been proposed that GSH combines with 1 to form 2c. A
variety of mechanisms have been suggested for this trans-
8
formation, including direct displacement of crotonate, two
9
stepwise 1,4-addition/â-elimination reactions, or 1,4-addition
8
and sigmatropic rearrangement. Such mechanistic specula-
tion seemed premature in the absence of conclusive confir-
mation of the structure of the GSH reaction product. Here
we report the isolation and purification of the GSH adduct
of (-)-COTC and subsequent characterization of that
structure as 2c. In addition, we present quantitative kinetic
information on the interaction of pure 2c with glyoxalase I.
Figure 1. Reciprocal plot of the velocity of the glyoxalase I
reaction (∆A240) versus the concentration of GSH-methylglyoxal
thiohemiacetal ([S]) in the presence and absence of 2c ([I]). In each
kinetic run, the concentration of free GSH was maintained at 0.2
mM by varying the total concentration of GSH and methylglyoxal
7
Freshly prepared synthetic (-)-1 (13.5 mg) was stirred
with GSH (15.4 mg) in sodium phosphate buffer (pH 7.5)
for 10 min at 37 °C and then poured onto an ion-exchange
10
column (2 cm × 2 cm Dowex-1 resin) and eluted with
on the basis of the dissociation constant of the hemithioacetal (Kdiss
11
) 2.2 mM).15 Conditions: 50 mM phosphate buffer, pH 7.0, 25
aqueous acetic acid to afford 2c in 93% yield. Besides the
loss of crotonate and gain of glutathionyl resonances, the
NMR spectrum of 2c featured a singlet at δ 6.74 for H3
that is characteristic of â-unsubstituted cyclohexenones such
as 1 and its congeners.
°
C.
that COTC functions simply by depleting levels of GSH
needed to form the methylglyoxal hemithioacetal substrate.8
Adduct 2c was a moderately potent competitive inhibitor
Studies with a variety of S-substituted glutathionyl deriva-
tives have established that K values decrease with increasing
i
of human erythrocyte glyoxalase I12 (K
) 183 ( 6 µM,
i
Figure 1). Moreover, the inclusion of 0.2 mM COTC 1 in
an assay cuvette did not reduce the initial rate of product
formation beyond what would be expected from a small
hydrophobicity of the sulfur substituent. These findings
suggest the presence of a hydrophobic binding pocket in the
active site of human glyoxalase I, which has recently been
confirmed using high-resolution X-ray crystallographic analy-
13
increase in the production of additional 2c, confirming that
COTC does not inhibit the enzyme.
14
sis. The consequent possibility that less polar analogues
The fact that adduct 2c is a moderately potent competitive
inhibitor of glyoxalase I argues against an earlier suggestion
of 2c may bind more tightly to the enzyme will be explored
further in ongoing structure-activity studies in our labora-
tories.
(
61.
9) Vasella, A.; Baudin, G.; Panza, L. Heteroatom Chem. 1991, 2, 151-
1
(
(
10) Furano, A. V. Methods Enzymol. 1971, 17B, 509-510.
11) For 2c: mp 210-220 °C dec; Rf 0.40 (10:1:5 n-PrOH:AcOH:H2O);
Acknowledgment. This work was supported in part by
grants from the NIH (GM 24054, to B.G.; CA 59612, to
D.J.C.) and the U.S. Army Medical Research and Materiel
Command (to D.J.C.). Support of the Cornell NMR Facility
has been provided by NSF and NIH.
1
H NMR (300 MHz, D2O) δ 6.74 (s, 1 H), 4.51 (dd, 1 H, J ) 4.8, 7.0 Hz),
4
(
.43 (m, 2 H), 3.84 (s, 2 H), 3.75 (dd, 1 H, J ) 6.4, 5.9 Hz), 3.41, 3.28
AB q, 2 H, J ) 14.0 Hz), 2.95, 2.76 (ABX, 2 H, JAB ) 14.0, JAX ) 9.1,
JBX ) 4.8 Hz), 2.48 (m, 2 H, J ) 3.8, 3.9, 2.7 Hz), 2.11 (dd, 2 H, J ) 7.5,
1
3
6
7
(
.5 Hz); C NMR (300 MHz, D2O) δ 196.3, 172.2, 169.6, 144.3, 130.6,
3.4, 72.5, 64.9, 51.4, 50.2, 39.6, 29.8, 28.5, 26.3, 23.3; MS ES m/z 464
OL006341Z
M + 1, 57%), 60 (100%).
12) Aronsson, A.-C.; Tibbelin, G.; Mannervik, B. Anal. Biochem. 1979,
2, 390-393.
13) The rise in [2c] in the assay cuvette was estimated from the second-
(
9
(14) Cameron, A. D.; Ridderstrom, M.; Olin, B.; Kavarana, M. J.;
Creighton, D. J.; Mannervik, B. Biochemistry 1999, 38, 13480-13490.
(15) Hamilton, D. S.; Creighton, D. J. J. Biol. Chem. 1992, 267, 24933-
24936.
(
-
1
order rate constant for formation of 2c from GSH and 1 (k ) 0.12 mM
min , pH 7, 25 °C).
-
1
3144
Org. Lett., Vol. 2, No. 20, 2000