Metallo-β-lactamase-catalyzed hydrolysis of cephalosporins: Some mechanistic insights into the effect of heterocyclic thiones on enzyme activity

Article abstract of DOI:10.1021/ic100253k

The hydrolysis of β-lactam antibiotics using zinc-containing metallo-β-lactamases (mβl) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic-SR side chains are less prone to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MTT, MDT, DMETT, and MMA, the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic-SR moieties eliminated from the β-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms, these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear zinc(II) thiolate complexes indicates that the-SR side chains eliminated from the antibiotics may interact with the zinc(II) metal center of mβl through their sulfur atoms.

Full text of DOI:10.1021/ic100253k

Inorg. Chem. 2011, 50, 749–756 749
DOI: 10.1021/ic100253k
Metallo-β-lactamase-Catalyzed Hydrolysis of Cephalosporins: Some Mechanistic
Insights into the Effect of Heterocyclic Thiones on Enzyme Activity
A. Tamilselvi and Govindasamy Mugesh*
Department of Inorganic & Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
Received February 8, 2010
The hydrolysis of β-lactam antibiotics using zinc-containing metallo-β-lactamases (mβl) is one of the major bacterial
defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation
of cephalosporins, cephamycins, and imipenem. It is shown in this paper that the cephalosporins having heterocyclic
-
SR side chains are less prone to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This
is partly due to the inhibition of enzyme activity by the thione moieties eliminated during hydrolysis. When the enzymatic
hydrolysis of oxacillin was carried out in the presence of heterocyclic thiones such as MTT, MDT, DMETT, and MMA,
the catalytic activity of the enzyme was inhibited significantly by these compounds. Although the heterocyclic -SR
moieties eliminated from the β-lactams upon hydrolysis undergo a rapid tautomerism between thione and thiol forms,
these compounds act as thiolate ligands toward zinc(II) ions. The structural characterization of two model tetranuclear
zinc(II) thiolate complexes indicates that the -SR side chains eliminated from the antibiotics may interact with the
zinc(II) metal center of mβl through their sulfur atoms.
4
Introduction
lyze serine-β-lactamase inhibitors such as clavulanic acid.
Therefore, the medicinal application of the most commonly
used antibiotics is severely compromised in bacteria that
produce the metalloenzymes.
Antibiotics based on β-lactams such as penicillins, cepha-
losporins, and penems are the most commonly used drugs for
bacterial infections. However, the medicinal applications of
1
The crystal structures of several mβl enzymes reveal a
binuclear zinc center in which the two zinc ions are connected
by a bridging hydroxide group. As the mβls are not suscep-
tible to inhibition by the classic serine-β-lactamase inhibitors
such as clavulanic acid, sulbactam, and tazobactam, con-
siderable attention has been directed toward the design and
synthesis of effective mβl inhibitors. These inhibitors include
biphenyl tetrazoles, N-arylsulfonyl hydrazones, β-lactams,
benzohydroxamic acid analogues, picolinic acids and its
derivatives, trifluoromethyl ketones, succinic acids, and5
thiols such as cysteinyl peptides and thiomandelic acid.
Payne et al. demonstrated that mercaptoacetic acid, a pro-
duct of the hydrolysis of mercaptoacetic acid thiol esters by
mβl, binds irreversibly to the enzyme through the formation
most of these antibiotics are limited due to the production of
β-lactamases that inactivate the antibiotics by hydrolyzing
the β-lactam ring (Scheme 1). The active site of these bacterial
enzymes contains either a serine residue (serine-β-lactamases)
2,3
or zinc(II) ions (metallo-β-lactamases, mβl). The metal-
loenzymes (mβl) represent a unique subset of zinc hydrolases
that can hydrolyze a wide range of β-lactam antibiotics,
including cephamycins and imipenems, both of which have
relatively more stable C-N bonds as compared to the
3
penicillin family of antibiotics. These enzymes also hydro-
*
To whom correspondence should be addressed. E-mail: mugesh@
ipc.iisc.ernet.in.
1) (a) Woodward, R. B. In Recent Advances in the Chemistry of b-Lactam
(
6
Antibiotics; Elks, J., Ed.; Chemical Society: London, 1977; pp 167-180. (b)
Waley, S. G. In The Chemistry of b-Lactams; Page, M. I., Ed.; Chapman and
Hall: London, 1992; pp 198-228.
of a disulfide bond with the active site cysteine residue. Page
and co-workers reported that the degradation of cephalos-
porins by mβl produces thiols that can bind to the zinc center
(
2) (a) Medeires, A. A. Br. Med. Bull. 1984, 46, 18–27. (b) Livermore, D. M.
Clinical Microbiol. Rev. 1995, 8, 557–584. (c) Rasmussen, B. A.; Bush, K.
Antimicrob. Agents Chemother. 1997, 41, 223–232. (d) Livermore, D. M.
J. Antimicrob. Chemother., Suppl. D 1998, 41, 25–41. (e) Fisher, J. F.; Meroueh,
S. O.; Mobashery, S. Chem. Rev. 2005, 105, 395–424.
(5) (a) Walsh, T. R.; Toleman, M. A.; Poirel, L.; Nordmann, P. Clin.
Microbiol. Rev. 2005, 18, 306–325. (b) Toney, J. H.; Moloughney, J. G. Curr.
Opin. Invest. Drugs 2004, 5, 823–826.
(6) Payne, D. J.; Bateson, J. H.; Gasson, B. C.; Proctor, D.; Khushi, T.;
Farmer, T. H.; Tolson, D. A.; Bell, D.; Skett, P. W.; Marshall, A. C.; Reid,
R.; Ghosez, L.; Combret, Y.; Marchand-Brynaert, J. Antimicrob. Agents
Chemother. 1997, 41, 135–140.
(
3) (a) Bush, K. Clin. Infect. Diseases 1998, 27, S48–S53. (b) Cricco, J. A.;
Orellano, E. G.; Rasia, R. M.; Ceccarelli, E. A.; Vila, A. J. Coord. Chem. Rev.
