Cadmium(II) complexes: Mimics of organophosphate pesticide degrading enzymes and metallo-β-lactamases

Article abstract of DOI:10.1021/ic300687y

Figure Persented: Cadmium(II) complexes of ethyl 4-hydroxy-3,5-bis(((2- hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate (CO₂EtH ₃L1) and ethyl 4-hydroxy-3,5-bis(((2-methoxyethyl)(pyridin-2- ylmethyl)amino)methyl)benzoate (CO

Full text of DOI:10.1021/ic300687y

Article
pubs.acs.org/IC
Cadmium(II) Complexes: Mimics of Organophosphate Pesticide
Degrading Enzymes and Metallo-β-lactamases
Lena J. Daumann,† Lawrence. R. Gahan,*,† Peter Comba,‡ and Gerhard Schenk†,§
†School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
‡Anorganisch-Chemisches Institut, Universitat Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
§Department of Chemistry, National University of Ireland, Maynooth, County Kildare, Ireland
S
* Supporting Information
ABSTRACT: Cadmium(II) complexes of ethyl 4-hydroxy-3,5-bis(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)-
benzoate (CO2EtH3L1) and ethyl 4-hydroxy-3,5-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate
(CO2EtHL2) are described. The two ligands possess an ethyl ester (CO2Et-) at the position para to the phenolic −OH;
CO2EtHL2, with methyl ether donors in contrast to potentially nucleophilic alkoxide donors in CO2EtH3L1, offers a direct
comparison of potential ligand-centered nucleophiles. The complex with CO2EtH3L1 was characterized using 1H and 13C NMR
spectroscopy, mass spectrometry and microanalysis; X-ray crystallography defined a tetranuclear structure
[Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2. Functional studies of the cadmium(II) complexes were undertaken
with the substrates bis(2,4-dinitrophenyl)phosphate (BDNPP), and nitrocefin to assess their phosphatase and β-lactamase
activities, respectively. The complexes with CO2EtH3L1 and CO2EtHL2 are competent phosphoesterase mimics with KM = 9.4
2.1 mM and 10.1 3.4 mM, kcat = 9.4 0.2 × 10−3 s−1 and 9.7 2.7 × 10−3 s−1, respectively. Use of a solvent mixture
containing H218O/H216O in the reaction with BDNPP showed that for the complex with CO2EtH3L1 the 18O label was
incorporated in the reaction product suggesting that the nucleophile involved is a Cd−OH moiety and not a metal bound
alkoxide; for CO2EtHL2 the presence of the methyl-ether dictates that the active nucleophile must also be a hydroxide. The
cadmium(II) complex with CO2EtH3L1 was furthermore found to be a competent β-lactamase mimic with kcat = 1.39 × 10−2
3
× 10−3 s−1, KM = 0.11 0.03 mM, and pKa = 7.9 0.1. Mass spectral evidence suggested that the active nucleophile in this
reaction is the alkoxide; lack of β-lactamase activity of the complex with CO2EtHL2 supports this assignment. Similar to enzyme-
catalyzed reactions, a blue reaction intermediate in the β-lactamase reaction of the CO2EtH3L1 complex was also identified. It is
proposed that the Cd(II) complexes of CO2EtH3L1 and CO2EtHL2 react identically as phosphatases, with a terminal hydroxide
as the nucleophile; the former exhibits β-lactamase activity with the alkoxide as a nucleophile, while the latter, without a
potentially nucleophilic alkoxide, is inactive.
entire family but exhibit variations in metal ion specificity and
basic mechanism.1 Thus, the identity of the metal ions can vary
from the well characterized Fe(III)−Fe(II) and Fe(III)−
Zn(II)/Mn(II) centers in PAPs and putative Fe(II)−Zn(II)
centers in phosphotriesterases to the di-Zn(II) combination in
MβLs.1,7−11 In some cases, the enzymes are promiscuous and
activity can be reconstituted with a range of divalent metal
INTRODUCTION
■
The structurally diverse dinuclear metallohydrolases include
ureases, the purple acid phosphatases (PAPs), phosphotries-
terases, exonucleases and metallo-β-lactamases (MβLs), among
others.1−4 These metalloenzymes employ a dinuclear metal ion
center to catalyze the hydrolysis of amides and esters of
carboxylic and phosphoric acids.1 Interest in these systems
arises from their potential as targets for drug design and
application in bioremediation.1,2,5,6 The metallohydrolases
display similarities in the first coordination sphere across the
Received: April 2, 2012
Published: July 3, 2012
© 2012 American Chemical Society
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Figure 1. Active site of GpdQ and part of the hydrogen bond network (left) and the active site of the metallo-β-lactamase from B. fragilis (right).
Chart 1. Substrates Commonly Used in β-Lactamase and Phosphatase Assays and the Structures of Co2EtH3L1 and CO2EtHL2
ions.12,13 Of particular interest in these systems is the identity
of the attacking nucleophile. Thus, for the PAPs proposals for
the nucleophile include a μ-OH, a quasi-terminal OH, a
terminal OH bound to the Fe(II) site and a second
coordination sphere nucleophile.1,14−16 For the phosphotries-
terases pathways involving μ- and terminal-OH are pro-
posed,13,17 and a metal bound OH is proposed as the
nucleophile in the MβLs.11
Our current interest is focused on organophosphate-
degrading phosphotriesterases and antibiotics-inactivating
MβLs. In the former case we are specifically interested in a
glycerophosphodiesterase from Enterobacter aerogenes (GpdQ),
a highly promiscuous phosphoesterase with activity toward
mono-, di- and triphosphoesters including the toxic EA 2192, a
product of VX degradation, and organophosphate pesti-
cides.5,18−22 The active site of GpdQ contains two metal ions
with a buried α site and a more solvent exposed β site.1,23,24
The metal ions are coordinated by four histidines, two
aspartates, and an asparagine residue.1,23,24 Enzymatic activity
in GpdQ can be reconstituted in the presence of Zn(II),
Co(II), Mn(II), and Cd(II).18,20,25,26 MβLs catalyze the
hydrolytic cleavage of the β-lactam ring of antibiotics, such as
the penicillins.27−29 The MβLs use zinc ions in vivo to gain full
catalytic activity. The two metal ions are usually bridged by a
hydroxide and have a 4-, 5-mixed coordination sphere, although
there is discussion as to the absolute requirement for the
second metal ion.11,30,31 The active site structures of GpdQ and
MβL, from B. fragilis, are shown in Figure 1.
terminal OH for the same substrate but different model
systems.38,39 Furthermore, examples of model systems with
either, or both, a metal−OH and a metal-OR implicated in the
mechanism have been reported.32,40−43
While the rationale for the use of zinc(II) in model systems is
obvious, cadmium(II) is not commonly considered to be a
biological metal ion. The recent discovery of a carbonic
anhydrase from the marine diatom Thalassiosira weissflogii with
cadmium bound in the active site has prompted interest in this
metal ion.44,45 Recently, we showed that (Cd2+)2-GpdQ and
[Cd2((HP)2B)(OAc)2(OH2)](PF6) (((HP)2B) = 2,6-bis([(2-
pyridylmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphe-
nol43) are active catalysts toward the phosphodiester substrates
bis-(4-nitrophenyl)phosphate (BNPP) and bis-(2,4-
dinitrophenyl)phosphate (BDNPP), and although not ap-
proaching the efficiency of the metalloenzyme itself, our
interest was aroused by the relative efficiency of the
cadmium(II) model system.25
To expand the study of cadmium model complexes the
synthesis and structural characterization of a cadmium
containing system based on ethyl 4-hydroxy-3,5-bis(((2-
hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)benzoate
(CO2EtH3L1) is reported. We discuss an investigation of the
phosphoesterase and MβL activity of the complex using the
substrates BDNPP and nitrocefin, a β-lactam compound,
respectively. In addition, a second ligand, ethyl 4-hydroxy-3,5-
bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-
benzoate (CO2EtHL2) and its cadmium(II) complex were
synthesized to investigate the mechanism in the absence of
potential alkoxide nucleophiles. The structures of the substrates
and ligands employed in this work are shown in Chart 1.
To study these metallohydrolases effective use has been
made of model systems. Model complexes are capable of
reproducing electronic, structural and reactivity characteristics
of metalloenzyme systems and although they generally exhibit
less substrate specificity they are often more robust than the
metalloenzymes themselves.32−37 The nucleophilic agent in
these models is typically a metal-bound hydroxide; there are
examples where the nucleophilic agent varies from μ−OH to
MATERIALS AND METHODS
■
General Methods. Nuclear magnetic resonance (NMR) spectra
were measured with Bruker AV300, AV400, AV500, and AV600
instruments. The spectra were recorded in CDCl3, (CD3)2CO,
(CD3)2SO, CD3OD, CD3CN, or D2O. Chemical shifts were
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determined in ppm, relative to known residual solvent peak references.
113Cd NMR was measured with the Bruker AV400 instrument and an
str); 2919 (m, C−H str); 2850 (m, C−H str); 1596 (m, CC str);
1433 (m, O−H def); 832, 763 (s, py-H).
Synthesis of 2-Methoxy-N-(pyridin-2-ylmethyl)-
aminoethanol. The compound was prepared according to a
previously published procedure.32
operating frequency of 89 MHz; Cd-shifts were referenced to
cadmium(II) acetate dihydrate in D2O (−46 ppm). Coupling
constants are given in Hz. 31P NMR spectra were recorded with a
600 MHz Bruker AV600 spectrometer in the digital acquisition mode
(operating frequency 242 MHz) at room temperature at the NMR
facilities of the Anorganisch-Chemisches Institut, University of
Heidelberg. Chemical shifts are reported in δ units relative to 85%
H3PO4 in D2O as external reference (δP = 0.00). Two-dimensional
correlation spectroscopy (COSY), heterobinuclear single quantum
correlation (HSQC) and heterobinuclear multiple bond connectivity
(HMBC) experiments were used to assign each signal in selected
spectra. Low resolution mass spectral data were collected with a
Bruker Esquire high capacity ion trap electrospray ionization mass
spectrometer (HCT ESI-MS), in methanol (MeOH), acetonitrile
(MeCN) or 50:50 MeCN:water with a Bruker ES source. Predicted
isotopic splitting patterns of peaks were calculated using the program
ChemCalc.46 Elemental analyses were performed with a Carlo Erba
NA 1500 Elemental Analyzer. UV−vis spectrophotometric experi-
ments were performed with a Varian Cary50 Bio UV/Visible
spectrophotometer with a Peltier temperature controller in 10 mm
quartz cuvettes. General FT-Infrared Spectroscopy was carried out
with a Perkin-Elmer FT-IR Spectrometer SPECTRUM 2000 with a
Smiths DuraSamplIR II ATR diamond window.
