A structural and catalytic model for zinc phosphoesterases

Article abstract of DOI:10.1039/b806391e

A structural model for the active site of phosphoesterases, enzymes that degrade organophosphate neurotoxins, has been synthesised. The ligand [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl) -5-methylbenzyl)(pyridin-2-ylmethyl)amino)a

Full text of DOI:10.1039/b806391e

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A structural and catalytic model for zinc phosphoesterases†
Rebecca R. Buchholz,a Morgan E. Etienne,a Anneke Dorgelo,a Ruth E. Mirams,a Sarah J. Smith,a
Shiao Yun Chow,a Lyall R. Hanton,b Geoffrey B. Jameson,c Gerhard Schenka and Lawrence R. Gahan*a
Received 24th April 2008, Accepted 4th August 2008
First published as an Advance Article on the web 23rd September 2008
DOI: 10.1039/b806391e
A structural model for the active site of phosphoesterases, enzymes that degrade organophosphate
neurotoxins, has been synthesised. The ligand [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-
ylmethyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)acetic acid] (H3L1) and
two Zn(II) complexes have been prepared and characterised as [Zn2(HL1)(CH3COO)](PF6)·H2O
and Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH). The ligand (H3L1) and complex
[Zn2(HL1)(CH3COO)](PF6)·H2O were characterised through 1H NMR, 13C NMR, mass
spectroscopy and microanalysis. The X-ray crystal structure of Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH)
revealed a tetramer of dinuclear complexes, bridged by two phosphate molecules and bifurcating acetic
acid arms. Functional studies of the zinc complex with the substrate bis(4-nitrophenyl)phosphate
(bNPP) determined the complex with HL12- to be a competent catalyst with
kcat = 1.26 0.06 ¥ 10-6 s-1.
P. diminuta (OPH), OpdA and GpdQ has been recorded with
Introduction
Co(II).13,15,16
Phosphoesterases hydrolyse the phosphorus oxygen bond of tri-,
di- or monophosphoesters.1–4 Among them, triesterases are
OPH and OpdA have high structural similarity and sequence
homology (~90% identity), and are assumed to have a similar
mechanism of action.9,10,17 The active sites of these enzymes provide
essentially the same N,O asymmetric coordination environment
for the two divalent metal ions (Fig. 1). The metal ions are
coordinated by nitrogen donors located on four histidine ligands,
an aspartate, a carboxylated lysine, and a metal ion-bridging
hydroxide ion.9,16,18–20 In GpdQ, the more buried a-metal is
coordinated in a distorted trigonal-bipyramidal arrangement by
the donor atoms from His197, His10, Asp8, the bridging Asp50
and hydroxide, whilst the more solvent exposed b-metal is ligated
by His156, His195, Asn80 and the two bridging donors.13
Both kinetic studies and kinetic isotope (18O) effect studies
have determined that the hydrolysis of the P–O bond of OPs by
phosphotriesterases is accompanied by an inversion of configu-
ration at the phosphorus atom.21 While this general mechanism
is widely accepted, some details such as the identity of the
attacking nucleophile remain subject to debate, with possibilities
including the bridging hydroxo, a terminal hydroxo or activated
uncoordinated water.13,17–22 While a mechanism involving the
bridging hydroxide was initially favoured, an alternative model has
gained credence, whereby a hydroxide that is terminally bound to
the a-metal ion attacks the phosphorus centre of the substrate,
which is monodentately bound to the b-metal ion.20
Biomimetics are useful for a number of reasons, including
that it is often easier to study structural and mechanistic
features of complex biological systems using a smaller and
simpler model system.3,23–27 Models may be structural, electronic
and/or functional mimics, although lower catalytic activity is
to be expected from the model complexes as they tend to be
poor functional mimics in comparison with their corresponding
enzyme systems.28–34 This approach is exemplified by recent
investigations into the structure and function of the binuclear
mono and diesterase purple acid phosphatase.27,31,33,35 We report
enzymes that have acquired the ability to degrade a range of
synthetic organophosphates (OP) including chemical warfare
agents such as sarin and VX, and insecticides such as paraoxon.5,6
The members of this group of enzymes have been isolated and
characterised from Pseudomonas diminuta, Flavobacterium sp. and
Agrobacterium radiobacter.7–10 The binuclear glycerophosphodi-
esterase from Enterobacter aerogenes (GpdQ)11,12 is structurally
unrelated to these triesterases but also has modest activity towards
OPs and diester products of VX degradation.4 Importantly,
recombinant Escherichia coli co-expressing the triesterase from
A. radiobacter (OpdA) and GpdQ is able to survive under
conditions where OPs are the only source of phosphate.12 Thus, this
bacterial system may be sufficiently robust to find an application
in the bioremediation of, for example, water supplies that are
contaminated with pesticide wastes.12
The in vivo metal ion compositions of metalloenzymes are often
diverse. Anomalous scattering analysis of OpdA has indicated
that its likely metal ion composition is of the Fe(II)–Zn(II) type,
whereas GpdQ is speculated to be of the di-Fe(II) type.13,14
However, the catalytic activity may be regenerated from the
apo-forms of both enzymes upon addition of various divalent
metals, including Mn(II), Co(II), Cd(II) and Zn(II).11,14,15 The
highest hydrolytic activity for the OP-degrading hydrolase from
aSchool of Molecular and Microbial Sciences, The University of Queensland,
Queensland, Australia 4072. E-mail: gahan@uq.edu.au
bDepartment of Chemistry, Te Tari Hua-Ruanuku, University of Otago, PO
Box 56, Dunedin, New Zealand
cMassey University, Palmerston North Campus, Private Bag 11222, Palmer-
ston North, 5301, New Zealand
† Electronic supplementary information (ESI) available: TOF MS data.
CCDC reference number 686253. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/b806391e
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Fig. 2 H3L1 and H3L2.
lmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphenol (H3L2)
(Fig. 2).28 The reaction of H2EtL1 with lithium hydroxide resulted
in a lithium salt of [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-
2-ylmethyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)-
amino)acetic acid] (LiH2L1).39
The nomenclature employed for the ligand refers to the number
of protons possibly available for removal upon complexation.
Thus, for H3L1, the inference is that the protons on the phenoxide,
the carboxylate and the pendant alcohol are candidates for depro-
tonation. Possible protonation sites on the pyridine nitrogen atoms
are not included in this nomenclature but these are considered in
the determination of the ligand pKa values (vide infra).
The reaction of LiH2L1 with zinc acetate in the presence of
sodium hexafluorophosphate resulted in a white solid, for which
microanalytical and spectroscopic data support the assignment as
[Zn2(HL1)(CH3COO)](PF6)·H2O. The crystal produced for the X-
ray structural study was isolated from the bulk reaction mixture
upon standing with sodium hexafluorophosphate. Subsequent
structural analysis indicated a complex structure (vide infra) most
likely resulting from the crystallisation methodology and not
necessarily representative of the initial product produced in the
reaction. The structure of this complex represents its only form of
characterization and is reported because of its high nuclearity and
some of its structural features.
