Journal of Inorganic Biochemistry 94 (2003) 263–271www.elsevier.com/ locate/ jinorgbio

D NA binding of iron(II) complexes with 1,10-phenanthroline and4,7-diphenyl-1,10-phenanthroline: salt effect, ligand substituent effect,

base pair specificity and binding strengtha a b b ,*Mudasir , Karna Wijaya , Naoki Yoshioka , Hidenari Inoue

aDepartment of Chemistry, Gadjah Mada University, Sekip Utara, P.O. Box Bls. 21, Yogyakarta 55281,IndonesiabDepartment of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

Received 18 August 2002; received in revised form 14 November 2002; accepted 17 December 2002

Abstract

The DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline(phen) and 4,7-diphenyl-1,10-phenanthro-21 21 21line(dip), [Fe(phen) ] , [Fe(phen) (dip)] and [Fe(phen)(dip) ] has been characterized by spectrophotometric titration and melting3 2 2

temperature measurements. The salt concentration dependence of the binding constant has allowed us to dissect the DNA-binding constantand free energy change of each iron(II) complex into the nonelectrostatic and polyelectrolyte contributions. A comparison of thenonelectrostatic components in the binding free energy changes among iron(II) complexes has made it possible to rigorously evaluate thecontribution of the ligand substituents to the DNA-binding event. The peripheral substitution of phen by two phenyl groups increases the

21nonelectrostatic binding constant of the iron(II) complex more than 20 times, which is equivalent to approximately 7.5 kJ mol of morefavorable contribution to the DNA binding. In general, the iron(II) complexes studied have higher affinity towards the more facile A–Tsequence than the G–C sequence. This preferential binding may be attributed to the steric effect induced by the ancillary part of theligands in the course of DNA binding. The binding of disubstituted iron(II) complex to DNA is quite strong as reflected in the modestincrease in the denaturation temperature (T ) of double helical DNA upon the interaction with the iron(II) complex.m

2003 Elsevier Science Inc. All rights reserved.

Keywords: Mixed-ligand complex; DNA-interaction; 1,10-Phenanthroline; Iron(II)

1 . Introduction bipyridine) and modified phen such as 4,7-diphenyl-1,10-phenanthroline (dip) [18–20] and dipyrido[3,2-a:29,39-

In the last decade the binding of small molecules to c]phenazine (dppz) [4–7,21–24]. The modified phen lig-DNA has been extensively investigated to develop novel ands are designed to achieve more effective bindingprobes of DNA structure [1–4], new therapeutic agents affinity of the complexes to DNA [23,24].which can recognize or cleave DNA [5–10] and DNA- In spite of this well-documented importance of the DNAmediated electron transfer reactions [11–13]. Metal com- binding of mixed-ligand complexes containing phen and its

n1plexes of the type [M(LL) ] , where LL is either 1,10- modified ligands, the DNA-binding studies of mixed-lig-3

phenanthroline (phen) or modified phen, are particularly and complexes having a central metal ion other thanattractive because they can effectively bind to DNA in ruthenium(II) ions, especially iron(II), have attracted muchdifferent modes of interactions [14–17]. The central metal less attention [2–6,25]. An exception to this are studiesor the ligands in these complexes may be varied in an reported by Norden and co-workers in which they thor-easily controlled manner to facilitate a certain application, oughly investigated ‘Pfeiffer effect’ induced by the inter-which provides an easy access for the detailed study of action of iron(II) complexes of bpy and phen with doubleDNA-binding mechanisms [5]. In recent years, much helical DNA [26–28]. In fact, further detailed studies usinginterest has been focused on the DNA binding of mixed- various central metal ions are needed to explore theligand complexes which contain both phen (or bpy52,29- influence of geometry, charge, spin state, redox potential,

etc., on the DNA binding of the mixed-ligand complexes.Moreover, studies of the interaction of metal complexes*Corresponding author. Fax:181-45-566-1551.

E-mail address: inoue@applc.keio.ac.jp(H. Inoue). with DNA have, to date, been concerned largely with

0162-0134/03/$ – see front matter 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0162-0134(03)00007-2

264 Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271

establishing their mode of the binding and with possible DNA, i.e.: poly[(dA–dT) ] and poly[(dG–dC) ] to eluci-2 2

structure of their DNA complexes. Apart from the de- date the sequence preference of the DNA binding. Thetermination of binding constants, the thermodynamics of DNA binding properties have been further investigated bytheir DNA binding, which is essential for a thorough thermal denaturation experiments of DNA, which provideunderstanding of the principle that governs the binding additional support for the DNA-binding strength of theevent, has not been studied in any detail. In addition to the mixed-ligand complexes. On the basis of the experimentalDNA binding of porphyrin and metalloporphyrin [29,30], data presented, thermodynamics, base-pair specificity andwe have recently reported the non-covalent interaction of strength of the DNA binding of the iron(II) mixed-ligandiron(II) mixed-ligand complexes with DNA [19,20]. The complexes have been discussed in detail.main purpose of the present work is to determine thecomplete thermodynamic profiles for the interaction of

