Deuterium NMR Spectroscopy - ACS Publications - American

6 Deuterium N M R Spectroscopy

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: July 9, 1982 | doi: 10.1021/bk-1982-0191.ch006

IAN C. P. SMITH and HENRY H. MANTSCH National Research Council of Canada, Ottawa, Canada, K1A 0R6

2

The enormous utility of H NMR was pointed out very early by Diehl and Leipert ( 1 ) . Due in part to lack of instrumentation, a considerable lag occurred in realizing this potential. However, with the advent of Fourier transform spectrometers, a great surge of activity ensued. In 1977 we attempted a comprehensive review of the literature on H NMR (2). The activity since then has been so great as to make impossible a thorough discussion of all reports. Instead we shall make extensive reference to our earlier review, and present some of the highlights of applications over the past four years. 2

Magnetic Properties of Deuterium 2

The fundamental properties of the H nucleus are listed in Table I. The low magnetogyric ratio of H leads to a somewhat low detection sensitivity, but this presents very little problem for a modern spectrometer. It also scales all couplings between H and other nuclei to 15% of the corresponding values for protons, as we have discussed in detail earlier ( 2 ) . The low natural abundance of H is an advantage, since labeling to 100% is relatively simple, and the negligible background signals at natural abundance w i l l not complicate the overall spectrum. The main novelty of H for high resolution NMR practitioners is the spin of 1. A feature of nuclei with I > 1/2 is an unsymmetrical charge distribution in the nucleus, Figure 1, and therefore an electrical quadrupole moment, Q. This moment can interact with the charge distribution about the nucleus and thus affect the energies of the nuclear spin states, Figure 2 . The magnitude of the interaction depends upon the second derivative of the electric potential at the nucleus, q £* and on the direction of the magnetic field with respect to the principal axes of the tensor q $. The energy of this interaction is given by the quadrupole coupling tensor, e q ^Q/h* Note that i f the q g are zero, there is no interaction and the nuclear spin energy levels are unaffected. This can arise in situations of high 2

2

2

2

a

a

2

a

a

0097-6156/82/0191-0097$06.25/0 © 1982 American Chemical Society

In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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Table I

Properties

of the Deuteron

N a t u r a l Abundance

0.015%

Resonance Frequency at 2.3487 Τ Spin

15.351 MHz 1

Sensitivity

1

Receptivity

2

2

r e l a t i v e to H

0.965% 6

1.45 χ 10"" %

Quadrupole moment ( x l O

2 8

2

m)

2.73 χ 10~

3

*For equal numbers of n u c l e i . 2

Sensitivity

r e l a t i v e to that of *H, a t n a t u r a l abundance.


6.

1*1/2, Q*0

1=1/2, Q = 0


99

Deuterium NMR Spectroscopy

SMITH AND MANTSCH

«Ρ " \3r 3r /r=0 a

P/

Quadrupole Coupling Constant*

Π

^

Figure 1. Representation of the properties of a nonquadrupolar and a quadrupolar nucleus. The charges around the nucleus represent those of its molecular environ ment. The electric field gradient tensor, q«0, is expressed in the coordinate system of the molecule. Usually, the z-axis lies along the carbon-deuterium bond; e is the charge on the electron, h is Planck's constant, and Q is the quadrupole moment. For axial symmetry, only q is required to express the quadrupole coupling constant. z z

χ

= Q

e qQ (3cos Q-1 4I(2I-1)h\ 2 2

2

[3m2-I(I D] +

m=-1 m=0 m=+1 Zeeman

Ζ e e m a n + Quadrupole

_Α_λ_ Figure 2. Effect of the quadrupole interaction on the Zeeman energy levels of an 1 = 1 nucleus with axial symmetry. I is the total spin, m its component, and θ is the angle between the applied magnetic field and the principal (z) axis of the quadru pole splitting tensor.


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symmetry, which are r a r e f o r deuterium. H o w e v e r , q u i t e commonly the tensor q ^ i s a x i a l l y - s y m m e t r i c , i . e . q = q y. In this case only q need be c o n s i d e r e d , and the s u b s c r i p t s are o f t e n neglected i n formulation of the quadrupole coupling constant, F i g u r e 1. D e v i a t i o n s from a x i a l symmetry a r e e x p r e s s e d i n terms of the asymmetry p a r a m e t e r , η = ( q - q )/q . For deuterium in sp bonds to c a r b o n , the asymmetry parameter i s u s u a l l y n e g l i g i b l e , and the quadrupole c o u p l i n g constant v a r i e s between 160 a n d 190 k H z , d e p e n d i n g u p o n t h e n a t u r e o f t h e o t h e r s u b s t i t u e n t s on the carbon atom (2). F i g u r e 2 shows t h e e f f e c t o f a f i n i t e q u a d r u p o l e c o u p l i n g c o n s t a n t ( i n t h e c a s e o f a x i a l s y m m e t r y ) , on t h e Zeeman l e v e l s o f deuterium. F o r a n i m m o b i l i z e d s p e c i e s , two t r a n s i t i o n s o f u n e q u a l energy are a l l o w e d , t h e i r energy d i f f e r e n c e s depending upon the magnitude of the quadrupole c o u p l i n g constant and the angle Θ between the m a g n e t i c f i e l d and the z - a x i s of the e l e c t r i c f i e l d gradient tensor q g , u s u a l l y the d i r e c t i o n of the carbondeuterium bond. a

