Targeted water soluble copper-tetrazolate complexes: Interactions with biomolecules and catecholase like activities

Article abstract of DOI:10.1039/c5dt01471a

Two new mononuclear water soluble copper(ii) complexes, [Cu{(5-pyrazinyl)tetrazolate}₂(1,10-phenanthroline)] 1 and [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)₂](NO₃)_0.5(N₃)_0.52, have been synthesized using the metal mediated [2 + 3] cycloaddition reaction between copper bound azide and pyrazinecarbonitrile. The interactions of these copper tetrazolate complexes 1 and 2 with biomolecules like DNA and bovine serum albumin (BSA) are studied and the catecholase like catalytic activity of compound 2 is also explored. Structural determination reveals that both compounds 1 and 2 are octahedral in nature. Screening tests were conducted to quantify the binding ability of complexes (1 and 2) towards DNA and it was revealed that complex 2 has a stronger affinity to bind to CT-DNA. DFT studies indicated that a lower HOMO-LUMO energy gap between the DNA fragment and metal complexes might be the reason for this type of stronger interaction. DNA cleavage activity was explored by gel-electrophoresis and moderate to strong DNA cleavage properties were observed in the presence and absence of co-reagents. Inhibition of cleavage in the presence of sodium azide indicates the propagation of the activity through the production of singlet molecular oxygen. Furthermore enzyme kinetic studies reflect that complex 2 is also effective in mimicking catecholase like activities. An ESI-MS spectral study indicates the probable involvement of dimeric species [(phen)₂Cu-(OH)₂-Cu(phen)₂]²⁺ in the catalytic cycle.

Full text of DOI:10.1039/c5dt01471a

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Targeted Water Soluble Copper-tetrazolate Complexes : Interactions with Biomolecules
and Catecholase like Activities
Manideepa Saha,a Mriganka Das,a Rajendar Nasani,a Indrani Choudhuri,a Muhammed
Yousufuddin,b Hari Pada Nayek,c Mobin M Shaikh,a Biswarup Pathak,a Suman
Mukhopadhyay*a
aDepartment of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
IET-DAVV Campus, Khandwa Road, Indore 452017, India. Tel : +91 731 2438 705 Fax:
+91 731 2361 482 E-mail: suman@iiti.ac.in
bUniversity of North Texas at Dallas, Life and Health Sciences Department,7400 University
Hills Blvd, 300 Founder's Hall, Dallas, TX 75241, USA.
cDepartment of Applied Chemistry, Indian School of Mines, Dhanbad, 826004, Jharkhand,
India, E-mail:
Abstract
Two
new
mononuclear
water
soluble
copper
(II)
complexes
[Cu{(5-
pyrazinyl)tetrazolate}2(1,10-phenanthroline)] 1 and [Cu{(5-pyrazinyl)tetrazolate}(1,10-
phenanthroline)2](NO3)0.5(N3)0.5 2 have been synthesized using the metal mediated [2 + 3]
cycloaddition reaction between copper bound azide and pyrazinecarbonitrile. The interaction
of these copper tetrazolate complexes 1 and 2 with biomolecules like DNA and bovine serum
albumin (BSA) are studied and catecholase like catalytic activity of compound 2 is also
explored. Structural determination reveals that both compounds 1 and 2 are octahedral in
nature. Screening tests were conducted to quantify the binding ability of complexes (1 and 2)
towards DNA and it was revealed that complex 2 has a stronger affinity to bind towards CT-
DNA. DFT studies indicated that a lower HOMO-LUMO energy gap between the DNA
fragment and metal complexes might be the reason for this type of stronger interaction. DNA
cleavage activity was explored through gel-electrophoresis and moderate to strong DNA
cleavage property was observed in the presence and absence of co-reagents. Inhibition of
cleavage in the presence of sodium azide indicates the propagation of the activity through
production of singlet molecular oxygen. Furthermore enzyme kinetic studies reflect that
1

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complex 2 is also effective in mimicking catecholase like activities. ESI-MS spectral study
indicates probable involvement of dimeric species [(phen)2Cu-(OH)2-Cu(phen)2]2+ in the
catalytic cycle.
Introduction
Interactions of various 5-substituted tetrazoles with copper ions have been widely
investigated by different researchers in the past few years.1-14 However, the prime focus of
these kinds of studies remain limited to generation of metal-organic frameworks, where the
tetrazole ligand is able to participate in nine different types of coordination, with a vast array
of structural diversities having various topologies. Solvothermal synthesis of copper-
tetrazolate complexes mostly have taken control in the arena of generation of polymeric
materials.1,4,6,7 Some of these synthesized frameworks or mononuclear compounds have
shown interesting magnetic,1,6,7,11,13 catalytic,2 photoluminescence5,6 or gas absorption3,8,12
properties.
The binding of DNA with tetrazolo metal complexes has attracted much attention
recently because of their site specific binding properties and wide range of applications. The
interaction between DNA and copper tetrazolate complex are relatively rare in literature,15
however to the best of our knowledge the interaction of copper-tetrazolate complexes with
bovine serum albumin (BSA) or any kind of bioinspired catalytic activities of these
complexes have not been explored yet. The rational reasoning might be lying in the fact that
the solvothermally generated uncontrolled copper-tetrazolate frameworks are mostly
insoluble in nature that make them unsuitable for this kind of investigation. However, as per
our continuous endeavor to generate metal tetrazolate complexes in a more controlled and
environmental friendly condition, herein we report the synthesis and structural
characterization
of
mononuclear
copper
tetrazolate
complexes
[Cu{(5-
pyrazinyl)tetrazolate}2(1,10-phenanthroline)] 1 and
[Cu{(5-pyrazinyl)tetrazolate}(1,10-
phenanthroline)2](NO3)0.5(N3)0.5 2. Both the complexes 1 and 2 exhibit interesting
interactions with biomolecules like DNA and serum albumin protein. Furthermore,
compound 2 has shown interesting catecholase like activity which is also reported here.
Interaction of the complexes with DNA are further investigated with Density functional
theory.