Fig. 1 Schematic of the active sites of OPH25 and GpdQ13
herein, the synthesis and characterisation of [2-((2-hydroxy-
3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)5-methyl-
benzyl)(pyridin-2-yl-methyl)amino)acetic acid] (H3L1) (Fig. 2)
and its di-Zn(II) complex, specifically as a structural model for
the GpdQ enzyme. The complex has been characterised spectro-
scopically, and its catalytic properties were investigated using the
substrate bis(4-nitrophenyl) phosphate (bNPP). In addition, an
octanuclear zinc(II) complex of HL12- has been characterised by
X-ray crystallography.
Results and discussion
Synthesis of the ligand and complex
The ethyl ester H2EtL1 was produced in a reaction sequence
(Scheme 1) involving a statistical reaction between N-(2-pyridyl-
methyl)glycine ethyl ester,36 N-(2-pyridylmethyl)-2-aminoe-
thanol37 and 2,6-bis(chloromethyl)-4-methyl-phenol38 in tetra-
hydrofuran in the presence of base. Chromatographic separation
of the mixture with silica readily produced ethyl 2-((2-hydroxy-
3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)amino)methyl)-5-methyl-
benzyl)(pyridin-2-ylmethyl)amino)acetate (H2EtL1) as well as
the symmetrical analogues [diethyl 2,20-(2-hydroxy-5-methyl-1,3-
phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)dia-
cetate] and the previously reported ligand 2,6-bis([(2-pyridy-
Solid state structure
The complex Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH) crystallised
as a tetramer of dinuclear [Zn2(HL1)]2+ units with two PO4
3-
molecules, one lithium ion and a molecule of methanol within
the complex cation. Diagrams of the complex cation are shown
in Fig. 3 and Fig. 4 with the crystal data listed in Table 1 with
important bond distances and angles listed in Table 2 (listed bond
lengths and angles involving disordered atoms refer to the main
3-
residue, i.e. the higher occupancy). The two PO4 anions are
6046 | Dalton Trans., 2008, 6045–6054
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Table 1 Crystallographic data for Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH)
Empirical formula
Formula weight
Temperature/K
C101H116F18LiN16O25P5Zn8
2980.85
90(2)
0.71073
˚
Wavelength/A
Crystal system
Space group
Monoclinic
P21/c
16.560(3)
40.130(2)
19.710(3)
104.92(2)
12657(3)
4
˚
a/A
˚
b/A
˚
c/A
b/◦
3
˚
V/A
Z
Absorption coefficient/mm-1
1.650
1.564
D
calcd/Mg m-3
Reflections collected/unique
Data/restraints/parameters
Final R indices [I > 2s(I)]
R indices (all data)
80 413/19 659 (Rint = 0.0414)
19 659/4700/2352
R = 0.0994, wR2 = 0.2348
R = 0.1195, wR2 = 0.2491
Fig. 3 ORTEP plot of the main residue of the disordered complex cation
of Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH). Thermal probability ellipsoids
are drawn at the 50% probability level. Hydrogen atoms are omitted for
clarity.
Scheme 1 Synthesis of ethyl-2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-
2-ylmethyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)-
acetate (H2EtL1) and the lithium salt (LiH2L1)
PF6 .41 In PO2F2 , the P–F and P–O bond lengths are different
-
-
42–44
˚
˚
(P–F, 1.49–1.55 A; P–O, 1.43–1.47 A)
whereas in the present
-
each connected to four Zn(II) ions. Three PF6 anions, two of
structure there is no difference between the two sets of bonds of
˚
them disordered, complete the structure. A significant disorder
was also observed in many of the ligand’s aromatic rings. The
electron density, apparent in the centre of the tetrameric structure,
was assigned to a lithium ion most likely arising from the use of
the lithium salt of the ligand in the synthetic step; the isotropic
atomic displacement parameters suggested that either a sodium
the tetrahedral anions (1.509–1.541 A). The charge balance also
3-
-
supports the presence of PO4 rather than PO2F2 .
There are four six-coordinate (Zn(1), Zn(4), Zn(5) and Zn(8))
and four five-coordinate zinc centres (Zn(2), Zn(3), Zn(6) and
Zn(7)) in the complex cation. The molecule is composed of two
halves, each composed of two HL12- ligands coordinated to four
Zn(II) ions. The halves are connected by the two PO4 anions,
separating the zinc atoms by approximately 4.72 A, and forming
3-
or a hydrogen ion was inappropriate. The lithium is strongly
3-
˚
˚
associated with the oxygen of the PO4 anions (1.95–2.06 A) and
a zipper-like connection with the Li+ cation in the centre of the
structure, but not present on a symmetry element. One side of the
zipper is composed of Zn(7), Zn(8), Zn(1), and Zn(2), the other
Zn(6), Zn(5), Zn(4) and Zn(3). The five-coordinate Zn(II) centres
(Zn(2), Zn(3), Zn(6) and Zn(7)) are at the end of the respective
zippers. The average Zn–Zn distance in the dinuclear complexes
˚
with the oxygen atom of a solvent methanol molecule (2.43 A).
