Design, synthesis, and evaluation of the transition-state inhibitors of coelenterazine bioluminescence: Probing the chiral environment of active site [13]

Full text of DOI:10.1021/ja005663v

J. Am. Chem. Soc. 2001, 123, 1523-1524
Design, Synthesis, and Evaluation of the Transition-
1523
State Inhibitors of Coelenterazine Bioluminescence:
Probing the Chiral Environment of Active Site
Hideshi Nakamura,†,‡,| Chun Wu,†,‡ Satoshi Inouye,*,§ and
Akio Murai‡
DiVision of Biomodeling
Department of Applied Molecular Biosciences
Graduate School of Bioagricultural Sciences
Nagoya UniVersity, Nagoya, 464-8601, Japan
DiVision of Chemistry, Graduate School of Science
Hokkaido UniVersity, Sapporo 060-0810, Japan
Yokohama Research Center, Chisso Co.
5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
ReceiVed October 3, 2000
Bioluminescence is one of the most attractive natural phenom-
ena, displayed by various different types of organisms from
bacteria to fishes.1 Recently, considerable attention has been given
to bioluminescence reactions for use as highly sensitive, nonde-
structive analytical tools, particularly in monitoring gene expres-
sions and studying protein-protein interactions by luminescence
energy transfer.2 Coelenterazine (1a) is well-known to be involved
in the luminescence reactions of various organisms, such as
jellyfishes,3 sea pansies,4 and deep-sea shrimps.5 It is widely
distributed among both bioluminescent and nonbioluminescent
marine organisms.6 The oxidation of coelenterazine results in light
emission either in the presence or absence of a protein factor,
providing a commonly used distinction between bioluminescence
and chemiluminescence. The quantum yield is much greater in
the bioluminescence that involves an enzyme than in the chemi-
luminescence.7 The luminescence reaction is an oxidation process
that takes place at the C-2 and C-3 positions of coelenterazine,
and it appears to involve several key peroxy intermediates or
transition states (3-4)7,8 (Figure 1).
Figure 1. Bioluminescence reaction of coelenterazine (1a) and its
possible intermediates and transition states (3, 4) and 3-enoyl sulfate of
coelenterazine (5).
Figure 2. Stable deazacoelenterazine analogue (6) and its transition-
state analogues (7, 8).
lography.10,11 Model studies on the peroxy intermediates of
coelenterazine analogues showed that peroxides decompose with
light emission at -50 °C and above, with lower efficiencies than
those found in bioluminescence.12 None of the peroxy intermedi-
ates or transition states of luciferase reactions have been
characterized, except for a 18O-labeling study of the luminescence
reaction of a shrimp luciferase.5a The inhibitory effects of several
coelenterazine analogues were investigated by Cormier et al.,13
revealing that the 3-enoyl sulfate of coelenterazine (5) was a
strong inhibitor (Ki ) 9.1 × 10-9 M). To investigate the enzymatic
reaction of coelenterazine bioluminescence that involves a highly
efficient chemical excitation step, we designed some inhibitors
that are stable and not altered by luciferases (6-8) (Figure 2).
The transition-state analogues synthesized inhibited the lumi-
nescence reaction of recombinant Renilla luciferase,9,14 apparently
reflecting the differences in the chirality of these inhibitors. The
results suggested for the first time that the mechanism-based
inhibitors of bioluminescence might be useful as the tools for
investigating the chiral environment of the active sites of enzyme
where the excited species is formed at a high efficiency.
Among the bioluminescence systems of coelenterazine, Renilla
luciferase has attracted much attention recently because of its
simplicity in the components required for luminescence reaction
and of usefulness as a reporter protein.2,9 The structure of
coelenterazine peroxide (3a) in aequorin was found to have 2S
configuration by 13C NMR experiments and X-ray crystal-
† Nagoya University.
‡ Hokkaido University.
§ Chisso Co.
| Deceased November 9, 2000.
(1) (a) Harvey, E. N. LiVing Light; Princeton University Press: Princeton,
1940. (b) Campbell, A. K. Chemiluminescence; VCH Publishers: New York,
1988.
(2) (a) Wilson, T.; Hastings, J. W. Annu. ReV. Cell DeV. Biol. 1998, 14,
197-230. (b) Naylor, L. H. Biochem. Pharmacol. 1999, 58, 749-757.
(3) (a) Shimomura, O.; Johnson, F. H. Nature 1970, 227, 1356-1357. (b)
Shimomura, O.; Johnson, F. H. Tetrahedron Lett. 1973, 31, 2963-2966. (c)
Shimomura, O.; Johnson, F. H. Nature 1975, 236-238. (d) Shimomura, O.;
Johnson, F. H. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 2611-2615.
(4) Matthews, J. C.; Hori, K.; Comier, M. J. Biochemistry 1977, 16, 5217-
5220.
(5) (a)Shimomura, O.; Masugi, T.; Johnson, F. H.; Haneda, Y. Biochemistry
1978, 17, 994-998. (b) Inouye, S.; Watanabe, K.; Nakamura, H.; Shimomura,
O. FEBS Lett. 2000, 481, 19-25.
(10) A photoprotein from a jellyfish contains a peroxide moiety and the
luminescence is triggered by calcium ion to emit blue light of 465 nm with
a high quantum efficiency of 0.23.3a
(11) (a) Musicki, B.; Kishi, Y.; Shimomura, O. J. Chem. Soc., Chem.
