NMR and Macromolecules - American Chemical Society

7 New NMR Experiments in Liquids

Downloaded by CORNELL UNIV on August 3, 2016 | http://pubs.acs.org Publication Date: March 28, 1984 | doi: 10.1021/bk-1984-0247.ch007

GEORGE A. GRAY Varian Associates, Palo Alto, CA 94303

The recent past has been the most e x c i t i n g and productive i n the short h i s t o r y of NMR as an a n a l y t i c a l technique. The previous developments of homogeneous and higher f i e l d magnets, multinuclear c a p a b i l i t e s and the introduction of pulsed and fourier methods were spaced over a much longer time and new experiments took years to gain widespread attention and use. New experiments were t i e d to new hardware and generation of new approaches r e l i e d on performing instrument development. Because of the "home-built" and specialized nature of technique development, rapid deployment of new experiments was impossible. P a r t i c u l a r l y within the l a s t five years, new instruments have been introduced which have accelerated the development of new experiments, and at the same time made t h e i r widespread use p o s s i b l e . The r e s u l t has been an explosion i n the number of strategies and applicable approaches. This chapter is intended to give an overview of these new developments and to provide general c l a s s i f i c a t i o n s by which advantages and drawbacks may be understood. On modern instruments the instrument time which any one experiment a c t u a l l y consumes i s a c t u a l l y shrinking, while the time necessary to i n t e l l i g e n t l y design and carry out the spectroscopic approach i s growing. Fortunately, the information gleaned from the e f f o r t i s growing at a much faster r a t e . Instrumental Requirements. The topic "New Experiments" i s s p e c i f i c a l l y intended to cover new ways of generating spectroscopic information. That i s , the techniques are not s p e c i f i c a l l y oriented toward one 0097-6156/84/0247 0097S06.50/0 © 1984 American Chemical Society Randall; NMR and Macromolecules ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


98

N M R A N D MACROMOLECULES

nucleus or type o f sample, but r e l y on fundamentally new m e t h o d s o f p e r t u r b i n g t h e n u c l e a r s p i n s y s t e m . The r e s u l t i n g s i g n a l s a r e d e t e c t e d i n t h e t r a d i t i o n a l manner. H e n c e , t h e f o c u s i n "New E x p e r i ments" i s on the events p r i o r to s i g n a l a c q u i s i t i o n . There i s nothing unique about pulses o f r f i n t h i s classification. CW m e t h o d s c a n a n d h a v e b e e n i m p l e mented. However, the i n s t r u m e n t a l c o n v e n i e n c e and the a b i l i t y to "instantaneously" affect nuclear s p i n s g i v e s h i g h - p o w e r , p u l s e d NMR a d e c i d e d a d v a n tage. T h u s , t h e t e r m " p u l s e s e q u e n c e " h a s come t o symbolize the r e c i p e for "preparing" a nuclear spin system. V a r i a t i o n s o f p l a c e m e n t , t i m i n g and phases o f t h e s e p u l s e s , on one o r more n u c l e i , p r o v i d e the r i c h n e s s and d i v e r s i t y upon w h i c h the r a p i d d e v e l o p ments o f the l a s t f i v e years rest. Spectral

Editing

Using

J-Modulated Spin-Echos

The f a v o r a b l e p r o p e r t i e s o f 1 8 0 ° r e f o c u s s i n g p u l s e s have been e x p l o i t e d i n two m a i n e f f o r t s ; o b t a i n i n g more s p e c t r a l i n f o r m a t i o n b y c a u s i n g s p e c t r a to depend on J and the number o f c o u p l e d n u c l e i , and secondly, discussed later, i n polarization transfer methods. Of c o u r s e , t h e s i m p l e s t way t o make s p e c t r a depend on J , e i t h e r homonuclear o r h e t e r o n u c l e a r , is t o r u n a c o u p l e d s p e c t r u m . The l o w s e n s i t i v i t y o f l ^ C , along w i t h the severe o v e r l a p i n a coupled s p e c t r u m , make t h i s a p p r o a c h n o t v e r y u n i v e r s a l . F r e q u e n t l y , an i n t e r m e d i a t e l e v e l o f i n f o r m a t i o n i s required, e.g., j u s t a k n o w l e d g e o f the number o f attached protons for the v a r i o u s carbons i n a NMR s p e c t r u m . F o r m a n y y e a r s t h e m e t h o d o f c h o i c e was t h e s i n g l e - f r e q u e n c y o f f - r e s o n a n c e d e c o u p l i n g ( S F O R D ) t e c h n i q u e (.1) w h i c h y i e l d s p a r t i a l l y c o l lapsed m u l t i p l e t s i n the X spectrum for X H spin systems. The m u l t i p l e t s a r e b r o a d due t o u n c o l l a p s e d l o n g - r a n g e c o u p l i n g s and are d i f f i c u l t to i n t e r p r e t i n complex m o l e c u l e s . The d e s i r a b l e features o f broadband-decoupled spectra are compell i n g , p a r t i c u l a r l y f o r small samples o r congested spectra. n

1

3

Fortunately, C s p i n echo p u l s e sequences have been developed (2-17) which a l l o w broadbandd e c o u p l i n g and c a r b o n m u l t i p l i c i t y s e l e c t i o n (the number o f a t t a c h e d p r o t o n s ) . Patterned after an experiment introduced for studying proton-proton

Randall; NMR and Macromolecules ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

7. G R A Y

New NMR Experiments in Liquids

c o u p l i n g s ( 1 J 3 ) , the c e n t r a l theme i s the amplitude and phase m o d u l a t i o n o f , f o r example signals, due t o h e t e r o n u c l e a r s p i n c o u p l i n g . This modulation describes the a c t i o n o f magnetization generated by a pulse under the c o n d i t i o n o f c o u p l i n g to another spin — therefore, the d e c o u p l e r must be t u r n e d o f f f o r some p e r i o d o f t i m e . The e f f e c t may be generated i n e i t h e r o f two ways, gated (inter rupted) decoupling or by applying simultaneous pro t o n and C 1 8 0 ° p u l s e s . The former p u l s e sequence i s noted i n Figure 1 along w i t h the effects o f v a r i a t i o n o f Τ w i t h i n the pulse sequence. Three useful c h a r a c t e r i s t i c s o f t h i s approach are obvious: (1) q u a t e r n a r y c a r b o n s a r e u n m o d u l a t e d , (2) a t = 1/2J a l l p r o t o n a t e d c a r b o n s a r e n u l l e d , a n d ( 3 ) a t t? = l / j q u a t e r n a r i e s and methylene carbons are at oppo s i t e phase from m e t h y l and m e t h i n e carbons.


1

3

The i n v e s t i g a t o r t h u s h a s t h e o p t i o n o f s e a r c h i n g f o r q u a t e r n a r i e s w h i c h might be masked by p r o tonated carbons by s e t t i n g = 1/2J; o r , i t might be more d e s i r a b l e t o s o r t b y CH/CH3 and CH2/C. It is a l s o p o s s i b l e t o combine n o r m a l s p i n - e c h o and modu lated spin-echo spectra to a r r i v e at edited subspec t r a c o n t a i n i n g o n l y carbons o f one type (6,14-17). P a t t and S h o o l e r y (8) have a d d r e s s e d the p r o b lem o f o p t i m i z i n g s e n s i t i v i t y i n t h i s experiment i n t h e i r APT ( A t t a c h e d P r o t o n T e s t ) e x p e r i m e n t . The normal use o f a 9 0 ° p u l s e t o b e g i n the sequence has t h e e f f e c t o f s a t u r a t i n g l o n g e r Τχ n u c l e i a n d t h e r e fore requires longer equilibrium delays. In normal e x p e r i m e n t s i t i s customary t o use an intermediate p u l s e angle set r e l a t i v e t o the expected Τ χ ' s and r e p e t i t i o n rate o f the experiment. By i n c o r p o r a t i n g a second 1 8 0 ° pulse j u s t p r i o r to a c q u i s i t i o n they show t h a t z - m a g n e t i z a t i o n l e f t a f t e r an initial s u b - 9 0 ° pulse i s restored to the z - a x i s by the last 180° pulse, thereby allowing faster repetition of the experiment. An example o f t h e use o f t h e APT i s g i v e n i n F i g u r e 1 f o r an e t h y l e n e - 1 - h e x e n e copolymer. I n v e r t e d s i g n a l s a r i s e f r o m C H a n d CH3 carbons. C o n t r a s t t h i s d i r e c t d e t e r m i n a t i o n t o the more p o o r l y - r e s o l v e d and l o w e r s e n s i t i v i t y o f f - r e s o n a n c e data also given. The d e l a y e d technique can be

detection aspects used to e l i m i n a t e

o f the spin-echo protonated


100

NMR A N D MACROMOLECULES

Ethylene-1 -Hexene Copolymer


(97%)

(3%)

15% in trichlorobenzene/C6D6 10mm/130C/XL-400

OFF-RESONANCE SPECTRUM

13C Chemical Shift (ppm)

F i g u r e 1. 1 0 0 MHz C spectra of ethylene-1hexene. The APT spectrum was o b t a i n e d w i t h a t a u o f 7 m i l l i s e c o n d s , i n v e r t i n g t h e methylene carbons. F i g u r e courtesy of V a r i a n A s s o c i a t e s . 1 3


7.

GRAY

New NMR

carbons from a C spectrum. However, no J m o d u l a t i o n p e r i o d need be u s e d . Rather, the proton d e c o u p l e r i s p l a c e d o f f - r e s o n a n c e a n d i s e i t h e r CW or broadband-modulated. The d i s t a n c e o f f - r e s o n a n c e must be enough t o s e v e r e l y b r o a d e n t h e r e s o n a n c e s o f protonated carbons, without s i g n i f i c a n t l y affecting t h e l i n e w i d t h ( o r T2) of nonprotonated carbons. By d e l a y i n g a c q u i s i t i o n f o r a s h o r t p e r i o d f o l l o w i n g an i n i t i a l 9 0 ° p u l s e the signals for protonated car bons d e c a y r a p i d l y and a r e n o t d e t e c t e d . The q u a t e r n a r y c a r b o n s a r e d e t e c t e d e a s i l y and p h a s i n g t h e s p e c t r u m i s made s i m p l e r b y i n s e r t i n g a r e f o c u s s i n g 1 8 0 ° p u l s e midway b e t w e e n t h e 9 0 ° p u l s e and a c q u i s i t i o n . C o o k s o n and S m i t h (14) h a v e applied t h i s technique to petroleum mixtures, c l e a r l y d e t e c t i n g the quaternary aromatic c a r b o n s . E x t e n s i o n o f t h i s method t o n o n - p r o t o n a t e d c a r b o n s i n p o l y m e r s i s d i r e c t and s t r a i g h t f o r w a r d , allowing d i r e c t d e t e c t i o n o f branching s i t e s , f o r example, which are hidden underneath a large protonated c a r bon e n v e l o p e . 1


Experiments in Liquids J

Beloeil, et. a l . ( 1 2 ) h a v e i n t r o d u c e d a new type o f spin-echo sequence which produces e i t h e r C/CH2 o r CH/CH3 C subspectra. It i n v o l v e s an i n i t i a l 4 5 ° p u l s e f o l l o w e d b y f - 1 8 0 ° - T p e r i o d and a f i n a l m o n i t o r i n g 4 5 ° p u l s e e i t h e r i n phase o r 1 8 0 ° out o f phase w i t h r e s p e c t to the o r i g i n a l 4 5 ° p u l s e . The p r o t o n d e c o u p l e r i s g a t e d o f f d u r i n g t h e s e c o n d *£(= l / j ) p e r i o d . 1

Polarization

3

Transfer

Methods

Those f a m i l i a r w i t h the r o u t i n e a c q u i s i t i o n o f C NMR s p e c t r a a r e a w a r e o f t h e c o n s e q u e n c e s o f t h e nuclear Overhauser e f f e c t (NOE). Saturation of pro tons has the e f f e c t o f i n c r e a s i n g the net C mag n e t i z a t i o n o f those carbons r e l a x e d by the protons o f up t o a f a c t o r o f t h r e e t i m e s t h e e q u i l i b r i u m magnetization. Most a n a l y t i c a l o r s u r v e y C spec t r a are obtained with continuous broadband proton d e c o u p l i n g and any r e s u l t a n t NOE. Characteristics o f t h i s mode o f o p e r a t i o n a r e , (1) t h e p o s s i b i l i t y o f v a r i a b l e NOE, (2) r e p e t i t i o n r a t e g o v e r n e d b y ^- C Τχ a n d (3) b o t h p r o t o n a t e d a n d n o n - p r o t o n a t e d c a r bons are d e t e c t e d . T h e f i r s t a s p e c t makes q u a n t i t a tion difficult. The s e c o n d a f f e c t s n e t sensitivity, and the t h i r d h a s t h e p r o s p e c t o f h a v i n g u n d e s i r a b l e signals in certain situations. 1

3

1 3

1 3

3


102

NMR

A N D MACROMOLECULES

S i n c e q u a n t i t a t i o n i s c r u c i a l i n many polymer a n a l y s e s , i t i s i m p o r t a n t t o o b t a i n d a t a w i t h Τχ and NOE i n mind. H i g h l y f l e x i b l e p o l y m e r s u n d e r g o i n g r a p i d segmental m o t i o n t y p i c a l l y g i v e narrow C l i n e s . O f t e n t h e s e c a r b o n s c a n have Τχ's o f s e v e r a l seconds and f u l l o r n e a r l y f u l l NOE. O t h e r , more r i g i d p o l y m e r s may e x h i b i t b r o a d l i n e s and l i t t l e NOE. I n t h e case o f e t h y l e n e - l - h e x e n e copolymer t h e r e i s c o n s i d e r a b l e NOE f o r t h e 1-hexene p o r t i o n ( F i g u r e 2 ) . R e l a t i v e peak a r e a s c a n produce good c o n c e n t r a t i o n s o n l y i f Τχ and NOE a r e p r o p e r l y con sidered . Downloaded by CORNELL UNIV on August 3, 2016 | http://pubs.acs.org Publication Date: March 28, 1984 | doi: 10.1021/bk-1984-0247.ch007

1 3

A fundamentally d i f f e r e n t approach t o s i g n a l e x c i t a t i o n i s present i n p o l a r i z a t i o n t r a n s f e r methods. These r e l y on t h e e x i s t e n c e o f a r e s o l v a b l e J c o u p l i n g between two n u c l e i , one o f w h i c h (normally t h e proton) serves as a p o l a r i z a t i o n s o u r c e f o r t h e o t h e r . The e a r l i e s t o f t h e s e type o f e x p e r i m e n t s were t h e SPI ( S e l e c t i v e P o p u l a t i o n I n v e r s i o n ) type ( 1 9 ) i n w h i c h low-power s e l e c t i v e pulses are applied to a s p e c i f i c X - s a t e l l i t e i n the p r o t o n spectrum f o r an X-H system. The r e s u l t a n t p o p u l a t i o n i n v e r s i o n p r o d u c e s an enhanced m u l t i p l e t i n t h e X spectrum i f d e t e c t i o n f o l l o w s t h e i n v e r s i o n . A b a s i c improvement w h i c h removes t h e need for s e l e c t i v e p o s i t i o n i n g o f t h e proton frequency was t h e i n t r o d u c t i o n o f t h e INEPT ( I n s e n s i t i v e Nucleus E x c i t a t i o n by P o l a r i z a t i o n Transfer) t e c h n i q u e b y M o r r i s and Freeman (20 ) . T h i s t e c h n i q u e uses s t r o n g n o n - s e l e c t i v e p u l s e s and g i v e s g e n e r a l s e n s i t i v i t y enhancement. The p u l s e sequence h a s a b a s i c p o l a r i z a t i o n t r a n s f e r p o r t i o n ( F i g u r e 3) w h i c h p r o d u c e s a n e t i n v e r s i o n o f one o f t h e p r o t o n s p i n s t a t e s . F o l l o w ing an X n u c l e u s 90° p u l s e t h e r e e x i s t s enhanced m a g n e t i z a t i o n i n t h e X m u l t i p l e t . The s i g n a l enhancement i s p r o p o r t i o n a l t o t h e r a t i o o f t h e magn e t o g y r i c r a t i o s o f t h e two n u c l e i i n v o l v e d , a f a c tor o f 4 f o r C and 10 f o r N f o r X- H e x p e r i ments. The r e p e t i t i o n r a t e o f t h e e x p e r i m e n t i s d i c t a t e d by t h e Τχ o f t h e p o l a r i z a t i o n s o u r c e nucleus. S i n c e t h i s i s t y p i c a l l y t h e p r o t o n , more s i g n a l p e r u n i t t i m e o f t e n c a n be o b t a i n e d t h a n b y NOE. S i n c e t h e r e l a x a t i o n mechanism f o r t h e X n u c l e u s i s n o t i n v o l v e d , v a r i a b l e o r n e g a t i v e NOE i s n o t a p r o b l e m , as c a n be t h e case f o r ^ N and 2 9 g i One drawback i s t h e e f f e c t o f s h o r t T 2 ' s on t h e sen s i t i v i t y improvement. S i n c e t h e sequence r e q u i r e s 1

3

1 5

1

1

β


7.

GRAY


Ethylene-1-Hexene Copolymer


15%in TCB/C6D6

ct£

10mm/130°C/XL-400 90

sec

delay

w/o NOE

1

3

F i g u r e 2. Comparison o f C s p e c t r a w i t h and w i t h o u t n u c l e a r o v e r h a u s e r enhancement. F i g u r e courtesy of Varian Associates.


103


104

NMR AND MACROMOLECULES

F i g u r e 3.

Polarization transfer

excitation

INEPT. F i g u r e c o u r t e s y o f V a r i a n A s s o c i a t e s .


using

7. G R A Y


delays on the order o f l / j before d e t e c t i o n , (X-H c o u p l e d systems w i t h J o f 10-100 Hz r e q u i r e d e l a y s o f f r o m 1 0 0 - 1 0 ms) s h o r t p r o t o n Τ 2 s o r X n u c l e u s T2's i n decoupled p o l a r i z a t i o n transfer experiments can r a d i c a l l y lower expected g a i n s . For example, i f t h e p r o t o n s h a v e a Τ 2 o f 3 0 ms ( l i n e w i d t h i n t h e p r o t o n s p e c t r a o f ^10 H z ) , 2/3 o f t h e p r o t o n magnet i z a t i o n w i l l d e c a y away i n 30 ms. T h i s would be the same t i m e n e c e s s a r y t o a t t a i n p o l a r i z a t i o n t r a n s f e r f o r a s y s t e m h a v i n g an X - H J c o u p l i n g o f 16 H z . For t y p i c a l one-bond c o u p l i n g s o f 40-250 Hz f o r a n d C , t h i s l o s s i s l e s s important because o f the l / J delay dependence. A l t h o u g h NOE f a l l s o f f a s field s t r e n g t h and m o l e c u l a r s i z e i n c r e a s e s , i t s t i l l may have a net advantage for macromolecules because o f 1

1


1 3

W h i l e t h e f i r s t r e p o r t e d INEPT e x p e r i m e n t d w e l t on a g e n e r a t i o n o f enhanced coupled s p e c t r a , later reports (21-23) extended the b a s i c sequence to allow broadband decoupling during d e t e c t i o n , realiz ing the g o a l o f g e n e r a l s e n s i t i v i t y enhancement i n a more w i d e l y u s e f u l form. As a s i d e b e n e f i t , through proper selection of a refocussing period, spectral editing is possible. No q u a t e r n a r y c a r b o n s a r e u s u a l l y observed since delays are t y p i c a l l y set for J's appropriate for protonated X nuclei. XH o n l y s p e c t r a c a n be o b t a i n e d e a s i l y , as w e l l as s p e c t r a i n w h i c h XH2 i s i n v e r t e d w i t h r e s p e c t t o X H a n d XH3. The c o m b i n a t i o n o f t h e s e two s p e c t r a t h e n a l l o w s d i r e c t i d e n t i f i c a t i o n o f X H , X H 2 a n d XH3 s i g n a l s i n even the most complex m o l e c u l e s i n a s i m i l a r manner as i n the J modulated s p i n echo e x p e r i m e n t s described above. An a n a l o g o u s c a s e i s p r e s e n t i n t h e l^C-^H INEPT e x p e r i m e n t (24 ) . Here, only deuterated car bons are o b s e r v e d . While the magnetogyric r a t i o f a c t o r s h o u l d g i v e o n l y a t h e o r e t i c a l 3/5 enhance ment, the s h o r t Τχ a l l o w s v e r y r a p i d a c c u m u l a tion. The t e c h n i q u e s h o u l d be u s e f u l f o r d e t e r m i n a tion of deuterated X nuclei spectra without i n t e r f e r e n c e s from p r o t o n a t e d X s i g n a l s . The s e n s i t i v i t y g a i n s h o u l d be v e r y d r a m a t i c f o r f u l l y deu t e r a t e d X n u c l e i where normal X nucleus Τ χ ' s can be extremely long because o f i n e f f i c i e n t d i p o l a r r e l a x a t i o n from H or other mechanisms. This experiment offers the c a p a b i l i t y o f d e t e c t i o n o f deuterated s i t e s at small concentration i n , for example, 2


