Influence of the coordination environment of zinc(II) complexes of designed mannich ligands on phosphatase activity: A combined experimental and theoretical study

Article abstract of DOI:10.1021/ic4015493

A mononucleating (HL¹) and a dinucleating (HL²) "end-off" compartmental ligand have been designed and synthesized by controlled Mannich reaction using p-cresol and bis(2-methoxyethyl)amine, and their formation has been rationalized. Six complexes have been prepared on treating HL¹ and HL² with Zn^IIX₂ (X = Cl^-, Br^-, I^-) with the aim to investigate their hydrolytic activity on phosphoester bond cleavage. Interestingly, the mononucleating ligand was observed to yield dinuclear complexes, [Zn ₂(L¹)₂X₂] (1-3), while the potential dinucleating ligand generated mononuclear complexes, [Zn(HL²)X ₂] (4-6). Four (1-4), out of six complexes studied, were characterized by single-crystal X-ray diffraction (XRD): the Zn ion exhibits trigonal bipyramidal and tetrahedral coordination spheres in the di- and mononuclear complex, respectively. The hydrolytic kinetics, followed spectrophotometrically with 4-nitrophenylphosphate (4-NPP) in buffered dimethylformamide (DMF) (97.5% DMF, v/v) because of solubility reasons, under excess substrate conditions (substrate:complex = 20:1), indicated that the complexes enormously accelerate the rate of phosphomonoester hydrolysis with first order rate constants (k_cat) in the range 2-10 s^-1 at 25 °C. In each case kinetic data analyses have been run by Michaelis-Menten treatment. The efficacy in the order of conversion of substrate to product (p-nitrophenolate ion) follows the trend 1 > 2 > 3 > 4 > 5 > 6, and the ratio of k_cat of an analogous dinuclear to mononuclear complex is ?2. An electrospray ionization-mass spectrometry (ESI-MS) study has revealed the dissociation of the centrosymmetric dinuclear complex to two mononuclear species instead of a syn-cooperative catalysis. Density functional theory (DFT) calculations have been performed to rationalize our proposed mechanistic pathway for phosphatase activity. The comparative analysis concludes the following facts under experimental conditions: (1) the halide bound to the active site affects the overall rate in the order: Cl^- > Br ^- > I^- regardless of nuclearity; (2) dinuclear complexes prevail over the mononuclear ones.

Full text of DOI:10.1021/ic4015493

Article
pubs.acs.org/IC
Influence of the Coordination Environment of Zinc(II) Complexes of
Designed Mannich Ligands on Phosphatase Activity: A Combined
Experimental and Theoretical Study
Ria Sanyal,† Averi Guha,† Totan Ghosh,† Tapan Kumar Mondal,‡ Ennio Zangrando,*,§
and Debasis Das*,†
†Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata 700009, India
‡Department of Chemistry, Jadavpur University, Jadavpur, Kolkata 7000032, India
§Dipartimento di Scienze Chimiche e Farmaceutiche, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy
S
* Supporting Information
ABSTRACT: A mononucleating (HL1) and a dinucleating (HL2) “end-
off” compartmental ligand have been designed and synthesized by
controlled Mannich reaction using p-cresol and bis(2-methoxyethyl)amine,
and their formation has been rationalized. Six complexes have been
prepared on treating HL1 and HL2 with ZnIIX2 (X = Cl−, Br−, I−) with the
aim to investigate their hydrolytic activity on phosphoester bond cleavage.
Interestingly, the mononucleating ligand was observed to yield dinuclear
complexes, [Zn2(L1)2X2] (1−3), while the potential dinucleating ligand
generated mononuclear complexes, [Zn(HL2)X2] (4−6). Four (1−4), out
of six complexes studied, were characterized by single-crystal X-ray
diffraction (XRD): the Zn ion exhibits trigonal bipyramidal and tetrahedral
coordination spheres in the di- and mononuclear complex, respectively.
The hydrolytic kinetics, followed spectrophotometrically with 4-nitro-
phenylphosphate (4-NPP) in buffered dimethylformamide (DMF) (97.5% DMF, v/v) because of solubility reasons, under excess
substrate conditions (substrate:complex = 20:1), indicated that the complexes enormously accelerate the rate of
phosphomonoester hydrolysis with first order rate constants (kcat) in the range 2−10 s−1 at 25 °C. In each case kinetic data
analyses have been run by Michaelis−Menten treatment. The efficacy in the order of conversion of substrate to product (p-
nitrophenolate ion) follows the trend 1 > 2 > 3 > 4 > 5 > 6, and the ratio of kcat of an analogous dinuclear to mononuclear
complex is ≃2. An electrospray ionization-mass spectrometry (ESI-MS) study has revealed the dissociation of the
centrosymmetric dinuclear complex to two mononuclear species instead of a syn-cooperative catalysis. Density functional
theory (DFT) calculations have been performed to rationalize our proposed mechanistic pathway for phosphatase activity. The
comparative analysis concludes the following facts under experimental conditions: (1) the halide bound to the active site affects
the overall rate in the order: Cl− > Br− > I− regardless of nuclearity; (2) dinuclear complexes prevail over the mononuclear ones.
both naturally occurring and synthetic phosphate esters.22,23
These zinc enzymes can use external water molecules or
INTRODUCTION
■
Zinc, being one of the most biocompatible metals,1−6 is present
internal alcoholic hydroxyl residues as nucleophiles to react
in all forms of life,7−10 with an average adult human content of
with electrophilic substrates. In case of dinuclear sites, both
about 3 g of this metal.11 This influence of zinc derives from its
metals are involved in the catalytic process: either one metal
role in enzymes, with functions that are both structural and
ion provides the nucleophile and the other coordinates the
catalytic. Excellent Lewis acidity, capability of undergoing rapid
ligand exchange, flat coordination hypersurface,12−15 and free
substrate or they may act cooperatively participating in
substrate binding, activation, and cleavage.24−26 This mutual
from any undesired redox activity make ZnII the most obvious
choice in hydrolytic enzymes.16,17 It is now well documented
action usually renders dinuclear complexes far more reactive
than their mononuclear analogues.27
that zinc enzymes play an important role in the cleavage of the
P-OR bond in phosphates, [(RO)PO3]2− and [(RO)2PO2]−, as
It is therefore evident that zinc plays multifaceted roles in
biological systems, and a detailed understanding of these roles
exemplified by their nuclease activity pertaining to the
hydrolysis of DNA and RNA.18,19 In nature, many enzymes
requires a correspondingly detailed appreciation of how the
chemistry of zinc is modulated by its coordination environ-
that catalyze phosphate ester hydrolysis are activated by two or
more metal ions. These include phosphate mono-, di-, and
triesterases, all pertaining to the class of phosphatase
Received: June 19, 2013
Published: December 19, 2013
enzymes,20,21 which have evolved to catalyze the cleavage of
© 2013 American Chemical Society
85
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Inorganic Chemistry
Article
C14H23N1O3: C 66.37, H 9.15, N 5.53, O 18.95; found C 65.99, H
9.75, N 5.80, O 19.18 1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.2(s,
3H; ph-CH3), 2.83(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), 7.2(d, 1H; Ar), 7.26(s,
1H; o to CH2−N), 7.43 ppm(d, 1H; o to phenolic−OH); 13C NMR
(300 MHz, DMSO-d6, 25 °C): δ = 20.13(1C, ph-Me), 52.61(2C, O-
Me), 56.47(1C, ph-CH2−N), 58.05(2C, N-CH2−CH2), 69.6(2C, N−
CH2-CH2), 115.08(1C, Ar), 127.07(1C, Ar), 128.57(1C, Ar),
129.49(1C, Ar), 129.74(1C, Ar), 154.97(1C, Ar−OH); IR data
(NaCl plate): γ bar = 768, 818, 1117, 1256, 1600, 2923 cm−1; UV/vis
(DMF): λmax(ε) = 253.1(2800), 284.3(5400), 339.4 nm (2 L mol−1
cm−1).