Synthesis of Ethyl 3,5-Bis(bromomethyl)-4-hydroxyben-
zoate. The compound was prepared by a previously published
procedure.51
Synthesis of Ethyl 4-Hydroxy-3,5-bis(((2-hydroxyethyl)-
(pyridin-2ylmethyl)amino) methyl)benzoate (CO2EtH3L1).
Ethyl 3,5-bis(bromomethyl)-4-hydroxybenzoate (0.50 g, 14 mmol)
in dichloromethane (4 mL) was added dropwise to N-(2-
pyridylmethyl)-2-aminoethanol (0.43 g, 28 mmol) and triethylamine
(0.80 g) in tetrahydrofuran (4 mL) at 0 °C. The resulting yellow
solution was stirred for 48 h, filtered to remove the white precipitate of
triethylamine hydrobromide and the solvent removed under vacuum.
The resulting brown oil was purified by flash column chromatography
(EtOAc/MeOH, 8:2, I2 stain, Rf = 0.44) giving a yellow oil (Yield 0.50
1
g, 1.0 mmol, 72%). H NMR (CDCl3, 500.13 MHz); δ 1.32 (t, 3H,
CH2CH3, J = 7.1 Hz); 2.67 (t, 4H, NCH2CH2, J = 5.0 Hz); 3.67 (t,
4H, NCH2CH2, J = 5.0 Hz); 3.77 (s, 4H, arCH2N); 3.83 (s, 4H,
NCH2py); 4.26 (q, 2H, CH2CH3, J = 7.1); 7.09 (m, 2H, pyH, J = 5.8,
0.8 Hz); 7.20 (d, 2H, pyH, J = 7.8 Hz); 7.54 (td, 2H, pyH, J = 7.6, 1.7
Hz); 7.64 (s, 2H, arH); 8.51 (dq, 2H, pyH, J = 4.8, 0.8 Hz). 13C NMR
(CDCl3, 100.62 MHz); δ 14.4 (CH2CH3); 55.8 (arCCH2), 56.5
(NCH2CH2); 58.5 (NCH2CH2); 58.8 (NCH2py); 60.6 (CH2CH3);
122.5 (pyC); 123.1 (arCCO2Et); 123.2 (arCCH2); 123.4 (pyC); 131.8
(arCH); 137.0 (pyC); 149.1 (pyC); 157.3 (pyCCH2); 160.9 (arCOH);
166.3 (CO2Et). FT-IR spectroscopy (v, cm−1) 3296 (m, O−H str);
2826 (m, C−H str); 1704 (m, CO str); 1595 (m, CC str); 1571
(m, CC str); 1304 (m, C−O sym def); 1204 (s, C−O asym def);
1026 (m, C−O str); 758 (s, py-H). ESI mass spectrometry (methanol)
m/z 495.19 [C27H34N4O5 + H]+.
Synthesis of Ethyl-4-hydroxy-3,5-bis(hydroxymethyl)-
benzoate. The compound was prepared by a modification of a
previously published procedure.47 To a cool 12% aqueous solution of
NaOH (70 mL, 0.21 mol), commercially available ethyl 4-
hydroxybenzoate (15 g, 0.09 mol) was added at 0 °C. An aqueous
formaldehyde solution (37%, 60 mL, 2.16 mol) was added and the
reaction was stirred at 55 °C for 3 days. The red solution was allowed
to reach room temperature and ethyl acetate (100 mL) was added.
The organic layer was discarded and ethyl acetate (100 mL) and a
saturated aqueous solution of NH4Cl were added to the remaining
aqueous phase. The organic phase was collected and dried over
Na2SO4. Removal of the solvent in vacuo left an orange solid which
was recrystallized from CHCl3/MeOH (1:1) to yield a pale yellow
Synthesis of Ethyl 4-Hydroxy-3,5-bis(((2-methoxyethyl)-
(pyridin-2-ylmethyl)amino) Methyl)Benzoate (CO2EtHL2). To
a solution of 2-methoxy-N-(pyridin-2-ylmethyl)aminoethanol (2.8 g,
16.8 mmol) and triethylamine (3.96 g) in tetrahydrofuran (25 mL) a
solution of ethyl-3,5-bis(bromomethyl)-4-hydroxybenzoate (2.5 g, 7.1
mmol) in dichloromethane (25 mL) at 0 °C was added dropwise. The
reaction mixture was stirred for 48 h, concentrated to 4 mL in vacuo
and filtered to remove the precipitated triethylamine hydrobromide.
Removal of the solvent resulted in a brown oil, which was further
purified by flash column chromatography (2.5 g crude ligand, l=30 cm,
Ø=1.5 cm, MeOH/EtOAc 2:8 (1 L), MeOH/EtOAc 2:1 (300 mL),
MeOH (300 mL), fraction volume 10 mL, 50 fractions collected.
FeCl3 stain, Rf = 0.46 in MeOH/EtOAc 2:8). The ligand was obtained
1
powder (Yield 11.7 g, 51.7 mmol, 57%). H- NMR (CD3OD, 400.13
MHz); δ 1.37 (t, 3H, CH2CH3, J = 7.1 Hz); 4.32 (q, 2H, CH2CH3, J =
7.1 Hz); 4.80 (s, 4H, CH2OH); 7.71 (s, 2H, Ar−H). 13C NMR
(CD3OD, 100.62 MHz) δ 14.7 (CH2CH3); 61.6 (CH2CH3); 61.8
(CH2OH); 122.4 (arCCO2Et); 128.4 (arCH); 129.6 (arCCH2OH);
159.4 (arCOH); 168.5 (CO2Et). ESI mass spectrometry (methanol)
m/z 153.10 [C8H8O3+H]+. FT-IR spectroscopy (v, cm−1) 3428 (m,
O−H str); 2985, 2942, 2871 (w, CH2 str); 1689 (s, CO str); 1206
(s, C−O str); 1010 (m, C−O str). Melting Point 137.6−139.4 °C;
139.0 °C (benzene/petroleum ether);47 137.0−138.0 °C (chloro-
form).48
Synthesis of N-(2-Pyridylmethyl)-2-aminoethanol. The com-
pound was prepared by a modification of previously published
procedures.49,50 Pyridine 2-carboxaldehyde (4.42 g, 0.04 mol) in
methanol (10 mL) was added dropwise to ethanolamine (2.42 mL,
0.04 mol) in methanol (20 mL) at 0 °C. The yellow solution was
brought to room temperature and stirred for two hours. Sodium
borohydride (3.60 g, 0.1 mol) was added in small portions at 0 °C and
the solution stirred for a further two hours. Water (30 mL) was added,
the reaction mixture concentrated to approximately 30 mL and the
remaining solution extracted with dichloromethane (3 × 20 mL). The
combined organic phases were dried over Na2SO4 and the solvent
removed in vacuo to yield a yellow oil (Yield 2.50 g, 16.4 mmol, 41%).
1H NMR (CDCl3, 400.13 MHz); δ 2.14 (s, 1H, NH); 2.91 (t, 2H,
NCH2CH2, J = 5.7 Hz); 3.79 (t, 2H, CH2OH, J = 5.1 Hz); 5.27 (s, 2H,
arCH2N); 5.40 (bs, 1H, OH); 7.26 (m, 1H, pyH, J = 6.2 Hz); 7.63 (d,
1H, pyH, J = 7.7 Hz); 7.69 (tt, 1H, pyH, J = 7.8, 1.8 Hz), 8.55 (dd, 1H,
pyH, J = 4.9 Hz). 13C NMR (CDCl3, 100.62 MHz); δ 56.9
(NCH2CH2); 58.2 (arCH2N); 59.7 (CH2 OH); 123.2 (pyCH); 123.2
(pyCH); 137.3 (pyCH); 149.4 (pyCH); 153.8 (pyCCH2). ESI mass
spectrometry (methanol) m/z 175.07 [C8H12N2O+Na]+; 153.11
[C8H12N2O+H]+. FT-IR spectroscopy (v, cm−1) 3214 (m, O−H
1
as a yellow oil (Yield 2.19 g, 4.2 mmol, 60%). H NMR (CDCl3,
300.13 MHz); δ 1.34 (t, 3H, CH2CH3, J = 7.1 Hz); 2.73 (t, 2H,
NCH2CH2, J = 5.7 Hz); 3.24 (s, 6H, OCH3); 3.49 (t, 2H, NCH2CH2,
J = 5.7 Hz); 3.81 (s, 2H, arCH2N); 3.85 (s, 2H, NCH2py); 4.30 (q,
2H, CH2CH3, J = 7.1 Hz); 7.10 (ddd, 2H, pyH, J = 7.4, 4.9, 1.1 Hz);
7.42 (d, 2H, pyH, J = 7.8 Hz); 7.57 (td, 4H, pyH, J = 7.7, 1.3 Hz); 7.83
(s, 2H, arH); 8.48 (dq, 2H, pyH, J = 4.9, 1.7, 0.8 Hz). 13C NMR
(CDCl3, 100.62 MHz); δ 14.2 (CH2CH3); 53.1 (NCH2CH2), 55.0
(arCCH2); 58.5 (OCH3); 60.4, 60.5 (CH2CH3/NCH2py); 70.7
(NCH2CH2); 120.4 (pyC); 121.5 (arCCO2Et); 122.9 (arCCH2);
123.6 (pyC); 130.4 (arCH); 136.4 (pyC); 148.6 (pyC); 158.5
(pyCCH2); 160.3 (arCOH); 166.5 (CO2Et). FT-IR spectroscopy (v,
cm−1) 2927 (m, C−H str); 1705 (m, CO str); 1602 (m, CC str);
1197 (m, C−O−C str): 1109 (m, C−O str); 832 (m, Ar−H); 762 (s,
py-H). ESI mass spectrometry (methanol) m/z 523.21 [C29H38N4O5
+ H]+.
Synthesis of [Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2.
CO2EtH3L1 (50 mg, 0.1 mmol) was dissolved in MeOH (2 mL) and a
methanol solution (2 mL) of cadmium(II) acetate dihydrate (50 mg,
0.2 mmol) with sodium acetate (8 mg, 0.1 mmol) was added dropwise.
