A fluorescence turn-on probe for the detection of thiol-containing amino acids in aqueous solution and bioimaging in cells

Article abstract of DOI:10.1016/j.tet.2014.01.060

A turn-on fluorescent probe, based on a water-soluble terphenyl derivative, for the detection of cysteine and homocysteine is reported. The aldehyde groups in the probe play crucial roles in providing reaction with thiol groups in the amino acids, leading

Full text of DOI:10.1016/j.tet.2014.01.060

Tetrahedron 70 (2014) 2034e2039
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A fluorescence turn-on probe for the detection of thiol-containing
amino acids in aqueous solution and bioimaging in cells
a
b
c
c
a
,
b
, ,
y
*
Sang-Ho Son , Yongkyun Kim , Min Beom Heo , Yong Taik Lim , Taek Seung Lee
a
Organic and Optoelectronic Materials Laboratory, Graduate School of Green Energy Technology, Chungnam National University, Daejeon 305-764,
Republic of Korea
b
Organic and Optoelectronic Materials Laboratory, Department of Advanced Organic Materials and Textile System Engineering, Chungnam National
University, Daejeon 305-764, Republic of Korea
c
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A turn-on fluorescent probe, based on a water-soluble terphenyl derivative, for the detection of cysteine
and homocysteine is reported. The aldehyde groups in the probe play crucial roles in providing reaction
with thiol groups in the amino acids, leading to a formation of thiazolidine (from cysteine) or thiazinane
ring (from homocysteine). As a result, the new formation of such rings alters the electronic property of
the conjugated system in the probe and results in emission enhancement. The probe in aqueous solution
exhibits a remarkable increase in its quantum yield upon exposure to cysteine (up to 20-fold) and to
homocysteine (up to 700-fold), while slight quenching is observed in the presence of glutathione.
Moreover, an investigation on time-resolved fluorescence spectra of the probe in the presence of cysteine
and homocysteine reveals potential discriminatory detection of cysteine and homocysteine. Bioimaging
of the thiols in live HeLa cells was successfully applied.
Received 17 December 2013
Received in revised form 16 January 2014
Accepted 21 January 2014
Available online 31 January 2014
Keywords:
Fluorescence
Fluorescent probes
Amino acids
Sensors
Turn-on sensor
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Hcy, based on the formation of thiazolidines and thiazinane rings
from the cyclization of Cys and Hcy with aldehyde, respectively. It
7
Cysteine (Cys) and homocysteine (Hcy) are small molecular
weight, biological thiol-containing amino acids, which play im-
portant roles in many biochemical and physiological processes. Cys
deficiency is closely related to diseases such as skin lesions, hair
is thought that both thiol and amino groups cooperate to cyclize,
which enables the selective detection of Cys and Hcy over other
amino acids. However, it has been known that the selective de-
tection of one of them is still challenging due to their similarity in
1
7a,c,8
depigmentation, liver damage, and slow growth in children. An
structure and reactivity of thiols.
abnormal level of Hcy in blood (>12
m
M) is a risk factor for Alz-
Compared to ‘turn-off’ quenching probes, the remarkable ad-
vantage of ‘turn-on’ fluorescence enhancing probes is related to the
ease of measuring low-concentrations in a dark background. This
ability reduces the possibility of false positive signals, and thus,
2
heimer’s and cardiovascular diseases. Thus, the quantitative de-
tection of these biothiols has drawn a great deal of attention for
human healthcare.
8
b,9
A number of detection methods for these biological thiols using
increases the selectivity.
3
various materials have been developed, including colorimetric,
Herein, we report a newly synthesized fluorescent, cationically
charged, conjugated probe with tertiary ammonium groups for
water-solubility and aldehyde groups for a reaction with biological
thiol amino acids. It is expected that the probe is reactive toward
thiol compounds, which, in turn, enhances the fluorescence in-
tensity of the probe (turn-on mode) due to disruption of the elec-
tronic structure in the probe.
phosphorescent,4 and fluorometric methods, with the aim of
attaining high sensitivity, low cost, and convenience in detection. In
some cases, a variety of chemical reactions with strong nucleophilic
5
6
thiol groups were explored including Michael addition and so on.
