Protein binding, DFT/TDDFT calculation and catecholase activity of five coordinated distorted square pyramidal/trigonal bipyramidal Cu(II) complexes

Article abstract of DOI:10.1016/j.poly.2018.04.008

Two Cu(II) complexes, [Cu(L¹)(OCN)(H₂O)] (1) and [Cu(L²)]·H₂O (2) [HL¹, bis(2-pyridyl carbonyl)amine; H₂L², 2{3-[5-(2-hydroxy-3methoxy)-pent-4-enylamino]-propylimino}methyl-6-methoxy phenol], were synthesized and characterized by single crystal X-ray crystallography, thermal analysis, FTIR and mass spectroscopic techniques. Complexes 1 and 2 show a square pyramidal and trigonal bipyramidal Cu(II) coordination sphere, respectively. In the solid state complex 1 forms hydrogen bonded 1D supramolecular structure and complex 2 forms 1D supramolecular structure with C–H…π interactions. Results of experimental electronic absorption and FTIR spectral studies have been compared with the calculated results obtained by density functional theory (DFT) and time dependent density functional theory (TD-DFT) using the CAM-B3LYP functional, with LanL2DZ basis set. Catecholase activity of the compounds have been studied using 3,5-di-tert butyl catechol (3,5-DTBC) as model substrate and found that both of these are active for catalyzing oxidation of 3,5-DTBC to 3,5-di-tert butyl benzoquinone (3,5-DTBQ) in presence of molecular oxygen. Calculated values of the turnover numbers (K_cat) are 29.2 ± 3 and 112.14 ± 5 h^?1 for 1 and 2, respectively. The interactions of complexes 1 and 2 with bovine serum albumin (BSA) and human serum albumin (HSA) were studied using electronic absorption and fluorescence spectroscopic methods and the results show that both the compounds interact with BSA/HSA with ground state association process.

Full text of DOI:10.1016/j.poly.2018.04.008

Accepted Manuscript
Protein binding, DFT / TDDFT calculation and catecholase activity of five co-
ordinated distorted square pyramidal / trigonal bipyramidal Cu(II) complexes
Kalipada Das, Samar Dolai, Pavel Vojtí šek, Subal Chandra Manna
PII:
S0277-5387(18)30175-X
https://doi.org/10.1016/j.poly.2018.04.008
POLY 13109
DOI:
Reference:
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3 April 2018
Please cite this article as: K. Das, S. Dolai, P. Vojtí šek, S.C. Manna, Protein binding, DFT / TDDFT calculation
and catecholase activity of five coordinated distorted square pyramidal / trigonal bipyramidal Cu(II) complexes,
Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.04.008
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Protein binding, DFT / TDDFT calculation and catecholase activity of five
coordinated distorted square pyramidal / trigonal bipyramidal Cu(II)
complexes
Kalipada Das,a Samar Dolai,a Pavel Vojtíšek,b Subal Chandra Manna*, a
aDepartment of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, India,
E-mail: scmanna@mail.vidyasagar.ac.in, Fax: (91) (03222) 275329, Tel. +91 9433414858.
bDepartment of Inorganic Chemistry, Charles University in Prague, Hlavova 2030/8, 128 43 Prague 2,
Czechia.
____________________________________________________________________________________
ABSTRACT
Two Cu(II) complexes, [Cu(L1)(OCN)(H2O)] (1) and [Cu(L2)]·H2O (2) [HL1, bis (2-pyridyl
carbonyl) amine; H2L2, 2{3-[5-(2-hydroxy-3methoxy)-pent-4-enylamino]-propylimino}methyl-
6-methoxy phenol], were synthesized and characterized by single crystal X-ray crystallography,
thermal analysis, FTIR and mass spectroscopic techniques. Complexes 1 and 2 show a square
pyramidal and trigonal bipyramidal Cu(II) coordination sphere, respectively. In the solid state
complex 1 form hydrogen bonded 1D supramolecular structure and complex 2 form 1D
supramolecular structure with C-H…π interactions. Results of experimental electronic
absorption and FTIR spectral studies have been compared with the calculated results obtained
by density functional theory (DFT) and time dependent density functional theory (TD-DFT)
using the CAM-B3LYP functional, with LanL2DZ basis set. Catecholase activity of the
compounds have been studied using 3,5-di-tert butyl catechol (3,5-DTBC) as model substrate
and found that both of these are active for catalyzing oxidation of 3,5-DTBC to 3,5-di-tert butyl
benzoquinone (3,5-DTBQ) in presence of molecular oxygen. Calculated values of the turnover
numbers (Kcat) are 29.2  3 and 112.14  5 h-1 for 1 and 2, respectively. The interactions of
complexes 1 and 2 with bovine serum albumin (BSA) and human serum albumin (HSA) were
studied using electronic absorption and fluorescence spectroscopic methods and the results show
that both the compounds interacts with BSA/HSA with ground state association process.
1

