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Spectral characteristics, DNA-binding and cytotoxicity of two functional Ru(II) mixed-ligand complexes.
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Cite this: Dalton Trans., 2012, 41, 4575
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Spectral characteristics, DNA-binding and cytotoxicity of two functional
Ru( ) mixed-ligand complexes
II
a,b a,c a a a a
Lifeng Tan,* JianliangShen, JingLiu, Leli Zeng, LianheJin andChaoWeng
Received12thDecember2011,Accepted13thJanuary2012
DOI:10.1039/c2dt12402e
TwofunctionalRu(II)mixed-ligandcomplexes,[Ru(phen)
2
(ttbd)]2+(1)(ttbd=4-(6-propenyl-pyrido
[3,2-a]phenzain-10-yl-benzene-1,2-diamine,phen=1,10-phenanthroline)and[Ru(bpy) (ttbd)]2+(2)
2
(bpy=2,2′-bipyridine),havebeensynthesizedandcharacterized.Thespectralcharacteristicsof
complexes1and2wereinvestigatedusingfluorescencespectroscopyandrevealedthatbothcomplexes
wereverysensitivetosolventpolarityandoxygenmoleculesinnonaqueoussolvents.Thebinding
propertiesofthetwocomplexestowardscalfthymusDNA(CT-DNA)wereinvestigatedwithdifferent
spectrophotometricmethods,viscositymeasurementsandquantumchemistrycalculations,indicatingthat
bothcomplexescouldenantioselectivelybindtoCT-DNAbymeansofintercalation,butwithdifferent
bindingstrengthsanddiscrimination.Ontheotherhand,thecytotoxicityofbothcomplexeshavebeen
evaluatedbyMTTassaysandGiemsastainingexperiments.Themainresultsrevealthatthe
hydrophobicityandsurfaceareaoftheancillaryligandshaveasignificanteffectontheirDNAbinding
behaviorandbothcomplexesarelikelytobeusefulforopticallyprobingnonaqueousandoxygen-free
environments.
1. Introduction understandthefactorsthatdeterminetheDNAbindingmodeare
necessary.
During the past two decades, the binding of transition metal On the other hand, ruthenium complexes are regarded as
complexestoDNAhasbeenextensivelystudied.1–8Inparticular,
promising alternatives to platinum complexes in cancer
ruthenium(II)complexeswithpolypyridineligandshaveattracted therapies.10–12Severalrutheniumcomplexeshavenowbeenpro-
considerable attention9 due to acombination of theireasily con- posed as potential anticancer substances.10 Some of the ruthe-
structable rigid chiral structures, which span all three spatial nium complexes demonstrate remarkable anticancer activity11,12
dimensionsandtheirrichphotophysicalrepertoire.Thus,agreat andthebestexamplethatdescribesthedependencyofruthenium
dealofattentionhasfocusedontheDNAbindingmechanismof compoundsonplatinumdrugscomesfromtheworkonsocalled
Ru(II) polypyridyl complexes with DNA.1,9 Previous studies RDCs (ruthenium derived complexes) by Prof. Pfeffer and co-
have suggested that Ru(II) polypyridyl complexes can bind to workers.12 In attempting to mimic cisplatin chemistry, they
DNA bynon-covalentinteractions, suchaselectrostaticbinding, obtained several compounds, which they ultimately patented as
groove binding, intercalative binding and partial intercalative potentcytotoxicagentsforbraintumors.13Ourrecentworkalso
binding.1 However, there is still no consensus regarding the showedthattheRu(II)complex,[Ru(bpy)
2
(hnip)]2+{bpy=2,2′-
orientation and/or the location (major or minor groove) of the bipyridine,hnip=2-(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]
enantiomers binding with DNA and the binding mode of the phenanthroline},possesseshighanticanceractivityagainstHeLa
prototypecomplex,[Ru(phen)
3
]2+(phen=1,10-phenanthroline), cells.13
remains an issue of vigorous debate.1,14,15 Therefore, further Herein, two new Ru(II) polypyridyl complexes, [Ru
studies using different structural ligands to evaluate and (phen) (ttbd)]2+(1;phen=1,10-phenanthroline,ttbd=4-(6-pro-
2
penyl-pyrido[3,2-a]phenzain-10-yl-benzene-1,2-diamine) and
[Ru(bpy) (ttbd)]2+ (2; bpy = 2,2′-bipyridine), have been syn-
2
aCollegeofChemistry,XiangtanUniversity,Xiangtan411105, thesized and characterized. Their DNA binding behavior was
P.R.China.E-mail:lfwyxh@yahoo.com.cn;Fax:+8673158292477; explored by spectroscopic titration, viscosity measurements and
Tel:+8673158293997
thermal denaturation. Theoretical methods were used to explain
bKeyLabofEnvironmentallyFriendlyChemistryandApplicationin
MinistryofEducation,XiangtanUniversity,Xiangtan411105, their different DNA binding affinities by applying density func-
P.R.China tional theory (DFT). Additionally, their antitumor cell activities
cMOELaboratoryofBioinorganicandSyntheticChemistry,StateKey
werepreliminarilyevaluatedbyMTT{3-(4,5-dimethylthiazol-2-
LaboratoryofOptoelectronicMaterialsandTechnologies,Schoolof
yl)-2,5-diphenyltetrazolium bromide} assays and Giemsa stain-
ChemistryandChemicalEngineering,SunYat-senUniversity,
Guangzhou,510275,P.R.China ingexperiments.
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2. Experimental used instead of cis-[Ru(phen) Cl ]·2H O. Yield: 82%, 272 mg.
