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UBC Theses and Dissertations

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 University, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of CHEMISTRY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March ,1972 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of (Lh4-t~\'i si~i The University of B r i t i s h Columbia Vancouver 8, Canada Date n 2. ? ; / f 7 Z - i i -ABSTRACT This thesis i s divided into three chapters; each involving a 1 19 different approach to the use of both proton ( H) and fluorine ( F) nuclear magnetic resonance (n.m.r.) to study large organic molecules i n solution. To some extent the three chapters represent an evolution i n technique of the s c i e n t i f i c world i n general and of our laboratory i n p a r t i c u l a r . Previous students i n this laboratory have used high resolution 1 31 19 n.m.r. spectroscopy (of H, P and F nuclei) combined with double i r r a d i a t i o n techniques to study progressively larger organic molecules. In the f i r s t chapter t h i s work has been extended to s "natural products" - molecules not previously very susceptible, because of their spectral complexity, to detailed n.m.r. studies. In i 19 par t i c u l a r ti and F n.m.r. have been employed i n conjunction with heteronuclear noise modulated decoupling and *H-{ >H} INDOR. A series of steroids substituted with fluorine i n the A, B or D rings have been examined as model systems: 2a-fluoro-cholestan-3-one, 6a- and 63-fluoro-cholest-4-en-3-one and 16,16-difluoro-androst-5-en-33-ol-17-one. In each case i t has been possible to obtain coupling constants and chemical s h i f t s for the nuclei i n the area of the fluorine atom and hence to derive s t r u c t u r a l data from the n.m.r. spectra i n spite of thei r complexity. The H-{ INDOR technique alone has been further applied to several problems posed by organic chemists involved with natural products. In each case this approach has been successful. - i i i -Recently very large molecules such as enzymes have been studied by n.m.r. via their effects on the chemical shift and line width of smaller molecules with which they interact. As part of a programme to investigate the application of heteronuclear n.m.r. to problems of biological interest, we have used the above technique to study the interaction of various N-trifluoroacetylated monosaccharides with 19 the enzyme, lysozyme. The F chemical shifts of N-trifluoroacetyl-D-glucosamine and i t s methyl glycosides have been studied as a function of enzyme concentration. The results suggest that the fluorine substituents affect the binding process to some extent but that such effects can ba informative. The possibility of using n.m.r. to study the effects 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 similar results. This work is treated in Chapter II along with a brief discussion of the conformations of the inhibitors involved. Very recently organic chemists and biochemists have begun employing pulsed n.m.r. equipment in a variety of problems. We have become interested in the applications of relaxation time measurements to 1 structural problems. In Chapter III pulsed H n.m.r. experiments involving several model systems are reported: mixtures of the cis and trans isomers of 1,2-dichloro and 1,2-dibromoethylene and of the ethyl esters of maleic and fumaric acids have been studied. The results of these experiments are encouraging, indicating that, in this case at least, relaxation times are sensitive to structure and substituent in a consistent fashion. We have experimented almost exclusively with - i v -selective pulse techniques and have built 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 for the experiments discussed in Chapter III. The same audiofrequency-pulse techniques have been applied to the measure-ment of nuclear relaxation times of individual protons in.the alkaloid, vindoline, and in the sugar, 3,4,6-tri-0_-acetyl-l-0-benzoyl-2-bromo-2-deoxy-g-D-glucopyranose and i t s 2-chloro analogue with less encouraging 19 results. In addition we have reported the use of F pulsed n.m.r. to calculate the rate constants, k and k^, for the association of the a-anomer of N-trifluoroacetyl-D-glucosamine with lysozyme. - v -TABLE 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 8 B. Application of 1H-{"hs> INDOR to Natural Products 31 Experimental 41 CHAPTER II. 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 Shift Reference 52 B. Experiments with N-Trifluoroacetyl-D-glucosamine 53 C. Experiments with C^-Substituted N-Acetyl-D-glucosamine-a-methyl glycosides 71 D. Conformation of Free Monosaccharide Inhibitors 83 E. N.M.R. as a Probe for Conformation of Bound Monosaccharide Inhibitors 92 Experimental 98 - v i -Page CHAPTER III. APPLICATION OF THE AUDIOFREQUENCY PULSE TECHNIQUE TO THE STUDY OF LARGE ORGANIC MOLECULES IN SOLUTION 105 Introduction • • • • ^05 Results and Discussion 113 A. A Case for the Applicability of T^ and to Structural Organic Chemistry 113 B. Measurement of Nuclear Relaxation Times of an Alkaloid by the Audiofrequency Pulse Technique 123 C. Measurement of Nuclear Relaxation Times of Carbohydrate Derivatives by the Audiofrequency Pulse Technique 131 D. The Audiofrequency Pulse Technique and Enzyme-Inhibitor Rate Constants 138 Experimental 145 APPENDIX A. PRINCIPLES OF PULSED N.M.R. SPECTROMETRY WITH PARTICULAR REFERENCE TO HIGH RESOLUTION EXPERIMENTS ... 148 APPENDIX B. AUDIOFREQUENCY PULSE SPECTROMETERS EMPLOYED 153 REFERENCES 162 - v i i -LIST OF TABLES Table Page CHAPTER I 1 N.M.R. Parameters for the Ring A Resonances of 2a-Fluorocholestan-3-one (1) 18 2 N.M.R. Parameters for Parts of Rings A and B of 6a- and 6g-Fluoro-cholest-4-en-3-one (3 and 4) ... 23 3 N.M.R. Parameters for the Ring D of 16,16-Difluoro-androst-5-en-3g-ol-17-one (5) 2 9 4 N.M.R. Parameters for Compounds 6 (Cyano-aldehyde), 9, 10, and 11 (Decalones) 36 CHAPTER I I 19 1 F Chemical Sh i f t Data for N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl Glycosides (3 and 4) i n the Presence of Lysozyme 2 Linewidth Data for N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl Glycosides (3 and 4) i n the Presence of Lysozyme 3 K^, Kg and A Values for Compounds 1 to 4; also ^ H Chemical Sh i f t Data for 3 and 4 Competing with N-Acetyl-D-glucosamine-a-methyl-glycoside for Lyzozyme 4 Chemical Shift Data for the N-Acetyl Protons of 7, 9, and 10 and for the C^-Methyl of 10 Interacting x^ith Lysozyme 63 69 66 78 - v i i i -Page Table 5 Kp, Kg and A Values for Compounds 7, 9, and 10; 1 also H Chemical Shift of Glycoside Methyl on Binding 79 6 N.M.R. Parameters for the 0-Acetate Derivatives of 3*, 4' , 7' , 9' and 10' 90 CHAPTER III 1 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) 136 2 Nuclear Relaxation Time Data for the a- and (3-Anomers of the Free Sugar, N-Trif luoroacetyl -D-glucosamine 142 - ix -LIST OF FIGURES Figure Page CHAPTER I 1 The "^H n.m.r. spectrum (100 MHz) of 2a-fluoro-cholestan-3-one (1) in CDCl^ solution. A. The normal spectrum; B. The spectrum measured with 19 simultaneous irradiation at the F resonance frequency 9 2 The J H - { >H} INDOR experiments performed on the 19 1 1 F decoupled spectrum of 1 i n CDCl^ solution .... ± x 3 The >H-{ .H} INDOR experiments performed on the normal spectrum of 1 in CDCl^ solution 13 4 Partial >H n.m.r. spectrum of the highfield region of 1 in CDCl^ solution. A. The normal spectrum; 19 B. the F decoupled spectrum 14 1 9 5 The F n.m.r. spectra (94.071 MHz) of 1 in CDC13 solution 16 6 The normal H^ n.m.r. spectra of 3 (A) and 4 (B) in CDCl^ solution 20 I 9 7 The F n.m.r. spectra (94.071 MHz) of 3 (A), 4 (B) and 5 (C) in CDC13 solution 24 I I 19 8 The H-{ H} INDOR experiments performed on the F decoupled spectrum of 5 in CDCl^ solution 27 9 The "*"H n.m.r. spectrum (100 MHz) of a steroid (6) reaction mixture in CDCl^ solution 32 10 The >H n.m.r. spectra of decalones 9 (A), 10 (B) , and 11 (C) at 100 MHz in CDC1, solution 38 - x -Figure Page CHAPTER II 1 The section of a natural polysaccharide substrate which would occupy lysozyme's active site during lysis 47 19 2 F n.m.r. spectra (94,071 MHz) of a mutarotating solution of N-trifluoroacetyl-D-glucosamine (a-anomer, 1 ) in pH 5 . 5 citrate buffer 55 19 3 F n.m.r. spectra recorded during a study of 1 and 2 (a- and g-anomers of N-trifluoroacetyl glucos-amine) with lysozyme 5 7 19 4 A. Graph of F chemical shift data from the study of 1 with lysozyme B. Increase in 6 as a function of time during the mutarotation of pure a-anomer ( 1 ) to an equilibrium mixture in the presence of lysozyme 61 5 ''"H n.m.r. spectra recorded during a study of C,.-methyl-N-acetyl glucosamine-a-methyl glycoside ( 1 0 ) with lysozyme 74 6 Graphs of chemical shift data for 1 0 , 7 and 9 interacting with lysozyme 77 7 A. T^I n.m.r. spectrum ( 1 0 0 MHz) of N-acetyl glucosamine a-methyl glycoside (7) in D2O B. Partial hi n.m.r. spectrum ( 1 0 0 MHz) of C,-o iodo-N-acetyl glucosamine-a-methyl glycoside diacetate (9') in C D C 1 „ solution 84 - x i -Figure Page 8 Part ia l >H n.m.r. spectra (100 MHz) of A. N-t r i f luoroacety l glucosamine-!*-methyl glycoside triacetate (3') and B. N-tr i f luoroacetyl glucos-amine g-methyl glycoside triacetate (4') in CDCl^ solution 86 9 Computer-simulated spectra of the H^ 'doublet' region of a hypothetical N-acetyl glucosamine-a-methyl glycoside in aqueous solution 94 CHAPTER III 1 The alkene proton regions of the n.m.r. spectra (100 MHz, solvent CDC13) of A. 1,2-dibromo- and 1,2-dlchloroethylene (cis and trans isomers) and B. maleic and fumaric acid diethyl esters (plus cis- l ,2-dichloroethylene) 115 2 Effect of a modified Carr -Purcel l sequence on the magnetization vector, M q 119 3 Effect of a T^ sequence on the magnetization vector, M 121 o 4 The 1H n.m.r. spectrum (100 MHz) of the a lkalo id , vindoline, in CDCl^ solution 125 5 A. Scope photograph of a typical Carr -Purcel l sequence on the C^-acetate resonance of vindoline. B. Scope photograph of a typical T^ pulse sequence on the same resonance 126 - x i i -Figure Page 6 Relaxation time data obtained on the a lkalo id , vindoline, in CDC13 solution 129 7 . Par t ia l 1H n.m.r. spectra (100 MHz) of 3 , 4 , 6 - t r i -0-acetyl- l -0-benzoyl-2-bromo-2-deoxy-B-D-gluco-pyranose (A) and i t s 2-chloro analogue (B) in C^D^ 133 8 A. Plots of 1/T and 1/T2 vs. [ E q ] for the N-tr i f luoroacetyl group of the a-anomer. B. Plot of (1/T--1/TJ vs. [E ] for the a-anomer 143 2 1 o APPENDIX 1 Scope traces of various pulses on the alkene resonance of the diethyl ester of maleic acid . . . . 151 2 Block diagram of the components used in the "Mark I" audiofrequency-pulse spectrometer 154 3 Block diagram of the components used in the "Mark I" pulse unit 156 4 Block diagram of the components used in the "Mark II" audiofrequency-pulse spectrometer 159 5 Block diagram of the components used in the "Mark II" pulse unit I6 0 - x i i i -ACKNOWLEDGEMENTS Fir s t l y , I would like to thank Dr. L.D. Hall for his direction in this work - not only as a source of information and ideas, but also for his guidance in developing a more mature s c i e n t i f i c philosophy. Secondly I would like to acknowledge my numerous helpful discussions with Ben Malcolm and Dr. P. Steiner of this laboratory regarding my early work, with Dr. A.G. Marshall about theoretical aspects and with Roland Burton concerning n.m.r. electronics. 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 is well known. But with increasing molecular size 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 particular interest is obscured by the overlapping transitions of other resonances, which i s the so-called "hidden-resonance" problem. 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 in the last decade and these can, in many cases, help considerably in 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 is 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 for InterNuclear Double Resonance. - 2 -specifically incorporated for that purpose or of heteronuclei already 31 13 present in the molecule (e.g. P or C). This laboratory has investigated both of the above techniques and their application to 2 3 4 ever-larger molecules, e.g. sugars ' and cyclic phenyl phosphates. Success in such studies encouraged us to investigate the potential of these methods with s t i l l larger and more complex molecules and "natural products" presented a next logical step in this direction. Various >H n.m.r. methods have already been tried in this area but have met with limited success."' In some areas researchers have looked for empirical chemical shift relationships; for example, in the steroid area the relationship between the methyl group and ring geometry has been studied.^''' More recently, however, shift 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 in the literature on steroids as yet. "Natural products" are of considerable importance, yet organic chemists who isolate and synthesize them often have considerable d i f f i c u l t y in learning their chemical identity or configuration. The n.m.r. problem in this case i s often that transitions of interest are located in the "methylene envelope" which generally extends from x 7.5 to T 9.0. Our f i r s t experiments involved the use of n.m.r. spectroscopy in an attempt to examine the configuration of the basic steroid 9 skeleton. Sites of interest were labelled with fluorine. 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 - 3 -could be used for ^ H-{^ H} INDOR. experiments, 2. 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 H- F coupling constant data used to augment the proton data. 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 basically a set of coupling constants and chemical shifts for the groups of nuclei investigated. Relative signs of coupling constants can in many cases also be obtained by the double resonance techniques used but were not investigated here. Small differences in chemical shift between different molecules provide a very limited structural probe because of the many un-predictable factors involved. Coupling constants, on the other hand, are a very useful structural probe. Vicinal ^H-"4l 1 19 and 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 in this work have been quite thoroughly described in the literature. They a l l involve the perturbing effect of a second radio-frequency f i e l d (i.e., in 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 is the nuclear gyromagnetic ratio and B.^ is the perturbing r.f. field) compared with i t s distance from a given line. Early use of double resonance was confined to perturbing fields 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 selectivity of the resultant experiment. Freeman and Anderson have discussed the theory and merits of using weak perturbing fields (yH^/^iT of the order of the transition 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 for ^ H-{^ "H} double resonance experiments, the lowest power (and hence most selective) form of INDOR is convenient. This technique involves observing one sharp, well-resolved peak in a spectrum while slowly scanning a weak perturbing f i e l d through other regions of the spectrum. The peak being observed corresponds to a transition between two nuclear energy levels: 1 1 a (8 m, . . , . , . , . . population g The intensity of this peak i s related to the ratio, -c—-—= ; . population a If the weak perturbing f i e l d reduces this ratio for the peak one is 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 nuclei. For instance, consider monitoring the peak corresponding to a transition A^ of the following two spin AX system:^ _ 5 -a a pa A , X 2 A ' 2 A i A- Xi X 2 The peak corresponds to a transition between levels g a and act. These levels are also involved with the and lines, but not with the A£ line, of the spectrum. Sweeping a weak perturbing radio-frequency f i e l d through X^ and should cause intensity changes in A^: a decrease as the a g - a a transition pumps spins into the act level, and an increase as the gg - get transition pumps spins into the g a level. A convenient way to run such experiments is to arrange the n.m.r. equipment so that the recorder pen Y-axis monitors the intensity of a particular peak while the X-axis corresponds to the position of the weak perturbing f i e l d as i t is swept through the spectrum. The result of the above experiment would be^^: I AT A 2 X, X 2 - 6 -This model is 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 transitions of other protons with which i t is spin coupled (e.g., in the previous diagram, the X lines would have been detected even had they been entirely hidden beneath the methylene envelope of a steroid). The INDOR technique is so selective as to be applicable as long as individual lines can be identified, preferably on a f i r s t order basis. The limitation i s that some proton connected with the site of interest must be visible 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-cholest-4-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 trans 5-10 and 13-14 ring junctions. An additional interest in these particular molecules is associated with the fluorine substituent i t s e l f since fluorine as a steroid "label" is interesting from a biological viewpoint (a number 20 of fluorinated steroids have shown biological activity - sometimes 21 enhanced ). Partly for this reason, the literature on introduction of 22 such a label is considerable. Nevertheless, in terms of the general applicability 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 labelling. We have found that in many cases the natural product site of interest, being the reaction site, i s marked by a double bond, an oxygen-containing group, or some other electronegative species. Such a group often 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. It can then usually be employed in INDOR experiments to pick out the transitions of neighbouring protons which are s t i l l buried in the methylene envelope. This technique was found to work consistently well in identifying the products of several new reactions on natural products. The results of some typical problems are included at the end of this chapter. - 8 -Results and Discussion A. Studies of Flubrostefoids 2a-Fluoro-choles tan-3-one (1) This compound w i l l be discussed in some detail as i t represents a "typical" case; the other molecules w i l l be more brie f l y dealt with except where they posed special problems. The normal "^H n.m.r. spectrum of 1 (Fig. 1A) clearly shows the 2, R = Br - 9 -9 10 11 12 C-18 J2/3F| '5/6//8 1 2 3 4 6.6 7.6 8.6 Fig. 1. The H n.m.r. spectra (100 MHz) of 2ct—f luoro—cholestan—3—one (1) in deuterochloroform solution. A. The normal spectrum B. The spectrum measured with simultaneous irradiation 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 19 the electronegative fluorine atom. Irradiation at the F resonance 19 frequency removes the 48 Hz geminal Y-Yl0 coupling, collapsing this low-field octet to a quartet (Fig. IB). Changes also occur in the methylene envelope region during fluorine decoupling and these w i l l be discussed later. Two series of ^H-{"'"H} INDOR measurements were made while monitoring transitions of the clearly-discernible proton. The f i r s t of these 19 was performed on the F decoupled spectrum. Fig. 2 shows the results of this experiment; thus, Fig. 2A is 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 transition 12 (using the numbering system of Fig. IB). Sequential monitoring of transitions 11 and 9 gave the responses shown in Fig. 2B and C. The frequency at which an INDOR response occurs marks the position of a transition connected to the one being monitored. The sense of the response (either up or down) contains information as to the relative signs of the coupling constants involved - but in this work the signs were not of interest. The effective summation of a l l the INDOR responses (shown diagrammatically above the normal spectrum in Fig. 2) picks out a l l transitions corres-ponding 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 higher-field set of INDOR responses now identify the transitions of the other C^ proton which are normally totally obscured by the resonances of other methylene protons. - 11 -. 