5-[4-(Dimethylamino)phenyl]-2-benzamidopyrazines: Fluorescent dyes based on Cypridina oxyluciferin

Article abstract of DOI:10.1007/s11164-012-0645-3

To clarify the acyl substitution effect on the fluorescent property of Cypridina oxyluciferin analogs, which have the 4-(dimethylamino)phenyl group at C5, we compared the spectroscopic properties of benzamide 1b and its 2,6-dimethylphenyl derivative 1c in various solvents with those of acetamide 1a. Similarity of the fluorescence wavelengths of 1a-c indicates that the π-electronic conjugation in the excited singlet (S₁) state is terminated at the acyl group and that the benzamide moiety in 1b has little effect on modulating the florescence color. The similar fluorescence solvatochromism of 1a-c reveals that their S₁ states have a similar intramolecular charge transfer character. The fact that 1a-c have various fluorescence quantum yields and lifetimes depending on solvent polarity and hydrogen bonding interactions in solutions provides information about the decay processes competing with the fluorescence process in the S₁ states. Among 1a-c, benzamide 1b exhibits the most sensitive variation of the fluorescence intensity depending on the solvent used. Similarity of the fluorescence spectra of 1a-c adsorbed in NaY zeolites was also observed, which indicates that the S₁ states of 1a-c give mobile conformers in the NaY supercages. The relationship between the molecular structures of 1a-c and their spectroscopic properties will provide a guide for designing a new fluorophore based on Cypridina oxyluciferin. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-012-0645-3

Res Chem Intermed (2013) 39:233–245
DOI 10.1007/s11164-012-0645-3
5-[4-(Dimethylamino)phenyl]-2-benzamidopyrazines:
fluorescent dyes based on Cypridina oxyluciferin
•
Takashi Hirano Tatsuki Nakagawa Ai Kodaka
•
•
•
Shojiro Maki Haruki Niwa Minoru Yamaji
•
Received: 9 October 2011 / Accepted: 21 November 2011 / Published online: 14 June 2012
Ó Springer Science+Business Media B.V. 2012
Abstract To clarify the acyl substitution effect on the fluorescent property of
Cypridina oxyluciferin analogs, which have the 4-(dimethylamino)phenyl group at C5,
we compared the spectroscopic properties of benzamide 1b and its 2,6-dimethylphenyl
derivative 1c in various solvents with those of acetamide 1a. Similarity of the fluores-
cence wavelengths of 1a–c indicates that the p-electronic conjugation in the excited
singlet (S1) state is terminated at the acyl group and that the benzamide moiety in 1b has
little effect on modulating the florescence color. The similar fluorescence solvatochro-
mism of 1a–c reveals that their S1 states have a similar intramolecular charge transfer
character. The fact that 1a–c have various fluorescence quantum yields and lifetimes
depending on solvent polarity and hydrogen bonding interactions in solutions provides
information about the decay processes competing with the fluorescence process in the S1
states. Among 1a–c, benzamide 1b exhibits the most sensitive variation of the fluores-
cence intensity depending on the solvent used. Similarity of the fluorescence spectra of
1a–c adsorbed in NaY zeolites was also observed, which indicates that the S1 states of
1a–c give mobile conformers in the NaY supercages. The relationship between the
molecular structures of 1a–c and their spectroscopic properties will provide a guide for
designing a new fluorophore based on Cypridina oxyluciferin.
Keywords Cypridina bioluminescence ꢀ Oxyluciferin ꢀ Fluorescence ꢀ
Solvatochromism ꢀ Intramolecular charge transfer ꢀ Zeolite
T. Hirano (&) ꢀ T. Nakagawa ꢀ A. Kodaka ꢀ S. Maki ꢀ H. Niwa
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
M. Yamaji
Department of Chemistry and Chemical Biology, Graduate School of Engineering,
Gunma University, Kiryu, Gunma 376-8515, Japan
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Introduction
Bioluminescence provides a photon-producing system based on a chemical reaction
[1]. The luminous ostracod Cypridina (presently Vargula), for example, produces
blue light via a luciferin–luciferase reaction [1–3] in which Cypridina luciferin (Ln)
reacts with O2 in luciferase to give the excited singlet (S1) state of Cypridina
oxyluciferin (OLn), and the excited OLn emits light (Chart 1). Because Ln and OLn
have potential as a chemiluminescence reagent [4] and a fluorescent dye [5],
respectively, they are attractive prototypes for photofunctional molecules. Based on
this idea, we investigated their photofunctional potentials by preparing chemically
modified analogs of Ln and OLn [6–10].