__________________________________________________________________________
Keywords: Cu(II) complex; crystal structure; DFT; serum albumin binding studies; catecholase
activity.
________________________________________________________________________
1. Introduction
Schiff base ligands are important in the medicinal chemistry due to their potential
chelating ability and diverse biological, pharmacological and antitumor activities [1]. Some
Schiff bases show greater biological activity when form complex with metal than as free organic
ligand [2]. Among the 3d metal Schiff base complexes, copper(II) Schiff base complexes are
important in medicinal inorganic chemistry due to their various geometries and coordination
numbers, various oxidation states, better solubility, higher affinity for the nucleobases [3].
Literature survey reveals that copper Schiff base complexes potentially show anticancer,
antibacterial, antiproliferative, and antimitotic activities. Moreover it is interesting to note that
some Schiff base copper complexes show relatively lower toxicity in comparison to well known
platinum compounds [4-7].
Serum albumin is the major soluble protein in human body which functions as carrier
and depositor of compounds like drugs, amino acids, fatty acids and pharmaceuticals etc.[8].
Therefore to develop efficient copper metal based pharmaceuticals, the study of kinetics of
interactions of copper metal complex and serum albumin is very important [9]. In addition to
the wide biological activities, Schiff base coordinate copper(II) complexes also function as
model compounds for showing catecholase activity [10]. On the other hand to explain electronic
and spectroscopic properties of coordination compounds, theoretical calculations implementing
density functional theory (DFT) and time dependent density functional theory (TD-DFT) are
widely used [11]. Inspite of potential utility of DFT, the use of DFT computation to explain
electronic properties of copper (II) complexes are less documented in the literature [12].
2

The present contribution reports the synthesis, crystal structure, kinetics of interaction
with serum albumins, DFT/TD-DFT calculation, catecholase activity of two distorted square
pyramidal / trigonal bipyramidal Cu(II) complexes, [Cu(L1)(OCN)(H2O)] (1) and [Cu(L2)]·H2O
(2).
2. Experimental
2.1.
Materials and physical measurements
High purity bis(3-aminopropyl) amine, pyridine-2-carboxylic acid, 2-pyridinecarboxamide,
bovine serum albumin, human serum albumin and 3,5-di tertiary butylcatechol were purchased
from Aldrich chemical Company. All other chemicals used were of analytical grade. Solvents
used for spectroscopic studies were purified and dried by standard procedures immediately
before use [13].
Elemental analyses (C, H and N) were performed using a Perkin-Elmer 240C elemental
analyzer. IR spectra were recorded as KBr pellets on a Bruker Vector 22FT IR
spectrophotometer operating from 400 to 4000 cm–1. Electronic absorption spectra were
obtained in methanol with a Shimadzu UV-1601 UV-vis spectrophotometer at room
temperature. Quartz cuvettes with a 1 cm path length and a 3 cm3 volume were used for all
measurements. Emission spectra were recorded on a Hitachi F-7000 spectrofluorimeter. Room
temperature (300 K) spectra were obtained in solution using a quartz cell of 1 cm path length.
The slit width was 5 nm for both excitation and emission. ESI-MS spectra of the compounds
were recorded in acetonitrile solution. Mass spectra were recorded on a Q-tof-micro quadruple
mass spectrometer by electron spray ionization (ESI) technique and the software used for mass
analysis is Mass hunter. Thermal analysis was done on a Shimadzu TGA 60 instrument under a
nitrogen environment.
3

2.2.
Synthesis
Caution! Metal perchlorate was used in small quantities and handled with care since
explosion may occur.
The complexes have been synthesized by adopting the procedure schematically given in Scheme
1.
Scheme 1. Synthesis of 1 and 2.
2.2.1. [Cu(L1)(OCN)(H2O)] (1)
The tridentate ligand, bis (2-pyridyl carbonyl) amine (HL1), was prepared by refluxing 2-
pyridine carboxamide (1 mmol; 0.122 g) and pyridine 2-carboxylic acid (1 mmol, 0.123 g) in
methanol (20 mL) according to literature method [14]. A methanolic solution (10 mL) of
copper(II) perchlorate hexahydrate (1 mmol; 0.371 g) was added to methanolic solution (20 mL)
of tridentate ligand HL1 (1 mmol) and the resulting clear deep green solution was stirred for 1
hour. An aqueous solution (5 mL) of sodium cyanate (1 mmol; 0.065 gm) was added dropwise
to it with continuous stirring and the resulting reaction mixture was stirred for additional 2 hours
at room temperature (25 ºC) and filtered. The filtrate was left for slow evaporation at room
temperature. Suitable green single crystals for structure determination were obtained from the
4

filtrate after a few days. Yield 81 %. Anal. Calc. C13H10CuN4O4 (349.80): C, 44.59; H, 2.85; N,
16.00 %. Found: C, 44.62; H, 2.89; N, 16.03 %. IR (cm-1): 3502 (s), 3031 (s), 2090 (vs), 1739
(s), 1699 (vs), 1636 (s), 1601 (s), 1569 (s), 1558 (s), 1556 (s), 1521 (vw), 1507 (vw), 1478
(vw), 1449 (s), 1365 (vs), 1349 (s), 1269 (w) , 1253 (w), 1228 (vw), 1217 (vw), 1159 (s), 1096
(s), 836 (w), 824 (w), 802 (s), 756 (vs), 702 (vs), 657 (s), 650 (s), 634 (vw), 549 (vw), 490 (s),
471 (vw). ESI-MS, in CH3CN, m/z: 350.89 [Cu(OCN)(L1)(H2O)+H]+, 332.92
[Cu(OCN)(L1)+H]+ and 288.94 [Cu(L1)]+. The ESI-MS spectrum of complex 1 is shown in
Fig.1S.
2.2.2. [Cu(L2)]·H2O (2)
The pentadentate Schiff base (H2L2) was prepared by refluxing 2-hydroxy-3-
methoxybenzaldehyde (2 mmol; 0.304 g) and bis(3-aminopropyl) amine (1 mmol; 0.130 g) for
1 h, in methanol (10 mL). A methanolic solution (5 ml) of Cu(ClO4)2·6H2O (1 mmol; 0.371g)
was added drop wise to a methanolic solution (10 ml) of mixture of H2L2 (1 mmol; 0.399 g) and
triethylamine (TEA) (2 mmol) with constant stirring at room temperature (25 ºC). The resulting
clear deep green mixture was stirred for 1.5 hours and filtered. Suitable shining green single
crystals for X-ray structural determination were obtained after a few days. Yield: 80 (%). Anal.
Calc. for C22H29CuN3O5 (479.02): C, 55.11; H, 6.05; N, 8.76 %. Found: C, 55.14; H, 6.03; N,
8.79 (%). IR (cm-1): 3432 (b), 2988 (vw), 2130 (vw), 1678 (w), 1634 (s), 1550 (s), 1468 (w),
1410 (s), 1373 (vw), 1304 (s), 1219 (s), 1164 (w), 1077 (w), 884 (vw), 749 (vw), 666 (vw), 460
(vw), 409 (vw). ESI-MS, in CH3CN, m/z: 480.16 {[Cu(L2)]·H2O + H}+, 462.16 [Cu(L2) + H]+
and 400.24 (H2L2 + H)+. The ESI-MS spectrum of complex 2 is shown in Fig.2S.
2.3.
Crystallographic data collection and refinement
Data collection for compounds 1 and 2 were carried out at 150(2) K with Mo-Kα radiation (λ =
0.71073 Å) on a Bruker Smart Apex diffractometer equipped with CCD. Cell refinement,
5

