Dinuclear zinc(II)-iron(III) and iron(II)-iron(III) complexes as models for purple acid phosphatases

Article abstract of DOI:10.1002/1099-0682(200106)2001:6<1457::AID-EJIC1457>3.0.CO;2-F

The heterodinuclear Zn^IIFe^III complex 1 and the isostructural Fe^IIFe^III complex 2 with the dinucleating ligand from 2,6-bis[{bis(2-pyridylmethyl)amino}methyl]-4-methoxyphenol (HBPMOP, 3) were prepared and charac

Full text of DOI:10.1002/1099-0682(200106)2001:6<1457::AID-EJIC1457>3.0.CO;2-F

FULL PAPER
Dinuclear Zinc(II)؊Iron(III) and Iron(II)؊Iron(III) Complexes as Models for
Purple Acid Phosphatases
Sabine Albedyhl,[c] Marie Therese Averbuch-Pouchot,[a] Catherine Belle,*[a] Bernt Krebs,*[c]
Jean Louis Pierre,[a] Eric Saint-Aman,[b] and Stephane Torelli[a]
Keywords: ZnIIFeIII complex / FeIIFeIII complex / Purple acid phosphatases / Phosphate ester hydrolysis
The heterodinuclear ZnIIFeIII complex 1 and the isostructural
FeIIFeIII complex 2 with the dinucleating ligand from 2,6-
bis[{bis(2-pyridylmethyl)amino}methyl]-4-methoxyphenol
(HBPMOP, 3) were prepared and characterized by X-ray
crystallography. Solution studies (UV/Vis spectroscopy;
electrochemistry) are described. A pH-induced change in the
phosphate (HPNP) was investigated in acetonitrile/water
(1:1) in the presence of complexes of the ligand BPMOP and
its methyl analogue BPMP with regards to its dependence on
the pH value. At the optimum pH value (8.5 ± 0.2), the ZnII-
FeIII complex from BPMOP shows a 2-fold higher rate accel-
eration compared with that of the complex containing BPMP.
coordination spheres of the metal centers is seen. These com- The diiron complex from BPMOP is 4-fold more reactive than
plexes serve as models for the mixed-valence oxidation state
in purple acid phosphatases. The cleavage acceleration of
the activated phosphodiester 2-hydroxypropyl p-nitrophenyl
the homologous complex from BPMP. The heterodinuclear
ZnIIFeIII catalysts are at least 10-fold more reactive than the
homonuclear FeIIFeIII catalysts.
Introduction
(the product of the reaction) and with tungstate (a strong
inhibitor), have also been determined[3] allowing the pro-
posal of a mechanism for the hydrolysis of phosphomonoe-
sters (Figure 1).
Purple acid phosphatases (PAPs) catalyse the hydrolysis
of activated phosphoric esters like p-nitrophenyl phosphate,
naphthyl phosphate or ATP, at a pH range from 4 to 7.[1]
The active site of red kidney bean purple acid phosphatase
(KBPAP) harbours an FeIII and a ZnII ion, while those of
the mammalian PAPs, isolated from bovine spleen (BSPAP)
or from porcine uterus, contain a diiron site in an FeIIFeIII
state for the active form. The inactive purple FeIIIFeIII
form, involves two high-spin iron ions which are antiferro-
magnetically coupled (ϪJ Ͼ 150 cmϪ1). In the active FeII-
FeIII pink form, the two iron ions are weakly antiferromag-
netically coupled (ϪJ ϭ 19.8 cmϪ1). The crystal structure
In the proposed mechanism, the phosphate group of the
substrate binds to the zinc ion by displacing the presumed
water ligand. The terminal FeIII-bound hydroxide ion at-
tacks the phosphorus atom to form a pentacoordinated
intermediate, and the PϪO bond, opposite from the hy-
droxide ion attack, breaks to form the leaving group and
phosphate which is consistent with the inversion at the
phosphorus atom. Another kinetically indistinguishable
mechanism has been suggested,[4] involving a nucleophilic
attack by a bridging oxide instead of iron-bound hydrox-
ide.
of KBPAP, firstly determined at a resolution of 2.9 A[2] and
˚
[3]
˚
further at 2.65 A, reveals a homodimeric 111 kDa en-
zyme, containing two domains in each subunit. In the active
site, located in the carboxyl-terminal domain, the two metal
In order to mimic the reactivity of PAPs, two isostructu-
ral dinuclear complexes, containing a ZnIIFeIII and an FeII-
FeIII center, respectively, have been prepared and studied
with the aim of comparing their activity towards phosphate
ester cleavage and to allow comparisons between KBPAP
and BSPAP. The dinucleating ligand from HBPMOP, which
is of the known HBPMP type[5,6] 2,6-bis[{bis(2-
pyridylmethyl)amino}methyl]-4-methylphenol has been
used. It bears a strong electron-donating methoxy group in
the para position to the bridging phenoxo group: 2,6-
bis[{bis(2-pyridylmethyl)amino}methyl]-4-methoxyphenol
(3). Heterodinuclear ZnIIFeIII complexes have been de-
scribed as models for the active site of KBPAP. The first
heterodinuclear phosphate-bridged model compound,
[FeZn(BPMP){(O2P(OPh)2}2], could be obtained by using
˚
ions are 3.26 A apart and bridged by Asp164, each one in-
volving octahedral coordination. The iron ion is further co-
ordinated by Tyr167, His325, and Asp135 residues, and the
zinc ion by His286, His323, and Asn201 residues. Three
exogenous ligands are also included in the X-ray model to
complete the coordination sphere: one terminal hydroxo li-
gand to the iron ion, one terminal water ligand to the zinc
ion, and one µ-hydroxo group between the metal ions. The
crystal structures of KBPAP complexed with phosphate
[a]
´
Laboratoire de Chimie Biomimetique, LEDSS, UMR CNRS
´
5616, Universite J. Fourier,
BP 53, 38041 Grenoble Cedex 9, France
E-mail: Catherine.Belle@ujf-grenoble.fr
HBPMP.[5a] The FeϪZn distance (3.695 A) in this complex,
˚
[b]
[c]
Laboratoire d’Electrochimie Organique et de Photochimie
´
Redox, UMR CNRS 5630, Universite J. Fourier,
is significantly larger than in the carboxylate-bridged com-
BP 53, 38041 Grenoble Cedex 9, France
[6]
˚
˚
plex (3.428 A) compared to the value of 3.33 A measured
on KBPAP in the presence of phosphate. Some other heter-
Anorganisch-Chemisches Institut der Universität Münster,
Wilhelm-Klemm-Strasse 8, 48149 Münster, Germany
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ1948/01/0606Ϫ1457 $ 17.50ϩ.50/0
1457

C. Belle, B. Krebs et al.
FULL PAPER
Figure 1. The active site of KBPAP and proposed mechanism of phosphate ester hydrolysis[3]
odinuclear ZnIIFeIII complexes have been described,[7Ϫ9] all Crystal Structures of 1 and 2
of them containing symmetrical ligands. A ZnϪFe complex
Compounds 1 and 2 are isostructural complexes and are
therefore described in comparison with each other. The tri-
clinic elementary cells of 1 and 2 contain two formulas. The
metal centers exhibit octahedral coordination. In the hetero-
dinuclear complex 1 (Figure 2), the metal atoms are linked
by a µ-phenoxo group from the ligand and by two µ-di-
involving a constitutionally unsymmetrical ligand, but with
identical chemical environments (two N3O coordination
spheres), has also been described in solution.[10] Mixed-val-
ence phenoxo-bridged diiron complexes have also been re-
ported as models for BSPAP by several authors.[5a,6,10Ϫ17]
A recent review has been published, describing structural
and functional model compounds of PAPs.[18]
˚
phenylphosphato bridges with a distance of 3.7030(7) A.
The angle over Zn(1)ϪO(1)ϪFe(1) is 122.9(1)°. The two
iron centers in the analogous complex 2 (Figure 3) have a
˚
distance of 3.6400(6) A, which is slightly shorter than the
Results and Discussion
corresponding distance in 1. The angle Fe(1)ϪO(1)ϪFe(2)
is 121.44(9)°. The nitrogen donor atoms from BPMOP
complete the coordination sphere per facial coordination,
of resulting in an equivalent N3O3 donor set. Selected bond
Synthesis of the Complexes
The
reaction
of
equimolecular
amounts
Zn(ClO4)2·6H2O and Fe(ClO4)3·H2O with HBPMOP lengths and angles are given in Table 1.
in
methanol,
yielded
blue-green
crystals
of
The structure of 2 confirms the mixed valence state. The
[ZnIIFeIIIBPMOP{O2P(OPh)2}2](ClO4)2·H2O (1) after the crystal structure of complex [ZnIIFeIIIBPMP{O2P-
addition of diphenyl phosphate. These were suitable for (OPh)2}2](ClO4)2·1.5CH3OH·H2O which involves the same
X-ray
crystallography.
Green
crystals
of coordination sphere as in 1 has been reported.[5a] The
[FeIIFeIIIBPMOP{O2P(OPh)2}2](ClO4)2·H2O (2) were ob- ZnϪFe distance of 3.695(1) A is in the range of the ZnϪFe
˚
˚
tained by the analogous reaction with
2
equiv. of distance in 1 [3.7030(7) A]. This distance is significantly
[6]
˚
Fe(ClO4)3·H2O. The only metal species isolated was the shorter in the carboxylate-bridged analogue [3.437(1) A]
mixed-valence diiron complex, indicating the participation and larger in the molybdato-bridged complex from 2,6-
of a redox process during complex formation. It is known bis[{bis(2-pyridylmethyl)amino}methyl]-4-tert-butylphenol
[9]
˚
that the diiron(III) species are strong oxidants, suggesting (HBPBP) [3.819(4) A]. This can be due to the different
that the reductant is the methanol solvent.[6]
1458
bite distance of the phosphate group and therewith indi-
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464

Models for Purple Acid Phosphatases
FULL PAPER
˚
Table 1. Selected bond lengths [A] and angles [°] in 1 and 2
1
2
Zn(1)ϪFe(1)
Zn(1)ϪO(1)
Zn(1)ϪO(3)
Zn(1)ϪO(7)
Zn(1)ϪN(1)
Zn(1)ϪN(2)
Zn(1)ϪN(3)
Fe(1)ϪO(1)
Fe(1)ϪO(4)
Fe(1)ϪO(8)
Fe(1)ϪN(4)
Fe(1)ϪN(5)
Fe(1)ϪN(6)
3.7030(7)
2.168(2)
2.042(2)
2.028(2)
2.183(3)
2.122(3)
2.124(3)
2.047(2)
1.974(2)
1.984(2)
2.168(3)
2.128(3)
2.125(3)
Fe(1)ϪFe(2)
Fe(1)ϪO(1)
Fe(1)ϪO(3)
Fe(1)ϪO(7)
Fe(1)ϪN(1)
Fe(1)ϪN(2)
Fe(1)ϪN(3)
Fe(2)ϪO(1)
Fe(2)ϪO(4)
Fe(2)ϪO(8)
Fe(2)ϪN(4)
Fe(2)ϪN(5)
Fe(2)ϪN(6)
3.6400(6)
2.215(2)
2.096(2)
2.060(2)
2.212(3)
2.166(3)
2.160(3)
1.958(2)
1.972(2)
1.956(2)
2.179(3)
2.147(3)
2.131(3)
O(1)ϪZn(1)ϪO(3)
O(1)ϪZn(1)ϪO(7)
O(1)ϪZn(1)ϪN(1)
O(1)ϪZn(1)ϪN(2)
O(1)ϪZn(1)ϪN(3)
O(3)ϪZn(1)ϪO(7)
O(3)ϪZn(1)ϪN(1)
O(3)ϪZn(1)ϪN(2)
O(3)ϪZn(1)ϪN(3)
O(7)ϪZn(1)ϪN(1)
O(7)ϪZn(1)ϪN(2)
O(7)ϪZn(1)ϪN(3)
N(1)ϪZn(1)ϪN(2)
N(1)ϪZn(1)ϪN(3)
N(2)ϪZn(1)ϪN(3)
O(1)ϪFe(1)ϪO(4)
O(1)ϪFe(1)ϪO(8)
O(1)ϪFe(1)ϪN(4)
O(1)ϪFe(1)ϪN(5)
O(1)ϪFe(1)ϪN(6)
O(4)ϪFe(1)ϪO(8)
O(4)ϪFe(1)ϪN(4)
O(4)ϪFe(1)ϪN(5)
O(4)ϪFe(1)ϪN(6)
O(8)ϪFe(1)ϪN(4)
O(8)ϪFe(1)ϪN(5)
O(8)ϪFe(1)ϪN(6)
N(4)ϪFe(1)ϪN(5)
N(4)ϪFe(1)ϪN(6)
N(5)ϪFe(1)ϪN(6)
Zn(1)ϪO(1)ϪFe(1)
86.52(9)
97.33(9)
87.0(1)
87.3(1)
162.4(1)
94.5(1)
93.5(1)
171.8(1)
86.5(1)
171.2(1)
91.7(1)
99.3(1)
80.8(1)
77.3(1)
97.9(1)
97.17(9)
88.3(1)
89.7(1)
88.6(1)
164.7(1)
95.2(1)
168.0(1)
90.9(1)
97.5(1)
94.8(1)
173.4(1)
86.1(1)
79.4(1)
76.6(1)
95.5(1)
122.9(1)
O(1)ϪFe(1)ϪO(3)
O(1)ϪFe(1)ϪO(7)
O(1)ϪFe(1)ϪN(1)
O(1)ϪFe(1)ϪN(2)
O(1)ϪFe(1)ϪN(3)
O(3)ϪFe(1)ϪO(7)
O(3)ϪFe(1)ϪN(1)
O(3)ϪFe(1)ϪN(2)
O(3)ϪFe(1)ϪN(3)
O(7)ϪFe(1)ϪN(1)
O(7)ϪFe(1)ϪN(2)
O(7)ϪFe(1)ϪN(3)
N(1)ϪFe(1)ϪN(2)
N(1)ϪFe(1)ϪN(3)
N(2)ϪFe(1)ϪN(3)
O(1)ϪFe(2)ϪO(4)
O(1)ϪFe(2)ϪO(8)
O(1)ϪFe(2)ϪN(4)
O(1)ϪFe(2)ϪN(5)
O(1)ϪFe(2)ϪN(6)
O(4)ϪFe(2)ϪO(8)
O(4)ϪFe(2)ϪN(4)
O(4)ϪFe(2)ϪN(5)
O(4)ϪFe(2)ϪN(6)
O(8)ϪFe(2)ϪN(4)
O(8)ϪFe(2)ϪN(5)
O(8)ϪFe(2)ϪN(6)
N(4)ϪFe(2)ϪN(5)
N(4)ϪFe(2)ϪN(6)
N(5)ϪFe(2)ϪN(6)
86.13(7)
97.77(7)
86.17(8)
87.33(8)
160.29(8)
97.40(8)
92.88(9)
170.37(9)
85.03(8)
170.13(9)
91.48(9)
100.70(9)
79.62(9)
76.72(9)
98.97(9)
99.41(8)
91.24(8)
90.44(8)
88.36(8)
165.42(9)
96.41(9)
165.30(8)
90.30(9)
95.08(9)
94.25(9)
173.26(9)
85.54(9)
79.03(9)
75.66(9)
93.17(9)
Figure 2. ORTEP molecular representation of complex 1 showing
50% probability displacement ellipsoids; H atoms, water molecule,
and perchlorate ions have been omitted for clarity
Fe(1)ϪO(1)ϪFe(2) 121.44(9)
[6]
˚
from BPMP [3.365(1) A] and slightly shorter than in the
non-bridged FeIIFeIII complex of the ligand BPBP
[9]
˚
[3.726(2) A].
Electronic Spectra
The main feature of the electronic spectra of 1 and 2 in
acetonitrile is the LMCT transition from phenolate to
iron(III) which is observed at λmax ϭ 654 nm (ε ഠ 900) in
1 and 673 nm (ε ഠ 900) in 2, respectively. In addition, a
Figure 3. ORTEP molecular representation of complex 2 showing
50% probability displacement ellipsoids; H atoms, water molecule,
and perchlorate ions have been omitted for clarity
cates a remarkable structural flexibility of the phenoxo- shoulder is observed in 2, at λmax ϭ 385 nm (ε ഠ 3000)
bridged intermetal(II,III) distance which may be important which can be assigned to a charge-transfer transition from
for the catalytic activity. The ZnϪFe distance in the native FeII to pyridine. The analogous complexes from HBPMP[5a]
˚
KBPAP is 3.33 A in the presence of phosphate. The show absorption maxima in methanol at λmax ϭ 569 nm
FeIIϪFeIII distance in 2 [3.6400(6) A] is also significantly (ε ഠ 800) for the ZnIIFeIII complex and at λmax ϭ 610 nm
˚
larger than in the carboxylate-bridged FeIIFeIII complex (ε ഠ 950) for the FeIIFeIII complex. A shoulder in the spec-
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464
1459