In this manuscript, we focus on an OLn analog (OLA) that will be used to
develop a fluorescent dye. OLn contains the amidopyrazine structure as the
fluorescent core together with three appendages: an (S)-2-butyl group at the acyl
part and 3-(1-guanidino)propyl and 3-indolyl groups at C3 and C5, respectively.
In these appendages, the 3-indolyl group exhibits p-electronic conjugation with the
amidopyrazine core and plays an important role in determining the fluorescence
property. We previously investigated a substituent effect of aryl groups at C5 on the
fluorescent property of amidopyrazines to confirm that the 3-indolyl group of OLA
acts as an electron-donating aryl group to develop tunable fluorescence properties
[8]. In addition, the combination of the electron-withdrawing amidopyrazine core
and the electron-donating 3-indolyl group induces intramolecular charge transfer
(ICT) in the S1 state, resulting in fluorescence solvatochromism. We also found that
modified OLA 1a, which has the 4-(dimethylamino)phenyl group instead of the
3-indolyl group at C5, shows a better fluorescence property, because of the stronger
electron-donating ability of the 4-(dimethylamino)phenyl group [8].
O
O
acyl
NH
N
N
N
N
H
N
H
N
3
5
NH
NH
2
2
N
H
NH
NH
N
H
N
H
Cypridina luciferin (Ln)
Cypridina oxyluciferin (OLn)
O
R
O
Me
NH
N
N
NH
N
N
5
5
Me
N
N
H
1a: R = Me
Me
OLA
1b: R = Ph
Me
1c: R =
Me
Chart 1
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Fluorescent dyes based on Cypridina oxyluciferin
235
To modify the fluorescent property of OLA, we designed benzamide derivative
1b, which incorporates expanded p-electronic conjugation in the acyl direction.
Additionally, we prepared 2,6-dimethylphenyl derivative 1c, in which the
p-electronic conjugation is inhibited by steric hindrance. Herein we describe the
electronic absorption and fluorescence properties of 1b and 1c in comparison with
those of 1a. The results obtained in the present work provide evidence for the
molecular structure of the S1 state of 1. The data also revealed that benzamide
modification is not suitable for expanding the p-electronic conjugation in the S1
state. We also describe our attempt to modulate the fluorescent properties of 1a–c by
adsorbing the compounds into zeolite supercages [11].
Experimental
General
Melting points were determined using a Yamato MP-21 apparatus. Infrared (IR)
spectra were measured with a Horiba FT-720 spectrometer. High-resolution electro-
spray-ionization (ESI) mass spectra were recorded with a JEOL JMS-T100LC mass
spectrometer. 1H nuclear magnetic resonance (NMR) spectra were recorded on a JEOL
ECA-500 (500 MHz) instrument. Ultraviolet (UV)/visible absorption spectra of
solutionsamplesweremeasuredwitha VarianCary50spectrophotometer(scanspeed,
600 nm/min; data interval, 1 nm). Optical diffuse reflectance spectra of zeolite
samples were recorded on a JASCO V-670 spectrophotometer (scan speed, 400 nm/
min; data interval, 1 nm) using an integrating sphere attachment, and are shown as the
Kubelka–Munk spectra using F(R) = (1 - R)2/2R, where R is the observed diffuse
reflectance of a sample with barium sulfate as the reference. Fluorescence spectra were
measured with a JASCO FP-6500 fluorescence spectrophotometer (excitation and
emission bandpasses, 3 and 10 nm, respectively; scan speed, 500 nm/min) and were
corrected according to the manufacturer’s instructions. Fluorescence quantum yields
(Uf) were determined relative to quinine sulfate in 0.10 M H2SO4 (Uf = 0.55,
kex = 366 nm [12]) as standard. Fluorescence lifetimes (sf) were measured with a
Hamamatsu Photonics TAU time-correlated single-photon counting fluorimeter
system. Spectral-grade solvents were used for measurements of UV/visible absorp-
tion and fluorescence. NaY (HSZ 302NAA) obtained from Tosoh Company (Tokyo)
was calcined at 500 °C in air for more than 2 h before use. Density functional
theory (DFT) calculations were performed using the Gaussian 09 program [13]. DFT
includes Beck’s three-parameter function combined 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.