21 21 21Fe[(phen) ] , Fe[(phen) (dip)] and Fe[(phen)(dip) ]3 2 2

2 . Experimental(Fig. 1) with DNA. Here, we have determined the bindingconstant of the interaction of each iron(II) complex with

2 .1. Chemicalscalf thymus DNA using spectrophotometric titration tech-niques and have examined the salt concentration depen-

Tris(phen)iron(II) perchlorate [Fe(phen) ](ClO ) wasdence of the binding constant. From the salt concentration 3 4 2

synthesized according to the literature procedure [31] anddependence of the DNA binding, it has been demonstratedconfirmed by elemental analysis and UV–Vis absorptionthat the polyelectrolyte theory enables us to separate thespectroscopy. Iron(II) mixed-ligand complexes, i.e.,free energy changes in the DNA binding into the nonelec-[Fe(phen) (dip)](ClO ) and [Fe(phen)(dip) ](ClO )trostatic and polyelectrolyte contributions and then to use 2 4 2 2 4 2

were prepared from tris-(phen)iron(II) perchlorate bythe nonelectrostatic part of the binding free energy changeligand substitution reaction as reported previously [19].to evaluate the effect of a specific substituent on theThe mixed-ligand complexes are very stable in the solidbinding free energy change. The advantage of this strategystate and show no changes in their absorption spectra asis that the influence of ligand charges on the binding freewell as their HPLC retention time (t ) in Tris–HCl bufferenergy change can be ruled out so that the comparison of R

solution even after 3 days of dissolution. This suggests thatthe binding free energy change is purely based on thewater molecules do not permanently substitute for phendifferences in substituents attached to the ligand of theand dip ligands. The concentration of iron(II) complexesmixed-ligand complexes. In addition, we have also de-for DNA-binding studies was determined spectrophoto-termined the binding constants of the interaction of

21 21 metrically using the molar absorptivity (´) at their metal-Fe[(phen) (dip)] and Fe[(phen)(dip) ] with synthetic2 2

to-ligand charge transfer (MLCT) bands:´ 511 900510 nm21 21 21 21 21M cm for [Fe(phen) ] ,´ 516 500 M cm3 517 nm

21 21 21for [Fe(phen) (dip)] and́ 519 100 M cm for2 525 nm21[Fe(phen)(dip) ] [19]. Tris buffer (2-amino-2-hydroxy-2

methyl-1,3-propandiol) was purchased from Junsei Chemi-cal (Tokyo, Japan) and sodium chloride (NaCl) for adjust-ing ionic strength was from Wako Pure Chemical Indus-tries (Japan). All chemicals and solvents were of analyticalgrade or higher and were used without further purification.

2 .2. DNA samples

Calf thymus DNA (CTDNA), poly[(dG–dC) ] and2

poly[(dA–dT) ] were obtained from Sigma (USA) and2

used as received. The solid sodium salts were stored below4 8C. A stock solution of CTDNA, poly[(dG–dC) ] and2

poly[(dA–dT) ] was prepared and stored in 5 mM Tris–2

HCl buffer at pH 7.2 (unless otherwise stated). A molar4 21 21absorptivity of ´ 51.31310 M cm , ´ 51.683260 254

4 21 21 4 21 2110 M cm and´ 51.32310 M cm was used262

to spectrophotometrically determine the concentrations of21 CTDNA [32], poly[(dG–dC) ] [33] and poly[(dA–dT) ]2 2Fig. 1. Chemical structures of iron(II) complexes: (1) [Fe(phen) ] , (2)3

21 21 [34] and the results were expressed in terms of base-pair[Fe(phen) (dip)] , (3) [Fe(phen)(dip) ] ; onlyL-enantiomers are2 2

viewed. equivalents per 1000 ml.

Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271 265

2 .3. Measurements of salt effect in DNA binding in our cases because we are working at the lower values ofR, i.e., 0.5–0.08, where the concentration of the iron(II)

25The binding equilibrium constant (K ) of each iron(II) complexes is kept constant at around 10 M. The saltb

complex to CTDNA was determined by spectrophoto- concentration dependence of the DNA binding of themetric titration over a range of NaCl concentrations of iron(II) complexes was then evaluated by plotting logKb

15–120 mM. A fixed amount of each iron(II) complex in 5 versus log [Na ]. Each measured point was the averagemM Tris–HCl buffer at pH 7.2 and in various con- value of at least three separate measurements with acentrations of NaCl was titrated with increasing amounts relative standard deviation (RSD) of less than 15%.

