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Metal_Ions_in_Biological_Systems/0824796888/files/cover.htmlcovernext page> title:Metal Ions in Biological Systems. Vol. 33, Probing of Nucleic Acids By Metal Ion Complexes of Small MoleculesMetal Ions in Biological Systems ; V. 33author:Sigel, Helmut.publisher:isbn10 | asin:0824796888print isbn13:9780824796884ebook isbn13:9780585378770language:subjectpublication date:lcc:ddc:subject:covernext page>

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Metal_Ions_in_Biological_Systems/0824796888/files/page_100.html Page 100mation. Whether the double helix can be considered as a catalyst is still a matter of discussion. It is safe to state that the double helix acts as a promoter and it seems important to devote further studies on these promoted reactions because of their fundamental interest in the chemistry of platinum(II) complexes as well as because of their potential applications.Recently, it was shown that the platinum-triamine complexes cis-[Pt(NH3)2(Am)Cl]+, in which Am is an amine ligand derived from pyrimidine, purine, and piperidine, are active against murine and human tumor systems [40]. This new series of platinum(II) antitumor agents, which initially form monofunctional adducts on DNA, violate the structure-activity relationship established for platinum complexes. According to the results presented in Sec. 4, one expects a two-step reaction (the cleavage of the Pt-Am bond which generates monofunctional cis-DDP adducts and then the formation of bifunctional crosslinks with adjacent bases) yielding the same adducts as those formed in the reaction between DNA and cis-DDP. It is not yet known which adducts (intrastrand and/or interstrand crosslinks) are involved in the antitumor activity of cis-DDP. It might be possible to form preferentially one kind of adduct by the right choice of Am, with Am favoring the recognition of sequences for intrastrand or interstrand crosslinks.It is important to explain the apparent disagreement in the rate of formation of trans-DDP adducts and their composition [79]. These experiments deal with samples containing different percentages of platinum residues. We find that in trans-DDP-modified DNA (0.005 platinum per nucleotide), the major adducts are the monofunctional adducts and the interstrand crosslinks, about 80% and 15%, respectively [32]. We exclude (G1,G3)- and (G1,G4)-intrastrand crosslinks as the major adducts. We exclude also that (G1,G3)-intrastrand crosslinks are first formed and then transformed into interstrand crosslinks. One can argue that in vivo even the interstrand crosslinks are hardly formed. The interstrand crosslinking reaction is slow and it is likely that most of the monofunctional adducts are trapped by compounds such as glutathione [79]. It is tempting to speculate that trans-DDP is clinically ineffective because it does not form bifunctional adducts with cellular DNA. Chemical modifications of the nonleaving groups which could favor the formation of bifunctional adducts, might be interesting to make derivatives of trans-DDP clinically active, as in fact shown in two recent reports [41,42].

Metal_Ions_in_Biological_Systems/0824796888/files/page_101.html Page 101Many of the small molecules used in human therapy, as cis-DDP, are not specific, i.e., they bind to several genes. The antisense or the antigene strategy appears very promising in the design of new specific drugs. In the antisense strategy, several studies have shown that oligonucleotides complementary to a given sequence of a mRNA, can control the stability and the function of the mRNA [43,44]. However, the binding of the oligonucleotides to their target sequences is reversible; thus it is difficult to completely block biological functions in a RNase-H-independent mechanism. To make the process irreversible, chemical and photoactivable reagents have been covalently linked to the oligonucleotides [43,44]. Non-sequence-specific reactions have often been observed for chemically induced crosslinks. Photochemical activation is difficult in the in vivo experiments. An advantage of the oligonucleotides containing trans-{Pt(NH3)2[d(GXG)-N7-G,N7-G]} intrastrand crosslinks is that under physiological conditions the crosslinks can be considered as stable as long as the oligonucleotides are single-stranded. The rearrangement into interstrand crosslinks occurs only when the oligonucleotides bind to their targets. The same goal can be achieved with the oligonucleotides containing cis-[Pt(NH3)2(Am)(dG)]n+ [reaction (4)]. However, the rates of the crosslinking reactions have to be increased to make this application useful in the in vivo experiments. Recent data (unpublished) indicate that under some conditions most of the intrastrand crosslinks are transformed in less than 1 hr. As concerns the antigene strategy, it is not yet known whether the triplex formed by the binding of the platinated strand to the complementary duplex promotes the interstrand crosslinking reaction.To conclude, it is tempting to speculate that the active participation of the DNA double helix in the reaction between DNA, cis- or trans-DDP, and heterocyclic amines is an example of reactions occurring in more elaborate systems such as the complexes between DNA, metal ions, and proteins.AcknowledgmentsThis work was supported in part by la Ligue contre le Cancer, l'Association pour la Recherche sur le Cancer, la Fondation pour la Recherche Mdicale, and the EU contracts (CHRX-CT92-0016, CHRX-CT 94-0482).

Metal_Ions_in_Biological_Systems/0824796888/files/page_102.html Page 102AbbreviationsAmheterocyclic amineDDPdiamminedichloroplatinum(II)MDAPN-methyl-2,7-diazapyreniumNMRnuclear magnetic resonancePupurinePypyrimidineXstands for A (adenine), G (guanine) or T (thymine) in several DNA sequencesReferences1. S. L. Bruhn, J. H. Toney, and S. J. Lippard in Progress in Inorganic Chemistry: Bioinorganic Chemistry, Vol. 38 (S. J. Lippard, ed.), John Wiley and Sons, New York, 1990, p. 477 ff.2. J. Reedijk, Inorg. Chim. Acta, 198, 873 (1992).3. A. Eastman, Pharmacol. Ther., 34, 155 (1987).4. M. Sip and M. Leng in Nucleic Acids and Molecular Biology, Vol 7 (F. Eckstein and D. M. J. Lilley, eds.), Springer-Verlag, Berlin, 1993, p. 1 ff.5. A. M. Fichtinger-Shepman, J. L. and van de Veer, P. H. Lohman, and J. Reedijk, Biochemistry, 24, 707 (1985).6. C. A. Lepre and S. J. Lippard in Nucleic Acids and Molecular Biology, Vol. 4 (F. Eckstein and D. M. J. Lilley, eds.), Springer-Verlag, Berlin, 1990, p. 9 ff.7. A. Eastman and M. A. Barry, Biochemistry, 26, 3303 (1987).8. A. Eastman, M. M. Jennerwein, and D. L. Nagel, Chem. Biol. Interact., 67, 71 (1988).9. D. P. Bancroft, C. A. Lepre, and S. J. Lippard, J. Am. Chem. Soc., 112, 6860 (1990).10. V. Brabec and M. Leng, Proc. Natl. Acad. Sci. USA, 90, 5345 (1993).11. J. M. Malinge and M. Leng, Nucl. Acids Res., 16, 7663 (1988).

Metal_Ions_in_Biological_Systems/0824796888/files/page_103.html Page 10312. D. Payet, F. Gaucheron, M. Sip, and M. Leng, Nucl. Acids Res., 21, 5846 (1993).13. S. K. C. Elmroth and S. J. Lippard, J. Am. Chem. Soc., 116, 3633 (1994).14. A. Pullman and B. Pullman, Quart. Rev. Biophys., 14, 289 (1981).15. P. J. Hagerman, Annu. Rev. Biochem., 59, 755 (1990).16. O. Kennard and W. N. Hunter, Quart. Rev. Biophys., 22, 327 (1989).17. A. A. Travers, Annu. Rev. Biochem., 58, 427 (1989).18. J. Ramstein and R. Lavery, J. Biomol. Struct. Dynam., 7, 915 (1990).19. J. L. Leroy, E. Charretier, M. Kochoyan, and M. Gueron, Biochemistry, 27, 8894 (1988).20. V. Brabec, M. Sip and M. Leng, Biochemistry, 32, 11676 (1993).21. V. Brabec, V. Kleinwchter, J. L. Butour and N. P. Johnson, Biophys. Chem., 35, 129 (1990).22. J. Kozelka and J. C. Chottard, Biophys. Chem., 35, 165 (1990).23. S. F. Bellon and S. J. Lippard, Biophys. Chem., 35, 179 (1990).24. F. Herman, J. Kozelka, V. Stoven, E. Guittet, J. P. Girault, T. Huynh-Dinh, J. Igolen, J. Y. Lallemand, and J. C. Chottard, Eur. J. Biochem., 194, 119 (1990).25. J. Kozelka, M-H. Fouchet, and J. C. Chottard, Eur. J. Biochem., 205, 895 (1992).26. J. M. Malinge, C. Perez, and M. Leng, Nucl. Acids Res., 22, 3834 (1994).27. K. M. Comess, C. E. Costello, and S. J. Lippard, Biochemistry, 29, 2102 (1990).28. R. Dalbis, M. Boudvillain, and M. Leng, Nucl. Acids Res., 23, 949 (1995).29. R. Dalbis, D. Payet, and M. Leng, Proc. Natl. Acad. Sci. USA, 91, 8147 (1994).30. C. A. Lepre, L. Chassot, C. E. Castello, and S. J. Lippard, Biochemistry, 29, 811 (1990).31. M. F. Anin and M. Leng, Nucl. Acids Res., 18, 4395 (1990).32. M. Boudvillain, R. Dalbis, and M. Leng Nucl. Acids Res., 23, 2381 (1995).

Metal_Ions_in_Biological_Systems/0824796888/files/page_104.html Page 10433. J. M. Malinge and M. Leng, Proc. Natl. Acad. Sci. USA, 86, 6317 (1986).34. J. M. Malinge, A. Schwartz, and M. Leng, Nucl. Acids Res., 15, 1779 (1987).35. J. M. Malinge, M. Sip, A. J. Blacker, J. M. Lehn, and M. Leng, Nucl. Acids Res., 18, 3887 (1990).36. W. I. Sundquist, D. P. Bancroft, L. Chassot, and S. J. Lippard, J. Am. Chem. Soc., 110, 8559 (1988).37. T. Ren, D. P. Bancroft, W. I. Sundquist, A. Masschelein, M. V. Keck, and S. J. Lippard, J. Am. Chem. Soc., 115, 11341 (1993).38. F. Gaucheron, J. M. Malinge, A. J. Blacker, J. M. Lehn, and M. Leng, Proc. Natl. Acad. Sci. USA, 88, 3516 (1991).39. D. Payet and M. Leng, in Structural Biology: the State of the Art, Vol. 2 (R. H. Sarma and M. H. Sarma, eds.), Adenine, Guilderland, NY, p. 325 ff.40. L. S. Hollis, A. R. Amundsen, and E. W. Stern, J. Med. Chem., 32, 128 (1989).41. N. Farrell, L. R. Kelland, J. D. Roberts, and M. Van Beusichen, Cancer Res., 52, 5065 (1992).42. M. Coluccia, A. Nassi, F. Loseto, A. Boccarelli, M. A. Maiggio, D. Giordano, F. P. Intimi, P. Caputo, and G. Natile, J. Med. Chem., 36, 510 (1993).43. C. Hlne and J. J. Toulm, Biochim. Biophys. Acta, 1049, 99 (1990).44. W. Marshall and M. H. Caruthers, Science, 259, 1564 (1993).

Metal_Ions_in_Biological_Systems/0824796888/files/page_105.html Page 1055Trans-Diammineplatinum(II):What Makes It Different from cis-DDP?Coordination Chemistry of a Neglected Relative of Cisplatin and Its Interaction with Nucleic AcidsBernhard LippertFachbereich Chemie, Universitt Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany1. Introduction1062. Basic Properties of trans-a2PtCl21072.1. Synthesis of trans-(NH3)2PtCl21072.2. Properties of trans-(NH3)2PtCl2 and Differentiation from Its cis Isomer1082.3. trans-(NH3)2PtCl2 Analogs; Complex Isomerization1092.4. Solvolysis of trans-(NH3)2PtCl21102.4.1. Mono- and Diaqua Species1102.4.2. Acid-Base Equilibria1112.4.3. Selected Examples1123. Biological Effects1133.1. Toxicity, Antitumor Activity, Mutagenicity of trans-DDP1133.2. Novel Active trans Compounds1134. Reactions with Nucleic Acids114

Metal_Ions_in_Biological_Systems/0824796888/files/page_106.html Page 1064.1. Kinetics of DNA Adduct Formation1144.2. Spectrum of DNA Adducts of trans-DDP1144.3. Macroscopic Effects of Transplatin Binding1154.4. Staining of tRNAs1165. Reactions with Defined Oligonucleotides1165.1. Single-Stranded Oligonucleotides1165.2. 1,3-Intrastrand Crosslinking in Double-Stranded Oligos1165.3. Interstrand G,C Crosslinking1185.4. Linkage Isomerization Reactions1186. Model Studies1196.1. Mono(nucleobase) Complexes1206.2. Bis(nucleobase) Complexes and Derivatives1226.2.1. Trans-a2PtL2 Compounds1226.2.2. Heteronuclear Derivatives of trans-[a2PtL2]n+1236.3. Mixed Nucleobase Complexes1256.3.1. Metal-Modified Base Pairs1256.3.2. Dimetalated Triples and Cyclic Quartets1266.4. Combining cis- and trans-DDP1286.5. Trans-a2Pt(IV) Nucleobase Complexes1296.6. Ternary Nucleobase/Amino Acid Complexes1296.7. Other trans-a2Pt(II) Nucleobase Complexes1296.8. Trans-a2Pd(II) Nucleobase Complexes1307. Summary130Abbreviations132References1331IntroductionThe high specificity of reactions between biomolecules depends on the proper chiralities of the partners. Thus the optical isomers of a certain molecule may have completely different effects, one being reactive, the other one being totally unreactive or a competitive inhibitor or even a

Metal_Ions_in_Biological_Systems/0824796888/files/page_107.html Page 107toxic agent. In many cases these differences can be explained on the basis of the ''key-and-lock" principle. Chiral metal complexes, when interacting with chiral biomolecules, are no exception to it.The dissimilar effects of cis- vs. trans-(NH3)2PtCl2 in living cells represent a rare case of geometrical isomers of a metal compound causing pronounced differences in biological systems [1]. While the cis isomer exhibits remarkable activity in many tumor systems and is now a clinically important antitumor drug, the trans isomer has a much lower cytotoxic potency and for this reason is not useful as a drug. This observation holds for many analogs of these two compounds, even though there seem to be occasional exceptions. The obvious question, "What makes the two isomers so different?" cannot be answered at this stage. Despite the fact that many comparative studies have been undertaken, no really satisfactory picture has emerged as yet. With DNA being widely considered the crucial target of antitumor-active cis-a2Pt(II) compounds, research has concentrated in particular on possible differences in reactivity of the two isomers with this molecule, on differences in adducts, as well as differential repair of DNA lesions.In this chapter, the attempt is made to survey literature data both on the basic chemistry of trans-(NH3)2PtCl2 and its relatives and their reactions with DNA, oligonucleotides, and model nucleobases that may be relevant to the question posed in the title.2Basic Properties of trans-a2PtCl22.1Synthesis of trans-(NH3)2PtCl2There are a number of ways according to which trans-diamminedichloroplatinum(II), trans-(NH3)2PtCl2 (transplatin), can be prepared [2]. The first documented synthesis of transplatin is by J. Reiset ("Reiset's second chloride") who obtained it by heating dry [Pt(NH3)4]Cl2 at 250C [3]. A major disadvantage of this methodextensive decomposition to Pt(0)can be overcome if the temperature is kept at 190195C and a reduced pressure applied [4]. This modification also allows the preparation of isotopically labeled compounds, e.g., of trans-Pt(15NH3)2Cl2. Alternatively, and probably still the most common procedure of preparation, is the action of concentrated HCl on an aqueous solution of

Metal_Ions_in_Biological_Systems/0824796888/files/page_108.html Page 108[Pt(NH3)4]Cl2, originally proposed by Peyrone [5] and later modified by Kauffman and Cowan [6]. It is still the method of choice to prepare transplatin analogs.2.2Properties of trans-(NH3)2PtCl2 and Differentiation from Its cis IsomerBasic-physico properties of trans-(NH3)2PtCl2, as determined prior to 1957 [2] and 1973 [7], respectively, have been compiled. They include, among others, solubility data, electronic spectra, infrared (IR) spectra, and a qualitative description of the bonding properties. More recent data have been obtained by luminescence, magnetic circular dichroism (CD), nuclear quadrupole resonance, and extended Hckel molecular orbital (EHMO) calculations [8]. X-ray crystallography has revealed the solid state structure of trans-(NH3)2PtCl2 [9,10]: The compound crystallizes in the monoclinic system, space group P21/a with a = 7.99(1) , b = 6.00(1) , c = 5.45(1) , b = 95.2(2), U = 260.2 3, Z = 2 (120 5 K). Bond lengths are 2.05(4) for PtN and 2.32(1) for PtCl. In many cases, a comparison with the corresponding cis analog has been made. A convenient method of differentiation of the two isomers has been Raman spectroscopy, due to differences in molecular symmetry (D2h for the trans isomer; C2v for the cis isomer) and characteristic differences in skeletal vibrational modes [11]. Alternative methods applied involve derivatization of either isomer (allyl alcohol [12] or thiourea [13,14]) and subsequent analysis by ultraviolet-visible (UV-vis) spectroscopy [12] or high-performance liquid chromatography (HPLC) [13,14]. Thiourea (tu) derivatization (''Kurnakow test" [15]) of cis- and trans-(NH3)2PtCl2 leads to yellow [Pt(tu)4]Cl2 and colorless trans-[Pt(NH3)2(tu)2]Cl2, respectively. Both compounds have been crystallized and X-ray structurally characterized [16].195Pt nuclear magnetic resonance (NMR) spectroscopy, despite its improvement in sensitivity in recent years, does not readily differentiate between the two isomers, e.g., d195Pt, 2101 ppm for trans-(NH3)2-PtCl2 and 2104 ppm for cis-(NH3)2PtCl2 [4]. Similarly, chemical shifts in the 15N NMR spectra differ by only 0.4 ppm (66.3 and 65.9 ppm, respectively [4]), but J(Pt-N) coupling constants are markedly different, 278 Hz for the trans isomer and 303 Hz for the cis form.

