Heteroleptic Cu(II)-polypyridyl complexes as photonucleases

Article abstract of DOI:10.1039/c6nj00409a

A series of heteroleptic Cu(ii)-polypyridyl complexes with terpyridine (3N)/imidazole (2N) backbones and appended with pyridyl, 2-naphthyl, 9-anthryl and 1-pyrenyl groups are synthesized and evaluated for their photonuclease activity. An array of techniques viz. UV-vis, fluorescence, circular dichroism and thermal denaturation established strong DNA binding affinity (K_b = ～10⁴-10⁶ M^-1) and the binding modes were correlated with molecular docking studies. Photonuclease efficiency exceeded 90% for all the complexes under identical conditions. Interestingly, DNA binding propensity and photonuclease efficiency followed the increasing size, planarity, aromaticity, π-Stacking ability and hydrophobicity of the peripheral moiety.

Full text of DOI:10.1039/c6nj00409a

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Heteroleptic Cu(II)–polypyridyl complexes as
photonucleases†‡
Cite this:DOI: 10.1039/c6nj00409a
ab
a
a
a
c
V. Singh, K. Sharma, B. Shankar, S. K. Awasthi and R. D. Gupta*
A series of heteroleptic Cu(II)–polypyridyl complexes with terpyridine (3N)/imidazole (2N) backbones and
appended with pyridyl, 2-naphthyl, 9-anthryl and 1-pyrenyl groups are synthesized and evaluated for
their photonuclease activity. An array of techniques viz. UV-vis, fluorescence, circular dichroism and
Received (in Victoria, Australia)
4
6
À1
6th February 2016,
thermal denaturation established strong DNA binding affinity (K
b
= B10 –10
M ) and the binding
Accepted 5th May 2016
modes were correlated with molecular docking studies. Photonuclease efficiency exceeded 90% for
all the complexes under identical conditions. Interestingly, DNA binding propensity and photonuclease
efficiency followed the increasing size, planarity, aromaticity, p-Stacking ability and hydrophobicity of the
peripheral moiety.
DOI: 10.1039/c6nj00409a
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Introduction
The stability of deoxyribonucleic acid (DNA) against potential
1,2
nucleophiles sustains biological life, while in vivo DNA cleavage
3–6
forms the basis of replication and transcription processes. In
this context, the manipulation of DNA with small molecules could
7
–9
10
11,12
be useful in biotechnology, medicine and cancer therapy.
In particular, phthalocyanine and porphyrin-based compounds are
13–15
proven to have strong photo-nuclease activity
but are known to
16
cause severe side effects. Metal-based nucleases, such as cisplatin,
are a huge success but are non-specific in action and lose effec-
tiveness, in the long run, owing to covalent interactions with DNA.
At present, much research is focused on developing metal-based
photonucleases, operating through non-covalent interactions, as
these can impart structural, optical and electronic advantages,
along with energetic contributions to their interactions with anionic
Fig. 1 Representation of Cu(II)–polypyridyl complexes, TPYC-1 to TPYC-4,
where TPYC-1 = pyridyl; TPYC-2 = 2-naphthyl; TPYC-3 = 9-anthryl and
TPYC-4 = pyrenyl-appended complexes (see the red part), respectively.
while functionalized pyridyl, naphthyl, anthryl and pyrenyl act
as active front-intercalators. Importantly, the ligand architecture
ensures a lower electron density at the metal centre, which allows
improved interaction with the anionic DNA, as confirmed by
density functional studies. This design strategy led to compara-
tively high DNA binding affinities through non-covalent inter-
actions, while proposed DNA binding modes were correlated
with molecular docking studies. Significantly, experimental as
well as theoretical evidence categorically suggested that DNA
binding and photocleavage follow the intrinsic properties of the
peripheral moieties.
17–19
DNA.
Therefore, and in this direction, a series of heteroleptic
Cu(II)–polypyridyl complexes are tested for their chemical as well
as photo-nuclease activity. These nucleases utilize a terpyridine/
imidazole spine (Fig. 1) for coordinating to a copper center,
a
Department of Chemistry, University of Delhi, Delhi-110 007, India
b
Department of Chemical Engineering, Pohang University of Science and
Technology, Pohang-790-784, South Korea
c
Faculty of Life Sciences and Biotechnology, South Asian University, Delhi-110 021,
India. E-mail: rdgupta@sau.ac.in
†
‡
Dedicated to Late Dr Tarkeshwar Gupta.
