A Spectroscopic Survey of Substituted Indoles Reveals Consequences of a Stabilized 1Lb Transition

Abstract

Although tryptophan is a natural probe of protein structure, interpretation of its fluorescence emission spectrum is complicated by the presence of two electronic transitions, (1)L(a) and (1)L(b). Theoretical calculations show that a point charge adjacent to either ring of the indole can shift the emission maximum. This study explores the effect of pyrrole and benzyl ring substitutions on the transitions' energy via absorption and fluorescence spectroscopy, and anisotropy and lifetime measurements. The survey of indole derivatives shows that methyl substitutions on the pyrrole ring effect (1)L(a) and (1)L(b) energies in tandem, whereas benzyl ring substitutions with electrophilic groups lift the (1)L(a)/(1)L(b) degeneracy. For 5- and 6-hydroxyindole in cyclohexane, (1)L(a) and (1)L(b) transitions are resolved. This finding provides for (1)L(a) origin assignment in the absorption and excitation spectra for indole vapor. The 5- and 6-hydroxyindole excitation spectra show that despite a blue-shifted emission spectrum, both the (1)L(a) and (1)L(b) transitions contribute to emission. Fluorescence lifetimes of 1(0) ns for 5-hydroxyindole are consistent with a charge acceptor-induced increase in the nonradiative rate (1).