In-cell Catalysis by Tethered Organo-Osmium Complexes Generates Selectivity for Breast Cancer Cells.

Anticancer agents that exhibit catalytic mechanisms of action offer a unique multi-targeting strategy to overcome drug resistance. Nonetheless, many in-cell catalysts in development are hindered by deactivation by endogenous nucleophiles. We have synthesised a highly potent, stable Os-based 16-electron half-sandwich ('piano stool') catalyst by introducing a permanent covalent tether between the arene and chelated diamine ligand. This catalyst exhibits antiproliferative activity comparable to the clinical drug cisplatin towards triple-negative breast cancer cells and can overcome tamoxifen resistance. Speciation experiments revealed Os to be almost exclusively albumin-bound in the extracellular medium, while cellular accumulation studies identified an energy-dependent, protein-mediated Os accumulation pathway, consistent with albumin-mediated uptake. Importantly, the tethered Os complex was active for in-cell transfer hydrogenation catalysis, initiated by co-administration of a non-toxic dose of sodium formate as a source of hydride, indicating that the Os catalyst is delivered to the cytosol of cancer cells intact. The mechanism of action involves the generation of reactive oxygen species (ROS), thus exploiting the inherent redox vulnerability of cancer cells, accompanied by selectivity for cancerous cells over non-tumorigenic cells.

Radiosensitisation of human colorectal cancer cells by ruthenium(II) arene anticancer complexes.

Some of the largest improvements in clinical outcomes for patients with solid cancers observed over the past 3 decades have been from concurrent treatment with chemotherapy and radiotherapy (RT). The lethal effects of RT on cancer cells arise primarily from damage to DNA. Ruthenium (Ru) is a transition metal of the platinum group, with potentially less toxicity than platinum drugs. We postulated that ruthenium-arene complexes are radiosensitisers when used in combination with RT. We screened 14 ruthenium-arene complexes and identified AH54 and AH63 as supra-additive radiosensitisers by clonogenic survival assays and isobologram analyses. Both complexes displayed facial chirality. At clinically relevant doses of RT, radiosensitisation of cancer cells by AH54 and AH63 was p53-dependent. Radiation enhancement ratios for 5-10 micromolar drug concentrations ranged from 1.19 to 1.82. In p53-wildtype cells, both drugs induced significant G2 cell cycle arrest and apoptosis. Colorectal cancer cells deficient in DNA damage repair proteins, EME1 and MUS81, were significantly more sensitive to both agents. Both drugs were active in cancer cell lines displaying acquired resistance to oxaliplatin or cisplatin. Our findings broaden the potential scope for these drugs for use in cancer therapy, including combination with radiotherapy to treat colorectal cancer.

Microfluidic integration of photonic crystal fibers for online photochemical reaction analysis.

Liquid-filled hollow-core photonic crystal fibers (HC-PCFs) are perfect optofluidic channels, uniquely providing low-loss optical guidance in a liquid medium. As a result, the overlap of the dissolved specimen and the intense light field in the micronsized core is increased manyfold compared to conventional bioanalytical techniques, facilitating highly-efficient photoactivation processes. Here we introduce a novel integrated analytical technology for photochemistry by microfluidic coupling of a HC-PCF nanoflow reactor to supplementary detection devices. Applying a continuous flow through the fiber, we deliver photochemical reaction products to a mass spectrometer in an online and hence rapid fashion, which is highly advantageous over conventional cuvette-based approaches.

In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model.

We have compared the organometallic arene complexes [(eta(6)-biphenyl)M(ethylenediamine)Cl](+) RM175 (M=Ru(II)) and its isostructural osmium(II) analogue AFAP51 (M=Os(II)) for their ability to induce cell detachment resistance from fibronectin, collagen IV and poly-l-lysine, and cell re-adhesion after treatment, their effects on cell migration and cell viability, on matrix metalloproteinases production, and on primary tumour growth of MCa mammary carcinoma, the effect of human serum albumin on their cytotoxicity. There are differences between ruthenium and osmium. The Os complex is up to 6x more potent than RM175 towards highly-invasive breast MDA-MB-231, human breast MCF-7 and human epithelial HBL-100 cancer cells, but whereas RM175 was active against MCa mammary carcinoma in vivo and caused metastasis reduction, AFAP51 was not. Intriguingly the presence of human serum albumin in the growth medium enhanced the cytotoxicity of both compounds. RM175 increased the resistance of MDA-MB-231 cells to detachment from substrates and both compounds inhibited the production of MMP-2. These data confirm the key role of ruthenium itself in anti-metastatic activity. It will be interesting to explore the activity of osmium arene complexes in other tumour models and the possibility of changing the non-arene ligands to tune the anticancer activity of osmium in vivo.

Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds.

Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium(II) organometallic arene complexes in human ovarian cancer. Br J Cancer 2002;86(10):1652-7]. [(eta6-C6H5C6H5)Ru(en)Cl]+ (as a PF6 salt, where en=ethylenediamine (RM175)) has been evaluated in a 13-cell line panel. Particular sensitivity (approximately 10-fold lower than mean IC50) was noted in breast cancer and non-small cell lung cancer cell lines. In addition, IC50 in the A549 was 2 microM and RM175 (25 mg kg-1, days 1 and 5, i.p.) caused a significant (p=0.004) growth delay in a xenograft model. HC11 [(eta6-tetrahydroanthracene)Ru(en)Cl]PF6 was more potent in the A549 cell line (IC50 0.5 microM). HC11 (25 mg kg-1, days 1, 8 and 15, i.p.) was also active in vivo. Following RM175 25 mg kg-1, days 1 and 5, and 15 mg kg-1, days 1-5, HC11 25 and 40 mg kg-1, day 1, elevated alanine transaminase levels were detected, suggesting hepatotoxicity. No changes were observed in kidney or haematological parameters. In liver sections, multi-focal hepatic necrosis was seen, becoming confluent at high doses of HC11. In vitro studies confirmed that HC11 was more toxic than RM175 to fresh human hepatocytes and equitoxic to mithramycin. Liver toxicity may be related to the arene ligand and modification may reduce the potential for hepatic toxicity, while maintaining the anti-tumour activity seen.

Analgesia during radial artery cannulation: comparison of the effects of lidocaine applied by local injection or iontophoresis.

We compared the effect of lidocaine injection with lidocaine iontophoresis for pain relief during radial artery cannulation prior to induction of anaesthesia. Patients were allocated randomly to one of two groups. Group 1 (n = 15) received iontophoresis for 10 min prior to cannulation, using 4% lidocaine 4 ml. Group 2 (n = 15) received local infiltration of 1% lidocaine 1 ml using a 25G needle. Pain scores were recorded immediately after cannulation using a 10-cm visual analogue scale (VAS). There was no difference in mean (SD) pain scores [group 1: 2.2 (1.5) cm; group 2: 2.3 (2.7) cm; 95% CI of difference -1.8 to 1.5 cm]. Lidocaine delivered by iontophoresis is an effective and painless method of providing analgesia for radial artery cannulation.

Sequence-selective metalation of double-helical oligodeoxyribonucleotides with PtII, MnII, and ZnII ions.

Reactions of [PtCl(dien)](+) (dien=diethylenetriamine), Mn(2+) and Zn(2+) ions with three different double-helical oligodeoxyribonucleotides, which contain the central sequence GGXY (XY=AT, TA or CC) have been monitored by NMR spectroscopy. 2 D [(1)H, (15)N] HSQC/HMQC NMR spectroscopy using (15)N-labeled Pt(dien) shows that the rate of formation of 3'-G-N 7 and 5'-G-N 7 platinated adducts is highly sequence dependent. The relative rates of platination of 5'-G versus 3'-G are largest for the sequence -GGCC-, for which only a small fraction of the 3'-G adduct is formed; for -GGTA-, the rate of 5'-G platination is about eight times that of 3'-G, and for -GGAT- the ratio is 1.2. These values are in qualitative agreement with those obtained for G-N 7/Mn(2+) selectivity as determined by paramagnetic line broadening of the adjacent G-H 8, and also G-N 7/Zn(2+) selectivity as determined by G-H 8 chemical shift changes. Fluctuation in the nucleophilicity of G-N 7 may be explained by variation of the pi-stacking interaction between base residues along the double helix. The reaction mixtures containing platinated 3'-G and 5'-G fractions were separated by HPLC. Since the duplexes are self-complementary, the platinated single strands were readily annealed to duplexes with twofold symmetry and analyzed by 2 D [(1)H, (1)H] NOESY NMR spectroscopy. Unexpectedly, the 5'-G-H 8 resonance signals of both 5'-G and 3'-G platinated duplexes showed large downfield shifts in the range delta=0.3-0.6 ppm, while the 3'-G-H 8 resonance signals in both cases exhibited no, or only slight, upfield shifts. Resonance signals for several other protons in the central region undergo large chemical shift variations induced by platination, indicating that monofunctional binding to DNA leads to appreciable conformational changes.

In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer.

Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen novel ruthenium(II) organometallic arene complexes have been evaluated for activity (in vitro and in vivo) in models of human ovarian cancer, and cross-resistance profiles established in cisplatin and multi-drug-resistant variants. A broad range of IC50 values was obtained (0.5 to >100 microM) in A2780 parental cells with two compounds (RM175 and HC29) equipotent to carboplatin (6 microM), and the most active compound (HC11) equipotent to cisplatin (0.6 microM). Stable bi-dentate chelating ligands (ethylenediamine), a more hydrophobic arene ligand (tetrahydroanthracene) and a single ligand exchange centre (chloride) were associated with increased activity. None of the six active ruthenium(II) compounds were cross-resistant in the A2780cis cell line, demonstrated to be 10-fold resistant to cisplatin/carboplatin by a mechanism involving, at least in part, silencing of MLH1 protein expression via methylation. Varying degrees of cross-resistance were observed in the P-170 glycoprotein overexpressing multi-drug-resistant cell line 2780AD that could be reversed by co-treatment with verapamil. In vivo activity was established with RM175 in the A2780 xenograft together with non-cross-resistance in the A2780cis xenograft and a lack of activity in the 2780AD xenograft. High activity coupled to non cross-resistance in cisplatin resistant models merit further development of this novel group of anticancer compounds.

Interactions of bismuth with human lactoferrin and recognition of the Bi(III)-lactoferrin complex by intestinal cells.

Several bismuth compounds are currently used as antiulcer drugs, but the mechanism of action still remains unclear. The antimicrobial activity of Bi(III) complexes toward Gram-negative bacteria is reported to be dependent on the iron uptake system [Domenico, P., et al. (1996) J. Antimicrob. Chemother. 38, 1031-1040]. Electronic absorption and 13C NMR spectroscopic data show that Bi(III) binds to human lactoferrin at the specific Fe(III) sites along with either carbonate or oxalate as the synergistic anion. The uptake of Bi(III) by apo-hLF was rapid [minutes in 10 mM Hepes buffer and 5 mM bicarbonate (pH 7.4)], and almost equal in both lobes. The presence of ATP facilitates the release of Bi(III) from the Bi2-hLF complex when the pH is lowered. The Bi2-hLF complex blocked the uptake of the radiolabeled 59Fe-hLF complex into rat IEC-6 cells. Surprisingly, apo-hLF (but not apotransferrin) was almost as effective in blocking 59Fe uptake as bismuth-loaded lactoferrin. These results suggest that Bi(III)-loaded hLF might be recognized by the lactoferrin receptor and be taken up into cells.

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride.

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.

Inhibition of cancer cell growth by ruthenium(II) arene complexes.

Inhibition of the growth of the human ovarian cancer cell line A2780 by organometallic ruthenium(II) complexes of the type [(eta(6)-arene)Ru(X)(Y)(Z)], where arene is benzene or substituted benzene, X, Y, and Z are halide, acetonitrile, or isonicotinamide, or X,Y is ethylenediamine (en) or N-ethylethylenediamine, has been investigated. The X-ray crystal structures of the complexes [(eta(6)-p-cymene)Ru(en)Cl]PF(6) (5), [(eta(6)-p-cymene)RuCl(2)(isonicotinamide)] (7), and [(eta(6)-biphenyl)Ru(en)Cl]PF(6) (9) are reported. They have "piano stool" geometries with eta(6) coordination of the arene ligand. Complexes with X,Y as a chelated en ligand and Z as a monofunctional leaving group had the highest activity. Complexes 5, 6 (the iodo analogue of 5), 9, and 10 (ethylethylenediamine analogue of 9) were as active as carboplatin. Hydrolysis of the reactive Ru-Cl bond in complex 5 was detected by HPLC but was suppressed by the addition of chloride ions. Complex 5 binds strongly and selectively to G bases on DNA oligonucleotides to form monofunctional adducts. No inhibition of topoisomerase I or II by complexes 5, 6, or 9 was detected. These chelated Ru(II) arene complexes have potential as novel metal-based anticancer agents with a mechanism of action different from that of the Ru(III) complex currently on clinical trial.

A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity.

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn(4)Cys(9)His(2) cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys(4) and two ZnCys(3)His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn(4)Cys(11) cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys(4)-coordinated zinc ions in SmtA readily exchanges with exogenous metal ((111)Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of beta-strand and alpha-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

Comparison of the effects of intravenous alfentanil and esmolol on the cardiovascular response to double-lumen endobronchial intubation.

We compared the effect of alfentanil 10 microg.kg-1 and esmolol 1.5 mg.kg-1 on the cardiovascular responses to laryngoscopy and double-lumen endobronchial intubation in two groups of 20 ASA 2-3 patients undergoing pulmonary surgery, in a randomised double-blind study. Arterial pressure and heart rate decreased after induction of anaesthesia and increased after intubation in both groups (p < 0.05) but remained at or below baseline values, and changes were comparable in both groups. Plasma catecholamine concentrations decreased after induction of anaesthesia in both groups (p < 0.05). Epinephrine concentrations increased in the esmolol group after intubation (p < 0.05) but remained below baseline in the alfentanil group (p < 0.05). Norepinephrine concentrations increased significantly in both groups after intubation but were higher in the esmolol group (p < 0.05). Although both esmolol 1.5 mg.kg-1 and alfentanil 10 microg.kg-1 similarly attenuated the arterial pressure and heart rate response to endobronchial intubation, plasma catecholamine concentrations increased in the esmolol group to values greater than previously reported after tracheal intubation.

Competitive binding of bismuth to transferrin and albumin in aqueous solution and in blood plasma.

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.

Cu2+ -induced low density lipoprotein peroxidation is dependent on the initial O2 concentration: an O2 consumption study.

Atherosclerotic plaques form in the arterial intima, where low density lipoprotein (LDL) is thought to be oxidatively modified at sites which may contain catalytic amounts of copper in the presence of low O2 tension. We have investigated O2 consumption during LDL peroxidation induced by Cu2+ ions in vitro and found two phases: a lag phase followed by a phase of rapid O2 consumption. The length of the lag phase was dependent on Cu2+ and on initial O2 concentrations; increasing either decreased the lag time; however, LDL. concentration had no effect. LDL-induced Cu2+ reduction, however, was not affected by low initial O2 concentrations, suggesting that O2 is not required for LDL-mediated reduction of Cu2+. Following the lag phase, O2 consumption was dependent upon LDL or initial O2 concentrations; Cu2+ concentrations had little effect, suggesting that the propagation phase is more dependent on the presence of LDL lipids and O2 as substrates for the reaction. In summary, LDL peroxidation takes place in the presence of Cu2+ at low O2 tension; however, the reaction is dependent upon initial O2 concentrations; increases shorten the lag phase and accelerate O2 consumption.

Sequence-dependent bending of DNA induced by cisplatin: NMR structures of an A.T-rich 14-mer duplex.

The NMR solution structure of the A.T rich DNA 14-mer duplex d(ATACATGGTACATA).d(TATGTACCATGTAT) is reported. This is compared with the NMR structure of the same duplex intrastrand cross-linked at the d(G*pG*) site by cis-(Pt(NH3)2¿2+, derived from the anticancer drug cisplatin. The unmodified duplex has B-DNA geometry, but there is a large positive base-pair roll (roll angle 24 +/- 2 degrees) at the T9-A10 step on the 3' side of the central GG site. Platination of the DNA duplex causes the adjacent guanine bases to roll toward one another (roll angle 44 +/- 4 degrees), leading to an overall helix bend of 52 +/- 9 degrees. The platinum atom is displaced from the planes of the coordinated G7* and G8* by 0.8 A and 0.3 A, respectively. The minor groove opposite the platinum lesion is widened and flattened, with geometric parameters similar to those of A-form DNA. The unwinding of the helix at the platination site is 26 degrees. Platination causes the DNA duplex to bend toward the 3'-end (with respect to the G*G* strand), in contrast to G C-rich structures reported previously, which bend toward the 5'-end. This difference can be attributed to the predisposition of the A.T rich duplex toward bending in this region. Protein recognition of bent platinated G*G* lesions may therefore exhibit a strong dependence on the local DNA structure.

Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride.

The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.

Bathocuproine-assisted reduction of copper(II) by human albumin.

