W. Lin et al.
Preparation of probe 1: 3-Acetyl-7-diethylaminocoumarin (2; 200.0 mg,
0.77 mmol) and 2-pyridinecarboxaldehyde (85.8 mg, 0.80 mmol) were dis-
solved in ethanol/acetonitrile (5 mL; 1:1), and then three drops of piperi-
dine were added as a catalyst. The mixture was heated to reflux for 12 h,
and the solvent was removed under reduced pressure. The resulting resi-
due was then purified by chromatography on silica gel with acetone/pe-
troleum ether (1:5) as the eluent to give 1 as a brick-red solid (114.3 mg,
0.328 mmol, 42.6%). M.p. 144–1468C; 1H NMR (400 MHz, [D1]CDCl3,
TMS): d=1.26 (t, 6H), 3.47 (q, 4H), 6.50 (d, J=2.4 Hz, 1H), 6.62–6.65
(dd, J1 =8.8 Hz, J2 =2.8 Hz, 1H), 7.30 (t, 1H), 7.44 (d, J=8.8 Hz, 1H),
7.66 (d, J=8.8 Hz, 1H) , 7.52–7.79 (t, 1H), 7.81–7.85 (d, J=15.2 Hz, 1H),
8.44–8.48 (d, J=15.6 Hz,1H), 8.56 (s, 1H), 8.70 ppm (d, J=4.0 Hz, 1H);
UV/Vis (water): lmax (log e)=466 nm (4.49); ESIMS: m/z (%): 349.2
(100) [M++H], 371.1 (23) [M++Na]; elemental analysis calcd (%) for
C21H20N2O3: C 72.40, H 5.79, N 8.04; found: C 72.77, H 5.90, N 7.68.
422–432; d) M. T. Heafield, S. Fearn, G. B. Stevenson, R. H.
Malinow, F. J. Nieto, M. Szklo, L. E. Chambless, G. Bond, Circula-
tion 1993, 87, 1107–1113; g) M. Soinio, J. Marniemi, M. Laakso, S.
Lehto, T. Ronnemaa, Ann. Intern. Med. 2000, 140, 94–101; h) E.
Sundaramoorthy, S. Maiti, S. K. Brahmachari, S. Sengupta, Proteins
ques, I. H. Rosenberg, R. B. D. Agostino, P. W. F. Wilson, P. A. Wolf,
Blom, D. van Oppenraaij, H. Przuntek, A. Kretschmer, T. Bꢄttner,
1405–1420; c) I. Rahman, W. MacNee, Am. J. Physiol. 1999, 277,
L1067–L1088; d) A. M. Cantin, S. L. North, R. C. Hubbard, R. G.
Crystal, J. Appl. Physiol. 1987, 63, 152–157; e) L. J. Smith, M. Hous-
ton, J. Anderson, Am. Rev. Respir. Dis. 1993, 147, 1461–1464.
[5] For selected examples, see: a) A. R. Ivanov, I. V. Nazimov, L. A.
Kaniowska, G. Chwatko, R. Glowacki, P. Kubalczyk, E. Bald, J.
wara, Y. Mukaib, T. Togawa, T. Suzuki, S. Tanabe, K. Ishii, J. Chro-
Spectral measurements: The amino acids (Cys, Hcy, Arg, Glu, Ala, Val,
Ser, Leu, and Lys), GSH, metal ions (Na+, K+, Ca2+, Mg2+, Fe3+, and
Zn2+), reactive oxygen species (hydrogen peroxide), reducing agent
(NADH), nucleosides (cytosine, thymine, adenine, and guanine), and glu-
cose stock solutions were prepared in twice-distilled water. Probe 1 was
dissolved in buffer solution (25 mm phosphate buffer, pH 7.4) at room
temperature to afford the probe stock solution (14 mm). Test solutions
were prepared by placing probe stock solution (1.5 mL) and an appropri-
ate aliquot of each analyte stock into a flask and then diluting the solu-
tion to a volume of 3 mL with buffer solution. The resulting solution was
shaken well before the absorption and emission spectra were recorded.
