Spectroscopic Properties of Amine‐substituted Analogues of Firefly Luciferin and Oxyluciferin

Abstract

Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an amine substituent (NH , NHMe and NMe ) at the C6' position were studied based on absorption and fluorescence measurements. Their π-electronic properties were investigated by DFT and TD-DFT calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An increase in the electron-donating strength of the substituent led to the bathochromic shift of the fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding oxyluciferin analogues in a solvent were well correlated with each other. Based on the obtained data, the polarity of a luciferase active site was explained. As a result, the maximum wavelength of bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence of the luciferin analogue in place of that of the corresponding oxyluciferin analogue.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases

“… 39 , showed that L. cruciata luciferase displays a close active site conformation with the critical luciferyl-adenylate intermediate analog, DLSA, and an open one with the products AMP and oxyluciferin. Later studies with 6′-aminoluciferin analogs supported the existence of two binding modes for oxyluciferin in firefly luciferases active site, an apolar (N) responsible for green light emission and another polar (P) responsible for red emission 21 , 22 . The active site in firefly luciferases may undergo conformational changes between these two extremes, and the light emitting step may take place somewhere between these two conformations.…”

“…Among the specific interactions, the presence of bases near the thiazinic side of the luciferin binding site assisting the tautomerization between a keto and enol forms has been originally claimed to explain green to red bioluminescence color change in firefly luciferases 9 , 13 . More recently, interactions influencing the resonance forms of excited oxyluciferin 20 , specific acid-base and finally electrostatic effects around oxyluciferin 6′ phenol group are being considered to determine bioluminescence colors 18 , 21 , 22 .…”

Computational investigation into the fluorescence of luciferin analogues

“…We therefore used the alternative 1b conformation in the remainder of the analyses. Kakiuchi and Hirano reported a similar near‐degeneracy for the two conformations in the ground state, but found a smaller difference of 5 nm when the S 0 ➔ S 1 absorption was considered . In Figure we show the experimentally measured emission wavelengths against the predicted wavelengths with the best performing level of theory, with the outlier indicated.…”

“…Chen and Lindh examined the effect of the medium on the spectra . More recently, a larger number of luciferin/oxyluciferin analogues have been studied, but comparison with experimentally measured emission wavelengths has been relatively limited …”

pH‐Dependent Absorption Spectrum of Oxyluciferin Analogues in the Presence of Adenosine Monophosphate

“…The extraction of the contribution due to each oxyluciferin form to the absorption or emission spectra is often tedious . For that reason, oxyluciferin analogues have been proposed and synthesized for inhibiting certain forms . In this work, we are interested in a particular set of analogues (Figure , right), studied both experimentally and theoretically .…”