ORIGINAL PAPER

Nucleic acid-binding comparative studies and cytotoxic propertiesof a Ru(II) complex

Beilei Li • Lifeng Tan • Xiao-Qin Zou

Received: 17 January 2011 / Accepted: 20 June 2011 / Published online: 10 January 2012

� Iranian Chemical Society 2012

Abstract The comparative studies on the interaction of a

ruthenium(II) complex [Ru(IP)2DPBPD(NH2)2]2? (1) {IP =

imidazole[4,5-f] [1,10] phenanthroline, DPBPD(NH2)2 =

2,4,5,6-tetraaminopyrimidine-[3,2-a:20,30-c]-phenazine}

with CT-DNA (calf thymus DNA) and yeast tRNA have

been investigated by different spectrophotometric methods

and viscosity measurements. The antitumor activities of

complex 1 have been evaluated by MTT {MTT = (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide}

method. On the basis of the spectroscopic results, the

binding mode of complex 1 to CT-DNA and yeast tRNA

are intercalation, and RNA binding of complex 1 is

stronger than DNA binding. Furthermore, complex 1 is a

better candidate for an enantioselective binder to yeast

tRNA than to CT-DNA. The results can be explained by

the different structure and configuration between CT-DNA

and yeast tRNA reasonably, suggesting that the configu-

ration and structure of nucleic acids have significant effects

on the binding behaviors of metal complexes. On the other

hand, the complex demonstrates different antitumor activ-

ity against selected tumor cell lines in vitro.

Keywords Ruthenium(II) complex � Nucleic

acid-binding behavior � Binding affinity � Cytotoxicity

Introduction

In the recent years, tremendous interest has been aroused to

explore the potential applications of metal complexes as

non-radioactive probes of nucleic acid structure and as

possible DNA cleaving agents [1, 2]. In particular, Ru(II)

complexes with polypyridyl ligands, due to a combination

of easily constructed rigid chiral structures spanning all

three spatial dimensions and a rich photophysical reper-

toire, have attracted considerable attention [3–6].

Binding studies of small molecules to DNA are very

important in the development of DNA structure probes,

molecular light switches, chemotherapy and photodynamic

therapy, and so forth [7–9]. Generally, Ru(II) polypyridyl

complexes can bind to DNA in a non-covalent interactions

fashion, such as electrostatic binding, groove binding [10],

intercalative binding and partial intercalative binding [11,

12]. Many useful applications of these complexes require

that the complexes bind to DNA through an intercalative

mode, and the intercalative ligand dominates the DNA-

binding modes and affinities. Therefore, the vast majority

of such studies have been focused on modifying the

intercalative ligand.

It is well known that RNA is a versatile molecule that

plays essential roles in many biological processes, and

consequently, it is an attractive target for potential thera-

peutics. The structure diversity present in RNA molecules

has led to specific drug recognition sites. Therefore, a

considerable amount of new information for RNA–metal

complex interactions has also emerged [13–15]. Metal

complexes were usually used as catalyst of RNA hydrolysis

probes of RNA tertiary structure [14], agents of RNA

oxidation cleavage [15], and recognition of mismatches

in RNA. However, investigations of the binding mode and

the enantioselectivity for the interaction between metal

B. Li � L. Tan (&) � X.-Q. Zou (&)

College of Chemistry, Xiangtan University,

Xiangtan 411105, People’s Republic of China

e-mail: lfwyxh@yahoo.com.cn

X.-Q. Zou

e-mail: zouxq2002@gmail.com

L. Tan � X.-Q. Zou

Key Lab of Environmentally Friendly Chemistry and

Application in Ministry of Education, Xiangtan University,

Xiangtan 411105, People’s Republic of China

123

J IRAN CHEM SOC (2012) 9:357–366

DOI 10.1007/s13738-011-0031-x

complexes and RNA have been relatively few. The future

development of RNA-targeting drugs will rely on a deeper

understanding of these binding processes.

When compared with each other, DNA and RNA are

different not only in the composition of bases but also in

structure. For example, CT-DNA (calf thymus DNA) has a

B-form configuration, while yeast tRNA has an A-form

configuration with an L-shaped tertiary structure. Although

some experimental investigations on RNA–metal complex

interactions have been carried out during the past decade as

described above, there are still some questions need to be

answered for a better understanding of the mechanism and

the biological implications of the interactions. For exam-

ple, how the structures of nucleic acids affect the binding

behaviors of metal complexes with them. Thus, a com-

parative study of the interactions of metal complexes with

CT-DNA and yeast tRNA in binding mode, binding

strength and enantioselectivity will be very important in

understanding the mechanism of the interactions and the

biological impact of metal complexes.

Herein, a new ligand DPBPD(NH2)2 have been syn-

thesized and characterized. The CT-DNA and yeast tRNA-

binding behaviors of complex 1 have been investigated by

different spectrophotometric methods and viscosity mea-

surements. The cytotoxicity of complex 1 has also been

evaluated by MTT method. We hope the results to be of

value in understanding the mechanism of the interactions

of metal complexes with nucleic acids, and may be useful

in the development of nucleic acid molecular probes and

new therapeutic regents for some diseases.

Experimental

Chemicals

1,10-phenanthroline-5,6-dione [16], cis-[Ru(IP)2Cl2]�2H2O

[17] and IP [18] were synthesized according to the litera-

ture procedures. Doubly distilled water was used to prepare

buffers. CT-DNA and the yeast tRNA were obtained from

the Sino-American Biotechnology Company. Other mate-

rials were commercially available and of reagent grade.

5 mM Tris–HCl buffer (pH 7.2) containing 50 mM NaCl

was used for DNA binding experiments. To avoid the

degeneration of RNA induced by metal ions, 5 mM Tris–

HCl buffer (pH 7.2) containing 50 mM NaCl and 0.1 mM

EDTA (pH 7.2) was used for RNA binding experiments.

Preparation of ligand DPBPD(NH2)2

A mixture of 2,4,5,6-tetraaminopyrimidine sulfate, (0.48 g,

2.0 mmol), 1,10-phenanthroline-5,6-dione (0.42 g, 2.0 mmol)

and glacial acetic acid (15 mL) was refluxed with stirring

at 130 �C under argon for 3 h. The cooled solution was

filtered, diluted with water (20 mL) and neutralized with

concentrated aqueous ammonia. The orange precipitate

was collected and purified by column chromatography on

alumina with ethanol–toluene (4:1, v/v) as eluent to give

the title compound as amorphous yellow solid. Yield:

0.55 g, 87%. Anal. Calc. for C16H10N8: Calc. C 61.14, H

3.21, N 35.65. Found: C 59.95, H 3.32, N 35.70%. m/z (FAB)

315 [M ? 1].

Preparation of [Ru(IP)2 DPBPD(NH2)2]

(ClO4)2�4H2O (1)

A mixture of cis-[Ru(IP)2Cl2]�2H2O (0.15 g, 0.25 mmol),

DPBPD(NH2)2 (0.08 g, 0.25 mmol) and ethylene glycol

(15 mL) was thoroughly deoxygenated. The purple mixture

was heated for 8 h at 150 �C under argon. When the

solution finally turned red, it was cooled to room temper-

ature and equal volume of saturated aqueous sodium per-

chlorate solution was added under vigorous stirring. The

red solid was collected and washed with small amounts of

water, ethanol and diethyl ether, then dried under vacuum,

and purified on a neutral alumina column with CH3CN–

toluene (2:1, v/v) as eluant. Yield: 0.18 g, 64%. Anal.

Calc. for C42H34N16Cl2O12Ru: C 44.77, H 3.04, N

19.89%. Found: C 44.57, H 3.32, N 19.71%. kmax/nm

(e/L mol-1 cm-1) (MeOH): 463 (13,000), 280 (23,000),

253 (56,000). m/z (ES-MS, positive mode, MeCN,): 955.7

([M–ClO4]?), 428.4 ([M–2ClO4]2?). 1H NMR (400 MHz,

d6-DMSO (dimethyl sulfoxide); d, doublet; s, singlet; t,

triplet): 9.09 (d, 6H), 8.67 (s, 2H), 7.98 (d, 6H), 7.61–7.70

(t, 6H), 3.51 (s, 4H).

Physical measurements

Microanalyses (C, H and N) were carried out on a Perkin–

Elmer 240Q elemental analyzer. 1H NMR spectra were

recorded on a Avance-400 spectrometer with d6-DMSO as

solvent at room temperature and TMS (tetramethylsilane)

as the internal standard. UV–vis spectra were recorded on a

Perkin–Elmer Lambda-25 spectrophotometer and emission

spectra were recorded on a Perkin–Elmer LS-55 lumines-

cence spectrometer at room temperature. Electrospray mass

spectrometry (ES-MS) data were recorded on a LQC sys-

tem (Finngan MAT, USA) using CH3CN as the mobile

phase. Circular dichroism (CD) spectra were measured on a

JASCO-J810 spectropolarimeter.

Solutions of yeast tRNA and CT-DNA gave ratios of UV

absorbance at 260 and 280 nm of over 2.0 and 1.8–1.9,

respectively, indicating that both nucleic acids were fully

free of protein [19]. The concentrations of CT-DNA and

yeast tRNA solutions were determined at 260 nm by

absorption spectroscopy using molar absorption coefficients

358 J IRAN CHEM SOC (2012) 9:357–366

123

of 6,600 M-1 cm-1 for CT-DNA [20] and 7,700 M-1 cm-1

for yeast tRNA [20].

Spectroscopic titration experiments were performed by

using a fixed Ru(II) complex concentration (20 lM), to

which CT-DNA and yeast tRNA stock solutions were

added, respectively. The Ru(II) complex-DNA or -RNA

solution was allowed to equilibrate for 5 min before

spectra were recorded.

