The role of Zn-OR and Zn-OH nucleophiles and the influence of para-substituents in the reactions of binuclear phosphatase mimetics

Article abstract of DOI:10.1039/c1dt11187f

Analogues of the ligand 2,2′-(2-hydroxy-5-methyl-1,3-phenylene) bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethanol (CH₃H ₃L1) are described. Complexation of these analogues, 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)m

Full text of DOI:10.1039/c1dt11187f

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PAPER
The role of Zn–OR and Zn–OH nucleophiles and the influence of
para-substituents in the reactions of binuclear phosphatase mimetics†
Lena J. Daumann,a Kristian E. Dalle,a Gerhard Schenk,a Ross P. McGeary,a Paul V. Bernhardt,a
David L. Ollisb and Lawrence R. Gahan*a
Received 23rd June 2011, Accepted 4th October 2011
DOI: 10.1039/c1dt11187f
Analogues of the ligand 2,2¢-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-
2-ylmethyl)azanediyl)diethanol (CH3H3L1) are described. Complexation of these analogues,
2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (CH3HL2),
4-bromo-2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (BrHL2),
2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-nitrophenol (NO2HL2) and
4-methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (CH3HL3) with zinc(II)
acetate afforded [Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(NO2L2)(CH3COO)2](PF6),
[Zn2(BrL2)(CH3COO)2](PF6) and [Zn2(CH3L3)(CH3COO)2](PF6), in addition to
[Zn4(CH3L2)2(NO2C6H5OPO3)2(H2O)2](PF6)2 and [Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2. The complexes
were characterized using 1H and 13C NMR spectroscopy, mass spectrometry, microanalysis, and X-ray
crystallography. The complexes contain either a coordinated methyl- (L2 ligands) or phenyl- (L3 ligand)
ether, replacing the potentially nucleophilic coordinated alcohol in the previously reported complex
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6). Functional studies of the zinc complexes with the substrate
bis(2,4-dinitrophenyl) phosphate (BDNPP) showed them to be competent catalysts with, for example,
[Zn2(CH3L2)]+, kcat = 5.70 0.04 ¥ 10-3 s-1 (Km = 20.8 5.0 mM) and [Zn2(CH3L3)]+, kcat = 3.60 0.04 ¥
10-3 s-1 (Km = 18.9 3.5 mM). Catalytically relevant pKas of 6.7 and 7.7 were observed for the zinc(II)
complexes of CH3L2- and CH3L3-, respectively. Electron donating para-substituents enhance the rate
of hydrolysis of BDNPP such that kcat p-CH3 > p-Br > p-NO2. Use of a solvent mixture containing
H2O18/H2O16 in the reaction with BDNPP showed that for [Zn2(CH3L2)(CH3COO)2](PF6) and
[Zn2(NO2L2)(CH3COO)2](PF6), as well as [Zn2(CH3HL1)(CH3COO)(H2O)](PF6), the 18O label was
incorporated in the product of the hydrolysis suggesting that the nucleophile involved in the hydrolysis
reaction was a Zn–OH moiety. The results are discussed with respect to the potential nucleophilic
species (coordinated deprotonated alcohol versus coordinated hydroxide).
combinations such as Zn(II)/Fe(II) likely.7 The enzymes are also
catalytically active as Co(II), Cd(II), Mn(II) or Ni(II) substituted
Introduction
forms.8,9 The active site is typically composed of a nitrogen/oxygen
coordination environment for two divalent metal ions coordinated
by nitrogen donors located on histidine ligands, oxygen donors
typically from an aspartate, and often including a metal ion-
bridging hydroxide ion.10–14
Model complexes capable of reproducing the electronic, struc-
tural and reactivity characteristics of OP-hydrolyzing metalloen-
zyme systems generally exhibit less substrate specificity and
are often more robust than the metalloenzymes themselves.15–18
The question of the nucleophilic agent in these models, and
often with the enzymes themselves, requires examination. In
most hydrolytic enzymes a metal-hydroxide is suggested as the
nucleophile in the key hydrolysis step, although exogenous alcohol
moieties are also implicated.19,20 Examples of model systems with
either or both a metal-OH and a metal-OR present, which are
Organophosphorous compounds (OPs) are amongst the most
commonly used pesticides.1 They act by inhibiting acetyl-
cholinesterase thus preventing proper functioning of nerve cells.
This property has resulted in OPs such as sarin, soman and VX
finding application as nerve agents.1–3 OP-hydrolyzing enzymes
are common2–5 and were initially thought to natively contain
a single Zn(II) in the active site,6 but more recent analysis by
atomic absorption spectroscopy and X-ray anomalous scattering
suggested that the active form of these enzymes is binuclear with
aSchool of Chemistry and Molecular Biosciences, The University of Queens-
land, Brisbane, QLD 4072, Australia. E-mail: gahan@uq.edu.au
bResearch School of Chemistry, Australian National University, Canberra,
ACT 0200, Australia
† CCDC reference numbers 830841–830846. For crystallographic data in
CIF or other electronic format see DOI: 10.1039/c1dt11187f
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implicated in the mechanism, have been reported.21–24 Interestingly,
a coordinated alcohol is a stronger nucleophile than a coordinated
hydroxide21,23–28 and there is evidence that the coordinated alcohol
is deprotonated at or below the same pH as a coordinated water
molecule, particularly in the case of zinc(II) complexes.22
methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-nitrophenol
(NO2HL2) are analogues of CH3HL2 with bromo- and nitro-
substituents in the para-positions of the phenoxide, respectively.
The syntheses are described in Scheme 1. The di-zinc(II) complexes
of these ligands were prepared after reaction with zinc acetate
and X-ray quality crystals were produced either on standing or
by recrystallization.
The nomenclature employed for the ligands denotes the number
of labile protons upon complexation and the substituent on the
para-position of the phenoxide. Hence NO2HL2 and BrHL2 are
derivatives of the methyl ether parent (CH3HL2) and have one
potential site for deprotonation, the phenolic OH; complexation
as NO2L2- and BrL2- implies a single deprotonation. Thus,
the previously reported complex with CH3H3L1, with a methyl
substituent in the para- position and with potential sites for de-
protonation being the phenol and the two pendant alcohol donors,
was characterized as [Zn2(CH3HL1)(CH3COO)(H2O)](PF6) in-
dicating complexation as CH3HL12- with deprotonation at the
phenol and one alcohol.24 The ligand CH3HL3 has a methyl
substituent, one site for deprotonation and phenyl-ether pendants.
Previous studies with the ligand CH3H3L1 have employed different
nomenclature (L);24 the current nomenclature attempts to make
more ready comparison between the various ligands reported
herein. Ligands employed in this work are shown in Chart 1.
In this work we have explored the role of the coordinated alcohol
and a coordinated water molecule using zinc(II) complexes of
the previously described ligand 2,2¢-(2-hydroxy-5-methyl-
1,3-phenylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)-
diethanol (CH3H3L1),24 and analogues of this ligand, 2,6-bis-
(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methyl-
phenol (CH3HL2) and 4-methyl-2,6-bis(((2-phenoxyethyl)-
(pyridin-2-ylmethyl)amino)methyl)phenol (CH3HL3). In addi-
tion, the chemistry of the substituted zinc(II) complexes
of 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-
4-nitrophenol (NO2HL2) and 4-bromo-2,6-bis(((2-methoxyethyl)-
(pyridin-2-ylmethyl)amino)methyl)phenol (BrHL2) has been
studied (Chart 1). The catalytic potential of these complexes
has been explored using the phosphodiester substrate bis(2,4-
dinitrophenyl) phosphate (BDNPP).
Solid state structures
The homologous series of binuclear complexes [Zn2(CH3L2)-
(CH3COO)2](PF6),
[Zn2(CH3L3)(CH3COO)2](PF6)·CH3OH,
[Zn2(BrL2)(CH3COO)2](PF6) and [Zn2(NO2L2)(CH3COO)2]-
(PF6) were isolated and characterized structurally. In all cases
the structures comprised the ligand monoanion, two zinc(II)
ions and two bridging acetates with a hexafluorophosphate
anion. The four complexes adopt the anti-configuration with
respect to the plane of the phenoxide ring,29 as does the
complex [Zn2(CH3HL1)(CH3COO)(H2O)](PF6).24 For the
complex with CH3L3 a methanol solvate was present. In
Chart 1 Ligands discussed in this work.
-
some instances the PF6 was resolved into two disordered
Results and discussion
octahedral anions sharing the same central atom. Selected
crystallographic data are shown in Table 1 and selected bond
lengths and angles are displayed in Table 2. ORTEP plots of
the complex cations are shown in Fig. 1.30 In all cases the
complexes have the phenolate bridging the two zinc(II) centers
which are six-coordinate. For [Zn2(CH3L2)(CH3COO)2](PF6),
Syntheses and nomenclature of ligands and complexes
The previously reported ligand 2,2¢-(2-hydroxy-5-methyl-1,3-phe-
nylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethan-
ol (CH3H3L1),24 in addition to 2,6-bis(((2-methoxyethyl)-
(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (CH3HL2)
[Zn2(BrL2)(CH3COO)2](PF6),
and [Zn2(CH3L3)(CH3COO)2](PF6)
environment of both metal ions is composed of the tertiary
[Zn2(NO2L2)(CH3COO)2](PF6)
and
amino)methyl)phenol (CH3HL3) have been synthesized through
substitution reaction between 2,6-bis(chloromethyl)-4-
4-methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)-
the coordination
˚
a
amine (Zn(1)–N(3): 2.184(3), 2.196(4), 2.193(3), 2.200(4) A;
˚
Zn(2)–N(1): 2.188(3), 2.206(4), 2.197(3), 2.187(4) A), the pyridine
methylphenol and 2-methoxy-N-(pyridin-2-ylmethyl)ethanamine
and 2-phenoxy-N-(pyridin-2-ylmethyl)ethanamine, respectively.
The 2-pyridylmethyl-2-hydroxyethylamine derivatives were
prepared by condensation of 2-methoxyethanolamine and
˚
nitrogen (Zn(1)–N(4): 2.123(3), 2.114(4), 2.134(4), 2.108(4) A;
˚
Zn(2)–N(2): 2.148(3), 2.127(4), 2.129(4), 2.123(4) A), and the
˚
ether oxygens (Zn(1)–O(5): 2.397(3), 2.433(4), 2.380(4), 2.807 A;
˚
2-phenoxyethanolamine
at room temperature, followed by reduction with sodium
borohydride. These ligands offer direct comparison of
with
pyridine-2-carboxaldehyde
Zn(2)–O(4): 2.467(3), 2.368(4), 2.323(3), 2.564 A), respectively, in
addition to the bridging acetates. The six-coordinate geometry
is completed by the bridging oxygen from the phenolate (Zn(1)–
a
˚
methyl and phenyl ether donors with the alkoxide donor
O(1): 2.006(2), 2.007(3), 2.043(3), 2.003(3) A; Zn(2)–O(1):
in
The
the
complex
ligands,
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6).24
2.024(3), 2.020(3), 2.041(3), 2.024(3) A) with Zn(1)–O(1)–Zn(2)
˚
4-bromo-2,6-bis(((2-methoxyethyl)(pyridin-
109.70(11)◦, 110.21(14)◦, 109.73(13)◦ and 107.49(16)◦ and Zn(1)–
˚
2-ylmethyl)amino)methyl)phenol (BrHL2) and 2,6-bis(((2-
Zn(2) distances of 3.296, 3.304, 3.341 and 3.247 A, respectively.
1696 | Dalton Trans., 2012, 41, 1695–1708
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Scheme 1 Synthesis of (a) and (b) 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol (CH3HL2) and 4-methyl-2,6-bis(((2-
phenoxyethyl)(pyridin-2-ylmethyl)amino)methyl)phenol (CH3HL3); (c) bis(bromomethyl)-4-nitrophenol; (d) 2,6-bis(((2-methoxyethyl)(pyridin-2-ylme-
thyl)amino)methyl)-4-nitrophenol (NO2HL2); (e) 4-bromo-2,6-bis(bromomethyl)phenol; and, (f) 4-bromo-2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)-
amino)methyl)phenol (BrHL2).
The Zn–O (phenolate) and Zn–N distances appear typical of those
reported in this type of complex. The Zn–O (ether) distances are at
the long end of the range seen previously for the Zn(II) complexes
has been reported and in that case the two alkoxide arms are
protonated, with the two nickel(II) sites bridged by two acetato
ligands.34
of
2,6-bis(bis(2-methoxyethyl)aminomethyl)-4-methylphenol
X-ray structural data were obtained for two other complexes,
(Hbomp; [Zn2(bomp)(CH3COO)2]BPh4; 2.214(5), 2.197(3),
bearing
tetranuclear
(dimer
of
dimers)
structures,
31
˚
2.362(4), 2.440(3) A),
and 1,2-dimethoxyethane (glyme)
[Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2 and [Zn4(CH3L2)2(NO2C6H5-
OPO3)2 (H2O)2](PF6)2. For [Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2
each dimer is composed of a [Zn2(BrL2)]+ fragment, the metal
˚
([Zn2Cl2(glyme)], 2.1286(11), 2.1216(10) A; [Zn2I2(glyme)],
32,33
˚
2.067(7) A)
and are significantly longer than those
observed for the deprotonated alkoxide (Zn–O-) distances
ions in each fragment Zn(1)/Zn(2) and Zn(3)/Zn(4) separated by
2-
˚
˚
˚
˚
(1.906(3) A) and coordinated water (2.074(3) A) reported
for [Zn2(CH3HL1)(CH3COO)(H2O)](PF6).24 The analogous
nickel(II) complex [Ni2(CH3H2L1)(CH3COO)2](PF6)·0.5H2O
3.724 A and 3.572 A, respectively. The two PO3F anions display
3
a m3h coordination mode35 and complete the coordination sphere
for Zn(3) and Zn(4) such that two of the oxygen donors of
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Table 2 Selected bond lengths (A) and angles (◦) for [Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(CH3L3)(CH3COO)2](PF6)·CH3OH and
[Zn2(NO2L2)(CH3COO)2](PF6), [Zn2(BrL2)(CH3COO)2](PF6)
˚
[Zn2(CH3L2)-
(CH3COO)2](PF6)
[Zn2(CH3L3)(CH3COO)2]-
(PF6)·CH3OH
[Zn2(NO2L2)-
(CH3COO)2](PF6)
[Zn2(BrL2)-
(CH3COO)2](PF6)
Zn(1)–O(1)
Zn(1)–O(2)
Zn(1)–O(5)
Zn(1)–O(6)
Zn(1)–N(3)
Zn(1)–N(4)
Zn(2)–O(1)
Zn(2)–O(3)
Zn(2)–O(4)
Zn(2)–O(7)
Zn(2)–N(1)
Zn(2)–N(2)
Zn(1) . . . Zn(2)
2.006(2)
2.049(3)
2.397(3)
1.997(3)
2.184(3)
2.123(3)
2.024(3)
1.992(2)
2.467(3)
2.076(3)
2.188(3)
2.148(3)
3.296
2.003(3)
1.979(3)
2.043(3)
1.992(3)
2.380(4)
2.055(3)
2.193(3)
2.134(4)
2.041(3)
2.076(3)
2.323(3)
1.997(3)
2.197(3)
2.129(4)
3.341
2.007(3)
2.000(4)
2.433(4)
2.067(3)
2.196(4)
2.114(4)
2.020(3)
2.068(4)
2.368(4)
1.991(3)
2.206(4)
2.127(4)
3.304
2.044(3)
2.200(4)
2.108(4)
2.024(3)
2.021(3)
1.982(4)
2.187(4)
2.123(4)
3.247
O(1)–Zn(1)–O(2)
O(1)–Zn(1)–O(5)
O(1)–Zn(1)–O(6)
O(1)–Zn(1)–N(3)
O(1)–Zn(1)–N(4)
N(3)–Zn(1)–N(4)
O(1)–Zn(2)–O(3)
O(1)–Zn(2)–O(4)
O(1)–Zn(2)–O(7)
O(1)–Zn(2)–N(1)
O(1)–Zn(2)–N(2)
N(1)–Zn(2)–N(2)
Zn(1)–O(1)–Zn(2)
91.69(11)
89.46(10)
99.21(11)
91.24(11)
167.50(13)
77.71(13)
100.72(11)
88.43(10)
92.69(11)
88.93(10)
167.01(11)
78.08(12)
109.70(11)
95.65(14)
98.64(13)
91.72(13)
91.30(13)
89.11(11)
167.04(13)
78.24(13)
92.03(13)
93.56(12)
98.56(12)
88.77(11)
166.63(13)
77.88(14)
109.73(13)
97.86(14)
91.14(15)
92.35(14)
89.66(13)
167.80(16)
78.25(15)
91.79(14)
95.33(14)
99.09(13)
89.33(13)
166.91(15)
77.66(16)
110.21(14)
95.01(13)
89.34(16)
167.69(17)
78.48(19)
93.12(12)
97.80(14)
89.24(15)
167.12(17)
78.02(18)
107.49(16)
˚
˚
˚
the anion are bidentate (Zn(3)–O(3B) 2.099(4) A, Zn(3)–O(6B)
O(3A), 2.080(3) A and Zn(2)–O(7A) 2.128(3) A. Other relevant
bond distances and angles for [Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2
and [Zn4(CH3L2)2(NO2C6H5OPO3)2(H2O)2](PF6)2 are reported in
Table 3. The bridged structure of the phosphate esters is typical
of other similar reported structures of di-zinc(II) complexes
with bridging phosphate esters, dibenzyl phosphate,40 diphenyl
phosphate41 and bis(4-nitrophenyl)phosphate.42 In terms of the
relevance to the catalytic studies (vide infra) the structure of
[Zn4(CH3L2)2(NO2C6H5OPO3)2(H2O)2](PF6)2, in line with previ-
ous studies,41 suggests that in these di-zinc(II) complexes bidentate
coordination of substrate occurs.
˚
˚
˚
1.962(4) A, Zn(4)–O(2B) 1.999(4) A and Zn(4)–O(7B) 2.078(5) A),
with the remaining oxygen donors coordinated monodentately to
the other zinc(II) pair (Zn(1)–O(3A) 2.015(4) A and Zn(2)–O(7A)
2.024(4) A). The coordination spheres of Zn(1) and Zn(2) are
completed by a terminal water molecule coordinated to each
metal atom, Zn(2)–O(2A) 2.083(4) A and Zn(1)–O(6A) 2.086(4)
A. The assignment of the PO3F2- is based on the overall charge of
the molecule, the P–F bond versus P–O bond distances (P–O(av.)
˚
˚
˚
˚
˚
˚
1.506(4) A; P–F(av.) 1.577(4) A) and comparison with previous
examples (P–F(av.) 1.568(2) A).36–38 The PO3F2- arose presumably
˚
from hydrolysis of the hexafluorophosphate on standing in
aqueous solution. The presence of apparent hydrolysis products
of NaPF6 is not without precedent as in a previous study we
characterized a zinc(II) complex with the PO4 anion arising as
an impurity in the NaPF6 sample.39
Mass spectrometry
3-
Mass spectral analysis, determined in both methanol and
H2O : MeCN (1 : 1), shows that the complexes exist as dinu-
clear zinc species with the isotopic patterns for Zn(II)2 species
being distinctly different from that observed for a mono-
zinc species.24 In methanol the complex with CH3HL2 shows
acetate present ([Zn(CH3L2)(CH3COO)2]+, exp m/z 711.20; calc.
m/z 711.15 (99.9%), 713.15 (100%)). Loss of acetate is con-
sidered a prerequisite for formation of the catalytically rele-
vant species in solution.43–46 Under the conditions employed for
the catalytic studies (H2O : MeCN, 1 : 1) and in the presence
of the slow substrate PNPP (the latter a hydrolysis product
of BPNPP) the mass spectrum showed the presence of a
tetrameric species [Zn4(CH3L2)2(PNPP)2(OH)(H2O)]+ (exp m/z
1659.14; calc. m/z 1657.22 (100%), 1659.22 (87%)), a dimeric
species [Zn2(CH3L2)(PNPP)2+2H]+ (exp m/z 1031.08; calc. m/z
1031.10 (100%)) as well as [Zn2(CH3L2)(PNPP)]+ (exp m/z 811.4;
calc. m/z 810.1 (97%), m/z 812.1 (100%)). Species such as
The crystallization of the [Zn4(CH3L2)2(NO2C6H5OPO3)2-
(H2O)2](PF6)2 complex arose from a deliberate attempt to probe
the structure of a substrate-bound complex using BPNPP as
a slow substrate. The resulting complex contained PNPP, a
hydrolysis product of the initial substrate. Each dimer is com-
posed of a [Zn2(CH3L2)]+ fragment, the metal ion separation
˚
˚
Zn(1)/Zn(2) and Zn(3)/Zn(4) being 3.747 A and 3.521 A,
-
3
respectively. Again, the NO2C6H5OPO3 moiety has a m3h
coordination mode35 and completes the coordination spheres
with bidentate coordination to one pair of Zn(II) ions (Zn(3)–
˚
˚
O(3B) 2.082(3) A, Zn(4)–O(2B) 1.993(3) A and Zn(3)–O(6B)
˚
˚
1.982(4) A and Zn(4)–O(7B) 2.065(4) A) and monodentate
˚
coordination to the other zinc(II) pair (Zn(1)–O(6A) 2.001(4) A
˚
andZn(2)–O(2A) 2.011(4) A). The coordination spheres of Zn(1A)
and Zn(2A) are completed by terminal aqua ligands, Zn(1)–
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Fig. 1 ORTEP plots of [Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(CH3L3)(CH3COO)2](PF6)·CH3OH, [Zn2(NO2L2)(CH3COO)2](PF6), [Zn2(BrL2)(CH3-
COO)2](PF6), [Zn4(CH3L2)2(NO2C6H5OPO3)2 (H2O)2](PF6)2 and [Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2 with 25% probability level of thermal ellipsoids.
Hydrogen atoms, counter ions and non-coordinated solvent molecules have been omitted for clarity, as have carbon atom labels.
[Zn2(CH3L2)(HCOO)-H]+ (exp m/z 639.4; calc. m/z 637.1 (98%),
m/z 639.1 (100%)) and [Zn2(CH3L2)(HCOO)2]+ (exp m/z 684.4;
calc. m/z 683.1 (98%), m/z 685.1 (100%)) are also present. The for-
mate ion is commonly found in the mass spectra of these types of
complexes39,47 arising either from fragmentation of the acetate ion
or adventitiously from the instrument. In the presence of BPNPP,
again in a mixed H2O : MeCN (1 : 1) solvent system, the mass spec-
trum shows the presence of [Zn2(CH3L2)(BPNPP)(H2O)(OCH3)]+
(exp m/z 979.1; calc. m/z 981.1 (96%), m/z 983.1 (100%)) as well
as evidence of BPNPP hydrolysis products [Zn2(CH3L2)(PNPP)]+
(exp m/z 811.4; calc. m/z 810.1 (97%), m/z 812.1 (100%)) sug-
gesting that under the conditions of the experiment the complex
is able to hydrolyze BPNPP. In addition, in each case under the
conditions of the mass spectral measurements, the predominant
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◦
˚
Table 3 Selected bond lengths (A) and angles ( ) for [Zn4(BrL2)2-
(PO3F)2(H2O)2](PF6)2 and [Zn4(CH3L2)2(NO2C6H5OPO3)2(H2O)2](PF6)2
[Zn4(BrL2)2(PO3F)2- [Zn4(CH3L2)2(NO2C6H5-
(H2O)2](PF6)2
OPO3)2(H2O)2](PF6)2
Zn(1)–O(6A)
Zn(1)–O(1A)
Zn(1)–O(3A)
Zn(1)–N(4A)
Zn(1)–N(3A)
Zn(2)–O(2A)
Zn(2)–O(1A)
Zn(2)–O(7A)
Zn(2)–N(2A)
Zn(2)–N(1A)
Zn(2)–O(4A)
Zn(3)–O(6B)
Zn(3)–O(1B)
Zn(3)–O(3B)
Zn(3)–N(3B)
Zn(3)–N(4B)
Zn(3)–O(5B)
Zn(4)–O(2B)
Zn(4)–O(1B)
Zn(4)–O(7B)
Zn(4)–N(2B)
Zn(4)–N(1B)
Zn(4)–O(4B)
O(1A)–Zn(1)–N(4A) 108.47(17)
O(1A)–Zn(1)–N(3A) 93.55(17)
N(4A)–Zn(1)–N(3A) 79.95(19)
N(2A)–Zn(2)–N(1A) 80.38(17)
N(2A)–Zn(2)–O(1A) 87.13(16)
N(1A)–Zn(2)–O(1A) 92.02(17)
O(1B)–Zn(3)–N(3B) 90.11(18)
O(1B)–Zn(3)–N(4B) 164.7(2)
N(3B)–Zn(3)–N(4B) 75.6(2)
O(1B)–Zn(4)–N(2B) 166.47(18)
O(1B)–Zn(4)–N(1B) 90.36(17)
N(2B)–Zn(4)–N(1B) 76.8(2)
Zn(1)–O(1A)–Zn(2) 125.56(19)
2.086(4)
2.028(4)
2.015(4)
2.118(5)
2.164(5)
2.083(4)
2.159(4)
2.024(4)
2.114(4)
2.156(4)
2.296(4)
1.962(4)
2.057(4)
2.099(4)
2.165(6)
2.173(5)
2.269(4)
1.999(4)
2.054(4)
2.078(5)
2.159(6)
2.182(5)
2.285(5)
2.001(4)
2.065(4)
2.080(3)
2.111(4)
2.154(4)
2.011(4)
2.096(3)
2.128(3)
2.129(5)
2.164(5)
2.374(4)
1.982(4)
2.050(4)
2.082(3)
2.170(5)
2.184(6)
2.262(4)
1.993(3)
2.013(4)
2.065(4)
2.126(6)
2.202(5)
2.389(4)
93.61(16)
93.81(18)
80.01(18)
80.2(2)
Fig. 2 1H NMR spectra of the aromatic region for [Zn2(CH3L2)(CH3-
COO)2](PF6) in CD3CN at four different temperatures.
observation was made for the complex with the CH3HL3 ligand
although in this case the higher field signals were more intense at
room temperature, again gaining intensity upon increasing the
temperature of the sample. The second set of resonances for
both complexes could thus be assigned to either (i) a reversible
interchange of the two geometric forms, either syn- or anti- with
respect to the plane of the aromatic ring, or (ii) to a species
with only partially coordinated ether (–OCH3 or –OPh) arms in
solution. The interchange of syn- and anti-forms would involve
a process necessitating inversion at the tertiary nitrogen donor.
On replacing the CD3CN solvent with (CD3)2SO the apparent
equilibrium position in solution was shifted, with the higher field
resonances becoming relatively more intense. In terms of the donor
number, DMSO would be expected to coordinate more strongly
than acetonitrile.48–51 Consideration of the effect of temperature
and solvent coupled with the pronounced effect on the 1H NMR
spectrum of replacing methyl- with phenyl-ether donors suggests
what is occurring in solution is more likely to be an effect of the
loosely bound ether pendants, in line with the solid state structures
of these complexes which display long zinc(II)–ether interactions.
Interestingly, for [Zn2(CH3HL1)(CH3COO)(H2O)](PF6), with Zn–
O- and Zn–OH donors, and crystallized as the anti-form, only one
set of resonance is reported in the 1H NMR spectrum.24
90.71(16)
93.23(17)
89.64(19)
165.5(2)
77.3(2)
167.4(2)
89.8(2)
77.6(2)
128.4(2)
120.1(2)
Zn(4)–O(1B)–Zn(3)
120.67(19)
species is the [Zn2(CH3L2)]n+ complex and substrate, the acetate
Phosphodiesterase-like activity
anions not being present.
Phosphatase-like activities of the complexes were measured at
various pH values ranging from 5 to 10.5 using the activated
substrate BDNPP. The data were consistent with the presence
of either one or two protonation equilibria and were thus fit to an
equation derived for a monoprotic (eqn (1)) or diprotic (eqn (2))
system.52
NMR solution studies
When one equivalent of zinc(II) acetate was added to a CD3CN
solution of the ligand CH3HL2 the resonances in the aromatic
region of the 1H NMR spectrum exhibited a downfield shift.
The spectrum showed the presence of free ligand and signals
identical to the spectrum when a second equivalent of zinc(II)
acetate was added. Thus the spectrum with one equivalent zinc(II)
added showed that only free ligand and ligand with two zinc ions
coordinated are present, suggesting that the acetates support a
cooperative binding of two zinc ions simultaneously to the ligand.
In the NMR spectrum with two equivalents of zinc a second set of
low intensity signals, but at higher field, was detected (also present
in the spectrum with one equivalent but partially overlapping
with free ligand signals) indicative of a minor, but different,
isomeric form. To investigate possible temperature effects, the
NMR spectrum of the complex was recorded at four different
temperatures (298, 316, 333 and 343 K; Fig. 2)). The intensities
of the resonances were temperature-dependent; at higher temper-
ature the higher field less intense signals gained intensity. The
process was reversible upon cooling of the sample. The same
V
max
V0 =
[H+ ]
Kes
(1)
1+
V
max
V0 =
[H+ ]
Kes
2
[H+ ]
(2)
1+
+
Kes
1
Here, V0 is the initial reaction rate, Vmax is the maximum rate
with BDNPP as substrate (5 mM), and Kes (= Ka) is the protonation
equilibrium constant relevant to catalysis. The resulting fits and
catalytic parameters are shown in Fig. 3 and Table 4, respectively.
Most notable were the kinetically relevant pKas for the complexes
with CH3HL12-, CH3L2- and CH3L3- (6.6; 6.7, 11.0; 7.7, 10.8;
respectively). The substrate concentration dependence of catalysis
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Table 4 Kinetic data for [Zn2(CH3HL1)(CH3COO)2](PF6), [Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(CH3L3)(CH3COO)2](PF6), [Zn2(NO2L2)(CH3COO)2]-
(PF6) and [Zn2(BrL2) (CH3COO)2](PF6)
Complex
kcat (s-1)
Km (mM)
pKa
[Zn2(CH3HL1)(CH3COO)2](PF6)
[Zn2(CH3L2)(CH3COO)2](PF6)
1.07 0.04 ¥ 10-3
5.70 0.04 ¥ 10-3
12.5 7.0
20.8 5.0
6.6
6.7
11.0
7.7
10.8
6.5
[Zn2(CH3L3)(CH3COO)2](PF6)
3.60 0.04 ¥ 10-3
18.9 3.5
[Zn2(NO2L2)(CH3COO)2](PF6)
[Zn2(BrL2)(CH3COO)2](PF6)
0.76 0.04 ¥ 10-3
1.90 0.04 ¥ 10-3
6.5 1.4
7.1 4.0
6.5
10.6
Fig. 4 Substrate concentration dependence for [Zn2(HL1)(CH3COO)-
Fig.
3
pH dependence of rate of BDNPP cleavage by
[Zn2(CH3L2)(CH3COO)2]-
(H2O)](PF6) ( ) [Zn2(CH3L2)(CH3COO)2](PF6) (ꢀ) and [Zn2(CH3L3)-
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6)
(
)
᭺
᭺
(CH3COO)2](PF6) (᭛); at 25 ◦C; aqueous multi-component buffer
(50 mM each of MES, HEPES and CHES), ionic strength controlled
by 250 mM LiClO4; 50 : 50 MeCN : buffer; 40 mM complex.
(PF6) (ꢀ) and [Zn2(CH3L3)(CH3COO)2](PF6) (᭛); 25 ◦C; aqueous
multi-component buffer (50 mM each of MES, HEPES and CHES), ionic
strength controlled by 250 mM LiClO4; 50 : 50 MeCN : buffer; 40 mM
complex; 5 mM BDNPP.
was also measured at pH 10.5 for the CH3HL12- and pH 8
for the CH3L1- and CH3L2- complexes. Michaelis–Menten type
behaviour was observed in each case (Fig. 4) and the data were fit
for each complex using non-linear least squares analysis (eqn (3))
to give the Michaelis constant, Vmax and kcat (Vmax/[complex]) for
each complex.52
V
max[S]
V0 =
(3)
Km +[S]
Here, Km is the Michaelis constant and [S] the substrate
concentration. Relevant kinetic data (kcat, Km, pKa) are reported
in Table 4. For all complexes the rate of hydrolysis of BDNPP
(5 mM) was linear for complex concentrations from 20–160 mM.
The [substrate] and [complex] dependence studies for the di-Zn(II)
complexes of CH3HL2 and CH3HL3 were carried out at pH 8.5,
for NO2HL2 pH 9.5 and BrHL2 pH 8.8.
Fig. 5 Linear correlation for the Hammett parameter (s) and log10(kcat
)
for [Zn2(CH3L2)(CH3COO)2](PF6), Zn2(NO2L2)(CH3COO)2](PF6) and
Inductive effects
[Zn2(BrL2)(CH3COO)2](PF6).
The linear plot of log10(kcat) versus s, the Hammett parameter,53
for the three complexes [Zn2(CH3L2)(CH3COO)2](PF6)·CH3OH,
[Zn2(NO2L2)(CH3COO)2](PF6) and [Zn2(BrL2)(CH3COO)2]-
(PF6) (Fig. 5) suggests that the substituent in the para-position
influences the rate of the hydrolysis reaction with the substrate
BDNPP. The sequence kcat p-CH3 > p-Br > p-NO2 is in accord
with a previous example, [Fe(III)Zn(II)(L1)(OAc)2]+ (H2L1 =
2--(((3-((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-me-
thylbenzyl)(pyridin-2-ylmethyl)amino)-methyl)phenol), where the
1702 | Dalton Trans., 2012, 41, 1695–1708
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same sequence of reactivity for the substrate BDNPP
was observed.54 The linear relationship between
and
In contrast to [Zn2(CH3HL1)]2+, [Zn2(CH3L2)(CH3-
COO)2](PF6) and [Zn2(CH3L3)(CH3COO)2](PF6) have only
one kinetically relevant pKa (6.76 and 7.72, respectively), and
the absence of the metal-bound alcohol allow the assignment of
this pKa to a zinc(II)-bound water molecule. Thus, this terminal
hydroxide is the likely nucleophile in the reaction catalyzed
by these two complexes. In order to explore the mechanism
employed by these three systems further the incorporation of
18O into the reaction product was monitored by NMR. Previous
studies have shown that incorporation of 18O (as Zn–18OH) into
a phosphate ester hydrolysis product results in a ~0.02 ppm shift
in the 31P NMR signal as a result of 18O incorporation into the
product.62–64 50% incorporation of the 18O would be expected to
result in a splitting of the original 31P resonance attributed to the
hydrolyzed substrate. Therefore a solvent mixture of H2O16/H2O18
(50 vol%) in acetonitrile was employed in the reaction of
[Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(NO2L2)(CH3COO)2](PF6)
and [Zn2(CH3HL1)(CH3COO)(H2O)](PF6) with BDNPP.
In the absence of the label, no splitting in the 31P NMR
signal of the coordinated product of the reaction was
observed in any sample. For [Zn2(CH3L2)(CH3COO)2](PF6)
and [Zn2(NO2L2)(CH3COO)2](PF6) in the presence of the
H2O16/H2O18 mixture the 31P NMR of the products showed a
~0.02 ppm splitting in resonances arising at -2.70 and -3.28
ppm, respectively. For [Zn2(CH3HL1)(CH3COO)(H2O)](PF6)
the 31P NMR spectrum again showed a ~0.02 ppm splitting,
the resonance arising at -10.9 ppm. The incorporation of
the 18O label in the product of the reaction suggests that
for all three complexes the Zn–OH moiety has a significant
nucleophilic role,64 but the different chemical shifts observed
for L1 and the two L2 systems suggest that the substrate in
[Zn2(CH3HL1)]2+ may be coordinated differently from the other
systems.
s
pKa2, the catalytically relevant dissociation constant, for
[Fe(III)Zn(II)(L1)(OAc)2]+ suggested that the para-substituent in-
fluenced the acidity of the Fe(III)-OH2 group.54 The small range
of pKa values, and inherent errors, and the lower charge for
di-Zn(II) complexes makes a similar analysis more problematic.
What is clear is that in the complexes based on L2 the hydrolase
activity towards BDNPP is enhanced by the introduction of an
electron donating substituent. There is no clear relationship for
[Fe(III)Zn(II)(L1)(OAc)2]+ nor the di-Zn(II) complexes between the
Hammett parameter, s, and Km suggesting that the effect of the
substituent is primarily on the nucleophile and not on the binding
of the substrate to the metal centre. For the di-Zn(II) complexes the
influence of the para-substituent accounts for less than an order
of magnitude change in kcat but this effect in combination with
previous studies which showed that second coordination sphere
hydrogen bonding effects can also improve the efficiency of model
systems55,56 suggests that an approach combining both influences
may be effective.
Interestingly, for the complex [Zn2(bomp)(CH3COO)2]BPh4
(bomp = 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphe-
nol), an aminopeptidase mimic, when the para-methyl group was
replaced by p-Cl and p-NO2 and the aminopeptidase activity stud-
ied using the substrate L-leucine-p-nitroanilide, the NO2 complex
was found to be the most active, the reactivity sequence being
NO2 > Cl > CH3.57 The difference between the two mechanisms
is that the hydrolase mimics suggest a terminal hydroxide whereas
the aminopeptidase mimic suggests a bridging hydroxide as the
nucleophile.54,57
Mechanism of reaction
The NMR results, however, do not entirely rule out a role for the
alkoxide in reactions of [Zn2(CH3HL1)(CH3COO)(H2O)](PF6).
Pathways involving both Zn–OH and Zn–OR nucleophiles
have been suggested previously.22,60,61 Thus, the di-zinc(II) com-
plex of 4-(2-hydroxyethyl)-1,4,7,16,19,22-hexaaza-10,13,25,28-
tetraoxacyclotriacontane, with both Zn–OR and Zn–OH moieties
present as potential nucleophiles, has been employed to investigate
the hydrolysis of carboxyester 4-nitrophenyl acetate (NA) and the
phosphate ester BPNPP.22 For NA hydrolysis it was suggested
that the Zn-alkoxide acted as a nucleophile to give an acetyl
derivative which was subsequently hydrolyzed to acetate by a
Zn–OH nucleophile, with regeneration of the catalyst.22 For
BPNPP hydrolysis nucleophilic attack of the alkoxide led to a
pendant alcohol phosphorylated intermediate which, after attack
of the Zn–OH nucleophile, led to a phosphomonoester complex.22
It may be that for [Zn2(CH3HL1)(CH3COO)(H2O)](PF6) when
both Zn(II)-alkoxide or Zn(II)–OH are present, similar sequential
involvement of the nucleophiles occurs offering a pathway for
incorporation of the 18O label22 and consequently a pathway
for regeneration of the active form (Fig. 6).19,22 In contrast, the
mechanism employed by the L2 and L3 systems is similar to that
proposed for related binuclear complexes such as the mimics of the
enzyme purple acid phosphatase (PAP).43,45,46,65,66 A nucleophilic
attack by the terminally-bound hydroxide initiates hydrolysis
and exchange of the metal-coordinated PNPP product by water
molecules regenerates the active site for the next catalytic cycle.
Comparison of the methyl and phenyl ether complexes
[Zn2(CH3L2)(CH3COO)2](PF6), [Zn2(CH3L3)(CH3COO)2](PF6)
and the analogue [Zn2(CH3HL1)(CH3COO)(H2O)](PF6) to-
wards the activated substrate BDNPP offers the opportu-
nity to investigate the relative activities of potential nucle-
ophilic zinc(II)-bound pendant alcohol (Zn–OR) or hydroxide
(Zn–OH) groups. Previous potentiometric studies have shown
that [Zn2(CH3HL1)(CH3COO)(H2O)](PF6) displays two pKas
at 7.2 and 8.1.24 Based on a comparison to other Zn(II)
complexes21–25,27,58,59 and theoretical studies,28 pKa = 7.2 was
assigned to the alkoxide group and pKa = 8.1 to the hy-
droxide. For monomeric Zn(II) systems it was shown that the
metal-bound alcohol nucleophile is more reactive than the
metal-bound hydroxide.26–28 Thus, the mechanism proposed for
[Zn2(CH3HL1)]2+ involves a nucleophilic attack by the zinc-
bound deprotonated alcohol group (pKa = 7.2), which leads to
the formation of a “transition complex”.24 Following breakage
of the P–O bond of the substrate BPNPP and release of a
nitrophenolate it remains unclear how the active site is regenerated.
The nucleophilic attack by the alkoxide is expected to result in a
complex with a covalently bound PNPP species, similar to that
suggested for other copper(II) and zinc(II) complexes.22,25,60,61 It
has been proposed that PNPP is replaced by acetate groups to
reform the catalytic site,24 but the precise details for this process
are currently unknown.
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product, that the three systems investigated here may employ
a similar mechanistic strategy with a metal-bound hydroxide as
nucleophile. The previously proposed model for the mechanism
of [Zn2(CH3HL1)]2+ may still be applicable but would likely
require the dual action of both the alkoxide and hydroxide
nucleophiles, similar to the reaction scheme proposed for alkaline
phosphatases.19 In contrast, the remaining complexes in this study
are good functional models for enzyme systems that do not have
covalent reaction intermediates, such as PAPs or OP-degrading
hydrolases.2,3,46
Experimental
Materials and methods
Nuclear Magnetic Resonance (NMR) spectra were measured
with Bruker AV300, AV400 and AV500 instruments. The spectra
were recorded in CDCl3, CD3OD, CD3COCD3, CD3CN or
(CD3)2SO. Chemical shifts were determined in ppm, relative
to known residual solvent peak references. Coupling constants
are given in Hz. Two-dimensional correlation spectroscopy
(COSY), heterobinuclear single quantum correlation (HSQC) and
heterobinuclear multiple bond connectivity (HMBC) experiments
were used to assign each signal in the spectra of the final ligands.
Low resolution mass spectral data were collected with a Bruker
Esquire high capacity ion trap electrospray mass spectrometer
(HCT ESI-MS), in methanol (MeOH) or acetonitrile (MeCN) or
50 : 50 MeCN : water, with a Bruker ES source. High resolution
mass spectra were obtained using a Bruker microTOFQ ESI-MS
in MeOH. The predicted isotopic splitting patterns of peaks were
calculated using the program Molecular Weight Calculator.67 Ele-
mental analyses were performed using the microanalysis facilities
at The University of Queensland.
Syntheses of ligands and complexes
2,6-Bis(chloromethyl)-4-methylphenol,68 2-(pyridyl-2-ylmethyl-
amino)ethanol,69 2,6-bis(bromomethyl)-4-nitrophenol,70 2,2¢-(2-
hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((pyridin-2-yl-
methyl)azanediyl)diethanol (CH3H3L1),24 and [Zn2(CH3HL1)-
(CH3COO)(H2O)](PF6) were prepared as described previously.24
Fig. 6 Key steps in the mechanism, similar to that proposed for the
alkoxide assisted hydrolysis mechanisms for the dinuclear zinc alka-
line phosphatase enzyme where both the nucleophilic RO–Zn(II) and
OH–Zn(II) are involved in the hydrolytic process.19
Synthesis of 2-methoxy-N-(pyridin-2-ylmethyl)ethanamine.
Pyridine 2-carboxaldehyde (2.25 g, 0.02 mol) in methanol (5 mL)
was added dropwise to 2-methoxyethanolamine (1.58 g, 0.02
◦
mol) in methanol (10 mL) at 0 C. The solution was brought to
room temperature and stirred overnight. Sodium borohydride
(0.83 g, 0.02 mol) was added in small portions at 0 ◦C and the
solution was subsequently stirred for a further 2 h. Water (30 mL)
was added and the reaction mixture was concentrated to 30 mL
in vacuo. The solution was extracted with dichloromethane (3 ¥
20 mL) and the combined organic extracts were washed three
times with brine, the solution was dried over Na2SO4 and the
solvent was removed under vacuum. The desired product was
obtained as a yellow oil in 90.6% yield (2.85 g). 1H NMR (CDCl3,
300.13 MHz); d 2.26 (s, 1H, NH); 2.72 (t, 2H, HN–CH2–, J =
5.4 Hz); 3.20 (s, 3H, OCH3); 3.43 (t, 2H, CH2–O, J = 5.4 Hz);
3.79 (s, 2H, HN–CH2–ar); 7.02 (dq, 1H, pyCH, J = 7.5, 4.9 Hz);
7.19 (dt, 1H, pyCH, J = 7.7, 1.0 Hz); 7.48 (td, 1H, pyCH, J
= 7.6, 1.8 Hz); 8.40 (dq, 1H, pyCH, J = 4.9, 1.73, 0.9 Hz). 13C
Conclusion
The di-zinc(II) complexes reported here possess properties anal-
ogous to binuclear metallohydrolases.3,5,65 The results obtained
for the hydrolysis of the activated substrate BDNPP suggest
that complexes with CH3L2- and CH3L3- were more effective
than with CH3HL1, but electron-withdrawing para-substituents
lower the rate of hydrolysis of BDNPP. Previous studies with
the complex [Zn2(CH3HL1)(CH3COO)(H2O)](PF6)24 and related
complexes with a coordinated alcohol moiety22,60,61 suggested that
the corresponding alkoxide was the nucleophilic agent in the
reaction of these model systems with phosphate esters. The present
study does not rule out this possibility but it indicates, based
on an analysis of the incorporation of 18O into the hydrolysis
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NMR (CDCl3, 100.62 MHz); d 48.6 (HN–CH2); 54.9 (OCH3);
58.8 (HN–CH2–ar); 71.8 (CH2–O); 121.7 (CH); 121.9 (CH);
136.2 (CH); 149.1 (CH); 159.6 (CH). ESI mass spectrometry
(methanol) m/z 167.10 [C9H14N2OH]+. FT-IR spectroscopy (v,
cm-1) 3318.5 (m, N–H str); 2889.1, 2830.3 (m, C–H str); 1592.2
(m, C C str); 1434.7 (m, C–H def); 1356.5 (w, C–H sym. def);
1110.0 (s, C–O–C str); 757.1 (s, py–H).
Ar–H); 755.9 (s, py–H). ESI mass spectrometry (methanol) m/z
487.18 [C27H36N4O3Na]+, 465.22 [C27H36N4O3H]+.
4-Methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)amino)-
methyl)phenol (CH3HL3). 2,6-bis(chloromethyl)-4-methylphe-
nol (0.16 g, 0.55 mmol) in dichloromethane (2 mL) was
added dropwise to
a mixture of 2-phenoxy-N-(pyridin-2-
ylmethyl)ethanamine (0.25 g, 1.09 mmol) and triethylamine (0.30
g) in tetrahydrofuran (2 mL) at 0 ◦C. The resulting yellow solution
was stirred for 48 h at room temperature then filtered to remove the
white precipitate of triethylamine hydrochloride and the solvent
was removed under vacuum. The yellow oil was purified by flash
column chromatography (EtOAc/MeOH, 9 : 1, FeCl3 stain, Rf =
Synthesis of 2-phenoxy-N-(pyridin-2-ylmethyl)ethanamine.
Pyridine 2-carboxaldehyde (0.39 g, 0.004 mol) in methanol (2 mL)
was added dropwise to 2-phenoxyethanolamine (0.5 g, 0.004
mol) in methanol (3 mL) at 0 ◦C. The solution was brought to
room temperature and stirred overnight. Subsequently sodium
borohydride (0.2 g, 0.005 mol) was added in small portions and
the solution was stirred for a further 4 h. Water (6 mL) was added
and the reaction mixture was concentrated to about 6 mL in vacuo.
The remaining solution was extracted with dichloromethane (3
¥ 10 mL) and the combined organic extracts were dried over
Na2SO4 and the solvent was removed under vacuum to yield 0.75
g of a yellow oil in 91.2% yield. 1H NMR (CDCl3, 500.13 MHz);
d 2.14 (s, 1H, NH); 3.04 (t, 2H, HN–CH2, J = 5.3 Hz); 3.98 (s,
2H, HN–CH2–ar); 4.09 (t, 2H, CH2–O, J = 5.3 Hz); 6.89 (m, 2H,
arCH); 6.91 (m, 1H, arCH); 7.13 (m, 1H, pyCH, J = 7.3, 4.9, 1.0
Hz); 7.24 (m, 2H, arCH, J = 7.4 Hz); 7.31 (d, 1H, pyCH, J = 7.8
Hz); 7.61 (td, 1H, pyCH, J = 7.7, 1.8 Hz); 8.54 (dq, 1H, pyCH,
J = 4.9, 1.7, 0.9 Hz). 13C NMR (CDCl3, 100.62 MHz); d 48.4
(HN–CH2); 55.1 (HN–CH2–ar); 67.3 (CH2–O); 114.5 (arCH);
120.7 (arCH); 121.9 (pyCH); 122.1 (pyCH); 129.3 (arCH); 136.4
(pyCH); 124.3 (pyCH); 158.8 (O–C); 159.6 (CH2–pyC). ESI mass
spectrometry (methanol) m/z 229.10 [C14H16N2OH]+. FT-IR
spectroscopy (v, cm-1) 3404.8 (m, N–H str); 2923.8 (m, C–H str);
1593.9 (m, C C str); 1510.1 (s, N–H def); 1448.1 (m, C–H def);
1331.0 (w, C–H sym. def); 1212.3 (s, C–O–C str); 777.0 (w, Ar–H);
745.5 (s, py-H); 692.0 (w, Ar–H).
1
0.75 in EtOAc/MeOH 9 : 1) (0.17 g, 52%). H NMR (CDCl3,
500.13 MHz); d 2.26 (s, 3H, CH3); 3.03 (t, 4H, CH2O, J = 5.8
Hz); 3.88 (s, 4H, arCH2N); 3.98 (s, 4H, NCH2py); 4.10 (t, 4H,
NCH2, J = 5.8 Hz); 6.85 (m, 4H, arCH, J = 8.7, 1.0 Hz); 6.92
(tt, 2H, arCH, J = 7.4, 1.0 Hz); 6.99 (s, 2H, arCH); 7.13 (ddd,
2H, pyCH, J = 7.4, 4.9, 1.1 Hz); 7.24 (m, 4H, CH, J = 7.4 Hz);
7.49 (d, 2H, pyCH, J = 7.8 Hz); 7.60 (td, 2H, pyCH, J = 7.6,
1.8 Hz); 8.53 (dq, 2H, pyCH, J = 4.8, 1.7, 0.8 Hz). 13C NMR
(CDCl3, 100.62 MHz); d 20.5 (CH3); 52.3 (CH2O), 55.2 (arCH2N);
60.4 (NCH2py); 65.5 (NCH2); 114.4 (arCH); 120.6 (arCH); 122.0
(pyCH); 123.2 (pyCH); 123.3 (arC); 127.6 (CH3C); 129.3 (arCH);
136.5 (pyCH); 148.8 (pyCH); 153.6 (COH); 158.6 (pyC). FT-IR
spectroscopy (v, cm-1) 2921.7, 2831.4 (m, C–H str); 1596.8 (m,
C
C str); 1475.4 (m, C–H def); 1241.6 (m, C–O–Ph str); 752.2 (s,
py- H); 729.9, 690.9 (s, Ar–H). ESI mass spectrometry (methanol)
m/z 611.23 [C37H40N4O3Na]+; 589.25 [C37H40N4O3H]+.
2,6-(((2-Methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-
nitrophenol
(NO2HL2). 2-Methoxy-N-(pyridin-2-ylmethyl)-
aminoethanol (0.51 g, 31 mmol) was dissolved with triethylamine
(0.7 g) in THF (5 mL) and cooled to 0 ◦C. Upon dropwise
addition of 2,6-bis(bromomethyl)-4-nitrophenol (0.5 g, 15 mmol)
in dichloromethane (7 mL) the solution turned bright yellow and
a precipitate formed immediately. The mixture was stirred for
3 days at room temperature, filtered and concentrated in vacuo.
After purification by column chromatography (EtOAc/MeOH,
8 : 1, FeCl3 stain, Rf = 0.76) a yellow oil (0.5 g) was obtained in
68% yield. 1H NMR (CDCl3, 500.13 MHz); d 2.80 (t, 3H, NCH2,
J = 5.6 Hz); 3.29 (s, 6H, OCH3); 3.52 (t, 4H, CH2OCH3, J = 5.6
Hz); 3.85 (s, 4H, arCH2N); 3.91 (s, 4H, NCH2py); 7.16 (dd, 2H,
pyCH, J = 6.9 Hz); 7.42 (d, 2H, pyCH, J = 7.8 Hz); 7.65 (td, 2H,
pyCH, J = 7.8, 1.7 Hz); 8.14 (s, 2H, arCH); 8.52 (d, 2H, pyCH,
J = 4.4 Hz). 13C NMR (CDCl3, 100.62 MHz); d 53.2 (NCH2);
54.6 (arCH2N); 58.8 (OCH3); 60.3 (NCH2py); 70.6 (CH2OCH3);
122.2 (pyCH), 123.0 (pyCH); 124.5 (pyCH); 125.0 (arCH); 136.7
(pyCH); 139.7 (CNO2); 148.9 (pyCH); 158.5 (pyC); 162.5 (COH).
FT-IR spectroscopy (v, cm-1) 3372.3 (m, O–H str); 2927.1, 2877.8,
Synthesis of 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)-
amino)methyl)-4-methylphenol (CH3HL2). To a solution of
2-methoxy-N-(pyridin-2-ylmethyl)aminoethanol (1.50 g, 0.009
mol) and triethylamine (0.91 g, 0.009 mol) in tetrahydrofuran
(4.5 mL) was added dropwise a solution of 2,6-bis(chloromethyl)-
4-methylphenol (0.94 g, 5 mmol) in dichloromethane (4 mL) at 0
◦C. A white precipitate formed immediately. The reaction mixture
was stirred for 48 h and then filtered to remove precipitated
triethylamine hydrochloride. Removal of the solvents left a
yellow oil (2.17 g) which was further purified by flash column
chromatography (EtOAc/MeOH 8 : 2 (900 mL), EtOAc/MeOH
1 : 1 (400 mL), I2 stain, Rf = 0.66 in EtOAc/MeOH 8 : 2). The
ligand was obtained as yellow oil in 65.4% yield (1.35 g). 1H NMR
(CDCl3, 500.13 MHz); d 2.21 (s, 3H, CH3); 2.75 (t, 2H, N–CH2, J
= 5.8 Hz); 3.25 (s, 6H, OCH3); 3.50 (t, 4H, CH2OCH3, J = 5.8 Hz);
3.77 (s, 4H, arCH2N); 3.84 (s, 4H, NCH2ar); 6.92 (s, 2H, arCH);
7.10 (ddd, 2H, pyCH, J = 7.4, 4.9, 1.1 Hz); 7.46 (d, 2H, pyCH, J =
7.8 Hz); 7.60 (td, 2H, pyCH, J = 7.6, 1.8 Hz); 8.47 (dq, 2H, pyCH,
J = 4.9, 1.7 Hz). 13C NMR (CDCl3, 100.62 MHz); d 20.6 (CH3);
53.8 (NCH2), 55.6 (arCH2N); 58.7 (OCH3); 60.9 (NCH2py); 71.0
(CH2OCH3); 123.0 (pyCH); 124.0 (pyCH); 124.7 (arC); 128.2
(arCCH3); 130.3 (arCH); 137.4 (pyCH); 149.8 (pyCH); 154.6
(arC–OH); 159.8 (pyC). FT-IR spectroscopy (v, cm-1) 2921.8,
2875.2, 2817.0 (m, C–H str); 1590.6 (m, C C str); 1434.1 (m,
O–H def); 1365.0 (m, C–O–C str): 1109.1 (m, C–O str): 843.8 (m,
2820.8 (m, C–H str); 1591.6 (m, C C str); 1513.7 (m, N
O
asym. str); 1471.4 (m, C–H def); 1329.8 (s, N O sym. str);
1094.5 (m, C–O str); 845.3 (w, Ar-H); 752.3 (s, py-H). ESI mass
spectrometry (methanol) m/z 496.25 [C26H33N5O5+H]+.
4-Bromo-2,6-bis(hydroxymethyl)phenol. 4-Bromo-2,6-bis(hy-
droxymethyl)phenol was prepared by a modification of a pre-
viously published procedure.71 4-Bromophenol (17.3 g, 0.1 mol)
in methanol (25 mL), aqueous sodium hydroxide solution (25%,
50 mL) and formaldehyde were combined and left at room
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temperature for 12 days. Subsequently water (50 mL) and glacial
acetic acid (15 mL) were added and after 2 h the solution was
concentrated to 50 mL in vacuo and then cooled on ice. The
solution was decanted from the orange oil/precipitate obtained
on standing and the remaining oil was dissolved in aqueous
sodium hydroxide solution (10%, 50 mL). Activated charcoal was
added and the suspension stirred overnight. After filtration the
solution was acidified with 2 M HCl to pH 5. The resulting yellow
precipitate was collected on a sintered glass funnel, recrystallized
from water, dried in air and washed with cold chloroform (~20 mL)
to yield 7 g of the desired product as a white powder in 23% yield.
