A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins.

Using a chemical approach to crosslink functionally versatile bioeffectors (such as peptides) to native proteins of interest (POI) directly inside a living cell is a useful toolbox for chemical biologists. However, this goal has not been reached due to unsatisfactory chemoselectivity, regioselectivity, and protein selectivity in protein labeling within living cells. Herein, we report the proof of concept of a cytocompatible and highly selective photolabeling strategy using a tryptophan-specific Ru-TAP complex as a photocrosslinker.

Photo-CIDNP Reveals Different Protonation Sites Depending on the Primary Step of the Photoinduced Electron-/Proton-Transfer Process with Ru(II) Polyazaaromatic Complexes.

The excited-state quenching of [Ru(TAP)(HAT)] (TAP = 1,4,5,8-tetraazaphenanthrene, HAT= 1,4,5,8,9,12-hexaazatriphenylene) by hydroquinone (HQ), N-acetyl-tyrosine (N-Ac-Tyr) or guanosine-5'-monophosphate (GMP) was investigated at various pH values. The quenching occurs via electron/proton transfer, as evidenced by transient absorption spectroscopy and confirmed by H photochemically induced dynamic nuclear polarization (photo-CIDNP). Reductive quenching also occurs in strongly acidic solution despite a much shorter lifetime of the protonated excited-state complex.

pH Dependence of Photoinduced Electron Transfer with [Ru(TAP)].

The quenching of the excited state of [Ru(TAP)] (TAP = 1,4,5,8-tetraazaphenanthrene) by guanosine-5'-monophosphate (GMP), N-acetyltyrosine (N-Ac-Tyr), and hydroquinone (HQ) has been studied in aqueous solution over a wide range of pH values including, for the first time, strongly acidic media. This quenching by electron transfer was examined by steady-state H photochemically induced dynamic nuclear polarization (photo-CIDNP) as well as by more conventional techniques, among which are pulsed laser-induced transient absorption and emission experiments. A deeper knowledge of the photochemical behavior of [Ru(TAP)] has been gained thanks to the combined use of these two approaches, photo-CIDNP and electronic spectroscopies, highlighting their complementarity.

Photoaddition of two guanine bases to single Ru-TAP complexes. Computational studies and ultrafast spectroscopies to elucidate the pH dependence of primary processes.

The covalent photoadduct (PA) between [Ru(TAP)3](2+) (TAP = 1,4,5,8-tetraazaphenanthrene) and guanosine monophosphate (GMP) opened the way to interesting photobiological applications. In this context, the PA's capability upon illumination to give rise to the addition of a second guanine base is especially interesting. The origins of these intriguing properties are for the first time thoroughly investigated by an experimental and theoretical approach.

Revisited photophysics and photochemistry of a Ru-TAP complex using chloride ions and a calix[6]crypturea.

The effects of the nonprotonated and protonated calix[6]crypturea 1/1(•)H(+) on the PF6(-) and Cl(-) salts of a luminescent Ru-TAP complex (TAP = 1,4,5,8-tetraazaphenanthrene) were investigated. Thus, the phototriggered basic properties of this complex were examined with 1(•)H(+) in acetonitrile (MeCN) and butyronitrile (BuCN). The Ru excited complex was shown to be able to extract a proton from the protonated calixarene, accompanied by a luminescence quenching in both solvents.

Selective DNA purine base photooxidation by bis-terdentate iridium(III) polypyridyl and cyclometalated complexes.

Two bis-terdentate iridium(III) complexes with polypyridyl and cyclometalated ligands have been prepared and characterized. Their spectroscopic and electrochemical properties have been studied, and a photophysical scheme addressing their properties is proposed. Different types of excited states have been considered to account for the deactivation processes in each complex.

Synthesis and electrochemical and photophysical properties of calixarene-based ruthenium(II) complexes as potential multivalent photoreagents.

