Synthesis of 13C-dehydrocoelenterazine and NMR studies on the bioluminescence of a Symplectoteuthis model

Article abstract of DOI:10.1016/S0040-4020(00)00163-0

The bioluminescence of luminous squid (Symplectoteuthis oualaniensis) is presumed to be initiated by the addition of the sulfhydryl residue of a photoprotein to dehydrocoelenterazine (DCL). To clarify this step, a novel synthetic route was established to label DCL with ¹³C. Dithiothreitol (DTT) and glutathione (GSH) were used as photoprotein models. The addition of DTT and GSH to ¹³C-labeled DCL gave luminous chromophores. Its structures were confirmed by NMR and MS spectrometry. The DTT adduct emitted light under alkaline condition to produce an oxidized compound. Thus we succeeded in modeling the bioluminescence of a photoprotein with DTT. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00163-0

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2629±2639
13
Synthesis of C-Dehydrocoelenterazine and NMR Studies on the
Bioluminescence of a Symplectoteuthis Model
*
Masaki Kuse and Minoru Isobe
Laboratory of Organic Chemistry, School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received 24 January 2000; accepted 21 February 2000
AbstractÐThe bioluminescence of luminous squid (Symplectoteuthis oualaniensis) is presumed to be initiated by the addition of the
sulfhydryl residue of a photoprotein to dehydrocoelenterazine (DCL). To clarify this step, a novel synthetic route was established to
label DCL with 13C. Dithiothreitol (DTT) and glutathione (GSH) were used as photoprotein models. The addition of DTT and GSH to
13C-labeled DCL gave luminous chromophores. Its structures were con®rmed by NMR and MS spectrometry. The DTT adduct emitted light
under alkaline condition to produce an oxidized compound. Thus we succeeded in modeling the bioluminescence of a photoprotein with
DTT. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction1
chromophore in 2 is the same as 4; this chemical change
was supported by UV and Fluorescence spectra.4
Molecular mechanisms of the bioluminescence and chemi-
luminescence have not been well-de®ned, although many
studies on chemi- and bioluminescence have been under-
taken in recent decades.2 We have studied a biolumi-
nescence system of an oceanic luminous squid,
Symplectoteuthis oualaniensis L. In 1981, Tsuji and
Leisman reported that a homogenate of the luminous
organ of this squid gave light in the presence of monocations
such as Na1, K1 etc. and molecular oxygen at pH 7.8.3 This
luminous system is potentially useful as a monitor of mono-
cations in single cells. For monitoring dications, an aequorin
system is especially famous for the determination of the
Ca21 concentration in living cells.4 In 1993, we succeeded
in the extraction of the photoprotein, named symplectin,
responsible for the bioluminescence in a 0.6 M KCl solution
from the same squid S. oualaniensis collected in Okinawa,
Japan. We have reported that this symplectin contains
dehydrocoelenterazine (1a), a similar chromophore to
coelenterazine (4), and that this chromophore may chemi-
cally be bound with the symplectin through a covalent bond
such as thioether.5 The major evidence for this chromophore
is that the mixing of dehydrocoelenterazine (1a) solution
into apoprotein of this squid enhanced the bioluminescence
in almost the same amount of light emission as from natural
symplectin, but mixing with the yellow coelenterazine (4)
solution did not show any bioluminescence. The reddish
solution of 1a mixed with aposymplectin solution
instantaneously changed into a yellowish color since the
The connecting position of this chromophore has been
suggested from an acetone adduct (3) to a dehydroco-
elenterazine (1a), which was isolated as an artifact from
this photogenic organ.4
A time course of ¯uorescence and absorption spectra were
recorded during the symplectin bioluminescence.6 As a
result of the spectra, the chromophore seemed to link with
the cysteine residue of the symplectin through a thioether as
illustrated in Scheme 1. As an evidence of this hypothesis,
model studies on the bioluminescence were undertaken with
DTT and GSH as a model for the symplectin. Firstly, the
predicted carbon connecting to the sulfhydryl residue was
labeled with 13C, and secondly, the chemical shift changing
was measured by high-®eld NMR.
Result and Discussion
Keywords: bioluminescence; photoprotein; dehydrocoelenterazine; nuclear
magnetic resonance.
* Corresponding author. Tel.: 181-52-789-4109; fax: 181-52-789-4111;
e-mail: isobem@agr.nagoya-u.ac.jp
The retrosynthetic analysis of the 13C-labeled dehydroco-
elenterazine (1ap) is shown in Scheme 2. (In order to indicate
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00163-0

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M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
Scheme 1. Initial step in the bioluminescence of the Symplectoteuthis photoprotein.
Scheme 2. Retrosynthetic analysis of 13C-labeled dehydrocoelenterazine (1a).
and to distinguish 13C-labeled from natural abundance
isotope compounds the asterisk is attached to the formula
number such as Np in text.) Dehydrocoelenterazine (1a)7 is
obtained from coelenterazine (4)8 by MnO2 oxidation, and 4
is synthesized from coelenteramine (5) and ketoaldehyde
(6) by reported procedures.9 For the preparation of the
13C-labeled compound 6p, it is necessary to establish a
novel synthetic route to 6. Ozonolysis of a,b-unsaturated
Scheme 3. Synthesis of 13C-labeled and non-labeled dehydrocoelenterazine.

M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
2631
ketone (7) will afford 6. The disconnection between
benzylic and phenyl carbon gives the critical two precursors
8 and 9. Halogenation of the diene (10) will give 8. After a
consideration of the availability of 13C sources, we decided
to use 13CH3I. A derived cyanohydrin (11) from cinnam-
aldehyde is a precursor of 10.
(1b±1d) with natural abundance isotopes (Eq. (1), equi-
librium between dehydrocoelenterazine (1) and DTT adduct
(21)) was studied prior to the use of 13C-labeled compounds.
The novel synthetic route to the 20-13C-dehydrocoelenter-
azine analog (1bp) is shown in Scheme 3. The cinnam-
aldehyde (12) was reacted with trimethylsilyl cyanohydrin
(TMS-CN)10 to give cyanohydrin (11). Methylation of 11
with 13CH3I and subsequent hydrolysis afforded benzal-
acetone (13) in 65% overall yield. To oxidize the methyl
group of 13, it was ®rst converted to vinylacetate (14p, 74%)
in isopropenylacetate in the presence of catalytic amounts of
conc. H2SO4. Oxidation of the methyl moiety of 13 was
accomplished by bromination of 14 with N-bromosuccini-
mide (NBS) to give bromoketone (15p, 81%). The critical
addition of p-methoxyphenylmagnesium bromide11 to 15
was carried out in diethyl ether ®rst at 2408C and then
08C to provide the bromohydrin (16p) as the sole product
in 60% yield. This bromohydrin (16) rearranged and then
converted into various products at higher temperatures. The
expected 1,2-rearrangement cleanly took place with silver
carbonate to give a,b-unsaturated ketone (17p, 80%).