C. Belle, B. Krebs et al.
FULL PAPER
trum of the latter is observed at λmax ϭ 365 nm (ε ഠ
3480).[6]
Electrochemistry
Although PAPs catalyse non-redox-active hydrolytic re-
actions, the catalytic activity is dependent on the oxidation
state of the iron active site, the mixed-valence oxidation
state being active towards the hydrolysis of the phosphate
esters while the oxidized form is inactive. The determination
of the electrochemical stability domain of synthetic models
thus appears of prime importance. Electrochemical investi-
gations by cyclic voltammetry (CV) of 1 in CH3CN (Fig-
ure 4, A, curve 1) show two reversible one-electron waves at
E1/2 ϭ 0.13 V and 1.36 V (vs. SCE). This is confirmed by
rotating disc electrode (RDE) experiments. The RDE curve
recorded under the same experimental conditions (Figure 4,
A, curve 2) displays two well-behaved waves of very close
intensity, without contamination of the FeIIFeIII complex 2
electrochemical response (see below). The cathodic wave at
E1/2 ϭ 0.13 V is attributed to the FeIIIZnII/FeIIZnII redox
couple while the anodic wave corresponds to the ligand-
based oxidation. The CV curve in CH3CN of the BPMP
analog
of
1,
[ZnIIFeIII
BPMP{O2P(OPh)2}2]-
(ClO4)2·1.5·CH3OH·H2O, shows a reversible one-electron
transfer at a potential of 143 mV (FeIIIZnII/FeIIIZnII redox
Figure 4. Voltammograms of 1 (A) and 2 (B), 0.9 m, in CH3CN
couple) and a weak signal at ϩ765 mV, which has been ϩ TBAP (0.1 ); E vs. Ag/AgNO3 (10 m) (ϩ339 mV vs. SCE);
attributed to a slight contamination with FeII.[5a] The li-
curve 1: CV at a vitreous carbon disc electrode (5 mm diameter),
v ϭ 0.1 VsϪ1, S (scale) ϭ 20 µA; curve 2: voltammetry at the RDE
(3 mm diameter), 600 rpm, S ϭ 10 µA
gand-based oxidation is not observed in the accessible po-
tential range. The electron-donating effect of the methoxy
group in para position of the bridging phenolic moiety
causes a stabilization of the oxidized form of the BPMOP
ligand compared to the non-substituted BPMP ligand. In
the case of 2, two reversible one-electron waves are observed
on the CV curve (Figure 4, B, curve 1) at E1/2 ϭ 0.12 V and
0.74 V (vs. SCE) which are assigned to the redox couples
FeIIFeII/FeIIFeIII and FeIIFeIII/FeIIIFeIII, respectively. The
RDE curve (Figure 4, B, curve 2) is characterized by one
cathodic and one anodic wave confirming the mixed-va-
lence character of 2.
main factors involved in the magnitude of Kcom although,
considering related complexes,[5b] the electron delocali-
zation energy has been found to be too small to contribute
significantly to the mixed-valence state stability. Electro-
static interactions may strongly influence the stability do-
main. Taking into account the close proximity of the two
metal ions, the II,II form of the complex will be more read-
ily oxidized than the II,III form leading to an increase in
the stability domain of 2. On the other hand, by comparing
other related synthetic models of PAPs,[11,13] it is shown that
the replacement of pyridyl groups in phenoxo-bridged di-
iron complexes by one and two phenolate terminal groups
leads to a cathodic shift in the redox potentials by ca. Ϫ0.4
V and Ϫ1.0 V, respectively, the stability domain ranging
between 0.5 and 1.0 V according to the substitution on the
phenoxo-bridging ligand and to the exogeneous ligands. It
must also be noticed that porcine purple acid phosphatase
(uteroferrin) has a reduction potential (FeIIIFeIII/FeIIFeIII
redox couple) of 367 mV[19] vs. NHE (i.e. 122 mV vs. SCE).
This is close to the value of 0.12 V found for 2.
Similar results have been found in the CV behavior
of
[FeIIFeIIIBPMP{O2P(OPh)2}2](ClO4)2·CH3OH·H2O
(E1/2 ϭ ϩ135 mV and ϩ755 mV, respectively[5a]). Compar-
ing the electrochemical features of 1 or 2 with those of their
BPMP analogs, only a weak cathodic shift, around 20 mV,
in the metal-centered electron transfers, are observed, i.e.
the substitution by the methoxy group in the para position
on the bridging phenolic moiety in 1 and 2 did not mark-
edly influence the metal-centered electron transfer poten-
tial. From the separation of the redox potentials in 2, we
have determined the stability of the mixed-valence form of
2 by evaluating the comproportionation constant, Kcom, for
the equilibrium according to Equation (1).
pH-Induced Changes in the Coordination Sphere
A UV/Vis titration (monitoring of the LMCT band) of a
solution of HBPMOP and equimolar amounts of zinc(II)
FeIIFeII ϩ FeIIIFeIII Ǟ 2 FeIIFeIII, ∆E1/2 ϭ (RT/F) · ln(Kcom
)
(1)
Using ∆E1/2 ϭ 0.62 V, a value of 3.0·1010 (∆G ϭ Ϫ59 kJ perchlorate and iron(III) perchlorate has been performed in
molϪ1) is found, which is close to the value of 3.2·1010 de- acetonitrile/water (1:9) upon addition of concentrated aque-
termined for the BPMP analog of 2. Electron delocali- ous solution of NaOH or HClO4 (Figure 5). In the pH
zation, electrostatic interaction and steric interaction are the range 1.95Ϫ3.35, an isosbestic point is observed at 575 nm
1460
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464

Models for Purple Acid Phosphatases
FULL PAPER
indicating the presence of only two absorbing species (Fig- trum of 4 exhibits a parent peak corresponding to the re-
ure 5, A). The spectrophotometric data were fitted and the spective heterodinuclear core (no peak corresponding to the
calculations afforded a pK1 value of 2.8Ϯ0.05. Another homodinuclear ZnϪZn or FeϪFe species was observed).
equilibrium occurs at higher pH values. At pH ഠ 5, the The molecular ion peak is m/z ϭ 681 and corresponds to
colour of the solution turns from blue to brown and a new [ZnFeBPMOP(OH)]ϩ. The electronic spectrum of 4 in ace-
isosbestic point is detected at 525 nm in pH range 4.5Ϫ6.1 tonitrile shows one absorption maximum at λmax ϭ 661 nm
(Figure 5, B).
(ε ϭ 2200); this band is shifted to 585 nm (ε ϭ 900) in a
solution of acetonitrile/water (1:9). In addition, the UV/Vis
spectra of the titration described above, in the intermediate
pH range 3Ϫ5 (Figure 6), exhibit the same features as the
spectrum of the isolated complex 4 for which we propose
a µ-OH-bridged heterodinuclear compound. At higher pH
values (Ͼ 6), the brown species is formed following pro-
gressive and reversible deprotonation of 4. This species will
prove to be the active species towards the cleavage of phos-
phate diesters (vide infra). One possibility is that this com-
plex has at least one pendant hydroxo ligand. Another pos-
sibility is that the bridging µ-OH (4) was deprotonated to
form a µ-oxo ligand between the two metallic ions.[4]
Cleavage of Phosphate Diesters
Model studies have shown that simple dinuclear metal
complexes can provide rate accelerations for hydrolyzing
phosphate esters, shedding light on how metal ions may
promote these reactions.[20Ϫ28] A heterodinuclear ZnIIFeIII
complex has been reported by Komiyama et al. which hy-
drolyses RNA.[8]
In order to test the activity of complexes towards phos-
function of the pH value: (A) in the range of 1.95 Ͻ pH Ͻ 3.35 phate ester hydrolysis or cleavage, activated phosphoric acid
Figure 5. UV/Vis titration curves in acetonitrile/water of 1 as a
and (B) in the range of 4.5 Ͻ pH Ͻ 6.1; arrows indicate the spectral
trends upon increasing the pH value
esters are used. In this work 2-hydroxypropyl p-nitrophenyl
phosphate (HPNP) was applied. In the absence of the
metallocatalysts, HPNP is very stable.[24] The pH depend-
The fitting of the values of absorbance vs. pH leads to a
ence of the HPNP cleavage acceleration, in the presence of
pK2 ϭ 4.9Ϯ0.26. The species distribution curves are shown
in Figure 6, providing evidence for three pH-dependent spe-
cies. It must be emphasized that the pH-driven conversion
between these species is reversible. Same results were ob-
tained by decreasing or increasing the pH value of the solu-
tion.
[29]
ZnIIFeIII or FeIIFeIII
complexes from the ligands
BPMOP and BPMP respectively, has been studied. The rate
increases corresponding to the pH and optimum values
were obtained at pH ϭ 8.5Ϯ0.2. The rate constant (kuncat
)
for background hydrolysis was similarly obtained. Some ob-
served rate constants are listed in Table 2. The rate accelera-
tion was also measured in dependence of the substrate and
complexes concentration, showing first order dependence in
each case.
Table 2. Observed pseudo-first-order rate constants for transes-
terification of HPNP hydrolysis in CH3CN/aqueous buffer CHES
(1:1) at pH ϭ 8.5 (Ϯ 0.2) and 25 °C with [catalyst] ϭ 0.5 m and
[HPNP] ϭ 0.82 m
Catalyst
kobs [10Ϫ5sϪ1
]
kobs/kuncat
Figure 6. Species distribution curves of 1 as a function of the pH
[ZnFe(BPMOP)]4ϩ
[ZnFe(BPMP)]4ϩ
[Fe2(BPMOP)]4ϩ
[Fe2(BPMP)]4ϩ
spontaneous
2.39
1.14
112
54
11
3
value
0.227[a]
0.065[a]
0.0213
Attempts to isolate the pure products involved in this ti-
tration have not yet proved successful. Nevertheless by inde-
pendent preparations, a blue product (4) was obtained by
addition of equimolecular amounts of Zn(ClO4)2·6H2O and
Fe(ClO4)3·H2O, dissolved in water, to a solution of
HBPMOP in acetone and precipitation as a powder after
1
[a]
10% of DMSO has been added to avoid precipitate.
The ZnIIFeIII complex of the ligand BPMOP shows a 2-
addition of diethyl ether. The positive ion FAB mass spec- fold higher rate acceleration compared with the complex
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464
1461

C. Belle, B. Krebs et al.
FULL PAPER
Vis spectra were obtained using a UVIKON 933 spectrometer of
Kontron instrument equipped with 1.0-cm matched quartz cells
containing the ligand BPMP. The diiron complex from
HBPMOP is 4-fold more reactive than the homologous
complex from BPMP. The heterodinuclear ZnIIFeIII com-
plexes from BPMOP and BPMP are 10-fold and 18-fold,
respectively, more reactive than the corresponding homodi-
nuclear FeIIFeIII complexes. These differences in reactivities
may be ascribed to the greater ability of the zinc center
compared with the iron center, to coordinate the substrate.
The optimum pH value for the hydrolysis of HPNP is no-
tably higher than that for the enzymes. This observation con-
firms the necessity of a deprotonation process as underlined
with the enzymes.[1b] The different activities of the com-
plexes of BPMOP and BPMP can be attributed to the
stronger electron donating effect of the methoxy group
compared with that of the methyl group in the para position
to the bridging oxygen atom.
and operating in the range 200Ϫ900 nm; ε values are given in
1

Ϫ1cmϪ1. Ϫ H NMR and 13C NMR spectra were recorded with
a BRUKER AC 200 spectrometer at 25 °C, in CDCl3 or
[D6]DMSO as solvent. Chemical shifts (ppm) were referenced to
residual solvent peaks.