Unless otherwise noted, for all measurements, the excitation wavelength
was 444 nm and the excitation and emission slit widths were 5 nm. The
thiol assay in biological fluids typically requires the reduction of disul-
fides to free thiols. This can be accomplished by using triphenylphosphi-
ne.[6a,8c] Thus, a newborn-calf serum solution obtained from a commercial
source or a human urine sample from a healthy volunteer was treated
with excess triphenylphosphine. Aliquots of the newborn-calf serum solu-
tion or the human urine sample after reduction were then added directly
to probe 1 (7 mm) in buffer solution (3 mL; 25 mm phosphate buffer,
pH 7.4), and the emission at 496 nm was recorded. The unknown amount
of thiols in the humane urine sample was estimated by using the standard
addition method with Cys as the standard.[11c]
[6] For selected examples, see: a) A. R. Ivanova, I. V. Nazimova, L. A.
[7] For selected examples, see: a) D. Potesil, J. Petrlova, V. Adam, J.
Vacek, B. Klejdus, J. Zehnalek, L. Trnkova, L. Havel, R. Kizek, J.
W. J. R. Santos, R. d. C. S. Luz, F. S. Damos, A. B. Oliveira, M. O. F.
[8] For selected examples, see: a) N. Shao, J. Y. Jin, S. M. Cheung, R. H.
Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F. B.
439; c) W. Wang, J. O. Escobedo, C. M. Lawrence, R. M. Strongin, J.
Kim, J. O. Escobedo, S. O. Fakayode, K. A. Fletcher, M. Lowry,
C. M. Schowalter, C. M. Lawrence, F. R. Fronczek, I. M. Warner,
Acknowledgements
Funding was partially provided by the Key Project of Chinese Ministry
of Education (grant no.: 108167), the National Science Foundation of
China (grant no.: 20872032), the Scientific Research Foundation for the
Returned Overseas Chinese Scholars, State Education Ministry (grant
no.: 2007-24), and the Hunan University research funds.
[1] a) R. Hong, G. Han, J. M. Fernꢃndea, B.-J. Kim, N. S. Forbes, V. M.
4911; c) R. O. Ball, G. Courtney-Martin, P. B. Pencharz, J. Nutr.
2006, 136, 1682S–1693S; d) R. Carmel, D. W. Jacobsen, Homocys-
teine in Health and Disease, Cambridge University Press, Cambridge,
143–153; f) P. J. Wlodek , M. B. Iciek , A. Milkowski , O. B. Smolen-
Sahbaz, F. N. Ertas, G. Nisli, Turk. J. Chem. 2003, 27 , 513–520;
i) N. F. Zakharchuk, N. S. Borisova, T. V. Titova, J. Anal. Chem.
2008, 63, 171–179; j) M. Kemp, Y.-M. Go, D. P. Jones, Free Radical
[9] For a selected example, see: Y. Sato, T. Iwata, S. Tokutomi, H. Kan-
[10] For selected examples, see: a) N. Burford, M. D. Eelman, D. E.
D. T. Mooney, G. Clark-Scannell, T. T.-H. Tong, J. Watson, T. M.
Hagen, J. F. Stevens, C. S. Maier, J. Pept. Res. 2005, 4, 1403–1412;
c) X. Guan, B. Hoffman, C. Dwivedi, D. P. Matthees, J. Pharm.
[11] For selected examples, see: a) M. Zhang, M. Yu, F. Li, M. Zhu, M.
Li, Y. Gao, L. Li, Z. Liu, J. Zhang, D. Zhang, T. Yi, C. Huang, J.
[3] a) H. Refsum, P. M. Ueland, O. Nygard, S. E. Vollset, Annu. Rev.
Med. 1998, 49, 31–62; b) O. Nekrassova, N. S. Lawrence, R. G.
5102
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 5096 – 5103