26 24of DNA stock solutions (10 –10 M) and the hypo-chromicity in the MLCT band due to complex-DNA 2 .4. Measurements of base pair specificity in DNAinteraction was monitored by a Jasco V-550 UV–Vis bindingspectrophotometer equipped with a Jasco ETC-505T cell-temperature controller and a cell magnetic stirrer. Cell The base-specific binding of the iron(II) complexes tocompartments were thermostated at 2560.18C. The K DNA was determined by evaluating the value of theb

values at various NaCl concentrations were calculated on equilibrium binding constant (K ) of each iron(II) com-b

the basis of the following equation [18,20]: plex. For such a purpose, the spectrophotometric titrationof each iron(II) complex using CTDNA and synthetic

[DNA] /(´ 2´ )5 [DNA] /(´ 2´ )1 1/K (´ 2´ ) oligonucleotides, poly[(dA–dT) ] and poly[(dG–dC) ],A F B F b B F 2 2

was performed in the Tris–HCl buffer at pH 7.2 and 50(1)mM NaCl at 258C and theK values were calculated byb

where ´ , ´ and ´ correspond toA / [complex], the Eq. (1). The differences inK values brought about by theA F B obsd b

molar absorptivity for the free iron(II) complex, and the interaction of each iron(II) complex with different kinds ofmolar absorptivity of the iron(II) complex in the fully DNA are associated with the selectivity of the iron(II)bound form, respectively. In plots of [DNA] /(´ 2´ ) complexes in their DNA binding. All data of theK valuesA F b

versus [DNA],K is given by the ratio of the slope to the obtained were the average values of three repeated mea-b

intercept. It should be noted that when ligands bind to surements and their RSD values are normally less thanDNA there might be a multiplicity of strengths of binding 15%.as the binding of the second ligand is affected by the first,especially in the concentration range where the ratioR 2 .5. Measurements of denaturation temperature(5[complex] / [DNA]) is high. The linearity of our plots(see Fig. 2) indicates that such phenomenon does not occur The melting of polynucleotide strand from a double

helical DNA manifests itself as absorption hyperch-romicities in the 260-nm wavelength region. The meltingtemperature (T ) of polynucleotide is generally increasedm

upon addition of DNA binders due to the stabilization ofthe duplex structure. TheT of CTDNA in the presencem

and absence of each iron(II) complex atR ([complex] /[DNA]) 50.5 was measured by a Jasco V-550 UV–Visspectrophotometer equipped with a Jasco ETC-505T cell-temperature controller and a cell magnetic stirrer. Thebuffer solution containing a mixture of iron(II) complexand CTDNA was stirred continuously and the temperaturewas elevated gradually from 25 to 958C at a speed of1 8C/min with a reading of absorbance taken automaticallyevery 10 s. The denaturation temperature (T ) was takenm

as the mid-point of the hyperchromic transition. Allmeasurements ofT were repeated three times for eachm

complex and the data presented are the average values withan RSD of lower than 5%.

3 . Results and discussion

Fig. 2. Typical plots of [CTDNA]/(́ 2´ ) versus [CTDNA] for theA F21 3 .1. Salt concentration dependence of DNA bindingspectrophotometric titration of [Fe(phen) (dip)] with increasing2

amounts of [CTDNA] in 5 mM Tris–HCl buffer (pH 7.2) containingvarious concentrations of NaCl at 258C. The typical plots of [CTDNA] versus [CTDNA]/(́ 2A

266 Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271

on the binding ligand,g is the mean activity coefficient at61a cation concentration of [M ], and the remaining terms

are constants for double-stranded B-form DNA,j54.2oandd50.56. In fact, the magnitude ofK indicates to whatt

extent the nonelectrostatic forces stabilize the ligand–DNAocomplex. Since theK is a parameter independent of saltt

concentrations, its value should be constant regardless ofthe concentration of NaCl. Our calculated results presented

oin Table 1 are consistent with the expectation that theK t

are constant within our experimental errors throughout theconcentration of NaCl. It is also noteworthy that thenonelectrostatic contribution to the overall binding con-

1stant is relatively small, i.e., less than 20% at [Na ]5

0.120 M. This suggests that the DNA-binding constants ofthe iron(II) complexes are largely resulting from electro-static interactions.

