Synthesis, spectral, DNA binding and cleavage properties of ruthenium(II) Schiff base complexes containing PPh3/AsPh3 as co-ligands

Article abstract of DOI:10.2298/JSC121201073S

Dihydroxybenzaldehyde-based Schiff base ligands (L¹-L ³) and their ruthenium(II) complexes were synthesized and characterized by elemental analysis, ¹H-, ¹³C- and ³¹P-NMR, UV-Vis, IR and mass spectros

Full text of DOI:10.2298/JSC121201073S

J. Serb. Chem. Soc. 79 (2) 151–165 (2014)
JSCS–4572
UDC 577.213.3+546.962+542.9+547.571+
547.551:543.42–74
Original scientific paper
Synthesis, spectral, DNA binding and cleavage properties of
ruthenium(II) Schiff base complexes containing PPh3/AsPh3
as co-ligands
SUBBAIYAN SATHIYARAJ, GANESAN AYYANNAN
*
and CHINNASAMY JAYABALAKRISHNAN
Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, Coimbatore – 641 020, Tamil Nadu, India
(Received 1 December 2012, revised 8 May 2013)
Abstract: Dihydroxybenzaldehyde-based Schiff base ligands (L1–L3) and their
ruthenium(II) complexes were synthesized and characterized by elemental
1
analysis, H-, 13C- and 31P-NMR, UV–Vis, IR and mass spectroscopy. The
DNA binding of the ruthenium(II) complexes was investigated by UV–Vis
absorption spectroscopy. The experiments revealed that all the compounds
could bind to DNA through electrostatic interactions and the intrinsic binding
constants (Kb) were estimated under similar sets of experimental conditions.
The absorption spectral study indicated that the ruthenium(II) complexes had
intrinsic binding constants in the range of (1.6–8.6)×104 mol-1 dm3. The com-
plex [Ru(CO)(PPh3)2(L3)] bound more strongly than the other complexes. In
addition, the DNA cleavage properties for all ruthenium(II) complexes were
tested.
Keywords: Schiff base; ruthenium(II) complexes; CT-DNA; nuclease activity.
INTRODUCTION
It is well known that deoxyribonucleic acid (DNA) plays an important role
in the life process since it contains all the genetic information for cellular func-
tion. However, DNA is the primary intracellular target of anticancer drugs due to
the interaction between small molecules and DNA, which cause DNA damage in
cancer cells, blocking the division of cancer cells and resulting in cell death. This
is due to their possible application as new therapeutic agents and their photoche-
mical properties that make them potential probes of DNA structure and confor-
1–3
mation.
The binding interaction of transition metal complexes with DNA is of
4
interest for both therapeutic and scientific reasons. Many transition metal com-
plexes are known to bind to DNA via both covalent and non-covalent interac-
*Corresponding author. E-mail: cjayabalakrishnan@gmail.com
doi: 10.2298/JSC121201073S
151
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SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
tions. In covalent binding, the labile ligand of the complexes is replaced by a
nitrogen base of DNA. On the other hand, the non-covalent DNA interactions
include intercalative, electrostatic and groove (surface) binding of cationic metal
complexes along the outside of the DNA helix, and the major or minor groove.
Schiff bases are potential anticancer drugs and when administered as their metal
complexes, the anticancer activity of these complexes is enhanced in comparison
to that of the free ligand. Schiff base complexes are considered the most impor-
tant stereochemical models in transition metal coordination chemistry due to their
preparative accessibility and structural variety. It was suggested that the azo-
methine linkage in Schiff bases is responsible for their biological activities, such
5
as antitumour, antibacterial, antifungal and herbicidal activities.
Metal complexes are employed in many fields of drug discovery. Platinum
coordination complexes are widely used as antitumour drugs. The first platinum
antitumour drug introduced into clinical practice was cis-diamminedichloroplati-
num(II) (cisplatin), which became the most widely used anticancer drug in the
world. However, intrinsic and acquired tumour resistance diminishes the clinical
efficacy of cisplatin and other platinum drugs. In addition cisplatin is of high
6
toxicity, leading to side effects that limit the administered dose. These limiting
issues have led to an intense effort to design new transition metal-based com-
pounds that are capable of overcoming problems associated with cisplatin while
maintaining the same level of activity and broadening the spectrum of the thera-
peutic effect. In attempts to find a new, metal-based anticancer drug with activity
complementary to cisplatin, several ruthenium complexes have recently been
7
investigated for their antitumour activity.
