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[EN] Pterin (Ptr) is a model photosensitizer that acts mainly through type I mechanism and is able to photoinduce the one-electron oxidation of purine and pyrimidine nucleobases. However, under anaerobic conditions Ptr reacts with thymine (T) to form photoadducts (Ptr-T) but does not lead to the photodegradation of guanine (G), which is the nucleobase with the lowest ionization potential. Accordingly, G is thermodynamically able to reduce the radicals of the other nucleobases and has been described in this sense as the "hole sink" of the DNA double helix. Here we analyze by steady-state and time-resolved studies the effect of G in the anaerobic photosensitization of T by Ptr, using nucleotides and oligonucleotides of different sequences. We demonstrated that G is able to reduce T radicals but does not prevent the formation of Ptr-T adducts. Our results suggest that after the encounter between the excited Ptr and T, and completion of the electron transfer step, part of the radicals escape from the solvent cage, to further react with other species. However, a proportion of radicals do not escape and evolve to photoadducts before separation. We provide new evidence that contributes to understand the photosensitizing properties of Ptr in the absence of O-2, the mechanism of formation of photoadducts in the DNA and the protective role of G towards the photodamage in other nucleobases., The present work was partially supported by Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET-Grant P-UE 2017 22920170100100CO) , Agencia de Promocion Cientifica y Tecnologica (ANPCyT-Grant PICT 2015-1988, and PICT 2017-0925) , Universidad Nacional de La Plata (UNLP-Grant 11/X840) . Funding from the Program CSIC de Cooperacion Cientifica para el Desarrollo (iCOOP-Light project ref 20105CD0017) and Spanish Government (PGC2018-096684-B-I00) are gratefully acknowledged. S.E.R. and J.R.N.R. thanks ANPCyT and CONICET for doctoral research fellowships. A.M.R. thanks Universidad de Antioquia (Colombia) for research fellowship. C.L. and A.H.T. are research members of CONICET. V.L.V. is research member of CSIC.

UV-A radiation (320-400 nm) induces modifications to different biomolecules through photosensitized reactions. Oxidized pterins are efficient photosensitizers that accumulate in the skin affected by vitiligo, and photoinduce the oxidation of guanine in a process initiated by an electron transfer from the nucleobase to the triplet excited state of the photosensitizer. In this work, we have investigated the degradation of 2′-deoxyguanosine 5′-monophosphate (dGMP) photosensitized by pterin (Ptr), the parent compound of oxidized pterins, in aqueous solutions under UV-A irradiation. We have identified five products containing the oxidized guanine moiety: the deoxyribonucleoside 5′-monophosphate derivatives of imidazolone, dehydroguanidinohydantoin, guanidinohydantoin, oxazolone and spiroiminodihydantoin. An additional product with a much higher molecular weight, denoted P680, was also detected. The MS/MS analyses show that this compound contains an intact guanine moiety and a modified one. The dependence of the rate of product formation in different experimental conditions was analyzed and a general mechanistic scheme is proposed. Fil: Serrano, Mariana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Estébanez, Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Vignoni, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Lorente, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Vicendo, Patricia. Université Paul Sabatier; Francia Fil: Oliveros, Esther. Université Paul Sabatier; Francia Fil: Thomas, Andrés Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina

The photochemistry of the DNA biomacromolecule is an issue of paramount importance as it is part of the etiology of skin cancer development, being ultraviolet sunlight radiation the most relevant environmental carcinogen. Herein, we demonstrate the potential of pterin, an endogenous compound, to form covalent adduct under UVA irradiation with a short thymine oligomer as well as with the whole DNA polymer. Our approach is based on the spectroscopic features of pterin, which allow, by monitoring specific absorption or emission wavelengths, the following-up of the covalent binding. The results are confirmed by HPLC coupled with mass spectrometry, revealing the attachment of one or two pterin units to the homothymine 5-mer oligonucleotide. Altogether the findings point toward the role of pterin as endogenous sensitizer and genotoxic compound., The present work was partially supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-Grant PIP 112-200901-0304), Agencia de Promoción Científica y Tecnológica (ANPCyT-Grant PICT-2012–0508), Universidad Nacional de La Plata (UNLP-Grant X712). Funding from the Programa CSIC de Cooperación Científica para el Desarrollo (iCOOPLight project ref 20105CD0017) and Spanish Government (CTQ2015-70164-P) are gratefully acknowledged. S.E.R. thanks ANPCyT and CONICET for doctoral research fellowships. M.G.T thanks CONICET for postdoctoral fellowship. C.L. and A.H.T. are research members of CONICET. The authors thank Dr. Mariana Vignoni (INIFTA, CONICET) and Nathalie Martins-Froment of the Service Commun de Spectrométrie de Masse (FR2599), Université de Toulouse III (Paul Sabatier) for their crucial contributions in mass spectrometry measurements.

El objetivo general de esta tesis doctoral es investigar los procesos fotosensibilizados por pterinas de bases pirimidínicas que forman parte de diferentes sustratos en condiciones aneróbicas. Para ello se trabajó con Ptr, que es el compuesto modelo no sustituido de las pterinas oxidadas. Como sustratos se emplearon nucleótidos, nucleósidos, oligonucleótidos y ADN eucariota. A continuación se detallan los objetivos específicos que han dirigido el desarrollo de la tesis: - Estudiar el proceso fotosensibilizado del nucleósido de timina por Ptr bajo diferentes condiciones experimentales y dilucidar el mecanismo de reacción. - Comparar la eficiencia de los procesos fotosensibilizados en nucleótidos y nucleósidos por Ptr en condiciones anaeróbicas. Concretamente, realizar un estudio sobre la generación de aductos fluorescentes entre el fotosensibilizador y el sustrato. - Caracterizar estructuralmente dichos aductos. - Analizar las propiedades fotofísicas de aductos pterina-timina. - Investigar la generación de aductos fluorescentes en oligonucleótidos, tanto de hebra simple como de hebra doble. - Estudiar el daño sufrido por moléculas de ADN eucariota en procesos fotosensibilizados por Ptr. - Realizar un análisis acerca de la formación de aductos pterina-timina en moléculas de ADN., Facultad de Ciencias Exactas

Pterin derivatives are heterocyclic compounds which are present in different biological systems. Neutral aqueous solutions of pterins present acid-base and keto-enol equilibria. These compounds, under UV-A radiation fluoresce, undergo photooxidation, generate reactive oxygen species and photoinduce the oxidation of biological substrates. As photosensitizers, they may act through different mechanisms, mainly through an electron transfer-initiated process (type-I mechanism), but they also produce singlet molecular oxygen (1 O2 ) upon irradiation (type-II mechanism). In general, upon UV-A excitation two triplet states, corresponding to the lactim and lactam tautomers, are formed, but only the last one is the responsible for the photosensitized reactions of biomolecules. We present a study of the photochemical properties of 3-methylpterin (3-Mep) which, in contrast to most pterin derivatives, exists only in the lactam form. Also an improvement in the synthesis of 3-Mep is reported. The spectroscopic properties 3-Mep in aqueous solution were similar to those of the unsubstituted pterin derivative (Ptr) in its acid form, such as absorption, fluorescent and phosphorescent emission spectra. Experiments using 2'-deoxyguanosine 5'-monophosphate (dGMP) as oxidizable target demonstrated that methylation at C-3 position of the pterin moiety does not affect significantly the efficiency of photosensitization, but results in a more photostable sensitizer.

A new fluorescent compound was isolated from UVA-irradiated aqueous solutions containing pterin (Ptr) and 2′deoxythymidine (dT) in anaerobic conditions. Pterins are widespread in living systems in small amounts, but they are accumulated in some pathological situations. Under UVA radiation, pterins are photochemically active, fluorescent, and photosensitize the generation of singlet oxygen [1O2(1Δg)]. The isolated compound was structurally characterized by using liquid chromatography coupled to tandem mass spectrometry, and its photophysical properties were studied with the time-correlated single-photon-counting technique. The molecular weight and the analysis of the fragmentation correspond to a molecule where the pterinic moiety is attached to the thymine nucleobase. The product exhibits photophysical properties similar to those of Ptr, including relatively high fluorescence and 1O2 production quantum yields., Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas