RAPTA-Decorated Polyacrylamide Nanoparticles: Exploring their Synthesis, Physical Properties and Effect on Cell Viability.

In this study, we report the first successful immobilisation of a known cytoactive [Ru(η6 -arene)(C2 O4 )PTA] (RAPTA) complex to a biologically inert polyacrylamide nanoparticle support. The nanoparticles have been characterised by zetasizer analysis, UV/Vis, ATR-FTIR, TGA and ICP-MS to qualitatively and quantitatively confirm the presence of the metallodrug on the surface of the carrier. The native RAPTA complex required a concentration of 50 µM to produce a cell viability of 47.1±2.1 % when incubated with human Caucasian colorectal adenocarcinoma cells for 72 h. Under similar conditions a cell viability of 45.1±1.9 % was obtained with 0.5 µM of RAPTA complex in its immobilised form. Therefore, conjugation of the RAPTA metallodrug to our nanoparticle carriers resulted in a significant 100-fold decrease in effective concentration of ruthenium required for a near identical biological effect on cell viability.

Homo- and Hetero-dinuclear Arene-Linked Osmium(II) and Ruthenium(II) Organometallics: Probing the Impact of Metal Variation on Reactivity and Biological Activity.

Dinuclear metallodrugs offer much potential in the development of novel anticancer chemotherapeutics as a result of the distinct interactions possible with bio-macromolecular targets and the unique biological activity that can result. Herein, we describe the development of isostructural homo-dinuclear OsII -OsII and hetero-dinuclear OsII -RuII organometallic complexes formed from linking the arene ligands of [M(η6 -arene)(C2 O4 )(PTA)] units (M=Os/Ru; PTA=1,3,5-triaza-7-phosphaadamantane). Using these complexes together with the known RuII -RuII analogue, a chromatin-modifying agent, we probed the impact of varying the metal ions on the structure, reactivity and biological activity of these complexes. The complexes were structurally characterised by X-ray diffraction experiments, their stability and reactivity were examined by using 1 H and 31 P NMR spectroscopy, and their biological activity was assessed, alongside that of mononuclear analogues, through MTT assays and cell-cycle analysis (HT-29 cell line). The results revealed high antiproliferative activity in each case, with cell-cycle profiles of the dinuclear complexes found to be similar to that for untreated cells, and similar but distinct profiles for the mononuclear complexes. These results indicate these complexes impact on cell viability predominantly through a non-DNA-damaging mechanism of action. The new OsII -OsII and OsII -RuII complexes reported here are further examples of a family of compounds operating via mechanisms of action atypical of the majority of metallodrugs, and which have potential as tools in chromatin research.

Synthesis and In Vitro Biological Evaluation of a Second-Generation Multimodal Water-Soluble Porphyrin-RAPTA Conjugate for the Dual-Therapy of Cancers.

In this study, we report the synthesis and biological evaluation of a novel cationic porphyrin-[Ru(η6-arene)(C2O4)PTA] (RAPTA) conjugate with potential as a multimodal dual-therapeutic agent. In the absence of high intensity light, relative to untreated cells our conjugate resulted in a 83% decrease in viable human adenocarcinoma cells at a concentration of 10 µM, which is significantly more active than the 57% decrease achieved with the same concentration of the unconjugated RAPTA complex alone. With a light dose of 20 J cm-2 (400-1200 nm) a reduction of 98% of viable cells was observed for the same concentration of conjugate. The conjugate is internalized by HT-29 cancer cells as proven by ICP-MS analysis and fluorescence microscopy; the latter result suggests that the conjugate has applications as a multimodal agent by acting as a fluorophore to obtain in vivo biodistribution data. Furthermore, the conjugate has an excellent relative singlet oxygen quantum yield, and the tetrapyrollic unit was found to be photostable under irradiation by either white light or red light.

The Application of Reversible Intramolecular Sulfonamide Ligation to Modulate Reactivity in Organometallic Ruthenium(II) Diamine Complexes.

