Metallo-β-lactamase and phosphotriesterase activities of some zinc(II) complexes

Article abstract of DOI:10.1016/j.ica.2011.03.064

Metallo-β-lactamases (mβl) and phosphotriesterase (PTE) are zinc(II) enzymes, which hydrolyze the β-lactam antibiotics and toxic organophosphotriesters, respectively. In the present work, we have synthesized a few asymmetric phenolate-based ligands by seq

Full text of DOI:10.1016/j.ica.2011.03.064

Inorganica Chimica Acta 372 (2011) 353–361
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Metallo-b-lactamase and phosphotriesterase activities of some zinc(II) complexes
⇑
Muthaiah Umayal, Govindasamy Mugesh
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 5 April 2011
Metallo-b-lactamases (mbl) and phosphotriesterase (PTE) are zinc(II) enzymes, which hydrolyze the
b-lactam antibiotics and toxic organophosphotriesters, respectively. In the present work, we have synthe-
sized a few asymmetric phenolate-based ligands by sequential Mannich reaction and their corresponding
zinc(II) complexes. These zinc(II) complexes were studied for their mbl and PTE activities. It is shown that
the zinc(II) complexes can hydrolyze oxacillin, the b-lactam antibiotic, at much higher rates as compared
to the hydrolysis of p-nitrophenyl diphenylphosphate (PNPDPP), the phosphotriester. Among the com-
plexes studied, the binuclear asymmetric complex 1 having a water molecule coordinated to one of
the zinc(II) ions exhibits much better mbl activity than the mononuclear complexes. However, the mono-
nuclear zinc(II) complexes having labile chloride ions exhibit significant PTE activity, which can be
ascribed to the replacement of chloride ions by hydroxide ions during hydrolysis reactions.
Ó 2011 Elsevier B.V. All rights reserved.
Dedicated to Prof. S.S. Krishnamurthy
Keywords:
Antibiotic resistance
Asymmetric ligands
Metallo-b-lactamase
Phosphotriesterase
Synthesis
Zinc(II) complexes
1. Introduction
essential for its hydrolytic activity. Currently, the inhibitors namely
clavulanic acid, sulbactam and tazobactam are known for sbl.
Zinc, the second most abundant transition metal in biology,
plays important roles in a variety of biological processes [1]. It is
an essential trace element, which is required for growth, develop-
ment and differentiation of all types of life [2]. It plays an impor-
tant role in transcription of DNA, translation of RNA, etc. The
strong Lewis acidity, rapid ligand exchange, flexible coordination
geometry, lack of redox property and ‘borderline’ hard–soft behav-
ior make zinc as a suitable element for biological systems [3]. Zinc
plays catalytic, cocatalytic, structural and protein interface roles in
proteins. The largest group of zinc enzymes is zinc hydrolases,
which are capable of hydrolyzing a broad spectrum of substrates
in different metabolic pathways [4]. The mononuclear zinc(II) en-
zymes such as carbonic anhydrase and carboxypeptidase A are well
studied. The study of multinuclear zinc(II) enzymes are of current
interest [5]. The zinc(II) bound water or hydroxide ion is essential
for the hydrolytic activity. In this regard, studies on zinc(II) en-
zymes such as metallo-b-lactamases (mbl) and phosphotriesterase
(PTE) attracted considerable attention as the binuclear active sites
of these enzymes are comparable and also possess metal-bound
water molecule (Fig. 1).
However, bacteria have evolved the zinc(II) containing metallo-
b-lactamases that are capable of hydrolyzing a variety of b-lactam
antibiotics including the latest generation of cephalosporins and
carbapenems (e.g. imipenem) [6,7]. These mbls exists as both
mono- and binuclear zinc(II) enzymes (Fig. 1). To date, there are
no clinically useful inhibitors for these enzymes. Therefore, under-
standing the mode of action of mbl and design of inhibitors are
important.
Synthetic organophosphates have been widely used as plasticiz-
ers, petroleum additives, pesticides (e.g. paraoxon and parathion)
and chemical warfare agents (e.g. sarin) [8–10]. These organophos-
phates are extremely toxic to mammals as they are known to
inhibit acetylcholine esterase (AChE) [11]. This leads to the accu-
mulation of the neurotransmitter, acetylcholine which results in
nerve failure, paralysis and ultimately death. The enzyme phos-
photriesterase (PTE) is known to hydrolyze these toxic organo-
phosphorus triesters to less toxic diesters (Scheme. 1) [12,13].
PTE has been isolated from soil bacteria and is a binuclear zinc(II)
enzyme (Fig. 1) [14]. To date, the natural substrate for PTE is not
known. As PTE degrades toxic organophosphorous triesters, the de-
sign of synthetic analougues for PTE may lead to the development
of catalysts for the degradation of such compounds. The active site
of PTE consists of a binuclear zinc(II) site with metal-bound water
molecule (Fig. 1). The ligand system employed in most of the
synthetic analogs known for these enzymes mbl and PTE are
symmetrical in nature [15–17]. As the metal ions are in different
coordination environment in natural systems, we have synthesized
some asymmetrical ligands and their corresponding zinc(II)
b-Lactam antibiotics are the most widely used class of antibiot-
ics, and the bacterial enzymes that hydrolyze these antibiotics are
known as b-lactamases (Scheme 1). b-lactamases are classified into
serine-b-lactamases (sbl) and metallo-b-lactamases (mbl). The ser-
ine-b-lactamases possess an active site serine residue which is
⇑
Corresponding author.
E-mail address: mugesh@ipc.iisc.ernet.in (G. Mugesh).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.03.064

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M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
Fig. 1. Active sites of metallo-b-lactamase and phosphotriesterase. (A) and (B) represents mono- and binuclear active sites of metallo-b-lactamase, respectively, from B.
cereus. (C) Represents the binuclear active site of phosphotriesterase from Pseudomonas diminuta. PDB codes for structures (A), (B) and (C) are 1BMC, 1BC2 and 1HZY,
respectively [18–20].
Scheme 1. Function of metallo-b-lactamase and phosphotriesterase. (A) Hydrolysis of b-lactam ring in antibiotics by metallo-b-lactamase. (B) Hydrolysis of
organophosphotriesters to diesters by phosphotriesterase.
complexes. The mbl and PTE activities of the synthesized zinc(II)
complexes were determined and compared with that of a known
synthetic mimic.
chased from local suppliers. Solvents were purified by standard
procedures and were freshly distilled prior to use. The chemical
reactions were carried out under nitrogen by using standard
vacuum-line techniques. 1H (400 MHz) and 13C (100 MHz) NMR
spectra were obtained by using a Bruker 400 NMR spectrometer.
Chemical shifts are cited in ppm with respect to SiMe4 as internal
(1H and 13C) standard. Mass spectral studies were carried out by
using either a Q-TOF micro mass spectrometer with ESI-MS mode
analysis or a Bruker ESI-MS ion-trap. In the case of isotopic
patterns, the value is given for the most intense peak. The synthesis
of HL3, HL4, complex 4 and p-nitrophenyl diphenylphosphate
(PNPDPP) were carried out by using reported procedures
[15,17,21].
2. Experimental
2.1. General procedure
Oxacillin (sodium salt) was obtained from Fluka; penicillanase
(b-lactamae II, BCII) from Bacillus cereus was obtained from Sigma.
Sodium hydride, HEPES sodium salt and CHES were obtained from
Sigma–Aldrich Chemical Company. HPLC grade solvents for the ki-
netic experiments were obtained from Merck. N,N-dimethylamine,
N,N-diethylamine and N,N-diproylamine, p-Cresol, paraformalde-
hyde and 37% formaldehyde solution were of highest purity pur-
Thin-layer chromatography analyses were carried out on pre-
coated silica gel plates (Merck) and spots were visualized by UV
irradiation. Column chromatography was performed on glass

M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
355
columns loaded with silica gel or an automated flash chromatogra-
2.5. Synthesis of 2-((dimethylamino)methyl)-6-((dipropylamino)-
methyl)-4-methyl phenol (HL2)
phy system (Biotage) by using preloaded silica cartridges. High
performance liquid chromatography (HPLC) experiments were car-
ried out on a Waters Alliance System (Milford, MA) consisting of a
2690 photodiode-array detector and a fraction collector. The assays
were performed in 1.8 ml sample vials and a built-in autosampler
was used for sample injection. The alliance HPLC system was con-
trolled with EMPOWER software (Waters corporation, Milford,
MA).
To a solution of 2-((dipropylamino)methyl)-4-methyl phenol
(1000 mg, 4.52 mmol) and 40% aqueous solution of N,N-dimethyl-
amine (0.57 mL, 4.52 mmol), 37% formaldehyde aqueous solution
(0.34 mL, 4.52 mmol) was added dropwise and the temperature
was maintained below 30 °C (overall time was 30 min). After the
mixture was stirred at room temperature for 1 h, the stirring was
continued at temperatures between 45–55 °C for another 24 h.
As the Mannich reaction was reversible under reaction conditions,
the formation of p-cresol was observed. This led to the formation of
the symmetrical ligands 2,6-((dipropylamino)methyl)-4-methyl
phenol and 2,6-((dimethylamino)methyl)-4-methyl phenol. To
minimize the reversibility, the reaction was carried out at lower
temperature. Sodium chloride was added and stirring was contin-
ued for additional 20 min. The resulting oil was subjected to col-
umn chromatography by using petroleum ether and ethyl acetate
as eluents. Yield: 0.2 g (16%). 1H NMR (CDCl3), d 0.89 (t, 3H,
J = 8 Hz), 1.57–1.66 (m, 4H), 2.24 (s, 3H), 2.53–2.57 (m, 10H),
3.81 (s, 2H), 3.89 (s, 2H), 6.93 (s, 2H), 7.07 (s, 2H); 13C NMR (CDCl3),
d 12.3, 19.8, 20.9, 45.3, 55.7, 57.3, 58.6, 122.8, 123.6, 127.7, 129.1,
130.4, 154.6; ESI-MS Anal. Calc. for C17H30N2O [M+H]+: 279.2431.
Found: 279.2436.