Results and discussions
Electronic supplementary information (ESI) available: Synthesis of ligands
1
(
TPY-1 to 4 and IMI-1); H NMR spectra of TPY-1 to 4; ESI-MS spectra of complexes Synthesis and characterization
TPYC-1 to 4; details of UV-vis studies, competitive displacement assay, circular
dichroism studies, thermal denaturation studies, chemical and photo-nuclease
activity and mechanistic experiments; Tables S1 and S2; Fig. S1–S16. See DOI:
Fig. 1 illustrate the ligand architecture (TPY-1 to 4; terpyridyl-based
ligand and IMI-1; imidazole-based ligand) and the corresponding
Cu(II)–polypyridyl complexes (TPYC-1 to 4).
1
0.1039/c6nj00409a
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Table 1 Selected physicochemical data for complexes TPYC-1 to 4
a
À1
4À
IR /cm
l
max/nm
E
(DE
1/2/V
Ipa/Ipc
2
À1
À1
d
À1
TPYC [v (ClO )] (log e/M cm
)
p
/V)
(D/cm s )
b
À4
À4
À4
1
2
3
4
1076
1082
1079
1092
744 (1.81)c
607 (2.36)c
671 (1.73)
0.0280 (0.34) 0.25 (2.8 Â 10
0.0025 (0.36) 0.86 (8.4 Â 10
0.0075 (0.41) 0.40 (1.0 Â 10
)
)
)
c
e
À5
594 (1.84)
À0.35 (NA) NA (5.0 Â 10
)
a
c
b
ATR-IR using ZnSe crystal. In 10% DMF, ligand field bands.
d
2+
+
In CH
3
CN. Redox couple (Cu /Cu ) in 30% aqueous DMF–0.1 mM
= (Epa À Epc), where Epa and Epc are the
anodic and cathodic peak potentials, respectively. The potentials are
TBAP, E1/2 = 0.5(Epa + Epc), DE
p
À1
e
vs. Ag/AgCl. Scan rate = 0.4 V s
. Epc (NA = not available).
Fig. 2 Cyclic voltammograms of copper complexes in 10% DMF (1 mM)
In a typical synthesis procedure for TPYC-1 to 4, 74 mg
0.02 mmol) Cu(ClO O was dissolved in 10 mL MeOH/
Á6H
CHCl (1 : 1, v/v) at room temperature. To the stirred solution,
.02 mmol of the respective TPY ligand in 10 mL MeOH/CHCl
1 : 4, v/v) was added dropwise. The solution was stirred for 2 h potential, E1/2 values close to 0.02 V, with no anodic response in
and then 59.4 mg (0.02 mmol) IMI-1 in 10 mL MeOH/CHCl3 the case of TPYC-4. An increasing trend was seen in DE values
4 : 1, v/v) was added dropwise to the stirred solution. Further with concomitant large negative values of Epc on increasing the
and TBAP as supporting electrolyte (0.1 mM) vs. Ag–AgCl at a scan rate
(
4
)
2
2
À1
of 100 mV s
.
3
0
3
(
p
(
À1
stirring for 1 h afforded a light to dark green coloured precipitate. scan rate from 100 to 1000 mV s (Fig. S12, ESI‡), which could
The precipitate was washed with ether, dried over anhydrous be attributed to the increasing size of the aromatic tail units and
2
+
calcium chloride and kept in a glovebox. Average yield: B40–55%. their electron donating ability. These factors stabilize the Cu
2
+
+
Detailed synthetic procedures and characterizations are provided oxidation state and make Cu - Cu conversion less feasible
in the ESI‡ (see Fig. S1–S9 and Table 1). The following sub-sections thermodynamically. Moreover, the observed cathodic and anodic
will provide detailed UV-vis absorption, electrochemical, and shifts with increasing scan rate implied non-Nernstian behaviour,
electron paramagnetic resonance characteristics, as well as DNA possibly attributed to heterogeneous electron transfer kinetics
21
binding and cleavage studies of these complexes, including and coupled chemical reaction(s). The plot of cathodic peak
1/2
molecular docking studies.
current vs. the square root of the scan rate (Ipc vs. v ) for each of
UV-vis absorption characteristics. The UV-vis spectra of hetero- the complexes gives linear fitting (R B 0.97 to 0.99) and is
leptic, penta-coordinated, 3d Cu(II)-complexes (see Fig. S10, ESI‡; indicative of a diffusion-controlled process (Fig. S13, ESI‡). The
Table 1) exhibited ligand-centred bands due to spin-allowed slope of each Ipc vs. v plot is calculated, and the diffusion
p - p* or n - p* transitions in the UV and the near UV region. constant, D (see Table 1) is calculated using the Randles–Sevcik
2
9
1/2
22
Additionally, characteristically broad and relatively weak metal- equation for diffusion-controlled electrochemical processes.
centred or d - d bands appeared, as signified by their log e values
EPR characteristics. EPR spectra for the complexes presented
see Table 1). These ligand field bands are intrinsically short-lived an oblate axial with g 4 g 4 g and a d ground state, that
because they involve the population of the strongly metal–ligand is, an unpaired electron resides in the d orbital and displays
antibonding orbital, usually designated as the d
orbital at the a typical four-line spectrum (three well-resolved and one over-
higher wavelengths (lmax 4 600 nm). Similar d - d transitions lapped with the g signal (Fig. 3 and Fig. S14, ESI‡)). For all
have been observed for such penta-coordinated Cu(II)-complexes complexes, g values are around 2.18, which signifies that Cu is
- E coordinated to N-atoms in a square pyramidal geometry as coor-
transition, which points to the square-pyramidal geometry of dination through O-atoms (possibly through H O or the perchlo-
these complexes. We further used the UV-vis technique to rate moiety), which would have resulted in larger g values (see
assess the stability of the complexes in 10% DMF/tris-HCl Table S1, ESI‡). Moreover, trigonal bipyramidal geometry (tbp) is
(
J
>
e
2 2
x –y
2 2
x –y
z –y
2 2
>
2
+
J
2
2
and have been attributed to the symmetry-allowed B
1
2
2
0
J
23
buffer solutions and found them to be stable for B1 week as not feasible, as the g
there were no observable changes in their respective UV-vis
spectra during this period (see Fig. S11, ESI‡).
o g
condition must have been satisfied.
J
>
Electrochemical characteristics. Cyclic voltammograms of
TPYC-1 to 4 were recorded in 10% DMF/tris-HCl buffer solution
and showed quasi-reversibility features (see Fig. 2; Table 1), that
is, the reduction half-wave is not entirely reproduced during the
oxidation process, as corroborated by the ratio of anodic to
2
+/+
cathodic current (Ipa/Ipc) values. For these complexes, the Cu
couple was observed at a cathodic peak potential, Epc B À0.14 V
values ranging from
to À0.35 V (vs. Ag/AgCl) with large DE
p
À1
Fig. 3 X-band EPR spectrum of complex TPYC-1 in DMF glass at 77 K.
.34 V to 0.41 V at a scan rate of 100 mV s and half-wave redox (frequency 9.1 GHz and 100 kHz field).
0
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G values in the range 3–4 further confirm the square-based
geometries of these complexes, which are retained in the solution
2
4
phase and allow for small exchange coupling. Hyperfine
À4
À1
coupling constant, A values are Z160 Â 10 cm for all the
J
complexes, which is expected for a square-based CuN chromo-
/A ), is considered as
J J
an index of deviation from the idealized geometry.
has values ranging from 110 to 120 cm
while 130–150 cm indicates slight to moderate distortion and
80–250 cm indicates considerable distortion from the ideal
5
2
5
phore. The degree of distortion, f (a) = (g
2
6,27
For planar
À1
complexes, g
J
/A
J
,
À1
À1
1
geometry. For the complexes studied, g /A values are in the range
J
J
1
20 to 140, which indicates slight to moderate distortions from
28
the square-based geometry; g values o 2.3 are indicative of
covalent bonding in these complexes. Kivelson and Nieman
J
29
suggested an equation for calculating the in-plane s-covalency
2
30
parameter (a ) (see eqn (1)).
Fig. 4 Representative example showing (A) spectral traces of TPYC-3
(10 mM) in 10% DMF upon gradual addition of ct-DNA (300 mM) in 50 mM
2
a = |A
J
/P| + (g
J
À 2.0023) + 3/7(g
>
À 2.0023) + 0.04
(1)
tris-HCl buffer; (B) non-linear least-squares fitted plot of Deaf/Debf  [DNA]
À1
2
where ‘P’ is the dipolar hyperfine coupling parameter (À0.036 cm ). vs. [DNA] for all complexes (Red sphere, TPYC-1, R = 0.98; Olive sphere,
2
2
2
The values of a lie between 0.5 and 1, the limits of pure covalent and TPYC-2, R = 0.98; Orange sphere, TPYC-3, R = 0.99; Blue sphere,
2
2
TPYC-4, R = 0.99) using MvH equation; (C) linear least-squares fitted plot
pure ionic bonding, respectively. For these complexes, a lies in the
range 0.67 to 0.74, which accounts for the fraction of the unpaired
electron density located on Cu and is indicative of considerable
covalency in the s-bond.
2
Deaf/Debf vs. [DNA] (Red circles, TPYC-1, R = 0.99; Olive circles, TPYC-2,
2
2
R
R
= 0.99; Orange circles, TPYC-3, R = 0.99; Blue circles, TPYC-4,
2+
2
= 0.99) using modified Mcghee von Hippel equation given by Bard
28–31
and co-workers.
DNA binding, molecular docking, chemical and photonuclease
activities
(see Fig. 5B and Table 2). Furthermore, Stern–Volmer plots
DNA binding. Complexes TPYC-1 to 4 showed quite similar showed linear dependencies (see Fig. 5C), which indicated
behaviour during absorption spectral titrations (AST) with one dominant quenching mechanism, while accessibility to
ct-DNA (see ESI‡ for experimental details). Perturbations in EB-DNA sites increased from TPYC-1 to TPYC-4, as suggested by
À1
ligand-centred bands were monitored during the addition of increasing KSV values. Furthermore, (KSV
)
suggests that anthryl
ct-DNA, where all complexes showed varying degrees of hypo- and pyrenyl-terminated complexes can more easily displace EB
chromism (B12 to 40%), accompanied by minor to significant from EB-DNA sites as larger concentrations of other complexes are
red shifts (B1 to 8 nm) in the respective absorption bands of required to induce a similar effect (see Table 2).
their UV-vis spectra, mostly ligand-centred bands (see Fig. 4,
Table 2 and Fig. S15, ESI‡). These features were strongly suggestive T , in the presence of TPYC-1 to 4, showed a change (DT) of +
In thermal denaturation studies, the ct-DNA melting point,
o
m
of non-covalent binding of complexes with DNA. The appearance 2.8 to +7.2 1C, where DT was found to be highest for TPYC-4 (see
of isosbestic points indicated two absorbing species, viz. complex Fig. 5D, E and Table 2). In the light of the DT values, it could be
and DNA, linearly related by stoichiometry, although ratios of more suggested that these complexes tend to prefer groove/partial
than 1 : 1 are possible. Moreover, uncoordinated –NH groups of the intercalation binding modes and play a more significant role
3
2
imidazole moiety and N-atoms of the pyridyl moiety could possibly in ds-DNA stabilization than simple mono-intercalation.
allow secondary interactions, such as hydrogen bonding with DNA, Notably, the effect is more pronounced for ‘G–C’-rich regions
which possesses several hydrogen bonding sites accessible both in of TPYC-3 and TPYC-4, as is evident from the denaturation
3
3
the minor and major grooves. This is supported by low batho- curves. Moreover, it can also be noted that DT values are not
chromic shifts in the case of TPYC-1 and higher ones for TPYC-3 significantly large, implying that covalent interactions with
4
6
À1
34
and TPYC-4. Intrinsic binding constants, K
Table 2), values suggested strong and increasing DNA binding
affinity from TPYC-1 to TPYC-4 and are thus consistent with the in the ellipticity of ct-DNA on complex addition (see Fig. 5F).
intercalative binding mode. Visible red/blue shifts were observed with respect to the positive
Competitive displacement assays using ethidium bromide (EB) band. TPYC-1 and TPYC-2 witnessed +36% and +34% changes in
see ESI‡ for experimental details) complement UV-vis studies ellipticity along with a minor red shift. For TPYC-3 and TPYC-4,
b
(B10 À 10 M ; see DNA are not a feasible option.