Metal_Ions_in_Biological_Systems/0824796888/files/page_109.html Page 1092.3trans-(NH3)2PtCl2 Analogs; Complex IsomerizationAnalogs of transplatin are generated by changing the ammonia ligands, the halogens, or the square planar metal ions. In the following, only Pt compounds containing N and Cl donor atoms will be considered, with a brief look at the Pd(II) analog of transplatin.The simplest Pt analogs of transplatin are the methylamine compound, trans-(MeNH2)2PtCl2 [17], the dimethylamine compound, trans-(Me2NH)2PtCl2 [18], as well as the trimethylamine species, trans-(Me3N)2PtCl2 [19]. X-ray crystal structure analyses are available for the MeNH2 [17] and Me2NH [18] compounds. A number of analogs with larger amine ligands, e.g., trans-(cyclohexylamine)2PtCl2 [20], has likewise been characterized. There appears to be no X-ray crystal structure analysis available of a mixed amine complex of the type trans-(a)-(a')PtCl2 except for an example with a = NH3 and a' = 1-methylcytosine [21] (see also Sec. 6.7). Finally, there is a long list of transplatin analogs containing N-heterocyclic ligands. For example, trans-(py)2PtCl2 is prepared in analogy to transplatin from [Pt(py)4]Cl2 in aqueous HCl [22]. Alternatively, it can also be obtained from the corresponding cis compound, prepared from K2PtCl4 and pyridine (py) in aqueous solution, upon isomerization in nonaqueous solvents such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) in the presence of free ligand (py) [23,24]. With substituted pyrimidines (pym), an analogous isomerization has been reported [24] and verified by X-ray analysis [25]. Interestingly, the reverse processisomerization from the trans to the cis formis accomplished in 4 M HCl in the heat [25]. This process involves protonation of the coordinated pym ligand and temporary displacement of pymH+.Isomerization reactions in solution (during simple recrystallization [26] or photochemically [27]) or in the solid state [28] are a common phenomenon in Pt(II) chemistry [29], but in the majority of cases cis isomers are more prone to isomerization than trans compounds.The Pd(II) analog of transplatin is prepared in analogy to the latter from [Pd(NH3)4]2+ upon precipitation with HCl [30]. While structural analogs of trans-(NH3)2PdCl2 with NH3 replaced by heterocyclic ligands [31], including nucleobases [32], are known and X-ray structurally characterized, it is well known that the corresponding diaqua

Metal_Ions_in_Biological_Systems/0824796888/files/page_11.html Page 11''particle-mesh-Ewald" method was recently successfully applied to MD simulations of fully solvated ubiquitin, and DNA and RNA oligonucleotides [42].2.2.3Representation of the SolventApplications of MD approaches to DNA can be classified into three groups [45]: (1) simulations with implicit inclusion of environment effects, e.g., by reducing the phosphate charge [46] or adding "hydrated" counterions [47]; (2) simulations using NOE constraints which reflect all effects, including those of the environment [26,27]; (3) simulations taking into account solvent and counterions explicitly. The most extended MD simulations of the latter type have been carried out by the Beveridge group, and the reader is referred to their recent review [48]. We will limit ourselves to emphasizing that the methodology is far from being well established, and so far no convincing accord between a detailed NMR study and an MD simulation has been achieved. The fact that in all studies (except one; see below) with explicit water representation artefactual base-pair dissociation occurred, unless the Watson-Crick hydrogen bonds were reinforced by (weak) harmonic constraints, suggests that there remain some fundamental problems of the force field to be solved. The recent communication by Cheatham et al., according to which the particle-mesh-Ewald technique yielded stable MD trajectories for solvated DNA and RNA oligonucleotides [42], hints that the truncation of long-range electrostatic interactions could have been a principal cause of the artefactual helix denaturation.One possible source of error is the water model itself. Most of the studies have employed the rigid body models SPC or TIP3P, which both fail to reproduce the experimental diffusion constant of water. Daggett and Levitt, who compared different water models [49], therefore raised the question of how reasonable the simulated motions of a solute can be when the water motion is 6075% too fast. Two features of the standard water models have been made responsible for the inappropriately high diffusion constant: the rigidity of the O-H bonds [49,50] and the neglect of atomic polarization [51,52]. Allowing for flexibility of the O-H bonds [49,50] or inclusion of atomic polarization in the electrostatic energy [51,52] decreased the diffusion constant toward the experimental value.

