9-Aryl-3-aminocarbazole as an Environment- and Stimuli-Sensitive Fluorogen and Applications in Lipid Droplet Imaging

Article abstract of DOI:10.1021/acs.joc.9b00493

Environment-sensitive luminophoric molecules have played an important role in the fields of smart materials, sensing, and bioimaging. In this study, it was demonstrated that depending on the substituents, 9-aryl-3-aminocarbazoles can display aggregation-induced emission and solvatofluorochromism, and the operating mechanism was clarified. The application of these compounds to lipid droplet imaging and fluorescent probes for cysteamine was demonstrated.

Full text of DOI:10.1021/acs.joc.9b00493

Subscriber access provided by UNIV OF LOUISIANA
Article
9-Aryl-3-aminocarbazole as an environment- and stimuli-
sensitive fluorogen and applications in lipid droplet imaging
Ryosuke Matsubara, Tomoaki Kaiba, Akito Nakata, Tatsushi Yabuta,
Masahiko Hayashi, Motonari Tsubaki, Takashi Uchino, and Eri Chatani
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00493 • Publication Date (Web): 11 Apr 2019
Downloaded from http://pubs.acs.org on April 12, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 14
The Journal of Organic Chemistry
1
2
3
4
5
6
9-Aryl-3-aminocarbazole as an environment- and stimuli-sensitive
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fluorogen and applications in lipid droplet imaging
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ryosuke Matsubara,* Tomoaki Kaiba, Akito Nakata, Tatsushi Yabuta, Masahiko Hayashi, Motonoari
Tsubaki, Takashi Uchino, Eri Chatani
Department of Chemistry, Graduate School of Science, Kobe University, Nada, Kobe 657-8501, Japan
ABSTRACT: Environment-sensitive luminophoric molecules have played an important role in the fields of smart materials, sensing,
and bioimaging. In this study, it was demonstrated that depending on the substituents, 9-aryl-3-aminocarbazoles can display
aggregation-induced emission and solvatofluorochromism, and the operating mechanism was clarified. The application of these
compounds to lipid droplet imaging and fluorescent probes for cysteamine was demonstrated.
INTRODUCTION
synthesis of 14 was not trivial. The initial attempt to directly
synthesize 14 from 3,6-dibromocarbazole (9) yielded a
moderate quantity, albeit as a mixture with byproducts derived
from the bond-forming reaction with carbazole nitrogen. The
separation of 14 from the other byproducts was exceptionally
challenging. Moreover, this reaction exhibited low
reproducibility depending on the reaction scale; this can be
attributed to the volatility of dimethyl amine at the applied
reaction temperature. Therefore, we adopted the protective
group strategy to overcome this issue. The NH group of 9 was
protected with benzyl-type protective groups to yield carbazole
10 and 11. The subsequent introduction of NMe
proceeded well; reasonable yields
bis(dimethylamino)carbazoles 12 and 13 were obtained. The
removal of the N-benzyl-type protection was initially attempted
by hydrogenation using transition metal catalysts such as Pd/C,
Environment-sensitive luminophoric properties have
attracted attention owing to their potential applicability to smart
materials, sensing, and bioimaging.
1
-5
Aggregation is an
environmental factor regulating the luminescent property.
Numerous luminophoric compounds generally exhibit
aggregation-caused quenching (ACQ) and emit reduced
fluorescence upon aggregation, which is ordinarily caused by
intermolecular – stacking interaction to quench the excited
6
-7
luminophore. In contrast, certain luminogenic molecules
enhance the emission upon aggregation; this phenomenon is
called aggregation-induced emission (AIE).
8-9
2
groups
of
Extensive
research has been performed since the development of the AIE
1
0
concept. Hexaphenylsilole and tetraphenylethene are the
1
1
prototypical molecules with AIE property, and numerous
other types of structural motifs have also emerged as AIE
1
2-13
2
Pd(OH) /C, Raney Ni, and Wilkinson’s catalyst with or without
luminophores.
Solvent polarity is another environmental
pressurizing hydrogen gas. All trials failed with the starting
material completely recovered; this was likely to be a result of
the considerable electron-richness of carbazole moiety. The
DDQ-oxidation for p-methoxybenzyl-protected carbazole 13
was unsuccessful owing to the lability of electron-rich
carbazole toward oxidation. Meanwhile, the treatment of 13
factor that dramatically influences the fluorescent attributes
such as color and intensity.
1
4-19
A majority of the molecules exhibiting AIE and/or a
solvatofluorochromic nature contain plural aromatic moieties
connected with each other via several single bonds, composing
an overall large -conjugated system. Herein, we report that a
relatively small unit, 9-aryl-3-aminocarbazole (the minimum
required unit size is ~300 Da), endows the molecules with both
AIE and solvatofluorochromic properties. The presence of an
amino group at the three-position of the carbazole and of an
electron-deficient aromatic substituent at the nine-position was
the prerequisites for the AIE property. The operating
mechanism of the observed AIE phenomenon is discussed. The
application of solvatofluorochromism was demonstrated
through light up detection of lipid droplets (LDs) in living cells
with low background noise.
with BCl
mono-NMe
3
provided a reasonable yield of 14. On the other hand,
carbazole 16 could be synthesized without
2
protective group manipulation. The mono-bromination of
parent carbazole using an equivalent of N-bromosuccinimide
(NBS) followed by palladium-catalyzed amination provided a
good yield of 16. The N-arylation of 14 and 16 was achieved by
copper-catalyzed coupling reaction, yielding NAC 1–5. NAC 6
and 7 were synthesized by the copper-catalyzed N-arylation of
the corresponding known N–H carbazoles. The synthesis of
julolidine-type NAC 8 was accomplished by nitration of NAC
6 followed by reduction, N-alkylation, and cyclization.
RESULTS
Synthesis
Photophysical properties
The photophysical properties of NACs 1–8 were analyzed by
UV-Vis and emission spectroscopies. The absorption and
emission maximum as well as the photoluminescence (PL)
quantum yields in solution state ( ) are presented in Table 1.
The PL quantum yields in solid state (
A series of N-arylcarbazoles (NACs) were synthesized as
shown in Figure 1. We set 3,6-bis(dimethylamino)carbazole
(14) as a key common intermediate, with which various
bis(dimethylamino)carbazole derivatives were synthesized.
Shortly after the investigation was started, we realized that the
ACS Paragon Plus Environment
l
2
0
s
) for the selected NACs
are also presented. The data, except those for 
s
, were obtained

The Journal of Organic Chemistry
Page 2 of 14
from deoxygenated THF solution. The solid-state quantum position of the carbazole unit exhibited strong emission in THF
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yields were measured for the powder materials obtained after
grinding the microcrystals obtained initially.
solution ( are 35% and 96%, respectively); this indicated that
l
an electron deficient aryl substituent at the nine-position is
2
required for manifesting AIE property. The NMe group at the
three-position is also necessary to quench the fluorescence in
the solution state: NACs 6 and 7 with electron deficient aryl
group at the nine-position albeit without the NMe
2
group at the
three-position, are strongly fluorescent in THF (
l
are 34% and
3
1%, respectively). For NAC 8 (similar in structure to NAC 4
except for a non-rotatable C–N single bond at the three-position
owing to the julolidine polycyclic structure), the PL quantum
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yield in THF solution was low (
l
= ~0%).
Table 1. Optical properties of NAC luminophores 1–8a
ab (nm)
337, 384
337, 379
327, 389
358
em (nm)b
578

l
(%)
s
 (%)
NAC
1
1.6
~0
35
1.5
96
34
31
~0
15
4.4
–c
2
3
4
5
6
7
8
n.d.
430
570
20
– c
5.1
14
– c
374
425
326, 336
351, 365
374
395
459
n.d.
a

ab = absorption maximum, em = emission maximum, 
l
=
fluorescence quantum yield in solution (solvent: THF),  = solid
s
b
state (powder) fluorescence quantum yield. Measured by exciting
at the absorption maxima. n.d. = Signal is very weak and not
c
detectable. Amorphous.
(A)
(B)
Figure 1. Synthesis of NAC 1–8. Conditions: a) BnBr (2.0 equiv),
K
2
CO
equiv), p-(MeO)C
Pd (dba) (1 mol%), Ruphos (2.4 mol%), LiHMDS (6.0 equiv),
Me NH Cl (3.0 equiv), tetrahydrofuran (THF), 80 C, 83% (12),
7% (13). d) BCl (6.0 equiv), CH Cl , rt, then 4 M HCl aq., 86%.
e) NBS (1.0 equiv), THF, rt, 89%. f) Pd (dba) (1 mol%), Ruphos
(2.4 mol%), LiHMDS (6.0 equiv), Me NH Cl (3.0 equiv), THF, 80
C, 73%. g) For 1, 2, and 4; ArI (1.1 equiv), LiCl (1.0 equiv), CuI
10 mol%), 1,10-phenanthroline (10 mol%), Cs CO (1.2–1.3
equiv), DMF, 150 C, 65% (1), 64% (2), 67% (4). For 3 and 5;
PhBr (10 equiv), CuI (1.1 equiv), K CO (3.9 equiv), AcNMe , 180
C, 76% (3), 80% (5). h) HNO (17 equiv), AcOH, 72%. i) Pd/C
3
(10 equiv), DMF, room temperature (rt), 71%. b) NaH (1.5
6
H
4
CH Cl (1.5 equiv), DMF, rt, 99%. c)
2
2
3
2
2
9
3
2
2
Figure 2. Representation of AIE nature of NAC 1. Photographs of
A) 10-M THF solution and (B) solid state of 1 under UV
2
3
(
2
2
irradiation (excited by 254 nm and black light irradiation,
respectively).

