T. Liu et al. / Dyes and Pigments 99 (2013) 537e542
539
Fig. 2. a) Time dependence of fluorescence intensity of NIeNHS (10
profiles of NIeNHS (10 M) with 20 equiv H2S.
m
M) at 550 nm with 20 equiv H2S (CH3CN:PBS ¼ 1:9, pH ¼ 7.4, 37 ꢁC). b) Time dependence of fluorescence
m
4.14 (m, 2H), 4.12 (s, 3H), 1.79e1.66 (m, 2H), 1.46 (m, 2H), 0.98 (t,
3H). HRMS (ESI) calcd for C17H17NO3 [MHþ] 283.1208, found
283.1205.
solid (0.91 g, 56.2%). 1HNMR (400 MHz, CDCl3)
8.71 (d, J ¼ 8.0, 1H), 8.59 (d, J ¼ 8.0 Hz, 1H), 8.48 (d, J ¼ 8.4 Hz, 1H),
8.44 (d, J ¼ 8.0 Hz, 1H), 7.85 (m, J ¼ 8.4 Hz, 1H), 7.23 (m, 2H), 4.28e
4.14 (m, 2H), 1.73 (m, 2H), 1.46 (m, 2H), 0.99 (t, 3H). 13C NMR
d
8.98 (d, J ¼ 8.0, 1H),
2.2.3. Synthesis of N- butyl -4-hydroxy-1,8-naphthalimide (1)
A mixture of compound 4 (1 g, 3.5 mmol) and 50 mL concen-
trated HI (57%) was refluxed for 6 h. After cooling and adjusting pH
to neutral, the precipitate was filtered to give compound 1 as yellow
(100 MHz, CDCl3) d 163.80, 163.18, 155.30, 153.97, 143.2, 140.65,
132.48, 131.89, 129.88, 129.27, 127.96, 127.61, 124.20, 123.18, 122.45,
121.06, 120.39, 114.46, 40.40, 30.20, 20.38, 13.84. HRMS (ESI) calcd
for C22H17N3O7 [MHþ] 435.1066, found 435.1069. Anal Calc for
needles (0.81 g, yield :86.2%). 1HNMR (400 MHz, DMSO)
d
11.85 (s,
C22H17N3O7: C 60.66; H 3.94; N 9.64; O 25.72. Found C 60.62; H
1H), 8.50 (d, J ¼ 8.3 Hz, 1H), 8.43 (d, J ¼ 7.2 Hz, 1H), 8.33 (d,
J ¼ 8.2 Hz, 1H), 7.73 (t, J ¼ 7.8 Hz, 1H), 7.14 (d, J ¼ 8.2 Hz, 1H), 4.01 (t,
J ¼ 7.3 Hz, 2H), 2.53 (s, 1H), 1.68e1.52 (m, 2H), 1.42e1.25 (m, 2H),
0.93 (t, 3H). HRMS (ESI) calcd for C16H15NO3 [MHþ] 269.1052, found
269.1051.
3.95; N 9.66; O 25.73.
2.3. Culture of MCF-7 cells and fluorescent imaging
MCF-7(human breast carcinoma) was cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Invitrogen) supplemented with
10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and 95%
air at 37 ꢁC. The cells were seeded in 24-well flat-bottomed plates
2.2.4. Synthesis of NIeNHS
Compound 1 (1 g, 3.7 mmol), 1-bromine-2,4-dinitrobenzene
(1.5 g, 6.14 mmol) and K2CO3 (0.848 g, 6.14 mmol) were dissolved
in anhydrous DMF (10 mL). The reaction mixture was then heated
at 90 ꢁC for 4 h under N2 atmosphere. Cooling to room temperature,
the reaction mixture was poured into ice water (100 mL). The crude
product was extracted with ethyl acetate (3 ꢀ 25 mL) and dried
over MgSO4, and purified by flash column chromatography (ethyl
acetate/CH2Cl2 ¼ 1/1) to obtain the compound NIeNHS as a white
and then incubated for 24 h at 37 ꢁC under 5% CO2. NIeNHS (5
mM)
was then added to the cells and incubation for another 30 min
followed. The cells were washed three times with phosphate-
buffered saline (PBS). Fluorescence imaging was observed under a
confocal microscopy (Olympus FV1000) with a 60 ꢀ objective lens.
3. Results and discussion
3.1. The spectroscopic properties of NIeNHS with H2S in aqueous
solutions
700
600
500
400
300
200
100
0
The absorption and fluorescence titration experiments of NIe
NHS with H2S were recorded in aqueous solution (CH3CN:PBS ¼ 1:9,
pH ¼ 7.4, 10
mM NIeNHS) (Fig. 1). In the absence of H2S, NIeNHS
presented a major absorption band at 358 nm. On addition of 0e20
equiv of H2S to the solution of NIeNHS, the absorbance at 358 nm
decreased sharply to its limiting value, while a new absorption band
centered at 438 nm developed which induced the colour change
from colourless to yellow (Fig. S1). The free NIeNHS displayed quite
weak fluorescence. Importantly, with the addition of NaHS, the
fluorescence intensity of NIeNHS increased significantly at 550 nm
(37 fold) due to the thiolysis of the dinitrophenyl ether by H2S. The
MS and HPLC analysis confirmed that the fluorescence emission and
enhancement was due to the formation of compound 1 (Fig. S2 and
3). The detection limit was calculated to be 0.18
(Fig. S4).
The influence of pH on the fluorescence of NIeNHS was deter-
mined by fluorescence titration (Fig. S5). The fluorescence at
550 nm of NIeNHS remains unaffected between pH 9-6.5, then
gradually decreases from pH 6.5 to pH 3, and below pH 3 slight
m
M (S/N ¼ 3)
Fig. 3. Fluorescence responses of 10 mM NIeNHS to various analytes in aqueous so-
lutions (CH3CN:PBS ¼ 1:9, pH ¼ 7.4, 37 ꢁC). lex ¼ 450 nm. Bars represent the final
fluorescence intensity of NIeNHS with 1 mM analytes over the original emission of
free NIeNHS. 1) free NIeNHS; 2) Na þ; 3) Kþ; 4) Mg2þ; 5) Ca2þ; 6) Zn2þ; 7) Fe; 8) Cle
;
;
9) Bre; 10) Ie; 11) CO32e; 12) H2O2; 13) SO42e; 14)HCO-3; 15) NO2e; 16) CH3COOe
17) HSO4e; 18) PO43e; 19) CH3COOe; 20) N3e; 21) S2O3e; 22) S2O4e; 23) S2Oe5 ; 24) homo-
cysteine; 25) ascorbic acid; 26) Cysteine; 27) Glutathione; 28) NaHS.