Spectroscopic and molecular docking studies on the interaction of human serum albumin with copper(II) complexes

Article abstract of DOI:10.1016/j.saa.2016.10.043

Two osazone based ligands, butane-2,3-dione bis(2′-pyridylhydrazone) (BDBPH) and hexane-3,4-dione bis(2′-pyridylhydrazone) (HDBPH), were synthesized out of the 2:1 M Schiff base condensation of 2-hydrazino pyridine respectively with 2,3-butanedione and 3,4-hexanedione. The X-ray crystal structures of both the ligands have been determined. The copper(II) complex of HDBPH has also been synthesized and structurally characterized. HDBPH and its copper(II) complex have thoroughly been characterized through various spectroscopic and analytical techniques. The X-ray crystal structure of the copper complex of HDBPH shows that it is a monomeric Cu(II) complex having ‘N₄O₂’ co-ordination chromophore. Interaction of human serum albumin (HSA) with these ligands and their monomeric copper(II) complexes have been studied by various spectroscopic means. The experimental findings show that the ligands as well as their copper complexes are good HSA binders. Molecular docking investigations have also been done to unravel the mode of binding of the species with HSA.

Full text of DOI:10.1016/j.saa.2016.10.043

Spectroscopic and molecular docking studies on the interaction of human
serum albumin with copper(II) complexes
Bhargab Guhathakurta, Ankur Bikash Pradhan, Suman Das, Nirmalya
Bandyopadhyay, Liping Lu, Miaoli Zhu, Jnan Prakash Naskar
PII:
DOI:
Reference:
S1386-1425(16)30638-2
doi:10.1016/j.saa.2016.10.043
SAA 14742
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15 July 2016
20 October 2016
21 October 2016
Please cite this article as: Bhargab Guhathakurta, Ankur Bikash Pradhan, Suman Das,
Nirmalya Bandyopadhyay, Liping Lu, Miaoli Zhu, Jnan Prakash Naskar, Spectroscopic
and molecular docking studies on the interaction of human serum albumin with copper(II)
complexes, (2016), doi:10.1016/j.saa.2016.10.043
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ACCEPTED MANUSCRIPT
Spectroscopic and molecular docking studies on the interaction of human serum
albumin with copper(II) complexes
Bhargab Guhathakurtaa, Ankur Bikash Pradhana, Suman Dasa, Nirmalya
Bandyopadhyaya, Liping Lub, Miaoli Zhub, Jnan Prakash Naskara,*
aDepartment of Chemistry, Jadavpur University, Kolkata-700 032, India. Fax:
+(91)(33)2414 6223 E-mail: jpnaskar@rediffmail.com
bInstitute of Molecular Science, Key Laboratory of Chemical Biology and Molecular
Engineering of the Education Ministry, Shanxi University, 92 Wucheng Road, Taiyuan,
Shanxi- 030006, People's Republic of China.
Abstract
Two osazone based ligands, butane-2,3-dione bis(2′-pyridylhydrazone) (BDBPH) and
hexane-3,4-dione bis(2′-pyridylhydrazone) (HDBPH), were synthesized out of the 2:1 molar
Schiff base condensation of 2-hydrazino pyridine respectively with 2,3-butanedione and 3,4-
hexanedione. The X-ray crystal structures of both the ligands have been determined. The
copper(II) complex of HDBPH has also been synthesized and structurally characterized.
HDBPH and its copper(II) complex have thoroughly been characterized through various
spectroscopic and analytical techniques. The X-ray crystal structure of the copper complex of
HDBPH shows that it is a monomeric Cu(II) complex having ‘N4O2’ co-ordination
chromophore. Interaction of human serum albumin (HSA) with these ligands and their
monomeric copper(II) complexes have been studied by various spectroscopic means. The
experimental findings show that the ligands as well as their copper complexes are good HSA
binders. Molecular docking investigations have also been done to unravel the mode of
binding of the species with HSA.
Key words: Osazone, Copper, Crystal structure, HSA binding, Molecular docking
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1. Introduction
In 1964, B. Chiswell and F. Lions reported metal complexes of an osazone based ligand,
butane-2,3-dione bis(2′-pyridylhydrazone). This planar quadridentate chelating agent was
generated through the condensation of biacetyl and 2-hydrazino pyridine [1]. α-Dicarbonyl
precursors like glyoxal, biacetyl and cyclohexane-1,2-dione are prone to yield osazones on
treatment with 2-hydrazino pyridine. On the contrary, α-diketones like benzil,
phenanthraquinone, acenaphthenequinone are averse to form osazones so much so that they
react with 2-hydrazino pyridine to give rise only the mono-(2-pyridylhydrazone) [2]. Osazone
of benzil and 2-hydrazino pyridine was first synthesized by F. H. Case and co-workers [3].
Osazones are analytical chromogenic reagents. Butane-2,3-dione bis(2′-pyridylhydrazone)
(BDBPH/L1H2) is a known colorimetric reagent, often used for the determination of Co(II)
and Pd(II) ions [2,4,5]. Osazones obtained from hydrazine/substituted hydrazine may or may
not undergo protonic dissociation of the two dissociable NH protons during the course of
their metal binding. For ready reckoning, BDBPH binds to copper(II) in protonated form [6];
while to nickel(II) only after its deprotonation [7]. The crystal structure of bare BDBPH is,
however, not known till date. The structure of the monomeric copper(II) complex of BDBPH,
as determined by us, reveals that the compound crystallizes in the monoclinic space group
[6]. This one-electron paramagnetic copper(II) complex is in Jahn-Teller distorted octahedral
geometry. The copper center resides in a ‘N4O2’ coordination chromophore.