Based on the polyelectrolyte theory, one may dissect thebinding free energy change at a given concentration of

Fig. 3. Salt concentration dependence of CTDNA-binding constants for NaCl into its nonelectrostatic and polyelectrolyte contribu-iron(II) complexes. The slopes of these plots correspond to the quantity

tions [37]. Table 2 summarizes the binding constantsSK listed in Table 2.obtained in 40 mM NaCl along with the salt concentration

1dependence of the binding constants (d lnK /d ln [Na ])b

´ ) for the determination of binding constants (K ) at obtained by linear regression of the data in Fig. 3. TheF b

various concentrations of NaCl and the salt concentration binding constants (K ) listed in Table 2 were used tobodependence of the interaction between iron(II) complexes calculate the total free energy change (DG ) based on the

and CTDNA are shown in Figs. 2 and 3, respectively. Fig. standard relation3 illustrates that the DNA-binding constant of each iron(II)

oDG 5 2RT ln K (3)bcomplex decreases with the concentration of sodium ions.

The binding constants were obtained in the concentrationwhere R is the gas constant andT is the temperature in

range from 5 to 120 mM NaCl to evaluate the contributionKelvin. Logarithmic plots of the binding constant lnKo bof the nonelectrostatic binding constant (K ) to the overall 1t versus ln [Na ] give the slope, SK:

binding constant (K ) in terms of the polyelectrolyte theorybo

1(Table 1). The values ofK were calculated at varioust SK 5 d ln K /d ln[Na ] (4)b1concentrations of NaCl ([M ]) based on Eq. (2) [35,36].where the value ofSK is equal to the absolute value ofZc

o 21 1ln K 5 ln K 1 Zj hln(g d )j1Zc(ln[M ]) (2) in Eq. (2). In the previous studies [37,38], theSK is usedb t 6

to calculate the polyelectrolyte contribution of the freeowhere Zc is the negative of the slope obtained from the energy change (DG ) to the overall free energy changepe

1 oplots of lnK versus ln[M ] (Fig. 2),c is the fraction of (DG ) at a given NaCl concentration by the relation:b

counterions associated with each DNA phosphate (c50.88o 1for double-stranded B-form DNA),Z is the partial charge DG 5 (SK)RT ln[Na ] (5)pe

Table 1a,bBinding constants of iron(II) mixed-ligand complexes to calf thymus DNA in 5 mM Tris–HCl buffer (pH 7.2) and various concentrations of NaCl

21 21 21[NaCl] /M [Fe(phen) ] [Fe(phen) (dip)] [Fe(phen)(dip) ]3 2 2

o o oK K K K K Kb t b t b t23 21 23 21 23 21 23 21 23 21 23 21(310 ) /M (310 ) /M (310 ) /M (310 ) /M (310 ) /M (310 ) /M

0.005 6.03 0.042 (0.700) 274.5 0.84 (0.306) 133.6 1.40 (1.05)0.020 1.43 0.039 (2.73) 60.57 0.92 (1.52) 40.95 1.51 (3.69)0.040 0.753 0.041 (5.44) 26.58 0.90 (3.39) 19.26 1.33 (6.91)0.080 0.441 0.048 (10.9) 11.74 0.89 (7.58) 11.31 1.49 (13.2)0.120 0.254 0.042 (16.5) 7.726 0.95 (12.3) 8.146 1.57 (19.3)

a K is the binding constant per DNA base pair and determined according to Eq. (1). All figures are average values of at least three separatebomeasurements with an RSD of less than 15%.K is the nonelectrostatic contribution to the overall binding constant and calculated based on Eq. (2). Thet

values ofZ used are as follows; 1.10 (tris-phen complex), 1.28 (mono-substituted complex) and 1.01 (di-substituted complex). All values are at 258C.b oThe values in parentheses represent the percentages of the non-electrostatic binding constant contribution (K ) to the overall binding constants (K ).t b

Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271 267

Table 2a,bComparison of thermodynamic parameters in CTDNA binding of iron(II) mixed-ligand complexes and other ligands

3 o o o 3 oBinding K /10 DG SK DG K /10 DGb pe t t21 21complex M bp M bp

21[Fe(phen) ] 0.753 216.3 0.97 27.9 0.041 (5.4) 28.40 (51.5)321[Fe(phen) (dip)] 26.6 225.1 1.1 29.2 0.90 (3.4) 215.9 (63.3)221[Fe(phen)(dip) ] 19.3 224.7 0.89 27.1 1.33 (6.9) 217.6 (71.3)2

bEthidium 494 232.2 0.75 25.0 61 (12.3) 227.2 (84.5)bDaunomycin 4900 237.7 0.84 25.9 422 (8.6) 231.8 (84.4)

