Functional models for type-2 and type-3 copper oxidases: Self-assembled molecular association in [Cu(L)(Hdpa)](ClO4) determines the catalytic activity

Article abstract of DOI:10.1016/j.ica.2019.119016

Mononuclear copper(II) complex [Cu(L)(Hdpa)](ClO₄) (1), where H(L) is tridentate NNO donor ligand and Hdpa is 2,2′-dipyridylamine has been isolated. The X-ray crystal structure of 1 possesses a CuN₄O chromophore with square pyramidal

Full text of DOI:10.1016/j.ica.2019.119016

Accepted Manuscript
Research paper
Functional models for type-2 and type-3 copper oxidases: Self-assembled mo-
lecular association in [Cu(L)(Hdpa)](ClO4) determines the catalytic activity
Velusamy Sathya, Mariappan Murali
PII:
S0020-1693(18)31343-4
https://doi.org/10.1016/j.ica.2019.119016
119016
DOI:
Article Number:
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ICA 119016
Inorganica Chimica Acta
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16 August 2018
10 June 2019
10 July 2019
Please cite this article as: V. Sathya, M. Murali, Functional models for type-2 and type-3 copper oxidases: Self-
assembled molecular association in [Cu(L)(Hdpa)](ClO4) determines the catalytic activity, Inorganica Chimica
Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119016
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Functional models for type-2 and type-3 copper oxidases: Self-assembled molecular
association in [Cu(L)(Hdpa)](ClO4) determines the catalytic activity
Velusamy Sathya and Mariappan Murali*
Coordination and Bioinorganic Chemistry Research Laboratory
Department of Chemistry
National College (Autonomous)
Tiruchirappalli 620 001
Tamil Nadu, India
e-mail: ma66mu@gmail.com
Phone: +91-431-2482995 / Fax: +91-431-2481997
Abstract
Mononuclear copper(II) complex [Cu(L)(Hdpa)](ClO4) (1), where H(L) is tridentate NNO donor
ligand and Hdpa is 2,2’-dipyridylamine has been isolated. The X-ray crystal structure of 1
possesses a CuN4O chromophore with square pyramidal (4+1) coordination geometry. It displays
self-assembled molecular association viz., (a) intermolecular N-HO hydrogen bonding and (b)
inter-pair π-π anti-parallel stacking. The complex is catalytically effective in the oxidation of
benzylamine to benzaldehyde and 3,5-di-tert-butylcatechol to 3,5-di-tert-butyl-o-quinone, thus
act as a functional model for amine oxidase and catechol oxidase respectively.
1. Introduction
Copper containing metalloproteins in the living system plays a fundamental role in
revealing its catalytic role as dioxygen and/or substrates activators towards different bio-function
[1]. In this context, chemists have significantly contributed in unveiling the mystery of nature,
especially by designing different metal complexes which can potentially mimic the active site
and/or function of different copper proteins in nature [2,3]. One of the important copper oxidases
is ascorbate oxidase (AO), consisting of three types of copper centers in which the type-2 copper
site (A|| > 140  10-4 cm-1) belongs to the functional trinuclear cluster [4], coordinated to two
histidine molecules and a water molecule. The primary role of the type-2 copper center is
electron transfer, in which ascorbate under physiological reaction conditions acts as an electron
donor. The reduced copper(I) sites activate dioxygen and convert it to water involving four
electrons and four protons in AO [5]. The amine oxidase (AmO) is another type-2 copper
oxidase, which arises as homodimers. The active site of each subunit contains one copper center
[6] coordinated with three histidine residues and two water molecules in a distorted square
pyramidal geometry. It is responsible for two-electron oxidation of primary amines to aldehydes
[7]. Catechol oxidase (CO), catalyzes the oxidation of catechol to o-quinone accompanying the
four electron reduction of O2 to H2O [8]. The CO is a type-3 copper protein containing dicopper
center coordinated by six histidine nitrogen atoms and -2:2 peroxo-bridged oxy form [9-11].