Among them, it was reported that fluorophores containing al-
dehyde groups can serve as fluorescent probes for both Cys and
2. Results and discussion
*
Corresponding author. E-mail addresses: tslee@cnu.ac.kr, leets84@yahoo.co.kr
0
0
0
Probe 4 (3 ,6 -bis(6-trimethylammoniumhexyloxy bromide)-p-
(
T.S. Lee).
y
terphenyl-4,4 -dialdehyde) was readily synthesized from dia-
www.onom.re.kr.
URL: http://www.onom.re.kr
0
0
0
ldehyde precursor 3 (3 ,6 -bis(6-bromohexyloxy)-p-terphenyl-4,4 -
0
040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.01.060

S.-H. Son et al. / Tetrahedron 70 (2014) 2034e2039
2035
with H NMR, 13C NMR, and elemental analysis. Compound 4 was
1
dialdehyde) and trimethylamine, resulting in ammonium salt
groups for water-solubility (Scheme 1). Precursor 3 was prepared
by the reaction of 2 and 4-formylphenylboronic acid in the pres-
ence of palladium catalyst via Suzuki coupling reaction. The
chemical structure of 3 and 4 was confirmed by characterization
found to be soluble in water, ethanol, and DMSO.
The absorption and fluorescence emission spectra of 4 in various
solvents are shown in Fig. 1. In aqueous solution, 4 exhibited a ab-
sorption at 341 nm and emission at 433 nm of deep blue fluores-
cence. Compared to the spectra from ethanol and DMSO solution,
they were blue-shifted. In DMSO solution, 4 showed an absorption
at 359 nm and emission at 490 nm, presumably due to the differ-
ence in polarity and aprotic property of the solvent.
As mentioned above, the aldehyde groups at the ends of 4 will
react specifically with Cys and Hcy to provide thiazolidine and
thiazinane rings, respectively. Consequently, as electron-accepting
aldehyde groups disappear, the optical properties of 4 will be al-
tered. Based on the formation of new rings at the ends of probe 4,
we demonstrated a new system to detect Cys or Hcy via emission
enhancement as shown in Scheme 2.
The fluorescence spectra clearly show an increase in the emis-
sion intensity of 4 in aqueous solution (100 mM) as the concentra-
tion of Cys and Hcy increases (Fig. 2). Notably, a blue shift of
emission maximum was observed upon exposure to Cys
(433e419 nm) or Hcy (433e404 nm). Nucleophilic attack of the
thiol is expected to shorten the conjugation length and restrict the
intramolecular charge transfer (ICT) to induce the blue shift of
3
c,10
emission.
The emission enhancement is also attributed to the
poor solubility of the resultant thiazolidine and thiazinane rings to
5
g,h
induce hydrophobic environment in aqueous solution.
Absorp-
Scheme 1. Synthetic route for 4.
tion spectral changes of 4 in the presence of Cys and Hcy are
Fig. 1. Absorption (a) and normalized fluorescence spectra (b) of 4 in aqueous solution (-), ethanol (B), and DMSO (:) at the concentration of 25 mM for absorption spectra.
Scheme 2. (a) Reaction of 4 with Cys and Hcy and (b) schematic representation of the reaction with fluorescent probe and biothiols.

2036
S.-H. Son et al. / Tetrahedron 70 (2014) 2034e2039
Fig. 2. Fluorescence spectral changes of 4 in aqueous solution (100 mM) upon addition of Cys (a) and Hcy (b). Two spectra were measured at different slit widths.
demonstrated in Figs. S1 and S2 (Supplementary data), re-
spectively. Clear absorption spectral changes of blue shift can be
observed, resulting from variations in conjugation and charge
transfer.