Sodium hexafluorophosphate (50 mg, 0.3 mmol) was added and the
resulting yellow solution left to evaporate at room temperature. A
white product was obtained after 5 days. Recrystallization from
methanol resulted in white crystals of the complex (Yield 40 mg, 0.02
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mmol, 66%). Microanalysis C61.5H81.25Cd4Cl0.25N8O19.5P2F12: calc. C:
37.06, H: 4.11, N: 5.62; found: C 37.43, H 4.28, N 5.63%. ESI mass
spectrometry (methanol) m/z: 837.0 Calc for [C31H39Cd2N4O9]+, m/
z 837.08. FT-IR spectroscopy (ν, cm−1): 3173 (b, O−H str in water)
2906 (w, C−H str), 1670 (m, CO str), 1604 (m, asym str OAc);
1546 (m, asym str OAc); 1413 (m, sym str OAc); 1373 (m, sym str
OAc). 1015 (w, C−OH str), 831 (s P−F str), 766 (m, py C−H def),
the formation of 2,4-dinitrophenol; the extinction coefficient for this
product at 400 nm, throughout the pH range studied, is 12,100 M−1
cm−1.55−57 An aqueous multicomponent buffer (50 mM in each of 2-
(N-morpholino)ethanesulfonic acid (MES; pH 3.50−6.50), 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.00−
8.50) and 2-(N-cyclohexylamino)ethane sulfonic acid (CHES; pH
9.00−10.00) and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS;
pH 10.5−11.0), with the ionic strength controlled by 250 mM
LiClO4) was used. Assays were carried out in 50:50 MeCN:buffer, with
substrate and complex initially dissolved in MeCN. The pH values
refer to the aqueous component, it should however be noted that the
pH of a solution of the buffer was the same within error as a 1:1
m i x t u r e o f b u ff e r a n d a c e t o n i t r i l e . S o l i d
[Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2 was dissolved
in MeCN to give a 1 mM stock solution. A 1 mM stock solution of
[Cd2(CO2EtL2)(CH3COO)2]+ was generated in MeCN in situ by
refluxing one equivalent ligand and two equivalents cadmium acetate
for 30 min and confirming the successful formation by mass
spectrometry. Assays conducted to investigate the pH dependence
of catalytic properties were 40 μM in complex and 5 mM in BDNPP
and showed no significant buffer effects. [Substrate] dependence
assays were 40 μM in complex and 1−11.5 mM in BDNPP, and
[complex] dependence assays were 20−120 μM in complex and 5 mM
in BDNPP. Background assays of autohydrolysis in the presence of
two equivalents of cadmium(II) acetate were subtracted from the data.
All data were fitted by nonlinear least-squares regression analysis.
Metallo-β-lactamase-like Activity. MβL assays were conducted
in the same aqueous multicomponent buffer as mentioned above with
the ionic strength controlled by 250 mM LiClO4. Nitrocefin was
purchased from Merck, Darmstadt, Germany (Cambiochem, ≥ 95%
purity). Assays were carried out at 37 °C in 50:50 MeCN:buffer, with
nitrocefin initially dissolved in MeCN (10 mM) and the complex
dissolved in MeCN:water (1 mM). Assays conducted to investigate
pH dependence were 5 μM in complex and 50 μM in nitrocefin.
[Substrate] dependence assays were 5 μM in complex and 10−50 μM
in nitrocefin, and [complex] dependence assays were 20−120 μM in
complex and 5 mM in nitrocefin. Background assays of autohydrolysis
in the presence of two equivalents of cadmium(II) acetate were
subtracted from the data. The assays were monitored for the hydrolysis
of the substrate at 390 nm for 5 min and left to equilibrate for 2 min
before each measurement. Since nitrocefin and its hydrolysis product
both absorb at 390 nm a corrected extinction coefficient of 13 415 M−1
cm−1 was used to calculate the rate of catalysis.38,39
1
556 (s, P−F). H NMR (CD3CN/D2O 1:1, 500.13 MHz, referenced
to D2O); δ 1.70 (t, 3H, CH2CH3, J = 7.12 Hz); 2.39 (s, 6H,
acetateCH3); 3.28 (t, 4H, NCH2CH2, J = 8.40 Hz); 3.43 (m, 2H, CH2,
J = 11.60 Hz); 4.13 (m, 4H, CH2CH2OH); 4.15−4.23 (m, 2H, CH2);
4.45 (m, 2H, CH2, J = 5.85 Hz); 4.59−4.66 (m, 4H, CH2/CH2CH3),
7.39 (d, 2H, pyCH, J = 7.85 Hz); 7.58 (t, 2H, pyCH, J = 6.10 Hz);
7.77 (s, 2H, arCH); 8.00 (t, 2H, pyCH, J = 7.72 Hz); 8.70 (d, 2H,
pyCH, J = 4.70 Hz). 13C NMR (CD3CN, /D2O 1:1, 100.62 MHz,
referenced to CD3CN); δ 14.5 (CH2CH3); 22.7 (acetateCH3); 56.9
(CH2); 57.7 (CH2); 58.3 (CH2); 58.7 (CH2); 61.6 (CH2); 124.4
(pyCH); 124.5 (pyCH); 125.5 (arC); 134.4 (arCH); 140.1 (pyCH);
148.8 (pyCH); 156.1(pyC); 161.1 (arCOH); 166.2 (CO2Et); 181.8
−
(CO2 ); (the arCCO2Et signal was not assigned due to overlapping
with the solvent signal).113Cd NMR (CD3CN/D2O 1:1, 89 MHz) δ
36.4.
Synthesis of [Cd2(CO2EtL2)(CH3COO)2](PF6). EtCO2HL2 (180
mg, 0.34 mmol) was dissolved in methanol and cadmium(II) acetate
dihydrate (183 mg, 0.68 mmol) and sodium acetate (56 mg, 0.68
mmol) added. The yellow solution was subsequently refluxed for 30
min and then allowed to cool to room temperature and sodium
hexafluorophosphate (115 mg, 0.68 mmol) added. After filtration, the
yellow solution was left to evaporate at room temperature to leave a
yellow oil. Attempts to crystallize the complex from a range of solvents
were unsuccessful. After repeated (5x) evaporations of the methanolic
complex solution a white powder was obtained. FT-IR spectroscopy
(ν, cm−1): 3385 (b, O−H str in water) 2927 (w, C−H str); 1702 (m,
CO str); 1603 (m, asym str OAc); 1574 (m, asym str OAc); 1441
(m, sym str OAc); 1368 (m, sym str OAc). 833 (s, P−F str). ESI mass
spectrometry (methanol) found m/z 864.0 (100%), 863.0 (90%)
[C33H43Cd2N4O9]+ calc m/z 865.11 (100.0%), 863.11 (88.3%), 864.11
(86.7%).1H NMR (CD3CN/D2O 1:1, 500.13 MHz, referenced to
D2O); δ 1.84 (t, 3H, CH2CH3, J = 7.12 Hz); 2.46 (s, 6H, acetateCH3);
3.33 (m, 2H, CH2); 3.43 (m, 1H, CH2); 3.63−3.77 (m, 7H, CH2);
3.79 (s, 6H, OCH3); 3.93−4.49 (m, 6H, CH2); 4.75 (q, 2H, CH2CH3,
J = 7.16 Hz); 7.57 (m, 1H, pyCH); 7.76 (m, 2H, pyCH); 7.87 (t, 1H,
pyCH, J = 6.79 Hz); 7.97, 8.12 (s, 2H, arCH); 8.19 (m, 1H, pyCH);
8.32 (m, 1H, pyCH); 8.83 (m, 1H, pyCH); 8.94 (m, 1H, pyCH). 13C
NMR (CD3CN, /D2O 1:1, 100.62 MHz, referenced to CD3CN); δ
14.6 (CH2CH3); 22.8 (acetateCH3); 56.3 (CH2); 59.9 (OCH3); 60.5
(CH2); 60.7 (CH2); 61.8 (CH2); 68.2 (CH2); 68.5 (CH2); 124.6
(pyCH); 124.9 (pyCH); 125.5 (arC); 125.7 (arC); 134.4 (arCH);
134.8 (arCH); 140.3 (pyCH); 140.6 (pyCH); 148.8 (pyCH); 149.3
(pyCH); 155.9 (pyC); 168.5 (arCOH); 171.0 (CO2Et); 181.1
Fluoride Inhibition Studies. Two thousand equivalents NaF
were added to a stock solution of [Cd2(CO2EtH2L1)(CH3COO)2]+.
The mixture was incubated at room temperature for 24 h prior to
activity measurements conducted as described for nitrocefin and
BDNPP hydrolysis.
18O-Labeling Studies. 18O-labeled water was purchased from
Novachem, Victoria, Australia (97% purity). The substrate bis(2,4-
dinitrophenyl)phosphate (BDNPP) was hydrolyzed in a 50:50 mixture
of MeCN:buffer (50 mM, CHES, pH 9.8) in the presence of one
equivalent complex. In the isotopic labeling studies the buffer solution
contained 50% 18O-labeled water (97% purity). For the unlabeled
experiment the solution was made up of 0.005 mmol of complex in
MeCN (0.3 mL), 100 mM CHES buffer pH 9.8 (0.15 mL) and
distilled water (0.15 mL). To this, one equivalent BDNPP (2.5 mg)
was added and the mixture was left for three days at room temperature
prior to spectra recording. For the 18O-labeled sample the solution was
made up from a solution of 0.005 mmol [Cd4(CO2EtH2L1)2CH3-
OO)3.75Cl0.25(H2O)2](PF6)2 complex in MeCN (0.3 mL), 100 mM
CHES buffer pH 9.8 (0.15 mL) and 18O-water (97%, 0.15 mL). To
this one equivalent BDNPP (2.5 mg) was added and the mixture was
again left for three days at room temperature prior to 31P NMR spectra
recording.
−
(CO2 ); 113Cd NMR (CD3CN/D2O 1:1, 89 MHz) δ 23.4.
Crystallographic Measurements. X-ray diffraction data for a
crystal of [Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2
were collected at room temperature with an Oxford Diffraction
Gemini Ultra dual source (Mo and Cu) CCD diffractometer with Cu
(λKα = 1.5418 Å) radiation. The structures were solved by direct
methods (SIR-92) and refined (SHELXL 97) by full matrix least-
squares methods based on F2.52,53 These programs were accessed
through the WINGX 1.70.01 crystallographic collective package.53 All
non-hydrogen atoms were refined anisotropically unless they were
disordered. Hydrogen atoms were fixed geometrically and were not
refined. Selected crystal data and some details of refinements are given
in Supporting Information Table S1; selected bond distances and
angles are presented in Table S2.