Preparation of amidopyrazines 1
Acetamidopyrazine 1a was prepared by the reported procedure [8]. Similarly, 1b
and 1c were synthesized from 5-[4-(dimethylamino)phenyl]aminopyarzine as
follows.
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Synthesis of 1b
Benzoyl chloride 17 lL (0.14 mmol) was added to a solution of 5-[4-(dimethyl-
amino)phenyl]aminopyarzine (31 mg, 0.14 mmol) in pyridine (1 mL) at room
temperature under Ar, and the mixture was stirred for 3.5 h. The reaction was
quenched by addition of water, and the resulting product was extracted with ethyl
acetate. The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel thin-layer chroma-
tography (TLC) [hexane/ethyl acetate (1:2)], yielding 1b (20 g, 43 %) as yellow
crystals: mp 216–217 °C. IR (KBr) 3,376, 1,670, and 1,610 cm-1
.
1H NMR
(500 MHz, CDCl3) dppm 3.04 (6 H, s), 6.81 (2 H, d, J = 8.6 Hz), 7.53 (2 H, t,
J = 7.5 Hz), 7.60 (1 H, t, J = 7.5 Hz), 7.92 (2 H, d, J = 9.1 Hz), 7.95 (2 H, d,
J = 7.4 Hz), 8.44 (1 H, s), 8.63 (1 H, d, J = 1.7 Hz), and 9.66 (1 H, d, J = 1.7 Hz).
HRMS (ESI, m/z) calcd. for C19H19N4O ([M ? H]?) 319.1559, found: 319.1537.
Synthesis of 1c
A solution of 2,6-dimethylbenzoic acid (307 mg, 2.04 mmol) and thionyl chloride
(147 lL, 2.04 mmol) in pyridine (1 mL) was stirred for 2.5 h at room temperature
under Ar. The mixture was concentrated in vacuo with an evaporator to give a
residue containing 2,6-dimethylbenzoyl chloride. To the residue diluted with
pyridine (1 mL), 5-[4-(dimethylamino)phenyl]aminopyarzine (151 mg, 0.70 mmol)
was added under Ar, and the mixture was heated at 95 °C for 3.5 h. After cooling,
the reaction was quenched by addition of water, and the product was extracted with
chloroform. The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel column chromatog-
raphy [chloroform/hexane/ethyl acetate (3:2:1–3:2:2)], and the resulting crystals
were washed with hexane, yielding 1c (100 mg, 41 %) as yellow powder: mp
208–209 °C. IR (KBr) 3,305, 1,689, and 1,610 cm-1. 1H NMR [500 MHz, dimethyl
sulfoxide (DMSO)-d6] dppm 2.26 (6 H, s), 2.95 (6 H, s), 6.79 (2 H, d, J = 8.6 Hz),
7.08 (2 H, d, J = 7.5 Hz), 7.20 (1 H, t, J = 7.5 Hz), 7.92 (2 H, d, J = 9.2 Hz), 8.84
(1 H, s), 9.37 (1 H, s), and 11.09 (1 H, s). HRMS (ESI, m/z) calcd. for C21H23N4O
([M ? H]?) 347.1872, found: 347.1852.
Spectroscopic measurements of 1a–c adsorbed in zeolites
Amidopyrazines 1a–c were adsorbed in zeolites (NaY, 100 mg) from their o-xylene
solutions (2 mL) at concentrations that would produce 1.0 % loading (molecules/
supercages) at room temperature for more than 2 h. After centrifugation and
removal of the supernatant solvent, the sample was further evaporated and degassed
under vacuum (ca. 10-2 Torr). UV absorptions of the supernatants determined the
amounts of unadsorbed 1a–c, and these data were used to calculate the loading
percentages. Zeolite samples were placed in a quartz vessel, and their steady-state
optical diffuse reflectance and fluorescence spectra were measured.
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Fluorescent dyes based on Cypridina oxyluciferin
237
Results and discussion
Spectroscopic and photophysical properties
We first investigated the spectroscopic properties of 1b and 1c together with those of
1a in various solvents. UV/visible absorption and fluorescence spectra of 1a–c were
measured in benzene, chloroform, dichloromethane (DCM), 2-propanol (2-PrOH),
acetonitrile (MeCN), and methanol (MeOH). Table 1 summarizes the spectroscopic
and photophysical data. The data are listed in order of orientation polarizability (Df) as
a solvent parameter estimated using Eq. 1:
À
Á À
Á
Df ¼ ðe ꢁ 1Þ=ð2e þ 1Þ ꢁ n2 ꢁ 1 = 2n2 þ 1 ;
ð1Þ
where e and n are the dielectric constant and refractive index of the solvent,
respectively [17]. Figure 1 shows the electronic absorption and fluorescence
emission spectra of 1b and 1c.
Electronic absorption maxima (kab) of 1c are similar to those of 1a. The kab
values of 1b show a bathochromic shift compared with those of 1a and 1c,
indicating that the benzoyl moiety of 1b works to expand the p-electronic
conjugation in the ground state. The molar absorptivities of 1a–c are in the
magnitude of 104 dm3 mol-1 cm-1, which indicates that the lowest energy bands
are assigned to p–p* transitions. The fact that the solvent-dependent differences in
the lowest energy bands of 1a–c are small (\8 nm) shows that the ground states of
1a–c have a small dipolar character. In contrast, the fluorescence emission maxima
(kf) of 1a–c are similar to one another, indicating that the p-electronic conjugation
of the S1 states of 1a–c is terminated at the acyl group. The kf values of 1a–c show
bathochromic shifts with increasing solvent polarity. The results clearly indicate
that the S1 states of 1a–c have an identical p-conjugating structure with an ICT
character that is stronger than that of the ground states.
The ICT property of the S1 state of 1a has been characterized using the Lippert–
Mataga equation [18, 19]. Similarly, we correlated solvent-dependent Stokes shifts
(Dm in cm-1) of 1b and 1c with the Df values using the same analysis. Figure 2
shows the Dm values plotted as a function of Df for 1a–c. The plots for aprotic
solvents are straight lines, whereas those for protic solvents (2-PrOH and MeOH)
deviate from the lines. This result indicates that hydrogen bonding interactions, in
addition to solvent polarity, strongly affect the stability of the S1 states of 1a–c. This
is a general phenomenon observed in fluorescence spectroscopy [20]. The slopes of
the linear relationships for 1a–c in aprotic solvents are identical to one another.
The values of Dm and Df are related using Eq. 2:
À
Á
2
Dm ¼ 2 le ꢁ lg Df=ð4pe0hca3Þ þ constant;
ð2Þ
where le and lg are the dipole moments of the excited and ground states, respec-
tively, e0 is the permittivity of the vacuum, h is the Planck constant, c is the velocity
of light, and a is the Onsager cavity radius. We assumed the a value to be 0.6 nm,
which was used previously for several fluorescent biaryl molecules [21, 22].
The (le - lg) values for 1b and 1c were estimated to be 16 7 and 15 6 D,
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T. Hirano et al.
