Real light emitter in the bioluminescence of the calcium-activated photoproteins aequorin and obelin: light emission from the singlet-excited state of coelenteramide phenolate anion in a contact ion pair

Article abstract of DOI:10.1016/j.tet.2006.04.044

Fluorescence of the phenolate anion (3(O)^-) and the amide anion (5(N)^-) of coelenteramide analogues in ion pairs with various counter cations was systematically investigated to elucidate the ionic structure of the light emitter in th

Full text of DOI:10.1016/j.tet.2006.04.044

Tetrahedron 62 (2006) 6272–6288
Real light emitter in the bioluminescence of the
calcium-activated photoproteins aequorin and obelin: light
emission from the singlet-excited state of coelenteramide
phenolate anion in a contact ion pair
Kotaro Mori,a Shojiro Maki,a Haruki Niwa,a Hiroshi Ikedab and Takashi Hiranoa,
*
aDepartment of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan
bDepartment of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan
Received 22 March 2006; revised 8 April 2006; accepted 18 April 2006
Available online 19 May 2006
Abstract—Fluorescence of the phenolate anion (3(O)ꢀ) and the amide anion (5(N)ꢀ) of coelenteramide analogues in ion pairs with various
counter cations was systematically investigated to elucidate the ionic structure of the light emitter in the bioluminescence of the calcium-
activated photoproteins aequorin and obelin. The fluorescent properties of 3(O)ꢀ in an ion pair with a conjugate acid of an organic base
(BASE–H+) were varied depending on the structural variation of the ion pair and the solvent polarity. In particular, the fluorescence of
3(O)ꢀ in the ion pair with the conjugate acid of n-butylamine (NBA–H+) indicates that the singlet-excited state of 3(O)ꢀ (13(O)ꢀ*) and
NBA–H+ make a contact ion pair in which the fluorescence emission maxima of 3(O)ꢀ is sensitive to the solvent polarity and the fluorescence
quantum yields of 3(O)ꢀ increase in a less polar solvent. The results also confirm that 13(O)ꢀ* is a twisted intramolecular charge transfer state.
By contrast, the fluorescence of 5(N)ꢀ in an ion pair depends little on the BASE–H+ or the solvent polarity. Based on these results, we conclude
that the light emitter in aequorin and obelin bioluminescences is the singlet-excited state of coelenteramide phenolate anion 2(O)ꢀ (12(O)ꢀ*
)
in a contact ion pair with an imidazolium side chain of a histidine residue, which is located at the less polar active sites of the photoproteins. We
also propose a mechanism for the bioluminescence reaction, including the chemiexcitation process to give 12(O)ꢀ*
Ó 2006 Elsevier Ltd. All rights reserved.
.
1. Introduction
obelin has also yielded important information on the charac-
ter of obelin’s active site.7
Calcium-activated photoproteins are light-generating mole-
cules for several marine bioluminescent organisms.1 The
biochemical aspects of the photoproteins aequorin2,3 and
obelin,4 isolated from the jellyfish Aequorea and Obelia,
respectively, have been particularly well investigated.
Chemical study on calcium-activated photoproteins was pio-
neered by Shimomura et al., who discovered aequorin.2a
Aequorin is made from apoaequorin (apoprotein), coelenter-
azine (1) (substrate), and molecular oxygen (O2). Chelation
of Ca2+ with the EF-hands of aequorin initiates the biolumi-
nescence reaction, which gives a fluorescent protein (FLP),5
CO2, and bluish light (Scheme 1). The crystal structure of
aequorin helped to clarify its supramolecular structure, con-
taining oxygenated coelenterazine in the active site.6 Recent
research on the crystal structures of the various forms of
Ca2+
fluorescent protein (FLP)
aequorin
+ CO2 + light
+ coelenterazine (1)
+ O2
− coelenteramide (2H)
apoaequorin
O
OH
OH
O
3 2
1
N
N1
4N
N
6
5
5
6
H
2
3
8
N
CH2Ph
7N
H
CH2Ph
4
HO
HO
coelenteramide (2H)
coelenterazine (1)
Scheme 1.
Keywords: Bioluminescence; Photoprotein; Aequorin; Obelin; Coelenter-
amide; Fluorescence; Solvent effect; Intramolecular charge transfer;
Hydrogen bond; Contact ion pair.
* Corresponding author. Tel./fax: +81 424 86 1966; e-mail: hirano@pc.uec.
ac.jp
The bioluminescence reaction of the calcium-activated
photoproteins is based on the reaction of 1 with O2, which
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.04.044

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6273
yields coelenteramide (2H), CO2, and light.8–10 Light emis-
sion occurs from the singlet-excited state of the anion spe-
cies of 2H (12ꢀ*) in apoprotein, to give FLP, a complex of
properties of 2(O)ꢀ in a chemically defined molecular envi-
ronment, such as an ion pair with a counter cation.
2H and apoprotein.11 The structure elucidation of 2ꢀ* in
In the previous study we reported the florescence of 3a(O)ꢀ,
an analogue of 2(O)ꢀ having an octanoyl group for improv-
ing solubility in various solvents, though we did not clarify
the influence of an ion pair of 13a(O)ꢀ* with a counter cation
on its fluorescence.17b To confirm the mechanism for light
emission from 12(O)ꢀ* in the photoprotein, here we reinves-
tigate the florescence of 3a(O)ꢀ in ion pairs with conjugate
acids of several organic bases as counter cations. The fluo-
rescence of methyl analogue 3b(O)ꢀ in an ion pair was
also investigated to clarify steric effects of the methyl group
at C6 on the p-conjugation between the pyrazine ring and
the 4-oxidophenyl (4-OꢀC6H4) group.24 Furthermore, we in-
vestigated fluorescence emission from 15(N)ꢀ* in an ion pair
generated by chemiluminescence reactions of coelentera-
zine analogue 4 in various solvents. Herein we report the
spectroscopic characteristics of 3H and those of 3(O)ꢀ and
5(N)ꢀ in an ion pair with a counter cation. We show that
the fluorescence of 3(O)ꢀ depends on a structural variation
of an ion pair, while that of 5(N)ꢀ does not. We also recon-
firm that 12(O)ꢀ* is the light emitter in the bioluminescence
of aequorin and obelin, and we propose a bioluminescence
reaction mechanism involving a chemiexcitation process to
1
apoprotein is one of the controversies in the reaction mech-
1
anism. Two structures are possible for 2ꢀ*: the singlet-
excited state of an amide anion 2(N)ꢀ (12(N)ꢀ*) and the
singlet-excited state of a phenolate anion 2(O)ꢀ (12(O)ꢀ*
)
(Scheme 2). Both anions have an anionic center conjugating
with the fluorescent core, the pyrazine ring.
R
O
R
O
N
N
NH
N
N
N
CH2Ph
CH2Ph
O
HO
−
−
2(N)
2(O)
Scheme 2. R¼(4-hydroxyphenyl)methyl or (4-oxidophenyl)methyl.
Excited amide anion 12(N)ꢀ* is the light emitter of a chemi-
luminescence reaction of 1 in an aprotic solvent such as
N,N-dimethylformamide (DMF) and dimethylsulfoxide
(DMSO).12,13 Becausetheemissionwavelengthsofthechemi-
luminescence of 1 and the bioluminescence of aequorin are
similar to each other, 12(N)ꢀ* has been believed as the light
emitter in aequorin bioluminescence.12–14 However, we
noticed that this explanation cannot account for the fact that
the fluorescence emission of FLP obtained from aequorin
occurs from common 12ꢀ* to the light emission in aequorin
bioluminescence.11a,15 Thus, we are the first to propose that
12(O)ꢀ* is the light emitter in aequorin biolumines-
give 12(O)ꢀ*
.
C7H15
NH
C7H15
NH
CH2Ph
O
O
1
R
N
2
R
N
N
6
5
3
N
CH2Ph
4
HO
O
3a(O)−: R = H
1
3aH: R = H
3bH: R = CH3
cence.15–17 The fact suggests the generation of 2(O)ꢀ* in
3b(O)−: R = CH3
aequorin bioluminescence, because it is conceivable that
2H eliminates a proton from the phenolic hydroxy group
with a moderate acidity18 rather than from the amide moiety
with a weak acidity.19–21 In addition, we found that 2(O)ꢀ in
benzene showed the fluorescence spectrum similar to the
emission spectrum of aequorin bioluminescence,17 support-
CH3
N
CH3
O
N
O
N
N
N
R
N
H
CH2Ph
CH2Ph
1
ing that the light emitter is 2(O)ꢀ* in apoaequorin, whose
CH3O
CH3O
active site has a polarity similar to benzene.
4
5H: R = H
5(N)−: R =
The mechanism involving a light emission from 12(O)ꢀ* in
aequorin is now applied to obelin bioluminescence.7b,22
There is confusion in some reports, where 12(O)ꢀ* in polar
and in less polar solvents has been categorized as a ‘quinoid
anion’ and an ‘ion pair,’ respectively.7b,22,23 However, the
quinoid form, shown in Scheme 3c, is only one of the several
2. Results and discussion
1
resonance structures of 2(O)ꢀ*. Similarly, we should not
2.1. UV–vis absorption spectra of coelenteramide ana-
logue 3H in the presence of organic bases
1
distinguish 2(O)ꢀ* in a less polar solvent as an ion pair,
1
because 2(O)ꢀ* is only a component of the ion pair. To
precisely understand the fundamental characteristics of
Coelenteramide analogue 3aH was prepared by the proce-
dure previously reported.17b Analogue 3bH was prepared
12(O)ꢀ*, we have to further establish the spectroscopic
O
O
R
O
R
R
N
N
NH
CH2Ph
N
N
NH
N
N
NH
CH2Ph
etc
CH2Ph
O
O
O
a
b
c (quinoid)
Scheme 3. R¼(4-hydroxyphenyl)methyl or (4-oxidophenyl)methyl.

