Fluorescence Properties of Diphenylthiazolo[4,5-b]pyrazines Tuned by Donor-Acceptor Substituent Effects

Article abstract of DOI:10.1111/php.12440

Fluorescence properties of 2,6- and 2,5-diphenylthiazolo[4,5-b]pyrazine (TPy) derivatives having an electron-donating substituent (methoxy and dimethylamino) on the 6- and 5-phenyl groups were studied. It was found that 2,6-diphenyl derivatives fluoresce

Full text of DOI:10.1111/php.12440

Photochemistry and Photobiology, 20**, **: *–*
Fluorescence Properties of Diphenylthiazolo[4,5-b]pyrazines Tuned by
Donor-Acceptor Substituent Effects
Tatsuki Nakagawa1, Minoru Yamaji2, Shojiro Maki1, Haruki Niwa1 and Takashi Hirano1*
1Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-
Communications, Chofu, Tokyo, Japan
2Division of Molecular Science, Graduate School of Science and Technology, Gunma University, Kiryu, Gunma, Japan
Received 27 December 2014, accepted 15 February 2015, DOI: 10.1111/php.12440
ABSTRACT
C5 and C6 positions of the TPy ring was investigated. We report
herein the fluorescent properties of 2,6- and 2,5-diphenyl TPy
derivatives 2 and 3 having an electron-donating substituent (dim-
ethylamino and methoxy) on the 6- and 5-phenyl groups
(Scheme 1), respectively, concluding that 2,6-diphenyl TPy with
an electron-donating substituent on the 6-phenyl group is a
prominent fluorescent core. For comparison, we have also devel-
oped TPy derivatives 4 having both electron-donating and
accepting substituents on the 6- and 2-phenyl groups, respec-
tively (Scheme 1).
Fluorescence properties of 2,6- and 2,5-diphenylthiazolo[4,5-
b]pyrazine (TPy) derivatives having an electron-donating
substituent (methoxy and dimethylamino) on the 6- and 5-
phenyl groups were studied. It was found that 2,6-diphenyl
derivatives fluoresce more efficiently than 2,5-diphenyl deriv-
atives. Furthermore, a 2,6-diphenyl derivative having an
additional cyano group on the 2-phenyl ring was an excellent
fluorophore showing a wide solvatochromism with great fluo-
rescence yields. Based on the obtained spectroscopic data and
mechanistic explanations concerning the substituent effects
on the fluorescence properties, useful information on design-
ing new TPy fluorophores is provided.
MATERIALS AND METHODS
General. Melting points were measured with a Yamato MP-21 apparatus.
IR spectra were obtained with a Horiba FT-720 spectrometer. High-reso-
lution ESI mass spectra were measured with a JEOL JMS-T100LC mass
spectrometer. 1H NMR spectra were recorded on a JEOL ECA-500
instrument (500 MHz). UV/visible absorption spectra were measured
INTRODUCTION
with a Varian Cary 50 spectrophotometer (scan speed, 600 nm minꢀ1
;
Because bioluminescence shows high performance in photon
production (1), we have paid attention to developing new artifi-
cial luminescent compounds mimicking bioluminescence fluoro-
phores. In particular, we have aimed to design new fluorophores
which are applicable to fluorescence functions such as fluores-
cent sensors for biological imaging and emitters for light-emit-
ting device technology (2–5). For this purpose, we have focused
on Cypridina oxyluciferin (Scheme 1), the light-emitter for the
bioluminescence in the ostracod Cypridina (the proper name is
Vargula), which is a natural fluorophore having an amidopyra-
zine structure (6,7). Then, we clarified the spectroscopic proper-
ties of amidopyrazine derivatives as Cypridina oxyluciferin
analogs (OCLA, Scheme 1) (8,9) and their modified fluorophores
such as difluoro[amidopyrazinato-O,N]boron derivatives (10). In
addition, we found that thiazolo[4,5-b]pyrazine (TPy), which is
readily prepared from amidopyrazine (Scheme 1) (11), has a
potential as a fluorophore (12). In particular, TPy derivatives 1
and 2a having the 4-(dimethylamino)phenyl group showed inten-
sive fluorescence from their excited singlet (S1) states having
charge transfer (CT) character. We provided a guide on design-
ing a TPy fluorophore by introducing an electron-donating sub-
stituent. In a previous report, we investigated the substituent
effect on the fluorescence property of TPy derivatives with the
2-phenyl group (12). For improving fluorescence performance of
TPy derivatives, the substituent effect of the phenyl group at the
data interval, 1 nm). Fluorescence spectra and fluorescence quantum
yields were obtained with a Hamamatsu Photonics Quantaurus-QY abso-
lute PL quantum yields measurement system. Fluorescence lifetimes (sf)
were measured with a Hamamatsu Photonics TAU time-correlated single-
photon counting fluorimeter system. Spectroscopic measurements were
performed using 1.0 9 10ꢀ5 mol Lꢀ1 sample solutions in a quartz cuv-
ette (1 cm path length) at 25 ꢁ 1°C. Spectral-grade solvents were used
for the measurements of UV/visible absorption and fluorescence. Density
functional theory (DFT) calculations were performed using the Gaussian
09 program (13). DFT includes Beck’s three-parameter function com-
bined with Lee, Yang, and Parr’s correlation function (B3LYP) along
with the 6-31G(d) basis set (14–16). Molecular graphics were generated
using GaussView, Version 5 (17).
Preparation of the TPy derivatives 2–4. According to the synthesis
method for 2a (12), the derivatives 2b, 3 and 4 were prepared from the
corresponding amidopyrazines, 6 and 8 with Lawesson’s reagent [2,4-bis
(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide] in one pot
in 30–90% yields (Scheme 2). 50-Arylamidopyrazines 6 were synthesized
by acylation of 5-arylpyrazinamines 5 (8,9,18,19). Similarly, 60-arylami-
dopyrazines 8 were synthesized by acylation of 6-arylpyrazinamines 7,
which were prepared by Suzuki coupling of 6-chloropyrazinamine with
the corresponding arylboronic acid.
6-(4-Dimethylaminophenyl)pyrazinamine (7a). To a solution of 6-chlo-
ropyrazinamine (100 mg, 0.77 mmol) in 1,4-dioxane (0.8 mL), 4-dim-
ethylaminophenylboronic
acid
(153 mg,
0.93 mol),
tetrakis
(triphenylphosphine)palladium (0) (27 mg, 0.023 mmol) and 2 M Na2CO3
aqueous solution (0.8 mL) were added under Ar, and the reaction mix-
ture was heated under reflux for 7.5 h. The mixture was diluted with
water (50 mL), and the product was extracted with chloroform (40 mL 9
4). The organic layer was washed with brine, dried over Na2SO4 and
concentrated in vacuo. The yellow residue was purified with silica gel
column chromatography [CH2Cl2/ethyl acetate (2:1)], to give 7a (91 mg,
55%) as yellow crystals: mp 189–190°C. dH (CDCl3) 3.02 (6 H, s), 4.50
*Corresponding author email: thirano@uec.ac.jp (Takashi Hirano)
© 2015 The American Society of Photobiology
1

2
Tatsuki Nakagawa et al.
Scheme 2. Synthesis of the TPy derivatives 2–4.
Scheme 1. Cypridina oxyluciferin, its analogue (OCLA) and the TPy
derivatives 1–4, together with a synthesis of TPy ring.
9.2 Hz), 7.54 (2 H, t, J 7.5 Hz), 7.61 (1 H, t, J 7.5 Hz), 7.91 (2 H, d, J
9.2 Hz), 7.98 (2 H, d, J 7.5 Hz), 8.47 (1 H, br s), 8.75 (1 H, s) and 9.47
(1 H, s). m (KBr)/cmꢀ1 3436, 1673, 1610, 1538, 1423 and 1269. m/z
(ESI) 319 ([M+H]+) and 341 ([M+Na]+).
(2 H, br s), 6.77 (2 H, d, J 9.2 Hz), 7.79 (1 H, s), 7.86 (2 H, d, J
8.6 Hz) and 8.31 (1 H, s). m (KBr)/cmꢀ1 3427, 3134, 1610, 1521, 1434
and 1286. m/z (ESI) 237 ([M+Na]+).
6-(4-Methoxyphenyl)pyrazinamine (7b). 96% yield. Yellow crystals,
mp 139–140°C. dH (CDCl3) 3.86 (3 H, s), 4.55 (2 H, br s), 6.98 (2 H, d,
J 8.6 Hz), 7.87 (1 H, s), 7.90 (2 H, d, J 9.2 Hz) and 8.32 (1 H, s). m
(KBr)/cmꢀ1 3438, 3110, 1608, 1533, 1434 and 1249. m/z (ESI) 202
([M+H]+).
60-(4-Methoxyphenyl)benzamidopyrazine (8b). Approximately 100%
yield. Yellow crystals, mp 149–150°C. dH (CDCl3) 3.88 (3 H, s), 7.03 (2
H, d, J 8.6 Hz), 7.54 (2 H, t, J 8.0 Hz), 7.62 (1 H, t, J 7.8 Hz), 7.94-
8.00 (4 H, m), 8.50 (1 H, br s), 8.78 (1 H, s) and 9.57 (1 H, s). m (KBr)/
cmꢀ1 3430, 3054, 1668, 1608, 1533, 1419 and 1259. m/z (ESI) 306
([M+H]+) and 328 ([M+Na]+).
6-(4-Methoxyphenyl)-2-phenylthiazolo[4,5-b]pyrazine (2b). To a solu-
tion of 50-(4-methoxyphenyl)benzamidopyrazine (90 mg, 0.30 mmol) in
chlorobenzene (1.0 mL), Lawesson’s reagent (72 mg, 0.18 mmol) was
added and the reaction mixture was heated under reflux overnight. For
completing the reaction, Lawesson’s reagent (72 mg, 0.18 mmol) was
added again, and the solution was heated under reflux for 4 h. The reac-
tion mixture was directly purified by silica gel thin layer chromatography
[CHCl3/ethyl acetate (15:1)] and the product was washed with n-hexane
to give 2b (35 mg, 37%) as yellow crystals: mp 191–192°C. dH (CDCl3)
3.90 (3 H, s), 7.06 (2 H, d, J 8.6 Hz), 7.53–7.59 (3 H, m), 8.08 (2 H, t,
J 8.6 Hz), 8.21 (2 H, m) and 9.08 (1 H, s). m (KBr)/cmꢀ1 2840, 1606,
1514, 1442 and 1325. m/z (ESI) Found: 342.0659 ([M+Na]+).
C18H13N3NaOS requires 342.0677.
5-(4-Dimethylaminophenyl)-2-phenylthiazolo[4,5-b]pyrazine (3a). 51%
yield. Orange crystals, mp 201–203°C. dH (CDCl3) 3.07 (6 H, s), 6.83 (2
H, d, J 8.6 Hz), 7.51-7.58 (3 H, m), 8.13 (2 H, d, J 9.2 Hz), 8.22 (2 H,
m) and 8.92 (1 H, s). m (KBr)/cmꢀ1 1610, 1514, 1466, 1369, 1236 and
1200. m/z (ESI) Found: 355.0946 ([M+Na]+). C19H16N4NaS requires
355.0993.
5-(4-Methoxyphenyl)-2-phenylthiazolo[4,5-b]pyrazine (3b). 59% yield.
Yellow crystals, mp 182–183°C. dH (CDCl3) 3.90 (3 H, s), 7.06 (2 H, d,
J 9.2 Hz), 7.51-7.60 (3 H, m), 8.16 (2 H, d, J 9.2 Hz), 8.23 (2 H, m)
and 8.95 (1 H, s). m (KBr)/cmꢀ1 1610, 1514, 1326, 1261 and 1201. m/z
(ESI) Found: 342.0639 ([M+Na]+). C18H13N3NaOS requires 342.0677.
2-(4-Cyanophenyl)-6-(4-dimethylaminophenyl)thiazolo[4,5-b]pyrazine
(4a). 30% yield. Orange crystals, mp >300°C. dH (CDCl3) 3.08 (6 H, s),
6.82 (2 H, d, J 8.6 Hz), 7.82 (2 H, d, J 8.0 Hz), 8.05 (2 H, d, J 8.6 Hz),
50-(4-Methoxyphenyl)benzamidopyrazine (6a). To a solution of 5-(4-
methoxyphenyl)pyrazinamine (150 mg, 0.75 mmol) in pyridine (1.0 mL),
benzoyl chloride (105 lL, 0.90 mol) was added under Ar, and the reaction
mixture was stirred at room temperature for 2 h. The reaction was
quenched by adding saturated NaHCO3 aqueous solution (50 mL), and
the product was extracted with chloroform (50 mL 9 3). The organic
layer was washed with brine, dried over Na2SO4 and concentrated in
vacuo, to give 6a (230 mg, 100%) as yellow crystals: mp 189–190°C. dH
(CDCl3) 3.88 (3 H, s), 7.02 (2 H, d, 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). m (KBr)/cmꢀ1 3369, 1668, 1610,
1513, 1419, 1353 and 1249. m/z (ESI) 306 ([M+H]+) and 328 ([M+Na]+).
4-Cyano-60-(4-dimethylaminophenyl)benzamidopyrazine (6b). ~100%
yield. Yellow crystals, mp 200°C (decomp.). dH (CDCl3) 3.04 (6 H, s),
6.80 (2 H, d, J 9.2 Hz), 7.83 (2 H, d, J 8.6 Hz), 7.92 (2 H, d, J 8.6 Hz),
8.05 (2 H, d, J 8.6 Hz), 8.43 (1 H, br s), 8.65 (1 H, d, J 1.7 Hz) and
9.62 (1 H, d, J 1.2 Hz). m (KBr)/cmꢀ1 3369, 2229, 1670, 1610, 1496
and 1357. m/z (ESI) 344 ([M+H]+) and 366 ([M+Na]+).
4-Cyano-60-(4-methoxyphenyl)benzamidopyrazine (6c). 95% yield.
Yellow crystals, mp 238–239°C. dH (CDCl3) 3.88 (3 H, s), 7.03 (2 H, d,
J 9.2 Hz), 7.84 (2 H, d, J 8.6 Hz), 7.97 (2 H, d, J 8.6 Hz), 8.06 (2 H, d,
J 8.6 Hz), 8.46 (1 H, br s), 8.68 (1 H, d, J 1.7 Hz) and 9.67 (1 H, d, J
1.2 Hz). m (KBr)/cmꢀ1 3307, 2229, 1654, 1610, 1542, 1498 1357, and
1250. m/z (ESI) 331 ([M+H]+) and 353 ([M+Na]+).
60-(4-Dimethylaminophenyl)benzamidopyrazine (8a). 88% yield. Yel-
low crystals, mp 200–201°C. dH (CDCl3) 3.05 (6 H, s), 6.79 (2 H, d, J

Photochemistry and Photobiology
3
8.29 (2 H, d, J 8.1 Hz) and 9.11 (1 H, s). m (KBr)/cmꢀ1 2225, 1610,
1533, 1425, 1373 and 1195. m/z (ESI) Found: 380.0964 ([M+Na]+).
C20H15N5NaS requires 380.0946.
2-(4-Cyanophenyl)-6-(4-methoxyphenyl)thiazolo[4,5-b]pyrazine (4b).
90% yield. Yellow crystals, mp 255–256°C. dH (CDCl3) 3.91 (3 H, s),
7.07 (2 H, d, J 8.6 Hz), 7.84 (2 H, d, J 8.6 Hz), 8.10 (2 H, d, J 9.2 Hz),
8.31 (2 H, d, J 8.6 Hz) and 9.15 (1 H, s). m (KBr)/cmꢀ1 2229, 1606,
1513, 1457, 1415, 1255 and 1200. m/z (ESI) Found: 383.0401 ([M+K]+).
C19H12KN4OS requires 383.0369.
various solvents. The data of UV/visible absorption and fluores-
cence are summarized in Tables 1 and 2, respectively, along with
the ET(30) index (20). The compounds 2b and 3 certainly
showed fluorescence as well as 2a. In particular, the quantum
yields (Φf) of 2,6-diphenyl derivatives 2a and b are larger than
those of the corresponding 2,5-diphenyl derivatives 3a and b in
all solvents. This tendency in Φf corresponds to that in the
extinction coefficients (e) of the lowest energy absorption bands.
The e values of 2a and b are larger than those of 3a and b. In
the case of 2,6-diphenyl derivatives, the Φf values of dimethyla-
mino derivative 2a were larger than 0.7 in middle and less polar
solvents, while those in polar solvents were less than 0.3. In con-
trast, methoxy derivative 2b shows large Φf values (>0.7) in sol-
vents other than cyclohexane. Thus, introduction of the methoxy
group on the 6-phenyl is appropriate for maintaining the efficient
RESULTS AND DISCUSSION
Spectroscopic properties of diphenyl TPy derivatives 2–4
Figure 1 shows absorption and fluorescence spectra of 2,6- and
2,5-diphenyl derivatives 2b and 3 together with 2a (12) in
Figure 1. UV/visible absorption (Abs) and fluorescence (Fl) spectra of 2a (A), 2b (B), 3a (C) and 3b (D) in cyclohexane (a), benzene (b), chloroform
(c), dichloromethane (d), acetonitrile (e), 2-propanol (f) and methanol (g) at 298 K. The spectra of 2a were reproduced using the data in Ref. (12).
Table 1. Electronic absorption of 2–4 in various solvents at 298 K.
kab/nm (e/104)†
Solvent [ET(30)*]
2a‡
2b
3a
3b
4a
4b
Cyclohexane [30.9]
407 (4.1)
295 (2.0)
367 (4.2)
398 (2.0)
317 (3.1)
283 (2.2)
410 (1.8)
321 (2.9)
287 (2.0)
412 (1.7)
322 (2.7)
288 (2.0)
412 (1.8)
319 (2.9)
287 (2.1)
407 (1.9)
317 (2.9)
286 (2.2)
411 (1.7)
317 (2.8)
286 (2.1)
413 (1.8)
317 (2.9)
286 (2.3)
365 (2.4)
290 (2.6)
263 (3.0)
370 (2.6)
294 (2.6)
433 (3.4)
312 (2.2)
380 (3.1)
270 (1.3)
Benzene [34.3]
418 (3.7)
304 (2.1)
371 (3.9)
372 (4.0)
370 (4.0)
368 (4.0)
372 (3.6)
371 (4.0)
443 (3.7)
315 (2.7)
384 (3.4)
Chloroform [39.1]
Dichloromethane [40.7]
Acetonitrile [45.6]
2-propanol [48.4]
Methanol [55.4]
422 (3.3)
306 (2.2)
370 (2.7)
294 (2.5)
266 (3.0)
368 (2.7)
293 (2.6)
265 (3.1)
364 (2.8)
289 (2.6)
263 (3.2)
368 (2.6)
292 (2.6)
265 (3.1)
367 (2.7)
292 (2.4)
264 (3.0)
449 (3.3)
316 (2.8)
384 (3.4)
273 (1.6)
420 (3.5)
305 (2.3)
445 (3.5)
315 (2.9)
382 (3.5)
271 (1.6)
417 (3.5)
303 (2.3)
437 (3.6)
312 (2.9)
378 (3.6)
271 (1.6)
424 (3.2)
306 (2.2)
445 (3.2)
315 (2.8)
383 (3.4)
273 (1.6)
424 (3.3)
307 (2.4)
444 (3.4)
314 (3.0)
381 (3.5)
273 (1.7)
*ET(30) in kcal molꢀ1 †Absorption maximum (kab) and extinction coefficient (e) in dm3 molꢀ1 cmꢀ1 in parenthesis. ‡Ref. (12).
.

4
Tatsuki Nakagawa et al.
Table 2. Fluorescence of 2–4 in various solvents at 298 K.
kfl/nm (Φf)†
Solvent [ET(30)*]
2a‡
2b
3a
3b
4a
4b
Cyclohexane [30.9]
Benzene [34.3]
Chloroform [39.1]
Dichloromethane [40.7]
Acetonitrile [45.6]
2-propanol [48.4]
Methanol [55.4]
449, 470 (0.89)
499 (0.78)
526 (0.81)
553 (0.78)
610 (0.28)
601 (0.11)
634 (0.008)
397, 416 (0.24)
416, 427 (0.76)
431 (0.85)
447, 468 (0.15)
519 (0.41)
552 (0.47)
585 (0.28)
654 (0.006)
638 (0.016)
n/a§
396, 413 (0.036)
419 (0.093)
429 (0.11)
478, 508 (0.86)
547 (0.88)
583 (0.85)
620 (0.57)
710 (0.012)
658 (0.035)
n/a§
415, 438, 462 (0.51)
455 (0.86)
467 (0.91)
475 (0.92)
499 (0.91)
490 (0.92)
437 (0.87)
448 (0.87)
447 (0.84)
461 (0.90)
437 (0.12)
456 (0.15)
453 (0.16)
473 (0.23)
509 (0.87)
*ET(30) in kcal molꢀ1 †Emission maximum (kfl) and quantum yield (Φf) in parenthesis. ‡Ref. (12). §Not observed.
.
<0.04). It is notable that the kfl value of 4a in acetonitrile is
the most red-shifted one (710 nm). Conversely, methoxy deriva-
tive 4b intensively fluoresced in the solvents other than cyclo-
hexane (Φf >0.8) and showed moderate fluorescence in
cyclohexane (Φf = 0.5). The kab values of the lowest energy
absorption bands of 4 also showed negligible solvent depen-
dency similarly to those of 2. The kab values of 4a and b are
red-shifted by ca. 20 and 10 nm compared to those of 2a and
b, respectively, indicating that an enhancement of the donor/
acceptor structure reduces the HOMO-LUMO gap. The varia-
tion ranges of the kfl values of 4a (478–710 nm) and 4b (415–
509 nm) are wider than those of the corresponding derivatives
2a (449–634 nm) and 2b (397–461 nm), respectively. To evalu-
ate fluorescence solvatochromic properties, the plots of the
energy values (Efl in kcal molꢀ1) estimated from kfl were made
as a function of ET(30). The Efl–ET(30) plots show linear corre-
lations with correlation coefficients (r) of the lines (Fig. 3);
Figure 2. UV/visible absorption (Abs) and fluorescence (Fl) spectra of 4a
(A) and 4b (B) in cyclohexane (a), benzene (b), chloroform (c), dichlorom-
ethane (d), acetonitrile (e), 2-propanol (f), and methanol (g) at 298 K.
Efl = ꢀ0.67ET(30) + 80 (r
ꢀ0.26ET(30) + 76 (r ꢀ0.98) for 2b, Efl = ꢀ0.98ET(30) +
88 (r
=
ꢀ0.96) for 2a (12), Efl
=
=
=
ꢀ0.94) for 4a and Efl = ꢀ0.37ET(30) + 76 (r
=
fluorescence property in polar media. In the case of 2,5-diphenyl
derivatives, moderate Φf values (>0.1) are provided from dimeth-
ylamino derivative 3a in less polar solvents, while those from
methoxy derivative 3b are shown in polar solvents.
ꢀ0.96) for 4b. The slopes of the linear correlations for 4a and
b are greater than those for the corresponding 3a and b, respec-
tively, indicating that the strength in polarized character of the
S1 states of 4a and b is enhanced compared to those of 2a and
b by introduction of the cyano group.
Fluorescence of 2 and 3 showed characteristic solvatochro-
mism, while their absorption maximum wavelengths (kab) of the
lowest energy absorption bands showed a negligible solvent-
dependent variation. The result indicates that the S1 states of 2
and 3 are polarized more than the corresponding ground (S0)
states. Polarized character of their S1 states is caused by CT
character of their S0 ? S1 transitions, which was shown in our
previous studies on the fluorescent properties of 1 and 2a (12).
The variation ranges of the kfl values of dimethylamino deriva-
tives 2a (449–634 nm) and 3a (447–654 nm) are wider than
those of methoxy derivatives 2b (397–461 nm) and 3b (396–
473 nm), indicating that a strength of electron donation of the
substituent effectively expands the variation range of the kfl
values in fluorescence solvatochromism.
2,6-Diphenyl TPy derivatives 2 having an electron-donating
substituent on the 6-phenyl group showed intensive fluorescence
compared to 2,5-diphenyl derivatives 3. Based on this finding,
2,6-diphenyl derivatives 4 having the cyano group on the 2-
phenyl ring were newly designed to enhance an electron-donor/
acceptor structure. The spectroscopic properties of 4 were inves-
tigated in the various solvents (Fig. 2, Tables 1 and 2). Dimeth-
ylamino derivative 4a showed intense fluorescence with
solvatochromic character in middle and less polar solvents (Φf
>0.5), while its fluorescence was reduced in polar solvents (Φf
Fluorescence lifetime measurements
Fluorescence lifetimes (sf) of 2b, 3 and 4 in cyclohexane were
measured to reveal a factor to govern their fluorescence yields
(Table 3) along with the data of 2a (12). By using the sf and Φf
values, the rate constants of the fluorescence emission process
(kf) and the quantum yields and the rate constants of nonradiative
decay processes (Φf and knr) were estimated using the equations,
kf = Φf sfꢀ1, Φnr = 1–Φf and knr = Φnr sfꢀ1, respectively. The kf
values of 2,6-diphenyl derivatives 2 and 4 are very close, indi-
cating that the electron-donating substitution on the 6-phenyl
group and the introduction of the cyano group on the 2-phenyl
group do not influence the fluorescence emission processes. It is
characteristic of the knr values of methoxy derivatives 2b and 4b
to be larger than those of dimethylamino derivatives 2a and 4a.
In contrast, 2,5-diphenyl derivatives 3 have the kf values slightly
smaller than those of the 2,6-diphenyl derivatives 2 and 4
whereas the knr values of 3 are greater than those of the 2,6-
diphenyl derivatives 2 and 4. There seems to be a tendency that
the knr value of dimethylamino derivative 2a, 3a and 4a are less
than those of the corresponding methoxy derivatives 2b, 3b and

Photochemistry and Photobiology
5
(Table 4). The results show that the S0 ? S1 and the S0 ? S2
transitions of 2–4 are of p,p* and n,p*, respectively, and have
main contribution of the HOMO ? LUMO and HOMO–2 ?
LUMO configurations, respectively. The relative variation in the
wavelengths (ktr) estimated from the calculated transition ener-
gies of 2–4 is similar to that in the observed kab values of the
lowest energy absorption bands from absorption measurements.
The calculated oscillator strengths (f) for the S0 ? S1 transitions
of 2 are larger than those of 3, predicting that the transitions
between the S0 and S1 states in 2 take place more efficiently than
those in 3. This prediction matches the observations showing rel-
ative differences in the e values of the lowest energy absorption
bands of 2 and 3 and in their kf values (21).
The difference in the f values of the S0 ? S1 transitions
between 2 and 3 may be originated from those in the structural
characteristics. For evaluating the optimized structures of 2 and
3, axes a and b are defined as the extended lines passing through
the C2–C10 and C6–C1″ (or C5–C1″) bonds in 2 (or 3), respec-
tively (Fig. 4). The two axes intersect each other with interior
angles 161° and 136° for 2 and 3, respectively. Because the thia-
zole part in the TPy ring is distorted by the long C–S bonds, the
structures of 3 are bent more than those of 2.
For understanding how the bent structures affect the p-elec-
tronic character, visualized electronic distributions of the HO-
MOs and LUMOs of 2 and 3 are depicted in Fig. 5. In the case
of 2, electronic distributions of the LUMOs spread throughout
Figure 3. Efl values of 2a (■), 2b (□), 4a (●) and 4b (○) plotted as a
function of ET(30).
4b, which is responsible for the difference in the Φf values
in cyclohexane between the dimethylamino and methoxy
substituents.
DFT calculations
For further understanding the relationship between the spectro-
scopic and the p-electronic properties, DFT and TDDFT calcula-
tions of 2–4 were carried out with the B3LYP/6-31G(d) method
Table 3. Photophysical properties of 2–4 in cyclohexane at 295 K.
kf ‡/108 sꢀ1
knr§/108 sꢀ1
†
Compounds
kab/nm
kfl/nm
kex */nm
Φnr
Φf
sf/ns
2ak
2b
3a
3b
4a
4b
407
367
398
365
433
380
449, 470
397, 416
447, 468
396, 413
478, 508
405
365
405
365
405
365
0.11
0.76
0.85
0.96
0.14
0.49
0.89
0.24
0.15
0.036
0.86
0.51
2.3
0.56
1.05
<0.3
2.54
1.14
3.9
4.2
1.4
>1.2
3.4
4.5
0.48
14
8.1
>30
0.55
4.3
415, 438, 462
*Excitation wavelengths. †Determined by Φnr = 1–Φf. ‡Determined by kf = Φf sfꢀ1
. .
§Determined by knr = Φnr sfꢀ1 kRef. (12).
Table 4. Calculation data of 2–4 with DFT and TD-DFT using B3LYP/6-31G(d).
Compound
HOMO/eV
LUMO/eV
n-orbital* (DEn†/eV)
Hꢀ2 (1.42)
Transitions‡
ktr§/nm (f)
Configuration*,k
H ? L (0.70)
H–2 ? L (0.69)
H–2 ? L + 1 (ꢀ0.11)
H ? L (0.70)
Hꢀ2 ? L (0.69)
Hꢀ2 ? L + 1 (ꢀ0.10)
H ? L (0.70)
2a¶
ꢀ5.12
ꢀ1.94
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
426 (0.77)
346 (0.001)
2b
ꢀ5.69
ꢀ2.10
Hꢀ2 (1.01)
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
377 (0.89)
346 (0.002)
3a
3b
4a
ꢀ5.09
ꢀ5.72
ꢀ5.32
ꢀ1.90
ꢀ2.04
ꢀ2.50
Hꢀ2 (1.46)
Hꢀ2 (1.00)
Hꢀ2 (1.50)
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
446 (0.14)
343 (0.002)
383 (0.25)
342 (0.0008)
479 (0.73)
362 (0.0003)
Hꢀ2 ? L (0.69)
H ? L (0.70)
Hꢀ2 ? L (0.69)
H ? L (0.71)
Hꢀ2 ? L (0.69)
Hꢀ2 ? L + 1 (0.14)
H ? L (0.70)
Hꢀ2 ? L (0.68)
Hꢀ2 ? L + 1 (0.14)
Hꢀ1 ? L (ꢀ0.12)
4b
ꢀ5.94
ꢀ2.64
Hꢀ2 (1.05)
S0 ? S1 (p,p*)
S0 ? S2 (n,p*)
412 (0.86)
362 (0.001)
*H, Hꢀn (n = 1 and 2), L, L + 1 denote the HOMO, HOMOꢀn (n = 1 and 2), LUMO, and LUMO+1, respectively. †Energy difference between
HOMO and n-orbital. ‡The p,p* and n,p* transitions to the excited singlet states with the lowest excitation energies. Character of the transitions is in
¶
the parentheses. §Wavelength estimated from transition energies. kConfiguration of excitation. Coefficients are in the parentheses. Ref. (12).

6
Tatsuki Nakagawa et al.
Figure 4. Optimized structures of 2 and 3.
The difference in the knr values observed in the present work
may be related to intersystem crossing (ISC) yields as previously
reported that the main nonradiative decay process from the S1
state of a 2-phenyl TPy derivative is ISC (12). We also proposed
that an energy difference (DEn) between HOMO and n-orbital is
a useful scale for predicting ISC efficiencies. In the case of 2-
phenyl derivatives, DEn values of a fluorescent derivative are
greater than those of the corresponding weakly fluorescent deriv-
atives. In the case of 2,5- and 2,6-diphenyl derivatives 2–4, the
HOMO–2s are shown to be of n-orbitals by DFT calculations.
The DEn values of dimethylamino derivatives 2a, 3a and 4a are
greater than those of the corresponding methoxy derivatives 2b,
3b and 4b, rationalizing the difference in the knr values.
CONCLUSION
Comparison of the fluorescence properties of 2,6- and 2,5-diphe-
nyl TPy derivatives (2 and 3) having the electron-donating sub-
stituent on the 6- and 5-phenyl groups, respectively, leads to the
finding that 2 fluoresces efficiently more than 3. In particular, the
methoxy derivative 2b shows large Φf values (>0.7) in solvents
other than cyclohexane, while the variation range of the kfl val-
ues of 2b is narrower than that of the dimethylamino derivative
2a, which shows a wide fluorescence solvatochromism (449–
634 nm) and great Φf values (>0.7) in middle and less polar sol-
vents. Based on these findings, 2,6-diphenyl derivatives 4 with
the cyano group added on the 2-phenyl ring were developed. It
is noteworthy that the methoxy derivative 4b is an excellent
fluorophore showing a wide solvatochromism (415–509 nm)
with great Φf values (>0.5) in all the used solvents, because of
its moderate CT character in the S1 state. The dimethylamino
derivative 4a is also a good solvatochromic fluorophore in mid-
dle and less polar solvents (Φf >0.5). While the Φf values of 4a
are small (<0.04) in polar solvents, it shows the most red-shifted
kfl value (710 nm) in acetonitrile. The difference in the position
(C5/C6) of the phenyl group with the electron-donating substitu-
ent effectively affects the fluorescence property. In particular,
2,5-diphenyl derivatives 3 show small Φf values, which are
Figure 5. HOMOs and LUMOs of 2 and 3.
the molecules, while the HOMOs have main electronic distribu-
tions on the 6-phenyl TPy parts with small distributions on the
2-phenyl moieties. On the other hand, electronic distributions of
the HOMOs and LUMOs of 3 localize in the 5-phenyl TPy and
2-phenyl TPy parts, respectively. The spatial overlaps between
the HOMOs and LUMOs of 3 are less than those of 2, leading
to small transition moments of the S0 ? S1 transitions for 3 rela-
tive to those for 2. Therefore, the difference in character of the
frontier orbitals between 2 and 3 reflects well on the property of
the electronic transitions between the S0 and S1 states.

Photochemistry and Photobiology
7
8. Hirano, T., T. Nakagawa, A. Kodaka, S. Maki, H. Niwa and M.
Yamaji (2013) 5-[4-(Dimethylamino)phenyl]-2-benzamidopyrazines:
Fluorescent dyes based on Cypridina oxyluciferin. Res. Chem. Inter-
med. 39, 233–245.
9. Hirano, T., Y. Takahashi, H. Kondo, S. Maki, S. Kojima, H. Ikeda
and H. Niwa (2008) The reaction mechanism for the high quantum
yield of the Cypridina (Vargula) bioluminescence supported by
chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-
ones (Cypridina luciferin analogues). Photochem. Photobiol. Sci. 7,
197–207.
10. Hachiya, S., T. Inagaki, D. Hashizume, S. Maki, H. Niwa and T.
Hirano (2010) Synthesis and fluorescence properties of difluoro
[amidopyrazinato-O, N]boron derivatives: A new boron-containing
fluorophore. Tetrahedron Lett. 51, 1613–1615.
11. Fruit, C., A. Turck, N. Ple and G. Queguiner (2002) Syntheses of
N-diazinyl thiocarboxamides and of thiazolodiazines. Metalation
studies. Diazines XXXIII. J. Heterocyclic Chem. 39, 1077–1082.
12. Nakagawa, T., M. Yamaji, S. Maki, H. Niwa and T. Hirano
(2014) Substituent effects on fluorescence properties of thiazolo
[4,5-b]pyrazine derivatives. Photochem. Photobiol. Sci. 13, 1765–
1772.
caused by the slightly smaller kf values and the larger knr values
compared to those of 2,6-diphenyl derivatives 2. One of the rea-
sons for the difference is suggested based on the DFT calculation
data that the f values for the S0 ? S1 transitions of 3 are less
than those of 2. Because compounds 3 have bended structures
more than 2, the electronic distributions of the HOMOs and LU-
MOs in 2 and 3 are different, leading to the different f values.
The obtained spectroscopic data and mechanistic explanations on
the fluorescent properties of 2–4 will be useful information on
designing new TPy fluorophores. At present, 4b provides the
best performance of fluorescence yields in any solvents and a
wide-ranged solvatochromism.
Acknowledgements—This work was supported by a Grant-in-Aid for
Scientific Research C (No. 22550031) from the Japan Society for the
Promotion of Science. Financial support was also provided by the Iketani
Science and Technology Foundation and Izumi Science and Technology
Foundation. We thank the Information Technology Center of the
University of Electro-Communications for technical assistance in
computing the quantum chemical calculations.
13. Frisch, M. J., G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.
A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A.
F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,
Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J.
E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N.
Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghava-
chari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,
N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bak-
ken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Ya-
zyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L.
Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Fluorescence decay curves of 2b (a), 3a (b), 3b
(c), 4a (d) and 4b (e) in cyclohexane at 295 K.
Figure S2. Lippert–Mataga plots for 2 and 4.
Table S1. Analysis of the electronic absorption and fluores-
cence of 2 and 4 in various solvents for Lippert–Mataga plots.
€
J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Fores-
man, J. V. Ortiz, J. Cioslowski and D. J. Fox (2009) GAUSSIAN 09
(Revision A.02). Gaussian, Inc., Wallingford, CT.
14. Becke, A. D. (1993) Density-functional thermochemistry 3. The role
of exact exchange. J. Chem. Phys. 98, 5648–5652.
15. Lee, C. T., W. T. Yang and R. G. Parr (1988) Development of the
Colle-Salventti correlation-energy formula into a functional of the
electron-density. Phys. Rev. B 37, 785–789.
16. Stephens, P. J., F. J. Devlin, C. F. Chabalowski and M. J. Frisch
(1994) Ab-initio calculation of vibrational absorption and circular-
dichroism spectra using density-functional force-fields. J. Phys.
Chem. 98, 11623–11627.
REFERENCES
1. Shimomura, O. (2012) Bioluminescence: Chemical Principles and
Methods, rev edn. World Scientific, Singapore.
2. Tsien, R. Y. (1993) Fluorescent Chemosensors for Ion and Molecule
Recognition (Edited By A. W. Czarnik), pp. 130–146. ACS, Wash-
ington, DC.
17. Dennington, R., T. Keith and J. Millam (2009) GaussView, Version
5. Semichem Inc., Shawnee Mission, KS.
3. Zollinger, H. (2003) Color Chemistry: Syntheses, Properties, and
Applications of Organic Dyes and Pigments, 3rd edn. Wiley-VCH,
Weinheim.
4. Hunger, K. (ed.) (2003) Industrial Dyes: Chemistry, Properties,
Applications. Wiley-VCH, Weinheim.
5. Valeur, B. and M. N. Berberan-Santos (2013) Molecular Fluores-
cence: Principles and Applications, 2nd edn. Wiley-VCH, Wein-
heim.
18. Toya, Y., T. Kayano, K. Sato and T. Goto (1992) Synthesis and
chemiluminescence properties of 6-(4-methoxyphenyl)-2-methylimi-
dazo[1,2-a]pyrazin-3(7H)-one and 2-methyl-6-(2-naphthyl)imidazo
[1,2-a]pyrazin-3(7H)-one. Bull. Chem. Soc. Jpn. 65, 2475–2479.
19. Cavalier, J.-F., M. Burton, F. Dussart, C. Marchand, J.-F. Rees and
J. Marchand-Brynaert (2001) Catechol derivatives of aminopyrazine
and cell protection against uvb-induced mortality. Bioorg. Med.
Chem. 9, 1037–1044.
6. Goto, T. (1968) Chemistry of bioluminescence. Pure Appl. Chem.
17, 421–441.
20. Reichardt, C. (2003) Solvents and Solvent Effects in Organic Chem-
istry, 3rd edn. Wiley-VCH, Weinheim.
7. Goto, T. and H. Fukatsu (1969) Cypridina bioluminescence 7.
Chemiluminescence in micelle solutions– A model system for Cypri-
dina bioluminescence. Tetrahedron Lett. 10, 4299–4302.
21. Strickler, S. J. and R. A. Berg (1962) Relationship between absorp-
tion intensity and fluorescence lifetime of molecules. J. Phys. Chem.
37, 814–822.