Kinetic analysis of the reaction between d(TTGGCCAA) and [Pt(NH3)3(H2O)]2+ by enzymatic degradation of the products and ESI and MALDI mass spectrometries.

The kinetics of the reaction between the octanucleotide d(TTGGCCAA) in the single-stranded form in pure water and the platinum complex [Pt(NH3)3(H2O)]2+ was investigated by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometries coupled with enzymatic degradation of the adducts. These methods led to the determination of specific rate constants of platination. The global rate constant characteristic of the formation of adducts on each 5'- or 3'-guanine were measured by electrospray ionization analysis. The ratios between the 5'- and 3'-adducts were determined from enzymatic degradation of the final reaction mixture and MALDI analysis. The platination in water is approximately eight times faster than in 0.1 M NaClO4. The selectivity of platination is a factor of 2 in favor of the 5'-guanine, and similar to that observed for the reaction between d(CTGGCTCA) and [Pt(NH3)3(H2O)]2+ in 0.1 M NaClO4.

How does cisplatin alter DNA structure? A molecular mechanics study on double-stranded oligonucleotides.

Molecular models for two double-stranded decanucleotides, d(GCCG*G*ATCGC)-d(GCGATCCGGC) (1) and d(GCTG*G*ATCGC)-d(GCGATCCAGC) (2), with the G* guanines cross-linked by a cis-Pt(NH3)2 moiety, were calculated using molecular mechanics. Nine models for 1 and eight models for 2 are reported; in all of them, the double helix is kinked by approx. 60 degrees towards the major groove and slightly unwound. The model building has been guided by comparison with the NMR data available for duplex 1. The influence of the base at the 5'-side of the coordinated G*G* dinucleotide is discussed.

Investigation of sulfur containing amino acids at the lipoxygenase active site using a platinum complex.

Inactivation of native soybean lipoxygenase-1 was observed upon preincubation with (NEt4)[PtCl3(P(Bun)3)]. Removal of the platinum complex(es) from the inactivated enzyme by treatment with sodium diethyldithiocarbamate (Naddtc) which reverses methionine but not cysteine binding, restores most of the activity. Linoleic acid, an enzyme substrate, protects it from inactivation. The quenching of the fluorescence of the putative active site tryptophans which accompanies inactivation disappears after Naddtc reactivation. The (NEt4)[PtCl3(P(Bun)3)]-inactivated enzyme iron(II) cannot be oxidized at variance with that of the native or Naddtc reactivated enzyme, as checked by EPR spectroscopy. These results show that at least one methionine is close to the iron binding site in soybean lipoxygenase-1.

Comparison between HPLC and capillary electrophoresis for the separation and identification of the platination products of oligonucleotides with cis-[Pt(NH3)2(H2O)2]2+ and [Pt(NH3)3(H2O)]2+.

HPLC and capillary electrophoresis (CE) were compared in order to separate the platinum adducts formed upon reaction of the double-stranded 18-mer d(AACGGTTAACCGTTAATT)2 I with the two complexes cis-[Pt(NH3)2(H2O)2]2+ 1 and [Pt(NH3)3(H2O)]2+ 2 and of the single-stranded octamer d(CTGGCTCA) II with complex 2. The GG sequence in both oligonucleotides is the only site of platination giving monoadducts on either of the two guanines or the diadduct which is the N7,N7 chelate of the Pt(NH3)(2+)2 moiety. The HPLC separation of the adducts was performed with a reverse-phase technique similar to that previously used for platinated oligonucleotides up to octamers. The adducts were identified by enzymatic digestion followed by mass spectrometry characterization of the digests collected at the outlet of the column. The CE separation of the platination products of I with 2 and 1 was performed using respectively a simple capillary zone electrophoresis technique and a micellar electrokinetic capillary chromatography technique. For the more difficult separation of the platination products of II with 2, a capillary gel electrophoresis techniques was required. In contrast to HPLC, CE gives better resolution the longer the oligonucleotide. Moreover, the separations are run with higher efficiency and time saving. However, with the present machines, CE precludes the recovery of sufficient amounts of material required by product identification.

H8 chemical shifts in oligonucleotide cross-linked at a GpG sequence by cis-Pt(NH3)2(2+): a clue to the adduct structure.