The most abundant protein in human blood plasma is human serum albumin (HSA). HSA
executes a plethora of crucial physiological functions aided by its enhanced solubility in
blood plasma. It regulates osmotic blood pressure and helps in the storage, transportation,
metabolism and distribution of biologically important compounds. HSA acts as the disposer
of different exogenous and endogenous species through plasma circulation. Again, it
deactivates radicals and delivers amino acids after hydrolysis for the synthesis of new
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proteins [8]. HSA plays an indispensible ubiquitous role in circulatory system to maintain
blood pH level [9]. Besides, HSA anchors and transports metal ions, fatty acids, hormones,
steroids, vitamins and a variety of pharmaceuticals including metal based drugs efficiently
with minimal toxicity. Thus, HSA has relevance in molecular pharmacology and
pharmacokinetics as well. HSA also reduces the chance of the oxidation of the bound drug
[10]. Tumour cells take up and accumulate HSA at an enhanced level in comparison to their
normal counterparts. Accordingly, HSA offers the efficacy to serve as carrier conjugate of
antimalignant drugs like paclitaxel and doxorubicin [11]. HSA is significant from clinical
oncological viewpoint too. Owing to these versatile yet benign myriad prospects, studies on
the binding of HSA encompass a burgeoning area of contemporary research in chemical
biology and pharmacology [12]. Again, from the pharmacological and toxicological view-
point the metal complexes show significant advantages over the organic compounds [13,14].
Metal complexes facilitate the transport processes through cell membranes and protect the
drug from enzymatic degradation [15]. Copper, a bio essential trace element, is notable in this
perspective. Copper complexes play a crucial role in different biological processes like
electron transfer, DNA damage and as anticancer agents [16]. These aspects kindled our
interest to undertake the studies on the interaction of copper(II) complexes derived from
osazone based ligands, L1H2 and L2H2 and to evaluate their subsequent HSA binding
prospects.
Here we wish to report the syntheses, characterization and structures of a novel ligand,
hexane-3,4-dione bis (2′-pyridylhydrazone) (HDBPH/L2H2) and its copper(II) complex,
CuL2H2(ClO4)2.H2O (2). The crystal structure of another analogous ligand system L1H2
butane-2,3-dione bis(2′-pyridylhydrazone) has also been determined. Syntheses of L1H2 and
its copper(II) complex, CuL1H2(ClO4)2 (1) have been reported previously by us [6]. The HSA
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binding and molecular docking aspects of the above ligands and their copper(II) complexes
have also been dealt with.
2. Experimental
2.1. Materials and measurements
All chemicals were of analytical reagent grade and were used as received without further
purification. 2,3-butanedione, 3,4-hexanedione and 2-hydrazino pyridine were procured from
Aldrich, USA. HSA was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).
Electro-thermal digital melting point apparatus (SUMSIM India) was employed for
determining the melting point of the ligand and it is uncorrected. C, H and N microanalyses
were done on a Perkin-Elmer 2400II elemental analyzer. Copper was estimated
gravimetrically as its white CuSCN complex. FT-IR spectra (KBr disc) [(vb) = very broad,
(b) = broad, (m) = medium, (s) = strong] of L2H2 and 2 were recorded employing a Nicolet
Magna-IR spectrophotometer (Series II). UV-Vis spectra (in MeOH) of L2H2 and 2 were
recorded on a Shimadzu UV-160A spectrophotometer. A Bruker DPX300 MHz spectrometer
1
was used to run the H NMR spectrum (in CDCl3: reference, TMS) of L2H2. ESI mass
spectra of both L2H2 and 2 were recorded on a Waters Q-TOF Micro YA263 spectrometer in
methanol. Electric conductivity measurements of 2 were performed at room temperature on a
Systronics (India) direct reading conductivity meter (model 304). A PAR 155 vibrating
sample magnetometer fitted with a walker scientific L75FBAL magnet, calibrated with
Hg[Co(SCN)4], was used to determine the magnetic susceptibility of 2 at room temperature.
The magnetic susceptibility data were corrected for diamagnetism using Pascal’s constants.
Caution: Perchlorate salts of metal complexes can be explosive [17]. Although no detonation
tendencies have been observed, care is advised and handling of only small quantity is
recommended.
2.2. Buffer and protein solutions
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Using the molar absorption coefficient value of 42 864 M−1 cm−1 at 280 nm [18], the
concentration of HSA solution was determined. 10 mM citrate-phosphate (CP) buffer of pH
7.0 was used to perform all the experiments. The CP buffer solution was made in quartz-
distilled deionized water from a Milli-Q source (Millipore, USA). Then the solution was
filtered to remove impurities with Millipore filters of 0.45 μm pore size.
The mother solutions of the ligands as well as their copper complexes were prepared in
water-methanol (4:1) mixed solvent. We chose this mixed solvent for the necessary inclusion
of CP buffer in the milieu to undertake the protein binding study under physiological
conditions. To check the stability of the ligands and their copper complexes in this mixed
solvent, we have taken the UV-Vis spectra of the ligands and their complexes maintaining the
physiological pH. The spectra of the ligands and their complexes correlate nicely with their
respective spectra, taken only in pure methanol, in terms of band positions with expected
features.