Phosphoesterase-like Activity. The phosphoesterase activity of
the complexes was determined by measuring hydrolysis of the
substrate BDNPP, which was synthesized after a previously published
method.54 The initial rate method was employed and assays were
measured such that the initial linear portion of the data was used for
analysis. Product formation was determined at 25 °C by monitoring
Binding of Phosphoesters in Solution. Two methods were
employed: (i) mass spectrometry and (ii) 113Cd NMR. Using the
solvent system employed for the kinetic studies the mass spectrum was
measured in order to monitor species forming during phosphate ester
hydrolysis. The complex [Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25
-
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Figure 2. Synthesis of the ligands CO2EtH3L1 and CO2EtHL2.
(H2O)2](PF6)2 was dissolved in MeCN/water 1:1 and the mass
spectra recorded with complex concentrations ranging from 0.1 mM to
10 μM. With the same solvent conditions the mass spectra were
recorded in the presence of either 20 equiv of PNPP (4-nitrophenyl
phosphate), a product mimic, or 25 equiv of DPP (diphenylphos-
phate), a substrate mimic lacking the activating nitro groups and which
is therefore not hydrolyzed by the model complex. The solutions were
left to incubate at room temperature for one hour prior to recording of
spectra. A 113Cd NMR spectrum of a 0.01 mM solution of
[Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2 in CD3CN/
D2O 1:1 was recorded after the addition of 10 equiv of DPP and
incubation for 6 h.
Binding of β-Lactams in Solution. Three methods were
employed: (i) solution IR measurements, (ii) 13C NMR, and (iii)
UV−vis studies. IR spectra were recorded at the University of
Heidelberg (Organisch-Chemisches Institut) with a ReactIR 45 m
from Mettler Toledo between 650 and 2800 cm−1. The spectra of
nitrocefin alone and in the presence of 1 equiv of cadmium(II)
complex were recorded in MeCN/water (50:50) at 293 K every 2 min
for two hours. The initial concentrations of complex and substrate
were 5 mM each. In the 13C NMR experiments the solution was
idin-2-ylmethyl)amino)methyl)benzoate (CO2EtHL2). An
overview of the syntheses is shown in Figure 2. The
nomenclature employed follows that used previously for these
types of ligands.32 Thus, CO2EtH3L1 indicates that the ligand
has an ethyl ester (CO2Et-) para to the phenolic −OH and has
potentially three sites for deprotonation, the phenol and the
two pendant alcohol donors. The second ligand, CO2EtHL2,
offers a direct comparison of methyl ether donors with the
alkoxide donor in CO2EtH3L1 (Figure 2). As only one proton
can be potentially subtracted from the former ligand upon
deprotonation, the nomenclature is adjusted accordingly. Upon
crystallization, the complex with CO2EtH3L1 was characterized
as [Cd4(CO2EtH2L1)2(CH3COO)3.75 Cl0.25(H2O)2](PF6)2, the
nomenclature indicating a single deprotonation had occurred.
Based on mass spectral characterization the complex with
CO2EtHL2 was formulated as [Cd2(CO2EtL2)(CH3COO)2]-
(PF6), where a single deprotonation of the ligand had occurred.
The crystallization of this complex proved to be difficult.
However after several recrystallization attempts the complex
could be obtained in low yield and was analyzed further with IR
and NMR spectroscopy.
initially 0.075
M in penicillin G and 0.075 M of
[Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25(H2O)2](PF6)2 in
(CD3)2CO/(CD3)2SO, 1:1. This solvent system and substrate were
employed in order to directly compare the chemical shifts obtained to
literature values. The spectrum was recorded at room temperature
after 24 h of mixing both components. To elucidate how many
molecules of nitrocefin could be hydrolyzed by each complex, a UV−
vis titration experiment was conducted where successively one to four
equivalents of nitrocefin were added to the complex under conditions
used for kinetic experiments (MeCN/buffer pH 8, 37 °C, 300−700
nm). After each addition the solution was scanned in intervals of one
minute for a total time of one hour. Initial concentrations of nitrocefin
and complex were 0.05 mM.
Intermediate Species formed during the Hydrolysis of
Nitrocefin. UV−vis experiments were conducted by measuring the
absorption spectrum of 0.05 mM nitrocefin in dry MeCN at 37 °C and
then adding one equivalent of cadmium(II) complex and recording the
spectrum again after 2 min. The same conditions were applied after
the addition of 10 uL water to the mixture. Mass spectra were also
recorded immediately after mixing equimolar amounts of nitrocefin
and cadmium(II) comlex in dry acetonitrile. Initial concentrations of
complex and substrate were 0.01 mM.
Crystal Structure of [Cd4(CO2EtH2L1)2(CH3COO)3.75
Cl0.25(H2O)2](PF6)2. X-ray quality crystals of a complex
subsequently identified as [Cd4(CO2EtH2L1)2(CH3COO)3.75
-
-
Cl0.25(H2O)2](PF6)2 were isolated after addition of methanol
solution of cadmium(II) acetate in the presence of sodium
acetate and sodium hexafluorophosphate and subsequent
evaporation of the solvent. The chloride ion is believed to
arise from contamination of the cadmium(II) starting material.
The structure and atomic numbering scheme are illustrated in
Figure 3.
The complex crystallized as a dimer of dimer structure with
disorder apparent around the chloride and acetate ligands. The
dimers are connected through the carbonyl oxygen donor of
the ethyl ester bonded to one of the two cadmium(II) ions
bridged by the CO2EtH2L1− ligand. The dimers are therefore
connected intermolecularly “head to tail” through the carbonyl
oxygen donors. The dimeric unit has two cadmium ions
coordinated by one ligand molecule. The seven-coordinate
Cd(1) has a distorted pentagonal bipyramidyl geometry while
Cd(2) has a distorted six-coordinate geometry. One formally
bidentate acetate ligand is coordinated to Cd(1) and Cd(2) is
coordinated by disordered chloride and acetate ligands. The
coordination sphere of Cd(1) is composed of two nitrogen
donors (Cd(1)−N(1), 2.412(4) Å and Cd(1)−N(2), 2.318(4)
Å), the protonated alcohol (Cd(1)−O(2), 2.475(4) Å), a water
molecule (Cd(1)−O(8), 2.273(4) Å), a bidentate acetate
ligand (Cd(1)−O(4), 2.384(4) Å and Cd(1)−O(5),
2.428(4)Å), the phenoxide oxygen that bridges both cadmium
ions with (Cd(1)−O(1), 2.368(3) Å, Cd(2)−O(1), 2.251(3)
RESULTS AND DISCUSSION
■
Ligand and Complex Syntheses. Reaction between ethyl
3,5-bis(bromomethyl)-4-hydroxybenzoate and two equivalents
of N-(2-pyridylmethyl)-2-aminoethanol in dichloromethane/
tetrahydrofuran in the presence of triethylamine, and
subsequent column chromatography, resulted in the desired
ligand, ethyl 4-hydroxy-3,5-bis(((2-hydroxyethyl)(pyridin-2-
ylmethyl)amino)methyl)benzoate (CO2EtH3L1). Reaction of
two equivalents of 2-methoxy-N-(pyridin-2-ylmethyl)-
aminoethanol with ethyl-3,5-bis(bromomethyl)-4-hydroxyben-
zoate yielded ethyl 4-hydroxy-3,5-bis(((2-methoxyethyl) (pyr-
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the Δνasym‑sym = 231 cm−1, while infrared bands from the
bidentate nonbridging acetate ligands were assigned to 1547
and 1413 cm−1, respectively, with Δνasym‑sym = 134 cm−1.62
Infrared Spectrum of [Cd2(CO2EtL2)(CH3COO)2(H2O)]-
(PF6). After repeated dissolution in hot MeOH and evaporation
a white powder was obtained and the IR spectrum recorded. In
addition to typical ligand and hexafluorophosphate bands, the
carbonyl stretch from the ethyl ester of the ligand was observed
at 1702 cm−1, not very different from the free ligand (1704
cm−1), suggesting that the complex exists as a single dinuclear
complex and that the ester carbonyl of another unit is not
coordinated to either of the metal ions. The asymmetric and
symmetric bands from the acetato ligands were, however,
similar to those observed for [Cd4(CO2EtH2L1)2-
(CH3COO)3.75Cl0.25(H2O)2](PF6)2 and are found at 1603,
1574, 1368, and 1441 cm−1, respectively. Here Δνasym and
Δνsym are 235 and 133 cm−1, respectively, which suggest the
presence of bidentate nonbridging, and unidentate acetate.
Moreover, the broad band at 3385 cm−1 also suggests the
presence of water.
Mass Spectral Characterization. The mass spectrum of
the complex with CO2EtH3L1 in methanol displays an isotope
pattern typical of a Cd(II)2 species,63 the most prominent
pattern being [C31H39Cd2N4O9]+ (m/z (100%) found 837.0,
calc. m/z 837.08) assigned as [Cd2(CO2EtH2L1)-
(CH3COO)2]+(Calculated and observed isotope patterns can
be found in Supporting Information Figure S1). Loss of an
acetate results in [Cd2(CO2EtH2L1)(CH3COO)-H]+ (found
m/z (100%) 777.0, calc. m/z 777.06 [C29H35Cd2N4O7]+),
while coordination of methanol results in [Cd2(CO2EtH2L1)-
(CH3COO)2(CH3OH)]+ (found m/z (100%) 867.0, calc. m/z
868.11 (100%), 867.1 (66%) [C32H42Cd2N4O10]+). A half mass
peak at 605.14 (calc. m/z 606.15 (100%), 605.15 (83%)) is
attributed to ([Cd2(CO2EtH2L1)2]2+; [C54H66Cd2N8O10]2+).
The base peak of the spectrum measured in MeCN/water (1:1)
again corresponds to ([Cd2(CO2EtH2L1)2]2+; found m/z
605.14, calc. m/z 606.15 (100%) and 605.15 (82.8%)). The
mass spectrum of the complex with CO2EtHL2 in methanol
showed the typical Cd2 pattern, the peak at m/z 864.0 (100%),
863.0 (90%) [C33H43Cd2N4O9]+ calc. m/z 865.11 (100.0%),
863.11 (88.3%), 864.11 (86.7%) attributed to [Cd2(CO2EtL2)-
(CH3COO)2]+ (Supporting Information Figure S2). The base
peak of the spectrum in MeCN/water 1:1 corresponds to a
doubly positive charged complex formed by two ligands and
two cadmium ions ([Cd2(CO2EtL2)2]2+; found m/z 634.2,
calc. m/z 634.18 (100.0%), 633.68 (91.5%),
[C58H74Cd2N8O10]2+).