Metallation of biomacromolecular species forms the basis for the anticancer activity of many metallodrugs. A major limitation of these compounds is that their reactivity is indiscriminate and can, in principle, occur in healthy tissue as well as cancerous tissue, potentially leading to side effects in vivo. Here we present pH-dependent intramolecular coordination of an arene-tethered sulfonamide functionality in organometallic ruthenium(II) ethylenediamine complexes as a route to controlling the coordination environment about the central metal atom. Through variation of the sulfonamide R group and the length of the tether linking it to the arene ligand the acidity of the sulfonamide NH group, and hence the pH-region over which regulation of metal coordination occurs, can be modulated. Intramolecular sulfonamide ligation controlled the reactivity of complex 4 within the physiologically relevant pH-region, rendering it more reactive towards 5'-GMP in mildly acidic pH-conditions typical of tumour tissue compared to the mildly alkaline pH-conditions typical of healthy tissue. However, the activation of 4 by ring-opening of the chelate was found to be a slow process relative to the timescale of typical cell culture assays and members of this series of complexes were found not to be cytotoxic towards the HT-29 cell line. These complexes provide the basis for the development of analogues of increased potency where intramolecular sulfonamide ligation regulates reactivity and therefore cytotoxicity in a pH-dependent, and potentially, tissue-dependent manner.

Recent progress in the development of organometallics for the treatment of cancer.

From their early successes in medicine, organometallic compounds continue to attract interest as potential chemotherapeutics to treat a range of diseases. Here, we show from recent literature selected largely from the last two years that organometallics offer unique opportunities in medicine and, increasingly, a mechanistic-based approach is applied to their development, which has not always been the case.

Nucleosome acidic patch-targeting binuclear ruthenium compounds induce aberrant chromatin condensation.

The 'acidic patch' is a highly electronegative cleft on the histone H2A-H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research.

An Organometallic Compound which Exhibits a DNA Topology-Dependent One-Stranded Intercalation Mode.

Understanding how small molecules interact with DNA is essential since it underlies a multitude of pathological conditions and therapeutic interventions. Many different intercalator compounds have been studied because of their activity as mutagens or drugs, but little is known regarding their interaction with nucleosomes, the protein-packaged form of DNA in cells. Here, using crystallographic methods and molecular dynamics simulations, we discovered that adducts formed by [(η(6) -THA)Ru(ethylenediamine)Cl][PF6 ] (THA=5,8,9,10-tetrahydroanthracene; RAED-THA-Cl[PF6 ]) in the nucleosome comprise a novel one-stranded intercalation and DNA distortion mode. Conversely, the THA group in fact remains solvent exposed and does not disrupt base stacking in RAED-THA adducts on B-form DNA. This newly observed DNA binding mode and topology dependence may actually be prevalent and should be considered when studying covalently binding intercalating compounds.

Potential of cycloaddition reactions to generate cytotoxic metal drugs in vitro.

Severe general toxicity issues blight many chemotherapeutics utilized in the treatment of cancers, resulting in the need for more selective drugs able to exert their biological activity at only the required location(s). Toward this aim, we report the development of an organometallic ruthenium compound, functionalized through a η(6)-bound arene ligand with a bicyclononyne derivative, able to participate in strain-promoted cycloaddition reactions with tetrazines. We show that combination of the ruthenium compound with a ditetrazine in biological media results in the in situ formation of a dinuclear molecule that is more cytotoxic toward cancer cells than the starting mononuclear ruthenium compound and tetrazine components. Such an approach may be extended to in vivo applications to construct a cytotoxic metallodrug at a tumor site, providing a novel approach toward the turn-on cytotoxicity of metallodrugs in the treatment of cancer.

Synthesis, molecular structure, computational study and in vitro anticancer activity of dinuclear thiolato-bridged pentamethylcyclopentadienyl Rh(III) and Ir(III) complexes.

Neutral dinuclear dithiolato-bridged pentamethylcyclopentadienyl Rh(III) complexes of the type (C5Me5)2Rh2(µ-SR)2Cl2 (R = CH2Ph, 1; R = CH2CH2Ph, 2) and cationic dinuclear trithiolato-bridged pentamethylcyclopentadienyl Rh(III) and Ir(III) complexes of the type [(C5Me5)2M2(µ-SR)3](+) (M = Rh, R = CH2Ph, 3; M = Rh, R = CH2CH2Ph, 5; M = Rh, R = CH2C6H4-p-(t)Bu, 7: M = Ir, R = CH2Ph, 4; M = Ir, R = CH2CH2Ph, 6; M = Ir, R = CH2C6H4-p-(t)Bu, 8) have been synthesized from the chloro-bridged pentamethylcyclopentadienyl Rh(III) and Ir(III) dimers (C5Me5)2M2(µ-Cl)2Cl2 by reaction with the corresponding thiol derivative (RSH). Complexes 3-8 were isolated as chloride salts. All complexes were obtained in good yield and were fully characterized by spectroscopic methods. The molecular structures of the neutral complexes (1 and 2) show interesting features: the two rhodium atoms are bridged by two thiolato ligands with no metal-metal bonds and the pentamethylcyclopentadienyl and chlorido ligands are oriented syn to each other, an uncommon conformation for such dinuclear complexes. These structural features were rationalized using DFT calculations. Additionally, the antiproliferative activity of the complexes was evaluated against the cancerous A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) human ovarian cell lines and on the noncancerous HEK293 human embryonic kidney cells. All complexes were found to be active and the cationic iridium complexes , and are particularly cytotoxic, with IC50 values in the nanomolar range (IC50 < 0.1 µM). The catalytic activity of the complexes for the oxidation of glutathione (GSH) to GSSG was evaluated by NMR spectroscopy.