2.2. Synthesis of 2-((diethylamino)methyl)-4-methyl phenol
To a solution of p-cresol (2.9 mL, 27.8 mmol) and N,N-diethyl-
amine (2.9 mL, 27.8 mmol) in 5 mL of ethanol, paraformaldehyde
(835 mg, 27.8 mmol) was added. The reaction mixture was stirred
at 80 °C for 3 h and it was monitored time to time by TLC for com-
pletion. The solvent was evaporated and the resulting oil was sub-
jected to column chromatography by using petroleum ether and
ethyl acetate as eluents. Yield: 2.8 g (52%). 1H NMR (CDCl3, ppm),
d 1.11 (t, 6H, J = 8 Hz), 2.25 (s, 3H), 2.62 (q, 4H, J = 4 Hz), 3.72 (s,
2H), 6.71 (d, 1H, J = 8 Hz), 6.78 (s, 1H), 6.96 (d, 1H, J = 8 Hz); 13C
NMR (CDCl3, ppm), d 11.7, 21.0, 46.7, 57.4, 116.2, 122.3, 128.3,
129.3, 129.4, 156.4.
2.6. Synthesis of complex 1
2.3. Synthesis of 2-((diethylamino)methyl)-6-((dimethylamino)
methyl)-4-methyl phenol (HL1)
Complex 1 was synthesized from HL1 by following the reported
procedure. The ligand HL1 (150 mg, 0.6 mmol) in dichloromethane
(5 ml) together with 60% NaH (21.6 mg, 0.54 mmol) in hexane
(15 ml) were mixed and stirred at room temperature for 1 h in
nitrogen atmosphere to obtain the corresponding sodium pheno-
late (NaL1). Zn(OAc)2ꢀ2H2O (197.6 mg, 0.9 mmol) was then added
and the stirring was continued for an additional 1 h. It resulted
To a solution of 2-((diethylamino)methyl)-4-methyl phenol
(1000 mg, 5.2 mmol) and 40% aqueous N,N-dimethylamine
(0.6 mL, 5.2 mmol), 37% formaldehyde aqueous solution (0.4 mL,
5.2 mmol) was added dropwise and the temperature was main-
tained below 30 °C (overall time was 30 min). After the mixture
was stirred at room temperature for 1 h, it was stirred again be-
tween 45–55 °C for another 24 h. As the Mannich reaction is
reversible, the formation of p-cresol was observed during the
reaction. This led to the formation of the symmetrical ligands
2,6-((diethylamino)methyl)-4-methyl phenol and 2,6-((dimethyl-
amino)methyl)-4-methyl phenol. To minimize the reversibility,
the reaction was carried out at lower temperature and allowed
to proceed for 48 h. To the resulting mixture, sodium chloride
was added and stirring was continued for 20 min. The resulting
oil was subjected to column chromatography by using petroleum
ether and ethyl acetate. Yield: 0.18 g (14%). 1H NMR (CDCl3), d
1.09 (t, 6H, J = 8 Hz), 2.22 (s, 3H), 2.34 (s, 6H), 2.62 (q, 4H,
J = 8 Hz), 3.58 (s, 2H), 3.72 (s, 2H); 13C NMR (CDCl3, ppm) d 11.5,
21.0, 44.9, 46.7, 55.9, 58.4, 122.4, 122.8, 128.0, 129.7, 130.9,
155.1; ESI-MS Anal. Calc. for C15H26N2O [M+H]+: 251.2118. Found:
251.0314.
in a turbid solution from which complex 1 [Zn2L1(l-OAc)(OA-
c)2(H2O)], was filtered off as a white solid. The product was dried
under vacuum to obtain a white crystalline solid. It was dissolved
in dichloromethane and kept for crystallization. On standing at
room temperature, colorless needle shaped crystals suitable for
X-ray analysis was obtained. Yield: 0.22 g (64%) 1H NMR (CDCl3),
1.35 (br s, 6H), 2.01 (br s, 9H), 2.19 (s, 3H), 2.38 (s, 6H), 3.09–
3.17 (m, 4H), 3.68 (s, 2H), 4.05 (s, 2H), 6.79 (s, 1H), 6.81 (s, 1H);
13C NMR (CDCl3), 8.91, 20.73, 23.24, 30.19, 46.71, 47.31, 55.68,
63.85, 118.35, 123.54, 124.11, 130.74, 133.7, 162.84, 180.0; ESI-
MS Anal. Calc. for [Zn2L1+4H]+: 381.0863. Found: 381.1214.
2.7. Synthesis of complex 2
Complex 2 was synthesized from HL2 by following the reported
procedure [15]. The ligand HL2 (50 mg, 0.18 mmol) in dichloro-
methane (5 ml) together with 60% NaH (6.5 mg, 0.16 mmol) in
hexane (10 ml) were mixed and stirred at room temperature for
1 h in nitrogen atmosphere to obtain the corresponding sodium
phenolate (NaL1). Anhydrous ZnCl2 (12.2 mg, 0.09 mmol) was then
added and the stirring was continued for an additional 1 h. It re-
sulted in a turbid solution from which complex 2 was filtered off
as a white solid. The product was dried under vacuum to obtain
a white crystalline solid. It was dissolved in dichloromethane and
kept for crystallization. On standing at room temperature, colorless
needle shaped crystals suitable for X-ray analysis was obtained.
Yield: 0.045 g (60%).1H NMR (CDCl3), d 0.98–1.02 (t, 6H), 1.76–
1.87 (m, 4H), 2.18 (s, 3H), 2.46 (s, 3H), 2.96–3.01 (m, 4H), 3.63 (s,
2H), 4.14 (s, 2H), 6.79 (s, 1H), 6.82 (s, 1H); 13C NMR (CDCl3), d
11.7, 17.7, 20.7, 47.7, 54.8, 58.2, 63.7, 117.8, 123.5, 125.0, 130.8,
133.8, 161.6; ESI-MS Anal. Calc. for [ZnClL2+4H]+: 381.1629.
