Thioester hydrolysis promoted by a mononuclear zinc complex

Article abstract of DOI:10.1021/ic902322h

The mononuclear zinc complex [(bpta)Zn](CIO₄)₂ · 0.5H₂O promotes the hydrolysis of the thioester PhCH(OH)C(O)SCD₃ when dissolved in CH₃CN:H₂O (50:50 buffered at pH 9.0). This reaction results in the formation of a mixture of CD₃SH and a zinc thiolate complex, the latter of which can be protonated to generate additional CD₃SH. Kinetic studies revealed an overall second-order reaction with an activation energy that is similar to that found for aqueous OH^- promoted thioester hydrolysis. These studies represent the first investigation of chemistry relevant to that occurring in the monozinc-containing form of human glyoxalase II.

Full text of DOI:10.1021/ic902322h

778 Inorg. Chem. 2010, 49, 778–780
DOI: 10.1021/ic902322h
Thioester Hydrolysis Promoted by a Mononuclear Zinc Complex
James J. Danford,† Atta M. Arif,‡ and Lisa M. Berreau*,†
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300 and
‡Department of Chemistry, University of Utah, Salt Lake City, Utah 84112.
Received November 23, 2009
The mononuclear zinc complex [(bpta)Zn](ClO4)2 0.5H2O pro-
containing nitrocefin has been investigated,6 there are no
3
motes the hydrolysis of the thioester PhCH(OH)C(O)SCD3 when
dissolved in CH3CN:H2O (50:50 buffered at pH 9.0). This reaction
results in the formation of a mixture of CD3SH and a zinc thiolate
complex, the latter of which can be protonated to generate
additional CD3SH. Kinetic studies revealed an overall second-
order reaction with an activation energy that is similar to that found
for aqueous OH- promoted thioester hydrolysis. These studies
represent the first investigation of chemistry relevant to that
occurring in the monozinc-containing form of human glyoxalase II.
reports in the literature of detailed studies of a thioester
hydrolysis reaction involving a mononuclear zinc complex.
Such studies would have relevance to human glyoxalase II.
Additionally, these investigations have relevance to a recently
reported Zn-OH-promoted hydrolysis of the thioester
compound thiocoumarin within the active site of carbonic
anhydrase (CA). This reaction results in the formation of a
ring-opened product that is a nanomolar inhibitor of three
CA isozymes.7
We have previously shown that use of an aliphatic,
deuterium-labeled thioester (PhCH(OH)C(O)SCD3), with
2H NMR as the monitoring method, is a feasible approach
for investigating thioester hydrolysis reactions promoted by
dizinc and Fe(III)Zn(II) complexes.8,9 These studies revealed
that the presence of a terminal Zn-OH species enhanced
thioester hydrolysis reactivity in both types of complexes. In
the research outlined herein, we have studied thioester
hydrolysis promoted by the mononuclear zinc complex
The glyoxalase pathway is ubiquitous in biological systems
and involves two metalloenzymes, glyoxalase I (GlxI) and
glyoxalase II (GlxII), with glutathione as an essential cofac-
tor.1 GlxI catalyzes the isomerization of a hemithioacetal of
methyl glyoxal to a thioester, and GlxII then catalyzes the
hydrolysis of the thioester to produce nontoxic products. The
glyoxalase pathway is involved in cellular detoxification and
is important in preventing the formation of advanced glyca-
tion end products, which are linked to aging and various
diseases.2 Because of its critical role in cellular detoxification,
the glyoxalase system is under investigation as a possible
antitumor target.3
Crowder and co-workers recently reported that human
glyoxalase II contains an Fe(II)Zn(II) center but is catalyti-
cally active as a mononuclear zinc enzyme.4 This is similar to
the reactivity found for some metallo-β-lactamases, which
catalyze the hydrolytic ring-opening of β-lactam antibiotics.5
In the monozinc form of both enzymes, a (His)3Zn site
catalyzes the hydrolysis of the substrate. While the reactivity
of a mononuclear Zn-OH complex with the β-lactam-
[(bpta)Zn](ClO4)2 0.5H2O (1). Nitrate and triflate analogs
3
of this complex have been previously reported.6 However,
both were prepared in solution, and no structural or spectro-
scopic data was reported. We have fully characterized 1 using
elemental analysis, 1H and 13C NMR, FTIR, mass spectro-
metry, and X-ray crystallography.10 When crystallized from
CH3CN/Et2O, the cationic portion exhibits facial coordina-
tion of the bpta ligand, with two acetonitrile donors and
one water molecule completing the coordination sphere
(Figure 1). The water ligand is positioned trans to a pyridyl
˚
nitrogen, with a Zn-O distance of 2.110(3) A. The two
coordinated acetonitrile ligands have Zn-N distances of
˚
2.221(3) and 2.185(2) A, respectively. Three distinct Zn-N
distances are found involving the bpta ligand, with the
˚
shortest being Zn(1)-N(3) (2.055 A), which is trans to the
*To whom correspondence should be addressed. E-mail: berreau@
cc.usu.edu.
