Dipolar Dyes with a Pyrrolo[2,3-b]quinoxaline Skeleton Containing a Cyano Group and a Bridged Tertiary Amino Group: Synthesis, Solvatofluorochromism, and Bioimaging

Article abstract of DOI:10.1002/asia.201600257

Two strongly polarized dipolar chromophores possessing a cyclic tertiary amino group at one terminus of the molecule and a CN group at the opposite terminus were designed and synthesized. Their rigid skeleton contains the rarely studied pyrrolo[2,3-b]quinoxaline ring system. The photophysical properties of these regioisomeric dyes were different owing to differing π conjugation between the CN group and the electron-donor moiety. These dipolar molecules showed very intense emission, strong solvatofluorochromism, and sufficient two-photon brightness for bioimaging. One of these regioisomeric dyes, namely, 11-carbonitrile-2,3,4,5,6,7-hexahydro-1H-3a,8,13,13b-tetraazabenzo[b]cyclohepta[1,2,3-jk]fluorene, was successfully utilized in two-photon imaging of mouse organ tissues and showed distinct tissue morphology with high resolution.

Full text of DOI:10.1002/asia.201600257

DOI: 10.1002/asia.201600257
Full Paper
Dipolar Dyes
Dipolar Dyes with a Pyrrolo[2,3-b]quinoxaline Skeleton
Containing a Cyano Group and a Bridged Tertiary Amino Group:
Synthesis, Solvatofluorochromism, and Bioimaging
[
a]
[b]
[a]
[c]
Łukasz G. Łukasiewicz, Irena Deperasi n´ ska, Yevgen M. Poronik, Yong Woong Jun,
[
b]
[b]
[c]
[a]
Marzena Banasiewicz, Bolesław Kozankiewicz,* Kyo Han Ahn,* and Daniel T. Gryko*
Abstract: Two strongly polarized dipolar chromophores pos-
sessing a cyclic tertiary amino group at one terminus of the
molecule and a CN group at the opposite terminus were de-
signed and synthesized. Their rigid skeleton contains the
rarely studied pyrrolo[2,3-b]quinoxaline ring system. The
photophysical properties of these regioisomeric dyes were
different owing to differing p conjugation between the CN
group and the electron-donor moiety. These dipolar mole-
cules showed very intense emission, strong solvatofluoro-
chromism, and sufficient two-photon brightness for bio-
imaging. One of these regioisomeric dyes, namely, 11-car-
bonitrile-2,3,4,5,6,7-hexahydro-1H-3a,8,13,13b-tetraazaben-
zo[b]cyclohepta[1,2,3-jk]fluorene, was successfully utilized in
two-photon imaging of mouse organ tissues and showed
distinct tissue morphology with high resolution.
Introduction
cial attention has been given to coumarins owing to their
large dipole moments and high photostability combined with
[
14]
The design of new organic chromophores for two-photon ex-
cited fluorescence microscopy (TPFM) of biological objects has
straightforward synthesis. New trends include studies of p-
[
15]
expanded coumarins
and redshifting the emission bands
[1,2]
attracted significant attention,
as this technique is an effi-
while combating the decrease in the fluorescence quantum
[
16]
cient means to study biological processes in live tissues and
yield in polar solvents by introducing superstrong acceptor-
[
3]
[17a–d]
[17e–h]
offers many advantages over traditional imaging methods. As
larger quadrupolar and octupolar, nonlinear absorbing dyes
have difficulty in crossing cell membranes, small molecules
tend to be more promising two-photon absorbing chromo-
s
or more powerful electron-donating groups
(includ-
[
18]
ing bridged, cyclic tertiary amines). Bridged and cyclic amine
donors in dipolar dyes restrict internal rotations, which thereby
stops nonradiative excited-state decay channels, but they re-
quire multistep syntheses. A solution to the synthetic difficulty
is to utilize the bidentate nucleophilic character of 1,8-diazabi-
cyclo[5.4.0]undec-7-en (DBU), which upon reacting with elec-
trophiles gives rise to compounds possessing cyclic tertiary
[4–8]
phores for fluorescence imaging purposes.
Push–pull chro-
mophores are the prevailing variety of smaller fluorophores, as
[9]
they have been known and studied for a long time and have
[
10]
still been a subject of active investigation in recent decades.
[11]
[19]
Derivatives of both aromatic hydrocarbons (especially 2,6-
amino groups directly attached to aromatic system.
[9a,11a,12]
disubstituted naphthalene, i.e., acedan)
and heterocyclic
The goal of this project was to further strengthen the dipo-
lar interactions in 2-amino-1,4-diazaindole derivatives, which
can be synthesized from 2,3-dichloroquinoxalines and DBU in
[
13]
systems have been intensively studied. In this last case, spe-
[
20]
one step according to a procedure reported by us. We rea-
soned that placing a cyano group at the opposite side of the
planar heterocycle from a tertiary amino group would further
polarize the whole system and lead to desirable optical re-
sponse. In combination with the small size of the molecules,
this approach should open an avenue to imaging studies of
cells and tissues. Herein, we reveal the synthetic and photo-
physical details of our study as well as two-photon excited
fluorescence microscopy experiments.