The grafting of photoreactive and photooxidizing Ru(II)(TAP) (TAP = 1,4,5,8-tetraazaphenanthrene) complexes on calix[4 or 6]arene molecular platforms is reported. Thus, either [Ru(TAP)2(phen)](2+) (phen = 1,10-phenanthroline) or [Ru(TAP)2(pytz)](2+) [pytz = 2-(1,2,3-triazol-4-yl)pyridine] complexes are anchored to the calixarenes. The data in electrochemistry, combined with those in emission under steady state and pulsed illumination and the determination of the associated photophysical rate constants, indicate the presence of intramolecular luminescence quenching by the phenol moieties of calixarene.

Peculiar properties of homoleptic Cu complexes with dipyrromethene derivatives.

In view of preparing Cu polynuclear complexes with dipyrromethene ligands, the mononuclear complexes [Cu(II)(dipy)2] (dipyH = 5-phenyldipyrromethene) and [Cu(II)(dpdipy)2] (dpdipyH = 1,5,9-triphenyldipyrromethene) have been prepared and characterized by X-ray crystallography, mass spectrometry and EPR spectroscopy. Their peculiar redox and spectroscopic (absorption/emission) behaviours are discussed. In contrast to Cu(II) complexes of 1,1'-bidypyrrin, the reduction electrolysis of [Cu(II)(dpdipy)2] leads to decomposition products on a time scale of a few hours.

Evaluation of a new biocompatible poly(N-(morpholino ethyl methacrylate)-based copolymer for the delivery of ruthenium oligonucleotides, targeting HPV16 E6 oncogene.

This study investigates the use of a new biocompatible block copolymer poly(2-(dimethylamino)ethyl methacrylate-N-(morpholino)ethyl methacrylate (PDMAEMA-b-PMEMA) for the delivery of a particular antisense oligonucleotide targeting E6 gene from human papilloma virus. This antisense oligonucleotide was derivatized with a polyazaaromatic Ru(II) complex which, under visible illumination, is able to produce an irreversible crosslink with the complementary targeted sequence. The purpose of this study is to determine whether by the use of a suitable transfection agent, it is possible to increase the efficiency of the antisense oligonucleotide targeting E6 gene, named Ru-P-4.

Ru-TAP complexes and DNA: from photo-induced electron transfer to gene photo-silencing in living cells.

In this review, examples of applications of the photo-induced electron transfer (PET) process between photo-oxidizing Ru-TAP (TAP = 1,4,5,8-tetraazaphenanthrene) complexes and DNA or oligodeoxynucleotides (ODNs) are discussed. Applications using a free Ru-TAP complex (not chemically anchored to an ODN) are first considered. In this case, the PET gives rise to the production of an irreversible adduct of the Ru complex on a guanine (G) base, with formation of a covalent bond.

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.

What does the future hold for photo-oxidizing Ru(II) complexes with polyazaaromatic ligands in medicinal chemistry?

Since the discovery of cisplatin, the search for diagnostic or therapeutic agents based on other metals, has expanded intensively owing to the numerous possibilities offered by coordination chemistry. This mini-review focuses on recent advances in the search for Ru(II) polyazaaromatic complexes of potential interest as molecular tools applied to cellular diagnostics or as specific cellular photo-reagents for future biomedical applications. The interaction of Ru(II) polyazaaromatic complexes with living cells is reported, as well as the photo-reaction mechanisms of photo-oxidizing Ru(II) complexes with nucleic acids.

Photocrosslinking between peptide-peptide or peptide-oligonucleotide by Ru(II)-TAP complexes.

Ru(II)-TAP complexes have been shown to be very attractive compounds in the frame of developments of new anticancer drugs targeting the genetic material. This increasing interest originates from observations of covalent bond formations, triggered by photo-induced electron transfer (PET) between Ru(II)-TAP complexes and guanine bases of DNA. This photoreaction has recently been extended to the tryptophan (Trp) amino acid for future applications involving peptides.

Trinuclear ruthenium dendrons based on bridging PHEHAT and TPAC ligands.

Novel polynuclear compounds, the trinuclear precursor complex cis-{[(phen)(2)Ru(PHEHAT)](2)Ru(CH(3)CN)(2)}(6+) 4 and the trinuclear TPAC (tetrapyrido[3,2-a:2',3'-c:3'',2'-h:2''',3'''-j]acridine) complex {[(phen)(2)Ru(PHEHAT)](2)Ru(TPAC)}(6+) 5 have been prepared. Their electrochemistry and photophysics indicate that the (3)MLCT (metal to ligand charge transfer) emissions involve the external {Ru(PHEHAT)} moieties for both complexes and there is no spectro-electrochemical correlation. The trinuclear dendron with the TPAC ligand represents a key compound for future constructions of much larger species thanks to the TPAC that could bridge another polynuclear precursor.

Ru-TAP complexes with btz and pytz ligands: novel candidates as photooxidizing agents.

Two ligands containing 1,2,3-triazole moieties 1 and 3 were easily prepared by a Cu(I)-catalysed "click reaction" between commercially available (trimethylsilyl)alkynes and benzyl azide. These ligands were used in the synthesis of Ru(II) complexes with TAP ligands, i.e.

Photoinduced electron transfer from tryptophan to Ru(II)TAP complexes: the primary process for photo-cross-linking with oligopeptides.

The photoreaction mechanism of [Ru(TAP)(2)(phen)](2+) and [Ru(TAP)(3)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene) with tryptophan (Trp), N-acetyl-Trp, and Lys-Trp-Lys is examined. The existence of a photoelectron-transfer process from the amino acid unit is demonstrated by laser flash photolysis experiments. The back electron transfer (BET) from the reduced complex to the oxidized amino acid, occurring at the microsecond time scale, corresponds approximately to an equimolecular-bimolecular process; however, it is disturbed by another reaction, originating from the oxidized Trp.

Photo-reactive Ru(II)-oligonucleotide conjugates: influence of an intercalating ligand on the inter- and intra-strand photo-ligation processes.

The damaging efficacy towards OligoDeoxyriboNucleotides (ODNs) of two photoreactive polyazaaromatic ruthenium(II) complexes, Ru(T) and Ru(D), has been evaluated. Both compounds correspond to the known [Ru(TAP)(2)(dppz)](2+) complex, but they are anchored differently to a guanine-containing single strand ODN (probe strand). This has allowed us to investigate the influence of the interactions existing between the tethered complexes and the single or double strand, on the photo-ligation processes.

Density functional theory interpretation of the 1H photo-chemically induced dynamic nuclear polarization enhancements characterizing photoreduced polyazaaromatic Ru(II) coordination complexes.

The unprotonated and protonated monoreduced forms of the polyazaaromatic Ru(II) coordination complexes [Ru(tap)(3)](2+) and [Ru(tap)(2)(phen)](2+) (tap = 1,4,5,8-tetraazaphenanthrene ; phen = 1,10-phenanthroline), that is, [Ru(tap)(3)](*+), [Ru(tap)(2)(phen)](*+), [Ru(tap)(2)(tap-H)](*2+), and [Ru(tap)(tap-H)(phen)](*2+), were studied by Density Functional Theory (DFT). The electron spin density of these radical cations, the isotropic Fermi-contact, and the anisotropic dipolar contributions to the hyperfine coupling constants of the H nuclei were calculated in vacuo and using a continuum model for water solvation. For [Ru(tap)(2)(phen)](*+), as well as for its protonated form, the DFT results show that the unpaired electron is not localized on the phen ligand.

A Ru(II)-TAP complex, photoreagent for tryptophan-containing peptides: structure of the covalent photoadduct.