Isomeric product was unexpectedly obtained in 20%
yield.12 We found that the cinnamyl moiety of 16 had also
an ability of rearrangement. A direct ozonolysis of 17 gave
some degradation products presumably due to its facile
enolization. The carbonyl moiety of 17 was temporally
protected as the ethylene ketal (18). The ozonide prepared
by ozonolysis of 18 was successfully reduced with triethyl-
amine13 to give the desired ketal±aldehyde (19p). Because
of an instability with silica gel, 19 was not isolated and was
directly reacted with aminopyrazine (5) at 808C in a mixture
of 10% HCl water and dioxane. The coelenterazine mono-
methyl ether (20p, d 20-C 33.3 ppm) was isolated in 34%
overall yield in 4 steps from the enone (17p). Oxidation
of 20p with manganese dioxide in a mixture of ethanol
and diethyl ether provided the dehydrocoelenterazine
mono-methyl ether (1bp, d 20-C 135.5 ppm) in 77% yield.
ꢀ1†
The questions in Eq. (1) are the effects of the dissociation of
the phenol groups in the equilibrium and also the relation-
ship to the pKa being 8 for the dihydromidazopyrazinone
N±H proton in 21. The dissociation of the N±H proton is the
critical step for the chemiluminescence in aprotic polar
solvents as DMSO with strong bases such as potassium
t-butoxide. When an aqueous solution of the compounds
21a or 21b was added to a solution of aposymplectin
prepared from symplectin (0.6 M KCl at pH 7.8 under air
oxygen atmosphere), both of them showed a biolumi-
nescence and the latter afforded about 40% of the light of
the former. On the contrary the chemiluminescence of 21a
in DMSO with t-BuOK yielded less than 10% of the light
amount of 21b as shown in Fig. 1. This striking difference
would be due to the dissociation of phenol extended to the
2-position of the imidazopyrazinone ring. A strong alkaline
condition for the chemiluminescence caused the equi-
librium to eliminate the sulfhydryl residue from 21a.
The equilibrium of the addition of DTT as a sulfhydryl
compound to dehydrocoelenterazine (1a) and its analogs
The equilibrium between compound 21 and 1 with DTT was
measured under slightly alkaline media between pH 6.8 and
9.2 since the optimum pH is known to be 7.8 for the
squid symplectin. The equilibrium is monitored by means
of UV±Vis spectra as shown in Fig. 2, where A±D are
corresponding to 1a±d (21a±d), respectively. The increase
Figure 1. Relative light amount (%) of bio- and chemiluminescence of 21a
and 21b.

2632
M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
Figure 2. UV spectra A±D of the equilibrium between dehydrocoelenterazine (1a±d) and its DTT adduct (21a±d), respectively, at various pH.
in the absorbance at around 550 nm in A indicates the
increase in the concentration of dehydrocoelenterazine
(1a). On the other hand, the decrease in the absorbance at
around 400 nm means the decrease in the amount of 21a.
(Arrows indicate the direction of increasing pH.) A similar
tendency is observed with 21c in C.
for 20 min the reaction mixture was acidi®ed to pH 3.0 with
1N HCl. The adduct 21bp is the most stable in this acidic
media. Concentration of the solution of the DTT adduct
(21bp) under reduced pressure gave a yellow oil. This
residue was puri®ed by HPLC to isolate the adduct 21bp
with a Develosilw ODS-UG-5 reversed-phase column
(10£250 mm) and 70% acetonitrile±water containing
0.1% TFA. As a mobile phase 70% methanol±water was
not suited for this puri®cation, since the high pressure in the
column decomposed all 21bp to DTT and 1bp. Isolated 21bp
was analyzed by NMR to afford two signals at d 45.2 and
45.4 ppm in the 13C spectra (Fig. 3); thus derived from d
135.3 ppm. Proton NMR also showed two doublets at ca.
5.6 ppm, and a C±H COSY spectrum showed the correla-
tion of the signals at 1H NMR d 5.67 and 5.62 ppm with the
signals at d 45.2 and 45.4 ppm, respectively, as shown
in Fig. 4 (left side). We found that the addition of DTT
dehydrocoelenterazine analog (1a) gave two diastereomers
of 21bp. Isolated DTT adduct 21b (natural abundance 13C)
gave m/zˆ590 (M1H)1 by FAB-MS spectrum. The
Those compounds (21b and 21d) having methyl ether on the
right phenol does not show such propensity in the spectrum
B and D. No elimination of SH in 21b and 21d happens
under these alkaline conditions. The equilibrium of 1 and 21
was not affected by the left phenol residue (B and D). As a
consequence the dehydrocoelenterazine analogs having
R1ˆMe (21b and 21d) can provide stable adducts with SH
compounds. Dehydrocoelenterazine mono-methyl ether 1bp
(20-carbon labeled with 13C) is the best candidate to study
the mimicking of the bioluminescence of the symplectin.
Mixing lbp and dithiothreitol (DTT) with 1:10 molar ratio
in a mixture of methanol and dichloromethane (1:1)
provided the adduct 21bp at room temperature. After stirring
Figure 3. 13C NMR spectra of adduct 21bp of 13C-labeled dehydrocoelenterazine analog 1bp and DTT.

M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
2633
Figure 4. C±H COSY spectrum and summary of the measurement of DTT adduct 21bp with NMR.
Figure 5. Structure of GSH adduct (23p) and HPLC chart of 23bp analyzed by reversed phase ODS colunm.
Figure 6. 13C NMR of adduct 23p of 13C-labeled dehydrocoelenterazine analog 1bp and GSH.

2634
M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
Figure 7. 2D (C±H COSY and HMBC) NMR spectrum of isolated GSH adduct 23bp.
absolute structure shown in Fig. 4 (right side) was con®rmed
by measurement of 21bp with 2D NMR (HMBC and
HOHAHA spectrum).
the puri®ed 23bp was analyzed by NMR as the diastereo-
meric mixture.
Two signals at d 44.6 and 45.3 ppm appeared in the 13C
NMR spectrum and two signals at d 5.69 and 5.66 ppm in
the 1H NMR spectrum, respectively, as shown in Figs. 6 and
7. The correlation between the 13C-labeled carbon and the
beta protons of the cysteine residue of GSH was observed in
the HMBC spectrum as shown in Fig. 7. (HMBC and
HOHAHA was measured in DMSO-d6.) These beta protons
of cysteine were con®rmed by HOHAHA spectrum as illu-
strated in Fig. 8. Isolated GSH adduct 23 (natural abundance
13C) gave m/zˆ743 (M1H)1 by FAB-MS spectrum.
