Catalytic promiscuity of a copper(II)-Mannich base complex having unprecedented radical pathway in catecholase activity

Article abstract of DOI:10.1016/j.ica.2020.119480

A flexible single-side Mannich base ligand namely 4-(tert-butyl)-2-((ethyl(2-hydroxyethyl)amino)methyl)phenol (H₂L) having O, N, O donor sites has been designed and synthesized using 4-(tert-butyl)phenol, formaldehyde and 2-(ethylamino)ethanol

Full text of DOI:10.1016/j.ica.2020.119480

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Research paper
Catalytic promiscuity of a copper(II)-Mannich base complex having unprece-
dented radical pathway in catecholase activity
Sanchari Dasgupta, Arnab Mandal, Debabrata Samanta, Ennio Zangrando,
Suvendu Maity, Debasis Das
PII:
S0020-1693(19)32025-0
https://doi.org/10.1016/j.ica.2020.119480
ICA 119480
DOI:
Reference:
Inorganica Chimica Acta
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25 December 2019
27 January 2020
27 January 2020
Please cite this article as: S. Dasgupta, A. Mandal, D. Samanta, E. Zangrando, S. Maity, D. Das, Catalytic promiscuity
of a copper(II)-Mannich base complex having unprecedented radical pathway in catecholase activity, Inorganica
Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119480
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Catalytic promiscuity of a copper(II)-Mannich base complex having
unprecedented radical pathway in catecholase activity
Sanchari Dasgupta,a,* Arnab Mandal,a Debabrata Samanta,b Ennio Zangrando,c
Suvendu Maity,d and Debasis Das a,*
a Department of Chemistry, University of Calcutta,92, A. P. C. Road, Kolkata 700009, India
b Department of Chemistry, Indian Institute of Technology, Kanpur, Kanpur-208016, India
c Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri
1, 34127 Trieste, Italy
d Department of Chemistry, R. K. Mission Residential College, Narendrapur, Kolkata 103, India
ABSTRACT
A
flexible single-side Mannich base ligand namely 4-(tert-butyl)-2-((ethyl(2-
hydroxyethyl)amino)methyl)phenol (H2L) having O, N, O donor sites has been designed and
synthesized using 4-(tert-butyl)phenol, formaldehyde and 2-(ethylamino)ethanol as precursors
with the view to prepare polynuclear complex(s). In the first attempt a tetranuclear copper (II)
.
complex, [Cu4(L)2(HL)2].(ClO4)2 H2O (1) has been synthesized employing H2L and copper(II)
perchlorate with the aim to explore catalytic promiscuity of the species. Detailed experimental
investigations suggest that in solution complex 1 exits as a dinuclear entity. The dinuclear
species is a square-planar CuII complex as is evident from EPR and UV-Vis spectral studies. It
exhibits excellent catalytic activities in catalyzing the oxidation of 3,5-di-tert-butyl catechol (3,5-
DTBC) to 3,5-di-tert-butyl quinone (3,5-DTBQ); oxidation of o-aminophenol to phenoxazinone
and P-O bond hydrolysis of 4-nitrophenyl phosphate. Amazingly, complex 1 catalyses the
aerobic oxidation of 3,5-DTBC not by well established metal centered redox participation, rather
via a ligand centered radical pathway, a completely new finding in copper(II) based catechol
oxidase modeling as are verified by experiments and seconded by DFT calculations.
Keywords: Copper(II)-Mannich Base complex, Catalytic promiscuity, Catecholase activity,
Phenoxazinone Synthase activity, Phosphatase like activity, DFT study
1

1. Introduction
‘Catalytic Promiscuity’, a new popular terminology is now widely used by bioinorganic
chemists. The term ‘Catalytic Promiscuity’ of an enzyme is defined as its ability to catalyze
multiple catalytic transformations which belong to different classes [1]. Another definition of
‘Catalytic Promiscuity’ is the capability of a single active site to catalyze more than one
chemical transformation as proposed by Romas J Kazlauskas [2]. Chymotrypsin (that catalyzes
hydrolysis of amides, esters, thiol esters, acid chlorides and anhydrides) [3], bovine carbonic
anhydrase II(catalyzes phosphotriesterase activity, carbon esterase activity and CO2 hydrates
activity)[4], carbonic anhydrase III (catalyzes hydrolysis of phosphomonoester monocation,
carbon ester and hydration of carbon dioxide)[5] are familiar enzymes showing catalytic
promiscuity.
In recent times activation of dioxygen by dinuclear transition metal complexes has been studied
comprehensively and preparation of bio-mimetic models of oxidases, oxygenases,
dehydrogenases and other metalloenzymes draws the immense attention of the bioinorganic
chemists.[6] Multicopper oxidase belongs to an important class of oxidase enzyme which
contains seven kinds of active sites depending on the structural and functional properties of
copper centre namely, Type-1, Type-2, Type-3, Type-4, CuA, CuB and CuZ active sites. Catechol
oxidase(CO) and phenoxazinone synthase (PHS) belongs to oxidases group of enzyme [7].
CO, a Type-3 copper protein, contains antiferromagnetically coupled dinuclear copper(II) centers
at their active site and catalyzes oxidation of catechol derivative to corresponding quinone [8-
14]. This oxidation is worthy for medical diagnosis of the hormonally active catecholamine,
adrenaline, noradrenaline and dopa [15,16]. Till date several bio mimicking models of CO have
been reported to have an insight about their mechanistic pathways. Generally two kinds of
pathways are operative, (i) metal centered redox participation [17-24] and (ii) ligand centered
radical pathway [25-32]. The ligand centered radicals till now demonstrated are of three types: (i)
imine radical, [25-30] found in Schiff base based models, (ii) arene/benzene anion radical
observed in reduced Schiff base complexes [31] and (iii) phenoxy radical for Mannich base
complexes[32].
PHS contains five different copper centers at active site namely type-1, type-2 and type-3 [33].
PHS which is very important in pharmaceutical industries being involved in the last step of
biosynthesis of antibiotic actinomycin D by Streptomyces antibioticus[34]. The mechanistic
2