2. The H-{ H} INDOR experiments performed on the F decoupled spectrum of 1 in deuterochloroform solution. Using the transition numbering system of Fig. 1, the spectrum in A corresponds to monitoring transition 12, B corresponds to monitoring transition 11, and C to transition 9. The composite assignment resulting from these spectra i s shown diagrammatically above the normal proton spectrum .CHCI3 was used as the internal-reference signal for the field-frequency lock. - 12 -The second series of INDOR measurements (Fig. 3) was done without 19 F decoupling. Five lines of the H 0 resonance were monitored i n turn z p to pick out a l l transitions belonging to the protons. Because of the decrease in intensity of the transitions being monitored, the effective signal-to-noise ratio for these responses is poorer than that of the responses shown in Fig. 2. Nevertheless i t i s possible to make a summation of the individual responses and obtain the complete set of transition frequencies for both of the protons. Due to the fortuitous, near equality of the spectral splittings, there i s considerable degeneracy for the upfield 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 transitions, two further proton multiplets were subject to some decoupling; li k e l y these were from the protons. Several of these transitions were clearly resolved and a series of INDOR experiments in which these transitions 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,.. In this latter part of the 1 19 work the a b i l i t y to remove or retain H- F couplings at w i l l was very useful for identifying transitions. Close inspection of the INDOR spectra shown in Fig. 2 and Fig. 3 reveals that some responses occur at frequencies corresponding to the C-methyl resonances. These seem to be instrumental artifacts arising from overloading of some amplifier stage i n the spectrometer. 19 The normal F spectrum of 1 is shown in Fig. 5. The comparatively Fig. 3. The H-{ H} INDOR experiments performed on the normal spectrum of 1 in deuterochloroform containing enough CHCl^ for a lock signal. The transitions were monitored as follows: A transition 8; B transition 6; C transition 7; D transition 2; E transition 3. A diagrammatic summary of these responses is given above the normal spectrum. - 1 4 -^4(3 ^ 4 a B J4/35oc J4a5ot J4a4/3 Fig. 4 . Partial H n.m.r. spectrum of the high f i e l d region of 1 in deuterochloroform solution (CHCI3 lock). The normal spectrum i s shown in A and the 1 9 F decoupled spectrum in B . The first-order assignments of these spectra were based on the INDOR experiments discussed in the text. - 15 -poor resolution of the undecoupled spectrum i s due to the fact that 19 the F substituent is part of a f a i r l y highly-coupled proton spectrum arid to the large number of small, long-range couplings. In addition to the large geminal coupling with H (ca. 48 Hz) a number of smaller Zp proton couplings are resolved (Fig. 5). The foregoing set of experiments served to identify the resonances of a l l the protons associated with ring A of derivative 1. Comparison 19 of the transitions 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 in reasonable accord with the 19 estimates obtained by direct measurement of the F spectrum (Fig. 5). These data are included in Table 1. Simulation of the spectra of the H and H 0 resonances using the parameters involving these three i zp nuclei and the T?^ substituent, gave calculated transition energies which were in close accord with the experimental values; on this basis i t was concluded that a f u l l , iterative analysis was unnecessary. Interestingly, the H^-"'"H couplings for the H^ and protons listed in Table 1 are identical, within experimental error, with those 23 previously reported by Allinger 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, in spite of the small chemical shift separation of the two C^ protons, a satisfactory estimate of the inter-proton couplings 19 was readily made by examination of the F decoupled spectra and by iterative, computer-based analysis involving the H^, and H,. resonances. Because of several fortuitous degeneracies, several - 16 -F H , 1 . • I . I . 1 . 1 . I , I 1 1,, 1 I -40 H Z + 194.5 +40 H Z PPM. FROM CFCI3 19 Fig. 5. The F n.m.r. spectra (94.071 MHz) of 1 in deuterochloroform solution. The field-frequency lock in this case was C F C I 3 . The normal spectrum is shown in the lower trace. The insert was recorded at the same gain but with simultaneous irradiation of the entire . H spectrum using a noise-modulated radio-frequency centered at 99, 997, 945.0 Hz. A partial f i r s t order assignment of the smaller couplings is shown. - 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 crucial for these assignments since in i t s absence i t would have been impossible to assign the and 19 resonances. The F-H. coupling was assigned an absolutely positive 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 rationalized the v i c i n a l H^-^ couplings of the 2a-bromo analog (2) of the derivative we were studying in terms of a slightly 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, indirectly, the validity of previous conclusions. Thus the ^ coupling of 14.4 Hz clearly accords with a trans-diaxial relationship between these two protons. 19 1 Furthermore, the magnitudes of the vicinal F- H couplings are only consistent with a gauche relationship between the F„ substituent and 2a the two protons at C^. Although there is a paucity of data concerning 4 J couplings, the values listed in Table 1 for the couplings between 24 F„ , H. and H, appear to be in reasonable accord with expectation. 2a 4 a 4g 1 h One of the more interesting points which we wished to evaluate in the course of this study was whether i t might be possible to use a fluorine substituent to label a specific site of a steroid molecule 19 for subsequent n.m.r. studies. It is obvious from the normal F spectrum of 1 (Fig. 5) that, for this molecule at least, conventional Table 1. N.M.R. Parameters for the Ring A Resonances of 2a-Fluoro-cholestan-3-one (1) Chemical shifts (x-values, <J> -values) la H13 H2B 4a 43 H5 F 8.52 7.52 5.01 7.79 7.65 8.47 <j> +194.5 c Coupling constants (Hz) H l a H 1 3 H l a H 2 3 H13 H23 V V V -12.2+ 12.3+ 6.9* 48.0+ 11.5* 4.5* R. H. H. H c H. H c H. F H. F H c F 4a 43 4a 5a 43 5a 4a 43 5a -13.9* 2.1* 14.4* 7.3* -2.0§ ^0 Measured in deuterochloroform solution containing CHCl^ or CFCl^ * Estimated error + 0.2 Hz + Estimated error + 0.4 Hz § Estimated error + 1.0 Hz Note: The error limits indicated by t and § represent the limits between which the coupling constants were found to vary when the individual transitions were varied over the maximum possible limit. 19 F measurements have l i t t l e to recommend them. However, the detection 19 of a F resonance while simultaneously irradiating the proton spectrum appears to be a more attractive 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 only resulted in partial collapse of 19 1 the proton couplings. However, noise-modulated F- H decoupling effectively removed a l l of the proton couplings and, as is indicated 19 in the insert of Fig. 5 , the F resonance was then detected as a reasonably sharp singlet. 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) in deutero-chloroform solution are shown for comparison in Fig. 6. A few drops of chloroform was used for the internal field-frequency lock. Both H. and H, appear to low f i e l d in each case. H, shows the characteristic b o 19 1 geminal F- H coupling of ca. 50 Hz. An interesting difference between the two spectra is the splitting of the C^Q methyl group into a doublet in the 6g-fluoro steroid (Fig. 6A) whereas i t is a sharp singlet in the 6«-fluoro analog (Fig. 6 B ) . H, H 6/5 4 . 0 7 . 0 1 0 . 0 Fig. 6. 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 irradiation 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 . 2 ^ a > ^ > c B o t h and C ^ methyl groups have been observed to be s p l i t into doublets under certain 19 conditions in steroids containing a F substituent. Cross and Landis 19 l rationalized the splittings in 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 F decoupling) in Fig. 6. Such splittings could be quite readily understood i f the 19 fluorine were close to the methyl group in question - but when the F is five or six 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) is s t i l l q u e s t i o n a b l e ^ 0 ' ^ but 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 in the direction of the proton, and originating at the methyl carbon." 19 In both 3 and 4, the resonance could be readily examined by F decoupling to determine the FH^ split t i n g . The H^ H^  s plitting was also clearly assignable during these decoupling experiments. In both anomers part of the highly coupled H^ H^  proton system i s visible 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. 19 The systems comprising H,, H,, H., , H,„, H„ and F were close r 4 6 7a 76' 8 - 22 -/ enough to f i r s t order to enable INDOR analysis. was the logical set of transitions to monitor during INDOR experiments designed to find the protons and Hg. Unfortunately the lines were broadened 19 and poorly-resolved for both isomers. With F decoupling i t was possible to monitor while running INDOR scans to find both protons (Fig. 6). However, the quality of the INDOR responses did not allow an unambiguous determination of the position of Hg. For both 3 and 4 the 7a-proton was entirely hidden i n the methylene envelope, but several transitions of the 7g-proton could be distinguished. 19 Without the F decoupling, the INDOR response quality was even lower. However, the H 7g~F splittings could be picked out for 3 and 4 by direct comparison between the decoupled and undecoupled spectra. 19 A good estimate of the H 7 -F couplings was also obtained from the F / a spectra. At this stage, the next step should have been to analyze the chemical shift and splitting data (with the help of a computer program) to obtain 'true' chemical shifts and coupling constants. This was not done here because a reliable value for the H_ chemical shift was not o found through the INDOR experiments. Nevertheless, the nuclei at C^, Cg, and can be seen (Fig. 6) to form a virtually f i r s t order system -and the H^  transitions found by INDOR seem to indicate that Hg is not very close to either. This means that one can f a i r l y safely take the observed chemical shifts and splittings 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 in Fig. 7A, and that of 4 in Fig. 7B. That of compound 3 in particular is broadened by - 23 -Table 2. N.M.R. Parameters for Farts of Rings A and B of 6 a - and 6g-Fluoro-cholest-4-en-3-one (3 and 4) First order chemical shifts (x-values, (j^-values) Compound H. H, F, H., H.,n Cir.-CH„ 4 6 6 7a 7g 10 J 3 ( 6 3-F) 4.16 5.05 $ + 165.61 8.81 7.84 8.70 4 (6a-F) 3.97 4.94 $ + 183.63 8.79 7.74 8.84 First order coupling constants (Hz) Compound 3 H.H, H. F H^  H_„ H^  H^  H^  H„ 4 oa 6a 6a 73 6a 7a 7a o 49.5 2.5±0.8 2.6±0.8 13.0±1.0 H-j H., H H_ Cin-CH F H^ F H., F ^,F 7a 73 73 8 10 3 7a 73 4 -13.5±1.0 3.0±0.8 1.9 47±3 13.6 5.0 Compound 4 H.H,D H,.F H,.H7. H,QH._ H 7 H Q r 4 66 63 63 73 63 7a 7a 8 1.8 47.8 5.8 11.8 11.4 H 7 H_. H_QHQ Cin-CH_F H.. F H..F H.F 7a 76 76 8 10 3 7a 73 4 -10.8 2.5 0 8.8 4.7 0.7 Measured in 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. 100 HZ t B 100 HZ p "1 i q 100.5 109.6 Fig. 7. The F n.m.r. spectra (94.071 MHz) of 3 (A), 4(B) and 5 (C) in deuterochloroform solution. CFCl^ was used for the internal field-frequency lock. - 25 1 19 unresolved splittings - but in both cases, the H- F couplings are recognizable in the fluorine spectra. 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. They showed that the 6g-fluorine is more strongly spin-coupled to than is a 6a-fluorine. We have observed the same phenomenon here. The INDOR technique in this case supplied several useful points. The location of the proton chemical shifts permitted a check on the f i r s t order nature of the observed H,-H_, splittings 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 vi c i n a l 1 1 H-F couplings as well as making the study possible. Both H- H 1 19 and H- F vincinal couplings in compounds 3 and 4 are indicative of an approximate chair conformation for ring B in spite of the double bond in ring A. 19 With regard to the possibility 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 is broad, that of 4 could be useful. 16,16-Difluoro-androst-5-en-3g-ol-17-one 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) in deuterochloroform solution: chloroform was used for - 26 -the i n t e r n a l f i e l d - f r e q u e n c y l o c k . The i n s e r t shows the C^g and 19 p r o t o n s : t h e i r normal spectrum on the l e f t and t h e i r F decoupled spectrum on the r i g h t . Here we have another example of the " c o n v e r g i n g -v e c t o r r u l e " but t h i s t ime i t i s the C^^ methyl group which i s s p l i t 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 to decouple 19 the F nucleus a l l o w e d an accura te measurement of J 1 D 18 O Once a g a i n , two se ts of da ta were a v a i l a b l e due to the s i m p l i c i t y 19 1 of n o i s e d e c o u p l i n g the F n u c l e i . H t r a n s i t i o n s a s s o c i a t e d w i t h 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 d e c o u p l i n g power on and o f f , and comparing the s p e c t r a . Then, a f i r s t , s imple set of INDOR experiments ( F i g . 8A, B, C, D) was run w h i l e i r r a d i a t i n g 19 both of the F n u c l e i . In t h i s case , t r a n s i t i o n s of b o t h H.. c and 15a H - ^ g cou ld be moni tored w h i l e scanning 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 set 19 of t r a n s i t i o n s because even w i t h o u t the F n u c l e i i t i s coupled i n t o f o u r other p r o t o n s . However, i t s chemica l s h i f t was r e a d i l y f o u n d . Note that m o n i t o r i n g l i n e 4 (a degenerate l i n e of H _ ) leads to I . I , 1 . I • 1 . ! . I . I • . I , . I 7.5 8.0 8.5 r 9.0 1 1 19 8. 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 monitored 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 these 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 normal p r o t o n spectrum. The i n s e r t shows the e f f e c t of - L 9 F d e c o u p l i n g on the C 1 0 and (^3 methyl groups . Decoupl ing 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. - 28 -responses for a l l four lines of H.^ .^  (Fig- 8C). The effective summation of the INDOR responses is shown above the spectrum and clearly 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 1 C„ which included l o o t i j p 1 19 H- F splittings. Note that the C-methyl resonances give rise to their usual artifact responses in the INDOR spectra. 19 The normal F spectrum of 5 is shown in Fig. 7C. The spectrum is sharp and well resolved. It displays the typical large (ca. 284.6 Hz) . . 19_ 19_ , . geminal F- F coupling. The experiments described above provided enough data to completely analyze the D-ring nuclei of 5. The data was analyzed with the help of a computer program to obtain the true coupling constants and chemical : shifts (Table 3). One would expect to be able to say something about the D-ring conformation at this stage. It is 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 hybridization amongst other things; furthermore, applications to five-membered rings appear to be particularly d i f f i c u l t . It 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. Thus J., . .. r_ = 14.0 Hz would definitely indicate a B 14,153 trans-diaxial relationship of the two protons and J.. . .. c = 5.9 Hz r v 14,15a - 29 -Table 3. N.M.R. Parameters for the Ring D of 16,16-Difluoro-androst-5-en-3g-ol-17-one (5) Chemical shifts (x-values, <j) -values) H14 H15a H15g F16a F16g C13~ C H3 8.55 7.60 8.04 <j> + 109.57 a) + 100.49 8.95 c c Coupling constants (Hz) H14 H15a H14 H15B H15aH15B H15a F163 ^15aF16a 5.9* 14.0+ -14.2+ -0.7* 19.6* H15gF16B H F 15p*16a CH 1 3-CH 3F 1 6 g 13 3 16a F16a F16g 16.3* 22.6* + 0.9 0 284.6* Measured in deuterochloroform containing CHCl^ or CFC1 Estimated error ±0.3 Hz. Estimated error ± 0.4 Hz. - 30 -indicates roughly a 60° dihedral angle for this pair. The F resonances are now readily identified as 16a upfield and 16|3 downfield. The -0.7 Hz coupling between H . a n d F., suggests a ^90° dihedral 1 5 a lop angle between them. The remaining three H-j.5^ 16 couplings agree with the same geometry. 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 different when carbon 17 is sp hydridized. In fact i t has been suggested that carbons 14, 15, 16 and 17 are in a plane for 7 28 the 17-OH and 176-acetyl analogues.' 19 It seems that in this compound at least, the F labels have very useful "normal" n.m.r. spectra. Nevertheless, they can be reduced to sharp singlets by noise modulated decoupling of the entire proton 19 region. Similarly, the F splittings 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 F-{ H} and 1 19 H-{ F} heteronuclear decoupling experiments can considerably f a c i l i t a t e n.m.r. studies of fluorinated steroidal systems. In particular, the observation that noise-modulated irradiation of the entire H^ spectrum 19 can effectively reduce a F resonance to a narrow singlet augurs well - 31 -for the future of fluorine as a probe for evaluating the interaction of steroidal derivatives with other systems of biochemical significance such as membranes. We also conclude that "^H-{^ H} INDOR measurements should find widespread application in a number of structural problems commonly encountered in natural product chemistry. For, although the 19 combination of F labelling and double resonance can be a powerful technique, the presence of a fluorine substituent is not a necessary prerequisite for such measurements. The next section w i l l discuss this. B. Application of 1H-{1H} INDOR to Natural Products The examples discussed in the previous section of this thesis were chosen with a variety of different points in mind; however they a l l indicated that "^-{"Si} INDOR measurements greatly faci 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 usefully applied routinely to solve the type of structural 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 in order to investigate this point. (i) A Steroidal Problem As part of another programme, Weiler and Paisley (private communica-tion) were attempting to perform the conversions indicated in 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 is shown in Fig. 9. The mixture contains starting material and at least H 2 • i NJ I 0.0 5.0 9.0 X Fig. 9. The h n.m.r. spectrum (100 MHz) of a steroid (6) reaction mixture in deuterochloroform solution. The position of the C H C I 3 internal field-frequency lock is shown. Chemical shift positions of the protons of interest are shown. - 33 -one side product. The only clear spectral features associated with the molecular site 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 in turn while sweeping a perturbing radio-frequency f i e l d through the methylene envelope. A pair of quartets was obtained by summing the INDOR responses: one at x 7.93 and the other at x 7.48 (Fig. 9 and Table 4). Both of these quartets were otherwise entirely hidden in 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 in differentiating amongst the various po s s i b i l i t i e s . Chemical confirmation was doubtful because of the known possibility of anomerization during reaction. Several other possible products could also have produced an aldehyde quartet e.g. (8 could have a quartet for the aldehyde via second order coupling to one of the C, protons). But neither of these compounds would have 6 produced the simple AMX system found by the INDOR experiments. Thus one very useful feat l i r e cf the INDOR method i s that i t can be applied to crude mixtures of compounds. ( i i ) 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 Phillips - private communication). It Proposed minor product Proposed major product was hoped that their n.m.r. spectra would provide a check of the above assignment. The n.m.r. spectrum of the minor product is given in Fig. 10A and that of the major product in Fig. 10B. Both samples were dissolved in deuterochloroform. In each case the methyl group is readily identified by comparison with other compounds and by double resonance experiments (Fig. 10) as the downfield doublet in the methyl region. 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 is resolved. For both compounds i t was possible to perform INDOR experiments in which the methyl group lines were monitored in 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. The information so obtained was a chemical shift value for (Table 4) and the fact that in both compounds the H. transitions were spread over at least 32 Hz. If the H.-H_ 4 4 5 coupling were not axial-axial (e.g. axial-equatorial) the H^  transitions would be spread over only 22-25 Hz at most. This result agrees with the proposed structures given above. The remaining v i s i b l e downfield transitions for 9 were assigned, on the basis of their chemical shift and small number of couplings, to B.^ and ^ monitoring these transitions in 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 relatively f i r s t order system (Fig. 10A and Table 4). H^  was not unambiguously located for 9 as only 8 mg of sample was available - 36 -Table 4. N.M.R. Parameters for Compounds 6 (Cyano-aldehyde), 9, 10, and 11 (Decalones) First order chemical shifts * (x-values) Compound % H l H2 6 7.48 7.93 0.14 C.-CH. 4 3 H4 H26 H2a H l 9 8.97 7.33 7.52 7.86 -10 8.99 7.88 7.93 7.66 8.50 C1-CH3 H l H2 1 H 2 11 9.08 8.08 7.20 7.93 First order coupling constants (Hz) Compound H1 2 H2 H H 1 2 -6 1.4* 4.0* 14.5+1.0 C.