z

x

x

y

z

x x

y

v

Z

2


3

a

In s o l u t i o n , where m o l e c u l a r r e o r i e n t a t i o n i s r a p i d r e l a t i v e to the r e c i p r o c a l of the quadrupole c o u p l i n g constant, these e f f e c t s are averaged and o n l y a s i n g l e t r a n s i t i o n at the frequency of the i s o t r o p i c s i t u a t i o n , ω , i s observed. The m o d u l a t i o n of the quadrupole i n t e r a c t i o n by molecular r e o r i e n t a t i o n manifests i t s e l f i n the r e l a x a t i o n behaviour of H , p r o v i d i n g the dominant mechanism (2). I n l i q u i d c r y s t a l s or biomembranes, where r a p i d motion occurs with l i m i t e d amplitude w i t h i n a large, slowly tumbling s u p e r s t r u c t u r e , p a r t i a l a v e r a g i n g of quadrupole s p l i t t i n g takes p l a c e , the r e s u l t a n t s p l i t t i n g b e i n g a measure of the degree of m o l e c u l a r o r d e r i n g w i t h i n the s u p e r s t r u c t u r e ( 2 , 3 ) . 0

2

H i g h R e s o l u t i o n D e u t e r i u m NMR H i g h r e s o l u t i o n H NMR i s e x t r e m e l y u s e f u l i n s t u d i e s o f small molecules i n s o l u t i o n (2). The c h e m i c a l s h i f t s of H are t h e same i n p a r t s p e r m i l l i o n as t h o s e o f * H ; t h e s e p a r a t i o n s i n frequency u n i t s are correspondingly less than those of H by the quotient of the magnetogyric r a t i o s , Υ2 /Υΐ 0-154. The r e d u c e d c o u p l i n g s t o *H a f f o r d an enormous s i m p l i f i c a t i o n of t h e s p e c t r a , and t h e s e can be removed by b r o a d band d e c o u p l i n g . 2

2

1

Η

Η

=

F i g u r e 3 shows t h e ^ - c o u p l e d and - d e c o u p l e d H NMR s p e c t r a of a m i x t u r e of s o l v e n t s , t a k e n a t n a t u r a l abundance of H (4). The only couplings that u s u a l l y appear i n the H s p e c t r a are those of g e m i n a l h y d r o g e n s , as s e e n i n the e x p a n s i o n of t h e a c e t o n e resonances (starred region of the l e f t of Figure 3). In very complex p r o t o n systems, the a b i l i t y to o b t a i n c h e m i c a l s h i f t s from NMR s p e c t r a o f H at n a t u r a l abundance, without r e s o r t to s p e c t r a l s i m u l a t i o n , i s a p o w e r f u l f i r s t s t e p towards u n d e r s t a n d i n g the *H spectra (2). There are circumstances where knowing the chemical s h i f t s i n 2

2

2

2


In NMR Spectroscopy: New Methods and Applications; Levy, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 1

2

m

Ή noise-decoupled

2

Figure 3. NMR spectra (15.4 MHz) of H at natural abundance for a mixture of common sol vents, as indicated from left to right: dimethylformamide (amide), benzene, ethanol (hydroxyl), ethanol (methylene), dimethylformamide (methyl), acetone, ethanol (methyl), tetramethylsilane. The D in each formula is to emphasize that for a molecular species containing H at natural abundance all other hydrogen is H. Key: A, proton-coupled; B, proton-decoupled. (From Ref. 4.)

no decoupling

Q


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H s p e c t r a i s not s u f f i c i e n t f o r assignment of resonances. An example i s the H NMR s p e c t r u m o f n i c o t i n e , F i g u r e 4. In this case s p e c i f i c l a b e l i n g w i t h d e u t e r i u m a l l o w e d b o t h measurement of c h e m i c a l s h i f t s and assignments f o r the methylene groups at p o s i t i o n s 3 , 4 ' , and 5 (5). Note the considerably greater widths of the H resonances r e l a t i v e to t h e i r H counterparts; t h i s i s due t o t h e e f f i c i e n t q u a d r u p o l a r r e l a x a t i o n mechanism o f H , which i s a p p r o x i m a t e l y 20 t i m e s m o r e e f f e c t i v e than that of ^ in a geminal p a i r of hydrogen. 1

X

!