The fluorescence intensity at 419 nm and 404 nm were plotted
as a function of Cys and Hcy concentrations, respectively, and the
plots show a proportional relationship between emission in-
tensities at 419 nm (for Cys) and at 404 nm (for Hcy) as well as the
data, Fig. S3). After the reaction with Cys, the aldehyde peak at
9.89 ppm in D O solution diminished. Along with the weak alde-
hyde peak, a new peak centered at 5.86 ppm was observed, which
could be assigned to the CH and CH protons of the thiazolidine
Besides, the formation of the thiazolidine and thiazinane
2
2
7
a,12
rings.
derivatives can be verified by the mass spectra as shown in Fig. S4
(Supplementary data), which are known to be formed via Schiff
5
h
base intermediates.
corresponding concentrations in Fig. 3. The limit of detection (3
slope, where is the standard deviation of four independent
measurements) was estimated according to a published method
and was determined to be 0.29 M for Cys and 0.14 M for Hcy.
s
/
To further understand the high selectivity of the probe for bio-
thiols such as Cys and Hcy over other amino acids, we examined the
changes in the emission ratio of 4 upon treatment of amino acids. As
shown in Fig. 4a, the emission enhancement can be clearly seen
upon addition of Cys, while other amino acids: such as lysine, ala-
nine, tryptophan, glycine, asparagine, valine, aspartic acid, arginine,
serine, isoleucine, threonine, methionine, glutamine, phenylalanine,
s
11
m
m
To gain an insight into the mechanism of Cys binding to 4
1
resulting in the formation of heterocycle, the H NMR spectra of 4
was monitored before and after the addition of Cys (Supplementary
Fig. 3. Changes in the fluorescence intensity of 4 in aqueous solution (100
mM) at 419 nm upon addition of Cys (a) and at 404 nm upon addition of Hcy (b).
Fig. 4. (a) Changes in fluorescence intensity and (b) I/I
0
of 4 (100 mM) upon addition of amino acids (8.0 mM) in aqueous solution. For Hcy detection, the concentration of 4 is 10 mM.
I
0
and I correspond to the intensities at 419 nm (404 nm for Hcy) before and after addition of amino acid, respectively.

S.-H. Son et al. / Tetrahedron 70 (2014) 2034e2039
2037
glutamic acid, and histidine, do not affect the emission of 4. The
emission ratio of Cys displays a great increase in intensity (about
eight fold). Hcy elicits an emission ratio enhancement of about 600
fold. However, 4 exhibits an essentially weak response to other
amino acids, and thus, shows negligible changes as shown in Fig. 4b.
The changes in emission intensity of 4 upon exposure to Cys and
Hcy are directly compared in Fig. 5a. By introduction of Hcy to 4,
a remarkable increase in the emission of 4 was observed and this
can be notably observed with the naked eye, as shown in Fig. 5b.
Probe 4 does not fluoresce to a sufficient degree in aqueous solution
with quantum yield (QY) of 0.08% (relative to Rhodamine B). Ad-
dition of Cys into aqueous solution of 4 induces a more than 20-fold
increase in the QY (1.76%). Surprisingly, the QY was greatly en-
hanced in the presence of Hcy, up to 61.5%. It is thought that the
thiazinane rings formed by the addition of Hcy restricts ICT more
than the thiazolidine rings formed by addition of Cys. It is likely that
the formation of six-membered (thiazinane) ring could provide
a comparably hydrophobic microenvironment to decrease charge
transfer between the media and the molecule. Thus, this inhibits
intra- and intermolecular interactions, leading to enhancement of
emission and QY. The presence of glutathione (GSH) did not affect
the emission intensity of 4, presumably due to the fact that GSH did
not react with aldehyde group in 4. GSH has thiol group but does
not react with aldehyde to form ring structure because amine
group, which is essential for ring formation, is not present in GSH.