Similarly glutathione (GSH) was added to 1bp with 1:5
molar ratio in a mixture of methanol and water (1:1) at
room temperature for 50 min, then the reaction mixture
was acidi®ed to pH 3.0, as shown in Fig. 5 (left side).
Concentration of the solution of the GSH adduct (23bp)
gave a yellow oil. Isolation of 23bp was performed with
reversed phase HPLC (30% acetonitrile±water containing
0.1% TFA) as shown in Fig. 5 (right side). Two peaks
corresponding to the diastereomers of 23bp appeared in
the chromatogram. But the peaks could not be separated,
due to only slight differences in their retention time. Thus
The structure of the GSH adduct 23bp was elucidated in
Figure 8. HOHAHA spectrum of adduct 23bp of 13C-dehydrocoelenterazine analog 1bp and GSH.

M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
2635
Figure 9. Summary of NMR measurements of GSH adduct (23bp) and the chemiluminescence product 22bp as Symplectoteuthis bioluminescence model.
detail as shown in Fig. 9 (left side) from a series of measure-
ment by NMR. Undoubtedly the sulfhydryl group of the
cysteine residue of GSH is connecting to the carbon labeled
with 13C of dehydrocoelenterazine (1bp).
and FAB mass spectra were measured with a JEOL JMS-
700 or JMS-600. High-resolution (HR) mass spectra were
measured with a JEOL JMS-700. Elemental analysis and
HRMS were performed by the Analytical Laboratory of
this school. Fluorescence spectra were measured with a
JASCO FP-777 spectrometer. Chemiluminescence was
recorded on an Otsuka Electronics MCPD- 110A, or a
JASCO FP-770 spectrometer without excitation beam.
Chemiluminescence pro®les were recorded on a Labo
Science Lumiphotometer TD-1000. Light yields of the
chemiluminescence were determined with a Labo Science
TD-4000 Lumiphotometer, or a TS-1000 Lumiphotometer
by integration of the total light emission. Ozone was
produced by a Nihon Ozone Corporation Ozonator, style
0-3-2, with the voltage set at 40 V and oxygen pressure at
2 kg cm22 to give approximately 0.3 g h21 ozone concen-
tration. The input oxygen was passed through a column of
Drierite to ensure dryness.
The chemiluminescence of DTT adduct 21bp emits light
(520 nm) in DMSO under basic condition (containing
10% of 1N t-BuOK/t-BuOH) as shown in Fig. 9 (right
side). After the chemiluminescence was completed, the
solution was neutralized with saturated ammonium
chloride, then the product (22b) was extracted and analyzed
to give m/zˆ577 (using natural abundance 13C) by FAB-MS
spectrum. In its 13C NMR spectrum two sets of signals at
d 42.5, 43.2 ppm appeared, presumably derived from
the two diastereomers of coelenteramide analog (22bp).
The molecular mechanism on the bioluminescence of
S. oualaniensis may be similar to the above model
studies. In the natural dehydrocoelenterazine (1a) which
exhibits ef®cient bioluminescence the local pH around the
phenol and dihydroimidazopyrazinone groups should be
presumably controlled by the protein surfaces. To get
further evidence the molecular mechanism should be
studied directly with the symplectin. Now structural
and cloning studies on this symplectin are also under
investigation.
Dichloromethane (CH2Cl2) was distilled from calcium
hydride. Tetrahydrofuran (THF) or 1,4-dioxane was distilled
from sodium metal in the presence of sodium benzophenone
ketyl as indicator. Dimethylsulfoxide (DMSO) was distilled
from calcium hydride under reduced pressure. Pyridine was
dried over NaOH pellets and used without distillation. The
other solvents were of reagent grade.
Analytical thin-layer chromatography (TLC) was conducted
on precoated TLC plates: silica gel 60 F-254 [E.Merck (Art
5715) Darmstadt, Germany], layer thickness 0.25 mm.
Silica gel column chromatography utilized Fuji Devision
(BW 820-MH) or Merck (Silica Gel 60).
Experimental
All melting points were measured on Yanaco MP-S3 and
uncorrected. UV spectra were taken on a JASCO U-best 50
spectrometer. IR spectra were determined on a JASCO FT/
IR-7000S spectrophotometer. Proton NMR spectra were
recorded on a JEOL EX 270 or GSX 270 for 270 MHz, a
JEOL JNML-500 for 500 MHz or a Bruker AMX-600 for
600 MHz. Chemical shifts (d) are given in parts per million
relative to tetramethylsilane (d 0.00) or CD3OD (d 3.30) or
DMSO-d6 (d 2.49) as internal standard and coupling
constants (J) in Hz. Carbon NMR were recorded on a
JEOL EX 270 or GSX 270 for 67.8 MHz or a JEOL
JNML-500 for 125.7 MHz or on a Bruker AMX-600 for
150.9 MHz. Chemical shifts are (d) given in parts per
million relative to CDCl3 (d 77.0) or CD3OD (d 49.0) or
DMSO-d6 (d 45.0) as internal standard. Coupling constants
(J) are given in Hz. The asterisks (p) represent signals of the
13C incorporated carbon. Low-resolution EI mass spectra
The HPLC analysis was carried out using a JASCO PU-980
pump system equipped with a JASCO UV-970 UV±Vis
detector and JASCO 807-IT integrator. An ODS column
of 5£250 mm (Develosil ODS-UG-5, Nomura Chemicals
Co., Ltd) was eluted with acetonitrile±water containing
0.1% of tri¯uoroaceticacid (TFA) at a ¯ow-rate of
0.5 mL min21 at room temperature. The puri®cation by
HPLC was performed with an ODS column 10£250 mm
(Develosil ODS-UG-5, Nomura Chemicals Co., Ltd).
Cinnam-cyanomethyl trimethylsilyl ether 11
To a suspension of cinnamaldehyde (12) (1.56 g, 8.0 mmol)

2636
M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
and ZnI2 (catalytic amount) in CH2Cl2 was added TMSCN
(1.18 ml, 8.8 mmol) at 08C. Then the mixture was allowed
to warm to room temperature and was stirred under argon
atmosphere for 2 h. The reaction mixture was concentrated
under reduced pressure to remove CH2Cl2. The resultant
residue was in almost pure state 11 in quantitative yield.
1H NMR (CDCl3, 270 MHz) d 0.27 (s, 9H), 5.13 (dd,
Jˆ5.9, 1.0 Hz, 1H), 6.21 (dd, Jˆ16.0, 5.9 Hz, 1H), 6.82
(dd, Jˆ16.0, 1.0 Hz, 1H), 7.3±7.43 (m 5H, Ar H) ppm.
13C NMR (CDC13, 67.8 MHz), d 20.1, 62.3, 118.4,
123.6, 127.0, 128.7, 128.8, 134.0, 135.1 ppm. EIMS m/z
205 (M12CN), 158 (M12TMS), 131 (M12TMSCN).
to room temperature, the reaction mixture was poured into
water (20 mL). The layer was separated, and the water layer
was extracted with diethyl ether (£3). The organic layer was
washed ®rstly with water, secondly with sat. NaCl aq., and
then dried over anhydrous Na2SO4. Evaporation under
reduced pressure gave a crude oil, and it was puri®ed by
chromatography on silica gel with diethyl ether±n-hexane
(1:5) to afford 14p (13C) (590 mg) in 74%. 1H NMR (CDCl3,
270 MHz), d 2.30 (s, 3H), 4.98 (dd, Jˆ160.0, 2.0 Hz, 1H),
5.13 (dd, Jˆ160.0, 2.0 Hz, 1H), 6.58 (d, Jˆ16.3 Hz, 1H),
6.66 (dd, Jˆ16.3, 1.5 Hz, 1H), 7.22±7.44 (m, 5H, Ar H)
p
ppm. 13C NMR (CDCl3, 67.8 MHz), d 106.2 ppm. EIMS
m/z 189 (M1), 147 (M1213CH3CO). HRMS (EI) calcd for
13C1,C11 H12O2 189.0871, found 189.0860.
Cinnam-methylketone 13, 13p (13C)
The compound 14 (natural isotope abundance) was prepared
according to the same procedure from 310 mg (2.1 mmol)
of cinnam-methylketone (13), 3.0 mL of isopropenyl-
acetate, and few drops of conc. H2SO4 in 69% yield (14,
280 mg). Mp 27±288C. IR (KBr) nmax 1757, 1640, 1607,
1448, 1372, 1210, 1022, 966, 870, 753, 694 cm21. 1H NMR
(CDCl3, 270 MHz), d 2.29 (s, 3H), 4.98 (d, Jˆ2.0 Hz, 1H),
5.12 (d, Jˆ2.0 Hz, 1H), 6.58 (d, Jˆ16.3 Hz, 1H), 6.64 (d,
Jˆ16.3 Hz, 1H),7.21±7.43 (m, 5H, Ar H) ppm. 13C NMR
(CDCl3, 67.8 MHz), d 20.9, 106.1 122.6, 126.9, 128.2,
128.6, 129.9, 135.9, 151.8, 168.7 ppm. EIMS m/z 188
(M1), 146 (M12CH3CO). Anal, calcd for C12H12O2; C,
76.57; H, 6.43%. Found C, 76.36; H, 6.41%.
A solution of the cyanohydrin TMS±ether (11) (ca. 1.9 g,
8.0 mmol) in 9 mL of THF was added dropwise over 20 min
to a solution of 8.8 mL (1.0 M hexane solution) of LiHMDS
in THF (35 mL) under argon atmosphere at 2788C. After
stirring this solution for 1 h, 13CH3I (1.0 g, 7.0 mmol) was
added dropwise to the solution over 5 min through a gas-
tight syringe under argon atmosphere. After being stirred for
25 min at 2788C, the reaction mixture was allowed to warm
to 08C over 1 h, then warmed up to room temperature. After
20 min later, the reaction mixture was cooled to 08C, then
slowly poured into 1N HCl (30 mL) and stirred for 1 min,
then allowed to warm to room temperature, and stirred over-
night at room temperature. The layers were separated, and
the water layer was extracted with CH2Cl2 (£3). The
combined organic layers were washed ®rstly with 1/10N
HCl twice, secondly with 1N NaOH twice, and then dried
over anhydrous Na2SO4. Evaporation under reduced
pressure gave a crude product (1.12 g), and it was puri®ed
by chromatography on silica gel with diethyl ether±n-
hexane (1:4 then 1:3) to afford 13p(13C) (660 mg) in 65%
(3 steps). 1H NMR (CDCl3, 270 MHz), d 2.38 (d,
Jˆ127.6 Hz, 3H), 6.71 (dd, Jˆ16.0, 1.5 Hz, 1H), 7.44
(brt, Jˆ2.5 Hz, 3H), 7.52 (d, Jˆ16.0 Hz, 1H), 7.55 (brd,
Jˆ9.4 Hz, 2H) ppm. 13C NMR (CDC13, 67.8 MHz), d
p27.5 ppm. EIMS m/z 147 (M1), 131 (M1213CH3).
HRMS (EI) calcd for 13C1C9H10O 147.0766, found
147.0755.
Bromomethyl-cinnamketone 15, 15p (13C)
To a solution of 1-acetoxy-l-cinnnam-ethene (14p) (13C)
(610 mg, 3.2 mmol) in 20 mL of THF was added NBS
(970 mg, 5.4 mmol) and water (94 mg, 5.4 mmol) at 08C.
After a few minutes, the reaction mixture was allowed to
warm to room temperature and stirred for 45 min under
argon atmosphere. Then the solution was cooled to 08C
and poured into 20 mL of sat. NaHCO3 aq. then diluted
with water. After extraction by diethyl ether (£3), the
organic layer was washed ®rstly with water, secondly with
brine, and then dried over anhydrous Na2SO4±SiO2±anhy-
drous Na2SO4 column. Evaporation under reduced pressure
gave a crude oil, and it was puri®ed by chromatography on
silica gel with diethyl ether±n-hexane (1:5) to afford 15p
The compound 13 (natural isotope abundance) was synthe-
sized according to the same procedure from ca. 0.46 g
(2.0 mmol) of the TMSCN-adduct (11), 2.3 mL (1.0 M
hexane solution) of LiHMDS and 0.26 mL of CH3I in
63% yield (13, 0.20 g). Mp 34±358C. IR (KBr) nmax 1682,
1
(13C) (570 mg) in 81%. H NMR (CDCl3, 270 MHz), d
4.09 (d, Jˆ151.4 Hz, 2H), 6.96 (dd, Jˆ15.8, 1.0 Hz, 1H),
7.39±7.62 (m, 5H, Ar H), 7.71 (d Jˆ15.8 Hz, 1H,) ppm. 13C
NMR (CDCl3, 67.8 MHz), d p33.1 ppm. EIMS m/z 227
(M1(81Br)), 225 (M1(79Br)), 146 (M12Br). HRMS (EI)
calcd for 13C1C9H9OBr 224.9871 (79Br) and 226.9850
(81Br), found 224.9871 (79Br) and 226.9831 (81Br).
1
1605, 1452, 1363, 1189, 985, 735 cm21. H NMR (CDCl3,
270 MHz), d 2.38 (s, 3H), 6.72 (d, Jˆ16.0 Hz, 1H), 7.40
(brt, Jˆ3.5 Hz, 3H), 7.52 (d, Jˆ16.0 Hz, 1H), 7.55 (brd,
Jˆ9.4 Hz, 2H) ppm. 13C NMR (CDCl3, 67.8 MHz), d
27.5, 127.1, 128.2, 128.9, 130.5, 134.4, 143.4, 198.3 ppm.
