Stereoselective incorporation of isoleucine into cypridina luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

Article abstract of DOI:10.1271/bbb.60066

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four 2H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.

Full text of DOI:10.1271/bbb.60066

Biosci. Biotechnol. Biochem., 70 (6), 1528–1532, 2006
Communication
Stereoselective Incorporation of Isoleucine into Cypridina Luciferin
in Cypridina hilgendorfii (Vargula hilgendorfii)
y
2
Shin-ichi KATO,1 Yuichi OBA,1 Makoto OJIKA,1; and Satoshi INOUYE
1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2Yokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received February 6, 2006; Accepted March 27, 2006; Online Publication, June 23, 2006
[doi:10.1271/bbb.60066]
The emission of light in the marine ostracod Cypri-
dina hilgendorfii (presently Vargula hilgendorfii) is
produced by the Cypridina luciferin-luciferase reaction
in the presence of molecular oxygen. Cypridina luciferin
has an asymmetric carbon derived from isoleucine, and
the absolute configuration is identical to the C-3 position
in L-isoleucine or D-alloisoleucine. To determine the
stereoselective incorporation of the isoleucine isomers
(L-isoleucine, D-isoleucine, L-alloisoleucine, and D-allo-
isoleucine), we synthesized four 2H-labeled isoleucine
isomers and examined their incorporation into Cypri-
dina luciferin by feeding experiments. Judging by these
results, L-isoleucine is predominantly incorporated into
Cypridina luciferin. This suggests that the isoleucine
unit of Cypridina luciferin is derived from L-isoleucine,
but not from D-alloisoleucine.
Isoleucine unit
H
O
N
N
H
N
NH2
N
H
NH
Arginine unit
HN
Tryptophan unit
Cypridina luciferin (1)
Fig. 1. Chemical Structure of Cypridina Luciferin and Its Biosyn-
thetic Units.
Key words: bioluminescence; biosynthesis; luciferin;
isotopic label; imidazopyradinone
Me
C2H(5-n)2Hn
Me
C2H(5-n)2Hn
HOOC
HOOC
The luminous marine ostracod Cypridina hilgendorfii
(presently Vargula hilgendorfii) lives near the coast
around Japan. The luminescence system has been
investigated extensively, since Harvey reported the
luciferin-luciferase reaction in 1917.1) When the speci-
men is stimulated physically or electrically, it expels
Cypridina luciferin (1) and Cypridina luciferase into
the seawater to produce a brilliant bluish luminescence.
The chemical structure of Cypridina luciferin (1), which
possesses an imidazopyrazynone skeleton with side
residues of isoleucine, arginine, and tryptophan, was
determined by Kishi et al. (Fig. 1).2–4) An asymmetric
carbon of the isoleucine unit in 1 was determined to be
(S)-configuration by chemical and enzymatic proce-
dures.3) Recently, it has been demonstrated that 1 can be
biosynthesized from three L-amino acids (L-arginine,
L-isoleucine, and L-tryptophan), but not from tryptamine
or D-tryptophan.5–8) But it was not determined whether
the origin of the single asymmetric carbon in 1 was from
L-isoleucine or D-alloisoleucine. In this study, we
NH2
NH2
L-[4,5-2H]Isoleucine
D-[4,5-2H]Isoleucine
(2; 2S,3S)
(3; 2R,3R)
Me
Me
HOOC
HOOC
C2H(5-n)2Hn
C2H(5-n)2Hn
NH2
NH2
L-[4,5-2H]Alloisoleucine
D-[4,5-2H]Alloisoleucine
(4; 2S,3R)
(5; 2R,3S)
Fig. 2. [4,5-2H]-Labeled Isoleucine Isomers Used for the Incorpo-
ration Studies.
2
examined the stereoselective incorporation of four H-
labeled isoleucine isomers 2–5 (Fig. 2) into Cypridina
luciferin by feeding experiments using live animals.
