by f i e l d inhomogeneity.  The proton resonances  corresponding to bromo analogues f a l l to lower f i e l d than those of chloro analogues and i n each case the c i s anomer i s to low f i e l d of the trans isomer ( i d e n t i f i c a t i o n of the bromo compounds was v i a scalar 13 couplings i n the  C satellites).  to s e l e c t i v e l y measure T^ and  I t was a r e l a t i v e l y simple matter for each resonance (as w i l l be  discussed i n more d e t a i l at the end of this section) although the separation between the chloro isomer resonances was only 6.3 Hz. values of T^ and 1^ found are l i s t e d below F i g . 1.  The  The error l i m i t s  involved i n such measurements are roughly + 10% for the T^ data but are probably considerably larger for T^ i n this p a r t i c u l a r case as w i l l be discussed l a t e r i n this section.  The notable features of  the data are that: (a)  i n each case T^ i s considerably longer than  (this d i f f e r -  ence i s more pronounced i n the case of the chloro compounds) (b)  i n each case the c i s isomer has shorter relaxation times  than the trans isomer. Fig. IB shows the "*"H n.m.r. spectrum of the alkene region of a second sample containing the esters of maleic and fumaric acids as well as the already measured c i s 1,2-dichloroethylene. This sample was made up i n an exactly analogous fashion to that of the sample just considered:  solvent CDCl^ with a few drops of TMS for a f i e l d -  frequency lock; and was degassed (6 freeze-pump-thaw cycles) p r i o r to being sealed o f f . The alkene proton resonance of fumaric acid d i e t h y l ester f a l l s to low f i e l d of i t s maleic acid counterpart.  - 117 -  Note the reduced ringing i n the cis-isomer.  Both of these acid esters  may have very small unresolved couplings between the ethylene protons and the ethyl protons.  This second sample (Fig. IB) contained a  somewhat lower concentration of protonated one  species than the previous  (Fig. IA) however i n each case the sample was  roughly 80% CDCl^  by volume. That there were no large differences i n intermolecular effects between the samples of F i g . IA and IB was  checked i n this case by  the i n c l u s i o n of some common species i n each sample. or  i n t e r n a l standard, was  The common species,  c i s 1,2-dichloroethylene.  The relaxation data for this new sample i s l i s t e d below that for the f i r s t sample i n F i g . 1.  Each of the seven spectral t r a n s i t i o n s i s  numbered and i t s corresponding compound indicated above the spectrum. Note that i n the second sample,  f o r c i s 1,2-dichloroethylene has  remained the same as i n the f i r s t sample within experimental error (however the s l i g h t increase may  r e f l e c t the more d i l u t e solution).  This use of a standard permits a certain amount of data comparison between samples.  Once again i t i s obvious that the c i s isomer has a  shorter relaxation time than the trans isomer.  However, i n this case,  T^ and T^ are much more nearly equal (in fact part of the difference may  be experimental, as w i l l be discussed l a t e r i n this section).  The T^  Experiment  The method of T^ measurement employed here was  that commonly  referred to as the "Gill-Meiboom modification of the Carr-Purcell pulse  -  118 -  86 87 99 sequence'.'  '  '  The result of such a measurement on resonance #7  (the d i e t h y l ester of maleic acid) i n F i g . IB i s shown i n F i g . 2 together with a diagrammatic explanation of the pulse sequence. consists of a Tr/2-pulse  (see Appendix A) along the X ' - a x i s  by a series of TT-pulses along the Y ' - a x i s frame.  This sequence  of the rotating  followed reference  Experimentally i t was convenient to make the spacing between  successive ir-pulses equal to twice that between the ir/2-pulse and the f i r s t rr-pulse.  The duration of a rr/2 pulse in the p a r t i c u l a r  trace shown i s 0.2 seconds.  The components of such a pulse sequence  are discussed i n d e t a i l i n Appendix A.  Here i t  s u f f i c e s to say that  the C a r r - P u r c e l l sequence removes the contribution of f i e l d inhomogeneity to the measured value of  and the Gill-Meiboom modification makes  less c r u c i a l the accurate setting of pulse durations.  The value of  can be readily calculated from the peak heights of the i n i t i a l  90°-pulse  and the ensuing echoes according to the exponential relationship M  =  -t/T M  e  q  2  (Appendix A) where M  vector along the Y ' - a x i s value at some l a t e r time,  q  i s the magnitude of the magnetization  immediately a f t e r the 9 0 ° - p u l s e and  i s the  t.  The T.^ Experiment The pulse sequence used to measure T^ was considerably simpler and involved less pulsing per unit time as w i l l be discussed l a t e r this s e c t i o n .  The sequence consisted of two pulses:  (a Tr-pulse along the X ' - a x i s ) ( M ) along the - Z ' - a x i s , q  l a t e r time, t ,  in  the f i r s t pulse  to prepare the magnetization vector  and a second pulse along the X ' - a x i s  to force the magnetization vector  at some  (M ) into or through  0 Fig.  20  TIME [sec]  2.  E f f e c t o f a m o d i f i e d C a r r - P u r c e l l sequence on the m a g n e t i z a t i o n v e c t o r , M . A . E q u i l i b r i u m ; B. i r / 2 - p u l s e a l o n g the X ' - a x i s ; C. B l o c h decay; D. ^ - p u l s e a l o n g the Y ' - a x i s c a u s i n g r e f o c u s s i n g of the f i e l d - i n h o m o g e n e i t y component; E . E c h o ; F . B l o c h decay; G. - r r - p u l s e ; H . Echo. The scope t r a c e f o r such such a sequence on the m a l e i c a c i d d i e t h y l e s t e r resonance of F i g . IB o f Chapter I I I i s shown. Q  40  - 120 -  the observation plane (X'Y*-plane) so that the receiver c o i l could detect i t . pulse was  In the set of experiments just described, this second a 27r-pulse (360°).  One  then simply waited for the system  to return to equilibrium and then repeated the two pulse sequence using a d i f f e r e n t time delay, t, between the f i r s t and second pulses. The result of such a measurement on resonance ill  (trans  1,2-  dibromoethylene) of F i g . 1A i s shown i n F i g . 3 together with a diagrammatic explanation. seconds.  The Tr-pulse length i n this case was  Note that, for convenience,  0.8  the i n i t i a l Tr-pulse of each  pulse pair has been triggered exactly on top of that from the previous pulse p a i r .  Hence the trace shown represents seven pulse pairs vn.th  varying values of the delay, t.  The value of T^ was  readily calculated  from the positive-going recorder peak heights according to the relationship M. = M t o  -t/T (l-2e 1) where M  o  i s the i n i t i a l magnitude of  the magnetization vector along the -Z'-axis and  i s i t s magnitude at  some l a t e r time, t. The results of the experiments just described and the  techniques  used for measurement of T^ and T^ deserve several comments.  