f

2

1

2

A n o b v i o u s a p p l i c a t i o n o f h i g h r e s o l u t i o n d e u t e r i u m NMR i s to s t u d y r e a c t i o n mechanisms i n v o l v i n g hydrogen. Rather than f o l l o w i n g t h e d i s a p p e a r a n c e o f one o f many H r e s o n a n c e s due t o i n c o r p o r a t i o n o f d e u t e r i u m , i t i s much more c o n v e n i e n t t o o b s e r v e the a p p e a r i n g d e u t e r i u m resonances d i r e c t l y , as o n l y t h e i n c o r p o r a t e d d e u t e r i u m atoms a r e o b s e r v e d . Furthermore, the deuterium resonances y i e l d v e r y a c c u r a t e i n t e g r a t e d i n t e n s i t i e s as t h e r e i s no s i g n i f i c a n t n u c l e a r O v e r h a u s e r e f f e c t t o accompany proton decoupling. A number o f s u c h k i n e t i c s t u d i e s , i n v o l v i n g b o t h s l o w and f a s t exchange p r o c e s s e s have been d e s c r i b e d i n r e f e r e n c e (2).


1

A more r e c e n t d e m o n s t r a t i o n of the u s e f u l n e s s of h i g h r e s o l u t i o n d e u t e r i u m NMR a s a n a n a l y t i c a l t e c h n i q u e i s i l l u s t r a t e d i n F i g u r e 5. Here deuterium resonances were used to characterize the r a d i c a l - and c a t i o n - d e r i v e d products obtained by anodic o x i d a t i o n of the [ 2 , 2 - d 2 ] b u t y r a t e i o n (6). The assignment of these r e s o n a n c e s w a s m a d e o n t h e b a s i s o f t h e 1:1 r e l a t i o n s h i p b e t w e e n the c h e m i c a l s h i f t s of *H and H , and the d e u t e r i u m l a b e l d i s t r i b u t i o n was d e t e r m i n e d f r o m t h e p r o t o n n o i s e - d e c o u p l e d H NMR spectra. A s c a n b e s e e n f r o m F i g u r e 5, p r o p e n e w a s l a b e l e d i n t h e terminal o l e f i n i c carbon but not i n the c e n t r a l o l e f i n i c carbon atom, w h i l e propane t u r n e d out to be d e u t e r i u m - l a b e l e d e x c l u s i v e l y at C j . 2

2

F u r t h e r i n f o r m a t i o n may b e e x t r a c t e d f r o m t h e a n a l y s i s o f the f i n e s t r u c t u r e of proton — coupled h i g h r e s o l u t i o n deuterium spectra. Thus, the proton-coupled deuterium spectrum of the m e t h y l group of propene appears as a d o u b l e t of d o u b l e t s (see i n s e t to F i g u r e 5), i n d i c a t i n g t h a t the p o s i t i o n must be doubly deuterated. A n o t h e r r e c e n t a p p l i c a t i o n o f h i g h r e s o l u t i o n H NMR w a s t o study t h e r e a c t i o n mechanism of the s o l v o l y s i s of norbornen-2-yl d e r i v a t i v e s (7). The a c e t o l y s i s of t h e m o n o - d e u t e r i u m compound i> exo-5-norbornen-2-y1 b r o s y l a t e , for instance l e d to the p r o d u c t s 2 t o .5. The d e u t e r i u m l a b e l d i s t r i b u t i o n of t h e s e prod u c t s w a s d e t e r m i n e d u n a m b i g u o u s l y ( F i g u r e 6) a n d t h u s a l l o w e d the e l i m i n a t i o n of a p r e v i o u s l y proposed mechanism w h i c h r e q u i r e d the i n t e r v e n t i o n of asymmetrical h o m o a l l y l i c c a t i o n i n t e r m e d i a t e s . 2

I n a p i o n e e r i n g e x p e r i m e n t C o x a n d S t y l e s (8) h a v e a p p l i e d h i g h r e s o l u t i o n d e u t e r i u m NMR t o w a r d s b i o c h e m i c a l i m a g i n g b y generating H NMR s p e c t r a c o l l e c t e d f r o m l o c a l i z e d r e g i o n s , that i s f r o m v o l u m e e l e m e n t s , s m a l l c o m p a r e d w i t h t h e NMR c o i l . 2



SMITH AND MANTSCH

103


6.

3

2

ppm Figure 4. NMR spectra of nicotine and specifically deuterated derivatives: top, *H at 100 MHz; others, H at 15.4 MHz. (Reproduced from Re]. 5. Copyright 197S American Chemical Society.) 2

y


I


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NMR SPECTROSCOPY

I

I

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PPM

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Figure 5. a, H NMR spectra (at 100 MHz, in CCl under pressure) of a mixture of gaseous propane, propene, and cyclopropane, b, Proton noise-decoupled H NMR spectra (at 15.4 MHz, in CCl under pressure) of some of the reaction products obtained from the electrolysis of the [2,2-d ] butyrate ion: Expansion, protoncoupled deuterium resonances. (Reproduced from Ref. 6. Copyright 1980, American Chemical Society.) 1

k

2

k

2


6.