21cD-[Ru(bpy) (ppz)] 2.80 219.7 1.20 28.8 0.10 (3.6) 210.9 (55.3)2

21cL-[Ru(bpy) (ppz)] 11.0 223.0 1.21 28.8 0.37 (3.4) 214.2 (61.7)2

a o 21In 40 mM NaCl, 5 mM Tris buffer (pH 7.2) at 258C. DG (kJ mol ) is the binding free energy change calculated from Eq. (3). The parameterSK iso o 21the absolute value of the slope obtained from the plots of Fig. 3.DG andDG (kJ mol ) are the polyelectrolyte and the ‘non-electrostatic’ contributions,pe t

1respectively. The polyelectrolyte contribution was calculated using Eq. (5) and evaluated at [Na ]540 mM. The ‘non-electrostatic’ portion of the freeenergy change was calculated by the difference.

b Taken from Ref. [39]; all values refer to the solution of 50 mM NaCl, 5 mM Tris buffer (pH 7.1) at 208C.c oTaken forK , SK andK from Ref. [36] and referred to the solution of 50 mM NaCl, 5 mM Tris buffer (pH 7.2) at 258C; other values were calculatedb t

o oas for iron(II) complexes: bpy, bipyridine; ppz, 4,7-phenanthrolino[6,5-b]-pyrazine. The values in parentheses forK and DG correspond to thet topercentage of the non-electrostatic contribution to the overall binding constants (K ) and free energy changes (DG ), respectively.b

o oThe difference betweenDG andDG is defined as the values. Such smaller values are rather common for thepeo‘nonelectrostatic’ free energy change (DG ): metal complexes of these types as can be seen in Table 2t

for the ruthenium(II) complexes with bpy and ppz [36].o o o

DG 5DG 2DG (6)t pe Moreover, the values ofSK51.38 and 1.24 also have been21reported for the interaction ofD-[Ru(phen) ] andL-3

o 21The quantityDG corresponds to the portion of the [Ru(phen) ] with calf thymus DNA [39]. The smallert 3

binding free energy change, which is independent of salt SK values observed could arise from coupled anion releaseconcentrations and contains a minimal contribution from from the ligand or from changes in the ligand or DNApolyelectrolyte effects such as coupled ion release. It is a hydration upon binding.useful parameter for the comparison of the effect ofdifferent substituents attached to the binding ligand on the 3 .2. Effect of ligand substituents on DNA bindingDNA-binding event because it contains only a smallcontribution from the charge of the binding ligand [37]. Since both binding constants and free energy changes inAlthough the iron(II) complexes studied have formally the the interaction of iron(II) complexes with calf thymussame charge, the local or partial charges involved in the DNA have been dissected into their electrostatic andinteraction are not exactly the same due to the difference in ‘nonelectrostatic’ contributions, the effect of ligand sub-structure and size of the iron(II) complexes. Therefore, the stituents on the binding event may be evaluated by

o o o opartitioning of K and DG values into the electrostatic comparing the quantities ofK and DG in the DNAt t t t

and nonelectrostatic contribution is necessary for a com- binding of the iron(II) complexes. A comparison of ligando oparison of the effect of certain substituents on the DNA substituent effects usingK andDG is more appropriatet t

binding event under the minimized influence of the ligand than that using overall binding constants and total freeo ocharge. energy changes becauseK and DG are free fromt t

Eqs. (3)–(6) were used to divide the binding free energy polyelectrolyte contributions and can be assumed as purelychange into its nonelectrostatic and polyelectrolyte contri- resulting from ligand substituent effects. Moreover, the

o o obutions. All the values ofSK, DG , DG andDG derived polyelectrolyte contribution of the iron(II) complexest pe

from experimental data shown in Fig. 3 are listed in Table studied varies considerably on going from one iron(II)o2 along with other data for ethidium, daunomycin [39] and complex to another as reflected in theirDG values, e.g.,pe

21the ruthenium complexes [36] for the purpose of com- from 7.1 to 9.2 kJ mol in 40 mM NaCl (see Table 2),parison. TheSK data shown in Table 2 indicate that both although the net charge of the iron(II) complexes isethidium and daunomycin carry a charge of approximately theoretically12. This suggests that two kinds of DNA-11, which is consistent with the known structures of these binders with the same charge do not always make the samecompounds. On the other hand, the expected value ofSK polyelectrolyte contribution to the binding event. There-

ofor all the iron(II) complexes would be (12)(0.88)51 fore, here we use the more appropriate parametersK andto1.76 because they theoretically have a net charge of12 at DG to evaluate ligand substituent effects on the bindingt

neutral pH. It is not surprising that ourSK values for the event.binding of the iron(II) complexes listed in Table 2 are The striking observation emerging from the data pre-

o omuch the same although they are smaller than the expected sented in Table 2 is that bothK and DG increaset t

