Highly selective colorimetric sensor for cysteine and homocysteine based on azo derivatives

Article abstract of DOI:10.1016/j.tetlet.2006.07.080

A simple colorimetric method for the determination of cysteine and homocysteine has been developed. The reaction of the azo dyes containing an aldehyde group with cysteine or homocysteine afforded very stable derivatives thiazolidines or thiazinanes under

Full text of DOI:10.1016/j.tetlet.2006.07.080

Tetrahedron Letters 47 (2006) 7093–7096
Highly selective colorimetric sensor for cysteine and
homocysteine based on azo derivatives
Dengqing Zhang, Meng Zhang, Zhiqiang Liu, Mengxiao Yu, Fuyou Li,*
Tao Yi and Chunhui Huang*
Department of Chemistry and Laboratory of Advanced Materials, Fudan University, Shanghai 200433, PR China
Received 9 May 2006; revised 15 July 2006; accepted 18 July 2006
Available online 14 August 2006
Abstract—A simple colorimetric method for the determination of cysteine and homocysteine has been developed. The reaction
of the azo dyes containing an aldehyde group with cysteine or homocysteine afforded very stable derivatives thiazolidines or thia-
zinanes under neutral pH conditions. The method is selective and sensitive for cysteine and homocysteine detection without the
interference of other amino acids. Importantly, the recognition of Cys and Hcy could be observed by naked eyes.
ꢀ 2006 Elsevier Ltd. All rights reserved.
As one kind of important amino acids, cysteine (Cys)
and homocysteine (Hcy) play a crucial role in living sys-
tems.1 The deficiency of Cys is associated with slowed
growth, hair depigmentation, edema, lethargy, liver
damage, muscle and fat loss, skin lesions, and weak-
ness.2 Additionally, Cys-induced hypoglycemic brain
damage has been studied as an alternative mechanism
to excitotoxicity.3 At elevated levels in plasma, Hcy is
a risk factor for Alzheimer’s,4 cardiovascular diseases,
neutral tube defects, and osteoporosis.5 Therefore, the
determination of Cys and Hcy in body fluids is very
important from the biological and pharmacological
stand points. The cysteine and homocysteine analyses
have been often carried out with high performance
liquid chromatography (HPLC),6 capillary electro-
phoresis (CE),7 electrochemical detection,8 UV–vis
detection,9 fluorescent detection,10 fourier transform
infrared (FTIR) detection,11 and mass spectrometry
identification.12
for free amino acids,14 amine,15 and thiols16 have been
reported, selective detection of Cys and Hcy by color
change is quite limited.17,18 Strongin’s group have
reported the xanthene dyes containing aldehydes that
afforded thiazolidines or thiazinanes by the reaction
with Cys or Hcy, resulting in a color change,17 however,
this detection of Cys or Hcy has to be carried out under
alkaline condition. Cys and Hcy usually are obtained
in physiological condition, therefore, the development
of selective colorimetric sensors for Cys and Hcy is
required under neutral pH. In this letter, we report a
naked-eye sensor for Cys and Hcy employing azo dyes
4-[[40-(N,N-dimethylamino)phenyl-10-]azo]benzaldehyde
(1) and 4-[[40-(bis(2-hydroxyethyl)amino)phenyl-10-]azo]-
3-nitrobenzaldehyde (2) (Scheme 1) under neutral pH
with high selectivity and sensitivity.
Sensor 1 was synthesized according to the previous
literature.19 Sensor 2 was prepared by diazo-coupling
of 4-formyl-2-nitroaniline20 to N-phenyldiethanolamine,
and was characterized by NMR and elemental
Recently, colorimetric sensors are popular due to their
capability to detect analyte by the naked eye without
resorting to any expensive instruments.13 One key prob-
lem for Cys and Hcy detections is the discrimination of
Cys and Hcy from other amino acids, amines, and thi-
ols. Although fluorescent and visual detection methods
1
OHC
OHC
N
N
N
N
N
OH
OH
2
N
Keywords: Colorimetric detection; Cysteine; Homocysteine.
NO2
*
Corresponding authors. Tel./fax: +86 10 6275 7156; e-mail
addresses: fyli@fudan.edu.cn; chhuang@pku.edu.cn
Scheme 1. Chemical structures of compounds 1 and 2.
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.07.080