Electrochemistry: Electrochemical experiments were carried out us-
ing a PAR model 273 potentiostat equipped with a Kipp-Zonen x-
y recorder. All experiments were run at room temperature under
argon. A standard three-electrode cell was used. For the experi-
ments performed in acetonitrile as solvent, potentials are refer-
enced to an Ag/10 m AgNO3 reference electrode with 0.1  tetra-
n-butylammonium perchlorate (TBAP) as supporting electrolyte.
Vitreous carbon disc electrodes for cyclic voltammetry (CV) (5 mm
diameter) and rotating disc electrode (RDE) (3 mm diameter) were
polished with 1 µm diamond paste.
X-ray Data Collection and Crystal Structure Determinations: The
main details dealing with the diffraction data collections and the
strategy used for the structure determinations are gathered in
Table 3. The diffraction data were processed through the texSan
software[30] for complex 1 and through the Collect Software Nonius
BV for complex 2.[31] Crystallographic data (excluding structure
factors) for the two structures reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication nos. CCDC-140082 (1) and -140083 (2).
Copies of the data can be obtained free of charge on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Fax:
Experimental Section
All chemicals were obtained from commercial sources and used as
received. Solvents were purified by standard methods before use.
Caution: Although no problems were encountered during the pre-
paration of perchlorate salts, suitable care should be taken when
handling such potentially hazardous compounds.
Spectrometry: Fast-atom bombardment (FAB) mass spectra in the
positive mode were recorded with a Nermag R 1010C apparatus
equipped with an M scan (Wallis) atom gun (8 kV, 20 mA). Ϫ UV/ (internat.) ϩ 44-1223/336-033; E-mail: deposit@ccdc.cam.ac.uk].
Table 3. Crystallographic data for 1 and 2
Complexes
1
2
Formula
Molecular mass
Cell dimensions: a, b, c [A];
α β γ [°]
Volume [A ]
Z
C57H53Fe1N6O10P2Zn1·2ClO4·H2O
C57H53Fe2N6O10P2·2ClO4·H2O
1382.18
1372.65
˚
10.237(5), 13.381(7), 23.899(10);
106.01(2), 94.08(2), 98.47(2)
3091(3)
2
10.269(5), 13.396(5), 23.991(9);
105.74(1), 94.26(1), 98.31(1)
3121(1)
2
3
˚
dcalcd.
System, space group
Diffractometer
1.483
triclinic/P1
Nonius-CAD4
Mo-Kα ϭ 0.7107
1.458
triclinic/P1
Nonius Kappa CCD
Ag-Kα ϭ 0.5608
¯
¯
˚
Wavelength [A]
Monochromator
Crystal size [mm]
Temperature
graphite plate
0.27 ϫ 0.31 ϫ 0.32
293 K
omega
graphite plate
0.30 ϫ 0.35 ϫ 0.35
293 K
Ϫ
Scan mode
Theta range [°]
Reflections measured
No of independent rfls.
I/σ(I)
2Ϫ27
12107
7625
2.2
2Ϫ30
21780
7713
3
µ [cmϪ1
]
8.31
3.57
Absorption correction
Solution
LS refinements
H-atoms location
Thermal parameters
none
none
direct methods (SIR92)[32]
on F/full matrix
geometry and Fourier methods
anisotropic for non-H atoms
isotropic for H atoms
9.2
direct methods (SIR92)[32]
on F/full matrix
geometry and Fourier methods
anisotropic for non-H atoms
isotropic for H atoms
9.5
Number of parameters/number of reflections
Final R[a]
0.055/0.062
0.053/0.055
and R(w)[b]
Program used
texSan software[30]
texSan software[30]
[a]
[b]
R ϭ Σ||Fo| Ϫ |Fc||/Σ|Fo|. Ϫ R(w) ϭ [Σ(|Fo| Ϫ |Fc|)2/wFo2]1/2] with w ϭ 1/[σ2(Fo) ϩ 3.4·10Ϫ4|Fo|2].
1462
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464

Models for Purple Acid Phosphatases
FULL PAPER
[ZnIIFeIIIBPMOP{O2P(OPh)2}2](ClO4)2·H2O (1): To a solution of
HBPMOP (23.5 mg, 0.043 mmol) in methanol (2 mL), a solution
of Zn(ClO4)2·6H2O (16.0 mg, 0.043 mmol) in methanol (1 mL) was
added dropwise. Upon the subsequent addition of Fe(ClO4)3·H2O
(15.2 mg, 0.043 mmol), dissolved in methanol (1 mL), the
solution turned blue-green. Diphenyl phosphate (21.6 mg, 0.086
mmol), dissolved in methanol (1 mL), was added and the solvent
was allowed to evaporate slowly at room temperature. Blue-
green crystals (13 mg, 0.010 mmol, 23%) suitable for X-ray
Syntheses
HBPMOP: 2,6-Bis[{bis(2-pyridylmethyl)amino}methyl]-4-meth-
oxyphenol has been synthesized in three steps from p-methoxy-
phenol.
2,6-Bis(hydroxymethyl)-4-methoxyphenol (5): To 4-methoxyphenol
(10 g, 80.6 mmol) in 25 mL of methanol was added drop by drop
a solution of sodium hydroxide (60 mL, 25%) under nitrogen at 0
°C. After 30 min of stirring, 120 mL of formaldehyde (37% in
methanol) was added dropwise. The resulting mixture was refluxed
during 12 h. Then the reaction mixture was neutralised at 0 °C to
pH ϭ 5 with 15 mL of pure acetic acid in 30 mL of water. Evapora-
tion of the solvent yielded a solid. After addition of 200 mL of
water, the product was extracted with ethyl acetate (3 ϫ 200 mL).