Bearing these facts in mind, our interest was focused on the synthesis of
ruthenium(II) complexes containing tridentate Schiff base ligands and triphenyl-
phosphine or triphenylarsine as co-ligands. The Schiff base ligands were derived
by the condensation of 2,4-dihydroxybenzaldehyde with 2-aminobenzothiazole,
2-amino-6-methylbenzothiazole or o-aminophenol (Scheme 1). Then the new
Schiff base ligands and their ruthenium(II) complexes were characterized by ele-
1
13
31
mental analysis, FT-IR, electronic, mass spectra, and H-, C- and P-NMR
spectroscopy. Additionally, a comparative study of the interaction of the ruthe-
nium complexes with CT-DNA has been employed in order to investigate the
potential mechanism of their biological properties using UV–Vis spectroscopic
and gel electrophoresis techniques.
EXPERIMENTAL
Materials and instrumentation
Reagent grade chemicals were used without further purification in all the synthetic work.
All solvents were purified by standard methods. RuCl3·3H2O, triphenylphosphine / arsine
were purchased from Himedia. Calf thymus DNA (CT-DNA) was purchased from Bangalore
Genei, Bangalore, India. Infrared spectra were recorded on a Perkin Elmer FT-IR spectro-
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
153
photometer (model RXI) as KBr pellets in the range 4000–400 cm-1. Elemental analyses were
performed with a Vario ELIII CHNS instrument at the Sophisticated Test and Instrumentation
Centre (STIC), Cochin University, Kerala, India. Electronic spectra were recorded in DMSO
solution in a Systronics 2202 double beam spectrophotometer in the 800–200 nm range. The
1H-, 13C- and 31P-NMR spectra were recorded on Bruker WM DCX 500 MHz instrument
using TMS and orthophosphoric acid as internal standards at SAIF, Indian Institute of
Technology, Chennai. The mass spectra were recorded using a JEOL GC mate instrument at
SAIF, Indian Institute of Technology, Chennai, India. The DNA cleavage studies were per-
formed using the gel documentation system, Gelstan. Melting points were recorded on a
Veego VMP-DS model heating table and are uncorrected. The metal precursors
[RuHCl(CO)(PPh3)3],8 [RuHCl(CO)(AsPh3)3],9 were prepared according to reported proce-
dures.
HO
N
S
Benzothiazol-2-amine
N
H
OH
L1
3
6
HO
OH
HO
N
Ethanol
6-methylbenzothiazol-2-amine
2-hydroxybenzaldehyde
4
2
N
H
O
S
OH
Reflux for 6 h
H3C
5
1
C
H
L2
OH
HO
N
OH
H
L3
Scheme 1. Preparation of the Schiff base ligands.
Preparation of the Schiff base ligands
A solution of 2,4-dihydroxybenzaldehyde (1.38 g, 10 mmol) in ethanol (15 ml) was
added to a stirred solution of 2-aminobenzothiazole/2-amino-6-methylbenzothiazole/o-amino
phenol (1.09–1.64 g, 10 mmol) in ethanol (20 mL). The mixture was refluxed for 6 h. Upon
cooling, a precipitate was formed, which was filtered off, dried and recrystallized from
ethanol (Scheme 1).
Preparation of the ruthenium(II) Schiff base complexes
All the new ruthenium(II) complexes were prepared by the following general procedure
(Scheme 2). A solution of [RuHCl(CO)(B)3] (0.190–0.216 g, 0.20 mmol) in benzene (20 mL,
B = PPh3/AsPh3), was added to a stirred solution of Schiff base ligands (0.045–0.056 g, 0.20
mmol) in methanol (15 mL). The mixture was refluxed for 12 h. The solvent was then eva-
porated under reduced pressure and the solid mass filtered and washed with petroleum ether.
The purity of the complexes was checked by thin layer chromatography and was further
purified by column chromatography using 1:10 CH2Cl2:n-hexane as an eluent. This solid was
recrystallized from CH2Cl2/n-hexane mixture. All attempts to obtain single crystals of the
complexes were unsuccessful.
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SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
DNA-binding and cleavage assay
Electronic absorption spectroscopy. Experiments involving the interaction of the ruthe-
nium(II) complexes with CT-DNA were realized in double distilled water containing tris(hyd-
roxymethyl)aminomethane (Tris, 5 mM) and sodium chloride (50 mM), with the pH adjusted
to 7.2 using hydrochloric acid. A solution of CT-DNA in the buffer gave a UV absorbance
ratio at 260 to 280 nm of about 1.9, indicating that the DNA was sufficiently free of protein.