3-
Although no PO4 was intentionally added to the crystallising
mixture, possible sources include an impurity in the sodium
hexafluorophosphate, or from hydrolysis of PF6 to PO4 by the
Zn(II) complex.40 The bond lengths and angles, as well as the
coordinative saturation, of this moiety support its assignment
-
3-
3-
-
˚
as PO4 as opposed to PO2F2 , another hydrolytic product of
is 3.55 A. Zn(1) and Zn(2) share a coordination environment
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◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for the main residue of the disordered complex Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH)
Molecule 1
Zn(1)–O(3)
Zn(1)–N(1)
Zn(1)–O(1)
Zn(1)–N(2)
Zn(1)–O(51)
Zn(1)–O(13)
Zn(2)–O(4)
Zn(2)–N(4)
Zn(2)–O(3)
Zn(2)–O(52)
Zn(2)–N(3)
Zn(3)–N(8)
Zn(3)–N(7)
Zn(3)–O(7)
Zn(3)–O(8)
Zn(3)–O(53)
2.137(11)
2.171(7)
2.312(6)
2.104(7)
1.959(5)
2.082(6)
2.051(7)
2.102(12)
1.954(7)
1.945(6)
2.128(15)
1.948(10)
2.201(15)
1.982(6)
2.213(14)
1.969(6)
1.541(8)
1.533(6)
1.532(6)
1.536(6)
89.8(3)
Zn(4)–O(7)
Zn(4)–N(6)
Zn(4)–O(54)
Zn(4)–O(9)
Zn(4)–N(5)
Zn(4)–O(5)
Zn(5)–O(5)
Zn(5)–O(43)
Zn(5)–O(11)
Zn(5)–N(9)
Zn(5)–N(10)
Zn(5)–O(9)
Zn(6)–O(11)
Zn(6)–N(11)
Zn(6)–O(12)
Zn(6)–N(12)
Zn(6)–O(44)
O(43)–P(4)
O(52)–P(5)
2.092(6)
2.107(8)
1.972(6)
2.085(13)
2.144(8)
2.659(18)
2.061(16)
2.030(7)
2.065(8)
2.225(15)
2.137(10)
2.318(13)
2.022(8)
2.196(14)
2.013(11)
2.089(9)
1.966(7)
1.542(7)
1.523(7)
Zn(7)–N(15)
Zn(7)–O(41)
Zn(7)–N(16)
Zn(7)–O(16)
Zn(7)–O(15)
Zn(8)–O(42)
Zn(8)–N(13)
Zn(8)–O(15)
Zn(8)–O(13)
Zn(8)–N(14)
Zn(8)–O(1)
Zn(1) ◊ ◊ ◊ Zn(2)
Zn(3) ◊ ◊ ◊ Zn(4)
Zn(4) ◊ ◊ ◊ Zn(5)
Zn(5) ◊ ◊ ◊ Zn(6)
Zn(7) ◊ ◊ ◊ Zn(8)
Zn(1) ◊ ◊ ◊ Zn(8)
O(44)–P(4)
2.246(13)
1.898(11)
2.167(14)
1.879(10)
2.139(10)
1.969(5)
2.181(8)
2.079(6)
2.514(6)
2.114(7)
2.070(6)
3.553(1)
3.529(1)
3.559(2)
3.561(2)
3.603(2)
3.479(1)
1.509(8)
1.526(6)
O(41)–P(4)
O(42)–P(4)
O(51)–P(5)
O(54)–P(5)
O(53)–P(5)
O(3)–Zn(1)–N(1)
O(3)–Zn(1)–N(2)
O(3)–Zn(1)–O(13)
N(1)–Zn(1)–N(2)
N(1)–Zn(1)–O(13)
O(1)–Zn(1)–O(51)
N(2)–Zn(1)–O(51)
O(51)–Zn(1)–O(13)
O(3)–Zn(2)–N(4)
O(3)–Zn(2)–O(52)
N(3)–Zn(2)–O(4)
N(4)–Zn(2)–O(4)
O(4)–Zn(2)–O(52)
O(7)–Zn(3)–N(7)
O(7)–Zn(3)–O(53)
N(7)–Zn(3)–O(8)
N(8)–Zn(3)–O(8)
O(8)–Zn(3)–O(53)
O(7)–Zn(4)–O(5)
O(7)–Zn(4)–N(6)
O(7)–Zn(4)–O(9)
N(5)–Zn(4)–O(54)
O(5)–Zn(4)–N(6)
O(5)–Zn(4)–O(9)
N(6)–Zn(4)–O(9)
O(11)–Zn(5)–N(9)
O(11)–Zn(5)–N(10)
O(11)–Zn(5)–O(5)
N(9)–Zn(5)–N(10)
N(9)–Zn(5)–O(5)
O(9)–Zn(5)–O(43)
N(10)–Zn(5)–O(43)
O(43)–Zn(5)–O(5)
O(11)–Zn(6)–O(12)
O(11)–Zn(6)–O(44)
N(11)–Zn(6)–O(12)
N(12)–Zn(6)–O(12)
O(12)–Zn(6)–O(44)
O(15)–Zn(7)–N(16)
O(15)–Zn(7)–O(41)
N(15)–Zn(7)–O(16)
N(16)–Zn(7)–O(16)
O(16)–Zn(7)–O(41)
O(15)–Zn(8)–O(13)
O(15)–Zn(8)–O(42)
N(13)–Zn(8)–O(13)
N(13)–Zn(8)–O(42)
O(13)–Zn(8)–N(14)
O(13)–Zn(8)–O(1)
O(3)–Zn(1)–O(1)
O(3)–Zn(1)–O(51)
N(1)–Zn(1)–O(1)
N(1)–Zn(1)–O(51)
O(1)–Zn(1)–N(2)
O(1)–Zn(1)–O(13)
N(2)–Zn(1)–O(13)
O(3)–Zn(2)–N(3)
O(3)–Zn(2)–O(4)
N(3)–Zn(2)–N(4)
N(3)–Zn(2)–O(52)
N(4)–Zn(2)–O(52)
O(7)–Zn(3)–N(8)
O(7)–Zn(3)–O(8)
N(7)–Zn(3)–N(8)
N(7)–Zn(3)–O(53)
N(8)–Zn(3)–O(53)
O(7)–Zn(4)–N(5)
O(7)–Zn(4)–O(54)
N(5)–Zn(4)–O(5)
N(5)–Zn(4)–N(6)
N(5)–Zn(4)–O(9)
O(5)–Zn(4)–O(54)
N(6)–Zn(4)–O(54)
O(54)–Zn(4)–O(9)
O(11)–Zn(5)–O(9)
O(11)–Zn(5)–O(43)
N(9)–Zn(5)–O(9)
N(9)–Zn(5)–O(43)
O(9)–Zn(5)–N(10)
O(9)–Zn(5)–O(5)
N(10)–Zn(5)–O(5)
O(11)–Zn(6)–N(11)
O(11)–Zn(6)–N(12)
N(11)–Zn(6)–N(12)
N(11)–Zn(6)–O(44)
N(12)–Zn(6)–O(44)
O(15)–Zn(7)–N(15)
O(15)–Zn(7)–O(16)
N(15)–Zn(7)–N(16)
N(15)–Zn(7)–O(41)
N(16)–Zn(7)–O(41)
O(15)–Zn(8)–N(13)
O(15)–Zn(8)–N(14)
O(15)–Zn(8)–O(1)
N(13)–Zn(8)–N(14)
N(13)–Zn(8)–O(1)
O(13)–Zn(8)–O(42)
N(14)–Zn(8)–O(42)
162.6(2)
85.9(3)
116.5(2)
79.9(3)
151.2(2)
90.0(2)
176.6(3)
93.5(2)
137.0(4)
98.5(4)
86.3(5)
107.9(8)
94.2(2)
89.9(3)
100.5(2)
78.2(4)
114.9(5)
90.3(3)
160.3(4)
93.2(3)
126.4(4)
96.1(3)
86.3(4)
73.2(4)
85.1(4)
90.3(4)
84.8(3)
114.7(5)
79.2(4)
151.5(5)
82.8(4)
171.0(4)
97.3(3)
108.9(4)
92.1(3)
81.2(4)
108.2(4)
98.8(4)
115.8(5)
98.2(5)
92.2(3)
72.9(2)
97.2(3)
91.0(2)
80.5(2)
89.9(3)
90.4(5)
112.9(8)
79.0(4)
170.1(3)
91.5(4)
139.0(4)
102.1(4)
81.3(4)
165.9(4)
96.6(4)
93.5(3)
95.3(4)
66.9(4)
80.4(3)
138.2(4)
84.7(4)
171.0(3)
92.1(4)
163.4(4)
97.8(3)
73.1(4)
92.1(3)
92.2(5)
81.5(5)
89.3(6)
89.6(4)
138.0(4)
77.3(4)
178.2(4)
101.0(4)
86.2(5)
109.7(5)
74.7(6)
171.4(5)
96.7(6)
92.9(3)
94.6(3)
121.5(2)
79.8(3)
144.1(3)
85.8(3)
170.1(3)
83.3(4)
127.1(5)
102.1(4)
162.4(3)
95.1(2)
69.6(3)
97.6(3)
84.4(3)
76.1(3)
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Table 2 (Contd.)
