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. Aside from the high selectivity, the photolabeling is blue light-driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light-photocrosslinking a UV-sensitive peptide to POI. Our visible light photolabeling can generate photocaged proteins for subsequent activity manipulation by UV light. Cytoskeletal dynamics regulation is demonstrated in living cells via the unprecedented POI photomanipulation and proves that our methodology opens a new avenue to endogenous protein modification.

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 in-cellulo protein labeling. Herein we report a highly selective photoaffinity labeling (PAL) method using a tryptophan-specific Ru-TAP complex as photocrosslinker (Trp-tag). Aside from the high selectivity, the PAL is blue light driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light photocrosslinking a UV-sensitive peptide to POI. The remote-control functionality of the peptide allows POI inhibition after blue light irradiation, and reactivation upon UV photolysis. Cytoskeletal dynamics regulation is demonstrated via the unprecedented in-cellulo POI photomanipulation, which opens a new avenue to endogenous protein modification for novel functions.

Two bis-terdentate cyclometalated Ir(III) complexes with polypyridyl (N^N^N) and cyclometalated (C^N^C) ligands [Ir-Py]+ and [Ir-Py-Me]2+ have been synthesized and characterized. Their absorption and emission properties have been examined, more particularly in MeCN as a function of the addition of different acids. Depending on the acid strength and concentration, two distinct effects have been observed. For acetic acid at high concentration or trifluoroacetic acid (TFA) at low concentration, the pyridine moiety of [Ir-Py]+ is protonated; its spectroscopic behaviour is then similar to that of [Ir-Py-Me]2+. Moreover at higher concentration in TFA, the methoxy group of both complexes is protonated. The spectroscopic and electrochemical data as well as the DFT and TD-DFT calculations support contributions of charge-transfer excited states in absorption and emission from metal-ligand/ligand based HOMOs to ligand-based LUMOs, i.e. MLLCT.

The excited-state quenching of [Ru(TAP)2(HAT)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene, HAT= 1,4,5,8,9,12-hexaazatriphenylene) by hydroquinone (H2Q), 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 1H 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. Photo-CIDNP revealed a different mechanism at low pH, involving protonation before electron transfer and yielding a distinct protonated monoreduced complex. The experimental photo-CIDNP patterns are consistent with density functional theory calculations. This work highlights the power of 1H photo-CIDNP for characterizing, at the atomic level, transient species involved in electron-transfer processes.

The quenching of the excited state of [Ru(TAP)3]2+ (TAP = 1,4,5,8-tetraazaphenanthrene) by guanosine-5'-monophosphate (GMP), N-acetyltyrosine (N-Ac-Tyr), and hydroquinone (H2Q) 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 1H 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)3]2+ has been gained thanks to the combined use of these two approaches, photo-CIDNP and electronic spectroscopies, highlighting their complementarity. In contrast to what was believed, it is found that the protonated excited state of [Ru(TAP)3]2+ may give rise to an electron transfer with N-Ac-Tyr and H2Q. Such a photoinduced electron transfer does not occur with protonated GMP, however. 1H photo-CIDNP experiments are expected to be particularly promising for characterization of the reductive quenching of excited-state ruthenium(II) polypyridyl complexes comprising several nonequivalent protonation sites.

The photoreactive [Ru(TAP)(2)(phen)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene; phen = 1,10-phenanthroline) complex tethered to the cell-penetrating peptide (CPP) TAT was studied in vitro and in cellulo. The tethering of the complex does not affect its behavior under blue-light irradiation in the presence of guanine-containing oligodeoxyribonucleotides (ODNG). Thus, the luminescence is quenched in the presence of ODNG, and gel electrophoresis experiments showed the appearance of products corresponding to the irreversible attachment of the conjugate to ODNG upon illumination. The cellular uptake of the conjugate was examined by flow cytometry, inductively coupled plasma mass spectrometry (ICP-MS), and confocal imaging microscopy. These experiments showed that the [Ru(TAP)(2)(phen-TAT)] conjugate is readily taken up by HeLa cells and, despite these favorable factors, the cellular survival was 100%, as measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A possible origin of the inactivity of [Ru(TAP)(2)(phen-TAT)] under irradiation is proposed on the basis of the fluorescence-activated cell sorting (FACS), ICP-MS, and confocal microscopy results.