Figure 3. Structure of the tetranuclear complex and a view of the first
coordination sphere. The disordered coordinating acetate oxygen and
the chloride are shown with dashed bonds. Counter ions and hydrogen
atoms have been omitted for clarity (25% ellipsoid probability in all
ORTEP plots).
Å), Cd(1)−O(1)−Cd(2) 128.59(14)°, and Cd(1)···Cd(2)
4.162 Å. For the second metal ion, the coordination sphere is
composed of two nitrogen donors (Cd(2)−N(3), 2.365(4) Å
and Cd(2)−N(4), 2.374(4) Å), the protonated alcohol
(Cd(2)−O(3), 2.367(4) Å) and a disordered chloride
(Cd(2)−Cl(1), 2.635(13) Å; 0.18) and monodentate acetate
(Cd(2)−O(6) 2.181(4) Å; 0.82). The coordination sphere of
Cd(2) is completed by an interaction with the carbonyl oxygen
atom of the neighboring dimer (Cd(2)−O(9), 2.413(4) Å),
resulting in the dimer of dimer configuration. For both Cd(1)
and Cd(2), the Cd−O bond lengths from the pendant alcohol
appear typical as do the Cd−N bond lengths.25,58−61 The
alcohol arm of the ligand is protonated and the charge is
−
balanced by two PF6 anions.
The structure of the cadmium(II) complex with CO2EtHL2
has been inferred based on spectroscopic techniques. A similar
zinc(II) complex exhibited long Zn(II)-OR interactions,32 and
it is likely that in solution the ether donors are not coordinated
to the cadmium(II) sites, making them more accessible to
substrate (vide infra) and the complex more difficult to
crystallize.
NMR Spectra. The 1H and 13C NMR spectra were recorded
for both complexes in a mixture of D2O/CD3CN 1:1. Only one
1
resonance for the acetate methyl group was found in the H
NMR of [Cd2 (CO2 EtH2 L1)(CH3 COO)2 ]+ and
[Cd2(CO2EtL2)(CH3COO)2]+ at 2.39 and 2.46 ppm,
respectively. Also the 13C resonances support the presence of
two equivalent acetate ligands in both complexes. A single
resonance at 22.7 ppm and 22.8 ppm, for the acetate-CH3, and
single acetate-carbonyl resonances at 181.8 ppm and 181.1 ppm
were observed for [Cd2(CO2EtH2L1)(CH3COO)2]+ and
[Cd2(CO2EtL2)(CH3COO)2]+, respectively. The spectra can
be found in the Supporting Information (Figures S3 and S4).
Analysis of the 113Cd NMR spectrum of ([Cd2(CO2EtH2L1)-
(CH3COO)2]+ suggests a similar coordination environment for
the two Cd(II) ions; only one resonance is found at 36.4 ppm.
For [Cd2(CO2EtL2)(CH3COO)2]+ the observation of only
Infrared Spectrum of [Cd4(CO2EtH2L1)2(CH3COO)3.75
-
Cl0.25(H2O)2](PF6)2. The IR spectrum of a solid sample of the
complex displays, in addition to typical ligand and hexafluor-
ophosphate bands, distinct carbonyl stretches from the ethyl
ester of the ligand and the acetate groups. The ester carbonyl
stretch at 1670 cm−1 is at lower frequency than observed for the
free ligand (1704 cm−1), attributed to the weakening of the
CO bond upon coordination to cadmium(II). The
asymmetric and symmetric bands from unidentate acetato
ligand were observed at 1604 and 1373 cm−1, respectively, with
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one cadmium resonance at 23.4 ppm also suggests two Cd(II)
ions in a similar environment.
In an attempt to monitor the binding of the carboxylate
group of penicillin to [Cd2(CO2EtH2L1)(CH3COO)2]+
equimolar amounts of penicillin and the complex were mixed
in (CD3)2CO:(CD3)2SO 1:1, and after one day the 13C NMR
spectrum was recorded (Supporting Information Figure S8).
The lactam carbonyl resonance of penicillin (173.5 ppm) had
disappeared suggesting that the complex cleaved the lactam
bond under these (nonbuffered, water-free) conditions.
Furthermore, an additional poorly resolved, broad (169.1−
171.7 ppm) resonance appeared, indicative of a newly formed
ester moiety, rather than a carboxylate (Supporting Information
Figure S8).
Stopped-flow measurements of nitrocefin hydrolysis by
MβLs have revealed that an open-ring intermediate forms
during the course of hydrolysis.68,69 This intermediate, “the
blue species”,68,70,71 has been shown in model studies with a
dizinc(II) complex and nitrocefin to be composed of a species
with a hydrolyzed β-lactam ring and the carboxylate thus
formed bound to the zinc(II) complex; the deprotonated
secondary amine of the open-ring substrate is also bound to a
zinc(II) ion.70 The blue color, with an intense absorbance at
665 nm in the MβL from Bacteroides fragilis68 and 640 nm in
the model system,70 has been suggested to arise from the
deprotonation of the amine nitrogen and the resulting extended
conjugation.
An intermediate with an absorbance at 640 nm was observed
in the UV−vis spectrum when [Cd2(CO2EtH2L1)-
(CH3COO)2]+ was added to a solution of nitrocefin in dry
acetonitrile. Upon addition of water the blue intermediate was
protonated and a characteristic absorption at 485 nm from
hydrolyzed nitrocefin was apparent (Supporting Information
Figure S9). The solution was further analyzed by mass
spectrometry. A peak at m/z 1313.8 is attributed to a species
comprised of one ligand, two cadmium ions, two acetonitrile
solvent molecules and the intermediate covalently bound to the
complex through the alcohol arm of the ligand (calc. m/z
1315.13 (100.0%), 1314.13 (83.5%) [C48H47Cd2N8O13S2-
(CH3CN)2]+ (Figure 4). As the color change suggested,
hydrolysis of the substrate occurred, but no additional
hydroxide was found in the mass spectrum, so it is concluded
that the substrate was hydrolyzed by the alcohol arm of the
ligand and thus covalently bound after hydrolysis. A peak at
m/z 1748.7 was assigned to a similar species as above but with
an additional unhydrolyzed nitrocefin bound and no solvent
molecules coordinated (calc. m/z 1749.12 (100.0%), 1748.12
(80.0%), [C48H47Cd2N8O13S2(C21H15N4O8S2)]+). Further ad-
dition of nitrocefin to the mass spectrum sample caused the
latter peak to become the major peak in the spectrum. A blue
species was not observed when [Cd2(CO2EtL2)(CH3COO)2]+
was added to a solution of nitrocefin in dry acetonitrile;
however, the complex with one or two intact nitrocefin
molecules bound could be detected in the mass spectrum
(found m/z 1320.9 and 1776.7).
Binding of Phosphoester and β-Lactam Substrates to
the Cadmium(II) Complex of (CO2EtH3L1) in Solution. To
characterize the substrate binding and species present during
catalysis, mass spectrometry, in-solution infrared measurements
UV−vis, 13C- and 113Cd NMR spectroscopy were carried out
with mixtures of the complex with substrates, substrate mimics
and products.
After addition of [Cd4(CO2EtH2L1)2(CH3COO)3.75Cl0.25
-
(H2O)2](PF6)2 to a solution of the product mimic 4-
nitrophenol phosphate (PNPP) in acetonitrile, the mass
spectrum of the mixture shows free ligand and a peak at m/z
936.03 arising from [Cd2(CO2EtH2L1)(PNPP)]+ (calc. m/z
(100%) 936.03; [C33H37Cd2N5O11P]+), in addition to the m/z
605 peak attributed to [Cd2(CO2EtH2L1)2]2+, suggesting that
the latter peak is not representative of the catalytically active
species but a species produced under the conditions of the mass
spectrometer (Supporting Information Figure S5). Under the
same experimental conditions using diphenyl phosphate
(DPP), a substrate mimic that lacks the activating nitro groups
and is therefore not hydrolyzed by the model complex, resulted
in a peak at m/z 1215.9 assigned to [Cd2(CO2EtH2L1)-
(DPP)2]+ (Supporting Information Figure S6, calc. m/z
(100%) 1216.11, (88.6%) 1215.11, [C51H52Cd2N4O13P2]+).
The 113Cd NMR of the cadmium(II) complex with
CO2EtH3L1 in CD3CN/D2O (1:1) displayed one resonance
at +36.4 ppm, assigned to the [Cd2(CO2EtH2L1)-
(CH3COO)2]+ species and suggesting that the two Cd(II)
ions have the same coordination environment in solution
(Supporting Information Figure S7). Upon addition of ten
equivalents of the substrate analogue DPP, the resonance
shifted to −22.6 ppm; the observation of only one resonance
indicates that the substrate, or substrates, are bound symmetri-
cally (Supporting Information Figure S7).