EIMS m/z 146 (M1), 131 (M12CH3), 103 (M12CH3CO),
77 (M1269). HRMS (EI) calcd for C10H10O 146.0732,
found 146.0743.
The compound 15 (natural isotope abundance) was synthe-
sized according to the same procedure from 210 mg
(1.1 mmol) of 1-acetoxy-1-cinnnam-ethene (14) in 6.3 mL
of THF, and NBS (330 mg, 1.9 mmol) and water (34 mg,
1.9 mmol) in 75% yield (15, 190 mg). Mp 44±458C. IR
(KBr) nmax 1694, 1613, 1496, 1449, 1388, 1333, 1157,
1-Acetoxy-1-cinnam-ethene 14, 14p (13C)
1
1069, 993, 892, 748, 688, 557 cm2l. H NMR (CDCl3,
To a solution of the cinnam-methylketone (13p) (13C)
(620 mg, 4.2 mmol) in 10 mL of isopropenylacetate was
added a few drops of conc. H2SO4, then the solution was
re¯uxed over 90 min under argon atmosphere. After cooled
270 MHz), d 4.09 (s, 2H), 6.95 (d, Jˆ16.0 Hz, 1H), 7.38±
7.45 (brdd Jˆ5.4, 1.5 Hz, 3H,), 7.55±7.61 (brdd, Jˆ6.4,
2.0 Hz, 2H), 7.70 (d, Jˆ16.0 Hz, 1H) ppm. 13C NMR
(CDCl3, 67.8 MHz), d 33.0, 122.2, 128.6, 129.0, 131.1,

M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
2637
134.0, 145.4, 191.0 ppm. EIMS m/z 226 (M1(81Br)), 224
(M1(79Br)), 145 (M12Br). Anal, calcd for C10H9OBr; C,
53.36; H, 4.03%. Found C, 53.26; H, 4.02%.
was chromatographed on silica gel with CH2Cl2 as an
eluent. 4-methoxyphenyl-cinnnamyl ketone 17p (13C) was
given in 80% (440 mg). H NMR (CDCl3, 270 MHz), d
1
3.80 (s, 3H), 3.87 (d, Jˆ127.6 Hz, 2H), 6.77 (d
Jˆ16.3 Hz, 1H,), 6.88 (d, Jˆ8.4 Hz, 2H), 7.18 (dd, Jˆ8.9,
4.0 Hz, 2H) 7.33±7.54 (m, 5H, Ar H), 7.60 (d, Jˆ16.3 Hz,
1H) ppm. 13C NMR (CDCl3, 67.8 MHz), d p47.6 ppm.
EIMS m/z 253 (M1), 131, 122, 103, 77. HRMS (EI) calcd
for 13C1C16H16O2 253.1184, found 253.1172. Isomer 24p
2-Bromo-1-cinnnam-1-(p-methoxyphenyl)-ethanol 16,
16p (13C)
To a solution of bromomethyl-cinnamketone (15p) (13C)
(540 mg, 2.4 mmol) in THF (18 mL) was added dropwise
over a few minutes 1.4 M p-methoxyphenylmagnesium
bromide freshly prepared in diethyl ether solution11
(1.9 mL, 2.6 mmol) under argon atmosphere at 2408C.
After being stirred for 20 min at 2408C, the reaction
mixture was poured into sat. NH4C1 aq. at 08C. The result-
ing mixture was extracted with diethyl ether (£3) and the
combined organic layers were washed with water (£1) and
sat. NaCl aq. (£1). After being dried over anhydrous
Na2SO4, the organic layer was evaporated under reduced
pressure, then the crude products obtained. They were puri-
®ed by using column chromatography on silica gel with
diethyl ether±n-hexane (1:3), the product 16p (13C) was
given in pure state (60%, 500 mg). 1H NMR (CDCl3,
270 MHz), d 2.79 (d, Jˆ2.0 Hz, 1H), 3.81 (s, 3H), 3.86
(dd, Jˆ153.5, 2.0 Hz, 2H), 6.48 (dd, Jˆ15.8, 3.0 Hz, 1H),
6.71 (d, Jˆ15.8 Hz, 1H), 6.91 (d Jˆ8.9 Hz, 2H) 7.21±7.40
(m, 5H, Ar H), 7.44 (d, Jˆ8.9 Hz, 2H) ppm. 13C NMR
1
(13C) in 20% (220 mg). H NMR (CDCl3, 270 MHz), d
3.85 (dd, Jˆ127.1, 5.4 Hz, 2H), 3.87 (s, 3H), 6.50 (m,
2H), 6.95 (d, Jˆ8.9 Hz, 2H), 7.20±7.45 (m, 5H, Ar H),
8.00 (d, Jˆ8.9 Hz, 2H) ppm. 13C NMR (CDCl3,
p
67.8 MHz), d 42.5 ppm. EIMS m/z 253 (M1), 131, 118.
HRMS (EI) calcd for 13C1C16H16O2 253.1184, found
253.1156.
The compound 17 (natural isotope abundance) was synthe-
sized according to the same procedure from 750 mg
(2.2 mmol) of bromohydrin (16) in 750 mL of CH2Cl2,
and silver carbonate (720 mg, 2.5 mmol) in 70% yield
(17, 400 mg). Mp 79±808C. IR (KBr) nmax 2364, 1665,
1617, 1513, 1341, 1247, 1177, 1034, 805, 750, 691 cm21
.
1H NMR (CDCl3, 270 MHz), d 3.80 (s, 3H), 3.88 (s, 2H),
6.77 (d, Jˆ16.0 Hz, 1H), 6.89 (d, Jˆ8.9 Hz, 2H), 7.19 (d,
Jˆ8.9 Hz, 2H) 7.37±7.59 (m, 5H, Ar H), 7.62, (d,
Jˆ16.0 Hz, 1H) ppm. 13C NMR (CDCl3, 67.8 MHz), d
47.5, 55.2, 114.2, 125.1, 126.3, 128.3, 128.9, 130.4, 130.5,
134.4, 143.2, 158.6, 197.6 ppm. EIMS m/z 252 (M1), 131,
121, 103. Anal, calcd for C17H16O2; C, 80.93; H, 6.39%.
Found C,80.49; H, 6.39%. Isomer 24 (natural abundance
13C) in 30% (171 mg). Mp 109±1108C. IR (KBr) nmax
(CDC13, 67.8 MHz),
d
p44.6 ppm. EIMS m/z 335
(M1(81Br)), 333 (M1(79Br)), 254 (M12Br). HRMS (EI)
calcd for 13C1C16H17O2Br 333.0446 (79Br) and 335.0426
(81Br), found 333.0460 (79Br) and 335.0426 (81Br).