ment. Many metal ion based model systems have been
reported, and in most of the cases the ligands are tridentate
or tetradentate Schiff-base ligands with free coordination sites
of the metal cation.28 Here our preliminary goal is to develop
reactive chemical systems employing comparatively less ex-
plored ligands, Mannich base ligands that efficiently hydrolyze
phosphate monoester bonds, and to study the roles of
nuclearity, labile groups, geometry of the complexes, and so
forth on the catalytic efficiency of the systems. In the present
work, we have synthesized two phenolic ligands, namely, 2-
[bis(2-methoxyethyl)aminomethyl]-4-methylphenol (HL1) and
2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol
(HL2),29−34 by properly tuning the progress of Mannich
reaction to achieve the insertion of one or two pendant
chelating arms. On complexation with ZnX2 salts (X = Cl−, Br−,
I−) the former ligand generates dinuclear and the latter
generates mononuclear complexes, a behavior that appears
apparently anomalous. The solid-state structures of four
complexes out of six have been authenticated by X-ray single
cryatal structural analyses, and their solution phase structures
have been established by UV−vis, electrospray ionization-mass
spectrometry (ESI-MS), NMR spectra (DMF-d7 and DMSO-d6
as d-solvents), and by molar conductivity studies. Finally,
phosphatase activity of those complexes has been thoroughly
investigated using 4-nitrophenylphosphate as substrate in
aqueous dimethylformamide (DMF) for all the complexes at
298 K. The role of halide ligands on the phosphatase activity
has been explored, and probable mechanistic pathways involved
in mono- and dinuclear ZnII complexes have been proposed on
the basis of density functional theory (DFT) calculations.
Synthesis of HL2. To an ethanolic solution (30 mL) of p-cresol (30
mmol, 3.24 gms), bis(2-methoxyethyl)amine (90 mmol, 11.99 gms)
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 an additional 1 h at room temperature and then
refluxed for 24 h. It was followed by the same workup as HL1. The
liquid ligand was further purified by flash column chromatography
(silica gel, hexane: ethylacetate: triethylamine =100: 5: 1) to afford the
desired β-aminophenol.
Yield = 3 g (25%). Elemental analysis calcd (%) for C21H38N2O5: C
63.29, H 9.61, N 7.03, O 20.07; found: C 63.42, H 9.49, N 7.16, O
19.93.
1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.22(s, 3H; ph-CH3),
2.83(t, 8H; N-CH2-CH2), 3.34(s, 12H; O-CH3), 3.55(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): δ = 20.27(1C, ph-Me), 52.41(4C, O-Me),
56.45(2C, ph-CH2−N), 58.10(4C, N-CH2−CH2), 69.50(4C, N−
CH2-CH2), 115.04(1C, Ar), 127.17(2C, Ar), 129.39(2C, Ar),
155.28(1C, Ar−OH); IR data (NaCl plate): γ bar= 773, 821, 1115,
1255, 1367, 1463, 1609, 2876 cm−1; UV/vis (DMF): λmax(ε) =
254.3(2400), 287.1(5600), 435.7 nm (2 L mol−1 cm−1).
2
Synthesis of Zinc(II) Complexes. Synthesis of [Zn2L1 Cl2] (1).
To a methanolic solution (25 mL) of HL1 (0.5 mmol, 126.5 mg), a
methanolic solution (10 mL) of anhydrous zinc chloride (0.5 mmol,
112.6 mg) was added. The light yellow reaction mixture was refluxed
for 2 h, cooled to room temperature, filtered and kept in a beaker.
After 1 day, two kinds of crystals were observed in the colorless
solution. They were carefully separated under a microscope with the
following result: (1) colorless needle-shaped and (2) colorless square-
shaped single crystals.
EXPERIMENTAL SECTION
■
Materials and Methods. (4-Nitrophenyl)phosphate was pur-
chased from Aldrich. 4-NPP was recrystallized from ethanol/water
before use. Reaction solutions for 4-NPP cleavage were prepared
according to the standard sterile techniques. DMF was dried over
CaH2 for 2 days and then distilled under reduced pressure prior to use.
The anhydrous zinc halide salts (analytical grade) were obtained from
Merck. Common organic reagents and solvents used for synthesis were
reagent grade which were obtained from commercial sources and
redistilled before use. Water used in all physical measurement
experiments was Milli-Q grade.
Yield = 72 mg (41%). Elemental analysis calcd (%) for
C28H44Cl2N2O6Zn2: C 47.62, H 6.28, Cl 10.04, N 3.97, O 13.59, Zn
18.52; found: C 47.69, H 6.13, Cl 10.31, N 3.90, O 13.29, Zn 18.68;
1H NMR (300 MHz, DMF-d7, 25 °C): δ = 2.25(s, 6H; ph-CH3),
2.84(t, 8H; N-CH2-CH2), 3.02(s, 12H; O-CH3), 3.38(t, 8H; N−CH2-
CH2), 3.92(s, 4H; ph-CH2-N), 6.8(d, 2H; Ar), 7.22(d, 2H; o to CH2−
N), 7.29 ppm(s, 2H; o to phenolic−OH); 13C NMR (300 MHz,
DMSO-d6, 25 °C): δ = 20.18(1C, ph-Me), 52.60(2C, O-Me),
56.40(1C, ph-CH2−N), 58.07(2C, N-CH2−CH2), 69.57(2C, N−
CH2-CH2), 115.33(1C, Ar), 122.88(1C, Ar), 127.10(1C, Ar),
128.59(1C, Ar), 129.53(1C, Ar), 154.91(1C, Ar−OH); ESI-MS m/
z: Calcd for [C14H23Cl1N1O3Zn1]+ = 354.148, Found = 353.98; FT-IR
data (KBr pellet): γ bar = 836, 1021, 1105, 1270, 1491, 2370, 2924
cm−1. UV/vis (DMF): λmax(ε) = 237.9(5000), 266.4(6600), 285.5 nm
(6000 L mol−1 cm−1).
Elemental analyses (carbon, hydrogen, and nitrogen) were
performed using a PerkinElmer 240C analyzer. Infrared spectra
(4000−400 cm−1) were recorded at 28 °C on a Shimadzu FTIR-8400S
and PerkinElmer Spectrum Express Version 1.03 using both KBr-
pellets and NaCl-plates as mediums. H and 13C NMR spectra (300
1
MHz) were recorded in CDCl3 and DCON(CD3)2 solvents at 25 °C
on a Bruker AV300 Supercon NMR spectrometer using the solvent
signal as the internal standard in a 5 mm BBO probe. UV−visible
spectra and kinetic traces were monitored with a Shimadzu 2450 UV/
vis spectrophotometer equipped with multiple cell-holders and
thermostat. 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.