β-Lactams can bind to the metal complexes through their
carboxylate moiety, the β-lactam amide, a sulfur atom or
terminal amides.37,39,64 A study employing the dizinc(II)
complex of a pyrazolate-based bioinspired ligand and a number
of β-lactam antiobiotics, as well as inhibitors including
sulbactam and 2-azeidinone, has shown that while the latter
was deprotonated and bound by the two zinc(II) ions the other
substrates bound preferentially by the carboxylate group.37 The
binding of nitrocefin to the cadmium(II) complex was
investigated by in situ FT-IR spectroscopy in MeCN/water
(50:50) solution. No change of the β-lactam carbonyl
absorption was observed on addition of [Cd2(CO2EtH2L1)-
(CH3COO)2]+; typically a shift of the carbonyl absorption of
20−50 cm−1 to lower wavenumbers would be expected upon
coordination to the metal ion.64−66 Hydrolysis of nitrocefin by
[Cd2(CO2EtH2L1)(CH3COO)2]+ was apparent through the
characteristic color change from yellow to orange and the
disappearance in the IR band at 1673 cm−1, attributed to the
lactam carbonyl group. Changes in the IR-spectrum, however,
could only be visualized in the difference spectra as the amide
and carboxylate bands overlap. Similar results have been
reported for the reaction of a dizinc(II) complex upon reaction
with penicillin G in DMSO/water.67 A separate investigation of
the hydrolysis of cephalothin, a more “nitrocefin-like” substrate,
in pure DMSO showed no significant change of the β-lactam
carbonyl IR absorptions, suggesting that this substrate is
binding solely via its carboxylate group to transition metals.38
Phosphatase Activity. BDNPP is an activated substrate
employed in studies of phosphoesterase models. The hydrolytic
reaction is followed by monitoring the formation of 2,4-
dinitrophenol which has an intense chromophore at 400
nm.55,56 The phosphatase-like activity promoted by
[Cd2(CO2EtH2L1)(CH3COO)2]+ in 50:50 MeCN/water was
measured at pH 10.4 with BDNPP (5 mM). The [complex]
dependence was linear in the region 0.005−0.05 mM. The
initial rate of BDNPP cleavage, determined at pH 10.4 as a
function of substrate concentration, reveals typical saturation
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where Vmax = kcat([complex]), [S] is the substrate concen-
tration, and Km is the Michaelis constant. Here, KM = 9.4 2.1
mM, Vmax = 3.76 × 10−7 M s−1, with kcat (=Vmax/[complex]) =
9.4 0.2 × 10−3 s−1. The dependence of catalysis on pH was
measured from pH 7.0−0.7 resulting in a typical monoprotic
curve exhibiting a limiting rate at high pH (Figure 5b). The
data were fit to eq 2,72
Vmax
V =
[H+]
1 +
Ka
(2)
where V is the initial rate, Vmax the limiting rate, and Ka the
kinetically relevant ionization constant. The fit resulted in pKa =
10.1
0.6. For [Cd2(CO2EtL2)(CH3COO)2]+ pKa = 8.7
0.6, with kcat = 9.7 2.7 × 10−3 s−1 and Km = 10.1 3.4 mM
(Figure S10).
Metallo-β-lactamase Activity. Nitrocefin is commonly
employed in studies of β-lactamase mimics; the hydrolytic
cleavage of the β-lactam ring results in a color change and the
reaction is followed at 390 nm.38,39 The pH-dependence data of
the reaction of [Cd2(CO2EtH2L1)(CH3COO)2]+ with nitro-
cefin was monitored and the data fitted to eq 1 (Figure 6a).
Figure 4. Mass spectrum of the blue intermediate (absorbance at 640
nm) when [Cd2(CO2EtH2L1)(CH3COO)2]+ was added to a solution
of nitrocefin in dry MeCN. (peak at m/z 1313.8 is attributed to a
species comprised of one ligand, two cadmium ions, two acetonitrile
solvent molecules and the intermediate covalently bound to the
complex through the alcohol arm of the ligand (calc. m/z 1315.13
(100.0%), 1314.13 (83.5%); peak at m/z 1748.7 assigned to a similar
species as above but with an additional not hydrolyzed nitrocefin
bound and no solvent molecules coordinated (calc. m/z 1749.12
(100.0%), 1748.12 (80.0%)).
behavior (Figure 5a). The data were fitted by nonlinear
regression using the Michaelis−Menten equation,72
V
max[S]
V =
[S] + Km
(1)
Figure 6. (a) Substrate concentration dependence for
[Cd2(CO2EtH2L1)(CH3COO)2]+ and (b) pH dependence of rate
of nitrocefin cleavage by [Cd2(CO2EtH2L1)(CH3COO)2]+, 25οC,
aqueous multicomponent buffer (50 mM each of MES, HEPES, and
CHES), ionic strength controlled by 250 mM LiClO4, 50:50 MeCN/
buffer, and 40 μM complex.
The pKa of 7.9 0.1, important for catalysis, is lower than that
determined for the BDNPP hydrolysis. The substrate depend-
ence was conducted at pH 8 and at 37 °C and resulted in Vmax
= 6.97 × 10−8 M s−1, with kcat = 1.39 × 10−2 3 × 10−3 s−1 and
KM = 0.11 0.03 mM (Figure 6b). Substrate concentrations
above 0.06 mM resulted in significantly decreased activity,
suggesting substrate inhibition.
UV−vis titration experiments with [Cd2(CO2EtH2L1)-
(CH3COO)2]+ and successively one, two, three, and four
equivalents of nitrocefin were undertaken (Figure 7). Reaction
between the complex and one equivalent of nitrocefin was
followed for approximately 60 min after which time spectral
Figure 5. (a) Substrate concentration dependence for
[Cd2(CO2EtH2L1)(CH3COO)2]+, and (b) pH dependence of rate
of BDNPP cleavage by [Cd2(CO2EtH2L1)(CH3COO)2]+, 25 °C,
aqueous multicomponent buffer (50 mM each of MES, HEPES, and
CHES), ionic strength controlled by 250 mM LiClO4, 50:50 MeCN/
buffer, 40 μM complex.
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Figure 7. UV−vis spectra of the hydrolysis of successively one and two equivalents of nitrocefin by [Cd2(CO2EtH2L1)(CH3COO)2]+, pH 8,
buffer:MeCN 1:1, (first ten minutes of the reaction are shown, initial concentration of complex and nitrocefin 0.05 mM; spectra recorded at 1 min
intervals), 37 °C. Middle: Species distribution. Bottom: Proposed species.
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Figure 8. Proposed mechanisms for the BDNPP and nitrocefin hydrolysis by [Cd2(CO2EtH2L1)(CH3COO)2]+ and [Cd2(CO2EtL2)-
(CH3COO)2]+. For the nitrocefin hydrolysis only the steps involving the first substrate molecule are included for clarity.
changes effectively ceased. Addition of a second equivalent of
substrate to the solution again initiated changes in the spectra,
which were followed until the reaction ceased. The spectral
changes for the first and second equivalent of substrate were
fitted separately to a model involving two consecutive reactions
(Figure 7), where the [Cd(II)-(nitrocefin)] species represent a
bound substrate complex and [product 1] represents a complex
with the substrate hydrolyzed but still coordinated to the metal
ion (product inhibition). Analysis of the data (300−700 nm;
[complex] 0.05 mM, [substrate] 0.05 mM; pH 8) based on this
model resulted in k1 = 4.8 × 103 8.7 M−1 min−1, k2 = 4.3 ×
10−2 1.4 × 10−4 min−1, and k1′ = 1.5 × 103 9.5 M−1 min−1,
k′2 = 6.1 × 10−2 3 × 10−4 min−1.73 The data predict that the
addition of the second substrate molecule is less facile and k2
and k2′ are of the same order as expected if the reaction
represents the hydrolysis of the lactam ring by the coordinated
nucleophile. Addition of, successively, a third and fourth
equivalent of nitrocefin resulted in very slow spectral changes
indistinguishable from those observed for the autohydrolysis
reaction. [Cd2(CO2EtL2)(CH3COO)2]+ showed no activity in
the hydrolysis of nitrocefin over the pH range studied (pH
4.5−10).
or Cd(II)−OH2 species.25,74−79 Interestingly the complex
[Cd2(CO2EtH2L1)(CH3COO)2]+ displays a pKa similar to
that reported for the Cd2-substituted enzyme GpdQ (pKa =
9.4).25 For the enzyme a terminal water ligand is the proposed
nucleophile.25 To further probe the identity of the nucleophile
in the phosphoester hydrolysis, an 18O labeling experiment was
conducted with [Cd2(CO2EtH2L1)(CH3COO)2]+. 31P NMR
can be used to determine the extent of incorporation of 18O-
labeled water/hydroxide into the hydrolysis product of BDNPP
as phosphorus signals display a high sensitivity to isotopic shifts
from an 18O instead of 16O bound to the phosphorus.80−82 The
phosphorus resonance arising from 18O−31P is shifted to lower
frequency, the shift dependent on the operating frequency.32,81
A solvent mixture of H2O16/H2O18 (50% vol) in MeCN was
employed in the reaction of [Cd2(CO2EtH2L1)(CH3COO)2]+
with one equivalent BDNPP. After standing for three days at
room temperature a resonance attributed to DNPP, the
hydrolysis product of BDNPP, was observed at −3.15 ppm in
the 31P NMR spectrum. If the nucleophile is a cadmium(II)
bound water/hydroxide, or a free water molecule from the
solvent, DNPP with 50% 18O incorporated is expected. This
was observed in the 31P-spectrum with the resonance for DNPP
split into a doublet with a peak separation of approximately
16.9 Hz.32,80−82 The result suggests that the active nucleophile
in BDNPP hydrolysis is a terminally bound Cd(II)−OH. A
bridging water/hydroxide as nucleophile was not considered as
previous studies have shown that for a Cd(II)−(μ-H2O)−
Cd(II) complex the pKa is <6.6.59 Further, in the present study,
a fluoride inhibition experiment did not alter the activity of the
complexes; fluoride is likely to assume a bridging rather than a
Mechanism of Phosphodiester Hydrolysis. The absence
of a metal bound alcohol in [Cd2(CO2EtL2)(CH3COO)2]+
allows the assignment of the kinetically relevant pKa (8.7) to a
metal-bound terminal water molecule. This pKa is similar to
that reported previously for a cadmium(II) complex with
pendant alcohol arms (pKa = 8.9).25 For [Cd2(CO2EtH2L1)-
(CH3COO)2]+ the pKa of 10.1 is within the range reported for
cadmium(II) complexes with either pendant hydroxyl groups
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terminal position when added to the complexes.83,84 Given that
the complex with CO2EtHL2 can only exhibit hydrolytic
activity through a coordinated hydroxide nucleophile it is
proposed that in both complexes the active nucleophile is a
terminal hydroxo group (Figure 8).
hydrolyzed nitrocefin sterically hinders coordination of
phosphoester substrate; it is possible as well that hydrolyzed
nitrocefin binds more tightly than phosphodiester substrates.
Finally, the rapid decrease in activity after the complete
hydrolysis of one equivalent of nitrocefin suggests the
hydrolyzed substrate is bound irreversibly to the complex. A
proposed mechanism for the hydrolysis of nitrocefin is shown
in Figure 8.
Other mechanistic scenarios are possible.32,38 One would be
where the alcohol arm of the ligand is actively involved in the
mechanism. In this case it could be envisaged that the alcohol
arm is deprotonated at pH 10.1 and acts as the primary
nucleophile to hydrolyze the substrate BDNPP. The ligand is
then recovered by an external water molecule and the substrate
is released. A second scenario is possible where, upon
deprotonation, the alkoxide acts as a general base to activate
an external water molecule which then hydrolyzes the substrate.