Molecule 1
N(14)–Zn(8)–O(1)
Zn(1)–O(3)–Zn(2)
Zn(5)–O(11)–Zn(6)
Zn(1)–O(1)–Zn(8)
Zn(5)–O(9)–Zn(4)
O(44)–P(4)–O(42)
O(42)–P(4)–O(41)
O(42)–P(4)–O(43)
O(52)–P(5)–O(53)
O(53)–P(5)–O(51)
O(53)–P(5)–O(54)
87.0(2)
120.5(6)
121.2(4)
105.0(2)
107.7(5)
110.3(4)
109.8(4)
102.9(4)
110.0(4)
108.6(4)
109.5(4)
O(42)–Zn(8)–O(1)
Zn(4)–O(7)–Zn(3)
Zn(8)–O(15)–Zn(7)
Zn(4)–O(5)–Zn(5)
Zn(8)–O(13)–Zn(1)
O(44)–P(4)–O(41)
O(44)–P(40–O(43)
O(41)–P(4)–O(43)
O(52)–P(5)–O(51)
O(52)–P(5)–O(54)
O(51)–P(5)–O(54)
89.8(2)
120.0(3)
117.3(4)
97.0(3)
98.0(3)
111.1(5)
114.6(4)
107.7(4)
114.4(4)
110.5(4)
103.6(3)
Hydrogen bonding
O(2) ◊ ◊ ◊ O(16)
2.574(10)
2.57(2)
165.4
168.6
O(6) ◊ ◊ ◊ O(12)
2.538(17)
2.598(18)
163.1
177.1
O(10) ◊ ◊ ◊ O(8)
O(14) ◊ ◊ ◊ O(4)
O(2) ◊ ◊ ◊ H–O(16)
O(10) ◊ ◊ ◊ H–O(8)
O(6) ◊ ◊ ◊ H–O(12)
O(14) ◊ ◊ ◊ H–O(4)
structures. The bifurcated nature of this interaction is reminiscent
of that seen for Asp50 in the structure of GpdQ.13
The five-coordinate Zn(2) (and by analogy Zn(3), Zn(6) and
Zn(7)) ions are trigonal-bipyramidal, where the amine nitrogen
and phosphate oxygen atoms are trans. The five-coordinate
geometry is comprised of bonds to the bridging phenoxide (Zn(2)–
O(3)), a pyridine nitrogen (Zn(2)–N(4), a tertiary nitrogen (Zn(2)–
N(3)), a hydroxyl oxygen (Zn(2)–O(4)) and a phosphate oxygen
(Zn(2)–O(52)). Strong hydrogen bonds link the hydroxyl oxygen
of one Zn2HL1 unit to the uncoordinated carboxylate oxygen
of a neighbouring Zn2HL1 unit ◦(for example: O(4)–H ◊ ◊ ◊ O(14)
˚
2.597(4) A, O(4)–H ◊ ◊ ◊ O(14) 177 ).
Mass spectrometry
The ligand precursor H2EtL1 displays the expected low-resolution
positive-ion mass spectrum with m/z 479.21. The ESI-MS
of the zinc complex with the HL12- ligand, characterised as
[Zn2(HL1)(CH3COO)](PF6) in acetonitrile indicates the pres-
ence of [Zn2(HL1)(CHOO)]+ (m/z 625.06, 623.06 and 621.06),
[Zn2(HL1)]+ (m/z 579.06, 577.06 and 575.06) with the most
prominent peaks typical of the isotopic pattern expected for a di-
Zn(II) complex. In addition, the species [Zn(HL1)]+ (m/z 517.14,
515.14 and 513.14) was also present. The ESI-MS in water–
acetonitrile (90 : 10) indicates the presence of [Zn(HL1) + H]+
(m/z 513.14) with an isotope pattern consistent with the presence
of only one metal ion. In the presence of two equivalents of
zinc acetate, in the same solvent mixture, peaks attributed to
[Zn(HL1)(CH3COO)(H2O)6 + 2H]+ (m/z 681.23, 682.23 and
683.23) and [Zn2(HL1)(CH3COO)(H2O)6]+ (m/z 743.14, 745.14,
747.14 and 748.14) were present, the latter with an isotope pattern
indicative of a di-Zn(II) complex (see ESI†). In order to probe the
hydrolysis reaction of [Zn2(HL1)(m-CH3COO)](PF6), the complex
was reacted with the substrate bNPP for 24 h at 50 ◦C in
a water–acetonitrile (90 : 10) solution and the mass spectrum
recorded. The most prominent peak was at m/z 513.14, assigned
to [Zn(HL1) + H]+; no peaks assigned to phosphate-containing
species were observed. The addition of excess Zn(II) (as Zn(II)
Fig.
4
ORTEP plot of a fragment of the complex cation of
Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH). Thermal probability ellipsoids are
drawn at the 50% probability level. Hydrogen atoms are omitted for clarity.
of an HL12- ligand with an unsymmetrical bridge through the
m-phenoxo oxygen donor O(3); likewise, Zn(3) and Zn(4), Zn(5)
and Zn(6), and Zn(7) and Zn(8) each share an HL12- ligand. The
six-coordinate geometry of Zn(1) and Zn(8) (and by analogy Zn(4)
and Zn(5)) is made up of bonds to the m-phenoxide (Zn(1)–O(3)),
the pyridine nitrogen (Zn(1)–N(2)), the tertiary nitrogen (Zn(1)–
N(1)), the PO43- oxygen (Zn(1)–O(51)), the oxygen of an adjacent
carboxylate (Zn(1)–O(13)) and the oxygen of the carboxylate from
the HL12- shared with Zn(8) (Zn(1)–O(1)). Thus, Zn(1) and Zn(8)
(and similarly Zn(4) and Zn(5)) are bridged through a bifurcation
of the oxygen donors of the acetic acid arms of the HL12- ligand,
one oxygen from each ligand. In each case, two of the interactions
are relatively long (Zn(1)–O(1), Zn(8)–O(13), Zn(4)–O(5), Zn(5)–
O(9)) whereas the others are significantly shorter (Zn(1)–O(13),
Zn(8)–O(1), Zn(4)–O(9), Zn(5)–O(5)). The Zn(4) and Zn(8) sites,
˚
which have the long (>2.5 A) Zn–O(carboxylate) bonds, are
noticeably more distorted from the octahedral geometry, and in
fact may be better described as edge-bridged trigonal-bipyramidal
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acetate) resulted in the appearance of more prominent di-Zn(II)
species (m/z 745.14 and 747.14).