999, 190-192, 519–535. (c) Walsh, T. R.; Toleman, M. A.; Poirel, L.;
1
Nordmann, P. Clin. Microbiol. Rev. 2005, 18, 306–325. (d) Crowder, M. W.;
Spencer, J.; Vila, A. J. Acc. Chem. Res. 2006, 39, 721–728 and references therein.
(
4) Higgins, P. G.; Wisplinghoff, H.; Stefanik, D.; Seifert, H. Antimicrob.
(7) Badarau, A.; Llinas, A.; Laws, A. P.; Damblon, C.; Page, M. I.
Biochemistry 2005, 44, 8578–8589.
Agents Chemother. 2004, 48, 1586–1592.
r 2011 American Chemical Society
Published on Web 01/06/2011
pubs.acs.org/IC

7
50 Inorganic Chemistry, Vol. 50, No. 3, 2011
Tamilselvi and Mugesh
Scheme 1. Enzymatic Hydrolysis of the β-Lactam Ring in Penicillin G
by β-Lactamases, Leading to the Formation of Penicilloic Acid
Scheme 2. Elimination of Heterocyclic Thione from the Hydrolysis of
the β-Lactam Ring in Certain Cephalosporins by β-Lactamases
7
and inhibit the enzyme activity. Kurosaki et al. have shown
that the thiols capable of blocking the dinuclear zinc center
can irreversibly inhibit the enzyme by forming stable thiolate-
8
bridged complexes.
to avoid any possible decomposition. The concentration of the
-
3
Recently, we have reported that the mβl-mediated hydro-
lysis of antibiotics having heterocyclic thiol side chains
eliminates thiones, leading to the generation of potent heme
stock solution of β-lactams was fixed at 5 ꢀ 10 M. In a typical
kinetic experiment, a sample vial containing 1.5 mL of the test
samples containing an appropriate concentration of β-lactam
and 0.44 nM BcII enzyme was incubated. At various time
intervals, 10 μL aliquots were removed from the reaction
mixture and injected directly into the HPLC column. The
compounds were eluted in the linear gradient mode with a
mixture of acetonitrile and 0.1% TFA (50% to 80% in the case
of penicillin G, oxacillin; 30% to 80% in case of moxalactam)
over 8.5 min at a flow rate of 1.0 mL/min. The chromatograms
were obtained at 254 nm for penicillin G and oxacillin and at 275
nm for moxalactam. The concentration of β-lactam or the
hydrolyzed product was determined for each injection from
the peak area by using a calibration plot.
9
peroxidase inhibitors. For example, the hydrolysis of cepha-
losporins having a thio-tetrazole side chain leads to the
formation of 1-methyl-1H-tetrazole-5-thione (MTT, 1;
Scheme 2), which effectively inhibits the lactoperoxidase
(
LPO)-catalyzed iodination reactions. However, it is un-
known whether the heterocyclic thiones eliminated from
cephalosporins by enzymatic hydrolysis can inhibit the mβl
activity. In this paper, we report that the heterocyclic thiones
such as MTT eliminated during hydrolysis by the metalloen-
zyme significantly inhibit the enzyme activity. We also show
that the thiones act as good ligands to produce zinc-thiolate
complexes, indicating that the inhibition of mβl is due to the
binding of thiones to the zinc(II) center.
Synthesis of Heterocyclic Disulfides. Synthesis of MTT Di-
sulfide (MTTox, 11). To a mixture of 1-methyl-1H-tetrazole-5-
thione (MTT, 0.58 g, 5.0 mmol) and sodium bicarbonate (0.63 g,
7
.5 mmol) was added dropwise a 40 mL solution of I (0.63 g, 2.5
2
mmol) in a 50 mM KI solution with constant stirring. The
reaction was stirred for 45 min, and the resulting mixture was
extracted with chloroform. The organic layer was washed with a
brine solution and dried over anhydrous Na SO . The solvent
Experimental Section
Chemicals. The sodium salt of penicillin G and oxacillin were
obtained from Fluka. Penicillinase (β-lactamase II, BcII) from
Bacillus cereus and deuterated solvents CDCl , D O, DMSO-d ,
3 2 6
2
4
was removed under vacuum conditions to afford a white solid.
The product was recrystallized from chloroform to afford MTT
disulfide as white crystals. Yield: 0.33 g (26%). H NMR
and CD OD were obtained from Sigma-Aldrich. Potassium
3
1
dihydrogen phosphate and dipotassium hydrogen phosphate
were obtained from other commercial sources and used as
received. HPLC-grade solvents for the kinetic experiments were
obtained from Merck.
1
3
(
CDCl ) δ (ppm): 4.19 (s, 6H). C NMR (CDCl ) δ (ppm):
3 3
þ
3
Found: 253.0059.
5.3, 151.4. ESI-MS (m/z) calcd for (M þ Na) : 253.0055.
Synthesis of MDT Disulfide (MDTox, 12). To a mixture of
General Procedure. All chemical reactions were carried out in
a room atmosphere, unless specifically mentioned otherwise.
Buffers of desired pH were freshly prepared prior to use. The
HPLC experiments were performed on an analytical HPLC
system fitted with a PDA detector controlled by the EMPOW-
ER software (Waters Corporation, Milford MA). The reaction
products were isolated and purified by using reverse-phase flash
chromatography (Biotage) and preparative HPLC (Waters
Corporation, Milford MA) systems. The final products were
obtained from the column fractions by freeze-drying the sam-
2
-methyl-1,3,4-thiadiazole-2-thione (MDT, 0.68 g, 5.1 mmol)
and sodium bicarbonate (0.63 g, 7.5 mmol) was added dropwise
a 40 mL solution of I (0.63 g, 2.5 mmol) in a 50 mM KI solution
2
with constant stirring. The reaction mixture was stirred for 45
min, and the resulting solution was extracted two times with
chloroform (2 ꢀ 50 mL). The combined organic layers was
2 4
washed with brine solution and dried over anhydrous Na SO .
Evaporation of the solvent afforded the product as a white solid.
The desired compound was obtained in pure form by recrystal-
lization of the crude solid from chloroform. Yield: 0.63 g (44%).
1
13
ples on a Lyophilizer (Eyela). H (400 MHz) and C (100 MHz)
NMR spectra were obtained on a Bruker-Avance 400 NMR
Spectrometer. Chemical shifts are cited in ppm with respect
1
13
H NMR (CDCl
3
) δ (ppm): 2.79 (s, 6H). C NMR (CDCl
3
) δ
þ
(
2
ppm): 16.1, 166.1, 168.9. ESI-MS (m/z) calcd for (M þ Na) :
84.9373. Found: 284.9357.
4
to SiMe as an internal standard. Mass spectral studies were
carried out on a Q-TOF micro mass spectrometer or with ESI-
MS mode analysis. Elemental analyses were performed on a
Thermo Finnigan CHN analyzer.
Synthesis of Zinc(II)-Thiolate Complexes. Synthesis of
Complex 19. The ligand 2,6-bis((dimethylamino)methyl)-4-
methyl phenol (HL1; 0.22 g, 1.0 mmol) in dichloromethane
(
(
5 mL) was mixed with 60% NaH (0.04 g, 0.9 mmol) in hexane
25 mL), and the mixture was stirred at room temperature for 1 h
HPLC Studies. The kinetics of the hydrolysis of β-lactams
were followed with HPLC by using a reverse-phase column. All
reactions with BcII were carried out in 50 mM HEPES buffer at
a pH of 7.5. The stock solutions of β-lactams and BcII were
prepared in a HEPES buffer and 50 mM phosphate buffer at a
pH of 7.5, respectively, and used immediately in the experiments
to obtain the corresponding sodium phenolate (NaL1). Zn-
OAc) 2H O (0.33 g, 1.5 mmol) and 1-(2-(dimethylamino)-
(
2
2
3
ethyl)-1H-tetrazole-5-thione (DMETT, 0.26 g, 1.5 mmol) were
then added, and the stirring was continued for an additional 3 h.
This resulted in a turbid solution from which the zinc complex
[
4 2 2 4
Zn L1 (μ-OH) (DMETT) ] was filtered off as a white solid.
(
8) Kurosaki, H.; Yamaguchi, Y.; Higashi, T.; Soga, K.; Matsueda, S.;
The product was dried under vacuum conditions to afford
a white crystalline solid. The solid product was dissolved
in a dichloromethane/methanol mixture and was kept for
Yumoto, H.; Misumi, S.; Yamagata, Y.; Arakawa, Y.; Goto, M. Angew.
Chem., Int. Ed. 2005, 44, 3861–3864.
(
9) Tamilselvi, A.; Mugesh, G. ChemMedChem 2009, 4, 512–516.

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 751
crystallization. Upon standing at room temperature, the
product crystallized out as white needles suitable for single
1
H
crystal X-ray diffraction analysis. Yield: 0.16 g (11%).
NMR (CD OD), δ (ppm): 2.21 (s, 3H), 2.27 (s, 12H), 2.54
3
(
6
s, 12H), 2.80 (t, J = 6.8 Hz, 2H), 3.30 (s, 2H), 4.38 (t, J =
.8 Hz, 2H), 7.49 (t, J = 3.2 Hz, 1H). C NMR (CD OD),
3
1
3
δ (ppm): 19.4, 21.5, 44.5, 44.8, 57.0, 61.1, 63.2, 119.4, 124.2,
þ
1
32.5, 162.8, 180.3. ESI-MS (m/z) calcd for complex 19 (M) :
1
O
420.3. Found: 1419.8. Elem anal. calcd (%) for C48
Zn : C, 38.71; H, 5.93; N, 23.56. Found: C, 39.21; H, 5.88;
N, 22.79.
84 24
H N -
4
S
4
4
Synthesis of Complex 20. The ligand 2,6-bis((dimethylamino)-
methyl)-4-methyl phenol (HL1; 0.22 g, 1.0 mmol) in dichloro-
methane (5 mL) and 60% NaH (0.04 g, 0.9 mmol) in hexane (25
mL) were mixed and stirred at room temperature for 1 h to obtain
the corresponding sodium phenolate (NaL1). Zn(OAc) 2H O
2
3
2
(
0.33 g, 1.5 mmol) and MDT (0.20 g, 1.5 mmol) were then added,
and the stirring was continued for an additional 3 h. This resulted in
a turbid solution from which the zinc complex [Zn L1 (μ-OH)
MDT) ] was filtered off as a white solid. The product was dried
Figure 1. Chemical structures of the cephalosporins having various
0
substitution at the 3 position.
4
2
2
-
13
views of the complexes were obtained using PLATON or
ORTEP.
(
14
4
under vacuum conditions to afford a white crystalline solid. The
solid product was dissolved in a dichloromethane/methanol mixture
and was kept for crystallization. Upon standing at room tempera-
ture, the product crystallized out as white needles suitable for single
Computational Methods
All calculations were performed using the Gaussian 03
suite of quantum chemical programs. The hybrid Becke-3-
1
15
crystal X-ray diffraction analysis. Yield: 0.19 g (15%). H NMR
(
CD
3
OD), δ (ppm): 2.21 (s, 3H), 2.36 (s, 3H), 2.80 (t, 2H), 3.28 (s,
Lee-Yang-Parr (B3LYP) exchange correlation functional
was applied for DFT calculations. Geometries were fully
13
16
12H). C NMR (CD OD), δ (ppm): 16.2, 20.6, 23.3, 46.5, 60.3,
3
1
20.0, 123.1, 132.5, 161.8, 176.3. ESI-MS (m/z) calcd for complex 20
þ Na]
optimized at the B3LYP level of theory using 6-31þG(d)
basis sets. All stationary points were characterized as minima
by corresponding Hessian indices. Transition states were
optimized using the TS keyword. Furthermore, the transition
states and the stable conformers were characterized by the
presence or absence of a single imaginary frequency. IRC
calculations were done at the B3LYP/6-31þG(d) level on
B3LYP/6-311þþG(d,p) level-optimized geometries to verify
a transition structure of the hydrogen transfer involved in the
interconversion of different tautomeric forms. The activation
energies are the difference in the electronic energy corrected
for zero-point vibrational energy between the transition state
and the stable conformations.
