Design and synthesis of novel photoaffinity probes for study of the target proteins of oleanolic acid

Article abstract of DOI:10.1016/j.bmcl.2011.11.123

To explore the molecular mechanisms of oleanolic acid, two novel photoaffinity probes were synthesized based on the structure-activity relationship reported previously. Their potency were evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa), a known target protein of oleanolic acid. The inhibitory activity of probe 2 was only about two-fold less potent than the mother compound oleanolic acid. The photoaffinity labeling experiments were also performed and two proteins were specifically tagged by probe 2. The results suggest that the synthesized probes could be used as powerful tools to isolate and identify the target proteins of oleanolic acid.

Full text of DOI:10.1016/j.bmcl.2011.11.123

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1036–1039
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel photoaffinity probes for study of the target
proteins of oleanolic acid
Liying Zhang a,b, Yingxia Zhang b, Jizhe Dong b, Jun Liu b, , Luyong Zhang b, Hongbin Sun b,
⇑
⇑
a Institute of Traditional Chinese Medicine, Chengde Medical College, Chengde 067000, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2011
Revised 24 September 2011
Accepted 29 November 2011
Available online 7 December 2011
To explore the molecular mechanisms of oleanolic acid, two novel photoaffinity probes were synthesized
based on the structure–activity relationship reported previously. Their potency were evaluated in an
enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa), a known target pro-
tein of oleanolic acid. The inhibitory activity of probe 2 was only about two-fold less potent than the
mother compound oleanolic acid. The photoaffinity labeling experiments were also performed and two
proteins were specifically tagged by probe 2. The results suggest that the synthesized probes could be
used as powerful tools to isolate and identify the target proteins of oleanolic acid.
Keywords:
Oleanolic acid
Target proteins
Photoaffinity labeling probe
Tag-free probe
Ó 2011 Elsevier Ltd. All rights reserved.
Click chemistry
The identification of target proteins for bioactive molecules is
always an essential task in modern drug development that can ex-
tend our understanding of the drug’s mechanisms and provide
information for designing new drug candidates.1 Furthermore,
the discovery of new druggable target proteins may provide the
biochemical evidence for their therapeutic effects in different
diseases.2
Oleanolic acid (OA, 1), a naturally occurring bioactive molecule,
has been successfully used as an anti-hepatitis drug in China for more
than 20 years.3 It has been reported to possess various biological
activities including antitumor, anti-inflammation, anti-oxidation,
anti-HIV, anti-hyperglycemia,hepatoprotective andcardioprotective
acitvities.4 Given the clinical significance and biological importance
of OA, the identification of its target proteins is highly desirable. To
date, partial target proteins of OA have been proposed, such as glyco-
For investigation of the potential target proteins of OA, two tri-
functional photoaffinity probes were designed based on the previ-
ous primary structure–activity relationship which indicated that
the modifications of OA at C-3 position had little effect on its activ-
ities.6 Consequently, we modified OA at C-3 position with a benzo-
phenone photophore for covalent labeling and a biotin reporter
group for detecting and purifying the target proteins. Moreover,
in order to avoid the sterically hindrance caused by the biotin
tag, we designed another tag-free probe which employed an azide
handle for downstream conjugation to reporter tag via the click-
chemistry reaction after proteome labeling. Herein, we describe
the synthesis of those probes and the evaluation by the glycogen
phosphorylase inhibition assay and photoaffinity labeling experi-
ments, the reaction of the tag-free probe 3 with the biotin-derived
alkyne on a simple model system was also studied.
gen phosphorylase, protein tyrosine phosphatase 1B (PTP1B) and
glycosidase5–8 in the modulation of glucose metabolism, and perox-
isome proliferators activated receptors (PPARs), nuclear factor
(NF-
B) and phospholipase A2 (PLA2) for its anti-cancer activity.9–12
Although the mechanisms mentioned above cover many therapeutic
facets of OA, the target proteins of OA are still not completelyrealized
so that many preventive and therapeutic functions of OA can not be
explained until now. Therefore, more studies in this area are
warranted.
a
-
The synthesis of intermediates 8 and 12 is outlined in Scheme 1.
Treatment of toluene 4 with 4-nitrobenzoyl chloride 5 in the pres-
ence of AlCl3 at room temperature afforded benzophenone 6 in 78%
yield. Acid-catalyzed oxidation of compound 6 with CrO3 gave the
corresponding acid 7. Acid 7 was treated with SOCl2 to yield acyl
chloride 8. On the other hand, reaction of 2-(2-aminoethoxy)etha-
nol 9 with benzyl bromide in the presence of K2CO3 in CH3CN at
50 °C gave amino–alcohol 10. Alkylation of 10 with bromoacetic
acid afforded the corresponding acid 11. Acyl chloride 12 was ob-
tained upon treating 11 with SOCl2.
jB
j
The synthesis of probe 2 is shown in Scheme 2. According to the
procedures reported previously,6 reaction of 1 (OA) with benzyl
chloride afforded benzyl ester 13 in high yield. Esterification of
⇑
Corresponding authors. Tel.: +86 25 83271043; fax: +86 25 83271198 (J.L.);
tel./fax: +86 25 83271198 (H.S.).
E-mail addresses: junliu@cpu.edu.cn (J. Liu), hbsun2000@yahoo.com (H. Sun).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.123

L. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1036–1039
1037
H
COOH
H
HO
H
(Oleanolic acid)
1
H
H
H
N
O
COOH
HN
S
H
O
H
H
O
O
O
H
O
H
N
N
O
H
O
2
H
COOH
O
H
O
O
O
H
O
N3
H
N
N
O
H
O
3
O
COCl
a
+
H3C
O2N
O2N
4
5
6
O
O
b
c
O2N
COOH
COCl
O2N
8
7
Bn
O
e
d
O
N
Bn
OH
H2N
OH
10
9
c
Bn
O
Cl
Bn
O
OH
O
N
Bn
N
Bn
O
O
O
12
11
Scheme 1. Reagents and conditions: (a) AlCl3, rt; (b) CrO3, H2SO4, HAc, 0 °C; (c) SOCl2, 80 °C; (d) BnBr, K2CO3, CH3CN, 50 °C; (e) BrCH2COOH, NaH, THF, reflux.
13 with 12 afforded ester 14. Hydrogenolysis of 14 over Pd-C in
THF furnished acid 15 in 47% yield. Amidation of 15 with 8 afforded
amide 16. Reduction 16 with stannous chloride afforded amine 17.
ification of 19 (biotin) with propargyl bromide afforded alkyne 20.
Treatment of 20 with probe 3 under typical click conditions (cop-
per sulfate, sodium ascorbate as reducing agent) resulted in the
ligantion product 21 in high yield.
Coupling of amine 17 with D-biotin in DMF in the presence of iso-
butyl chloroformate and N-methylmorphorpholine as the condens-
ing agents gave the probe 2.
The synthesis of tag-free probe 3 is summarized in Scheme 3.
Amidation of 17 with chloroacetyl chloride afforded compound
18, which was converted to probe 3 via nucleophilic substitution
reaction with sodium azide.
It is obviously important that the synthesized probes retain po-
tency in bioassays. Therefore, probes 2 and 3 were evaluated in an
enzyme inhibition assay against rabbit muscle glycogen phosphor-
ylase a (RMGPa), a known target protein of OA. The activity of
RMGPa was measured through detecting the release of phosphate
from glucose-1-phosphate in the direction of glycogen synthesis
based on the published method.13 The results are summarized
in Table 1. The newly synthesized probes exhibited moderate
Next, we attempted to study the click-chemistry process about
the tag-free probe 3 on a simple model system (Scheme 4). Ester-

1038
L. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1036–1039
H
H
H
COOBn
O
COOH
a
COOBn
H
b
H
H
O
O
H
HO
HO
O
H
H
Bn
N
Bn
14
1
13
H
H
COOH
COOH
O
O
H
O
H
d
c
O
O
O
O
H
H
O
O
H
N
NH2
NO2
O
15
16
H
H
H
H
N
O
COOH
COOH
HN
S
H
O
O
H
H
H
e
f
O
O
O
O
O
H
H
O
O
H
N
H
N
NH2
O
N
O
H
O
O
17
2
Scheme 2. Reagents and conditions: (a) BnCl, K2CO3, DMF, 60 °C; (b) 12, Et3N, CH2Cl2, rt; (c) H2, 10% Pd-C, THF, rt; (d) 8, pyridine, rt; (e) SnCl2, ethanol, reflux; (f) D-biotin,
isobutyl chloroformate, HMM, DMF.
H
H
COOH
COOH
O
O
H
O
H
O
a
O
O
O
O
O
O
H
H
Cl
O
O
O
H
N
H
N
N
H
NH2
18
17
H
COOH
O
H
b
O
O
H
N3
O
H
N
N
O
O
H
O
3
Scheme 3. Reagents and conditions: (a) chloroacetyl chloride, Et3N, CH2Cl2, rt; (b) NaN3, DMF, 55 °C.
inhibitory activity against RMGPa in spite of the incorporation of
benzophenone photophore and reporter group. Especially, probe
2 (IC50 = 41.4
lM) exhibited only about two-fold less potent than
the mother compound OA. Therefore, the probe 2 may be very

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
the online version, at doi:10.1016/j.bmcl.2011.11.123.
References and notes
1. Han, S.-Y.; Kim, S. H. Arch. Pharm. Chem. Life Sci. 2007, 340, 169.
2. Rix, U.; Superti-Furga, G. Nat. Chem. Biol. 2009, 5, 616.
3. Liu, J. J. Ethnopharmacol. 1995, 49, 57.
4. Dazubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.; Biedermann, D.;
Markova, L.; Urban, M.; Sarek, J. Nat. Prod. Rep. 2006, 23, 394.
5. Chen, J.; Liu, J.; Zhang, L.; Wu, G.; Hua, W.; Wu, X.; Sun, H. B. Bioorg. Med. Chem.
Lett. 2006, 16, 2915.
6. Wen, X. A.; Sun, H. B.; Liu, J.; Cheng, K. G.; Zhang, P.; Zhang, L. Y.; Hao, J.; Ni, P.
Z.; Zographos, S. E.; Leonidas, D. D.; Alexacou, K. M.; Gimisis, T.; Hayes, J. M.;
Oikonomakos, N. G. J. Med. Chem. 2008, 51, 3540.
7. Na, M. K.; Cui, L.; Min, B. S.; Bae, K.; Yoo, J. K.; Kim, B. Y.; Oh, W. K.; Ahn, J. S.
Bioorg. Med. Chem. Lett. 2006, 16, 3273.
8. Ali, M. S.; Jahangir, M.; Hussan, S. S.; Choudhary, M. I. Phytochemistry 2002, 60,
295.
9. Jeong, H. G. Toxicol. Lett. 1999, 105, 215.
10. Lim, S. W.; Hong, S. P.; Jeong, S. W.; Kim, B.; Bak, H.; Ryoo, H. C.; Lee, S. H.; Ahn,
S. K. J. Dermatol. 2007, 34, 625.
11. Choi, C. Y.; You, H. J.; Jeong, H. G. Biochem. Biophys. Res. Commun. 2001, 288, 49.
12. Laszczyk, M. N. Planta Med. 2009, 75, 1549.
13. Martin, W. H.; Hoover, D. J.; Armento, S. J.; Stock, I. A.; McPherson, R. K.;
Danley, D. E.; Stevenson, R. W.; Barrett, E. J.; Treadway, J. L. Proc. Natl. Acad. Sci.
1998, 95, 1776.
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