7094
D. Zhang et al. / Tetrahedron Letters 47 (2006) 7093–7096
analysis.21 The complexation abilities of 1 and 2 with
Cys and Hcy were investigated by the UV–vis absorp-
tion technique.
HO2C
H2N
b
n
SH
H
N
a
CO2—
N
N
N
CHO
N
N N
n
H
S
1a n = 1
1b n = 2
1
As shown in Figure 1, the DMF solution of 1 showed
maximum absorbance at 465 nm with the extinction
coefficient of 3.4 · 104 molꢀ1 L cmꢀ1, corresponding to
the intramolecular charge-transfer character (ICT) of
the chromophore due to the push–pull effect.22 Upon
addition of Cys, the absorption maximum of 1 shifted
from 465 to 442 nm (Fig. 1a), corresponding to an
isosbestic point at 416 nm, indicating the formation of
thiazolidine 1a (Scheme 2). Furthermore, this transfor-
HO2C
H2N
HO
HO
O2N
N N
O2N
N
n
SH
H
N
CO2—
N
N
CHO
N
H
n
S
HO
HO
2a n = 1
2b n = 2
2
Scheme 2. Reactions of 1 and 2 with Cys or Hcy to afford thiazolidines
(1a, 2a) or thiazinanes (1b, 2b), respectively.
1
mation was confirmed by H NMR and FT-IR spectra.
Upon addition of Cys, the aldehyde signal at 10.05 ppm
disappeared and the thiazolidine resonances (protons a
and b, Scheme 2) centered at d 5.74 and 5.57 ppm
appeared (see ESI). Moreover, FT-IR spectra showed
that the strong vibration band at 1701 cmꢀ1, the signal
ascribed to C@O stretching of the aldehyde group of
1, disappeared upon addition of Cys, indicating the
interaction of aldehyde group with Cys. Similar spectral
changes were also observed upon addition of Hcy (see
Fig. 1b). These data indicated that 1 could be used as
a chemosensor for Cys or Hcy.
character, and give rise to red-shift in the absorption
spectrum compared with 1. The absorption spectrum
of 2 showed a band centered at 515 nm with an extinc-
tion coefficient of 3.6 · 104 molꢀ1 L cmꢀ1, indicating
that 2 had a strong ability of absorbing light. The
absorption spectrum of 2 and its titration with Cys
and Hcy were recorded in DMF–H2O solution (9:1,
v/v) at pH 7 maintained with HEPES buffer (50 mM).
As shown in Figure 2a, upon addition of Cys, the
absorption intensity at 515 nm decreased with the con-
comitant formation of a hypsochromically shifted band
peaked at 475 nm, corresponding to a color change from
pink to yellow, which were clearly visible to the naked
eyes (see Fig. 3). Moreover, a clear isosbestic point at
488 nm was observed, indicating thiazolidine 2a was
In the light of the insolubility of 1 in water, 2 with two
hydroxyl groups was synthesized to increase the solubil-
ity in water, furthermore, the presence of a nitro group
in 2 would enhance the intramolecular charge-transfer
0.2
a
1.6
1.2
0.8
a
1.08
0.3
0.2
0.1
1.04
1.00
0.96
0.92
0
20
40
60
C (Cys) / uM
0
20
60
C (Cys) /4u0M
0.1
0.0
0.2
0.0
0.4
1.5
1.0
b
b
1.08
1.02
0.96
0.90
0.3
0.2
0.1
0.0
0
40
80
120
C (Hcy) / uM
0
60
120
180
0.1
0.0
C (Hcy) / uM
400
500
600
700
400
500
600
700
Wavelength
Wavelength
Figure 2. UV–vis spectra of 2 (5.6 lM) in the presence of different
concentrations of Cys or Hcy in DMF–H2O solution (9:1, v/v, 50 mM
HEPES, pH 7.0) at room temperature. Each spectrum is recorded
5 min after Cys or Hcy addition. As the concentration of Cys or Hcy
increases, a blue shift from 515 to 475 nm was observed. Inset:
ratiometric calibration curve A475/A515 as a function of Cys or Hcy
concentration.
Figure 1. UV–vis spectra of 1 (10 lM) in the presence of different
concentrations of Cys or Hcy in DMF solution at room temperature.
Each spectrum is recorded 5 min after Cys or Hcy addition. As the
concentration of Cys or Hcy increases, a blue shift from 465 to 442 nm
was observed. Inset: ratiometric calibration curve A442/A465 as a
function of Cys or Hcy concentration.

