NMR Spectroscopy - American Chemical Society

3 N M R Spectroscopy at 600 M H z

Downloaded by CORNELL UNIV on July 21, 2016 | http://pubs.acs.org Publication Date: July 9, 1982 | doi: 10.1021/bk-1982-0191.ch003

A. A. BOTHNER-BY and J. DADOK Carnegie-Melon University, Department of Chemistry, Pittsburgh, PA 15213

In December 1979, the NMR Facility for Biomedical Studies formally opened service on the 600 MHz NMR spectrometer assembled there, and the intervening year has seen a variety of applications to structure made possible by the high magnetic field (14.1 Tesla) employed. The first advantage which comes to mind when considering the use of high magnetic fields for NMR is the increased spectral dispersion which results, and the possibility of separating and observing signals from complex molecules which are not resolved at lower fields. This advantage is well understood and widely appreciated, and only a few illustrative examples will be given here. An example of the degree of complexity which can be sorted out is Palytoxin, a water-soluble toxin obtained from marine coelenterates of the genus Palythoa. The molecular formula of the compound is C H N O . The complete gross structure for Palytoxin has been elucidated by R. E. Moore and G. Bartolini of the University of Hawaii and reported in a recent communication (1). The structural elucidation involved extensive proton spectroscopy at 600 MHz, together with homonuclear decoupling, to establish the sequence and stereochemistry in degradation frag ments as well as the entire molecule. The structure deduced is displayed in Figure 1, with the spectrum of the intact toxin shown beneath. An expanded view of a portion of this spectrum is displayed in Figure 2. The structure of an N-terminal degradation product and its spectrum is displayed in Figure 3. A second example illustrating the greater dispersion at high field is bradykinin, a nonapeptide with sequence arg-pro-pro-glyphe-ser-pro-phe-arg. The spectrum of this peptide and of a number of analogs has been investigated in a collaborative effort with Dr. G. Fisher of the University of Miami. The spectrum with assignments is shown in Figure k. The separate observation of β protons from each of the three proline residues has made it possible, for example, to determine the degree of puckering and preference for Ramachandran A or Β forms in proline rings. Analysis of coupling constants in the side chains, amide proton 129

221

3

54

0 0 9 7 - 6 1 5 6 / 8 2 / 0 1 9 1 -0031 $ 0 6 . 0 0 / 0 © 1982 A m e r i c a n C h e m i c a l Society

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

NMR

SPECTROSCOPY


32

I

I

4000

I

I

I

3000

I

I

I

I

2000

I

I

I

1

1

1000

Figure 1. A 600-MHz high resolution spectrum of palytoxin, a marine toxin isolated from a species of Palythoa. The gross structure deduced for the toxin also is shown.



3.

B O T H N E R - B Y A N D DADOK

4000

NMR

Spectroscopy

at 600

3500

Figure 2.

33

MHz

3000

Expanded view of a small region of the spectrum of palytoxin shown in Figure 1.



Figure 3.

Structure of terminal fragment of palytoxin obtained from the N-p-bromobenzoyl rivative via periodate cleavage and spectrum thereof.


de-

NMR

Spectroscopy

at 600

MHz



15ÔÔ

'

1400

Figure 4.

'

1300

'

1200

'

1100

'

1000

'

A 600-MHz spectrum of bradykinin in D O solution. z


900

N M R SPECTROSCOPY


36

s h i f t s , pH t i t r a t i o n , use o f paramagnetic s h i f t and broadening reagents, and other standard NMR techniques have been a p p l i e d to demonstrate that b r a d y k i n i n i s a r a p i d l y i n t e r c o n v e r t i n g mixture of many conformers, w i t h no h i g h l y p r e f e r r e d conformation i n s o l u t i o n , but that complexes are formed w i t h l i p i d s , p a r t i c u l a r l y at pro^ and phe° which impose some conformational r e s t r a i n t on the molecule ( 2 ) . Other c l a s s e s o f compounds f o r which the e x t r a r e s o l v i n g power at h i g h f i e l d has been u s e f u l i n c l u d e DNA r e s t r i c t i o n f r a g ments ( i n v e s t i g a t e d by T. A. E a r l y , D. R. Kearns, W. H i l l e r and R. D. Wells (2); a l k a l o i d s (S. Danishefsky (kj), and sugars (£). A second advantage o f h i g h e r f i e l d s i s that r a p i d chemical exchange processes ( f o r example between conformational s t a t e s ) are more r e a d i l y apparent. L i n e broadening i n the r a p i d exchange l i m i t i s given by: Δν