6274
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
from 6-methylpyrazinamine by the procedure shown in
Scheme 4. Pyrazinamine precursor 3-benzyl-5-(4-benzyloxy-
phenyl)-6-methylpyrazinamine was prepared by bromina-
tion of 6-methylpyrazinamine with tetrabutylammonium
tribromide followed by selective Stille and Suzuki cou-
plings. Acylation of the pyrazinamine precursor and the
following hydrogenolysis gave 3bH.
UV–vis absorption spectra of 3H were measured in p-
xylene, toluene, benzene, benzene/chloroform (20:1), chloro-
benzene, 1-chloropropane, benzene/chloroform (2:1),
1,2-dichlorobenzene, chloroform, dichloromethane, 1,2-
dichloroethane, benzonitrile, N,N-dimethylacetamide (DMAc),
DMF, propionitrile, DMSO, and acetonitrile in the absence
and presence of an organic base. In this study, n-butylamine
(NBA), 1,1,3,3-tetramethylguanidine (TMG), and 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) were used as an organic
base (BASE). Thus, their conjugate acids (BASE–H+) will
become a counter cation for 3(O)ꢀ in an ion pair. Selected
spectra are shown in Figure 1 and the spectral data are sum-
marized in Tables 1 and 2, where the data are listed in order of
the solvent polarity scale ET(30) (in kcal molꢀ1).25
CH3
Br
N
N
NH2
CH3
N
N
NH2
CH3
Br
N
N
NH2
Br
i)
ii)
CH2Ph
O
C7H15
CH3
N
NH
CH3
N
NH2
CH2Ph
iii)
iv)
As reported previously,17b two types of characteristic spec-
tral changes of the lowest energy band of 3H were observed
in the presence of BASE. One is type I, which caused the
small shift (Dl<12 nm) in a less polar solvent. Type II is
the other, causing a large bathochromic shift (Dl>20 nm)
in a more polar solvent. Type I and II spectral changes
were caused by the formation of a 1:1 hydrogen-bonded
complex of 3H and BASE ([BASE/3H]) and the formation
of an ion pair of 3(O)ꢀ with BASE–H+ ([BASE–H+/3(O)ꢀ]),
respectively (Scheme 5).
N
N
CH2Ph
RO
BnO
R = Bn
R = H (3bH)
v)
Scheme 4. Reagent and conditions: (i) (C4H9)4NBr3, pyridine, CHCl3,
reflux, 46%; (ii) Bn(C4H9)3Sn, PdCl2(PPh3)2, DMF, 130 ꢁC, 36%; (iii)
BnOC6H4B(OH)2, Pd(PPh3)4, 1,4-dioxane, 2 mol Lꢀ1 Na2CO3 aq, 110 ꢁC,
94%; (iv) C7H15COCl, pyridine, CH2Cl2, 0 ꢁC, 52%; (v) H2, Pd–C,
C2H5OH/CH3CO2C2H5, 67%.
(i) benzene
(ii) dichloromethane
(iii) DMF
A
A
A
b c d
0.2
0.1
0.2
0.1
0.2
0.1
b c
a
a
c d
d
a
0.0
0.2
0.0
0.2
0.0
0.2
B
B
B
b c d
a
b c
a
0.1
0.0
0.1
0.0
0.1
0.0
c d
d
a
300
350
400
450
300
350
400
450
300
350
400
450
500
wavelength / nm
(iv) DMSO
wavelength / nm
wavelength / nm
(v) acetonitrile
A
A
0.2
0.2
d
d
c
a
0.1
0.1
0.0
a
c
0.0
0.2
B
B
0.2
0.1
0.0
0.1
0.0
d
c
d
a
c
a
300
350
400
450
500
300
350
400
450
500
wavelength / nm
wavelength / nm
Figure 1. UV–vis absorption spectra of 3aH (A, 1.5ꢂ10ꢀ5 mol Lꢀ1) and 3bH (B, 1.5ꢂ10ꢀ5 mol Lꢀ1) in selected solvents in the absence and presence of
organic bases (1.0ꢂ10ꢀ2 mol Lꢀ1) at 25 ꢁC; organic base: none (a), NBA (b), TMG (c), and DBU (d). The selected solvents are benzene (i), dichloromethane
(ii), DMF (iii), DMSO (iv), and acetonitrile (v).

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6275
Table 1. UV–vis absorption data of 3aH (1.5ꢂ10ꢀ5 mol Lꢀ1) in various solvents in the absence and presence of organic bases (NBA, TMG, and DBU) at 25 ꢁC
Solvent
ET(30)/kcal molꢀ1
lmax/nm (3/104)a
lmax/nm (absorbance)
3aH+TMGb
3aH
3aH+NBAb
3aH+DBUb
p-Xylene
33.1
33.9
34.3
36.4
36.8
37.4
38.0
332 (1.05)
335 (0.164)
340 (0.170)
300 (0.176)
340 (0.228)
342 (0.188)
302 (0.198)
342 (0.222)
302 (0.242)
343 (0.237)
301 (0.258)
342 (0.242)
302 (0.261)
343 (0.218)
304 (0.233)
340 (0.221)
301 (0.249)
390 (0.035, sh)
340 (0.228)
302 (0.261)
343 (0.222)
304 (0.249)
380 (0.072, sh)
331 (0.227)
302 (0.212)
380 (0.102, sh)
339 (0.258)
307 (0.213)
375 (0.047, sh)
338 (0.227)
303 (0.227)
390 (0.063, sh)
341 (0.219)
400 (0.010, sh)
337 (0.224)
297 (0.276)
410 (0.024, sh)
335 (0.215)
297 (0.221)
385 (0.084, sh)
337 (0.243)
299 (0.218)
Toluene
332 (1.24)
335 (0.212)
Benzene
331 (1.44)
294 (1.48)
331 (1.46)
295 (1.50)
331 (1.16)
291 (1.28)
329 (1.16)
293 (1.22)
331 (1.49)
294 (1.56)
335 (0.224)
297 (0.237)
335 (0.228)
297 (0.242)
336 (0.186)
298 (0.200)
332 (0.185)
295 (0.203)
334 (0.221)
296 (0.230)
340 (0.233)
299 (0.255)
340 (0.239)
300 (0.257)
340 (0.212)
300 (0.239)
339 (0.212)
299 (0.237)
338 (0.228)
299 (0.243)
Benzene/chloroform (20:1)
Chlorobenzene
1-Chloropropane
Benzene/chloroform (2:1)
1,2-Dichlorobenzene
Chloroform
38.0
39.1
332 (1.14)
339 (0.176)
342 (0.198)
302 (0.221)
338 (0.234)
298 (0.246)
331 (1.33)
294 (1.33)
333 (0.231)
295 (0.237)
Dichloromethane
40.7
41.3
330 (1.52)
291 (1.75)
333 (0.233)
295 (0.239)
338 (0.245)
298 (0.258)
1,2-Dichloroethane
331 (1.43)
293 (1.45)
334 (0.219)
295 (0.224)
390 (0.014, sh)
338 (0.227)
299 (0.242)
400 (0.010, sh)
338 (0.207)
336 (0.222)
298 (0.248)
Benzonitrile
DMAc
41.5
42.9
334 (1.36)
337 (0.204)
c
—
335 (1.54)
296 (1.83)
c
—
DMF
43.2
43.6
336 (1.47)
296 (1.58)
430 (0.014, sh)
335 (0.209)
296 (0.219)
385 (0.023, sh)
333 (0.227)
296 (0.233)
276 (0.210)
426 (0.078)
343 (0.183)
299 (0.159)
410 (0.066, sh)
336 (0.212)
296 (0.185)
273 (0.177)
Propionitrile
330 (1.46)
292 (1.56)
274 (1.52)
334 (0.224)
296 (0.246)
278 (0.224)
c
—
DMSO
45.1
45.6
337 (1.38)
297 (1.47)
423 (0.081)
344 (0.186)
299 (0.153)
400 (0.105, sh)
339 (0.242)
302 (0.185)
c
—
Acetonitrile
327 (1.44)
293 (1.50)
272 (1.47)
Extinction coefficient in L molꢀ1 cmꢀ1
.
Absorption spectra of 3aH did not change in the presence of NBA.
.
a
Concentrations of the organic bases were 1.0ꢂ10ꢀ2 mol Lꢀ1
b
c
The type I spectral change of 3H was observed in the pres-
ence of BASE in solvents from p-xylene (top) to benzonitrile
and propionitrile, listed in Tables 1 and 2. In the presence of
DBU in benzene/chloroform (2:1), 1,2-dichlorobenzene,
chloroform, dichloromethane, 1,2-dichloroethane, benzo-
nitrile, and propionitrile, both the type I and II spectral
changes were observed. Typical spectra are shown in
Figure 1i and ii. Formation of [BASE/3H] was mainly ob-
served in the solvents with low ionizing power. A partial
generation of [DBU–H+/3(O)ꢀ] in these solvents was also
observed, because DBU has high enough basicity to elimi-
nate a proton from the phenolic hydroxy groups of 3H.
The order of Dl, DBU>TMG>NBA, corresponds to the re-
ported acidities (pKa) of their conjugated acids, 24.1, 23.3,
and 18.1, respectively, in acetonitrile.26 The order of Dl
also corresponds to the formation constants (K, Scheme 5)
of [DBU/3aH], [TMG/3aH], and [NBA/3aH]: 7800,
1430, and 22017b molꢀ1 L, respectively, in benzene at 25 ꢁC.
Therefore, the more basic an organic base becomes, the
stronger 3H makes a hydrogen bond with the organic base
and the more Dl and K increase.
The type II spectral changes for 3H were observed in the
presence of TMG and DBU in DMAc, DMF, DMSO, and
acetonitrile, while the addition of NBA did not affect the
original spectra of 3H (Fig. 1iii–v). These solvents have
enough ionizing power to generate [BASE–H+/3(O)ꢀ]. In
addition, TMG and DBU have high enough basicities to gen-
erate 3(O)ꢀ from 3H, but NBA does not. Absorption spectra
of [TMG–H+/3(O)ꢀ] and [DBU–H+/3(O)ꢀ] were similar to
each other, suggesting that the interaction of 3(O)ꢀ with
TMG–H+ in the ion pair is similar to that of DBU–H+. One
ofthecharacteristicsofthespectralchangesisthatagrowthof
the absorption of [TMG–H+/3b(O)ꢀ] or [DBU–H+/3b(O)ꢀ]

