Journal of Inorganic Biochemistry 102 (2008) 1892–1900

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Journal of Inorganic Biochemistry

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Interaction of the DNA bases and their mononucleotides withpyridine-2-carbaldehyde thiosemicarbazonecopper(II) complexes. Structure of the

cytosine derivative

Begoña García a,*, Javier Garcia-Tojal a,*, Rebeca Ruiz a, Ruben Gil-García a, Saturnino Ibeas a,Bruno Donnadieu b, José M. Leal a

a Departamento de Química, Universidad de Burgos, 09001 Burgos, Spainb Department of Chemistry, University of California, Riverside, CA 92521, USA

a r t i c l e i n f o

Article history:Received 1 April 2008Received in revised form 20 June 2008Accepted 23 June 2008Available online 1 July 2008

Keywords:CopperThiosemicarbazoneNucleobaseNucleotideBinding

a b s t r a c t

Experimental studies of the binding interactions of [CuL(NO3)] and [{CuL0(NO3)}2] (HL = pyridine-2-carb-aldehyde thiosemicarbazone, and HL0 = pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) withadenine, guanine, cytosine, thymine and their mononucleotides (dNMP), 2-deoxyadenosine-50-mono-phosphate, (dAMP), 20-deoxyguanosine-50-monophosphate, (dGMP), 20-deoxycytidine-50-monophps-phate (dCMP), and thymidine-50-monophosphate (dTMP) have been carried out in aqueous solution atpH 6.0, I = 0.1 M (NaClO4) and T = 25 �C. The complexation constants of these compounds, calculatedby Hildebrand–Benesi plots for the dye binding, D, ([CuL] or [CuL0]) to the nucleobases or nucleotides(P), have shown two linear stretches in adenine, guanine, dAMP and dGMP. The data were analyzed interms of formation of 1:1 DP and 1:2 DP2 complexes with increasing purine base or nucleotide content.For cytosine and dCMP only 1:1 complexes have been observed, whereas for thymine and dTMP suchcomplex structures were not observed. The [CuL(Hcyt)](ClO4) cytosine derivative has been isolated andcharacterized. The crystal structure consists of perchlorate ions and [CuL(Hcyt)]+ monomers attachedby hydrogen bond, chelate p�ring and anion–p interactions. The Cu2+ ions bind to the NNS chelating moi-ety of the thiosemicarbazone ligand and the cytosine N13 site (N3, most common notation) yielding asquare-planar geometry. A pseudocoordination to the cytosine O12 site (=O2) can also be considered.

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1. Introduction

The interaction of divalent metal cations with nucleic acids hasbeen the subject of intense research due to their significance forthe DNA replication and transcription [1], and has a great deal ofpromise for a number of metabolic processes. Despite its toxicityin the pure form, copper plays a fundamental role for the activityof many enzymes that bear importance in processes such as oxy-gen transport and insertion, electron transfer and oxidation–reduc-tion, among others.

Thiosemicarbazones display a wide variety of biological proper-ties, depending on the parent aldehyde or ketone involved [2]. Overthe last decades, the Cu2+ complexes of pyridine-2-carbaldehydethiosemicarbazone have attracted attention in part because theyexhibit relevant biological properties related to the cytotoxic ef-fects against tumoral cells [3–6] and also because of its chemicaladsorbing ability on gold electrodes that enhances the cytochrome

0162-0134/$ - see front matter � 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jinorgbio.2008.06.013

* Corresponding authors. Tel.: +34 947 258819; fax: +34 947 258831 (B. Garcia),tel.: +34 947 258035; fax: +34 947 258831 (J. Garcia-Tojal).

E-mail addresses: begar@ubu.es (B. García), qipgatoj@ubu.es (J. Garcia-Tojal).

c redox reactions [4,5]. It has been suggested that in aqueous solu-tion the pyridine-2-carbaldehyde thiosemicarbazonecopper(II)system gives rise to [CuL(H2O)n]+ species (Scheme 1), which con-tain [CuL]+ planar entities, with the thiosemicarbazone moiety act-ing as a NNS tridentate ligand [6,7]; the log constant for formationcomplex is 16.90 [7]. The ligand coordination by Cu2+ ions is main-tained even below pH 1.4 [8].

The spectrophotometric studies have suggested the existence ofthe [Cu(HL)]2+, [CuL]+ and [CuL(OH)] species corresponding to twoacid–base equilibria with proton dissociation constants pKa,1 = 2.4and pKa,2 = 8.3, respectively. The latter constant is presumed tocharacterize the formation of [CuL(OH)] from [CuL]+ [8]. The smal-ler constant is attributed to the protonation of [CuL]+ at the hydra-zine N site of the thiosemicarbazone moiety, since the pyridinering nitrogen is bound to copper. Inspection of the acid–base con-stants has led us to conclude that, at pH 6.0, the only stable speciespresent in solution is [CuL]+ (Scheme 1), whose planarity and posi-tive charge would, in principle, make it a privileged candidate tointeract with nucleic acids either by intercalation or by externalbinding. Similar results have been reported for the [CuL0]+ entities,with proton dissociation constants pKa,1 = 2.7 and pKa,2 = 8.9 [9].