105


SMITH AND MANTSCH

D


3

R e a c t i o n mechanism of the s o l v o l y s i s derivatives·

of

norbornen-2-yl

CDCl

4,5

***** Β

PPm

L 7

J

L

5

3

1

Figure 6. Proton noise-decoupled H NMR spectra (at 15.4 MHz) of the reaction products obtained from the acetolysis of [2-d]-exo-5-norbornen-2-yl brosylate in the absence (A) and presence (B) of Eu (fod) . (Reproduced, with permission, from Ref. 7. Copyright 1980, Swiss Chemical Society.) 2

3


NMR SPECTROSCOPY

106


Deuterium Nuclear

Relaxation

As d e s c r i b e d e a r l i e r , the d e u t e r i u m n u c l e u s has a s i g n i f i c a n t q u a d r u p o l e moment w h i c h i n t e r a c t s w i t h t h e e l e c t r i c f i e l d gradient at the nucleus. The i n t e r a c t i o n between the d e u t e r o n n u c l e a r e l e c t r i c q u a d r u p o l e moment a n d t h e e l e c t r o s t a t i c field gradient at the nucleus i s at l e a s t an order of magnitude greater than e i t h e r the deuteron d i p o l e - d i p o l e i n t e r a c t i o n or the s p i n r o t a t i o n i n t e r a c t i o n C2). D e u t e r i u m NMR r e l a x a t i o n t h u s r e s u l t s predominantly from the i n t r a m o l e c u l a r quadrupole i n t e r a c t i o n , making deuterium an i d e a l nucleus f o r the study of i n t r a m o l e c u l a r r o t a t i o n a l motion (2,3). Due t o t h e s t r e n g t h o f t h i s i n t e r a c t i o n , deuterium r e l a x a t i o n times are short r e l a t i v e to those of s p i n i n u c l e i r e o r i e n t i n g w i t h t h e same c o r r e l a t i o n t i m e . In processes w h e r e a H n u c l e u s i s r a p i d l y e x c h a n g i n g b e t w e e n two s i t e s of d i f f e r e n t c o r r e l a t i o n times (for example a l i g a n d exchanging between b u l k s o l u t i o n and the b i n d i n g s i t e of a p r o t e i n , the d i f f e r e n c e i n c o r r e l a t i o n time between the f r e e and the bound l i g a n d i s r e f l e c t e d i n a g r e a t l y increased l i n e w i d t h of the o b s e r v e d d e u t e r i u m NMR s i g n a l r e l a t i v e t o t h a t o f t h e f r e e l i g a n d 2

(2). A m i s c o n c e p t i o n o f t e n e n c o u n t e r e d among s p e c t r o s c o p i s t s is t h a t i n h i g h r e s o l u t i o n d e u t e r i u m NMR t h e r e s o n a n c e s a r e s o broadened by d e u t e r i u m quadrupole r e l a x a t i o n as to be of l i t t l e use. While t h i s i s c e r t a i n l y true for heavier quadrupole n u c l e i s u c h as N or C 1 , the l i n e widths of deuterium resonances, p a r t i c u l a r l y those of s m a l l e r molecules, are often s m a l l r e l a t i v e to the c h e m i c a l s h i f t s e p a r a t i o n s of i n t e r e s t . In the case of CDCI3 t h e d e u t e r i u m r e s o n a n c e h a s a w i d t h a t h a l f h e i g h t o f only 0.5 Hz ( 2 ) , and the a p p a r e n t w i d t h o f s u c h n a r r o w s i g n a l s i s o f t e n determined by the inhomogeneity of the e x t e r n a l magnetic f i e l d or by incomplete coalescence of m u l t i p l e t s a r i s i n g from geminal or v i c i n a l H - H coupling. The most r e l i a b l e way t o determine the t r u e l i n e w i d t h of deuterium resonances i s by measuring the s p i n - l a t t i c e (T ) or s p i n - s p i n (T ) r e l a x a t i o n time; under the conditions of extreme narrowing which are u s u a l l y e n c o u n t e r e d f o r s m a l l m o l e c u l e s , Δνι = Ι / τ τ ^ = 1 / π Τ . The T j v a l u e o f CDCI3 30°C i s 1.6 s , c o r r e s p o n d i n g t o a n i n t r i n s i c l i n e w i d t h of 0.2 H z . 1

4

3

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2

x

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a

t

B o t h d i r e c t and i n d i r e c t methods have been used to measure d e u t e r i u m r e l a x a t i o n Ç2); the i n v e r s i o n - r e c o v e r y or p a r t i a l l y r e l a x e d F o u r i e r t r a n s f o r m method i s most w i d e l y employed. Deuterium s p i n - l a t t i c e r e l a x a t i o n times f o r a v a r i e t y of deutera t e d compounds h a v e b e e n r e p o r t e d i n t h e l i t e r a t u r e ( 2 , 1 0 , 1 1 ) . D e u t e r i u m atoms i n h i g h l y m o b i l e p o s i t i o n s have l o n g r e l a x a t i o n times w h i l e those i n a s s o c i a t e d or very r i g i d systems have short relaxation times. The r a n g e o f d e u t e r i u m r e l a x a t i o n t i m e s r e p o r t e d i n s o l u t i o n extends from v e r y l o n g (10), such as t h a t of t h e m e t h y l g r o u p i n n e a t C D B r ( 7 . 4 3 s ) , t o v e r y s h o r t (11) such as i n aqueous [3d] N - a c e t y l g l u c o s a m i n e (0.017 s ) . In the l i m i t 3


6.