þ
2þ
(
(
(
M þ Na) : 1278.9. Found: 1131.0 ([C35
H
52
N
10
O
3
S
6
Zn
4
one MDT and one hydroxide ligands eliminated). Elem anal. calcd
%) for C H N O S Zn : C, 36.14; H, 4.47; N, 13.31. Found: C,
38
56 12
4
8
4
35.86; H, 5.02; N, 13.05.
X-Ray Crystallography. X-ray crystallographic studies were
carried out on a Bruker CCD diffractometer with graphite-
monochromatized Mo KR radiation (λ = 0.71073 A) controlled
by a pentium-based PC running the SMART software
package. Single crystals were mounted at room temperature
on the ends of glass fiber, and data were collected at 293 K.
Intensity data were measured in frames with increasing ω (width
of 0.3°/frame) at a scan speed of 18 s/frame, and the SMART
and SAINT software were used for data acquisition and data
extraction, respectively. The structures were solved and refined
using the SHELXTL software package. All non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were as-
signed at idealized locations. Empirical absorption corrections
17
˚
10
Results and Discussion
1
1
Hydrolysis of Cephalosporins by Metallo-β-lactamase.
It is known that the hydrolysis of the penicillin family
of antibiotics (e.g., penicillin G and oxacillin (2)) by
β-lactamases is generally much faster than that of most
of the cephalosporins (e.g., cefazolin (3), moxalactam (4),
and cefmetazole (5)) and penem-based compounds. It has
also been shown that the hydrolysis of cephalosporins by
either mβls or transpeptidase leads to the elimination of
1
2
were applied to all structures using SADABS. The perspective
(10) SMART, version 5.05; Bruker AXS: Madison, WI, 1998.
(11) (a) Sheldrick, G. M. SHELXTL, V97-2; Siemens Industrial Automa-
tion Inc.: Madison, WI, 1997. (b) Sheldrick, G. M. SHELX-97; University of
G €o ttingen: G €o ttingen, Germany, 1997.
(
12) Sheldrick, G. M. SADABS, Version 2; University of G €o ttingen:
0
the side chain at the 3 position with the formation of
G €o ttingen, Germany, 2001.
1
8
(
(
13) Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7–13.
cephalosporonic acid (7) (Figure 1). Page et al. have
reported an interesting observation that the hydrolysis of
cephalosporins generates dihydrothiazines, which undergo
isomerization at C-6 by the cleavage of the C-S bond to
14) Johnson, C. K. ORTEP, Report ORNL-5138; Oak Ridge National
Laboratory: Oak Ridge, TN, 1976.
(
15) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T., Jr.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada,
M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian,
H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala,
P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.;
Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.;
Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.;
Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill,
P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian 03, Revision C.01; Gaussian, Inc.: Wallingford, CT, 2004.
7
produce the corresponding thiol intermediates. They
have shown that these thiol intermediates generated
during the reaction can be trapped by β-lactamase
from Bacillus cereus, causing inhibition of the enzyme.
(16) (a) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785–789. (b)
Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652.
(17) (a) Fukui, K. Acc. Chem. Res. 1981, 14, 363–368. (b) Gonzalez, C.;
Schlegel, H. B. J. Chem. Phys. 1989, 90, 2154–2161. (c) Gonzalez, C.; Schlegel,
H. B. J. Chem. Phys. 1991, 95, 5853–5860.
(18) (a) Buckwell, S. C.; Page, M. I.; Longridge, J. L.; Waley, S. G. J.
Chem. Soc., Perkin Trans. 2 1988, 1823–1827. (b) Mustafi, D.; Knock, M. M.;
Shaw, R. W.; Makinen, M. W. J. Am. Chem. Soc. 1997, 119, 12619–12628.

7
52 Inorganic Chemistry, Vol. 50, No. 3, 2011
Tamilselvi and Mugesh
0
Table 1. The Initial Rate (v ) and % Conversion for the Hydrolyses of Penicillins and Cephalosporins by BcII at Room Temperature
(μM min-
1 a
)
time (min)
b
conversion (%)
entry
substrate
[BcII] (M)
v
0
c
-8
2
a
b
c
d
e
penicillin G
oxacillin (2)
3.04 ꢀ 10
3.04 ꢀ 10
3.04 ꢀ 10
3.04 ꢀ 10
2.36 ꢀ 10
4.675 ꢀ 10
80
412
487
666
50
100
50
10
05
50
c
-8
-7
-7
-7
2.022
0.927
c
moxalactam (4)
cefmetazole (5)
nitrocefin (6)
c
0.354
0.449
d
a
b
Calculated from the initial 5-10% of the reaction by following the decrease in the peak area due to substrates (β-lactam). Time required for the %
conversion of the hydrolysis of the substrate was determined in 50 mM HEPES buffer, pH 7.5, by following the decreasein the peak area due to β-lactams.
[β-lactam]: 1.00 ꢀ 10 M. The hydrolysis was followed by UV-vis spectrophotometry under identical conditions. [nitrocefin]: 0.05 ꢀ 10 M. DMSO
was used to dissolve nitrocefin, but its concentration does not exceed 2.5% in the reaction mixture.
c
-3
d
-3
Table 2. Inhibition of Metallo-β-lactamase Activity by Various Heterocyclic
Thiones
a
inhibition of mβl
b
s. no.
compound
IC50 (μM)
i
K (μM)
1
2
3
4
5
6
7
8
MTT (1)
MDT (8)
65.78 ( 3.55
47.55 ( 0.02
36.64 ( 2.21
43.64 ( 2.89
47.40 ( 4.80
23.75 ( 0.16
0.68 ( 0.01
2.98 ( 0.35
18.33 ( 1.24
15.07 ( 0.48
9.77 ( 0.45
15.57 ( 1.12
17.51 ( 0.74
3.73 ( 0.12
0.25 ( 0.02
0.94 ( 0.30
DMETT (9)
MMA (10)
MTTox (11)
MDTox (12)
MAA (13)
MPA (14)
Figure 2. Thione and disulfide forms of the side chains eliminated
during the hydrolysis of various cephalosporins.