Viscosity experiments were carried out using an Ub-

belodhe viscometer maintained at a constant temperature

(28.0 ± 0.1 �C) in a thermostatic water-bath. CT-DNA

samples approximately 200 base pairs in average length

were prepared by sonicating in order to minimize com-

plexities arising from DNA flexibility [21]. Flow time was

recorded with a digital stopwatch and each sample was

measured thrice, then an average flow time was calculated.

Data were presented as (g/g0)1/3 versus binding ratio [22],

where g is the viscosity of CT-DNA in the presence of

complex, and g0 is the viscosity of CT-DNA alone.

Equilibrium dialyses were conducted at ambient tem-

perature with 10 mL DNA or RNA (1.0 mM) sealed in a

dialysis bag and 10 mL of complex 1 (20 lM) outside the

bag, then, the system was agitated on a shaker bath. After

16 h the CD (CD circular dichroism) spectrum of the

dialysate outside the bag was measured on a on a JASCO-

J810 spectropolarimeter.

Measurement of tumour cell toxicity

MTT assay procedures were used [23]. Cells (HeLa, hepG2,

BEL-7402 and MCF-7) were cultured in Dulbecco’s modi-

fied Eagle’s medium (DMEM) with 10% (v/v) fetal bovine

serum, 100 U/ml penicillin, and 100 lg mL-1 streptomycin

at 37 �C in a humidified atmosphere (90%) containing 5%

CO2. For MTT assay, cells were seeded in 96-well micro-

assay culture plates at a density of 20–30 cells/lL (volume

of 200 lL/well) and incubated overnight at 37 �C in a 5%

CO2 incubator. Test complexes were then added to the wells

to achieve final concentrations ranging from 10-6 to

10-4 M. Control wells were prepared by addition of culture

medium (200 lL). Wells containing culture medium without

cells were used as blanks. The plates were incubated at

37 �C in a 5% CO2 incubator for 48 h. Upon completion

of the incubation, stock MTT dye solution (20 lL,

5 mg mL-1) was added to each well. After 4 h incubation,

N,N-dimethylformamide (150 lL) was added to solubilize

the MTT formazan. The optical density of each well was

then measured on a microplate spectrophotometer at a

wavelength of 490 nm. IC50 values of the target compounds

were calculated using Sigmaplot software and expressed as

mean ± SD of triplicate experiments.

Results and discussion

Synthesis and characterization

An outline of the synthesis of the ligand DPBPD(NH2)2

and its Ru(II) complex is presented in Scheme 1. The

ligand DPBPD(NH2)2 was prepared through condensation

of 1,10-phenanthroline-5,6-dione with 2,4,5,6-tetraamino-

pyrimidine sulfate on the basis of the method for DPPZ

(DPPZ dipyrido-[3,2-a:20,30-c]-phenazine) ring according

to the literature methods [24]. The complex [Ru(IP)2

2+

N

N

N

N

N

NH2

NH2

N

NH2N

H2N

H2N

NH2

cis-[Ru(IP)2Cl2]

DPBPD(NH2)2

N

DPBPD(NH2)2

N

N

O

O

N

N KBr, reflux, 3 h

HNO3/H2SO4

ethylene glycol, Ar, reflux, 8 h

glacial acetic acid reflux, 3 h

NN

NHN

N

N N

N

N

N

NH2

NN

NH2

HNN

Ru

ab

c

a'

b'c'

d

e

e'

Scheme 1 Synthesis of the

ligand DPBPD(NH2)2 and its

Ru(II) complex [Ru(IP)2

DPBPD(NH2)2]2?

J IRAN CHEM SOC (2012) 9:357–366 359

123

DPBPD(NH2)2]2? was prepared by direct reaction of

DPBPD(NH2)2 with the appropriate mole ratios of cis-

[Ru(IP)2Cl2]�2H2O in ethylene glycol in relative high

yield. In the ES-MS spectra of [Ru(IP)2DPBPD(NH2)2]2?,

both [M–ClO4]? and [M–2ClO4]2? were observed, and

the determined molecular weights were consistent with

expected values.

The well-defined 1H NMR spectra of complex 1 was

given in Fig. 1, which permitted unambiguous identifica-

tion and assessment of purity. The 1H-NMR chemical

shifts were assigned by the aid of a 1H, 1H-COSY exper-

iments, and by comparison with the values of similar

compounds [11, 25–27]. The proton resonance on the

nitrogen atom of the imidazole ring was not observed, and

it has also been proposed that the proton exchanges quickly

between the two nitrogens of the imidazole ring and it is a

characteristic of an active proton [27]. On the other hand,

the resonance peaks of different protons completely

overlaps between IP and BPD(NH2)2, this phenomenon has

also been found with other analogues [14]. One set corre-

sponds to the ligands IP, and the other set corresponds to

the ligand DPBPD(NH2)2.