Found: 381.0720.
2.4. Synthesis of 2-((dipropylamino)methyl)-4-methyl phenol
To a solution of p-Cresol (2.9 mL, 27.8 mmol) and N,N-dipropyl-
amine (3.8 mL, 27.8 mmol) in 5 mL of ethanol, paraformaldehyde
(835 mg, 27.8 mmol) was added. The reaction mixture was stirred
at 80 °C for 5 h and it was monitored by TLC till completion. The
solvent was evaporated and the resulting oil was subjected to col-
umn chromatography by using petroleum ether and ethyl acetate
as eluents. Yield: 3.67 g (60%). 1H NMR (CDCl3, ppm), d 0.9 (t, 6H,
J = 8 Hz), 1.52–1.59 (m, 4H), 2.24 (s, 3H), 2.45–2.49 (m, 4H), 3.71
(s, 2H), 6.71 (d, 1H, J = 8 Hz), 6.77 (s, 1H), 6.96 (d, 1H, J = 8 Hz);
13C NMR (CDCl3, ppm) d 12.3, 20.0, 21.0, 55.9, 58.8, 116.2, 122.5,
128.4, 129.3, 129.4, 156.2.

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M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
2.8. Synthesis of complex 3
obtained by injecting known concentrations of p-nitrophenol.
The initial rate 0 and t50 value (the time required for the hydroly-
m
Complex 3 was synthesized from HL3 by following the reported
procedure [15]. The ligand HL2 (50 mg, 0.18 mmol) in dichloro-
methane (5 ml) together with 60% NaH (6.5 mg, 0.16 mmol) in
hexane (10 ml) were mixed and stirred at room temperature for
1 h in nitrogen atmosphere to obtain the corresponding sodium
phenolate (NaL1). Anhydrous ZnCl2 (12.2 mg, 0.09 mmol) was then
added and the stirring was continued for an additional 1 h. It re-
sulted in a turbid solution from which complex 3 was filtered off
as a white solid. The product was dried under vacuum to obtain
a white crystalline solid. It was dissolved in dichloromethane and
kept for crystallization. On standing at room temperature, colorless
needle shaped crystals suitable for X-ray analysis was obtained.
Yield: 0.06 g (80%). The crystal structure of complex 3 reveals the
formation of an identical complex prepared by different procedure
[21].
sis of 50% of the substrate) for the hydrolysis of p-nitrophenol by
synthesized zinc(II) complexes 1–3 were determined and com-
pared with that of the known synthetic mimic for PTE, complex
4. In a typical experiment, the incubation mixtures for the HPLC
analysis contained PNPDPP (10ꢂ3 M) and zinc(II) com-
plexes(0.2 ꢁ 10ꢂ3 M) in 35% EtOH and 20 ꢁ 10ꢂ3 M N-cyclohexyl-
2-aminoethanesulfonic acid (CHES) buffer at pH 9.0. The mixture
was incubated at room temperature and aliquots (10 ll) injected
onto the HPLC column and eluted with isocratic solvent system
(80% Methanol and 0.1% TFA in water) using a C18 reverse-phase
column. The p-nitrophenol, one of the reaction products (retention
time, 3.78 min) and PNPDPP (retention time, 5.27 min) are separa-
ble. The increase in the amount of p-nitrophenol (lg) was calcu-
lated from the calibration plot. The chromatograms were
extracted at 305 nm.
2.9. Single crystal X-ray crystallography
2.12. Comparison of metallo-b-lactamase and phosphotriesterase
activities
X-ray crystallographic studies were carried out on a Bruker CCD
diffractometer with graphite-monochromatized Mo K
a radiation
In order to compare the mbl and PTE activities of the synthe-
sized zinc(II) complexes, both the assays were carried out under
identical conditions by using CHES buffer at pH 9.0 [17]. The mbl
activities of complexes were determined by using 10ꢂ3 M oxacillin
and 0.2 ꢁ 10ꢂ3 M zinc(II) complexes in 20 ꢁ 10ꢂ3 M CHES buffer at
pH 9.0. The PTE activities of complexes were determined by using
10ꢂ3 M PNPDPP and 0.2 ꢁ 10ꢂ3 M zinc(II) complexes in 35% EtOH
and 20 ꢁ 10ꢂ3 M CHES buffer at pH 9.0.
(k = 0.71073 Å) controlled by a Pentium-based PC running on the
SMART software package (Bruker AXS: Madison, WI) [22]. Single
crystals were mounted at room temperature on the ends of glass
fibers and data were collected at room temperature. The structures
were solved by direct methods and refined using the SHELXTL soft-
ware package (Siemens Industrial Automation Inc., Madison, WI)
[23]. In general, all non-hydrogen atoms were refined anisotropi-
cally. Hydrogen atoms were assigned idealized locations. Empirical
absorption corrections were applied to all structures using SADABS
(Siemens Industrial Automation Inc., Madison, WI) [24].
3. Results and discussion
2.10. Metallo-b-lactamase assay
3.1. Synthesis
The hydrolysis of oxacillin, the b-lactam substrate, by the zin-
c(II) complexes were determined by HPLC method [15]. The de-
crease in the concentration of the oxacillin was followed by
measuring the peak area of oxacillin at 254 nm and the amount
of oxacillin present in the solution at a given time is determined
from the calibration plot obtained by injecting known concentra-
The asymmetric phenolate-based ligands can be prepared
either by sequential Mannich reaction of para-substituted phenol
[25] or by the selective oxidation of one of the hydroxyl groups
of 2,6-bis (hydroxymethyl)-4-methyl phenol [26]. We have synthe-
sized the asymmetric ligands by the sequential Mannich reaction
of para-substituted phenol, p-cresol with formaldehyde and two
different amines. The reported procedure was slightly modified
and ligands were prepared by following the method described in
Scheme 2. As discussed earlier, the synthesis is complicated by
the reversibility of Mannich reactions, and therefore, the formation
of corresponding two symmetrical ligands was observed. The
asymmetric ligand HL1, 2-((diethylamino)methyl)-6-((dimethyl-
amino)methyl)-4-methyl phenol, was prepared as follows. In the
first step, N,N-diethylamine, paraformaldehyde and p-cresol were
refluxed in ethanolic solution. This led to the formation of both
mono- and bi-aminoalkylated products, from which the mono-
aminoalkylated product was separated by column chromatogra-
phy. To the pure mono-aminoalkylated product, the second amine,
N,N-dimethylamine and formaldehyde solution were added and
the reaction was carried out under mild conditions and allowed
to proceed for longer time. The second step was carried out under
mild condition in order to minimize the reversibility of Mannich
reaction that leads to the formation of the p-cresol under reaction
conditions, which can possibly react with the amines present in the
reaction medium to produce the corresponding symmetrical li-
gands. The ligand HL1 was separated from the corresponding sym-
metrical ligands by means of column chromatography. The
asymmetric ligand HL2 was also prepared by using N,N-dipropyl-
amine and N,N-dimethylamine. However, the symmetric ligands
HL3 and HL4 were prepared by following the reported procedures
[21]. The complexes 1–3 were prepared from ligands HL1–HL3,
tions of oxacillin. The initial rate m0 and t50 value (the time required
for the hydrolysis of 50% of the substrate) for the hydrolysis of oxa-
cillin by synthesized zinc(II) complexes 1–3 were determined and
compared with that of the known synthetic mimic for mbl,
complex 4. In a typical experiment, the incubation mixtures for
the HPLC analysis contained oxacillin (1.06 ꢁ 10ꢂ3 M) and zinc(II)
complexes(2.1 ꢁ 10ꢂ5 M) in 9:1 mixture of N-(2-hydroxyethyl)
piperazine-N0-ethanesulfonic acid (HEPES) buffer/dimethyl sulfox-
ide (DMSO) at pH 7.5. The mixture was incubated at room temper-
ature and aliquots (10 ll) injected onto the HPLC column and
eluted with gradient solvent system (0.1% TFA in water–MeCN)
using a C18 reverse-phase column. The oxacillin (retention time,
4.24 min) and the reaction product (oxacillin turnover product,
retention time, 3.66 min) are separable. The decrease in the
amount of oxacillin (lg) was calculated from the calibration plot.
The chromatograms were extracted at 254 nm.
2.11. Phosphotriesterase assay
The hydrolysis of p-nitrophenyl diphenylphosphate (PNPDPP)
by the zinc(II) complexes were determined by HPLC method [17].
The increase in the concentration of the hydrolyzed product,
p-nitrophenol was followed by measuring the peak area of p-nitro-
phenol at 305 nm and the amount of p-nitrophenol present in the
solution at a given time is determined from the calibration plot

M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
357
Scheme 2. Synthesis of symmetrical and unsymmetrical ligands by Mannich-type reactions.
Fig. 2. Chemical structures of ligands HL1–HL4 and their corresponding zinc(II)complexes 1–4.
respectively, by using reported procedure with slight modifications
[15]. Complex 4, which has been shown to be a synthetic mimic for
mbl and PTE, was prepared for a comparison purpose [15]. The
chemical structures of the ligands HL1–HL4 and complexes 1–4
are shown in Fig. 2.
a symmetrical ligand system. However, the co-ordination environ-
ment around Zn1 differs slightly in these two complexes, as the
nature of coordination of the terminal acetate ligand is different.
In complex 1, the first zinc(II) ion (Zn1) is four co-ordinate with
distorted tetrahedral geometry and is co-ordinated to three oxygen
atoms and a nitrogen atom. The phenolate oxygen and an acetate
ion act as bridging ligands, whereas the other asymmetric acetate
(mono-dendate) and amino nitrogen act as terminal ligands. In
both these complexes, the second zinc(II) ion (Zn2) is six co-
ordinate with distorted octahedral geometry and is co-ordinated
to five oxygen atoms and a nitrogen atom. In Zn2, the terminal
acetate ion acts as a bidendate ligand and the sixth co-ordination
is completed by a water molecule. The ZnꢀꢀꢀZn distance in complex
1, 3.398 Å is comparable to the ZnꢀꢀꢀZn distances in complex 4
(3.355 Å) [15], mbl enzyme from species such as Stenotrophomonas
3.2. X-ray crystal structure
The single crystals suitable for X-ray diffraction for complexes
1–3 are obtained from dichloromethane solution. The X-ray crystal
structures of complexes 1–3 are shown in Fig. 3 and their crystal-
lographic data are given in Table 1. The X-ray crystal structure of
complex 1 reveals a binuclear zinc(II) complex. The two zinc(II)
ions are in different co-ordination environments. The structure of
this complex is similar to that of reported complex 4 obtained from

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M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
Fig. 3. X-ray crystal structures of complexes 1–3. In 1, the water molecule is hydrogen bonded to one of the acetate ligand, whereas in 3 and 4, the amino nitrogen is hydrogen
bonded to phenolate oxygen.
