Solvent dependent ligand transformation in a dinuclear copper(ii) complex of a compartmental Mannich-base ligand: Synthesis, characterization, bio-relevant catalytic promiscuity and magnetic study

Article abstract of DOI:10.1039/c5ra05776k

An "end-off" pentadentate compartmental ligand HL has been synthesized by Mannich base condensation using p-cresol and 2-benzyl amino ethanol and structurally characterized. A dinuclear copper(ii) complex, namely [Cu₂(L)(μ-OH)(H₂O)(C

Full text of DOI:10.1039/c5ra05776k

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Solvent dependent ligand transformation in a dinuclear copper (II)
complex of a compartmental Mannichꢀbase ligand: synthesis,
characterization, bioꢀrelevant catalytic promiscuity and magnetic
study
Ishani Majumder,a Prateeti Chakraborty,a Sudhanshu Das,a Hulya Kara,b Shymal Kumar
Chattopadhyay, c Ennio Zangrando,d and Debasis Das*,a
aDepartment of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata – 700009, India
b
Department of Physics, Faculty of Science and Art, Balikesir University, 10145 Balikesir,
Turkey
cDepartment of Chemistry, IIEST Shibpur, Howrah, India
dDepartment of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1,
34127 Trieste, Italy
Abstract
An “endꢀoff” pentadentate compartmental ligand HL has been synthesized by Mannich base
condensation using pꢀcresol and 2ꢀbenzyl amino ethanol and structurally characterized. A
dinuclear copper(II) complex, namely [Cu2(L)(µꢀOH)(H2O)(ClO4)2], has been prepared by
.
treating HL with Cu(ClO4)2 6H2O in methanolic solution with the aim to investigate its catalytic
promiscuity. Single crystal structural analysis reveals that CuꢀCu separation is of 2.9 Å.
Catecholase activity of the complex has been investigated in anhydrous DMSO as well as in
DMSOꢀwater mixture with progressive increasing quantity of water up to a 1:1 volume ratio in
order to assess the bio compatibility of the catalyst using 3,5ꢀDTBC as model substrate. In
anhydrous DMSO the catalytic activity reaches its peak and decreases with increasing the water
concentration, a feature most likely due to insolubility of 3,5ꢀDTBQ, the product formed in the
catalysis, in water. The complex also shows excellent phosphataseꢀlike activity by exploiting the
Lewis acidity, the necessary requirement for that activity, under different pH. Thorough
investigation reveals that no activity is observed at pH 6 but the activity increases with
increasing pH and attains maximum at pH 9. Variable temperature magnetic study shows that the
two Cu centers are antiferromagnetically coupled at low temperature with J value
1

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–78.63±1.30cm−1. In acetonitrile medium the complex shows very exciting behavior. A new
transformed ligand is generated that has been assigned as a Schiffꢀbase ligand, 2,6ꢀbisꢀ[(2ꢀ
hydroxyꢀethylimino)ꢀmethyl]ꢀ4ꢀmethylphenol. The genesis of the new ligand is a consequence of
dealkylation from HL followed by oxidation. This oxidation is counterbalanced by reduction of
Cu(II) to Cu(I) as it is evidenced from isolation of [Cu(MeCN)4](ClO4) from the mixture
followed by Xꢀray structural characterization of the species.
Introduction
Catalytic promiscuity, defined as the ability of a single active site to catalyze more than one
chemical transformation, constitutes a very important property of many enzymes.1ꢀ2 An
illustrative example of this interesting feature is chymotrypsin, which exhibits catalytic
promiscuity by catalyzing both amidase and phosphotriesterase reaction at its active site.3 Many
metalloenzymes containing two copper ions in their active sites can operate cooperatively.4
Consequently diꢀnuclear copper complexes with two metal ions are of great interest to the
researchers in relation to their potential use as bimetallic catalysis, mimicking the active site of
enzymes.5ꢀ6 Only a few dinuclear copper complexes are known which may show catalytic
promiscuity in important bioꢀrelevant systems involving two important bioꢀrelevant material
such as H2O and O2.1ꢀ2 Our laboratory is actively engaged in developing the structure function
relationship in synthetic analogues of some metallobiosites like catechol oxidase, phosphatase
etc 5,7,8. The bioꢀcompatibility of catechol oxidase study is hampered mainly due to the
insolubility and/or dissociation of the catalyst in water medium. On the other hand, it is well
known that natural phosphatases accomplish the hydrolysis of phosphateꢀ ester bond using redox
innocent Zn(II) in their active sites, employing Lewis acidity, rapid ligand exchange behavior
and coordination flexibility of Zn(II).9 To give a breakthrough in the new arena of catecholase
activity we have synthesized a water soluble dinuclear copper(II) complex of a symmetric
Mannich base ligand as a model for the met form of the active site of catechol oxidase. In
addition to exhibit catecholase activity the complex is also able to catalyze the hydrolysis
reaction of phosphateꢀester bond when the substrate is 4ꢀnitro phenyl phosphate (4ꢀNPP). Along
with the versatile catalytic activity, variable temperature magnetic study and electrochemical
behavior of the complex has been investigated. More interestingly, in acetonitrilic medium a
unique ligand transformation was observed, which was coupled with dealkylation and
2

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concomitant redox reaction. All these interesting features have been vividly portrayed in this
manuscript.
Keywords: Mannichꢀbase, Cu(II) complex, Catecholase activity, Phosphatase activity, Ligand
transformation, Magnetic property.
