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Synthesis, characterization, DNA binding, light switch "on and off", docking studies and cytotoxicity, of ruthenium(II) and cobalt(III) polypyridyl complexes.
JFluoresc
DOI10.1007/s10895-014-1355-6
ORIGINALPAPER
Synthesis, Characterization, DNA Binding, Light Switch
“ ”
On and Off , Docking Studies and Cytotoxicity,
of Ruthenium(II) and Cobalt(III) Polypyridyl Complexes
M.RajenderReddy&PuttaVenkatReddy&
YataPraveenKumar&A.Srishailam&
NavaneethaNambigari&S.Satyanarayana
Received:22September2013/Accepted:27January2014
#SpringerScience+BusinessMediaNewYork2014
Abstract The novel ligand (dmbip) 2-(4-N, N- Introduction
dimethylbenzenamine)1H-imidazo[4, 5-f][1,
10]phenanthroline and its complexes [Ru(phen) dmbip]2+ During recent decades a variety of luminescent polypyridyl
2
(1), [Ru(bpy) dmbip]2+ (2), [Co(phen) dmbip]3+ (3) and complexes employing a range of transition metal ions and
2 2
[Co(bpy) dmbip]3+ (4) [where phen=1, 10-phenanthroline, ligands architectures have been reported. The luminescent
2
′
bpy=2, 2-bipyridine], have been synthesized and charac- and redox properties of Ru(II) and Co(III) complexes of 1,
terized by elemental analysis, IR, UV-Vis, 1H NMR, 13C 10-phenanthroline (phen), 2, 2′ bipyridine(bpy), and related
NMRandMassspectra.TheDNAbindingpropertiesofthe bidentate ligands have been studied due to their significant
complexes were investigated by absorption, emission, MLCT absorption in the visible region [1–3]. The clinical
quenching studies, light switch “on and off”, salt depen- utility of transition metal complexes binding to DNA has
dent,sensor(cationandanion)studies,viscositymeasure- inspired a great interest in the design and development of
ments, cyclic voltammetry, molecular modeling and novelcomplexesthatcanbeappliedinDNA-structureprobes,
docking studies. The four complexes were screened for DNA “molecular light switches”, DNA-photocleavage re-
Photo cleavage of pBR322 DNA, antimicrobial activity agents, anticancer drugs and so forth [4–7]. In recent years,
andcytotoxicity.Theexperimentalresultsindicatethatthe Ru(II) and Co(III) polypyridyl complexes have been
four complexes can intercalate into DNA base pairs. The employed in studies with DNA, with a view to design and
DNA-bindingaffinitiesofthesecomplexesfollowtheorder develop synthetic restriction enzymes, new drugs and DNA
[Ru(phen) dmbip]2+ > [Co(phen) dmbip]3+ > footprinting agents [8–12]. The electron rich DNA bases, or
2 2
[Ru(bpy) dmbip]2+>[Co(bpy) dmbip]3+. phosphate groups are available for direct covalent coordina-
2 2
tion to a metal center. There are also non covalent binding
modes,suchashydrogenbondingandelectrostaticbindingto
Keywords Ru(II)&Co(III)complexes .CalfthymusDNA . groovedregionsoftheDNAandintercalationofplanararo-
Photocleavage .Antimicrobialactivity.Sensors .Cyclic maticligandsintothestackedbasepairs.Varyingsubstitutive
voltammetry.Cytotoxicity group(or)substituentpositionintheintercalativeligandscan
createsomeinterestingdifferencesinthespaceconfiguration
and electron density distribution of Ru(II) and Co(III)
polypyridyl complexes. The ancillary ligands of Ru(II) and
: : : : Co(III)polypyridylcomplexescanhaveasignificanteffecton
M.R.Reddy P.V.Reddy Y.P.Kumar A.Srishailam
the spectralpropertiesand theDNAbinding behaviorofthe
S.Satyanarayana(*)
complexes[13,14].
DepartmentofChemistry,OsmaniaUniversity,Hyderabad500007,
India Recently,ourgrouphasbeenreportedseveralpolypyridyl
e-mail:ssnsirasani@gmail.com ligand complexes of Ru(II) and Co(III) [15–27]. The aim of
thepresentinvestigationistostudymoreindetailtheeffectof
N.Nambigari
Ru(II)andCo(III)polypyridylcomplexeswithDNA.Herein,
DepartmentofChemistry,NizamCollege,OsmaniaUniversity,
Hyderabad500007,India we report the synthesis and characterization ofa new ligand
JFluoresc
dmbip (2-(4-N, N-dimethylbenzenamine)1H-imidazo[4, 5- carriedoutonOstwaldViscometer,immersedinthermostated
f][1, 10]phenanthroline) and its complexes waterbathmaintainedat30±0.1°C.CT-DNAsamplesapprox-
[Ru(phen) dmbip]2+ (1), [Ru(bpy) dmbip]2+ (2), imately 200 base pairs in average length were prepared by
2 2
[Co(phen) dmbip]3+ (3) and [Co(bpy) dmbip]3+ (4) [where sonication in order to minimize the complexities arising from
2 2
′
phen=1, 10-phenanthroline, bpy=2, 2-bipyridine] (Fig. 1). DNAflexibility.CyclicvoltmeterwascarriedoutonWonATech
ThebindingpropertiesonRu(II)andCo(III)complextocalf multichannelpotentiostat/galvanostat(WMPG1000,Gyeonggi-
thymusDNAinphysiologicalbuffer(pH7.4)wasinvestigat- do, Korea), the Patch dock server tool was used to perform
ed by multi-spectroscopic methods. The results showed that dockingcalculationsandmolecularmodelingstudieswerecar-
spectroscopic techniques could provide a convenient way to riedoutusingtheHyperChem7.5software.
characterizeboththebindingmodeandtheinteractionmech-
anismofRu(II)andCo(III)polypyridylcomplextoDNA.All
the four complexes were screened for Photo cleavage of
MaterialsandMethods
pBR322DNAandantimicrobialactivity.Thecytotoxicityof
complexes 1, 2, 3 and 4 has been evaluated by MTT RuCl CoCl , 1, 10-phenanthrolinemonohydrate 2, 2′-
3, 2
{MTT=(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium
bipypridine and DNA, were purchased from Merck(India).
bromide)}assay.Webelievethattheknowledgegainedfrom
1, 10-phenanthroline-5, 6-dione was synthesized according
this study will be helpful to further understand the binding
to literature procedure [28]. Dimethyl sulfoxide (DMSO)
mechanisms and can provide much fruitful information for
and RPMI 1640 were purchased from Sigma Aldrich.
designinganewtypeofhighlyeffectiveantitumordrugs.