Circular dichroism experiments produced noticeable changes
(
(
Fig. 5A and Fig. S16 and S17, ESI‡). Apparent binding constant, parallel changes in ellipticity were of negative order, that is, À30%
Kapp values followed the same order as for K . Additionally, DC , and À40%, along with pronounced blue shifts in the positive
b
50
the concentration of the complex at 50% EB displacement, values band. For all complexes except TPYC-4, a negative band exhibited
for TPYC-3 and TPYC-4 were significantly low at B2 mM and an increase in the intensity. The variations in the CD spectra of
are in line with structural characteristics of ancillary ligands ct-DNA with the addition of small molecules are suggestive of
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Table 2 Comparative chart for absorption spectral titrations of TPYC-1 to 4 (10 mM) against ct-DNA (300 mM) in 50 mM tris-HCl buffer; competitive
binding and thermal denaturation studies
À1 a
À1 b
app/M
c
À1
d
TPYC
K
b
/M
(s, Gmax
)
K
(DC50/mM)
K
SV (KSV
)
m
DT /1C
4
6
1
2
3
4
1.80 Æ 0.20 Â 10 (0.119, 4.20)
1.94 Æ 0.35 Â 10 (6.70 Æ 0.22)
1.50 (0.66)
1.61 (0.62)
3.60 (0.27)
11.06 (0.09)
+2.8
+3.2
+4.8
+7.2
5
6
1.23 Æ 0.15 Â 10 (0.116, 4.31)
1.39 Æ 0.20 Â 10 (9.30 Æ 0.30)
5
6
1.43 Æ 0.24 Â 10 (0.121, 4.13)
2.71 Æ 0.30 Â 10 (4.80 Æ 0.12)
6
6
1.05 Æ 0.10 Â 10 (0.125, 4.000)
7.70 Æ 0.15 Â 10 (1.68 Æ 0.25)
a
b
c
d
Intrinsic binding constant. Apparent binding constant. Stern–Volmer quenching constant. Change in DNA melting point.
Fig. 5 Top: (A) Representative example of addition of complex (TPYC-3) to EB-bound DNA solution; (B) plot of percent displacement of EB from DNA on
2
2
2
gradual addition of complexes (red circles, TPYC-1, R = 0.99; olive circles, TPYC-2, R = 0.99; orange circles, TPYC-3, R = 0.99; blue circles, TPYC-4,
2
2
2
R = 0.99); (C) Stern–Volmer plots for calculation of quenching constants (red circles, TPYC-1, R = 0.97; olive circles, TPYC-2, R = 0.98; orange circles,
TPYC-3, R = 0.98; blue circles, TPYC-4, R = 0.99). Bottom (D) DNA melting curves for ct-DNA alone (130 mM) and DNA + complexes TPYC-1 to 4
2
2
(33 mM); NA = normalized absorbance; (E) first derivative of DNA melting curves for calculation of T
m
; (F) circular dichroism spectra of DNA alone
(100 mM, black line) in 50 mM tris HCl buffer and in the presence of complex solutions (10 mM, coloured circles).
subtle changes in the DNA double helix promoted by stabilization (see Fig. 6A). It is interesting to note that the energy gap
of its secondary structure upon DNA–metal complex interaction, decreases and that the charge transfer process becomes easier
which could be due to either partial intercalative or groove from the pyridyl to pyrenyl tail units and correlates well with the
35–37
binding to DNA.
However, this evidence cannot be used alone UV-vis data, where LMCT bands shift to lower energy for the
to classify the mode of binding.
anthryl and pyrenyl moieties.
DFT and molecular docking studies. A careful analysis of the
Furthermore, docking experiments were performed in order
optimized geometries of TPYC-1 to 4 suggests that pyridyl, to find out the chosen binding site, along with the preferred
naphthyl, anthryl and pyrenyl units are in the plane perpendi- orientation of the ligand inside the DNA minor groove (Fig. 6A
cular to the terpyridyl unit and that, as suggested by EPR and Fig. S19, ESI‡). Computational docking studies were carried
studies, there is distortion from the ideal square-based geometry out to gain a theoretical insight into the interactions between
(see Fig. S18, Table S2 and S3, ESI‡). As confirmed by the DNA
binding studies, with an increase in the hydrophobicity, planarity
and aromaticity of the appended ligand, the DNA-binding
propensity increases. DFT studies indicate that these moieties
actually take the whole complex to the DNA binding regions.