Firstly,  the pulse sequence used to measure T^ i s more laborious than others available.  For instance, since the 2u-pulse used for monitoring  magnitude of M  the  leaves the vector i n i t s o r i g i n a l p o s i t i o n , there  should be no need to perform a T^ experiment by a series of pulse  99 pairs with waiting periods i n between. have pointed this out and suggested  Freeman and Wittekoek  that T^ can be measured by a pulse  sequence consisting of one Tr-pulse followed by a series of equally spaced 2Tr-pulses (in R.F. pulse work this corresponds sequence").  to the  "triplet  This technique has the drawback of requiring long,  0 Fig. 3 .  50  100  150  T I M E [sec] Effect of a T^ sequence on the magnetization vector, M Q . A. Equilibrium; B. Preparatory u-pulse along the X'-axis; C . A 2TTmonitoring pulse along the X'-axis; D. E. and F. show 2iT-pulses which would occur at progressively longer time delays. A sequence on the trans 1,2-dibromoethylene resonance of F i g . IA of Chanter I I I .  - 122 frequent pulses and i n our experience the slower method gives somewhat better data. In the case of  there i s no alternative to measurement with one  pulse sequence because d i f f u s i o n effects i n solution cause "phase memory l o s s " of the spin isochromats which can only be minimized frequent ( r e l a t i v e to the d i f f u s i o n rate) echo generation. the Carr-Purcell technique involves a minimum of pulsing. i s considerably longer than T^,  ^  c a n  by  Nevertheless, But when  contribute appreciably to the 99  observed length of the spin-echo decay during pulses.  Hence i n the  case of samples such as the halo-ethylenes, one has to choose between too many and too few pulses i n the mentioned e a r l i e r , the as the  sequence.  For this reason, as  data i n this case are l i k e l y not as accurate  data although they were quite reproducible within a reasonable  range of pulse r e p e t i t i o n rate.  Loss of magnetization between pulses  can become more serious i f a resonance contains small, unresolved 99 splittings between  and this could account for some of the observed difference and  for the maleic and fumaric acid derivatives where  the pulse i n t e r v a l s have been kept long f o r comparison to the other compounds.  I t would seem desirable when  =  to generate frequent  echoes. The very large difference between T^ and T^ i n the case of the  84 halogenated  species i s l i k e l y due to scalar coupling  protons to the halogen quadrupole moment.  of the ethylene  The shorter relaxation times  of the c i s isomers r e l a t i v e to the trans i n each case i s quite possibly due to the intramolecular dipole-dipole coupling mechanism: i . e . , the ethylene protons are closer together i n the c i s isomer and  - 123 84 6 the expression for dipole-dipole coupling contains a factor of 1/r • However, without knowing more about the system, i t i s not possible to rule out chemical s h i f t anisotropy and spin-rotation interactions as sources of the difference. The point we wish to make i s that there are differences amongst the relaxation times and they appear to be systematic.  Furthermore,  the techniques of dissolving samples i n the same solution and of using an i n t e r n a l standard between d i f f e r e n t samples seem to be useful for purposes of comparison.  B.  Measurement of Nuclear Relaxation Times of an A l k a l o i d by the Audiofrequency Pulse Technique The above experiments demonstrated  that i t i s possible to  s e l e c t i v e l y pulse small organic molecules with long relaxation times i n order to calculate T^ and  using Freeman and Wittekoek's audio-  99 frequency-pulse technique.  We found that, i n the cases studied at  least, chemical changes were systematically reflected i n changes i n T^ and T ^ .  In this section we report the use of s e l e c t i v e pulse  techniques  to measure T^ and  for i n d i v i d u a l protons of an a l k a l o i d ,  vindoline.  Vindoline was chosen to test the s u i t a b i l i t y of audio-  frequency-pulse techniques for relaxation time measurements with The apparatus used for experiments on vindoline was the "Mark I" pulse spectrometer (see Appendix B) designed and constructed i n collaboration with Mr. Roland Burton of this Department.  - 124 substances having the molecular complexity commonly associated with "natural products".  I t was of additional i n t e r e s t to us because of  i t s r e l a t i o n with the anti-tumour p r i n c i p l e s obtained from Vinca rosea l i n . " * " ^  The important point which we wished to e s t a b l i s h  was  whether i n d i v i d u a l protons of a substance of t h i s complexity would s t i l l have d i f f e r i n g nuclear relaxation times. Because we were interested i n b i o l o g i c a l systems where i t may often be inconvenient or undesirable to degass samples, t h i s p a r t i c u l a r set  of experiments was performed i n non-degassed solution ( i t was  subsequently shown that other relaxation mechanisms were so e f f e c t i v e as to make the presence of atmospheric oxygen r e l a t i v e l y unimportant). The normal  n.m.r. spectrum of vindoline i n deuterochloroform  solution (100 MHz) TMS  i s shown i n F i g . 4 along with a s t r u c t u r a l formula.  dissolved i n the same sample was  used to generate a s i g n a l f o r  the field-frequency lock. I n i t i a l l y , the relaxation times of the acetate-methyl and N-methyl singlets were determined.  Measurement of the spin-spin relaxation  times, T^, for these two resonances  followed the usual Meiboom-Gill  modification of the Carr-Purcell experiment. from such an experiment Fig.  A t y p i c a l scope trace  on the C^-acetate methyl peak i s shown i n  5A (7r/2-pulse duration = 0.10  sec).  The echoes are seen as broad  humps between the i n f l e c t i o n s caused by the 180°-pulses.  Because of the  rather short relaxation times, T^, of these protons, the spin-echo trace f a l l s to zero rather rapidly and only the f i r s t few echoes can be used for measurement purposes.  However, i t was  always possible to  vary the delay between pulses during the course of several d i f f e r e n t  0.0  1  5.0  10.0  Fig. 4. The H n ,m. r. ' spectrum (100 MHz) of the a l k a l o i d , vindoline, i n deuterochlorof orm solution using TMS f o r the i n t e r n a l field-frequency lock. A s t r u c t u r a l formula for vindoline i s given and the resonances pulsed are indicated.  X  Fig. 5  A.  Scope photograph of a t y p i c a l Carr-Purcell sequence on the C^-acetate resonance of vindoline. Duration of •rr/2-pulse = 0 . 1 sec.  B.  Scope photograph of a t y p i c a l T pulse sequence on the same resonance. Tf/2-pulse duration = 0 . 1 sec. x  - 127 -  Carr-Purcell sequences and so obtain s u f f i c i e n t points f o r an acceptable determination of T^.  