D. Zhang et al. / Tetrahedron Letters 47 (2006) 7093–7096
7095
and thiazinane 2b was a key for the selective recognition
of Cys and Hcy. Furthermore, compared with the assays
using thiol reagents designed for the detection of thiols
by the Michael addition,16b,23 no response of 2 upon
addition of glutathione was observed (see ESI). These
phenomena suggested the high selectivity of 2 for Cys
and Hcy over other amino acids.
Achieving high selectivity for the analyte of interest over
a complex background of potentially competing species
is a challenge in sensor development. Thus, the compe-
tition experiments were conducted in the presence of
50 equiv of one of the above other amino acids or the
mixture of them, at 5.6 lM of 2 mixed with Cys or
Hcy (20 equiv), respectively. No significant variation in
absorption intensity was found in comparison of that
containing only Cys or Hcy (Fig. 4). Moreover, no obvi-
ous interference in its absorption was observed when
performing the titrations with Cys or Hcy in the mixture
of other amino acids. These facts indicated a high selec-
tivity of 2 in sensing Cys and Hcy.
Figure 3. The color change of 2 (5.6 lM) in DMF–H2O 9:1 (v/v) in the
presence of 20 equiv of certain amino acids. From left to right and top
to bottom: absence of amino acid, phenylalanine, threonine, arginine,
histidine, asparagine, leucine, alanine, proline, valine, glycine, lysine,
glutamine, methionine, isoleucin, serine, cysteine, tryptophan, homo-
cysteine, and aspartic acid.
formed by the interaction of aldehyde with Cys, reduc-
ing the electron-withdrawing ability of 2, resulting in a
relative weak ICT of the chromophore (see Scheme 2).
The similar experimental phenomena were also observed
when Hcy was added into the solution of 2 (see Fig. 2b).
As shown in Figure 2, the dependence of the absorption
ratios at 475 nm to that of 515 nm (A475/A515) on the
concentration of Cys or Hcy indicated the obvious
change upon addition of Cys or Hcy. In the presence
of electrolyte (phosphate buffer, pH = 7.2, NaCl, 0.145
mol Lꢀ1, K2CO3, 5.5 mmol Lꢀ1, CaCl2, 12 mmol Lꢀ1),
a similar spectral shift of 2 was observed upon addition
of Cys or Hcy (see ESI).
In conclusion, we have developed a highly selective col-
orimetric chemosensor for Cys and Hcy under neutral
pH. The recognition of Cys and Hcy gave obvious color
changes from pink to yellow, which was clearly visible to
the naked eyes. Moreover, the competition experiments
showed that the interference from other amino acids was
minimal. Due to the simplicity and sensitivity of the
analysis, this sensor would have many opportunities in
a variety of settings requiring rapid and accurate Cys
and Hcy analysis. This preliminary understanding on
the Cys and Hcy sensing mechanism would actually help
to find possible structural modification to achieve new
probes that show Cys and Hcy sensing capacity in pure
water.
For an excellent chemosensor, high selectivity is a mat-
ter of necessity. Photophysical properties of 2 in DMF–
H2O (9:1, v/v) were also investigated upon addition of
other amino acids (phenylalanine, threonine, arginine,
histidine, asparagine, leucine, alanine, proline, valine,
glycine, lysine, glutamine, methionine, serine, isoleucine,
tyrosine, tryptophane, glutamic acid, and aspartic acid).
As shown in Figures 3 and 4, no obvious color changes
were observed upon addition of other amino acids com-
pared with the hypsochromic shift of 2 with Cys and
Hcy, indicating that the formation of thiazolidine 2a
Acknowledgments
The authors thank the National Science Foundation
of China (20490210 and 20501006), Huo Yingdong
Education Foundation (104012), and Shanghai Sci.
Tech. Comm. (05DJ14004 and 06QH14002) for finan-
cial support.
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.tetlet.
2006.07.080.
References and notes
1. (a) Wood, Z. A.; Schro¨der, E.; Harris, J. R.; Poole, L. B.
Trends Biochem. Sci. 2003, 28, 32; (b) Homocysteine in
Health and Disease; Carmel, R., Jacobsen, D. W., Eds.;
Cambridge University Press: Cambridge, UK, 2001.
2. Shahrokhian, S. Anal. Chem. 2001, 73, 5972.
Figure 4. A475/A515 change profile of 2 (5.6 lM) in DMF–H2O
solution (9:1, v/v, 50 mM HEPES, pH 7.0) in the presence of various
amino acids (20 equiv) (black bars), one of other amino acids or the
mixture of them with Cys (white bars) and with Hcy (gray bars).
3. Gazit, V.; Ben-Abraham, R.; Coleman, R.; Weizman, A.;
Katz, Y. Amino Acids 2004, 26, 163.