Supercoiled pBR322 DNA was obtained from Bangalore
Genie.Doublydistilledwaterwasusedforpreparingvarious
buffers. The DNA had a ratio of UVabsorbance at 260 and
280nmof1.8–1.9:1,indicatingthattheDNAwassufficiently
Experimental
freefromprotein[29].DNAconcentrationpernucleotidewas
determined by using a molar absorption coefficient
PhysicalMeasurements
[6,600 M
−1
cm
−1]
at 260 nm [30]. The perchlorate salts of
metalcations(Cd2+,Zn2+andFe2+)andtetrabutylammonium
UV-Visible spectra were recorded with an Elico Bio-
salts ofanions (F¯,Br¯, Cl¯ and CH COO¯) purchased from
spectrophotometer, model BL 198. IR spectra were recorded 3
commercialsuppliersandusedasthroughouttheexperiment.
in KBr discs on a Perkin-FT-IR-1605 spectrophotometer 1H
NMR and 13C NMR spectra were measured on a Bruker Z-
Gradient 400 MHz spectrophotometer using DMSO-d as the SynthesisofLigand
6
solvent. Electrospray ionization mass spectrometry (ESI-MS)
was recorded on a LQC system (Finnigan MAT, USA) using Theligandwassynthesizedaccordingtotheprocedureinthe
CH CN as mobile phase. Fluorescence spectra were recorded literature[31].Amixtureofphen-dione(0.53g,2.50mM),N,
3
with SL 174 spectrofluorometer, Viscosity experiments were N-Dimethylaminobenzaldehyde (0.675 g, 3.50 mM),
Fig.1 Procedureforsynthesisof
ligandandcomplexes N O
[Ru(phen)(dmbip)]2+(1),
2 N O
[Ru(bpy)(dmbip)]3+(2),[Co
2
(phen)(dmbip)]2+(3)and
[Co(bp 2 y)(dmbip)]3+(4) Gla. AcOH H
2 NH OAc N
4 O
Reflux 4h
N
H
N N N H N [Ru(phen) 2 Cl 2 ] [Ru(bpy) 2 Cl 2 ] N Ru N N N
Ru N N N
N N N N N
N H
N N 2
1
N
N N
dmbip
N N
H H
N N N N N N
Co N Co N
N N N [Co(phen) 2 Cl 2 ] [Co(bpy) 2 Cl 2 ] N N N
N N
3 4
JFluoresc
ammoniumacetate(3.88g,50.0mM)andglacialaceticacid Table2 FT-IRdataofRu(II)&Co(III)complex
(15ml)wasrefluxedfor4h.Theabovesolutionwascooledto Compound FTIR-data(Cm−1)
roomtemperature and diluted with water, drop wise addi-
tion of Conc. NH gave a yellow precipitate, which was C=C C=N M-N(L) M-N(dmbip)
3
collected, washed with H O and dried. The crude product
2
dmbip 1,468 1,588 734 633
recrystallized with C H N. H O and dried (yield: 71 %).
5 5 2 [Ru(phen)(dmbip)]2+ 1,460 1,581 728 629
Anal.dataforC H N :calc.C,74.32;H,5.05;N,20.63; 2
21 17 5
found: C, 74.29; H, 5.01; N, 20.61. ES+-MS Calc: 339; [Ru(bpy) 2 (dmbip)]2+ 1,603 1,445 722 623
[Co(phen)(dmbip)]3+ 1,541 1,430 714 624
found: 340. 2
[Co(bpy) (dmbip)]3+ 1,560 1,448 718 620
2
SynthesisofComplexes
[Co(phen) (dmbip)](ClO ) .3H O
2 4 3 2
[Ru(phen) (dmbip)](ClO ) .2H O
2 4 2 2 A mixture of [Co(phen) Br ]Br. 3H O (0.531 g, 1.0 mM),
2 2 2
dmbip(0.489g,1.5mM)in50mlethanolwasrefluxedfor4h
A mixture of [Ru(phen) Cl ] 2H O (0.531 g, 1.0 mM),
2 2 2 togiveayellowsolution.Afterfiltration,thecomplexeswere
dmbip(0.489g,1.5mM)andethanol(15ml)wasrefluxed
precipitated by addition of saturated ethanol solution of
for 8 h under N atmosphere. When the light purple color
2 NaClO . The complex was filtered and further dried under
solutionisobtained,itwascooledtoroomtemperatureand 4
vacuumbeforerecrystallization(ethanol),(yield:76%)Anal.
anequalvolumeofsaturatedaqueousNaClO solutionwas
4 data for CoC H N Cl O : C, 48.64; H, 3.54; N, 11.35;
added under vigorous stirring.The red solid was collected 45 39 9 3 15
found: C, 48.60; H, 3.51; N, 11.34. ES+-MS: calc: 1109;
and washed with small amounts of water, ethanol and
found:1110.
diethylether,thendriedundervacuum(yield:62%).Anal.
data for RuC H N Cl O : cal. C, 52.18; H, 3.60; N,
45 37 9 2 10 [Co(bpy) (dmbip)](ClO ) .3H O
12.17; found: C, 52.14; H, 3.58; N, 12.11. ES+-MS cal: 2 4 3 2
1035;found:1036.
Thiscomplexwassynthesizedasdescribedabovebytakinga
mixtureof[Co(bpy) Br ]Br.3H O(0.531g,1.0mM),dmbip
2 2 2
(0.489 g, 1.5 mM) (yield: 65 %) Anal. data for
[Ru(bpy) (dmbip)](ClO ) .2H O
2 4 2 2 CoC H N Cl O :calc.C,46.32;H,3.70;N,11.86;found:
41 39 9 3 15
C,46.30;H,3.68;N,11.82.ES+-MScalc:1061;found:1062.
Thiscomplexwassynthesizedasdescribedabovebytakinga
mixture of [Ru(bpy) Cl ] 2H O (0.531 g, 1.0 mM), dmbip
2 2 2 SpectroscopicCharacterization
(0.489 g, 1.5 mM) (yield: 63 %). Anal. data for
RuC H N Cl O :calc.C,49.85;H,3.78;N,12.76;found:
41 37 9 2 10 The IR spectral data for the complexes exhibit bands at
C,49.81;H,3.72;N,12.71.ES+-MScalc:987;found:988.