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Fluorescent dyes based on Cypridina oxyluciferin
239
3.0
Abs (1b)
a−f
FL (1b)
a bc e d f
1.0
0.5
0.0
2.0
1.0
0.0
300 350 400 450 500
400
500
600
700
wavelength / nm
wavelength / nm
3.0
Abs (1c)
a−f
FL (1c)
a bc e d f
1.0
0.5
0.0
2.0
1.0
0.0
300 350 400 450 500
400
500
600
700
wavelength / nm
wavelength / nm
Fig. 1 Electronic absorption (Abs) and fluorescence emission (FL) spectra of 1b and 1c in benzene (a),
chloroform (b), DCM (c), 2-PrOH (d), MeCN (e), and MeOH (f)
respectively, which is similar to that of 1a (16 6 D) [8]. Thus, the S1 states of 1a–
c have a common, strong, ICT character of the p-conjugating structure, in which the
4-(dimethylamino)phenyl group and the amidopyrazine function as the electron-
donating and electron-accepting moieties, respectively.
While the kf values of 1a–c are similar to one another, the fluorescence quantum
yields (Uf) exhibit drastic differences depending on the solvents used and their
molecular structures. In benzene and chloroform, 1a–c produce intense fluores-
cence, and their Uf values are over 0.2. Amidopyrazines 1a and 1c also show strong
fluorescence (Uf [ 0.37) in DCM and MeCN, but the Uf values for 1c in these
solvents are \0.1. In 2-PrOH and MeOH, fluorescence of 1a–c is significantly
diminished (Uf \ 0.03). The Lippert–Mataga plots in Fig. 2 indicate that the S1
states of 1a–c are stabilized by hydrogen bonding interactions in the protic solvent
molecules, as described above. These hydrogen bonding interactions play the role of
quenching the S1 states of 1a–c. These evaluations illustrate that 1b can show
sensitive dependence of the fluorescence intensity on solvent polarity and hydrogen
bonding interactions.
To evaluate the difference in the Uf values, we measured fluorescence lifetimes sf
and estimated the rate constants (kf) of the fluorescence emission process using Eq. 3:
kf ¼ Ufsꢁf 1
ð3Þ
In aprotic solvents, the sf values for 1a and 1c are in the range of 3.5–6.5 ns and
vary with a similar solvent dependency. The kf values from the S1 states of 1a and
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T. Hirano et al.
Fig. 2 Lippert–Mataga plots
for 1a (filled diamonds),
1b (filled squares), and 1c
(open squares)
MeOH
2-PrOH
10000
9000
8000
7000
6000
5000
ν
0.00
0.10
0.20
0.30
Δf
1c are ca. 1 9 108 s-1. In contrast, the sf values for 1b in aprotic solvents are
smaller than those of 1a and 1c, which suggests that the benzoyl moiety affects the
decay process of the S1 state of 1b. The estimated kf values for 1b in benzene,
chloroform, and DCM are similar to those for 1a and 1c, which suggests that the S1
state of 1b may have fast decay pathways, such as intersystem crossing and/or
chemical reactions competing with the fluorescence process. This may explain why
the fluorescence intensity of 1b varies sensitively depending on the solvent polarity.
In 2-PrOH and MeOH, 1a–c have very short fluorescence lifetimes (sf \ 0.5 ns),
which is indicative of the presence of fast decay processes from their S1 states
competing with fluorescence emission. Hydrogen bonding interactions in the S1
state of 1a–c may play a role of enhancing the fast decay processes.
DFT calculations
To evaluate the effect of the structural difference in p-electronic conjugation of 1b
and 1c on their spectroscopic properties, we carried out DFT calculations of 1b and
1c along with 1a using the B3LYP/6-31G(d) method. We previously reported a
locally energy-minimized conformer of 1a that is close to the structure of a luciferin
derivative (conformer I in Fig. 3) [8], although another conformer (II) is the most
energetically stable structure. The difference in the heat of formation between
conformers I and II for 1a is 32 kJ mol-1. Similarly, conformer II is the optimum
structure of 1b and 1c (Fig. 3). Table 2 summarizes the calculation data: dihedral
angles (h) of the pyrazinyl/4-(dimethylamino)phenyl moieties and of the CO/
benzene ring in the acyl parts; dipole moments (l); total Mulliken charge densities
(q) of the atoms constituting the acyl, NH, pyrazine, and 4-(dimethylamino)phenyl
parts; and highest occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) levels. In the geometry of the ground states of 1a–c,
the coplanarity between the pyrazinyl and 4-(dimethylamino)phenyl moieties is
identical for all three compounds. The 2,6-dimethylphenyl group of 1c is largely
twisted (67°) against the amide carbonyl compared with the corresponding twisting
123

Fluorescent dyes based on Cypridina oxyluciferin
241
in 1b (19°). Thus, the p-electronic conjugation of 1b is larger than that of 1c. The
slightly smaller l value of 1b compared with those of 1a and 1c might be caused by
the difference in p-conjugation. In fact, the positive and negative charges of the acyl
and NH parts of 1b are larger than those of 1a and 1c. The extended p-electronic
conjugation in 1b also causes a drop of the LUMO level compared with those of 1a
and 1c, whereas the HOMO levels of 1a–c are all similar.
Table 3 summarizes the results of the time-dependent (TD)-DFT calculations:
excitation wavelengths (kex), oscillator strengths (f), and configurations for the
allowed S0 ? S1 transitions. Because the LUMO of 1b is lower than those of 1a and
1c, 1b has the lowest kex value among 1a–c. HOMO ? LUMO transitions mainly
contribute to the electronic excitations of 1a–c. Electron distributions of the HOMOs
and LUMOs of 1a–c are shown in Fig. 4. These HOMOs and LUMOs are in the
p-bonding orbitals, and the assignment of electronic excitations of 1a–c due to p–p*
transitions is supported by this calculation result. Only the LUMO of 1b has bonding
interaction at the C–C bond between the C=O and phenyl groups in the benzoyl part,
which results in lowered LUMO energy compared with those of 1a and 1c. Electrons
of the HOMOs and LUMOs of 1a–c are mainly localized at the 4-(dimethyl-
amino)phenyl and pyrazinyl parts, respectively, indicating that the electronic
top view
top view
side view
side view
1a (conformer I)
1a (conformer II)
top view
top view
side view
side view
1b
1c
Fig. 3 Optimized geometries of 1a–c calculated using the B3LYP/6-31G(d) method
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T. Hirano et al.
Table 2 Dihedral angles (h), dipole moments (l), total Mulliken charge densities (q) of the atoms
constituting the partial structures, and HOMO and LUMO levels of 1a–c calculated using the B3LYP/
6-31G(d) method
Compound hAr (°)a hRCO (°)b l/D
qcRCO
qcNH
qcPy
qcAr
HOMOd LUMOd
1a
1b
1c
a
12.9
12.8
12.0
–
3.71 0.093 -0.304 0.114 0.097 -4.99
3.30 0.109 -0.325 0.119 0.097 -4.98
3.79 0.076 -0.290 0.116 0.098 -5.00
-1.17
-1.42
-1.23
19.3
67.3
Dihedral angles between the pyrazinyl and 4-(dimethylamino)phenyl moieties
b
c
Dihedral angles between the CO and benzene ring in the acyl parts
Total Mulliken charge densities of the atoms constituting the acyl (RCO) (qRCO), NH (qNH), pyrazine
(C4H2N2) (qPy), and 4-(dimethylamino)phenyl (qAr) parts
d
HOMO and LUMO levels in units of eV
Table 3 Excitation wavelengths (kex), oscillator strengths (f), and configurations of the allowed S0 ? S1
transitions for 1a–c calculated with TD-DFT using B3LYP/6-31G(d)
Compound
kex/nm
f
Configurationa
1a
1b
1c
a
363
384
367
0.30
0.58
0.43
H ? L (0.67), H ? L ? 1 (-0.21)
H ? L (0.67)
H ? L (0.68), H ? L ? 1 (-0.18)
Configuration of excitation. H, L, and L ? 1 denote the HOMO, LUMO, and LUMO ? 1, respectively
excitations induce ICT. The S1 states of 1a–c have, thus, increased positive and
negative charges at the 4-(dimethylamino)phenyl and pyrazinyl parts, respectively, as
shown in Scheme 1. Fluorescent properties of 1a–c suggested that their S1 states have
a similar p-conjugating structure, in which the p-electronic conjugation is terminated
at the acyl group. The C(Ph)–C(C=O) bond in the benzoyl part of the LUMO of 1b has
a bonding character, and all LUMOs of 1a–c have a node at the nitrogen atom of the
amide moieties. Accordingly, the p-electronic conjugation in the S1 states of 1a–c will
be broken at the C(pyrazine)–N or N–C(CO) bond in the amide moieties (Scheme 1).
