Chemi- and bioluminescence of coelenterazine analogues with a conjugated group at the C-8 position

Article abstract of DOI:10.1016/S0040-4039(01)00340-9

The chemiliminescent compound coelenterazine is involved in various bioluminescence reactions as the substrates, causing the luminescence with an emission peak in the range of 450-475 nm. Anticipating the introduction of a bathochromicshift of the lumines

Full text of DOI:10.1016/S0040-4039(01)00340-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2997–3000
Chemi- and bioluminescence of coelenterazine analogues with a
conjugated group at the C-8 position
Chun Wu,a,* Hideshi Nakamura,a,† Akio Muraib and Osamu Shimomurac
aDivision of Biomodeling, Department of Applied Molecular Bioscience, 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, MA 02543, USA
Received 18 January 2001; revised 21 February 2001; accepted 23 February 2001
Abstract—The chemiliminescent compound coelenterazine is involved in various bioluminescence reactions as the substrates,
causing the luminescence with an emission peak in the range of 450–475 nm. Anticipating the introduction of a bathochromicshift
of the luminescence, several new coelenterazine analogues that have conjugated olefins or aromatic groups at the 8-position of
imidazopyrazinone ring were synthesized. In the chemiluminescence reaction, the emission spectra of a majority of the compounds
synthesized showed a bathochromic shift, giving an emission peak in the range of 520–580 nm. In the bioluminescence catalyzed
by Oplophorus luciferase, the bisthienyl analogue of coelenterazine emitted a moderate intensity of luminescence (5% of
coelenterazine) with an emission maximum at 528 nm, which was the longest wavelength of all the analogues tested. © 2001
Elsevier Science Ltd. All rights reserved.
Coelenterazine 1 is well known as a chromophoric
compound of aequorin and also as the luciferins of
various bioluminescent marine organisms such as the
sea pansy Renilla reniformis and the deep-sea shrimp
Oplophorus gracilirostris. The bioluminescence of coe-
lenterazine is highly efficient, thus the luciferases
involved may be usable as reporter proteins. The emis-
sion maxima of the bioluminescence of coelenterazine
have been reported in the range 450–475 nm, showing
slight differences by the luciferase species used.1 The
amide anion 2 is believed to be the light emitter (see
Fig. 1).2 We have recently reported that an imidazopy-
razinone having chlorostyryl functionality at the 8-posi-
tion displayed a large bathochromic shift in the
chemiluminescence in neutral condition,3 showing that
the color of bioluminescence can be spectrally shifted
by the introduction of a conjugated group at 8-position.
In the present study we synthesized several new coelen-
*
O
X
O
-
N
N
N
X
N
N
Luciferase
O2
Light
N
H
Y
-CO2
Y
2
coelenterazine 1a: X=Y=OH
2-deoxycoelenterazine 1b: X=H, Y=OH
bisdeoxycoelenterazine 1c: X=Y=H
Figure 1. The light emitters involved in the bioluminescence of coelenterazine (1a), 2-deoxy-coelenterazine (1b) and bisdeoxycoe-
lenterazine (1c).
Keywords: coelenterazine; bathochromic shift; Oplophorus luciferase; chemiluminescence; bioluminescence.
* Corresponding author. Tel.: (81)52-789-4280; fax: (81)52-789-4280; e-mail: i993001d@mbox.media.nagoya-u.ac.jp
†
Deceased November 9, 2000.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00340-9