1,438 cm −1 and at 1,548–1,585 cm −1 due to C=C and C=N
vibrationsoftheringrespectively.Bandswerepresentaround
567 cm −1 and 578 cm −1 corresponding to Co–N (phen) and
Table1 1HNMRdataofligandandRu(II)&Co(III)complexes
Co–N of (bpy) respectively. In the 1H NMR spectra of the
Compound 1HNMR(400MHz,ppm,DMSO-d,TMS)
6
Table3 13C[1H]-NMRdataofRu(II)&Co(III)complexes
dmbip 9.03(d,2H);8.95(d,2H);8.24(d,2H);
7.21(d,2H);6.95(d,2H);2.10(s,6H,–CH 3 ). Complex 13CNMR(100MHz,DMSO-d 6, majorpeaks)
[Ru(phen)(dmbip)]2+ 9.16(d,6H),8.71(d,6H),8.26(s,4H),8.11
2
(t,6H),7.77(d,2H),6.84(d,2H), [Ru(phen)(dmbip)]2+ 154.62,153.16,150.30,147.83,137.23,130.92,
2
2.76(s,6H,–CH). 128.42,126.25,117.26,112.52,34.25.
3
[Ru(bpy)(dmbip)]2+ 9.13(d,2H),8.89(d,4H),8.83(d,4H),8.23 [Ru(bpy)(dmbip)]2+ 157.43,154.74,152.04,151.83,149.91,
2 2
(d,2H),8.12(t,4H),7.61(t,2H),7.35(t,4H), 138.39,130.99,128.60,128.23,124.81,
6.91(d,2H)6.74(d,2H),2.78(s,6H,–CH). 117.36,33.45.
3
[Co(phen)(dmbip)]3+ 9.15(d,6H),8.60(d,6H),8.56(s,4H), [Co(phen)(dmbip)]3+ 155.53,153.96,146.22,143.68,136.15,
2 2
7.98(t,6H),7.66(d,2H),6.78(d,2H), 133.07,131.75,129.68,129.11,124.96,
2.71(s,6H,–CH). 116.53,112.56,33.53.
3
[Co(bpy)(dmbip)]3+ 9.10(d,2H);8.79(d,4H);8.58(d,4H); [Co(bpy)(dmbip)]3+ 152.82,151.63,150.79,148.52,146.35,
2 2
8.21(d,2H);8.20(t,4H);7.50(t,2H); 136.66,130.45,128.35,126.51,117.22,
7.38(t,4H);6.87(d,2H);2.78(s,6H,–CH). 112.36,33.47.
3
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0.0
300 325 350 375 400 425 450 475 500 525 550
complexesthepeaksduetovariousprotonsofligandshifted constant K , with CT-DNAwas obtained by monitoring the
b
downfield compared to the free ligand indicating complexa- change in the absorbance at metal to ligand charge transfer
tion. As expected, the signals for the ligand appeared in the (MLCT) band, with increasing concentration of DNA at
range around 7 to 9.8 ppm and the 6 hydrogens of the N- 25°C.TheintrinsicbindingconstantK ,wascalculatedfrom
b
methylgrouppeaksappearedat1.7–1.9ppm,13CNMRpeaks Eq.1[32].
ofthecomplexesappearedaround154.62to112.52ppmand
N-Methyl carbon peaks appeared around 34.25 to 33.53
½DNA(cid:2)=ðε –ε Þ¼½DNA(cid:2)=ðε –ε Þþ1=K ðε –ε Þ ð1Þ
(showinTables1,2,and3). a f b f b b f
DNABindingStudies where [DNA] is the concentration of DNA, ε , ε and ε
a f b
corresponds to the apparent absorption coefficient A /
obsd
The interaction of Ru(II) and Co(III) complexes with DNA [complex], the extinction coefficient for the free complex,
were studied in tris-buffer (5 mM Tris-HCl, 10 mM NaCl and the extinction coefficient for the complex in the fully
pH=7.1). The absorption titrations of these complexes were bound form, respectively. In plots of [DNA]/(ε –ε) vs
a f
performed by treating fixed concentration of complexes [DNA],K isgivenbytheratioofslopetotheintercept.
b
(10 μM) with varying concentration (0–150 μM) of DNA. Intheemissionstudiesfixedmetalcomplexconcentration
Complex-DNAsolutionswereallowedtoincubatefor5min (6 μM) was taken and to this varying concentration (0–
before recording the absorption spectra. In order to evaluate 150 μM) of DNA was added. The excitation wavelength
the binding strength of the complex, the intrinsic binding was fixed and the emission range was adjusted before
ecnabrosbA
3.50E-06 3.00E-06 2.50E-06
2.00E-06
1.50E-06
1.00E-06
5.00E-07
0.00E+00
0.00E+00 1.00E-05 2.00E-05 3.00E-05 4.00E-05 [DNA]
Wavelength (nm)
) −
(/]AND[
f a
Fig.2 Absorptionspectraof
[Ru(phen)(dmbip)]2+intris-
2
bufferinabsenceandpresenceof CT-DNAthe[complex]=10–
15μM;[DNA]=0–126μM.
Insertplotsof[DNA]/(ε–ε)Vs
a f
[DNA]forthetitrationofDNA
withcomplex.Thearrowshows
changeinabsorptionwith
increasingDNAconcentration
7500
7000
6500
6000
5500
5000
4500
570 580 590 600 610 620 630 640 650
Wavelength (nm)
ytisnetnI
4.00E+03
3.00E+03
2.00E+03
1.00E+03
0.00E+00
0.01 0.015 0.02
C/r f
JFluoresc
Fig.3 Fluorescencespectraof
[Ru(phen)(dmbip)]2+complexin
2
Tris-HClbufferwithincreasing
concentrationofCT-DNA.The
arrowshowsthefluorescence
intensitychangeuponincreaseof r
DNAconcentration.Inset:
Scatchardplotofr/C vsr
f
Table4 Intrensicbinding
constantsofabsorptionand Complex Hyperchromism(%) AbsorptionΔ λ Absorptionbinding Emission
emissionstudiesofcomplexes constantK b constant
[Ru(phen)(dmbip)]2+ 21 14 2.2×105 7.3×105
2
[Ru(bpy) (dmbip)]2+ 18 11 1.3×105 5.5×105
2
[Co(phen)(dmbip)]3+ 19 9 1.7×105 7.1×105
2
[Co(bpy) (dmbip)]3+ 16 7 1.1×105 4.3×105
2
measurements.Thefractionoftheligandboundwascalculat- [DNA],whereηistheviscosityofDNAinthepresenceofthe
edfromtherelationC =C[(F−F )/F −F )],whereC isthe complex, and η is the viscosity of DNA alone. Viscosity
b t 0 max 0 t 0
total complex concentration, F is the observed fluorescence values were calculated from the observed flow time of
emission intensity at a given DNA concentration, F is the DNA-containing solutions (t>100 s) corrected for the flow
0
intensityintheabsenceofDNAandF isthefullybound timeofthebufferalone(t )[35].
max 0
DNAtocomplex.Bindingconstant(K )wasobtainedfroma Cyclic voltammogram recorded by using standard three
b
modified Scatchard equation [33]. From a Scatchard plot of electrodecellcontainingaplatinumfoilasworkingelectrode,
r/C vsr,whereristheC /[DNA]andC istheconcentration platinum wire as counter electrode and saturated calomel
f b f
offreecomplex. electrode (SCE) as reference electrode, 50 mg of the active
Sensorstudieswereperformedasfollows,initiallyprepare compound(writeyourmetalcomplexnameinsteadofactive
stocksolutionofcomplexesinabsoluteCH CNdiluteintris- compound)dissolvedin100mlofacetonitrilecontaining1M
3
HCl buffer solution (10 mM; PH=7.0). Aliquots of Et NBF at10mg/sscanrate.