(1) Mannervik, B. Drug Metabol. Drug. Interact. 2008, 23, 13.
(2) Thornalley, P. J. Drug Metabol. Drug. Interact. 2008, 23, 125.
(3) More, S. S.; Vince, R. J. Med. Chem. 2009, 52, 4650 and references cited
therein.
(4) Limphong, P.; McKinney, R. M.; Adams, N. E.; Bennett, B.; Makar-
off, C. A.; Gunasekera, T.; Crowder, M. W. Biochemistry 2009, 48, 5426.
(5) (a) Hu, Z.; Gunasekera, T. S.; Spadafora, L.; Bennett, B.; Crowder,
M. W. Biochemistry 2008, 47, 7947. (b) Tamilselvi, A.; Mugesh, G. J. Biol.
Inorg. Chem. 2008, 13, 1039.
(6) (a) Kaminskaia, N. V.; He, C.; Lippard, S. J. Inorg. Chem. 2000, 39,
3365. (b) Kaminskaia, N. V.; Spingler, B.; Lippard, S. J. J. Am. Chem. Soc. 2000,
122, 6411.
coordinated water molecule. Upon crushing and drying of
the crystals, the acetonitrile ligands and half of the water is
(7) Maresca, A.; Temperini C.; Pochet, L.; Masereel, B.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. ASAP, 11/13/09.
(8) Berreau, L. M.; Saha, A.; Arif, A. M. Dalton Trans. 2006, 183.
(9) Danford, J. J.; Dobrowolski, P.; Berreau, L. M. Inorg. Chem. 2009, 48,
11352.
(10) 1: C20H29Cl2N5O9Zn, M = 619.75, orthorhombic, Pbca, colorless plate,
3
˚
˚
˚
˚
a = 10.8255(2) A, b = 14.0872(2) A, c = 32.7227(8) A, V = 5245.30(18) A ,
Z = 8, T = 150(1) K, 10038 total reflections, 5736 independent {Rint = 0.0295,
R1 [I > 2sigma(I)] = 0.0443, wR2 (all data) = 0.1160}.
r
pubs.acs.org/IC
Published on Web 12/29/2009
2009 American Chemical Society

Communication
Inorganic Chemistry, Vol. 49, No. 3, 2010 779
Table 1. Rate Constants for the Hydrolysis of PhCH(OH)C(O)SCD3 Promoted
by 1a
temperature (K)
k2 (M-1 s-1
)
299.5
309.5
319.5
329.5
1.87(26) Â 10-3
4.69(24) Â 10-3
1.06(11) Â 10-2
2.44(6) Â 10-2
a [PhCH(OH)C(O)SCD3] = 0.0018 M; [1] = 0.0019-0.027 M; 50:50
CH3CN:H2O [CHES buffer, 0.36 M, I = 0.61 M (NaNO3)].
