Rathindra N. Bose

HPLC Determination of Binding of Cisplatin to DNA in the Presence of Biological Thiols: Implications of Dominant Platinum-Thiol Binding to Its Anticancer Action

AbstractPurpose. The purpose of this work is to evaluate the extent of the binding of cisplatin (cis-diamminedichloroplatinum(II)) to DNA in the presence and absence of biological thiols, glutathione, and cysteine, and to test the hypothesis whether the platinum-thiol complexes can serve as a drug reservoir for subsequent binding to DNA. Methods. Reactions of cisplatin (50 μM to 1.0 mM) with calf thymus DNA (870 μM to 6.75 mM) in the presence and absence of glutathione and cysteine (0 to 10mM) were carried out at pH 4.4, 7.0, and 7.3. Following the reactions, the DNA was enzymatically digested with nucleases, separated by RP HPLC, and analyzed to determine the extent of DNA binding. The method was independently verified by proton NMR measurements. Results. At neutral pH, and equimolar concentrations of DNA and thiols, only a very small amount of platinum (<5%) was coordinated to DNA, and most of the platinum was coordinated to the thiols. At pH 4.4, binding to DNA was dominant over the binding to thiols. No conversion of platinum-thiol to platinum-DNA complexes was observed up to 7 days of incubation. Conclusion. At physiological pH, the cisplatin was exclusively coordinated to biological thiols and platinum-DNA was a minor adduct. Data presented in this paper does not support the “drug reservoir” hypothesis.

Kinetic analysis of the cis-diamminedichloroplatinum(II)--cysteine reaction: implications to the extent of platinum--DNA binding.

The reaction between cis-diamminedichloroplatinum(II) (cis-DDP) and L-cysteine was examined at neutral pH at 37 degrees C. The reaction proceeds through a Pt(NH3)2 (cys)Cl intermediate which undergoes parallel reactions with a second molecule of cysteine to form a bis(cysteine) complex, Pt(NH3)2(cys)2 and with the starting platinum complex to form a cysteine-bridged dinuclear complex. In the presence of excess cysteine, the product is predominantly the bis(cysteine) complex. The intermediate is formed by the direct reaction of the platinum complex with cysteine with a bimolecular rate constant 2.2 +/- 0.2 x 10(-2) M-1.s-1 at 37 degrees C as well as through a rapid reaction with the mono aqua-platinum complex. The rate constant for the formation of the dimer was evaluated to be 0.24 +/- 0.4 M-1.s-1, an order of magnitude higher than that for the mononuclear complex formation. The intermediate reacts with a second cysteine molecule with a bimolecular rate constant, 5.6 +/- 0.4 x 10(-2) M-1.s-1. The rate constant for the equation of Pt(NH3)2(cys)Cl was evaluated to be 1.8 +/- 0.2 10(-4) s-1. The Pt-195 chemical shifts for the mono(cysteine), bis(cysteine), and cysteine bridged dimer were found to be -3308, -3705, and -3104 ppm. The bis(cysteine) complex at neutral pH undergoes slow reaction (t1/2 approximately equal to four days) to form a secondary product, presumably Pt(NH3)(cys)2, in which one cysteine acts a bidentate chelating agent. In acidic solution, with equimolar concentrations of cysteine and diaqua-platinum complex, the reaction predominantly yielded a cysteine bridged dimeric complex. When cysteine concentration was increased fourfold over the platinum complex, the bis(cysteine) chelate with complete removal of coordinated ammonia appeared as the dominant product. The platinum-195 chemical shift for this chelate was found to be -3290 ppm. Considering the abundance of thiols in amino acids/peptides and replication enzymes in the cellular milieu, it remains to be seen how platinum complexes react with DNA. Direct platination to replication enzymes as a possible mechanism for antineoplactic activity is yet to be ruled out.

Platinum(II) catalysis and radical intervention in reductions of platinum(IV) antitumor drugs by ascorbic acid.

