Fluorescence following Excited-State Protonation of Riboflavin at N(5)

Excited-state protonation of riboflavin in the oxidized form is studied in water. In the -1 < pH < 2 range, neutral and N(1)-protonated riboflavin coexist in the electronic ground state. Transient absorption shows that the protonated form converts to the ground state in <40 fs after optical excitation. Broadband fluorescence upconversion is therefore used to monitor solvation and protonation of the neutral species in the excited singlet state exclusively. A weak fluorescence band around 660 nm is assigned to the product of protonation at N(5). Its radiative rate and quantum yield relative to neutral riboflavin are estimated. Protonation rates agree with proton diffusion times for H+ concentrations below 5 M but increase at higher acidities, where the average proton distance is below the diameter of the riboflavin molecule.