2998
C. Wu et al. / Tetrahedron Letters 42 (2001) 2997–3000
terazine analogues having a conjugated group at the
8-postion by the use of Pd-cross coupling reactions,4
then examined the luminescence characteristics of the
products.
N-anions of the amide compounds produced by a
chemiluminescence reaction.7
Bioluminescence properties of these analogues were
investigated using recombinant Renilla luciferase,1
Oplophorus luciferase1 and apoaequorin.1 In the pres-
ence of Renilla luciferase, the luminescence of the ana-
logues having a 6-phenyl group, 4a–c and 1c, were
poor.8 The Ca-triggered luminescence of the aequorins
that were regenerated with analogues 4a–c were also
very weak,8 although the aequorin regenerated with 3c
emitted a total light corresponding to 5.5% of that
regenerated with coelenterazine, with an emission maxi-
mum at 438 nm accompanying two very weak peaks at
545 and 610 nm.
Introduction of a conjugated chromophore at the 8-
position of coelenterazine was easily achieved by the
method previously reported, using 2-amino-3,5-dibro-
mopyrazine and tin reagents, as shown in Fig. 2.4 This
cross coupling reaction, which gave yields of 52–74%, is
regioselective due to the chelation of an amino group
and the electron deficiency at the 3-position. The sec-
ond cross coupling reactions at the 5-position were
performed using an excess of tin reagents. The deriva-
tives having a benzyl substituent at 2-position5,6 were
synthesized by the condensation of 2-ketoaldehyde with
3,5-disubstitued-2-aminopyrazine.
Oplophorus luciferase was previously found to catalyze
the luminescence of bisdeoxycoelenterazine 1c with high
efficiency.8 The luminescence of the new coelenterazine
analogues was considerably more efficient with
Oplophorus luciferase than with Renilla luciferase or
apoaequorin. For example, the analogue 3b having one
conjugated double bond at the 8-postion emitted
luminescence at 482 nm, which was moderately strong
in both the intensity and the total light. The lumines-
cence of the analogue 4a having an aromatic group at
the 8-position was similar to that of the analogue 3b,
whereas the analogue 3c that also has an aromatic
group at the 8-position emitted only feeble lumines-
cence at 476 nm, although the total light emitted from
this compound was close to those emitted from the
analogues 4a and 3b. The analogues 3c and 4a, both
with an aromatic group at the 8-position, showed their
chemiluminescence maxima at the wavelengths consid-
erably longer than their bioluminescence maxima, for
reasons which are unclear. A significant bathochromic
Coelenterazine analogues synthesized showed chemilu-
minescence in polar aprotic solvents. In chemilumines-
cence, the analogue 3a having a styryl group showed
the largest bathochromic shift under neutral conditions,
giving a peak at 580 nm. A comparison of the analogue
3c (peak at 519 nm) and the analogue 4a (peak at 520
nm) suggests that the electron rich function group may
not enhance the bathochromic effect. Two analogues 4b
and 4c having hetero rings showed slightly larger shifts
than the analogue 4a having aromatic rings. The chemi-
luminescence spectra of 4a–c were superimposable with
the fluorescence spectra of these compounds after
chemiluminescence reaction (Table 1), showing that
their light emitters are amide anion. The chemilumines-
cence spectra of the analogues 3a–c in the presence of
alkali did not match the fluorescence spectra of the
spent solution after chemiluminescence, suggesting that
the fluorescence is emitted probably from the pyrazine-
R1SnBu3 (1 eq)
Pd(PPh3)4 (2 mol%)
LiCl (3eq)
t-BuO-C6H4-SnBu3
O
EtO
EtO
or PhSnBu3 (3 eq)
N
N
NH2
R1
(2.4 eq)
N
N
NH2
Br
N
N
NH
R1
2 Pd(PPh3)4 (5 mol%)
3 or 4
LiCl (3eq)
Hunig Base (2.5 eq)
DMF, Reflux, 2-3 h
R2
HCl, EtOH, Reflux
2 h, Y. 45-70%
Br
Br
5
3
Hunig Base (2.5 eq)
DMF, Reflux, 2-7 h,
Y. 52-60%
O
O
O
N
N
H
N
N
N
N
H
N
N
H
N
H
H
HO
OH
HO
H3C
CH3
HO
C6H5
H
3c
3a
3b
O
O
O
N
N
N
N
N
N
S
S
S
N
H
N
H
N
H
4c
4a
4b
Figure 2. Synthesis of coelenterazine analogues 3 and 4 by Pd-mediated cross couplings.