The UV–vis absorption spectra of the complex showed

four well-resolved bands in the range 200–600 nm range

(Fig. 2), characterized by intense p ? p* ligand transi-

tions in the UV region, as well as by metal-to-ligand charge

transfer (MLCT) transition in the visible region. The broad

MLCT absorption band appeared at 463 nm for complex 1,

and is attributed to Ru(dp) ? DPBPD(NH2)2 (p*) transi-

tions. The MLCT band is bathochromically shifted relative

to those of [Ru(phen)3]2? (448 nm) [28] and [Ru(bpy)3]2?

(452 nm) [28], in accordance with the extension of the

corresponding framework. The peak below 400 nm was

assigned to internal p ? p* transition of the ligands,

by comparison with the spectra of [Ru(phen)3]2? and

[Ru(bpy)3]2?.

Fig. 1 Aromatic region (top) and integration (bottom) of the peaks in 1H NMR spectra of complex I in d6-DMSO

360 J IRAN CHEM SOC (2012) 9:357–366

123

Nucleic acid-binding studies

UV–vis titration

The application of electronic absorption spectroscopy in

DNA/RNA-binding studies is one of the most useful

techniques [29]. Complex binding with DNA/RNA through

intercalation usually results in hypochromism and batho-

chromism, due to the intercalative mode involving a strong

stacking interaction between an aromatic chromophore and

the base pairs of DNA/RNA. In general, the extent of the

hypochromism commonly parallels the intercalative bind-

ing strength.

Figure 3 shows the electronic spectral traces of complex

1 titrated with CT-DNA and yeast tRNA, respectively. As

can be seen from Fig. 3, upon increasing the concentrations

of CT-DNA or yeast tRNA, the UV–vis spectra of complex

1 shows clearly hypochromism and red shift in absorbance

bands. For CT-DNA binding of complex 1, the pronounced

hypochromism in the intraligand (IL) band reaches as high

as 15.7% at 279 nm with a red shift of 2 nm at a [DNA]/

[Ru] ratio of 3.9. The MLCT band at 459 nm shows hyp-

ochromism by about 9.1% and a red shift of 1 nm under the

same experimental conditions. For yeast tRNA binding of

the complex, with increasing the yeast tRNA concentra-

tions, each absorption bands’ position changes signifi-

cantly, the pronounced hypochromism in the IL band

reaches as high as 23.4% at 279 nm with a red shift of

2 nm at a [RNA]/[Ru] ratio of 4.8. The MLCT band at

459 nm shows hypochromism by about 13.4% and a red

shift of 1 nm under the same experimental conditions.

Obviously, these spectral characteristics suggested that

complex 1 could interact with CT-DNA and yeast tRNA,

and the hypochromism of complex 1 binding with yeast

tRNA is higher than that of complex 1 binding with

CT-DNA. On the other hand, compared the hypochromism

of the UV–visible spectra for both CT-DNA and yeast

tRNA binding to complex 1 with that of CT-DNA binding

of its parent complex [Ru(phen)3]2? (hypochromism in

MLCT) band at 445 nm is 12% and the red shift is 2 nm

[27], which interacts with CT-DNA through a semiinter-

calation or quasi-intercalation [30], and considering that

the UV–vis spectrum of [Ru(bpy)3]2? (bpy = 2,20-bipyri-

dine), a typical electrostatic binding complex, has been

demonstrated to be unchanged upon the addition of CT-DNA,

these spectroscopic characteristics obviously suggest that

240 320 400 480 560 6400.00

0.25

0.50

0.75

1.00

Abs

orba

nce

Wavelength (nm)

Fig. 2 Absorption spectra of complex 1 in MeOH

240 320 400 480 560 6400.0

0.2

0.4

0.6

0.8

1.0

1.2

Wavelength (nm)

Abs

orba

nce

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

-[R

NA

]/ (

ε a−εf )

× 1

09 [m

ol L

-1 c

m]

[RNA] ×10 5 [mol L-1]

240 320 400 480 560 6400.0

0.2

0.4

0.6

0.8

1.0

0.0 0 .5 1.0 1 .5 2 .0 2 .5 3 .0

3

4

5

6

7

8

-[D

NA

]/ ( ε

a−εf )

× 10

9 [m

ol L

-1 c

m]

[D N A ] ×10 5 [m o l L -1]

Wavelength (nm)

Abs

orba

nce

(a)

(b)

Fig. 3 UV and visible spectra of complex 1 upon the addition of CT-

DNA (a) and yeast tRNA (b) in Tris–HCl buffer (pH 7.2) at 25 �C.

[Ru] = 2.0 9 10-5 M, [DNA] = (0 - 3.0) 9 10-5 M, [RNA] =

(0 - 3.9) 9 10-5 M. Arrow shows the absorbance changing upon

increasing DNA or RNA concentrations. Inset: plots of -109 [Nucleic

acid]/(ea - ef) (in (mol L-1)2 cm] versus [Nucleic acid] (in mol L-1)

for the titration of DNA or RNA with complex 1 for the determination

of the binding constant Kb

J IRAN CHEM SOC (2012) 9:357–366 361

123

complex 1 interacts with DNA and yeast tRNA most likely

through an intercalative mode. These spectral characteristics

obviously suggest that complex 1 binding with CT-DNA and

yeast tRNA are most likely through a mode that involved a

stacking interaction between the aromatic chromophore

DPBPD(NH2)2 and the base pairs of CT-DNA and yeast

tRNA.