2

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Results and Discussion
To tackle the problem of lower solubility of copper-tetrazolate complex and to explore the
possibilities in the domain of interaction with biomolecules, pyrazinecarbonitrile was chosen
as a prospective precursor of tetrazolate ligand with extra hydrogen bonding site in the form
of pyrazinyl nitrogen. A copper azide polymer complex [Cu(phen)(N3)2]n was chosen as the
starting material where the ratio between copper to azide was 1:2 inducing the possibility for
cycloaddition with two molecules to pyrazine nitrile to generate two chelating bidentate 5-
pyrazinyltetrazole ligands which along with one ancilliary 1,10-phenanthroline ligand
completes the octahedral geometry surrounding the copper center and helps to break down
the polymeric complex to a monomeric entity. Reaction between [Cu(phen)(N3)2]n and excess
of pyrazine carbonitrile in DMF at refluxing condition provides a mixture of compound 1 and
2 upon addition of NaNO3. ESI-MS indicates the presence of two separate species viz.
[Cu(phen)(pzta)2] 1 and [Cu(phen)2(pzta)]+ 2 [phen = 1,10-phenanthroline and pzta = 5-
pyrazinyltetrazolate] (Scheme 1 & Figure 1). The cationic complex 2 was solidified upon
addition of NaNO3 as [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)2](NO3)0.5(N3)0.5
2
(scheme 1). The mixture of compounds 1 and 2 were further separated by cation-exchange
chromatography using SP-Sephadex C25. The neutral complex 1 was obtained by eluting
with methanol where as the cationic complex 2 was isolated by elution with aqueous 0.1 M
NaNO3 solution. The compound 2 was also prepared through different method which
provides [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)2](NO3)0.5(N3)0.5
2
as the
exclusive product only. The [2 + 3] dipolar cycloaddition reaction between copper ligated
azide [Cu(phen)2N3](NO3) with pyrazine carbonitrile under refluxing condition in DMF
yielded a green residue upon concentration. This residue was dissolved in acetonitrile and
layered with diethylether to obtain a relatively small amount of green coloured crystals,
which were physically separated. Those crystals were then dissolved in acetonitrile and
layered with ether. This procedure was repeated several times to obtain an increasingly more
pure compound 2 and the remaining mother solution was rejected. Both the compounds ( 1
and 2) were characterized by IR, ESI-MS spectroscopy, elemental analyses and single crystal
X-ray diffraction method.
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N
N3
N3
N
N
N
N
N
Cu
N
N
N
N
N
Cu
N
N
N
Cu
N
N
N
N
N
N
N
N
N
N
N
1
N
N
N
N
CN
DMF , REFLUX
1 + 2
NO3
N3
Cu
NaNO3 , DMF ,
REFLUX
SEPARATED BY
N
ION EXCHANGE
RESIN COLUMN
N
CHROMATOGRAPHY
N
CN
N
N
(NO3)0.5 (N3)0.5
N
Cu
N
N
N
N
N
N
N
2
Scheme 1. Schematic representation for the synthesis of complex [Cu(phen)(pzta)2] 1
and [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)2] (NO3)0.5(N3)0.5 2.
4

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Figure 1: ESI-MS shows the presence of two separate species viz. [Cu(phen)(pzta)2 + Na]+ 1
and [Cu(phen)2(pzta)]+ 2 [phen = 1,10-phenanthroline and pzta = 5-pyrazinyltetrazolate]
Compound 1 shows a typical band at 1681 cm-1 for tetrazolate ligand instead of two
bands around 2057 cm-1 for ligated azide in starting compound.16 Compound 2 also confirms
the presence of free azide, tetrazolate and nitrate ion through their signature bands at 2111
cm-1 , 1628 cm-1 and 1377 cm-1 in IR spectrum, respectively.17 The ESI-MS spectrum for the
mixture of compounds shows two main peaks at 570.116 and 560.0786 corresponding to the
cationic complex of [Cu(phen)2(pzta)]+ 2 and molecular ion peak of [Cu(phen)(pzta)2 +
Na]+ 1, respectively (Figure 1). However after separation of the mixtures, the respective pure
compounds were further characterized through ESI-MS spectra (as shown in Figure S1 for
complex 1 and Figure S2 for complex 2). As the compounds 1 and 2 obtained show good
solubility in common polar solvents including water, we were tempted to check their
interaction with different biomolecules and explore the catecholase like activity since these
areas are relatively unexplored for copper tetrazolate complexes.
The exact structures of the copper complexes were determined by single crystal X-ray
crystallographic study. Compound 1 crystallizes in the space group P-1 (Table 1) and
displays two molecules with very similar geometries (Figure 2). The central ion is situated in
the distorted octahedral environment where both the tetrazole ligands act as chelating
bidentate fashion (Figure 2). The N-Cu-N bond angle in tetrazole ligands are found to be in
the range of 72-74° (Table 2), because of the very small bite. The Cu-N bond distances are
found to be within the expected range except the pyrazinyl nitrogen which are coordinated
axially and making distance around 2.4-2.5 Å, because of Jahn-Teller distortion.18
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Figure 2: Molecular structure of [Cu(phen)(pzta)2](H2O) 1 with the atomic numbering
scheme.
Compound 2 crystallizes in the space group P-1 with one Cu complex, one-half
azide, and one-half nitrate in the asymmetric unit for a Z value of 2 (Figure S3). The azide
and nitrate sit on inversion centers and are disordered. The disorder was treated with ISOR
restraints. The central atom exists in the center of a highly distorted octahedral geometry and
crystallizes in the space group P-1 (Table 1). The pyrazine nitrogen is found to be
coordinated to the metal center at a distance of 2.678 Å generating a semi-coordinated bond
(Table 3).19 Moreover, the small bite angle of the tetrazole ligand (70.8°) makes the molecule
further distorted (Table 2). The nitrate and azide counterion are further enjoined with each
other through complicated hydrogen bonding to form a three dimensional hydrogen bonded
network (Figure S4).
The crystal structure of complex 2 contains two disordered water molecules which
could not be modeled by discrete atoms. Their contribution was subtracted from the
diffraction pattern by the “SQUEEZE” method.
To ascertain the number of water molecules in the complex thermogravimetric
analysis was carried out. Complex 2 shows first weight loss of 5% within the range of 25-70
°C (Figure S5). This corresponds to the loss of four molecules of water of crystallization for
one unit cell.