indexing and scaling of the data sets were done by using Bruker Smart Apex, BrukerSaint
packages [15] and CrysAlisPro program [16]. The structures were solved by direct methods and
subsequent Fourier analyses [17] and refined by the full-matrix least-squares method based on
F2 with all observed reflections [18]. All hydrogen atoms for the compounds were placed at
calculated positions. The publishing data were prepared by using the WinGX System, Ver
1.80.05 [19] and Diamond [20]. Crystal data and details of refinements of the structures reported
are given in Table 1.
2.4. Theory and computational methods
All computations were performed using the Gaussian 09 (G 09) software package [21]. by using
the by using the Coulomb-attenuating method Becke’s three-parameter hybrid exchange and the
Lee-Yang-Parr non-local correlation functional (CAM-B3LYP) [22]. In the calculation 6-31G
(d-p) basis set was assigned to all elements with the exception of copper for which the Los
Alamos effective core potentials plus the Double Zeta (LanL2DZ) [23] basis set were used. The
ground state (doublet) structurees of the complexes were entirely optimized at the CAM-B3LYP
level. The vibration frequency calculations were performed to make sure that the optimized
geometries correspond to local minima associated with positive eigen values only.
Time-dependent density functional theory (TD-DFT) method [11d,11e] in methanol, using the
conductor-like polarisable continuum model (CPCM) [24] was employed to get theoretically
possible vertical electronic excitations based on CAM-B3LYP functional. GaussSum [25] was
used to calculate the fractional contributions of various groups to each molecular orbital.
Calculated coordination geometries of complexes are shown in Table 2.
2.5. Protein binding studies
Stock solutions human serum albumin (HSA, 7.69 × 10-7 M) and bovine serum albumin
(BSA, 8.9 × 10-7 M) were prepared in 55 mM HEPES buffer (pH 7.2). Aqueous solution of the
6

copper(II) compounds were prepared by dissolving the compounds in water: HEPES buffer
(1:99). The interactions of compounds with serum albumins were studied by recording room
temperature tryptophan fluorescence of HSA / BSA. Fluorescence spectra were recorded in the
range 290 - 450 nm at an excitation wavelength of 280 nm. To the solutions of serum albumins,
copper(II) compounds were added, and the quenching of emission intensities at 340 nm (λex, 280
nm) were recorded after gradual addition (10 µL, 0.11 mM) of aqueous solution of compounds.
The Stern-Volmer constants (Ksv) were calculated [26] using the equation I0/I = 1 + Ksv
[complex], where I0 and I are the fluorescence intensities in the absence and in the presence of
the complex, respectively. To calculate the binding constant of the compound with serum
albumins, following Scatchard equation [27] have been used,
log[(Io–I)/I] = log Kb + n log[complex], where Kb is the binding constant of the
compound with serum albumin and n is the number of binding sites per albumin.
3. Results and discussion
3.1.
Molecular structures of [Cu(L1)(OCN (H2O)] (1) and [Cu(L2)]∙H2O (2)
Crystal structure determination reveals that both 1 and 2 are mononuclear, five-coordinated
Cu(II) complexes. The ORTEP drawing of the metal centers of 1 and 2 with the atom numbering
scheme are shown in Figs.1 and 2, while selected geometrical parameters are given in Table 2.
The complex 1 crystallizes in the centrosymmetric space group P-1 and the copper atom exhibits
a slightly distorted square pyramidal geometry with a N4O chromophore environment. The
metal is coordinated by tridentate (N, N, N) chelating ligand (L1)ˉ, and completes the distorted
square pyramidal geometry with one cyanato nitrogen atom and another oxygen atom comes
from coordinated water molecule. The N1, N2 and N3 donors of chelating ligand (L1)ˉ and
cyanato N21 define the equatorial plane of distorted square pyramid. The axial position is
7

