Solvent and substituent effects on the fluorescent properties of coelenteramide analogues

Article abstract of DOI:10.1039/a701156c

Coelenteramide 1 is the light emitter in aequorin bioluminescence. To establish the fluorescent character of 1, the fluorescence properties of 1 and a series of its analogues, 3a-f, possessing a substituent R [= CF₃, F, H, OCH₃, OH, N(CH₃)₂] at the para-position on the 5-phenyl group have been investigated in solvents of various polarity. The fluorescence emission maxima of 1 and 3d-f, possessing an electron-donating group R [= OCH₃, OH, N(CH₃)₂] shift to lower energy with increasing solvent polarity, while those of the analogues 3a-c (R = CF₃, F, H) are independent of the solvent polarity. The linear correlation between the fluorescence maxima of 1 and 3d-f and the solvent polarity scales can be explained by formation of the singlet excited state with a charge-transfer (CT) character. The quantum yields of CT fluorescence of 1 and 3d-f have been found to be higher than those of 3a-c. These results indicate that the solvatochromic fluorescence of 1 originates from the CT excited state and the existence of an electron donating hydroxy group on the 5-phenyl group is essential for determining a wavelength and a high fluorescence quantum yield of aequorin bioluminescence.

Full text of DOI:10.1039/a701156c

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Solvent and substituent effects on the fluorescent properties of
coelenteramide analogues
Ryota Saito, Takashi Hirano,* Haruki Niwa and Mamoru Ohashi
Department of Applied Physics and Chemistry, The University of Electro-Communications,
Chofu, Tokyo 182, Japan
Coelenteramide 1 is the light emitter in aequorin bioluminescence. To establish the fluorescent character
of 1, the fluorescence properties of 1 and a series of its analogues, 3a–f, possessing a substituent R [= CF 3,
F, H , OCH 3, OH , N (CH 3)2] at the para-position on the 5-phenyl group have been investigated in solvents
of various polarity. The fluorescence emission maxima of 1 and 3d–f, possessing an electron-donating
group R [= OCH 3, OH , N (CH 3)2] shift to lower energy with increasing solvent polarity, while those of the
analogues 3a–c (R = CF 3, F, H ) are independent of the solvent polarity. The linear correlation between
the fluorescence maxima of 1 and 3d–f and the solvent polarity scales can be explained by formation of the
singlet excited state with a charge-transfer (CT) character. The quantum yields of CT fluorescence of 1
and 3d–f have been found to be higher than those of 3a–c. These results indicate that the solvatochromic
fluorescence of 1 originates from the CT excited state and the existence of an electron donating hydroxy
group on the 5-phenyl group is essential for determining a wavelength and a high fluorescence quantum
yield of aequorin bioluminescence.
O
Introduction
A photoprotein, aequorin (AQ) isolated from the jellyfish
N
N
NH
OH
Aequorea victoria, emits blue light by a reaction with calcium
ions.1 AQ is composed of apoaequorin (an apoprotein), coel-
enterazine and a molecular oxygen.2 The binding of calcium
ions to AQ triggers the transformation of AQ into an excited
state of a blue fluorescent protein (BFP), resulting in emission
of blue light (λmax = 465 nm). BFP consists of coelenteramide
1 bound to apoaequorin noncovalently. Coelenteramide 1 is an
oxidation product from coelenterazine (Scheme 1). The singlet
excited state of 1 in BFP is the light emitter in the biolumin-
escent reaction.3 Coelenteramide 1 has two phenolic hydroxy
groups and an amide group, and can exist as the ionic structures
II and III as well as the neutral structure I(1) under appropriate
HO
I
O
N
N
OH
N
HO
Ca2+
CO2 Light (λmax = 465 nm)
+
Aequorin
+
Blue Fluorescent Protein
II
O
O
N
N
N
N
NH
OH
OH
N
H
HO
O
O2
O
– CO2
Coelenterazine
III
N
N
NH
OH
O
N
N
N
CH3
OH
HO
HO
Coelenteramide (1)
Scheme 1
2
conditions.4,5 From the results of studies on a chemilumin-
escence reaction of coelenterazine derivatives in an aprotic
solvent, it has been suggested that the structure of the excited
coelenteramide 1 in aequorin bioluminescence is the amide
anion II.6 However, we proposed that the emitting structure
is the phenolate anion III on the basis of studies on the
fluorescence properties of a semi-synthetic BFP regenerated
J. Chem. Soc., Perkin Trans. 2, 1997
1711

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from apoaequorin and the N-methylcoelenteramide analogue
2.5 To confirm this hypothesis, we have been studying system-
atically the fluorescent properties of 1 and its derivatives under
various conditions. For the neutral structure I, Shimomura
has shown that the fluorescence maxima of 1 is changed with
a change in solvent polarity,7 in a similar manner to the
fluorescence spectra of Cypridina (Vargula) oxyluciferin.8 This
paper deals with our studies of the solvent and substituent
effects on the fluorescence character of the neutral structure, 1,
by comparison with the fluorescence spectra of coelenteramide
analogues 3a–f, possessing a substituent at the para-position on
the 5-phenyl group. From these results, it is clear that the
solvent-dependent fluorescence of the neutral structure I has
charge transfer (CT) character in the singlet excited state.
