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Chemical and spectroscopic studies of complex organic molecules Bernstein, Michael Alec 1984

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c •< CHEMICAL AND SPECTROSCOPIC STUDIES OF COMPLEX ORGANIC MOLECULES by MICHAEL ALEC BERNSTEIN B.Sc. (Hons.), University of Cape Town, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1983 © Michael Alec Bernstein, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of C^^Ti^/  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 9e*T.; IE-6 (3/81) - i i -ABSTRACT This thesis concerns i t s e l f mainly with carbohydrates--particularly their physical properties and structural elucidation by spectroscopic methods. Within this broad area a variety of topics are pursued. The findings are divided into two sections; the f i r s t deals with several spectroscopic approaches applicable to the study of carbohydrates attached to proteins (glycoproteins) and the second concentrates on the use of recently devised NMR experiments which assist in the determination of molecular structure and conformation. The studies in Section I depend on methods for "activating" specific l o c i in glycoprotein glycans. These materials were then "tagged" with nitroxide spin-labels, which reported on the motional f l e x i b i l i t y of the glycans, which were found to be relatively mobile, 2 and hydrated. These data were corroborated in an analogous H NMR relaxation study, and this (superior) method reported very similar motional behavior to that derived from ESR. Finally in this section, the u t i l i t y of synthetic glycoproteins in the analysis of 1 3C NMR spectra of glycoproteins was studied. First a useful method for neoglycoprotein synthesis was devised, and proteins bearing homogeneous 13 and known sugars were prepared. It was determined that the C chemical shifts of the pendant sugars are comparable with those of the corresponding glycoside. Enzymic oxidation studies suggested that this approach may also greatly assist with the assignment of such carbohydrate molecules. - i i i -The second section begins with a detailed survey of recent advances in high-resolution NMR spectroscopy which may be useful in organic chemistry. The experiments are explained in a simple conceptual fashion with i l l u s t r a t i v e data for trideuteriomethyl 2,3,4,6-tetra-0-(trideuterioacetyl)-a-D-glucopyranoside given throughout. This chapter i s intended to be of assistance to the practicing organic chemist who has l i t t l e experience in the areas of two-dimensional (2D) and other multi-pulse NMR procedures. A limited number of these experiments are used extensively in later chapters. Chapter II.3 details a new procedure whereby an oligosaccharide may be characterized using NMR spectroscopy, only. The method is "de  novo" in that l i t t l e need be known about the molecule prior to analysis; i t relies on the characterization of a l l coupling pathways within each constituent monosaccharide and the determination of linkage order by inter-residue nuclear Overhauser enhancements (nOe's). To increase the chemical shift dispersion, the molecule is derivatized - here as a per-O-acetate. 13 1 The C and H NMR spectra of the plant alkaloid, brucine, were then studied. The emphasis here was on the choice and utilization of selected 1- and 2D NMR methods to determine the molecular structure and conformation of a complex organic molecule in the minimum of time. Elucidation of the f i n a l conformational detail required a strong emphasis to be placed on nOe experiments. - iv -Chapter II.4 details an NMR study on a cardenolide glycoside, digoxin. Using the strategies devised earlier, the molecule was studied at 500 MHz with the aim of complete spectral assignment. The spectrum is complicated by extensive signal overlap and the molecule's self-associative properties. For this reason, the lipophilic (steroidal) moiety displays broader lines which resist analysis by spin-echo experiments. The homonuclear and heteronuclear chemical shift correlation experiments performed well under these conditions, allowing the assignment of a l l sugar and some steroidal protons/carbons. The genin resonances proved relatively intractable, even with the powerful nOe experiment. - v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS xix CHAPTER I.1 - MOTIONAL STUDIES ON GLYCOCONJUGATES 2 1. 1 . 1 Background 2 References 12 CHAPTER 1.2 - ELECTRON SPIN RESONANCE STUDIES ON SPIN LABELLED GLYCOCONJUGATES 14 1. 2 . 1 The ESR Spin Label Method 14 1. 2 . 2 Spin Labelling of Glycoconjugates 2 0 1 . 2 . 3 Results and Discussion 2 5 1.2.4 Information Derived from Spin Label Studies of Glycoconjugates • 2 8 References 3 2 CHAPTER 1.3 - DEUTERIUM MAGNETIC RESONANCE STUDIES ON GLYCOCONJUGATES 3 4 References 4 3 CHAPTER 1.4 - NEOGLYCOPROTEINS: SYNTHESIS AND M4R STUDIES ... 44 1.4.1 Introduction 44 1.4.2 Neoglycoprotein Synthesis 46 1.4.3 C NMR of Neoglycoproteins 5 5 1.4.4 *H NMR of Neoglycoproteins 7 3 1.4.5 Conclusions • 8 0 References.. 8 2 - v i -Page CHAPTER 1.5 - EXPERIMENTAL FOR SECTION I 85 1.2 ESR Experiments 85 1.3 2H NMR 87 1.4 Neoglycoproteins 88 1.4.2 1 3C NMR 91 1.4.3 XH SEAS Experiments 92 CHAPTER II. 1 - INTRODUCTION 94 References 99 CHAPTER II.2 - HIGH RESOLUTION NMR METHODS 100 11.2.1 General Theory 100 11.2.2 Spectral Simplification 109 11.2.2.1 Via Ti in NMR Spectroscopy.... 109 11.2.2.2 Via in C NMR Spectroscopy 115 11.2.3 Spin Decoupling-Difference Spectra (SDDS) 119 11.2.4 Nuclear Overhauser Effect 125 11.2.4.1 Steady-State nOe 130 11.2.4.2 Transient Overhauser Effect (TOE) 135 11.2.4.3 Truncated Driven NOE Difference Spectroscopy 140 II. 2.5 Two-Dimensional Experiments 142 11.2.5.1 Basic Concepts 142 11.2.5.2 Homonuclear J-Modulated Spectroscopy (2D J-resolved) 154 11.2.5.3 Jeener Experiment: J-Correlated 2D NMR... 164 11.2.5.3.1 COSY 168 11.2.5.3.2 SECSY 175 11.2.5.3.3 Delayed COSY - Detection of Long-Range Couplings 178 11.2.5.3.4 "Decoupled" COSY 180 11.2.5.4 Heteronuclear Chemical Shift Correlation.. 184 11.2.5.5 Relayed Coherence Transfer 192 11.2.5.6 2D Nuclear Overhauser Enhancement Spectroscopy 194 II.2.6 Conclusions 201 References 204 - v i i -Page CHAPTER II.3 - OLIGOSACCHARIDE CHARACTERIZATION DSING NHR ... 209 References 229 CHAPTER II.4 - NMR SPECTROSCOPIC ASSIGNMENT OF BRDCINE 232 References 257 CHAPTER II. 5 - DIGOXIN 258 References 284 CHAPTER II. 6 - EXPERIMENTAL FOR SECTION II 285 - v i i i -LIST OF TABLES Table Page Chapter 1.2 1.2.1 Parameters characterizing spin label probes attached to various substrates. Percentage of possible sites spin labelled. N.D.: not determined due to low signal-to-noise 27 Chapter 1.3 2 1.3.1 Comparison of T c determined from H NMR relaxation measurements, and nitroxide ESR. a. T c in ns. b. Reductive amination of protein lysyl 6-amine groups with hexadeuterioacetone, or 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) 41 Chapter 1.4 1.4.1 Table of sugars coupled to BSA and their efficiencies. BSA bears 55, lysyl 6-amino groups per molecule. Expressed as mole/mole 52 1 3 1.4.2 Comparison of C chemical shifts of the carbohydrate resonances of neoglycoproteins with the methyl glycoside of the attached sugar. The chemical shifts of neoglycorpteins are with an error of ± 0.1 ppm. A6 represents the difference in chemical shift betwen a sugar resonance of a neoglycoprotein and i t s corresponding methyl glycoside... 62 Chapter II.2 II.2.1 An indication of the relative number of publications relating to 2D NMR in the literature, categorized by year 203 Chapter II.3 II.3.1 Comparison of expected and obtained vicinal ring proton coupling constants for (A) a 8-gluco-pyranoside, and (B) 8-galactopyranoside ring 220 - i x -T a b l e P a g e Chapter II.4 1 1 . 4 . 1 P a r a m e t e r s u s e d i n a u t o m a t e d '^ su r v e y c o n d i t i o n s " c o l l e c t i o n a n d p r o c e s s i n g o f H COSY a n d 2D J _ - r e s o l v e d e x p e r i m e n t s a t 360 MHz 2 4 0 1 1 . 4 . 2 S u m m a r i z e d *H NMR c h e m i c a l s h i f t a s s i g n m e n t s a n d c o u p l i n g c o n s t a n t s o f b r u c i n e i n CDCI3 ( c a . 0 . 0 2 M ) . 2 51 13 1 1 . 4 . 3 T a b l e o f a s s i g n e d C c h e m i c a l s h i f t s f o r b r u c i n e , c o m p a r e d w i t h t h e l i t e r a t u r e . C h e m i c a l s h i f t s a r e r e f e r e n c e d t o t h e c e n t r a l t r a n s i t i o n o f CDCI3 ( 6 7 7 . 0 ) . T h e s o l u t i o n wa s c a . 0 . 3 M i n C D C l 3 : C D 3 0 D ( 1 0 : 1 ) a n d t h e i n s t r u m e n t a B r u k e r W H - 4 0 0 , o p e r a t i n g a t 1 0 0 . 6 MHz f o r C 254 Chapter II.5 3 I I . 5 . 1 E x p e c t e d J_ v a l u e s f o r a 8 - d i g i t o x o s e s u g a r r e s i d u e 2 6 3 x -LIST OF FIGURES Figure Page Chapter 1.2 1.2.1 Solution ESR spectrum of a rapidly tumbling nitroxide spin label i l l u s t r a t i n g the measurement of parameters (From Ref. 10) 16 1.2.2 Solution ESR spectra of a nitroxide spin label as a function of molecular motion, controlled by altering solvent viscosity. (Most mobile, bottom spectrum; least mobile, top spectrum. From Ref. 3) 18 1.2.3 0- and N-glycosides of fetuin. (Frm Ref. 17) 21 1.2.4 Oxidation of gactose termini by galactose oxidase 23 1.2.5 Reductive amination of an aldehyde with TEMPAMINE, using NaCNBH 3 24 1.2.6 ESR spectra of nitroxide-labelled materials. Concentration and relative gain are given in parentheses. A. Periodate-activated fetuin (3.24 mg in 50 pi H20; gain 1). B. Galactose oxidase-activated asialo-BSM (2.67 ul In 50 ul H20; gain 6.4). C. Periodate-activated, intact erythrocytes (73 ul packed cells in phosphate-buffered saline; gain 187.5). D. Fetuin perfused with 1 and NaCNBH3 for 2 h (3.00 mg in 50 ul H20; gain 62.5) (From Ref. 14) 26 Chapter 1.3 1.3.1 Procedure for the specific deuteration of s i a l i c acid termini of glycoproteins at the C-7 position 38 1.3.2 Procedure for the specific deuteration of galactose termini of asialo-glycoprotein6 at the C-6 position 38 - xi -Figure Page 1.3.3 NMR spectra measurement at 61.4 MHz. Samples were dissolved in «* 1.5 ml H2O. (A) BSM deuterated at C-7 on s i a l i c acid residues (145 mg; 2700 transients). (B) Asialofetuin deuterated at C-6 position on terminal galactose residues (190 mg). (C) Fetuin reductively aminated with hexadeuterioacetone on lysine residues (240 mg; 550 transients). The HOD resonance was arbi t r a r i l y assigned a shift of 5 ppm. From Ref. 7 39 Chapter 1.4 1.4.1 Gray's procedure for the attachment of a reducing disaccharide to a protein (P) via i t s lysyl 6-amino groups 46 1.4.2 Neoglycoprotein synthesis protocol. From Ref. 7... 48 1.4.3 Reaction mechanism for the reductive ozonolysis of an alkene in methanol, using dimethyl sulphide.. 50 1.4.4 1 3C NMR spectra at 100.3 MHz of (A) native BSA in D2O, and (B) the denatured protein in 8 M urea/D20 56 1.4.5 1 3C NMR spectra at 100.3 MHz, showing the "carbohydrate region" only. (A) Native BSA. (B).(A), superimposed with the spectrum of methyl 6-D-glucoside, with line-widths broadened by 2 Hz. D: internal dioxane reference ( 6 67.4)... 58 13 1.4.6 C NMR spectrum of a neglycoprotein (8-glucose attached to BSA), with the "carbohydrate region" expanded and assigned. Lines aboves the resonances indicate the literature chemical shifts of methyl 6-D-glucoside 60 1.4.7 The "carbohydrate region" of the 100.3 MHz 1 3C NMR spectrum of three neoglycoproteins: (A) 6-glucose, (B) a-galactose, and (C) B-lactose attached to BSA... 61 1.4.8 The action of D-galactose oxidase on methyl a-D-galactopyranoside. 64 - x l i -Figure Page 1.4.9 1 3C NMR spectrum (100.3 MHz) of (A) methyl o-galactoside, and, (B), the same after oxidation at C-6 by g-galactose oxidate, at 20% efficiency... 65 1.4.10 The 100.3 MHz 1 3C NMR spectrum of B-lactose attached to BSA. (A) Unoxidized. (B) After oxidation by galactose oxidase overnight. (C) After reduction of (B) with NaBHi* 67 1.4.11 The 100.3 MHz 1 3C NMR spectrum of a mixture of BSA and a l l y l 6-lactoside, (A) after enzymic oxidation by galactose oxidase, and (B), after NaBH4 reduction of (A) 68 1.4.12 The 270 MHz *H spectra of BSA in D20 (14 mg/6.4 ml). (A) Single pulse experiment (B) -(F) SEAS experiments with the indicated T values. The HOD was supressed by 3 s presaturation 76 1.4.13 The 270 MHz XH SEAS spectra with i = 40 ms. (A) BSA. (B) BSA plus a l l y l B-acetoglucosaraine. (C) 8-AcetoglcNAc attached to BSA. (D) a- and 8-glcNAc attached to BSA 77 Chapter II.2 11.2.1 The laboratory (A) and rotating-reference frame (B) 102 11.2.2 A stylized AX spin system 102 11.2.3 The action of an AX spin system's vectors, (A) at equilibrium, (B) after a 90° pulse and (C) after some time to allow dephasing. (Solid line denotes A spin vectors and broken lines, X)... 104 11.2.4 Energy level diagrams of AX spin-system (A) showing transition probabilities (B). 0 and ±6 represent relative equilibrium populations 104 11.2.5 The rotating reference frame vector model of the Tj inversion-recovery experiment. Vectors are shown for a short t^  value (A,B,C,E) and a relatively long one (A,B,D,F) 110 11.2.6 Plot of z-magnetization vs time in a Tj inversion recovery experiment, indicating the null point..... 110 - x i i i -Figure Page II.2.7 400 MHz T1IR experiment on 1 (0.1 M in C6D6). The relaxation delay was 12 s, and t^  values noted next to each spectrum... 112 II.2.8 Spectral editing of 1 by T1IR. The control spectrum is C. t B 0.7s is a "methine sub-spectrum", with methylene signals nulled. _t • 2.5s gives the "methylene sub-spectrum", with methine signals partially nulled. Other parameters as In Fig. II.2.7. The asterisk marks an artifact at the carrier position 113 11.2.9 DEPT performed on 2 (0.4 M; CDC13) at 100 MHz 118 11.2.10 SDDS on 1 at 400 MHz. The irradiated proton is indicated in each case. False responses arising from Bloch-Ziegert effects are labelled "B.Z." 122 11.2.11 SDDS irradiating H-l [ ( Y B 2 / 2 H ) = 17 Hz], and varying the frequency difference (Av) between the decoupler on resonance (fixed) and the control, off-resonance (varied) 124 11.2.12 Energy levels, transition probabilities and relative populations (A) for an IS system at equilibrium, and (B) for an l{s} situation (where S is saturated) 126 11.2.13 SSN0EDS on 1 (400 MHz). The irradiated proton is indicated next to each difference spectrum. The irradiation time was 6s 134 11.2.14 (A) The equilibrium z-magnetization of 1 (H-5). The z-magnetization immediately after (B) a single selective 180° pulse, and (C) a compositive 180° pulse 136 11.2.15 Difference TOE on 1 (400 MHz), selectively inverting H-5 with a composite pulse (see text). The time, t^, between selective population inversion and total signal acquisition is given with each spectrum , 138 - x l v -Figure Page 11.2.16 Data from Fig. II.2.15, showing the nOe build-ups of H-l and H-3 with time 139 11.2.17 Schematized data matrices ill u s t r a t i n g the stages of processing of a 2D data-set 143 11.2.18 Effect of apodization functions on line-shapes in phase-sensitive (column 2) and magnitude mode (column 3) 146 11.2.19 Comparison of stacked (A) and contour plots (C). B. The Fj projection of the t i l t e d data-set. The stipled peak arises through strong coupling between 6' and 6'. Data are from the same region A of a 2D J-resolved experiment of a disaccharide.... 149 11.2.20 A. Stacked-plot of schematized 2D data set. B. Contour plot i l l u s t r a t i n g f u l l 0° and 90° projections (top and right), and "partial" 90° projections (bottom) 150 11.2.21 Vector diagram model of the 2D J-resolved experiment. With the X transitions in an AX spin system (A-E), both spins feel both pulses; in A,B,C,F,G, only spin X is subjected to the refocussing pulse (From Freeman ) 157 11.2.22 Schematized 2D j-resolved contour plot output of AX spin system without (A) and with (B) t i l t i n g 159 11.2.23 270 MHz 2D J-resolved experiment on 1. See text for details 162 11.2.24 Data taken from Fig. II.2.23B, now plotted in the contour-plot mode. Quadrature images for the intense H-2 resonance are marked with an asterisk.. 163 11.2.25 Stylized jUF^.F^) contour-plot output of an AX spin system (A) with COSY and (B) with SECSY experiments • 165 11.2.26 Plot of sin (TIJAXII) exp(-£ 1/T 2) vs. t j for J - 4.0 and 0.5 Hz 167 11.2.27 Schematized density matrix formalism of COSY experiment for an AX spin-system 172 11.2.28 COSY spectrum of 1 (270 MHz) 174 - xv -Figure Page 11.2.29 SECSY spectrum of 1 (400 MHz). The sweep-width in J_i was slightly small, causing aliasing of part of the J(4,5) connectivity; the aliased portion is marked with an asterisk......... 156 11.2.30 Delayed COSY experiment on 1 (270 MHz). A • 0.4 s. Responses from long-range couplings are shaded 179 11.2.31 Pulse scheme for the COSY experiment with homodecoupling in Fj 181 11.2.32 Decoupled COSY experiment with 1 at 270 MHz. t^ • 182 ms and A ° 0. (A), contour-plot of 2D spectrum. (B), J j projection. (C), control spectrum 183 11.2.33 Energy levels of a CH fragment at equilibrium (A) and after a proton population inversion (B). (From a review by R. Freeman, Ref. 45) 186 II.2.34 The affect of a proton 90° - t_i - 90° pulse sequence on proton vectors, and their inter-pretation in terms of proton spin state populations is given in (A) and (B). (C) maps the corresponding change in population differences across the C transitions. (Adapted from Freeman and Morris. ) 186 11.2.35 CSCM on a 0.5 M solution of 2, at 100.3 MHz for 1 3C i ~. 189 11.2.36 Steps of magnetization transfer In the relayed coherence transfer experiment 192 11.2.37 Relayed coherence transfer experiment performed on n-propanol ( H frequency 400 MHz). Peaks present in the CSCM experiment are labeled with an asterisk 194 11.2.38 Schematized NOESY experiment and vector model 196 11.2.39 2D NOESY spectrum of 1 at 400 MHz. A mixing time of 0.3 s was used, randomized ± 10% 200 - x v i -Figure Page Chapter II.3 11.3.1 With a 6(1-4) glycosidic linkage, irradiation of the glycosidic proton (H-l') induces intra-ring ( ) and a single inter-ring ( ) nOe 215 11.3.2 270 MHz spectra of (A) B-allyl lactoside In D2O, and (B) 0 - a l l y l aceto(d_6) lactoside 215 II. 3.3 Contour plot of 2D J_-resolved NMR spectrum of 2 (270 MHz; 0.2 M in CsT>e). The assigned, resolution-enhanced spectrum i s plotted above 217 11.3.4 400 MHz COSY spectrum of 2 (0.2 M in C6D6). Connectivities within the glycopyranosyl ring are shown ( ) in the top le f t half, and the galactopyranosyl ( ) in the bottom right half. A l l y l connectivities are shown ( — • — • — ) . . 218 11.3.5 400 MHz SSN0EDS of 2 (0.2 M in C6D 6). Intra-ring nOe's are shown with a solid line ( ) and inter-ring nOe's with a broken line ( ) 221 11.3.6 NOESY of 2 (0.2 M in C6D6) at 400 MHz. The mixing time, = 0.75 s ± 15%. Intra-ring nOe's for the glucosyl ( ) and galactosyl ( ) rings are drawn in the bottom right half, and inter-ring ( ) in the top le f t half... 222 11.3.7 Combined NOESY (top left) and COSY (bottom right) data taken from Figs. II.3.4. and II.3.6, respectively 223 Chapter II.4 11.4.1 400 MHz *NMR spectrum of brucine (ca. 0.02 M in CDCI3), showing the assignment of non-aromatic protons 237 11.4.2 360 MHz COSY spectrum of brucine in the region between 6 4.5 and 1.0. Connectivities to the vinylic proton are indicated, though they are not on the diagram 239 11.4.3 SSNOEDS experiments performed on brucine at 400 MHz 243 - x v i i -Figure Page 11.4.4 Conformations of rings IV and V (A), VI (B), VII (C) and II (D) in brucine, indicating some of the results from the SSNOEDS experiments. The irradiated proton is indicated with (•) and nOe's ( ) 245 11.4.5 400 MHz NOESY spectrum of brucine. The 256 * IK data-matrix was collected in ca. 8 h, with relaxation delay 3.0 s, mixing time 0.6 s ± 10% and 32 scans per t± interval. The data were processed and plotted using a Nicolet 1280 computer and Zeta 8 recorder. The fin a l d i g i t i -zation was 5.8 Hz pt~ in both dimensions, after z e r o - f i l l i n g J j; the "sinebell" multiplier was used in both dimensions, and magnitude spectra calculated 248 11.4.6 The high-field region of the NOESY plot in Fig. II.4.5 249 11.4.7 360 MHz (1H) CSCM experiment with brucine (ca. 0.3 M in C6D6/CD3OD, 10:1). 6 C 22-80 and 6JJ 1.0-4.5 is plotted in the contour mode. Fj and F_£ projections are on the abcissa and ordinate, respectively 253 Chapter II.5 II.5.1 Solid-state conformations of digoxigenin (lower) and digoxin glycone (upper). (From Go et a l . ).... 260 II. 5.2 A. 500 MHz *H NMR spectrum of digoxin in DMS0-d_6. B. Same molecule following a D20 exchange. Hydroxy protons are marked (•) in A 261 11.5.3 Expansions of the low-field region of the 500 MHz H NMR spectrum of digoxin in DMS0-d_6i with a drop of D20 added 264 11.5.4 500 MHz COSY spectrum of digoxin (1) in DMS0-d6, after deuterium exchange. The HOD signal was suppressed by preirradiation, and the 512 * 1024 word data set symmetrized prior to display in the contour mode 269 11.5.5 Expansion of region A in Fig. II.5.4 270 11.5.6 Expansion of region B in Fig. II.5.4 271 - x v i i i -F i g u r e P a g e 1 1 . 5 . 7 D r i v e n nOe e x p e r i m e n t s p e r f o r m e d o n d i g o x i n , s h o w i n g n O e ' s i n d u c e d i n t o t h e m e t h y l e n e / m e t h i n e r e g i o n 2 7 3 1 1 . 5 . 8 D r i v e n nOe e x p e r i m e n t s s h o w i n g n O e ' s i n d u c e d i n t o d i g i t o x o s e H - 5 p r o t o n s b y i r r a d i a t i n g H - l ' , o r H - l " a n d H - l " » 2 7 4 1 1 . 5 . 9 D r i v e n nOe e x p e r i m e n t s w i t h s t e r o i d a l m e t h y l p e a k s i r r a d i a t e d ; t h e d i s p l a y e d r e g i o n i s t h a t o f t h e m e t h y l e n e / m e t h i n e p r o t o n s , a l t h o u g h o t h e r n O e ' s a r e e v i d e n t t o l o w e r f i e l d 276 1 1 . 5 . 1 0 CSCM e x p e r i m e n t ^ H , 3 6 0 M H z ) p e r f o r m e d o n d i g o x i n . T h e l o w - f i e l d r e g i o n s a r e p r e s e n t e d a s a s t a c k e d - p l o t 2 78 1 1 . 5 . 1 1 T h e h i g h - f i e l d r e g i o n o f t h e CSCM d a t a - s e t i n F i g . I I . 5 . 1 0 i s p l o t t e d b e t w e e n t h e f r e q u e n c y l i m i t s i n d i c a t e d 279 - xix -ACKNOWLEDGEMENT It is a pleasure to acknowledge the guidance, encouragement and friendship of Dr. L.D. Hall. It is impossible to thank a l l those who contributed to my education, but a number of individuals stand out. Work in neoglycoprotein synthesis was with the assistance of Ms. L. Darge (formerly, Evelyn), Dr. J.M. Berry and Dr. J.D. Stevens. The glycoconjugate work was assisted by Drs. P.E. Reid, J.D. Aplin, D.E. Brooks, the late Prof. C.F.A. Culling and Mr. R. Snoek. Drs. J.C. Waterton, S. Sukumar and G.A. Morris provided helpful discussions in the i n i t i a l NMR studies. It is a pleasure to thank Drs. R.E. Hurd and L.F. Johnson of Nicolet Magnetics for their contributions and use of equipment and Dr. G. Pouzard for discussions on NMR theory. Dr. W.E. Hull recorded the H NMR spectra. Finally, any instrument-oriented study such as this relies on expert technical back-up, and Mr. T. Marcus and others in the electrical shop of this department are to be thanked. Ms. E. Jensen provided fast, accurate and cheerful art-work. - 1 -SECTION I - 2 -CHAPTER 1.1 MOTIONAL STUDIES ON GLYCOCONJUGATES I.I.I Background It has been known for some time that carbohydrates are found in Nature covalently linked to proteins and l i p i d s ; 1 such molecules are commonly called "glycoproteins" and "glycolipids", respectively and collectively called glycoconjugates. Owing to the important part they play in l i f e processes, a tremendous effort has been placed in the characterization and determination of the function and structure of such molecules. In what follows here we w i l l concentrate on glycoproteins; spectroscopic studies have already played an important part in the understanding of their function, and part of the purpose of the work described in this chapter is to develop methods for chemically "tagging" specific carbohydrates to act as "reporters" on their microenvironment. The literature on glycoproteins w i l l not be reviewed in any great 2 3 detail. Recent reviews by Montreuil, and Wagh and Bahl are highly recommended to the interested reader. Although somewhat dated now, a book by Sharon** covers most topics in an "easy to read" style. Membrane glycoproteins' structure and function are insightfully reviewed by Hughes.5 Glycoproteins have been found in virtually a l l forms of l i f e , from microorganisms to man. They serve a wide range of functions, some of which we w i l l soon high-light. The contribution of carbohydrate to the total mass of a glycoprotein varies considerably; thus, lysozyme has no detectable carbohydrate, and some blood-group substances are more - 3 -than 80% carbohydrate. An important class of structural molecules, the "proteoglycans", 6 is predominantly polysaccharide with a small proportion of protein. Three classes of monosaccharides are commonly found in mammalian glycoproteins:- (1) neutral sugars, such as g-galactose (gal), g-mannose (man), g-glucose (glc), and L-fucose (fuc), (2) N -acetyl amino sugars, N-acetyl glucosamine (glcNAc) and N-acetylgalactosaraine (galNAc), and (3) acidic sugars such as the N_-acetylneuraminic acids (NANA). The neuraminic acids may be O-acetylated. Plant and microbial glycoproteins may contain deoxy sugars: g-xylose, L~arabinose and L-rhamnose. Although this diverse selection of monosaccharides in glycoproteins can, in principle, be joined in an almost infinite number of ways, consideration of the carbohydrate units and the nature of their linkage to the protein allows for a certain degree of classification. The glycans may be conjugated to the protein in two ways, and this is the f i r s t basis for classification. (1) The O-glycoproteins are O-glycosidically linked to L-serine or L-threonine units in the protein back-bone; (2) the N-glycoproteins are N-glycosidated to L-asparagine. With O-glycoproteins, the sugar residue involved in the linkage is often galNAc, and the glycan can be detached from the protein by a l k a l i treatment, through a 8-elimination reaction. N-glycoproteins involve so far only glcNAc attached to Jj-asparagine, and the glycan is usually isolated as a glycopeptide, following extentensive proteolysis; they are more abundant than O-glycoproteins. A proposed classification scheme has been put forward by Montreuil, where he draws comparisons between - A -glycoprotein glycans and the immunoglobulins; in the glycoproteins he identifies certain non-specific "invariable", or "core" arrangements to which are attached additional carbohydrates coding the "variable" fraction. The existance of certain "core" structures suggests a limited number of biosynthetic pathways. A key step in biosynthesis must be the attachment of the glycan to the protein. Thus, the amino-acid sequence around the glycan attachment has been extensively studied. N-Glycoproteins Invariably are linked to the peptide sequence Asn*-X-Thr (Ser), where Asn is asparagine, Thr, is threonine and Ser is serine. But the presence of this sequence does not guarantee glycosylation, nor does the polarity of "X" determine the nature of carbohydrate units attached. Such an invariant amino-acid sequence i s not clear with fj-glycoproteins. Many details of the biosynthesis of glycoproteins are 8 9 known and the elegant results are reviewed. » Structure determination methods have developed considerably. Classical "wet" chemical methods are now augmented with GLC-MS (of 1 3 methylated derivatives), enzymic (glycosidase) methods and NMR ( C and 1H). The interested reader is referred to the l i t e r a t u r e 1 0 for further details, and discussions in Ch. II.3. Whilst the fundamental aspects of glycoproteins, outlined above, have concerned some workers, others have asked why such molecules even exist. Glycoproteins were i n i t i a l l y thought to be metabolic accidents playing no important biological role. In fact, glycoproteins play two major roles. The f i r s t is physico-chemical. Glycoproteins such as the - 5 -"mucins" lubricate the gastro-intestinal tract, protecting i t from mechanical abrasion and proteolytic attack. Such viscous glycoproteins also lubricate the eye-socket and form a barrier at the cervix, preventing bacteria from entering the uterus (and abdominal cavity). Upon ovulation, the mucin changes Its physical properties so as to permit spermatazoa to enter the uterus and effect conception. The freezing point of certain Arctic fish's serum is depressed by special "anti-freeze" glycoproteins.* 1 . With a l l these glycoproteins, the integrity of the glycan i s essential for proper activity. (With the enzyme ribonuclease B, however, the glycoprotein has the same activity as the carbohydrate-free counterpart, ribonuclease A). The second major role of glycoproteins concerns molecular interactions. Here the carbohydrate units play a very Important role. For example, the ABO and Lewis blood-group classification results from 12 the constituent sugar residues of the glycan. These glycans have received wide interest and an understanding of their properties has benefitted greatly from synthetic and conformational studies performed by Leraieux 1 3 and co-workers. The influenza virus is known to agglutinate red blood-cells, unless s i a l i c acid is removed from the c e l l membranes. The removal of the carbohydrate from the hormone, human chorionic gonadotropin, attenuates i t s biological activity, but does not affect i t s immunological activity. The former implies a contribution of the carbohydrate to the biological activity, in either an active or passive way. The latter is in accord with the well-known fact that glycan - 6 -moieties of glycoproteins are usually weakly immunogenic - the obvious exception being the blood-group substances. Glycan termini, often s i a l i c acids, serve as determinants of recognition for the clearance of some plasma glycoproteins. Ashwell and Ik * Morell showed that deslalized ceruloplasmin, now bearing terminal galactose residues, is rapidly removed from circulation by the l i v e r . Although this clearance mechanism is believed to be f a i r l y general, asialo-transferrin appears to have an alternative catabolic pathway. Similarly, the presence of s i a l i c acid on circulating mammalian erythrocyte membranes Is essential for their s u r v i v a l . 1 5 The glycoproteins on cell-surfaces are believed to play an important role in the way the c e l l interacts with both small molecules in i t s surroundings and, indeed, other c e l l s . 1 6 The importance of cell-surface glycoproteins is perhaps exemplified by the changes occuring upon malignant or v i r a l transformation. The evidence for the Involvement of glycoproteins in c e l l - c e l l Interactive phenomena is far less detailed, but this topic s t i l l must be regarded as pivotal to the understanding of how cells grow to form tissues, and what processes can result in the loss of contact inhibition and the onset of uncontrolled cancerous growth. Most studies on glycoprotein functions have been phenomeno-logical. For example, Ashwell and Morell labelled ceruloplasmin with a radionuclide and injected (a) the intact (sialo-) compound, and (b) the asialo-compound into an animal, and measured the plasma h a l f - l i f e * *The time required to reduce the plasma concentration by 50%. - 7 -In each case. In that the asialoceruloplasmin had a much shorter plasma h a l f - l i f e , i t was obviously being cleared from circulation faster than the intact glycoprotein and this indicated that the s i a l i c acid guarantees the glycoprotein's circulatory survival. The radioactivity accumulated in the l i v e r , from whence the receptors for asialoceruloplasmin were later purified. The work of Ashwell and Morell is now considered a "classic" in studies on the role of the carbohydrate moiety of glycoproteins. Others, however, have focussed their attention on the ways in which these phenomena occur at the molecular level; the purpose of this section of the thesis is to investigate some of the "tools" available for these studies, and the chemistry required to make them feasible. Such studies involve spectroscopy, 1 7 and nitroxide ESR (electron spin resonance) and NMR (nuclear magnetic resonance) spectroscopy w i l l be used here. A variety of methods and procedures have been investigated, and each chapter in this section w i l l be introduced later in this discussion. F i r s t , we briefly discuss each spectroscopic method, to define i t s potential information content, and state the underlying prerequisites for i t s use. The principal useful spectroscopic techniques available are fluorescence studies, ESR and NMR. Fluorescence and ESR studies invariably require chemical attachment ("labelling") of the macromolecule with a reporter probe; NMR may u t i l i z e naturally occuring nuclides (e.g. 1H, 1 3C or 3 1 P ) , or isotopic incorporation (e.g. 2H, 1 9 F , 13 or isotope enrichment of C). - 8 -How do these methods provide information with atomic detail? The ESR of spin-labels has been used to yield different types of information, but we concentrate on just two. F i r s t l y , the "g-value" of a nitroxide spin-label - analogous to chemical shift in NMR - is an indication of the polarity of the microenvironment in which the probe i s resident. For this information to be of any value, site-specific incorporation i s essential. This Introduces an important concept underlying the studies in this chapter: chemical or enzymic methods are necessary to activate unique sites in a complex macromolecule for the derived data to be of significance. Secondly, ESR, NMR relaxation and fluorescence studies share a common feature in that they can potentially provide the "correlation time", T £ - a measure of the motional libration of the reporter group. Roughly, the correlation time i s the time the probe takes to rotate in solution through one radian, and typical values encountered here range between 10 (fast rotation) and 10 s. (medium rotation). Rather than placing too much significance on actual derived numbers, most studies rely on comparisons of upon perturbation of the system. For example, preparation of a range of fatty acids bearing reporter groups at different positions along the acyl chain, Incorporation into a model membrane system and measurement of might allow one to come to some conclusions regarding the relative mobility of different points of the chain. Incorporating the previous concept of - 9 -site-specific modification, a person wishing to study the interacion between a specific carbohydrate residue and a macromolecule to which i t i s bound might label that residue specifically and monitor any changes In x £ as the binding protein is added. One advantage of attaching a spin-, fluorescent- or isotope-probe to a molecule is that the remainder of the molecule w i l l be 8pectroscopically "silent" and interpretation of data may be greatly simplified. S t i l l , a good case can be made for the observation of a glycoprotein's NMR spectrum at natural abundance. With proton spectra, the problem li e s in the very large number of protein resonances overlapping with those of carbohydrate origin, and their assignment. Concerning the latter, a rich legacy of experimental data stems from the efforts of Vliegenthart'8 group in Holland, where the focus has been on the sequencing of glycopeptide carbohydrates. With carbon spectra, the carbohydrate resonances f a l l into a "window" of the spectrum having very few protein resonances. Thus, the resonances are easily detectable (within the usual constraints of low inherent sensitivity), but assignment i s s t i l l a problem. Although the topics of this section easily f a l l into three distinct categories, i t should be emphasized that the overlap i s considerable, at the conceptual level. In fact, many concepts share elements in common with those described in Section II. Chapter 1.2 deals with nitroxide spin-label studies on Intact and Isolated glycoproteins. Methods were developed to specifically label - 10 -carbohydrate termini of sialo- and asiologlycoproteins,* and correlation time measurements made. Similarly, red blood c e l l surface glycoproteins and glycolipids were tagged in the same way and the data compared. Whilst the spin-labelling technique has many advantages, It is 20 also fraught with uncertainties. Deuterium NMR is recognized as a reliable source of correlation times, and in Ch. 1.3 the synthetic scheme was altered for the site-specific introduction of deuterons, and correlation time measurements were made. This study was the f i r s t of it s kind in this area and, in addition to proving f e a s i b i l i t y of the technique, provided motional correlation times which compared favourably with those from the ESR studies in Ch. 1.2. Next, the question of the observation of ^•H and 1 3C NMR spectra of glycoprotein glycans was addressed (Ch. 1.4). Since most isolated glyco-proteins bear a variety of different glycans on a single protein 2 2 core, i t was decided that "synthetic glycoproteins" - neoglycoproteins - would be useful in that one could attach any (predetermined) sugar and so simplify the analysis. Several schemes for neoglycoprotein synthesis existed in the literature which failed to meet a l l the cr i t e r i a we considered necessary for a useful protocol, and we therefore developed our own. Some of these materials were analysed by 1H NMR, where a multi-pulse experiment was used to assess the possibility of differ e n t i a l l y observing carbohydrate resonances under the broad protein *It i s these carbohydrate residues which are responsible for the recognition phenomena involved in the clearance of some plasma glycoproteins and ce l l s . - 11 -envelope. With C NMR, assignments were made by comparisons of chemical shifts of the attached and unattached sugar glycoside, and chemical shift changes induced by enzymic modification. This also provided some interesting information on the requirements of the enzyme. It w i l l become evident in the following chapters that a variety of methodologies exist for the study of glycoproteins by spectroscopic methods, each with i t s particular benefits and shortcomings. The handling of biological materials also provides new technical challenges for most spectroscopists and organic chemists, but the restriction to relatively simple systems s t i l l allows conceptually important hypotheses to be tested. - 12 -REFERENCES 1. N. Sharon. Scientific American. 230 (5), 78-86 (1974); Ibid. 243 (5), 90-116 (1980). 2. J. Montreuil. Adv. Carbohydr. Chem. Biochem. 37, 157-223 (1980). 3. P.V. Wagh and O.P. Bahl. CRC C r i t . Rev. Biochem. 10, 307-377 (1981). 4. N. Sharon. Complex carbohydrates: their chemistry, biosynthesis and functions. Addison-Wesley Publishing Co., Mass. 1975. 5. R.C. Hughes. Membrane glycoproteins: a review of structure and function. Butterworths, London, 1976. 6. V.C. Hascall. Biology of carbohydrates, Vol. 1. Edited by V. Ginsburg and P. Robbins. John Wiley and Sons, New York. 1981. pp 1-49. 7. N. Parthasarathy and S.M. Bose. J. Sclent. Ind. Res. 37, 305-316 (1978) . 8. A.P. Cornfield and R. Schauer. Biol. Cellulaire. 36, 213-226 (1979) . 9. H. Schachter. The glycoconjugates, Vol. 2. Edited by M.I. Horowitz and W. Pigman. Academic Press, New York. 1978. pp 87-181. 3 10. Relevant references may be found in the review by Wagh and Bahl. 11. R.E. Feeney. Amer. Scientist. 62, 712-719 (1974). 12. See Chaps 12 and 13 in Ref. 4. 13. R.U. Lemieux. Chem. Soc. Rev. 7, 423-452 (1978). 14. G. Ashwell and A.G. Morell. Adv. Enzymol. 41, 99-128 (1974). 15. D. Aminoff, W.C. Bell, and W.G. Vorder Bruegge. Prog. Clin. Biol.  Res. 23, 569-581 (1978). 16. K.M. Yamada and J. Pouyssegur. Biochimie. 60, 1221-1233 (1978). 17. See, for example, H.C. Anderson. Ann. Rev. Biochem. 47, 359-383 (1978), and Ref. 3. 18. See, for example, A.D. Keith, M. Sharnoff, and G.E. Cohn. Biochim. Biophys. Acta. 300, 379-419 (1973), and other reviews cited in Ch. 1.2. - 13 -19. J.F.G. Vliegenthart, H. van Halbeek, and L. Dorland. Pure Appl.  Chem. 53, 45-77 (1981). 20. S. Schreler, CF. Polnaszek, and I.CP. Smith. Biochim. Biophys.  Acta. 515, 375-436 (1978). 21. A. Allerhand, K. D i l l , and W.J. Goux. NMR Biochem., Symp. Edited by S.J. Opella and P.Lu. Marcel Dekker, New York. 1978 (Pub. 1979). pp 31-50. 22. J.D. Aplin and J.C. Wriston, Jr. CRC Cr i t . Rev. Biochem. 10, 259-306 (1981). - 14 -CHAPTER 1.2 ELECTRON SPIN RESONANCE STUDIES ON SPIN LABELLED GLYCOCONJUGATES 1.2.1 The ESR Spin Label Method E l e c t r o n Spin Resonance* (ESR) i s an important spectroscopic t o o l f o r the study of b i o l o g i c a l systems; some systems n a t u r a l l y have unpaired e l e c t r o n s (e.g. those having some t r a n s i t i o n m e t a l s ) , while other r e q u i r e i n c o r p o r a t i o n of a paramagnetic r e p o r t e r group such as a "sp i n l a b e l " . ESR s p i n l a b e l l i n g s tudies hae con t r i b u t e d enormously to the understanding of such b i o l o g i c a l systems as membranes and enzymes, and a number of e x c e l l e n t reviews and monographs cover many aspects of the theory, a p p l i c a t i o n s and p r a c t i c a l problems associated w i t h the t e c h n i q u e . 1 - 1 1 Spin l a b e l s can be considered as probes, or re p o r t e r groups designed to be s p e c i f i c a l l y placed In a molecule, whose spec t r a would d e t a i l the environment of the probe and, h o p e f u l l y , that of the 12 13 molecule to which I t i s attached. E a r l y work i n our lab o r a t o r y ' s e n s i t i z e d us to the many problems and d i f f i c u l t i e s a s s o c i a t e d w i t h the approach. Discu s s i o n s l a t e r i n the chapter w i l l h i g h - l i g h t some of these i n the context of t h i s study. Spin l a b e l s have the bas i c s t r u c t u r e given below, where Rj and R 2 are chosen to protect the n i t r o x i d e f u n c t i o n a l i t y from d i s p r o p o r t i o n a t i o n , and provide a means of attachment to the molecule under study. *Also c a l l e d E l e c t r o n Paramagnetic Resonance (EPR). - 15 -o The basic resonance equation defines the large interaction of the electron spin with the laboratory magnetic f i e l d , and provides the central resonance frequency:-hv - g 0 B B 0 [1.2.1] h is Planck's constant and v the microwave frequency of the ESR spectrometer (of the order of G H z ) . B is the a constant for the electron, the Bohr magneton, Bn i s the applied magnetic f i e l d (e.g. 0.34 T) and go is the isotropic g-value of the system. The unpaired electron in a spin label i s largely confined to the N-0 group, and largely resides in a it-orbital on the nitrogen. In that N has a nuclear spin quantum number, I_ » 1, i t w i l l have three nuclear spin quantum states (m • 1, 0, -1) with which the unpaired electron may interact. This is called the electron-nuclear hyperfine interaction, and results in a three-line spectrum being observed, with the lines having equal intensity and separated by the hyperfine coupling, a (ca. 0.17 mT). Fig. 1.2.1 shows a typical ESR spectrum of a nitroxide tumbling rapidly In solution, and shows the measurements of g„, a n and peak-to-peak line widths, WQ» Hi an<* Ji-1 (corresponding to the three - 16 -labelled transitions). The recorded lines are first-derivative and require integration to yield absorption spectra. The spectrum in Fig. 1.2.1 represents isotropically averaged values of a_ and g. By "doping" a crystal of the diamagnetic analogue (reduced) of a spin label with the paramagnetic molecule and orienting i t in the applied magnetic f i e l d , an angular dependence is observed in the g-value and coupling constant (a). The extremes in g and ji l i e •i o -| - - W., 1 -Wo - -W H Fig. 1.2.1. Solution ESR spectrum of a rapidly tumbling nitroxide spin label i l l u s t r a t i n g the measurement of parameters. (From Ref. 10). along the principal molecular x, y and z axes, where the x-axis lies parallel to the N-0 bond, and the z-axis along the nitrogen 2p ( f t ) orbital. For each nitroxide, the three values of g and a_ can be measured; a > a , a , and a * ' —zz —xx —yy' —xx —yy The principal uses of nitroxide spin labels pertinent to this - 17 -chapter l i e in their a b i l i t y to report on (a) polarity (by means of a_u) and (b) molecular motion (by mean of line widths and heights). In solution, the nitroxide can be considered to be made up of the following resonance forms, with the zwitterion species favoured in a polar environment:-N 0* « • +N* 0" Fig. 1.2.2 illustrates the basic changes in line-shapes as the motion of a spin label is restricted. We have seen that a freely tumbling nitroxide spin label in a non-viscous solvent displays a symmetrical pattern of three lines of equal intensity (Fig. 1.2.1). As the motion i s further restricted (here, by increasing the solvent viscosity) i t i s seen that f i r s t the high-field line broadens, then the low-field and, fi n a l l y , the central line. Additionally, spectral features begin to appear at frequencies distant from the original ones ("outer extrema"). Ultimately, a "glass" spectrum is observed, and any further restriction of motion w i l l not be apparent in the spectrum: the ESR spin labelling technique selectively reports on motions ("correlation times") between 11 8 1 10" and 10" s rad~ - a useful range for most biological studies. The derivation of accurate correlation times from such spectra i s d i f f i c u l t , and only totally convincing when substantiated by spectral simulations. However, an approximate value i s often a l l that i s necessary when two spectra are to be compared where the introduction of a pertubatlon induces large changes in probe mobility. For such cases, 12 simplified equations may be derived and these have been used in this chapter:-- 18 -(rigid) */N^T *f<sk Mobility T / S Approx. O • , 5 0 G , ° - 1 0 0 ' C - ' ^ - / > k - ^ _ _ _ ^ - Strongly immobilized 2x l0-« -Mr— 0°C —S\j i - 3 6 ' C — — -\\\—— 8x 10" Moderately immobilized Sx IO - * Weak! > immobi lized 8 x 10 " 1 0 Freely tumbling 5 x 10" " Fig. 1.2.2. Solution ESR spectra of a nitroxide spin label as a function of molecular motion, controlled by altering solvent viscosity. (Most mobile, bottom spectrum; least mobile, top spectrum. From Ref. 3). W -1 * 6.6 * 10 -10 P.] 1/2 - 1 [1.2.2] - 19 --.1/2 i - W * 6.8 * 10 c -0 -10 [1.2.3] - i 1/2 T - W~ * 6.7 * 10 c —0 -10 + - 2 [1.2.4] Generally, xcwas calculated by a l l three equations and the average reported as the correlation time. Aside from its high signal-to-noise sensitivity (concentrations — 5 —7 between 10 and 10 M are the lower limit of detectability, depending on the line broadening) a reflection of motion and polarity, nitroxide spin labels have the further benefit that the spin concentration may be determined. Double integration of the spectrum is necessary, as i t is the area under the absorption peaks which varies linearly with concen-tration. By knowing the spin concentration, the efficacy of a chemical coupling procedure may be assessed. As mentioned previously, the spin label must provide the means for attachment to the biological substrate. Although several basic skeletons exist, the studies in this chapter involve the spin label based on the 2,2,6,6-tetramethylpiperidine-N-oxyl structure, bearing substituents at C-4. - 20 -H NH 2 x O 1 With R representing a secondary amine, the molecule is 2,2,6,6-tetra-methylpiperidine-4-amino-N-oxyl (1)*. 