Phosphatase models: Synthesis, structure and catalytic activity of zinc complexes derived from a phenolic Mannich-base ligand

Article abstract of DOI:10.1016/j.poly.2015.05.013

A series of dinuclear [Zn₂(L1)₂X₂] (1-3) and mononuclear [Zn(HL2)X₂] complexes (4-6), (X = Cl, Br, I) were synthesised from two Mannich-base compartmental ligands, namely [bis(2-methoxyethyl)aminomethyl]-4-chlor

Full text of DOI:10.1016/j.poly.2015.05.013

Polyhedron 97 (2015) 55–65
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Phosphatase models: Synthesis, structure and catalytic activity of zinc
complexes derived from a phenolic Mannich-base ligand
a,
Ria Sanyal a, Prateeti Chakraborty a, Ennio Zangrando b, , Debasis Das
⇑
⇑
a Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700009, India
b Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dinuclear [Zn2(L1)2X2] (1–3) and mononuclear [Zn(HL2)X2] complexes (4–6), (X = Cl, Br, I)
were synthesised from two Mannich-base compartmental ligands, namely [bis(2-methoxyethyl)amino-
methyl]-4-chlorophenol (HL1) and 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-chlorophenol (HL2),
respectively. They were characterised by routine physicochemical techniques (CHN, UV, IR, ESI-MS and
NMR) and complex 2–5 was further structurally characterised by single crystal X-ray analysis where
the Zn. . .Zn bond-distance is 3.10–3.12 Å. All the quintessential complexes exhibit excellent phosphatase
activity and the experimental first order rate constant values (kcat) for the hydrolysis of 4-nitrophenyl
phosphate ester (PNPP) reaction in methanol are in the range from 1.05 to 214 sꢀ1 at 25 °C evaluated
by monitoring spectrophotometrically the gradual release of p-nitrophenolate (kmax = 427 nm,
Received 20 December 2014
Accepted 10 May 2015
Available online 19 May 2015
Keywords:
Mannich-base
Zinc(II)
Phosphatase
Mechanistic pathway
Halide
e
= 18500 Mꢀ1 cmꢀ1). The coordinated X halides affect the phosphatase activity in the order Br > Cl > I
(in dinuclear complexes) and Cl > Br > I (in mononuclear) and the trend in the two cases has been well
recognised to be due to a different rate determining step. Moreover the influence of chloro atom in
para-position of the phenol ring and the role of solvent have been rationalised by comparing the kinetic
parameters with those obtained for the corresponding methyl analogues having reasonably close struc-
tural resemblance as reported by Sanyal et al. (2014).
Ó 2015 Published by Elsevier Ltd.
1. Introduction
catalytically promiscuous Zn(II) derivatives [13–29] some com-
plexes show low activity, others require rigorous synthetic condi-
Among the model metalloenzymes studied till date, those con-
taining zinc(II) constitute by far the widest category [1,2]. Indeed,
there has been a remarkable success in the identification of zinc-
dependent enzymes attributing to emerging crystallographic and
spectroscopic techniques [3]. As a matter of fact, each of the funda-
mental enzyme class, namely, oxidoreductases, transferases,
hydrolases, lyases, isomerases, and ligases, is a benchmark of a
zinc(II) active site [4–6]. Undoubtedly, phosphatase activity is
one of the outstanding functions among the versatile range of
enzymatic behaviour of divalent zinc ion [7–10] paving the path
for an extensive study of various functional mimics of phospho-
esterases. Over the years, synthetic Zn(II) (and also other transition
metal) complexes have been judiciously studied as phosphate ester
models taking into account their extraordinary Lewis acidity, redox
rigidity, nucleophile generation, leaving group stabilization and
physiological relevancy [11,12]. However, among the numerous
tions or have high toxicity. Therefore the development of new
efficient metallocatalysts is still a tough challenge in the field of
biocatalysis.
The aim of this work is neatly directed to expand the compre-
hension of the structure–function relationship of zinc-based model
systems showing phosphatase activity (Scheme 1). Recently we
have reported that mono- and dinuclear Zn(II) complexes derived
from two novel synthesised Mannich-base ligands are not only
capable of promoting the hydrolysis of activated phosphate
monoesters, but likewise were found to possess promising cat-
alytic activity [30]. In this context, here we report the synthesis
and characterisation of a series of similar methoxyamine based
mono- and dinuclear ZnII complexes, where the ligands differ from
those previously reported for the presence in para position of the
phenol ring of a chloro instead of a methyl group (Scheme 2).
The electronic effects of the substituent in para-position and of
the solvent in which the hydrolysis is carried out have been eval-
uated. More importantly, a possible mechanism for the PNPP cleav-
age promoted by Zn2L and the concomitant halide role is proposed
on the basis of kinetic and spectral analysis.
⇑
Corresponding authors.
E-mail addresses: zangrando@units.it (E. Zangrando), dasdebasis2001@yahoo.
com (D. Das).
http://dx.doi.org/10.1016/j.poly.2015.05.013
0277-5387/Ó 2015 Published by Elsevier Ltd.

56
R. Sanyal et al. / Polyhedron 97 (2015) 55–65
NO2
Yield = 6.57 g
(80%).
Elemental
analysis
calcd
for
NO2
ZnII-Complex
MeOH / RT
C13H20N1O3Cl1: C, 57.04; H, 7.36; N, 5.12; O, 17.53; Cl, 12.95; found
C, 57.17; H, 7.43; N, 5.45; O, 17.46; Cl, 12.49%; 1H NMR (300 MHz,
CDCl3, 25 °C): d = 2.79 (t, 4H; N-CH2-CH2), 3.33 (s, 6H; O-CH3), 3.54
(t, 4H; N-CH2-CH2), 3.81 (s, 2H; ph-CH2-N), 6.65 (d, 1H; Ar), 6.88 (s,
1H; o to CH2-N), 7.1 ppm (d, 1H; o to phenolic-OH); 13C NMR
(300 MHz, DMSO-d6, 25 °C): d = 52.66 (2C, O-Me), 56.50 (1C, ph-
CH2-N), 58.05 (2C, N-CH2-CH2), 69.6 (2C, N-CH2-CH2), 117.11 (1C,
Ar), 127.57 (1C, Ar), 128.57 (1C, Ar), 129.49 (1C, Ar), 129.77 (1C,
ONa
O
O
P O
ONa
(4-nitrophenyl)phosphate
disodiumsalt
(PNPP, UV inactive)
4-nitrophenolate
(UV band=400-430 nm)
Ar), 154.97 (1C, Ar-OH); IR data (NaCl plate):
c bar = 644, 831,
1018, 1106, 1256, 1272, 1468, 1581, 2891 cmꢀ1
.
Scheme 1. A schematic depiction of the hydrolysis reaction of PNPP-catalysed by
our Mannich-based mono- and dinuclear zinc(II) complexes.
2.2.2. Synthesis of HL2
To an ethanolic solution (30 ml) of p-chlorophenol (30 mmol,
3.86 g), bis(2-methoxyethyl)amine (90 mmol, 11.99 g) was added
dropwise with constant stirring. After 30 min, 37% (w/v) formalin
solution (90 mmol, 7.5 ml) was added to it. The resulting mixture
was stirred for 1 h at room temperature and then refluxed for
24 h. Then the same procedure-was followed as for HL1. The liquid
ligand was further purified by flash column chromatography (silica
gel, hexane:ethylacetate:triethylamine = 100:5:1) to afford the
desired b-aminophenol.