occupied by the oxygen atom O(99), from coordinated water molecule. The axial Cu-O(99)
bond length (2.3855(14) Å) is longer in comparison to equatorial bonds (1.9213(18) to
2.0238(14) Å; Table 2) and this is probably due to the Jahn-Teller distortion. The bond angles
between the successive coordinating equatorial atoms with central atom are 80.83(6), 81.47(6),
98.06(6) and 98.95(7)°, and the angle between axial atom, central atom and equatorial atoms
are 91.55(5), 95.41(6), 93.17(5) and 93.52(6)°. Complex 1 form 1D supramolecular structure
2
2
2
(Fig 3) with R4 (8), R2 (12) and R1 (6) H-bonding synthons (dH…A = 2.06, 2.13 and 2.41 Å;
Table 1S). The crystallographic independent unit (Fig.2) of complex 2 comprises the metal unit
[Cu(L2)] and a lattice water molecule, and crystallizes in the the non-centrosymmetric space
group Pna21. The copper atom is coordinated by penta-dentate (O, O, N, N, N) chelating Schiff
base (L2)2- and form slightly distorted trigonal bipyramidal geometry. Cu-O /Cu-N bond lengths
fall in the range 1.9869(18) to 2.138(2) Å (Table 2). The Schiff base O1, O2 and N2 donors
define the triagonal plane whereas the axial positions are occupied by the N1 and N3 atoms of
the Schiff base. The bond angles between the successive coordinating equatorial atoms with
central atom are 112.79(8), 122.8(8) and 124.06(8)°, the angle between axial atom N1, central
atom and equatorial atoms are 88.11(7), 88.15(7) and 87.84(8)° and the angle between axial
atom N3, central atom and equatorial atoms are 97.85(8), 89.65(7) and 88.56(9)°. The bond
angle between two axial atoms and central atom is 173.88(9)°. Complex 2 form 2D
supramolecular architecture (Fig. 4) through C-H…π interactions (Table 2S) [28].
To evaluate the deviations of structures from regular trigonal bipyramidal (TBP) and square
pyramidal (SP) geometries, the trigonality τ parameters [29] were calculated as (α-β)/60, where
α and β are the two largest coordination bond angles. Hence a regular TBP structure with D3h
symmetry has τ = 1 and for a regular C4v SP geometry τ = 0. Calculated τ values were 0.1344
and 0.6938 for complexes 1 and 2, respectively. The calculated results clearly confirms the
presence of distorted square pyramidal and trigonal bipyramidal geometries for complexes 1,
and 2, respectively.
8

3.2.
IR spectral studies
IR spectra of complexes 1-2 are shown in Figs. 3S-4S and the more significant bands are
summarized in Tables 3S-4S. Broad bands in the region 3300 – 3600 cm-1 (for 1) and 3100 -
3600 cm-1 (for 2) are assigned to be stretching vibrations of water molecules [30]. The
weak bands in the region 2988 - 3031 cm-1 correspond to aromatic (C-H) stretching vibrations
in 1-2, and imine Csp2-H stretching vibration in 2. For complex 2, the band at 2910 cm−1 is due
to the ν(Csp3−H) stretching vibration. Aromatic (C=C) and aliphatic (C=N) stretching
vibrations are appears in the region 1349-1569 cm-1 and 1373-1550 cm-1, for 1 and 2,
respectively. For complexes 1 and 2, the bending vibration of water molecule,
appears at 1636 and 1634 cm-1, respectively. For complex 1, stretching vibrations at
2090 [for υ(CN)], 1365 [for υ(CO)] and 634 cm-1 [for δ(NCO)] corroborate the presence of N-
coordinated cyanato ligand [30].
3.3.
ESI mass spectrometry
The ESI mass spectra (Figs 1S and 2S) of complexes 1 and 2 were recorded using acetonitrile
solutions. The ESI mass spectrum of complex 1 displays peaks at m/z = 350.89, 332.92 and
288.94, corresponding to [Cu(OCN)(L1)(H2O) + H]+ (calc. m/z = 350.0), [Cu(OCN)(L1) + H]+
(calc. m/z = 331.99) and [Cu(L1)]+ (calc. m/z = 288.99). On the other hand for 2, the peaks are
appeared at 480.16, 462.16 and 400.24, corresponding to {[Cu(L2)]·H2O + H}+ (calc. m/z =
479.15), [Cu(L2) + H]+ (calc. m/z = 462.15) and (H2L2 + H)+ (calc. m/z = 400.22). This result
evidence that in methanolic solution, the penta coordinated coordination environment of Cu(II)
centers of both the complexes are same as observed in their solid state structures.
9

3.4.
Geometrical optimization and electronic structure
DFT and TD-DFT calculations of optimized structures of [Cu(L1)(OCN)(H2O)] and [Cu(L2)]
(complex 2, excluding lattice water molecule) were performed to explain their electronic
structure and spectral transitions. The complexes 1 and 2 were fully optimized at the Coulomb-
attenuating method Becke’s three-parameter hybrid exchange and the Lee-Yang-Parr non-local
correlation functional (CAM-B3LYP) level in the ground state (doublet). The optimized
structures of ligands and complexes along with their Mulliken charge distribution are shown in
Figs.5S-7S. The theoretically possible spin-allowed (doublet-doublet) electronic transitions with
the their assignments are listed in Table 3. Calculated bond lengths and angles along with the
crystallographically determined bond lengths and angles along for 1 and 2 shows sufficient
agreement between calculated and experimental values (Table 2). Calculated trigonality τ
parameters for complexes 1 and 2 are 0.1344 and 0.6938, respectively. Theoretically calculated
τ parameters corroborate crystallographically characterized square pyramidal and trigonal
bipyramidal geometries in complexes 1 and 2, respectively. The orbital diagram along with
their energies and contributions from the ligands and metal, are given in Tables 5S-8S and Figs.
5 and 8S-11S. For α-MOs of 1, the energies of highest singly occupied molecular orbital
(SOMO) and lowest unoccupied molecular orbital (LUMO) are - 8.63 eV and - 1.45 eV,
respectively. Whereas for complex 2, corresponding values are - 6.57 eV and - 0.32 eV,
respectively. For β-MOs of 1, the energies of SOMO and LUMO are - 8.56 eV and - 1.44 eV,
respectively, and for 2 the corresponding values are - 6.53 eV and - 0.75 eV, respectively. This
results evidence that SOMO-LUMO energy difference of α-MOs are larger [ΔE = 7.18 eV (for
1); 6.25 eV (for 2)] compare to corresponding values of β-MOs [ΔE = 7.12 eV (for 1); 5.78 eV
(for 2)]. For complex 1, contribution of chelating ligand (L1), cyanato (OCN), water (H2O) and
copper (Cu) to SOMO and LUMO of α-MOs are [7 % L1, 89 % OCN, 0 % H2O, 4% Cu] and
10