pathway of phenoxazinone synthase, involves the formation of organic radical with concomitant
reduction of either metal or ligand centre [30, 35,36].
On the other hand designing of metal complexes as bio-mimicking models of phosphatase, a
class of hydrolysis enzymes became an imperative field to bio-inorganic chemists. These
enzymes catalyse the hydrolysis of P-O bond of phosphate monoesters, di-esters, tri-esters,
fluorophosphates, fluorophosphonates, phosphoric anhydrides etc. and the hydrolysis reaction
plays a significant role in life processes, medical field and agriculture[37-42]. It is well known
that bi-valent metal cations play a vital role towards the nucleophilic substitution of naturally
occurring phosphate ester bond in most of the cases. It is already well documented in literature
that the rate of hydrolysis of phosphate ester bond increases several times in presence of
mononuclear model complexes in compare to the uncatalysed reaction and that rate enhance
appreciably when dinuclear catalysts are used. Most of the reported models contain zinc(II) as
metal centre. However, Sigel et al. and Trogler et al. reported that copper(II) metal ion can also
efficiently catalyse the hydrolysis of P-O bond of triphosphates [43-44]. It is already established
that Cu2+ and Ni2+ metal based models exhibit the highest catalytic activity among the first row
transitional metals[44]. Thus in past few decades Cu2+ has been used as a good alternative to
Zn2+ in model complexes[45-47].
In the present work we report a tridentate Mannich base ligand with O, N, O donor set, which is
designed and synthesized for the flexibility of the CH2-NR moiety (Scheme 1), that may help to
produce complexes of different nuclearity [48-49]. In the first attempt, a tetranuclear complex,
.
[Cu4(L)2(HL)2].(ClO4)2 H2O (1) has been synthesized (Scheme 1) purposefully to explore its
catalytic promisicuty in terms of catecholase activity, phenoxazinone synthase activity and
phosphatase activity. Complex 1 acts as the catalyst precursor to generate dinuclear active
catalyst in solution. Complex 1 appears not only as a highly efficient oxidizing catalyst but also
as a potential hydrolytic agent. It catalyses the aerobic oxidation of the model substrate 3,5-
DTBC to 3,5-DTBQ in excellent rate. The reaction proceeds not via metal centered redox
participation but through ligand centered radical pathway. It is a completely new finding in
catecholase activity study in dinuclear copper(II) system as is established by combined
experimental and theoretical investigations. The mechanistic pathways of phenoxazinone
synthase activity and phosphatase activity of complex 1 have also been extensively studied. All
those findings have been vividly portrayed in this communication.
3

Scheme 1. Synthetic Route of Complex 1.
2. Experimental section.
2.1. Physical methods and materials
Elemental analyses (carbon, hydrogen, and nitrogen) were carried out using a Perkin–Elmer
240C elemental analyzer. Fourier Transform Infrared spectra were recorded on KBr disks and
NaCl disks (400-4000 cm-1) and a Perkin-Elmer RXI FTIR spectrophotometer. Electronic spectra
and kinetic studies (200-900 nm) were measured at room temperature with a Shimadzu UV
3101PC instrument and dry DMF as solvents. 1H NMR spectra (300 MHz) were recorded in d6-
DMSO as solvents and TMSCl as standard at 25 °C with a Bruker AV300 Supercon NMR
spectrometer and use of the solvent signal as the internal standard in a 5 mm BBO probe.
Electrospray mass spectra were recorded on a WATERS Xevo G2-S Q Tof mass spectrometer
using HRMS-grade acetonitrile as solvent. The cyclic voltammograms were obtained with a Basi
C-3 cell voltameter under nitrogen with a glassy carbon electrode as working electrode, Ag/AgCl
as reference electrode and tetrabutylammonium perchlorate as supporting electrolyte.
High-purity 4-(tert-butyl)phenol, 2-(ethylamino)ethanol and 37% (w/v) formalin solution,
copper(II) perchlorate hexahydrate were purchased from commercial sources (Alfa Aesar,
Sigma-Aldrich, Merck) and used as received. Solvents were dried by standard procedures and
distilled prior to use.
Caution! Perchlorate salts are explosive. They should be handled with great care and in small
amounts.
4