Besides, poorer water-solubility was observed upon formation
of six-membered ring (by addition of Hcy), which, in turn, resulted
in considerable increase in emission. The average hydrodynamic
The cytotoxicity of 4 was evaluated by metabolic viability of
HeLa cells after incubation with different concentrations of 4. Fig. 6
shows the cell viability after incubation with 4 at the concentration
of 0, 1.3, 2.6, 6.5, 13, and 26 mM for 24 and 48 h, respectively. Cell
viabilities of more than 95% were observed during the tested time,
indicative of low cytotoxicity of 4. The low toxicity of 4 is essential
for its application in cell imaging. To investigate the ability of 4 for
monitoring intracellular biothiol levels in live cells, HeLa cells were
incubated with 4 and subsequently analyzed with a fluorescence
Fig. 6. Cell viability values (%) of 4. HeLa cells were cultured in the presence of 4
ꢀ
(1.3e26
m
M at 37 C for 24 h (black) and 48 h (gray)).
Fig. 5. (a) Fluorescence spectra of 4 (100 mM) after addition of 80 equiv of Cys and Hcy and 100 equiv of GSH in water and (b) the photographs of changes in fluorescence emission.
diameter of 4 estimated from dynamic light scattering (DLS) was
78 nm. By addition of Cys and Hcy to 4, the hydrodynamic di-
ameter of 4þCys and 4þHcy in water measured by DLS increased to
12 nm and 1066 nm, respectively, indicating that the solubility
microscope. The HeLa cells incubated with 4 for 30 min exhibited
strong emission, indicating that 4 was expressed ratiometrically in
cytoplasm as shown in Fig. 7.
4
6
was responsible for the increase in emission intensity. Though both
Cys and Hcy provide blue-shifted emission enhancement of 4, they
can be recognized one from the other not only by the increase in
emission intensity but also by the fluorescence decay time as
shown in Table 1 and Fig. S4 (Supplementary data). As tabulated in
Table 1, the lifetime of 4 was decreased more in the presence of Hcy
than the case of Cys, indicating that electronic perturbations in the
presence of Cys and Hcy are different.
3
. Conclusion
We have synthesized a fluorescent probe 4 for the specific de-
tection of thiol-related compounds of Cys and Hcy and explored
in vitro bioimaging application. The probe 4 was easily synthesized
on the basis of Suzuki coupling reaction. Based on the reaction
between aldehyde groups in 4 and thiol groups in Cys and Hcy, the
probe exhibits a blue-shift in emission (14 nm for Cys and 29 nm for
Hcy) and a drastic emission increase upon addition of Cys and Hcy.
Importantly, the increase in QY of Cys is completely different from
that of Hcy, in which 20-fold (for Cys) and 700-fold (for Hcy) were
shown, while other amino acids exhibited negligible increases in
emission intensity of 4. In terms of fluorescence decay lifetime, 4
with Hcy exhibited different lifetimes, compared to 4 with Cys due
Table 1
Time-resolved fluorescence decay data of 4, 4þCys, and 4þHcy
4
4þCys
4þHcy
s
s
1
2
(ns)
(ns)
0.76 (3.9%)
3.81 (96.1%)
0.85 (8.9%)
3.31 (91.1%)
0.032 (35.6%)
2.54 (64.4%)

2038
S.-H. Son et al. / Tetrahedron 70 (2014) 2034e2039
removed by evaporation and the resulting brown solid was then
taken up in methylene chloride and washed with water
3ꢁ200 mL), 3 N aqueous sodium hydroxide solution (3ꢁ200 mL),
M aqueous hydrochloric acid solution (3ꢁ200 mL), and brine
. The solvent was re-
(
3
(
3ꢁ200 mL) and, finally, dried over MgSO
4
moved on the rotary evaporator to yield a brown solid that was
crystallized from ethyl acetate to yield 8.76 g (17%) of 1. H NMR
1
(
4
1
300 MHz, CDCl
H), 1.78 (m, 4H), 1.5 (m, 8H). C NMR (75 MHz, CDCl
47.86, 116.02, 69.24, 33.84, 32.98, 30.21, 28.74, 25.74. Anal. Calcd
for C18 : C, 49.56; H, 6.47. Found: C, 49.88; H, 6.19.
3
) d/ppm: 6.8 (s, 4H), 3.9 (t, 4H), 3.4 (t, 4H), 1.89 (m,
13
3
) d/ppm:
2 2
H28Br O
4.3. Synthesis of 1,4-bis(6-bromohexyloxy)-2,5-dibromoben
zene (2)
Compound 1 (8.76 g, 20 mmol) was dissolved in methylene
chloride (50 mL) in a three-neck flask. A mixture of bromine
(
15 mL) and methylene chloride (100 mL) was added dropwise into
ꢀ
the flask over 1 h at 0 C. After 1 h, the temperature was increased
to room temperature and the mixture was stirred for 14 h. The
mixture was poured into a separatory funnel and was washed with
saturated NaHSO
over MgSO and the solvent was removed on the rotary evaporator.
The resulting brown solid was recrystallized from n-hexane to yield
3
solution and brine. The organic phase was dried
4
1
7
(
(
.13 g (60%) of 2. H NMR (300 MHz, CDCl
t, 4H), 3.44 (t, 4H), 1.92 (m, 4H), 1.83 (m, 4H), 1.54 (m, 8H). C NMR
75 MHz, CDCl /ppm: 146.56, 119.87, 111.02, 67.39, 33.04, 31.89,
8.78, 27.38, 24.11. Anal. Calcd for C18
Found: C, 36.51; H, 4.45.
3
) d/ppm: 7.09 (s, 2H), 3.97
13
3
) d
2
H28Br
4
O
2
: C, 36.4; H, 4.41.
Fig. 7. Live-cell fluorescence images of HeLa cells incubated with (a) 1.3
c) 26 M, and (d) 52 M of 4 for 30 min.
mM, (b) 13 mM,
(
m
m
0
0
0
4
.4. Synthesis of 3 ,6 -bis(6-bromohexyloxy)-p-terphenyl-4,4 -
dialdehyde (3)
to formation of more hydrophobic six-membered ring. The probe 4
shows low cytotoxicity on live cells, which enables in vivo cell
imaging. This reveals that 4 could serve as a naked-eye detectable
probe for Cys and Hcy with good selectivity.
Compound 2 (3 g, 5.05 mmol), and 4-formylphenylborinic acid
(1.74 g, 11.6 mmol) were added to a 500 mL three-neck round-bot-
tomed flask with a condenser under a nitrogen atmosphere. Tetra-
hydrofuran (THF, 50 mL) and 2 M aqueous potassium carbonate
solution (3 mL) were injected into the flask, and a small amount of
Aliquat 336 and tetrakis(triphenylphosphine)palladium(0) (0.289 g,
0.25 mmol) was then added. The reaction mixture was refluxed for 2
days. The reaction mixture was then cooled to room temperature
and was filtered under vacuum. The filtrate was taken up in meth-
ylene chloride and washed with aqueous NH
The organic phase was dried over MgSO and the solvent was re-
moved in vacuo. The product was purified by column chromatog-
3
raphy to yield 0.498 g (15%) of 3. H NMR (300 MHz, CDCl ) d/ppm:
10.09 (s, 2H), 7.96 (d, 4H), 7.77 (d, 4H), 7.01 (s, 2H), 3.96 (t, 4H), 3.35
4
4
. Experimental section
.1. Reagents and instrumentation
Hydroquinone and potassium carbonate were purchased from
Duksan. 4-Formylphenylboronic acid, trimethylamine, 1,6-
dibromohexane, and tetrakis(triphenylphosphine)palladium(0)
were purchased from Aldrich and used as received. Bromine was
obtained from Junsei. Cys, Hcy, and other amino acids were sup-
plied from Sigma. All chemicals were used without further purifi-
4
Cl solution and brine.
