Intensely blue-fluorescent 2,5-bis(benzoimidazol-2-yl)pyrazine dyes with improved solubility: Their synthesis, fluorescent properties, and application as microenvironment polarity probes

Article abstract of DOI:10.1016/j.tet.2010.08.036

In order to improve the solubility of intensively fluorescent 2,5-bis(benzimidazol-2-yl)pyrazine (BBIP), we synthesized new BBIP derivatives (2, 3a,b, and 5a,b) possessing two alkyl chains at the N-1 and N-1′ positions of the two benzimidazole moieties. C

Full text of DOI:10.1016/j.tet.2010.08.036

Tetrahedron 66 (2010) 8273e8279
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Tetrahedron
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Intensely blue-fluorescent 2,5-bis(benzoimidazol-2-yl)pyrazine dyes with
improved solubility: their synthesis, fluorescent properties, and application
as microenvironment polarity probes
a
,
b
a
b
*
Ryota Saito , Yuriko Matsumura , Saori Suzuki , Naoki Okazaki
a
Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kitamachi, Musashino 180-8633, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2010
Received in revised form 12 August 2010
Accepted 14 August 2010
Available online 21 August 2010
In order to improve the solubility of intensively fluorescent 2,5-bis(benzimidazol-2-yl)pyrazine (BBIP), we
synthesized new BBIP derivatives (2, 3a,b, and 5a,b) possessing two alkyl chains at the N-1 and N-1 positions
of the two benzimidazole moieties. Characterization of these compounds demonstrated that they exhibit
high fluorescence intensity even in protic solvents, as well as solvatochromic fluorescence, in which their
0
r
fluorescence maxima in aqueous methanol exhibited bathochromic shift with increasing 3 value of the
medium. We utilized 5a as a microenvironment polarity probe to indicate the variation inpolarity around the
backbone of the temperature-sensitive poly(N-isopropylacrylamide) by measuring the spectral change
caused by the thermal phase transition of the polymer.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM)
hydrogel. PNIPAM exhibits a thermo-responsive phase transition
2
The design and synthesis of intensely fluorescent organic mole-
culeshasbeenanattractivefieldbecauseoftheincreasingdemandfor
developing effective light emitting devices and bioimaging probes.
We have previously reported that 2,5-bis(benzimidazol-2-yl)pyr-
azine (BBIP, 1) exhibited extremely intensive blue-fluorescence with
maximum emission at 444 nm and a fluorescence quantum yield
at the critical solution temperature in aqueous solution. Upon the
phase transition, the microenvironmental polarity around the
polymer backbone alters from a polar condition to a less polar
one. The fluorescence spectrum of the fluorophore introduced
into the polymer should be influenced by the polarity change, and
consequently the thermal phase transition of the polymer should
be detected by the spectral change. Here we describe details of the
synthesis and quantitative photophysical properties of the new
BBIP derivatives.
3
,4
1
of 0.90 in dimethyl sulfoxide (DMSO). In the presence of sodium
methoxide as a base, the fluorescence maximum of 1 showed
a bathochromic shift to 477 nm with a fluorescence quantum yield of
almost unity.
Despite these fascinating properties as a blue-fluorescent dye, 1
has the fatal drawback of poor solubility in most conventional
solvents except for DMSO and N,N-dimethylformamide (DMF).
Successful application of the superior luminescent property of 1 to
useful materials, such as organic luminescent devices and probes,
requires that the solubility of 1 be improved. One of the universal
methods to improve the solubility of organic molecules, especially
in volatile organic solvents, is to introduce alkyl groups. Thus, in the
present study, we synthesized new BBIP derivatives possessing two
2. Results and discussion
2.1. Synthesis
2,5-Bis(1-butylbenzimidazol-2-yl)pyrazine(2)wassynthesizedas
depicted in Scheme 1. 2-Bromonitrobenzene was reacted with
butylamine to give 2-butylaminonitrobenzene, which was converted
1
5
into N -butylbenzene-1,2-diamine by catalytic hydrogenation. The
phenylenediamine was then reacted with pyrazine-2,5-dicarboxylic
acid in polyphosphoric acid to afford the benzimidazolylpyrazine 2.
1-Butyl-2-(pyrazin-2-yl)benzimidazole 6 was also synthesized as
a spectral reference compound by reacting pyrazinecarboxylic acid
and N-butylphenylenediamine in a manner similar to the synthesis
of 2.
0
alkyl chains at the N-1 and N-1 positions of the two benzimidazole
moieties (2, 3, and 5) and investigated their fluorescent properties
in various organic solvents. We also achieved successful application
of the compound 5a, which was introduced into a polymer chain of
BBIP derivatives with
u-aminoalkyl groups (5a, b) were prepared
*
Corresponding author. E-mail address: saito@chem.sci.toho-u.ac.jp (R. Saito).
as shown in Scheme 2. The direct alkylation of 1 with Boc-protected
0
040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.08.036

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R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
H
N
H
N
Br
i)
ii)
iii)
N
N
N
N
N
NO2
NO2
NH2
N
2
ꢀ
2
Scheme 1. Reagents and conditions: (i) butylamine in DMSO, (ii) H PdeC in MeOH, (iii) pyrazine-2,5-dicarboxylic acid in PPA at 140 C.