1.2.2 Spin Labelling of Glycoconjugates In this chapter a modest start is made towards answering the questions of how carbohydrates attached to proteins reorient in solution. 14 To il l u s t r a t e the potential of site-specific spin labelling of the carbohydrate portion of glycoconjugates, we chose a plasma glycoprotein, f e t u i n 1 5 (isolated from foetal calf serum), a mucin, 1 6 bovine submaxillary mucin (BSM, isolated from boxine submaxillary glands), and human erythrocytes (red blood cells = RBC's). In a l l cases, the goal was the labelling of the glycoconjugate sugar termini. A l l three materials are known to be rich in s i a l i c acids (N-acetyl neuraminic acid = NANA), usually at the "non-reducing" terminus, attached to gal or galNAc. Fetuin bears O-glycosides and *This is often called "TEMPAMINE" and indicated in synthetic pathways as SL-NH2. - 21 -NANA NANA NANA NANA Ja2-»-3 ja2-»3 Ja2->-3 Gal Gal Gal Gal JB1-M JB1+4 JB1+4 GlcNAc GlcNAc GlcNAc NANAa(2-*6) GalNAc jBl+3(4) JB1*2(4,6) J o Mana(l-»-2 (6) ) Mana(l-+3) Man (l->4) Gl cNAcB (l-*4) Gl cNAcBAsn Ser (Thr) Fig. 1.2.3. 0- and N-glycosides of fetuin. (From Ref. 17). N-glycosides of the form depicted in Fig. 1.2.3;17 the outer trisaccharide structure, NANA-a-(2->3)-gal-8-(l-»'4)-glcNAc, is common in animal glycoproteins. The mucins are extensively sialated, and the following sequence occurs several hundred times in each molecule: 1 7 NANA-a-(2->-6)-galNAc-a-(l-»- )-Ser-(or Thr). The exocyclic t r i o l of NANA may be fJ-acylated at one or more position. RBC's obviously present a highly complex and varied carbohydrate code to interacting species. A 18 major RBC membrane protein is glycophorin A, which is a highly sialated trans-membrane protein bearing the carbohydrate determinant for the MN blood-group classification. Although some doubt s t i l l exists as to the detailed structure of the carbohydrate moieties, the terminal sequence of NANA-a-(2-*x)-gal is known (x = 3 or 6). It has been mentioned that asialoglycoproteins* are an important sub-set of the sialoglycoproteins, as the terminal galactose marks a plasma protein for circulatory clearance and catabolism by the liv e r . With glycophorin A, terminal s i a l i c acid is essential for the exhibition •Sialoglycoproteins having been desialated in some way. - 22 -of MN blood-group activity. Thus, we recognize the need for the labelling of NANA termini of sialoglycoproteins, and galactose termini of the asialo analogues, i f their roles at the molecular level are to be monitored. Precedent exists in the literature for the generation of an aldehyde group either at NANA19 or g a l 2 0 termini and we considered this to be potentially useful. The reductive amination procedure had been 21 used to chemically modify protein lysyl 6-NH2 groups with methanal, using NaBH^ to reduce the intermediate Schiff base; Borch et a l . described 2 2 a superior reductant for this purpose, sodium cyanoborohydride (NaCNBH3). It was determined by Van Lenten and Ashwell that the exocyclic t r i o l of NANA may be selectively oxidized by periodate under mild conditions.* Strong oxidative conditions could result in the oxidative cleavage of a l l vicinal diols, thus destroying the integrity of the •Periodate is classically used in the "Smith degradation" to determine intersugar linkages in a polysaccharide. 2 3 Its reactivity towards vicinal diols varies in the order: extracyclic > intracyclic c i s - > intracyclic trans-carbohydrate chain and the specificity of the reaction. The procedure originally called for the addition of 10 moles of NalOi, per mole of s i a l i c acid, and reaction for 10 min at 0°C and pH 5.6. Later, Jourdian 24 et a l . quantified the reaction and i t is clear from their data that reaction for 35 min at 0°C yields good selectivity and oxidized NANA for isolated glycoproteins. With RBC's, the total NANA content is d i f f i c u l t to estimate, and the reaction time is accordingly reduced to 15 min. to limit possible oxidation of terminal gal residues (which are the most susceptible to cleavage, after the NANA exocyclic t r i o l ) . The oxidation of galactose is best performed enzymatlcally. Galactose oxidase (E.C. No. 1.1.3.9) effects the oxidation of the 6-CH2OH to an aldehyde (Fig. 1.2.4). The enzyme w i l l act on terminal gal or galNAc residues, and H 20 2 i s produced. Although not done in this spin-label study, i t is advisable to remove the H 20 2 as i t i s formed (with a second enzyme, catalase), as this inhibits the forward reaction of the enzyme by the classical feed-back control mechanism. Fig. 1.2.4. Oxidation of galactose termini by galactose oxidase. - 24 -To expose subtenninal galactose residues, i t i s necessary that NANA be removed; this may be effected by mild acid hydrolysis, or enzymatically using an exoglycosidase specific to NANA - neuraminidase (B.C. No. 3.2.1.18). Having generated aldehyde functionalities on NANA or gal termini, the spin label TEMPAMINE (1) was reductively aminated onto this point, using NaCNBH3. Unreacted, low molecular weight components were separated from the glycoprotein by size-exclusion chromatography (Sephadex G-25), and by exhaustive dialysis in the case of RBC's. P I" ~1 NaCNBH, • . I U c i —C. + N H 2 = [ -C=N—SL j • — C K 2 - N H - S L " SL + H 2 0 Fig. 1.2.5. Reductive amination of an aldehyde with TEMPAMINE, using NaCNBH3. Major advantages of the use of NaCNBH3 over NaBHi* include i t s resistance to aqueous hydrolysis and specificity for the intermediate Schiff base over the starting aldehyde.* * NaBH^ w i l l also reduce the aldehyde to a primary alcohol. - 25 -1.2.3 Results and Discussion Fetuin, BSM and RBC's were spin labeled at their NANA termini using the above-mentioned procedure based on specific activation under mild periodate oxidative conditions, and coupling of the amine spin label, TEMPAMINE, by reductive araination using NaCNBH3. Subterminal galactose residues of fetuin and BSM were exposed by neuraminidase treatment, and oxidized by galactose oxidase/catalase action. TEMPAMINE was then attached as before. Representative ESR spectra are given in Fig. 1.2.6. In a l l cases the level of incorporation was high enough to easily detect an ESR spectrum with milligram quantities of glycoprotein, and a partially immobilized spin label was indicated. Amplification of the high-field region (outer extrema) did not reveal any signal from a rigid component to any spectrum. Motional correlation times were determined from peak heights and widths using Eqns. 1.2.2-4. Isotropic hyperfine couplings, a_Q, were measured from spectra, and the percentage derivatization determined by double integration of the ESR signal. The relevant data are tabulated (Table 1.2.1). Control experiments involved the incubation of NH2-SL and NaCNBH3 with the unactivated glycoprotein, and Sephadex separation. Non-specific adsorption was found to contribute < 0.5% of the total signal and was not pH dependent. With the erythrocytes, spin labelling In physiological saline resulted in a minimum of c e l l l y s i s , and the spin labels were found exclusively in glycoconjugates associated with the c e l l wall, after RBC "ghosts" were prepared. The coupling reagents - 26 -F i g . 1 . 2 . 6 . E S R s p e c t r a o f n i t r o x i d e - l a b e l l e d m a t e r i a l s . C o n c e n t r a t i o n a n d r e l a t i v e g a i n a r e g i v e n i n p a r e n t h e s e s . A . P e r i o d a t e - a c t i v a t e d f e t u i n ( 3 . 2 4 mg i n 50 u l H 2 0 ; g a i n 1 ) . B . G a l a c t o s e o x i d a s e - a c t i v a t e d a s i a l o - B S M ( 2 . 6 7 u l i n 50 u l H2O; g a i n 6 . 4 ) . C . P e r i o d a t e - a c t i v a t e d , i n t a c t e r y t h r o c y t e s ( 7 3 u l p a c k e d c e l l s i n p h o s p h a t e - b u f f e r e d s a l i n e ; g a i n 1 8 7 . 5 ) . D . F e t u i n p e r f u s e d w i t h 1 a n d NaCNBH3 f o r 2 h ( 3 . 0 0 mg i n 50 u l H 2 0 ; g a i n 6 2 . 5 ) . ( F r o m R e f . 1 4 . ) Table 1.2.1 Parameters characterizing spin label probes attached to various substrates. aPercentage of possible sites spin labelled. N.D.: not determined due to low signal-to-noise. Material FETUIN ASIALO-FETUIN BSM ASIALO-BSM RBC Activation Procedure NalO^, 0°C, 35 min. Neuraminidase, galactose oxidase NalCH, 0°C 35 min. Neuraminidase, galactose oxidase NalO^, 0°C 15 rain. T c (ns) (±0.05 ns) a^ (mT) % Activation Number spins (± 0.02 mT) (±5%) per Molecule 0.79 0.52 0.35 0.49 1.0 1.68 1.68 1.68 1.67 1.65 ca. 20 50 33 N.D. 2.5 6.0 115.5 7.0 N.D. - 28 -w e r e r e a c t e d w i t h t h e a c t i v a t e d g l y c o c o n j u g a t e f o r 2 h a t r o o m - t e m p e r a t u r e - n o i n c r e a s e i n l a b e l l i n g e f f i c i e n c y was o b s e r v e d b y e x t e n s i o n o f t h i s p e r i o d . T u r n i n g now t o t h e E S R d a t a , t h e a_0 v a l u e s , a r o u n d 1 . 67 mT, i n d i c a t e a s p i n l a b e l i n a p o l a r e n v i r o n m e n t . T h i s i s c o n s i s t e n t w i t h w h a t one m i g h t e x p e c t , a s t h e ( h y d r o p h i l i c ) s u g a r r e s i d u e s w o u l d be e x p e c t e d t o be h i g h l y s o l v a t e d a n d e x p o s e d t o t h e m e d i u m . T h e c o r r e l a t i o n t i m e s i n d i c a t e a m o d e r a t e l y i m m o b i l i z e d s p i n l a b e l a n d i t i s i n t e r e s t i n g t o n o t e t h a t t h e c o r r e l a t i o n t i m e s o f f e t u i n (MW 48 0 0 0 amu) a n d BSM (MW 1 . 3 * 1 0 6 amu) s p i n l a b e l l e d a t t h e i r s u g a r t e r m i n i a r e q u i t e c o m p a r a b l e . T h i s may s u g g e s t t h a t t h e b u l k o f t h e c o n t r i b u t i o n t o t h e o b s e r v e d T c s t e m s f r o m t h e m o b i l i t y a b o u t t h e b o n d s j o i n i n g t h e s p i n l a b e l t o t h e t e r m i n a l s u g a r r e s i d u e , o r i t c o u l d be t h a t , i n b o t h c a s e s , t h e p e n d a n t s u g a r r e s i d u e s a r e e q u a l l y m o b i l e w i t h r e s p e c t t o t h e p r o t e i n b a c k - b o n e . D a t a o b t a i n e d b y H NMR ( C h a p t e r 1 . 3 ) s u p p o r t t h e s e c o n d h y p o t h e s i s . 1.2.4 Information Derived from Spin Label Studies of Glycoconjugates O u r s t u d i e s i n d i c a t e t h a t NANA o r g a l t e r m i n i o f c o m p l e x g l y c o c o n j u g a t e s may be e f f i c i e n t l y a c t i v a t e d a n d s p i n l a b e l l e d u s i n g t h e p r o c e d u r e s d i s c u s s e d . T h e s p i n l a b e l e x i s t s i n a h y d r o x y l a t e d , p o l a r e n v i r o n m e n t a n d i s m o b i l e c o m p a r e d w i t h t h e g l y c o p r o t e i n a s a w h o l e . T h e s e a d d i t i o n a l m o t i o n a l d e g r e e s - o f - f r e e d o m c o u l d r e f l e c t t h e e n h a n c e d m o b i l i t y o f t h e p e n d a n t s u g a r s r e s u l t i n g f r o m r o t a t i o n a b o u t g l y c o s i d i c b o n d s , o r c o u l d be d o m i n a t e d b y m o t i o n a b o u t t h e c h e m i c a l b o n d s l i n k i n g t h e n i t r o x i d e s p i n l a b e l t o t h e s u g a r t e r m i n u s . - 29 -Since the work described here was completed, others found application for this and related procedures to study the role of sugars In binding events. A discussion of these papers is in order as they serve to highlight some of the d i f f i c u l t i e s with which the technique i s fraught. Grant and co-workers have performed an extensive study • of the headgroup dynamics and carbohydrate role in lectin binding processes with the transmembrane glycoprotein, glycophorin A (vide supra). When terminal NANA residues are specifically spin-labelled, the probe is confined to a highly polar milieu, and the motion is that of a moderately immobilized spin, even when constituted into a model membrane. That which reduces NANA mobility (as reflected in the ESR correlation time) is a specific binding event. Wheat-germ lectin (WGA) binds to glycophorin A, and the terminal NANA has been implicated in the binding process. As WGA i s added, a dramatic increase in i s observed which i s a function of the amount of lectin added. This study was 27 extended to gangliosides, labelled in an analogous fashion. Here, upon addition of WGA an i n i t i a l decrease in T £ was observed, followed by (the expected) increase as the WGA concentration was further raised. Such observations were explained in terms of randomizing an i n i t i a l patch-wise distribution of gangliosides, although this was not 2 8 positively determined as i t can be with spin labels. 29 30 Studies on a similar system by Butterfield and co-workers » are In contradiction to those of Grant. These workers used cyanoborohydride to couple spin label with periodate-activated - 30 -erythrocyte ghosts, which was found to be more efficient than NaBR\ (used by Grant). Upon addition of lectins which bind glycophorin A (WGA and Phaseolus vulgaris agglutinin, PHA) an increase in mobility was 30 observed which was interpreted in terms of conformational changes exposing the spin label to a less restricted environment upon lectin binding. It i s important to note that these two sets of data are not s t r i c t l y comparable, since Lee and Grant worked with a specifically labelled glycophorin either in solution or a model membrane, while Butterfield and co-workers used intact erythrocyte ghost membranes, where several questions arising from the complexity of the system can be raised. A third study 3 1 of WGA binding to spin labeled erythrocytes i s in accord with the findings of Lee and Grant; special pains were taken to purify the commercial lectins, and the activation was of the intact erythrocytes resulting in very low degree of ganglioside labelling (cf. Butterfield). 3 2 Finally on this subject, a French study was concerned with the f l e x i b i l i t y of isolated branched glycopeptides; NANA was spin labelled by periodate oxidation and reductive amination. Concanavalin A* was added resulting in a slight immobilization of the spin label - this i s not altogether surprising, as the binding site for the lectin i s distant from the labelled termini. However, It does suggest a certain r i g i d i t y of the oligosaccharide chain in i t s a b i l i t y to propagate such effects. Since the inception of the studies presented in this chapter, two other *A lectin with a specificity for mannose, present in the glycan. - 31 -groups have reported on the use of similar methods to spin label the carbohydrate moiety of immunoglobulins, and their fractions. Both used 3 3 non-specific oxidation - 8 hours at room temperature, and 16-24 hours at room temperature or 0° C. 3 1 t» 3 5 One group explicitly states that the selectivity for NANA was only 60%, and i t is clear from the pertinent literature reports that the labelling cannot be selective in either case. The Russian group i n i t i a l l y reported a highly mobile spin 34 label and then published revised data showing a more immobilized spin q c label; the i n i t i a l error was caused through carry over of non-specifically adsorbed label. The facts emerging from this chapter are that (a) NANA or galactose termini of glycoproteins and erythrocytes can be specifically labelled (b) the spin labels are mobile relative to the protein as a whole, and in a polar environment, and (c) extreme caution should be exercised when drawing conclusions from a complex system, when biological perturbants are introduced. That is not to say that the spin label method i s without use, but the experiments should be very carefully designed and controlled. - 32 -REFERENCES 1. I.CP. Smith. Biological applications of electron spin resonance. Edited by H.M. Schwartz, J.R. Bolton, and D.C. Borg. John Wiley, New York. 1972. pp. 483-539. 2. A.D. Keith, M. Sharnoff, and G.E. Cohn. Biochim. Biophys. Acta. 300, 379-419 (1973). 3. R.A. Dwek. Nuclear magnetic resonance in biochemistry: applications to enzyme systems. Clarendon Press, Oxford. 1975. Chapter 12. 4. G.I. Likhtenstein. Spin labeling methods in molecular biology. John Wiley, New York. 1976. 5. B.J. Gaffney and D.C. Lin. The enzymes of biological membranes. Vol. 1. Edited by A. Martonosi. Plenum Press, New York. 1976. pp 71-90. 6. P.M. Vignais and P.F. Devaux. Ibid, pp. 91-117. 7. Spin labeling: theory and applications. Edited by L.J. Berliner. Academic Press, New York. 1976. 8. Spin labeling II: theory and applications. Edited by L.J. Berliner. Academic Press, New York. 1979. 9. S. Schreier, C. Polnaszek, and I.CP. Smith. Biochim. Biophys.  Acta. 515, 375-436 (1978). 10. P.C. Jost and O.H. G r i f f i t h . Methods Enzymol. 49, 369-418 (1978), 11. L.J. Berliner. Methods Enzymol. 49, 418-480 (1978). 12. J.D. Aplin. PhD. Thesis. University of British Columbia. 1979, and references therein. 13. M. Yalpani. Ph.D. Thesis. University of British Columbia. 1980, and references therein. 14. J.D. Aplin, M.A. Bernstein, C.F.A. Culling, L.D. Hall, and P.E. Reld. Carbohydr. Res. 70, C9-C12 (1979). 15. E.R.B. Graham. Glycoproteins: their composition, structure and function. Edited by A. Gottschalk. Elsevier, New York. 1972. pp. 716-731. 16. W. Pigman and A. Gottschalk. Ibid, pp. 817-821. - 33 -17. N. Sharon. Complex carbohydrates: their chemistry, biosynthesis, and functions. Addison-Wesley, Reading, Mas. 1975. 18. H. Furthmayr. Biology of carbohydrates, Vol. 1. Edited by V. Ginsburg and P. Robbins. John Wiley, New York. 1981. Chapter 4. 19. L. Van Lenten and G. Ashwell. Methods Enzymol. 28, 209-211 (1972), and references therein. 20. A.G. Morell and G. Ashwell. Ibid. 205-208, and references therein. 21. G.E. Means and R.E. Feeney. Chemical Modification of Proteins. Holden-Day, San Francisco. 1971. 22. R.F. Borch, M.D. Bernstein, and H.D. Durst. J. Amer. Chem. Soc. 93, 2879-2904 (1971). 23. P.V. Wagh and O.P. Bahl. C.R.C. Crit. Rev. Biochem. 10, 307-377 (1981), and references therein. 24. G.W. Jourdian, L. Dean, and S. Roseman. J. Biol. Chem. 246, 430-435 (1971). 25. P.M. Lee and C.W.M. Grant. Biochem. Biophys. Res. Comm. 90, 856-863 (1979). 26. P.M. Lee and C.W.M. Grant. Can. J. Biochem. 58, 1197-1205 (1980). 27. P.M. Lee, N.V. Ketis, K.R. Barber, and C.W.M. Grant. Biochim.  Biophys. Acta. 601, 302-314 (1980). 28. L.D. Hall and J.C. Waterton. J. Amer. Chem. Soc. 101, 3697-3698 (1979). 29. J.B. Felx and D.A. Butterfield. FEBS Lett. 115, 185-188 (1980). 30. J.B. Feix, L.L. Green, and D.A. Butterfield. Life Sciences. 31, 1001-1009 (1982). 31. R. Snoek. Personal communication. 32. J. Devoust, V. Michel, G. Spik, J. Montreuil, and P.F. Devaux. FEBS Lett. 125, 271-276 (1981). 33. K.J. Willan, B. Golding, D. Givol, and R.A. Dwek. FEBS Lett. 80, 133-136 (1977). 34. R.S. Nezlln, V.P. Timofeev, Y.K. Sykulev, and S.E. Zurabyan. Immunochem. 15, 143-144 (1978). 35. V.P. Timofeev, I.V. Dudich, Y.K. Sykulev, and R.S. Nezlln. FEBS  Lett. 89, 191-195 (1978). CHAPTER 1.3 DEUTERIUM MAGNETIC RESONANCE STUDIES ON GLYCOCONJUGATES Chapter 1.2 in this thesis detailed ESR stdies on the carbohydrate moiety of glycoproteins, which provided information on the relative mobility of the glycan. Although the ESR method has advantages - chiefly, sensitivity and the absence of "background" signals from the molecule i t s e l f - the physical bulk of the nitroxide probe i t s e l f may perturb the system and lead to erroneous conclusions. In addition, where one is interested in the motional correlation time (T c) of a sugar to which the spin label i s attached, the measured value may largely reflect a high degree of mobility of the probe about the bonds attaching i t to the sugar, rather than the sugar per se. Alternatives exist for the extraction of motional information from such systems - preferably, these should be "non-invasive" in that they do not introduce an extraneous bulky "reporter" group. In this light, NMR relaxation studies are the most promising. To simplify matters, i t i s desirable to attach a "probe" isotopically enriched in a nuclide with low natural abundance, as such a procedure w i l l obviate complications arising from overlap with signals from the system ("back-ground"). Enrichment in 1 3C i s one possible approach; numerical values for T c may be calculated once the relaxation behaviour and the chemical system are known. An alternative approach i s *H NMR; substitution of a proton with a deuteron i s clearly a minor perturbation to the system. Deuterium, having nuclear spin, I_ • 1, i s quadrupolar, meaning that i t s nuclear - 35 -charge i s asymmetrically distributed about the nucleus. This leads to some broadening of the resonances but, most importantly to this discussion, the relaxation mechanism for the deuteron i s dominated by this quadrupole effect, and a simplification of the determination of motional information results. Working in i t s disfavour i s the low 2 1 inherent sensitivity of H (< 1% that of H): this factor can be partially offset by the use of high magnetic fields (B 0 9.4 T in this case), and the Fourier transform method, now so ubiquitous in heteronuclear NMR studies. o The application of high-resolution H NMR (and some broad-line studies) was exhaustively reviewed by Mantsch et a l . 1 in 1977, and 2 up-dated in 1982. These two treatises are mandatory reading for the 2 applicant of H NMR, and the reader wishing a broader view on the subject should consult these. In the carbohydrate area, a l l related reports have been with isotopically enriched monosaccharides. In 1973, 3 13 Brewer et^ a l . studied the binding of C-enriched oc-methyl-D-glucopyranoside to the lecti n , * Concanavalin A. Their mapping of the binding site was found to differ considerably from previous X-ray studies, where an iodophenyl glucoside was used. Few challenge the validity of the NMR data, and this example amply i l l u s -trates the d i f f i c u l t i e s which may arise when large reporter groups are i Q l» used. Alter and Magnuson used F NMR to study the same system, and Neurohr et a l . studied 5 the binding of isotopically labelled methyl o-*A lectin i s a protein which binds specific carbohydrate(s). - 36 -13 and 8-D-galactopyranosides to peanut agglutinin (another lectin) by C NMR relaxation. The same group6 studied the molecular dynamics of the sugar binding process to the lectin, wheat germ agglutinin. In this case, the sugar was isotopically enriched in H and this satisfying relaxation study includes an analysis for the case where a contribution to the motion from internal rotation is present in the bound state. In the case of rapid (where to T < 1) and isotropic motion, the o c dominant quadrupole relaxation mechanism is given by:1 2 2 ( I i ) - 1 = ( T 2 r 2 - J (1 + U_) (i-39.) 2 T [1.3.1] — 8 3 h c n i s the asymmetry parameter of the f i e l d gradient (and may be neglected), and e qQ/h is the quadrupole coupling constant. Although T_i or T_2 could be measured by multi-pulse methods, i t is justifiable to neglect contributions to the line-width from magnetic inhomogeneities and iH-2H couplings, and assume Tj, " l / 1 1 ^ V0.5 (where AVQ #5 is the peak width at half-height). Equation 1.3.1 reduces to: ( T 2 ) " 1 - , A v Q > 5 - |(4^ )2 xc H.3.2] By measuring AVQ.S and using a reasonable value for the quadrupole coupling constant, xc may be easily calculated. In Chapter 1.2 we described two methods by which aldehyde functionalities may be introduced at specific l o c i in a glycoprotein glycan. Our approach to the specific incorporation of H into glycoprotein rests on the use of these same procedures to generate the - 37 -specific aldehyde, and the regeneration of an alcohol using the ^ - l a b e l l e d reductant, sodium borodeuteride. The specific deuteration of terminal s i a l i c acid or galactosyl units is illustrated in Figs. 1.3.1 and 1 . 3 . 2 , respectively. With s i a l i c acid, the sugar is modified in that the exocyclic t r i o l i s shortened by two carbons; with galactose, the labelled sugar matches the native sugar almost exactly. The exocyclic -CHDOH unit i s racemic, and this, too, w i l l contribute to the observed line-width. In light of our previous successes with the reductive amination procedure using sodium cyanoborohydride, i t was apparent that proteins could be labelled with deuterons by performing such a reaction with hexadeuterioacetone and lysyl 6-amino groups: / C D 3 H 6-NHU+ 0=C NaCNBH3 6-N1C1H - \ C D * | or 2\ C D 3 The above-mentioned procedures were used to label the s i a l i c acid termini of bovine submaxillary mucin ( B S M ) , galactose termini of asialofetuin and lysyl groups of fetuin. The H NMR spectra were recorded at 61.4 MHz (BQ 9.4 T) as solutions in d i s t i l l e d water, and are shown in Fig. I.3.3.* The partial overlap of the sugar signal(s) *Spectra were recorded by Dr. W.E. Hull of Bruker, West Germany. - 38 -Fig. 1.3.1 Procedure for the specific deuteration of s i a l i c acid termini of glycoproteins at the C-7 position. Fig. 1.3.2. Procedure for the specific deuteration of galactose termini of asialo-glycoproteins at the C-6 position. - 39 -Fig. 1.3.3. H NMR spectra measurement at 61.4 MHz. Samples were dissolved in = 1.5 ml H2O. (A) BSM deuterated'at C-7 on s i a l i c acid residues (145 mg; 2700 transients). (B) Asialofetuin deuterated at C-6 position on terminal galactose residues (190 mg). (C) Fetuin reductively aminated with hexadeuterioacetone on lysine residues (240 mg; 550 transients). The HOD resonance was arbitrarily assigned a shift of 5 ppm. From ref. 7. - 40 -with the residual HOD in d i s t i l l e d water is undesirable and the problem was largely alleviated by using deuterium-depleted water as a solvent (data not shown). For the determination of T c» line-widths were measured and inserted in Equation [1.3.2]. A t y p i c a l 1 quadrupole coupling constant (165 kHz) was used throughout. The correlation times from H NMR are compared with those for the analogous molecule from ESR measurements (Ch. 1.2), in Table 1.3.1. The quoted errors are that in measuring peak widths - especially d i f f i c u l t when the signal-to-noise is low and overlap with the HOD resonance is significant (as in the case with Fig. I.3.3.B). The correlation between the measurements is good, and these data suggest that x c values derived from nitroxide ESR studies are representative in such systems. BSM labelled at s i a l i c acid "appears" from the H NMR measurements to be roughly twice as mobile as from the ESR, and this should serve to caution the reader not to take the figures too l i t e r a l l y . Indeed, this may be a real effect, reflecting a somewhat restricted motion of the spin label with BSM, where the glycan is short (a disaccharide), and the carbohydrate substitution level high. As mentioned in the previous chapter, useful results may arise from ESR studies where changes in T C resulting from the perturbation of the system are considered, and this conviction i s supported by the literature. We conclude that, given ca. 100 milligrams of material, one may successfully label specific sites on a glycoprotein with deuterons, and - 41 -Table 1.3.1 Comparison of x determined from H NMR relaxation measurements, and nitroxide ESR. a. x in ns. b. Reductive amination of protein lysyl 6-amine groups with hexadeuterioacetone, or 2,2,6,6-tetramethylpiperidine -4-keto-N-oxyl (TEMPONE). Activation Substrate Procedure x& from 2H NMR x from ESR c c FETDIN PERIODATE 0.73 ± 0.13 0.79 ASIALOFETUIN GALACTOSE OXIDASE 0.57 ± 0.03 0.52 BSM PERIODATE 0.12 ± 0.07 0.35 ASIALO-BSM GALACTOSE OXIDASE Not Determined 0.49 FETUIN REDUCTIVE AMINATIONb 0.5 0.77 - 42 -observe the H NMR resonances on a high-field spectrometer. Measurement of line-widths yields a correlation time for the sugar very similar to that derived from an analogous nitroxide spin-label study. While this is Interesting and, perhaps, useful Information, i t should be cautioned that in neither study have we perturbed the system with, for example, a molecule which should bind the sugar (i.e. a lectin) to evaluate the possible effect of the bulky spin label on the binding process. It is our belief that ^ NMR studies of the kind detailed here w i l l be useful in understanding molecular interactions and physical properties of biological macromolecules. - 43 -REFERENCES FOR CHAPTER 1.3 1. H.H. Mantsch, H. Saito, and I.CP. Smith. Prog. Nuclear Magn.  Reson. Spectrosc. 11, 211-271 (1977). 2. I.CP. Smith and H.H. Mantsch. NMR spectroscopy: new methods and applications. Edited by G.C Levy. ACS Symposium Series 191. 1982. pp. 97-117. 3. CF. Brewer, H. Sternlicht, D.M. Marcus, and A.P. Grollman. Proc.  Nat. Acad. Sci. USA. 70, 1007-1011 (1973). 4. CM. Atler and J.A. Magnuson. Biochemistry. 13, 4038-4045 (1974). 5. K.J. Neurohr, N.M. Young, I.CP. Smith, and H.H. Mantsch. Biochemistry. 20, 3499-3504 (1981). 6. K.J. Neurohr, N. Lacelle, H.H. Mantsch, and I.CP. Smith. Biophysical J. 32, 931-938 (1980). 7. M.A. Bernstein, L.D. Hall, and W.E. Hull. J. Amer. Chem. Soc. 101, 2744-2746 (1979). - 44 -CHAPTER 1.4 NEOGLYCOPROTEINS: SYNTHESIS AND NMR STUDIES 1.4.1 Introduction At the time when the work described in this chapter was performed (1979/1980), much use had been made of 1 3C and *H NMR in the study of oligo- and polysaccharides (see Ch. II.3), but l i t t l e in a second important class of glycoconjugates, glycoproteins (Ch. 1.2). At natural abundance the scarcity of studies can, in part, be attributed to the complexity of the material and the lack of model compounds. In addition to contending with protein resonances (overlapping with carbohydrate peaks to a greater or lesser extent), glycoproteins often display microheterogeneity in their glycans, making i t impossible to write a single chemical structure for the carbohydrate moiety.* This added complexity obviously compounds the d i f f i c u l t y of analysis. Glycoproteins cannot always be isolated in appreciable quantities, and this could preclude 1 3C NMR studies. One approach to the development of simple model systems for NMR studies i s to liberate the glycans from the protein (or perform an enzymic protein digest), separate the fragments, and perform NMR experiments on these purified materials. This solves the problem of microheterogeneity and eliminates the bulk of the protein resonances, and could be a worthwhile f i r s t approach. We considered this to be somewhat undesirable, as the low molecular weight fractions were too far *An obvious exception to the rule Is the Arctic fish "antifreeze" glycoproteins, which are extremely homogeneous. - 45 -from the "real" situation. An alternative exists and we have exploited this: known, characterized sugars can be covalently attached to a (hitherto sugarless) protein to create a "neoglycoprotein". The bulk of protein resonances can potentially be identified by observing the protein before glycan attachment. In essence, one can characterize both the protein and carbohydrate independently of one another, and then observe changes when the neoglycoprotein i s studied. This allows one to make assignments of neoglycoprotein glycans and evaluate such concepts as the potential use of chemical shift data of carbohydrate standards. With the purpose of spectral assignment we evaluated the use of enzymic (and, in principal, chemical) perturbations. We fe l t that the neoglycoprotein approach had the further advantage that the material is of similar physical form to a glycoprotein, and this was crucial in our AH NMR studies where use was made of the differential in mobility between the glycan and protein back-bone. The structure and complexity of the glycone would be restricted by the lengths an investigator i s prepared to go in oligosaccharide synthesis*. The neoglycoprotein synthetic protocol allows also for f l e x i b i l i t y in the nature and linkage length of the glycone to protein, and this could be useful to one interested in dif f e r e n t i a l mobility or enzymic studies (vide infra). At this time, other groups have proceeded to successfully employ A 3C NMR to study a variety of aspects of the structure and form of glycoprotein glycans, and these w i l l be reviewed later in the chapter. *This i s , by no means, a t r i v i a l task! In this study, monosaccharides and purchasable disaccharides were considered. - 4 6 -1.4.2 Neoglycoprotein Synthesis The f i r s t neoglycoproteins were synthesized by Goebel and Avery 1 in 1929, and their procedure is s t i l l popular. A sugar i s 0_ -glycosylated with para-aminophenol, and the p-aminophenyl glycoside converted into the diazonium salt (nitrous acid, 0°, 15 min.). This i s then mixed with the protein, in basic solution for another 15 min.; substitution primarily occurs at histidine, lysine and tyrosine amino acid l o c i . This procedure has a number of major draw-backs:- the specificity i s low, which can cause problems in the interpretation of results, and the introduction of the bulky aromatic group can i t s e l f e l i c i t an antibody response, reducing the specificity of the antibody to the sugar hapten. Literature precedent existed for the selective amination of methanal by lysyl amino acids, at the 6-amine position. » Reductive amination had been described by Gray and co-workers between lysyl amines and reducing sugars (Fig. 1.4.1), using sodium cyanoborohydride as the reductant. Although the reaction i s controllable and mild, i t has the disadvantages that i t introduces a polyol (to which antibodies have been shown to direct), destroys one sugar ring (which is disadvantageous when the attached product i s the result of a long synthetic route) and the R - Sugar Fig. 1.4.1. Gray's procedure for the attachment of a reducing disaccha-ride to a protein (P) via i t s lysyl 6-amino groups. - 47 -An encyclopaedic review of methods described for neoglycoprotein synthesis is not in order here - two excellent reviews have appeared recently. b» 6 Our survey of these methods for neoglycoprotein synthesis did not point to an ideal procedure, compared with the c r i t e r i a we considered necessary:-1. The method should be directly compatible with the reagents and protecting groups used in oligosaccharide synthesis. 2. It should cause no destruction of the integrity of the reducing (terminal) sugar ring. 3. It should have in-built v a r i a b i l i t y of the "spacer" between the terminal sugar and the point of attach-ment on the protein, in terms of length and hydro-p h i l i c i t y . 4. The coupling reaction should be convenient, and rapid at, or near, physiological pH in aqueous media, using chemically stable reagents. 5. The reaction should give high-yielding coupling to a specific amino acid via a hydrolytlcally stable, covalent linkage. 6 . The surface charge of the protein should not be significantly altered by carbohydrate attachment. In consideration of the above-mentioned c r i t e r i a , and the two protocols illustrated above, i t seemed that the most obvious route was the glycosidatlon of a sugar such that an aldehyde could be generated at a specific point on the aglycone. Our (see Ch. 1.2) and Gray's - 48 -experience with the reductive amination reaction using sodium cyanoborohydride encouraged us to consider this method for the coupling reaction. Further, we wanted a protocol which relied on previously characterized reactions. Our protocol 7 is illustrated in Fig. 1.4.2 for the attachment of a-g-glucose to bovine serum albumin (BSA). The procedure involves three steps:- glycosidation of the parent sugar with an alkenyl alcohol, reductive ozonolysis in methanol to generate an aglycone aldehyde (not isolated), and reductive amination with lysyl 6-amino groups on the protein using sodium cyanoborohydride. A l l steps have been well characterized in the literature. Fig. 1.4.2. Neoglycoprotein synthesis protocol. From Ref. 7. - 49 -The a l l y l group is well known for i t s use as a protecting group 8 9 in carbohydrate synthesis, and Lee and Lee have described the glycosidation of sugars with a l l y l alcohol. The glycosidation procedures we used were either those of Lee, or variations. 1 0 o-Glycosides were generally prepared by heating the free sugar in a l l y l alcohol under reflux, and B-glycosides prepared from the a-bromo sugar per-acetate and alkenyl alcohol via a Koenigs-Knorr 1 1 related procedure. To illustrate the vers a t i l i t y of spacer arms, glucose was also glycosidated with undecenyl alcohol - H0-(CH2)9CH=CH2, thereby introducing a long (relatively hydrophobic) spacer arm between the "nascent aldehyde" (i.e. the alkene) and the sugar. The prepared a l l y l glycosides a l l had melting points in close agreement with the literature, and their NMR spectra were consistent with their structures. 1 2 They are a l l stable molecules which can, as far as we know, be stored indefinitely at room temperature. Although alternate routes exist for the oxidative cleavage of the alkene aglycone (e.g. periodate-ruthenium dioxide oxidation 1 3), we 14 found the reductive ozonolysis reaction to be fast, specific, and high-yielding (essentially quantitative in a total of ca. 2 hours). The sugar is dissolved in a methanolic solvent (pure methanol in the case of a free sugar or 4:1 MeOH-CH2Cl2 with protected sugars) and purged with ozone at reduced temperature (5-10 min./mmole). The reaction i s "self-indicating", as excess ozone Is manifested by the clear solution turning blue. This step produces the hydroperoxide (8) which is reduced in situ with dimethyl sulphide (DMS). The generally accepted mechanism15 for the reaction i s given in Fig. 1.4.3. - 50 -Fig. 1.4.3. Reaction mechanism for the reductive ozonolysis of an alkene in methanol, using dimethyl sulphide. After adding an excess of DMS, the solution is allowed to reach room temperature and l e f t for ca. 2 hours. Methanol, methanal and unreacted DMS can be removed by rotary evaporation under reduced pressure and, in the case of the protected sugar, DMSO removed by repeated washing of a - 51 -chloroform solution with water. The aldehydo sugar (3) was only isolated for the purpose of characterization, and the reaction routinely monitored for completion by TLC. The aldehydo sugar was then dissolved with BSA at a molar ratio of ca. 5 (sugar:lysyl 6-amine). The pH was adjusted to 9.0, and a ca. 20-fold molar excess of NaCNBH3 added. In the case of the coupling of a blocked sugar, a heterogeneous reaction was performed in a water/methanol solvent pair (85:15). The mixture was shaken and coupling allowed to proceed for 18-48 hours, at which time the neoglycoprotein was isolated. The homogeneous solution was applied to a Sephadex G-25 column and the excluded volume collected. In some cases this was further purified by dialysis against running water, and then the solution was lyophilized. The solid neoglycoprotein was stored at -4°C. Sugar analyses were performed on milligram quantities using the standard phenol-sulphuric acid assay, 1 6 with a calibration curve prepared from freshly prepared standard solutions of the appropriate sugar. The protein was not found to interfere with the assay, and accuracies are within 10%. Using the above-mentioned procedures, neoglycoproteins (10-17) were prepared. The coupling conditions chosen were, largely, those of 17 Schwartz and Gray. The use of ratios of sugar/amine (5:1), should, 18 based on other workers' experience, result predominantly in 2° amine formation at the ~35 easily accessible 6-amino groups on BSA. The higher levels of incorporation observed with the attachment of acetylated sugars to BSA, in methanollc/aqueous solutions, could either - 52 -T a b l e 1 . 4 . 1 - T a b l e o f s u g a r s c o u p l e d t o BSA a n d t h e i r e f f i c i e n c i e s . BSA b e a r s 5 5 , l y s y l 6 - a m i n o g r o u p s p e r m o l e c u l e . a E x p r e s s e d a s m o l e / m o l e C o u p l i n g S u g a r s p e r S u g a r G l y c o s i d e A g l y c o n e E f f i c i e n c y BSA M o l e c u l e 3 10 a - g a l a c t o s e a l l y l 36 20 11 o - g l u c o s e a l l y l 36 20 12 8 - N - a c e t y l g l u c o s a m i n e a l l y l 5 3 13 6 - l a c t o s e a l l y l 24 13 14 c t - a c e t o g l u c o s e a l l y l 36 20 15 8 - a c e t o g l u c o s e a l l y l 6 9 - 8 2 3 8 - 4 5 16 8 - a c e t o - N - a c e t y l - a l l y l 24 13 g l u c o s a m i n e 17 8 - a c e t o g l u c o s e u n d e c e n y l 15 8 - 53 -result from the "exposure" of formerly inaccessible amines, or 3° amine formation (i.e. attachment of >1 sugars to each amine) resulting, perhaps, from a decrease in aldehydrol concentration caused by the reduced water concentration. Although extensive pH profile studies were not performed here, the reaction was found to proceed with greater efficiency in non-buffered solutions. This could result from the anionic form of the buffer associating with the cationic lysyl 6-amino group (pH < 10) in an ion pair, thereby "shielding" i t from collision with the aldehyde, and possible aldimine formation. Coupling reactions were often performed overnight (18 hrs) but satisfactory levels of derivatization were possible in 2-4 hrs. Rather than extensively characterize an already well-studied reaction, we were content with the levels of substitution and made no attempts to "fine-tune" the coupling procedure. Similarly, we make no attempt to interpret the varying coupling levels too closely. Neutral free sugars appear to give a substitution level of coupling of ca. 20 sugars per BSA*, and N-acetyl glucosamine appears to give lower levels. Lactose coupled to BSA at a lower level of efficiency caused, perhaps, by i t s greater bulk. The highly efficient coupling of 8-acetoglucose (38-45 sugars/BSA) cannot be explained without further experiments. The relatively low efficiency of coupling the long-chain B-acetoglucose molecule could result from Its reduced solubility In aqueous media, and the possibility of i t s self-association. *With much longer coupling times, up to 32 sugars may be attached. - 54 -In summary, we believe we have developed a useful protocol for neoglycoprotein synthesis. By capitalizing on prior detailed analyses of a similar coupling reaction, such studies were obviated here. The limitations of Gray's procedure are circumvented by using a sugar glycoside bearing a "nascent" aldehyde on the aglycone in the form of an alkene. Protected* or free sugars have been attached to lysyl 6-amine groups of a common test protein, BSA. It is appropriate to mention related developments which have occurred since the inception of our studies. Lee and Lee have 18 19 reported » the synthesis of 1-thioglycosides bearing co-aldehydo groups, stored as a dimethyl acetal. Acid hydrolysis of the acetal (0.05 M HC1, 100°, 20 min.) is required before the coupling stage, and we draw the reader's attention to the possibility that acid-sensitive glycosidic bonds (e.g. fucosyl) may be hydrolysed at this stage, should these sugars be present. Jentoft and Dearborn performed a detailed analysis of the reductive amination reaction with raethanal and BSA - a close comparison between their observations and ours is dangerous, due to the very different nature of the aldehyde. Lee and Lee recently des-20 cribed the use of 1-thioaldehydo sugars very similar to ours; they ob-served that at very high sugar/protein molar ratios (ca. 1000:1) as many as 70 sugars can be attached to each BSA molecule. Obviously, in this case, a large number of 3° amines are formed, and the authors also post-ulate the existence of -35 "accessible" lysyl 6-amine groups, and the rest relatively hidden. One might postulate that the latter are perhaps involved in ionic bonds with acidic amino acids, to stabilize the *Neoglycoproteins of this kind can be de-O-acetylated, i f required. - 55 -protein's tertiary structure. Finally, Friedman et a l . have reported 21 the reductive amination of a protein and an aromatic aldehyde; the reaction proceeds in very good yield (ca. 90% of lysyl 6-amines modified), suggesting that this non-polar aldehyde can penetrate the protein to the lysyl amines previously determined "inaccessible". This finding is in accord with ours for the neoglycoprotein (15), where a very high yield was observed when coupling a protected sugar, which is relatively apolar, to a methanol!c/aqueous solution of B S A . 