FLUORESCENCE QUENCHING IN INDOLES BY EXCITED-STATE PROTON-TRANSFER

Indoles undergo two isotopically sensitive temperature-dependent fluorescence quenching processes: solvent quenching and excited-state proton transfer. Fluorescence quantum yields of simple indoles in protium and deuterium solvents were measured in the absence and presence of glycine. Photochemical H-D exchange was monitored by H-1 NMR and mass spectrometry. Although the fluorescence quantum yield and lifetime of 2-methylindole show large deuterium isotope effects in aqueous solutions, photochemical H-D exchange was not detected after extensive irradiation, whereas, H-D exchange is readily observed for 2- and 3-methylindole in solutions containing glycine. Stern-Volmer plots of glycine quenching data give bimolecular rate constants k(q) from (0.5-3) x 10(8) M-1 s-1 for indoles in water. The k(q) values of 2- and 3-methylindole are faster in protium than in deuterium solvents. The isotope effect on k(q) implicates excited-state proton transfer in the collisional quenching mechanism. This contrasts with iodide quenching which has no isotope effect on k(q). A glycine derivative lacking the ammonium protons, N,N,N-trimethylglycine, does not quench indole fluorescence. The intermolecular excited-state reaction of 2- and 3-methylindole with 0.3 M glycine-d5 in 50% D2O/CD3OD induces H-D exchange at three ring carbons. In 2-methylindole the exchange is fastest at C3 and occurs with similar rates at C4 and C7 on the indole ring. The temperature dependence of 3-methylindole fluorescence in 0.5 M glycine was also determined. The large difference in temperature dependence for solvent quenching and glycine quenching causes curvature in the Arrhenius plot. The frequency factor A2 = 7.2 X 10(10) s-1 and activation energy E2* = 3.6 kcal/mol for glycine quenching are similar to the values for intramolecular excited-state proton transfer in tryptamine. Possible mechanisms for the excited-state proton transfer reaction and the implications of this reaction for tryptophan fluorescence in proteins are discussed.