6276
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
Table 2. UV–vis absorption data of 3bH (1.5ꢂ10ꢀ5 mol Lꢀ1) in various solvents in the absence and presence of organic bases (NBA, TMG, and DBU) at 25 ꢁC
Solvent
ET(30)/kcal molꢀ1
lmax/nm (3/104)a
lmax/nm (absorbance)
3bH+TMGb
3bH
3bH+NBAb
3bH+DBUb
p-Xylene
Toluene
Benzene
Benzene/chloroform (20:1)
Chlorobenzene
1-Chloropropane
33.1
33.9
34.3
36.4
36.8
37.4
323 (1.35)
322 (1.32)
321 (1.33)
321 (1.32)
322 (1.43)
320 (1.38)
324 (0.201)
324 (0.203)
324 (0.203)
323 (0.201)
325 (0.218)
322 (0.207)
329 (0.206)
328 (0.209)
328 (0.210)
327 (0.198)
330 (0.225)
327 (0.210)
275 (0.206)
327 (0.198)
331 (0.207)
329 (0.209)
330 (0.206)
329 (0.204)
331 (0.225)
328 (0.213)
277 (0.216)
380 (0.017, sh)
331 (0.192)
331 (0.215)
370 (0.038, sh)
322 (0.180)
370 (0.048, sh)
328 (0.191)
275 (0.192)
326 (0.192)
276 (0.186)
385 (0.029, sh)
329 (0.185)
324 (0.198)
400 (0.005, sh)
333 (0.189)
295 (0.140)
375 (0.037, sh)
323 (0.186)
412 (0.024)
325 (0.186)
380 (0.045, sh)
322 (0.170)
271 (0.165)
Benzene/chloroform (2:1)
38.0
321 (1.30)
324 (0.198)
1,2-Dichlorobenzene
Chloroform
38.0
39.1
321 (1.31)
319 (1.28)
325 (0.209)
322 (0.195)
330 (0.216)
326 (0.194)
Dichloromethane
40.7
319 (1.26)
321 (0.197)
325 (0.198)
274 (0.192)
1,2-Dichloroethane
Benzonitrile
41.3
41.5
320 (1.31)
323 (1.21)
322 (0.197)
325 (0.183)
327 (0.203)
273 (0.200)
390 (0.007, sh)
325 (0.183)
326 (0.197)
410 (0.004, sh)
333 (0.192)
294 (0.140)
380 (0.008, sh)
321 (0.188)
416 (0.020)
326 (0.188)
390 (0.016, sh)
321 (0.170)
294 (0.129)
265 (0.156)
c
DMAc
DMF
42.9
43.2
323 (1.39)
323 (1.27)
294 (0.89)
—
—
c
Propionitrile
DMSO
43.6
45.1
45.6
320 (1.24)
324 (1.27)
321 (0.188)
c
—
c
—
Acetonitrile
318 (1.16)
295 (0.89)
263 (1.10)
Extinction coefficient in L molꢀ1 cmꢀ1
.
Absorption spectra of 3bH did not change in the presence of NBA.
.
a
Concentrations of the organic bases were 1.0ꢂ10ꢀ2 mol Lꢀ1
b
c
was smaller than that of [TMG–H+/3a(O)ꢀ] or [DBU–H+/
3a(O)ꢀ]. The K value for the formation of [TMG–H+/
3b(O)ꢀ] in DMSO, 28 molꢀ1 L at 25 ꢁC, is much smaller
than that of [TMG–H+/3a(O)ꢀ], 480 molꢀ1 L at 25 ꢁC,17b
clearly indicating that the generation of 3b(O)ꢀ from 3bH
is less favorable than that of 3a(O)ꢀ from 3aH. This is
explained by the methyl group at C6 in 3b(O)ꢀ sterically in-
hibiting the p-electronic conjugation between the 4-oxido-
phenyl and amidopyrazine moieties (vide infra) to decrease
the molecular stability.
type I
C7H15
O
C7H15
O
K
NH
CH2Ph
N
N
R
NH
CH2Ph
N
N
R
BASE +
HO
O
BASE
H
3H
[BASE···3H]
hydrogen-bonded complex
free molecule
type II
C7H15
O
C7H15
O
K
+
-
NH
CH2Ph
BASE H
N
N
R
NH
CH2Ph
N
N
R
BASE +
HO
O
3H
free molecule
+
−
[BASE-H / 3(O) ]
ion pair
Scheme 5.

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6277
fluorescence emission from 13(O)ꢀ* in an ion pair has an in-
tramolecular charge transfer (ICT) nature, energies EFL (in
kcal molꢀ1) calculated from the lFL of [BASE–H+/3(O)ꢀ]
show a linear correlation to ET(30).17b Thus, we made
EFL–ET(30) plots of 3(O)ꢀ in the ion pairs with NBA–H+,
TMG–H+, and DBU–H+, as shown in Figure 3. The EFL–
ET(30) correlations were EFL¼ꢀ1.04ET(30)+97 (r¼ꢀ0.96),
EFL¼ꢀ0.89ET(30)+88 (r¼ꢀ0.98), and EFL¼ꢀ0.90ET(30)+
89 (r¼ꢀ0.98), for [NBA–H+/3a(O)ꢀ], [TMG–H+/3a(O)ꢀ],
and [DBU–H+/3a(O)ꢀ], respectively.27 Similarly, EFL¼
ꢀ1.40ET(30)+111 (r¼ꢀ0.99), EFL¼ꢀ1.00ET(30)+92 (r¼
ꢀ0.97), and EFL¼ꢀ1.02ET(30)+93 (r¼ꢀ0.98) were for
[NBA–H+/3b(O)ꢀ], [TMG–H+/3b(O)ꢀ], and [DBU–H+/
3b(O)ꢀ], respectively.
H
H
H
O
C
H
C7H15
N
O
NH
N
H
CH2Ph
−
3b(O)
2.2. Fluorescent properties of phenolate anions 3(O)L in
ion pairs with conjugate acids of organic bases
Growth of the fluorescence emission band of 3(O)ꢀ was ob-
served along with decreasing fluorescence intensity of the
neutral molecule 3H in the presence of NBA,17b TMG,17b
and DBU, as shown in the selected spectra (Fig. 2). We
also confirmed that fluorescence excitation spectra of 3(O)ꢀ
in the presence of organic bases correspond to their ab-
sorption spectra. Corresponding to the type I and II spectral
changes in the UV–vis absorption, there are two possible
processes to generate 13(O)ꢀ* in an ion pair with a BASE–H+
(Scheme 6).17 One is a stepwise process via [BASE/13H*]
generated by electronic excitation of [BASE/3H], as shown
in Scheme 6a. The other is a direct electronic excitation
process from [BASE–H+/3(O)ꢀ] (Scheme 6b). Both pro-
cesses show the fluorescence from [BASE–H+/13(O)ꢀ*].
The EFL–ET(30) correlations for [TMG–H+/3(O)ꢀ] and
[DBU–H+/3(O)ꢀ] were similar to each other, while the
slopes and intercepts of the linear EFL–ET(30) correlations
for [NBA–H+/3(O)ꢀ] were larger than those for [TMG–
H+/3(O)ꢀ] or [DBU–H+/3(O)ꢀ]. Similarly, the Ff of
[TMG–H+/3(O)ꢀ] and [DBU–H+/3(O)ꢀ] were similar to
each other, while most of the Ff of [NBA–H+/3(O)ꢀ] were
larger than those of [TMG–H+/3(O)ꢀ] or [DBU–H+/3(O)ꢀ].
These results indicate that there are two modes of interaction
between 13(O)ꢀ* and a BASE–H+, which may be caused by
a structural variation of an ion pair: a solvent separated ion
pair (SSIP) for [TMG–H+/13(O)ꢀ*] or [DBU–H+/13(O)ꢀ*],
and a contact ion pair (CIP) for [NBA–H+/13(O)ꢀ*].25,29
The selectivity to form an SSIP or a CIP is determined by
Fluorescence emission maxima (lFL) and quantum yields
(Ff) are summarized in Tables 3 and 4. The Ff values
of [BASE–H+/3(O)ꢀ] were obtained by subtracting the
fluorescences of 3H from the total spectra. Because the
1
the basicity of BASE. Because the SSIP of 3(O)ꢀ* and
(i) benzene
(ii) dichloromethane
(iii) DMF
a
A
A
A
a
a
c
d
(×5)
b
d c
b
d
c
B
B
B
a
b
a
a
d
(×10)
c
d
b
d c
c
400
500
600
700
400
500
600
700
400
500
600
700
wavelength / nm
wavelength / nm
wavelength / nm
(iv) DMSO
a
(v) acetonitrile
a
A
A
d
c
(×10)
(×50)
d
c
B
B
a
a
(×10)
d
c
(×15)
d
c
400
500
600
700
400
500
600
700
wavelength / nm
wavelength / nm
Figure 2. Fluorescence emission spectra of 3aH and [BASE–H+/3a(O)ꢀ] (A) and 3bH and [BASE–H+/3b(O)ꢀ] (B) in various solvents in the absence and
presence of organic bases (1.0ꢂ10ꢀ2 mol Lꢀ1) at 25 ꢁC; organic base: none (a), NBA (b), TMG (c), and DBU (d). Selected solvents were benzene (i), dichloro-
methane (ii), DMF (iii), DMSO (iv), and acetonitrile (v). The initial concentrations of 3H were 1.5ꢂ10ꢀ6 mol Lꢀ1, except for those (1.5ꢂ10ꢀ5 mol Lꢀ1) in
DMF, DMSO, and acetonitrile containing TMG and DBU. Excitation wavelengths (lex) were 320 nm in benzene and dichloromethane. The lex in DMF,
DMSO, and acetonitrile were also 320 nm in the absence of the organic bases. The lex in DMF and acetonitrile containing bases and in DMSO containing bases
were 400 and 425 nm, respectively.