1.4.3 13C NMR of Neoglycoproteins Literature precedent has established 1 3C NMR spectroscopy as a useful tool in the study of proteins in solution, within the restraints of the necessary large sample size and high solubility (caused by the 13 low inherent sensitivity of C). Comparing the spectra of native and denatured proteins, i t became clear in the late 1970's that i t would be 22 possible to observe single carbon sites in the native protein. The relaxation behaviour of 1 3C nuclei is of great importance as i t has the potential to report on the mobility of the protein at a specific site, once the relaxation mechanisms are understood. Therefore, i t is common to report T_i, nOe and line-width (Avo.5) measurements in an attempt to derive conclusions on this fundamental aspect of protein behaviour in solution (vide infra). To simplify analysis the protein's spectrum is divided into three regions:- aliphatic region (6 10-75), aromatic region (6 105-160) and the carbonyl region (6 170-185). The 100.3 MHz 1 3C NMR spectra of native and denatured BSA are shown in Fig. 1.4.4. F i g . 1 . 4 . 4 1 3 C NMR s p e c t r a a t 1 0 0 . 3 MHz o f ( A ) n a t i v e BSA i n D 2 0 , a n d ( B ) t h e d e n a t u r e d p r o t e i n i n 8 M u r e a / D 2 0 . - 57 -It i s well known that the C NMR spectra of common neutral 23 carbohydrates is divisible into two regions:- the O-glycosidic anomeric carbon region (6 95-105) and the region in which the rest of the hydroxylated and N-glycosldic anomeric carbons resonate (6 60-80). Clearly, the anomeric carbons f a l l into a spectroscopically silent "window" of the protein spectrum, and the other carbohydrate resonances overlap slightly with a few protein aliphatic resonances (mainly, serine and threonine CP). Hence i t would seem reasonable to postulate that anomeric carbons w i l l be uniquely discernible and other carbohydrate 13 carbons somewhat less so, in the C NMR spectrum of natural or synthetic glycoproteins. A possible factor in the assignment of carbohydrate resonances between 6 60-80 might be the relative mobility of the overlapping resonances. Our previous studies (Chs 1.2 and 1.3) indicate that the terminal sugar has at least an order of magnitude of rotational freedom greater than the protein and this, reflected In their line-widths, could prove sufficient to differentiate the two. To il l u s t r a t e this point, Fig. 1.4.5 shows the result of superimposing the spectrum of native BSA with that of B-O-methyl glucopyranoside. In Fig. I.4.5B, the sugar resonances are broadened by 2 Hz with an Exponential f i l t e r , and in Fig. I.4.5C, by 15 Hz; only the "carbohydrate region" (6 60-110) is 23 plotted, and the carbohydrate peaks assigned. Clearly, a large number of relatively mobile carbohydrate resonances can be readily distinguished from the broader peaks originating from protein carbons, in Fig. I.4.5B. - 58 -B. 100 80 6(ppm) Fig. 1.4.5 1 3C NMR spectra at 100.3 MHz, showing the "carbohydrate region" only. (A) Native BSA. (B).(A), superimposed with the spectrum of methyl B-D-glucoside, with line-widths broadened by 2 Hz. (C).(B), with line-widths broadened by 15 Hz. D: internal dioxane reference (6 67.4). - 59 -These elementary " f e a s i b i l i t y studies" bode well for the 13 possibility of observing C sites on a (neo)glycoprotein, but they 1 3 cannot predict the possible effects the protein may have on the C chemical shifts of the sugar(s). 1 3C resonances are notoriously d i f f i c u l t to assign and the most common resource is to make comparisons between the chemical shifts of known, assigned molecules and those of the "unknown". Thus, we might consider the methyl glycosides of mono-, d i - and oligosaccharides to be useful model compounds in the assignment 1 3 of (neo)glycoproteins' C NMR spectra, and we went about testing this theory. Pfeffer et a l . have used deuterium isotope-induced shifts (DIS) 13 23 to assign the C NMR spectra of a range of mono- and disaccharides, and their data was used throughout this study. This was the rationale for studying neoglycoproteins as model glycoproteins: the carbohydrate moieties have been assigned independently of the protein and, by 1 3 recording the C NMR spectra of the (known) same sugar, now attached to the protein, useful generalizations might be derived concerning assignment in the glycoconjugate. Fig. 1.4.6 shows the 100.3 MHz 1 3C NMR spectrum of a neoglycoprotein comprising 8-glucose attached to BSA* (11, having 14 glucose molecules per BSA), comparing i t with the spectrum of BSA. The carbohydrate region is expanded above the f u l l spectrum and the chemical shifts of methyl 6-g-glucoside indicated above the peaks. We immediately note that the carbohydrate peaks are sharp in comparison *A11 neoglycoproteins were prepared by the procedure detailed in the previous section (1.4.2). - 60 -C-6 150 100 50 5(ppm) Fig. 1.4.6 1 3C NMR spectrum of a neoglycoprotein (B-glucose attached to BSA), with the "carbohydrate region" expanded and asigned. Lines aboves the resonances indicate the literature chemical shifts of methyl 8-D-glucoside. - 61 -C-1' C-1 C-3 —1 C-5 - , C-5'n ' l C-4 C-2,-3' C-2' C-6' C-6 C-4' C-3,-4 C-1 B. C-5 V C-2 C-6 C-5 C-1 C-3 C-2 C-4 C-6 100 Fig. 1.4.7. 80 6 (ppm) The "carbohydrate region" of the 100.3 MHz 1 3C NMR S d ^ T 8 1 7 ^ * 1 - 8 ( A ) P " g l u c o - (B) ^ a l a c osT and (C) B-lactose attached to BSA. galactose, Table 1.4.2 Comparison of C chemical shifts of the carbohydrate resonances of neoglycoproteins with the methyl glycoside of the attached sugar. The chemical shifts of neoglycoproteins are with an error of ± 0 . 1 ppm. A6 represents the difference in chemical shift between a sugar resonance of a neoglycoprotein and its corresponding methyl glycoside. C-l C-2 C-3 C-4 C-5 C-6 C-l' C-2» C-3' C-4' C-5' C-6' 8-glc-BSA B-Me-glc A6 103.1 103.4 -0.3 74.0 74.1 -0.1 76.6 76.8 -0.2 70.6 70.7 -0.1 76.9 76.8 -0.1 61.7 61.8 -0.1 oc-gal-BSA o-Me-gal A6 99.6 100.1 -0.5 69.2 69.2 0.0 70.2 70.5 -0.3 70.2 70.4 -0.2 72.1 71.6 -0.5 62.1 62.2 -0.1 B-lac-BSA B-Me-lac A6 103.3 103.8 -0.5 73.8 73.8 0.0 75.4 75.3 0.1 79.7 79.3 0.4 76.0 75.6 0.4 61.4 61.1 0.3 104.1 103.8 0.3 72.1 71.9 0.2 73.8 73.5 0.3 69.7 69.5 0.2 76.4 76.2 0.2 62.0 62.0 0.3 - 63 -with those of the protein, and the chemical shift correlation is 13 excellent. Fig. 1.4.7 shows the C NMR spectrum of the carbohydrate region of the three neoglycoproteins indicated. Sharp resonances are seen in every case and the measured chemical shifts are compared with those of their parent methyl glycosides in Table 1.4.2. Within the limits of ± 0.5 ppm, the agreement in chemical shifts between the methyl glycoside and the corresponding neoglycoprotein sugar resonances is excellent, with the constraints of this s t a t i s t i c a l l y small survey. Except for a trend for C-l to be slightly deshielded (=0.5 ppm) in the neoglycoprotein compared with the methyl glycoside, the differences in chemical shift (A6) are often within the experimental error of the measurement. Hence, we conclude that the methyl glycoside of a sugar may be used to give chemical shifts which can be compared closely (± 0.5 ppm) with the chemical shifts of the sugar attached to a protein. This is an important finding and is analogous to the use of small saccharides' chemical shifts to assign and determine the structure. Oh of polysaccharides, which is now a relatively common procedure. We then decided to extend the study and determine whether some of the technologies used in previous chapter could be applied to these systems to assist in carbohydrate structure determination. Specifically, we considered D-galactose oxidase which catalyses the oxidation of, e.g., methyl o-D-galactose to the molecule having an aldehyde at C-6; in aqueous solution, methyl q-D-galacto-hexodialdo-1,5-pyranoside (19), predominates. This reaction has been studied by *H NMR by Maradufu and P e r l i n , 2 6 and Whyte and Englar. 2 7 The 1 3C chemical - 64 -Fig. 1.4.8. The action of D-galactose oxidase on methyl a-D-galactopyranoside. 2 8 shift of an aldehydrol may be typified by a study on streptomycins in D2O; C-13 (aldehydrol) has a chemical shift of 90-96 ppm. The chemical shift of the hydrated aldehydic carbon of acetaldehyde is 88.9 ppm. Hence, we might confidently anticipate that the 1 3C chemical shift of C-6 of a terminal galactosyl residue on a glycoprotein might change by ~ 30 ppm after oxidation with galactose oxidase and the aldehydrol should be easily detectable, as i t resonates in a spectroscopically "silent" region. To test this, and ascertain the effect of oxidation at C-6 on the other ring carbons' chemical shifts, we subjected a 0.2 M solution of methyl a-g-galactopyranose to such an oxidation, measuring the 1 3C NMR spectrum before and after the reaction (Fig. 1.4.9). Fig. I.4.9A shows the 1 3C NMR spectrum before, and Fig. I.4.9B after ca. 20% galactose oxidase oxidation. The shift In resonant frequency (A6) is - 65 -C-1 C-3-, C-5-B. r- C6(0) -27.4 C-5(0) -, -1.7 C-4 -C-4(0) 0.5 C-2 C-6 C-2(0) 0.1 C-1 C-5 r-C-3 rC-4 r-C-2 C-6 u 100 80 5 (ppm) Fig. 1.4.9 1 3C NMR spectrum (100.3 MHz) of (A) methyl a-galactoslde, and, (B), the same after oxidation at C-6 by D-galactose oxidase, at 20% efficiency. - 66 -recorded for each carbon. As might be expected, C-6 suffers the largest shift (-27.6 ppm); C-5 and C-4 are also shifted significantly. Only C-1 and C-3 did not shift measurably. It i s interesting to note that the relative peaks heights of C-6 and C-3 do not change significantly with oxidation, but the decreased intensity is measurable in peak areas of C-2, C-4, C-5 and C-6. This pilot experiment bodes well for the use of galactose oxidase in the assignment of C-4, -5 and -6 terminal galactosyl residues, especially when an accurate integration is possible. Galactose-containing neoglycoproteins were then reacted with galactose oxidase. Compound 13 bears terminal galactose residues (lactose bound to BSA) and, after treatment with the enzyme, the spectrum in Fig. 1.4.10B was obtained. The aldehydrol C-6' is clear (6 98.8), but the level of oxidation is low, as the large galactosyl methylene C-6' peak suggests. This peak is narrower, (indicating less area), and so we may only conclude from this experiment that (a) the pendant sugar bears terminal galactose residues, and, (b) the galactosyl C-6 resonates at 6 62.0. Shifts in resonance frequency of other carbon centres are small, overlap with other peaks, or are lost in the noise. Whether this would be the case with complete oxidation i s not possible to predict, but the model study with the a-galactoslde, above, suggests that significant shifts in the resonance frequencies of C-5' and C-4' would occur, assisting in their assignment, too. Two control experiments were performed. F i r s t l y , the oxidized sample which gave the spectrum of Fig. 1.4.10B was reduced with sodium - 67 -100 80 8 (ppm) F i g . 1 . 4 . 1 0 T h e 1 0 0 . 3 MHz 1 J C NMR s p e c t r u m o f 6 - l a c t o s e a t t a c h e d t o BSA. ( A ) . U n o x i d i z e d . (B) A f t e r o x i d a t i o n b y g a l a c t o s e o x i d a s e o v e r n i g h t . ( C ) A f t e r r e d u c t i o n o f (B) w i t h NaBHi*. - 68 -c-6'n 100 80 8 (ppm) F i g . 1 . 4 . 1 1 T h e 1 0 0 . 3 MHz 1 3 C NMR s p e c t r u m o f a m i x t u r e o f BSA a n d a l l y l 8 - l a c t o s i d e , (A) a f t e r e n z y m i c o x i d a t i o n b y g a l a c t o s e o x i d a s e , a n d (B), a f t e r NaBHit r e d u c t i o n o f (A). - 69 -borohydride (Fig. I.4.IOC). By this time the sample was quite degraded, and this is evident in the line-widths in Fig. 1.4.IOC, which reflect the fact that some sample had precipitated by the end of the measurement. However, the aldehydrol peak is no longer present and the spectrum bears the features of that of the starting material, as might be predicted. To determine whether the protein was in some way inhibiting the action of the enzyme, a l l y l B-g-lactoslde was mixed with BSA in a mole ratio comparable with the neoglycoprotein. Galactose oxidase treatment almost completely oxidized C-6' as indicated by i t s large down-field resonance shift (Fig. I.4.11A,B). Similarly with the monosaccharide, C-5' shifts down-field 1.6 ppm, C-4' up-field 0.8 ppm and the carbon involved in the glycosidic linkage (C-4) down-field by a surprisingly large 0.9 ppm. The last observation has important implications as i t suggests that the galactose oxidase treatment gives information on the point of attachment of the terminal galactose, too. It is interesting to note the changes in the spectrum between 6 65 and 80 - many extra peaks appear after oxidation; i t is possible that some of these result from side-reactions galactose oxidase is known to •t 26 produce. Again, borohydride reduction resulted in the loss of the aldehydrol peak, and a reappearance of the galactosyl methylene C-6'. This second experiment is not a perfect control, but unambiguously shows that galactose oxidase can oxidize C-6' galactose groups on lactose in the presence of BSA, and that diagnostically useful chemical shift changes occur with f u l l oxidation. - 70 -Galactose oxidase failed to react with 10, a-galactose attached to BSA. It is tempting to postulate that the free substrate ( a l l y l 8-D-lactopyranoside) can easily f i t in the enzyme's active site and is effici e n t l y oxidized. Lactosyl-BSA (13) is partially oxidized due to somewhat restricted " f i t " , or accessibility of substrate to enzyme caused by the proximity of BSA. With galactosyl-BSA (10), the substrate cannot f i t into the active site at a l l , and no reaction can occur. To test this hypothesis i t would be interesting to synthesize a series of alkenyl glycosides having spacer arms of varying length, attach each one to BSA and determine the accessibility of the sugar to oxidation by galactose oxidase. If our reasoning is correct, an increase in level of oxidation should occur as the spacer arm is lengthened. It i s interesting to speculate on the effect specific chemical 13 modifications might have on the C NMR spectra of (neo)glycoproteins. It i s probable that such approaches might usefully augment the enzymatic approach which we have evaluated and found potentially useful. 13 In summary, studying the C spectra of neoglycoproteins might prove useful in the investigation of glycoproteins. The carbohydrate resonances are sharper than most protein resonances, reflecting the influence of their greater mobility on T_2. The corresponding methyl 13 glycoside of the attached sugar appears to be a good model for C assignment. That is to say, i f a sugar glycoside Is characterized, and 13 i t s C NMR spectrum assigned, i t is reasonable to expect that the spectrum of the same sugar attached to a protein w i l l have peaks at the same frequencies (± 0.5 ppm). Finally, except with only galactose attached to a protein, terminal galactosyl residues present w i l l be - 71 -determinable from their susceptibility to oxidation by galactose oxidase. This procedure not only identifies the presence of terminal galactose, but also clearly assigns a number of i t s resonances, and the attaching carbon in the sub-terminal sugar residue. 13 Finally, we make mention of the relevant C NMR studies of glycoconjugates performed in other laboratories after the inception of our work; much of these come from the laboratory of Allerhand. Harris 29 and Thornton studied carbohydrate head-group dynamics of glycolipids in various phases; only anomeric resonances were assigned and It was concluded that the pentasaccharide had no differential in mobility along i t s chain, indicating a closely hydrogen-bonded entity. D i l l and Allerhand 3 0 reported the f i r s t 1 3C NMR spectrum of a glycoprotein, glucoamylase. Few specific assignments were possible due to the complex nature of the carbohydrate, but certain generalizations were possible. 3 1 A study on the highly regular and simple antifreeze glycoproteins yielded beautiful 1 3C NMR spectra; a differential in mobility is quite clear from the carbohydrate line-widths and explicit assignments were possible by comparison of chemical shifts of low molecular weight analogues. The anomeric carbons were assigned on the basis of *J_CH measurements. Jennings et_ a l . used chemical and enzymic methods to 13 assist in the assignment of the C NMR spectrum of a streptococcal polysaccharide. Ribonuclease (RNA'se) Is found in glycosylated (RNA'se B) and non-glycosylated forms (RNA'se A), and both were studied by Allerhand 33 and co-workers. Protein folding was found to have small effects on the chemical shifts of close carbohydrate centres. Comparison of model - 72 -oligosaccharides allowed the structure determination of the complex glycoprotein carbohydrate. Arm!tage and co-workers characterized the glycopeptide of glycophorin A, a red-blood-cell membrane glycoprotein. Assignments were largely based on procedures common in polysaccharide studies - model compound chemical shifts plus additivity rules. Calcium ion titrations 13 induced shifts in N-acetylneuraminic acid C resonances. Several unusual shifts are explained by the involvement of hydrogen bonding in the maintenance of a fixed secondary structure. Berman and Allerhand 3 5 studied the hydrolytic action, specificity, and kinetics of an a-mannosidase (endoglycosidase enzyme) on the glycopeptide of ovalbumin, where they found that most Information came from a study of the anomeric region. The rationale for their assignments has not, yet, been published. 3 6 Goux et a l . perfomed a detailed relaxation study on uniformly 13 C-enriched galactose, attached by enzymatic methods to the carbohydrate chain of hen ovalbumin. The overall motion of the terminal residue was found to be anisotropic, comprising a slow (23 ns) isotropic contribution (arising from the motion of the glycoprotein as a whole) and a fast (40-80 ps) contribution arising from rotation of the carbohydrate C-H vector rotating at an angle of 30° about the effective axis of rotation. 37 13 Finally, Bedford and co-workers have reported the C NMR spectra of canine mucins. Purified materials yield reasonably sharp resonances in the carbohydrate region of the spectrum, which further sharpen when the material is treated with 8-mercaptoethanol (which - 73 -reduces p r o t e i n c y s t e i n y l d i s u l p h i d e b r i d g e s ) . T h e i r m a t e r i a l was found to have a very low s i a l i c a c i d content, which was s u r p r i s i n g . 1.4.4 *H NMR of Neoglycoproteins 13 The previous s e c t i o n i l l u s t r a t e d C NMR as having the p o t e n t i a l to be of considerable u t i l i t y i n the study of g l y c o p r o t e i n s . Two major d i f f i c u l t i e s remain:- the large amounts of sample required (> 120 mg) and the d i f f i c u l t y of a s s i g n i n g complex glycans. The f i r s t problem may be p a r t i a l l y solved by observing a more s e n s i t i v e n u c l i d e , such as 1H, and i n Chapter I I . 3 we describe how *H NMR may be used de_ novo to a s s i g n o l i g o s a c c h a r i d e s . When studying the *H NMR spectrum of g l y c o p r o t e i n s , one major d i f f i c u l t y l i e s i n the extensive overlap of glycan and p r o t e i n s i g n a l s . Our previous studies have i n d i c a t e d that the glycans are at l e a s t an order of magnitude more mobile than the p r o t e i n , and t h i s f a c t was seen to be p i v o t a l In the route to the "sp e c t r o s c o p i c f a c t o r i z a t i o n " of the sugar resonances. L i t e r a t u r e precedent e x i s t s f o r the observation of r a p i d l y tumbling molecules i n the presence of immobile ones, where a l l methods r e l y on the d i f f e r e n t i a l i n transverse r e l a x a t i o n times (T_2) between the two e n t i t i e s . These w i l l be reviewed at the end of the chapter. A l l methods f o r such d i f f e r e n t i a t i o n come under the heading of "spin-echo" techniques. E s s e n t i a l l y , a 90° pulse c r e a t i n g magnetization i n the x'-y' plane i s followed by a delay T, and a second pulse i s used to induce the spin-echo, having a maximum at 2T. The choice of the delay Is c r u c i a l , since t h i s i s where the slow and f a s t tumbling molecules are "sorted". Protons having long T^'s ( r a p i d l y - 74 -tumbling) w i l l s t i l l have magnetization after the refocussing pulse (180°), and w i l l , therefore, be observed; those with short T_2's (slowly tumbling) w i l l have lost their transverse magnetization by the time the 180° pulse is applied and, therefore, w i l l not be seen when the receiver i s turned on. The classical experiment is the Hahn spin-echo, comprising a (90°-T-180°-X-AQN) pulse-sequence. A spin-echo maximum is generated at 2x, and the following half-echo is then acquired (see explanation of 2D J-resolved spectroscopy, Ch. II.2.5.2). The amplitude of the refocussed echo i s determined by a T_2 term, and in some cases, diffusion rates and magnetic f i e l d f i e l d gradients w i l l enhance the decay of the spin-echo amplitude. With homonuclear spin-coupled spins, the peaks' phases w i l l be "J-modulated", meaning that the peaks may be partially or fully Inverted. In some instances, such as when J_ is known, this may be of u t i l i t y for assignment purposes, but this is not often the case. One recourse is to calculate the phase-insensitive spectrum (magnitude, or power mode), but contributing dispersive components lead to broad lines being observed with this approach. If a high-resolution spin-echo spectrum with no J-modulation is required, one of two approaches may be taken. A train of closely spaced 180° pulses can be used to refocus the magnetization (rather than a single 180° pulse) and this removes the dependencies on J_ and the 3 9 diffusion and magnetic f i e l d gradient terms. Phase-sensitive display is used and narrow lines are observed. Alternately, one can make use of SEAS1*0 (spin-echo absorption spectroscopy), which requires collection of - 75 -the f u l l Hahn-echo (90° - x - 180°^ - AQN). With a fully symmetrical echo, Fourier transform (FT) and phase-insensitive display removes J_-modulation and gives absorption-like peaks. Although the two methods were not c r i t i c a l l y compared in our laboratory, we chose to use SEAS for these studies. The basic experiment was modified slightly to include a solvent null procedure -3 s presaturation prior to the 90° pulse. A time-domain window function which insured that there was zero intensity at the beginning and end of the acquisition* improved spectral quality. The f i r s t test of the technique was on a D20 solution of BSA (14 mg/0.4 ml). Fig. 1.4.12A shows the normal spectrum, with HOD solvent nulling; only broad spectral features are discernible. The SEAS experiment was then performed, with the indicated x delays (Fig. I.4.12B-F). Using x = 40 ms results In very l i t t l e signal intensity arising from the BSA protons, and so i t was decided to perform a l l subsequent experiments using this delay time. Results for the SEAS experiment (x • 40 ms) for a variety of molecules are given in Fig. I. 4.13. The question was: can SEAS be used to yield the spectrum of the glycan portion of a glycoconjugate with sufficient detail for this to be of u t i l i t y in their assignment? F i r s t l y , BSA and the B-allyl glycoside of acetylated glucosamine were co-dissolved in D20. SEAS (x = 40 ms) yielded spectrum 1.4.13B, which compares favourably with the spectrum of the monosaccharide in D20 *The Nicolet (NTCFTB) "trapezoidal multiplication" (TM) command was used. - 76 -5 0 6(ppm) Fig. 1.4.12 The 270 MHz AH spectra of BSA in D20 (14 mg/6.4 ml). (A) Single pulse experiment. (B)-(F) SEAS experiments with the indicated x values. The HOD was supressed by 3 8 presaturation. - 77 -C. x B. 1 - LJVAAJHA _J OH H O - ^ L^-^-o -BSA NHAc ^ O A c AcO-NHAc BSA .OAc AcO-+BSA NHAc S(ppm) Fig. 1.4.13 The 270 MHz XH SEAS spectra with T»40 ms. (A) BSA. (B) BSA plus a l l y l 8-acetoglucosamine. (C) B-AcetoglcNAc attached to BSA. (D) o- and B-glcNAc attached to BSA. - 78 -(data not shown). The carbohydrate spectrum is s t i l l clear even when using T up to 100 ms. We conclude that SEAS can be used to yield the spectrum of a rapidly tumbling monosaccharide in the presence of a protein macromolecule. Next, the neoglycoprotein having aceto-glucosamine attached to BSA (12) was studied and i t s SEAS spectrum is given in Fig. 1.4.13C* A pattern of peaks similar to the monosaccharide is evident, but much of the detail in coupling constants has been lost to the increased int r i n s i c line-widths. With x > 40 ms, the carbohydrate ring protons are rapidly lost, leaving only the acetyl peaks. The B-anomeric proton i s , as with a l l these spectra, hidden under the incompletely suppressed HOD peak. Next, a neoglycoprotein was prepared, bearing a 1:1 mixture of o- and 8-N-acetyl glucosamine. The SEAS spectrum of this material is given in Fig. 1.4.13D. Broad "humps" are visible in the carbohydrate region, but, again, l i t t l e detail i s evident upon close inspection, and the two anomeric proton signals cannot be detected, as hoped. Our data indicate that SEAS can be used to obtain a *H spectrum of the carbohydrate moiety of a neoglycoprotein. The line-widths of the signals appear to be broadened slightly in comparison with the monosaccharide, to the point where they obscure the details of the coupling constants in the sugar spectrum. This is unfortunate, as part of the great strength of *H NMR as a method of structure determination l i e s in this information. Whether this situation could be remedied by 41 the application of time-domain resolution enhancement functions was •Neoglycoproteins were synthesized as described in Ch. 1.4.2. - 79 -not determined, but might be a possibility. The solvent peak overlap with the anomeric protons was a problem which could probably be part i a l l y alleviated by elevation of temperature. This would also increase the mobility (and sharpen the lines of) the carbohydrate molecules, which would be an added bonus. In short, the spin-echo experiments described here bode well for the Incorporation of *H NMR in the scheme of procedures available for the study of glycan components of glycoconjugates. However, much work s t i l l has to be done before the u t i l i t y of the procedure can be accurately assessed. In 1975, Campbell et^ a l . described the use of spin-echo experiments to simplify protein spectra. Egmond et a l . studied the *H u 3 NMR spectrum of the sialo-glycoprotein, glycophorin. The broad, protein component was partially removed by the use of a convolution-difference time-domain function. Several aspects of the molecule's macroscopic behaviour were studied, and some sialo-sugar assignments were possible in the high-field region; the sugars were mobile, relative to the protein. Several workers have used the Hahn spin-echo to study liv i n g erythrocytes. Brown and co-workers assigned some of the unassociated, low molecular weight components of red blood cells (RBC's), and later 39 US *+6 went on to study RBC membrane transport using NMR. > Rabenstein in Alberta has reported on several aspects of the chemistry of small molecules inside and out of RBC's. ••7 The SEAS experiment was f i r s t described by Bax jet a l . and later by Hall and Sukumar who applied i t to a mixture of carbohydrates, lysozyme1*0 and RBC's.1*8 - 80 -1.4.5 Conclusions To sum up, we have described an efficient route to the synthesis of a r t i f i c i a l glycoproteins (neoglycoproteins). The method Is quick and high-yielding, and we anticipate i t could be of use, too, in the synthesis of carbohydrate haptens. Our use for this technology has been In NMR studies of conjugated sugars. 13 The study of neoglycoproteins by C NMR is facilitated by the fact that the sugar resonances resonate, largely, in a spectroscopically "silent" region, and, through their enhanced mobility, may be identified. It was determined that the corresponding methyl glycoside of the sugar(s) attached to the protein acts as a good source of reference for assigning 1 3C resonances, with our neoglycoproteins. No significant shifts in resonance frequency are observed when we compared three neoglycoproteins this way. Oligosaccharides bearing terminal galactose residues can be identified by oxidation with D-galactose oxidase. Significant shifts occur in galactosyl C-6, C-5 and C-4, and the carbon to which the galactosyl anomeric carbon is attached. With an ot-galactoside and 8-lactoslde, the shifts were very similar in their direction and magnitude. This approach would appear to be of great potential in the assignment of sugars bearing terminal galactose, provided a reasonable level of oxidation can be achieved. *H NMR studies of neoglycoproteins require that special steps be taken to observe the sugar resonances; this can be done by a spin-echo experiment, SEAS. The method relies on differentials in mobilities (and T 2) and can easily give the spectrum of a monosaccharide with a - 81 -protein present. When the monosaccharide i s attached to the protein, the differentiation is not as dramatic, and line broadening of the sugar resonances occurs to the point where i t obscures many J^-couplings. Several possibilities exist to remedy this and the other major limitation, viz., the overlap of the solvent resonance with the anomeric proton, but these were not assessed. We feel that these studies are, in their own right, of interest, but the hope is that the information derived w i l l ultimately be of use in the study of glycoproteins using NMR. - 82 -REFERENCES 1. W.F. Goebel and O.T. Avery. J. Exp. Med. 50, 521-531 (1929). 2. G.E. Means and R.E. Feeney. Biochemistry. 7, 2191-2201 (1968). 3. N. Jentoft and D.G. Dearborn. J. Bio l . Chem. 254, 4359-4365 (1979) . 4. G.R. Gray. Methods Enzymol. 50, 155-160 (1978), and references cited therein. 5. J.D. Aplin and J.C. Wriston, Jr. CRC C r i t . Rev. Biochem. 10, 259-306 (1981). 6. CP. Lee and Y.C. Lee. Adv. Carbohydr. Chem. Biochem. 37, 225-281 (1980) . 7. M.A. Bernstein and L.D. Hall. Carbohydr. Res. 78, C1-C3 (1980). 8. P.A. Gent and R. Gigg. J. Chem. Soc. Perkln Trans. 1. 361-363 (1975), and others in the series. 9. R.T. Lee and Y.C. Lee. Carbohydr. Res. 37, 193-201 (1974). 10. From the series:- Methods in carbohydrate chemistry. Academic Press, New York, Vols. 2 and 5. 1963 and 1965, respectively. 11. K. Igarashi. Adv. Carbohydr Chem. Biochem. 34, 243-283 (1977). 12. J. Balatoni. B.Sc. Thesis. University of British Columia. 1981. 13. H.O. House. Modern synthetic reactions. Benjamin, New York. 1972. Chapter 7, and references cited therein. 14. J.J. Pappas, W.P. Rlareney, E. Gancher, and M. Berger. Tetrahedron  Lett. 4273-4278 (1966). P.S. Bailey and R.E. Erickson. Org.  Synthesis. 41, 41-45 (1961). 15. P.S. Bailey. Chem. Rev. 58, 925-1010 (1958). 16. M. Dubois, K.A. Gill e s , J.K. Hamilton, P.A. Rebers, and F. Smith. Anal. Chem. 28, 350-356 (1956). 17. B. Schwartz and G.R. Gray. Arch. Biochem. Biophys. 181, 542-549 (1977). 18. R.T. Lee and Y.C. Lee. Carbohydr. Res. 77, 149-156 (1979). 19. R.T. Lee and Y.C. Lee. Biochemistry. 19, 156-163 (1980). 20. R.T. Lee and Y.C. Lee. Carbohydr. Res. 101, 49-55 (1982). - 83 -21. M. Friedman, L.D. Williams, and M.S. Misri. Int. J. Peptide Protein Res. 6, 183-185 (1974). 22. A. Allerhand. Accts. Chem. Res. 11, 469-474 (1978), and references cited therein. 23. P.E. Pfeffer, K.M. Valentine, and F.W. Parrish. J. Amer. Chem.  Soc. 101, 1265-1274 (1979); Ibid., 7438 (correction). 24. P.A.J. Gorin. Adv. Carbohydr. Chem. Biochem. 38, 13-104 (1981). 25. See Chaps 1.2 and 1.3, and references cited therein. 26. A. Maradufu and A.S. Perlin. Carbohydr. Res. 32, 93-99 (1974). 27. J.N.C. Whyte and J.R. Englar. Carbohydr. Res. 57, 273-280 (1977). 28. K. Bock and C. Pedersen. J. Antibiotics. 27, 139-140 (1974). 29. P.L. Harris and E.R. Thornton. J. Amer. Chem. Soc. 100, 6738-6745 (1979). 30. K. D i l l and A. Allerhand. J. Biol. Chem. 254, 4524-4531 (1979). 31. E. Berman, A. Allerhand, and A.L. De Vries. J. Biol. Chem. 255, 4407-4410 (1980). 32. H.J. Jennings, C. Lugowski, and D.L. Kasper. Biochemistry. 20, 4511-4518 (1981). 33. E. Berman, D.E. Walters, and A. Allerhand. J. Biol. Chem. 256, 3853-3857 (1981). 34. R. Prohaska, T.A.W. Koerner, Jr., I.M. Armltage, and H. Furthmayr. J. Biol. Chem. 256, 5781-5791 (1981). 35. E. Berman and A. Allerhand. J. Biol. Chem. 256, 6657-6662 (1981). 36. W.J. Goux, C. Perry and T.L. James. J. Biol. Chem. 257, 1829-1835 (1982). 37. K. Barrett-Bee, G. Bedford, and P. Loftus. Bioscience Reports. 2, 257-263 (1982). 38. R. Freeman and H.D.W. H i l l . Determination of spin-spin relaxation times in high-resolution NMR. Edited by L.M. Jackman and F.A. Cotton. Academic Press, New York. 1975. pp. 131-162. 39. K.M. Brindle, F.F. Brown, I.D. Campbell, C. Grathwohl, and P.W. Kuchel. Biochem. J. 180, 37-44 (1979), and references cited therein. - 84 -40. L.D. Hall and S. Sukumar. . J. Magn. Reson. 38, 559-564 (1980). 41. J.C. Lindon and A.G. Ferrige. Prog, in NMR Spectroscopy. 14, 27-66, 1980. 42. I.D. Campbell, CM. Dobson, R.J.P. Williams, and P.E. Wright. FEBS  Lett. 57, 96-99 (1975). 43. M.R. Egmond, R.J.P. Williams, E.J. Welsh, and D.A. Rees. Eur. J.  Biochem. 97, 73-83 (1979). 44. F.F. Brown, I.D. Campbell, P.W. Kuchel, and D.C. Rabenstein. FEBS  Lett. 82, 12-16 (1977). 45. F.F. Brown and I.D. Campbell. Phil. Trans. R. Soc. Land. B. 289, 395-406 (1980). 46. D.L. Rabenstein, S.J. Backs, and A.A. Isab. J. Amer. Chem. Soc. 103, 2836-2841 (1981), and others in the series. 47. A. Bax, A.F. Mehlkopf, and J. Smidt. J. Magn. Reson. 35, 373-377 (1979). 48. S. Sukumar. Ph.D. Thesis. University of British Columia. 1980. - 85 -CHAPTER 1.5 EXPERIMENTAL FOR SECTION I 1.2 ESR Experiments 1. Reagents A l l r e a g e n t s w e r e p u r c h a s e d a n d u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n : f e t u i n ( T y p e I V , S i g m a ) , BSM ( B o e h r i n g e r M a n n h e i m ) , s o d i u m p e r i o d a t e ( F i s h e r ) , s o d i u m c y a n o b o r o h y d r i d e ( A l d r i c h ) , TEMPAMINE ( A l d r i c h ) , S e p h a d e x G - 2 5 m e d i u m ( P h a r m a c i a ) , V i b r i o c h o l e r a n e u r a m i n i d a s e ( B e h r i n g w e r k e , 500 U / m l ) , g a l a c t o s e o x i d a s e ( S i g m a , 227 U/mg p r o t e i n ) . C e n t r i f l o CF 25 c o n e s ( r e t e n t i o n l i m i t 2 5 0 0 0 amu) w e r e p u r c h a s e d f r o m A m i c o n . 2. ESR Measurements E S R s p e c t r a w e r e r e c o r d e d a t X - b a n d u s i n g a V a r i a n E - 3 s p e c t r o m e t e r i n t h e d e r i v a t i v e a b s o r p t i o n m o d e . D e r i v a t i v e s i g n a l s w e r e i n t e g r a t e d w i t h a P a c i f i c P r e c i s i o n C o . M P - 1 0 1 2 A i n t e g r a t o r ; s e c o n d d e r i v a t i v e s w e r e c a l c u l a t e d b y c u t t i n g o u t a n d w e i g h i n g t h e p e a k a r e a s , a n d c o m p a r i s o n w i t h a s t a n d a r d i z a t i o n c u r v e d e r i v e d f r o m ESR s p e c t r a o f f r e s h l y p r e p a r e d s a m p l e s o f s p i n l a b e l o f k n o w n c o n c e n t r a t i o n . S p e c t r o m e t e r s e t t i n g s - m o d u l a t i o n a m p l i t u d e , f i l t e r t i m e c o n s t a n t a n d s c a n r a t e - w e r e c h o s e n i n e a c h c a s e t o a v o i d s p e c t r a l d i s t o r t i o n s , a n d p o w e r l e v e l s w e r e n o n - s a t u r a t i n g . A l l a q u e o u s s a m p l e s ( 5 0 - 7 3 u l w e r e p l a c e d I n a f l a t , h i g h - q u a l i t y g l a s s c e l l ( J . S c a n l o n C o . - c a p a c i t y 7 3 u l ) . - 86 -3. Fetuin-SL 15 mg of fetuin (4.14 umoles NANA) was dissolved in 2.7 ml H20, to which was added 14.6 umoles NalO^ and the solution kept at 0° for 35 minutes. The reaction was quenched with an excess of Na2S203 and KI. The solution was concentrated using CF 25 membrane cones. A 15 molar excess of SL-NH2 and 100 molar excess of NaCNBH3 were added in 1-2 ml of solution at pH 8-9; this was kept at room temperature for 2 hours. The solution was passed through a Sephadex G-25 column (eluant, 0.02% azide; column dimensions, 2.2 cm (i.d.) * 20 cm) and the excluded volume collected. The sample was freeze-dried and stored at -4°C unt i l spectroscopic measurements were made. 4. Asialofetuln -SL 15 mg of fetuin was dissolved in 5 ml of buffer (pH 6.9, 0.2 M NaOAc, 0.15 M NaCl, 0.002 M CaCl 2), to which was added ca. 20 units of galactose oxidase and 30 units of neuraminidase. The latter was added in equal aliquots, one at the start of the experiment, and the second after 24 hours. The solution was maintained at 37° for 48 hours. After Centriflo CF 25 concentration, the coupling procedure was as for sialo-fetuin (3). 5. BSM-SL 15 mg of BSM (16 umoles s i a l i c acid) was f i r s t saponified (removal of 0_-acetyl substituents on NANA extracyclic t r i o l ) by exposure to 1.5 ml of 1 M NaOH for 30 min. at room temperature, and neutralized with 1 M HC1. Desalting was affected by Centriflo CF 25 membranes, and the material exposed to NalO^ (43 umoles) for 35 min. at 0°. A l l other procedures were as for fetuin-SL (3). - 87 -6. AslaloBSM-SL The native BSM was saponified as above (5) and the neuraminidase and galactose oxidase treatment performed as in (4). The coupling of TEMPAMINE proceeded as in (3). 7 . Erythrocytes-SL Freshly drawn venous blood from a healthy donor was packed, and to 7 ml of packed erythrocytes, 0.5 ml of 0.01 M NalOi^ was added. The solution was kept at room temperature for 15 min. The oxidation was terminated by the removal of reagents - three washes with physiological saline solution, with RBC precipitation by centrifugation (400 g, 10 min). A 25 fold molar excess of SL-NH2 and 100 fold molar excess of NaCNBH3 were added and the saline solution (pH 7.5-8) kept at room temperature for 2 hours. With centrifugation and washing, some c e l l l y s i s was indicated by a slightly red supernatant. The labelled erythrocytes were dialysed against PBS buffer for 3 days at 4° - the buffer was changed daily. The cells were spun down and ESR spectra run on packed erythrocytes. 1.3 *H NMR 2H NMR spectra were recorded at 61.4 MHz (B Q 9.4 T) on a Bruker WH-400 instrument (Karlsruhe, West Germany, and U.B.C.). Samples were either dissolved in d i s t i l l e d water (spectra in Fig. 1.3.3) or deuterium-depleted water (Aldrich). The ca. 1.5 ml sample, in a 10 mm tube, was run at room temperature without field-frequency lock and proton decoupling. A 25 us pulse was used and the acquisition time was 0.85 s. BSM and fetuin were activated by the procedure detaile in - 88 -Chapter 1.2. Typically, 125 mg of material was used. For the deuteration, the aldehyde-containing compound was dissolved at pH 9, and ca. 2.5 mg NaBDH (MSD) added to the solution at 0°. The solution was allowed to reach room temperature and l e f t for one hour, when the pH was lowered to ca. 5 to decompose unreacted NaBD^. Desalting was performed either on a Sephadex G-25 (medium) column, or using Centriflo PM 10 size-exclusion membranes (Amicon). With asialofetuin, catalase was not used in conjunction with galactose oxidase, and this probably accounts for the rather low signal-to-noise (low level of activation) in this instance. Fetuin was labelled at lysine residues with deuterium by dissolving the glycoprotein in a borate buffer solution (pH 9.0, 0.1 M) at a concentration of ca. 100 mg/200 ml. Sodium cyanoborohydride (15 moles/mole lysine) was added, followed by a ca. 200 fold molar excess of hexadeuterioacetone, added drop-wise to prevent protein precipitation. The solution was stirred overnight at room temperature, and unreacted hexadeuterioacetone removed carefully under reduced pressure. Further desalting procedures were as described previously. 1.4 Neoglycoproteins Chemicals were often used as supplied from the chemical companies. A l l y l alcohol (Aldrich) was dried over calcium sulphate and stored under N 2. Chloroform was rendered alcohol-free by washing with cone. I^SOit, neutralizing with a l k a l i washes and dried over 4 A molecular sieves. Mercuric oxide and bromide were from BDH and mercuric cyanide from ICN. Acid resins were from Bio-Rad. Melting points were determined on a Fisher-Johns apparatus and are uncorected. - 89 -Ozone was generated by a Welsbach Ozonator (90 V with 2 p.s.i. Input 0 2 pressure), dimethyl sulphide was from Sigma and sodium cyanoborohydride from Aldrich. Bovine serum albumin was purchased from Miles and passed down a Sephadex G-25 column prior to use, to remove low molecular weight impurities. A l l a l l y l glycosides were prepared by standard methods and checked for purity by TLC,  lB. NMR and melting points. O-Acetylations were performed with pyridine and acetic anhydride in the cold. Acetoglycosyl bromides were prepared from the per-O-acetylated sugar using 30% HBr in acetic acid. Koenigs-Knorr glycosidations were performed either using silver carbonate (undecenyl 8-D-acetoglucopyranoside), mercuric cyanide (2-acetamido-3,4,6-tri-0_-acetyl allyl-8-D-glycopyranose), or mercuric oxide and mercuric cyanide ( a l l other a l l y l p-D-acetoglycosides). De-O-acetylations were with a methanolic solution of the sugar to which was added a freshly prepared solution of 0.1 M sodium methoxide in methanol. For example, a l l y l a-D-galactopyranose was prepared thus. One gram of Dowex 50W-X8 (H +) resin was prepared by washing several times with methanol (to remove high molecuar weight impurities), and then washed several times with a l l y l alcohol. Galactose (1.8 g) was dried (over NaOH in a vacuum desiccator) and to this was added the resin and 20 ml of dry a l l y l alcohol. The apparatus was fitted with a reflux condenser and heated under reflux (B.Pt. 98°) for 20 hours, by which time a l l the galactose had dissolved and the reaction judged completed - 90 -by TLC (eluant, CHCl3/MeOH/HOAc/H20, 25:15:4:2). The a l l y l alcohol was removed by rotary evaporation at reduced pressure, and the a-glycoside precipitated preferentially from an ethanolic solution of the o i l at -4°. The isolated yield was 10%, and the melting point 146-148° ( L i t . 143-145°). The synthesis of a B-glycoside is typified by a l l y l B-fi-glucopyranoside. 2,3,4,6-Tetra-O-acetyl-a-g-glucopyranosyl bromide (1.5 g) was added to a mixture of 10 ml dry a l l y l alcohol, 10 ml alcohol-free chloroform, 1.0 g "Drierite", 0.65 g HgO and 0.05 g HgB r2 , and stirred for 48 hours. The mixture was filtered through a layer of Cellte, and evaporated to dryness. The sugar was extracted into dry chloroform; any remaining mercuric salts were precipitated and removed by f i l t r a t i o n . The f i l t r a t e was evaporated to dryness and the product crystallized from an ethanolic solution at -4°, in 70% overall yield. M.Pt. 86° (Lit. 86/88°). The ozonolysis was performed as follows. The sugar was dissolved in methanol (unprotected) or MeOH/CH2Cl2, 4:1 (protected sugar) at a concentration of 1 mmole per 5 ml solvent. The solution was cooled to -60° (acetone/dry ice) and ozone-enriched oxygen bubbled through (5-10 min/mmole) until the methanolic solution went pale blue, indicating an excess of ozone. The solution was flushed with N2 to remove this ozone, and excess dimethyl sulphide (DMS; > 2 mole/mole sugar) added. The solution was removed from the coolant and allowed to reach room temperature, where i t was stirred for 2 hours. Excess DMS, methanal and methanol were removed under reduced pressure. With blocked sugars, the - 91 -sirup was dissolved in methylene chloride and washed several times with water to remove DMSO. In a typical reductive amination reaction, BSA (18 mg; 0.015 mmole lysine), aldehydo sugar (16.2 mg; 0.074 mmole) and NaCNBH3 (22.8 mg; 0.38 mmole) were dissolved in 6 ml water, and the pH adjusted to 9.0 with 0.1 M NaOH. The solution was lef t overnight, and then passed down a Sephadex G-25 column (0.02 % sodium azide eluant), where the excluded volume was collected. This was freeze-dried and the neoglycoprotein analyzed by the phenol-sulphuric acid method, measuring the absorption at 490 nm. With a blocked sugar, the procedure was identical except that the reaction solution was H20/MeOH, 3:1. The blocked aldehydo sugar (In MeOH/H20, 2:1) was added over 10 min. Methanol was removed under reduced pressure taking care to minimize frothing. If any solids were suspended in the solution, they were filtered off prior to desalting, usually by dialysis (3 days against d i s t i l l e d H 20). Note that 2-acetamido-3,4,6-trio-0_-acetyl allyl-8-D-glucopyranose Is moderately soluble in water, and methanol was not necessary In the reaction solution. 