Deuterium exchange as an indicator of hydrogen bond donors and acceptors

Article abstract of DOI:10.1021/ja076185s

Hydrogen-deuterium exchange has been used to determine the hydrogen bonding preferences of a select group of small peptides in organic solvents. This kinetic analysis permitted comparison of hydrogen bond strengths in relation to controls and also indicated the identity of both hydrogen bond donors and hydrogen bond acceptors. Copyright

Full text of DOI:10.1021/ja076185s

Published on Web 10/04/2007
Deuterium Exchange as an Indicator of Hydrogen Bond Donors and
Acceptors
Lauren R. Steffel, Timothy J. Cashman, Michael H. Reutershan, and Brian R. Linton*
Department of Chemistry, Bowdoin College, Brunswick, Maine 04011
Received August 16, 2007; E-mail: blinton@bowdoin.edu
Hydrogen bonding is a fundamental feature in the structured
Table 1. Half-lives of H/D Exchange in 10% CD3OD/CDCl3 for
R CONHR Derivatives (1-10)
1
2
1
folding of proteins and synthetic foldamers. As interest grows in
2
peptidomimetic design and peptide catalysts, a heightened under-
t
1
/
2
t
1/2
R1
R2
(min)
R1
R2
(min)
standing of the hydrogen bonding properties of these molecules is
essential. Herein we demonstrate that hydrogen/deuterium exchange
is a valuable tool in determining the relative strengths of hydrogen
bonds in relation to controls. Additionally, it provides a means to
elucidate the separate roles of both hydrogen bond donors and
hydrogen bond acceptors.
1
2
3
4
5
Me
Et
i-Pr
t-Bu
Me
n-Bu
n-Bu
n-Bu
n-Bu
i-Pr
14
20
42
6
7
8
9
10
Me
t-Bu
n-Bu
i-Pr
t-Bu
t-Bu
110
1200
1220
1450
24
tBuO
tBuO
tBuO
CF3
14500
14
Hydrogen/deuterium (H/D) exchange is an analytical technique
that has been used to correlate hydrogen bond strength with the
rate of chemical exchange of the participating hydrogen to
in Figure 1. More striking is the comparison with control molecules
that contain similar functional groups: amide 2 (O) and carbamate
7 (0). The glycine amide shows a significant decrease in the rate
of exchange in comparison with the control, consistent with this
hydrogen serving as a hydrogen bond donor. Interestingly, the
carbamate hydrogen shows a significant acceleration in the rate of
H/D exchange. This is indicative of the carbamate serving as a
hydrogen bond acceptor. Presumably, the presence of the hydrogen
bond serves to accelerate the H/D exchange of the carbamate proton
by stabilizing the increased electron density on the carbamate that
occurs when the proton is removed.9
Two additional N-methylated glycine derivatives (12, 13) were
created to further probe the role of hydrogen bonding and gauge
possible electronic and field effects. The H/D exchange kinetics of
these derivatives are also shown in Figure 1, and the corresponding
rate data are listed in Table 2. Boc-N-Me-Gly-NHBu 12 (×)
exhibited a rate of exchange that was similar to the non-methylated
derivative, suggesting comparable hydrogen bonding in both
3
deuterium. While this technique has been widely used to evaluate
4
protein dynamics in water, there has been minimal work involving
5
small molecules in organic solvents. In an effort to demonstrate
how this method can be employed to measure intramolecular
hydrogen bonding, H/D exchange was applied to a series of model
compounds chosen to distinguish the participation of hydrogen
bonding from other effects.
Englander has shown that steric and electronic environments
affect H/D exchange rates.6 As a result, any consideration of
hydrogen bonding must be in the context of comparison with similar
controls that are unlikely to form intramolecular hydrogen bonds.
1
2
Amide derivatives 1-10 (R CONHR ) vary in their amine and
carboxyl substituents, and the half-lives of their H/D exchange in
0% CD OD/CDCl
3 3
are listed in Table 1.7 Steric bulk of the
1
carbonyl and nitrogen substituents affects the baseline rate of H/D
exchange to differing degrees. A comparison of 1-4 shows that
1
changes to the carbonyl substituent (R ) from methyl through tert-
butyl produced exchange rates that differed by 1000-fold. Deriva-
2
tives that differ only in their nitrogen substituent (R ) showed a
lesser difference in their exchange rates. This can be seen by
comparing amides 1, 5, and 6, as well as carbamates 7-9. Electronic
effects have also been shown to influence H/D exchange rates.
Derivatives 6, 9, and 10 were all formed from tert-butyl amine but
contain differing carbonyl functional groups. The electron with-
drawal of the trifluoromethyl group accelerated H/D exchange,
while the electron donation of the carbamate slowed the rate of
exchange. Both the electronic and steric results point to a dissocia-
tive mechanism where removal of the proton is rate-determining
and inhibited by greater electron density or by increased steric bulk,
most appreciably on the carbonyl substituent.8
With the establishment of baseline exchange rates for controls,
hydrogen/deuterium exchange was measured for fundamental
molecules capable of making only a select number of intramolecular
hydrogen bonds and with minimal steric differences. Boc-Gly-
NHBu 11 was chosen to provide two hydrogen bond donors and
two hydrogen bond acceptors, with the possibility of forming
hydrogen bonds through either a five-membered or a seven-
membered ring. On first inspection, the H/D exchange data for 11
Figure 1. H/D exchange kinetics comparing glycine derivatives 11 (Boc-
NH ) 9, NHBu ) b), 12 (×) and 13 (+) with controls 2 (O), 7 (0).
Arrows indicate the differences in rates between non-hydrogen-bonding
