6416 J. Am. Chem. Soc., Vol. 122, No. 27, 2000
Kaminskaia et al.
Hydrolysis of Penicillin G and Penicillin G Methyl Ester. These
reactions were studied by H NMR spectroscopy. The solvent was a
stituted phenol, provide an environment for two zinc(II) ions
that resembles those in the active sites of the three metallo-â-
lactamases depicted in Scheme 1. Two zinc(II) ions at the active
sites are coordinated predominantly by histidine residues14-16
and are bridged by a hydroxide ion postulated to serve as the
nucleophile in hydrolysis. The bridging phenolate and the
restricted separation between the two ethylenediamine arms in
these ligands strongly favor the formation of dimetallic com-
plexes.33-35 Although several copper(II) and manganese(II)
complexes of ligands in this class, including HL1, have been
synthesized and employed as biomimetic models,33-36 only a
few phenolate-bridged dizinc(II) complexes are known. Included
is a dizinc(II) complex having four methoxyethyl chelating
arms.37,38 In the present study, four novel dizinc(II) complexes
of HL1 and HL2, [Zn2L1(µ-NO3)NO3)2], [Zn2L1(µ-OMe)(NO3)],
[Zn2L2(NO3)3], and [Zn2L1(µ-OH)(NO3)2] were prepared and
characterized by X-ray crystallography. The first three were also
1
1:1 mixture of D2O and acetone-d6 and the temperature was 313 K.
The initial concentrations of [Zn2L1(µ-NO3)(NO3)2] and a substrate
(penicillin G, sodium salt; and penicillin G, methyl ester) were 0.025
M each. Substrate hydrolysis was followed by monitoring the C(5)-H
and C(6)-H resonances at 4.96 and 4.46 ppm in (5R)-penicilloic acid,
respectively, and at 5.10 and 4.50 ppm in (5R)-penicilloic acid-1-methyl
ester, respectively.
Kinetics of Hydrolysis. The kinetics of substrate hydrolysis was
followed by UV-visible spectrophotometry. The temperature inside
the cuvettes was measured directly with a thermocouple. The solvent
was either a 9:1 mixture of 0.05 M HEPES buffer and DMSO or a 9:1
mixture of acetone and DMSO. The reported pH values of the solutions
correspond to the aqueous component. The substrate used for the kinetic
studies was nitrocefin. Its extinction coefficient (ꢀ) at 390 nm was
determined by measuring the absorbance of solutions containing known
concentrations in 9:1 mixtures of 0.05 M HEPES buffer and DMSO at
pH values 6.95 and 8.59 and in a 9:1 mixture of acetone and DMSO.
The values of ꢀ were the same within experimental error in all three
media, the average being 21 000 M-1 cm-1. Extinction coefficients of
the product at 390 and 486 nm were determined in “aged” reaction
mixtures and assuming that nitrocefin is completely converted to its
hydrolysis product. The values are 7585 M-1 cm-1 and 16 000 M-1
cm-1 at 390 and 486 nm, respectively. Stock solutions of the complexes,
nitrocefin, and the inhibitor (sodium succinate and sodium acetate) were
prepared in the appropriate solvent and used immediately to avoid
possible decomposition. The concentrations of stock solutions of
complex and nitrocefin were 0.010 M each. The concentrations of the
stock solutions of the inhibitors were varied and are specified in each
particular experiment. Unless stated otherwise, the concentration of
[Zn2L1(µ-NO3)(NO3)2] in kinetic studies was 5.0 × 10-4 M. In a typical
kinetic run all of the reagents were mixed in a spectrophotometric cell
and allowed to equilibrate for 2 min inside the temperature-controlled
UV-visible spectrophotometer before data collection began. Data were
usually collected at 390 nm, the wavelength that corresponds to the
maximum absorbance of nitrocefin. Because the reaction product also
absorbs at 390 nm (ꢀP ) 7585 M-1 cm-1), the extinction coefficient of
nitrocefin was corrected, as shown in eqs 7-9 to account for product
absorbance.
1
characterized in solution by H NMR spectroscopy.