Synthesis, Molecular Structure and Cytotoxicity of Molecular Materials Based on Water Soluble Half-Sandwich Rh(III) and Ir(III) Tetranuclear Metalla-Cycles.

The neutral dinuclear complexes [(η5-C5Me5)2Rh2(µ-dhnq)Cl2] (1) and [(η5-C5Me5)2Ir2(µ-dhnq)Cl2] (2) (dhnqH2 = 5,8-dihydroxy-1,4-naphthoquinone) were obtained from the reaction of [(η5-C5Me5)M(µ-Cl)Cl]2 (M = Rh, Ir) with dhnqH2 in the presence of CH3COONa. Treatment of 1 or 2 in methanol with linear ditopic ligands L (L = pyrazine, 4,4'-bipyridine or 1,2-bis(4-pyridyl)ethylene), in the presence of AgCF3SO3, affords the corresponding tetranuclear metalla-rectangles [(η5-C5Me5)4M4(µ-dhnq)2(µ-L)2]4+ (L = pyrazine, M = Rh, 3; M = Ir, 4; L = 4,4'-bipyridine, M = Rh, 5; M = Ir, 6; L = 1,2-bis(4-pyridyl)ethylene, M = Rh, 7; M = Ir, 8). All complexes were isolated as their triflate salts and were fully characterized by infrared, ¹H and 13C NMR spectroscopy, and some representative complexes by single-crystal X-ray structure analysis. The X-ray structures of 3, 5 and 6 confirm the formation of the tetranuclear metalla-cycles, and suggest that complexes 5 and 6 possess a cavity of sufficient size to encapsulate small guest molecules. In addition, the antiproliferative activity of the metalla-cycles 3-8 was evaluated against the human ovarian A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) cancer cell lines and on non-tumorigenic human embryonic kidney HEK293 cells. All cationic tetranuclear metalla-rectangles were found to be highly cytotoxic, with IC50 values in the low micromolar range.

Evidence for the optical signalling of changes in bicarbonate concentration within the mitochondrial region of living cells.

Image and spectral intensity from bicarbonate-selective europium(III) probes localised in the mitochondria of cells is modulated reversibly by variation of external pCO(2), and is suppressed by addition of the carbonic anhydrase inhibitor, acetazolomide.

Templating a polymer-scaffolded dynamic combinatorial library.

A water soluble polymer-scaffolded dynamic combinatorial library whose members can interconvert through acylhydrazone exchange was prepared and shown to re-equilibrate in the presence of macromolecular templates.

Reactive thermoresponsive copolymer scaffolds.

Thermoresponsive copolymer scaffolds containing reactive aldehyde functions were prepared and a selection of organic residues conjugated to these copolymer scaffolds through oxime/hydrazone formation. The conjugation of hydrophobic residues affords copolymers whose lower solution critical temperatures are in most cases higher than that of the parent copolymer scaffold.

Electron-capture dissociation and collision-induced dissociation of lanthanide metal-ligand complexes and lanthanide metal-ligand complexes bound to phosphopeptides.

Collision-induced dissociation (CID) and electron-capture dissociation (ECD) of lanthanide metal(III)-ligand complexes and lanthanide metal(III)-ligand-phosphopeptide complexes have been investigated using a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). Ternary adducts were formed for [LnL(3+)+solvent anion(s)(n-)]((3-n)+) [Ln=europium, terbium and ytterbium, L=heptadentate ligand, solvent anion=acetate or trifluoromethane-sulphonate (triflate)]. Results show that ECD provides much more diagnostically useful information than CID. ECD was found to systematically cleave N-C bonds in the "arms" of the ligand, similar to the N-C(alpha) cleavage of peptides, generating two fragmentation sites per arm of the ligand. The most intense and informative fragment ions were obtained from the terbium complex and it is believed that this is a consequence of terbium's greater reduction potential: the greater the reduction potential, the greater the ligand fragmentation; the lower the reduction potential, the more likely the molecule is to relinquish the solvent anion. The choice of solvent is also shown to be important. In general, the complexes studied fragment easily to lose CH(3)CO(2)H; however, particularly for ECD, the complexes retain the triflate anion causing the ligand to fragment instead, thus providing much more useful information. The triflate anion can be displaced by a phosphopeptide to create a lanthanide metal-ligand-phosphopeptide adduct. ECD is able to sequence the phosphopeptide, locating the site of phosphorylation bound to [LnL](3+) and also confirm the identity of the ligand. Small differences in the fragmentation of the lanthanide metal-ligand-phosphopeptide adducts follow the trend Eu