The dye binding to mononucleosides and mononucleotides(Fig. 1) has been extensively used as a model for nucleic acids[10]; however, their behavior noticeably differs from that of poly-nucleotides in issues such as the effect of polyelectrolytes or theabsence of grooves. Nucleotides play a key role in the cell metabo-lism and their concentration may reach pretty high values; for in-stance, in the chromaphin granules of the adrenal medulla, ATPmay reach a concentration as high as 0.15 M [11].

The X-ray studies carried out with guanine in the presence ofCu-complexes with tridentate ligands have shown that the bindingoccurs preferentially through the N7 site [12,13]. The binding ofdivalent metals to the N7 guanine site could in principle induce arather high rate of spontaneous formation of ion-pair structuresof the GC base pair, due to a substantial destabilization of the H1guanine ring hydrogen; this would, in turn, originate a highlymutagenic process. However, further consideration shows that thisprocess is basically always eliminated by the environment [14]. Anumber of complexes with Cu attached to the adenine N1 [15]and N7 [16,17] sites are well described.

Regarding cytosine, four coordinate complexes between Cu-tri-dentate ligand fragments and cytosine through the N3 site [18,19]five coordinate compounds with N3 and carbonyl oxygen as donoratoms [20,21] have been identified in solid state by X-ray measure-ments. Very few references have been found in the literature onthymine–Cu2+ complexes, and only a five coordinate complex

containing Cu2+ ions linked to a tridentate ligand and thyminethrough the N1 site has been identified by X-ray measurements[22].

With the aim of shedding light into the interaction mechanismof Cu–DNA complexes, the interaction of the nucleobases (adenine,guanine, cytosine and thymine) and their monophosphatenucleotides (dAMP, dGMP, dCMP and dTMP) with pyridine-2-carb-aldehyde thiosemicarbazonatocopper(II), [CuL]+, and pyridine-2-carbaldehyde 4N-methylthiosemicarbazonatocopper(II), [CuL0]+,were looked into in aqueous solution pH 6.0, I = 0.1 M (NaClO4)and T = 25 �C by spectrophotometric measurements and theoreti-cal calculations. Moreover, the [CuL(Hcyt)](ClO4) compound wassynthesized and characterized by CHNS microanalysis, IR spectros-copy and single crystal X-ray diffraction measurements. To ourknowledge, it represents the first X-ray structural evidence for athiosemicarbazone metal-nucleobase adduct.

2. Experimental section

2.1. Materials

Copper(II) nitrate trihydrate, copper(II) perchlorate hexahy-drate, sodium hydroxide, thiosemicarbazide and pyridine-2-carb-aldehyde were purchased from commercial sources and used asreceived. Published methods were used to synthesize HL [23],HL0 [24,25], CuL(NO3) [26] and [{CuL0(NO3)}2] [27]. In solutionthese complexes were used as dyes, and their concentration wasexpressed as CD.

Solutions were kept in the dark at 4 �C. The nucleobases andnucleotides were purchased as the lyophilized sodium salts andused without further purification. The concentration is expressedin molarity and is indicated as CP. Solutions were prepared withdoubly distilled deionized water, from a Millipore Q apparatus(APS; Los Angeles, California), containing NaClO4 to attain 0.1 M io-nic strength; Sodium cacodylate, Na(CH3)2AsO2, 0.0025 M wasused to keep up the pH constant at 6.0.

Caution: Perchlorate salts and their respective metal complexescan be explosive and use of only small amounts of reactants isstrongly recommended.

2.2. Physical measurements

Microanalyses were performed with a LECO CHNS-932 ana-lyzer. IR spectra were obtained with samples prepared as KBr pel-lets in the 400–4000 cm�1 region on a Nicolett Impact 410 FTIRspectrophotometer. The intensities of the reported IR bands are de-fined as vs = very strong, s = strong, m = medium, and w = weak,whereas b denotes a broad band. The pH measurements were per-formed using a CRISON micro pH 2002 pH-meter outfitted with acombined glass electrode with a 3 M KCl solution as a liquidjunction.

The spectrophotometric titrations were performed on a spectro-photometer fitted out with diode array detection and computer-as-sisted temperature control systems. The titrations were carried outby direct addition of increasing amounts of the nucleobase ornucleotide (P) into the cell containing the Cu-complex, D, (CuL orCuL0) solution, and also adding increasing D amounts into the cellcontaining P. No noticeable dye adsorption on the spectrophotom-eter cells was detected.

2.2.1. X-ray crystallographic studiesCrystal data collections were carried out on an X8 APEXII Bruker

Nonius diffractometer equipped with a four circles Kappa goniom-eter and an APEX II detector. The WinGX-Version 1.70.01 set ofprograms [28] was used for solving, refining and analyzing the sin-

N3

N1

N9

N7

N6

R

HH

N3

N1

N4

O2

HH

R

N3

N1

N9

N7

N2

O8

R

H

H

HN1

N3

O4

O2

CH3 H

R

Adenine Guanine ThymineCytosine

HO

O

OH

OH

OH

O

P

R: ;

Fig. 1. Chemical structure of DNA bases (R = H) and their nucleotides.

N C

N

N

N

S

HR

CuH 2O

H

+

Scheme 1. Structure of thiosemicarbazonecopper(II) complexes, with R = H, [CuL]+

or R = CH3, [CuL0]+.