Reductions of four platinum(IV) amine complexes, cis-diamminetetrachloroplatinum(IV), tetraammine-cis-dichloroplatinum(IV), cis,cis,trans-diamminedichlorodihydroxoplatinum(IV), and cis,trans,cis-dichloro-dihydroxo-bis(isopropylamine)platinum(IV) by ascorbic acid were catalyzed by platinum(II) at pH 7.3 and 22 degrees C. Except for the first mentioned compound, initial slow uncatalyzed reductions yielded platinum(II) products which served as catalyst as revealed by the presence of induction periods and their disappearance by the addition of the platinum(II) products. The platinum(II) catalysis generated ascorbate bound platinum(IV) intermediates. An internal electron transfer process within these intermediates led to the formation of platinum(II) complexes. Although the rate constants for the uncatalyzed reductions vary greatly depending on the nature of the ligands and their spatial arrangements, the magnitudes of the platinum(II) catalyzed rate constants fall in the narrow range, 100 to 300 M(-2) s(-1). The values of the uncatalyzed reductions lie in the range 5 x 10(-2) to 15 M(-1) s(-1), the tetrachloroplatinum(IV) complex suffered the faster reduction. The reduction of iproplatin with two hydroxide ligands in trans configuration was the slowest. The internal electron transfer rate constants span two orders of magnitude, from 0.15 to 4 x 10(-3) s(-1). These reactions were accompanied by the formation of the ascorbate radical which persists throughout the entire reaction. Although the tetrachloro species exhibited simple second order reduction, first order in each of the reactants, the rate of reduction was also accelerated by the addition of cis-diamminedichoroplatinum(II) indicating the presence of catalysis in this reaction as well.

TEABr directed synthesis of ZSM-12 and its NMR characterization

Abstract Synthesis of ZSM-12 using tetraethylammonium bromide (TEABr) as the template was investigated. Among the various parameters that affect the crystallization of ZSM-12, aluminum content of the gel, OH − /SiO 2 and TEA/SiO 2 ratios were the important determinants. Systematic variations of these parameters revealed that the TEABr-assisted synthesis had many similarities to the synthesis using TEAOH template reported earlier, with the exception that the OH − /SiO 2 ratios had to be maintained at lower values. Furthermore, the OH − /SiO 2 ratios favorable for ZSM-12 formation lie in a very narrow range. The source of alkalinity also affected the rate of crystallization and the composition of the product. The crystallization was found to be faster and better incorporation of aluminum in the zeolite framework was obtained when NaOH was used to provide alkalinity rather than KOH. Successful synthesis of highly crystalline ZSM-12 samples with Si/Al ratio around 30 was achieved using a minimal amount of relatively inexpensive TEABr (TEA/SiO 2 =0.125). Aluminum-27 NMR spectroscopy unambiguously revealed that all aluminum atoms are incorporated in the zeolite framework in tetrahedral coordination. The NMR line-widths of aluminum signals of the calcined samples were significantly larger than those with template incorporated samples. Spin-lattice relaxation times, conventional and rotating frame, as well as magic angle spinning (MAS) cross-polarization data with variable contact time support that there is a significant proton reservoir in the aluminum framework. The NMR data indicate that many distorted tetrahedral sites are formed upon removal of the template and some of these sites contain Al–OH moieties.

Mechanisms of formation and decomposition of hypervalent chromium metabolites in the glutathione-chromium (VI) reaction.

A long-lived chromium(IV) intermediate is generated during the reaction between Cr(VI) and glutathione in glycine below pH 3. The intermediate reacts with the tripeptide to produce Cr(III) and oxidized glutathione. A dynamic magnetic susceptibility measurement based on a nuclear magnetic resonance method yielded a 2.8 microB magnetic movement for the chromium(IV) species. The intermediate is formed by parallel third-order and second-order processes. The third-order process (k = 5.9 x 10(2) M-2 s-1) involves first-order participation by each of the oxidant, reductant, and hydrogen ions. A hydrogen ion independent pathway leads to a sluggish second-order process (k = 0.11 M-1 s-1) that is first order with respect to reduced glutathione [GSH] and [Cr(VI)]. Chromium(IV) species is reduced to Cr(III) by a second-order process (k = 0.13 M-1 s-1) that is first order in each of [Cr(IV)] and [GSH] and does not depend on [H+]. At pH 3.4, a chromium(V) species was detected as a minor intermediate as well. In the pH range 6.5-7.5, three dominant chromium(V) intermediates were detected. The existence of Cr(IV) in low pH offers an opportunity to examine the mechanism of DNA damage by this rare oxidation state.

Synthesis, X-ray Crystallographic, and NMR Characterizations of Platinum(II) and Platinum(IV) Pyrophosphato Complexes †