These C–N bonds might be twisted in the S1 states.
Spectroscopic properties of 1a–c adsorbed in zeolites
To modulate the spectroscopic properties of 1a–c further, we attempted to use a
supramolecular environment [11]. For this purpose, we chose a faujasite zeolite, NaY,
˚
which is a porous crystalline material that contains supercages (diameter, ca. 13 A)
˚
connected to four other supercages through 8-A windows [11, 23]. The supercage
density in NaY is 6.27 9 10-4 mol g-1. If the conformations of 1a–c were tightly
fixed in NaY supercages, it is expected to observe the difference in the fluorescence
spectra of 1a–c, which is caused by their different p-electronic conjugation. Amides
1a–c were adsorbed into NaY supercages from their o-xylene solutions for 1.0 %
loading for 2 h. The process produced NaY samples adsorbing 1a–c (1a–c@NaY)
with 0.93, 0.99, and 0.93 % loadings. 1a–c@NaY showed absorption maxima at 333,
123

Fluorescent dyes based on Cypridina oxyluciferin
243
HOMO (1a)
LUMO (1a)
LUMO (1b)
LUMO (1c)
HOMO (1b)
HOMO (1c)
Fig. 4 HOMOs and LUMOs of 1a-c
Scheme 1 Properties of the S1
states of 1a–c
break of
π-electronic conjugation
H
N
1
R
N
*
δ−
N
O
δ+
N
b
0.2
A
B
1.0
c
a
0.1
0.0
0.5
c
b
a
0.0
300
400
500
400
500
600
wavelength / nm
wavelength / nm
Fig. 5 Diffuse reflectance (A) and fluorescence emission (B) spectra of 1a (a), 1b (b), and 1c
(c) adsorbed in NaY (1 % loading)
354, and 342 nm, respectively (Fig. 5A). The difference in these absorption maxima
indicates that the p-electronic conjugations of 1a–c@NaY in the ground states differ
from one another. On the other hand, fluorescence emission spectra of 1a–c@NaY
(Fig. 5B) were similar to one another and showed similar emission maxima at 541 nm.
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T. Hirano et al.
The amide moiety is conformationally
mobile in the S1 state.
Scheme 2 The S1 states of
1a–c adsorbed in NaY
supercages
1
N
H C
3
*
NH
O
N
R
N
H C
3
The fluorescence features are similar to those in solutions, indicating that adsorption in
NaY supercages did not fix conformation of the S1 states of 1a–c, as shown in
Scheme 2. In particular, the amide moieties of the excited 1a–c will be flexible in NaY
supercages. To achieve conformational regulation within a zeolite environment, we
have to use a zeolite with an adsorption space smaller than that of NaY.
Conclusions
To determine if the benzamide moiety can modulate the fluorescent property of
amidopyrazine with the 4-(dimethylamino)phenyl group at C5 by expanding
p-electronic conjugation, we investigated the fluorescent properties of 1b and its
2,6-dimethylphenyl derivative 1c in various solvents and compared these properties
with those of acetamide 1a [8]. The kf values of 1a–c are similar to one another,
which illustrates that the p-electronic conjugation in the S1 state is terminated at the
acyl group and that the benzamide moiety in 1b has little effect on modulating the kf
values by p-electronic expansion. DFT calculation results suggest that the C–N
bonds of the amide moieties might be twisted in the S1 states of 1a–c. The
fluorescence-solvatochromic features of 1a–c are also similar to one another,
indicating that their S1 states have a similar strong ICT character of p-conjugating
structure, in which the 4-(dimethylamino)phenyl and pyrazinyl parts have increased
positive and negative charges, respectively. The Uf and sf values for 1a–c vary
sensitively depending on the solvent polarity and hydrogen bonding interactions in
the solutions; these results provide information about the decay processes that
compete with the fluorescence process from the S1 states. In particular, hydrogen
bonding interactions in protic solvents enhance the fast decay pathways from the S1
states of 1a–c, resulting in low Uf values and small sf values. Among the derivatives
1a–c, benzamide 1b exhibits the most sensitive solvent-dependent variation of
the fluorescence intensity. Thus, 1b is one of the candidates for solvatochromic
fluorophores like anilinonaphthalenesulfonic acid (ANS) derivatives [20], which
have the ON/OFF function sensing the polarity and the hydrogen bonding ability of
solvents. The fluorescence of 1a–c adsorbed in NaY zeolites also showed emission
maxima that were similar to one another, indicating that the NaY supercages cannot
fix the conformation of the S1 states of 1a–c. The relationship between the
molecular structures of 1a–c and their spectroscopic properties will provide a guide
for designing a new fluorophore based on Cypridina OLn.