Metal_Ions_in_Biological_Systems/0824796888/files/page_110.html Page 110species, trans-[(NH3)2Pd(H2O)2]2+, undergoes ligand disproportionation to [(NH3)Pd(H2O)3]2+ and [(NH3)3Pd(H2O)]2+ as well as isomerization to cis-[(NH3)2Pd(H2O)2]2+ [33]. For nucleobase chemistry related to this aspect, see also Sec. 6.8.2.4Solvolysis of trans-(NH3)2PtCl22.4.1Mono- and Diaqua SpeciesSolvolysis of the Cl ligands of trans-(NH3)2PtCl2 in water takes place in two steps (Fig. 1). Reported equilibrium constants for K1 range from 23.9 105 M [34], 32 105 M [35], and 48 12105 M [36] to 62.2 105 M [37]. K2 values are estimated to be 2 105 M [35,36]. These values indicate that, compared to cisplatin, spontaneous hydrolysis of the trans isomer is considerably reduced, at least by a factor of 10 for the first step and a factor of 20 for the second one. For practical purposes this means that chloride hydrolysis of trans-(NH3)2PtCl2 is only significant in very dilute solutions free of added Cl. In an acidic aqueous solution containing transplatin at a concentration of 102 M, for example, some 20 3% (depending on K1 values used) of trans-[(NH3)2PtCl(H2O)]+ exists if equilibrium is reached.From the kinetic measurements [3437] that were used for calculating the thermodynamic equilibrium constants, it is evident that, although the rate constant k1 for the first hydrolysis step is faster for the trans isomer as compared to the cis isomer (consequence of the higher kinetic trans effect of Cl over NH3), the reverse reaction (k1) is likewise faster for the trans isomer, thus leading in essence to a smaller thermodynamic equilibrium constant K1. The second hydrolysis step k2 is faster for the cis isomer. Reported rate constants for transplatin are as follows: k1 [s1], 9.8 105 [35], 1.9 105 [37], k1 [M1 s1], 3.05 Fig.1.Stepwisehydrolysisoftrans-(NH3)2PtCl2.

Metal_Ions_in_Biological_Systems/0824796888/files/page_111.html Page 111102 [37]; k2 [s1], Cd2+ >> Mn2+.The transition metal ions Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ can also stabilize intermolecular PuPuPy triple-stranded structures of the type d(AG)nd(GA)nd(TC)n [42]. As already indicated, Mg2+ and Ca2+ ions stabilize only the sequences which contain the GGC triplets. In contrast, transition metal ions enhance the stability of the structures containing both the GGC and AAT triplets. An interesting hypothesis [44] has been put forward to explain the observed difference. According to this hypothesis, divalent metal cations can stabilize the PuPuPy triplexes by phosphate charge screening and enhancement of Hoogsteen hydrogen bonds. Transition metal ions with higher affinity to the purine bases are able to polarize both adenine and guanine (Fig. 6). The polarization would strengthen the Hoogsteen hydrogen bond in which the bases participate, leading to a greater overall stabilization of both GGC and AAT triplets. The effect of polarization by metal ions on the stability of Watson-Crick base pairs in the double-helical DNA has already been substantiated by recent theoretical calculations [45].4Quadruplexes4.1Telomeres and AptamersThe existence of planar 6-oxopurine quartets, held together by four pairs of hydrogen bonds in a cyclic arrangement, appears to have been first proposed in the late 1950s for polyI [46] and a few years later for monomeric guanine nucleosides and nucleotides (for reviews, see

Metal_Ions_in_Biological_Systems/0824796888/files/page_155.html Page 155Fig.6.StrengtheningoftheHoogsteen-typehydrogenbondinducedbymetalionbindingtoN7ofpurinesofthethirdstrand.(Reproducedwithpermissionfrom[44].)[47,48]). By use of X-ray fiber diffraction methods, this structural motif was eventually confirmed also for polyG [49,50]. Meanwhile a number of NMR solution studies on guanine-rich oligos is available, which conclusively demonstrate the existence of guanine quartets [51,52], as do two high-resolution single-crystal X-ray structures of the Oxytricha telomere repeat d(GGGGTTTTGGGG) [53] and of a parallel-stranded tetraplex formed by d(TGGGGT) [54]. Both X-ray and NMR solution studies reveal a surprising structural variability around the central guanine quartets (Fig. 7), which refers to strand and glycosidic bond orientation, (anti or syn, for a review, see [55]). Guanine quartets can form within a folded single oligonucleotide molecule [56,57] by association of four single strands (all parallel) [54,58] or by association of two DNA hairpins in antiparallel orientation [53,59]. Moreover, solid state and solution structure may differ with respect to loop topologies [60] and complicated equilibria between various forms may exist in solution.Interest in the biological significance of guanine quartets primarily stems from suggestions that the G-rich 3' single-strand overhangs of chromosome ends (''telomeres") can associate to quadruplex structures