Data were found to be consistent over a  range of pulse i n t e r v a l s . The method used to measure T^ was  e s s e n t i a l l y that described i n  the previous section which involved a repeated series of two-pulse experiments. was  However, i n this case,the monitoring second pulse  a ir/2-pulse.  This was  chosen as the monitoring pulse since i t  required the least quantity of energy to be put into the system and hence had the greatest " s e l e c t i v i t y " .  Because of the short relaxation  times involved here i t was not inconvenient to wait f o r the system to relax completely between pulse p a i r s .  A sequence of such  experiments gives the entire T^-decay envelope including the zeropoint as i s i l l u s t r a t e d with an example i n F i g . 5B.  This figure  shows the result of such a measurement on the C^-acetate methyl resonance (ir/2-pulse duration = 0.10  second).  Instrumentally the measurement of (T^) straightforward by the audiofrequency-pulse  values i s p a r t i c u l a r l y method.  Thus the i n i t i a l  Tf/2-pulse i s r e a d i l y phase-shifted by p r e c i s e l y 90° and the  (T ) n  1  decay detected.  P  Unfortunately, these p a r t i c u l a r measurements appear  to be s u f f i c i e n t l y prone to interference from neighbouring  lines  that t h e i r general application to complex organic substances may somewhat limited.  For example, the (T.) 1  be  measurement shown below P.  c l e a r l y f a i l s to return to the baseline as i t should due to the interference of near neighbour l i n e s .  Nevertheless  there are p o t e n t i a l  advantages to the (T^)p method as mentioned previously, since the pulse power i s maintained  constantly.  In a l l the preliminary work  - 128 -  A TT/2 pulse (0.1 sec) and a  (T..) experiment on the C.-proton 1 p 4 Note the extra height of the (T,) trace  resonance of vindoline.  1 P  (upper) and i t s f a i l u r e to return to the baseline.  discussed here we have adhered  to those pulse methods requiring the  least amount of pulse power i n order to achieve high s e l e c t i v i t y . Further work on p r a c t i c a l samples w i l l be required i n order to test the a p p l i c a b i l i t y of (T ) . 1 P 1  Following the comparatively simple experiments  on the intense  singlet resonances, attention was next directed to the other  resonances.  The two methoxyl groups gave two resonances which were only separated by ca. 1 Hz and i t proved impossible to effect any recognizable experiments with them. single  proton was  On the other hand, the singlet due to the  readily amenable to measurement of T^ and T^.  the many spin-multiplets, only the sharp doublet due to the proved suitable for relaxation measurements.  This doublet has a  Of  proton  T,  =  1 . 3 5 ± Q 1 5  T  =  1 . 4 0 ± 0 . 1 5  2  T,  = 1 . 4 5 :  0.15  T = \AO  0.15  2  downfield  upfield  transition  transition  H  vo  OCOCH, "  CH 0 CQ CH 2  Ti T  6.  T  2  , 2 0  -  Q 1  = 1 . 2 0 ± 0 . 1  ~n = i.5o±ai  3  Fig.  V  = 2  T  2  = 1 . 3 0 ± 0 . 1  3  1.15±0.1  = 0 . 9 0 ± 0 . 1  Relaxation time data obtained on the a l k a l o i d , vindoline, i n deuterochloroform solution, shown associated with the protons to which they correspond.  Values are  - 130 s p l i t t i n g of 8.0 Hz and i t was possible to perform measurements on both of the t r a n s i t i o n s . We were disappointed  that i t was not  possible to make measurements on the other well resolved multiplets: unfortunately  the small peak-separations and the rather short  relaxation times (accompanied by large half-height widths and necessitating  short pulses) e f f e c t i v e l y precluded meaningful experiments.  Degassing of the solution did very l i t t l e to lengthen the relaxation times. It does not seem appropriate  to make any lengthy discussion of  the relaxation times obtained, beyond a b r i e f comment that even though they are a l l quite short some d i f f e r e n t i a l s t i l l remains.  As can be  seen fromthe data summarized i n F i g . 6, the N-methyl group has the shortest relaxation time.  This might have been expected from the  84 scalar coupling e f f e c t both transitions of the  of the nitrogen quadrupole moment.  Note that  proton have the same relaxation times,  and note also that i n every case T^ - T^.  The problem of pulsing  i n d i v i d u a l lines i n a multiplet i s not t r i v i a l from a t h e o r e t i c a l viewpoint, e s p e c i a l l y i f the system involves high order  coupling. 102  This has been discussed i n some d e t a i l by Freeman et a l .  In a l l  cases studied i n this work only r e l a t i v e l y f i r s t order multiplets have been pulsed and the early parts of the T^ decay curves have been used to calculate T^ as suggested by the above workers.  The  experiments on vindoline gave values which probably have an accuracy i n the neighbourhood of + 10% i n terms of r e p r o d u c i b i l i t y . In conclusion then, i t seems that s e l e c t i v e measurements of  -  131 -  relaxation times of i n d i v i d u a l protons of reasonably complex organic substances are f e a s i b l e .  The audiofrequency pulse technique i s  compatible with measurements of transitions separated by 5 Hz or more from a neighbouring resonance, providing that the transitions question are sharp ( i . e . , 1^ > 3 s e c ) :  in  for broader t r a n s i t i o n s a  separation of 8-10 Hz i s a more r e a l i s t i c one.  C.  Measurement of Nuclear Relaxation Times of Carbohydrate Derivatives by the Audiofrequency Pulse Technique We report here a study v i a pulsed "''H n.m.r. of two closely  related saccharides.  The compounds studied were 3 , 4 , 6 - t r i - O -  acetyl-l-o-benzoyl-2-bromo-2-deoxy-g-D-glucopyranose 2-chloro analogue (2).  (1) and i t s  Both had been previously prepared by Dr. John 2  Manville i n this laboratory  and were c r y s t a l l i z e d from aqueous  ethanol p r i o r to use. The compounds were studied separately in C^D^ solution using TMS for an i n t e r n a l field-frequency lock.  Each sample was c a r e f u l l y  f i l t e r e d and then degassed (6 freeze-pump-thaw cycles) p r i o r to being sealed o f f .  For purposes of intercomparison, both samples were made  up to v i r t u a l l y the same concentration (concentration of bromo compound, 0.20 M; concentration of chloro compound, 0.23 M) and were treated i d e n t i c a l l y .  In order to confirm that the solution  characteristics were the same i n each case, a small amount of acetone was added to each sample p r i o r to degassing.  By measuring the  relaxation time of this i n t e r n a l standard a cross check was possible between samples.  - 132 -  The  two compounds, 1 and 2, were chosen for this study because i t  was already known that they had well resolved "htl n.m.r. spectra and because they had the same configuration. provided a further opportunity  Thus these substances  to test the v i a b i l i t y  technique with "complex" organic systems.  