Influence of electron-donating and electron-withdrawing substituents on the Chemiluminescence behavior of Coelenterazine analogs

Article abstract of DOI:10.1246/bcsj.20100185

Coelenterazine analogs 3a3e possessing various substituents at the para-position of the 6-phenyl group (R = CF₃, F, H, OMe, and NMe ₂) were synthesized, and the chemiluminescent properties of 3a3e in dimethyl sulfoxide (DMSO) were investigated quantitatively. Chemiluminescence maxima observed in the range of 454478nm showed a bathochromic shift as the electron-donating ability of R increased. Chemiluminescence quantum yields (φ_CL) were obtained in the range of 0.00060.0018. The analog 3a possessing the electron-withdrawing CF₃ group showed a slight decrease in its φ_CL value. The fluorescence quantum yields (φ_F) of the light emitter, 2-acetamidopyrazine anions 4a ^--4e^- , were observed in the range of 0.0050.21 and showed substituent dependency in that the increment in the electronwithdrawing ability of R decreases the φ_F value. The efficiency of generation of the singlet-excited 4a^--4e^- (φ_S) showed a small change in the range of 0.0080.015. From these quantitative analyses of the quantum efficiencies, we found that an electron-donating substituent R is not required for the efficient generation of a singlet-excited light emitter, but is required for the high φ_F of the emitter.

Full text of DOI:10.1246/bcsj.20100185

90
Bull. Chem. Soc. Jpn. Vol. 84, No. 1, 90–99 (2011)
© 2011 The Chemical Society of Japan
Influence of Electron-Donating and Electron-Withdrawing Substituents
on the Chemiluminescence Behavior of Coelenterazine Analogs
Ryota Saito,*1 Takashi Hirano,2 Shojiro Maki,2 Haruki Niwa,2 and Mamoru Ohashi2,#
1Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510
2Department of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585
Received July 5, 2010; E-mail: saito@chem.sci.toho-u.ac.jp
Coelenterazine analogs 3a-3e possessing various substituents at the para-position of the 6-phenyl group (R = CF3,
F, H, OMe, and NMe2) were synthesized, and the chemiluminescent properties of 3a-3e in dimethyl sulfoxide (DMSO)
were investigated quantitatively. Chemiluminescence maxima observed in the range of 454-478 nm showed a
bathochromic shift as the electron-donating ability of R increased. Chemiluminescence quantum yields (ΦCL) were
obtained in the range of 0.0006-0.0018. The analog 3a possessing the electron-withdrawing CF3 group showed a slight
¹
decrease in its ΦCL value. The fluorescence quantum yields (ΦF) of the light emitter, 2-acetamidopyrazine anions 4a -
¹
4e , were observed in the range of 0.005-0.21 and showed substituent dependency in that the increment in the electron-
¹
¹
withdrawing ability of R decreases the ΦF value. The efficiency of generation of the singlet-excited 4a -4e (ΦS) showed
a small change in the range of 0.008-0.015. From these quantitative analyses of the quantum efficiencies, we found that
an electron-donating substituent R is not required for the efficient generation of a singlet-excited light emitter, but is
required for the high ΦF of the emitter.
Coelenterazine (1) (Chart 1) is well known as a common
CH2X
N
O
O
CH2X
NH
luminescent substrate of various luminescent marine organ-
isms.1-5 A photoprotein called aequorin, isolated from the
jellyfish Aequorea aequorea, is one such representative
luminescent system that contains 1. Aequorin is made up of
an apoprotein (apoaequorin), 1, and oxygen. In aequorin, 1
exists as a peroxide which is formed by the reaction of 1 with
O2 at the C2 position.6 Upon binding with calcium ions, a
conformational change is induced in the protein, after which
the peroxide intermediate undergoes the luminescent reaction
in the polypeptide environment to yield coelenteramide (2) and
carbon dioxide. During this bioluminescence reaction, the
singlet-excited state of the phenolate anion of 2 is generated
and emits blue light (-max = 465 nm). As a chemiluminescence
process, 1 also undergoes a reaction with molecular oxygen in
an aprotic solvent such as dimethyl sulfoxide (DMSO) without
any support of a protein. It has been suggested that the bio- and
chemiluminescence reaction of 1 follows the same molecular
mechanism as that shown in Scheme 1, in which the thermal
decomposition of the 1,2-dioxetanone intermediate (I) is an
important chemical process in the generation of the electroni-
cally excited state of the emitter (II). Although the same
mechanism is adopted for explaining the bio- and chemilumi-
nescence of 1, there is a remarkably substantial difference
between their luminescence efficiencies. That is, the chemi-
luminescence efficiency of 1 (Φ = 0.002 in DMSO) is
considerably lower than that of aequorin bioluminescence
(Φ = 0.2). Quantitative analyses of chemiluminescence quan-
tum yields of coelenterazine-related compounds has revealed
that quantum efficiencies in the steps of singlet-excited emitter
generation (from I to II) are very small (0.2-1.5%),7,8 while in
bioluminescence the singlet-excited emitter is believed to be
N
N
N
N
H
R
R
1: X = C6H4(p-OH), R = OH 2: X = C6H4(p-OH), R = OH
3a: X = H, R = CF3
3b: X = H, R = F
3c: X = H, R = H
3d: X = H, R = OMe
3e: X = H, R = NMe2
4a: X = H, R = CF3
4b: X = H, R = F
4c: X = H, R = H
4d: X = H, R = OMe
4e: X = H, R = NMe2
Chart 1.
formed quantitatively.9 In addition, there have been papers
pointing out that the high-quantum-yield bioluminescence
involves a high efficiency in the singlet-excited emitter
generation.10 The reason behind the difference in the efficacies
of singlet-excited emitter generation has long been the main
focus of bio- and chemiluminescence studies and remains a
subject of controversy.
To explain the effective generation of singlet-excited
molecules in the decomposition of dioxetane compounds,
Schuster proposed a chemically initiated electron exchange
luminescence (CIEEL) mechanism that involves complete one-
electron transfer, followed by the annihilation of the generated
radical ions (Scheme 2a).11 Several reports, however, have
demonstrated that CIEEL reactions do not necessarily produce
Published on the web January 1, 2011; doi:10.1246/bcsj.20100185

R. Saito et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
91
O
O
*
R1
N
O
2
O
R1
O
R1
R1
O
N
–H+
O2
– CO2
N
N
N
N
N
N
N
N
2
6
5
R3
N
R2
R3
N
R2
R3
R2
R3
R2
H
hν
dioxetanone
intermediate (I)
singlet-excited state
of amide anion (II)
amide anion in
ground state (III)
Scheme 1.
CIEEL mechanism
O
O
O
R1
one-electron
transfer
CO2
R1
O
N
annihilation
decomposition
N
N
N
N
a
R3
R2
R3
N
R2
radical-ion pair
O
O
*
R1
O
R1
O
N
N
N
N
N
5
R3
R2
R3
N
R2
singlet-excited state of
coelenteramide anion
dioxetanone intermediate
δ–
O
O
b
R1
O
N
N
δ+
decomposition
charge transfer
(CT)
R3
N
R2
CT state
Scheme 2.
singlet-excited molecules in good yields.12 As an alternative
explanation, it has been suggested that the decomposition of
dioxetanone intermediates proceeds via charge-transfer transi-
tion states, and not via the completely separated ion pairs, as in
the CIEEL mechanism (Scheme 2b).12a,13 Although it has not
been determined which explanation is correct, both explan-
ations suggest the necessity of a strong electron donor within
a molecule for an effective luminescence reaction. Therefore,
it is important to determine the bio- and chemiluminescence
efficiency of the electron-donating hydroxy group on the 6-
phenyl group of 1 by regulating the electronic properties of the
reaction intermediates in Scheme 1. In order to experimentally
clarify the relevance of an electron-donating substituent at the
para-position (C4¤) of the 6-phenyl group for the efficient
generation of a singlet-excited emitter in the bioluminescent
reactions of 1, we synthesized coelenterazine analogs 3a-3e
possessing various substituents R (CF3, F, H, OMe, and NMe2),
whose Hammett substituent constant ·P was systematically
varied, and reported the preliminary results of their chemi-
luminescence.8 Herein, we report the detailed spectroscopic
and chemiluminescent properties of 3a-3e, and discuss whether
or not the electron-donating or electron-withdrawing property
of the substituent R essentially effects the efficacy of generating
a singlet-excited-state emitter during the oxidative lumines-
cence reaction of 3a-3e.
Results and Discussion
Synthesis and Electronic Absorption Properties of
Coelenterazine Analogs. Coelenterazine analogs 3a-3e were
synthesized by the condensation of pyruvaldehyde and the
corresponding coelenteramine derivatives 5a-5e,14 as shown in
Scheme 3. Compounds 6a, 6c,15 and 6d16 were also synthe-
sized as references for discussing the electronic absorption
properties of 3a-3e (Scheme 4).
In order to elucidate the electronic effect of the substituent
R on the central imidazopyrazinone ³-system, electronic
absorption spectra of 3a-3e were measured. The results are
listed in Table 1 (see also Figure S1). Each analog showed four
transition bands in the region of 200-500 nm. The lowest
transition band around 420 nm showed a slight bathochromic

92
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
Chemiluminescence of Coelenterazine Analogs
Table 1. Electronic Absorption Data of Coelenterazine Analogs 3a-3e, 6a, 6c, and 6d in Methanol
Experimental
max/nm (¾/103 dm3 mol¹1 cm
Calculateda)
-max/nm (oscillator strength)
Compound (R)
¹1
-
)
3a (CF3)
3b (F)
3c (H)
3d (OMe)
3e (NMe2)
6a (CF3)
6c (H)
420 (sh 0.67) 388 (0.74) 265 (2.19)
413 (0.65)
426 (0.84)
427 (0.82)
432 (0.86)
352 (0.56) 279 (sh 1.55) 254 (1.97)
358 (0.54) 280 (sh 1.32) 246 (2.12)
349 (0.47) 260 (2.23)
304 (2.57) 276 (sh 1.40) 223 (sh 1.34)
428 (sh 0.64) 377 (0.91) 256 (2.17)
414 (0.27) 361 (0.26) 251 (0.22) 223 (0.50) 204 (0.38)
422 (0.29) 352 (0.25) 248 (0.26) 223 (0.46) 202 (0.40)
432 (0.30) 346 (0.18) 257 (0.17) 230 (0.33) 205 (0.40)
424 (0.61)
428 (0.73)
355 (0.55) 248 (1.80)
351 (0.55) 263 (2.07)
6d (OMe)
a) Calculated as in methanol (dielectric constant ¾ = 32.7) using the INDO/S method. Transitions with oscillator strength less than 0.2
are omitted.
N
N
NH2
N
N
NH2
H
O
Me
O
H
O
Me
O
HCl
+
+
3a–e
EtOH
R
Me
N
R
O
5a: R = CF3
5b: R = F
HCl
N
5c: R = H
5d: R = OMe
5e: R = NMe2
EtOH
N
H
R
Scheme 3.
6a: R = CF3
6c: R = H
shift with increasing electron-donating ability of the substituent
R. In contrast, the second-lowest-energy transition band around
350 nm is markedly sensitive to the substituent constant of R
and showed a hypsochromic shift accompanied by a decrease
in the absorption coefficient with increasing electron-donating
ability of R. Two other transition bands below 300 nm, which
could be assigned to the ³-³* transitions of the 6-phenyl
group, tend to separate with increasing electron-donating
ability of R. Compounds 6a, 6c, and 6d exhibited the same
behavior, suggesting that these absorption bands originate in
the ³-conjugate system composed of the central imidazopyr-
azinone skeleton and the 6-phenyl group. Table 1 also lists the
computer-simulated electronic transitions of 6a, 6c, and 6d in
methanol. In these calculations, the optimized structures of 6a,
6c, and 6d in methanol were first calculated using the AM1-
COSMO method.17 According to these calculations, 6a, 6c, and
6d were found not to take planar structures, and the dihedral
angles between the 6-phenyl groups and the central pyrazine
were estimated to be 39.0, 40.9, and 41.5°, respectively. With
these optimized structures, their electronic excitation spectra
were simulated using INDO/S (intermediate neglect of the
differential overlap model parameterized for spectroscopy),18
which is a convenient and widely used method for simulating
electronic transition properties of organic dyes.19 As a result,
the second transition band was shifted hypsochromically with
an increase in the electron-donating ability of R, a result that
coincides with the experimental data. This indicates that these
semiempirical calculations provide us with adequate data to
understand the substituent effect on the electronic absorption of
the imidazopyrazinones. Thus, in order to assign the observed
lowest- and second-lowest-energy transition bands for the
imidazopyrazinones, the electronic transition of 6c was further
6d: R = OMe
Scheme 4.
investigated using the INDO/S method. The results are
summarized in Table 2. According to this calculation, the
lowest-energy transitions around 420 nm are assignable as the
transition between HOMO and LUMO, and the second-lowest-
energy transition around 350 nm is assignable as that between
HOMO and LUMO+1. Figure 1 shows the HOMO, LUMO,
and LUMO+1 of 6c as obtained by the AM1-COSMO
calculation. LUMO+1 has a considerably large electronic
distribution on the 6-phenyl group, while HOMO and LUMO
have only a negligible electronic distribution on the 6-phenyl
group. In particular, LUMO+1 has a large coefficient at C4¤ of
the 6-phenyl group, which indicates that the energy level of
LUMO+1 is affected by the substituent R. The LUMO+1 level
of 6c would be elevated by introducing an electron-donating
substituent R, thus causing a marked hypsochromic shift of the
second-lowest-energy transition. These calculated results well
coincide with the observations. From these results, it can be
safely said that the second-lowest-energy transitions of 3 and 6
are directly related to the ³-conjugations between the central
imidazopyrazinone and the 6-phenyl groups. To support this
consideration, the effect of the dihedral angle between the
imidazopyrazinone skeleton and the 6-phenyl group in 6c on its
absorption maxima was simulated by INDO/S. The results are
listed in Table 3. Obviously, the second-lowest-energy transi-
tion band was shifted bathochromically with a decrease in the
dihedral angle. Similar shifts in the second-lowest-energy ab-
sorption bands have been observed with a ring-fused coelenter-
azine analog, 7 (Chart 2).20 This compound exhibited second-

R. Saito et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
93
Table 2. Computed Electron Transition Spectra for Singlet State of 6c in Methanol Calculated by INDO/S Methoda)
Transition energy
State
Oscillator strength
Main CSFs MOb)
CI coefficientc)
Energy/eV
Wavelength/nm
1
2
2.937
3.524
422
352
0.289
0.247
HOMO ¼ LUMO
0.923
0.856
0.346
0.820
0.290
0.744
0.535
HOMO ¼ LUMO+1
HOMO ¼ LUMO+3
HOMO¹1 ¼ LUMO
HOMO ¼ LUMO+4
HOMO¹1 ¼ LUMO+1
HOMO ¼ LUMO+4
3
4
4.365
4.994
284
248
0.163
0.264
a) Transitions with oscillator strength less than 0.1 are omitted. b) Main configuration state function’s molecular orbital;
electronic configurations that dominate the corresponding transition energy, as described in terms of their molecular orbitals.
c) Absolute configuration interaction coefficient for the CSF.
–0.152
O
–0.151
O
0.244
0.019N
–0.339
O
0.263
0.320 N
–0.023
–0.127
0.401
–0.400
0.370
–0.072
0.220
–0.230N
N
N
N
–0.483
0.389
0.203
0.204
–0.310
–0.058
–0.458
–0.199
–0.099
0.029
–0.010
–0.008
–0.278
–0.049
–0.014
–0.125
0.603
–0.0864
N
0.021 N
N
0.363
0.277
–0.087
0.057
0.053
–0.018
–0.357
0.287
0.053
–0.005
0.018
–0.187
–0.070
–0.111
HOMO
LUMO
LUMO+1
Figure 1. Pertinent molecular orbitals for lowest-energy transition (HOMO ¼ LUMO) and second-lowest-energy transition
(HOMO ¼ LUMO+1) of imidazopyrazinone derivative 6c, calculated using AM1 method. The solvent effect was simulated by
use of the COSMO keyword.
Table 3. Electronic Absorption Spectra as a Function of Dihedral Angle (ª) between the Imidazopyrazinone Skeleton
and the 6-Phenyl Group of 6c Simulated by the INDO/S Method
ª/dega)
-max/nm (oscillator strength)
0
15
30
40
50
60
75
90
421 (0.28) 368 (0.30) 288 (0.16) 256 (0.55) 240 (0.14) 219 (0.25) 202 (0.15)
422 (0.28) 365 (0.29) 288 (0.16) 256 (0.37) 254 (0.16) 241 (0.20) 221 (0.24) 203 (0.15)
422 (0.28) 358 (0.26) 286 (0.17) 251 (0.37) 241 (0.18) 222 (0.34) 201 (0.19)
422 (0.29) 352 (0.25) 284 (0.16) 249 (0.27) 241 (0.17) 223 (0.45) 201 (0.39)
422 (0.29) 346 (0.23) 282 (0.16) 246 (0.16) 241 (0.17) 222 (0.56) 203 (0.14) 202 (0.40)
422 (0.30) 340 (0.21) 281 (0.15) 240 (0.18) 220 (0.61) 202 (0.48)
422 (0.31) 332 (0.18) 279 (0.15) 240 (0.22) 215 (0.51) 203 (0.30)
422 (0.31) 329 (0.18) 278 (-.16) 239 (0.20) 210 (0.30)
a)
Me
O
N
N
θ
N
H
6c
lowest-energy absorption bands with maxima at longer wave-
lengths than that of 1, which indicates that this absorption band
is strongly affected by the degree of ³-conjugation between the
central imidazopyrazinone ring and the 6-phenyl group.
Chemiluminescent Property of 3a-3e in DMSO. Chemi-
luminescence Emission Maxima: The chemiluminescence of
3a-3e in DMSO was observed at 25 °C under an aerated
condition. In this study, as a solvent we used DMSO, which is
frequently used as a solvent for the chemiluminescence of
imidazopyrazinones. The observed emission maxima (CLmax
and quantum yields (ΦCL) are summarized in Table 4,
accompanied by the fluorescence emission maxima (FLmax
of amide anions and neutral forms of the corresponding
coelenteramide analogs 4a-4e.14 Figure 2 shows representative
chemiluminescence spectra of the analogs 3a, 3c, and 3e as
well the fluorescence spectra of the amide anions of 4a, 4c, and
)
)

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Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
Chemiluminescence of Coelenterazine Analogs
Table 4. Chemiluminescence Properties of Coelenterazine Analogs 3a-3e in DMSO and Fluorescence
Properties of Coelenteramide Analogs 4a-4e in DMSO and DMSO Containing 1 M NaOH (0.5% v/v)
under Aerated Condition at 25 °C
FLmaxb)/nm
Quantum yield
R
CLmaxa)/nm
In DMSO
d)
e)
f)
g)
In DMSO
ΦCL
ΦR
ΦF
ΦS
+ NaOHaq
CF3
F
H
OMe
NMe2
454
467
462
473
479
450
466
462
466
476
388c)
376c)
375c)
396c)
505c)
0.0006
0.0015
0.0015
0.0018
0.0015
0.78
1.00
0.93
0.95
0.96
0.05
0.12
0.18
0.21
0.19
0.015
0.012
0.008
0.009
0.008
a) Chemiluminescence maxima of coelenterazine analogs 3a-3e. b) Fluorescence maxima of
coelenteramide analogs 4a-4e. c) Values taken from Ref. 14. d) Chemiluminescence quantum yield of
coelenterazine analogs 3a-3e determined on the basis of luminol. e) Chemical yield of the
chemiluminescence emitter as determined by HPLC analyses. f) Fluorescence quantum yield of
coelenteramide analogs 4a-4e in DMSO containing 1 M NaOH (0.5% v/v). g) Quantum efficiency of
chemical generation of the singlet-excited state of 4a-4e anion.
CL of 3e
CL of 3c
CL of 3a
(A)
(B)
(C)
454
462
479
FL of 4e–
FL of 4c–
FL of 4a–
400
500
600 400
500
Wavelength/nm
600 400
500
600
Figure 2. Selected chemiluminescence spectra (CL) of coelenterazine analogs 3a, 3c, and 3e in DMSO and fluorescence spectra
(FL) of coelenteramide analogs 4a, 4c, and 4e in DMSO containing 1 M aqueous NaOH (0.5% v/v): (A) 3a and 4a, (B) 3c and 4c,
and (C) 3e and 4e.
ability of R. In order to evaluate the substituent effect of R
OH
O
on the CLmax value more quantitatively, the energies (ECL in
kcal mol¹1) of the chemiluminescence maxima are plotted
against the Hammett substituent constants ·P of R, as shown in
Figure 3. Interestingly, a good correlation between the ·P and
the ECL values is obtained.
N
N
N
H
To rationalize the observed substituent effect on CLmax, the
molecular dipoles and the MOs of anionic 2-formamido-5-
phenylpyrazines 8 having substituent R (=CF3, H, and OMe),
HO
¹
¹
simple model compounds of the amide anion 4a , 4c , and
7
¹
4d , were calculated (Figure 4). In these calculations, the
Chart 2.
geometries of the anionic 8 in the lowest singlet-excited (S1)
states in DMSO were first optimized using the AM1-COSMO
method, and then the energy levels of the S1 state and the
corresponding ground states that have the same geometry as the
S1 (S0FC) state were estimated. Table 5 displays the calculated
S1 and S0FC levels for the anionic 8 having substituent R (=CF3,
H, and OMe), the differential energies (¦E in eV) for their
4e. The result that the CLmax values of 3a-3e matched well
with the FLmax values of the amide anions of 4a-4e (Table 4)
indicates that the light emitters involved in the chemilumines-
cent reactions are amide anions III, as shown in Scheme 1. The
CLmax values showed a small but apparent dependency on a
change in the substituent R. The CLmax value shifted to a longer
wavelength region with an increment of the electron-donating
FC
S1 ¼ S0 transitions, and the molecular dipole moments for
the optimized geometries. The computed ¦E value increased

R. Saito et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
95
Table 5. Calculated Molecular Dipole Moments, Energy Levels of the Lowest-Singlet-Excited States (S1)
and the Ground States with Unrelaxed Franck-Condon Geometries (S0FC), and the Differential Energies
FC
(¦E) for the S1 ¼ S0 Transitions for the Model Compound 8
Energy level/eVa)
Structure of 8
Substituent (R)
Dipole moment/Da)
¦E/eVa)
FC
S0
S1
CF3
H
6.522
12.635
16.841
¹5.971
¹5.788
¹5.722
¹3.228
2.880
2.711
2.692
O
H
N
N
N
¹3.140
¹3.119
R
OMe
a) Calculated as in DMSO (dielectric constant ¾ = 45.0) by the AM1-COSMO method.
-2
S1
-3.119
-3
-3.140
-3.228
-4
2.692
2.711
2.880
-5
-5.722
-5.788
-6
-5.971
S0FC
¹1
Figure 3. Chemiluminescence energy ECL (in kcal mol
)
-7
of coelenterazine analogs 3a-3e plotted against Hammett
substituent constant ·P.
R = CF3
R = H
R = OMe
FC
Figure 4. Energy levels of S0 and S1 for 8 (R = CF3, H,
and OMe) calculated as in DMSO (¾ = 45.0) by the AM1-
COSMO method. The S0FC-S1 energy gaps are shown with
arrows.
with the electron-withdrawing trifluoromethyl group and
decreased with the electron-donating methoxy group. These
results correspond well to the experimentally observed sub-
stituent effect. The calculated molecular dipole moment
increased sequentially with an increase in the electron-donating
ability of the substituent R. The S1 energies were estimated in
Chemiluminescence Efficiency: The chemiluminescence
quantum yields (ΦCL) of 3a-3e, obtained by the measurement
of the total number of photons emitted during the course of the
chemiluminescence reactions at 25 °C under air, are listed in
Table 4. The ΦCL values obviously increased with the electron-
donating ability of R. The ΦCL value is defined as the product
of three yields: the chemical yield of the chemiluminescence
emitter (ΦR), the quantum yield of the formation of a singlet-
excited light emitter from a high-energy intermediate (ΦS), and
the fluorescence quantum yield of a light emitter (ΦF).9
FC
the range of ¹3.228 to ¹3.119 eV (Δ = 0.109 eV), and the S0
energies in the range of ¹5.971 to ¹5.722 eV (Δ = 0.249 eV).
Both the S0FC and S1 energies decreased with an increase in the
electron-withdrawing ability of R, which indicates that the
introduction of an electron-withdrawing substituent R stabilizes
both HOMO and LUMO levels. The larger energy variation in
FC
S0 (Δ = 0.249 eV) than in S1 (Δ = 0.109 eV) indicates that
FC
the S0 states are more susceptible to the electronic effect of
substituent R than the S1 states, and the larger stabilization with
trifluoromethyl group enlarges the energy gap between S0FC and
S1 resulting in hypsochromic shift in the emission wavelength.
These computational results strongly indicate that the
observed substituent-dependent CLmax can be ascribed not to
the molecular dipole change of the amide anions but to large
variation in the HOMO levels of the emitters depending on the
electronic properties of the substituents.
ꢀCL ¼ ꢀR ꢀ ꢀS ꢀ ꢀF
ð1Þ
To clarify the effect of the substituent R on these efficiencies
(ΦR, ΦS, and ΦF), we estimated these values for the chemi-
luminescence reactions of 3a-3e. The ΦR values were deter-
mined as the yields of acetamidopyrazine 4a-4e by HPLC
analyses of the spent products obtained after the chemilumi-
nescence reactions of 3a-3e. The ΦF values were measured as

96
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
Chemiluminescence of Coelenterazine Analogs
O
O
Me
O
Table 6. Absolute HOMO Coefficients, the Formal Charges
and the Atomic Electron Densities on the Nitrogen Atom
of the Pyrazylaminide Anion (N7) of the Model
Compound 9
Me
N
N
N
N
N
7
Absolute HOMO Formal Atomic electron
Substituent (R)
N
coefficienta)
chargea)
densitya)
R
R
CF3
H
F
OMe
NMe2
0.475
0.464
0.480
0.473
0.463
¹0.450
¹0.458
¹0.466
¹0.469
¹0.470
5.451
5.458
5.466
5.469
5.470
9
Chart 3.
the fluorescence quantum yields of the amide anions of 4a-4e in
DMSO containing 1 M NaOH aqueous solution (0.5% v/v). By
substituting the ΦCL, ΦR, and ΦF values in eq 1, the ΦS values
were calculated. These ΦR, ΦS, and ΦF values are compiled in
Table 4. Each analog underwent a chemiluminescent reaction to
give 4a-4e in good yield in DMSO under air. The fluorescence
quantum yields ΦF of the 4c-4e anions were ca 0.2, while the
a) Calculated as in DMSO (dielectric constant ¾ = 45.0) by the
AM1-COSMO method.
O
O
O
O
H
O
Φ
FL of 4a and 4b were lower than those of 4c-4e. Thus, it was
N
N
NH
(or CH2Ph)
N
N
N
H
proved that an electron-withdrawing R substantially decreases
the fluorescence efficiency of the singlet-excited amide anion
III. Unlike the substituent-sensitive ΦF, the estimated ΦS values
showed only a small variation in the range of 0.008-0.015 by
changing substituents. From these results, it can safely be said
that the observed substituent effect on the ΦCL value predom-
inantly reflects the ΦF change by R.
(or CH2Ph)
H
R
R
O
10 (R = Br, H, Me, NMe2)
11 (R = Br, H, Me, NMe2)
O
O
(CH2)n
In order to correlate the obtained ΦS values with the
electronic properties of the central 5-aryl-3-benzylpyrazylami-
nide anion moiety of the dioxetane intermediate, the net atomic
charges, electron densities, and the HOMO coefficients of the
nitrogen atom of the aminide anion (designated as N7) of the
corresponding model structure 9 (Chart 3) with the substituent
R were calculated using the AM1 method. The results are given
in Table 6. The absolute HOMO coefficients are estimated in
the range of 0.463 to 0.480 eV (Δ = 0.017 eV). The calculated
net atomic charges and the electron densities are in the range of
¹0.450 to ¹0.470 (Δ = 0.020) and 5.451 to 5.470 (Δ = 0.019),
respectively. Thus, in the present imidazopyrazinone chemi-
luminescence system, the electronic effect of R does not reach
the N7, and the small change in the electronic properties of the
pyrazylaminide anion moiety can be attributed to the substitu-
ent-insensitive ΦS.
Teranishi and Goto reported the chemiluminescence of 5-(5-
aryl-2-pyrazinylamino)-1,2,4-trioxane derivatives 10 (Chart 4),
which chemiluminesced in the presence of a base by way of the
dioxetane intermediate 11, and the observed ΦS values for this
system were in the range of 0.0026 to 0.0067.7 Based on these
results, they claimed that the electron-donating ability of the
nitrogen anion in the intermediate 11 was strong enough to
accomplish the dioxetane decomposition, which is why no
distinct substituent effect was observed. Our present ΦS values
for the chemiluminescence of 3a-3e demonstrated trends
similar to those reported for 10.
O–
n = 0, 1, 2
12
Chart 4.
essential for the high ΦS value. The importance of the effective
electron donation toward the dioxetane ring has already
been systematically proven by Schaap et al. for CIEEL-type
chemiluminescent 1,2-dioxetanes.21 Therefore, there should be
another factor that causes such a high ΦS value in aequorin
bioluminescence. One possible explanation for this is that the
reactivity of the dioxetanone intermediate II is regulated not
only by the electronic properties of the 5-arylpyrazylaminide
anion moiety, but also by the conformation of the dioxetanone
intermediate. With regard to this, Head and co-workers
discovered that the coelenterazine 1 in aequorin is incorporated
in the hydrophobic active site of the protein backbone in a
peroxidize form, and its orientation is fixed by numerous
hydrogen bondings.6 In addition, Matsumoto and co-workers
reported that the conformation of the electron donor moiety
relative to the dioxetane ring in 12 is an important factor for
determining the chemiluminescence quantum yield, and they
demonstrated that the ΦS values varied drastically with changes
in the dihedral angle between the dioxetane rings and the
phenolate anion moiety.22 The importance of the fixed
conformation was also pointed out by an ab initio calculation.23
In light of these facts, we considered the difference between
the ΦS values of aequorin bioluminescence and imidazopyr-
azinone chemiluminescence systems. We considered that the
polypeptide matrix of apoaequorin may effectively regulate the
Aequorin bioluminescence shows a high luminescence
quantum yield (Φ = 0.2), and therefore the ΦS value is larger
than 0.2. This value is considerably higher than those obtained
in the present chemiluminescence system (ΦS = 0.008-0.015).
According to both the CIEEL and the CT mechanisms, strong
electron donation from the 6-position in 1 is considered to be

R. Saito et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
97
conformation of the reaction intermediate with the hydrogen-
bonding network in such a way that the interaction between the
pyrazylamino moiety and the dioxetanone ring is appropriate
for a high ΦS value. However, under chemiluminescence
conditions, the most probable conformation of the pyrazyl-
aminide anion relative to the dioxetanone ring could be
inappropriate for a high ΦS value, perhaps owing to the
electrostatic repulsion between the anion on the nitrogen and
the dioxetanone ring, which is responsible for the observed
small ΦS values and the negligible substituent effect.
(CAChe Scientific Inc.). INDO/S calculations18 were per-
formed on the ZINDO program incorporated in the CAChe
system. For CI calculations, nine occupied molecular orbitals
(HOMO¹8 to HOMO) and nine unoccupied molecular orbitals
(LUMO to LUMO+8) were taken into account. All conven-
tional chemicals used in the present study are commercially
available, and were used as received.
Chemiluminescence Spectra. A solution for chemilumi-
nescence measurement was prepared by mixing a stock
solution (100 ¯L) of 1.0 mM 3a-3e in methanol and DMSO
(2.0 mL) within a quartz cuvette at room temperature. A PMA-
10 photonic multichannel analyzer (Hamamatsu Photonics
K. K.) was used for recording the chemiluminescence spectra.
Chemiluminescence Quantum Yields. The chemilumi-
nescence quantum yields of 3a-3c at 25 °C were determined by
comparing their luminescence intensities to that of luminol as a
standard (ΦCL = 0.028). The luminescence intensities were
recorded on a TD-4000 lumiphotometer (Laboscience Co.,
Tokyo, Japan). The conditions for this experiment were as
follows: a 15-¯L methanolic solution of 3a-3c (4.0 ¯M) was
placed in the lumiphotometer, and the chemiluminescence
reaction was initiated by the injection of 300 ¯L of dehydrated
DMSO.
UV-vis Absorption and Fluorescence Spectrometry.
Spectral grade solvents were used for the measurement of
UV-vis absorption and fluorescence spectra. UV-vis spectra
were recorded on a Model 320 (Hitachi Co., Ltd., Japan) or a
V-550 spectrophotometer (JASCO Co., Ltd., Japan). Fluores-
cence spectra were measured with a Model F-4010 (Hitachi
Co., Ltd., Japan) or a Model F-777 fluorescence spectropho-
tometer (JASCO Co., Ltd., Japan) and corrected according
to the manufacturer’s instructions (excitation bandpass: 5 nm;
emission band pass: 5 nm; response: 2.0 s; scan speed: 60
nm min¹1). A solution of compound 4a-4e for fluorescence
measurement was prepared by mixing a stock solution (100 ¯L)
of 4a-4e in DMSO (2.0 mM) and a solvent (2.0 mL) within a
1-cm2 quartz cuvette at 25 °C. The reproducibility of each
measurement was definitively verified.
Conclusion
We carried out a systematic study of the substituent effect on
the chemiluminescent properties of coelenterazine analogs 3a-
3e in DMSO. A quantitative investigation of ΦCL revealed that
the ΦCL change was predominantly caused by the variation of
the fluorescence efficiency (ΦF) of the corresponding light
emitter, 4a-4e anion. The ΦF value showed strong substituent
dependency and decreased with the electron-withdrawing
ability of R. The generation efficiency of the singlet-excited
4a-4e anions, on the other hand, was independent of the
substituent, and this efficiency was considerably lower than the
bioluminescence efficiency of coelenterazine (1). These results
led us to conclude that it is difficult to develop an efficient
imidazopyrazinone-chemiluminescence system that is compa-
rable to aequorin bioluminescence, exploiting only the elec-
tronic substituent effect. The chemiluminescence maxima
(CLmax) were substituent dependent and exhibited a good
correlation with the Hammett substituent constant (·p) of R.
Thus, we found that CLmax can be arbitrarily controlled by
varying the ·p value of R.
Experimental
General. All melting points were measured on a MP-21
(Yamato Scientific Co., Ltd., Japan) in open capillary tubes; the
1
values are uncorrected. H NMR spectra were recorded on a
JNM-GX270 spectrometer (JEOL Ltd., Japan). Chemical shifts
(¤) are reported in ppm using tetramethylsilane or an
undeuterated solvent as internal standards in the deuterated
solvent used. Coupling constants (J) are given in Hz. Chemical
shift multiplicities are reported as s = singlet, d = doublet,
t = triplet, q = quartet, and m = multiplet. Infrared (IR) spec-
tra were obtained using IR-810 spectrophotometers (JASCO
Co., Ltd., Japan). Fast-atom-bombardment (FAB) mass spectra
were obtained on a JMS-600-H mass spectrometer (JEOL Ltd.,
Japan). Xenon was used as a bombardment gas, and all
analyses were carried out in positive mode with the ionization
energy and the accelerating voltage set at 70 eV and 3 kV,
respectively. A mixture of dithiothreitol (DTT) and ¡-thiogly-
cerol (TG) (1:1 or 1:2) or m-nitrobenzyl alcohol (mNBA) was
used as a liquid matrix. High- and low-resolution electron
impact (EI) mass spectra were obtained with a M-80B mass
spectrometer (Hitachi Co., Ltd., Japan). The ionization energy
and the accelerating voltage were 70 eV and 3 kV, respectively.
Column chromatography was carried out on silica gel (63-210-
¯m particle size; Kanto Chemical Co.). Semiempirical calcu-
lations for ground and singlet-excited states were carried out
with the AM1 method17a,17b and the COSMO model17c,17d of
MOPAC version 94 incorporated in CAChe software system
Fluorescence Quantum Yields. The fluorescence quantum
yields at 25 °C were measured relative to quinine bisulfate in
0.1 M sulfuric acid14 and calculated on the basis of the
following equation:
R
nref ODref Ifsamð-f Þd-fsam
sam
2
ꢀsfam ¼ ꢀrfef
R
Ifrefð-rfefÞd-rfef
2
nsam ODsam
where nref and nsam are the refractive indices of the solvents,
ODref and ODsam (¯0.02) are the optical densities, Φref (=0.52)
and Φfsam are the quantum yields, and the integrals denote the
(computed) area of the corrected fluorescence spectra, each
parameter for the standard (ref) and sample (sam) solutions,
respectively. The reproducibility of each measurement was
definitively verified.
Determination of ΦR Values.
The ΦR values were
determined by comparing the HPLC chromatogram of the
luminescence-spent product with that of the corresponding
synthesized acetamidopyrazine 4a-4e. The HPLC analyses
were performed on an Inertsil ODS column (4.6 © 250 mm; GL
Sciences, Inc., Japan) with an acetonitrile/water solvent system
as a mobile phase.

98
Bull. Chem. Soc. Jpn. Vol. 84, No. 1 (2011)
Chemiluminescence of Coelenterazine Analogs
8-Benzyl-2-methyl-6-(4-trifluoromethylphenyl)imidazo-
solids (83%): mp 134 °C (decomp); 1H NMR (270 MHz,
CD3OD): ¤ 2.45 (s, 3H), 7.83 (d, J = 8.4 Hz, 2H), 7.90-8.05
(m, 4H); IR (¯max(KBr)/cm¹1): 3420 (br, ¯NH), 3080, 2930
(¯CH), 1675 (¯C=O), 1620 (¯C=C), 1326 (¯CF). HRMS
(positive-EI) Calcd for C14H10F3N3O: 293.0776. Found:
293.0763.
[1,2-a]pyrazin-3(7H)-one (3a).
To a degassed ethanol
solution (4.5 mL) of 5a (100 mg, 0.30 mmol) was added
pyruvaldehyde (40 wt % solution in water, 106 mg, 0.58 mmol)
and conc. HCl (50 ¯L). The mixture was heated at 80 °C in a
sealed vessel under argon atmosphere for 2.5 h. After cooling to
room temperature, the solvent was removed under reduced
pressure. The resulting yellow residue was purified by column
chromatography on silica gel with 10% methanol in dichloro-
methane as a mobile phase to yield 3a as yellow solids: yield,
95 mg (83%); mp 134 °C (decomp); 1H NMR (270 MHz,
CD3OD): ¤ 2.46 (s, 3H), 4.42 (s, 2H), 7.21-7.43 (m, 5H),
7.78 (d, J = 8.4 Hz, 2H), 7.92 (br m, 3H); IR (¯max(KBr)/
cm¹1): 3358 (br, ¯NH), 3066, 2914 (¯CH), 1638 (¯C=O),
1614, 1552, 1513, 1491 (¯C=C; ring stretching), 1319 (¯CF).
HRMS (positive-EI) Calcd for C21H16F3N3O: 383.1245.
Found: 383.1246.
The authors gratefully acknowledge support for this research
by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(MEXT). Financial support to R.S. by “High-Tech Research
Center” Project for Private Universities: matching fund subsidy
from the MEXT and by the Faculty of Science Special Grant
for Promoting Scientific Research at Toho University are also
gratefully appreciated.
Supporting Information
8-Benzyl-6-(4-fluorophenyl)-2-methylimidazo[1,2-a]pyra-
zin-3(7H)-one (3b). This was prepared from 5b in a procedure
analogous to that used to obtain 3a. The product 3b was
obtained as yellow solids (86%): mp 175 °C (decomp);
1H NMR (270 MHz, CD3OD): ¤ 2.47 (s, 3H), 4.40 (s, 2H),
7.20-7.41 (m, 7H), 7.72 (br m, 3H); IR (¯max(KBr)/cm¹1):
3382 (br, ¯NH), 3062, 2914 (¯CH), 1670 (¯C=O), 1594, 1557,
1509 (¯C=C; ring stretching), 1180 (¯CF). HRMS (positive-
EI) Calcd for C20H16FN3O: 333.1277. Found: 333.1278.
8-Benzyl-2-methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-
one (3c). This was prepared from 5c in a procedure analogous
to that used to obtain 3a. The product 3c was obtained as
yellow solids (83%): mp 115 °C (decomp); 1H NMR (270 MHz,
CD3OD): ¤ 2.48 (s, 3H), 4.41 (s, 2H), 7.24-7.62 (m, 11H); IR
(¯max(KBr)/cm¹1): 3418 (br, ¯NH), 3044, 2914 (¯CH), 1671
(¯C=O), 1631, 1556, 1493 (¯C=C; ring stretching), 1180
(¯CF), 872, 694 (£CH). HRMS (positive-EI) Calcd for
C20H17N3O: 315.1372. Found: 315.1369.
8-Benzyl-6-(4-methoxyphenyl)-2-methylimidazo[1,2-a]-
pyrazin-3(7H)-one (3d). This was prepared from 5d in a
procedure analogous to that used to obtain 3a. The product 3d
was obtained as yellow solids (67%): mp 130 °C (decomp);
1H NMR (270 MHz, CD3OD): ¤ 2.47 (s, 3H), 3.85 (s, 3H), 4.40
(s, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.23-7.41 (m, 5H), 7.56-7.59
(m, 3H); IR (¯max(KBr)/cm¹1): 3426 (br, ¯NH), 3164, 3052
(¯CH), 1664 (¯C=O), 1628, 1604, 1538, 1509 (¯C=C; ring
stretching), 1024 (¯ArOC). HRMS (positive-EI) Calcd for
C21H19N3O2: 345.1477. Found: 345.1473.
Absorption spectra of 3a-3e in methanol (Figure S1) are
available. This material is available free of charge on the web at
http://www.csj.jp/journals/bcsj/.
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