Highly selective fluorescent chemosensor with red shift for cysteine in buffer solution and its bioimage: symmetrical naphthalimide aldehyde

Article abstract of DOI:10.1016/j.tetlet.2008.09.021

A new fluorescent probe 1, N-butyl-4, 5-(p-aldehyde)phenyl-1,8-naphthalimide, was designed and synthesized for the determination of the cysteine (Cys). Upon addition of Cys, the emission of 1 was enhanced with about 25 nm red-shift in the emission maximum (from 455 to 480 nm), accompanied with the fluorescent color change from blue to cyan, which was attributed to the reaction of the aldehyde groups in 1 with cysteine to form very stable thiazolidines derivative. Compound 1 was highly selective for cysteine detection without the interference of other amino acids and can be used for bioimaging of Cys.

Full text of DOI:10.1016/j.tetlet.2008.09.021

Tetrahedron Letters 49 (2008) 6624–6627
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Highly selective fluorescent chemosensor with red shift for cysteine in
buffer solution and its bioimage: symmetrical naphthalimide aldehyde
*
Liping Duan, Yufang Xu, Xuhong Qian , Fang Wang, Jianwen Liu, Tanyu Cheng
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 June 2008
Revised 12 August 2008
Accepted 5 September 2008
Available online 10 September 2008
A new fluorescent probe 1, N-butyl-4, 5-(p-aldehyde)phenyl-1,8-naphthalimide, was designed and syn-
thesized for the determination of the cysteine (Cys). Upon addition of Cys, the emission of 1 was
enhanced with about 25 nm red-shift in the emission maximum (from 455 to 480 nm), accompanied
with the fluorescent color change from blue to cyan, which was attributed to the reaction of the aldehyde
groups in 1 with cysteine to form very stable thiazolidines derivative. Compound 1 was highly selective
for cysteine detection without the interference of other amino acids and can be used for bioimaging of
Cys.
Ó 2008 Published by Elsevier Ltd.
Cysteine deficiency causes many diseases such as slowed
growth in childhood, depigmentation of hair, edema, lethargy, liver
damage, and loss of muscle.1–5 In light of the important roles of Cys
in a variety of fundamental physicological processes in organisms,
selective and sensitive detections for Cys have gained tremendous
attentions. The determination of Cys is generally accomplished by
high-performance liquid chromatography (HPLC) or high-perfor-
mance capillary electrophoresis (HPCE).6–12 There are also some
examples of fluorescence quenching chemosensors and UV/vis
spectroscopic response for Cys in organic solution.13–16 However,
there are a few chemosensors with fluorescence enhancement
(FE) for Cys in organic solutions, and only two reports on fluores-
cence turn-on sensing and bioimaging of Cys in aqueous solu-
tions.17 Therefore, we stress to pay more attention to novel
selective chemosensors with FE for Cys in aqueous solution.
Electron donor (D)–electron acceptor (A) interactions play a
fundamental role in organic chemistry. Reactivities and physical
properties of organic compounds largely depend on the strength
of the D–A interactions. The guest-binding will induce changes in
two-channel output signals (color change and fluorescence varia-
tion), which are convenient and sensitive for practical utilization,
especially when the substituent in the D–p–A system was strongly
electron-withdrawing or electron-acceptor. 4,5-Substituted-1,8-
naphthalimide, a frequently used fluoroionophore, possesses desir-
able spectroscopic properties. In addition, the special reaction of
aldehydes with N-terminal cysteines to form thiazolidines has
O
N
O
O
N
O
O
N
O
O
N
O
c
a
b
NH2
I
NO2
Br
Br
Br
O
O
2
4
3
1
Scheme 1. Synthesis of 1. Reagents and conditions: (a) SnCl2ꢀ2H2O, AcOH, HCl (g), room temperature, yield: 90%; (b) NaNO2, HCl, KI, ꢁ5 to 0 °C, yield: 48%; (c) p-
aldehydephenylboric acid, Pd(PPh3)4, toluene, N2, reflux, yield: 85%.
* Corresponding author.
E-mail address: xhqian@ecust.edu.cn (X. Qian).
