Yuri P. Tsentalovich

Ultrafast Excited-State Dynamics of Kynurenine, a UV Filter of the Human Eye

The excited-state dynamics of kynurenine (KN) has been examined in various solvents by femtosecond-resolved optical spectroscopy. The lifetime of the S(1) state of KN amounts to 30 ps in aqueous solutions, increases by more than 1 order of magnitude in alcohols, and exceeds 1 ns in aprotic solvents such as DMSO and DMF, internal conversion (IC) being shown to be the main deactivation channel. The IC rate constant is pH independent but increases with temperature with an activation energy of about 7 kJ/mol in all solvents studied. The dependence on the solvent proticity together with the observation of a substantial isotope effect indicates that hydrogen bonds are involved in the rapid nonradiative deactivation of KN in water. These results give new insight into the efficiency of KN as a UV filter and its role in cataractogenesis.

pH-Sensitive C―ON Bond Homolysis of Alkoxyamines of Imidazoline Series with Multiple Ionizable Groups As an Approach for Control of Nitroxide Mediated Polymerization

Recently, a new concept of pH-switchable agents for reversible addition-fragmentation chain transfer (RAFT) polymerization has been introduced by Benaglia et al. (J. Am. Chem. Soc.2009, 131, 6914-6915). In this paper we extended the concept of pH-switchable mediators to nitroxide mediated polymerization (NMP) by employing nitroxides with basic or acidic groups as controlling agents. Four alkoxyamines, the derivatives of 2-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-2-(pyridin-4-yl)-2,5-dihydro-1H-imidazol-1-oxyl and 2-(2-carboxyethyl)-5,5-diethyl-2,4-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, have been prepared. The influence of pH on alkoxyamine homolysis rate constants (k(d)) and on the nitroxide-alkyl radical recombination rate constants (k(c)) was studied. All alkoxyamines under study as well as the parent nitroxides have several basic groups, which under pH variation can undergo consecutive protonation. It was shown that the k(d) value under basic conditions are significantly (up to 15-fold) higher than in acidic solution at the same temperature, whereas the k(c) value in basic solutions decrease by a factor of 2 only. The efficiency of NMP is known to be dependent on k(d) and k(c), both constants being dependent on the monomer structure; therefore the performance of NMP of different monomers in the controlled mode requires different conditions. It is shown that the pH value crucially affects the polymerization regime, changing it from the controlled to the uncontrolled mode. The controlled regime of NMP of different hydrophilic monomers (sodium 4-styrenesulphonate and acrylamide) in aqueous solution under mild conditions (90 °C) can be achieved using the same alkoxyamine by the variation of the pH value. The chain length of polymers depends on pH value during the polymerization.

Photochemical Properties of UV Filter Molecules of the Human Eye

PURPOSE To compare the photochemical properties of UV filter molecules present in the human lens (kynurenine, KN; 3-hydroxykynurenine, 3OHKN; 3-hydroxykynurenine O-β-D-glucoside, 3OHKG; 4-(2-aminophenyl)-4-oxobutanoic acid, AHA; and glutathionyl-kynurenine, GSH-KN) with the use of the following parameters: excited singlet lifetime τ(S), fluorescence quantum yield Φ(fl), triplet quantum yield Φ(T), and photodecomposition quantum yield Φ(dec). METHODS The excited singlet lifetimes were measured with the use of fluorescence upconversion (time resolution, 210 fs) and pump-probe transient absorption (time resolution, 200 fs) methods. The fluorescence quantum yields were determined relative to an aqueous solution of quinine bisulfate. The triplet quantum yields were measured with the use of nanosecond laser flash photolysis. The photodecomposition quantum yields were determined by steady state photolysis followed by the high-performance liquid chromatography analysis. RESULTS The secondary UV filters--AHA and GSH-KN are better photosensitizers than the primary ones--KN, 3OHKN and 3OHKG: the singlet state lifetimes of the secondary UV filters are longer, and the quantum yields of fluorescence and triplet state formation are higher. CONCLUSIONS With aging, the ratio primary/secondary UV filters in the human lens decreases from approximately 10:1 to 2:1. The obtained results demonstrate that the quality of the secondary UV filters is inferior compared to the primary ones, which may result in a higher susceptibility of old lenses to UV light. That might be an important factor for the development of the age-related cataract.