[100 % L1, 0 % OCN, 0 % H2O, 0% Cu], respectively. For β-MOs corresponding values are [6
% L1, 89 % OCN, 0 % H2O, 5% Cu] and [99 % L1, 0 % OCN, 0 % H2O, 1% Cu], respectively.
For 2, contribution of Schiff base (L2) and copper to both SOMO and LUMO of α-MOs is [99 %
L2, 1 % Cu] and [98 % L2, 2 % Cu] respectively, whereas for SOMO and LUMO of β-MOs are
[98 % L2, 2 % Cu] and [32 % L2, 68 % Cu], respectively. In summary for complex 1, SOMO of
α-MO and β-MOs are characterized by the cyanato orbitals (89 %), while LUMO of α-MO and
β-MO are characterized by the chelating ligand (L1) orbitals (≥ 99 %). On the other hand for 2,
SOMO and LUMO of α-MO, and SOMO β-MO are characterized by the Schiff base (L2)
orbitals, but LUMO β-MO is characterized by both Schiff base (L2) and copper orbitals. From
TD-DFT calculations, the theoretically possible spin allowed doublet–doublet electronic
transitions of complexes 1 and 2 with their assignment are listed in Table 9S. For 1, the TD-
DFT results show that experimental electronic transition (in methanol) at 210 nm may be assign
as SOMO -7 (β) → LUMO (β) transition. On the other hand for 2, spectral transition at 248 nm
is due to HOMO-3 (β) → LUMO+1 (β) transition (Table 3). Both these transitions are intra
ligand charge transfer transition (in chelating ligand) in nature.
3.5.
Catechol oxidase studies and kinetics
The catecholase activities of complexes 1 and 2 have been investigated according to the
following reaction, using 3,5-di-tert-butyl catechol (3,5-DTBC) as model substrate in oxygen
saturated methanol medium. In this reaction, complexes 1 and 2 function as catalyst:
O
O
O H
O H
c a t., O 2
m e th a n o l
3 ,5 -D T B C
3 ,5 -D T B Q
At room temperature 1×10-4 M methanolic solution of complexes were added to the 1 × 10-2 M
methanolic solution of 3,5-DTBC. The UV-vis spectra of the mixture were recorded at a six
11

minute interval for up to 1 hour. A new band at 400 nm corresponding to 3,5-di-tert-
butylquinone (3,5-DTBQ) [31] appeared after addition of complexes to the solution of 3,5-
DTBC (Fig.6). log[(A∞-Ao)/(A∞-At)] vs time (t) plots (where A∞ and At are the absorbance at
infinite time and ‘t’ time, respectively) give straight lines (Fig. 7) passing through the origin
with 9.26 ×10-3 and 8.08 ×10-3 min-1 as slope, for complexes 1 and 2, respectively. The rate
constants for complex-substrate mixture were determined from the slope of these curves and the
calculated values were 9.26 ×10-3 and 8.08 ×10-3 min-1, for 1 and 2, respectively. Fig.8, shows
the change of rate of reaction with concentration of substrate. From this figure it is clear that at
low concentration of 3,5-DTBC a first order dependence of the rate on the substrate
concentration were observed, whereas a saturation kinetics were observed at higher
concentration of 3,5-DTBC. Since complex showed saturation kinetics, we have used Michaelis-
Menten model for this system. The values of the Michaelis binding constant (KM), maximum
velocity (Vmax) and the rate constant for dissociation of substrates (i.e., turnover number, Kcat)
were calculated for complex from the graph of vs
Burk graph using the equation
(inset of Fig.8), known as Lineweaver-
and the kinetics parameters are given in Table 4.
The turnover numbers (Kcat) were calculated by dividing Vmax with concentration of complexes
and calculated values of turn over numbers are 29.2  3 and 112.14  5 h-1 for 1 and 2,
respectively, which are relatively small to those reported values for copper(II) complexes [32].
From the above result it is clear that in between two five coordinated complexes, compound
having triagonal bipyramidal geometry shows higher rate of catalytic activity than that of
compound having square pyramidal geometry.
12