Cell-penetrating metal complex optical probes: targeted and responsive systems based on lanthanide luminescence.

To understand better the structure and function of biological systems, cell biologists and biochemists would like to have methods that minimally perturb living systems. The development of emissive optical probes is essential for improving our observation of intracellular signaling and recognition processes. Following excitation of the probe, photons emitted from the probe may be observed by spectroscopy or microscopy and encode information about their environments in their energy, lifetime, and polarization. Such optical probes may be based on organic fluorophores, quantum dots, recombinant proteins, or emissive metal complexes. In this Account, we trace the emergence of lanthanide coordination complexes as emissive optical probes. These probes benefit from sharp emission bands and long lifetimes. We can design these complexes to report on the concentration of key biochemical variables by modulation of spectral form, lifetime, or circular polarization. These properties allow us to apply ratiometric methods of analysis in spectroscopy or microscopy to report on local pH, pM (M = Ca, Zn), or the concentration of certain anionic metabolites, such as citrate, lactate, bicarbonate, or urate. For optical microscopy studies in living cells, these probes must be cell-permeable and, ideally, should localize in a given cell organelle. We undertook systematic studies of more than 60 emissive complexes, examining the time dependence of cellular uptake and compartmentalization, cellular toxicity, protein affinity, and quenching sensitivity. These results and their relationship to probe structure have allowed us to identify certain structure-activity relationships. The nature and linkage mode of the integral sensitizing group-introduced to harvest incident light efficiently-is of primary importance in determining protein affinity and cellular uptake and trafficking. In many cases, uptake may occur via macropinocytosis. We have defined three main classes of behavior: complexes exhibit predominant localization profiles in protein-rich regions (nucleoli/ribosomes), in cellular mitochondria, or in endosomes/lysosomes. Therefore, these systems offer considerable promise as intracellular optical probes, amenable to single- or two-photon excitation, that may report on the local ionic composition of living cells subjected to differing environmental stresses.

Critical evaluation of five emissive europium(III) complexes as optical probes: correlation of cytotoxicity, anion and protein affinity with complex structure, stability and intracellular localisation profile.

Five structurally related europium(iii) complexes of heptadentate macrocyclic ligands bearing azaxanthone or azathiaxanthone chromophores have been evaluated comparatively as responsive probes of the intracellular environment. Protein binding (HSA) and oxy-anion binding constants have been measured by titrimetric analysis, examining ratiometric emission changes for three examples, and systems exhibiting selectivity for citrate or hydrogencarbonate identified. The cytotoxicity of each ligand and complex has been assessed and correlated with the observed chemical stability profile in competitive aqueous media. The europium complex of L(2) is non-toxic, exhibits a large change in its emission spectral profile to variation in HCO(3)(-) (or citrate) concentration allowing ratiometric analysis, and localises in cellular mitochondria. Such features augur well for its future application as a responsive probe in microscopy to monitor local changes in pHCO(3).

Two-photon absorption and photoluminescence of europium based emissive probes for bioactive systems.

Observation of two-photon excitation (760 nm) and emission of two responsive water soluble europium complexes is reported with cross-sections of up to 2 GM. Two-photon excitation spectra have also been measured, acquisition being achieved by the use of a cavity-dumped mode locked Ti-sapphire laser. Time-gated detection is used to differentiate the ligand fluorescence and metal centred emission in these europium complexes.

A cationic lanthanide complex binds selectively to phosphorylated tyrosine sites, aiding NMR analysis of the phosphorylated insulin receptor peptide fragment.

The binding of two cationic europium complexes to a differentially phosphorylated insulin receptor peptide has been studied by emission spectroscopy and (31)P NMR and (1)H NMR TOCSY methods. Analysis of the europium emission and NMR spectral data was consistent with the presence of species in slow exchange on the NMR and emission timescales, in agreement with selective binding of the lanthanide ion to the phospho-tyrosine site, allowing such complexes to be considered as prototypical chemoselective paramagnetic derivatising agents.