SMITH AND MANTSCH

Deuterium

NMR

107

Spectroscopy

of r a p i d i s o t r o p i c motion these correspond to deuterium resonances having l i n e widths at h a l f height of r e s p e c t i v e l y 0.043 and 18.7 Hz. In r i g i d s o l i d s where r e l a x a t i o n i s o u t s i d e the motional narrowing range, deuterium T\ values can be very l o n g . G e n e r a l l y , molecular r e - o r i e n t a t i o n i n l i q u i d s can be d e s c r i b e d i n a simple way by i s o t r o p i c Brownian r o t a t i o n , although t h i s i s r i g o r o u s l y a p p l i c a b l e only to s p h e r i c a l l y - s y m m e t r i c molecules. For the study of a n i s o t r o p i c molecular motion s p i n l a t t i c e r e l a x a t i o n times of C have been used i n the past as the standard technique, but s p i n - l a t t i c e r e l a x a t i o n of deuterium i s becoming i n c r e a s i n g l y more important, s i n c e i t i s dominated by a s i n g l e i n t r a m o l e c u l a r quadrupole mechanism and does not r e q u i r e v e r i f i c a t i o n of the r e l a x a t i o n mechanism. Deuterium a l s o has the advantage of s h o r t r e l a x a t i o n times and t h e r e f o r e measurements i n v o l v e c o n s i d e r a b l y l e s s instrument time. We have s t u d i e d the stereochemical dependence of deuterium r e l a x a t i o n times f o r a wide range of deuterated compounds (12). In [ds] p y r i d i n e f o r example,the deuterons at the α(1.2 s ) , 6(1-1 s) and γ(1.3 s) p o s i t i o n s have almost i d e n t i c a l Τχ v a l u e s , i n d i c a t i n g that p y r i d i n e undergoes e s s e n t i a l l y i s o t r o p i c motion. In [dy-] . dimethylformamide, on the other hand, the l a r g e d i f f e r e n c e between the r e l a x a t i o n times of the c i s (3.0 s) and t r a n s (1.6 s ) , deuteriomethyl groups i n d i c a t e s a p r e f e r r e d a x i s of r o t a t i o n .


1 3

E q u a l l y i n f o r m a t i v e are the deuterium s p i n - s p i n r e l a x a t i o n times T ( u s u a l l y obtained from l i n e width measurements), e s p e c i a l l y when studying low deuterium c o n c e n t r a t i o n s . Due to the s e n s i t i v i t y of l i n e widths to s u b t l e changes i n c o r r e l a t i o n times, H NMR can g i v e a d e t a i l e d p i c t u r e of r o t a t i o n a l motion of s p e c i f i c a l l y deuterium-labeled r e p o r t e r groups. This technique has, f o r i n s t a n c e , been used to study i n t e r n a l motions of deuterium-labeled molecules bound to p r o t e i n s (2,9,11,13). We have used t h i s technique to d e l i n e a t e the molecular dynamics of sugars bound to a l e c t i n (11). The b i n d i n g to wheat germ a g g l u t i n i n of N-acetylglucosamine deuterated s p e c i f i c a l l y i n the N - a c e t y l group and i n the pyranoside r i n g (C3) was i n v e s t i gated v i a the l i n e broadening of the deuterium resonances as a f u n c t i o n of the t o t a l sugar c o n c e n t r a t i o n (Figure 7). Due to the short exchange r a t e s ( l a r g e d i s s o c i a t i o n r a t e constants) of Nacetylglucosamine with the l e c t i n , t h i s system was i d e a l l y s u i t e d f o r study by deuterium NMR. The c o r r e l a t i o n time of the bound [3d] N-acetylglucosamine (3xl0"" s) was found to be i d e n t i c a l to that of the p r o t e i n , i n d i c a t i n g that the six-membered r i n g has n e g l i g i b l e motional freedom r e l a t i v e to the p r o t e i n ; the c o r r e l a t i o n time of the bound [ d ] N-acetylglucosamine ( 1 . 7 x l 0 ~ s) i n d i c a t e s that the N - a c e t y l s i d e c h a i n i s a l s o immobilized, the only motion a v a i l a b l e being the r o t a t i o n of the CD3 group around i t s threefold axis. 2

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0.5

1.0

1.5

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Ι/Δΐ/(Ηζ ') _

Figure 7. Binding of N-acetylglucosamine to wheat germ agglutinin. Plot of the total sugar concentration vs. the reciprocal linewidth of the indicated deuterated sites. The intercepts yield the dissociation constant and the slopes yield the linewidth of the bound species. (Reproduced, with permission, from Ref. 11. Copy right 1980, Biophysical Society.)