(
2
3
2
3
2
The AIE properties of NAC 1 are further investigated in

3
THF/water mixed solutions with different water fractions (f
The emissions from the pure THF solution (f = 0%) were weak
albeit visible (Figure 3A). However, after the addition of water
yielding an f of 10%), the emissions were quenched. The non-
emissive behavior continued until the f reached 90%. Further
addition of water at the f of 99% caused the precipitation of
w
).
(
10 mol%), H
2
(1 atm), MeOH, rt, 33%. j) 1-Br-3-Cl-pentane (15
w
equiv), Na
2
CO
3
(4.0 equiv), 100 C. k) DMF, 160 C, 26% (2 step).
(
w
w
AIE properties were evident in NACs 1, 2, and 4, which have
electron deficient aryl substituents at the nine-position and
w
NAC 1 in the quartz cell, making the solution slightly opaque.
Then, the emissions resumed, although becoming blue-shifted
compared to those obtained in pure THF. The fluorescence
change was visually observable, as shown in Figure 3B. The
dynamic light scattering (DLS) measurement of the solution
NMe
quantum yields in THF solution were low (
and 1.5%, respectively), whereas they were emissive in the
solid state ( were 15%, 4.4%, and 20%, respectively). The PL
difference between the solution and solid states of NAC 1 is
visually observable (Figure 2). In contrast, NACs 3 and 5 with
an electronically neutral arene (phenyl group) at the nine-
2
group at the three-position of the carbazole unit; their
l
were 1.6%, ~0%,
s
with an f
w
of 99% revealed the formation of particles with
dimensions of submicrometer order (Figure 3C), which was
ACS Paragon Plus Environment

Page 3 of 14
The Journal of Organic Chemistry
microcrystals initially obtained after the recrystallization, the
visually evidenced by the Tyndall scattering observed using a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
laser pointer (Figure 3C, inset) and field emission scanning
electron microscopy (FE-SEM) analysis (Figure S7).
difference between the microcrystals and ground solids was
investigated (Figure 5). The microcrystals and powders
exhibited similar patterns in the fluorescence spectroscopy and
powder X-ray diffraction (PXRD) analysis. These results
indicate that the grinding did not change the morphology of the
microcrystals significantly, ensuring the validity of basing the
discussion of solid fluorescent property on the SCXRD
structure.
(A)
(B)
100
- fw = 0
fw = 10
-
80
60
40
20
0
- fw = 30
- fw = 50
- fw = 70
-
fw = 90
- fw = 95
fw = 99
-
400
500
wavelength (nm)
600
(A)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(C)
50
40
30
20
60
Ex. light
-
crystal state
40
- powder state
10
0
0.1
1
10
100 1000 10000
20
size (nm)
Figure 3. Fluorescence examination of the 10 M solution
of 1 in the THF/water mixed solvents with various water
fractions. (A) Luminescence spectra. (B) Photographs
under UV irradiation (black light). (C) DLS measurement
300
400
500
600
700
wavelength (nm)
of the solution of 1 in the THF/water mixed solvent (f
w
:
(B)
99%). (inset) Tyndall scattering seen using a laser pointer.
3
16x10
Single crystal X-ray diffraction (SCXRD) analysis revealed
the molecular structure of the fluorescent solid NAC 1 (Figure
). A unit cell contained the two independent molecules; one of
- crystal state
powder state
12
8
-
4
these is depicted in Figure 4A. The nitrogen atoms of the two
dimethyl-amino groups exhibit almost planar geometry (the
sum of the three bond angles around the concerned nitrogen is
4
3
50.2); this indicated that the lone pair on the nitrogen of the
dimethyl amino groups is conjugated with the -electrons on
the carbazole to a considerable extent. The aryl group on the
nine-position nitrogen twisted against the carbazole -plane
with a dihedral angle of 43.1. With regard to the molecular
packing, the molecules are positioned close to each other
0
1
0 20 30 40 50 60
angle (2θ)
Figure 5. Comparison between the as-prepared microcrystals and
ground powder of NAC 1. (A) Solid PL spectra. (B) PXRD spectra.
(
Figure 4B): the carbazole and benzonitrile units are placed one
above the other with a mutual distance of 3.4Å, indicating the
presence of – stacking.
The PL properties of NAC 1 as well as of NAC 3 in various
solvents are presented in Table 2. The results demonstrate that
NAC 1 rather than NAC 3 exhibits solvatofluorochromism
nature. The absorption spectra of NAC 1 and 3 exhibited
negligible change in solvents with different polarities (which
can be quantified by orientation polarizability f); this indicated
that the ground states of NAC 1 and 3 are negligibly affected by
the solvent (Figure S2). However, the fluorescence of NAC 1
was sensitive to the solvent; as the solvent polarity increased,
the fluorescence maximum wavelength lengthened (Figure 6A),
and the fluorescence quantum yield decreased. The color
change of fluorescence could be observed by the naked eyes
(A)
(B)
side view
.4Å
.4Å
3
3
f = 43.1
top view
(
Figure 6C). In contrast, the fluorescent property of NAC 3 was
insensitive to the solvent polarity (Figure 6BD).
Figure 4. Single-crystal structure of NAC 1. (A) ORTEP
representation of one of the two independent molecules in the unit
cell. (B) Packing pattern of molecules.
Because the solid-state fluorescence was measured for the
powder materials that were prepared by grinding the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 14
(
A)
(B)
at its absorption maxima. PL spectra of (A) NAC 1 and (B) 3.
1
1
0
0
0
.2
.0
.8
.6
.4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
- c-hexane
- toluene
THF
- CH CN
Photographs of (C) NAC 1 (ex = 365 nm) and (D) 3 (ex= 254 nm).
1
.2
1.0
.8
0.6
.4
-
- c-hexane
- toluene
- THF
3
0
- CH3CN
0
To exhibit the solvent polarity effect on the fluorescence of
NAC 1, we gradually changed the hydrophobicity of the solvent
by increasing the hexane fraction (f ) against THF in the
h
solution of 1. The non-normalized luminescence spectra
demonstrated that as the hydrophobicity increased, the
spectrum was blue-shifted, and the intensities of the peaks
increased (Figure 7A and B). The luminescence color of 1
changed from yellow to green to blue as the solution
hydrophobicity increased (Figure 7C). According to the DLS
0.2
.0
0.2
0.0
0
4
00
500
wavelength (nm)
600
700
350
400
450
500
550
600
wavelength (nm)
(D)
CH CN THF
(
C)
CH3CN THF
Tol
c-hex
Tol
c-hex
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
h
measurement, the solution of 1 with the f of 99% did not
contain aggregates (Figure 7D); this indicated that the blue
color emission was derived from the isolated form rather than
the aggregated state.
Figure 6. PL properties of NAC 1 and 3 in solution state in various
solvents. Solution concentration: 10 M. Each solution was excited
Table 2. Solvent effects on PL properties of 1 and 3
1
3
fa
ab (nm)
335, 382
339, 387
337, 384
337, 381
em (nm)b
458
 (%)
ab (nm)
324, 383
329, 390
327, 389
323, 387
em (nm)b  (%)
l
solvent
l
25
25
1.6
~0
c-hexane
toluene
THF
0
420
430
430
439
37
49
35
39
0.014
0.210
0.306
512
578
CH
3
CN
n.d.
a
b
Orientation polarizability. Measured by exciting at 335 nm. n.d. = Signal is very weak and undetectable.
To demonstrate the solvent polarity effect on the fluorescence
of NAC 1, we gradually changed the hydrophobicity of the
solvent by increasing the hexane fraction (f ) against THF in the
solution of 1. The non-normalized luminescence spectra
demonstrated that as the hydrophobicity increased, the
spectrum was blue-shifted, and the intensities of the peaks
increased (Figure 7A and B). The luminescence color of 1 was
changed from yellow to green to blue as the solution
hydrophobicity increased (Figure 7C). According to the DLS
measurement (Figure 7D) and atomic force microscopy (AFM)
Luminescence spectra. (B) Plot of maximum emission wavelength.
Solution concentration: 10 M. ex = 400 nm. (C) Photographs
under UV irradiation (black light). (D) DLS measurement of the
h
solution of 1 in the THF/hexane mixed solvent (f
figure shows no Tyndall scattering observed (compare with Figure
C).
h
: 98%). Inset
3
Applications
With the knowledge that NAC 1 exhibits polarity-sensitive
fluorescence, we investigated its capability to stain LDs in
h
analysis (Figure S8), the solution of 1 with the f of 99% did not
2
1
living cells (Figure 8). For comparison, Nile Red,
a
contain a large size of aggregates; this indicated that the blue
color emission was derived from the isolated form rather than
the aggregated state.
commercially available fluorescent stain for LDs, was used in
parallel with NAC 1. The living HeLa cells were pre-incubated
2
2
with oleic acid to form LDs. Then, Nile Red and NAC 1 were
incubated in independent batches of cultured cells, followed by
washing with PBS buffer. The fluorescence from each batch
was analyzed by a confocal fluorescence microscope. The LDs
were lit up by using Nile Red (Figure 8A and C). However, the
whole cytoplasm was vaguely stained, resulting in the low S/N
(
A)
(B)
6
00
4
00 - f
- f
h
h
h
h
h
h
h
= 0
= 10
= 30
= 50
= 70
= 90
= 98
-
f
- f
- f
550
3
00
2
00 - f
5
4
4
00
50
00
-
f
100
0
2
3
ratio, as indicated previously. NAC 1 was cell membrane
permeable and absorbed in the cell. Moreover, NAC 1
selectively lit up the LDs with low background fluorescence
3
50 400 450 500 550 600 650
wavelength (nm)
0
20
40
60
80
100
fh
(
D)
(
C)
5
4
3
2
1
0
(
Figure 8B and D); this is likely to be because of the strong
0
0
0
0
0
dependence of the fluorescence intensity of NAC 1 on the
surrounding polarity (Figure 3 and Figure 6). No noticeable
cytotoxicity of 1 was observed at the tested concentration (12

M) and further cytotoxicity investigation with higher
concentration was hampered owing to the poor solubility of 1.
0
.1
10
1000
size (nm)
To further explore the application of the unique
photophysical properties of the developed NAC, we designed a
stimuli-sensitive turn-on fluorescent molecule based on the
Figure 7. Fluorescence examination for the solution of 1 in
THF/hexane mixed solvents with various hexane fractions. (A)
ACS Paragon Plus Environment