Commun. 1986, 1566. (b) Head, J. F.; Inouye, S.; Teranishi, K.; Shimomura,
O. Nature 2000, 405, 372-376.
(12) (a) Teranishi, K.; Ueda, K.; Nakao, H.; Hisamatsu, M.; Yamada, T.
Tetrahedron Lett. 1994, 35, 8181-8184. (b) Usami, K.; Isobe, M. Tetrahedron
Lett. 1995, 36, 8613-8617, Chem. Lett. 1996, 215-216, and Tetrahedron
1996, 52, 12061-12090. (c) Teranishi, K.; Hidamatsu, M.; Yamada, T.
Tetrahedron Lett. 1997, 38, 2689-2692.
(13) Matthews, J. C.; Hori, K.; Cormier, M. J. Biochemistry 1977, 16,
5217-5220.
(14) Lorenz, W. W.; MaCann, R. O.; Longiaru, M.; Cormier, M. J. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 4438-4442.
(6) (a) Shimomura, O.; Inoue, S.; Johnson, F. H.; Haneda, Y. Comp.
Biochem. Physiol. 1980, 65B, 435-437. (b) Shimomura, O. Comp. Biochem.
Physiol. 1987, 86B, 361-363. (c) Campbell, A. K.; Herring, P. J. Mar. Biol.
1990, 104, 219-225.
(7) (a) Goto, T. In Marine Natural Products, Chemical and Biological
PerspectiVe; Scheuer, P. J., Ed.; Academic Press: New York, 1980; Vol. 3,
pp 179-222. (b) Hori, K.; Wampler, J. E.; Matthew, J. C.; Cormier, M. J.
Biochemistry 1973, 12, 4463-4468.
(8) (a) MaCapra, F. EndeaVour 1973, 32, 139-145. (b) Shimomura, O.;
Johnson, F. H. Biochem. Biophys. Res. Commun. 1971, 44, 340-346. (c)
Fujimori, K.; Nakajima, H.; Akutu, K.; Mitani, M.; Sawada, K.; Nakayama,
M. J. Chem. Soc., Perkin Trans. 2 1993, 2405-2409.
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10.1021/ja005663v CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/25/2001

1524 J. Am. Chem. Soc., Vol. 123, No. 7, 2001
Communications to the Editor
Scheme 1a
Table 1. Inhibitory Effects of the Transition-State Analogues on
the Bioluminescence Reaction of Recombinant Renilla Luciferase
and Benzylcoelenterazine (1b)
cmpd
Ki/M
cmpd
Ki/M
deaza (6)
1.3 × 10-7 benzylcoelenteramide (2b) 2.3 × 10-8a
S-TS-1 (7-S) 4.0 × 10-8 R-TS-1 (7-R)
5.0 × 10-8
9.3 × 10-9
S-TS-2 (8-S) 2.1 × 10-7 R-TS-2 (8-R)
a Data from ref 13.
stronger inhibitory effects than the sp2 analogue, however with
little difference between the two in the extents of inhibition. The
results suggest that the p-hydroxybenzyl group of the bound
enantiomers might be located at a similar position in the active
site of the enzyme, in resemblance to the finding in the
acetylcholine esterase inhibition.19 To examine the effect of the
peroxide moiety which might enhance selective binding, a
hydroxymethyl group was introduced at position 2 by an aldol
reaction. The enantiomers (TS-2) were separated with a chiral
column, and their configurations were determined by comparing
their CD spectra with those of model compounds.18,20,21 An
enantiomer (8-R) with an R-configuration gave the strongest
inhibition with a large enantioselectivity (about 22) (Table 1).
The mechanism of imidazopyrazinone bioluminescence in-
volves several transition states and intermediates, despite the
apparent simplicity of the reaction system that requires only
luciferin and luciferase in addition to oxygen. Although the 3D
structures of imidazopyrazinone luciferases are still unknown
except for the photoprotein aequorin, it seems most likely that
the luciferases serve to stabilize the structure of coelenterazine
substrate while catalyzing the oxidation reaction that results in a
highly efficient luminescence. The structure of the enantiomer
(R)-TS-2 (8-R) might represent the structure of one of the
transition states involved in the luminescence reaction. Interest-
ingly, opposite enantioselectivity was observed in the inhibition
of aequorin regeneration with benzylcoelenterazine (1b) and the
enantiomer (S)-TS-2 (8-S).
a (a) 4-MeO-C6H4B(OH)2, 2 M Na2CO3, Pd(PPh3)4, benzene-MeOH,
80 °C, 24 h, 99%; (b) sec-BuLi, TMEDA, -78 °C, then s.m, THF, 1 h,
benzaldehyde, -78 °C, 30 min; (c) toluene, 110 °C, 24 h, 80% for 2
steps; (d) DIBAL, -78 °C, CH2Cl2, 90%.; (e) Ph3P-CHCOOBn, benzene,
80 °C, 1 h, 95%; (f) H2, Pd/C, EtOH-EtOAc, 23 °C, 36 h, 94%; (g) SOCl2
80 °C, 3 h; (h) TiCl4, CH2Cl2, 12 h, 0 °C to 5 °C, 50% for 2 steps; (i)
4-methoxybenzaldehyde, 2.5 M NaOH, EtOH, 24 °C, 20 h, 74%; (j) BBr3
, -78 °C to rt, 24 h, 52%; (k) PtO2, H2, EtOH, 23 °C, 48 h, 70%; (l)
BBr3 , -78 °C to rt, 48 h, 50%; (m) NaOH, HCHO, THF-H2O, 23 °C,
2 h, 60%.
In summary, we have established a novel and effective route
to synthesize stable, chiral analogues of coelenterazine for
studying transition states and unstable intermediates involved in
the bioluminescence of coelenterazine. Among the analogues
studied, the hydroxymethyl model of the hydroperoxide structure,
(R)-TS-2, showed the most potent inhibition of recombinant
Renilla luciferase. Efforts to elucidate the key structure required
for efficient luminescence and the identification of catalytic site
are in progress.
Figure 3. Lineweaver-Burk plot of the results of Renilla luciferase inhi-
bition assay for the synthesized inhibitors, deazacoelenterazine analogue
(6, [) and the transition-state analogues, R-TS-1 (*), S-TS-1 (9), R-TS-2
(b), and S-TS-2 (O) at a concentration of 1.1 × 10-7 M. The initial rate
of luminescence of a substrate, benzylcoelenterazine (1b, X ) H), with
the recombinant enzyme (0.5 µg/mL) was measured in 25 mM Tris buffer
pH 7.5 containing 0.1 M NaCl at 23 °C with or without inhibitors (0).
Our first object in this study was the synthesis of a stable core
structure of coelenterazine, for which we selected a deaza-
analogue of coelenterazine (Scheme 1). The synthesis was started
with 3-bromobenzamide (9), and a p-hydroxyphenyl group was
introduced by Suzuki coupling15 to yield a biphenyl compound
(10). A benzyl group was added by the coupling reaction of an
ortho-metalated carbanion of 10 with benzaldehyde.16 After
lactonization, a two-step transformation provided an acid which
was converted to an indanone (13) by standard procedure.
A second p-hydroxyphenyl moiety was introduced to the
indanone by aldol condensation with p-methoxybenzaldehyde to
form the olefin (14) with an E-exo configuration. After depro-
tection, a stable deazacoelenterazine analogue (6) with an isomeric
double bond was obtained. The compound showed a moderate
inhibition of recombinant Renilla luciferase in a competitive
manner (Figure 3).
Acknowledgment. A Grant-in-Aid from the Ministry of Education,
Science, Sports and Culture of Japan is gratefully acknowledged. We
thank financial support from the Naito Foundation and a JSPS fellowship
to C.W.
Supporting Information Available: Syntheses and characterization
of the compounds (PDF). This material is available free of charge via
the Internet at http://acs.pubs.org.
JA005663V
(17) The S [CD (EtOH): 270 (∆ꢀ +7.4), 330 (0.0), and 350 nm (-1.1)]
and R [CD (EtOH) 270 (∆ꢀ -7.4), 330 (0), and 350 nm (+1.1)] of 7 were
eluted from an OC column (4.6 × 250 mm) at 20 and 24 min, respectively,
with a mobile phase of hexane-ethanol (2:1, 0.5 mL/min).
(18) These absolute configurations were assigned by comparing their CD
spectra and [R]D with those of similar indanone compounds, which were
consistent with the sector rule for indanones: Lucke, M. I.; Marguet A.; G.
Snatzke, Tetrahedron 1972, 28, 1677.
For the synthesis of transition-state models having a chiral sp3
carbon at position 2, the olefin 14 was subjected to hydrogenation
with PtO2 followed by deprotection and separation on a chiral
column (DAICEL OC), affording two enantiomers, (S)-TS-1 (7-
S) and (R)-TS-1 (7-R).17,18 As expected, these compounds showed
(19) Kawakami, Y.; Inoue, A.; Kawai, T.; Wakita, M.; Sugimoto, H.;
Hopfinger, A. J. Bioorg. Med. Chem. 1996, 4, 1429-1446.
(20) The S [R]25 +108 (c 0.02, EtOH), CD (EtOH) 263 (∆ꢀ +10), 330
D
(0.0), 350 nm (-1.1) and R [R]25 -108(c 0.02, EtOH)CD (EtOH) 263 (∆ꢀ
D
-10) 330 (0.0) 350 nm (+1.1). Enantiomers of 8 were separated by the same
OC column with a mobile phase of hexane-ethanol (6:1, 0.5 mL/min) at 32
and 39 min, respectively.
(15) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457-2483.
(16) Beak, P.; Brown R. A. J. Org. Chem. 1982, 47, 34-46.
(21) Kuroyanagi, M.; Fukuoka, M.; Yoshida, K.; Natori, S. Chem Pharm.
Bull. 1979, 27, 731-741.