Homodinuclear [Fe(III)?Fe(III)] and [Zn(II)?Zn(II)] complexes of a binucleating [N4O3] symmetrical ligand with purple acid phosphatase (PAP) and zinc phosphoesterase like activity

Article abstract of DOI:10.1016/j.poly.2018.01.029

The homodinuclear [Fe(III)–Fe(III)] (2) and [Zn(II)–Zn(II)] (3) complexes of binucleating [N₄O₃] symmetrical ligand (1) of the formulation, H₃L (L = 2,6-bis{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]methyl}-4-t-butylphenolate), display purple acid phosphatase (PAP) and zinc phosphoesterase like activity. For the two complexes, the spectrophotometric titration and the ESI-MS studies supported the existence of an equilibrium between a triaquated species, {L[(H₂O)Fe^III(μ-H₂O)Fe^III(H₂O)]}³⁺ (2A) and {L[(H₂O)Zn^II(μ-H₂O)Zn^II(H₂O)]}⁺ (3A), a diaquated and monohydroxo species, {L[(H₂O)Fe^III(μ-H₂O)Fe^III(OH)]}²⁺ (2B) and {L[(H₂O)Zn^II(μ-H₂O)Zn^II(OH)]} (3B), a monoaquated and dihydroxo species, {L[(H₂O)Fe^III(μ-OH)Fe^III(OH)]}⁺ (2C) and {L[(H₂O)Zn^II(μ-OH)Zn^II(OH)]}^? (3C), and a trihydroxo species, {L[(HO)Fe^III(μ-OH)Fe^III(OH)]} (2D) and {L[(HO)Zn^II(μ-OH)Zn^II(OH)]}^2? (3D), depending upon the pH of the solution. Of these, the active species namely, a monoaquated and dihydroxo species, {L[(H₂O)Fe^III(μ-OH)Fe^III(OH)]}⁺ (2C) and {L[(H₂O)Zn^II(μ-OH)Zn^II(OH)]}^? (3C), hydrolyzed an activated substrate bis(2,4-dinitrophenyl)phosphate at the pH range of 5.5–10.5, displaying Michealis–Menton kinetics. Significantly enough, the catalyst-substrate adduct of the type {L[Fe^III(μ-OH)(μ-RPO₄)Fe^III]} (2F) has been detected by ESI-MS for the dinuclear [Fe(III)–Fe(III)] complex (2).

Full text of DOI:10.1016/j.poly.2018.01.029

Polyhedron 145 (2018) 88–100
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Homodinuclear [Fe(III)ꢀFe(III)] and [Zn(II)ꢀZn(II)] complexes of a
binucleating [N4O3] symmetrical ligand with purple acid phosphatase
(PAP) and zinc phosphoesterase like activity
⇑
Chandni Pathak, Manoj Kumar Gangwar, Prasenjit Ghosh
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The homodinuclear [Fe(III)–Fe(III)] (2) and [Zn(II)–Zn(II)] (3) complexes of binucleating [N4O3] symmet-
rical ligand (1) of the formulation, H3L (L = 2,6-bis{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]methyl}-
4-t-butylphenolate), display purple acid phosphatase (PAP) and zinc phosphoesterase like activity. For
the two complexes, the spectrophotometric titration and the ESI-MS studies supported the existence
Received 25 October 2017
Accepted 28 January 2018
Available online 08 February 2018
of an equilibrium between a triaquated species, {L[(H2O)FeIII
ZnII(
(2B) and {L[(H2O)ZnII(
-OH)FeIII(OH)]}+ (2C) and {L[(H2O)ZnII(
FeIII -OH)FeIII(OH)]} (2D) and {L[(HO)ZnII(
tion. Of these, the active species namely, a monoaquated and dihydroxo species, {L[(H2O)FeIII
FeIII(OH)]}+ (2C) and {L[(H2O)ZnII( -OH)ZnII(OH)]}ꢀ (3C), hydrolyzed an activated substrate bis(2,4-dini-
(
l
-H2O)FeIII(H2O)]}3+ (2A) and {L[(H2O)
-H2O)FeIII(OH)]}2+
monoaquated and dihydroxo species, {L[(H2O)FeIII
-OH)ZnII(OH)]}ꢀ (3C), and
trihydroxo species, {L[(HO)
-OH)ZnII(OH)]}2ꢀ (3D), depending upon the pH of the solu-
-OH)
-H2O)ZnII(H2O)]}+ (3A), a diaquated and monohydroxo species, {L[(H2O)FeIII
-H2O)ZnII(OH)]} (3B),
(l
Keywords:
l
Homodinuclear complexes
Magnetic properties
Enzyme models
Zinc phosphoesterase
Purple acid phosphatase
l
a
(
l
l
a
(
l
l
(
l
l
trophenyl)phosphate at the pH range of 5.5–10.5, displaying Michealis–Menton kinetics. Significantly
enough, the catalyst-substrate adduct of the type {L[FeIII
(l
-OH)(
l
-RPO4)FeIII]} (2F) has been detected
by ESI-MS for the dinuclear [Fe(III)–Fe(III)] complex (2).
Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction
tidine, aspartate and a hydroxo moiety and the other divalent
metal center M(II) [M = Fe, Zn, Mn] bound to an asparagine, two
histidines and a H2O molecule, with an aspartate and a hydroxo
moiety bridging between the two metal centers [7–12]. Similarly,
the dinuclear active site of the zinc phosphoesterase, as isolated
from Pseudomonas diminuta, contains a homodinuclear [Zn(II)–Zn
(II)] core with one of the Zn(II) center bound to two histidine moi-
eties and the other Zn(II) center bound to two histidine and an
aspartate residue while a carbamylated lysine and a hydroxo moi-
ety bridging between the two metal centers [4,7,13].
Extensive active site modelling studies with transition metal
coordination complexes have been performed for obtaining an
understanding of the enzyme mode of action. In this regard many
polypodal symmetrical [14,15] [16–25] and unsymmetrical ligands
[17,26–34], containing a bridging phenoxide moiety and different
N/O donor atoms representing a variety of {[N3O], [N3O]} [16–
18,35–37], {[N3O], [N2O2]} [26–28,38–46], {[N3O], [NO3]} [47,48],
{[N3O], [N2O]} [30], {[N2O2], [NO3]} [31] and {[N2O2], [N2O]}
[34,49–54] {[N2O2], [N2O2]} [54–60] cores have been reported.
Though much of the study has been performed with the objective
of getting insight on the reactive intermediate of the complexes
responsible for the phosphoester bond cleavage using various
The cleavage of the phosphoester (PAO) bonds is an important
transformation in biology being involved in the energy production
in the ATP to ADP conversion and in the various DNA and RNA
cleavage activities [1]. Nature successfully partakes the phospho-
ester bond cleavage with the aid of the metalloenzymes like, the
phosphodiesterase and the phosphatase that mainly contain dinu-
clear metal centers working in unison in activating and hydrolyz-
ing the PAO bond of a phosphoester substrate [1–3]. In this
context, notable are the enzymes like, the purple acid phosphatase
(PAP) containing a heterodinuclar [M(II)–Fe(III)] (M = Fe, Zn, Mn)
core that carry out the hydrolysis of different phosphomonoesters
and amide substrates [4,5] and the zinc phosphoesterase that con-
tain a homodinuclear [Zn(II)–Zn(II)] core, and which perform the
hydrolysis of the tri-, di- and mono-phosphoester substrates
[2,4,6].
The dinuclear active site of PAP, as isolated from mammals,
plant and fungi, contain a Fe(III) center bound to a tyrosine, his-
⇑
Corresponding author. Fax: +91 22 2572 3480.
E-mail address: pghosh@chem.iitb.ac.in (P. Ghosh).
https://doi.org/10.1016/j.poly.2018.01.029
0277-5387/Ó 2018 Elsevier Ltd. All rights reserved.