—CH„H, 4 3 4 H2a H2B H2B H13 H2a H16 9 6.4* t 13.0 + 3.9 13.0* C, — CH _H, 4 3 4 H2a H23 H 2 B H l a ^2a^la 10 6.4* + 12.6 t 12.6 t 3.8T H H 1 2 H H 2 11 7.2* 14.5* 1.4* t 6.2* Measured in deuterochr'oroform solution containing CHC1 Estimated error I 0,2 Hz. * Estimated error + 0.4 Hz. - 37 -and the signal -to-noise ratio 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 shif t value for H^. In the case of 10, only one proton was v is ib le at the low-f ield end of the methylene envelope and i t s transitions were monitored during INDOR experiments to f ind the others (Fig. 10B and Table 4). The spl i t t ings quoted above are certainly close enough to f i r s t order values to be used for gross conformational analysis. The results are interesting. From the structure proposed for 9 one would expect J 0 - and J < 6 Hz. But the values in Table 4 suggest Za ,J-p Zp,lp that H^ is axial rather than equatorial. Thus the n.m.r. data agree with the structure proposed for 10 but not for 9. Partly as a result of this confl ict ing n.m.r. data, Piers and Ph i l l ips were led to propose that the AB ring junction had formed cis instead of trans during the Birch reduction of a 4,5 double bond. This would avoid the ster ic strain caused by an axial isopropyl group. It should be noted that the n.m.r. INDOR data does not distinguish between 9 and 10. It only says that 9 i s not the compound i t was 8.0 9.0 10. The % n.m.r. spectra of decalones 9 (A), 10 (B) and 11 (C) at 100 MHz' in 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 -originally thought to be. But once again, INDOR provided important structural information. Fig. IOC shows the "^H n.m.r. spectrum of a decalone produced via Birch reduction in a manner very similar 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 vicinal proton. Both of these other protons were hidden in 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 above-mentioned vicinal partner was shown to be H^. The data shown in Table 4 support structure (a). Structure (b) would be expected to have one of the H^ -H^  splittings greater than 10 Hz. The protons have not been assigned as a or 3 in the table. However, i t seems quite l i k e l y that the downfield proton is a n <^ the upfield one is H . The axial C. methyl group might be expected Za 1 to distort the A-ring in 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 find considerable application as an analytical 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 in 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 H-{ INDOR spectra has also been given previously."^ (c) A l l n.m.r. measurements were made in deuterochloroform solution. Since the spectrometer gain is 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 possible. For natural products CHCl^ was found to be useful . Proton chemical shifts are reported in the T scale. (d) Where possible, analyses of the n.m.r. spectra were made with a modified version of the LA0C00N III program and an I.B.M. 360-67 computer in the U.B.C. Computer Centre. 2ot -Fluoro-cholestan-3 -one (1) was prepared as described in the 32 l i terature . 6g- and 6q -Fluoro-cholest-4 -en-3 -one (3 and 4) were prepared 20 32 33 according to general procedures described in the l i terature . ' ' 16,16 -D i f luoro-andros t-5-en-3-ol-17^-one (5) was prepared as 34 described in the l i terature . The non-fluor'inated natural products studied were obtained from L. Weiler and K. Paisley (steroid) and E. Piers and N. Ph i l l ips (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 in biological systems are becoming increasingly widespread. Perhaps this should not be surprising as i t is one of the only two methods capable of detecting individual atoms in macromolecular systems - the other method being X-ray diffraction 35 studies of crystals. In cases where a very large molecule (for instance a protein) can be isolated in a pure, crystalline form, X-ray diffraction can yield uniquely valuable information concerning i t s fi 36a,b,c , . 37a,b , , ^ .38 geometry (lysozyme, carboxypeptidase A, and chymotrypsm being cases in point). N.m.r. is attractive because i t offers, at least in principle, straightforward techniques for the detailed investigation of the structure of large molecules in solution as well as techniques for studying the dynamic aspects of molecular structure and interaction. An enzyme found in 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 articles in the recent literature dealing with the uses of n.m.r. spectroscopy in biological 39-43 systems. There is no one,universal n.m.r. approach to such problems. A whole area has grown up around the use of paramagnetic 40 probes in n.m.r. studies of enzyme-substrate complexes. A more obvious approach is the use of normal high resolution n.m.r. spectra to study structure, and pH effects thereon, of 'small' biological building 42 blocks such as amino acids, peptides etc. A recent, but very important, area of endeavour is the application of n.m.r. to the study of rate phenomena in biochemical systems. Chemists have been using 44 n.m.r. to study rate processes for a good ten years, but biological 45 applications have been longer in appearing. Recent technological advances have opened up whole new areas to the n.m.r. spectroscopist 41 interested in biochemical studies. Typical examples include 13 Fourier transform methods for measuring natural abundance C n.m.r. spectra, superconducting magnets for 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 in applying n.m.r. to the study of biopolymers is that the combination of large linewidths due to slow reorientation, and the presence of many closely spaced lines in 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 indirect approaches. Obviously spectrometers with higher magnetic fields 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., 46 the use of n.m.r. in c e l l membrane studies ). In favourable cases, the n.m.r. spectrum of a biopolymer may contain peaks or regions identifiable with some site of interest and such spectral features provide probes of their surroundings (e.g., lysozyme, ^ a>^ ribonuclease^) . In less favourable cases, selective deuteration or heteronuclear labelling may be tried. 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 in line positions and/or relaxation times which can be related to phenomena of interest. The use of ions in such experiments generally involves relaxation time measurements - the results being typically treated as outlined in Stengle and Baldeschwieler's 49 ion-probe method. The technique of comparing the n.m.r. spectrum of a small molecule before and after addition of some biopolymer i s an indirect but very promising one and is the method considered in 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 in such a way as to reflect upon some site of interest. A good kinetic model for many problems is the "two site case"^'"^ The small molecule is thought of as existing either free in solution (site A) or associated in some way with the biopolymer Relaxation phenomena w i l l be discussed in Chapter III. - 45 -molecule (site B). Thus E + I f = [EI] ^ [E][I] E = enzyme I = inhibitor EI Kg = the binding constant There may be considerable information on the rates of the exchange processes involved contained in the relaxation phenomena associated with such a system (see Chapter III). In general, the two sites available to a small molecule may be characterized by different resonance frequencies, to. (bound) and UL (free). This i s so because A B in general the magnetic properties of s i te A w i l l d i f fer from those of s ite B. This may be part icular ly noticeable i f the bound molecule is proximal to an aromatic system or the e lect r ic f ie ld of a polar group or metal ion. Here i t suffices to say that i f the rate of exchange (k_^sec )^ is very small compared to the difference in frequency, (u>^ -u)g) f then separate resonances w i l l occur at and uv,. If the reverse is true, then a single average resonance w i l l D appear between the two frequencies at a distance 6 = P^A from ujA/2ir where P„ is the fraction of the small molecule which i s bound A B at a given time and A is the chemical shift (io^ -u>g) /2TT between bound and unbound species. This latter situation has been found to occur regularly for reversible inhibitors interacting with lysozyme. The chemical shi f t of an observed inhibitor as a function of concentration, t See Experimental Section for a more complete description. - 46 -pH or temperature yields information which can be used to calculate K^, A and thermodynamic constants. Lysozyme 54 Like a l l enzymes, lysozyme is a globular protein - a series of amino acids bound together in a chain by peptide bonds and folded into a complex three-dimensional structure. Lysozyme consists of 129 amino acid subunits of 20 different kinds and has a molecular weight of about 14,400."^ Thus i t is a very small enzyme. It i s also exceptionally stable in aqueous solution and is readily purified and crystallized. 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 diffrac-36a,b,c,56,57,. _ . , . _ , , , tion ). Lysozyme provides then a very useful model enzyme system for solution study. Lysozyme's biological function in animals (where i t is found in many tissues) i s to destroy the c e l l walls of certain bacteria by catalysing the hydrolysis of the carbohydrate component, specifically a g-l,4-linked polysaccharide with alternate N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) residues. This reaction proceeds with cleavage of a C^-oxygen bond in the substrate. Fig. 1 shows the section of such a polysaccharide which would be bound to the enzyme during l y s i s . A, C and E are NAG residues, B, D and F are NAM residues. - 47 -\ \ The enzyme's a b i l i t y to perform this catalytic 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 in 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 overall folding of the lysozyme polypeptide chain has led to a structure with a deep cleft running up one side. It is this cleft which contains the 'active site'. It is not possible to perform X-ray studies of lysozyme interacting with a bacterial c e l l wall. But i t has been possible to grow crystals of the enzyme Interacting with small "inhibitor" sugar molecules. These are species which can inhibit the action of lysozyme by themselves bind-ing to the active site in a manner similar to that of the true substrate but which themselves react only slowly i f at a l l . Lysozyme's action is known to be inhibited 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, 5 8 chitobiose (di-NAG) and the trisaccharide, chitotriose (tri-NAG). 36c It was primarily X-ray studies of lysozyme interacting with tri-NAG that led to the binding array shown in Fig. 1. This array places a monosaccharide subunit in each of 6 subsites (A to F) in the cleft of lysozyme. The bond cleaved is shown by a dotted line. Polysaccharides consisting of NAG up to the tetrasaccharide are known to bind with 58 59 their reducing end in subsite C ' and consecutive sugar rings in subsites B and then A (hence the monosaccharide NAG occupies subsite C). From the proximity of the sugar residues to polypeptide side chains in the cleft (as indicated by X-ray studies), modes of bonding have been postulated"^'"^° for a l l six subsites. Workers have been quite specific about likely hydrogen bonding interactions but less specific about nonpolar ones. Nevertheless nonpolar binding seems likely to play in important part in 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 " ^ 0 ' " ^ involves the catalytic effect of the proximate peptide side chains of residue 35 (glutamic acid) and 52 (aspartic acid) on the C-0 bond in question (Fig. 1). It has been suggested that of - 49 -ring D passes through a carbonium ion intermediate and that binding of the substrate distorts ring D. X-ray data on lysozyme has formed a basis for a l l the more recent solution work. Lysozyme and the Chemical Shift Technique Previous studies from this laboratory have shown^^3'^ that ~^F n.m.r..parameters are more sensitive to changes in chemical environment than are n.m.r. parameters. Because of this we have been interested in the poss ib i l i t y of using fluorine substituents as structural 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 is part icular ly so in the study of biomolecular associations where i t i s often d i f f i c u l t to identify unequivocally a part icular proton resonance. In view of the strong stereoelectronic preference of the carbon-62 63 fluorine bond, ' i t seems l ike ly that in 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 ie ld valuable information as we shal l show. Lysozyme seemed to be an ideal model for studying this phenomenon because of the detailed X-ray and n . m . r . ' ^ ' ^ ^ data which are already available. For these studies four N- t r i f luoro -acetyl derivatives (1-4) of D-glucosamine were synthesized St r i c t l y these are derivatives of 2-deoxy-2-trifluoroacetamido-D-glucopyranose. - 50 -C H 2 O H which correspond to four known inhibitors (5-8) of lysozyme. These latter compounds (5-8) have been extensively studied via n.m.r. by Raftery et a l . ^ ^ and by Sykes et a l . ^ ' ^ (both of whom monitored the N-acetate peaks). Values of and A obtained for such compounds necessarily have a high experimental error (typically + 10-20%). Hence one must be careful not to place undue emphasis on small differences in measured values between different compounds. Our studies of the trifluoro derivatives (in which we have monitored the N-trifluoroacetate peaks) subsequently led us to investigate two other problems: (a) The effect of substituents at C, (b) The conformation of inhibitors. The former problem was approached by synthesizing and studying the following series of compounds: C H 2 R NHCOCH3 Inhibitor conformations were studied by analysis of the T^i n.m.r. spectra of their O-acetates. We have also briefly considered the possibility of studying the conformation of bound inhibitors. - 52 -Results and Discussion A. Choice of a Suitable Chemical Shift Reference The most accurate way to measure small chemical shifts in n.m.r. spectroscopy is by comparison with some reference compound. When using organic solvents i t i s common practice to employ tetramethylsilane (TMS) as an internal reference for measuring proton chemical shifts, and freon 11 (CFCl^) for fluorine work. However, enzyme studies are carried out i n aqueous solution. Theoretically one could use a capillary of TMS or freon 11 held concentric with the n.m.r. tube 69 (see Experimental Section). But i t has been shown that chemical shifts measured relative to such external standards are very prone to bulk magnetic susceptibility effects - especially when the temperature is varied between samples. For proton work i t i s convenient to use the water peak for a f i e l d -frequency lock; but this peak is notoriously temperature and pH dependent. Hence a small quantity of some other species must be added for use as an internal standard. For lysozyme work, both acetone^ ^ and t_-butanol^ have been employed. 19 For a F field-frequency lock a capillary of trifluoroacetic acid was used. This was found to be very suitable for work with a tr i f l u o r o -acetyl label as extensive f i e l d offsets'were not required. We have considered the possibility of using sodium trifluoroacetate (NaOCOCF^) as an internal reference. It i s very soluble and quite inert. Unfortunately, i t was found to interact with lysozyme in such a way as to shift and broaden. Other internal references tried were trifluoro-ethanol (CFoCH„0H) and hexafluoroacetone sesquihydrate. The former I - 53 -appears as a very sharp triplet quite close to the region of the t r i -fluoroacetate label (Fig. 2). It shows no sign of broadening or shifting on addition of lysozyme (Table 2) and was used in a l l fluorine chemical shift studies. Trifluoroethanol is somewhat acidic. For this reason a check was made to see that i t was not overloading the 0.1 M pH 5.5 citrate buffer used for this work. Twice the concentration of trifluoroethanol actually employed during enzyme runs was seen to have no noticeable effect on the buffer pH, hence no problem is to be expected. B. Experiments with N-Trifluoroacetyl-D-glucosamine The monosaccharide, N-acetyl-D-glucosamine (NAG-anomers 5 and 6) are known"^'"^'^ from X-ray studies to bind specifically and reversibly at subsite C on the enzyme surface (although the a-anomer is a special case). The methyl glycosides (7 and 8) seem l i k e l y " ^ ' ^ to behave in a similar fashion to 6 with respect to binding to lyso-60,58 zyme. In a l l four cases, the resonance of the N-acetyl group shows a definite upfield s h i f t ^ ' ^ and broadening on binding of the sugar to lysozyme. This shift has been attributed to the proximity of the N-acetyl methyl protons in the bound sugar to the aromatic portion of a tryptophan residue (residue 108) in l y s o z y m e . ' ^ In the case of the free sugar, NAG, the a- and B-anomers have been observed to 58 59 show'a degenerate N-acetyl resonance when no lysozyme is present ' but this singlet gradually becomes a doublet as enzyme is added. This 58 59 68 degeneracy has considerably hampered ' ' accurate chemical shift - 54 -measurements on the system and no doubt partly explains why Raftery et a l . and Sykes et a l . get somewhat different values for K and A . D We have synthesized (see Experimental section) the following series of compounds in which the normal N-acetate group of N-acetyl-D-glucosamine has been replaced by an N-trifluoroacetyl group. It was expected that the fluorine resonances of 1 and 2 might not be degenerate C H 2 O H as in the H case. Moreover, i t was hoped that fluorine chemical shifts on association with- the enzyme would be greater than proton 19 shifts. At the same time we expected that the F nuclei might well have some perturbing effect on the interaction. When a mixture of the a- and g-anomers of the free sugar was allowed to crystallize slowly from water, the a-anomer was obtained as a pure, crystalline hydrate. The mutarotation of this species to 19 i t s equilibrium mixture could be followed by F n.m.r. (Fig. 2) and was observed to take about 45 minutes in pH 5.5 citrate buffer at 31.5°C. The f i n a l ratio 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 in which the H 1-H„ - 55 -Time (min.) .a r m 12 3 0 0 200 Hz. From TFA 19 Fig. 2 F n.m.r. spectra (94.071 MHz) of a mutarotating solution of N-trifluoroacetyl-D-glucosamine (a-anomer, 1) in pH 5.5 citrate buffer (0.1 M). ~The internal standard, trifluoroethanol, i s seen as a sharp tr i p l e t some 100 Hz to higher f i e l d . A capillary of TFA was used for the field-frequency lock. The time after dissolving the pure a-anomer is shown to the right of the spectra. - 56 -sp l i t t ing i s clearly v is ib le in the downfield C^-proton. It should be noted from Fig . 2 that, as was hoped, the N-tr i f luoroacetyl groups of 1 and 2 are non-degenerate (separation ca. 27.0 Hz), and that they f a l l in a convenient position relat ive to the internal refer -ence compound, CF^Cr^OH. Unfortunately the mutarotation of the pure a-anomer i s suf f ic ient ly fast to result in an appreciable amount of the g-anomer (2) only several minutes after dissolution. The excellent 19 separation of the F peaks of the two anomers permitted accurate integration of the intensi t ies . 19 F ig . 3 shows the effect 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 section). In F ig . 3A no enzyme has been added and the shifts of a - and g-anomers are shown relative to the centre l ine of the trif luoroethanol t r ip le t (204.5 Hz downfield from the tr i f luoroacet ic acid capi l lary lock s ignal ) . Internal standard concentration was 0.05 M. F ig . 3B displays the spectrum obtained when _3 a sample of sugar at the same concentration (0.10 M) i s made 3 x 10 M in lysozyme. The a-anomer (1) i s seen to broaden considerably and to shi f t upfield toward the internal reference signal . The g-anomer (2) shows no appreciable change. When the sugar concentration i s lowered to 0.04 M (Fig. 3C) the compound 1 shows even greater changes in chemical shi f t and l i n e -width , whereas the spectrum of 2 reamins re lat ively unchanged. The chemical shif t results of this experiment are l i s ted in Table 1. The data were treated, as described in the experimental section, according to the 66 method of Dahlquist and Raftery in order to obtain the dissociation - 57 -0.10 M. 0.10 M. + LYS. 0.04 M. + LYS. H Z . F R O M T F A 19 Fig. 3 F n.m.r. spectra recorded during a study of 1 and 2 (a- and 3 -anomers of N-trifluoroacetyl glucosamine) with lysozyme in pH 5.5 citrate 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 line of the trifluoroethanol standard triplet is shown to highfield. - 58 -constant, K = ^— , and the bound chemical s h i f t , A. For this purpose D KB i t was i n i t i a l l y assumed that because the g-anomer does not shift or broaden appreciably, that therefore i t does not bind to lysozyme. Making this assumption (Fig. 4A, f i l l e d circles) 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 in linewidth observed 19 for the F resonances of 1 and 2 and for the centre line of the CF^ CrL^ OH t r i p l e t . Such changes w i l l be discussed at length in Chapter III, but i t should be noted here that although the resonance of 1 broadens considerably (Table 2), that corresponding to 2 broadens negligibly and the reference remains as sharp as i t was before lysozyme was added. However, lack of a detectable chemical shift or broadening in the spectrum of an inhibitor in contact with an enzyme does not preclude the possibility of i t s binding. For one thing, the fraction of bound inhibitor i s low at any one time. Also there are several mechanisms for broadening and, as w i l l be shown in Chapter III, that operating in the case of the a-anomer (1) is 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. N-acetate resonances of the proton analogues of compounds 1 to 4. Exchange is a mechanism for broadening i f i t occurs between sites of different chemical s h i f t . In other words, the observed lack of 19 change in the F spectrum of 2 could be explained by i t s binding to lysozyme in such a way that the N-trifluoroacetyl group does not experience a very great change in magnetic f i e l d . It might in fact be i - 59 -a stronger inhibitor of lysozyme than 1 and yet appear not to bind at a l l from chemical shift and linewidth data. 66 Dahlquist and Raftery have derived equations permitting one to calculate = 1/Kg and A for two interconverting anomers, both of which can be seen to shift on binding. They had limited success in applying these equations to the compounds 5 and 6 because of the small CH 2 OH H O \ O H 5r R 1 = H R 2 = OH R 1 = OH R 2 = H Is R1 = H R 2 = OCH3 R 1 = O C H 3 R 2 = H shift 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 partial answer to this impasse was to make use of the pure a-anomer (1) by dissolving weighed amounts in thermostated solutions 19 just prior to recording their F n.m.r. spectra (see experimental section). It was then possible to monitor spectral changes during anomerization. This was done repeatedly for several i n i t i a l concentra-tions of sugar ([a Q] = 0.10 M and [O.Q] = 0.06 M) and the results were compared with the results at mutarotation equilibrium to decide whether or not the g-anomer (2) was binding effectively. Perhaps this i s best illustrated by example. Suppose an i n i t i a l concentration, -3 0.10 M, of pure a-anomer with 3 x 10 M lysozyme shows a chemical shift 6 = x Hz. As mutarotation occurs, the concentration of a-anomer - 6b -(1) w i l l drop to 0.046 M and hence the bound f ract ion, P „ , w i l l 13 increase and 6 w i l l increase ( i . e . , as shown in F ig . 4A, smaller sugar concentrations give larger sh i f ts ) . 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 sites on the enzyme and at anomeric equilibrium the number of available enzyme sites 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 shi f t 6 = x Hz w i l l remain v i r tual ly the same (note that at these sugar concentrations nearly a l l of the sugar i s in the unbound form). F ig . 4B shows the results of these mutarotation experiments. The data so obtained suffered from the fact that a f in i te 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 in the probe mutarotation took place rapidly relative to the time required to make careful measurements. Nevertheless, the data show clearly that the a-anomer (1) does undergo an upfleld shi f t during mutarotation (note that, conversely, a downfield shi f t would indicate that the g-anomer was bound more strongly than the a) . Moreover the magnitude of this mutarotation sh i f t (^2.7 Hz when [a ] = 0.06 M and ^2.1 Hz when [a ] = o o 0.10 M) i s roughly within experimental error of the sh 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 1 i s considerably larger than for 2. But from these experiments i t i s impossible to r e a l i s t i c a l l y - 61 -Fig. 4. 19T t ( m i n . ) A. Graph of " F chemical shift data from the study of 1 with lysozyme: f i l l e d circles - mutarotated mixture; X's - maximum error from the study of pure a-anomer. The Y-axis i s in units of molarity of 1. B. Increase in 6" (5') as a function of time during the mutarotation of pure a-anomer (1) to an equilibrium mixture in the presence of lysozyme: f i l l e d circles - [a ] = 0.10 M, X's - [ a j = 0.06 M. - 62 -estimate a for 2. It ij; possible to use the results of the muta-rotation studies to estimate a maximum correction to the original graph for the a-anomer. This is done in Fig. 4A: the line through the f i l l e d circles representing equilibrium data, and the line through the "X's" indicating an approximate maximum correction to the pure oranomer data. As shown in Fig. 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 this w i l l change for 1 from 0.0103 M to roughly 0.012 M. However, in view of the error limits involved in the mutarotation studies (and indeed in the technique i t s e l f ) such a correction is not warranted. It should be emphasized here that there remains the possibility that the g-anomer is binding to the enzyme in such a way that i t neither shifts on binding nor interferes with the binding of the a-anomer or that i t binds non-competitively in the slow exchange limit. Lysozyme and the Methyl Glycosides of N-Trifluoroacetyl-D-glucosamine As mentioned previously, the methyl glycosides (3 and 4) were made as part of the series of fluorinated compounds. These were purified and separated by column chromatography on s i l i c a gel (see C H 2 O H O, N H C O C F 3 3/ R 1 = H R 2 = O C H 3 4/ R-, = O C H 3 R 2 = H 19 Table 1. F Chemical Shift Data for N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl _3 Glycosides (3 and 4) in the Presence of Lysozyme (3 x 10 M) Total Sugar Concentration [io3 (M) Chemical Shift from Trifluoroethanol (Hz) Changes in Shift for 1 6 (Hz) 1/6 (Hz ) 0.10 (no lys.) 129.85 0.10 125.41 0.08 124.72 0.06 123.45 0.04 121.55 0.02 117.88 102.96 0 103.21 4.57 0.219 103.41 5.27 0.190 103.47 6.53 0.153 103.57 8.43 0.119 103.60 12.11 0.0826 Sugar Concentration [I Q] (M) Chemical Shift from Trifluoroethanol (Hz) 0.10 0.10 0.08 0.06 0.04 0.02 (no lys.) 130.95 131.43 131.40 131.46 131.65 131.75 Sugar Concentration [I Q] 00 Chemical Shift from Trifluoroethanol (Hz) 4 0.10 0.10 0.08 0.06 0.04 0.02 (no lys.) 99.82 100.20 100.68 100.28 100.45 100.35 - 64 -Table 2. Linewidth Data for the N-Trifluoroacetate resonances of N-Trifluoroacetyl-D-glucosamine (1 and 2) and i t s Methyl _3 Glycosides (3 and 4) in the Presence of Lysozyme (3 x 10 M) Total Sugar Concentration [I ] (M) o Transition Linewidth at Half Height 1 2 Standard 0.10 (no lys.) 0.70 0.70 0.60 0.10 1.10 0.75 0.60 0.08 1.15 0.76 0.61 0.06 1.28 0.80 0.59 0.04 1.50 0.80 0.61 0.02 2.00 0.79 0.60 Sugar Transition Linewidth at Concentration Half Height  [ I o ] ( M ) 3 Standard 0.10 (no lys.) Q.10 0.08 0.06 0.04 0.02 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 Standard 0.10 (no lys.) 0.69 0.48 0.10 0.74 0.49 0.08 0.77 0.49 0,0.6 0.75 0.50 0.04 0.75 0.49 0.02 0.75 0.50 - 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 shift relative to the internal standard, trifluoroethanol, while varying the concentration of sugar from 0.10 to 0.02 M) in the presence of a constant concentration of enzyme (3 x ^3 10 M). The chemical shift results are tabulated (Table 1) with those for the free sugar. The chemical shift data for 3 and 4 is very similar to that for 2. That i s , there is only a very slight (~Q.5 Hz) downfield shift as compared to the 12.0 Hz upfield shift of the a-anomer, 1, This similarity amongst 2, 3 and 4 also shows up in the line broadening data: in each case a very slight broadening (ca. 0.1 Hz) is apparent over the total range of concentration data (Table 2) compared to a broadening of well over 1.0 Hz for 1. The standard (trifluoroethanol) can be seen not to broaden detectably. In the case of the glycosides i t is 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 inhibitor,1. The known inhibitor, 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 later in this chapter) and found i t to be 22.9 M _ 1 (K^ = 0.0437 M) . Th,is inhibitor is assumed to bind at subsite C."^ ^ The experiment performed in each case invplved the same approach. 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 Shifts (A) for Compounds 1 to 4 K D (M) , -1 . Compound K B (M ) A(ppm) 1 0.0103 96.7 0.947 2 >>0.0103 <<96.7 3 >0.0437 <22.9 ^0 4 >0.0437 <22.9 ' ^0 Competition of the N-Trifluoroacetyl-a- and g-methyl Glycosides (3 and 4) for Subsite C as Indicated by the N-Acetyl Proton Chemical Shift of 7 Concentration [E ] (M) Other Sugar Observed Shift of 7 (M) u Added ( 6 ) for 7 (Hz) 0.49 0.003 none 2.2 T I Cpd. 3 M).49 M 1.7 I I I I none 2.2 I I I I Cpd. 4 <v0.49 M 1.5 - 67 -concentration of the sugar, 7, and enzyme plus an equal concentration of either 3 or 4. In each case the observed shift, 6 , was seen to decrease by roughly 1/4 (Table 3). This indicates that 3 and 4 bind to subsite C (or at least inhibit binding at subsite C) less strongly than N-acetyl-glucosamine-a-methy1 glycoside for which we have calculated = 22.9 M (literature values ^ 20 M ^ ^ ) . We have at several points mentioned the problem that i t is d i f f i c u l t to separate the phenomenon of binding at one specific site from that of a multiple equilibrium with several sites. Our 59 64—66 data cannot resolve this d i f f i c u l t y . Both Dahlquist and Raftery ' and Sykes and Parravano^ ^ have been quick to realize the problem. The best information on the subject is that provided by the X-ray work 57 58 of Blake et a l . ' which indicates that monosaccharide NAG binds at subsite C (although a-NAG binds in a different orientation from 5 8 p-NAG ). It is certainly possible to state from our results that the fluorinated analogues 1, 3 and 4 compete with normal NAG for sites on the enzyme surface - but this is not concrete proof of subsite C occupation. It seems appropriate at this point to consider our results in the ILght of present theories regarding binding of inhibitors and substrates to lysozyme. Since the original X-ray studies of Blake ^ .. 36,57,58 , e ... j . 60 * - , „ , ^ 35 et a l . a number of solution studies and further X-ray data have appeared which have to a large extent borne out the i n i t i a l claims. However, there is s t i l l uncertainty as to the actual importance of the various possible binding modes. Workers in this area have generally assumed that contributions from each of the six 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 inhibitors have been studied to date and intercomparisons permit several conclusions. It is generally accepted that the mpst important contribution to binding at subsite C arises from interactions of the acetamido group with the enzyme. In particular i t has been postulated to form hydrogen bonds between i t s NH and CO groups and 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 is a strong nonpolar association of the acetamido methyl group with the aromatic indole ring of tryptophan 108. It is of course the proximity to this latter side chain which is proposed to account for the large value of A for acetamido methyl protons of sacpharides bound at subsite C. There are in fact a total o • of 30 proposed Van der Waals contacts (< 4 A) with the enzyme for a 58 sugar residue at subsite C. Blake et a l , have reported a slightly different bound orientation for the a- and 3-anomers of NAG. They suggest that the g-anomer occupies subsite C in a manner typical 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 the NH groups of the tryptophan side chains 62 and 63 respectively. The a-anomer on the other hand is thought to be subject to the above mentioned interactions except those involving 0(6) and 0(3) and is bound in such a way as to achieve a hydrogen bond between 0(1) (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 5 8 bond v ia 0(1) but rather to bind as the 3~anomer of the free 58 sugar - Blake et a l . suggest that this is due to the glycoside methyl group's interfering s ter ica l l y with the formation of such a bond. Certainly the qualitative trend of our results (that 1 binds differently than do 2, 3 and 4 and that 2, 3 and 4 behave similar ly to one another) is in agreement with what would be predicted from known facts. However, there are several discrepancies. Perhaps the most glaring is that substitution of NHC0CF3 for NHC0CH3 has greatly 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 variation 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 for 1 and something considerably less than 96.7 M ^ for 2, 3 and 4. Average l i terature 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) is 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 i t s F analogue has disrupted (or even destroyed) certain modes of binding v ia this group ( i .e . the two hydrogen bonding sites and the Van der Waals interactions). Our previously mentioned observation that the NH proton is more readily exchanged for deuterium in the trifluoroacetate derivatives would support such a postulate. It is - 70 -already widely accepted that binding via the acetamido group of NAG derivatives accounts for most of the free energy of association at subsite C. This same lack of association of the N-trifluoroacetyl group with tryptophan 108 would explain the lack of chemical shift on binding (particularly in the case of 3 and 4 which seem to bind x^eakly at least). However, such a simple argument does not explain the relatively strong binding (for monosaccharide inhibitors) of the a-anomer, 1. If replacement of the N-acetyl group of 5 by an N-trifluoroacetyl group served only to weaken bonding via this group we would certainly not expect to observe K_, = 96.7 M An observation which may provide a clue is that the total bound chemical shift for 1 was found to be A = 95 Hz which is not much more than the proton shifts (A = 70 Hz) observed for monosaccharides binding to subsite C. This apparent 19 lack of F chemical shift sensitivity to environment may simply reflect (as with 2, 3 and 4) a lack of binding via the trifluoro-acetamido group. The fact that a shift is seen here at a l l may result from a strong 0(1) hydrogen bond to the NH of residue 109 which holds the inhibitor in position. 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 is B suggested in a recent note by Kent and Dwek^ who report high binding constants for several halogenated monosaccharide inhibitors. Since we completed our work a communication has appeared which reports the inhibition 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, 3, and 4 interacting with lysozyme and report that for 1 is 0.0091 M and A = 78 Hz (at 100 MHz). Both of these values agree within experimental error with our own. These workers also report no n.m.r. chemical shift for 2, 3, and 4 interacting with the enzyme. 72 73 Although there are few examples in the literature ' of the 19 use of F as a probe for investigating enzyme-inhibitor interactions, the technique does seem to have potential benefits. For instance, the a- and g-anomers (1,2) were well separated from one another in their spectra in contrast to their proton analogues (5,6). It 19 also seems likely that the failure of the F probe to display a large bound shi f t , A, is a peculiarity of this system as mentioned previously. Certainly the effects observed point out dramatically that in biological systems a CF bond can have specific effects which are different from those of a CH bond, and these effects may throw light on the mechanisms involved. C. Experiments with C^-Substituted N-Acetyl-D-glucosamine-a-methyl glycosides 19 In the previous section we reported studies via F n.m.r. of the interaction of lysozyme with various monosaccharide inhibitors. These studies were carried out partly to study the applicability of fluorine labels to such systems and partly in an effort to learn more about the nature of the interaction. One of the conclusions we came to was that the replacement of CH^ by CF^ in 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 in the normal fashion have been postulated to have 30 Van der Waals contacts 'and 4 hydrogen bonding sites (2 of the latter 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 in this section an attempt to guage 58 the importance of the hydrogen bonding contact postulated between 0(6) and the NH group of tryptophan side chain 62 using n.m.r. Our approach has been to synthesize a series of C^-substituted mono-5 8 saccharide inhibitors, one of which has been previously studied by X-ray diffraction and is known to bind in the 'typical' way at subsite C (compound 9), another which is assumed to bind at subsite C in the same way (compound 7), and a third which is a previously unknown inhibitor of lysozyme (compound 10). These three inhibitors are shown below: CH 2 R N H C O C H 3 - 73 -The proton spectra of each of these compounds contained sharp peaks corresponding to the N-acetyl and methoxyl groups - these were suitable for studies with lysozyme. The new inhibitor, 10, also displayed a sharp doublet corresponding to the protons and this proved to be useful as a further probe of the bound environment. It was hoped that the changes at in going from 7 to 9 to 10 would be specific in their effects as measured by K and A. Compound 10 The study of this compound's interaction with lysozyme may be taken as typical of the proton work done here. The compound i t s e l f was produced by catalytic hydrogenation at atmospheric pressure of the C,-iodo derivative 9, as described in 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 shift of sharp inhibitor 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) in pH 5.5 citrate buffer (0.1 M). Sample spectra from the experiment with 10 are shown in Fig. 5. Fig. 5A displays the condition, 6 = 0 , in which no enzyme is present. Spectral features shown are the N-acetate peak, the C^-methyl doublet and the internal reference compound, 0.025 M tertiary butanol. The large water peak was used for a field-frequency lock. 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 shift o (and broadening). When the sugar concentration is decreased to 0.04 M, the value of 6 is increased as illustrated in Fig. 5C. Note that - 74 -NAc C 5 C H 3 . Std. 0.10 M C 5 Hz 0.10 M. + LYS. 0 . 0 4 M + LYS. i r 269.9 - ' 340.4 349.0 H Z . F R O M H O D Fig. 5 H n.m.r. spectra recorded during a study of C^-methyl-N-acetyl glucosamine-a-methyl glycoside (10) with lysozyme in pH 5.5 citrate buffer (0.1 M). A. Sugar concentration 0.10 M, no enzyme -3 Sugar concentration 0.10 M, 3 x 10 M enzyme B . C . .-3 Sugar concentration 0.04 M, 3 x 10 "" M enzyme The acetate resonance, C^-methyl resonance and the tertiary butanol internal standard are shown. - 75 -the Cg protons sh i f t less than the acetyl protons. The shifts (relative to the internal standard) of both the N-acetate peak and the C^-methyl doublet were large enough to be readily measured during the course of the experiment. In F ig . 6A, 1/6 i s plotted vs. the i n i t i a l sugar concentration for both of these spectral 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 rc les) and that for the C^-methyl peak (X's) intersect at very nearly the same point. Naturally this is as should be because the intersection point, -(Kp + [ E q ] ) , should be the same for a l l peaks in the same compound as described in the experimental section. 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 is 26.4 M . The former i s probably more accurate in 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 in solution with lysozyme previously^ with the result that i t s values i for Kg and A are already in the l i terature . 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 possible. 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 in F ig . 6B and are l is ted in Table 4. Shift values were again measured relative to the internal standard, tert iary butanol. The shifts given in the table and plotted - 76 -in F ig . 6B are those observed for the N-acetyl peak. The values calculated for Kg and 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 shi f t studies because of the complexity of their spectrum. Compound 9 58 Although X-ray studies have been made of the compound, 9, interacting with lysozyme, no solution data seem to have been reported. The compound was synthesized from the a-methyl glycoside of NAG (7) v ia the C^-tosylate by displacement with Nal as described in the experimental section. This part icular compound was physically the most d i f f i c u l t to work with because of i t s low so lubi l i ty in the citrate bufer used. Partly for this reason i t was considered a good system on which to test the repeatabil ity of measurements. The results of two separate studies are shown graphically in F ig . 6C and the data is l i s ted in Tables 4 and 5. As with 7, the N-acetate peak alone was used to calculate Kg. The runs were performed in 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 internal reference, tert iary 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 60 31.5°C. The results calculated from the two separate runs are Kg 35.0 M _ 1 and A = 0.631 ppm (X's),  = 0.583 ppm ( f i l l e d circ les) and Kg = 31.8 M 1 and A = 1 (Hz1) Fig. 6 Graphs of chemical shift data for the N-acetate protons of 10, 7, and 9 ( f i l l e d circles - 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 later. - 78 -Table 4. Chemical Shift Data for the N-Acetyl Protons of Compounds 7, 9 and 10 and for the C^-Methyl of 10 Interacting with -3 Lysozyme (3 x 10 M) Compound 7 [I Q] (M) 6 (Hz) 1/6 (Hz l) .10 1.44 0.694 .08 1.70 0.588 .06 2.01 0.498 .04 2.45 0.408 .02 3.17 0.316 Compound 9 (data from two runs) [I ] (K) - 6 (Hz) 1/6 (Hz X) o .10 1.29 .775 .08 1.58 .633 .06 1.90 .526 .04 2.46 .406 .02 3.21 .312 .10 1.34 . 749 .08 1.57 .637 .06 1.95 .513 .04 . 2.43 .412 .02 3.25 .308 Compound 10 [ I o ] ( M ) 6-NHC0CH3 1/6-NHC0CH3 6-CH 3 1 / 6-CH 3 "(Hz) (Hz"1) (Hz) (Hz"1) .10 1.51 0.662 0.53 1.888 .08 1.74 0.575 0.60 1.670 .06 2.17 0.461 0.74 1.351 .04 2.62 0.382 0.87 1.149 .02 3.50 0.286 1.26 0.794 - 79 -Table 5. Dissociation Constants (K_), Binding Constants (K D) and D D Bound Chemical Shifts (A) for Compounds 7, 9 and 10 Compound K (M) IC, (M_1) A(ppm) D B 7 0.0437 22.9 0.734 9 0.0314 31.8 0.631 (data from two 0.0286 35.0 0.583 runs) 10 NHC0CH3 0.0370 27.0 0.728 Cr-CH3 0.0378 26.4 0.254 Shift of Glycoside Methyl on Binding (+ = upfield, - = downfield). Compound [ 1 ^ (M) [ E q ] (M) «_ NHC0CH 3 6 ~ O C H 3 7 0.10 0 0 0 0.05 0.003 + 2.2 - 0.2 9 0.10 0 0 0 0.05 0.003 + 2.2 + 0.2 10 0.10 0 0 0 0.05 0.003 + 2.4 - 0.1 - 80 -A further technique-checking experiment performed with this sugar was one designed to test for a sugar concentration dependence in the shift between the standard and the acetate peak. In this experiment no enzyme was added but the sugar concentration was varied over i t s f u l l range. Without lysozyme being present the chemical shift between the N-acetate peak and the internal standard (tertiary 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 potentially suitable for chemical shift 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 shift 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 is encouraging that the NHCOCH^  6 values found here f a l l exactly on graphs of data taken several months earlier. 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 inhibitors. Raftery et a l . ^ have used the N-deuteroacetyl analogue of NAG (see experimental section) for - 81 -similar problems because, although not a strong inhibitor, i t has the advantage of not displaying an interfering N-acetate resonance. It was indeed found that this compound affected 7, 9 and 10 to roughly the same extent in 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 identical and the value for 9 is not very different (perhaps not significantly different) - however, i t may indicate that the bound orientation of 9 is very slightly different from that of 7 and 10. The Kg values for the three compounds are also too similar to allow any clear distinctions. Nevertheless i t may be significant that the order of relative magnitudes, IH (compound 9) > K (compound a a 10) > K (compound 7), is 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 for 7 is not larger than K for 10 and 9. This suggests that either: D (a) hydrogen-bonding via the C^-oxygen i s unimportant or (b) there is also some potential for Van der Waals interactions involving the Cg region and that these make up for the loss of a hydrogen-bonding site. This latter possibility is attractive in view of the appreciable bound shift seen for the C^-methyl group of 10. We have already 3 mentioned that an appreciable Van der Waals interaction i s postulated - 82 -between the N-acetate methyl of NAG and the indole ring of tryptophan 108 and that i t is the aromatic f i e l d of this ring which causes the large bound shift of the N-acetate peak. There are in 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 is postulated to be involved in hydrogen-bonding to 0(6) of sugars in subsite C, and tryptophan 63 which i s postulated*^ to be involved in hydrogen-bonding to 0(3) of sugars in subsite C. The imidazole ring contains an NH group which is the proposed hydrogen-bonding moiety; Van der Waals interactions via 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 biological systems. These have been pointed out in several review papers mentioned at the beginning of this chapter. One really noteworthy advantage i s the abil i t y to investigate individual parts of complex systems (e.g. individual anomers in mixtures and individual protons in the same molecule - examples of both of which have been included in this chapter). A noteworthy drawback to n.m.r. in biological systems is the low signal-to-noise ratio of the technique. The recent introduction of Fourier transform n.m.r. spectroscopy represents a quantum 5 u mP i n t n e solution of this latter problem, but i t requires expensive equipment. The search for 19 n.m.r. probes such as F which potentially display a large bound chemical shift, A , is sensible because a large A permits the use of very dilute enzyme solutions in studies such as those performed here. - 83 -D. Conformation of Free Monosaccharide Inhibitors The binding of an i n h i b i t o r or substrate to an enzyme active s i t e i s often highly s p e c i f i c and depends, not only on the substituent groups,but also on the configuration and conformation of the molecule 74 75 involved. ' Also mechanisms for enzyme action 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 interest. The conformations i n solution of the 2-deoxy-2-amino sugars studied here have not been previously reported. Also, quite apart from determining the absolute conformations of NAG derivatives, we wanted to either confirm or rule out the p o s s i b i l i t y of conformational differences being the cause of va r i a t i o n i n and A between i n h i b i t o r s . The n.m.r. spectra of sugars i n aqueous solution are generally unsuitable for conformational studies. This i s a result of the fact that the bulk of the ring protons (often a l l but H^) f a l l close together i n the region about x 6.0 to x 6.5 (e.g. Fig. 7A). A standard method of getting around this problem i s to make the completely ()-acetylated derivative 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 ring proton spectrum over a wider energy range. I t i s then often possible to find a solvent i n which the n.m.r. spectrum i s partly or wholly analysable. The assumption i s made that the data so derived can be reasonably extrapolated to the non-acetylated species i n water. For the molecules studied here this assumption could be checked v i a the coupling J.. ~. We report here l , z the results of such a study on the completely O-acetylated derivatives of - 84 -OMe NAc 4.0 6.0 8.o r Ho H 4 OMe H 6 H 6 7 A. 5.0 H n . m . r . g l y c o s i d e 1 6.0 7.0 T B. P a r t i a l spectrum (100 MHz) of N - a c e t y l glucosamine a -methyl (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 . 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 -amine-a-methyl 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 order 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 their parent compounds as follows: CH 2 OAc N H C O C F 3 CH 2 R N H C O C H 3 The H n.m.r. spectra (100 MHz) of the ring proton regions of 3' and 4' are shown in Fig. 8A and B respectively. In each case the solvent is 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 in the spectra shown in Fig. 8 by exchanging the nitrogen proton with deuterium. This was accomplished by adding several drops of to the n.m.r. tube containing the sample dissolved in deuterochloroform-TMS and to this adding a tiny drop - 86 -Hi 5.0 5.5 6.0 6.5 T Fig. 8 Partial H n.m.r. spectra (100 MHz) of A. N-trifluoroacetyl glucosamine-a-methyl glycoside triacetate (3') and B. N-trifluoroacetyl glucosamine-g-methyl glycoside triacetate (4'), both in CDC13 solution using TMS for a field-frequency lock. - 87 -of triethylamine. For the N-tr l f luoroacetyl compounds this exchange occurred very readi ly , presumably due to the acidity of the -NH group brought about by the electronegative fluorine atoms. Both 3' and 41 show a well 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^ sp l i t t ing of the a-compound. B.^ was readily assigned in each case by INDOR experiments performed while monitoring H^. Subsequent INDOR experiments permitted a l l transitions to be assigned as shown diagrammatically in F ig . 8A and B. In each case the C^-methoxyl group occurs at highest f i e l d . H^ and H^ form a s l ight ly coupled system to lowfield. The protons form a f a i r l y highly coupled system at highf ield and H,. can be seen at x 6.02 (3') and T 6.18 (41) as an octet. The l ine positions for compounds 3' and 4' were used to calculate coupling constants and chemical shifts by means of i te rat i ve , computer-based analyses. Both compounds could be treated as seven spin systems. The data so obtained are l i s ted in Table 6. The completely O-acetylated derivatives of 7, 9 and 10 (7 ' , 91 and 10') were treated in the same way as 3' and 4 ' . For example, the ring proton spectrum of 9' i s shown in F ig . 7B. Once again the characterist ic H^ resonance can be ident i f ied (at x 5.24). ^H-{"^ H} INDOR experiments run while monitoring these transitions enabled one to pick out the B^ transit ions. The B^ transitions 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 shift to highfield. 7' and 10' were treated i n exactly the same fashion. As with the N-trifluoroacetyl compounds i t was found convenient to simplify the spectrum somewhat by exchanging the C 2 _nitrogen proton for deuterium as described earlier. 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 trifluoro-substituted group as pointed out previously. In fact a remnant of the R^ -NH coupling can be seen i n Fig. 7B as weak satellites of since the exchange was not 100%. In general the C2~nitrogen proton appeared to lowfield when not replaced by deuterium (e.g. for 10' -NH occurred at T 3.99 and the I^-NH spli t t i n g was 9.5 Hz). There i s no point in discussing each of these three compounds in detail. In each case, transitions were assigned and used as the basis of an iterative calculation (by computer) of the true coupling constants and chemical shifts (see Table 6). The proton spectra of a l l compounds 3', 4', 7', 9' and 10' were very similar with two exceptions: (a) 4' has a larger H-^-^ c o u p l i n g (axial-axial 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 for the acetates should be compared to that shown in Fig. 7A. It is the H"*" n.m.r. spectrum of 7 in D2O - obviously very l i t t l e information is available from such a spectrum. The only clearly identifiable features are H. (which suffers a degeneracy with - 8 9 -the HOD peak) at x 4.5 and the methoxyl and N-acetate peaks to highfield. 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 capillary 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 in the coupling constants ^, ^, ^, and ^. The conformations suggested by the data in Table 6 are shown below - one very important feature being that, regardless of their absolute conformation, each of the five sugars studied seems to have the same conformation as indicated by the close similarity of the values. In each case (except that of 4') J „ would suggest an equatorial-axial coupling with a dihedral angle in the neighbourhood of 60°. In the case of 4', the H -H„ relationship seems to approach axial-axial. 0 M e - 90 -Table 6. N.M.R. Parameters for the O-Acetate Derivatives 3', 4', 7', 9' and 10' Chemical Shifts (x-values) H l H2 H3 H4 H5 61 H f i 62 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 -t Coupling Constants (Hz) H1 H2 H2 H3 H3 H4 H4 H5 5 6 1 61 62 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 - -* Measured in deuterochloroform solution containing TMS. t The errors in these coupling constants are +0.2 Hz - 91 -The values of the ^-H^, H-j-H^  a n d H4~ H5 couplings ( a l l - 10 Hz) are in close accord with a trans-diaxial relationship in each case (i.e. the 180° vicinal angle indicated in the sugar diagrams). As mentioned previously, i t is not immediately obvious that the conformations arrived at for the above compounds may be meaningfully extrapolated to those of their un-acetylated parent compounds in water. However i t i s possible to check the observed splitting of H^ for a sugar dissolved in water (e.g. Fig. 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^  splittings in the neighbourhood of 3.1 Hz. At f i r s t sight this would suggest that their conformations are slightly different from those of their O-acetylated derivatives in deuterochloroform. However, as w i l l be discussed in more detail in the rext section, and H^ f a l l quite close together in the parent sugars (Fig. 7A) and are strongly coupled (J„ „ = 10-11 Hz ^ their chemical shift separation). In fact a theoretical calculation of the H^ spectrum assuming ^ = 3.6 Hz but taking into account the close coupling of H^  and H^ gives a splitting for H^ of 3.1 Hz (see Fig. 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 steric 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 for Conformation of Bound Monosaccharide  I n h i b i t o r s There has been considerable speculation concerning the r o l e of conformational changes on binding of substrates (or i n h i b i t o r s ) to enzymes. Perhaps the best known proponent of enzyme conformational changes on binding i s K o s h l a n d ^ whose "induced f i t theory" has been put forward as a mechanism of enzyme action. In the case of lysozyme -a r e l a t i v e l y small, r i g i d enzyme - X-ray work has shown that very l i t t l e 35 36 d i s t o r t i o n of the enzyme occurs on binding. ' On the other hand, the same workers have postulated that during cleavage of oligosaccharides the sugar unit bound i n subsite D of the active 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. This conclusion was drawn from molecular models. No concrete proposals have been made regarding 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 possible. In p r i n c i p l e n.m.r. chemical s h i f t s and coupling constants could be used to measure d i s t o r t i o n on binding. The l a t t e r p o s s i b i l i t y w i l l be considered here. Since, i n the fast exchange l i m i t , the i n h i b i t o r resonances represent a population-weighted average of the bound and unbound species, i t should be possible to extrapolate from the observed coupling constants to those of the t o t a l l y bound species. However, as has been emphasized previously (Chapter I ) , observed s p l i t t i n g s can only be taken as true coupling constants i n very s p e c i a l circumstances (the basic condition for t h i s assumption to be v a l i d being that a l l n u c l e i be seprated by s h i f t s large with respect to the coupling constants). - 93 -In none of the inhibitors examined here i s this true. For instance, the n.m.r. spectrum of 7 dissolved in D2O.is shown in Fig. 7A. H^ can be seen to lowfield (T 5.25) and the splitting due to coupling with E^ is clearly v i s i b l e . and are known to f a l l generally in the lowfield region of the ring proton envelope for free sugars dissolved ±1 aqueous media. Indeed INDOR experiments run while monitoring 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 is known from the last section to be 10.9 Hz. By inspection i t is possible to guess a rough chemical shift position for H^. Armed with this data i t is a simple matter to calculate theoretical spectra for the system formed by H^, E^ a n c* H^' This should give a f a i r l y reliable representation of H^ to be compared with that observable experimentally. A computer plot programme was used which actually drew out the calculated H^ spectrum. Fig. 9A shows the result of aich a computer print out for H^ when ^ = 3.6 Hz and J„ „ = 10.9 Hz (i.e., the coupling constants calculated in the previous section for 7'), and when the separation between H^  and H^ is taken as 12.0 Hz (see Table in Fig. 9). The splitting seen in the simulated spectrum is found to be 3.1 Hz. Fig. 9B shows the result of allowing H^  to shift upfield toward H^ by 1.7 Hz: the observed splitting in H^ has now become 3.0 Hz. In Fig. 9C, H^  has been allowed to shift another 1.0 Hz toward H^ with the result that the H splitting becomes 2.9 Hz. The point we have tried to make here is that rather small changes in the chemical shifts of H^  or H^ (changes which are on the scale of those expected from binding to the enzyme) can cause appreciable 10 H z C H E M I C A L SHIFT T H H; H, 1 5.253 6.103 6.223 B 5.253 6.120 6.223 5.253 6.130 6.223 D 5.253 6.183 6.223 A P P A R E N T J 1 2 Hz 3.1 3.0 2.9 A C T U A L '12 Hz 3.6 3.6 3.6 3.6 Fig. 9. Computer-simulated spectra of the 'doublet' region of a hypothetical N-acetyl glucosamine-a-methyl glycoside in 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 shift values shown above. Note that only the chemical shift of Hj is varied. - 95 -changes in the splitting observed in H^. Fig. 9D emphasizes the point by showing what happens to the 'doublet' when E^ has shifted 8.0 Hz toward H^ - obviously there i s now no simple interpretation of the H^ splitting in terms of J, „. In fact the splitting observed experi-mentally (Fig. 7A) in H^ was found to be 3.1 Hz which agrees well with the proposed arrangement of H^, E^ and H^ in Fig. 9A. In spite of what has been said above, i t would be good evidence i ! for inhibitor distortion on binding 'to see a large change i n the observed splitting of H^ . We have calculated an order of magnitude for errors l i k e l y to be introduced by chemical shift changes in E. and H^ (this error being roughly + 0.2-0.3 Hz). It is now a simple matter to calculate the changes to be expected in the H^ spli t t i n g of an inhibitor with K - 50 M and A - 0.70 ppm. J3 K - [ E I ] - 50 M"1 h ~ [ E ] [ I ] - 5 0 M where [EI] = concentration of bound inhibitor [E] = concentration of free enzyme [I] = concentration of free inhibitor i f [I] - [I ] = 0.04 M (where [I ] is the i n i t i a l inhibitor concentration) o o and [E] = 0.003 - [EI] [EI] t h 6 n (0.003-[EI])(0.04) 5 U - 96 -/. [ E I ] = 0.002 M Fraction of inhibitor bound = I = 1/20 and Fraction of inhibitor free = I = 19/20 Now in the fast exchange limit the observed s p l i t t i n g , J ^ g » is a sum of two terms: Jobs. TB Jobs.B + r F Jobs.F where ^ g - splitting in totally bound case J', „ = splitting in totally free case. obs.F ° If we assume quite a large distortion on binding so that J' _ = 6 Hz and J' _ = 3.1 Hz obs.B obs.F Then J' = (1/20) 6 + (19/20) 3.1 ODS . = 0.3 + 2.95 = 3.25 Hz It should be obvious then that to use spectral sp l i t t i n g changes to measure inhibitor distortion on binding w i l l require careful spectral analysis. Nevertheless, the technique should be viable provided the following steps are followed: (a) Obtain a high f r a c t i o n of bound i n h i b i t o r - t h i s w i l l involve d i l u t e solutions and broad l i n e s . (b) Use a time averaging technique (esp. Fourier transform) to achieve good signal-to-noise. 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 at higher temperatures) - t h i s w i l l greatly increase l i n e sharpness and accuracy, (d) Choose a system whose spectrum i s eit h e r f i r s t order or can be analysed to avoid ambiguities due to high order e f f e c t s . Another p o t e n t i a l technique f o r the i n v e s t i g a t i o n of conformational changes on binding i s one which t h i s laboratory has been working 19 toward for some years. It involves the extreme s e n s i t i v i t y of F 76 chemical s h i f t s to intramolecular e f f e c t s (such as conformation or 2 configuration changes ). For instance, i n the system studied here 19 one would observe a F substituent on the r i n g . The a b i l i t y to "noise decouple" the ring protons would be very useful i n any such project. - 98 -Experimental  General Methods (a) A l l *H n.m.r. spectra were measured with a Varian HA-100 spectrometer operating in the frequency sweep mode. For enzyme studies the water resonance was used for the field-frequency lock. Sugar solutions in CDCl^ were run locked on tetramethylsilane (TMS). A l l "^H chemical shifts are reported on the x scale. 