2 2 2
Elemental analysis (%) Calcd for C H N F O P Ru: C,
44 36 10 12 2 2
2.1. Materials 46.80; H, 3.22; N, 12.41; found: C, 46.69; H, 3.34; N, 12.31.
UVλ /nm (ε/M −1cm −1,MeCN): 414(30435),293(81770),
All solvents were of analytical reagent grade. 1,10-phenanthro- max
line-5,6-dione,14 cis-[Ru(bpy) Cl ]·2H O and cis-[Ru 261 (40340). 1H NMR (400 MHz, d 6 -DMSO (dimethyl sulfox-
2 2 2 ide);d,doublet;s,singlet;t,triplet;m,multiplet):9.67(m,2H),
(phen) Cl ]·2H O were prepared according to the literature pro-
cedures 2 .15 2 3,3′- 2 Diaminobenzidine and K PtCl were purchased 9.22(s,1H), 8.97(d,2H, J=8.4),8.90 (m, 4H), 8.75(d,1H, J 2 4 = 8.8), 8.64 (d, 1H, J = 9.2), 8.57 (m, 1H), 8.26 (m, 2H), 8.16
fromSigmaChemicalCompany(St.Louis,MO,USA).Double-
(m, 2H), 8.06 (m, 2H), 7.83 (d, 4H, J = 3.2), 7.71 (d, 2H, J =
stranded calf thymus DNA (CT-DNA) was obtained from the
Sino-American Biotechnology Company. The Tris–HCl buffer 8.8), 7.62 (t, 2H), 7.41 (t, 2H). ESI-MS (MeCN): m/z 947.5
solution (5 mM Tris–HCl, 50 mM NaCl, pH 7.0, Tris = tris
([M–PF
6
]+),401.4([M–2PF
6
]2+).
(hydroxymethyl)aminomethane) was prepared using doubly dis-
tilled water. A solution of CT-DNA in Tris–HCl buffer gave a
2.3. Physicalmeasurements
ratioofUVabsorbanceat260and280nmof1.8–1.9:1,indicat-
ing that the DNAwas sufficiently free of protein.16 The DNA 2.3.1. General methods. Microanalyses (C, H and N) were
concentrationpernucleotidewasdeterminedbyabsorptionspec- carried out onPerkin–Elmer 240Q elementalanalyzer.1HNMR
troscopy using the molar absorption coefficient (6600 M −1 spectra were recorded on an Avance-400 spectrometer with d -
6
cm −1)at260nm.17Stocksolutionswerestoredat4°Candused DMSO (dimethyl sulfoxide) as the solvent at room temperature
within4days. and TMS (tetramethylsilane) as the internal standard. UV–vis
(UV–visible)spectrawererecorded onaPerkin–ElmerLambda-
25spectrophotometerandtheemissionspectrawererecordedon
2.2. Synthesis aPerkin–ElmerLS-55luminescencespectrometeratroomtemp-
2.2.1. Synthesis of 4-(6-propenyl-pyrido[3,2-a]phenzain-10- erature. Fast atom bombardment mass spectroscopy (FAB-MS)
yl-benzene-1,2-diamine. A mixture of 3,3′-diaminobenzidine was performed on a VG ZAB-HS spectrometer in a 3-nitroben-
zyl alcohol matrix. Electrospray mass spectra (ES-MS) were
(0.43 g, 2.0 mmol), 1,10-phenanthroline-5,6-dione (0.42 g,
2.0mmol)andglacialaceticacid(40mL)wasrefluxedwithstir- recorded on a LQC system (Finngan MAT, USA) using CH 3 CN
ring for 2 h. The cooled solution was filtered and diluted with asthemobilephase.Thesprayvoltage,tubelensoffset,capillary
voltage and capillary temperature were set at 4.50 kV, 30.00 V,
water (10 mL) and then neutralized with concentrated aqueous
ammonia. The yellow precipitate was collected and purified by 23.00 Vand 200 °C, respectively, and the quotedm/z values are
column chromatography(CC, Alox;EtOH–toluene3:1)togive for the major peaks in the isotope distribution. Circular dichro-
ism (CD) spectra were measured on a JASCO-J810
apureyellowcompound.Yield:76%,0.57g.Elementalanalysis
spectropolarimeter. (%) Calcd for C H N O :C,67.91; H,4.75; N, 19.80;found:
24 20 6 2
C,67.69;H,4.82;N,19.71.FAB-MS:m/z=389.4[M+1].
2.3.2. DNA binding experiments. All DNA binding exper-
2.2.2. Synthesis of [Ru(phen) (ttbd)](PF ) ·2H O (1). A
imentswereperformedinTris–HClbufferat25°C.Theabsorp-
mixture of cis-[Ru(phen) Cl ]·2H O 2 (0.18 g, 6 0 2 .30 m 2 mol) and tion titrations were performed at a fixed complex concentration,
2 2 2
ttbd (0.10 g, 0.30 mmol) in 20 mL of ethylene glycol was towhich the DNA stock solution was graduallyadded up to the
thoroughlydeoxygenated.Thepurplemixturewasheatedfor8h point of saturation. The mixture was allowed to equilibrate for
at 150 °C with stirring under an argon atmosphere, after which 5 min before the spectra were recorded. The intrinsic binding
time the the solution finally turned red. After it was cooled to constants,K b ,oftheRu(II)complexesboundtoDNAwerecalcu-
room temperature, an equal volume of saturated aqueous KPF
latedfromeqn(1):18
6
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
solution was added under vigorous stirring. The red solid was
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ifi
an
e
d
d ε
ε a (cid:1)
(cid:1)
ε
ε
f ¼ b(cid:1)ðb2(cid:1)
2K
2K
C
b 2C t ½DNA(cid:3)=sÞ ð1aÞ
b f b t
on a neutral alumina column with MeCN–toluene (2:1, v/v) as
an eluant. Yield: 74%, 0.26 g. Elemental analysis (%) Calcd for b¼1þK C þK ½DNA(cid:3)=ð2sÞ; ð1bÞ
b t b
C H N F O P Ru: C, 49.03; H, 3.09; N, 11.91; found: C,
48 36 10 12 2 2
48.96; H, 3.21; N, 11.83. UV λ /nm (ε/L mol −1 cm −1, where [DNA] is the concentration of DNA in the nucleotide
MeCN): 405 (28000), 311 (47500 m ), ax 262 (75000). 1H NMR phosphate and ε a , ε f and ε b are the apparent, free and bound
metal complex extinction coefficients, respectively. K is the
(400 MHz, ppm, d -DMSO (dimethyl sulfoxide); d, doublet; s, b
6
equilibrium binding constant in M
−1,
C is the total metal
singlet; m, multiplet.): 9.67 (t, 2H), 9.21 (s, 1H), 8.97 (d, 2H, t
complex concentration and s is the average binding size. When
J = 8.4), 8.82 (t, 4H), 8.74 (d, 1H, J = 8.8), 8.43 (s, 4H), 8.31
plotting(ε −ε )/(ε −ε )vs.[DNA],K isgivenbytheratioof
(s, 2H), 8.24 (t, 2H), 8.08 (d, 2H, J = 5.6), 7.97 (m, 2H), 7.82 a b f b b
theslopetotheintercept.
(m, 6H). ESI-MS (m/z, positive-ion mode, MeCN): 995.2
([M–PF ]+),425.5([M–2PF ]2+). Viscosity measurements were carried out using an Ubbelohde
6 6
viscometer maintained at a constant temperature of 25 ± 0.1 °C
2.2.3. Synthesis of [Ru(bpy) (ttbd)](PF ) ·2H O (2). The in a thermostatic bath. DNA samples of ca. 200 bp average
2 62 2
proceduretopreparecomplex2wassimilartothatofcomplex1, length were prepared by sonication.19 The flow time was
except that cis-[Ru(bpy) Cl ]·2H O (130 mg, 0.25 mmol) was measured with a digital stopwatch and each sample was tested
2 2 2
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three times to get an average calculated time. Data were pre- 2.5. Cytotoxicityassays
sentedas(η/η )1/3vs.thebindingratio,20whereηistheviscosity
ofDNAinthe 0 presenceofthecomplexandη istheviscosityof Standard MTT assay procedures26 were performed using the
0 methodology described in detail previously.13 Two different
freeDNA.
tumor cell lines (HeLa and HepG2) were the subjects of this
ThermalDNAdenaturationexperimentswerecarriedoutwith
aPerkin–ElmerLambda850spectrophotometerequippedwitha study. To study the apoptosis of the cancer cells induced by
either complex 1 or 2, HepG2 cells treated with the compounds
Peltier temperature-control programmer (±0.1 °C). The tempera-
for 48 h were stained with Giemsa and then observed by
ture of the solution was increased from 50 to 95 °C at a rate of
1 °C min
−1
and the absorbance at 260 nm was continuously
microscopy.
monitoredforsolutionsofCT-DNA(42μM)intheabsenceand
presenceoftheRu(II)complex(20μM).Thedatawerepresented
3. Resultsanddiscussion
as(A−A )/(A −A )vs.thetemperature,whereA,A andAare
0 f 0 f 0
the final, initial and observed absorbances at 260 nm, 3.1. Synthesisandcharacterization
respectively.
Equilibriumdialyseswereconductedwith10mLofCT-DNA
The synthetic routes to ttbd and its Ru(II) complexes, 1 and 2,
are presented in Scheme 1. The ligand, ttbd, is synthesized by
(1.0 mM) sealed in a dialysis bag and 10 mL of the complex
(50 μM) outside the bag with stirring of the solution for 36 h at condensation of 1,10-phenanthroline-5,6-dione with the appro-
priate mole ratio of 3,3′-diaminobenzidine on the basis of a
25°C.
methodusedforthepreparationofpyrazinerings.27Usingethyl-
ene glycol as a solvent, complexes 1 and 2 were then prepared 2.4. Theoreticalcalculations
by direct reaction of ttbd with the appropriate mole ratios of the
The structural schemes of complexes 1 and 2 are shown in precursor complexes, cis-[Ru(phen) Cl ]·2H O and cis-[Ru
2 2 2
Scheme 1. Each of them forms from the Ru(II) ion with one (bpy)
2
Cl
2
]·2H
2
O, and obtained in yields of 76% and 74%,
main (intercalative) ligand (ttbd) and two ancillary ligands respectively. The desired Ru(II) complexes were isolated astheir
(either phen or bpy). Full geometry optimization computations hexafluorophosphates and then purified by column chromato-
were performed by applying the DFT-B3LYP method and graphy. Both complexes were characterized by elemental analy-
Land2DZ basis set21,22 and assuming a single state for the sis, mass spectroscopy and NMR spectroscopy. In the ESI-MS
ground state of the complexes.23 All computations were per- spectra of complexes 1 and 2 two signals of [M–PF ]+ and
6
formed withtheG98quantumchemistryprogrampackage.24To [M–2PF ]2+ were observed and the determined molecular
6
vividly depict the details of the frontier molecular orbital inter- weightswereconsistentwiththeexpectedvalues.
actions,thestereographsofsomerelatedfrontiermolecularorbi- Complexes 1 and 2 give well defined 1H NMR spectra
tals of the complexes were drawn with the Molden v3.7 (Fig. 1), which further permit unambiguous identification and
program25basedontheobtainedcomputationalresults. assessment of their purity. In comparison to those of similar
Scheme1 Synthesisoftheligand,ttbd,anditsRu(II)complexes,1and2.
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compounds,28theprotonchemicalshiftswereassignedbyallow- attributed to intraligand π → π* transitions.29 The lowest energy
ing for the influence of the steric, inductive and conjugative band, at about 410 nm, is assigned to the metal–ligand charge
effects. For each of the complexes, two sets of NMR signals transfer (MLCT) transition, whichis attributed to Ru(dπ) →ttbd
were observed. Because of the shielding influences of the adja- (π*) transitions. Obviously, this band is blue shifted in compari-
cent ttbd and bpy (or phen) moieties, the two halves of each sontothoseof[Ru(phen) ]2+(λ =448nm)and[Ru(bpy) ]2+
3 max 3
phen are not chemically and magnetically equivalent, which (λ =452 nm), which maybe due to the increased π delocali-
max
leads to eight signals that correspond to the bpy (or phen) zation and, thus, the π-acceptor capacity of the ttbd ligand,
protons:onesetoffourisassociatedwiththebpy(orphen)half resulting in a decreased electron density on the central Ru(II)
near thettbd and theotherset offour is associated with thebpy and,inturn,stabilizationofthemetaldπorbital.
(or phen) portion nearthe other bpy(or phen).Sincethe shield-
ingeffectofttbdisobviouslygreaterthanthatofbpy(orphen),
3.2.2. Emission spectra. There has been increasing demand
the chemical shifts of the latter protons appear more downfield
for the design and development of transition metal complexes
thanthoseoftheformer.
that can act as luminescent probes for use in various environ-
ments.1Complexes1and2showednoemissioninaqueoussol-
utions at 25 °C in air. In contrast, distinct photoluminescence
3.2. Spectralcharacteristics
behaviorwasobservedforcomplexes1and2innonaqueoussol-
3.2.1. Absorption spectra. The absorption spectra of com- vents, with emission maxima shifting over a range of ∼30 nm
plexes1and2,upondissolutioninvariousnonaqueoussolvents around the generic ∼665 nm peak in air at 25 °C. In addition,
(DMF,DMSO,MeCNandacetone),areverysimilarandconsist theemissionspectraaresomewhatsolvatochromic(Fig.2),indi-
of three well resolved bands at about 410, 310 and 260 nm, cating that the more polar the solvent is, the smaller the relative
respectively. The bands at about 310 nm and 260 nm are intensityis.30
Fig.1 The1HNMRaromaticregionsinthespectraofcomplexes1(top)and2(bottom)ind -DMSO(400MHz).
6
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Notably, the luminescence of complexes 1 and 2 in organic localenvironmentandbothcomplexesarelikelytobeusefulfor
solvents is very sensitive to oxygen molecules (Table 1 and the optical probing of nonaqueous and oxygen-free
Fig.3).Ifairisremovedfromthesolutionofcomplex1(anitro- environments.
gen atmosphere), for example, a greater relative intensity is
observed; while if the oxygen-free system is replaced with air,
thegreaterrelativeluminescentintensityobservedintheabsence
of oxygen is brought back to the original level. This indicates
that oxygen-free radicals may be involved in the process of
complex light emission. Thus, both complexes can serve as
“oxygen molecule light switch” complexes. In particular, by the
successive introduction of nitrogen and air atmospheres a
cycling oxygen molecule light switch (with an “off and on”
effect)canbeaccomplishedusingbothcomplexes.Ontheother
hand, the addition of low concentrations of H O to both com-
2
plexes dissolved in nonaqueous solvents leads to their lumines-
cence being almost quenched. Fig. 4 shows the progressive
decrease in the emission intensity of complex 1 in MeCN upon
the addition of H O. This result may indicate that the emission
2
quenching of the ruthenium complex proceeds via hydrogen
bonding to nearest-neighbor water molecules and may explain
the lack of emission for both complexes in aqueous solutions.31
Thus, the emission of both complexes is very sensitive to the
Fig.3 Thefluorescencespectraofcomplex1(a)and2(b)(2μM)in
Fig.2 The fluorescence spectra of complex 1 (20 μM) in CH CN, MeCN at 25 °C in air-saturated solutions and oxygen-free solutions,
3
DMFandDMSOat25°C. respectively.
Table1 Theemissioncharacteristicsofcomplexes1and2(2μM)innonaqueoussolventsat25°C
Solvent
CH CN DMF DMSO
3
λ em,nm I(a.u.) λ em,nm I(a.u.) λ em,nm I(a.u.)
max max max
Complex Aira N b Aira N b Aira N b Aira N b Aira N b Aira N b
2 2 2 2 2 2
1 666 649 17 37 682 660 7 15 697 620 3 8
2 654 627 14 40 662 653 12 23 672 636 6 10
a,bThephotoluminescencewasdeterminedforbothcomplexesinair-saturatedsolutionsandoxygen-freesolutions.
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Fig.4 The fluorescence spectra of complex 1 (2 μM) in MeCN at
25 °C in the absence (dotted line) and presence (solid lines) of water.
Increasingamountsofwaterupto200μLwereadded.Thearrowshows
theintensitychangesupontheadditionofincreasingamountsofwater.
3.3. ElectronicabsorptiontitrationofRu(II)complexesand
DNA
The application of electronic absorption spectroscopy in DNA
binding studies is one of the most useful techniques.32 Complex
binding with DNAvia intercalation usually results in hypochro-
mism and bathochromism, due to the intercalative mode invol-
ving a strong stacking interaction between the aromatic
chromophore and the base pairs of DNA. The extent of the
hypochromismandredshiftiscommonlyrelatedtotheintercala-
tivebindingstrength.Theabsorptionspectraofcomplexes1and
2 in the absence and presence of CT-DNA are given in Fig. 5.