6278
(a)
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
(b)
C7H15
C7H15
O
O
*
*
+
-
BASE H
NH
CH2Ph
N
N
R
NH
CH2Ph
N
N
R
+
−
1
[BASE-H / 3(O) *]
O
O
BASE
H
1
+
−
1
[BASE··· 3H*]
[BASE-H / 3(O) *]
fluorescence
emission
fluorescence
emission
electronic
excitation
electronic
excitation
C7H15
O
C7H15
O
+
-
BASE H
NH
CH2Ph
N
N
NH
N
N
R
R
+
−
[BASE-H / 3(O) ]
CH2Ph
O
O
BASE
H
+
−
[BASE···3H]
hydrogen-bonded complex
[BASE-H / 3(O) ]
ion pair
Scheme 6.
TMG–H+ or DBU–H+ is a loose ion pair separated by solvent
molecules, the Coulomb force between 13(O)ꢀ* and TMG–
H+ or DBU–H+ is weak and the fluorescence of 3(O)ꢀ is
not much affected by the choice of BASE–H+. On the other
[NBA–H+/13(O)ꢀ*] in a less polar solvent is favorable for
increasing the Ff of 3(O)ꢀ.
The slopes of the EFL–ET(30) correlations for [BASE–H+/
3b(O)ꢀ] are larger than the corresponding correlations
for [BASE–H+/3a(O)ꢀ]. This indicates that the degree of
ICT in methyl derivative 13b(O)ꢀ* is enhanced by the
twisted structure between 4-oxidophenyl and amidopyrazine
moieties, as shown in Scheme 7. The stability of the twisted
13b(O)ꢀ* is more sensitive to the solvent polarity than is that
of 13a(O)ꢀ*, giving a steeper slope for the EFL–ET(30)
correlation for [BASE–H+/3b(O)ꢀ] than for [BASE–H+/
3a(O)ꢀ]. On the other hand, the Ff values of 3a(O)ꢀ and
1
hand, the CIP of 3(O)ꢀ* and NBA–H+ holds together by
an effective Coulomb force, and the degree of binding be-
tween 13(O)ꢀ* and NBA–H+ varies with the solvent polarity.
Therefore, the molecular stability of [NBA–H+/13(O)ꢀ*] is
sensitive to the solvent polarity, resulting in steep slopes of
the EFL–ET(30) correlations for [NBA–H+/3(O)ꢀ]. Among
a variety of combinations of 3(O)ꢀ and BASE–H+, [NBA–
H+/3(O)ꢀ] showed the highest Ff values (>0.1) in the sol-
vent, with ET(30)<38 kcal molꢀ1. This indicates that
Table 3. Fluorescence of 3aH and its phenolate anion 3a(O)ꢀ in ion pairs with NBA–H+, TMG–H+, and DBU–H+ in various solvents at 25 ꢁC
Solvent
ET(30)/kcal molꢀ1
lFLa/nm (Ffb)
3aHc
3aHc+NBAd
3aHc+TMGd
3aHc+DBUd
[NBA–H+/3a(O)ꢀ]
[TMG–H+/3a(O)ꢀ]
[DBU–H+/3a(O)ꢀ]
p-Xylene
Toluene
Benzene
Benzene/chloroform (20:1)
Chlorobenzene
Chloropropane
Benzene/chloroform (2:1)
1,2-Dichlorobenzene
Chloroform
Dichloromethane
1,2-Dichloroethane
Benzonitrile
DMAc
DMF
33.1
33.9
34.3
36.4
36.8
37.4
38.0
38.0
39.1
40.7
41.3
41.5
42.9
43.2
43.6
45.1
45.6
383 (0.14)
383 (0.17)
383e (0.19)e
384 (0.19)
385 (0.15)
385 (0.17)
387 (0.19)
385 (0.16)
389 (0.23)
388 (0.18)
388 (0.18)
394 (0.23)
401 (0.22)
402 (0.24)
392 (0.20)
408 (0.22)
394 (0.20)
452 (0.24)
466 (0.23)
469e (0.27)e
473 (0.23)
501 (0.35)
498 (0.19)
499 (0.20)
505 (0.34)
500 (0.16)
518 (0.15)
520 (0.17)
546 (0.03)
490 (0.04)
495 (0.03)
498 (0.03)
504 (0.03)
542 (0.09)
526 (0.10)
527 (0.03)
537 (0.14)
542 (0.03)
557 (0.03)
554 (0.05)
562 (0.04)
575 (0.07)
584e (0.04)e
582 (0.02)
591e (0.02)e
615e (0.01)e
486 (0.06)
491 (0.06)
490 (0.06)
503 (0.05)
531 (0.15)
521 (0.12)
522 (0.03)
530 (0.17)
542 (0.02)
553 (0.04)
544 (0.05)
554 (0.03)
575 (0.06)
583 (0.04)
582 (0.01)
590 (0.03)
598 (0.01)
f
—
—
f
Propionitrile
DMSO
Acetonitrile
554 (0.02)
f
—
—
f
a
Fluorescence emission maxima. In the presence of the organic bases, only the lFL of the generated anion species are shown.
Fluorescence quantum yields.
b
c
d
e
f
The initial concentration of 3aH was 1.5ꢂ10ꢀ6 mol Lꢀ1
.
The initial concentration of the organic base was 1.0ꢂ10ꢀ2 mol Lꢀ1
.
The value revised from the previous report (Ref. 28).
No new emission band was observed in the presence of NBA.

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6279
Table 4. Fluorescence of 3bH and its phenolate anion 3b(O)ꢀ in ion pairs with NBA–H+, TMG–H+, and DBU–H+ in various solvents at 25 ꢁC
Solvent
ET(30)/kcal molꢀ1
lFLa/nm (Ffb)
3bHc
3bHc+NBAd
3bHc+TMGd
3bHc+DBUd
[NBA–H+/3b(O)ꢀ]
[TMG–H+/3b(O)ꢀ]
[DBU–H+/3b(O)ꢀ]
p-Xylene
Toluene
Benzene
Benzene/chloroform (20:1)
Chlorobenzene
1-Chloropropane
Benzene/chloroform (2:1)
1,2-Dichlorobenzene
Chloroform
Dichloromethane
1,2-Dichloroethane
Benzonitrile
DMAc
DMF
33.1
33.9
34.3
36.4
36.8
37.4
38.0
38.0
39.1
40.7
41.3
41.5
42.9
43.2
43.6
45.1
45.6
383 (0.027)
383 (0.031)
384 (0.030)
384 (0.029)
384 (0.046)
383 (0.026)
385 (0.032)
385 (0.039)
385 (0.032)
386 (0.025)
384 (0.031)
394 (0.080)
395 (0.052)
396 (0.052)
391 (0.027)
402 (0.065)
391 (0.025)
444 (0.24)
451 (0.30)
451 (0.32)
472 (0.16)
480 (0.23)
487 (0.09)
504 (0.10)
501 (0.19)
514 (0.07)
530 (0.04)
527 (0.07)
557 (0.02)
480 (0.02)
489 (0.02)
494 (0.02)
504 (0.02)
522 (0.08)
529 (0.04)
540 (0.01)
542 (0.11)
560 (0.01)
578 (<0.005)
562 (0.01)
570 (0.01)
572 (0.01)
602 (0.01)
585 (0.01)
610 (0. 01)
600 (<0.005)
479 (0.04)
481 (0.04)
488 (0.04)
500 (0.03)
515 (0.11)
521 (0.05)
534 (0.02)
531 (0.10)
556 (0.01)
552 (<0.005)
552 (0.02)
557 (0.02)
582 (0.01)
597 (0.01)
576 (0.01)
607 (0.01)
595 (<0.005)
e
—
—
e
Propionitrile
DMSO
Acetonitrile
566 (<0.005)
—
—
e
e
a
Fluorescence emission maxima. In the presence of the organic bases, only the lFL of the generated anion species are shown.
Fluorescence quantum yields.
b
c
d
e
The initial concentration of 3bH was 1.5ꢂ10ꢀ6 mol Lꢀ1
.
The initial concentration of the organic base was 1.0ꢂ10ꢀ2 mol Lꢀ1
.
No new emission band was observed in the presence of NBA.
A
B
C
65
60
55
50
45
65
60
55
50
45
65
60
55
50
45
32
36
40
44
32
36
40
44
32
36
40
44
−1
−1
−1
E (30)/kcal mol
E (30)/kcal mol
E (30)/kcal mol
T
T
T
Figure 3. Plots of EFL (in kcal molꢀ1) for 3a(O)ꢀ (A) and 3b(O)ꢀ (>) in ion pairs with NBA–H+ (A), TMG–H+ (B), and DBU–H+ (C) against ET(30) (in
kcal molꢀ1).
3b(O)ꢀ were similar to each other, while 3aH and 3bH had
different Ff values. This result indicates that the methyl
group at C6 in 3b(O)ꢀ does not enhance a non-radiative pro-
the EFL–ET(30) correlations for [BASE–H+/3a(O)ꢀ] with
that for a dimethylamino analogue 6, EFL¼ꢀ0.64ET(30)+
85 (r¼ꢀ0.98).16 The slopes of the EFL–ET(30) correlations
for [BASE–H+/3a(O)ꢀ] are all steeper than those for 6, while
the intercepts of the EFL–ET(30) correlations for [TMG–H+/
3a(O)ꢀ] or [DBU–H+/3a(O)ꢀ] and 6 are similar to each
other. The different slopes for [BASE–H+/3a(O)ꢀ] and 6
are related to the electron-donating abilities of the oxido
and dimethylamino groups, respectively. The modified
Hammett constant s+p for the oxido group (ꢀ2.30) is smaller
than that of the dimethylamino group (ꢀ1.70).30,31 There-
fore, the oxido group plays an important role as an electron
donor for stabilizing the ICT state of 13a(O)ꢀ* (Scheme 7).
1
cess from the twisted 3b(O)ꢀ*, which competes with the
fluorescence emission process. Excited neutral molecules
13H*, however, are affected by the methyl substitution,
whose steric effect causes a decrease of Ff.
*
R
O
H
C7H15
N
O
NH
N
H
CH2Ph
CH3
O
1
3a(O)−*: R = H
1
3b(O)−*: R = CH
3
N
N
NH
Scheme 7.
CH2Ph
Because the EFL–ET(30) correlations for 3a(O)ꢀ in the ion
(CH3)2N
pairs were revised from the previous one,27 we recompared
6