1.4.2 1 3C NMR Most neoglycoprotein spectra were recorded on solutions (ca. 150 mg/1.8 ml D20) at 308 K. The spectrometer used was a Bruker WH-400, equipped with a single-frequency probe for 1 3C (100.6 MHz). The spectral width was 200 ppm and the recycle time ca. 0.8 s. An excitation pulse (18 us for neoglycoproteins or 12 us for - 92 -m o n o s a c c h a r i d e s ; 9 0 ° «* 22 u s ) was f o l l o w e d b y t h e a c q u i s i t i o n o f 16 K d a t a - p o i n t s , w h i l e b r o a d - b a n d e d *H d e c o u p l i n g was e m p l o y e d . S i g n a l - a v e r a g i n g g e n e r a l l y t o o k p l a c e o v e r n i g h t ( c a . 160 0 0 0 s c a n s ) . F o r t h e e n z y m e s t u d i e s , g a l a c t o s e o x i d a s e ( S i g m a o r W o r t h i n g t o n ) w a s a d d e d ( c a . 20 U / 1 5 0 mg n e o g l y c o p r o t e i n ) t o g e t h e r w i t h 5 u l c a t a l a s e ( S i g m a ; 6 0 , 0 0 0 U ) t o c a . 150 mg o f n e o g l y c o p r o t e i n . T h e s o l u t i o n was l e f t o p e n t o t h e a t m o s p h e r e ( 0 2 ) o v e r n i g h t a t r o om t e m p e r a t u r e , a n d t h e 13 C NMR s p e c t r u m was r e c o r d e d . W h e r e s p e c i f i e d , s o d i u m b o r o h y d r i d e r e d u c t i o n was p e r f o r m e d (2 m g , f r o m M C B ) . 1.4.3 *H SEAS Experiments F o r t h e *H NMR ( S E A S ) e x p e r i m e n t s , s a m p l e s w e r e p r e p a r e d b y d i s s o l v i n g i n D 2 0 a n d f r e e z e - d r y i n g s e v e r a l t i m e , t o r e m o v e H 2 0 a n d e x c h a n g e a b l e p r o t o n s . T h e s p e c t r a w e r e r e c o r d e d a t 270 MHz u s i n g a h o m e - b u i l t s p e c t r o m e t e r , b a s e d o n a n O x f o r d s u p e r c o n d u c t i n g m a g n e t (BQ 6 . 3 5 T ) , a B r u k e r W P - 6 0 c o n s o l e , a n d N i c o l e t 1 180 c o m p u t e r a n d 2 9 3 B p u l s e - p r o g r a m m e r . T h e d e u t e r i u m l o c k , q u a d r a t u r e d e t e c t i o n , a n d p r o t o n d e c o u p l e r u n i t s w e r e m a n u f a c t u r e d i n t h e D e p a r t m e n t o f C h e m i s t r y , U . B . C . ( D r . G . A . M o r r i s a n d M r . T . M a r c u s ) . S p e c t r a l w i d t h s w e r e 3 0 1 2 H z , w i t h 8K d a t a - p o i n t s c o l l e c t e d . P r e s a t u r a t l o n a t t h e HOD r e s o n a n c e f r e q u e n c y wa s f o r 3 s e c o n d s ; no r e l a x a t i o n d e l a y was u s e d . T y p i c a l l y , 4 0 0 s p e c t r a w e r e a c q u i r e d f o r e a c h t a u v a l u e . D a t a - p r o c e s s i n g i n v o l v e d t h e u s e o f a t r a p e z o i d a l w i n d o w ( N T C F T B " T l - 5 5 " ) a n d 1 .2 H z e x p o n e n t i a l l i n e - b r o a d e n i n g . M a g n i t u d e - c a l c u l a t i o n s p e c t r a w e r e r e c o r d e d . T h e 9 0 ° a n d 180 ° p u l s e s w e r e c h e c k e d b e f o r e e a c h e x p e r i m e n t , a n d t y p i c a l v a l u e s a r e 10 u s a n d 21 u s , r e s p e c t i v e l y . - 93 -SECTION II - 94 -CHAPTER II.1 INTRODUCTION High-resolution nuclear magnetic resonance (NMR) spectroscopy has deservedly earned a place as one of the most universal and powerful tools in organic structural determination. The NMR phenomenon was f i r s t reported for bulk solids by Bloch and coworkers1 and Purcell and coworkers2 In 1945, who were jointly awarded in 1952 the Nobel prize for their discovery. Improvements in magnetic f i e l d homogeneity soon resulted in measurement of higher resolution spectra, which revealed f i r s t the chemical shift, and then the spin coupling phenomena. In 1966 Ernst and Anderson3 demonstrated the application of Fourier transformation (FT) of pulse responses, and along with signal averaging this led to further increases in sensitivity. With the concomitant integration of di g i t a l computers, insensitive but important nuclei such 13 15 as C and N could be routinely observed. The next major land-mark in the development of high-resolution NMR was the application of super-conducting materials to magnet construction. Starting In the late 1960's the then common 100 MHz instruments were soon superceded by 200-360 MHz devices, and by the late 1970'8, 400 MHz instruments were common, and 500 and 600 MHz instruments were being described at NMR congresses. These high f i e l d instruments gave the anticipated increases In dispersion and inherent signal-to-nolse. At the conceptual level, perhaps the most startling development came in 1971, when Jeener suggested the possibility of two-dimensional - 95 -(2D) NMR experiments. Interestingly, although i t was five years before 5 the detailed quantum-mechanical theory of the experiment was presented, many other experiments were described in the literature, most from the research groups of Ernst, in Switzerland, and Freeman, in England. Although the technological capability to perform most of these experiments has been commercially available for at least five years (since 1978), surprisingly few reports on their applications have appeared. In the biolgical area, the collaborative efforts of Ernst and Wuthrich have amply demonstrated the potential for studies of small proteins. 6 Hall's group in Canada had an early interest in the evaluation of the technology as applied to structural organic chemistry; this thesis forms a part of that tradition. In this work, the author has undertaken a systematic, objective appraisal of some of the available 2D NMR experiments, in the light of their application to structural investigations in organic and natural-products chemistry. It became evident early on that 2D experiments were not a panacea to a l l the problems associated with the investigation of complex molecules, and modified forms of certain ID experiments found an important place in the general protocols which emerged. In a l l cases, the fundamental objective was the efficient integration of spectroscopic methods, the most expeditious route to the required answers, and the conditions to which each experiment was best suited. It was decided that before attempting to apply the methods to complex molecules, a thorough assessment of each experiment using a - 96 -single simple exemplar would prove beneficial. This lengthy investigation Is detailed in Chapter II.2, where the data are presented along with theoretical aspects. A simple protected o-glucopyranoside was chosen, since i t is conformationally rigid and has a reasonably simple, first-order spectrum at 270 or 400 MHz. As w i l l be seen, these experiments are an important, integral component of the research of the thesis, since an equivalent array of experiments has not previously been systematically applied to a single molecule. Furthermore, because this i n i t i a l investigation of the procedures so successfully provided a broad base of expertise and experience, a l l subsequent studies could be performed rather eff i c i e n t l y , and the descriptions of subsequent chapters are correspondingly concise. This was a very welcome development. Chapter II.3 describes a protocol' based on NMR for the de novo sequencing of oligosaccharides. Vliegenthart's and Dabrowski's groups have already exposed some of the power of NMR in the structural analysis of glycopeptldes and ceramlde oligosaccharides, respectively, where they found great u t i l i t y . Dabrowski's more recent work has elements In common with our procedures in that reliance on chemical shifts Implicit in Vliegenthart's approach as a basis for assignment was avoided. The intention was to develop a general procedure whereby the complete primary structure of an oligosaccharide could be determined by NMR with only reliance on typical vicinal coupling constants for the carbohydrate ring protons. As w i l l be seen, the combination of ID and 2D methods found suitable proved more than adequate for the disaccharide glycoside, which was used as a model. A more complex biological material, digoxin, - 97 -was studied in Chapter II.5. Chapter II.4 deals with the assignment and structural investigation of brucine. The main objective here was to find the most expeditious route to the assignment of i t s *H and i 3C NMR spectra, and conformational information on the molecule. Further, the author also attempted to place himself in the position of a professional applied spectroscopist to evaluate the performance of the required experiments under "survey conditions". This required the minimum knowledge of the molecule's spectroscopic characteristics before performing experiments under "routine" conditions where demands on instrument time is a major consideration. This is in sharp contrast to the developmental situation in Ch. II.2, which is conceptually akin to a spectroscopy research study. The final study in Chapter II.5 is on a steroidal glycoside, 1 13 digoxin. Here again, the object was to assign the H and C NMR spectra using the currently available techniques. The glycone is considered as a c r i t i c a l test of the oligosaccharide sequencing methodology described in Ch. II.3. Although i t consists of only three sugars, the situation is complicated by the fact that they are identical In nature and linkage, and are 2-deoxy sugars with methylene resonances overlapping with the bulk of the steroid protons. The cardenolide (steroid genin) is equally Intractable because of i t s low extent of substitution and conformation. As w i l l be seen, these complexities are compounded by the fact that the molecule self-associates in a polar solvent, giving broad lines for the steroid moiety, especially - 98 -troublesome In the H NMR spectrum, the molecule was studied at 500 MHz and, for the f i r s t time in this work, significant limitations became evident In some 2D experiments. Anticipating somewhat the conclusions of Section II, the author believes that this thesis points to an increasingly bright future for NMR spectroscopy. It is clear that certain protocols for structure determination are extremely powerful under certain regimes; thus, rapidly tumbling, medium molecular weight molecules can most certainly be studied under "survey conditions", demanding comparatively l i t t l e instrument time. However, although some larger molecules can definitely benefit from use of the highest obtainable static f i e l d and a modified approach, some intrinsic limitations exist. - 99 -REFERENCES 1. F. Bloch, W.W. Hansen, and M. Packard. Phys. Rev. 69, 127 (1946). 2. E.M. Purcell, H.C. Torrey, and R.V. Pound. Phys. Rev. 69, 37-38 (1946). 3. R.R. Ernst and W.A. Anderson. Rev. Sci. Instr. 37, 93-102 (1966). 4. J. Jeener. Ampere International Summer School, Basko Polje, Yugoslavia. 1971. 5. W.P. Aue, E. Batholdi, and R.R. Ernst. J. Chem. Phys. 64, 2229-2246 (1976). 6. G. Wagner, A. Kumar, and K. Wuthrich. Eur. J. Biochem. 114, 375-384 (1981), and others in the series. 7. M.A. Bernstein and L.D. Hall. J. Amer. Chem. Soc. 104, 5553-5555 (1982). 8. J.F.G. Vliegenthart, H. van Halbeek, and L. Dorland. Pure Appl.  Chem. 53, 45-77 (1981), and references therein. 9. J. Dabrowski, P. Hanfland, and H. Egge. Methods Enzymol. 83, 69-86 (1981), and references therein. - 100 -CHAPTER II.2 HIGH RESOLUTION NMR METHODS II.2.1 General Theory Although some understanding by the reader of pulsed Fourier Transform (FT) high resolution NMR w i l l be assumed, i t i s necessary to briefly review a few relevant concepts to ensure that the reader has access to the relevant nomenclature. In what follows below, we Introduce three models, each of which gives different insight to the effect of a pulse of radlofrequency energy on an ensemble of nuclear spins. We consider an ensemble of Identical spin-1/2 nuclei in a static magnetic f i e l d , JJ 0. Two energy levels, o and B, w i l l exist, differing in energy by AE: AE - yfc B 0 [II.2.1] Y is the magnetogyric ratio of the nuclide, and fi i s the reduced Planck's constant (h/2n). At thermal equilibrium, nuclei are distributed between the two energy levels by a Boltzmann distribution, with the resultant net macroscopic magnetization termed My. It i s convenient to define a frame of reference as shown in Fig. II.2.1A. The applied magnetic f i e l d , BJJ i s defined to l i e parallel to the z-axis. Magnetic moments (vectors), u, are required to precess about JB0 at the Larmor angular velocity, co0: too " "Y B 0 [II.2.2] Expressed in frequency units, the Larmor precessional frequency, v 0, - 101 -is given by: vo - -y Bo/2 n [II.2.3] There is no phase coherence in the x-y plane and consequently no resultant magnetization; the resultant magnetization, MQ, lies on the z-axis. To induce resonance, a second radio frequency (RF) f i e l d , j3\, must be applied in the x-y plane. Resonance is attained when the frequency of B>i exactly matches VQ. It is now convenient to redefine the terms of reference in Fig. II.2.1.A.; now the reference frame i t s e l f is rotating about the z axis with the same sense and frequency, v, as the RF f i e l d , JJi. When v » vo, magnetic moments are effectively motionless, or moving slowly (vo - v). The x and y axes are re-labelled x' and y' to make the distinction, as in Fig. II .2.1.B (where v «• vo). This is the "rotating reference frame". Let us now consider the effect of B_i on Mn> placed along the x' axis, with the resonance condition met. Bj exerts a torque on MQ, tipping i t away from the z axis. If B^ is applied for a time, tp* (the "pulse length"), M0 rotates about x' by an angle G, defined by: *A square pulse of length jtp (time domain) of frequency vo corresponds, in frequency space, to a range of frequencies centred at vo and extending from V n - t " 1 to vo + t " 1 . Hence, with t typically of ~ P ~ P ~P the order of microseconds, a range of frequencies extending over tens or hundreds of KHz w i l l exist. - 102 -F i g . I I . 2 . 2 A s t y l i z e d AX s p i n s y s t e m - 103 -9 - Bi t Y/2H [II.2.4] — -p There i s now a component of M along the y' axis, My. This perturbs the spin system from i t s thermal equilibrium with respect to i t s surrounding (the l a t t i c e ) . The Boltzmann equilibrium state is re-established by leakage of magnetization into the lattice; the first-order rate-constant for this process is Tj, the spin-lattice (longitudinal) relaxation rate. A second rate constant, T_2 (the spin-spin, or transverse relaxation rate) describes the exponential decay of transverse magnetization in the x'-y' plane. Following application of the perturbing pulse, measurement of the magnetization in the x'-y' plane as a function of time produces the free-induction decay (FID) - this measure of My vs. time, has the form Mo cos(tot) exp(-£Ar2). Fourier-transform (FT) of this time-dependent function yields the frequency-space spectrum as a single Lorentzian line. This is the key to pulsed FT NMR spectroscopy. Let us now briefly look at a homonuclear AX system, In the rotating reference frame, with the spectrometer frequency, to, placed close to that of the high-field transition of spin A; the four line, frequency-domain spectrum i s stylized in Fig. II.2.2. Fig. II.2.3 follows the behavior of the magnetization vectors in the x-y plane, Ignoring T_j relaxation. At thermal equilibrium, a l l four vectors are aligned along the z-axis (Fig. II.2.3A). After application - 104 -c. Fig. II.2.3 The action of an AX spin system's vectors, (A) at equilibrium, (B) after a 90° pulse and (C) after some time to allow dephasing. (Solid line denotes A spin vectors and broken lines, X). A. B. Fig. II.2.4 Energy Level diagrams of AX spin-system (A) showing transition probabilities (B). 0 and ±6 represent relative equilibrium populations. - 105 -of a perfect 90° pulse to a l l spins, the vectors l i e along y' (Fig. II.2.3B). Since a l l four vectors have a different angular velocity than that (to) of the rotating reference frame, with time these precess in the x'-y' plane (Fig. II.2.3C), and dephase with respect to each other. The average of lines t o i 2 and to 3 1 + w i l l precess at angular velocity, 0^ (the difference between to and 10*) and similarly, to,, and to„, precess slower at Each doublet w i l l have a relatively slow (s) and fast (f) precessing component. As we shall see later, this model provides a convenient description of the concepts of spin precession and phase coherence. We now consider the same spin system using the "energy-level" model. The homonuclear AX system has four energy levels: oca (aB, pa) and 88 in increasing energy,* as depicted in Fig. II.2.4A. In this model, the 90° pulse Induces the four single-quantum transitions, (Fig. II.2.4B) and i t i s these which are detected. Note that the zero- and double quantum transitions (W0 and W2 respectively) are not induced by this single pulse experiment. Although the rotating reference frame and energy level models discussed above are simple and provide reassuring, pic t o r i a l explanations for many NMR experiments, they are often i l l - s u i t e d for explanations of many processes. For that reason, the density matrix formalism, which i s more complicated but exact in a l l cases, w i l l now be *In the heteronuclear case, the aP energy state does not have the same energy as 6a. In the homonuclear case their energies are almost equal, as indicated by their equal equilibrium populations. - 106 -brie f l y introduced. For simplicity, i t is worthwhile using a diagrammatic formalism. We w i l l ignore relaxation effects, as this simplifies the discussion. An AX system is generally represented by a four-by-four matrix, a, constituting a l l the possible products of the four energy states: a a a B B a B B o a o i l a 1 2 °13 a\u, a B °21 a ^ 0 2 3 a 2 1 + 8a °"31 a 3 2 a 3 3 a 3 4 B B a m o-»*2 °"<+3 [II.2.5] At thermal equilibrium, the density matrix, c(0), w i l l have unequal Boltzmann population terms (P°) along the diagonal, as in Eq. II.2.6a. Inserting the relative populations (see Fig. II.2.4A) gives the matrix Eq. II.2.6b, in which 6 is the population difference between the a B or B a state, and a a or B B . o(0) p° 0 0 0 0 pO 0 0 0 0 p° 0 0 0 0 p° [II.2.6a] - 107 -a(0) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -6 [II.2.6b] Consider now the effect of the pulse (rotation) on o(0). The new density matrix, a', is given by: a' = R_ o(0) R + [II.2.7] 90 where R + are rotational matrices. For a 90° pulse, R + w i l l take on a special form and, omitting the details, we obtain, ex p l i c i t l y , Eq. II.2.8a which may be stylized as in Eq. II.2.8b. 0 216 2i6 0 -216 0 0 2i6 -216 0 0 2i6 0 -216 -216 0 [II.2.8a] [II.2.8b] - 108 -If the reader compares Eq. II.2.8a with Eq. II.2.5, i t w i l l be clear that only single-quantum transitions have non-zero values, and hence are excited. The zero- (o-23» ^32^ o r double- ( o ^ . a^\) transitions have zero values. Consider now the behaviour of these components as the matrix is allowed to "evolve" with time, t. The 012 term, for example, previously 2i6 now becomes 2i6 exp ( i (012 t ) . The exponential term contains sin and cos contributions and we may loosely represent the "detected" matrix, o'(t), as: o'(t)» [II.2.9] - 109 -In 2D experiments at least 2 pulses are necessary and where necessary, these w i l l be discussed in light of the density matrix formalism introduced in i t s s t y l i s t i c form here. II.2.2 Spectral Simplification The purpose of this section i s to introduce some of the methods which are available to "simplify" otherwise complex NMR spectra. II.2.2.1 Via T^ i n *H NMR Spectroscopy 5* 6 Spectra can often by simplified by taking advantage of the fact that different types of protons in a molecule frequently have widely different spin-lattice relaxation times. For example methylene signals can quite easily be "separated" from methine, when overlap causes complications and impedes spectral assignment. The concept rests on the fact that methylene protons w i l l relax faster than methine protons, and w i l l a l l relax at approximately the same rate, since their mutual relaxation is so dominant. Consider the Inversion recovery T_x experiment (T1IR); the pulse sequence i s : (RD - 180° - ^  - 90° - AQN)n, where RD i s a relaxation delay which Is set >^  5 * T_i (longest). According to the rotating reference frame model (Fig. II.2.5), the 180° pulse inverts a l l spin populations, placing the Mo vector along the -z axis. During the delay time, t, M^  returns towards i t s equilibrium - 110 -D. F. Fig. II.2.5 The rotating reference frame vector model of the T_i inversion-recovery experiment. Vectors are shown for a short t_ value (A,B,C,E) and a relatively long one (A,B,D,F). Fig. II.2.6 Plot of z-magnetlzation vs time in a Ti inversion recovery experiment, indicating the null point. - I l l -position at the rate, expC-t/T^). Since only x'-y' magnetization can be detected, a second 90° ("read") pulse i s applied to determine the magnitude of Mg, the z magnetization. This two-pulse sequence i s performed for a number of different t_ values. Results for trldeuteriomethyl 2,3,4,6-tetra-0j-(trideuterioacetyl)-a-I>-glucopyranoside (1) are given in Fig. II.2.7.* We can see that for short t_ values the peaks are negative-going (Figs. II.2.5C, E) eventually 1 : R - C0.CD3, R* - CD3 2 : R - CO.CH3, R' - CH3 becoming positive-going (Figs. II.2.5D,F). A t^  value exists where 1 ^ • 0 at the time when the "read" pulse Is applied, I.e. the "null condition" (Fig. II.2.6). This i s the basis for this form of "spectral *At this stage we are not concerned with the quantification of J_i although i t is worth mentioning that these data may be used to gain useful information concerning inter-proton distances for molecules in solution (vide infra). - 112 -F i g . I I . 2 . 7 400 MHz T 1 I R e x p e r i m e n t o n 1 (0 .1 M i n C 6 D 6 ) . T h e r e l a x a t i o n d e l a y was 12 s , a n d v a l u e s n o t e d n e x t t o e a c h s p e c t r u m . - 113 -METHYLENE Fig. II.2.8 Spectral editing of 1 by T1IR. The control spectrum is C. jt • 0.7s Is a "methine sub-spectrum", with methylene signals nulled. _t • 2.5s gives the "methylene sub-spectrum", with methine signals partially nulled. Other parameters as in Fig. II.2.7. The asterisk marks an artifact at the carrier position. - 114 -editing". A preliminary T1IR experiment with a series of arbitrarily selected t_ values Is performed to determine the null time for the - C H 2 resonances, for example. One of the resultant spectra might accidentally give a good nulling of the - C H 2 resonances under consideration. If not, a single experiment may be performed to achieve good nulling; under these conditions the more slowly relaxing -CH resonances w i l l then have negative amplitudes. An attempt to edit methine peaks i s not as easy, since their T_i's may vary considerably. Fig. II. 2 . 8 illustrates these statement. The normal spectrum of 1 is shown in Fig. I I . 2 . 8 C . Choosing _t « 0 . 7 s gives a "methine sub-spectrum", with methylene signals nulled (Fig. I I . 2 . 8 A ) . With t • 2 . 5 s , both methylene protons are almost f u l l y relaxed whilst methine protons are either nulled (H-4) partly inverted (H-l, H-3) or partly upright (H - 2 ) , (H-5) (Fig. I I . 2 . 8 B ) . Thus, a suitable choice of _t allows the creation of a spectrum lacking methylene peaks. This procedure has been used effectively in the assignment of the complex spectrum of a steroid. Although i t i s generally impossible to null a l l methine signals simultaneously, It Is possible to do a number of separate experiments and choose the _t values which null overlapping resonances. This method constitutes a simple and easy, yet little-used solution to the hidden resonance problem. With 1 3C NMR, this approach may be used to determine the number of attached protons to a carbon 9 (Chapter II.4). - 115 -II.2 . 2 . 2 Via J C T In 1 3C NMR Spectroscopy 13 Although broad-band proton decoupled C NMR spectra are attractive in their simplicity, a price is paid for the removal of the 13 1 C- H coupling information in that the spectra are d i f f i c u l t to 13 assign. The f i r s t steps towards assignment of C NMR spectrum involves determining the chemical shifts, and the number of protons attached to each carbon. Single-frequency-off-resonance-decoupling 1 0 (SFORD)* is widely used to achieve the latter purposes; however, i t has serious limitations in crowded regions of a spectrum, is inherently insensitive and can produce misleading r e s u l t s . 1 1 12— 18 Fortunately, a number of multi-pulse experiments exist ~ which are easy to use, are not as time consuming as SFORD and give a much clearer representation of the information. The f i r s t of these i s 12 "refocussed INEPT " (insensitive nuclei enhanced by polarization transfer); although this works well with decoupled spectra, the observed transitions are distorted when the 1JJTJH couplings are preserved. A number of other variants have been described; however, the author has 13 19 20 had the opportunity to try only one of these, dubbed DEPT » » (distortionless enhancement by polarization _transfer). This experiment gives undistorted coupled spectra, as well as good editing. "Polarization transfer" i s a process in which magnetization from the protons, which are high natural abundance and sensitive nuclides, is *SFORD employs a single *H decoupler frequency placed (usually) to high-field of the H spectrum. This causes suppression of the one-bond C-1!! couplings - 1 1 6 -transferred to the relatively insensitive C nuclei. The maximum intensity enhancement is Y H ^ Y C " compared with the maximum nOe attainable by broad-band lH decoupling ( 1 + ( 1 / 2 ) Y H / Y C " ^ ) T N I S may not seem to be a significant improvement. However, a further advantage lies in the fact that the repetition rate for the experiment i s governed by the spin-lattice relaxation rates of the protons rather 13 1 than of the C spins; given that H relaxation rates are typically 1 3 faster than those of C , 2-3 times as many scans can be obtained in unit time. Other advantages of DEPT over INEPT are that the former i s unhindered by overlap of solute and solvent peaks (which is particularly important when working at high dilution) and that i t has a reduced dependence on the magnitude of ^JjZE' H e n c e » a n "average" value for 1J_CH may be chosen which produces good results for a l l carbon sites in a molecule. The pulse sequence for the DEPT experiment is presented below. Proton pulses are applied through the *H decoupler coils of the 1 3 C probe, at the normal offset frequency for broadband (BB) decoupling (i.e. near 6JJ = 5 ) . An average value of the * J coupling is used in the calculation of the ( 2 J ) - 1 value, giving 3.6 ms. A l l pulses, and the *H: 90°(«i) 1 3 C : - ( 2 J ) _ 1 - 1 8 0 ° ( $ 2 ) - ( 2 J ) - 1 - e ( $ 3 > " (2J)" 1 - (BB) 90°($3) 1 8 0 o ( $ i t ) AQN($5) [II.2.10] - 117 -Phase cycling: Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 * 1 X X X X X X X X -x -x -x -x -x -x -x -x $ 2 X -x X -x X -x X -x X -x X -x X -x X -x $ 3 y y y y y y y y y y y y y y y y *4 X X -x -x y y - y - y X X -x -x y y - y - y *5 X X -x -x y y - y - y X X -x -x y y - y - y [ I I . 2 . 1 1 ] receiver are phase-cycled.($i - $5) through sixteen steps for the best cancellation of artifacts. This appears complex on paper, but is quite simple to programme on most modern spectrometers. Acquisition of the 1 3C FID ("AQN") is often with broad-band proton decoupling ("BB").* Spectral editing is achieved by acquiring three spectra, for ( 1 ) 0 i - 4 5 ° (TC /4) , ( 2 ) 0 2 - 9 0 ° ( T I / 2 ) and ( 3 ) 0 3 = 135 ° ( 3 i t / 4 ) . If n scans are acquired for 0 i and 0 3 , 2_n should be acquired for 0 2 . The free induction decay signals are Fourier transformed, phased, and the edited spectra obtained by linear combinations of the three spectra: -CH : 0 2 -CH2 : 0 1 - 0 3 -CH3 : 01 + 03 - 0 . 7 0 7 02 [ I I . 2 . 1 2 ] *The abbreviations "AQN" (acquisition), BB (broad-band proton decoupling) and $ x (phase-cycling programme) w i l l be freely used in pulse schemes in the rest of this chapter. - 118 -CH. CH 2 CH C H X J •OCH 3 100 90 80 70 60 F i g . I I . 2 . 9 DEPT p e r f o r m e d o n 2 ( 0 . 4 M; C D C 1 3 ) a t 100 M H z . - 119 -These additions/subtractions may not be totally exact; for example, the -CH2 subspectrum is more correctly given by ©i - a. 63, where "a" varies between 1.00 and 1.10. Most modern spectrometers have a "real time" subtractive routine which allows the operator to "fine-tune" these values to achieve most efficient editing. One instrument manufacturer 20 boasts an iterative routine which finds the best subtracted specta. Although the question of proton-pulse calibration is deferred until Section II.2.5.4, It is important to point out the -CH sub-spectrum (62) is reasonably sensitive to mlsset in the proton f l i p angles (vide infra) and as a result can be used to determine *H pulse lengths with reasonable accuracy. In practice, the experiment is repeated for a number of pulse lengths close to the approximate value, and the transformed spectra inspected for the most efficient cancellation of a l l but methine carbon peaks. Results are presented in Fig. II.2.9 for DEPT performed for a =0.4 M solution of methyl 2,3,4,6-tetra-O-acetyl-a-D-glucopyranoside, 2 in CDCI3. The -OMe peak appears as a weak, aliased peak at 6 63.2. However, we see that i t Is clearly visible in the -CH3 sub-spectrum; nulling i s excellent. II.2.3 Spin Decoupling-Difference S p e c t r a 6 * 2 1 - 2 3 (SDDS) This i s a variant of the well known spin decoupling experiment, in which responses are observed only from those resonances directly - 120 -involved in spin coupling; a l l others are "nulled" by the "difference" method. The advantages of this mode of presentation only become obvious when one considers crowded spectra, which have overlapping resonances. In general, interpretation is limited to observation of spectral changes; detailed analysis of the decoupled difference peaks may not be easy by inspection, but may be computer-simulated for verification. In practice two spectra are acquired for each decoupling experiment, one with the decoupler set to the precise resonance frequency and the other, the "control", with i t set just off resonance. To minimize instrumental d r i f t over a long period of time, the data are collected in blocks, which may be repeated i f long-term signal-averaging is necessary to build up adequate signal-to-noise. This process is termed "interleaving". If a series of separate experiments are to be performed at one time, the appropriate decoupling frequencies are listed in the computer memory, together with decoupler power settings adequate for homonuclear decoupling. A typical sequence of events would be: 1. Where 11 resonances require decoupling, create on disc 2n blank f i l e s , specifying decoupler frequencies and power settings. 2. Read into core memory f i l e number 1. 3. Perform 2-4 "dummy" scans (discarded), to establish a steady-state. 4. Acquire ^ scans (8 or 16) with the decoupler on resonance. - 121 -5. Save the (on-resonance) decoupled free induction decay on disc. 6. Read into core memory f i l e 2. 7. Acquire ^ scans with the decoupler set just off resonance. 8. Save the (off-resonance) "control" free induction decay on disc. 9. Repeat steps 2-8 (n-2) times, reading in each appropriate f i l e . 10. Repeat 2-9 m times, so as to build up sufficient signal-to-noise. The total number of scans for each difference spectrum i s 2(A * m). Note that the total number of scans (A * m) need only be equal or slightly more than that required to obtain a normal spectrum with adequate signal-to-noise. A l l free induction decays are then exponentially weighted (line broadening, 1.5 - 2.0 Hz), Fourier transformed, phased, and the frequency-domain spectra subtracted. The phase of the subtracted spectrum may require minor alteration before plotting. Figure II.2.10 illu s t r a t e s SDDS on 1. Several points arise. We see that in crowded regions (H-l, -2, -4, and H-5, -6 and -6•) the results are not always clear-cut, due to Bloch-Slegert effects.* *In the presence of the decoupling f i e l d B 2 , a l l resonance frequencies are shifted from their "true" frequency by an amount which i s proportional to the decoupler power (yB^/ZTt), and inversely proportional to their frequency separation. This is part of the Bloch-Slegert effect. - 122 -nli" T 6.0 4--AA A-B.S. H-3 H-4 H^ 2H-1 JL_JULL 5.0 JI 41—1*-~}\— H-6 H-6 H-5 H-4 H-3 H-2 H-l H-6 H-6' i±5L 4.0 II.2.10 SDDS on 1 at 400 MHz. The irradiated proton i s indicated in each case. False responses arising from Bloch-Siegert effects are labelled "B.S." - 123 -Computational solutions to these problems have been proposed. With {H-2}, for example, the calculated shift in H-4 i s 0.87 Hz with the decoupler on-resonance. In general, the decoupler can be moved either to high or low f i e l d for measurement of the control spectrum. In this case It was moved to low f i e l d as H-4 i s farther from H-2 than H-l and this minimized Bloch-Siegert distortions. For the control experiment, the decoupler was 285.4 Hz from H-4 and now the induced Bloch-Siegert shift Is 0.93 Hz. The difference, 0.06 Hz between these two values, accounts for the false "response" at H-4 In the SDDS spectrum. We note that a false response due to this phenomenon i s very different from the genuine decoupling-difference response ( c f . H-3). Having made this point, consider now the |H-6'} experiment (top trace). Protons 6' and 5 are coupled (J • 2.3 Hz), yet the "response" at H-5 looks more like a Bloch-Siegert artifact than a decoupling difference peak; in contrast, the {H-3} experiment i s entirely unambiguous. We conclude then that SDDS is a powerful tool, but that i t should be approached with caution in spectrally crowded regions. If this method alone is used to establish J_-connectivity, several experiments may be necessary, varying decoupler pulse power and/or control spectrum frequency. An ill u s t r a t i o n i s given in Fig. II.2.11 where, with a constant decoupler power (yB?/2n • 17 Hz), the control offset i s sequentially moved from 50 to 10 Hz from vn_i ( v c o n t r o i -Vfj.j i s termed Av). With Av - 50 Hz, Bloch-Siegert effects are seen for every proton; in the top trace (Av - 10 Hz), Bloch-Siegert shifts are insignificant. The line-shape of H-2 is poorer than with Av - 15 - 1 2 4 -10 Hz 15 Hz T 20 Hz 25 Hz 30 Hz -vvv- 40 Hz H-2 H-l Av =50 Hz 6.0 5.0 4.0 F i g . I I . 2 . 1 1 S D D S i r r a d i a t i n g H - l [(YB2/2TI) = 17 H z ] , a n d v a r y i n g t h e f r e q u e n c y d i f f e r e n c e (Av) b e t w e e n t h e d e c o u p l e r o n r e s o n a n c e ( f i x e d ) a n d t h e c o n t r o l , o f f - r e s o n a n c e ( v a r i e d ) . - 125 -Hz; H-3 is long-range coupled to H-l and the response i s probably genuine. II.2.4 Hudear Overhauser E f f e c t 2 5 ^ 2 8 The nuclear Overhauser effect (nOe) is of great u t i l i t y in 1 13 routine H and C NMR spectroscopy. Classically, one considers a saturating RF f i e l d , B_2, and two spins, I and S.* The nOe specifies a variation in Intensity (= integrated area) of spin I when spin S i s saturated. In this section, discussion is restricted to a homonuclear steady-state experiment involving intramolecular nOe's between weakly coupled (or coupled) spins (such as ^H^H} in rig i d molecules, in the absence of chemical exchange phenomena). The nOe, defined as the fractional change in intensity of the signal of spin 1 when spin j (or group of spins j) is saturated, is given by: <I .> - I , f.(j> — [II.2.13] oi <IZ^> is the average magnetization of i in the presence of saturation of spin(s) j , and I 0 ^ the equilibrium magnetization in the absence of I$2« The nOe phenomenon involves population transfer resulting from relaxation effects between spins. If i s the total direct relaxation rate of spin 1, i t may be considered as: *In keeping with the literature nomenclature, the two spins are designated I and S (with S irradiated), rather than A and X. - 126 -R = Z p + p * [II.2.14] i * j 2 where p ^ j is the dipolar relaxation rate of spin i with j , and p j * i s the direct relaxation rate due to mechanisms other than dipolar (which for small molecules is often negligible in comparison with p ) . At this stage a feel for the populations and transition probabilities i s useful. Consider the energy level diagram for the A. B. Fig. II.2.12 Energy levels, transition probabilities and relative populations (A) for an IS system at equilibrium, and (B) for an l { s } situation (where S is saturated). weakly coupled IS spin system at equilibrium (Fig. II.2.12A). F_x is the equilibrium population of the x-state, and W the transition probability. The intensity of the I spins w i l l be proportional to (P3 -P_l) + (Pi^. - P_2). Measurement of single-quantum W_i transitions has no information on dipolar-relaxation and, hence, distances; this information i s in the zero - (Wo) and double-quantum (W2) transition - 127 -probabilities and can only be determined when the spin populations have been prepared in some suitable fashion (e.g. saturation or population inversion). When S i s saturated (Fig. II.2.12B), populations of levels 1 and 2, 3 and 4 are equalized: (P 3 - P^) - (P^ - P 2), and (P^ 0 - P_i°) = 2(P1+ - P_i). This change in the energy level populations w i l l be negated by the "cross-relaxation" processes: W_2 w i l l attempt to re-establish equilibrium by increasing Pj and decreasing Pj,. The effect of WQ w i l l be to increase P3 and decrease P_2« These cross-relaxation terms contribute to a cross-relaxation rate, o"ij : o ± j - wJ D ( i j ) - W?D ( i j ) [II.2.15] For the totally dlpole-dipole (DD) relaxation mechanism, p^j can be written in terms of transition probabilities: P1;J =H.0D (il) + 2W.1D (ij) +W? D (ij) [II.2.16] Eq. II.2.13 can be expressed in terms of a and p: f i ( J > * °U/Pii [H.2.17] Expressions may also be derived for pj^j and ojj which interest us more, as they contain the dipolar distance information for the homonuclear experiment (as mentioned). Under extreme narrowing conditions, the frequency terms in the f u l l e r expressions can be Ignored - 128 -and i t can be shown that: Pij " Yi Yj h ' Tc rIj UL2.18] o±i = (1/2) Y2 h 2 T c r~J [II.2.19] where r^j is the internuclear separation between spins i and j , zc i s the motional correlation time* and n, Planck's constant divided by 2 it. We now return to the measurable fractional nOe, f ^ ( j ) , and relate this to the concepts introduced above as we work towards a practical use for the experiment. The measured f^(j) has (the f i r s t ) direct polarization of spin i by spin j , minus the indirect polarization of spin I by spin j through the other spins, n, in a multispin system: f Yj o±j Z Yn cr i n J n ( j ) f i ( j ) - ^Tli ~ 7R UI.2.20] The second term In Eq. 1.2.20 accounts for the "three-spin effect" which is the indirect interaction between i and j, mediated by n. This i s discussed later. The next stage requires developing a working protocol for the determination of internuclear distances involves consideration of a three-spin AMX system. From Eq. II.2.14 we get: *Very roughly, the motional (= rotational) correlation time is the time taken for the molecule to tumble in solution and rotate by one radian. - 129 -= PAM + PAX + P A PAS + PMX + PX L I 1- 2- 2 1 1 = PMX + PAM + P M * * * Assuming » « p^, and given that R^, R^ and were measurable q u a n t i t i e s , the three unknowns i n these three equations can e x p l i c i t l y be determined, and i n t e r n u c l e a r d i s t a n c e s c a l c u l a t e d from Eq. II.2.18. This i n v o l v e s a f u r t h e r assumption that the c o r r e l a t i o n times of the AM, 13 29 AX and MX vectors (which could be q u a n t i f i e d from C r e l a x a t i o n data ) are equal, Hence, we a r r i v e at a master equation of t h i s form: f,(M) + f,(X) f_(M) _2 £ ± [ T T o 221 f A ( X ) + f A ( M ) f M ( X ) U l . ^ . ^ J With the two-spin approximation, only the f i r s t terms [f^(M) and f ^ ( X ) ] are used. I f one of the two distances i s e i t h e r known or can be assumed, the other can be c a l c u l a t e d . A l s o , i f the l o c a t i o n of A i s sought, i t should not be i r r a d i a t e d , but the A{M} and Afx} experiments should be performed. In terras of experimental p r a c t i c e , the power l e v e l s have to be c a r e f u l l y set to ensure the same percentage s a t u r a t i o n of each s p i n ( s ) , or normalized. These are the fundamental tenets of the process of determining i n t e r n u c l e a r distances i n small organic m o l e c u l e s . 3 0 31 As described i n some d e t a i l by Mersh and Sanders, i t i s w e l l known that the 3-spin case can lead to the observation of negative nOe's - 130 -"3 o i n roughly linear systems when long (2-3 T_T) irradiation times are used. The authors explain the phenomenon and i t s potential diagnostic uses. In such cases, more explicit equations (e.g. Eq. II.2.20) are used which take into account the positive "direct effects" and "indirect" negative ones. It i s interesting to note that, under some conditions, these two contributions can almost cancel, leading to a small induced nOe in a proton which i s geometrically close in space to the one being irradiated; this is an unpleasant possibility. II.2.4 .1 Steady-State nOe This i s the simplest nOe experiment and has been alluded to in the preceding remarks. With two spins I and S, radiofrequency f i e l d ( B 2 ) is applied to one resonance long enough (= 3 T_i) for the nOe to build up in the other. Immediately prior to acquisition, the decoupler is turned off so as to retain spin-spin coupling and preclude Bloch-Ziegert effects. Schematically: &2 • 5TJ (RD) - 3Tj - 90° - AQN I - 1 [II.2.22] The experiment i s repeated to obtain a good signal-to-nolse ratio and i s then repeated with B_2 placed off resonance, and the integrals of the two spectra compared. Several practical considerations warrant expansion. When the - 131 -frequencies of I and S are close (say, < 1 ppm) It Is d i f f i c u l t to saturate one spin without radiation leakage into the other. In order to maintain the required frequency selectivity the strength of the irradiating f i e l d iy^/Zn) should be reduced so as to effect 60-80% saturation. A less than saturating decoupler f i e l d does not invalidate the measurement since the magnitude of the induced nOe for a particular spin i s proportional to the extent of saturation of i t s neighbour.3** Use of a weaker B_2 f i e l d reduces the magnitude of induced nOe's and this could demand a significantly larger signal-to-noise ratio i f these (small) changes in intensity are to be reliably measured. In practice, the frequency selectivity can be checked by performing a number of experiments varying the decoupler power. To minimize nOe build-up, the decoupler i s le f t on for a short period of time (ca. 0.01s). Comparison of peak Intensities of resonances close to the decoupler frequency with those in a "control" experiment (decoupler off-resonance) w i l l ascertain whether the desired frequency selectivity i s achieved. It i s invariably convenient to perform the experiment in the difference mode3** (nOe difference spectroscopy = NOEDS). For n irradiation experiments, (n_ + 1) experiments are required, including one "control" where the decoupler frequency i s chosen distant from the spectral region - say, 5 or 10 p.p.m. to high f i e l d of the highest-field resonance. Again, the signal acquisition 6 i s interleaved: 1. Create on disc (n_ + 1) blank f i l e s , with the decoupler frequencies and power settings specified. - 132 -2. Read into core memory f i l e number 1. 3. With the decoupler set to u>i, obtain 2-4 "dummy" scans (discard), to establish a steady-state. 4. Acquire scans (8 or 16). 5. Save the spectrum on disc. 6. Repeat 2-5, now with f i l e number 2 ((1)2). 7. Continue until (n_ + 1) spectra have been saved on disc, reading in the appropriate f i l e each time. 8. Repeat 2-7, m times so as to build up a good signal-to-noise. The total number scans for each experiment * * m. Again, the FID's are exponentially weighted (using 1.5 - 2.0 Hz line-broadening), Fourier transformed and phase corrected. Each spectrum i s then subtracted from the "control", and plotted.* Overhauser enhancements are best measured by dig i t a l integration of signals (and not by measuring peak heights). Subtraction errors, usually obvious with intense singlets, may be reduced by small (0.001 Hz) shifts of the 24 spectra. When plotted with the irradiated peak negative-going in intensity, positive going peaks indicate a positive nOe (fast molecular tumbling) and negative peaks indicate a negative nOe (usually indicating slow tumbling). Care should be exercised in applying this rule, since radiation leakage into a nearby signal may falsely indicate a negative *Small phase adjustments in the difference spectrum may Improve i t s quality. - 1 3 3 -nOe, and, as has been discussed previously, certain linear spin-systems can produce negative nOe's in the extreme narrowing limit. We reiterate that, in practice, excellent signal-to-noise ratios are required for each spectrum, especially when (a) one is using significantly less than a saturating decoupling f i e l d , and (b) the expected nOe's are small. With care, enhancements down to 0.5% are measurable, as seen in later chapters. It i s not always necessary to attempt a l l the nOe experiments which are possible for the molecule of interest. Choice of which signals to saturate i s important. It is d i f f i c u l t (but not impossible) to induce and detect nOe's into a methyl resonance because the methyl protons relax one another ef f i c i e n t l y without requiring significant relaxation pathways from other protons in the molecule; furthermore, induced nOe's are d i f f i c u l t to detect since methyl signals are often sharp singlets and, therefore, readily show small frequency offsets when displayed in the difference mode. In short, for qualitative data one should irradiate the proton having the fewest alternative relaxation pathways, since this w i l l be dominated by dlpole-dipole relaxation from the smallest number of protons and therefore show maximal nOe's. It has 6 31 been noted > that irradiation of steroid methyl groups is useful in providing information on 6-axial protons In the v i c i n i t y . Finally, we note that tailored excitation sequences 3 5* 3 0 could prove useful in (a) their frequency selectivity and (b) the a b i l i t y to "irradiate" simultaneously serveral chosen multlplets in a spectrum. Fig. II.2.13 illustrates a series of NOEDS(SS) experiments Fig. II.2.13 SSNOEDS on 1 (400 MHz). The irradiated proton Is indicated next to each difference spectrum. The irradiation time was 6s. - 135 -performed on 1. It i s clear that It i s d i f f i c u l t to get the required frequency selectivity given the small frequency separation (Av) between H-l and H-2 (75% saturation; Av - 64 Hz). {5} is an interesting experiment as i t illustrates both the strong nOe induced in i t s 1,3 trans diaxial neighbour (H-3), and the comparatively small nOe into the methylene protons (which have an effic i e n t , mutual relaxtion patheway). II.2.4.2 Transient Overhauser E f f e c t 2 8 . 