As the DNA concentrations were gradually increased, the
Fig.5 The absorption spectra of complexes 1 (a) and 2 (b) in Tris–
absorptionspectraofcomplexes1and2showed significantper- HClbufferuponadditionofCT-DNAat25°C.[Ru]=20μM,[DNA]=
turbation. Forcomplex1, thehypochromism in theMLCT band (0–42)μM.Thearrowsshowtheabsorbancechangeuponanincreasing
reaches about 33% at 415 nm with a red shift of 12 nm at a DNA concentration. Insets: the plots of [DNA] − (ε − ε) vs. [DNA]
a f
[DNA]–[Ru] ratio of 1.84. With an increasing DNA concen- forthetitrationofDNAwithcomplexes1and2.
tration,complex2showedahypochromismofabout22%inthe
MLCTband(414nm)witharedshiftof7nmata[DNA]–[Ru]
ratio of 1.58. Comparing the hypochromism and red shifts of (1.16 ± 0.24) × 106 M −1 (s = 0.24 ± 0.01), respectively. The K
b
bothcomplexeswiththatof[Ru(phen) ]2+(12%hypochromism values of both complexes are close to that of [Ru(phen)
3
for the MLCT band at 445 nm and a 2 nm red shift),33 which (ppd) ]2+ with two intercalative ligands (1.55 × 106 M −1),36 but
2
interacts with DNA through a semi-intercalation or quasi-inter- smaller than those of [Ru(phen) (dppz)]2+ and [Ru
2
calation,34 and [Ru(bpy) ]2+, which is a typical electrostatic (bpy) (dppz)]2+ (dppz = dipyrido-[3,2-a-2′,3′-c]phenazine, >106
3 2
binding complex whose absorption was demonstrated to be M −1), which are DNA intercalative Ru(II) complexes.37
unchanged upon the addition of DNA,35 indicates that these However, the K values of complexes 1 and 2 are stronger than
b
spectral characteristics suggest that complexes 1 and 2 interact thoseofothertypicalDNAintercalativeRu(II)complexes(1.1×
with DNA. This most likely proceeds through a mode that 104 – 4.8 × 104 M −1)38 and arealso stronger than the K values
b
involves a stacking interaction between the aromatic chromo- oftheseRu(II)complexeswithtwointercalativeligands,e.g.[Ru
phoreandthebasepairsofDNA. (phen)(dicnq) ]2+ (dicnq = 6,7-dicyanodipyrido[2,2-d-2′,3′-f]
2
To quantitativelycomparethe binding affinityof complexes 1 quinoxaline, 3.0 × 104 M −1),39 [Ru(phen)(pztp) ]2+ (4.8 × 104
2
and 2 towards DNA, the intrinsic binding constants, K , were M −1) and [Ru(bpy)(pztp) ]2+ (1.4 × 104 M −1).40 In particular,
b 2
determinedtobe(1.61±0.34)×106M −1(s=0.42±0.01)and the intrinsic binding constants of complexes 1 and 2 are much
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stronger than those of their parent complexes, [Ru(bpy) ]2+
3
(4.7 × 103 M −1) and [Ru(phen) ]2+ (5.5 × 103 M −1),41 which
3
can be explained as follows: firstly, the planarity area (S) of the
intercalatedligandisS >S /S ;secondly,theintercalated
ttbd phen bpy
ligand, ttbd, contains two free amino groups, which may form
intermolecular hydrogen bonds with the base pairs. These two
factors are advantageous for the binding of complexes 1 and 2
with DNA. These results also indicatethatthesizeandshape of
the intercalated ligand has a significant effect on the strength of
DNA binding and the most suitable intercalating ligand leadsto
the highest affinity of the complexes for DNA. Additionally,
from these results, we could deduce that both 1 and 2 bind to
DNAbyintercalationandtheirdifferentDNAbindingproperties
maybeduetotheancillaryligands.Comparingphenandbpy,it
is clear that the hydrophobicity and the surface area decrease in
bpy,resultinginaweakerDNAbindingaffinityforcomplex2.
3.4. Competitivebinding
Luminescencespectroscopyisoneofthemostcommonandsen-
sitive methods to analyze drug–DNA interactions. Support for
the aforementioned intercalative binding mode also comes from
theemissionmeasurementsofbothcomplexes.Unfortunately,in
Tris–HCl buffer at 25 °C, both complexes showed no fluor-
escenceemission,neitheralonenorinthepresenceofCT-DNA.
The mechanism of the “light switch” effect for [Ru
(bpy) (dppz)]2+ (dppz = dipyrido[3,2-a-2′,3′-c]phenazine) and
2
[Ru(phen) (dppz)]2+ has been intensively studied and all of the
2
evidence pointstohydrogenbondingand/orexcited-statehydro-
genatomtransfertothephenazinenitrogenatomasthemechan-
ism of deactivation of the complexes’ excited state.42 Upon
intercalation,DNAprovidesthemetal-bounddppzligandwitha
hydrophobic environment, which in turn protects the Ru(II)
complex from the quenching effect of water.42 Comparing com-
plexes 1 and 2 with their parent complexes, [Ru(bpy) (dppz)]2+
2
and[Ru(phen) (dppz)]2+,mayexplainwhytheycouldnotserve
2
asmolecular“lightswitches”forDNA.Themainreasonmaybe
that when the ligand, ttbd, intercalates into the DNA helix, with
Fig.6 The emission spectra of EB bound to DNA in the presence of
either the two nitrogen atoms on the pyrazine ring or the two complex1(a,λ =458nm)and2(b,λ =457nm)at25°C.[EB]=
ex ex
amino groups on the benzene ring, outside the DNA helix. In 20 μM, [DNA] = 100 μM; [Ru]–[DNA] = 0.00, 0.04, 0.08, 0.12 and
this case, they would not be protected by DNA and could still 0.16.Thearrowsshowtheintensitychangesuponanincreasingconcen-
form hydrogen bonds with solvent water molecules, resulting in trationofthecomplexes.Inset:theplotsofI 0 /Ivs.[Ru]–[DNA].
the fluorescence emission of both complexes being fully
quenchedbythesolventwatermolecules.
a [Ru]–[DNA] ratio of 0.16 occurred. This indicates that both
A steady-state competitive binding experiment using either
complexes could compete with EB in binding to DNA and
complex 1 or 2 as the quencher may afford further information
complex 2 binds to DNA slightly stronger than complex 1.