Moreover, the highest occupied molecular orbital (HOMO) in
each case resides/is localized over pyridyl, naphthyl, anthryl
and pyrenyl units of the terpyridyl unit. The lowest unoccu-
pied molecular orbital (LUMO) is distributed over the metal
centre and the terpyridyl unit for all the complexes. The HOMO–
LUMO energy gap is a critical parameter for eventual charge
3
8
transfer interactions within the molecules. The HOMO–LUMO
energy gap for TPYC-1 is calculated to be 2.44 eV, for TPYC-2
Fig. 6 (A) HOMO–LUMO of TPYC-1 to 4 (left to right) along with energy
gap; (B) docked structure of TPYC-4 with d(CGCGAATTCGCG) strands of
is 2.27 eV, for TPYC-3 is 1.75 eV and for TPYC-4 is 1.77 eV ct-DNA.
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TPYC-1 to 4 and DNA. The optimized structures of the metal
complexes were docked with a B-DNA structure (protein data
bank; 1BNA) using Autodock 4.2.6. The docked model indicated
that TPYC-1 to 4 favoured intercalation into the minor groove of
ct-DNA. Structural analysis of the docked structures provided
minimum binding energy (BE) values as À36.6, À34.1, À35.4 and
À1
À46.2 kJ mol , respectively, where more negative values indicated
more potent binding between DNA and target molecules and
correlated well with the experimental evidence; vide supra.
Furthermore, we analysed B50 positions for each docked
structure. As discussed above, we chose the position that had
the highest negative value (minimum energy value). In the case
of TPYC-1 and TPYC-4, hydrogen bonding with DNA base pairs
was evident (see Fig. 6B) but TPYC-2 and TPYC-3 did not show
hydrogen bonding; instead they exhibited van der Waal’s
interactions with the nearest base pairs. Nonetheless, TPYC-1
Fig. 8 (A) Gel electrophoresis diagrams showing chemical nuclease
activity of TPYC-1 to 4 (1) TPYC-1, (2) TPYC-2, (3) TPYC-3, (4) TPYC-4
with increasing concentration of complexes as we move from lane 2 to
lane 7. Lane 1: DNA control (0.5 mL in each case); Lane 2: DNA + 5 mM
is hydrogen-bonded with the guanine 114 (H-bond distance TPYC + MPA; Lane 3: DNA + 6.67 mM TPYC + MPA; Lane 4: DNA + 8.33 mM
2
1
.159 Å) of the B chain of DNA, and TPYC-4 with the cyanine TPYC + MPA; Lane 5: DNA + 11.11 mM TPYC + MPA; Lane 6: DNA +
2.22 mM TPYC + MPA; Lane 7: DNA + 13.89 mM TPYC + MPA. (B) Gel
electrophoresis diagram showing chemical nuclease activity of TPYC-1 to
(10 mM) in the presence of various controls. Lane 1: DNA control (0.5 mL in
each case); Lane 2: DNA + TPYC + MPA; Lane 3: DNA + TPYC + MPA +
1
11 (H-bond distance 2.187 Å) of the A chain of DNA (see
Fig. 6B). This further supported the strong DNA binding affinity
of the complexes.
4
DNA cleavage activity. For the chemical nuclease activity, the NaN ; Lane 4: DNA + TPYC + MPA + D O; Lane 5: DNA + TPYC + MPA +
3
2
presence of 3-MPA as co-reductant was necessary for pUC19 DMSO; Lane 6: DNA + TPYC + MPA + KI. (C) Gel electrophoresis diagrams
of photonuclease activity of complexes TPYC-1 to 4. (1) TPYC-1; (2) TPYC-2;
DNA cleavage as no significant formation of nicked circular
(
3) TPYC-3; (4) TPYC-4. Lane 1: DNA control + UV light; Lane 2: DNA +
TPYC + UV light; Lane 3: DNA + TPYC + NaN + UV light; Lane 4: DNA +
TPYC + D O + UV light; Lane 5: DNA + TPYC + DMSO + UV light; Lane 6:
DNA (o4 Æ 2%) was observed (see Fig. S20, ESI‡), while due
3
corrections were made for low levels of nicked circular DNA
2
3
9
already present. This suggested that TPYC-1 to 4 were unable DNA + TPYC + KI + UV light.