Page 5 of 14
The Journal of Organic Chemistry
deficient aryl group at the nine-position to obtain AIE and
NAC architecture. Considering that an electron deficient
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituent on the phenyl group at the nine-position is required
for the emission quenching of the solution in a polar solvent, we
synthesized NAC 20 with an electrophilic formyl group on the
phenyl group (Figure 9). The formyl group is adequately
electron-withdrawing to ensure the emission quenching of 20 in
solvatofluorochromic capabilities.
As shown in Figure 1, their synthesis is straightforward,
short-step, and modular; here, N-H carbazole 14 or 16 can be
used as a common intermediate for a variety of NACs with
potential environment-sensitive fluorescence property.
the solution state in CH
NAC 20 was smoothly converted to fluorescent 1,3-thiazolidine
1; thus, NAC 20 functioned as a turn-on fluorescent probe for
3
CN. Upon the addition of cysteamine,
NAC 1, 2, and 4 exhibit a low intensity of fluorescence in
THF solution and are bright in the solid state (Table 1); i.e., they
2
8
are AIE molecules. Carbazole is an intrinsically luminogenic
cysteamine.
molecular unit and is wide applied in organic light emitting
2
4
diodes (OLED) and chemical probes; this is consistent with
the fluorescence emission of NAC 1, 2, and 4 in the solid state.
However, their indistinct fluorescence in the solution state is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DISCUSSION
Environment-sensitive luminophoric molecular units can
operate, by themselves or by being incorporated into the
molecules of interest, as a switching module for the fabrication
of the molecular sensing probes. Because they are generally
used in biological systems, the minimum structural motif
required for the manifestation of the switching function should
be minimized to secure cell permeability and to minimize the
interference in the original function of the parent molecule. This
report illustrates a simple 3-aminocarbazole with an electron
2
5-27
uncommon.
Strong fluorescence was observed from their
variant NAC 3 and 5–7, where an electron withdrawing
substituent on the nine-position aryl group or an amino group
on the carbazole is omitted (Table 1). These results indicate that
a combination of strongly electron-rich carbazole and electron
deficient aryl group results in the quenching of the potential
fluorescence of carbazole in the solution state.
(
A)
(B)
(
C)
(D)
Figure 8. Confocal fluorescence images of living HeLa cells pre-incubated with 62.5-M oleic acid. (A) and (B) After incubation with Nile
Red (30 M) (A) and NAC 1 (12 M) (B). Left, bright-field image; middle, fluorescence image; right, merged image. (C) and (D) Magnified
images of (A) and (B), respectively. Left, bright-field image; right, fluorescence image. Red staining was produced by exciting at 545  13
nm with the emission filter set at 605  35 nm. Blue staining was produced by exciting at 360  20 nm with the emission filter set at 460 
2
5 nm.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 14
solvents becomes almost non-fluorescent. Meanwhile, in the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aggregated state, the molecule cannot relax to a further twisted
geometry upon photoexcitation; this is owing to the motionally-
restricted situation and absence of interaction with solvent
molecules (Figure 11B). Accordingly, 
significantly higher than  (Table 1); this implies that NAC 1
is an AIE molecule. The still modest value of  of NAC 1 is
s
of NAC 1 is
l
s
probably owing to the presence of – stacking in the packing
geometry (Figure 4). The observation of – stacking can be
easily rationalized by considering that the carbazole is electron
rich and the benzonitrile is electron deficient; there should be
electrostatic interaction between the carbazole and benzonitrile
units.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NAC 6 and 7 can potentially acquire TICT excited states;
however, their 
l
in THF are high (Table 1). The degree to
state compared
which the twisted geometry contributes to the S
1
to the more planar geometry is determined by the balance
between the stabilization effect by the enhanced dipole moment
in polar solvents and the destabilization outcome by the charge
2
9
separation and the loss of mesomeric effect. A hydrogen or
MeO group is a weaker electron-donating group than NMe
2
;
therefore, the radical cation of carbazole unit in the expected
TICT excited states of NAC 6 and 7 are not likely to be
adequately stabilized. Thus, NAC 6 and 7 acquire the S states
1
with a less twisted geometry and exhibit stronger fluorescence
Figure 9. Fluorescence change of 20 upon treatment with
cysteamine. ex = 335 nm.
It is established that molecules having an electron donor (D)
and an electron acceptor (A) tend to generate the intramolecular
charge transfer (ICT) excited state. NAC 1, 2, and 4 belong to
this category. The ground states of NAC 1, 3, and 6 were
theoretically simulated using DFT at the B3LYP level of theory
employing the 6-31G(d,p) basis set in THF (Figure 10). With
regard to NAC 1 and 6, the HOMO is located on the carbazole
part and the LUMO on the nine-position aryl group. In contrast,
both the HOMO and LUMO of NAC 3 are distributed on the
carbazole part. Therefore, it is likely that the transition from
HOMO to LUMO upon photoirradiation would result in the
ICT excited state for NAC 1 and 6, and locally excited (LE)
state for NAC 3.
than NAC 1.
We first surmised the rotation of the single bond between the
carbazole and NMe
responsible for the TICT excited-state formation.
2
group (bond b in Figure 11C) to be
3
2-33
To verify
this hypothesis, NAC 8 was designed, in which the single bond
34
of concern cannot rotate. As a result, NAC 8 exhibits weak
fluorescence in THF similarly as NAC 1 or 4 (Table 1); this
indicates that contrary to our initial conjecture, the TICT
excited state is formed via twisting between the carbazole and
nine-position aryl group rather than between the carbazole and
2
NMe group (Figure 11C).
Because of the charge-separated nature, the ICT excited state
exhibits a large dipole moment and is stabilized by polar
solvents. Therefore, the more polar the solvent is, the smaller
the energy gap between S
0 1
and S is, and the more red-shifted
the fluorescence becomes (Figure 7 and Table 2). According to
2
8
the energy gap law, a smaller 
reasonable; however, the observed 
l
in polar solvents is
of NAC 1 in the polar
l
l
solvents is still excessively marginal (e.g.,  is approximately
zero in acetonitrile, Table 2).
This magnified luminescence-quenching phenomenon can be
explained by considering a twisted intramolecular charge
2
9-31
transfer (TICT) excited state.
NAC 1 would acquire the ICT
excited states with the -planes of the D and A units being more
twisted via the single bond rotation (bond a in Figure 11C) than
it is in the ground state. In this TICT excited state, the orbitals
on the D and A units are less conjugated, resulting in the
Figure 10. Electron cloud distribution of HOMO and LUMO of
NAC 1, 3, and 6 calculated using DFT at B3LYP level of theory
employing 6-31G(d,p) basis set in THF modeled by PCM approach.
+

enhanced charge separation (D and A ); this enable further
stabilization of the excited state by polar solvents. The oscillator
1 0
strength of the transition from S to S is marginal owing to less
conjugation of the two SOMOs in the TICT excited state.
Moreover, the contribution of the nonradiative pathway, such
as the energy loss by the interaction with solvent molecules,
becomes dominant owing to the smaller excited energy of the
1
solvent-stabilized S (energy gap law). Overall, NAC 1 in polar
ACS Paragon Plus Environment

Page 7 of 14
The Journal of Organic Chemistry
signal transduction and inflammation.38 Therefore, the
(
a)
(b)
(c)
toluene
d-
THF
d-
CH
d-
3
CN
solid
1
2
3
4
5
6
7
8
9
development of selective detection method for LDs is of great
importance. Nile Red is a traditional fluorescent probe for LDs
but notorious due to a high background noise. Indeed, the low
S/N ratio was also observed in our experiments (Figure 8AC).
In contrast, NAC 1 is non-emissive in polar environments such
as cytoplasm, but becomes fluorescent in lipophilic media such
as LD. Expectedly, LDs can be lit up by NAC 1 with a low
background noise (Figure 8BD).
A
S
1
A
A
A
D
A
S
1
0
A
D
A
23
+
d+
S
1
S
0
d+
d
S
0
S
D
D
D
D
D