2.3.Methods for HSA Binding
2.3.1. UV−Visible Spectroscopy
Shimadzu (model UV-1800) spectrophotometer (Shimadzu Corporation, Japan) was used to
measure all the UV−Vis absorption spectra. Matched quartz cuvettes having 1 cm path length
were used throughout the study. During the experiment, a thermoprogrammer was attached to
the spectrophotometer via Peltier effect to maintain the temperatures of the sample and
reference cuvettes. Keeping the sample concentration fixed at 4.9 μM and with the rising
concentration of HSA until the saturation point is reached, the spectrophotometric titrations
were performed. At the λmax value, the changes in the absorption spectra were noted for each
sample until saturation was obtained.
2.3.2. Spectrofluorimetric studies
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Shimadzu RF-5301PC spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan) was used to
analyse the steady-state fluorescence spectral data for the samples. Free quartz cell of 1 cm
path length was used to record the fluorescence data. The excitation and emission bandpass
were respectively of 3 and 5 nm. A fixed amount of ligands as well as complexes (9.2 μM)
was titrated with increasing concentration of HSA at the same excited λmax of 285 nm.
2.3.3. Fluorescence lifetime measurements
In the absence and in the presence of rising concentration of HSA in 10 mM CP buffer of pH
7.0 at 25 °C for the fluorescence decay of the samples, time correlated single photon counting
(TCSPC) measurements were done. The photoexcitation was performed at 300 nm using a
picosecond diode laser (IBH Nanoled-07) in an IBH fluorocube apparatus. Using IBH DAS6
software [from the equation (1)], fluorescence decay data were analysed which were
collected on a Hamamatsu MCP photomultiplier (R3809).
t
F(t)   e( )

i

i
(1)
Here αi represents the ith pre-exponential factor and τ stands for the decay time. The lifetime
of the excited species is referred to as decay time.
2.3.4. Thermodynamic studies: temperature dependent spectrofluorometry
Temperature dependent emission spectra were recorded in a Horiba-Jobin-Yvon Fluoromax-4
spectrofluorimeter attached with thermometric cell temperature programmer and temperature
controller. These measurements were performed at 15, 25 and 35 °C.
The values of K′ were determined at different temperatures. Thermodynamic parameters were
estimated by analysis of van’t Hoff plot (ln K′ versus 1/T) obtained over the temperature
range of the study. The slope of the plot gives the binding enthalpy change (H0) as
Ho
R

ln K /  1/T  
(2)




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The Gibbs free energy change (G0) was determined from the binding constant at a particular
temperature according to this relation
0

(3)
G  RT ln K


The entropy change (S0) was then estimated from the following equation
S0  H0 G0 /T
(4)


2.4.
Molecular docking studies
AutoDock (version 4.2) was used to find out the binding interaction and the probable binding
sites of ligands as well as complexes with HSA. The X-ray crystal structure of HSA was
taken from RCS Protein Data Bank having PDB code 1AO6. 3D structure of the four samples
was created in Chem3D Ultra 8.0. Using two well-known software packages Gaussian 09W
and AutoDock 4.2, necessary modification was done. The DFT//B3LYP/6-31G level of
theory was used to optimize the geometry of the samples using the Gaussian 09W set and the
Gauss view 5.0 software in a compatible file format was used to exploit the optimized
geometry and to generate the required file in AutoDock 4.2. Polar hydrogen atoms and
Gasteiger charges were added to the protein and ligand/complex. To perform the docking
simulations, a grid box with dimensions of 120, 120 and 120 Å and a grid spacing of 0.403 Å
were assigned. Other parameters were taken as usual as the default values set by the
AutoDock program. AutoGrid was used to deliberate the grid maps for energy and the
docking calculations were done by Lamarckian genetic algorithm (LGA). The best optimized
docked model having lowest energy was chosen for further analysis of docking simulations
which was best viewed in PyMOL and Mercury software.
2.5.
Synthesis of ligands and complexes
2.5.1. Preparation of Ligand, BDBPH (L1H2)
The synthesis and general characterization of L1H2 was reported in our previous
communication [6].
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Needle-shaped yellow single crystals of L1H2, fit for X-ray crystallography, were grown
during the course of slow evaporation of a moderately concentrated solution of it in
methanol.
2.5.2 Preparation of Ligand, HDBPH (L2H2)
0.03 mL (0.25 mmol) of 3,4-hexanedione was dissolved in 15 mL of dry methanol. Then 0.56
mg (0.51 mmol) of 2-hydrazinopyridine was added to it all at a time. The resulting yellow
orange mixture was refluxed under heating for 4 h. After refluxing, the resulting yellow
solution was kept in a refrigerator for slow evaporation. After 10 days, the separated ligand
was filtered and was washed with ice-cold diethyl ether thoroughly. The ligand is soluble in
methanol, acetonitrile and chloroform but insoluble in benzene, petroleum ether, diethyl ether
and water. Yield: 48 mg (64%), m. p.: 216-220 °C. C16H20N6 (296.16): Anal. Calc. for
C16H20N6: C, 64.83; H, 6.81; N, 28.36. Found: C, 64.96; H, 6.76; N, 28.42 %. FT-IR (KBr)
v/cm-1: 3197vb (NH), 1676vb (C=N imine), 1598, 1575, 1436s (aromatic C=C ring vibration
frequencies), 1575b (amine), 987m (pyridine ring), 773s (four adjacent H on aromatic ring)
and 738m (mono-substituted pyridine). UV-Vis (MeOH): λmax (ε/M-1 cm-1) 273 (11 240), 292
(37 810) and 329 (39 680). 1H NMR (CDCl3): δ/ppm 8.32 (2H, s, –NH– proton), 8.13 (2H, d,
o-proton of pyridine ring), 7.59 (2H, t, p-proton of pyridine ring), 7.32 (2H, d, m-proton of
pyridine ring near –NH– group), 6.79 (2H, t, m-proton of pyridine ring), 3.48 (4H, t, ethyl
protons), 2.17 (6H, d, ethyl protons). ESI-MS (positive ion mode) (m/z): 297.1304 (calcd.