to cleave DNA hydrolytically. Concentration optimization studies
revealed that, for all complexes, concentrations as low as B10 mM
were able to achieve 490% cleavage and at B15 mM, DNA of KI did not produce any significant variation in the nuclease
cleavage exceeds B99% with B2–3% deviation from the average activity (B1–3% decline in cleavage activity). Furthermore, the
value (see Fig. 7(left) and 8A).
linear form (form III) could not be discerned in any of the
Furthermore, mechanistic studies (see ESI‡) advocated the chemical nuclease experiments. Therefore, an oxidative cleavage
involvement of reactive oxygen species (ROSs) in the nuclease mechanism following type-I processes for DNA cleavage was
+
activity (see Fig. 7(centre) and 8B). In this connection, there was found to be active for the complexes under study, where Cu ,
1
2+
no effect from the addition of a O quencher, NaN (B1–3% being less stable than Cu , most probably helps in reducing
2
1
3
decrease in cleavage activity) or a O lifetime enhancer, D O molecular oxygen to reactive/reduced oxygen species/intermediates
B2–3% enhancement in cleavage activity), which suggested (ROS’s), viz., OH . Based on literature and experimental evidence,
2
in the cleavage reactions. Moreover, the a DNA cleavage mechanism is proposed. At first, copper complexes
addition of the hydroxyl radical scavenger, DMSO, reduced the bind to DNA. Then, 3-MPA reduces the DNA-bound Cu to Cu .
nuclease activity to B5–10%, which unambiguously recommended The H
the involvement of hydroxyl radicals in DNA cleavage. The addition Cu -complex, leading to the formation of OH at the binding sites,
2
2
ꢀ
(
1
little or no role of O
2+
+
+
2
O
2
formed oxidizes the DNA-bound Cu -complex to the
2+
ꢀ
Fig. 7 Bar diagram showing the % DNA cleavage upon treating with complexes TPYC-1 to 4. (left) % DNA cleavage at various concentrations of different
complexes. (middle) % DNA cleavage at 10 mM complex and various controls. (right) % DNA cleavage during photonuclease activity of complexes.
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thus initiating the chain reaction, which relaxes the supercoiled DNA was procured from Genei, Bangalore, and kept at À20 1C
40
form to nicked circles (Scheme S1, ESI‡).
Photo-exposure of TPYC-1 to 4 + DNA to UV-A light at 365 nm
2
inside an N filled glovebox.
and 12 W power (UV-A source) produced significant nuclease Physical characterization
activity as B80–90% of SC DNA converted to NC DNA within
Optical measurements. All UV/vis spectra were recorded on
a JASCO UV-vis-NIR spectrophotometer (Model V-670). For
measurements, a pair of quartz cuvettes with an optical path
length of 1 cm was used. Every measurement and DNA binding
studies were performed at room temperature and suitable
baselines were recorded for preliminary adjustments. A Perkin
Elmer fluorescence spectrometer (Model LS45) was employed
for carrying out competitive DNA binding studies with a
quartz cuvette with an optical path length of 1 cm. Thermal
denaturation studies were performed on a Perkin Elmer UV/vis
spectrophotometer (Model Lambda 45) equipped with a Peltier
temperature-controlling programmer (PTP-6) functionality and
a water circulation system. A Thermo Scientific NanoDrop 2000
spectrophotometer was used for determining DNA purity.
A JASCO circular dichroism spectropolarimeter (Model J-815)
was used for determining structural changes in DNA upon DNA
binding with copper complexes. A quartz cuvette with a 1 cm
path length was used for carrying out measurements with
proper preliminary adjustments.
0
.5 h of irradiation (see Fig. 7(right) and 8C). The addition of
NaN reduced the cleavage of SC DNA to a mere B5–10%. In
consonance with this, the presence of D O enhanced the
3
2
cleaving activity by up to B5–12%. The presence of either
DMSO or KI did not have any noticeable effect on photonuclease
activity. These results were found to be consistent with the
formation of O species and further highlighted the necessity of
molecular oxygen in photonuclease activities. Mechanistically,
UV-A light seemed to sensitize the complexes to an excited state,
which upon efficient energy transfer appeared to activate oxygen
from its stable triplet (O
state and thus followed type II processes for effecting DNA cleavage
1
2
3
1
2
, SgÀ) to the highly toxic singlet (O
2 g
, D )
22
(Scheme S2, ESI‡).