2. Experimental
2.1. Materials and methods
The anhydrous zinc halide salts (analytical grade) were
obtained from Merck. Common organic reagents and solvents used
for the syntheses were reagent grade and were obtained from com-
mercial sources and redistilled before use. Water used in all phys-
ical measurement experiments was Milli-Q grade. (p-Nitrophenyl)
phosphate (PNPP) was purchased from Sigma–Aldrich and
recrystallized from ethanol/water before use. Elemental analyses
(carbon, hydrogen, and nitrogen) were performed using
PerkinElmer 240C analyzer. Infrared spectra (4000–400 cmꢀ1
were recorded at 28 °C on Shimadzu FTIR-8400S and
PerkinElmer Spectrum Express Version 1.03 using both KBr pellets
and NaCl-plates as mediums. 1H and 13C NMR spectra (300 MHz)
were recorded in CDCl3 and DCON(CD3)2 solvents at 25 °C on a
Bruker AV300 Supercon NMR spectrometer using the solvent sig-
nal as the internal standard in a 5 mm BBO probe. UV–Vis spectra
and kinetic traces were monitored with a Shimadzu 2450 UV–Vis
spectrophotometer equipped with multiple cell-holders and ther-
mostat. Electrospray mass spectra were recorded on a Micromass
Q-TOF mass spectrometer. Molar conductances were measured
with a Systronics conductivity meter 306 at 25 °C.
Yield = 7.1
g
(56.5%). Elemental analysis calcd for
C20H35N2O5Cl1: C, 57.34; H, 8.42; N, 6.69; O, 19.09; Cl, 8.46; found:
C, 57.96; H, 8.11; N, 6.77; O, 18.78; Cl, 8.38%.
1H NMR (300 MHz, CDCl3, 25 °C): d = 2.81 (t, 8H; N-CH2-CH2),
3.32 (s, 12H; O-CH3), 3.53 (t, 8H; N-CH2-CH2), 3.8 (s, 4H; ph-CH2-
N), 6.96 ppm (s, 2H; Ar); 13C NMR (300 MHz, DMSO-d6, 25 °C):
d = 52.41 (4C, O-Me), 56.45 (2C, ph-CH2-N), 58.10 (4C, N-CH2-
CH2), 69.51 (4C, N-CH2-CH2), 117.12 (1C, Ar), 127.21 (2C, Ar),
a
)
a
129.30 (2C, Ar), 155.31 (1C, Ar-OH); IR data (NaCl plate):
c
bar = 644, 831, 1107, 1272, 1468, 1581, 2891 cmꢀ1
2.3. Synthesis of zinc(II) complexes
2.3.1. [Zn2(L1)2Cl2] (1)
.
To a methanolic solution (20 mL) of HL1 (0.5 mmol, 136 mg),
anhydrous zinc chloride (0.5 mmol, 112.6 mg) in 10 mL of metha-
nol was added. The yellow reaction mixture was refluxed for 1 h,
cooled to room temperature, filtered and subsequently kept in a
beaker. Good quality crystalline compound was obtained from
the filtrate.
2.2. Syntheses of ligands
2.2.1. Synthesis of HL1
Yield = 116 mg
(62%).
Elemental
analysis
calcd
for
To an ethanolic solution (50 ml) of p-chlorophenol (30 mmol,
3.86 g), bis(2-methoxyethyl)amine (30 mmol, 3.99 g) was added
dropwise with constant stirring. After 30 min, 37% (w/v) formalin
solution (30 mmol, 2.43 ml) was added to it. The resulting mixture
was stirred for 1 h at room temperature and then refluxed for 8 h.
It was evaporated under reduced pressure and the yellow oil was
extracted with saturated brine solution and diethyl-ether several
times. The organic phase was separated, dried with anhydrous
MgSO4, concentrated by evaporation of ether and subsequently
vacuum-dried for the removal of last traces of water.
C26H38N2Cl4O6Zn2: C, 41.79; H, 5.13; N, 3.75; Cl, 18.98; O, 12.85;
Zn, 17.50; found: C, 41.56; H, 5.28; Cl, 19.05; N, 3.76; O, 12.83;
Zn, 17.52%; 1H NMR (300 MHz, DMF-d7, 25 °C): d = 2.85 (t, 8H; N-
CH2-CH2), 3.00 (s, 12H; O-CH3), 3.38 (t, 8H; N-CH2-CH2), 3.92 (s,
4H; ph-CH2-N), 6.8 (d, 2H; Ar), 7.31 (d, 2H;
o to CH2-N),
8.15 ppm (s, 2H; o to phenolic-OH); 13C NMR (300 MHz, DMSO-
d6, 25 °C): d = 52.60 (2C, O-Me), 56.40 (1C, ph-CH2-N), 58.07 (2C,
N-CH2-CH2), 69.58 (2C, N-CH2-CH2), 117.30 (1C, Ar), 122.88 (1C,
Ar), 127.11 (1C, Ar), 128.58 (1C, Ar), 129.50 (1C, Ar), 154.91 (1C,
Ar-OH); ESI-MS m/z: Calcd for [C13H20Cl2NO3Zn]+ = 374.691,
Found = 374.691; FT-IR data (KBr pellet):
c bar = 662, 784, 834,
1020, 1112, 1270, 1470, 1592, 2932 cmꢀ1
.
Cl
Cl
2.3.2. [Zn2(L1)2Br2] (2)
The complex was prepared by the same procedure as above by
using anhydrous zinc bromide as the metal salt. After 1 day,
colourless square single crystals appeared in a yellow oil and were
washed with cold methanol and ether to obtain good crystals of X-
ray crystallographic quality.
N
O
OH
N
O
N
O
OH
O
O
O
Yield = 102 mg
(49%).
Elemental
analysis
calcd
for
C
26H38N2Br2Cl2O6Zn2: C, 37.35; H, 4.58; N, 3.35; Br, 19.11; Cl,
HL1
HL2
8.48; O, 11.48; Zn, 15.65; found: C, 37.42; H, 4.41; N, 3.43; Br,
Scheme 2. Mannich-base ligands used in the present study.
19.28; Cl, 8.44; O, 11.44; Zn, 15.58%; 1H NMR (300 MHz, DMF-d7,

R. Sanyal et al. / Polyhedron 97 (2015) 55–65
57
25 °C): d = 2.88 (t, 8H; N-CH2-CH2), 3.08 (s, 12H; O-CH3), 3.42 (t,
FT-IR data (KBr pellet):
c
bar = 664, 785, 833, 1019, 1111, 1270,
8H; N-CH2-CH2), 3.97 (s, 4H; ph-CH2-N), 6.55 (d, 2H; Ar), 7.23 (d,
2H; o to CH2-N), 8.01 ppm (s, 2H; o to phenolic-OH); 13C NMR
(300 MHz, DMSO-d6, 25 °C): d = 52.61 (2C, O-Me), 56.35 (1C, ph-
CH2-N), 58.04 (2C, N-CH2-CH2), 69.55 (2C, N-CH2-CH2), 117.39
(1C, Ar), 122.84 (1C, Ar), 127.13 (1C, Ar), 128.59 (1C, Ar), 129.45
(1C, Ar), 154.93 (1C, Ar-OH); ESI-MS m/z: Calcd for
[C13H20ClBrNO3Zn]+ = 419.044, Found = 419.59; FT-IR data (KBr
1470, 1592, 2932 cmꢀ1
.
2.3.6. [Zn(HL2)I2] (6)
It was also synthesised by following the same method as for
complex 4 by using zinc iodide. A colourless crystalline compound
of good transparency was obtained and well characterised.
Yield = 502 mg
(68%).
Elemental
analysis
calcd
for
pellet):
c
bar = 661, 1268, 1470, 1592 cmꢀ1
.
C20H35N2I2ClO5Zn: C, 32.54; H, 4.78; N, 3.79; I, 34.38; Cl, 4.80; O,
10.84; Zn, 8.87; found: C, 32.58; H, 4.76; N, 3.84; I, 34.41; Cl, 4.77;
O, 10.80; Zn, 8.84%. 1H NMR (300 MHz, DMF-d7, 25 °C): d = 2.78 (t,
8H; N-CH2-CH2), 3.31 (s, 12H; O-CH3), 3.50 (t, 8H; N-CH2-CH2),
4.02 (s, 4H; ph-CH2-N), 6.98 ppm (s, 2H; Ar); 13C NMR (300 MHz,
DMSO-d6, 25 °C): d = 52.44 (4C, O-Me), 56.45 (2C, ph-CH2-N),
58.21 (4C, N-CH2-CH2), 69.54 (4C, N-CH2-CH2), 117.32 (1C, Ar),
127.17 (2C, Ar), 129.37 (2C, Ar), 155.60 (1C, Ar-OH); ESI-MS m/z:
Calcd for [C20H35ClIN2O5Zn]+ = 611.21, Found = 612.37; FT-IR data
2.3.3. [Zn2(L1)2I2] (3)
Anhydrous zinc iodide (0.5 mmol, 319.19 mg) in methanol
(5 mL) was added dropwise to a methanolic solution (10 mL) of
the ligand L1 (0.5 mmol, 136 mg), and the mixture was stirred
for 2 h. The resulting yellow solution was filtered. Single crystals
suitable for X-ray diffraction experiment were collected after the
evaporation of solution.
Yield = 163 mg
(70%).
Elemental
analysis
calcd
for
(KBr pellet):
c .
bar = 641, 761, 1117, 1197, 1458, 1589, 2877 cmꢀ1
C26H38Cl2N2I2O6Zn2: C, 33.58; H, 4.12; I, 27.29; Cl, 7.62; N, 3.01;
O, 10.32; Zn, 14.06; found: C, 33.48; H, 4.27; N, 3.06; I, 27.31; Cl,
7.66; O, 10.28; Zn, 13.94%; 1H NMR (300 MHz, DMF-d7, 25 °C):
d = 2.86 (t, 8H; N-CH2-CH2), 3.06 (s, 12H; O-CH3), 3.39 (t, 8H; N-
CH2-CH2), 3.95 (s, 4H; ph-CH2-N), 6.79 (d, 2H; Ar), 7.28 (d, 2H; o
2.4. X-ray data collection
Data collection for compounds 2–5 were carried out at room
temperature on a Bruker Smart Apex diffractometer equipped with
CCD and monochromatized Mo Ka radiation (k = 0.71073 Å). Cell
to CH2-N), 8.19 ppm (s, 2H;
o
to phenolic-OH); 13C NMR
(300 MHz, DMSO-d6, 25 °C d = 52.66 (2C, O-Me), 56.34 (1C, ph-
CH2-N), 58.03 (2C, N-CH2-CH2), 69.50 (2C, N-CH2-CH2), 117.42
(1C, Ar), 122.89 (1C, Ar), 127.03 (1C, Ar), 128.55 (1C, Ar), 129.48
(1C, Ar), 154.97 (1C, Ar-OH); ESI-MS m/z: Calcd for
[C13H20ClINO3Zn]+ = 466.134, Found = 465.98; FT-IR data (KBr pel-
refinement, indexing, and scaling of the data sets were done by
using the Bruker Smart Apex and Bruker Saint packages [31]. An
absorption correction was applied to all the data sets [32]. The
structures were solved by direct methods and subsequent Fourier
analyses and refined by the full-matrix least squares method based
let):
c
bar = 665, 786, 835, 879, 957, 1022, 1109, 1185, 1470, 1590,
on F2 with all observed reflections [33]. The
DF map of 3 revealed a
2884 cmꢀ1
.
disordered methoxy group O(6)–C(34), which could not be suitably
modelled. Crystal of 5 (of small dimensions) was low diffracting,
but attempts to obtain crystals of better quality were fruitless.
Hydrogen atoms were fixed at geometrical positions. All the calcu-
lations were performed using the WinGX System, Ver 1.80.05.37
[34].
2.3.4. [Zn(HL2)Cl2] (4)
To a solution of ligand HL2 (1 mmol, 418 mg) in acetonitrile
(25 mL), anhydrous ZnCl2 (2.5 mmol, 340.5 mg) dissolved in ace-
tonitrile (10 mL) was added and refluxed for 30 min. The mixture
was filtered and kept in a beaker for slow evaporation. Colourless
rectangular crystals, formed after 2 weeks, were washed with ether
to obtain single crystals of good crystallographic quality.
2.5. Kinetic measurements of the hydrolysis of PNPP in methanol
Yield = 327 mg
(59%).
Elemental
analysis
calcd
for
Disodium (4-nitrophenyl)phosphate hexahydrate (PNPP) was
used as substrate and the solvents chosen for this study were aque-
ous DMF and methanol. The solutions of substrate PNPP and Zn
complexes in each solvent were freshly prepared maintaining the
total volume of the reaction mixture at 2 mL. An initial screening
of the hydrolytic propensities of all the metal-complexes was per-
formed till the formation of 2% of p-nitrophenolate (ꢁ2 h) before to
collect kinetic data. The hydrolysis rate of PNPP in the presence of
complexes 1–6 was measured by an initial rate method following
the absorption increase at 427 nm due to the released 4-nitrophe-
C20H35N2Cl3O5Zn: C, 43.26; H, 6.35; Cl, 19.16; N, 5.04; O, 14.41;
Zn, 11.78; found: C, 43.28; H, 6.41; N, 5.11; Cl, 19.09; O, 14.35;
Zn, 11.76%. 1H NMR (300 MHz, DMF-d7, 25 °C): d = 2.78 (t, 8H; N-
CH2-CH2), 3.31 (s, 12H; O-CH3), 3.50 (t, 8H; N-CH2-CH2), 4.02 (s,
4H; ph-CH2-N), 6.98 ppm (s, 2H; Ar); 13C NMR (300 MHz, DMSO-
d6, 25 °C): d = 52.44 (4C, O-Me), 56.42 (2C, ph-CH2-N), 58.20 (4C,
N-CH2-CH2), 69.58 (4C, N-CH2-CH2), 117.30 (1C, Ar), 127.13 (2C,
Ar), 129.33 (2C, Ar), 1 55.59 (1C, Ar-OH); ESI-MS m/z: Calcd for
[C20H35Cl2N2O5Zn]+ = 520.03, Found = 520.03; FT-IR data (KBr pel-
let):
c
bar = 665, 786, 833, 882, 958, 1020, 1112, 1271, 1471, 1593,
nolate ion in aqueous DMF (e
, 18500 Mꢀ1 cmꢀ1) at 25 °C. All the
2933 cmꢀ1
.