C. Pathak et al. / Polyhedron 145 (2018) 88–100
89
Fig. 1. Dinuclear {L[FeIII
(l
-OAc)2FeIII]}ClO4 (2) and {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) complexes supported over symmetrical [N4O3] type ligand 1.
Scheme 1. Synthesis of the symmetrical [N4O3] type ligand 1.
techniques namely, electronic spectroscopy, magnetism and X-ray
diffraction studies [4,7], the mass spectrometric detection of such
intermediate have been rare and hence, we became interested in
designing synthetic small molecule mimics of these enzymes using
dinuclear complexes supported over binucleating phenoxy bridge
N/O donor ligand system and undertake the mass spectrometric
identification of these reactive intermediates.
sponding homodinuclear zinc complex, {L[ZnII(H2O)2ZnII(H2O)]}
ClO4 (3), in 58% yield (Scheme 2). The iron complex (2) and the zinc
complex (3) have been characterized by various techniques.
The electronic spectrum of the iron complex (2) and the zinc
complex (3) were recorded in CH3CN at room temperature. Both
of the complexes, 2 and 3, showed ligand based transitions in the
range of ca. 211–219 nm and 268–272 nm. In addition, the zinc
complex (3) showed another ligand based transition at ca. 286
nm. The iron complex (2) showed a transition at ca. 600 nm
With our larger goal remaining in designing synthetic analogs
of various metalloenzymes like, the catechol oxidase [61], the cat-
echol dioxygenase [62] and the galactose oxidase [63] with
mononuclear model complexes, we became interested in designing
synthetic analogs of phosphoester cleaving enzymes that contain
dinuclear active sites. In this regard we have recently reported
heterodinuclear M(II)–Fe(III) (M = Zn, Ni, Co and Cu) complexes
as the small molecule models for the PAP enzyme [64] and pro-
ceeding further along this line of study we decided to explore sim-
ilar catalytic potential of the related homodinuclear cores.
Here in this manuscript we report homodinuclear complexes of
(e
= 4896 Mꢀ1 cmꢀ1
charge-transfer transition (LMCT) at ca. 546 nm (
cmꢀ1) in PAPs enzyme [66,67] and other related complexes
)
consistent with the tyrosine-to-Fe(III)
e
= 2000 Mꢀ1
[26,56]. Another high energy transition at ca. 334 nm (e = 5045
Mꢀ1 cmꢀ1), attributed to another phenolate to Fe(III) charge-trans-
fer transition [56]. (Supporting Information Figs. S7, S11, S76 and
Table 1)
The molecular structures of the iron complex (2) and the zinc
complex (3) as determined by the single crystal X-ray diffraction
studies confirmed the bimetallic nature of these two complexes
with the metal centers bridged by a phenolate oxygen atom
(Figs. 2,3 and Table 2). In particular, in the iron complex (2), the
two iron centers are hexacoordinated and were bridged by endoge-
nous phenolate oxygen and two exogenous acetate moieties. Fur-
ther confirmation of the presence of acetate bridge came from
the IR spectroscopy which showed the presence of unsymmetrical
CAO stretching band at 1549 cmꢀ1 and symmetrical CAO stretch-
ing band at 1447 cmꢀ1 exhibiting a difference of ca. 102 cmꢀ1
between the two stretching frequencies in agreement with the
ionic carboxylate moieties bridging the two metal centers (Sup-
porting Information Fig. S8) [56,68]. The overall coordination envi-
ronments in both of these two distorted octahedral iron sites are of
the [N2O4] type with the Fe(III) metal centers being attached to a
tertiary amine-N, a pyridyl-N, a terminal phenolate-O atoms along
with one bridging phenoxy-O and two bridging acetate-O atoms.
The bond distances around the hexacoordinated iron centers were
found to be, Fe1–N2 (tertiary amine) [2.177(4) Å], Fe1–N4(pyridyl)
[2.160(4) Å], Fe1–O2 (terminal phenolate) [1.873(3) Å], Fe1–O7
(phenoxy bridge) [2.062(3) Å], Fe1–O3 (acetate) [1.985(4) Å] and
the type {L[FeIII( -OAc)2FeIII]}ClO4 and {L[ZnII(H2O)2ZnII(H2O)]}
l
ClO4 supported over a symmetrical [N4O3] ligand framework as a
small molecule functional mimic of the PAP and the zinc phospho-
esterase enzymes (Fig. 1).
2. Results and discussion
The phenoxy bridged binucleating ligand containing
a 2-
(methyl)pyridine and 2-(methyl)phenol side arms namely,
a
2,6-bis{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]methyl}-4-
t-butylphenol (H3L) (1) was synthesized from the reaction of
2,6-bis(chloromethyl)-4-t-butylphenol [65] with N-(2-hydroxy-
benzyl)-N-(2-pyridylmethyl)amine) [26] in 26% yield (Scheme 1).
Furthermore, the reaction of the ligand (H3L) (1) with the metal
precursor Fe(ClO4)3ꢁxH2O in CH3OH in the presence of NaOAc as
a base led to the precipitation of the homodinuclear iron complex,
{L[FeIII( -OAc)2FeIII]}ClO4 (2), in 82% yield (Scheme 2). Likewise,
l
the reaction of the ligand (H3L) (1) with the zinc precursor Zn
(ClO4)2ꢁ6H2O in the presence of Et3N as a base gave the corre-

90
C. Pathak et al. / Polyhedron 145 (2018) 88–100
Scheme 2. Synthesis of the dinuclear {L[FeIII
(l
-OAc)2FeIII]}ClO4 (2) and {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) complexes supported over symmetrical [N4O3] type ligand 1.
Table 1
Electronic spectral data for 1, 2 and 3 in CH3CN at room temperature.
S. No.
1.
Compound
kmax (nm)
e
(Mꢀ1 cmꢀ1
)
Transitions
1
211
262
268
281
211
272
334
600
219
268
286
24190
6320
6490
5737
2.
3.
2 [Fe(III)–Fe(III)]
3 [Zn(II)–Zn(II)]
30395
14568
5045
4896
27861
7788
LMCT
8264
Fe1–O5 (acetate) [2.011(4) Å] while the other iron center having
Fe2–N1 (tertiary amine) [2.204(4) Å], Fe2–N3(pyridyl) [2.156(4)
Å], Fe2–O1 (terminal phenolate) [1.878(3) Å], Fe2–O7 (phenoxy
bridge) [2.064(3) Å], Fe2–O4 (acetate) [1.979(4) Å] and Fe2–O6
(acetate) [2.018(4) Å] bond distance Fig. 3.
[2.106(3) Å], Zn1–O3 (terminal phenolate) [1.983(2) Å], Zn1–O1
(phenoxy bridge) [2.019(2) Å] and Zn1–O4 (terminal water)
[2.039(2) Å] whereas, the bond distances around the hexacoordi-
nated zinc center were found to be, Zn2–N2 (tertiary amine)
[2.157(3) Å], Zn2–N1 (pyridyl) [2.133(3) Å], Zn2–O2 (terminal
phenolate) [2.034(2) Å], Zn2–O1 (phenoxy bridge) [2.171(2) Å],
Zn2–O5 (terminal water) [2.059(2) Å] and Zn2–O6 (terminal
water) [2.255(2) Å].
Of particular relevance is the Mꢁ ꢁ ꢁM distance between the two
metal sites in the binuclear iron (2) complex and the binuclear zinc
(3) complex as the same in the active sites of the dinuclear PAP and
zinc phosphoesterase work in synchronicity to bring about the
hydrolysis of phosphoester (PAO) substrates. In this regard it is
worth noting that the Feꢁ ꢁ ꢁFe distance of 3.563(14) Å in the iron
complex (2) is slightly longer than the Mꢁ ꢁ ꢁM distance of 3.1 Å
between the Feꢁ ꢁ ꢁZn centers in the Red kidney bean purple acid
phosphatase[69] and of 3.31 Å between the Feꢁ ꢁ ꢁFe distance in
the Mammalian purple acid phosphatase [70]. The comparatively
In case of the zinc complex (3), the absence of the exogenous
acetate bridges was confirmed by both, the IR spectroscopy and
the X-ray diffraction results. In this complex, two types of the zinc
centers were present, one hexacoordinated and the other pentaco-
ordinated. The hexacoordinated distorted octahedral zinc center
had a [N2O4] type coordination formed from a tertiary amine-N,
a pyridyl-N, a terminal phenolate-O, two H2O molecules and a
bridging phenoxy-O atom and the pentacoordinated distorted trig-
onal bipyramidal zinc center had a [N2O3] type coordination
formed from a tertiary amine-N, a pyridyl-N, a terminal pheno-
late-O, a H2O molecule and a bridging phenoxy-O atom. The bond
distances around the pentacoordinated zinc center were found to
be, Zn1–N3 (tertiary amine) [2.143(3) Å], Zn1–N4 (pyridyl)

C. Pathak et al. / Polyhedron 145 (2018) 88–100
91
well with that of the other related [Fe(III)Fe(III)] (3.528(8) Å) [56]
and [Zn(II)Zn(II)] (3.620 Å) complexes [71] known in the literature.
A glimpse of the nature of interaction occurring between the
two dinuclear [Fe(III)ꢀFe(III)] sites in the iron complex (2) can be
obtained by variable temperature magnetic susceptibility mea-
surements performed at 500 Oe in the temperature range of 4–
300 K. The magnetic data were fitted for the iron complex (2) using
the PHI software [72]. The temperature dependence of vMT for the
powder samples of the complexes is shown in Fig. 4 and Support-
ing Information Figs. S12–S14 and the parameters from the fitted
data are shown in Supporting Information Tables S1–S2. In partic-
ular, the variable temperature magnetic susceptibility experiment
showed a weak antiferromagnetic coupling with an effective mag-
netic moment (leff) of 7.84 lB at room temperature, occurring
between the two dinuclear Fe(III)ꢀFe(III) sites in the iron complex
(2) containing five unpaired electrons (high spin, S = 5/2) at each Fe
(III) center. The decrease in
K supports the antiferromagnetic coupling occurring in this com-
v
MT to a value of 0.04 cm3 molꢀ1 K at 4
Fig. 2. ORTEP of 2 with thermal ellipsoids drawn at the 50% probability level. A
ClOꢀ4 anion has been omitted for the sake of clarity. Selected bond lengths (Å) and
angles (°) : Fe1–O2 1.873(3), Fe1–O3 1.985(4), Fe1–O5 2.011(4), Fe1–O7 2.062(3),
Fe1–N4 2.160(4), Fe1–N2 2.177(4), Fe2–O1 1.878(3), Fe2–O4 1.979(4), Fe2–O6
2.018(4), Fe2–O7 2.064(3), Fe2–N3 2.156(4), Fe2–N1 2.204(4), Fe1–O7ꢀFe2 119.43
(16).
plex. The curve fitting of variable temperature vMT plot gave g val-
ues of gFe = 2.00 and an exchange coupling of J = ꢀ5.87 cmꢀ1 which
are comparable with the earlier observed gFe (2.00) [56,73] and the
exchange coupling in the range of J [ꢀ(6.0–6.4) cmꢀ1] for related
high spin [Fe(III), Fe(III)] complexes known in the literature
[56,73].
larger Mꢁ ꢁ ꢁM distance in the iron complex (2) as compared to that
in the enzyme active site is due to the presence of two bridging
acetate moieties in the model iron complex (2) as compared to
one hydroxide moiety bridging the two metal centers in the
enzyme active site.
The Mꢁ ꢁ ꢁM distance of 3.695(8) Å in the zinc complex (3) is
marginally larger than that of the iron complex (2) [3.563(14) Å],
and this may be due to the absence of any bridging acetate or
hydroxo moieties between the two zinc centers in the zinc com-
plex (3). The Znꢁ ꢁ ꢁZn distance of 3.695(8) Å in the zinc complex
(3) is also slightly longer than the Znꢁ ꢁ ꢁZn distance of 3.4 Å in the
active site of zinc phosphoesterase as isolated from P. diminuta
bacteria [13]. Lastly the Mꢁ ꢁ ꢁM distance of 3.563(14) Å in the iron
complex (2) and of 3.695(8) Å in the zinc complex (3) also correlates
An insight in to the nature of the equilibria that exist in aqueous
medium for the iron complex (2) and the zinc complex (3) were
obtained from spectrophotometric titration performed under
kinetic conditions in a mixed medium of CH3CN/H2O (v/v 1:1) over
a pH range of ca. 5.50–10.2 (Tables 3 and 4 and Supporting Infor-
mation Table S3). In this regard, the characteristic LMCT band of
the iron complex (2) showed a hypsochromic shift from kmax value
at ca. 600 nm in CH3CN to ca. 543.5 nm in the mixed medium of
CH3CN/H2O (v/v 1:1) indicating a change in the coordination envi-
ronment of the dinuclear [Fe(III)-Fe(III)] sites as a result of the
replacement of two kinetically labile bridging acetate moieties
with three H2O molecules, leading to the formation of different
aquated species in solution. The zinc (3) complex however, did
not show any such significant hypsochromic shift as it did not
Table 2
X-ray crystallographic data for 2 and 3.
Compound
2
3
Lattice
Formula
Formula weight
Crystal size (mm)
Space group
a (Å)
monoclinic
C44H45ClFe2N4O13
984.99
monoclinic
C38H47ClN4O11Zn2
902.03
0.39 ꢂ 0.27 ꢂ 0.26
0.44 ꢂ 0.35 ꢂ 0.28
P21/n
P21/c
16.545(7)
12.367(4)
20.968(8)
90
14.523(4)
14.419(4)
19.663(6)
90
b (Å)
c (Å)
a
(°)
b (°)
92.037(6)
90
4288(3)
95.336(5)
90
4100(2)
c
(°)
V (Å3)
Z
4
4
T (K)
100(2)
100(2)
Radiation (k, Å)
0.71075
1.526
0.811
4.43 to 25.36
32117
0.71075
1.461
1.298
4.37 to 25.33
30589
q
(calcd.), (g cmꢀ3
)
Absorption coefficient (mmꢀ1
h range (deg.)
)
Reflections collected
Data/restraints/parameters
Independent reflections [Rint
Completeness to h = 25.000
7773/0/577
7773 [0.0472]
99.1%
7482/0/515
7482 [0.0422]
98.4%
]
Final R indices [I > 2(
R indices (all data)
r
)]
R1 = 0.0769, wR2 = 0.1621
R1 = 0.0785, wR2 = 0.1629
1.184
0.878 and ꢀ0.774
R1 = 0.0473, wR2 = 0.1109
R1 = 0.0528, wR2 = 0.1145
1.065
0.962 and ꢀ0.719
Goodness-of-fit (GOF) on F2
Largest difference in peak and hole (e Ǻꢀ3
)

