NMR Spectroscopy - American Chemical Society

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N M R of C Enriched Amino Acids and Peptides

Downloaded by CORNELL UNIV on July 21, 2016 | http://pubs.acs.org Publication Date: July 9, 1982 | doi: 10.1021/bk-1982-0191.ch007

ROBERT E. LONDON University of California, Los Alamos National Laboratory, Los Alamos, NM 87545

The combination of isotopic enrichment and nuclear magnetic resonance spectroscopy provides an extremely powerful biochemical/biophysical probe. The magnetic properties (e.g. chemical shift, relaxation behavior) and the metabolic fates of individual nuclei can be studied in systems as complex as whole cells. Thus, the advantages of sensitivity gains, spectral simplification and spin labeling are the primary motivations for isotopic enrichment. In addition to these more obvious aspects of isotopic labeling, there are unique problems of interpretation and sources of information which require careful analysis and which represent important considerations in experimental design. Some of these aspects of the problem can be approached most directly by considering the spectral characteristics of relatively simple systems such as labeled amino acids and peptides. Such studies thus lay the groundwork for more sophisticated applications of isotopic labeling. The primary objective of the present article is to outline some of the characteristics of labeled systems, with particular emphasis on the relation of information content to labeling pattern. S p e c t r a l C h a r a c t e r i s t i c s oL c E n r i c h e d Amino A c i d s The s p e c t r o s c o p i c c h a r a c t e r i s t i c s o f C e n r i c h e d amino a c i d s a r e most conveniently discussed by c o n s i d e r i n g s e p a r a t e l y the three categories of isotopic labeling: (1) S p e c i f i c e n r i c h m e n t , u s u a l l y o f a s i n g l e c a r b o n p o s i t i o n ; (2) u n i f o r m e n r i c h m e n t w i t h t h e l a b e l i n g o f each c a r b o n s t a t i s t i c a l l y independent; (3) general enrichment w i t h the carbon l a b e l i n g nonrandom. The f i r s t type o f l a b e l i n g i s t y p i c a l l y o b t a i n e d v i a organic s y n t h e s i s and i s o f p a r t i c u l a r value f o r l a b e l i n g s t u d i e s of macromolecules i n which s p e c t r a l s i m p l i c i t y i s a p r i m a r y concern. The second c a t e g o r y o f l a b e l e d amino a c i d s i s most f r e q u e n t l y o b t a i n e d b i o s y n t h e t i c a l l y from algae grown f o r many 3

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0097-6156/82/0191 -0119$ 10.25/0 © 1982 American Chemical Society

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

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generations on ^ C 0 so that a l l carbons become e q u a l l y enriched. The l a s t c a t e g o r y i n c l u d e s p r i m a r i l y m o l e c u l e s p r o d u c e d i n b i o s y n t h e t i c s t u d i e s i n w h i c h the l a b e l i n g p a t t e r n itself c o n t a i n s i n f o r m a t i o n o f v a l u e f o r the e l u c i d a t i o n o f the b i o s y n t h e t i c pathways i n v o l v e d . The s p e c t r a l c h a r a c t e r i s t i c s of each type of l a b e l e d amino a c i d are considered below.