[
a] Ł. G. Łukasiewicz, Dr. Y. M. Poronik, Prof. D. T. Gryko
Institute of Organic Chemistry
Polish Academy of Sciences
Kasprzaka 44/52, 01-224 Warsaw (Poland)
E-mail: dtgryko@icho.edu.pl
[
b] Dr. I. Deperasi n´ ska, Dr. M. Banasiewicz, Prof. B. Kozankiewicz
Institute of Physics
Polish Academy of Sciences
Al. Lotników 32/46, 02-668 Warsaw (Poland)
E-mail: kozank@ifpan.edu.pl
[
c] Y. W. Jun, Prof. K. H. Ahn
Department of Chemistry
POSTECH
Results and Discussion
7
7 Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk 37673 (Korea)
2
,3-Dichloro-6-cyanoquinoxaline (2) was chosen as the precur-
Supporting Information for this article can be found under http://
dx.doi.org/10.1002/asia.201600257.
sor for the designed dipolar heterocyclic system. This com-
Chem. Asian J. 2016, 11, 1718 – 1724
1718
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[
20]
1
reaction
and fully consistent analytical data ( H NMR and
13
C NMR spectroscopy, HRMS), in conjunction with X-ray as-
signment for compound 4, made it possible to unambiguously
assign the structure of the second product as 5.
The absorption spectra of both regioisomers in cyclohexane,
toluene, and methanol are shown in Figure 2. The lowest
energy absorption maxima (labs) and extinction coefficients (e)
are collected in Table 1. Clearly, the labs of both isomers
showed a bathochromic shift from approximately l=426–
4
4
28 nm (i.e., violet photons) in nonpolar solvents to l=443–
49 nm in polar solvents.
Scheme 1. Synthesis of compounds 4 and 5.
pound was obtained in a two-step reaction according to a pre-
[21]
viously described method in 57% yield (Scheme 1).
The reaction was conducted by following the general experi-
[
20]
mental procedure
under previously optimized conditions
(
1158C, 20 min). According to our expectations, 2,3-dichloro-6-
Figure 2. Absorption spectra of isomers 4 (dotted line) and 5 (solid line) in
cyclohexane, toluene, and methanol at room temperature.
cyanoquinoxaline (2) was treated with DBU to provide a mix-
ture of two regioisomers (4 as the major product at the ex-
pense of compound 5) (Scheme 1).
Despite very similar retardation factors, the mixture was suc-
cessfully separated by column chromatography (4/5=6:1). The
nonstatistical ratio of the reaction products can be rationalized
by the electronic structure of quinoxaline 2, which consists of
two CÀCl bonds with different reactivities. The cyano group at
the 6-position exerts a mesomeric effect that strongly affects
the 2-position towards nucleophilic attack. The fact that dye 4
formed preferentially suggests that contrary to what we hy-
The fluorescence spectra of isomers 4 and 5 are shown in
Figure 3. Both compounds strongly emit green-yellow light in
various solvents. In nonpolar solvents, the fluorescence spectra
are composed of two bands [maxima of the bands (l ) are
fl
given in Table 1]. Dyes 4 and 5 demonstrated a strong fluores-
cence response in nonpolar solvents. As predicted by the
[
22]
Strickler–Berg equation,
the fluorescence quantum yields
(F ) of heterocycles 4 and 5 decrease in polar solvents as
fl
[
20]
pothesized earlier, initial attack takes place on the nitrogen
atom of DBU (and not on the b-carbon atom).
a result of the bathochromic shift in the emission band, which
induced both an increase in the rate of nonradiative decay and
Spectroscopic analyses confirmed the identity of regioiso-
meric products 4 and 5 but did not allow for structure assign-
ment. Consequently, X-ray analysis of a more polar product
was performed, which revealed its structure as 4 (Figure 1). In
[
20]
analogy to previously studied pyrrolo[2,3-b]quinoxalines, the
molecule is essentially planar. The well-known character of this
Figure 3. Normalized fluorescence spectra of compounds 4 and 5 in cyclo-
Figure 1. X-ray structure of compound 4 (CCDC 1456392).
hexane, dichloromethane, and methanol.
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1719
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Table 1. Photophysical properties of compounds 4 and 5.
Compd
Solvent
l
abs
e
Dn
l
em
F
À3 À1
À1
À1
[a]
[
nm]
[10
m
cm
]
[cm
]
[nm]
[%]
C
6
H
12
428
427
436
443
443
449
426
426
436
443
443
447
4.7
nd
2100
2100
3000
4300
4500
4400
2000
2000
2700
3000
3450
3800
470/498
469/497
502/524
546
552
559
466/492
465/493
494/515
511
523
539
75
68
45
18.5
6.5
2.5
94
80
77
61
37
n-nonane
toluene
CH
MeCN
MeOH
11.4
12.7
12.8
12.6
15.3
nd
19.1
22.6
20.2
18.1
4
2
Cl
2
C
6
H
12
n-nonane
toluene
CH
MeCN
MeOH
5
2
Cl
2
11.5
[
a] Relative fluorescence quantum yields were determined by using perylene in cyclohexane as the reference. nd: not determined.
a reduction in the rate of radiative decay. Notably, fluorescence
quantum yields are higher for isomer 5 than for isomer 4. The
values of the Stokes shifts for compounds 4 and 5 in cyclohex-
ane and n-nonane, respectively, were very similar; however,
with the increase in solvent polarity, the Stokes shift tended to
increase (Figure 4, Table 1). This observation may be attributed
to charge redistribution that occurred during the excitation
process, which formed the excited state with higher charge
separation in chromophores 4 and 5. Polar solvents decreased
the energy of the excited state, which resulted in the increased
Stokes shift. For dye 4 in polar solvents, the values were all no-
ticeably higher.
on solvatofluorochromism. Whereas F for a series of previous-
fl
ly studied pyrrolo[2,3-b]quinoxalines was approximately 40%
[
20]
in nonpolar solvents, for heterocycles 4 and 5 it was equal
to 75 and 94%, respectively. A previously studied DBU-fused
[
20a]
pyrrolo[2,3-b]quinoxaline
displayed relatively weak solvato-
fluorochromism (F =8% in MeOH), whereas for compounds 4
fl
and 5 their emission intensities decreased more than one
order of magnitude.