3 6 * 3 7 (TOE) This experiment is similar to the steady-state nOe experiment in that the populations of one spin multiplet are selectively inverted (using a selective 180° pulse) and the recovery of the spin system i s followed. To obtain frequency selectivity, the duration of the decoupler pulse i s , typically, 0.01 to 0.03 s. The experiment i s depicted below: 5TX(RD)- - t - 90° - AQN 1_JL a) [II.2.23] n An i n i t i a l relaxation delay i s ended by the selective 180° pulse, which is applied through the decoupler channel. A variable time-delay (t) follows (cf. T1IR experiment), with a non-selective 90° "read" pulse and data acquisition. For each jt value, ri scans (8-16) are i n i t i a l l y - 136 -collected, and the cycle repeated in a fashion analogous to the SSNOEDS and SDDS experiments. To achieve a more selective 180° pulse, a composite pulse comprising a "sandwich" of three pulses, 90°+ x 240°+ y 9 0 ° ± x is highly recommended, as this gives excellent population inversion and removes errors caused by resonance offset. A comparison of the effect of using a single selective 180° pulse vs. the composite pulse i s given in Fig. II.2.14. A. B. C. Fig. II.2.14 (A) The equilibrium z-magnetizatlon of 1 (H-5). The z-magnetizatlon immediately after (B) a single selective 180° pulse, and (C) a compositive 180° pulse. Conceptually, the TOE experiment can be explained by invoking similar arguments to those proposed in II.2.4.1. In general magnetization of the inverted resonance w i l l follow a multiexponential recovery, while the unperturbed spin suffers a transient change in intensity, which depends on the extent of i t s relaxation with the one perturbed. Attention is directed to the growth in intensity of the - 1 3 7 -unperturbed spin (displayed in the difference mode) as a function of time, _t. As we shall see, comparisons of absolute nOe's for different protons do not necessarily give the correct distance information; a 26 28 more precise evaluation comes from the i n i t i a l slope » of the TOE build-up curve, which is approximately equal to a. Using a two-spin approximation and assuming p^j ™ pj^, 0"AM/a^ X = ^rAX^rAM)6, Although not absolutely precise, this gives us a maximum figure for r, the internuclear separation. Fig. I I . 2 . 1 5 illustrates a difference TOE experiment conducted on 1; H-5 is selectively inverted by a composite pulse ( 1 8 0 ° = 20 ms). t_ values are In seconds. A relatively fast build-up of nOe in H-3 is observed, while, for example, H-l builds up a smaller nOe at a slower rate. Fig. I I . 2 . 1 6 shows plots of the nOe build-ups for H-3 and H-l. Consider now the nOe build-ups in H-3 and H-l. The ratios fH_l(H-5)/fH-3(H-5) vary from 0.25 - 0.41 and cannot be used for distance calculations. From the i n i t i a l slopes', a\/03 " 0.26. Knowing that (01/03) = ( r 5 _ 3 / r 5 _ 1 ) + 6 , and assuming a typical H-3, H-5 internuclear separation of 2.64A, we calculate r = 3.32 A ( ± . 1 A ) . - 138 -H-3 k. H-l X JJ^ * * • —f^-1 8.00 4.75 3.50 ^ 2 . 5 0 1 7 5 - ^ 0 7 5 1 0 . 2 5 S E L . 1 8 0 0 C O N T R O L JLOL Fig. II.2.15 Difference TOE on 1 (400 MHz), selectively inverting H-5 with a composite pulse (see text). The time, j t , between selective population inversion and total signal acquisition i s given with each spectrum. - 139 -Fig. II.2.16 Data from Fig. II.2.15, showing the nOe build-ups of H-l and H-3 with time. Considering the small size of OR-I and the resultant error In Its calculation, we note a close agreement to the distance obtained by g neutron-diffraction for a similar system, of 3.33 A. Note also that errors in the measurement of a translate to sixth-root of errors in distances. This example illustrates the ease with which approximate distance measurements may be obtained for rig i d systems. The interested g reader i s referred to a thesis on this subject. Distance information derived from dynamic methods such as TOE is much more useful than SSNOEDS, since the method automatically gives a - 140 -feel for the rate at which these population transfer processes occur, and this gives a better picture of the system. TOEDS eliminates the guess-work involved with choice of the irradiation time in the SSNOE experiment. Results are found in build-up rates, rather than SSNOE peak Integrals. We also draw attention to the potential frequency selectivity through the use of a selective 180° pulse. If further selectivity Is demanded, one may make use of a tailored excitation pulse sequence 3 5 or DANTE**1. For the author's purposes, the composite 180° pulse has proved satisfactory. In conclusion we note the potential of the experiment with 37 39 •*() slowly tumbling molecules » » (spin-diffusion limit; vide  infra). II.2.4.3 Truncated Driven HOE Difference Spectroscopy For studies of molecules outside of the extreme narrowing limit, the results from steady-state NOE measurments no longer bear a direct relation to interproton distances; Instead, the behaviour depends on "spin diffusion". It i s general practice to study biologically important molecules - even as small as an octapeptlde - dissolved in viscous solvents (e.g. DMS0-d 6^) and observed at as high a field-strength as possible (to obtain maximum dispersion). These two factors combine to place the system in the spin diffusion regime where a maximum nOe of -1 i s attainable. Two alternative, one-dlmensional experiments may be used to study molecules in this regime: transient nOe experiment (vide supra) and the more - 141 -u s e f u l t r u n c a t e d d r i v e n nOe e x p e r i m e n t 2 9 • ** 2 - 4 ** ( " d r i v e n n O e " ) . T h e p u l s e s e q u e n c e f o r d r i v e n nOe r e q u i r e s a s e l e c t i v e , n e a r - s a t u r a t i n g t o be a p p l i e d a t cog f o r v a r y i n g t i m e s , j t . * T h i s i s f o l l o w e d b y a " r e a d " p u l s e a n d s i g n a l a c q u i s i t i o n . A g a i n , t h e e x p e r i m e n t i s c o l l e c t e d i n b l o c k s , t o g e t h e r w i t h a b l a n k , a n d t h e d a t a p r e s e n t e d i n t h e d i f f e r e n c e m o d e . A t e a c h f r e q u e n c y , a n u m b e r o f R D ( 5 T 1 ) - -90° - AQN B , _ r i £2 t [ I I . 2 . 2 4 ] e x p e r i m e n t s i s p e r f o r m e d , v a r y i n g t^. F o r e a c h e x p e r i m e n t t h e i n d u c e d nOe i s p l o t t e d a g a i n s t J t , f o r e a c h s p i n s h o w i n g a n n O e . T h e nOe c u r v e f o r e a c h p r o t o n b u i l d s up t o a max imum v a l u e a n d t h e n l e v e l s o f f . A g a i n , i t i s t h e i n i t i a l s l o p e o f e a c h c u r v e w h i c h g i v e s t h e d i s t a n c e i n f o r m a t i o n . I f s p i n i i s i r r a d i a t e d a n d t h e nOe b u i l d - u p c u r v e f o r s p i n s p l o t t e d , t h e i n i t i a l s l o p e o f t h e c u r v e • - 0 ~ i j ' T h i s c a n be t r a n s l a t e d i n t o d i s t a n c e i n f o r m a t i o n u s i n g t h e c o m p a r a t i v e m e t h o d s d i s c u s s e d e a r l i e r . I f o n e i s f a m i l i a r w i t h t h e s y s t e m u n d e r i n v e s t i g a t i o n a n d k n o w s t h a t f o r a p a r t i c u l a r i r r a d i a t i o n t i m e o n e i s i n t h e a p p r o x i m a t e l y l i n e a r , " r i s i n g " p o r t i o n o f t h e b u i l d - u p c u r v e , o n e may p e r f o r m j u s t o n e e x p e r i m e n t , w i t h t h i s i r r a d i a t i o n t i m e a n d c o m p a r e s i n g l e n O e ' s r a t h e r *t_ I s s h o r t c o m p a r e d w i t h t h e i r r a d i a t i o n t i m e s u s e d i n SSNOEDS « 0.5 s ) . - 142 -than the slopes, to obtain the distance information. Examples of this experiment appear in Chapter II.5. II.2.5 Two-Dimenslonal Experiments'*5"'17 II.2.5.1 Basic Concepts Most readers are familiar with the concept of normal pulse FT NMR. A pulse is applied to the system and a signal is detected as a function of time, js(jt). The process of Fourier transformation converts this time-dependent function into a frequency-dependent one, S(F) -which is the familiar display of absorption jvs frequency. With two dimensional ("2D") NMR spectroscopy the magnetization is detected as a function of two time Intervals; one a linearly incremented time delay, jt i , and detection occurring during the acquisition time, labelled tjj, resulting in a two dimensional data array, J5(t_i, Jt^). Usually, ii experiments are performed, each with an Increasing value of t_i, and the ii data sets saved on a mass data storage device, such as a hard disc. Each signal detected in t2 corresponds basically to a normal single-quantum spectrum of the molecule. However, each resonance has a "memory" of processes occurring during t_i and this w i l l result in a modulation of the signals - either by phase or amplitude. The analogy has been drawn between 2D NMR and two-dimensional paper chromatography. Chromatographic separation of similar compounds is improved by using one solvent system to disperse components along one axis of the paper which is then turned 90° and developed with a different solvent system in the second dimension to provide enhanced dispersion. If the paper is - 143 -A. s ( \ ] t \ 2 ) 2m i 1 n -A /- ^ B. 5(t,Jc2) C. S(F 2 , t i ) D. S(F 2 /Fi) .2m. J L _ L A n i E. S(Fi ,F 2) F i g . I I . 2 . 1 7 S c h e m a t i z e d d a t a m a t r i c e s i l l u s t r a t i n g t h e s t a g e p r o c e s s i n g o f a 2D d a t a - s e t . - 144 -sprayed to visualise the compounds, a plot of colour intensity over the eluted area would produce diagrams similar to those presented in this chapter! With 2D NMR, the NMR responses are spread over two frequency axes to improve dispersion. The "solvent" used in J_2 ^ s almost always chemical shift, but Fj may characterise a variety of parameters (e.g. homonuclear J_'s), depending on the pulse-sequence used to e l i c i t the NMR responses. As mentioned, ti experiments are performed and the FID's (2m words) sequentially stored on disc (Fig. II.2.17.A.). This data matrix of n_ rows and 2m columns Is denoted sCti.t^). Next, a l l FID's are Fourier transformed, yielding (Fig. II.2.17.B.) the sCtj.F^) matrix; this comprises a set of spectra, the signals of which are modulated in either their amplitude or phase. Next, a transposition is performed giving a data-set which indicates the dependence of the magnetization at a particular point in T^, as a function of t^; j5(F2»t.l) * n (Fig* II.2.17.C). This generates a series of interferograms* - one for each absorption frequency in F^. These are traces of columns taken from the s(t.l»F.2) data set and indicate the "spectrum" of amplitude or phase modulation in tj. The interferograms are Fourier transformed, to give jS(F_2,j?i) in Fig. II.2.17.D.; these signals are often displayed in a phase-insensitive mode (vide infra) since phasing in both frequency dimensions i s d i f f i c u l t . Finally, this matrix is transposed again to give a S(Fj ,F ?) data set (Fig. II.2.17.E.). This process i s simpler in *An interferogram is essentially an "FID" of magnetization as a function of t^, given another name to differentiate i t from _t2 signals. - 145 -practice than i t may appear here, and can be performed relatively easily with standard soft-ware packages included in most modern spectrometers. Some programmes require the operator f i r s t to specify a l l parameters and then with a single command, the above steps are performed automatically, transparent to the operator, while others require operator intervention at each step. As mentioned previously, 2D spectra are normally presented in either power- or magnitude-mode (i.e. phase insensitive,* as this obviates the necessary complex phase adjustments required over two dimensions). Unfortunately this procedure creates a problem, since the phase-insensitive display of a Lorentzlan line has very wide "wings" -intensity distant from the resonance frequency. These dramatically reduce the base-line separation between individual signals, and can give rise to artifacts - false signals arising from interference between the wings of two peaks. Fig. II.2.18 illustrates the problem and some of the solutions. The f i r s t column shows the appearance of several weighting (apodisation) functions,** the second shows the derived phase-sensitive frequency-domain spectra and the last is the magnitude calculation of the absorptions in the second column on the same frequency scale. Fig. II.2.18A shows the exponential f i l t e r *Fourier transformation of a time-domain signal yield a "real" (u) and imaginary (v) component. Usual (phase-sensitive) detection requires observation of the "real" component, with some phase adjustments. The "magnitude" (or "absolute") display calculates the modulus of the signal, (u + v )*' 2, and the "power" mode is i t s square (u 2 + v 2 ) . - 146 -"FID" •PHASE-SENSITIVE MAGNITUDE SPECTRUM . II.2.18 Effect of apodization functions on line-shapes in phase-sensitive (column 2) and magnitude mode (column 3). - 147 -(line-broadening of 0.75 Hz) applied to a sine-wave. FT yields a Lorentzian absorption line-shape which, when subjected to magnitude calculation yields the very broad "wings" (^''tailing"), indicated with arrows. Converting the sine-wave into a Gaussian function, as in Fig. II.2.18B, yields an improved situation in both regimes. Fig. II.2.18C illustrates the use of the "sinebell" function. An exponential-shaped FID (Fig. II.2.18A) is apodised with a sine-function of period twice the acquisition time, and zero phase shift (i.e. zero at jt=0 and _t=AT). Its phase-sensitive FT shows decreased line-width accompanied by significant distortion at the base of the peak. A significant loss in signal-to-noise i s incurred, not obvious from the second frame. Magnitude calculation produces a line-shape which approximates to an absorption line and is comparable with the phase-sensitive Lorentzian signal. This form of modulation is termed a "pseudo-echo", as i t is roughly symmetical about AT/2 and zero at both start and finish. The pseudo-echo shaping may be further improved by f i r s t multiplying the decaying exponential function by a rising exponential to a r t i f i c i a l l y eliminate the decay, and then applying the sinebell function (or a similar symmetrical Guassian envelope). A similar protocol, less drastic in i t s attenuation of signal-to-noise i s the "double exponential" f i l t e r , shown in Fig. II.2.18D. Here, the FID is weighted with a rising exponential f i l t e r and a f a l l i n g function for Gaussian line shape. The result i s a compromise - the resolution enhancement is poorer, but there is less peak-base distortion and degradation of slgnal-to-noise. The magnitude - 148 -calculation spectrum shows less t a i l i n g . The display of 2D data sets is a non-trivial problem. Early experiments were invariably displayed as a "stacked plot"; this consists of a series of conventional S(J_2) spectra, one for each regular interval of J_ i (or, vice versa). The three-dimensional impression is further enhanced by use of the "white-wash" routine, whereby peaks from previous traces are not overwritten (the pen l i f t s up and skips over them). Although the three-dimensional effect Is excellent and the plots are aesthetically very pleasing, i t is often d i f f i c u l t to extract the s c i e n t i f i c information. Furthermore, the process is unecessarily time-consuming because the majority of the traces only display noise. A far more efficient routine employs the "contour plot" display which is similar to that used for geographical maps; contour lines link l o c i of, say, equal elevation. With 2D NMR, the F_i/J_2 plane Is viewed from above, along the amplitude axis; contours join points with the same amplitude. Data are easily extracted from such displays. Problems arise in choosing the contour levels when a large dynamic-range exists between different peaks; In fact, a series of peaks could be entirely missed i f the threshold level were set too high - hence, caution should be exercised. Fig. II.2.19 illustrates the benefit of the contour plot over the stacked plot. The diagrams are from the 2D homonuclear J_-correlated spectrum of a derivatized disaccharide. The relevant parameters can often be extracted from the data set without the need for the 3D representation. Here, use is made of p r o j e c t i o n s 5 0 * 5 1 (or sums) either taken over the entire frequency - 149 -4.20 4.15 4.10 8 (ppm) J(Hz) Fig. II.2.19 Comparison of stacked (A) and contour plots (C). B. The J_i projection of the t i l t e d data-set. The stlpled peak arises through strong coupling between 6' and 6^ . Data are from the same region of a 2D J-resolved experiment of a disaccharide. - 150 -Fig. II.2.20 A. Stacked-plot of schematized 2D data set. B. Contour plot i l l u s t r a t i n g f u l l 0° and 90° projections (top and right), and "partial" 90° projections (bottom). I - 151 -range in £2 or Fj (0° or 90° projection, respectively) or a single trace, or " s l i c e " may be taken out at a particular J_2 value and viewed at 90° (Fig. II.2.20). The latter are called JFJ traces and generally contain a l l the information needed. An almost overwhelming variety of 2D experiments has already been published and a l l indications are that this rapid proliferation w i l l continue. An objective inspection of the recent literature indicates that the major experiments have already been developed; what is now evident Is a second generation of pulse sequences in which existing ones are either refined (see, for example, ref. 46, pp. 50-65) or two experiments are combined to either yield additional information (e.g. Section II.2.5.3.4) or improve the experiment (e.g. ref. 52). It i s useful at this juncture to broadly classify the available 2D experiments: A. Resolved NMR In this case, J_2 Is the chemical shift axis of the observed nucleus and Fj may represent a variety of "resolving" parameters such as homonuclear J-coupling (vide infra), heteronuclear J_-coupling, heteronuclear chemical shift (vide  infra) or dipolar coupling. B. Correlated NMR Also termed "autocorrelation" the same chemical shift axis i s plotted along both dimensions and spins are correlated either through J_-coupling (vide infra) or spin exchange (nOe or chemical exchange). - 152 -C. Indirect Detection Multiple quantum transitions (MQT) cannot be observed directly, but experiments have been developed to allow for their indirect detection. Indirect detection of rare nuclei also f a l l s In this category. D. Combination of A, B and C, and Others Before commencing with more detailed comments on some selected experiments, a few general points are appropriate. A major experimental restriction is often digitization, since one is often limited by available data storage space and time. As a result, the minimum sweep-widths in and J_2 should be selected. Any aliasing in F_2, due to insufficient sweep-width can usually be chosen so that these peaks f a l l into a blank region of the spectrum. They can be attenuated by use of the frequency f i l t e r i f they have no information.* It Is not possible to perform frequency f i l t e r i n g in and the experimenter i s advised either to avoid aliasing by choosing a suitable sweep-width, or exercise caution by being aware of the way in which this occurs. The number of experiments performed, n, is usually a binary number (6A, 128, 256, etc). The smaller n, the less machine time w i l l be required to obtain _s(ti, t ^)* Digitization in Fj can usually be improved by z e r o - f i l l i n g , but caution should be exercised in trying to •Solvent signals, impurities and acetate singlets can often be treated in this way. - 153 -let a very small ri suffice, since this may lead to severe truncation of the interferograms, and associated line-shape distortions. Having chosen a suitable block-size and sweep width (in Yj), one can calculate the acquisition time and set up the experiment such that the effective acquisition time in r j , (ti * Ati) i s of similar duration. The incremental delay-time, Ati is easily calculated from the required SW in Fj, 0.5 Ajti used in spin-echo experiments is given by expression II.2.24, and A_ti Is given by II.2.25: 0.5 Ati - (4 * ± SW„ ) _ 1 [II.2.24] — £.1 Ati - (2 * ± SW,, ) - 1 [II.2.25] — £.1 Finally the total experimental time may be estimated from equation II.2.26. Total time - n.NA[l/2 ( t - 1 ) m a x + AT + RD] [II.2.26] n is the number of increments in j t i , AT is the acquisition time (_t_2^» ^ is the relaxation delay between acquisition and the f i r s t pulse in the sequence, and NA i s the number of acquisitions for each jti value (including dummy scans). Finally, we address the topic of signal-to-noise in 2D experiments, which has been ex p l i c i t l y formulated by Ernst and 53 co-workers. It happens that the overall demands of the method are reasonable, since both the signals and the noise are spread over the (F_l ,F_2)-plane. Obviously a loss in signal-to-noise i s incurred as a - 154 -result of signal decay during j t j , but this only becomes c r i t i c a l when dealing with molecules with very short T^'s (see Chapter II.5). Some autocorrelation experiments suffer a further degradation in sensitivity because each conventional resonance generates -2E~1 components, where m i s the number of weakly-coupled non-equivalent spins. This is offset by an increase in sensitivity resulting from symmetrization procedures (discussed in II.2.5.3.1). The author's "working rule" i s that i f signals can be seen upon FT of the f i r s t of the n_ intervals (blocks) of the jsCti.t^) data-set, the overall experiment stands a good chance of succeeding. Of course, i f resolution enhancement is necessary for the resolution of closely spaced peaks, the slgnal-to-noise requirements of the original data set w i l l be correspondingly higher. I I . 2 . 5 . 2 Homonuclear J-Modulated Spectroscopy51* ( 2 D JHresolved) The key to this experiment i s the basic Hahn spin-echo, which has been documented55 for over 30 years. Hahn used a 90°-T-180°-T-AQN pulse-sequence; he noted the narrower line widths attainable and used this very early on in making some of the f i r s t scalar J_ measurements. He analysed plots of echo-amplitude vs i in the frequency domain and came to some anticipatory conclusions. A modulation of the echo envelope which depends on both and the chemical shift separation was observed. Further, he noted that " . . . i f only one of two coupled nuclei 19 1 Is subjected to resonance (e.g. between F and H) no echo envelope modulation w i l l appear for either of such coupled nuclei, although the steady state resonance w i l l s t i l l reveal the J_ sp l i t t i n g . " - 155 -Herein l i e the concepts of 2D J-resolved spectroscopy, albeit without the complex two-dimensional FT. The above quotation has also been documented in the light of 2D J-resolved spectroscopy. 5 6 The basic 2D J-resolved pulse-sequence i s : RD - 90°($i) - 0.5ti - 180°($2) - 0.5ti - AQN($3) [II.2.27] Phase cycling of the pulses and receiver is important. The EXORCYCLE scheme proposed by Bodenhausen, Freeman and Turner 5 7 (II.2.28) 58 eliminates so-called "ghosts" and "phantoms" arising from pulse Imperfections and residual transverse magnetization. Further improvements in the quality of the fin a l spectrum may be o ± X 180°^ 90° ± x; 180+y = 90° v 180°-x 90°.y and (b) extending the phase-cycling to 16 steps by incorporating the well known CYCLOPS scheme (II.2.29) to reduce quadrature Images in F 2 . achieved by (a) using a composite 180° refocussing pulse (180°^ = 90 Cycle 1 2 3 4 *1 X X X X $2 X y -x -y [ 1 1 . 2 . *3 X -x X -x Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 *1 X X X X y y y y -x -x -x -x -y -y -y -y $2 X y -x -y y -x -y X X -y x y -y X y -x $3 X -x X -x y -y y -y X -x X -x y -y y -y [II.2.29] 156 -The 16-cycle scheme is preferable should one require much signal-averaging, especially for studies of dilute solutions. If only four pulses yield sufficient signal-to-noise, then phase-cycling according to II.2.28 with a composite 180° pulse should be sufficient, but one should be aware of very strong signals which may s t i l l show quadrature images in F_2 & / (vide infra). The 2D J-resolved experiment may be explained in terms of vector diagrams. Consider the X nucleus of an AX spin system. The relaxation-delay (RD) i s terminated by the application of a 90^ pulse, which tips the equilibrium magnetization onto the y_'-axis (Fig. II.2.21A). The X magnetization has a fast (f) and slow (s) vector*, corresponding to the two possible spin states of A to which It i s coupled. With time, the vectors w i l l precess in the (x'-y_')-plane (Fig. II.2.21B) (as depicted previously in Fig. II.2.3) mainly under the influences of chemical shift and J. A 180° (refocussing) pulse is now _ y applied which takes the vectors to their mirror-image positions about the y'-axis in the rotating reference frame. Consider the situation where both A and X experience the 180° pulse, which gives the situation in Fig. II.2.21C. As well as changing the position of the vectors *Here, we are using the terms "fast" and "slow" to describe two different transitions, as in Fig. II.2.3. Note that in discussions on the removal of magnetic inhomogenelty effects by this experiment, these terms pertain to a single resonance and have a different meaning. - 1 5 7 -Fig. II.2.21 Vector diagram model of the 2D J-resolved experiment. With the X transitions in an AX spin system (A,B,E,F), both spins feel both pulses; in A-D, only spin X i s subjected to the refocussing pulse (From Freeman**'5). - 158 -representing X, the labels of the two X transitions are changed (F <-> jS) as A changes i t s spin state (o <-> B). Thus, during the second 0.5 _ti period the vectors continue to diverge and the echo consists of two components (Fig. II.2.21F); that i s , only the chemical shift effects are refocussed and not spin-spin coupling. The overall result i s a modulation in phase of the signals by J_, as a function of j t i . The phase of a signal w i l l be a function of i t J t^. A second situation is possible when A i s a heteronuclear species 19 ( F, for example), for the 180° pulse affects the X spins but not A. Under these conditions the X vectors are flipped through 180°, but their "labels" remain unchanged (Fig. II.2.21C). Therefore, after O.St^ both the X transitions w i l l be exactly refocussed (Fig. II.2.21D); the echo is modulated by neither chemical shift nor J_ effects, and would be seen to decay in amplitude due to transverse relaxation effects (T^) only. We have seen that the usual 2D J_ experiment (Figs. II.2.21A,B,E,F) leads to a Jj-modulated spin echo. With the step-wise incrementation of t_i one builds up a data matrix which can be handled in the usual way (Section II.2.1) and the FT in jt x w i l l be a function of J . Hence, this i s called the "J_-diraensIon". One further point not apparent from the above discussion i s that line-widths in the 2D * J_-resolved experiment w i l l approach the natural line-width (T_2 •*• T_2'» i.e. the contribution of magnetic inhomogeneities to line-widths w i l l be very small, compared with a normal FT experiment. This arises from the fact that magnetic f i e l d inhomogeneity effects also refocus at t^, the time of the echo. - 159 -A generalized out-put is given in Fig. II.2.22A, depicted in the contour mode. Well resolved 2D peaks hold a one-to-one correspondence to the resonance lines observed in ID NMR, in the absence of strong coupling effects. One notes that a line drawn through a multlplet Is at 45° to J_2, when J_i and F_2 are drawn to the same scale. It i s mandatory to remove the 6 contribution from J_2 by "shearing", or " t i l t i n g " the data matrix according to (0)1,0.12') = (coi, 012 ~ " l ) , to give the plot shown in Fig. II.2.2.22B. Now J_2' is a function of 6 only and J_i (the J_-dimension) encodes only the scalar *H - *H couplings. 5 A 8 X 8 A 8X F2(5J) F2(6) A. B. Fig. II.2.22 Schematized 2D J-resolved contour plot output of AX spin system without (A) and with (B) t i l t i n g . The u t i l i t y of this experiment becomes apparent when one 51 considers projections and slices taken as outlined In Fig. II.2.20. The 0° projection (onto J^') of the complete data matrix w i l l given one line for each spin-multiplet - this has the appearance of a "broad-band decoupled" proton spectrum and gives the *H shifts. "Slices" taken at - 160 -the frequency corresponding to each shift, and viewed at 90°, w i l l give the "J-spectrum" for each resonance. These are referred to as "partial J_" spectra. In the absence of either strong coupling or very short T_2's, homonuclear 2D J-resolved spectroscopy must be the ultimate answer to the "hidden resonance" problem. Resonances need only be removed from one another by a few Hz (= 0.01 ppm at high field) for their J-spectra to be completely resolvable. The question of line-shapes in the 2D J-resolved experiment has received much attention. Because the method is based on phase modulation, the resonance lines have a phase-twisted shape which is very d i f f i c u l t to phase c o r r e c t 6 0 in two dimensions. With a phase-sensitive S(F_i,F_2) matrix one can almost always phase correct individual partial J_-spectra using the conventional A and B phase-corrections. A phase-sensitive t i l t e d ,FJ> ) matrix w i l l always yield J_-spectra with distortions at the base of resonance lines; however, the resultant absorption-like spectrum is obtainable 6 1 with significantly better line-width than those from the more commonly used phase-insensitive spectrum (power- or magnitude-spectrum). Homonuclear 2D J_-resolved spectroscopy is known to produce 62 63 additional transitions for strongly coupled » spin systems, which increases the complexity of the interpretation. Fortunately, these effects are generally predictable; additional transitions arising from strong coupling w i l l often l i e midway between the coupled resonances and usually (but not always) appear as a broad hump in the 0° projection of - 161 -the t i l t e d data-set. As such, they actually have diagnostic u t i l i t y in the sense that they provide warning of the presence of strong coupling. A note of caution should be sounded in the interpretation of such strongly-coupled systems, since the observable resonances may not be truly depicted in _Fi. Of course, one can make use of a two-dimensional 6 3 spectral simulation program as a check, but this is unlikely to be a routine procedure. Strong coupling effects in the J_-resolved spectra of amino acids have been studied in detail, 6** as have other spin systems. 6 3 The question of digital f i l t e r i n g (apodization) deserves special mention. The use of a pseudo-echo f i l t e r 6 5 w i l l produce absolute value lines with absorptive Gaussian character; that i s , narrower lines than what one would obtain using an exponential envelope (giving Lorentzian lines in the frequency domain). However, this is not ideal as i t attenuates signal-to-noise, causes severe intensity distortions and is particularly destructive when a many-lined multiplet resonates close to a strong signal. In a situation where signal-to-noise is at a premium, the usual exponential f i l t e r may be used in t_2' *f possible, i t is recommended to use a pseudo-echo f i l t e r in the j t 2 dimension. Line-narrowing in _tj may then be effected by a Lorentzian-to-Guassian transformation (a double-exponential function). If one is optimising the experiment for resolution in F j , a large number of experiments (n) should be performed to yield an acquisition time in Fi of several T_2's. Tilted proton 2D J_-resolved spectra should obviously be symmetrical about Fi • 0 , and a symmetrization procedure has been proposed 6 6 which improves spectral quality and signal-to-noise by H-4 H-2 H-1 H-6 Figure II.2.23 270 MHz 2D J-resolved experiment on 1. details. See text for - 163 -4- UM.W • > • M«r«- • I 1 1 1 I ' 1 1 I 1 1 1 I 6.0 i 1 1 1 i 4.0 5.0 * 1 k * • i 1 ft < j * ft i l 1 i • I !) i i i i 1 i I * t I * (<5 p p m ) J(Hz) F i g . I I . 2 . 2 4 D a t a t a k e n f r o m F i g . I I . 2 . 2 3 B , now p l o t t e d i n t h e c o n t o u r - p l o t m o d e . Q u a d r a t u r e i m a g e s f o r t h e i n t e n s e H - 2 r e s o n a n c e a r e m a r k e d w i t h a n a s t e r i s k . - 164 -elminating much of the "tailing" in F_ , and is especially useful in the case of strong singlet resonances. Fig. II.2.23 contains phase-insensitive displays of the homonuclear 2D J-resolved spectrum of 1. Fig. II.23A is the ID spectrum, showing seven multiplets. Fig. II.2.23B is the stacked plot representation of the 2D J-resolved experiment, performed with Exorcycle phase cycling and a single 180° pulse. Sinebell resolution enhancement was used in both dimensions. Fig. II.2.23C is the 0° projection of the ti l t e d data matrix - the "proton-decoupled" proton spectrum. From this the chemical-shifts are measured and the seven corresponding partial J-spectra are displayed. Their enhanced resolution is evident, especially for the H-l and H-5 resonances which are long-range coupled to one another, and other protons. Alternately, one may use a contour mode of display, as demonstrated in Fig. II.2.24. The resolution-enhanced ("sinebell") ID spectrum is above the contour plot. Most of the splittings are clearly discernable in the contour-plot and J_ measurements may be made directly from i t with adequate accuracy. I I . 2 . 5 . 3 Jeener Experiment: J-Correlated 2D NMR The "autocorrelation" 2D experiment was one of the f i r s t to 67 be mathematically explained and documented, yet did not gain wide acceptance until relatively recently when i t was improved to allow 68 quadrature detection in both dimensions. Now i t is perceived as a very useful experiment, which is quick and easy to perform, and whose - 165 -F i g . I I . 2 . 2 5 S t y l i z e d S_(F_i,F_2) c o n t o u r - p l o t o u t p u t o f a n AX s p i n s y s t e m ( A ) w i t h COSY a n d ( B ) w i t h SECSY e x p e r i m e n t s . - 166 -informational content is high and predictably extractable. Two major variants on the experiments exist, which w i l l be discussed separately. The 2D correlation spectroscopy (COSY) experiment involves a (90° — JtA — 90° - j t 2 ) pulse sequence and the idealized out-put (AX spin-system) i s given in Pig. II.2.25A. The spin-echo correlation spectroscopy (SECSY) variant involves a (90° - 0.5 t_i - 90° 69 - 0.5tj - _t 2) pulse sequence, and i t s stylized output i s shown in Fig. II.2.25B. The COSY experiment displays both chemical shift and couplings along both frequency axes, and gives rise to two types of peaks: "diagonal" peaks (8) along the line J _ i - J_2 (labelled "D") and "correlation", or "cross" peaks (8) between pairs of spins which are scalar coupled (labelled "C"). The latter are symmetrically disposed with respect to the principal diagonal. The SECSY experiment displays chemical shift and scalar couplings along F^, and A6/2 along F^. Thus, the informational content i s the same as COSY in that diagnoal- and cross peaks are both present - but the display i s slightly different. For both experiments, optimum magnetization transfer in t^i between coupled spins (AX) occurs at the maximum of s i n ( i t J j ^ t,i) exp ( - j t 1/T 2), while optimum detection of the transferred magnetization occurs at the maximum of the corresponding expression for jt 2. The expression i s plotted for a typical T^ (1.5s) and J_'s of 0.5 and 4 Hz i n Fig. II.2.26. This topic w i l l be raised later in Section II.2.5.3.3. A f i n a l general note concerns apodisatlon. With the Jeener experiment a pseudo-echo shaping function (e.g. "sinebell") i s recommended for use in both dimensions. As usual, absolute value mode - 167 -- 168 -display i s chosen and the reader interested in phase-sensitive 67 68 line-shapes i s referred to the literature. • II.2.5.3.1 COSY The basic COSY experiment uses the pulse sequence given below, and the phase-cycling of II.2.31. The latter i s required to differentiate between the so-called N-type (echo) and P-type (anti-echo) signals which must be differentiated when quadrature detection is used. RD - 90°(x) - Jtj - 90°($ 1) - AQN (_t2; $ 2 or $ 3) [II.2.30] Cycle 1 2 3 4 $1 x y —x -y * 2 (»> x -x x -x [II.2.31] $3 (P) x x x x The f i r s t preparatory pulse (90£) i s followed by the evolution period ( t i ) and the second mixing pulse (90°), and detection ( t 2 ) . The preparatory pulse has constant phase and the phase of the mixing pulse Is Incremented In 90° steps ($i). The receiver phase chosen, d>2 or $ 3 , selects the coherence transfer echo or anti-echo, respectively, and cancels the axial peaks at F t • 0 (which have Tj information and are relatively uninteresting). This phase-cycling scheme may be expanded to incorporate CYCLOPS, which removes Images resulting from quadrature detection. The basic four-cycles are repeated three times, incrementing - 169 -a l l phases by 90° to give a 16-cycle scheme. A satisfactory explanation of the experiment i s not possible without a density-matrix formalism, and even this becomes rather unwieldy when considering more than two or three spins. We limit discussion here to a highly stylized density-matrix formalism (see Fig. II.2.27) for an AX spin-system, with relaxation effects ignored. States A, B and C were described at the beginning of the chapter, in equations II.2.5 - II.2.9. A rotational matrix changes the i n i t i a l Boltzmann state density matrix, OQ into o"i which has eight non-zero terms from single-quantum transitions. With time, the terms in the density matrix evolve, and after jt x may be represented by o"2» Application of a 90° x 90° x Preparation Evolution Ct.i> Detection (t^) A. Boltzmann State 00 P 0 0 0 p 0 0 0 0 Pc 0 0 0 0 - 170 B . A f t e r P r e p a r a t o r y P u l s e «1 0 0 0 0 C . A f t e r E v o l u t i o n - 171 -D. M i x i n g P u l s e 0 o 0 0 A ^ 0 0 o A/> M i l A ^ H i 0 - 172 -F i g . I I . 2 . 2 7 S c h e m a t i z e d d e n s i t y m a t r i x f o r m a l i s m o f COSY e x p e r i m e n t f o r a n AX s p i n - s y s t e m - 173 -second rotational operator (i.e. a 90° pulse) on o 2 has the effect of mixing the elements in the two areas denoted by broken lines* Now each element Is a sum of four frequencies. The observable (single-quantum) coherences are depicted whilst generated zero- and multiple- quantum coherencies are ignored and indicated by parentheses. Each observable resonance frequency w i l l be detected during modulated by events in jti which are due to scalar coupling. The experiment Is performed with incremental delays in Jtj in the usual way, and subjected to 2D complex FT. The diagonal and cross peaks are symmetrical about the F_j • j ? 2 axis. Noise in the J j dimension, especially from strong solvent peaks, i s not symmetric and may be removed by "symmetrization" procedures described by Wuthrich and co-workers; 7 0* 7 1 an increase in signal-to-noise automatically results. For the symmetrization, each of the pairs of points in the 2D matrix which should be Identical are compared in turn; either the geometric average of the two points i s calculated and used in place of both values, or the smaller of the two i s taken as correct and i s substituted for the larger. A much "cleaner" and unambiguous display results. Fig. II.2.28 i s the 2D COSY spectrum of 1. Digitization i s f a i r l y good (2.7 Hz p t - 1 in F_j and F^), and considerable detail i s obvious within each response. In general practice one may often have to tolerate much coarser digitization (« 10 Hz p t - 1 ) but this does not appear to significantly affect the information content;* detail within *The antiphase nature of peaks in a correlation suggests that course digitization w i l l lead to their cancellation and poor intensity seen; this conviction does not appear to born out in the experiments performed in this thesis. F i g . I I . 2 . 2 8 COSY s p e c t r u m o f 1 ( 2 7 0 M H z ) . - 175 -c r o s s - p e a k s may n o t b e i d e n t i f i a b l e b u t " t h e c r o s s p e a k " i t s e l f w i l l s t i l l b e c l e a r . T h i s p o i n t w i l l be i l l u s t r a t e d i n l a t e r s e c t i o n s . T h e d i s p l a y e d d a t a - s e t wa s c o l l e c t e d u s i n g t h e f o u r c y c l e s e q u e n c e w i t h a n t i - e c h o s e l e c t i o n . T h e jS(F_2,F_i) d i s p l a y was n o t s y m m e t r i z e d . 3 C o r r e l a t i o n s a r e c l e a r f o r a l l J_ p a i r s a n d t h e c o u p l i n g p a t t e r n i s e a s i l y mapped o u t . A s u r p r i s i n g l y s h o r t r e l a x a t i o n d e l a y o f c a . 0 . 1 s i s u s u a l l y a l l t h a t i s n e c e s s a r y , m a k i n g t h e d a t a c o l l e c t i o n t i m e q u i t e s h o r t . II.2.5.3.2 SECSY T h e p u l s e s e q u e n c e u s e d f o r t h i s e x p e r i m e n t i s g i v e n b y E q . I I . 2 . 3 2 . RD - 9 0 ° ( x ) - 0 . 5 ^ - 9 0 ° ( d> i ) - 0.5_t_i - A Q N U 2 ; $ 2 ) [ I I . 2 . 3 2 ] P h a s e c y c l i n g i s a s f o r t h e COSY e x p e r i m e n t , w i t h e c h o s e l e c t i o n . T h e e x p e r i m e n t h a s t h e p o t e n t i a l a d v a n t a g e o v e r COSY t h a t t h e F j d i m e n s i o n r e p r e s e n t s d i f f e r e n c e s i n c h e m i c a l s h i f t , r a t h e r t h a n c h e m i c a l s h i f t i t s e l f . A s a r e s u l t , i t may be p o s s i b l e t o s u b s t a n t i a l l y r e d u c e t h e s w e e p - w i d t h i n F i w i t h o u t a l i a s i n g , p r o v i d e d t h a t t h e h i g h e s t - f i e l d r e s o n a n c e s a r e n o t c o u p l e d t o t h o s e a t l o w e s t - f i e l d . T h i s s m a l l e r s w e e p - w i d t h m e a n s t h a t f e w e r d a t a p o i n t s ( n ) i n F_i a r e n e c e s s a r y , w h i c h t r a n s l a t e s i n t o a s m a l l e r e x p e r i m e n t a l t i m e b o t h i n t e r m s o f d a t a a c q u i s i t i o n a n d c o m p u t a t i o n . A l t h o u g h t h i s i s a n a t t r a c t i v e o p t i o n , f o r many o r g a n i c m o l e c u l e s i t i s n o n - v i a b l e s i n c e i t i s q u i t e f e a s i b l e t h a t t w o r e s o n a n c e s a t d i f f e r e n t e x t r e m e s o f t h e s p e c t r u m may b e c o u p l e d . U n d e r t h e s e c i r c u m s t a n c e s , t h e t h e o r e t i c a l max imum s w e e p - w i d t h i n j ? i s h o u l d b e c h o s e n , s o a s t o a v o i d a l i a s i n g , a n d t h e p o t e n t i a l a d v a n t a g e s - 176 -o f SECSY o v e r COSY d i s c u s s e d e a r l i e r a r e i n a p p l i c a b l e . T h i s i s n o t t h e c a s e w i t h SECSY p e r f o r m e d o n p r o t e i n s , h o w e v e r , a n d i t i s w i t h t h i s c l a s s o f c o m p o u n d s t h a t t h e e x p e r i m e n t s hows m o s t p r o m i s e . T h i s t o p i c i s c o v e r e d i n d e t a i l i n t h e r e v i e w o f N a g a y a r a a . F i g . I I . 2 . 2 9 s hows t h e SECSY s p e c t r u m o f 1 . A s l i g h t l y r e d u c e d s w e e p w i d t h i n F j wa s p o s s i b l e i n t h i s c a s e , s i n c e t h e c o n n e c t i v i t i e s H-3 H-4 H-2 H-l H-6 H-6' H-5 E 2 F i g . I I . 2 . 2 9 SECSY s p e c t r u m o f 1 ( 4 0 0 M H z ) . T h e s w e e p - w i d t h i n _F_i was s l i g h t l y s m a l l , c a u s i n g a l i a s i n g o f p a r t o f t h e J _ ( 4 , 5 ) c o n n e c t i v i t y ; t h e a l i a s e d p o r t i o n i s m a r k e d w i t h a n a s t e r i s k . w e r e a l r e a d y k n o w n ; i f t h i s w e r e n o t t h e c a s e , i t w o u l d be i n a d v i s a b l e . D a t a - r e t r i e v a l f o r SECSY i s m a r g i n a l l y l e s s c o n v e n i e n t t h a n w i t h C O S Y . T h e J_i • 0 l i n e c o r r e s p o n d s t o t h e d i a g o n a l i n COSY . L i n e s j o i n i n g c o r r e l a t i o n s l i e a t a c o n s t a n t a n g l e w i t h t h e F_i = 0 l i n e ( 1 3 5 ° , i f p l o t t e d t o t h e same s c a l e ) ; t h i s p r o c e s s i s i l l u s t r a t e d i n F i g . I I . 2 . 