Figure 1. Cationic portion of [(bpta)Zn(CH3CN)2(H2O)](ClO4)2 (1
2CH3CN 0.5H2O). Hydrogen atoms other than those of the coordinated
3
3
formation of two -SCD3-containing products as identified
by 2H NMR (Figure 2). The more abundant product has a 2H
NMR signal at 1.77 ppm,11 and a smaller signal is found at
2.12 ppm. The latter signal corresponds to CD3SH (pKa =
10.4).9 We note that in the absence of 1, the hydrolysis of
PhCH(OH)C(O)SCD3 under identical conditions results in
the formation of CD3SH as the only sulfur-containing pro-
duct.9 We propose that the species at 1.77 ppm is a zinc
thiolate complex, with an analytical formulation such as
[(bpta)Zn-SCD3(Sol)n]X (sol = H2O or CH3CN).12 The rela-
tive amounts of CD3SH and the zinc thiolate complex
produced in the reaction mixture change as a function of
the 1:thioester ratio (Figure 2), with more of the zinc thiolate
species being produced in solutions having a higher initial
concentration of zinc complex. Using the 10:1 ([1]:thioester)
reaction mixture, which following stoichiometric thioester hy-
drolysis contains only a trace amount of CD3SH (Figure 2),
lowering of the pH to ∼8.6 resulted in the formation of
additional CD3SH (2.12). Further lowering of the pH to 8.3
resulted in additional CD3SH being generated. This data is
consistent with the presence of a Zn-SCD3 complex that can
undergo protonation to release CD3SH. Additional evidence
that the 1.77 ppm species contains a Zn-SCD3 moiety comes
from treatment of the reaction mixture with excess CH3I,
which results in the formation of the sulfonium salt [(CD3)S-
(CH3)2]X (2H NMR 2.59 ppm; Supporting Information).
At pH 9.0, the thioester hydrolysis reaction promoted by 1
is catalytic, with 10 turnovers requiring ∼12 days to go to
completion at 329.5 K. Monitoring the loss of thioester in
single turnover reactions as a function of time at specific con-
centrations of 1 yielded plots from which pseudo first-order
rate constants (kobs) were determined.13 Varying of the con-
centration of 1 from 1.9 to 27 mM, with [PhCH(OH)C(O)-
SCD3]=1.8 mM, produced linear plots of kobs versus [1] from
which second-order rate constants (k2) were determined
(Figure S1 of the Supporting Information; Table 1). Variable
temperature studies in the range of 299.5-329.5 K, and con-
struction of an Eyring plot (Figure S2 of the Supporting
Information), yielded ΔH‡ = 13.6(6) kcal/mol, ΔS‡ =
-25.5(2.0) cal/mol K, and Ea=14.1(6) kcal/mol.
water molecule have been omitted for clarity. Selected bond dis-
˚
tances (A) and angles (°): Zn(1)-N(3) 2.055(2), Zn(1)-O(1) 2.1103(3),
Zn(1)-N(2) 2.130(2), Zn(1)-N(5) 2.185(2), Zn(1)-N(4) 2.221(3),
Zn(1)-N(1) 2.254(2), N(1)-Zn(1)-N(2) 77.48(9), N(2)-Zn(1)-N(3)
100.02(9), and N(1)-Zn(1)-N(3) 80.66(9).
Figure 2. 2H NMR spectra obtained following hydrolysis of PhCH-
(OH)C(O)SCD3 promoted by 1 at pH 9.0. The species at 2.12 ppm has
been identified as CD3SH. The resonance at 1.77 ppm is proposed to be
the signal for [(bpta)Zn-SCD3(Sol)n]X (Sol = CH3CN or H2O; X =
anion present in solution).
lost, leading to an analytical formulation for the dried solid
as [(bpta)Zn](ClO4)2 0.5H2O (1).