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The electronic absorption spectra for both the complexes were recorded in methanol.
Complex 1 reveals a shoulder around 660 nm and a low intensity peak for complex 2 was
2
observed at 662 nm, characteristic of d-d transition band arising due to B1g
→
2Eg transition
of tetragonally distorted octahedral geometry.20 The presence of other electronic transitions
which appear in the UV region can be correlated to the π→π* transition in the ligand (Table
4).21
To explore the possibility of these copper complexes binding with CT-DNA (which
has been found to be a critical step for the chemical nuclease activities of metal complexes)
DNA was added over several steps with increasing amounts while changes in the absorption
spectrum of the metal complex were monitored. It has been reported that interaction between
metal complexes and DNA bases may lead to intercalation where hypochromic shift in the
UV region was found to be a manifestation of that phenomena.22,23 Furthermore a
bathochromic shift indicates the decrease in the energy gap between the highest occupied and
lowest unoccupied molecular orbital when the complex binds to DNA.24 Absorption spectral
titration carried out for compounds 1 and 2 reveals a decrease in molar absorptivity with a
slight bathochromic shift indicating a possible interaction between aromatic moieties of the
complex with DNA base pairs and stabilization of CT-DNA duplex. In the presence of DNA,
complex 1 exhibited hypochromism of 10% at 275 nm (Figure S6) where as for complex 2 it
increased to 20% (Figure S7), revealing considerable interaction between complexes and
DNA. These interactions are attributed to π→π* transition of the complex. The
hypochromicity and bathochromic shift indicates the intercalative mode of binding and
considerable stacking interaction between aromatic chromophore and base pairs of DNA. The
plot of [DNA]/[εa - εf] versus [DNA] (where εa = Aobs/[compound] and extinction coefficient
of the free compound) gives a straight line and the ratio of the slope to the intercept gives the
intrinsic binding constant Kb. The values of the intrinsic binding constant for 1 (Figure S8)
and 2 (Figure S9) were found to be 2.02 X 104 M-1 and 2.88 X 104 M-1 respectively. These
values are moderate in nature with respect to the other ternary copper complexes reported so
far. 25
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240
200
160
120
80
40
0
550
600
650
700
750
800
Wavelength (nm)
Figure 3: ETBr displacement assay by change in fluorescent intensity of EB with respect to
conc.of complex 1.
To order to obtain further information and investigate the interaction mode between the
complex and CT-DNA, fluorescence titration experiments were carried out using ethidium
bromide (EB) as a spectral probe. EB shows reduced emission intensity in buffer because of
significant solvent quenching, however in presence of CT-DNA the emission intensity gets
enhanced because of intercalative binding of EB to DNA. In presence of metal complex
which competes with EB to bind the DNA a noteworthy reduction of emission intensity are
generally observed.24 In our experiment the fluorescence intensity at 607 nm showed
significant decreasing trend with increasing concentration of complex 1 (Figure 3) and 2
(Figure 4) due to the displacement of DNA bound EB by copper complex. The Kq value
which was obtained from Stern-Volmer plot was found to be 4.18 X 103 M-1and 3.30 X 104
M-1 for complex 1 (Figure S10) and 2 (Figure S11), respectively. This shows a greater
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interaction for compound 2 over compound 1. The corresponding Kb values obtained from
the Scatchard plot (Figure S12 and Figure S13) where found to be 1.27 X 104 M-1 and 1.73 X
105 M-1, respectively. The values obtained are consistent with other values reported for
similar copper complexes.26,27
240
200
160
120
80
40
0
550
600
650
700
750
800
Wavelength (nm)
Figure 4: ETBr displacement assay by change in fluorescent intensity of EB with respect to
conc. of complex 2.
DFT Study:
In order to understand the DNA binding with the octahedral Cu-complexes (1 and 2), we
have performed the density functional theory (DFT) study to investigate the role of the
frontier orbitals towards such binding. The DNA structure is modelled as a deoxy-guanosine
monophosphate (dGMP)28 unit where one of the phosphate oxygen is methylated. Therefore,
the total charge of the dGMP unit is negative. All the calculations are carried out in water
solvent using B3LYP level of theory as implemented in the Gaussians 09 package.29 The 6-
311++G** basis set is used for the main group elements (C, N, O, P, and H) and LANL2DZ
ECP basis set is used for Cu.30,31 The solvent calculations are carried out using the polarizable
continuum model32 as implemented in the Gaussian 09.
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The optimized Cu complexes (1, 2) and dGMP structures are presented in S14. We have
done the time dependent density functional theory (TDDFT) study33-39 on the Cu complexes
to understand their absorption spectrum. Our calculated results (Figure 5) show that the
complex 1 and 2 absorbs at 288.38 nm and 296.31 nm respectively. The oscillator strength
and expansion coefficients calculated for these two transitions are 0.057 and 0.68 for
complex 1 and 0.031 and 0.36 for complex 2 respectively. The calculated values are very
much in agreement with the experimental absorption spectra of the metal complexes. Our
molecular orbital study shows that such transition corresponds to the ligand to metal charge
transfer (LMCT) transition (Figure 5), which agrees well with our experimental findings.
Figure 5: The calculated absorption (λmax) corresponds to the LMCT of the Cu-complexes.
We have studied the frontier orbitals (Figure 6) of the dGMP and the Cu-complexes (1
and 2) to understand their role towards the binding. It is well established that the HOMO of
the DNA fragments donate electron to the LUMO of the metal complexes.40,41 So, we have
calculated and presented (Figure 6) the HOMO energy of the dGMP structure with respect to
the LUMO energies of the Cu-complexes (1-2). The HOMO orbital energy of the dGMP
fragment is -3.66 eV and LUMO energies of the 1 and 2 are -3.26 eV and -3.65 eV
respectively. Therefore the HOMO orbital energy of the dGMP fragment (-3.66 eV) is very
close to the LUMO energy of 2 (-3.65 eV) than 1 (-3.26 eV). So the electron transfer from
dGMP fragment to metal complexes is easier for 2 than 1. Such finding agrees well with the
experimental DNA binding constant of complexes 1 and 2.
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Figure 6: The position of the frontier orbitals of dGMP fragment and Cu-Complexes (1 and
2).
We have also modelled the complexes (1 and 2) bonded to the dGMP fragment (Figure 7).
Here we consider, the dGMP fragment attacks the metal centre via the negatively charged O
-
atom of the PO4 group as shown in figure 7. Both the dGMP-bonded Cu-complexes are
relaxed and found to be minima in their potential energy surfaces. We find the Cu-dGMP
complex 2 is more distorted (Figure 7) than complex 1. We have calculated the binding
energy of the Cu complexes with dGMP fragments using the following equation.
EB = E(Cu-dGMP) – (EdGMP + ECu-complex
)
The energy of the E(Cu-dGMP) is taken from the energy of the optimized structure of Cu- dGMP
complex and the energy of the EdGMP and ECu-complex are taken from the single point energy of
the fragments within the geometry of the Cu-dGMP complex. Hence the single point energy
is calculated by deleting the fragments one by one from the optimized geometry of the Cu-
dGMP complexes.
The calculated binding energy between the dGMP and complex 1 and 2 are –0.59 Kcal/mol
and –2.51 Kcal/mol respectively. Therefore the binding energy of the complexes (1 and 2)
show similar trend as we found through our frontier orbitals study, which in turn agrees well
with the experimental trend of the DNA binding constant.
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(b)
(a)
4.76 Å
Cu
O
O
4.71 Å
Cu
O
P
O
P
O
O
O
1
2
Figure 7: Optimized geometry of the Cu-dGMP complexes (1 and 2).
As both the complexes have shown considerable DNA binding ability therefore we
started to explore the DNA cleavage activities for both the Cu tetrazolate complexes 1 and 2.
Plasmid pBR322 DNA was taken as the substrate in a medium of 50 mM Tris-HCl buffer
(pH= 7.2) and the DNA cleavage activity was studied under physiological conditions. It is
well known that circular plasmid DNA under conduction by electrophoresis splits into several
bands. Fastest migration happens for supercoiled form (Form I). If one strand gets cleaved
then nicked circular form is produced which moves in a slower manner with respect to
supercoiled form (Form II). A linear form (Form III) is obtained when both strands are
cleaved and it migrates between Form I and Form II.42 Cleavage of supercoiled (SC) DNA
(Form I) to nicked circular (NC) DNA (Form II) was observed for both the complexes by
varying the concentration of the test solutions. The experiments were carried out in the
concentration range of 50μM-400μM for the complexes 1 and 2 and the results obtained after
1 hour of incubation at a temperature of 40 °C are displayed in Figure 8a and Figure 8b
respectively. Examination of these figures revealed that in control experiment no measurable
DNA cleavage occurred when supercoiled pBR322 plasmid DNA was incubated whereas in
presence of copper complex substantial cleavage of DNA was observed even in the absence
of any co-reagents. This cleavage is more prominent in the case of complex 2 ( Figure 8b)
where almost 91% of the DNA is cleaved to form nicked circular strands even in the absence
of oxidizing agent H2O2 at 1μM concentration. With increasing concentration of copper
complexes an enhanced DNA cleavage was observed. This cleavage may be due to the
increased reaction of copper ions with H2O2, thereby producing diffusible hydroxyl radicals
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or molecular oxygen, both of which are capable of damaging DNA by Fenton-type
mechanism.43 In the range of 50μM complex concentration, complex 2 possesses a superior
DNA cleavage capability than complex 1 where the percentage of nicked circular is 46.2 for
complex 1 (Table 5) and 94.8 in case of complex 2 (Table 6). It was also observed that higher
concentration range of metal complexes 1 and 2, promotes depletion of the supercoiled DNA
form, with the consequent formation of linear DNA as a result of double stranded breaks. To
explore the cleavage mechanism DNA cleavage was investigated with different potential
radical scavangers to find out the intermediate which are possible reactive oxygen species
(ROS) in presence and absence of external cofactors. DMSO and ethanol were used as a
hydroxyl radical (HO˙) scavenger and sodium azide was used as a singlet oxygen (1O2)
scavenger and SOD was used as a Superoxide radical Scavenger. The results indicate that
NaN3 inhibits more than the other two scavengers to cleave DNA from SC form to NC form
(Figure S15). This noticeable inhibition was observed with NaN3, indicating the involvement
of the Cu(II) complexes to cleave the SC form of pBR322 DNA by producing singlet
molecular oxygen. 44
The groove binding ability of complex 2 was studied in the presence of minor groove binder
DAPI and major groove binder methyl green. As shown in figure S15, in presence of methyl
green the band darkness for nicked circular DNA is very less. So it may be concluded that
methyl green inhibits the cleavage of SC DNA to NC DNA, suggesting that the Cu complex
2 preferentially interacts through the major groove of the DNA helix.
Figure 8a: Agarose gel electrophoresis cleavage showing cleavage of pBR322 supercoiled
DNA by complex 1 after 1 hr of incubation at different concentration; Lane 1: DNA control;
Lane 2: DNA+H2O2; Lane 3: DNA+H2O2+1(50µM); Lane 4: DNA+ H2O2+1(100µM); Lane
5: DNA+ H2O2+1(200µM); Lane 6 : DNA+ H2O2+1(400µM); Lane 7 : DNA+ 1(50µM) .
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Figure 8b: Agarose gel electrophoresis cleavage showing cleavage of pBR322 supercoiled
DNA by complex 2 after 1 hr of incubation at different concentration; Lane 1: DNA control;
Lane 2: DNA+H2O2; Lane 3: DNA+H2O2+2(50µM); Lane 4: DNA+ H2O2+2(200µM); Lane
5: DNA+ H2O2+2(400µM); Lane 6 : DNA+ 2(50µM) .