2 9 . - 177 -Two disadvantages of the experiment merit note. F i r s t l y , since correlations in J_i occur at half the difference in chemical shifts (vs the f u l l chemical shift difference with COSY) the spectral resolution in F_l is halved. As molecular complexity increases, the number of cross-peaks increase too and with SECSY the "frequency area" (Fi*_F 2) i s half that of COSY, Increasing the likelihood of cross-peak overlap, and the resultant ambiguity. The second point arises from considerations of transverse relaxation times, and signal-to-noise. When performing any echo experiment on a large molecule with broader lines (shorter J J J ' S ) , significant intensity loss can occur during the refocussing period and the detected signal may be severely attenuated. This is less of a limitation with SECSY than with horaonuclear 2D J-correlated spectroscopy, as the following sample figures i l l u s t r a t e . Assume a half-height line-width (Avo.5) of 5 Hz with negligible contribution from magnetic inhomogeneity; from _T2 " 1/HAVQ 5 we get T_2 » 64 ms. The amplitude of a signal w i l l be attenuated by 75% after T_2*An 4 = 88 ms. Now, assuming a sweep-width in ¥_\ of ± 1000 Hz (10 ppm for a 4.7 Tesla magnet), the incremental delay required w i l l be 0.25 ms and the evolution period w i l l reach 88 ms after about 350 experiments. With a 2D J_-resolved experiment, the sweep-width in F_i might be ± 20 Hz, giving an incremental delay of 12.8 ms. One T_2 period w i l l have passed after 5 experiments. The signal w i l l be 75% attenuated in intensity after < 7 increments which is a small number compared with the number (32) required to give a digitization of 1.25 Hz per point. Of course, this disadvantage would be offset by the fact that resonances with broad - 178 -line-widths do not require very fine digitization, and 1.25 Hz per point could be more than adequate. These arguments hold in practice, as we 8ha11 see in later chapters. This question of sensitivity losses in the spin-echo experiment i s addressed briefly by Morris in his discussion on SECSY.72 SECSY appears to be a viable alternative to COSY when cross-peak overlap and broad line-widths are not evident. Whilst i t i s true that, in principle, SECSY should be faster to perform than COSY, this may not always be the case since signal degradation during the second 0.5 _ti period may demand more scans to be collected than would be the case with COSY. II.2.5.3.3 Delayed COSY - Detection of Long-Range Couplings As mentioned in the introduction to this section, the observed J_'s in a COSY experiment are a function of t^ and t^. If one i s Interested in detecting small J_'s (< 1 Hz), such as those which arise from long-range coupling, the data for a COSY experiment should be sampled around t% m 0.5 - 1.0s after the mixing pulse. Using the pulse scheme in Section II.2.5.3.1, to have an acquisition time <*> Is in both dimensions would require the acquisition and storage of prohibitive amounts of data and one resorts to a variant on the COSY experiment, 6 8 where a fixed delay, A, i s inserted after each pulse. RD - 90* - A - - 90°(*i) - A - A0N(t_2J *2 or » 3) [II.2.33] Practically, A i s set to approximately 0.3 s, which offers the best compromise between observation of correlations between protons coupled by small J_'s, and loss in signal amplitude during A. - 179 -F i g . I I . 2 . 3 0 D e l a y e d COSY e x p e r i m e n t o n 1 ( 2 7 0 M H z ) . A = 0 . 4 s . R e s p o n s e s f r o m l o n g - r a n g e c o u p l i n g s a r e s h a d e d . - 180 -Fig. II.2.30 shows this version of the COSY experiment, again using 1 as the exemplar. The delay, A, was 0.4 s. Responses corresponding to a l l J_ couplings are visi b l e , in addition to those from a wealth of long-range couplings (which are shaded in the diagram). H-l appears to be the most richly coupled proton, showing long-range responses to H-3, H-5 and H-6. The long-range coupling, J(H-1, H-6), is quite interesting and presumably indicates a reasonably "rigid orientation" of the acyclic C-6 moiety. H-2 i s conservative in i t s coupling - only to H-l and H-3. We draw the reader's attention to the 2D J-resolved experiment, Fig. II.2.24, where in the 0° projection (a 73 sky-line silhouette ) i t i s clear that H-2 i s the most intense signal, and H-l the weakest. We attribute this to the fact that H-2 has only four, well digitized lines, whilst H-l must have at least sixteen, poorly digitized. II.2.5.3.4 "Decoupled" COSY An interesting variant on the basic COSY experiment has been 48 suggested which has the net result that a l l couplings in Fj are collapsed, and the autocorrelation map i s considerably simplified. Its potential merits are obvious. The experimental scheme i s given in Fig. II.2.31. The 90° (preparatory) and 45° (mixing) pulses represent a variant on the 2-pulse COSY experiment and are phase-cycled in exactly the same way. Now, however, the time between these pulses Is held constant (t^) and a reduced mixing pulse of 45° i s used. Increments of 0.5t± separate the preparatory pulse from the refocussing 180° pulse.* The period between •Again, a composite pulse 3 8 i s used: 180°. = 90° 180° x 90° - 181 -90° 180° 45° 1$,) 1^3) (<I>2) Fig. II.2.31 Pulse scheme for the COSY experiment with homodecoupling in F j . Cycle 1 2 3 4 5 6 7 8 *1 X X X X X X X X $2 X y -x -y X y -x -y * 3 y y y y -y -y -y -y $1+ X -x X -x X -x -x -x [II.2.34] the refocusing and mixing pulses, t^ - 0.5_tj has to be decremented. At this time a decremental t\ is not available in an instrument 74 manufacturer's standard soft-ware, and must be programmed. If - 182 -long-range couplings are of interest, a delay, A, i s Inserted prior to acquisition. The phase-cycling is according to Eq. II.2.34. Usually the last value of 0.5t± has the composite 180° pulse just before the 45° pulse; that i s , the value of i s determined by the sweep-width and digitization in Pj. Now, the magnitude of t d w i l l favour cross-peaks arising from a certain J . / 5 With ** 1 s, J_ - 0.25 Hz i s favoured, and with Jt^  - 200 ms, £ - 1.25 Hz i s favoured. A i s non-zero only when long-range couplings are of interest. Fig. II.2.32A shows a contour plot of the decoupled COSY spectrum of 1. Sinebell resolution enhancement was utilized in both dimensions. A sweep-width in J_i of ± 350 Hz, and the 256 x 512 complex point data matrix resulted in t^ - 182 ms. Decoupling is seen to be good; the only visible imperfections in this plot i s a set of spurious responses at the mid-point between the geminal protons in F_2, which arises through strong coupling. Other smaller imperfections are not visible in the contour plot, but are seen in the Fj projection - they are signals aliased in J_2 about J_2 - 0 and are called "quadrature images". These are presumably due to imperfect phase settings for the pulses and residual transverse magnetization, and could possibly be eliminated by extending the phase cycling to include CYCLOPS. Once again the J(4,5) correlation i s weak in this experiment, as i t was in a l l other previous Jeener experiments. •Again, a composite p u l s e 3 8 is used: 180° = 90° 180° x 90° - 183 -F i g . I I . 2 . 3 2 D e c o u p l e d COSY e x p e r i m e n t w i t h 1 a t 2 7 0 M H z . t j - 182 ms a n d A • 0 . ( A ) , c o n t o u r - p l o t o f 2D s p e c t r u m . ( B ) , F j p r o j e c t i o n . ( C ) , c o n t r o l s p e c t r u m . - 184 -The experiment clearly has elements of COSY and homonuclear 2D correlated spectra, and i f the digitization in F_2 were fine enough, this single experiment could replace both these experiments, with the added advantage of the simplification of the COSY content. Of course, caution would have to be exercised with the detailed interpretation of line-shapes in F 7. II.2.5 .4 Heteronuclear Chemical Shift Correlation This very powerful 2D experiment correlates the chemical shifts of heteronuclei. Usually F_j i s chosen as the chemical shift axis 13 for protons, and as C. Although i t B uses with other heteronuclei such as 1 1B, 1 5N and 3 1P have been reported,**5 we shall restrict our discussion to the 1 3C - *H chemical shift correlation map (CSCM) 7 6 77 experiment » which correlates carbon resonances with the directly bound proton(s), as this i s the most common and diagnostically useful one. The pulse-sequence i s best designed such that C resonances (J_2) are broad-band decoupled, whilst 1H resonances (F^) show homonuclear coupling. These couplings turn out to be useful in interpretation of data, even though (a) a detailed examination of the proton multiplicities can lead to errors arising from i n f i d e l i t i e s inherent to the experiment, and (b) the digitization in F^ is seldom good enough to permit the observation of fine detail in homonuclear proton couplings. The experiment involves a magnetization-transfer step (see DEPT, II.2.2.2) and i s therefore inherently quite sensitive. Typically, 512 experiments are performed, collecting 2K complex data points in _t2» The minimum sweep-width in J_2 i s chosen and one can alias quaternary signals since they w i l l give no correlations. (Similarly, deuterated solvent peaks w i l l be absent). - 185 -The pulse-sequence has been refined to one having quadrature detection in both dimensions and the characteristics listed above. A minimum of four phase-cycles are mandatory, but this is often extended to eight (Eq. II.2.35). XH: RD - 90°(.^ - 0.5tj - - 0.5tj - A x - 90°($ 2) - A 2 - BB 1 3C: 180°($3) 90°(*O - AON (t 2;*5> [II.2.34] Cycle 1 2 3 4 5 6 7 8 $1 X X X X -x -x -x -x $2 X y -x - y -x - y X y $ 3 X X X X -x -x -x -x $ 4 X X X X X X X X &5 X y -x - y X y -x - y [II.2.35] The experiment may be understood by considering the populations for an isolated C - H fragment, as given in Fig. II.2.33A; the relative spin populations reflect the four-fold sensitivity difference between * H and C , viz. YJJ = 4y^. Now, we may ignore the 1 8 0 ° , C pulse at this stage, and concentrate on the proton pulses: 9 0 ° - t^ - 9 0 ° . A population inversion across one of the proton transitions results in the situation in Fig. II.2.33B. The affect of two 9 0 ° ( * H ) pulses, separated by tl to produce "population inversion" is shown in Fig. TI.2.34A and B. The i n i t i a l 9 0 ° * H pulse creates transverse magnetization which precesses in the x'-y' plane according to jJ_c-H* Depending on i t s position after t_i, the second 9 0 ° pulse may, in the limits, cause population inversion, - 186 -B. Fig. II .2.33 Energy levels of a CH fragment at equilibrium (A) and after a proton population inversion (B). (From a review by R. Freeman, Ref. 45). Wtio! 90* proton pulse Second 9 0 ' proton pulse Proton populations Change in °C moanetization PROTON FREE PRECESSION (U) diverted Saturated Boftzmam Saturated inverted Fig. II.2.34 The affect of a proton 90° - _t i - 90° pulse sequence on proton vectors, and their interpretation in terms of proton spin state populations is given in (A) and (B). ( C ) maps the corresponding change in population differences across the C transitions. (Adapted from Freeman and Morris. ) - 187 -saturation, or regeneration of Boltzmann state populations. The proton populations follow a cosine modulation after the second 90° pulse. Since the i 3C transitions share an energy level In common with this 13 modulated proton transition, the population differences between C levels are necessarily affected and this can be monitored with a 90° C "read" pulse. Four antiphase signals would be detected from such a pulse-sequence (90° - t^ - 90° 90° - AQN), showing heteronuclear coupling in both frequency dimensions. It would be desirable to remove the heteronuclear coupling; this i s accomplished in by employing 1 13 broad-band H decoupling during C acquisition, and in J_i by inserting 1 3 a C, 180° pulse halfway through the _tj period. This experiment might be expected to result in one transition for a C-H fragment,* but would actually result in no signal at a l l , due to the exact cancellation of the four antiphase signals. Two delays, A} and A 2 are Inserted to allow 180° relative phase rotation between these components (which differ in frequency by Jp_ H) and this provides a practical solution to the problem described above. This i s the experiment described in Eq. II.2.34. For a value of (2 * ^J^VE) - 1 effects the required phase 13 rotation of the proton vectors. Considering the C vectors, the situation i s more complex and calls for a compromise, since the time for the phase rotation w i l l depend on the number of attached protons. Bax has shown**6 that a good compromise is to select A 2 • 0,3/^ Jj^* Further, two refinements exist. To improve the i 3C decoupling, a composite 180° pulse 3 8 (180° - 90° 180° 90° ) is recommended. It •This would be located at 6 C (in F 2 ) and fig (in _F_i). - 188 -has also been demonstrated that broad-band C decoupling during t i may 78 be effected with an MLEV-16-type procedure giving good results without 7 9 the problem of excessive sample heating. Let us now pause to consider the merits and shortcomings of the experiment. Its strengths l i e in the relative ease with which i t is performed with a l l variables being determinable before the 2D experiment i s performed. The sensitivity i s good, since one benefits from the nOe as well as the polarization transfer. Further, the repetition rate i s based on proton relaxation parameters as opposed to the (generally 13 longer) C values. A relaxation delay of ca. 1.3 Tj is optimal. One may find spurious peaks at 6Q when the protons attached to the carbon form a tightly-coupled AB pair. Secondly, since the experiment relies on polarization transfer, this must be effi c i e n t . Hence the condition (1/2J) < T_2 i s necessary, which may restrict work with very large, conformationally rigid macromolecules. S t i l l , one major factor makes the experiment very attractive; one uses the very high dispersion of the 1 3C spectrum as a lever for the *H spectrum. Fig. II.2.35 shows the result of an experiment performed on a 0.5 M solution of 2 in C 6D 6, displayed in the contour plot mode. Details of the proton spectrum are not clear even when these are taken out as slices (not shown). In that the assignment of the proton 1 ^ spectrum is known, the C assignment immediately follows (going from low- to high-field: C-1, C-2, C-3, C-4, C-5, C-6). We conclude with a few remarks addressed to some practical considerations and suggest a general protocol. The experiment may be 13 1 performed with a C probe, using the H decoupler c o i l to pulse the proton spectrum. - 189 -1. 2. Tune the 1 3 C and ^ c o i l s and shim the magnet c a r e f u l l y , since proton spinning side-bands can be bothersome. Record the 1 3 C spectrum (quadrature detection; broad-band 1H decoupling, with the minimum sweep-width necessary -quaternary peaks may be folded over). Select a block si z e which gives an a c q u i s i t i o n time (AT) > 1T_2 and adequate d i g i t i z a t i o n f o r c l o s e l y spaced 1 3 C resonances. Save a sample spectrum on d i s c , with very good d i g i t i z a t i o n and signal-to-noise. 435-E, CH -435-C-1 100 C-3 C-5 C-2 c-4 J L 90 80 70 C-6 1 • -1 1 • 1 1 60 6 C H-5 H-6' -H-4 -H-3 E2(6c) F i g . II.2.35 CSCM on a 0.5 M solu t i o n of 2, at 100.3 MHz for 1 3 C . - 190 -3. Determine C pulse-lengths.* If these measurements are made on the sample Itself, i t may be best to observe the fastest relaxing carbon (e.g. -CH3). 4. Set up the spectrometer to observe protons, pulsing and observing with the decoupler c o i l . Set the decoupler offset to the middle of the *H spectrum and choose a minimum sweep-width without introducing aliasing. Save a sample spectrum on disc. Note the dwell-time (DW) for the particular sweep-width. 5. Measure the *H t_go (90° pulse-length), observing *H. 6. Change back to 1 3C observe. 7. Determine an accurate *H 90° pulse length: a. By residual coupling in a single frequency off-resonance decoupled (SFORD) experiment. 2 ^ (YB 2) - AV F T 1) Hz [II. 2.36] —RES = 1/4( YB 2) s [II.2.37] Av = distance (Hz) the decoupler is placed from the proton resonance frequency. J_0 = J_(C-H) for the attached carbon in the absence of continuous-wave decoupling. - residual J_(C-H) for the attached carbon with continuous-wave decoupling during acqusition. *For determination of a l l pulse lengths, one may choose to use a simpler sample at higher concentration, such as methanol. - 1 9 1 -With this experiment i t is necessary to know at least one 1 3 C - *H assignment and determine the *H frequencies 13 before returning to C observe. Two experiments are performed, one with the decoupler off during acqusition (to give Jfl) and one with i t on at i t s highest power level during acqusition (to give J R E S ^ * Knowing Av, too is now easily calculated. (Eqns. 1 1 . 2 . 3 6 , 3 7 ) . b. By a simple coherence-transfer experiment. 1 3 C : RD - 9 0 ° - 1 / 2 J - - AQN XH: 9 - BB [ I I . 2 . 3 8 ] This simple pulse scheme is performed after any J (C-H) has been measured. When 8 M 9 0 ° , no signal is seen for 13 this C resonance. 8 . The C S C M experiment is now set up. 9 . Ai, A2 are selected for a typical J£H» E * 8 * 1 3 5 Hz.* 1 0 . The incremental delay in _ti i s set to half the proton dwell time (see 4 ) or calculated from I I . 2 . 2 4 . The number of experiments to be performed (typically, 5 1 2 ) should give an acqusition time in _ti of ca. 1 0 0 ms. 1 1 . The number of scans at each jti value should be set to the value which gives "reasonable" signal-to-noise in the ID 1 3 C experiment. 1 2 . A relaxation delay of ca. 1 . 3 * T_i (1H) is chosen. *Ai ° 3 . 7 ms and A 2 ™ 2 . 2 ms. - 192 -13. Data are now c o l l e c t e d (minimum time f o r experiment « ca. 1 hour). 14. The data are processed with as much resolution-enhancement i n both dimensions as the signal-to-noise permits. A double-exponential or s h i f t e d s i n e b e l l m u l t i p l i e r should be used i n the data manipulation. II.Z.5.5 Relayed Coherence Transfer 8 0 This 2D experiment was suggested by Bolton as an assignment a i d , having elements of CSCM and homonuclear c o r r e l a t i o n experiment. A CSCM-like d i s p l a y having, i n addition, c o r r e l a t i o n s when the attached protons are scalar coupled. Consider an AMX fragment as i n F i g . II.2.36A. The f i r s t stage of the experiment involves transfer of coherence between H A and H^. This may then be relayed to the heteronucleus, In a CSCM experiment. C C C C C C X X X A B C F i g . II.2.36 Steps of magnetization transfer i n the relayed coherence transf e r experiment. The pulse sequence and phase-cycling are as per II.2.39 and II.2.40, r e s p e c t i v e l y : - 193 -XH: RD-90°-0. 51^- -0.5_ti~90 0 (*i)-0.5 tffi-l80°-0.5t^-Ai-900 ($ 2)-A 2-BB 1 3C: 180° 90°($ 2)- AQN <t 2) [II.2.39] Cycle 1 2 3 4 5 6 7 8 9 10 n 12 13 14 15 16 *1 X -x y -y X -x y -y X -x y - y X -x y -y $ 2 X X X X -x -x -x -x y y y y - y - y - y -y [II.2.40] The f r e e i n d u c t i o n decays are co-added and ( t m + A i ) ™ ca. 33 ms, favouring JAM = 7.7 Hz (1/4JAM). The experiment was f i r s t performed on a concentrated sample of n-propanol, d i s p l a y e d as a stacked-plot i n F i g . II.2.37. Peaks a r i s i n g from heteronuclear c o r r e l a t i o n s are l a b e l l e d (*) and the other symmetrical c o r r e l a t i o n s i d e n t i f y neighboring carbons/protons. Hence, the one experiment has the p o t e n t i a l to act as a t o o l i n determining heteronuclear c o r r e l a t i o n s and coupling pathways w i t h i n a molecule. The 13 1 C and H assignments are obvious. The experiment w i l l not be d e a l t w i t h at any l e n g t h , since i t was observed i n the author's hands to be l i m i t e d to simple molecules having weakly-coupled proton s p e c t r a . When s o l u t i o n s of sucrose and 2 were t r i e d , the r e s u l t s were l e s s than s a t i s f a c t o r y . An analogous homonuclear c o r r e l a t i o n experiment which has a COSY-llke d i s p l a y 81 i d e n t i f i e s a f a m i l y of s c a l a r coupled s p i n s . The output i s q u i t e - 1 9 4 -messy even for the single AMQ3X system illustrated, and one is led to speculate that even two such overlapping systems would be d i f f i c u l t to decipher. Fig. II.2.37 Relayed coherence transfer experiment performed on n-propanol (*H frequency 400 MHz). Peaks present in the CSCM experiment are labelled with an asterisk. 82 83 II.2.5.6 2D Nuclear Overhauser Enhancement Spectroscopy > A 2D experiment exists which explores magnetization transfer (via chemical exchange or nOe) and, in the nOe sense, i s called 2D nuclear Overhauser enhancement spectroscopy or "NOESY". The experiment bears many similarities to COSY/SECSY; i t involves a three-pulse - 195 -sequence and i s an autocorrelation experiment, correlating spins involved in magnetization transfer, such as nOe. The display of the experimental output may be chosen as COSY-like (II.2.47A) or SECSY-like (II.2.41B). RD - 90°($ 1) - t_i - 90 8($ 2) ~ ^ i x " 9 0 ^ ^ 5 " A0N(_t2;^) [II.2.41A] RD - 90°($i) - 0.5t\ - 90°($ 2) - f m l x-90°($3) - 0.5t^ - AQN(t2;5>0 [II.2.41B] For sequence II.2.41A the display shows a plot of 6 vs 6 with the normal spectrum along the diagonal, and cross-peaks indicating magnetization transfer ( c f . COSY). With sequence II.2.41B, Fj. represents A6/2 and the normal spectrum li e s at F_x - 0 ( c f . SECSY). Phase-cycling Is important in this experiment and achieves two purposes: (a) quadrature detection in Fj with quadrature image supression and (b) elimination of multiple quantum coherence (MQC) contributions to cross-peaks (see later). Hull proposed the following scheme. Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 * l X X X X X X X X X X X X X X X X $2 X y -x - y X y -x -y -x - y X y -x - y X y $3 X - y X - y y X y X -x y -x y - y -x -y -x *4 X X -x -x y y - y - y X X -x -x y y - y - y tH.2.42] - 196 -Since the experiment was f i r s t suggested, several detailed papers have emerged from the same group concentrating on theoretical a s p e c t s , 8 2 * 8 3 ' 8 5 refinements 8 6 - 8 9 and a p p l i c a t i o n s . 9 0 - 9 2 A brief description is given in Fig. II.2.38. 9 0 X 9 0 X 9 0 X Preparation Evolution •A B' Mixing •C ( Tmix ) Detection (t^) •E Fig. II.2.38 Schematized NOESY experiment and vector model. - 197 -We consider two (homonuclear) spin-1/2 nuclei, close in space and not scalar coupled. The "preparation" period is terminated by the f i r s t 90° "preparatory" pulse, which creates transverse magnetization (Fig. II.2.38A). The "evolution" period (t^i)* serves to frequency-label the vectors ( c f . COSY) and at the end of this period they may be represented by Fig. II.2.38B. A second "mixing" pulse now generates z-magnetization (Fig. II.2.38C); transverse magnetization also exists at this stage but is undesirable and removed by phase-cycling and, therefore, omitted from the diagram. During the "mixing" period, %ix> magnetization transfer (e.g. dipole-dipole or cross relaxation) processes occur where possible. The end of the mixing period (Fig. II.2.38D) is marked by a fin a l 90° "detection" pulse, which recreates detectable transverse magnetization (Fig. II.2.38E) which is sampled during the "detection" period, t_2> Normal 2D FT yields a COSY-like plot with a diagonal bearing the ID spectrum and cross-peaks arising where magnetization transfer processes occurred during t ^ x (ideally). We now look at the experiment in the light of the motion of the molecule. A rapidly tumbling, low molecular weight molecule (extreme-narrowing limit) w i l l behave differently from a large, high molecular weight molecule at high-field (spin diffusion l i m i t ) . In the extreme-narrowing limit, the dipole-dipole relaxation mechanism often dominates and NOESY cross-peaks would be negative going compared to the diagonal peaks ( i f phase-sensitive display were used) and small - a maximum of ca. 10% of diagonal peak height ( t ^ x = T n e c r i t i c a l case of uin^c ° 1*12 leads to zero cross-peak intensity. In the spin *_ti i s systematically incremented in the usual fashion. - 198 -diffusion regime, cross relaxation dominates and cross peaks have the same phase as diagonal peaks. Moreover, cross- and diagonal-vpeak intensities can be quite comparable under favourable conditions. The degree of intragroup relaxation i s an important factor in the two regimes. If one were considering two close methyl groups in a molecule, these protons would have strong intragroup relaxation and under extreme narrowing conditions the cross-peaks would amost certainly be undetectably small; in the spin diffusion limit, however, strong cross-peaks could be expected. When attempting a NOESY experiment, a decision has to be made regarding the choice of T^^* The variation of nOe cross-peak intensity as a function of Tmi x i s well understood. In the extreme-narrowing limit*, the cross-peak build up occurs as a function of Tmix i s roughly analogous to those seen in the TOE experiment (II.2.4.2) and nOe builds up linearly with to a maximum value, and then tapers off. In such a case an optimal i m i x would be » 1T_J. In the spin diffusion limit,* the build up curve i s entirely analogous 9 3 to that obtained from a driven nOe experiment (1.2.4.3). Generally, a mixing time can be selected by doing a t r i a l driven nOe experiment; an irradiation time which produces a strong negative nOe can be used as % i x ' *In the extreme narrowing limit w i l l be ca. Is, and in the spin diffusion limit, ca. 100 ms. - 199 -A complication with the NOESY experiment, alluded to earlier, is that more than one mechanism can give rise to cross-peaks. Zero (Z-) and MQC give rise to cross-peaks with scalar coupled spins, analogous to COSY. These are referred to as J_ cross-peaks and arise from coherent magnetization transfer (vs. incoherent magnetization transfer in the case of nOe). Several schemes have been put forward to supress these contributions. F i r s t - , second- and third order MQC may be easily 8 3 86 removed by phase-cyling. » The problem remains with ZQC. Several 8 5 87 ideas have been suggested » in this respect but have not been systematically evaluated: a. Digital apodization can be used to discriminate against J_ cross-peaks. b. Randomized T m j y « Instead of using exactly the same Tmix in each experiment, T ^ X is randomly varied 10-20% (e.g. 0.9 < T m <. 1.1 is a 10% randomization of x^x • 1.0 s). NOe cross-peaks are hardly affected by this, but J_ cross-peak intensity behaves in an oscillatory fashion during Xmix a n (* t n e randomization causes a smearing of J_ cross-peaks in the Fj axis, resulting in an additional noise band significantly reduced In intensity and, hopefully, below the contour level threshold. The noise band could, however, obscure a small nOe response of Interest. c. Partial refocussing. Introduction of a refocussing 180° pulse placed at random positions during T ^ X n a s been suggested to result in a similar effect to (b). - 200 -II.2.39 2D NOESY spectrum of 1 at 400 MHz. A mixing time of 0.3 was used, randomized ± 10%. - 201 -d. Sequential variation of Y . In this experiment, x m i x i s progressively incremented in parallel with the evolution time, jtj. The mixing time is set - x^^ + X t.i, where x° Is the i n i t i a l value and x * s chosen such that mix Tmix ^ (ac3u*s:'-t:|l-on t-^we i - n £.].)• ^ symmetrization procedure is imperative to eliminate ZQC and MQC peaks which are not symmetrical about the line Fj = J_2. Here, JT cross-peaks are not smeared out as in the case of (b), but i t was advised that the experiment be repeated for a second Tmix t o r e m o v e a nY doubts of ambiguities arising from accidental symmetrical disposition of Z- or MQC peaks. In the author's hands, the simplest procedure has been the u t i l i z a t i o n of phase-cycling, the randomized x^x and sinebell apodization in both jt x and _t2« Fig. II.2.39 shows a contour plot of the NOESY experiment for 1, which should be compared with the J_-correlated spectrum (Fig. II. 2.28). II.2.6 Conclusions The reader has been introduced to a large number of the NMR experiments which we believe w i l l find greater u t i l i t y in the years to come. Where possible, the theory has been explained at as pic t o r i a l a level as possible since the assumption has been made that the reader w i l l not have had extensive experience in these areas. Except where an experiment has failed (relayed coherence - 2 02 -t r a n s f e r ) , t h e s e h a v e b e e n v i e w e d i n a s i m p a r t i a l a l i g h t a s p o s s i b l e , s i n c e t h e i n t e n t i o n w i t h t h i s c h a p t e r i s t o i d e n t i f y , e x p l a i n a n d i l l u s t r a t e p o t e n t i a l l y u s e f u l e x p e r i m e n t s . I n t h e f o l l o w i n g c h a p t e r s , s e v e r a l o f t h e s e e x p e r i e n t s a r e u s e d a s we w o r k o u r way t o w a r d s a p r a c t i c a l e v a l u a t i o n o f t h e c o m b i n a t i o n o f e x p e r i m e n t s t o y i e l d a n e f f e c t i v e p r o t o c o l f o r t h e s t r u c t u r e e l u c i d a t i o n o f o r g a n i c m o l e c u l e s u s i n g NMR s p e c t r o s c o p y . The g r a p h , b e l o w , s h o w s t h e r e l a t i v e n u m b e r o f p u b l i c a t i o n s o f 2D NMR r e l a t e d p a p e r s s i n c e I t s c o n c e p t i o n 1 9 7 5 . T h e f i r s t 3 o r 4 y e a r s s aw a s t e a d y r a t e o f 2D NMR p u b l i c a t i o n , d o m i n a t e d b y a h a n d f u l o f w o r k e r s c o n c e n t r a t i n g o n f u n d a m e n t a l d e v e l o p m e n t . T h e i n c o r p o r a t i o n b y i n s t r u m e n t m a n u f a c t u r e r s o f t h e n e c e s s a r y s o f t - w a r e i n t o s t a n d a r d s p e c t r o m e t e r s , a n d t h e e x c i t e m e n t g e n e r a t e d I n t h e s c i e n t i f i c c o m m u n i t y i n t h e p r e c e d i n g y e a r s , l e d t o a r a p i d g r o w t h p h a s e f r o m 1979 t o 1 9 8 1 . I n t e r e s t i n g l y , s t i l l r e l a t i v e l y f e w g r o u p s w e r e i n v o l v e d w i t h t h e a s s e s s m e n t o f t h e f i r s t e x p e r i m e n t s , a n d t h e i r a p p l i c a t i o n t o " r e a l - l i f e " p r o b l e m s . I n t h e a u t h o r s ' s o p i n i o n , 1 9 8 3 a n d s u b s e q u e n t y e a r s w i l l w i t n e s s a f u r t h e r e x p l o s i o n o f l i t e r a t u r e a r t i c l e s d r a w i n g o n 2D NMR f o r r e s u l t s . I n c r e a s i n g n u m b e r s o f p a p e r s w i l l h a v e a s t h e i r f o c u s t h e d a t a f r o m t h e e x p e r i m e n t s , r a t h e r t h a n t h e i r e v a l u a t i o n a s a s o u r c e o f i n f o r m a t i o n . T h i s t h e s i s w i l l h o p e f u l l y a s s i s t i n t h e a c c e p t a n c e o f 2D NMR w i t h a new a i r o f c o n f i d e n c e . T h i s c h a p t e r , s p e c i f i c a l l y , s h o u l d s e r v e a s a c o m p i l a t i o n o f ID a n d 2D NMR e x p e r i m e n t s w h i c h a n o r g a n i c c h e m i s t o r s p e c t r o s c o p t i s t s h o u l d f i n d u s e f u l w h e n c o n s i d e r i n g a p a r t i c u l a r e x p e r i m e n t . - 203 -Y e a r R e l a t i v e N u m b e r o f P u b l i c a t i o n s 1975 * 1976 * 1977 *** 1978 *** 1979 ********* 1930 ************ 1981 ******************** 1982 ********************** T a b l e I I . 2 . 1 A n i n d i c a t i o n o f t h e r e l a t i v e n u m b e r o f p u b l i c a t i o n s r e l a t i n g t o 2D NMR i n t h e l i t e r a t u r e , c a t e g o r i z e d by y e a r . - 204 -REFERENCES 1. M.L. Martin, J.-J. Delpeuch, and G.J. Martin. Practical NMR spectroscopy. Heyden and Son Ltd., Philadelphia. 1980. 2. T.C. Farrar and E.D. Becker. Pulse and Fourier transform NMR. Introduction to theory and methods. Academic press, N.Y. 1971. 3. D. Shaw. 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Phys. Chem. 84, 2880-2883 (1980). 40. S.L. Gordon and K. Wiithrich. J. Amer. Chem. Soc. 100, 7094-7096 (1978) . 41. G.A. Morris and R. Freeman. J. Magn. Reson. 29, 433-462 (1978). 42. G. Wagner and K. Wiithrich. J. Magn. Reson. 33, 675-680 (1979). 43. F.M. Poulsen, J.C. Hoch, and CM. Dobson. Biochem. 19, 2597-2607 (1980). 44. CM. Dobson, E.T. Olejniczak, F.M. Poulsen, and R.G. Ratcliffe. J. Magn. Reson. 48, 97-110 (1982). 45. Recent reviews: G.A. Morris. Fourier, Hadamard, and Hilbert transforms in chemistry. Edited by A.G. Marshall. Plenum Press, New York. 1982. pp. 271-305. K. Nagayama. Adv. Biophys. 14, 139-204 (1981). R. Freeman. Proc. R. Soc. Lond. A. 373, 149-178 (1980). R. Freeman and G.A. Morris. Bull. Magn. Reson. 1, 5-26 (1979) . 46. A. Bax. Two-dimensional nuclear magnetic resonance in liquids. D. Reidel, Holland. 1982. 47. S. Sukumar. Ph.D. Thesis, University of British Columbia. 1981. 48. G. Bodenhausen, R. Freeman, R. Niedermeyer, and D.L. Turner. J.  Magn. Reson. 26, 133-164 (1977). 49. Ref. 46, pp. 41-43. 50. S. Brownstein. J. Magn. Reson. 42, 150-154 (1981). 51. K. Nagayama, P. Bachman, K. Wiithrich, and R.R. Ernst. J. Magn.  Reson. 31, 133-148 (1978). 52. D.L. Turner. J. Magn. Reson. 49, 175-178 (1982). 53. W.P. Aue, P. Bachmann, A. Wokaun, and R.R. Ernst. J. Magn. Reson. 29, 523-533 (1978). 54. Ref. 46, pp. 99-101. 55. E.L. Hahn and D.E. Maxwell. Phys. Rev. 88, 1070-1084 (1952). 56. L.D. Hall and S. Sukumar. J. Amer. Chem. Soc. 101, 3120-3121 (1979). 57. G. Bodenhausen, R.Freeman, and D.L. Turner. J. Magn. Reson. 27, 511-514 (1977). - 207 -58. G. Bodenhausen and D.L. Turner. J. Magn. Reson. 41, 200-206 (1980) . 59. L.D. Hall, G.A. Morris, and S. Sukumar. Carbohydr. Res. 76, C7-C9 (1979). 60. M. Levitt and R. Freeman. J. Magn. Reson. 34, 675-678 (1979). 61. L.D. Hall and S. Sukumar. J. Magn. Reson. 38, 555-558 (1980). 62. A. Kumar. J. Magn. Reson. 30, 227-249 (1978). 63. G. Bodenhausen, R. Freeman, G.A. Morris, and D.L. Turner. J.  Magn. Reson. 31, 75-95 (1978). 64. G. Wider, R. Bauraann, K. Nagayama, R.R. Ernst, and K. Wiithrich. J. Magn. Reson. 42, 73-87 (1981). 65. A. Bax. R. Freeman, and G.A. Morris. J. Magn. Reson. 43, 333-338 (1981) . 66. J.D. Mersh and J.K.M. Sanders. J. Magn. Reson. 50, 171-174 (1982). 67. W.P. Aue, E. Bartholdi, and R.R. Ernst. J. Chem. Phys. 64, 2229-2246 (1976). 68. A. Bax and R. Freeman. J. Magn. Reson. 44, 542-561 (1981). 69. K. Nagayama, A. Kumar, K. Wiithrich, and R.R. Ernst. J. Magn.  Reson. 40, 321-334 (1980). 70. R. Bauman, G. Wilder, R.R. Ernst, and K. Wiithrich. J. Magn. Reson. 44, 402-406 (1981). 71. R. Bauman, A. Kumar, R.R. Ernst, and K. Wiithrich. J. Magn. Reson. 44, 76-83 (1981). 72. Morris' review, ref. 45. 73. B. Bliimich and D. Ziessow. J. Magn. Reson. 49, 151-154 (1982). 74. S. Sukumar, personal communication. 75. A. Bax, personal communication. 76. A. Bax and G.A. Morris. J . Magn. Reson. 42, 501-505 (1981), and others in the series. 77. Ref. 46, pp. 50-65, and references cited therein. 78. J. S. Waugh. J. Magn. Reson. 49, 517-521 (1982). - 208 -79. P.H. Bolton. J. Magn. Reson. 51, 134-137 (1983). 80. P.H. Bolton. J. Magn. Reson. 48, 336-340 (1982). 81. G. Eich, G. Bodenhausen, and R.R. Ernst. J. Amer. Chem. Soc. 104, 3731-3732 (1982). 82. J. Jeener, B.H. Meier, P. Bachman, and R.R. Ernst. J. Chem. Phys. 71, 4546-4553 (1979). 83. S. Macura and R.R. Ernst, Molec. Phys. 41, 95-117 (1980). 84. W.E. Hull. Two-dimensional NMR. Aspect 2000. Bruker Report (1982). 85. S. Macura, Y. Huang, D. Suter, and R.R. Ernst. J. Magn. Reson. 43 259-281 (1981). 86. G. Bodenhausen and R.R. Ernst. Molec. Phys. 47, 319-328 (1982). 87. S. Macura, K. Wiithrich, and R.R. Ernst. J. Magn. Reson. 47, 351-357 (1982). 88. S. Macura, K. Wiithrich, and R.R. Ernst. J. Magn. Reson. 46, 269-282 (1982). 89. G. Bodenhausen and R.R. Ernst. J. Amer. Chem. Soc. 104, 1304-1309 (1982). 90. A. Kumar, R.R. Ernst, and K. Wiithrich. Biochem. Biophys. Res.  Comm. 95, 1-6 (1980). 91. A. Kumar, G. Wagner, R.R. Ernst, and K. Wiithrich. Biochem  Biophys. Res. Comm. 96, 1156-1163 (1980). 92. G. Wagner, A. Kumar, and K. Wiithrich. Eur. J. Biochem. 114, 375-384 (1981). 93. A. Kumar, G. Wagner, R.R. Ernst, and K. Wiithrich. J. Amer. Chem.  Soc. 103, 3654-3658 (1981). - 209 -CHAPTER II.3 OLIGOSACCHARIDE CHARACTERIZATION USING NMR Complex carbohydrates are widely dispersed in Nature where they play a variety of roles. Often in polymerized form, they constitute structural materials (e.g. cellulose, bacterial cell-walls), or energy storage molecules (e.g. starch, glycogen). With bacterial cell-walls they can serve the additional purpose of coding the immunogenicity the organism presents. An equally important role is played by carbohydrates conjugated to protein or l i p i d - glycoproteins or glycolipids respectively. Again these serve the gamut of functions, as structural molecules (e.g. collagen), the "letters" of the blood-group substance's "alphabet", lubricants (e.g. gastro-intestinal tract mucins), "anti-freeze" agents (in Arctic fish) and bound to a host of proteins (e.g. some enzymes and the immunoglobulin proteins). It has even been speculated that in time, a l l proteins w i l l be found to contain some carbohydrate! The ubiquity and importance of complex carbohydrates in biological systems has, not surprisingly, attracted much attention. I n i t i a l investigations tended to focus on primary structure - which sugars are present and the details of the ordering. However, in the last few years people have begun to try to relate structure with function, and to this end knowledge of the secondary, three-dimensional picture i s sought. Prior to this study, a wide range of technologies existed for sequencing complex carbohydrates, which w i l l be briefly reviewed below. - 210 -The last few years has seen a demonstration of the tremendous potential of NMR in this f i e l d and this chapter discusses the development of one such protocol which we believe to be of some potential. Pivotal to the procedure is the use of recently developed techniques detailed in the preceding chapter; an understanding of these w i l l henceforth be assumed. NMR has the broad advantages of being a non-destructive technique with several extractable parameters, each with i t s own information content. As a counterpoint, the method is inherently insensitive and demands on sample quantity may totally preclude i t s use for many biologically important systems. Before the 1970's, almost a l l structural studies hinged on classical "wet" chemical analyses. 3 These demand relatively large amounts of sample, are labour intensive and have even been found to be incorrect when re-examined. The early seventies saw the Infusion of spectroscopic techniques and at this time, such methods must be considered indispensable. Although the focus here is primarily on NMR, the u t i l i t y of mass-spectroscopy cannot be overlooked, especially in light of i t s signal-to-noise sensitivity. 13 C NMR spectroscopy has been of great u t i l i t y in the area, (often in conjunction with "wet" methods), and recent reviews have been presented by Gorin and Barker jat a l . Nevertheless C NMR spectra remain d i f f i c u l t to ful l y assign and structural assignments are totally 7 8 convincing only when a series of molecules is synthesized ' (or produced by degradation) and a systematic investigation is pursued. Anomeric configuration may confidently be assigned on the basis of - 211 -e i t h e r C c h e m i c a l s h i f t s o r JJCI-HI* T n e p o t e n t i a l e x i s t s f o r t h e 1 13 e x t r a c t i o n o f c o n f o r m a t i o n a l i n f o r m a t i o n , b a s e d o n i n t e r - r i n g H - C 9 c o u p l i n g s . H o w e v e r , t h e s e a r e o f t e n s m a l l (<_ 6 H z ) , u n r e s o l v e d a n d o n e may h a v e t o s y n t h e s i z e 1 0 t h e m o l e c u l e e n r i c h e d i n 1 3 C ; a s a r e s u l t t h i s a p p r o a c h h a s l i m i t e d p o t e n t i a l . N e v e r t h e l e s s , t h e o v e r a l l s i m p l i c i t y o f 13 C NMR s p e c t r a i s a p p e a l i n g a n d t h e t e c h n i q u e i s f i n d i n g a l a r g e r p l a c e a s s u p e r i o r e x p e r i m e n t s a n d p r o t o c o l s a r e d e s i g n e d . I n t h e l i m i t 13 t h o u g h , t h e f a c t t h a t C NMR i s i m p r a c t i c a l w i t h s u b - m i l l i g r a m q u a n t i t i e s may s e r v e t o l i m i t i t s u t i l i t y . A n u m b e r o f E u r o p e a n a n d C a n a d i a n g r o u p s h a v e a l r e a d y d e d i c a t e d some c o n s i d e r a b l e e f f o r t t o t h e a p p l i c a t i o n o f ^H NMR t o o l i g o p e p t i d e s a n d c e r e b r o s i d e s . T h e F r a n c o - D u t c h g r o u p o f V l i e g e n t h a r t a n d M o n t r e u i l 2 h a s f o c u s s e d o n t h e s t r u c t u r a l l y r e l a t e d o l i g o m a n n o s i d e g l y c o p e p t i d e s , i s o l a t e d f r o m t h e u r i n e o f p a t i e n t s w i t h m a n n o s i d o s i s . T h e m a n n o s e a n o m e r i c a n d H - 2 p r o t o n s r e s o n a t e d o w n - f i e l d f r o m t h e b u l k o f t h e r i n g p r o t o n s . T h e i r c h e m i c a l s h i f t s a n d c o u p l i n g c o n s t a n t s w e r e a c c u r a t e l y m e a s u r e d a n d , b y r e f e r e n c e t o d a t a f r o m k n o w n c o m p o u n d s , a s s i g n m e n t s 11 12 w e r e m a d e . I n a b r o a d e r s t u d y b y C a r v e r a n d G r e y , u s e f u l c o n c l u s i o n s w e r e d r a w n a n d t h e m e t h o d e x t e n d e d . A l t h o u g h t h i s a p p r o a c h i s p r o b a b l y w h o l l y a p p r o p r i a t e f o r m o l e c u l e s o f t h i s t y p e , we d r a w a t t e n t i o n t o i t s l a c k o f g e n e r a l i t y b e c a u s e o f i t s r e l i a n c e o n a " l i b r a r y " o f r e f e r e n c e d a t a . F u r t h e r m o r e , v i r t u a l c o u p l i n g may 13 c o m p l i c a t e a n o m e r i c p r o t o n s i g n a l s . D a b r o w s k i ' s g r o u p i n G e r m a n y h a s c o n c e n t r a t e d o n g l y c o s p h i n g o l l p i d s a n d h i s w o r k h a s r e c e n t l y b e e n 14 r e v i e w e d . C o m p o u n d s w e r e d i s s o l v e d I n DMSO-d^ a n d p r o t o n m e a s u r e m e n t s - 212 -made on sub-milligram quantities. Extensive ID NMR measurements, together with catalogue data, allowed almost complete assignment of cerebrosides having up to ten sugar residues. At the time our study was performed, no 2D NMR had been used In such structural studies, and the developments are presented chronologically. 15 18 Hall and co-workers have put some considerable effort ~ into the use of proton Jj's as a measure of inter-proton distances in carbohydrate molecules. These, together with nOe methods described 19 earlier, have been capitalized on by Lemieux and co-workers in the complex carbohydrate area. An elegant study 7 used such NMR techniques to validate carbohydrate secondary structures determined by hard-sphere molecular modelling calculations, taking into account the exo-anomeric effect (HSEA). A series of blood-group determinant oligosaccharides 1 13 were synthesized and systematically studied by H and C NMR. This paper represents a land-mark in both spectral assignment and conformational analysis of complex oligosaccharides. Cerebrosides have been derivatised and studied in organic solvents by NMR. Martin-Lomas and Chapman made per-O-acetylated 2 0 galactocerebrosldes and noted large chemical shift changes upon varying solvents; this allowed them to make partial first-order assignments and confirm the anomeric configuration, and conformation of 21 the pyranose ring. Falk et a l . studied more complex Lewis-like cerebrosides as their (>-methyl ethers. Six compounds were considered, including a heptaglycosylcerebroslde. The fact that the O-CH3 resonances resonate at roughly the same chemical shift as the other ring - 213 -protons restricted their study mainly to anomeric protons at 6 > 4.0. A method was developed for differentiating Type I and Type II saccharide chains. 1 R = H 2 R = C O . C D 3 We now propose a protocol for the structure elucidation of 22 complex oligosaccharides, using NMR. As a model compound we chose a l l y l B-D-galactopyranosyl-B-(l->-4)-D-glucopyranoside ("8 a l l y l 23 lactoside"), 1, which we prepared using standard procedures. The oligomer is i n i t i a l l y considered as a mixture of glycosides of the constitutent monosaccharides, and we aim for the f u l l assignment of carbohydrate ring protons, and the determination of linkage order by Inter-ring nOe measurements. Our protocol is as follows: 1. Maximize signal dispersion. Use of a very high-field spectrometer with the parent compound may be adequate, but most times derivatization w i l l be necessary. Preferably the - 214 -derivatization reagents should be fully deuterated and, hence, spectroscopically silent, to preclude introduction of additional resonances which may overlap with those of the parent solute resonances. 2. A solvent, pure or mixed, should be found which minimizes signal overlap. 3. The assignment process i s begun with ID techniques, such as partially Tj relaxed spectra, SDDS and NOEDS. Should these prove sufficient to complete the assignment, steps 4 and 5 are omitted. 27 4. The 2D J-resolved experiment i s used to locate the resonance frequencies of a l l ring protons. Partial J_ spectra allow the measurement of a l l coupling constants. 5. Next, the J_ connectivities are established by the 2D J_ correlation experiment, COSY (or SECSY); each ring acts as an isolated spin system oblivious to it s partners, since inter-ring coupling w i l l be indetectably small or zero. 6. A few very specific ID experiments (3) may be necessary to resolve any outstanding ambiguities in assignment. 