In order to further elucidate quantitatively the affinity of

complex 1 binding to CT-DNA and yeast tRNA, the

intrinsic binding constants Kb were obtained by monitoring

the changes in absorbance at 279 nm with increasing

concentration of CT-DNA or yeast tRNA using the fol-

lowing equation [31]:

½Nucleic acid�=ðea � efÞ ¼ ½Nucleic acid�=ðeb � efÞþ 1=½Kbðeb � efÞ� ð1Þ

Where [Nucleic acid] is the concentration of DNA or RNA

in base pairs, and ea, ef and eb are the extinction coefficients

of the apparent, free, and bound metal complex, respec-

tively. Kb is the equilibrium binding constant in M-1.

When plotting [Nucleic acid]/(ea - ef) versus [Nucleic

acid], Kb is obtained by the ratio of the slope to the

intercept. Thus, the intrinsic binding constants Kb of

complex 1 binding with CT-DNA and yeast tRNA

were determined as (6.7 ± 0.50) 9 104 and (1.7 ±

0.1541 9 105 M-1, respectively. Comparing the binding

constants obtained with those of typical DNA-intercalative

Ru(II) complexes (1.1 9 104–4.8 9 104 M-1) [32], and

the complexes [Ru(bpy)3]2? (4.7 9 103 M-1) and

[Ru(phen)3]2? (5.5 9 103 M-1) [33], we could deduce that

complex 1 binds to CT-DNA and yeast tRNA by interca-

lation. The intrinsic binding constants of complex 1 are

bigger than those of [Ru(phen)3]2? and [Ru(bpy)3]2?, which

can also be interpreted by the planarity area of the interca-

lated ligand and the hydrophobicity effect (HE) of the

ancillary ligand. The planarity area (S) is SDPBPDðNH2Þ2 [Sphen and Sbpy), on the other hand, the hydrophobicity effect

is HEDPBPDðNH2Þ2 [ HEphen and HEbpy. In general, both the

extend p system of the intercalative ligand and the increased

hydrophobicity of the ancillary ligand will increase the

strength of interaction of the complexes with nucleic acid

[34].

However, the intrinsic binding constant Kb of complex 1

binding with CT-DNA is smaller than that of [Ru(IP)2

DPPZ]2? (2.1 9 107 M-1) [17]. Since the ancillary ligands

of complex 1 and [Ru(IP)2DPPZ]2? are the same, the dif-

ference of the DNA-binding affinity should be attributed to

the intercalative ligand effects. Comparing the ligand

DPBPD(NH2)2 with the ligand DPPZ, the latter is the

parent ligand of the former. Upon ammoniation and azo-

tizing in the ligand DPPZ, it is very interesting that the

planarity of the new ligand DPBPD(NH2)2 is not changed.

Theoretically, the introductions of –NH2 group and N atom

into DPPZ can form hydrogen bonds with basic groups of

DNA, which are advantageous to the DNA-binding of

complex 1. However, upon the introductions of two –NH2

functional groups into DPPZ, the steric hindrance of the

new ligand DPBPD(NH2)2 in complex 1 is obviously

substantial. Generally, an intercalative ligand containing a

steric hindrance group results in lower DNA-binding

affinity relative to the parent ligand [35]. Therefore, syn-

thetically considering these factors, the difference of DNA-

binding affinity of complex 1 is smaller than that of

[Ru(IP)2DPPZ]2? can be well understood.

Compared the affinity of complex 1 binding to DNA

with that of complex 1 binding to yeast tRNA, we can

demonstrate that the binding of complex 1 to yeast tRNA is

stronger than that of CT-DNA. A possible explanation for

this trend may be due to the A-form configuration and the

L-shaped tertiary structure of yeast tRNA, in which the

major grove is wide and shallow, thus its base pairs are

well exposed and can be attacked by complexes easily. In

addition, complex 1 may bind to the bugle region of yeast

tRNA, which may be another reason why the affinity of

complex 1 binding with RNA is greater.