3
Kinetic studies of nitrocefin hydrolysis promoted by
[(bpta)Zn](NO3)2 nH2O yielded a kinetic pKa of 7.84(2) for
3
the Zn-OH2 moiety.6 For the thioester hydrolysis studies
reported herein, we have performed all reactions at pH 9.0 to
ensure that a Zn-OH species is present. Admixture of 1.05
equivalents of 1 and one equivalent of PhCH(OH)C(O)SCD3
in 50:50 CH3CN:H2O (buffered at pH 9.0), followed by
heating of this solution at 329.5 K for ∼12 h, results in the
The kinetic data for the hydrolysis of PhCH(OH)C-
(O)SCD3 promoted by 1 revealed that the reaction is second-
order overall with an associative type mechanism that likely
involves nucleophilic attack of the Zn-OH moiety on
the thioester, with no formation of a precursor complex
(Scheme 1). This differentiates thioester hydrolysis promoted
by the mononuclear zinc complex from that found for a
(11) The CDH2CN signal is positioned under the 1.77 ppm resonance.
(12) Mononuclear zinc alkyl thiolate complexes of N3 donor ligands have
been previously isolated and characterized. However, the properties of such
complexes in aqueous solution have not been reported. (a) Brombacher, H.;
Vahrenkamp, H. Inorg. Chem. 2004, 43, 6050. (b) Notni, J.; Gorls, H.; Anders,
E. Eur. J. Inorg. Chem. 2006, 1444. (c) Puerta, D. T.; Cohen, S. M. Inorg. Chem.
2002, 41, 5075. (d) Tekeste, T.; Vahrenkamp, H. Inorg. Chem. 2006, 45, 10799.
(e) Boerzel, H.; Koeckert, M; Bu, W.; Spingler, B.; Lippard, S. J. Inorg. Chem.
2003, 42, 1604. (f) Brand, U.; Rombach, M.; Seebacher, J.; Vahrenkamp, H.
Inorg. Chem. 2001, 40, 6151. (g) Matsunaga, Y.; Fujisawa, K.; Amir, N.;
Miyashita, Y.; Okamoto, K. Trans. Met. Chem. 2006, 31, 897. (h) Ruf, M.;
Burth, R.; Weis, K.; Vahrenkamp, H. Chem. Ber. 1996, 129, 1251. (i) Walz, R.;
Weis, K; Ruf, M.; Vahrenkamp, H. Chem. Ber. 1997, 130, 975. (j) Calvo, J. A. M.;
Vahrenkamp, H. Inorg. Chim. Acta 2006, 359, 4079. (k) Alsfasser, R.; Powell,
A. K.; Vahrenkamp, H.; Trofimenko, S. Chem. Ber. 1993, 126, 685.
(13) Rates for the buffer promoted thioester hydrolysis reaction were
determined at temperatures of 299.5-329.5 K. These values were subtracted
from those obtained for the reactions involving 1 as described in the
literature. diTargiani, R. C.; Chang, S.; Salter, M. H., Jr.; Hancock,
R. D.; Goldberg, D. P. Inorg. Chem. 2003, 42, 5825.

780 Inorganic Chemistry, Vol. 49, No. 3, 2010
Danford et al.