19 (b) A l l F spectra were measured at 94.071 MHz using a Varian HA-100 spectrometer operating in the frequency sweep mode. A capillary of trifluoroacetic 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 capillary -m.p. apparatus and are corrected for thermometer error. (d) The HA-100 probe temperature was 31.5°C for a l l enzyme experiments. (e) Two different internal reference compounds x^ere used for the measurement of chemical shifts (<5) in enzyme studies: for proton work, tertiary butanol and for fluorine work, trifluoroethanol (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 in 0.1 M citrate buffer at pH 5.5 (Documenta Geigy^). (h) The technique of measuring 6 relative to the internal standards in enzyme runs was as follows. The spectrum of the standard was recorded and calibrated followed by that of the sugar resonance of interest. Then the standard spectrum was run again and the results - 99 -were averaged. This was repeated several times for each sample. (i) A l l enzyme measurements were made with the same batch of hen egg white lysozyme (Worthington Biochemical - salt-free, twice recrystallized). Solutions were made up freshly immediately prior to use. Enzyme concentrations were calculated by removing a 25 y£ aliquot and diluting to 25.00 ml with buffer. The absorbance was then read at 280 my on a double beam Bausch a Lomb Spectronic 600 78 and used to calculate [E ] from the known extinction coefficient. o 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 trifluorothiolacetate (Pierce Chemical Co.) on D-79 glucosamine in methanol as described in the literature. D-glucos-amine i t s e l f was produced in the reaction vessel from glucosamine hydrochloride (Aldrich) and sodium methoxide prior to adding the trifluorothiolacetate. The a-anomer (1) crystallized selectively 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-trifluoroacetyl glucosamine via the general method of acid catalysis in methanol using a resin (Dowex-Ag 50 W X8, Bio Rad) as catalyst. The resin in 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 starting material was dissolved in about 50 ml of dry methanol. To this x^ as added 30 ml of resin and the mixture was refluxed with stirring for 3 hours. The mixture was then fil 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 ratio). Column chromatography on s i l i c a gel (Mallinckrodt "SilicAR" CC-7), using f i r s t 10% MeOH/CHCl3 and then 20% MeOH/CHCl3 gave the pure anomers in roughly 60% yield. M.p. of 3 was 196.5-197.5°C (with decomp.), m.p. of 4 was 216.0-217.0°C (with decomp. Both were white solids. 2-Amino-2-deoxy-N-acetyl-D-glucose-a-methyl glycoside (7) was prepared from N-acetyl glucosamine (Pfanstiel Lab.) as described in 80 the literature using Dowex resin in the acid form and MeOH. Purification was achieved by means of column chromatography on s i l i c a gel using 10% MeOH/CHCl3 as eluent. The m.p. of the white solid was 192-193°C. 2-Amino-2-deoxy-N-acetyl(d3)-D-glucose was prepared by selective N-acylation of glucosamine with acetic anhydride-d^ (Merck, Sharp o 80 and Dohme) as described in the literature. 2-Amino-2-deoxy-N-acetyl-Cg-0-p-tolylsulfonyl-D-glucose-a-methyl-glycoside was produced in a fashion similar to that described in the i - 101 -literature for i t s glucose analogue. 3 . 9 g of 7 was dissolved in dry pyridine (55 ml) in a flask equipped with drying tube. 1.1 mole equivalents of p-tolylsulfonyl chloride (Eastman) was dissolved in 8 ml of dry pyridine. The solution of 7 was cooled to 0°C in a salt-ice bath. The p-tolylsulfonyl 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. The mixture was then l e f t to warm up to room temperature. After 48 hours the solvent was stripped off. The resulting o i l was dissolved in CHCl^ and washed with an aqueous solution of KHCO^ . The yield was 50-60% of a light 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 in the literature for i t s glucose analogue. The o i l obtained as described above (4 g) was dissolved in dry acetone (40 ml). 3 mole equivalents of dry Nal (Allied Chemical) was then added and the solution was refluxed with stirring for 3 hours after which the platey crystals of sodium p-tolylsulfonate were fi l t e r e d off. Workup afforded a slightly tacky powder. Purification was achieved by column chromatography on s i l i c a gel eluted with 5% MeOH/ CHCly The result was a good yield of a white solid, m.p. 162-164°C. 2-Amino-2-deoxy-N-acetyl-C^-methyl-D-glucose-a-methyl glycoside (10) was prepared by catalytic hydrogenation of 9 at atmospheric pressure. The general technique for hydrogenation of alkyl halides i s discussed - 102 -82 elsewhere. 2.0 g of 9 was dissolved In 25 ml of MeOH in a 50 ml Erlenmeyer flask. 3-4 equivalents (1 g) of solid KOH was added and 300 mg of palladium on powdered charcoal (Matheson, Coleman and Bell -10% catalyst). 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 identification via their n.m.r. They were produced by reaction with excess acetic anhydride (Baker Chemical Co.) in dry pyridine. Treatment of Data The correlation of chemical shift ( 6 ) and inhibitor concentration ([I ]) with bound chemical shift (A) and dissociation constant o 1 66 (K = ——) has been described by Dahlquist and Raftery in some D KB detail. The basic treatment is shown below for an inhibitor interacting reversibly with an enzyme. E + I y EI K = (1) [E] = enzyme concentration D [hi J [I] = inhibitor concentration [EI] = concentration of enzyme-inhibitor complex In the fast exchange limit we have: - 103 -6 = P_ A (2) where 6 is the observed shift B of a particular sugar peak from i t s unbound position and P is D the fraction of total inhibitor bound at a given time from (2) 6 = j y ^ j - A o • [EI] = [I o] | but [E] = [ E l - [EI] = [E ] - [I ] | o o o A and [I] = [ l o ] - [EI] = [I o] - [I o] | where the subscript 'o' indicates i n i t i a l concentration. Substitution into equation (1) gives D o 5 o o o A Making the following approximations that: (a) 6 << A and (b) K Q is of the order of [I ] we may drop the term — [I Q] to get: [E ]A U O O O [E J A or [ I J | ~ ~ [ V - 104 -Hence a plot of [I ] vs. 1/6 should yield a straight line of slope [E ]A and intercept -(K + [E ]). o D o CHAPTER III 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 in solution have already been discussed. Magnetic resonance methods (both e.s.r. and n.m.r.) involve relatively low energy spectroscopic processes and, hence, reflect in their spectra phenomena of great interest to the chemist. Previously in this thesis we have made use of the well known spectral features: chemical sh i f t , scalar coupling and integrated intensity. A fourth feature, the relaxation times associated with a given resonance, w i l l now be considered. Until quite recently this last feature has been used primarily by physicists and physical chemists in problems involving small molecules. The term relaxation here refers to the transfer of spin populations between nuclear energy levels as a result of their energy differential in a magnetic f i e l d . Obviously i f there is some mechanism for this transfer there w i l l be a tendency to reach a Boltzmann distribution such that a resultant macroscopic magnetic moment vector w i l l exist along the magnetic f i e l d axis. At temperatures greater than a few degrees - 106 -Kelvin, the ratio of lower to upper state population can be written N. 2yH _± = ! + o N kT T is the Boltzmann spin temperature: the spins are immersed in a thermal bath (the bulk solution) at roughly room temperature. However, there is 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 irradiation at the resonance frequency. This weak coupling arises from time-dependent local magnetic fields (the time dependence being derived from molecular motion). These fluctuating local magnetic fields to which a molecule in solution is exposed are responsible for a l l nuclear relaxation processes but there are some half dozen major mechanisms for 51 83 84 production aid interaction of such fields. ' ' Moreover there are two macroscopically observable features of relaxation processes: the ease of saturation of a transition (related to the above spin-temperature discussion) and the linewidth of the resonance corresponding to the transition (related to the nuclear spin state lifetime). In dealing with nuclear relaxation, i t i s generally convenient to consider the above-mentioned macroscopic magnetic moment which is experimentally observable and which at equilibrium li e s along the Z-axis (H -axis). The behaviour of this vector can be described by classical 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 transition in a n.m.r. spectrum there i s associated such a vector. When this vector i s disturbed from i t s equilibrium position (say by irradiation at i t s resonance frequency, oi) i t recovers exponentially at a characteristic rate upon removal of the perturbation. The time constant for i t s recovery parallel to the Z-axis is called T^ and that for i t s collapse in the XY-plane i s called T^. Experimentally both T^ and 1^ can be measured by a variety of techniques. is reflected in the linewidth of a single transition according to the relationship T^ = 1/TTAV (where Av is the linewidth at half height). Unfortunately, for most common organic molecules in solution the 'true' value of Av is obscured by f i e l d inhomogeneity 86 87 and/or small, unresolved splittings. Spin-echo methods ' and the 88 89 T 1 technique ' are more generally applicable for measurement of T„. lp z The spin-echo technique was chosen for use in this work, although both 89 other methods have been tried. T, can be measured by T, , adiabatic 1 lp 90 91a rapid passage, saturation recovery and progressive 91b saturation techniques. In this work T^ was measured by a very simple pulse technique in which a single pulse is used to invert the magnetization along the Z-axis (see Appendix A) and i t s return to equilibrium is monitored at intervals. Biological Applications Because T^ and T^ are determined by time-dependent inter- and intramolecular relationships, they contain information about relative rates of motion, orientations and distances in the molecules involved. Although the biological applications of relaxation studies are not as - 108 -numerous to date as those of chemical shift and coupling constant data, they are very important and becoming more so. A very general review 41 of the area has been included in the art i c l e by Allerhand and T r u l l . More specific review articles exist on various aspects of the use of nuclear relaxation rates to study macromolecules. In particular, Mildred Cohn has described in detail the use of paramagnetic 40 probes. For instance considerable work has been done by introducing paramagnetic species at the active site of an enzyme for enzyme-inhibitor interaction studies. The effect of the unpaired electron spins on the relaxation rates of nuclear spins i s a function of the distance between the two interacting species and of the motional freedom in the region of the paramagnetic species (the reason for using a paramagnetic probe in the f i r s t place is that unpaired electrons are extremely effective in causing rapid relaxation of neighbouring nuclei). Thus the exploitation of relaxation rate changes effected by paramagnetic probes at the active site provides the possibility of estimating interatomic distances and of mapping the substrates at the active site in solution as well as characterizing the molecular motion of highly localized regions of the active s i t e . The same approach may permit calculation of the rate of chemical exchange between the 'enzyme-bound' and 'unbound' substrate conditions. This rate is related to the rate of the f i r s t elementary step in the reaction sequence of enzyme catalysis. A well known example of the paramagnetic probe technique is the study of creatine kinase using paramagnetic 40 manganous ions and a stable nitroxide free radical. - 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 in biological systems exclusive of paramagnetic probe methods. They point out that there are two approaches whereby biological systems have been studied for kinetic data. Both approaches involve solution of the phenomenological Bloch equations for the 92 nuclear magnetization, as modified by McConnell to include the effects of chemical exchange, for a spin system where a nucleus is transferred back and forth between two distinct environments, A and B, with f i r s t order lifetimes, x^ and i . Perhaps the most obvious technique is that applicable when both sites are approximately equally populated. 44 45 51 It i s then possible to derive equations describing the lineshape ' ' for a variety of exchange lifetimes and to compare these to the observed spectrum. With this approach i t i s desirable to be able to TA TB vary the exchange lifetime, x = ( — — — ) , over a f a i r l y wide range TA TB relative to the difference in resonance frequencies (u> -uO i f this A B is non-zero or relative to the relaxation rates of the sites i f to. = A V In the system (lysozyme and inhibitor) studied in this chapter, the condition of equal site 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) is the study of helix-coil transitions 45 94 in 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 for calculation of forward and reverse exchange rate constants is applicable when - 110 -the population of one site is much greater than that of the other site. This is a common situation in enzyme-substrate and enzyme-inhibitor studies where the concentration of free substrate or inhibitor i s much larger than the concentration of bound species. In fact in these cases only one resonance is observed even in the slow exchange limit 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, spectro-meter frequency or relative population of sites A and B. In our case we have used the latter approach by varying the enzyme concentration. 45 Sykes and Scott point out that for the case of Population A >> 95 Population B ,Swift and Connick have derived a general solution to the modified Bloch equation for T^ and CJ which is valid for a l l exchange rates. A corresponding equation for T^ has been derived by Luz and Meiboom^a and by O'Reilly and Poole. 97a 97b Sykes and Sykes and Parravano have manipulated these equations into forms suitable for dealing (via n.m.r.) with the case of an enzyme-inhibitor system in which the bound magnetic environment of some portion of the inhibitor differs 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 inhibitors of lysozyme seems to be 97a 97b in this intermediate range. Sykes and Sykes and Parravano have applied their equations to the interaction 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 similar measurement on trifluoroacetyl-D-phenylalanine interacting - I l l -with a-chymotrypsin. In this chapter, amongst other uses of the audiofrequency pulse 99 technique, 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 particular interest 19 to us because we were curious as to the effects of F labels with regard to the work of Chapter II. Applications to Large Organic Molecules Although the phenomenon of nuclear relaxation is of fundamental importance to the measurement of n.m.r. spectra, and has as such received a great deal of attention from physicists, i t has been largely neglected by organic chemists. There are several good reasons for this lack of interest, of which the most cogent is that until recently, instrumentation suitable for the routine and selective measurement of the nuclear relaxation times of anything other than very simple systems has been unavailable. However, the development of the selective, audiofrequency-pulse (n.m.r.) technique by Freeman and 99 Wxttekoek has made possible, at least in principle, relaxation studies of complex organic systems. This is an extremely new area about which very l i t t l e is known. 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 in dealing with them. The work reported here (and indeed elsewhere in the literature) is only a scratch on the surface of the f i e l d of selective relaxation studies of large - 112 -organic molecules; eventually i t may be possible to relate the results of such studies to intramolecular phenomena of interest. 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 closely related simple compounds (the cis and trans isomers of 1,2-dichloroethylene, 1,2-dibromoethylene and the ethyl esters of maleic and fumaric acids). As w i l l be shown, the results of these experiments are encouraging. We have also gone on to apply similar methods to more practical examples: the measurement of proton relaxation times of various nuclei in an alkaloid and several sugars. - 113 -Results and Discussion A. A Case for the Applicability of and to Structural Organic  Chemistry Physicists and physical chemists have measured nuclear relaxation times of small molecules in attempts to elucidate the mechanisms of relaxation. ^ ' ^ ' " ^ 53,100 ^ typical approach is 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 in a solvent possessing no hydrogen mclei or in a deuterated solvent and the data extrapolated to i n f i n i t e dilution to rule out intermolecular effects. 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 willing to settle for empirical relationships. For instance, are and sensitive to changes in substituent groups or orientation and proximity of substituents? If not, a number of potential uses of pulsed n.m.r. would be closed to us. * As a f i r s t step in this direction we have measured the relaxation times of a series of substituted cis and trans isomers of ethylene: The apparatus used for pulse experiments described in this chapter (with the exception of those in Section B) was the "Mark II" pulse spectrometer (see Appendix B) designed and constructed in collabora-tion with Mr. Roland Burton of this Department. c \ R = CI 1,2-dichloroethylene (M.W. = 96.96) 1,2-dibromoethylene (M.W. = 185.88) R =. Br R = COOEt maleic (cis) and fumaric (trans) acid diethyl esters (M.W. = 172.20) Since we were interested primarily in changes in and brought about by intramolecular chemical differences rather than in 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 in the same sample - a method which is beautifully compatible with selective pulse techniques. As a typical example Fig. 1A shows the ~^H n.m.r. spectrum (100 MHz) of a mixture of the cis and trans isomers of 1,2-dichloro- and 1,2-dibromoethylenes. The solvent used was deuterochloroform with a few drops of TMS for a field-frequency lock. Although the bromo-compounds were purchased as a mixture of isomers, the concentrations of the four compounds were approximately equal (= 0.5 M). The sample was carefully 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 EtOOC B ^COOEt E tOOC Cl V - / / " " \ COOEt " \ 7 i 1. 3.0 L i n e ' as T-\ T 2 (sec.) 1 2 3 4 58.5 15.7 95.4 28.0 78.4 6.7 114.9 11.9 r 5 28.8 20.1 6 - 7.5 7 12.5 9.3 The alkene proton regions of the n.m.r. spectra (100 MHz) of degassed samples (solvent deuterochloroform) of A. 1,2-dibromo-and 1,2-dichloroethylene (cis and trans isomers) and B. maleic and fumaric acid diethyl esters (plus cis 1,2-dichloroethylene as a reference). TMS was used for the internal 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 is tabulated below the spectra. - 116 -is 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 in each case the cis anomer is to low f i e l d of the trans isomer (identification of the bromo compounds was via scalar 13 couplings in the C s a t e l l i t e s ) . It was a relatively simple matter to selectively measure T^ and for each resonance (as w i l l be discussed in more detail at the end of this section) although the separation between the chloro isomer resonances was only 6.3 Hz. The values of T^ and 1^ found are l i s t e d below Fig. 1. The error limits involved in such measurements are roughly + 10% for the T^ data but are probably considerably larger for T^ in this particular case as w i l l be discussed later in this section. The notable features of the data are that: (a) in each case T^ is considerably longer than (this d i f f e r -ence is more pronounced in the case of the chloro compounds) (b) in each case the cis 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 cis 1,2-dichloroethylene. This sample was made up in 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) prior to being sealed off. The alkene proton resonance of fumaric acid diethyl 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 in 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 species than the previous one (Fig. IA) however in each case the sample was roughly 80% CDCl^ by volume. That there were no large differences in intermolecular effects between the samples of Fig. IA and IB was checked in this case by the inclusion of some common species in each sample. The common species, or internal standard, was cis 1,2-dichloroethylene. The relaxation data for this new sample is listed below that for the f i r s t sample in Fig. 1. Each of the seven spectral transitions is numbered and i t s corresponding compound indicated above the spectrum. Note that in the second sample, for cis 1,2-dichloroethylene has remained the same as in the f i r s t sample within experimental error (however the slight increase may reflect the more dilute solution). This use of a standard permits a certain amount of data comparison between samples. Once again i t is obvious that the cis isomer has a shorter relaxation time than the trans isomer. However, in 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 later in 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 diethyl ester of maleic acid) in F ig . IB is shown in F ig . 