297.3781) for [L2H2+H+] (100%), 295.1235 (calcd. 295.2908) for [L2H2–2H·+H+] (30%) and
319.1163 (calcd. 319.3599) for [L2H2+Na+] (50%). We got yellow needle-shaped single
crystals of L2H2, suitable for X-ray crystal structure determination, by slow aerial
evaporation of the mother liquor of L2H2 in methanol.
2.5.3. Preparation of CuL1H2(ClO4)2 (1)
The preparation and thorough characterization of 1 was reported earlier by us [6].
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2.5.4. Preparation of CuL2H2(ClO4)2.H2O (2)
12 mg (0.04 mmol) of L2H2 was dissolved in 15 mL of MeOH. Then 15 mg (0.04 mmol) of
solid Cu(ClO4)2.6H2O was added all at a time with constant stirring to the yellow ligand
solution. The colour of the solution changed to brick red almost instantly. The resulting red
reaction mixture was stirred for 2 h. After stirring, the resulting solution was kept in a
refrigerator for slow evaporation. After 7 days, the separated dark red compound was filtered
and washed with ice-cold diethyl ether thoroughly. Then it was dried in a vacuum desiccator
over fused CaCl2. The compound is soluble in methanol, acetonitrile and acetone but
insoluble in benzene, petroleum ether, diethyl ether, dichloromethane and water.
Yield: 16 mg (66%). C16H22N6CuO9Cl2 (576.67): Anal. Calc. for C16H22N6CuO9Cl2: C,
33.29; H, 3.84; N, 14.57; Cu, 11.01; Found C, 33.21; H, 3.75; N, 14.71; Cu, 10.92%. FT-IR
(KBr): v 3414vb (OH), 1614s (C=N of imine), 1485s (C=N of oxime), 1230s (N-O), 1113vs
(ClO4) and 631s (ClO4) cm-1. UV-Vis (MeOH): λmax (ε/M-1 cm-1) 273 (1 502), 338 (6 078),
366 (6 850) and 446 (1 136) ESI-MS (positive ion mode) (m/z): 358.0315 (calcd. 358.3623)
for [Cu63(L2H2)–2H++H+] (100%), 360.0375 (calcd. 360.3623) for [Cu65(L2H2)–2H++H+]
(40%) and 357.0356 (calcd. 357.3543) for [Cu63(L2H2)–H·–H+] (30%). M (MeOH): 207.37
cm2ohm-1mol-1 (1:2 electrolyte), M (MeCN): 235.23 cm2ohm-1mol-1 (1:2 electrolyte).
eff/B: 1.94 (at 298 K).
We got needle-shaped red single crystals of this compound fit for X-ray structure
determination by slow evaporation of the moderately concentrated solution of the compound
in acetone on standing in a refrigerator.
2.6. Crystal structure determination
Single crystals suitable for X-ray crystallographic analyses were selected after examination
under a microscope. A Bruker Smart APEX II diffractometer equipped with 1K charge-
coupled device (CCD) area detector and graphite monochromated MoKα radiation (λ =
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0.71073 Å) was used for the data collection of L1H2, L2H2 and CuL2H2(ClO4)2.H2O
respectively at 273, 298 and 100 K. The cell parameters were determined using SMART
software [19]. SAINTPlus [19] was used for data reduction and correction. Absorption
corrections were made by SADBAS [20]. The structures were solved by direct methods with
SHELXL-97 program [21] and also were refined by full-matrix least-squares methods on all
F2 data by SHELXL-97. The anisotropic refinement was done over all non-hydrogen atoms.
Subsequently, the additional H-atoms were placed at their respective parent atoms and also
were refined using riding model. Lastly, the cycle of full-matrix least-squares refinement was
made on the basis of observed reflections and variable parameters. All structural refinement
values of L1H2, L2H2 and CuL2H2(ClO4)2.H2O are given in Table 1. Some selected bond
lengths, bond angles and hydrogen-bond geometries of them are tabulated respectively in
Tables 2 and 3. The cif files of L1H2, L2H2 and 2 were deposited with the Cambridge
Crystallographic Data Centre (CCDC) bearing CCDC Nos. 1446043, 1446044 and 1446045
respectively.
3. Results and discussion
3.1.
Synthesis and formulation
L1H2 is the di-condensate Schiff base adduct of diacetyl with 2-hydrazino pyridine. L2H2, an
analogous osazone of L1H2, is derived from 3,4-hexanedione with 2-hydrazino pyridine.
They are obtained in copious yields out of the reaction of respective diketone with 2-
hydrazino pyridine in 1:2 stoichiometric proportions in dehydrated methanol (Scheme. 1).