C. Wu et al. / Tetrahedron Letters 42 (2001) 2997–3000
2999
Table 1. Bioluminescence, chemiluminescence and fluorescence of coelenterazine analogues
1a
1b
3a
3b
3c
1c
4a
4b
4c
Aequorina
Total light (%)
Emission max. (nm)
100
82
0.4
ND
1.5
455
5.5
0.018
ND
ND
0
ND
ND
ND
465f 464f
438 (1):545 (0.2):610 (0.06) 450g
Oplophorus luciferaseb
Initial intensity (%)
Total light (%)
100
100
97
75
0.001
0.007
ND
16
31
482
0.43
20
476 (1):545 (0.2)
79
66
9.4
26
483
0.057
3
510
0.18
5.5
528
Emission max. (nm)
452f 457f
448g
Renilla luciferasec
Initial intensity (%)
100f
57f
ND
580
0.12
0.017
519
0.32g
0.0025
ND
0.0026
534
Chemiluminescenced
Emission max. (nm)
465h 466
461
453
520
525
Fluorescencee
Emission max. in neutral (nm) 411h 412
Emission max. with base (nm) 524h 526
540
612
425
531
420
541
405
453
448
520
423
525
430
534
a Regenerated from 10 mg analogues and recombinant apoaequorin (0.5 mg) in 0.5 ml of 10 mM Tris–HCl/2 mM EDTA/5 mM 2-mercapto-
ethanol, pH 7.5 for overnight, then luminescence was measured by adding 10 ml of the solution to 3 ml of 10 mM calcium acetate.
b Analogue (0.24 nmol) was added to Oplophorus luciferase (10 mg) in 3 ml of 50 mM NaCl/15 mM Tris–HCl, pH 8.3.
c Analogue (0.24 nmol) was added to recombinant R. luciferase in 3 ml of 0.1 M NaCl/25 mM Tris–HCl, pH 7.5.
d 10 mM ethanol solution of analogue (30 ml) was added to DMSO (2 ml) and chemiluminescence was triggered by the addition of 0.1 M acetate
buffer pH 6.5 (100 ml).
e Fluorescence was measured with the spent solution of chemiluminescence under neutral condition or with addition of 1 M KOH.
f Data from Ref. 1.
g Data from Ref. 8.
h Data from Ref. 9. ND: not determined due to low light intensity.
shift was found with the low intensity luminescence of
analogue 4b having a compact thienyl group at the
8-position, and a further red-shift was observed with
the bisthienyl compound 4c that showed an emission
maximum at 528 nm.
1973, 492–493.
3. Nakamura, H.; Wu, C.; Takeuchi, D.; Murai, A. Tetra-
hedron Lett. 1998, 39, 301–304.
4. Nakamura, H.; Takeuchi, D.; Murai, A. Synlett 1995,
1227–1228.
5. Compound 3a: brown solid; mp 137–138°C; 1H NMR (400
MHz, 9:1 CDCl3–CD3OD 12 M HCl): l 4.35 (2H, s), 6.97
(2H, d, J=8 Hz), 7.95 (2H, d, J=8 Hz), 7.27 (1H, d,
J=16 Hz), 7.76 (1H, d, J=16 Hz), 7.28–7.40 (4H, m),
7.45–7.48 (4H, m), 7.78–7.80 (2H, m), 8.50 (1H, s); HR-
FABMS, m/z 420.1648 (calcd for C27H22N3O2 420.1633).
Compound 3b: brown solid; mp 145–146°C; 1H NMR (400
MHz, 9:1 CDCl3–CD3OD 12 M HCl): l 2.13 (3H, s), 2.37
(3H, s), 4.27 (2H, s), 6.73 (1H, s), 8.40 (1H, s), 7.20–7.40
(5H, m), 7.80 (2H, d, J=8 Hz), 6.87 (2H, d, J=8 Hz);
HR-FDMS, m/z 371.1663 (calcd for C23H21N3O2
371.1633). Compound 3c: red solid; mp 129–131°C; 1H
NMR (400 MHz, 9:1 CDCl3–CD3OD): l 8.15 (1H, s), 4.36
(2H, s), 7.20–7.40 (5H, m), 7.62 (2H, d, J=8 Hz), 6.98
(2H, d, J=8 Hz), 6.85 (2H, d, J=8 Hz), 7.86 (2H, d, J=8
Hz); HR-FABMS, m/z 410.1440 (calcd for C25H19N3O3
410.1426).
It has been reported that Oplophorus luciferase could be
a candidate for useful reporter protein because of its
favorable properties.1 The present study shows that, in
the bioluminescence of coelenterazines catalyzed by
Oplophorus luciferase, a conjugated group can induce a
bathochromic shift. Efforts are in progress to improve
the efficiency of the coelenterazine–luciferase biolu-
minescence reactions for practical applications.
Acknowledgements
We acknowledge financial support from Grants-in-Aid
for Scientific Research in priority areas (A) from the
Ministry of Education, Sciences, Sports and Culture,
Japan and from Naito Foundation. We would also like
to give thanks for the JSPS scholarship which was
awarded to C. Wu.
6. Compound 4a: brown solid; mp 101–102°C; 1H NMR (300
MHz, CD3OD): l 4.15 (2H, s), 7.90 (1H, s), 7.14–7.58
(9H, s), 7.84 (2H, d, J=6 Hz), 7.47 (2H, d, J=6 Hz), 8.06
(2H, d, J=6 Hz); HR-FABMS, m/z 378.1542 (calcd for
C25H20N3O 378.1542). Compound 4b: brown solid; mp
1
152–154°C; H NMR (300 MHz, DMSO): l 4.12 (2H, s),
References
8.50 (1H, s), 7.16–7.53 (9H, s), 7.30–7.35 (5H, s), 7.80 (1H,
d, J=6 Hz), 8.69 (2H, d, J=6 Hz), 8.11 (1H, s, J=6 Hz);
HR-EIMS, m/z 383.1075 (calcd for C23H17N3OS
1. Inouye, S.; Shimomura, O. Biochem. Biophys. Res. Com-
mun. 1997, 233, 349–353.
2. Hori, K.; Wampler, J. E.; Comier, M. J. Chem. Commun.
1
383.1058). Compound 4c: brown solid; mp 122–124°C; H

3000
C. Wu et al. / Tetrahedron Letters 42 (2001) 2997–3000
NMR (300 MHz, CD3OD 12 M HCl): l 4.45 (2H, s), 8.70
(1H, s), 7.18–7.20 (2H, m), 7.30–7.35 (5H, s), 7.61 (1H, s,
J=6 Hz), 7.86–7.90 (2H, m), 8.11 (1H, d, J=6 Hz);
HR-FABMS, m/z 390.0728 (calcd for C12H16N3OS2
390.0696).
51–58.
8. Nakamura, H.; Wu, C.; Murai, A.; Inouye, S.; Shimo-
mura, O. Tetrahedron Lett. 1997, 38, 6405–6406.
9. Hirano, T.; Mizoguchi, I.; Yamaguchi, M.; Chen, F.;
Ohashi, M.; Ohmiya, Y.; Tusji, F. J. Chem. Soc., Chem.
Commun. 1994, 165–167.
7. Shimomura, O.; Teranishi, K. Luminescence 2000, 15,
.
.