Probes for Vicinal-Dithiol-Containing Proteins
FULL PAPER
a HP 1100 LC-MS spectrometer. Melting points were determined by an
X-6 micro-melting point apparatus and were uncorrected. IR spectra
were recorded on a Nicolet Nexus 770 spectrometer. All pH measure-
ments were performed by using a Sartorius basic pH-Meter PB-20. Fluo-
rescence spectra were determined using a Varian Cary Eclipse fluores-
cence spectrometer. Absorption spectra were determined by a Varian
Cary 100 UV/Vis spectrophotometer.
Synthesis of target probes: The detailed syntheses of VTA2, VTA4,
VTA6, F2, and other related compounds are described in the Supporting
Information according to published procedures.[12]
Figure 8. In-situ-imaging of VDPs in Chang liver cells with S1 (5 mm).
Images were taken with an Olympus IX71 microscope at 10ꢁ, 20ꢁ and
40ꢁ magnification, respectively. Scale bar: 100, 50, 10 mm, respectively.
N-[4-(1,3,2-Dithiarsolan-2-yl)phenyl]-6-[(7-nitrobenzo[c]ACTHNUTRGNE[UNG 1,2,5]oxadiazol-
4-yl)amino]hexanamide (S1): Et3N (55 mL, 0.393 mmol) was added to a
solution of VTA4 (122 mg, 0.328 mmol) in dry dichloromethane, followed
by addition of 4-chloro-7-nitrobenzo[c]ACTHNUGTRNEUNG[1,2,5]-oxadiazole (NBD-Cl,78 mg,
0.393 mmol) in dry dichloromethane (or another procedure was that
VTA4 was dissolved in dry acetonitrile, added to potassium carbonate,
then NBD-Cl in dry acetonitrile was added dropwise to the reaction mix-
ture) at room temperature under an Ar atmosphere overnight. TLC
showed that the reaction was complete, and then the solvent was re-
moved under reduced pressure. The crude product was purified by chro-
matography on a silica gel column (CH2Cl2/methanol, 25:1, v/v) to give
S1 as a brown powder (58 mg, 33% yield). S1 was also can be prepared
through the conjugation of NBD derivative N1 with VTA2 in a relatively
high yield (detailed procedures was described in the Supporting Informa-
tion). 1H NMR (400 MHz, CDCl3): d=8.50 (d, J=8.8 Hz, 1H), 7.63 (d,
J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.21 (s, 1H), 6.43 (br, 1H), 6.20
(d, J=8.8 Hz, 1H), 3.56 (q, J=6.4 Hz, 2H), 3.41–3.35 (m, 2H), 3.22–3.15
(m, 2H), 2.46 (t, J=7.2 Hz, 2H), 1.93–1.82 (m, 4H), 1.64–1.56 ppm (m,
2H); 13C NMR (100 MHz, CDCl3): d=170.7, 144.2, 143.9, 139.0, 138.5,
136.4, 131.6, 123.9, 119.4, 98.5, 43.5, 41.8, 37.0, 28.0, 26.2, 24.3 ppm; IR
according to the features of the probes (such as the fluores-
cence response of the probes at different pH values, differ-
ent logP values, and electrostatic potentials, etc.) in living
cells. Meanwhile, these probes afford convenient tools for
the ongoing proteomics study of various disease-related
VDPs.
Conclusion
In summary, we have expanded the conjugation approaches
to afford a series of chemical probes through attachment of
various functional tags (naphthalimide, NBD, fluorescein,
and biotin) to different linkers (6-aminocaproic acid, succin-
ic acid and piperazine) attached to VTA2. These different
linkers were introduced to make the synthesis more facile.
Most importantly, the property of the probes (such as logP
value, rigidity and biocompatibility, etc.) can be tuned
through these linkers. Additionally, the different characteris-
tics of the fluorophores (different pH response, hydropho-
bicity, etc.) make the probes suitable for potential labeling
of a variety of vicinal dithiols in VDPs. These new probes
will be utilized to label various types of VDPs in different
microenvironments in living cells and to identify new endog-
enous VDPs in subsequent proteomics analyses. Especially,
the fluorescence characteristics of probes S1 and S2 may
make in situ imaging of vicinal thiols existing in different re-
gions of VDPs in living cells possible through direct fluores-
cence readout. This versatile chemical strategy provides an
alternative to antibody-based methods and affords a rapid,
effective and convenient tool for the in situ imaging of
VDPs and the ongoing proteomics study of various disease-
related VDPs.
(KBr): n˜ =3334, 3269, 2940, 1667, 1596, 1513, 1294, 1246, 1181 cmÀ1
;
HRMS (ES+): m/z calcd for C20H22N5O4NaS2As: 558.0227 [M++Na];
found: 558.0222.
Alternative approach for the synthesis of S1: HATU (76 mg, 0.2 mmol),
VTA2 (51 mg, 0.2 mmol) and DIPEA (35 mL, 0.2 mmol) were added to a
solution of N1 (58 mg, 0.2 mmol) in anhydrous DMF (10 mL). The mix-
ture was stirred at room temperature for about 12 h until TLC showed
that the reaction was complete. The crude product was obtained after the
solvent was removed under reduced pressure and then it was purified by
chromatography on a silica gel column (CH2Cl2: methanol, 25:1, v/v) to
afford brown powder (70 mg, 65% yield). 1H NMR (400 MHz, CDCl3):
d=8.50 (d, J=8.8 Hz, 1H), 7.63 (d, J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz,
2H), 7.21 (s, 1H), 6.43 (br, 1H), 6.20 (d, J=8.8 Hz, 1H), 3.56 (q, J=
6.4 Hz, 2H), 3.41–3.35 (m, 2H), 3.22–3.15 (m, 2H), 2.46 (t, J=7.2 Hz,
2H), 1.93–1.82 (m, 4H), 1.64–1.56 (m, 2H); 13C NMR (100 MHz,CDCl3):
d=170.7, 144.2, 143.9, 139.0, 138.5, 136.4, 131.6, 123.9, 119.4, 98.5, 43.5,
41.8, 37.0, 28.0, 26.2, 24.3 ppm; IR (KBr): 3334, 3269, 2940, 1667, 1596,
1513, 1294, 1246, 1181 cmÀ1
C20H22N5O4NaS2As: 558.0227 [M++Na]; found: 558.0222.
6-[(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]-N-phenylhexanamide
(SC1): Compound SC1 was prepared by the reaction of VTAC2 with 4-
chloro-7-nitrobenzo[c][1,2,5]- oxadiazole (NBD-Cl) to give a dark red
;
HRMS (ES+): m/z calcd for
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
solid (0.23 g, 88% yield). The procedure was the similar as synthesis of
1
S1. H NMR (400 MHz, [D6]DMSO): d=9.85 (s, 1H), 9.58 (br, 1H), 8.49
(d, J=8.8 Hz, 2H), 7.55 (d, J=7.6 Hz, 2H), 7.28–7.24 (m, 2H), 7.02–6.98
(m, 1H), 6.41 (d, J=7.2 Hz, 1H), 3.49–3.46 (m, 2H), 2.31 (t, J=7.2 Hz,
2H), 1.75–1.60 (m, 4H), 1.45–1.37 ppm (m, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=171.5, 139.7, 138.4, 134.9, 129.0, 123.3, 119.4, 111.1, 99.6,
43.6, 36.7, 27.9, 26.5, 25.2 ppm; MS (ESI): m/z (%): 370 (100) [M++H].
Experimental Section
N-[4-(1,3,2-Dithiarsolan-2-yl)phenyl]-5-{(3aS,4S,6aR)-2-oxohexahydro-
Materials and apparatus: All chemical reagents and solvents were pur-
chased from Sigma–Aldrich and were used without further purification
except for THF and N,N-dimethylformamide (DMF), which were puri-
fied according to the literature.[26] Thin-layer chromatography (TLC) was
performed on silica gel plates. Column chromatography was performed
by using silica gel (Hailang, Qingdao) 200–300 mesh. 1H and 13C NMR
spectra were recorded employing a Bruker AV-400 spectrometer with
chemical shifts expressed in parts per million (Me4Si as internal stand-
ard). Electrospray ionization (ESI) mass spectrometry was performed in
1H-thieno
ACHTUNGTRENNUNG
hexahydro-1H-thieno
AHCTUNGTRENNUNG
244 mg, 1.0 mmol) and hydroxybenzotriazole (HOBt, 27 mg, 0.2 mmol)
were suspended in anhydrous DMF (10 mL), and heated until a clear sol-
ution was obtained. Then the reaction mixture was cooled to room tem-
perature and a solution of DCC in CH2Cl2 (2.7 mL of a 1.0m solution in
CH2Cl2, 2.7 mmol) was added dropwise and the mixture remained stirring
at room temperature for another 3 h. VTA2 (311 mg, 1.2 mmol) and
Chem. Eur. J. 2013, 19, 7739 – 7747
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7745