268 Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271

significantly as the phen ligand is substituted by the dip 3 .3. Base pair specificity in DNA bindingligand. The substitution of one phen by dip ligand, e.g., on

21 21 In order to gain more insights into the possibility ofgoing from [Fe(phen) ] to [Fe(phen) (dip)] , gives rise3 2o 21 1 specific binding of iron(II) complexes to various DNA,to the increase inK from 40 M for [Fe(phen) ] tot 3

2 21 21 spectrophotometric titration using synthetic oligonucleo-9.0310 M for [Fe(phen) (dip)] or more than 20-2o tides poly[(dA–dT) ] and poly[(dG–dC) ], in addition tofold. This increase inK is comparable to approximately 2 2t

21 native DNA (CTDNA), has been carried out for7.5 kJ mol of free energy change and more favorable for21 21[Fe(phen) (dip)] and [Fe(phen)(dip) ] . Fig. 4 illus-DNA binding. In other words, the substitution of two 2 2

trates an example of the absorption spectral traces forhydrogen by phenyl substituents in the phen ligand makes21[Fe(phen) (dip)] in the metal-to-ligand charge transferthe DNA binding of the iron(II) complex more favorable 2

o (MLCT) band upon increasing addition of poly[(dA–dT) ](more negative inDG ), as compared to that of the Tris 2t21 o in 5 mM Tris–HCl buffer and 50 mM NaCl at pH 7.2 andcomplex [Fe(phen) ] . Such a large increase inK and3 t

o 25 8C. The MLCT bands of both iron(II) complexesDG is probably attributed to the fact that the phenyl grouptexperience hypochromism and red shift (ca. 2–3 nm foris a planar aromatic substituent with delocalizedp-elec- 21 21[Fe(phen) (dip)] and ca. 6–9 nm for [Fe(phen)(dip) ] )2 2trons and hence there are more facilities for the DNAin the presence of the synthetic oligonucleotides, sug-binding via, for example, hydrophobic interaction andp–pgesting that these complexes bind effectively to both kindselectron stacking. The substitution by another dip ligand as

21 of synthetic DNA.in [Fe(phen)(dip) ] consistently increases the value of2 The typical plots of [DNA] versus [DNA]/(́ 2´ ) areo 2 3 21 A FK from 9.0310 to 1.33310 M and thus stabilizest shown for the titration of poly[(dA–dT) ] and poly[(dG–21 2the DNA binding by 1.7 kJ mol , although the effect isdC) ] with the iron(II) complexes in Figs. 5 and 6,2not linear. This non-linearity is probably due to the morerespectively. The plots were constructed according to Eq.

complicated structure of the mixed-ligand complexes so(1), affording the ratio of the slope to the intercept, i.e.,

that steric effect plays a prominent role in the DNA- binding constant (K ). The K determined for the bindingb bbinding process. It is also noteworthy that the racemic of the iron(II) complexes to synthetic oligonucleotides aremixtures of iron(II) complexes investigated are relatively summarized in Table 3. In view of theK values obtained,blabile towards racemization and upon binding to DNA, it is concluded that the iron(II) complexes investigated,

21diastereomeric shift occurs in the solution, resulting in an especially [Fe(phen)(dip) ] , have higher affinity towards2enrichment of one enantiomer which is more favorable to the adenine and thymine (A–T) pairs of DNA. Similarthe DNA binding [20,26–28]. It could be anticipated that specificity to the A–T pairs has been also observed for

1the bulkier and more hydrophobic complex such as [Cu(bcp) ] (bcp5bathocuproine52,9-dimethyl-4,7-221[Fe(phen)(dip) ] may undergo slower inversion upon2

binding to DNA and therefore the smaller the increase inoK .t

A further examination of Table 2 reveals that theocontribution of the nonelectrostatic binding constant (K )t

to the overall binding constant (K ) is relatively small evenb

in common intercalators such as ethidium anddaunomycin, i.e., 3.4–12.3% ofK . In contrast, such ab

osmall contribution ofK results in a large contributions tot

the total free energy changes in the DNA binding. Asoshown in Table 2, the large contribution toDG rangest