Results and Discussion
Design, Rationalization, and Characterization of Ligand. 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 natural products.10 Foremost we had aspired to the synthesis of a
conventional phenolic ligand with N, N, O donor sites by this ageꢀold reaction with maximum
yield and purity. For Mannich condensation a secondary amine [2ꢀbenzyl amino ethanol] and
formaldehyde on one side and an activated phenyl ring [pꢀcresol] on the other side are generally
chosen for the occurrence of aromatic electrophilic substitution reaction. The interaction between
formaldehyde and 2ꢀbenzyl amino ethanol first of all generates methylene iminium intermediate
which subsequently attacks pꢀcresol at two ortho positions (with respect to phenolic –OH) and
thereby generates an endꢀoff compartmental ligand, HL, having N2O3 donor set (Fig. 1). Since
some amount of the intermediate (methylene iminium) still remains in the equilibrium of the
reaction, column chromatographic separation is required to obtain the pure ligand.
Fig. 1 Designed ligand with N2O3 donor sets obtained by proper tuning of the Mannich reaction.
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Synthesis, Rationalization, and Characterization of the MetalꢀComplex. The complex was
synthesized adopting sequential or stepwise synthesis technique i.e. the Mannichꢀbase ligand HL
was synthesized and characterized first, then it was treated with a methanolic solution of copper
(II) perchlorate dihydrate. Complex 1 was obtained after recrystallization of the product obtained
from the above reaction in ethanol medium. The IR spectrum of the complex shows a band due
to CꢀN stretch at 1576 cmꢀ1 and skeletal vibration at 1496 cmꢀ1. Broad band centered at 1078 cmꢀ
indicates the presence of perchlorate ion in the complex.11 Additionally, the observed bands at
1
1132, 1281, 897 cmꢀ1 confirm the presence of bridging perchlorate ion in the complex.11
Solution Studies: Electronic Spectra, Potentiometric Titration and Mass Spectrometry. In
order to characterize the catalytically relevant species for hydrolase like activity UVꢀVis study,
potentiometric titration and ESIꢀMS study are carried out in DMSOꢀwater mixtures (3:1).
Electronic spectral study of the complex 1 was performed at different pH (Fig S7). The band
around (600ꢀ650) nm composed of dꢀd transition changes significantly and thus suggests that the
coordination environment around the Cu(II) centers must be considerably distinct at different
pH.
By examining the electronic spectrum of 1 as a function of pH an interesting spectral
change that involves the species in equilibrium could be observed. At very lower pH ∼ (3ꢀ4) the
potentiometric titration indicates the pKa which corresponds to the deprotonation of phenolic ꢀ
OH (Fig S8; Table S1)) and the phenoxo ligand thus obtained helps to generate the dinuclear
species as 1a [Cu2L(H2O)2]3+. When the pH of the solution is raised to 5 another protonationꢀ
deprotonation equilibrium is observed involving one of the coordinated H2O, with corresponding
pka of 6.1 (Table S1) and the species is most like to be [Cu2L(µꢀOH)(H2O)]2+ (1b) (Fig 2.) The
second pKa is calculated as 8.15 and it can be attributed to the formation of [Cu2L(µꢀOH)(OH)]+
(1c) by deprotonation of the bound water molecule from the species 1b. All this protonationꢀ
deprotonation phenomena is depicted in the scheme1.
4

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∼
Scheme 1. Structural representation of the pHꢀdependent aqueous equilibria of 1.
Fig. 2 Spectrophotometric titration of 1 (a) (pH value: 5.54ꢀ6.85; pKa = 6.10) and (b) (pH value:
7.15ꢀ9.23; pKa = 8.15) Conditions: Complex = 10ꢀ3 [M]; [KCl] = 0.100 mol Lꢀ1; [KOH] = 0.100
mol Lꢀ1; in solution DMSO /water (75:25%v/v; 50 mL) at 25ºC.
From the above discussion it is clear that at nearly neutral pH our complex should exists as 1b
and that very proposition is supported by ESIꢀMS study of complex 1 (Fig S9 ) in DMSOꢀwater
medium (pH=6.7) where we observed the base peak at ~ 297.78 amu (calc. 297.71 amu) matches
well with the m/z value of [Cu2L(µꢀOH)(H2O)]2+ (1b).
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Crystal Structure Description. The ORTEP diagram of complex 1 with atom numbering
scheme is depicted in Fig. 3, and a selection of bond lengths and angles is given in Table 1. The
crystal structure reveals that in the dinuclear complex the copper atoms Cu1 and Cu2 possess a
distorted square pyramidal and octahedral geometry, respectively. The doubly bridging phenoxo
and hydroxo oxygen atoms occupy the basal plane, and the metals complete their coordination
geometry through the amineꢀnitrogen and hydroxyꢀoxygen donors of the pentadentate chelating
ligand.
In the basal plane the CuꢀO bond distances are comparable in lengths ranging from 1.912(6) to
1.963(7)Å (Table 1), the shorter values for the hydroxo oxygen O1. On the other hand the Cuꢀ
N(amino) bond distances are slightly longer, of 2.004(8) and 1.988(8) Å. The bridging bond
angles subtended by the phenoxo and hydroxo groups, of Cu1ꢀO1ꢀCu2 and Cu1ꢀO4ꢀCu2 of
99.7(3) and 97.1(3)°, respectively, lead to a metalꢀmetal separation of 2.926(2) Å. The apex of
the square pyramidal geometry at Cu1 is occupied by a perchlorate oxygen (Cu1ꢀO10 =
2.561(11) (Å). On the other hand, the axial positions in the octahedral geometry of Cu2 are
occupied by an aqua ligand and an oxygen atom of the second perchlorate as shown in Fig. 3
(Cu2ꢀO1w = 2.519(12);Cu2ꢀO6 = 2.590(10)Å). All these CuꢀO bond lengths are longer with
respect to the other coordination bond distances as consequence of the JahnꢀTeller effect.