0
0
u-aminoalkylbromides gave 3a and 3b, which were, respectively,
N,N,N -N -tetramethylethylenediamine as the accelerator, and po-
tassium persulfate as the initiator in DMFewater-mixed solvent
under a nitrogen atmosphere (Scheme 3).
deprotected by trifluoroacetic acid followed by condensation with
acrylic acid to afford 5a,b.
Scheme 2. Reagents and conditions: (i) Boc-protected
2 2
u-aminoalkylbromide and NaH in DMSO, (ii) TFA, and (iii) acrylic acid, EDCl, and HOBt in DMFeCH Cl .
CH CH2
O
CHCH2 y' CH CH2 x'
z'
O
O
HN
O
HN
(
CH2)
3
HN
CH2
O
HN
N
N
i)
N
N
5a
+
+
HN
HN
O
N
N
(
CH2)3
NH
HN
NH
O
O
O
CH CH2 x CH CH2 y
CH CH2 z
ꢀ
2
Scheme 3. Reagents and conditions: (i) TMEDA and KPS in DMFewater(2/3) at 4 C under N for 24 h.
The structures of all synthesized compounds were assigned on
the basis of 1H NMR, IR, and mass spectral data as well as com-
bustion analyses.
2.2. Electronic absorption properties of 2, 3, and 5
The electronic absorption spectra of 2, 3, and 5 in various solvents
are summarized in Table 1, and selected absorption spectra are
shown in Figure 1.
It is noteworthy that the measurements in non-polar solvents,
such as cyclohexane and benzene, were successful. This apparently
indicates that the solubility of BBIP was improved by introducing
alkyl groups. Compound 2 showed an absorption maximum at
around 375 nm accompanied by a shoulder at around 400 nm. A
comparison of the absorption spectra of 2 with those of mono-
substituted pyrazine 6 and N-butylbenzimidazole 7, displayed in
Figure 2, revealed that these absorption bands were attributable to
the absorption of the whole molecule 2, as was the case for 1. An
Et
N
N
N
N
N
Et
N
N
N
N
N
N
N
6
7
8
6
A PNIPAM hydrogel containing BBIP fluorophore was prepared
by free radical copolymerization of N-isopropylacrylamide and 5a
as the monomers, N,N-methylenebisacrylamide as the crosslinker,

R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
8275
Table 1
Electronic absorption data of 2, 3, and 5 in various solvents
4
3
ꢁ1
ꢁ1
)
Compound
Absorption maximum wavelength/nm (
3
/10 dm mol cm
Dioxane (2.21)a
Cyclohexane (2.02)a
Benzene (2.27)a
2-PrOH (19.92)a
Acetone (20.56)a
MeOH (32.66)a
MeCN (35.94)a
DMSO (46.45)a
2
392 (4.49)
393c (3.76)
378 (5.17)
396c (3.54)
394c (3.46)
374 (5.17)
391c (3.85)
375 (4.42)
394c (3.00)
374 (3.95)
391c (3.77)
374 (4.84)
393c (3.28)
375 (4.29)
395c (3.04)
374 (4.91)
390 (3.09)
392c (3.24)
371 (4.92)
396 (3.53)
390c (2.80)
369 (4.62)
389 (2.69)
389c (2.99)
369 (4.72)
388 (3.03)
397c (2.80)
375 (4.80)
393c (2.85)
376 (3.80)
395c (2.73)
375 (3.62)
395c (3.36)
375 (4.66)
393c (2.97)
375 (3.99)
3
79 (5.71)
b
c
c
c
c
3
3
5
5
a
b
a
b
d
d
d
d
3
78 (4.15)
375 (4.03)
372 (4.29)
371 (3.86)
371 (3.86)
c
c
c
c
c
396 (3.03)
78 (3.87)
393 (2.69)
389 (3.07)
390 (2.46)
388 (2.72)
3
374 (3.71)
372 (3.95)
370 (3.59)
370 (3.59)
c
c
c
c
c
396 (3.58)
77 (4.74)
392 (3.44)
389 (3.72)
388 (3.42)
388 (2.49)
3
374 (3.74)
371 (4.64)
371 (4.64)
370 (4.63)
c
c
c
c
c
396 (3.26)
78 (4.25)
394 (2.94)
389 (3.14)
388 (2.76)
389 (1.92)
3
374 (4.11)
372 (4.11)
372 (4.11)
370 (3.88)
a
b
c
Dielectric constant.
Not measured.
Shoulder peak.
investigation of derivatives 3 and 5 showed the same results. The
absorption data of 3 and 5 are also listed in Table 1.
benzimidazolylpyrazine unit of 2) in the ground state were calcu-
lated by the AM1 method and estimated to be 0.51 and 3.55 Debye
7
Careful investigation of the observed spectra revealed that each
compound exhibited a slightly hypsochromic shift of the absorp-
tion bands with increasing solvent polarity. For a concrete example,
the absorption bands were observed near 380 nm in benzene,
while those observed around 370 nm in acetonitrile. To identify the
cause of this behavior, the dipole moments of 2 and 7 (the partial
for 2 and 7, respectively. These data clearly indicate that 2 has
a partially polarized structure at the benzimidazoleepyrazine
moieties (i.e., 7), and that these partial dipole moments cancel each
other out in the whole structure. The results are similar to those we
1
previously obtained for 1.