Many model complexes with dicopper center (CuCu  5 Å) mimic the copper oxidases [12-
14], both structurally and functionally whereas the same for the monocopper center counterparts
remain uncommon. Several works in the literature indicates that the efficiency of a mononuclear
copper(II) complex as catalyst in copper oxidase activity [15-20] have been influenced by a
number of factors such as nature of square pyramidal (4+1) coordination geometry, labile
binding sites, chelate ring size, nature of the donor groups, electronic properties of the ligands,
existence of self-assembled molecular association to form mononuclear pairs, CuCu
separation, redox potentials from electrochemical studies and steric match between the substrate
and catalyst. All these facts reflect that explorations of the possibility of exhibiting copper
oxidase activity by new types of compounds are very much demanding, and our research group
is engaged in this endeavor. In the present work, green colored mononuclear copper(II) complex
(Scheme 1) has been synthesized, characterized and its potential catalytic activity has been tested
with an aim to mimic functional sites of ascorbate, amine and catechol oxidases.
2. Experimental Section
2.1. Materials
Copper(II) acetate monohydrate, 2,2’-dipyridylamine, 2-hydroxy-1-naphthaldehyde,
sodium perchlorate, N,N-dimethylethylenediamine, 3,5-di-tert-butylcatechol, tetra-N-
butylammonium bromide (Sigma-Aldrich), L-ascorbic acid (Fisher Scientific), benzylamine
(Avra), KI, ammonium molybdate and hydrogen peroxide solution (30% w/v) (Merck) were used
as received. HPLC grade methanol, N,N-dimethylformamide, ethyl acetate, hexane,
dichloromethane and acetonitrile and reagent grade diethyl ether were purchased from Merck.
2.2. Physical Measurements
Microanalyses (C, H, and N) were carried out with a Vario EL elemental analyzer. The
conductivity is measured using EQUIPTRONICS EQDCMP bridge with a solute concentration
of 110-3 M in MeOH. Mass spectrometry was performed on a ZQ ESI-MS spectrometer.
Magnetic susceptibility data at 27 °C were obtained for polycrystalline samples using George
Associates Inc. FTIR spectra were recorded using a Perkin Elmer Spectrum RX1 FTIR
spectrophotometer in the range 400-4000 cm-1 with a sample prepared as KBr disc. UV-Visible
spectroscopy of reflectance spectra was recorded using Shimadzu UV-2450 UV-VIS
spectrophotometer and solution spectra were recorded using Perkin Elmer Lambda-365 UV-VIS
spectrophotometer using cuvettes of 1 cm path length. X-band EPR spectra of the complex in
DMF, MeOH and MeCN at liquid nitrogen temperature (77 K) and polycrystalline at room

1
temperature (RT) were recorded on a JEOL JES-FA200 ESR spectrometer. H NMR spectra
were recorded on a Bruker 300 MHz with AVANCE II NMR spectrometer in CDCl3. Cyclic
voltammetry (CV) and differential pulse voltammetry (DPV) on glassy carbon electrode were
performed in MeOH 25  0.2 C. The voltammograms were generated using CH instruments
620C electrochemical analyzer. A three electrode system has been used to study the
electrochemical behavior of complexes (0.001 M) consisting of a glassy carbon working
electrode (A = 0.0707 cm2), a platinum wire auxiliary electrode and saturated calomel reference
electrode and TBAP (0.1 M) is used as a supporting electrolyte. Solutions were deoxygenated by
purging with nitrogen gas for 15 min prior to the measurements.
2.3. Synthesis of [Cu(L)(Hdpa)](ClO4) (1)
The complex 1 was prepared [21] by the reaction of Cu(O2CMe)2.H2O (0.20 g, 1 mmol)
with 2,2’-dipyridylamine (Hdpa; 0.17 g, 1 mmol) in methanol (15 mL). The mixture was left
stirring for 1 h at 25 °C at which point the yellow solution of H(L), obtained from the
condensation of 2-hydroxy-1-naphthaldehyde (0.17 g,
1
mmol) and N,N-
dimethylethylenediamine (0.09 g, 1 mmol) [22,23], was added. The resultant green solution was
refluxed for 3 h. The green solids obtained after the addition of a methanolic solution of NaClO4
(0.122 g, 1 mmol) were filtered and dried in vacuo over P4O10. Yield: 0.35 g (61 %). Anal.
Calcd. for C25H26N5O5ClCu (1): C, 52.18; H, 4.55; N, 12.17 %. Found: C, 52.24; H, 4.46; N,
12.13 %. M: 75 (MeOH) -1 cm2 mol-1. ESI-MS (MeCN): m/z 475.17 [M+-ClO4]. eff (27 C):
1.8 B. FT-IR (KBr, cm-1) selected bands: 1622 νimine(C=N), 1584 νpy(C=N), 1305 ν(naph-O),
-
1092, 621 ν(ClO4 ). Electronic spectrum in solid/MeOH [λmax/nm (εmax/dm3 mol-1 cm-1)]: 653/651
(202), 897 sh (70), 393 (6802), 310 (28702), 260 (41118), 249 (44597). Electronic spectrum in
MeOH:H2O (4:1 v/v) [λmax/nm (εmax/dm3 mol-1 cm-1)]: 649 (179), 891 sh (64), 389 (6414), 310
(26739), 261 (37863), 249 (40542). Polycrystalline EPR spectrum at RT: giso = 2.078. EPR
spectrum in DMF solution at 77 K: g|| = 2.227, g = 2.045, A|| = 188 × 10-4 cm-1, g||/A|| = 118 cm,
G = 5.2, ² = 0.80, ² = 0.65, ² = 0.50, K|| = 0.72, K = 0.63. EPR spectrum in MeOH solution at
77 K: giso = 2.128. EPR spectrum in MeCN solution at 77 K: giso = 2.083. Redox behavior in
MeOH (0.1 M TBAP): CV, E1/2 = 0.159 V, ΔEp = 67 mV, ipa/ipc = 2.4 (decreases towards unity
with increase in scan rate), D = 5.2 × 10-6 cm2 s-1; DPV, E1/2 = 0.152 V.
2.4. X-ray structure determination
The green single crystal of [Cu(L)(Hdpa)](ClO4) (1) suitable for X-ray structural
determination was obtained by cooling a solution of 1 in MeOH:MeCN at 5 °C for five days. A
green single crystal of 1 with dimensions 0.30 × 0.25 × 0.20 mm3, was selected under the