Metabolomic composition of normal aged and cataractous human lenses

Quantitative metabolomic profiles of normal and cataractous human lenses were obtained with the combined use of high-frequency nuclear magnetic resonance (NMR) and high-performance liquid chromatography with high-resolution mass-spectrometric detection (LC-MS) methods. The concentration of more than fifty metabolites in the lens cortex and nucleus has been determined. For the majority of metabolites, their concentrations in the lens cortex and nucleus are similar, which confirms low metabolic activity in the lens core. The difference between the metabolite levels in the cortex and nucleus of the normal lens is observed for antioxidants and UV filters, which demonstrates the activity of redox processes in the lens. A huge difference is found between the metabolomic compositions of normal and age-matched cataractous lenses: the concentrations of almost all metabolites in the normal lens are higher than in the cataractous one. The most pronounced difference is observed for compounds playing a key role in the lens cell protection and metabolic activity, including antioxidants, UV filters, and osmolytes. The results obtained imply that the development of the age-related cataracts might originate from the metabolic dysfunction of the lens epithelial cells.

Photophysics and Photochemistry of the UV Filter Kynurenine Covalently Attached to Amino Acids and to a Model Protein

The photophysics and photochemistry of kynurenine (KN) covalently bound to the amino acids lysine, cysteine, and histidine, the antioxidant glutathione, and the protein lysozyme have been studied by optical spectroscopy with femto- and nanosecond time resolution. The fluorescence quantum yield of the adducts of KN to amino acids is approximately 2 times higher than that of the free KN in solution; KN attached to protein exhibits a 7-fold increase in the fluorescence quantum yield. The S(1) state dynamics of KN-modified lysozyme reveals a multiphasic decay with a broad dispersion of time constants from 1 ps to 2 ns. An increase of the triplet yield of KN bound to lysozyme is also observed; the triplet state undergoes fast intramolecular decay. The obtained results reveal an increase of the photochemical activity of KN after its covalent attachment to amino acids and proteins, which may contribute to the development of oxidative stress in the human lenses-the main causative factor for the cataract onset.

Alkoxyamine re-formation reaction. Effects of the nitroxide fragment: a multiparameter analysis.

A few years ago, Studer and co-workers (Macromolecules 2006, 39, 1347-1352) reported the dramatic effect of the reaction of re-formation of alkoxyamines on the fate of the nitroxide-mediated polymerization (NMP) of styrene. This prompted us to investigate more carefully the effects of the nitroxide structure on the re-formation rate constant k(c). Ten new values of k(c) were obtained for the reaction of imidozalidine nitroxide and the phenethyl radical. These values were combined with the 21 values of k(c) reported in the literature for a multiparameter analysis (log(k(c)/M(-1) s(-1)) = (10.22 ± 0.10) + (0.46 ± 0.02)E(s) + (0.41 ± 0.17)σ(I)) using the electrical Hammett constant σ(I) to describe both the stabilization and polar effects as well as the modified Taft steric constant E(s) of the nitroxide. The same analysis was performed for the k(c) values of the cross-coupling reaction of nitroxides with tert-butoxylcarbonyl-2-prop-2-yl radical (log(k(c)/M(-1) s(-1)) = (11.10 ± 0.25) + (0.57 ± 0.05)E(s) + (1.42 ± 0.18)σ(I)) and tert-butoxycarbonylethyl radical (log(k(c)/M(-1) s(-1)) = (10.23 ± 0.16) + (0.35 ± 0.03)E(s) + (0.93 ± 0.25)σ(I)). These correlations were applied for the analysis of the NMP of styrene controlled by 6π(•), 6θ(•), and 6ρ(•) using a Fischer phase diagram.

Metabolomics of the rat lens: a combined LC-MS and NMR study.

This work is the first comprehensive report on the quantitative metabolomic composition of the rat lens. Quantitative metabolomic profiles of lenses were acquired with the combined use of high-frequency nuclear magnetic resonance (NMR) and high-performance liquid chromatography with high-resolution mass-spectrometric detection (LC-MS) methods. More than forty low molecular weight compounds found in the lens have been reliably identified and quantified. The most abundant metabolites in the 3-month-old Wistar rat lens are taurine, hypotaurine, lactate, phosphocholine and reduced glutathione. The analysis of age-related changes in the lens metabolomic composition shows a gradual decrease of the content of most metabolites. This decrease is the most pronounced between 1 and 3 months, which probably corresponds to the completion of the lens maturation in one-month-old rats and to the high rate of the young lens growth. The enhanced levels of tryptophan, tyrosine, carnitine, glycerophosphate, GSH and GSSG were found in lenses of senescence-accelerated OXYS rats; for some metabolites, this effect may probably be attributed to the compensatory response to oxidative stress.

Experimental and quantum chemical study of photochemical properties of 4-hydroxyquinoline

UV irradiation of aqueous 4-hydroxyquinoline (4HQN) solutions results in the formation of the triplet states with the quantum yields 30%, 35%, and 7.5% in acidic, neutral, and basic solutions, respectively. In neutral solutions, the keto form is the major tautomeric structure for ground, excited singlet and triplet states of 4HQN. Triplet 4HQN reacts with amino acids tryptophan and tyrosine and with antioxidant ascorbate via the mechanism of electron transfer. The individual experimental and calculated UV-Vis spectra of different acid-base forms of 4HQN in the ground and triplet excited states, as well as reduced and oxidized forms of 4HQN, are reported. Under intense laser irradiation 4HQN undergoes biphotonic ionization, the excited singlet state being the precursor for photoionization.

Deaminated UV filter 3-hydroxykynurenine O-β-d-glucoside is found in cataractous human lenses

Analysis of UV filter levels in 48 cataractous human lenses was performed with the use of HPLC. A new chromophore with the absorption maximum at 410nm and molecular mass of 369Da was detected and assigned as deaminated 3-hydroxykynurenine O-beta-D-glucoside (3OHCKAG). Cataractous lenses are characterized by the wide range of the UV filter concentrations and remarkably lower levels of UV filters and glutathione than published for the normal lenses. No correlation between the lens age and the level of UV filters has been found in cataractous lenses.

Time-resolved CIDNP and laser flash photolysis study of the photoreaction between triplet 2,2′-dipyridyl and guanosine-5′-monophosphate in water

Laser flash photolysis and time-resolved CIDNP have been applied to the investigation of the kinetics and the mechanism of the photoreaction between triplet 2,2′-dipyridyl (DP) and guanosine-5′-monophosphate (GMP) over a wide pH range in aqueous solution. The pH dependence of the rate constant kq of quenching the triplet dipyridyl by the nucleotide has been measured. Upon pH titration, four pairs of the reacting species contribute to the observed value of kq: pH 9.4, TDP and G(−H)−, with the corresponding quenching rate constants k1=1.3×109 M−1 s−1, k2=2.7×109 M−1 s−1, k3=1.6×108 M−1 s−1, k4=1.1×109 M−1 s−1. Based on LFP and CIDNP data, the established mechanism of the quenching reaction is hydrogen atom transfer in neutral solution (5.8<pH<9.4), and electron transfer in all other pH regions. Kinetic CIDNP measurements reveal that in acidic and basic solutions the CIDNP kinetics for GMP is determined by the degenerate electron exchange between the GMP radical and its parent molecule with the rate constants 1.3×108 M−1 s−1 (acidic conditions) and 4.0×107 M−1 s−1 (basic conditions). The nuclear paramagnetic relaxation time for the proton H8 of GMP, T1=20±5 μs, obtained from the simulations of the CIDNP kinetics, is found to be independent of the protonation state of the radical.