7. Next, each unit is identified (e.g. 8-galactopyranosyl) by 29 comparison of the measured J_-values with reference values. This identifies the sugars, their anomeric configurations and ring sizes. 8. The final task is the determination of the sequence along the chain; this i s done by determining the specific inter-ring relaxation effects. With ID nOe experiments i t is necessary - 215 -Fig. II.3.1 With a B(l-4) glycosidic linkage, irradiation of the glycosidic proton (H-l 1) induces intra-ring ( ) and a single inter-ring ( ) nOe. that a l l the anomeric protons be separately resolved (which may be possible by changing solvent). Aside from known intra-ring nOe's (1,3 diaxial protons, e.g.), each Fig. II.3.2. 270 MHz spectra of (A) 8 - a l l y l lactoside in D2O, and (B) p- a l l y l aceto(d_a) lactoside. - 216 -i r r a d i a t i o n should induce a unique i n t e r - r i n g nOe (see F i g . II.3.1). Since a l l protons have been assigned, the linkage can be e a s i l y deduced. The same information may be obtained from a 2D NOESY experiment. 3 0 In t h i s case the advantage i s that the anomeric signals may be hidden; t h i s may be of greatest u t i l i t y with more complex structures. We i l l u s t r a t e t h i s approach using B - a l l y l l a c t o s i d e , 1, as the exemplar. The 270 MHz 1H NMR spectrum of a D 20 so l u t i o n of the molecule i s shown i n F i g . II.3.2.A. The anomeric protons are c l e a r l y discernable as the doublets near 6 4.5. The bulk of the ring protons a l l resonate between 6 3.9 and 3.5 and only a few are separately discernable. The corresponding per-O-trideuterlo-acetylated d e r i v a t i v e , 2, was synthesized by standard methods 3 1 using perdeuterio-acetic anhydride. Its spectrum (0.2 M i n CgD 6*) i s shown i n F i g . II.3.2B. As expected, many signals ( a r i s i n g from O-acetylated centres) are deshielded and the spectrum i s considerably s i m p l i f i e d . In t h i s simple instance, spin-decoupling experiments almost s u f f i c e d to determine the f u l l assignment. 2D NMR in v e s t i g a t i o n s began with the 2D J-resolved experiment which allowed the detection of ten carbohydrate r i n g protons and the f i v e a l l y l protons. A contour p l o t of the 2D J-resolved experiment i s given i n F i g . II.3.3; t h e i r chemical s h i f t s and coupling constants were recorded. Next, the COSY experiment was performed at 400 MHz; the data are shown i n F i g . II.3.4, i n the contour-plot mode again. I t i s *In t h i s case, t h i s balance of concentration and solvent was found to minimize signal overlap. Fig. II.3.3 Contour plot of 2D J-resolved NMR spectrum of 2 (270 MHz; 0.2 M in CgDg). The assigned, resolution-enhanced spectrum is plotted above. - 218 -I I . 3 . 4 4 0 0 MHz COSY s p e c t r u m o f 2 ( 0 . 2 M i n C 6 D 6 ) . C o n n e c t i v i t i e s w i t h i n t h e g l u c o p y r a n o s y l r i n g a r e s hown ( ) i n t h e t o p l e f t h a l f , a n d t h e g a l a c t o p y r a n o s y l ( ) i n t h e b o t t o m r i g h t h a l f . A l l y l c o n n e c t i v i t i e s a r e s h o w n ( — • — • — ) . - 219 -convenient to begin tracing connectivities with the anomeric protons. For example, starting with H-l, the connectivities to the other glucosyl ring protons are clear from an inspection of Fig. II.3.4. In this way, i t was now possible to trace out a l l connectivities. The only ambiguity arose from the galactosyl H-2', H-3' and H-41 where cross-peak contour overlap resulted from H-2' and H-4' resonating very close to one another and both being coupled with H-3'. The assignment was made after careful Inspection of an expansion of this region of the COSY plot, and confirmed by matching coupling constants from the 2D J-resolved data. Comparison of these coupling constants with standard literature values in Table II.3.1, confirmed the existence of a 6-galactopyranoside and a 8-glucopyranoside moiety. The identification of the constituents monosaccharides and their anomeric configuration was complete. Next, the linkage sequence was determined from the nOe experiments involving the anomeric protons. The SSNOEDS experiment was just feasible at 400 MHz with adequate decoupler-frequency selectivity. The results, shown In Fig. II.3.5, clearly indicate a single "inter-ring" nOe In each case. As expected, irradiation of the galactosyl H-l' resonance induces an nOe into the glycosyl H-4 proton (as well as some lntra-rlng nOe's), and irradiation of the glucosyl H-l resonance induces an nOe into the a l l y l methylene pair, H-l^ and H-l". Similar information i s obtained from the 2D NOESY experiment, shown as the contour plot in Fig. II.3.6. The unique inter-ring nOe between H-l' and H-4 (nji_^) is clear, but the nOe into the methylene pair is small and below the contour level threshold. - 220 -Table II.3.1 - Comparison of expected and obtained vicinal ring proton coupling constants for (A) a 8-glucopyranoside, and ( B ) 8-galactopyranoside ring. Expected J ( H z ) 2 9 Measured J(Hz) A: (1,2) 7.8 8.0 (2,3) 9.6 9.9 (3,4) 8.8 8.9 (4,5) 9.5 10.4 B: (l'-2') 7.8 8.1 (2'-3') 9.8 11.0 (3'-4') 3.4 3.7 (4'-5«) 1.0 1.7 - 221 -6.0 5.0 4.0 8 (ppm) Fig. II.3.5 400 MHz SSNOEDS of 2 (0.2 M in C 6D 6). Intra-ring nOe's are shown with a solid line ( ) and lnter-rlng nOe's with a broken line ( ). - 222 -Fig. II'.3.6 NOESY of 2 (0.2 M in C 6D 6) at 400 MHz. The mixing time, Tmix "0.75 8 ± 15%. Intra-ring nOe's for the glucosyl ( ) and galactosyl ( ) rings are drawn in the bottom right half, and inter-ring ( ) in the top left half. F i g . I I . 3 . 7 C o m b i n e d NOESY ( t o p l e f t ) a n d COSY ( b o t t o m r i g h t ) d a t t a k e n f r o m F i g s . I I . 3 . 4 . a n d I I . 3 . 6 , r e s p e c t i v e l y . - 224 -S i n c e b o t h t h e COSY a n d NOESY d i s p l a y s a r e s i m i l a r a n d s y m m e t r i c a l a b o u t t h e p r i n c i p a l d i a g o n a l , t h e y c a n be c o m b i n e d i n t o a 3 2 s i n g l e d i s p l a y w h i c h h a s a l l t h e i n f o r m a t i o n . T h i s i s s h o w n i n F i g . I I . 3 . 7 . A n u m b e r o f p o i n t s w a r r a n t d i s c u s s i o n . A l t h o u g h t h i s p r o t o c o l d o e s n o t u s e a n y new NMR e x p e r i m e n t s , t h e i r i n t e g r a t i o n i n t o t h i s s c h e m e p r o v i d e s a n a p p r o a c h w h i c h w i l l f i n d a p p l i c a t i o n w i t h many c o m p l e x m o l e c u l e s o f b i o l o g i c a l i n t e r e s t . We f e e l t h e f a c t t h a t t h e a p p r o a c h h a s a m i n i m a l r e l i a n c e o n r e f e r e n c e d a t a w i l l make i t a p p l i c a b l e t o a w i d e a r r a y o f m o l e c u l e s . I t I s i m p o r t a n t t o n o t e t h a t t o t h e a u t h o r ' s k n o w l e d g e , n o i n s t a n c e s i n N a t u r e h a v e b e e n r e p o r t e d w h e r e s u g a r r i n g s e x i s t i n u n u s u a l c o n f o r m a t i o n s - h o w e v e r , I f s u c h a s i t u a t i o n w e r e t o o c c u r , i t w o u l d be i m m e d i a t e l y o b v i o u s f r o m t h e v i c i n a l c o u p l i n g s , a n d e x t r a s t e p s c o u l d be n e c e s s a r y t o r e s o l v e s u c h a n a n o m a l y . B y r e s t r i c t i n g t h e p r o t o c o l t o *H e x p e r i m e n t s , d emand s o n s a m p l e s h o u l d n o t p r o v e e x c e s s i v e . B o t h O - r a e t h y l a t i o n a n d O - a c e t y l a t i o n a r e e f f i c i e n t r e a c t i o n s . COSY a n d 2D J - r e s o l v e d s p e c t r o s c o p y a r e r e l a t i v e l y e a s y e x p e r i m e n t s t o s e t up i n t h a t m o s t p a r a m e t e r s ( d e l a y s , s w e e p - w i d t h s e t c . ) c a n b e r e l i a b l y c h o s e n b e f o r e p e r f o r m i n g t h e e x p e r i m e n t a n d t h e n e e d o f r e p e t i t i o n i s s m a l l ; f u r t h e r m o r e , n e i t h e r o f t h e s e e x p e r i m e n t s h a s d e m a n d i n g s i g n a l - t o - n o i s e p r e r e q u i s i t e s . T h e nOe e x p e r i m e n t s demand c o n s i d e r a b l y m o r e m a c h i n e t i m e . T h e SSNOEDS t e c h n i q u e r e q u i r e s c a r e f u l c a l i b r a t i o n o f t h e d e c o u p l e r p o w e r a n d p o s i t i o n , a n d h a s h i g h s i g n a l - t o - n o i s e d e m a n d s ; b e c a u s e i t i s a " d i f f e r e n c e " t e c h n i q u e , t h e d emand s o n i n s t r u m e n t s t a b i l i t y a r e u n u s u a l l y h i g h o v e r w h a t may be a p e r i o d o f up t o 2 d a y s , i f o n e w e r e - 2 25 -w o r k i n g w i t h e x t r e m e l y s m a l l ( m i c r o g r a m ) q u a n t i t i e s . NOESY h a s , i n a d d i t i o n t o s i g n a l - t o - n o i s e d e m a n d s , a v a r i a b l e - t h e m i x i n g t i m e , T j n i x . A s m e n t i o n e d i n S e c t i o n I I . 2 . 5 . 6 , t h e c h o i c e o f tm±x d e p e n d s o n t h e r a t e a t w h i c h t h e m o l e c u l e i s t u m b l i n g a n d nOe b u i l d - u p o c c u r s . A l t h o u g h n o t n e c e s s a r y e v e r y t i m e a new m o l e c u l e i s e n c o u n t e r e d , t h e e x p e r i m e n t e r may w i s h t o p e r f o r m some e x p l o r a t o r y ID nOe e x p e r i m e n t s b e f o r e v e n t u r i n g i n t o t h e 2D f i e l d . We o b s e r v e d i n o u r e x p e r i m e n t s a s t r o n g nOe b e t w e e n H - l ' a n d H - 4 , b u t a w e a k e r one b e t w e e n H - l a n d t h e a l l y l m e t h y l e n e p a i r . T h i s i s i n a c c o r d w i t h w h a t m i g h t be e x p e c t e d , s i n c e t h e m e t h y l e n e p r o t o n s w i l l r e l a x o n e a n o t h e r q u i t e e f f i c i e n t l y t o t h e e x c l u s i o n o f " e x t e r n a l " c o n t r i b u t i o n s . NOESY a p p e a r e d t o f a i l i n t h i s r e s p e c t , a l t h o u g h a c r o s s - s e c t i o n t a k e n a t t h e c h e m i c a l s h i f t o f H - l ( n o t s h o w n ) d i d s h o w e v i d e n c e f o r a s m a l l nOe i n t o H - l " a n d H - l " . T h e r e i s a m p l e p r e c e d e n t I n t h e l i t e r a t u r e f o r u s i n g c h e m i c a l m e a n s t o i m p r o v e s p e c t r a l d i s p e r s i o n . T h e q u e s t i o n o f w h e t h e r o r n o t t o d e r i v a t i z e t h e m o l e c u l e r e s t s o n a n u m b e r o f f a c t o r s . An i d e a l s i t u a t i o n a r i s e s when t h e " n a t u r a l " m o l e c u l e i t s e l f g i v e s s h a r p , w e l l d i s p e r s e d r e s o n a n c e s s i n c e , i n a d d i t i o n t o t h e i n f o r m a t i o n o n p r i m a r y s t r u c t u r e , T j o r nOe e x p e r i m e n t s h a v e t h e p o t e n t i a l t o y i e l d t h e d e s i r a b l e s e c o n d a r y s t r u c t u r e i n f o r m a t i o n . I n t h e e v e n t t h a t s u c h d a t a h a v e t o be o b t a i n e d i n s t e a d f r o m t h e " d e r i v a t i z e d " m o l e c u l e , i t i s f a c t t h a t t h e e x t r a p o l a t i o n o f a n y c o n f o r m a t i o n a l I n f o r m a t i o n a b o u t t h e d e r i v a t i z e d b a c k t o t h e u n p r o t e c t e d m a t e r i a l i n t r o d u c e s u n c e r t a i n t i e s . I n s p i t e o f t h i s , t h e c a s e f o r d e r i v a t i z a t i o n i s s t i l l a s t r o n g o n e - 226 -because, In addition to the increased spectral dispersion, derivatization of amphipathic molecules can often reduce the tendency of the unprotected molecules to associate in polar solvents to form micelles, thereby decreasing the effective molecular weight and narrowing the lines observed (lengthening T ? ) . In the case of oligosaccharides, i t i s d i f f i c u l t to decide whether the O-acetylated or C^-methylated derivative should be chosen for study. If mass spectrometrlc analyses are planned, the 0_-methyl derivative w i l l probably have to be made in any case. However, i f the parent material bears 0_-acetate groups, d i f f i c u l t i e s with 0_-acetyl migration may arise during the derivatization procedure. Furthermore, since 0_-methyl groups resonate in the region of Interest, the deuterated methyl iodide used for the derivatization should be of the highest lsotopic purity obtainable. Experience in this laboratory has shown 3 3 that f u l l (>-acetylation of polysaccharides i s non-trivial and demands 34 that conditions be optimised for each substrate. On this basis, O-methylatlon of polysaccharides may be a more general procedure, 3 5 but this i s not substantiated with experimental evidence. Since the completion of this study, other groups have found u t i l i t y for 2D NMR in structural studies on complex oligosaccharides. More recently two dimensional J_-resolved spectroscopy and SECSY have been integrated into Dabrowski's studies (vide supra) to allow the 3 6 extension of the work to a ceraraide pentadecasaccharide. Conceptually, resonance frequencies are determined from the 2D J-resolved experiment, and Jj-connectivlties established by taking - 2 2 7 -s e c t i o n s t h r o u g h t h e SECSY d a t a - s e t . W h i l s t D a b r o w s k i d r a w s a t t e n t i o n t o t h e u t i l i t y o f t h e 2D e x p e r i m e n t s , h e a l s o c a u t i o n s t h e y a r e " . . . b y n o mean s a p a n a c e a f o r a l l s i g n a l o v e r l a p p r o b l e m s . . . " . T h i s i s i n a g r e e m e n t w i t h o u r c o n v i c t i o n s . F i n a l l y , we d r a w a t t e n t i o n t o t h e b r o a d n e s s o f some o f t h e s i g n a l s ( s h o r t T_ 2 ) w h i c h we k n o w t o p r e s e n t p r o b l e m s i n s p i n e c h o - t y p e 2D e x p e r i m e n t s ( s e e II .5). A n I n d e p e n d e n t s t u d y by P r e s t e g a r d et_ a l . f o u n d u t i l i t y f o r t h e 2D c h e m i c a l s h i f t - a n d nOe c o r r e l a t e d e x p e r i m e n t s ( S E C S Y a n d NOESY) i n 3 7 t h e e l u c i d a t i o n o f p r i m a r y s t r u c t u r e o f a g l y c o l i p i d a t 500 M H z . I t i s k n o w n t h a t c e l l u l o s e c a n be " f u l l y " O - a c e t y l a t e d a n d y i e l d a s p e c t r u m 3 8 i n CDCI3 w i t h d i s c e r n a b l e v i c i n a l c o u p l i n g s . G a g n a i r e ' s g r o u p i n 39 F r a n c e h a s r e p o r t e d 2D J - r e s o l v e d s p e c t r o s c o p y o n i n t a c t a n d d e r i v a t i z e d p o l y s a c c h a r i d e s . T h i s e x p e r i m e n t i s n o t e a s y t o p e r f o r m , d u e t o t h e r a p i d T_2 r e l a x a t i o n d u r i n g t h e r e f o c u s s i n g p e r i o d . We s u g g e s t t h a t s u c h 2D NMR a n a l y s e s o n p o l y s a c c h a r i d e s may be i m p r o v e d b y p a r t i a l o r c o m p l e t e d e p o l y m e r i z a t i o n e i t h e r b y m i l d a c i d h y d r o l y s i s o r k 1 b a c t e r i o p h a g e d e g r a d a t i v e p r o c e d u r e s . I f p a r t i a l d e p o l y m e r i z a t i o n y i e l d s a d e q u a t e l y n a r r o w l i n e - w i d t h s , t h i s w o u l d be p r e f e r a b l e s i n c e o n e c o u l d e f f e c t i v e l y d i s r e g a r d t h e t e r m i n a l r e d u c i n g s u g a r u n i t w h i c h w o u l d c o m p l i c a t e t h e a n a l y s i s o f t h e s i n g l e r e p e a t i n g o l i g o s a c c h a r i d e u n i t . 42 B r u c h a n d B r u c h h a v e a l s o r e p o r t e d t h e a p p l i c a t i o n o f 2D J - r e s o l v e d s p e c t r o s c o p y t o g l y c o p e p t i d e s ; s e v e r a l b r o a d a n o m e r i c s i g n a l s r e s p o n d e d p o o r l y t o t h e a n a l y s i s . P a t t h a s d e s c r i b e d t h e u s e o f 2D 43 13 44 INADEQUATE t o a s s i g n t h e C s p e c t r a o f a m o n o s a c c h a r i d e a n d a d i s a c c h a r i d e , 1 * 5 b u t t h e p u n i t i v e d emand s o n s a m p l e c o n c e n t r a t i o n make - 228 -t h e e x p e r i m e n t o f l i t t l e m o r e t h a n a c a d e m i c i n t e r e s t i n t h e p r e s e n t c o n t e x t . One f i n a l p o i n t o n t h e g e n e r a l i t y o f o u r s e q u e n c i n g p r o t o c o l i s t h a t we b e l i e v e i t t o b e e x t e n d a b l e t o a l m o s t a n y c o m p l e x m o l e c u l e c o n s i s t i n g o f u n i t s o f c o u p l e d s p i n s . W h i l s t c o u p l i n g t h r o u g h a p e p t i d e b o n d i s m e a s u r a b l e a t 500 M H z 1 * 6 i t i s s t i l l s m a l l ( 5 J a = c a . 0 . 5 H z ) a n d o n e c a n s p e c u l a t e o n t h e a p p l i c a t i o n o f s u c h t e c h n o l o g y t o o l i g o p e p t i d e s . - 229 -REFERENCES 1. N. Sharon. Complex carbohydrates - their chemistry, biosynthesis and functions. Addison-Wesley Reading, Mass. 1975. 2. J. Montreuil. Adv. Carb. Chem. Biochem. 37, 157-223 (1980), and references cited therein. 3. G.O. Aspinall and A.M. Stephen. In MTP International reviews of science. Organic chemistry, series one. Edited by G.O. Aspinall. University Park Press, Baltimore, Maryland. 1973. Vol. 7, pp. 285-317. 4. G.G.S. Dutton and E.H. Merrifield. Carbohydr. Res. 1 0 5 , 1 8 9 - 2 0 3 (1982). 5. P.A.J. Gorin. Adv. Carbohydr. Chem. Biochem. 38, 13-104 ( 1 9 8 1 ) . 6. R. Barker, H.A. Nunez, P. Rosevear, and A.S. Serianni. Methods  Enzymol. 83, 58-69 (1982). 7. R.V. Lemieux, K. Bock, L.T.J. Delbaere, S. Koto, and V.S. Rao. Can. J. Chem. 58, 631-653 (1980). 8. L.O. Sillerud, R.K. Yu, and D.E. Schafer. Biochem. 21, 1260-1271 (1982). 9. J.A. Schwarcz and A.S. Perlin. Can. J. Chem. 50, 3667-3676 (1972). 10. P.R. Rosevear, H.A. Nunez, and R. Barker. Biochem. 21, 1421-1431 (1982). 11. J.F.G. Vliegenthart, H. van Halbeek, and L. Dorland. Pure Appl.  Chem. 53, 45-77 (1981), and references therein. 12. J.P. Carver and A.A. Grey. Biochem. 20, 6607-6616 (1981). 13. J-R. Brisson and J.P. Carver. J. Biol. Chem. 257, 11207-11209 (1982). 14. J. Dabrowski, P. Hanfland, and H. Egge. Methods Enzymol. 83, 69-86 (1982), and references therein. 15. J.M. Berry, L.D. Hall, D.G. Welder, and K.F. Wong In Anomeric effect, origins and consequences. Edited by W.A. Szarek and D. Horton. ACS Symposium Series, No. 87. 1979. pp. 30-49. 16. K.F. Wong, Ph.D. Thesis, University of British Columbia (1979), and references therein. 17. L.D. Hall and CM. Preston. Carbohydr. Res. 29, 522-524 (1973). - 230 -18. L.D. Hall and H.D.W. H i l l . J. Amer. Chem. Soc. 98, 1269-1270 (1976). 19. See section II.2.A. 20. M. Martin-Lomas and D. Chapman. Chem. Phys. Lipids. 10, 152-164 (1973) . 21. K-E. Falk, K-A. Karlsson, and B.E. Samuelsson. Arch. Biochem.  Biophys. 192, 191-202 (1979). 22. M.A. Bernstein and L.D. Hall. J. Amer. Chem. Soc. 104, 5553-5555 (1982). 23. V . Horejsi and J . Kocourek. Biochim. Biophys. Acta. 336, 338-343 (1974) ; H.M. Flowers. Methods Carbohydr. Chem. 6, 474-480 (1972). 24. See section II.2.2.1. 25. See section II.2.3. 26. See section II.2.4. 27. See section II.2.5.2. 28. See section II.2.5.3. 29. C. Altona and C.A.G. Haasnoot. Org. Magn. Reson. 13, 417-429 (1980) . 30. See section II.2.5.6. 31. M.L. Wolfrom and A. Thompson. Methods Carbohydr. Chem. 2, 211-215 (1973). 32. K. Nagayama and K. Wuthrich. Eur. J. Biochem. 114, 375-384 (1981) . 33. J. Hoffman and B. Lindberg. Methods Carbohydr. Chem. Biochem. 8, 117-122 (1980). 34. K.R. Holme, private communication, 35. H.E. Conrad. Methods Carbohydr. Chem. 7, 361-364 (1972), and references cited therein. 36. J. Dabrowski and P. Hanfland. FEBS Lett. 142, 138-142 (1982). 37. J.H. Prestegard, T.A.W. Koerner, Jr., P.C. Detnou, and R.K. Yu. J.  Amer. Chem. Soc. 104, 4993-4995 (1982). 38. H. Friebolin, G. Keilich, and E. Siefert. Angew. Chem. Int. Ed. 8, 766-767 (1969). - 231 -39. D.Y. Gagnaire, F.R. Taravel, and M.R. Vignon. Macromol. 15, 126-129 (1982). 40. M.L. Wolfram and M.E. Franks. Methods Carbohydr. Chem. 5, 276-280 (1965), and others in the same section. 41. G.G.S. Dutton, J.L. DiFabio, D.M. Leek, E.H. Merrifield, J.R. Nunn, and A.M. Stephen. Carbohydr. Res. 97, 127-138 (1981). 42. R.C. Bruch and M.D. Bruch. J. Biol. Chem. 257, 3409-3414 (1982). 43. A. Bax, T.A. Frenkiel, R. Freeman, and M.H. Levitt. J. Magn.  Reson. 43, 478-483 (1981). 44. S.L. Patt, Carbohydr. Res., in press. 45. S.L. Patt, F. Sauriol, and A.S. Perlin. Carbohydr. Res. 197, C1-C4 (1982). 46. H. Kessler, W. Bermel, A. Friedrich, G. Krack, and W.E. Hull. J.  Amer. Chem. Soc. 104, 6297-6304 (1982). - 232 -CHAPTER II.4 NMR SPECTROSCOPIC ASSIGNMENT OF BRUCINE Interest in the Strychnos alkaloids, of which strychnine (1) and brucine (2) are members, dates from their isolation in 1819, and 1946 when the correct skeletal structure was fi n a l l y proposed. Although of "medium" molecular weight, the skeleton Is disproportionately complex, having six chiral centres in a 24 atom skeleton. NMR spectroscopists have also been Interested in these alkaloids for some time, and the relevant literature is reviewed below. 1 : R - H 2 : R - OCH3 - 2 3 3 -The object of the exercise detailed in this chapter i s to determine the generality of the NMR methods presented in Chapter II. 2, and to determine which i s the most expeditious route to the fi n a l assignment of the H and C spectra of complex molecules. An important aspect of this study was an evaluation of the time required to perform the experiments, and optimum ways to integrate data-acquisition and processing. It should be stressed that the experiments were intentionally carried out using the minimum of literature data; thus, only brucine*s skeletal structure and configuration, and an early 13 1 (erroneous) table of C chemical-shift assignments was used as reference data. No data that could lead to subtle chemical shift arguments were sought, since the Intention was to keep the reliance on such tacks to a minimum. Wherever possible, spectra were acquired under "survey conditions", that i s , using the fullest possible sweep-widths In both dimensions for the 2D experiments, with "typical" delays. In this way, an attempt was made to simulate conditions which an "applied" spectroscopist might encounter when analyzing the structure of a molecule for which l i t t l e or no prior spectroscopic knowledge was available. The questions are: which experiments should be performed, In what order and how much instrument time w i l l they demand? The considerable body of classical chemistry performed on the Strychnos alkaloids has been reviewed. After uncountable man-hours of degradative analyses by numerous groups the correct skeletal structure was proposed in 1946 by R. Robinson and colleagues. 3 The absolute configuration at the asymmetric centres was determined by X-ray - 234 -diffraction analysis, and Woodward succeeded in synthesizing strychnine in 1954. The i n i t i a l NMR spectroscopic interest focussed on the 1 3C spectrum, probably because of the complexity of the 1H spectrum at the fi e l d strength of the then common magnets (< 100 MHz for 1H). Since the 6 7 i n i t i a l report by Wehrli, • some considerable controversy has 13 occurred in the literature over the C assignments, which has only recently reached f u l l consistency. In 1973, Wehrli described 6 the assignment of the seven quaternary carbons in brucine. This was based on chemical shift additivity rules and T A relaxation data. In that the relaxation pathway is dipolar, quaternary carbons are relaxed by protons on a carbon atoms. The Tj's vary from 2.62 to 11 .75 sec. Shortly thereafter, 13 / Wehrli published the complete C assignment of brucine. Again, much 1 3 attention was focussed on C Tj relaxation rates. Single frequency off-resonance decoupling (SFORD) was used to determine 1 3C multiplicities and further assignments were based on chemical shift and residual splitting (1JXH) arguments, and comparisons with related molecules. This assignment was correct, but numerous "reassignments" 1 8 were made over the following years. Srlnivasan and Lichter, and Singh et a l . , followed with some erroneous "reassignments,"' later summarized and c l a r i f i e d by Verpoorte 9 and coworkers. 1 0 Leung and Jones reported 1 1 on the 1 3C NMR spectrum of strychnine, which needed only one reassignment. Wenkert et a l . , reported 1 2 the 13CNMR (and some % NMR) assignments of a number of related alkaloids of this genre, which were - 235 -in agreement with Verpoorte. The above workers relied on SFORD to provide the information on the number of attached protons, various derivatives, chemical shift arguments, and (reduced) ^^JCH V A* U E 8 f°T their assignments. Many of the discrepancies between the various assignments can be traced to the data from the SFORD experiment. In the case of an {AB}X experiment with a methylene carbon, complexities may arise and this i s well documented by Roth and Bauer, 1 3 using strychnine as an exemplar. Depending on the decoupler position and power, C-15 is seen to show 3 or 4 lines (6 transitions, maximum). Careful analysis can yield 1J^ X» 1J£X and 2J^B. This was extended in Roth's description 1** of NORD (noise-modulated off-resonance decoupling) which i s similar in concept and potential information-yield. D i f f i c u l t i e s can arise in the interpretation of the SFORD spectrum where lines are crowded; Wehrli has published the SFORD spectrum of brucine, 7 and the complexity Is evident. Martin suggested 1 5 an extension of Wehrli*s Tj relaxation ideas. In that the relaxation mechanism is predominantly dipole-dipole (even at 1.3 M) and mediated by the directly attached protons, carbons bearing two protons w i l l relax at about twice the rate of those bearing one. Quaternary carbons, as mentioned, relax at a rate proportional to the number of a protons. Methyl carbons do not always conform because they can have additional internal reorientation, but s t i l l relax much faster than methine or methylene carbons. These concepts are similar to those presented in II.2.2.1. Additionally, SFORD experiments were - 236 -p e r f o r m e d w i t h s e l e c t i v e e x c i t a t i o n o f t h e C s i g n a l s . T h i s i s o n e way a r o u n d t h e s o m e t i m e s m i s l e a d i n g { A B } X s y s t e m d i s c u s s e d a b o v e . I n t h i s w a y , M a r t i n was a b l e t o c o r r e c t l y d e t e r m i n e t h e m u l t i p l i c i t y o f e a c h c a r b o n i n t h e m o l e c u l e , a n d a s s i g n t h e m u s i n g t h e t a c t i c s o u t l i n e d p r e v i o u s l y . T h e 250 MHz *H NMR s p e c t r u m o f s t r y c h n i n e was f u l l y a s s i g n e d b y C a r t e r e t a l . i n 1 9 7 4 . 1 6 S p e c i f i c a l l y d e u t e r a t e d a n a l o g u e s w e r e p r e p a r e d a n d t h i s , i n c o n j u n c t i o n w i t h d o u b l e - r e s o n a n c e e x p e r i m e n t s , s p e c t r a l s i m u l a t i o n a n d s o l v e n t i n d u c e d s h i f t s , r e s u l t e d i n t h e t o t a l a s s i g n m e n t . W e n k e r t et^ a l . s u b s t a n t i a t e d t h e a s s i g n m e n t s w i t h some 12 s m a l l m o d i f i c a t i o n s - r e v e r s a l o f a s s i g n m e n t w i t h i n e a c h p a i r o f g e m i n a l p r o t o n s a t C - 1 5 , C - 1 8 a n d C - 2 0 . * T h e i r w o r k was b a s e d o n c o n f o r m a t i o n a l a r g u m e n t s u s i n g c o u p l i n g c o n s t a n t s ; h o w e v e r t h e r e s u l t s w e r e n o t c o m p a r e d w i t h s o l i d - s t a t e c o n f o r m a t i o n a l d a t a . C a r t e r et^ a l . , made t h e s e a s s i g n m e n t s b a s e d o n s o l v e n t i n d u c e d s h i f t s . I n a p r o t o n 17 r e l a x a t i o n s t u d y b y C o l e b r o o k a n d H a l l , t h e a r o m a t i c p r o t o n s o f b r u c i n e w e r e a s s i g n e d . F i n a l l y , a *H nOe s t u d y b y B o t h n e r - B y a n d 18 c o - w o r k e r s u s e d b r u c i n e d i s s o l v e d i n a v i s c o u s s o l v e n t a s a m o d e l c o m p o u n d f o r t h e d r i v e n nOe e x p e r i m e n t ( S e e I I . 2 . 4 . 2 ) . B e f o r e p r e s e n t i n g t h e s p e c t r o s c o p i c s t u d i e s o f t h i s t h e s i s , a n u m b e r o f p o i n t s s h o u l d be r e i t e r a t e d . T h e m a j o r i t y o f t h e c o n s i d e r a b l e l i t e r a t u r e d e t a i l e d a b o v e was d e l i b e r a t e l y c o n s u l t e d p o s t f a c t o , a n d i n 21 * A r e c e n t s t u d y b y C o l e b r o o k a n d c o - w o r k e r s c o n f i r m s t h i s r e a s s i g n m e n t , a n d c o r r e c t s t h a t o f H - 1 7 a a n d 8 . NOEDS a n d 7\ m e a s u r e m e n t s , a l o n g w i t h s p e c t r a l s i m u l a t i o n w e r e u s e d . - 237 -our reasoning, a minimal reliance was made on chemical shift arguments. As the emphasis was on the spectroscopic aspects, access to derivatized materials and data on analogues were also excluded. The overall strategy chosen was f i r s t l y to fully assign the proton NMR spectrum and then use this to assign the non-quaternary signals in the carbon-13 spectrum. The former was achieved primarily using COSY (II.2.5.3.2), with some assistance from 2D J-resolved spectroscopy (II.2.5.2), and 1- and 2-D nOe experiments. With this completed, the 1 3C NMR spectrum was easily assigned using the CSCM experiment (II.2.5.4), with spectral editing performed using DEPT (II.2.2.2). Fig. II.4.1 400 MHz NMR spectrum of brucine (ca. 0.02 M in CDC13), showing the assignment of non-aromatic protons. - 238 -Portions of the H NMR spectrum of brucine are presented in Fig. II.4.1. Two singlets in the aromatic region are omitted, and the multiplet in the vinyl region i s shown in an inset. Analysis at 360 MHz began with the COSY spectrum. The experiment was performed with coherence transfer echo selection, on a ca. 0.01 M solution in CDC13. A relaxation delay of only 0.2s, and the minimum of 4 acquisitions per t^ interval meant that 20 min was required for the collection of a 512 * IK data-set; the sweep-width was 10 ppm in both dimensions. Next, a 2D J-resolved experiment was set up; this experiment required ca. 45 min for collection of the data - this was somewhat longer than that of the COSY due to the necessarily longer relaxation delay and more scans. Whilst these J_-resolved data were being collected, the COSY data-set was processed and plotted. At this stage, acquisition of the 2D J-resolved data set was complete, and data processing was performed. A l l the above operations were performed automatically in a MACRO program on the Nlcolet system.* Minimal operator intervention was required, except at the plotting stage, where decisions were made to expand certain spectral regions. Details of the "survey-condition" parameters are in Table II.4.1. The COSY spectrum of brucine Is shown in Fig. II.4.2. Only the region between 64.5 and 1.0 i s plotted here, for the sake of c l a r i t y . The analysis began with the realization that four "pockets" of coupled spins exist in the molecule, with l i t t l e or no coupling between them *A11 360 MHz experiments were performed at Nicolet Magnetics Corp., Fremont, Ca. USA. - 239 -F i g . I I . 4 . 2 3 6 0 MHz COSY s p e c t r u m o f b r u c i n e i n t h e r e g i o n b e t w e e n 6 4.5 a n d 1.0. C o n n e c t i v i t i e s t o t h e v i n y l i c p r o t o n a r e i n d i c a t e d , t h o u g h t h e y a r e n o t o n t h e d i a g r a m . Table II.4.1. Parameters used in automated "survey conditions" collection and processing of H COSY and 2D J_-resolved experiments at 360 MHz. a F u l l sweep-width (Hz). ^Bioc^-gf z e s (words). Relaxation delay (s). dApodization function: EM = exponential multiplier; DM = double exponential multiplier; MS » sinebell multiplier. With the J-resolved experiment, the strongest resolution enhancement function is chosen for t_2 which the signal-to-noise permits. eSymmetrization was not possible with the 2D J-resolved data, but would be recommended. Total Time (min) Sweep Width 3 Block Size b R e l a x J 0 , Apodization d Z e J ° 7 Symm.e  r Scans f i l l F 2 Fi F_2 £i Delay 0 Per _t i t_i tj, tj? Collect ID spectrum - Calibrate pulse widths 0 Begin collecting COSY data 20 Begin collecting 2D Process and plot COSY 65 Commence processing and plotting of 2D J-resolved experiment 16K 3,600 3,600 1,024 512 3,600 29 4,096 64 0.2 1.0 4 16 MS EM DM MS MS DM MS No Yes Yes No 120 END - 2A1 -(cf. II.3):namely (a) H-20o and H-20B, (b) H-17a, H-17B, H-18a and H-18B, (c) H-22, H-23o and H-238, and (d) H-lla, H-11B, H-12, H-13, H-8, H-14, H-15o, H-15B and H-16. COSY succeeded in assigning a l l 18 of these protons, although i t was not possible at this stage to differentiate a from 8 geminal protons.* The connectivities are indicated along with the assignments in the Figure; these were made without ambiguity. The large -OCH3 peaks which resonate very close to H-8 and H-16 could have hindered the assignment had H-8 and H-16 been scalar coupled as they might then have obscured a set of cross-peaks; this was not the case. Special considerations were required for the tightly coupled geminal protons, which were assigned to the H-23 and H-17 pairs. Distortion of peak intensities indicated tight coupling, especially in the case of the H-17 pair. To assess the r e l i a b i l i t y of the COSY spectrum, i t i s interesting to consider H-15B (ca. 6 1.4), which shows one large splitting and several smaller (<_ 1.5 Hz) ones. In the COSY spectrum, the expected three connectivities are clear, even though the digitization i s less than 7 Hz per point. The 2D J_-resolved experiment did not prove very useful and these data are not included here. It would have been particularly useful in the -OCH3 signal region. However, the overlapping H-16 resonance remained obscured by the familiar " t a i l i n g " in Fj from the intense -OCH3 singlets. The partial J_-spectrum of the hidden H-l8 resonance was not *The molecule i s not planar and hence, an upper (p) and lower (a) face is not clear. For this reason, geminal protons are defined (See 1 and 2) for the purpose of this study. - 242 -useful, since i t suffered from the same virtual coupling effects as the other H-18 resonance at 6 2.83, making the extraction of accurate coupling constant data impossible without spectral simulation. (The geminal H-17 protons, to which they are vicinal, are tightly coupled). The assignment of the aromatic, geminal and ^ -methyl protons was now sought. The solid-state conformation as determined by X-ray crystallography was used to build a molecular model having the correct configuration. This was found to be a rigid structure, suggesting that the solution conformation might be very similar to that in the solid-state. The experiments required to complete these *H assignments also served to corroborate this postulated solution conformation. In the determination of conformation, techniques relying on "through-space" phenomena (e.g. T_i, nOe) prove very useful, often in conjunction with (through-bond) coupling-constant information. Axial protons take on unusual importance in an nOe experiment. The 1,3 diaxial interaction provides important information in a six-membered ring, and with a boat conformation, the "flagpole" protons are close to one another in spite of their being 5 bonds removed; an nOe experiment w i l l also detect this. The SSNOEDS results are presented In Fig. II.4.3. No special effort was made to optimise the experiment and the data were collected In approximately three hours. A single decoupler power setting was used (which resulted in 40-80% saturation, depending on the total multiplet width). In a l l cases a check was made for decoupler power leakage into - 243 -Fig. II.4.3. SSNOEDS experiments performed on brucine at 400 MHz. - 244 -neighbouring resonances ("spillover"), and was found to be absent. The irradiation time was set at 0.5 s*, and the experiments "interleaved" by collecting scans in blocks of 16, repeated 30 times. Let us consider ring IV, which Is in a chair conformation in the solid-state (Fig. II.4.4.A). Evidence for this also being so in solution comes from a consideration of coupling-constants, and nOe experiments. J_ (8, 13) = 10.5 Hz, which indicates a trans or cis arrangement of the two protons. In keeping with the chair conformation, H-l4 and H-l6 are judged to be roughly co-planar since (14, 15a) « J (16, 15a), and J (14, 158) « J (16, 156).** Strong confirmatory evidence for the proposed conformation comes from the SSNOEDS experiment where H-15 (6 1.46) was irradiated, inducing an nOe in H-13; the two protons must assume axial orientations In a ring having a chair conformation, and the irradiated proton is accordingly assigned to H-l5p. Irradiation of the higher-field of the two H-l8 protons induced an nOe in H-8, thereby identifying the proton irradiated as H-18B and H-8 to be axial, as depicted in Fig. II.4.4A. Consider now ring VI. Since the H-20 protons have no vicinal neighbours, no conformational information can be sought from arguments based on vicinal coupling-constnts. The nOe experiment where H-15a was *This i s far less than the time usually used for nOe build-up (ca. 3* Tj)» and was chosen to minimize the experimental acquisition time and "three spin effects", without jeopardizing the observation of "direct" interactions. **The H-15 protons are assigned here to allow the reader to check the statement with the reported data in Table II.4.2, but the absolute assignment of the H-15 protons Is not necessary at this stage. - 245 -A. B. C. D. o Fig. II.4.4 Conformations of rings IV and V (A), VI (B), VII (C) and III (D) in brucine, indicating some of the results from the SSNOEDS experiments. The irradiated proton i s indicated with (•) and nOe's ( ). - 246 -irradiated induced a strong nOe in the lower-field of the two H-20 resonances. This confirms the approximate "boat" conformation of this ring (Fig. II.4.4.B), which places the "flagpole" protons, H-15o and H-20a, close to one another; this i s a "bowsprit-flagpole" interaction. Hence, this single experiment confirms the expected conformation of ring VI, and identifies H-20oc (and H-20B, by default). The analysis of ring VII was complicated somewhat by the fact that i t is a seven-membered ring, with a double bond and ring hetero-atom.* Two nOe experiments were performed to support the structure shown in Fig. II.4.4C. Irradiation of the vinylic H-22 induced an nOe into the pseudo-equatorial proton H-238, the lower-field of the two H-23 protons. Additionally, an nOe is induced in H-208. Although not depicted in this diagram, H-22 and H-20B are close to one another and this confirms the assignment of the geminal H-20 protons based on the irradiation of H-l5a. Irradiation of H-l2 induced an nOe into other axial or axial-like protons: H-14 and H-23a.** The assignment of the H-23 protons i s now confirmed. The conformational analysis of ring III is more complex. Here the H-lla and H-lIB protons must be assigned. The molecular model favours a boat-like conformation. A pure boat conformation would make H-l2 and H-lip trans diaxial, and one would anticipate a large vicinal coupling constant here on the basis of the Karplus relationship; using *This i s a tetrahydrooxepin ring. **Here, a negative nOe is Induced in H-23 p due to the "three-spin effect". - 247 -this argument, the lower f i e l d of the two H-ll protons would be assigned to H-11B. The geminal coupling constant J_ (11a, 116) i s , at 17.4 Hz, unusually large. This suggests that the carbonyl on C-10 roughly 19 bisects the angle between the geminal protons and C - l l , and this would not be the case i f ring III were to be in the pure boat conformation considered above. Satisfying the condition of the relationship between the carbonyl and geminal protons "flattens" one end of the boat, placing N-9, C-10, C - l l , C-12 and C-13 approximately co-planar. In this situation H-l2 and H-lla have a small dihedral angle (ca. 10°), and, hence, a large vicinal coupling constant would be anticipated. This argument assigns the lower f i e l d of the two H-ll protons as H-l16 - a reversal of assignment. It is clear that in both cases H-8 and H-l16 are close in space, and an nOe experiment would not suffer from these ambiguities. The H-ll protons could be assigned, and on this basis the ring conformation determined from the coupling-constant arguments. The SSNOEDS experiment (vide supra) was not possible here, since a l l three protons - H-8, H-lla and H-lIB - overlap with other resonances, precluding irradiation selectivity. A 2D NOESY experiment was performed, and the data are given in Figs. II.4.5 and .6.* Fig. II.4.5 shows the NOESY spectrum over the entire spectral width, and Fig.II.4.6 shows the high-field portion of this data in more detail; we concentrate on the latter, for the moment. Before searching for the *Here, the principal diagonal runs from the top left-hand corner to the bottom right; a mirror image of the displays previously shown, but entirely analogous. Fig. II.4.5 400 MHz NOESY spectrum of brucine. The 256 * IK data-matrix was collected in ca. 8 h, with relaxation delay 3.0 s, mixing time 0.6 s ± 10% and 32 scans per t_i interval. The data were processed and plotted using a Nicolet 1280 computer and Zeta 8 recorder. The fin a l digitization was 5.8 Hz pt in both dimensions, after z e r o - f i l l i n g Jj; the "sinebell" multiplier was used in both dimensions, and magnitude spectra calculated. - 249 -Fig. II.4.6 The high-field region of the NOESY plot In Fig. II.4.5 - 250 -8-1 ip nOe, i t i s worth checking the experiment by looking for the nOe's determined previously using SSNOEDS. These are a l l clearly discernable and are indicated in the upper half of Fig. II.4.6. A number of nOe's with H-16 are clear here which were not in the ID experiments, as a result of being obscured by subtraction errors with the overlapping sharp 0-CH_3 singlets. The 8-1lp nOe correlation is discernable, assigning H-lip as the higher f i e l d of the H-ll resonances. This is in accord with the second argument above using vicinal and geminal J_-coupling magnitudes and suggests a boat-like conformation, flattened at C - l l (See Fig. II.4.4.D). This i s confirmed by the observation of a larger nOe between H-12 and H-llo than H-12 and H-llp. The NOESY spectrum also provides the means for the assignment of the aromatic and O-methyl signals. The relaxation of H-4 must be dominated by the 0-CH3 protons on C-3, and H-l by those on C-2. H-l has additional relaxation contributions from H-16 and H-17a. Fig. II.4.5 shows the nOe between the aromatic and the O-methyl protons. Correlations between an aromatic proton and protons on ring V are below the contour threshold; slices taken at each aromatic proton's chemical shift (not shown) identify the higher f i e l d of the two aromatic resonances as that of H-l, by the above reasoning. The nOe with H-17 is not clear, as i t overlaps with the strong correlation to the O-methyl resonance. Further, the O-methyl resonances are easily assigned; the higher f i e l d of the two singlets corresponds to the O-methyl on C-2 and the lower f i e l d to that on C-3. - 251 -T a b l e I I . 4 . 2 S u m m a r i z e d H NMR c h e m i c a l s h i f t a s s i g n m e n t s a n d c o u p l i n g c o n s t a n t s o f b r u c i n e i n C D C I 3 ( c a . 0 . 0 2 M ) . a b s t r o n g c o u p l i n g . C o u p l i n g p a t t e r n c o m p l i c a t e d b y v i r t u a l c o u p l i n g . P r o t o n 6 ( ppm) J ( H z ) 1 6 . 6 6 -4 7 . 8 0 -8 3 . 8 3 ( 8 - 1 8 ) = 1 0 . 5 1 1 a 3 . 0 9 ( 1 1 a , 1 1 8 ) - 1 7 . 4 ( 1 1 a , 1 2 ) = 8 . 5 1 1 8 2 . 6 4 ( l i p , 1 2 ) - 3 . 3 12 4 . 2 7 ( 1 2 , 1 3 ) = 3 . 3 13 1 . 2 6 ( 1 3 , 1 4 ) •= 3 . 2 14 3 . 1 4 ( 1 4 , 1 5 a ) - 4 . 0 ( 1 4 , 15 p ) = 1 .0 1 5 a 2 . 3 4 ( 1 5 a , 1 5 p ) = 1 4 . 2 ( 1 5 a , 1 6 ) - c a . 4 . 9 156 1 . 4 6 ( 1 5 p , 1 6 ) - 2 . 1 16 3 . 9 1 7 a , p a 1 . 9 , 1 . 8 ( 1 7 a , 1 7 p ) - c a . 1 2 . 0 1 8 a b 3 . 1 5 1 8 B b 2 . 8 3 2 0 a 3 . 6 8 ( 2 0 a , 2 0 p ) = 1 4 . 8 ( 2 0 a , 2 2 ) = 1 .6 20 8 2 . 7 0 2 3 a 4 . 0 5 ( 2 3 a , 2 3 p ) = 1 3 . 8 ( 2 3 a , 2 2 ) - 6 . 3 2 3 8 4 . 1 3 ( 2 3 p , 2 2 ) = 6 . 9 ( C - 2 ) - O C H 3 3 . 8 5 -( C - 3 ) - O C H 3 3 . 8 9 -- 252 -The fin a l assignments of the H NMR spectrum of brucine are compiled in Table II.4.2. These data are in complete accord with those 12 of Wenkert e>t a l . , for strychnine. We have seen that the COSY experiment was sufficient to perform the bulk of the analysis of the *H NMR spectrum of brucine. The ID and 2D nOe experiments assigned individual geminal protons and provided valuable conformational information. 13 Now, the C spectrum was analysed. A ca. 0.3 M solution of brucine in CDCl3:CD3OD (10:1) was prepared. F i r s t , the number of protons attached to each carbon was determined using DEPT. This might have been redundant because a CSCM experiment was planned; however in this case not a l l methylene protons had very different chemical shifts (vide infra). The CSCM experiment required 45-60 min. for data collection. The CSCM spectrum of the interesting regions of 6c 22-80 and 6H 1.0-4.5 is shown in Fig. II.4.7. (A l l assignments outside this region are unambiguous). The DEPT information is included. Knowing the proton assignments, the indicated carbon assignments were made. In only two cases was there any uncertainty. H-8 and H-16 resonate very close to one another making the assignment of the carbon signals around 6 59 d i f f i c u l t . The carbon signals around 6 41 are very close and insufficiently digitized in this case. Hence, C - l l and C-16 cannot be distinguished. No attempt was made to assign quaternary carbons since this would not f a l l within the scope of the present design protocol. The data from the CSCM is compared with that of Srinivasan and Lichter 1 -- 253 -C-12 \ DEPT-CH -OCH. C-8 C-16 f 2 PROJECTION «5H C-23 A C-18 C-20 C-13 C-11 C-17 A/ A. iV 8 CH , CH CH, CH, CH CH. s I I I—I—I—I—I—I—I—I—I—I 80 70 60 - i — i — I — r E C-14 £ 1 C-15 -j\ A — CH CH„ 8 ^ < 5 I I—i—r 50 40 30 ppm o l C N C N O CO CO o 5 C Fig. II.4.7 360 MHz (iH) CSCM experiment with brucine (ca. 0.3 M in C6D6/CD3OD, 10:1). 