The effect of complex concentration, based on [Zn2(HL1)(m-
CH3COO)](PF6)·H2O (pH 9.0; 50 ◦C), on the rate of hydrolysis
of bNPP (1 mM) was linear for concentrations of complex from
0.05–0.25 mM (Fig. 5), with a second order rate constant of 7.55
0.26 ¥ 10-3 M-1 s-1. The addition of extra Zn(II) had no effect on
the rates of the reactions. The pH dependence on the rate of bNPP
cleavage by the complex displays a sigmoidal behaviour in the pH
range 7–9.5. The data were fitted using an equation derived for a
monoprotic system,47 yielding a pKa value of 7.87 (Fig. 6) for the
catalytically relevant agent in the model substrate complex.
Potentiometric studies
The pKa values of H3L1 were determined from the potentiometric
titration curve obtained when the ligand was titrated in an
acetonitrile–water solution (1 : 4, v/v) at 25 ◦C, I = 0.1 (Et4NClO4)
with Et4NOH. The pKa values at low (<3) and high (>9) pH
were determined iteratively after fitting the mid-pH range and
then holding the obtained fitted parameters fixed in order to fit
the extremes of the curve. Once a satisfactory fit was obtained,
all the pKa values were allowed to refine freely in order to
minimise the complete set. The pKa values determined were 2.95,
assigned to the carboxylic acid, 4.97 and 7.53, assigned to the two
pyridine nitrogen atoms,45 and 10.97, which corresponds to the
deprotonation of the alcoholic oxygen.
Potentiometric titrations of Zn(II) and H3L1 with base in the
pH range 2.0–8.5 in an acetonitrile–water solution (1 : 4, v/v)
yielded logK1 = 5.98 and logK2 = 3.35 as the simplest model.
The fit to the two binding constants was reproducible over a
number of different titrations and, when more complicated models
including hydroxo ligands were added these were routinely rejected
as valid models unless the most rigorous fitting conditions were
applied. The simplest model was therefore accepted. The relative
magnitudes of the binding constants suggest that one Zn(II) is
bound relatively tightly to HL12-, and the second more loosely.
Fig. 5 Dependence of the pseudo-first order rate constant on the
concentration of [Zn2(HL1)]+ in 88 mM CHES buffer at pH 9.0, 50 ◦C,
containing 88 mM LiClO4.
Phosphodiesterase-like activity
The phosphatase-like activity of the proposed [Zn2(HL1)-
(CH3COO)](PF6)·H2O was measured under conditions of ex-
cess substrate, using bNPP (bis-nitrophenyl phosphate). The
species tested was the initially prepared Zn2 complex and
not the supramolecular Zn8 complex, which resulted dur-
ing growth of diffraction-quality crystals. Under the condi-
tions of the mass spectrometer for the complex formulated as
[Zn2(HL1)(CH3COO)](PF6)·H2O, the acetate is retained in the
absence of substrate, however exposure to bNPP results in a
species in which the acetate is lost, albeit on prolonged standing. A
number of assumptions can be made concerning the catalytically
active species. It could be assumed that, to produce a catalytically
active species, theacetate ligand partiallyor completelydissociates,
resulting in a complex with either terminal or bridging aqua
ligands, [Zn2(HL1)(OH2/OH)x]m+ 28–31,33–35 Alternatively, the active
.
species may be an aqua complex [Zn2(HL1)(m-CH3COO)(OH2)x]+
in which the m-acetato ligand is retained.
Fig. 6 pH dependence on the reaction rate for [Zn2(HL1)]+.
The hydrolysis of bNPP was examined in the presence of added
zinc ions and as a function of the concentration of [Zn2(HL1)(m-
CH3COO)]+. In all experiments, the effect of added Zn(II) was
negligible up until the addition of two equivalents when a slight
rate enhancement was observed. This observation suggests that the
most significant active species in solution is a di-Zn(II) complex
based on [Zn2(HL1)(m-CH3COO)n(OH2/OH)x]m+ (n = 0 or 1).
It is possible that the presence of substrate stabilises or aids in
the assembly of the di-Zn(II) site, analogous to the suggestion
made that in some phosphoesterase systems the reconstitution of
a catalytically competent enzyme may only occur in the presence
of substrates.2,46
The initial rate of bNPP cleavage has also been determined as
a function of substrate concentration, revealing typical saturation
behaviour (Fig. 7). The data were fitted by non-linear regression,
using the Michaelis–Menten equation,
Vmax[S]
Rate =
,
[S] + KM
where Vmax = kcat[Zn2(complex)]
The substrate concentration at half-maximal rate (KM) is 1.96
0.25 ¥ 10-3 M with Vmax = 4.37 0.51 ¥ 10-11 mol L s-1 and
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amongst others, some purple acid phosphatases, arginases and
ureases.31,65–69 In contrast, a recent crystallographic study showing
trapped substrates bound to the active site of OpdA suggests that
in this enzyme the nucleophile is monodentately bound to the
a-metal site (Fig. 1).20 Clearly, there is substantial ambiguity and
variety with respect to assigning nucleophiles. In an attempt to
reconcile these different strategies, it has been suggested that the
m-hydroxo is the actual source of the nucleophile, but that substrate
binding triggers the displacement of the m-OH to form a quasi-
terminal hydroxide, coordinated predominantly to the more buried
or a- site.70,71
While it is no trivial task to deconvolute the precise details
of the catalytic mechanism(s) employed by binuclear hydrolytic
systems, it has emerged that subtle variations in the first and
second coordination environment and differences in the metal
ion composition have less subtle effects on that mechanism(s).2
For instance, for purple acid phosphatase extracted from the pig
uterine fluid (uteroferrin) the likely nucleophile for the FeFe and
FeZn metal ion combinations is a terminally bound hydroxide,72
while for the GaZn, FeNi and possibly FeMn combinations, the
m-hydroxide is the likely candidate nucleophile.31,35 Similarly, dif-
ferent nucleophiles are proposed by different groups for the highly
homologous OPH and OpdA (~90% sequence identity).9,10,17
In summary, despite the ambiguity of the assignment of
the catalytically relevant pKa values for the Zn(II) complex of
HL12-, the mechanistic features of the model system are in good
agreement with those of the corresponding enzymatic systems.