Metal_Ions_in_Biological_Systems/0824796888/files/page_156.html Page 156Fig.7.HydrogenbondingpatternwithinaGquartet.Thecationnecessaryforstabilizationmaybeeitherinthecenterofthequartetorbetweenadjacentquartets.[48,61]. Moreover, tetraplex formation has been implicated in the HIV-1 genome dimerization [62], in the recombination of certain regions of immunoglobulin genes [58], as well as in a number of other instances [60]. Finally, in vitro selected DNA oligomers sharing a highly conserved G-rich region of 1417 nucleotides, with a high affinity for the blood-clotting protein thrombin (''thrombin aptamers") [63], fold back in a way as to generate compact structures containing two guanine quartets [56,57].4.2Role of Metal IonsEven at an early stage the importance of metal cations was recognized in the formation and stabilization of guanine quartets (with monomeric building blocks) as well as guanine quadruplexes (with oligonucleotides; "G4-DNA") [47]. While Na+ K+, and Rb+ salts were found to be essential for the formation of ordered G tetrads, with K+ being most effective, Li+ and Cs+ salts did not generate G tetrads. This finding clearly

Metal_Ions_in_Biological_Systems/0824796888/files/page_157.html Page 157pointed to a size effect of the cation rather than a charge effect and suggested that metal cations are integral parts of guanine quartets. Among other methods, 1H NMR was instrumental in proving this point [64]. While it has generally been accepted that the steric fit of the cation, either in the G4 plane or, more likely, in the cavity between stacked G quartets, is the sole determinant of the ion selectivity, recent free energy perturbation calculations have been interpreted in terms of an additional metal-specific electronic effect also being important [65].Definitive evidence for the location of metal cations in G-quadruplex structures comes from the two single-crystal X-ray structure analyses mentioned above [53,54]. Although the positions are not well defined due to disorder, in the Oxytricha telomere structure potassium ions are somewhat asymmetrically located between two levels of G quartets [53]. The parallel-stranded TG4T tetraplex structure has been solved at 1.2- resolution [54]. In this structure, sodium ions are identified as lying along the axis of the tetraplex, coordinated by eight O6 oxygens of guanines. Interestingly, aquated Ca2+ ions also present in the crystal are not directly involved in binding to the tetraplex but rather interact with the phosphate oxygens of the DNA backbone in an outer-sphere fashion via their aqua ligands.The G-quadruplex-forming effect of K+ can lead to the paradoxical situation of preventing the formation of G4-DNA in certain G-rich sequences by overstabilizing transient guanine quartets, which then do not permit formation of long G4 structures [66]. In contrast, Na+ and Rb+ do not show this effect but rather allow formation of extended G4 DNA structures.There are relatively few studies on the effects of divalent alkaline earth metal cations on G4-DNA formation [6771]. The G4-DNA stabilizing effect of these divalent cations follows the order Sr2+ > Ba2+ > Ca2+ > Mg2+ with Sr2+ being the most effective ion of all cations studied thus far [71]. Concentrations required for the formation of G tetrads in DNA oligomers containing terminal TGTG3TGTGTGTG3 sequences are in the millimolar range for Sr2+, compared to 100-fold higher concentrations in the case of alkali cations.Virtually nothing is known about the effect of other metal cations, in particular exogenous ones with a high binding preference for N7 of guanine. It is tempting to speculate on the possible effect of a cationic Pt species binding to these sensitive G regions. It is certainly feasible that quadruplex formation is seriously hampered or even prevented.

Metal_Ions_in_Biological_Systems/0824796888/files/page_158.html Page 1584.3Other Four-Stranded Helices and SuperstructuresSupported by the presence of G quartets, quartet formation of bases other than guanine may be anticipated. For the RNA tetraplex (UGGGGU)4 the existence of a uracil quartet at either end of the four central G quartets has been postulated [72]. Similarly, cyclic thymine quartets have been suggested to be present in quadruplexes formed by d(G5T5) [73]. Quartet formation between pairs of A and T oligonucleotides has likewise been suggested [7477] and theoretical calculations on conformational parameters and potential energies of several feasible arrangements of (AT)4 quartets have been performed [76].Finally, parallel-stranded guanine tetraplexes are able to form superstructures with two, three, and even four tetraplexes bonded front-to-back [78]. While a metal cation effect has been noted for the latter phenomenon, the possible role of metal cations in stabilizing, for example, (AT)4 structures appears not to have been studied as yet.5Four-Way DNA JunctionsFor processes leading to a rearrangement of DNA such as genetic recombination, a central intermediate, the so-called Holliday junction, has been postulated, in which the four strands of two recombining helices cross [79]. This junction, with the help of recombination proteins, can migrate along DNA before being cleaved to regenerate two separate DNA duplexes. According to a model proposed by Lilley and colleagues [8082], the helical arms of the four-way junction are arranged in a stacked, X-shaped structure (Fig. 8). It is important to recognize that two of the four DNA strands undergo a dramatic change of direction (bending) at the exchange point.This specific tertiary structure crucially depends on the presence of certain metal cations: The dipositive aqua cations of Mg2+, Ca2+, and Ni2+ and in particular the tripositive [Co(NH3)6]3+ stabilize this arrangement very efficiently, whereas the monovalent Na+ and K+ induce a partial folding of the junction only [80,83]. Similarly, if Mg2+ is complexed by ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to give the anionic [Mg(EDTA)]2 species, the structure is completely different,

