Synthesis,characterization and DNA binding studies of ruthenium(II)complexes:[Ru(bpy)2(dtmi)]2+and [Ru(bpy)2(dtni)]2+
Yun-Jun Liu*,Xin Yu Wei,Wen-Jie Mei and Li-Xin He
School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,P.R.China
Received 22March 2007;accepted 27March 2007手持身份证照
Abstract
Two new ruthenium(II)polypyridyl complexes ½Ru(bpy)2(dtmi) ðClO 4Þ2(1)(dtmi =3-(pyrazin-2-yl)-as-triazino [5,6-f]-5-methoxylisatin)and ½Ru(bpy)2(dtni) ðClO 4Þ2(2)(dtni =3-(pyrazin-2-yl)-as-triazino[5,6-f]-5-nitroisatin)have been synthesized and characterized by elemental analysis,FAB-MS,ES-MS The DNA-binding patterns of complexes were investigated by spectroscopic titration,viscosity measurements and thermal denatur-ation.The results indicate that the complexes (1)and (2)interact with calf thymus DNA (CT-DNA)by inter-calative mode.Due to the withdrawing electronic substitutent in the intercalative ligand,ptni,the DNA-binding affinity of the complexes (2)is larger than that complex (1)does.
Introduction
In past decades,Ru II complexes have been the subject of much rearch becau of their properties as photoprobes and photoreagents of DNA [1–7].A majority of the reported ruthenium complexes contain-ing two bipyridine or phenanthroline as ancillary li-gand,some of complexes exhibit unique characteristics,for example,½Ru(phen)2(DPPZ) 2þ(DPPZ =dipyrido[3,2-a;2¢,3¢-c]phenazine)and
½Ru(bpy)2(ppd) 2þ
(ppd =pteridino[7,6-f][1,10]phe-nanthroline-1,13(10H,12H)-dione)[8]show no lumi-nescence in aqueous solution at ambient temperature,but luminesce brightly upon binding intercalatively with the DPPZ and ppd ligands between the adjacent DNA ba pairs,displaying the characteristic of ‘‘molecular light switches’’.The complex
½Ru ðNH 3Þ4(dppz) 2þ
has been reported to exhibit no luminescence either in the abnce or in the prence of double-helical DNA [9].Studies of DNA-binding show that Ru II complexes can bind DNA in a non-covalent i
nteractions fashion,such as electrostatic binding,grooving binding,and intercalation.Many applications require complex binding to DNA by in-tercalative interaction.Intercalation,a strongly favor-able binding mode,involves the p -stacking of a ligand between the adjacent ba pairs of DNA.This stack-ing interaction requires the intercalative ligand to be a flat,extended aromatic system,which is annulated with heterocyclic ring,e.g .pyridine and pyrazine [10],the studies on the complex containing pyrazine ring have been paid great attention in recent years.Herein,in order to explore the DNA-binding behaviors of two
Ru II complex containing pyrazine ring,½Ru(bpy)2(dtmi) ðClO 4Þ2(1)(dtmi =3-(pyrazin-2-yl)-as-triazi-no[5,6-f]-5-methoxylisatin)and ½Ru(bpy)2(dtni) ðClO 4Þ2(2)(dtni =3-(pyrazin-2-yl)-as-triazino[5,6-f]-5-nitroisatin)have been synthesized and characterized by elemental analysis,FAB-MS,ES-MS The DNA-binding properties have been investigated by spectroscopic methods,viscosity measurements and thermal denaturation.The results show that complexes (1)and (2)can intercalate the DNA ba pairs (Scheme 1).
Experimental Materials and method
Calf thymus DNA (CT-DNA)was obtained from the Sino-American Biotechnology Company.Doubly di
s-tilled water was ud to prepare buffers (5mM tris (hy-droxymethylaminomethane)–HCl,50mM NaCl,pH =7.2).A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260and 280nm of ca .1.8–1.9:1,indicating that the DNA was sufficiently free of protein [11].The DNA concentra-tion per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient ð6600M À1cm À1Þat 260nm [12].Physical measurements
Microanalysis (C,H,and N)was carried out with a Perkin-Elmer 240Q elemental analyzer.Fast atom bombardment (FAB)mass spectra were recorded on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol
*Author for correspondence:E-mail:
Transition Metal Chemistry (2007)32:762–768ÓSpringer 2007
DOI 10.1007/s11243-007-0246-y
matrix.Electrospray mass spectra(ES-MS)were re-corded on a LCQ system(Finnigan MAT,USA)using methanol as mobile pha.The spray voltage,tube lens offt,capillary voltage and capillary temperature were t at 4.50KV,30.00V,23.00V and200°C, respectively,and the quoted m/z
values are for the ma-jor peaks in the isotope spectra were recorded on a Varian-500spectrometer.All chemical shifts were given relative to tetramethylsilane (TMS).u.v.–vis spectra were recorded on a Shimadzu UV-3101PC spectrophotometer at room temperature. Cyclic voltammetric measurements were performed on a CHI660A Electrochemical Workstation.All samples were purged with nitrogen prior to measurements.A standard three-electrode system comprising of plati-num microcylinder working electrode,platinum-wire auxiliary electrode and a saturated calomel reference electrode(SCE)was ud.
Preparation
Cis-½Ru(bpy)2Cl2 Á2H2O[13]and pyrazine-2-carboxa-mide hydrazone[14]were prepared according to the literature procedures,and other chemicals were com-mercially available.
悬赏执行
3-(pyrazin-2-yl)-as-triazino[5,6-f]-5-methoxylisatin (dtmi)
A mixture of pyrazine-2-carboxamide hydrazone
(0.272g,2mmol)and5-methoxylisatin(0.354g, 2mmol)was refluxed with stirring in ethanol(60cm3) under argon for5h.The cooled solution was evapo-rated to remove the solvent to10cm3,then the solu-
tion was placed in a refrigerator overnight:a yellow precipitate appeared.The insoluble material wasfil-tered,washed with cool ethanol and dried at50°C in vacuo.Yield:61%.(Anal.Found:C,60.4;H,3.65; N,30.2.calcd.:C14H10N6O C60.4,H3.6,N30.2%. FAB-MS:m/z=279[M+1]+.
3-(pyrazin-2-yl)-as-triazino[5,6-f]-5-nitroisatin(dtni)
A mixture of pyrazine-2-carboxamide hydrazone
(0.272g,2mmol)and5-nitrolisatin(0.384g,2mmol) in glacial acetic acid(30cm3)was refluxed for5h un-der argon,then returned to room temperature.Drop-wi addition of concentrated aqueous ammonia gave red precipitate,which was collected and washed with cool ethanol.Anal.Found:C,53.2;H,2.4;N,33.5. calcd.:C13H7N7O2C53.25,H2.4,N33.4%);FAB-MS:m/z=294[M+1]+.
½Ru(bpy)2(dtmi) ðClO4Þ2(1)
A mixture of Cis-½Ru(bpy)2Cl2 Á2H2O(0.260g,
0.5mmol)and dtmi(0.139g,0.5mmol),ethanol (30cm3)and water(15cm3)was refluxed under argon for
8h to give a clear red solution.Upon cooling,a brown red precipitate was obtained by dropwi addi-tion of a saturated aqueous NaClO4solution.The crude product was purified by column chromatogra-phy on neutral alumina with CH3CN-toluene(3:1,v/ v)as eluant.The mainly brown red band was col-lected.The solvent was removed under reduced pres-sure and a brown red powder was obtained.Yield: 64%.Anal.Found:C,45.7;H,3.0;N,15.6;calcd.: C34H26N10Cl2O9Ru C45.9,H2.9,N15.7%. (DMSO-D6,500MHz)d:11.50(s,1H),8.84(d,2H, J=8.6Hz),8.80(d,2H,J=8.5Hz),8.77(d,1H, J=8.3Hz),8.29(s,1H),8.19(t,1H,J=7.6Hz), 7.93(t,2H,J=7.7Hz),7.78(t,2H,J=7.5Hz), 7.57(d,2H,J=8.2Hz),7.50(d,2H,J=8.5Hz), 7.45(t,1H,J=7.4Hz),7.32(d,2H,J=8.0Hz), 7.26(d,2H,J=8.2Hz),7.16(d,1H,J=8.5Hz), 7.05(d,1H,J=8.3Hz), 3.30(s,3H).ES-MS ðCH3CNÞ:m/z690.5ð½M-2ClO4-H þÞ,345.6ð½M-2ClO4 2þÞ:
甲减检查什么项目½Ru(bpy)2(dtni) ðClO4Þ2(2)
This complex was synthesized using the same proce-dure described for complex(1).Yield:63%.Anal. Found:C,44.0;H, 2.7;N,17.2;calcd.: C33H23N11Cl2O10Ru C43.8,H 2.6,N17.0%.(DMSO-D6,500MHz)d:9.88(s,1H),8.87(d, 1H,J=8.6Hz),8.82(d,2H,J=8.5Hz),8.75(d, 2H,J=8.5Hz),8.39(d,1H,J=8.6Hz),8.35(d, 2H,J=8.6Hz),8.32(d,2H,J=8.5Hz),8.30(s, 1H),8.25(t,2H,J=7.
7Hz),8.19(t,2H, J=7.5Hz),7.97(d,1H,J=8.4Hz),7.79(t,1H, J=7.6Hz),7.68(d,1H,J=8.7Hz),7.50(t,2H, J=7.5Hz),7.44(t,2H,J=7.6Hz).ES-MS (CH3CN):m/z705.3ð½M-2ClO4-H þÞ,353.4ð½M-2ClO4 2þÞ:
海底两万里感悟
2+
[Ru(bpy)2(dtmi)]2+
2+
[Ru(bpy)2(dtni)]2+
Scheme1.Structure of complexes½Ru(bpy)2(dtmi) 2þand
½Ru(bpy)2(dtni) 2þ:
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DNA-binding studies
The absorption titrations of Ru II complex in buffer were performed by using afixed ruthenium complex concentration(20l M)to which the DNA stock solu-tion were added.Ruthenium-DNA solutions were al-lowed to incubate for5min before the absorption spectra were recorded.In order to further illustrate the binding strength of the complex,the intrinsic binding constant K with CT-DNA was obtained by monitoing
the change in the absorbance at metal-to-ligand trans-fer(MLCT),with increasing the concentration of DNA,The following equation was applied[15–17].
ðe aÀe fÞ=ðe bÀe fÞ
¼ðbÀðb2À2K2C t½DNA =sÞ1=2Þ=ð2KC tÞ
ð1Þb¼1þKC tþK½DNA =2sð2Þwhere[DNA]is the concentration of CT-DNA in ba pairs,the apparent absorption coefficients e a;e f and e b correspond to A obd=½Ru ,the absorbance for the free ruthenium complex,and the absorbance for the ruthe-nium complex in fully bound form,respectively.K is the equilibrium binding constant,C t is the total metal com-plex concentration in nucleotides and s is the binding site size.
Viscosity measurements were carried out using an Ubbelodhe viscometer maintained at a constant temper-ature at28.0(±0.1)°C in a thermostat bath.DNA samples,approximately200bp in average length,were prepared by sonicating in order to minimize complexi-ties arising from DNAflexibility[18].Flow time was measured with a digital stopwatch,and each sample was measured three times,and an averageflow time was cal-culated.Data were prented as(g/g)1/3versus binding ratio[19],where g is the viscosity of DNA in the pres-ence of complex and g0is the viscosity of DNA alone. Thermal denaturation studies were carried out with a Perkin-Elmer Lambda35spectrophotometer equip-ped with a Peltier temperature-controlling programmer (±0.1°C).The melting curves were obtained by mea-suring the absorbance at260nm for solutions of CT-DNA(80l M)in the abnce and prence of different concentrations of the Ru II complex as a fun
ction of the temperature.The temperature was scanned from 50°C to90°C at a speed of1°C min)1.The melting temperature(T m)was taken as the mid-point of the hyperchromic transition.
Equilibrium dialysis was carried out in the dark for 24h with5cm3of DNA solution and10cm3of the complex(100l M)outside the bag.
Results and discussion
Electrochemistry
The electrochemical behavior of the two complexes has been studied in acetonitrile.The results are listed in Table1.The Ru II complex exhibits well-defined waves corresponding to the metal-bad oxidation and three successive ligand-bad reduction in the sweep range from)1.8V to1.8V.The oxidation potentials
1.28V and1.30V(versus SCE)of complexes(1)and
(2),respectively,are similar to that of½Ru(bpy)3 2þ(1.29V)[20].Thefirst reduction,which is usually con-trolled by the intercalative ligand having most popula-tion in the lowest unoccupied molecular orbital (LUMO)[21],should be attributed to the ligand(dtmi, dtni)-centered reduction.The latter two successiv
e reductions are characteristic of co-ligand(bpy)[22].
Absorption spectroscopic studies
The electronic absorption spectra of complexes(1) and(2)mainly consist of two or three resolved bands. The low energy absorption band centered at 450–470nm is assigned to metal-to-ligand charge transfer(MLCT)transition,the band at362–385nm is attributed to pfip*transition and the other band below300nm is attributed to intraligand(IL)pfip*transition by comparison with the spectrum of other polypyridyl Ru II complexes[23–26].
The electronic spectra traces of the two Ru II com-plexes titrated with calf thymus DNA(CT-DNA)are shown in Figure1.As increasing the concentration of CT-DNA,the MLCT transition bands of complexes (1)at448nm and(2)at456nm exhibit hypochro-mism of about16.0%and22.5%,and bathochromism of2nm and3nm,respectively.Comparing the hypo-chromism of the two complexes with that of ½Ru(phen)3 2þ(hypochromism in the MLCT band at 445nm of12%and red shift of2nm)[27],which interacts with DNA through a mi-intercalation or quasi-intercalation[28],the spectral characteristics obviously suggest that complexes(1)and(2)interact with DNA most likely through a mode that involves a stacking interaction between the aromatic chromo-phore and the ba pairs of DNA.
In order to further illustrate the binding strength of the complex,the intrinsic constants K were determined by monitoring the changes of absorbance in the MLCT band with increasing concentration of DNA, the intrinsic binding constants K of complex(1)and (2)were8.3Â104M)1(s=1.69)and3.0Â105M)1 (s=1.84),respectively.The values are larger than that of complexes½Ru(bpy)2L 2þ[L=ptdb(1.9ÂTable1.Electrochemical data of the Ru II complexes
Complexes E ox=V E red=V
I II III
½Ru(bpy)2(dtmi) 2þ 1.28)0.83)1.41)1.64½Ru(bpy)2(dtni) 2þ 1.30)1.18)1.36)1.61½Ru(bpy)3 2þ 1.29)1.31)1.50)1.77 Redox potentials were quoted versus SCE in0.1M TBAH-MeCN. Scan rate¼200mV SÀ1.
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104),ptda (3.1Â104)and ptdp (5.9Â104
)][29],but smaller than that obrved for
½Ru(bpy)2(dppz) 2þ
ð106Þ[30].The difference between the two intrinsic constants is owing to different substit-utents.The electron-withdrawing substituent (–NO 2in dtni)on the intercalative ligand can improve the DNA binding affinity,whereas the electron-releasing sub-stituent (–OCH 3in dtmi)decreas the DNA affinity.The results show that the DNA binding affinities of the complexes cloly correlate with the electronic ef-fects of their intercalative ligands.Fluorescence spectroscopic titration
Complexes (1)and (2)can emit luminescence in the abnce of CT-DNA in Tris buffer,with a maximum appearing at 595nm and 599nm.Fixed amounts (5l M )of complexes (1)and (2)were respectively ti-trated with increasing amounts of CT-DNA.The re-sults of the emission titration curves are shown in Figure 2.Upon addition of CT-DNA,for complexes
(1)and (2),the emission intensity grows to around 7.33and 8.27times larger than that in the abnce of DNA and saturates at a [DNA]/[Ru]ratio of 30.This implies that both complexes can strongly interact with DNA and be protected by DNA efficiently,since the hydrophobic environment inside the DNA helix re-duces the accessibility of solvent water molecules to the complex and the complex mobility is restricted at the binding site,leading to decrea of the vibrational modes of relaxation.Thermal denaturation studies
The melting temperature T m ,which is defined as the temperature where half of the total ba pairs are un-bond,is determined from the thermal denaturation curves of DNA by monitoring the changes of absorp-tion spectra of DNA bas (k =260nm).The melting of DNA can be ud to distinguish between tho mol-ecules which bind via intercalation and tho which bind externally,and thermal denaturation behavior of DNA can offer information about the interaction
Wavelength/nm
Wavelength/nm
A b s o r b a n c e
炒饼怎么做简单又好吃A b s o r b a n c e
Fig.
1.Absorption spectra of complex in Tris–HCl buffer upon addition of CT-DNAh开头单词
in the prence of complexes (1)(a)and (2)(b).[Ru]¼20l M .Arrow shows the absorbance changing upon the increa of DNA concentration.Plots of ðe a Àe f Þ=ðe b Àe f Þversus [DNA]for the titration of DNA with Ru II complex.