Interactions of bovine serum albumin (BSA) with copper complexes were studied
through spectroscopic methods. It is well known that the interaction between metal complex
and protein leads to the quenching of the fluorescence of the BSA protein and it may happen
because of dynamic or static interaction.45 In a dynamic mechanism the fluorescent molecule
undergoes change only in the excited state whereas in a static quenching mechanism the
interaction takes place at the ground state leading to considerable change in the absorption in
UV-vis spectroscopy. The absorption peak for BSA is visible at 278 nm arising from the
presence of aromatic amino acids (Trp, Tyr and Phe),46 while with addition of both the
complexes 1 and 2, individually the absorption increases with a concomitant slight blue shift
accompanied by it (Figure S16 and Figure S17). This is an indication of the interaction
between BSA and the complex at ground state. This leads to conformational changes in BSA
and induces a change in the polarity of the microenvironment around Tyr and Trp residues of
BSA.47
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80
70
60
50
40
30
20
10
800
600
400
200
0
0
0
20
40
60
-6
80
100
[ Q ] X 10 (M)
300
320
340
360
380
400
420
440
460
Wavelength (nm)
Figure 9: Fluorescence quenching of BSA by complex 1. Stern–Volmer plot is in the inset.
To investigate further Trp-emission quenching experiments were carried out using BSA
in the presence of increasing amounts of complexes 1 and 2. A fixed amount of BSA solution
was titrated with an increasing concentration of each complex. A continuous decrease of
fluorescence intensity for the fluorophore was observed accompanied with a red shift (Figure
9 and Figure 10). The fluorescence emission was observed at 340 nm upon excitation at 290
nm. To get more insight concerning the interaction between complex and BSA the obtained
result was analysed by the Stern-Volmer equation48 :
Fꢀ
ꢂ ꢃ
= 1 + kꢁτꢀ Q = 1 + Kꢄꢅ
ꢂ ꢃ
Q
F
where F0 and F are the fluorescence intensities in the absence and the presence of a quencher,
kq is the bimolecular quenching rate constant, τ0 is the average lifetime of fluorophore in the
absence of a quencher and [Q] is the concentration of a quencher (Metal complexes). In our
case a non-linear plot with an increasingly upward trend was obtained (Figure 9 and Figure
10).
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1000
800
600
400
200
0
350
300
250
200
150
100
50
0
-50
0
20
40
60
80
100
-6
[ Q ] X 10 (M)
300
350
400
450
Wavelength (nm)
Figure 10: Fluorescence quenching of BSA by complex 2. Stern–Volmer plot is in the inset.
This positive deviation indicates a probable two way quenching by collision and as well
as by complex formation with the same quencher.48 KSV is the Stern–Volmer quenching
constant in M−1 which can be determined by linear regression of F0/F against [Q]. The
calculated value of the Stern–Volmer quenching constant (KSV), the bimolecular quenching
rate constant (kq), and the number of binding sites (n) for both the complexes 1 and 2 are
listed in Table 7. The values of the quenching constant from Stern–Volmer plot were found to
be in the order of 1013. This value also indicates that there is a larger possibility that the
quenching mechanism of fluorescence of BSA by two different complexes is initiated by
static quenching process.47 The binding constant value (Kb) and the number of binding sites
(n) from Scatchard plot were obtained from the equation :
Fꢀ − F
log ꢆ
ꢇ = logKꢈ + n logꢂQꢃ
F
The value of n was found to be more than 2 indicating that there might be two probable
binding sites available in BSA for all the complexes (Figure S18 and Figure S19). The value
of Kb indicates a very strong interaction between copper complexes and BSA.
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To explore the possible catecholase like activity of the copper complex 3,5-di-tert-
butylcatechol (3,5-DTBC) was taken as the prospective substrate as the compound shows a
low quinone-catechol reduction potential.48 The reactions were carried out at 25°C in
presence of air and monitoring was done by UV-vis spectroscopy. The experiments revealed
that whereas complex 2 is quite active towards catalytic oxidation of 3,5-DTBC, complex 1
has not shown any such tendency. In case of complex 2 appearance of a new band at 440 nm
occurred instantaneously upon addition of catechol which obscures the peak which is
supposed to be observed at around 400 nm for the formation of benzoquinone. The peak
observed at 440 nm may be attributed to ligand to metal charge transfer band because of the
binding of phenoxo moiety to copper ion (Figure 11).21 However, the reaction was monitored
for 90 minutes and a hint of another peak gradually appears at 402 nm with time.
0.4
3,5 DTBC
Complex
0.3
Increment of quinone
band for 3,5 DTBQ
0.2
0.1
0.0
300
350
400
450
500
Wavelength (nm)
Figure 11: Spectral pattern of catecholase activity over time for complex 2 after addition of
3,5-DTBC.
To obtain the rate constant for the catalyst complex traditional initial rate method was
employed and on the basis of the Michaelis–Menten approach of enzymatic kinetics, the
observed rate versus substrate concentration data were then analyzed (Figure S20).
Linearization of Lineweaver−Burk plot (Figure S20) provides the value of Michaelis−Menten
constant (KM) and maximum initial rate (Vmax) which were found to be 0.00367 (M) and
0.0022 M min-1. The turnover number values (kcat) obtained was 1.32 X 104 h-1. These results
clearly show the ability of complex 2 to effectively catalyse the oxidation of 3,5-DTBC . All
the obtained kinetic parameters are presented in Table 8.
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To look further into the probable mechanism, compound 2 was subjected to
electrospray ionization mass spectrum in methanol solution which exhibits three assignable
peaks at 570.45, 423.36 and 390.32 arising out of [Cu(phen)2(pzta)]+, [Cu(phen)2]+ (copper
(II) gets reduced to copper (I) at ESI-mass spectroscopy condition) and [Cu(phen)(pzta)]+
(Figure S2). To find out the possible complex-substrate intermediate ESI-MS positive
spectrum was taken for a mixture of complex 2 and 3,5-DTBC in 1:100 molar ratio and
recorded in 5 minutes interval. The spectra obtained was having lot of peaks indicating
extensive fragmentation of the complex, substrate or complex-substrate intermediate.
However, the peak at 243.1 is assigned as quinone-sodium aggregate [(3,5-DTBQ)Na]+.
Among the remaining peaks the peak at 301.1 could be assigned for {[(phen)2Cu-(OH)2-
Cu(phen)2]2+ + Na+} and 339.2 could be for [Cu(phen)2(3,5-DTBCH)(O2)-H]2+ (Figure S21).