Table 1
The phenolic proton is abstracted by the amino nitrogen atom of
the non-coordinated arm of the ligand, which forms a positively
Crystallographic data for complexes 1–3.
charged ammonium group. The charge balance is provided by the
negatively charged zinc(II) co-ordination sphere. The positively
charged amino group is hydrogen bonded to the phenolate oxygen
ion with N–HꢀꢀꢀO distances 2.636 Å and 2.696 Å in complexes 2 and
3, respectively.
Crystal data
Complex 1
Complex 2
Complex 3
Empirical formula
Formula Weight
Crystal system
Space group
a (Å)
C
21H36N2O8Zn2 C17H30N2OClZn C17H30N2OClZn
575.3
monoclinic
C2/c
414.7
monoclinic
P2(1)/c
414.7
monoclinic
P2(1)/c
26.3168(38)
14.2710(21)
17.6770(26)
90.000(0)
125.532(3)
90.000(0)
5402.68(143)
8
9.1765(6)
20.2727(12)
11.0961(6)
90.000(0)
97.590(2)
90.000(0)
2046.15(6)
4
17.9451(55)
8.0921(24)
14.4635(43)
90.000(0)
103.380(5)
90.000(0)
2043.29(27)
4
b (Å)
c (Å)
3.3. Metallo-b-lactamase activity of zinc(II) complexes
a
(°)
b (°)
c
(°)
The mbl activities of the zinc(II) complexes were studied by fol-
lowing the hydrolysis of oxacillin, a b-lactam substrate. The mbl
activities of the newly synthesized complexes 1–3 have been com-
pared with that of complex 4. The plots of % oxacillin vs time for
the hydrolysis by zinc(II) complexes, free ligand, HL4 (one of the li-
gands) and zinc(II) acetate are given in Fig. 4 and the time required
for the 50% hydrolysis of the substrate is given in Table 2. These
data suggest that complex 1 is able to hydrolyze oxacillin com-
pletely with a t50 value of 158 min, which is very similar to that ob-
served for complex 4 (126 min). This value is much lower than that
of the control reaction or the reaction mediated by HL4 or zinc(II)
acetate. The free ligand, HL4, does not show appreciable hydrolysis
and only 24% hydrolysis was observed even after 1940 min.
Although zinc(II) acetate showed a appreciable initial rate in
V (Å3)
Z
Dcalc (g cm3)
1.41
1.819
1.35
1.467
1.35
1.469
Absorption coefficient
(mmꢂ1
)
Reflections collected/
unique
F(0 0 0)
Rint
23145/6361
16160/3828
16731/4818
2399.5
0.099
0.055
0.094
0.944
871.8
0.042
0.064
0.099
1.163
ꢂ0.341, 0.377
871.8
0.089
0.090
0.235
1.076
ꢂ0.735, 1.822
R
Rw
Goodness-of-fit on F2
D
qmin and
D
qmax
ꢂ0.344, 0.276
(e Åꢂ3
)
maltophilia (3.459 Å) [27], Bacteroides fragilis (3.457 Å) [28] and PTE
(3.46 Å) [20]. However this ZnꢀꢀꢀZn distance in complex 1 is shorter
than that of binuclear mbl enzyme from B. cereus (3.848 and
4.365 Å) [29]. Complex 1 appears to be a good model for binuclear
zinc(II) enzymes mbl and PTE as this complex posses a metal-
bound water molecule similar to that in the active site of these en-
zymes. The metal-bound water molecule is hydrogen bonded to
one of the acetate ion (O–HꢀꢀꢀO distance 2.624 Å). The crystal struc-
tures of complexes 2 and 3 obtained from asymmetric ligand HL2
and symmetric ligand HL3, respectively, inidicate the formation of
mononuclear complexes. Although a different procedure was used,
the structure of complex 3 was found to be identical to that re-
ported by Habbadi et al. [21] The crystal structures of complexes
2 and 3 are similar to those obtained by Habbadi et al. for similar
symmetrical ligand systems [21]. In these complexes, the zinc(II)
ion is four co-ordinate with distorted tetrahedral co-ordination.
Zinc(II) ion is co-ordinated to two chloride ions and phenolate oxy-
gen and amino nitrogen from one of the arms of the ligand system.
100
80
60
40
20
0
a
b
c
d
e
f
0
200
400
600
time (min)
800 1000
Fig. 4. Hydrolysis of oxacillin (1.06 ꢁ 10ꢂ3 M) by HL4 (a), zinc acetate (b), 2 (c), 3
(d), 1 (e) and 4 (f) (2.1 ꢁ 10ꢂ5 M) in DMSO:HEPES buffer (1:9), pH: 7.5. The reaction
was followed by reverse-phase HPLC and the amount of oxacillin hydrolyzed at a
given time was calculated from calibration plot.

M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
359
Table 2
Table 3
The t50 values and initial rates
Zn(OAc)2ꢀ2H2O and complexes 1–4 at room temperature.