It is possible that each of the partially polarized structures in 2
are somewhat stabilized in polar solvents in order to lower the
HOMO energy. To test this conjecture, the effect of the solvent on
8
the HOMO energy level was simulated by AM1-COSMO calcula-
tions with various relative dielectric constant (
calculations, we simulated the effects of 2-propanol (
DMSO ( ¼78.4). The results are listed in Table
¼46.5), and water (
. The calculated heat of formation ( H) decreased with increasing
, suggesting that 2 is more stable in polar solvents than in less
polar ones. The HOMO and LUMO were also stabilized with
increments of , but the degree of stabilization in HOMO
¼0.416 eV) was larger than that in LUMO ( ¼0.095 eV). Conse-
quently, the differential energies ( E) between the HOMO and
LUMO increased with higher values. These calculation results
3
r
) values. In these
3
r
¼19.9),
3
r
3
r
2
D
3
r
3
r
(D
D
D
3
r
closely match the observed solvent effects, in which higher tran-
sition energies were required in solvents with higher polarity.
2.3. Fluorescent properties of 2, 3, and 5
The fluorescence of 2, 3, and 5 was measured in various solvents,
Figure 1. Absorption spectra of 2 in DMSO (black) or benzene (red), 3a in benzene
and the observed fluorescence maxima are summarized in Table 3,
accompanied by the fluorescence quantum yields. Figure 3 shows
the fluorescence spectra of 5a as a typical example. In each solvent,
we observed two distinct emission bands at around 410 and
(
blue), and 3b in benzene (purple); c¼10
mM.
430 nm, as well as a shoulder at around 470 nm. High fluorescence
quantum yields ( ) were observed in all solvents employed, and
F
F
were maintained even in polar protic solvents, such as methanol.
Spectral features of each compound were found to change slightly
with solvent polarity. The fine structure observed in non-polar
r r
solvents with low 3 values became ambiguous with increasing 3 . In
Table 2
Simulation of the effect of solvent’s dielectric constant on the heat of formation (
DH),
the HOMO and LUMO energies, and the differential energies (
DE) between the
HOMO and LUMO for 2 calculated by the AM1-COSMO method
H/kcal mol 1
ꢁ
Energy level/eV
DE/eV
Dielectric constant (
3
r
)
D
HOMO
LUMO
19.9
46.5
78.4
149.81
149.20
149.00
e9.129
e9.154
e9.159
e1.339
e1.342
e1.346
7.792
7.812
7.813
Figure 2. Absorption spectra of 2 (black), 6 (blue), and 7 (red) in DMSO; c¼20
mM.

8276
R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
Table 3
Fluorescence maxima (
l
FL) and fluorescence quantum yields (
F
FL) of 2, 3, and 5 in various solvents
3a 3b
Solvent (Dielectric constant)
2
5a
5b
a
a
a
a
a
lFL/nm
F
FL
l
FL/nm
F
FL
lFL/nm
F
FL
lFL/nm
F
FL
lFL/nm
F
FL
b
Cyclohexane
Benzene
Dioxane
(2.024)
406,430
414,436
414,434
413,434
418,433
433
0.62
0.83
0.80
0.83
0.79
0.77
0.81
0.84
d
d
d
d
d
d
d
d
(2.274)
(2.209)
(19.92)
(20.56)
(32.66)
(35.94)
(46.45)
420,440
417,435
416,434
417,433
434
1.00
1.00
0.95
0.96
0.95
0.96
0.93
417,437
415,434
414,433
417,437
434
1.00
0.95
0.90
0.95
0.87
0.93
0.92
423,438
418,435
414,433
417,433
434
0.90
0.95
0.84
0.88
0.79
1.00
0.82
418,438
416,435
414,434
418,433
434
0.98
0.93
0.88
0.94
0.86
0.94
0.89
2-PrOH
Acetone
MeOH
MeCN
431
441
433
440
433
440
433
440
432
440
DMSO
a
10
These values were determined on the basis of the fluorescence efficiency of quinine sulfate in 0.1 M sulfuric acid (
Data were not measured.
F
FL¼0.55).
b
9
general, spectral broadening is caused by solvent interactions. The
excited state is stabilized in a polar solvent, resulting in a red-shifted
fluorescence. Therefore, the singlet-excited 2 would be stabilized
9
b
observed broadening in the emission spectra suggests that in-
teractions between the solvent and 2 become stronger in solvents
in polar solvents so as to lower the LUMO level.
with higher
The fluorescence maximum tended to shift toward longer
wavelengths in solvents with larger values. To investigate this
spectral shift in detail, the observed fluorescence energies were
plotted against the values of the solvents employed (Fig. 4).
According to this figure, the variation in emission energy appears
to be independent of solvent polarity. It is likely that this scattering is
due to the solvation strength and structure being different for each
individual solvent molecule. In order to reduce the specific in-
teractions of the solvent molecules with different structures and to
glean more precise information on the effect of a solvent’s dielectric
constant on the fluorescence shift, the fluorescence spectra of BBIP
derivatives were measured in aqueous methanol with different
water ratios. Compounds 5a and 5b were targeted in these mea-
surements, taking account of the fact that these compounds would
be applied in the subsequent polymerization experiments. A typical
result obtained for 5a is depicted in Figure 5.
3
r
values.
In order to simulate this possibility, the effect of
level of 8dan ethyl analogue of 2din the S state and in the cor-
responding ground state with unrelaxed FranckeCondon geometry
r
3 on the energy
1
3
r
FC
(S
geometry of 8 in the ground state within a dielectric of a given
was first optimized, and then the optimized geometry in the S
0
) was estimated by AM1-COSMO. In these calculations, the
3
r
3
r
1
state in the same dielectric was calculated. The butyl groups were
replaced with ethyl groups for ease of calculation. Table 4 shows the
FC
calculated S
eV) for the S
seen from these results, the S
with increasing . This trend closely matches the observed bath-
ochromic shift in Figure 5, and implies that the fluorophore in the
state becomes polar in solvents with larger values.
To test this conjecture, the dipole moments of 8 in the S
aqueous methanol were also simulated. The results are compiled in
Table 4. The dipole moment in 100% methanol (
¼32.7) was cal-
culated to be 23.67 Debye, and its value increased with the value,
1
and S
0
levels for 8 and the differential energies (
conditions. As can be
transition energy decreased
DE in
FC
1
0 r
/S transition under various 3
FC
1
/S
0
3
r
S
1
3
r
1
state in
3
r
Both 5a and 5b showed bathochromic shifts in their fluorescence
maxima by increasing the solvent’s water ratio. Plots of the observed
3
r
i.e., the water ratio of the solvent system. Thus the central bis-
benzimidazolylpyrazine chromophore was estimated to have
a large dipole moment of over 20 Debye in polar solvents.
According to the AM1-COSMO calculations, the optimized geome-
try was found to take on a conformation in which the two benz-
ꢁ
1
fluorescence energies of 5a and 5b (in cm ), respectively, against
the dielectric constant of the wateremethanol binary solvent system
gave straight lines (Fig. 6).
These results clearly demonstrate that the 2,5-bis(benzimidazol-
2
-yl)pyrazine fluorophore is applicable as a fluorescent polarity
probe. The observed red-shift in fluorescence with increasing sol-
vent polarity indicates that the S -state fluorophore becomes more
polar in a solvent with a larger value. Generally, a molecule in the
state is more polar than one in the ground state, and the polar
imidazoles are twisted against the central pyrazine ring in
0
1
1
a direction such that the ethyl groups on the N and N atoms of the
imidazole rings are oriented with one another in a syn configura-
tion. This deviation from a symmetrical structure in polar solvents
may account for the large dipole. It is likely that the conformational
1
3
r
S
1
r
change is induced to adapt to the change in 3 of the surroundings.
Figure 3. Fluorescence spectra of 5a in DMSO (black), cyclohexane (purple), acetoni-
trile (red), and methanol (blue); c¼10 M.
Figure 4. Plots of the fluorescence maximum,
organic solvents against the solvent’s relative dielectric constants (3
l
FL (in cmꢁ1), of 2 observed in various
).
m
r

R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
8277
7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
Table 4
Effect of relative dielectric constant,
2) the energy levels of S and the corresponding ground state that have unrelaxed
Franck-Condon geometry (S ), and (3) the differential energies (DE in eV) for the
r 1
3 , on (1) the dipole moment in the S state (mS1),
(
1
FC
0
FC
S1eS
0
transition of 8
3
r
m
S1 /debye
Energy level/eV
DE/eV
FC
S
0
S
1
32.7
42.6
51.7
23.665
23.771
23.877
23.932
23.929
e6.816
e6.806
e6.798
e6.794
e6.795
e3.408
e3.412
e3.417
e3.421
e3.424
3.407
3.394
3.381
3.374
3.370
60.9
0.0
7
400
450
500
550
unit and the fluorescence exhibited a hypsochromic shift from
ꢀ
ꢀ
4
42 nm at 23 C to 433 nm at 38 C as shown in the inset of Figure 7,
Wavelength /nm
which plots the fluorescence maximum wavelength (
l
FL) against
ꢀ
Figure 5. Fluorescence spectra of 5a in methanolewater-mixed solvent. Water con-
tent: 0% (black), 25% (blue), 50% (light blue), 65% (green), 70% (purple), and 75% (red).
the temperature. On increasing the temperature from 23 C to
ꢀ
31 C, lFL showed a continuous hypsochromic shift. This shift was
ꢀ
ꢀ
markedly accelerated from 31 C to 35 C, and became almost
constant at 433 nm above 36 C. The hypsochromic shift reflects an
ꢀ
The magnitude of the resulting dipole moment could depend on
the polarity of the surroundings.
increase of hydrophobicity around the 5a unit as discussed above.
ꢀ
When the PNIPAM hydrogel is in its swollen state below 34
C
(volume phase transition temperature; VPTT), it contains much
water inside the gel. With increasing temperature, this water was
expelled from the gel in concert with the volume phase transition.
Therefore, the hydrophilicity inside the hydrogel was decreased.
The fluorescence maximum wavelength correlated linearly with
the solvent polarity (3 ). The following equation was obtained for 5a
r
by least-squares analysis of the data in Figure 6 (the r-value is the
correlation coefficient).
2
.4. Application as a microenvironment polarity probe
The general application of a fluorescent dye as a probe requires
a characterization of its polarity, mobility, and other properties in
diverse media. Since the fluorescence probing method was first
used to study the solution behavior of a thermo-responsive poly-
mer in 1982, there have been many reports investigating dynamics
of molecular assemblies, such as micelles and polymers, by moni-
toring the microenvironments around the assemblies with this
technique.
Poly(N-isopropylacrylamide) (PNIPAM) is a well-known and
typical thermo-responsive polymer, and many researchers have
investigated its thermo-responsive properties. It exhibits a phase
ꢀ
ꢁ
ꢁ
1
fluorescence maximum in cm
4
2
¼
ꢁ20:91
3
r
þ 2:382ꢂ10 ; r ¼ 0:98
(1)
The microenvironmental polarity near the BBIP units in the
hydrogel was estimated from the observed
l
FL value using Eq. (1).
ꢀ
transition at ca. 32 C in water and its hydrogel shows volume
phase transition at ca. 34 C. We used a crosslinked PNIPAM
hydrogel to investigate the application of the BBIP derivative
ꢀ
The estimated value above the VPTT (at 38 C,
r
3 ¼33) was small
ꢀ
2
ꢀ
compared to that below the VPTT (at 23 C,
3
r
¼60), suggesting that
the hydrophobicity around the BBIP units increased in concert with
volume phase transition.
5
adwhich has two double bonds on its structure at the N-1 and
0
N-1 positions of the benzimidazol moietydas a microenvironment
polarity probe.
The 5a-labeled PNIPAM hydrogel, which was prepared as shown
in Scheme 3, exhibited a broad emission (390e550 nm) from the 5a
4
4
4
4
4
4
42
40
38
36
34
32
a
b
4
00
450
500
Wavelength /nm
16
20
24
28
32
36
40
Temperature /ºC
Figure 6. Plots of
circle) and 5b (open square). The
were taken from literatures.
l
FL as a function of relative dielectric constant (
3
r
) for 5a (closed
Figure 7.
l
FL values for the 5a-labeled PNIPAM hydrogel as a function of tem-
ꢀ
3
r
values for methanol-water binary solvent system
perature. Inset: fluorescence spectra for the 5a-labeled PNIPAM hydrogel at 23
(a) and 38 C (b).
C
11
ꢀ

8278
R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
As discussed above, BBIP derivatives with double bonds on their
A solution of 2, 5a or 5b for fluorescence measurement was
prepared by mixing a stock solution (100 L) of 1.0 mM of 2, 5a or
structure can be incorporated in a thermo-responsive polymer
chain, and thus they can sense and report microenvironmental
changes in the polymer.
m
5b in dimethyl sulfoxide and a solvent (2.0 mL) within a quartz
cuvette at room temperature. For measuring fluorescence of 2 in
cyclohexane, chloroform was used for preparing the stock solution.
The fluorescence quantum yields were determined using quinine
3
. Conclusion
10
sulfate in 0.1 M sulfuric acid as a standard.
New BBIP derivatives possessing two alkyl chains at the N-1 and
4
.2. Synthesis
0
N-1 positions of the two benzimidazole moieties (2, 3a,b, and 5a,b)
were successfully synthesized. The N-alkylation markedly im-
proved their solubility in organic solvents in comparison to the
parent BBIP (1), which is soluble only in DMSO. In addition, the
electronic absorption and fluorescence spectra of these compounds
were successfully measured even in non-polar cyclohexane. Sub-
sequent characterization of these compounds demonstrated that
their fluorescence efficiencies were preserved at high levels (>0.7)
even in protic solvents. In addition, both 5a and 5b exhibited sol-
vatochromic fluorescence, and their fluorescence maxima in
0
0
1
4.2.1. 2,5-Bis(1 -n-butylbenzimidazol-2 -yl)pyrazine
(2). N -Butyl-
benzene-1,2-diamine (1.06 g, 6.45 mmol) and pyrazine-2,5-di-
carboxylic acid dihydrate (556 mg, 2.72 mmol) were added to
polyphosphoric acid (14 g), and the mixture was stirred at 140
under argon for 24 h. Diphosphorus pentaoxide (1.95 g, 13.7 mmol)
was added to the mixture and the stirring was continued for 5 days.
After cooling to room temperature, water (60 mL) was added to the
reaction mixture and the pH of the solution was adjusted to 10 with
ꢀ
C
1
0% NaOH. Organic materials were then extracted with chloroform
50 mLꢂ3). After evaporation of the solvent, the obtained red solid
was purified by column chromatography on a silica gel (46e50 m,
5 g) with chloroformemethanol (30/1 to 5/1; v/v) as eluents, fol-
lowed by recrystallization from ethyl acetate to afford 2 as yellow
aqueous methanol were bathochromicly shifted with increasing 3
r
(
value of the medium. With this solvatochromic property, 5a was
utilized as a microenvironment polarity probe to successfully in-
dicate the polarity variation around the backbone of the tempera-
ture-sensitive polymer PNIPAM by the spectral change upon the
thermal phase transition of the polymer.
m
3
ꢀ
1
needles (52 mg, 4%). Mp 195.0e195.5 C. H NMR (CDCl
/ppm 0.98 (6H, t, J¼7.3 Hz), 1.44 (4H, sextet, J¼7.3 Hz), 1.91 (4H,
quint, J¼7.3 Hz), 4.85 (4H, t, J¼7.7 Hz), 7.34e7.41 (4H, m), 7.50 (2H, d,
3
, 400 MHz)
d
ꢁ1
4
4
. Experimental
J¼7.4 Hz), 7.88 (2H, d, J¼7.3 Hz,), 9.73 (2H, s). IR (KBr)
nmax/cm
CeH). MS (FAB , mNBA)
þ
3056, 3018 (
nCeH), 2924, 2955 (
n
CeH), 743 (
g
þ
.1. General
425 [MþH] . HRMS (positive EI) calcd for C26
28 6
H N
424.2376, found:
4
24.2374.