of the audio pulse  The ring proton region of  the normal "4i n.m.r. spectrum of 1 i n C,D, solution i s shown i n F i g . 7A.  The i d e n t i f i c a t i o n of the various multiplets i s shown above the spectrum i n F i g . 7A.  I n i t i a l l y pulse experiments were performed on  the resonances of the c l e a r l y resolved  doublet.  The method of measuring T^ was that previously discussed with reference to the a l k a l o i d , vindoline:  a series of ir-pulse, ir/2-pulse  pairs interspersed with waiting periods f o r e q u i l i b r a t i o n . The scope trace f o r a Carr-Purcell sequence used to measure 1^ (upper f i e l d t r a n s i t i o n #2 i n F i g . 7A; TT/2 pulse duration = 0.1 sec) i s shown below.  - 133 -  Fig. 7. P a r t i a l H n.m.r. spectra (100 MHz) of 3,4,6-tri-i0-acetyl-l-0-benzoyl-2bromo-2-deoxy-8-D-glucopyranose (A) and i t s 2-chloro analogue (B) i n deuterobenzene (degassed). Proton assignments follow those of reference 2 and are the same i n each case. Transitions are numbered for discussion i n the text. TMS was used f o r a lock signal.  -  I  1  134  -  1  4  lines.  1  SEC.  Note the spikes between echoes. from near-neighbour  1  These are caused by i n t e r f e r e n c e  However, during the echoes  themselves  the pulse causing the i n t e r f e r e n c e i s shut o f f . As i n the case of the a l k a l o i d experiments of the previous s e c t i o n , the need f o r high s e l e c t i v i t y necessitated the use of as long pulses as p o s s i b l e ( i . e . , low pulse power), and because of the short r e l a x a t i o n times, only a few echoes were obtained i n each experiment.  Several  experiments were performed on each l i n e with d i f f e r e n t delays between pulses.  The values of T^ and 1^ so obtained f o r the resonances of H^  are l i s t e d i n Table 1 . S i m i l a r experiments were performed on the outer t r a n s i t i o n s , //3 and #6,  of the H  quartet ( F i g . 7 A ) .  The inner t r a n s i t i o n s , #4 and  #5,  - 135 were resolved into separate l i n e s too close f o r selective pulse work. S i m i l a r l y , of the four t r a n s i t i o n s making up H^-, only the outer ones, #7 and #10, were made the subject of pulse experiments t r a n s i t i o n s , #8 and #9, were nearly degenerate. T^ and  during these experiments  - the inner  The values found f o r  are also l i s t e d i n Table 1.  As observed f o r the a l k a l o i d of the previous section, these relaxation times are short. experiments  Another disappointing feature of these  i s that i t was not possible to make s a t i s f a c t o r y measure-  ments of T^ and T^ on any of the other resonances. of the H  Even the transitions  quartet (#11-#14) were too close together f o r r e a l i s t i c  ft  1 measurements.  T^ f o r the acetone i n t e r n a l standard i n this sample  was found to be 15.2 seconds. The p a r t i a l hi n.m.r. spectrum of the chloro compound (2) i n CgDg i s shown i n F i g . 7B.  The assignment i s the same as that indicated  fori  Although the spectra i n F i g . 7A and B are very s i m i l a r , the  and  "quartets" are somewhat closer together i n the case of the  chloro compound.  Pulse experiments  on this compound were very  similar to those on i t s bromo analogue. and  The transitions 6 and 7 of  respectively were deemed too close together (ca. 6 Hz) f o r good  measurements on such broad lines (half height width _ca. 0.8-1.0 Hz due to small unresolved s p l i t t i n g s ) .  Nevertheless, measurements of T^  and T„ were made on transitions of H , H„ and H. and are l i s t e d i n 2 1 3 4 n  Table 1 along with those for compound 1.  Note that i n this case the  reasonably sharp peak corresponding to t r a n s i t i o n s #8 and #9 was pulsed s a t i s f a c t o r i l y .  The value of T^ for the i n t e r n a l standard,  acetone, was found to be 15.8 seconds i n this second sample.  This of  - 136 Table 1.  Relaxation Time Data f o r Sugar Derivatives 1 and 2  1 Bromo analogue l i n e //  1/T  (sec" ) 1  2  T (sec) 2  1/T  (sec" ) 1  1  T  1  (sec)  1  0.52  1.9  0.52  1.9  2  0.54  1.8  0.55  1.8  3  0.58  1.7  0.53  1.9  6  0.66  1.5  0.54  1.8  7  0.84  1.2  0.70  1.4  10  0.83  1.2  0.69  1.4  15.2  -  -  Acetone  0.0660  2 Chloro analogue line #  1/T  (sec ) -1  2  T (sec) 2  1/T  (sec" ) 1  1  T (sec) x  1  0.56  1.8  0.51  2.0  2  0.54  1.8  0.53  1.9  3  0.45  2.2  0.50  2.0  8 &9  0.92  1.1  0.72  1.3  10  0.85  1.2  0.80  1.2  Acetone  0.0634  15.8  —  _  - 137 -  course Is well within the + 10% error l i m i t of that found for the f i r s t sample.  It should be pointed out that i n spin coupled systems of  complex molecules each spectral l i n e i s usually broadened by unresolved s p l i t t i n g s so that the observed value of  i s generally greater than  that indicated by the linewidth from the relationship Av For  instance i f  =  TTT2  i s greater than 1.5 second, a single resonance would  be expected to have a half-height width of less than 0.22 Hz. for  Allowing  a f i e l d inhomogeneity of up to 0.3 Hz, one would expect linewidths  of 0.5 Hz - i n fact most resonances are broader than t h i s . At of  f i r s t sight the close s i m i l a r i t y between the relaxation times  the various protons studied here may  seem to be rather disappointing;  however i t should be noted that a l l the protons have an a x i a l orientation and this may be a c o n t r o l l i n g feature of the relaxation processes.  It  i s also possible that the acetate methyl protons contribute s i g n i f i c a n t l y to the o v e r a l l relaxation of the ring protons. for  Note that the values  compound 1 are closely similar to those for the corresponding  protons of compound 2 (Table 1) - this i s not surprising i n view of the close s t r u c t u r a l s i m i l a r i t y between the molecules.  As  mentioned  previously, one of the penalties f o r using the low energy Carr-Purcell  99 sequence to measure  can be a loss of magnetization between pulses.  It may be this phenomenon which accounts f o r the fact  that i n the H^  resonances of 1 and 2 (which are braoder than those of H^ and H^) i s s l i g h t l y longer than T^even for the  T^  However the differences between T^ and  resonances are small r e l a t i v e to the + 10% experimental  error involved and may by Freeman et al."**^  arise from the spin multiplet e f f e c t s  mentioned  - 138 -  Clearly i t w i l l be necessary to study other examples to see i f any d i f f e r e n t i a l exists between the relaxation times of protons i n more d i f f e r e n t chemical environments.  In this regard a measurement  technique having an even higher degree of s e l e c t i v i t y would be desirable; i f this cannot be developed, then the p o t e n t i a l of this area v i l l be severely r e s t r i c t e d .  I t would also seem desirable i f  any improvements i n s e l e c t i v i t y could be accompanied by attempts to increase the o v e r a l l magnitude of the relaxation times.  