B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900 1893

gle crystal X-ray diffraction data. In particular, SIR92 [29] wasemployed to solving for the structure and then was refined byfull-matrix least-squares method, using the SHELXL-97 computerprogram [30]. The scattering factors and anomalous dispersioncoefficients were taken from International Tables of X-ray Crystal-lography [31]. Refinements on F2

0 were made for all reflections. Allnon-hydrogen atoms were assigned anisotropic thermal parame-ters. The hydrogen atoms were constrained to ideal geometriesand refined with fixed isotropic displacement parameters. Thespace group of [CuL(Hcyt)](ClO4) is non-centrosymmetric and, inthis case, BASF and TWIN instructions were used in the last stepsof refinement. The Flack parameter [32] obtained was 0.347.Crystallographic details are shown in Table 1. Different views ofthe unit cell are given as Supporting material in Figs. S1–S3.

2.3. Preparation of the [CuL(Hcyt)](ClO4)

Solid HL (0.5 mmol, 0.090 g) was added to a solution ofCu(ClO4)2 � 6H2O (0.5 mmol, 0.185 g) in 20 ml water. The resultingdark green solution was stirred at room temperature for 30 minand filtered over an aqueous suspension of cytosine (0.25 mmol,0.028 g).1 Then, the acidity was adjusted at pH 7.4 by addition ofdiluted NaOH. The reaction was kept by stirring for 1.5 h and a darkgreen precipitate was filtered off (0.011 g); after 3 weeks, slow evap-oration of mother liquors yielded single crystals suitable for X-raydiffraction measurements. Anal. found: C, 28.08; H, 2.58; N, 21.37;S, 6.67. Calc. for C22H24Cl2Cu2N14O10S2 (906.65 g/mol): C, 29.15; H,2.67; N, 21.63; S, 7.07. IR bands (cm�1, KBr pellet): 3433 b,s, 3320s, 3114 s, 2961 w, 2927 w, 1683 s, 1634 vs, 1602 s, 1557 w, 1511m, 1482 m, 1384 w, 1321 w, 1296 w, 1230 m, 1172 m, 1150 s,1121 vs, 1109 s, 1082 s, 915 w, 883 w, 775 w, 736 w, 636 w, 627m, 454 w.

3. Results and discussion

3.1. Molecular structure

3.1.1. Crystal structure of [CuL(Hcyt)](ClO4)The crystal structure of [CuL(Hcyt)](ClO4) contains [CuL(Hcyt)]+

monomer units and perchlorate counterions. The two independentcationic crystallographic entities present in the asymmetric unitare related through a pseudo inversion center, as shown in Fig. 2.The selected distances and angles summarized in Table 2 are rathersimilar for both cations, which only differ by a flip of the cytosinering around the Cu–N13 bond.

The coordination around the metal center shapes a distortedsquare-planar topology. The thiosemicarbazone ligand exhibitsthe usual tridentate behavior through the pyridine N1, azomethineN2 and S atoms, while the cytosine binds to the Cu2+ ion throughthe N13 atom. Notwithstanding, the intramolecular 2.687(5) Åand 2.726(5) Å Cu� � �O12 distances for B and A, respectively, couldbe interpreted as a (4 + 1) pseudocoordination. In fact, theselengths fall inside the 2.55–2.80 Å range reported in the literaturefor the cytosine Cu–O bonds [21,33–36]. This feature is also sup-ported by the Cu–N13–C12 and Cu–N13–C14 angles: 108.1(4)/129.1(4)� and 106.3(4)/132.6(4)� for A and B, respectively. Thesevalues reflect the approach of the cytosine ring to the metal centerthrough the O atom. Nevertheless, the C12–O12 bond lengths donot significantly differ from that of 1.237(2) Å in the free cytosine[37,38].

The thiosemicarbazone ligand can be regarded as quasi-planar,with major deviations from the pyridine plane of 0.430 and 0.505 Åfor the N4(A) and N4(B) atoms, respectively. Considering the basalplane as that defined by the N1N2S donor set, we found that the Cu

Table 1Crystallographic data for [CuL(Hcyt)](ClO4)

Empirical formula C22H24Cl2Cu2N14O10S2

Formula weight 906.65Temperature 100(2) KWavelength 0.71073 ÅCrystal system OrthorhombicSpace group Pca21

Unit cell dimensions a = 25.975(5) Å a = 90.00�b = 7.5450(16) Å b = 90.00�c = 16.665(4) Å c = 90.00�

Volume 3266.0(12) Å3

Z 2Density (calculated) 1.844 Mg/m3

Absorption coefficient 1.671 mm�1

F(000) 1832Crystal size 0.22 � 0.07 � 0.06 mmh range for data collection 1.99–26.45�Index ranges �32 6 h 6 32, �7 6 k 6 9, �20 6 l 6 20Reflections collected 22,304Independent reflections 6656 [Rint = 0.1031]Absorption correction Semi-empirical from equivalentsData/restraints/parameters 6656/1/470Goodness-of-fit on F2 S = 0.979R indices [I > 2r(I)] R1 = 0.0512, wR2 = 0.0823R indices (for all 8825 data) R1 = 0.0939, wR2 = 0.0949Largest diffraction peak and hole 0.423 and �0.432 eÅ�3

Fig. 2. ORTEP drawing of the two independent [CuL(Hcyt)]+ entities in [CuL(Hcyt)](ClO4). Only fragment A is labeled. Thermal ellipsoids are drawn at the 50%probability level. Note that the labeling scheme of the cytosine adds ‘‘1” before thecorresponding usual notation, given in Fig. 1 (e.g. N13 = N3, etc. . .).