2 together with a diagrammatic explanation of the pulse sequence. This sequence consists of a Tr/2-pulse (see Appendix A) along the X' -ax is followed by a series of TT -pulses along the Y ' -ax is of the rotating reference frame. 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 part icular trace shown is 0.2 seconds. The components of such a pulse sequence are discussed in detai l in Appendix A. Here i t suff ices to say that the Carr -Purcel l sequence removes the contribution of f i e l d inhomogeneity to the measured value of and the Gill-Meiboom modification makes less crucial 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°-pu lse and the ensuing echoes according to the exponential relationship M = -t/T M q e 2 (Appendix A) where M q i s the magnitude of the magnetization vector along the Y ' -ax is immediately after the 90°-pulse and i s the value at some later time, 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 later in this section. The sequence consisted of two pulses: the f i r s t pulse (a Tr-pulse along the X' -axis) to prepare the magnetization vector (M q ) along the - Z ' - a x i s , and a second pulse along the X' -ax is at some later time, t , to force the magnetization vector (M ) into or through 0 2 0 TIME [sec] 4 0 F i g . 2. E f f e c t of 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 Q . A . E q u i l i b r i u m ; B. i r/2 -pulse a long the X ' - a x i s ; C. B loch decay; D. ^ - p u l s e a long the Y ' - a x i s caus ing 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 . Echo; F . B loch 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 of Chapter I I I i s shown. - 120 -the observation plane (X'Y*-plane) so that the receiver c o i l could detect i t . In the set of experiments just described, this second pulse was a 27r-pulse (360°). One then simply waited for the system to return to equilibrium and then repeated the two pulse sequence using a different 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 Fig. 1A is shown in Fig. 3 together with a diagrammatic explanation. The Tr-pulse length in this case was 0.8 seconds. Note that, for convenience, 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 pair. 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 -t/T relationship M. = M (l-2e 1) where M is the i n i t i a l magnitude of t o o the magnetization vector along the -Z'-axis and is i t s magnitude at some later time, t. The results of the experiments just described and the techniques used for measurement of T^ and T^ deserve several comments. F i r s t l y , the pulse sequence used to measure T^ is more laborious than others available. For instance, since the 2u-pulse used for monitoring the magnitude of M leaves the vector in i t s original position, there should be no need to perform a T^ experiment by a series of pulse 99 pairs with waiting periods in between. Freeman and Wittekoek have pointed this out and suggested 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 to the " t r i p l e t sequence"). This technique has the drawback of requiring long, 0 50 100 150 T I M E [sec] Fig. 3 . Effect of a T^ sequence on the magnetization vector, M Q . A. Equilibrium; B. Preparatory u-pulse along the X'-axis; C . A 2TT-monitoring 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 Fig. IA of Chanter III. - 122 -frequent pulses and in 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 diffusion effects i n solution cause "phase memory loss" of the spin isochromats which can only be minimized by frequent (relative to the diffusion rate) echo generation. Nevertheless, the Carr-Purcell technique involves a minimum of pulsing. But when is considerably longer than T^, ^ c a n contribute appreciably to the 99 observed length of the spin-echo decay during pulses. Hence in the case of samples such as the halo-ethylenes, one has to choose between too many and too few pulses i n the sequence. For this reason, as mentioned earlier, the data in this case are li k e l y not as accurate as the data although they were quite reproducible within a reasonable range of pulse repetition rate. Loss of magnetization between pulses can become more serious i f a resonance contains small, unresolved 99 splittings and this could account for some of the observed difference between and for the maleic and fumaric acid derivatives where the pulse intervals have been kept long for comparison to the other compounds. It would seem desirable when = to generate frequent echoes. The very large difference between T^ and T^ in the case of the 84 halogenated species is li k e l y due to scalar coupling of the ethylene protons to the halogen quadrupole moment. The shorter relaxation times of the cis isomers relative to the trans in each case is quite possibly due to the intramolecular dipole-dipole coupling mechanism: i.e., the ethylene protons are closer together i n the cis 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 is not possible to rule out chemical shift anisotropy and spin-rotation interactions as sources of the difference. The point we wish to make is that there are differences amongst the relaxation times and they appear to be systematic. Furthermore, the techniques of dissolving samples in the same solution and of using an internal standard between different samples seem to be useful for purposes of comparison. B. Measurement of Nuclear Relaxation Times of an Alkaloid by the  Audiofrequency Pulse Technique The above experiments demonstrated that i t is possible to selectively pulse small organic molecules with long relaxation times in order to calculate T^ and using Freeman and Wittekoek's audio-99 frequency-pulse technique. We found that, in the cases studied at least, chemical changes were systematically reflected in changes in T^ and T ^ . In this section we report the use of selective pulse techniques to measure T^ and for individual protons of an alkaloid, vindoline. Vindoline was chosen to test the su 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 in collaboration with Mr. Roland Burton of this Department. - 124 -substances having the molecular complexity commonly associated with "natural products". It was of additional interest to us because of i t s relation with the anti-tumour principles obtained from Vinca rosea li n . " * " ^ The important point which we wished to establish was whether individual protons of a substance of this complexity would s t i l l have differing nuclear relaxation times. Because we were interested in biological systems where i t may often be inconvenient or undesirable to degass samples, this particular set of experiments was performed in non-degassed solution ( i t was subsequently shown that other relaxation mechanisms were so effective as to make the presence of atmospheric oxygen relatively unimportant). The normal n.m.r. spectrum of vindoline i n deuterochloroform solution (100 MHz) is shown in Fig. 4 along with a structural formula. TMS dissolved in the same sample was used to generate a signal for 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. A typical scope trace from such an experiment on the C^-acetate methyl peak is shown in Fig. 5A (7r/2-pulse duration = 0.10 sec). The echoes are seen as broad humps between the inflections 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 different 0.0 1 5 .0 10 .0 X Fig. 4. The H n ,m. r. ' spectrum (100 MHz) of the alkaloid, vindoline, in deuterochlorof orm solution using TMS for the internal field-frequency lock. A structural formula for vindoline is given and the resonances pulsed are indicated. Fig. 5 A. Scope photograph of a typical Carr-Purcell sequence on the C^-acetate resonance of vindoline. Duration of •rr/2-pulse = 0 .1 sec. B. Scope photograph of a typical T x pulse sequence on the same resonance. Tf/2-pulse duration = 0.1 sec. - 127 -Carr-Purcell sequences and so obtain sufficient points for an accept-able determination of T^. Data were found to be consistent over a range of pulse intervals. The method used to measure T^ was essentially that described in the previous section which involved a repeated series of two-pulse experiments. However, in this case,the monitoring second pulse was 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 "selectivity". Because of the short relaxation times involved here i t was not inconvenient to wait for the system to relax completely between pulse pairs. A sequence of such experiments gives the entire T^-decay envelope including the zero-point as i s illustrated with an example in Fig. 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^) values is particularly straightforward by the audiofrequency-pulse method. Thus the i n i t i a l Tf/2-pulse i s readily phase-shifted by precisely 90° and the (T n) 1 P decay detected. Unfortunately, these particular measurements appear to be sufficiently prone to interference from neighbouring lines that their general application to complex organic substances may be somewhat limited. For example, the (T.) measurement shown below 1 P . clearly f a i l s to return to the baseline as i t should due to the interference of near neighbour lines. Nevertheless there are potential advantages to the (T^)p method as mentioned previously, since the pulse power is 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 resonance of vindoline. Note the extra height of the (T,) trace 1 P (upper) and i t s failure to return to the baseline. discussed here we have adhered to those pulse methods requiring the least amount of pulse power in order to achieve high selectivity. Further work on practical samples w i l l be required in order to test the applicability of (T 1) . 1 P 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. On the other hand, the singlet due to the single proton was readily amenable to measurement of T^ and T^. Of the many spin-multiplets, only the sharp doublet due to the proton proved suitable for relaxation measurements. This doublet has a T, = 1 . 3 5 ± Q 1 5 T 2 = 1 . 4 0 ± 0 . 1 5 T , = 1 . 4 5 : T2 = \AO 0 . 1 5 0 . 1 5 d o w n f i e l d t r a n s i t i o n u p f i e l d t r a n s i t i o n H CH 30 vo OCOCH, V , 2 0 - Q 1 " T 2 = 1 . 2 0 ± 0 . 1 C Q 2 C H 3 ~n = i.5o±ai T 2 = 1 . 3 0 ± 0 . 1 T i = 1 . 1 5 ± 0 . 1 T 2 = 0 . 9 0 ± 0 . 1 Fig. 6. Relaxation time data obtained on the alkaloid, vindoline, in deuterochloroform solution, shown associated with the protons to which they correspond. Values are - 130 -spl i t t i n g of 8.0 Hz and i t was possible to perform measurements on both of the transitions. 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 necessitat-ing short pulses) effectively 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 brief comment that even though they are a l l quite short some differential s t i l l remains. As can be seen fromthe data summarized in Fig. 6, the N-methyl group has the shortest relaxation time. This might have been expected from the 84 scalar coupling effect of the nitrogen quadrupole moment. Note that both transitions of the proton have the same relaxation times, and note also that in every case T^ - T^. The problem of pulsing individual lines in a multiplet is not t r i v i a l from a theoretical viewpoint, especially i f the system involves high order coupling. 102 This has been discussed in some detail by Freeman et a l . In a l l cases studied in this work only relatively 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 in the neighbourhood of + 10% in terms of reproducibility. In conclusion then, i t seems that selective measurements of - 131 -relaxation times of individual protons of reasonably complex organic substances are feasible. The audiofrequency pulse technique is compatible with measurements of transitions separated by 5 Hz or more from a neighbouring resonance, providing that the transitions in question are sharp ( i . e . , 1^ > 3 s e c ) : for broader transitions 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 ia 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 (1) and i t s 2-chloro analogue (2). Both had been previously prepared by Dr. John 2 Manville in this laboratory and were crystal l ized from aqueous ethanol prior to use. The compounds were studied separately in C^D^ solution using TMS for an internal field-frequency lock. Each sample was carefully f i l te red and then degassed (6 freeze-pump-thaw cycles) prior to being sealed off . For purposes of intercomparison, both samples were made up to v i r tua l ly the same concentration (concentration of bromo compound, 0.20 M; concentration of chloro compound, 0.23 M) and were treated ident ica l ly . In order to confirm that the solution characteristics were the same in each case, a small amount of acetone was added to each sample prior to degassing. By measuring the relaxation time of this internal 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. Thus these substances provided a further opportunity to test the v i a b i l i t y of the audio pulse technique with "complex" organic systems. The ring proton region of the normal "4i n.m.r. spectrum of 1 in C,D, solution is shown in Fig. 7A. The identification of the various multiplets i s shown above the spectrum in Fig. 7A. I n i t i a l l y pulse experiments were performed on the resonances of the clearly resolved doublet. The method of measuring T^ was that previously discussed with reference to the alkaloid, vindoline: a series of ir-pulse, ir/2-pulse pairs interspersed with waiting periods for equilibration. The scope trace for a Carr-Purcell sequence used to measure 1^ (upper f i e l d transition #2 in Fig. 7A; TT/2 pulse duration = 0.1 sec) is shown below. - 133 -Fig. 7. Partial H n.m.r. spectra (100 MHz) of 3,4,6-tri-i0-acetyl-l-0-benzoyl-2-bromo-2-deoxy-8-D-glucopyranose (A) and i t s 2-chloro analogue (B) in deuterobenzene (degassed). Proton assignments follow those of reference 2 and are the same in each case. Transitions are numbered for discussion in the text. TMS was used for a lock signal. - 134 -I 1 1 1 1 4 S E C . Note the spikes between echoes. These are caused by interference from near-neighbour l i n e s . However, during the echoes themselves the pulse causing the interference i s shut off. As i n the case of the alkaloid experiments of the previous section, the need for high s e l e c t i v i t y necessitated the use of as long pulses as possible ( i . e . , low pulse power), and because of the short relaxation times, only a few echoes were obtained i n each experiment. Several experiments were performed on each l i n e with different delays between pulses. The values of T^  and 1^ so obtained for the resonances of H^ are listed i n Table 1 . Similar experiments were performed on the outer t r a n s i t i o n s , //3 and #6, of the H quartet (Fig. 7 A ). The inner t r a n s i t i o n s , #4 and #5, - 135 -were resolved into separate lines too close for selective pulse work. Similarly, of the four transitions making up H^ -, only the outer ones, #7 and #10, were made the subject of pulse experiments - the inner transitions, #8 and #9, were nearly degenerate. The values found for T^ and during these experiments are also lis t e d in Table 1. As observed for the alkaloid of the previous section, these relaxation times are short. Another disappointing feature of these experiments i s that i t was not possible to make satisfactory measure-ments of T^ and T^ on any of the other resonances. Even the transitions of the Hft quartet (#11-#14) were too close together for r e a l i s t i c 1 measurements. T^ for the acetone internal standard in this sample was found to be 15.2 seconds. The partial hi n.m.r. spectrum of the chloro compound (2) in CgDg is shown in Fig. 7B. The assignment is the same as that indicated f o r i Although the spectra in Fig. 7A and B are very similar, the and "quartets" are somewhat closer together in the case of the chloro compound. Pulse experiments on this compound were very similar to those on i t s bromo analogue. The transitions 6 and 7 of and respectively were deemed too close together (ca. 6 Hz) for good measurements on such broad lines (half height width _ca. 0.8-1.0 Hz due to small unresolved splittings). Nevertheless, measurements of T^ and T„ were made on transitions of Hn, H„ and H. and are listed in 2 1 3 4 Table 1 along with those for compound 1. Note that in this case the reasonably sharp peak corresponding to transitions #8 and #9 was pulsed satisfactorily. The value of T^ for the internal standard, acetone, was found to be 15.8 seconds in this second sample. This of - 136 -Table 1. Relaxation Time Data for Sugar Derivatives 1 and 2 1 Bromo analogue line // 1/T2 (sec" 1) T 2 (sec) 1/T1 (sec" 1) T1 (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 Acetone 0.0660 15.2 - -2 Chloro analogue line # 1/T2 (sec - 1) T 2 (sec) 1/T1 (sec" 1) T x (sec) 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 limit of that found for the f i r s t sample. It should be pointed out that in spin coupled systems of complex molecules each spectral line is usually broadened by unresolved splittings so that the observed value of is generally greater than that indicated by the linewidth from the relationship Av = TTT2 For instance i f is greater than 1.5 second, a single resonance would be expected to have a half-height width of less than 0.22 Hz. Allowing for a f i e l d inhomogeneity of up to 0.3 Hz, one would expect linewidths of 0.5 Hz - in fact most resonances are broader than this. At f i r s t sight the close similarity between the relaxation times of 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 axial orientation and this may be a controlling feature of the relaxation processes. It is also possible that the acetate methyl protons contribute significantly to the overall relaxation of the ring protons. Note that the values for compound 1 are closely similar to those for the corresponding protons of compound 2 (Table 1) - this is not surprising in view of the close structural similarity between the molecules. As mentioned previously, one of the penalties for 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 for the fact that in the H^  resonances of 1 and 2 (which are braoder than those of H^ and H^ ) T^ is slightly longer than T^- However the differences between T^ and even for the resonances are small relative to the + 10% experimental error involved and may arise from the spin multiplet effects mentioned by Freeman et al."