TICT contribution
 Dipole moment in S
small
large
1
TICT
major
Stimuli-sensitive turn-on fluorescent probes can be
straightforwardly designed by locating a switchable substituent
on the N-aryl group, which loses or weakens its electron-
withdrawing property upon stimulation. As a proof of concept,
NAC 20 with a formyl group on the 9-phenyl ring was designed
for detecting cysteamine (Figure 9). The formyl group reacts
with cysteamine without a catalyst to form 1,3-thiazoline,
which is electronically neutral. As expected, the resulting 21
minor
strong
blue
Emission
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
weak
red
Figure 11. Rationale for environment-sensitive fluorescence
property of 1.
The fluorescence behavior of NAC 1 in the THF/water mixed
was emissive in CH CN in contrast to 20, and the increase in
the fluorescent intensity of the solution was observed as the
amount of adopted cysteamine increased.
3
solvents with various water fractions (Figure 3) can be
3
5-36
rationalized as follows:
w
In a pure THF solution (f = 0%),
the fluorescence intensity is weak owing to the TICT excited
state; however, they are still visible (
increase in the solvent polarity (e.g., f
l
= 1.5 %). A marginal
= 10%) is adequate to
CONCLUSION
w
In this work, we demonstrated 9-aryl-3-aminocarbazole to be
an environment- and stimuli-sensitive fluorogen. When the
substituent on the 9-aryl group is an electron-withdrawing
group such as the cyan and ester groups, the molecules exhibit
a highly weak or an absence of fluorescence in polar solvents.
This phenomenon could be originating from the TICT excited
state, in which the -planes of the 9-aryl and carbazole units are
more twisted than its ground state. In less polar environments,
the
completely shut down the fluorescence of NAC 1. This
quenching is persistent until f is 90%. A further increase in the
water fraction (f = 99%) results in the precipitation of NAC 2;
w
w
then, the fluorescence derived from the aggregates appears.
LDs are reservoirs of neutral lipids.37 They are mainly found
in adipose tissue but can exist in nearly all cells. Recent studies
have shown that LDs not only function as an energy storage but
also are involved in other physiological events including
membrane formation and maintenance, protein degradation,
solution
solid state
in polar solvent
in less polar solvent
short-WL
R  EWG
short-WL
emissive
(but dependent on
packing mode)
non-emissive
or weak long-WL
R = EWG
short-WL
stimuli sensitive
polarity sensitive
AIE (WL = wavelength)
Figure 12. Summary of environment-responsive nature of 9-aryl-3-aminocarbazole
TICT excited state is less dominant in the S state, and the
1
detection in living cells. The molecule also exhibits AIE
property; it becomes emissive in the aggregated form because it
cannot generate the TICT excited state. When the substituent on
the 9-aryl group is not an electron-withdrawing group, the
molecule is emissive regardless of which solvent is used.
Exploiting the fluorescence variation depending on the 9-aryl
fluorescence intensity increases. Moreover, the fluorescence
becomes blue-shifted owing to the raised energy gap of S and
; this results from the decrease in the polarity-assisted solvent
stabilization of the CT S state. That is, the molecule is endowed
with solvatofluorochromism and was therefore applied to LD
0
S
1
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 14
substituents, we designed the turn-on fluorescent probe for stirred in hexane (200 mL). The suspension was filtrated to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cysteamine detection. The fluorescent behavior of 9-aryl-3-
aminocarbazole is summarized in Figure 12. These multi-mode
fluorescence switchability based on a relatively simple
molecular framework should encourage further development of
bioimaging materials and other luminescence applications.
afford the title product (4.51 g, 71% yield) as a white solid. Mp:
1
162.4–162.7 C; H NMR (400 MHz, CDCl ): d = 8.17 (d, J =
3
1.6 Hz, 2H), 7.52 (dd, J = 8.4, 2.0 Hz, 2H), 7.28–7.22 (m, 5H),
1
3
7.04 (dd, J = 6.4, 2.4 Hz, 2H), 5.46 (s, 2H) ppm; C NMR (100
MHz, CDCl ): d = 139.6, 136.3, 129.4, 129.0, 127.9, 126.3,
3
1
1
1
7
C
23.7, 123.4, 112.6, 110.8, 46.8 ppm; IR (neat):  = 3401, 3049,
694, 1621, 1599, 1574, 1467, 1444, 1332, 1269, 1238, 1200,
133, 1110, 1054, 1018, 1003, 928, 879, 843, 809, 766, 756,
EXPERIMENTAL SECTION
General. Unless otherwise noted, all reactions were carried
out in well cleaned glasswares with magnetic stirring.
Operations were performed under an atmosphere of dry argon
using Schlenk and vacuum techniques, unless otherwise noted.
All starting materials were obtained from commercial sources
or were synthesized using standard procedures. Melting points
were measured on a Yanaco MP-500D and are not corrected.
–1
45, 723, 678 cm ; HRMS (DART): m/z calcd for
79
81
+
19
H
3
14 Br BrN: 415.9473 [M+H ]; found: 415.9446.
,6-Bis(dimethylamino)-9-benzylcarbazole (12) A solution
of Pd (dba) (100 mg, 0.109 mmol, 1.0 mol%) and Ruphos (120
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
mg, 0.257 mmol, 2.4 mol%) in THF (50 mL) was stirred for 30
min. To the solution were added 3,6-dibromo-9-
benzylcarbazole (4.50 g, 10.8 mmol, 1.0 equiv), LiHMDS in n-
hexane (1.17 M, 55.6 mL, 65.0 mmol, 6.0 equiv), THF (50 mL)
and dimethylamine hydrochloride (2.66 g, 32.6 mmol, 3.0
equiv). The mixture was stirred for 5 h at 80 C then cooled to
rt. The reaction was quenched by adding 1 M HCl aq. (65 mL)
then 20 mL of sat. NaHCO
added and the mixture was washed with water three times, dried
over anhydrous Na SO , filtered, and concentrated in vacuo.
The residue was purified by recrystallization in hexane/ethyl
acetate to afford the title product (3.08 g, 83% yield) as a green
solid. Mp: 212.8–213.4 C; H NMR (400 MHz, (CD
=
1
13
H and C NMR spectra (400 and 100 MHz, respectively) were
recorded on a Bruker Avance III HD 400 using TMS (0 ppm),
and CDCl (77.16 ppm) or (CD CO (29.84 ppm) as an internal
3
3 2
)
standard, respectively. The following abbreviations are used in
connection with NMR; s = singlet, d = doublet, t = triplet, q =
quartet, quin = quintet, sep = septet, and m = multiplet. Mass
spectra were measured using a JEOL JMS-T100LP (DART
3
aq. (20 mL). Ethyl acetate was
2
4
method,
ambient
ionization).
Preparative
column
chromatography was performed using Kanto Chemical silica
gel 60 N (spherical, neutral), Fuji Silysia BW-4:10MH silica gel
or YMC_GEL Silica (6 nm I-40-63 um). Thin layer
chromatography (TLC) was carried out on Merck 25 TLC silica
gel 60 F254 aluminium sheets. Single crystal X-ray diffraction
analyses were conducted using Bruker Apex-II diffractometer
equipped with a CCD detector. Powder X-ray diffraction (XRD)
1
3
)
2
CO)): d
7.55 (d, J = 2.0 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.27–7.19
(m, 3H), 7.55 (dd, J = 7.2, 1.6 Hz, 2H), 7.02 (dd, J = 9.0, 2.4
1
3
Hz, 2H), 5.51 (s, 2H), 2.94 (s, 12H) ppm; C NMR (100 MHz,
(CD CO): d =146.2, 139.6, 135.9, 129.3, 127.9, 127.5, 124.4,
115.7, 110.2, 105.5, 46.8, 42.7 ppm; IR (neat):  = 2873, 2797,
3 2
)
patterns were obtained with
a diffractometer (Rigaku,
1
1
7
615, 1572, 1493, 1480, 1450, 1436, 1340, 1327, 1264, 1213,
167, 1142, 1122, 1056, 1029, 965, 949, 903, 842, 823, 793,
79, 751, 737, 721, 695, 650 cm ; HRMS (DART): m/z calcd
SmartLab) using Cu Kα radiation. Steady state
photoluminescence spectra of liquid samples were recorded on
a spectrofluorometer (JASCO FP-8300) with a 1-cm-square
quartz fluorescence cuvette. Steady state photoluminescence
spectra of solid samples were recorded on a spectrofluorometer
–
1
+
for C23
H
26
N
3
: 344.2127 [M+H ]; found: 344.2155.
3
,6-Dibromo-9-(4-methoxybenzyl)carbazole (11) Sodium
hydride (60% in mineral oil, 1.818 g, 45.52 mmol, 1.5 equiv)
was washed using 20 mL of hexane, then the solution of 3,6-
dibromocarbazole (9.625 g, 29.62 mmol, 1.0 equiv) in 60 mL
of dimethylformamide (DMF) was added. To the solution was
added p-methoxybenzylchloride (6.1 mL, 44.4 mmol, 1.5
equiv) and the solution was stirred for 3 h at rt. The reaction
was quenched by adding water (70 mL). To the resultant
mixture was added ethyl acetate and the organic layer was
(
JASCO FP-6600) with a quartz fluorescence cuvette and
integrating sphere (Otsuka Electronics QE-2000). UV–visible
spectra were recorded on a Shimadzu UV-1800 spectrometer
and a JASCO V-770 spectrometer with a 1-cm-square quartz
absorption cuvette (light path: 1 cm). In order to estimate
particle sizes, DLS experiments were performed using Zetasizer
Nano-S (Malvern Instruments, Worcestershire, UK). FE-SEM
images were obtained using a JEOL JSM-7100F thermal field
emission electron microscope instrument operated at 5.0 kV
unless otherwise noted. The specimens for FE-SEM were
prepared by placing a drop of the solution on N-type silicon
wafer (thickness: 0.5 mm, orientation: <1 0 0>, resistivity: 8–
2 4
washed with brine six times, dried over anhydrous Na SO ,
filtered, and concentrated in vacuo. To the residue was added
hexane (100 mL) and the mixture was stirred for 2 h, then
filtered to afford the title product (13.15 g, >99% yield) as a
1
white solid. Mp: 155.7–156.1 C; H NMR (400 MHz, CDCl
3
):
1
2 Ohm cm) and drying under vacuum overnight. AFM images
d = 8.15 (d, J = 8.8 Hz, 2H), 7.51 (dd, J = 8.8, 2.0 Hz, 2H), 7.23
were obtained in dynamic mode using NanoNavi II/IIe and
SPA-400 (Hitachi High-Tech Science Corporation, Tokyo,
Japan) equipped with an OMCL-AC160TS-C3 micro cantilever
(
2
d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 9.6 Hz,
H), 5.39 (s, 2H), 3.74 (s, 3H) ppm; C NMR (100 MHz,
1
3
CDCl
3
): d = 159.2, 139.5, 129.3, 128.3, 127.7, 123.7, 123.4,
(Olympus Corporation, Tokyo, Japan). The specimens for AFM
were prepared by placing a drop of the solution on a cleaved
mica plate and drying under vacuum overnight.
114.4, 112.4, 110.8, 55.4, 46.3 ppm; IR (neat):  = 2934, 2835,
1611, 1587, 1512, 1469, 1448, 1433, 1374, 1334, 1314, 1302,
1289, 1246, 1203, 1176, 1145, 1112, 1059, 1033, 1017, 977,
3,6-Dibromo-9-benzylcarbazole (10) To a solution of 3,6-
dibromocarbazole (5.00 g, 15.4 mmol, 1.0 equiv) and potassium
carbonate (21.3 g, 154 mmol, 10 equiv) in DMF (69 mL) was
added benzyl bromide (3.7 mL, 30.8 mmol, 2.0 equiv) and the
reaction mixture was stirred for 3 h at room temperature (rt).
The reaction was quenched by being poured into 250 mL of ice
water. A mixture was filtered and the solid was triturated and
–
887, 878, 846, 828, 815, 796, 788, 757, 737, 708, 648, 635 cm
1
;
8 9
HRMS (DART): m/z calcd for C H O: 121.0653
+
[(MeO)C
3,6-Bis(dimethylamino)-9-(4-methoxybenzyl)carbazole
(13) A solution of Pd (dba) (273 mg, 0.298 mmol, 1.0 mol%)
6 4 2
H CH ]; found: 121.0629.
2
3
and Ruphos (335 mg, 0.718 mmol, 2.4 mol%) in THF (100 mL)
was stirred for 30 min. To the solution were added 3,6-dibromo-
ACS Paragon Plus Environment