2
Four stereoisomers of H-labeled isoleucine (Fig. 2)
were synthesized chemically and enzymatically from
y
To whom correspondence should be addressed. Tel/Fax: +81-52-789-4284; E-mail: ojika@agr.nagoya-u.ac.jp
Abbreviations: HPLC, high performance liquid chromatography; ESI, electrospray ionization; IT, ion trap; MS, mass spectrometry; U, units

Stereoselective Incorporation of Isoleucine into Luciferin
1529
A
Me
Me
a
HOOC
HOOC
C2H(5-n)2Hn
98%
NHAc
NHAc
(2S*,3S*)-7
(2S*,3S*)-6
Me
C2H(5-n)2Hn
Me
C2H(5-n)2Hn
b
HOOC
HOOC
+
NH2
NHR
L-[4,5-2H]Isoleucine (2)
(2R,3R)-7: R = Ac 37%
D-[4,5-2H]Isoleucine (3): R = H 91%
c
49%
B
Me
Me
a
HOOC
HOOC
C2H(5-n)2Hn
NHAc
100%
NHAc
(2S*,3R*)-6
(2S*,3R*)-7
Me
C2H(5-n)2Hn
Me
HOOC
b
HOOC
C2H(5-n)2Hn
+
NH2
NHR
L-[4,5-2H]Alloisoleucine (4)
(2R,3S)-7: R = Ac 43%
D-[4,5-2H]Alloisoleucine (5): R = H 73%
c
49%
Scheme 1. Synthesis of 2H-Labeled Isoleucines.
(A) 2H-labeled L- and D-isoleucines, (B) 2H-labeled L- and D-alloisoleucines. Reagents: (a) Platinum black, 2H2, r.t., 40 min, (b)
aminoacylase I, 0.1 M sodium phosphate buffer (pH 7.0), r.t., 24 h, (c) D-aminoacylase, 0.1 M sodium phosphate buffer (pH 7.5), 37 ꢂC, 12 h.
racemic mixtures of (2Sꢀ,3Sꢀ)-6 or (2Sꢀ,3Rꢀ)-6, which
were prepared according to the previous report9)
(Scheme 1). In brief, compound (2Sꢀ,3Sꢀ)-6 was labeled
using platinum black under a deuterium atmosphere to
give (2Sꢀ,3Sꢀ)-7. Enantioselective hydrolysis of (2Sꢀ,
3Sꢀ)-7 was accomplished using aminoacylase I to give
L-[4,5-2H]isoleucine (2) and (2R,3R)-7. D-[4,5-2H]Iso-
leucine (3) was given by hydrolysis of (2R,3R)-7 using
D-aminoacylase. L-[4,5-2H]Alloisoleucine (4) and D-
[4,5-2H]alloisoleucine (5) were prepared from (2Sꢀ,
3Rꢀ)-6 by the same procedures as above. The structures
and purity of the stereoisomers were determined by
NMR. The enantio purity of the stereoisomers was
analyzed by HPLC with a chiral column: Chiralpak
MA(+) (4:6 ꢁ 50 mm, Daicel Chemical Industries,
Osaka, Japan); mobile phase, 2 mM CuSO4 aqueous
solution; flow rate, 0.5 ml/min; UV detection, 254 nm.
Each product contained trace of the diasteromer (0.4%
in 2, 0.9% in 3, and 1.6% in 4 and 5), but not the
Billerica, MA) in the positive mode, and the mobile
phase was 50% methanol containing 0.1% formic acid at
a flow rate of 0.2 ml/min (Table 1).
For feeding experiments, the specimens were collect-
ed at Mukaishima, Hiroshima, Japan on 25 July 2005,
and were kept in an aquarium at 25 ꢂC. Each isomer of
2H-labeled isoleucine (25 mg) was dissolved in aqueous
extracts of porcine liver (1 ml) and gelled in 3% agarose
(Type VII, Sigma, St. Louis, MO). After feeding of a
piece of the gel for 5 d, three specimens were frozen in
liquid nitrogen and extracted in 3 times weight volume
of ethanol. A portion (1 ml) of the extract was analyzed
on LC/ESI-IT-MS (Table 2): Agilent 1100 HPLC
system with Esquire 3000; column, a Cadenza CD-
C18 (2:0 ꢁ 75 mm; Imtakt, Kyoto, Japan) with an
Unison US-C18 precolumn (2:0 ꢁ 5 mm, Imtakt); mo-
bile phase, methanol-water containing 0.1% formic acid,
linear gradient from 25% to 65% methanol for 20 min;
flow rate, 0.2 ml/min; UV detection, 280 nm; MS
analysis, ESI-IT-MS in the positive ion mode. The
results of the mass spectral analyses showed that the
incorporation efficiency of L-[4,5-2H]isoleucine (2), L-
[4,5-2H]alloisoleucine (4), and D-[4,5-2H]alloisoleucine
(5) into Cypridina luciferin were 20.1, 1.1, and 0.3%
2
enantiomer (Fig. 3). The efficiency of H-labeling was
determined by flow injection analysis on an ESI-ion
trap-mass spectroscopy (ESI-IT-MS): Agilent 1100
HPLC system (Agilent Technologies, Palo Alto, CA)
with an Esquire 3000 (ESI-IT-MS; Bruker Daltonics,

1530
S. KATO et al.
5
3
4
2
0
10
20
Time (min)
30
Fig. 3. HPLC Analysis of the Synthetic [4,5-2H]Isoleucines on the Chiral Column.
