Full Color Tunable Aggregation-Induced Emission Luminogen for Bioimaging Based on an Indolizine Molecular Framework

Article abstract of DOI:10.1021/acs.bioconjchem.0c00467

By taking advantage of a unique mechanism of aggregation-induced emission (AIE) phenomena, AIE luminogens (AIEgens) have been provided as a solution to overcome the limitations of conventional fluorophores bearing the feature of aggregation-caused quenching (ACQ) phenomena. Especially, AIEgens paved the way to develop fluorogenic probes ideal for fluorescent imaging in live cell conditions. Despite the high demand for discovery of new AIEgens, it is still challenging to find a versatile molecular platform to generate diverse AIEgens. Herein, we report a new colorful molecular framework, Kaleidolizine (KIz), as a molecular platform for AIEgen generation. The KIz system allows systematic tuning of the emission wavelength from 455 to 564 nm via perturbation of the electron density of substituents on the indolizine core. Increasing the water fraction of the KIz solution in the THF/water mixture induces the fluorescence intensity increase up to 120-fold. Crystal structure analysis, computational calculations, and solvatochromism studies suggest that a synergistic effect between the intramolecular charge transfer and restriction of intramolecular rotation acts as the AIE mechanism in the KIz system. Conjugation of the triphenylphosphonium moiety to KIz allows successful development of triphenylphosphonium (TPP)-KIz for real-time bioimaging of innate mitochondria in live cells, thereby revealing the potential of KIz as a versatile molecular platform to generate fluorogenic probes based on AIE phenomena. We do believe the KIz system could serve as a new, reliable, and generally applicable molecular platform to develop various AIEgens having desired photophysical properties along with an excellent signal-to-noise ratio and with experimental convenience especially for fluorogenic live cell imaging.

Full text of DOI:10.1021/acs.bioconjchem.0c00467

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Article
A full color tunable aggregation-induced emission luminogen
for bio-imaging based on an indolizine molecular framework
Sang-Kee Choi, Jun Gi Rho, Sang Eun Yoon, Jin-Hong Seok, Hyungi Kim, Junsik Min, Woojin
Yoon, Sanghee Lee, Hoseop Yun, O-Pil Kwon, Jong H. Kim, Wook Kim, and Eunha Kim
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.0c00467 • Publication Date (Web): 28 Sep 2020
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Bioconjugate Chemistry
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A full color tunable aggregation-induced emission luminogen for bio-im-
aging based on an indolizine molecular framework
Sang-Kee Choi1, Jungi Rho1, Sang Eun Yoon1, Jin-Hong Seok1, Hyungi Kim1, Junsik Min1, Woojin
Yoon2, Sanghee Lee3, Hoseop Yun2, O-Pil Kwon1, Jong H. Kim1, Wook Kim1, Eunha Kim1,*
1Department of Molecular Science and Technology, Ajou University Suwon, 16499, Korea
2Department of Chemistry, Ajou University Suwon, 16499, Korea
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3Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea
KEYWORDS aggregation induced emission, indolizine, intramolecular charge transfer, restriction of molecular rotation,
mitochondria
ABSTRACT: By taking advantage of unique mechanism of aggregation-induced emission (AIE) phenomena, AIE Luminogen
(AIEgen) have been provided a solution to overcome the limitations of conventional fluorophores bearing the feature of aggregation-
caused quenching (ACQ) phenomena. Especially, AIEgens paved the way to develop fluorogenic probes ideal for fluorescent imaging
in live cell condition. Despite high demand for discovery of new AIEgens, it is still challenging to find a versatile molecular platform
to generate diverse AIEgens. Herein, we report a new colorful molecular framework Kaleidolizine (KIz) as a molecular platform for
AIEgen generation. The KIz system allows systematic tuning of emission wavelength from 455 nm to 564 nm via perturbation of the
electron density of substituents on the indolizine core. Increase the water fraction of KIz solution in THF/water mixture induces
fluorescence intensity increase up to a 120-fold. Crystal structure analysis, computational calculations, and solvatochromism studies
suggest that a synergistic effect between the intramolecular charge transfer and restriction of intramolecular rotation acts as the AIE
mechanism in the KIz system. Conjugation of the triphenylphosphonium moiety to KIz allows successful development of TPP–KIz
for real-time bioimaging of innate mitochondria in live cells, thereby revealing the potential of KIz as a versatile molecular platform
to generate fluorogenic probes based on AIE phenomena. We do believe KIz system could serve as new reliable and generally appli-
cable molecular platform to develop various AIEgens having desired photophysical properties along with excellent signal to noise
ratio and with experimental convenience especially for fluorogenic live cell imaging.
Organic fluorescent materials have received significant atten-
tion from the scientific community as an attractive and versatile
research tool for studying the nanoscopic1,2, microscopic3–5, and
macroscopic world6,7 for pathogen detection8–10, disease diag-
nosis11,12, life sciences13–16, and modern biomedical research17–
21. Most conventional fluorophores are highly fluorescent in
lower concentrations or in the solution state, but weakly fluo-
rescent or non-emissive at higher concentrations or in the solid
state. This phenomenon is generally referred to as aggregation-
caused quenching (ACQ)22–27. In contrast to conventional fluor-
ophores, certain molecules show enhanced emissions in their
aggregated or solid state forms, which is referred to as aggrega-
tion-induced emission (AIE)28–32. Since their discovery28, many
of fluorophore exhibiting AIE properties are discovered,
generally referred as AIE luminogens (AIEgens). Therefore,
AIEgens have provided a solution to the limitation of ACQ phe-
nomena in conventional fluorophores, especially in terms of
fluorogenic probe development. AIE features a minimized
background, an enhanced signal fidelity, and an increased sig-
nal-to-noise (S/N) ratio, which is ideal for biosensing and bi-
oimaging28–32. Despite a relatively limited number of AIEgens,
applications of AIE phenomenon are remarkably increasing in
the fields of biomedical research and life science28–32. Research-
ers are in pursuit of AIEgens to tailor their unique light-up char-
acteristics for innovative applications28–32. Therefore, the dis-
covery of new AIEgens has tremendous potential for applica-
tions in biomedical research and life science. For the cellular
heterogeneity study33, integrative single cell analysis34 and mul-
tiplexed profiling of the biological system18,35, on the other hand,
there are growing interest on development of molecular system
having diverse emission properties35. However, it is still chal-
lenging to control photophysical properties because of complex
underlying photophysical phenomena and lack of synthetic
flexibility of the known fluorescent compounds. Therefore, it is
desirable to find single fluorescent core skeleton having con-
trollable and modulable emission properties of fluorescent com-
pound.
