Convergent and short-step syntheses of dl-Cypridina luciferin and its analogues based on Pd-mediated cross couplings

Article abstract of DOI:10.1016/S0040-4039(00)00131-3

dl-Cypridina luciferin and its analogues were synthesized from 2- aminopyrazine by an eight-step method that included two regio-selective Pd- mediated cross couplings, and their chemi- and bioluminescent activities were compared. Analogues having a 3-benzofuranyl or a 3-benzothienyl group in the place of a 3-indolyl group showed luciferase affinities similar to Cypridina luciferase but with a lower luminescent efficiency, suggesting that the NH group is unimportant for molecular recognition whereas the indolyl group is crucial for efficient luminescence. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00131-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2185–2188
Convergent and short-step syntheses of dl-Cypridina luciferin and
its analogues based on Pd-mediated cross couplings
Hideshi Nakamura,a,∗ Mihoko Aizawa,b Daisuke Takeuchi,b Akio Murai b and
Osamu Shimoura c
aDivision of Biomodeling, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya 464-8601, Japan
bDivision of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
cMarine Biological Laboratory, Woods Hole, Massachusetts 02543, USA
Received 13 December 1999; revised 13 January 2000; accepted 14 January 2000
Abstract
dl-Cypridina luciferin and its analogues were synthesized from 2-aminopyrazine by an eight-step method that
included two regio-selective Pd-mediated cross couplings, and their chemi- and bioluminescent activities were
compared. Analogues having a 3-benzofuranyl or a 3-benzothienyl group in the place of a 3-indolyl group showed
luciferase affinities similar to Cypridina luciferase but with a lower luminescent efficiency, suggesting that the NH
group is unimportant for molecular recognition whereas the indolyl group is crucial for efficient luminescence.
© 2000 Elsevier Science Ltd. All rights reserved.
Keywords: benzofurans; bioluminescence; benzothiophenes; indoles.
Cypridina luciferin (1a) is an imidazopyrazinone and involved in the bioluminescence of the crusta-
cean Cypridina (Vargula) hilgendorfii and deep-sea fishes. Cypridina luciferin shows a typical lucife-
rin–luciferase reaction to emit a blue light of 465 nm from an oxyluciferin–luciferase complex.1 Bio-
luminescent imidazopyrazinones were synthesized from properly substituted 2-aminopyrazines, which
are synthesized by the pyrazine ring construction of a coupling pair of a glyoxal, an α-aminoamidine
or an α-aminonitrile and an α-ketooxime (Fig. 1). The first total synthesis of Cypridina luciferin was
achieved by Kishi et al. in 19662 by using a former coupling pair and the synthesis of ethioluciferamine
was reported in 1971 by White and Karpetsky3 by a TiCl4-mediated coupling of an α-aminonitrile and
an α-ketooxime. Several analogues were prepared based on Kishi’s method and the structure–activity
relationship and mechanistic aspects of bio- and chemiluminescence were investigated extensively by
Goto.4
In the course of our studies on bio- and chemiluminescent imidazopyrazinones, we have developed
a new synthetic approach to imidazopyrazinones by a Pd-mediated cross coupling, and achieved the
∗
Corresponding author. Tel/fax: 81-52-789-4284; e-mail: hnak@agr.nagoya-u.ac.jp (H. Nakamura)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00131-3
tetl 16408