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Before the advent of F o u r i e r transform spectrometers, widel i n e H NMR was done by sweeping the magnetic f i e l d and observing the d i s p e r s i o n s i g n a l , or by p u l s i n g the radiofrequency and observing the f r e e i n d u c t i o n decay without t r a n s f o r m a t i o n . The very broad s p e c t r a l widths have caused problems with b a s e l i n e s and f a i t h f u l r e p r e s e n t a t i o n s of the e n t i r e l i n e s h a p e s . Various techniques, such as the quadrupole echo (14), p r o g r e s s i v e phase a l t e r n a t i o n of the e x c i t a t i o n pulse and d e t e c t o r , short s p e c t r o meter dead times, and p o s t - a c q u i s i t i o n s p e c t r a l c o r r e c t i o n (15) have circumvented most of these. Wide-line s p e c t r a are observed f o r s o l i d s , or f o r systems i n s o l u t i o n with very long (with respect to the i n v e r s e of the quadrupole s p l i t t i n g ) c o r r e l a t i o n times f o r r e - o r i e n t a t i o n . Examples of the l a t t e r are c o l l a g e n f i b r e s , membranes, and l i q u i d c r y s t a l s . As many examples of t h i s type of study were given i n our e a r l i e r review (2), we s h a l l c o n f i n e ourselves to c o l l a g e n and membranes.


2

2

S i n g l e c r y s t a l s give simple H NMR s p e c t r a whose quadrupole s p l i t t i n g s depend on the angles between the a p p l i e d magnetic f i e l d and the p r i n c i p a l axes of the s p l i t t i n g tensor. Polycrystalline samples present a l l angles simultaneously, and t h e r e f o r e t h e i r s p e c t r a are s u p e r p o s i t i o n s of a l l those f o r the v a r i o u s angles (2,16). They are r e f e r r e d to as powder s p e c t r a ; from t h e i r d i s t i n c t i v e shapes the quadrupole s p l i t t i n g s f o r two p r i n c i p a l values of Θ (0° and 90°, see F i g . 2 and r e f e r e n c e (16)) can be e a s i l y obtained. Rapid a n i s o t r o p i c motion leads to r e d u c t i o n i n the magnitudes of the p r i n c i p a l s p l i t t i n g s , and these reduced values l e a d to estimates of the degree of molecular order (3); t h i s assumes t h a t other motions are too slow to a f f e c t l i n e shapes or apparent quadrupole s p l i t t i n g s (16). S i m i l a r s p e c t r a a r i s e f o r very l a r g e molecules, or molecular assemblies, which r e - o r i e n t slowly i n s o l u t i o n . The s y n t h e t i c polypeptide p o l y b e n z y l glutamic a c i d can be a l i g n e d i n a magnetic f i e l d such that o v e r a l l motion of the h e l i c a l s t r u c t u r e i s very slow. Rapid a n i s o t r o p i c motion of the pendant s i d e chains leads to reduced quadrupole s p l i t t i n g s f o r these l a b e l e d p o s i t i o n s , the magnitudes of which were used to d e r i v e i n f o r m a t i o n on the amplitudes of the a n i s o t r o p i c motions (17). A much more complex system i s c o l l a g e n , a t r i p l e polypep t i d e h e l i x of dimensions 1.5 χ 300 nm and molecular weight 285,000. J e l i n s k i et a l . produced c o l l a g e n c o n t a i n i n g e i t h e r [ 3 , 3 , 3 - d ] a l a n i n e or [ d 7 ! v i a t i s s u e c u l t u r e i n the presence of l a b e l e d amino a c i d s (JL8, 19). F i g u r e 8 shows both experimental and simulated H NMR s p e c t r a f o r t h i s system c o n t a i n ing [3,3,3-d ]alanine. The top spectrum i s t h a t of the p o l y c r y s t a l l i n e amino a c i d ; i t has a reduced quadrupole s p l i t t i n g of 38.8 kHz due to r a p i d r o t a t i o n of the methyl group. Frozen c o l l a g e n f i b r i l s (Figure 8b), c o n t a i n i n g the same labe" l e u c i n e

3

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3


NMR SPECTROSCOPY

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EXPERIMENTAL

-40

«20

0 kHz

20

CALCULATED

40

-40

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-20

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40

Figure 8. Ή NMR spectra (33.78 MHz) of [3,3,3-d ] alanine and of collagen labeled with this amino acid. Key: a, poly crystalline [3,3,3-d ] alanine; b, labeled collagen fibrils, —18°C; c, as in b, but 18°C; d, labeled collagen in solution, 18°C; and e-h, calculated spectra for a-d, respectively. (Reproduced, with permission, from Ref. 18. Copyright 1980, MacMillan Journals.) 3