Dimeric species similar to [(phen)2Cu-(OH)2-Cu(phen)2]2+ are also reported elsewhere as a
probable intermediate for catcholase mimicking activity.49 Identification of these two
probable species helps us to understand and propose a possible mechanism for the catalytic
process. To get further insight into the probable mechanism we have carried out qualitative as
-
well as quantitative detection of I3 band (~353nm) by UV-Vis spectroscopy for indication of
formation of H2O2 during catalytic oxidation procedure. The experiment indicates the
-
oxidation of I- to I2 followed by the generation of I3 ,which was qualitatively detected by UV-
Vis spectral study of solution which is the outcome of the reduction of dioxygen to H2O2 as
reported before (Figure S22).48 Quantitative analysis of H2O2 indicates that 0.85 mol (≈1) of
H2O2 was shown to be produced per mol of 3,5 DTBC along with formation of 1 mol 3,5
DTBQ, which strongly supports the mechanism of reaction involving a two electron
reduction process of areal oxygen. Taking account of all the above experimental results a
reaction mechanism is proposed (Scheme 2) in which the oxidation process occurs through
catecholate bound copper(I) center (A) which gets generated by elimination of tetrazolate ion
from the copper center and subsequent coordination by catechol in an unidentate manner
keeping the option open for aerial oxygen to get attached to the copper(I) center to reoxidize
it to copper (II) superoxide intermediate (B). The next step involves the intramolecular proton
transfer process from oxygen atom of catechol moiety to the superoxide oxygen atom to
produce a peroxo-intermediate (C) which upon reacting with moisture of the solvent
molecule is generating the dihydroxo bridged copper (II) intermediate [(bipy)2Cu-(OH)2-
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Cu(bipy)2]2+ (D) as evidenced by ESI-MS spectroscopy. Intermediate (D) upon further
reaction with DTBC regenerates the copper (I) center (A) to complete the catalytic cycle.
Conclusion
In conclusion two new octahedral water soluble copper complexes [Cu{(5-
pyrazinyl)tetrazolate}2(1,10-phenanthroline)]
1
and [Cu{(5-pyrazinyl)tetrazolate}(1,10-
phenanthroline)2](NO3)0.5(N3)0.5 2 were synthesized by [2 + 3] dipolar cycloaddition between
copper ligated azide and pyrazinecarbonitrile. Single crystal X-ray diffraction (XRD) results
for the complexes 1 and 2 revealed distorted octahedral geometry around the metal ion.
Interaction with DNA and BSA binding studies of water soluble copper tetrazolate
compounds have been explored. Both the complexes have shown binding ability to CT-DNA
where compound 2 was found to be a stronger binder. DFT studies indicated that a lower
HOMO-LUMO energy gap between DNA fragment and metal complexes might be the
reason behind this kind of stronger interaction. Furthermore both the complexes 1 and 2 have
shown DNA nuclease property in presence and absence of additional co-reagents.
Inhibitation of DNA cleavage activity in presence of sodium azide indicates the involvement
of singlet molecular oxygen in mechanistic pathway. The interaction of compound 1 and 2
with albumin protein show intense interaction between metal complex and protein with very
high fluorescence quenching. The Stern–Volmer quenching constant value obtained was to
the tune of 1013 M-1 showing that both static and dynamic quenching occurring
simultaneously. Apart from the above mentioned interaction with biomolecules complex 2
exhibited promising catecholase like activity with TON value to the order of 104 h-1. ESI-MS
study indicates the mechanistic pathway of the reaction probably involves a dimeric copper
intermediate [(phen)2Cu-(OH)2-Cu(phen)2]2+ as cited in various previous reports.
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t
Bu
N
2
N
N
N
N
N
N
t
2
HO
Bu
CuII
N
CuII
CuI
N
2
OH
2
N
N
N
N
N
O
O
N
- 2C5H4N6
t
t
N
Bu
Bu
N
N
HO
HO
t
t
t
Bu
Bu
Bu
( A )
2
2 O2
t
HO
Bu
OH
N
N
H
CuII
N
CuII
N
N
2
O
( B )
N
N
N
CuII
N
N
O
O
H
N
O2
t
Bu
N
HO
t
Bu
( D )
t
Bu
O
O
H O
2
2
2
2
t
Bu
N
N
H
2O
2
CuII
2
N
N
O
O
t
Bu
HO
O
t
Bu
( C )
Scheme 2: Probable catalytic cycle of oxidation of 3,5-DTBC by Cu(II) octahedral
complex 2.
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Experimental Section
Materials and physical measurements
Reagents and Materials. All reagents and solvents were purchased commercially and were
used as received. Copper(II) nitrate trihydrate, 1,10-phenanthroline monohydrate, sodium
azide and pyrazine carbonitrile were purchased from Merck-India Chemical Company. Calf
thymus DNA, bovine serum albumin (BSA, fraction V powder), ethidium bromide (EB),
Agarose Low EEO Superior grade type II gel, supercoiled plasmid pBR322 DNA, DAPI
dihydrochloride (4’,6-diamidino-2-phenylindole dihydrochloride), methyl green and
superoxide dismutase (SOD) were purchased from SRL (India). Tris(hydroxymethyl)
aminomethane-hydrochloride (Tris-HCl) buffer was prepared using distilled water.
Methods and Instrumentation. Infrared spectra (4000 to 500 cm−1) were recorded with a
BRUKER TENSOR 27 instrument in KBr pellets. Mass spectrometric analyses were done
on Bruker-Daltonics, microTOF-Q II mass spectrometer and elemental analyses were carried
out with a Thermo Flash 2000 elemental analyzer. Spectrophotometric measurements were
performed on a Varian UV-Vis spectrophotometer (Model: Cary100) (for absorption) and a
Fluoromax-4p spectrofluorometer from Horiba JobinYvon (Model: FM-100) (for emission)
using a quartz cuvette with path length of 1 cm.
Caution! Azide and tetrazolate compounds are potentially explosive. Only a small amount of
material should be prepared and handled with care.