106

NMR

AND

MACROMOLECULES


s t u d i e s o f t h e mechanism o f d e u t e r a t i o n ( o r r e a c t i o n u s i n g d e u t e r a t e d r e a c t a n t s , e.g., o x i d a n t s ) o f macromolecules. W h i l e p o s s e s s i n g s e v e r a l a d v a n t a g e s , INEPT has p r o p e r t i e s w h i c h a r e u n d e s i r a b l e : (1) c o u p l e d spec t r a have, a t t i m e s , m i s s i n g l i n e s and a n t i p h a s e com p o n e n t s ; (2) t h e r e i s a f a i r l y s t r o n g J dependence i n c a s e s where s i g n a l s s h o u l d n u l l , l e a d i n g t o r e s i d u a l unwanted s i g n a l s ; and, (3) r e f o c u s s e d INEPT d e l a y p e r i o d s a r e v a r i a b l e , depending on m u l t i p l i c i t y desired, leading to p o s s i b l e v a r i a t i o n i n i n t e n s i t y due t o v a r i a t i o n s i n T2 among the r e s o nances. D o d d r e l l , Pegg and B e n d a l l ( 2 5 , 2 6 ) have d e v e l o p e d a D i s t o r t i o n l e s s Enhancement by P o l a r i z a t i o n T r a n s f e r (DEPT) p u l s e sequence ( F i g u r e 4) w h i c h a d d r e s s e s t h e s e problems t o a l a r g e d e g r e e . Coupled X n u c l e i DEPT s p e c t r a have t h e normal b i n o m i a l d i s t r i b u t i o n o f i n t e n s i t i e s , t h e J dependence f o r XH s e l e c t i v i t y i s b e t t e r than f o r INEPT, and the m u l t i p l i c i t y s e l e c t i o n r e l i e s on a v a r i a b l e p r o t o n f l i p angle r a t h e r than v a r i a b l e d e l a y s , thus f a c t o r i n g out T2 dependence. By c h o o s i n g θ = 45°, 90°, and 135° i n s e p a r a t e s p e c t r a , s p e c t r a l e d i t i n g i s p o s s i b l e through proper combinations o f these s p e c t r a to g i v e XH, X H and XH3 s u b s p e c t r a . DEPT i s more sen s i t i v e o v e r a l l t o s p i n r e l a x a t i o n d u r i n g the p u l s e t r a i n s i n c e the r e l e v a n t d e l a y i s o f magnitude 3/2J w h i l e c o r r e s p o n d i n g p e r i o d s i n INEPT r a n g e s from l / J t o 5/4J t y p i c a l l y . 2

Two-Dimensional

NMR

P r o b a b l y t h e g r e a t e s t r e c e n t change i n the p r a c t i c e o f NMR has been the e x p l o s i v e growth i n t e c h n i q u e s and a p p l i c a t i o n s o f 2D NMR. 2D NMR i s an e x t e n s i o n o f o r d i n a r y NMR. The b a s i c p r i n c i p l e was i n v e n t e d by J e n n e r (27) and cov e r s e s s e n t i a l l y a l l 2D e x p e r i m e n t s i n NMR, as w e l l as o t h e r a r e a s . The g e n e r a l 2D NMR e x p e r i m e n t i s c h a r a c t e r i z e d by up t o f o u r t i m e p e r i o d s : Preparation...Evolution(t^)... Mixing(t )...Detection(t2) m

P r e p a r a t i o n t i m e i s n e c e s s a r y t o b r i n g t h e system t o a known s t a t e , e.g., e q u i l i b r i u m m a g n e t i z a t i o n , and i s u s u a l l y a f i x e d d e l a y t i m e . At t h e b e g i n n i n g o f t h e E v o l u t i o n p e r i o d , t h e s p i n s a r e p e r t u r b e d and


GRAY


DEPT Pulse Sequence ο

·

90 equil.

1/2J

Ο

1/2J ο

90


·

180

1/2J

180

J U U

Ί

Γ

10

D

F i g u r e 4. The e d i t e d s p e c t r a a r e t h e r e s u l t s p e c i f i c combinations o f d i f f e r e n t spectra o b t a i n e d f o r θ = 4 5 ° , 90° a n d 135© u s i n g a solution of cholesteryl acetate i n CDCI3. Figure courtesy of Varian Associates.


108

NMR

A N D

M A C R O M O L E C U L E S


H O M O N U C L E A R C H E M I C A L S H I F T 2D

hDD

HDD

I EDO

1DDD

BDD

bDD

CORRELATION

HDD

NMR

DDD

F i g u r e 5. The d a t a f r o m t h e 2D e x p e r i m e n t i s d i s p l a y e d i n contours o f i n t e n s i t y , each a x i s represents a proton s h i f t axis. The o f f - d i a g o n a l i n t e n s i t i e s show h o m o n u c l e a r c o u p l i n g s . For i n s t a n c e , t h e p r o t o n a t 950 Hz ( a s i n d i c a t e d b y an a r r o w ) h a s o f f - d i a g o n a l i n t e n s i t y s h o w i n g c o u p l i n g s to the four other i n d i c a t e d protons. Figure

courtesy

of Varian

Associates.


•

7.

GRAY


109


a l l o w e d t o e v o l v e w i t h i n a s p e c i f i c environment t h a t may be d i f f e r e n t from any environment i n t h e o t h e r time p e r i o d s . This E v o l u t i o n time i s r e g u l a r l y v a r i e d t h r o u g h a range o f v a l u e s from z e r o t o some maximum, each r e s u l t i n g i n a s e p a r a t e FID r e c o r d e d f o r a f i x e d time d u r i n g the d e t e c t i o n p e r i o d (Cer t a i n 2D e x p e r i m e n t s use a m i x i n g , o r s p i n exchange period). The r e s u l t i s a c o l l e c t i o n o f FID's, t h e number o f w h i c h e q u a l s t h e number o f d i f f e r e n t v a l u e s o f t h e E v o l u t i o n t i m e p e r i o d . A l l FID's a r e t h e n t r a n s f o r m e d i n t h e u s u a l way, r e s u l t i n g i n a c o l l e c t i o n o f spectra (the f a m i l i a r inversionr e c o v e r y Τχ e x p e r i m e n t i s an example o f t h i s s t a g e o f t h e p r o c e s s ) . A new c o l l e c t i o n o f FID's i s assembled b y t a k i n g d a t a p o i n t s from each spectrum at given frequency v a l u e s . For example, v a l u e s o f s p e c t r a l i n t e n s i t y i n each spectrum found a t a f r e q u e n c y f a r e t a k e n and arranged i n a data t a b l e . This i s repeated f o r e v e r y f v a l u e i n t h e spectrum, r e s u l t i n g i n Ν FID's when Ν i s t h e number o f p o i n t s i n t h e o r i g i n a l spec trum. These Ν FID's a r e t h e n f o u r i e r t r a n s f o r m e d , r e s u l t i n g i n Ν s p e c t r a . These Ν s p e c t r a , p r e s e n t e d on a t w o - d i m e n s i o n a l p l o t , a r e now c h a r a c t e r i z e d b y two f r e q u e n c y a x e s . One f r e q u e n c y (F2) i s always t h a t o f t h e normal o b s e r v e spectrum s i n c e i t r e s u l t s from FT o f normal FID's. The s i g n i f i c a n c e o f t h e remaining ( F l ) frequency i s determined by t h e p u l s e sequence used. There have been m a j o r advances i n t h e l a s t few y e a r s b o t h i n t h e number and v a r i e t y o f t h e p u l s e sequences used t o p e r t u r b t h e s p i n s , and i n t h e com p u t i n g power and d a t a p r o c e s s i n g t e c h n i q u e s employed. A l l two d i m e n s i o n a l e x p e r i m e n t s use e s s e n t i a l l y t h e same d a t a r e d u c t i o n p r o c e s s . S e v e r a l y e a r s ago t h e d a t a a c q u i s i t i o n phase o f 2D e x p e r i m e n t s was u s u a l l y the s h o r t e s t , w i t h double t r a n s f o r m a t i o n r e q u i r i n g up t o s e v e r a l h o u r s f o l l o w e d by, a g a i n , comparable p l o t t i n g t i m e . O f f - l i n e d a t a p r o c e s s i n g was o f t e n used f o r f a s t e r p r o c e s s i n g , b u t t h i s s e v e r e l y l i m i t e d any w i d e s p r e a d use o f 2D methods. 2D p r o c e s s i n g c a p a b i l i t i e s became g e n e r a l l y a v a i l a b l e on commercial s p e c t r o m e t e r s a f t e r 1979 and w i t h t h e i n t r o d u c t i o n o f f l e x i b l e p u l s e programmers t h e