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a c i d , y i e l d a very s i m i l a r spectrum. However, the same f i b r i l s at 18°C (Figure 8c) y i e l d e d a spectrum o f reduced o v e r a l l i n t e n s i t y with a reduced quadrupole s p l i t t i n g ; t h i s i s due to the onset of s i d e c h a i n motions other than r o t a t i o n of the methyl group about i t s C 3 a x i s . C o l l a g e n i n s o l u t i o n a t 18°C y i e l d e d F i g . 8d, w i t h a quadrupole s p l i t t i n g of only 10 kHz. T h i s further r e d u c t i o n i n quadrupole s p l i t t i n g was a t t r i b u t e d to r o t a t i o n of the c o l l a g e n molecule about i t s long a x i s , w i t h the angle between the CD3-CH bond of a l a n i n e and the long molecular a x i s having a value of roughly 70°. The shapes of the s p e c t r a were simulated (Figure 8, r i g h t s i d e ) i n terms of a model where t h i s bond jumps between two o r i e n t a t i o n s . For the c o l l a g e n f i b r i l s i t was concluded that the component c o l l a g e n molecules undergo r a p i d ( 1 0 s"" ) a n i s o t r o p i c motion with a range o f azimuthal angles from 30-40°. S i m i l a r s t u d i e s have been reported f o r a r t i f i c i a l l y deuterated (deuteromethylation of methionine to i t s dimethyl sulfonium analog) heme p r o t e i n c r y s t a l s o r i e n t e d i n a magnetic f i e l d (20). A f i e l d i n which H NMR has c o n t r i b u t e d extremely v a l u a b l e i n f o r m a t i o n i s b i o l o g i c a l membranes. E a r l y s t u d i e s concentrated on development of l a b e l i n g methods, spectrometer techniques, and e l u c i d a t i o n of the p r o p e r t i e s of l i p i d s i n model membranes (2,J3, 16). The experience gained with the models i s now a c t i v e l y under a p p l i c a t i o n to i n t a c t b i o l o g i c a l membranes. E a r l y success i n the study of b i o l o g i c a l membranes was a t t a i n e d by use of the r e l a t i v e l y simple organism Acholeplasma l a i d l a w i i (21). I t has a s i n g l e membrane, the plasma membrane, and w i l l accept l a b e l e d f a t t y acids from the growth medium without s i g n i f i c a n t a l t e r a t i o n . For some f a t t y a c i d s enrichment of the membranes to greater than 95% can be achieved by use of the p r o t e i n a v i d i n i n the growth medium (22). F i g u r e 9 shows the H NMR s p e c t r a of the membranes of t h i s organism, e n r i c h e d i n e i t h e r [ l 2 - d ] - o l e i c a c i d or [ l 3 - d ] - p a l m i t i c a c i d , at 37°C, the growth temperature. The narrow resonances a t the centres of the quadrupolar p a t t e r n s are due to H at n a t u r a l abundance i n the surrounding water. This can be avoided by the use of deuteriumdepleted water. The presence of the c i s double bond i n o l e i c a c i d leads to r e l a t i v e l y f l u i d , l i q u i d c r y s t a l l i n e membranes a t 37°. The quadrupole s p l i t t i n g i s a measure of the degree of molecular o r d e r i n g a t the 1 2 - p o s i t i o n w i t h i n the membrane. In c o n t r a s t , the s t r a i g h t chain p a l m i t i c a c i d r e s u l t s i n more ordered, l e s s f l u i d membranes a t t h i s temperature (note the l a r g e r quadrupole s p l i t t i n g o f the narrow p a t t e r n i n the c e n t r e of the spectrum). Furthermore, the broad s p e c t r a l components on the wings o f the spectrum i n d i c a t e the presence of a second l i p i d phase, the s o - c a l l e d g e l phase, i n which the a c y l chains a r e e s s e n t i a l l y a l l - t r a n s , and molecular motion i s of l e s s e r amplitude and r a t e than i n the l i q u i d c r y s t a l l i n e phase. With both f a t t y a c i d s the optimum growth temperature of A. l a i d l a w i i i s 35 ± 1 ° , whereas the l i q u i d c r y s t a l to g e l t r a n s i t i o n s , T , occur a t widely 7

