Low-barrier hydrogen bonding in aqueous and aprotic solutions of dicarboxylic acids: Spectroscopic characterization

Article abstract of DOI:10.1006/bioo.1998.1102

We present additional spectroscopic evidence for the formation of low- barrier hydrogen bonds (LBHBs) within vicinal and geminal dicarboxylic acid monoanions. Hydrogen cis-cyclohexane 1,2-dicarboxylate, displays low-field ¹H NMR signals in apro

Full text of DOI:10.1006/bioo.1998.1102

BIOORGANIC CHEMISTRY 26, 213–219 (1998)
ARTICLE NO. BH981102
Low-Barrier Hydrogen Bonding in Aqueous and Aprotic
Solutions of Dicarboxylic Acids: Spectroscopic Characterization
Constance S. Cassidy, Jing Lin, John B. Tobin, and Perry A. Frey
Institute for Enzyme Research, The Graduate School, and Department of Biochemistry,
College of Agricultural and Life Sciences, University of Wisconsin–Madison,
Madison, Wisconsin 53705
Received May 5, 1998
We present additional spectroscopic evidence for the formation of low-barrier hydrogen
bonds (LBHBs) within vicinal and geminal dicarboxylic acid monoanions. Hydrogen cis-
1
cyclohexane 1,2-dicarboxylate, displays low-field H NMR signals in aprotic solvents at 19.3
to 19.7 ppm, depending on the solvent. The LBHB in hydrogen 2,2-dimethylmalonate is
further characterized by the observation of a positive value for the deuterium isotope effect
1
on the chemical shift, ⌬[ͳ
H
Ϫ ͳ
D
], of 0.8 Ϯ 0.3 ppm. Low-field H NMR signals are not
observed for hydrogen trans-cyclohexane 1,2-dicarboxylate, hydrogen succinate, or hydrogen
malonate under the same conditions. These compounds lack internal structural constraints
forcing close contact between the caboxylic acids, although close contacts are possible,
confirming that compression of carboxylic acid groups facilitates LBHB formation. Internally
1
strained dicarboxylic acid monoanions also display low field H NMR signals in aqueous
6 2
solutions (90/10, acetone-d /H O) at low temperatures, at which proton exchange is slowed.
The low field signals at Ϫ50ЊC are centered at 20.2 ppm for hydrogen maleate, 19.0 ppm for
hydrogen 2,2-dimethylmalonate, and at 19.2 ppm for hydrogen cis-cyclohexane 1,2-
dicarboxylate.
 1998 Academic Press
Low-barrier hydrogen bonds (LBHBs) have been postulated to stabilize transition
states and intermediates in enzymatic catalysis (1–4). LBHBs have been observed
in enzymes (4–9), and the conditions required for their formation, as well as their
functional significance when formed, are currently controversial issues (10–13). It
has been postulated in the case of chymotrypsin that the LBHB arises from compres-
ͳ
1
ͳ
1
sion between N of His 57 and O of Asp 102 in the active site brought about by
a substrate-induced conformational change (7).
Physical chemists have classified hydrogen bonds as three types: weak, strong,
and very strong (14). Very short hydrogen bonds exist under special conditions,
and in the gas phase they may be stronger than weak covalent bonds (14, 15);
however, it is questionable whether they can be very strong in solution (16). Weak
hydrogen bonds are the widely observed, conventional variety. LBHBs are of the
intermediate, strong type, in which the barrier in the double minimum potential is
low and near the vibrational frequency for hydrogen (14). LBHBs display very low
1
field H NMR chemical shifts, positive deuterium isotope effects (⌬[ͳ Ϫ ͳ ]) on
H
D
the chemical shifts, deuterium isotope effects on proton stretching frequencies, and
low fractionation factors in D O/H O mixtures (14). We here report a positive
2
2
deuterium isotope effect on the low field proton NMR signal in hydrogen 2,2-
2
13
0
045-2068/98 $25.00
Copyright  1998 by Academic Press
All rights of reproduction in any form reserved.

2
14
CASSIDY ET AL.
1
dimethylmalonate and very low field H NMR signals in hydrogen cis-cyclohexane
1
,2-dicarboxylate dissolved in aprotic solvents. We further report the observation
1
of downfield H NMR signals characteristic of low barrier hydrogen bonding in
aqueous acetone solutions of highly strained vicinal and geminal dicarboxylic acids
at Ϫ50ЊC.
EXPERIMENTAL PROCEDURES
Materials. cis-Cyclohexane 1,2-dicarboxylic anhydride, 2,2-dimethylmalonic acid,
maleic acid, and trans-cyclohexane 1,2-dicarboxylic acid were purchased from Al-
drich and succinic acid from Fisher. All were recrystallized before use, and their
1
purity was verified by H NMR. Solutions of tetrabutylammonium hydroxide (1.0
M) in methanol (Aldrich or Sigma) in sealed vials were opened under nitrogen.
Anhydrous CHCl and CDCl (Aldrich) in sealed vials were opened in a dry box
3
3
and protected from moisture throughout the experiments. Acetonitrile-d (CD CN)
3
3
dimethylsulfoxide-d (DMSO-d ), and tetrahydrofuran-d (THF-d ) were purchased
6
6
8
8
from Aldrich in boxes of sealed ampoules.
Preparation of tetrabutylammonium salts of dicarboxylic acids and analysis by
H NMR. cis-Cyclohexane 1,2-dicarboxylic acid was prepared by reaction of the
1
anhydride with 1.0 M acetic acid in an ice bath overnight. The solvent was removed
1
in vacuo, and the compound was recrystallized from water. H NMR (500 MHz)
of cis-cyclohexane 1,2-dicarboxylic acid in DMSO-d , ambient temperature, recycle
6
time ϭ 1 s: ͳ 12.03 (2H, s); 2.67 (2H, m); 1.86 (2H, m); 1.66 (2H, m); 1.37 (4H, m).
The tetrabutylammonium (NBu ) salts of cis- and trans-cyclohexane 1,2-dicarbox-
4
ylic acid, succinic acid, 2,2-dimethylmalonic acid, and succinic acid were prepared
by dissolving the purified acids in methanol, adding one equivalent of tetrabutylam-
monium hydroxide from a 1.0 M stock solution, and stirring for 20 min. The solvent
was removed by rotary evaporation in vacuo, and the salt was dried under vacuum.
1
Preliminary H NMR samples were prepared at 15 mM in anhydrous CDCl or
3
DMSO-d and diluted to 1.5 or 0.5 mM. Samples of 15 mM hydrogen cis-cyclohexane
6
1
1
,2-dicarboxylate were also prepared in acetonitrile-d and THF-d . H NMR (500
3
8
MHz) of 15 mM hydrogen cis-cyclohexane 1,2-dicarboxylate in CDCl , ambient
3
temp., recycle time ϭ 1s: ͳ 19.75 (1H, s); 3.29 (8H, t, NBu CH ’s); 2.74 (2H, m);
4
2
2
1
.00 (4H, m); 1.66 (8H, q, NBu CH ’s); 1.44 (8H, m, NBu CH ’s); 1.44 (2H, m);
4 2 4 2
1
.00 (12H, t, NBu CH ’s). H NMR of 15 mM hydrogen 2,2-dimethylmalonate in
4
3
CDCl : ͳ 19.6 (1H, s); 3.26 (8H, t, NBu CH ’s); 1.16 (8H, m, NBu CH ’s), 1.44
3
4
2
4
2
(
8H, m, NBu CH ’s and 6H (acid 2-CH ’s)); 1.04 (12H, t, NBu CH ’s).
4 2 3 4 3
Preparation of O-deuterated 2,2-dimethylmalonate salt. The O-deuterated tetrabu-
tylammonium hydrogen 2,2-dimethylmalonate was prepared by dissolving hydrogen
,2-dimethylmalonic acid in D O, freezing the sample, and drying under vacuum.
2
2
Exchange of deuterium into the sample was verified by the disappearance of the
1
1
2
H NMR signal for –COOH in the H NMR spectrum. The H NMR spectrum was
obtained on the 500 MHz DMX Bruker NMR spectrometer through the BroadBand
2
channel with the H lock channel detuned. A 1% solution of CDCl in CHCl was
3
3
used as an external reference. The low-field deuterium signal was not observable

LOW-BARRIER HYDROGEN BONDING
215
TABLE 1
Values of Chemical Shift for the Low-Field Proton of Hydrogen cis-Cyclohexane
1
,2-Dicarboxylate in Various Organic Solvents
Solvent
Concentration (mM)
ͳ
H
(ppm)a
cis-Cyclohexane 1,2-dicarboxylate
CDCl
CDCl
DMSO-d
DMSO-d
THF-d
3
CD CN
3
15
1.5
15
1.5
15
15
19.75
19.71
19.77
19.76
19.35
19.34
3
6
6
8
a
Spectra were accumulated at ambient temperature with a delay time of 1 s.
at 15 mM, but at 0.1 M a broad resonance was observed at 18.83 ppm (␯1/2 ϭ 1366
Hz) at 25ЊC. The line width of this signal was decreased to 784 Hz at 5ЊC and the
chemical shift of the low-field deuteron was 19.2 ppm. The difference in the values
of chemical shift at 25 and 5ЊC was used to estimate the error on ͳD .
RESULTS AND DISCUSSION
The deuterium isotope effect on the chemical shift of the low field proton in
hydrogen 2,2-dimethylmalonate. Hydrogen 2,2-dimethylmalonate was previously
studied in CDCl , DMSO-d , CD CN, and THF-d , and it displayed a low field
3
6
3
8
resonance at ͳH 19.4 to 19.6 ppm in these solvents (4). We have confirmed the
value of ͳ 19.6 ppm for this compound at higher concentrations (100 and 15 mM).
H
We have now observed a broad signal for the low field deuteron in the deuterium
NMR spectrum of hydrogen O-deutero-2,2-dimethylmalonate (100 mM). The relax-
ation rate T is a function of the electric quadrupole moment and the molecular
1
symmetry. Deuterium is a quadrupolar nucleus with a spin of one, and the short
relaxation time may result in significant line broadening relative to the correspond-
ing proton signal. The width of the signal may be accentuated by chemical exchange
of the deuteron with adventitious D O in the samples. The width of the low-field
2
2
H NMR signal at 25ЊC (ͳ 18.8 ppm; ␯ ϭ 1366 Hz) made it difficult to obtain
D
1/2
an accurate value for ⌬[ͳ Ϫ ͳ ]. The line width at 5ЊC was narrower (ͳ ϭ 19.2,
H
D
D
␯1/2
ϭ 784 Hz), allowing a calculation of ⌬[ͳ Ϫ ͳ ] with a reasonable estimate on
H D
the error for the ͳ measurement. The deuterium isotope effect, ⌬[ͳ Ϫ ͳ ], was
D
H
D
estimated to be ϩ0.8 Ϯ 0.3 ppm. The positive isotope effect is in the range for
LBHBs (14) and supports the assignment of the bridging proton in hydrogen 2,2-
dimethylmalonate as an LBHB.
Observation of low field protons in hydrogen cis-cyclohexane 1,2-dicarboxylate.
A low field proton NMR signal was observed for tetrabutylammonium hydrogen
cis-cyclohexane 1,2-dicarboxylate in aprotic solvents (Table 1). The signal in CDCl3

216
CASSIDY ET AL.
and DMSO was nearly concentration-independent in the mM range, and the signal
was also present in THF-ͳ and CD CN.
8
3
Low field proton NMR signals were not observed in CDCl solutions of tetra-
3
butylammonium hydrogen trans-cyclohexane 1,2-dicarboxylate or of tetrabutyl-
ammonium hydrogen succinate. The signals for COOH appeared at 12–13 ppm.
Similar, negative results were reported for hydrogen malonate (4). The carboxylic
acid and carboxylate groups of these dicarboxylic acids can, in principle, interact
with each other, although they are not forced into contact by internal structural
constraints. Inasmuch as intramolecular LBHBs are not found, the potential strength
of an intramolecular LBHB must not be sufficient to draw these molecules into
internally hydrogen bonded conformations in competition with intermolecular, hy-
drogen bonded complexes. In the cases of hydrogen 2,2-dimethylmalonate and
hydrogen cis-cyclohexane 1,2-dicarboxylate, internal structural factors tend to force
the carboxylate and carboxylic acid groups into close contact, and these constraints
appear to be necessary to induce the formation of the LBHB. The role of compres-
sion in the induction of LBHBs has been discussed elsewhere (7, 17).
The hydrogen cyclohexane 1,2-dicarboxylates offer an interesting case of transi-
tion between LBHB formation in the cis-isomer and conventional hydrogen bonding
in the trans-isomer. In the trans-isomer 1 the preferred geometry is diequatorial
(
18–20), in which the carboxylate and carboxylic acid groups can potentially interact,
˚
with the carboxyl-carbons separated by only 3.0 A. However, an LBHB is not
observed. Molecular models of hydrogen cis-cyclohexane 1,2-dicarboxylate 2 show
that the carbon atoms of the carboxylate and carboxylic acid groups are closer
˚
(
2.8 A) than in the trans-isomer, and the NMR spectrum indicates the presence of
an LBHB. It appears that the small difference in carboxyl-separation for the cis-
and trans-isomers may be a factor in LBHB formation. Owing to free rotation
about the bonds to the cyclohexane backbone, the carboxylic and carboxylate
groups in the trans-isomer should be able to assume an appropriate contact geometry
for strong hydrogen bonding, but a potential LBHB is not strong enough to bring
the two groups close enough together for it to exist. In the case of the cis-isomer,
the cyclohexane backbone appears to force the two groups sufficiently close together
for LBHB formation.
The significance of internal compression in the formation of LBHBs is further
highlighted by the observation of an LBHB in hydrogen 2,2-dimethylmalonate but
not in hydrogen malonate (4). Similar enforced interactions of surface carboxylic

LOW-BARRIER HYDROGEN BONDING
217
FIG. 1. Low field, low temperature 1H NMR spectra of dicarboxylic acid monoanions in aqueous
acetone. The tetrabutylammonium salts of hydrogen maleate, hydrogen 2,2-dimethylmalonate, hydrogen
cis-cyclohexane 1,2-dicarboxylate, and hydrogen trans-cyclohexane 1,2-dicarboxylate were dissolved at
0
.1 to 0.2 M concentrations in 90% acetone-d
6
1
/H
2
O, with 2,2-dimethyl-2-silapentane-5-sulfonate as the
internal standard. The low field region of the H NMR spectra showed no signals at room temperature.
The low field spectra obtained at Ϫ55ЊC are shown in the figure. The low field signals are centered at
2
0.2 ppm for hydrogen maleate, 19.0 ppm for hydrogen 2,2-dimethylmalonate, and at 19.2 ppm for
hydrogen cis-cyclohexane 1,2-dicarboxylate. Hydrogen trans-cyclohexane 1,2-dicarboxylate does not
show a low field signal.
acid groups in proteins have recently been analyzed (21), and substrate-induced
compression between His 57 and Asp 102 is thought to be essential in the formation
and function of the LBHB in chymotrypsin (7).
Low field protons in aqueous solutions of strained dicarboxylic acid monoanions.
Low field protons assigned as LBHBs in NMR spectra are commonly observed for
the monoanions of strained vicinal and geminal dicarboxylic acids dissolved in
aprotic solvents (4, 13, 14). However, they are not observed in aqueous solutions
of the same species. We have considered whether they might exist in aqueous
solutions but are not easily observable in NMR spectra owing to fast exchange with
water protons. In such cases, low field protons might be observed if their exchange