6280
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
2.3. Luminescent properties of amide anion 5(N)L:
chemiluminescence of coelenterazine analogue 4 in
various solvents
was observed, as shown in Figure 4. In benzene, an emission
from excited neutral molecule 5H* also appeared around
1
400 nm. The lFL values and chemiluminescence quantum
yields (Fcl) are summarized in Table 5. The Fcl values in
benzene, dichloromethane, and acetonitrile were smaller
than those in DMSO and DMF. The lFL values of [BASE–
H+/5(N)ꢀ] were in the range of 464–476 nm, which is less
than that of [BASE–H+/3a(O)ꢀ]. The lFL values of
[BASE–H+/5(N)ꢀ] did not depend on the BASE–H+, which
were DMSO–H+, DMF–H+, and TMG–H+. Therefore, the
fluorescence of 5(N)ꢀ was not significantly affected by the
interaction with BASE–H+ in the ion pair or the solvent
polarity.
To consider the difference in the fluorescent properties be-
tween 2(O)ꢀ and 2(N)ꢀ, we investigated the fluorescence
of amide anion 5(N)ꢀ generated from 5H with a strong
base, such as NaOH, in a polar solvent with strong ionizing
power, such as DMSO.32 Unfortunately, reproducible fluo-
rescence data were not obtained for 5(N)ꢀ in a solvent
with a low ionizing power such as benzene, because of inter-
ferences from side reactions. The difficulty in observing
5(N)ꢀ is caused by the low acidity of the amide moiety in
5H.19–21
Table 5. Emission maxima (lFL) and quantum yields (Fcl) for the chemilu-
minescence of 4 in various solvents under air
To evaluate the fluorescent properties of 5(N)ꢀ in an ion pair
in various solvents, we investigated the chemiluminescence
of 4, which showed the fluorescence emission from [BASE–
H+/15(N)ꢀ*] (Scheme 8). Chemiluminescence of 4 was
easily observed in aerated DMSO and DMF (Fig. 4), and
the fluorescence emission maxima (lFL) of [DMSO–H+/
5(N)ꢀ] and [DMF–H+/5(N)ꢀ] almost reproduced the
reported data.12,32–34 On the other hand, 4 did not react in
aerated benzene, dichloromethane, or acetonitrile in the ab-
sence of a base. TMG was used as an organic base in these
solvents to initiate a chemiluminescence reaction of 435
Solvent
Additive
lFL/nm
Fcl/10ꢀ3
Benzene
TMGa
TMGa
None
464
470
471
476
473
0.005
0.02
1.5
1.1
0.06
Dichloromethane
DMF
DMSO
None
Acetonitrile
TMGa
The concentration of TMG was 1.0ꢂ10ꢀ2 mol Lꢀ1
.
a
2.4. Molecular orbital calculations for the ground and
the singlet-excited states of anion species of a coelenter-
amide analogue
and the fluorescence emission from [TMG–H+/15(N)ꢀ*
]
To gain insight into the structural characteristics of 2(O)ꢀ
and 2(N)ꢀ, we carried out PM5 semi-empirical molecular
orbital (MO) calculations for the ground and the singlet-
excited states of 7(O)ꢀ and 7(N)ꢀ, which are the anion spe-
cies of an acetamide analogue (Tables 6 and 7). Because the
stabilities of ionic molecules are affected by dielectric mo-
lecular environments, we performed the calculations with
the COSMO method36 using several dielectric constants
(3).25
CH3
+
O
*
-
BASE H
O2
N
N
N
+
−
[BASE-H /5(N) ] +
h
4
BASE
CH2Ph
CH3O
15(N)−*
Scheme 8.
9
O
9
CH3
H
CH3
8
O
8
N7
1
1
N
N7
N
6
5
6
5
2
2
3
3
11
11
10
10
12
12
N
N
4
4
H
13
13
O
O
14
14
−
−
7(O)
7(N)
a
In the ground state, it is reasonable that the heats of forma-
tion (DHf) of 7(O)ꢀ are smaller than those of 7(N)ꢀ under
various dielectric conditions, because the acidity of a pheno-
lic hydroxy group is higher than that of an amide moiety in
general.18,19 The HOMO–LUMO energy gaps of 7(O)ꢀ
were predicted to be smaller than those of 7(N)ꢀ. The torsion
angles C6–C5–C10–C11 (q) and N1–C2–N7–C8 (f) of
7(O)ꢀ and 7(N)ꢀ indicate that the conformations of the
pyrazine rings with the 4-oxido- or 4-hydroxyphenyl group
and with the amide moiety are twisted, but neither perpen-
dicular nor coplanar. As the characteristic bond lengths in
7(O)ꢀ and 7(N)ꢀ, the C13–O14 of 7(O)ꢀ is shortened by
the conjugation of the oxido group to the phenyl group,
while the C8]O9 of 7(N)ꢀ is elongated by the conjugation
b
c
d
e
400
500
600
700
wavelength / nm
Figure 4. Chemiluminescence spectra of 4 (1.5ꢂ10ꢀ5 mol Lꢀ1) in various
solvents under air. Solvents were benzene containing TMG (a), dichloro-
methane containing TMG (b), DMF (c), DMSO (d), and acetonitrile con-
taining TMG (e). Concentrations of TMG were 1.0ꢂ10ꢀ2 mol Lꢀ1
.

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6281
˚
Table 6. Calculated properties of 7(O)ꢀ and 7(N)ꢀ in the ground state with the PM5-COSMO method
Compounds 3a
7(O)ꢀ
DHfb/kJ molꢀ1 HOMO/eV LUMO/eV DEHOMO–LUMOc/eV qd/ꢁ
fd/ꢁ
r(C]O) /A r(C–O) /A r(C–C) /A
e
e
e
˚
˚
2.27 ꢀ405
8.93 ꢀ593
ꢀ6.06
ꢀ8.09
ꢀ8.76
ꢀ8.81
ꢀ6.92
ꢀ8.53
ꢀ8.95
ꢀ9.01
0.61
ꢀ0.73
ꢀ1.14
ꢀ1.17
1.07
ꢀ0.33
ꢀ0.85
ꢀ0.90
6.67
7.26
7.62
7.64
155.5 113.1 1.235
1.271
1.298
1.309
1.309
1.444
1.461
1.464
1.465
142.7
140.5
141.0
42.4 1.247
37.8 1.253
39.7 1.254
35.94 ꢀ664
46.45 ꢀ669
7(N)ꢀ
2.27 ꢀ396
8.93 ꢀ567
35.94 ꢀ627
46.45 ꢀ632
7.99
8.20
8.10
8.11
43.6 114.0 1.262
1.358
1.354
1.353
1.352
1.468
1.469
1.468
1.468
47.6
46.3
45.5
73.0 1.278
68.4 1.284
75.1 1.285
a
Dielectric constants at 25 ꢁC of benzene (2.27), dichloromethane (8.93), acetonitrile (35.94), and DMSO (46.45) from Ref. 25.
Heat of formation.
HOMO–LUMO energy gap.
The angles q and f are torsion angles C6–C5–C10–C11 and N1–C2–N7–C8, respectively.
The r(C]O), r(C–O), and r(C–C) are the bond lengths of C8–C9, C13–O14, and C5–C10, respectively.
b
c
d
e
of the amide anion. The C5–C10 of 7(O)ꢀ and 7(N)ꢀ are the
4-oxidophenyl (A), pyrazine (B), acetamide (C), and benzyl
(D). The negative charge in 7(O)ꢀ is mainly localized at the
4-oxidophenyl group. On the other hand, the q values at
the pyrazine and the acetamido parts of 17(O)ꢀ* were below
ꢀ0.86 and ꢀ0.13, respectively, indicating that the negative
lengths of typical single bonds. The calculated structures
of 7(O)ꢀ with a twisted conformation between the 4-oxido-
phenyl and the pyrazine moieties and the C5–C10 single
bond predict that 2(O)ꢀ has little proportion of a quinoid
form (Scheme 3c) in the resonance structures.
P
P
Table 8. Summations of the net atomic charges ( q) at the 4-oxidophenyl
The data of 17(O)ꢀ* and 17(N)ꢀ* calculated with the
PM5-CI-COSMO method (Table 7) indicate that the DHf
(A), pyrazine (B), acetamide (C), and benzyl (D) parts of 7(O)ꢀ and
17(O)ꢀ*, calculated with the PM5-COSMO and the PM5-CI-COSMO
methods, respectively
1
1
values of 7(O)ꢀ* are much smaller than those of 7(N)ꢀ*
under various dielectric conditions. The difference in DHf
CH3
O
between 7(O)ꢀ* and 7(N)ꢀ*, 105–159 kJ molꢀ1, is much
1
1
larger than that between 7(O)ꢀ and 7(N)ꢀ, 9–37 kJ molꢀ1
,
B
N
N
N
H
H
C
D
suggesting that the difference in the acidities between the
phenolic hydroxy group and the amide moiety in the sin-
glet-excited state is much larger than that in the ground
state. Thus, the phenolic hydroxy group is the most acidic
moiety in 12H*.18 As structural characteristics, the q values
of 17(O)ꢀ* and 17(N)ꢀ* are close to 90ꢁ, except the q value
H
H
H
H
O
A
−
1
−
*
7(O) and 7(O)
1
of 7(N)ꢀ* with 3¼2.27 (benzene). The C8]O9 and C5–
Compounds
3a
Summations of the net atomic
charges ( q)
P
1
1
C10 of 7(O)ꢀ* and 7(N)ꢀ* show a small difference from
those of the corresponding ground states, while their C13–
O14 becomes slightly short. The calculated structure of
17(O)ꢀ* with the perpendicular conformation between the
4-oxidophenyl and the pyrazine moieties is consistent with
the twisted structure of 13(O)ꢀ* (Scheme 7) concluded
from the fluorescence observations. This result also indicates
A
B
C
D
7(O)ꢀ
2.27
8.93
35.94
46.45
ꢀ0.814
ꢀ0.898
ꢀ0.917
ꢀ0.918
ꢀ0.056
ꢀ0.045
ꢀ0.034
ꢀ0.028
ꢀ0.228
ꢀ0.199
ꢀ0.203
ꢀ0.204
ꢀ0.863
ꢀ0.920
ꢀ0.949
ꢀ0.957
ꢀ0.082
ꢀ0.051
ꢀ0.039
ꢀ0.037
ꢀ0.157
ꢀ0.137
ꢀ0.134
ꢀ0.133
+0.124
+0.148
+0.158
+0.159
17(O)ꢀ*
2.27
8.93
35.94
46.45
+0.076
+0.102
+0.117
+0.118
1
that 2(O)ꢀ* does not have the property of a quinoid form
(Scheme 3c).
a
Dielectric constants at 25 ꢁC of benzene (2.27), dichloromethane (8.93),
acetonitrile (35.94), and DMSO (46.45) from Ref. 25.
Table 8 shows the summations of the net atomic charge
q) at the four parts of 7(O)ꢀ and 17(O)ꢀ*: the
P
(
Table 7. Calculated properties of 7(O)ꢀ and 7(N)ꢀ in the singlet-excited states (17(O)ꢀ* and 17(N)ꢀ*) with the PM5-CI-COSMO method
d
r(C]O) /A
d
r(C–O) /A
d
r(C–C) /A
3a
DHfb/kJ molꢀ1
qc/ꢁ
fc/ꢁ
˚
˚
˚
Compounds
17(O)ꢀ*
2.27
ꢀ243
ꢀ413
ꢀ470
ꢀ476
ꢀ84
ꢀ261
ꢀ363
ꢀ371
90.2
92.3
91.8
91.7
93.8
1.238
1.252
1.254
1.255
1.243
1.259
1.267
1.268
1.461
1.455
1.453
1.452
8.93
35.94
46.45
64.5
49.9
51.0
17(N)ꢀ*
2.27
8.93
35.94
46.45
26.5
92.8
94.1
94.0
44.9
114.6
114.9
115.4
1.250
1.283
1.287
1.287
1.357
1.295
1.291
1.290
1.437
1.453
1.457
1.457
a
Dielectric constants at 25 ꢁC of benzene (2.27), dichloromethane (8.93), acetonitrile (35.94), and DMSO (46.45) from Ref. 25.
Heat of formation.
The angles q and f are torsion angles C6–C5–C10–C11 and N1–C2–N7–C8, respectively.
The r(C]O), r(C–O), and r(C–C) are the bond lengths of C8–C9, C13–O14, and C5–C10, respectively.
b
c
d

6282
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
charge localized at the amidopyrazine moiety in the singlet-
excited state. These results predict that the negative charge
of 7(O)ꢀ is transferred from the 4-oxidophenyl group to
the amidopyrazine moiety upon electronic excitation, and
strongly support that 12(O)ꢀ* is the twisted ICT state
(Scheme 7). The amidopyrazine moiety of 2(O)ꢀ is the
center of the fluorescent chromophore and has a localized
negative charge in the singlet-excited state. Therefore, a
solvation of the amidopyrazine moiety of 12(O)ꢀ* by polar
solvent molecules will effectively increase its molecular
stability, resulting in a decreased transition energy from
12(O)ꢀ* to 2(O)ꢀ. This causes the solvent-dependent spec-
tral shift of the fluorescence of 2(O)ꢀ, which is observed
as the steep slopes of the EFL–ET(30) correlations for
[BASE–H+/3(O)ꢀ].
of imidazole: 14.2 in acetonitrile40), 12(O)ꢀ* and the imida-
zolium cation will make a CIP such as [NBA–H+/13(O)ꢀ*] at
the active sites in aequorin and obelin. By applying the EFL
–
ET(30) correlation for [NBA–H+/3a(O)ꢀ] to evaluate the
lBL of aequorin bioluminescence (465–470 nm), we can pre-
dict that the active site in aequorin has a polarity similar to
benzene (ET(30)¼34.3), as predicted in previous reports.17
The EFL–ET(30) correlation for [NBA–H+/3b(O)ꢀ] is also
useful for evaluating the reported lBL of a semi-synthetic ae-
quorin, m(5)-aequorin, prepared using 5-methylcoelentera-
zine analogue. The lBL of m(5)-aequorin (438–440 nm)24
is similar to the lFL (444 nm) of [NBA–H+/3b(O)ꢀ] in p-xy-
lene (ET(30)¼33.1), also supporting the less polar character
of the active site in aequorin. The lBL of obelin biolumines-
cence (485–495 nm) predicts that the active site has a polar-
ity intermediate between a mixed solvent of benzene/
chloroform (20:1) (ET(30)¼36.4) and 1-chloropropane
(ET(30)¼37.4). Thus, aequorin and obelin have less polar
active sites, where the CIP of 12(O)ꢀ* and the imidazolium
side chain show a high Ff. Based on these evaluations and
the X-ray crystal structures of aequorin6,39 and obelin,7 we
can draw a schematic representation of the important inter-
actions of 12(O)ꢀ* with the active site in aequorin or obelin,
shown in Scheme 9.
2.5. Bioluminescence light emitter and the reaction
mechanism of the calcium-activated photoproteins
The fluorescent properties of [BASE–H+/3a(O)ꢀ] and
[BASE–H+/5(N)ꢀ] are summarized in Table 9. The reported
emission maxima (lBL) of the bioluminescence of aequorin
and obelin are in the ranges of 465–470 nm11a,37 and 485–
495 nm38, respectively. Both ranges are covered by the lFL
range of [BASE–H+/3a(O)ꢀ], 452–615 nm, but not by that
of [BASE–H+/5(N)ꢀ], 464–476 nm. The lFL range of
[NBA–H+/3a(O)ꢀ], 452–554 nm, especially covers these
lBL ranges, while that of [TMG–H+/3a(O)ꢀ] and [DBU–
H+/3a(O)ꢀ], 486–615 nm, is red shifted compared with the
lBL range of aequorin. These results support the fact that
the real ionic structure of the excited coelenteramide in the
bioluminescence of aequorin and obelin is 12(O)ꢀ* in
a CIP, with a counter cation composed of a side chain of
an amino acid. The large spectral variation in the fluores-
cence of 3a(O)ꢀ is caused by the molecular stability change
The conclusion that 12(O)ꢀ* in the CIP is the light emitter in
aequorin and obelin bioluminescence leads us to propose
a bioluminescence reaction mechanism (Scheme 10) includ-
ing the active site structures of aequorin6,39 and obelin.7
Because 12(O)ꢀ* in the CIP is the primary excited product,
we can propose that the chemiexcitation process is a thermal
decomposition of the NH-form of dioxetanone intermediate
11ꢀ possessing a 4-oxidophenyl group.41 The chemiexcita-
tion from 11ꢀ to 12(O)ꢀ* in the CIP would be an important
process to achieve the high quantum yield in the biolumines-
cence reaction. On the whole, a structural change of the ac-
tive site due to chelation of Ca2+ ions with the EF-hands
induces the conversion of coelenterazine 2-hydroperoxide
8 to a reactive peroxide anion (92ꢀ), followed by a cycliza-
tion to an unstable amine anion form of dioxetanone inter-
mediate 102ꢀ. Dioxetanone dianion 102ꢀ receives a proton
from a neighboring 4-hydroxyphenyl group of a tyrosine resi-
due (Y132 and Y138 for aequorin and obelin, respectively)
in the active site to give 11ꢀ. Then, the chemiexciation
1
of 3a(O)ꢀ* at the twisted ICT state, depending on the ion
pair structure and the solvent polarity.
The X-ray crystal structures of aequorin6,39 and obelin7 indi-
cate that their active sites are composed of hydrophobic
a-helices and the substrates are surrounded by tryptophans,
tyrosines, histidines, and phenylalanines. Furthermore, the
imidazolium side chains of His 16 in aequorin and His 22
in obelin are able to be counter cations for the 4-oxidophenyl
group of 12(O)ꢀ*. Because the imidazole part of the histidine
side chain is a weak organic base (pKa of the conjugated acid
1
1
from 11ꢀ to 2(O)ꢀ* and the light emission from 2(O)ꢀ*
lead to FLP, which contains the hydrogen-bonded complex
Table 9. Comparison of the fluorescent properties of [BASE–H+/3a(O)ꢀ] and [BASE–H+/5(N)ꢀ]
C7H15
CH3
O
O
BASE-H+
+
BASE-H
N
N
N
N
N
NH
CH2Ph
CH2Ph
CH3O
O
[BASE–H+/3a(O)ꢀ]
[BASE–H+/5(N)ꢀ]
Character of the singlet-excited state of the anion
Variation of lFL depending on the solvent
polarity (observed lFL range)
Twisted ICT state
Large (452–615 nm)
Normal p–p* state
Small (464–476 nm)
Variation of lFL depending on the ion pair structure
High Ff condition
Large (SSIP vs CIP)
In a CIP with a conjugate acid of a weak base
such as n-butylamine in a less polar solvent
Small
0.2 in DMSO

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6283
Y184 (AQ),
Y190 (OB)
W129 (AQ)
less polar character of
apophotoprotein active site
H169 (AQ),
H175 (OB)
less polar like solvations
F113 (AQ)
in benzene for aequorin and
in a benzene/chloroform
mixed solvent for obelin
H
O
N
N
O
H
N
H
W173 (AQ),
W179 (OB)
H
H3C
O
O
T166 (AQ),
T172 (OB)
H
N
H
H
H
O
a conjugate acid of
a weak organic base
H
O
O
*
H
I105 (AQ),
I111 (OB)
O
H
N
H
or OH
H16 (AQ),
H22 (OB)
N
N
N
Y132 (AQ),
Y138 (OB)
O
H
N
H
H
Coulomb interaction
in the contact ion pair
O
O
H
F92 (OB)
H
12(O)−*
H
N
H
N
N
NH
W86 (AQ),
W92 (OB)
Y82 (AQ),
F88 (OB)
W108 (AQ),
W114 (OB)
H169 (AQ),
H175 (OB)
Scheme 9. (AQ¼aequorin, OB¼obelin).
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+ Ca2+
N
N
H
O
N
H
N
O
O
H
N
H
N
H
O
O
O
H
O
N
H
N
R
R
O
R
H
N
H
N
O
O
+ Ca2+
O
H
O
N
N
N
N
N
N
N
H
H
N
N
O
O
O
H
H
H
N
H
H
H
H
H
N
H
O
O
O
92−
102−
8
photoprotein (aequorin, obelin, etc)
protonation at the amine-anion moiety
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+ Ca2+
N
N
H
N
N
N
N
H
N
H
N
O
O
H
H
H H
N
H
H
R
O
R
O
R
R
O
H
N
H
N
H
N
O
CO2
*
O
high Φs
N
N
NH
N
N
NH
N
N
N
N
NH
N
O
O
H
O
N
N
N
− CO2
H
O
O
H
H
O
O
H
H
N
H
H
H
H
H
O
H
O
H
O
O
hydrogen-bonded
complex
ion-pair
ion-pair
12(O)−*
2H
11−
2(O)−
the NH-form of dioxetanone intermediate
bearing the 4-oxdophenyl group
the singlet-excited phenolate anion
(light-emitter)
fluorescent protein (FLP)
Scheme 10. R¼(4-hydroxyphenyl)methyl or (4-oxidophenyl)methyl.
of 2H with an imidazole side chain of a histidine residue
(H16 and H22 for aequorin and obelin, respectively).
Electronic excitation of FLP gives 12(O)ꢀ* in the CIP with
the imidazolium side chain by the process illustrated in
Scheme 6a, and shows a fluorescence emission spectrum
identical to the bioluminescence spectrum. The molecular
mechanism shown in Scheme 10 is common in the biolumi-
nescence system of calcium-activated photoproteins.1,42
elucidate the ionic structure of the light emitter in aequorin
and obelin bioluminescences. The fluorescence of [BASE–
H+/3(O)ꢀ] was observed by an electronic excitation of
[BASE–H+/3(O)ꢀ] or [BASE/3H] in a polar or a less polar
solvent (Scheme 6). Because the fluorescence emission from
13(O)ꢀ* in an ion pair has an ICT nature, the lFL of [BASE–
H+/3(O)ꢀ] was evaluated with linear EFL–ET(30) correla-
tions. A comparison of the EFL–ET(30) correlations and
the Ff values among the ion pairs of 3(O)ꢀ with NBA–H+,
TMG–H+, and DBU–H+ clarified that the fluorescence of
3(O)ꢀ was affected by a structural variation of an ion pair:
an SSIP for [TMG–H+/13(O)ꢀ*] or [DBU–H+/13(O)ꢀ*],
3. Conclusion
The fluorescent properties of [BASE–H+/3(O)ꢀ] and
[BASE–H+/5(N)ꢀ] were systematically investigated to
and a CIP for [NBA–H+/13(O)ꢀ*]. [NBA–H+/13(O)ꢀ*
]

6284
K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
showed especially steep EFL–ET(30) correlations and high
Ff compared with those of [TMG–H+/13(O)ꢀ*] and
[DBU–H+/13(O)ꢀ*]. Comparison of the fluorescent pro-
perties of 3a(O)ꢀ and 3b(O)ꢀ in ion pairs clarified that
13(O)ꢀ* is the twisted ICT state with a localized negative
nescence reaction was monitored using a Hamamatsu
R5929 photomultiplier tube powered by a Hamamatsu
C4900 power supply. The signal from the photomultiplier
was collected on a PC computer and the data were analyzed
with the graphic program Igor Pro, Version 4.0.8.0 (Wave
Metrics, Inc.). Semi-empirical MO calculations for ground
and singlet-excited states were carried out with the PM5
and PM5-CI methods43 and the COSMO model36 of
CAChe Ver. 5.04 for Windows (Fujitsu Ltd, Tokyo, Japan,
2002). The geometries of ground state molecules were fully
optimized by the MM2 calculations before PM5 calcula-
tions. Geometries of singlet-excited molecules were opti-
mized using the keywords excited singlet cisd c.i.¼4
root¼2.
charge at the amidopyrazine moiety (Scheme 7). This was
1
supported by PM5-CI-COSMO calculations for 7(O)ꢀ*
.
The fluorescence emission from [BASE–H+/15(N)ꢀ*] gener-
ated by the chemiluminescence reactions of 4 indicates that
the fluorescence of [BASE–H+/5(N)ꢀ] is not significantly af-
fected by interaction with BASE–H+ in the ion pair or the
solvent polarity. These results confirm that 12(O)ꢀ* in
a CIP is the light emitter in the bioluminescence of the rep-
resentative calcium-activated photoproteins, aequorin and
obelin. The EFL–ET(30) correlations for [NBA–H+/3(O)ꢀ]
were applied to evaluate the lBL values of aequorin and obe-
lin bioluminescences, suggesting that the active sites of
these photoproteins have a polarity similar to a less polar sol-
vent such as benzene. The CIP of 12(O)ꢀ* with an imidazo-
lium side chain of a histidine residue in the less polar active
site is a good state for a high Ff of 2(O)ꢀ in apoprotein. The
predicted character of the active sites of aequorin and obelin
is consistent with their structural characteristics established
by X-ray crystallographic analyses of aequorin6,39 and obe-
4.2. Preparation of coelenteramide and coelenterazine
derivatives
Coelenteramide analogue 3aH [3-benzyl-5-(4-hydroxy-
phenyl)-2-octanamidopyrazine] and coelenterazine analogue
4 [8-benzyl-6-(4-hydroxyphenyl)-2-methylimidazo[1,2-a]-
pyrazin-3(7H)-one] were prepared by reported proce-
dures.16,17b,44 Coelenteramide analogue 3bH [3-benzyl-
5-(4-hydroxyphenyl)-6-methyl-2-octanamidopyrazine] was
synthesized from 6-methylpyrazinamine as follows
(Scheme 4).
1
lin7 (Scheme 9). The conclusion that 2(O)ꢀ* in the CIP is
the light emitter indicates that 12(O)ꢀ* is the primary prod-
uct of the bioluminescence reaction. Therefore, we propose
a bioluminescence reaction mechanism including the chem-
4.2.1. 3,5-Dibromo-6-methylpyrazinamine. To a solution
of 6-methylpyrazinamine45 (0.97 g, 8.8 mmol) in CHCl3
(30 mL) and pyridine (3.5 mL) was added tetra-n-butyl-
ammoniumtribromide (10.5 g, 0.21 mol) under Ar. Thereac-
tion mixture was heated under reflux for 2 h. After cooling,
the mixture was concentrated under reduced pressure. The
residue was diluted with water (100 mL) and the product
was extracted with ether (200 mLꢂ4). The ether phase was
washed with saturated brine, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (CHCl3),
yielding 3,5-dibromo-6-methylpyrazinamine (1.10 g, 46%)
1
iexcitation from dioxetanone intermediate 11ꢀ to 2(O)ꢀ*
(Scheme 10). Further study to establish the reaction mecha-
nism is now in progress in our lab.
4. Experimental
4.1. General
Melting points were obtained with a Yamato MP-21 appara-
tus and were uncorrected. IR spectra were measured with
a Horiba FT-720 spectrometer. Electron ionization (EI)
mass spectra were recorded with a JEOL JMS-600 mass
spectrometer. High-resolution EI and electro-spray ioniza-
tion (ESI) mass spectra were recorded with a JEOL HX-
110 and a Bruker-Daltonics APEX III mass spectrometer,
respectively, at the Research Centre for Giant Molecules,
1
as pale yellow cubes: mp 167–168 ꢁC; H NMR (CDCl3)
d 4.93 (br s, 2H), 2.46 (s, 3H); IR (KBr) 3499, 3292, 3155,
1632, 1540, 1448 cmꢀ1; MS (EI, 70 eV) m/z (%) 269 (49),
267 (M+, 100), 265 (51); HRMS (ESI) calcd for
C5H6Br2N3 265.8928; found 265.8923 (M+H+).
1
Graduate School of Science, Tohoku University. H and
4.2.2. 3-Benzyl-5-bromo-6-methylpyrazinamine. A solu-
tion of 3,5-dibromo-6-methylpyrazinamine (114 mg,
0.43 mmol), benzyltri-n-butyltin (195 mg, 0.51 mmol) and
PdCl2(PPh3)2 (15 mg, 5 mol %) in DMF (1 mL) was heated
at 130 ꢁC for 2.5 h under Ar. After cooling, the solution was
concentrated under reduced pressure. The residue was dis-
solved in a saturated KF solution in methanol (7 mL) and
the mixture was stirred for 1 h at room temperature. The re-
action mixture was directly adsorbed on silica gel (5 g) and
purified by silica gel column chromatography (CHCl3/ethyl
acetate¼20:1), yielding 3-benzyl-5-bromo-6-methylpyra-
zinamine (43 mg, 36%) as pale yellow cubes: mp 94.5–
13C NMR spectra were recorded on a JEOL GX-270 instru-
ment (270 and 67.8 MHz, respectively). UV–vis absorption
spectra were measured with a Varian Cary 50 spectrophoto-
meter (scan speed, 200 nm minꢀ1; data interval, 0.33 nm).
Fluorescence spectra were measured with a JASCO FP-
6500 fluorescence spectrophotometer (excitation and emis-
sion bandpasses, 3 nm; scan speed, 200 nm minꢀ1) and
were corrected according to manufacturer’s instructions.
Fluorescence quantum yields were determined relative to
quinine sulfate in 0.10 mol Lꢀ1 H2SO4 (FF¼0.55, lex
366 nm) as the standard. Chemiluminescence spectra were
measured with an ATTO AB-1850 spectrophotometer.
Spectroscopic measurements were made in a quartz cuvette
(1 cm path length) at 25ꢃ1 ꢁC. Spectral-grade solvents were
used for measurements of UV–vis absorption, fluorescence,
and chemiluminescence emission spectra. The intensity
of the total light (400–700 nm) emitted from a chemilumi-
1
95.5 ꢁC; H NMR (CDCl3) d 7.20–7.36 (m, 5H), 4.28 (br
s, 2H), 4.06 (s, 2H), 2.49 (s, 3H); IR (KBr) 3465, 3305,
3165, 1633, 1602, 1533, 1429 cmꢀ1; MS (EI, 70 eV) m/z
(%) 279 (98), 277 (M+, 100), 276 (41), 198 (25), 130 (26);
HRMS (ESI) calcd for C12H13BrN3 278.0293; found
278.0287 (M+H+).