The origin of the anomalous H8 chemical shifts observed in 1H-NMR spectra of oligonucleotides cross-linked at a GpG sequence with cis-[Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum-ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 +/- 0.07 ppm (M. H. Fouchet, D. Lemaire, J. Kozelka and J.-C. Chottard, unpublished results); (b) the ring-current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5'-H8 and positive (deshielding) for the 3'-H8 in single-stranded adducts, but has the opposite sign in double-stranded adducts; (c) a deshielding polarization effect of the phosphate 5' to the GpG unit. The different signs of the ring-current effects in single-stranded and double-stranded oligonucleotides originate from the orientation of the guanines in the cis-[Pt(NH3)2(Gua)2]2+ moiety (Gua, guanine), which is left-handed helicoidal in single strands and right-handed helicoidal in double strands. In the platinated dinucleotides (cis-[Pt(NH3)2(GpG)]+, cis-[Pt(NH3)2(d(GpG))]+ and cis-[Pt(NH3)2(d(GpG))]), the guanines assume either the left-handed or the right-handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data.

[Modality of the antimitotic acitivity of the diamine cis-dichloroplatinum (II) studied on the plant cell]. / Modalités de l'activité antimitotique du cis-dichloro diammine platine (ii) étudiées sur la cellule végétale

In the order of decreasing doses, cis-platinum (II) diamine dichloride acting on root meristem of Allium sativum produces first an irreversible metostatic effect quickly followed by lethal effect, then a variable mitodepression accompanied by important chromosomal alterations: numerous disruptions and stickiness. This chromatoclasic effect persists at very low concentrations but stathmodieretic effect is not observed. The chromatoclasic activity of the compound is compared to that of other antitumoral drugs.

Probing the mechanism of an Mn2+-dependent ribozyme by means of platinum complexes.

The smallest ribozyme system known is the pentanucleotide GAAACp, which is specifically cleaved by Mn2+, in the presence of poly(U), generating guanosine 2',3'-cyclic phosphate and AAACp. A plausible mechanism has been proposed, involving the participation of two Mn2+ with structural and catalytic roles, the first one cross-linking the two N7 atoms of G1 and A4, and the other binding to the N7 atom of A2 and activating the 2'-OH group of G1 [Kazakov, S. & Altman, S. (1992) Proc. Natl Acad. Sci. USA 89, 7939-7943]. In the present work, we have utilized the high affinity of Pt(II) complexes for N7 atoms of purines in an attempt to form a stable active ribozyme by replacing the structural Mn2+ by Pt2+. We thus replaced the proposed kinetically labile G1N7-Mn2+-A4N7 cross-link by an inert N7-trans-Pt(NH3)(2)(2+)-N7 cross-link. In a complementary investigation, the N7 atoms of the individual purines of GAAACp were selectively blocked by a Pt(NH3)(3)(2+) residue to determine which of the N7 sites participate in the Mn2+-mediated cleavage. Other N7-Pt(II)-N7 crosslinks were also investigated. Accordingly, we have prepared four monoadducts, each bearing the Pt(NH3)(3)(2+) residue on one of the purines and a series of chelates of trans-Pt(NH3)(2)(2+) and cis-Pt(NH3)(2)(2+) and have tested them for Mn2+-induced cleavage. Binding of Pt(NH3)(3)(2+) to G1 or A4 did not alter the efficiency of the specific cleavage between G1 and A2 catalyzed by Mn2+/poly(U), whereas cross-linking of G1 and A4 by trans-Pt(NH3)(2)(2+) inhibited it completely. Hence, a cross-link between G1 and A4 is not required for the site-specific cleavage. Binding of Pt(NH3)(3)(2+) to A2 or A3 strongly inhibits the G1/A2 cleavage, suggesting that these bases are likely to be involved in manganese coordination in the cleaving complex. A site-specific Mn2+-dependent cleavage between A4 and C5 was observed for the G1-A4 and G1-A3 adducts cross-linked by trans-Pt(NH3)(2)(2+), the G1-A2 adduct cross-linked by cis-Pt(NH3)(2)(2+), and the three monoadducts bearing the Pt(NH3)(3)(2+) residue on G1, A2 or A3; poly(U) did not exert any influence on this cleavage.

Nitrosoalkanes as Fe(II) ligands in the hemoglobin and myoglobin complexes formed from nitroalkanes in reducing conditions.