In this  99 regard decoupling experiments should be h e l p f u l . It i s worth remarking  that from the data given here i t would seem  that no great d i f f e r e n t i a l exists between the relaxation times of the various resonances  associated with any one proton.  However, the  spectral features studied were f a i r l y f i r s t order and i n general 102 differences may be expected. D.  The Audiofrequency  Pulse Technique and Enzyme-Inhibitor  Rate  Constants We have just discussed to some extent the p o s s i b i l i t y of deriving s t r u c t u r a l information from n.m.r. pulse studies of 'large' organic molecules  i n solution.  Although this area i s as yet almost  totally  uninvestigated, there has been considerable introductory work done on the use of pulse techniques i n studying rate processes involving b i o l o g i c a l systems.  In p a r t i c u l a r , enzyme-inhibitor interactions have  been studied v i a the technique of observing nuclear relaxation phenomena associated with the i n h i b i t o r .  - 139  -  Sykes has done excellent work i n this area and has technique to systems involving chymotrypsin^ and Sykes  97a  97b  and Sykes and Parravano  k ^ and k^,  applied  the  lysozyme. ^ 'k 9  a  have measured the rate constants,  for the N-acetyl-D-glucosamine derivatives, 5-8,  of  the previous chapter. k  = rate constant for d i s s o c i a t i o n of the enzyme-inhibitor complex  k  = rate constant for complex formation  K ^  = tEHi] [EI]  _  ^=1 k x  19 We have already shown that on the binding  The  some effect  of N-trifluoroacetylated monosaccharides to lysozyme.  We were curious inhibitors.  F labels used i n Chapter II had  therefore as to the rate constants for our  fluorinated  h  exchange of a nucleus, or group of equivalent  n u c l e i , between  s i t e s of d i f f e r e n t l o c a l magnetic environment (characterized  by  d i f f e r e n t resonance frequencies) i s a relaxation mechanism for the nuclear spin system.  If the rate of exchange i s less than the resonance  frequency of the nuclei involved  (- lO^MHz here), the exchange w i l l  shorten the transverse relaxation time, T^, but w i l l not a f f e c t the longitudinal relaxation time, T^. has been shown  In fact this exchange mechanism  97a b ' to be the dominant relaxation mechanism of  the  N-acetyl protons brought about by addition of lysozyme to aqueous fi solutions of monosaccharide NAG NAG  derivatives.  Of. the fluorinated  derivatives studied i n Chapter I I , only the a-anomer of the  sugar was  free  found to show an appreciable bound chemical s h i f t which would  permit an accurate c a l c u l a t i o n of the rate constants  involved.  - 140 -  CH OH 2  1 = H  R  2  R  1= OH  R  2  R  1= H  R  2  =  R  1= O C H 3  R  2  = H  R  HO  = OH = H  OCH3  NHCOCF3  97a As mentioned i n the introduction, Sykes and Sykes and Parravano have put the Bloch equation solutions into a form very 97b convenient for r e l a t i n g the difference •ence between constant, k_^.  In the case where  [EI]  [I]  and  to the rate  << 1 and [I] ~ [I ] the  expression i s :  T  2  T  where  _1_ T„ l  %  +  and A  I N  • [  V  k  (r—)  - l  .2 A  are i n seconds  i s i n radians/sec concentrations are i n molarity.  Hence i f  and A have been calculated from chemical s h i f t studies, i t  i s a r e l a t i v e l y simple matter to arrive at k_^ from a plot of (—— - Y~) vs. [ E ] while holding [ I ] constant. q  In this study,  q  and  for the N - t r i f l u o r o a c e t y l group were  measured for each of four d i f f e r e n t values of [ E ] (Fig. 8A and Table 2) A l l measurements were made i n 0.1 M c i t r a t e buffer at pH 5.5. The  - 141 "Mark I I " pulse unit attached to a Varian HA-100 spectrometer operating at 94.071 MHz 1^.  (probe temperature 31.5°C) was  used to measure  and  A c a p i l l a r y of t r i f l u o r o a c e t i c acid held concentric with the  n.m.r. sample tube was  used f o r a field-frequency lock.  sugar concentration (0.0775 M) was  The  the same i n each sample.  total Hence  the concentration of a-anomer (compound 1 of Chapter II) i n each case was  0.0356 M.  Since, as was  determined Chapter I I , the e f f e c t of the  g-anomer on the a-anomer binding was error, the e f f e c t of the g-anomer was  no larger than neglected.  In each case the measurement of T^ was the conventions  experimental  straightforward and  followed  described for the 'large' organic molecules of the  previous two sections (a series of ir-, 7r/2-pulse pairs with varying delays between the f i r s t and second pulse).  As can be seen from the  data (Table 2 and F i g . 8A - s o l i d l i n e through X's), T^ decreases s l i g h t l y with increasing enzyme concentration.  only  A similar s l i g h t  97a decrease has been noted by Sykes. The measurement of methods.  was  quite simple but was  checked by several  The spin-echo method described previously was suitable  for measurement of short on the remaining  on the f i r s t two samples.  But  samples (Table 2) that f i e l d  only a small correction to the Bloch decay generated  became so inhomogeneity became by a 7r/2-pulse.  The value of T^ obtained from the time constant for the Bloch decay of a ir/2-pulse i s the same as that obtained from the expression Av —jjj—  provided there are no unresolved  of the f i e l d  couplings.  =  The actual magnitude  inhomogeneity correction term can be estimated  from the  linewidth of an i n t e r n a l standard for which T. i s long or from the  Table 2.  Nuclear Relaxation Time Data f o r the ct- and g-Anomers of the Free Sugar, N-Trifluoroacetyl-DGlucosamine  Sample #  [E ] (M)  T  ±  (sec)  1/T  (sec ) 1  T  (sec)  1/T  (sec" ) 1  <i- - i - ) (sec" ) 2 l 1  T  a  !-  0  3 2  a  0.95 x l O  - 3  o  a  3  2.85 x 10 ^  4  a  4.75 x 10  [ a ] = 0.0356 M o [3 ] = 0.0419 M o  3  8 7  0.534  1.58  0.634  T  0.100  1-82  0.549  1.52  0.658  1.62  0.617  0.830  1.20  0.587  1-32  0.755  0.364  2.75  1.994  1-63  0.612  1.06  0.940  0.960  1.04  0.234  4.27  3.226  Fig. 8.  A. Plots of 1/T  (solid l i n e through X's) and 1/T  N - t r i f l u o r o a c e t y l group of the a-anomer.  (solid l i n e through f i l l e d c i r c l e s ) vs. [E ] for the  Several data points for 1/T^  (dotted l i n e through triangles)  and l / T ^ (dotted l i n e through open c i r c l e s ) for the 3-anomer are shown for comparison. B. Plot of (1/T„-1/T ) vs. [E ] for the a-anomer N-trifluoroacetate group.  - 144 effect of spin-echo generation on the same sample or on a s i m i l a r sample with longer T^.  Neither technique i s extremely accurate i n the  intermediate range of linewidths  less than 2 Hz but more than 0.8  Hz.  Fig. 8A ( s o l i d l i n e through f i l l e d c i r c l e s ) c l e a r l y shows the sharp decrease i n  for the N - t r i f l u o r o a c e t y l resonance of the free  sugar a-anomer upon addition of lysozyme.  