about the binding of the complex to DNA. Ethidium bromide
Additionally, the quenching plots (inset in Fig. 5) illustrate that
(EB) emits intense fluorescence light in the presence of DNA,
the removal of EB bound to DNA by either complex 1 or 2 is
due to its strongintercalation between adjacentDNAbase pairs.
non-linear and not in agreement with the linear Stern–Volmer
Itwaspreviouslyreportedthattheenhancedfluorescencecanbe
equation, which implies that the competitive binding process is
quenched, at least partially, by the addition of a second mol-
bothdynamicandstatic.44
ecule.43 The extent of fluorescence quenching an EB–DNA
system is used to determine the extent of binding of a second
molecule to DNA. The emission spectra of EB–DNA in the
3.5. Metaliontitration
absence and presence of both complexes are shown in Fig. 6.
Fig. 6 indicates that, upon addition of either complex 1 or 2 to Theabovediscussionmentionsthatbothcomplexesshownoflu-
the EB–DNA system, an appreciable reduction in the emission orescence emission even in the presence of DNA. Considering
intensityofabout84%forcomplex1and82%forcomplex2at the structure of the intercalative ligand, ttbd, it’s obvious that
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either the two nitrogen atoms on the pyrazine ring or the two [PtCl
]2−
into the solution of DNA-free complex 1, a gradual
4
amino groups on the benzene ring (but not both) are inside the decrease in the absorption intensity is observed as a function of
DNA helix when the complex binds with DNA. Additionally, if [PtCl
]2−
,whichindicatesthat [PtCl
]2−
coordinatestottbd.The
4 4
the two amino groups in ttbd are outside the DNA helix when decrease reaches a maximum at a [Ru]–[ PtCl ] ratio of 1:1
4
the complex binds, the two amino groups become coordination when essentially all of complex 1 is bound to [PtCl ]2− , reflect-
4
sites and can bind with other metal cations, such Pt2+ and Cu2+. ing that the coordination ratio of [PtCl ]2− to complex 1 is
4
Thus, in the absence and presence of DNA, a foreign metal ion 1.Notethattheintroduced[PtCl
]2−
speciesmaynotcoordinate
4
may disturb the absorption of the complexes to some extent, to the two nitrogen atoms on the pyrazine ring because of the
affordingfurtherinformationtoexplainthelackofluminescence stereo-hindrance effect. In this case, the coordination ratio of
forcomplexes1and2inthepresenceofDNA.Therefore,metal [PtCl
]2−
to complex 1 is beyond 1. In other words, [PtCl
]2−
4 4
iontitrationwascarriedoutwithcomplex1astherepresentative binding to the two nitrogen atoms on the pyrazine ring may not
example. occur. As Cu2+ has a relativelysmall size, it was used to further
The absorption spectra for the titration of complex 1 with confirm that the coordination site presented by the two amino
[PtCl ]2− is presented in Fig. 7. Clearly, upon addition of groupsisthepreferentialsiteofmetalioncoordinationcompared
4
to the two nitrogen atoms on the pyrazine ring. Similarly, the
Cu2+titrationintothesolutionofDNA-freecomplex1indicated
thatthemaximaldecreaseinthehypochromismalso occurredat
a ratio of ca. 1:1 [Ru(phen) (ttbd)2+]–[Cu2+], which further
2
suggeststhatthetwonitrogenatomsonthepyrazineringarenot
thepreferentialcoordinationsites.
The absorption spectra in Fig. 8 show DNA-bound [Ru
(phen) (ttbd)]2+ titrated with and without either [PtCl ]2− or
2 4
Cu2+.Clearly,noobviousdecreaseinabsorptionintensityofthe
DNA-bound [Ru(phen) (ttbd)]2+ system upon addition of either
2
[PtCl ]2− orCu2+isobserved,whichfurtheraffirmsthatthetwo
4
amino groups on the benzene ring are inside the DNA helix,
suggesting intercalation as a probable binding mode of both
complexes toward DNA. It is apparent that the two amino
groups,asthecoordinatedsites,becomeunavailable inthepres-
ence of DNA. Therefore, the reason why complexes 1 and 2
show no fluorescence emission in the presence of CT-DNA is
thatwhenbothcomplexesbindtoDNA,thetwonitrogenatoms
on the pyrazine ring are outside the DNA helix and can still
form hydrogen bonds with water molecules, resulting in the flu-
orescence emission of both complexes being fully quenched by
thewatermolecules.
3.6. Viscositymeasurements
Further clarification of the interaction between the complexes
andDNAwascarriedoutbyviscositymeasurements.Photophy-
sical probes provide necessary, but not sufficient, clues to
support a binding model. Hydrodynamic measurements that are
sensitive to the length change (i.e. viscosity and sedimentation)
areregardedastheleastambiguousandmostcriticaltestsofthe
binding model in solution, in the absence of crystallographic
structural data.51 A classical intercalation model results in
lengthening of the DNA helix as the base pairs are separated to
accommodate the binding ligand, which leads to an increase in
the viscosity of DNA. However, a partial and/or non-classical
intercalation of the complex, such as with [Ru(phen) ]2+, may
3
bend(orlink)theDNAhelixandreduceitseffectivelengthand,
concomitantly,itsviscosity.45Inaddition,somecomplexes,such
Fig.7 The absorption spectra of complex 1 (20.0 μM) in Tris–HCl as [Ru(bpy) 3 ]2+, which interacts with DNA by an electrostatic
bindingmode,havenoobviousinfluenceonDNAviscosity.46
bufferwithandwithoutmetalionsaddedat25°C.(a)Withandwithout
20.0 μM PtCl 4 2− added. (b) With and without 20.0 μM Cu2+− added. The effects of complexes 1 and 2, together with [Ru(bpy) 3 ]2+
The spectrawere collected after 5 min at 298 K. A reagent blank con- and EB, on the viscosity of rod-like DNA are shown in Fig. 9.