Metal_Ions_in_Biological_Systems/0824796888/files/page_159.html Page 159Fig.8.SchematicrepresentationofHollidayjunction.OfthefourDNAstrands,twoundergosharpchangesindirectionandaremostlikelytorequireMg2+forstabilization.(Reproducedwithpermissionfrom[80].)with the four arms unstacked and fully extended. These conclusions are the result of electrophoretic mobility studies of digests of Holliday junctions in the presence of a variety of cations [83]. The situation with DNA four-way junctions is thus reminiscent of that of tRNAs where Mg2+ stabilizes in particular regions where bends and turns occur. In the absence of Mg2+, both the characteristic tertiary structure and the biological activity of tRNAs are lost. From model building it is evident that negatively charged clefts, generated by phosphate oxygens near the four-way junction, call for specific metal binding as opposed to simple charge neutralization [80,83]. The high efficiency of [Co(NH3)6]3+ (effective concentration 2 M as compared to 25 M for spermine and 100 M for Ca2+) points to hydrogen bonding between metal ligands (here NH3) and phosphate oxygens and/or nucleobase donor sites as being another important factor besides charge.Some insight into the possible role of hydrated Ca2+ ions in the stability of four-way junctions has been provided by a very recent crystallographic research on the B-DNA decamer CTCTCGAGAG [84]. The structure of this decamer shows a crossed arrangement of helices in the

Metal_Ions_in_Biological_Systems/0824796888/files/page_16.html Page 16tional force fields, the atomic charges are not explicitly supplied in a parameter file but derived from two fundamental atomic parameters, the electronegativity ci and the hardness hi, corresponding to the first and second derivatives of the electrostatic energy with respect to the atomic charge qi, respectively. The charges are determined at the initial geometry by minimizing the electrostatic energy (9) with the constraint that the total charge is equal to the charge of the system.A disputable point is the fact that the charges are calculated only once, at the initial geometry. During the energy minimization or MD simulation, the geometry can change and therefore the initially determined charges may no longer be adequate. As pointed out by Shi [65], recalculation of charges during the calculation is, on the one hand, desirable, but on the other hand, could be dangerous, since the structure may become temporarily distorted during the energy minimization or MD simulation, which could lead to physically unreasonable charges.2.3.3.4ValidationThe ESFF force field has been validated by comparing energy-minimized structures with X-ray data of 579 compounds covering the first six rows of the periodic table [64]. However, the really stringent test on organic macromolecules, including their adducts with metal complexes, which will prove whether ESFF allows the correct identification of low-energy conformations, remains to be carried out. The novel energy functional terms will be implemented in the Discover 95.0 version of the Biosym software.3Results Overview:Modeling of Platinum-Oligonucleotide ComplexesAs we have seen in the preceding section, modeling of both nucleic acids and transition metal complexes are domains in development. It is therefore not astonishing that efforts to model transition metal complexes with nucleic acids or their constituents have been scarce so far. The only area where extensive force field calculations were applied to a

Metal_Ions_in_Biological_Systems/0824796888/files/page_160.html Page 160crystal lattice, producing a feasible model for the Holliday junction. The interactions between the helices are facilitated by a cluster of hydrated Ca2+ ions, which bind in the minor groove on either side of a close contact with a neighboring phosphate group.6Strand Crosslinking by Metal Ions6.1Interstrand CrosslinkingAs pointed out in the preceding sections, cationic metal species in virtually all cases are essential for stabilizing multistranded nucleic acid structures. Metal cations discussed so far either are coordinatively saturated and inert, e.g., [Co(NH3)6]3+, or form kinetically labile adducts with nucleic acids (alkali and alkaline earth ions, Zn2+,...). Apart from outer-sphere phosphate binding, metal binding to donor sites of the heterocyclic part of nucleobases (e.g., O6 and N7 of guanine) appears to also take place. Superficially, the cations stabilizing G4-DNA may be considered as crosslinking strands but nevertheless should not be compared with those transition metal cations that form strong covalent bonds between bases of different strands.The earliest studies on crosslinking of two DNA strands involved Hg2+. There have been many suggestions concerning the structure of DNA-Hg2+ interstrand crosslinks. These include the ''slippage model" with selective T-N3, T-N3 binding [86]; less selective binding to a variety of bases, including the amino groups of C and A [8789]; and insertion into A,T base pairs with T-O4, L-N6 binding [90]). Despite work that goes back to a time when the DNA double-helical structure was still unknown [85], and despite the many suggestions on how DNA-Hg2+ interstra