The DNA concentration per nucleotide was determined by absorption spectroscopy using the
molar extinction coefficient value of 6600 dm3 mol-1 cm-1 at 260 nm. Electronic absorption
titration experiments were performed by maintaining the concentration of the complexes
constant (25 μM) but with variable nucleotide concentrations from 0 to 25 μM. While mea-
suring the absorption spectra, equal amounts of DNA were added to both compounds and
reference solutions to eliminate the absorbance of DNA itself. The data were then fitted into
the following equation and the intrinsic binding constant Kb was calculated in each case:10
[DNA]/(εa–εf) = [DNA]/(εb–εf) + (1/Kb)(εb–εf)
(1)
where [DNA] is the concentration of DNA in base pairs. The apparent absorption coefficients
εa, εf and εb correspond to Aobsd/[complex], the extinction coefficient of the free compound
and the extinction coefficient of the compound when fully bound to DNA, respectively. In
plots of [DNA]/(εa–εf) versus [DNA], Kb is given by the ratio of the slope to the intercept.
DNA cleavage studies. The DNA cleavage activity of the ruthenium(II) complexes was
monitored by agarose gel electrophoresis on CT-DNA. Each reaction mixture contained 30
µM of CT-DNA, 30 and 60 µM of each complex in 50 mM Tris–HCl, (pH 7.1). The reaction
was incubated at 37 °C for 2 h. After incubation, 1µL of loading buffer (0.25 % bromophenol
blue, 0.25 % xylene cyanol and 60 % glycerol) was added to the reaction mixture and loaded
onto a 1 % agarose gel containing 1.0 µg mL-1 of ethidium bromide (3,8-diamino-5-ethyl-6-
-phenylphenanthridinium bromide). The electrophoresis was performed for 2 h at 50 V in
Tris–acetic acid–EDTA (ethylenediaminetetraacetic acid) buffer, pH 7.1. The bands were
visualized under UV light and photographed.
RESULTS AND DISCUSSION
A new series of ruthenium(II) dihydroxybenzaldehyde Schiff base com-
plexes were synthesized, stable in air at room temperature, non-hygroscopic in
nature and soluble in common solvents such as dichloromethane, dimethylform-
amide and dimethyl sulphoxide. The analytical data (Table I) of the ligands and
complexes are in good agreement with the calculated values, thus confirming the
proposed molecular formulae (Scheme 2).
IR spectra
The IR spectra of the complexes, in comparison with those of the free
ligands, display certain changes, which gives an indication about the mode of co-
ordination and their structure. The significant IR spectral bands of the ligands
and the complexes are listed in Table II. The free Schiff bases show a very strong
−1
absorption around 1636–1626 cm , which is the characteristic of the azomethine
11
(>C=N) group. Coordination of the Schiff bases to the ruthenium ion through
the azomethine nitrogen atom is expected to reduce the electron density in the azo
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
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TABLE I. Physicochemical analysis data of the ligands and ruthenium(II) Schiff base com-
plexes
Found (Calcd.), %
Yield, M.p.
%
Ligands and complexes Empirical formula Colour
°C
Yel- 82 221 62.56 3.81 10.11 12.14
low (62.21)(3.73)(10.36)(11.86)
Yel- 78 212 63.42 4.48 9.44 11.49
C
H
N
S
L1
L2
L3
C14H10N2O2S
C15H12N2O2S
C13H11NO3
low
Or-
(63.34)(4.24) (9.85) (11.28)
72 245 67.80 4.51 6.37
(68.11)(4.84) (6.11)
–
ange
[RuCl(CO)(PPh3)(L1)] C33H24ClN2O3PRuS Pink 66 285 56.62 4.03 4.28 4.73
(1)
(56.94)(3.69) (3.94) (4.52)
[RuCl(CO)(PPh3)(L2)] C34H26ClN2O3PRuS Pink 62 275 57.12 4.01 4.04 4.42
(2)
(57.50)(4.15) (4.17) (4.55)
C51H41NO4P2Ru Green 61 293 68.12 4.51 1.05
(68.45)(4.62) (1.57)
[Ru(CO)(PPh3)2(L3)]
–
(3)
[RuCl(CO)(AsPh3)(L1)] C33H24ClN2O3AsRuS Brown 68 267 53.21 3.70 4.02 4.54
(4)
(53.56)(3.27) (3.79) (4.33)
[RuCl(CO)(AsPh3)(L2)] C34H26ClN2O3AsRuS Brown 64 271 53.40 3.01 3.92 4.30
(5)
(54.15)(3.48) (3.71) (4.25)
C51H41NO4As2Ru Black 63 288 62.67 4.42 1.67
(62.33)(4.20) (1.43)
[Ru(CO)(AsPh3)2(L3)]
–
(6)
Scheme 2. Formation of the new ruthenium(II) Schiff base complexes.
methine link and thus lower the ν(C=N) absorption frequency. Hence these bands
−1
undergo shift to lower frequencies (1621–1594 cm ) after complexation,
12,13
indicating coordination of the azomethine nitrogen to ruthenium.