L-[4,5-2H]Isoleucine (2), D-[4,5-2H]isoleucine (3), L-[4,5-2H]alloisoleucine (4), and D-[4,5-2H]alloisoleucine (5).
Table 1. Relative Intensity of Mass Ion Peaks in [4,5-2H]-Labeled Isoleucines
Relative peak intensity (%)
No. of stable
isotope atom
Non-labeling
L-Ile
L-[4,5-2H]Ile
(2)
D-[4,5-2H]Ile
(3)
L-[4,5-2H]alloIle
(4)
D-[4,5-2H]alloIle
(5)
+0
+1
+2
+3
+4
+5
100:00 ꢃ 0:38
16:13 ꢃ 0:33
56:33 ꢃ 0:61
95:75 ꢃ 0:78
100:00 ꢃ 0:43
58:46 ꢃ 0:82
15:56 ꢃ 0:32
16:22 ꢃ 0:56
56:28 ꢃ 0:92
98:21 ꢃ 0:82
100:00 ꢃ 0:25
57:07 ꢃ 0:96
15:49 ꢃ 0:36
8:69 ꢃ 0:66
40:53 ꢃ 0:92
89:55 ꢃ 1:00
100:00 ꢃ 0:69
60:36 ꢃ 0:87
14:40 ꢃ 0:17
8:19 ꢃ 0:55
38:14 ꢃ 1:77
83:80 ꢃ 0:86
100:00 ꢃ 2:20
56:71 ꢃ 0:73
14:33 ꢃ 0:39
7:17 ꢃ 0:35
0:90 ꢃ 0:06
—
—
—
Data are expressed as the mean ꢃ standard error of three independent measurements. Ile, isoleucine; alloIle, alloisoleucine.
Table 2. Incorporation of [4,5-2H]-Labeled Isoleucines into Cypridina Luciferin (Divalent ion)
Relative peak intensity (%)
No. of stable
isotope atom
L-[4,5-2H]Ile
(2)
D-[4,5-2H]Ile
(3)
L-[4,5-2H]alloIle
(4)
D-[4,5-2H]alloIle
(5)
Non-feeding
+0
+1
+2
+3
+4
+5
100:00 ꢃ 0:46
24:82 ꢃ 0:35
3:29 ꢃ 0:29
0:37 ꢃ 0:09
—
100:00 ꢃ 0:93
30:76 ꢃ 0:70ꢀꢀ
12:43 ꢃ 0:54ꢀꢀ
9:99 ꢃ 0:68ꢀꢀ
5:75 ꢃ 0:45
100:00 ꢃ 0:74
22:99 ꢃ 0:68
3:38 ꢃ 0:18
0:32 ꢃ 0:06
—
100:00 ꢃ 0:54
24:13 ꢃ 0:40
3:99 ꢃ 0:18ꢀ
1:11 ꢃ 0:04ꢀ
0:48 ꢃ 0:12
0:24 ꢃ 0:02
100:00 ꢃ 0:88
24:43 ꢃ 0:61
3:45 ꢃ 0:24ꢀ
0:62 ꢃ 0:06
0:23 ꢃ 0:06
0:09 ꢃ 0:04
—
1:86 ꢃ 0:12
—
Total
128.48
160.79
126.69
—
129.95
128.82
Incorporationa
20.1%
1.1%
0.3%
Data are expressed as the mean ꢃ standard error of three independent measurements. Asterisks indicate statistical significance compared to the non-feeding
specimens group at: ꢀP < 0:05, ꢀꢀP < 0:01. Ile, isoleucine; alloIle, alloisoleucine. aIncorporation efficiency (%) = (Totalfeeding ꢄ Totalnon-feeding)/Totalfeeding ꢁ 100
respectively (Table 2), whereas no detectable incorpo-
ration of D-[4,5-2H]isoleucine (3) was observed. The
small incorporation of 4 might be attributed to L-[4,5-
2H]isoleucine (2) in synthesized 4 (Fig. 3). In conclu-
sion, L-isoleucine is stereoselectively incorporated into
Cypridina luciferin (1). This suggests that the isoleucine
unit of Cypridina luciferin (1) is derived from L-
isoleucine, but not from D-alloisoleucine.