21from 51.5% in [Fe(phen) ] to 84.5% in ethidium and3odaunomycin. In case of theDG for the DNA binding oft

o othe iron(II) complexes, the contribution ofDG to DGt

increases consistently about 10% with the number of thedip ligand in the iron(II) complexes, e.g., 51.5% in

21 21[Fe(phen) ] and 71.3% in [Fe(phen)(dip) ] . The large3 2o ocontribution ofDG to DG suggests that in the moleculart

design of a more stable DNA binder we should take intoaccount not only the charge of the binding ligands but also

Fig. 4. Variation in metal-to-ligand charge transfer spectra of 12mMthe modification of the substituents of the binding ligands 21[Fe(phen) (dip)] upon increasing addition of poly[(dA–dT) ] in 5 mM2 2because the charge of the DNA binder makes but a smallTris–HCl buffer (pH 7.2) and 50 mM NaCl at 258C. The concentrationscontribution to the stabilization energy of the DNA of poly[(dA–dT) ] added were 0.0, 1.2, 2.4, 4.8, 7.2, 10.7, 15.4, 20.4,2

binding. 35.4, 49.4, 65.7, 81.8 and 97.8mM.

Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271 269

21diphenyl-1,10-phen) [40], [Ru(phen) ] and31[Re(py)(CO) (dppz)] (py5pyridine) [6]. This preferen-3

tial binding to poly[(dA–dT) ] can be explained by taking2

into account that propeller twisting of the base pairs isrelatively facile for an A–T sequence and that the ligandsize of the iron(II) complexes becomes larger on goingfrom tris- to disubstituted complexes. If it is the only case,the level of specificity towards the A–T sequence wouldnot differ between the two complexes significantly. Sur-

21prisingly, while [Fe(phen) (dip)] exhibits only 1.5-fold2

higher affinity towards the A-T sequence than towards theguanine–cytosine (G–C) sequence, in contrast

21[Fe(phen)(dip) ] shows a remarkable level of specificity2

towards the A–T sequence, i.e., approximately 12-foldspecificity compared with that towards the G–C sequence.Therefore, there must be structural differences in theirinteraction with DNA between these two iron(II) complex-es. In other words, this phenomenon may be explained byFig. 5. Typical plots of [poly[(dA–dT) ]] / (́ 2´ ) versus [poly[(dA–2 A F

21 the structurally induced difference in the binding mode ofdT)]] for spectrophotometric titration of [Fe(phen) (dip)] (12.0mM)221and [Fe(phen)(dip) ] (13.0mM) with increasing amounts of [poly[(dA– the iron(II) complex to double helical DNA. One of the2

dT) ]] in 5 mM Tris–HCl buffer (pH 7.2) containing 50 mM NaCl at2 possible explanation is that the iron(II) complex may be25 8C.

partially intercalated into the base-pairs of DNA. If such abinding mode occurs, it is required that the non-intercala-tive ligands around the metal center are located in such away that the steric effect is minimized. The relativelyfacile A–T sequence is much more ready to alleviate thesteric effect associated with the non-intercalative ligandsthan the G–C sequence. Hence, it is quite reasonable that

21[Fe(phen)(dip) ] , in which one of the ligands is possibly2

partially intercalated into DNA base-pairs [20,41], hasrelatively high affinity towards the A–T sites of DNA.However, further thorough studies involving physicochem-ical measurements and other techniques must be done toconfirm such explanation.

3 .4. Effects on thermal denaturation profiles of DNA

The denaturation of polynucleotide strand from double-stranded to single-stranded nucleic acid manifests itself asabsorption hyperchromism around 260 nm. The binding ofmetal complexes to the double-stranded DNA usuallyFig. 6. Typical plots of [poly[(dG–dC) ]] / (́ 2´ ) versus [poly[(dG–2 A F

21 stabilizes the duplex structure to some extent depending ondC) ]] for spectrophotometric titration of [Fe(phen) (dip)] (5.5mM)2 221 the strength of their interaction with nucleic acid. Theand [Fe(phen)(dip) ] (4.6mM) with increasing amounts of [poly[(dG–2

dC) ]] in 5 mM Tris–HCl buffer (pH 7.2) containing 50 mM NaCl at binding should lead to an increase in the melting tempera-2

25 8C. ture of DNA as compared with DNA alone (see Fig. 7).

Table 3Binding affinity of iron(II) mixed-ligand complexes to various DNA

a 21DNA Binding constant (K ) (M )b

21 21 21[Fe(phen) ] [Fe(phen) (dip)] [Fe(phen)(dip) ]3 2 2

2 4 4Calf thymus DNA 4.70310 1.75310 1.723104 5Poly[(dA–dT) ] – 2.71310 2.5831024 4Poly[(dG–dC) ] – 1.92310 2.183102

a Average values of three separate measurements with an RSD of less than 15%, determined in 50 mM NaCl, 5 mM Tris buffer (pH 7.2) at 258C.