Structural Studies. The structures of [Zn2L1(µ-NO3)(NO3)2],
[Zn2L1(µ-OMe)(NO3)2], [Zn2L2(NO3)3], and [Zn2L1(µ-OH)-
(NO3)2] are presented in Figure 1, and selected bond lengths
and angles are provided in Table 2. The complexes [Zn2L1(µ-
NO3)(NO3)2], [Zn2L1(µ-OMe)(NO3)2], and [Zn2L1(µ-OH)-
(NO3)2] have similar structures. Each zinc(II) ion has a distorted
octahedral environment with a bidentate nitrate serving as the
terminal ligand. The C2 symmetry along the phenoxide-4-methyl
axis in [Zn2L1(µ-OH)(NO3)2] complex leads to identical coor-
dination spheres of the two zinc atoms. The presence of the
two bridging groups in each complex, the phenolate oxygen
and either a hydroxide, methoxide, or nitrate ion, results in
relatively short Zn‚‚‚Zn distances of 3.115, 3.109, and 3.294
Å, respectively. The bridging ligand and terminal nitrate ion
occupy coordination positions cis to one another. The chirality
at N2 and N2A in [Zn2L1(µ-OH)(NO3)2] and at N3 and N2 in
[Zn2L1(µ-OMe)(NO3)2] and [Zn2L1(µ-NO3)(NO3)2] could, in
principle, result in the formation of the four diastereoisomers
for each complex. The solid-state structures revealed the
formation of only RR and SS isomers in each case.
AT ) AS + AP ) ꢀScS + ꢀPcP
(7)
The asymmetric complex [Zn2L2(NO3)3] contains two
zinc(II) ions, Zn1 and Zn2, having distorted octahedral and
distorted tetrahedral environments, respectively. The most
striking feature of this complex is its lack of an exogenous
bridging group; it therefore has a longer Zn‚‚‚Zn distance
compared to the other three compounds, 3.394(3) Å.
∆AT ) ∆AS + ∆AP ) AS(f) - AS(i) + AP(f) - AP(i) )
ꢀS{cS(f) - cS(i)} + ꢀPcP(f) ) ꢀS{cS(f) - cS(i)} +
ꢀP{cS(i) - cS(f)} ) {ꢀS - ꢀP}{cS(f) - cS(i)} (8)
∆AT
cS(f) - cS(i) )
(9)
ꢀS - ꢀP
Solution Structures. The structures of [Zn2L1(µ-NO3)(NO3)2]
1
In the equations, AT, AS, and AP are the total, substrate, and product
absorbances at a given wavelength; ꢀS and ꢀP are the extinction
coefficients of the substrate and the product at a given wavelength;
cS and cP are the concentrations of the substrate and the product at a
given time; and c(f) and c(i) stand for final and initial concentrations.
Data were collected only during the first 3-5% of conversion in each
reaction to avoid significant changes in the concentrations of the starting
materials. The observed rate constants were then determined from
the initial rates, the known concentration of the substrates, and the
corrected extinction coefficient of nitrocefin at 390 nm. The rate
constants for the formation (kform) and disappearance (kdis) of the anionic
intermediate were obtained by fitting the experimental data to eq 10
and [Zn2L2(NO3)3] in solution were investigated by H NMR
spectroscopy. The resonances of the two methylene groups in
HL1 appear as a singlet for the free ligand. Upon binding to
zinc(II), the R and â protons in each methylene group undergo
chemical shift changes and become inequivalent. Two pairs of
methylene proton resonances occur for [Zn2L1(µ-NO3)(NO3)2]
in aqueous solution, one as well-resolved doublets at 4.25 and
3.32 ppm and the other as broad peaks at 4.00 and 3.60 ppm.
We assign the former to the NMR-equivalent RR and SS
isomers based upon the following two findings. First, [Zn2L1-
(33) Sorrell, T. N.; O’Connor, C. J.; Anderson, O. P.; Reibenspies, J. H.
J. Am. Chem. Soc. 1985, 107, 4199-4206.
(34) Mallah, T.; Boillot, M.-L.; Kahn, O.; Gouteron, J.; Jeannin, S.;
Jeannin, Y. Inorg. Chem. 1986, 25, 3058-3065.
(35) Eduok, E. E.; O’Connor, C. J. Inorg. Chim. Acta 1984, 88, 229-
233.
At ) a{exp(-kformt) - exp(-kdist)}
(10)
where At is the absorbance at 640 nm and a is kform[N]0/ꢀ(kdis - kform
)
([N]0 - initial concentration of nitrocefin). Nitrocefin and hydrolyzed
nitrocefin do not absorb at 640 nm.
(36) Nasir, M. S.; Cohen, B. I.; Karlin, K. D. J. Am. Chem. Soc. 1992,
114, 2482-2494.
Results and Discussion
(37) Uhlenbrock, S.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1992, 31,
1647-1648.
Preparation of Ligands and Complexes. The dinucleating
ligands HL1 and HL2 (Scheme 2), derived from 2,4,6-trisub-
(38) Sakiyama, H.; Mochizuki, R.; Sugawara, A.; Sakamoto, M.; Nishida,
Y.; Yamasaki, M. J. Chem. Soc., Dalton Trans. 1999, 997-1000.