Oxidative damage to proteins leads to a variety of modifications that are markers of pathogenesis. One of the most important modifications is the dityrosine (Tyr₂ ) cross-link, resulting from an oxidative covalent bond between two tyrosines (Tyr). An optimized methodology for preparation of pure Tyr₂ is important to investigate in detail its physicochemical properties and reactivity. Pterin (Ptr), the parent and unsubstituted compound of oxidized pterins, is able to photosensitize the cross-linking of free tyrosine (Tyr) and tyrosine residues of peptides and proteins through a photoinduced electron transfer mechanism. We have optimized a simple, one-step photocatalyzed formation of Tyr₂ , using Ptr as photocatalyst. Our procedure is carried out in aqueous solutions under UV-A radiation for few minutes. The purification of Tyr₂ is performed by reverse-phase chromatography. The obtained highly pure solution is used to fully characterize the Tyr₂ (exact mass and ¹H, ¹H- ¹H COSY; DEPT; HSQC and HMBC NMR experiments) and to deeper study its fluorescence properties.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas