Michael J. Clarke

Reduction and Subsequent Binding of Ruthenium Ions Catalyzed by Subcellular Components

The reduction of Cl(NH3)5Ru(III) and subsequent binding of heterocyclic ligands by the resultant (H2O)(NH3)5Ru(II) ion is shown to be catalyzed by components of rat-liver cells. The presence of air significantly decreases the rate of heterocyclic ligand binding. In the case of microsome and soluble component catalysis, this is probably due to oxidation of the Ru(II) ion prior to complexation. Various inhibitors of electron-transfer proteins were employed in an effort to determine the preferred reducing species. These results lend support to the hypothesis that the antitumor activity of acido ruthenium(III) ammine complexes involves activation by reduction in vivo prior to metal coordination to nucleic acids. Anticancer drugs functioning by this mechanism may be preferentially toxic to or may localize in hypoxic areas of tumors.

Electrochemistry, synthesis, and spectra of pentaammineruthenium(III) complexes of cytidine, adenosine, and related ligands

Abstract: The synthesis of pentaammineruthenium(II1) complexes of cytidine, adenosine, and tubercidin by redox catalysis is reported. The spectroscopic and chemical properties of these complexes favor coordination site assignments as the exocyclic nitrogens of cytidine, adenosine, and tubercidin, but do not preclude binding to the adjacent pyrimidine ring nitrogens for the corresponding Ru(I1) complexes. An N-7 coordinated complex of guanine is also reported. These complexes exhibit strong li- gand to metal charge transfer bands in the visible and near-ultraviolet. The cytidine and adenosine complexes are remarkably acidic (having pK, values of 3.15 and 3.64, respectively) with displacement of a proton from the exocyclic amine. The acidity of these complexes is attributed to the close juxtaposition of the Ru(II1) to the ionizable proton and to intramolecular hydrogen bonding between a coordinated ammine and an adjacent nitrogen following proton loss. Values of pK, for the Ru(I1) com- plexes have also been measured. Cyclic voltammetry studies indicate that the cytidine and adenosine complexes dissociate or isomerize following reduction to the Ru(I1) state. Implications of the chemistry of these complexes regarding their formation and behavior in biochemical systems are discussed.

Mutagenic and toxic effects of ruthenium.

Selected platinum and ruthenium complexes were tested for their ability to cause Salmonella typhimurium strains TA98 and TA100 to revert to histidine independence. The results indicate that ruthenium compounds are capable of reverting both strains while cis-Cl2(NH3)2Pt primarily causes reversions in strain TA100. In addition, cis-platinum is an order of magnitude more mutagenic and toxic than are the ruthenium complexes. Selected compounds were also tested for their ability to induce the bacterial SOS system in the Bacillus subtilis Comptest. In this system, cis-platinum similarly showed greater inducing ability than did the ruthenium complexes. These results also demonstrated that the nature of the sixth ligand in the ruthenium compounds has a significant effect on the mutagenic capacity of these agents.

Biochemical effects of binding [(H2O)(NH3)5RuII]2+ to DNA and oxidation to[(NH3)5RuIII]n—DNA.

Abstract The reaction of [(H 2 O)(NH 3 ) 5 Ru II ] 2+ with calf thymus and salmon sperm DNA has been studied over a wide fange of transition metal ion concentrations. Kinetic studies revealcd a biphasic reaction with an initial fairly rapid coordination of the metal ion being followed by slower reactions. Binding studies were done under pseudo-equilibrium conditions following completion of the initial rapid reaction. Spectra and HPLC of acid-hydrolyzed samples of [(NH 3 ) 5 Ru II ] n -DNA prepared by incubation of [(H 2 O)(NH 3 ) 5 Ru II ] 2+ with DNA (where [P DNA ] = 1.5 mM and reactant [Ru II ]/[P DNA ] ratios were in the fange 0.1 to 0.3) followed by air oxidation showed the predominant binding site on helical DNA to be in the major groove at the N-7 of guanine. The equilibrium constant for [(H 2 O)(NH 3 ) 5 Ru II ] 2+ binding to the G 7 site in helical CT DNA is 5.1 x 103. Differential pulse voltammetry exhibited a single peak at 48 mV, which is attributed to the reduction of Rum on the G 7 sites. At [Run]/[P DNA ] Tm values for the DNA decreased linearly with increasing ruthenium concentration and an increase in the intensity of the 565 nm dG→ Ru(III) charge transfer band was noted upon melting. The UV and CD spectra of these samples indicated no extensive destacking or alteration in geometry (B family) compared to unsubstituted DNA. At [Run]/[P DNA ]〉 0.5 or when single-stranded DNA was used, increased absorbance at 530 nm and 480 nm suggested additional binding to the exocyclic amine sites of adenine and cytosine residues. HPLC and individual spectrophotometric identification of the products derived from hydrolysis of these spec~es yielded both [(Gua)(NH 3 ) 5 Ru III ] and [(Ade)(NH 3 ) 5 Ru III ]. Earlier studies have established the cytidine and adenosine binding sites of [(NH 3 ) 5 Ru III ] to be at their exocyclic amines (C4 and A6). Coordination to these positions indicates disruption of the double helix since these amines are involved in hydrogen bonding on the interior of B-DNA. Agrose gel electrophoresis of superhelical pBR322 plasmid DNA after exposure to various complexes of [(nh 3 ) 5 Ru iii ] in the presence of a reductant and air generally revealed moderately efficient cleavage of the DNA, presumably due to the generation of hydroxyl radical via Fentons chemistry. However, similar studies involving [(NH 3 ) 5 Ru III ] directly coordinated to the DNA showed no strand cutting above background. Polyacrylamide gel electrophoresis of a 381 bp, 3′- 32 P-labeled fragment of pBR322 plasmid DNA containing low levels of bound [(NH 3 ) 5 Ru III ] further indicated negligible DNA cutting by the coordinated metal ion.

Pentaammineruthenium-guanine complexes

The synthesis of several pentaammineruthenium(I1 and 111)-guanine complexes is reported, in which the metal is believed to be bound to Ni. The Ru(II1) compounds exhibit a broad low energy guanine-to-metal chargetransfer absorption, while the Ru(1I) complexes show a metal-to-ligand charge transfer in the ultraviolet. The effect of Ru(I1 and 111) on the acidity of the protons at N1 and Ng is investigated. Electrochemical potentials are reported for the complexes over a broad pH range. At low pH the Ru(II1) nucleoside complexes undergo acidcatalyzed hydrolysis of the sugar-purine bond at a much slower rate than do the corresponding free nucleosides. he interaction of metal ions with nucleotides has T been the subject of considerable investigation for the past several years. Areas of interest include: the effect of metal ions on the stability of nucleic acids, 2, synthesis of heavy-atom derivatives as aids in determining the structure of RNA by X-ray crystallogr a ~ h y , ~ the participation of metal ions in the biological function of nucleic acids,5 the use of heavy metal derivatives of nucleosides as cytological stains,6 and the sequencing of nucleic acids by electron microscopy with the aid of a metal ion complex binding selectively to sites along the polynucleotide chain.7~8 (1) R. M. Izatt, J. J. Christensen, and J. H. Rytting, Chem. Rev., 71, (2) G. L. Eichhorn, eral., Adaan. Chem. Ser., No. 100,135 (1971). (3) G. L. Eichhorn, E. Tarien, and J. Butzow, Biochemistry, 10,2014 (4) S. H. Kim, G. J. Quigley, F. L. Suddath, A. McPherson, D. ( 5 ) W. Szer and S. Ochoa, J . Mol. Biol., 8 , 823 (1964). (6) R. J. Barrnett, J. Roy. Microsc. SOC., 83, 143 (1964). (7) M. Beer and I. Moudrianakis, Proc. Nut. Acad. Sci. U. S., 48, (8) D. Gibson, M. Beer, and R. Barrnett, Biochemistry, 10, 3699 439 (1971).

Ruthenium Chemistry Pertaining to the Design of Anticancer Agents

Ruthenium compounds hold particular promise in the design of new anticancer agents, including: a) chemotherapeutic drugs, b) radiopharmaceuticals for diagnostic imaging, and, c) most recently, radiosensitizers for radiotherapy. With regard to chemotherapeutic agents, complexes with nitrogen ligands and anionic leaving groups appear to be the most active as cytotoxic agents, with nuclear DNA usually assumed as the target site. Binding to DNA may occur in a variety of modes with ion-pairing and covalent bonding probably being the most important for active agents, often with the former occurring before the latter. After injection as a Ru(III) prodrug, selective localization in the tumor may take place by in vivo reduction to the more actively binding Ru(II) complex. Transferrin may also mediate the transfer of some ruthenium complexes to the tumor site. At least in the case of [(H2O)(NH3)5Ru]2 +, coordination to DNA occurs initially at G7 sites in the major groove of the DNA.. However, higher concentrations appear to cause uncoiling with subsequent binding to the exocyclic amines of A and C. On coordination to nucleosides, ammineruthenium ions can migrate between nitrogen sites or exist as rotamers on exocyclic ammines. Such metal ion movements can be choreographed by controlling the pH and electrochemical potential of the media. Square-wave voltammetry promises to be a sensitive method for probing the redox chemistry of ruthenium both on free nucleosides and on DNA, and for monitoring isomerization reactions following electron transfer. At high pH, Ru(III) induces the rapid autoxidation of nucleosides, in a manner reminiscent of xanthine oxidase, to yield 8-keto-purines. The anticancer activities of some selected ruthenium compounds are surveyed as to possible mechanisms consistent with their chemistry.

The influence of NO-containing ruthenium complexes on mouse hippocampal evoked potentials in vitro.