1 This precipitate composition differs from that of the crystals studied here. Duringthe evaporation process, other green powdered solids were filtered off prior toattainment of the crystals. All these compounds seem to contain anionic cytosineligands, and the CHNS analysis on one of them matched fairly well with a[CuL]2(cyt)(ClO4)(H2O)2 formula.

Table 2Selected bonds (Å) and angles (�)

A B A B

Cu–S 2.274(2) 2.260(2) N4–C7 1.306(8) 1.342(9)Cu–N2 1.940(5) 1.952(5) C7–N3 1.352(9) 1.337(9)Cu–N13 1.976(5) 1.950(5) N2–N3 1.381(7) 1.374(8)Cu–N1 2.039(5) 2.007(6) N2–C6 1.290(9) 1.296(9)S–C7 1.738(7) 1.730(7)S–Cu–N2 85.2(2) 84.3(2) N2–Cu–N13 176.4(2) 178.4(2)S–Cu–N13 97.1(2) 96.4(2) N2–Cu–N1 81.0(2) 81.4(2)S–Cu–N1 162.6(2) 163.6(2) N13–Cu–N1 97.2(2) 98.0(2)

1894 B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900

and N13 atoms lie at 0.176 Å and 0.400 Å above for molecule A, and0.226 Å and 0.556 Å for B. Thiosemicarbazones show an E configu-ration with respect to the C6–N2 double-bond and undergo confor-mational changes upon coordination. The N2 and S atoms are antiwith respect to the N3–C7 linkage in the free ligands. However, theN1 and N2 atoms are anti with respect to the C5–C6 bond for com-pounds HL � nH2O (n = 1 [39], 1.75 [40] and 2 [41]), while being synfor n = 2.75 [3] and the pyridinium salts [42,43]. Both conforma-tions become syn after chelation.

The C–C and C–N thiosemicarbazone lengths are similar tothose in the free ligand except for N3–C7, N4–C7 and C7–S bonds,which are 1.358(4), 1.317(4) and 1.698(3) Å, respectively [39]. TheN(3)–C(7) and C(7)–S bond lengths diminish and rise, respectively,upon coordination. This feature has been put down, respectively, tothe gain and loss in double-bond character of the mentioned dis-tances upon complexation. The selected parameters in Table 3are in good agreement with those expected for compounds con-taining HL in the anion form [44,45], except for small divergencesin non-bonding distances related with the B molecule.

The dihedral angles between the thiosemicarbazone NNS donorplanes and cytosine ligands are 96.6� and 93.1� for A and B, respec-tively. An analysis of the molecular packing suggests the stabiliza-tion of the lattice by several hydrogen bonds, mainly involving theN3, N4, N11, N14, O12 and the perchlorate O1, O2, O3 atoms (seeTable S1 in Supplementary material, which also indicates the con-sidered symmetry transformations). Monomers stack formingpseudochains in the [010] direction; these are represented inFig. S4, where adjacent monomers of the asymmetric unit are closetogether, giving rise to CuA� � �N3B and CuB� � �N3A distances of2.927(6) and 2.919(6) Å, respectively. These lengths are longer

than those corresponding to the Cu–N bonds, but below the sumof the van der Waals radii (3.55 Å) [46]. Other short distances be-tween both monomers inside the asymmetric unit are N3B� � �N2A,N3A� � �N2B, N4B� � �N1A and C7B� � �N2A of 3.165(8), 3.172(8),3.172(8) and 3.24(1) Å, respectively. These distances relating paral-lel fragments suggest the existence of p–p stacking in the struc-ture. A chelate ring can be defined on the basis of the Cu, N2, N3,C7 and S atoms, namely the ‘‘Cu–thiosemicarbazone ring”. The cen-troid� � �centroid distance between Cu–thiosemicarbazone rings inthe same asymmetric unit is 3.25 Å, concurrent with previouslypublished results for intermolecular interactions involving metal-chelate rings with delocalized p-bonds [47–49]. Note that alsoother chelate rings could be defined as well, such as those formedby the Cu, N2, C6, C5 and N1 atoms. The shortest distances be-tween monomers of different asymmetric units correspond toC5B� � �N2Ai and C7B� � �N3Ai with 3.353(9) and 3.19(1) Å, respec-tively. In addition, perchlorate ions are surrounded by [CuL(Hcyt)]+

cations, which may form cavities where the minimum distancesbetween perchlorate O atoms and the cytosine rings areO2B� � �C16B 2.860 Å (O3B� � �centroid of cytosine B 3.568 Å) andO3A� � �N11A 3.067 Å (O2A� � �centroid of cytosine A 3.637 Å), seeFigs. S5 and S6. These data fall inside the range reported for an-ion–p interactions [50–53]. A plot showing the complexity of thenon-covalent interactions in the structure is given in Fig. 3. A struc-tural analysis of the p–p stacking in this compound is summarizedin Table S2.