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(a) S p e c i f i c a l l y E n r i c h e d Amino Acids, Specifically e n r i c h e d amino a c i d s a r e o f use i n m e t a b o l i c s t u d i e s and f o r macromolecular l a b e l i n g s t u d i e s , and a wide v a r i e t y of i s o t o p i c isomers i s now a v a i l a b l e commercially. The ready a v a i l a b i l i t y o f t h e s e amino a c i d s makes them a t t r a c t i v e f o r NMR analysis, although r e l a t i v e l y l i t t l e work has been reported. Examination o f the u n e n r i c h e d c a r b o n r e s o n a n c e s p r o v i d e s d a t a on the s c a l a r c o u p l i n g constants which are o f use t h e o r e t i c a l l y f o r the i n t e r p r e t a t i o n o f the molecular o r b i t a l s t r u c t u r e , as w e l l as f o r assignment purposes. For example, we have r e c e n t l y s t u d i e d the c a r b o n - c a r b o n c o u p l i n g s [ 90$-3i ]-L-tryptophanLH which had been used f o r a l a b e l i n g study of the enzyme, d i h y d r o f o l a t e reductasei£l ( F i g . 1). The observed c o u p l i n g p a t t e r n was used to h e l p e s t a b l i s h the a s s i g n m e n t s . ( 1 ) I n t h i s c a s e , the r i n g c a r b o n s were o r i g i n a l l y a s s i g n e d c o r r e c t l y by B r a d b u r y and N o r t o n ( ^ ) but s u b s e q u e n t w o r k e r s based the a s s i g n m e n t s on an i n c o r r e c t i n d o l e a s s i g n m e n t ^ ) l e a d i n g to some confusion i n the literature. C o r r e c t a s s i g n m e n t s o f b o t h the i n d o l e ( 5 ) and t r y p t o p h a n i i L L have s i n c e been made. S p e c i f i c '^C e n r i c h m e n t combined w i t h NMR a n a l y s i s o f the u n l a b e l e d c a r b o n s has a l s o proven o f value f o r the assignment o f carbohydrate resonances.Xli We note f u r t h e r that although t h i s c o u p l i n g i n f o r m a t i o n i s a l s o a v a i l a b l e from the s p e c t r a o f [U- ^C] enriched amino a c i d s , the much g r e a t e r complexity of the s p e c t r a o c c a s i o n a l l y obscures the i n f o r m a t i o n , p a r t i c u l a r l y i n the c a s e o f l o n g r a n g e c o u p l i n g s . The a v a i l a b i l i t y o f s p e c i f i c a l l y e n r i c h e d amino a c i d s f o r coupling constant analysis represents an u n d e r u t i l i z e d s p e c t r o s c o p i c resource, and a d d i t i o n a l s t u d i e s may be a n t i c i p a t e d i n the f u t u r e . c

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(b) r u J 3 - C l E n r i c h e d Amino A c i d s . The s p e c t r a o f [ U - C ] amino a c i d s are i n many r e s p e c t s analogous to t y p i c a l H spectra. However, s i n c e e n r i c h m e n t l e v e l s a r e t y p i c a l l y l e s s t h a n 100?, the s p e c t r a corresponding to a s u p e r p o s i t i o n of i s o t o p i c isomers are obtained. T h i s c h a r a c t e r i s t i c i s i l l u s t r a t e d f o r the C-2 carbon o f g l y c e r o l i n F i g . 2(8) Although the l a r g e range of s h i f t s f r e q u e n t l y j u s t i f i e s f i r s t order a n a l y s i s o f the spectrum, t h i s i s not a l w a y s the c a s e , as i s a p p a r e n t f r o m the peak a s y m m e t r i e s i n F i g . 2. An i n t e r e s t i n g a s p e c t o f t h i s a n a l y s i s w h i c h has been d i s c u s s e d by Bengsch and Ptak(Q) i s t h a t the v a r i o u s sub-spectra may be o f d i f f e r e n t order. Thus, some o f the i s o t o p i c isomers w i l l e x h i b i t strong c o u p l i n g and some may not. T h i s c o m p l i c a t i o n can be u t i l i z e d f o r s p e c t r a l i n t e r p r e t a t i o n 1

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S .


t" S s? •5 .a

I" 0

R «

^

Si § 5 s **

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* Y


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GLYCEROL C-2 RESONANCE

CORRESPONDING ISOTOPE ISOMER

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Co

C3

Ci

Co

C3

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Ci

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_jjiWL 77

75 73 71 ppm

Figure 2. Proton decoupled C-2 resonances of [U- C ] glycerol biosynthesized by a marine red alga. The observed resonances represent a superposition of singlet, doublet, and quartet corresponding to the isotope isomers illustrated in the figure. The relative intensities of the lines in each multiplet were calculated using the J and Av values obtained from the spectrum. (Reproduced, with permission, from Ref. 8. Copyright 1979, John Wiley & Sons, Ltd.) 13