For purposes of comparison,  several points are plotted f o r the g-anomer (1/T^ as dotted l i n e through t r i a n g l e s ; l / T ^ as dotted l i n e through open c i r c l e s ) . Fig. 8B i s a plot of (1/T  - 1/T ) vs. [E ].. The slope of this  2  ^  ——7—? r and was found to be 6.72 x 10^ D o - l The calculated values of and k^ for the a-anomer of the  l i n e i s equal to 1  sec  M  .  [ a  ]  k  free sugar are k 1  k. 1  4 -1 = 1.1 x 10 sec v —1 6 —1 —1 = - — = 1.1 x 10 M sec D K  The error l i m i t s involved are at least + 30% i n view of the errors i n the numbers used for the c a l c u l a t i o n .  It i s i n t e r e s t i n g that for the  proton analogue (compound 5 of Chapter II) of the above N - t r i f l u o r o acetate, Sykes and Parravano^^^ quote k_^ = 8.5 + 2.5 x lO^sec ^ and for k^ they report 3.5 + 1.2 x 10  5  molal "'"sec . 1  Hence, the replacement  of the N-acetyl protons by fluorines seems not to have affected k_^ but may have s l i g h t l y increased k^.  - 145 -  Experimental General Methods 1 (a)  A l l H and  19 F spectra were run on the same spectrometer  (Varian HA-100) and .under the same conditions as described i n Chapter I and Chapter I I . (b)  Enzyme handling techniques were the same as those described  in Chapter I I . (c)  The "Mark I" pulse unit was used for the experiments on  vindoline.  A l l other pulse work was done with the "Mark I I " unit.  Compounds The a l k a l o i d , vindoline, was obtained from Professor J.P. Kutney of this  department.  Ethylene derivatives were obtained from the Aldrich Chemical Co. and were used without further p u r i f i c a t i o n .  3,4,6-Tri-0-acetyl-l-0-benzoyl-2-bromo-2-deoxy-3-D-glucopyranose (1) m.p. = 161-162°C and i t s 2-chloro analogue (2) m.p. = 160-161°C were made previously by Dr. J . Manville of this laboratory and were r e c r y s t a l l i z e d from aqueous ethanol p r i o r to use.  Pulse Unit Operation Each i n d i v i d u a l relaxation time measurement requires the sequential adjustment of the correct observing frequency, of the amount of power contained i n a single pulse, and of the phase of the i r r a d i a t i n g  field.  - 146 -  In general i t i s convenient to set up these parameters i n the order: (1) pulse phasing (2) exact frequency  determination  (3) pulse power With the apparatus available to us at this juncture we have found i t convenient to correctly adjust the phase of the observing f i e l d i n the fashion generally used i n high resolution n.m.r. experiments:  by  using the sweep-oscillator of the frequency synthesizer i t i s convenient to sweep repeatedly through the resonance of interest and to make adjustments with the phasing control of the P.A.R. lock-in amplifier. With the "Mark I I " pulse spectrometer  i t i s necessary to make this  adjustment only once since the pulse frequency i s held constant. Freeman and Wittekoek  99  have described previously how (T-)  J- P  can be used f o r optimising the phasing controls.  experiments  We have used t h e i r  method and find i t to be quite convenient providing that a (T^) experiment can be meaningfully performed on the p a r t i c u l a r sample: however, the d i f f i c u l t i e s already mentioned when a near neighbour resonance i s present tend to complicate the method. The precise frequency difference between the field-frequency lock and the pulse signal needed for a p a r t i c u l a r experiment can be obtained i n either of two ways.  Either by direct measurement and  c a l i b r a t i o n of the high resolution spectrum, or by observation of the decay occurring after a pulse.  In the l a t t e r case the l i n e p o s i t i o n  i s quickly measured to within 0.5 Hz and the frequency i s adjusted i n small increments after each pulse.  to achieve a smooth exponential decay to baseline The off-resonance condition i s marked by the trace  - 147 -  of the magnetization  vector either continuing through the baseline  before returning to i t s equilibrium p o s i t i o n or by a hump i n the otherwise smooth decay curve.  This, method i s very fast and quite  general aid can give the frequency to within + 0.02 Hz (depending on the linewidth). The amount of power required to produce a p a r t i c u l a r pulse i s conveniently ascertained by s e t t i n g up the condition f o r a iT-pulse (or 2TT-pulse) ; i t i s then a simple matter to a r r i v e at the power l e v e l or pulse duration f o r a 7r/2-pulse.  - 148 -  APPENDIX A PRINCIPLES OF PULSED N.M.R. SPECTROMETRY WITH PARTICULAR REFERENCE TO HIGH RESOLUTION EXPERIMENTS  Nuclei having a spin quantum number greater than zero possess both spin angular momentum and a magnetic moment, u = yh I (where y i s the nuclear gyromagnetic  r a t i o and I i s the nuclear spin quantum number).  Magnetic moments give n u c l e i certain properties c h a r a c t e r i s t i c of gyroscopes.  For instance the nuclear spin isochromats precess about  an external magnetic f i e l d , H , at a frequency c h a r a c t e r i s t i c of the type of nucleus:  to o  = v H ' o  radians/sec  w  o  =  Larmor freauency ••  In the powerful magnetic f i e l d of a n.m.r. spectrometer there i s a resultant macroscopic magnetization (M ) along the f i e l d axis (Zaxis) at equilibrium. By b r i e f l y applying a new magnetic f i e l d , H^, perpendicular to H , i t i s possible to turn the vector M o o r  s p e c i f i c angle away from the Z-axis.  through a  The resultant magnetic vector,  M , w i l l then precess l i k e the i n d i v i d u a l spin isochromats about the Z-axis at the Larmor frequency.  For this reason, i t i s standard  practice to transform from the XYZ laboratory frame to a rotating reference frame, X'Y'Z', which s t i l l has H along the Z'-axis, but o which rotates about that axis at the Larmor frequency, to .  In this  new reference frame, neither the spin isochromats, nor their resultant M , precess about the Z'-axis.  Short-lived H  f i e l d s can be conveniently  - 149 -  produced by bursts of radiation at the Larmor frequency, a ) . Q  radiofrequency magnetic f i e l d , H^,  The  can be thought of as made up of  two counter-rotating f i e l d s ; one of which synchronizes with the precessing nuclear magnetic moments and the other of which has a negligible effect. Thus, i f  i s directed along the X'-axis i t w i l l cause a  rotation of the resultant, magnetization vector i n the Y'Z' plane at a frequency:  u  i  =  y i H  If H, i s l e f t on for a time, t seconds, then the vector, M , w i l l turn 1 o through an angle:  - 150 a  =  t o)^ radians  It i s possible to vary the strength of the duration  of i r r a d i a t i o n (pulse duration) to achieve varying  degrees of vector r o t a t i o n . powerful  (the pulse power) and/or  In radiofrequency pulse experiments,  f i e l d s are generally employed:  this permits one to pulse  extremely broad lines and to measure very short relaxation times. high resolution n.m.r.,  and  In  are generally of the order of 1  second or longer and very homogeneous magnetic f i e l d s are employed: this permits the use of long but very s e l e c t i v e (narrow band) pulses allowing one to measure relaxation times of i n d i v i d u a l resonances i n complex spectra. The receiver c o i l of the spectrometer used here i s oriented so as to detect any non-zero resultant magnetization  i n the X'Y'  plane - and  the larger that component, the larger the recorder response. pulses employed are so weak that the magnetization actually be monitored during the pulse.  