Page 9 of 14
The Journal of Organic Chemistry
-(4-methoxybenzyl)carbazole (13.15 g, 29. 62 mmol, 1.0
9
Methyl 4-[3,6-bis(dimethylamino)carbazol-9-yl]benzoate
(2) A solution of 3,6-bis(dimethylamino)-9H-carbazole (300
mg, 1.18 mmol, 1.0 equiv), methyl 4-iodobenzoate (342 mg,
1.31 mmol, 1.1 equiv), LiCl (52 mg, 1.2 mmol, 1.0 equiv), CuI
(26 mg, 0.14 mmol, 10 mol%), 1,10-phenanthroline (25 mg,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
equiv), LiHMDS (29.72 g, 177.6 mmol, 6.0 equiv), THF (200
mL) and dimethylamine hydrochloride (7.332 g, 89.92 mmol,
3.0 equiv). The mixture was stirred for 28 h at 80 C then cooled
to rt. The reaction was quenched by adding 1 M HCl aq. (178
mL) then sat. NaHCO
ethyl acetate and the mixture was washed with brine six times,
dried over anhydrous Na SO , filtered, and concentrated in
vacuo to afford the title product (10.70 g, 97% yield) as a yellow
3
aq. (178 mL). To the solution was added
0.13 mmol, 10 mol%) and Cs
equiv) in DMF (5.0 mL) was stirred for 12 h at 150 C. After
cooled to rt, the reaction was quenched by adding NH Cl aq.
(17 mL). Ethyl acetate was added and the solution was washed
with brine five times, dried over anhydrous Na SO , filtered and
2 3
CO (464 mg, 1.42 mmol, 1.2
2
4
4
1
solid. Mp: 144.5–145.0 C; H NMR (400 MHz, CDCl
.54 (d, J = 2.4 Hz, 2H), 7.32 (d, 8.8 Hz, 2H), 7.09 (d, J = 8.8
Hz, 2H), 7.02 (dd, J = 8.8, 2.4 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H),
3
): d =
2
4
7
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 1/1) to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
5
.42 (s, 2H), 3.70 (s, 3H), 2.94 (s, 12H) ppm; C NMR (100
MHz, (CD CO): d = 159.8, 146.0, 136.0, 131.3, 128.7, 124.4,
15.7, 114.6, 110.3, 105.5, 55.4, 46.3, 42.7 ppm; IR (neat):  =
afford the title product (294 mg, 64% yield) as a yellow solid.
1
3
)
2
Mp 146.4–146.9 C; H NMR (400 MHz, CDCl
3
): d = 8.23 (dt,
1
J = 8.8, 1.9 Hz, 2H), 7.66 (dt, J = 8.8, 2.1 Hz, 2H), 7.44 (d, J =
2.4 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.01 (dd, J = 9.0, 2.6 Hz,
2795, 1613, 1572, 1499, 1482, 1435, 1327, 1297, 1246, 1214,
1172, 1137, 1109, 1055, 1029, 965, 948, 845, 820, 808, 786,
1
3
2H), 3.97 (s, 3H) 3.03 (s, 12H) ppm; C NMR (100 MHz,
–
1
7
75, 754, 731, 666, 650, 634 cm ; HRMS (DART): m/z calcd
CDCl ): d = 166.7, 146.3, 143.1, 134.2, 131.3, 127.2, 125.3,
3
+
for C24
H
28
N
3
O: 374.2232 [M+H ]; found: 374.2253.
124.9, 115.0, 110.4, 104.5, 52.4, 42.6 ppm; IR (neat):  = 2988,
2937, 2874, 2842, 2791, 1707, 1606, 1573, 1496, 1473, 1433,
1348, 1326, 1277, 1220, 1190, 1173, 1106, 950, 817, 797, 782,
2
0
3
,6-Bis(dimethylamino)-9H-carbazole (14) To a solution
of 3,6-dimethylamino-9-(4-methoxybenzyl)carbazole (100 mg,
.268 mmol, 1.0 equiv) in CH Cl (2.1 mL) was dropwise added
over 5 minutes a solution of 1M BCl in heptane (1.61 mL, 1.61
–
1
0
2
2
769, 703, 584 cm ; HRMS (DART): m/z calcd for C24
H
26
N
3
O
2
:
+
3
388.2025 [M+H ]; found: 388.2010.
mmol, 6.0 equiv) at –78 C and the mixture was stirred for 30
min, then stirred for 1 h at –20 C. The solution was stirred for
3,6-Bis(dimethylamino)-9-phenylcarbazole (3) Asolution of
3,6-bis(dimethylamino)-9H-carbazole (300 mg, 1.18 mmol, 1.0
equiv), bromobenzene (1.24 mL, 11.8 mmol, 10 equiv), CuI
(237 mg, 1.24 mmol, 1.05 equiv), K CO (639 mg, 4.62 mmol,
2 3
3.9 equiv) in dimethyl acetoamide (DMA, 6 mL) was stirred for
9
0 h at rt with protection from light. The reaction was quenched
by adding methanol (5.2 mL), and concentrated in vacuo. To the
solid residue was added 4 M HCl aq. (9 mL) and the mixture
was stirred at 70 C. The resultant mixture was cooled to rt. 4
M NaOH aq. (20 mL) was added to basify the mixture. The
18 h at 180 C with protection from light. The reaction was
quenched by being poured into water (100 mL). Conc. NH
was added and the mixture was extracted with CH Cl three
times. The organic layer was washed with brine three times,
dried over anhydrous Na SO , filtered. The filtrate was
3
aq.
resultant mixture was extracted with CH
organic layer was dried over anhydrous Na
concentrated in vacuo. The residue was purified by column
chromatography on NH -silica gel (hexane/ ethyl acetate = 1/ 1)
2
Cl
2
three times. The
2
2
2
SO , filtered,
4
2
4
concentrated in vacuo and purified by column chromatography
on silica gel (hexane/ethyl acetate = 1/1) to give the product
2
to afford the title product (58 mg, 86% yield) as a green yellow
solid.
1
(295 mg, 76% yield) as a brown oil. H NMR (400 MHz,
3
,6-Bis(dimethylamino)-9-(4-cyanophenyl)carbazole (1) A
solution of 3,6-bis(dimethylamino)-9H-carbazole (285 mg,
.13 mmol, 1.0 equiv), 4-iodobenzonitrile (285 mg, 1.24 mmol,
CDCl
3
): d = 7.57 (d, J = 4.4 Hz, 4H), 7.51 (d, J = 2.4 Hz, 2H),
7.42–7.40 (m, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.04 (dd, J = 9.2,
2.4 Hz, 2H), 3.04 (s, 12H) ppm; C NMR (100 MHz, CDCl ):
3
1
3
1
1.1 equiv), LiCl (48 mg, 1.1 mmol, 1.0 equiv), CuI (23 mg, 0.12
mmol, 10 mol%), 1,10-phenanthroline monohydrate (23 mg,
d = 145.8, 138.7, 135.3, 129.8, 126.5, 126.5, 124.2, 115.3, 110.3,
104.8, 42.9 ppm; IR (neat):  = 2842, 2794, 1618, 1591, 1573,
1486, 1475, 1435, 1354, 1325, 1280, 1220, 1165, 1148, 1110,
1079, 1056, 1024, 968, 949, 908, 894, 860, 836, 816, 795, 783,
0.12 mmol, 10 mol%) and Cs
equiv) in DMF (5.0 mL) was stirred for 19 h at 150 C. After
cooled to rt, the reaction was quenched by adding NH Cl aq.
17 mL). Ethyl acetate was added and the mixture was washed
with brine seven times, dried over anhydrous Na SO , filtered
2 3
CO (485 mg, 1.49 mmol, 1.3
–
1
4
761, 739, 715, 700, 666, 659, 631, 616 cm ; HRMS (DART):
+
(
24 3
m/z calcd for C22H N : 330.1970 [M+H ]; found: 330.1997.
3
9
2
4
3-Bromo-9H-carbazole (15) A solution of carbazole (1.00 g,
6.00 mmol, 1.0 equiv) in THF (12 mL) was cooled in ice bath.
To the mixture was added N-bromosuccinimide (1.07 g, 6.00
mmol, 1.0 equiv) slowly with protection from light. After
stirring for 30 min at 0 C, the solution was stirred for 17 h at
rt. The reaction was quenched by adding sat. Na SO aq. (10
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 3/1) to
afford the title product (258 mg, 65% yield) as a yellow solid.
Single crystals of 1 suitable for X-ray diffraction analysis were
obtained by recrystallization from hexane/1,2-dichloroethane
2
3
1
by vapor diffusion. Mp: 254.8–255.2 C. H NMR (400 MHz,
mL) and the solution was concentrated in vacuo to remove THF.
Ethyl acetate was added and the solution was washed with brine
four times, dried over anhydrous Na SO , filtrate and
2 4
concentrated in vacuo to afford the title product (1.31 g, 89%
yield) as a white solid.
CDCl
3
): d = 7.83 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H),
7.41 (s, 2H), 7.39 (d, J = 9,0 Hz, 2H), 6.99 (dd, J = 9.0, 2.4 Hz,
2H), 3.04 (s, 12H) ppm; C NMR (100 MHz, CDCl
143.2, 133.8, 133.7, 125.8, 125.3, 118.9, 114.8, 110.3, 108.6,
1
3
3
): d = 146.6,
3
-Dimethylamino-9H-carbazole (16) A solution of Pd
2 3
(dba)
1
1
04.4, 42.5 ppm; IR(neat):  = 2840, 2792, 2216, 1603, 1578,
512, 1498, 1471, 1435, 1372, 1329, 1218, 1171, 1148, 1132,
(11 mg, 12 μmol, 1.0 mol%) and Ruphos (14 mg, 30 μmol, 2.4
mol%) in THF (13.2 mL) was stirred. To the solution were
added 3-bromo-9H-carbazole (300 mg, 1.22 mmol, 1.0 equiv),
LiHMDS (1.22 g, 7.31 mmol, 6.0 equiv) and dimethylamine
hydrochloride (297 mg, 3.64 mmol, 3.0 equiv). The mixture
was stirred for 65 h at 120 C then cooled to rt. The reaction
1
6
106, 1061, 968, 947, 910, 858, 826, 798, 748, 716, 661, 643,
–
1
23 4
02 cm . HRMS(DART) = m/z calcd for C23H N : 355.1923
+
[
M+H ]; found: 355.1904.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 14
was quenched by adding 1 M HCl aq. (7.3 mL) then sat.
NaHCO aq. (7.3 mL). Ethyl acetate was added and the solution
was washed with brine three times, dried over anhydrous
Na SO , filtered, concentrated in vacuo. Another two sets of
reactions were conducted under the same conditions. The
obtained crude materials were combined and purified by
column chromatography on silica gel (hexane/ ethyl acetate) to
(100 MHz, CDCl ): d = 146.3, 141.2, 138.3, 134.8, 129.9, 127.0,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
126.9, 125.8, 124.1, 123.5, 120.3, 119.4, 115.3, 110.3, 109.8,
104.7, 42.7 ppm; IR (neat):  = 2788, 1673, 1594, 1492, 1451,
2
4
1438, 1363, 1326, 1228, 1134, 1103, 1074, 1025, 953, 926, 835,
–
1
794, 759, 741, 697, 684, 643, 619 cm ; HRMS (DART): m/z
+
calcd for C H N : 287.1548 [M+H ]; found: 287.1548.
20 19
2
4
0
9-(4-Cyanophenyl)carbazole (6)
A solution of 9H-
afford the title product (563 mg, 73% yield) as a green yellow
carbazole (837 mg, 5.00 mmol, 1.0 equiv), 4-iodobenzonitrile
(1.260 g, 5.50 mmol, 1.1 equiv), LiCl (212 mg, 5.50 mmol, 1.0
equiv), CuI (96 mg, 0.50 mmol, 10 mol%), 1,10-phenanthroline
monohydrate (99 mg, 0.50 mmol, 10 mol%) and Cs CO (2.120
1
solid. Mp: 114.7–115.0 C; H NMR (400 MHz, (CD
3
)
2
CO): d
=
9.97 (s, 1H), 8.07 (dd, J = 7.8, 1.2 Hz, 1H), 7.54 (d, J = 2.4
Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33
td, J = 7.4, 1.2 Hz, 1H), 7.11 (td, J = 7.4, 0.8 Hz, 1H), 7.06 (dd,
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
g, 6.51 mmol, 1.3 equiv) in DMF (20.5 mL) was stirred for 22
1
3
J = 8.8, 2.4 Hz, 1H), 2.97 (s, 6H) ppm; C NMR (100 MHz,
h at 150 C. The reaction was quenched by adding sat. NH Cl
4
(CD CO): d = 146.5, 141.6, 134.6, 125.9, 124.6, 124.1, 120.8,
3
)
2
aq. (20.5 mL). Ethyl acetate was added and the organic layer
118.8, 116.0, 111.9, 111.5, 105.0, 42.6 ppm; IR (neat):  = 3025,
2948, 2863, 2835, 2788, 1609, 1585, 1496, 1484, 1471, 1450,
1435, 1328, 1306, 1276, 1241, 1184, 1158, 1135, 1105, 1045,
was washed with brine five times and sat. NH
4
Cl aq. four times,
dried over anhydrous Na SO , filtered and concentrated in
2
4
vacuo to afford the title product (1.329 g, 99% yield) as a white
solid.
1
6
2
019, 1007, 946, 934, 888, 870, 848, 804, 767, 751, 735, 714,
–
1
37, 613 cm ; HRMS (DART): m/z calcd for C14
H
15
N
2
:
3,6-Dimethoxy-9-(4-cyanophenyl)carbazole (7) A solution
of 3,6-dimethoxy-9H-carbazole (455 mg, 2.00 mmol, 1.0 equiv),
4-iodobenzonitrile (504 mg, 2.20 mmol, 1.1 equiv), CuI (38 mg,
0.20 mmol, 10 mol%), LiCl (85 mg, 2.00 mmol, 1.0 equiv),
1,10-phenanthroline monohydrate (40 mg, 0.20 mmol, 10
+
11.1235 [M+H ]; found: 211.1242.
3
-Dimethylamino-9-(4-cyanophenyl)carbazole
solution of 3-dimethylamino-9H-carbazole (250 mg, 1.19 mmol,
.0 equiv), 4-iodobenzonitrile (300 mg, 1.31 mmol, 1.1 equiv),
LiCl (50 mg, 1.18 mmol, 1.0 equiv), CuI (23 mg, 0.12 mmol,
0 mol%), 1,10-phenanthroline monohydrate (24 mg, 0.12
mmol, 10 mol%) and Cs CO (504 mg, 1.55 mmol, 1.3 equiv)
in DMF (5.3 mL) was stirred for 41 h at 150 C. After cooled to
rt, the reaction was quenched by adding NH Cl aq. (17 mL).
Ethyl acetate was added and the solution was washed with brine
five times, dried over anhydrous Na SO , filtered and
(4)
A
1
2 3
mol%) and Cs CO (847 mg, 2.60 mmol, 1.3 equiv) in DMF
1
(8.2 mL) was stirred for 21 h at 150 C. The reaction was
2
3
quenched by adding sat. NH Cl aq. (8.2 mL). Ethyl acetate was
4
added and the organic layer was washed with sat. NH
four times and brine three times, dried over anhydrous Na
4
Cl aq.
SO
4
2
4
,
filtered and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate =
5/1) to afford the title product (508 mg, 77% yield) as a white
2
4
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 3/1) to
1
solid. Mp: 173.6–173.8 C; H NMR (400 MHz, CDCl ): d =
3
afford the title product (247 mg, 67% yield) as a yellow solid.
7.87 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.54 (d, J =
2.4 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.05 (dd, J = 8.8, 2.4 Hz,
1
Mp: 134.3–135.2 C; H NMR (400 MHz, CDCl
3
): d = 8.09 (d,
1
3
J = 7.4 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz,
2H), 3.95 (s, 6H) ppm; C NMR (100 MHz, CDCl ): d = 154.8,
3
2
7
2
H), 7.47 (d, J = 2.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.42–
142.6, 135.1, 134.0, 126.4, 124.6, 118.6, 115.5, 110.7, 109.7,
.36 (m, 2H), 7.28 (dt, J = 1.2, 7.4 Hz, 1H), 7.02 (dd, J = 8.8,
103.2, 56.1 ppm; IR(neat):  = 2988, 2953, 2933, 2828, 2226,
1626, 1601, 1506, 1488, 1462, 1449, 1428, 1371, 1328, 1291,
1263, 1202, 1184, 1175, 1160, 1111, 1040, 1028, 957, 943, 910,
13
.4 Hz, 1H), 3.04 (s, 6H) ppm; C NMR (100 MHz, CDCl
3
): d
= 146.9, 142.7, 140.2, 133.9, 133.3, 126.5, 126.2, 125.0, 124.4,
120.6, 118.7, 114.8, 110.2, 109.6, 104.2, 42.3 ppm; IR (neat): 
= 3048, 2840, 2796, 2222, 1667, 1626, 1599, 1497, 1454, 1440,
1367, 1328, 1296, 1225, 1173, 1151, 1132, 1104, 1067, 1028,
–
1
849, 810, 824, 793, 749, 726, 663, 641, 605 cm ;
+
HRMS(DART) = m/z calcd for C21
found: 329.1308.
17 2 2
H N O : 329.1290 [M+H ];
–
1
9
55, 914, 842, 831, 795, 761, 744, 735, 665, 631, 620 cm ;
3-Nitro-9-(4-cyanophenyl)carbazole (17) A solution of 9-(4-
cyanophenyl)carbazole (531 mg, 2.00 mmol, 1.0 equiv) in
AcOH (3.5 mL) was stirred. To the solution was added nitric
acid (1.50 mL, 33.8 mmol, 17 equiv). The mixture was stirred
for 1 hour at rt. The mixture was cooled in ice bath and the
suspension was filtrated, washed with ice-cooled distilled water
thirty times. The remaining water of the obtained solids was
removed with papers to give the crude material (528 mg, 72%).
+
HRMS (DART): m/z calcd for C21
found: 312.1523.
18 3
H N : 312.1501 [M+H ];
3
-Dimethylamino-9-phenylcarbazole (5) A solution of 3-
dimethylamino-9-carbazole (200 mg, 0.95 mmol, 1.0 equiv),
bromobenzene (1.00 mL, 9.55 mmol, 10 equiv), CuI (195 mg,
1
2 3
.02 mmol, 1.07 equiv), K CO (513 mg, 3.71 mmol, 3.9 equiv)
in DMA (4.8 mL) was stirred for 18 h at 180 C with protection
from light. After cooled to rt, the reaction was quenched by
1
The yield was determined by H NMR analysis with durene as
1
being poured into water (240 mL). Conc. NH
and the solution was extracted with CH Cl
organic layer was concentrated in vacuo in order to remove
CH Cl . Ethyl acetate was added and the organic layer was
washed with brine five times, dried over anhydrous Na SO
3
aq. was added
an internal standard. Mp: 256.2–256.6 C; H NMR (400 MHz,
2
2
five times. The
CDCl ): d = 9.07 (d, J = 2.4 Hz, 1H), 8.35 (dd, J = 8.8 Hz, 1H),
3
8.23 (dd, J = 7.2, 1.6 Hz, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.75 (d,
J = 8.8 Hz, 2H), 7.56 (dt, J = 1.2, 8.0 Hz, 1H), 7.47–7.42 (m,
2
2
1
3
2
4
,
3H) ppm; C NMR (100 MHz, CDCl ): d = 143.2, 142.2, 141.5,
3
filtered. The filtrate was concentrated in vacuo and purified by
140.7, 134.4, 128.2, 127.7, 123.9, 123.5, 122.6, 122.3, 121.4,
column chromatography on silica gel (hexane/ethyl acetate =
1
1
18.0, 117.5, 112.3, 110.5, 109.5 ppm; IR (neat):  = 3085, 2227,
601, 1584, 1505, 1471, 1448, 1334, 1286, 1270, 1242, 1224,
1186, 1166, 1113, 1088, 1022, 915, 893, 860, 847, 835, 817,
1
1
0/1) to give the product (218 mg, 80% yield). H NMR (400
MHz, CDCl
3
): d = 8.11 (d, J = 7.6 Hz, 1H), 7.61–7.56 (m, 4H),
.53 (d, J = 2.4 Hz, 1H), 7.45–7.40 (m, 2H), 7.38 (td, J = 6.8,
.2 Hz, 1H), 7.35 (d, J = 6.8 Hz, 1H), 7.24 (td, J = 7.4, 1.2 Hz,
–
1
7
1
1
788, 767, 746, 727, 718, 703, 658, 636, 623 cm ; HRMS
(DART): m/z calcd for C H N O : 314.0930 [M+H ]; found:
+
1
9
12
3
2
1
3
H), 7.05 (dd, J = 2.4, 8.8 Hz, 1H), 3.03 (s, 6H) ppm; C NMR
314.0943.
ACS Paragon Plus Environment

Page 11 of 14
The Journal of Organic Chemistry
-Amino-9-(4-cyanophenyl)carbazole (18) A mixture of
3
NMR (400 MHz, CDCl ): d = 10.06 (s, 1H), 8.07 (dt, J = 8.4,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the crude material of 3-nitro-9-(4-cyanophenyl)carbazole
obtained above (366 mg, purity 86%, 1.0 mmol, 1.0 equiv) and
10wt% Pd/C (106 mg, 0.10 mmol, 10 mol%) in MeOH (10 mL)
was stirred under hydrogen gas (1 atm). After 7 h, the reactant
mixture was filtrated on celite. After the concentration of the
filtrate, the residue was purified by chromatography on silica
gel (hexane/ ethyl acetate = 1/ 3) to afford the title product as a
2.0 Hz, 2H), 7.77 (dt, J = 8.8, 2.0 Hz, 2H), 7.46 (d, J = 9.2 Hz,
2H), 7.43 (d, J = 2.4 Hz, 2H), 7.00 (dd, J = 9.2, 2.4 Hz, 2H),
1
3
3.04 (s, 12H) ppm; C NMR (100 MHz, CDCl ): d = 191.1,
3
146.5, 144.6, 133.9, 133.4, 131.5, 125.5, 125.2, 114.8, 110.5,
104.4, 42.5 ppm; IR (neat):  = 2842, 2799, 1679, 1596, 1578,
1
1
7
558, 1511, 1495, 1472, 1435, 1391, 1368, 1343, 1323, 1302,
213, 1169, 1149, 1105, 1063, 965, 949, 909, 860, 831, 799,
1
–
1
white solid (93 mg, 33%). Mp: 176.0–176.7 C; H NMR (400
87, 744, 727, 719, 677, 658, 631 cm ; HRMS (DART): m/z
+
MHz, CDCl
3
): d = 8.03 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 8.8 Hz,
23 24 3
calcd for C H N O: 358.1919 [M+H ]; found: 358.1949.
2
H), 7.70 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.43 (d,
3,6-Bis(dimethylamino)-9-[4-(1,3-thiazolidin-2-yl)phenyl]-
9H-carbazole (21) A solution of 3,6-bis(dimethylamino)-9-(4-
formylphenyl)carbazole (71.5 mg, 0.200 mmol, 1.0 equiv) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J = 2.0 Hz, 1H), 7.39 (td, J = 8.4, 1.2 Hz, 1H), 7.29 (d, J = 6.0
Hz, 1H), 7.27 (dt, J = 1.2, 7.2 Hz, 1H), 6.85 (dd, J = 8.6, 2.4 Hz,
1
3
1
H), 3.70 (s, 2H) ppm; C NMR (100 MHz, CDCl
3
): d = 142.5,
3
cysteamine (154 mg, 2.00 mmol, 10 equiv) in CH CN (8 mL)
141.0, 140.2, 134.0, 133.9, 126.6, 126.3, 125.1, 123.9, 120.6,
120.6, 118.7, 115.8, 110.4, 109.7, 109.6, 106.0 ppm; IR (neat):
 = 3384, 3197, 2223, 1626, 1599, 1511, 1491, 1454, 1361,
was stirred at rt for 20 h then at 40 C for 29 h. The reaction was
cooled to rt and quenched by adding brine and ethyl acetate.
After concentration in vacuo, ethyl acetate was added and the
mixture was washed by brine three times, dried over anhydrous
Na SO , filtered and concentrated in vacuo. The residue was
2 4
purified by column chromatography on silica gel (hexane/ethyl
1
8
338, 1317, 1235, 1207, 1169, 1025, 936, 911, 884, 864, 856,
–
1
26, 806, 765, 742, 718, 700, 646, 632, 609, 600 cm ; HRMS
+
(DART): m/z calcd for C19
H N
14 3
: 284.1188 [M+H ]; found:
2
84.1202.
-(2,3,6,7-Tetrahydro-1H-quinolizino[1,9-bc]carbazol-
(5H)-yl)benzonitrile (8) mixture of 3-amino-9-(4-
acetate = 1/3) to afford the title product (33 mg, 40% yield) as
1
4
yellow oil. H NMR (400 MHz, CDCl
3
): d = 7.68 (d, J = 8.4 Hz,
9
A
2H), 7.54 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 2.4 Hz, 2H), 7.35 (d,
J = 8.8 Hz, 2H), 7.01 (dd, J = 8.4, 2.4 Hz, 2H), 5.67 (s, 1H),
3.70–3.61 (m, 1H), 3.23–3.13 (m, 3H), 3.02 (s, 12H), 2.04 (s,
cyanophenyl)carbazole (57 mg, 0.20 mmol, 1.0 equiv) and
Na CO3 (85 mg, 0.80 mmol, 4.0 equiv) in 1-bromo-3-
2
13
chloropentane (0.3 mL, 3.05 mmol, 15 equiv) was stirred for 1
1H) ppm; C NMR (100 MHz, CDCl ): d = 145.9, 138.6, 138.2,
3
h at 70 C, then for 24 h at 100 C. The reaction was cooled to
135.1, 128.7, 126.3, 124.3, 115.3, 110.3, 104.7, 73.1, 53.0, 42.8,
36.8 ppm; IR (neat):  = 2957, 2925, 2855, 1726, 1700, 1598,
1489, 1466, 1369, 1259, 1231, 1189, 1162, 1107, 1070, 1025,
rt. To the reactant mixture were added CH
mixture was extracted with CH Cl three times, dried over
anhydrous Na SO , filtered. The filtrate was concentrated in
2 2
Cl and water and the
2
2
-
1
2
4
960, 796, 753, 688 cm ; HRMS (DART): m/z calcd for
+
vacuo. To the crude material was added DMF (2 mL). The
mixture was stirred for 90 h at 160 C. The reaction was cooled
to rt. Ethyl acetate was added and the mixture was washed with
23 24 3 1
C H N O : 358.1919 [M+H ]; found: 358.1930. In the mass
analysis only the ion peaks derived from compound 20 were
observed instead of those of 21, probably owing to the rapid
hydrolysis under the ionization conditions.
Fluorescence spectra of 1 in the THF/water mixed solvents
with various water fraction (Figure 3) Samples for
fluorescence measurements in the THF/water mixed solvents
were made as follows. In all cases the concentration of 1 was
10 M. (a) 1 mM THF solution of 1 (A) 0.03 mL + THF 2.97
2 4
brine eight times, dried over anhydrous Na SO , filtered and
concentrated in vacuo. The residue was purified by
chromatography on silica gel to afford the title product as a
1
yellow amorphous (19 mg, 26% yield). H NMR (400 MHz,
CDCl
3
): d = 8.13 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H),
7
.69 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.34 (t, J =
7.2 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 6.97 (s, 1H), 3.40 (t, J =
6.8 Hz, 2H), 3.19–3.12 (m, 4H), 2.94 (t, J = 6.8 Hz, 2H), 2.23
mL (푓 = 0) (b) A 0.03 mL + THF 2.67 mL + water 0.3 mL
w
(푓 = 10) (c) A 0.03 mL + THF 2.07 mL + water 0.9 mL (푓 =
w
w
1
3
(quin, J = 6.8 Hz, 2H), 2.06 (quin, J = 6.4 Hz, 2H) ppm;
NMR (100 MHz, CDCl ): d = 142.7, 140.2, 139.1, 133.9, 133.1,
27.2, 125.1, 123.5, 123.1, 121.1, 120.3, 118.7, 117.2, 109.8,
C
30) (d) A 0.03 mL + THF 1.47 mL + water 1.5 mL (푓 = 50)
w
3
(e) A 0.03 mL + THF 0.87 mL + water 2.1 mL (푓 = 70) (f) A
w
1
1

1
1
9
6
3
0.03 mL + THF 0.27 mL + water 2.7 mL (푓 = 90) (g) A 0.03
w
09.1, 107.4, 51.2, 50.5, 29.0, 26.1, 22.7, 22.6 ppm; IR (neat):
= 3053, 2929, 2837, 2221, 1599, 1507, 1487, 1479, 1466,
450, 1437, 1425, 1398, 1366, 1327, 1315, 1293, 1270, 1241,
215, 1201, 1186, 1175, 1155, 1117, 1086, 1070, 1055, 1028,
mL + water 2.97 mL (푓 = 99). The sample preparation and
w
fluorescence measurements were conducted under ambient
atmosphere. Excitation light wavelength: 335 nm.
Solid sample preparation of 1 for comparison of as
prepared microcrystal and ground powder (Figure 5) The
microcrystal of NAC 1 was prepared according to the following
recrystallization procedure. After purification by column
chromatography, NAC 1 (258 mg) was dissolved in ethyl
acetate (43 mL) and hexane (75 mL) at 60 C, then the solution
was cooled to –20 C and left for overnight. The mixture was
filtered and the solids were washed by the hexane/ethyl acetate
(2/1) mixed solvents (10 mL) to afford the microcrystals of
NAC 1 (174 mg). The powder material of NAC 1 was prepared
by grinding the microcrystals with a pestle and mortar.
59, 929, 916, 886, 858, 843, 836, 795, 769, 743, 726, 682, 666,
–1
54, 631, 618 cm ; HRMS (DART): m/z calcd for C25
H
22
N
3
:
+
64.1814 [M+H ]; found: 364.1805.
3
,6-Bis(dimethylamino)-9-(4-formylphenyl)carbazole (20)
A solution of 3,6-bis(dimethylamino)-9H-carbazole (900 mg,
3.55 mmol, 1.0 equiv), 4-iodobenzaldehyde (907 mg, 3.91
mmol, 1.1 equiv), LiCl (157 mg, 4.28 mmol, 1.0 equiv), CuI
(69.2 mg, 0.363 mmol, 10 mol%), 1,10-phenanthroline
monohydrate (70.7 mg, 0.357 mmol, 10 mol%) and Cs
2
CO
3
(1.34 g, 4.28 mmol, 1.2 equiv) in DMF (15 mL) was stirred for
5
h at 150 C. The reaction was quenched by adding sat. NH
4
Cl
Fluorescence spectra of 1 in the THF/hexane mixed
solvents with various hexane fraction (Figure 7) Samples for
fluorescence measurements in the THF/hexane mixed solvents
were made as follows. In all cases the concentration of 1 was
aq. (51 mL). Ethyl acetate was added and the mixture was
washed with brine six times, dried over anhydrous Na SO
filtered and concentrated in vacuo to afford the title product
2
4
,
1
(
1.223 g, 96% yield) as a red solid. Mp: 192.3–193.3 C; H
ACS Paragon Plus Environment

