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Abstract: We report isolation and structure elucidation of
AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs
from the bioluminescent earthworm Fridericia heliota. They
were found to be highly unusual modified peptides, com-
prising either of the two tyrosine-derived chromophores,
CompX or CompY and a set of amino acids, including
threonine, gamma-aminobutyric acid, homoarginine, and un-
symmetrical N,N-dimethylarginine. These natural compounds
represent a unique peptide chemistry found in terrestrial an-
imals and rise novel questions concerning their biosynthetic
origin.
Introduction
Results and Discussion
Bioluminescence—emission of “cold light” by a live organ-
ism—generally results from oxidation of a small organic mole-
cule called luciferin catalyzed by a specific enzyme luciferase.[1]
Until recently, structures of only seven natural luciferins were
known. 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 g-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, Mg2+ ions,
atmospheric oxygen, and a specific (although still not fully
characterized) luciferase.[4,5]
Specimens of F. heliota were individually hand-collected in
darkness from the forest soil near Krasnoyarsk (Russia) from
June to November 2012. Anion-exchange chromatography of
the extract prepared from 40 g of frozen homogenized worms
followed by two rounds of RP-HPLC allowed isolation of four
novel individual compounds, designated AsLn5, AsLn7, AsLn11,
and AsLn12 (ꢀ0.05, 0.04, 0.005 and 0.04 mg, respectively,
Figure S1 in the Supporting Information).
All four compounds showed UV/Vis absorption spectra simi-
lar to Fridericia luciferin. However, two distinct spectral types
were observed: AsLn7 had UV absorption identical to luciferin,
whereas AsLn5, AsLn11, and AsLn12 showed broader absorp-
tion peaks and the different line-shapes at short wavelengths,
indicating a different chromophore (Figure 2).
In the course of the isolation and purification of Fridericia
luciferin we encountered a number of highly unusual peptidic
components of the worm biomass that showed chromato-
graphic and UV spectral properties similar to luciferin. In our
previous publications we reported structures of two such com-
ponents, designated CompX[6,7] and AsLn2[8,9] (Figure 1). Here
we present structure elucidation of four novel luciferin
analogues from F. heliota: AsLn5, AsLn7, AsLn11, and AsLn12
which reveal an unprecedented peptide chemistry found in
terrestrial animals (Figure 1).
Structure elucidation of AsLn7
ESI-HRMS spectra of AsLn7 showed an [M+H]+ molecular ion
with m/z=324.1068, to which the closest molecular formula is
+
C15H18NO7 (calcd m/z=324.1078). (Figure S2 in the Support-
ing Information). Since the quantity of natural AsLn7 was low
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(ca. 40 mg), only H, DQF-COSY, and H,13C-HSQC NMR spectra
could be obtained for its solution in D2O using a 700 MHz in-
strument equipped with a cryogenically cooled probe. Analysis
of the NMR spectra (Figure 3, and Figure S3–S5 in the Support-
ing Information) revealed the presence of an aliphatic chain
1
(CH2-CH2-CH2) with H and 13C chemical shifts characteristic for
g-aminobutyric acid (GABA) and four protons with chemical
shifts and multiplicities very close to those observed for the
previously described CompX fragment.[2,7,8] This observation
allowed us to presume that AsLn7 is the peptide formed by
GABA’s amino group and one of the two carboxyls of CompX.
The exact position of the peptide bond was determined by an
NMR pH titration experiment, in which pH was gradually
[a] M. A. Dubinnyi, A. S. Tsarkova, Z. M. Kaskova, S. I. Kovalchuk,
R. H. Ziganshin, M. S. Baranov, K. S. Mineev, I. V. Yampolsky
Institute of Bioorganic Chemistry, Russian Academy of Sciences
Miklukho-Maklaya 16/10, Moscow 117997 (Russia)
1
decreased from 5.0 to 3.0 and changes in H chemical shifts
[b] A. S. Tsarkova, Z. M. Kaskova, M. S. Baranov, I. V. Yampolsky
Pirogov Russian National Research Medical University
Ostrovitianov 1, Moscow 117997 (Russia)
were monitored (Figure 3).
The titration experiment revealed that H-3 proton is much
more pH-sensitive than H-5 (Dd 0.31 vs. 0.06 ppm, respective-
ly), implying that the C-1 carboxylic group is free, while the C-
10 carboxyl is occupied by the GABA residue. In order to verify
the assumed structure of AsLn7 and to perform full characteri-
zation of this substance by NMR spectroscopy we performed
its synthesis (see Supporting Information). NMR investigation
of the synthetic sample (D2O, pH 5.0) demonstrated its exact
coincidence in all chemical shifts and multiplicities with the
[c] V. N. Petushkov, N. S. Rodionova
Laboratory of Photobiology, Institute of Biophysics
Siberian Branch of the Russian Academy of Sciences
Akademgorodok, Krasnoyarsk 660036 (Russia)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201406498. It contains NMR and HRMS
spectra and chromatographic data of natural and synthetic AsLn5, AsLn7,
AsLn11 and AsLn12, synthetic procedures for both double bond isomers of
CompY.
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Chem. Eur. J. 2015, 21, 1 – 7
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!