CN
NOH
O
H2N
+
R
4a : R = CF3
5
4b : R = F
4c : R = H
i
4d : R = OCH3
4f : R = N(CH3)2
O–
N+
N
N
NH2
NH2
O
CH3
ii
N
N
N
NH
R
R
5
6a : R = CF3
6b : R = F
6c : R = H
R
7a : R = CF3
7b : R = F
7c : R = H
3a : R = CF3
3b : R = F
6d : R = OCH3
7d : R = OCH3
7e : R = OH
6f : R = N(CH3)2
iii
3c : R = H
7f : R = N(CH3)2
3d : R = OCH3
3e : R = OH
3f : R = N(CH3)2
iv
O
CH3
Results and discussion
N
N
NH
Coelenteramide 1 was prepared by acylation of coelenteramine
7e using a previously reported procedure.3,9 Coelenteramide
analogues 3a–f were synthesized by acetylation of the corre-
sponding coelenteramine analogues 7a–f, which were prepared
by the coupling of oximino ketone 4a–d, f and α-amino nitrile 5
followed by reduction with Raney Ni, as illustrated in Scheme
2.9–12
The UV–VIS absorption spectra of 1 and 3a–f in various
solvents are summarized in Table 1. Each compound showed
several bands in the region of 250–370 nm. The lowest energy
bands of 1 and 3a–f were virtually independent of solvent
polarity, suggesting that the lowest energy transition does not
have a charge transfer (CT) character.
R
3a : R = CF3
3b : R = F
3c : R = H
3d : R = OCH3
3e : R = OH
3f : R = N(CH3)2
In contrast to the absorption spectra, the fluorescence spec-
tra of 1 were dependent on solvent polarity as shown in Fig. 1.
The fluorescence spectra of selected coelenteramide analogues
3a, 3d and 3f in various solvents are also depicted in Fig. 1. The
fluorescence maxima of 1 and 3a–f are summarized in Table 2,
accompanied by the quantum yields. Coelenteramide 1 showed
a bathochromic shift of the fluorescence maxima with increas-
ing solvent polarity. This result reproduces the fluorescent
character of 1 reported by Shimomura.7 Similarly, the fluor-
escence maxima of 3d–f, which possess an electron donating
substituent R [R = OCH3, OH, N(CH3)2], showed a solvent-
dependent bathochromic shift, while 3a–c showed negligible
solvent dependency. The fluorescence maxima of 3e matched
exactly those of 1. The solvatochromic fluorescence of 1 and
3d–f is a typical characteristic of fluorescent compounds which
consist of an electron-donating moiety and an electron-
accepting moiety, such as 2(arylamino)naphthalene-6-sulfon-
ates (ANS)13 and N-phenyl-4-methylenepiperidine derivatives.14
These donor–acceptor compounds have a CT state, labeled
S1,ct, as the lowest excited state, whose stability is dependent on
solvent polarity.