Competitive binding experiments

Complex 1 fails to show steady-state emission in various

organic solvents at room temperature and also lacks

emission in aqueous solution even in the presence of

CT-DNA or yeast tRNA. Synthetically considering the

structural characteristics of complex 1, why this complex

shows no emission under the above conditions can be well

explained by the following two reasons. First, there are too

many free N atoms (the uncoordinating N atoms) in the

ligands IP and DPBPD(NH2)2, especially in the ligand

DPBPD(NH2)2. Upon increasing the uncoordinating N

atoms in the ligand, the channels of the energy loss of

the complex will increase, which will result in reducing the

emission intensity of the complex, to a point that the

complex completely loses its emission. Therefore, for

complex 1, no emission was observed in various organic

solvents. Similar results have also been reported for this

type of Ru(II) complexes [36, 37]. Second, the mechanism

of the ‘‘light switch’’ effect for [Ru(bpy)2(DPPZ)]2? and

[Ru(phen)2(DPPZ)]2? have been studied intensively and all

evidence points to H-bonding and/or excited-state H-atom

transfer to the phenazine N-atom as the mechanism of

deactivation of the complexes’ excited state. Upon inter-

calation, DNA provides the metal-bound DPPZ ligand with

a hydrophobic environment, which in turn protects the

Ru(II) complex from the quenching effect of water [29, 31,

38]. Herein, why complex 1 could not show emission in the

presence of CT-DNA or yeast tRNA? The main reason

may be that when complex 1 intercalate into the bases of

362 J IRAN CHEM SOC (2012) 9:357–366

123

DNA or yeast tRNA, not all the uncoordinating N atoms

could be protected by DNA or yeast tRNA. Having said

that some uncoordinating N atoms (especially –NH group

in the ligand IP) still can form H-bonding with water

molecules. Therefore, the formed hydrogen-bonds can

intensively reduce the luminescence intensity of com-

plexes, even completely quench the emission of complexes

[35, 39, 40].

It is well known that EB (ethidium bromide) emits

intense fluorescence in the presence of DNA due to its

strong intercalation between the base pairs of DNA. It also

has been reported that the enhanced fluorescence can be

quenched, at least partially by the addition of a second

molecule. Since complex 1 does not exhibit emission in the

presence of DNA and yeast tRNA, respectively, and also

shows no influence on the emission intensity of free EB in

the absence of DNA or yeast tRNA, the competitive DNA-

and yeast tRNA-binding of complex 1 with EB could

provide further information regarding its nature of DNA-

and yeast tRNA-binding. The extent of quenching fluo-

rescence of DNA- and yeast tRNA-bound EB can be used

to determine the extent of binding affinity between the

second molecule and DNA or RNA. If the intense fluo-

rescence is reduced by adding a second intercalative mol-

ecule, it will be an evidence of the intercalation of the

second molecule between DNA and RNA base pairs.

The emission spectra of DNA- and yeast tRNA-bound

EB in the absence and the presence of the title complex are

presented in Fig. 4. As shown in Fig. 4, we find that the

addition of complex 1 to the DNA- and yeast tRNA-EB

system causes obvious reduction of 24.7 and 39.6% in

fluorescence intensity, respectively, indicating the interca-

lation of the complex accompanied by the replacement of

the EB molecules. In addition, the fluorescence quenching

curves (inset in Fig. 4) illustrate that the fluorescence

quenching of DNA- and RNA-bound EB by complex 1 are

in good agreement with the linear Stern–Volmer equation

with Ksv values of 1.78 and 3.12 for DNA and yeast tRNA,

respectively, indicating that complex 1 exerts stronger

yeast tRNA-binding affinity than that of CT-DNA.

Viscosity measurements

Hydrodynamic measurements sensitive to length changes,

as reflected in viscosity and sedimentation, are regarded as

the least ambiguous and the most critical tests of a binding

model in solution in the absence of crystallographic

structural data [41]. A classical intercalation model

demands that the DNA helix lengthens as base pairs are

separated to accommodate the binding ligand, which, in

turn, leads to an increase in the viscosity of DNA. In Fig. 5,

the changes in viscosity for rod-like DNA is shown in the

presence of complex 1, [Ru(bpy)3]2? and EB. Where EB, a

well-known DNA intercalator, gives rise to a strong change

in DNA viscosity upon complexation; [Ru(bpy)3]2?, which

binds by electrostatic interactions only, exerted essentially

no such effect. As can be seen from Fig. 5, upon increasing

the amounts of the title complex, the relative viscosity of

DNA increases steadily, which is similar to the case of EB.

The increase in relative viscosity, which is expected to

correlate with the compound DNA-intercalating potential,

followed the order EB [ complex 1 [ [Ru(bpy)3]2?. The

0

50

100

150

200

250

300

0.0 0.1 0.2 0.31.0

1.2

1.4

1.6

1.8 Ksv

= 3.12

I 0 / I

[Ru]/[RNA]

Wavelength (nm)

Inte

nsity

(a.

u.)

500 550 600 650 700 750 8000

100

200

300

400

500

600

700

0 .0 0 0 .0 8 0 .1 6 0 .2 4 0 .3 2

1 .0 8

1 .1 7

1 .2 6

1 .3 5K

s v = 1 .7 8

I 0/I

[R u ]/ [D N A ]

Wavelength (nm)

Inte

nsity

(a.

u.)

500 550 600 650 700 750 800

(a)

(b)

Fig. 4 Emission spectra of EB bound to CT-DNA (a) or yeast tRNA

(b) in presence of complex 1 in Tris–HCl buffer (pH 7.2) at 25 �C.