2.2. Synthesis of ligand (H2L)
To an ethanolic solution of 4-(tert-butyl)phenol (25 mmol, 3.75 g), ethanolic solution of 2-
(ethylamino)ethanol (25 mmol, 2.22 g) is added. Then 37% (w/v) formalin solution (25 mmol,
2.1 mL) has been added and the resulting yellow colored solution is refluxed for 10 hours. The
reaction mixture is evaporated under reduced pressure. The oil-like residue is extracted with
brine solution and dichloromethane for three times. The organic layer has been collected and
dried with anhydrous MgSO4. The ligand has been collected after complete evaporation of
dichloromethane.Yield: 90%; FT-IR (NaCl): ν (C˭N) 1583 cm-1; ν (skeletal vibration) 1472 cm-1
1
(Fig. S1). H NMR (300 MHz, d6-DMSO, 25 °C) : δ 1.124 (s, 9H, C(CH3)3); δ 3.630 (s, 2H, Ar-
CH2-N); δ 2.463 (q, 2H, N-CH2-CH3 and 2H, N-CH2-CH2-OH ); δ 3.449 (q, 2H, N-CH2-CH2-
OH); δ 0.899 (t, 3H, N-CH2-CH3); δ 3.630 (q, 1H, N-CH2-CH2-OH); δ 7.078 (d,1H,Ar) ; δ 6.973
(d,1H,Ar); δ 6.598 (d,1H,Ar) (Fig. S2). 13C NMR (75 MHz, d6-DMSO, 25 °C): δ 31.85
(3C,C(CH3)3); δ 34.01(1C, C(CH3)3); δ 155.47, 141.29, 126.32, 124.99, 122.48, 115.25 (6C,Ar) ;
δ 54.95 (1C,Ar-CH2-N); δ 47.20 (1C,N-CH2-CH3); δ 11.26 (1C,N-CH2-CH3); δ 58.79 (1C,N-
CH2-CH2-OH); δ 56.96 (1C,N-CH2-CH2-OH) (Fig. S3). ESI-MS (ES+, Acetonitrile) calcd. m/z
value for C15H25NO2 [M+H+] : 252.1964 amu, found 252.1986 amu (Fig. S4).
.
2.3. Synthesis of [Cu4(L)2(HL)2].(ClO4)2 H2O (1)
To a methanolic solution of H2L (0.251 g, 1 mmol) copper(II) perchlorate hexahydrate (0.365, 1
mmol) is added and resulting green colored solution is stirred very slowly for 15 min. The
solution is filtered and kept in desiccators. Green colored needle shaped crystals suitable for X-
Ray diffraction is obtained after three days. Yield: 85%, Elemental analysis calcd. (%) for
C60H96N4O17Cl2Cu4: C, 49.01; H, 6.58, N 3.81; found: C 49.05, H 6.62, N 3.82; FT-IR (KBr):
ν(C-N) 1606 cm-1; ν(skeletal vibration) 1495 cm-1; ν(perchlorate) 1090 cm-1; ν(tertiary amine)
2960 cm-1 (Fig. S5); UV-Vis (DMF): λmax(ε) = 421 nm(716 L mol-1 cm-1), 710 nm (170 L mol-1
+
cm-1 ) (Fig. S6). ESI-MS (ES+, Acetonitrile) calcd. m/z for C30H47Cu2N2O4 : 625. 2128 amu,
found 625. 2158 amu (Fig. S7).
2.4. X-ray crystallography
Intensities data for crystal structure analysis of complex 1 are collected at room temperature with
Mo-Kα radiation (λ = 0.71073 Å) on a Bruker Smart Apex diffractometer equipped with CCD.
5

Cell refinement, indexing, and scaling of the data set were performed using the Bruker Smart
Apex, and Bruker Saint packages [51]. The structure is solved by direct methods and subsequent
Fourier analyses [52] and refined by the full-matrix least-squares method based on F2 with all
observed reflections[52]. Hydrogen atoms are placed at calculated positions and included in final
cycles of refinement. A perchlorate anion was found disordered over two positions of refined
occupancies 0.574/0.426(16). All calculations are performed using the WinGX System,
Ver2013.3[53]. Crystal data and details of refinements are given in Table S1.
2.5. Catecholase activity and kinetics study of complex 1
The catecholase activity of complex 1 is performed using 3,5-DTBC as model substrate because
the presence of two bulky tert-butyl groups lowers the catechol-quinone redox potential and it
also inhibits polymerization of oxidized product. The catalytic reaction is monitored by adding
110-4 (M) complex solution to 110-2 (M) 3,5-DTBC solution under aerobic condition at 25 °C
in N,N-dimethylformamide(DMF) medium. The progress of the reaction is monitored by time
dependent wavelength scan for 2 hours at 10 min interval of time.
The kinetic study of the oxidation of 3,5-DTBC by complex 1 have been performed by initial
rate method at 25 °C. Solutions of concentration varying from 0.001 to 0.05 (M) have been
prepared from a freshly prepared concentrated substrate solution. The dependence of the initial
rate on the concentration of the substrate is monitored by UV-Vis spectroscopy at the respective
wavelength by maintaining the fixed catalyst concentration [110-4(M)] for each set. The
complexes show saturation kinetics, and a treatment based on the Michaelis-Menten found to be
the most appropriate. The binding constant (KM), maximum velocity (Vmax), and rate constant for
dissociation of the substrates (i.e. turn over number, kcat) are calculated for the complexes using
1
the Lineweaver-Burk graph of V versus [1], with the equation (2),
S
1
KM
(Vmax)
1
1
Vmax
(2)
=
×
+
[ ]
S
V
2.6.Detection of hydrogen peroxide in the catalytic reaction
In order to detect the formation of hydrogen peroxide during the catalytic cycle reaction mixtures
have been prepared as in kinetic experiments. After 1 hour of reaction equal volume of water is
added and 3,5-DTBQ formed is extracted with dichloromethane. pH of the aqueous layer is fixed
6

at 2 with H2SO4. 1 mL of a 10% solution of KI and three drops of 3% solution of ammonium
molybdate are added. In the presence of hydrogen peroxide, the reaction, H2O2 + 2I- + 2H+→
2H2O + I2 occurs, and with an excess of iodide ions, the triiodide ion is formed according to the
-
reaction I2(aq) + I- = I3 .The reaction rate is slow but increases with increasing concentrations of
acid, and the addition of an ammonium molybdate solution renders the reaction almost
-
instantaneous. The formation of I3 can be monitored spectrophotometrically due to the
-
development of the characteristic I3 band (λmax= 353 nm, ε = 26000 L mol-1 cm-1).
2.7. Computational methods
To further understand a possible mechanistic pathway for catalytic catechol oxidation by
dinuclear copper complex (1b), density functional theoretical (DFT) computations are performed
using Gaussian09 program package [54]. The Minnesota functional, M06-2X, in conjugation
with 6-31G (d) basis set, is employed for ground state geometry optimizations [55]. M06-2X
functional is found to estimate energy of metal complex catalyzed catechol oxidations,
satisfactorily [31]. For triplet state optimizations, unrestricted broken spin symmetry approach is
utilized to break the spin and spatial symmetries by mixing frontier molecular orbitals. Grimme’s
d3 dispersion is incorporated to consider dispersive interactions in all computations[56]. The
solvent effect (DMF) is also introduced in computations by polarizable continuum model.
Calculated minima have been confirmed by the absence of imaginary frequency (NIMAG=0) in
harmonic vibrational frequency analysis. Gibbs free energies including thermally corrected
unscaled zero point vibrational energies are reported in kcal mol−1 relative to the corresponding
starting materials the dinuclear copper complex (1b). In computations, perchlorate/perchloric
acid was used for proton acceptor/donor.
2.8. Phenoxazinone synthase like activity and kinetics study of complex 1
The phenoxazinone synthase like activity of complex 1 has been monitored by the reaction of
110-4 (M) solution of the complex with 110-2 (M) solution of o-aminophenol (OAP) in aerobic
condition in DMF at 25°C. The course of the reaction is followed by time dependent wavelength
scan. An absorption band at λmax ~ 433 nm (ε = 7400 M-1 cm-1) signature of phenoxazinone
(APX) chromophore is monitored with time. The progress of the reaction is observed by time
dependent wavelength scan for 30 min at 5 min interval of time.
7

