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obtained with an FLS-920 Fluores-
cence Spectrometer (Edinburgh In-
struments, UK). Absorption spectra
were recorded on a Varian Cary
100 UV/Vis spectrophotometer.
The fluorescence images of living
cells were obtained with
a
confocal laser scanning micro-
scope (TCS SP5, Leica, Wetzlar,
Germany). The MTT analysis was
recorded on a microplate reader
(Bio-Tek ELx800, USA).
Synthesis
Synthesis of o-Dm-Ac: First, com-
pound I was synthesized according
to our previous report.[30] In brief,
Figure 6. Confocal fluorescence images of HepG2 cells. a) Cells without o-Dm-Ac as a control. c) Cells incubated
with 2 mm o-Dm-Ac for 1 min. e) Cells treated with 1 mm BSO for 24 h without o-Dm-Ac at 378C. g) Cells
pretreated with 1 mm BSO for 24 h at 378C and then incubated with 2 mm o-Dm-Ac for 1 min. b), d), f), and
h) Corresponding bright-field images.
o-phenylenediamine
(0.7 g,
6.5 mmol), acridine hydrochloride
(0.7 g, 3.2 mmol), and sulfur
(0.45 g) were placed in a flask, and
then the mixture of solids was
slowly heated to 1408C with vigo-
match the distance between sulfydryl groups in a protein mol-
rous stirring for about 4 h until no more H2S gas was emitted.
Then the melt was cooled and washed with diethyl ether (2ꢁ
50 mL). The black solid was extracted with 10% HCl (100 mL) to
give a dark brown solution. The solution was basified with NH3
(aq) and a brown solid was precipitated. The solid was collected by
filtration and dried under vacuum at 508C to give I as a yellow-
brown powder. The vicinal maleimide compound was prepared ac-
cording to a previously published synthetic route.[17] Diamine I
(373 mg, 1.31 mmol) and maleic anhydride (385 mg, 3.93 mmol)
were placed in a dry 100 mL round-bottom flask. Chloroform
(12 mL) was added and the solution was heated to reflux for 20 h.
The mixture was then filtered to give II as a yellow powder, which
was rinsed liberally with acetone and dried under vacuum. Acetic
anhydride (10 mL) and sodium acetate (43 mg, 0.524 mmol) were
added to this solid and the reaction was sustained for 2 h at
1008C. The mixture was then cooled to 48C, stirred vigorously for
2 h, and filtered. The brown solid obtained was dried under
vacuum and purified by recrystallization from DMF/H2O to give o-
~
ecule. Thus, two active-site-matched fluorescent probes, o-Dm-
Ac and m-Dm-Ac, were synthesized by regulating the spatial
separation of two maleimide groups in a fluorescent dye to
match that of two active cysteine residues in the conserved
amino acid sequence (–CGPC–) of human thioredoxin. Experi-
mental results showed that the two probes can specifically
detect dicysteine-containing motifs in peptides and proteins,
and o-Dm-Ac exhibits a higher sensitivity to W-7 and rTrx,
which implies a positive correlation between active-site match-
ing and sensitivity of detection. More importantly, o-Dm-Ac
was successfully used to image VSPPs rapidly and directly both
in vitro and in living cells. The probe-design strategy is instruc-
tive for the design of other protein probes, and our probe will
provide a good chemical tool to help explore the potential
function of VSPPs in living cells.
Dm-Ac (0.5 g, 85%). IR (KBr): n=3100, 1717 cmÀ1
;
1H NMR
(300 MHz, DMSO): d=7.18–7.24 (d, 2H, J=18 Hz), 7.59–7.65 (m,
5H), 7.71–7.75 (d, 4H, J=12 Hz), 7.86–7.91 (d, 2H, J=15 Hz), 8.23–
8.26 ppm (d, 2H, J=9 Hz); HRMS (ESI-TOF): m/z [M+H]+ calcd:
446.4266; found: 446.4257; elemental analysis (%) calcd for
C27H15N3O4: C 72.80, H 3.39, N 9.43; found: C 72.82, H 3.40, N
9.47%.
Experimental Section
Materials and apparatus
Bovine serum albumin (BSA), thioredoxin (Trx), l-buthionine sulfoxi-
mine (BSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), and tris(2-carboxyethyl)phosphine (TCEP) were pur-
chased from Sigma-Aldrich. GSH-Glo Glutathione Assay was ob-
tained from Promega. All other chemicals and solvents used were
local products of analytical grade. Small peptides WCGPCK,
WCGGPCK, and WCGGGPCK were provided by GL Biochem Ltd
(Shanghai, P. R. China). Stock solutions (1.00 mm) of o-Dm-Ac and
m-Dm-Ac were prepared by dissolution in DMSO. Ultrapure water
(18.2 MWcm) was used throughout the analytical experiments.
HepG2 cells were purchased from the Committee on Type Culture
Collection of the Chinese Academy of Sciences. Elemental analysis
was performed on an Element Analyzer (CHNS/O II, PerkinElmer,
Synthesis of m-Dm-Ac: The protocol used to obtain m-Dm-Ac was
followed, except that m-phenylenediamine was used instead of o-
1
phenylenediamine. IR (KBr): n=3100, 1713 cmÀ1; H NMR (300 MHz,
~
DMSO): d=6.74 (s, 1H), 7.18–7.30 (m, 4H), 7.48–7.59 (m, 4H), 7.70–
7.81 (m, 4H), 8.13–8.16 ppm (d, 2H, J=9 Hz); HRMS (ESI-TOF): m/z
[M+H]+ calcd: 446.4257; found 446.4251; elemental analysis (%)
calcd for C27H15N3O4: C 72.80, H 3.39, N 9.43; found: C 72.79, H
3.42, N 9.47.
SDS-PAGE and fluorescence imaging of gels
1
USA). H NMR spectra were recorded on an AVANCE 300 spectrom-
The selectivity of the two probes was confirmed by 12% SDS-
PAGE. Samples were labeled in PBS at 378C for 5 min with
a probe-to-protein ratio of 4:1. After labeling, 20 mg of the protein
per well was loaded in SDS-PAGE gel. Then, electrophoresis was
eter (Bruker, Switzerland). ESI mass spectra were recorded on
a QTOF Micro YA 263 mass spectrometer. IR spectra were recorded
on a Nicolet Nexus 770 spectrometer. Fluorescence spectra were
Chem. Eur. J. 2015, 21, 2117 – 2122
2121
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