[EB] = 20 lM, [Nucleic acid] = 100 lM (Nucleic acid = CT-DNA

or yeast tRNA), [Ru]/[Nucleic acid] = 0.05, 0.10, 0.15, 0.20, 0.25.

The arrows show the intensity changes upon increasing concentra-

tions of complex 1. Inset: fluorescence quenching curve of Nucleic

acid-bound EB by complex 1

J IRAN CHEM SOC (2012) 9:357–366 363

123

result suggests that complex 1 binds to DNA through a

classical intercalation model. However, viscosity cannot be

used to study the interaction between yeast tRNA and

metal complexes, since yeast tRNA is not a linear structure.

In fact, no obvious change in viscosity was observed for

yeast tRNA in the presence of complex 1.

Reverse salt titration

At neutral pH, Ru(II) complexes have a dipositive charge,

and it may therefore be expected that the interaction

between the Ru(II) complex and nucleic acids would be

influenced by such factors as the presence of other cations

or the ionic strength of the solution. In general, binding of

Ru (II) complexes to nucleic acids may lead to the release

of sodium ion (Na?) from the nucleic acid. The sensitivity

to ionic strength (Na?) is expected to decrease in the order

of the binding modes: electrostatic [ groove-binding [intercalative. Herein, because complex 1 is a dication, its

binding to DNA and RNA is thermodynamically linked to

Na? binding to DNA and RNA. As a result, the DNA and

RNA binding affinity will depend on the total Na? con-

centration. Therefore, the effect of increasing concentra-

tions of NaCl on the absorption spectra was tested.

Figure 6 shows the dependence of the absorbance spectra

of complex 1 on the concentration of Na? as determined by

reverse salt titrations. It is clear from this diagram that the

absorbance spectra of complex 1 decrease with increasing

salt concentration. This is due to a stoichiometric amount of

counterion release that accompanies the binding of a

charged ligand [42]. In the presence of CT-DNA, with

increasing concentrations of NaCl from 0.050 M to 0.18 M,

the IL band at 279 nm exhibits a hypochromism as high as

17.3% with 2 nm red shift at a ratio [NaCl]/[Ru] of 17.8.

However, in the presence of yeast tRNA, with increasing

concentrations of NaCl from 0.050 to 0.18 M, the IL band at

279 nm exhibits a hypochromism of about 11.6% with a

1-nm red shift at a ratio [NaCl]/[Ru] of 12.3. From these

result results we may conclude that the complex 1–yeast

tRNA interaction is not as sensitive to ionic strength,

implying that complex 1 binds more tightly to yeast tRNA

in a potentially intercalative mode.

0.00 0.02 0.04 0.06 0.081.00

1.05

1.10

1.15

1.20

1.25

1.30(η

/η0)1/

3

[RU]/[DNA]

Fig. 5 Effect of increasing amounts of ethidium bromide (filledcircle), [Ru(bpy)3]2? (filled square), the complex 1 (filled triangle),

on the relative viscosity of calf thymus DNA in Tris–HCl buffer (pH

7.2) at 28 ± 0.1 �C. Total DNA concentration 0.5 mM

320 400 480 560 6400.0

0.2

0.4

0.6

0.8

1.0

17.3%

Abs

orba

nce

wavelength (nm)

0.0

0.2

0.4

0.6

0.8

1.0 11.6%

Abs

orba

nce

wavelength (nm)320 400 480 560 640

(a)

(b)

Fig. 6 The effects of successive additions of NaCl ([NaCl] =

0.05 - 0.18 M) on UV–vis spectra of complex 1 ([Ru] = 2.0 9

10-5 M) in the presence of CT-DNA (a 3.0 9 10-5 M) and yeast

tRNA (b 3.9 9 10-5 M) in Tris–HCl buffer (pH 7.2)

364 J IRAN CHEM SOC (2012) 9:357–366

123

Enantioselective binding

Equilibrium-dialysis experiments may offer the opportu-

nity to examine the enantioselectivity of complexes bind-

ing to nucleic acids. According to the proposed binding

model by Barton and co-workers [29], the D enantiomer of

the complex, a right-handed propeller-like structure, dis-

plays a greater affinity than the K enantiomer with the

right-handed CT-DNA helix due to more- appropriate

steric matching. We, thus, decided to test if the racemic

complexes could be (partly) resolved in the presence of

chiral CT-DNA or yeast tRNA. To this end, racemic

solutions of complex 1 were dialyzed against CT-DNA or

yeast tRNA for 20 h, and then subjected to circular-

dichroism (CD) analysis. During the course of the dialysis

at certain time intervals, the CD signals started from none,

increased to the maximum magnitude after 16 h dialysis of

complex 1, then no longer changed. That is to say, the final

CD signals of complex 1 neither increased nor decreased.