(from Escherichia coli, EC 3.5.1.81, 10.1 MU/g) were
2
purchased from Wako Pure Chemical (Tokyo), and H2
gas was purchased from Shoko (Tokyo). 1H-NMR
(600 MHz) and 13C-NMR (150 MHz) spectra were
recorded on a Bruker AMX2-600 spectrometer. Chemi-
1
cal shifts of H-NMR were given in ppm relative to the
solvent peak of CD2HOD (ꢀ 3.5) or the internal standard
of 1,4-dioxane (ꢀ 3.75). Chemical shifts of 13C-NMR
were given in ppm relative to the solvent peak of
CD3OD (ꢀ 49.0) or the internal standard of 1,4-dioxane
(ꢀ 69.3). Elementary analyses were performed on a
Experimental
Platinum black, aminoacylase I (from porcine kidney,
EC 3.5.1.14, 4300 U/mg) and D-aminoacylase amano

Stereoselective Incorporation of Isoleucine into Luciferin
1531
Series II CHNS/O Analyzer 2400 (Perkin Elmer,
Wellesley, MA). Optical rotations [ꢁ]D were determined
on a DIP-370 polarimeter (Jasco, Tokyo).
MHz, D2O) ꢀ: 13.6 (m), 17.4, 27.1 (m), 38.6, 62.4,
177.4. Found: C, 54.93, H, 9.98, N, 10.65. Calcd. for
C6H13NO2: C, 54.94; H, 9.99; N, 10.68.
[4,5-2H]-(2Sꢀ,3Sꢀ)-2-Acetamido-3-methylpentanoic
acid ((2Sꢀ,3Sꢀ)-7): Procedure I. A mixture of (2Sꢀ,3Sꢀ)-
2-acetamido-3-methylpent-4-ynic acid ((2Sꢀ,3Sꢀ)-6; 365
mg, 2.16 mmol) and platinum black (19.1 mg) in ethyl
[4,5-2H]-(2Sꢀ,3Rꢀ)-2-Acetamido-3-methylpentanoic
acid ((2Sꢀ,3Rꢀ)-7). Compound (2Sꢀ,3Rꢀ)-7 was synthe-
sized from (2Sꢀ,3Rꢀ)-6 (211 mg, 1.25 mmol) according
to Procedure I and was obtained as a white solid in
2
1
acetate (11 ml) was stirred under H2 at room temper-
quantitative yield (222 mg, 1.25 mmol). H-NMR (600
ature for 40 min. The mixture was filtered, and the
filtrate was evaporated under reduced pressure. The
residue was dissolved in MeOH and evaporated again to
give 374 mg (98% yield) of [4,5-2H]-(2Sꢀ,3Sꢀ)-2-acet-
amido-3-methylpentanoic acid ((2Sꢀ,3Sꢀ)-7). 1H-NMR
(600 MHz, CD3OD) ꢀ: 1.11 (1.7H, m), 1.14 (3H, d, J ¼
6:6 Hz), 1.43 (0.5H, m), 1.70 (0.5H, m), 2.06 (1H, m),
2.19 (3H, s, CH3CO), 4.55 (1H, d, J ¼ 6:0 Hz). 13C-
NMR (150 MHz, CD3OD) ꢀ: 11.4 (m), 16.0, 22.3 (m),
38.2, 58.3, 173.4, 174.9.
MHz, CD3OD) ꢀ: 1.10 (1.6H, m), 1.13 (3H, d, J ¼
6:6 Hz), 1.42 (0.4H, m), 1.59 (0.4H, m), 2.15 (1H, m),
2.20 (3H, s), 4.71 (1H, s). 13C-NMR (150 MHz,
CD3OD) ꢀ: 11.6 (m), 15.1, 22.3, 27.0 (m), 38.1, 56.9,
173.6, 175.3.