**^ - 138 -Clearly i t w i l l be necessary to study other examples to see i f any differential exists between the relaxation times of protons i n more different chemical environments. In this regard a measurement technique having an even higher degree of selectivity would be desirable; i f this cannot be developed, then the potential of this area v i l l be severely restricted. It would also seem desirable i f any improvements in selectivity could be accompanied by attempts to increase the overall magnitude of the relaxation times. In this 99 regard decoupling experiments should be helpful. It is worth remarking that from the data given here i t would seem that no great differential 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 in general 102 differences may be expected. D. The Audiofrequency Pulse Technique and Enzyme-Inhibitor Rate  Constants We have just discussed to some extent the possibility of deriving structural information from n.m.r. pulse studies of 'large' organic molecules in solution. Although this area is as yet almost totally uninvestigated, there has been considerable introductory work done on the use of pulse techniques in studying rate processes involving biological systems. In particular, enzyme-inhibitor interactions have been studied via the technique of observing nuclear relaxation phenomena associated with the inhibitor. - 139 -Sykes has done excellent work in this area and has applied the technique to systems involving chymotrypsin^ and lysozyme.9^a'k 97a 97b Sykes and Sykes and Parravano have measured the rate constants, k ^ and k^, for the N-acetyl-D-glucosamine derivatives, 5-8, of the previous chapter. k = rate constant for dissociation of the enzyme-inhibitor complex k = rate constant for complex formation K = t E H i ] _ ^=1 ^ [EI] k x 19 We have already shown that F labels used in Chapter II had some effect on the binding of N-trifluoroacetylated monosaccharides to lysozyme. We were curious therefore as to the rate constants for our fluorinated inhibitors. h The exchange of a nucleus, or group of equivalent nuclei, between sites of different local magnetic environment (characterized by different resonance frequencies) is a relaxation mechanism for the nuclear spin system. If the rate of exchange is 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 affect the longitudinal relaxation time, T^. In fact this exchange mechanism 97a b has been shown ' to be the dominant relaxation mechanism of the N-acetyl protons brought about by addition of lysozyme to aqueous fi solutions of monosaccharide NAG derivatives. Of. the fluorinated NAG derivatives studied in Chapter II, only the a-anomer of the free sugar was found to show an appreciable bound chemical shift which would permit an accurate calculation of the rate constants involved. - 140 -C H 2 O H H O N H C O C F 3 R 1 = H R 2 = O H R 1= O H R 2 = H R1= H R 2 = O C H 3 R1= O C H 3 R 2 = H 97a As mentioned in the introduction, Sykes and Sykes and 97b Parravano have put the Bloch equation solutions into a form very •ence [ E I ] convenient for relating the differen between and to the rate constant, k_^. In the case where expression i s : [I] << 1 and [I] ~ [I ] the _1_ T„ T2 T l % + [ V k - l • I N .2 ( r — ) A where and are in seconds A is in radians/sec concentrations are in molarity. Hence i f and A have been calculated from chemical shift studies, i t is a relatively simple matter to arrive at k_^ from a plot of (—— - Y~) vs. [E q] while holding [ I q] constant. In this study, and for the N-trifluoroacetyl group were measured for each of four different values of [ E ] (Fig. 8A and Table 2) A l l measurements were made in 0.1 M citrate buffer at pH 5.5. The - 141 -"Mark II" pulse unit attached to a Varian HA-100 spectrometer operating at 94.071 MHz (probe temperature 31.5°C) was used to measure and 1^. A capillary of trifluoroacetic acid held concentric with the n.m.r. sample tube was used for a field-frequency lock. The total sugar concentration (0.0775 M) was the same in each sample. Hence the concentration of a-anomer (compound 1 of Chapter II) in each case was 0.0356 M. Since, as was determined Chapter II, the effect of the g-anomer on the a-anomer binding was no larger than experimental error, the effect of the g-anomer was neglected. In each case the measurement of T^ was straightforward and followed the conventions 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 Fig. 8A - solid line through X's), T^ decreases only slightly with increasing enzyme concentration. A similar slight 97a decrease has been noted by Sykes. The measurement of was quite simple but was checked by several methods. The spin-echo method described previously was suitable for measurement of on the f i r s t two samples. But became so short on the remaining samples (Table 2) that f i e l d inhomogeneity became only a small correction to the Bloch decay generated by a 7r/2-pulse. The value of T^ obtained from the time constant for the Bloch decay of a ir/2-pulse is the same as that obtained from the expression Av = —jjj— provided there are no unresolved couplings. The actual magnitude of the f i e l d inhomogeneity correction term can be estimated from the linewidth of an internal standard for which T. is long or from the Table 2. Nuclear Relaxation Time Data for the ct- and g-Anomers of the Free Sugar, N-Trifluoroacetyl-D-Glucosamine Sample # [E ] (M) T± (sec) 1/T (sec 1) T (sec) 1/T (sec" 1) <i- - i - ) (sec" 1) T2 T l a 0 !- 8 7 0.534 1.58 0.634 0.100 3 1-82 0.549 1.52 0.658 2 a 0.95 x l O - 3 1.62 0.617 0.830 1.20 4 a 4.75 x 10 3 0.960 1.04 0.234 4.27 0.587 a o 1-32 0.755 0.364 2.75 1.994 3 2.85 x 10 ^ 1-63 0.612 1.06 0.940 3.226 [ a ] = 0.0356 M o [3 ] = 0.0419 M o Fig. 8. A. Plots of 1/T (solid line through X's) and 1/T (solid line through f i l l e d circles) vs. [E ] for the N-trifluoroacetyl group of the a-anomer. Several data points for 1/T^ (dotted line through triangles) and l/T^ (dotted line through open circles) 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 similar sample with longer T^. Neither technique is extremely accurate in the intermediate range of linewidths less than 2 Hz but more than 0.8 Hz. Fig. 8A (solid line through f i l l e d circles) clearly shows the sharp decrease in for the N-trifluoroacetyl resonance of the free sugar a-anomer upon addition of lysozyme. For purposes of comparison, several points are plotted for the g-anomer (1/T^ as dotted line through triangles; l/T^ as dotted line through open ci r c l e s ) . Fig. 8B i s a plot of (1/T - 1/T ) vs. [E ].. The slope of this 2 ^ line is equal to — — 7 — ? r and was found to be 6.72 x 10^ 1 D [ a o ] k - l sec M . The calculated values of and k^ for the a-anomer of the free sugar are 4 -1 k = 1.1 x 10 sec 1 v —1 6 —1 —1 k. = - — = 1.1 x 10 M sec 1 KD The error limits involved are at least + 30% in view of the errors in the numbers used for the calculation. It i s interesting that for the proton analogue (compound 5 of Chapter II) of the above N-trifluoro-acetate, Sykes and Parravano^^^ quote k_^ = 8.5 + 2.5 x lO^sec ^ and for k^ they report 3.5 + 1.2 x 105 molal "'"sec 1. Hence, the replacement of the N-acetyl protons by fluorines seems not to have affected k_^ but may have slightly increased k^. - 145 -Experimental General Methods 1 19 (a) A l l H and F spectra were run on the same spectrometer (Varian HA-100) and .under the same conditions as described in Chapter I and Chapter II. (b) Enzyme handling techniques were the same as those described in Chapter II. ( c ) The "Mark I" pulse unit was used for the experiments on vindoline. A l l other pulse work was done with the "Mark II" unit. Compounds The alkaloid, 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 purification. 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 recrystallized from aqueous ethanol prior to use. Pulse Unit Operation Each individual relaxation time measurement requires the sequential adjustment of the correct observing frequency, of the amount of power contained in a single pulse, and of the phase of the irradiating f i e l d . - 146 -In general i t i s convenient to set up these parameters in 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 in the fashion generally used in 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 II" pulse spectrometer i t i s necessary to make this adjustment only once since the pulse frequency is held constant. 99 Freeman and Wittekoek have described previously how (T-) experiments J- P can be used for optimising the phasing controls. We have used their method and find i t to be quite convenient providing that a (T^) experiment can be meaningfully performed on the particular sample: however, the d i f f i c u l t i e s already mentioned when a near neighbour resonance is present tend to complicate the method. The precise frequency difference between the field-frequency lock and the pulse signal needed for a particular experiment can be obtained in either of two ways. Either by direct measurement and calibration of the high resolution spectrum, or by observation of the decay occurring after a pulse. In the latter case the line position i s quickly measured to within 0.5 Hz and the frequency i s adjusted in small increments to achieve a smooth exponential decay to baseline after each pulse. The off-resonance condition is marked by the trace - 147 -of the magnetization vector either continuing through the baseline before returning to i t s equilibrium position or by a hump in the otherwise smooth decay curve. This, method is 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 particular pulse is conveniently ascertained by setting up the condition for a iT-pulse (or 2TT-pulse) ; i t is then a simple matter to arrive at the power level or pulse duration for 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 is the nuclear gyromagnetic ratio and I is the nuclear spin quantum number). Magnetic moments give nuclei certain properties characteristic of gyroscopes. For instance the nuclear spin isochromats precess about an external magnetic f i e l d , H , at a frequency characteristic of the type of nucleus: to = v H radians/sec w = Larmor freauency o ' o o •  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 (Z-axis) at equilibrium. By brie f l y applying a new magnetic f i e l d , H^, perpendicular to H , i t is possible to turn the vector M through a r o o specific angle away from the Z-axis. The resultant magnetic vector, M , w i l l then precess like the individual 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 fields can be conveniently - 149 -produced by bursts of radiation at the Larmor frequency, a ) Q . The radiofrequency magnetic f i e l d , H^, can be thought of as made up of two counter-rotating fields; one of which synchronizes with the precessing nuclear magnetic moments and the other of which has a negligible effect. Thus, i f is directed along the X'-axis i t w i l l cause a rotation of the resultant, magnetization vector in the Y'Z' plane at a frequency: u i = y H i If H, is 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 pulse power) and/or the duration of irradiation (pulse duration) to achieve varying degrees of vector rotation. In radiofrequency pulse experiments, powerful fields are generally employed: this permits one to pulse extremely broad lines and to measure very short relaxation times. In high resolution n.m.r., and are generally of the order of 1 second or longer and very homogeneous magnetic fields are employed: this permits the use of long but very selective (narrow band) pulses allowing one to measure relaxation times of individual resonances in complex spectra. The receiver c o i l of the spectrometer used here is oriented so as to detect any non-zero resultant magnetization in the X'Y' plane - and the larger that component, the larger the recorder response. The pulses employed are so weak that the magnetization vector can actually be monitored during the pulse. Fig. 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 Fig. IA shows the characteristic sharp rise in recorder response as M q is forced maximally into the X'Y' plane, followed by an exponential Bloch decay of M q along the Y'-axis. Fig. IB clearly demonstrates the effect of a 180°-pulse (n-pulse) on the same resonance after i t has been allowed to come to equilibrium: M has been forced from the positive Z'-axis into the X'Y'-plane o (giving a sharp recorder response) and then on through the X'Y' plane I I I 1 1 1 1 8 sec. Fig. 1. Scope traces of various pulses on the alkene resonance of the diethyl ester of maleic acid in the sample shown in Fig. IB of Chapter III. 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 , is 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 slight extent. This is 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 distribution -the result being that the central part of a resonance receives slightly too strong a pulse and the wings of the resonance receive slightly too weak a pulse. Fig. IC illustrates the effect of a 360°-pulse (27r-pulse) on the M vector at equilibrium: note that a maximum positive recorder response is observed as M passes through the positive Y'-axis and a maximum negative response as i t passes through the negative Y'-axis. Obviously the recorder response is zero again at the end of a '2Tr-pulse (except for the f i n i t e linewidth effect mentioned above) and the overall effect of a perfect 2-fr-pulse is negligible. A l l pulses shown in Fig. 1 were performed on the alkene resonance of diethoxy maleic acid (resonance #7 of Fig. IB of Chapter III) and were done with the same pulse power but differing pulse durations - the pulse durations being 0.2, 0.4 and 0.8 seconds in Fig. IA, B and C respectively. It should be noted that i f the pulse duration is not << T^ and T^, then an appreciable amount of decay of the vector M 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 9 9 used in 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 ig i ta l computer to control a l l pulse durations and intervals. It should be emphasized that the components which we used as the basis for the gate and other c i rcui ts were selected solely because they were readily available in our laboratory at the time we started these experiments; more versat i le , 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, is given in Fig. 2., The two major components of the system are a JEOLCO computer (model JRA-5) and a Hewlett Packard Frequency Synthesizer (model 5110B), which is driven by a "master osc i l la tor" (Hewlett-Packard, model 5105B). The principle components of the pulse-unit are summarized in F ig . 3. Br ief ly , the c i rcuit ry used here serves the following functions: -(a) In the lock-channel, i t takes a 1 MHz signal from the master * This device was designed and bui l t in collaboration with Mr. Roland Burton of this Department. - 154 -M A S T E R D I G I T A L F R E Q U E N C Y S Y N T H E S I S E R O S C I L L A T O R P U L S E U N I T O R A - 5 C O M P U T E R V - 4 3 5 4 L O C K — B O X J L V A R I A N L O C K S Y S T E M M O D U L A T I O N C O I L S R E C E I V E R C O I L S V - 4 3 1 1 R E C E I V E R P A R - 121 L O C K —IN A M P L I F I E R T E K T R O N I X - R 5 6 4 B S T O R A G E O S C I L L O S C O P E Fig. 2. Block.diagram of the components used in the "Mark I" audio-frequency-pulse spectrometer. - 155 -oscillator and reduces i t by di g i t a l division to a 2.5 KHz square wave. This signal (at 1 V p.p.) is then used in the Varian lock-box (V-4354) instead of the signal normally provided by the manual oscillator (card 910868). We have made this interchange switchable so that i t does not require physical removal of the manual oscillator card. A l l subsequent operations of the lock-circuitry are unaltered by this interchange except that lock-offsets are not possible. (b) In the observation channel the circuitry provides the audio-modulation frequency required for the detection of resonances. At the same time this channel requires provision for switching, phase shifting and f i l t e r i n g . In order to obtain a sufficiently fine (+ 0.01 Hz) control of the frequency of the observation side-band, the dig i t a l frequency synthesizer is generally run at ca. 1 MHz and the required audiofrequency (ca. 2.5 KHz) is obtained by di g i t a l division as for the lock-channel. Since the phase-shifts required in pulse experiments are integral units of 90°, these are conveniently obtained during the above division. Sequential division -r 10, •=• 10, 2, invert, v 2 gives the required audiofrequency square-wave which is used in a line A (Fig. 3) as the reference signal to the Princeton Applied Research (PAR) lock-in amplifier and. to the gate. Using the f i r s t three stages of the above sequence ( v l O , * 10,^ 2) followed by a separate T 2 stage gives a second audiofrequency square-wave in line B. This square-wave has precisely the same frequency as that of line A but differs from i t and the signal in the lock-in amplifier by a precisely 90° phase-shift. Either of these two audiofrequencies can then be connected to the modulation coils via a f i l t e r and an attenuator. The latter is a 10-turn - 156 -L O C K - C H A N N E L 1 M H z S I N E — W A V E 2 .5 K H z S Q U A R E - W A V E M A S T E R O S C I L L A T O R / - H O , - M O , - 4 / M A N U A L O S C I L L A T O R O F V - 4 3 5 4 O B S E R V A T I O N - C H A N N E L D I G I T A L F R E Q U E N C Y S Y N T H E S I S E R - H O , - M O — 2 I N V E R T E R -2 G A T E — 2 ® R E F E R E N C E I N P U T O F P A R — 121 T J R A - 5 G A T E C O M P U T E R T O D .C M O D U L A T I O N C O I L S V I A V - 4 3 9 1 F I L T E R A N D A T T E N U A T O R g. 3. Block diagram of the components used in the "Mark I" pulse unit. - 157 -200 ohm helipot. The switching of both line A and line B i s controlled by the JEOLCO JRA-5 computer which is programmed to perform the following:-(a) To accept positive integers (<32,000) from the teletype; nine of these integers may be used. Each of these controls a specific switching operation. (b) To turn on, or off, either line A or line B of the observation channel. When line A of Fig. 3 is "on" the phase-shift of the irradiating f i e l d - i s zero, and when line B is "on" the phase-shift is 90°. (c) To pause for a time-interval which is numerically equal to the integer selected in (a) above, multiplied by 10 milliseconds. It i s , of course, a t r i v i a l matter to vary the pulse-lengths, pulse-intervals, and phase sequences in any of a wide variety of ways. The spectra obtained from the pulse spectrometer are conveniently recorded on a storage oscilloscope (Tektronix, model R56A B) and a ** permanent record obtained with a homemade scope camera using Polaroid Film. "Mark II" The "Mark II" pulse spectrometer is an improved version of "Mark I". More accurately, the phase-shift between the reference signal going to the PAR-121 lock-in amplifier and the signal going to the modulation c o i l . ** Derived from a Polaroid camera (model 210). It is necessary to divide the original 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 in the original camera. The total cost of our camera was less than $50. + This device also was designed and built in collaboration with Mr. Roland Burton of this Department. - 158 -It is not only more convenient from the operator's point of view, but i t contains i t s own solid state pulse sequence generator which obviates the need for an expensive computer. The choice of pulse durations (both pulse #1 and pulse #2) is d i g i t a l l y variable over the following range: .025, .050, .10, .20, .40, .80, 1.60, oo seconds. The delay between pulse #1 and pulse #2 is continuously variable. A block diagram of the "Mark II" pulse spectrometer together with the intersystem connections required for interface with a Varian HA-100 spectrometer, is given in Fig. 4. As mentioned above, although the '-. Hewlett-Packard Frequency Synthesizer (model 5110 B) driven by a "master oscillator" (Hewlett Packard, model 5105 B) is s t i l l used as a source of stable frequencies, the JEOLCO computer has been replaced with solid state circuitry built into the pulse unit. The principle components of this new pulse-unit are summarized in Fig. 5. There are basic differences in the circuitry used for "Mark II" i as follows:-(a) The lock channel frequency is variable and hence is taken from the d i g i t a l frequency synthesizer. For high accuracy in setting the frequencies, the lock signal of ca. 2.5 KHz was obtained by digital division (see Fig. 5) of a radiofrequency in 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 oscillator (card 910868). With this arrangement any lock offset is readily achievable. (b) As in the "Mark I" unit, the observation channel circuitry provides the audio-modulation frequency required for detection of - 159 -DIGITAL FREQUENCY SYNTHESIZER MASTER OSCILLATOR MARK II PULSE UNIT V - 4 3 5 4 LOCK - BOX VARIAN LOCK SYSTEM MODULATION COILS RECEIVER COILS V-43II RECEIVER PAR-121 LOCK IN AMPLIFIER TEKTRONIX-R 5 6 4 B S T O R A G E OSCILLOSCOPE Fig. 4. Block diagram of the components used in the "Mark II" audio-frequency-pulse spectrometer. - 160 -LOCK CHANNEL D I G I T A L F R E Q U E N C Y S Y N T H E S I Z E R 1 . 5 - 3 . 5 KHz T I O , T I O , T 5 , - 2 F I L T E R S I N E - W A V E ( I V p-p) M A N U A L O S C I L L A T O R O F V 4 3 5 4 OBSERVATION CHANNEL M A S T E R O S C I L L A T O R f 2 P U L S E D U R A T I O N A N D S P A C I N G r IO, f IO r 2 I N V E R T E R r 2 I 8 0 0 >zT=90 0 r 2 T O D . C . M O D U L A T I O N C O I L S V I A V - 4 3 9 1 H E W L E T T -P A C K A R D 4 6 5 A A M P L I F I E R I N V E R T E R 1 G A T E 1 G A T E 2 F I L T E R A N D A T T E N U A T O R Fig. 5. Block diagram of the components used in the "Mark II" pulse unit. - 161 -resonances. The observation frequency of 2.5 KHz is derived by dig i t a l division of the master oscillator (1 MHz) and is fixed. Hence i t is sufficient to tune the PAR lock-in-amplifier to one frequency. The 2.5 KHz pulse #1 is obtained by a simple sequential division,-f 10, -MO, f2, il of the master oscillator frequency. Pulse #2 has exactly the same frequency as pulse #1 (see Fig. 5) but may be switched to a variety of different phase relations to pulse #1 (0°, 90° or 180°). This switching is 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. As with the "Mark I" unit, these pulse sequences are fed to the D.C. modulation coils and the demodulated output of the receiver c o i l (demodulated in the V-4311 receiver unit) is fed to the PAR lock-in-amplifier. The Tektronix, model R 564 B, storage oscilloscope is used to record the D.C. output. The same Polaroid camera is used to record storage scope traces. - 162 -REFERENCES 1. E . B . Baker , J . Chem. P h y s . , 37.. 911 (1962). 2. J . M a n v i l l e , P h . D . T h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1967), and re ferences t h e r e i n . 3. P . S t e i n e r , M . S c . T h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1969) , and re ferences t h e r e i n ; P h . D . T h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1971), and re ferences t h e r e i n . 4. R . B . M a l c o l m , M . S c . T h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1969), and re ferences t h e r e i n . 5 . D . ! ' . W i l l i a m s and N . 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