The crystal packing shows the complexes paired about a crystallographic center of symmetry
leading to the formation of strong Hꢀbonds between the alcoholic groups (O4…O3’ = 2.571 Å,
Fig. 4).
Table 1. Selected Coordination Bond Lengths (Å) and Angles (deg) for complex 1 with esds in
Parentheses.
Cu1ꢀO1
Cu1ꢀO2
Cu1ꢀO4
Cu1ꢀN1
Cu1ꢀO10
Cu1…Cu2
1.916(7)
1.962(8)
1.940(7)
2.004(8)
2.561(11)
2.926(2)
Cu2ꢀO1
Cu2ꢀO3
Cu2ꢀO4
Cu2ꢀN2
Cu2ꢀO1w
Cu2ꢀO6
1.912(6)
1.924(7)
1.963(7)
1.988(8)
2.519(12)
2.590(10)
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O1ꢀCu1ꢀO4
O1ꢀCu1ꢀO2
O4ꢀCu1ꢀO2
O1ꢀCu1ꢀN1
O4ꢀCu1ꢀN1
O2ꢀCu1ꢀN1
O1ꢀCu1ꢀO10
O4ꢀCu1ꢀO10
O2ꢀCu1ꢀO10
N1ꢀCu1ꢀO10
O1ꢀCu2ꢀO3
O1ꢀCu2ꢀO4
O3ꢀCu2ꢀO4
81.7(3)
174.0(4)
97.5(3)
94.7(3)
174.9(3)
85.6(3)
87.8(4)
86.1(4)
98.2(4)
97.5(4)
176.7(3)
81.3(3)
100.2(3)
O1ꢀCu2ꢀN2
O3ꢀCu2ꢀN2
O4ꢀCu2ꢀN2
O1ꢀCu2ꢀO1w
O3ꢀCu2ꢀO1w
O4ꢀCu2ꢀO1w
N2ꢀCu2ꢀO1w
O1ꢀCu2ꢀO6
O3ꢀCu2ꢀO6
O4ꢀCu2ꢀO6
N2ꢀCu2ꢀO6
O1wꢀCu2ꢀO6
Cu1ꢀO1ꢀCu2
Cu1ꢀO4ꢀCu2
93.2(3)
85.5(3)
174.1(3)
100.2(4)
82.9(4)
88.7(3)
90.4(4)
92.3(3)
85.0(3)
82.9(3)
99.3(3)
163.8(4)
99.7(3)
97.1(3)
Fig. 3 ORTEP drawing (ellipsoid probability 30%) of complex 1 (C atoms not labelled for sake
of clarity).
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Fig. 4 Crystal packing: dinuclear complexes paired through strong hydrogen bonds involving the
alcoholic groups (O4…O3’ = 2.571 Å; O4ꢀH…O3’ = 167°). Perchlorate and water molecules are
not shown for clarity.
PXRD Study of the Complex 1. In order to gain a better understanding of the uniformity of the
complex 1, we performed a PXRD study (Fig. S10) and compared the observed pattern with the
PXRD pattern generated from the CIF file of the crystal. The two patterns are nearly
superimposable and thereby suggests that the crystalline nature of the species remains intact in
bulk form. Therefore it is reasonable to explore all the catalytic activities with this bulk.
Catecholase Activity.
Catechol oxidase, a typeꢀ3 copper protein can bind oxygen reversibly at room temperature and so
it can be employed to oxidize phenols to respective oꢀbenzoquinones. As a model of the enzyme
we have taken one dinuclear hydroxo bridged complex of Cu(II) and have studied it’s efficiency
towards the oxidation of 3,5ꢀdiꢀtertꢀbutylcatechol (3,5ꢀDTBC) to 3,5ꢀdiꢀtertꢀbutylbenzoquinone
(3,5ꢀDTBQ). Interestingly, it displays significant catalytic activity towards the oxidation of 3,5ꢀ
DTBC to 3,5ꢀDTBQ in DMSO medium. Before proceeding into detailed kinetic study we have
checked the ability of the complex to mimic the active site of catechol oxidase by treating 1×10ꢀ4
mol dm3 solutions of complex 1 with 1×10ꢀ2 mol dmꢀ3 (100 equivalents) of 3,5ꢀDTBC under
aerobic condition. Fig. 5 shows the spectral change for the complex upon addition of 100ꢀfold
3,5ꢀDTBC (1×10ꢀ2 M ) observed at an interval of 5 mins in DMSO medium. The kinetics of the
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oxidation of 3,5ꢀDTBC was determined by monitoring the increase of the concentration of the
product 3,5ꢀ DTBQ and the experimental conditions were the same as we reported earlier.7 All
the complexes showed saturation kinetics and a treatment based on the Michaelis−Menten model
seemed to be appropriate. The binding constant (KM), maximum velocity (Vmax), and rate
constant for dissociation of substrates (i.e., turnover number, kcat) were calculated for the
complex by using the Lineweaver−Burk graph of 1/V vs 1/[S] , using the equation 1/V =
{KM/Vmax}{1/[S]} + 1/Vmax,}, and the kinetic parameters are presented in Table 2 and in Table
S2.
Fig. 5 Changes observed in UV−vis spectra of complex 1 for 120 minutes (conc. 1 × 10−4 M)
upon addition of 100ꢀfold 3,5ꢀDTBC (1 × 10−2 M).(inset) Absorption profile due to the formation
of 3,5ꢀDTBQ (λmax = 393 nm) on addition of 3,5ꢀDTBC to complex 1.
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Fig. 6 Dependence of rate of reaction on substrate concentration for complex 1(100 ꢁM) at 25°C
in DMSO for oxidation of Catechol.
Fig. 7 Lineweaver−Burk plots for complex 1 in DMSO medium for oxidation of Catechol
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Table 2. First Order Rate Constants for the Oxidation of Catechols by the Complex 1 and the
Previously Reported analogous Dinuclear Cu(II) Complexes using 3,5ꢀDTBC as substrate.