6 C 22-80 and 6 H 1.0-775 is plotted in the contour mode. Fj and F2 projections are on the abcissa and ordinate, respectively. - 254 -Table II.4.3 Table of assigned C chemical shifts for brucine, compared with the literature. Chemical shifts are referenced to the central transition of CDCI3 (6 77.0). The solution was ca. 0.3 M in CDCl3:CD30D (10:1) and the instrument a Bruker WH-400, operating at 100.6 MHz for 1 3C. a'bmay be interchanged. Carbon Ref. 1 Ref. 10 This Study 1 105.78 105.7 106.4 4 101.00 101.1 101.5 8 59.84 60.3 60.2a 11 50.09 42.3 42.2b 12 77.64 77.8 77.5 13 48.18 48.3 48.1 14 31.50 31.5 31.4 15 26.73 26.8 26.5 16 60.28 59.9 59.6a 17 42.28 42.3 41.9b 18 52.57 50.1 49.9 20 42.28 52.7 52.4 22 127.14 127.2 127.6 23 64.47 64.6 64.4 OCH3 56.43; 56.08 56.4; 56.2 56.6; 56.0 - 255 -that referred to whilst the work was in progress - and the now accepted a assignments of Verpoorte. Our data are in complete agreement with the latter. Several conclusions can be drawn from this study. F i r s t , only modest amounts of spectrometer time are necessary for the partial assignment of the *H NMR spectrum of a medium sized molecule such as brucine; in this case a total of ca. 2 hours for the 2D J-resolved and COSY experiments and an additional 3-8 hours for the complete spectroscopic and conformational assignment. Additional conformational information, plus the assignment of Individual geminal protons results from nOe experiments. Where the selective irradiation experiments can be performed, the SSNOEDS experiment i s probably the most expeditious route. In the case of signal overlap, the 2D NOESY experiment w i l l provide a l l the necessary Information in a single experiment, but with greater demands on spectrometer time. This investment of time i s offset by the fact that a l l possible nOe experiments are "simultaneously" performed, resulting in a high return being realized in terms of information yield. With the proton spectrum assigned at least to the point before 13 nOe experiments are performed, non-quaternary sites in the C NMR spectrum can be routinely assigned providing their resonances are not too close (ca. > lppm) and the attached proton resonances are not badly overlapping. We believe that the 2D J_-resolved and COSY experimental data can be collected and processed In a highly efficient, integrated fashion, - 256 -with the prior knowledge of the chemical system. NOe data might require some educated guess-work in the selection of an irradiation time (SSNOEDS experiment) or the mixing time (NOESY), or a series of exploratory experiments could be performed. A l l the experiments described In this chapter provided the required information from the data collected the f i r s t time i t was performed. Hurd 2 0 has independently reported on the u t i l i t y of the combination of COSY with the CSCM experiment. It i s advantageous to use a 1 3C probe to measure both the 1 3C and *H spectra, the latter via the XH decoupler c o i l . In this way the COSY assignment (and NOESY, i f required) i s for the molecule at the same concentration as for the CSCM experiment and no time i s expended changing probes. This approach has the further merit that i f i t were possible to automate changeover of the probe cir c u i t s , i t would be possible to automate the acquisition and processing of a l l *H and 1 3C data into a simple (overnight) run. Given i t s high efficiency, i t seems probable that the mode of analysis described in this chapter w i l l find a place in organic structural investigations which warrant more than a few, unrelated NMR measurements. - 257 -REFERENCES 1. P.R. Srinlvasan and R.L. Lichter. Org. Magn. Reson. 8, 198-201 (1976). 2. G.F. Smith. The alkaloids. Edited by R.H.F. Manske. Academic Press, New York, Vol. 8, 1965, pp. 591-671. 3. L.H. Briggs, H.T. Openshaw, and R. Robinson. J. Chem. Soc. 903-908 (1946), and others in the series. 4. A.F. Peerdeman. Acta Cryst. 9, 842 (1956). 5. Ref. 2, pp. 642-647, and references therein. 6. F.W. Wehrli. J. Chem. Soc. Chem. Commun. 379-380 (1973). 7. F.W. Wehrli. Adv. Mol. Relaxation Processes. 6, 139-151 (1974). 8. S.P. Singh, V.I. Stenberg, S.S. Parmar, and S.A. Farnum. J.  Pharm. Sci. 68, 89-92 (1979). 9. R. Verpoorte. J. Pharm. Sci. 69, 865-867 (1980). 10. R. Verpoorte, P.J. Hylands, and N.G. Bisset. Org. Magn. Reson. 9, 567-571 (1977). 11. J. Leung and A.J. Jones. Org. Magn. Reson. 9, 333-337 (1977). 12. E. Wenkert, H.T.A. Cheung, H.E. Gottieb, M.C. Koch, A. Rabaron, and M.M. Plat. J. Org. Chem. 43, 1099-1105 (1978). 13. K. Roth and H. Bauer. Org. Magn. Reson. 15, 331-332 (1981). 14. K. Roth. J. Magn. Reson. 42, 132-140 (1981). 15. G.E. Martin. J. Pharm. Sci. 70, 81-84 (1981). 16. J.C. Carter, G.W. Luther, III, and T.C. Long. J. Magn. Reson. 15, 122-131 (1974). 17. L.D. Colebrook and L.D. Hall. Can. J. Chem. 58, 2016-2023 (1980). 18. N.M. Szeverenyl, A.A. Bothner-By, and R. Bittner. J. Phys. Chem. 84, 2880-2883 (1980). 19. R.R. Fraser and B.F. Raby. J. Amer. Chem. Soc. 94, 3458-3463 (1972). 20. R.E. Hurd. Relaxation Times, January 1982. An in-house publication of Nicolet Magnetic Corp., Fremont, California, U.S.A. 21. W.J. Chazin, J.T. Edward, and L.D. Colebrook. Submitted for publication. - 258 -CHAPTER I I .5 DIGOXIN The studies on oligosaccharide sequencing (Ch. II.3) and brucine (Ch. II.4) suggested to us that the NMR spectroscopic methods detailed in Ch. II.2 are effective for molecules which tumble rapidly in solution* These encouraging results prompted us to see i f specific problems might be associated with the analysis of NMR spectra of more complex, higher molecular weight molecules. Our laboratory's experience with carbohydrates and steroids led us to the naturally occurring steroidal glycosides. 1 Of the extensive group of such plant natural products, 2 we chose digoxin (1) - f i r s t isolated from the leaves of Digitalis lanata by Smith3 in 1930. The molecule i s of more than academic Interest due to i t s important pharmacological place as the most frequently prescribed d i g i t a l i s steroid for heart congestion therapy. 1 - 259 -The molecule is highly complex. The glycone consists of three B-D-digitoxose units, identically linked (1 •* 4). From the NMR viewpoint one could anticipate in advance of any experiment, that this molecule poses some stringent tests. Thus, the steroid moiety (viz. digoxigenin) i s substituted by hetero-atoms at only three positions (C-3, C-12 and C-14), which implies that only a few steroid protons w i l l resonate outside the "methylene hump". (It i s advantageous to have as many steroid protons deshielded from this region as possible, since they are of tremendous assistance in assignment). Furthermore, digoxin i s amphlpathic and would be expected to aggregate (form micelles) in polar solvents such as DMSO (which i s one of the few good solvents for digoxin). This would increase the effective molecular weight, adding to the already broader lines resulting from the high monomer molecular mass of 780 (C 1 | 1H 6» t0 1 4). Owing to the higher viscosity of DMSO (cf. CDC13), molecules dissolved therein are expected to display broader lines, even in the absence of molecular aggregation. A l l JH NMR investigations* were performed at 500 MHz, and 1 3C at 90 MHz (360 MHz for 1H), using NIcolet spectrometers. As In previous chapters, reliance on chemical shift arguments was kept to a minimum, and l i t t l e chemical derivatization was performed. Our goals were to assign as many of the 1H and 1 3C resonances as possible, draw conformational conclusions and, most importantly, determine the limits *The findings of this chapter resulted from a collaborative study with Dr. R.E. Hurd of NIcolet Magnetics Corp., Fremont, Ca. U.S.A. - 260 -of the procedures hitherto extensively relied upon. If an experiment were to f a i l , could a shift in emphasis make up for this? The proton NMR spectrum was investigated f i r s t using the procedures found useful in Chaps. II.3 and II.4. As the assignments progressed, the emphasis was allowed to find i t s balance; the chapter presented largely chronologically. The digitoxose rings have the Cj conformation, with the - C H 3 substituents equatorial. The conformation of the glycoside and genin are depicted below. Fig. II.5.1 Solid-state conformations of digoxigenin (lower) and the digoxin glycone (upper). (From Go et a l . ) - 261 -Fig. II.5.2 A. 500 MHz AH NMR spectrum of digoxin in DMS0-d6. B. Same molecule following a D20 exchange. Hydroxy protons are marked (•) In A. - 262 -A solution of 19 mg digoxin in 0.4 ml DMSO-dg was used throughout the *H NMR studies. Some line-narrowing was induced by heating the sample to ca. 70°C and allowing to cool to room temperature. We assume that this treatment results In the molecules forming a more homogeneous solution. The spectrum reveals a low-field region with relatively sharp, overlapping lines, whereas those in the the methylene region appear broader and l i t t l e detail i s evident. This line-width (T2) phenomenon can be postulated to result from micelle formation, with the lipophilic steroid moieties in the aggregated phase, and solvated, more mobile, pendant sugars. If this were the case, of the sugar rings one would expect ring III to have the greatest mobility, and ring I the least; conclusions to support this hypothesis appear later. The original spectrum in DMS0-d_6 required some simplification, and a D2O exchange was performed. The spectrum of the parent compound is shown in Fig. II.5.2 A with the -OH resonances marked (•), and that of the -0D compound shown in Fig. II.5.2 B; the latter was used for a l l subsequent studies. At the outset, the probable vicinal coupling constants for the digitoxose groups were estimated, 5 and these are listed in Table II.5.1. - 263 -Protons Expected J_ (Hz) (1,2a) 10.0 (l,2e) 2.5 (2a,3) 2.6 (2e,3) 3.1 - 3.6 (3.4) 2.6 - 3.1 (4.5) 9.3 - 9.6 (5.6) 6.2 Table II.5.1 Expected J_ values for a B-digitoxose sugar residue. These data enabled a surprising number of assignments to be made by direct inspection of the spectrum. We concentrate f i r s t on the region corresponding to 6 > 2, where twelve carbohydrate and six steroidal protons are expected to resonate. Five groups of resonances can be distinguished In this region, and these are shown expanded in Fig. II.5.3 (after resolution enhancement with a double-exponential time-domain f i l t e r ) . "Ladder" assignments indicate the resonances which require assignment.* On the basis of i t s chemical shift, we assign the most low-field proton (6 5.81) to H-22 on ring E of the steroid, which has a small (1.83 Hz) long-range coupling to the geminal H-21 protons. Inspection of the region 6 4.92 - 4.72 (Fig. II.5.3. B) reveals, amongst other resonances, the AB portion of an ABX system; a large (geminal) coupling *The f i n a l assignments are included, for reference later in the chapter. - 264 -I I . 5 . 3 E x p a n s i o n s o f t h e l o w - f i e l d r e g i o n o f t h e 5 0 0 MHz H NMR s p e c t r u m o f d i g o x i n i n DMS0-d_6, w i t h a d r o p o f D2O a d d e d . - 265 -T | I I 1 | >~ 1 I J ' T T T j - T T T p l T T T I 1 | 1 I I | 1 I" I ~[ I T I T " T " I ' *[ fT I | 4.06 4.02 3.98 3.94 3.90 3.86 p p m 5 F i g . I I . 5 . 3 c o n t . - 266 -constant J_ (A,B) i s indicated by the intensity build-up and direct measurement. The smaller coupling constant matches that in H-22. Accordingly, these protons are assigned to the geminal H-21 protons. Further "general" assignments of digitoxose protons may be made on the basis of a comparison of vicinal coupling constants (Table II.5.1) and the simplification effected by O-deuteration. Based on the magnitude of their two coupling constants to the geminal H-2 protons, three sets of anomeric protons are distinguishable in Fig. II.5.3 B. Between 6 A.07 and 3.84 (Fig. II.5.3 C) the almost identical digitoxose H-3 resonances occur. This i s based on their three very similar coupling constants (giving rise to a coupling pattern resembling a quartet) and their simplification upon O-deuteration (data not shown). By virtue of their unique coupling to the methyl groups, the digitoxose H-5 protons can be identified to resonate between 6 3.74 and 3.57 (Fig. II.5.3 D)* Fig. II.5.3 E and F show the digitoxose H-4 protons, identified by matching coupling constants and comparison with data in Table II.5.1. Two other protons resonate in this region and, by default, must be steroidal in origin. The proton at 63.01 can be assigned to H-4"', as ring III i s the only digitoxose residue having an exchangeable hydroxyl proton at position 4 and this agrees with the results of the deuterium exchange experiment. Thus, the simple procedure of matching spectral splittings and a deuterium exchange *The HOD peak at 6 3.66 has been eliminated by presaturation. - 267 -experiment led to significant steps being taken in the assignment of the "low-field" region of the spectrum (6 > 2). The above assignments were made on the underivatized material, and encouraged us to proceed with a f u l l assignment of the spectrum. Although this was in apparent conflict with the conclusions of Chap. II.3, the temptation to proceed with this assignment was strong since any conformational deductions would be of considerable Interest. A proton 2D J_-resolved experiment was performed in an attempt to unravel the high-field methylene/methlne resonances (24 protons extensively overlapping between 61 and 2.1). Unfortunately this failed to produce useful information for the high-field region due to the protons' short J_2's (See Discussion in II.2.5.2). With this experiment, most of the transverse magnetization had relaxed beyond the point of detection after only six or seven experimental increments (ti/2) had been performed. Some data were extractable for the resonances of the low-field region, but these provided no additional information over what had been gained by straight inspection of the 500 MHz *H NMR spectrum. We believe this to be the f i r s t report of the failure of the 2D J-resolved experiment. Attempts to improve the efficacy of this experiment included the use of a much larger than necessary sweep-width in the J-dimension, to shorten At_i/2 and, hence, improve the digitization of the i n i t i a l portion of the interferograra. Also, the experiment was attempted at elevated temperature (60°C) in an effort to lengthen J_2's; both attempts were to no avail. - 268 -Next the COSY experiment (II.2.5.3.1) was performed to establish proton coupling connectivities. (SECSY was avoided as i t Is a spin-echo experiment and would be expected to suffer similar limitations to those described for the 2D J_-resolved experiment). A contour plot of the COSY spectrum of the deuterium exchanged digoxin is shown in Fig. II.5.4. Aside from a confirmation of assignment for the lactone, ring E, not much is clear until we consider expansions of the two regions indicated. Between 6 4.2 and 2.8 the digitoxose 3,4 and 5 protons resonate, and the COSY plot for this region is given in Fig. II.5.5. With H-4''' previously assigned, H-3''1 and H-5 1" are readily assigned as the most high-field resonances In each group of resonances. The lowest f i e l d of the carbohydrate H-4 resonances is coupled to the lowest f i e l d of the H-5 resonances, but these cannot yet be unambiguously assigned to H-4' and H-5', or H-4" and H-5". The connectivities do not distinguish H-3' from H-3". Next, the connectivities into, and out of, the high-field region are considered in Fig. II.5.6. With H-3'" assigned, H-2'" and H-2'" and H-2"' can be identified in the overlapping high f i e l d region, and these lead in turn to the assignment of H-l' " , which lies between H-l' and H-l''. The highest f i e l d anomeric proton (which we later assign to H-l') has two clear correlations into the methylene region, and those protons are coupled to the highest f i e l d of the carbohydrate H-3 protons (H-3'). Similarly, the anomeric proton at lowest-field is on the same ring as the low-field carbohydrate 3 proton (H-3"). Hence, we have now - 2 6 9 -r-—i— i—i—i i u r 1 1 I T I 1 T I 1 1 1 1 1 ! ! I I 1 I JUL 4 1 ppm 22 a i B A r • - - y / • • c£7 I I 22 Fig. II. 5 . 4 5 0 0 MHz COSY spectrum of digoxin ( 1 ) in DMS0-c[ 6, after deuterium exchange. The HOD signal was suppressed by preirradiation, and the 5 1 2 * 1 0 2 4 word data set symmetrized prior to display in the contour mode. - 270 -F i g . I I .5.5 E x p a n s i o n o f r e g i o n A i n F i g . I I . 5 . A . F i g . I I . 5 . 6 . E x p a n s i o n o f r e g i o n B i n F i g . I I . 5 . 4 . - 272 -determined a l l connectivities of the ring protons of the digitoxose units I and II, but we cannot assign a particular set of resonances to either unit. Connectivities between the carbohydrate methyl-groups and the H-5 protons are present, but unassignable due to extensive cross-peak overlap. Similarly, couplings between the steroid protons (H-3, H-12 and H-17) and the methylene/methine region are evident, but not fully assignable. The H-12 and H-17 resonances are each expected to have two correlations, and H-3, four. The resonance at 6 3.88 cannot be either H-12 or H-17 and must, by default, be H-3e. However, only three correlations are clear, and the fourth must either be below the contour threshold or overlap with another cross-peak. The next stage of the assignment involved a series of "driven nOe" experiments (II.2.A.3); these proved pivotal in the differentiation of ring I and ring II protons, and several steroidal protons. It turns out that the molecule i s in the spin-diffusion regime and a l l observed nOe's were strong and negative. The highest f i e l d anomeric proton i s sufficiently far from the other two that i t could be irradiated with reasonable selectivity. This Induced a strong inter-ring nOe into the steroid proton at 6 3.88 (data not shown) which was thereby assigned as H-3. Since only H-l' i s expected to induce an nOe into a steroid proton, this assignment provided confirmation of the assignment of H-3; numerous other nOe's were induced in the aliphatic region - presumably into the H-2 and H-4 protons. With this second c r i t i c a l assignment (the f i r s t being H-4'"), H-2', H-2', H-3', H-4' and and H-5' may be 6 8 identified from the COSY connectivities. Since these resonances, and - 2 7 3 -i' I ' I 1 1 1 ' ' 1 1 1 1 1 • i' 1 1 1 1 1 ' > 1 1 1 {H-3',3"} i ' ' ' ' i •1'' i ' ' ' ' i ' ' ' ' i 2.1 1.9 1.7 1.5 1.3 1.1 ppm 2.1 1.9 1.7 1.5 1.3 1.1 ppm CONTROL 2.1 1.9 1.7 1.5 1.3 1.1 ppm F i g . I I . 5 . 7 D r i v e n nOe e x p e r i m e n t s p e r f o r m e d on d i g o x i n , s h o w i n g n O e ' s i n d u c e d i n t o t h e m e t h y l e n e / m e t h i n e r e g i o n . - 274 -hence, their c r o 6 S-peaks are so close, verification for these assignments was sought from the nOe experiments. Confirmation of the digitoxose H-2 protons' assignments comes from the three nOe experiments, illustrated in Fig. II.5.7. Irradiation of (equatorial) H-3'" clearly shows H-2'" and H-2"'. Irradiation of e a H-3' and -3" induces nOe's in the indicated geminal protons; H-2^ and H-2", and H-2' and H-2" are differentiated by the COSY experiment 6 3 9. (Fig. II.5.6) which shows the connectivities from H-l 1 to H-2g to high f i e l d of H-2e' and H-2^" (which are coincident) and H-2^ to high-field of H-2", based on the connectivities from H-3', 3". The experiment involving irradiating of H-l' is included (vide supra). The carbohydrate H-5 region assignment was confirmed by the nOe experiments indicated in Fig. II.5.8. By irradiating H-l', or H - l " r > < i l i i i I i i i I i i i I i « • l • i i l • • i I « > 3.74 3.70 3.66 3.62 ppm Fig. II.5.8 Driven nOe experiments showing nOe's induced into digitoxose H-5 protons by irradiating H-l', or H-l'' and H - l ' " - 275 -Table II.5.2 Average interproton distances between -Me(18) and -Me(19), and steroid ring protons. (A ) C(18)-H3 H-ll H-8 H-17 H-l 2 H-23 H-22 a a.e 2.868 3.193 3.398 3.680 3.088, 4.410 3.982 C(19)-H3 H-l a.e H-8 H-5 H-ll i H-6 2.621, 2.825 2.732 2.762 2.887 2.999 - 276 -plus H-l''* an nOe is induced in the corresponding H-5 with which i t has a 1,3-diaxial relationship. A number of further checks were performed which confirmed the previous assignments. Irradiation of H-5' and -5'" induced nOe's into H-4'" and the lower-field of the H-4'/H-4'* overlapping resonances, and confirmed assignments of H-l' and H - l ' " (data not shown). This complemented the experiment where H-5" was irradiated. It is clear that a carefully selected array of irradiation experiments has, in addition to providing assignments, the added bonus of enabling previous assignments to be checked or confirmed; hence, such a series of experiments provides a "network" of assignment information. These nOe experiments shed some light on the steroid assignment, but far less than one might have anticipated. It is well known10 that the axial methyl groups (C-18 and C-19) can act as "transmitter beacons" to induce responses from axial protons which are near them in space. When the C(18)-H3 protons are irradiated, one might expect nOe's with H - l l a , H-8 on the p-face of the steroid, and H-12 and H-17. Further, one might expect an nOe into ring E, depending on i t s orientation. The cis C,D ring-fusion results in the protons on C-15 and C-16 being too far from -Me(18) to have an nOe induced. Irradiation of C(19)-H3 is unlikely to be of much use in assigning protons on ring A, as a result of the cis A/B ring junction. Protons referred to in the following discussion are listed in Table II.5.2, with the internuclear separations calculated from the X-ray data . Irradiation of -Me(18) induces the expected nOe's. At 6 < 2.1, we note two nOe's - one into H-8 (6 1.48) and the other, H-lip (6 - 277 -I i I I i l i i i i I i i i i I i i i i l i i i i I i i i i l i i i i I i i i i l i i i i l i i i i l i i i i I i i 2.1 1.9 1.7 1.5 1.3 1.1 Fig. II.5.9 Driven nOe experiments with steroidal methyl peaks irradiated; the displayed region 1B that of the methylene/methine protons, although other nOe's are evident to lower f i e l d . - 278 -1.12). This assignment is consistent with the COSY data, where a correlation exists between H-17 and H-16, but none between H-17/H-12 and H-8. Since both proton multiplets are overlapping with other signals, the enhancement factor cannot be reliably quantified. An nOe is seen at 6 3.2 where H-12 and H-17 overlap, but this is likely to include nOe's into both protons. (Based on their solid-state interproton distances, H-12 would be expected to show 70% the nOe of H-17). Significant nOe's are seen in a l l three protons on ring E. Irradiation of -Me(19) also reveals significant nOe's in H-8 and H-ll8. A total of six protons in the methylene/methine region display nOe's, consistent with the six protons having interproton separations < 3.0 A, listed in Table II.5.2. No cross checks can be made, since the multiplets overlap with the signals from their closest neighbours. Irradiation of H-22 results In an nOe in H-17, allowing i t to be distinguished from H-12 (data not shown). The proton assignment is far from complete, s t i l l much information has been gleaned. With the assignments resulting from the nOe experiments, l i t t l e i s to be gained by returning to the COSY map for the crowded methylene/methine region. Far fewer cross-peaks are distinct than anticipated; this w i l l be discussed later In the chapter, along with other observations relating to the performance of a l l the experiments described. Before leaving the *H NMR data It is interesting to look at the dlgltoxose sugar assignments in the light of predictions on the self association of the molecule in solution. Such a system would have relatively mobile, solvated sugars extending from the aggregated phase. - 279 -3.0 ppm 6 H 8 c F i g . I I . 5 . 1 0 CSCM e x p e r i m e n t N F i g . I I . 5 . 1 1 T h e h i g h - f i e l d r e g i o n o f t h e CSCM d a t a - s e t i n f i g . I I . 5 . 1 0 i s p l o t t e d b e t w e e n t h e f r e q u e n c y l i m i t s i n d i c a t e d . - 281 -Hence, one might expect the terminal digitoxose ring III to have more mobility (and, therefore, narrower lines) than ring II, and likewise even more mobility than ring I. Since the coupling pattern for each proton at a particular position on a l l rings i s the same, we can consider peak height to be a reflection of peak width (and T_2). With the eye of faith, i t i s clear that this is so for a l l the observable resonances to lower f i e l d ; ring III appears to have greater mobility than ring II and s t i l l greater than ring I. 1 3 We now turn our attention to the C data. The broad-band 13 decoupled C NMR spectrum of digoxin in DMS0-d_6 is well dispersed with few overlapping lines. The CSCM experiment was performed and parts of the resultant data-table are presented in Figs. II.5.10 and 11. Those 1 13 H assignments made were used to assign the corresponding C signals. C signals between 6 40 and 120 produced correlations in the H spectrum between o„ 2.9 and 6.0; a l l protons in this region were a. 1 3 assigned. The C NMR assignments followed smoothly provided i t was not the case that both the *H signals and the 1 3C signals were very close. 13 The C assignments are given in Figs. II.5.10 and 11. In the proton methylene/methine region, eleven assignments were made and the C signals were readily correlated. The steroid methyl carbons were readily identified (data not shown). A l l the above assignments were checked with the l i t e r a t u r e , 5 and were in agreement. Whilst the exercise of extracting an F^ (AH) trace for each 1 3C signal was performed, no proton assignments were made on this basis. Our spectra were run with the molecule dissolved in DMSO-dj, whilst Brown et a l . 6 used CDCI3:DMS0-d_e (1:2); small but sometimes significant - 282 -d i f f e r e n c e s i n c h e m i c a l s h i f t s w e r e a p p a r e n t , m a k i n g s u c h c o m p a r i s o n s 13 d a n g e r o u s . I t I s s i g n i f i c a n t t o n o t e t h a t i f t h e C NMR s p e c t r u m w e r e f u l l y a s s i g n e d , t h e *H NMR a s s i g n m e n t w o u l d e a s i l y f o l l o w f r o m t h e CSCM e x p e r i m e n t . T h e l a r g e d i s c u s s i o n o n t h e *H NMR v s . t h e 1 3 C NMR d a t a i n t h i s c h a p t e r i n v i t e s a f e w r e m a r k s . A l t h o u g h t h e *H s p e c t r a a r e m o r e c o m p l i c a t e d ( i n c l u s i o n o f J_; s m a l l e r i n t r i n s i c d i s p e r s i o n ) t h e y h a v e t h e h i g h e r i n f o r m a t i o n c o n t e n t . I n t h a t mo r e p a r a m e t e r s a r e r e p r e s e n t e d , a w i d e r r a n g e o f e x p e r i m e n t s i s p o s s i b l e ; h o w e v e r , t h e s e h a v e a l i m i t w h i c h , i n t h i s c a s e , i s d e t e r m i n e d b y t h e e x t e n s i v e o v e r l a p o f Tj>-b r o a d e n e d s i g n a l s i n t h e m e t h y l e n e / m e t h i n e a r e a . T h e *H NMR s p e c t r u m w o u l d h a v e b e e n e a s i e r t o a s s i g n h a d t h e A / B a n d C /D r i n g j u n c t i o n s b e e n t r a n s , s i n c e t h i s w o u l d h a v e p e r m i t t e d c o n s i d e r a b l y mo r e i n f o r m a t i o n t o r e s u l t f r o m t h e nOe e x p e r i m e n t s w h e r e m e t h y l g r o u p s w e r e i r r a d i a t e d . On 1 3 t h e o t h e r h a n d , a l t h o u g h t h e C NMR s p e c t r u m i s much s i m p l e r , i t i s e q u a l l y d i f f i c u l t t o a s s i g n i n i s o l a t i o n , f o r d i f f e r e n t r e a s o n s . T_2 l i n e b r o a d e n i n g no l o n g e r s eems t o p o s e a s e r i o u s p r o b l e m , b u t now t h e d i f f i c u l t y i n a s s i g n m e n t s eems t o l i e i n t h e a v a i l a b l e p r o c e d u r e s . We 1 13 h a v e s e e n t h a t a n a s s i g n e d H NMR s p e c t r u m c a n be u s e d t o a s s i g n a C NMR s p e c t r u m ( C h a p t e r I I . 4 ) a n d t h e same i s t r u e h e r e , w h e r e *H a s s i g n m e n t s a r e k n o w n . H o w e v e r , f o r t h o s e *H - 1 3 C p a i r s f o r w h i c h t h e Hi a s s i g n m e n t i s n o t a v a i l a b l e , f u r t h e r e x p e r i m e n t a t i o n i s s o m e w h a t l i m i t e d , a n d o ne i s d r i v e n t o c o n s i d e r o t h e r p r o c e d u r e s , s u c h a s c o m p a r i s o n s o f a s e r i e s o f s t r u c t u r a l l y r e l a t e d m o l e c u l e s . 13 A l t h o u g h i t i s p r o b a b l e t h a t f u r t h e r s p e c t r o s c o p i c a i d s t o C NMR a s s i g n m e n t s w i l l e m e r g e , t h e r e i s o n l y o n e c o n t e n d e r a t p r e s e n t . - 2 83 -T h e 2D INADEQUATE e x p e r i m e n t c a n u n d o u b t e d l y p r o v e u s e f u l . T h e o b v i o u s p r o b l e m f o r d i g o x i n i s t h e p r e p a r a t i o n o f a s u f f i c i e n t l y c o n c e n t r a t e d s o l u t i o n t o e n a b l e t h e e x p e r i m e n t t o be p e r f o r m e d I n a r e a l i s t i c t i m e p e r i o d . A l e s s o b v i o u s p r o b l e m may be a s s o c i a t e d w i t h s e c o n d o r d e r d i s t o r t i o n s r e s u l t i n g f r o m t h e r e l a t i v e l y s m a l l c h e m i c a l s h i f t r a n g e I n w h i c h t h e ( c o u p l e d ) ^ 3 C r e s o n a n c e s o c c u r . I t h a s b e e n d e m o n s t r a t e d w i t h c a r b o h y d r a t e s p e c t r a t h a t t h e e x p e r i m e n t may r e q u i r e c o m p e n s a t i o n f o r 8 2 13 s u c h e f f e c t s ; t h i s i s n o t u n r e a s o n a b l e when one c o n s i d e r s t h a t J_ ( C - C ) = c a . 40 H z , a n d c a r b o n r e s o n a n c e s f r o m s i m i l a r c e n t r e s f a l l i n t o f a i r l y n a r r o w r a n g e s ( e . g . 3 ' , 3 " , 3 * " , 5 ' , 5 " a n d 5 * " w i t h i n 4 p . p . m . ) . I n c o n c l u d i n g t h i s c h a p t e r i t i s i m p o r t a n t t o c o n s i d e r t h e l i m i t s o f t h e v a r i o u s e x p e r i m e n t s a c t u a l l y u s e d a n d t h e e f f e c t s o f t h o s e l i m i t s o n t h e g e n e r a l s t r a t e g y p o s t u l a t e d i n e a r l i e r c h a p t e r s . T h e 2D J - r e s o l v e d e x p e r i m e n t w o r k e d s a t i s f a c t o r i l y f o r t h o s e r e s o n a n c e s h a v i n g l o n g e r j T 2 v a l u e s w h i c h w e r e , m o s t l y , p r o t o n s a t t a c h e d t o h e t e r o a t o m - s u b s t i t u t e d c a r b o n s . U n f o r t u n a t e l y , t h e s e w e r e p r e c i s e l y t h e r e s o n a n c e s w h i c h w e r e n o t d i f f i c u l t t o d i s t i n g u i s h i n t h e ID e x p e r i m e n t , a s t h e y w e r e d e s h i e l d e d a n d r e s o n a t e d o u t o f t h e m e t h y l e n e / m e t h i n e r e g i o n . A n d u n f o r t u n a t e l y t h e r e s o n a n c e s i n t h e r a n g e 6 1 . 0 t o 2 . 1 h a d T_2 v a l u e s w h i c h w e r e t o o s h o r t . T h u s , i n c o n t r a s t t o e a r l i e r e x p e r i e n c e s , t h e 2D J _ - r e s o l v e d e x p e r i m e n t p r o v e d t o be d i s a p p o i n t i n g . T h e COSY e x p e r i m e n t was v e r y u s e f u l f o r t h e l o w - f i e l d r e g i o n . N o t w i t h s t a n d i n g t h e c o a r s e d i g i t i z a t i o n ( c a . 10 Hz p t - 1 ) , c o r r e l a t i o n s - 284 -to protons only a few Hz apart could be reliably differentiated. It i s c r i t i c a l to note that this was only possible with like protons (e.g. H-3 * and H-31') where the two correlation peaks have very similar patterns. By comparison, i t was not possible on a f i r s t inspection to discern correlations between H-12 or H-17 and the high-field region. Also, coupling pathways to (broader) proton resonances to high f i e l d in the methylene/methine region were clear (e.g. H-3 e). In the high-f i e l d region, far fewer cross-peaks appeared than expected. This i s probably the result of two factors, cross-peak overlap and the attenuation of the (broad) high-field signals by the necessary sinebell apodization function. Unfortunately without the latter, extensive " t a i l i n g " occurs which can lead to false "cross-peaks" where they Q interfere. The question of attenuation by the apodization function may be partly remedied by using a less severe function (e.g. the double-exponential function), and by using a smaller block-size and ze r o - f i l l i n g to give adequate digitization. The driven nOe experiment proved quite useful as a qualitative rather than quantitative tool. Quantitations were limited by unfortunate signal overlap in the low-field region even at 500 MHz which made selective irradiation d i f f i c u l t , and overlap in the high-field region which made the determination of fractional enhancements nigh on impossible. The procedure of assigning, e.g. the Irradiated methyl protons an area of 300 "units", in the difference mode, and induced intensity enhancement calculated by area relative to this, did not prove successful when compared with expected ratios based on known - 285 -d i s t a n c e s f r o m t h e s o l i d - s t a t e . T h i s c o u l d e i t h e r b e a r e s u l t o f t h e b r e a k - d o w n o f t h e s i m p l i f i e d e q u a t i o n s , r e s u l t i n g f r o m t h e a n i s o t r o p i c m o t i o n , o r t h e d i f f i c u l t y i n m e a s u r i n g an nOe o f a p r o t o n d i r e c t l y u n d e r t h e d i g i t o x o s e m e t h y l s i g n a l s . N e v e r t h e l e s s , q u a l i t a t i v e l y , t h e e x p e r i m e n t p e r f o r m e d w e l l , w i t h i n t h e u s u a l r e s t r i c t i o n s o f n o n - o v e r l a p p i n g r e s o n a n c e s i n t h e ID e x p e r i m e n t . T h e CSCM e x p e r i m e n t w o r k e d e x t r a o r d i n a r i l y w e l l a n d seems w e l l s u i t e d t o a s y s t e m o f t h i s n a t u r e . W i t h i n t h e l i m i t s o f d i g i t i z a t i o n , t h e *H l i n e - s h a p e s ( F j ) w e r e e a s i l y r e c o g n i z a b l e . I t i s w i t h o u t d o u b t 13 t h a t a l m o s t a l l p r o t o n a s s i g n m e n t s c o u l d be made i f t h e C s i g n a l s w e r e c r e d i b l y a s s i g n e d . I n some w a y s t h i s i s t h e m o s t i m p o r t a n t f a c t t o e m e r g e f r o m t h i s d i g o x i n s t u d y . I t c a l l s f o r a c h a n g e i n t h e p r o t o c o l f o u n d m o s t e f f e c t i v e w i t h d e r i v a t i z e d o l i g o s a c c h a r i d e s a n d b r u c i n e . T h e r e t h e i n i t i a l e m p h a s i s was on t h e t o t a l a s s i g n m e n t o f t h e 1 H NMR 13 s p e c t r u m , f o l l o w e d b y a s s i g n m e n t o f t h e C NMR s p e c t r u m o n t h e b a s i s o f t h e CSCM e x p e r i m e n t . W i t h d i g o x i n , w h e r e t h e 1 H NMR s p e c t r u m i s d i f f i c u l t t o f u l l y a s s i g n e v e n a t 5 0 0 M H z , a m o r e e f f e c t i v e a p p r o a c h w o u l d p r o b a b l y be t o f i r s t a s s i g n a s much o f t h e *H NMR s p e c t r u m a s p o s s i b l e u s i n g t h e m e t h o d s d e s c r i b e d a b o v e , a n d t h e n c o n c e n t r a t e o n t h e 13 t o t a l a s s i g n m e n t o f t h e C NMR s p e c t r u m . To t h i s e n d , t h e 2D INADEQUATE e x p e r i m e n t s h o u l d be p e r f o r m e d , b u t a p a u c i t y o f i n s t r u m e n t t i m e p r e v e n t e d t h i s i n a r e a s o n a b l e t i m e p e r i o d . W i t h t h i s i n f o r m a t i o n , c o m p l e t i o n o f t h e *H NMR a s s i g n m e n t w o u l d be t r i v i a l . I n c o n c l u s i o n , t h i s s t u d y o n d i g o x i n i n d i c a t e s t h a t n o t a l l t h e p r o c e d u r e s d e s c r i b e d i n p r e v i o u s c h a p t e r s c a n be r e l i e d u p o n f o r s t u d i e s - 286 -o f m o l e c u l e s w h i c h h a v e b r o a d , s t r o n g l y o v e r l a p p i n g r e s o n a n c e s . I n p a r t i c u l a r , t h e h o m o n u c l e a r 2D e x p e r i m e n t s a p p e a r t o be l e s s t h a n o p t i m a l i n t h i s r e g a r d . O f t h e a l t e r n a t i v e e x p e r i m e n t s , CSCM o p e r a t e s 13 b e s t i n t h i s m i l i e u , w i t h t h e f u n d a m e n t a l l i m i t a t i o n t h a t t h e C NMR s p e c t r u m mus t be a s s i g n a b l e . T o t h i s e n d , t h e 2D INADEOUATE e x p e r i m e n t s h o u l d be p e r f o r m e d t o c o m p l e t e t h i s c h a p t e r . - 287 -REFERENCES 1 . L . F . F i e s e r a n d M . F i e s e r . S t e r o i d s . R e i n h o l d P u b l i s h i n g C o r p o r a t i o n , New Y o r k . 1 9 5 9 . p p . 7 5 2 - 7 5 4 . 2 . J . H . H o c h . A s u r v e y o f c a r d i a c g l y c o s i d e s and g e n i n s . U n i v e r s i t y o f S o u t h C a r o l i n a P r e s s . 1 9 6 1 . 3. S . S m i t h . J . C h e m . S o c . 5 0 8 - 5 1 0 ( 1 9 3 0 ) . 4 . K. G o , G . K a r t h a , a n d J . P . C h e n . A c t a C r y s t . B 3 6 , 1 8 1 1 - 1 8 1 9 ( 1 9 8 0 ) . 5 . C . A l t o n a a n d C . A . G . H a a s n o o t . O r g . M a g n . R e s o n . 1 3 , 4 1 7 - 4 2 9 ( 1 9 8 0 ) . 6 . L . B r o w n , H . T . A . C h e u n g , R. T h o m a s , T . R . W a t s o n , a n d J . L . E . N e m o r i n . J . C h e m . S o c , P e r k i n T r a n s . 1 . 1 7 7 9 - 1 7 8 1 ( 1 9 8 1 ) . 7 . A . B a x , T . A . F r e n k i e l , R. F r e e m a n , a n d M . H . L e v i t t . J . M a g n .  R e s o n . 4 3 , 4 7 8 - 4 8 3 ( 1 9 8 1 ) . 8 . S . L . P a t t . C a r b o h y d r . R e s . I n p r e s s . 9 . A . B a x a n d R. F r e e m a n . J . M a g n . R e s o n . 4 4 , 5 4 2 - 5 6 1 ( 1 9 8 1 ) . - 288 -II.6 EXPERIMENTAL FOR SECTION II In general, a l l spectra reported at 270 MHz were recorded on a home-built spectrometer based on an Oxford Instruments superconducting magnet (B^ j 6.35 T), a Bruker WP-60 console, and NIcolet 1180 computer and 293B pulse-programmer. Unless stated otherwise, standard NTCFTB soft-ware was used with this instrument. Spectra at 400 MHz (AH) were recorded on a Bruker WH-400 instrument, having a 9.4 T magnet. The operating program in routine use was DISNMRP. Five hundred and 360 MHz spectra were performed using Nicolet spectrometers at the factory In California. It i s the author's opinion that a detailed documentation of a l l parameters for each experiment would not be useful, since these w i l l vary widely depending on the instrument being used. It ±e_ useful, however, to document any extra pieces of information which are relevant to a particular experiment, and not mentioned in the text. For H^ experiments, the 180° pulse-length was determined (zero intensity cross-over), and a l l pulse-lengths calculated from this. For 13 C observe the procedure was the same, often using a reasonably concentrated "dummy" sample in a similar solvent to that in which the molecule of interest was dissolved. Proton pulses through the decoupler channel on the 1 3C probe were determined by the methods outlined in the text. When attempting an experiment for the f i r s t time, i t i s common to begin with a very simple molecule at high concentration. Knowing the molecule's geometry, T A parameters, etc. can greatly assist in the - 289 -I n i t i a l evaluation, or "debugging" of an experiment. With heteronuclear correlation experiments in this thesis, for example, a concentrated solution of n-propanol was used to familiarize the operator with important parameters and experimental set-up. For DEPT (II.2.2.2), the 1 3C parameters were selected in the usual manner, and A H pulse-lengths "fine-tuned" by performing the experiment with 6 a 9 0 ° . When perfect cancellation of - C H 2 and - C H 3 peaks was achieved, the proton pulse lengths were correctly set. With a l l homonuclear decoupling experiments (including nOe), a c r i t i c a l factor i s the choice of decoupler power and position. Even with sufficient decoupler power, Incomplete decoupling may result from the resonance frequency of the saturated spin changing while the decoupler is on. In most cases this 1B not too important a consideration, but i t may be necessary to slowly move the decoupler frequency (in 0.5 Hz steps) to find the optimal position to effect perfect decoupling. This Is particularly a consideration in spectrally crowded regions where decoupler selectivity is d i f f i c u l t to achieve. To achieve this selectivity Is most d i f f i c u l t with the SDDS experiment (II.2.3) and i t i s fortunate that reliable two-dimensional experiments having none of these problems (e.g. COSY) may be resorted to. With nOe experiments i t i s important to come as close to 100% saturation as possible, without leakage (or "spill-over") of decoupler power Into spectrally close spins. This i s easily checked before the experiment i s done: the irradiation of the spin in the most crowded spectral region i s used to determine the maximum permissible power, - 290 -without spill-over. No excessive time-averaging is necessary here, as one is not looking for very small (< 1%) changes in peak height. Such checks not only give the experimenter a degree of confidence, but the percentage saturation In each experiment may be useful information i f certain comparisons are to be made. This practice of collecting only the FID comprising the difference spectrum, and not both spectra individually, i s not recommended, since (a) information i s needlessly discarded, and (b) where multiple nOe experiments are necessary, more machine time w i l l be required to accumulate the same data. With the procedure for SSNOEDS (II. 2.4.1) in the text (ri + 1) experiments are necessary for ii nOe irradiations; i f only the difference FID is collected, 2n_ experiments would be necessary to derive the same information. When the sample concentration is low, demanding a very large number of scans to build up signal-to-noise, the author has found i t useful to increase the repetition rate by (a) using no relaxation delay (b) decreasing the period of irradiation to ca. 1 or 2 T j , and, to compensate for these alterations in the experimental scheme, (c) decreasing the tip-angle (from ca. 80° to 60°). In such a way, four nOe experiments were possible in an overnight run with ca. 2 mg of sample on a Bruker WH-400, allowing the detection of < 1% nOe's with confidence. With the one-dimensional experiments discussed above, data storage space on hard disc is seldom limiting, and spectra can be acquired with good digitization. In the case of two-dimensional (2D) NMR experiments the reverse is often the case: long "acquisition times" - 291 -in t^i may require large amounts of instrument time to acquire, large blocks of data to store, and long computation times for data-processing. It i s therefore common to restrict the sweep-widths in both dimensions to the minimum possible value. If, for example, acetate peaks to high f i e l d have no information, they may be "folded" into the spectrum where they do not overlap with any other peaks. With this approach, the minimum block-size can be chosen in both dimensions and experimental times reduced. With the 2D J-resolved experiment (II.2.5.2), fine digitization Is not necessary in F_2, and the experiment should be optimized for digitization in F j , the "J_-dimenslon". In the interest of minimizing instrument time, i t i s common to collect data for 32 t_i increments and z e r o - f i l l in JFj_ to give the necessary digitization to resolve small couplings. Although the author has not had opportunity to assess symmetrization procedures in this experiment, i t i s quite l i k e l y they w i l l be extremely useful since second-order artefacts are seldom symmetrical about F_| • 0 and such a procedure should prove useful in identifying such peaks and simplifying the spectrum. Other advantages of this procedure are mentioned in the text. We have had cause to use the COSY experiment (II.2.5.3) quite extensively in this study, and have found i t remarkably resistant to abuse. Pulse-angles need only be set to ± 10%, and coarse digitization (e.g. 5 Hz/pt) i s often sufficient. The data may be acquired quite rapidly (ca. 0.2 sec. relaxation delay) and the sinebell apodization function is almost always appropriate for both dimensions. The Jeener - 292 -experiment i s so powerful that, given a state-of-the-art spectrometer, i t i s arguable whether SDDS methods are ju s t i f i a b l e . The important heteronuclear chemical shift correlation (CSCM: II.2.5.4) i s described in some detail in the text and a discussion on a "typical" set-up procedure i s included. The author has not had time to thoroughly evaluate the 2D nOe experiment (NOESY: II.2.5.6). Probably the most d i f f i c u l t decision with this experiment is choosing an optimal mixing time. In the slow tumbling regime, a "crude" driven nOe experiment (II.2.4.3) should indicate the rate of nOe build-up and allow an optimal xn^x to be chosen. In the fast tumbling regime, a valid approach might be to f i r s t perform a "crude" transient nOe experiment (II.2.4.2) in order to characterize a "typical" nOe bulld-up curve and f a c i l i t a t e the choice of a useful "%£ x. Cross-peaks are small in this latter experiment, and symmetrlzatlon of the data-set w i l l almost certainly be necessary. As is the case with COSY, coarse digitization w i l l often suffice, thereby reducing the time required to collect the data for this somewhat long experiment. 

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