The combined organic layers were dried with sodium sulfate and
concentrated to yield the crude product which was purified by flash
chromatography (silica gel, ethyl acetate/cyclohexane, 1:1) and af-
crystallography were obtained after
2
d.
Ϫ
UV/Vis
(acetonitrile): λmax ϭ 654 (ε ഠ 900) nm. Ϫ FAB-MS (Matrix
NBA): m/z ϭ 1264 [{ZnIIFeIIIBPMOP[O2P(OPh)2]2}ClO4]ϩ. Ϫ
C57H53FeN6O10P2Zn·2ClO4·H2O (1382.18): calcd. C 48.53, H 4.01,
N 6.08; found C 48.59, H 3.98, N 5.85.
[FeIIFeIIIBPMOP{O2P(OPh)2}2](ClO4)2·H2O (2): Complex 2 was
prepared in a similar manner to complex 1. To a solution of
HBPMOP (23.5 mg, 0.043 mmol) in methanol (2 mL) a solution of
Fe(ClO4)3·H2O (32 mg, 0.086 mmol) in methanol (2 mL) was ad-
ded dropwise. Upon the addition, the solution turned dark green.
Diphenyl phosphate (21.6 mg, 0.086 mmol), dissolved in methanol
(1 mL), was added and the solvent was allowed to evaporate slowly
at room temperature. Green crystals suitable for X-ray crystallog-
1
forded 5 as a white powder (11.2 g, 75% yield); m.p. 114 °C. Ϫ H
NMR (200 MHz, [D6]DMSO): δ ϭ 3.66 (s, 3 H, OCH3), 4.51 (s, 4
H, CH2), 5.19 (s, 2 H, OH), 6.73 (s, 2 H, CH). Ϫ 13C NMR
(50 MHz, [D6]DMSO): δ ϭ 55.8, 59.9, 111.6, 130.4, 145.6, 153.0.
Ϫ C9H12O4 (184.19): calcd. C 58.69, H 6.57; found C 58.75, H
6.60. Ϫ MS (DCI, NH3, isobutane): m/z ϭ 202 [Mϩ ϩ NH4ϩ],
184 [Mϩ].
raphy were obtained after 1 d. Ϫ UV/Vis (acetonitrile): λmax
385 (ε ഠ 3000), 673 (ε ഠ 900) nm. Ϫ FAB-MS (Matrix NBA):
m/z 1155
[FeIIFeIIIBPMOP{O2P(OPh)2}2]ClO4]ϩ.
ϭ
ϭ
Ϫ
2,6-Bis(chloromethyl)-4-methoxyphenol
(6):
2,6-Bis(hydroxy-
C57H53Fe2N6O10P2·2ClO4·H2O (1372.65): calcd. C 49.88, H 4.04,
methyl)-4-methoxyphenol (2.43 g, 13.2 mmol), suspended in 20 mL
of dry dichloromethane under nitrogen atmosphere, was cooled
with an ice bath. A solution of freshly distilled thionyl chloride
(16 g, 132 mmol) in dry dichloromethane (20 mL) was added drop-
wise. The reaction was complete after 30 min. The excess thionyl
chloride was removed in a rotary evaporator. The remaining solid
was washed several times with dry pentane and 2.85 g (12.9 mmol,
98%) of 6 as a white powder was obtained. The product was used
without any purification. Ϫ 1H NMR (200 MHz, CDCl3): δ ϭ 3.77
(s, 3 H, OCH3), 4.66 (s, 4 H, CH2), 6.84 (s, 2 H, CH). Ϫ 13C NMR
(50 MHz, CDCl3): δ ϭ 42.4, 55.8, 116.1, 126.0, 146.7, 152.9.
N 6.12; found C 49.63, H 3.92, N 6.16.
Blue Powder (4): To a solution of HBPMOP (150 mg, 0.275 mmol)
in acetone (2 mL) a solution of Zn(ClO4)2·6H2O (100.5 mg,
0.275 mmol) in water (1 mL) was added dropwise. Upon the addi-
tion of Fe(ClO4)3·H2O (95.7 mg, 0.275 mmol), dissolved in water
(1 mL), the solution turned blue-green and was stirred for 1 h. The
acetone was removed in a rotary evaporator and diethyl ether
(100 mL) was added in small portions. The solution was placed
in a refrigerator and after a few days a blue powder (134.0 mg,
0.120 mmol, 31%) precipitated which was filtered and washed with
diethyl ether. Ϫ UV/Vis (acetonitrile): λmax ϭ 661 nm (ε ഠ 2200).
Ϫ FAB-MS (matrix NBA): m/z ϭ 681 [ZnIIFeIIIBPMOP(OH)]ϩ.
2,6-Bis[bis(2-pyridylmethyl)aminomethyl]-4-methoxy-
phenol
(3):
2,6-Bis(chloromethyl)-4-methoxyphenol
(5.0 g,
The Deprotonation Constants by UV/Vis Spectroscopy: UV/Vis ti-
22.6 mmol) and dry tetrahydrofuran (50 mL) were placed in a two-
necked round-bottomed flask under nitrogen and the mixture was
cooled with an ice bath. A solution containing dipicolylamine
(9.0 g, 45.2 mmol), distilled triethylamine (15 mL, 90.4 mmol) and
dry tetrahydrofuran (20 mL) was added dropwise. The reaction was
controlled by thin-layer chromatography (ethyl acetate/dichloro-
methane, 1:1). After 2.5 d of stirring at room temperature, the reac-
tion mixture was filtered, washed with tetrahydrofuran and the fil-
trate was concentrated to dryness. The resulting oil was dissolved
in dichloromethane (150 mL) and water was added (100 mL). The
organic phase was separated and the aqueous phase was extracted
three times with dichloromethane (100 mL). The organic phases
were combined and dried with sodium sulfate. Evaporation of the
solvent yielded a yellow oil, which was further purified by flash
chromatography (silica gel, acetone). Removal of the solvent af-
forded 8.9 g (16.3 mmol, 72%) of 3 as a pale yellow powder, m.p.
tration (monitoring of the LMCT band) of a solution of the ligand
HBPMOP (2.5·10Ϫ4

Ϫ1) and equimolar amounts of zinc(II) per-
chlorate and iron(III) perchlorate in acetonitrile/water (1:9) with an
aqueous solution of sodium hydroxide leads to a formation of new
species. The ionic strength was fixed at 0.1  with sodium perchlo-
rate. The reaction is reversible by addition of an aqueous solution
of perchloric acid. The spectrophotometric data were fitted with
the LETAGROP-SPEFO program.[33] The program uses a non lin-
ear least-squares method and calculates the thermodynamic con-
stants of the absorbing species and their corresponding electronic
spectra. The calculations were done from absorbance data of seven
wavelengths (between 400 and 800 nm) for 15 solutions in the pH
range of 1.95 Ͻ pH Ͻ 3.35 and for eight solutions in the pH range
of 4.5 Ͻ pH Ͻ 6.1.
HPNP (2-Hydroxypropyl p-Nitrophenyl Phosphate) was prepared
1
76 °C. Ϫ H NMR (300 MHz, CDCl3): δ ϭ 3.75 (s, 3 H, OCH3),
according to a known procedure.[34]
3.80 (s, 4 H, CH2), 3.88 (s, 8 H, CH2), 6.85 (s, 2 H, CH), 7.08Ϫ7.14
(m, 4 H, Py-H), 7.47Ϫ7.51 (m, 4 H, Py-H), 7.55Ϫ7.64 (m, 4 H,
Kinetic Investigations of the HPNP Cleavage by UV/Vis Spectro-
Py-H), 8.52 (d, J ϭ 4.8 Hz, 4 H, Py-H), 10.9 (s, 1 H, OH). Ϫ 13C scopy: The kinetic investigations were made at 25 °C in buffered
NMR (75 MHz, CDCl3): δ ϭ 54.6, 55.5, 59.6, 114.1, 121.8, 122.7, solutions of acetonitrile/water (1:1). HEPES {2-[4-(2-hydroxyethyl)-
124.7, 136.4, 148.7, 149.5, 151.8, 159.0. Ϫ C33H34N6O2 (546.68): piperazin-1-yl]ethanesulfonic acid} and CHES [2-(cyclohexylami-
calcd. C 72.50, H 6.27, N 15.37; found C 72.55, H 6.25, N 15.41.
no)ethanesulfonic acid] were used as buffers. The ionic strength
was fixed at 0.1  with sodium perchlorate. The complexes were
Ϫ MS (DCI, NH3, isobutane): m/z ϭ 547 [Mϩ ϩ Hϩ].
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464
1463

C. Belle, B. Krebs et al.
FULL PAPER
[11]
[12]
[13]
B. Krebs, K. Schepers, B. Bremer, G. Henkel, E. Althaus, W.
Müller-Warmuth, K. Griesar, W. Haase, Inorg. Chem. 1994,
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Engl. 1994, 33, 887Ϫ889.
synthesized in situ by adding an aqueous solution of
Zn(ClO4)2·6H2O and Fe(ClO4)3·H2O to a solution of the ligand in
acetonitrile. The spontaneous hydrolysis under these conditions
was measured in a buffered acetonitrile/water (1:1) solution con-
taining the substrate and 0.1  sodium perchlorate. The pH value
varied less than 0.1 unit during the experiments. In a typical experi-
ment, an aqueous solution (40 µL, 0.82 m) of HPNP was injected
into a cuvette which contained an aqueous solution of the buffer
(400 µL, 0.01 ), an aqueous solution of perchlorate (200 µL, 0.1
), a solution of the complex in acetonitrile/water (10:2) (120 µL,
8.25 mmol), acetonitrile (990 µL), and water (340 µL). In order to
determine the activity towards phosphate ester cleavage, the in-
crease of the concentration of p-nitrophenolate (PNPat), was fol-
lowed by UV/Vis spectroscopy at 400 nm, (ε ϭ 18500 Ϫ1cmϪ1).
All kinetic experiments were run twice and the obtained values
were merged. In respect to a pH-dependent equilibrium between p-
nitrophenol (PNP) and p-nitrophenolate (PNPat), the increase of
the concentration of the reaction product can be calculated (pH ϭ
pKS ϩ log[PNPat]/[PNP]). The kobs values were obtained by Equa-
tion (2).
E. Lambert, B. Chabut, S. Chardon-Noblat, A. Deronzier, G.
Chottard, A. Bousseksou, J. P. Tuchagues, J. Laugier, M. Bar-
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kobs·dt ϭ d([PNP]total)/[HPNP]0
(2)
[21]
Acknowledgments
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The authors are grateful to Dr. A. Deronzier for laboratory facilit-
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Prof. G. Serratrice for fruitful discussions. S. A. and B. K. thank the
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Electrochemical investigations by RDE voltammetry experi-
ments performed under similar conditions than HPNP cleavage
catalysis tests [preparation in situ of the complexes in a mixture
acetonitrile/water buffered at pH ϭ 8.5 with CHES (0.1 )]
show that the mixed-valence state for the FeIIFeIII complexes
is unambiguously conserved in these conditions.
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[9]
[34]
[10]
Received October 30, 2000
[I00412]
1464
Eur. J. Inorg. Chem. 2001, 1457Ϫ1464