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. 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.

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)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.

pH Dependence of Photoinduced Electron Transfer with [Ru(TAP)3]2.

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.

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. 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.

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. 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.

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. 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.

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. From transient absorption studies under pulsed laser irradiation, it is concluded that the quenching originates from a par proton-coupled electron transfer (PCET) process. Such an intramolecular quenching is absent when the phenol groups of the calixarene platform are derivatized by azido arms.

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. In a recent study, we showed that Oligofectamine transfected Ru-P-4 antisense oligonucleotide failed to inhibit efficiently the growth of cervical cancer cell line SiHa, contrarily to the Ru-P-6 antisense oligonucleotide, another sequence also targeting the E6 gene. The ability of PDMAEMA-b-PMEMA to form polyplexes with optimal physicochemical characteristics was investigated first. Then the ability of the PDMAEMA-b-PMEMA/Ru-P-4 antisense oligonucleotide polyplexes to transfect two keratinocyte cell lines (SiHa and HaCat) and the capacity of polyplexes to inhibit HPV16+ cervical cancer cell growth was evaluated. PDMAEMA-b-PMEMA base polyplexes at the optimal molar ratio of polymer nitrogen atoms to DNA phosphates (N/P), were able to deliver Ru-P-4 antisense oligonucleotide and to induce a higher growth inhibition in human cervical cancer SiHa cells, compared to other formulations based on Oligofectamine.

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. 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.

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. After absorption of a second photon, this adduct can generate a bi-adduct, whereby the same complex binds to a second G moiety. These bi-adduct formations are responsible for photo-cross-linking between two strands of a duplex, each containing a G base, or between two G moieties of a single strand such as a telomeric sequence, as demonstrated by polyacrylamide gel electrophoresis analyses or mass spectrometry. Scanning force microscopy also allows the detection of such photobridgings with plasmid DNA. Other applications, for example with Ru-ODN, i.e. ODN with chemically anchored Ru-TAP complexes, are also discussed. It is shown that such Ru-ODN probes containing a G base in their own sequences are capable of photo-cross-linking selectively with their targeted complementary sequences, and, in the absence of such targets, they self-photo-inhibit. Such processes are applied successfully in gene photo-silencing of human papillomavirus cancer cells.

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.

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. The novel strategies currently developed to improve their reactivity and specificity towards DNA, more particularly in the gene-silencing framework, are also discussed.

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. Thus, a double photo-addition of Trp residues of peptides on Ru(II) complexes is demonstrated by mass spectrometry with some structural issues. Such bi-adduct formations offer the possibility of photocrosslinking two Trp-containing biomolecules, which is investigated in this study. Thus, photocrosslinking between two complementary oligonucleotides (ODNs) derivatized by Trp-containing tripeptides is demonstrated by polyacrylamide gel electrophoresis (PAGE) in the presence of Ru(II)-TAP complexes. Both PAGE and MS indicate that such photocrosslinkings arise from two reaction pathways: either via the double addition of Trp residues on the Ru complex or from dimerization of Trp radicals. The competition between these two pathways depends on the experimental conditions. Heterobridgings between guanine bases and tryptophan residues mediated by Ru(II)-TAP complexes is also examined, opening the way to ODN-peptide photocrosslinkings.

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. For decreasing the length of preparation of these compounds, microwave assisted syntheses have been tested and used not only for the targeted complexes but also for the precursors ((phen)(2)RuCl(2), {(phen)(2)Ru(phendione)}(2+), {(phen)(2)Ru(PHEHAT)}(2+) (PHEHAT = 1,10-phenanthrolino[5,6-b]1,4,5,8,9,12-hexaazatriphenylene), (DMSO)(4)RuCl(2)), and for the bridging TPAC ligand itself. The microwave method allows a drastic decrease of the preparation times, especially in the case of the TPAC, from 8 days to 60 min.

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. [Ru(TAP)(2)btz](2+)2 and [Ru(TAP)(2)pytz](2+)4. The electrochemical and photophysical properties of these complexes were investigated. The data show that both complexes should behave as highly oxidizing agents under illumination. However, complex 4 displays more attractive photophysical properties than complex 2 and constitutes thus a Ru-TAP compound that can be easily derivatized for photodamaging biomolecules.

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. Moreover, in competition with the BET, the reduced and oxidized intermediates give rise to an adduct. The latter is clearly detected by gel electrophoresis experiments in denaturing conditions, with a system composed of an oligonucleotide derivatized at the 3' end by the Ru(II)TAP complex and hybridized with the complementary sequence functionalized at the 5' end by the tripeptide Lys-Trp-Lys. Thus, upon illumination, a cross-linking between the two strands is observed, which originates from the presence of a Trp residue.

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. From melting temperature measurements of the duplex formed between these Ru-ODNs and their complementary sequence (target strand), it has been found that Ru(T) anchored via the TAP ligand interacts with the duplex by means of the intercalating dppz ligand (head on geometry), while Ru(D) anchored via the dppz ligand likely adopts a side on geometry without intercalation. Both single stranded Ru conjugates self-inhibit in the absence of their target ODN by forming exclusively a cyclic "seppuku" photo-adduct (intra-molecular photoreaction). In contrast, this intra-molecular photo-product is precluded in presence of the target strand, and the Ru-ODN sequence photo-crosslinks with the latter (inter-molecular photoreaction). Both intra- and inter-molecular processes with both complexes are efficient (80% yields) and lead to stable photo-adducts. Interestingly, detailed studies have revealed that the similar photo-damaging efficacy of crosslinking by Ru(T) and Ru(D) is a consequence of a cascade of events with compensatory effects, originating from the different geometry of interaction of the tethered complexes. Notably, antagonistic effects are present when the complex is intercalated, the guanine oxidation step being highly favoured and the recombination of the quenching products being hindered.

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. For both [Ru(tap)(3)](*+) and [Ru(tap)(2)(phen)](*+), they reveal high electron spin density in the vicinity of tap H-2 and tap H-7 (the H atoms in the ortho position of the tap non-chelating N atoms). These results are in full agreement with recent steady-state (1)H photo-Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP) measurements. The DFT calculations performed for the protonated species also predict major (1)H photo-CIDNP enhancements at these positions. Interestingly, they indicate significantly different polarization for tap H-9,10, suggesting that the occurrence of a photoinduced electron transfer with protonation of the reduced species might be detected by high-precision photo-CIDNP experiments.

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. The yield of photocrosslinking of the two strands of duplexes by 1 is the highest when the G bases of each strand are separated by three to four base pairs. This corresponds with each Ru(tap)(2) moiety of complex 1 forming an adduct with the G base. This separation distance of the G units of a duplex could be determined thanks to the rigidity of complex 1. On the basis of results of gel electrophoresis, mass spectrometry, and molecular modelling, it is suggested that such photocrosslinking can also occur intramolecularly in the human telomeric quadruplex d(T(2)AG(3))(4).