The  vector can  F i g . 1 shows a series of scope  presentations for the various constituents of a l l pulse sequences together with a diagrammatic representation of each.  The 90°-pulse  (•rr/2-pulse) of F i g . IA shows the c h a r a c t e r i s t i c sharp r i s e i n recorder response as M  q  i s forced maximally into the X'Y'  followed by an exponential Bloch decay of M  q  plane,  along the Y'-axis.  Fig. IB c l e a r l y demonstrates the e f f e c t of a 180°-pulse  (n-pulse)  on the same resonance after i t has been allowed to come to equilibrium: M  o  has been forced from the p o s i t i v e Z'-axis i n t o the X'Y'-plane  (giving a sharp recorder response) and then on through the X'Y'  plane  I  Fig. 1.  I  I  1  1  1  1  8 sec.  Scope traces of various pulses on the alkene resonance of the d i e t h y l ester of maleic acid i n the sample shown i n F i g . IB of Chapter I I I . A. a/2-pulse = 0.2 sec; B. u-pulse = 0.4 sec; C. 2ir-pulse = 0.8 sec. The effect of each pulse on the equilibrium magnetization vector, M , i s shown to the right of the trace.  - 152 -  to the negative Z'-axis where i t gives a zero recorder response.  Note  that the recorder response actually returns through the baseline to a s l i g h t extent.  This i s because the resonance pulsed has a f i n i t e  linewidth and the pulse i t s e l f has a bell-shaped power d i s t r i b u t i o n the result being that the central part of a resonance receives s l i g h t l y too strong a pulse and the wings of the resonance receive s l i g h t l y too weak a pulse. on the M  F i g . IC i l l u s t r a t e s the effect of a 360°-pulse (27r-pulse)  vector at equilibrium: note that a maximum p o s i t i v e recorder  response i s observed as M  passes through the p o s i t i v e Y'-axis and a  maximum negative response as i t passes through the negative Y'-axis. Obviously the recorder response i s zero again at the end of a '2Tr-pulse (except for the f i n i t e linewidth effect mentioned above) and the o v e r a l l effect of a perfect 2-fr-pulse i s n e g l i g i b l e .  A l l pulses shown i n F i g . 1  were performed on the alkene resonance of diethoxy maleic acid (resonance #7 of F i g . IB of Chapter III) and were done with the same pulse power but d i f f e r i n g pulse durations - the pulse durations being 0.2,  0.4 and 0.8 seconds i n F i g . IA, B and C respectively.  be noted that i f the pulse duration i s not << T^ and appreciable amount of decay of the vector M  I t should  T^, then an  can occur during the pulse.  -  153 -  APPENDIX B AUDIOFREQUENCY PULSE UNITS EMPLOYED "Mark  I"* The basic format of both "Mark I"  and "Mark II"  pulse spectrometers 99  used i n these experiments follows that of Freeman and Wittekoek. Because we were uncertain as to which pulse durations would be most suitable for studies of complex organic substances we  chose to use a  small d i g i t a l computer to control a l l pulse durations and i n t e r v a l s . It  should be emphasized that the components which we used as the basis  for the gate and other c i r c u i t s were selected s o l e l y because they were readily available i n our laboratory at the time we started these experiments; more v e r s a t i l e , and cheaper, components are now available from a variety of manufacturers. A block diagram of the instrument, together with the intersystem connections required for interface with a Varian HA-100 spectrometer, i s given in F i g . 2., The two major components of the system are a JEOLCO computer (model JRA-5) and a Hewlett Packard Frequency Synthesizer  (model 5110B), which i s driven by a "master o s c i l l a t o r "  (Hewlett-Packard, model 5105B). The principle components of the pulse-unit are summarized i n F i g . 3. Briefly, (a)  the c i r c u i t r y used here serves the following In the lock-channel, i t  functions:-  takes a 1 MHz signal from the master  * This device was designed and b u i l t i n collaboration with Mr. Roland Burton of this Department.  - 154 DIGITAL FREQUENCY SYNTHESISER  MASTER OSCILLATOR  PULSE  UNIT  ORA- 5 COMPUTER  MODULATION COILS RECEIVER COILS  V-4354 LOCK—BOX  V-4311 RECEIVER  P A R - 121 L O C K —IN AMPLIFIER  JL  VARIAN LOCK SYSTEM  Fig. 2.  TEKTRONIX-R 564B STORAGE OSCILLOSCOPE  Block.diagram of the components used i n the "Mark I" audiofrequency-pulse spectrometer.  - 155 -  o s c i l l a t o r and reduces i t by d i g i t a l d i v i s i o n to a 2.5 KHz square wave. This signal (at 1 V p.p.) i s then used i n the Varian lock-box  (V-4354)  instead of the signal normally provided by the manual o s c i l l a t o r (card 910868).  We have made this interchange switchable so that i t does not  require physical removal of the manual o s c i l l a t o r card. operations of the l o c k - c i r c u i t r y are unaltered by this  A l l subsequent interchange  except that lock-offsets are not possible. (b)  In the observation channel the c i r c u i t r y provides the audio-  modulation frequency required f o r the detection of resonances.  At  the same time this channel requires provision f o r switching, phase s h i f t i n g and f i l t e r i n g .  In order to obtain a s u f f i c i e n t l y fine (+ 0.01  Hz) control of the frequency of the observation side-band,  the d i g i t a l  frequency synthesizer i s generally run at ca. 1 MHz and the required audiofrequency  (ca. 2.5 KHz) i s obtained by d i g i t a l d i v i s i o n as f o r  the lock-channel.  Since the phase-shifts required in pulse experiments  are i n t e g r a l units of 90°, these are conveniently obtained during the above d i v i s i o n .  Sequential d i v i s i o n -r 10, •=• 10,  gives the required audiofrequency  2, invert, v 2  square-wave which i s used i n a l i n e A  (Fig.  3) as the reference signal to the Princeton Applied Research  (PAR)  lock-in amplifier and. to the gate.  of  Using the f i r s t three stages  the above sequence ( v l O , * 10,^ 2) followed by a separate T 2 stage  gives a second audiofrequency  square-wave i n l i n e B.  This square-wave  has p r e c i s e l y the same frequency as that of l i n e A but d i f f e r s from i t and the signal i n the l o c k - i n amplifier by a p r e c i s e l y 90° phase-shift. Either of these two audiofrequencies can then be connected to the modulation c o i l s v i a a f i l t e r and an attenuator.  The l a t t e r i s a 10-turn  - 156 -  LOCK-CHANNEL 2.5 K H z  1 M H z SINE —WAVE  /  MASTER OSCILLATOR  OBSERVATION -  DIGITAL FREQUENCY SYNTHESISER  /  -HO,-MO,-4  SQUARE-WAVE MANUAL OSCILLATOR V-4354  CHANNEL  -HO,-MO  -2  — 2  — 2  INVERTER  ®  REFERENCE INPUT O F P A R — 121  GATE JRA-  5  COMPUTER  T O D.C MODULATION COILS VIA V-4391  g. 3.  T  GATE  FILTER AND ATTENUATOR  Block diagram of the components used i n the "Mark I" pulse unit.  OF  - 157 -  200 ohm h e l i p o t . The switching of both l i n e A and l i n e B i s controlled by the JEOLCO JRA-5 computer which i s programmed to perform the following:(a) To accept p o s i t i v e integers (<32,000) from the teletype; nine of these integers may  be used.  Each of these controls a s p e c i f i c  switching  operation. (b) To turn on, or o f f , either l i n e A or l i n e B of the channel. field-is  observation  When l i n e A of F i g . 3 i s "on" the phase-shift of the i r r a d i a t i n g zero, and when l i n e B i s "on" the phase-shift  i s 90°.  (c) To pause for a time-interval which i s numerically equal to the integer selected i n (a) above, m u l t i p l i e d by 10 milliseconds. It i s , of course, a t r i v i a l matter to vary the pulse-lengths,  pulse-  i n t e r v a l s , and phase sequences i n any of a wide variety of ways. The spectra obtained recorded  from the pulse spectrometer are  conveniently  on a storage oscilloscope (Tektronix, model R56A B) and a  ** permanent record obtained with a homemade scope camera  using Polaroid Film.  "Mark I I " The "Mark I I "  **  +  pulse spectrometer i s an improved version of "Mark I".  More accurately, the phase-shift between the reference s i g n a l going to the PAR-121 l o c k - i n amplifier and the s i g n a l going to the modulation c o i l . Derived from a Polaroid camera (model 210). It i s necessary to divide the o r i g i n a l camera into two parts, the back-film holder and the lens-shutter mechanism. The two parts are then remounted approximately twice as far apart as i n the o r i g i n a l camera. The t o t a l cost of our camera was less than $50. This device also was designed and b u i l t i n collaboration with Mr. Roland Burton of this Department.  - 158 -  It i s not only more convenient i t contains i t s own  from the operator's point of view, but  s o l i d state pulse sequence generator which  obviates the need for an expensive  computer.  The choice of pulse  durations (both pulse #1 and pulse #2) i s d i g i t a l l y v a r i a b l e over the following range: .025,  .050,  .10,  .20,  .40,  .80, 1.60,  oo  seconds.  The delay between pulse #1 and pulse #2 i s continuously v a r i a b l e . A block diagram of the "Mark I I " pulse spectrometer  together with  the intersystem connections required for interface with a Varian HA-100 spectrometer,  i s given i n F i g . 4.  As mentioned above, although the '-.  Hewlett-Packard Frequency Synthesizer (model 5110 B) driven by a "master o s c i l l a t o r " (Hewlett Packard, model 5105  B) i s s t i l l used as a  source of stable frequencies, the JEOLCO computer has been replaced with s o l i d state c i r c u i t r y b u i l t into  the pulse unit.  The p r i n c i p l e components of this new Fig. 5.  pulse-unit are summarized i n  There are basic differences i n the c i r c u i t r y used for "Mark I I " i  as follows:(a)  The lock channel frequency i s variable and hence i s taken  from the d i g i t a l frequency synthesizer.  For high accuracy i n setting  the frequencies, the lock signal of ca. 2.5 KHz was  obtained by  d i g i t a l d i v i s i o n (see F i g . 5) of a radiofrequency i n the 1 MHz  range  and f i l t e r i n g to obtain a sine wave at (1 V p.p.) whose frequency  can  be set with extreme accuracy and which can be simply switched to replace the ordinary manual o s c i l l a t o r (card 910868).  With this arrangement  any lock offset i s readily achievable. (b)  As i n the "Mark I" unit, the observation channel  provides the audio-modulation  circuitry  frequency required for detection of  - 159 -  DIGITAL FREQUENCY SYNTHESIZER  MASTER OSCILLATOR  MARK II PULSE UNIT  MODULATION COILS RECEIVER COILS  V-4354 LOCK - BOX  VARIAN LOCK SYSTEM Fig. 4.  V-43II RECEIVER  PAR-121 LOCK IN AMPLIFIER  TEKTRONIX-R 564B STORAGE OSCILLOSCOPE  Block diagram of the components used i n the "Mark I I " audiofrequency-pulse spectrometer.  - 160 -  LOCK  CHANNEL  1 . 5 - 3 . 5 KHz DIGITAL FREQUENCY SYNTHESIZER  TIO,TIO,T5,  - 2  OBSERVATION  MASTER OSCILLATOR  f 2  FILTER  INVERTER 0  MANUAL OSCILLATOR OF V 4 3 5 4  CHANNEL  r IO, f IO  I80  SINE-WAVE ( I V p-p)  r  2  r  2  >zT=90  INVERTER  0  1 PULSE DURATION AND SPACING  r  2  GATE  1 GATE 2  TO D.C. MODULATION COILS VIA V - 4 3 9 1  HEWLETTPACKARD 465A AMPLIFIER  FILTER AND ATTENUATOR  Fig. 5. Block diagram of the components used i n the "Mark I I " pulse unit.  - 161 -  resonances.  The observation frequency of 2.5 KHz i s derived by  d i g i t a l d i v i s i o n of the master o s c i l l a t o r (1 MHz) i t i s s u f f i c i e n t to tune the PAR  and i s fixed.  lock-in-amplifier to one  Hence  frequency.  The 2.5 KHz pulse #1 i s obtained by a simple sequential division,-f 10, -MO,  f2,  il of the master o s c i l l a t o r frequency.  Pulse #2 has exactly  the same frequency as pulse #1 (see F i g . 5) but may be switched to a variety of d i f f e r e n t phase r e l a t i o n s to pulse #1 (0°, 90° or 180°). This switching i s manual.  The pulses #1 and //2 are gated according  to switch settings on the front panel of the pulse unit to give any sequence desired. are  As with the "Mark I" unit, these pulse sequences  fed to the D.C.  modulation  c o i l s and the demodulated  output of  the receiver c o i l (demodulated i n the V-4311 receiver unit) i s fed to the PAR lock-in-amplifier.  The Tektronix, model R 564 B, storage  oscilloscope i s used to record the D.C.  output.  camera i s used to record storage scope traces.  The same Polaroid  - 162 -  REFERENCES 1.  E . B . Baker,  J.  Chem. P h y s . , 37.. 911 ( 1 9 6 2 ) .  2.  J . 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CM  .CA*..  Qato/wetr-  ^7 .... ..3 3*7.  X . -C.4. .€.**>..,  Cr.ci.c:«if-n.e.c_ _jE_i_X  _C  C. L .  /  X...  7 o) .  J  £ / )  Z  S  ~  ?  3  .(/ iJo). c  t  W ... n .........6r/i.<*.«./_ .a.w.e/...._.4.'._ t>.....-hb>> ii C_«*.  . X . . _ . C4  O  —  -<M-7. _XjS.  7 2J_  AJ^LXKV^CL. S- i / 9Q <f- - JS~  IU  -ir  L3.LM—JL  / 9&ty-~ 6 S~  Ohi<*r,'  T A g.  S_cXo_L<*xz.s AJ.  Cf Que,r-  TLe  D g.<a m  o  1  S  ^  or-  >  /ioisf o Uis~  13 dcm ~f~^s  g  SC  i\  o  I  &f£jS_ly.JL.jQ.  L'J-JS—t.--  -CojAvy JSji^.Lc>.CJ*.  of~  C fri^a J a  _$c l\-0-La._*zsbJ.y9.. JJJ-JLJ^JJL  11U.—6.7  TUn.  j , L , uy,  . _ U . €  _/V_._/^-.-C-, /_ ?7_Z_r <  ./V_t  c.a,_c_ ISA.2  Grijl  S c U o Ift*~ s L ;p  &?JjLg, $c_dtut - ^ € _  ILoyJ  e skip  Sic,L  o./..cicsALja.  

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