controls and similar hydrogen-bonding groups.
(Figure 1) showed a slower rate of exchange for the amide hydrogen
(b) than the carbamate hydrogen (9), consistent with preferential
formation of a seven-membered ring γ-turn hydrogen bond shown
12956
9
J. AM. CHEM. SOC. 2007, 129, 12956-12957
10.1021/ja076185s CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Half-lives of H/D Exchange in 10% CD3OD/CDCl3 for
Peptides (11-15) Capable of Making Intramolecular Hydrogen
Bonds
in 15 is not accelerated to the same extent as 11 may indicate that
its role as a hydrogen bond acceptor is diminished by comparison.
Both of these effects point to the presence of conformation 15b.
These simple molecules demonstrate the usefulness of hydrogen/
deuterium exchange in the assessment of hydrogen bonding.
Relative rates of H/D exchange can be correlated with the presence
of hydrogen bonds, given comparison to controls that account for
inherent steric and electronic effects. While a number of existing
techniques indicate the role of hydrogen bond donors, this is one
of only a few techniques that directly illuminates the participation
of individual hydrogen bond acceptors.13 This approach is currently
being employed in ongoing investigations of larger hydrogen
bonding molecules.
t
1/2
exchanging NH
(min)
11
11
12
13
14
14
15
15
15
BocNHCH2CONHBu
BocNHCH2CONHBu
BocNMeCH2CONHBu
BocNHCH2CONMe2
BocNHCH2CH2NHAc
BocNHCH2CH2NHAc
BocNHCH2CONHCH2CONHBu
BocNHCH2CONHCH2CONHBu
BocNHCH2CONHCH2CONHBu
40
132
150
800
570
33
63
13
180
derivatives. The exchange kinetics of Boc-Gly-NMe
2
13 (+) showed
a rate of exchange that was similar to that of the carbamate control,
suggesting that when the γ-turn was unavailable there was no
significant hydrogen bonding present in this derivative.
A comparison with other techniques indicates the sensitivity of
H/D exchange in illustrating these somewhat weak hydrogen
7
bonding interactions. Both N-H protons of 11 show significant
Acknowledgment. This work was supported by an award from
Research Corporation. The authors are grateful to the NSF for
financial support (MRI-0116416).
changes in NMR chemical shift (g2 ppm) upon the addition of a
hydrogen bonding solvent,10 making it difficult to accurately
describe the presence of hydrogen bonding. Analysis of the infrared
spectra of 11-13 does indicate the presence of a hydrogen-bonded
amide for 11 and 12, but even 12 exhibits predominantly a non-
hydrogen-bonded amide stretch.11
Supporting Information Available: Synthetic details, NMR
spectra, and kinetic data for H/D exchange. This material is available
free of charge via the Internet at http://pubs.acs.org.
The selectivity of hydrogen bond formation is not purely
dependent on preferred ring size; it is also a matter of the functional
groups involved. Molecule 14 was designed to permit two possible
seven-membered ring hydrogen bonds 14a and 14b, each involving
an amide and a carbamate functional group. The H/D kinetics
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control 7, while the amide exchanged more slowly than control 2.
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bond donor and the carbamate the hydrogen bond acceptor. In
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tion, the exchange kinetics likely represent the average of the
dynamic equilibrium between 14a and 14b. This equilibrium would
appear to favor conformation 14a, presumably due the difference
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groups.
2
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(
(
7) Detailed data can be found in the Supporting Information.
8) H/D exchange shows a minimal rate at pH 3-5, suggesting that with no
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Englander, S. W.; Sosnick, T. R.; Englander, J. J.; Mayne, L. Curr. Opin.
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(
Dipeptide Boc-Gly-Gly-NHBu 15 possesses an additional hy-
3
7, 1885.
drogen bond donor and acceptor, and hydrogen/deuterium exchange
(
(
(
(
10) (a) Pitner, T. P.; Urry, D. W. J. Am. Chem. Soc. 1972, 94, 1399. (b)
Venkatachalapathi, Y. V.; Prasad, B. V. V.; Balaram, P. Biochemistry
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12
is helpful in illuminating the preferred conformations. The kinetic
7
profile showed that, once again, the butyl amide (b) exchanged
11) (a) Gellman, S. H.; Dado, G. P.; Liang, G.-B.; Adams, B. R. J. Am. Chem.
Soc. 1991, 113, 1164. (b) D ´ı az, H.; Espina, J. R.; Kelly, J. W. J. Am.
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more slowly than control 2 (O) but also slightly more slowly than
the butyl amide in 11 (see Table 2). The carbamate of 15 (9)
exchanges at a rate that is much faster than control 7 (0) but not
as fast as the carbamate in 11. The central amide exchanges at a
rate slightly faster than control 2. As a whole, these three rates are
most consistent with a â-turn conformation 15a, in equilibrium with
at least one other conformation. The slight acceleration in the
exchange of the central amide suggests that it serves to some extent
as a hydrogen bond acceptor. The observation that the carbamate
12) For seminal work on the hydrogen bonding patterns of peptide turns, see:
Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1994, 116, 1054 and
related references.
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stretching frequencies that accompany hydrogen bonding. (a) Compagnon,
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