218
CASSIDY ET AL.
rates could be lowered enough. This approach has succeeded in the observation of
a downfield proton in the case of 4,5-dihydroxynaphthalene-2,7-disulfonate in 10/90
water/acetone-d at low temperature (22). To examine this issue, we prepared the
6
tetrabutylammonium salts of dicarboxylic acid monoanions in aqueous acetone
(
10/90 water/acetone-d ) and recorded the low field proton NMR spectra at temper-
6
atures ranging from ambient to Ϫ50ЊC. The low field regions at Ϫ50ЊC are shown
in Fig. 1 for hydrogen maleate, hydrogen 1,2-dimethylmalonate, hydrogen cis-cyclo-
hexane 1,2-dicarboxylate, and hydrogen trans-cyclohexane 1,2-dicarboxylate. Low
1
field H NMR signals were observed in all cases except trans-cyclohexane 1,2-
dicarboxylate. Therefore, LBHBs appear to form in strained dicarboxylic acids in
aqueous solutions. The signals in aqueous acetone are broadened relative to those
in aprotic solutions, which may be attributed to chemical exchange with water. The
field positions of the low field protons in aqueous acetone are 0.2 to 0.4 ppm upfield
from those found in aprotic solvents, which may represent the effects of variant
solvation in aqueous and aprotic solvents. However, the signals in Fig. 1 are clearly
low field and well within the range observed in aprotic solvents for low barrier
hydrogen bonding (14).
The possibility of strong intramolecular hydrogen bonding in aqueous solutions
has been discussed elsewhere (17). Strong intramolecular hydrogen bonding in
water was postulated on the basis of an analysis of the large differences between
values of pK and pK for internally strained, geminal and vicinal dicarboxylic acids.
1
2
1
The downfield H NMR signals of hydrogen maleate, hydrogen cis-cyclohexane-
,2-dicarboxylate, and hydrogen 2,2-dimethylmalonate in Fig. 1 confirm these expec-
tations.
1
ACKNOWLEDGMENTS
Supported by Grant GM 51806 and C.S.C. was supported by the predoctoral training Grant GM
8505 from the National Institute of General Medical Sciences. This study made use of the National
0
Magnetic Resonance Facility at Madison, which is supported by NIH Grant RR02301 from the Biomedical
Research Technology Program, National Center for Research Resources. Equipment in the facility was
purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program
(Grant DMB-8415048), NIH Biomedical Research Technology Program (Grant RR02301), NIH Shared
Instrumentation Program (Grant RR02781), and the U.S. Department of Agriculture.
REFERENCES
1
2
3
4
5
6
7
8
. Cleland, W. W. (1992) Biochemistry 31, 317–319.
. Gerlt, J. A., and Gassman, P. G. (1993) Biochemistry 32, 11943–11952.
. Cleland, W. W., and Kreevoy, M. M. (1994) Science 264, 1887–1890.
. Frey, P. A., Whitt, S. A., and Tobin, J. B. (1994) Science 264, 1927–1930.
. Halkides, C., Wu, Y. Q., and Murray, C. J. (1996) Biochemistry 35, 15941–15948.
. Markley, J. L., and Westler, W. M. (1996) Biochemistry 35, 11092–11097.
. Cassidy, C. S., Lin, J., and Frey, P. A. (1997) Biochemistry 36, 4576–4584.
. Zhao, Q., Abeygunawardana, C., Gittis, A. G., and Mildvan, A. S. (1997) Biochemistry 36, 14616–
1
4626.

LOW-BARRIER HYDROGEN BONDING
219
9
. Kim, S. W., Cha, S.-S., Cho, H.-S., Kim, J.-S., Ha, N.-C., Cho, M.-J., Joo, S., Kim, K. K., Choi, K. C.,
and Oh, B.-H. (1997) Biochemistry 36, 14030–14036.
1
1
1
1
1
1
1
1
1
1
2
0. Guthrie, J. P. (1996) Chem. Biol. 3, 163–170.
1. Warshel, A., Papazyan, A., and Kollman, P. A. (1995) Science 269, 102–104.
2. Shan, S., and Herschlag, D. (1996) Proc. Natl. Acad. Sci. USA 93, 14474.
3. Perrin, C. L. (1997) Annu. Rev. Phys. Chem. 48, 511–544.
4. Hibbert, F., and Emsley, J. (1990) Adv. Phys. Org. Chem. 26, 255–379.
5. Kreevoy, M. M., and Liang, T. M. (1980) J. Am. Chem. Soc. 102, 3315–3322.
6. Perrin, C. L., and Thoborn, J. D. (1992) J. Am. Chem. Soc. 114, 8589–8565.
7. Frey, P. A., and Cleland, W. W. (1998) Bioorganic Chemistry, Submitted.
8. Benedetti, E., Corradine, P., Pedone, C., and Post, B. (1968). Chem. Commun. 1626–1627.
9. Abraham, R. J., Hudson, B. D., and Thomas, W. A. (1986) J. Chem. Soc. Perkin Trans. II 1635–1640.
0. Samoshin, V. V., Chertkov, V. A., Vatlina, L. P., Dobretsova, E. K., Simonov, N. A., Kastorsky,
L. P., Gremyachinsky, D. E., and Schneider, H.-J. (1996) Tetrahedron Lett. 37, 3981–3984.
1. Flocco, M. M., and Mobray, S. L. (1995) J. Mol. Biol. 254, 96–105.
2
2
2. Zhao, Q., Abeygunawardana, C., Talalay, P., and Mildvan, A. (1996) Proc. Natl. Acad. Sci. USA
9
3, 8220–8224.