The kinetic study of phenoxazinone synthase activity has been performed by adopting the same
procedure as followed in catecholase activity.
2.9. Phosphatase like activity and kinetics study of complex 1
The phosphatase like activity of complex 1 is studied by monitoring hydrolysis of phosphate-
ester bond of model substrate Disodium (4-nitrophenyl)phosphate (4-NPP) hexahydrate. The
hydrolytic property of complex 1 is studied by monitoring the hydrolysis of a solution in
97.5%(v/v) DMF-H2O containing 1×10-3 (M) 4-NPP and 5×10-5 (M) complex for 2 h with the
help of UV-Vis spectroscopy at 25 °C. The course of the reaction has been observed by
increment of a peak at λmax ~ 425 nm characteristics of 4-nitrophenolate anion which is
hydrolyzed product of 4-NPP.
The Kinetics study is performed in seven sets having a catalyst of 5×10-5 (M) concentration and
substrate concentrations of 0.5 (10 equiv), 0.7 (14 equiv), 1.0 (20 equiv), 1.2 (24 equiv), 1.5
mmol (30 equiv), 1.7 mmol (34 equiv) and 2.0 mmol (40 equiv). The reactions were initiated by
addition of 0.06 mL of metal complex (2.5×10-3 M) into 2.94 mL of a 4-NPP solution, and the
spectrum is recorded only after complete mixing at 25 °C. The rates of reaction for various
substrate concentrations are interpreted with the help of Michaelis-Menten approach and the
kinetic parameters are calculated from Lineweaver-Burk plot.
3. Results and discussions.
3.1. Synthesis of ligand and complex 1
A single-side Mannich product 4-(tert-butyl)-2-((ethyl(2-hydroxyethyl)amino)methyl)phenol
(H2L) having O, N, O donor sites has been purposefully synthesized using 4-(tert-butyl)phenol
(an activated phenol), 2-(ethylamino)ethanol (secondary amine), 37% (w/v) formalin solution as
precursors. The method is similar as reported earlier.50 H2L has been characterized by FT-IR,
1
13
UV-Vis, H and C NMR, and ESI-MS spectral studies (data are in experimental section, Fig.
S1-S4). A tetranuclear copper(II) complex has been prepared by treating methanolic solution of
copper(II) perchlorate hexahydrate with the methanolic solution of H2L.
3.2. FT-IR and UV-Vis spectra of complex 1
8

The FT-IR spectrum of complex 1 contains a sharp band at 1606 cm-1 due to C-N stretching. The
bands at 1495 cm-1 and 2960 cm-1 are observed due to skeletal vibration and tertiary amine
respectively. The broad band at 1090 cm-1 is the signature of perchlorate ion (Fig. S5)[57]. The
UV-Vis spectral study has been performed in DMF medium. The bands at λmax = 421 nm (ɛ =
716 L mol-1 cm-1) is observed due to ligand to metal charge transfer but its intensity is relatively
lower than Schiff base analogue since tertiary amine is present in this case. The band at λmax
=
710 nm (ɛ = 170 L mol-1 cm-1) is observed due to d-d transition (Fig. S6)[58]. CuII is a d9 system
2
2
and if it is in an octahedral coordination environment the electronic transition Eg → T2g is
expected to take place at around 800 nm and this band will undergo a significant blue shift on
distortion of octahedral coordination to a square-pyramidal and square-planar geometry. Thus,
coordination environment around copper centres is square-pyramidal or square-planar rather than
octahedral.
3.3. Structural description
The X-ray crystal structural analysis of complex 1 reveals that asymmetric unit contains one
tetranuclear copper(II) di-cationic complex having a pseudo center of symmetry (Fig. 1a). The
cationic unit is counterbalanced by two perchlorate anions and a lattice water molecule is
present.
The complex shows a zig-zag chain of four copper atoms where Cu1/Cu2 and Cu3/Cu4 metal
pairs are doubly bridged by an alkoxy and phenoxide oxygen atoms, while the central Cu2/Cu3
are doubly bridged by the participation of two phenoxide oxygen atoms to form three irregular
Cu2O2 rhomboids (Fig. 1b). Of the four copper(II) atoms, Cu(1) and Cu(4) exhibit a square
planar geometry, while Cu(2) and Cu(3) adopt a distorted square pyramidal geometry (Fig. 1c) as
indicated by their trigonal index τ values[59] [τ for Cu(2) =0.29, Cu(3) = 0.22].
The coordination sphere of square planar copper centers is built by phenoxy oxygen, amine
nitrogen, and the alcoholic oxygen atom of ligand HL, and the fourth site is occupied by
deprotonated alkoxy oxygen atom of second L. On the other hand the square pyramidal positions
in the base are occupied by the phenoxy oxygen, amine nitrogen, and the deprotonated alkoxy
oxygen atom of L, and the alcoholic oxygen of nearby HL. A phenoxy oxygen is present at the
apex.
9

The Cu-O bond distances in square planar and in square pyramidal base of all copper centers are
close comparable as reported in Table S2, falling in a range 1.877(11)-1.996(10) Å. The Cu-N
distances vary from 1.984(13) to 2.020(15) Å. The range of cisoid angles 77.9(4) - 101.2(4)° and
those of transoid angles157.6(5) -176.1(5)° give indications of the distortion from ideal values.
The axial Cu-phenoxide bond lengths are significantly longer, of 2.271(11)and 2.245(11)Å, as
expected for Jahn-Teller effect. Finally the distance between pair of copper centers in the
tetranuclear chain is 3.037(3) and 3.001(3), with an intermetallic angle averaging to 119°. All the
coordination distances, although at lower accuracy, are comparable with those of analogous
derivatives reported by others [22,60].
(a)
(b)
(c)
Fig. 1. (a) ORTEP view (30% probability) of complex 1 cation, H-atoms have been omitted (for
clarity); (b) Zig-Zag chain of copper centers and (c) coordination geometry around two different
CuII atoms
10

3.4. ESI-MS study complex 1
To get an idea of solution phase structure of complex 1 ESI-MS study is performed in positive
mode using acetonitrile as solvent. The base peak observed at m/z = 625.2158 amu (Fig. S7)
corroborates well with monocationic dinuclear species having molecular formula
+
C30H47Cu2N2O4 (calcd. m/z = 625.2128 amu). The dinuclear species (Fig. 2) is regarded as
active species during all the catalytic activities.
1b
Fig. 2. Probable composition of dinuclear species in solution as evidenced from ESI-MS study.
3.5. Elecrochemical study of complex 1
The cyclic voltammetry of complex 1 is carried out in DMF medium at a scan rate of 100
mV/sec. The CuII/CuI redox couple appeared at ~0.0 V (0.0078 volt) with respect to Ag/AgCl
reference electrode and the cathodic peak at -0.48 V was attributed to CuI/Cu0 redox couple. The
restricted scan from -0.6 volt to 0.6 volt resulted in a quasi-reversible redox couple as the
observed Epc = -0.422 volt with a ΔEp value of 0.502 volt and ipc / ipa = 2.15 (Fig. 3).
(a)
(b)
11

Fig. 3. Cyclic voltammogram of complex 1 in DMF medium during (a) cathodic scan and (b)
anodic scan
3.6. EPR study
X-band EPR spectrum of complex 1 is recorded in DMF medium at room temperature. The
spectrum consists of hyperfine lines which are due to 63Cu and 65Cu in the highfield (3/2)
transition as is depicted in Fig. 4. It exhibited signals at g┴ = 2.07 and the values of g║ are 2.12,
2.17, 2.21. The greater g║ value as compare to g┴ suggests that the unpaired electron is present at
2
2
dx -y orbital. The spectral pattern reveals that metal center in solution phase adopts square planar
geometry as is presented in Fig. 2 on the basis of ESI-MS study [61,62].
Fig. 4. X-band EPR spectrum of complex 1 at 298 K in DMF medium.
3.7. Catecholase activity
From the above discussion it is clear that complex 1 is the catalyst precursor and the dinuclear
obtained on dissolution of 1, most probably a square planar dinuclear CuII complex is the active
catalyst. The catalytic activity of 1 towards the oxidation of 3,5-DTBC, a model substrate
generally used to investigate the catecholase activity has been monitored by time dependent UV-
Vis spectral scan in DMF medium. The change in spectral pattern of complex 1 in presence of
3,5-DTBC has been depicted in Figure 5. The peak at λmax ~ 400 nm characteristics of 3,5-DTBQ
(ε = 1900 L mol-1 cm-1) increases with time which proves that complex 1 is a potential catalyst
12

for the oxidation of 3,5-DTBC. It is generally demonstrated by nearly all the investigators
working on modeling of catechol oxidase that during catalysis by dinuclear copper(II) complexes
the substrate catechol interact with the copper center(s). Then two electron reduction of the two
copper centers occurred to generate copper(I) species with concomitant oxidation of catechol to
quinone [63]. The copper(II) species is re-generated by reaction of oxygen with copper(I)
species. Now two situations may be visualized in UV-Vis spectral study: (i) disappearance of d-d
band indicating the formation of CuI with fast electron transfer and the re-oxidation by oxygen of
the formed copper(I) complex which is often the rate-determining step; (ii) the d-d band remains
intact or a blue or red shifting, indicating intramolecular electron transfer should be the rate-
determining step[64]. In case of complex 1 d-d band undergoes red shift to λmax ~ 752 nm upon
addition of 3,5-DTBC. Therefore it may be presumed that intramolecular electron transfer is
likely to be the rate-determining step. A striking feature has been noticed along with the above
spectral changes is the gradual increase of a new band at λmax ~ 565 nm. The previous reports
suggest that the increment of peak at λmax ~ 565 nm is the signature of phenoxyl radical
formation [32, 65-67]. Therefore, this particular band makes us curious to find out the origin of
the catalysis: is there any possibility of radical pathway in copper based model system in
catechol oxidation?
(a)
(b)
Fig. 5. (a) Oxidation of 3, 5-DTBC by species 1b (substrate: catalyst = 100:1) observed in UV-
Vis spectroscopy with time in DMF medium at 25 οC, (b) change in d-d band of species 1b with
oxidation of 3, 5-DTBC.
13