Recently, we have shown that the mβl-catalyzed hydro-
lysis of antibiotics having heterocyclic -SR side chains
eliminates the thiol moieties in addition to the C-N bond
cleavage at the β-lactam ring. These thiols undergo a
rapid tautomerism to produce the corresponding
a
Inhibition studies were carried out for the mβl-mediated hydrolysis
-3
of oxacillin. [BcII] = 44.0 nM; [oxacillin] = 1 ꢀ 10 M; 50 mM HEPES,
b
pH = 7.5. The IC50 values were determined from the inhibition plots
obtained by plotting the percentage control activity against the concen-
tration of inhibitors.
9
thiones, which may bind to the enzyme active site,
was much faster than that of moxalactam and cefmeta-
zole. Although moxalactam, cefmetazole, and nitrocefin
belong to the family of cephalosporins, nitrocefin does
leading to a significant product inhibition at higher
concentrations of the products. To test this hypothesis,
we have studied the hydrolysis of various β-lactams by the
binuclear metallo-β-lactamase from Bacillus cereus (BcII)
using a HPLC method. The amounts of products formed
at a given time were determined from the peak areas. For
the hydrolysis of the chromogenic substrate nitrocefin (6),
0
not contain any heterocyclic thiols at the 3 position.
These observations indicate that the thiols/thiones elimi-
nated during the hydrolysis may inhibit the enzyme
activity by coordinating to the metal center at the active
site.
an UV-vis method was employed. The initial rates (v )
0
When the hydrolysis of oxacillin by mβl was carried out
in the presence of heterocyclic thiones, the rate of hydro-
lysis decreased with an increase in the concentration of
thiones. This indicates that the thiones inhibit the mβl
activity, and the inhibition potency depends on the nature
of thiones (Figure 2). The variation in the IC50 values for
the inhibition of mβl activity by various thiones (Table 2)
is probably due to the differences in the structure of the
thiones. For example, MDT having a thiadiazole moiety
is more efficient (IC : 47.50 ( 0.02 μM) than MTT that
for the hydrolysis were calculated by following the reac-
tions during the first 5-10% of the conversion, where the
concentrations of the products were relatively low.
As expected, penicillin G and oxacillin were hydrolyzed
by mβl much faster than the cephalosporins (Table 1).
The initial rate (v ) for the hydrolysis of penicillin G was
0
found to be almost 2 orders of magnitude higher than that
of oxacillin (Table 1, entry a and b). In contrast to
oxacillin and cephalosporins, a complete hydrolysis was
observed when penicillin G was used as the substrate.
Although the BcII enzyme was able to hydrolyze the
5
0
contains a tetrazole ring (IC : 65.78 ( 3.55 μM). Simi-
5
0
larly, DMETT (9) and 2-(2-mercapto-4-methylthiazol-
-yl)acetic acid (MMA, 10), which are present in the
extended spectrum third generation antibiotics cefo-
-
7
cephalosporins at higher concentrations (3.04 ꢀ 10
5
M), the initial rates were found to be much lower than
that observed for penicillin G and oxacillin. However, a
significant decrease in the reaction rates was observed
after ∼5-10% of the hydrolysis. For example, moxalac-
tam (Table 1, entry c), which belongs to the latest
generation of the cephalosporin family of antibiotics,
was hydrolyzed much faster in the beginning of the
1
9
20
dizime and cefotiam, respectively, are more effective
than MTT. Although DMETT contains a tetrazole ring
similar to that of MTT, the presence of a -CH CH -
2
2
NMe group may increase the reactivity of the sulfur
2
moiety. Furthermore, the amino group may provide a
further stabilization to the enzyme-inhibitor complex if
the inhibition is due to the coordination of the sulfur
-
1
reaction (v : 0.927 μM min ), but the reaction rate
0
became very slow after ∼10% of the substrate was
hydrolyzed. Similarly, the hydrolysis of cefmetazole
(
(
19) Klesel, N.; Limberti, M.; Seeger, K.; Seibert, G.; Winkler, I.;
Schrinner, E. J. Antibiot. 1984, 37, 901–909.
20) Tsuchiya, K.; Kida, M.; Kondo, M.; Ono, H.; Takeuchi, M.; Nishi,
T. Antimicrob. Agents Chemother. 1978, 14, 557–568.
Table 1, entry d) was also found to be very slow, and
only 5% hydrolysis was observed after 666 min. In
contrast, the hydrolysis of nitrocefin (Table 1, entry e)
(

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 753
Figure 3. Chemical structures of some aliphatic thiols used as inhibitors
for mβls.
moiety to the zinc(II) center. It has been previously shown
that the N,N-dimethyl amino group deprotonates thiols/
selenols to generate more reactive thiolates/selenolates,
2
1
which can enhance the nucleophilicity of thiols/selenols.
Figure 4. Inhibition of β-lactamase activity by MTT (a) for serine-β-
-8
The disulfides MTTox (11) and MDTox (12) also inhibited
the enzyme activity, as these disulfides are converted
rapidly to the corresponding thiones MTT and MDT,
respectively, in the assay mixture (Figure S11, Supporting
Information). As cleavage of the disulfide bond can
produce two thiones, the IC50 values for the disulfides
were found to be lower than that of the corresponding
thiones.
The irreversible inhibition of mβl from Bacillus cereus
BcII) by mercapto acetic acid thiol ester has been
ascribed to the cleavage of the thiol ester bond in this
compound during the hydrolysis to produce mercapto
acetic acid (13, MAA, Figure 3). While compound 13 has
been shown to form a disulfide bond with the active site
lactamase and (b) metallo-β-lactamase. Conditions: [mβl] = 4.44 ꢀ 10
- -3
7
M; [sβl] = 1.67 ꢀ 10 M; [oxacillin] = 1 ꢀ 10 M; 50 mM HEPES, pH
7
.5.
