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.

Determination by electrospray mass spectrometry of the outersphere association constants of DNA/platinum complexes using 20-mer oligonucleotides and ([Pt(NH(3))(4)](2+), 2Cl(-)) or ([Pt(py)(4)](2+), 2Cl(-)).

The cytotoxic effects of cisplatin, cis-diamminedichloroplatinum(II), are generally ascribed to the formation of DNA adducts. Several in vitro as well as in vivo studies showed that cisplatin binds preferentially to guanines belonging to (G)(n) sequences (n > or = 2). After mono- or diaquation of cisplatin, giving the cationic complexes cis-[PtCl(NH(3))(2)(H(2)O)](+) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), DNA platination occurs in two steps: the cationic complex gives an outersphere association with DNA and the actual coordination then occurs by substitution of one aqua ligand by guanine-N7. For a better understanding of the (G)(n) selectivity of cisplatin giving the biologically active adducts, also necessary for the conception of new platinum drugs, the respective contribution of the outersphere association and actual guanine platination must be investigated. To check the role of outersphere association in the overall platination process, we used electrospray mass spectrometry (ESMS) to detect and quantify outersphere association between 20-mer oligonucleotides and platinum complexes. The 20-mer oligonucleotides were single- or double-stranded, with the same number of guanines either isolated or adjacent to each other. To deal only with outersphere association and check the influence of platinum ligands, the [Pt(NH(3))(4)](2+) and [Pt(py)(4)](2+) complexes were used. We characterized by ESMS all the different outersphere association species formed during titration of each oligonucleotide with the various platinum complexes and evaluated their affinity constants. The ESMS results demonstrate that the outersphere association depends on electrostatic interactions and on the ability of the platinum ligands to participate to hydrogen bonding, particularly within the duplex form.

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.

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.

Platination of a GG site on single-stranded and double-stranded forms of a 14-base oligonucleotide with diaqua cisplatin followed by NMR and HPLC -- influence of the platinum ligands and base sequence on 5'-G versus 3'-G platination selectivity.

Detailed studies of the kinetics of platination of the single-stranded 14-base DNA oligonucleotide d(ATACATGGTACATA) and the corresponding duplex by cis-[Pt(NH3)2(H2O)2]2+ show that HPLC and NMR are complementary methods which provide similar results. The 5'-G and 3'-G monofunctional intermediates were trapped, separated and characterized by NMR (via 15NH3 labeling) and enzymatic digestion followed by mass spectrometry. The kinetic data are compared with those for the corresponding reactions of cis-[PtCl2(NH3)2] (cisplatin) and its monohydrolysed analogue. For both single and double strands of the oligonucleotide, the aqua complex shows little selectivity for the 5'-G or the 3'-G in the initial platination step, whereas the chloro-complex preferentially platinates the 3'-G. The base on the 3' side of the GG sequence appears to play an important role in controlling this selectivity; replacement of T by C increases the selectivity of duplex platination by the diaqua complex by a factor of about 6, and the selectivity of chelation of the 3'-G monofunctional adduct by a factor of about 3. In general the reactivity of the 5'-G in a GG sequence appears to be enhanced in a duplex compared with a single-strand. For both the aqua-monoadduct and chloro-monoadduct, cis-[Pt(NH3)2(N7G)(H2O or Cl)], the 5'-G monoadduct is much longer lived (t1/2 approximately 4 h at 288 K for aqua, 80 h at 298 K for chloro) than the 3'-G monoadduct (t1/2 < or = 45 min at 288 K for aqua, 6 h at 298 K for chloro). Inspection of molecular mechanics models of the end states of various monofunctional adducts provided insight into H-bonding and destacking interactions in these adducts and the sequence selectivity observed in their formation. Such adducts may play an important role in the mechanism of action of platinum anticancer drugs.

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.

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.

Binding of the Escherichia coli UvrAB proteins to the DNA mono- and diadducts of cis-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II ) and cisplatin. Analysis of the factors controlling recognition and proof of monoadduct-mediated UvrB-DNA cross-linking.

The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatin um(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t 1/2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleo-protein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.

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.

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.

Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).

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.

Soybean lipoxygenases-1, -2a, -2b and -2c do not contain PQQ.