A series of mononuclear cis-diamineplatinum(II) pyrophosphato complexes containing ammine (am), trans-1,2-cyclohexanediamine (dach), and 1,2-ethanediamine (en) as the amine ligands were synthesized and characterized by (31)P and (195)Pt NMR spectroscopy. Chemical shifts of (31)P NMR resonances of these completely deprotonated complexes appear at 2.12, 1.78, and 1.93 ppm, indicating a coordination chemical shift of at least 8 ppm. The (195)Pt NMR chemical shifts for the am and dach complexes were observed at -1503 and -1729 ppm. The complexes are highly stable at neutral pH; no aquation due to the release of either phosphate or amine ligands was observed within 48 h. Furthermore, no partial deligation of the pyrophosphate ligand was detected within several days at neutral pH. At lower pH, however, release of a pyrophosphate ion was observed with concomitant formation of a bridged pyrophosphatoplatinum(II) dinuclear complex. The extended crystal structure containing the dach ligand revealed a zigzag chain stacked in a head-to-tail fashion. Moreover, two zigzag chains are juxtaposed in a parallel fashion and supported by additional hydrogen bonds reminiscent of DNA structures where two strands of DNA bases are held by hydrogen bonds. Theoretical calculations support the notion that the two dinuclear units are held together primarily by hydrogen bonds between the amine and phosphate moieties. Platinum(II) pyrophosphato complexes were readily oxidized by hydrogen peroxide to yield cis-diamine-trans-dihydroxopyrophosphatoplatinum(IV) complexes. Two of these complexes, containing am and en, were characterized by X-ray crystallography. Notable structural features include Pt-O (phosphate) bond distances of 2.021-2.086 A and departures from 180 degrees in trans-HO-Pt-OH bond angles, >90 degrees in O-Pt-O, and >90 degrees in cis-N-Pt-N bond angles. The departure in the trans-HO-Pt-OH angle is more pronounced in the 1,2-ethanediamine complex compared to the dach analogue because of the existence of two molecules possessing enantiomeric conformations within the asymmetric unit. (31)P NMR spectra exhibited well-resolved (195)Pt satellites with coupling constants of 15.4 Hz for the ammine and 25.9 Hz for both the 1,2-ethanediamine and trans-1,2-cyclohexanediamine complexes. The (195)Pt NMR spectrum of the ammine complex clearly showed coupling with two equivalent N atoms.

DNA oxidation by peroxo-chromium(V) species: oxidation of guanosine to guanidinohydantoin

Reactions of peroxo-chromium(v) complexes with DNA afforded mainly guanine oxidation yielding, a four-electron oxidation product, guanidinohydantoin, and exhibited extensive base labile strand scission.

Potentially Deadly Carcinogenic Chromium Redox Cycle Involving Peroxochromium(IV) and Glutathione

Peroxochromium(IV) complexes are putative DNA-damaging and mutagenic agents in chromium(VI)-mediated carcinogenesis. The reaction between aquaethylenediaminebis(peroxo)chromium(IV) and glutathione at neutral pH exhibits a cyclic redox process displaying a persistent recycling of Cr(IV) and Cr(VI) with the intervention of chromium(V) intermediates. The coordination by a glutathione molecule triggers an autooxidation of the Cr(IV)-peroxo complex to Cr(VI) via an internal electron-transfer process followed by reduction to Cr(IV) via metastable chromium(V) intermediates. The cycle is repeated by the second peroxo species. The Cr(V) and -(IV) intermediates have been characterized as mono- and bisglutathionato complexes with or without a coordinated peroxo moiety. These intermediates are capable of damaging DNA, as evidenced by single strand breaks and DNA oxidation. The implication here is that the potential for a persistent, if not perpetual, deadly chromium carcinogenic cycle exists in the cellular milieu through the assistance of molecular oxygen and glutathione.

Phosphonato complexes of platinum(II): Kinetics of formation and phosphorus-31 NMR characterization studies

Reactions of cis-diamminedichloroplatinum(II) with phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and methylenediphosphonic acid (MDP) yield various phosphonatoplatinum(II) chelates which were characterized by phosphorus-31 NMR spectroscopy. The P-31 resonances for the chelates appear at 6-12 ppm downfield as compared to the uncomplexed ligands. All complexes exhibit monoprotic acidic behavior in the pH range 2-10. The chemical shift-pH profiles yielded acidity constants, 1.0 x 10(-4), 1.5 x 10(-4), and 1.3 x 10(-6) M-1, for the PFA, PAA, and MDP chelates. In addition to the monomeric chelate, MDP formed a bridged diplatinum(II,II) complex when it reacted with cis-Pt (NH3)2(H2O)2(2)+. The P-31 resonance for this binuclear complex appears at 22 ppm downfield from the unreacted ligand. Rate data for the complexation reactions of the phosphonate ligands with the dichloroplatinum complex are consistent with a mechanism in which a monodentate complex is formed initially through rate-limiting aquation process of the platinum complex, followed by a rapid chelation. For the PFA and PAA complexes, initial binding sites are the carboxylato oxygens. Implications of the various binding modes of the phosphonates in relationship to their antiviral activities are discussed.

Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase‐I were examined in the presence of the anti‐tumor drug cis‐diaminedichloroplatinum(II) and its inactive geometric isomer trans‐diaminedichloroplatinum(II). The trans‐isomer did not inhibit the enzyme activity. The anti‐tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis‐diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective pre‐incubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05–3.0 mM. A mechanism that deals with the formation of an initial platinum–polymerase‐I complex with a binding constant >105 M−1 followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.