L-[4,5-2H]Alloisoleucine (4) and D-[4,5-2H]alloiso-
leucine (5). Compound 4 was synthesized from
(2Sꢀ,3Rꢀ)-7 according to Procedure II and was obtained
as a white solid in 49% yield (75 mg). [ꢁ]D +37.0ꢂ (c
27
0.1, 6 M HCl). 1H-NMR (600 MHz, D2O) ꢀ: 0.92 (3H, d,
J ¼ 7:2 Hz), 0.93 (1.8H, m), 1.30 (0.4H, m), 1.40 (0.4H,
m), 2.05 (1H, m), 3.71 (1H, d, J ¼ 3:6 Hz). 13C-NMR
(150 MHz, D2O) ꢀ: 13.4 (m), 16.0, 28.1 (m), 38.2, 61.3,
177.5. Found: C, 54.95; H, 9.95; N, 10.56. Calcd. for
C6H13NO2: C, 54.94; H, 9.99; N, 10.68. D-[4,5-
2H]Alloisoleucine (5) was obtained as white solid in
L-[4,5-2H]Isoleucine (2) and D-[4,5-2H]isoleucine
(3): Procedure II. Compound (2Sꢀ,3Sꢀ)-7 (282 mg,
1.59 mmol) was dissolved in 0.1 M sodium phosphate
buffer (16 ml, pH 7.0). Aminoacylase I (3 mg, 12,900 U)
was added, and the mixture was incubated at room
temperature for 24 h. After incubation, the solution was
adjusted to pH 5 with 1 M HCl, and the enzyme was
denatured at 60 ꢂC for 10 min and then filtered. The
filtrate was acidified to pH 1 with 1 M HCl and washed
with ethyl acetate. The aqueous layer was applied to a
cation exchange column (20 ꢁ 20 mm, Biorad AG 50W-
X8 resin, Hþ-form), which was rinsed with water to
make it neutral, and then eluted with 1 M NH4OH. The
eluate was evaporated under reduced pressure to give
L-[4,5-2H]isoleucine (2; 105 mg, 49%) as a white solid.
31% yield. (2 steps, 48 mg) [ꢁ]D ꢄ32:0ꢂ (c 0.1, 6 M
27
HCl). 1H-NMR (600 MHz, D2O) ꢀ: 0.92 (3H, d, J ¼
7:2 Hz), 0.93 (2.2H, m), 1.29 (0.4H, m), 1.39 (0.4H, m),
2.00 (1H, m), 3.64 (1H, d, J ¼ 4:2 Hz). 13C-NMR (150
MHz, D2O) ꢀ: 13.5 (m), 16.0, 28.2 (m), 38.6, 61.4,
178.7. Found: C, 54.92; H, 9.85; N, 10.59. Calcd. for
C6H13NO2: C, 54.94; H, 9.99; N, 10.68.
Acknowledgments
[ꢁ]D +38.7ꢂ (c 0.1, 6 M HCl). 1H-NMR (600 MHz,
27
D2O) ꢀ: 0.91 (1.8H, m), 1.00 (3H, d, J ¼ 6:6 Hz), 1.23
(0.4H, m), 1.44 (0.5H, m), 1.97 (1H, m), 3.65 (1H, d,
J ¼ 3:6 Hz). 13C-NMR (150 MHz, D2O) ꢀ: 13.4 (m),
17.4, 27.1 (m), 38.5, 62.3, 176.9. Found: C, 54.96; H,
9.78; N, 10.52. Calcd. for C6H13NO2: C, 54.94; H, 9.99;
N, 10.68. The ethyl acetate layer was washed with 1 M
HCl and dried over Na2SO4. The solvent was evaporated
under reduce pressure to give [4,5-2H]-(2R,3R)-2-acet-
amido-3-methylpentanoic acid ((2R,3R)-7; 103 mg,
37%) as a white solid. Compound (2R,3R)-7 (103 mg,
0.58 mmol) was dissolved in 0.1 M phosphate buffer
(pH 8.0). D-Aminoacylase amano (1 mg, 10.1 kU) was
added, and the mixture was incubated at 37 ꢂC for 12 h.
After incubation, the solution was adjusted to pH 5 with
1 M HCl, and the enzyme was denatured at 60 ꢂC for
10 min and then filtered. The filtrate was acidified to
pH 1 with 1 M HCl and extracted with ethyl acetate. The
aqueous layer was applied to the cation exchange
column as described above. The eluate was evaporated
under reduced pressure to give D-[4,5-2H]isoleucine
The authors thank Dr. K. Yasui and Dr. N.
Yamaguchi of Mukaishima Marine Biological Labora-
tory of Hiroshima University in Japan for assistance in
the collection of animals, and Mr. S. Kitamura of the
analytical laboratory of Nagoya University for elemen-
tary analyses. This work was supported in part by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan, and by a JSPS fellowship (to S.K.).
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