0040-4039/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2008.09.021

L. Duan et al. / Tetrahedron Letters 49 (2008) 6624–6627
6625
150
100
Figure 3. 1H NMR spectra of 1 in DMSO-d6 in the absence (a) and the presence of
40 equiv of Cys at 25 °C (b).
2
4
6
8
10
12
PH
Figure 1. Influence of pH on the fluorescence of 1 in the ethanol–water (60:40, v/v)
solution, IF = fluorescence intensity, excitation wavelength = 365 nm, emission
wavelength = 455 nm, [1] = 10 lm.
1.8-naphthalimide 320 and N-butyl-4-bromo-5-iodo-1.8-naphthal-
imide 4,21 the target compound N-Butyl-4, 5-(p-aldehyde)phenyl-
1,8-naphthalimide was obtained (Scheme 1);22 its fluorescence
quantum yield is 0.30 in absolute ethanol, which was determined
by using quinine sulphate as standard.
The fluorescence of sensors is usually disturbed by proton in the
detection of metal ions, so their low sensitivity to pH is desired.
The effect of pH on the fluorescence of 1 was determined in etha-
nol–water (60:40, v/v) solution. The emission spectra of 1 have no
been used to label and immobilize peptides and proteins.18 So we
designed a simple and aqueous-soluble chemosensor 1, which is
composed of the naphthalimide fluorophore and the two alde-
hydes as Cys receptors.
From the starting material N-butyl-4-bromo-5-nitro-1, 8-naph-
thalimide,19 through the intermediate N-butyl-4-bromo-5-amino-
300
0.3
200
100
0
0.2
Cys
Cys
0.1
0.0
300
400
500
450
500
550
600
Wavethlength (nm)
Wavelength (nm)
Figure 2. UV–vis and emission spectra of 1 (10
HEPES buffer (50 mM) at 25 °C. kEX = 375 nm.
lm) with the addition of Cys (0–400
lm) in a mixed solution of ethanol and water (60:40, v/v) at pH 7.2 maintained with
O
N
O
O
N
O
O
HO
SH
H2N
NH
HO
HN
S
S
O
O
O
O
5
1
OH
Scheme 2. The change in fluorescent color and possible reaction.

6626
L. Duan et al. / Tetrahedron Letters 49 (2008) 6624–6627
that thiazolidine was formed by the interaction of aldehyde with
Cys.
A possible mechanism was proposed as shown in Scheme 2.
When Cys was added, the CHO group reacted with Cys to form
thiazolide (compound 5).
350
300
250
200
150
100
50
Selectivity is an important issue in the probe application of ami-
no acids detection. The effects of some relative amino acids on the
fluorescence spectra of 1 were determined. As shown in Figure 4,
the emission spectra of 1 in a mixed solution of ethanol and water
(60:40, v/v) at pH 7.2 maintained with HEPES buffer (50 mM) were
also investigated with the addition of other amino acids. The re-
sults showed that upon addition of other amino acids, slight fluo-
rescence quenching was observed without any color change,
which indicated that the CHO group only reacted with Cys, which
was the key for the selective recognition of Cys. Thus, we con-
firmed that this new probe offers good selectivity.
We also investigated the living cell bioimaging of Cys by using
DMSO–PBS(1:80, v/v, pH 7) as a staining medium. V79 cells were
used for the evaluation of 1 as potential cell imaging agent for
Cys, which were maintained as exponentially growing suspension
cultures in Eagle’s minimal essential medium with Earle’s salts,
modified for suspension cultures with 7.5% fetal calf serum. After
incubation with 1 for 2 h, it showed an intense intracellular fluo-
rescence (1 was added to cell suspension to give the appropriate
0
400
450
500
550
600
Wavelength(nm)
Figure 4. Fluorescence spectra of 1 (10 lm) in a mixed solution of ethanol and
water (60:40, v/v) at pH 7.2 maintained with HEPES buffer (50 mM) in the presence
of different amino acids (40 equiv) at 25 °C, and nearly no response to other amino
acids detected (alanine, arginine, asparagine, glutamine, glycine, histidine, isoleu-
cine, leucine, lysine, methionine, proline, serine, threonine, tyrosine, and valine).
obvious change between pH 12.0 and 2.00 (Fig. 1). So the detec-
tions of metal cations were operated in the ethanol–water
(60:40, v/v) solution at pH 7.2 maintained with HEPES buffer
(50 mM).
