Syntheses and evaluation of the bioluminescent activity of (S)-Cypridina luciferin and its analogs

Article abstract of DOI:10.1016/j.tetlet.2005.11.119

Cypridina luciferin is the substrate in the bioluminescence of a luminous ostracod Cypridina (Vargula) hilgendorfii. Cypridina luciferin contains a chiral center in the sec-butyl moiety. Here, we report a convenient method for the preparation of (S)-Cypridina luciferin by the condensation of (S)-1,1-diethoxy-3-methylpentan-2-one with ethioluciferin. The light yield of the synthesized (S)-luciferin in the presence of Cypridina luciferase was about 1.7 times as active as that of racemic form. Furthermore, several luciferin analogs prepared by the same condensation with different α-ketoacetal derivatives showed moderate light yield with Cypridina luciferase. These readily available Cypridina luciferin and analogs are applicable to the bioluminescent detection of Cypridina luciferase.

Full text of DOI:10.1016/j.tetlet.2005.11.119

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 753–756
Syntheses and evaluation of the bioluminescent activity of
(
a
S)-Cypridina luciferin and its analogs
b
b
a,
*
Chun Wu, Kosei Kawasaki, Satoru Ohgiya and Yoshihiro Ohmiya
a
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan
b
Research Institute for Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology,
Sapporo 062-8517, Japan
Received 24 October 2005; revised 9 November 2005; accepted 18 November 2005
Abstract—Cypridina luciferin is the substrate in the bioluminescence of a luminous ostracod Cypridina (Vargula) hilgendorfii. Cyp-
ridina luciferin contains a chiral center in the sec-butyl moiety. Here, we report a convenient method for the preparation of (S)-Cyp-
ridina luciferin by the condensation of (S)-1,1-diethoxy-3-methylpentan-2-one with ethioluciferin. The light yield of the synthesized
(
S)-luciferin in the presence of Cypridina luciferase was about 1.7 times as active as that of racemic form. Furthermore, several luci-
ferin analogs prepared by the same condensation with different a-ketoacetal derivatives showed moderate light yield with Cypridina
luciferase. These readily available Cypridina luciferin and analogs are applicable to the bioluminescent detection of Cypridina
luciferase.
ꢀ
2005 Elsevier Ltd. All rights reserved.
Cypridina luciferin, which was first isolated from a lumi-
nous ostracod Cypridina hilgendorfii (presently Vargula
hilgendorfii), is a substrate in an enzymatic oxidation
that generates visible light. Cypridina luciferin (1) was
shown to be an imidazopyrazinone compound having
racemic keto acetal 5 provided the racemic form 1 in
24% yield and the synthesized 1 showed a light yield
of about 60% of that of (S)-1 in the bioluminescence
of Cypridina luciferase. A practical synthetic method
for (S)-1 or optical resolution of the racemic form 1
4
three functional groups at the C2, C6, and C8 positions
has not to our knowledge been reported yet.
1
(
Fig. 1). It is obvious that luciferin 1 derives biogeneti-
cally from three amino acids or its equivalent: L-argi-
nine, L-isoleucine, and L-tryptophan (or tryptamine).
Recently, a feeding experiment has indicated that Cypri-
dina luciferin may be biosynthesized from these three
The secreted Cypridina luciferase has a big advantage as
a non-destructive bioluminescent reporter. However,
there are only a few examples in the literature of this
application because of the problem of the supply of
2
5
amino acids, but the enzymes involved in the biosynthe-
the luciferin. Recently, Cypridina luciferase from Cyp-
6
a
sis of 1 are still unknown. Cypridina luciferin (1) con-
tains a chiral center in the sec-butyl moiety at the C2
ridina noctiluca has been cloned and functionally
6
a
6b
expressed in mammalian cells and yeast. This live
yeast cell-based bioluminescent assay system is very use-
ful for high-throughput screening for drugs or hormone-
disrupting chemicals. Therefore, we are interested in
developing an efficient synthetic method for (S)-1 for
the Cypridina luciferase bioluminescent assay. Here,
we reported our recent results in developing a simple
method of preparing (S)-1 and syntheses of several Cyp-
ridina luciferin analogs having different alkyl groups at
the C2-position. Bioluminescent activities of the synthe-
sized (S)-1 and its analog were evaluated with recombi-
1
position. The natural luciferin is the (S)-configuration.
The first total synthesis of (S)-Cypridina luciferin (1)
was achieved by the condensation of reduced ethioluci-
1
ferin (2) with (+)-a-keto acid (3) in three steps, but the
yield (ꢀ1%) is too low to use this route to prepare (S)-
1
in a large quantity (Fig. 1). A new route using the con-
densation of 2 with racemic ketoaldehyde (4) in place of
3
1
in the presence of acidic catalyst gave the racemic form
in a moderate yield (70%). Another route by using a
3
6
nant Cypridina luciferase.
Keywords: Cypridina luciferin; Luciferase; Bioluminescence;
Imidazopyrazinone.
The approach taken for the preparation of (S)-1 luciferin
is shown in Figure 1. We chose the chiral (S)-ketoacetal
*
Corresponding author. E-mail: y-ohmiya@aist.go.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.11.119

754
C. Wu et al. / Tetrahedron Letters 47 (2006) 753–756
O
H
HO
O
2
H
O
NH2
N
N
N
N
H
3
N
NH2
NH
H
N
NH2
NH
N
6
8
O
H
N
H
OHC
N
H
2
4
1
OEt
H
H
EtO
1
3
O
5
OEt
A
H
OEt
OEt
H
+
Grignard reagents
H
H
EtO
EtO 1
3
O
B
6
O
S-5
HO
O
7
Figure 1. Three routes for the synthesis of Cypridina luciferin (1).
1
2
. SOCl2 r.t.
.TMSCHN2, r.t.
OEt
H
H
H
HO
EtO
3
. t-BuOCl, EtOH, r.t.
O
3
steps 39%
O
7
5
H
O
N
NH2
N
N
5
, H+
NH2 reflux
H
N
N
H
N
NH2
NH
NH
50%
N
H
N
H
N
H
2
1
OEt
H
R
O
N
R
NH2
EtO
N
N
H
N
O
N
NH2
NH
9
a-f
H
N
+
NH2
NH
H , reflux
N
H
N
H
N
H
8
a-f
yield
R
%
a Methyl
b ethyl
3
4
4
6
3
7
8
2
7
2
2
4
c iso-propy
d iso-butyl
e tert-butyl
f heptafluoropropyl
Scheme 1. Synthesis of 1 and its analogs.

C. Wu et al. / Tetrahedron Letters 47 (2006) 753–756
755
compound 5 because the acetal group was expected to
The bioluminescent activities of 1 and its analogs were
measured in the presence of recombinant Cypridina
luciferase (Table 1). The light yield of (S)-1 was 1.7 times
greater than that of (dl)-1. The light yield generated
from the analog having an isopropyl group was a little
higher than that of the racemic 1. Analogs with ethyl
and isobutyl groups showed about 10% of the light yield
of (S)-1. Analogs with methyl, butyl, and heptafluoro-
propyl group showed 1–3% of the light yield of (S)-1.
The wavelength of the light emission maxima of 1 and
its analogs were similar to each other. In previous stud-
ies, the change of indolyl group and the chain length of
alkylguaydinyl group of 1 caused a dramatic decrease in
avoid the racemization due to the difference of pK val-
a
ues of protons between the C1 and C3 positions. The
general approach for the preparation of a-ketoacetal
derivatives is via a Grignard reaction from esters 6 or
7
its amides (route A). However, this route is not suitable
for the preparation of (S)-5, which was prepared from
the commercially available reagent 7 (route B).8
Reaction of the starting material 7 with thionyl chloride
followed by treatment of the resulting acid chloride with
TMSCHN provided a homologation product (Scheme
2
1
). Treatment of the homologation product with tert-
4
,12
butylhyperchlorite in the presence of ethanol gave com-
the light yield.
Our results indicated that the light
pound 5. The specific optical rotations of (S)-5 was +22
yield also depends on the chirality and the size of the
substituent at the C2 position. This high substrate speci-
ficity should contribute to the highly efficient biolumi-
nescence of Cypridina luciferase, which is about 0.28
absolute light yield.
(
in dichloromethane), which was almost the same as that
of the starting material +19 (in dichloromethane). Com-
pound 2 was prepared according to the method
4
described by Nakamura et al. Reaction of 2 with 5
proceeded smoothly in the presence of acidic catalyst
9
affording (S)-1 in moderate yield (50%). Luciferin 1
was purified on a reversed-phase HPLC column
The commercially available firefly luciferase and Renilla
luciferase from the sea pansy are widely used to monitor
gene expression in eukaryotic cells. The secreted Cypri-
dina luciferase has a potentially large advantage over
these luciferases as a non-destructive genetic reporter.
Our method of preparing (S)-Cypridina luciferin is sim-
ple and efficient. These readily available Cypridina luci-
ferin and analogs are applicable to the bioluminescent
detection of Cypridina luciferase.
(
(
Fig. 2). The specific optical rotation of the synthetic
S)-1 was +22, which was almost the same as that of
1
0
the natural luciferin 1+19. Therefore, the optical pur-
ity of the synthetic 1 could be 100%. This is the first re-
port on the optical rotation of Cypridina luciferin. The
(
dl)-Cypridina luciferin was synthesized by using a race-
4
mic ketoacetal 5 under the same conditions. Condensa-
tion of 2 with ketoacetal derivatives 9a–f that were
prepared from commercially available acids by using
the same route afforded these desired Cypridina luciferin
Acknowledgments
1
1
analogs 8a–f in 32–74% yield (Scheme 1).
We acknowledge the financial support from the Ministry
of Economy, Trade, and Industry, Japan.
References and notes
1
. (a) Shimomura, O.; Goto, T.; Hirata, Y. Bull. Chem. Soc.
Jpn. 1957, 30, 929–933; (b) Kishi, Y.; Goto, T.; Hirata, Y.;
Shimomura, O.; Johnson, F. H. Tetrahedron Lett. 1966, 7,
3
427–3436; (c) Kishi, Y.; Goto, T.; Inoue, S.; Sugiura, S.;
Kishimoto, H. Tetrahedron Lett. 1966, 7, 3445–3450.
2
3
4
5
. Kato, S.; Oba, Y.; Ojika, M.; Inouye, S. Tetrahedron 2004,
6
0, 11427–11434.
. Inoue, S.; Sugiura, S.; Kakoi, H.; Goto, T. Tetrahedron
Lett. 1969, 20, 1609–1610.
. Nakamura, H.; Aizawa, M.; Takeuchi, D.; Murai, A.;
Shimomura, O. Tetrahedron Lett. 2000, 41, 2185–2188.
. (a) Thompson, E. M.; Nagata, S.; Tsuji, F. I. Gene 1990,
2
.00
4.00
6.00
Time (min)
8.00
10.00
Figure 2. A HPLC profile of the synthetic 1. (Cosmosil C18-column
.6 · 250 mm, 69:30:1 water/acetonitrile/10% TFA, 1.0 mL/min,
detected at 280 nm).
4
9
6, 257–262; (b) Inouye, S.; Ohmiya, Y.; Toya, Y.; Tsuji,
F. I. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9584–9587; (c)
Thompson, E. M.; Adenot, P.; Tsuji, F. I.; Renard, J. P.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1317–1321; (d)
Misenbock, G.; Rothman, J. E. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 3402–3407.
Table 1. Luminescent properties of Cypridina luciferin (1) and its
analogs (8a–f)
6
7
. (a) Nakajima, Y.; Kobayashi, K.; Yamagishi, K.; Eno-
moto, T.; Ohmiya, Y. Biosci. Biotechnol. Biochem. 2004,
(
S)-1 (dl)-1 8a
8b
12
8c
67
8d
10
8e
8f
Relative
light yield
100
58
3
2
2
6
8, 565–570; (b) To express Cypridina luciferase in yeast
will be reported elsewhere.
. Adamczyk, M.; Johnson, D.; Mattingly, P. G.; Pan, Y.;
Reddy, R. E. Synth. Commum. 2002, 32, 3199–3205.
kmax/nm
460
460
460 460 460 460 N.D. N.D.
ꢁ7
Bioluminescence was performed at 1 · 10 M of luciferins in 50 mM
Tris–HCl pH 8 with recombinant Cypridina luciferase (10 ng/mL).
N.D. not determined.
8. Kuse, M.; Doi, I.; Kondo, N.; Kageyama, Y.; Isobe, M.
Tetrahedron 2005, 61, 5754–5762.

756
C. Wu et al. / Tetrahedron Letters 47 (2006) 753–756
ꢁ
1
9
. A mixture of ethioluciferin 2 (0.03 mmol) and (S)-5
0.02 mL) in 1:50:49 47% aq HBr/ethanol/water (0.2 mL)
FABMS m/z 376 (M ); H NMR (500 MHz, CD OD) d
3
(
1.41 (3H, t, J = 8 Hz), 2.31 (2H, quintet, J = 8 Hz), 2.95
(2H, q, J = 8 Hz), 3.35 (2H, t, J = 8 Hz), 3.43 (2H, t,
J = 8 Hz), 7.16–7.22 (2H, m), 7.47 (1H, d, J = 8 Hz), 7.99
(1H, s), 8.02 (1H, d, J = 8 Hz), 8.35 (1H, s); Compound
8c: LR-FABMS m/z 390 (M ); H NMR (500 MHz,
CD OD) d 1.50 (6H, t, J = 8 Hz), 2.29 (2H, quintet,
J = 8 Hz), 3.32 (1H, sextet, J = 8 Hz), 3.37 (2H, q,
J = 8 Hz), 3.43 (2H, q, J = 8 Hz), 7.11–7.14 (2H, m),
7.40 (1H, d, J = 8 Hz), 7.96 (1H, s), 7.94 (1H, d, J = 8 Hz),
8.22 (1H, s); Compound 8d: LR-FABMS m/z 404 (M );
H NMR (500 MHz, CD OD) d 1.03 (6H, t, J = 8 Hz),
3
were heated to reflux for 1 h. The mixture was cooled to
room temperature and was purified by RP-HPLC. (Cos-
mosil C18-column 4.6 · 250 mm, 69:30:1 water/aceto-
2
D
4
ꢁ
1
nitrile/10% TFA, 1.0 mL/min.) The synthetic (S)-1: ½aꢂ
+
22 (0.046, acetonitril/water/10% TFA 39:60:1); The
3
1
0
24
natural Cypridina luciferin 1 ½aꢂ +19 (0.026, acetoni-
D
ꢁ
tril/water/10% TFA 39:60:1); LR-FABMS m/z 404 (M );
1
H NMR (500 MHz, CD OD) d 0.95 (3H, t, J = 8 Hz),
3
ꢁ
1.45 (3H, d, J = 8 Hz), 1.80–1.99 (2H, m), 2.33 (2H,
1
quintet, J = 8 Hz), 3.17 (1H, sextet, J = 8 Hz), 3.41 (2H,
q, J = 8 Hz), 3.44 (2H, q, J = 8 Hz), 7.18–7.21 (2H, m),
2.18 (1H, m), 2.33 (2H, quintet, J = 8 Hz), 2.78 (2H, d,
J = 8 Hz), 3.35 (2H, t, J = 8 Hz), 3.43 (2H, t, J = 8 Hz),
7.16–7.22 (2H, m), 7.47 (1H, d, J = 8 Hz), 7.99 (1H, s),
8.04 (1H, d, J = 8 Hz), 8.36 (1H, s); Compound 8e: LR-
7.50 (1H, d, J = 8 Hz), 8.01 (1H, s), 8.07 (1H, d,
J = 8 Hz), 8.44 (1H, s); IR (KBr) 3376, 3175, 1652,
ꢁ
1
1
545, 1446 cm .
ꢁ
1
1
0. Extraction and purification of natural Cypridina luciferin
was according to the below reference: Haneda, Y.;
Johnson, F. H.; Masuda, Y.; Saiga, Y.; Shimomura, O.;
Sie, H.-C.; Sugiyama, N.; Takatsuki, I. J. Cell. Comp.
Physiol. 1961, 57, 55. The purity of the natural luciferin 1
FABMS m/z 404 (M ); H NMR (500 MHz, CD OD) d
3
1.55 (9H, s), 2.33 (2H, quintet, J = 8 Hz), 3.41 (2H, t,
J = 8 Hz), 3.43 (2H, t, J = 8 Hz), 7.16–7.22 (2H, m), 7.47
(1H, d, J = 8 Hz), 8.00 (1H, s), 8.02 (1H, d, J = 8 Hz), 8.39
1
(1H, s); Compound 8f: H NMR (500 MHz, CD OD) d
3
9
was judged by the HPLC analysis.
2.29 (2H, quintet, J = 8 Hz), 3.01 (2H, d, J = 8 Hz), 3.41
(2H, t, J = 8 Hz), 7.14–7.20 (2H, m), 7.43 (1H, d,
J = 8 Hz), 7.87 (1H, s), 8.02 (1H, s), 8.08 (1H, d,
J = 8 Hz).
ꢁ
1
1
1. Compound 8a: LR-FABMS m/z 362 (M ); H NMR
(
3
500 MHz, CD OD) d 2.30 (2H, quintet, J = 8 Hz), 2.54
(
3H, s), 3.30 (2H, t, J = 8 Hz), 3.43 (2H, t, J = 8 Hz),
7
8
.17–7.22 (2H, m), 7.47 (1H, d, J = 8 Hz), 7.98 (1H, s),
.01 (1H, d, J = 8 Hz), 8.31 (1H, s); Compound 8b: LR-
12. Goto, T.; Isobe, M.; Coviello, D. A.; Kishi, Y.; Inoue, S.
Tetrahedron 1973, 29, 2035–2039.