The influence of different, nitric oxide-containing ruthenium complexes on the evoked potentials recorded from the CA1 region of the mouse hippocampus in vitro has been investigated. Of the compounds tested, only trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 (1-2.5 mM) exerted a strong facilitatory action on the population spike, the EPSP, and the spontaneous activity. Its activity probably depends upon its ability to release NO following reduction. The phosphito ligand is important both in terms of adjusting the reduction potential of the complex to be biologically accessible and in labilizing the coordinated NO. The effects of this compound could not be reversed by perfusion. Scavenging NO in slices preincubated with oxyhemoglobin prior to the addition of this compound eliminated its neurophysiological effects. The control molecules trans-[(P(OEt)3)2(NH3)4Ru](PF6)2, trans-[(H2O)(P(OEt)3) (NH3)4Ru](PF6)3, and [(NO)(NH3)5Ru]Cl3, which are structurally similar, but unable to generate NO, were ineffective. NaNO2 suppressed neuronal firing. Attempts to induce Long-Term Potentiation (LTP) at the time of maximal effect of trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 were unsuccessful, suggesting that the mechanism of amplification induced by trans-[(NO)(P(OEt)3)(NH3)4Ru](PF6)3 and LTP may share common pathways.

Cellular effects of transferrin coordinated to [Cl(NH3)5Ru]Cl2 and cis-[Cl2(NH3)4Ru]Cl

Abstract Estimates of the net equilibrium binding constants for [(H 2 O)(NH 3 ) 5 Ru II ] 2+ , [Cl(NH 3 ) 5 Ru III ] 2+ , cis -[(H 2 O) 2 (NH 3 ) 4 Ru II ] 2+ and cis -[Cl 2 (NH 3 ) 4 Ru III ] + with apotransferrin (Tf) and holotransferrin (Fe 2 Tf) suggests that Ru III , but not Ru II complexes bind with a higher affinity to the iron binding sites. Several other presumably histidyl imidazole sites bind with approximately the same affinity ( K eff =10 2 to 10 3 M −1 ) to both Ru II and Ru III . Compared to HeLa cells, an order of magnitude higher level of nuclear DNA binding ([Ru] DNA /[P] DNA ) was required to achieve the same level of toxicity in Jurkat T ag cells, which probably relates to the substantially higher levels of cis -[Cl 2 (NH 3 ) 4 Ru] + needed to inhibit 50% of the cell growth in the Jurkat T ag cell line. Against Jurkat T ag cells, the toxicity of the pentaammineruthenium(III) group is enhanced by approximately two orders of magnitude upon binding primarily to the Fe-sites in apotransferrin, whereas the toxicity of the tetraammineruthenium(III) moiety is only marginally increased. Binding to Fe 2 Tf does not increase the toxicity of either group. Significant dissociation over 24 h of the ammineruthenium(III) ions from apotransferrin requires reduction to Ru II .

Synthesis and structure of trans-[O2(Im)4Tc]Cl•2H2O, trans-[O2(1-meIm)4Tc]Cl•3H2O and related compounds

Abstract The synthesis, spectra and HPLC behavior of a series of trans-dioxotechnetium(V) complexes with imidazole and bidentate nitrogen ligands are reported. Crystals of trans-[O2(Im)4Tc]Cl·2H2O were found to form in the monoclinic space group C2/c with unit cell parameters a = 13.249 (3) A, b = 11.239 (2) A, c = 14.358 (3) A and β = 115.56 (2)°. The cell volume is 1929 (1) A3 with Z = 4. The similar compound trans-[O2(1-melm)4Tc]Cl·3H2O crystallized in the same space group with a = 20.980 (5) A, b = 8.599 (3) A, c = 16.105 (3) A and β = 124.22 (2)°. The cell volume is 2402 (1) A3 with Z = 4. The unweighted R-factors for the two structures are 0.091 and 0.122, respectively. The TcO and TcN bond distances are essentially identical in the two structures and are 1.71 A and 2.15 A, respectively. The imidazole ligands are canted slightly by an average angle of 15° off a plane perpendicular to that of the technetium and nitrogens. Infrared absorbances assigned to the asymmetric OTcO stretch occur in the range 810–830 cm−1. The compounds are all diamagnetic due to the strong tetragonal distortion exerted by the oxo ligands. Proton NMR resonances are deshielded slightly relative to the free ligands. The imidazole complexes are unstable in aqueous solution and the bidentate amine complexes are unstable at low pH. HPLC capacity factors by reverse-phase ion-pair chromatography varied in an unexpected manner and, in general, decreased with increasing aliphatic chain length of the eluant anion.

Binding of pentaammineruthenium(III) to double-helical and single-stranded DNA

Aufgrund der in vitro biochemischen Bedeutung wird die Wechselwirkung eines oktaedrischen Metallionsmamlich (NH3)5Ru(OH2)2 mit helicaler und denaturierter DNA mit Hilfe absorptionsspektroskopischer Untersuchungen im sichtbaren Bereich diskutiert.