The resulting absorption and fluorescence spectra are shown in
Figure 2. Upon the addition of Cys to the probe solution, the absor-
bance at 375 nm decreased and the maximum of the absorption
peak shifted to the shorter wavelength (to 370 nm). At the same
time, its emission spectra also displayed a significant change
(Fig. 2): the fluorescence intensity increased with about 25 nm
red-shift in emission band (from 455 to 480 nm), accompanied
with a fluorescent color change from blue to cyan. The fluorescence
quantum yield of 1 increased from 0.25 to 0.40.
concentration at 10 lM) (Fig. 5).
In summary, an aqueous soluble fluorescent chemosensor 1 for
recognition of Cys was designed and synthesized. It showed high
selectivity for Cys by fluorometric enhancement with red-shift in
aqueous solution. Moreover, fluorescence images indicate that 1
can be used for bioimaging of Cys in living cell.
Acknowledgments
Compound 1 is a A–F–A0
p–p conjugation system (A—electron
This work is supported by the National Natural Science Founda-
tion of China (20536010), Program of Shanghai Subject Chief Scien-
tist and Shanghai Leading Academic Discipline Project(B507), and
the National High Technology Research and Development Program
of China (863 Program 2006AA10A201).
accepting group, imide; F—aromatic fluorophore; A0—electron
accepting group, phenyl formaldehyde) due to the electron pull
effects of imide group and phenyl formaldehyde. When 1 reacts
with Cys to form compound 5, the
p–p conjugation will transform
to A–F–D conjugation system (A—electron accepting group,
p–p
imide; F—aromatic fluorophore; D—electron donating group, alkyl
substituent with sulfur and amino group) because of the slight
electron push effect of alkyl group. Therefore, the push–pull effect
is more efficient for compound 5, which leads to higher fluores-
cence yield accompanied with red-shift in emission spectrum.
Similar results were obtained when Cys was added to 0.1 M car-
bonate buffer solution of 1, which revealed that the Cys detection
could be operated in aqueous solution.
References and notes
1. (a) Lentz, S. R.; Haynes, W. G. Clin. J. Med. 2004, 71, 730; (b) Tang, B.; Xing, Y.; Li,
P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666.
2. Steegerstheunissen, R. P. M.; Boers, G. H. J.; Trijbels, F. J. M.; Eskes, T. K. A. B. N.
N. Engl. J. Med. 1991, 324, 199.
3. Ueland, P. M.; Vollset, S. E. Clin. Chem. 2004, 50, 1293.
4. (a) Fu, Y.; Li, H.; Hu, W.; Zhu, D. Chem. Commun. 2005, 25, 3189; (b) Li, S.; Yu, C.;
Xu, J. Chem. Commun. 2005, 4, 450; (c) Tanaka, F.; Mase, N.; Barbas, C. F., III.
Chem. Commun. 2004, 15, 1762.
5. (a) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew.
Chem., Int. Ed. 2005, 44, 2922; (b) Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T.
Angew. Chem., Int. Ed. 2006, 45, 1810.
6. (a) Chwatko, G.; Bald, E. Talanta 2000, 52, 509; (b) Tcherkas, Y. V.; Denisenko, A.
D. J. Chromatogr. A 2001, 913, 309; (c) Kaniowska, E.; Chwatko, G.; Glowacki, R.;
Kubalczyk, P.; Bald, E. J. Chromatogr. A 1998, 798, 27.
The 1H NMR experiments were carried out to confirm the
formation of thiazolidine derivative 1-Cys (Fig. 3). In neutral
condition, when excess Cys (in DMSO) was added into solution
of 1, the aldehyde resonance peak (9.84 ppm) of 1 disappeared
after 5 min and two new equal peaks centered at 5.63 and
5.35 ppm appeared, which could be assigned to the methine
protons of the thiazolidine diastereomer.15a These results indicated
7. Inoue, T.; Kirchhoff, J. R. Anal. Chem. 2000, 72, 5755.
8. (a) Potesil, D.; Petrlova, J.; Adam, V.; Vacek, J.; Klejdus, B.; Zehnalek, J.; Trnkova,
L.; Havel, L.; Kizek, R. J. Chromatogr. A 2005, 1084, 134; (b) Fei, S. D.; Chen, J. H.;
Figure 5. Fluorescence images of V79 cells incubated with(c, d) or without (a, b) 10
l
M compound 1 at 37 °C After 2 h incubation, scanning was taken. Magnification was
1000ꢂ. (a) Brightfield image of cells; and (b) excited in 365 nm, no obvious fluorescence was observed; (c) scanning was taken on brightfield; (d) excited at 365 nm.