In this paper, we introduce for the first time a new AIEgen
molecular platform, Kaleidolizine (KIz), based on the
indolizine structure. The molecular framework reveals
systematic tunability for the emission wavelength, which is
controlled by changing the electron density of substituents in
the chemical core skeleton. We confirm that the AIE of
Kaleidolizine derivatives results in a 119.5-fold increase in
fluorescence. Crystal structure analysis, computational
calculations, and solvatochromism studies suggest a synergistic
effect between the intramolecular charge transfer (ICT)36,37 and
restriction of intramolecular rotation (RIR)38,39 as the AIE
mechanism for the KIz system. Finally, conjugation of the
triphenylphosphonium (TPP) moiety to KIz allows successful
development of TPP–KIz as highly specific and sensitive
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fluorogenic probes for real-time bio-imaging of mitochondria color range under equal irradiation conditions (Figure 1c). Con-
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in cells.
sidering this observation, we hypothesized that the KIz fluores-
cent core skeleton could function as a molecular platform to
generate diverse AIE luminogens (AIEgens) using combinato-
rial chemistry along with emission color tunability. To confirm
AIE phenomenon in the KIz core skeleton, we measured emis-
sion properties of KIz solutions in THF/water mixture (20 µM)
with increased water fraction (ꢀw). A solution of each repre-
sentative KIz derivative in pure THF showed negligible emis-
sion. However, increasing the ꢀw in THF/water mixture from 0%
to 90% induced a gradual increase in fluorescence intensity
(Figure 1d), along with micron-sized aggregate formation (Fig-
ure S1). Most of the KIz derivatives exhibited strong fluores-
cence when ꢀw exceed 70~80% and the turn-on ratio for KIz 11,
KIz 1, KIz 3 and KIz 5 was 41.3, 119.5, 18.3, and 28.8 fold,
respectively (Figure 1d).
RESULTS AND DISCUSSION
As a continuation of our efforts to study the structure-photo-
physical property relationship (SPPR) of the indolizine fluores-
cent core skeleton, we discovered that the molecular framework,
Kaleidolizine (KIz) (Figure 1a) exhibits full color, tunable
solid-state fluorescence, and AIE phenomena. First, we selected
4 different KIz derivatives, including KIz 1, KIz 3, KIz 5, and
KIz 11 as representative KIz derivatives for the AIE study (Fig-
ure 1a). One of the most interesting properties of the KIz fluo-
rescent molecular framework is a tunable emission wavelength
(λem) in the solid state, which can be easily tuned by simple
changes of the electron density on the specific position of the
core skeleton via changing the substituent. For example, chang-
ing the substituents on the R2 position of trifluoromethyl (KIz
11) to hydrogen (KIz 1) induces a bathochromic shift of the
solid-state λem from 455 to 486 nm (Figure 1b). Likewise,
changing the substituent on the R1 position of KIz 1 from tri-
fluoromethyl to hydrogen (KIz 3) and to the dimethylamino
(KIz 5) group induces a bathochromic shift of the solid-state λem
from 486 to 507 and to 564 nm, respectively (Figure 1b). Inter-
estingly, solutions (20 µM) of KIz 11, KIz 1, KIz 3, and KIz 5
in tetrahydrofuran (THF) were weakly fluorescent under 365
nm irradiation, and the absolute fluorescence quantum yields
(φF) of the solutions were 1.6 %, 0.2 %, 0.3 %, and 0.5%, re-
spectively. In contrast, spin-coated solid films of the four dif-
ferent derivatives exhibit vivid colorful emission in the full
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Several different AIE mechanisms have been proposed, includ-
ing restriction of intramolecular rotations (RIR), vibrations
(RIV), and motions (RIM)30. To evaluate the mechanism of AIE
on the KIz core skeleton, single crystals of KIz 1 and KIz 63—
having equal chemical core skeleton structures shared by other
derivatives (KIz 11, KIz 3, and KIz 5, respectively)—were
grown by the slow diffusion-induced crystallization method
(Supporting Information). The single crystal structures of KIz 1
and KIz 63 are plotted in Figure 2, and their numerical data
given in Table S1.Based on X-ray diffraction (XRD) pattern
analysis, it is evident that the crystal system is triclinic (Figure
S2). The dihedral angle of KIz 63 between the central indolizine
ring and phenyl (φ) was 38.8° and that of benzoyl (ψ) was 55.8°
(Figure 2a).
Figure 1. Aggregation-Induced Emission (AIE) of Kaleidolizine. a) The chemical core skeleton structure of Kaleidolizine (KIz). b) The
emission spectra of KIz 11 (blue), KIz 1 (green), KIz 3 (orange), and KIz 5 (red) in the solid state. c) Photos of the compounds KIz 11, KIz
1, KIz 3, and KIz 5 (from left to right) under ambient light (up) and 365 nm UV light (down) both of solution (THF, vial) and of solid film.
d) Emission spectra of solutions of KIz 11, KIz 1, KIz 3, and KIz 5 (from left to right) in THF/water mixed solvents (20 µM) with different
water fractions (0-90%).
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Bioconjugate Chemistry
The KIz 1 molecule exhibited a conformation similar to that of 5 (Figure S4). Therefore, the AIE of KIz is not solely deter-
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KIz 63, but the phenyl and benzoyl were twisted out of the plane
of the central indolizine ring to a lesser extent (φ = 26.8° or
37.1°; ψ = 49.1° or 43.9°, Figure 2b and Figure S3). Crystal
mined by the RIR process. Based on the DFT calculations with
Gaussian 09 at the B3LYP/6-31G(d) level, it was predicted that
there was a clear separation between the highest occupied mo-
lecular orbital (HOMO) and lowest unoccupied molecular or-
bital (LUMO) electron distribution (Figure 2c and Figure S5)
that could trigger an intramolecular charge transfer (ICT)36,37
process in the molecule. Since most ICT molecules have re-
duced fluorescence intensity under polar conditions41, we antic-
ipated that aggregates of KIz in polar solvents could provide a
hydrophobic molecular environment, inducing a fluorescence
intensity increase in the KIz system via ICT. To determine the
ICT phenomena as another AIE mechanism in the KIz system,
we investigated solvatochromism38,41,42 of each compound (Fig-
ure 2d). The stock solutions of KIz 11, KIz 1, KIz 3, and KIz 5
in THF (10 mM) were diluted with four different solvents
(1000-fold dilution) with different solvent polarity indexes (P),
including hexane (P = 0.1), THF (4.0), ACN (5.8), and DMSO
(7.2). Compared to the DMSO solvent, all KIz solutions in hex-
ane are highly fluorescent (Figure 2d). Therefore, the solvato-
chromism study implied that the ICT process is a companion
AIE mechanism for the KIz system with the RIR process. To
systematically study the effect of hydrophobicity on the photo-
physical property of the KIz system, solutions of KIz with dif-
ferent hexane fractions (ꢀh) in the THF/Hexane mixture,
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structure analysis revealed more dynamic rotation of CF3 at
the R1 and R3 positions than at the R2 position of KIz 1 (Figure
2b). Considering that the turn-on efficiency appears in the order
of KIz 1 (3 different CF3 at the R1, R2, and R3 positions), KIz
11 (2 different CF3 at the R1 and R3 positions), and KIz 3 (2
different CF3 at the R2 and R3 positions) (Figure 1d), we hy-
pothesized that the introduction of a molecular rotor at the R1,
R2, and R3 positions triggers non-radiative decay of the KIz sys-
tem, and the molecular rotor at the R1 and R3 positions resulted
in a higher fluorescent quenching efficiency than at the R2 po-
sition. Therefore, we anticipated that the formation of aggre-
gates induced the RIR molecular process that eventually trig-
gered AIE in the KIz system. To evaluate the RIR mechanism
as an AIE phenomena in the KIz system, four different KIz so-
lutions (10 µM) in a THF/ethylene glycol mixture (Figure S4a)
or MeOH/glycerol mixture (Figure S4b) with different percent-
ages of ethylene glycol or glycerol, respectively, were prepared
to restrict the molecular motion by increasing the viscosity of
the solution without aggregate formation. Surprisingly, there
was no fluorescence increase in KIz 11, KIz 1, KIz 3, and KIz
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Figure 2. Crystal structure analysis and solvatochromism studies of KIz. a,b) Single crystal structure of KIz 63 (a) and of KIz 1 (b). c)
Electron density distributions in the HOMO and LUMO were predicted by DFT calculation using Gaussian 09 at the B3LYP/6-31G(d) level
with the conformations defined by crystal structure analysis of KIz 1 (left) and of KIz 63 (right). d) Emission spectra for solutions (20 µM)
of KIz 11, KIz 1, KIz 3, and KIz 5 in hexane (red), THF (blue), ACN (green), and DMSO (purple).
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Page 4 of 11
molecular rotor, we reasoned that non-radiative decay through
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intramolecular motion is more favorable in the order of KIz 1,
KIz 11 and KIz 3 in the solution state. Therefore, fluorescence
turn-on efficiency with an increase of solvent hydrophobicity is
diminished by non-radiative decay that is permissible in the so-
lution state. In contrast, fluorescence turn-on efficiency with ag-
gregate formation is amplified by blocking molecular rotation,
which reduces non-radiative decay in the solid state. Collec-
tively, we propose the AIE mechanism of the KIz system as a
synergistic effect between the RIR and ICT process. Consider-
ing the increased interest in fluorescent mitochondrial pheno-
typing43,44, we synthesized triphenylphosphonium45 conjugated
KIz (TPP–KIz) to develop a wash-free fluorogenic mitochon-
dria bioprobe to demonstrate the potential of AIE in the KIz
core skeleton for practical biomedical imaging applications
(Figure 4). For the synthesis of TPP–KIz (Scheme S1), we first
utilized the Suzuki coupling reaction between IM-B and 4-
methoxycarbonyl boronic acid with Pd(PPh3)4. Saponification
of the resulting compound 1 and subsequent amide coupling re-
action with boc-protected ethylenediamine generated com-
pound 3. The deprotected boc group with 4M HCl in 1,4-diox-
ane was followed by an amide coupling reaction to (5-carboxy-
pentyl)triphenylphosphine bromide for the synthesis of the
TPP–KIz compound (Scheme S1). More detailed information
on the synthetic procedure and full characterization of all new
compounds are available in the supporting information. First,
TPP–KIz (Figure 4a) had a larger Stokes shift (λem: 387 nm, λem:
507 nm), a typical characteristic of a fluorescent compound
having an ICT tendency36,41,42 that results in a high S/N ratio. To
confirm that the TPP–KIz could be used as a fluorogenic mito-
chondrial bioprobe in live cells, cytotoxicity of the probes was
evaluated. Chang liver cells were incubated with TPP-KIz at
different concentrations (from 1 to 100 nM) and cytotoxicity
was measured by a mitochondrial respiration viability assay,
confirming the lack of acute cytotoxicity by TPP-KIz up to 24
h time point (Figure S6). For biological investigation of TPP-
KIz, Chang liver cells expressing mitochondrial yellow fluores-
cent protein (Mito-YFP) were washed with PBS 1X, incubated
with Hoechst and TPP–KIz (1 nM) in full-growth media, and
imaged without washing using a Leica DMi8 epifluorescence
microscope. There was an excellent colocalization signal be-
tween the Mito-YFP and TPP–KIz channels (Figure 4b), and
the calculated Pearson’s R value was 0.95 (Figure S7). Plot in-
tensity values of pixels along a white line revealed not only a
sharp contrast between the mitochondrion and other areas, but
also excellent colocalization of the fluorescent signal between
Mito-YFP and TPP–KIz (Figure 4c). In addition, time-lapse flu-
orescent
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Figure 3. Emission spectra of KIz 11, KIz 1, KIz 3 and KIz 5
solutions (20 µM) in THF/hexane mixtures with different hexane
fractions (ꢀh). Each solution was excited with the absorption
maxima corresponding to each solvent condition.
from 0 to 90%, was prepared (Figure 3 and Table 1). In general,
there were negligible changes in λabs, whereas there were hyp-
sochromic shifts of λem (from 490, 535, 539; and from 533 to
456, 484, 492; and to 502 in the case of KIz 11, KIz 1, KIz 3
and KIz 5, respectively) with increasing solvent hydrophobicity
without any aggregate formation (Figure 3 and Table 1). There-
fore, this solvatochromism study confirmed the existence of the
ICT process in the KIz system. Interestingly, fluorescence turn-
on efficiency of the KIz solutions during solvatochromism
study is in reverse order of the turn-on efficiency observed with
ꢀw increase. For example, increasing hydrophobicity of KIz 1,
KIz 11 and of KIz 3 solutions by changing the solvent from
DMSO to hexane induces a 34.7, 93.0, and 170.4-fold fluores-
cence intensity increase (Figure 2d). However, increasing the
ꢀw of KIz 1, KIz 11 and of KIz 3 solution in the THF/water
mixture induces a 119.5, 41.3 and 18.3-fold fluorescence inten-
sity increase (Figure 1d). Since the difference between these
three different compounds is the number and position of CF3
Table 1. Photophysical properties and particle sizes of KIz 11, KIz 1 KIz 3 and KIz 5 in THF/Hexane Mixtures
KIz 11
λem
KIz 1
λem
KIz 3
λem
KIz 5
λem
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c
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c
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λab
dd
λab
dd
λab
dd
λab
dd
ꢀh [%]a
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547
502
n.a.
n.a.
n.a.
n.a.
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100
aHexane fractions in THF/Hexane mixture. bThe longest absorption wavelength (nm) . cMaximal emission wavelength (nm) . dParticle
size analysis with DLS (dynamic Light Scatteting). n.a. is not available.
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Bioconjugate Chemistry
imaging of Chang liver cells before and after treatment with
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TPP–KIz demonstrated the potential of the compound as a flu-
orogenic mitochondrial bioprobe (Figure 5). Most of the mito-
chondria were stained after treatment with TPP–KIz for 1 min
(Figure 5a), and the fluorescence intensity reached a maximum
within 5 min (Figure 5b). Furthermore, mitochondria were
stained with reasonable S/N ratios at very low concentrations
(down to 1 nM, Figure S8), highlighting the potential of
AIEgens based on the KIz core skeleton for biomedical appli-
cations. Therefore, we successfully developed highly sensitive
and specific fluorogenic bioprobes for mitochondria by utiliz-
ing the AIE phenomenon in the KIz molecular core skeleton
system.
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CONCLUSION
In summary, we studied the AIE phenomena and their bioimag-
ing application of the KIz core skeleton. The compounds KIz
11, KIz 1, KIz 3, and KIz 5 are weakly fluorescent in the solu-
tion state; however, they are highly fluorescent in the solid state.
Increasing the ꢀw in the THF/water mixture triggered aggregate
formation along with increased fluorescent intensity. By simple
changes of the substituent in the R1, R2 and R3 position of the
core skeleton, emission wavelengths of KIz system were easily
tunable in the visible color range. Crystal structure analysis,
computational calculation and solvatochromism studies sug-
gested a synergistic effect between ICT and RIR as an AIE
mechanism for the KIz system. We demonstrated that the AIE
phenomena in the KIz system allowed successful development
of fluorescent bioprobes TPP–KIz via conjugation of the TPP
moiety to KIz, thereby revealing the potential of KIz as a ver-
satile molecular platform to generate fluorogenic probes based
on AIE phenomena. Collectively, we do believe KIz system
could serve as new reliable and generally applicable molecular
platform to develop various AIEgens having desired photo-
physical properties along with excellent signal to noise ratio and
with experimental convenience especially for fluorogenic live
cell imaging. Following progress will be reported in due course.
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Figure 4. Fluorogenic imaging of mitochondria with TPP–KIz in
live cell without washing. a) chemical structure (left) and absorp-
tion & emission spectrums (right) of TPP–KIz. b) Chang liver cells
expressing mitochondrial Yellow Fluorescent protein (Mito-YFP)
were incubated with TPP–KIz (1 nM) and Hoechst dye and imaged
with DMi8 fluorescence microscopy without washing. c) Plot in-
tensity values of pixels along a white line of ROI, region of interest
of (b) were analyzed with the ImageJ program.
Figure 5. Time lapse imaging of live cell with TPP–KIz without washing. a) Chang liver cells expressing Mito-YFP were imaged with DMi8
fluorescence microscopy before and after the addition of TPP–KIz (1 nM) without washing. b) Fluorescence intensity of the images were
analyzed with the ImageJ program. Scale bar: 10 μm
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(DFT) at the B3LYP/6-31G* level. The energy levels of the
EXPERIMENTAL PROCEDURES
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highest occupied molecular orbital (HOMO) and the lowest un-
occupied molecular orbital (LUMO) were calculated with the
optimized ground state molecular geometry.
1
Compound characterization. H NMR and 13C NMR data
were recorded on Varian Mercury plus (400 MHz) spectrometer
and a JEOL JNM-ECZ 600R (600 MHz) at Ajou University.
Chemical shifts were reported in parts per million (δ) and cali-
brated using an internal tetramethylsilane (TMS) standard or re-
Photophysical property study depending on viscosity. Fluo-
rescence emission spectra and UV absorption spectra of KIz so-
lutions (20 µM) in THF/Ethylene glycol mixture or
MeOH/glycerol mixture were recorded on JASCO FP-8200
spectrofluorometer and on JASCO V-670 spectrophotometer
with different amount of ethylene glycol or glycerol (0-90%).
Dynamic Light Scattering (DLS) was measured by Otsuka
Zeta-potential & Particle size Analyzer ELS-Z.
1
sidual undeuterated solvent for H NMR spectra (CDCl3 7.26
ppm; CD3OD 3.31 ppm) and 13C NMR spectra (CDCl3 77.16
ppm; CD3OD 49.00 ppm). Multiplicity is indicated as follows:
s (singlet), d (doublet); t (triplet); q (quartet); Quin (quintet); m
(multiplet); dd (doublet of doublet); brs (broad singlet). Cou-
pling constants were reported in Hz. Low-resolution mass spec-
trometry (LRMS) was performed using a LCMS-2020 (Shi-
madzu) and compact mass spectrometer (Advion).
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Crystal study. KIz11 and 65 single crystals were grown by the
slow evaporation method in a solvent mixture of methanol and
dichloromethane (3.0 mL of methanol : 7.0 mL of dichloro-
methane = 3:7 v/v (1:1.33 mol/mol)). The powders were dis-
Materials. All chemicals were purchased from Sigma-Aldrich,
Tokyo Chemical Industry Co. Ltd, Alfa Aesar, or Thermo
Fisher Scientific, and used without further purification unless
otherwise specified. The progress of the reaction was monitored
using thin-layer chromatography (TLC) (silica gel 60, F254 0.25
mm), and components were visualized by observation under
UV light (254 and 365 nm) or by treating the TLC plates either
with KMnO4, phosphomolybdic acid (PMA), or ninhydrin fol-
lowed by heating. Solvents were purchased from commercial
vendors and used without further purification. Reagent syno-
nyms: N,N-dimethylformamide (DMF), methanol (MeOH), di-
chloromethane (DCM), triphenylphosphine (TPP), diisopro-
pylethylamine (DIPEA), triethylamine (TEA), benzotriazol-1-
yl-oxytripyrrolidinophosphonium hexafluorophosphate (Py-
BOP). Cell culture reagents including fatal bovine serum (FBS),
Dulbecco’s modified Eagle’ s culture medium (DMEM), non-
essential amino acid (NEAA), and antibiotic-antimycotic solu-
tion were purchased from GIBCO, Invitrogen. Phosphate-buff-
ered saline (PBS) was purchased from WELGENE. A cover-
glass-bottomed confocal dish was purchased from SPL Life
Sciences Co. Ltd., and a 100-mm culture dish was purchased
from CORNING.
solved in
a methanol/dichloromethane mixture (1:1.33
mol/mol) in 10.0 mL, and the solution was left to evaporate
slowly at 35 °C in an oven for two days. When the solution was
completely evaporated, the grown crystals were filtered and
washed with hexane.
Fluorogenic mitochondrial live cell imaging with TPP-KIz.
Human Chang Liver cells stably overexpressing mitochondria-
targeted yellow fluorescence protein (Mito-YFP) were cultured
in DMEM containing 10 % FBS, 1% NEAA, and 25.0 μg/mL
G418 at 37 °C in a humidified incubator with 5% CO2. Cells
were harvested using TrypLE™ Express (GIBCO) and sus-
pended in fresh culture medium. Harvested cells at a density of
1 × 104 cells were seeded on a confocal dish (SPL) and incu-
bated for 48 h. For staining the mitochondria, different concen-
trations of TPP-KIz were added. Cells were observed (without
washing) with an inverted fluorescence microscope (DMi8,
LEICA). Each experiment was observed using a 325 − 375 nm
exciter, 435 − 485 nm emitter for DAPI channel; 460 − 500 nm
exciter, 512 − 542 nm emitter for KIz channel; 541 − 551 nm
exciter and 565 − 605 nm emitter for the YFP channel. Images
were analyzed with ImageJ software from the National Insti-
tutes of Health.
Solution state photophysical property measurement. Fluo-
rescence emission spectra were recorded on a JASCO FP-8200
spectrofluorometer, and UV absorption spectra were recorded
on a JASCO V-670 spectrophotometer. All spectra experiments
were performed in a 1x1 cm quartz cuvette. Absolute quantum
yield was measured by QE-2000 (Otsuka Electronics). Each
product was dissolved with chloroform at a 20 mg/mL concen-
tration. The glass substrates were cleaned with deionized water,
acetone, and isopropanol for 10 min in an ultrasonic bath and
treated with UV for 20 min. The stock solutions were spin-
coated on glass substrates at 1500 rpm for 1 min.
Cytotoxicity Test Chang Liver cells (5 x 103 cells/well) were
seeded into 96-well cell culture plates per well and allowed to
adhere to the plates for 24 hr. The cells were incubated with cell
culture media containing each concentration of TPP-KIz (1 nM
to 100 nM) for 6, 12 or 24 h. After incubation, CellTiter 96
Aqueous One Solution Cell Proliferation Assay (Promega) was
added into each well. After 2 h, 490 nm absorption was meas-
ured with Cytation 3 microplate reader (BioTek).
Chemical synthesis and compound characterization
Solid-state photophysical property measurement. The glass
substrates were cleaned with deionized water, acetone, and iso-
propanol for 10 min each in an ultrasonicator. After drying, the
substrates were treated with UV light (365 nm) for 20 min. The
KIz solution in chloroform (200 µL, 20.0 mg/mL) was added to
the glass substrates and spin-coated at 1500 rpm for 60 s. Ab-
sorption and emission spectra were recorded using a JASCO
FP-8200 spectrophotometer and JASCO V-670 UV-Vis spec-
trometer, respectively.
IA-E. Reaction mixture of 4-(trifluoromethyl)pyridine (637.0
µL, 5.50 mmol) and 2-bromo-1-[4-(trifluoromethyl)-phe-
nyl]ethane-1-one (1.54 g, 5.70 mmol) in DMF (10 mL) was
stirred at 100 °C for overnight. After reaction completion, ethyl
acrylate (293.0 µL, 2.75 mmol), copper (II) acetate monohy-
drate (1.64 g, 8.25 mmol) and sodium acetate (1.35 g, 16.5
mmol) were added to the reaction mixture. Resulting reaction
mixture was stirred at 100 °C for additional 16 hrs. After the
reaction completion, monitored with TLC, the copper acetate
was removed by filtration through celite pad and the resulting
filtrate was concentrated in vacuo. Resulting crude product was
washed with water and organic material was extracted with
Quantum mechanical calculations. All quantum mechanical
calculations were performed in a Gaussin09W. The ground
state structures were optimized using density functional theory
ACS Paragon Plus Environment

Page 7 of 11
Bioconjugate Chemistry
DCM for 3 times. Combined organic material was dried over organic material was extracted with DCM for 3 times. Com-
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anhydrous Na2SO4 and concentrated. Combined organic phase
was purified with silica-gel flash column chromatography
(EA:Hexane = 1:6 — ethyl acetate 15%) to afford IA-E (0.52
mg, 43.8% yield) as a yellow solid; 1H NMR (400 MHz, CDCl3)
δ 10.0 (d, J = 7.2 Hz, 1H), 8.70 (s, 1H), 7.91 (d, J = 7.6 Hz, 2H),
7.82 (s, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.2 Hz, 1H),
4.40 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H); 13C NMR (100
MHz, CDCl3), δ 184.0, 162.8, 141.9, 137.7, 133.3, 132.6, 129.3,
128.9, 125.3, 124.7, 124.0, 122.6, 122.0, 121.2, 117.1, 110.9,
108.9, 60.6, 14.4; HRMS (ESI) m/z calcd for C20H14F6NO3
[M+H]+: 430.0878; found: 430.0872.
bined organic material was dried over anhydrous Na2SO4 and
concentrated. Crude product was purified with silica-gel flash
column chromatography (EA:Hexane = 1:4 — ethyl acetate
25%) to afford KIz 3 (48.7 mg, 96.0% yield) as a yellow solid;
1H NMR (400 MHz, CDCl3) δ 10.05 (d, J = 7.6 Hz, 1H), 8.14
(s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.55
(s, 1H), 7.53 (d, J = 3.6 Hz, 2H), 7.49 (d, J = 3.2 Hz, 2H), 7.39
(d, J = 7.2 Hz, 1H), 7.14 (dd, J = 7.4 Hz, 2.4 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 183.7, 143.1, 134.4, 133.3, 133.2, 132.9,
129.3, 129.2, 128.1, 127.5, 126.6, 125.8, 125.5, 125.4, 125.1,
124.7, 122.9, 122.0, 120.9, 115.7, 110.1; HRMS (ESI) m/z calcd
for C23H14F6NO [M+H]+: 434.0980; found: 434.0979.
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IA-B. To a solution IA-E (517.9 mg, 1.2 mmol) in methanol
(10 mL) was added KOH (5.39 g, 96.0 mmol). The reaction
mixture was stirred at room temperature for overnight. Reaction
mixture was acidified with 6 N HCl and resulting solid was col-
lected by filtration, washed with water and dried in drying oven
to afford IA-A as a brown solid. Resulting product was used in
the next step without further purification. To a slurry of result-
ing IA-A in DMF (10 mL) was added sodium bicarbonate
(302.4 mg, 3.6 mmol) and NBS (320.3 mg, 1.8 mmol) was
added to the reaction mixture portionwise at 0 °C. The reaction
mixture was stirred at ambient temperature for 12 hrs. Resulting
crude product was washed with water and organic material was
extracted with DCM for 3 times. Combined organic material
was dried over anhydrous Na2SO4 and concentrated. Crude
product was purified with silica-gel flash column chromatog-
raphy (EA:Hexane = 1:8 — ethyl acetate 12%) to afford IA-B
(434.1 mg, 82.9% yield) as a yellow solid; 1H NMR (400 MHz,
CDCl3) δ 9.95 (d, J = 7.2 Hz, 1H), 7.90 (s, 2H), 7.87 (s, 1H),
7.78 (d, J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.13 (dd, J = 7.2 Hz, 2.0
Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 183.1, 142.4, 135.0,
133.1, 129.2, 127.6, 126.6, 125.6, 125.5, 124.4, 123.0, 121.7,
115.4, 110.4, 93.0; LRMS (ESI) m/z calcd for C17H9BrF6NO
[M+H]+: 435.97; found: 436.05.
KIz 5. To a solution of IA-B (51.4 mg, 0.11 mmol) in
DMF:H2O = 2:1 mixture was added 4-(dimethylamino)phenyl
boronic acid (78.0 mg, 0.47 mmol), tetrakis(triphenyl phos-
phine)palladium(0) (231.0 mg, 20.0 mol%) and sodium car-
bonate (50 mg, 0.47 mmol). Resulting reaction mixture was
stirred at 100 °C for additional 12 hrs. After the reaction com-
pletion, monitored with TLC, the resulting crude product was
washed with water and organic material was extracted with
DCM for 3 times. Combined organic material was dried over
anhydrous Na2SO4 and concentrated. Crude product was puri-
fied with silica-gel flash column chromatography (EA:Hexane
= 1:4 — ethyl acetate 25%) to afford KIz 5 (51.5 mg, 91.6%
yield) as an orange solid; 1H NMR (400 MHz, CDCl3) δ 10.01
(d, J = 7.6 Hz, 1H), 8.12 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.78
(d, J = 8.0 Hz, 2H), 7.41 (d, J = 5.0 Hz, 2H), 7.40 (s, 1H), 7.09
(d, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 2H), 3.02 (s, 6H); 13
C
NMR (100 MHz, CDCl3) δ 183.4, 149.8, 143.3, 134.3, 132.9,
132.6, 129.2, 129.1, 128.8, 125.9, 125.7, 125.5, 125.1, 124.7,
122.6, 122.4, 122.0, 121.5, 121.4, 121.0, 116.0, 115.9, 112.9,
109.7, 40.6; LRMS (ESI) m/z calcd for C25H19F6N2O [M+H]+:
477.13; found: 477.35.
Compound IC-E. Reaction mixture of 4-(trifluoromethyl)pyr-
idine (348.0 µL, 3.00 mmol) and 2-bromoacetophenone (0.63
mg, 3.15 mmol) in DMF (7.0 mL) was stirred at 100 °C for
overnight. After reaction completion, ethyl acrylate (160.0 µL,
0.50 mmol), copper (II) acetate monohydrate (0.89 g, 1.50
mmol) and sodium acetate (0.74 g, 9.00 mmol) was added to
the reaction mixture. Resulting reaction mixture was stirred at
100 °C for additional 16 hrs. After the reaction completion,
monitored with TLC, the copper acetate was removed by filtra-
tion through celite pad and the resulting filtrate was concen-
trated in vacuo. Resulting crude product was washed with water
and organic material was extracted with DCM for 3 times. Com-
bined organic material was dried over anhydrous Na2SO4 and
concentrated. Combined organic phase was purified with silica-
gel flash column chromatography (EA:Hexane = 1:6 — ethyl
acetate 15%) to afford IC-E (0.48 g, 88.9% yield) as a yellow
solid; 1H NMR (400 MHz, CDCl3) δ 9.88 (d, J = 7.6 Hz, 1H),
8.59 (s, 1H), 7.78 (s, 1H), 7.74 (d, J = 6.8 Hz, 2H), 7.53 (t, J =
7.4 Hz, 1H), 7.44 (d, J = 7.6 Hz, 2H), 7.10 (dd, J = 7.4 Hz, 2.0
KIz 1. To a solution of IA-B (50.4 mg, 0.11 mmol) in
DMF:H2O = 2:1 mixture was added 4-trifulorophenyl boronic
acid (88.0 mg, 0.46 mmol), tetrakis(triphenyl phosphine)palla-
dium(0) (231.0 mg, 20.0 mol%) and sodium carbonate (49.0 mg,
0.46 mmol). Resulting reaction mixture was stirred at 100 °C
for additional 12 hrs. After the reaction completion, monitored
with TLC, the resulting crude product was washed with water
and organic material was extracted with DCM for 3 times. Com-
bined organic material was dried over anhydrous Na2SO4 and
concentrated. Crude product was purified with silica-gel flash
column chromatography (EA:Hexane = 1:4 — ethyl acetate
25%) to afford KIz 1 (50.0 mg, 86.7% yield) as a yellow solid;
1H NMR (400 MHz, CDCl3) δ 10.07 (d, J = 7.6 Hz, 1H), 8.11
(s, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.74
(d, J = 8.4 Hz, 2H), 7.66 (s, 1H), 7.64 (s, 1H), 7.52 (s, 1H), 7.18
(dd, J = 7.4 Hz, 2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 183.9,
142.9, 137.1, 134.4, 133.4, 133.1, 129.5, 129.2, 128.3, 127.3,
127.0, 126.2, 126.0, 125.6, 125.1, 124.5, 123.2, 122.8, 122.4,
121.8, 119.2, 115.3, 110.4; LRMS (ESI) m/z calcd for
C24H13F9NO [M+H]+: 502.08; found: 502.10.
Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) δ 185.3, 163.0, 138.8, 137.2, 131.7,
129.2, 128.8, 128.5, 128.3, 128.2, 128.0, 124.2, 123.2, 121.5,
117.0, 110.4, 108.5, 60.5, 14.5; LRMS (ESI) m/z calcd for
C19H15F3NO3 [M+H]+: 362.09; found: 362.10.
KIz 3. To a solution of IA-B (51.2 mg, 0.11 mmol) in
DMF:H2O = 2:1 mixture was added phenyl boronic acid (57.0
mg, 0.46 mmol), tetrakis(triphenyl phosphine)palladium(0)
(231 mg, 20.0 mol%) and sodium carbonate (49.0 mg, 0.46
mmol). Resulting reaction mixture was stirred at 100 °C for ad-
ditional 12 hrs. After the reaction completion, monitored with
TLC, the resulting crude product was washed with water and
IC-B. To a solution IC-E (481.9 mg, 1.3 mmol) in methanol
(10 mL) was added KOH (519.0 mg, 10.0 mmol). The reaction
mixture was stirred at room temperature for 4 hrs. Reaction
mixture was acidified with 6 N HCl and resulting solid was col-
lected by filtration, washed with water and dried in drying oven
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 11
to afford IC-A as a white solid. Resulting product was used in IM-B. To a solution IM-E (616 mg, 2.10 mmol) in methanol
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the next step without further purification. To a slurry of result-
ing IC-A in DMF (10.0 mL) was added sodium bicarbonate
(162.4 mg, 1.9 mmol) and NBS (172.0 mg, 0.9 mmol) was
added to the reaction mixture portionwise at 0 °C. The reaction
mixture was stirred at ambient temperature for 12 hrs. Resulting
crude product was washed with water and organic material was
extracted with DCM for 3 times. Combined organic material
was dried over anhydrous Na2SO4 and concentrated. Crude
product was purified with silica-gel flash column chromatog-
raphy (EA:Hexane = 1:8 — ethyl acetate 12%) to afford IC-B
(219.2 mg, 99.2% yield) as a yellow solid; 1H NMR (400 MHz,
CDCl3) δ 9.96 (d, J = 7.6 Hz, 1H), 7.90 (s, 1H), 7.79 (d, J = 8.0
Hz, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.51 (t, J = 7.4 Hz, 2H), 7.46
(s, 1H), 7.09 (dd, J = 7.6 Hz, 2.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 185.4, 139.1, 134.2, 131.7, 128.9, 128.8, 128.3, 127.4,
126.1, 125.7, 124.4, 123.2, 121.7, 115.1, 109.8, 92.3; LRMS
(ESI) m/z calcd for C16H10BrF3NO [M+H]+: 367.98; found:
368.12.
(20 mL) was added KOH (7.06 g, 126 mmol). The reaction mix-
ture was stirred at room temperature for 4 hrs. Reaction mixture
was acidified with 6 N HCl and resulting solid was collected by
filtration, washed with water and dried in drying oven to afford
IM-A as a white solid. Resulting product was used in the next
step without further purification. To a slurry of resulting IM-A
in DMF (10.0 mL) was added sodium bicarbonate (529 mg,
6.30 mmol) and NBS (561 mg, 3.15 mmol) was added to the
reaction mixture portionwise at 0 °C. The reaction mixture was
stirred at ambient temperature for 12 hrs. Resulting crude prod-
uct was washed with water and organic material was extracted
with DCM for 3 times. Combined organic material was dried
over anhydrous Na2SO4 and concentrated. Crude product was
purified with silica-gel flash column chromatography
(EA:Hexane = 1:8 — ethyl acetate 12%) to afford IM-B (590
mg, 93.6% yield) as a yellow solid; 1H NMR (400 MHz, CDCl3)
δ 10.04 (d, J = 6.8 Hz, 1H), 7.78 (d, J = 6.4 Hz, 2H), 7.61 (d, J
= 8.8 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.50 (d, J =7.6 Hz, 2H),
7.37 (s, 1H), 7.30 (dt, J = 7.7 Hz, 1.2 Hz, 1H), 7.00 (dt, J = 7.0
Hz, 1.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 184.0, 140.3,
136.9, 131.3, 129.0, 128.8, 128.5, 127.2, 125.4, 122.3, 117.3,
114.8, 89.9, 47.1, 34.8; LRMS (ESI) m/z calcd for C15H11BrNO
[M+H]+: 290.99; found: 290.95.
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KIz 11. To a solution of IC-B (51.2 mg, 0.14 mmol) in
DMF:H2O = 2:1 mixture was added 4-trifulorophenyl boronic
acid (106 mg, 0.55 mmol), tetrakis(triphenyl phosphine)palla-
dium(0) (231.0 mg, 20.0 mol%) and sodium carbonate (59.0 mg,
0.55 mmol). Resulting reaction mixture was stirred at 100 °C
for additional 5 hrs. After the reaction completion, monitored
with TLC, the resulting crude product was washed with water
and organic material was extracted with DCM for 3 times. Com-
bined organic material was dried over anhydrous Na2SO4 and
concentrated. Crude product was purified with silica-gel flash
column chromatography (EA:Hexane = 1:4 — ethyl acetate
25%) to afford KIz 11 (54.0 mg, 89.0% yield) as a yellow solid;
1H NMR (400 MHz, CDCl3) δ 10.05 (d, J = 7.6 Hz, 1H), 8.09
(s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.66
(d, J = 8.0 Hz, 2H), 7.60 (t, J = 6.8 Hz, 1H), 7.57 (s, 1H), 7.54
(d, J = 7.6 Hz, 2H), 7.14 (dd, J = 7.4 Hz, 2.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 185.5, 139.8, 137.5, 133.9, 131.8, 129.5,
129.1, 128.6, 128.3, 126.4, 126.2, 126.2, 126.0, 125.8, 123.8,
118.7, 115.3, 110.0, 110.0, 47.1, 34.7; LRMS (ESI) m/z calcd
for C23H14F6NO [M+H]+: 434.09; found: 434.30.
KIz 63. To a solution of IM-B (61.5 mg, 0.21 mmol) in
DMF:H2O = 2:1 mixture was added phenyl boronic acid (100
mg, 0.82 mmol) tetrakis(triphenyl phosphine)palladium(0)
(231.0 mg, 20.0 mol%) and sodium carbonate (87.0 mg, 0.82
mmol). Resulting reaction mixture was stirred at 100 °C for ad-
ditional 9 hrs. After the reaction completion, monitored with
TLC, the resulting crude product was washed with water and
organic material was extracted with DCM for 3 times. Com-
bined organic material was dried over anhydrous Na2SO4 and
concentrated. Crude product was purified with silica-gel flash
column chromatography (EA:Hexane = 1:4 — ethyl acetate
25%) to afford KIz 63 (56.2 mg, 89.9% yield) as a yellow solid;
1H NMR (400 MHz, CDCl3) δ 10.04 (d, J = 6.8 Hz, 1H), 7.89
(d, J = 9.2 Hz, 1H), 7.84 (d, J = 6.8 Hz, 2H), 7.19 (m, 7H), 7.31
(t, J = 7.4 Hz, 1H), 7.27 (d, J = 6.6 Hz, 1H), 7.25 (d, J = 6.6 Hz,
1H), 6.99 (t, J = 6.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
183.9, 140.3, 135.9, 134.2, 130.4, 128.5, 128.4, 127.8, 127.5,
126.1, 125.0, 124.6, 121.8, 117.2, 117.1, 113.8; LRMS (ESI)
m/z calcd for C21H16NO [M+H]+: 298.12; found: 298.10.
Compound IM-E. Reaction mixture of pyridine (564 µL, 7.00
mmol) and 2-bromoacetophenone (1.46 g, 7.35 mmol) in DMF
(15.0 mL) was stirred at 100 °C for overnight. After reaction
completion, ethyl acrylate (373 µL, 3.50 mmol), copper (II) ac-
etate monohydrate (2.09 g, 10.5 mmol) and sodium acetate
(1.72 g, 21.0 mmol) was added to the reaction mixture. Result-
ing reaction mixture was stirred at 100 °C for additional 16 hrs.