The compound 16 (natural isotope abundance) was synthe-
sized according to the same procedure from 660 mg
(3.0 mmol) of bromomethyl-cinnamketone (15) in 20 mL
of diethyl ether, and 1.4 M p-methoxyphenylmagnesium
bromide in diethyl ether solution (2.2 mL, 3.0 mmol) in
76% yield (16, 750 mg). IR (CHCl3) nmax 3024, 2942,
1609, 1511, 1461, 1419, 1304, 1250, 1180, 1034, 971,
2362, 1679,1598, 1256, 1215, 1172, 1027, 985, 690 cm21
.
1H NMR (CDCl3, 270 MHz), d 3.87 (d, Jˆ5.4 Hz, 2H), 3.88
(s, 3H), 6.46 (dt, Jˆ15.8, 6,4 Hz, 1H), 6.55 (d, Jˆ15.8 Hz,
1H), 6.96 (d, Jˆ8.9 Hz, 2H), 7.21±7.40 (m, 5H, Ar H), 8.00
(d, Jˆ8.9 Hz, 2H) ppm. 13C NMR (CDCl3, 67.8 MHz), d
42.5, 55.5, 113.8, 123.0, 126.2, 127.4, 128.5, 129.6, 130.6,
133.3, 137.0 163.5, 196.6 ppm. EIMS m/z 252 (M1), 135,
107. Anal, calcd for C17H16O2; C, 80.93; H, 6.39%. Found
C,80.79; H, 6.38%.
1
832 cm21. H NMR (CDCl3, 500 MHz), d 2.78 (s, 1H),
3.81 (s, 3H), 3.85 (d, Jˆ4.9 Hz, 2H), 6.47 (d, Jˆ16.0 Hz,
1H), 6.70 (d, Jˆ16.0 Hz, 1H), 6.91 (d, Jˆ8.8 Hz, 2H) 7.24
(t, Jˆ5.4 Hz, 1H), 7.31 (t, Jˆ7.8 Hz, 2H), 7.39 (d,
Jˆ7.3 Hz, 2H), 7.43 (d, Jˆ8.8 Hz, 2H) ppm. 13C NMR
(CDC13, 67.8 MHz), d 44.6, 55.3, 75.3, 113.9, 126.7,
127.0, 128.0, 128.6, 130.8, 132.0, 134.5, 136.2,
159.2 ppm. EIMS m/z 334 (M1(81Br)), 332 (M1(79Br)),
253 (M12Br). HRMS (El) calcd for C17H17O2Br
332.0412 (79Br) and 334.0393 (81Br), found 332.0422
(79Br) and 334.0373 (81Br).
Cinnam-( p-methoxyphenyl)-1,3-dioxolane 18. 18p (13C)
A mixture of 59 mg (0.23 mmol) of cinnnam-(p-methoxy-
phenyl)-ketone (17p) (13C), 2 mg of p-toluenesulfonic acid,
0.2 mL of ethylene glycol, 0.2 mL of 2-ethyl-2-methyl-1,3-
dioxolane, and 5.0 mL of dry benzene was heated at re¯ux
for 4.5 h in an apparatus equipped with a water separator
(Dean±Stark condenser). After having been cooled, the
reaction mixture was added to 20 mL of saturated sodium
bicarbonate and extracted with AcOEt (£3). The combined
extracts were washed with water and saturated brine, dried
over anhydrous Na2SO4, and evaporated to afford 69 mg
Cinnnam-( p-methoxyphenyl)-ketone 17, 17p (13C)
Silver carbonate (480 mg, 1.6 mmol) was added to a
solution of bromohydrin (16p) (13C) (500 mg, 1.5 mmol) in
CH2Cl2 (500 mL) at 08C. Then the reaction mixture was
allowed to warm to room temperature, and stirred overnight
at room temperature. The reaction mixture was ®ltered
through a pad of Celite and concentrated under reduced
pressure to a volume of ca. 20 mL. The solution was diluted
with AcOEt (100 mL) and the organic layer washed with
sat. NaHCO3 aq.(£1), water (£1), and brine (£1). After
dried over anhydrous Na2SO4, the organic layer was evapo-
rated under reduced pressure to give a crude oil. The residue
1
(quantitative yield) of 18p (13C) in almost pure state. H
NMR (CDCl3, 270 MHz), d 3.03 (d, 2H, Jˆ128.1 Hz),
3.77 (s, 3H), 3.79±3.88 (m, 4H), 6.11 (d, Jˆ15.8 Hz, 1H),
6.65 (d, Jˆ15.8 Hz, 1H), 6.81 (d, Jˆ8.9 Hz, 2H), 7.21 (dd,
Jˆ8.9, 5.1 Hz, 2H), 7.25±7.36 (m, 5H, Ar H) ppm. 13C
NMR (CDCl3, 67.8 MHz), d p44.3 ppm. EIMS m/z 297
(M1), 175, 122, 103. HRMS (EI) calcd for 13C1C18H20O3
297.1446, found 297.1432.

2638
M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
The compound 18 (natural abundance 13C) was synthesized
according to the same procedure described above for 17
(37 mg) in quantitative yield (18, 47 mg). 1H NMR
(CDCl3, 270 MHz), d 3.03 (s, 2H), 3.78 (s, 3H), 3.75±3.91
(m, 4H), 6.11 (d, Jˆ15.8 Hz, 1H), 6.65 (d, Jˆ15.8 Hz, 1H),
6.81 (d, Jˆ8.9 Hz, 2H), 7.20 (d, Jˆ8.9 Hz, 2H), 7.23±7.38
(m, 5H, Ar H) ppm. 13C NMR (CDCl3, 67.8 MHz), d 44.3,
55.2, 64.7, 108.8, 113.3, 126.7, 127.8, 128.0, 128.5, 129.2,
130.2, 131.7, 136.3, 158.3 ppm. EIMS m/z 296 (M1), 175,
121, 103. HRMS (EI) calcd for C19H20O3 296.1412, found
296.1419.
0.02 mmol) in diethyl ether (25 mL) and ethanol (5 ml) at
08C. The mixture was stirred at 08C for 3 h. The solution
was ®ltered through a pad of Celite and then concentrated
under reduced pressure to afford 1bp (13C) (7 mg, 77%). 1H
NMR (CDCl3, 500 MHz), d 3.92 (s, 3H), 4.38 (s, 2H), 6.89
(d, Jˆ8.8 Hz, 2H), 7.03 (d, Jˆ8.8 Hz, 2H), 7.24 (brs, 1H),
7.33 (t, Jˆ7.3 Hz, 2H), 7.50 (d, Jˆ156.0 Hz, 1H), 7.57 (d,
Jˆ7.3 Hz, 2H), 7.65 (s, 1H), 7.72 (d, Jˆ8.8 Hz, 2H), 8.31
(dd Jˆ8.8, 4.7 Hz, 2H,) ppm. 13C NMR (CDCl3,
125.7 MHz), d p134.6 ppm. FABMS (NBA) m/z 437
(MH1). HRMS (FAB/NBA) calcd for 13C1C26H21O3N3
436.1615, found 436.1620 (M1).