Conclusions
The heteroleptic copper(II)–polypyridyl complexes were found
to be avid DNA binders with binding affinity either comparable
or better than existing copper(II)–polypyridyl-based artificial
Electrochemical measurements. All cyclic voltammograms
were recorded on a CH Instruments electrochemical workstation
41–46
nucleases.
Binding characteristics favoured a groove/partial
(Model 660D). A single-compartment cell with a three-electrode
intercalative mode for interacting with DNA, while nuclease activity
appeared efficient under chemical as well as light stimulus. It is
concluded that peripheral ligand architecture influences the extent
of DNA binding and cleavage activity, where more planar, hydro-
phobic and aromatic ligands appeared to be more effective DNA
binders as well as cleavers under chemical or light stimulus.
Apparently, molecular docking studies correlated well with the
experimental evidence and favoured the DNA binding trends as
observed.
setup was used to perform the experiments. A glassy carbon
electrode, a Pt wire and an Ag/AgCl electrode (3 M KCl) were used
as working, counter and reference electrode respectively. TBAP
(
tetra butyl ammonium perchlorate) (0.1 M) was used as supporting
electrolyte. Complex solutions (1 mM in 30% DMF) were bubbled
with N gas for B20 min prior to recording cyclic voltammograms.
2
Electron paramagnetic resonance measurements. EPR mea-
surements were carried out on a JEOL JES-FA series EPR
spectrometer. All measurements were carried out in DMF at
77K (DMF glass) with 2,2-diphenyl-1-picrylhydrazyl (DPPH) as
an internal standard. The instrument was operated at X-band
frequencies (9.1 GHz) and a 100 kHz field.
Nuclear magnetic resonance measurements. All H-NMR spectra
were recorded on a JEOL JNMECX 400p spectrometer at room
temperature using a suitable deuterated solvent. All chemical shifts
Experimental section
Materials and methods
1
Copper(II) perchlorate hexahydrate, 2-acetyl pyridine, polyethylene
glycol (PEG-300), 4-pyridine carboxaldehyde, 2-naphthaldehyde,
(d) were recorded in ppm with reference to tetramethylsilane (TMS),
9-anthraldehyde, 1-pyrene carbaldehyde, 1,10-phenanthroline
and coupling constant ( J) values are provided in Hz.
monohydrate, potassium tert. butoxide, sodium perchlorate,
tetrabutylammonium perchlorate (TBAP), 3-mercaptopropionic
acid (3-MPA), ethylenediaminetetraacetic acid (EDTA) and tris-HCl
base were purchased from Sigma-Aldrich and used as received.
Sodium hydroxide, ethidium bromide (EB), ammonium acetate,
aqueous ammonia, and sodium sulphate were purchased from
SD Fine Chemicals, India and used as received. All solvents, viz.
ethanol, methanol, dichloromethane, and chloroform, were
purchased from Rankem, India and were distilled and degassed
Density functional studies. The ground state geometry opti-
mizations of TPYC-1 to 4 were carried out in the gas phase
47,48
using the B3LYP method.
The LANL2DZ basis set with
effective core potential was used for the copper atom. The
-31G* basis set was used for carbon, hydrogen and nitrogen
6
49
atoms. The initial geometry was obtained from the standard
geometrical parameters. All calculations were performed using
50
the Gaussian 09 program package.
following established procedures and kept in an N -filled glove-
2
box (O
2
o 0.5 ppm). Ultra-pure Milli-Q water (r = 18.2 MO cm at
Conflicts of interest
À1
25 1C) was used in all experiments. A 1 mg mL solution of calf
À1
thymus DNA (ct-DNA) and a 250 mg mL solution of pUC19 The authors declare no competing or financial conflict.
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Acknowledgements
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This work was financially supported by the University Grants
Commission (F-37-407/2009(SR)), New Delhi, India. VS thanks
University Grants Commission (UGC), India for a Senior
Research Fellowship. RDG thanks the Department of Science
and Technology (SERB/F/1448/2012-13).
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