X-ray crystallography
Single crystal X-ray structural studies of compound 1 and 2 were performed on a CCD
Agilent Technologies (Oxford Diffraction) SUPER NOVA diffractometer. Data for both the
complex 1 and 2 were collected at 150(2) K using graphite-monochromatic Mo Kα radiation
(λα = 0.71073 Å) and Cu Kα radiation (λα = 1.54184 Å) respectively. The strategy for the data
collection was evaluated using the CrysAlisPro CCD software. The data were collected by
the standard phi-omega scan techniques and were scaled and reduced using the CrysAlisPro
RED software. The structure was solved by direct methods using the SHELXS-97 and refined
by full matrix least-squares with the SHELXL-97 for compound 1 and SHELX-2014 for
compound 2, refining on F2. 50
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The positions of all the atoms were obtained by direct methods. All non-hydrogen
atoms were refined anisotropically. The remaining hydrogen atoms were placed in
geometrically constrained positions and refined with isotropic temperature factors, generally
1.2Ueq of their parent atoms. The contribution of solvent electron density was removed by the
SQUEEZE routine in PLATON.51
Synthesis. [Cu(phen)(N3)2]n and [Cu(phen)2N3](NO3)·(H2O) was synthesized according to
the literature procedure. 52, 53
Synthesis of [Cu(phen)(pzta)2]·(H2O) (1·H2O) and
[Cu(phen)2(pzta)](NO3)0.5(N3)0.5·2(H2O) (2·2H2O) :
A mixture of [Cu(phen)(N3)2]n (120 mg, 0.367 mmol), 2 mL of pyrazine carbonitrile and 3
mL of DMF was refluxed for 12 hrs . 30 mg of NaNO3 was added and the reaction mixture
was stirred for 30 mins at room temperature. The solvent was then removed in vacuo and the
resulting residue was dissolved in methanol and filtered. The filtrate was concentrated to 2-3
ml. This greenish brown compound was found to be a mixture of two compounds viz.
[Cu(phen)(pzta)2] and [Cu(phen)2(pzta)](NO3)0.5(N3)0.5. The mixture was separated by ion-
−
exchange chromatography using SP-Sephadex C-25, which is a strongly acidic (–SO3 Na+ )
cation exchanger based on cross-linked dextran. Methanol was used to elute the neutral
complex [Cu(phen)(pzta)2] 1 and 0.1 M NaNO3 solution was used as the elution solvent to
run the column for the elution of the charged complex [Cu(phen)2(pzta)](NO3)0.5(N3)0.5 2.
Complex 1·H2O was recrystallized from methanol–diethyl ether mixture to get suitable X-ray
diffraction-quality crystals. Anal. Calc. for compound 1.H2O: C44H32Cu2N28O2 (1112.02) : C
47.52; H 2.90; N 35.27%. Found: C 46.89; H 2.45; N 34.95%. ESI-MS (positive ion,
CH3OH) m/z: 537.7552 [Cu(phen)(pzta)2]+ (calcd 537.0822). IR (cm−1, KBr): 3426,
1681,1642, 1523, 1023. Complex 2·2H2O was recrystallized from acetonitrile–diethyl ether
mixture to get suitable X-ray diffraction-quality crystals. Anal. Calc. for compound 2·2H2O
: [C29H19N10Cu](NO3)0.5(N3)0.5·2(H2O) (659.13) : C 52.79; H 3.48; N 25.48%. Found: C
52.38; H 3.25; N 24.96%. ESI-MS (positive ion, CH3OH) m/z: 570.1 [M–NO3]+ (calcd
570.109). IR (cm−1, KBr, selected peak): 3414, 1624, 1385. IR (cm−1, CH2Cl2, selected peak):
3424, 2111, 1628, 1377.
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Synthesis of exclusive [Cu(phen)2(pzta)](NO3)0.5(N3)0.5·2(H2O) (2·2H2O) :
A mixture of [Cu(phen)2N3](NO3)·(H2O) (120 mg, 0.367 mmol), 2 mL of Pyrazine
carbonitrile and 3 mL of DMF was refluxed for 12 hrs to get a clear green colour solution.
The solvent was then removed in vacuo to obtain green colour residue upon concentration.
This residue was dissolved in acetonitrile and layered with diethylether to obtain a relatively
small amount of green coloured crystals, which were physically separated. Those crystals
were then dissolved in acetonitrile and layered with ether. This procedure was repeated thrice
to obtain an increasingly more pure compound 2 and the remaining mother solution was
rejected. Anal. Calc. for compound 2·2H2O : [C29H19N10Cu](NO3)0.5(N3)0.5·2(H2O) (659.13)
: C 52.79; H 3.48; N 25.48%. Found: C 52.38; H 3.25; N 24.96%. ESI-MS (positive ion,
CH3OH) m/z: 570.1 [M–NO3]+ (calcd 570.109). IR (cm−1, KBr, selected peak): 3414, 1624,
1385. IR (cm−1, CH2Cl2, selected peak): 3424, 2111, 1628, 1377.
Solubility and Stability. The complexes are highly soluble in H2O and MeOH . They were
stable in the solid and solution phases.
DNA binding experiments. The experiments involving the interaction of the complexes with
calf thymus (CT) DNA were carried out in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4). A
solution of calf thymus DNA dissolved in this buffer gave a ratio of UV absorbance at 260
and 280 nm of about 1.8 : 1, suggesting the CT-DNA are sufficiently free from protein. The
concentration of CT-DNA was estimated from its absorption intensity at 260 nm with a
known molar extinction coefficient value (ε) of 6600 M-1cm-1 after 1 : 100 dilution. The stock
solution of CT-DNA in buffer was stored at 4 °C and used within 4 days. Absorption titration
experiments were carried out by varying the concentration of CT-DNA from 0-200ꢀM while
keeping the metal complex concentration constant at 10ꢀM. Due correction were made for
the absorbance of CT-DNA by adding an equal quantity of CT-DNA to both the complex
solution and reference solution during titration. Moreover, DNA binding for both the
complexes was measured by a special fluorescence spectral technique; ETBr displacement
assay from ETBr bound CT-DNA in Tris-HCl buffer at biological pH 7.4. The fluorescence
intensities of ethidium bromide (20 ꢀM) at 605 nm (520 nm excitation) bound to DNA were
measured with respect to concentration of the complex (0 to 100 ꢀM). There was no apparent
emission of Ethidium bromide in Tris-buffer medium (pH 7.4) because of fluorescence
quenching of the free ethidium bromide by the solvent molecules. But in the presence of CT-
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DNA, ethidium bromide shows appreciably improved emission intensity due to its
intercalative binding to DNA. The competitive binding of the copper complexes to CT-DNA
resulted the displacement of the bound EtBr which was manifested in decreasing its emission
intensity.