4 4
cations(Cd2+, Zn2+, and Fe2+) and anions(F¯, Br¯, Cl¯ and For the gel electrophoresis experiments, Supercoiled
CH COO¯)werepreparedindoubledistilledwater.Bytaking pBR322DNA(100μM)wastreated withRu(II) complexes
3
theappropriateconcentrationofcationsandanionsinjectedto (40 and 80 μM) and the samples were irradiated at room
metalcomplexes,thenobservechangeinemissionspectra. temperature with a UV lamp (365 nm) for 30 min. The
Viscosity experiments were carried out on Ostwald vis- samples were analyzed by electrophoresis for 2.5 h at 40 V
cometer,immersedinthermostatedwaterbathmaintainedat ona0.8%agarosegelinTris-aceticacid-EDTAbuffer.The
30±0.1°C.CT-DNAsamplesapproximately200basepairsin gel was stained with 1 μg/ml ethidium bromide and then
averagelengthwerepreparedbysonicationinordertomini- photographedunderUVlight.
mize the complexes arising from DNA flexibility [34]. Data Microbial activity was performed by the standard disc
were presented as (η/η )1/3 versus concentration of [Ru(II)]/ diffusion method [36]. The complexes were screened for
0
3.5
3.0
2.5
2.0
1.5
1.0
0.00000 0.00008 0.00016 0.00024
I/I 0
3.5
a b 3.0
c
2.5
2.0
1.5
1.0
0.00000 0.00008 0.00016 0.00024
[Fe(CN)]4–
6
I/I 0
a b
c
[Fe(CN)]4–
6
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.00000 0.00008 0.00016 0.00024
I/I 0
2.8 a
2.6
b
2.4
c 2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.00000 0.00008 0.00016 0.00024
[Fe(CN)]4–
6
I/I 0
JFluoresc
Fig.4 Emissionquenchingof 1. 2.
complexes[Ru(phen)(dmbip)]2+
2 (1),[Ru(bpy)(dmbip)]2+(2) 2
[Co(phen)(dmbip)]3+(3)and 2
[Co(bpy)(dmbip)]3+(4)with
2
[Fe(CN)]4−intheabsenceof 6 DNA(a),presenceofDNA1:30
(b)and1:200(c).[Ru]and[Co]=
10μM,[Fe(CN)]4−=0.1M
6
3. 4.
a
b
c
[Fe(CN)]4–
6
antibacterialactivityagainststandardmicroorganismssuchas 1600 B
E.coli,Pseudomonasaeruginosa,Staphylococcusaurousand D
1400
antifungal activity against Neurospora crassa, C
Aspergillusniger,Aspergillusflavus.TheMuellerHintonagar 1200
was prepared and poured fresh into sterile Petri plates and A
1000
allowedtodry,andinoculate0.2mlofbacterialculturewhich
has106cells/mlconcentrations.Thecomplexwasdissolvedin 800
DMSO to get a final concentration of 100 μl per disc. Each
600
plate contains standard microorganisms with three different
complexes(5μleachcompound)andstandardantibioticswere
400
alsotestedonthesestandardmicroorganismsascontrols,and
555 570 585 600 615 630 645
keptintherefrigeratorfor5minandtheseweretransferredto
Wavelength (nm)
the incubator at 37 °C. After 24 h of incubation,the zoneof
inhibitionofthecomplexesaswellasstandardantibioticson
standardmicroorganismswerechecked.Theminimuminhib-
itoryconcentrationsforthesecomplexesweremeasured.
Cytotoxicity was assessed using standard 3-(4, 5-
dimethylthiazole)-2, 5-diphenyltetraazolium bromide (MTT)
(1–4complexes),whilethemetaltoligandchargetransfer
assay [37]. Cells were placed in 96-well microassay culture (MLCT) bands (metal dπ orbital to ligand π* orbital) ap-
plates(8×103cellsperwell)andgrownovernightat37°Cina pearat447,452,318and327nm,forcomplexes1,2,3and
5 % CO incubator. The complexes tested were dissolved in 4, respectively. The change in absorbance of the MLCT
2
DMSO and diluted with RPMI 1640 and then added to the bands with increasing amount of CT-DNA was used to
wells to achieve final concentrations ranging from 10 −6 to derive the intrinsic binding constants (K b ) [38, 39]. The
10 −4 M. Control wells were prepared by addition of culture valuesofK b forcomplexes1,2,3and4are2.2×105,1.3×
medium(100μL).Thewellscontainingculturemediumwith- 105,1.7×105and1.1×105,respectively.Hence,thebinding
out cells were used as blanks. The plates were incubated at constantsshowthefollowingorder:[Ru(phen) 2 dmbip]2+>
37°Cina5%CO 2 incubatorfor48h.Uponcompletionofthe [Co(phen) 2 dmbip]3+ > [Ru(bpy) 2 dmbip]2+ >
incubation, stock MTT dye solution (20 μL, 5 mg/ml) was [Co(bpy) 2 dmbip]3+. The complexes [Ru(phen) 2 dmbip]2+
addedtoeachwell.After4h,buffer(100μL)containingN,N- and [Co(phen)
2
dmbip]3+ posses more binding constants
dimethylformamide(50%)andsodiumdodecylsulfate(20%) than their respective bipyridyl complexes, because from
wasaddedtosolubilizetheMTTformazan.Theopticaldensity bpytophenplanarareaandhydrophobicityincrease,which
of eachwell was then measuredona microplate spectropho- wouldleadtoagreaterbindingaffinityforDNA.
tometer at a wavelength of 490 nm. The IC values were
50
determinedbyplottingthepercentageviabilityagainstconcen- FluorescenceSpectroscopy
trationonabargraphandreadingoftheconcentrationatwhich
50 % of cells remain viable relative to the control. Each The fluorescence spectroscopy gives further clarification to in-
experiment was repeated at least three times to get the mean vestigate the interaction between complex and DNA. Figure 3
values.HelaandA549celllineswerethesubjectsofthisstudy.
ResultsandDiscussion
AbsorptionSpectralStudies
The application of electronic absorption spectroscopy in
DNA-binding studies are one of the mostuseful techniques.