spectra were recorded for 2 h from a solution containing 1 mmol
PNPP and 0.05 mmol Zn complex. Kinetic experiments were per-
formed both at excess substrate and excess Zn complex keeping
constant the other conditions. Herein we report only the former
data. The study comprised 5 sets having catalyst of 0.05 mmol
and substrate concentration of 0.5 (10 equiv), 0.7 (14 equiv), 1.0
(20 equiv), 1.2 (24 equiv) and 1.5 mmol (30 equiv). The reactions
were initiated by injecting 0.04 mL of metal complex
(2.5 ꢂ 10ꢀ3 M) into 1.96 mL of PNPP solution, and the spectrum
was recorded only after fully mixing at 25 °C. The visible absorp-
tion increase was recorded for a total period of 30 min at regular
intervals of 5 min. All measurements were performed in triplicate,
and the average value was assumed. The reactions were corrected
for the degree of ionisation of the 4-nitrophenol at 25 °C using its
2.3.5. [Zn(HL2)Br2] (5)
It was synthesised by the same procedure as for complex 4 by
using zinc bromide. Suitable colourless rhombic-shaped single
crystals were obtained for X-ray data collection.
Yield = 367 mg
(57%).
Elemental
analysis
calcd
for
C
20H35N2Br2ClO5Zn: C, 37.29; H, 5.48; N, 4.35; Br, 24.81; Cl, 5.50;
O, 12.42; Zn, 10.15; found: C, 37.33; H, 5.43; N, 4.29; Br, 24.83;
Cl, 5.51; O, 12.49; Zn, 10.12%. 1H NMR (300 MHz, DMF-d7, 25 °C):
d = 2.77 (t, 8H; N-CH2-CH2), 3.34 (s, 12H; O-CH3), 3.48 (t, 8H; N-
CH2-CH2), 4.07 (s, 4H; ph-CH2-N), 6.97 ppm (s, 2H; Ar); 13C NMR
(300 MHz, DMSO-d6, 25 °C): d = 52.49 (4C, O-Me), 56.40 (2C, ph-
CH2-N), 58.25 (4C, N-CH2-CH2), 69.55 (4C, N-CH2-CH2), 117.28
(1C, Ar), 127.18 (2C, Ar), 129.35 (2C, Ar), 155.66 (1C, Ar-OH); ESI-
MS m/z: Calcd for [C20H35ClBrN2O5Zn]+ = 564.12, Found = 565.09;
molar extinction coefficient at 427 nm. The final A value for each
1
set was obtained after 2 days (at 25 °C).

58
R. Sanyal et al. / Polyhedron 97 (2015) 55–65
mono-nuclear complexes with one or two coordination sites on
3. Results and discussion
each metal centre, available for substrate binding in appropriate
fashion.
3.1. Synthetic aspects and characterisation of compounds
Thus here we have chosen the Mannich-based symmetrical din-
uclear and unsymmetrical zinc(II) complexes possessing pendant
ethylmethoxy chelating arms to investigate the hydrolysis mecha-
nisms of phosphomonoester PNPP. The chloro group at the termi-
nus of the phenolic ‘‘end-off’’ compartmental ligand has been
chosen appropriately for the sake of interesting comparative
analysis.
The ‘‘end-off’’ compartmental ligands used in this work are pure
Mannich-type bases and have been synthesised almost by the
same procedure as described in our previous work [30], which
reports the first observations on anomalous Mannich ligand reac-
tion. The precursors p-chlorophenol, bis(2-methoxyethyl)amine
and formalin were mixed together under appropriate conditions
and refluxed for a prolonged period of time. However certain alter-
ations were detected in the ratio of the component reactants. For
the monocondensation reaction, the ratio implemented was
1:0.9:0.9 while the dicondensation pathway required 1:2.2:2.2
that can be attributed to two opposing factors. First and foremost,
p-chlorophenol is a deactivated benzene ring. It is much less prone
to electrophilic substitution reaction as compared to p-cresol and
obviously it requires a greater concentration of electrophile. On
the other hand there is an increasing tendency of getting mixture
of the two compounds that makes column chromatographic sepa-
ration very tough. Thus single-side condensed product (HL1) was
almost purely isolated by employing the components in the ratio
as mentioned above. Further purification of ligand definitely needs
crucial separation by column chromatography. The complexes
were synthesised by using anhydrous zinc–halide salts, where
the halides were chloro, bromo and iodo. Ligand HL1 gives rise to
pentacoordinated dinuclear ZnII-complexes (1–3) with two halides
in a pseudo centrosymmetric arrangement (despite being clearly
mononucleating), while the other apparent dinucleating ligand
turns out to form mononuclear complexes (4–6) as depicted in
Fig. 1. This is undeniably an anomalous observation since HL1 is
an ideal ligand for the synthesis of mononuclear metallocomplexes
and at the same time HL2 is a conventional dinucleating ligand
[35–40]. Thus it would not be an overestimation to remark that
these kind of Mannich-base compartmental ligand system will
generate reverse nuclearity pattern with zinc–halides [30].
Unfortunately, complex 1 and 6 could not be structurally estab-
lished by repeated endeavours.
3.2. NMR spectral characterisation of ligands and complexes
The ligands (Figs. S9–S12, Supporting information) and com-
plexes (Figs. S13–S16, Supporting information) have been charac-
terised by both proton and carbon NMR spectroscopy in CDCl3
and DMSO-d6 solvent, respectively. The basic difference in the aro-
matic region of the two ligands is that the abnormal Mannich pro-
duct, HL1, displays three signals owing to three non-equivalent
protons while the conventional Mannich product shows one peak
which can be ascribed to the two equivalent aromatic protons in
the benzene ring.
3.3. UV–Vis spectra
The absorption spectra of the ligands in DMF (Supporting infor-
mation, Fig. S17) show one/two higher energy bands of high inten-
sity in the region 200–250 nm which is attributed to the
intraligand charge transfer of the open and closed forms of hydro-
gen-bonded species. In addition a lower energy band (ꢁ280 nm) of
lower intensity owing to the zwitterionic form of the ligand. The
corresponding spectra in methanol are shown in Fig. S18. The spec-
tra in methanol and DMF can be distinguished by the fact that two
high energy band is observed in methanol while it is one for DMF
solvent.
The complexes display one/two intraligand charge transfer
band(s) around 250–287 nm (high energy) and a ligand to metal
charge transfer band at 290–350 nm (low energy) in DMF which
is probably due to PhOꢀ?ZnII or to a mixture of the former and
Xꢀ?ZnII (Fig. 2). The spectra remain almost unaffected when mea-
sured in excess methanolic solution and therefore is not given sep-
arately. The solid-state absorption spectra of the complexes exhibit
peaks in the same region (Supporting information, Fig. S19). All the
UV-bands and their molar extinction coefficients are tabulated in a
list in Table S1 in Supporting information.