2-( p-Methoxybenzyl)-6-( p-methoxyphenyl)-8-benzyl-
3,7-dihydroimidazo[1,2-a]pyrazine-3-one 20, 2p (13C)
The compound 1b (natural isotope abundance) was prepared
according to the same procedure described above for 20 in
94% yield. Mp 198±1998C (decomp.). UV (EtOH) lmax
(log e), 529 (3.77), 402 (4.12), 270 (2.20) nm. IR (KBr)
nmax 3250, 1697, 1586, 1509, 1446, 1404, 1263,
Cinnam-(p-methoxyphenyl)-1,3-dioxolane (18p) (13C)
(48 mg, 0.16 mmol) was dissolved in CH2Cl2 (20 mL) and
the solution was cooled to 2788C. Ozone gas was bubbled
through the reaction mixture until the color of the solution
became blue. Nitrogen gas was bubbled through the solution
to remove excess amount of ozone gas. Then triethylamine
was added and stirred at room temperature for 1 h. The
volatiles were evaporated under reduced pressure and the
resulting residue 19p (13C) (60 mg) was used in the follow-
ing reaction without further puri®cation. A mixture of crude
residue of the ozonolysis products 19p (13C) (60 mg), amino-
pyrazine 5 (90 mg, 0.32 mmol), 10% HC1 (1.2 mL), water
(1.0 mL), and dioxane (5.0 mL) was heated at 808C for
90 min under argon atmosphere. After cooling this mixture
in an ice bath, the reaction mixture was diluted with water
(15 mL) and extracted with AcOEt (20 mL£3). The organic
layer was washed with brine, and dried over anhydrous
Na2SO4, and evaporated to dryness. The residue was
chromatographed on silica gel [MeOH±CH2Cl2 (5:95)]
1
1155 cm21. H NMR (DMSO-d6, 500 MHz), d 3.87 (s,
3H), 4.30 (s, 2H), 6.80 (d, Jˆ8.8 Hz, 2H), 7.13 (d, Jˆ
9.3 Hz, 2H), 7.23 (brt, Jˆ7.3 Hz, 1H), 7.33 (t, Jˆ7.3 Hz,
2H), 7.49 (d, Jˆ6.9 Hz, 2H), 7.51 (s, 1H), 7.76 (d,
Jˆ8.8 Hz, 2H), 7.91 (s, 1H), 8.40 (d, Jˆ8.8 Hz, 2H), 9.61
(s, 1H) ppm. 13C NMR (CDCl3, 125.7 MHz), d 33.3, ppm.
FABMS (NBA) m/z 436 (MH1). HRMS (FAB/NBA) calcd
for C27H21O3N3 435.1:583, found 435.1592 (M1).
DTT adduct 21b, 21bp (13C)
Dithiothreitol (2.0 mg, 0.012 mmol) was added to a solution
of dehydrocoelenterazine mono-methyl ether (1bp) (13C)
(2.7 mg, 0.0062 mmol) in MeOH (1.5 mL) and CH2Cl2
(1.5 mL) at room temperature under argon atmosphere.
After stirring for 20 min at room temperature, the reaction
solution was adjusted to pH 3 by using 1N HCl aq. A few
minutes later, the reaction mixture was concentrated under
reduced pressure to give a DTT adduct 21bp (13C). The
adduct was puri®ed using reversed-phase HPLC (ODS-
1
afford 20p (13C) in 34% yield (24 mg, 3 steps). H NMR
(CD3OD, 500 MHz), d 3.72 (s, 3H), 4.09 (d, Jˆ128.4 Hz,
2H), 4.29 (s, 2H), 4.38 (s, 2H), 6.81 (d, Jˆ8.8 Hz, 2H), 6.86
(d, Jˆ8.3 Hz, 2H), 7.23 (t, Jˆ7.4 Hz, 2H), 7.36 (d,
Jˆ7.4 Hz, 1H), 7.56 (t, Jˆ7.4 Hz, 2H), 8.00 (d, Jˆ8.3 Hz,
2H), 8.01 (d, Jˆ8.3 Hz, 2H) ppm. 13C NMR (CD3OD,
125.7 MHz), d p33.4 ppm. FABMS (NBA) m/z 439
(MH1). HRMS (FAB/NBA) calcd for 13C1C26H23O3N3
438.1771, found 438.1771 (M1).
1
UG-5 column, mobile phase; MeCN:H2Oˆ1:1) H NMR
of imidazopyrazinone core (CD3OD, 500 MHz), d 4.67 (s,
2H), 5.62 (d, Jˆ141.1 Hz, 0.5H), 5.67 (d, Jˆ141.1 Hz,
0.5H), 6.90 (d, Jˆ8.8 Hz, 2H), 6.94 (d, Jˆ8.8 Hz, 2H),
7.26±7.34 (m, 4H), 7.49 (d, Jˆ7.4 Hz, 2H), 7.54±7.58
(m, 2H), 7.62 (d, Jˆ8.8 Hz, 2H) ppm. 13C NMR (CD3OD,
p
p
The compound 20 (natural isotope abundance) was synthe-
sized according to the same procedure described above in
67% yield (3 steps from 18). UV (EtOH); lmax (log e), 434,
349, 265 nm. FL (EtOH) Em l 528 nm, Ex l 419 nm. IR
(CHCl3) nmax 3697, 3171, 2950, 2362, 1611, 1513, 1242,
1216, 1178, 838 cm21. 1H NMR (CDCl3, 500 MHz), d 3.61
(s, 3H), 4.03 (s, 2H), 4.29 (s, 2H), 6.71 (d, Jˆ9.0 Hz, 2H),
6.77 (d, Jˆ8.5 Hz, 2H), 7.10 (brt, Jˆ7.5 Hz, 1H), 7.17 (brt,
Jˆ7.5 Hz, 5H), 7.23 (brd, Jˆ7.5 Hz, 4H) ppm. 13C NMR
(CDCl3, 125.7 MHz), d 33.3, 35.0, 55.7, 107.8, 114.8,114.9,
11639. 124.8, 128.2, 129.3, 129.4 129.6, 130.6, 130.8,
131.3, 131.9, 133.5, 138.1, 159.8, 160.2 ppm. FABMS
(NBA) m/z 438 (MH1). HRMS (FAB/NBA) calcd for
C27H23O3N3 437.1739, found 437.1716 (M1).