110



u s e o f 2D m e t h o d s h a s g r o w n q u i c k l y . Very recently, new c o m p u t e r h a r d w a r e , i n c l u d i n g A r r a y P r o c e s s o r s , h a s r e v o l u t i o n i z e d 2D d a t a p r o c e s s i n g , a l l o w i n g c o m p l e t e d a t a r e d u c t i o n , i n c l u d i n g f u l l 2D d i s p l a y i n l e s s t h a n 30 s e c o n d s ( f o r a t y p i c a l 512 χ 512 matrix). This speed, coupled w i t h responsive c o l o r graphics, has permitted d i r e c t , o n - l i n e data pro c e s s i n g and removed the f o r m i d a b l e t i m e - b a r r i e r to f u l l u s e o f 2D NMR s p e c t r o s c o p y . J - R e s o l v e d 2D N M R . T h e k e y t o t h e n a t u r e o f t h e F χ domain l i e s i n the environment i n which the spins p r e c e s s d u r i n g t h e e v o l u t i o n p e r i o d t]_. This e n v i r o n m e n t may i n c l u d e t h e i n t e r a c t i o n s o f s p i n coupling, the chemical s h i f t — which governs the r a t e o f p r e c e s s i o n ( r e l a t i v e to the t r a n s m i t t e r fre quency as zero) — and magnet i n h o m o g e n e i t y w h i c h causes i d e n t i c a l n u c l e i to have d i f f e r e n t precession rates. R e f o c u s s i n g p u l s e s c a n remove some, o r a l l , of these interactions. In p a r t i c u l a r , after a 9 0 ° p u l s e use o f a s t r o n g n o n - s e l e c t i v e 1 8 0 ° r e f o c u s s i n g pulse on the observe nucleus midway through the evo l u t i o n period eliminates the effect o f chemical s h i f t d i f f e r e n c e s f o r a l l observe s p i n s as detected i n t-2 f o r a n y t ^ . H e n c e , t h e r e s u l t a n t 2D s p e c t r u m might be thought t o be r a t h e r s i m p l e , as i t is indeed for non-coupled n u c l e i - - singlets are cen tered at coordinates ( F j = 0 , F2 = v g ) . However, t h e m u t u a l J c o u p l i n g o f s p i n s d u r i n g t^ does affect t h e p h a s e o f t h e s i g n a l w h e n d e t e c t e d i n t2# and hence J i n f o r m a t i o n i s encoded i n the detected sig nals . T h e h e t e r o n u c l e a r J - r e s o l v e d 2D e x p e r i m e n t p r o duces c o m p l e t e l y separate i n f o r m a t i o n i n both domains, X n u c l e u s c h e m i c a l s h i f t i n F2 (since b r o a d b a n d d e c o u p l i n g i n t2 removes t h e e f f e c t o f J c o u p l i n g ) and AX c o u p l i n g i n F j . This is particu l a r l y v a l u a b l e , for example, i n C - H cases since complete c o u p l i n g p a t t e r n s can be e x t r a c t e d for each C without overlap of adjacent patterns. Informa t i o n can be e x t r a c t e d e a s i l y , i n c o n t r a s t to d i r e c t o b s e r v a t i o n o f h i g h l y o v e r l a p p e d c o u p l e d ID s p e c t r a . 1

3

1

1 3

T h e h o m o n u c l e a r J - r e s o l v e d 2D s p e c t r u m i s dif f e r e n t i n t h a t AX c o u p l i n g i s a c t i v e i n b o t h t^ and 2, and thus i s r e f l e c t e d i n the e x i s t e n c e o f s p i n multiplets i n both dimensions. t


7.

GRAY

New NMR

Experiments in Liquids


E a c h s p i n p a t t e r n i s p r e s e n t a l t h o u g h no one s l i c e p e r p e n d i c u l a r to F2 c o n t a i n s a whole p a t t e r n . A c o m p u t e r p r o c e s s known a s " t i l t i n g " , "rotating", o r " s h e a r i n g " can be used to r e a l i g n the s p i n p a t terns ( 4 5 ° t i l t ) perpendicular to F 2 · This permits d i s p l a y a n d p l o t t i n g o f " s l i c e s " o f t h e 2D d a t a — a n F± s p e c t r u m a t some s p e c i f i e d F 2 v a l u e . These s l i c e s are the f u l l y coupled s p i n p a t t e r n s . The spin-echo nature o f the experiment produces narrow l i n e s , and t h u s v e r y h i g h l y r e s o l v e d m u l t i p l e t s . In highly congested, but f i r s t - o r d e r , proton spectra t h i s t e c h n i q u e c a n b e o f immense v a l u e . P o l a r i z a t i o n t r a n s f e r may b e u s e d i n h e t e r o n u c l e a r J - r e s o l v e d 2D NMR ( 2 8 ) f o r s i g n a l e n h a n c e m e n t , s h o r t e r e q u i l i b r a t i o n p e r i o d s and e l i m i n a t i o n o f n o n - p r o t o n a t e d r e s o n a n c e s - - a l l t h e same f e a t u r e s a s i n INEPT o r D E P T . In t h i s e x p e r i m e n t t h e refocussing period following polarization transfer becomes the e v o l u t i o n t i m e , r a t h e r t h a n the 1/4J, 1/2J, a n d 3/4J v a l u e s n o r m a l l y u s e d f o r r e f o c u s s e d INEPT o r v a r i a b l e f l i p a n g l e # f o r D E P T . The p o l a r i z a t i o n t r a n s f e r p a r t o f the sequence i n c l u d e s a 1/2J p e r i o d , a s u s u a l , a n d t h u s t h e 2D s e q u e n c e c a n be t a i l o r e d to s p e c i f i c types (or J ' s ) o f XH c o u p l e d p a i r s , e . g . , c e r t a i n long-range couplings for q u a t e r n a r y c a r b o n s whose n o r m a l C Τ χ ' s may b e t o o l o n g t o p e r m i t o r d i n a r y h e t e r o n u c l e a r 2D J e x p e r i ments . 1

3

C h e m i c a l S h i f t C o r r e l a t i o n 2D NMR. Probably the m o s t w i d e l y u s e d 2D NMR e x p e r i m e n t s a r e t h o s e w h i c h r e l a t e chemical s h i f t s of d i f f e r e n t n u c l e i . That i s , F]_ a n d F 2 r e p r e s e n t c h e m i c a l s h i f t a x e s a n d t h e c l a s s i n c l u d e s b o t h h o m o n u c l e a r and h e t e r o n u c l e a r categories. A g a i n , the environment d u r i n g the e v o l u t i o n t i m e d i c t a t e s t h e i n t e r p r e t a t i o n o f t h e new information. The s i m p l e s t t o c o n s i d e r i s t h e homonuclear chemical s h i f t c o r r e l a t i o n (27,29-31), a l s o known i n o n e f o r m a s COSY ( c o r r e l a t i o n s p e c troscopy) . The most w i d e l y u s e d p u l s e s e q u e n c e i s r a t h e r s i m p l e , c o n s i s t i n g of 9 0 ° - t x - 9 0 ° followed by acquisition. T h e r e s u l t i n g p e a k s i n t h e 2D p l o t f a l l i n s e v e r a l c a t e g o r i e s : a x i a l , d i a g o n a l and off-diagonal. T h e a x i a l p e a k s l i e a l o n g F^ =0 a n d r e s u l t from l o n g i t u d i n a l m a g n e t i z a t i o n sampled by the l a s t p u l s e . The d i a g o n a l p e a k s a r e due t o m a g n e t i z a t i o n s w h i c h r e m a i n a s s o c i a t e d w i t h t h e same s p i n s b e f o r e and a f t e r t h e s e c o n d 9 0 ° p u l s e , t h a t i s t h e m a g n e t i z a t i o n h a s t h e same f r e q u e n c y d u r i n g t^