Human albumin (studied here as the recombinant protein rHA), a copper-binding protein in blood plasma, is shown to reduce Cu(II) to Cu(I) in the presence of a Cu(I) chelator, bathocuproinedisulfonate (BD). This reaction was accelerated at low pH, when there was little binding of Cu(II) to rHA. The addition of a competitive metal ion, Ni(II), or an increase in the concentration of BD, enhanced the reduction of Cu(II) to Cu(I). It was concluded that the oxidant was the Cu(II) complex of BD, which is likely to bind strongly to albumin. The free thiol at Cys34 was ruled out as the sole reducing agent, since Cys34-blocked albumin also gave rise to Cu(I) in the presence of BD. Reactions with amino acids and peptides suggested that Tyr and possibly His side-chains are potential reductants. BD and its homologues are frequently used as Cu(I)-specific chelators in biological experiments, but the strong oxidant activity of [Cu(II)(BD)2]2- and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.

Stereoselective formation of seven-coordinate titanium(IV) monomer and dimer complexes of ethylenebis(o-hydroxyphenyl)glycine.

Reactions between the antitumor agent titanocene dichloride (Cp2TiCl2) and the hexadentate ligand N,N'-ethylenebis-(o-hydroxyphenylglycine) (H4ehpg) have been investigated in aqueous solution and the solid state. The racemic ligands give crystals of the monomer [Ti(ehpg)(H2O)] x (11/3)H2O (1), while the meso ligand gives the oxo-bridged dimer [[Ti(Hehpg)(H2O)]2O] x 13H2O (2). Complex 1 crystallizes in the monoclinic space group C2/c with a = 24.149(4) A, b = 14.143(3) A, c = 19.487(3) A, beta = 105.371(13) degrees, V = 6417.7(19) A3, Z = 12, and R(F) = 0.0499 for 4,428 independent reflections having I > 2sigma(I), and contains seven-coordinate pentagonal-bipyramidal TiIV with two axial phenolate ligands (Ti-O, 1.869(2) A). The pentagonal plane contains the two N-atoms at 2.210(2) A, two carboxylate O-atoms at 2.061(2) A, and a water molecule (Ti-OH2, 2.091(3) A). Complex 2 crystallizes as an oxygen-bridged dimer in the triclinic space group P-1 with a = 12.521(6) A, b = 14.085(7) A, c = 16.635(8) A, alpha = 80.93(2) degrees beta = 69.23(2) degrees, gamma = 64.33(2) degrees , V = 2472(2) A3, Z = 4, and R(F) = 0.0580 for 5956 independent reflections having I > 2sigma(I). Each seven-coordinate, pentagonal-bipyramidal TiIV has a bridging oxide and a phenolate as axial ligands. The pentagonal plane donors are H2O, two carboxylate O-atoms, and two NH groups, which form H-bonds to O-atoms both in the same half-molecule (O...N, 2.93-3.13 A) and in the other half-molecule (O...N, 2.73-2.75 A); the second phenoxyl group of each Hehpg ligand is protonated and not coordinated to TiIV, but H-bonds to a nearby amine proton (O...N, 2.73-2.75 A) from the same ligand and to a nearby H2O (O...O, 2.68 A). In contrast to all previously reported crystalline metal-EHPG complexes containing racemic ligands, in which the N(S,S)C(R,R) or N(R,R)C(S,S) form is present, complex 1 unexpectedly contains the N(S,S)C(S,S) and N(R,R)C(R,R) forms. This is attributed to the presence of ring strain in seven-coordinate TiIV complexes. Moreover, the rac ligands selectively form crystals of monomeric 1, while the meso ligand selectively forms crystals of the dimer 2 (N(R,R)C(R,S) or N(S,S)C(S,R)). Complexes 1 and 2 exhibit phenolate-to-TiIV charge-transfer bands near 387 nm, and 2D NMR studies indicate that the structures of 1 and 2 in solution are similar to those in the solid state. Complex 1 is stable over the pH range 1.0-7.0, while 2 is stable only between pH 2.5 and pH 5.5. Cp2TiCl2 reacts with EHPG at pH* 7.0 to give complex 1 with a t 1/2 of ca. 50 min (298 K), but complex 2 was not formed at this pH* value. At pH* 3.7, the reaction is very slow: 1 forms with a half-life of ca. 2.5 d, and 2 after ca. 1 week at ambient temperature. The relevance of these data to the possible role of serum transferrin as a mediator for the delivery of TiIV to tumor cells is discussed.

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.