Scheme 2 Reagents and conditions: (i) TiCl4, pyridine, 80–85 ЊC, 2–3 h;
(ii) Raney–Ni (W-2), EtOH, reflux, 1.5 h; (iii) pyridinium chloride,
200 ЊC, 40 min; (iv) AcCl, pyridine, CHCl3, room temp., 30 min
were correlated with the ET(30) solvent polarity scale, which is
used to evaluate the solvatochromism of electronic transitions
with CT character,15 as shown in Fig. 2. For all three com-
pounds, a good proportional relationship between EF and
ET(30) was observed. The regression lines of 1 and 3d–f support
the proposal that their excited states are CT states. The slopes
for 1 and 3d–f were estimated as 0.31, 0.24, 0.28 and 0.63,
respectively. The slope value increases with an increasing
electron-donating ability of the substituent R. In particular,
the large gradient of 3f indicates that the CT excited state of 3f
is highly polar, similar to that of 2-[(2,6-dimethylphenyl)-
amino]naphthalene-6-sulfonate, whose slope was reported as
0.62 obtained from the EF –ET(30) plot in a dioxane–water
solvent system.16
In contrast to 1 and 3d–f, analogues 3a–c showed no solvato-
chromic fluorescence. To evaluate the substituent effect, the
fluorescence emission energies EF of 3a–f in cyclohexane,
chloroform and propan-2-ol were plotted as a function of the
In order to obtain a quantitative evaluation of the solvent
effect, the fluorescence emission energies EF of 1, 3d and 3f
1712
J. Chem. Soc., Perkin Trans. 2, 1997

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Table 1 Electronic absorption data of coelenteramide 1 and its analogues 3a–f in various solvents
Absorption maximum/nm (ε/104 dm3 molϪ1 cmϪ1
)
Solvents
1
3a
3b
3c
3d
3e
3f
Cyclohexane
332 (1.70)
291 (1.80)
277 (1.80)
333 (1.63)
294 (1.66)
279 (1.69)
333 (1.68)
294 (1.78)
277 (1.81)
319 (1.41)
292 (1.52)
266 (1.26)
320 (1.39)
292 (1.44)
321 (1.48)
287 (1.57)
260 (1.71)
323 (1.25)
288 (1.29)
321 (1.26)
288 (1.27)
262 (1.40)
322 (1.31)
292 (1.24)
330 (1.61)
294 (1.78)
275 (1.74)
332 (1.63)
295 (1.71)
278 (1.60)
331 (1.62)
294 (1.70)
274 (1.60)
333 (1.63)
295 (1.84)
277 (1.75)
334 (1.61)
295 (1.72)
280 (1.59)
333 (1.64)
295 (1.78)
276 (1.67)
355 (2.11)
323 (1.99)
266 (0.91)
364 (2.15)
331 (1.90)
Benzene
Diglyme
318 (1.36)
304 (sh, 1.37)
292 (1.46)
321 (1.43)
286 (1.42)
258 (1.66)
320 (1.24)
291 (1.19)
260 (1.35)
360 (2.06)
328 (1.87)
266 (0.84)
262 (1.21)
Chloroform
DMSO
333 (1.59)
294 (1.61)
278 (1.67)
336 (1.63)
295 (1.73)
280 (1.75)
319 (1.38)
294 (1.39)
262 (1.34)
320 (sh, 1.26)
305 (sh, 1.32)
295 (1.37)
322 (1.49)
287 (1.44)
260 (1.71)
321 (1.34)
288 (1.33)
264 (1.39)
321 (1.27)
291 (1.16)
261 (1.43)
321 (1.20)
294 (1.15)
261 (1.31)
332 (1.60)
295 (1.61)
278 (1.57)
333 (1.55)
295 (1.56)
275 (1.49)
333 (1.63)
295 (1.69)
277 (1.65)
336 (1.63)
297 (1.74)
279 (1.60)
365 (1.96)
331 (1.79)
268 (0.91)
366 (2.01)
333 (1.81)
272 (0.80)
262 (1.17)
Acetonitrile
Propan-2-ol
Methanol
329 (1.57)
292 (1.66)
274 (1.72)
316 (sh, 1.29)
303 (sh, 1.35)
292 (1.40)
317 (1.38)
285 (1.41)
255 (1.70)
316 (1.17)
291 (1.14)
256 (1.38)
328 (1.53)
294 (1.55)
274 (1.53)
329 (1.55)
293 (1.66)
273 (1.62)
359 (2.02)
331 (1.85)
266 (0.89)
257 (1.28)
333 (1.67)
294 (1.77)
278 (1.75)
315 (sh, 1.28)
301 (sh, 1.39)
293 (1.46)
317 (1.41)
285 (1.43)
256 (1.68)
317 (1.21)
292 (1.20)
257 (1.40)
329 (1.59)
294 (1.64)
274 (1.52)
333 (1.65)
295 (1.77)
277 (1.60)
361 (1.99)
329 (1.85)
267 (0.92)
258 (1.27)
332 (1.61)
293 (1.67)
277 (1.69)
314 (sh, 1.30)
301 (sh, 1.41)
293 (1.46)
316 (1.40)
285 (1.37)
255 (1.68)
316 (1.18)
292 (1.13)
257 (1.37)
330 (1.57)