Table 3. Calculated Energy for Thiol-Water, Transition State-Water, and
Thione-Water Complexes
Δ(E þ ZPE)
Δ(H þ ZPE)
Δ(G þ ZPE)
-
H
2
O
þ H
2
O
- H
2
O
þ H
2
O
- H
2
O
2
þ H O
1
1
1
-thiol
-TS
-thione
0
0
0
0
0
0
(
32.12
-7.81
0
28.75
-9.24
0
6.40
-8.74
0
6.92
-9.96
0
31.78
-8.10
0
37.90
-9.52
0
5.42
-8.86
0
32.22
-7.58
0
37.97
-8.96
0
7.59
8.49
0
8.33
-9.76
0
8-thiol
8-TS
8
9-thiol
9
9
5.88
-thione
-10.07
0
5.47
6
cysteine, larger mercaptocarboxylates such as 15 have
been shown to induce conformational changes at the
-TS
-thione
31.89
-7.54
6.48
-8.69
31.47
-7.91
32.55
-6.69
7.84
-8.49
-8.78
2
2
active site of IMP-1 mβl from Pseudomonos aeruginosa.
This conformational change allows the thiolates to bridge
between two zinc(II) ions and the carboxylates to bind to
for MAA (0.68 ( 0.10 μM) and MPA (2.98 ( 0.35 μM)
are much lower than that of MTT (65.78 ( 3.55 μM) and
other heterocyclic thiones, as shown in Table 2. This
indicates that the sulfur moiety in MTT and related
compounds may be different from that of aliphatic thiols.
As previously mentioned, the aliphatic thiols 13 and 14
have been shown to inhibit the β-lactamase activity by
forming a disulfide bond with the active site cysteine
2
2
Lys224 residue at the active site. Recently, Kurosaki et
al. have shown that the pentafluoro phenyl-based thiols
6 and 17 inhibit the mβl from Pseudomonoas aeruginosa
1
8
by binding to the zinc(II) sites in a bridging mode. These
observations lead to an assumption that MMA (10),
which contains both thione and carboxylic acid moieties,
may inhibit mβl in a similar way. However, the IC50 value
obtained for MMA (43.64 ( 2.89 μM) indicates that this
compound is a relatively poor inhibitor of mβl. This is
probably due to the rigidity of the heterocyclic ring, which
may not allow the sulfur moiety to act as a bridging ligand
for the bimetallic center. The thiol-thione tautomerism
in this compound may also reduce its possibility of acting
as a thiolate bridge.
Although MTT and the related thiones inhibit mβl-
mediated hydrolysis, these compounds are found to be
less effective as compared to other aliphatic thiols such as
mercapto acetic acid (MAA, 13) and mercapto propionic
acid (MPA, 14) that have been shown to be tight-binding
6
residue under aerobic conditions. To understand whe-
ther the heterocyclic thiones inhibit the mβl activity by a
similar mechanism, we have studied the inhibition of a
serine-β-lactamase, which also has cysteine residues in the
active site. These experiments reveal that the heterocyclic
thiones do not inhibit the serine-β-lactamase (Figure 4),
indicating that the inhibition of metallo-β-lactamase by
heterocyclic thiones is not due to the binding of these
compounds with cysteine residues, but it is due to the
coordination of the thiones to the metal center.
Theoretical Studies on Thiol-Thione Tautomerism.
From Table 2, it is clear that the nature of the heterocycle
plays an important role in the inhibition of mβl activity.
For example, DMETT, having a tert-amino group, and
MDT, having a thiadiazole moiety, are more efficient
than MTT, having an N-methyl tetrazole moiety. In
addition to the substituents, the thiol-thione tautomer-
ism may also play an important role in the inhibition as
the mβl-catalyzed hydrolysis of cephalosporins (e.g.,
6
8
inhibitors for mβl. For example, the IC50 values observed
(
21) (a) Iwaoka, M.; Tomoda, S. J. Am. Chem. Soc. 1994, 116, 2557–2561.
b) Bhabak, K. P.; Mugesh, G. Chem.;Eur. J. 2008, 14, 8640–8651. (c) Bhabak,
K. P.; Mugesh, G. Chem.;Eur. J. 2009, 15, 9846–9854. (d) Bhabak, K. P.;
Mugesh, G. Inorg. Chem. 2009, 48, 2449–2455.
22) Concha, N. O.; Janson, C. A.; Rowling, P.; Pearson, S.; Cheever,
(
(
4-6) eliminates heterocyclic thiols, which undergo a
tautomerism in solution. To understand the tautomeric
C. A.; Clarke, B. P.; Lewis, C.; Galleni, M.; Frere, J. M.; Payne, D. J.;
Bateson, J. H.; Abdel-Meguid, S. S. Biochemistry 2000, 39, 4288–4298.

7
54 Inorganic Chemistry, Vol. 50, No. 3, 2011
Tamilselvi and Mugesh
Figure 5. The potential energy profile for the conversion of MTT-thiol and MDT-thiol to MTT-thione and MDT-thione, respectively.
Figure 6. The potential energy profile for the water-assisted conversion of MTT-thiol and MDT-thiol to MTT-thione and MDT-thione, respectively.
behavior of MTT, MDT, and DMETT, we have per-
formed density functional theory (DFT) calculations.