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s i n c e the magnitude o f some couplings can be deduced from f i r s t order sub-spectra, and used f o r the subsequent i n t e r p r e t a t i o n o f higher order sub-spectra y i e l d i n g a d d i t i o n a l couplings and s i g n s of c o u p l i n g constants. Among t h e o t h e r c o n s e q u e n c e s o f t h e h i g h o r d e r n a t u r e o f some s u b - s p e c t r a a r e : (1) '^C - '^C s a t e l l i t e s w i l l n o t be s y m m e t r i c a l l y disposed about the center resonances; and (2) s m a l l i s o t o p e e f f e c t c o n t r i b u t i o n s t o the s h i f t can be overwhelmed by s h i f t s due to high order coupling. The s i m p l e s t i l l u s t r a t i o n o f t h i s e f f e c t can be observed i n an AB system. The c e n t e r s o f the d o u b l e t s c o r r e s p o n d i n g t o t h e [A,B- ^Cg] m o l e c u l e w i l l n o t c o i n c i d e w i t h the s i n g l e t s a r i s i n g from the [A- ^ C j ] i [B- ^] l a b e l e d m o l e c u l e s , b u t a r e s h i f t e d away f r o m t h e s i g n a l o f t h e a d j a c e n t carbon. R e l a t i v e t o t h e c e n t e r o f t h e AB p a t t e r n , t h e d o u b l e t / s i n g l e t s h i f t d i f f e r e n c e s w i l l have magnitude = 1/2 [(Δν + J ) ' _A ] where Δν = \> - Vg. T h i s s i t u a t i o n i s r e a l i z e d f o r t h e c a s e o f [ 1 , 2 - ^Cg] t a u r i n e . ( 1 0 ) The o b s e r v e d d o u b l e t / s i n g l e t s h i f t d i f f e r e n c e s o f 0.116 and 0.119 ppm f o r the S - m e t h y l e n e and N - m e t h y l e n e r e s o n a n c e s , r e s p e c t i v e l y , a r e i n c l o s e agreement w i t h the magnitude o f 0.119 ppm due t o the high o r d e r e f f e c t s w h i c h c a n be c a l c u l a t e d f r o m t h e s p e c t r a l parameters. Thus, there i s a net 0.003 ppm u p f i e l d i s o t o p e s h i f t o f t h e S - m e t h y l e n e r e s o n a n c e p r o d u c e d by t h e N - m e t h y l e n e resonance, and no measurable i s o t o p e s h i f t f o r the r e v e r s e case. In perhaps the most c a r e f u l a n a l y s i s c a r r i e d out t o date, Bangsch and PtakXjil f i n d no i s o t o p e s h i f t f o r t h e carb^qxyl c a r b o n 1


a n (

#

2

1

2

2

v

A

1 J

r e s o n a n c e s due t o ot-carbon e n r i c h m e n t o f s e v e r a l C e n r i c h e d amino a c i d s and peptides, but an average 0.010 ppm u p f i e l d s h i f t f o r the C / C s u b s t i t u t i o n at other positions. In contrast, H/'H and ' N / ' \ produced l a r g e r i s o t o p e s h i f t s o f 0.25 ppm and 0.020 ppm, r e s p e c t i v e l y . Although high order a n a l y s i s i s c l e a r l y i n d i c a t e d f o r s t r o n g l y coupled n u c l e i , the a p p r o p r i a t e a n a l y s i s o f weakly c o u p l e d n u c l e i i s more s u b t l e and has f r e q u e n t l y c o n f u s e d even e x p e r i e n c e d s p e c t r o s c o p i s t s . The most t h o r o u g h l y d i s c u s s e d example o f t h e need f o r u s i n g h i g h o r d e r a n a l y s i s f o r t h e i n t e r p r e t a t i o n o f resonances corresponding t o weakly coupled n u c l e i has been l a b e l e d " v i r t u a l c o u p l i n g " ^ 11 ) T h i s e f f e c t i s most commonly c o n s i d e r e d f o r an ABX s y s t e m i n w h i c h t h e X r e s o n a n c e s c a n appear as a 1:2:1 t r i p l e t d e s p i t e t h e f a c t t h a t A X ^ BX* s p e c t r a have been t e r m e d " d e c e p t i v e l y s i m p l e " due t o the l i k e l i h o o d o f o b t a i n i n g i n c o r r e c t r e s u l t s based on a f i r s t o r d e r a n a l y s i s w h i c h n e g l e c t s t h e e f f e c t o f s t r o n g AB coupling. I n o b s e r v i n g t h e w e a k l y c o u p l e d r e s o n a n c e s o f amino a c i d s (most f r e q u e n t l y c a r b o x y l g r o u p s ) , we have f r e q u e n t l y n o t e d a marked asymmetry o f resonances ( F i g . 3)«112l T h i s asymmetry i s o c c a s i o n a l l y suggestive o f a s t r o n g c o u p l i n g i n t e r a c t i o n d e s p i t e the o b v i o u s f a c t t h a t t h e «ϊχ£/(ν - v ) v a l u e s f o r a l l c o u p l i n g i n t e r a c t i o n s i n v o l v i n g X are s m a l l . Numerical s i m u l a t i o n o f the 1

2

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Figure 3. Proton decoupled C NMR spectra of the C-5 resonances of [90%-UC ] glutamic acid: a, pH 2.5; c, pH 10.6 (this pH is below the asymptotic value corresponding to essentially complete amino deprotonation, because under such conditions the C-l and C-5 resonances overlap completely). (Continued on next page.) 13

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10 H z

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10 H z Figure 3. Continued. Theoretical transitions corresponding to the fully labeled molecule are illustrated in b and d. The coupling constants indicated in a are apparent first-order constants, which is substantially in error for ' J ^ . The coupling constants used in the simulations are: *J = 55.0 Hz (pH 2.5) and 51.5 Hz (pH 10.6), and the remote coupling constants are given accurately from a first-order analysis of the center resonances corresponding to C-5, C-3, and C-2 but not C-4 labeled. The additional observable splitting at high pH reflects the increase in Jsi/kvsi from 0.31 to 0.43. The average observed coupling for the upfleld and downfield doublets in a is equal to the actual J value [Reproduced, with permission, from Ref. 12. Copyright 1981, Heyden (London).] H

3

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s p e c t r a I n d i c a t e s that the observed s p e c t r a l c h a r a c t e r i s t i c s r e f l e c t the presence o f unresolved t r a n s i t i o n s due t o high order i n t e r a c t i o n s i n the s p i n system, w i t h the apparent peak h e i g h t s r e l a t e d t o the s e p a r a t i o n o f these unresolved t r a n s i t i o n s . Such e f f e c t s a r e n o t o b s e r v e d i n a t h r e e s p i n ABX s y s t e m , b u t c a n a r i s e i n a f o u r s p i n s y s t e m w i t h a t l e a s t one s t r o n g c o u p l i n g i n t e r a c t i o n , i . e . , an ABMX s y s t e m . The X r e s o n a n c e s o f an ABX s y s t e m w i l l n e v e r be a s y m m e t r i c , however, t h e r e l a t i v e a r r a n g e m e n t s o f the two ABX s u b - s p e c t r a o f the ABMX s y s t e m c a n g i v e r i s e t o t h i s apparent asymmetryXl2l The C-5 c a r b o x y l resonances o f [90$-U- C ]-glutamate: ,J


5

0 C 2

-

CH

2

-

CH

2

-

3? C 0 CH -

2

p r o v i d e a good i l l u s t r a t i o n o f t h e s e e f f e c t s ( F i g . 3)· A t l o w pH, the C-5 resonances i n c l u d e f o u r c e n t e r peaks corresponding t o C-4 u n l a b e l e d , and two s e t s o f s a t e l l i t e s c o r r e s p o n d i n g t o C-4 labeled. I t i s apparent that these s a t e l l i t e resonances a r e s t r u c t u r a l l y d i f f e r e n t than the c e n t e r resonances, i.e., the introduction of a l a b e l a t C-4 has two e f f e c t s : (1) introduction of a large c o u p l i n g i n t e r a c t i o n ; and (2) i n t r o d u c t i o n o f a moderately strong i n t e r a c t i o n between C-3 and C-4 ( « ^ / Δ χ ^ = 0.31) w h i c h i n d i r e c t l y a l t e r s the C-3 - C-5 and C-4 - C-5 I n t e r a c t i o n s . I t c a n be s e e n f r o m F i g . 3a t h a t t h e c e n t e r r e s o n a n c e s a r e f i r s t o r d e r and c a n be used t o o b t a i n t h e two s m a l l remote carbon-carbon c o u p l i n g constants, Jco and Jc2^ The glutamate spectrum i s a c t u a l l y an ABMRX system w i t n o n l y the one s t r o n g c o u p l i n g i n t e r a c t i o n between C-3 and C-4, however the C-5 resonances can be i n t e r p r e t e d n e g l e c t i n g C-1 s i n c e i t i s not c o u p l e d t o C-5 and s i n c e i t i s o n l y w e a k l y c o u p l e d t o t h e o t h e r n u c l e i . The t h e o r e t i c a l s p e c t r u m shown i n F i g . 3b i l l u s t r a t e s the c l o s e l y s p a c e d component r e s o n a n c e s w h i c h g i v e r i s e t o the a p p a r e n t peak h e i g h t a s y m m e t r i e s i n F i g . 3a. A t h i g h pH, t h e s h i f t d i f f e r e n c e between C-3 and C-4 d e c r e a s e s , e n h a n c i n g t h e anomalous a p p e a r a n c e o f t h e C-5 r e s o n a n c e s ( F i g s . 3c, 3d). I n t h i s c a s e , some o f t h e component r e s o n a n c e s a r e s u f f i c i e n t l y separated t o appear as separate resonances. B o t h t h e v i r t u a l c o u p l i n g e f f e c t and t h e a s y m m e t r i c appearance o f weakly coupled n u c l e i are p r e d i c t e d d i r e c t l y from a high order a n a l y s i s as can be c a r r i e d out r o u t i n e l y by numerical s o l u t i o n o f the Hamiltonian.( 13) I t i s h e l p f u l , however, t o have a g e n e r a l u n d e r s t a n d i n g t h a t such e f f e c t s e x i s t and under what c o n d i t i o n s they a r i s e t o f a c i l i t a t e s p e c t r a l i n t e r p r e t a t i o n and to h e l p avoid m i s i n t e r p r e t a t i o n .


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(c) G e n e r a l l y Labeled Amino Acids. The proton decoupled C spectrum of glutamate b i o s y n t h e s i z e d from a [ 9 0 ί - 1 - Ο , ] glucose p r e c u r s o r by M*. ammoniaphilumf14) i s i l l u s t r a t e d i'n F i g . 4. A n a l y s i s of the b i o s y n t h e t i c pathways i n v o l v e d i n d i c a t e s that the l a b e l i n g occurs p r i m a r i l y at C-2 and C-4, with reduced enrichment a t C-3 and C-1, and n e g l i g i b l e C-5 e n r i c h m e n t . T h i s p a t t e r n i s c o n s i s t e n t w i t h the resonance i n t e n s i t i e s , s u b j e c t to the usual c a u t i o n r e q u i r e d i n comparing r e s o n a n c e s w i t h d i f f e r e n t T^ and NOE values. Absolute enrichment values may be obtained from the H NMR s p e c t r u m o f the C e n r i c h e d g l u t a m a t e ( F i g . 4a) s u b j e c t t o c a r e not to o v e r p u l s e the s p e c t r u m w h i c h can l e a d to c o n s i d e r a b l e o v e r e s t i m a t i o n of the enrichment due to the s h o r t e r T

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