329 (1.58)
275 (1.50)
332 (1.61)
293 (1.67)
277 (1.56)
363 (1.96)
330 (1.79)
267 (0.92)
255 (1.34)
Fig. 1 Fluorescence spectra of coelenteramide 1 and its selected analogues 3a, 3d and 3f in solvents of different polarities: (a) in cyclohexane, (b) in
benzene, (c) in diglyme, (d) in chloroform, (e) in DMSO, ( f ) in acetonitrile, (g) in propan-2-ol and (h) in methanol
Hammett σp substituent constant of a substituent R,17 as shown
in Fig. 3.16 For each solvent two lines were plotted. The high
slope lines are made by 3d–f, whose excited states are CT states,
S1,ct, and the horizontal lines are made by 3a–c. The lowest
excited states of 3a–c, whose stability is insensitive to a solvent
polarity, are assigned as the locally excited states S1. The
polarity of the S1 state of 3a–c is modestly different from
that of the ground state.
The difference in character between S1 and S1,ct in the coel-
enteramide analogues was also distinguished by the fluor-
escence quantum yields ΦF compiled in Table 2. The ΦF values
of 3a–c are smaller than 0.15 and are insensitive to solvent
polarity. On the other hand, the ΦF values of 1 and 3d–f are
larger than 0.28 in an aprotic solvent. In propan-2-ol and
methanol, 3d maintained its high ΦF values, while the ΦF values
of 1, 3e and 3f were smaller than those in the aprotic solvents.
This result suggests that a specific interaction between the solv-
ating alcohol and a hydroxy group (or dimethylamino group) of
1 and 3e (or 3f) quenches the emission process from the S1,ct
state. The decrease of the ΦF value of 1, 3e and 3f in a protic
solvent is a characteristic similar to those of the CT fluorescent
compounds, such as the ANS derivatives18 and the N-phenyl-4-
methylenepiperidines.14 One explanation of this quenching pro-
cess was given by Kosower, who elucidated that the fluorescence
quenching of an ANS derivative in polar protic solvents results
from the promotion of nonradiative electron-transfer by the
solvent.18
In conclusion, solvatochromic fluorescence of coelenter-
amide 1 and its analogues 3d–f is emitted from the CT excited
state S1,ct. On the other hand, fluorescence of 3a–c from the S1
state is insensitive to solvent polarity. The S1,ct state is stabilized
by the electron donating character of the para-substituted
phenyl group at the 5-position of coelenteramide 1. The elec-
tron donation from R [R = OCH3, OH and N(CH3)2] is enough
to stabilize the S1,ct state more than the S1 state. The CT fluor-
escence of 1 and 3d–f influences the value (ΦF > 0.28) of the
J. Chem. Soc., Perkin Trans. 2, 1997
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Table 2 Fluorescence data of coelenteramide 1 and its analogues 3a–f in various solvents
Fluorescence maximum/nm (quantum yield)
Solvents
1
3a
3b
3c
3d
3e
3f
Cyclohexane
Benzene
Diglyme
Chloroform
DMSO
Acetonitrile
Propan-2-ol
Methanol
383 (0.25)
389 (0.32)
389 (0.33)
403 (0.37)
409 (0.30)
397 (0.30)
418 (0.13)
428 (0.02)
367 (0.067)
369 (0.061)
372 (0.039)
367 (0.080)
376 (0.025)
370 (0.023)
369 (0.032)
371 (0.027)
370 (0.074)
376 (0.13)
370 (0.046)
371 (0.11)
374 (0.044)
370 (0.043)
370 (0.054)
374 (0.061)
369 (0.014)
369 (0.10)
370 (0.067)
370 (0.15)
372 (0.063)
370 (0.062)
369 (0.076)
372 (0.084)
380 (0.30)
387 (0.30)
391 (0.30)
392 (0.44)
396 (0.36)
391 (0.33)
403 (0.44)
420 (0.52)
387 (0.28)
390 (0.33)
390 (0.30)
403 (0.38)
407 (0.30)
396 (0.29)
417 (0.11)
426 (0.02)
438 (0.38)
441 (0.54)
481 (0.33)
473 (0.40)
505 (0.35)
503 (0.33)
525 (0.08)
n.d.a
a
Fluorescence could not be detected.
the phenolate anion III.5 Because the Hammett σp values of OϪ
(σp = Ϫ0.81) and N(CH3)2 (σp = Ϫ0.83) are similar, the fluor-
escent behaviour of III is predicted using that of coelenter-
amide analogue 3f [R = N(CH3)2]. The wavelength region of the
CT fluorescence of 3f contains the bioluminescence maximum,
supporting our hypothesis. Further, we need to evaluate the
influence of the ionic character of III on the fluorescence prop-
erties. To clarify this problem, characterization of the fluor-
escence of the anion species of 1 is now in progress.
To design a coelenterazine derivative with highly efficient bio-
or chemi-luminescence, a CT fluorescent coelenteramide ana-
logue with high fluorescence quantum yield should be con-
sidered as the light emitter. Because the efficiency of a bio- and
chemi-luminescence reaction is a product of three efficiencies,
which are (i) the fraction of reacting molecules that pursue the
correct chemical pathway (Φr), (ii) the efficiency of chemical
generation of a singlet excited state (Φs) and (iii) the fluor-
escence quantum yield of the light emitter (Φfl), it is also neces-
sary to design for increasing Φr and Φs accompanied by Φfl. On
this basis, we have been investigating substituent effects on the
chemiluminescence behaviour of a 7H-imidazo[1,2-a]pyrazin-
3-one derivative and the results will be reported elsewhere.
Our results are also useful as a standard scale of the substitu-
ent effect on the 5-position on the fluorescence emission of
amidopyrazine derivatives. Cypridina oxyluciferin 8 possessing
Fig. 2 Plots of fluorescence emission energies EF (in kcal molϪ1) for
1 (ᮀ), 3d (᭹) and 3f (᭜) against the solvent polarity parameter ET(30)
(in kcal molϪ1
)
O
N
N
NH
H
N
NH2
NH
N
H
8
an indol-3-yl group at the 5-position, for instance, shows a fluor-
escence maximum at 420 nm in diglyme.6c,8,19 This maximum of
8 is longer than that of 3e (R = OH, λmax = 390 nm) and is
shorter than that of 3f [R = N(CH3)2, λmax = 481 nm]. Therefore,
the order of the electron donating ability is estimated as p-
dimethylaminophenyl > indol-3-yl > hydroxyphenyl. This order
matches the order of the ionization potentials of these groups.20
Using the table of Hammett σp values,17 it is predicted that the
fluorescence maximum of 8 matches that of an acetamido-
pyrazine derivative possessing p-hydrazinophenyl group at
the 5-position.
Fig. 3 Plots of fluorescence emission energies EF (in kcal molϪ1) for
3a–f against Hammett substituent constant (a) in cyclohexane, (b) in
chloroform and (c) in propan-2-ol
Experimental
fluorescence quantum yield in an aprotic solvent. Therefore, in
aequorin bioluminescence the CT fluorescent character of co-
elenteramide 1 may have an important role in determining the
emission wavelength and the value of the quantum yield. We
proposed a hypothesis that blue light emission (λmax = 465 nm)
in aequorin bioluminescence originates from the S1,ct state of
All melting points were measured on a Yamato Model MP-21
apparatus and are uncorrected. 1H NMR spectra were recorded
on a 270 MHz JEOL JNM-GX270 spectrometer. Chemical
shifts (δ) are reported in ppm using as an internal standard
tetramethylsilane or an undeuteriated solvent in the deuteriated
1714
J. Chem. Soc., Perkin Trans. 2, 1997

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solvent used. Coupling constants (J ) are given in Hz. IR spectra
were obtained using a JASCO IR-810 spectrophotometer. High
and low resolution electron impact (EI) mass spectra were
measured on a Hitachi Model M-80B mass spectrometer with a
Hitachi M-0101 data system. The ionization energy and the
accelerating voltage were 70 eV and 3 kV, respectively.
mmol), EtOH and Raney Ni (W-2, 6.31 g) was heated to reflux
under H2 atmosphere for 1.5 h. After cooling to room temper-
ature, the reaction mixture was filtered through Celite and the
Celite bed was washed thoroughly with EtOH. The filtrate was
concentrated in vacuo and purified by recrystallization from
methanol to give 7a (280 mg, 83%) as tan needles; mp 154.5–
156 ЊC; δH (CDCl3) 4.21 (s, 2 H), 4.53 (s, 2 H), 7.28–7.37 (m, 5
H), 7.71 (d, J 8.4, 2 H), 8.06 (d, J 8.1, 2 H) and 8.44 (s, 1 H);
νmax(KBr)/cmϪ1 3482, 3294, 3142, 1637, 1463, 1327 and 1112; m/z
(P-EI) (relative intensity) 329 (Mϩ, 100) and 91 (24) [HRMS
(positive EI) Calc. for C18H14F3N3 329.1141. Found 329.1126].
UV–VIS absorption and fluorescence spectrometry
Spectral grade solvents were used for the measurement of
UV–VIS absorption and fluorescence spectra except diglyme.
Diglyme was passed through an alumina column, dried over
CaH2 and purified by distillation under reduced pressure. UV–
VIS absorption spectra were obtained using a Hitachi Model
320 spectrophotometer. Fluorescence spectra were measured
with a Hitachi Model F-4010 fluorescence spectrophotometer
(excitation bandpass, 5 nm; emission bandpass, 5 nm; response,
2 s; scan speed, 60 nm minϪ1) and corrected according to manu-
facturer’s instructions. A solution of coelenteramide 1 or its
analogues 3a–f for fluorescence measurement was prepared by
mixing a stock solution (100 µl) of 2.0 × 10Ϫ5 mol dmϪ3 1 or
3a–f in diglyme and a solvent (2.0 ml) within a quartz cuvette
at room temperature. The fluorescence quantum yields were
determined using quinine sulfate in 0.1  H2SO4 (ΦF = 0.55,
excitation wavelength: 355 nm, room temperature) as a
standard.
Synthesis of coelenteramine analogues 7b and c
Coelenteramine analogues 7b and c were prepared by an analo-
gous procedure to that for 7a.
2-Amino-3-benzyl-5-(4-fluorophenyl)pyrazine 7b. 72% Yield,
pale yellow needles (from methanol); mp 129.5–130 ЊC;
δH (CDCl3) 4.20 (s, 2 H), 4.66 (s, 2 H), 7.12–7.34 (m, 7 H), 7.89–
7.94 (m, 2 H) and 8.31 (s, 1 H); νmax(KBr)/cmϪ1 3480, 3301,
3142, 1628 and 1461; m/z (P-EI) (relative intensity) 280 (18),
279 (Mϩ, 100) and 278 (54) [HRMS (positive EI) Calc. for
C17H14FN3 279.1172. Found 279.1166].
2-Amino-3-benzyl-5-phenylpyrazine 7c. 77% Yield, pale yel-
low needles (from methanol); mp 138–139 ЊC; δH (CDCl3) 4.24
(s, 2 H), 4.54 (s, 2 H), 7.26–7.49 (m, 8 H) and 7.93–7.95 (m,
2 H); νmax(KBr)/cmϪ1 3484, 3288, 3118, 1634 and 1447; m/z
(P-EI) (relative intensity) 261 (Mϩ, 100) and 260 (68) [HRMS
(positive EI) Calc. for C17H15N3 261.1267. Found 261.1264].
Synthesis of coelenteramide 1, 2-acetamidopyrazine 3e,
2-aminopyrazine 1-oxide 6d and 2-aminopyrazines 7d–f
1, 3e, 6d and 7d–f were synthesized by a previously reported
procedure.3,9–12
Synthesis of 2-acetamido-3-benzyl-5-(4-trifluoromethylphenyl)-
pyrazine 3a
To a solution of 7a (103 mg, 0.31 mmol) and anhydrous pyri-
dine (0.65 ml, 8.04 mmol) in anhydrous CHCl3 (2.0 ml), acetyl
chloride (150 mg, 2.31 mmol) was added at 0 ЊC, giving a color-
less precipitate. After stirring for 30 min at room temperature,
the reaction was quenched by the addition of saturated aqueous
NaHCO3, and extracted three times with CHCl3. The organic
layer was dried over MgSO4, concentrated, and purified by
recrystallization from methanol, affording 3a (88.8 mg, 74%) as
colorless needles; mp 241–242 ЊC; δH ([2H6]DMSO) 2.08 (s, 3 H),
4.22 (s, 2 H), 7.19–7.31 (m, 5 H), 7.88 (d, J 8.4, 2H), 8.30 (d, J
7.9, 2 H), 9.05 (s, 1 H) and 10.38 (s, 1 H); νmax(KBr)/cmϪ1 3442,
3234, 1672, 1493, 1333 and 1117; m/z (P-EI) (relative intensity)
372 (24), 371 (Mϩ, 100), 329 (74), 328 (85), 91 (32), 77 (13), 65
(10) and 43 (93) [HRMS (positive EI) Calc. for C20H16F3N3O
371.1246. Found 371.1231].
Synthesis of 2-amino-3-benzyl-5-(4-trifluoromethylphenyl)-
pyrazine 1-oxide 6a
To a solution of p-trifluoromethyl-2-hydroxyiminoacetophen-
one 4a (1.99 g, 9.36 mmol) and 2-amino-3-phenylpropionitrile
hydrochloride 5 (2.35 g, 13.8 mmol) in dried pyridine (35 ml)
was added dropwise TiCl4 (0.5 ml, 4.56 mmol) in an ice bath
under N2 and the mixture was heated at 82–83 ЊC for 3 h. After
cooling to room temperature, the reaction mixture was filtered
through Celite. The filtrate was concentrated under reduced
pressure and the black residue was partitioned between CH2Cl2
and water. The organic phase was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (1:1 AcOEt–CHCl3) and
recrystallization from methanol, affording 6a (1.15 g, 33%) as
brownish plates; mp 155–157 ЊC; δH (CDCl3) 4.30 (s, 2 H), 5.60
(s, 2 H), 7.29–7.37 (m, 5 H), 7.74 (d, J 8.4, 2 H), 8.20 (d, J 9.2, 2
H) and 8.55 (s, 1 H); νmax(KBr)/cmϪ1 3439, 3282, 3136, 1612,
1482, 1324 and 1116; m/z (P-EI) (relative intensity) 345 (Mϩ, 8),
329 (100) and 91 (29).
Synthesis of coelenteramide analogues 3b–f
Coelenteramide analogues 3b–f were prepared by an analogous
procedure to that for 3a.
2-Acetamido-3-benzyl-5-(4-fluorophenyl)pyrazine 3b. 60%
Yield, colorless needles (from methanol); mp 218–218.5 ЊC;
δH ([2H6]DMSO) 2.06 (s, 3 H), 4.18 (s, 2 H), 7.18–7.39 (m, 5 H),
8.11–8.16 (m, 2 H), 8.95 (s, 1 H) and 10.32 (s, 1 H); νmax(KBr)/
cmϪ1 3444, 3256, 1668 and 1499; m/z (P-EI) (relative intensity)
322 (22), 321 (Mϩ, 100), 279 (78), 278 (77), 134 (19), 120 (23), 91
(20), 77 (10), 65 (6) and 43 (43) [HRMS (positive EI) Calc. for
C19H16FN3O 321.1278. Found 321.1286].
2-Acetamido-3-benzyl-5-phenylpyrazine 3c. 66% Yield, color-
less needles (from methanol); mp 206.5–207 ЊC; δH ([2H6]-
DMSO) 2.07 (s, 2 H), 4.19 (s, 2 H), 7.19–7.31 (m, 5 H), 7.44–
7.55 (m, 3 H), 8.06–8.10 (m, 2 H), 8.94 (s, 1 H) and 10.29 (s, 1
H); νmax(KBr)/cmϪ1 3432, 3278, 1668, 1505 and 1492; m/z (P-EI)
(relative intensity) 304 (24), 303 (Mϩ, 100), 261 (73), 260 (86),
91 (31), 77 (23), 65 (10) and 43 (79) [HRMS (positive EI) Calc.
for C19H17N3O 303.1372. Found 303.1381].
Synthesis of coelenteramine 1-oxide derivatives 6b and c
Coelenteramine 1-oxide derivatives 6b and c were prepared by
an analogous procedure to that for 6a.
2-Amino-3-benzyl-5-(4-fluorophenyl)pyrazine 1-oxide 6b. 48%
Yield, tan needles (from methanol); mp 163.5–164.5 ЊC;
δH (CDCl3) 4.26 (s, 2 H), 5.40 (s, 2 H), 7.13–7.37 (m, 7 H), 7.84–
7.89 (m, 2 H) and 8.39 (s, 1 H); νmax(KBr)/cmϪ1 3310, 3086,
1611, 1599, 1509, 1477, 1343 and 1227; m/z (P-EI) (relative
intensity) 296 (21), 295 (Mϩ, 100), 278 (80) and 91 (48).
2-Amino-3-benzyl-5-phenylpyrazine 1-oxide 6c. 38% Yield,
slightly yellow needles (from methanol); mp 164.5–165.5 ЊC,
δH (CDCl3) 4.27 (s, 2 H), 5.36 (s, 2 H), 7.30–7.52 (m, 8 H), 7.88–
7.91 (m, 2 H) and 8.44 (s, 1 H); νmax(KBr)/cmϪ1 3406, 3266, 3108,
3062, 1618, 1585, 1570, 1479, 1351 and 1128; m/z (P-EI) (rela-
tive intensity) 278 (21), 277 (Mϩ, 100), 261 (42), 260 (99), 233
(34) and 91 (26).
2-Acetamido-3-benzyl-5-(4-methoxyphenyl)pyrazine 3d. 41%
Yield, colorless needles (from methanol); mp 150.5–152 ЊC;
δH ([2H6]DMSO) 2.05 (s, 2 H), 3.82 (s, 3 H), 4.16 (s, 2 H), 7.06 (d,
J 8.9, 2 H), 7.16–7.30 (m, 5 H), 8.04 (d, J 8.9, 2 H), 8.87 (s,
1 H) and 10.24 (s, 1 H); νmax(KBr)/cmϪ1 3450, 3256, 1670, 1496
Synthesis of 2-amino-3-benzyl-5-(4-trifluoromethylphenyl)-
pyrazine 7a
A mixture of the aminopyrazine 1-oxide 6a (354 mg, 1.02
J. Chem. Soc., Perkin Trans. 2, 1997
1715

View Article Online
and 1174; m/z (P-EI) (relative intensity) 334 (24), 333 (Mϩ, 100),
291 (88), 290 (54), 91 (25), 77 (14) and 43 (64) [HRMS (positive
EI) Calc. for C20H19N3O2 333.1478. Found 333.1479].
2-Acetamido-3-benzyl-5-(4-dimethylaminophenyl)pyrazine 3f.
45% Yield, pale yellow needles (from methanol); mp 211.5–
212.5 ЊC; δH ([2H6]DMSO) 2.04 (s, 3 H), 2.97 (s, 6 H), 4.11 (s, 2
H), 6.79 (d, J 8.9, 2 H), 7.19–7.30 (m, 5 H), 7.93 (d, J 8.9, 2 H)
and 10.17 (s, 1 H); νmax(KBr)/cmϪ1 3438, 3248, 1662, 1609,
1529, 1496, 1439 and 1363; m/z (P-EI) (relative intensity) 347
(25), 346 (Mϩ, 100), 305 (22), 304 (93), 303 (26) and 43 (52)
[HRMS (positive EI) Calc. for C21H22N4O 346.1795. Found
346.1778].
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Acknowledgements
The authors are grateful for research support provided by a
Grant-in-Aid for Scientific Research in Priority Areas by the
Ministry of Education, Science, Sports, and Culture of Japan
(No. 06240221 and 06239106).
15 C. Reichardt, Solvents and Solvent Effects in Organic Chemistry,
VCH, Weinheim, 2nd edn., 1988.
16 E. M. Kosower, H. Dodiuk, K. Tanizawa, M. Ottolenghi and
N. Orbach, J. Am. Chem. Soc., 1975, 97, 2167.
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18 E. M. Kosower, Acc. Chem. Res., 1982, 15, 259.
19 Y. Toya, T. Kayano, K. Sato and T. Goto, Bull. Chem. Soc. Jpn.,
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Paper 7/01156C
Received 18th February 1997
Accepted 15th April 1997
1716
J. Chem. Soc., Perkin Trans. 2, 1997