Cécile Moucheron

Photoreactions of ruthenium(II) and osmium(II) complexes with deoxyribonucleic acid (DNA).

The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.

Ru(II) polypyridine complexes with a high oxidation power. Comparison between their photoelectrochemistry with transparent SnO2 and their photochemistry with desoxyribonucleic acids

Abstract The compounds which are discussed in the present review are highly oxidizing Ru(II) complexes, based on various polyazaaromatic ligands, and acting as efficient electron acceptors in the excited state. The photoinduced charge transfer process and the following associated kinetic steps are characterized for the whole series of complexes by quite different techniques and methods. Thus their behaviour in the presence of reductants such as hydroquinone and mononucleotides (guanosine-5′-monophosphate and adenosine-5′-monophosphate) are examined by flash photolysis, spectroelectrochemistry and photoelectrochemistry. It is explained how the light-initiated electron transfer process can be applied for spectral supersensitization of wide band gap SnO 2 semiconductor electrodes. Moreover, it is shown that such a knowledge of the behaviour of these photoredox reactions leads to interesting applications of these oxidizing complexes in a biological area, i.e. for the study of nucleic acids. Thus it is illustrated how these compounds can be used as promising photoreagents of DNA. The easy modulation of their size and shape, and their irreversible anchoring on the DNA bases, triggered by the reductive photoelectron transfer process from the guanine bases to the excited complex, allow one to regard these complexes as attractive molecular tools for DNA study and maybe as future possible drugs activatable under visible light.

From cisplatin to photoreactive Ru complexes: targeting DNA for biomedical applications

Since the discovery of cisplatin, the search for therapeutic agents based on other metallic compounds developed rapidly thanks to the versatility of coordination chemistry. This short review focuses on advances in research for new complexes based on Pt(IV), Ru(II) and Ru(III) either as anticancer drugs or as molecular (photo)reagents for biomedical applications. The DNA binding mechanisms of these compounds are highlighted alongside with the novel strategies developed to improve their reactivity and/or specificity towards DNA.

Development of a quantitative LC-MS/MS method for the analysis of common propellant powder stabilizers in gunshot residue.

Abstract:  In traditional scanning electron microscopy/energy dispersive X‐ray analysis of gunshot residue (GSR), one has to cope more and more frequently with limitations of this technique due to the use of lead‐free ammunition or ammunition lacking heavy metals. New methods for the analysis of the organic components of common propellant powder stabilizers were developed based on liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). A multiple reactions monitoring scanning method was created for the screening of akardite II, ethylcentralite, diphenylamine, methylcentralite, N‐nitrosodiphenylamine, 2‐nitrodiphenylamine, and 4‐nitrodiphenylamine, present in standards mixtures. Five out of seven of these target compounds can be selectively identified and distinguished from the two others with a high accuracy. Samples from the hands of a shooter were collected by swabbing and underwent solid phase extraction prior to analysis. Detection limits ranging from 5 to 115 μg injected were achieved. Results from several firing trials show that the LC‐MS/MS method is suitable for the detection of stabilizers in samples collected following the firing of 9 mm Para ammunitions.

A Photoreactive Ruthenium(II) Complex Tethered to a Guanine‐Containing Oligonucleotide: A Biomolecular Tool that Behaves as a “Seppuku Molecule”

The design of specific DNA or RNA damaging agents may be achieved by anchoring a reactive species to an oligonucleotide (ODN) probe which, by duplex or triplex formation, should direct the irreversible chemical modification towards the targeted sequence. This approach is certainly interesting in the context of gene silencing, particularly for the development of anticancer agents. However, the achievement of a high level of selectivity remains a challenge because many side reactions such as interaction with proteins can occur in biological systems. In this context, the tethering of a transition metal complex to an ODN strand and subsequent activation by light is an attractive strategy. Indeed, the reactivity of the metallic compound towards the genetic material can be 1) triggered by light to act on specific tissues and 2) directed towards a DNA or RNA target sequence to cause damage. We have prepared oligonucleotides labeled with ruthenium(II) complexes (Ru-ODNs) that are able to photo-cross-link with their complementary strand under visible irradiation (Figure 1 a). The different parameters that govern the photo-cross-linking efficiency have been extensively studied, 6] these conjugates could be promising as anticancer agents based on gene silencing. Indeed, this sequence-specific photo-cross-linking process has been shown to inhibit in vitro DNA polymerase with a high efficiency. The prerequisites for this photochemical reaction are 1) the presence of at least two p-deficient polyazaaromatic ligands such as 1,4,5,8-tetraazaphenanthrene (tap) in the tethered metallic complex and 2) the presence of at least one guanine base (G) in the target sequence, in the vicinity of the tethered complex after hybridization. The mechanism involves a photoinduced electron transfer (PET) from the G base towards the oxidizing excited Ru compound with a recombination of the produced radicals, which covalently links one of the tap ligands to the G unit [Figure 1b, Eqs. (1)–(3)]. The structure of the photoadduct (Figure 1b) has been determined by ESI mass spectrometry and NMR spectroscopy. This strategy was thought to be restricted to target sequences without cytosine in order to avoid the presence of a guanine in the Ru-ODN probe, which would be unfavorable for the photo-cross-linking. In spite of these limitations, we decided to investigate the effect of the presence of a G base in the Ru-ODN probe sequence on the photoreactivity of such systems. Herein, we report the unique photochemical behavior of a new generation of photoreactive G-containing Ru-ODN probes that can selfinhibit in the absence of their specific target strands. We have named these molecules “seppuku molecules” because their photochemical behavior reminds us of the suicide of someone who has not accomplished his attributed duty in antic Japanese society. The Ru complexes contain a phenanthroline ligand derivatized by a linker, namely phen’’ (phen’’= N-(2-(1,10phenanthrolin-5-ylamino)-2-oxoethyl)-2-(aminooxy)acetaFigure 1. a) Photo-cross-linking processes that occur between an ODN probe labeled with a suitable Ru complex and the complementary G-containing ODN strand and b) formation and structure of the photoadduct produced upon illumination of [Ru(tap)3] 2+ in the presence of a guanine residue in GMP (guanosine-5’-monophosphate) or DNA after acid hydrolysis.

New DNA-binding ruthenium(II) complexes as photo-reagents for mononucleotides and DNA

The spectroscopic properties of two photoprobes for DNA, Ru(phen)2(PHEHAT)2+ and Ru(TAP)2(PHEHAT)2+ (phen = 1,10-phenanthroline, TAP = 1,4,5,8-tetraazaphenanthrene, PHEHAT = 1,10-phenanthrolino[5,6-b]-1,4,5,8,9,12-hexaazatriphenylene), were examined and compared with those of complexes containing either an extended planar ligand (DPPZ) or π-acceptor ligands. The orbitals involved in the absorption and emission processes for Ru(phen)2(PHEHAT)2+ imply the PHEHAT ligand, whereas the chromophore and luminophore for Ru(TAP)2(PHEHAT)2+ are associated with the Ru(II)  TAP MLCT transition. The two complexes exhibit completely different behaviour in the presence of DNA. Whereas Ru(phen)2(PHEHAT)2+, which does not emit in water, luminesces upon intercalation between the DNA base pairs, the luminescence of Ru(TAP)2(PHEHAT)2+ is quenched by binding to DNA. Emission quenching is also observed in the presence of GMP, with a quenching rate constant of 1.25 × 109 l mol−1 s−1. This strongly suggests the presence of a photo-induced electron transfer from the guanine residues of GMP or DNA to the excited complex and leads to the conclusion that this complex is a good DNA photoreagent.

Tetrapyrido[3,2-a:2′,3′-c:3″,2″-h:2‴,3‴-j]acridine (tpac): a new extended polycyclic bis-phenanthroline ligand

A new heptacyclic planar molecule has been synthesized in a very efficient one-pot reaction from 5-aminophenanthroline. Ru(II) mono- and dinuclear complexes based on this new bridging ligand, Tpac, have been prepared.

Photocrosslinking in ruthenium-labelled duplex oligonucleotides.

The formation of a photoadduct between a [Ru(1,4,5,8‐tetraazaphenanthrene)24,7‐diphenylphenanthroline]2+ complex chemically attached to a synthetic oligonucleotide, and a guanine moiety in a complementary targeted single‐stranded DNA molecule was studied for ten 17‐mer duplexes by denaturing gel electrophoresis. This photoadduct formation leads to photocrosslinking of the two strands. The percentage quenching of luminescence of the complex by electron transfer was compared to the resulting yield of photocrosslinked product. This yield does not only depend on the ionisation potential of the guanine bases, which are electron donors, but also on other factors, such as the position of the guanine bases as compared to the site of attachment of the complex. The photocrosslinking yield is higher when the guanine moieties are towards the 3′ end on the complementary strand as compared to the tethering site. Computer modelling results are in agreement with this preference for the 3′ side for the photoreaction. Interestingly, the photocrosslink is not alkali labile. Moreover, a type III exonuclease enzyme is blocked at the position of photocrosslinking.

Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking.

The influence of the distance between the anchoring site of the tethered [Ru(TAP)2dip]2+ complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5′-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5′-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3′-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

Mesoscale DNA Structural Changes on Binding and Photoreaction with Ru[(TAP)2PHEHAT]2+

We used scanning force microscopy (SFM) to study the binding and excited state reactions of the intercalating photoreagent Ru[(TAP)(2)PHEHAT](2+) (TAP = 1,4,5,8-tetraazaphenanthrene; PHEHAT = 1,10-phenanthrolino[5,6-b]1,4,5,8,9,12-hexaazatriphenylene) with DNA. In the ground state, this ruthenium complex combines a strong intercalative binding mode via the PHEHAT ligand, with TAP-mediated hydrogen bonding capabilities. After visible irradiation, SFM imaging of the photoproducts revealed both the structural implications of photocleavages and photoadduct formation. It is found that the rate of photocleaving is strongly increased when the complex can interact with DNA via hydrogen bonding. We demonstrated that the photoadduct increases DNA rigidity, and that the photo-biadduct can crosslink two separate DNA segments in supercoiled DNA. These mechanical and topological effects might have important implications in future therapeutic applications of this type of compounds.