The IR
–1
spectrum of the ligands revealed a medium intensity band at 1599–1598 cm
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SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
(C=N) of the thiazole ring, which were shifted to lower frequencies (1586–1580
–1
cm ) after complexation in the spectra of complexes 1, 2, 4 and 5, which also
indicated that the thiazole ring was affected upon coordination to the ruthenium
14
−1
metal ion. The free ligands exhibit a broad band at 3426–3370 cm , which
may be assigned to the phenolic ν(OH) and this band was absent in the spectra of
all the complexes, implying deprotonation of the Schiff bases prior to coordi-
nation. The hydroxy protons were displaced by the metal leading to higher ν(C–O)
−1
−1
values (1285–1252 cm ) compared to those of the free ligands (1274–1242 cm ),
suggesting that the other coordinating atom was the phenolic oxygen. The
15
binding of the metal to the ligand through nitrogen and oxygen atoms was further
−1
supported by the appearance of new bands in the 460–400 cm and 540–510
−1
16
cm ranges due to ν(M–N) and ν(M–O), respectively, in the spectra of all the
−1
complexes. A strong band for all the complexes in the region 1957–1931 cm is
−1
due to terminally coordinated carbonyl groups. The ν(C–S–C) at 743 cm of the
thiazole ring remained unchanged, which demonstrated that the sulphur atom of
the thiazole group does not coordinate to the ruthenium metal. In addition, the
Schiff base complexes showed strong vibrations near 520, 695, 740 and 1430 cm ,
which are attributed to the triphenylphosphine or triphenylarsine fragments.
−1
17
TABLE II. FT-IR and electronic spectral data for the ligands and ruthenium(II) Schiff base
complexes
FT-IR, ν / cm-1
C=N Ph-OH Ph-CO C≡O C=N thiazole
UV–Vis
λmax / nm
Ligands and complexes
L1
1649 3390 1254
1626 3426 1242
1651 3370 1274
–
–
–
1598
1599
–
309, 368, 416, 448
307, 368, 420, 442
310, 366, 392, 435
308, 365, 421,
450, 486
308, 362, 425,
452, 481
308, 368, 420,
452, 480
306, 368, 416,
441, 481, 524
304, 363, 412,
442, 483, 521
307, 369, 420, 454
L2
L3
[RuCl(CO)(PPh3)(L1)] (1)
1597
1597
1611
–
–
–
–
–
–
1285 1941
1258 1931
1313 1943
1284 1939
1252 1957
1309 1943
1573
[RuCl(CO)(PPh3)(L2)] (2)
[Ru(CO)(PPh3)2(L3)] (3)
1575
–
[RuCl(CO)(AsPh3)(L1)] (4) 1594
[RuCl(CO)(AsPh3)(L2)] (5) 1594
1568
1582
–
[Ru(CO)(AsPh3)2(L3)] (6)
1621
Electronic spectra
The electronic spectra of all the ligands and complexes in DMSO showed
four to six bands in the 306–524 nm regions as given in Table II. The electronic
spectra of all the free ligands showed two types of transitions, the first one
appeared in the range 309–368 nm that could be assigned to π–π* transitions,
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
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which were due to transitions involving molecular orbitals located on the phe-
nolic chromophore. These peaks were shifted in the spectra of the complexes.
This may be due to the donation of a lone pair of electrons by the oxygen of the
18
phenoxy group to the central metal atom. The second type of transitions
appeared at the range 309–448 nm that could be assigned to n–π* transitions,
which were due to transitions involving the molecular orbitals of the C=N chro-
mophore. These bands were also shifted upon complexation, indicating that the
19
imine group nitrogen atom could be coordinated to the metal ion.