polarizing microscope and then mounted on the glass fiber. The crystal data collections were
performed through a Bruker AXS-KAPPA APEX II diffractometer using graphite
monochromated Mo-Kα radiation (λ = 0.71073 Å). Cell parameters were retrieved using Bruker
SMART software and refined using Bruker SAINT on all observed reflections [24]. Absorption
corrections were performed using SADABS [24]. Structure solution was performed using direct
methods with the program SHELXT-2014/4 and refined by full-matrix least squares on F2 using
SHELXL-2014/7 [25,26]. The C(1), C(2), C(3) and C(4) atoms in N,N-dimethylethylenediamine
moiety of H(L) ligand and O(2), O(3), O(4) and O(5) atoms in perchlorate anion are twofold
disordered. The components of disorder were located in difference Fourier map and refined with
restraints on bond distances. Also, the sum of occupancies of the components (C, 0.544(6) and
0.456(6) and O, 0.342(9) and 0.658(9)) was restrained as 1. The non-hydrogen atoms were
refined with anisotropic displacement parameters. All hydrogen atoms except the one belonging
to a nitrogen atom (N4) were geometrically fixed at chemically meaningful positions. The N4
hydrogen atom could be located in a difference map and was refined isotropically without
restraints.
2.5. Oxidation of ascorbic acid
The ascorbate oxidase activity of the complex is evaluated by reaction with 1 (3 × 10-3
M) and different concentration of ascorbic acid (H2A). The experiments were run under aerobic
conditions in MeOH:H2O (4:1 v/v) medium and monitored using UV-Vis spectroscopy.
2.6. Oxidation of benzylamine
The catalytic action of 1 (1 × 10-3 M) in the H2O2 (1 ml of 30% w/v) dependent
deamination of benzylamine (100 × 10-3 M) in 10 ml MeOH:H2O (4:1 v/v) results in the
oxidation of benzylamine to benzaldehyde. The organic products were isolated from the reaction
mixture by solvent extraction with diethyl ether after removal of methanol. Diethyl ether was
removed by rotary evaporation and the residue was dissolved in CDCl3 and MeOH for 1H NMR
and ESI-MS measurements.
2.7. Kinetics of 3,5-di-tert-butylcatechol oxidation
To determine the catecholase activity of the complex, 2.9 × 10-5 M solution of the copper
complex (1) in methanol was treated with 50 equivalents of 3,5-di-tert-butylcatechol (3,5-
DTBC) in methanol under aerobic condition. The UV spectra of the solution were recorded
directly after the addition and subsequently after regular intervals of 30 min and absorption value
at 393 nm were measured as a function of time over a period of 3 h. To determine the
dependence of the rates on the substrate concentration and various kinetic parameters, 2.9 × 10-5

