L. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1036–1039
1039
promising tool for isolation of the target proteins of OA. To our sur-
prise, the incorporation of azide moiety resulted in greatly dimin-
ished activity in the assay.
We next tested the synthesized probe 2 in photoaffinity labeling
experiments to detect the target proteins of OA. The soluble prote-
O
O
NH
S
NH
HN
HN
H
H
a
H
H
S
S
HO
O
H
H
omes prepared from HepG2 cells were incubated with 10 lM
O
O
probe 2 and then exposed to UV light for 30 min, and separated
by SDS–PAGE. The results showed that two proteins, whose molec-
ular weights were about 40–50 kDa, were tagged by probe 2. This
labeling was specific since it was competed by OA and no such
labeling was seen in the absence of UV irradiation condition
(Fig. 1). These results demonstrated that the synthesized probe 2
might be used to label the target proteins of OA.
In conclusion, the aim of this study was to develop specific re-
agents for isolation of OA target proteins. We have synthesized
two photoaffinity probes and evaluated their potency in an enzyme
inhibition assay against RMGPa. The results showed that the probe
2 exhibited inhibitory activity against RMGPa with an IC50 value of
19
20
H
HN
N
H
H
O
H
O
COOH
O
O
b
H
O
3 + 20
O
O
O
H
N
N
O
N
H
N
N
H
O
41.4 lM. Photoaffinity labeling experiments were also performed
and two proteins with 40–50 kDa in MW were specifically tagged
by probe 2. These data suggest that the synthesized probes might
be used to label, identify and purify the target proteins of OA.
We are now in the process of isolating the protein bands and
microsequencing.
21
Scheme 4. Reagents and conditions: (a) propargyl bromide, K2CO3, DMF, rt; (b)
CuSO4Á5H2O, sodium ascorbate, CH2Cl2–H2O, rt.
Table 1
Acknowledgments
Inhibition of rabbit muscle GPa by compounds 1–3
Compound
RMGPaa IC50
(lM)
We gratefully acknowledge the following agencies for funding:
European Foundation for the Study of Diabetes (2007 Grant
Awards for Collaborative Diabetes Research between China and
Europe) and National Natural Science Foundation of China (grants
30672523 and 90713037). This work was also supported by the
‘‘111 Project’’ from the Ministry of Education of China and the State
Administration of Foreign Expert Affairs of China (No. 111-2-07).
1
2
3
22.9 1.8
41.1 3.4
114.9 10.5
75.3 6.6
Caffeineb
a
Each value represents the mean SD of three
experiments.
b
Caffeine was used as a positive control.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
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Figure 1. Results of SDS–PAGE analysis of the photoaffinity labeling experiment by
synthesized probe 2. Photoaffinity labeling of the soluble proteomes prepared from
HepG2 cells followed SDS–PAGE electrophoresis and then transfer onto PVDF
membrane and detection with streptavidin-HRP. Samples were prepared by
incubating 2.0 mg/mL proteomes at different conditions: (A) with 0
and exposed to UV for 30 min; (B) with 10 M probe and exposed to UV light for
0 min; (C) with 10 M probe and exposed to UV light for 30 min; (D) with 10
probe and 1 mM OA and then exposed to UV light for 30 min.
lM probe 2
l
l
lM