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6285
4.2.3. 3-Benzyl-5-(4-benzyloxyphenyl)-6-methylpyrazin-
amine. A solution of 3-benzyl-5-bromo-6-methylpyrazin-
amine (64 mg, 0.23 mmol), 4-benzyloxyphenylboronic
acid (78 mg, 0.34 mmol), and Pd(PPh3)4 (13 mg, 5 mol %)
in 1,4-dioxane (1.2 mL) and 2 mol Lꢀ1 Na2CO3 aqueous so-
lution (1.2 mL) was heated at 110 ꢁC for 2 h under Ar. After
cooling, the mixture was diluted with water and the product
was extracted with ethyl acetate (20 mLꢂ3). The organic
phase was washed with saturated brine, dried over anhy-
drous Na2SO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromato-
graphy (CHCl3/ethyl acetate¼5:1), yielding 3-benzyl-5-(4-
benzyloxyphenyl)-6-methylpyrazinamine (58 mg, 94%) as
pale yellow powder: mp 125.5–126.5 ꢁC; 1H NMR
(CDCl3) d 7.50 (AA0BB0, 2H), 7.21–7.45 (m, 10H), 7.06
(AA0BB0, 2H), 5.13 (s, 2H), 4.29 (br s, 2H), 4.19 (s, 2H),
2.45 (s, 3H); IR (KBr) 3478, 3303, 3168, 1633, 1610,
1449, 1425 cmꢀ1; MS (EI, 70 eV) m/z (%) 382 (19), 381
(M+, 65), 290 (100); HRMS (ESI) calcd for C25H24N3O
382.1919; found 382.1914 (M+H+).
m/z (%) 418 (30), 417 (M+, 100), 291 (72), 290 (44);
HRMS (EI, 70 eV) calcd for C26H31N3O2 417.2416; found
417.2424 (M+).
4.3. Formation constant K of the 1:1 hydrogen-bonded
complexes of 3aH with TMG and DBU in benzene
The equilibrium to form the 1:1 hydrogen-bonded complex
[BASE/3aH] is shown in Scheme 5.17b To estimate the K of
[TMG/3aH] and [DBU/3aH] in benzene, we measured
UV–vis absorption spectra of 3aH in benzene in the absence
and the presence of various concentrations of TMG and
DBU, as shown in Figure 5. The concentrations of TMG
and DBU were in the ranges of 2.0ꢂ10ꢀ5–0.050 and
1.0ꢂ10ꢀ5–0.010 mol Lꢀ1, respectively.
The observed spectral changes with clear isosbestic points
were analyzed by Eq. 1 below.46 The value A0 is absorbance
of 3aH at a chosen wavelength in the absence of BASE.
After addition of BASE, absorbance at the chosen wave-
length is observed as Aobs. The difference between Aobs
and A0 is given by
4.2.4. 3-Benzyl-5-(4-benzyloxyphenyl)-6-methyl-2-octa-
namidopyrazine. To a solution of 3-benzyl-5-(4-benzyl-
oxyphenyl)-6-methylpyrazinamine (45 mg, 0.12 mmol) and
pyridine (0.2 mL) in dichloromethane (2 mL) was added
octanoyl chloride (70 mL, 0.42 mmol) at 0 ꢁC under Ar, and
allowed to warm up to ambient temperature for 90 min.
The reaction was quenched by the addition of saturated
NaHCO3 aqueous solution and the product was extracted
with CHCl3. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3/ethyl
acetate¼10:1), yielding 3-benzyl-5-(4-benzyloxyphenyl)-6-
methyl-2-octanamidopyrazine (31 mg, 52%) as colorless
powder: mp 156–157 ꢁC; 1H NMR (270 MHz, CDCl3)
d 7.54 (AA0BB0, 2H), 7.16–7.48 (m, 10H), 7.07 (AA0BB0,
2H), 5.14 (s, 2H), 4.23 (s, 2H), 2.55 (s, 3H), 2.36 (t,
J¼7.6 Hz, 2H), 1.30 (m, 10H), 0.89 (t, J¼6.9 Hz, 3H); IR
ꢀ
ꢁ
Aobs ꢀ A0 ¼ 0:5D3 ½3aHꢄ þ ½BASEꢄ þ 1=K ꢀ ð½3aHꢄ
ꢃ
ꢂ
1=2
2
þ ½BASEꢄ þ 1=KÞ ꢀ4½3aHꢄ½BASEꢄ
;
ð1Þ
where [3aH] and [BASE] are the initial concentrations of
3aH and BASE, respectively, and D3 is the difference in
the molar absorptivity between 3aH and [BASE/3aH] at
the chosen wavelength. The (AobsꢀA0) values were plotted
against [TMG] and [DBU], as shown in Figure 5B, D. The
data were analyzed by a nonlinear least squares curve fitting
Eq. 1, giving the K values 1430ꢃ40 and 7800ꢃ400 molꢀ1 L
at 25 ꢁC, respectively, accompanied by the D3 values
6680ꢃ40 (350 nm) and 8300ꢃ100 (355 nm) molꢀ1 L cmꢀ1
,
respectively.
(KBr) 3274, 2926, 2854, 1668, 1608, 1567, 1496 cmꢀ1
;
MS (EI, 70 eV) m/z (%) 508 (38), 507 (M+, 100), 290
(51); HRMS (ESI) calcd for C33H38N3O2 508.2964; found
508.2962 (M+H+).
4.4. Formation constant K of the ion pair of 3b(O)L with
TMG–H+ in DMSO
The formation constant K (Scheme 5) of [TMG–H+/3b(O)ꢀ]
was estimated by an analysis of an absorption spectral
change of 3bH (1.5ꢂ10ꢀ5 mol Lꢀ1) in DMSO in the ab-
sence and the presence of various concentrations of TMG
(2.0ꢂ10ꢀ3–0.25 mol Lꢀ1), as shown in Figure 6.
4.2.5. 3-Benzyl-5-(4-hydroxyphenyl)-6-methyl-2-octa-
namidopyrazine (3bH). To a solution of 3-benzyl-5-(4-
benzyloxyphenyl)-6-methyl-2-octanamidopyrazine (31 mg,
0.062 mmol) in 20 mL of ethanol/ethyl acetate (1:1) was
added 12 mg of Pd/C powder. The suspension was stirred
at room temperature overnight under H2 and was filtered
through Celite. The filtrate was concentrated in vacuo and
the residue was purified by recrystallization, yielding 3-
benzyl-5-(4-hydroxyphenyl)-6-methyl-2-octanamidopyrazine
(3bH) (17 mg, 67%) as colorless cubes: mp 176.5–177 ꢁC;
1H NMR (CDCl3) d 7.41 (AA0BB0, 2H), 7.16–7.30 (m, 5H),
6.82 (AA0BB0, 2H), 6.12 (br s, 1H), 4.27 (s, 2H), 2.50 (s,
3H), 2.36 (t, J¼7.8 Hz, 2H), 1.64 (quint, 2H), 1.28–1.31 (m,
8H), 0.89 (t, J¼8.9 Hz, 3H); 13C NMR (CDCl3) d 173.0 (s),
156.6 (s), 150.5 (s), 147.4 (s), 142.0 (s), 140.3 (s), 138.0 (s),
130.6 (d, 2C), 130.1 (s), 128.7 (d, 2C), 128.6 (d, 2C), 126.7
(d), 115.3 (d), 40.7 (t), 36.7 (t), 31.6 (t), 29.2 (t), 29.0 (t),
25.1 (t), 22.6 (q), 22.4 (t), 14.1 (q); IR (KBr) 3293, 2927,
2854, 1663, 1611, 1569, 1496, 1400 cmꢀ1; MS (EI, 70 eV)
The observed spectral change gave the differences between
obs and A0, which were analyzed by Eq. 2:
A
ꢀ
ꢁ
Aobs ꢀ A0 ¼ 0:5D3 ½3bHꢄ þ ½TMGꢄ þ 1=K ꢀ ð½3bHꢄ
ꢃ
ꢂ
1=2
2
þ ½TMGꢄ þ 1=KÞ ꢀ4½3aHꢄ½TMGꢄ
;
ð2Þ
where [3bH] and [TMG] are the initial concentrations and
D3 is the difference in the molar absorptivity between 3bH
and [TMG–H+/3b(O)ꢀ] at a chosen wavelength.46 The
(AobsꢀA0) values were plotted against [TMG], as shown in
Figure 6B, and were analyzed by a nonlinear least squares

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K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
0.8
0.6
0.4
0.2
0.0
A
B
0.3
0.2
0.1
0.0
300
350
400
−5
−4
−3
−2
−1
wavelength / nm
log[TMG]
D
C
0.2
0.1
0.0
0.10
0.05
0.00
−5
−4
−3
−2
300
350
400
wavelength / nm
log[DBU]
Figure 5. UV–vis absorption spectra of 3aH in benzene containing various concentrations of TMG (A) and DBU (C) at 25 ꢁC and plots of (AobsꢀA0) at 350 nm
versus log[TMG] (B) and (AobsꢀA0) at 355 nm versus log[DBU] (D) for 1:1 hydrogen-bonded complexation. The initial concentrations of 3aH were 5.0 and
1.5ꢂ10ꢀ5 mol Lꢀ1 for A and B, respectively. The concentration ranges of TMG and DBU were 2.0ꢂ10ꢀ5–0.050 and 1.0ꢂ10ꢀ5–0.010 mol Lꢀ1, respectively.
The dotted lines in B and D are the fitted curves corresponding to 1:1 complexation.
curve fitting Eq. 2, giving the K value 28ꢃ3 molꢀ1 L at
25 ꢁC, accompanied with the D3 value 4480ꢃ160
and the methanol was removed in vacuo. The cuvette con-
taining 4 was placed in a luminometer with a photomultiplier
tube and an aerated organic solvent (2.0 mL) was injected to
the cuvette at 25ꢃ1 ꢁC. The chemiluminescence reactions of
4 were traced by monitoring intensity of the total emitted
light. The Fcl values were determined as values relative
to the Fcl value (0.013) of luminol in DMSO containing
t-BuOK/t-BuOH under air.47 The experimental errors of
Fcl were within ꢃ10%.
(420 nm) molꢀ1 L cmꢀ1
.
4.5. Quantum yields of the chemiluminescence reactions
of 4
A small portion (10 or 20 mL) of a stock solution of 4
(2.0ꢂ10ꢀ3 mol Lꢀ1) in methanol was put in a Pyrex cuvette
0.2
0.1
0.0
B
A
0.06
0.04
0.02
0.00
−3
−2
−1
300
350
400
450
500
wavelength / nm
log[TMG]
Figure 6. UV–vis absorption spectra of 3bH (1.5ꢂ10ꢀ5 mol Lꢀ1) in DMSO containing various concentrations of TMG (2.0ꢂ10ꢀ3–0.25 mol Lꢀ1) at 25 ꢁC (A)
and plot of (AobsꢀA0) at 420 nm versus log[TMG] for ion pair generation (B). The dotted line in B is the fitted curve for ion pair generation.

K. Mori et al. / Tetrahedron 62 (2006) 6272–6288
6287
16. Saito, R.; Hirano, T.; Niwa, H.; Ohashi, M. J. Chem. Soc.,
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We thank Mr Toshiteru Enomoto at ATTO Co. and Dr
Yoshihiro Ohmiya at the National Institute of AIST for their
excellent technical assistance in measuring chemilumines-
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Professors Mamoru Ohashi and Frederick I. Tsuji for their
encouragement of this research. This work was supported
by grants from the Ministry of Education, Culture, Sports,
Science, and Technology [No. 15404009 for H.N. and No.
14050008 (Priority Area No. 417) for H.I.]. H.I. gratefully
acknowledges financial supports from the Izumi Science
and Technology Foundation and the Shorai Foundation.
We thank Professor Minoru Ueda of Tohoku University for
his generous considerations.
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a luciferin. At present our molecular mechanism for calcium-
activated photoproteins (Scheme 10) is not applicable to the
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41. Very recently, a reaction mechanism similar to ours was pro-
posed based on evaluating the active site structure of FLP of
obelin.7d Our mechanism contains the chemiexcitation process
from dioxetanone intermediate 11ꢀ possessing a 4-oxido-
1
phenyl group to 2(O)ꢀ*, while their mechanism contains the
chemiexcitation process from a neutral dioxetanone inter-
mediate to neutral excited molecule 12H*. Thus, there is an im-
portant difference in the chemiexcitation processes between
the two mechanisms.
42. The authors thank one of the referees for his/her valuable com-
ment on the other bioluminescence using coelenterazine as