3.1.2. Infrared characterization of [CuL(Hcyt)](ClO4)An unambiguous assignment of the IR bands in this compound

is rather difficult mainly because of the overlapping of the cytosine,thiosemicarbazone and perchlorate vibrations. Following pub-lished studies [21,33,54], a tentative proposal for the selectedbands has been carried out. Absorptions around 3430–2900 cm�1

are attributed to the m(NH2), m(NH) and m(CH) vibrations of the thi-osemicarbazone and cytosine ligands. Strong bands at 1683, 1511and 1603 cm�1 can be due mainly to the cytosine d(N14H2),d(N11H) and amide I [d(NH) + m(CO)] modes, respectively. The verystrong band at 1634 cm�1 can be assigned to the m(CO) on com-plexation and shifts to higher energies (1664 cm�1) in free cytosine[55]. The strong absorption at 1121, 1109 and 1082 cm�1 corre-spond to m3(ClO4), and those at 920 and 470 cm�1 are assigned tothe m2 and m1 perchlorate modes, respectively. The weak bandaround 454 cm�1 can be ascribed to the m(CuN) vibrations. Otherabsorptions corresponding to the pyridine-2-carbaldehyde thi-osemicarbazonatocopper(II) fragment have been discussed in de-tail elsewhere [56,57].

3.1.3. Biological significanceTo our knowledge, [CuL(Hcyt)](ClO4) represents the first X-ray

structural evidence for a thiosemicarbazonemetal-nucleobase ad-duct. The previous closest crystallographic works deal with thio-semicarbazone ligands obtained by condensation of nucleobases(mainly uracyl derivatives) and thiosemicarbazide [58–60]. Thetitle structure proves the affinity of the [CuL]+ cations for cyto-sine nucleobases and the relevance of the N13 atom in the link-age. In fact, the important role as donor site of the cytosine N3 inthe Cu-complexes has been previously shown [18–20,33–35,61–63]. On the other hand, the existence of multiple non-covalentinteractions spreads the options of interaction for the pyridine-2-carbaldehyde thiosemicarbazonatocopper(II) entities withnucleotides and DNA, including mono- or polynuclear p–p stack-ing and anion–p interactions with phosphate groups. In addition,other modes of action should not be ruled out, e.g. coordinationto other nucleobases or even phosphates. In this regard, struc-tures of different phosphate derivatives of [CuL]+ have been re-ported [64,65].

Table 3Selected structural differences between Cu2+ complexes containing neutral andanionic HL (Å, �)

Molecule N2–N3–C7 S–C7–N3 S–C7–N4 Cu� � �N3 N2� � �C7

A 111.3(5) 125.1(5) 118.8(5) 2.939(5) 2.238(9)B 111.0(5) 125.5(5) 118.5(5) 2.928(5) 2.252(9)HL range 116–118 121–122 121–123 2.89–2.90 2.28–2.32L� range 109–114 123–127 117–119 2.94–3.00 2.22–2.23

Fig. 3. A portion of the structure containing the main intermolecular non-covalentinteractions in [CuL(Hcyt)](ClO4): hydrogen bonds, chelate ring-p and anion–pinteractions. For the sake of clarity, most of the hydrogen atoms are omitted. Blackspheres represent the cytosine centroids.

B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900 1895

3.2. Binding of [CuL]+ and [CuL0]+ to DNA bases and mononucleotidesin solution

3.2.1. Fulfilment of Lambert–Beer LawThe spectra of [CuL]+ recorded upon stepwise increase of dye

concentrations at pH 6.0 and I = 0.10 M, shows that the profile ofthe absorption band changes; at high dye levels, the predominantpeak observed at the smaller wavelength reveals the formation ofan aggregate species. This finding was confirmed by checking therange of linearity of the absorbance vs dye concentration plot.Deviations from linearity indicate that the aggregate formation of[CuL]+ occurs already at dye concentrations above 5 � 10�5 M. Thisfeature was not observed for [CuL0],+ as Fig. 4 shows, where the A vsCD linearity was unaffected by concentration. The absorptivity val-ues, evaluated in the concentration range where the Lambert–Beerfulfils are listed in Table 4; the values show the considerable effectexerted by the methyl group on the molar absorptivity. It can beassumed that the stacking hydrophobic interactions, responsiblefor the aggregate formation in [CuL]+, do have a similar orientationas those in Figs. 3 and S4. Especially noteworthy is that the methylgroup effect on [CuL0]+ prevents from aggregate formation, at leastunder the experimental concentration conditions of the UV–Viscurves recorded.

3.2.2. EquilibriaElectronic absorption spectroscopy is universally employed to

determine the modes of binding of complexes with DNA. The spec-tra of the two Cu2+-complexes, [CuL]+ and [CuL0]+, were recorded inthe presence of increasing amounts of nucleoside and nucleotide.Fig. 5 displays the behavior of the different systems; little differ-ences were observed between the spectral curves of nucleosides

and their corresponding nucleotide (e.g., Fig. 5B and C shows thesimilarity between guanine and dGMP bases) and between [CuL]+

and [CuL0]+ in each case (little differences are observed in thecharge transfer band at 325 nm). If a single binding process occurs,this can be represented by the apparent reaction (1)

Dþ P ¡ DP ð1Þ

where D is the free dye ([CuL]+ or [CuL0]+), P the nucleobase ornucleotide, and DP the complex formed. Equilibrium (1), could bedescribed by the binding constant, Kapp, defined in Eq. (2):