L. Duan et al. / Tetrahedron Letters 49 (2008) 6624–6627
6627
Yao, S. Z.; Deng, G. H.; He, D. L.; Kuang, Y. F. Anal. Biochem. 2005, 339, 29; (c)
Houze, P.; Gamra, S.; Madelaine, I.; Bousquet, B.; Gourmel, B. J. Clin. Lab. Anal.
2001, 15, 144.
collected by filtration and washed with water. Crystallization from acetic acid
gave 4 as yellow needles: 3.42 g (98.8%), mp 210.5–211.2 °C, IR (KBr) v: 3460,
3370, 1700, 1650, 1630, 1560, 1420, 1280, 1150, 1030. 1H NMR (CDCl3,
400 MHz) d 0.975 (t, J = 7.2 Hz, 3H), 1.43–1.45 (m, 2H), 1.70–1.75 (m, 2H), 4.14
(t, J = 7.2 Hz, 2H), 6.20–6.40 (br s, 2H), 6.85 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz,
1H), 8.34 (d, J = 8 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) d
13.83, 20.38, 30.15, 40.12, 112.12, 112.16, 117.52, 122.74, 125.46, 131.56,
131.79, 132.49, 134.23, 150.52, 163.61, 164.01, MS (ESI+) C16H15BrN2O2
[M+H+]: 374.0.
9. (a) Amarnath, K.; Amarnath, V.; Valentine, H. L.; Valentine, W. M. Talanta 2003,
60, 1229; (b) Chwatko, G.; Bald, E. J. Chromatogr. A 2002, 949, 141.
10. Daskalakis, I.; Lucock, M. D.; Anderson, A.; Wild, J.; Schorah, C. J.; Levene, M. I.
Biomed. Chromatogr. 1996, 12, 205.
11. Sato, Y.; Iwata, T.; Tokutomi, S.; Kandori, H. J. Am. Chem. Soc. 2005, 127, 1088.
12. Wang, H.; Wang, W.; Zhang, H. Talanta 2001, 53, 1015.
13. Burford, N.; Eelman, M. D.; Mahony, D. E.; Morash, M. Chem. Commun. 2003, 2,
146.
21. Preparation of N-butyl-4-bromo-5-iodo-1.8-naphthalimide (4): 30 mL of NaNO2
(1.6 g, 0.023 mmol) aqueous solution was added dropwise to the HCl (22%,
25 mL) aqueous solution of 3 (3.5 g, 0.01 mol) at 0 °C. The reaction mixture was
stirred for 2 h at this temperature. The resulting precipitate was added to
40 mL of KI (8.85 g, 0.05 mol) aqueous solution and the mixture reacted for 1 h,
after which NaHSO3 was added to destroy excessive I2. The resulted solid was
collected by filtration and then purified by column chromatography (SiO2,
CHCl3) to give (5) as a white solid in 60% yield (2.7 g), mp 165.1–166.4 °C, IR
14. (a) Anzenbacher, P., Jr.; Try, A. C.; Miyaji, H.; Jursikova, K.; Lynch, V. M.;
Marquez, M.; Sessler, J. L. J. Am. Chem. Soc. 2000, 122, 10268; (b) Miyaji, H.;
Sessler, J. L. Angew. Chem., Int. Ed. 2001, 40, 154; (c) Miyaji, H.; Sato, W.; Sessler,
J. L. Angew. Chem., Int. Ed. 2000, 39, 1777; (d) Xu, S.; Chen, K.; Tian, H. J. Mater.
Chem. 2005, 15, 2676; (e) Han, M. S.; Kim, D. H. Tetrahedron 2004, 60, 11251; (f)
Li, S.; Yu, C.; Xu, J. Chem. Commun. 2005, 450.
15. (a) Rusin, O.; Luce, N. N. S.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I.
