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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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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].
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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
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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
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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
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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