Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by dinuclear mimics of metallohydrolases

Article abstract of DOI:10.1016/j.jinorgbio.2016.02.008

di-Zinc(II) complexes of the ligands 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1), 2,6-bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2) and 2,6-bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-4-methylphenol (HL3) have been prepared a

Full text of DOI:10.1016/j.jinorgbio.2016.02.008

JIB-09922; No of Pages 10
Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate
☆
esters catalyzed by dinuclear mimics of metallohydrolases
b
a
a
Joshua J. Brown a, Lawrence R. Gahan a, , Anne Schöffler , Elizabeth H. Krenske , Gerhard Schenk
⁎
a
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia
Institute of Organic Chemistry, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
di-Zinc(II) complexes of the ligands 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1), 2,6-
bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2) and 2,6-bis((hydroxyethyl)(methoxyethyl)-
aminomethyl)-4-methylphenol (HL3) have been prepared and characterized. The three ligands differ in their
donor types, having ether donors (HL1), alkoxido donors (HL2) and both ether and alkoxido donors (HL3). These
differences allowed an investigation into the role of the potential nucleophiles in the hydrolysis reaction with the
phosphodiester substrate bis(2,4-dinitrophenyl)phosphate (BDNPP). In addition, the di-Mg(II) complex of ligand
HL2 was prepared in order to examine the potential for Mg(II) to replace Zn(II) in these biomimetic systems.
Kinetically relevant pKa values for the three di-Zn(II) complexes were determined to be 7.14 and 9.21 for
[Zn2(L1)(CH3COO)2](PF6), 7.90 and 10.21 for [Zn2(L2)(CH3COO)2](BPh4) and 8.43 and 10.69 for
Received 9 October 2015
Received in revised form 17 January 2016
Accepted 10 February 2016
Available online xxxx
Keywords:
Di-zinc(II) complexes
Nucleophile
Hydrolysis reaction
Phosphodiester
Computational methods
[Zn2(L3)(CH3COO)2](BPh4). At the respective pH optima the relevant catalytic parameters are kcat
=
5.44(0.11) × 10−5 s−1 (Km = 5.13(0.92) mM), 2.60(0.87) × 10−4 s−1 (Km = 5.49(1.51) mM) and
1.53(0.27) × 10−4 s−1 (Km = 2.14(0.50) mM) for [Zn2(L1)(CH3COO)2](PF6), [Zn2(L2)(CH3COO)2](BPh4)
or [Zn2(L3)(CH3COO)2](BPh4), respectively. The di-Mg(II) complex was found to be unreactive in the hydrolysis
reaction with BDNPP under the conditions employed. Computational methods using the [Zn2(L2)(CH3COO)2](BPh4)
complex were used to discriminate between different possible mechanistic pathways. The DFT calculations indicate
that an alkoxido-mediated pathway in the complexes formed with ligands L2 or L3 is unlikely, because it induces
significant distortion of the Zn2(L) unit; a direct attack by a coordinated hydroxide is preferred in each of the
three systems studied here. The calculations also revealed the important role of ligand structural rigidity.
Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
1. Introduction
there are examples of both complexes that use a metal ion-
coordinated hydroxide as nucleophile, and those that use an alkoxide
We and others, have explored extensively the functional roles that
model complexes can play in reproducing the electronic, structural
and reactivity characteristics of organophosphate-hydrolyzing
metalloenzyme systems [1–26]. One of the prominent issues targeted
in these studies is the identity of the nucleophilic agent(s) in the models
and in the corresponding metalloenzymes [27]. It is generally accepted
that in the key chemical step in hydrolytic enzymes a hydroxide acts as
the pertinent nucleophile [27]. This hydroxide may be coordinated di-
rectly to a metal ion in the active site, or be located in the second coor-
dination sphere, activated through hydrogen bonding interactions with
amino acid side chains and/or water molecules lining the active site of
the enzyme [27–29]. Exogenous moieties (e.g. serine residues) have
also been implicated as potential nucleophiles in enzymes such as alka-
line phosphatases [27]. Among the various described model systems
moiety for this role [1,30]. In the case of a number of zinc(II) complexes
it has been proposed that a coordinated alcohol group is a stronger nu-
cleophile than a coordinated hydroxide [1,30,31]; in addition, it has
been suggested that the coordinated alcohol is deprotonated at or
below the same pH as a coordinated water molecule [31]. However, it
should be pointed out that in metallohydrolases, enzymes and corre-
sponding model complexes, the unambiguous identification of relevant
nucleophiles and hence the elucidation of the mechanistic pathway is
difficult [25,30].
Here, in order to address the issue of identifying relevant
nucleophiles, we describe the synthesis and characterization of the
di-zinc(II) complexes of three closely related ligands, 2,6-
bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1), 2,6-
bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2), and 2,6-
bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-4-methylphenol
(HL3) (Chart 1) [20,32–40]. These ligands provide ether donor (HL1), or
alkoxido donor ligands (HL2), or an asymmetric combination (HL3)
with both ether and alkoxido ligands. The catalytic potential of these
complexes has been explored using the phosphodiester substrate
☆
In memory of Professor Graeme Hanson and his contributions to bioinorganic
chemistry and electron paramagnetic resonance.
⁎
Corresponding author.
E-mail address: gahan@uq.edu.au (L.R. Gahan).
http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008
0162-0134/Crown Copyright © 2016 Published by Elsevier Inc. All rights reserved.
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
dinuclear mimics of me..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008

2
J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Chart 1. Ligands discussed in this work.
bis(2,4-dinitrophenyl)phosphate (BDNPP). In addition, we have
employed computational methods in an attempt to discriminate be-
tween different mechanistic pathways possible for these systems. As
part of this study the di-Mg(II) complex of ligand HL2 has also been pre-
pared with a view to examining a previous suggestion that Mg(II) is
able to replace Zn(II) in these hydrolytic enzyme model systems
[41–43].
was recorded with an Agilent 8453 UV–visible Spectrophotome-
ter, IR spectra were recorded with a Perkin-Elmer Frontier FT-IR/
NIR spectrometer SPECTRUM 400 (mode: MIR) with a Smiths
DuraSamplIR II ATR diamond window. The software used for data
processing was Perkin Elmer Spectrum version 10.03.09. 1H NMR
spectra were recorded at room temperature with a 300 MHz
Bruker AV 300/400 spectrometer. The abbreviations s = singlet,
d = doublet, t = triplet, m = multiplet have been employed. 13C
NMR spectra were recorded at room temperature with a 100 MHz
Bruker AV 400 spectrometer. Chemical shifts are reported relative
to d4-MeOD (δC = 49.0(7)). The software used for data processing
was TOPSPIN 3.0 from Bruker. For column chromatography silica
gel (SiO2, grain size 0.04–0.06 nm) produced by Scharlan was used.
Solvent mixtures of methanol and ethyl acetate mobile phases
were used.
2. Experimental
2.1. General methods and materials
Elemental microanalyses (C, H, N, S) were performed by the
Micro-analytical service at the School of Chemistry and Molecular
Biosciences, the University of Queensland. UV–vis spectroscopy
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
dinuclear mimics of me..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008

J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
3
2.2. X-ray crystallography
(10 mL) at 0 °C. The resulting crude oil was purified in methanol with
column chromatography (yield, 1.58 g, 46.1%). 1H NMR (300.13 MHz,
D2O): δ = 6.92 (s, 2H), 3.76 (s, 4H), 3.64 (t, 3JH,H = 5.7 Hz, 8H), 3.24
(s, MeOH), 2.73 (t, 3JH,H = 6.0 Hz, 8H), 2.12 (s, 3H) ppm.
Crystallographic data for the complexes were collected unless other-
wise stated at 293(2) K with an Oxford Diffraction Gemini Ultra dual
source (Mo and Cu) CCD diffractometer with Mo (λKα = 0.71073 Å)
or Cu (λKα = 1.5418 Å) radiation. The structures were solved by direct
methods (SIR-92) and refined (SHELXL 97) by full matrix least square
methods based on F2 [44]. These programs were accessed through the
WINGX 1.70.01 crystallographic collective package [45]. All non-
hydrogen atoms were refined anisotropically unless they were disor-
dered. Hydrogen atoms were fixed geometrically and were not refined.