1
The ground state of ruthenium(II) in an octahedral environment is A ,
1g
6
3
3
1
arising from the t
configuration. The excited state terms are T , T , T
2g
1g
1
2g
1g
1
3
and T . Hence four bands corresponding to the transition A → T ,
2g
1g
1g
1
3
1
1
1
1
A → T
A → T and A → T in order of increasing energy are pos-
1g 1g 1g 2g
1g
2g,
sible. The bands around 521–524 nm and 392–486 nm are assigned to
1
1
12,20
A → T and charge transfer (CT) transitions, respectively.
The charge
1g
1g
transfer bands observed in all the complexes due to M→L transitions are possible
in the visible region. Moreover, the presences of carbonyl, triphenylphos-
phine/arsine and heterocyclic bases as ligands, which are capable of producing
*
strong ligand field e levels place these levels relatively high in energy. There-
g
fore, the lowest charge bands due to excitation of an electron from the metal t
2g
level to an unfilled molecular orbital derived from the π* level of the ligands
should appear in the relatively high energy region compared to those due to
*
20–22
t → e transitions.
The other high intensity bands in the region 306–369
2g
g
nm region were assignable to ligand centred (LC) transitions and have been
designated as π–π* and n–π* transition. The pattern of the electronic spectra of
all the complexes indicated the presence of an octahedral environment around the
23
ruthenium(II) ion, similar to that of other ruthenium(II) octahedral complexes.
Mass spectra
The EI mass spectra of the ligands and complexes were recorded. The
2
maximum peaks are observed at m/z, 284, 230, 696, 755 for the ligands L and
3
1
2
L and for complexes [RuCl(CO)(PPh )(L )] and [RuCl(CO)(AsPh )(L ), res-
3
3
pectively, which match well with the corresponding calculated masses.
NMR spectra
1
The H-NMR spectra of ligands and complexes were recorded in DMSO-d
6
solution for confirming the binding mode of the Schiff base to ruthenium ion and
the values are given in Table III and the spectra are shown in Figs. 1 and 2. The
aromatic proton for the ligands appears as a multiplet at δ 6.24–8.03 ppm. On
complexation, the protons on the phenyl ring remain more or less unchanged in
the complexes, even if there are slight variation in their resonances due to the
24
delocalization of electron density in the system, and these signals in the com-
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SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
plexes cannot be distinguished from the aromatic signals of PPh /AsPh due to
3
3
25
their extensive overlap appearing at δ 6.22–8.24 ppm. The protons of the
hydroxyl groups appear as broad singlets at δ 11.64–11.98 (2-OH), δ 9.79–9.92
ppm (4-OH) and 14.27 ppm (Ph–OH) for the free Schiff base ligands. In the
spectra of the complexes, the resonances arising from the hydroxyl (2-OH) and
(Ph–OH) proton were not observed, indicating the coordination of the hydroxyl
14
oxygens to the metal ion. The signal due to the azomethine proton (–HC=N)
was found to be considerably deshielded at δ 9.12–9.35 ppm relatively to that of
the free Schiff base ligand δ 8.77–9.24 ppm as a consequence of electron dona-
2
tion to the metal centre. The methyl proton for L and its complexes appeared as
a singlet at δ 2.90–2.34 ppm.
TABLE III. 1H-NMR data for the ligands and ruthenium(II) Schiff base complexes
Ligands and complexes
L1
1H-NMR data, δ / ppm
6.39–8.03 (m, Ar), 9.24 (s, HC=N), 11.98 (s, 2-OH),
9.92 (s, 4-OH)
6.20–7.65 (m, Ar), 9.07 (s, HC=N), 11.90 (s, 2-OH),
9.79 (s, 4-OH), 2.29 (s, CH3)
6.24–7.43 (m, Ar), 8.77 (s, HC=N), 11.60 (s, 2-OH),
9.92 (s, 4-OH), 14.27 (s, OH)
L2
L3
[RuCl(CO)(PPh3)L1] (1)
[RuCl(CO)(PPh3)L2] (2)
[Ru(CO)(PPh3)2L3] (3)
[RuCl(CO)(AsPh3)L1] (4)
7.15–7.67 (m, Ar), 9.32 (s, HC=N), 9.94 (s, 4-OH)
7.26–7.70 (m, Ar), 9.12 (s, HC=N), 9.84 (s, 4-OH), 2.34 (s, CH3)
6.62–8.23 (m, Ar), 9.14 (s, HC=N), 9.92 (s, 4-OH)
6.92–7.76 (m, Ar), 9.35 (s, HC=N), 10.14 (s, 4-OH),
[RuCl(CO)(AsPh3)L2] (5) 6.26–7.83 (m, Ar), 9.32 (s, HC=N), 9.91 (s, 4-OH), 2.28 (s, CH3)
[Ru(CO)(AsPh3)2L3] (6)
6.82–7.66 (m, Ar), 9.16 (s, HC=N), 9.85 (s, 4-OH)
Fig.1. 1H-NMR spectrum of ligand L2.