(
m0
)
for the hydrolysis of oxacillin by HL4,
The t50 values and initial rates (
1–4 at room temperature.
m
0) for the hydrolysis of PNPDPP by zinc(II) complexes
Catalyst
Oxacillin
Catalyst
PNPDPP
e
c
t50 (min)a
m0
(
l
M minꢂ1
)
t50 (min)a
m0 (l )
M minꢂ1
Control
HL4
>6300b
>1940c
>750d
158.1
299.0
262.1
125.6
0.76
0.32
0.81
4.12
2.35
2.79
4.79
Control
>7094b
6180.3
4731.4
5174.9
5912.2
0.020
1
2
3
4
0.1389
0.1611
0.1490
0.1535
Zn(OAc)2ꢀ2H2O
1
2
3
4
a
The time required for the 50% hydrolysis of the substrate t50 was determined by
monitoring the increase in the peak area of p-nitrophenol. [PNPDPP]: 0.2 ꢁ 10ꢂ3 M;
[catalyst]: 0.2 ꢁ 10ꢂ3 M in 35% ethanol and CHES buffer, pH 9.0.
a
The time required for the 50% hydrolysis of the substrate t50 was determined by
monitoring the decrease in the peak area of oxacillin. [oxacillin]: 1.06 ꢁ 10ꢂ3 M;
After 7094 min, only 30% hydrolysis was observed.
b
[catalyst]: 2.12 x 10ꢂ5 M in DMSO/HEPES buffer (1:9), pH 7.5.
The initial rate m0 was calculated from initial 5–10% of the reaction, by moni-
c
b
After 6300 min, only 36% hydrolysis was observed.
After 1940 min, only 24% hydrolysis was observed.
After 750 min, only 36% hydrolysis was observed.
The initial rate m0 was calculated from initial 5–10% of the reaction, by moni-
toring the increase in the peak area of p-nitrophenol.
c
d
e
toring the decrease in peak area of oxacillin.
e
100
d
c
comparison with metal-free systems, it is capable of hydrolyzing
only 36% even after 750 min. This can be attributed to the inhibi-
tion of free zinc(II) ions in solution by the products. However, no
such inhibition was observed in the case of dinuclear zinc(II) com-
plex 1 as the metal ions are tightly bound in the well-organized li-
gand system.
80
b
60
a
40
20
0
The efficient hydrolysis of oxacillin by complex 1 can be attrib-
uted to the two tightly bound zinc(II) ions. As in the case of 4, com-
plex 1 also possesses the metal-bound water molecule, which is
essential for the catalytic activity. The Zn2-bound water molecule
is activated through a hydrogen bonding (O-HꢀꢀꢀO, 2.624 Å) with
the oxygen atom of the acetate ion coordinated to Zn1. This is in
accordance with the activation of metal-bound water molecule in
the active site of the mbl enzyme by amino acid residue such as
Asp-120, which acts as a general base. The minor variation in the
structure of ligands in complexes 1 and 4 does not appear to
change the catalytic activity significantly. Therefore, the metal-
bound water molecule in both these complexes is responsible for
their catalytic efficiency. The zinc(II) complexes 2 and 3, which
lacks metal-bound water molecule, exhibited almost two-times
higher t50 values as compared to complexes 1 and 4 having
metal-bound water molecule. However, the ability of mononuclear
zinc(II) complexes 2 and 3 to hydrolyze oxacillin suggests that the
presence of second zinc(II) ion is not important for the initial
hydrolysis of b-lactam. This is in accordance with the observation
of Kaminskaia et al. that the presence of second zinc ion is not
crucial for the b-lactamase activity [30].
0
4000
8000
time(min)
12000 16000
Fig. 5. Hydrolysis of PNPDPP (0.20 ꢁ 10ꢂ3 M) by control (a), 4 (b), 1 (c), 3 (d) and 2
(e) (2.1 ꢁ 10ꢂ5 M) in 35% ethanol and 20 mM CHES buffer, pH 9.0. The reaction was
followed by reverse-phase HPLC and the amount of p-nitrophenol formed at a given
time was calculated from the calibration plot.
can be ascribed to the inhibition of the complex by the hydrolyzed
product, diphenyl phosphate (DPP) [17]. The replacement of bridg-
ing acetate by DPP has been previously observed in complex 4 [17].
As in the case of mbl activity, the slight change in ligand system on
moving from 1 to 4 or 2 to 3 does not show significant change in
PTE activity.
Table 4
Comparison of mbl and PTE activity of the synthesized zinc(II) complexes.