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Scheme. 1. Synthetic scheme of the ligands
Our subsequent reaction of L1H2 and L2H2 with copper(II)perchlorate hexahydrate in 1:1
stoichiometric proportion in methanol at room temperature yielded monomeric copper(II)
complexes, 1 and 2, with moderate yields. Preparation of L1H2 and 1 had been reported
earlier by us. Both 1 and 2 are air stable for indefinite period. In the IR spectrum of L2H2 a
new bond stretching frequency is observed at 1676 cm-1 which is indicative of (C=N imine)
bond. This is indicative of the formation of Schiff base [22]. At 1113 cm-1 a broad double
split band is observed in the IR spectrum of 2. This is assigned to the coordinated perchlorate
with the copper center [23]. The ESI-MS mass spectra of L2H2 and 2 show the presence of
two dissociable protons on the ligand skeleton. This feature is akin to the earlier work on this
class of ligand [7].
3.2. Crystal structures of L1H2, L2H2 and 2
L1H2 crystallizes in the monoclinic crystal system having space group P2(1)/c, with z value
of 2. Centrosymmetric L2H2 also crystallizes in the monoclinic space group, C2/c, with four
molecular weight units accommodated per unit cell. The C-N bonds are found to be 1.2880
[N3–C6] and 1.284 Å [C1–N1] respectively for L1H2 and L2H2. These values are almost
similar. The corresponding N–N bond distances in L1H2 and L2H2 are found to be 1.3002 and
1.3654 Å. Both the ligands exist in stereochemical anti conformational preference over their
cis disposition to get rid of steric strain. The crystal structures of L1H2 and L2H2 are shown
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respectively in Fig. 1 and Fig. 2. The crystal structure of 2 (Fig. 3) reveals that the copper
centre of this mononuclear copper(II) complex is in ‘N4O2’ octahedral coordination
environment. The tetradentate ligand, L2H2 provides four nitrogen donor centers [two pyridyl
nitrogen donors, N1 and N6; two nitrogen donors from the hydrazine residue, N3 and N4].
The two oxygen donors from the coordinating perchlorate, O1 and O5, complete the ‘N4O2’
core. The Cu1–N3, Cu1–N4, Cu1–N6 and Cu1–N1 bond distances are respectively of
1.9577(12), 1.9585(12), 1.9913(12) and 1.9953(11) Å. Accordingly, they are of the same
order of magnitude. On the contrary, the two axial bonds, Cu1–O1 and Cu1–O5. are
respectively of 2.5753(13) and 2.5456(13) Å. These data are similar with that reported
previously for other mononuclear octahedral Cu(II) complex [24].The axial bond lengths are
of higher magnitude as expected in Jahn-Teller distorted octahedral copper(II) system. This
axial elongation is the natural consequence of an octahedral copper(II) complex [6].
Table1
Crystal data and structure refinement for L1H2, L2H2 and 2
L1H2
L2H2
2
CCDC No.
1446043
C14H16N6
268.33
1446044
C16H20N6
296.38
1446045
C16H22Cl2CuN6O9
576.83
Empirical formula
Formula weight
Temperature [K]
Wavelength [Å]
Crystal system
Space group
273(2)
298(2)
100(2)
0.71073
Monoclinic
P2(1)/c
0.71073
Monoclinic
C2/c
0.71073
Monoclinic
P2(1)/c
Unit cell dimensions
a [Å]
8.8092(4)
4.7855(2)
13.3888(6)
13.9584(6)
8.0850(16)
10.191(2)
b [Å]
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c [Å]
16.1183(8)
94.457(4)
677.44(5)
8.9084(3)
26.913(5)
95.79(3)
 [o]
100.667(3)
1636.09(12)
Volume [Å3]
2206.2(8)
Absorption coefficient
[mm-1]
0.085
0.077
1.295
Absorption correction
Z
multi-scan
2
multi-scan
4
multi-scan
4
ρCalcd [g/cm3]
1.315
1.203
1.737
F(000)
284
632
1180
Crystal size [mm]
Reflections collected
Parameters
0.21×0.06×0.04
9159
0.24×0.07×0.05
11694
0.29×0.05×0.07
10523
92
101
309
R1, all data, R1 [I >2σ(I)]
wR2,all data, wR2 [I >2σ(I)]
SonF2
0.0422,0.1104
0.0535,0.1169
1.076
0.0433,0.1207
0.0524,0.1304
1.033
0.0379,0.1196
0.0389,0.1213
1.034
Largest diff. peak
Largest diff. hole [eÅ-3]
0.148
0.201
0.491
-0.179
-0.156
-0.919
Table2
Selected bond distances (Å) and angles (o)
For L1H2
N1–C1
N1–C5
N2–N3
N2–C1
1.3333(19)
1.341(2)
C1–C2
C2–C3