The Stokes shift values between the absorption and fluores-
cence spectra can be analyzed with the Lippert–Mataga equa-
[
23,24]
tion [Eq. (1)]:
À
Á
2
2
À
Á
2 m À m
e À 1
n À 1
vac
abs
vac
e
g
The presence of a cyano group at a peripheral position rela-
tive to the aminopyrrole moiety has a non-negligible effect on
the optical behavior: the absorption band in hexane is batho-
chromically shifted versus that of the parent heterocycle pre-
nabs À n ¼ n À nfl
þ
ð
À
Þ
ð1Þ
fl
3
2
4
pe hca 2e þ 1 2n þ 1
0
in which Dn=nabsÀnfl is the solvatochromic Stokes shift, Dm=
Àm is the change in the electric dipole moment in the elec-
tronic excited (S ) and ground (S ) states of the molecule, h is
the Planck constant, e is the vacuum permittivity, c is the ve-
[21]
pared from 2,3-dichloroquinoxaline and DBU
by approxi-
m
e
g
mately 20 nm. However, the emission is not shifted: it is locat-
1
0
ed at approximately l=470 nm in all three cases. The pres-
0
ence of the cyano group has a tremendous effect on F and
locity of light, a is the radius of the Onsager cavity, e is the die-
lectric constant, and n is the refractive index of the solvent.
From the plots of the linear dependence of the Stokes shift as
fl
2
a function of the solvent parameter (eÀ1)/(2e+1)À(n À1)/
2
(
2n +1), which are presented in Figure 4, we calculated slope
À1
values of (3924Æ708) and (2764Æ455) cm for 4 and 5, re-
spectively. Assuming the same cavity radius for both isomers,
a=5 Š, we obtained Dm=6.98 and 5.86 D for 4 and 5, respec-
tively.
Fluorescence decay curves were measured by the “time cor-
related” single photon counting technique. The curves in non-
polar solvents were well fitted to a single-exponential depend-
ence (the calculated decay times, t , are collected in Table 2),
fl
but in polar solvents they appeared to follow at least a two-ex-
ponential function. Taking into account these data and the
fluorescence quantum yields, we calculated the radiative rate
constants for the S !S transition (according to the formula:
1
0
k =f /t ), which are listed in Table 2. Notably, the experimen-
r
fl fl
Figure 4. Plot of the spectral shift (Dn) as a function of solvent parameter. 4
tally determined rate constant k is higher for isomer 5 than for
(
&
), 5 (*). Linear fit to the experimental data for isomer 4 (red line) gave
r
Dm=6.98 D; for isomer 5 (blue line) gave Dm=5.86 D.
4. This observation is consistent with the fact that the absorp-
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1720
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
dipole moment change (Dm) for isomer 4 than for 5 (4: Dm=
[a]
r
Table 2. Fluorescence decay times (tfl) and radiative rate constants (k ).
6
.5 D vs. 5: Dm=5.0 D), in agreement with experimentally de-
Compound
Solvent
t
fl
k
r
termined values.
7
À1
[
ns]
[10 s ]
The electronic configurations of the HOMO–LUMO of both
isomers, describing the transition between the S and S states,
cyclohexane
n-nonane
toluene
cyclohexane
n-nonane
toluene
8.1
7.4
5.8
6.5
6.4
6.1
9.2
9.2
7.8
0
1
4
5
are also presented in Table 3. The shapes of the HOMO and
LUMO of both isomers differ depending on the side of the CN
group in the compounds. Interestingly, the HOMO of isomer 4
(in the part containing the benzonitrile moiety) resembles the
14.4
12.5
12.7
orbital of the benzonitrile molecule of the symmetry a (in the
2
[
r
a] Calculated according to the formula: k =ffl/tfl.
symmetry group C ), whereas the HOMO of isomer 5 is similar
2v
to the orbital b of benzonitrile. Orbitals for the LUMO of both
1
tion extinction value for isomer 5 is higher than that for 4 (5:
e=19100 vs. 4: e·=11 400, in toluene). Slightly stronger
solute–solvent dependence for isomer 4 (especially in weakly
polar toluene, m=0.36 D) may be an indication of its more
polar character relative to that of isomer 5 (see theoretical con-
siderations below).
isomers 4 and 5, in the area of the benzonitrile moiety, are
well described by the benzonitrile orbital of symmetry b1.
On the basis of the orbitals presented in Table 3, we con-
cluded that the differences in the optical properties between
isomers 4 and 5 are consequences of the different shapes of
the HOMO orbitals in the benzonitrile part of the molecule. In
Table S1 and Figure S1 in the Supporting Information, we show
that the different shapes of the HOMOs of both isomers find
reflection in the extinction ratio between the first (lꢀ420 nm)
and the second (lꢀ295 nm) absorption bands. This ratio is
considerably smaller for experimentally studied isomer 4 than
for isomer 5 (see Figure 2), and the same is observed for calcu-
lated isomers 4 and 5.
To better understand the photophysical properties of the in-
vestigated compounds, we performed quantum-chemical cal-
culations by using the DFT and time-dependent (TD) DFT
B3LYP/6-31G(d,p) methods. Some important results of these
calculations are displayed in Table 3. The wavelengths (l) and
oscillator strengths (f) for the absorption S !S (labs) and the
0
1
fluorescence emission S !S (l ) in n-nonane solvent were ob-
0
1
fl
tained for the structures optimized in the S and S states. The
The two-photon absorption cross-sections were measured
for compounds 4 and 5 by two-photon excited fluorescence in
the l=700–900 nm range. In the case of 4, the two-photon
action cross-section was not registered, whereas 5 presented
a value of approximately 2.3 GM.