Before to address this intriguing issue, it is essential to know the fate of dioxygen in the catalytic
cycle. During the re-generation of catalyst dioxygen may be converted to either water (4
electrons are involved) or hydrogen peroxide (2 electrons are involved). Spectroscopic study in
-
presence of the catalyst, KI and catechol (3,5-DTBC) detects I3 , which is a clear indication of
-
H2O2 formation. The band at λmax = 356 nm is observed due to generation of I3 and the plot of
absorption maxima at 356 nm at different time interval is presented in Fig. S8. Therefore a
radical pathway is most probably involved in this catalysis.
A controlled experiment has been carried out without using catalyst i.e. species 1b and the
absorbance of the characteristics band of 3,5-DTBQ around 400 nm is not observed to increase
with time (Fig. S9).
3.8. ESI-MS study
In order to identify the intermediates formed during the course of catecholase activity ESI-MS
study was carried out in positive mode. A small amount of reaction mixture was taken and
diluted with acetonitrile to record the mass spectrum. The base peak at m/z = 866.7215 amu
corresponds to 1:1 adduct of dinuclear species and 3,5- DTBC which is consistent with
+
molecular formula C44H69Cu2N2O6 + H2O (Calcd. m/z = 867.1357 amu). The peak at m/z =
624.0012 amu corresponds to dinuclear entity of complex 1. The peak at m/z = 243.1362 amu
represents the sodium adduct of 3,5-DTBQ (Calcd. m/z = 243.1361 amu) (Fig. S10).
3.9. Cyclic voltammetry Study
Cyclic voltammetry study was performed to monitor the redox reaction occurring during the
oxidation of catechol catalyzed by complex 1. Extensive analysis of cyclic voltammogram of
species 1b in presence of 3,5-DTBC showed that a new cathodic peak was generated at -0.06 V
and a new anodic peak at 0.5 V. Cathodic peak at -0.06 V can be tentatively assigned to the
reduction of free 3,5-DTBQ to copper(II) bound deprotonated 3,5-DTBC and the anodic peak at
0.5 V is due to oxidation of 3,5-DTBC to free 3,5-DTBQ (Fig. 6) [18].
14

Fig. 6. Cyclic voltammogram of reaction mixture of complex 1 and 3,5-DTBC in DMF medium.
3.10. EPR study
Finally to confirm the formation of organic radical generated during catalytic cycle X-band EPR
study has been carried out at 298 K. When 3,5-DTBC is added to the solution of complex 1 a
new sharp peak is observed to originate at g = 2.05 as a signature of organic radical formation
with the retention of the peaks observed for complex 1 only (Fig. 7). This observation becomes a
guiding factor to establish a new finding in catecholase activity study catalyzed by dicopper(II)
model system i.e. feasibility of radical pathway. Now as far as the nature of the radical is
concerned there is two possibilities, either arene anion radical or phenoxyl radical [31,32]. On
the basis of the EPR spectral pattern and the electronic spectral band at ~565 nm (vide supra) it
may be stated that the radical is phenoxy radical and that very proposition has been authenticated
by DFT calculations (vide infra).
15

Fig. 7. X-band EPR spectrum of reaction mixture of complex 1 and 3,5-DTBC in DMF medium
at 298 K.
3.11. Mechanistic pathway of catecholase activity and DFT study
In case of catecholase activity, metal centre redox participation likely to be occurred when redox
active metal centre(s) such as copper(II), manganese(III) is present in the active site of bio-
mimicking models as observed from earlier reports. Based on the experimental findings such as;
i) the UV-Vis band corresponding to the d-d transition of CuII (d9) at 752 nm remained intact,
indicating that CuII (d9) → CuI (d10) reduction do not occur during the course of catalytic cycle;
ii) observation of phenoxy radical in the UV-Vis (565 nm) and EPR spectral studies during
catalysis and iii) production of hydrogen peroxide in the reaction. Hence it is evident that
phenoxyl radical is formed during oxidation of 3,5-DTBC and thus ruling out the probability of
redox participation. These unusual experimental findings intrigued us to perform DFT
calculations. We have envisioned the catalytic cycle, as represented in Scheme 2, based on the
foregoing experimental observations for our DFT calculations.
16

1+
OH
OH
H
O
-
N
O
Cu Cu
O
ClO4
+
O
N
Proton
transfer
0
OH
HO
O
H
O
O
O
N
HClO4
1+
1b
Cu Cu
O
OH
O
O
N
H
O
O
N
Cu Cu
O
OH
O
O
A
S → T
Intersystem
crossing
N
O
O
OH
H2O2
D
E O
+HClO4
-ClO4
0
-
O
O
OH
0
SET
O
H
O
OH
O
N
O
H
O
Cu Cu
O
O
N
O2
O
N
Cu Cu
O
N
O
B
C
Scheme 2. Plausible catalytic cycle of catecholase activity catalyzed by active species 1b.
1+
OH
OH
O
O
E
+catechol
+ClO4
-
2.45 Å
-HClO4
0
1+
1a
2.21 Å
1b
0.0 kcal/mol
A
0
D
1.58 Å
+4.6 kcal/mol
0
+1.9 kcal/mol
SET
S → T
+HClO4
-
-ClO4
O2
triplet
C
B
-11.7 kcal/mol
+17.7 kcal/mol
Fig. 8. M06-2X optimized geometries for the intermediates of catalytic catechol oxidation by the
dimeric species 1b.
17

The optimized structures of the intermediates A, B, C and D have been presented in Fig. 8.
Density functional theoretical computations have been performed at M06-2X/6-31G* level to
compute Gibbs free energy of the intermediates for the evaluation of reaction feasibility at room
temperature. In the first step (1b→A) of the catalytic cycle, one molecule of catechol binds to the
dinuclear complex (S0) through bridging fashion with concomitant deprotonation of catechol.
The process is energetically facile as the catechol addition raises free energy of the system by 4.6
kcal/mol only. In the intermediate A, CuII-O (catechol) bond distances are 2.21 and 2.45 Å. In
the next step, A undergoes spin flipping from singlet to triplet state B and this intersystem
crossing is energetically favorable by 16.3 kcal/mol. The frontier molecular orbital analysis
(FMO) of the triplet state displayed two singly occupied molecular orbitals (SOMOs), one
situated on the ligand (-6.37 eV) and the other one on catechol moiety (-6.67 eV) (Fig. 9). At this
juncture, the catechol moiety can covalently combine with dioxygen in its triplet state to produce
intermediate C and the process raises the system’s energy to 17.7 kcal/mol. The C-O (O2) bond
distance is 1.58 Å. The FMO analysis of C revealed that the two SOMOs are situated on the
ligand with -6.68 eV and -6.36 eV of energy whereas the LUMO is located over catechol and
dioxygen units (Fig. 9). Therefore, a single electron transfer (SET) process can occur from the
ligand to the dioxygen unit accompanied by a proton transfer process yielding intermediate D.
The transformation is thermodynamically favourable as it brings down the system’s energy to 1.9
kcal/mol. In the last step, catechol fragment (E) release occurs easily to complete the catalytic
cycle. The intermediate E eventually produces benzoquinone and hydrogen peroxide.
a)
b)
-6.68 eV
-6.36 eV
-1.98 eV
LUMO
-6.67 eV
-6.37 eV
SOMOs
SOMOs
Fig. 9. Frontier molecular orbital plots for intermediate a) B and b) C.
18

3.12. Phenoxazinone Synthase Activity
Phenoxazinone synthase activity of complex 1 has been monitored using ortho-Aminophenol
(OAP) as model substrate in DMF medium under aerobic condition. The course of the reaction is
monitored by time dependent UV-Vis spectroscopy at 25 °C. The broad peak at λmax = 422 nm,
signature of phenoxazinone (APX) increases with time (Fig. 10), indicating that complex 1 is a
potential catalyst for the oxidation of ortho-aminophenol. The changes noticed in the d-d bands
which have been shown in Fig. 10 during the course of the reaction are noteworthy.
During oxidation of ortho-aminophenol the d-d band of complex 1 is disappeared immediately
after the start of oxidation. A plateau like nature of d-d band is observed in the region of 664 nm
but the enhancement of absorbance value is not appreciable. This phenomenon indicates that the
copper(II) centre is like to underwent reduction to copper(I).
In this case a controlled experiment is also carried out without using catalyst and the
characteristics band of APX around λmax ~ 422 nm was not found to increase with time (Fig.
S11).
Fig. 10. Oxidation of ortho-aminophenol catalysed by complex 1 (substrate: catalyst = 100:1)
observed in UV-Vis spectroscopy with time in DMF medium at 25 °C. (b) Change in d-d band of
complex 1 with oxidation of ortho-aminophenol.
3.13. ESI-MS Study
In order to detect the composition of the intermediate forming during phenoxazinone synthase
activity ESI-MS study has been performed in positive mode. A very small amount of reaction
mixture has been taken and diluted with acetonitrile. The peak present at m/z = 625.2156 amu is
19

signature of a monocationic dinuclear species as discussed earlier. The base peak is present at
m/z = 843.1345 amu which corroborated with the molecular formula [C42H60Cu2N4O6 + H+] and
is consistent with 1:2 adduct between dinuclear species and ortho-aminophenol. The peak at m/z
= 213.2041amu is observed due to formation of phenoxazinone, the peak being indicative of H
adduct to phenoxazinone (Calcd. m/z = 213.2121amu)(Fig. S12).
3.14. EPR study
X-band EPR study has been performed to detect the intermediate formed during catalytic cycle.
Complex 1 contains four line EPR signal. When ortho-aminophenol was added to complex 1 in
DMF medium an isotropic signal at g = 2.12 (Fig. 9) appears which is attributed to formation of
organic radical during catalytic cycle. The characteristics peak of complex 1 disappears upon
addition of OAP thus suggesting that metal center may undergo reduction. Previous reports
suggest that when redox active metal centre is present in the active site metal centered reduction
took place with generation of radical although ligand centered radical formation is another
alternative of metal centered redox participation during catalysis[68,69].
Fig. 11. X-band EPR spectrum of reaction mixture of complex 1 and ortho-aminophenol in DMF
medium at 298 K.
3.15. Cyclic voltammetry study
For complete assessment of the redox reaction occurring during catalysis, cyclic voltammetry of
the dinuclear copper species 1b in presence of ortho-amino phenol has been performed in DMF
medium at 100 mV/sec scan rate. The cyclic voltammogram of the reaction mixture consists of
20

two adjacent cathodic peaks at Epc = 0.11 V and Epc = -0.18 V, which may be attributed to
reduction of CuII to CuI during the catalytic cycle (Fig. 10).
Fig. 12. Cyclic voltammogram of reaction mixture of complex 1 and ortho-aminophenol in DMF
medium
3.16. Mechanistic pathway of Phenoxazinone Synthase activity
On combining the experimental results obtained from ESI-MS spectrum, EPR spectrum and
cyclic voltammogram of reaction mixture of complex 1 and OAP a mechanistic pathway has
been proposed. The catalytic cycle is initiated by the formation of 1:2 adduct between complex 1
and ortho-aminophenol. In the next step there occurs the formation of an organic radical located
on the ortho-aminophenol moiety with simultaneous reduction of copper(II) centre. In the next
step ortho-aminophenol is converted to phenoxazinone (APX) in presence of aerial oxygen. The
plausible mechanistic pathway of phenoxazinone synthase activity catalyzed by complex 1 is
presented in Scheme 3.
21