4
1
1
13
13
cation unless otherwise noted. H NMR and C NMR spectra were
recorded on a Bruker DRX-300 spectrometer (Korea Basic Science
Institute). Elemental analysis was performed with a CE Instruments
EA-1110 elemental analyzer. UVevis absorption spectra were
recorded on a PerkinElmer Lambda 35 spectrometer. Photo-
luminescence spectra were taken from a Varian Cary Eclipse
equipped with a xenon lamp excitation source. Fluorescence life-
times were measured with a time-resolved fluorescence spec-
trometer FL 900 equipped with Mira Model 900-P laser.
(t, 4H), 1.82 (m, 4H), 1.72 (m, 4H), 1.41 (m, 8H). C NMR (75 MHz,
3
CDCl ) d/ppm: 191.02, 146.87, 141.99, 136.11, 130.78, 128.69, 125.62,
113.24, 69.68, 33.85, 32.77, 29.80, 28.33, 25.14. Anal. Calcd for
32 2 4
C H36Br O : C, 59.64; H, 5.63. Found: C, 60.02; H, 5.74.
0
0
4.5. Synthesis of 3 ,6 -bis(6-trimethylammoniumhexyloxy
0
bromide)-p-terphenyl-4,4 -dialdehyde (4)
Compound 3 (0.18g, 0.28 mmol), THF (10 mL), and water (2 mL)
were charged in 100 mL three-neck round-bottomed flask with
a cold finger condenser. Trimethylamine gas was added into the
flask through the condenser. The reaction mixture was stirred for
24 h. Yellow precipitates in the reaction flask were isolated by fil-
4
.2. Synthesis of 1,4-bis(6-bromohexyloxy)benzene (1)
Hydroquinone (13.33 g, 0.12 mol), 1,6-dibromohexane
1
(
55.84 mL, 0.36 mol), potassium carbonate (77.25 g, 0.56 mol),
tration and were washed with THF to yield 51.7 mg (30%) of 4. H
and acetone (200 mL) were added to a 500 mL three-neck round-
bottomed flask with a condenser. The reaction mixture was
refluxed for 2 days. The reaction mixture was then cooled to room
temperature and filtered under vacuum. The solvent of filtrate was
NMR (300 MHz, D
2
O) d/ppm: 9.89 (s, 2H), 7.85 (d, 4H), 7.56 (d, 4H),
6.86 (s, 2H), 3.73 (s, 4H), 3.1 (t, 4H), 2.99 (s, 18H), 1.51 (m, 8H), 1.19
13
(m, 8H). C NMR (75 MHz, D
2
O) d/ppm: 190.61, 146.24, 141.91,
135.68, 130.02, 127.99, 124.78, 112.81, 69.14, 66.72, 54.55, 29.59,

S.-H. Son et al. / Tetrahedron 70 (2014) 2034e2039
2039
2
7.85, 25.44, 20.50. Anal. Calcd for C38
H
54Br
2
N
2
O
4
: C, 59.84; H, 7.14;
Program (2012R1A2A2A01004979) and Human Resource Training
Project for Regional Innovation (2012H1B8A2025978) is gratefully
acknowledged.
N, 3.67. Found: C, 59.99; H, 7.11; N, 3.59.
4
.6. Detection of amino acids
Amino acids were added to the aqueous solution of 4 and the
Supplementary data
resulting mixtures were analyzed using a fluorescence spectrom-
eter in a 10 mm quartz cuvette after 24 h. To investigate the re-
1H NMR, mass, time-resolved fluorescence spectra of 4 in the
presence and absence of amino acid. . Supplementary data associ-
ated with this article can be found in the online version, at http://
dx.doi.org/10.1016/j.tet.2014.01.060
sponse to amino acids, various concentrations of amino acids
ꢂ4
(
0e100 equiv) were added to the aqueous solution of 4 (1.0ꢁ10
M
ꢂ5
for Cys and other amino acids and 1.0ꢁ10 M for Hcy).