taining5mMTrisand20.0μMPtCl 2−orCu2+wasused. EB,awellknownDNAintercalator,resultsinastrongchangein
4
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Fig.9 TheeffectofincreasingamountsofEB(○),[Ru(bpy) ]2+(■),
3
complex 1 (▼) and complex 2 (▲) on the relative viscosity of
CT-DNAat25±0.1°C.ThetotalconcentrationofDNAis0.5mM.
leading to a greater DNA binding affinity for complex 1. Thus,
complex1isprobablymoredeeplyintercalatedandmoretightly
boundtoadjacentDNAbasepairsthancomplex2,whichiscon-
sistentwiththeforegoinghypothesis.
3.7. Thermaldenaturationstudy
DNA melting experiments are useful to establish the extent of
intercalation because the intercalation of the complex into DNA
basepairscausesstabilizationofthebasestackingand,therefore,
raises the melting temperature of double-stranded DNA.47 It is
wellknownthat,whenthetemperatureofthesolutionincreases,
double-stranded DNA gradually dissociates into single strands,
which generates ahyperchromiceffect onthe absorption spectra
of the DNA bases (λ = 260 nm). To identify this transition
Fig.8 Theabsorptionspectraofcomplex1–DNA([DNA]–[Ru]=1.9, max
[Ru] = 20 μM) in Tris–HCl buffer with and without metal ions added. process, the melting temperature, T m , which is defined as the
(a) With and without 20.0 μM PtCl 2− added. (b) With and without temperature when half of the total base pairs are unbonded, is
4
20.0μMCu2+−added.Thespectrawerecollectedafter5minat25°C. usuallyintroduced.Accordingtoapreviousreport,48theinterca-
Areagentblankcontaining5mMTris–HClbufferand42μMDNAwas lationofacomplexintoDNAgenerallyresultsinaconsiderable
used. increaseintheT .ThemeltingcurveofCT-DNAintheabsence
m
and presence of either complex 1 or 2 is presented in Fig. 10.
Here the T of metal complex-free CT-DNAwas determined to
m
the DNA viscosity upon complexation and, upon increasing be 75.11 ± 0.16 °C. Upon increasing the concentration of either
amounts of eithercomplex 1 or 2,the relativeviscosityof DNA complex1or2,theT increasessuccessivelyandreaches81.20
m
increases steadily, similar as in the case of EB. The increase in ± 0.11 °C and 83.49 ± 0.20 °C, respectively, at a [Ru]–[DNA]
the relative viscosity, which is expected to correlate with the ratio of 10:21. The ΔT values (6.09 °C and 8.28 °C) of
m
compounds DNA intercalating potential, followed the order EB complex 1/2–DNA adducts are bigger than those of some Ru(II)
>1>2>[Ru(bpy) ]2+.Theresult suggeststhatbothcomplexes intercalators,49 but smaller than those of DNA-intercalating Ru
3
bind to DNA through intercalation and the difference in the (II) polypyridyl complexes.6,7,49,50 However, the increases
binding strength is probably caused by the different ancillary (6.18 °C and 8.50 °C) in T are comparable to those observed
m
ligand. Comparing bpy to phen, it is clear that the surface area forclassicalintercalators,51whichrevealsthatthemodesofboth
and the hydrophobicity of the ancillary ligand increase in phen, complexes’bindingwithDNAareintercalation.
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Fig.10 Melting temperature curves of DNA in the absence (○) and
presence of complex 1 (●) and complex 2 (▲). [Ru] = 20 μM,
[DNA]=42μM.
Fig.11 ThCDspectraofcomplexes1(solidline)and2(dottedline)
after 32 h of dialysis against CT-DNA in stirred aqueous solutions at
3.8. Equilibrium-dialysisexperiments 25°C.[Ru]=20.0μM,[DNA]=1.0mM.
Equilibrium-dialysis experiments may offer the opportunity to
examine the enantioselectivity of complexes binding to DNA.52 titration indicate that the DNA binding affinity of complex 1 is
TheCDspectraintheUVregionofcomplexes1and2,afterits greater than that of complex 2, which further suggests that the
racemic solution has been dialysed against CT-DNA, are shown LUMO energy is not a decisive factor having an effect on the
inFig.11.Thedialysateofcomplex1(solidline)showsaposi- affinities of complexes 1 and 2 and their binding with DNA.
tive peak at 289 nm and a negative peak at 313 nm, while the Since the intercalative ligands of complexes 1 and 2 are the
dialysate of complexes 2 (dotted line) shows a positive peak at same, the difference in the DNA binding affinity should be
279nmandanegativepeakat304nm,indicatingthatbothcom- attributed to the ancillary ligand effects. Clearly, one is the
plexes can interact enantioselectively with CT-DNA. On the hydrophobic effect of the ancillary ligands and the other is the
otherhand,thestrongerCDsignalofcomplex1suggestsalarge surfaceareaoftheancillaryligands.Thehydrophobicityandthe
DNA binding discrimination between its two antipodes. Since surface area of phen in complex 1 are greater than those of bpy
the intercalative ligands of complexes 1 and 2 are the same, the in complex 2. The results may reflect the two factors playing a
difference should, again, be attributable to the ancillary ligands more important role than the LUMO and NLUMO energies of
and, more precisely, to the different hydrophobicity and surface the metal complex in the DNA binding affinity.54 In consider-
areaofthephenandbpymoieties.Theseresultsindicatethatthe ationofthesefactors,thedifferenceintheDNAbindingaffinity
hydrophobicity and surface area of the ancillary ligands have a ofcomplexes1and2canbewellunderstood.
significanteffectontheDNAbindingdiscrimination.