The geometries of thiols, thiones, and transition states
were optimized in the gas phase at the B3LYP/6-311-
þþG(d,p) level of theory. The relative electronic energies
the formation of MTT (1)-TS from MTT (1)-thiol in the
presence of a water molecule (6.40 kcal mol ) is almost 5
-
1
-
1
times lower than the energy required (32.12 kcal mol ) in
the absence of water. Although the relative energy differ-
ences between the thiol and thione tautomers are almost
identical in the presence and absence of water, the hydro-
gen transfer in the presence of a water molecule proceeds
via a low energy six-membered transition state, as shown
in Figure 6. This is in agreement with the report of Trujillo
et al. that the presence of water molecules considerably
reduces the energy barriers for the tautomerism involving
(
including the ZPVE correction) for the tautomerism are
summarized in Table 3. It has been proposed that the
conversion of thiols to the correponding thiones in mer-
capto thiadiazoles takes place in-plane via an intramole-
2
3
cular 1,3 hydrogen shift. However, the activation
energy barriers for the conversion of thiols to thiones
are very high, although the thione tautomers are more
stable than the thiols (Table 3). This indicates that the
transfer of hydrogen from the exocyclic sulfur atom to
the ring nitrogen via a 1,3 shift is not a favored process, as
this process produces an unfavored, high energy transi-
tion state involving a four-membered ring, as shown in
Figure 5.
2
5
selenouracils. Although the relative energies of the
thiones have been shown to be much lower than that of
2
6
the corresponding thiols, the thiones may have signifi-
cant thiolate character after binding to the metal ion.
The thiol-thione tautomerism in DMETT (9) is parti-
cularly interesting, as the tertiary amino group attached
to one of the nitrogen atoms may abstract the proton to
produce a reactive thiolate anion, which could also be
responsible for the inhibition. It is expected that both the
thiol-thione conversion and thiolate formation lead to
the generation of nucleophilic sulfur species. It has been
shown previously that the deprotonation of selenols by N,
N-dialkylamino substituents leads to the formation of
more reactive selenolates, which significantly enhances
Kryachko et al. have reported that the keto-enol
tautomerism in uracil-based compounds can be facili-
2
4
tated by water molecules. In this particular work, the
authors have shown that water molecules transfer hydro-
gen between oxygen and nitrogen atoms of different
2
4
uracil tautomers. As the tautomerism of thiols to
thiones in the heterocycles eliminated during the hydro-
lysis of antibiotics by mβl can also be mediated by water
molecules, we have studied the effect of a water molecule
on the thiol-thione tautomerism. Interestingly, the in-
troduction of one water molecule in the calculations
dramatically reduced the energy barriers for the tauto-
merism (Table 3). For example, the energy required for
2
1
the biological activity of selenium compounds. There-
fore, we have performed theoretical calculations to find
(
25) Trujillo, C.; M oꢀ , O.; Y ꢀa n~ ez, M. Org. Biomol. Chem. 2007, 5, 3092–
3099.
(26) (a) Katritzky, A. R.; Baykut, G.; Rachwal, S.; Szafran, M.; Caster,
K. C.; Eyler, J. J. Chem. Soc., Perkin Trans. 2 1989, 1499–1506. (b) Katritzky,
A. R.; Szafran, M. J. Chem. Soc., Perkin Trans. 2 1990, 871–876. (c) Les, A.;
Adamowicz, L. J. Am. Chem. Soc. 1990, 112, 1504–1509. (d) Leszczyiiski, J.;
Lammertsma, K. J. Phys. Chem. 1991, 95, 3128–3132. (e) Lamsabhi, M.;
Alcamí, M.; M ꢀo , O.; Bouab, W.; Esseffar, M.; Abboud, J. L. M.; Y ꢀa ~n ez, M.
J. Phys. Chem. A 2000, 104, 5122–5130.
(
23) Hipler, F.; Winter, M.; Fischer, R. A. J. Mol. Struct. 2003, 658, 179–
91.
24) Kryachko, E. S.; Nguyen, M. T.; Zeegers-Huyskens, T. J. Phys.
Chem. A 2001, 105, 1934–1943.
1
(

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 755
Figure 7. (A) Possible tautomeric conversions in DMETT (9). (B) Water-assisted thiol-thione tautomerism in DMETT.
out whether the transfer of hydrogen from the thiol
moiety to ring nitrogen is more favored than the hydrogen
abstraction by a basic amino group. These calculations
suggest that the water-assisted tautomerism of DMETT
(
9)-thiol to DMETT (9)-thione (Figure 7) is more favored
than the formation of DMETT (9)-zwitterion via abstrac-
tion of the S-H proton by the tertiary amino group. This
is probably due to the formation of a less favored transi-
tion state involving a seven-membered ring and the
comparable energies between the thione and DMETT
(
9)-zwitterion.
Synthesis and Structural Characterization of Model
Zinc(II)-Thiolate Complexes. To understand the coordi-
nation properties of heterocyclic thiones toward zinc(II)
ions, we have studied the interactions of MDT and
DMETT with the binuclear zinc(II) complex 18 by using
ESI-MS spectrometric techniques. It should be noted that
complex 18 has been shown to mimic the activity of mβl
2
7
by hydrolyzing a variety of β-lactam substrates. When
complex 18 was treated with DMETT in methanol,
formation of the tetranuclear zinc(II) complex 19 was
observed (Figure S12, Supporting Information). Simi-
larly, the addition of MDT to complex 18 led to the
formation of another tetranuclear zinc(II) complex 20.
However, the stability of complexes 19 and 20 was found
to be poor, and ligand dissociation was observed under
mass spectrometric conditions. In the presence of water,
complexes 19 and 20 underwent rapid ligand exchange
reactions to produce complex 21 (Figure 8). Although the
mass spectral data indicated the formation of complex 21,
this complex could not be isolated.
Figure 8. Chemical structures of the dinuclear complex 18, tetranuclear
zinc(II)-thiolate complexes 19 and 20, and tetranuclear hydoxo
complex 21.
located at the corners of a nearly planar rectangle, and the
two zinc ions in each subunit are bridged by two pheno-
late oxygen atoms. The coordination arrangement
around each zinc(II) ion is tetrahedral with one amino
coordination, bridging phenolate and hydroxo ligands
and a terminal thiolate ligand. Interestingly, the -NCH -
2
CH NMe group present in the DMETT moiety is not
2
2
involved in coordination to the metal ion. The Zn-O
bond lengths of the phenolate oxygen atom O(1) and two
zinc(II) ions are similar to that of complex 18 and are
within the usual range observed for other zinc-phenolate
Our attempts to crystallize the tetranuclear complexes
9 and 20 from the reactions of complex 18 with DMETT
1
2
7,28
and MDT, respectively, were unsuccessful. Therefore, we
have approached the synthesis of these complexes by
following a different route. Treatment of the sodium salt
of 2,6-bis((dimethylamino)methyl)-4-methyl phenol
complexes.
