A Spectroscopic Survey of Substituted Indoles Reveals Consequences of a Stabilized ¹L_b 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, 1La and 1Lb. 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 1La and 1Lb energies in tandem while benzyl ring substitutions with electrophilic groups lift the 1La/1Lb degeneracy. For 5- and 6-hydroxyindole in cyclohexane, 1La and 1Lb transitions are resolved. This finding provides for 1La 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 1La and 1Lb transitions contribute to emission. 10 0 ns fluorescence lifetimes for 5-hydroxyindole are consistent with a charge acceptor-induced increase in the nonradiative rate (1).

Explaining Level Inversion of the La and Lb States of Indole and Indole Derivatives in Polar Solvents

“…Therefore, we give strong theoretical evidence for level inversion between the L a and L b states in indole derivatives and we outline the underlying mechanism for the process. Our results are discussed in view of recent experimental work [12] and we find good agreement between the two.…”

“…Moreover, while the lowest emitting state is the L a state at its optimized geometry when ADC(2)/cc-pVDZ is used, the lowest emitting state is L b at its optimized geometry when ADC(3)/cc-pVDZ is used. The experimental fluorescence emission energy of indole vapor is 4.20 eV [12] and this is in excellent agreement with the 4.42 eV value calculated for the L b state at its equilibrium geometry using ADC(3)/cc-pVDZ. Considering the theoretical results for indole in the gas phase both for absorption and emission, it is found that ADC(2)/cc-pVDZ gives consistently higher energies for the L a state by about 0.2-0.3 eV, which is taken into account in the interpretation of the results to follow.…”

“…[12] While in general, the theoretical values are higher than the experimental values by about 0.5 eV, the trends in both data sets are in good agreement. A similar solvent shift in all indole derivatives in the series is found that is larger for water than for cyclohexane.…”

“…[12] We chose a series of indole derivatives that differ in the substitution pattern of a methyl group in the pyridine ring, that is, 2M (2-methyl indole), 3M (3-methyl indole) and 12M (1,2-dimethyl indole), according to the numbering scheme in Figure 1. We focused on this series, as the methyl substitution is expected to perturb the relative positions of the L a and L b states while keeping the non-specific solute-solvent interactions similar.…”

“…[10][11][12][13][14][15][16] Also, indole displays an unusually large fluorescence Stokes shift in polar solvents. [10][11][12] It was suggested that indole forms an excited-state complex (exciplex) with solvent molecules, and that stabilization of the exciplex is achieved by charge transfer states. [13,14] A different explanation is level inversion, which proposes that due to its polar nature, the L a state is stabilized in the presence of a polar solvent, and becomes the lowest emitting state in polar solvents.…”