1H NMR (CD3COCD3, 300.13 MHz); d 4.73 (s, 4H, CH2); 7.28 (s,
2H, arCH) 13C NMR (CD3COCD3, 100.62 MHz); d 61.4 (CH2);
111.6 (arCBr); 129.5 (arCH); 130.5 (arCCH2); 153.4 (arCOH).
FT-IR spectroscopy (v, cm-1) 3400.8, 3300.3 (s, O–H str); 2965.6,
2911.5, 2886.2 (w, CH2 str); 1649.0, 1610.3, 1586.9 (w, C C str);
1453.5 (s, CH2 def); 1331.3 (s, O–H def); 1064.7 (s, C–OH def);
870.0 (m, Ar–H def).
[Zn2(CH3L2)(CH3COO)2](PF6). CH3HL2 (0.250 g, 0.5 mmol)
was dissolved in MeOH (20 mL), and a solution of zinc acetate
dihydrate (0.240 g, 0.9 mmol) in MeOH (10 mL) was added
dropwise. The resulting pale yellow solution was then heated
under reflux for 0.5 h. The solution was permitted to cool to
room temperature and NaPF6 (0.140 g, 0.8 mmol) was added.
Upon standing colourless crystals were deposited and these were
collected by filtration (0.31 g, 71%). Anal. calc. for C31H41Zn2N4O7
PF6: C 43.42, H 4.82, N 6.53% Found: C 43.25, H 4.79, N 6.53%.
ESI mass spec (methanol) m/z: 711.2 [C31H41N4O7Zn2]+. FT-IR
spectroscopy (n, cm-1): 2924.0, 2850.5 (w, C–H str), 1593.8 (m,
C
O asym str, acetate); 1476.2 (m, C O sym str, acetate); 1427.7
(m, C–H def); 1092.5 (w, C–O str), 830.8 (s P–F str), 555.4 (s, P–F
str). 1H NMR (CD3CN, 500.13 MHz); d 1.90 (s, 6H, acetateCH3);
2.22 (s, 3H, CH3); 2.52 (m, 4H, NCH2, J = 5.7 Hz); 2.71 (bm,
4H, CH2CH2O); 2.98 (s, 6H, OCH3); 3.63 (d, 2H, arCH2N, J
= 11.9 Hz); 3.85 (d, 2H, NCH2py, J = 14.7 Hz); 4.39 (d, 2H,
NCH2py, J = 13.8 Hz); 4.48 (d, 2H, arCH2N, J = 9.3 Hz); 7.03
(s, 2H. arCH); 7.49–7.51 (m, 4H, pyCH); 7.99 (t, 2H, pyCH, J =
7.4 Hz); 8.74 (d, 2H, pyCH, J = 4.6 Hz). 13C NMR (CD3CN,
100.62 MHz); d 20.4 (CH3); 24.6 (acetateCH3); 55.1 (NCH2);
59.0 (OCH3); 61.3 (arCH2N); 61.6 (NCH2py); 69.7 (CH2O); 125.2
(pyCH); 125.8 (arC); 126.8 (CCH3); 132.8 (arCH); 140.8 (pyCH);
4 - Bromo - 2, 6 - bis(bromomethyl)phenol. 4-Bromo-2,6-bis(hy-
droxymethyl)phenol (3 g, 13 mmol) was added to hydrogen
bromide in acetic acid (30%, 15 mL) and stirred for 10 min until
all the starting material had dissolved. Upon addition of water the
clear orange solution formed a precipitate which was collected and
dried on a filter and then washed thoroughly with cold petroleum
spirit. The desired product was obtained as yellow powder, 3.3 g,
-
148.6 (pyCH); 155.8 (pyC); 161.1 (COH); 177.7 (CO2 ). Second
set of low intensity (25%) signals: 1H NMR (CD3CN, 500.13 MHz)
H,H-COSY; d 2.07 (s, 6H); 3.12 (s, 6H); 3.27 (d, 2H, J = 5.4 Hz);
3.74 (m, 4H); 3.74 (s, 2H); 3.86 (d, 2H, J = 15.8 Hz); 4.01 (s, 2H);
6.77 (s, 2H); 7.21 (d, 2H, J = 6.1 Hz); 7.37 (t, 2H, J = 5.8 Hz); 7.81
(t, 2H, J = 7.3 Hz); 8.51 (bs, 2H).
1
in 70.5% yield. H NMR (CDCl3, 300.13 MHz); d 4.49 (s, 4H,
CH2); 5.66 (bs, 1H, OH); 7.39 (s, 2H, arCH) 13C NMR (CDCl3,
100.62 MHz) d 27.9 (CH2); 112.7 (arCBr); 127.2 (arCCH2); 133.7
(arCH); 152.3 (arCOH).FT-IR spectroscopy (v, cm-1) 3537.1 (s,
[Zn2(CH3L3)(CH3COO)2](PF6)·CH3OH. This complex was
O–H str); 3050.3, 2979.8 (w, CH2 str); 1647.9, 1603.6 (w, C
str); 1467.1 (s, CH2 def); 868.8 (m, Ar–H def).
C
prepared in
a
similar manner to that described for
[Zn2(CH3L2)(CH3COO)2](PF6); (0.31 g, 71%). Anal. calc. for
C41H45Zn2N4O7PF6CH3OH: C 49.77, H 4.87, N 5.53% Found:
1
C 49.50, H 4.60, N 5.55%. H NMR (CD3CN, 400.13 MHz); d
4-Bromo-2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)-
methyl)phenol (BrHL2). To a solution of 2-methoxy-N-(pyridin-
2-ylmethyl)aminoethanol (0.925 g, 5.6 mmol) and triethylamine
(1.25 g) in tetrahydrofuran (10 mL) was added dropwise a
solution of 4-bromo-2,6-bis(bromomethyl)phenol (1 g, 2.7 mmol)
in dichloromethane (10 mL) at 0 ◦C. The reaction mixture was
stirred for 72 h and then filtered to remove the precipitate of
triethylamine hydrobromide. Removal of the solvent resulted
in a yellow oil which was further purified by flash column
chromatography (1 g crude ligand, l = 30 cm, Ø = 1.5 cm, EtOAc
(until first band is eluted) then MeOH/EtOAc 1 : 5, FeCl3 stain,
Rf = 0.55 in EtOAc). The ligand was obtained as a yellow oil in
63% yield (930 mg). 1H NMR (CDCl3, 300.13 MHz) d 2.79 (t, 4H,
NCH2CH2); 3.26 (s, 6H, OCH3); 3.52 (t, 4H, CH2OCH3); 3.79 (s,
4H, CH2N); 3.96 (s, 4H, NCH2py); 5.17 (s, 1H, OH); 7.08 (m, 2H,
pyCH); 7.19 (s, 2H, arCH); 7.44 (m, 2H, pyCH), 7.64 (m, 2H,
pyCH); 8.50 (m, 2H, pyCH). 13C NMR (CDCl3, 100.62 MHz);
d 52.8 (CH2); 54.6 (CH2); 58.7 (CH3); 59.8 (CH2); 69.9 (CH2);
110.7 (arCH); 122.45 (pyCH); 123.6 (pyCH); 123.8 (arCH);
131.9 (arCH); 136.8 (pyCH); 148.9 (pyCH); 156.0 (arCH); 156.8
(pyCH). FT-IR spectroscopy (v, cm-1) 3230.6 (m, O–H str); 2927.3,
2879.1 (m, CH2 str); 1591.8, 1572.1 (m, C C str); 1435.2 (s,
CH2 def); 1111.8 (s, C–O str); 831.5, 755.7 (s, Ar–H). ESI mass
spectrometry (methanol) m/z 529.10, 531.07 [C26H33BrN4O3+H]+;
551.16, 553.16 [C26H33BrN4O3+Na]+.
1.72 (s, 6H, acetateCH3); 2.24 (s, 3H, CH3); 2.77 (m, 3H); 3.04 (m,
3H); 3.37 (d, 4H); 3.54 (m, 3H); 3.65 (d, 2H, J = 12.3 Hz); 3.77 (d,
2H, J = 11.8 Hz); 3.94 (m, 5H); 4.18 (m, 5H); 7.21 (m, 4H, arCH);
6.94 (m, 2H, arCH); 6.62 (d, 4H, J = 8.1 Hz, arCH); 7.41–7.50
(m, 4H, pyCH); 7.97 (td, 2H, J = 7.7, 1.4, pyCH); 8.71 (d, 2H, J =
5.1, pyCH). Second set of low intensity aromatic proton signals:
7.21 (m, 4H); 6.94 (m, 2H); 6.73 (d, 4H, J = 8.6 Hz); 7.41–7.50
(m, 4H); 7.93 (td, 2H, J = 7.7, 1.6 Hz); 8.75 (d, 2H, J = 5.3 Hz).
ESI mass spectrometry (methanol) m/z: 855.7 [C41H47N4O8Zn2]+;
825.7 [C40H47N4O7Zn2]+. FT-IR spectroscopy (n, cm-1): 2927.4 (w,
C–H str), 1593.9 (m, C O asym str, acetate); 1482.6 (m, C
O
sym str, acetate); 1433.7 (m, C–H def); 1224.6 (w, C–O str), 832.5
(s P–F str), 760.3 (s, Ph–H); 694.1 (s, Ph–H); 555.4 (s, P–F str).