92
C. Pathak et al. / Polyhedron 145 (2018) 88–100
the diaquated and monohydroxo species, {L[(H2O)ZnII(
(OH)]}, (3B), to the monoaquated and dihydroxo species, {L[(H2O)
ZnII( -OH)ZnII(OH)]}ꢀ, (3C), to the trihydroxo species, {L[(HO)ZnII
-OH)ZnII(OH)]}2ꢀ, (3D), (Scheme 4) and which corresponds for
l
-H2O)ZnII
l
(l
the pKa values of 5.59 (pKa1), 6.78 (pKa2) and 8.70 (pKa3) for the iron
(2) complex and a pKa of 7.00–7.58 for the zinc (3) complex. Quite
significantly, for the iron (2) complex the diaquated and monohy-
droxo species {L[(H2O)FeIII( -H2O)FeIII(OH)]}2+, (2B) (Table 5, Sup-
l
porting Information Fig. S15), the monoaquated and dihydroxo
species, {L[(H2O)FeIII( -OH)FeIII(OH)]}+, (2C) (Table 5, Supporting
Information Fig. S16) and for the zinc (3) complex the monoa-
quated and dihydroxo species {L[(H2O)ZnII( -OH)ZnII(OH)]}ꢀ, (3C)
l
l
(Table 5, Supporting Information Fig. S77), have been detected by
ESI-MS studies.
Considering the active site of PAP [66] and zinc phospho-
esterase [4,7] and their corresponding activity studies, the monoa-
quated and dihydroxo species for the iron complex (2), {L[(H2O)
FeIII(
l
-OH)FeIII(OH)]}+, (2C), and the zinc complex (3), {L[(H2O)
ZnII(
l
-OH)ZnII(OH)]}ꢀ, (3C), displaying pKa of 6.78–7.55 in the pH
range of ca. 6.00–8.00 is proposed to be the active species of the
phosphoester cleavage reaction.
Both of the iron (2) complex and the zinc (3) complex efficiently
hydrolyzed the activated bis(2,4-dinitrophenyl)phosphate (2,4-
BDNPP) substrate in the mixed medium of CH3CN/H2O (v/v 1:1)
and the pH range of 5.5–10.5 at room temperature similar to that
of the PAP and zinc phosphoesterase enzymes [4]. The hydrolysis
reaction was monitored spectrophotometrically at 400 nm
Fig. 3. ORTEP of 3 with thermal ellipsoids drawn at the 50% probability level. A
ClOꢀ4 anion has been omitted for the sake of clarity. Selected bond lengths (Å) and
angles (°) : Zn1–O3 1.983(2), Zn1–O1 2.019(2), Zn1–O4 2.039(2), Zn1–N4 2.106(3),
Zn1–N3 2.143(3), Zn2–O2 2.034(2), Zn2–O5 2.059(2), Zn2–N1 2.133(3), Zn2–N2
2.157(3), Zn2–O1 2.171(2), Zn2–O6 2.255(2), Zn1–O1ꢀZn2 123.73(10).
(e
= 12100 Mꢀ1 cmꢀ1 of the 2,4-DNPꢀ anion) at 25 °C, as reported
in literature [39] and which yielded a curve with a maxima at pH
6.5 for the iron complex (2) (Fig. 5). Likewise, the pH profile curve
for the zinc complex (3) in the pH range of 5.5–10.5, displayed a
maxima at pH 7.5 (Fig. 5). These pH profile curves observed for
the iron complex (2) and the zinc complex (3) are analogous to that
of the related homodinuclear complexes [34,58] and the PAP [74]
and zinc phosphoesterase [6] enzymes [4,7]. A sigmoidal fitting
to the declining part of the pH profile curve of the iron complex
(2) gave the pKa values of 8.74 which are in good agreement with
the pKa3 value of 8.70 obtained by spectrophotometric titration
studies (Supporting Information Fig. S51). Similarly, a sigmoidal
curve fitting to the pH profile of the zinc complex (3) yielded the
values of 7.05 and 8.44, in good agreement with the pKa values
of 7.00 and 7.58 as obtained by spectrophotometric titration stud-
ies (Supporting Information Figs. S112 and S113).
It is worth mentioning that for both of the iron complex (2) and
the zinc complex (3), the maximal rates were observed in the pH
range that is at the pH range that fall between the pKa1 and pKa2,
where the concentration of the monoaquated and dihydroxo spe-
Fig. 4. Variable temperature
(the solid line represents the best fit curve).
vMT plot of powder sample of 2 measured at 500 Oe
cies of the type {L[(H2O)FeIII(
l
-OH)FeIII(OH)]}+, (2C) and {L[(H2O)
ZnII(
l
-OH)ZnII(OH)]}ꢀ (3C), are significant. Since the initial rates
(V0) are proportional to the concentration of the active species,
the maximum value of the initial rate (V0) at a particular pH
implies the highest concentration of the active species at that pH
thereby indicating that the monoaquated and dihydroxo species
to be the active species in the hydrolysis reaction analogous to that
of mammalian [74–77] and plant PAPs [11] and the zinc phospho-
esterase enzyme [4] and to a small-molecule enzyme model con-
sisting of a dinuclear [Fe(III), Fe(III)] [58] and [Zn(II),Zn(II)] [34]
complexes.
contain any kinetically labile bridging acetate moieties and did not
undergo any change in the coordination environment of the zinc
centers.
In the spectrophotometric titration studies, both of the com-
plexes exhibited three isobestic points representing the an equilib-
rium between the four aquated species as a function of pH for
example, the iron complex (2), showed isobestic points in the pH
range of 5.50–5.75, 6.00–8.00 and 8.40–10.2, representing a con-
version between the triaquated species, {L[(H2O)FeIII(
(H2O)]}3+
{L[(H2O)FeIII(
dihydroxo species, {L[(H2O)FeIII(
trihydroxo species, {L[(HO)FeIII( -OH)FeIII(OH)]}, (2D), (Scheme 3).
l
-H2O)FeIII
(2A), to the diaquated and monohydroxo species,
-H2O)FeIII(OH)]}2+, (2B), to the monoaquated and
-OH)FeIII(OH)]}+, (2C), to the
Another explanation that indicates the monoaquated and dihy-
droxo species of the type {L[(H2O)FeIII( -OH)FeIII(OH)]}+, (2C), and
l
,
{L[(H2O)ZnII( -OH)ZnII(OH)]}ꢀ (3C), to be the active species, is the
l
l
l
presence of a labile Fe(III)ꢀ(H2O) moiety in (2C) and Zn(II)ꢀ(H2O)
moiety in (3C) which provides a binding spot for the bis(2,4-dini-
trophenyl)phosphate (2,4-BDNPP) substrate, and the hydroxo moi-
ety bound to the adjacent metal center undergoes intramolecular
nucleophilic attack towards the P atom of the adjacent metal
l
Similarly, the zinc complex (3), showed the three isobestic points
in the pH range of 6.8–10.2, representing a conversion between
the triaquated species, {L[(H2O)ZnII( -H2O)ZnII(H2O)]}+, (3A), to
l

C. Pathak et al. / Polyhedron 145 (2018) 88–100
93
Table 3
Electronic spectrum (Column 1) in different pH ranges [(a) 5.50–5.75, (b) 6.00–8.00, (c) 8.40–10.2] and the corresponding titration plot (Column 2)
performed at 543.5 nm (LMCT band) as a function of pH with incremental additions of 0.1 M NaOH [5 lL in case of (a) and 20 lL in case of (b) and (c)] in an
CH3CN/H2O (v/v 1:1) solution of 2 (1.5 ꢂ 10ꢀ4 M) containing I = 0.1 M (KCl).
Table 4
Spectrophotometric titration data of 2 and 3.
S. No.
1.
Metal complex
kmax(nm)
pKa
2 [Fe(III)–Fe(III)]
pKa1
pKa2
pKa3
pKa
pKa
pKa
5.59
6.78
8.70
7.58
7.55
7.00
543.5
2.
3 [Zn(II)–Zn(II)]
216
264
287

94
C. Pathak et al. / Polyhedron 145 (2018) 88–100
Scheme 3. Equilibrium of chemical species in CH3CN/H2O solution proposed for the dinuclear {L[FeIII
ligand 1.
(l
-OAc)2FeIII]}ClO4 (2) complex supported over symmetrical [N4O3] type
Scheme 4. Equilibrium of chemical species in CH3CN/H2O solution proposed for the dinuclear {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) complex supported over symmetrical [N4O3]
type ligand 1.
bound bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) substrate
leading to phosphoester bond cleavage. Under acidic conditions
i.e., at a pH lower than pKa1, the Fe(III)ꢀ(OH) moiety in the iron
species (2C) and Zn(II)ꢀ(OH) moiety in the zinc species (3C) gets
protonated, preventing the intramolecular nucleophilic attack,
thereby decreasing the reaction rate, whereas under basic condi-
tions at a pH higher than pKa3, the Fe(III)ꢀ(H2O) in the iron species
(2C) and Zn(II)ꢀ(H2O) moiety in the zinc species (3C) gets deproto-

C. Pathak et al. / Polyhedron 145 (2018) 88–100
95
Table 5
Table represents the chemical species in equilibrium in CH3CN/H2O (v/v 1:1) as detected by ESI-MS experiments. The red and black line represents the simulated and the
experimental data respectively.
It is interesting to note that the substrate binding affinity as
obtained from the respective Km and Kass (Kass ffi 1/Km) values of
these two complexes were significantly different. The iron (2) com-
plex showed better substrate binding affinity as evident from its
low Km value of 7.8 ꢂ 10ꢀ4 M and a high Kass (Kass ffi 1/Km) value
of 1282 Mꢀ1 in comparison to the zinc (3) complex exhibiting a
higher Km value of 4.7 ꢂ 10ꢀ3 M and a lower Kass value of 210
Mꢀ1. The kcat values, representing, the rate of the hydrolysis reac-
tion of the bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) substrate
as catalyzed by the iron complex (2) [2.9 ꢂ 10ꢀ4 sꢀ1] and the zinc
complex (3) [0.8 ꢂ 10ꢀ5 sꢀ1] were smaller than that of a related
[Fe(III), Fe(III)] type complex (1.2 ꢂ 10ꢀ3 sꢀ1) [58] and a related
[Zn(II), Zn(II)] type complex (2.4 ꢂ 10ꢀ3 sꢀ1) [49]. These kcat values
were however, ca. 200–9000 times faster than the uncatalysed
reaction [78], with the iron complex (2) being more active. Addi-
tionally, the kcat value of 2.9 ꢂ 10ꢀ4 sꢀ1 for the iron (2) complex
was found to be higher as compared to the heterodinuclear com-
Fig. 5. A pH profile overlay of the initial rates for the hydrolysis of bis(2,4-
dinitrophenyl)phosphate (2,4-BDNPP) as catalyzed by {L[FeIII -OAc)2FeIII]}ClO4 (2)
(l
plexes reported by us for a related [M(II)–Fe(III)] {M = Zn (0.40 ꢂ
and {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) type complexes in CH3CN/H2O (v/v1:1).
10ꢀ4 sꢀ1), Ni (1.90 ꢂ 10ꢀ4 sꢀ1), Co (0.40 ꢂ 10ꢀ4 sꢀ1
) and Cu
(0.014 ꢂ 10ꢀ4 sꢀ1)} type complexes [64].
nated decreasing lability of the-OH moiety at the iron and the zinc
sites. This prevents the bis(2,4-dinitrophenyl)phosphate (2,4-
BDNPP) substrate from coordinating to the iron and the zinc sites.
Saturation kinetic data were obtained at pH 6.5 for the iron
complex (2), and at pH 7.5 for the zinc complex (3) and the data
so obtained were fitted to Michaelis-Menten equation. Specifically
the linear fitting of the curves using the Lineweaver-Burk method
gave the Km, Vmax and kcat parameter (Table 6 and Fig. 6 and Sup-
porting Information Figs. S70, S71 and S129, S130).
Based on the spectrophotometric titration data and the kinetic
data, a mechanism have been proposed for the hydrolysis of bis
(2,4-dinitrophenyl)phosphate (2,4-BDNPP) substrate by iron com-
plex (2) and the zinc complex (3) (Scheme 5,6).
Equation 1. Hydrolysis of 2,4-bis(dinitrophenyl)phosphate as
catalyzed by dinuclear {L[FeIII( -OAc)2FeIII]}ClO4 (2) and {L[ZnII
l
(H2O)2ZnII(H2O)]}ClO4 (3) complexes supported over symmetrical
[N4O3] ligand 1.