Comparing the IR spectra of these compounds to the spectra of
analogous compounds and available literature data on metal com-
pounds with a coordination environment comprised of ligands rel-
evant to the present study, viz., of other Mannich and reduced
Schiff-base complexes, fairly reliable empirical assignments could
be derived for the characteristic IR bands observed for the these
compounds. The IR spectra of the ligands (Figs. S1 and S2 in
Supporting information) and complexes (Figs. S3–S8 in
Supporting information) indicate a sharp signal at ꢁ1100 cmꢀ1
for C–N stretching vibration and ꢁ1580 cmꢀ1 for benzene skeletal
vibration. Thus it has been proved that these type of ligands can
afford kinetically and thermodynamically stable di- and
3.4. ESI-MS study
To elucidate the structure of the complexes in solution and the
active species present in the catalytic pathway during phosphomo-
noester hydrolysis, the electrospray-ionisation mass spectrometry
of the complexes has been performed in the positive mode. The
mass spectra of pure complexes, recorded in DMF medium (actu-
ally a mixture of DMF–dichloromethane), show a prominent base
peak at m/z 374.691 for complex 1 and at m/z 520.03 for complex
4, as shown in Figs. S20 and S21, respectively. The dinuclear com-
plexes were found to dissociate into two mononuclear moieties;
and we report only the mass spectrum of 1 ([C13H20NO3Cl2Zn]+,
theoretical m/z = 374.601), while for mononuclear series, a single
halide ion gets disconnected from the zinc centre to give rise to a
unipositive species as proved by the spectrum of complex 4
([C20H35N2O5Cl2Zn]+, theoretical m/z = 396.83). In order to get an
accurate picture of the structure of the enzyme–substrate adduct
in the phosphatase process and the nature of binding of PNPP to
the ZnII-complexes, we have done further ESI-MS investigations.
The mixture solutions of complex and PNPP of ratio 1:100, run
Cl
Cl
B
A
X
O
HN
O
N
O
O
O
N
O
O
N
O
Zn
X
Zn
Zn
O
O
O
X
X
Cl
Fig. 1. Chemical drawing of the complexes synthesised from ligands HL1 and HL2.
(A) 2:2 dinuclear metal-complex [Zn2(L1)2X2] (1–3) and (B) 1:1 mononuclear
metal-complex [Zn(HL2)X2] (4–6), X = Clꢀ (1, 4), Brꢀ (2, 5), Iꢀ (3, 6).

R. Sanyal et al. / Polyhedron 97 (2015) 55–65
59
Fig. 2. UV–Vis spectra of the metal-complexes 1–3 (A) and 4–6 (B) in DMF at 5 ꢂ 10ꢀ5 M concentration.
after 10 min of mixing in methanol solvent, showed the arising of
new peaks. In case of 1 a peak detected at 633.48 can be ascribed to
the cationic species [C20H27N2O10ClPZnNa2]+ (theoretical
m/z = 633.141) where a substrate molecule is attached to the cata-
lyst in monodentate fashion along with a methanolic moiety
(Fig. S22). For 4, a similar adduct is trapped by mass spectral anal-
ysis which gives a signal at 778.56, assigned to the complex cation
[C27H42N3O12ClPO12ZnNa2] (theoretical m/z = 778.325) as shown in
Fig. S23. In both the cases, the pure zinc(II) complex is maintained
in solution along with the adducts formed with PNPP.
The initial temperature Ti of decomposition of dinuclear and
mononuclear zinc complexes follow the trend 1 > 2 > 3 and
4 > 5 > 6 indicating highest thermal stability of Zn-chloro complex
irrespective of the nuclearity of the system. It may be due to better
match in size and energy of atomic orbitals between Zn and Cl with
respect to other halogens.
3.7. Crystal structure determinations
3.7.1. Dinuclear complexes 2 and 3
The X-ray structural analysis revealed that in complex 2 the
metal ions are bridged by two phenolato units in a centrosymmet-
ric fashion forming a four-membered Zn2O2 core (ORTEP drawing
shown in Fig. 4). Complex 3 shows a similar geometry although
it lacks a crystallographic centre of symmetry (Fig. 5). A selection
of coordination bond lengths and angles is reported in Tables S3
and S4.
In each complex the Zn atoms complete their coordination
sphere with the amino nitrogen, a halide and an oxygen from
one of the methoxyethyl arms, in a distorted trigonal bipyramidal
3.5. Solution state structures
The structure of the ZnII complexes in solution as concluded
from the ESI-MS investigations is depicted in Fig. 3. It is evident
that the dinuclear complexes (1–3) dissociates into two equivalent
monomeric species, while the mononuclear derivatives (4–6)
remain intact except for the loss of one of the coordinating halide
ions. The molar conductance values (as supplied in Table S2,
Supporting information) also support the above fact by proving
that they are non-electrolytes.
geometry for which a trigonality
s index [41] of 0.53 is derived for
2 and 3. Thus the Mannich ligand acts as a chelating tridentate spe-
cies towards a Zn ion, and in addition the phenoxo oxygen con-
nects the metal of another unit, so that one methoxyethyl arm at
each ligand remains uncoordinated. In complex 3 one methox-
yethyl chain evidences disorder and large thermal ellipsoids for
its atoms. Due to the local symmetry, the halide ligands as well
as the amine N atoms are trans located with respect to the Zn2O2
rhomboid mean plane.
In 2–3 the Zn–N and Zn–O distances show decreasing values
ongoing from the bromo to the iodo derivative (Table S3).
However, it is worth noting that the Zn–O(2) bond distances
(involving the coordinated methoxy group) are significantly longer
by 0.2–0.3 Å with respect to the other Zn–O distances of the bridg-
ing phenolato oxygens. On the other hand, as expected the Zn–
halide bond lengths augment from 2.3427(6) to 2.525–2.522(3) Å
in 2 and 3, respectively. The coordination geometry of Br and I
derivatives 2 and 3 (the latter with two independent Zn ions has
a lower accuracy) appears to follow a trend similar to the corre-
sponding dinuclear complexes synthesised from p-cresol recently
reported [30]. Thus the electronic property of 4-chlorophenol
3.6. Thermal analyses
Thermal analyses of all the six complexes have been performed
in order to (i) compare their thermal stability by understanding
their thermal decomposition patterns, (ii) to verify the molecular
composition of the complexes, and (iii) to synthesise the thermally
stable end products. The analyses performed in the temperature
range 30–700 °C suggest that all of them are stable up to nearly
180 °C. All of them yield ZnO as thermally stable end product.
Figs. S24–S26 and S27–S29 depict the thermogram of dinuclear
complexes 1–3 and mononuclear complexes 4–6, respectively.
Table 1 reports the calculated and experimental weight losses,
temperature range of decomposition for all the complexes studied.