2
Synthesis of [Zn2L1 Br2] (2). This was prepared by the same
procedure as above by using anhydrous zinc bromide as the metal salt.
After 3 days, colorless and rhombic single crystals appeared in an
yellow oil. It was washed with cold methanol and ether to obtain good
crystals of X-ray crystallographic quality. Yield = 89 mg (45%).
Elemental analysis calcd (%) for C28H44Br2N2O6Zn2: C 42.29, H 5.58,
Br 20.10, N 3.52, O 12.07, Zn 16.45; found: C 42.18, H 5.65, Br 20.35,
N 3.49, O 11.81, Zn 16.52; 1H NMR (300 MHz, DMF-d7, 25 °C): δ =
2.24(s, 6H; ph-CH3), 2.86(t, 8H; N-CH2-CH2), 3.04(s, 12H; O-CH3),
3.38(t, 8H; N−CH2-CH2), 3.95(s, 4H; ph-CH2-N), 6.77(d, 2H; Ar),
7.2(d, 2H; o to CH2−N), 7.28 ppm(s, 2H; o to phenolic−OH); 13C
NMR (300 MHz, DMSO-d6, 25 °C): δ = 20.10(1C, ph-Me),
52.65(2C, O-Me), 56.57(1C, ph-CH2−N), 58.13(2C, N-CH2−CH2),
Syntheses of Ligands. Synthesis of HL1. To an ethanolic solution
(50 mL) of p-cresol (30 mmol, 3.24 g), bis(2-methoxyethyl)amine (30
mmol, 3.99 gms) 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 an additional 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. Yield = 3.04 g (40%). Elemental analysis calcd (%) for
86
dx.doi.org/10.1021/ic4015493 | Inorg. Chem. 2014, 53, 85−96

Inorganic Chemistry
Article
Table 1. Crystallographic Data and Details of Refinement for Complexes 1−4
1
2
3
4
empirical formula
C28H44Cl2N2O6Zn2
706.29
C28H44Br2N2O6Zn2
795.21
C28H44I2N2O6Zn2
889.19
C21H38Cl2N2O5Zn
534.80
Fw
crystal system
space group
a, Å
monoclinic
P21/n
monoclinic
P21/c
monoclinic
P21/c
triclinic
P1
̅
9.9384(3)
15.8724(5)
11.2234(3)
9.936(3)
9.929(5)
9.161(4)
9.568(4)
16.493(7)
84.493(5)
88.106(5)
63.801(4)
1291.0(9)
2
b, Å
15.907(4)
11.429(3)
15.766(8)
11.506(6)
c, Å
α, deg
β, deg
γ, deg
V, Å3
115.3530(10)
115.753(3)
114.326(5)
1599.93(8)
2
1626.9(8)
2
1641.3(14)
2
Z
D
calcd, g cm−3
1.466
1.623
1.799
1.376
M(Mo−Kα), mm−1
F(000)
1.708
3.971
3.383
1.190
736
808
880
564
θ range, deg
2.30−30.51
21033
4878
2.28−24.97
10834
2.33−26.23
9950
1.24−24.72
8143
no. of reflns collcd
no. of indep reflns
Rint
2839
3048
4239
0.0244
4051
0.0635
2337
0.1269
2183
0.0445
no. of reflns (I > 2σ(I))
refined params
goodness-of-fit (F2)
3304
181
181
184
288
1.030
1.048
1.024
1.043
a
R1, wR2 (I > 2σ(I))
residuals, e/Å3
R1 = ∑||Fo| − |Fc||/∑|Fo|, wR2 = {∑[w(Fo − Fc2)2]/∑[w(Fo )2]}1/2
0.0334, 0.0884
0.0450, 0.1160
0.901, −0.728
0.0615, 0.1336
1.393, −0.997
0.0605, 0.1564
0.638, −0.402
0.711, −0.522
a
2
2
.
69.41(2C, N−CH2-CH2), 115.19(1C, Ar), 127.07(1C, Ar),
128.48(1C, Ar), 129.56(1C, Ar), 129.69(1C, Ar), 154.88(1C, Ar−
OH); ESI-MS m/z: Calcd for [C14H23Br1N1O3Zn1]+ = 398.613,
Found = 398.02; FT-IR data (KBr pellet): γ bar = 796, 1022, 1109,
1271, 1491, 1610, 2927 cm−1. UV/vis (DMF): λmax(ε) = 239.5(4200),
266.7(6800), 285.8 nm (5800 L mol−1 cm−1).
3.92(s, 4H; ph-CH2-N), 6.94 ppm(s, 2H; Ar); 13C NMR (300 MHz,
DMSO-d6, 25 °C): δ = 20.20(1C, ph-Me), 52.41(4C, O-Me),
56.42(2C, ph-CH2−N), 58.23(4C, N-CH2−CH2), 69.58(4C, N−
CH2-CH2), 115.34(1C, Ar), 127.07(2C, Ar), 129.37(2C, Ar),
1 5 5 . 6 1 ( 1 C , A r − O H ) ; E S I - M S m / z : C a l c d f o r
[C21H38Cl1N2O5Zn]+=499.347, Found = 499.257; FT-IR data (KBr
pellet): γ bar = 563, 1117, 1197, 1266, 1370, 1475, 2925 cm−1; UV/vis
(DMF): λmax(ε) = 234.4(3200), 284.9(5000), 317.4 nm (1100 L
mol−1 cm−1).
2
Synthesis of [Zn2L1 I2] (3). Anhydrous zinc iodide (1 mmol, 319.19
mg) in acetonitrile (5 mL) was added dropwise to an acetonitrile
solution (10 mL) of the ligand L1 (1 mmol, 253 mg), and the mixture
was stirred very slowly for 30 min. The resulting yellow solution was
filtered. After 6 h, off-white colored microcrystalline square-shaped
crystals were formed in it. Yield = 112 mg (50%). Elemental analysis
calcd (%) for C28H44I2N2O6Zn2: C 37.82, H 4.99, I 28.54, N 3.15, O
10.80, Zn 14.71; found: C 37.63, H 5.16, I 29.42, N 2.76, O 10.52, Zn
Synthesis of [Zn(HL2)Br2] (5). It was synthesized by the same
procedure as complex 4. The crystalline compound was recrystallized
from DMF-H2O twice and used for further analysis as no single crystal
was obtained in this case. Yield = 217 mg (35%). Elemental analysis
calcd (%) for C21H38Br2N2O5Zn: C 40.44, H 6.14, Br 25.62, N 4.49, O
12.83, Zn 10.48; found: C 40.34, H 6.25, Br 25.75, N 4.41, O 12.74,
1
14.51; H NMR (300 MHz, DMF-d7, 25 °C): δ = 2.24(s, 6H; ph-
1
CH3), 2.85(t, 8H; N-CH2-CH2), 3.03(s, 12H; O-CH3), 3.37(t, 8H;
N−CH2-CH2), 3.91(s, 4H; ph-CH2-N), 6.83(d, 2H; Ar), 7.24(d, 2H;
o to CH2−N), 7.25 ppm(s, 2H; o to phenolic−OH); 13C NMR (300
MHz, DMSO-d6, 25 °C): δ = 20.14(1C, ph-Me), 52.68(2C, O-Me),
56.30(1C, ph-CH2−N), 58.09(2C, N-CH2−CH2), 69.71(2C, N−
CH2-CH2), 114.98(1C, Ar), 126.91(1C, Ar), 128.64(1C, Ar),
129.33(1C, Ar), 129.78(1C, Ar), 154.91(1C, Ar−OH); ESI-MS m/
z: Calcd for [C14H23I1N1O3Zn1]+ = 445.603, Found = 445.09; FT-IR
data (KBr pellet): γ bar = 743, 1022, 1115, 1271, 1492, 1610, 2877
cm−1; UV/vis (DMF): λmax(ε) = 238.6(5200), 266.4(7400), 285.8 nm
(6400 L mol−1 cm−1).
Zn 10.51. H NMR(300 MHz, DMF-d7, 25 °C): δ = 2.24(s, 3H; ph-
CH3), 2.86(t, 4H; N-CH2-CH2), 3.04(s, 6H; O-CH3), 3.38(t, 4H; N−
CH2-CH2), 3.95(s, 2H; ph-CH2-N), 6.96 ppm(s, 2H; Ar); 13C NMR
(300 MHz, DMSO-d6, 25 °C): δ = 20.32(1C, ph-Me), 52.45(4C, O-
Me), 56.29(2C, ph-CH2−N), 58.21(4C, N-CH2−CH2), 69.47(4C,
N−CH2-CH2), 115.30(1C, Ar), 126.91(2C, Ar), 129.39(2C, Ar),
155.70(1C, Ar−OH); ESI-MS m/z: Calcd for [C21H38Br1N2O5Zn]+ =
543.796, Found = 542.17; FT-IR data (KBr pellet): γ bar = 551, 1100,
1183, 1253, 1362, 1461, 2910 cm−1; UV/vis (DMF): λmax(ε) =
234.7(3000), 284.9(4800), 340.1 nm (600 L mol−1 cm−1).
Synthesis of [Zn(HL2)I2] (6). It was also synthesized by the same
procedure as complex 4. A good quality crystalline compound was
obtained and well characterized. Yield = 387 mg (54%). Elemental
analysis calcd (%) for C21H38I2N2O5Zn: C 35.14, H 5.34, I 35.36, N
3.90, O 11.15, Zn 9.11; found: C 35.09, H 5.28, I 35.41, N 3.93, O
11.09, Zn 9.20. 1H NMR(300 MHz, DMF-d7, 25 °C): δ = 2.24(s, 3H;
ph-CH3), 2.85(t, 4H; N-CH2-CH2), 3.03(s, 6H; O-CH3), 3.37(t, 4H;
N−CH2-CH2), 3.91(s, 2H; ph-CH2-N), 6.98 ppm(d, 1H; Ar); 13C
NMR (300 MHz, DMSO-d6, 25 °C): δ = 20.15(1C, ph-Me),
52.55(4C, O-Me), 56.48(2C, ph-CH2−N), 58.17(4C, N-CH2−CH2),
69.50(4C, N−CH2-CH2), 115.39(1C, Ar), 127.08(2C, Ar),
129.29(2C, Ar), 155.60(1C, Ar−OH); ESI-MS m/z: Calcd for
[C21H38I1N2O5Zn]+ = 590.798, Found = 592.08; FT-IR data (KBr
Synthesis of [Zn(HL2)Cl2] (4). To a solution of ligand L2 (1 mmol,
398 mg) in methanol (25 mL), methanolic anhydrous ZnCl2 (2.5
mmol, 340.5 mg) was added (10 mL) and refluxed for 1 h. It was
filtered, and ∼500 mg of white powder was collected. It was air-dried
and redissolved in the mixed solvent dimethylformamide/water (9:1).
Colorless rhombic crystals were formed after 1 month which were
washed with ether to obtain single crystals of good crystallographic
quality. Yield = 213 mg (40%). Elemental analysis calcd (%) for
C21H38Cl2N2O5Zn: C 47.16, H 7.16, Cl 13.26, N 5.24, O 14.96, Zn
12.23; found: C 47.10, H 7.25, Cl 13.21, N 5.15, O 15.0, Zn 12.29. 1H
NMR(300 MHz, DMF-d7, 25 °C): δ = 2.25(s, 3H; ph-CH3), 2.84(t,
8H; N-CH2-CH2), 3.02(s, 12H; O-CH3), 3.38(t, 8H; N−CH2-CH2),
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pellet): γ bar = 798, 1021, 1115, 1271, 1491, 1349, 2900 cm−1; UV/vis
(DMF): λmax(ε) = 234.4(3400), 284.9(5400), 316.5 nm (1200 L
mol−1 cm−1).
X-ray Data Collection. Crystal structure analyses of all
compounds reported (1−4) were carried out at room temperature
on a Bruker Smart Apex diffractometer equipped with CCD and Mo−
Kα radiation (λ = 0.71073 Å). Cell refinement, indexing, and scaling of
the data sets were done by using the Bruker Smart Apex and Bruker
Saint packages.35 All the structures were solved by direct methods and
subsequent Fourier analyses35 and refined by the full-matrix least-
squares method based on F2 with all observed reflections.36 The ΔF
map revealed the methoxy group -O(3)−C(13)H2- of 1 and 2
disordered over two positions of occupancy x and 1 − x. The x
parameter was refined to 0.540(6) and 0.534(15), respectively,
keeping these atoms isotropically. The H atom of the protonated
amine in 4 was located on the Fourier map, all the other were fixed at
geometrical positions. All the calculations were performed using the
WinGX System, Ver 1.80.05.37 Crystal data and details of refinements
are given in Table 1.
Figure 1. Designed ligands with NO3 and N2O5 donor sets obtained
by proper tuning of the Mannich reaction.
Scheme 1. Proposed Mechanistic Pathway for the Synthesis
of Ligands HL1 and HL2 in Dry Ethanol by Controlled
Mannich Reaction
Kinetic Measurements of the Hydrolysis of 4-NPP in 97.5%
(v/v) DMF-H2O. Disodium (4-nitrophenyl)phosphate (4-NPP)
hexahydrate was used as the substrate. The solutions of substrate 4-
NPP and Zn complexes in the mixed solvent were freshly prepared,
and the total volume of the reaction mixture was maintained as 2 mL.
An initial screening of the hydrolytic tendencies of all the metal-
complexes was performed till 2% formation of p-nitrophenolate (∼2
h) and then their kinetic data was collected. The hydrolysis rate of 4-
NPP in the presence of complexes 1−6 was measured by an initial rate
method following the increase in the 427 nm absorption due to the
released 4-nitrophenolate ion in aqueous DMF (ε, 18500 M−1 cm−1)
at 25 °C . For wavelength scan, spectra were recorded from a solution
having 1 mmol 4-NPP and 0.05 mmol Zn complex for 2 h. Kinetic
experiments were done both at conditions of excess substrate and
excess Zn complex keeping the other constant. Herein we report only
the former data. The study comprised 5 sets having the catalyst
concentration as 0.05 mmol and substrate as 0.5 (10 equiv), 0.7 (14
equiv), 1.0 (20 equiv), 1.2 (24 equiv) and 1.5 (30 equiv) mmol. The
reactions were initiated by injecting 0.04 mL of complex (2.5 × 10−3
M) into 1.96 mL of 4-NPP solution, and the spectrum was recorded
only after fully mixing at 25 °C. The visible absorption increase was
recorded for a total period of 30 min at an interval of 5 min. All
measurements were performed in triplicate, and the average values
were adopted. The reactions were corrected for the degree of
ionization of the 4-nitrophenol at 25 °C using the molar extinction
coefficients for 4-nitrophenolate at 427 nm.38 The final A∞ values for
each set were obtained after 2 days at 25 °C.
Scheme 2. Metal Incorporation Reaction Leading to the 2:2
Dinuclear [Zn2(L1)2X2] (1−3) and 1:1 Mononuclear Metal-
Complexes [Zn(HL2)X2] (4−6)
Computational Method. Geometry optimizations of complex 1
and 4 along with the possible intermediates in the proposed
mechanisms were carried out using the DFT computational technique
at the B3LYP level39 in the Gaussian03 (G03)40 software. For C, H, N,
O, P, and Cl the 6-31G(d) basis sets were used, while for Zn the
LANL2DZ basis set with effective core potential was employed.41 The
vibrational frequency calculations were performed to ensure that the
optimized geometries represent the local minima and there are only
positive eigenvalues.
occurrence of aromatic electrophilic substitution reaction. On
monitoring the status of the reaction repeatedly for 30 h at an
interval of 2 h, we found that the reaction proceeds through an
intermediate that can be isolated and is a likely potent
pentadentate ligand. Henceforth we proceeded in obtaining the
two products independently in pure form.
Thus, starting from p-cresol with bis(2-methoxyethyl)amine
and appropriate reaction conditions described below, two
different types of end-off compartmental ligands have been
synthesized, HL1 and HL2, having a NO3 and N2O5 donor set,
respectively (Figure 1).
RESULTS AND DISCUSSION
■
Design, Rationalization, and Characterization of
Ligands. Mannich reaction is a relevant organic reaction for
C−C bond formation widely used in the synthesis of
nitrogenous molecules especially secondary and tertiary amine
derivatives and applied as a key step in the synthesis of many
bioactive molecules and complex natural products.42,43 Fore-
most we had aspired to the synthesis of a conventional phenolic
ligand by this age-old reaction with maximum yield and purity.
The protocol requires a secondary amine [bis(2-methoxyethyl)-
amine in our case] and formaldehyde on one side and an
activated phenyl ring [p-cresol in our case] on the other for the
These Mannich base ligands are actually produced in the
reaction mixture by mono- and dicondensation of the
electrophilic species generated in situ in presence of a protic
solvent like ethanol. When the methylene iminium ion and p-
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Table 3. Selected Bond Distances (Å) and Angles (deg) for
Mononuclear Compound 4
4 (X = Cl)
Zn−N(1)
2.092(4)
Zn−O(1)
1.926(3)
Zn−X(1)
Zn−X(2)
2.2304(17)
2.2289(17)
93.86(15)
106.40(12)
115.14(13)
114.57(13)
112.01(12)
N(1)−Zn−O(1)
N(1)−Zn−X(1)
N(1)−Zn−X(2)
O(1)−Zn−X(1)
O(1)−Zn−X(2)
Table 4. Hydrogen Bond Geometry (Å/deg) in Complex 4
complex
D−H
d(D−H) d(H··A)
0.85(6) 1.96(6)
∠DHA
d(D··A)
A
Figure 2. ORTEP view (35% ellipsoid probability) of compound 1
with atom labels of the crystallographic independent part. Of the
disordered O3/C13 fragments, atoms at higher occupancy are shown
(53%). The same numbering scheme of the ligand is applied to
complexes 2 and 3.
4
N2−H
139(6)
2.659(6)
O1
Table 2. Selected Bond Distances (Å) and Angles (deg) for
a
Compounds 1−3
1 (X = Cl)
2 (X = Br)
3 (X = I)
Zn−N(1)
Zn−O(1)
Zn−O(1)#1
Zn−O(2)
Zn−X(1)
Zn−Zn#1
2.1157(14)
2.0643(11)
1.9727(12)
2.3025(13)
2.2068(6)
3.1081(4)
2.114(4)
2.059(3)
1.967(3)
2.296(3)
2.3412(9)
3.1032(11)
2.093(6)
2.032(5)
1.941(5)
2.283(5)
2.5085(13)
3.071(2)
N(1)−Zn−O(1)
N(1)−Zn−O(1)#1
N(1)−Zn−O(2)
N(1)−Zn−X(1)
O(1)−Zn−O(1)#1
O(1)−Zn−O(2)
O(1)−Zn−X(1)
O(1)#1−Zn−O(2)
O(1)#1−Zn−X(1)
X(1)−Zn−O(2)
93.08(5)
113.80(5)
75.84(5)
124.13(4)
79.34(5)
160.20(6)
106.17(4)
90.06(5)
121.02(4)
93.61(4)
93.15(13)
114.69(14)
76.10(12)
124.09(10)
79.19(13)
160.05(13)
105.05(10)
89.96(12)
120.45(10)
94.88(9)
93.3(2)
Figure 4. UV−vis spectra of the ligands in DMF at 5 × 10−5(M). Inset
116.1(2)
76.3(2)
shows the UV spectra at 0.1(M) concentration.
123.63(16)
78.8(2)
cresol are present in equimolar amount (p-cresol: amine:
formalin =1: 1: 1) and the reflux time is limited to a few hours,
the product is exclusively the single-side Mannich product. This
can be explained assuming that the product attains a high
degree of stability through intramolecular hydrogen bonding
leading to a 6-membered chelate. Thus, the product in this case
is less reactive than the reactant and hence reaction proceeds
on the latter. On the other hand with a large excess of the
electrophile generating species in the reaction mixture (p-
cresol:amine:formalin =1:3:3) and a prolonged reaction time
(ca. 1 day), the reaction proceeds further on the former single-
arm Mannich molecule leading to a double-side condensation,
which is actually the conventional product. Since some amount
of the intermediate still remains in equilibrium despite
enforcing the reaction, column chromatographic separation is
required to obtain the pure ligand.
159.2(2)
104.12(16)
89.5(2)
119.69(16)
96.64(16)
a
Primed atoms at −x,−y,−z+2 for 1; at −x+1,−y,−z for 2; at −x+1,−
y,−z+1 for 3.
Synthesis, Rationalization, and Characterization of
ZnII Metal-Complexes. The treatment of the two ligand
systems with anhydrous Zn-dihalide salts (i.e., ZnCl2, ZnBr2,
and ZnI2) led to the isolation of six complexes: four of which
were structurally characterized by single crystal X-ray
diffraction, and two were confirmed by elemental analysis and
conventional spectroscopic techniques. The structure analyses
(vide infra) reveal that the ligand HL1, which is apparently
mononucleating, gave rise to dinuclear Zn-complexes of close
comparable structure, irrespective of the anion used. On the
contrary, HL2 which is distinctly a dinucleating ligand,
Figure 3. ORTEP view (35% ellipsoid probability) of compound 4.
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Figure 7. Wavelength scan for the hydrolysis of 4-NPP in the absence
and presence of complex 1 (substrate:catalyst =20:1) in 97.5% DMF
recorded at 25 °C at an interval of 5 min for 2 h. [4-NPP] = 1 ×
10−3(M), [Complex] = 0.05 × 10−3(M). Arrow shows the change in
absorbance with reaction time.
Figure 5. UV−vis spectra of the metal-complexes 1−3 (A) and 4−6
(B) in DMF at 5 × 10−5(M) concentration.
Figure 8. Absorption profile on addition of 4-NPP to complex-1 due
to the formation of p-nitrophenolate (λmax = 427 nm) studied for 2 h
(Least-squares fit, R2 = 0.998). Inset shows the plot of log [A∞/(A∞
At)] versus time for 30 min (R2 = 0.99).
−
Figure 6. Probable solution state structures of complex 1−3 (A) and
4−6 (B) in DMF where X = Cl− (1, 4), Br− (2, 5), and I− (3, 6).
produced mononuclear Zn-complexes. We presume that the
complex nuclearity is addressed by the strength of haloic and
nitric acids (compared to acetates) and hence a high
concentration of protons in the vicinity of the second nitrogen
donor. Thus this atom becomes deactivated by intra- or
intermolecular proton transfer (the former being kinetically and
thermodynamically more favorable) because of hard−hard
interaction hampering its coordination to another metal ion.
Moreover in the case of the reaction of HL1 with ZnCl2 two
crystal forms were obtained. Since these are close comparable
dinuclear complexes, we have reported only the structure of
that having one independent centrosymmetric complex. Since
the strength of haloic acids follow the trend HI > HBr > HCl,
we can be sure about the mononuclearity of complex 5 and 6
(no crystal structure given) in addition to the other analyses
done, ruling out any similarity with the complex reported by
Yamasaki et al.29
Figure 9. Plot of enzymatic kinetics for complex-1 (V vs S, R2 =
0.975). Inset shows the Lineweaver−Burk plot (1/V vs 1/S) having
intercept = 35.62867 (error = 5.59117), slope = 0.02896 (error =
0.00437) and R2 = 0.936.
Crystal Structure Determinations. Dinuclear Complexes
1−3. All dinuclear complexes are isostructural, and compounds
2−3 are also isomorphous, crystallizing in the same space group
P21/c. The X-ray structural analysis revealed that in complexes
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Table 5. First-Order Rate Constants for Phosphatase
Activity Obtained by Michaelis−Menten Treatment of
Complex 1−6
complex
kcat (sec−1
)
1
2
3
4
5
6
9.35
8.80
8.33
4.68
3.29
2.88
Figure 12. Overlay of the Lineweaver−Burk curve of mononuclear
series (4, 5, and 6).
Figure 10. Overlay of the Lineweaver−Burk curve of a dinuclear (1)
and a mononuclear (4) complex having the same halide substitution
(Cl−).
Figure 11. Overlay of the Lineweaver−Burk curve of dinuclear series
(1, 2, and 3).
Figure 13. Schematically depicted plausible mechanism for the
complex 4 as a representative of mononuclear (4−6) catalyzed
hydrolytic pathway.
1−3 the metal ions are bridged by two phenolato units in a
centrosymmetric fashion forming a four-membered Zn2O2 core.
The ORTEP drawing of complex 1 is shown in Figure 2
(ORTEP of 2 and 3 is given in Supporting Information, Figure
S18 and S19). A selection of coordination bond lengths and
angles are reported in Table 2.
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
geometry for which a trigonality τ index44 of 0.60 is derived
for 1−3. Thus, the organic species acts as a chelating tridentate
ligand toward each Zn ion, and in addition the phenoxo oxygen
connects the other metal, so that one of the methoxyethyl arms
remains uncoordinated evidencing a slight disorder as found in
the crystal structure of 1 and 2 (see Experimental Section) or
large thermal ellipsoids in 3 of these chain atoms. Because of
the local symmetry, the halide ligands as well as the amine N
atoms are located trans with respect to the Zn2O2 rhomboid
mean plane.
In 1−3 the Z-N and Zn−O distances follow a regular trend
showing decreasing values ongoing from the chloro to the iodo
derivative (Table 2). However, it is worth noting that the Zn−
O(2) bond distances (involving the coordinated methoxy
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Figure 15. Energy profile diagrams: (a) mononuclear complex 4 and
(b) dinuclear complex 1.
angles fall in a range from 106.40(12) to 116.24(5)° in the
three complexes. The metal ions reside almost in the phenolato
plane (max deviation of 0.161(5) Å in 4), and the coordinated
amino N results displaced by about 0.75 Å from it. The
structural determination confirms that the amino nitrogen on
the other arm of the ligand is protonated and hampers, as
suggested in the discussion, the coordination of a second metal
ion. Although the geometry does not indicate a suitable
alignment of the N−H···O fragment, the N(2)···O(1) distance
is of about 2.67 Å in all cases favoring an intramolecular
interaction (Table 4). The crystal packing does not manifest
any peculiar feature.
NMR Spectral Properties. The proton NMR spectra of
both ligands HL1 and HL2 in CDCl3 show 5 signals in the
aliphatic region consisting of 3 singlets and 2 triplets. At the
aromatic region, for HL1 one singlet peak at δ7.26 and two
doublets at δ7.2 and δ7.43 are observed, whereas for HL2 the
two equivalent aromatic protons resonate at δ6.96 (Supporting
Information, Figure S11−14). The proton spectra of the metal-
complexes 1−6 were taken in DMF-d7 which has a base peak at
δ8.1 and δ3.7. The dinuclear ones (1−3) have similar type of
spectra as HL1 with slightly shifted values (4 singlets, 2
doublets, and 2 triplets) and mononuclear (4−6) are
comparable to HL2 (5 singlets and 2 triplets). The proton
which binds to the uncoordinated tertiary nitrogen in 4−6 is
probably abstracted by the basic solvent and does not appear in
the deshielding zone (Supporting Information, Figures S15−
S18). Comparing the spectra of ligand and complex we find
that there is not much difference because not only that zinc is a
diamagnetic center, the coordinated and uncoordinated
methoxy groups have almost the same kind of magnetic
environment. Thus from all the assignments it appears that the
solution state structural formulations are in good agreement
with their crystalline form.
Figure 14. Schematically depicted plausible mechanism for complex 1
as a representative of the dinuclear (1−3) catalyzed hydrolytic
pathway.
group) are significantly longer by 0.3 Å with respect to the
other Zn−O distances of the bridging phenolato oxygens. On
the other hand, the Zn-halide bond lengths augment along the
series, following the expected trend of 2.2068(6), 2.3412(9),
and 2.5085(13) Å in 1−3, respectively. The decreasing values
of coordination distances is also reproduced in the metal−metal
distance: these values vary from 3.1081(4) and 3.1032(11) Å in
1 and 2, respectively, and to 3.071(2) Å measured in 3.