2186
Fig. 1.
synthesis of coelenterazine and its analogues.5 We have extensively applied the new method to Cypridina
luciferin as well as its analogues having different substituents at the 6-position. Here we would like to
report novel short-step syntheses of Cypridina luciferin and its analogues having 3-benzofuranyl or 3-
benzothienyl chromophores together with their bioluminescent activities.
The synthesis was started with 2-amino-3,5-dibromopyrazine prepared from commercially available
2-aminopyrazine by bromination with Br2.6 Regio-selective introduction of a C3 unit was achieved with
N-Boc-propargylamine under Sonogashira coupling7 conditions to yield an alkynyl compound (5) which
was hydrogenated with PtO2 to form bromopyrazine 6 (Fig. 2). A coupling reaction of bromopyrazine
6 with an indole was achieved by Suzuki coupling8a,b with N-tosylindolyl-3-boronic acid [7, X=NTs,
Y=B(OH)2]9 to afford indolylaminopyrazine 8a in 80% yield after hydrolysis with a 1:1 mixture of 5 M
NaOH aq. and 1,4-dioxane. Deprotection reaction with TFA afforded ethioluciferamine hydrochloride
in 87% yield. Total yield of ethioluciferamine in six steps was 30% from commercially available 2-
aminopyrazine.
Fig. 2.
Ethioluciferamine was converted to ethioluciferin by using a modified method10 in 96% yield.
Cyclization of ethioluciferin with a keto-acetal afforded dl-Cypridina luciferin11,12 in 24% yield.
Cypridina luciferin analogues (1b and 1c) with an oxygen or a sulfur atom instead of the NH group
were synthesized by a similar manner. Coupling reactions were achieved smoothly with tributylstannanes
under Stille coupling conditions.5a Compounds 8 (X=O and X=S) were obtained in a yield of 47 and
72%, respectively. A three-step transformation of 8b and 8c was carried out under the same conditions

2187
to afford dl-Cypridina luciferin analogues (1b: X=O and 1c: X=S) at moderate yields of 16 and
29%, respectively.11,12 The luminescence properties of dl-Cypridina luciferin and the analogues with
luciferase (bioluminescence) or without luciferase (chemiluminescence) are summarized in Table 1. The
analogues were bioluminescent but with lower efficiencies. However, the kinetic parameters, as well as
the enhancement of luminescence by luciferase (BL/CL), were similar each other, suggesting that the
NH group of the indolyl group in luciferin is not essential for binding luciferase13 but important for
high luminescent efficiency. The reason for low chemiluminescent efficiency of the analogues is not
completely clear. Detailed study of the structure–activity relationship is in progress in our laboratory.
Table 1
Luminescence properties of synthesized dl-Cypridina luciferin (1a) and analogues (1b and 1c) and
natural S-Cypridina luciferin at 23°C
Bioluminescence14 is a highly sensitive and non-destructive detection method applicable to monitor
various transient biological events such as a calcium wave during fertilization.15 Recently, genes of
bioluminescent proteins including apoaequorin, firefly luciferase, Renilla luciferase, and Cypridina
luciferase have been cloned and used as a reporter gene studying critical biological events. Cypridina
luciferase was shown to be useful for monitoring gene expression in measuring a secreted enzyme.16 Our
synthetic method may be applicable in future for the preparation of various types of Cypridina luciferin
in sufficiently large quantities for use in the area of life sciences.
Acknowledgements
This research was partly supported by a Grant-in-Aid from the Ministry of Education, Science, Sports
and Culture, Japan. The generous gift of Pd(PPh3)4 from Kawaken Co. is greatly acknowledged. We
thank the Naito Foundation for financial support.

2188
References
1. Shimomura, O.; Johnson, F. H.; Masugi, T. Science 1969, 164, 1299–1300.
2. Kishi, Y.; Goto, T.; Inoue, S.; Sugiura, S.; Kishimoto, H. Tetrahedron Lett. 1966, 3445–3450.
3. Karpetsky, T. P.; White, E. H. J. Am. Chem. Soc. 1971, 93, 2333–2334. Idem, Tetrahedron 1973, 29, 3761–3773.
4. Goto, T. Pure Appl. Chem. 1968, 17, 421–441.
5. (a) Nakamura, H.; Takeuchi, D.; Murai, A. Synlett 1995, 1227–1228. (b) Nakamura, H.; Aizawa, M.; Murai, A. Synlett 1996,
1015–1017. (c) Nakamura, H.; Wu, C.; Murai, A.; Inouye, S.; Shimomura, O. Tetrahedron Lett. 1997, 38, 6405–6406.
6. Sato, N. J. Heterocycl. Chem. 1982, 673–674.
7. Sonogashira, K. Comprehensive Organic Synthesis; Trost, B. M.; Fleming, I., Eds.; Pergamon Press: Oxford, 1991; Vol. 3,
Chapter 2.3, pp. 521–529.
8. (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457–2483. (b) It could not be achieved by Stille coupling with 1-
t-butyldimethylsilyl-3-tributylstannylindole but might be possible with 1-tosyl-3-trimethylstannylindole: Cianttini, P. G.;
Morera, E.; Ortar, G. Tetrahedron Lett. 1994, 35, 2405–2408.
9. Zheng, Q.; Yang, Y.; Martin, A. R. Heterocycles 1994, 37, 1761–1772.
10. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem. 1992, 57, 2497–2502.
11. The reaction conditions were not optimized yet at small scale reactions, and the condensation with a ketoaldehyde was
reported as good (80%) at a several hundred mg scale. Sugiura, S.; Inoue, S.; Goto, T. Yakugaku Zasshi 1970, 90, 707–710.
12. All compounds gave satisfactory spectral data. Compound 1a: pale orange crystals (HBr salts); mp 168–172; HR-FDMS
m/z 405.2279 (M+); calcd for C22H27N7O: 405.2277; 1H NMR (400 MHz, CD3OD) 0.95 (3H, t, J=7 Hz), 1.45 (3H, d, J=7
Hz), 1.80–1.99 (2H, m), 2.33 (2H, quintet, J=7 Hz), 3.17 (1H, sextet, J=7 Hz), 3.41 (2H, q, J=7 Hz), 3.44 (2H, q, J=7 Hz),
7.18–7.21 (2H, m), 7.50 (1H, d, J=7 Hz), 8.01 (1H, s), 8.07 (1H, d, J=7 Hz), 8.44 (1H, s); UV (1:1 EtOH:H2O) λmax 434
nm (ε 8800). Compound 1b: pale yellow crystals (HCl salts); FABMS m/z 407 (M+H)+; 1H NMR (400 MHz, CD3OD) 0.95
(3H, t, J=7 Hz), 1.47 (3H, d, J=7 Hz), 1.80–1.99 (2H, m), 2.33 (2H, quintet, J=7 Hz), 3.18 (1H, sextet, J=7 Hz), 3.40 (2H, q,
J=7 Hz), 3.43 (2H, q, J=7 Hz), 7.39–7.45 (2H, m), 7.62 (1H, d, J=7 Hz), 8.15 (1H, s), 8.49 (1H, d, J=7 Hz), 8.60 (1H, s); UV
(1:1 EtOH:H2O) λmax 424 nm (ε 6800). Compound 1c: pale yellow crystals (HCl salts); HR-FABMS m/z 422.1921 (M+);
calcd for C22H26N6OS: 422.1889; 1H NMR (400 MHz, CD3OD) 0.96 (3H, t, J=7 Hz), 1.48 (3H, d, J=7 Hz), 1.80–2.00 (2H,
m), 2.32 (2H, quintet, J=7 Hz), 3.20 (1H, sextet, J=7 Hz), 3.44 (2H, q, J=7 Hz), 3.45 (2H, q, J=7 Hz), 7.41–7.52 (2H, m),
8.00 (1H, d, J=7 Hz), 8.19 (1H, s), 8.30 (1H, d, J=7 Hz), 8.61 (1H, s); UV (1:1 EtOH:H2O) λmax 430 nm (ε 10400).
13. For a recent review, see: Wilson, T.; Hastings, J. W. Annu. Rev. Cell Dev. Biol. 1998, 14, 197–230.
14. Similar findings in an indolactam–protein kinase C interaction were reported by: Kozikowski, A. P.; Ma, D.; Du, L.; Lewin,
N. E.; Blumberg, P. M. J. Am. Chem. Soc. 1995, 117, 6666–6672.
15. Kubota, H. Y.; Yoshimoto, Y.; Yoneda, M.; Hiramoto, Y. Dev. Biol. 1987 19, 129–136.
16. (a) Inouye, S.; Ohmiya, Y.; Toya, Y.; Tsuji F. I. Proc. Natl. Acad. Sci. USA 1992, 89, 9584–9587. (b) Thompson, E. M.;
Adenot, P.; Tsuji, F. I.; Renard, J. P. Proc. Natl. Acad. Sci. USA 1995, 92, 1317–1321. (c) Miesenböck, G.; Rothman, J. E.
Proc. Natl. Acad. Sci. USA 1997, 94, 3402–3407.