[Zn2(NO2L2)(CH3COO)2](PF6). This
complex
was
prepared in similar manner to that described for
a
[Zn2(CH3L2)(CH3COO)2](PF6); (0.31 g, 71%). Anal. calc.
for C30H38Zn2N5O9PF6·CH3OH: C 40.45, H 4.60, N 7.61%
Found: C 39.36, H 4.27, N 7.67%. ESI mass spectrometry
(methanol) m/z: 744.0 [C30H38N5O9Zn2]+. FT-IR spectroscopy
(n, cm-1) 2931.1 (w, C–H str); 1596.3 (m, C O asym str, acetate);
1505.9 (w, NO2 asym str); 1426.3 (m, C O sym str, acetate);
1320.4 (m, NO2 sym str); 1091.8 (m, C–O str); 832.8 (s P-F str);
1
755.2 (m, py C–H def); 659.3 (w, Ar–H def); 556.1 (s, P-F). H
NMR (CD3CN, 500.13 MHz); d 1.91 (s, 6H, acetateCH3); 2.57
1706 | Dalton Trans., 2012, 41, 1695–1708
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(m, 4H, NCH2); 2.74 (bm, 4H, CH2CH2O); 2.99 (s, 6H, OCH3);
3.82 (d, 2H, arCH2N, J = 12.3 Hz); 3.90 (d, 2H, NCH2py, J = 14.8
Hz); 4.42 (d, 2H, NCH2py, J = 15.1 Hz); 4.48 (d, 2H, arCH2N,
J = 12.7 Hz); 7.52 (m, 4H, pyCH); 8.01 (t, 2H, pyCH, J = 6.9
Hz); 8.2 (s, 2H. arCH); 8.72 (m, 2H, pyCH).
can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Catalytic studies
The phosphoesterase-like activity of each complex was determined
by measuring hydrolysis of the substrate BDNPP. A Varian Cary50
Bio UV/Visible spectrophotometer with a Peltier temperature
controller was used to measure changes in absorbance values in
10 mm quartz cuvettes. The initial rate method was employed and
assays were measured such that the initial linear portion of the data
was used for analysis. Product formation was determined at 25 ◦C
by monitoring the formation of 2,4-dinitrophenol. Throughout the
pH range studied the extinction coefficient of this product at 400
nm is 12 100 M-1 cm-1.45,74 For each assay corrections for the rate of
autohydrolysis were applied. An aqueous multi-component buffer
(50 mM in each of 2-(N-morpholino)ethanesulfonic acid (MES),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and
2-(N-cyclohexylamino)ethane sulfonic acid (CHES), with the
ionic strength controlled by 250 mM LiClO4) was used. Assays
were carried out in 50 : 50 MeCN : buffer, with substrate and com-
plex initially dissolved in MeCN. Assays conducted to investigate
pH dependence were 40 mM in complex and 5 mM in BDNPP
and showed no significant buffer effects. Substrate dependence
assays were 40 mM in complex and 1–11.5 mM in BDNPP, and
complex dependence assays were 20–120 mM in complex and
5 mM in BDNPP. Background assays of autohydrolysis were
subtracted from the data. The change in absorbance produced
by hydrolysis of BDNPP by free Zn(II) (from Zn(ClO4)2), in the
same concentration as the complex did not differ significantly
from autohydrolysis values. All data were fitted by non-linear
least squares regression analysis. Mass spectral studies of the
hydrolysis reaction were conducted by mixing one equivalent of
[Zn2(CH3L1)(CH3COO)2]+ with twenty-five equivalents of either
bis(4-nitrophenyl)phosphate (BPNPP) or 4-nitrophenylphosphate
(PNPP), the latter a hydrolysis product of the former, in 1 : 1
MeCN : H2O, and recording the spectrum of the resulting mixture
after 1 h. BPNPP and its hydrolysis product PNPP were used as
they are slower substrates and permitted more ready analysis than
the substrate used for the kinetic studies (BDNPP).
[Zn2(BrL2)(CH3COO)2](PF6). BrHL2 (100 mg) was dissolved
in methanol (8 mL) with zinc acetate dihydrate (82 mg). The
mixture was heated under reflux for 30 min, permitted to
cool to room temperature and dry sodium hexafluorophosphate
(95 mg) was added. After filtration the solution was left to
stand at room temperature. Colourless crystals, suitable for X-
ray crystallography, emerged after 2 h (62 mg). Anal calc. for
C30H38BrN4O7Zn2PF6 C: 39.07, H: 4.15, N: 6.07% Found: C
39.33, H 4.13, N 6.14% ESI mass spectrometry (methanol)
m/z: 749.0 [C29H38BrN4O6Zn2]+, 707.3 [C27H36BrN4O5Zn]+, 593.1
[C26H32BrN4O3Zn]+. FT-IR spectroscopy (v, cm-1) 2930.0 (w, CH2
str); 1596.9 (s, bridging acetate antisym. str); 1424.4 (s, bridging
acetate sym. str); 829.4 (s, P–F str); 760.0, 657.6 (m, Ar–H def);
555.1 (m, P–F). 1H NMR (CD3CN, 500.13 MHz); d 1.99 (s, 6H,
acetateCH3); 2.54 (m, 4H, NCH2); 2.72 (bm, 4H, CH2CH2O); 2.99
(s, 6H, OCH3); 3.64 (d, 2H, arCH2N, J = 12.1 Hz); 3.84 (d, 2H,
NCH2py, J = 14.7 Hz); 4.38 (d, 2H, NCH2py, J = 14.4 Hz); 4.44
(d, 2H, arCH2N, J = 11.3 Hz); 7.38 (s, 2H. arCH); 7.48–7.50 (m,
4H, pyCH, J = 5.4 Hz); 7.99 (t, 2H, pyCH, J = 7.1 Hz); 8.72 (m,
2H, pyCH).
[Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2. This complex was pre-
pared in a similar manner to [Zn2(BrL2)(OAc)2](PF6) complex
but the sodium hexafluorophosphate employed for the crystal-
lization was wet. Colourless crystals, suitable for X-ray crystal-
lography, were obtained upon standing (40 mg). Anal calc. for
C52H68Br2N8O14Zn4P4F14: C: 33.94, H: 3.72, N: 6.09% Found:
C 33.35, H 3.61, N 5.99%. ESI mass spectrometry (methanol)
m/z: 754.9 [C26H32BrN4O3Zn2PFO3]+, 593.1 [C26H32BrN4O3Zn]+.
FT-IR spectroscopy (v, cm-1) 2854.3, 2928.9, 2892.97 (w, CH2
str); 1460.1, 1445.2 (m, CH2 def); 1182.6 (m, P O, str); 1121.0
(s, C–O–C str); 830.7 (s, P–F str); 774.4 (m, Py–H def); 555.1
(m, P–F).
X-ray crystallography
X-ray diffraction data for crystals of [Zn2(CH3L2)(CH3COO)2]-
(PF6), [Zn2(CH3L3)(CH3COO)2](PF6)·CH3OH, [Zn2(NO2L2)-
(CH3COO)2](PF6), [Zn2(BrL2)(OAc)2](PF6) and [Zn4(CH3L2)2-
(NO2C6H5OPO3)2(H2O)2](PF6)2 were collected at 293(2) K whilst
data for [Zn4(BrL2)2(PO3F)2(H2O)2](PF6)2 were collected at 150(2)
18O labelling Studies
31P NMR spectra were recorded with a 400 MHz Bruker AV400
spectrometer at room temperature in the digital acquisition mode
(operating frequency 161.9 MHz). Chemical shifts are reported
in d units relative to 85% H3PO4 in D2O as external reference
(dP = 0.00). For the 18O-labeled sample (50% 18O; 97% purity)
(Novachem, Victoria, Australia) the solution was made up from
a solution of complex (0.01 mmol) in acetonitrile (0.3 mL), 100
mM HEPES buffer pH 8 (0.15 mL) and 18O-water (97%, 0.15 mL)
(50 : 50 mixture of acetonitrile : buffer). To this, one equivalent
BDNPP (5.1 mg) was added and the mixture left for one week
at room temperature prior to recording the 31P spectra. For
experiments with 16O-water the solution was composed of complex
(0.01 mmol) in acetonitrile (0.3 mL), 100mM HEPES buffer pH
8 (0.15 mL) and distilled water (0.15 mL). To this, one equivalent
BDNPP (5.1 mg) was added and the mixture left for one week at
room temperature prior to recording the 31P NMR spectra.
K. An Oxford Diffraction Gemini Ultra dual source (Mo and Cu)
˚
CCD diffractometer with Mo (lKa = 0.71073 A) or Cu (lKa
=
˚
1.5418 A) radiation was used. The structures were solved by direct
methods (SIR-92) and refined (SHELXL 97)72 by full matrix least
squares methods based on F2. These programs were accessed
through the WINGX 1.70.01 crystallographic collective package.73
All non-hydrogen atoms were refined anisotropically unless they
were disordered. Hydrogen atoms were fixed geometrically and
were not refined. Drawings of molecules were produced with
ORTEP.30 Selected crystal data and some details of refinements are
given in Table 1. Selected bond distances and angles are presented
in Tables 2 and 3. X-ray data were deposited with the Cambridge
Crystallographic Data Centre CCDC 830841–830846. These data
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The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 1695–1708 | 1707
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son, G. Schenk and L. R. Gahan, Dalton Trans., 2008,
6045.
Acknowledgements
40 J. C. Mareque Rivas, R. T. M. de Rosales and S. Parsons, Dalton Trans.,
This work was funded by a grant from the Australian Research
Council (DP0986613).
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1708 | Dalton Trans., 2012, 41, 1695–1708
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