M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126, 438; (b)
Wang, W. H.; Escobedo, J. O.; Lawrence, C. M.; Strongin, R. M. J. Am. Chem. Soc.
2005, 127, 15949.
16. (a) Tanaka, F.; Mase, N.; Barbas, C. F., III. Chem. Commun. 2004, 1762; (b) Wang,
W. H.; Escobedo, J. O.; Lawrence, C. M.; Strongin, R. M. J. Am. Chem. Soc. 2004,
126, 340; (c) Zhang, M.; Li, M.; Zh, Q.; Li, F.; Zhang, D.; Zhang, J.; Huang, C.
Tetrahedron Lett. 2007, 48, 2329; (d) Shao, N.; Jin, J.; Cheung, S.; Yang, R.; Chan,
W.; Mo, T. Angew. Chem., Int. Ed. 2006, 45, 4944.
17. (a) Shibata, A.; Furukawa, K.; Abe, H.; Tsunedab, S.; Itoa, Y. Bioorg. Med. Chem.
Lett. 2008, 18, 2246; (b) 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.
18. (a) Tolbert, T. J.; Wong, C.-H. Angew. Chem., Int. Ed. 2002, 41, 2171; (b)
Fourneau, J. P.; Efimovsky, O.; Gaignault, J. C.; Jacquier, R.; LeRidant, C. C.R.
Acad. Sci. Ser. C 1971, 272, 1982; (c) Cooper, A. J. L.; Meinster, A. J. Biol. Chem.
1982, 257, 816.
(KBr) v: 2950, 1695, 1647, 1630, 1560, 1430, 1280, 1150, 1035 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz) d 0.985 (t, J = 7.2 Hz, 3H), 1.42–1.47 (m, 2H), 1.69–1.75 (m,
2H), 4.16 (t, J = 7.2 Hz, 2H), 8.18 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.41
(d, J = 8 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) d 13.83,
20.29, 30.20, 40.36, 107.12, 125.87, 126.67, 128.99, 135.28, 136.66, 136.87,
139.15, 139.87, 141.65, 161.56, 163.86. MS (ESI) C16H13BrINO2 [M+H+]: 457.9.
22. The preparation of N-butyl-4,5-(p-aldehyde)phenyl-1,8-naphthalimide (1): To a
solution of 46 mg (0.1 mmol) N-butyl-4-bromo-5-iodo-1,8-naphthalimide in
10 mL toluene under dry N2, 45 mg (0.3 mmol) p-aldehydephenylboric acid
and catalytic amounts of Pd(PPh3)4 were added. The mixture was then heated
at reflux for 6 h at 110 °C and monitored by TLC. After the reaction was
completed, the solvent was removed under reduced pressure. The crude
product was then purified by column chromatography (SiO2, CHCl3) to give 1
as
a
white solid in 90% yield (40 mg). Mp 174–175 °C, 1H NMR (CDCl3,
400 MHz) d 1.04 (t, J = 7.2 Hz, 3H), 1.47–1.55 (m, 2H), 1.75–1.82 (m, 2H), 4.26
(t, J = 7.2 Hz, 2H), 7.20 (d, J = 8.0 Hz, 4H), 7.52 (d, J = 8.0 Hz, 4H), 7.73 (d,
J = 7.8 Hz, 2H), 8.76 (d, J = 7.8 Hz, 2H), 9.83 (s, 2H). 13C NMR (CDCl3, 100 MHz) d
13.83, 20.39, 30.20, 40.46, 123.12, 127.57, 128.99, 130.20, 130.36, 130.77,
131.65, 134.87, 145.15, 147.24, 163.86, 191.13. HRMS (ESI) calcd for C30H23NO4
[M+Na+]: 484.1511, found: 484.1511.
19. Xu, Z.; Xiao, Y.; Qian, X.; Cui, J.; Cui, D. Org. Lett. 2005, 7, 889.
20. Preparation of N-butyl-4-bromo-5-amino-1.8-naphthalimide (3): Compound 2
(3.76 g, 0.01 mol) was added to a previously prepared solution of glacial acetic
acid (40 ml) containing 15.3 g of SnCl2 (0.08 mmol) and aerated with hydrogen
chloride. The reaction mixture was stirred for 1 h. The precipitated solid was