Complex binding with DNA through intercalation usually
results in hypochromisum and bathochromisum, because of
theintercalativemodeinvolvingastrongstackinginteraction
between an aromatic ligands and DNA base pairs. The ab-
sorption spectra of complex [Ru(phen) dmbip]2+ in the ab-
2
senceandpresenceofCT-DNAisshowninFig.2.Thebands
below300nmareattributedtointraligandπ→π*transitions
ytisnetnI
Fig. 5 The luminescence changes upon addition of Co2+, EDTA to
[Ru(phen)dmbip]2+ + DNA. [Ru(phen)dmbip]2+ complex alone (A),
2 2
[Ru(phen) dmbip]2+ + CT-DNA (B), [Ru(phen) dmbip]2+ + DNA +
2 2
Co2+(C)and[Ru(phen)dmbip]2++DNA+Co2++EDTA(D)
2
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
-2.0 -1.8 -1.6 -1.4 -1.2 -1.0
KgoL
qe
JFluoresc
a
b
c
d
Log[Na+]
Fig. 6 Salt dependence studies of [Ru(phen) (dmbip)]2+ (a),
2 [Co(phen) (dmbip)]3+ (b), [Ru(bpy) (dmbip)]2+ (c) and
2 2
[Co(bpy)(dmbip)]3+(d)complexes
2
Wavelength (nm)
showedthefluorescencespectraof[Ru(phen) (dmbip)]2+com- [Co(phen) dmbip]3+and[Co(bpy) dmbip]3+intheabsenceof
2 2 2
plex in the absence and in the presence of CT-DNA. The DNAwere32,24,30and23,respectively.Inthepresenceof
complexes1,2,3and4canemitfluorescenceinTrisbufferat DNA, the K values were 23, 17, 21 and 15, respectively.
sv
ambient temperature with a maximum appearing at 607, 610, Hence the K values are smaller in the presence of DNA.
sv
425and403nm,respectively.Theemissionintensitiesofcom- From Quenching studies, it is clear that the DNA binding
plexes1,2,3and4increasedsharplyandreachashighas1.72, abilityofcomplexesfollowstheorder[Ru(phen) dmbip]2+>
2
1.65,1.68and1.59timesoftheoriginalintensities,respectively. [Co(phen) dmbip]3+ > [Ru(bpy) dmbip]2+ >
2 2
Thisimpliestheabovefourcomplexescanstronglyinteractwith [Co(bpy) dmbip]3+.Suchatrendisconsistentwiththeobser-
2
DNA and can be protected by DNA efficiently, because the vationinelectronicabsorptiontitrations.
hydrophobic environment inside the DNA helix reduces the
accessibilityofsolventwatermoleculestothecomplexandthe LightSwitch“OnandOff”Experiment
complex mobility is restricted at the binding site, leading to
decreaseof thevibration modeofrelaxation.The binding data Above studies show that the presence of CT–DNAvaries the
(Table4)werecastintotheformofaScatchardplotofr/C vsr, photoluminescence intensity of Ru(II) and Co(III) complexes.
f
where ‘r’ is the binding ratio of C /[DNA] and C is the free Photoluminescence intensity of complexes could be tuned
b f
ligandconcentration.Thebindingconstantsare7.3×105,5.5× by introduction of Co2+ ions. The addition of Co2+
105, 7.1×105 and 4.3×105 for complexes 1, 2, 3 and 4, (0.01 mM) to the [Ru(phen) dmbip]2+ complex bound to
2
respectively.
EmissionQuenchingStudies
The quenching experiments may give further information
about the binding ability of complex with DNA. In the ab-
sence of DNA, the emission of complexes were efficiently
quenchedbyquencher[Fe(CN)
]4−
.Emissionquenchingwith
6
[Fe(CN)
]4−
inthepresenceofDNAareshownin(Fig.4)for
6
four complexes. The Stern–Volmer quenching constant K
sv,
canbedeterminedbyusingStern–Volmerequation[40].
I0 =I¼1þK
sv
½Q(cid:2);
whereI andIaretheemissionintensitiesintheabsenceand
0
presence of quencher [Fe(CN) ]4− , K is the linear Stern–
6 sv
Volmerconstantand[Q]isthequencherconcentration.Inthe
quenchingplotofI /IVs[Q],slopeistheK .TheK values 0 sv sv
for the complexes [Ru(phen) dmbip]2+, [Ru(bpy) dmbip]2+,
2 2
ytisnetnI
1000 A
B 900 C
800 D
E 700
600
500
400
300
200
400 415 430 445 460 475 490 505 520 535
Wavelength (nm)
ytisnetnI
Fig.7 aAnionsensorstudiesof a b A
[Ru(phen)dmbip]2+complex B
2
alone(A),[Ru(phen)
2
dmbip]2++
450
C
Cl¯(B),[Ru(phen)dmbip]2++ D
2
Br¯(C),[Ru(phen)dmbip]2++
CHCOO−(D)and 2 400
3 [Ru(phen)dmbip]2++F¯(E). 2 350
bCationsensorstudiesof
[Ru(phen)dmbip]2+complex
alone(A), 2 [Ru(phen)dmbip]2++ 300
2
Cd2+(B),[Ru(phen)dmbip]2++
2
Zn2+(C)and[Ru(phen)dmbip]2+ 250
2
+Fe2+(D)
200
400 415 430 445 460 475 490 505 520
4.5
4
3.5
3
2.5
2
1.5
1
0.01 0.03 0.05 0.07 0.09 0.11
3/1)
(
0
JFluoresc
a
b
c
d
e
[Complex]/[DNA]
Fig.8 EffectofincreasingamountsoftheRu(II)andCo(III)complexes
of a). Ethidium bromide, b). [Ru(phen) (dmbip)]2+, c).
2
[Co(phen) (dmbip)]3+, d). [Ru(bpy) (dmbip)]2+ and e).
2 2 [Co(bpy)(dmbip)]3+complexesontherelativeviscosityofCT-DNAat 2
27±0.1°C
JFluoresc
Table5 Minimuminhibition
concentration(MIC)ofcomplexes S.no Complex E.coli(mM) S.aureus(mM)
andligand(μg/ml)withEcoliand
S.aureusbacterias
5μl 10μl 15μl 5μl 10μl 15μl
1. dmbip(ligand) 2.0 2.2 2.5 2.0 2.1 2.6
2. [Ru(phen)dmbip]2+ 4.0 6.0 8.0 4.5 7.0 8.2
2
3. [Ru(bpy)dmbip]2+ 3.9 5.8 7.6 3.9 5.9 7.8
2
4. [Co(phen)dmbip]3+ 2.8 5.9 7.4 2.9 6.0 7.7
2
5. [Co(bpy)dmbip]3+ 2.8 5.8 7.3 2.7 5.9 7.6
2
DNAresultsinlossofluminescenceduetotheformationof cation, and anion sensors. The emission spectrum of Ru(II)
Co2+-[Ru(phen) dmbip]2+.AsshowninFig.5whenEDTAis complexinCH CNshowedabroademissionbandat460nm.
2 3
addedtothebuffersystemcontainingCo2+-[Ru(phen) dmbip]2+ The effect of the anions (F¯, Br¯, Cl¯ and CH COO¯)
2 3
the emission intensity of the complex is recovered again (light interactingwith[Ru(Phen) dmbip]2+complexwasinvestigat-
2
switch on) [41]. This indicates that the heterometallic complex edinCH CN.Figure7a,showedthattheadditionofanexcess
3
(Co2+-[Ru(phen) dmbip]2+)becomesfreeagainduetoformation of anions (F¯, Br¯, Cl¯ and CH COO¯) as their tetrabutyl
2 3
of EDTA-Co2+ complex. By repeating this titration with equi- ammonium(TBA) salts to [Ru(Phen) dmbip]2+ complex re-
2
molarconcentrations(0.01mM)ofCo2+decreasedtheintensity sultedindramaticchangestothe emissionspectra;theaddi-
of[Ru(phen) dmbip]2+,andonaddingequimolarconcentrations tion of, Cl¯ Br¯and CH COO¯ anions slightly quenched the
2 3
(0.01mM)EDTAphotoluminescencerecovered.Similarresults emissionspectrum,whereastheadditionofF¯anionwasquite
areobtainedforremainingcomplexes. significant, giving riseto a large degree ofquenching of the
emissionspectrum.Hencetheorderofquenchingofanionsto
Salt-DependentStudies [Ru(Phen) dmbip]2+ complex Cl¯ < Br¯ < CH COO¯ < F¯.
2 3
Similarly, the effect of the cations (Cd2+, Zn2+ and Fe2+)
The polyelectrolyte theory quantitatively describes the ther- interactingwith[Ru(Phen) dmbip]2+complexwasinvestigat-
2
modynamiclinkagebetweencationandchargedligandbind- edinCH CN.Figure7b,showedthattheadditionofanexcess
3
ingtotheDNAlattice.Thedependencyofthecomplex-DNA ofcations (Cd2+, Zn2+and Fe2+) as their perchlorate salts to
bindingconstantoncationconcentrationsisamanifestationof [Ru(Phen) dmbip]2+complexresultedindramaticchangesto
2
the thermodynamic linkage. As the concentration of salt the emission spectra, the addition of Cd2+ and Zn2+ slightly
(NaCl)increases,thebindingconstantdecreases.Fromthe quenched the emission spectrum, whereas the addition of
polyelectrolyte theory, the slope of the lines in Fig. 6 pro- ferrous(Fe2+) cation effectively quenched. Hence the order
videsanestimateofZΨ.WhereΨisthefractionofcounter of quenching of cations to [Ru(Phen) dmbip]2+ complex
2
ions associated with each DNA phosphate (Ψ=0.88 for Cd2+<Zn2+<Fe2+.Itiscleartheselectivityisanimportant
double-stranded B-form DNA) and Z is the charge on the characteristicofchemosensors,wehavefurtherevaluatedthe
complex. The data in Fig. 6 indicate that the data in Ru selectivityof[Ru(Phen) dmbip]2+complexforF¯(anion)and
2
complexes carry a net charge of +2 and cobalt complexes Fe2+(cation)overtheotheranionsandcations[43,44].
carryanetcharge+3.Consequently,theslopesofthelinesare
−1.363, –1.274, –1.171 and −1.142 for [Ru(phen) dmbip]2+,
2
[Co(phen) dmbip]3+, [Ru(bpy) dmbip]2+ and
2 2
[Co(bpy) dmbip]3+ complexes, respectively. These values are
2
less than the theoretically expected value of ZΨ (Ru(II) 2×
0.88=1.76andCo(III)3×0.88=2.64).Suchlowervaluescould
arisefromthecoupledanionrelease(fromtheligand)orfrom
changesinligandorDNAhydrationuponbinding.Byincreas-
ingtheNa+ concentration, theknowledge of ZΨallows fora
quantitative estimation of the non electrostatic contribution to
theDNAbindingconstantfortheabovefourcomplexes[42].
SensorStudies
Fluorescencespectroscopystudieswerecarriedoutinorderto Fig.9 Cyclicvoltametry,Ais[Ru(phen)dmbip]2+complexalone,Bis
2
evaluatetheabilityofthereceptorstooperateasafluorescent [Ru(phen)dmbip]2+complexwithCT-DNA
2
JFluoresc
ViscosityMeasurements DNAhelixlengthensasbasepairsareseparatedtoaccommo-
date the bound ligand, which leads to an increase in the
The viscosity studies provide a strong evidence for mode of viscosityofDNA.Incontrast,apartial,non-classicalinterca-
binding.TheviscositymeasurementswerecarriedoutonCT– lationofligandcouldbend(orkink)theDNAhelix,reducing
DNA by the addition of increasing of concentration of the itslength[45].ViscosityofDNAinthepresenceofcomplexes
complex. A classical intercalation model demands that the 1,2,3,4andethidiumbromide(EB)areshowninFig.8.All
Fig.10 Geometricallyoptimisedmodelofthecomplex[Ru(phen)dmbip]2+(a);complex[Co(phen)dmbip]3+(b)
2 2
JFluoresc
Table6 Electrochemicalbehaviourofcomplexesaloneandcomplexes Table8 Patchdockscoreanddesolvationenergiesofthemetalcomplex-
withCT-DNA DNAsolution
Complex Complexalone ComplexwithCT-DNA S.no. Complex Patchdockscore −ACEa
Anodoic Cathodoic Anodoic Cathodoic 1. [Ru(phen)dmbip]2+ 5,350 −498.33
2
(Epa) (Epc) (Epa) (Epc) 2. [Co(phen)dmbip]3+ 5,222 −716.83
2
[Ru(phen)dmbip]2+ 0.23V −0.01V 0.28V −0.09V 3. [Ru(bpy) 2 dmbip]2+ 5,438 −467.09
[Ru(bpy) 2 dmbip]2+ 0.21V −0.03V 0.27V −0.07V 4. [Co(bpy) 2 dmbip]3+ 5,286 −478.22
2
[Co(phen) 2 dmbip]3+ 0.19V −0.02V 0.22V −0.035V aDesolvationenergy
[Co(bpy)dmbip]3+ 0.18V −0.025V 0.21V −0.03V
2
voltammograms before and after addition of DNA to
[Ru(phen) dmbip]2+complexinsolution.Hencepositiveshift
2
the four complexes of Ru(II) and Co(III) leads to increased
of peak potential indicates this interaction mode may be
relative viscosity of DNA. These results suggest that the intercalation between [Ru(phen) dmbip]2+ and DNA. The
2
complexes intercalate between the base pairs of DNA and
Ru(II) complex showing the anodic and cathodic peaks at
the difference in the binding strength is probably caused by 0.23 and −0.01 Vrespectively, after loading with DNA, the
differentancillaryligands.Comparingbpytophen,itisclear
oxidation and reduction potentials are changed to 0.28 and
thatthesurfaceareaandthehydrophobicityofancillaryligand −0.09VwhichareshowninFig.9.Basedonaboveoxidation
increaseinphen,leadingtoagreaterDNAbindingaffinityfor
andreductionpotentialshifting,itcanbeconcludedthatDNA
complex1.Thus,complex1isprobablymoredeeplyinterca-
binds to the Ru(II) complex through intercalation [46]. The
latedandmoretightlyboundtoadjacentDNAbasepairsthan
decrease in current may be attributed to the diffusion of the
othercomplexes.Hence,followstheorderEB>1>3>2>4.