Cl
Cl
A
B
(pKa = 9.43),
a
weaker base compared to the p-cresol
N
O
O
N
O
O
N
O
(pKa = 10.26), does not seem to affect the coordination bond dis-
tances in the present complexes with respect to the series of
methyl analogues. Finally, the Zn–Zn distances are slightly longer
(3.1254(8) and 3.101(3) Å, in 2 and 3, respectively) confirming a
short value for the iodine derivative.
Zn
Zn
O
O
O
X
X
Fig. 3. Chemical drawing of the probable structures of the complexes (A) 1–3, (B)
4–6 in solution.

60
R. Sanyal et al. / Polyhedron 97 (2015) 55–65
Table 1
Theoretical and experimental weight loss of the complexes (from thermogram).
Complex End product
(assumed)
Initial temperature of decomposition
(in °C)
End temperature (in Theoretical weight loss Experimental weight loss (%) (from
°C)
(%)
thermogram)
1
2
3
ZnO
220
210
190
630–640
79.2818
89.0011
86.20868
85.4046
87.753
82.6209
4
5
6
ZnO
190
180
170
660
90.3578
87.3752
88.2368
83.0179
94.57
97.3885
Fig. 6. ORTEP view (35% ellipsoid probability) of compound 4.
Fig. 4. ORTEP view (35% ellipsoid probability) of compound 2 with atom labels of
crystallographic independent part.
3.7.2. Mononuclear complexes 4 and 5
The mononuclear entity of complexes 4 and 5 was verified by X-
ray structural analysis. The compounds are isomorphous and an
ORTEP view of these is shown in Figs. 6 and 7, respectively. The
metal in both the complexes presents a distorted tetrahedral coor-
dination environment being chelated by the Mannich ligand
through the phenolato oxygen and amino nitrogen and in addition
by two monocoordinated halides. On the other hand the ethyl-
methoxy fragments of the organic ligands point far away and do
not interact with the metal centre. The Zn–O and Zn–N bond
lengths are close comparable within their esd’s being of
1.9408(14) and 1.937(5) Å and 2.0917(17)–2.096(5) Å, in 4 and 5,
respectively, despite the different halide bound at zinc atom
(Table S5). The chelating angle N(1)–Zn–O(1) of ca. 94° deviates
Fig. 7. ORTEP view (35% ellipsoid probability) of compound 5.
significantly from the ideal tetrahedral value, while the other coor-
dination angles in the two complexes are in the range 106.92(5)–
116.24(5)°. The metal ion resides almost in the phenolato plane
and the coordinated amino nitrogen is displaced by ca. 0.24 Å from
it. The structural determination confirms that the amino nitrogen
on the other arm of the ligand is protonated and impedes the coor-
dination of a second metal ion. Complex 4 is isomorphous and thus
close comparable to the p-cresol derivative reported [30]. The pro-
tonated nitrogen is at 2.68 Å in both cases from the phenolato oxy-
gen favouring an intramolecular interaction (Table S6). The crystal
packing does not show any peculiar feature. The crystallographic
data and details of refinement for complexes are listed in Table 2.
3.8. Phosphatase activity
The efficiency of phosphatase activity of the complexes was
determined by using the phosphomonoester, p-nitrophenylphos-
phate (PNPP), a common substrate reported in previous investiga-
tions. While the study was performed in methanol solvent, the
complex was dissolved and added in DMF for solubility reasons.
We have deliberately avoided a drastic solvent and in this study
Fig. 5. ORTEP view (35% ellipsoid probability) of compound 3.

R. Sanyal et al. / Polyhedron 97 (2015) 55–65
61
Table 2
Crystallographic data and details of refinement for complexes 2–5.
2
3
4
5
Empirical formula
Fw
C
26H38Br2Cl2N2O6Zn2
C26H38Cl2I2N2O6Zn2
930.02
C20H35Cl3N2O5Zn
555.22
C20H35Br2ClN2O5Zn
644.14
836.04
Crystal system
Space group
a (Å)
b (Å)
c (Å)
monoclinic
P21/c
9.8712(14)
7.9016(12)
21.320(3)
90.0
96.794(2)
90.0
1651.3(4)
2
1.681
4.074
840
1.92–26.54
10785
triclinic
P1
triclinic
P1
triclinic
P1
ꢀ
ꢀ
ꢀ
10.134(3)
10.886(5)
15.148(6)
85.109(14)
87.185(14)
82.346(11)
1649.0(12)
2
1.873
3.529
912
1.35–21.92
5330
9.1482(4)
9.5543(4)
16.5944(7)
83.508(2)
87.016(2)
63.9680(10)
1294.92(10)
2
1.424
1.289
580
1.23–27.04
18946
9.285(3)
9.699(3)
16.762(6)
83.871(4)
87.107(4)
63.486(4)
1343.1(8)
2
1.593
4.019
652
1.22–22.98
5308
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dcalc (g cmꢀ3
)
l
(Mo K
F(000)
h range (°)
a
) (mmꢀ1
)
No. of reflections collected
No. of independent reflections (Rint
)
3159 (0.0319)
2384
181
1.039
0.0314, 0.0663
0.579, ꢀ0.427
3642 (0.0476)
2064
366
1.036
0.0732, 0.1943
1.821, ꢀ0.945
5578 (0.0263)
3481 (0.0406)
2731
283
1.037
0.0613, 0.1637
1.350, ꢀ1.069
No. of reflections (I > 2
Refined parameters
Goodness-of-fit (F2)
r(I))
287
1.040
0.0358, 0.0946
0.481, ꢀ0.274
R1, wR2 (I > 2
r
(I))a
Residuals (e/Å3)
a
R1
=
R
||Fo| ꢀ |Fc||/
R
|Fo|, wR2 = [
R
w(Fo2 – Fc2)2/ w(Fo2)2]1/2
R .
the hydrolytic activity of the complexes was estimated in a com-
mon solvent like methanol. Initial measurements for the hydrolysis
were run in 97.5% DMF–water solvent mixture, the same used in
the previous study with methyl para-substituted complexes [30].
However the result patterns were almost the same as those previ-
ously reported under similar experimental conditions. The hydrol-
ysis of PNPP was conveniently monitored through UV–Vis
spectrophotometry by following the increase of absorbance of
the band at 427–430 nm corresponding to the production of 4-ni-
trophenolate. The preliminary activity of complexes 1–6 were
examined by following the protocol earlier reported [30]. The
wavelength scan of the complexes were recorded as a mixture of
complex and PNPP where the substrate was 20-fold that of the
complex in methanol for 2 h at regular time intervals of 10 min
as illustrated in Fig. 8 for complex 2 (Figs. S30–S34 in Supporting
information for five other complexes).
The absorption profile of the complex-PNPP reaction mixture at
the kmax nearing about 430 nm is represented in Fig. 9 for complex
2 and the others in Supporting information, Figs. S35–S39. It shows
that the pathway follows a sigmoidal increase in absorbance as the
rate of increase decreases as the reaction approaches to a point of
maximum conversion. But this S-shaped nature of the graph does
not remain intact for complex 4 and 5 as the rate of increase in
absorbance remains equal over the time range of 2 h. The catalytic
activity on the hydrolysis of PNPP ester was investigated although
the complexes 1–6 in their present structure do not possess any
potential nucleophile like metal-coordinated water molecule.
Despite the matter that the methyl compounds had shown an
extraordinary mechanism for phosphatase activity (in DMF), it
was predicted that in case of methanol solvent, the two Zn(II) ions
would work cooperatively in the hydrolytic mechanism, through a
bridging interaction of the phosphate substrate with the two met-
als and a simultaneous nucleophilic attack of a Zn–OH function on
the substrate by the addition of an external water molecule on one
of the zinc ions. But the results point towards a different story
where the nature of nucleophilic reagent is probably a methanolic
group instead of a water molecule.
3.9. Control experiments
Control experiments have been performed in order to examine
whether there is any role of the ligand used for metallation and the
metal-salt themselves in the hydrolytic activity shown. We arrive
at the result that the metal-complex is solely responsible for the
phosphatase activity, i.e., the catalysis is zinc-ion mediated. The
control experiments are represented in Fig. S40 (A, B and C) in
Supporting information. Although the control with zinc–halide
metal-salts tend to portray
a band maxima at near about
430 nm, we can consider it as too negligible to be taken into
account.
3.10. Kinetics
The accumulated kinetic data were analysed on the basis of
Michaelis–Menten equation. The enzymatic kinetics plot (sub-
strate–catalyst interdependence) and the double-reciprocal plots
are shown in Fig. 10 for complex 2 and for the rest of the
Fig. 8. Wavelength scan for the hydrolysis of PNPP in the absence and presence of
complex 2 (substrate:catalyst = 20:1) in methanol recorded at 25 °C at an interval of
10 min for 2 h. [PNPP] = 1 ꢂ 10ꢀ3 M, [Complex] = 0.05 ꢂ 10ꢀ3 M. The arrow shows
the change in absorbance with reaction time.

62
R. Sanyal et al. / Polyhedron 97 (2015) 55–65
Table 3
First order rate constant values (sꢀ1) for phosphatase activity of complexes 1–6 in the
present in our previous study (Ref. [30]).
Complex kcat
kcat (sꢀ1) values of analogous para-substituted methyl
derivatives (Ref. [30])
(sꢀ1
)
1
2
3
4
5
6
8.94
13.06
5.10
1.05
1.79
4.35
9.35
8.88
8.33
4.68
3.29
2.88
results have been postulated from the previous representative
mechanisms. The substrate molecule binds as a dianionic monon-
itrophenyl phosphate to the complex having phosphatase activity.
An altered mechanism of hydrolysis for the metal-complexed
phosphate monoester have been demonstrated for the methanol
as compared to the reaction pathway in DMF solvent as reported
earlier [30]. As depicted in Fig. 12, for the mononuclear series of
complexes (4–6), RDS is the one where halide ion gets dissociated
from the metal-centre after the addition of PNPP which is probably
in a bidentate fashion. A methanol molecule gets associated with
the ZnII ion leading to the intermediate III which is deprotonated
to yield a methoxide ion as the potent nucleophile in our case
(intermediate IV). IV has been identified in ESI-MS study as a major
signal at an m/z of 778.56. It is followed by the formation of an
organophosphorus intermediate by nucleophilic substitution reac-
tion with the concomitant expulsion of 4-nitrophenolate ion. It
then gradually breaks down into the original complex and phos-
phoric acid. In case of dinuclear complexes (1–3), the picture is
slightly different in the perspective that the addition of PNPP and
dissociation of halide ion occurs in a simultaneous fashion, i.e., in
a well-concerted pathway, as vividly represented in Fig. S46 in
Supporting information. The complex–substrate adduct is attacked
by a methanol molecule and that intermediate has been success-
fully trapped by electrospray-ionisation mass spectrometry pro-
viding an obvious peak at m/z 633.48 which is followed by a
similar series of reactions to complete the catalytic cycle. Thus
the initial phosphorylation rate is straight-away portrayed in the
experimental rate constant magnitude.
Clearly, we have assumed a bipoint binding mode in the key
step of the pathway over a monopoint addition picture. Now some
intrinsic questions concerning the phosphatase mechanism arises
in our mind, such as (i) what is the special advantage in forming
the phosphoryl intermediate for indirect hydrolysis and (ii) how
does an external methanol molecule associated with the ZnII ion
become a nucleophile over the internally present methoxy group?
This can be attributed to the fact that after the substrate has been
recognised and associated via coordination linkages and electro-
static interactive forces, a proper coordination pattern grows up
whereby the configuration is just ideal for a nucleophilic attack
from the metal-centre to the electrophilic phosphorus site. It is
only after that step that a chain of events follow by which 4-nitro-
phenolate gets departed from the reaction-complex which proves
that substrate coordination is a general pre-requisite. Moreover,
the conjugate-base of p-nitrophenol is a very weak base and hence
p-nitrophenolate is a poor leaving group and so direct hydrolysis
would never proceed in absence of a phosphoryl intermediate.
The second point raised although seems contradictory to the fact
that intramolecular reactions are always preferred over inter-
molecular reactions, is quite justified from this perspective that
the methoxy group suffers a considerable amount of steric hin-
drance in our coordination sphere than the methanol moiety or
any other general base and hence lacks sufficient flexibility to
attack the phosphorus atom embedded in the cavity throughout
the reaction ordinate. Therefore that possibility is clearly ruled
Fig. 9. Absorption profile on addition of PNPP to complex 2 due to the formation of
p-nitrophenolate (kmax = 427 nm) monitored for 2 h. Inset shows the plot of log [A
/
1
(A1 ꢀ At)] vs. time for 1 h (R2 = 0.955, standard deviation = 764.5).
complexes in Figs. S41–S45 in Supporting information. The first-
order rate constant values are given in Table 3 and the rest of
the parameters are supplied in Table S7 in Supporting information.
The kinetic results indicate that the degree of catalytic power of
the
complexes
in
methanol
follows
the
order
of
2 > 1 > 3 > 6 > 5 > 4. Thus there are two vital points to be empha-
sised from the trend: firstly, the dinuclear complexes (1–3) are
more reactive than the mononuclear ones (4–6), as previously
anticipated. Secondly, the halide effect is not homogeneous in
the two group of complexes, being Br > Cl > I in case of the dinu-
clear complexes (Fig. 11A) while the trend for mononuclear is
I > Br > Cl (Fig. 11B).
3.11. Mechanistic implications
The coordination patterns and binding models of the catalyst–
substrate complex have been largely explored in literature
[15,16,20,30,42] and after rigorous considerations two plausible
catalytic cycles having reasonably good agreement with our kinetic
Fig. 10. Plot of enzymatic kinetics for complex 2 (V vs. [S]). Inset shows the
Lineweaver–Burk plot (1/V vs. 1/[S]) having intercept = 93.5806 (error = 9.11),
slope = 3.9859 (error = 0.7129), R2 = 0.955 and standard deviation = 764.54.

R. Sanyal et al. / Polyhedron 97 (2015) 55–65
63
Fig. 11. Overlay of the Lineweaver–Burk curves of (A) dinuclear complexes 1–3 and (B) mononuclear ones 4–6.