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
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Fig. 2. 1H-NMR spectrum of [RuCl(CO)(PPh3)L2].
13
The C-NMR data were recorded in DMSO-d solution and the assign-
6
ments for the ligands and the complexes are listed in Table IV and a respective
13
spectrum is shown in Fig. 3. The C-NMR spectra of all the Schiff base ligands
26
displayed a single resonance at δ 150–152 ppm, showing that the azomethine
carbon atoms were equivalent, which also confirms the structure of the ligands.
27
The signal at δ 164–165 ppm corresponds to thiazolic C=N carbon. The down-
field shift of these two signals at δ 164–165 and 169–172 ppm clearly indicates
28
that both the C=N carbons were affected by coordination. The aromatic car-
bons of the free ligands and the corresponding complexes show signals in the
2
region δ 102–139 ppm. The signal due to the methyl carbon of L and the cor-
responding complexes appeared at δ 21–22 ppm. For all the complexes, the ter-
29
minal carbonyl carbon appeared in the range δ 191–194 ppm.
TABLE IV. 13C-NMR data for the ligands and ruthenium(II) Schiff base complexes
Ligands and complexes
13C-NMR data, δ / ppm
117–137 (Ar C), 152 (C=N), 164 ( thiazole, C=N)
102–134 (Ar C), 151 (C=N), 165 ( thiazole, C=N), 21 (CH3)
107–134 (Ar C), 150 (C=N),
128–133 (Ar C), 164 (C=N), 170 ( thiazole, C=N), 194 (C≡O)
122–134 (Ar C), 165 (C=N), 169 ( thiazole, C=N),
191 (C≡O), 21 (CH3)
L1
L2
L3
[RuCl(CO)(PPh3)L1] (1)
[RuCl(CO)(PPh3)L2] (2)
[Ru(CO)(PPh3)2L3] (3)
[RuCl(CO)(AsPh3)L1] (4)
[RuCl(CO)(AsPh3)L2] (5)
122–139 (Ar C), 165 (C=N), 191 (C≡O)
112–134 (Ar C), 165 (C=N), 172 ( thiazole, C=N), 191 (C≡O)
127–133 (Ar C), 164 (C=N), 171 ( thiazole, C=N),
192 (C≡O), 22 (CH3)
[Ru(CO)(AsPh3)2L3] (6)
128–139 (Ar C), 164 (C=N), 193 (C≡O)
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SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
Fig. 3. 13C-NMR spectrum of [RuCl(CO)(PPh3)L2].
In order to confirm the presence of triphenylphosphine groups and to deter-
31
31
mine the geometry of the complexes P-NMR spectra were recorded. The P-
1
3
-NMR spectra of the complexes [Ru(CO)Cl(PPh )L ] and [Ru(CO)(PPh ) L ]
3
3 2
were recorded in DMSO-d solution. The observation of a sharp singlet at δ 28.2
6
1
3
and 32.2 ppm for the complexes [Ru(CO)Cl(PPh )L ] and [Ru(CO)(PPh ) L ],
3
3 2
respectively, confirmed the presence of only one triphenylphosphine group. The
3
appearance of only one signal for [Ru(CO)(PPh ) L ] suggests that the two tri-
3 2
phenylphosphine groups are magnetically equivalent and hence, they must be
trans to each other in the complex.
DNA binding study
The interactions of metal complexes with DNA are of interest for the deve-
lopment of effective chemotherapeutic agents. Transition metal centres are parti-
cularly attractive moieties for such research since they exhibit well-defined coor-
dination geometries and often possess distinctive electrochemical or photophy-
2
sical properties, thus enhancing the functionality of the binding agent. Electro-
nic absorption spectroscopy is one of the most useful techniques for DNA-bind-
ing studies of metal complexes. The interactions of the ruthenium complexes with
CT-DNA were investigated by UV–Vis absorption titrations. The binding of the
ruthenium(II) complexes to DNA helices were characterized by following the
changes in the absorbance and shift in wavelength on each addition of DNA
solution to the complex. Upon addition of increasing amounts of CT-DNA from
0 to 25 μM, a significant “hyperchromic” effect of the intraligand bands at 252–
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
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–323 nm was observed accompanied by a moderate red shift of 2–3 nm, indi-
cative of the breakage of the DNA helix. However, there were no appreciable
wavelength shifts in the charge transfer band. These spectral characteristics sug-
gest that the complexes and ligand bind either to the external contact (electro-
static binding) or to the major and minor grooves of DNA. As the concentration
2
of the DNA was increased, the absorption bands of the [RuCl(CO)(PPh )L ]
3
complex initially showed hyperchromism but on further increasing, hypo-
chromism with a blue shift of 5 nm was observed. An isosbestic point was
observed at 295 nm. This behaviour reveals an electrostatic association of the
30,31
complex with the helix surface.
Generally, hypochromism and hyperchro-
mism are the two spectral features which are closely connected with the double
helix structure of DNA. The observation of hypochromism is indicative of inter-
calative mode of binding of DNA to the complexes along with the stabilization of
32
the DNA double helix structure. On the other hand, the observation of hyper-
33
chromism is indicative of breakage of the secondary structure of DNA. The
2
binding constant of the complex [RuCl(CO)(PPh )L ] could not be evaluated due
3
to the random changes in the absorption on the addition of DNA. Hence, the
observation of hyperchromism with a red shift for the ligand and complexes
showed that they interact with the secondary structure of CT-DNA by breaking
its double helix structure.
In order to compare the DNA-binding affinity of the ruthenium(II) com-
plexes quantitatively, their intrinsic binding constants were calculated by moni-
toring the changes in absorption of the higher energy band with increasing con-
centration of DNA(Eq. (1) and Fig. 4). The intrinsic binding constants K were
b
4
–1
3
calculated and were found to be (1.6–8.6)×10 mol dm , for the ruthenium(II)
complexes (Fig. 4 and Table V). The magnitude of the binding constant clearly
3
showed that complex [Ru(CO)(PPh ) (L )] bound more strongly with CT-DNA
3 2
than the other complexes. The significant difference in DNA-binding affinity of
the ruthenium(II) complexes may be a result of the fact that complex with dif-
ferent co-ligands shows different binding affinity with DNA. Interestingly, the
K values obtained for the above ruthenium(II) complexes are comparable with
b
2+
those for another known complex [Ru(dmp) (APIP)] , APIP = 2-(2-amino-
phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, (2.3–3.3)×10 mol dm .
2
4
–1
3 34
DNA cleavage activity
To assess the DNA cleavage ability of the new ruthenium(II) complexes,
calf thymus DNA was incubated with two different concentrations of the
complexes in 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2 for 2 h without the
addition of a reductant. Upon gel electrophoresis of the reaction mixture, con-
centration-dependent DNA cleavage was observed. When the concentration of
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162
SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
Fig. 4. Plots of [DNA]/(εa–εf)
versus [DNA] for the titration of
the prepared compounds with
CT-DNA.
TABLE V. Binding constant value for interaction of the ruthenium(II) complexes with
CT-DNA
Complex
Kb×104 / mol-1 dm3
[RuCl(CO)(PPh3)(L1)] (1)
[RuCl(CO)(PPh3)(L2)] (2)
[Ru(CO)(PPh3)2(L3)] (3)
[RuCl(CO)(AsPh3)(L1)] (4)
[RuCl(CO)(AsPh3)(L2)] (5)
[Ru(CO)(AsPh3)2(L3)] (6)
8.2
–
8.6
5.6
3.3
1.6
the complexes 1–6 was increased from 30 to 60 µM, the production of Form II of
DNA increased, (Fig. 5 for complexes 1–3, and Fig. 6 for complexes 4–6). No
DNA cleavage was observed for the control in which metal complex was absent
(Fig. 5, lane 1, and Fig 6, lane 8). With increasing concentration of the
ruthenium(II) complexes (Fig. 5, lanes 2 and 3 for complex 1; lanes 4 and 5 for
complex 2; lanes 6 and 7 for complex 3 and Fig. 6, lanes 9 and 10 for complex 4;
lanes 11 and 12 for complex 5; lanes 13 and 14 for complex 6) the amount of
Form I of CT-DNA diminished gradually and the amount of the nicked circular
DNA (Form II) increased remarkably. When the concentration was increased to
60 μM for all the complexes, the DNA was completely converted from Form I to
Form II, showing the potential chemical nuclease activity of the complexes.
1
Moreover, complex [RuCl(CO)(PPh )(L )] (1) exhibited greater cleavage
3
efficiency than other complexes, which could be attributed to the longer metal-to-
1
ligand charge transfer (MLCT) excited state lifetime of [RuCl(CO)(PPh )(L )].
3
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DNA BINDING AND CLEAVAGE PROPERTIES OF RUTHENIUM(II) SCHIFF BASE COMPLEXES
163
Fig. 5. Gel electrophoresis showing the chemical nuclease activity of CT-DNA incubated at
37 °C for 2 h with different concentrations of complexes 1–3; lane 1, DNA control; lane 2,
DNA + complex 1 (30 µM); lane 3, DNA + complex 1 (60 µM); lane 4, DNA + complex 2
(30 µM); lane 5, DNA + complex 2 (60 µM); lane 6, DNA + complex 3 (30 µM);
lane 7, DNA + complex 3 (60 µM).
Fig. 6. Gel electrophoresis showing the chemical nuclease activity of the CT-DNA incubated
at 37 °C for 2 h with different concentrations of complexes 4–6; lane 8, DNA control; lane 9,
DNA + complex 4 (30 µM); lane 10, DNA + complex 4 (60 µM); lane 11, DNA + complex 5
(30 µM); lane 12, DNA + complex 5 (60 µM); lane13, DNA + complex 6 (30 µM);
lane 14, DNA + complex 6 (60 µM).
CONCLUSION
Three novel Schiff base ligands and their ruthenium(II) complexes were
designed, synthesized and characterized by elemental analyses, stoichiometric
and spectroscopic studies. Based on the characterization, an octahedral geometry
was tentatively proposed for all the new ruthenium(II) complexes. Furthermore,
in vitro DNA binding studies were performed for complexes 1–6 using the
absorption titration technique. These complexes are unique and act synergis-
tically at the molecular level but with different binding modes. This study
revealed that the complexes bind electrostatically to the DNA double helix sur-
4
–1
3
face. The binding constants were found to be (1.6–8.6)×10 mol dm for com-
plexes 1 and 3–6. Interestingly, the K values obtained for the above ruthe-
b
2+
nium(II) complexes were comparable to those for other known [Ru(dmp) (APIP)]
2
complexes (APIP = 2-(2-aminophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline),
4
–1
3
(2.3–3.3)×10 mol dm . From the values of the binding constant, it was
inferred that the triphenylphosphine complexes bind more with CT-DNA than the
corresponding triphenylarsine complexes. The DNA cleavage study revealed that
all ruthenium complexes had the ability to cleave nucleic acids and the extent of
the cleavage was found to be dose dependent. The information obtained in this
study could be helpful in the understanding of the mechanism of interactions of
ruthenium(II) complexes with nucleic acids and should be useful in the develop-
ment of potential probes for investigation of the structure and conformation of
DNA, or new therapeutic agents for some diseases.
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164
SATHIYARAJ, AYYANNAN and JAYABALAKRISHNAN
Acknowledgement. We sincerely thank the University Grants Commission (UGC), New
Delhi, India, for financial support (MRP Scheme, No. 38-222/2009 (SR)).
И З В О Д
СИНТЕЗА И СПЕКТРАЛНА КАРАКТЕРИЗАЦИЈА КОМПЛЕКСА РУТЕНИЈУМА(II) СА
ШИФОВИМ БАЗАМА КОЈИ САДРЖЕ PPh3/AsPh3 ЛИГАНДЕ И ИСПИТИВАЊЕ
ЊИХОВИХ ИНТЕРАКЦИЈА СА ДНК
SUBBAIYAN SATHIYARAJ, GANESAN AYYANNAN и CHINNASAMY JAYABALAKRISHNAN
Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of Arts
and Science, Coimbatore - 641 020, Tamil Nadu, India
У овом раду су синтетизоване Шифове базе полазећи од дихидроксибензалдехида
(L1–L3), као и одговарајући рутенијум(II) комплекси. Сва једињења су окарактерисана
1
применом елементалне микроанализе, H-, 13C- и 31P-NMR, и IR спектроскопије, UV–
–Vis спектрофотометрије и масене спектрометрије. Применом UV–Vis спектрофотомет-
рије испитиване су интеракције комплекса рутенијума(II) и ДНК. На основу добијених
резултата, закључено је да се сви комплекси електростатички везују за ДНК, при чему су
одговарајуће константе везивања (Kb) у опсегу (1,6–8,6)×104 mol-1 dm3. Додатно је испи-
тивано раскидање веза у молекулу ДНК помоћу комплекса рутенијума(II).
(Примљено 1. децембра 2012, ревидирано 8. маја 2013)
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