Primary and secondary aliphatic nitro compounds, R2CHNO2, react with myoglobin and hemoglobin, in the presence of sodium dithionite, leading to new complexes with Soret peaks respectively at 425 and 421 nm. These complexes are very stable even after disappearance of the starting nitro compounds and do not exchange their exogenous ligand after 10 h under 1 atm (101 325 Pa) CO. They are low-spin hexacoordinated myoglobin or hemoglobin complexes, as shown by the resonance Raman spectrum of the nitromethane-derived human hemoglobin complex which is similar to those of the known hemoglobin complexes with O2, CO, NO and nitrosobenzene. Evidence has been produced to show that the nitro compounds themselves do not bind to the hemoproteins; we propose that among the reduction derivatives produced in situ by dithionite, the corresponding unstable nitroso monomers, whose nitroso group is isoelectronic with dioxygen, are the actual ligands of the 425-nm or 421-nm-absorbing complexes.

A complete kinetic study of GG versus AG plantination suggests that the doubly aquated derivatives of cisplatin are the actual DNA binding species.

The hairpin-stabilized double-stranded oligonucleotides d(TATGGTATT4ATACCATA) (I) and d(TATAGTATT4ATACTATA) (II) were allowed to react with the three aquated forms of the antitumor drug cisplatin (cis-[PtCl2(NH3)2], 1) which are likely candidates for DNA binding, that is, cis-[PtC1(NH3)2(H2O)]+ (2), cis-[Pt(NH3)2(H2O)2]2+ (3), and its conjugate base cis-[Pt(OH)(NH3)2(H2O)]+ (4). The reaction between I and [Pt(NH3)3(H2O)]2+ (5) was also studied for comparison. All reactions were monitored by HPLC. The platination reactions of I and II were carried out in NaClO4 (0.1M) at 293 K and at a constant pH of 4.5 +/- 0.1 for 2, 3, and 5. The data relative to the platination by 4 were obtained from measurements in unbuffered NaClO4 solutions (0.1M) at a starting pH close to neutrality, where 3 and 4 are present in equilibrium. In this case, a fit function describing the pH-time curve allowed the determination of the actual concentrations of 3, 4, and the dihydroxo complex. The platination rate constants characterizing the bimolecular reactions between either I or II and 2, 3, and 4 were individually determined along with the rate constants for hydrolysis of the chloro-monoadducts and for the chelation reactions of the aqua-monoadducts. The reactivity of compounds 2-5, which have the general formula cis-[Pt(NH3)2(H2O)(Y)]2+/-, decreases in the order 3>4>5>>2, that is, Y= H2O > OH- >NH3 >> Cl-, which is the order of decreasing hydrogen-bond donating ability of Y. Deprotonation of 3 to 4 reduces the reactivity of the platinum complex only by a factor of approximately equals 2, and both complexes discriminate between the different purines of I and II in the same manner. Whereas 3 and 4 react approximately three times faster with the GG sequence of I than with the AG sequence of II, 2 shows a similar reactivity towards both sequences. In view of the well-established preferential binding of cisplatin to GG sequences of DNA in vivo and in vitro, this result suggests that the actual DNA platination species are derived from double hydrolysis of cisplatin.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of DNA-Pt(II) complexes.

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been used to characterize the reaction products of the 18-mer deoxyribonucleotide d(AACGGTTAACCGTTAATT) with [Pt(NH3)3(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+. Characteristic peaks corresponding to different monofunctional adducts (18-mer+n[Pt(NH3)3]) (n = 1, 2, 3 and 4) have been observed with the triamino-monoaqua complex. With the diamino-diaqua cis-Pt complex, formation of a chelate (18-mer+[Pt(NH3)2]) involving two adjacent guanines has been demonstrated. A good correlation between MALDI and polyacrylamide gel electrophoresis results is observed.

Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli.

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP[d(ApG)] adducts, although they account for only 25% of the lesions formed, are approximately 5 times more mutagenic than the major GG adduct. We report the construction of vectors bearing a single cisDDP[d(ApG)] lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A----T transversions are mainly observed (80%), whereas A----G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP[d(ApG)] adducts are not blocking lesions. The high mutation specificity of cisDDP[d(ApG)]-induced mutagenesis is discussed in relation to structural data.

Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation.

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.

Nitrosoalkanes as Fe(II) ligands in the 455-nm-absorbing cytochrome P-450 complexes formed from nitroalkanes in reducing conditions.