3.6.
Protein binding studies
Fig.9 and Fig.12S show the change of electronic absorption spectra of Bovine Serum Albumin
(BSA) (3 ml, 8.89 × 10-7 (M) aqueous solution) and Human Serum Albumin (HSA) (3 ml, 7.69
× 10-7 (M) aqueous solution) in presence of different concentration of complexes at pH 7.2
(using HEPES buffer). In presence of complexes, the absorption intensities of BSA and HSA
were enhanced with a little blue shift. For BSA, blue shifting occur by 6 nm (280 nm → 274
nm) and 11 nm (280 nm → 269 nm) with gradual addition of 10 µL 1.1×10-4 (M) aqueous
solutions of complexes 1 and 2, respectively and finally concentration of complexes in solution
mixture bebome 5.44×10-6 M (for 1) and 4.97×10-6 M (for 2). On the other hand, for HSA, blue
shift of spectral band observed by 6 nm (280 nm → 274 nm) and 13 nm (280 nm → 267 nm)
with gradual addition of 10 µL 1.1×10-4 (M) aqueous solutions of complexes 1 and 2,
respectively, and and finally concentration of complexes in solution mixture bebome 5.44×10-6
M (for 1) and and 4.01×10-6 M (for 2). Blue shift at 280 nm confirms the ground state
association of complexes 1 and 2 with serum albumins. The apparent association constants
(Kapp) were calculated adopting the following equation
where Aobs is the observed absorbance at 280 nm of the solution containing different
concentrations of the complex, A0 is the absorbance of serum albumin only and Ac is the
absorbance of serum albumin with complexes. The calculated values of apparent association
constants are 0.429 L mol-1 (for 1-BSA), 0.614 L mol-1 (for 1-HSA), 0.554 L mol-1 (for 2-BSA),
and 0.859 L mol-1 (for 2-HSA).
To examine the interaction of serum albumin (SA) with complexes 1 and 2, the fluorescence
spectroscopic studies were carried out. In aqueous solution (pH 7.2, HEPES buffer) complex 1
exhibits fluorescence (λem) at 335 nm, and 398 nm; complex 2, exhibits fluorescence (λem) at
13

309 nm, and 469 nm at room temperature upon excitation (λex) at 280 nm. On the other hand
both BSA and HSA exhibit luminescence at 330 nm, on excitation at 280 nm (in aqueous
solution, pH 7.2, HEPES buffer) at room temperature. The change of the fluorescence spectra of
BSA [3 ml 8.89 × 10-7 (M) aqueous solution] and HSA [3 ml 7.69 × 10-7 (M) aqueous solution]
uopn the addition of increasing concentration of complexes 1 and 2 are shown in Figs.10 and
13S. Upon gradual addition of 10 µL 1.1×10-4 M aqueous solutions of complexes to the solution
of serum albumins, significant decrease in fluorescence intensities (up to 50 and 26 % for BSA
and HSA, respectively, with complex 1; up to 58 and 50 % for BSA and HSA, respectively,
with complex 2) were observed at 330 nm (Figs.10 and 13S). The final concentration of
complex 1 in solution mixture became 3.33×10-6 M (for BSA titration) and 2.24×10-6 M (for
HSA titration) and for 2 the final concentration became 2.37×10-6 M (for BSA titration) and
3.03×10-6 M (for HSA titration). It is interesting to note that, in addition to fluorescence
quenching, slight red shifting of 330 nm fluorescence bands were also observed.
The observed quenching of fluorescence intensities of serum albumins with addition of
complexes mainly due to association of serum albumins to complexes, serum albumins
denaturation or conformational change of the serum albumins [33]. Insets of Figs.10 and 13S
show linear Stern-Volmer relationships [26] (R = 0.9993 for 1-BSA curve; R = 0.9976 for 1-
HSA curve; R = 0.9967 for 2-BSA curve; R = 0.9980 for 2-HSA curve) for the titration of
serum albumins using the complexes 1 and 2 as quencher. The calculated values of Stern-
Volmer quenching constants (Ksv) are (33.360.4) × 104 L mol-1 (for 1-BSA), (63.74±2.3) × 104
L mol-1 (for 2-BSA), (16.0 ±0.4) × 104 L mol-1 (for 1-HSA) and (34.6±0.8) × 104 L mol-1 (for 2-
HSA). The Ksv values indicate that complexes have good serum albumin fluorescence
quenching ability. From the UV-vis spectral study it is found that the absorption spectra of
BSA/HSA influenced by the addition of complexes. This phenomenon evidence the existence of
static interaction between BSA/HSA and complexes.
14

Since complexes 1 and 2 bind independently in a static mode to BSA/HSA, Scatchard equation,
[27]
log [(I0 – I)/I ] = logkb + n log [complex]
(where kb is the binding constant of complex with serum albumin, n is the number of binding
sites per serum albumin molecule) has been used to calculate number of binding sites of serum
albumins. The plots of log (I0 - I)/I vs log[complex] give straight lines (Figs.14S-15S) with n
and logkb as slope and the intercept, respectively. Calculated values of kb and n are given in
Table 5. The values of n for BSA and HSA are (1.179±0.02) (for 1-BSA), (0.935±0.01) (for 2-
BSA), (0.978±0.03) (for 1-HSA) and (0.917±0.02) (for 2-HSA), which indicates the existence
of static binding sites in BSA/HSA for complexes.
3.7. TGA study
To evaluate the thermal stability of the complexes 1 and 2, thermogravimetric analysis (TGA)
was carried out at a heating rate of 10 ºC min-1 under a N2 atmosphere in the temperature range
35 - 600 ºC (Fig.11). Upon heating, complex 1 shows loss of coordinated water molecule
(expected weight loss 5.15% and observed 5.2%) within the temperature range 75 - 150 ºC and
after dehydration, it gradually collapsed. This indicate the un-stability of four coordinated
dehydrated species, [Cu(L1)(OCN)]. The green colour complex 2 started to lose its lattice water
molecules at ~120 ºC and becomes deaquated (expected weight loss 3.76% and observed 3.8%)
at 160 ºC, yielding the species [Cu(L2)] (2a, black), which is stable up to ~ 265 ºC, and on
further heating decomposed to an unidentified product. Range of temperature of dehydration of
coordinated (in 1; 75 - 150 ºC) and lattice (in 2; 120 - 160 ºC) water molecules corroborate the
involvement water molecules in H-bonding with compounds.
15