X-ray data of the published structures were deposited with the
Cambridge Crystallographic Data Centre CCDC1429598-1,429,601.
Crystal data are given in Table 1 and selected bond distances and bond
angles in Table 2.
2.3.3. 2,6-bis(bis(methoxyethyl)-aminomethyl)-4-methylphenol (HL1) and
2,6-bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-4-methylphenol
(HL3)
2,6-chloromethyl-4-methylphenol (3.08 g, 15.0 mmol) in dichloro-
methane (12 mL) was added slowly to a solution of dihydroxyethylamine
(2.6 mL 27.0 mmol) and dimethoxyethylamine (3.95 mL 27.0 mmol),
triethylamine (3.75 mL) and tetrahydrofuran (13.5 mL) at 0 °C. The
resulting crude oil was purified in methanol with three rounds of column
chromatography, each ligand isolated separately (HL1 yield 0.18 g, 3.35%;
HL2 yield 0.17 g, 3.67%; HL3 yield 0.42 g, 7.55%). HL3: 1H NMR
(300.13 MHz, D2O): δ = 6.93–6.91 (d, 2JH,H = 6.0 Hz, 2H), 3.71 (s, 4H),
3.63 (t, 3JH,H = 6.0 Hz, 4H), 3.50 (t, 3JH,H = 5.7 Hz, 4H), 3.25 (s, MeOH),
3.22 (s, 6H), 2.72–2.70 (m, 5.4 Hz–5.7 Hz, 8H), 2.13 (s, 3H).
2.3. Syntheses
The 2,6-bis(chloromethyl)-4-methylphenol precursor was prepared
as previously described [46]. The preparation of ligand HL1, and the
characterization of a number of complexes of this ligand, has been re-
ported previously [32–40].
2.4. Syntheses of the metal complexes
2.4.1. [Zn2(L1)(CH3COO)2](PF6)
2.3.1. 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol (HL1)
A solution of bis(2-methoxyethyl)amine (2.66 g, 20.0 mmol) in
triethylamine (2.77 mL, 20.0 mmol) and tetrahydrofuran (10 mL) was
prepared and cooled to 0 °C. A solution of 2,6-bis(chloromethyl)-4-
methylphenol (2.05 g, 10.0 mmol) in dichloromethane (8 mL) was
added drop wise. The mixture was stirred at room temperature for 48 h
and then filtered to remove the colorless precipitate of triethylamine
hydrochloride. Removal of the solvents left a yellow oil which was
purified by column chromatography (ethyl acetate/methanol 4:1)
(yield 1.52 g, 37%). 1H NMR (300.13 MHz, CDCl3): δ = 7.10 (s, 2H), 4.10
(s, 4H), 3.67 (s, 8H), 3.31 (s, 12H), 3.07 (s, 8H), 2.22 (s, 3H) ppm.
2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol
2
(0.10 g, 0.25 mmol) was dissolved in methanol (5 mL) and a solution
of zinc acetate dihydrate (0.11 g, 0.50 mmol) in methanol (5 mL) was
added drop wise. The colorless solution was refluxed (70 °C) for 0.5 h
and then cooled to room temperature. Sodium hexafluorophosphate
(0.11 g, 0.60 mmol) was added. The mixture was filtered to remove
the excess sodium hexafluorophosphate and the solution was left in a
beaker so that the solvent evaporated slowly. Colorless crystals were ob-
tained after 7 days (yield 188 mg, 95%). 1H NMR (300.13 MHz, MeOD):
δ = 7.00 (s, 2H), 3.82 (s, 4H), 3.50 (broad s, 4H), 3.36 (s, 12H), 3.07
(broad s, 8H), 2.91 (m, 4H), 2.23 (s, 3H), 2.01 (s, 6H, CH3COO−) ppm.
13C NMR (100.62 MHz, MeOD): δ = 177.6 (CH3COO−), 161.4, 133.2,
127.7, 125.0, 68.9, 62.7, 59.7, 24.4, 20.4 ppm. ESI-MS (methanol) found:
2.3.2. 2,6-bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol (HL2)
2,6-chloromethyl-4-methylphenol (2.06 g, 10.0 mmol) in dichloro-
methane (9 mL) was added slowly to a solution of dihydroxyethylamine
(1.9 mL, 19.8 mmol), triethylamine (2.8 mL) and tetrahydrofuran
m/z = 645.1 [C25H43N2O9Zn2]+, 617.1 [C23H39N2O9Zn2]+
, 589.2
[C22H39N2O8Zn2]+. IR ν = 2937 (w, br), 1599 (m), 1481 (w), 1428 (m),
1327 (w), 1278 (w), 1099 (m), 1030 (w), 1003 (w), 826 (s), 665 (m),
Table 1
Crystal data.
[Zn2(L1)(CH3COO)2]PF6
[Zn2(L2)(CH3COO)2]BPh4.CH3CN
[Zn2(L3)(CH3COO)2]BPh4.2CH3OH
[Mg2(L1)(CH3COO)2]BPh4
Empirical formula
Formula weight
Wavelength (Å)
Crystal system
Space group
Crystal size (mm)
T (K)
C25H43F6N2O9PZn2
791.32
1.54180
Triclinic
P-1
C47H58BN3O9Zn2
950.51
1.54180
Monoclinic
P21/n
0.5 × 0.5 × 0.5
190(2)
C49H67BN2O11Zn2
1001.6
1.54180
Monoclinic
C2/c
0.4 × 0.4 × 0.3
190(2)
C46H59BMg2N2O10.5
867.38
1.54180
Monoclinic
P21/c
0.5 × 0.5 × 0.5
190(2)
0.3 × 0.05 × 0.05
190(2)
a (Å)
b (Å)
c (Å)
α (°)
10.6176(6)
12.3509(6)
12.6678(6)
85.694(4)
86.610(4)
85.979(4)
1650.03(15)
2
15.0013(5)
13.4784(4)
23.7110(10)
28.7875(9)
10.8171(3)
31.8882(10)
29.581(2)
13.6638(4)
25.722(2)
β (°)
96.725(3)
91.280(3)
115.559(7)
γ (°)
Vol (Å3)
4761.2(3)
4
1.692
1992
1.326
9927.4(5)
8
1.673
4224
1.34
9379.2(10)
8
0.936
3696
1.229
Z
μ (mm−1
F (000)
)
3.007
816
1.593
ρ (mg/m3)
Ind. Reflns
5232
7557
7826
14,711
θ range (°)
3.50–62.50
1.026
3.78–62.43
1.046
4.09–62.47
1.051
3.31–62.45
1.09
GOOF on F2
Final R indices[l N 2σ(i)]
R1 = .0350
wR2 = .0853
R1 = .0424
wR2 = .0911
R1 = .0525
wR2 = .1485
R1 = .0599
wR2 = .1591
R1 = .0362
wR2 = .0922
R1 = .0406
wR2 = .0988
R1 = .1013
wR2 = .2569
R1 = .1141
wR2 = .2649
R indices (all data)
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
dinuclear mimics of me..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008

4
J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Table 2
Selected bond lengths (Å) and angles (°).