Complex
Solvent Cu…Cu kcat in hꢀ1 Ref.
(Å)
[Cu2(L)(µꢀOH)(H2O)(ClO4)2]
DMSO
2.926
0.76×102
Present
work
0.28× 102 12
[Cu2(H2bbppnol)(µꢀ
MeOH
3.400
OAc)(H2O)2]Cl2.2H2O
[Cu2(H2btppnol)(µꢀOAc)](ClO4)2.
[Cu2(L1)(
µꢀOAc)](ClO4)2.(CH3)2CHOH
[Cu2(L2)(µꢀ
OAc)](ClO4).H2O.(CH3)2CHOH
[Cu2(tppnol)(OAc)(ClO4)2
[Cu2L3H(OH)]
MeOH
MeOH
3.400
3.350
0.28× 102 12
0.90×102
1.83×102
2
2
MeOH
MeOH
MeOHꢀ 3.500
water
3.560
3.360
0.11×102
0.02×102
13
14
mixture
Acetone 2.980
[Cu2(LOCH3)(µꢀOH)]2+
[Cu2(LCH3)(µꢀOH)]2+
[Cu2(LF)(µꢀOH)]2+
0.21×102
0.10×102
0.05×102
15
15
15
Acetone 2.966
Acetone 2.969
Mechanistic interpretation on catecholase activity exhibited by complex 1.
The mechanistic interpretation of catecholase activity of synthetic analogues of catechol oxidase
divulges that substrateꢀcatalyst interaction is the key step of the mechanism. Catechol may bind
16
the catalyst in a variety of coordination mode starting from monodentate asymmetric type to
bridging bidentate17a or via combination of both.5,7 However in the present case the spectral
study is not distinct enough to assign the binding mode of catechol to the copper center as we
noticed in our earlier studies.5,7 However, a preꢀequilibrium of the free complex and the substrate
exists preceding to the intramolecular electron transfer in the key step of the mechanism
followed by reduction of Cu(II) to Cu(I) with concomitant oxidation of catechol to quinone. Now
the rate determining step of the mechanism may be either the reꢀoxidation of Cu(I) by dioxygen
or the intramolecular electron transfer. Since in our case the dꢀd band remains intact with slight
blue shift (Fig. S11) during the catalytic reaction, and the detection of the intermediate by mass
spectral study after one hour of the mixing (Fig S12) supports the fact that the intramolecular
electron transfer is most probably the rate determining step. In the catalytic cycle the role of
dioxygen is very crucial for regeneration of the catalyst with its concomitant reduction of either
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kind: (i) two electron reduction to H2O2 or (ii) four electron reduction to water.16ꢀ17 But in our
case the spectral study after treatment with iodide fails to detect I3ꢀ (Fig. S13) and thereby clearly
excludes the possibility of dioxygen reduction to H2O2.
Study of Catecholase Activity in water medium: Searching for a BioꢀCompatible Catalyst.
There are plenty of reports in the literature of synthetic analogues of catechol oxidase in different
7,12ꢀ18
solvents,
but only few reports deal with the activity of catalyst in water medium.14 Metal
complexes of Schiff base cannot mimic catecholase activity in water medium as they have a
imine bond which may get hydrolyzed in presence of water. So we have designed an ammine
complex of Mannich base ligand which is soluble in water and therefore suitable to explore its
bio compatibility mode. Our synthesized complex 1 shows good catalytic efficiency up to a
50:50 solvent mixture of DMSO:water medium (Fig. S14 and S15). The catalytic efficiency
decreases with increasing the water quantity which may be due to the precipitation of DTBQ and
thereby the contribution of the increasing concentration of 3,5ꢀDTBQ may not be assessed by
spectral scan. However, at this point further extensive work is necessary to get deep insight on
role of water in catecholase activity as done by other investigator.14
Phosphatase Activity.
There are plenty of literature reports in which labile coordination sites of dinuclear transition
metal complexes assist the hydrolysis of phosphate esters.8a The criteria for the molecular
species to function as an efficient catalyst towards the hydrolysis is to provide the H2O/OHꢀ
species as nucleophile. Since our complex possesses a nucleophile constituted by metal bridging
hydroxyl group and a bound water molecule it would be reasonable to explore its phosphatase
activity following the hydroxylation of 4ꢀNPP (4ꢀnitrophenyl phosphate salt). The catalytic
efficiency of the complex was determined using the method of pseudoꢀfirst order rate constant by
monitoring the growth of 4ꢀnitrophenolate absorption band at 425 nm in 75% DMSO solution at
pH 9.0 at 25°C. The concentration of the substrate 4ꢀNPP was always kept at least 10 times
larger than that of the Cu(II) complex to maintain the pseudoꢀfirst order condition. Initially a
series of solutions of substrate 4ꢀNPP, having 5 different concentrations, were prepared from
concentrated stock solution of substrate using DMSO and buffered water in 3:1 ratio. After the
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addition of substrate to the solution of the catalyst a new band corresponding to 4ꢀnitrophenolate
appeared at 425 nm. The course of this typical reaction at pH 9.0 is shown as.
Fig. 8 Wavelength scan for the hydrolysis of 4ꢀNPP in the absence and presence of complex 1
(substrate:catalyst = 20:1) in 75% DMSOꢀbuffer medium at pH 9.0 recorded at intervals of 5
minutes for 90 minutes (pH= 7.0, T = 25°C); [4ꢀNPP]=1 × 10ꢀ3(M), [Complex]=0.05 × 10ꢀ3(M).
The arrow shows the change in absorbance with reaction time. (inset) Absorption profile due to
the formation of 4ꢀnitrophenolate (λmax = 425 nm) on addition of 4ꢀNPP to complex 1.