Primary and secondary aliphatic nitroalkanes RR'CHNO2, react with microsomal cytochrome p-450 in the presence of dithionite leading to new complexes with a Soret peak at 455 nm. The formation of these complexes is inhibited completely by CO and partially by metyrapone. However, once formed, their exogenous ligand is not displaced by excess CO. By deoxycholate treatment they are transformed into 423-nm-absorbing cytochrome P-420 complexes, which are spectrally similar to the corresponding RR'CHNO2-derived myoglobin complexes. The 455-nm-absorbing complexes are equally produced from RR'CHNO2 reduction, microsomal NADPH-dependent oxidation of the corresponding hydroxylamine RR'CHNHOH, or interaction of the nitrosodimer (RR'CHNO)2 with reduced cytochrome P-450. All the reported results are consistent with the involvement of a new class of ligands of cytochrome P-450-Fe(II), which are the unstable aliphatic nitrosomonomers RR'CHNO, whose nitroso group is isoelectronic with dioxygen and whose stabilisation results from their strong binding to heme-Fe(II), thus explaining the observed inhibition of the hydroxylating function of cytochrome P-450.

Formation of complexes between microsomal cytochrome P-450-Fe(II) and nitrosoarenes obtained by oxidation of arylhydroxylamines or reduction of nitroarenes in situ.

A cytochrome P-450 complex exhibiting a Soret peak at 454 nm is formed by direct interaction of nitrosobenzene with NADPH-reduced rat liver microsomes in anaerobic conditions, by reaction of phenylhydroxylamine with aerobic microsomes or during nitrobenzene reduction by NADPH-reduced or dithionite-reduced microsomes. In the latter conditions, the complex formation is only transient as it is unstable to dithionite. Analogous reactions with myoglobin lead to the previously described myoglobin-Fe(II)-nitrosobenzene complex which has similar properties to those of the 454-nm-absorbing cytochrome P-450 complex. This analogy, together with the various conditions of its formation, strongly indicates that it is a cytochrome-P-450-Fe(II)-nitrosobenzene complex. The corresponding complex with the 4-chloro-nitrosobenzene ligand is formed in similar conditions. Cytochrome P-450-Fe(II) complexes with nitrosoarenes seem less stable than the previously described complexes with nitrosoalkanes.

Platination of the (T2G4)4 telomeric sequence: a structural and cross-linking study.

The telomeric sequence (T(2)G(4))(4) was platinated in aqueous solutions containing 50 mM LiClO(4), NaClO(4), or KClO(4). The identification of the guanines which reacted with [Pt(NH(3))(3)(H(2)O)](2+) revealed that the same type of folding exists in the presence of the three cations and that the latter determine the relative stabilities of the G-quadruplex structures in the order K(+) > Na(+) >> Li(+). The tri-ammine complex yielded ca. 40--90% of adducts, mono- and poly-platinated, bound to 4 guanines out of the 16 guanines in the sequence, in the decreasing amounts G9 > G15 >> G3 > G21. The formation of these adducts was interpreted with a G-quadruplex structure obtained by restrained molecular dynamics (rMD) simulations which confirms the schematic model proposed by Williamson et al. [(1989) Cell 59, 871--880]. The bifunctional complexes cis- and trans-[Pt(NH(3))(2)(H(2)O)(2)](2+) also first reacted with G9 and G15 and gave cross-linked adducts between two guanines, which did not exceed 5% each of the products formed. Both the cis and trans isomers formed a G3-G15 platinum chelate, and the second also formed bis-chelates at both ends of the G-quadruplex structure: G3-G15/G9-G21 and G3-G15/G9-G24. The rMD simulations showed that the cross-linking reactions by the trans complex can occur without disturbing the stacking of the three G-quartets.

Interaction of cis-[Pt(NH3)2(H2O)2](NO3)2 with ribose deoxyribose diguanosine phosphates.

The three diguanosine phosphates GpG (4 X 10(-4) M), d(GpG) (10(-5) M), and d(pGpG) (10(-5) M) have been reacted with cis-[Pt(NH3)2(H2O)2](NO3)2 (1 Pt/dinucleotide) in water at pH 5.5 and 37 degrees C. In each case a single product is formed. The three complexes have been characterized by proton nuclear magnetic resonance (1H NMR) and circular dichroism (CD) analyses. They are N(7)-N(7) chelates of the metal with an anti-anti configuration of the bases. They present a conformational change upon deprotonation of guanine N(1)H whose pKa is ca. 8.7 (D2O). Their CD spectra, compared to those of the free dinucleotides, exhibit an increase of ellipticity in the 275-nm region, which can be qualitatively related to the characteristic increase reported for platinated DNA and poly(dG) . poly(dC). These results are in favor of the hypothesis of intrastrand cross-linking of adjacent guanines, by the cis-PtII(NH3)2 moiety, after a local denaturation of DNA.