270 Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271

obviously cannot intercalate into double helix DNA [43].When two phen ligands of the three are replaced by twodip ligands, the modest increase inDT value (by 5.58C)m

was observed. This value is quite similar to that measured21for [Ru(NH ) (dppz)] , i.e., DT 5.28C in 5 mM3 4 m5

phosphate buffer at pH 7.1 [43] which has been suggestedto interact with DNA in an intercalation mode, but it is stilllower than those of metal-complex intercalators such as

21[Ru(phen) (dppz)] (9.18C) [43] or organic intercalators2

such as ethidium bromide (138C) [44]. Since the bulkier21complex [Fe(phen)(dip) ] is more hydrophobic than the2

two other complexes, an aggregation of the complex alongthe DNA strands is likely [33]. This could also give rise tothe increase inDT value of the double helical DNA.m

Therefore, without any other supporting data, it is difficultto interpret the data presented here as well as to concludethe DNA-binding mode of the iron(II) complexes from theDT value alone. Nevertheless, based on the thermalm

denaturation profiles presented here, it is evident that theFig. 7. Thermal denaturation profiles of CTDNA in the absence (1) and binding of disubstituted iron(II) complex to the double21presence of [Fe(phen)(dip) ] (2). They were measured in 5 mM2 helical DNA is stronger than those of two other complexes.Tris–HCl buffer (pH 7.2) containing 50 mM NaCl at R ([complex] /

This result is consistent with their relatively high binding[CTDNA])50.5 and the completed results are collected in Table 4.affinity estimated from the spectrophotometric titration.

Therefore, the thermal denaturation profile together withother supporting data is a convenient tool for identifying 4 . ConclusionDNA-binding mode and also predicting relative bindingstrengths [42]. Under the present experimental conditions, The DNA binding of iron(II) mixed-ligand complexesthe melting temperature of free double helical CTDNA is containing phen and dip ligands is strongly influenced by75.58C regardless of the concentration of nucleic acid. the salt concentration in the buffer solution. Based on theThis melting temperature corresponds to the dissociation of experimental data of the salt concentration dependence ofthe Watson–Crick base-paired duplex. As expected, the the DNA binding, the DNA-binding constants and DNA-binding of all the iron(II) complexes studied results in a binding free energy changes have been dissected intorise in melting temperatures of CTDNA to varying extents polyelectrolyte and nonelectrostatic components. The latter(see Table 4). The data presented in the table are the component provides a good means for rigorously evaluat-average values of three separate measurements with an ing the contribution of ligand substituents to the bindingRSD of less than 5%. The tris- and monosubstituted event among the iron(II) mixed-ligand complexes. Thecomplexes show a relatively small value ofDT (2.0– results of quantitative analysis have revealed that them

2.58C), which is consistent with their simple electrostatic peripheral substitution of phen by two phenyl groupsbinding and with their relatively low binding affinity increases the nonelectrostatic binding constant of theestimated from the spectrophotometric titration. Such a iron(II) complexes 20 times as much, which is equivalent

21low value ofDT (1–28C) has been also observed for the to 7.5 kJ mol of stabilization energy for the DNAm21calf thymus DNA binding of [Ru(NH ) Cl] which binding. The iron(II) complexes in the DNA binding3 5

exhibit a slight base-pair specificity to prefer the relativelyTable 4 facile A–T sequence to the G–C one. The thermalMelting temperature (T ) of calf thymus DNA in the absence andm denaturation experiments have demonstrated that the dis-apresence of iron(II) mixed-ligand complexes

ubstituted iron(II) complex stabilizes the double helicalbAdded complex T / 8C DT / 8Cm m DNA significantly. This is consistent with their relatively

No addition 75.5 – high binding affinity estimated from the spectrophoto-21[Fe(phen) ] 77.5 2.03 metric titration.

21[Fe(phen) (dip)] 78.0 2.5221[Fe(phen)(dip) ] 81.0 5.52

a Average values of at least three separate measurements with an RSDA cknowledgementsof less than 5%, determined in 50 mM NaCl, 5 mM Tris–HCl buffer atpH 7.2.

b The first author (M) acknowledges financial supportAt R ([complex] / [CTDNA])50.5. The concentrations of CTDNA arein the range of 40–52mM. from The Hitachi Scholarship Foundation, Tokyo, Japan

Mudasir et al. / Journal of Inorganic Biochemistry 94 (2003) 263–271 271

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