Both scenarios are consistent with the finding of a 50% 18O
labeled DNPP (Supporting Information Figure S11).
CONCLUSIONS
■
The cadmium(II) complexes of two new ligands, ethyl 4-
hydroxy-3,5-bis(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)-
methyl)benzoate (CO2EtH3L1) and ethyl 4-hydroxy-3,5-bis-
(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-
benzoate (CO2EtHL2) have been prepared. These complexes
possess functional properties analogous to dinuclear phos-
phoesterases and β-lactamases. The results obtained for the
hydrolysis of the activated substrate BDNPP suggest a terminal
water is the active nucleophile, a conclusion based on an
analysis of the incorporation of 18O into the hydrolysis product
and on the fact that the pKa of a Cd(II) bound water molecule
can range from 7.6−10.3.87 Participation of the ligand alkoxide
can, however, not be ruled out. The cadmium(II) complex with
CO2EtH3L1 also exhibits β-lactamase activity toward the
substrate nitrocefin, mass spectral evidence suggesting that in
this case the active nucleophile is the alkoxide group. An
intermediate with an absorbance at 640 nm was observed in the
UV−vis spectrum when [Cd2(CO2EtH2L1)(CH3COO)2]+ was
added to a solution of nitrocefin in dry acetonitrile. A similar
intermediate has been observed previously in zinc(II) model
systems and in the reaction of the MβL enzyme. The
cadmium(II) complex with CO2EtHL2, without an alkoxide
nucleophile is inactive toward this substrate under identical
conditions.
The differences in the pKa observed for the complexes with
CO2EtHL2 and CO2EtH3L1, although both are assigned to a
Cd−OH2 moiety, can be ascribed to the effect of ligand and
substrate on the magnitude of the kinetically relevant pKa. This
has been illustrated previously in studies of the dizinc(II)
complexes of the ligands 2,2′-(2-hydroxy-5-methyl-1,3-
phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)-
diethanol (CH3H3L1), 2,6-bis(((2-methoxyethyl)(pyridin-2-
ylmethyl)amino)methyl)-4-methylphenol (CH3HL2) and 4-
methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)amino)-
methyl) phenol (CH3HL3).32,43 For [Zn2(CH3HL1)-
(CH3COO)(H2O)](PF6) with the substrate bis(4-
nitrophenyl)phosphate (BNPP) the pKa was reported as
7.13;43 with substrate BDNPP the pKa was reported to be
6.6.32 Upon replacement of the alkoxide ligand in
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6) with a methyl- or
phenyl-ether ([Zn2(CH3L2)(CH3COO)2](PF6) and
[Zn2(CH3L3)(CH3COO)2](PF6), respectively) and with
BDNPP as substrate, the kinetically relevant pKa values were
6.7 and 7.7, respectively.32
Mechanism of β-Lactam Hydrolysis. The active
nucleophile for the β-lactam hydrolysis by the complex formed
with the ligand CO2EtH3L1 could be a bridging or terminal
Cd−OH2/OH or a Cd-alkoxide, given the similarities in the
pKa values for these species.25,74−79 The μ-OH was considered
less likely given the lack of inhibitory effect of added fluoride
(vide supra) and the Cd(II)···Cd(II) separation (4.162 Å). A
number of findings suggest that the nucleophile is a metal
bound alkoxide rather than a terminal Cd(II)−OH. First,
[Cd2(CO2EtL2)(CH3COO)2]+ was inactive toward lactam
substrates. Second, in the mass spectrum a peak at m/z
1313.8, is assigned to a species comprised of CO2EtH2L1−, two
cadmium ions, two acetonitrile solvent molecules, and
hydrolyzed nitrocefin, the latter proposed to be bound through
the alkoxide arm of the ligand. The formation of an ester rather
than a carboxylic acid was also found in the 13C NMR of
penicillin with the complex [Cd2(CO2EtH2L1)(CH3COO)2]+
under water-free conditions. Alkoxide nucleophiles have been
reported previously in the methanolysis of β-lactams,85,86 and in
an infrared study of the reaction of a dinuclear zinc complex
with penicillin G in methanol it was observed that ester
formation due to alcoholysis of zinc bound methoxide had
occurred.67 In addition, the addition of two equivalents of
nitrocefin to [Cd2(CO2EtH2L1)(CH3COO)2]+ and subsequent
incubation at 37 °C for one hour resulted in a significant
decrease in the ability of the complex to degrade the phosphate
ester substrate BDNPP, suggesting that the bound and
ASSOCIATED CONTENT
* Supporting Information
■
S
Tables S1 and S2, crystallographic data, and Figures S1−S11.
This material is available free of charge via the Internet at
http://pubs.acs.org. Crystallographic data (without structure
factors) for the structure reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication number CCDC 873960. Copies of
the data can be obtained free of charge from the CCDC {12
Union Road, Cambridge CB2 1EZ, U.K.; phone (+44) 223−
336−408; fax (+44) 1223−336−003; email deposit@ccdc.cam.
ac.uk; Web site www.ccdc.cam.ac.uk].
AUTHOR INFORMATION
Corresponding Author
*E-mail: gahan@uq.edu.au. Telephone ++61 7 3365 3844. Fax:
++61 7 3365 4299.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by a grant from the Australian Research
Council (DP0986613). L.J. Daumann is supported by IPRS and
UQCent Scholarships. A DAAD travel grant is gratefully
acknowledged. We also thank Prof. Dr. Markus Enders,
Anorganisch-Chemisches Institut, Universitat Heidelberg for
̈
access to the NMR instrument.
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(33) Gahan, L. R.; Smith, S. J.; Neves, A.; Schenk, G. Eur. J. Inorg.
Chem. 2009, 19, 2745−2758.
REFERENCES
■
(1) Mitic,
́
N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.;
(34) Kirby, A. J. Angew. Chem., Int. Ed. 1996, 35, 707−724.
(35) Komiyama, M. J. Biochem. 1995, 118, 665−670.
(36) Bashkin, J. K. Curr. Opin. Chem. Biol. 1999, 3, 752−758.
(37) Wockel, S.; Galezowska, J.; Dechert, S.; Meyer, F. Inorg. Chem.
2012, 51, 2486−2493.
Schenk, G. Chem. Rev. 2006, 106, 3338−3363.
(2) Wilcox, D. E. Chem. Rev. 1996, 96, 2435−2458.
(3) Sogorb, M. A.; Vilanova, E. Toxicol. Lett. 2002, 128, 215−228.
(4) Umayal, M. T., A.; Mugesh, G.;, Metallo-beta-lactamases and
Their Synthetic Mimics: Structure, Function, and Catalytic Mecha-
nism. In Progress in Inorganic Chemistry; Karlin, K. D., Ed.; John Wley
and Sons, Inc.: Hoboken, NY, 2012; Vol. 57, pp 395−443.
(5) Raushel, F. M. Curr. Opin. Microbiol. 2002, 5, 288−295.
(6) Ely, F.; Foo, J. L.; Jackson, C. J.; Gahan, L. R.; Ollis, D.; Schenk,
G. Curr. Top. Biochem. Res. 2007, 9, 63−78.
(38) Kaminskaia, N. V.; He, C.; Lippard, S. J. Inorg. Chem. 2000, 39,
3365−3373.
(39) Kaminskaia, N. V.; Spingler, B.; Lippard, S. J. J. Am. Chem. Soc.
2000, 122, 6411−6422.
(40) Li, S. A.; Yang, D. X.; Li, D. F.; Huang, J.; Tang, W. X. New J.
Chem. 2002, 26, 1831−1837.
́
(7) Schenk, G.; Gahan, L. R.; Carrington, L. E.; Mitic, N.; Valizadeh,
(41) Bazzicalupi, C.; Bencini, A.; Berni, E.; Bianchi, A.; Fedi, V.; Fusi,
V.; Giorgi, C.; Paoletti, P.; Valtancoli, B. Inorg. Chem. 1999, 38, 4115−
4122.
M.; Hamilton, S. E.; de Jersey, J.; Guddat, L. W. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 273−278.
(8) Dumas, D. P.; Caldwell, S. R.; Wild, J. R.; Raushel, F. M. J. Biol.
Chem. 1989, 264, 19659−19665.
(42) Yang, D.; Li, S.; Li, D.; Xia, J.; Yu, K.; Tang, W. Dalton Trans.
2002, 4042−4047.
(43) Chen, J.; Wang, X.; Zhu, Y.; Lin, J.; Yang, X.; Li, Y.; Lu, Y.; Guo,
Z. Inorg. Chem. 2005, 44, 3422−3430.
(9) Jackson, C.; Carr, P. D.; Kim, H. K.; Liu, J.-W.; Herrald, P.; Mitic,
́
N.; Schenk, G.; Smith, C. A.; Ollis, D. L. Biochem. J. 2006, 397, 501−
508.
(44) Lane, T. W.; Saito, M. A.; George, G. N.; Pickering, I. J.; Prince,
R. C.; Morel, F. M. M. Nature 2005, 435, 42−42.
(45) Lane, T. W.; Morel, M. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
4627−4631.
(10) Concha, N. O.; Rasmussen, B. A.; Bush, K.; Herzberg, O.
Structure 1996, 4, 823−836.
(11) Crowder, M. W.; Spencer, M. G.; Vila, A. J. Acc. Chem. Res.
2006, 39, 721−728.
(46) Krampiec, M.; Noga, M.; Patiny, L. Chemcalc; Ecole
(12) Omburo, G.; J., K.; Mullins, L.; Raushel, F. J. Biol. Chem. 1992,
267, 13278−13283.
́ ́
Polytehnique Federale de Lausanne and Institute of Chemical Sciences
and Engineering: Lausane, Switzerland, 2011; http://www.chemcalc.
org
(47) Zinke, A.; Ott, R.; Leggewie, E.; Hassanein, A.; Zankl, G.
Monatsh. Chem. 1956, 87, 552−559.
(48) Tsue, H.; Enyo, K.; Hirao, K.-i. Org. Lett. 2000, 2, 3071−3074.
(49) Striegler, S.; Dittel, M. Inorg. Chem. 2005, 44, 2728−2733.
(50) Elslager, E. F.; Benton, E. L.; Short, F. W.; Tendick, F. H. J. Am.
Chem. Soc. 1956, 78, 3453−3457.
(51) Boehmer, V.; Marschollek, F.; Zetta, L. J. Org. Chem. 1987, 52,
3200−3205.