4
.7. QY measurement
References and notes
The relative QYs were determined by comparing the
1
. (a) Shahrokhian, S. Anal. Chem. 2001, 73, 5972; (b) Reddie, K. G.; Carroll, K. S.
wavelength-integrated intensity of the samples to that of a stan-
dard (Rhodamine B) in water. The QYs of the samples were calcu-
lated using:
Curr. Opin. Chem. Biol. 2008, 12, 746; (c) Weerapana, E.; Wang, C.; Simon, G.
M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker,
D.; Cravatt, B. F. Nature 2010, 468, 790; (d) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J.
Chem. Soc. Rev. 2010, 39, 2120; (e) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41,
5
2.
2
I OD n
R
ꢀ
2
. (a) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998, 49,
31; (b) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D’Ag-
ostino, R. B.; Wilson, P. W. F.; Wolf, P. A. N. Engl. J. Med. 2002, 346, 476.
. (a) Zeng, Y.; Zhang, G.; Zhang, D. Anal. Chim. Acta 2008, 627, 254; (b) Huo, F.-J.;
Sun, Y.-Q.; Su, J.; Chao, J.-B.; Zhi, H.-J.; Yin, C.-X. Org. Lett. 2009, 11, 4918; (c) Isik,
M.; Ozdemir, T.; Turan, I. S.; Kolemen, S.; Akkaya, E. U. Org. Lett. 2013, 15, 216;
(d) Yao, Z.; Bai, H.; Li, C.; Shi, G. Chem. Commun. 2011, 7431.
. (a) Ji, S.; Guo, H.; Yuan, X.; Li, X.; Ding, H.; Gao, P.; Zhao, C.; Wu, W.; Wu, W.;
Zhao, J. Org. Lett. 2010, 12, 2876; (b) Chen, H.; Zhao, Q.; Wu, Y.; Li, F.; Yang, H.; Yi,
T.; Huang, C. Inorg. Chem. 2007, 46, 11075.
5. (a) Yao, Z.; Feng, X.; Li, C.; Shi, G. Chem. Commun. 2009, 5886; (b) Lee, J. H.; Lim,
C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc. 2010, 132, 1216; (c) Zhu, B.;
Zhang, X.; Li, Y.; Wang, P.; Zhang, H.; Zhuang, X. Chem. Commun. 2010, 5710; (d)
Liu, Y.; Yu, Y.; Lam, J. W. Y.; Hong, Y.; Faisal, M.; Yuan, W. Z.; Tang, B. Z. Chem.
dEur. J. 2010, 16, 8433; (e) Bouffard, J.; Kim, Y.; Swager, T. M.; Weissleder, R.;
Hilderbrand, S. A. Org. Lett. 2008, 10, 37; (f) Kwon, N. Y.; Kim, D.; Jang, G.; Lee, J.
H.; So, J.-H.; Kim, C.-H.; Kim, T. H.; Lee, T. S. ACS Appl. Mater. Interfaces 2012, 4,
Q ¼ Q
RI OD n2
R
R
3
where Q is the quantum yield, I is the integrated fluorescence in-
tensity, n is the refractive index, and OD is the optical density. The
subscript R refers to the reference fluorophore of known QY
4
(
Rhodamine B).
4
.8. Cell viability assay
HeLa cells were incubated with 4 (1, 2, 5, 10, and 20 mg/mL) for
4 and 48 h in flat-bottom 96-well plates (Corning Costar, Cam-
2
3
bridge, MA, USA) at a density of 5ꢁ10 cells per well (100
mL). For
the MTS assay, the Cell Titer 96 Aqueous One Solution kit (Promega,
1
2
429; (g) Mei, J.; Tong, J.; Wang, J.; Qin, A.; Sun, J. Z.; Tang, B. Z. J. Mater. Chem.