Scheme 1. Comparison of the Thioester Hydrolysis Reaction
Pathways Involving Binuclear Fe(III)Zn(II) and Mononuclear Zn(II)
Complexes
Scheme 2. Thioester Hydrolysis Promoted by 1
Table 2. Activation Energies for OH- and Zn-OH-Promoted Thioester and
Ester Hydrolysis Reactions
nucleophile
thioester or ester
Ea (kcal/mol)
Fe(III)Zn(II) complex, which mimics a common binuclear
core found in glyoxalase II enzymes.9 In reactions involving
the Fe(III)Zn(II) complex, saturation kinetic behavior was
interpreted as indicating equilibrium coordination of the
deprotonated R-hydroxy group of the thioester to the Zn(II)
prior to nucleophilic attack by an Fe(III)-OH moiety
(Scheme 1). This coordination was proposed to occur via
reaction of a Zn-OH moiety with the R-hydroxy group of
the thioester.9 With this in mind, we propose that the lack of
thioester coordination to 1, and the observation of a simple
second-order reaction, is due to differences in the basicity of
the Zn-OH moieties of 1 and the Fe(III)Zn(II) complex,
with the latter being a better base, thus enabling deprotona-
tion of the thioester R-hydroxyl group. The thioester hydro-
lysis reaction promoted by the Fe(III)Zn(II) complex is >17-
fold faster than the reaction performed under identical
conditions using 1.9 With 10 equiv of Fe(III)Zn(II) complex
or 1 present, the rate of thioester hydrolysis is enhanced over
the background reaction by approximately 670- or 38-fold,
respectively. Thus, the equilibrium coordination of the de-
protonated thioester to the Zn(II) center of the Fe(III)Zn(II)
complex facilitates the hydrolysis reaction by positioning the
thioester carbonyl for nucleophilic attack by the Fe(III)-OH
moiety. In terms of leaving group stabilization, for the reac-
tion involving the Fe(III)Zn(II) complex, thiolate coordina-
tion was proposed to occur at the Fe(III) center on the basis
of the formation of a disulfide side product that likely results
from redox chemistry involving an Fe-SCD3 moiety. For
the thioester hydrolysis reaction promoted by 1, the thiolate
leaving group is stabilized via zinc coordination. For both
systems, catalytic turnover presumably occurs via protona-
tion of the M-SCD3 species and generation of the reactive
metal-hydroxide species. The proposed pathway for the
thioester hydrolysis reaction promoted by 1 is shown in
Scheme 2.
OH-a
1d
n-BTAb
TEe
13.9c
14.1(6)
10.3(1)
11.7(1)
10.8(1)
OH-h
4-NPAf
4-NPA
4-NPA
g
[([12]aneN3)Zn-OH]ClO4h
[([12]aneN4)Zn-OH]ClO4
a Ref 14. b n-BTA = n-butylthioacetate. c Temperature = 298 K.
d This work. e TE = PhCH(OH)C(O)SCD3. f 4-NPA = 4-nitrophenyl-
acetate. g Ref 15. h Ref 16.
suggests that both reactions proceed via a rate-determining
nucleophilic attack of hydroxide (either free or zinc-bound)
on the thioester carbonyl carbon. Similar results have been
reported for the hydrolysis of 4-nitrophenylacetate promoted
by OH- or mononuclear zinc complexes (Table 2).15,16
In summary, we report studies of thioester hydrolysis
promoted by a mononuclear zinc complex in a mixed
organic:aqueous environment. This reaction is relevant to
the chemistry of the monozinc-containing form of human
GlxII. In comparing the results of this study with those
derived from an investigation involving a Fe(III)Zn(II)
complex,9 we found that the binuclear metal complex more
effectively promotes thioester hydrolysis. In a broader con-
text, our results provide evidence to support the notion that
Zn-OH promoted ester and thioester hydrolysis reactions
can proceed via similar reaction pathways. This is relevant to
the hydrolysis of coumarin and thiocoumarin derivatives
within the active site of CA enzymes.7
Acknowledgment. Funding in support of this research
was provided by the Herman Frasch Foundation (501-
HF02).
Supporting Information Available: Synthetic and character-
ization details for 1, experimental details for thioester hydrolysis
reactions, plot of kobs versus [1] at 299.5-329.5 K, and Eyring
plot. This material is available free of charge via the Internet at
http://pubs.acs.org.
We note that the activation energy for the thioester
hydrolysis reaction promoted by 1 is similar to that found
for the alkaline hydrolysis of n-butylthioacetate.14 This
(15) Kimura, E.; Nakamura, I.; Koike, T.; Shionoya, M.; Kodama, Y.;
Ikeda, T.; Shiro, M. J. Am. Chem. Soc. 1994, 116, 4764.
(16) Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc. 1994, 116,
8443.
(14) Noda, L. H.; Kuby, S. A.; Lardy, H. A. J. Am. Chem. Soc. 1953, 75,
913.