A Novel Type of Luciferin from the Siberian Luminous Earthworm Fridericia heliota: Structure Elucidation by Spectral Studies and Total Synthesis

Abstract

We report structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota. This luciferin represents a key component of a novel ATP-dependent bioluminescence system. The UV, fluorescence, NMR and HRMS spectral studies were performed on 5 mkg of the isolated substance, and gave four isomeric structures, conforming with spectral data. These isomers were chemically synthesized and one of them was found to produce light in the reaction with a protein extract from Fridericia. The novel luciferin was found to have an unusual deeply modified peptidic nature, implying an unprecedented mechanism of action.

Novel Mechanism of Bioluminescence: Oxidative Decarboxylation of a Moiety Adjacent to the Light Emitter of Fridericia Luciferin

“…Adownfield shift of the CompX H3p roton by approximately 0.2 ppm was also observed, while all other protons remained intact and showed unchanged NMR signals.T aken together, these data provide unambiguous evidence that Fridericia oxyluciferin is produced through oxidative decarboxylation of the lysine moiety, contrary to our initial hypothesis that the oxalate fragment is oxidized during bioluminescence. [2] Them echanism of light production by the bioluminescence system of Fridericia heliota seems to be highly similar to that of the well-studied firefly bioluminescence (Scheme 2). Thereaction probably starts with the formation of aluciferin adenylate at the lysine carboxy group,f ollowed by deprotonation of the neighboring lysine a-CH, addition of an oxygen molecule to the carbanion to give ap eroxide,n ucleophilic attack of the peroxy group at the adenylated carboxy group with the release of AMP and formation of the dioxetanone cycle,a nd finally,e lectrocyclic scission of the dioxetanone ring to release aC O 2 molecule and form oxyluciferin, in amanner analogous to the firefly system.…”

“…[1,6] However,inthe firefly bioluminescence mechanism, the newly formed carbonyl group becomes part of the conjugated p system of the light emitter, while in the Fridericia mechanism, the p system of the light emitter (CompX fragment) remains unchanged during oxidation. Ther ole of the CompX moiety as the light emitter is supported by the close similarity of the luciferin fluorescence emission spectrum to its bioluminescence emission spectrum (l max 466 and 480 nm, respectively) [2] and also to the fluorescence emission spectrum of oxyluciferin (Figure S3). Thef luorescence quantum yield of oxyluciferin in water at pH 5.7 was determined to be as low as 0.16 %.…”

“…[1] We recently reported the structure of an ovel luciferin from the Siberian bioluminescent earthworm Fridericia heliota. [2] We found this luciferin to be ak ey component of an ovel ATPdependent bioluminescent system, along with as pecific luciferase,A TP,M g 2 + ions,a nd molecular oxygen [3] The Fridericia luciferin turned out to be an unusual peptide formed from oxalic acid, l-lysine,m odified tyrosine (CompX [4] ), and g-aminobutyric acid residues.Herein, we report the structural elucidation of the Fridericia luciferin oxidation product, i.e., the oxyluciferin. Ther esults provide as tructural basis for understanding the novel light generation mechanism underlying the bioluminescence of Fridericia heliota.…”

“…Oxyluciferin was prepared by mixing 0.18 mg of synthetic Fridericia luciferin [2] with excess ATPa nd crude Fridericia luciferase obtained through partial purification of protein extract from 40 go ft he worm biomass [5] (Scheme 1, see the Supporting Information for details). Ther eaction mixture produced constant eye-visible luminescence and the reaction course was monitored by HPLC analysis of aliquots (Figure 1), which showed formation of am ajor product of luciferin oxidation with ar etention time of 18.2 min.…”

Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota

“…In 2014 we have reported structure elucidation of a novel luciferin from the Siberian bioluminescent oligochaete earthworm Fridericia heliota. 2 Fridericia luciferin turned out to be an unusual peptide formed by the residues of oxalic acid, L ‐lysine, modified tyrosine, and γ‐aminobutyric acid. In contrast to other known earthworm bioluminescence mechanisms, which utilize hydrogen peroxide to oxidize N ‐isovaleryl‐3‐amino‐propanal in an ATP‐independent manner,3 Fridericia ’s bioluminescent system was found to utilize ATP, Mg 2+ ions, atmospheric oxygen, and a specific (although still not fully characterized) luciferase 4.…”