complexboundtothelarge,slowlydiffusingDNAmolecule
(Table5).The decreases inthe peakcurrentsare ascribedto
the stronger binding between Ru(II) and Co(III) complexes
CyclicVoltammetry
withDNA.
The application of cyclic voltammetry (CV) to the study of
binding ofmetal complexes toDNAprovidesa usefulcom- MolecularModeling
plement tothemethodfor the investigation ofUV-Visspec-
troscopy and fluorescence spectroscopy. The interaction of Molecularmodelinghasbecomeavitaltechniqueinchemis-
DNA with [Ru(phen) dmbip]2+ complex was characterized try and challenging in the modeling of d and f-block com-
2
by the anodic peak current difference (ΔIpa) in cyclic plexes [47]. Molecular modeling studies were carried out
Table7 Bondlengthsofthe3Dconformersofmetalpolypyridylcomplexes
S.no. Metalcomplex Bondlengths(Å)
M-N1a M-N2a M-N3a M-N4a M-N5b M-N6b
1. [Ru(phen)dmbip]2+ 1.9508 1.9517 1.9476 1.9476 1.9471 1.9470
2
Totalenergy 264.86kcal/mol
Metal-intercalatorlength(Å) 12.8569
2. [Co(phen)dmbip]3+ 1.8683 1.8683 1.8692 1.8685 1.8677 1.8676
2
Totalenergy 275.87kcal/mol
Metal-intercalatorlength(Å) 12.7832
3. [Ru(bpy)dmbip]2+ 1.9507 1.9477 1.9435 1.9435 1.9476 1.9475
2
Totalenergy 267.38kcal/mol
Metal-intercalatorlength(Å) 12.8538
4. [Co(bpy)dmbip]3+ 1.9091 1.8364 1.8364 1.9082 1.9138 1.9396
2
Totalenergy 313.71kcal/mol
Metal-intercalatorlength(Å) 12.7920
aN1,N2,N3&N4arePolypyridyl(phen/bpy)nitrogenbondedtometal
bN5&N6,Nofdmbipligandbondedtometal
JFluoresc
Fig.11 Photocleavagestudiesof 0 1 2 3
pBR322DNA,intheabsenceand a
presenceofcomplexes FORM-II
[Ru(phen)(dmbip)]2+(a),
2 FORM-I
[Co(phen)(dmbip)]3+(b),
2
[Ru(bpy)(dmbip)]2+(c)and
[Co(bpy) 2 (dmbip)]3+(d)after 0 1 2 3
2 b
irradiationat365nmfor30min. FORM-II
Lane0wascontrol(DNAalone),
lanes1→3wereadditionof FORM-I
complexes20,40and60μM,
respectively 0 1 2 3
c
FORM-II
FORM-I
0 1 2 3
d
FORM-II
FORM-III
FORM-I
using Hyper Chem 7.5 software [48]. The 3D structures of [49]. The 3D model built is subjected to a combination of
[Ru(phen) dmbip]2+ (1), [Ru(bpy) dmbip]2+ (2), optimization methods to search the potential energy matrix
2 2
[Co(phen) dmbip]3+ (3) and [Co(bpy) dmbip]3+ (4) com- based on the contributions of a stretch, bending, dihedrals,
2 2
plexes were built using drawing tools of the Hyper Chem Vander Waals and electrostatic interactions to the molecular
model builder. Figure 10 shows 3D structures of energy(Eq.2).Acombination ofoptimization methods was
[Ru(phen) dmbip]2+ (1) and [Co(phen) dmbip]3+ (3). The used to search for the potential energy surface for energy
2 2
complexes were sketched individually in two dimensions minima.
(2D)andlaterconvertedintothreedimensional(3D)entities
usingtheconversiontoolintheHyperChemModelBuilder E total ¼E BL þE DA þE VDW þE SBI þE EE ð2Þ
Table9 Hydrogenbonding
interactionsinvolvingthedocked S.no. Complex H-bond Bond VanderWaals Bond
posesofdsDNAwithmetal Donorgroup–acceptor length(Å) interactions(metal length(Å)
complexes group complex-DNA)
1. [Ru(Phen)dmbip]2+ N53–ADG7:O3′ 1.6493 N2–BDG22:O4′ 3.0385
2
N53–ADT8:OP1 2.2031 N2–BDG22:O3′ 3.0031
N53N–ADT8:OP2 0.9258 N45–ADC6:O3′ 2.7403
N53N–ADT8:O5′ 1.9451 N45–ADG7:O5′ 2.9141
2. [Co(Phen)dmbip]3+ N3–BDG22:O5′ 2.3839 N3–BDG22:O3′ 3.0752
2
N3–BDG23:O4′ 1.8503 N4–BDG22:O4′ 2.7184
N5–BDG22:O3′ 2.0077 N6–BDG22:N3 2.9447
N6–BDG22:O4′ 1.5532
N40:H1–BDC21:O4′ 2.4976
3. [Ru(Bpy)dmbip]2+ N41–ADG7:O3′ 2.2589 N41–ADT8:OP1 2.9349
2
N52–ADT8:O3′ 1.5576 N41–ADT8:O5′ 2.8412
N52–ADC9:OP2 2.0410 N52–ADC9:OP1 2.7273
N52–ADC9:O5′ 1.3268 N52–ADC9:O4′ 3.1606
4. [Co(Bpy)dmbip]3+ N3–ADA5:O4′ 2.1554 N3–ADA5:O4′ 3.1374
2
N3–ADA5:O3′ 2.4765 N4–ADC6:OP1 3.0878
N4–ADA5:O3′ 1.8565 N4–ADC6:O5′ 3.0732
N40–BDG23:O4′ 3.2086
N53–ADA5:O3′ 2.6779
T v a al b u l e e s 1 f 0 or T co h m e p IC le 5 x 0 es Compound IC 50 (μM) i w n e v r o e lv r i e n p g or t t h e e d d in oc T k a e b d le po 7 s a e n s d of H d y s d - r D o N ge A n w bo it n h d f in o g ur in c t o e m ra p c l t e io x n es s
againstA549andHeLa
celllines A549 HeLa werereportedinTable8.Theresultshaveshownthatallfour
complexescanbindtoDNAthroughintercalativemodeand
[Ru(phen) 2 dmbip]2+ 30 28 complexes1and3arestrongbindingabilitythan2and4.