Kapp ¼ ½DP�=½D�½P� ð2Þ

The data analyses were carried out with the Hildebrand–Benesimethod [66], Eq. (3),

CPCD=DAþ DA=De2 ¼ 1=DeKapp þ ðCD þ CPÞ1=De ð3Þ

where CP and CD denote the total monomer and dye concentrations,respectively,

DA ¼ A� ‘eDCD � ‘ePCP and De ¼ ‘ðePD � eDCD � epCpÞ ð4Þ

As the De term is unknown (‘ = 1 cm), the DA/De2 term was disre-garded, and had to be calculated by iteration from the slope of thestraight line obtained by plotting the data according to Eq. (3); nor-mally, the convergence is attained after only few iterations. Theanalysis was focused on the 200–300 nm region, where DA andDe were greater than those for k > 300 nm. For thymine and dTMP,the DA values were zero over the whole wavelength range.

The plot of the first term of Eq. (3) versus the (CP + CD) sum waslinear up to a certain dye content for purine bases and their mono-nucleotides, and over the whole concentration range for cytosineand dCMP (Fig. 7). The Kapp and De values are provided for all sixcases. A linear behavior often is put down to 1:1 binding as theonly existing species [67], the curvature of the plots with risingnucleotide concentration implying that higher order complexesare being formed [68].

Table 5 shows the Kapp values of 1:1 complex formation for thedifferent equilibria, as well as the CD/CP boundary from which Eq.(3) did not fitted the data. Adenine, guanine, cytosine, and theircorresponding nucleotides, may form stable complex species with[CuL]+ and [CuL0]+, whereas thymine and dTMP are unreactive.Complex species may arise from either covalent, non-covalention-dipole and stacking interactions. On the basis of ab initio cal-culations, Sponer et al. have suggested possible cation–p interac-tions between hydrated divalent cations and DNA bases. Thecalculations have demonstrated that aromatic base rings are, inprinciple, capable of forming cation–p complexes of similarstrength as that of benzene. However, aromatic base rings haveN and O sites with lone pairs and, in contrast to benzene, in-planebinding of cations to these nucleobase sites is highly preferredover any cation–p stacking [69]. On the other hand, the p–p*

transition exhibits a red shifted band with respect to the donoror acceptor; absence of these features in our systems (Fig. 5) isa clear indication that such complexes cannot be formed bystacking interactions.

At the working acidity levels used (pH 6.0), the bases are in theneutral form and the nucleotides are in equilibrium between theNMP�2 and H(NMP)�, the proton being released from the phos-phate group [70–72]. Differences in the basicity of the N3 atomin thymine (pK = 9.79) compared with the endocyclic N1 and N7atoms of adenine characterized by pK = 3.92, and the cytosine N3(pK = 4.61) and their monophosphates (pK = 4.02 and pK = 4.48,respectively) [72] have enabled us to observe systems where thepossible N atoms involved in the formation of metal complexesare the N unbounded to H (Fig. 1). The rather high pK values alongwith the absence of interaction with thymine and dTMP (Table 5)support the interaction of the Cu-complexes with the free N.

0 10 15 200.0

0.5

1.0

1.5

2.0

2.5

Abso

rban

ce

105CD, M5

Fig. 4. Absorbance values as a function of CD, the total dye concentration. At k = 280nm (j [CuL]+ and h [CuL0]+); at k = 400 nm (d [CuL]+ and s [CuL0]+) .

Table 4Absorptivity values of dyes at different wavelengths

k, (nm) [CuL]+ [CuL0]+

e, (M�1 cm�1) e, (M�1 cm�1)

230 12,103 10,529250 7270 7718258 6819 5771280 14,374 10,789320 10,456 11,627380 9318 11,009386 9360 11,289400 7184 9361

1896 B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900

The apparent constants (Kapp) of the 1:1 complexes with dAMP,dGMP, dCMP listed in Table 5 are one order of magnitude lowerthan those of the corresponding bases; this feature reveals thatthe presence of the phosphate group is responsible for the diminu-tion of the base affinity for the complex. This behavior can beaccounted for: (1) by complex formation, where the Cu atom bindsto solely the nucleotide phosphate group, an option that should notbe ruled out; however, absence of reaction with dTMP makes itnecessary to consider other possible routes; (2) by the formationof chelates, where the phosphate group and the N atom are in-volved; Sigel et al. [73] have shown that the purine-nucleotidecomplexes may adopt two conformations in solution: an openform, where the metal ion is only phosphate-coordinated, and aclose form (or chelate form), where the phosphate-bound metalion bridges the N7 of the purine-nucleobase (Fig. 7) thus giving riseto the M(purine fosfate)op M M(purine fosfate)cl intramolecular

equilibrium. For adenosine monophosphate (AMP) and M = Cu2+,the constant unity for the former equilibrium, indicates that 50%of each form is present; for the close form 54% is present asCuðADPÞ�Cl and 67% as CuðADPÞ2�Cl . Although not too big, this differ-ence shows that the properties of the N7 site are mainly responsi-ble for the extent of macrochelate formation [74]. The chelateformation does not account for the stabilization of the cytosinecomplexes, because the absence of the N7 site prevents from itsformation; in the [CuL(Hcyt)]+ X-ray structure (Fig. 2) it is difficultto realize how the phosphate could interact with the metal (anal-ogy to Fig. 7).