(52) Sheldrick, G. M., SHELXL97: Program for the Refinement of
(13) Ely, F.; Hadler, K. S.; Gahan, L. R.; Guddat, L. W.; Ollis, D. L.;
Schenk, G. Biochem. J. 2010, 432, 565−573.
(14) Mitic,
J. Am. Chem. Soc. 2010, 132, 7049−7054.
(15) Schenk, G.; Mitic, N.; Hanson, G. R.; Comba, P. Coord. Chem.
́
N.; Hadler, K. S.; Gahan, L. R.; Hengge, A. C.; Schenk, G.
́
Rev. 2012, DOI: 10.1016/j.ccr.2012.03.020.
(16) Wang, X. D.; Ho, R. Y. N.; Whiting, A. K.; Que, L. J. Am. Chem.
Soc. 1999, 121, 9235−9236.
́
(17) Ely, E.; Hadler, K. S.; Mitic, N.; Gahan, L. R.; Ollis, D. L.; Plugis,
N. M.; Russo, M. T.; Larrabee, J. A.; Schenk, G. J. Biol. Inorg. Chem.
2011, 16, 777−787.
́
(18) Hadler, K. S.; Tanifum, E. A.; Yip, S. H.-C.; Mitic, N.; Guddat,
Crystal Structures; University of Gottingen: Gottingen, Germany, 1997.
̈
̈
L. W.; Jackson, C. J.; Gahan, L. R.; Nguyen, K.; Carr, P. D.; Ollis, D.
L.; Hengge, A. C.; Larrabee, J. A.; Schenk, G. J. Am. Chem. Soc. 2008,
130, 14129−14138.
(53) Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837−838.
(54) Bunton, C. A.; Farber, S. J. J. Org. Chem. 1969, 34, 767−772.
(55) Batista, S. C.; Neves, A.; Bortoluzzi, A. J.; Vencato, I.; Peralta, R.
A.; Szpoganicz, B.; Aires, V. V. E.; Terenzi, H.; Severino, P. C. Inorg.
Chem. Commun. 2003, 6, 1161−1165.
(19) Jackson, C. J.; Hadler, K. S.; Carr, P. D.; Oakley, A. J.; Yip, S.;
Schenk, G.; Ollis, D. L. Acta Crystallogr., Sect. F 2008, 64, 681−685.
́
(20) Hadler, K.; Mitic, N.; Ely, F.; Hanson, G.; Gahan, L.; Larrabee,
́
(56) Smith, S. J.; Casellato, A.; Hadler, K. S.; Mitic, N.; Riley, M. J.;
Bortoluzzi, A. J.; Szpoganicsz, B.; Schenk, G.; Neves, A.; Gahan, L. R. J.
Biol. Inorg. Chem. 2007, 12, 1207−1220.
J.; Ollis, D.; Schenk, G. J. Am. Chem. Soc. 2009, 131, 11900−11908.
(21) Hadler, K. S.; Mitic, N.; Yip, S. H.-C.; Gahan, L. R.; Ollis, D. L.;
́
Schenk, G.; Larrabee, J. A. Inorg. Chem. 2010, 49, 2727−2734.
(22) Hadler, K. S.; Gahan, L. R.; Ollis, D. L.; Schenk, G. J. Inorg.
Biochem. 2010, 104, 211−213.
(57) Schenk, G.; Peralta, R. A.; Batista, S. C.; Bortoluzzi, A. J.;
Szpoganics, B.; Dick, A. K.; Herrald, P.; Hanson, G. R.; Szilagyi, R. K.;
Riley, M. J.; Gahan, L. R.; Neves, A. J. Biol. Inorg. Chem. 2008, 13,
139−155.
(23) Jackson, C. J.; Carr, P. D.; Liu, J. W.; Watt, S. J.; Beck, J. L.;
Ollis, D. L. J. Mol. Biol. 2007, 367, 1047−1062.
(58) Zompa, L. J.; Diaz, H.; Margulis, T. N. Inorg. Chim. Acta 1995,
232, 131−137.
(24) Ghanem, E.; Li, Y. C.; Xu, C. F.; Raushel, F. M. Biochemistry
2007, 46, 9032−9040.
(59) Tan, L. H.; Taylor, M. R.; Wainwright, K. P.; Duckworth, P. A.
Dalton Trans 1993, 2921−2928.
(25) Mirams, R.; Smith, S. J.; Hadler, K. S.; Ollis, D. L.; Schenk, G.;
Gahan, L. R. J. Biol. Inorg. Chem. 2008, 13, 1065−1072.
(26) Daumann, L. J.; McCarthy, B. Y.; Hadler, K. S.; Murray, T.;
Gahan, L. R.; Larrabee, J. A.; Ollis, D. L.; Schenk, G. Biochim. Biophys.
Acta 2012, DOI: 10.1016/.bbapap.2012.02.004.
(60) Allred, R. A.; Huefner, S. A.; Rudzka, K.; Arif, A. M.; Berreau, L.
M. Dalton Trans. 2007, 351−357.
(61) He, H.; Martell, A. E.; Motekaitis, R. J.; Reibenspies, J. H. Inorg.
Chem. 2000, 39, 1586−1592.
(27) Spencer, J.; Walsh, T. Angew. Chem., Int. Ed. 2006, 45, 1022.
(28) Ambler, R. P. Philos. Trans. R. Soc. London Ser. B 1980, 289, 321.
(29) Felici, A.; Amicosante, G.; Oratore, A.; Strom, R.; Ledent, P.;
Joris, B.; Fanuel, L.; Frere, J. M. Biochem. J. 1993, 291, 151.
(30) Cricco, J. A.; Orellano, E. G.; Rasia, R. M.; Ceccarelli, E. A.; Vila,
A. J. Coord. Chem. Rev. 1999, 190−192, 519−535.
(31) Heinz, U.; Adolp, H.-W. Cell. Mol. Life Sci. 2004, 61, 2827−
2839.
(32) Daumann, L. J.; Dalle, K. E.; Schenk, G.; McGeary, R. P.;
Bernhardt, P. V.; Ollis, D. L.; Gahan, L. R. Dalton Trans. 2012, 41,
1695−1708.
(62) Nakamoto, K. Infrared and Raman Spectra of Inorganic and
Coordination Compounds Part B: Applications in Coordination.
Organometallic, and Bioinorganic Chemistry Part B, 5th ed.; John Wiley
& Sons, Inc.: New York, 1997.
(63) Kang, D.; Seo, J.; Lee, S. Y.; Lee, J. Y.; Choi, K. S.; Lee, S. S.
Inorg. Chem. Commun. 2007, 10, 1425−1428.
(64) Assos, M.; Panossian, R.; Guiliano, M. Spectrosc. Lett. 1984, 17,
271−278.
(65) Kupka, T. Spectrochim. Acta Part A 1997, 53, 2649−2658.
(66) Anacona, J. R.; Serrano, J. J. Coord. Chem. 2003, 56, 313−320.
7680
dx.doi.org/10.1021/ic300687y | Inorg. Chem. 2012, 51, 7669−7681

Inorganic Chemistry
Article
(67) Bauer-Siebenlist, B.; Dechert, S.; Meyer, F. Chem.Eur. J. 2005,
11, 5343−5352.
(68) Wang, Z.; Fast, W.; Benkovic, S. J. J. Am. Chem. Soc. 1998, 120,
10788−10789.
(69) McMannus-Munoz, S.; Crowder, M. W. Biochemistry 1999, 38,
1547−1553.
(70) Kaminskaia, N. V.; Spingler, B.; Lippard, S. J. J. Am. Chem. Soc.
2001, 123, 6555−6563.
(71) Wang, Z.; Fast, W.; Benkovic, S. J. Biochemistry 1999, 38,
10013−10023.
(72) Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid
Equilibrium and Steady State Enzyme Systems, 2nd ed.; Wiley-
Interscience: New York, 1975.
(73) Maeder, M. ReactLabTM Kinetics, 1.1; Jplus Consulting:
Palmyra, WA, Australia, 2011.
(74) Walters, K. M.; Buntine, M. A.; Lincoln, S. F.; Wainwright, K. P.
J. Chem. Soc., Dalton Trans. 2002, 3571−3577.
(75) Davies, P. J.; Wainwright, K. P. Inorg. Chem. Acta 1999, 294,
103−108.
(76) Davies, P. J.; Taylor, M. R.; Wainwright, K. P.; Harriott, P.;
Duckworth, P. A. Inorg. Chem. Acta 1996, 246, 1−6.
(77) Turonek, M. L.; Duckworth, P. A.; Laurence, G. S.; Lincoln, S.
F.; Wainwright, K. P. Inorg. Chim. Acta 1995, 230, 51−57.
(78) Amorim, M. T. S.; Chaves, S.; Delgado, R.; Frausto da Silva, J. J.
R. Dalton Trans. 1991, 3065−3072.
(79) Xavier, F. R.; Peralta, R. A.; Bortoluzzi, A. J.; Drago, V.;
Castellano, E. E.; Haase, W.; Tomkowicz, Z.; Neves, A. J. Inorg.
Biochem. 2011, 105, 1740−1752.
(80) Cohn, M.; Hu, A. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 200−
203.
(81) Mitchell, M. C.; Taylor, R. J.; Kee, T. P. Polyhedron 1998, 17,
433−442.
(82) Williams, N. H.; Lebuis, A.-M.; Chin, J. J. Am. Chem. Soc. 1999,
121, 3341−3348.
(83) Elliott, T. E.; Mitic,
J. Braz. Chem. Soc. 2006, 17, 1558−1565.
(84) Schenk, G.; Elliott, T. W.; Leung, E. W. W.; Mitic,
Carrington, L. E.; Gahan, L. R.; Guddat, L. W. BMC Struct. Biol. 2008,
8 (6), No. 10.1186/1472-6807-8-6.
́
N.; Gahan, L. R.; Guddat, L. W.; Schenk, G.
́
N.;
(85) Montoya-Pelaez, P. J.; Brown, R. S. Inorg. Chem. 2002, 41, 309−
316.
(86) Montoya-Pelaez, P. J.; Gibson, G. T. T.; Neverov, A. A.; Brown,
R. S. Inorg. Chem. 2003, 42, 8624−8632.
(87) Burgess, J. Metal Ions in Solution; Ellis Horwood: Chichester,
U.K., 1978.
7681
dx.doi.org/10.1021/ic300687y | Inorg. Chem. 2012, 51, 7669−7681