Chemiluminescent 2,6-diphenylimidazo[1,2-a]pyrazin-3(7H)-ones: A new entry to Cypridina luciferin analogues

Article abstract of DOI:10.1039/c3pp50197c

An investigation of the chemiluminescent properties of 2,6- diphenylimidazo[1,2-a]pyrazin-3(7H)-one derivatives (1), having substituted phenyl groups, is described. Among the derivatives 1, the 6-[4-(dimethylamino) phenyl] derivatives (1a,d-f) gave a high quantum yield (Φ_CL ≥ 0.0025) in diglyme/acetate buffer, which is a model reaction condition for the Cypridina bioluminescence. Their efficient chemiluminescence is mainly caused by the electronic effect of the substituent at C6. In particular, the electron-donating 4-(dimethylamino)phenyl group at C6 of 1a,d-f plays an essential role in increasing the chemiexcitation efficiency (Φ_S) by the charge transfer-induced luminescence (CTIL) mechanism. The results provide useful information for designing new Cypridina luciferin analogues showing efficient chemiluminescence. The Royal Society of Chemistry and Owner Societies.

Full text of DOI:10.1039/c3pp50197c

Photochemical &
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Chemiluminescent 2,6-diphenylimidazo[1,2-a]pyrazin-
3(7H)-ones: a new entry to Cypridina luciferin
analogues†
Cite this: Photochem. Photobiol. Sci.,
2014, 13, 182
Yuki Ishii, Chihiro Hayashi, Yoshihisa Suzuki and Takashi Hirano*
An investigation of the chemiluminescent properties of 2,6-diphenylimidazo[1,2-a]pyrazin-3(7H)-one
derivatives (1), having substituted phenyl groups, is described. Among the derivatives 1, the 6-[4-(di-
methylamino)phenyl] derivatives (1a,d–f) gave a high quantum yield (ΦCL ≥ 0.0025) in diglyme/acetate
buffer, which is a model reaction condition for the Cypridina bioluminescence. Their efficient chemilumi-
nescence is mainly caused by the electronic effect of the substituent at C6. In particular, the electron-
donating 4-(dimethylamino)phenyl group at C6 of 1a,d–f plays an essential role in increasing the chemi-
excitation efficiency (ΦS) by the charge transfer-induced luminescence (CTIL) mechanism. The results
provide useful information for designing new Cypridina luciferin analogues showing efficient
chemiluminescence.
Received 28th June 2013,
Accepted 13th August 2013
DOI: 10.1039/c3pp50197c
www.rsc.org/pps
Introduction
Cypridina luciferin (Ln) is the substrate for the bio-
luminescence of the luminous ostracod Cypridina (Scheme 1),
and produces light by a reaction with molecular oxygen
(3O2).1,2 One of the characteristics of the bioluminescence is
its high efficiency at light production (ΦBL ≈ 0.3).3 The
imidazo[1,2-a]pyrazin-3(7H)-one (imidazopyrazinone) ring is
the core for the luminescence reactivity of Ln. Imidazopyrazi-
none derivatives, including Ln, exhibit chemiluminescence
reactivity with 3O2 in aprotic solvents.4,5
To establish the mechanism of the bioluminescence, the
chemiluminescence of imidazopyrazinone derivatives has
been investigated as a model of the Cypridina bioluminescence
reaction.5–9 For this purpose, 6-aryl-2-methylimidazopyrazi-
nones such as a Cypridina luciferin analogue (CLA) and its di-
methylamino derivative (dm-CLA), whose π-electronic
properties were modulated by a substituent at C6, have been
employed (Scheme 1).5c,d,6 The reason to choose 6-aryl imida-
zopyrazinone derivatives is that the 3-indolyl group at C6 on
Ln is a π-electronically conjugated appendage. DMSO contain-
ing a base (DMSO/base)4 and diglyme containing an acetate
buffer (diglyme/acetate buffer)5 have been used as the standard
reaction conditions for imidazopyrazinone chemilumines-
cence. The diglyme/acetate buffer condition is predicted to be
Department of Engineering Science, Graduate School of Informatics and Engineering,
The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan.
E-mail: hirano@pc.uec.ac.jp
Scheme 1 Cypridina luciferin (Ln), its analogues CLA, dm-CLA and
1a–f, and Cypridina oxyluciferin analogues dm-OCLA and 2a–f.
†In memory of the late Professor Nicholas J. Turro.
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a favorable condition for the chemiluminescence reaction of by a Hamamatsu C4900 power supply. The signal from the
Ln because it allows elementary processes similar to those of luminometer was collected on a PC and the data were analyzed
the bioluminescence reaction.2a,5 Among the 6-aryl-2-methyl- using the graphics program Igor Pro, Version 4.0 (Wave
imidazopyrazinones investigated, dm-CLA having an electron- Metrics, Inc.). Spectroscopic measurements were made in a
donating 4-(dimethylamino)phenyl group gives the highest quartz cuvette (1 cm path length) at 25 1 °C. Spectral-grade
chemiluminescence quantum yield (ΦCL = 0.015) in diglyme/ solvents were used for the measurement of UV/visible absorp-
acetate buffer.6c,d A mechanistic analysis of these substituent tion, fluorescence and chemiluminescence. HPLC analyses
effects led to the conclusion that the 3-indolyl group of Ln were carried out using a JASCO 980 HPLC system with a Kanto
plays an essential role as an electron-donating substituent that Chemical Mightysil RP-18 GP-II column (250 mm × 4.6 mm,
accelerates the oxygenation process and increases the chemi- 5 μm). Density functional theory (DFT) calculations were per-
excitation efficiency.
formed using the Gaussian 09 program.11 DFT includes Beck’s
Despite the success described above, the chemilumines- three-parameter function combined with Lee, Yang and Parr’s
cence of an imidazopyrazinone still gives an efficiency lower correlation function (B3LYP) along with the 6-31G(d) basis
than the ΦBL value of the Cypridina bioluminescence. In an set.12 Transition state structures were located using the Berny
attempt to solve this problem, new imidazopyrazinone deriva- algorithm until the Hessian matrix had only one imaginary
tives 1, with phenyl groups added at C2 and C6 to electroni- eigenvalue. Molecular graphics were generated using Gauss-
cally modulate the chemiluminescence, were designed as new View, Version 5.13
Cypridina luciferin analogues (Scheme 1). It has been found
that benzamidopyrazine 2a, which is the product of the chemi-
Preparation of imidazopyrazinones 1 and amidopyrazines 2
luminescence reaction of 1a, functions as a fluorophore.10
This encouraged us to study the chemiluminescence of 2,6- Imidazopyrazinone 1c and amidopyrazine 2a were prepared
diphenylimidazopyrazinones. We investigated the chemilumi- by previously reported procedures.10,14 Other derivatives
nescent properties of a series of substituted 2,6-diphenyl (1a,b,d–f) were synthesized from the corresponding 5-aryl-
derivatives 1 in DMSO/base and in diglyme/acetate buffer, and aminopyrazines and arylglyoxals as follows. Amidopyrazines
found that the derivatives having an electron-donating 4-(di- 2b–f were also synthesized by acylation of the corresponding
methylamino)phenyl group at C6 show efficient chemilumines- 5-arylaminopyrazines.
cence in diglyme/acetate buffer. To understand the origin of
Synthesis of 1a. To a solution of 5-[4-(dimethylamino)-
the efficient chemiluminescence of the 6-[4-(dimethylamino)- phenyl]aminopyrazine (40 mg, 0.20 mmol) and phenylglyoxal
phenyl] derivatives, the factors that determine the ΦCL values, monohydrate (92 mg, 0.60 mmol) in 1,4-dioxane (2.0 mL) was
including the reaction efficiency, the chemiexcitation added conc. HCl (0.2 mL) under Ar. The reaction mixture was
efficiency and the fluorescence quantum yield, were evaluated heated at 100 °C for 1 h. After cooling, the mixture was concen-
mechanistically. We report herein the chemiluminescent pro- trated under reduced pressure. The red residue was purified by
perties of 1, which can provide a guideline for designing new silica gel column chromatography four times [CHCl3–metha-
Cypridina luciferin analogues with efficient chemilumine- nol (20 : 1 and 5 : 1)], yielding 1a (38 mg, 57%) as a brown
scence.
powder: mp 256–257 °C. δH (CD3OD) 3.00 (6 H, s), 6.83 (2 H, d,
J 9.2 Hz), 7.37 (1 H, br t), 7.44 (2 H, br t), 7.52 (2H, d, J 8.6 Hz),
7.72 (1 H, br s), 8.00 (1 H, br s) and 8.41 (2 H, d, J 5.2 Hz).
ν (KBr)/cm−1 3056, 2927, 1608 and 1516. m/z (ESI) Found:
331.1597 ([M + H]+). C20H19N4O requires 331.1559.
Experimental
General
1b: orange powder, mp 89–90 °C. δH (CD3OD) 3.85 (3 H, s),
Melting points were obtained using a Yamato MP-21 appar- 7.08 (2 H, d, J 8.6 Hz), 7.32 (2 H, t, J 7.2 Hz), 7.38 (1 H, t, J
atus. IR spectra were measured using a Horiba FT-720 spectro- 7.5 Hz), 7.66 (2 H, d, J 8.6 Hz), 7.78 (1 H, s), 8.03 (1 H, br s)
meter. High-resolution electro-spray ionization (ESI) mass and 8.41 (2 H, d, J 7.5 Hz). ν (KBr)/cm−1 3411, 3060, 1610 and
spectra were recorded using a JEOL JMS-T100LC mass spectro- 1508. m/z (ESI) Found: 318.1263 ([M + H]+). C19H16N3O2
meter. 1H NMR spectra were recorded using a JEOL ECA-500 requires 318.1243.
instrument (500 MHz). UV/visible absorption spectra were
1d: dark red powder, mp >300 °C. δH (CD3OD) 3.28 (6 H, s),
measured using a Varian Cary 50 spectrophotometer (scan 3.87 (3 H, s), 7.09 (2 H, d, J 9.2 Hz), 7.53 (2 H, d, J 8.6 Hz),
speed, 600 nm min−1; data interval, 1 nm). Fluorescence 7.98 (2 H, d, J 8.6 Hz), 8.22 (2 H, d, J 8.6 Hz), 8.28 (1 H, s)
spectra and fluorescence quantum yields were obtained using and 8.49 (1 H, s). ν (KBr)/cm−1 3401, 1608 and 1508. m/z
a Hamamatsu Photonics Quantaurus-QY absolute PL quantum (ESI) Found: 361.1698 ([M
yield measurement system. Chemiluminescence spectra and 361.1665.
+
H]+). C21H21N4O2 requires
chemiluminescence quantum yields were measured using an
1e: dark red powder, mp >300 °C. δH (CD3OD) 3.32 (6 H, s),
ATTO AB-1850 luminescence detecting spectrophotometer. 3.82 (3 H, s), 3.94 (6 H, s), 7.64 (2 H, s), 7.66 (2 H, d, J 9.2 Hz),
The intensity of the total light (400–700 nm) emitted from a 8.04 (2 H, d, J 8.6 Hz), 8.33 (1 H, s) and 8.54 (1 H, s). ν (KBr)/
chemiluminescence reaction was monitored using a lumino- cm−1 3401, 1646, 1616 and 1508. m/z (ESI) Found: 421.1907
meter with a Hamamatsu R5929 photomultiplier tube powered ([M + H]+). C23H25N4O4 requires 421.1876.
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1f: dark red powder, mp >300 °C. δH (CD3OD) 3.26 (6 H, s), Analysis of the chemiluminescence reactions of
3.92 (3 H, s), 3.95 (3 H, s), 3.97 (3 H, s), 7.02 (1 H, d, J 9.2 Hz), imidazopyrazinones
7.52 (2 H, d, J 8.1 Hz), 7.98 (1 H, d, J 9.2 Hz), 8.12 (2 H, d, J
A small portion (20 μL) of a stock solution of imidazopyrazi-
none 1 (1.0 × 10–3 M) in methanol was added into a quartz
cuvette placed in a luminometer and was mixed with aerated
DMSO (2.0 mL) containing 0.10 M NaOH aqueous solution
(1.0% v/v) or aerated diglyme (2.0 mL) containing 0.10 M of
acetate buffer (acetic acid–sodium acetate buffer, pH 5.6,
0.66% v/v) at 25 1 °C. The chemiluminescence reactions of 1
were traced by monitoring the intensity (I) of the emitted light.
The pseudo-first-order rate constant (kCL) was obtained by ana-
lysis using the equation: ln It = −kCLt + lnI0, where I0 and It are
the intensities at t = 0 and t, respectively. The ΦCL values were
determined as values relative to the ΦCL (0.013) of luminol in
DMSO containing t-BuOK/t-BuOH under air.15
The yields of amidopyrazines 2 obtained after chemilumi-
nescence reactions of 1 in diglyme/acetate buffer were deter-
mined by HPLC. A spent solution (10 mL) obtained by a
chemiluminescence reaction for 24 h was mixed with 2-ethyl-
naphthalene (1.0 μL) as an internal standard, and was ana-
lyzed three times by reverse phase HPLC with a H2O–CH3CN
(1 : 4) eluent (1.0 mL min−1). Yields of 2 were determined by
relative peak integrations (monitoring the absorbance at
370 nm) and individual response factors against 2-ethyl-
naphthalene.
8.6 Hz) 8.69 (1 H, s) and 8.98 (1 H, s). ν (KBr)/cm−1 2937, 1585
and 1499. m/z (ESI) Found: 421.1875 ([M + H]+). C23H25N4O4
requires 421.1876.
Synthesis of 2c. A solution of 5-phenylaminopyrazine
(69 mg, 0.41 mmol) and benzoyl chloride (70 μL, 0.60 mmol)
in pyridine (1 mL) was stirred at room temperature under
Ar for 2.5 h. The reaction was quenched by the addition of
brine (20 mL) and the product was extracted with chloroform
(20 mL × 3). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by recrystalli-
zation from a mixture of CHCl3 and ethyl acetate (10 : 1), yield-
ing 2a (50 mg, 45%) as colorless cubes, mp 210–211 °C. δH
(CDCl3) 7.46 (1 H, t, J 7.5 Hz), 7.52 (2 H, t, J 8.0 Hz), 7.55 (2 H,
t, J 8.0 Hz), 7.62 (1 H, t, J 7.5 Hz), 7.97 (2 H, d, J 7.5 Hz), 8.01 (2
H, d, J 7.5 Hz), 8.53 (1 H, br s), 8.72 (1 H, br s) and 9.77 (1 H,
d, J 0.9 Hz). ν (KBr)/cm−1 3366, 3053, 1668, 1540 and 1501. m/z
(ESI) Found: 276.1149 ([M
276.1137.
+
H]+). C17H14N3O requires
2b: yellow cubes, mp 189–190 °C. δH (CDCl3) 3.88 (3 H, s),
7.02 (2 H, t, J 8.6 Hz), 7.53 (2 H, t, J 8.0 Hz), 7.61 (1 H, t, J 6.9
Hz), 7.95–7.97 (4 H, m), 8.49 (1 H, br s), 8.66 (1 H, d, J 1.8 Hz)
and 9.71 (1 H, d, J 1.8 Hz). ν (KBr)/cm−1 3369, 1668, 1610 and
1513. m/z (ESI) Found: 306.1266 ([M + H]+). C18H16N3O2
requires 306.1243.
Synthesis of 2d. A solution of 5-[4-(dimethylamino)phenyl]-
aminopyrazine (41 mg, 0.19 mmol), 4-methoxybenzoic acid
(29 mg, 0.19 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide (109 mg, 0.57 mmol) and 4-dimethylaminopyridine
Results and discussion
Chemiluminescence of 1a–c in DMSO/NaOH (aq.) and in
diglyme/acetate buffer
(23 mg, 0.19 mmol) in dichloromethane (2 mL) was heated To investigate the chemiluminescent activity of a 2,6-diphenyl-
under reflux under Ar for 3.5 h. The reaction was quenched by imidazopyrazinone derivative, the substituent effect of the
the addition of water (60 mL) and the product was extracted 6-phenyl group in 1a–c was studied first. Chemiluminescence
with chloroform (70 mL × 3). The organic layer was washed reactions of 1a–c in aerated DMSO containing 0.10 M NaOH
with brine, dried over Na2SO4 and concentrated in vacuo. The aqueous solutions were examined at 25 °C. The reactions of
residue was purified by silica gel TLC [CHCl3–ethyl acetate 1a–c showed only a weak luminescence, and the ΦCL values
(5 : 1)], yielding 2d (18 mg, 28%) as a yellow powder: mp were below 10−4. A chemiluminescence reaction of an imidazo-
219–220 °C. δH (CDCl3) 3.04 (6 H, s), 3.89 (3 H, s), 6.81 (2 H, d, pyrazinone in basic DMSO gives light emission from the
J 9.2 Hz), 7.00 (2 H, d, J 8.6 Hz), 7.92 (4 H, m), 8.35 (1 H, br s), excited singlet (S1) state of the amide anion of an amidopyra-
8.62 (1 H, d, J 1.7 Hz) and 9.65 (1 H, d, J 1.8 Hz). ν (KBr)/cm−1 zine product.5–7 Fluorescence measurements of the amide
3392, 1670, 1608 and 1540. m/z (ESI) Found: 371.1516 anions of 2a–c in DMSO containing 0.10 M NaOH aqueous
([M + Na]+). C20H20N4NaO2 requires 371.1484.
solutions showed quantum yields (ΦF) below 0.005. Therefore,
2e: yellow cubes, mp 177–178 °C. δH (CDCl3) 3.04 (6 H, s), the low luminescence of 1a–c was due to the fact that the
3.92 (3 H, s), 3.95 (6 H, s), 6.81 (2 H, d, J 8.6 Hz), 7.16 (2 H, s), amide anions of 2a–c were not fluorescent.
7.92 (2 H, d, J 8.6 Hz), 8.36 (1 H, br s), 8.64 (1 H, d, J 1.2 Hz)
Next, chemiluminescence reactions of 1a–c in aerated
and 9.65 (1 H, br s). ν (KBr)/cm−1 1646, 1610 and 1533. m/z diglyme containing acetate buffer (pH 5.6, 0.66% v/v) were
(ESI) Found: 431.1732 ([M + Na]+). C22H24N4NaO4 requires examined at 25 °C. While 1b,c showed only a weak chemilumi-
431.1695.
nescence (ΦCL < 10−4), 1a gave an estimated ΦCL value of
2f: yellow cubes, mp 212–213 °C. δH (CDCl3) 3.04 (6 H, s), 0.0025. The chemiluminescence emission spectrum of 1a
3.92 (3 H, s), 3.95 (3 H, s), 4.13 (3 H, s), 6.81 (2 H, d, J 9.2 Hz), showed the maximum (λCL) at 521 nm. This coincides with the
6.85 (1 H, d, J 9.2 Hz), 7.91 (2 H, d, J 9.2 Hz), 8.03 (1 H, d, J fluorescence spectrum of the neutral form of amidopyrazine
9.2 Hz), 8.65 (1 H, d, J 1.7 Hz), 9.68 (1 H, d, J 1.2 Hz) and 2a, indicating that light emission occurred from the S1 state of
10.52 (1 H, br s). ν (KBr)/cm−1 3332, 2974, 2939 and 1657. m/z 2a. The difference in chemiluminescence behavior between
(ESI) Found: 431.1684 ([M + Na]+). C22H24N4NaO4 requires 1a–c in diglyme/acetate buffer can be explained by the substi-
431.1695.
tuent effect of the 6-phenyl group with the reaction
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Table 1 Chemiluminescent properties of 1a,d–f and dm-CLA in
aerated diglyme/acetate buffera at 25 °C
Substrate λCLb/nm kCLc/10−4 s−1 ΦCLd/10−2 ΦR
ΦF
ΦS
e
f
g
1a
1d
1e
1f
521
524
523
518
1.3
3.1
1.2
4.5
0.25
1.2
0.62
1.4
0.89 0.16 0.018
0.45 0.34 0.078
0.32 0.29 0.067
0.37 0.44 0.086
0.62 0.33 0.073
dm-CLAh 491
23
1.5
a Diglyme containing acetate buffer (0.10 M, pH 5.6, 0.66% v/v).
b Emission maxima of the chemiluminescence spectra. c Pseudo-first-
order rate constants. d Chemiluminescence quantum yields.
e Chemical efficiencies to give the corresponding amidopyrazine
products by the chemiluminescence reactions. f Fluorescence quantum
yields of the corresponding amidopyrazines. g Chemiexcitation
efficiencies. h Ref. 6d.
Scheme 2 Reaction mechanism of the chemiluminescence of 1.
mechanism reported previously (Scheme 2).6 In DMSO/NaOH
(aq.), the chemiluminescence reactions of 1a–c give the S1
states of the amide anions of 2a–c via anionic dioxetanones
(D−). On the other hand, the chemiluminescence reaction of
1a in diglyme/acetate buffer gives the S1 states of the neutral
form of 2a via neutral dioxetanone (DH), while the chemilumi-
nescence reactions of 1b,c still give the excited amide anions
of 2b,c via D−. The difference between 1a–c is because of com-
petition between the decomposition of D− to give the excited
amide anion and the protonation of D− to give DH. Because
the 4-(dimethylamino)phenyl group is a good electron-donat-
ing substituent, the basicity of the anionic N atom of D− is
increased, which leads to the preferential protonation of D−.
In the case of 1b,c, the electron-donating properties of the
4-methyoxyphenyl and phenyl groups are not sufficient for the
preferential protonation of D−. Therefore, 1b,c gave negligible
chemiluminescence in diglyme/acetate buffer in a manner
similar to that in DMSO/NaOH (aq.). Only 1a, with the elec-
tron-donating 4-(dimethylamino)phenyl group, is chemilumi-
nescent in diglyme/acetate buffer.
Fig. 1 Chemiluminescence emission spectra of 1a,d–f (1.0 × 10–5 M) in
aerated diglyme/acetate buffer at 25 °C.
observed for 1a, indicating that light emitters are the S1 states
of 2d–f. The λCL values for 1a,d–f are similar to one another,
indicating that the S1 states of 2a,d–f have a similar π-elec-
tronic properties which are not affected by the structure of the
acyl groups. These π-electronic properties of the S1 states of 2a,
d–f are consistent with our previous report.10
Decay curves of chemiluminescence intensities (I) over time
for 1a,d–f gave pseudo-first-order rate constants (kCL) (Table 1).
The chemiluminescence reaction rate of an imidazopyrazinone
is regulated by the oxygenation process of an imidazopyrazi-
none anion as the rate-determining step (Scheme 2).5a,6b In
particular, thermal electron transfer from an imidazopyrazi-
3
6b
none anion to O2 determines the value of kCL
.
Because the
kCL values of 1a,d–f are smaller than that of dm-CLA, the
π-electronic conjugation of the phenyl group at C2 of the
Chemiluminescence of 1a,d–f in diglyme/acetate buffer
Based on the chemiluminescent structure of 1a, with the anions of 1a,d–f reduces the reaction rate. The difference in
4-(dimethylamino)phenyl group at C6, we investigated the the kCL value between 1a,d–f is explained by the introduction
effect on chemiluminescence of substituents on the phenyl of the methoxy group. The Hammett σp and σm constants of
group at C2 with 1d–f, all of which showed chemilumines- the methoxy group are −0.27 and 0.12, respectively.16 Thus,
cence in diglyme/acetate buffer. The observed properties are methoxy groups at the ortho and para positions of a phenyl
summarized in Table 1, together with those of 1a and group are electron donating and a methoxy group at the meta
dm-CLA, for comparison.
position is electron withdrawing. The order of the strength of
Chemiluminescence emission spectra for 1a,d–f are shown electron donation, 2,3,4-trimethoxyphenyl (1f) > 4-methoxy-
in Fig. 1. The λCL values for 1d–f are also coincident with the phenyl (1d) > 3,4,5-trimethoxyphenyl (1e) ≈ phenyl (1a), corres-
fluorescence emission maxima of the neutral forms of 2d–f, as ponds to the order of the kCL values, indicating that the
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Scheme 3 Three factors to make chemiluminescence quantum yield (ΦCL).
electron-donating properties of the phenyl group at C2 of an
imidazopyrazinone anion effectively modulate the oxygenation
reactivity.
The ΦCL values for 1d–f are in the range of 0.006–0.014 and
are similar to that (0.015) for dm-CLA and higher than that
(0.0025) for 1a (Table 1). The value of ΦCL tends to increase
with increasing electron donation from the phenyl group at C2
of 1a,d–f. To confirm the origin of the high ΦCL values for 1a,
d–f, the elements of the ΦCL value were evaluated. A ΦCL value
is a product of three efficiencies, ΦCL = ΦR × ΦS × ΦF, where ΦR
is the chemical efficiency to produce 2 by an appropriate
chemiluminescence reaction pathway, ΦS is the chemiexcita-
tion efficiency for generating the S1 state of 2 from a neutral
dioxetanone DH and ΦF is the fluorescence quantum yield of 2
(Scheme 3). The ΦR values were determined by product ana-
lyses of the chemiluminescence reactions of 1a,d–f in diglyme/
acetate buffer, which indicated that 2a,d–f were obtained in
32–89% yields. The ΦF values of 2a,d–f measured in diglyme/
acetate buffer exceeded 0.16. Consequently, the ΦS values were
estimated to be greater than 0.018. It is noteworthy that the
chemiluminescence of 1d,f in diglyme/acetate buffer gives
higher ΦS values than that of dm-CLA.
Fig. 2 Optimized geometries of DH[1a,e,f] and TS[1a,e,f] calculated by
the R- and UB3LYP/6-31G(d) methods, respectively.
The ΔE values for TS[1a,d–f] are similar to that of TS[dm-
CLA]. Because the value of ΔE corresponds to the activation
energy of the decomposition of DH, the reaction rates of the
chemiexcitation processes from DH[1a,d,e] and DH[dm-CLA]
will be similar to one another. The dipole moments, molecular
geometries and charge distributions of DH[1a,d,e] and
TS[1a,d,e] are also similar to those of DH[dm-CLA] and TS[dm-
CLA], respectively. On the other hand, TS[1f] has a character-
istically long O–O bond (2.25 Å) and the negative and positive
charges in the structure of TS[1f] are localized at the dioxeta-
none and 5-[4-(dimethylamino)phenyl]pyrazinylamino moi-
eties, respectively. In fact, the qDo value (−0.344) of TS[1f] is
much less than that (−0.156) of TS[1a] and the summation of
the qNH, qPy and qAr values (+0.335) for TS[1f] is much greater
than that (+0.249) for TS[1a]. TS[1f] has a larger dipole
moment (14.7 D) than the other species (8.4–10.0 D). These
characteristic properties of TS[1f] originate from the strong
electron-donating properties of the 2,3,4-trimethoxyphenyl
group and the steric hindrance of its ortho substituent, yet the
properties of TS[1f] do not cause a drastic change in the ΦS
value. The differences in the qAr′ values between TS[1a,d–f]
and DH[1a,d–f] are small (around −0.05). Thus, the phenyl
(Ar′) groups at C2 of 1a,d–f have only a small electronic effect
on the properties of the corresponding TS. That is, TS[1a,d–f]
Analysis of the chemiexcitation mechanism in the
chemiluminescence of 1a,d–f
To understand the cause of efficient chemiexcitation in the
chemiluminescence of 1a,d–f, we analyzed the chemiexcitation
processes mechanistically. The thermal decomposition of a
1,2-dioxetanone for efficient chemiexcitation can be explained
by the charge transfer-induced luminescence (CTIL) mechan-
ism.6d,17 In the CTIL mechanism, a high ΦS value requires that
both the transition state (TS) of decomposition of DH for
chemiexcitation and the S1 state of 2 have a strong intramole-
cular charge transfer (ICT) character. Electronic properties of
DH and TS for the chemiluminescence of 1a,d–f and dm-CLA
were predicted by DFT calculations with the restricted (R) and
unrestricted (U) B3LYP/6-31G(d) methods, respectively. Fig. 2
shows selected optimized geometries of DH and TS, and calcu-
lation data are summarized in Table 2. The value of ΔE is the
relative heat of formation of TS compared to that of the corres-
ponding DH. The rCC and rOO values are the C–C and O–O
bond lengths, respectively, and the qDo, qNH, qPy, qAr and qAr′
values are the total Mulliken charge densities of the atoms
constituting the dioxetanone (C2O3), NH, pyrazine (C4H2N2)
and two aryl parts, respectively (Scheme 4).
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Table 2 Relative energies (ΔE), dipole moments (μ), C–C and O–O bond distances (rCC and rOO) and Mulliken charge densities (q) for dioxetanones
(DH) and transition states (TS) of the dioxetanone decompositions for chemiluminescence of 1a,d–f and dm-CLA calculated by the R- and UB3LYP/
6-31G(d) methods
Dioxetanone or
transition statea
ΔEb/
rCC/Å
(ΔrCC
rOO/Å
(ΔrOO
c
c
kcal mol−1
μ/D (Δμ)c
)
)
qDod (Δq)c
qNHd (Δq)c
qPyd (Δq)c
qArd (Δq)c
qAr′d (Δq)c
DH[1a]
TS[1a]
0.0
17.5
4.1
1.546
1.565
(+0.019)
1.545
1.566
(+0.021)
1.546
1.565
(+0.019)
1.551
1.557
(+0.006)
1.538
1.492
1.963
(+0.471)
1.492
1.962
(+0.470)
1.492
1.961
(+0.469)
1.493
2.250
(+0.757)
1.492
0.014
−0.156 (−0.170)
−0.321
−0.291
(+0.030)
−0.323
−0.293
(+0.030)
−0.322
−0.289
(+0.033)
−0.297
−0.194
(+0.103)
−0.313
−0.293
(+0.020)
0.119
0.196
(+0.077)
0.117
0.190
(+0.073)
0.120
0.199
(+0.079)
0.102
0.257
0.099
0.209
(+0.110)
0.097
0.197
(+0.100)
0.097
0.209
(+0.112)
0.091
0.272
0.088
0.042
(−0.046)
0.100
0.062
(−0.038)
0.082
0.036
(−0.046)
0.064
10.0
(+5.9)
3.4
8.4
(+5.0)
3.2
8.7
(+5.5)
3.8
14.7
(+11.0)
4.3
DH[1d]
TS[1d]
0.0
17.6
0.009
−0.156
(−0.165)
0.022
−0.155
(−0.177)
0.041
−0.344
(−0.385)
0.044
DH[1e]
TS[1e]
0.0
17.4
DH[1f]
TS[1f]
0.0
18.7
0.008
(+0.155)
0.115
0.179
(+0.182)
0.099
0.192
(−0.056)
0.056e
0.019e
(−0.036)e
DH[dm-CLA]
TS[dm-CLA]
0.0
18.3
9.2
(+4.9)
1.558
(+0.020)
1.983
(+0.491)
−0.098
(−0.142)
(+0.064)
(+0.093)
a DH and TS denote dioxetanone and the transition state of the dioxetanone decomposition, respectively. DH and TS were calculated by
the R- and UB3LYP/6-31G(d) methods, respectively. b Energy of a transition state relative to that of the corresponding dioxetanone. c Differences
between the values for TS and the corresponding DH. d Skeletal parts for the Mulliken charge density are shown in Scheme 4. e Mulliken charge
densities of the methyl group.
Scheme 4 Definitions of the C–C and O–O bond lengths (rCC and rOO) and the total Mulliken charge densities (q) of the skeletal parts of DH[1]
and TS[1].
Scheme 5 The CTIL mechanism for the chemiexcitation process from DH[1] to 12* via TS[1] in the chemiluminescence of 1a,d–f.
maintain a favorable ICT character, like TS[dm-CLA], for mechanism in a manner similar to the decomposition of
efficient chemiexcitation, because they have the common 4-(di- DH[dm-CLA] (Scheme 5).
methylamino)phenyl group at the pyrazinylamino moiety. It
has already been shown that 2a has a large dipole moment
(μ, 16 D) in the S1 state.10 Similarly, the S1 states of 2d–f will
have large μ values. Thus, the S1 states of 2a,d–f, like the S1
Conclusion
state of dm-OCLA, have a strong ICT character. Therefore, the
decomposition of DH[1a,d–f] proceeds to give the S1 states of
2a,d–f via TS[1a,d–f] efficiently by following the CTIL
We have demonstrated that 1a having the electron-donating
4-(dimethylamino)phenyl group at C6 shows moderate chemi-
luminescent activity in diglyme/acetate buffer, while 1a gives
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negligible chemiluminescence in DMSO/NaOH (aq.). The
chemiluminescence reaction of 1a proceeds to give the S1 state
of the neutral form of 2a. Because 1b,c having the 4-methoxy-
phenyl and phenyl groups at C6 are non-chemiluminescent
substrates in DMSO/NaOH (aq.) and in diglyme/acetate buffer,
the introduction of the dimethylamino group to the phenyl
group at C6 is essential for the chemiluminescent activity of
2,6-diphenyl derivatives 1 via the generation of the S1 state of a
neutral amidopyrazine from a neutral dioxetanone DH. The
derivatives 1d–f having methoxy substituent(s) on the phenyl
group at C2 together with the 4-(dimethylamino)phenyl group
at C6 also show chemiluminescence in diglyme/acetate buffer,
and their ΦCL values are similar to the high ΦCL value of dm-
CLA. In the elements to determine the ΦCL values of 1a,d–f,
the ΦS values were greater than 0.018. The chemiexcitation
processes for 1a,d–f are explained by the efficient decompo-
sitions of DH to yield the S1 states of 2a,d–f with a strong ICT
character via ICT TS through the CTIL mechanism, as in the
case of dm-CLA. While the ΦCL values of 1a,d–f are still
smaller than the ΦBL value (ca. 0.3),3 1a,d–f are in the group of
derivatives with a high ΦCL value greater than 0.001 in
diglyme/acetate buffer like dm-CLA.6c,d Interestingly, the
methoxy substituted phenyl groups at C2 of 1d–f serves to
maintain or slightly increase the ΦS value, therefore contribut-
ing to a high ΦCL value. This finding provides a guideline for
designing a new Cypridina luciferin analogue with a ΦCL value
close to the ΦBL value. To the 6-[4-(dimethylamino)phenyl]-2-
phenylimidazopyazinone structure, for instance, an alkoxy-arm
group can be introduced to the phenyl group at C2, which will
have a supramolecular photochemical function of intramole-
cularly regulating the reactivity of the imidazopyrazinone
core.18 Based on this idea, we are studying the reaction mecha-
nism of an efficient Cypridina bioluminescence, in which
Cypridina luciferase has supramolecular functions that regu-
late the luminescence reaction and increase the ΦBL value.
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Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research C (no. 22550031) from the Japan Society for the Pro-
motion of Science (JSPS). We acknowledge technical assistance
in computing the quantum chemical calculations from the
Information Technology Center of UEC. We also thank Pro-
fessors Mamoru Ohashi (UEC), Haruki Niwa (UEC) and
Shojiro Maki (UEC) for their generous assistance and valuable
discussion.
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