The first order rate constants for catalytic phosphate ester cleavage were determined from the
slopes of the plots of log[A∞/A∞ꢀAt] vs time for different sets. In order to determine the rate
dependence on the substrate concentration, the complex is treated with different concentrations
of 4ꢀNPP. Initially a first order dependence of the substrate concentration was observed, while
saturation kinetics was observed at higher concentration (Fig. 9).
The rates of reaction for various substrate concentrations were interpreted with Michaelisꢀ
Menten approach and the data fitted by means of LineweaverꢀBurk plot to calculate the kinetic
parameters (Fig. 10).
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Fig. 9 Dependence of rate of reaction on substrate concentration for complex 1 (50 ꢁM) at 25°C
in 75% DMSO (pH 9) for hydroxylation of 4ꢀNPP.
Fig. 10 Lineweaver−Burk plot for the 4ꢀNPP hydroxylation by complex 1.
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Table 3. FirstꢀOrder Rate Constants for the Hydrolysis of Phosphate Esters by by the complex 1
and the Similar Previously Reported Cu(II) Complexes.
Complex
Conditions
kcat in sꢀ1 Substrate Ref.
[Cu2(L)(µꢀOH)(H2O)(ClO4)2]
3:1 DMSO:Water, pH 1.69
9.0, 25ºC.
4ꢀNPP
Present
work
2
[Cu2(L1)(
1:1 MeCN:Water, pH 2.97×10ꢀ4 2ꢀ4
µꢀOAc)](ClO4)2.(CH3)2CHOH
[Cu2(L2)(µꢀ
8.0, 50ºC.
BDNPP
1:1 MeCN:Water, pH 5.16×10ꢀ4 2ꢀ4
2
OAc)](ClO4).H2O.(CH3)2CHOH 8.0, 50ºC.
BDNPP
MeCN:Water, 5.30×10ꢀ4 2ꢀ4
BDNPP
MeCN:Water, 2.10×10ꢀ2 2ꢀ4
BDNPP
[Cu2L (Cl3)]
1:1
19
1
50ºC.
1:1
[Cu2(L)(µꢀOH)]2+
50ºC.
For correction of spontaneous cleavage of 4ꢀNPP in presence of the buffer, each reaction was
carried out against a reference cell that was identical to the sample cell in composition except for
the absence of complex 1. The time dependent spectral scan (Fig. 21) clearly suggests that the
rate of spontaneous hydrolysis of 4ꢀNPP is negligible in absence of catalyst and hence the rate of
spontaneous hydrolysis of 4ꢀNPP is not taken into account in the kinetic measurements. To
confirm that the synthesized ligands and the copper perchlorateꢀsalt used in the reaction do not
participate in the catalytic process, rigorous control experiments have been performed to intake
their role if any (Fig S22ꢀ23).The results indicated no appreciable change as we noticed in our
earlier experiments also.8
The effect of pH on catalytic activity was also studied in the pH range 6ꢀ9 (Fig 8 and Fig. S17ꢀ
S20). It is observed that the reaction rate increases on increasing the pH and finally gets saturated
at high pH 8.5. The complex shows no activity below pH 6.5, and the activity slightly increases
at pH 7.0 and becoming maximum at pH 8.5ꢀ9.0. The phosphatase activity of complex 1 is thus
strongly influenced by pH of the reaction mixture and reveals sigmoidal shape profiles (Fig. 11).
Here the data were fitted using a Boltzman model, resulting pKa =7.8 ± 0.2.Which is in fair
agreement with the value (pKa =8.15) found from potentiometric titration [Fig 2(b)]. Slight
difference in the value may be explained by the substrateꢀcatalyst interaction, which in some
how affects the Lewis acidity of the Cu (II) centers. Although such interpretation should be
considered as speculative because no further experimental and theoretical evidence is available at
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present. However, deprotonation of the Cu(II) coordinated water molecule may occurs at higher
pH to generate the catalytically active species [Cu2(L)(µꢀOH)(OH)]+ and consequently the rate of
hydrolysis of 4ꢀNPP increases at higher pH (>7.5). This pH ꢀdependent rate constant suggests
that deprotonation of a metalꢀbound water molecule favors the generation of catalytically active
nucleophile.20ꢀ23
Fig. 11 Dependence of the rate of 4ꢀNPP hydroxylation on pH by complex 1 in 75% DMSOꢀ
buffer at 25 °C.
As far as mechanistic interpretation of phosphateꢀester bond hydrolysis is concerned it
may be stated that the incoming substrate first of all binds effectively to the catalyst followed by
nucleophilic attack by the metal bound hydroxo group present in the complex to cleave PꢀO bond
with subsequent release of the product in the form of 4ꢀnitrophenolate ion.
Cyclic Voltammetric Study.
In DMSO solution the complex 1 undergoes two reductive responses at Epc values of ꢀ0.05 V and
ꢀ0.51V, with the later peak having current height 2.5 times that of the first peak (Fig. S28). On
reverse scan a strong stripping peak is detected at 0V, along with an oxidative response at 0.1V,
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which is probably coupled to the reductive peak at ꢀ0.05 V. On restricting the scan in the
negative side up to ꢀ0.2 V a quasireversible couple is detected with E1/2 value of 0.03 V, peak to
peak separation of 172 mV and ipc / ipa = 1.17; no stripping current is observed on scan reversal
in this case (Fig. 12). In differential pulse voltammetry (DPV) experiments two reductive peaks
are detected at Ep = ꢀ0.064 V and ꢀ0.316 V corresponding to E1/2 values of ꢀ0.04V and ꢀ0.29 V
respectively (Ep = E1/2 – ꢂE/2; where ꢂE is the pulse amplitude which in our experiment is 0.05
V). However, in this case the first peak has almost 3.4 times the current height of the second
peak. We assign the quasiꢀreversible reduction at 0.03 V observed in CV experiments to
Cu(II)/Cu(I) reduction of one of the Cu(II) center, while the irreversible reduction at ꢀ0.5V is
assigned to the Cu(II)/Cu(0) reduction of the second center along with Cu(I)/Cu(0) reduction of
the first center. Though the mixed valent Cu(I)Cu(II) species is stable in cyclic voltammetry time
scale, it probably undergoes slow decomposition due to strain suffered by the relatively rigid
ligand to accommodate a tetrahedral Cu(I) and another square planar Cu(II) center, and this may
account for the abnormally low current height of the peak at ꢀ0.316 V in DPV. The cyclic
voltammogram and differential pulse voltammogram of the complex is invariant with respect to
time in DMSO solvent.
Fig. 12 CV Cyclic voltammogram of complex 1, in DMSO, at the GC electrode at 100 mV s−1
scan rate as representative.
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On addition of 3,5ꢀDTBC to a DMSO solution of the complex, the Cu(II)/Cu(I) couple at 0.3 V
is replaced by a new couple with Epc = ꢀ0.02 V and Epa = +0.3 V and the current height of the
reductive peak being much larger than the metal based couples. We tentatively assign the
cathodic peak at ꢀ0.02 V to the reduction of free 3,5ꢀDTBQ to Cu(II)2 bound diꢀdeprotonated
3,5ꢀDTBC, and the anodic peak at +0.3 V to oxidation of Cu(II)2 bound 3,5ꢀDTBC to free 3,5ꢀ
DTBQ (Fig. 13).
Fig. 13 CV spectrum of complex 1 after addition of 3,5ꢀDTBC solution at the GC electrode at
100 mV s−1 scan rate as representative.
Magnetic Study.
The magnetic properties of complex 1, in the form of χM and χMT (χM is the susceptibility per
dinuclear unit) vs. T plots, are shown in Fig. 14 in a temperature range 2ꢀ300K. The χmT values
at 300 K, 0.828 emu K molꢀ1 (µeff = 2.57 µB) for 1, which is characteristic for two nonꢀcoupled
copper (II) ions with an average g factor of 2.10 (χMT = N ꢀ
B2 g2/2k). As the temperature is
lowered, the χmT values decrease in a monotonous manner and become 0.073 emu K molꢀ1 for 1
at 2 K. The curves are typical for antiferromagnetically coupled systems; the rise of the
susceptibility at low temperature is caused by paramagnetic impurities.24ꢀ26 Without these
paramagnetic impurities χM should tend to zero at 0 K.
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For dinuclear Cu (II) complexes (S1=S2=1/2), the theoretical expression of the magnetic
susceptibility based on the Heisenberg Hamiltonian (H=−2JS1S2) is:
2Ng2ꢀB2
[
Ng2ꢀB2
2kT
χ
=
(1−
ρ
)+
ρ
+ Nα
Eq.1
kT
3+ e−2J / kT
]
In this expression
ρ
present the fraction of paramagnetic impurity in the sample, Nα a
temperature independent paramagnetism and the other symbols have their usual meanings. The
paramagnetic impurity was assumed to be a mononuclear copper(II) species. To determine the
exchange parameters, χM was fitted for the range 2–300 K (solid curve in Fig. 14) for 1 gives the
best agreement with the experimental data for J = – 78.63±1.30, g=2.13±0.03, ρ= 0.08±0.01 and
Nα=0.00024±0.00008 cm3molꢀ1 (R2 = 0.99). The obtained magnetic parameters, J, g and ρ are
compatible with the values observed for other Cu (II) dimers. 27ꢀ36
0,04
0,03
0,02
0,01
0,00
1,0
0,8
0,6
0,4
0,2
0,0
0
50
100 150 200 250 300
T (K)
Fig. 14 Temperature variation of the magnetic susceptibilities of 1 as χ and χT versus T plots
(The solid line represents the best fit of the experimental data based on the Heisenberg model).
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Special characteristic feature of complex 1 in acetonitrile: Dealkylation followed by
oxidation to an imine ligand.
An unexpected characteristic feature of this complex takes place when the synthesis of complex
1, or its dissolution, is done in acetonitrile medium, but not in other solvents like MeOH, EtOH,
DMF or DMSO. The green coloration of the solution gradually decolorizes and the color of the
solution turns yellowish white. On keeping the reaction mixture for a few days white crystals
suitable for Xꢀray diffraction were obtained. The colorless crystals are found to be
ꢀ
[Cu(MeCN)4]+ ClO4 salt by Xꢀray analysis.37ꢀ38 The observation reveals that discoloration is due
to the reduction of Cu(II) ions to Cu(I) and the generated Cu(I) gets stabilized via softꢀsoft
interaction with N of CH3CN, where N site attains softness due to the presence of electron
donating methyl group (symbiotic effect). On the other hand, solvents like MeOH, EtOH, DMF
and DMSO have hard O as donor site and thereby fails to stabilize soft Cu(I) species.
Scheme 2. Proposed scheme for the Synthesis of Transformed Ligand TL in MeCN
Along with this reduction a concomitant oxidation should be natural consequence and it is most
probably the ligand moiety which should take part in the process. In order to characterize the
newly formed ligand it is essential to remove the complex as well as any unreacted metallic part
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which has been done according to the following manner. After complete precipitation of the
Cu(I)ꢀacetonitrile salt the remaining portion is treated with Na2S for complete separation of
copper to make the solution Copper free. After filtration the yellow oil was extracted with
saturated brine solution and chloroform for several times. The organic phase was separated, dried
with anhydrous MgSO4, concentrated by the evaporation of chloroform and subsequently
vacuumꢀdried for the removal of last trace of water. ESIꢀMS study of the reaction mixture after
30 min of addition of the reactants shows a base peak at m/z 91.10 amu that may be assigned to
the dealkylated fragment PhCH2ꢀ (calc. m/z= 91.126 amu), a peak at m/z 249.10 amu
corresponds to the TL species (calculated. m/z=249.283 amu), another peak at 327.42
corresponds to Cu(MeCN)4ClO4 (calc. m/z=327.202 amu) ,two small peaks at m/z 434.77 due to
unreacted ligand HL (calc.m/z=434.559 amu) and at m/z 262.49 to unreacted copper perchlorate
(calc. m/z=262.434 amu). However, other peaks cannot be identified accurately. Then we
1
perform H NMR study with the purified form in D6ꢀDMSO medium and a sharp peak at δ 8.1
confirms the formation of an imine bond. Thereby the newly formed ligand a Schiffꢀbase ligand
was proposed as depicted in Scheme 2. In addition the IR spectral study shows a newly
generated band at 1629 cmꢀ1, previously absent in the pure HL and characteristic for a C=N
bond. Our proposition has further been authenticated by ESIꢀMS study where a base peak
observed at m/z 249.12 corresponds to the composition of the TL species (calc. m/z=249.283
amu) along with a peak at m/z 91.51 amu as observed in earlier study.
We also monitored spectrophotometrically the whole reaction sequence. A 3 mL solution of
Cu(ClO4)2 in acetonitrile is taken in quartz cell and to this solution a stochiometric amount of the
ligand HL is added and the whole course of the reaction is monitored up to 2 hrs at the interval of
5 min. The characteristic dꢀd band for Cu(II) at 600nm gradually decreases and practically
disappeared after 2 hrs, and this observation supports the reduction of Cu(II) to Cu(I) during the
course of the reaction.
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Fig. 15 The decreasing band at 600 nm during the course of reaction as the Cu(II) is converted to
Cu(I).
To follow the transformation process we performed cyclic voltammetry study in MeCN solution.
The cyclic voltammogram shows irreversible reductions at 0.065, ꢀ0.40 and ꢀ0.80V (Fig. S29
and S30). In DPV experiments the reductive peaks are obtained at 0.18, ꢀ0.32, ꢀ0.49 and ꢀ0.61V.
However, all these peaks in either CV or DPV experiments undergo decay with time (Fig. 16),
which is consistent with the results obtained from other experiments that the complex undergoes
degradation in MeCN solution. The free ligand in MeCN solution shows reductive peaks at ꢀ0.81
and ꢀ1.74V in DPV experiments, the later peak have much stronger current height compared to
the peak at less cathodic potential. On addition of Cu(ClO4)2.6H2O to this solution in 2:1
metal:ligand ratio, the DPV undergoes dramatic change with a very strong peak appearing at ꢀ0.4
V which is characteristic of the complex and another doublet at ꢀ1.00V and ꢀ1.14V. The last two
peaks which are neither due to the complex nor due to the original ligand may be ascribed to the
reductions of the Schiff base ligand formed during degradation of the complex.
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Fig. 16 Time dependent CV and DPV spectrum of complex 1 at the GC electrode in Acetonitrile
medium at 100 mV s−1 scan rate as representative.
Experimental Section
Physical methods and materials. 3,5 diꢀtertꢀbutyl catechol and (4ꢀNitrophenyl)phosphate
were purchased from SigmaꢀAldrich. Reaction solutions for 3,5ꢀDTBC and 4ꢀNPP were
prepared according to the standard sterile techniques. DMSO was dried over CaH2 for 2 days and
then distilled under reduced pressure prior to use. All other 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 and experiments was MilliꢀQ grade.
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 FTIRꢀ8400S
1
and PerkinElmer Spectrum Express Version 1.03 using KBr pellets as mediums. H and 13C
NMR spectra (300MHz) were recorded in CDCl3 and D6DMSO 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 UVꢀ2450PC
spectrophotometer equipped with multiple cellꢀholders and thermostat. Magnetic susceptibility
measurements over the temperature range 2−300 K were performed at a magnetic field of 0.0750
T using a Quantum Design SQUID MPMSXLꢀ5 magnetometer. Correction for the sample
holder, as well as the diamagnetic correction, which was estimated from the Pascal constants39ꢀ40
was done. Electrospray mass spectra were recorded on a MICROMASS QꢀTOF mass
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spectrometer. Cyclic voltammetric and DPV measurements were performed by using a
CH1106A potentiostat with glassy carbon (GC) as working electrode, Ptꢀwire as counter
electrode and Ag, AgCl/sat KCl as reference electrode. All solutions were purged with dinitrogen
prior to measurements.
Synthesis of ligand HL. To an ethanolic solution (30 mL) of pꢀcresol (50 mmol, 5.407 g), 2ꢀ
benzyl amino ethanol (110 mmol, 16.6 g) was added dropwise with constant stirring. After 30
min, 37% (w/v) formalin solution (115 mmol, 9.3 mL) was added to it. The resulting mixture
was stirred for an additional 1 h at room temperature and then refluxed for 72 h. It was
evaporated under reduced pressure, and the yellow oil was extracted with saturated brine solution
and diethylꢀether for several times. The organic phase was separated, dried with anhydrous
MgSO4, concentrated by evaporation of ether and subsequently vacuumꢀdried for the removal of
last traces of water. The liquid ligand was further purified by flash column chromatography
(silica gel, hexane: ethylacetate: triethylamine =100: 5: 1) to afford the desired βꢀaminophenol.
Yield = 8.68 g (40%). Anal. Calcd for C27H34N2O3: C (74.64%); H (7.83%); N (6.45%); O
(11.05%); Found C (75.99%); H (7.75%); N (6.80%); O (11.18%);
1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.242(s, 3H; phꢀCH3), ~2.7(t, 4H; NꢀCH2ꢀCH2), ~3.7(t,
4H; N−CH2ꢀCH2), ~3.8(s, 4H; phꢀCH2ꢀN), 4.37(s,2H;CH2ꢀCH2ꢀOH), ~6.9 (s, 2H; Ar), ~(7.22ꢀ
7.35)(m, 10H; Ar); 13C NMR (300 MHz, CDCl3, 25 °C): δ = 20.52(1C, phꢀMe), 58.65(2C, Arꢀ
CH2−N), 51.90(2C, phꢀCH2−N), 63.32(2C, NꢀCH2−CH2), 86.40(2C, N−CH2ꢀCH2), 116.01(1C,
Ar), 127.50(2C, Ar), 129.39(2C, Ar),127.49ꢀ127.86(4CꢀAr), 155.18(1C, Ar−OH); IR data (NaCl
plate): ν bar=1602,1361,1498,1256,824,734 cmꢀ1; UV/vis (DMSO): λmax(ε) =282.43(6840 L
mol−1 cm−1).
Synthesis of the Complex [Cu2(L)(µꢀOH)(H2O)(ClO4)2] (1). To a methanolic solution (25 mL)
of HL (0.5 mmol, 0.217 g), a methanolic solution (10 mL) of copper perchlorate (0.5 mmol,
0.18 g) was added and the resulting mixture was stirred for 15 min. The solution thus obtained
was then filtered and the filtrate was kept in a CaCl2 dessicator. After 1 day solid green product
were obtained. The product was then dissolved in ethanol and after 1 week green needle shaped
single crystals suitable for Xꢀray analysis were obtained. (Yield 70%). Anal. Calcd for
C27H34Cu2N2O7Cl2 : C (46.55%); H (4.8%); N (4.02%); Found: C (46.94%); H (4.89%); N
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(4.1%); FTꢀIR data (KBr pellet): I.R: ν(CꢀN) 1576 cmꢀ1; ν(skeletal vibration) 1496 cmꢀ1; ν(ClO4)
1078 cmꢀ1; ν(ClO4 coordinated) 674 cmꢀ1. UV/vis (DMSO): λmax(ε) = 279(3560), 300(3520),
704nm (120 L mol−1 cm−1).
Xꢀray Data Collection and Structure Determination. Data collection of the structure reported
was carried out on a Bruker Smart CCD diffractometer equipped with graphiteꢀmonochromated
MoꢀKα radiation (λ=0.71073 Å) at room temperature. Cell refinement, indexing and scaling of
the data set were carried out using Bruker Smart Apex and Bruker Saint packages.41 The structure
was solved by direct methods and subsequent Fourier analyses 42 and refined by the fullꢀmatrix
leastꢀsquares method based on F2 with all observed reflections.42 A perchlorate anion was found
disordered by rotation about the Cl2ꢀO10 bond (oxygen atoms O11/O12/O13 refined at two
positions with occupancies 0.48(4)/0.52(4)). Two residuals were interpreted as a disordered
lattice water molecule occupancy refined at 0.35(5)/0.65(5). All the calculations were performed
using the WinGX System, Ver 1.80.05.43 Pertinent crystallographic data and refinement details
are summarized in Table 4.
CCDCꢀ1053269 contains the supplementary crystallographic data for this paper.
Table 4. Crystallographic data and refinement details of complex 1.
Empirical
formula
C27H38Cl2Cu2N2O14 F(000)
1672
Formula weight
Crystal system
Space group
a (Å)
812.57
θmax (°)
23.23°
4388
monoclinic
P 21/n
Reflns collected
Unique reflections 3842
16.6577(14)
14.6519(14
16.6876(14)
118.176(2)°
3590.3(6)
4
Rint
0.0531
b (Å)
Observed I> 2σ(I) 2944
c (Å)
Parameters
Goodness of fit(F2) 1.2020
452
β (°)
Volume (Å3)
R1 (I> 2σ(I))
0.0834
a
Z
wR2
0.2285
Dcalcd (g/cm3)
1.503
ꢂρ (e/Å3)b
ꢀ0.574, 0.636
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Conclusion
A diꢀcopper(II) complex of a Mannich base ligand HL has been synthesized and structurally
characterized with the aim to explore its bioꢀrelevant catalytic promiscuity by exploiting the
solubility of the complex in water. Xꢀray structural analysis reveals that the complex resembles
the active site of the catechol oxidase to a great extent having close comparable CuꢀCu
separation of 2.9 Å as in the enzyme. The present complex shows interesting properties not only
as catechol oxidase mimic in oxidation of 3,5ꢀDTBC and pH dependent phosphataseꢀlike
activity, but it also reveals antiferromanetically coupled copper centers at low temperature. To
the best of our knowledge the present study represents the most suitable bioꢀrelevant catalyst
behavior reported till date in DMSO/water solvent mixture. In addition a surprising feature was
observed when the complex is dissolved (or synthesized) in acetonitrile. In fact it was observed
that the reduction of Cu(II) to Cu(I) (monitored by electrochemical analysis) is accompanied
with dealkylation followed by concomitant oxidation of the ligand (HL) that resulted a Schiffꢀ
base species, 2,6ꢀbisꢀ[(2ꢀhydroxyꢀethylimino)ꢀmethyl]ꢀ4ꢀmethylphenol.
Acknowledgement.
The authors wish to thank University of Calcutta for providing the facility of the singleꢀcrystal
Xꢀray diffractometer from the DST FIST program. I.M is thankful to UGC [UGC/729/Jr
Fellow(Sc)] for providing fellowship.
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