96
C. Pathak et al. / Polyhedron 145 (2018) 88–100
Table 6
Kinetic parameters of complexes 2 and 3.
S. No.
Complex
KM (mM)
Kass (mMꢀ1
)
Vmax (10ꢀ8 ꢂ Msꢀ1
)
kcat (10ꢀ4 ꢂ sꢀ1
)
kcat/KM (10ꢀ4 ꢂ Mꢀ1 sꢀ1
)
kcat/kuncat (104)
1.
2.
2 [Fe(III)–Fe(III)]
3 [Zn(II)–Zn(II)]
0.78
4.75
1.28
0.21
1.47
0.04
2.94
0.08
3.72
0.02
0.90
0.02
Reaction condition: 0.05 mM catalyst (2/3), 0.5–5.0 mM bis(2,4-dinitrophenyl)phosphate (for 2) and 0.5–4.0 mM bis(2,4-dinitrophenyl)phosphate (for 3) and 0.05 M buffer
(pH 6.5 for 2, and pH 7.5 for 3) with ionic strength of 0.1 M NaClO4 in a total 2.5 ml CH3CN/H2O (v/v 1:1) at 25 °C. kuncat = 3.2 ꢂ 10ꢀ8 sꢀ1 [78].
Both of the iron complex (2) and the zinc complex (3) were catalyt-
ically active towards the phosphodiester bond cleavage of bis(2,4-
dinitrophenyl)phosphate (2,4-BDNPP) substrate in CH3CN/H2O (v/v
1:1) medium analogous to that of the PAP and zinc phospho-
esterase enzymes.
A
monoaquated and dihydroxo species,
{L[(H2O)FeIII( -OH)FeIII(OH)]}+, (2C), and {L[(H2O)ZnII(
l
l
-OH)ZnII
(OH)]}ꢀ (3C), is proposed to be the catalytically active species for
the hydrolysis reaction based on the spectrophotometric titration
data and the kinetic data. A catalytic pathway has been proposed
that involves the intramolecular nucleophilic attack by the adja-
cent Fe(III)ꢀ(OH) moiety in (2C) and by the adjacent Zn(II)ꢀ(OH)
moiety in (3C) on to the P atom of the of the metal bound 2,4-
BDNPP species, yielding the hydrolyzed 2,4-dinitrophenol product.
Finally, it can thus be concluded that the homodinuclear iron (2)
and the zinc (3) complexes successfully hydrolyzed the cleavage
of phosphoester bond and therefore, mimics the functional charac-
teristics of the PAP and the zinc phosphodiesterase enzyme.
Fig. 6. An overlay of the Lineweaver Burk plots for the hydrolysis of bis(2,4-
dinitrophenyl)phosphate (2,4-BDNPP) as catalyzed by {L[FeIII -OAc)2FeIII]}ClO4 (2)
(l
and {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) type complexes in CH3CN/H2O (v/v1:1).
4. Experimental
As mentioned earlier in the discussion on the aquated species in
equilibria for the iron complex (2) and the zinc complex (3), a
monoaquated and dihydroxo species of the type {L[(H2O)FeIII
4.1. General procedures
All manipulations were carried out using a combination of a
(l
l
-OH)FeIII(OH)]}+, (2C), for the iron complex (2) and {L[(H2O)ZnII
glovebox
and
standard
Schlenk
techniques.
2-Ami-
(
-OH)ZnII(OH)]}ꢀ (3C), for the zinc complex (3) is presumed to
nomethylpyridine, Fe(ClO4)3ꢁ9H2O and Zn(ClO4)3ꢁ6H2O were
obtained from Sigma Aldrich Chemical Co., while all the other
chemicals used are from spectrochem (India) were used without
any further purification. 2,6-bis(hydroxymethyl)-4-t-butylphenol
[79], 2,6-bis(chloromethyl)-4-t-butylphenol [65], N-(2-hydroxy-
benzyl)-N-(2-pyridylmethyl)amine) [26] were synthesized accord-
ing to modified literature procedures. 1H and 13C{1H} NMR spectra
were recorded on a Bruker 400 MHz and Bruker 500 MHz NMR
spectrometer. 1H NMR peaks are labeled as singlet (s), doublet
(d), triplet (t), quartet (q), quartet of doublets (qd), doublet of dou-
blets (dd) septet (sept) and broad (br). Mass spectrometry mea-
surements were done on a Micromass Q-Tof spectrometer and
Bruker maxis impact spectrometer. The electronic spectra of 2
and 3 complexes are recorded in CH3CN using a UV 3600 Shimadzu
and Cary Bio spectrophotometer. Elemental Analysis was carried
out on Thermo Finnigan FLASH EA 1112 SERIES (CHNS) Elemental
Analyzer. The variable temperature magnetic experiments were
executed in SQUID-VSM, quantum design, USA and the data was
fitted using PHI software [72].
be the active species that react with the bis(2,4-dinitrophenyl)
phosphate (2,4-BDNPP) substrate in the pH range of ca. 6.00–8.00.
The bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) substrate would
replace a metal bound H2O molecule [Fe(III)ꢀ(H2O)] from the
Fe(III) center in (2C) and a metal bound H2O molecule [Zn(II)ꢀ
(H2O)] from the Zn(II) center in (3C) resulting in the formation of
a substrate–catalyst complex of the type {L[(R2PO4)FeIII(
l
-OH)
(3E).
FeIII(OH)]} (2E), and {L[(R2PO4)ZnII( -OH)ZnII(OH)]}2ꢀ
l
,
Intramolecular nucleophilic attack by the adjacent Fe(III)ꢀ(OH)
moiety in (2C) and by the adjacent Zn(II)ꢀ(OH) moiety in (3C) on
to the P atom of the metal bound 2,4-BDNPP species would result
in {L[FeIII( -RPO4)FeIII]} (2F) and {L[ZnII(
l-OH)(l l-OH)(l-RPO4)
ZnII]}2ꢀ (3F), type intermediates and the hydrolysis product 2,4-
dinitrophenolate (2,4-DNP). Quite significantly, the substrate–
catalyst adduct for the iron species {L[FeIII(
l
-OH)(
(2F) has been identified using mass spectroscopy (Fig. 7, Support-
ing Information Fig. S72). Lastly, the iron species {L[FeIII(
-OH)
-RPO4)
l
-RPO4)FeIII]}
l
(l l-OH)(l
-RPO4)FeIII]} (2F) and the zinc species {L[ZnII(
ZnII]}2ꢀ (3F) would further react with water resulting in the regen-
eration of the monoaquated and dihydroxo species active species
of the type {L[(H2O)FeIII( -OH)FeIII(OH)]}+, (2C), and {L[(H2O)ZnII
l
4.2. X-ray crystallography
(l
-OH)ZnII(OH)]}ꢀ (3C).
X-ray diffraction data for compounds 2 and 3 were collected on
a Rigaku Hg 724+ diffractometer for unit cell determination and
intensity data collection. Data integration and indexing were car-
ried out using Rigaku suite of programs CrystalClear and Crys-
talStructure. Structure refinement and geometrical calculations
were carried out using programs in the WinGX module [80]. The
final structure refinement was carried out using full least squares
methods on F2 using SHELXL-2014 [81]. Crystal data collection and
refinement parameters are summarized in Table 2.
3. Conclusion
In summary, the homodinuclear complexes of the type {L[FeIII
(l
-OAc)2FeIII]}ClO4 (2) and {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3)
supported over a symmetrical [N4O3] ligand has been synthesized
and structurally characterized. The temperature dependent mag-
netic susceptibility measurement of the iron (2) complex, reveals
a weak antiferromagnetic coupling between the Fe(III) centers.

C. Pathak et al. / Polyhedron 145 (2018) 88–100
97
Scheme 5. Proposed mechanism for the hydrolysis of 2,4-bis(dinitrophenyl)phosphate as catalyzed by a dinuclear {L[FeIII
[N4O3] type ligand 1.
(l
-OAc)2FeIII]}ClO4 (2) supported over symmetrical
Scheme 6. Proposed mechanism for the hydrolysis of 2,4-bis(dinitrophenyl)phosphate as catalyzed by a dinuclear {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3) supported over
symmetrical [N4O3] ligand 1.

98
C. Pathak et al. / Polyhedron 145 (2018) 88–100
Fig. 7. ESI-MS spectra of the substrate–catalyst adduct {L[FeIII -RPO4)FeIII]} (2F). The red and the black lines represent the simulated and the experimental data
(l-OH)(l
respectively. (Colour online.)
4.3. Synthesis of 2,6-bis{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]
methyl}-4-t-butylphenol (1)
g, 1.06 mmol) in MeOH (ca. 10 mL) and the resulting deep blue
solution was stirred for 10 minutes at 40 °C. Upon cooling to room
temperature, the formation of a blue precipitate was observed. The
precipitate was isolated by filtration and washed with 2-propanol
and Et2O to get the product 2 as a blue microcrystalline solid
(0.316 g, 82%). IR data (KBr pellet) cmꢀ1: 3788 (w), 3454 (w),
3055 (w), 2926 (w), 1606 (w), 1569 (s), 1479 (m), 1447 (m),
1292 (w), 1280 (w), 1218 (w), 1121 (w), 1092 (w), 1023 (w), 955
(w), 935 (w), 890 (w), 831 (w), 790 (w), 762 (w), 731 (w), 704
(w), 650 (w), 634 (w), 622 (w), 576 (w). HRMS (ES): m/z
829.1983 [M-ClO4]+, calcd. 829.1983. Anal. Calc. for
A round bottom flask was charged with a solution of N-(2-
hydroxybenzyl)-N-(2-pyridylmethyl)amine (1.93 g, 9.00 mmol)
and Et3N (0.489 g, 4.83 mmol) in MeOH (ca. 10 mL) and cooled to
0 °C. A solution of 2,6-bis(chloromethyl)-4-t-butylphenol (1.00 g,
4.04 mmol) in MeOH (ca. 10 mL) was then added to the reaction
mixture in a dropwise fashion. The resulting solution was stirred
at room temperature overnight. The volatiles were then removed
under vacuum. The residue was dissolved in CHCl3 (ca. 10 mL)
and an equivalent volume of H2O (ca. 10 mL) was added to it.
The reaction mixture was extracted with CHCl3 (ca. 3 ꢂ 20 mL),
dried over anhydrous Na2SO4, filtered, and the solvent was reduced
in vacuum to give the crude product, which was purified by recrys-
tallization from MeOH to yield the product 1 as a white solid
(0.634 g, 26%). 1H NMR (CDCl3, 400 MHz, 25 °C): d 10.88 (br, 3H,
C
42H45ClFe2N4O11ꢁ2H2O: C, 52.27; H, 5.12; N, 5.81. Found: C,
51.87; H, 4.96; N, 5.87%.
4.5. Synthesis of {L[ZnII(H2O)2ZnII(H2O)]}ClO4 (3)
A
solution of 2,6-bis{[(2-hydroxybenzyl)(2-pyridylmethyl)
amino]methyl}-4-t-butylphenol (0.250 g, 0.415 mmol) 1 in MeOH
(ca. 20 mL) was heated to 50 °C, to it was added a solution of Zn
(ClO4)2ꢁ6H2O (0.309 g, 0.829 mmol) in MeOH (ca. 10 mL) and
Et3N (0.084 mg, 0.829 mmol). The resulting solution was allowed
to reflux for 3 hours after which it was filtered. The volatiles were
partially reduced in vacuo to a minimum volume (ca. 10 mL),
which upon standing in air for 12 h yielded pure product as white
crystals. The crystals were isolated by filtration and vacuum dried
to obtain the product 3 as a white crystalline solid (0.211 g, 58%).
IR data (KBr pellet) cmꢀ1: 3409 (w), 2955 (w), 2859 (w), 1608
(m), 1571 (w), 1484 (s), 1448 (m), 1392 (w), 1369 (w), 1282 (w),
1261 (m), 1223 (w), 1114 (s), 1022 (w), 970 (w), 941 (w), 886
(w), 828 (w), 762 (m), 733 (w), 625 (m), 591 (w), 529 (w), 489
(w). HRMS (ES): m/z 727.1609 [M-3H2O-ClO4]+, calcd. 727.1600.
Anal. Calc. for C38H45ClN4O10Zn2ꢁH2O: C, 50.60; H, 5.25; N, 6.21.
Found: C, 50.48; H, 5.32; N, 6.02%.
3
C6H2OH and 2C6H4OH), 8.62 (dd, 2H, JHH = 5 Hz,4JHH = 1 Hz,
3
4
C5H4N), 7.66 (td, 2H, JHH = 8 Hz, JHH = 2 Hz, C5H4N), 7.24 (d, 2H,
3JHH = 8 Hz, C5H4N), 7.22–7.19 (m, 2H, C5H4N), 7.15 (td, 2H, JHH
3
4
3
= 8, JHH = 1 Hz, C6H4OH), 7.07 (s, 2H, C6H2), 7.03 (dd, 2H, JHH = 7
Hz,4JHH = 1 Hz, C6H4OH), 6.82 (dd, 2H, JHH = 8 Hz,4JHH = 1 Hz,
3
3
4
C6H4OH), 6.75 (dt, 2H, JHH = 7, JHH = 1 Hz, C6H4OH), 3.84 (s, 4H,
CH2), 3.80 (s, 4H, CH2), 3.78 (s, 4H, CH2), 1.24 (s, 9H, C(CH3)3). 13C
{1H} NMR (CDCl3, 100 MHz, 25 °C):
d 157.7 (C5H4N), 156.3
(C6H4OH), 153.7 (C6H2), 148.6 (C5H4N), 141.4 (C6H2), 137.1
(C5H4N), 130.1 (C6H4OH), 129.1 (C6H4OH), 127.5 (C6H2), 123.7
(C5H4N), 122.5 (2C6H4OH), 122.3 (C6H2), 119.1 (C5H4N), 116.73
(C6H4OH), 57.7 (CH2), 56.7 (CH2), 54.9 (CH2), 34.0 (C(CH3)3), 31.7
(C(CH3)3). IR data (KBr pellet) cmꢀ1: 3789 (w), 3050 (w), 2952
(w), 2820 (s), 2706 (w), 1597 (m), 1487 (s), 1434 (w), 1375 (w),
1252 (w), 1152 (w), 1095 (w), 1041 (w), 1005 (w), 974 (w), 894
(w), 754 (m). HRMS (ES): m/z 603.3335 [M+H]+, calcd. 603.3330.
Anal. Calc. for C38H42N4O3ꢁ0.5H2O: C, 74.60; H, 7.08; N, 9.16. Found:
C, 74.81; H, 6.52; N, 9.49%.
4.6. General procedure for spectrophotometric titration
1.5 ꢂ 10ꢀ4 M solution of 2 and 1.3 ꢂ 10ꢀ4 M of 3 in CH3CN/H2O
(1:1 v/v) with the ionic strength of 0.1 M KCl was titrated with 0.1
M NaOH solution and change in the position of kmax was observed
with the change in pH of the solution.
4.4. Synthesis of {L[(FeIII( -OAc)FeIII)]}ClO4 (2)
l
4.7. General procedure for phosphoester hydrolysis
NaOAcꢁ3H2O (0.269 g, 1.97 mmol) and 2,6-bis{[(2-hydroxyben-
zyl)(2-pyridylmethyl)amino]methyl}-4-t-butylphenol1 (0.250 g,
0.415 mmol) were added to a solution of Fe(ClO4)3ꢁXH2O (0.377
By using a UV 3600 Shimadzu spectrophotometer increase in
the concentration of the product 2,4-dinitrophenolate (BNP), was