After the reaction completion, monitored with TLC, the copper
acetate was removed by filtration through celite pad and the re-
sulting filtrate was concentrated in vacuo. Resulting crude prod-
uct was washed with water and organic material was extracted
with DCM for 3 times. Combined organic material was dried
over anhydrous Na2SO4 and concentrated. Combined organic
phase was purified with silica-gel flash column chromatog-
raphy (EA:Hexane = 1:6 — ethyl acetate 15%) to afford IM-E
Compound 1. To a solution of IM-B (200.0 mg, 0.7 mmol) in
DMF and H2O mixture (v:v = 10:1) was added 4-methoxycar-
bonyl boronic acid (359.7 mg, 2.0 mmol), tetrakis(tri-
phenylphosphine)palladium (154.1 mg, 0.13 mmol) and sodium
carbonate (352.8 mg, 3.33 mmol). The reaction mixture was
stirred at 100 °C for 8 hours in argon atmosphere. After the re-
action completion, monitored with TLC, the reaction mixture
was concentrated in vacuo, and crude product was distributed
with DCM and water. Organic phase was extracted with DCM
for 3 times, dried over anhydrous Na2SO4, and was evaporated
under reduced condition. The resulted crude residue was puri-
fied with silica-gel flash chromatography (EA:Hexane = 1:9 -
ethyl acetate 20% ) to afford compound 1 (155.2 mg, 65.6 %
1
(616 mg, 60% yield) as a yellow solid; H NMR (400 MHz,
CDCl3) δ 9.83(d, J = 7.2 Hz, 1H), 8.25(dd, J = 9.0 Hz, 1.2 Hz,
1H), 7.73(d, J = 7.2 Hz, 2H), 7.71(s, 1H), 7.46(d, J = 7.2 Hz,
1H), 7.40(t, J = 7.2 Hz, 2H), 7.29(t, J = 8.0 Hz, 1H), 6.93(t, J =
7.0 Hz, 1H), 4.29(q, J = 7.0 Hz, 2H), 1.31(t, J = 7.0 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 184.8, 163.4, 139.5, 139.3,
131.1, 128.7, 128.6, 128.4, 128.0, 127.2, 122.1, 121.2, 119.0,
114.9, 105.9, 59.9, 14.5; LRMS (ESI) m/z calcd for C18H16NO3
[M+H]+: 294.11; found: 294.15.
1
yield) as a yellow solid; H NMR (600 MHz, CDCl3) δ 10.05
(td, J = 4.2 Hz, 1.2 Hz, 1H), 8.11 (td, J = 5.1 Hz, 1.8 Hz, 2H),
7.93 (td, J = 5.1 Hz, 1.2 Hz, 1H), 7.86 -7.84 (m, 2H), 7.65 (dt,
J = 5.1 Hz, 1.8 Hz, 2H), 7.58 (tt, J = 7.2 Hz, 1.8 Hz, 1H), 7.31
(td, J = 6.0 Hz, 3H), 7.35- 7.32 (m, 1H), 7.05 (td, J = 6.9 Hz,
1.8 Hz, 1H), 3.95 (s, 3H); 13C NMR (150 MHz, CDCl3), δ 184.2,
140.3, 136.9, 131.2, 128.9(2), 128.8(3), 127.2, 125.3, 117.3(3),
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114.6, 89.7; LRMS (ESI) m/z calcd for C23H18NO3 [M+H]+ :
Supporting Information.
1
2
The Supporting Inforrmaation is available free of charge via the
Internet at http://pubs.acs.org.”
356.12, found : 356.07
Compound 2. Compound 1 (20 mg, 56 µmol) and potassium
hydroxide(94.7 mg, 1.7 mmol) was dissolved in 1.0 mL of
MeOH and stirred for 5 hours at reflux condition. After the re-
action completion, motioned with TLC, the reaction mixture
was cooled down to room temperature and 6 N HCl was added
to the mixture slowly. Resulting precipitate was collected by
filtration and washed with water. The crude residue was used
for next reaction without further purification
3
4
5
6
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8
9
Supporting figures, tables and scheme
Copies of 1H and 13C NMR Spectra of a New Compounds
AUTHOR INFORMATION
Corresponding Author
*ehkim01@ajou.ac.kr
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Notes
Compound 3. To a solution of compound 2 (145.3 mg, 0.43
mmol) in DMF was added TEA (118.7 µL, 0.85 mmol) and re-
sulting mixture was stirred at ambient temperature for 30 min.
N-(tertbutoxycarbonyl)-1,2-diaminoethane (100.2 µL, 0.64
mmol) and PyBOP (443.1mg, 0.85 mmol) were added to the
reaction mixture and the mixture was stirred at ambient temper-
ature. After the reaction completion, monitored with TLC, the
resulting crude product was washed with water and organic ma-
terial was extracted with DCM for 3 times. Combined organic
material was dried over anhydrous Na2SO4 and concentrated in
vacuo. The resulted crude residue was purified with silica-gel
flash chromatography (EA:Hexane = 9:1 - ethyl acetate 90% )
to afford compound 3 (131.5 mg, 63.2 % yield) as a yellow solid;
1H NMR (600 MHz, CDCl3) δ 10.01 (d, J = 12.0 Hz, 1H), 7.88
(d, J = 12.0 Hz 3H), 7.86 (d, J = 6.0 Hz, 1H), 7.82 (d, J = 6.0
Hz, 2H), 7.58 (d, J = 6.0 Hz, 2H), 7.55 (t, J = 6.0 Hz, 2H), 7.49
(t, J = 6.0 Hz, 1H), 7.47 (s, 1H), 7.31 (brs, 1H), 7.27 (t, J = 6.0
Hz, 1H), 7.00 (t, J = 6.0 Hz, 1H), 5.09 (s, 1H), 3.57 (q, J = 6.0
Hz, 2H), 3.42 (q, J = 6.0 Hz, 2H), 3.63 (s, 9H); 13C NMR (150
MHz, CDCl3), δ 184.8, 171.3, 167.5, 140.7, 138.0, 136.5, 131.2,
129.2, 129.0, 128.4, 127.8, 127.7, 122.6, 117.5, 116.5, 114.6,
80.0, 42.3, 40.1, 28.5(2), 28.4(2), 21.1, 14.3; LRMS (ESI) m/z
calcd for C29H30N3O4 [M+H]+ : 484.22, found : 484.15.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was supported in part by the Creative Materials
Discovery Program through the National Research Foundation
(2019M3D1A1078941) and by the National Research Foundation
of Korea(NRF) grant funded by the Korean government (MSIT)
(NRF-2020R1C1C1010044) (NRF- 2019R1A6A1A11051471). X-
ray structural analysis was supported by Basic Science Research
Program through the National Research Foundation of Korea(NRF)
funded by the Ministry of Education (2019R1I1A2A01058066).
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Hz, 1H),7.93 - 7.89 (m, 3H), 7.81 - 7.75 (m, 6H), 7.66 - 7.60
(m, 15H), 7.42 (t, J = 6.0 Hz, 1H), 7.37 (s, 1H), 7.15 (t, J = 12.0
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137.8 136.7, 134.9 (5), 134.8, 133.5 (2), 133.4 (3), 133.3 (3),
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115.8, 63.2, 38.7 (3), 37.4, 35.2, 29.0; LRMS (ESI) m/z calcd
for C48H46N3O3P+ [M+H]+ : 743.32, found : 743.23
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