C3–C4
C4–C5
1.391(2)
1.364(2)
1.372(2)
1.361(3)
1.3628(17)
1.3785(19)
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N3–C6
1.2880(18)
1.470(3)
C6–C7
1.488(2)
C6–C6#1
C1–N1–C5
C6–N3–N2
N1–C1–N2
N2–C1–C2
For L2H2
N1–C1
C2–H2
0.9300
116.55(14)
118.67(12)
115.34(13)
121.94(14)
N3–N2–C1
C3–C2–C1
N1–C1–C2
118.10(12)
118.41(15)
122.72(14)
1.284(2)
C1–C2
1.503(2)
N1–N2
1.3654(16)
1.3777(19)
1.345(2)
C2–C3
1.492(3)
N2–C4
N3–C4
1.3279(18)
1.389(2)
N3–C8
C4–C5
C6–C7
1.372(3)
C7–C8
1.360(3)
C1–C1#2
C1–N1–N2
C4–N3–C8
N1–C1–C2
C3–C2–C1
For 2
1.475(3)
N2–H2
0.8600
119.40(13)
116.67(14)
125.77(13)
112.47(17)
N1–N2–C4
N1–C1–C1#1
C1#1–C1–C2
N3–C4–N2
117.02(12)
114.85(17)
119.32(17)
114.90(13)
Cu1–N3
Cu1–N6
Cu1–O1
N1–C1
1.9577(12)
1.9913(12)
2.5753(13)
1.3454(17)
1.3002(18)
1.3516(15)
1.3500(17)
Cu1–N4
Cu1–N1
Cu1–O5
N1–C5
1.9585(12)
1.9953(11)
2.5456(13)
1.3558(18)
1.2999(17)
1.3883(17)
1.3570(17)
N2–N3
N4–C7
N3–N5
N5–C12
N6–C16
N6–C12
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Cl1–O4
1.4402(11)
91.67(5)
86.70(5)
78.74(5)
92.33(5)
81.18(5)
159.85(5)
174.70(4)
84.17(5)
95.66(5)
83.81(5)
128.84(9)
109.07(7)
112.21(9)
Cl1–O1
1.4662(11)
122.51(12)
118.49(13)
120.80(13)
122.47(13)
120.08(13)
111.94(11)
117.20(12)
111.09(12)
123.93(12)
116.74(12)
124.01(12)
109.18(7)
O1–Cu1–N3
O1–Cu1–N4
N3–Cu1–N4
O1–Cu1–N1
N3–Cu1–N1
N4–Cu1–N1
O5–Cu1–O1
N3–Cu1–O5
N4–Cu1–O2
N1–Cu1–O5
C1–N1–Cu1
O3–Cl1–O1
C5–N1–Cu1
N1–C1–C2
C3–C2–C1
N2–C5–C4
N1–C5–C4
C2–C3–C4
N4–C7–C6
N6–C12–N5
C6–C8–C9
N4–C7–C10
N1–C5–N2
N3–C6–C8
O4–Cl1–O1
C12–O5–Cu1
122.57(7)
#1 -x, -y+2, -z+1; #2 -x+3/2, -y+1, -z
Table3
Hydrogen bonds (Å and o)
D–H···A
For L1H2
d(D–H)
d(H···A)
d(D···A)
(D–H–A)
Symmetry code
C7–H7A...N1#2
N2–H2A...N1#2
For L2H2
0.96
0.86
2.64
2.63
3.356(2)
132.0
159.9
-x, y+1/2, -z+1/2
-x, y+1/2, -z+1/2
3.4531(19)
3.2176(18)
3.0219(17)
N2–H2...N3#2
For 2
0.86
0.82
2.45
2.21
148.2
170.5
-x+1, y, -z+1/2
O9–H9E...O1#1
-x+1, y+1/2,-z+1/2
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O9–H9D...O3
C16–H16...O2#1
C8–H8A...O6#2
C1–H1...O2#1
N5–H5...O9#3
N2–H2...O6#2
0.82
0.95
0.99
0.95
0.88
0.88
2.19
2.49
2.52
2.46
1.96
2.00
2.9969(18)
3.3370(18)
3.355(2)
168.0
148.9
141.4
156.5
164.9
169.1
-x+1,y+1/2,-z+1/2
-x+1,-y+1,-z+1
3.3561(18)
2.8231(16)
2.8700(17)
-x+1,y+1/2,-z+1/2
-x+2,y-1/2,-z+1/2
-x+1,-y+1,-z+1
Fig. 1. ORTEP of L1H2 with 30% probability ellipsoids. Symmetry code i) -x, -y+2, -z+1
Fig. 2. ORTEP of L2H2 with 30% probability ellipsoids. Symmetry code i) -x+3/2, -y+1, -z
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Fig. 3. ORTEP of 2 with 30% probability ellipsoids
3.3. HSA binding study
3.3.1. Absorption spectroscopic study
Fig. 4 shows the absorption spectra of L1H2 and L2H2 and their complexes in aqueous and
protein (HSA) environments. It is evident that the absorption of both the ligands decreases
upon addition of HSA; while the reverse is true for their complexes. The perturbation in
absorption in presence of HSA was more pronounced in case of the complexes. The UV data
were used to construct the Benesi-Hilderbrand plot for 1:1 complexation using the following
equation:
1
1
1


(5)
A Amax KAmax[HSA]
Here, ∆A = |Ax- A0| and ∆Amax = |A∞ - A0|, where A0, Ax and A∞ are the absorbances of the
ligand or complex in the absence of HSA, at an intermediate HSA concentration, and at a
concentration of complete interaction with HSA respectively. K is the association constant
and [HSA] is the protein concentration. A plot of ∆Amax/∆A would give a straight line with
the intercept of 1 and the slope 1/[HSA]. The inverse of slope would yield the association
constant. The association constants were found to be 2.16±0.18 × 104 M-1 and 3.78±0.29 ×
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104 M-1 for L1H2 and L2H2 respectively. For 1 and 2, it was found respectively to be
6.36±0.47 × 104 M-1 and 8.25±0.55 × 104 M-1. These results clearly demonstrate the stronger
binding ability of the metal complexes with HSA compared to their ligand counterparts.
Fig. 4. Absorbance titration profiles of L1H2, L2H2, 1 and 2 with increasing concentration of
HSA. Panel A: Curve 1-5 represents absorption spectra of free L1H2 (4.9 M, curve 1)
treated with 6.34, 12.66, 25.32 and 42.2 M of HSA; Panel B: Curve 1-6 represents
absorption spectra of free 1 (4.9 M, curve 1) treated with 1.58, 3.17, 6.34, 12.66 and 25.32
M of HSA; Panel C: Curve 1-3 represents absorption spectra of free L2H2 (4.9 M, curve
1) treated with 25.32 and 42.2 M of HSA; Panel D: Curve 1-5 represents absorption spectra
of free 2 (4.9 M, curve 1) treated with 3.17, 6.34, 12.66, 25.32 and 42.2 M of HSA. Inset
of each figure shows the respective Benesi-Hilderbrand plot.
3.3.2. Fluorescence spectroscopic studies
Addition of HSA results in small changes in the absorption spectra of the systems. Now,
since all the systems are fluorescence inactive itself, here fluorescence titrations were carried
out by titrating HSA with increasing concentration of ligand/complex. Interestingly, HSA
induced changes in the fluorescence spectra of the systems (spectral shift or intensity
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distribution) are much more pronounced. This is evident from some of the representative
spectra shown in Fig. 5. Significant red shift as well as enhancement in emission intensity is
observed in all cases, though the extent of enhancement is more pronounced in case of the
complexes. Benesi-Hilderbrand plots (not shown) were constructed for all the cases. The
association constants were found to be 2.02±0.15 × 104 and 3.51±0.24 × 104 M-1 for L1H2 and
L2H2 respectively. For 1 and 2, those were found respectively to be 6.21±0.42 × 104 and
8.01±0.53 × 104 M-1. Thus, the fluorescence spectral data are found to be in good agreement
with the absorption results.
Fig. 5. Emission spectra of L1H2, L2H2, 1 and 2 with increasing concentration of HSA. Panel
A: Fluorescence spectra of free L1H2 (9.2 M, curve 1) treated with 1.56, 3.13, 4.69, 6.24,
7.79, 12.43, 18.58, 30.72 and 48.58 M of HSA; Panel B: Fluorescence spectra of free 1 (9.2
M, curve 1) treated with 0.78, 1.56, 3.13, 4.69, 6.24, 7.79, 10.89, 15.51, 21.63 and 30.72
M of HSA; Panel C: Fluorescence spectra of free L2H2 (9.2 M, curve 1) treated with 1.56,
3.13, 4.69, 6.24, 7.79, 12.43, 18.58, 24.67, 30.72 and 44.16 M of HSA; Panel D:
Fluorescence spectra of free 2 (9.2 M, curve 1) treated with 0.78, 1.56, 3.13, 4.69, 6.24,
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7.79, 9.34, 12.43, 18.58, 24.68 and 30.72 M of HSA in 10 mM CP buffer of pH 7.0 at 25
°C.
The tryptophan residue of HSA shows a strong fluorescence emission with a peak at 338 nm
when its excitation is performed at 280 nm. In Fig. 5, it is not clearly visible since the current
profile depicts the titration where the emission intensity increases abruptly. Consequently,
this emission has been obscured in the envelope of the final spectrum. The spectrum of native
HSA has been shown in Fig. 6.
Fig. 6. Fluorescence spectrum of free HSA in 10 mM CP buffer of pH 7.0 at 25 °C.
The spectrum clearly manifests a maximum around 338 nm. According to Lakowicz [25],
fluorescence spectra of protein in presence of small molecules undergo changes due to a good
number of phenomenon like excited state reactions, molecular rearrangements, energy
transfer, ground state complexation, collision quenching etc. Probably, the excited state
energy transfer is taking place between the tryptophan residue and the ligands as well as
metal complexes. Several examples are known in the literature where the metal complexes
even give rise to quenching of fluorescence emission on binding to HSA [26,27]. However,
mere residing near the Trp residue does not always result in quenching. Recently, Dey et al
has shown that the emission of albumin proteins increases significantly in presence of some
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carbazole containing probes [28]. In our present case, the observed enhancement in
fluorescence intensity owing to HSA binding of the ligands as well as their complexes is
most likely due to the concomitant change in microenvironment around Trp residues [28].
Our molecular docking study further reveals that the HSA binding occurs preferentially near
Trp residue.
3.3.3. Fluorescence lifetime studies
Fluorescence lifetime measurement of the ligands as well as their complexes in presence of
HSA was performed by using TCSPC technique. Fluorescence decay profiles are represented
in Fig. 7. Fluorescence lifetime measurement often serves as an indicator of the local
environment of a fluorophore and its response towards excited state interactions. Lifetime
data obtained from the best fittings of the decay profiles and goodness of fits has been
established from the 2 criterion (2 will be within 0.95-1.05). The nature of decay of all these
systems was bi-exponential in nature and remained the same in presence of HSA. The
lifetime value was increased upon addition of HSA and the enhancement was more
pronounced in case of the complexes compared to their respective ligand counterparts.
Enhancement in the lifetime value is an indication of strong binding of the small molecule to
the polynucleotide [29].
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Fig. 7. Time-resolved fluorescence decay profile of L1H2, L2H2, 1 and 2 with increasing
concentration of HSA. Panel A: decay profile of free L1H2 (9.2 M,) treated with 12.66
M () and 42.2 M () of HSA; Panel B: decay profile of free 1 (9.2 M,) treated with
6.34 M (), 25.32 M () of HSA; Panel C: decay profile of free L2H2 (9.2 M,) treated
with 12.66 M () and 42.2 M () of HSA; Panel D: decay profile of free 2 (9.2 M,)
treated with 3.17 M (), 6.34 M () and 25.32 M () of HSA in 10 mM CP buffer of pH
7.0 at 25 C.
3.3.4. Thermodynamics of the interaction
Thermodynamic parameters for the association of present ligands and their copper(II)
complexes were calculated from the temperature dependent binding constants using
fluorescence spectroscopic studies in 10 mM CP buffer of pH 7.0. The values of the
thermodynamic parameters are tabulated in Table 4. The van’t Hoff plot for binding of L1H2
with HSA is shown in Fig. 8.
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Fig. 8. van’t Hoff plot for binding of L1H2 with HSA
Linear fit of the data indicates a very small value of heat capacity change (CP0). It can be
seen that in each case the binding was characterized by both negative enthalpy and entropy
changes.
Table4
Thermodynamic parameters for the interaction of ligands and complexes with HSA in 10 mM
CP buffer, pH 7.0 obtained from fluorescence spectrophotometry.a
Temperature
(in K)
288
K´ x 104
M-1
G° (kJ mol-1) H°(kJ mol-1)
TS° (kJ mol-1)
Sample
L1H2
1
at 25 ºC
at 25 ºC
at 25 ºC
4.67±0.28
2.02±0.15
1.10±0.09
11.92±0.58
6.21±0.42
-24.78±1.04
-51.64±1.93
-26.86±1.21
-25.71±1.09
298
308
288
-27.40±1.25
-53.11±2.01
298
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3.01±0.21
308
288
298
308
288
298
308
7.01±0.48
L2H2
3.51±0.24
1.63±0.13
15.83±0.72
8.01±0.53
3.74±0.25
-26.11±1.14
-28.05±1.29
-51.89±1.95
-53.76±2.03
-25.78±1.11
-25.71±1.09
2
a Average of three determinations
Our thermodynamic data revealed that the binding process was facilitated by negative
enthalpy change and was opposed by negative entropy change. The negative enthalpy change
for the binding process may be explained in the spirit of van der Waals’ stacking interactions
[11,30], hydrophobic as well as weak electrostatic interactions. Negative entropy change for
the interaction process may be attributed to the increase in order of the formation of rigid
complex.
3.4. Molecular docking study
We have docked 4 systems into the 3D structure of HSA using AutoDock Tools. These
models provide the most probable binding sites and poses in the protein that have not resulted
in structural alteration of HSA. The location of the fluorescence active Trp-214 moiety of
HSA is shown in Fig. 9. Trp-214 moiety resides in domain II of HSA. From Fig. 9, it is
obvious that all the systems reside near the Trp residue. As a result, so much perturbation of
fluorescence spectra of HSA has been observed in presence of all the systems. It is a point to
note that the difference in anchoring position between the ligands and their complexes was
not so significant. Here, molecular docking study was undertaken only to show the probable
location of binding of the ligands and their complexes.
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Fig. 9. Stereo view of the docked conformation of the systems with HSA in near vicinity (4
Å).
For copper(II) complexes, HSA offers two specific binding sites — the N-terminal site
(NTS) and the multi-metal binding site (MBS) [31]. The NTS strongly binds Cu(II) ions
preferentially over the MBS. This NTS comprises Asp-Ala-His, the first three amino acid
residues of the HSA sequence [31]. Employing nitrilotriacetic acid (NTA) as a low molecular
weight competitor, Rózga et al had earlier demonstrated experimentally through competitive
spectroscopic evaluation of conditional stability constant that the HSA binds copper(II) ions
at the NTS of HSA. From this study it is apparent that the conditional stability of a copper(II)
system is crucial for its binding at the NTS of HSA. To augment our present situation, we
have taken recourse to check the stability of L1H2, L2H2, 1 and 2 through absorption
spectrophotometric means maintaining the physiological pH under the same experimental
conditions of our HSA binding studies. Our exercise prompts us to predict that the observed
binding is most likely to the NTS of HSA.
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4. Conclusions
In this work, a novel osazone type Schiff base ligand, hexane-3,4-dione bis(2′-
pyridylhydrazone) and its monomeric copper(II) complex have been synthesized and
characterised through the available spectroscopic and analytical means. The X-ray crystal
structures of them have also been determined. The X-ray crystal structure of an analogous
ligand system, butane-2,3-dione bis(2′-pyridylhydrazone), has also been provided. Our
spectroscopic investigations reveal that these ligands and their Cu(II) complexes bind to
HSA, an important highly stable yet flexible blood plasma protein having 3D structure.
Thermodynamic parameters for the interaction of ligands and their complexes with HSA have
also been determined. Molecular docking studies also corroborate the binding of HSA with
them.
Acknowledgements
Financial support received from the Science & Engineering Research Board (SERB), DST,
New Delhi, Govt. of India under its Empowerment and Equity Opportunities for Excellence
in Science Scheme (F. No. SB/EMEQ-029/2013) is humbly acknowledged. M.L.Z and L.P.L.
appreciated the NSFC (Grant Nos. 21171109, 21271121 & 21571118), SRFDP (Grant Nos.
20111401110002 & 20121401110005), the Natural Science Foundation of Shanxi Province
of China (Grant Nos. 2010011011-2 & 2011011009-1) and the Shanxi Scholarship Council
of China (2012-004). We are thankful to the reviewers for their valuable suggestions. Thanks
are also due to Prof. Nitin Chattopadhyay, Department of Chemistry, Physical Chemistry
Section, Jadavpur University for various help.
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