Dityrosine: photochemical synthesis, isolation and characterization

REID, LARA O.; THOMAS, ANDRÉS H.; LHIAUBET-VALLET,VIRGINIE; MIRANDA, MIGUEL A.; MARIN, M. LUISA; DANTOLA, MARIA LAURA

Villa CarlosPaz

Congreso; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología (XIII ELAFOT); 2017

Oxidative damage to proteins leads to a variety of modifications which are markers of pathogenesis. One of the most important modifications is the dityrosine cross-link, an oxidative covalent bond between two tyrosine (Tyr) residues, which is known to occur in many diseases like amyloid fibril formation, Parkinson?s disease and epidermoid carcinoma. The mechanism of dityrosine (Tyr2) linkage starts with the one-electron oxidation of Tyr, which leads to the long-lived tyrosyl radical (Tyr(-H)?). Two Tyr(-H)? are able to react yielding the Tyr2. Radiation can induce the dimerization of Tyr by direct light absorption or by photosensitization process.[1] The cross-linking of free Tyr and tyrosyl groups in proteins was reported for reactions photosensitized by pterins derivatives,[2,3] a family of heterocyclic compounds which are present in a wide variety of living systems and participate in relevant biological functions. Various attempts to synthesize Tyr2 using different oxidizing agents were unsuccessful. However, oxidation of Tyr with peroxidase and hydrogen peroxide was shown to be the method of choice for in vitro production of Tyr2.[4] The most important difficulty of this method is the isolation of pure Tyr2 from the incubation mixture, which requires many steps. Despite of that, this method is still the main way to obtain Tyr2 and although it has been improved, the purification step is still difficult. Taking into account the biomedical importance of Tyr2, and the lack of studies on its properties due to the drawbacks of the synthesis and purification, the main aim of this work is to develop a new economic and simple method to prepare aqueous solutions of Tyr2 with high purity. Our procedure is based on the dimerization of Tyr photosensitized by pterin (Ptr) in aqueous solutions under UV-A radiation. To develop this, air equilibrated acid aqueous solution of Tyr and Ptr were exposed to UV-A radiation at different concentrations of each reactant for different irradiation time. After the experimental conditions were optimized in order to produce the maximum amount of Tyr2, the compound was isolated from HPLC runs by collecting the mobile phase after passing through the high perfomance liquid cromatography photodiode array (HPLC-PDA) detector. A C18 column was used for isolation of Tyr2 and NH4Ac 1 mM (pH 6.0) was used as eluent. The isolated fraction was analyzed by fluorescence spectroscopy, UPLC coupled to ESI mass spectrometry (UPLC-MS) and Proton Nuclear Magnetic Resonance (1RMN). The mass chromatograms of isolated fraction showed a unique peak with a m/z value of 359.1239 Da which correspond to [2Tyr-2H-H]ˉ. In agreement with this, the mass chromatograms registered for the specific ion mass of 359.1239 Da showed that this molecular weight was observed at only one retention time, indicating that only one isomer of Tyr2 is formed. The generation of this compound could be verified by means of its 1RMN spectrum. The fluoresence quantum yield of the acid and basic forms were determined to be 0.25±0.03 and 0.46±0.07, respectively. The mass of Tyr2 in the isolated sample was calculated taking into account the volume obtained during the isolation of the sample and its concentration. The result was compared to the initial mass of Tyr , and a value of 9 (±1) % was obtained for the overall yield of the process. Acknowledgments: L.O.R., A.H.T, M.L.D. knowledges UNLP, CONICET and ANPCyT for financial support. M.A.M., V. L.-V. and M.L.M. knowledges Spanish Government for financial contribution.References [1] G. Boguta and A. M. Dancewicz ,Nuleonika, 1981, 26, 11 [2] C. Castaño, M. L. Dántola, E. Oliveros, A. H. Thomas and C. Lorente Photochem. Photobiol., 2013, 89, 1448[3] L. O. Reid, E. A. Roman, A. H. Thomas and M. L. Dántola, Biochemistry, 2016, 55 (34), 4777[4] A. J. Gross and I. W. Sizer, J. Biol. Chem., 1959, 234, 1611