Fig. 7 Dependence on the rate of bNPP cleavage by [Zn2(HL1)]+
(0.02 mM) at pH 9.0, 50 ◦C, 88 mM LiClO4, [CHES buffer] 88 mM.
the corresponding kcat = 1.26 0.06 ¥ 10-6 s-1. The indicative
substrate binding constant (Kb = 1/KM) is 510 M-1, suggesting
that bNPP is a stronger ligand for [Zn2(HL1)]2+ than it is for the
H2L2- complex (Kb = 16.3 M-1).28 For the analogue [Zn2(HL2)(m-
CH3COO)(H2O)](PF6)28 (2,6-bis([(2-pyridylmethyl)(2-hydroxy-
ethyl)amino]methyl)-4-methylphenol (H2L2-)) under similar
reaction conditions KM = 6.15 ¥ 10-2 M and kcat = 4.60 ¥ 10-6 s-1
have been reported.28
Mechanistic implications
Conclusions
[Zn2(HL1)(CH3COO)](PF6) and [Zn2(HL2)(m-CH3COO)(H2O)]-
(PF6)·H2O display catalytically relevant pKa values of 7.87 and
7.13, respectively.28 Theoretical studies have indicated that the
Zn(II)–alcohol has a lower pKa than the Zn(II)–water in the same
molecule.48 This, combined with studies in non-aqueous solvents,
led to the suggestion that for [Zn2(HL2)(m-CH3COO)(H2O)](PF6),
the alcohol hydroxyl, with a pKa of 7.20, is the nucleophile.28
The identity of the nucleophile is less clear for [Zn2(HL1)(m-
CH3COO)](PF6)·H2O. From the pH dependence studies, the active
species was determined to display a pKa of 7.87, typical of that
for the deprotonation of a terminal aqua ligand,49 although the
pKa for the deprotonation of a bridging hydroxide is also within
this range.50–52 The assignment of the catalytic pKa is further
complicated by the observation that the magnitude of the Zn(II)–
aqua pKa depends on, amongst other factors, the stereochemistry
around the Zn(II) ion, the metal–metal ion separation, and the
extent of hydrogen bonding. Thus, pKa values as low as ~4.44
and as high as 9 are observed with apparently little discrimination
between the pKa exhibited by the monomeric and dimeric Zn(II)
complexes.28,48,49,52–62
Effective biomimetics are expected to display both the structural
and functional characteristics of the metallobiosite.24–29 In the
case of both HL12- and H2L2- 28, the zinc complexes mimic
aspects of the features of the metallobiosites of OPH, OpdA
and GpdQ, HL12-, in particular, appearing to present a strong
and loose binding site, analogous to the situation observed in the
enzymes. Modelling the activity and the exact nucleophilic agent
is more problematic. The catalytic activity of the Zn(II) complexes
of HL12- and HL2- appears typical of that exhibited by similar
complexes, although it is difficult to make definitive comparisons
due to the different reaction conditions employed.28,52 Whilst
the kcat of ~1.5 ¥ 10-6 s-1 represents considerable enhancement
over the uncatalyzed rate (1.1 ¥ 10-11 s-1)73 the enhancement
does not approach the catalytic rate of the metalloenzymes (the
catalytic rates for OPH and OpdA are > 1000 s-1).13,15,16 There are
strategies, however, that reportedly lead to an enhanced activity
for biomimetic systems.59,74 Considering the potential applications
of OPH, OpdA and GpdQ for bioremediation, it is an attractive
endeavour to develop more robust biomimetic systems exhibiting
sufficient structural and functional stability to be used practically.
The assignment of the catalytically relevant pKa of the Zn(II)
complex of HL12- to either a terminal or metal ion bridg-
ing hydroxide is in agreement with the corresponding assign-
ments in OPH, OpdA, GpdQ and various other binuclear
metallohydrolases.2,17,19,31,63 A combination of the experimental
and theoretical (computational) studies of OPH suggests that the
bridging hydroxide with a pKa of 8.4 is the nucleophile in phos-
phate ester hydrolysis.20,63,64 Other binuclear metallohydrolases that
are reported to initiate hydrolysis with the m-hydroxide include,
Experimental
Instrumental methods
NMR spectra were measured using Bruker AV400 (400 MHz)
and AV500 (500 MHz) instruments. The spectra were re-
corded in CDCl3, with chemical shifts reported in parts per
million (ppm) and calibrated using the resonances for CDCl3,
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dH(CHCl3) = 7.24 ppm and dC(CDCl3) = 77.0 ppm. Low and
high-resolution positive-ion mass spectra were obtained using
either a Q-Star time-of-flight mass spectrometer, in methanol or
acetonitrile, using 10% acetic or formic acid carrier liquid or a
Finnigan MAT 900 XL mass spectrometer and methanol solutions
for the ligand, and acetonitrile for the metal complex. All samples
were subjected to electrospray ionisation, with voltages tuned
to optimise the signals. The predicted isotopic splitting patterns
of peaks were calculated using the program Molecular Weight
Calculator.75 Infrared spectroscopy was performed with a Perkin
Elmer FT-IR SPECTRUM 2000 spectrometer with a Smiths
DuraSamplIR II ATR diamond window. Elemental analyses were
performed using the microanalysis facilities at The University of
Queensland.
t, J = 5.28 Hz, CH2OH), 3.72 (2H, s, CH2), 3.79 (2H, s, CH2),
3.80 (2H, s, CH2), 3.92 (2H, s, CH2), 4.12 (2H, q, J = 7.14 Hz,
CH2CH3), 6.78 (1H, d, J = 1.90 Hz, Ar–H), 6.85 (1H, d, J =
1.85 Hz, Ar–H), 7.03 (1H, qd, J = 1.67 Hz, 1.05, py–CH), 7.11
(1H, qd, J = 1.63 Hz, 1.03, py–CH), 7.34 (1H, dt, J = 7.88 Hz,
1.01, py–CH), 7.44 (1H, td, J = 3.88 Hz, 1.85, py–CH), 7.59 (1H,
td, J = 3.84 Hz, 1.82, py–CH), 8.40 (1H, dq, J = 4.93 Hz, 0.86,
py–CH), 8.50 (1H, dq, J = 4.75 Hz, 0.83, py–CH). NMR dC/ppm
(500 MHz, CDCl3): 14.08 (CH2CH3), 20.29 (Ar–CH3), 53.96,
54.50, 55.05, 55.70, 58.87, 59.09, 59.41, 60.52 (CH2), 121.84,
121.99 (py–CH), 122.19, 122.36 (Ar–C), 122.99, 123.08 (py–CH),
127.45 (Ar–C), 129.88, 130.70 (Ar–CH), 136.38, 136.70, 148.46,
=
148.80 (py–CH), 153.51 (Ar–C), 157.96 (py–C), 171.03 (C O).
m/z: 479.22 (C27H35N4O4, M+), 388.22 (C21H30N3O4), 327.17
(C19H23N2O3), 285.17 (C17H21N2O2), 242.29 (C15H18N2O). The
third product eluted (Rf: 0.30) was symmetric H3L2 (0.33 g).
NMR dH/ppm (500 MHz, CDCl3): 2.17 (3H, s, Ar–CH3); 2.69
(4H, t, J = 5.20 Hz, 2 ¥ NCH2CH2OH); 3.64 (4H, t, J = 5.23 Hz,
2 ¥ NCH2CH2OH); 3.72 (4H, s, 2 ¥ CH2); 3.84 (4H, s, 2 ¥ CH2);
6.79 (2H, s, 2 ¥ Ar–H); 7.10 (2H, qd, J = 1.70 Hz, 1.10, py–CH),
7.26 (2H, dt, J = 7.88 Hz, 1.19, py–CH), 7.54 (2H, td, J = 3.85 Hz,
1.85, py–CH), 8.49 (2H, dq, J = 5.13 Hz, 0.85, py–CH). NMR
dC/ppm (500 MHz, CDCl3): 20.38 (Ar–CH3), 55.75, 55.99, 59.31
59.34 (CH2), 122.14, 123.16 (py–CH), 123.70, 127.56, 130.32
(Ar–CH), 136.68, 148.81 (py–CH), 153.71 (Ar–CH), 158.79
(py–CH). m/z: 437.21 (C25H33N4O3, M+), 285.17 (C17H21N2O2),
242.29 (C15H18N2O), 153.11 (C8H13N2O).
Syntheses of the ligands and metal complex
2,6-Bis(chloromethyl)-4-methyl-phenol, N-(2-pyridylmethyl) gly-
cine ethyl ester, N-(2-pyridylmethyl)-2-aminoethanol were pre-
pared as described previously.36–38
[Ethyl-2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-ylmethyl)-
amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)acetate]
(H2EtL1). H2EtL1 was synthesised from 2,6-bis(chloromethyl)-
4-methyl-phenol, N-(2-pyridylmethyl)glycine ethyl ester and
N-(2-pyridylmethyl)-2-aminoethanol using a statistical reaction.
Thus, N-(2-pyridylmethyl) glycine ethyl ester (1.12 g, 5.76 mmol)
and N-(2-pyridylmethyl)-2-aminoethanol (0.88 g, 5.8 mmol)
were dissolved in
9 mL of tetrahydrofuran (THF) with
Lithium salt of [2-((2-hydroxy-3-(((2-hydroxyethyl)(pyridin-2-yl-
methyl)amino)methyl)-5-methylbenzyl)(pyridin-2-ylmethyl)amino)-
acetic acid] (LiH2L1). A methanol solution of LiOH (0.1 M)
was prepared from 0.2115 g LiOH in 100 mL methanol. H2EtL1
(0.1087 g, 0.227 mmol) was dissolved in MeOH (1 mL), 2.27 mL
of the LiOH–methanol solution was added and the mixture
stirred for 48 h. Removal of the solvent in vacuo giving LiH2L1
as a yellow oil (0.10 g). NMR dH/ppm (400 MHz, CDCl3): 2.54
(2, NCH2CH2), 3.30–3.85 (12, CH2), 4.12, 6.61–6.71 (2, Ar–H),
6.99–8.33 (8, py–CH). NMR dC/ppm (400 MHz, CDCl3): 19.69
and 19.87 (Ar–CH3), 51.00–59.27 (CH2), 60.61 (CH2), 121.64
(py–CH), 121.88 (py–CH), 122.02 (Ar–tert-C), 122.69 (py–CH),
122.80 (py–CH), 136.39 (Ar–CH), 136.43 (py–CH), 136.69
(py–CH), 148.33 (py–CH), 157.51 (Ar–C), 158.30 (py–C), 159.61
1.15 g triethylamine (TEA) (11.48 mmol). Separately, 2,6-bis-
(chloromethyl)-4-methyl-phenol (1.18 g, 5.76 mmol) was dissolved
in 8 mL dichloromethane (DCM) and added dropwise under
stirring into the TEA solution at 0 ◦C. The mixture was stirred at
room temperature for 48 h, the precipitated TEA·HCl was filtered
off, and the solvent was removed by rotary evaporation. The
residue was taken up in DCM (50 mL), washed with concentrated
aqueous NaCl (20 mL), the organic layer dried over anhydrous
sodium sulfate, filtered and the solvent removed in vacuo, to
yield an orange oil (2.35 g). NMR and TLC of the mixture
revealed a combination of three products. The separation of
the products was achieved with column chromatography (silica;
80 : 20, ethyl acetate–methanol). The first product eluted from
the column (Rf: 0.89) was determined to be [diethyl-2,20-(2-
hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-2-
ylmethyl)azanediyl)diacetate] (0.4 g). NMR dH/ppm (500 MHz,
CDCl3): 1.21 (6H, t, J = 7.15 Hz, CH2CH3), 2.18 (3H, s, Ar–CH3),
3.37 (4H, s, CH2), 3.84 (4H, s, CH2), 3.93 (4H, s, CH2), 4.12 (2H,
q, J = 7.22 Hz, CH2CH3), 6.91 (2H, s, Ar–H), 7.09 (2H, qd, J =
1.65 Hz, 1.17, py–CH), 7.45 (2H, dt, J = 7.89 Hz, 1.01, py–CH)
7.59 (2H, td, J = 3.85 Hz, 1.80, py–CH) 8.47 (2 H, dq, J =
4.93 Hz, 0.91, py–CH). NMR dC/ppm (500 MHz, CDCl3): 14.12
(CH2CH3), 20.39 (Ar–CH3), 54.32, 59.41, 60.43 (CH2), 122.02
(Ar–C), 123.10, 123.14 (py–CH), 127.56 (Ar–CH), 130.05 (Ar–C),
=
(py–C), 171.20 (C O).
[Zn2(HL1)(CH3COO)](PF6)·H2O and Li[Zn2(HL1)]4(PO4)2-
(PF6)3·(CH3OH). LiH2L1 (0.10 g, 0.219 mmol) was dissolved
in methanol (1 mL). To this was added zinc acetate (0.0481 g,
0.219 mmol) dissolved in methanol dropwise with stirring.
The resulting mixture was refluxed gently for 30 min then
permitted to cool to room temperature. NaPF6 (0.0550 g,
0.327 mmol) was dissolved in methanol (4 mL) and added
and the solution was left to sit at room temperature. Slow
evaporation and subsequent filtration produced a creamy white
solid (53.6 mg, 77%). Found: C 40.75, H 4.11, N 7.00%. Calcd
for [Zn2(C25H28N4O4)(CH3COO)](PF6)·H2O: C 40.47, H 4.15,
N 7.00%. NMR dH/ppm (400 MHz, DMSO): 1.98 (Ar–CH3),
2.06 (NCH2CH2OH), 2.78 (CH2OH), 2.16–3.23 (CH2), 3.51–
4.04 (CH2), 6.54 and 6.66 (ArH), 6.80–8.55 (py–CH). NMR
dC/ppm (400 MHz, DMSO): 19.70 and 19.77 (CH3), 54.65,
55.33, 55.95, 56.72, 58.25, 58.85, 60.95 (CH2), 121.76 (Ar–C),
136.56, 148.77 (py–CH), 153.48 (Ar–C), 158.74 (py–C), 171.31
+
=
(C O). m/z: 521.22 (C29H37N4O5, M ), 479.22 (C27H35N4O4),
327.17 (C19H23N2O3), 286.16 (C17H22N2O2), 195.13 (C10H15N2O2).