ι / 2

=

ν

"ρ!ρ|(ν - Β

) 2 τ

Α

where increased l i n e width, P A and p are the f r a c t i o n s o f the two conformers, (v^-Vg) the chemical s h i f t i n frequency u n i t s between n u c l e i i n the two conformers, and τ the average r e s i d e n c e time i n one conformer. Increasing the f i e l d by a f a c t o r of t e n w i l l increase the l i n e broadening by a f a c t o r of one hundred; r a p i d exchange processes which cause a broadening o f 0 . 1 Hz at 60 MHz and might escape n o t i c e , w i l l cause a broadening of 10 Hz at 600 MHz and w i l l be very n o t i c e a b l e . Thus P r o f e s s o r J . G l a s e l ( 6 ) noted that the 600 MHz spectrum of morphine at p h y s i o l o g i c a l pH was broad ( F i g u r e 5 ) , (although the low f i e l d spectrum appeared normal). This suggested a con formational e q u i l i b r i u m (axial-N-methyl Ζ equatorial -N-methyl). The c o n c l u s i o n was confirmed by observing the spectrum at pH 1. 5 , which slowed the i n t e r c o n v e r s i o n and gave a sharp spectrum show ing s i g n a l s from each conformer. The i n t e n s i t i e s were i n the r a t i o 1 : 5 , i n d i c a t i n g an energy d i f f e r e n c e of o n l y about 1 K c a l / mole, although e a r l i e r c a l c u l a t i o n s (7) had p r e d i c t e d the Ne q u a t o r i a l conformer to be more s t a b l e by about 6 Kcal/mole. The i n t e r e s t i n g question a r i s e s as to which conformer i s bound i n the r e c e p t o r s i t e , and the r o l e o f the lone p a i r i n binding. The a v a i l a b i l i t y of h i g h e r f i e l d s a l s o opens the door to the o b s e r v a t i o n o f features i n the NMR spectrum which depend quadrati c a l l y on the f i e l d strength. In p a r t i c u l a r , the f i e l d i s s u f f i c i e n t l y strong so that even r e l a t i v e l y small diamagnetic and paramagnetic molecules w i t h a n i s o t r o p i c magnetic s u s c e p t i b i l i t i e s are s i g n i f i c a n t l y o r i e n t e d , and the o r d e r i n g produces observable e f f e c t s i n the spectrum. Such e f f e c t s have been observed i n the past w i t h molecules i n macroscopic c r y s t a l s which are e i t h e r p h y s i c a l l y o r i e n t e d , or are o r i e n t e d by the f i e l d , ( 8 ) or by observing the s p e c t r a of the molecules d i s s o l v e d i n l i q u i d cry s t a l s ( i n which the l i q u i d c r y s t a l domains are o r i e n t e d by the f i e l d ) , (2) o r by the a p p l i c a t i o n o f a strong e l e c t r i c f i e l d t o s o l u t i o n s of p o l a r molecules ( 1 0 ) . i s

t l i e

B


NMR Spectroscopy at 600


3.

MHz

37

:


(a)

_aJl

jui

K-IOOHz t

t

ι

i

ι

ι

I

ι

ι

1

1

1

1

1

— 1

(t» 10

X Ο I

I

I

ο χ

CO

° (Ο

σ a>

χ

ω χ

JJL —I

I.

1

I

I

I

L.

σ α)

X

σ Ο

ο

ΙΟ X

ο

X

Ϊ Ι

Jill,

. J U L . _l

I

1

1—

Figure 5. Spectra of morphine at neutral and low pH demonstrating the occur rence of exchange phenomena at neutral pH (a) and slow inter conversion of axial and equatorial N-methyl isomers at low pH (b).


38

NMR

SPECTROSCOPY

F o l l o w i n g the treatment o f Lohman and MacLean, (jLl) the o r d e r i n g of the molecule may be described i n terms of two order ing parameters S and K, which are defined by the r e l a t i o n s S = 2


2 Κ = ( s i n θ cos 2 φ ) The angular brackets denote an e x p e c t a t i o n or time-averaged value, and θ and φ are the p o l a r angles g i v i n g the d i r e c t i o n of the s t a t i c magnetic f i e l d i n the molecule-fixed coordinate frame x,y,z. These axes are chosen to be the p r i n c i p a l axes of the magnetic s u s c e p t i b i l i t y tensor o f the molecule. The energy o f the molecule i n a magnetic f i e l d i s given by the expression Ε = - 1/2 Η· χ· Η The o r d e r i n g parameters S and Κ may be c a l c u l a t e d assuming a Boltzmann d i s t r i b u t i o n , and are then given by S = ΔχΗ^/^Τ

Κ = ALx«P/l5kT where Δχ the s u s c e p t i b i l i t y anisotropy i s given by χ -l/2 (Xxx Xyy)> Χ s u s c e p t i b i l i t y asymmetry i s given by Xxx " Xyy' po ^. For aromatic molecules, Δχ amounts to about 1 χ 1 0 " ^ ° cnP/mol per benzene r i n g ( 1 2 ) , w h i l e Δ χ i s expected to be an order of magnitude smaller, so the o r d e r i n g parameter S f o r benzene at room temperature and lkl k i l o g a u s s i s expected to be about 3 χ ΙΟ- . ζ ζ

+

a

n

d

Δ

χ

t l i e

χ

6

The parameter S f o r paramagnetic molecules

S = 1/I5(f^)

i s given by

2

S u b s t i t u t i n g t y p i c a l values of Δ μ , the parameter i s found to be only one to two orders o f magnitude l a r g e r than those of aromatic compounds. The occurrence o f t h i s o r d e r i n g can be detected i n s e v e r a l ways u s i n g magnetic techniques. In the case of quadrupolar n u c l e i , such as deuterium, the o r d e r i n g w i l l give r i s e to a quadrupole s p l i t t i n g of the deuteron s i g n a l , as i s w e l l known from experiments with l i q u i d c r y s t a l s (Γ5). The o b s e r v a t i o n of l a r g e s p l i t t i n g s i n a small paramagnetic molecule a r i s i n g from t h i s o r i g i n have been reported by P. J . Domaille (1*0, example showing such s p l i t t i n g s i s given i n Figure"^. The o b s e r v a t i o n of deuteron quadrupole s p l i t t i n g s i n the deuteron s p e c t r a of deuterated aromatic compounds has a l s o been reported i n a s e r i e s o f a r t i c l e s by Lohman and Maclean ( l l , l j ? , l 6 ) . a n d


a

n

BOTHNER-BY

NMR Spectroscopy at 600

A N D DADOK

MHz

A s s i g n m e n t s of B r a d y k i n i n Arg^Pro - Pro -Gly -Phe -Ser -Pro -Phe -Arg


2

4500

4300

4400

3

4200"

4

4100

5

6

7

"4000

8

9

"3900

a I

NMR Spectroscopy - American Chemical Society

Recommend Documents