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. The PA's spectroscopic and redox data combined with TDDFT results corroborated with resonance Raman data show that the properties of this PA (pKa around 7) depend on the solution pH. Theoretical results indicate that the acid form PA.H(+) when excited should relax to MLCT (metal-to-ligand charge transfer) excited states, in contrast to the basic form PA whose excited state should have LLCT/ILCT (ligand-to-ligand charge transfer/intra ligand charge transfer) characteristics. Ultrafast excitation of PA.H(+) at pH 5.9 produces continuous dynamic processes in a few hundred picoseconds involving coupled proton-electron transfers responsible for luminescence quenching. Long-lived species of a few microseconds capable of reacting with GMP are produced at that pH, in agreement with the formation of covalent addition of a second GMP to PA, as shown by mass spectrometry results. In contrast, at pH 8 (mainly nonprotonated PA), other ultrafast transient species are detected and no GMP biadduct is formed in the presence of GMP. This pH dependence of photoreaction can be rationalized with the different nature of the excited states, thus at pH 8, unreactive LLCT/ILCT states and at pH 5.9 reactive MLCT states.

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. However, in BuCN, the Cl(-) salt of the complex exhibited a surprising behavior in the presence of 1/1(•)H(+). Although an emission decrease was observed with the protonated calixarene, an emission increase was evidenced in the presence of nonprotonated 1. As the Cl(-) ions were shown to inhibit the luminescence of the complex in BuCN, this luminescence increase by nonprotonated 1 was attributed to the protection effect of 1 by encapsulation of the Cl(-) anions into the tris-urea binding site. The study of the luminescence lifetimes of the Ru-TAP complex in BuCN as a function of temperature for the PF6(-) and Cl(-) salts in the absence and presence of 1 led to the following conclusions. In BuCN, in contrast to MeCN, in addition to ion pairing, because of the poor solvation of the ions, the luminescent metal-to-ligand charge transfer ((3)MLCT) state could reach two metal-centered ((3)MC) states, one of which is in equilibrium with the (3)MLCT state during the emission lifetime. The reaction of Cl(-) with this latter (3)MC state would be responsible for the luminescence quenching, in agreement with the formation of photosubstitution products.

A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and one phenanthroline (phen) ligands was synthesized and then tethered to (antisense) oligonucleotides (RuASO) to target a destabilized GFP (dGFP). The specificity of the photoreaction of this RuASO conjugate was studied in vitro by polyacrylamide gel electrophoresis (PAGE) experiments in denaturing conditions. Other nonspecific RuASO conjugates were also prepared and evaluated with human keratinocytes that expressed dGFP. An illumination-dependent cytotoxicity was observed for most RuASO conjugates that varied from 10 to almost 40?%, but only the specific RuASO conjugate was able to significantly reduce GFP expression in illuminated cells.

Under irradiation, Ru-TAP (TAP = 1,4,5,8-tetraazaphenanthrene) complexes tethered to oligonucleotides (Ru-ODN) hybridized to their complementary target sequences produce irreversible adducts with guanine (G) bases. The interesting feature is that the G bases that react in the double strand (DS) topology belong exclusively to the complementary strand; thus, no G base of the Ru-ODN strand participates in the production of these adducts. This highly specific photoreaction in the DS topology is demonstrated by gel electrophoresis analyses of different duplex sequences in which the positions of the G bases are varied. Atomistic molecular modelling simulations indicate that this specificity is due to important steric constraints for the intrastrand adduct formation in the duplex. Luminescence lifetime measurements furnish more detailed information on these different duplex systems.

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. Interestingly, in the presence of mono- or polynucleotides, a photoinduced electron-transfer process from a DNA purine base (i.e., guanine or adenine) to the excited complex is shown through luminescence quenching experiments. For the first time, this work reports evidence for selective DNA purine bases oxidation by excited iridium(III) bis-terdentate complexes.

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. Moreover in relation to this observation, [Cu(I)(dpdipy)2](-) could not be synthesized in spite of the Cu(I) core protection by the phenyl substituents in ortho position of the nitrogen atoms. Theoretical calculations provide some explanations for this instability. Interestingly [Cu(II)(dipy)2] and [Cu(II)(dpdipy)2] display weak luminescence at room temperature, attributed to a ligand centered emission.