C. Pathak et al. / Polyhedron 145 (2018) 88–100
99
monitored at 25 °C by UV–Vis spectroscopy at 400 nm in quartz
Acknowledgement
cuvettes. The kinetics measurements were done in CH3CN/H2O
(v/v 1:1). For the study of the pH dependence, substrate and com-
plex concentrations were 0.005 M and 3.90 ꢂ 10ꢀ5 M for complex
2 and 3.75 ꢂ 10ꢀ4 M for complex 3, respectively. The ionic strength
and pH were kept constant by using total concentrations of 0.1 M
NaClO4 and 0.05 M buffer MES (2-(N-morpholino)ethanesulfonic
acid) (pH 5.5–6.5), HEPES (4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid) (7.0–8.0), CHES (N-Cyclohexyl-2-
aminoethanesulfonic acid) (8.5–9.0) and CAPS (N-cyclohexyl-3-
aminopropanesulfonic acid) (9.5–10.5). The pH of the buffer was
adjusted in standard solutions using a calibrated pH meter. Each
We thank the Department of Science and Technology
(EMR/2014/000254), New Delhi, India, for financial support of this
research. We gratefully acknowledge the Single Crystal X-ray
diffraction Facility, Department of Chemistry, IIT Bombay, Mumbai,
India, for the crystallographic characterization data and central
facility, Industrial Research and Consultancy Center (IRCC), IIT
Bombay for Physical Property Measurement System (PPMS). C.P.
thanks UGC, New Delhi, India, for research fellowships. M.K.G.
thanks CSIR, New Delhi, India, for research fellowships.
cuvette was prepared by successive addition of 156
l
L of a 0.080
Appendix A. Supplementary data
M
standard solution of bis(2,4-dinitrophenyl)phosphate (2,4-
BDNPP) in CH3CN/H2O (v/v 1:1), 1250
containing 0.2 M NaClO4, 1029 lL of CH3CN for 2 and 969 l
l
L of a 0.1 M buffer solution
L of
CH3CN for 3. After mixing, the background absorption was mea-
sured. Then 65 L of a 0.0015 M standard solution of 2 or 125
CCDC 938705 (for 2) and 968273 (for 3) contains the supple-
mentary crystallographic data. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
l
lL
of a 0.0075 M standard solution for 3 in CH3CN were added and
the increase in absorption overtime was measured at 400 nm.
The initial rates were calculated by fitting a linear line to the curve
corresponding to absorbance for 15 minutes for 2 and 30 minutes
for 3. The total concentration of phenol from the absorption of the
phenolate at 400 nm was calculated from the molar extinction
Supplementary data (1H NMR, 13C{1H} NMR, IR spectra, HRMS
and elemental analysis data of 2,6-bis{[(2-hydroxybenzyl)(2-
pyridylmethyl)amino]methyl}-4-t-butylphenol, (H3L) (1) and com-
plexes 2 and 3.) associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.poly.2018.01.029.
coefficient
e
of BNP (12100 Mꢀ1 cmꢀ1).
For the study of dependence of substrate concentration on the
initial rate, varying volume of stock solution of 2,4-bis-dinitro-
phenyl phosphate (0.08 M) was added to the cuvette to give a total
concentration of 0.5–5.0 mM for 2 and 0.5–4.0 mM for 3, while the
complex concentration was kept constant at 0.05 mM. Kinetic
measurements were done using Michaelis–Menten equation and
the results were evaluated from Lineweaver–Burk plots.
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