7096
D. Zhang et al. / Tetrahedron Letters 47 (2006) 7093–7096
˚
4. Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E.
A. P.; Sandanayake, K. R. A. S. Angew. Chem., Int. Ed.
Engl. 1990, 29, 1173; (f) Mizutani, T.; Wada, K.; Kita-
gawa, S. J. Am. Chem. Soc. 1999, 121, 11425; (g) Mohr, G.
J.; Demuth, C.; Spichiger-Keller, U. E. Anal. Chem. 1998,
70, 3868; (h) Reetz, M. T.; Sostmann, S. Tetrahedron 2001,
57, 2515; (i) Hirose, K.; Ogasawara, K.; Nishioka, K.;
Tobe, Y.; Naemura, K. J. Chem. Soc., Perkin Trans. 2
2000, 1984.
Annu. Rev. Med. 1998, 49, 31.
5. Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.;
Rosenberg, I. H.; D’Agostino, R. B.; Wilson, P. W. F.;
Wolf, P. A. N. Engl. J. Med. 2002, 346, 476.
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.
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.;
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.
9. (a) Amarnath, K.; Amarnath, V.; Amarnath, K.; Valen-
tine, H. L.; Valentine, W. M. Talanta 2003, 60, 1229; (b)
Chwatko, G.; Bald, E. J. Chromatogr. A 2002, 949, 141.
10. (a) Daskalakis, I.; Lucock, M. D.; Anderson, A.; Wild, J.;
Schorah, C. J.; Levene, M. I. Biomed. Chromatogr. 1996,
10, 205; (b) Wang, H.; Wang, W. S.; Zhang, H. S. Talanta
2001, 53, 1015.
16. (a) Okabe, K.; Wada, R.; Ohno, K.; Uchiyama, S.;
Santa, T.; Imai, K. J. Chromatogr. A 2002, 982, 111; (b)
Kanaoka, Y. Angew. Chem., Int. Ed. Engl. 1977, 16,
137.
17. (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.; Rusin, O.; Xu, X. Y.; 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.
18. (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, 3400.
11. Sato, Y.; Iwata, T.; Tokutomi, S.; Kandori, H. J. Am.
Chem. Soc. 2005, 127, 1088.
19. Singh, A. K.; Das, J.; Majumdar, N. J. Am. Chem. Soc.
1996, 118, 6185.
12. Burford, N.; Eelman, M. D.; Mahony, D. E.; Morash, M.
Chem. Commun. 2003, 146.
20. Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano,
T. J. Am. Chem. Soc. 2004, 126, 3357.
13. (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. H.; Yu, C. W.; Xu, J. G. Chem. Commun.
2005, 450.
14. (a) Imai, H.; Misawa, K.; Munakata, H.; Uemori, Y.
Chem. Lett. 2001, 688; (b) Ait-Haddou, H.; Wiskur, S. L.;
Lynch, V. M.; Anslyn, E. V. J. Am. Chem. Soc. 2001, 123,
11296; (c) Feuster, E. K.; Glass, T. E. J. Am. Chem. Soc.
2003, 125, 16174.
21. Data for 1: Yield 85%. 1H NMR (400 MHz, CDCl3) d
10.06 (s, 1H), 7.90–7.99 (m, 6H), 6.76 (q, 2H), 3.12 (s, 6H);
13C NMR (100 MHz, CDCl3): 191.9, 157.1, 153.3, 143.9,
136.3, 130.9, 125.9, 122.9, 111.7, 40.5; Anal. Calcd for
C15H15N3O (253.12): C, 71.13; H, 5.97; N, 16.59. Found:
C, 71.00; H, 5.90; N, 16.62.
Data for 2: Yield 40%. 1H NMR (400 MHz, CDCl3): d
10.06 (s, 1H), 8.32 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.84–
7.89 (m, 3H), 6.76 (d, J = 9.2 Hz, 2H), 3.97 (t, 4H), 3.76 (t,
4H), 3.03 (s, 2H); 13C NMR (100 MHz, DMSO-d6): 191.7,
153.8, 148.5, 147.5, 143.6, 135.9, 133.6, 127.5, 125.9, 119.8,
112.7, 58.9, 54.1; Anal. Calcd for C17H18N4O5 (358.13): C,
56.98; H, 5.06; N, 15.63. Found: C, 56.88; H, 5.00; N,
15.61.
15. (a) Kubo, Y.; Maeda, S.; Tokita, S.; Kubo, M. Nature
1996, 382, 522; (b) Kaneda, T.; Ishizaki, Y.; Misumi, S.;
Kai, Y.; Hirao, G.; Kasai, N. J. Am. Chem. Soc. 1988, 110,
2970; (c) Fuji, K.; Tsubaki, K.; Yanaka, K.; Hayashi, N.;
Otsubo, T.; Kinoshita, T. J. Am. Chem. Soc. 1999, 121,
3807; (d) Wong, W. L.; Huang, K. H.; Teng, P. F.; Lee, C.
S.; Kwong, H. L. Chem. Commun. 2004, 384; (e) de Silva,
22. Gunnlaugsson, T.; Leonard, J. P.; Murray, N. S. Org.
Lett. 2004, 6, 1557.
23. (a) Tanaka, F.; Thayumanavan, R.; Barbas, C. F., III.
J. Am. Chem. Soc. 2003, 125, 8523; (b) Mase, N.; Tanaka,
F., ; Barbas, C. F., III. Org. Lett. 2003, 5, 4369; (c) Tanaka,
F.; Thayumanavan, R.; Mase, N.; Barbas, C. F., III.
Tetrahedron Lett. 2004, 45, 325.