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u .a (y t i s n e t n I )
u .a (y t i s n e t n I Wavelength/nm
Wavelength/nm
Fig.2.Emission spectra of complex (1)(a)and (2)(b)in Tris–HCl buffer in the abnce and prence of CT-DNA.Arrow shows the intensity change upon increasing DNA concentrations.
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strength of complexes with DNA.According to the lit-erature [31–33],the intercalation of natural or synthe-sized organics and metallointercalators generally results in a considerable increa in melting tempera-ture (T m ).Here,a DNA melting experiment revealed that T m of calf thymus DNA is 74.2±0.5°C in the abnce of the complexes (Figure 3)under our experi-mental conditions.The obrved melting temperature in the prence of complexes (1)and (2)were 78.7±0.5°C and 79.8±0.5°C,respectively.The moderate increas in T m of the two Ru II complexes (the D T m is 4.5and 5.6°C for (1)and (2),respec-tively)are comparable to that obrved for classical in-tercalators [31–33]and lend strong support for intercalation into the helix.The experimental results also indicate that complex (2)exhibits larger DNA-binding affinity than complex (1)does.The binding constants of complex (1)and (2)to CT-DNA at T m were determined by McGee’s equation [34,35].
1=T 0m À1=T m ¼ðR =D H m Þ½ln ð1þK b L Þ
1=ndnf解除安全模式官网
ð3Þ
where T m 0and T m are the melting temperatures of CT-DNA alone and in the prence of complex,respectively.D H m is the enthalpy of DNA melting (per bp),R is the gas constant.K b is the DNA binding constant at T m ,L is the free Ru II complexes concen-tration,and n is the binding site size.According to document [34],D H m =6.9Kcal Æmol )1,the binding constants K b for complex (1)and (2)at T m were cal-culated to be 1.57Â103and 1.72Â103M )1,respec-tively.According to the van’t Hoffs Equations (4),(5)and (6)[36]
ln ðK 2=K 1Þ¼ðD H 0=R Þ½ðT 2ÀT 1Þ=T 1T 2 ð4ÞD G 0T ¼ÀRT ln K
ð5ÞD G 0T ¼D H ÀT D S
0ð6Þ
where K 1and K 2are the DNA binding constants of the complex at the temperature of T 1and T 2,respec-
tively.R is the gas constant,D H 0;D G 0T and D S 0
are the changes of standard enthalpy,standard free energy and standard entropy of binding of the complex to
CT-DNA.The values of D G 0T ;D H 0
,and D S 0are shown in Table 2.The negative binding free energies suggest that the sum of the free energies of the free complex ½Ru(bpy)2(dtmi) 2þand ½Ru(bpy)2(dtni) 2þto DNA are higher than that of the adduct.It is obvi-ous that the binding of complexes to CT-DNA is ener-getically highly favorable at room temperature and the binding reaction was enthalpically driven.While large negative entropy changes are unfavorable for com-plexes binding to CT-DNA [37],the changes of D G 0T suggest that the complex (2)has larger DNA-binding affinity than that of complex (1).
Viscosity measurements
The viscosity measurements of DNA are regarded as the least ambiguous and the most critical test of a DNA binding model in solution and provides strong arguments for intercalative DNA binding mode [38,39].A classical intercalation mode requires that the DNA helix lengths are parated to accommodate the binding ligand,leading to an increa in the DNA vis-cosity.A partial,non-classical intercalation of the li-gand could bend (or kink)the DNA helix,reduce its effective length and,concomit
antly,its viscosity [38,39].Figure 4shows the effects of complexes (1)and (2),together with ½Ru(bpy)3 2þand ethidium bromide on the viscosity of rod-like DNA.Ethidium bromide is a known DNA classical intercalator and increas the
Table 2.D H 0ðKJ mol À1Þ,D G 0T ðKJ mol À1Þand D S 0
ðJ mol À1K À1Þfor (1)and (2)
Complex
D H 0D G 0T D S 0½Ru(bpy)2(dtmi) 2þ)64.38)28.09)121.7½Ru(bpy)2(dtni) 2þ
)82.33
)31.25
)171.4
(η/η0)
3
/1[ R u ] / [ D N A ]
Fig.4.Effect of increasing amounts of ethidium bromide (n ),com-plex (1)(•),(2)(m )and ½Ru(bpy)3 2þ(r ),on the relative viscosity of calf thymus DNA at 28(±0.1)°C.[DNA]=0.5mM.
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