M = Zn(II) or Mg(II)
[Zn2(L1)(CH3COO)2]PF6
[Zn2(L2)(CH3COO)2]BPh4·CH3CN
[Zn2(L3)(CH3COO)2]BPh4·2CH3OH
[Mg2(L1)(CH3COO)2]BPh4
M(1)-O(1)
M(1)-O(2)
M(1)-O(3)
M(1)-O(6)
M(1)-N(1)
M(2)-N(2)
M(2)-O(1)
M(2)-O(4)
2.0119(18)
2.1692(19)
2.005(2)
2.242(3)
2.096(2)
2.088(2)
2.163(3)
2.145(3)
1.990(2)
2.224(3)
2.139(3)
2.053(3)
3.320
1.9982(15)
2.1843(16)
2.3747(16)
1.9897(17)
2.1166(19)
2.1473(19)
2.0475(15)
2.1603(17)
2.2703(19)
2.1463(17)
3.282
2.017(4)
2.138(4)
2.085(5)
2.023(4)
2.218(5)
2.198(5)
2.013(4)
2.133(4)
2.085(5)
2.070(5)
3.366
1.9600(19)
2.127(2)
2.142(2)
2.0216(18)
M(2)-O(5)
M(2)-O(7)
M(1)⋯M(1)
2.166(2)
2.053(2)
3.270
O(1)-M(1)-O(2)
O(1)-M(1)-O(3)
O(1)-M(1)-N(1)
O(2)-M(1)-O(3)
O(3)-M(1)-N(1)
O(2)-M(1)-N(1)
O(4)-M(2)-N(2)
O(4)-M(2)-O(5)
O(1)-M(2)-O(4)
O(1)-M(2)-O(5)
O(1)-M(2)-N(2)
O(5)-M(2)-N(2)
M(1)-O(1)-M(2)
171.07(8)
163.77(11)
96.68(11)
91.50(10)
93.51(12)
77.60(10)
78.37(11)
80.05(11)
87.18(12)
169.48(11)
98.22(11)
92.21(10)
77.87(11)
112.43(11)
171.88(7)
165.02(19)
95.05(19)
90.19(17)
92.75(19)
77.20(18)
79.08(17)
78.40(18)
92.9(2)
165.1(2)
94.91(19)
91.00(18)
77.04(19)
113.33(19)
93.47(6)
93.33(7)
89.49(6)
75.95(7)
80.05(7)
76.78(7)
90.30(8)
164.45(7)
94.67(7)
89.90(7)
77.51(7)
108.40(7)
92.36(8)
78.85(9)
85.29(9)
167.93(8)
91.14(8)
108.34(8)
621 (w), 555 (s) cm−1. Anal calc. for C25H43N2O9Zn2PF6 3H2O: C 35.52, H
5.84, N 3.31; found: C 35.83, H 5.34, N 3.21%.
2.4.4. [Mg2(L2)(CH3COO)2](BPh4)
The complex was prepared following a similar procedure as de-
scribed for complex with ligand HL2 except magnesium acetate
tetrahydrate (0.22 g, 1.0 mmol) was employed. Colorless crystals were
obtained after 2–3 days and were left in solution before X-ray diffraction
data collection (yield, 290 mg, 70%). 1H NMR (300.13 MHz, MeOD):
2.4.2. [Zn2(L2)(CH3COO)2](BPh4)
2,6-bis((bis(2-Hydroxyethyl)amino)methyl)-4-methylphenol
(0.17 g, 0.5 mmol) was dissolved in methanol (3 mL) and a solution of
zinc acetate dihydrate (0.22 g, 1.0 mmol) in methanol (3 mL) was
added. The colorless solution was refluxed (65 °C) for 0.75 h and then
cooled to room temperature. Sodium tetraphenylborate (0.205 g,
0.60 mmol) was added, the mixture filtered and the solution was left
in a beaker so that the solvent evaporated slowly. Colorless crystals
were obtained after 2–3 days (yield, 340 mg, 79%). 1H NMR
(300.13 MHz, MeOD): [−BPh4 Counterion] δ = 7.30 (broad s, 8H),
6.98 (t, 3JH,H = 7.5 Hz, 8H), 6.84 (t, 3JH,H = 7.2 Hz, 4H) ppm. [Complex]
δ = 6.94 (s, 2H), 3.82 (broad s, 4H), 3.62 (broad s, 4H), 3.37 (broad s,
4H), 2.96 (broad s, 4H), 2.74 (broad s, 4H), 2.22 (s, 3H), 1.98 (s,
3
[−BPh4 Counterion] δ = 7.28 (broad s, 8H), 6.96 (t, JH,H = 7.5 Hz,
3
8H), 6.82 (t, JH,H = 7.2 Hz, 4H) ppm. [Complex] 6.90 (s, 2H), 3.91
3
3
(s, 4H), 3.70 (t, JH,H = 5.4 Hz, 8H), 2.83 (t, JH,H = 5.4 Hz, 8H), 2.29
(s, 3H), 1.92 (s, 6H). 13C NMR (100.62 MHz, MeOD): δ = 134.2
(−BPh4), 123.2(−BPh4), 119.6 (−BPh4), 55.4, 54.5 ppm. IR ν =
~3240 (w, broad), 3055(m), 1579(s), 1478(m), 1424(s), 1324(m),
1273(m), 1084(s), 847(m), 806(w), 733(s), 704(s), 611(s) cm−1. Anal
calc. for (C21H35N2O9Mg2BPh4: C 65.33, H 6.70, N 3.39; found: C 65.67,
H 6.72, N 3.42%.
2.4.5. Attempted synthesis of [Mg2(L1)(CH3COO)2](PF6)
CH3COO−
) δ = 178.6
ppm. 13C NMR (100.62 MHz, MeOD):
2,6-bis((bis(2-Methoxyethyl)amino)methyl)-4-methylphenol
(0.11 g, 0.25 mmol) was dissolved in methanol (5 mL) and a solution of
magnesium acetate tetrahydrate (0.11 g, 0.50 mmol) in methanol
(5 mL) was added drop wise. The colorless solution was refluxed
(70 °C) for 0.5 h and then cooled to room temperature. 2.50 eq sodium
hexafluorophosphate (0.11 g, 0.60 mmol) were added subsequently. The
mixture was filtered to remove the excess sodium hexafluorophosphate
and the solution was left in a beaker. Evaporation of the solvent left a
colorless oil (54.1 mg, 31%). All attempts to crystallize the complex
from a range of solvents were unsuccessful.
(CH3COO−), 135.9 (−BPh4), 130.7, 125.7, 124.9 (−BPh4), 123.9,
121.3 (−BPh4), 60.1, 56.6, 22.6 (CH3COO−), 18.9 ppm. IR ν =
3493(m), ~3250(w, broad), 3058(m), 1609 (s), 1578 (m), 1475(w),
1427(s), 1308(w), 1284(w), 1032(M), 889(m), 731(m), 710(s),
620(s) cm−1. Anal calc. for C21H35N2O9Zn2BPh4: C 59.42, H 6.09, N
3.08; found: C 59.42, H 6.12, N 3.08%.
2.4.3. [Zn2(L3)(CH3COO)2](BPh4)
The complex was prepared following a similar procedure as de-
scribed for complex with ligand HL2. Colorless crystals were obtained
with tetraphenylborate anion after 2–3 days but were maintained in
the mother liquor prior to collection of X-ray diffraction data (yield,
204 mg 43%). 1H NMR (300.13 MHz, MeOD): δ = [BPh4 Counterion]
ESI ꢀ MS ðmethanolÞ found : m=z
¼ 563:3 ½C25H43N2O9Mg2ꢁþ; 535:3 ½C23H39N2O9Mg2ꢁþ:
3
3
δ = 7.25 (broad s, 8H), 6.31 (t, JH,H = 7.2 Hz, 8H), 6.80 (t, JH,H
=
7.2 Hz, 4H) ppm. [Complex] δ = 3.79 (broad s, 4H), 3.57 (broad s,
4H), 3.46 (broad s, 4H), 3.32 (s, 6H), 3.05 (broad s, 4H), 2.75 (broad s,
4H), 2.19 (s, 3H), 1.96 (s, 6H, CH3COO−) ppm. 13C NMR (100.62 MHz,
MeOD): δ = 179.0 (CH3COO−), 136.0 (−BPh4), 131.7, 125.7, 124.9
(−BPh4), 123.6, 121.2(−BPh4), 58.0, 22.8 (CH3COO−) ppm. IR ν =
~3300(w, broad), 3055(m), 1578(s), 1478(m), 1423(s), 1327(m),
2.5. Phosphatase activity measurements
Kinetic studies of the phosphatase-like activity of the complexes
towards the substrate BDNPP were carried out by monitoring the
formation of 2,4-dinitrophenolate (DNP) anion at 400 nm. A Varian
Cary50 Bio UV/Visible spectrophotometer was employed with
10 mm quartz cuvettes and a Peltier temperature controller to main-
tain a temperature of 298 K. Measurements were taken using a 50:50
buffer/acetonitrile solution, 40 μM complex and 5 mM BDNPP in
1273(m), 1098(m), 1088(m), 849(w), 732(s), 704(s), 620(s) cm−1
.
Anal calc. for C23H39N2O9Zn2BPh4.MeOH: C 59.46, H 6.55, N 2.89;
found: C 59.35, H 6.44, N 2.76%.
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J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
5
acetonitrile. The aqueous buffer consisted of 50 mM MES (2-(N-
morpholino)ethanesulfonic acid) (pH 5.50–6.70), HEPES (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.00–8.50),
CHES (2-(N-cyclohexylamino)ethane sulfonic acid) (pH 9.00–
10.00) and CAPS (N-cyclohexyl-3-aminopropanesulfonic acid)
(pH 10.5–11) at constant ionic strength using 250 mM LiClO4. The pH
was adjusted using NaOH to give buffers ranging from pH 5.0 to 10.5.
These were then treated with Chelex for 24 h and filtered through a
0.45 μm Millex syringe-driven filter. For the kinetic measurements the
complex and buffer were mixed and left for 1 min before the addition
of the substrate; after a short period for equilibration in the reaction
chamber the initial rates were determined over a period of 3 min. As-
says were carried out in 50:50 MeCN:buffer, with substrate and com-
plex initially dissolved in MeCN. The pH values reported are those of
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 mixture of
buffer and acetonitrile [47,48]. Assays performed to investigate the ef-
fect of pH (over a pH range of 5.5–11.0) on the hydrolytic properties
of the complexes contained 250 μM of the Zn(II) complexes and
750 μM of the Mg(II) complex with 5 mM of BDNPP; no buffer effects
were detected. Assays to assess the effect of the substrate concentration
used the same complex concentrations with 1–10 mM in BDNPP. Back-
ground assays for autohydrolysis were subtracted from the data. The
change in absorbance produced by the hydrolysis of BDNPP by free
Zn(II) (from Zn(CH3COO)2·2H2O), using similar concentrations as
with the complexes, did not vary significantly from the measured
autohydrolysis rates. All data were fitted by non-linear least square re-
gression analysis.
attempts to prepare the [Mg2(L1)(CH3COO)2](PF6) complex resulted in
an oil which was not able to be obtained in solid form. However, the
mass spectrum in methanol suggested the presence of the desired com-
pound. The crystals of the zinc complexes with HL2 and HL3 as well as
the magnesium complex were found to desiccate upon separation from
the mother liquor, and were therefore kept in solution prior to structural
analysis.
The nomenclature employed for these types of ligands (HL1, HL2
and HL3, Chart 1) follows that described previously [48]. The nomencla-
ture denotes the number of labile protons upon complexation; there are
a number of deprotonations possible — from the phenolic –OH and from
the alcohol pendant arms of the ligands. The analyses of the complexes
suggest that in all cases a single anionic entity is present (PF−6 or BPh4−)
and that a single deprotonation of the ligand has occurred on complex-
ation. Therefore, designation of the ligand as L1, L2 and L3 implies a
single deprotonation and a monoanionic ligand.
3.2. X-ray structures
The X-ray crystal structures of the four complexes all show the
presence of a complex cation composed of the respective ligand,
and two metal ions triply linked by the deprotonated phenol moiety
from the ligand and by two bridging acetate groups, with either
tetraphenylborate or hexafluorophosphate as the uncoordinated
anion. Selected crystallographic data are shown in Table 1, selected
bond lengths and angles are displayed in Table 2. ORTEP plots of
the four complex cations are shown in Fig. 1(a)–(d).
For the complexes [Zn2(L2)(CH3COO)2](BPh4), [Zn2(L3)
(CH3COO)2](BPh4) and [Mg2(L2)(CH3COO)2](BPh4) each six coordi-
nate metal ion environment is composed of a NO5 chromophore con-
taining a tertiary amine, two alcohol and/or ether-O donors, and two
oxygens from the bridging acetates. The six-coordinate geometry is
completed by the bridging oxygen from the phenoxide in each
case. The Zn–O and Zn–N distances are typical of those reported for
this type of complex [1,23,30,35,56]. The structure of the zinc(II)
complex with ligand HL1 has been reported previously as the
tetraphenylborate salt, [Zn2(L1)(CH3COO)2]BPh4 [35]; in our case
the PF−6 salt, [Zn2(L1)(CH3COO)2](PF6), was isolated. Both structures
2.6. Computational modeling
Density functional theory (DFT) calculations were performed in
Gaussian 09 [49]. Geometry optimizations employed the B3LYP func-
tional [50–53] and a mixed basis set consisting of LANL2DZ on Zn and
6-31G(d) on other atoms. Harmonic vibrational frequency calculations
were used to identify whether stationary points were local minima
(zero imaginary vibrational frequencies) or transition states (one imag-
inary frequency) and to obtain zero-point energy and thermochemical
corrections. Solvation energies in water were computed with the SMD
implicit solvent model [54] using the same functional and basis set.
Single-point energies were then calculated with the M06 functional
[55] and a mixed basis set consisting of LANL2DZ on Zn and 6-
31G(d,p) on other atoms. Gibbs free energy in solution was calculated
by adding the B3LYP zero-point energy, thermochemical corrections
and solvation energy to the M06 potential energy. A standard state of
298.15 K and 1 mol/L was used. Certain transition states for P–O bond
formation or P–O bond cleavage could not be located by direct optimi-
zation to a first-order saddle point. In these cases the procedure for
identification of the transition state involved stretching the relevant
P–O bond of the phosphorane intermediate in 0.1 Å increments and
locating the maximum in ΔG(B3LYP) along the bond-stretching
coordinate.
exhibit two short Zn(II)–O–CH3 bonds (2.166 and 2.1669 Å, PF6−
;
2.214(5) and 2.197(3) Ǻ, BPh4−) and two are considerably longer
(2.523 and 2.528 Å, PF−6 ;2.362(4) and 2.440(3) Ǻ, BPh−4 ) [35]. This
has also been observed previously for similar zinc complexes [1].
The metal–metal distances range from 3.270 Ǻ to 3.366 Ǻ, the longest
being that for the di-Mg(II) complex.
There are numerous reports of di-Mg(II) complexes in the literature
with many examples of phenoxo-bridged macrocycles [57–72]. Of
relevance to this work is the reported structure of the complex
hemi[hexaaquamagnesium(II)](μ-2,6-
bis[(bis(carboxylatomethyl)amino)methyl]-4-chloropheno-
lato)bis[diaquamagnesium(II)] decahydrate (Chart 1, I) [70], which
has Mg–N distances of 2.210(2) Ǻ and 2.201(2) Ǻ compared to
2.197(5) Ǻ and 2.218(5) Ǻ in [Mg2(L2)(CH3COO)2](BPh4). Furthermore,
for [Mg2(L2)(CH3COO)2](BPh4) Mg-μ-O(phenoxide) 2.016(4) Ǻ and
2.013(4) Ǻ, Mg…Mg 3.366 Ǻ and Mg–O–Mg 113.37(19)° whilst for
the previously reported structure [70], Mg-μ-O(phenoxide) 2.062(2) Ǻ
and 2.072(2) Ǻ, Mg…Mg 3.787(1) Ǻ, and Mg–O–Mg 132.72(9)°.