The CD spectra in the region of 230–350 nm for complex 1

after its racemic solution had been dialyzed against

CT-DNA and yeast tRNA are shown in Fig. 7. As can be

seen from Fig. 7, the dilysates of 1 (solid line) dialyzed

against CT-DNA shows two CD signals with a positive

peak at 295 nm and a negative peak at 276 nm, while

complex 1 (doted line) the dilysates of 1 (solid line) dia-

lyzed against yeast tRNA shows weak CD signals with a

positive peak at 269 nm and a negative peak at 297 nm,

respectively. The stronger CD signals of complex 1 dia-

lyzed against yeast tRNA suggest that complex 1 binding

with yeast tRNA shows more enantioselectivity than that

with CT-DNA. Therefore, for yeast tRNA, complex 1 is a

better enantioselective binder.

Cytotoxicity assay

Cytotoxicity tests were performed using the MTT assay,

following the exposure of four tumor cell lines HeLa

(cervical), HepG-2 (hepatocellular), BEL-7402 (hepato-

cellular) and MCF-7 (breast cancer) to the synthesized

complexes at increasing concentrations for 48 h. Because

the solubility of the compounds is limited in water, a

DMSO stock solution is used for all compounds to perform

a proper comparison among the complexes, and blank

samples containing the same amount of DMSO are taken as

controls. Cis-Pt(NH3)2Cl2 has been included as the control,

and it shows high cytotoxicity, which is in accordance with

the literature reports [42]. Table 1 demonstrates the IC50

values obtained from non-linear regression analysis of dose

response data for complex 1 tested. As shown in Table 1,

complex 1 displayed difference in the antitumor activity

against the tumor cells tested. The HeLa cells are more

sensitive to complex 1 than the other tumor cell lines, the

cytotoxicities of complex 1 against MCF-7, Hep-G2 and

BEL-7402 are rather moderate, with IC50 values ranging

between 24 and 38 lM, which are higher than those of cis-

Pt(NH3)2Cl2, meaning that the complex 1 is less effective

in terms of tumoricidal effect as compared to cis-

Pt(NH3)2Cl2, but considering that Ru(II) drugs are char-

acterized by their low toxicities, the prospect of clinical

applications still exists for complex 1. It is well known that,

due to presence of leaving group (chloride ion) in cis-

Pt(NH3)2Cl2, its cytotoxic effects are exerted through

covalent binding to DNA thereby forming cis-DDP-DNA

adducts, which interfere with DNA replication and tran-

scription and ultimately induces cell death. However, no

leaving group exists in complex 1. Therefore, the antitumor

240 260 280 300 320 340

-6

-3

0

3

6

9

Elli

ptic

ity /

(°m

)

Wavelength (nm)

Fig. 7 CD spectra of complex 1 after 16 h of dialysis against

CT-DNA (dotted line) and yeast tRNA (solid line) in stirred in

Tris–HCl buffer (pH 7.2) at 25 �C. [Ru] = 20.0 lM, [Nucleic

acid] = 1.0 mM (Nucleic acid = CT-DNA or yeast tRNA)

Table 1 IC50 (lM) of complex 1 (10-4 M) and drug against different tumor cell at 25 �C

Complexes IC50 (lM)

HeLa Hep-G2 BEL-7402 MCF-7

1 18.06 ± 5.41 32.37 ± 5.30 24.27 ± 4.01 37.30 ± 4.54

cis-Pt(NH3)2Cl2 5.85 ± 0.6 11.18 ± 2.48 17.15 ± 3.45 35.07 ± 4.52

Each IC50 is the mean ± standard error obtained from at least three independent experiments

J IRAN CHEM SOC (2012) 9:357–366 365

123

mechanism of complex 1 may not occur with the DNA

cross-linking reaction, further studies in detail are currently

underway to determine the antitumor mechanism of com-

plex 1.

Conclusions

In conclusion, the novel complex of [Ru(IP)2DPBPD

(NH2)2]2? has been synthesized and characterized. The

results suggest that the binding mode of the complex 1 to

CT-DNA and yeast tRNA is through intercalation, RNA

binding of the complex is stronger than DNA binding; the

D enantiomer of the complex may bind more favorably to

both CT-DNA and yeast tRNA, and complex 1 is a better

candidate for an enantioselective binder to yeast tRNA than

to CT-DNA. The results can be explained by the different

structure and configuration between CT-DNA and yeast

tRNA reasonably, suggesting that the configuration and

structure of nucleic acids have significant effects on the

binding behaviors of metal complexes. The cytotoxicity of

the complex has been evaluated by MTT assay, which

suggests that the human HeLa cells are more sensitive to

complex 1 than the other cells in vitro. Altogether, the

information obtained from the present study may be of

value in further understanding the mechanism of the

binding of metal complexes to nucleic acids. This in turn

may help the development of nucleic acid molecular probes

and new therapeutic reagents for some diseases.

Acknowledgments The authors are grateful to the supports of the

National Natural Science Foundation of the People’s Republic of

China (21071120) and the Scientific Research Foundation of Hunan

Provincial Education Department (11A117).

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