M solution of 1 was treated with 5-45 equivalents of 3,5-DTBC in methanol under aerobic
condition.
To detect the formation of hydrogen peroxide during the catalytic reaction, reaction
mixtures were prepared as in the kinetic experiments. During the course of the oxidation
reaction, the solution was acidified with H2SO4 to pH 2 to stop further oxidation after a certain
time and an equal volume of water was added. The formed quinine was extracted three times
with dichloromethane. To the aqueous layer were added 1 mL of a 10% solution of KI and three
-
drops of a 3% solution of ammonium molybdate. The formation of I3 could be monitored
-
spectrophotometrically because of the development of the characteristic I3 band ( = 353 nm, 
= 26000 M-1 cm-1).
3. Results and Discussion
3.1. Synthesis and general aspects
Complex 1 was synthesized from 2,2’-dipyridylamine (Hdpa), Schiff base ligand H(L)
(derived
from
the
condensation
of
2-hydroxy-1-naphthaldehyde
and
N,N-
dimethylethylenediamine) and Cu(O2CMe)2.H2O and isolated as green colored crystals in good
yield (60%). The ESI-MS data in MeCN (m/z [M+-ClO4], 475.17) reveal that the complex
retains its identity even in solution and this is supported by the value of molar conductivity in
methanol (75 -1cm2mol-1) for 1:1 electrolyte [20]. The complex exhibits infrared spectral band
due to imine(C=N) (1622 cm-1), py(C=N) (1584 cm-1) and (naph-O) (1305 cm-1), implying
direct coordination of imine and pyridine nitrogen and naphtholate oxygen donors to copper(II).
A broad intense band (1092 cm-1) and a strong sharp band (621 cm-1) are observed, which are
characteristic of non-coordinated perchlorate ion. Based on the elemental analysis the complex
was formulated as [Cu(L)(Hdpa)](ClO4), which was confirmed by single crystal X-ray
determination of 1.
3.2. Description of the structure
The crystal refinement data and bond lengths and bond angles are listed in Tables 1 and 2
respectively and an ORTEP drawing of the complex cation [Cu(L)(Hdpa)]+ (1) is shown in Fig.
1a. The Cu(II) cation is coordinated by naphtholate oxygen atom O(1) and two nitrogen atoms
N(1) and N(2) of the Schiff base ligand, H(L) and two nitrogen atoms N(3) and N(5) of the Hdpa
co-ligand. The value of the trigonality index  (0.12) reveals that the coordination geometry
around copper(II) is best described as distorted square pyramidal [20, 27]. The imine N(1) and
amine N(2) nitrogen atoms and the naphtholate oxygen atom O(1) of the meridionally

coordinated Schiff base ligand and one of the imine nitrogen atom N(3) of Hdpa occupy the
corners of the CuN3O square plane of this geometry. The copper is displaced by 0.278(9) Å from
the N3O plane towards the axially coordinated nitrogen atom N(5) of Hdpa. Thus, axial position
is occupied by nitrogen atom N(5) at a distance 2.1805(18) Ǻ, longer than equatorial atoms
(Cu(1)-O(1), 1.9567(15) Å; Cu(1)-N(1), 1.9230(18) Å; Cu(1)-N(2), 2.1338(19) Å; Cu(1)-N(3),
1.9979(18) Å) as a consequence of the presence of two electrons in dz² orbital of copper(II). The
improper orientation of the lone pair on N(2) nitrogen of -NMe2 group towards the dx²-y² orbital
also would contribute to the longer Cu(1)-N(2) bond distance. The Cu-Namine bond (Cu(1)-N(2),
2.1338(19) Å) is longer than the Cu-Nimine bond (Cu(1)-N(1), 1.9230(18) Å) formed by the
Schiff base ligand, which is expected of sp3 and sp2 hybridizations of the amine N(2) and imine
N(1) nitrogen atoms respectively. The degree of distortion is more towards square pyramidal due
to smaller trigonality index, which could be related to (i) the two six-membered chelate rings
Cu(1)O(1)C(15)C(6)C(5)N(1) and Cu(1)N(3)C(20)N(4)C(21)N(5) and the chelate ring,
Cu(1)O(1)C(15)C(6)C(5)N(1) consists of Cu center bonded by the oxygen atom O(1) and
nitrogen atom N(1) of the H(L) Schiff base ligand is nearly coplanar (0.011(3) Å) and (ii) the
smaller dihedral angle (5.41(3)) between the two coordinated pyridine rings of Hdpa.
Crystal packing reveals that cations of adjacent molecules are arranged in dimeric
association (Fig. 1b). These pairs are held together by (a) intermolecular N-HO hydrogen
bonding and (b) inter-pair - interaction. The significant structural observation is the strongest
intermolecular interaction leading to a centrosymmetric dimer (symmetry code: 2-x, -y, 1-z) in
the crystal, is a N(4)-H(4)O(1) hydrogen bond existing between the amine proton, H(4)(N4) of
Hdpa and coordinated naphtholate oxygen O(1) (N(4)-H(4)O(1) = 2.114(18) Å, N(4)O(1) =
2.945(2) Å, N(4)-H(4)O(1) = 160(2)). The dimeric association is supported by inter-pair -
interactions brought on by intermolecular anti-parallel stacking involving Hdpa ligands. The -
stacking between C(20) of py and C(21) of py (C(20)C(21) = 3.281(4) Å) rings of neighboring
coordinated Hdpa ligand (Cg(p)Cg(p) (3.502(4) Å) is expected to stabilize the complex in the
solid state. At a centroid contact of 3.502(4) Å and angle of 21.99(4) between the ring normal
and the centroid vector corresponds to horizontal displacement of 1.383(4) Å reveals the shift of
almost one C-C bond length face-to-face alignment (Cg(p)). Therefore, the stacked Hdpa ligands
show the CuCu separation as 6.551(3) Å.
3.3. Electronic spectra
The electronic spectra of 1 in MeOH and MeOH:H2O (4:1 v/v) are very similar and
display a broad ligand-field band (max, 651 nm) with a weak low-energy shoulder (max, 897