(2.090 and 2.119 A
complex 18 (Zn-N: 2.068 and 2.202 A
distances (3.422 and 3.603 A) are longer than those
observed for other related zinc-phenolate complexes,
The N-Zn bond lengths in this complex
˚
) are also comparable to that of
2
8
˚
). The Zn Zn
3 3
3
˚
(
HL1) with Zn(OAc) 2H O and DMETT afforded com-
2 2
3
2
8
plex 19. Crystals suitable for single-crystal X-ray diffrac-
tion studies were obtained from a methanolic solution.
The synthesis of complex 19 was found to be reproduci-
ble, and the crystallization in dichloromethane/MeOH by
using different concentrations of the reaction product
invariably afforded complex 19. The X-ray crystal struc-
ture of complex 19 reveals that the two identical dinuclear
which normally vary from 3.1 to 3.4 A
lengths (2.292 and 2.306 A) are comparable to those of
similar zinc-thiolate complexes (2.282 and 2.314 A
˚
. The Zn-S bond
˚
˚
)
2
9
reported by Seebacher et al.
Treatment of the sodium salt of HL1 with Zn-
(OAc) 2H O followed by the addition of MDT instead
of DMETT resulted in the formation of complex 20.
2
3
2
Zn (L1)(DMETT) subunits are connected to each other
2
2
Crystals suitable for X-ray diffraction studies were obtained
via hydroxo bridges (Figure 9). The four zinc(II) ions are
(28) Kaminskaia, N. V.; Spingler, B.; Lippard, S. J. J. Am. Chem. Soc.
2
000, 122, 6411–6422.
(
27) Tamilselvi, A.; Nethaji, M.; Mugesh, G. Chem.;Eur. J. 2006, 12,
(29) Seebacher, J.; Shu, M. H.; Vahrenkamp, H. Chem. Commun. 2001,
7
797–7806.
1026–1027.

7
56 Inorganic Chemistry, Vol. 50, No. 3, 2011
Tamilselvi and Mugesh
Figure 9. Crystal structures of the tetranuclear zinc complexes 19 and 20 having four DMETT and MDT ligands, respectively.
from a methanolic solution. The X-ray diffraction
studies reveal that complex 20 is structurally similar
to complex 19, and each of the two zinc ions within the
subunit is bridged by phenolate oxygen atoms
moieties eliminated during the hydrolysis. Although the
heterocyclic thiols eliminated from the β-lactams upon hydro-
lysis undergo a rapid tautomerism to produce the correspond-
ing thiones, these compounds act as thiolate ligands toward
zinc(II) ions. The structural characterization of two model
tetranuclear zinc(II) complexes indicates that the thiolates can
form reasonably stable complexes with a zinc(II) metal center.
This study suggests that the introduction of -SR moieties in
cephalosporins that can strongly inhibit the β-lactamase
activity upon cleavage may help in designing new β-lactam
antibiotics with enhanced resistance to β-lactamases.
(
Figure 9). The tetrahedral arrangements of ligands
around the metal center in this complex are almost
identical to that of complex 19. Although DMETT and
MDT can act as N-donor or bridging ligands, no such
coordination modes were observed in complexes 19 and
3
0
2
0. These observations indicate that the inhibition of
mβl activity by the heterocyclic thiols may be due to the
coordination of thiolates to the zinc(II) centers.
Although the thiolate ligands in complexes 19 and 20
are very labile and can be readily replaced by other
ligands, the significant inhibition of mβl activity by
these thiolates indicates that some additional factors
such as hydrogen bonding may influence the thiol/
Acknowledgment. This study was supported by the
Department of Science and Technology (DST), New
Delhi, India. G.M. acknowledges the DST for the award
of Ramanna and Swarnajayanti fellowships, and A.T.
thanks the University Grants Commission (UGC), India
for research fellowship.
3
1
thione binding to the enzyme active site.
Supporting Information Available: More details on the effect of
various thiones on the β-lactamase activity of metallo-β-lactamase,
the HPLC chromatograms of the MTTox and MDTox at physio-
logical pH, and the ESI-MS spectrum obtained from reaction of
dinuclear zinc(II) complex 18 with DMETT in methanol. This
material is available free of charge via the Internet at http://pubs.
acs.org/.
Conclusions
In this paper, we have shown that the metallo-β-lactamase
mβl)-catalyzed hydrolysis of the cephalosporins having
(
heterocyclic thiol side chains is much slower than that of
the β-lactam antibiotics lacking such side chains. This is
partly due to the inhibition of enzyme activity by the thiol
(31) For the influence of hydrogen bonding on thiolate coordination, see: (a)
Yoshioka, S.; Tosha, T.; Takahashi, S.; Ishimori, K.; Hori, H.; Morishima, I.
J. Am. Chem. Soc. 2002, 124, 14571–14579. (b) Simonson, T.; Calimet, N.
Proteins: Struct. Funct. Genet. 2002, 49, 37–48. (c) Parkin, G. Chem. Rev. 2004,
104, 699–768. (d) Smith, J. N.; Hoffman, J. T.; Shirin, Z.; Carrano, C. J. Inorg. Chem.
2005, 44, 2012–2017.
(
2 3 2
30) It has been shown that the reaction of MTT with PdCl (CH CN)
produces a mononuclear Pd(II) complex in which the thione is coordinated
to the metal center in a neutral form: Wang, Y.-L; Shi, Q.; Bi, W.-H.; Li, X.;
Cao, R. Z. Anorg. Allg. Chem. 2006, 632, 167–171.