Vibrational Spectra of Chemical and Isotopic Variants of Oxyluciferin, the Light Emitter of Firefly Bioluminescence

Abstract

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-(13)C]-OxyLH2, [3-(15)N]-OxyLH2, [4-(13)C]-OxyLH2, [5-(13)C]-OxyLH2, [2'-(13)C]-OxyLH2, [3'-(15)N]-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.

Firefly Luciferin‐Inspired Biocompatible Chemistry for Protein Labeling and In Vivo Imaging

“…This is an advantage for buildings ensing probesb ecause of the wide use of bioluminescence in imaging area. [58,124] For example, Chang's group have shown the controlled synthesis of firefly luciferini ns itu for dual-analyte bioluminescence imagingo fr eactive oxygen species (ROS) and caspase-8 activity during acute inflammation processes in living mice. [125] Caspase-3 controlled building of firefly luciferin for bioluminescence imaging of tumor apoptosis in mice [126] and d-Cys-dependentp roduction of firefly luciferinf or bioluminescence detection of d-amino acid oxidase activity [127] have been also reported based on this reaction.…”

“…Compared to other biocompatible reactions, the reaction between CBT and d ‐Cys comes from the nature, and produces the firefly luciferin that can emit bioluminescence when catalyzed by the luciferase. This is an advantage for building sensing probes because of the wide use of bioluminescence in imaging area . For example, Chang's group have shown the controlled synthesis of firefly luciferin in situ for dual‐analyte bioluminescence imaging of reactive oxygen species (ROS) and caspase‐8 activity during acute inflammation processes in living mice .…”

Advances in computational photochemistry and chemiluminescence of biological and nanotechnological molecules

“…Actually, both of them are very limited, compared with the numerous bioluminescent systems in the nature. The years 2014 and 2015 have seen the reports of theoretical studies of firefly, [163][164][165][166][167][168][169][170][171] Cypridina, 172,173 bacteria, 174,175 and obelin. 176 The impressive contributions are briefly summarized below.…”

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase

“…According to the reaction mechanism of the firefly BL that was originally advanced, OxyLH 2 is generated via thermal decomposition of a chemiexcited unstable intermediate in its (ionic) keto form, although there are also arguments that point to emission from its enol tautomer and/or the neutral form. ,,, The “keto hypothesis” clearly contrasts the tautomeric form, which was observed in a single crystal and as the major component in polar solutions, where the molecules are stabilized and exist as neutral hydrogen-bonded dimers of the enol tautomer. ,, Furthermore, inspection of the bond distances of the emitter in the crystal structure of luciferase from Luciola cruciata shows that in the ground state OxyLH 2 exists as the neutral enol form . Although simulations of the molecular dynamics indicate that any significant reorganization of the microenvironment would occur at ultrafast time scales, stabilization of the enol form by a nearby leaving adenosine monophosphate (AMP, Figure ) could account for the enol form being the resting (ground) state of the emitter within the enzyme …”