123

Fluorescent dyes based on Cypridina oxyluciferin
245
Acknowledgments 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 Casio Science Promotion Foundation and the Iketani 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.
References
1. O. Shimomura, Bioluminescence: Chemical Principles and Methods (World Scientific, Singapore,
2006)
2. T. Goto, Pure Appl. Chem. 17, 421–441 (1968)
3. F.H. Johnson, O. Shimomura, Methods Enzymol. 57, 331–364 (1978)
4. K. Teranishi, Bioorg. Chem. 35, 82–111 (2007)
5. T. Goto, H. Fukatsu, Tetrahedron Lett. 49, 4299–4302 (1969)
6. T. Hirano, Y. Gomi, T. Takahashi, K. Kitahara, F.Q. Chen, I. Mizoguchi, S. Kyushin, M. Ohashi,
Tetrahedron Lett. 39, 5771–5774 (1992)
7. R. Saito, T. Hirano, H. Niwa, M. Ohashi, J. Chem. Soc. Perkin Trans. 2, 1711–1716 (1997)
8. T. Hirano, Y. Takahashi, H. Kondo, S. Maki, S. Kojima, H. Ikeda, H. Niwa, Photochem. Photobiol.
Sci. 7, 197–207 (2008)
9. S. Hachiya, T. Inagaki, D. Hashizume, S. Maki, H. Niwa, T. Hirano, Tetrahedron Lett. 51,
1613–1615 (2010)
10. R. Saito, T. Hirano, S. Maki, H. Niwa, M. Ohashi, Bull. Chem. Soc. Jpn. 84, 90–99 (2011)
11. N.J. Turro, V. Ramamurthy, J.C. Scaiano, Modern Molecular Photochemistry of Organic Molecules,
Chap 13 (University Science Books, Sausalito, 2010)
12. D.F. Eaton, Pure Appl. Chem. 7, 1107–1114 (1988)
13. M.J. Frisch, 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. Raghavachari, 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. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski,
R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich,
¨
A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision
A.1 (Gaussian, Inc., Wallingford, 2009)
14. A.D. Becke, J. Chem. Phys. 98, 5648–5652 (1993)
15. C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37, 785–789 (1988)
16. P.J. Stephens, F.J. Devlin, C.F. Chabalowski, M.J. Frisch, J. Phys. Chem. 98, 11623–11627 (1994)
17. C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, 3rd edn. (Wiley-VCH, Weinheim,
2003)
18. E. Lippert, Z. Electrochem. 61, 962–975 (1957)
19. N. Mataga, Y. Kaifu, M. Koizumi, Bull. Chem. Soc. Jpn. 29, 465–470 (1956)
20. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn. (Springer, New York, 2006)
21. F. Lahmani, E. Breheret, A. Zehnacker-Rentien, C. Amatore, A. Jutand, J. Photochem. Photobiol. A
70, 39–49 (1993)
22. M. Maus, W. Rettig, D. Bonafoux, R. Lapouyade, J. Phys. Chem. A 103, 3388–3401 (1999)
23. D.W. Breck, Zeolite Molecular Sieves–Structure, Chemistry and Use (Wiley, New York, 1974)
123