4. Conclusion
Two distorted square pyramidal / trigonal bipyramidal Cu(II) complexes were
synthesized and characterized by spectroscopic methods, single crystal X-ray diffraction studies
and thermogravimetric analysis. Both the complexes are mononuclear and in the solid state
stabilized by non-covalent interactions. DFT / TD-DFT calculations were performed using
CAM-B3LYP functional and theoretically possible spin allowed doublet-doublet electronic
transitions along with their assignments have been discussed. Bovine serum albumin / human
serum albumin binding of the copper(II) complexes were investigated using electronic
absorption and fluorescence spectroscopic techniques. Results of the fluorescence spectroscopic
studies reveal that interactions of serum albumins with the complexes occur through ground
state association process. Both the complexes are active for catalyzing oxidation of 3,5-DTBC to
3,5-DTBQ in presence of molecular oxygen and catalytic activity is more for compound having
triagonal bipyramidal geometry than the compound with square pyramidal geometry.
Acknowledgements
Authors gratefully acknowledge the financial assistance given by the DST, Govt. of India, under
the SERB Fast Track Scheme to Dr. Subal Chandra Manna (Grant No. SR/FT/CS-131 /2010).
K. D. acknowledges CSIR, Govt. of India for fellowship (09/599(0063)/2015-EMR-I).
Supplementary information
CCDC 1544345 and 1544346 contain the supplementary crystallographic data for complexes 1
and
2.
These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk. IR and DFT /TD-DFT results have also been deposited.
16

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Table 1 Crystal data and details of structure refinement of complexes 1 and 2.
1
2
Empirical formula
Formula Weight
Crystal System
Space group
a , Å
C13H10CuN4O4 C22H29CuN3O5
349.80
Triclinic
P-1
479.02
Orthorhombic
Pna21
14.5785(3)
10.8756(2)
13.4619(3)
90
7.3069(3)
10.1652(5)
10.3162(4)
112.059(2)
95.855(2)
109.385(2)
646.99(5)
2
b, Å
c, Å
α, deg
β, deg
90
γ, deg
V , (Å3)
90
2134.38(8)
4
Z
D(calc) [g/cm3]
(Mo-Kα), mm–1
F(000)
1.796
1.491
1.714
1.063
354
1004
θ range (°)
No. of collected data
No. of unique data
R(int)
2.2, 27.6
11302
2.3, 27.5
15693
4812
2999
0.019
0.022
Observed data [I > 2.0 sigma(I)]
2803
4515
20

Goodness of fit (F2)
R1 (all data)
1.016
0.995
0.0233
0.0632
-0.26, 0.42
0.0220
0.0544
-0.22, 0.24
wR2 (all data)
Min. and Max. Resd. Dens. e Å–3
½
aR1 = Fo–Fc / Fo, wR2 = [w (Fo2 – Fc 2) 2/ w (Fo2) 2 ]
Table 2 Experimental and calculateda coordination bond lengths (Ǻ) and angles (º) for
complexes 1-2.
Complex 1
Exp
Complex 2
Exp
Parameters
Calc
Parameters
Calc
Bond lengths
Cu(1)-O(99 )
Cu(1)-N(1)
2.3855(14) 2.227
2.0238(14) 2.025
1.9483(16) 1.968
2.0187(14) 2.024
1.9213(18) 1.946
Cu(1)-O(1)
Cu(1)-O(2)
Cu(1)-N(1)
Cu(1)-N(2)
Cu(1)-N(3)
2.0063(18) 2.137
1.9869(18) 1.996
Cu(1)-N(2)
2.092(2)
2.138(2)
1.989
2.128
Cu(1)-N(3)
Cu(1)-N(21)
2.0732(18) 1.968
Bond angles
O(99)-Cu(1)-N(1)
O(99)-Cu(1)-N(2)
O(99)-Cu(1)-N(3)
91.55(5)
95.41(6)
93.17(5)
95.101
97.310
95.305
93.302
81.653
O(1)-Cu(1)-O(2) 124.06(8)
O(1)-Cu(1)-N(1) 88.11(7)
O(1)-Cu(1)-N(2) 112.79(8)
O(1)-Cu(1)-N(3) 97.85(8)
O(2)-Cu(1)-N(1) 88.15(7)
O(2)-Cu(1)-N(2) 122.80(8)
O(2)-Cu(1) -N(3) 89.65(7)
N(1)-Cu(1)-N(2) 87.84(8)
N(1)-Cu(1)-N(3) 173.88(9)
N(2)-Cu(1)-N(3) 88.56(9)
120.263
85.939
105.452
91.061
88.352
134.183
90.645
92.185
175.814
91.426
0.6938
O(99)-Cu(1)-N(21) 93.52(6)
N(1)-Cu(1)-N(2)
N(1)-Cu(1)-N(3)
N(1)-Cu(1)-N(21)
N(2)-Cu(1)-N(3)
N(2)-Cu(1)-N(21)
N(3)-Cu(1)-N(21)
τ5 parameter[29]
80.83(6)
162.03(6) 161.321
98.95(7)
81.47(6)
97.415
81.669
171.07(7) 169.386
98.06(6)
0.1506
97.423
0.1344
τ5 parameter[29]
0.8303
a Using conductor-like polarizable continuum model (CPCM) in methanol; basis set, LanL2DZ;
CAM-B3LYP functional.
21

Table 3 Selected list of excitation energies of the complexes 1 and 2 in methanol.
State λcal (nm), εcal
Oscillator λexp (nm), εexp Key transition
Characterb
(M−1
(eV)
cm−1), strength
(M−1 cm−1),
(f)
(eV)
210,
ILCT
D46
211.22,
0.10
SOMO-7(β)→LUMO(β)
1
3.00 × 104,
6.99 × 104,
(24%)
(5.86)
(5.90)
ILCT
D30
248.59,
0.13
238,
SOMO-3(β)→LUMO+1(β)
2
2.63 × 104, (4.98)
5.47 × 104,
(14%)
(5.20)
b
ILCT = intra ligand charge transfer transition in L1; ILCT = intra ligand charge transfer
transition in L2 .
Table 4 Kinetics parameters for the catecholase activity of the complexes.
Complex
Vmax (M min-1)
KM (M)
Kcat(h-1)
29.23
Kcat/KM (M-1h-1)
(8.552.5) × 104
(2.620.83) × 104
(2.760.35) × 10-7 (3.410.02) × 10-4
(9.730.56) × 10-7 (4.260.01) × 10-3
1
2
112.145
Table 5 Kinetics parameters for the protein binding activity of the complexes.
Compounds
1+BSA
Ksv (M-1)
kb (M-1)
n
(314.9±0.1)×104
(28.13±0.1)×104
(12.4±0.1)×104
(12.53±0.1)×104
(1.179±0.02)
(0.935±0.01)
(0.978±0.03)
(0.917±0.02)
(33.360.4)×104
(63.74±2.3)×104
(16.0 ±0.4)× 104
(34.6±0.8)×104
2+BSA
1+HSA
2+HSA
Captions of the figures
Fig.1. ORTEP diagram of complex 1 (30 % probability ellipsoid).
Fig. 2. ORTEP diagram of complex 2 (30% probability ellipsoid).
Fig. 3. 1D supramolecular structure of complex 1 formed with hydrogen bonding interactions.
Fig.4. 1D supramolecular structure of complex 2 formed with C-H…π interactions.
22

Fig. 5. Surface plot of the frontier orbitals (α MOs) of 1 along with their energies and
compositions (Using CAM-B3LYP functional).
Fig.6. Increase in the absorbance around 400 nm after the addition of 100 equiv of 3,5-DTBC to
a methanolic solution of the complex for 1 and for 2. The spectra were recorded in every 6 min.
Fig.7. Plots of log(A∞-Ao)/(A∞-At) vs time for the catalyzed oxidation of 3,5-DTBC for
complexes 1 and 2.
Fig.8. Initial rates versus substrate concentration for the 3,5-DTBQ to 3,5-DTBQ oxidation
reaction catalyzed by the complexes 1 and 2 in methanol. Inset pictures show Lineweaver-Burk
plots.
Fig.9. Change of electronic absorption spectra of BSA (3 ml, 8.89 × 10-7 (M) aqueous solution)
upon gradual addition of complexes 1 and 2. Concentration of complexes varies from 0 to
5.44×10-6 M (for 1) and 0 to 4.97×10-6 M (for 2).The arrows indicate the change upon
increasing the complex concentration. Inset picture shows the plot of 1/Aobs-Ao vs 1/[Complex].
Fig.10. Change of emission spectra of BSA (3 ml, 8.89 × 10-7 (M) aqueous solution) upon
gradual addition of 1 and 2. Concentration of complexes varies from 0 to 3.33×10-6 M (for 1)
and 0 to 2.37×10-6 M (for 2).The arrows indicate the effect of metal complexes on the emission
spectra. Inset picture shows the plot of Io/I vs [Complex].
Fig. 11. TGA of the complexes 1 and 2.
23

Fig.1
Fig. 2
24

Fig. 3
Fig.4
25

SOMO-5
SOMO-4
SOMO-3
SOMO-2
E = - 9.42 eV
E = - 9.1 eV
E = - 8.9 eV
E = - 8.69 eV
Cu 0%, L1 100%, H2O Cu 3%, L1 92%, H2O 1%, Cu 0 %, L1 93 %, H2O Cu 15%, L1 79%, H2O
0%, OCN 0%
OCN 4%
0 %, OCN 7 %
0%, OCN 6%
SOMO-1
E = - 8.63 eV
SOMO
LUMO
E = - 1.45 eV
LUMO + 1
E = - 0.84 eV
E = - 8.63 eV
Cu 2%, L1 9%, H2O
0%, OCN 89%
Cu 4%, L1 7%, H2O 0%, Cu 0%, L1 100%, H2O
Cu 2%, L1 98%, H2O
0%, OCN 0%
OCN 89%
0%, OCN 0%
LUMO + 2
E = - 0.27 eV
LUMO + 3
E = - 0.04 eV
LUMO + 4
E = 0.99 eV
LUMO + 5
E = 1.23 eV
Cu 0%, L1 100%, H2O Cu 2%, L1 98%, H2O 0%, Cu 92%, L1 0%, H2O
Cu 99%, L1 0%, H2O
1%, OCN 0%
0%, OCN 0%
OCN 0%
8%, OCN 0%
Fig. 5
26

Fig.6
Fig.7
27

Fig.8
Fig.9
28

Fig.10
Fig. 11
29