The Journal of Organic Chemistry
0 M. (a) 0.5 mM THF solution of 1 (B) 0.06 mL + THF 2.94
Page 12 of 14
1
(5) Han, J.; Burgess, K., Fluorescent Indicators for Intracellular pH.
Chem. Rev. 2010, 110, 2709-2728.
6) Förster, T.; Kasper, K., Ein Konzentrationsumschlag der
Fluoreszenz. Phys. Chem. (Muenchen, Ger.) 1954, 1, 275-277.
7) Birks, J. B., Photophysics of Aromatic Molecules. Wiley: New
York, 1970.
8) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang,
B. Z., Aggregation-Induced Emission: Together We Shine, United
We Soar! Chem. Rev. 2015, 115, 11718-11940.
(9) He, Z.; Ke, C.; Tang, B. Z., Journey of Aggregation-Induced
Emission Research. ACS Omega 2018, 3, 3267-3277.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mL (푓 = 0) (b) B 0.06 mL + THF 2.64 mL + hexane 0.3 mL
h
(
(푓 = 10) (c) B 0.06 mL + THF 2.04 mL + hexane 0.9 mL (푓 =
h
h
30) (d) B 0.06 mL + THF 1.44 mL + hexane 1.5 mL (푓 = 50)
h
(
(e) B 0.06 mL + THF 0.84 mL + hexane 2.1 mL (푓 = 70) (f) B
h
0
.06 mL + THF 0.24 mL + hexane 2.7 mL (푓 = 90) (g) B 0.06
h
(
mL + hexane 2.94 mL (푓 = 98). The sample preparation and
h
fluorescence measurements were conducted under ambient
atmosphere. Excitation light wavelength: 335 nm.
Lipid droplet detection with Nile Red and NAC 1 (Figure 8)
HeLa cells (Riken BioResource Center) were cultured in high
glucose DME medium (Sigma-Aldrich) containing 10 %(v/v)
FBS, 1 %(v/v) Penicillin-Streptomycin (P/S) (Sigma-Aldrich)
(10) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z., Aggregation-
induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem.
Commun. 2001, 1740-1741.
(11) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z.,
Aggregation‐Induced Emission: The Whole Is More Brilliant than
the Parts. Adv. Mater. 2014, 26, 5429-5479.
2 2
at 37 C in a CO incubator (5% CO ). Then, the HeLa cells
5
were seeded at a density of 2  10 cells/mL in DME medium
in a 35 mm glass bottom dish. After 24 hours of culture, to
increase triacylglycerol synthesis and storage in cells, 62.5 μM
oleic acid complexed to albumin (OA/BSA) was added to the
medium and incubated overnight. To visualize LDs using dyes,
the cells were incubated with 30-300 M Nile Red and/or NAC
(
12) Kang, X.; Wang, S.; Zhu, M., Observation of a new type of
aggregation-induced emission in nanoclusters. Chem. Sci. 2018, 9,
062-3068.
13) Zheng, Z.; Zhang, T.; Liu, H.; Chen, Y.; Kwok, R. T. K.; Ma,
3
(
1
2 2
for 15 min in a CO incubator (5 % CO ) at 37 C. After the
C.; Zhang, P.; Sung, H. H. Y.; Williams, I. D.; Lam, J. W. Y.; Wong,
K. S.; Tang, B. Z., Bright Near-Infrared Aggregation-Induced
Emission Luminogens with Strong Two-Photon Absorption,
Excellent Organelle Specificity, and Efficient Photodynamic
Therapy Potential. ACS Nano 2018, 12, 8145-8159.
(14) Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence,
D. S., Real Time Visualization of Protein Kinase Activity in Living
Cells. J. Biol. Chem. 2002, 277, 11527-11532.
incubation, the cells were washed thrice with PBS buffer. The
fluorescence derived from Nile Red or NAC 1 was then
analyzed using
a fluorescence microscope (BZ-X700;
KEYENCE) equipped with BZ-X filter TRITC or DAPI and a
4
0x objective lens with the exposure time indicated.
Fluorescence measurements of NAC 20 in the presence of
cysteamine (Figure 9) Samples were made by adding the
designated amount of 1 M aqueous solution of cysteamine to 25
(15) Parisio, G.; Marini, A.; Biancardi, A.; Ferrarini, A.; Mennucci,
B., Polarity-Sensitive Fluorescent Probes in Lipid Bilayers:
Bridging Spectroscopic Behavior and Microenvironment
Properties. J. Phys. Chem. B 2011, 115, 9980-9989.
mM CH CN solution of NAC 20 (1 mL) and stirring at 40 C
3
for 12 h. The amount of added cysteamine solution is as
follows: (a) 2.5 L (0.1 equiv) (b) 5.0 L (0.2 equiv) (c) 7.5 L
(16) Toutchkine, A.; Kraynov, V.; Hahn, K., Solvent-Sensitive Dyes
(0.3 equiv) (d) 10 L (0.4 equiv) (e) 12.5 L (0.5 equiv) (f)
to Report Protein Conformational Changes in Living Cells. J. Am.
Chem. Soc. 2003, 125, 4132-4145.
(17) Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T., Design and
Synthesis of a Library of BODIPY-Based Environmental Polarity
Sensors Utilizing Photoinduced Electron-Transfer-Controlled
Fluorescence ON/OFF Switching. J. Am. Chem. Soc. 2007, 129,
1
8.75 L (0.75 equiv) (g) 25 L (1.0 equiv). Each sample was
diluted to 10 M and subjected to fluorescence measurements.
Excitation light wavelength: 335 nm, Slit width: 2.5 nm/2.5 nm,
Sensibility: high, Under ambient atmosphere.
5
597-5604.
ASSOCIATED CONTENT
Supporting Information.
(18) Duan, X.; Li, P.; Li, P.; Xie, T.; Yu, F.; Tang, B., The synthesis
of polarity-sensitive fluorescent dyes based on the BODIPY
chromophore. Dyes Pigments 2011, 89, 217-222.
(19) Klymchenko, A. S., Solvatochromic and Fluorogenic Dyes as
Environment-Sensitive Probes: Design and Biological
Applications. Acc. Chem. Res. 2017, 50, 366-375.
CIF file for NAC 1, absorption and fluorescence spectra,
experimental procedure for quantum yield determination, FE-SEM
and AFM images, calculation details, X-ray crystallographic details
1
13
and H and C NMR spectra (PDF)
(20) Matsubara, R.; Yabuta, T.; Md Idros, U.; Hayashi, M.; Ema,
This material is available free of charge via the Internet at
http://pubs.acs.org.
F.; Kobori, Y.; Sakata, K., UVA- and Visible-Light-Mediated
Generation of Carbon Radicals from Organochlorides Using
Nonmetal Photocatalyst. J. Org. Chem. 2018, 83, 9381-9390.
(21) Greenspan, P.; Mayer, E. P.; Fowler, S. D., Nile red: a selective
fluorescent stain for intracellular lipid droplets. J. Cell Biol. 1985,
REFERENCES
(1) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E., Signaling
Recognition Events with Fluorescent Sensors and Switches. Chem.
Rev. 1997, 97, 1515-1566.
(2) Callan, J. F.; de Silva, A. P.; Magri, D. C., Luminescent sensors
and switches in the early 21st century. Tetrahedron 2005, 61, 8551-
1
00, 965-973.
(22) Smirnova, E.; Goldberg, E. B.; Makarova, K. S.; Lin, L.;
Brown, W. J.; Jackson, C. L., ATGL has a key role in lipid
droplet/adiposome degradation in mammalian cells. EMBO reports
2
006, 7, 106-113.
8
(
588.
3) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V.,
Teaching Old Indicators New Tricks. Acc. Chem. Res. 2001, 34,
63-972.
4) Klymchenko, A. S.; Mely, Y., Chapter Two - Fluorescent
(23) Fam, T. K.; Klymchenko, A. S.; Collot, M., Recent Advances
in Fluorescent Probes for Lipid Droplets. Materials (Basel,
Switzerland) 2018, 11, 1768.
9
(
(24) Hongji, J.; Jian, S.; Jinlong, Z., A Review on Synthesis of
Carbazole-based Chromophores as Organic Light-emitting
Materials. Curr. Org. Chem. 2012, 16, 2014-2025.
Environment-Sensitive Dyes as Reporters of Biomolecular
Interactions. In Prog. Mol. Biol. Transl. Sci., Morris, M. C., Ed.
Academic Press: 2013; Vol. 113, pp 35-58.
(25) Xu, S.; Liu, T.; Mu, Y.; Wang, Y.-F.; Chi, Z.; Lo, C.-C.; Liu,
S.; Zhang, Y.; Lien, A.; Xu, J., An Organic Molecule with
ACS Paragon Plus Environment

Page 13 of 14
The Journal of Organic Chemistry
Asymmetric Structure Exhibiting Aggregation-Induced Emission,
Cabrera, E.; Tang, B. Z., Twisted Intramolecular Charge Transfer
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Delayed Fluorescence, and Mechanoluminescence. Angew. Chem.
Int. Ed. 2015, 54, 874-878.
and Aggregation-Induced Emission of BODIPY Derivatives. J.
Phys. Chem. C 2009, 113, 15845-15853.
(37) Murphy, D. J., The biogenesis and functions of lipid bodies in
animals, plants and microorganisms. Prog. Lipid Res. 2001, 40,
325-438.
(38) Fujimoto, T.; Ohsaki, Y.; Cheng, J.; Suzuki, M.; Shinohara, Y.,
Lipid droplets: a classic organelle with new outfits. Histochem. Cell
Biol. 2008, 130, 263-79.
(39) Huang, L.; Ma, P.; Deng, G.; Zhang, K.; Ou, T.; Lin, Y.; Wong,
M. S., Novel electron-deficient quinoxalinedithienothiophene- and
phenazinedithienothiophene-based photosensitizers: The effect of
conjugation expansion on DSSC performance. Dyes Pigments 2018,
159, 107-114.
(26) Chen, S.; Qin, Z.; Liu, T.; Wu, X.; Li, Y.; Liu, H.; Song, Y.; Li,
Y., Aggregation-induced emission on benzothiadiazole dyads with
large third-order optical nonlinearity. Phys. Chem. Chem. Phys.
2
013, 15, 12660-12666.
(27) Chen, S.; Chen, N.; Yan, Y. L.; Liu, T.; Yu, Y.; Li, Y.; Liu, H.;
Zhao, Y. S.; Li, Y., Controlling growth of molecular crystal
aggregates for efficient optical waveguides. Chem. Commun. 2012,
4
8, 9011-9013.
(28) Bixon, M.; Jortner, J.; Cortes, J.; Heitele, H.; Michel-Beyerle,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
M. E., Energy Gap Law for Nonradiative and Radiative Charge
Transfer in Isolated and in Solvated Supermolecules. J. Phys. Chem.
1
994, 98, 7289-7299.
(40) Mann, G.; Hartwig, J. F.; Driver, M. S.; Fernández-Rivas, C.,
2
(29) Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W., Structural
changes accompanying intramolecular electron transfer: focus on
twisted intramolecular charge-transfer states and structures. Chem.
Rev. 2003, 103, 3899-4032.
Palladium-Catalyzed C−N(sp ) Bond Formation:ꢀ N-Arylation of
Aromatic and Unsaturated Nitrogen and the Reductive Elimination
Chemistry of Palladium Azolyl and Methyleneamido Complexes.
J. Am. Chem. Soc. 1998, 120, 827-828.
(30) Sasaki, S.; Drummen, G. P. C.; Konishi, G.-i., Recent advances
in twisted intramolecular charge transfer (TICT) fluorescence and
related phenomena in materials chemistry. J. Mater. Chem. C 2016,
4, 2731-2743.
(31) Rettig, W., Charge Separation in Excited States of Decoupled
Systems—TICT Compounds and Implications Regarding the
Development of New Laser Dyes and the Primary Process of Vision
and Photosynthesis. Angewandte Chemie International Edition in
English 1986, 25, 971-988.
AUTHOR INFORMATION
Corresponding Author
* matsubara.ryosuke@people.kobe-u.ac.jp
ACKNOWLEDGMENT
This work was carried out by the joint research program of
Molecular Photoscience Research Center, Kobe University
(32) Rotkiewicz, K.; Grellmann, K. H.; Grabowski, Z. R.,
Reinterpretation of the anomalous fluorescense of p-n,n-
dimethylamino-benzonitrile. Chem. Phys. Lett. 1973, 19, 315-318.
(H29001, H30021). We thank Profs. Atsunori Mori and Kentaro
(33) Reinterpretation of the anomalous fluorescence of p-N,N-
Okano for their help on mass analysis, and Profs. Yasuhiro Kobori,
Manabu Sakurai and Naoki Yamamoto for their helpful discussion.
Prof. Atsuo Tamura and Haruka Sakurai are acknowledged for the
help on DLS analysis, and Profs. Satoshi Minakata and Yohei
Takeda for the help on fluorescence measurements. Financial
supports from JSPS KAKENHI Grant Number JP16K18844,
Shorai Foundation for Science and Technology, and Research
Foundation for Opto-Science and Technology are greatly
appreciated.
dimethylamine-benzonitrile: Chem. Phys. Letters 19 (1973) 315.
Chem. Phys. Lett. 1973, 21, 212 (erratum).
(34) Jones, G.; Jackson, W. R.; Halpern, A. M., Medium effects on
fluorescence quantum yields and lifetimes for coumarin laser dyes.
Chem. Phys. Lett. 1980, 72, 391-395.
(35) Hamada, H.; Tsuji, H.; Nakamura, E., Aggregation-responsive
ON–OFF–ON fluorescence-switching behaviour of twisted
tetrakis(benzo[b]furyl)ethene made by hafnium-mediated
McMurry coupling. Mater. Chem. Front. 2018, 2, 296-299.
(36) Hu, R.; Lager, E.; Aguilar-Aguilar, A.; Liu, J.; Lam, J. W. Y.;
Sung, H. H. Y.; Williams, I. D.; Zhong, Y.; Wong, K. S.; Peña-
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 14
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
ACS Paragon Plus Environment