Scheme 3. Plausible catalytic cycle of phenoxazinone synthase activity catalyzed by species 1b.
3.17. Phosphatase Like Activity
To explore the hydrolytic property of complex 1, phosphatase like activity is studied in 97.5 %
(v/v) DMF-H2O mixture using disodium salt of (4-nitrophenyl)phosphate(4-NPP)hexahydrate (4-
NPP) as the model substrate and progress of the reaction has been monitored by time dependent
UV-Vis spectral scan at 25 °C. The complex 1 is capable to catalyze the hydrolysis of the
phosphate-ester bond of the substrate as is evident from the time dependent UV-Vis spectral
study. The spectrum of reaction mixture shows an increment of peak at λmax ~ 424 nm with time,
characteristics of 4-nitrophenolate ion (Fig. 11).
A controlled experiment has been carried out with copper(II) perchlorate hexahydrate and ligand
H2L and in both the cases hydrolysis of phosphate ester bond did not occur (Fig. S11).
22

Fig. 13. Hydrolysis of 4-NPP catalyzed by complex 1 (substrate: catalyst = 20:1) in 97.5% (v/v)
DMF-H2O observed in UV-Vis spectroscopy with time at 25 °C at intervals of 5 min.
3.18. ESI-MS Study
A very small amount of reaction mixture has been taken and diluted with acetonitrile and ESI-
MS spectrum was recorded in positive mode. The base peak is observed at m/z = 667.1119 amu
which corroborates exactly with the molecular formula C30H47Cu2N2O4+ CH3CN (Calcd. m/z =
666.8491 amu) i.e. it represents the acetonilrile adduct of dinuclear entity. The peak at m/z =
862.1069 amu (Calcd. m/z = 862.2171 amu) corresponds to hydroxo bridged dinuclear species
having molecular formula [C36H54Cu2N3O11P](2H+) (Fig. S14) and this is supposed to be the key
intermediate in P-O bond hydrolysis reaction[43].
3.19. Mechanistic pathway of Phosphatase activity
The catalytic cycle is likely to be initiated by attack of water and 4-nitrophenylphosphate (4-
NPP) followed by the conversion of water molecule to hydroxo species. In the next step
nucleophilic attack of hydroxo to the phosphorus centre takes place and this step is believed to be
the rate determining step.[70] Actually, this step involves the formation of dihydrogen phosphate
species with the concomitant liberation of 4-nitrophenolate ion. In the last step
dihydrogenphosphate is released from dinuclear copper center with regeneration of the catalyst,
1b.
23

Scheme 4. Plausible catalytic cycle of phosphatase activity catalyzed by dinuclear species.
3.20. Kinetics Study of complex 1 for all three catalytic activity
The kinetic studies of complex 1 for catecholase activity, phenoxazinone synthase activity and
phosphatase have been performed to quantify the catalytic efficacy of the newly synthesized
complex. Michaelis-Menten approach is found to be the best because the complex showed
saturation kinetics and the kinetic parameters were calculated from Lineweaver Burk plot (Fig.
S15-S17). The kcat/ KM value dictates the catalytic efficiency of the enzyme and it is observed
that in case phosphatase activity kcat/KM value is the highest among the all three activity. The
kinetic parameters of complex 1 are presented in Table 1.
Table 1
Kinetic parameters of complex 1 for various catalytic activity
Vmax (M s-1)
KM (M)
2.38×10-3 23 h-1
3.55×10-3 38.52 h-1 3.01
kcat
kcat/ KM (M-1 s-1) S.D
R2
Catecholase activity
6.44×10-7
2.70
0.153 0.999
0.988 0.981
1.040 0.995
Phenoxazinone synthase activity 1.07×10-6
Phosphatase activity
2.66×10-4
8.58×10-4 5.22 s-1
6.08×103
24

The rate constant values of some catalytic promiscuous homometallic and heterometallic
complexes are presented in Table S3. On considering the kcat value of catecholase activity with
the previously reported complexes it may be stated that complex 1 is moderately active catalyst
but the mechanistic pathway of 1 makes it unique from the reported catalysts. It is also capable in
catalyzing the oxidation of ortho-aminophenol with moderate efficiency. The species show
appreciable hydrolyzing property, only fewer zinc(II) based models showed higher activity than
this newly synthesized species. Hence, it may be concluded that the newly synthesized complex
1 is a unique species having the capability to catalyze oxidation of catechol, oxidation of ortho
amino phenol and hydrolysis of phosphate monoester dianion.
4. Conclusions
Flexible ligand design by utilizing Mannich base ligand synthesis becomes fruitful in making
polynuclear complex of our interest. An exciting catalytic promiscuity has been demonstrated in
the present report where our synthesized tetranuclear CuII complex is observed to act as catalyst
precursor. The dinuclear species, which has been originated from the catalyst precursor in
solution, is actually the active catalyst. 1 has shown its potential as hydrolytic catalyst with high
efficiency to catalyze P-O bond hydrolysis and thereby acts as a functional model of phosphatase
enzyme. Activity of 1 as redox catalyst to catalyze phenoxazinone synthases is moderate. On the
other hand, 1 becomes a unique member in the model study of catechol oxidase. A completely
new finding of radical pathway in copper based models, rather than copper centered redox
participation has been explored by combined experimental and theoretical investigation.
Acknowledgements
The authors wish to thank University of Calcutta for providing the facility of ESI-MS
spectrophotometer from the DST-PURSE program. S.D [09/028(1023)/2018-EMR-I] and A.M
[09/028(0959)/2015-EMR-I] is thankful to CSIR-India for providing fellowship. Financial
assistance from DST-WB [Sanction memo no. 327(sanc)/ST/P/S&T/15G-8/2016 dt 06/03/2019]
to DD has been duly acknowledged. The authors wish to thank Professor Prasanta Ghosh of
Narendrapur Ramakrishna Mission Residential College for their endless help regarding EPR
facility. The authors wish to thank Professor A. Ghosh for providing CV facility.
25

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