012, 22, 17063; (h) Mei, J.; Wang, Y.; Tong, J.; Wang, J.; Qin, A.; Sun, J. Z.; Tang,
Madison, WI, USA) was used according to the manufacturer’s pro-
tocols. Briefly, the MTS reagent was added (10
plates were incubated for 2 h at 37 C. Absorbance was measured at
m
L per well), and the
B. Z. Chem.dEur. J. 2013, 19, 613; (i) Lee, K.-S.; Kim, T.-K.; Lee, J. H.; Kim, H.-J.;
Hong, J.-I. Chem. Commun. 2008, 6173; (j) Kim, D.; Jang, G.; Kim, J.; Seo, S.; Park,
W. H.; Lee, T. S. Macromol. Rapid Commun. 2012, 33, 1510.
ꢀ
4
90 nm with a microplate reader (VersaMax, Molecular Devices,
6
. (a) Tang, B.; Yin, L.; Wang, X.; Chen, Z.; Tong, L.; Xu, K. Chem. Commun. 2009,
Sunnyvale, CA, USA).
5293; (b) Sreejith, S.; Divya, K. P.; Ajayaghosh, A. Angew. Chem., Int. Ed. 2008, 47,
7883; (c) Yang, Y.-K.; Shim, S.; Tae, J. Chem. Commun. 2010, 7766; (d) Zhang, M.;
Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.;
Huang, C. J. Am. Chem. Soc. 2007, 129, 10322; (e) Kim, G.-J.; Lee, K.; Kwon, H.;
Kim, H.-J. Org. Lett. 2011, 13, 2799.
4
.9. Analysis of cellular uptake
7
. (a) Rusin, O.; Luce, N. N. S.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.;
To determine intracellular delivery of 4, HeLa cells were incubated
Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126, 438; (b) Wang,
W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.;
Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.;
Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949; (c) Yang, X.; Guo, Y.; Strongin,
R. M. Angew. Chem., Int. Ed. 2011, 50, 10690.
with 4 (1.3,13, 26, 52
mg/mL) in a
m
-slide 8-well microscopy chamber
3
ꢀ
at a density of 5ꢁ10 cells/well for 30 min at 37 C. The cells were
then washed in cold PBS and fixed with 4% (w/v) paraformaldehyde
solution for 20 min at room temperature. Fluorescence images were
obtained using a DeltaVisionÔ PD (Applied Precision Technologies,
Issaquah, WA, USA) with a filter set (DAPI: excitation 360/40, emis-
sion 455/50, Omega Optical, Brattleboro, VT, USA).
8
9
. (a) Li, H.; Fan, J.; Wang, J.; Tian, M.; Du, J.; Sun, S.; Sun, P.; Peng, X. Chem.
Commun. 2009, 5904; (b) Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tunga, C.-
H.; Yang, Q.-Z. Chem. Commun. 2013, 1294; (c) Wang, H.; Zhou, G.; Gai, H.; Chen,
X. Chem. Commun. 2012, 8341; (d) Xu, Y.; Li, B.; Xiao, L.; Li, W.; Zhang, C.; Sun,
S.; Pang, Y. Chem. Commun. 2013, 7732.
. (a) McQuade, D. T.; Hegedus, A. H.; Swager, T. M. J. Am. Chem. Soc. 2000, 122,
Acknowledgements
12389; (b) Jun, M. E.; Roy, B.; Ahn, K. H. Chem. Commun. 2011, 7583.
1
0. Lin, W.; Long, L.; Yuan, L.; Cao, Z.; Chen, B.; Tan, W. Org. Lett. 2008, 10,
577.
1. Thomsen, V.; Schatzlein, D.; Mercuro, D. Spectroscopy 2003, 18, 112.
12. Zhang, X.; Ren, X.; Xu, Q.; Loh, K. P.; Chen, Z. Org. Lett. 2009, 11, 1257.
5
Financial support from National Research Foundation of Korea
1
(
NRF) funded by Korean government via Basic Science Research