1

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SPECTROSCOPY

d i f f e r e n t temperatures. These s p e c t r a have been discussed i n d e t a i l i n earlier publications (23,24). The v a r i a t i o n of molecular order with p o s i t i o n i n the membranes of A. l a i d l a w i i has been s t u d i e d i n d e t a i l with both p a l m i t i c (25) and o l e i c (24) a c i d s . For the s a t u r a t e d a c i d at temperatures above the phase t r a n s i t i o n , the order i s of i n t e r mediate and constant magnitude f o r the f i r s t 1 0 - 1 2 carbon atoms from the c a r b o x y l group, dropping r a p i d l y t h e r e a f t e r to very low values f o r the t e r m i n a l methyl group. The p a t t e r n f o r the unsaturated a c i d i s shown i n F i g u r e 1 0 ; i t i s s i m i l a r to that f o r the s a t u r a t e d a c i d except f o r a l a r g e drop near the c i s double bond. T h i s i s l a r g e l y a geometric f a c t o r ; when the p a r t i c u l a r angles of the double bond are considered, i t i s shown to be a l s o h i g h l y ordered ( 2 4 ) . The c i r c l e s i n F i g u r e 10 i n d i c a t e the e f f e c t of i n c o r p o r a t i n g 20 mole % c h o l e s t e r o l i n t o the membranes. Note that t h i s i n c r e a s e s the quadrupole s p l i t t i n g (thus the order parameter) at a l l p o s i t i o n s , with the l a r g e s t i n c r e a s e coming f o r p o s i t i o n s near the carboxyl group. A s i m i l a r response to c h o l e s t e r o l a d d i t i o n was found f o r A. l a i d l a w i i membranes c o n t a i n ing p a l m i t i c a c i d Ç 2 6 ) . F i g u r e 11 shows the temperature dependence of the H NMR s p e c t r a of A. l a i d l a w i i membranes enriched to 90% i n the fourteen carbon s a t u r a t e d f a t t y a c i d l a b e l e d at the t e r m i n a l methyl group. We see again the presence of a l i q u i d c r y s t a l to g e l phase t r a n s i t i o n f o r the membrane l i p i d s , centered at 4 0 ° C as determined by scanning c a l o r i m e t r y . Note the l a r g e d i f f e r e n c e i n quadrupole s p l i t t i n g s , f o r even the t e r m i n a l p o s i t i o n of the chains, between the two s t a t e s , i n d i c a t i n g a high degree of o r d e r i n g i n the g e l phase. A d i f f i c u l t problem with such two-component H NMR s p e c t r a i s q u a n t i t a t i n g the r e l a t i v e proportions of the components. Recently J a r r e l l et a l . have shown that the moments of the spectra can be used to d e r i v e r e l i a b l e values f o r these f r a c t i o n s ( 2 7 ) . A p p l i c a t i o n s of t h e i r method to the data of F i g u r e 11 y i e l d s the f r a c t i o n of l i q u i d c r y s t a l l i n e l i q u i d as a f u n c t i o n of temperat u r e , as shown i n F i g u r e 1 2 . The parameter Δ , which i s a measure o f the heterogeneity of the system ( 2 7 ) , i s a l s o shown i n F i g u r e 12. I t need not pass through a maximum at the midpoint of the t r a n s i t i o n ; the temperature of the maximum depends upon the p r o p e r t i e s of the two component s t a t e s ( 2 7 ) . 2

2

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Many of the p r o p e r t i e s of b i o l o g i c a l membranes, i n c l u d i n g those of JE. c o l i , have been found to be very s i m i l a r to those of model l i p i d systems. T h i s , and a survey of other systems s t u d i e d by NMR, has been d i s c u s s e d r e c e n t l y by S e e l i g and S e e l i g ( 2 8 ) . The H NMR s t u d i e s of model and b i o l o g i c a l membranes by O l d f i e l d and co-workers have a l s o been reviewed r e c e n t l y ( 2 9 ) . The accomplishments of Bloom and co-workers have a l s o been d i s c u s s e d i n the recent reviews, and are e x e m p l i f i e d by r e f e r e n c e ( 3 0 ) . As a l a s t example of H NMR of s o l i d s the recent work of Pines and co-workers must be mentioned. They have shown that use of m u l t i p l e quantum t r a n s i t i o n s and c r o s s - p o l a r i z a t i o n , magic 2

2



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SMITH AND MANTSCH

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Figure 9. H NMR spectra of the plasma membranes of Acholeplasma laidlawii enriched in palmitic acid labeled at the 13-position (13-d 16:0) and in oleic acid labeled at the 12-position (12-d 18:1). Spectra were obtained at the growth tem perature, 37°C. The temperatures of optimal growth, T , and the calorimetric gel to liquid crystal phase transition of the lipids in the membranes, T , are indicated. Details of sample preparation and spectral acquisition are given in Ref. 23 and 24. 2

2

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c

m

30 h

• • • o

20 h

•.

KHZ

Θ

10 .0.

6

10

us

If

Carbon, atom. Figure 10. Variation of the quadrupole splitting (T) , proportional to the order parameter) with position of labeling of the oleoyl chains in the membranes of Acholeplasma laidlawii, in the absence (\J) and in the presence (Q) of 20 mol % cholesterol at 25°C. Data for membranes without cholesterol are from Ref. 24; those with cholesterol are the unpublished data of M. Ranee, K. R. Jeffrey, A. P. Tulloch, K. W. Butler, and I. C. P. Smith. n


NMR SPECTROSCOPY


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20 10

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Figure 11. H NMR spectra (46.1 MHz) of the plasma membranes of Acholeplasma laidlawii enriched in myristic acid labeled at the terminal methyl group (C-14:0-

Deuterium NMR Spectroscopy - ACS Publications - American

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