NMR A N D MACROMOLECULES

F i g u r e 6. E a c h p e a k i n t h e 2D s p e c t r u m a r i s e s from a C - H bond. Extrapolation to the relevant axes g i v e the c o r r e s p o n d i n g chemical s h i f t s . The d a t a was o b t a i n e d o n 90 mg i n 1 0 . 5 h o u r s u s i n g a 1 0 mm p r o b e a t 75 M H z ( X L - 3 0 0 ) . F i g u r e c o u r t e s y Varian A s s o c i a t e s .


of


7. G R A Y

New NMR


and t 2 · The o f f - d i a g o n a l p e a k s c o n f i r m t h e f a c t t h a t m a g n e t i z a t i o n p r e s e n t a t o n e f r e q u e n c y i n t^ i s t r a n s f e r r e d to another frequency a f t e r the second 9 0 ° "mixing" pulse. This i s o n l y p o s s i b l e i f the two n u c l e i a t t h e two f r e q u e n c i e s s h a r e t h e same spin system, t h a t i s , they are J - c o u p l e d . This last f e a t u r e forms the b a s i s o f the w i d e s p r e a d use o f h o m o n u c l e a r s h i f t c o r r e l a t e d 2D NMR s i n c e i t gives equivalent information to spin-decoupling. The s p r e a d o f i n f o r m a t i o n i n two d i m e n s i o n s a c t u a l l y makes t h e i n t e r p r e t a t i o n e a s i e r . P r o p e r p h a s e c y c l i n g (31) c a n remove t h e a x i a l p e a k s , c o n s i d e r a b l y s i m p l i f y i n g the experiment. V a r i a t i o n s on the b a s i c experiment i n c l u d e the 9 0 ° - t x - 4 5 ° - a c q u i r e e x p e r i m e n t (29 ) . T h i s v e r s i o n n a r r o w s t h e p a t t e r n s on the d i a g o n a l p e r m i t t i n g a n a l y s i s o f c o r r e l a t i o n s o f n u c l e i c l o s e l y spaced i n chemical s h i f t . T h e s e c o n d m a j o r f o r m o f s h i f t c o r r e l a t i o n 2D NMR i s h e t e r o n u c l e a r 2D s h i f t c o r r e l a t i o n . The e x p e r i m e n t as p r o p o s e d b y M a u d s l e y and E r n s t (32) can be v i s u a l i z e d as g e n e r a t i n g p r o t o n m a g n e t i z a t i o n , f o r e x a m p l e , b y a 9 0 ° p u l s e and l e t t i n g t h e m a g n e t i z a t i o n p r e c e s s f o r a time t^. The e x t e n t o f p r e c e s s i o n ( i n the r o t a t i n g frame) i s p r o p o r t i o n a l t o the d i s t a n c e o f f - r e s o n a n c e from the p r o t o n t r a n s m i t t e r f r e q u e n c y and t h e r e f o r e i t s phase i s proton s h i f t dependent. Simultaneous 9 0 ° p u l s e s on t h e p r o t o n and X o b s e r v e n u c l e u s t r a n s f e r m a g n e t i z a t i o n t o X — j u s t a s i n t h e INEPT e x p e r i m e n t . The phase o f the X n u c l e u s m a g n e t i z a t i o n i s hence coded with the chemical s h i f t o f the attached p r o t o n . The e x p e r i m e n t i s c a r r i e d o u t f o r t h e f u l l r a n g e o f t^ v a l u e s t o p r o d u c e t h e 2D s p e c t r u m . Since the X n u c l e u s s h i f t i s p r e s e n t i n F2# t h e XH p a i r h a s b o t h chemical s h i f t s i d e n t i f i e d by e x t r a p o l a t i o n to the i n d i v i d u a l a x e s f r o m t h e XH p e a k i n t h e 2D d a t a . Broader u t i l i z a t i o n o f heteronuclear chemical s h i f t 2D h a s r e a l l y j u s t b e g u n . Amman e t . a l . , (33) h a v e i l l u s t r a t e d how 2D m e t h o d s c a n b e u s e d t o a s s i g n p r o t o n s a n d c a r b o n s i n l u p a n e , a C30 triterpene c o n t a i n i n g o n l y c a r b o n and h y d r o g e n . Heteronuc l e a r J - r e s o l v e d 2D was u s e d t o a s s i g n t h e number o f p r o t o n s t o e a c h c a r b o n , and h e t e r o n u c l e a r c h e m i c a l s h i f t c o r r e l a t i o n allowed assignment o f the a s s o c i a t e d p r o t o n s h i f t s a n d many o f t h e h o m o n u c l e a r H-H coupling constants. I k u r a and H i k i c h i (34) u s e d t h e same t e c h n i q u e s on d - b i o t i n . A mixture of a l l y l n i c k e 1 c o m p l e x e s was a l s o s t u d i e d by Benn (3JO , and


113

114

NMR AND

MACROMOLECULES


M o r r i s and H a l l (35) have examined a s e r i e s o f c a r bohydrates. Larger molecules are, of course, feasi ble. Chan and M a r k l e y (36) have a s s i g n e d h i s t i d i n e , t y r o s i n e and p h e n y l a l a n i n e p r o t o n a t e d c a r b o n r e s o n a n c e s i n u n i f o r m l y e n r i c h e d (20%) o x i d i z e d ferrod i x i n b y h e t e r o n u c l e a r H / C 2D s h i f t c o r r e l a t i o n N M R . Polypeptide resonances have a l s o been assigned using C / H 2D NMR ( 3 7 , 3 8 ) . I n t h e s e s y s t e m s Η / Η h o m o n u c l e a r , H / C h e t e r o n u c l e a r a n d Ν / Η h e t e r o n u c l e a r 2D techniques have allowed d i r e c t c o n f i r m a t i o n o f a s s i g n m e n t s a n d c o n n e c t i v i t i e s , i n p a r t i c u l a r t h e NH n i t r o g e n s and p r o t o n s . Heteronuclear chemical shift correlation tech niques can be used to i n f e r s p i n - l a t t i c e r e l a x a t i o n times o f the protons attached to the observe nucleus (39.) · T h i s i s a c c o m p l i s h e d b y s a t u r a t i o n o f t h e p r o tons and observe n u c l e u s f o l l o w e d by a v a r i a b l e time t (saturation recovery) during which the observe nucleus i s c o n t i n u e d at s a t u r a t i o n (by repeated p u l s i n g ) and the a t t a c h e d p r o t o n r e m a g n e t i z e s . This p r o c e s s i s f o l l o w e d b y t h e n o r m a l H / X 2D s h i f t correlation experiment. T h e t - d e p e n d e n c e o f t h e 2D peak i n t e n s i t i e s i s then used to e x t r a c t ^H Τχ s by exponential analysis. This approach can be used to extract proton T i ' s i n polymers v i a observation of t h e i r attached ^ C ' s , obtaining motional data nor m a l l y i m p o s s i b l e t o o b t a i n b y p r o t o n NMR. 1

A l l o f the above h e t e r o n u c l e a r s h i f t c o r r e l a t i o n techniques produce spectra which have H-H spin m u l t i p l e t s i n the dimension. These can be i n v a l u a b l e i n c e r t a i n s i t u a t i o n s where the pattern i s o b s c u r e d i n t h e ID s p e c t r u m . At other times sen s i t i v i t y c o n s i d e r a t i o n s would argue for c o l l a p s i n g these m u l t i p l e t s , thereby gaining at least a factor o f two i n i n t e n s i t y . In other cases, h i g h l y con g e s t e d 2D s p e c t r a c o u l d h a v e o v e r l a p p e d H / C c o r r e l a tions. Bax (40) has d e v e l o p e d a method f o r c o l l a p s ing these m u l t i p l e t s which e s s e n t i a l l y replaces the s i n g l e X nucleus 180° pulse at the m i d - p o i n t o f the e v o l u t i o n time w i t h the element 90° (H)-l/2J180° (H)180° (X)-1/2J-90° (H). T h e r e s u l t a n t 2D s h i f t c o r r e l a t i o n spectra are characterized by s i n gle peaks for a X - H bond. S l i c e s i n F^ show p r o t o n s i n g l e t s at the appropriate chemical s h i f t . Of c o u r s e , a p r o j e c t i o n o n t o t h e F]_ a x i s w o u l d g i v e a proton "stick" spectrum. x

X

X

X



7.

GRAY

New NMR Experiments

115

in Liquids

M a g n e t i z a t i o n T r a n s f e r 2D N M R . O n e o f t h e m o r e e x c i t i n g a s p e c t s o f 2D NMR i s t h e c l a s s o f e x p e r i ments which can probe i n t r a - m o l e c u l a r interactions, p r o x i m i t y o f n u c l e i and c h e m i c a l exchange. As o p p o s e d t o s o m e o f t h e a b o v e 2D e x p e r i m e n t s where transverse (X,Y) m a g n e t i z a t i o n t r a n s f e r occurs, t h e r e are e x p e r i m e n t s w h e r e i n l o n g i t u d i n a l (Z) magnetization is transferred, i . e . , energy l e v e l popul a t i o n s change. The n u c l e a r O v e r h a u s e r e f f e c t is one example o f p o p u l a t i o n r e d i s t r i b u t i o n w i t h i n a s p i n system v i a an i n c o h e r e n t p r o c e s s . Other types i n c l u d e s a t u r a t i o n t r a n s f e r experiments where the mechanism i s c h e m i c a l exchange. O t h e r means o f s t u d y i n g c h e m i c a l exchange o r NOE h a v e i n c l u d e d l i n e s h a p e a n a l y s i s a t o r n e a r c o a l e s c e n c e and s e l e c t i v e s a t u r a t i o n o r i n v e r s i o n with subsequent following of propagation o f magnetiz a t i o n throughout the spin system. The f o r m e r technique can be v e r y d i f f i c u l t f o r broad coalescence peaks and r e q u i r e c e r t a i n t e m p e r a t u r e s t o be e s t a blished. The l a t t e r i s v e r y u s e f u l f o r a l i m i t e d number o f l i n e s t o be a f f e c t e d b u t g e t s t i m e c o n s u m i n g f o r more t h a n a few l i n e s and v e r y d i f f i cult for closely-spaced lines. T h e 2D m e t h o d r e q u i r e s no p a r t i c u l a r s p e c i a l c o n d i t i o n s and t h e r e fore has a g r e a t deal o f a t t r a c t i v e n e s s for studies o f c h e m i c a l and b i o c h e m i c a l d y n a m i c s . This class of e x p e r i m e n t s was p r o p o s e d b y J e n n e r e t . a l . (41,42) and e l a b o r a t e d on b y M a c u r a and E r n s t ( 4 3 ) . The b a s i c pulse sequence i s 9 0 ° - t - 9 0 ° - t ( M I ) C T - 9 0 . The second 9 0 ° p u l s e can be thought o f as r e s t o r i n g to the Z - a x i s transverse magnetizations created by the first 90° pulse. The component and d i r e c t i o n o f t h e resultant Z - a x i s magnetization i s a function o f the p r e c e s s i o n o f the spins during t^. Some c o m p o n e n t s w i l l b e p o s i t i v e , some n e g a t i v e a n d some z e r o . These components w i l l o s c i l l a t e as a f u n c t i o n o f t j . The r e s u l t a n t Z - m a g n e t i z a t i o n s w i l l be i n a n o n e q u i l i b r i u m s t a t e and s p i n - l a t t i c e r e l a x a t i o n processes w i l l work toward e q u i l i b r i u m . For the case of protons r e l a x a t i o n i s p r i m a r i l y d i p o l a r v i a other protons, this non-equilibrium magnetization w i l l then be r e d i s t r i b u t e d by mutual s p i n f l i p to other protons. The f i n a l 9 0 ° p u l s e m o n i t o r s the extent of magnetization transfer. T h e 2D e x p e r i m e n t samples a l l d e g r e e s o f m a g n e t i z a t i o n t r a n s f e r as i t increments t^. o

1



116


T h e 2D s h i f t c o r r e l a t i o n s p e c t r u m t h u s produced is c h a r a c t e r i z e d by the usual diagonal peaks coming f r o m m a g n e t i z a t i o n r e m a i n i n g a t t h e same frequency i n ti and t 2 · P h a s e - c y c l i n g can remove the axial p e a k s , j u s t as i n homonuclear s h i f t c o r r e l a t i o n 2D NMR. The o f f - d i a g o n a l p e a k s come f r o m magnetization which has been t r a n s f e r r e d i n a s p i n - l a t t i c e relaxat i o n sense from one t y p e o f s p i n t o a n o t h e r . This c o u l d be because o f t r u e c h e m i c a l exchange o c c u r r i n g d u r i n g the mix t i m e , o r i t c o u l d be s i m p l y from s p i n s c l o s e enough i n space to p r o v i d e mutual dipolar relaxation. T h i s sequence has been used to e s t a b l i s h NOE's i n p o l y p e p t i d e s and p r o t e i n s (45,46). It i s p a r t i c u l a r l y e f f e c t i v e i n macrom o l e c u l e s where t h e r e i s much s l o w e r m o l e c u l a r tumb l i n g and more f a v o r a b l e NOE s. V a r i a t i o n of t(MIX) allows probing of the rates o f magnetization t r a n s f e r and t h u s p r o x i m i t y o f p r o t o n s f o r NOE, o r chemical k i n e t i c s for true exchange (47). 1

Carbon-13 chemical exchange networks have been e x p l o r e d by Huang, Macura and E r n s t (48) u s i n g the above t e c h n i q u e s . Normal 9 0 - t - 9 0 ° - t T M I X ) - 9 0 sequences were used, i n the presence o f proton d e c o u p l i n g ( f o r NOE, s e n s i t i v i t y and simplicity). R e f o c u s s e d INEPT was a l s o used t o p r e p a r e the init i a l magnetization, i n place o f the f i r s t 9 0 ° pulse, to gain additional s e n s i t i v i t y . This extension sugg e s t s f u t u r e f l e x i b i l i t y and s e l e c t i v i t y s i n c e the INEPT p o r t i o n o f the sequence c a n be t a i l o r e d for specific J's ( l o n g - r a n g e and d i r e c t ) , t h u s a l l o w i n g v e r y p r e c i s e c o n t r o l over the s i t e from w h i c h magn e t i z a t i o n can evolve. Huang, e t . a l (48) applied these s t r a t e g i e s to the c l a s s i c exchange problems o f r i n g - p u c k e r i n g i n d e c a l i n , bond s h i f t i n b u l l v a l e n e and s o l v a t i o n s h e l l exchange i n aluminum complexes. The l o n g e r r e l a x a t i o n t i m e s o f c a r b o n - 1 3 c a n actua l l y be put to advantage i n these s t u d i e s s i n c e t h e y permit longer mix times for slower exchange processes. T h e o t h e r m a j o r a d v a n t a g e o f t h e 2D t e c h n i q u e i s t h a t i t can be performed on a v e r y s l o w l y e x c h a n g i n g system whose l i n e s a r e s t i l l narrow. o

o

1


7.

GRAY

New NMR


L i t e r a t u r e Cited 1.

2. 3. Downloaded by CORNELL UNIV on August 3, 2016 | http://pubs.acs.org Publication Date: March 28, 1984 | doi: 10.1021/bk-1984-0247.ch007

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

E r n s t , R . R . , J. Chem. Phys. 1966, 45, 3845; Reich, H.J.; J a n t e l a t , M . ; Meese, M . T . ; Weigert, F.J.; J . D . Roberts, J . Am. Chem. Soc. 1969, 91, 7445. Anet, F.A.L.; J a f f e r , N.; Strouse, J., 21st Experimental NMR Conference, 1980, Tallahassee, FL. Rabenstein, D . L . ; Nakashima, T . K . , A n a l . Chem. 1979, 51, 1465A. L e v i t t , M . H . ; Freeman, R . , J. Magn. Reson. 1980, 39, 533. LeCocq, D . ; Lallemand, J.-Υ., J. Chem. Soc. Chem. Commun. 1981, 150. Cookson, D.J.; Smith, B.E., Org. Magn. Reson. 1981, 16, 11. Brown, D.W.; Nakashima, T . K . ; Rabenstein, D . L , J. Magn. Reson. 1981, 45, 302 P a t t , S . L . ; Shoolery, J.N., J. Magn. Reson. 1982, 46, 535. Cookson, D.J.; Smith, B.E., Fuel 1983, 62, 34. P e i , Feng-Kui, Freeman, R . , J. Magn. Reson. 1982, 48, 318. Jakobsen, H.J.; Sorensen, O.W.; Brey, W.S.; Kanyha, P . , J. Magn. Reson. 1982, 48, 328. B e l o e i l , J.-C.; LeCocq, C . ; Lallemand, J.-Υ., Org. Magn. Reson. 1982, 19, 112. Nakashima, T . K . ; Rabenstein, D . L . , J. Magn. Reson. 1982, 47, 339. Cookson, D.J.; Smith, B.E., Fuel 1983, 62, 39. B e n d a l l , M . R . ; D o d d r e l l , D . M . ; Pegg, D . T . , J . Amer. Chem. Soc. 1981, 103, 4603. B e n d a l l , M . R . ; Pegg, D . T . ; D o d d r e l l , D . M . ; Johns, S . R . ; Willing, R . , J . C . S . Chem. Commun., 1982, 1138. B e n d a l l , M . R . ; Pegg, D . T . ; D o d d r e l l , D . M . ; W i l l i a m s , D . H . , J. Org. Chem. 1982, 47, 3021. Campbell, I . D . ; Dobson, C . M . ; W i l l i a m s , R . J . P ; Wright, P.E., FEBS L e t t . 1975, 57, 96. Pachler, Κ.G.R.; Wessels, P.L., J. Magn. Reson. 1977, 28, 53; Jakobsen, H.J.; W.S. Brey, J . Amer. Chem. Soc. 1979, 101, 760. M o r r i s , G . A . , J. Amer. Chem. Soc. 1980, 102, 428. Burum, D . P . ; E r n s t , R . R . , J. Magn. Reson. 1980 39, 163. D o d d r e l l , D . M . ; Pegg, D . T . , J. Amer. Chem. Soc. 1980, 102, 6388. B o l t o n , P . H . , J. Magn. Reson. 1980, 41, 287.


118 24. 25. 26. 27.


28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

NMR AND MACROMOI.ECULES

R i n a l d i , P.L.; Baldwin, N.J., J. Amer. Chem. Soc. 1982, 104, 5791. B e n d a l l , M . R . ; Pegg, D . T . ; D o d d r e l l , D . M . ; Field, J., J. Amer. Chem. Soc. 1981, 103, 934. D o d r e l l , D . M . ; Pegg, D . T . ; B e n d a l l , M . R . , J . Magn. Reson. 1982, 48, 323. Jeener, J.; Ampere I n t e r n a t i o n a l Summer School, Basko P o l j e , Yugoslavia 1971. Thomas, D . M . ; B e n d a l l , M . R . ; Pegg, D . T . ; D o d d r e l l , D . M . ; Field, J., J . Magn. Reson. 1981, 42, 298, Rutar, V.; Wong, T.C., J. Magn. Reson. 1983, 53, 495. Aue, W.P.; B a r t h o l d i , Ε . ; E r n s t , R . R . , J. Chem. Phys. 1976, 64, 2229. Nagayama, N.; Kumar, A.; Wuthrich, K . ; E r n s t , R . R . , J . Magn. Reson. 1980, 40, 321. Bax, Α . ; Freeman, R . ; M o r r i s , G . , J. Magn. Reson. 1981, 42, 164. Maudsley, A.A.; E r n s t , R . R . , Chem. Phys. L e t t . 1971, 50, 368. Ammann, W.; R i c h a r z , R.; W i r t h l i n , T . ; Wendisch, D . , Org. Magn. Reson. 1982, 20, 260. Ikura, M . ; Hikichi, Κ., Org. Magn. Reson. 1982, 20, 266. Benn, R . , Z. Naturforsch 1982, 37b, 1054. M o r r i s , G . A . ; Hall, L.D., J. Amer. Chem. Soc. 1981, 103, 4703. Chan, T.-M.; Markley, J.L., J. Amer. Chem. Soc. 1982, 104, 4010. K e s s l e r , H . ; H e h l e i n , W.; Schuck, R . , J. Amer. Chem. Soc. 1982, 104, 4534. Gray, G . A . , Org. Magn. Reson. 1983, 21, 111. Avent, A.G.; Freeman, F., J. Magn. Reson. 1980, 39, 169. Bax, Α . , J. Magn. Reson. 1983, 53, 517. Jeener, J.; Meier, B . H . ; Bachmann, P . ; Ernst, R.R. J . Chem. Phys. 1979, 71, 4546. Meier, B.H.; Ernst, R . R . ; J. Amer. Chem. Soc. 1979, 101, 6441. Macura, S . ; E r n s t , R . R . , M o l . Phys. 1980, 41, 95. Kumar, Α . ; E r n s t , R . R . ; Wutrich, Κ., Biochem. Biophys. Res. Commun. 1980, 95, 1. Bosch, C . ; Kumar, A.; Baumann, R.; E r n s t , R . R . ; Wutrich, Κ., J . Magn. Reson. 1981, 42, 159. Kumar, A.; Wagner, G . ; E r n s t , R . R . ; Wutrich, Κ., J. Amer. Chem. Soc. 1981, 103, 3654. Huang, Y.; Macura, S . ; E r n s t , R . R . , J . Amer. Chem. Soc. 1981, 103, 5327.

RECEIVED

October 31, 1983


NMR and Macromolecules - American Chemical Society

Recommend Documents