Catalyst
mbl activity
t50 (min)a
PTE activity
3.4. Phosphotriesterase activity of zinc(II) complexes
c
f
m0
(l
M minꢂ1
)
t50 (min)d
m0 (l )
M minꢂ1
Control
>1885b
156.8
170.3
317.5
103.8
0.09
13.54
12.60
8.44
>2800e
6180.3
4731.4
5174.9
5912.2
0.020
As mbl and PTE possess similar active sites with metal-bound
water molecule, we have studied the synthesized zinc(II) com-
plexes for their ability to hydrolyze organophosphotriester,
1
2
3
4
0.1389
0.1611
0.1490
0.1535
PNPDPP. The initial rate (m0) and t50 values for the hydrolysis of
18.23
PNPDPP by zinc(II) complexes to produce p-nitrophenol are given
in Table 3, and the plots of % p-nitrophenol vs time are shown in
Fig. 5. Interestingly, complexes 3 and 4 having zinc-bound chloride
ions exhibited much better activity than complexes 1 and 2 having
zinc-bound water molecule. This can be attributed to the replace-
ment of labile chloride ions in 3 and 4 by hydroxide ion in CHES
buffer at pH 9.0. This is in accordance with our previous observa-
tions that the metal-bound chloride ions are replaced by hydroxide
ions in CHES buffer [17]. These metal-bound hydroxide ions act as
nucleophile in the hydrolytic reactions. The binuclear complex 1
exhibited lower PTE activity as that of reported complex 4, which
a
The time required for the 50% hydrolysis of the substrate t50 was determined by
monitoring the decrease in the peak area of oxacillin. [oxacillin]: 1.0 ꢁ 10ꢂ3 M;
[catalyst]: 0.2 ꢁ 10ꢂ3 M in CHES buffer, pH 9.0.
b
After 1885 min, only 29% of hydrolysis of oxacillin was observed.
The initial rate m0 is calculated from the initial 5–10% of the reaction by fol-
c
lowing the decrease in peak area of oxacillin.
d
The time required for the 50% hydrolysis of the substrate t50 was determined by
monitoring the increase in the peak area of p-nitrophenol. [PNPDPP]: 1.0 ꢁ 10ꢂ3 M;
[catalyst]: 0.2 ꢁ 10ꢂ3 M in 35% ethanol and CHES buffer, pH 9.0.
e
After 2880 min, only 71% of hydrolysis of PNPDPP was observed.
The initial rate m0 is calculated from the initial 5–10% of the reaction by fol-
f
lowing the increase in peak area of p-nitrophenol.

360
M. Umayal, G. Mugesh / Inorganica Chimica Acta 372 (2011) 353–361
100
80
60
40
20
0
100
(A)
(B)
80
a
a
60
40
20
b
b
0
0
4000
8000
time (min)
12000
0
4000
8000
time (min)
12000
100
80
60
40
20
0
(C)
100
80
60
40
20
0
(D)
a
b
a
b
0
4000
8000
12000
0
4000
8000
12000
time (min)
time(min)
Fig. 6. Comparison of PTE and mbl activities of (A) complex 1 (B) complex 2 (C) complex 3 and (D) complex 4. Plots a and b represent PTE and mbl activities, respectively.
Hydrolysis of oxacillin and PNPDPP were carried out in 20 mM CHES buffer, pH 9.0. For PTE assay 35% ethanol was used to dissolve PNPDPP. [oxacillin] or [PNPDPP]:
1 ꢁ 10ꢂ3 M and [catalyst]: 0.2 ꢁ 10ꢂ3 M.
3.5. Comparison between mbl and PTE activities of zinc(II) complexes
much higher PTE activity, indicating that the replacement of the
metal-bound labile chloride ions by hydroxide ions is important
for the PTE activity. The poor PTE activity of the binuclear zinc(II)
complexes can be ascribed to the inhibition by the hydrolyzed
product, diphenyl phosphate.
To compare the mbl and PTE activities of complexes 1–4, both
the mbl and PTE assays were carried out under identical conditions
in CHES buffer at pH 9.0. The substrate:catalyst concentration was
kept at 5:1. The initial rates (m0) and t50 values for the hydrolysis of
PNPDPP or oxacillin by zinc(II) complexes are given in Table 4, and
the plots of % conversion vs time is shown in Fig. 6. In the presence
of complexes 1–4, the hydrolysis of oxacillin was found to be much
higher than that of PNPDPP, indicating that complexes 1–4 are bet-
ter mbl mimics than PTE mimics. Among these four complexes, the
mbl activity of 1 and 4 was found to be higher than that of com-
plexes 2 and 3. This can be ascribed to the two tightly bound zin-
c(II) ions and also to the presence of activated water molecules.
However, the increase in PTE activity of 2 and 3 can be attributed
to the replacement of labile chloride ion by hydroxide ion under
assay condition. As discussed earlier, the decrease in PTE activity
of 1 and 4 is possibly due to an inhibition by DPP [17].
Acknowledgements
This study was supported by the Department of Science and
Technology (DST), New Delhi. GM acknowledges the DST for the
award of Ramanna and Swarnajayanti fellowships. MU thanks
the Indian Institute of Science (IISc) for a research fellowship.
Appendix A. Supplementary material
The supplementary data include 1H NMR, 13C NMR and mass
spectra of the ligands. CCDC 797091, 797090 and 797092 contain
the supplementary crystallographic data for complexes 1, 2 and
3, respectively. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.ica.2011.03.064.
4. Conclusions
In this paper, we have described the synthesis of a few asym-
metric phenolate-based ligands and their corresponding zinc(II)
complexes. These complexes were studied for their mbl and PTE
activities. In agreement with our previous studies on mbl mimics,
a binuclear zinc(II) complex having a water molecule exhibits high
mbl activity. Although the mononuclear zinc(II) complexes 2 and 3
exhibit considerable mbl activity, the activity is almost two times
lower than that of the binuclear zinc(II) complexes. As the active
sites of both mbl and PTE possess zinc(II)-bound water molecule,
these complexes were also studied for their PTE activity. All the
four complexes exhibited higher mbl activity than PTE activity. In
contrast to the mbl activity, the mononuclear complexes exhibited
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