[Ru(bpy)dmbip]2+ 27 35
2
[Co(phen)dmbip]3+ 42 39
2 PhotocleavageofpBR322DNA
[Co(bpy)dmbip]3+ 46 52
2
After establishing the binding abilities of Ru(II) and Co(III)
complexes with DNA, the photocleavage experiments were
The conjugate gradient method was chosen for the mo-
performedbyagarosegelelectrophoresisusingplasmidDNA
lecularmechanicscalculationtoobtainenergyminimawith
(pBR322DNA)irradiatedfor60minat302nm.ThepBR322
theAMBERforcefield.Geometricoptimization iscarried
plasmid DNA can exist in three different forms supercoiled,
out by Polak–Ribiere algorithm [50]. Unit final conver-
nicked, andlinear.These threedifferentformscanbedistin-
gencecriteriaof1×10
−5K.cal/molperǺisobtained.Bond
guishedbygelelectrophoresis.WhencircularplasmidDNA
lengths of the 3D conformers of [Ru(phen) dmbip]2+ (1),
2 issubjectedtoelectrophoresis,relativelyfastmigrationwillbe
[Ru(bpy) dmbip]2+ (2), [Co(phen) dmbip]3+ (3) and
2 2 observed in the intact supercoiled form (Form I) [51, 52]. If
[Co(bpy) dmbip]3+(4)complexeswerereportedinTable6.
2 one strand is cleaved, the supercoil will relax to generate a
slower moving open circular form (Form II). If both strands
DockingStudies are cleaved, a linear form (Form III) that migrates between
Form-I and Form-II will be generated. Figure 11 shows, no
Patchdockservertoolwasusedtoperformdockingcalcula- obvious DNA cleavage was observed for control (lane-0) in
tions between the metal complexes (ligand) and B–DNA whichcomplexwasabsent,orincubationoftheplasmidwith
(Receptor) sequence. Input used for the docking is the B– the complex in darkness, lanes 1→3 were added of com-
DNAsequence51–D(AP CPCPGPAPCP GPTPCP GP plexes20,40and60μM,respectively.Withincreasingcon-
GP T)–31 which is obtained from Protein Data Bank (PDB centration of Ru(II) and Co(III) complexes the amount of
id:423D) at a resolution of 1.6Ǻ and the 3D models of the form-I pBR322 DNA was diminished gradually, whereas
metal complexes built using Hyper Chem 7.5 are used. The Form-IIincreasesandForm-IIIisalsoproduced.
receptorispreparedbydeletingalltheheteroatomsincluding
water, Mg2+ ion and the polar hydrogen atoms were added. AntimicrobialStudiesofLigandandComplexes
The PDB files of both DNA and metal complexes were
uploaded.TheprogramparametersweresettoRMSDof4Ǻ The antimicrobial screening data (Table 9) show that the
and all other parameters wereatdefault settings. Patchdock Ru(phen) dmbip]2+, [Ru(bpy) dmbip]2+, [Co(phen) dmbip]3+
2 2 2
resultswereobtainedasasetofscoringfunctionsbasedonthe and [Co(bpy) dmbip]3+ complexes possess good antibacterial
2
shapecomplementarityandtheACE,theatomicdesolvation properties.Chelatingtendstomakethechelatingligandsmore
energy of the transformed complex is evaluated. The ACE potent bactereostatic agent, thus inhibiting the growth of
desolvationscoreisbasedonthesumoftheACEscoresofall bacteria upon complexation the lipophilic character in-
ligandatom-receptoratompairsincontact.PatchDockScore creased and favours the permeation through the layer of
and Desolvation energies of four complexes-DNA solution the bacterial membranes. A zone of inhibition was
120
A
B 100 C
D
80
60
40
20
0
0 3.25 6.5 12.5 25 50 100
)%(
ytilibaiv
lleC
1.
120
A
B 100 C
D
80
60
40
20
0
0 3.25 6.5 12.5 25 50 100
Concentration (µM)
)%(
ytilibaiv
lleC
JFluoresc
Fig.12 Cellviabilityofcelllines 2.
HeLa(1)andA549(2)invitro
aftertreatmentwithcomplexes
[Ru(phen)(dmbip)]2+(A), 2
[Co(phen)(dmbip)]3+(B),
2
[Ru(bpy)(dmbip)]2+(C)and 2
[Co(bpy)(dmbip)]3+(D)
2
Concentration (µM)
JFluoresc
measured for Ru(phen) dmbip]2+, [Ru(bpy) dmbip]2+, Ru(II)andCo(III)complexesexhibitedtheDNAlightswitch
2 2
[Co(phen) dmbip]3+and[Co(bpy) dmbip]3+complexesas onand off effect. Inthe cyclicvoltammogramofRu(II) and
2 2
wellasligandagainstthebacteriaEscherichiacoli(E.coli) Co(III) polypyridyl complex the cathodic peak current de-
andStaphylococcusaureus(S.aureus)at1mg/mlconcen- creasedgraduallywiththeadditionofDNA,thedecreasesin
trationbythepaperdisc methodusingnutrientagarasthe thepeakcurrentsareascribedtothestrongerbindingbetween
medium. DMSO control has negligible activity compared the complex and DNA. Furthermore, the binding mode be-
to the metal complexes. The complex 1 exhibited more tween the complexes and DNA was studied by molecular
antibacterial activity against both bacteria than other com- modeling and docking, the effects of the complexes on cell
plexes and ligand. The antimicrobial activity increased as viabilityweretestedusingtheMTTassayandresultsindicat-
theconcentrationofthecompoundsincreased.Anincrease edthatthefourcomplexeshadcertaineffectoncancercells.
inthelipophiliccharacterofthecomplexfavorsitsperme-
ationthroughthelipidlayerofthebacterialmembrane,and
Acknowledgements WearegratefultoDBT,NewDelhiforfinancial
thereforeshowshigheractivity. support and CFRD, Osmania University, Hyderabad-07, for recording
NMRspectra.
Cytotoxicity
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