On the other hand, the formation of chelates normally stabilizesthe complex and, unlike the observed behavior, Kapp would be lar-ger than that for the respective nucleobases; however, the inherententropy diminution might play a critical role for the smaller equi-librium constant of the Cu–nucleotide complex. On the basis of the

2000.0

0.5

1.0

1.5

2.0

2.5Ab

sorb

ance

λ, nm λ, nm

λ, nmλ, nm

0.0

0.5

1.0

1.5

2.0

2.5

Abso

rban

ce

0.0

0.5

1.0

1.5

2.0

2.5

Abso

rban

ce

0.0

0.5

1.0

1.5

2.0

2.5

Abso

rban

ce

250 300 350 400 450 200 250 300 350 400 450

200 250 300 350 400 450 200 250 300 350 400 450

A

DC

B

Fig. 5. Absorbance spectra recorded during the titration of: (A) adenine with [CuL]+; (B) guanine with [CuL]+; (C) dGPM with [CuL0]+; and (D) dCPM with [CuL]+. pH 6.0,I = 0.1 M (NaClO4), T = 25 �C. CD = 2.80 � 10�5 M at k = 250 nm, from bottom to top CP/CD = 0–18. D represents [CuL]+ or [CuL0]+ and P represents nucleobase or nucleotide.

Table 5Equilibrium constants 10�6Kapp/M�1 and CD/CP ratio (parenthesized) from which Eq. (3) do not fit the experimental data of purine bases

Dye Adenine Guanine Cytosine Thymine dAMP dGMP dCMP dTMP(CD/CP) (CD/CP) (CD/CP) (CD/CP)

[CuL]+ 4.0 ± 0.5 5.4 ± 0.7 6.0 ± 0.8 – 0.62 ± 0.06 0.89 ± 0.04 0.73 ± 0.06 –(0.11) (0.12) (0.22) (0.28)

[CuL0]+ 0.96 ± 0.09 2.1 ± 0.3 1.1 ± 0.9 – 0.4 ± 0.03 0.71 ± 0.05 0.44 ± 0.02 –(0.15) (0.10) (0.25) (0.35)

T = 25 �C.

B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900 1897

van’t Hoff equation, comparison between the Kapp values at 25 �C(Table 5) and 30 �C for the [CuL]/(adenine) and [CuL]/(dAMP) sys-tems ((2.8 ± 0.3) � 106 M�1 and (3.5 ± 0.4) � 105 M�1, respec-tively), and as preliminary conclusions, shows that the reactionenthalpy with dATP is �86 kJ/mol, roughly some 50% more exo-thermic than with adenine (�54 kJ/mol), and the entropy is about2-fold more negative for dATP (�117 kJ/mol) than adenine (�53 kJ/mol), a feature consistent with chelation as a plausible route forthe [CuL]+/dAMP reaction. Other effects responsible for the loweredaffinity for the dNMP might be: (3) the inductive �I effect exertedby the phosphate group on the base and the basicity lowering of

the reactive nitrogen, which implies an inherent diminution ofthe Kapp values while keeping unaltered the structure of the nucle-otide complexes regarding that of the nucleobases; (4) the stericeffect; (5) the interactions p–anion with ClO�4 persist in solution(analogy to Fig. 3) and charge repulsion occurs between the phos-phate and the perchlorate lowering the affinity of the dNMP versusthe bases.

Fig. 6 shows that, starting from a certain CD/CP ratio (Table 5),the purine bases and the corresponding nucleotides did not followEq. (3) (valid for 1:1 complexes). However, Eq. (3) nicely fits cyto-sine and dCMP over the whole concentration range, indicating that

0 4 8 12 160

2

4

6

8

10

108 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

107 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

107 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

108 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

107 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

105 (CD+C P)

105 (CD+C P) 105 (CD+C P)

105 (CD+C P)

105 (CD+C P)

CD /C P =0.11A

0 8 12 16 20-3

-2

-1

0

CD /CP=0.25

B

0

1

2

3

4

5CD /CP= 0.10

C

0 4 8 12 16 20 24 28-20

-16

-12

-8

-4

0CD /C P=0.35

D

0 8 12 16 20 24 280.0

0.5

1.0

1.5

2.0

2.5

3.0E

0 4 8 12 16-2.0

-1.6

-1.2

-0.8

-0.4

0.0F

4

0 8 12 16 20 24 284

4

107 (C

DC

P/Δ

A+Δ

A/Δ

ε2)

104 (CD+C P)

Fig. 6. Spectrophotometric titration of: (A) adenine with [CuL]+, k = 260 nm; (B) dAMP with [CuL0]+, k = 277 nm; (C) guanine with [CuL0]+, k = 246 nm; (D) dGPM with [CuL0]+,k = 250 nm; (E) cytosine with [CuL]+, k = 230 nm ; (F) dCPM with [CuL0]+, k = 266 nm. I = 0.1 M (NaClO4), pH 6.0, T = 25 �C evaluated according to the Hildebrand–Benesimethod, Eq. (3). (The P concentration rises along the x axis).