Cl
Cl
NPP2-
Cl
X
slow, RDS
2
1
N
O
N
O
N
O
N
O
O
O
N
O
N
O
O
X
Zn
X
Zn
O-
O-
O
Zn
O
O
O
O
O
O
O
P
P
O
R
O
R
(I)
(II)
phosphoric acid
MeOH
3
O-
R=
O2N
7
Cl
H2O/X
Cl
O
N
N
O
O
N
O
HN
Zn OCH3
O
Zn
O-
P
O
O
O
O
O-
H
O-
O-
O
(III)
R
O
P
OCH3
(VI)
NO2
O
4
O
6
Cl
Cl
intramolecular nucleophilic attack
on phosphorus atom
5
N
O
N
HN
Zn OCH3
HN
O
O
Zn
O-
O
R
O-
O
O-
O
O
O
O
O
O-
P
P
OCH3
O2N
O
O
O-
(V)
(IV)
m/z=778.56
Fig. 12. Plausible mechanism for PNPP hydrolysis promoted by mononuclear complexes 4–6.

64
R. Sanyal et al. / Polyhedron 97 (2015) 55–65
out as it appears to be electronically and geometrically fictitious as
demonstrated in Fig. 13. Here it is necessary to say that a methano-
lic nucleophile is more accurate than a classical aqueous nucle-
ophilic reagent despite the fact that the pKa value of the two are
very comparable (pKa water = 15.7, pKa MeOH = 16), since we have
avoided any deliberate inclusion of water in any proportion to
our catalytic system. The above postulated mechanistic pathway
is reasonably consistent with, and can be used to systematically
interpret, the experimental observations reached in our case.
The dinuclear compounds are expectedly more catalytically
active than their mononuclear analogues [43–47,24,48], although
the former provide effectively mononuclear species in solution.
This is primarily due to the fact that the total concentration of cat-
alytically active species is twice in case of dinuclear complexes
independent of the halide ion. Many literature studies have
explored coordinating ligands that act to sterically constrain the
ZnII geometry, therefore limiting the extent of the geometrical flat-
tening for appropriate enzyme–substrate interaction. In our case,
the steric factor acts only in case of ligand HL2 due to the second
methoxyethyl chelating arm which is the secondary reason for
the lower reactivity of the mononuclear compounds as compared
to the dinuclear ones.
Fig. 14. Overlay of the Lineweaver–Burk plots of complexes 1–6 with those of the
methyl analogues in our previous work.
Thus if we compare our catalytic activity results with that of our
previous work by Sanyal et al. [30] (Table 3), we observe specific
changes, both qualitatively and quantitatively, viz., there is a huge
change in the nature of halide dependence on kinetic behaviour
and the rate constant value of complex 2 which is a dinuclear com-
plex of HL1 and ZnBr2 metal-salt. The overlay of the Line-weaver
Burk plots of the ten complexes (1, 3–6 from both series) generated
by Michaelis Menten analysis has been represented in Fig. 14A and
complex 2 has been plotted separately in Fig. 14B since 2 of chloro
series has an extremely high catalytic efficiency. Although the
dual-point binding model has been kept unaltered, there have been
complete change in the nature of solvent used along with the alter-
ation in the para-group. Previously we had opted for 97.5% DMF
which is roughly a non-aqueous catalytic medium, the catalytic
amount of water present in the system is potent enough to render
nucleophilicity for solvolysis of P–O bond. So there occurs a basic
change in the actual nucleophile participating in the catalysis.
Now the magnitude of rate of hydrolysis, in terms of kcat values,
is essentially lower than the methyl-compounds except in two
cases, which are complex 2 and 6. This is probably because DMF
being an aprotic but highly polar solvent can stabilise the ionic
intermediates to a larger extent than methanol solvent by ion–
dipole interaction. However methanol can display some amount
hydrogen-bonding interaction which cannot exceed the degree of
stabilisation attained from ion–dipole interaction. The discrepancy
which is shown for 2 and 6 must be for the chloro-group in the
para-position of the benzene ring which activates the metal-cata-
lyst by its electron-withdrawing inductive effect and renders the
zinc–ion more lewis-acidic making the hydrolytic assay much
more favourable. The Znꢃ ꢃ ꢃZn intermetallic bond-distances of the
two series of complexes have not been dealt with for comparative
study since the bimetallic complexes tend to dissociate into
mononuclear entities and lose their dimeric nature in the process.
3.12. Influence of electronegativity and bond energy
There are two governing parameters which rule the phos-
phatase activity of zinc-halido metal-complexes, namely elec-
tronegativity trend and the zinc–halogen bond energy value. The
electronegativity factor influences the reaction rate in a fashion
such that the pathway should be preferred for the coordinated
halide which is more electronegative and this is because the more
electronegative halide increases the Lewis acidity of the zinc metal
ion (Fig. S47). In the later case, the bond energy value is inversely
proportional to the rate since higher value implies poorer leaving
group ability (Fig. S48). A conclusion which should be apparent
from the foregoing is that both these parameters are meaningful
in determining the catalytic power of a complex and our observa-
tions are a combination of both these characteristic property. A
similar situation did not arise for the methyl-substituted com-
plexes where only electronegativity factor ruled the entire kinetics
and it was computationally verified. The matter is very compli-
cated to elucidate the reason behind the relative governance of
these two proposed factors where the possibility of a substantial
solvent role cannot be ruled out which needs further theoretical
modelling.
4. Conclusions
The cleavage of phosphoester PNPP mediated by Mannich-
based di- and mononuclear zinc(II) complexes have been investi-
gated. It has been discussed that although monomeric entities
can perform as active catalyst, the dinuclear ones are more com-
petitive than their mononuclear counterparts in P–O bond-fission.
To accommodate all the kinetic data, we have established that
while the dinuclear catalysts prefer a concerted catalytic mecha-
nism, the mononuclear counterparts religiously follow a step-wise
A
B
O
X
X
N
O
O
O
O
II
O
II
N
O
O
II
Zn
Zn
Zn
N
X
O
O
O
O
SN2-type
nucleophilic
addition–substitution
pathway.
O
O
O
P
P
O2N
Conclusively, this research definitely provides certain insights into
the solvent effect, halide role, nuclearity influence and their inter-
relation in synthetic phosphatase model zinc complexes with
Mannich base ligands leading to a more or less perfect functional
mimic. Although no absolute correlation could be drawn between
O
O2N
O
Fig. 13. Possible intermediates with the methoxy group as the effective nucleophile
during phosphatase activity promoted by (A) dinuclear complexes 1–3 (B) and
mononuclear ones 4–6.

R. Sanyal et al. / Polyhedron 97 (2015) 55–65
(d) S.D. Aubert, Y. Li, F.M. Raushel, Biochemistry 43b (2004) 5707;
65
the topological architecture of a molecule and its efficiency of
phosphoester hydrolysis, few accumulated facts appear salient
and define the crucial factors in designing efficient artificial phos-
phatases. In particular, we can end this project in this noble antic-
ipation that the knowledge acquired is quite satisfactory to obtain
more attractive phosphoesterase mimics in the future. Finally,
these being Zn(II) compounds provide a rational alternative to
expensive, less abundant, and more toxic heavy-metal-containing
complexes. We can end here with this note that though this field
has attracted significant research attention, a lot more job remains
behind.
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New
Delhi
[CSIR
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No.
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