3.10. Invitrocytotoxicity
3.9. AtheoreticalexplanationforthedifferenceinDNA
MTT experiments were performed on two cancer cell lines,
bindingofcomplexes1and2
HeLa and HepG2, using cis-Pt(NH ) Cl as the control.6 The
3 2 2
It has been documented that the LUMO energy of a metal IC values of complexes 1 and 2 are presented in Table 3,
50
complexisanimportantfactor(butnottheonlyfactor)indeter- whichindicatethatHepG2cellsaremoresensitivetocomplexes
mining the DNA binding constant because a lower LUMO 1 and 2 than the HeLa cancer cell line. In particular, the IC
50
energy of a metal complex is advantageous for an interaction valueofcomplex1againstHepG2cellsisclosetothatofcis-Pt
between the intercalative ligand and the double helical DNA.53 (NH ) Cl .6Notably,cis-Pt(NH ) Cl exertsitscytotoxiceffects
3 2 2 3 2 2
DFT calculations show that complex 2 is thermodynamically through covalently binding to DNA to form cis-DDP–DNA
more unstable than complex 1 since the computed total energy adducts, which interferes with the DNA replication and tran-
of complex 1 is lower than that of complex 2 (see Table 2). In scription and ultimately induces cell death.55 We speculate that
contrast, the LUMO and NLUMO energies of complex 2 are the anticancer activity of complexes 1 and 2 may not only be
lower than those of complex 1 to some extent (see Table 2 and related to intercalation, but also related to the specific molecular
Fig. 12–14), which is favorable for the electron flow from the shape of the complex, the chemical structure and the nature of
base pairs of DNA to the metal complex. Thus, the greater theintercalativeligand.
LUMO energy of complex 2 is surely not advantageous to its To further study the apoptosis of the cancer cells induced by
DNA binding affinity. In contrast, the above absorption spectra complexes 1 and 2, Giemsa staining experiments were carried
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Table2 Somefrontiermolecularorbitalenergies(ε/atomicunit)ofcomplexes1and2(1atomicunit=27.21eV)
i
Compound H-2 NHOMOa HOMO LUMO Virb Δε c Δε Δε E d
L-H L-NH L-(H-2) Total
1 −0.3769 −0.3396 −0.3014 −0.2608 −0.2576 0.0406 0.0788 0.1161 −2489.2155
2 −0.3764 −0.3392 −0.3001 −0.2640 −0.2613 0.0361 0.0752 0.1124 −2308.4489
aOcc=occupiedmolecularorbital;HOMO(orH)=thehighestOcc.NHOMO(orNH)=thenextHOMO(orH-1).bVir=virtualmolecularorbital;
LUMO(orL)=thelowestVir.cΔε L–H =energydifferencebetweentheLUMOandHOMO,etc.dE Total =thetotalenergyofthecomplex.
out on HepG2 cells. Fig. 15 indicatesthat, similar to those cells
treated with cis-Pt(NH ) Cl , the majority of the HepG2 cells
3 2 2
treatedwitheithercomplex1or2displayedtheclassicmorpho-
logicalfeaturesofapoptosisafterexposureto20μMofthecom-
plexes, including nuclear condensation, cell shrinkage,
cytoplasmic concentration, which is indicated by a dark red
color, and the formation of apoptotic bodies, which is indicated
by a purple color (Fig. 15c, d). In contrast, no obvious changes
wereobservedinthecontrolcells(Fig.15a).Theresultssuggest
that both complexes can induce HepG2 cell death in viable cell
numbers, partially through induction of HepG2 cells apoptosis.
Further in detail studies are under-way to quantitatively deter-
minetheeffectoncellapoptosis.
4. Conclusions
In conclusion, two novel Ru(II) complexes, [Ru(phen)
2
(ttbd)]2+
(1)andRu(bpy) (ttbd)]2+(2),havebeensynthesizedandcharac-
2
terized. The photoluminescence of both complexes were very
Fig.12 Aschematicdiagramoftheenergiesandrelated1MLCTtran- sensitive to solvent polarity and oxygen molecules in nonaqu-
sitionsofcomplexes1and2. eous solvents and, by cycling the environmentbetween oxygen-
Fig.13 Somerelatedfrontiermolecularorbitalstereographsofcomplexes1and2.
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rich (air) and oxygen-deficient (nitrogen), an “off/on light
switch” could be accomplished for both complexes, revealing
that both complexes are likely to be useful in optically probing
nonaqueousandoxygen-freeenvironments.Competitivebinding
andviscositystudiesyieldconvincingevidenceforthetrueinter-
calative binding of both complexes to CT-DNA, while complex
1possessesagreaterbindingaffinitywithDNAthancomplex2,
as inferred by spectroscopic studies and DFT, which reveal that
the surface area and the hydrophobicity of the ancillary ligand
haveasignificanteffectontheDNAbinding.TheIC valueof
50
complex 1 against HepG2 cells is close to that of cis-Pt
(NH ) Cl , and both complexes can induce HepG2 cell apopto-
3 2 2
sis. These results further advance our knowledge of the inter-
action of metal complexes with DNA and may be useful in
developing DNA-targeted therapeutics, as well as optical probes
fornonaqueousandoxygen-freeenvironments.
Fig.14 TheDFT-optimizedstructuresandvisualizationoftheorbitals
Acknowledgements
ofcomplexes1(left)and2(right).
The authors are grateful for the support of the National Natural
Science Foundation of the People’s Republic of China
(21071120) and the Scientific Research Foundation of Hunan
Table3 IC values of cisplatin and complexes 1 and 2 towards
50 Provincial Education Department (11A117). We also thank the
differentcelllinesdeterminedbyMTTassayfollowingexposureof72h
reviewers for their comments that enabled us to improve the
IC (μM) manuscript.
50
Complex HeLa HepG2
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