1898 B. García et al. / Journal of Inorganic Biochemistry 102 (2008) 1892–1900

only a single type of complex species has been formed. Fig. 2 showsthe solid complex structure, [CuL(Hcyt)]+, where the N13 site (cor-responding to N3 in Fig. 1) coordinates the Cu atom represented inScheme 1, forming an 1:1 complex species. The same structure isfeasible also in solution, where p–p interactions with [CuL] aggre-gate formation (as in the solid state) has been observed (Figs. 3 and4 and S4).

For the purine complexes, the observed curvature starting froma certain CD/CP level cannot be justified in a straightforward man-ner. The CP values rose along the x-axis, that is, Eq. (3) fulfils in thefirst stretch, and the curvature appears under excess of P. Someconsiderations can be excerpted on the possible reasons:

(A) Aggregates formation from a given CP value for the base ornucleotide. In vitro studies have shown that self-associationmust be considerable; it occurs via base-stacking and devel-ops beyond the dimer stage, i.e., oligomers are formed[12,73]. If one takes for granted the dimerization constantof uncharged guanosine as a reference value (K = 8 M�1)[73], then 99% of the species is present in the monomerform, even at the highest base concentration used(�10�4 M). The self-association equilibrium constant forGMP2� was determined in aqueous solution (K = 1.3 M�1)[71]. Assuming K = 2 M�1 for nucleotides it follows that, for0.3 mM solutions over 99% of de NMP species is present inthe monomer form. The 99% limiting value can still bereached if K were equal to 16 M�1, a value close to that mea-sured for adenosine (K = 15 M�1) [12]. Hence, all the resultspresent below apply to monomer base and nucleotide spe-cies, and self-association does not account for the observedcurvature upon increase of the base or nucleotideconcentration.

(B) Tautomers present in solution which, depending on theirdifferent affinity for thiosemicarbazonecopper(II), couldyield different types of complexes. Marino et al. [75] havestudied with the B3LYP/6-311+G(2df,2p) density functionallevel the interaction of isolated Cu2+ cation with the moststable DNA tautomers. From the calculated DG values itcan be concluded that the DlogK values between the twoedges (the most and the less stable tautomers) are: thymine(�0.02) < cytosine (0.02) < guanine (0.05) < adenine (0.07).Extrapolation for free Cu2+ to the [CuL]+ and [CuL0]+ species,such small differences cannot justify the observed behaviorfor the purine complexes.

(C) The formation of DP2 complexes may account for theobserved curvature with increasing base concentration[76–78]. The complex formation may occur by adopting Pan apical position, thus giving rise to a square-pyramidalpentacoordinated structure. For nucleotides, a number ofdifferent DP2-like structures have been described whereCu2+, either free or coordinated, binds two N atoms of a basecontaining phosphate groups similar to nucleotides [79], a Natom and a phosphate group [80], and two phosphate groupsof nitrogenated bases [81], indicating that any of these com-binations is feasible. Nevertheless, the formation of DP2

occurs in the nucleotides at a CP concentration lower thanthat in the corresponding base (Fig. 6); this feature denotesthat the phosphate group is involved in the new species.Unfortunately we were unable to evaluate the stability con-stant of the DP2 complexes, because the closeness of thisvalue and the DP formation constant (Kapp) requires knowl-edge of the absorptivity coefficients for D, (DP) and (DP2)[10], which were unavailable.

To ensure the non existence of D2P complex species, where Pacts as a ligand bridge between two D entities, a set of experimentswas repeated by adding the tridentate Cu thiosemicarbazonecop-per(II) complex to the base or nucleotide; a single straight stretchwas observed whose data pairs were nicely fitted by Eq. (3) overthe whole concentration range; the resulting Kapp values were sim-ilar to those of the DP complexes (Table 5).

Finally, it was observed that the equilibrium constants evalu-ated with [CuL]+ were always somewhat larger than that with[CuL0]+ (Table 5); the donor character of the methyl group andthe pronounced ligand conjugation justifies the observed behavior.The same effect would be observed if the formation constant of[CuL0]+ from Cu2+ and L0 were higher than 16.90, the value for theformation constant of [CuL]+ [7]. This would, in turn, originate alarger Cu2+ affinity for the ligand L0 than for L and, hence, a lesser[CuL0]+ affinity for a second ligand (either base or nucleotide) thanin the case of [CuL]+.

Acknowledgments

The financial support by Ministerio de Educación y Ciencia, Pro-ject CTQ2006-14734/BQU (Cofinanced by Feder), Junta de Castilla yLeón, Spain, Projects BU 001A-06 and BU033A06, and DGESIC(Grant # PM98-0073), Spain, are gratefully acknowledged. R. G.-G.is indebted to Junta de Castilla y León for a Doctoral fellowship.

Appendix A. Supplementary material

Crystallographic data for the structure of [CuL(Hcyt)](ClO4)have been deposited with the Cambridge Crystallographic DataCentre, CCDC–676447. Copies of the data can be obtained free ofcharge on application to The Director, CCDC, 12 Union Road, Cam-bridge CB2 1EZ, UK [Fax: (internat.) + 44-1223/336-033; E-mail:deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk]. Views ofthe crystal packing, p–p stacking and the cavities around the per-chlorate anions are given in Figures S1–S6. Tables of selectedhydrogen bonds and p–p stacking are also given (Tables S1 andS2). Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.jinorgbio.2008.06.013.

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