Vibrational spectra of chemical and isotopic variants of oxyluciferin, the light emitter of firefly bioluminescence

The chemical complexity of oxyluciferin (OxyLH₂), the light-emitting molecule in the bioluminescence of fireflies, originates from the possibility of keto/enol tautomerism and single or double deprotonation. Herein, we present detailed infrared spectroscopic analysis of OxyLH₂ and several of its chemical isomers and isotopomers. To facilitate the future characterization of its biogenic forms, we provide accurate assignments of the solid-state and solution FTIR spectra of OxyLH₂ based on comparison to six isotopically labeled variants ([2-¹³C]-OxyLH₂, [3-¹⁵N]-OxyLH₂, [4-¹³C]-OxyLH₂, [5-¹³C]-OxyLH₂, [2′-¹³C]-OxyLH ₂, [3′-¹⁵N]-OxyLH₂), five closely related structural analogues, and theoretically computed spectra. The computed DFT harmonic vibrational force fields (B3LYP and M06 functionals with basis sets of varying flexibility up to 6-311++G) reproduce well the observed shifts in the IR spectra of both isotopically labeled and structurally related analogues. Illuminating IR spectra: A detailed IR spectroscopic study of oxyluciferin, the light emitter of firefly bioluminescence, has been performed, which reveals the most accurate vibrational assignments to date (see figure). To that end, a set of oxyluciferin analogues and isotopomers were prepared, and their spectra were recorded in the solid state and in acetonitrile solution.