Interaction between DNA and complexes. Absorption titration method has been used to
monitor the mode of interaction of the complexes with CT-DNA. The intrinsic equilibrium
DNA binding constants (Kb) of the complexes with DNA are determined by monitoring the
change in the absorption intensity of the charge-transfer spectral bands of the compounds
with increasing concentration of the DNA. In the experiment, there is addition of CT-DNA
from 0-200 ꢀM to the aqueous solution of complex 1(12ꢀM) and complex 2(10ꢀM)
respectively. An intercalative binding of the complexes to DNA normally leads to
hypochromism along with a bathochromic ( red) shift of the electronic spectral bands. From
the changing tendency of UV spectras and the binding constants, we can conclude that the
intercalative binding of complex 2 is more than complex 1.
BSA Interaction Studies. The protein interaction study was performed by tryptophan
fluorescence quenching experiments using BSA in TRIS-HCl buffer (pH
∼ 7.4) with
excitation at 295 nm and the corresponding emission at 340 nm, using a Fluoromax-4p
spectrofluorometer [from Horiba JobinYvon (Model: FM-100)] with a rectangular quartz
cuvette of 1 cm path length. Concentrated stock solutions of complexes 1 and 2 were
prepared by dissolving them separately in TRIS-HCl buffer and diluted suitably with TRIS-
HCl buffer to get the required concentrations. Quenching of the emission intensity of
tryptophan residue of BSA (2 ml, 10ꢀM) was monitored using complexes 1 and 2 (0-100
ꢀM) as quenchers with increasing complex concentration. Furthermore, the interaction with
proteins was also studied by measuring the increment in the absorption band at 278 nm in
UV-Vis spectroscopy through successive addition of 0 to 100ꢀM of complex 1 and 2 in 10
ꢀM protein solution.
Catecholase activity study :.
In order to study the ability of the Cu(II) complexes to oxidize 3,5-DTBC, 2 × 10−5
M
methanolic solutions of complexes 1 and 2 were treated with 2 × 10−3 M (100 equiv) of 3,5-
DTBC under aerobic conditions at room temperature. In the UV−vis spectroscopy, where the
absorbance of the resultant reaction mixture was plotted with respect to wavelength 300-500
nm, the time-dependent spectral scans for the complex 2 is plotted in Figure 11 . From the
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figure, it is apparent that a band
of 3,5-DTBC because of the gradual increment of the concentration of 3,5-DTBQ (3,5-DTBQ
exhibits λmax 400 nm in methanol) while no change is noticed in the spectral pattern with
∼398 nm is observed to increase with time after the addition
∼
complex 1. These data definitely reveal that complex 2 is an active catalysts for the aerial
oxidation of 3,5-DTBC to 3,5-DTBQ, whereas complex 1 turns out to be inactive.
For the complex 2, the kinetics for the oxidation of the substrate 3,5-DTBC was
determined by the initial rate method at 25 °C. The substrate solutions of concentration
3
ranging from 0.002 to 0.01 mol dm− were prepared in methanol from a concentrated stock
solution. A total of 2 mL of the substrate solution was poured into a 1 cm spectrophotometer
quartz cell thermostatted at 25 °C. Then 1 mL of a 0.00002 M methanolic solution of
complex 2 was quickly added to it so that the ultimate concentration of the complex became 1
5
3
× 10− mol dm− . The dependence of the initial rate on the concentration of the substrate was
spectrophotometrically monitored at the respective wavelength. Furthermore, the initial rate
method display a first-order kinetics on the complex concentration and shows a saturation
kinetics at higher substrate concentrations based on Michaelis
constant (KM), maximum velocity (Vmax), and rate constant for dissociation of the substrates
(i.e., turnover number, kcat) were calculated for the complex 2 using the Lineweaver Burk
−Menten model. The binding
−
graph of 1/V versus 1/[S], (FigureS18) , with the equation 1/V = (KM/Vmax)(1/[S]) + 1/Vmax
,
and all the obtained kinetic parameters are presented in table 8.
Iodometric method was followed to detect the formation of hydrogen peroxide during
the catalytic reaction. The reaction mixtures were used as it was prepared in the kinetic
experiments. After 45 mins of the reaction, an equal amount of water was added, to prevent
further oxidation. Then the formed quinone was extracted three times with dichloromethane
and the solution was acidified to pH = 2 with H2SO4. To the aqueous layer, 1 mL of 10% KI
-
solution and two drops of 3% ammonium molybdate solution was added. The formation of I3
-
was monitored by UV-Visible spectrophotometer due to the formation of the characteristic I3
band at λ = 353 nm. ( ε = 14100 M-1 cm-1).
Acknowledgement
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We are grateful for the financial support received from the Council of Scientific and
Industrial Research, New Delhi. M. S. thanks CSIR for the award of SRF. We are also
thankful Sophisticated Instrument Center, IIT Indore for the structure elucidation.
Notes and references
aDepartment of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
IET-DAVV Campus, Khandwa Road, Indore 452017, India; Fax : (+91)-731-2361482; Tel:
(+91)-731-2438705; E-mail : suman@iiti.ac.in
bUniversity of North Texas at Dallas, Life and Health Sciences Department,7400 University
Hills Blvd, 300 Founder's Hall, Dallas, TX 75241, USA. E-mail: myousuf@untdallas.edu
c Department of Applied Chemistry, Indian School of Mines, Dhanbad, 826004, Jharkhand,
India, E-mail: hpnayek@yahoo.com
† Electronic Supplementary Information (ESI) available: Cif files for the complexes 1 and 2
′
and other necessary supplementary figures are provided. CCDC number: 1039303 ( for the
normal structure ) and 1430170 (for the “squeezed” refined structure ) respectively.
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