Soybean isoenzymes lipoxygenases-1, -2a, -2b and -2c were examined spectroscopically for the presence of covalently bound pyrrolo quinoline quinone (PQQ) after derivatization by phenylhydrazine (PH), 2,4-dinitrophenylhydrazine (DNPH) and 3-methyl-2-benzothiazolinone hydrazone (MBTH). DNPH derivatization of PQQ after a pronase digestion step of lipoxygenase-1 in the presence of an anion exchange gel fixing the cofactor was also investigated. None of these experiments provided evidence for the presence of PQQ contrary to previous report by Van der Meer et al (1). We have checked, by EPR spectroscopy, that the three reactants used were able to reduce the active site ferric iron. Our results were confirmed by the absence of enzyme inhibition by cis- and trans-1,2-diaminocyclohexane or benzylamine in the presence of NaBH3CN which have been reported to react with PQQ and to inactivate quinoproteins (2,3).

A d(GpG)-platinated decanucleotide duplex is kinked. An extended NMR and molecular mechanics study.

A conformational study of the double-stranded decanucleotide d(GCCG*G*ATCGC).d(GCGATCCGGC), with the G* guanines chelating a cis-Pt(NH3)2 moiety, has been accomplished using 1H and 31P NMR, and molecular mechanics. Correlation of the NMR data with molecular models has disclosed an equilibrium between several kinked conformations and has ruled out an unkinked structure. The deformation is localized at the CG*G*.CCG trinucleotide where the helix is kinked by approximately 60 degrees towards the major groove and unwound by 12-19 degrees. The models revealed an unexpected mobility of the cytosine complementary to the 5'-G*. This cytosine can stack on either branch of the kinked complementary strand. The energy barrier between the two positions has been calculated to be less than or equal to 12 kJ/mol. The NMR data are in support of rapid flip-flopping of this cytosine. An explanation for the strong downfield shift observed in the 31P resonance of the G*pG* phosphate is given.

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.

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.

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.

Hexanal phenylhydrazone is a mechanism-based inactivator of soybean lipoxygenase 1.

Hexanal phenylhydrazone (1; 70:30 E:Z mixture) at micromolar concentration irreversibly inactivates soybean lipoxygenase 1 (L-1) in the presence of dioxygen. L-1 catalyzes the oxidation of 1 into its alpha-azo hydroperoxide 2 [C5H11CH(OOH)N = NC6H5]. 2 is an efficient inactivator of L-1. The aerobic reaction between 1 and L-1 follows a branched pathway leading to the release of 2 into the medium or to L-1 inactivation. The respective parameters corresponding to this inactivation by the (E)-1 and (Z)-1 isomers are Ki = 0.25 and 0.40 microM and kinact = 0.8 and 2.1 min-1. Linoleic acid protection agrees with a mechanism-based inactivation process. The oxidation of a minimum of 13 +/- 3 molar equiv of 1 is required for complete L-1 inactivation, but up to 70 equiv is necessary in the presence of a very large excess of 1. The inactivation is actually the result of two pathways: one is due to a reaction of 2 as soon as it is formed at the active site (20%); the other is due to 2 released into the medium and coming back to the active site (80%). The inactivation is accompanied by the oxidation of 1.8 +/- 0.8 methionine residues of the protein into the corresponding sulfoxide. The inactivated L-1 is electron paramagnetic resonance (EPR) silent with an effective magnetic moment of mu = 5.0 +/- 0.1 Bohr magnetons corresponding to an S = 2 spin state. An inactivation mechanism is proposed on the basis of EPR and magnetic susceptibility data obtained from the anaerobic and aerobic reactions of L-1 with 1 and 2.

A high melting cis-[Pt(NH3)2[d(GpG)]]adduct of a decanucleotide duplex.

The [cis-Pt(NH3)2(d(GCCGGATCGC)-N7(4), N7(5))]-d(GCGATCCGGC) duplex has been prepared with Tm = 49 degrees C (vs 58 degrees C for the unplatinated form). NMR of the ten observable imino protons supports a kinked structure with intact base pairing of the duplex on the 3'-side of the d(GpG).cis-Pt chelate (relative to the platinated strand) The modification of the B-DNA type CD spectrum, due to the platinum chelate, is comparable to that observed for the platination (at a 0.05 Pt:base ratio) of the Micrococcus Lysodeikticus DNA (72% GC).