Mononuclear Complex 4. The mononuclear entity of
complex 4 was verified by the X-ray structural analysis. The
ORTEP view of 4 is shown in Figure 3. The metal presents in
the complex a distorted tetrahedral coordination environment
being chelated by the phenolato ligand and by two
monocoordinated halides. The ethylmethoxy fragments of the
organic ligands point far away from the metal center. The Zn−
O and Zn−N bond lengths and their esd’s are 1.926(3) Å and
2.0917(17) Å, respectively (Tables 2 and 3). It is worth to
compare these distances with those found in the dinuclear
species [Zn2(L2)(MeCO2)2]+.29 Here the phenolato oxygen
bridge two hexa-coordinated zinc ions with Zn−O distances
averaging to 1.999(4) Å, and also the Zn−N distances are
significantly longer 2.147(4) Å likely to allow the methoxy
oxygen atoms to be coordinated.
The two Zn−Cl distances, averaging at 2.23 Å, are just
slightly longer than that found in the dinuclear species 1. The
chelating angle N(1)−Zn−O(1) of about 94° deviates
significantly from the ideal tetrahedral value, while the other
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a
Table 6. First-Order Rate Constants for the Hydrolysis of Various Phosphate Esters by Previously Reported ZnII Complexes
complex
substrate
conditions
kcat (s‑1)
reference
[Zn2L]
NPP
acetonitrile−water, pH 6.8, 25 °C
pH 6−6.5, 25 °C
methanol−water (33%, v/v), 30 °C
water−methanol (93:7), 40 °C
water, 35 °C
methanol−water (1:5), pH 7.2, 50 °C
DMSO/buffered water (1:1), 50 °C
water, 25 °C
476−203
50
55
51
56
57
52
58
59
60
[Zn2(L2O)]
HPNP
HPNP
BNPP
BNPP
BNPP
HPNP
BNPP
BNPP
4.10 × 10−3
3.44 × 10−4
3.60 × 10−5
2.26 × 10−6
4.60 × 10−6
4.00 × 10−7
5.74 × 10−7
2.30 × 10−8
[ZnII2(L2)-(μ-O2CMe)2(MeCN)2][PF6]
[MPGN-ZnII]
[Zn2(LH−2)]2+
[Zn2(L2H)(AcO)(H2O)](PF6)] 0.2H2O
[Zn2complex]
[Zn(bpy)Cl2]
[Zn2PBTPA]+
30% DMSO, 55 °C
a
BNPP = Bis(4-nitrophenyl)phosphate. HPNP = 2-Hydroxypropyl(4-nitrophenyl)-phosphate.
Electronic Properties. The absorption spectra of the
Kinetic Studies. The kinetic study of complexes 1−6 was
done by the initial slope method following the rate of increase
in absorption of the band at ∼427 nm corresponding to a rise
in 4-nitrophenolate concentration. The initial first-order rate
constants, V (min−1), for the cleavage of NPP were obtained
directly from the plot of log[A∞/(A∞α − At)] values versus
time (through origin) which were linear with R2 ≥ 0.99. A
typical plot of that kind is represented in the inset of Figure 8
for complex 1 (and Supporting Information, Figure S24 for
complex 4) along with the change in its absorbance profile at
427 nm for the concentration used in the wavelength scan
(Figure 7). To determine the dependence of rate constants on
substrate concentration, 1−6 were treated with five different
concentrations of substrate. As can be observed for each
complex, initially the cleavage rate increases linearly with the
increase of 4-NPP concentration but deviates gradually from
linearity and finally tends toward a saturation curve (Figure 9).
In other words, it is a first order kinetics at lower concentration
which gradually differs from unity at higher concentration. The
kinetic parameters (Vmax, kM, kcat) for the catalyzed reactions
were determined from the linear plots of 1/V versus 1/[NPP]
values (Lineweaver−Burk plot) as per the Michaelis−Menten
approach of enzymatic kinetics. Similar plots for 2−6 are given
in Supporting Information, Figures S25−S29. The results of
calculation remained the same when the experiment was done
by varying the [complex] under investigation at constant
[NPP]. All the kinetic parameters are given in Supporting
Information, Table S2, and only the kcat values are summarized
in Table 5.
To correct for the spontaneous cleavage of 4-NPP, each
reaction was measured against a reference cell that was identical
to the sample cell in composition except for the absence of Zn
complex. The spontaneous hydrolysis of 4-NPP had a much
lower reaction rate when compared to the metal catalyzed
reaction and hence the rate of spontaneous hydrolysis of 4-NPP
was too low to be taken into account in the kinetic
measurements. Therefore, the general rate of spontaneous
hydrolysis was not separately determined.49To confirm that the
synthesized ligands and the zinchalide-salts used do not
participate in the catalytic reaction, rigorous control experi-
ments have been performed to intake their role if any
(Supporting Information, Figures S30−S32). The results
indicated no appreciable change.
ligands in DMF (Figure 4) show two higher energy bands of
high intensity in the region 250−285 nm which is attributed to
the intraligand charge transfer of the open and closed forms of
hydrogen-bonded species and also a lower energy band (330−
430 nm) of lower intensity owing to the zwitterionic form of
the ligand. The complexes display one/two intraligand charge
transfer band(s) in 235−265 nm (high energy) and a ligand to
metal charge transfer band at ∼285 nm (low energy) which is
probably due to PhO−→ZnII or admixture of the former and
X−→ZnII (Figure 5).45−47 The spectra remains almost same
when measured in 97.5% DMF and therefore is not given
separately.
The solid-state absorption spectra of the complexes exhibit
peaks in the same region (Supporting Information, Figure S19
and S20).
Solution State Structures. ESI-MS spectra of the
complexes indicate a base peak corresponding to the presence
of a mononuclear unit for the dinuclear complexes (1−3) and a
monodehalogenated species for the mononuclear ones (4−6)
as depicted in Supporting Information, Figure S21 and S22 for
complex 1 and 4, respectively. Molar conductance studies in
97.5% DMF at pH 6 prove all the complexes are nonelectrolyte
(values are provided in Supporting Information, Table
S1).46,48,49 The UV−vis spectra of the complexes in the solid
and solution phase remains more or less unaltered suggesting
no major change in the basic chromophore of the molecules.
Nevertheless the spectral nature of a dinuclear and a
mononuclear complex in DMF is very similar emphasizing a
close resemblance in their structures in solution. Thus
according to our observations, in solution, a dinuclear complex
probably dissociates into two equivalent mononuclear moieties
whereas a mononuclear complex loses an HX molecule (X =
Cl−/Br−/I−, proton from the uncoordinated nitrogen and
halide ion from the zinc) having structures as shown in Figure
6. Thus in either case the absorption spectra is not much
perturbed.
Hydrolysis of Phosphomonoester in DMF-Buffered
H2O. To study the phosphatase activity of the zinc complexes,
disodium salt of (4-nitrophenyl)phosphate hexahydrate was
chosen as the substrate. Their hydrolytic tendency was detected
spectrophotometrically by monitoring the time evolution of p-
nitrophenolate (λmax = 427 nm) through wavelength scan from
200 to 800 nm in aqueous DMF (DMF:water = 97.5:2.5)
where substrate was in 20 equivalents of the catalyst, till
roughly 2% reaction conversion. The change in spectral
behavior of complex 1 is shown in Figure 7 as a representative
of dinuclear complexes and of 4 (Supporting Information,
Figure S23) for mononuclear ones.
The kinetic data shows very clearly that the first-order rate
constants (kcat) follow the order of 1 > 2 > 3 > 4 > 5 > 6. This
indicates that the dinuclear compounds (1−3) are better
catalysts for phosphate ester hydrolysis than mononuclear ones
(4−6) (Figure 10) as supported by Konig et al.,21 Kandaswamy
et al.,50 Mukherjee et al.51 and more interestingly, in both cases,
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halide dependence follows the order chloride > bromide >
iodide (Figure 11 and 12).
To get a better visualization of the energetically viable
mechanistic pathway proposed by us we have represented the
relative energies of the intermediates as the conventional
energy-profile diagrams, which are depicted in Figure 15a and
15b (for mononuclear complex 4 and dinuclear complex 1,
respectively).
Understandably, the kcat value of a dinuclear:mononuclear is
approximately 2 for all the three halide groups. For 1:4 it is
exactly equal to 2, for 2:5 it is 2.7, and for 3:6 it is 2.9.
Moreover the ratio of kcat for 1:2:3 = 1.12:1.06:1 whereas for
4:5:6 = 1.62:1.14:1. This is quite anticipated as reactivity has an
inverse relationship with selectivity, and as the halides move
toward electropositive character, the halide effect becomes
more and more prominent and hence the rate of decrease in kcat
value for mononuclear complexes increases.
It is now essential to compare the catalytic efficiency of our
Zn-complexes as hydrolytic catalyst with the previously
reported analogous Zn complexes in terms of kcat values.
Table 6 represents the hydrolytic rate constants of all recently
reported Zn complexes along with their relevant references.
Now on comparing Table 5 and Table 6 it can be safely
commented that our Zn complexes belong to the group of
complexes showing the highest catalytic efficiency reported till
date.
The commonly accepted mechanism of the metal ion
catalyzed hydrolysis of phosphate esters involves the following
key steps: deprotonation of a metal-bound species to form the
effective nucleophile, binding of the substrate to the metal
center(s), intramolecular nucleophilic attack on the electro-
philic phosphorus atom with the release of the leaving group,
and possibly regeneration of the catalyst.51 Thus to begin with,
first and foremost, the zinc(II) core recognizes and binds the
phosphate bond through coordinate linkage and electrostatic
interaction. Then it activates and nucleophilically attacks the
central phosphorus atom by a metal-bound external water/
hydroxide ion or the proximally bound methoxy group
generating a trigonal bipyramid phosphorus intermediate
from where the P−O ester bond is finally cleaved. Now the
latter possibility of the nucleophilic attack is geometrically and
electronically much less favorable. Therefore we have neatly
established a classical Lewis acid activation mechanism where
Zn(II)-coordinated H2O/OH− is the sole nucleophile, and an
increase in Lewis-acidity of the metal ion enhances the acidity
of the MII-coordinated water which in turn magnifies the
propensity of the metal ion to catalyze the hydrolysis of an
activated phosphate ester 4-NPP.
To rationalize the mechanistic pathway involved in
phosphatase activity we have performed detailed DFT
calculations considering complex 1 and complex 4 as
representatives of di- and mononuclear series respectively.
Along with the knowledge gathered from the literature
survey24,46−54 and the results obtained from DFT calculations
we have put forward the most probable mechanistic pathway
involved in mono- and dinuclear complex catalyzed reactions as
depicted in Figures 13 and 14, respectively. In case of a
mononuclear complex, the reaction is initiated by an association
of the substrate and metallocatalyst (intermediate II) with a
loss of halide ion. For the dinuclear complexes, the reaction is
initiated by the dissociation of the dinuclear (I) to mononuclear
species (II), association of the substrate molecule in a cis-
bidentate fashion (III) and loss of the halide ion (IV). This is
followed by an intramolecular nucleophilic attack by a pendant
methoxy group on the zinc center (III for monomer and X for
dimer) or the direct addition of a water molecule on the zinc
ion (IV for monomer and XI for dimer). Formations of IV and
XI (in case of mono- and dinuclear complexes, respectively) are
energetically more favorable than their respective counterparts
III and X, formed by intramolecular nucleophilic attack by a
pendant methoxy group. In the final step P−O bonds present
in IV and XI undergo hydrolytic cleavage due to nucleophilic
attack by internal water molecules leading to elimination of p-
nitrophenolate and simultaneous aquation generating the
intermediates V and XII. The subsequent dissociation of the
reactive species from V and XII regenerate the active catalyst to
continue the catalytic cycle. The concentration of active species
involved in case of the dinuclear complex is double that of the
mononuclear complex since one molecule of dinuclear species
generates two mononuclear species and that particular
consequence is supposed to be responsible for nearly double
reactivity (in terms of kcat) of dinuclear complexes over their
respective mononuclear counterparts. The role of halide is
attributed to their electron-withdrawing inductive effect which
improves the electrophilicity of the metal center thus enhancing
its hydrolytic reactivity. So the halide effect is actually their
electronegativity order.
CONCLUSIONS
■
An unprecedented Mannich reaction has been identified while
synthesizing the ligand where one of the two products is a
conventional intermediate in this age-old synthetic reaction. It
can henceforth be applicable to generate a mononucleating and
dinucleating ligand simultaneously on a similar organic
backbone. The metalation strategy also depicted an interesting
phenomenon where the nuclearity feature was prominently
reversed. The three dinuclear and three mononuclear ZnII
complexes so formed have been found to be efficient
monophosphatase catalysts. The kinetic parameters are quite
higher than most of the values reported till date under our
experimental conditions. The role of the coligand which is a
halide has been investigated, established, and reasoned. The
influence follows the order: Cl− > Br− > I−, irrespective of the
nuclearity of the catalysts in solid state. A classical hydrolytic
pathway for the phosphatase activity has been proposed on the
basis of our experimental observations, and DFT analyses have
been performed to rationalize our proposition. Influences of
other metal ions in the same ligand environment on
phosphatase-like activity are also under process.
ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray crystallographic data in CIF format of complexes 1−4,
ORTEP diagrams, kinetic plots, and FTIR, NMR, UV−vis, ESI-
MS spectroscopic data and DFT optimized energies of different
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: zangrando@units.it (E.Z.).
*E-mail: dasdebasis2001@yahoo.com (D.D.).
Notes
The authors declare no competing financial interest.
94
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Inorganic Chemistry
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ACKNOWLEDGMENTS
■
This research is funded by the Council of Industrial and
Scientific Research, New Delhi [CSIR project no. 01(2464)/
11/EMR-II dated 16-05-2011 to D.D.]. R.S. is thankful to
CSIR-SRF fellowship for financial support and Pujarini
Banerjee of IACS for ESI-MS measurements. We are also
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