nm) in the visible region and the intensity of higher energy band (max, 202 M-1cm-1) is
significantly higher than the intensity of lower energy shoulder (max, 70 M-1cm-1), which is
characteristic for distorted square pyramidal copper(II) complexes [28,29]. The diffuse
reflectance spectrum of 1 exhibits the broad ligand-field band (max, 653 nm) suggesting the
possibility of an oxygen atom coordinated to copper(II). The position of the band remains almost
unchanged in the solid-state when compared with the solution spectrum indicating that the
complex retains in solutions its solid-state coordination geometry [30] determined by X-ray
crystallography (distorted square pyramidal). The intense absorption band observed in the UV
region (max, 249-310 nm) is attributed to the intraligand -* transition in the coordinated
diimines. The low-intensity band (max, 393 nm) is assigned to naphtholate anion to copper(II)
ligand-to-metal charge-transfer (LMCT) transition, revealing the involvement of the naphtholate
oxygen atom in coordination even in solution.
3.4. EPR spectra
The EPR spectrum of 1 exhibit one broad singlet (giso = 2.078) in the polycrystalline state
at 298 K arising from dipolar broadening and enhanced spin-lattice relaxation. In frozen
solutions of 1, three of the four parallel hyperfine features were well separated while the fourth
one overlapped with the perpendicular features in DMF whereas the parallel feature was very
weakly resolved and the perpendicular feature was moderately broader in MeOH (giso, 2.128)
and in MeCN (giso, 2.083), the signals were significantly broader (Fig. 2). This suggests that the
dimeric intermolecular association is retained in weak coordinating solvents like MeCN and
MeOH [31] while strong coordinating solvent like DMF, the association is disturbed due to
solvolysis.
The frozen DMF solution EPR spectrum of 1 shows axial spectral features typical of
mononuclear Cu(II) species (g||>g>2.0; G = (g||-2) / (g-2) = 5.2) suggesting the presence of
dx²-y² ground state in copper(II) located in square-based geometries [18]. Further, a square-based
CuN4 chromophore is expected [32,33] to show g|| (2.200) and A|| (180-200  10-4 cm-1) values
and the replacement of a nitrogen atom in this chromophore by an oxygen atom and
incorporation of strong axial interaction has been found to increase the g|| and decrease the A||
values [34]. Therefore, the observed g|| (2.227) and A|| (188  10-4 cm-1) values are consistent
with the presence of a square-based CuN3O chromophore with strong axial interaction (cf.
above). The value of g||/A|| (118 cm) quotient indicates appreciable distortion of the structure,
which is borne out by the value of the trigonality index, . Molecular orbital coefficients [35], 2
(a measure of the covalency of the in-plane -bonding between 3d orbital and the ligand orbitals)

and 2 (covalent in-plane -bonding) were estimated and the 2 (0.80) and 2 (0.65) values
indicate that there is a substantial interaction in the in-plane -bonding whereas the in-plane -
bonding is almost covalent. For complex 1, it is observed that K|| > K [36] (K|| (0.72) and K
(0.63) are orbital reduction factors), showing the significant out-of-plane -bonding.
3.5. Redox properties
Cyclic (CV) and differential pulse voltammetric (DPV) responses of 1 obtained from
MeOH reveal the electrochemically and chemically reversible CuII/CuI redox behavior, as
apparent from the linear plot of ipc versus 1/2 (D, 5.2  10-6 cm2 s-1) and the values acquired for
peak current ratio (ipa/ipc, 2.4; reaches unity by increasing the scan rate) respectively. However,
the value of the limiting peak-to-peak separation (Ep, 67 mV), which is smaller than that for the
Fc/Fc+ couple (Ep, 86 mV; E1/2, 0.406 V vs SCE) under identical conditions. Interestingly, it
exhibits low Ep (67 mV) and positive Cu(II)/Cu(I) redox potential (E1/2, 0.159 V vs SCE),
which signifies that the structural reorganization between their copper(II) and copper(I) species
is minimal leading to a facile heterogeneous electron transfer [37] possibly due to the formation
of boat conformation in six-membered chelate ring (Cu-O-C-C-C-N) induced by the steric clash
between -NMe2 group in H(L) and non-planar Hdpa ligand (cf.  values, 0.12).
3.6. Ascorbic acid oxidation
Interestingly, the addition of ascorbic acid (H2A) to the green solution of catalyst 1 (3 
10-3 M) did not change to the brown solution and no new CT band is observed (Fig. 3). Instead,
the d-d band undergoes blue shift (max: 649 to 634 nm), indicating the coordination of one of the
oxygen donors (H2A) with a square pyramidal complex 1 (ESI-MS positive (Fig. S6): m/z,
673.35 confirms the formation of an adduct, {Na-[CuII(L)(Hdpa)(ascorbate ion)]}+ and the peaks
are found at m/z, 474.50 and 950.85 corresponds to monomeric unit, [CuII(L)(Hdpa)]+, and
dimeric aggregate, 2[CuII(L)(Hdpa)]+, of the complex respectively), which is consistent with
octahedral geometry. This is possible because of the centrosymmetric dimer consists of two
stable six-membered chelate rings (Cu(1)N(3)C(20)N(4)C(21)N(5)) in the anti-parallel slip
orientation due to the rapid flapping of the py rings of Hdpa in the opposite direction. This could
destroy the steric match between H2A and catalyst (1) in order to prevent the oxidation of
ascorbic acid to dehydroascorbic acid.
3.7. Benzylamine oxidation
The catalytic reaction of benzylamine (100  10-3 M) using complex 1 (1  10-3 M) as a
catalyst in the presence of H2O2 (1 ml of 30% w/v) in 10 ml of MeOH:H2O (4:1 v/v) causes the
complete oxidation of benzylamine to benzaldehyde (PhCH2NH2  PhCHO + NH3). The

formation of the reaction products is evidenced by the 1H NMR (Fig. S7: benzylamine, -CH2-, 
3.872 ppm; benzaldehyde, -CHO,  10.032 ppm) and mass (Fig. S8: shows two peaks at m/z,
105.9 and 107.0 assignable to [PhCO++H] and [PhCHO+H] respectively) spectral studies. The
proposed catalytic cycle reveals that the initial step of the catalytic reaction is the formation of an
adduct showing a significant shift of the visible band (max: 643 to 595 nm) to higher energy
(Fig. 4) by the addition of benzylamine to the solution of 1. This adducts species react with H2O2
to form an initial brown color solution that does not show any d-d band. It indicates the one-
electron reduction of copper along with the concomitant protonation of the L ligand by
benzylamine adduct with copper. Thus, the copper(I) species is reactive towards benzylamine
which undergoes oxidation to benzylimine and the presence of water hydrolyzes benzylimine to
benzaldehyde.
3.8. Catechol oxidation
The reactivity of copper(II) complex, 1 (2.9  10-5 M) in methanol towards the aerobic
oxidation of 3,5-di-tert-butylcatechol (3,5-DTBCH2; 50 equivalent) was repeatedly monitored by
the improvement of the strong band at 393 nm for every 30 min time interval during the course
of a reaction (Fig. 5). It reveals the development of a strong absorption band at 393 nm
characteristic of quinone chromophore (3,5-di-tert-butyl-o-quinone; 3,5-DTBQ), which prompt
us to conclude that 1 is reactive towards catecholase activity. The same reaction was done by
scaling up the reactants and the desired 3,5-DTBQ was isolated column chromatographically
using 10% mixture of ethyl acetate and hexane as eluent. The isolated o-quinone was identified
by 1H NMR spectra in CDCl3 (Fig. S9: -CH3 (C), s,  1.229 ppm, 9H; -CH3 (D), s,  1.272 ppm,
9H; H (B), s,  6.220 ppm, 1H; H (A), s,  6.936 ppm, 1H). The kinetic data were determined by
the method of initial rates by monitoring the growth of the 393 nm band of the product 3,5-
DTBQ for the first 3 h, formed due to the oxidation of 3,5-DTBCH2 (5-45 equivalents) in the
presence of 1 (2.9  10-5 M). A treatment on the basis of the Michaelis-Menten model is
appropriate and Lineweaver-Burk plot for 1 gave straight line (Fig. 6), from which the kinetic
parameters such as maximum velocity (Vmax, 6.9  10-4 M min-1), Michaelis binding constant
(KM, 12.2  10-5 M) and the turnover number (kcat, 1424 h-1) were evaluated. Interestingly, the
kcat is superior to the other mono and binuclear complexes (kcat, 33-925 h-1) [38]. Also, we found

the band at 353 nm which is due to the existence of I3 [39], provides evidence for the formation
of H2O2 from the reaction mixture. In order to understand the catalytic oxidation of 3,5-DTBCH2
to 3,5-DTBQ, the electrospray ionization mass spectrum (ESI-MS positive) of a mixture of 1 and
3,5-DTBCH2 (molar ratio, 1:10) was recorded after 30 min in methanol solution (Fig. 7). The

most abundant species found at m/z = 476.75 arises from a monomeric unit of the complex and
are assigned to [CuII(L)(Hdpa)]+. The low intense peak at m/z = 952.45 appears due to the
dimeric aggregate of the complex, which can be assigned to 2[CuII(L)(Hdpa)]+ indicating the
existence of the self-assembled molecular association of 1 in solution and substantiating the
results from the frozen solution EPR spectra in MeOH and MeCN (cf. above). The peak at
243.30 is well assignable to the [3,5-DTBQ-Na]+. The remaining peak at m/z = 721.80 including
two additional peaks at m/z = 753.35 and 1197.30 are observed, which clearly indicate that these
peaks arise from complex-substrate aggregates and can be assigned to {Na-[CuI(HL)(Hdpa)(3,5-
DTBCH)]}+, {Na-[CuII(HL)(Hdpa)(O2)(3,5-DTBCH)]}+ and {Na-[CuII(HL)(Hdpa)]-(3,5-
DTBC)-[CuII(HL)(Hdpa)]}3+ respectively.
4. Conclusion
Most noteworthy about the catalytic results is the fact that the efficient catalysis is
influenced by square pyramidal (4+1) coordination geometry with the labile binding site of the
complex. It is also assumed that a close steric match between the substrate and the copper ion is
of crucial importance for efficient catalysis to occur [40]. It has been shown that electron transfer
from substrate to copper(II) can begin only after the substrate and copper(II) species form
copper(II)-substrate intermediate like ‘enzyme-substrate’ complex [41]. The copper(II) complex
(1) possesses square pyramidal (4+1) coordination geometry and its crystal packing reveals the
formation of the centrosymmetric dimer held together by self-assembled molecular association
(N-HO and inter-pair -). Interestingly, the existence of the dimeric intermolecular
association in solution is evidenced by the frozen EPR and ESI-MS solution studies. The dimeric
aggregate of the complex does not have the steric match between the bulky ascorbic acid and
copper(II) ion, which prevents the formation of copper(II)-ascorbate intermediate. However, the
enhanced catalytic activity takes place in the case of compact benzylamine or catechol with 1
due to the easy formation of copper(II)-substrate intermediate, indicating the close steric match
between the substrate and the catalyst. Thus, the self-assembled molecular association
determines that 1 behaves as a functional model for amine and catechol oxidases but not for
ascorbate oxidase.
Acknowledgments
We thank the Science and Engineering Research Board, New Delhi (Grant No.
EMR/2016/007756) for financial support. Thanks are due to STIC, Cochin University of Science

and Technology for X-ray crystal structure data. We thank SAIF, Indian Institute of Technology
Madras for structure solution and refinement (Dr. P. K. Sudhadevi) and for recording EPR
spectra. This paper is dedicated to Dr. R. Brakaspathy, (Former Secretary, SERB, New Delhi,
India) Research and Development Advisor, Indian Institute of Technology Madras, Chennai,
India on the occasion of his 62nd birthday.
Appendix A. Supplementary data
Crystallographic data for the structural analysis of [Cu(L)(Hdpa)](ClO4) (1) have been deposited
with Cambridge Crystallographic Data Center, CCDC No. 1861819. Copies of this information
may be obtained free of charge from http://www.ccdc.ac.uk/const/retrieving.html or from the
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (email: deposit@ccdc.cam.ac.uk).
Supplementary data associated with this article can be found, in the online version, at …….
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H I G H L I G H T S
 Possess square pyramidal (4+1) coordination geometry
 Display two types of self-assembled intermolecular associations
 No catalytic activity towards the oxidation of ascorbic acid
 Effective catalyst towards the oxidation of benzylamine and 3,5-di-tert-butylcatechol
 Functional Model for Cu(II) sites in type-2 and type-3 copper oxidases
Graphical Abstract
Mononuclear copper(II) complex [Cu(L)(Hdpa)](ClO4) possesses CuN4O chromophore. It shows
good catalytic activity towards the oxidation of benzylamine and 3,5-di-tert-butylcatechol due to the
existence of self-assembled molecular association, in turn, favors steric match between catalyst and
substrate.
Amine oxidase
Catechol oxidase
Ascorbate oxidase

Legends for figures
Scheme 1 Structure of ligands (H(L) obtained from the condensation of 2-hydroxy-1-
naphthaldehyde and N,N-dimethylethylenediamine and Hdpa is 2,2’-dipyridylamine)
Fig. 1 (a) ORTEP diagram of [Cu(L)(Hdpa)]+ (1) showing 40% probability thermal
ellipsoids with the atom labeling scheme for the metal, heteroatoms and disorded C(1),
C(1)’, C(2), C(2)’, C(3), C(3)’, C(4), C(4)’ atoms in N,N-dimethylethylenediamine moiety
and (b) Packing diagram viewed down the a-axis showing intermolecular interactions
(Symmetry code: -x+1, -y, -z+2) of [Cu(L)(Hdpa)]+ (Green, N-H∙∙∙O bonding; Red, π-π
stacking). The disordered perchlorate anion is omitted for clarity.
Fig. 2 EPR spectra for frozen solutions of [Cu(L)(Hdpa)](ClO4) (1) at 77K: (a) DMF, (b) MeOH
and (c) MeCN.
Fig. 3 Visible spectral traces showing the conversion of [Cu(L)(Hdpa)](ClO4) (1) (3 × 10-3 M)
(a) to (b) on coordination with ascorbic acid (3 × 10-3 M) in MeOH:H2O (4:1 v/v).
Inset: No conversion of the copper(I) species.
Fig. 4 Visible spectra of [Cu(L)(Hdpa)](ClO4) (1) in MeOH:H2O (4:1 v/v) (a), in presence of
benzylamine (b) and after treatment with benzylamine and H2O2 (c).
Fig. 5 Oxidation of 3,5-DTBCH2 by [Cu(L)(Hdpa)](ClO4) (1) in MeOH monitored by UV-Vis
spectroscopy.
Fig. 6 Dependence of reaction rates on the 3,5-DTBCH2 concentration for the oxidation reaction
catalyzed by [Cu(L)(Hdpa)](ClO4) (1). Inset: Lineweaver-Burk plot.
Fig. 7 ESI-Mass spectrum recorded for the reaction between 3,5-DTBCH2 and 1, after 30
minutes.

Scheme 1
H
N
N
N
N
N
OH
Hdpa
H(L)

Fig. 1
(a)
(b)

Fig. 2
a
b
g
gII
giso
c
giso
3000
2500
3500
4000
H (G)

Fig. 3
1.0
0.5
0.0
1.5
1.0
0.5
0.0
649 nm a
450
600
750
Wavelength (nm)
634 nm b
450
600
750
Wavelength (nm)

Fig. 4
1.0
0.5
0.0
c
a
b
450
600
750
900
Wavelength (nm)

Fig. 5
0.6
0.4
0.2
0.0
3,5-DTBQ
complex
3,5-DTBCH2
400
300
500
Wavelength (nm)

Fig. 6
8
6
4
2
0
0.30
0.25
0.20
0.15
0.0
0.2
0.4
0.6
0.8
1/[substrate] 10-4 (M-1)
0
5
10
15
[substrate] 10-4 (M)

Fig. 7

Table 1
Selected crystal data and structure refinement parameters for [Cu(L)(Hdpa)](ClO4) (1)
Formula
C25H26ClCu N5O5
575.50
296(2)
0.71073
Monoclinic
P21/c
9.0434 (2)
17.7067 (5)
15.8463 (4)
90
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
 ()
 ()
 ()
100.2410 (10)
90
2497.02 (11), 4
1.531
1.029
V (Å)3, Z
Dcalc (g cm-3)
µ (mm-1)
F(000)
1188
Crystal size (mm)
 ()
Index ranges
0.30 × 0.25 × 0.20
2.56-28.17
-11h9,
-23k23,
-20l20
28648
6049
4513
0.0324
Reflections collected
Independent reflections
Reflections observed [I > 2(I)]
Rint
GOOF
1.015
R1 [I > 2(I)]
wR2 [I > 2(I)]
R1/wR2 all data
0.0359
0.0951
0.0564/0.1070
Table 2
Selected bond lengths (Å) and bond angles (°) for [Cu(L)(Hdpa)](ClO4) (1)
Cu(1)-N(1)
Cu(1)-N(3)
Cu(1)-O(1)
1.9230(18)
1.9979(18)
1.9567(15)
Cu(1)-N(2)
Cu(1)-N(5)
2.1338(19)
2.1805(18)
O(1)-Cu(1)-N(1)
O(1)-Cu(1)-N(3)
91.04(7)
88.54(7)
O(1)-Cu(1)-N(2)
O(1)-Cu(1)-N(5)
160.32(7)
103.05(7)

N(1)-Cu(1)-N(2)
N(1)-Cu(1)-N(5)
N(2)-Cu(1)-N(5)
83.38(8)
103.26(7)
96.60(7)
N(1)-Cu(1)-N(3)
N(2)-Cu(1)-N(3)
N(3)-Cu(1)-N(5)
167.40(8)
92.85(7)
89.09(7)