3. Results and discussion
3.1. Syntheses
The ligands described were prepared by the reaction between
2,6-bis(chloromethyl)-4-methylphenol precursor and either bis(2-
methoxyethyl)amine, bis(2-hydroxyethyl)amine, or both, the synthetic
approach to HL3 utilizing a statistical reaction resulted in HL1, HL2 and
HL3 which were separated through column chromatography. The metal
complexes were prepared by the reaction of the appropriate ligands
with zinc(II) acetate resulting in complexes [Zn2(L1)(CH3COO)2](PF6),
[Zn2(L2)(CH3COO)2](BPh4) and [Zn2(L3)(CH3COO)2](BPh4), or the reac-
tion with magnesium(II) acetate to produce [Mg2(L2)(CH3COO)2](BPh4);
3.3. Phosphatase-like activity
Phosphatase-like activities of the zinc complexes [Zn2(L1)
(CH3COO)2](PF6), [Zn2(L2)(CH3COO)2](BPh4), [Zn2(L3)(CH3COO)2]
(BPh4) were measured using the activated substrate BDNPP. Based on a
series of previous studies [30,73–80] the underlying assumption of our
approach is that the two metal ion-bridging acetate groups dissociate in
aqueous environment, thus providing binding sites for the substrate
and water molecules from the solvent. While other scenarios are, in
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
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6
J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Fig. 1. ORTEP plot of (a) [Zn2(L1)(CH3COO)2]+; (b) [Zn2(L2)(CH3COO)2]+ (disorder is present in one chelate ring); (c) [Zn2(L3)(CH3COO)2]+; (d) [Mg2(L2)(CH3COO)2]+. Hydrogen atoms,
counter ions and labels of non-coordinating atoms have been omitted for clarity.
principle, possible (e.g. partial dissociation of one or both acetate groups)
there is currently no evidence to support these alternatives. For each
complex the dependence of the reaction rate on pH was determined in
the range between pH 5 to 10.5. The pH-rate profiles for each of the
three Zn complexes follow a bell-shaped curve but their pH maxima differ
(Fig. 2a). [Zn2(L1)(CH3COO)2](PF6) exhibits a rate maximum at pH 8
whereas [Zn2(L2)(CH3COO)2](BPh4) and [Zn2(L3)(CH3COO)2](BPh4)
display maxima at pH 9 and 9.5, respectively. The pH profiles were
fitted using Eq. (1) [81]:-
2.60(0.87) × 10−4 s−1 or 1.53(0.27) × 10−4 s−1, and Km
=
5.13(0.92) mM, 5.49(1.51) mM or 2.14(0.50) mM for [Zn2(L1)
(CH3COO)2](PF6), [Zn2(L2)(CH3COO)2](BPh4) or [Zn2(L3)(CH3COO)2]
(BPh4), respectively. In each case the kcat value shows an acceleration
of the reaction rate of at least ~103 fold over the uncatalyzed hydrolysis
of BDNPP (kuncat = 3.88 × 10−7 s−1) [82].
The [Mg2(L2)(CH3COO)2](BPh4) complex displayed very low activi-
ty over the entire pH range and was thus not further investigated. A DFT
study of the hydrolysis mechanisms of the phosphomonoester p-
nitrophenylphosphate catalyzed by unsymmetrical dinuclear Zn(II)2
and Mg(II)2 complexes [41] concluded that, based on its hard base
properties, Mg(II) ion should be able to substitute for other divalent
ions in dinuclear phosphatases [41]. However, as demonstrated above,
and at least for the systems reported in this work, Mg(II) is not capable
of effectively replacing Zn(II) to promote the hydrolysis of substrates
such as the activated phosphoester BDNPP.
νmax
!
ν0
¼
ð1Þ
ꢀ
ꢁ
Hþ
Ka2
ꢀ
ꢁ
1 þ
þ
Hþ
Ka1
where ν0 is the rate of formation of DNP (M s−1) and νmax is the maximal
(pH independent) rate. From the fitted data it is possible to extract
the kinetically relevant pKa values for the three complexes, i.e. pKa1
=
The pH rate profiles for the zinc complexes are consistent with the
existence of two protonation equilibria relevant for the reaction; for
each complex the deprotonation associated with pKa1 leads to an activa-
tion of the reaction, whereas the deprotonation associated with pKa2
has the opposite effect. The magnitude of the pKa1 values are consistent
with their assignment to either a zinc(II)-bound pendant alcohol or a
zinc(II)-bound water molecule [1]. Both are potential nucleophiles in
these complexes and both have been suggested previously to be active
in such systems [1,31]. Studies have suggested that a zinc(II)-
7.14, 7.90 or 8.43, and pKa2 = 9.21, 10.21 or 10.69 for [Zn2(L1)
(CH3COO)2](PF6), [Zn2(L2)(CH3COO)2](BPh4) or [Zn2(L3)(CH3COO)2]
(BPh4), respectively.
For each of the three Zn complexes the dependence of the reaction
rate on the substrate (BDNPP) concentration was measured at the re-
spective pH optima (i.e. pH 8, 9.0 and 9.5; Fig. 2b). The data were fitted
using non-linear least square analysis to provide an estimate for the kcat
value and Michaelis constant (Km): kcat = 5.44(0.11) × 10−5 s−1
,
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J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
7
3.4. Computational modeling
Density functional theory (DFT) calculations were performed to ex-
plore the roles of coordinated hydroxide and alkoxide nucleophiles in
the mechanism of phosphorolysis; specifically, the calculations focused
on the di-zinc complex of L2 (Chart 1), in which either a metal-bound
water molecule or one of the alcohol ligands could give rise to the cata-
lytic nucleophile. bis(para-Nitrophenyl)phosphate (BNPP) was used as
the model substrate. The computations were performed at the M06/6-
31G(d,p)-LANL2DZ//B3LYP/6-31G(d)-LANL2DZ level of theory [50–53,
55], simulating an aqueous medium with the SMD implicit solvent
model [54]. Density functional theory calculations have previously
been used to study phosphate and amide hydrolysis by related dizinc
complexes [21,40,41,85–89].
Generation of the catalytically active complex from [Zn2(L2)
(CH3COO)2]+ is thought to require the initial dissociation of the acetate
ligands [27,90]. One or more of the coordination sites made available by
the loss of these acetate groups is likely to be occupied by water mole-
cules, but their exact number is not known (and may also vary in differ-
ent systems). Therefore, a range of possible pathways were modeled,
involving zero, one or two water ligands, coordinated in either a
monodentate or metal-bridging mode. Different binding modes for the
phosphate ester substrate (i.e. mono-, bi- or tridentate) were also ex-
plored, with either a coordinated hydroxide or alkoxide acting as the
catalytic nucleophile.
Scheme 1 shows the lowest-energy reaction mechanisms that were
identified for hydroxide (Path A) and alkoxide (Path B) nucleophiles.
Both pathways originate from the same starting complex (1), which
contains one water molecule bound to zinc. The water is deprotonated
to form the catalytic nucleophile in the hydroxide-mediated pathway
(Path A), and is a spectator in the alkoxide-mediated pathway (Path
B). The kinetically relevant pKa1 for this complex (7.91) is consistent
with deprotonation of either a metal-bound water or alkoxide and it is
likely that both of the monodeprotonated species, 1a− and 1b−, may
be capable of formation at pH 9, the conditions of the experiment. The
calculations suggest that in Path A, the nucleophilic hydroxide prefers
to be bound to a single zinc centre only, rather than bridging between
the two zinc ions. Geometry optimizations commencing with a bridging
hydroxide converged to a monodentate complex. The geometries of the
transition states for nucleophilic attack on BNPP by the coordinated hy-
droxide (TSA) or alkoxide (TSB) are shown in Fig. 3.
Fig. 2. (a) Plot of pH dependence of initial rate of BDNPP hydrolysis (5 mM) by
[Zn2(L1)(CH3COO)2](PF6) (●), [Zn2(L2)(CH3COO)2](BPh4) (x), and [Zn2(L3)(CH3COO)2]
(BPh4) (♦): the fit to the data using Eq. (1) is shown as the solid line. (b) Plot of
substrate dependence of initial rate of BDNPP hydrolysis (5 mM) by [Zn2(L1)
(CH3COO)2](PF6) (●) (pH 8), [Zn2(L2)(CH3COO)2](BPh4) (x) (pH 9), [Zn2(L3)
(CH3COO)2](BPh4) (♦) (pH 9.5); the fit to the data using the Michaelis–Menten
equation is shown as the solid lines.
Importantly, the metal ion harboring the nucleophile is six-
coordinate, while the other one has five ligands only. In both hydrolysis
pathways, the nucleophile prefers to occupy a coordination site in cis
position relative to the nitrogen and the phenolate oxygen ligands.
Attack on BNPP by the hydroxide nucleophile (TSA) is computed to
have an activation barrier ΔG‡ of 23.9 kcal/mol, while attack by the
alkoxide nucleophile (TSB) has a barrier of 34.5 kcal/mol. These ΔG‡
values are computed with respect to the most stable form of the
monodeprotonated reactant complex 1− (see the Supporting Informa-
tion). The immediate products of hydroxide and alkoxide attack are
phosphoranes 2 and 3, respectively. These phosphoranes are predicted
to occupy shallow energy wells, where the barrier for the loss of one
of the nitrophenolate leaving groups is small or nonexistent. The
phosphorane intermediate 2 in the hydroxide-mediated pathway
(Path A) loses one nitrophenolate moiety to form 4, which is a complex
of Zn2(L2) with the product, i.e. (p-NO2C6H4O)P(OH)O−2 . Ligand ex-
change with BNPP and water is proposed to complete the catalytic
cycle. In the alkoxido-mediated pathway (Path B), after the loss of the
nitrophenolate leaving group from phosphorane 3 the phosphorus
remains covalently bound to L2 (see 5). Completion of the catalytic
cycle requires hydrolysis of the covalent alkoxido–phosphate bond.
The mechanism of this step is unknown and requires a second nucleo-
phile. While the identity of this nucleophile remains obscure, the
10.6 kcal/mol difference in activation barriers between TSA and TSB in-
dicates that the alkoxide-mediated pathway (Path B) is highly
coordinated alcohol has a lower pKa than a zinc(II)-coordinated water
[30,31,83–85], however the differences may not be significant and the
unambiguous identification of the relevant nucleophile is frequently
dependent on additional data such as those from labeling studies
with H2O16/18 [1]. However, even then the possibility of a mechanism
involving both a Zn–OH moiety and a bound alkoxide in a two-step
reaction may not be discounted [1]. In the case of [Zn2(L1)
(CH3COO)2](PF6) the assignment of the nucleophile (i.e. pKa1) is
unambiguous in that the molecule does not contain a coordinated alk-
oxide (Chart 1) and the ether donors are not nucleophilic; hence, here
pKa1 is assigned to a Zn–OH2 moiety. For the other two complexes,
[Zn2(L2)(CH3COO)2](BPh4) and [Zn2(L3)(CH3COO)2](BPh4), the situa-
tion is less straightforward. The catalytic parameters of all three com-
plexes are similar to those of various examples of di-Zn(II) complexes
of this type [25,78]. For [Zn2(L1)(CH3COO)2](PF6) a coordinated
hydroxido ligand can be proposed as the active nucleophile; as for the
other complexes [Zn2(L2)(CH3COO)2](BPh4) has a symmetric donor
set and alkoxide donors, and [Zn2(L3)(CH3COO)2](BPh4) is an example
of a system with an asymmetric donor set and these are predicted to be
more realistic models for the enzyme systems [23]. In order to under-
stand more of the potential mechanistic pathways for the reactions of
these complexes computational studies were undertaken.
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
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8
J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Scheme 1. Computed reaction mechanisms for hydrolysis of BNPP by the dizinc complex of L2 involving either a coordinated hydroxide nucleophile (Path A) or a coordinated alkoxide
nucleophile (Path B). Gibbs free energies in water computed with M06/6-31G(d,p)-LANL2DZ//B3LYP/6-31G(d)-LANL2DZ are shown (kcal/mol).
unfavorable and is thus unlikely to play any significant role in phos-
phate ester hydrolysis at 25 °C.
the transition state was the same as in TSA. DFT calculations predicted
that for the macrocyclic complex, direct attack by a coordinated hydrox-
ide was preferred relative to an alternative mechanism where the Zn–
OH group deprotonated a nearby water molecule to generate the active
nucleophile.
The different barrier heights for hydroxido- and alkoxido-mediated
attacks reflect the different degrees of structural reorganization that
the Zn2(L2) unit undergoes when forming the respective transition
states TSA and TSB. This is illustrated by the top-down views of the
precatalyst and the transition states, shown in Fig. 3(b). In the favored
TSA, the Zn2(L2) unit has a similar conformation to that in precatalyst
[Zn2(L2)(CH3COO)2](BPh4). The phosphate is bound in such a way
that two of its oxygen atoms are positioned, approximately, in the
same coordination sites formerly occupied by the acetate groups. By
contrast, in the alkoxido-mediated pathway, significant reorganization
of the Zn2(L2) core occurs. One of the phosphate oxygen atoms in TSB
is located underneath the phenolate group, where it binds to both
metal centers. To accommodate this bridging interaction, the Zn2(L2)
unit needs to undergo significant distortions. The structural reorganiza-
tion is perhaps most evident in the angle between the Zn–Zn axis and
the plane of the central phenolate ring (Fig. 3(b)); this angle is about
45° in both [Zn2(L2)(CH3COO)2](BPh4) and in TSA, but has twisted to
20° in TSB. The structural reorganization of the Zn2L2 unit in TSB in-
duces strain within the L2 ligand and distortion of the zinc coordination
spheres, which together represent the main reason for the high energy
of TSB relative to TSA.
The calculations therefore reveal the important role of ligand struc-
tural rigidity. A related computational study revealing the influence of
catalyst flexibility on the rates and mechanisms of phosphodiester hy-
drolysis by a di-zinc(II) complex of bismacrocyclic ligand II (Chart 1)
was recently reported by Maxwell, Mosey, and Brown [91]. The mode
of substrate binding and hydrolysis computed for the di-zinc(II) com-
plex of L2 (TSA) resembles the mechanism recently proposed by Zhao
et al. for the di-zinc(II) complex of macrocyclic oxyimine/phenolate li-
gand III (Chart 1) [41,89]. In that case, no alcohol donor was present
but the binding mode of the phosphate between the metal centers in
The calculated barrier (ΔG‡) of 23.9 kcal/mol for hydroxide-
mediated BNPP hydrolysis via TSA corresponds to
a kcat of
2 × 10−5 s−1, which is in good agreement with the experimental kcat
of 2.60 × 10−4 s−1 for the hydrolysis of the more reactive substrate
BDNPP (vide supra). The predicted lack of involvement of any
alkoxide-mediated pathway agrees well with the experimental kcat
values for the di-Zn(II) complexes of L1, L2, and L3, which vary by a fac-
tor of only five. The similar kcat values are consistent with a common
catalytic mechanism, where the small differences in rates for the com-
plexes of L1–L3 would reflect small differences in the nucleophilicity
of the coordinated hydroxide in each complex, rather than a role for
alkoxido-mediated pathways in the L2 and L3 complexes.
4. Conclusions
The synthesis and characterization of the di-zinc(II) complexes of the
ligands 2,6-bis((bis(2-methoxyethyl)amino)methyl)-4-methylphenol
(HL1), 2,6-bis(bis(hydroxyethyl)aminomethyl)-4-methylphenol
(HL2), and 2,6-bis((hydroxyethyl)(methoxyethyl)-aminomethyl)-
4-methylphenol (HL3) has been investigated. The ligands differ in
their donor types with HL1 having ether donors, HL2 alkoxido do-
nors and HL3, being an asymmetric ligand, possessing both ether
and alkoxido donors. The goal was to investigate the role of the po-
tential nucleophiles in the hydrolysis reaction with the phosphodies-
ter substrate BDNPP. In addition the di-Mg(II) complex of ligand HL2
was prepared in order to examine the potential for Mg(II) to replace
Zn(II) in these biomimetic systems. The three di-zinc(II) complexes
were found to display very similar pKa values irrespective of the
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J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
9
Fig. 3. (a) Transition states for hydrolysis of BNPP by the dizinc complex of L2 in water, computed at the M06/6-31G(d,p)-LANL2DZ//B3LYP/6-31G(d)-LANL2DZ level of theory (bond
distances in Å, ΔG‡ in kcal/mol). (b) Top-down views showing the conformation of the Zn2(L2) unit in TSA and TSB as compared with the X-ray structure of [Zn2(L2)(CH3COO)2](BPh4).
presence of potential hydroxido or alkoxido nucleophiles; Computa-
tional methods were used in an attempt to discriminate between the
mechanistic pathways possible for these systems. The DFT calcula-
tions indicate that an alkoxido-mediated pathway in the complexes
formed with ligands L2 or L3 is unlikely, because it induces signifi-
cant distortion of the Zn2(L) unit; a direct attack by a coordinated hy-
droxide is preferred in each of the three systems studied here.
Interestingly, and in contrast to a previous theoretical study, Mg(II)
was not able to reconstitute significant catalytic activity.
References
[1] L.J. Daumann, K.E. Dalle, G. Schenk, R.P. McGeary, P.V. Bernhardt, D.L. Ollis, L.R.
Gahan, Dalton Trans. 41 (2012) 1695–1708.
[2] F. Meyer, P. Rutsch, Chem. Commun. (1998) 1037–1038.
[3] H. Daiyasu, K. Osaka, Y. Ishino, H. Toh, FEBS Lett. 503 (2001) 1–6.
[4] F. Meyer, Eur. J. Inorg. Chem. (2006) 3789–3800.
[5] D.L. Tierney, G. Schenk, Biophys. J. 107 (2014) 1263–1272.
[6] B. Bauer-Siebenlist, F. Meyer, E. Farkas, D. Vidovic, S. Dechert, Chem. Eur. J. 11 (2005)
4349–4360.
[7] C. Bazzicalupi, A. Bencini, C. Bonaccini, C. Giorgi, P. Gratteri, S. Moro, M. Palumbo, A.
Simionato, J. Sgrignani, C. Sissi, B. Valtancoli, Inorg. Chem. 47 (2008) 5473–5484.
[8] N.J. Cosper, D.L. Bienvenue, J.E. Shokes, D.M. Gilner, T. Tsukamoto, R.A. Scott, R.C.
Holz, J. Am. Chem. Soc. 125 (2003) 14654–14655.
Acknowledgments
[9] M. Egertova, G.M. Simon, B.F. Cravatt, M.R. Elphick, J. Comp. Neurol. 506 (2007)
604–615.
This work is financially supported by the Australian Research Coun-
cil, Discovery Projects Scheme (DP120104263 and DP150104358). G.S.
and E.H.K. also acknowledge the receipt of ARC Future Fellowships
(FT120100694 to G.S. and FT120100632 to E.H.K.). This work was sup-
ported by computational resources provided by the Australian Govern-
ment through the National Computational Infrastructure National
Facility under the National Computational Merit Allocation Scheme,
and by the University of Queensland Research Computing Centre. We
are grateful to Prof. Paul Bernhardt (UQ) for his help with the crystal
structure analysis.
[10] C. He, S.J. Lippard, J. Am. Chem. Soc. 122 (2000) 184–185.
[11] N.V. Kaminskaia, C. He, S.J. Lippard, Inorg. Chem. 39 (2000) 3365–3373.
[12] B. Malfroy, H. Kado-Fong, C. Gros, B. Giros, J.C. Schwartz, R. Hellmiss, Biochem.
Biophys. Res. Commun. 161 (1989) 236–241.
[13] A.L. Stamp, P. Owen, K. El Omari, C.E. Nichols, M. Lockyer, H.K. Lamb, I.G. Charles, A.R.
Hawkins, D.K. Stammers, Protein Sci. 19 (2010) 1897–1905.
[14] S. Viars, J. Valentine, M. Hernick, Biomolecules 4 (2014) 527–545 (519 pp.).
[15] M.L. Zastrow, A.F.A. Peacock, J.A. Stuckey, V.L. Pecoraro, Nat. Chem. 4 (2012)
118–123.
[16] Z. Zhang, J. Liu, Q. Luo, J. Zhang, J. Xu, X.X. Zhu, Org. Biomol. Chem. 9 (2011)
8220–8223.
[17] M. Zhao, L. Zhang, H.-Y. Chen, H.-L. Wang, L.-N. Ji, Z.-W. Mao, Chem. Commun. 46
(2010) 6497–6499.
[18] Y.-H. Zhou, H. Fu, W.-X. Zhao, M.-L. Tong, C.-Y. Su, H. Sun, L.-N. Ji, Z.-W. Mao, Chem.
Eur. J. 13 (2007) 2402–2409.
[19] Y.-H. Zhou, M. Zhao, H. Sun, Z.-W. Mao, L.-N. Ji, J. Mol. Catal. A Chem. 308 (2009)
61–67.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2016.02.008.
[20] H. Sakiyama, Y. Igarashi, Y. Nakayama, M.J. Hossain, K. Unoura, Y. Nishida, Inorg.
Chim. Acta 351 (2003) 256–260.
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
dinuclear mimics of me..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008

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J.J. Brown et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
[21] N. Guo, J.-Y. Zhong, S.-L. Chen, J.-Q. Liu, Q. Min, R.-X. Shi, Chem. Phys. 457 (2015)
70–77.
[22] M.J. Hossain, A. Wada, Y. Igarashi, K.-i. Aimono, K. Suzuki, K. Tone, H. Sakiyama, Int. J.
[54] A.V. Marenich, C.J. Cramer, D.G. Truhlar, J. Phys. Chem. B 113 (2009) 6378–6396.
[55] Y. Zhao, D.G. Truhlar, Theor. Chem. Accounts 120 (2008) 215–241.
[56] K.E. Dalle, L.J. Daumann, G. Schenk, R.P. McGeary, L.R. Hanton, L.R. Gahan, Polyhe-
dron 52 (2013) 1336–1343.
Inorg. Chem. (2011) 395418 (395414 pp.).
[23] L.J. Daumann, L. Marty, G. Schenk, L.R. Gahan, Dalton Trans. 42 (2013) 9574–9584.
[24] L.J. Daumann, P. Comba, J.A. Larrabee, G. Schenk, R. Stranger, G. Cavigliasso, L.R.
Gahan, Inorg. Chem. 52 (2013) 2029–2043.
[57] P.K. Saini, C. Romain, Y. Zhu, C.K. Williams, Polym. Chem. 5 (2014) 6068–6075.
[58] P.K. Saini, C. Romain, C.K. Williams, Chem. Commun. 50 (2014) 4164–4167.
[59] W. Yi, H. Ma, Dalton Trans. 43 (2014) 5200–5210.
[25] L.J. Daumann, G. Schenk, D.L. Ollis, L.R. Gahan, Dalton Trans. 43 (2014) 910–928.
[26] L.J. Daumann, L.R. Gahan, P. Comba, G. Schenk, Inorg. Chem. 51 (2012) 7669–7681.
[27] N. Mitić, S.J. Smith, A. Neves, L.W. Guddat, L.R. Gahan, G. Schenk, Chem. Rev. 106
(2006) 3338–3363.
[28] R.R. Buchholz, M.E. Etienne, A. Dorgelo, R.E. Mirams, S.J. Smith, S.Y. Chow, L.R.
Hanton, G.B. Jameson, G. Schenk, L.R. Gahan, Dalton Trans. (2008) 6045–6054.
[29] G. Schenk, N. Mitić, L.R. Gahan, D.L. Ollis, R.P. McGeary, L.W. Guddat, Acc. Chem. Res.
45 (2013) 1593–1603.
[60] W. Yi, H. Ma, Inorg. Chem. 52 (2013) 11821–11835.
[61] M.R. Kember, C.K. Williams, J. Am. Chem. Soc. 134 (2012) 15676–15679.
[62] C.-Y. Li, C.-R. Wu, Y.-C. Liu, B.-T. Ko, Chem. Commun. 48 (2012) 9628–9630.
[63] S. Dutta, P. Biswas, U. Florke, K. Nag, Inorg. Chem. 49 (2010) 7382–7400.
[64] V. Chandrasekhar, B. Murugesapandian, T. Senapati, P. Bag, M.D. Pandey, S.K.
Maurya, D. Goswami, Inorg. Chem. 49 (2010) 4008–4016.
[65] A.C. Raimondi, D.J. Evans, T. Hasegawa, S.M. Drechsel, F.S. Nunes, Spectrochim. Acta
A 67A (2007) 145–149.
[30] J. Chen, X. Wang, Y. Zhu, J. Lin, X. Yang, Y. Li, Y. Lu, Z. Guo, Inorg. Chem. 44 (2005)
[66] D. Zhang, H. Kawaguchi, Organometallics 25 (2006) 5506–5509.
[67] A.C. Raimondi, V.R. de Souza, H.E. Toma, D.J. Evans, T. Hasegawa, F.S. Nunes,
Spectrochim. Acta A 61A (2005) 1929–1932.
3422–3430.
[31] C. Bazzicalupi, A. Bencini, E. Berni, A. Bianchi, V. Fedi, V. Fusi, C. Giorgi, P. Paoletti, B.
Valtancoli, Inorg. Chem. 38 (1999) 4115–4122.
[68] V. Chandrasekhar, R. Azhakar, J.F. Bickley, A. Steiner, Chem. Commun. (2005)
[32] H. Sakiyama, Inorg. Chim. Acta 360 (2007) 715–716.
459–461.
[33] H. Sakiyama, R. Ito, H. Kumagai, K. Inoue, M. Sakamoto, Y. Nishida, M. Yamasaki, Eur.
[69] A.C. Raimondi, V.R. de Souza, H.E. Toma, A.S. Mangrich, T. Hasegawa, F.S. Nunes,
Polyhedron 23 (2004) 2069–2074.
J. Inorg. Chem. (2001) 2705.
[34] H. Sakiyama, R. Ito, H. Kumagai, K. Inoue, M. Sakamoto, Y. Nishida, M. Yamasaki, Eur.
J. Inorg. Chem. (2001) 2027–2032.
[70] F. Gao, B.H. Meng, Y.B. Wei, Acta Crystallogr. Sect. C Cryst. Struct. Commun. C60
(2004) m360–m362.
[35] H. Sakiyama, R. Mochizuki, A. Sugawara, M. Sakamoto, Y. Nishida, M. Yamasaki, J.
[71] A.C. Raimondi, P.B. Hitchcock, G.J. Leigh, F.S. Nunes, J. Chem. Crystallogr. 34 (2004)
83–87.
Chem. Soc. Dalton Trans. (1999) 997–1000.
[36] H. Sakiyama, A. Sugawara, M. Sakamoto, K. Unoura, K. Inoue, M. Yamasaki, Inorg.
Chim. Acta 310 (2000) 163–168.
[37] H. Sakiyama, T. Suzuki, K. Ono, R. Ito, Y. Watanabe, M. Yamasaki, M. Mikuriya, Inorg.
[72] R.L. Dutta, R.K. Ray, J. Indian Chem. Soc. 60 (1983) 185–186.
[73] E. Lambert, B. Chabut, S. Chardon-Noblat, A. Deronzier, G. Chottard, A. Bousseksou,
J.-P. Tuchagues, J. Laugier, M. Bardet, J.-M. Latour, J. Am. Chem. Soc. 119 (1997)
9424–9437.
Chim. Acta 358 (2005) 1897–1903.
[38] H. Sakiyama, M. Yamasaki, T. Suzuki, Y. Watanabe, R. Ito, A. Ohnishi, Y. Nishida,
Inorg. Chem. Commun. 9 (2006) 18–20.
[39] R. Sanyal, S.K. Dash, S. Das, S. Chattopadhyay, S. Roy, D. Das, J. Biol. Inorg. Chem. 19
[74] B. Das, H. Daver, A. Singh, R. Singh, M. Haukka, S. Demeshko, F. Meyer, G. Lisensky,
M. Jarenmark, F. Himo, E. Nordlander, Eur. J. Inorg. Chem. (2014) 2204–2212.
[75] F.R. Xavier, A.J. Bortoluzzi, A. Neves, Chem. Biodivers. 9 (2012) 1794–1805.
[76] F.R. Xavier, A. Neves, A. Casellato, R.A. Peralta, A.J. Bortoluzzi, B. Szpoganicz, P.C.
Severino, H. Terenzi, Z. Tomkowicz, S. Ostrovsky, W. Haase, A. Ozarowski, J.
Krzystek, J. Telser, G. Schenk, L.R. Gahan, Inorg. Chem. 48 (2009) 7905–7921.
[77] F.R. Xavier, R.A. Peralta, A.J. Bortoluzzi, V. Drago, E.E. Castellano, W. Haase, Z.
Tomkowicz, A. Neves, J. Inorg. Biochem. 105 (2011) 1740–1752.
[78] L.J. Daumann, Spectroscopic and Mechanistic Studies of Dinuclear
Metallohydrolases and their Biomimetic Complexes, Springer International Publish-
ing, Switzerland, 2014.
(2014) 1099–1111.
[40] R. Sanyal, A. Guha, T. Ghosh, T.K. Mondal, E. Zangrando, D. Das, Inorg. Chem. 53
(2014) 85–96.
[41] X. Zhang, Y. Zhu, X. Zheng, D.L. Phillips, C. Zhao, Inorg. Chem. 53 (2014) 3354–3361.
[42] M.M.E. de Backer, S. McSweeney, P.F. Lindley, E. Hough, Acta Crystallogr. Sect. D Biol.
Crystallogr. D60 (2004) 1555–1561.
[43] P. Gettins, J.E. Coleman, Fed. Proc. 41 (1982) 2966–2973.
[44] G.M. Sheldrick, SHELXL97: Program for the refinement of crystal structures, Univer-
sity of Gottingen, Germany, 1997.
[79] S. Drueke, K. Wieghardt, B. Nuber, J. Weiss, H.P. Fleischhauer, S. Gehring, W. Haase, J.
[45] L.J. Farrugia, J. Appl. Crystallogr. 32 (1999) 837–838.
Am. Chem. Soc. 111 (1989) 8622–8631.
[80] M. Lanznaster, A. Neves, A.J. Bortoluzzi, B. Szpoganicz, E. Schwingel, Inorg. Chem. 41
(2002) 5641–5643.
[46] R.T. Paine, Y.-C. Tan, X.-M. Gan, Inorg. Chem. 40 (2001) 7009–7013.
[47] N.V. Kaminskaia, B. Spingler, S.J. Lippard, J. Am. Chem. Soc. 122 (2000) 6411–6422.
[48] L.J. Daumann, J.A. Larrabee, P. Comba, G. Schenk, L.R. Gahan, Eur. J. Inorg. Chem.
(2013) 3082–3089.
[81] I.H. Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady
State Enzyme Systems, Wiley-Interscience, New York, 1975.
[49] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark,
J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.
Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M.
Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L.
Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S.
Dapprich, A.D. Daniels, Ö Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox,
Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford CT, 2009.
[50] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B Condens. Matter 37 (1988) 785–789.
[51] A.D. Becke, J. Chem. Phys. 98 (1993) 1372–1377.
[82] A. Greatti, M. Scarpellini, R.A. Peralta, A. Casellato, A.J. Bortoluzzi, F.R. Xavier, R.
Jovito, M. Aires de Brito, B. Szpoganicz, Z. Tomkowicz, M. Rams, W. Haase, A.
Neves, Inorg. Chem. 47 (2008) 1107–1119.
[83] E. Kimura, Y. Kodama, T. Koike, M. Shiro, J. Am. Chem. Soc. 117 (1995) 8304–8311.
[84] M. Livieri, F. Mancin, U. Tonellato, J. Chin, J. Chem. Soc. Chem. Commun. (2004)
2862–2863.
[85] J. Xia, Y.B. Shi, Y. Zhang, Q. Miao, W.X. Tang, Inorg. Chem. 42 (2003) 70–77.
[86] R. Sanyal, X. Zhang, P. Kundu, T. Chattopadhyay, C. Zhao, F.A. Mautner, D. Das, Inorg.
Chem. 54 (2015) 2315–2324.
[87] H. Gao, Z. Ke, N.J. DeYonker, J. Wang, H. Xu, Z.-W. Mao, D.L. Phillips, C. Zhao, J. Am.
Chem. Soc. 133 (2011) 2904–2915.
[88] S. Ishiyama, C. Kaneda, J. Shibayama, K. Suzuki, R. Yamaguchi, H. Sakiyama, J.
Comput. Chem. Jpn. 13 (2014) 157–158.
[89] X. Zhang, X. Zheng, D.L. Phillips, C. Zhao, Dalton Trans. 43 (2014) 16289–16299.
[90] L.R. Gahan, S.J. Smith, A. Neves, G. Schenk, Eur. J. Inorg. Chem. (2009) 2745–2758.
[91] C.I. Maxwell, N.J. Mosey, R.S. Brown, J. Am. Chem. Soc. 135 (2013) 17209–17222.
[52] A.D. Becke, J. Chem. Phys. 98 (1993) 5648–5652.
[53] P.J. Stephens, F.J. Devlin, C.F. Chabalowski, M.J. Frisch, J. Phys. Chem. 98 (1994)
11623–11627.
Please cite this article as: J.J. Brown, et al., Investigation of the identity of the nucleophile initiating the hydrolysis of phosphate esters catalyzed by
dinuclear mimics of me..., J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.02.008