125.7 MHz), d 45.2, 45.4 ppm.
The compound 21b (natural isotope abundance) was
prepared according to the same procedure described above
from dehydrocoelenterazine mono-methyl ether (1b)
(3.0 mg, 0.0069 mmol), DTT (1.8 mg, 0.012 mmol),
MeOH (1.5 mL), and CH2Cl2 (1.5 mL). FABMS (NBA)
m/z 590 (MH1).
Glutathione adduct 23b, 23bp (13C)
The solution of GSH (3.0 mg, 0.0046 mmol) in water
(0.5 mL) was added to a solution of dehydrocoelenterazine
1bp (13C) (2.7 mg, 0.0062 mmol) in MeOH (1.0 mL) at
room temperature. After stirring for 50 min at room
temperature, the reaction solution was adjusted to pH 3
by using 1N HCl aq. A few minutes later, the reaction
mixture was concentrated under reduced pressure to give
a GSH adduct (23bp) (13C). The adduct was puri®ed with
Dehydrocoelenterazine mono-methyl ether 1b, 1bp (13C)
Manganese (II) oxide (110 mg, 1.3 mmol) was added to the
solution of methoxy coelenterazine 20p (13C) (9.0 mg,

M. Kuse, M. Isobe / Tetrahedron 56 (2000) 2629±2639
2639
2. (a) Isobe, M.; Fujii, T.; Swan, S.; Kuse, M.; Tsuboi, K.;
Miyazaki, A.; Feng, M. C.; Li, J. Pure Appl. Chem. 1998, 70,
2085±2092. (b) Isobe, M.; Takahashi, H.; Usami, K.; Hattori,
M.; Nishigohri, Y. Pure Appl. Chem. 1994, 66, 765±772.
(c) Usami, K.; Isobe, M. Tetrahedron 1996, 52, 12061±12090.
(d) Usami, K.; Isobe, M. Tetrahedron Lett. 1995, 36, 8613±8616.
3. Tsuji, F. I.; Leisman, G. Proc. Natl. Acad. Sci. USA 1981, 78,
6719±6723.
reverse-phased HPLC (ODS-UG-5 column, mobile phase;
MeCN:H2Oˆ3:7). 1H NMR of imidazopyrazinone core
(CD3OD, 500 MHz), d 5.66 (d, Jˆ141.1 Hz, 0.5H), 5.69
(d, Jˆ141.1 Hz, 0.5H), 6.90 (dd, Jˆ8.8, 3.9 Hz, 2H), 6.95
(d, Jˆ8.8 Hz, 2H), 7.25±7.34 (m, 4H), 7.50 (t, Jˆ7.4 Hz,
2H), 7.55 (dt, Jˆ8.8, 2.9 Hz, 2H), 7.63 (dd, Jˆ8.8, 2.9 Hz,
2H) ppm. 13C NMR (CD3OD, 125.7 MHz), d p44.6,
p45.3 ppm.
4. (a) Shimomura, O.; Johnson, F. H.; Saiga, Y. J. Cell. Comp.
Physiol. 1962, 59, 223±240. (b) Shimomura, O.; Johnson, F. H.
Proc. Natl. Acad. Sci. USA 1978, 75, 2611±2615.
The compound 23b (natural isotope abundance) was
prepared according to the same procedure described above
from dehydrocoelenterazine mono-methyl ether (1b) and
GSH. FABMS (NBA) m/z 743 (MH1), 436 (M12GSH),
308 (M12DCL).
5. Takahashi, H.; Isobe, M. BioMed. Chem. Lett. 1993, 3, 2647±
2652.
6. Takahashi, H.; Isobe, M. Chem. Lett. 1994, 843±846.
7. (a) Goto, T.; Iio, H.; Inoue, S.; Kakoi, H. Tetrahedron Lett.
1974, 2321±2324. (b) Inoue, S.; Taniguchi, H.; Murata, M.;
Kakoi, H.; Goto, T. Chem. Lett. 1977, 259±262.
Chemiluminescence of DTT adduct 21b, 21bp (13C)
Through a solution of 21bp (13C) (ca. 1 mg.) in DMSO
(1.0 mL) containing 0.10 mL of 1N t-BuOK in t-BuOH,
molecular oxygen was bubbled. After 25 min later, the
mixture was neutralized with sat. NH4Cl aq., then diluted
with water (5 mL). The solution was extracted with AcOEt
(20 mL£3) the organic lever was washed with water (£1)
and brine (£1). The solution was dried over anhydrous
Na2SO4, then concentrated by evaporating under reduced
pressure. The resultant residue (22b) was analyzed by
NMR without further puri®cation. 13C NMR (CD3OD,
8. Recently novel synthetic routes to coelenterazines were
reported in the following papers. (a) Keenan, M.; Jones, K.;
Hibbert, F. Chem. Commun. 1997, 323±324. (b) Hirano, T.;
Negishi, R.; Yamaguchi, M.; Chen, F. Q.; Ohmiya, Y.; Tsuji,
F. I.; Ohashi, M. Tetrahedron 1997, 53, 12 903±12 916.
9. Kishi, Y.; Tanino, H.; Goto, T. Tetrahedron Lett. 1972, 2747,
2750.
10. Gassman, P. G.; Talley, J. J. Tetrahedron Lett. 1978, 3773±
3776. Trimethylsilyl cyanide was prepared according to the
method described in Ref. 2c.
p
125.7 MHz), d 42.5, p43.2 ppm.
11. The Grignard reagent was prepared from bromoanisole and
magnesium turnings in diethyl ether.
12. Compound 24p was obtained as the isomeric product in
the rearrangement reaction in 20% yield from bromohydrin 16p.
The spectroscopic data of 24 and 24p were described in detail in the
experimental section of this paper.
The compound 22b (natural isotope abundance) was
prepared according to the same procedure described
above. FABMS (NBA) m/z 577 (M1).
Acknowledgements
The authors are grateful for ®nancial support from JSPS±
RFTF 96L00504. They also thank Dr T. Kondo and Mr K.
Koga for technical assistance in NMR measurements. M.
Kuse thanks JSPS (Japan Society for the Promotion of
Science) for granting a Research Fellowship for Young
Scientists. We also express our thanks to Dr T. Franz for
assistance in checking the manuscript.
References
1. Preliminary communication about this paper was reported in the
next literature. Isobe, M.; Kuse, M.; Yasuda, Y.; Takahashi, H.
BioMed. Chem. Lett. 1998, 8, 2919±2924.
13. Isobe, M.; Iio, H.; Kawai, T.; Goto, T. Tetrahedron Lett. 1977,
703±706. (b) Iio, H.; Isobe, M.; Kawai, T.; Goto, T. Tetrahedron
1979, 35, 941±948.