Ple Na es we dJ oo u nr no at l ao df jCu hs et mm i as tr rgy ins
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ARTICLE
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emit biological background auto-fluorescence that greatly salts (Na
2
S, Na
2
S
2
O
4
, KNO
, NaI, Na
3
, KSCN, NaHSO ) and biological thiols (Cys,
1
8,
DOI: 10.1039/C9NJ00210C
affects the sensitivity and accuracy of fluorescence analysis
NaNO
2
, NaN
, Na
3
, K
SO
2
P
2
O
7
2 4 3
SO , CH COONa, KF, NaCl,
2
2-23
.
The near-infrared (NIR) fluorescent probes, which have NaH
2
PO
4
2
3
2
4
fluorescence emission in the region of 650 to 900 nm , have GSH, Hcy) were purchased from Aladdin co. (Shanghai, China).
attracted increasing research interest due to their high signal- Cell lines including human ovarian adenocarcinoma (SKOV-3),
to-noise ratio, compatibility with biological samples, and deep human breast cancer (MDA-MB-231), human cervical cancer
tissue penetrability 25. Hence, developing more near-infrared (HeLa), and human liver cancer cells (HepG2) were purchased
reliable fluorescence probes for the sensitive detection and from Cell Bank of the Chinese Academy of Sciences (Shanghai,
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endogenous visualization of H
2
S in vivo is of great significance China). The UV-Vis spectra were recorded on a Beckman DU
to better understand the biological function of hydrogen 800 spectrometer (USA) and fluorescent spectra were
sulfide.
collected on a SPEX Flurolog 3-TCSPC instrument (USA) with 1
1
13
cm path length cuvettes. H and C NMR spectra was acquired
on a Bruker AVIII-400 (Germany) spectrometer and mass
spectra were acquired by AB Qstar HR-ES (USA). Fluorescent
images were taken by confocal microscopy (Olympus FV-1000-
IX81). MTT experiments were measured on 96-well plates with
Tecan M1000pro reader (Switzerland). The deionized water
was prepared on a milli-Q water purification system and used
throughout all experiments.
Dicyanoisophorone-based dyes are usually treated as
fluorophores due to their preeminent sensing abilities, such as
NIR fluorescence, remarkable large Stokes shift, great
25-26
photostability and excellent transmembrane properties
.
The 7-nitro-1, 2, 3-benzoxasiazole (NBD) moiety is generally
used as an electron-withdrawing group to effectively switch off
the inherent fluorescence of fluorescent dyes that contain
specific recognition sites for H S via thiolysis even in the
2
presence of biothiols like GSH . Therefore, the enhanced Synthesis of REDCP
18
fluorescent response of such probes can be observed after
The REDCP was synthetized according to previous report 15
.
2
cleaving the NBD moiety via H S-specific thiolysis NBD amines.
Compound
Considering the advantages of the dicyanoisophorone and malononitrile) and Compound
NBD structures, we designed a novel near-infrared fluorescent benzaldehyde) were synthesized according to the literatures
1
(2-(3, 5, 5-trimethylcyclohex-2-enylidene)
2
(4-(piperazin-1-yl)
2
7-
2
8
probe (NIR-NP) composed of these two moieties for the highly
selective detection of H S. The structure of NIR-NP and the mg, 0.54 mmol) and 4-(piperazin-1-yl) benzaldehyde (102 mg,
proposed reaction mechanism between NIR-NP and H S was 0.54 mmol) were dissolved in anhydrous ethanol (15 ml). Then,
outlined in the Scheme 1. In the presence of H S, the NBD five drops of piperidine was added to the solution under argon
moiety could be cleaved from the probe by H S-induced atmosphere. After heating at reflux for 5 h, the mixture
thiolysis of the NBD amine bond, then the dicyanoisophorone solution was cooled to room temperature and the solvent was
moiety could display the inherent fluorescence. Detection removed under reduced pressure. The crude product was
results showed a large Stokes shift (186 nm), near-infrared purified by silica gel column chromatography with
emission (λem: 670 nm), and bright turn-on fluorescence. dichloromethane/ methanol (200:1) as the eluent to obtain
. 2-(3, 5, 5-trimethylcyclohex-2-enylidene) malononitrile (100
2
2
2
2
1
Based on this strategy, we successfully applied the NIR-NP to REDCP (119 mg, 0.33 mmol) as a red solid in 61% yield. H
image and monitor endogenous hydrogen sulfide in different NMR (400 MHz, DMSO-d6): δ (ppm) = 7.56 (d, J = 8.0 Hz, 2H),
tumor cells and in tissue.
7.22 (d, J = 4.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 21H), 6.78 (s, 1H),
.19 (m, 4H), 2.81 (m, 4H), 2.59 (s, 2H), 2.53 (s, 2H), 1.01 (s,
H). C NMR (100 MHz, DMSO-d6):δ (ppm) = 170.53, 157.53,
52.81, 139.17, 130.06, 126.04, 125.84, 121.33, 114.84, 114.77,
14.02, 74.35, 48.53, 45.88, 42.88, 40.73, 40.52, 40.31, 40.10,
3
6
1
1
3
1
3
9.90, 39.69, 39.48, 38.79, 32.15, 27.98. And HRMS m/z Calcd
+
for C23
H
26
N
4
: 358.2175 [M+H] +; found: 359.2230 (Figure S4).
Synthesis and characterization of NIR-NP
.1 mL N,N-Diisopropylethylamine (DIPEA) was added into a
solution of REDCP (90 mg, 0.25 mmol) in anhydrous
0
dichloromethane (15 ml) under an argon atmosphere. After
Scheme 1 The structure of the NIR-NP (a) and the response mechanism of NIR-NP stirring for 15 min, 5 ml NBD-Cl solution (60 mg, 0.3 mmol) in
towards for recognizing H S (b).
2
anhydrous dichloromethane was added dropwise at 0°C.The
reaction was stirred at room temperature overnight. Removal
of solvent under reduced pressure and purification by silica gel
column chromatography (eluted with dichloromethane:
petroleum ether = 3/1, v/v) to afford the pure product NIR-NP
Experimental
Materials and Apparatus
1
(
110 mg, 0.21 mmol) as a red solid in 84% yield. H NMR (600
All of the materials were obtained from commercial suppliers
and used without further purification. The experimental
reagents contain dimethyl sulfoxide (DMSO) and all inorganic
MHz, CDCl3): δ 8.47 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.2 Hz, 2H),
.01 (m, J = 14.6 Hz, 1H), 6.92 (d, J = 8.2 Hz, 2H), 6.87 (d, J =
7
2
| J. Name., 2012, 00, 1-3
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