The second product to be eluted (Rf: 0.59) was identified as
the target product (0.65 g). NMR dH/ppm (500 MHz, CDCl3):
1.19 (3H, t, J = 7.15 Hz, CH2CH3), 2.15 (3H, s, Ar–CH3), 2.70
(2H, t, J = 5.18 Hz, NCH2CH2), 3.33 (2H, s, CH2), 3.66 (2H,
6052 | Dalton Trans., 2008, 6045–6054
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123.05, 123.31, 123.81, 124.61 (py–CH), 131.79 (Ar–CH), 139.02,
139.74, 146.55, 147.26 (py–CH), 154.96 (Ar–C), 159.65 (py–C),
0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 mM, respectively, leading to a 1–
10-fold excess of zinc. The effect of pH on the hydrolytic reaction
was studied in the pH range 6.5–10 with the Zn2H2L1, and no
excess zinc. The reactions were followed to 5% of bNPP hydrolysis.
The kinetic experiments, as a function of substrate concentration,
were performed at pH 9. The concentration of bNPP in the final
3 mL reactions was 0.5, 1.0, 1.5 and 2.0 mM, respectively.
=
173.05 (C O), 177.75 (Ar–C). ES-MS m/z: 623.10 and 625.1
[Zn2C25H28N4O4(CHOO)]+; 577.09 and 579.1 [Zn2C25H27N4O4]+;
513.17 [ZnC25H29N4O4]+. FT-IR spectra (u/cm-1): 1605.23 (m,
=
=
C O str), 1568.99 (m, phenol and py C C str), 1478.21 (m, py
CH str), 1433.29 (m, py CH stretch), 1397.61 (m, CH2CO def),
1267.72 (w, tertiary NC str), 1057.55, 1019.42 (w, COH str) 824.80
-
-
Potentiometric titrations
(vs. PF6 ), 797.90, 765.92 (m, m, py CH def), 554.96 (s, PF6 ).
Crystallisation of the complex proved to be difficult. A screening
method was devised using crystallisation wells to screen solution
environments for precipitating crystals. Acetate, nitrate, sulfate
and phosphate anions were screened with differing quantities
of the counter ion PF6 or ClO4 . The only environment to
produce crystals was in the presence of hexafluorophosphate.
Thus, the bulk reaction mixture was left to stand in the presence
Titrant solutions of acid (HClO4) were standardised against Borax
and titrant solutions of Et4NOH were standardised against the
previously standardised HClO4. Potentiometric titrations were
performed with a Metrohm 665 Dosimat. The e.m.f. was measured
with an ORION model 720A pH meter with a glass electrode. All
titrations were carried out at a constant ionic strength of 0.1 M
Et4NClO4 and were performed under a nitrogen atmosphere at
25 ◦C. For each titration the pH meter was calibrated using
pH 6.88 and 4.00 buffers and a calibration titration was performed
to determine Eo and pKw values. Potentiometric titrations were
performed on both the free ligands and in the presence of zinc
perchlorate under a MeCN–H2O saturated nitrogen atmosphere
at 25 ◦C. The reaction vessel contained 2 mL of product (5 mM)
in acetonitrile, acidified with 8 mL HClO4 (6.8 mM). The
final concentration of the product was therefore 1 mM in an
acetonitrile–water (1 : 4, v/v) solution. The ionic strength was
kept constant at 0.1 M with Et4NClO4. Calibration titrations were
conducted using constant volume increments (0.02 mL) of 0.109 M
Et4NOH solution. Reactant titrations were performed by keeping
the mV change constant (-4 mV). The curve fitting of all the data
was performed using SUPERQUAD80 and all pKa values were
reproducible within 0.5 units.
-
-
-
of PF6 resulting, after 4 d, a small crystal suitable for X-ray
diffraction studies. This complex was subsequently characterised
crystallographically as Li[Zn2(HL1)]4(PO4)2(PF6)3·(CH3OH).
Crystallographic measurements
X-Ray diffraction data for a crystal of Li[Zn2(HL1)]4(PO4)2(PF6)3·
(CH3OH) were collected with a Bruker APEX II diffractometer
˚
with graphite monochromated MoKa (l = 0.71073 A) radiation.
The structure was solved by direct methods using SIR9776 and
refined on F2 using SHELXL-9777 running within the WinGX
interface.78 Plots were drawn using the ORTEP program.79 The
hydrogen positions for the alcohol, methylene and coordinated
water molecules were calculated and included in the final refine-
ment cycle. All non-hydrogen atoms were refined with anisotropic
thermal parameters. Selected crystal data and details of refine-
ments are given in Table 1.
Acknowledgements
Catalytic studies
This work was funded by a grant from the Australian Research
Council (DP0664039).
Catalytic studies on the zinc complexes of H3L1 were performed
with bNPP, which hydrolyses to form 4-nitrophenolate and 4-
nitrophenyl phosphate. The hydrolysis was followed by monitoring
the formation of 4-nitrophenol at 50 ◦C at 400 nm. The extinction
coefficient at 400 nm for 4-nitrophenol is pH dependent and
was experimentally determined for each relevant pH to kinetic
experiments. All studies were performed in 3 mL reaction aliquots
using the substrate bNPP (30 mM stock), buffers (MES, HEPES,
Tris, CHES, 100 mM, aq.), and catalytic species Zn2H2L1 and
Zn(NO3)2 in acetonitrile (all at 1 mM). All experiments were run in
triplicate. The protocol for initialising the hydrolysis reaction was
to add 0.3 mL of an acetonitrile solution of the zinc complex to the
calculated amount of buffer in the test tubes. Where appropriate,
excess Zn(NO3)2 was added to the test tubes. 0.1 mL bNPP was
added, the solution was mixed and the absorbance at 400 nm
measured. The reaction vessels were placed in a 50 ◦C water bath
and the absorbance recorded at specified time intervals. The zinc
dependence of bNPP hydrolysis by the Zn2H2L1 was investigated
by performing kinetic experiments at varying molar equivalents of
Zn(NO3)2 added to the reaction mixture. Control experiments were
also run in identical environments in the presence of Zn(NO3)2
and absence of complex. The final concentrations in 3 mL were as
described above and the final concentrations of added zinc were
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