0
1
Stokes shifts (Dn) and the dipole moment changes (Dm) were
obtained by using the following formulae [Eqs. (2) and (3)]:
1
1
labs lfl
Dn ¼
À
ð2Þ
ð3Þ
Compound 5 was chosen for tissue imaging of different
mouse organs (e.g., brain, lung, liver, kidney, and spleen) to
observe their tissue morphology by TPFM. One advantageous
feature of compound 5 is that it can be excited at l=900 nm,
which can reduce the autofluorescence in TPFM tissue imaging
1
Dm ¼ m Àm0
According to the calculations, dye 5 was characterized by
considerably higher oscillator strength (f) than compound 4 (5:
f=0.4266 vs. 4: f=0.1954) in absorption as well as in emission.
This result correlated well with the experimentally determined
larger extinction coefficient (e) and larger rate constant of fluo-
rescence (k ) for isomer 5 than for isomer 4 (5: k =12.7 ns vs.
[
25]
while also permitting a deeper penetration depth. Figure 5
shows that all of the tested organs were stained clearly and
uniformly at a depth of 100 mm or deeper (if a higher laser
power could be applied). The close-up image of brain provides
the high-resolution morphology of neurons along the hippo-
campus (Figure 5 f). Furthermore, the brighter spots that
r
r
4
: k =7.8 ns, in toluene). We also noticed a higher calculated
r
Table 3. Results of the quantum chemistry calculations for isomers 4 and 5.
Compd
l
abs
f
m
0
l
fl
f
m
1
Dn
Dm[D]
LUMO
HOMO
À1
[nm]
[D]
[nm]
[D]
[cm ]
S
0
S
1
4
5
418.0
414.6
0.1954
0.4266
13.4
538.2
505.9
0.0944
0.2366
19.9
5343
4353
6.5
13.2
18.2
5.0
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1721
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
determined with Me Si as the internal reference; J values are given
4
in Hz. Chromatography was performed on silica gel (Kieselgel 60,
2
00–400 mesh).
Procedure for the synthesis of 4 and 5
A sealed tube containing 2,3-dichloroquinoxalin-6-carbonitrile (2;
1
2
2
.6 g, 7 mmol) and 1,8-diazobicyclo[5.4.0]undec-7-ene (3.2 g,
1 mmol) was placed in a preheated oil bath at 115 8C. After
5 min, the mixture was cooled to RT. Then, CH Cl was added and
2
2
column chromatography on silica gel (CH Cl /acetone=97:3) was
2
2
performed to obtain the pure products as orange crystals. Yields:
8% (for 4), 9% (for 5).
4
Characterization data
0-Carbonitrile-2,3,4,5,6,7-hexahydro-1H-3a,8,13,13b-tetraazaben-
1
zo[b]cyclohepta[1,2,3-jk]fluorene (4): R =0.66 (SiO ; CH Cl /EtOAc=
f
2
2
2
1
9
:1); m.p. 265–266; H NMR (500 MHz, CDCl , Me Si): d=8.29 (d, J=
3 4
Figure 5. Two-photon microscopy tissue imaging of five different mouse
organs stained by compound 5 (10 mm). All the images were captured at
a depth of approximately 100 mm of the sectioned tissues (thickness
1.8 Hz, 1H), 7.91 (d, J=8.5 Hz, 1H), 7.53 (dd, J=8.5, 1.8 Hz, 1H),
4.18 (t, J=6.0 Hz, 2H), 3.45 (m, 4H), 2.95 (t, J=6.0 Hz, 2H), 2.28 (m,
2H), 2.00 (m, 2H), 1.87 ppm (m, 2H); C NMR (125 MHz, CDCl3,
13
~
400 mm). lex =900 nm; emission filter=500–605 nm. Images show sec-
Me Si): d=21.7, 23.0, 26.9, 29.5, 38.1, 49.8, 56.4, 90.3, 108.1, 119.7,
4
tioned tissue images of a) brain, b) lung, c) liver, d) kidney, and e) spleen.
Close up images of f) brain and g) liver. Laser power was 28.3 mW at the
focal point. Scale bar in panels a–e is 250 mm and for panels f and g is
1
25.0, 128.5, 132.8, 139.4, 140.2, 142.7, 145.2, 153.0 ppm; HRMS
+
(
EI): m/z: calcd for C H N : 303.1484 [M ]; found: 303.1484.
18 17 5
7
5 mm.
11-Carbonitrile-2,3,4,5,6,7-hexahydro-1H-3a,8,13,13b-tetraazaben-
zo[b]cyclohepta[1,2,3-jk]fluorene (5): R =0.20 (SiO ; CH Cl /EtOAc=
f
2
2
2
1
9
1
4
2
:1); m.p. 256–257; H NMR (500 MHz, CDCl , Me Si): d=8.22 (d, J=
3 4
spread out throughout the high-resolution liver tissue image
suggest sublocalization of the compound (Figure 5g).
.8 Hz, 1H), 7.96 (d, J=8.5 Hz, 1H), 7.60 (dd, J=8.5, 1.8 Hz, 1H),
.18 (t, J=6.0 Hz, 2H), 3.47 (m, 4H), 2.97 (t, J=6.0 Hz, 2H), 2.29 (m,
H), 2.00 (m, 2H), 1.88 ppm (m, 2H); C NMR (125 MHz, CDCl3,
13
Me Si): d=21.7, 22.9, 26.8, 29.5, 38.00, 49.8, 56.23, 91.1, 105.7,
4
Conclusions
1
20.0, 126.9, 128.1, 133.1, 136.1, 142.7, 143.4, 145.2, 153.9 ppm;
+
HRMS (EI): m/z: calcd for C H N : [M ] 303.1484; found: 303.1484.
18
17
5
In conclusion, we showed that replacing a hydrogen atom by
an electron-withdrawing cyano substituent led to substantial
variations in the optical properties of pyrrolo[2,3-b]quinoxa-
lines. This effect was particularly visible in stronger solvato-
fluorochromism and higher fluorescence quantum yields. The
combined effects of 6-cyanoquinoxaline as an electron-defi-
cient moiety and a bridged and fully conjugated tertiary amino
group led to absorption of violet photons and emission of
green-yellow light, depending on the solvent polarity. As a con-
sequence of the different shapes of the HOMO orbitals in the
benzonitrile part of the molecule, depending on the position
of the cyano group in the target dye molecules, their spectro-
scopic properties were different. The new push–pull molecules
showed marginal but sufficient two-photon brightness. One of
these dyes was successfully applied for two-photon tissue
imaging of mouse organs, and it afforded high-resolution
tissue morphology of brain hippocampus neurons and subloc-
alization of the compound in liver tissue.
Optical properties
The absorption and fluorescence spectra of dyes 4 and 5 in liquid
solutions of cyclohexane, n-nonane, toluene, dichloromethane, ace-
tonitrile, and methanol (all spectroscopic grade) were measured at
room temperature with the aid of a PerkinElmer UV/Vis Lambda 35
absorption spectrometer and a Hitachi F-7000 fluorescence spec-
trometer, respectively. Fluorescence quantum yields (F ) were de-
termined with respect to perylene in cyclohexane (F =0.94).
The error inherent with the F estimation did not exceed 10%.
fl
[
26]
fl
fl
Fluorescence spectra of both compounds in n-nonane matrix at
K were monitored with the aid of a home-built spectrometer
5
equipped with a McPherson 207 monochromator, an EMI9659 pho-
tomultiplier, and an EasyScan PC module.
Fluorescence kinetics studies were performed with the aid of the
“
time correlated” single photon counting technique (in the invert-
ed time mode). Excitation pulses were provided by the second har-
monics of a mode-locked Coherent Mira-HP femtosecond laser
pumped by a Verdi 18 laser. Original repetition rate of a Mira laser
was reduced with the aid of an APE Pulse selector to 2 MHz. Fluo-
rescence photons were dispersed with a McPherson 207 mono-
chromator and detected with a HMP-100–50 hybrid detector and
SPC-150 module inserted into a PC, both from Becker&Hickl GmbH.
Fluorescence decays were long relative to the detected time width
of the excitation pulses (ꢀ200 ps), and therefore, the decay curves
(delayed by 1 ns with respect to the excitation) were fitted to
Experimental Section
General methods
All chemicals were used as received unless otherwise noted. Re-
agent-grade solvents (i.e., CH Cl , hexane, toluene) were distilled
2
2
1
13
prior to use. All reported H NMR and C NMR spectra were record-
ed with a Varian 500 MHz spectrometer. Chemical shifts (d) were
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1722
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
single or double exponential dependences without using a decon-
volution procedure. Estimated precision of the decay time determi-
nation was 10 ps.
(2014K1A1A2064569) through the National Research Founda-
tion (NRF) funded by Ministry of Science, ICT & Future Planning
(
Korea) is acknowledged. Theoretical calculations were per-
formed at the Interdisciplinary Center of Mathematical and
Computer Modeling (ICM) of the Warsaw University (Poland)
under the computational grant No. G-32-10. We thank
W. Justin Youngblood for amending the manuscript.
The two-photon absorption measurements were performed by
using an IsoPlane SCT 320 fluorescence spectrometer (Princeton In-
strument) equipped with an RMS4X-PF (Olympus) objective lens by
[27]
using methods described earlier.
Samples were measured in
1
.0”1.0 cm quartz cuvettes in a 908 setup with direct excitation
from a femtosecond (<100 fs) titan-sapphire-laser (Chameleon
Compact OPO-Vis, Coherent) with 80 MHz repetition rate. The
measurements were performed in the wavelength range from 740
to 880 nm. Details of the calculations are given in the Supporting
Information.
Keywords: dyes/pigments · fluorescence · fused-ring systems ·
heterocycles · photophysics
[
1] a) M. Pawlicki, H. A. Collins, R. G. Denning, H. L. Anderson, Angew. Chem.
Int. Ed. 2009, 48, 3244–3266; Angew. Chem. 2009, 121, 3292–3316;
b) H. M. Kim, B. R. Cho, Chem. Commun. 2009, 153–164; c) G. S. He, L.-S.
Tan, Q. Zheng, P. N. Prasad, Chem. Rev. 2008, 108, 1245–1330; d) H. M.
Kim, B. R. Cho, Acc. Chem. Res. 2009, 42, 863–872.
Calculations
All calculations within this work were made with the aid of the
[2] a) S. Yao, H.-Y. Ahn, X. Wang, J. Fu, E. W. Van Stryland, D. J. Hagan, K. D.
Belfield, J. Org. Chem. 2010, 75, 3965–3974; b) C. D. Andrade, C. O.
Yanez, L. Rodriguez, K. D. Belfield, J. Org. Chem. 2010, 75, 3975–3982;
c) D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez,
F. W. Wise, W. W. Webb, Science 2003, 300, 1434–1437; d) L. Ventelon, S.
Charier, L. Moreaux, J. Mertz, M. Blanchard-Desce, Angew. Chem. Int. Ed.
[28]
Gaussian 09 package. Optimization of the molecular geometry in
the electronic ground (S ) and lowest excited (S ) states were per-
0
1
formed with the DFT and TD DFT B3LYP/6-31G(d,p) methods. The
spectra of molecules in solutions were calculated within the frame-
work of the PCM model with the use of the default options of this
model implemented in the Gaussian package. The vibrational
structures of the electronic spectra were calculated with a proce-
dure included in Gaussian 09, which used the Franck–Condon fac-
tors and the Duchinsky matrix.
2001, 40, 2098–2101; Angew. Chem. 2001, 113, 2156–2159; e) T. R.
Krishna, M. Parent, M. H. V. Werts, S. Gmouh, A.-M. Caminade, L. Mor-
eaux, S. Charpak, J.-P. Majoral, M. Blanchard-Desce, Angew. Chem. Int.
Ed. 2006, 45, 4645–4648; Angew. Chem. 2006, 118, 4761–4764.
[3] a) H. M. Kim, B. R. Cho, Chem. Rev. 2015, 115, 5014–5055; b) A. Ustione,
D. W. Piston, J. Microsc. 2011, 243, 221–226; c) D. Kim, H. G. Ryu, K. H.
Ahn, Org. Biomol. Chem. 2014, 12, 4550–4566.
Tissue imaging for organs of mice
[4] a) H. M. Kim, B. R. Cho, Chem. Asian J. 2011, 6, 58–69; b) H.-Y. Ahn, K. E.
Fairfull-Smith, B. J. Morrow, V. Lussini, B. Kim, M. V. Bondar, S. E. Bottle,
K. D. Belfield, J. Am. Chem. Soc. 2012, 134, 4721–4730.
A C57L6-type mouse (6 weeks, male) was used for this experiment.
Experiments were done under light-protected conditions, in a dark-
room, and with the use of aluminum foil. The mouse was dissected
after dislocation of the cervical vertebral. Blood perfusion with
phosphate-buffered saline (PBS, 1X solution) was performed for
elimination of blood. The organs were dissected, washed with PBS
buffer, and then sliced with a vibrating blade microtome (VT1000S,
Leica, Germany) with 400 mm thickness. Tissue slice samples were
immersed in 4% aqueous paraformaldehyde for 1 h to fix the
tissue. After the fixation, the tissues were immersed in the dye so-
lution (10 mm) for 30 min at 378C. Stained samples were placed on
the glass slide for imaging, after washing with PBS several times.
Two-photon microscopy equipped with a Ti-Sapphire laser (Chame-
leon Vision II, Coherent) at 140 fs pulse width and 80 MHz pulse
repetition rate was used (TCS SP5 II, Leica, Germany). The excita-
tion laser was tuned to l=900 nm and emission light was collect-
ed at l=500–605 nm in the case of compound 5. Prepared tissue
slice samples were mounted on the tight-fitting holder. The power
of the excitation laser was approximately 28.3 mW at the focal
point. The imaging resolution was 1024”1024 pixels and scanning
speed was 200 Hz all the way through the imaging. Acquired
images were processed by using LAS AF Lite (Leica, Germany).
[
5] a) E. W. Seo, J. H. Han, C. H. Heo, J. H. Shin, H. M. Kim, B. R. Cho, Chem.
Eur. J. 2012, 18, 12388–12394; b) C. H. Heo, S. K. Park, C. S. Lim, M. K.
Park, H. J. Kim, H. M. Kim, B. R. Cho, Chem. Eur. J. 2012, 18, 15246–
15249.
[
[
[
6] X. Dong, J. H. Han, C. H. Heo, H. M. Kim, Z. Liu, B. R. Cho, Anal. Chem.
2
012, 84, 8110–8113.
7] S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han, B. R. Cho, Chem. Commun. 2012,
8, 8395–8397.
8] a) S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E.-H. Joe, B. R. Cho, H. M. Kim,
J. Am. Chem. Soc. 2013, 135, 9915–9923; b) A. R. Morales, A. Frazer,
A. W. Woodward, H.-Y. Ahn-White, A. Fonari, P. Tongwa, T. Timofeeva,
K. D. Belfield, J. Org. Chem. 2013, 78, 1014–1025; c) A. R. Morales, C. O.
Yanez, Y. Zhang, X. Wang, S. Biswas, T. Urakami, M. Komatsu, K. D. Bel-
field, Biomaterials 2012, 33, 8477–8485.
4
[9] a) H. Meier, Angew. Chem. Int. Ed. 2005, 44, 2482–2506; Angew. Chem.
2
005, 117, 2536–2561; b) Z. R. Grabowski, K. Rotkiewicz, W. Rettig,
Chem. Rev. 2003, 103, 3899–4032; c) G. Weber, F. J. Farris, Biochemistry
979, 18, 3075–3078; d) D. J. Cowley, Nature 1986, 319, 14–15; e) Z.
Yang, J. Cao, Y. He, J. H. Yang, T. Kim, X. Peng, J. S. Kim, Chem. Soc. Rev.
014, 43, 4563–4601.
1
2
[
10] a) Q. Verolet, A. Rosspeintner, S. Soleimanpour, N. Sakai, E. Vauthey, S.
Matile, J. Am. Chem. Soc. 2015, 137, 15644–15647; b) A. S. Klymchenko,
R. Kreder, Chem. Biol. 2014, 21, 97–113; c) H. M. Kim, M. J. An, J. H.
Hong, B. H. Jeong, O. Kwon, J.-Y. Hyon, S.-C. Hong, K. J. Lee, B. R. Cho,
Angew. Chem. Int. Ed. 2008, 47, 2231–2234; Angew. Chem. 2008, 120,
CCDC 1456392 (4) contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre.
2263–2266; d) H. M. Kim, B. H. Jeong, J.-Y. Hyon, M. J. An, M. S. Seo,
J. H. Hong, K. J. Lee, C. H. Kim, T. Joo, S.-C. Hong, B. R. Cho, J. Am. Chem.
Soc. 2008, 130, 4246–4247; e) M. K. W e˛ cławski, T. T. Meiling, A. Leniak,
P. J. Cywi n´ ski, D. T. Gryko, Org. Lett. 2015, 17, 4252–4255; f) B. E. Cohen,
T. B. McAnaney, E. S. Park, Y. N. Jan, S. G. Boxer, L. Y. Jan, Science 2002,
296, 1700–1703; g) K. Gaus, E. Gratton, E. P. W. Kable, A. S. Jones, I. Gel-
issen, L. Kritharides, W. Jessup, Proc. Natl. Acad. Sci. USA 2003, 100,
Acknowledgements
5554–15559; h) A. Petri cˇ , S. A. Johnson, H. V. Pham, Y. Li, S. Ceh, A. Go-
ˇ
1
Support from the National Science Centre, Poland (Grants
Maestro - 2012/06/A/ST5/00216 and Maestro - 2012/04/A/ST2/
lobi cˇ , E. D. Agdeppa, G. Timbol, J. Liu, G. Keum, N. Satyamurthy, V.
Kepe, K. N. Houk, J. R. Barrio, Proc. Natl. Acad. Sci. USA 2012, 109,
16492–16497; i) M. Cui, M. Ono, H. Watanabe, H. Kimura, B. Liu, H. Saji,
0
0100)
and
Global
Research
Laboratory
Program
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1723
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
J. Am. Chem. Soc. 2014, 136, 3388–3394; j) M. Grzybowski, A. Je z˙ ewski,
I. Deperasi n´ ska, D. H. Friese, M. Banasiewicz, V. Hugues, B. Kozankiewicz,
M. Blanchard-Desce, D. T. Gryko, Org. Biomol. Chem. 2016, 14, 2025–
Org. Chem. 2011, 76, 8113–8116; f) A. R. Sarkar, C. H. Heo, H. W. Lee,
K. H. Park, Y. H. Suh, H. M. Kim, Anal. Chem. 2014, 86, 5638–5641; g) M.
Tasior, D. Kim, S. Singha, M. Krzeszewski, K. H. Ahn, D. T. Gryko, J. Mater.
Chem. C 2015, 3, 1421–1446.
2
033; k) L. D. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142–155; l) Y.
Zhang, M. Jiang, G.-C. Han, K. Zhao, B. Z. Tang, K. S. Wong, J. Phys.
Chem. C 2015, 119, 27630–27638.
11] a) L. Ji, A. Lorbach, R. M. Edkins, T. B. Marder, J. Org. Chem. 2015, 80,
[16] S. Singha, D. Kim, B. Roy, S. Sambasivan, H. Moon, A. S. Rao, J. Y. Kim, T.
Joo, J. W. Park, Y. M. Rhee, T. Wang, K. H. Kim, Y. H. Shin, J. Jung, K. H.
Ahn, Chem. Sci. 2015, 6, 4335–4342.
[
[
5
658–5665; b) W. C. W. Leu, A. E. Fritz, K. M. Digianantonio, C. S. Hartley,
[17] a) X. Bulliard, Y. W. Jin, G. H. Lee, S. Yun, D.-S. Leem, T. Ro, K.-B. Park, C.-J.
Heo, R.-I. Satoh, T. Yagi, Y. S. Choi, S.-J. Lim, S. Lee, J. Mater. Chem. C
2016, 4, 1117; b) S. Shen, P. Jiang, C. He, J. Zhang, P. Shen, Y. Zhang, Y.
Yi, Z. Zhang, Z. Li, Y. Li, Chem. Mater. 2013, 25, 2274–2281; c) M.-A.
Tehfe, F. Dumur, B. Graff, F. Morlet-Savary, J.-P. Fouassier, D. Gigmes, J.
LalevØe, Macromolecules 2013, 46, 3761–3770; d) M. Hauck, M. Stolte, J.
Schçnhaber, H.-G. Kuball, T. J. J. Miller, Chem. Eur. J. 2011, 17, 9984–
9998; e) K. Nagy, E. Orbµn, S. Bçsze, P. Kele, Chem. Asian J. 2010, 5,
773–777; f) M. Leung, C.-C. Chang, M.-H. Wu, K.-H. Chuang, J.-H. Lee, S.-
J. Shieh, S.-C. Lin, C.-F. Chiu, Org. Lett. 2006, 8, 2623–2626; g) R. Nazir,
T. T. Meiling, P. J. Cywi n´ ski, D. T. Gryko, Asian J. Org. Chem. 2015, 4, 929–
935; h) I. Jung, J. K. Lee, K. H. Song, K. Song, S. O. Kang, J. Ko, J. Org.
Chem. 2007, 72, 3652–3658.
[18] C. Hu, F. Liu, H. Zhang, F. Huo, Y. Yang, H. Wang, H. Xiao, Z. Chen, J. Liu,
L. Qiu, Z. Zhen, X. Liu, S. Bo, J. Mater. Chem. C 2015, 3, 11595–11604.
[19] a) C. Zhou, Y. Li, Y. Zhao, J. Zhang, W. Yang, Y. Li, Org. Lett. 2011, 13,
292–295; b) A. P. Kozynchenko, Y. M. Volovenko, V. K. Promonenkov,
A. V. Turov, F. S. Babichev, Chem. Heterocycl. Compd. 1988, 24, 923–927;
c) Y. M. Poronik, D. T. Gryko, Chem. Commun. 2014, 50, 5688–5690.
[20] a) D. T. Gryko, J. Piechowska, M. Tasior, J. Waluk, G. Orzanowska, Org.
Lett. 2006, 8, 4747–4750; b) D. T. Gryko, J. Piechowska, V. Vetokhina, D.
Wójcik, Bull. Chem. Soc. Jpn. 2009, 82, 1514–1519.
J. Org. Chem. 2012, 77, 2285–2298.
12] a) X. Dong, C. H. Heo, S. Chen, H. M. Kim, Z. Liu, Anal. Chem. 2014, 86,
3
08–311; b) Y. Niko, S. Sasaki, S. Kawauchi, K. Tokumaru, G.-i. Konishi,
Chem. Asian J. 2014, 9, 1797–1807; c) H. J. Kim, C. H. Heo, H. M. Kim, J.
Am. Chem. Soc. 2013, 135, 17969–17977; d) S.-S. Li, K.-J. Jiang, C.-C. Yu,
J.-H. Huang, L.-M. Yang, Y.-L. Song, New J. Chem. 2014, 38, 4404; e) K.
Rathore, C. S. Lim, Y. Lee, H. J. Park, B. R. Cho, Asian J. Org. Chem. 2014,
3
, 1070–1073.
[
13] a) K. Yamaguchi, T. Murai, S. Hasegawa, Y. Miwa, S. Kutsumizu, T. Mar-
uyama, T. Sasamori, N. Tokitoh, J. Org. Chem. 2015, 80, 10742–10756;
b) T. Murai, K. Yamaguchi, F. Hori, T. Maruyama, J. Org. Chem. 2014, 79,
4
930–4939; c) R. Orłowski, M. Banasiewicz, G. Clermont, F. Castet, R.
Nazir, M. Blanchard-Desce, D. T. Gryko, Phys. Chem. Chem. Phys. 2015,
7, 23724–23731; d) E. Genin, V. Hugues, G. Clermont, C. Herbivo,
1
M. C. R. Castro, A. Comel, M. M. M. Raposo, M. Blanchard-Desce, Photo-
chem. Photobiol. Sci. 2012, 11, 1756–1766; e) I. López-Duarte, P. Chaira-
tana, Y. Wu, J. PØrez-Moreno, P. M. Bennett, J. E. Reeve, I. Boczarow, W.
Kaluza, N. A. Hosny, S. D. Stranks, R. J. Nicholas, K. Clays, M. K. Kuimova,
H. L. Anderson, Org. Biomol. Chem. 2015, 13, 3792–3802; f) D. Alonso
Doval, S. Matile, Org. Biomol. Chem. 2013, 11, 7467.
[
14] a) H. Von Pechmann, C. Duisberg, Ber. Dtsch. Chem. Ges. 1883, 16, 2119;
b) C. Murata, T. Masuda, Y. Kamochi, K. Todoroki, H. Yoshida, H. Nohta,
M. Yamaguchi, A. Takadate, Chem. Pharm. Bull. 2005, 53, 750–758;
c) M.-S. Schiedel, C. A. Briehn, P. Bäuerle, Angew. Chem. Int. Ed. 2001, 40,
[21] a) C. A. Obafemi, W. Pfleiderer, Helv. Chim. Acta 1994, 77, 1549–1556;
b) D. R. Romer, J. Heterocycl. Chem. 2009, 46, 317–319.
[22] S. J. Strickler, R. A. Berg, J. Chem. Phys. 1962, 37, 814–822.
[23] E. Lippert, Z. Naturforsch. A 1955, 10, 541.
4
677–4680; Angew. Chem. 2001, 113, 4813–4816; d) R. M. Christie,
K. M. Morgan, M. S. Islam, Dyes Pigm. 2008, 76, 741–747; e) D. J. Yee, V.
Balsanek, D. Sames, J. Am. Chem. Soc. 2004, 126, 2282–2283; f) K. Siva-
kumar, F. Xie, B. M. Cash, S. Lung, H. N. Barnhill, Q. Wang, Org. Lett.
[24] N. Mataga, Y. Kaifu, M. Koizumi, Bull. Chem. Soc. Jpn. 1955, 28, 6.
[25] D. Kim, H. Moon, S. H. Baik, S. Singha, Y. W. Jun, T. Wang, K. H. Kim, B. S.
Park, J. Jung, I. Mook-Jung, K. H. Ahn, J. Am. Chem. Soc. 2015, 137,
6781–6789.
2004, 6, 4603–4606; g) A. Elangovan, J.-H. Lin, S.-W. Yang, H.-Y. Hsu, T.-I.
Ho, J. Org. Chem. 2004, 69, 8086–8092; h) M.; Tasior, Y. M. Poronik, O.
Vakuliuk, B. Sadowski, M. Karczewski, D. T. Gryko, J. Org. Chem. 2014, 79,
[26] B. Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,
Academic Press, New York, 1971.
8
723–8732; i) X. Liu, J. M. Cole, P. G. Waddell, T.-C. Lin, J. Radia, A. Zei-
[27] a) R. K. Wang, Y. Wickramasinghe, Appl. Opt. 1998, 37, 7342–7351; b) C.
Xu, W. W. Webb, J. Opt. Soc. Am. B 1996, 13, 481–491.
dler, J. Phys. Chem. A 2012, 116, 727.
[
15] a) D. Kim, Q. P. Xuan, H. Moon, Y. W. Jun, K. H. Ahn, Asian J. Org. Chem.
[28] See the Supporting Information for more details.
2014, 3, 1089; b) D. Kim, S. Singha, T. Wang, E. Seo, J. H. Lee, S.-J. Lee,
K. H. Kim, K. H. Ahn, Chem. Commun. 2012, 48, 10243–10245; c) D. Kim,
S. Sambasivan, H. Nam, K. H. Kim, J. Y. Kim, T. Joo, K.-H. Lee, K.-T. Kim,
K. H. Ahn, Chem. Commun. 2012, 48, 6833–6835; d) D. Guo, T. Chen, D.
Ye, J. Xu, H. Jiang, K. Chen, H. Wang, H. Liu, Org. Lett. 2011, 13, 2884–
Manuscript received: February 28, 2016
Revised: March 18, 2016
Accepted Article published: March 30, 2016
Final Article published: May 11, 2016
2
887; e) J. H. Son, C. S. Lim, J. H. Han, I. A. Danish, H. M. Kim, B. R. Cho, J.
Chem. Asian J. 2016, 11, 1718 – 1724
www.chemasianj.org
1724
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim