Biosynthesis of luciferin in the sea firefly, Cypridina hilgendorfii: L-tryptophan is a component in Cypridina luciferin

Article abstract of DOI:10.1016/S0040-4039(02)00257-5

Feeding experiment to the marine ostracod crustacean, Cypridina (Vargula) hilgendorfii, using L-tryptophan labeled with deuterium at indole ring revealed that the labeled L-tryptophan was incorporated into Cypridina luciferin as a component and the animals were able to synthesize the luciferin in a de novo synthetic pathway.

Full text of DOI:10.1016/S0040-4039(02)00257-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2389–2392
Biosynthesis of luciferin in the sea firefly, Cypridina hilgendorfii:
L
-tryptophan is a component in Cypridina luciferin†
Yuichi Oba,a,* Shin-ichi Kato,a Makoto Ojikaa and Satoshi Inouyeb
aGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
bYokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received 7 January 2002; accepted 1 February 2002
Abstract—Feeding experiment to the marine ostracod crustacean, Cypridina (Vargula) hilgendorfii, using
L-tryptophan labeled
with deuterium at indole ring revealed that the labeled -tryptophan was incorporated into Cypridina luciferin as a component
L
and the animals were able to synthesize the luciferin in a de novo synthetic pathway. © 2002 Elsevier Science Ltd. All rights
reserved.
The bioluminescence system in the marine ostracod
crustacean, Cypridina hilgendorfii (presently Vargula
hilgendorfii) living along Japanese coast, has been well
investigated since Harvey reported the luciferin–luci-
ferase reaction with the extracts of the animals in 1917.1
The luciferin from Cypridina hilgendorfii (1: hereafter
called Cypridina luciferin) was isolated2 and the chemi-
cal structure was determined by studying the degrada-
tion products3 and total synthesis4 in 1966. On the
other hand, Cypridina luciferase has been purified and
its cDNA has been cloned5 and used as a reporter
protein in living cells.6 On the basis of the chemical
structure of Cypridina luciferin 1,3,4 the biosynthetic
pathway of Cypridina luciferin has been proposed to be
synthesized from three amino acids or their equivalents:
arginine, isoleucine and tryptophan (or tryptamine)
(Scheme 1).3,4,7 However, no clear experimental evi-
dence has been exhibited in living animals.
Here we reported the feeding study in vivo using the
deuterium-labeled
ponent of Cypridina luciferin and identified the incor-
poration of the deuterium atoms in -tryptophan into
L
-tryptophan 2 as an expected com-
L
Cypridina luciferin by LC/ESI-TOF MS analyses.
The synthesis of 2 was accomplished by the deuterium-
exchange method with D2O and DCl (Scheme 2).
Briefly,
L-tryptophan (300 mg, Wako Pure Chemical
Industries, Ltd.) was dissolved in 2.0 ml of D2O (99.9
atom% D, Cambridge Isotope Laboratories, Inc.) con-
taining 0.4 ml of DCl (20 wt% solution, 99.5 atom% D,
Aldrich) and 0.1 ml of mercaptoacetic acid (Aldrich).
Isoleucine
H
O
Isoleucine
N
N
-CO2, -3H2O, -6H+
Tryptophan
H
N
Arginine
NH2
NH
N
H
Arginine
Tryptophan
N
H
1: Cypridina luciferin
Scheme 1. Structure of Cypridina luciferin 1 and its predicted biosynthesis. The closed circle represents the a-carbon of arginine,
isoleucine and tryptophan.
Keywords: bioluminescence; biosynthesis; imidazopyrazinone; isotopic labeling; ostracod.
* Corresponding author. Tel./fax: +81-52-789-4284; e-mail: oba@agr.nagoya-u.ac.jp
† This paper is dedicated to the late Professor Hideshi Nakamura (deceased 9th November 2000).
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00257-5

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Y. Oba et al. / Tetrahedron Letters 43 (2002) 2389–2392
HOOC
NH2
HOOC
NH2
X
DCl, HSCH2COOH in D2O
110 °C, 3.5-4 h, >99 %
X
X
N
H
X
N
H
X
2: X=H or D
Deuterium-labeled L-tryptophan
(ee = 95 %)
L-Tryptophan
(ee = 98 %)
Scheme 2. Preparation of deuterium-labeled
L
-tryptophan 2.
The mixture was degassed three times in a methanol-
dry ice bath, and was heated in an oil bath at 110°C
for 3.5–4 h. After removing mercaptoacetic acid by
the extraction with ethyl acetate, the reaction mixture
was evaporated and this labeling procedure was
repeated. The resultant solution was dried up by
evaporation, resolved in 2.0 ml of H2O and dried up
iments, the deuterium-labeled 2 (50 mg) dissolved in a
water-extract from the porcine liver was gelled in 3%
agarose (Type VII: Sigma) as the food and was fed
to the animals in a small partition of the aquarium at
22°C. After 6 days feeding, the animals were quickly
frozen by liquid nitrogen. For extraction of Cypridina
luciferin, six frozen animals (wet weight; 41.6 mg)
again. The white powder of
2
(360.1 mg) was
were homogenized in 0.12 ml of ethanol by a
obtained as a hydrochloride form and its yield was
>99%, which was determined by using the data of the
deuterium content of 2 from ESI-TOF-MS analysis as
described below. The purity of 2 was determined by
reversed phase-HPLC conducting on a Jasco 1500
system equipped with a Develosil ODS-SR-5 column
(4.6×250 mm; Nomura Chemicals) and a multiwave-
length detector (195–650 nm; MD-2010 plus, Jasco).
Any products except 2 were not detected in this analy-
sis (data not shown). The absolute configuration of
tryptophan was determined by the method of Mar-
micropestle on an ice bath and centrifuged at 12,000×
g for 10 min. The resultant supernatant was filtrated
using an Ultrafree-MC 0.45 mm filter (Millipore), and
then was analyzed by LC/ESI-TOF-MS with an
Agilent 1100 HPLC system (Hewlett–Packard) con-
nected to a Mariner Biospectrometry Workstation.10
The retention time of the extracted Cypridina luci-
ferin on HPLC (asterisk in Fig. 2) was identical with
that of the chemically synthesized dl-Cypridina luci-
ferin as a standard.10,11 Mass spectrum of the sepa-
rated Cypridina luciferin showed the significant
[D5]-luciferin peak (m/z 410 (M+), Fig. 3), compared
to the control luciferin that was extracted from the
animals without feeding. The peak ratio of [D4]- and
[D5]-luciferin was 40:100 which was identical to that
fey.8,9 The optical purity of
labeling was 98% ee and the deuterium labeled 2 was
95% ee with the -configuration. Further, to confirm
L-tryptophan used for the
L
the number of deuterium atoms in 2, ESI-TOF-MS
analysis was carried out with a Mariner Biospec-
trometry Workstation (Applied Biosystems). The
of the labeled [D4]- and [D5]-L-tryptophan as a feed-
ing source. The labeling efficiency of 2 was 9.2% of
the total luciferin in vivo. This evidence strongly indi-
results exhibited that [D4]- and [D5]-
L
-tryptophan (m/z
209 and 210 (M+H+), respectively) were most abun-
cated that
L-tryptophan was one of the components
dant (Fig. 1) and the peak ratio of [D4]- and [D5]-
L
-
in the Cypridina luciferin biosynthesis (Scheme 3). C.
hilgendorfii is the first example that the animals can
produce the imidazopyrazinone-type luciferin from
free amino acid by a de novo biosynthetic pathway in
marine organisms including jellyfishes12, shrimps13 and
fishes.14
tryptophan was 40:100.
Specimens of C. hilgendorfii were collected at
Mukaishima, Hiroshima in Japan on 27 Sept. 2001,
and were kept in an aquarium. For the feeding exper-
Figure 1. Mass spectrum of deuterium labeled L-tryptophan 2.

Y. Oba et al. / Tetrahedron Letters 43 (2002) 2389–2392
2391
Figure 2. HPLC analysis of an ethanol-extracted fraction from C. hilgendorfii fed with 2. The asterisk and the closed circle
represent Cypridina luciferin and -tryptophan, respectively.
L
Figure 3. Mass spectrum of Cypridina luciferin isolated from C. hilgendorfii fed with 2. Inset: magnification of the isotope peaks
due to incorporation.
H
O
N
N
D
D
H
N
D
D
NH2
NH
C. hilgendorfii
N
H
2
de novo synthesis
N
H
D
[D5]-Cypridina luciferin
m/z 410.2586 (calcd)
Scheme 3. [D5]-Cypridina luciferin synthesis from 2.
Acknowledgements
2. Shimomura, O.; Goto, T.; Hirata, Y. Bull. Chem. Soc.
Jpn. 1957, 30, 929–933.
We thank Dr. T. Uyama, Mukaishima Marine Biologi-
cal Laboratory, Hiroshima University, Japan for col-
lecting the animals. This work was supported in part by
a Grant-in-Aid for Scientific Research on Priority
Areas (A) from the Ministry of Education, Culture,
Sports, Science and Technology.
3. Kishi, Y.; Goto, T.; Hirata, Y.; Shimomura, O.; Johnson,
F. H. Tetrahedron Lett. 1966, 7, 3427–3436.
4. Kishi, Y.; Goto, T.; Inoue, S.; Sugiura, S.; Kishimoto, H.
Tetrahedron Lett. 1966, 7, 3445–3450.
5. Thompson, E. M.; Nagata, S.; Tsuji, F. I. Proc. Natl.
Acad. Sci. USA 1989, 86, 6567–6571.
6. Inouye, S.; Ohmiya, Y.; Toya, Y.; Tsuji, F. I. Proc. Natl.
Acad. Sci. USA 1992, 89, 9584–9587.
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