All melting points were measured on an MP-21 (Yamato Sci-
0
00
entific Co., Ltd., Japan) in open capillary tubes; the values were
uncorrected. 1H NMR spectra were recorded on a JNM-ECP400
spectrometer (JEOL Ltd., Japan) or a JNM-LA400D spectrophotom-
eter (JEOL Ltd., Japan). Chemical shifts (d) are reported in parts per
million using tetramethylsilane or an undeuterated solvent as in-
ternal standards in the deuterated solvent used. Coupling constants
4.2.2. 2,5-Bis{1 -(3 -tert-butoxycarbamoylpropyl)benzimidazol-
0
0
2 -yl}pyrazine (3a). A suspension of 2,5-bis(benzimidazol-2 -yl)
pyrazine (340 mg, 1.1 mmol) in dehydrated DMSO (13 mL) was
added to a slurry of sodium hydride (55% in oil,100 mg, 2.3 mmol) in
dehydrated DMSO (4 mL). After stirring for 30 min, tert-butyl-N-(3-
bromopropyl)carbamate (529 mg, 2.3 mmol) in dehydrated DMSO
(3 mL) was added dropwise to the mixture and stirring was con-
tinued for 23 h. After filtration of the reaction mixture, the collected
residue was successively washed with CHCl
pure product as a greenish amorphous (280 mg, 45%). H NMR
(CDCl , 400 MHz)
(
J) are given in hertz. Chemical shift multiplicities are reported as
s¼singlet, d¼doublet, t¼triplet, q¼quartet, quint¼quintet, and
m¼multiplet. Infrared (IR) spectra were obtained using the spec-
trophotometers FT/IR-4100, FT/IR-460plus, or FT/IR-660plus (JASCO
Co., Ltd., Japan). Fast-atom-bombardment (FAB) mass spectra were
taken 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 dithio-
3
and AcOEt to give the
1
3
d
/ppm 1.48 (18H, s), 2.18 (4H, quint, J¼7.3 Hz), 3.26
(4H, t, J¼7.3 Hz), 4.87 (4H, t, J¼7.3 Hz), 5.18 (2H, s), 7.34e7.42 (4H, m),
7.50 (2H, d, J¼7.6 Hz), 7.86 (2H, d, J¼7.6 Hz), 9.67 (2H, s). IR (KBr)
ꢁ1
n
max/cm 3359 (
CeN), 1172 ( CeO), 740 (
6.75, N 17.88, found: C 64.96, H 7.01, N 17.62.
n
NeH), 2971 (
nCeH), 1685 (nC]O), 1533 (nCeH), 1251
(n
n
42 8 4
gCeH). Anal. Calcd for C34H N O : C 65.16, H
threitol (DTT) and
a-thioglycerol (TG) (1:1 or 1:2) or m-nitro-
benzylalcohol (mNBA) was used as a liquid matrix. High- and
low-resolution electron impact (EI) mass spectra were obtained
with a JMS-AM II 50 mass spectrometer (JEOL Co., Ltd., Japan). The
ionization energy was 70 eV, and the accelerating voltages were 0.3
and 0.5 kV for the low- and high-resolution analyses, respectively.
Fluorescence spectra were recorded on an F-777 fluorescence
spectrophotometer (JASCO Co., Ltd., Japan) and corrected according
to the manufacturer’s instructions. Absorption spectra were mea-
sured on a V-650 spectrophotometer (JASCO Co., Ltd., Japan), and
combustion analyses were performed on an MT-6 analyzer (Yanaco
New Science Inc., Japan). Column chromatography was carried out
0
00
0
4.2.3. 2,5-Bis{1 -(3 -tert-butoxycarbamoylbutyl)benzimidazol-2 -yl}-
pyrazine (3b). Compound 3b was prepared from 1 and tert-butyl-
N-(3-bromobutyl)carbamate by a procedure analogous to that for
obtaining 3a, in 42% yield. H NMR (CDCl
1
3
, 400 MHz)
d
/ppm 1.44
(18H, s), 1.61 (4H, quint, J¼7.3 Hz), 1.98 (4H, quint, J¼7.3 Hz), 3.21
(4H, t, J¼7.3 Hz), 4.85 (4H, t, J¼7.3 Hz), 7.34e7.41 (4H, m), 7.51 (2H,
ꢁ1
d, J¼7.6 Hz), 7.86 (2H, d, J¼7.6 Hz), 9.67 (2H, s). IR (KBr)
n
max/cm
3406 (
CeH). Anal. Calcd for C36
found: C 64.24, H 6.92, N 16.65.
nNeH), 3061 (nCeH), 2982 (nCeH), 1710 (nC]O), 1173 (nCeO), 743
(g
H
46
N
8
O
4
$H
2
O: C 64.27, H 7.19, N 16.65,
on a silica gel (63e210-mm particle size; Kanto Chemical Co.). Semi-
0
00
0
empirical molecular orbital (MO) calculations for ground and sin-
glet-excited states were carried out with AM1 method and the
4.2.4. 2,5-Bis{1 -(3 -aminopropyl)benzimidazol-2 -yl}pyrazine
(4a). A solution of 3a (250 mg, 0.40 mmol) and trifluoroacetic acid
(6 mL) was stirred for 22 h. After evaporation of the solvent, H O
2
12
COSMO model on the MOPAC2009 program.
All conventional chemicals used in the present study are com-
mercially available and were used as received. Spectral grade solvents
were used for the measurement of electronic absorption and fluo-
rescence spectra.
(10 mL) was added to the residue and the pH of the solution was
adjusted to 11 with 10% NaOH. After filtration, the resulting crude
product (190 mg) was given quantitatively as a brown solid. This
material was used directly in the next step without further

R. Saito et al. / Tetrahedron 66 (2010) 8273e8279
ꢁ1
8279
1
þ
purification. H NMR (DMSO-d
6
, 400 MHz)
d
/ppm 1.91e1.93 (4H,
cm 3052 (
n
CeH), 2957 (
n
CeH), 742 (
g
CeH). MS (FAB , DTT/TG¼1:2)
þ
m), 2.58e2.65 (4H, m), 4.88 (4H, t, J¼7.3 Hz), 7.30e7.40 (4H, m),
m/z 253 [MþH] . HRMS (positive EI) calcd for C15
16 4
H N 252.1375,
ꢁ
1
7
.76e7.80 (4H, m), and 9.62 (2H, s). IR (KBr)
n
max/cm 3365(
n
NeH),
found: 252.1374.
2
940(
n
CeH), 743 (
g
CeH).
4.3. Synthesis of hydrogel
0
00
0
4
.2.5. 2,5-Bis{1 -(3 -aminopropyl)benzimidazol-2 -yl}pyrazine
(
4b). Compound 4b was synthesized from 3b quantitatively by
A solution of isopropylacrylamide (440 mg, 3.9 mmol), methyl-
0
0
a procedure analogous to that for obtaining 4a, and used directly in
the next step without further purification as was 4a. H NMR
enebisacrylamide (7.4 mg, 0.48 mmol), and N,N,N ,N -tetramethyl-
ethylenediamine (2.2 L, 14.8 mol) in water (2.8 mL) was added to
1
m
m
(
(
(
(
CDCl
3
, 400 MHz)
d
/ppm 1.55e1.61 (8H, m), 1.94e2.02 (4H, m), 2.76
a solution of compound 5a in DMF. After degassing the solution
4H, t, J¼7.3 Hz), 4.86 (4H, t, J¼7.3 Hz), 7.34e7.41 (4H, m), 7.49e7.52
(4.5 mL) by bubbling N gas for 20 min on an ice bath, a potassium
2
ꢁ
1
2H, m), 7.87e7.89 (2H, m), 9.74 (2H, s). IR (KBr)
CeH), 2930 ( CeH), 742 (
n
max/cm 3409
persulfate aqueous solution (8.8 mM, 0.5 mL) and capillary tube
n
n
g
CeH).
(1.54 mm in diameter) were added to the degassed solution. After
gelation in therefrigeratorfor 24h, the gelswereimmersed in alarge
amount of water to wash away residual chemicals.
0
0
0
4
.2.6. 2,5-Bis{1 -(3 -acrylamidepropyl)benzimidazol-2 -yl}pyrazine
(5a). Under argon, EDCI hydrochloride (180 mg, 0.94 mmol) in
dehydrated dichloromethane (3 mL) was added to a solution of 4a
Acknowledgements
(
(
205 mg, 0.47 mmol), HOBt (127 mg, 0.94 mmol) and acrylic acid
0.64 mL, 9.4 mmol) in dehydrated DMF (12 mL), and the mixture
This research was supported by ‘High-Tech Research Center’
Project for Private Universities: matching fund subsidy from the
Ministry of Education, Culture, Sports, Science and Technology of
Japan (MEXT), and KAKENHI (Grant-in-Aid for Scientific Research)
on Priority Area ‘Strong Photon-Molecule Coupling Fields (No. 470)’
from the MEXT. Financial support to R.S. by the Faculty of Science
Special Grant for Promoting Scientific Research at Toho University
is also gratefully appreciated.
was stirred at room temperature for 93 h. After evaporation of the
solvent, the residue was dissolved in water (50 mL) and extracted
with CHCl
NaHCO (30 mL) and dried over anhydrous Na
ration of the solvent, recrystallization from CHCl
3
(50 mLꢂ2). The organic layer was washed with 5%
SO . After evapo-
gave the pure
3
2
4
3
ꢀ
1
product as a yellow solid (34 mg, 13%). Mp 186 C (decomp.). H
NMR (CDCl
3
, 400 MHz)
d/ppm 2.24 (4H, quint, J¼7.2 Hz), 3.45 (4H, t,
J¼7.2 Hz), 4.91 (4H, t, J¼7.2 Hz), 5.67 (2H, d, J¼10.7 Hz), 6.11 (2H, dd,
J¼10.2 and 17.1 Hz), 6.26 (2H, d, J¼17.1 Hz), 7.38e7.42 (4H, m), 7.51
Supplementary data
(
cm
2H, d, J¼7.6 Hz), 7.88 (2H, d, J¼7.6 Hz), 9.70 (2H, s). IR (KBr)
max
n /
ꢁ
1
3280 (nNeH), 3062 (nCeH), 2935 (nOeH), 1655 (nC]O), 738
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.08.036.
(g
CeH). HRMS (positive EI) calcd for C30
H
30
N
8
O
2
: 534.2492, found:
5
34.2497.
References and notes
0
0
0
4
.2.7. 2,5-Bis{1 -(3 -acrylamidepropyl)benzimidazol-2 -yl}pyrazine
(
4
(
5b). Compound 5b was obtained as a pale yellow solid (42%) from
1. Saito, R.; Machida, S.; Suzuki, S.; Katoh, A. Heterocycles 2008, 75, 531e536.
2. Schild, H. G. Prog. Polym. Sci. 1992, 17, 163e249.
3. Winnik, F. M. Macromolecules 1990, 23, 233e242.
ꢀ
b by a procedure analogous to that for obtaining 5a. Mp 197
C
decomp.). 1H NMR (CDCl
3
, 400 MHz)
d
/ppm 1.67 (4H, quint,
4
5
. Fujimoto, K.; Nakajima, Y.; Kashiwabara, M.; Kawaguchi, H. Polym. Int. 1993, 30,
237e241.
. (a) Blatch, A. J.; Chetina, O. V.; Howard, J. A. K.; Patrick, L. G. F.; Smethurst, C. A.;
Whiting, A. Org. Biomol. Chem. 2006, 4, 3297e3302; (b) Hérault, D.; Aelvoet, K.;
Blatch, A. J.; Al-Majid, A.; Smethurst, C. A.; Whiting, A. J. Org. Chem. 2007, 72, 71e75.
J¼7.2 Hz), 1.99 (4H, quint, J¼7.2 Hz), 3.40 (4H, t, J¼7.2 Hz), 4.87 (4H,
t, J¼7.2 Hz), 5.64 (2H, d, J¼10.1 Hz), 6.06 (2H, dd, J¼10.1 and
1
6.8 Hz), 6.29 (2H, d, J¼16.8 Hz), 7.24e7.41 (4H, m), 7.50 (2H, d,
ꢁ
1
J¼7.6 Hz), 7.87 (2H, d, J¼7.6 Hz), 9.72 (2H, s). IR (KBr)
n
max/cm
6. Liu, Q.-X.; Yin, L.-N.; Feng, J.-C. J. Organomet. Chem. 2007, 692, 3655e3663.
7. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J. Am. Chem. Soc. 1985,
3
294 (nNeH), 2933 (nCeH), 1677 (nC]O), 741 (gCeH). HRMS (positive
107, 3902e3909.
EI) calcd for C32
34
H N
8
O
2
562.2805, found: 562.2807.
8
9
. Klamt, A.; Schüürmann, G. J. Chem. Soc., Perkin Trans. 2 1993, 799e805.
. (a) Turro, N. J. Modern Molecular Photochemistry; University Science Books:
Sausalito, CA, 1991; (b) Lakowicz, J. R. Principles of Fluorescence Spectroscopy,
3rd ed.; Springer: New York, NY, 2006.
1
0
4
.2.8. N -Butyl-2-(pyrazin-2 -yl)benzimidazole (6). Compound 6 was
1
prepared from pyrazinecarboxylic acid N -butylbenzene-1,2-di-
amine by a procedure analogous to that for obtaining 2 with an yield
1
0. (a) Saito, R.; Hirano, T.; Niwa, H.; Ohashi, M. J. Chem. Soc., Perkin Trans. 2 1997,
711e1716;(b)Saito, R.; Hirano, T.;Niwa, H.; Ohashi,M. Chem.Lett.1998, 27, 95e96.
11. (a) Albright, P. S.; Gosting, L. J. J. Am. Chem. Soc. 1946, 68, 1061e1063;
b) Uchiyama, S.; Matsumura, Y.; de Silva, A. P.; Iwai, K. Anal. Chem. 2003, 75,
926e5935.
2. MOPAC2009 Stewart, J. J. P. Stewart Computational Chemistry; Colorado Springs:
CO, USA, 2008; http://OpenMOPAC.net.
1
ꢀ
1
of 8%. Mp 66.0e67.0 C H NMR (CDCl
J¼7.3 Hz), 1.39 (2H, sextet, J¼7.3 Hz),1.87 (2H, quintet, J¼7.3 Hz), 4.79
2H, t, J¼7.3 Hz), 7.32e7.39 (2H, m), 7.47 (1H, d, J¼8.0 Hz), 7.87 (1H, d,
J¼8.0 Hz,), 8.61e8.64 (2H, m), 9.68 (1H, d, J¼1.1 Hz). IR (KBr)
3
, 400 MHz) d/ppm 0.95 (3H, t,
(
5
(
1
max
n /