We report the first structure determination of a covalent photoadduct between a Ru(II)-tap complex and a tryptophan-containing peptide (AlaTrpAla) by mass spectrometry and NMR spectroscopy. Ru(II)-tap complexes could thus be exploited as photodamaging agents of Trp-containing polypeptides or proteins.

A rigid dinuclear ruthenium(II) complex as an efficient photoactive agent for bridging two guanine bases of a duplex or quadruplex oligonucleotide.

The rigid dinuclear [(tap)(2)Ru(tpac)Ru(tap)(2)](4+) complex (1) (TAP=1,4,5,8-tetraazaphenanthrene, TPAC=tetrapyridoacridine) is shown to be much more efficient than the mononuclear bis-TAP complexes at photodamaging oligodeoxyribonucleotides (ODNs) containing guanine (G). This is particularly striking with the G-rich telomeric sequence d(T(2)AG(3))(4). Complex 1, which interacts strongly with the ODNs as determined by surface plasmon resonance (SPR) and emission anisotropy experiments, gives rise under illumination to the formation of covalent adducts with the G units of the ODNs.

Oxidizing Ru(II) complexes as irreversible and specific photo-cross-linking agents of oligonucleotide duplexes.

Oxidizing polyazaaromatic Ru(II) complexes containing two TAP ligands (TAP = 1,4,5,8-tetraazaphenanthrene) are able under illumination to cross-link irreversibly the two strands of an oligonucleotide (ODN) duplex by covalent bond formation. The cross-linking proceeds by two successive absorptions of a photon. An adduct of the metallic complex on a guanine (G) base of one ODN strand is first photoproduced, followed by a second photoaddition of the same Ru species to a G base of the complementary strand, provided that the two G moieties are separated by 0 or 1 base pair.

Investigation of the resonance Raman spectra and excitation profiles of a monometallic ruthenium(II) [Ru(bpy)2(HAT)]2+ complex by time-dependent density functional theory.

The resonance Raman (RR) properties of the [Ru(bpy)(2)(HAT)](2+) (where bpy = 2,2'-bipyridine and HAT = 1,4,5,8,9,12-hexaazatriphenylene) complex have been investigated by means of time-dependent density functional theory calculations employing the hybrid B3LYP-35 XC functional and by including the effects of the solvent within the polarizable continuum model approach. Analysis of the electronic excited-state energies has demonstrated that mainly four different metal-to-ligand charge-transfer excitations contribute to the first absorption band in vacuo and water. The simulation of the absorption spectra by including the vibronic structure of the states has shown a general agreement with the experimental spectrum recorded in water.

Exploring photoreactions between polyazaaromatic Ru(II) complexes and biomolecules by chemically induced dynamic nuclear polarization measurements.

Steady-state (1)H photo-chemically induced dynamic nuclear polarization (CIDNP) experiments were conducted at 14.1 T on deoxygenated (buffered pH 7) aqueous solutions of [Ru(phen)(3)](2+), [Ru(tap)(2)(phen)](2+), and [Ru(tap)(3)](2+) (tap = 1,4,5,8-tetraazaphenanthrene; phen = 1,10-phenanthroline) in the presence of guanosine-5'-monophosphate or N-acetyltyrosine. For the first time, CIDNP arising from photo-oxidation by polyazaaromatic Ru(II) complexes is reported.

Photo-cross-linking between polymers derivatized with photoreactive ruthenium-1,4,5,8-tetraazaphenanthrene complexes and guanine-containing oligonucleotides.

We have shown previously that complexes containing 1,4,5,8-tetraazaphenanthrene (TAP) ligands are able to form photoadducts with the guanine bases of DNA and oligonucleotides. In this work, we have exploited this specific photoreaction for carrying out photo-cross-linkings between guanine-containing oligonucleotides (G-ODNs) and biodegradable polymers derivatized with the photoreactive Ru(II) compounds. The aim in the future is to use these polymer conjugates as vectorizing agents of the metallic compounds inside the cells.