Design and synthesis of biotinylated troglitazone as an active affinity probe

Article abstract of DOI:10.1002/cjoc.201090370

A biotin-tagged analogue of troglitazone was designed, synthesized and applied to affinity chromatography to pulldown EGFR from 293T cell lysates overexpressing EGFR, a membrane protein assumed to be troglitazone's direct binding target by previous work.

Full text of DOI:10.1002/cjoc.201090370

FULL PAPER
Design and Synthesis of Biotinylated Troglitazone as an Active
Affinity Probe
,
a
Li, Xina,b(李新)
Li, Xiaoqic(李肖琦)
Tang, Hongc(唐宏)
Nan, Fajun (南发俊)
*
a Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Shanghai 201203, China
b College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
c Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
A biotin-tagged analogue of troglitazone was designed, synthesized and applied to affinity chromatography to
pulldown EGFR from 293T cell lysates overexpressing EGFR, a membrane protein assumed to be troglitazone’s
direct binding target by previous work. The results indicate the feasibility of the biotinylated probe as a vigorous
tool to clarify the molecular mechanisms for troglitazone’s antitumor activities.
Keywords antitumor agent, molecular mechanism, structure-activity relationship, biotinylated probe, affinity
chromatography
Introduction
Erk1/2 pathway.16-18
Troglitazone (TGZ) is a synthetic high-affinity
ligand of peroxisome proliferator-activated receptor γ
(PPARγ),1 a nuclear transcription factor that controls
genetic programs involved in glucose and lipid homeo-
stasis, energy metabolism, adipocyte differentiation, and
maturation.2 Recent studies have well established that
TGZ, like other members of the thiazolidinedione class
of drugs, causes growth inhibition, induces differentia-
tion, and triggers apoptosis of various human malignant
cells in addition to its well-known activity to increase
insulin sensitivity.3-6 However, the underlying mecha-
nism remains inconclusive.7 As TGZ has already shown
anticancer efficacy in humans and represents a promis-
ing new anticancer agent, molecular mechanism for its
anti-tumor activity has become the focus of investiga-
tion.5,8 To date, numerous studies have shown that TGZ
exerts growth inhibitory effects independently of
PPARγ and may result from multiple mechanisms.9-14 In
the long term, validating TGZ’s target proteins may
help to define its molecular mechanisms and facilitate
the design of improved therapeutic agents. The modifi-
cation of TGZ with biotin is among the most direct
strategies to address this question.15 By streptavidin-
biotin affinity chromatography, the biotinylated probe
should be suitable for purification of TGZ’s target pro-
teins. In this paper, we reported the design, synthesis
and preliminary evaluation of a biotin-tagged analogue
of troglitazone (TGZ-biotin) as an activity-based probe
to study the molecular mechanism of thiazolidinedione
class of drugs related to cross-activation of EGFR-
Results and discussion
Design and synthesis of TGZ-biotin
To ensure that the biotinylated probe would maintain
sufficient potency, design of the probe was based on the
structure-activity relationship of TGZ and its analogues
(Figure 1). Since all TGZ analogues with antiprolifera-
tive activities have the thiazolidinedione motif in their
structures, it seems that this moiety is essential to keep
the desired probe’s activity. On the other hand, it has
been reported that protection of the hydroxyl group in
the chroman ring of TGZ with benzyl retains its eugly-
cemic and hypolipidemic activities in vivo.19 This fact,
together with the observation that tocopherol moiety
alone does not induce physiological response similar to
TGZ in HCT-116 cells,10 suggests that the modification
of the hydroxyl group in the chroman moiety would be
well tolerated. To ensure a large conformational free-
dom of the bioactive TGZ motif, a poly(ethylene glycol)
(PEG) linker with rational length was introduced to link
it with the biotin tag.20
Preparation of the probe was commenced with
(R/S)-Trolox.19 Reduction of the carboxyl acid group
with LiAlH4 yielded 1, which was then attached with
the PEG linker to give 2. Reaction of 2 with 4-fluoro-
benzaldehyde afforded 3 with low yield, owing to par-
tially denatured KOBu-t. Aldol reaction of 3 with thia-
zolidine-2,4-dione gave 4, which was reduced with
MeOH-Mg to furnish 5 as a mixture of two di-
astereomer pairs (2R-5R/2S-5S and 2R-5S/2S-5R). Re-
*
E-mail: fjnan@mail.shcnc.ac.cn; Tel./Fax: 0086-021-50800954
Received February 22, 2010; revised May 21, 2010; accepted July 8, 2010.
Project supported by the National Natural Science Foundation of China (Nos. 30725049, 90813037, 30721005).
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Chin. J. Chem. 2010, 28, 2240— 2244

Design and Synthesis of Biotinylated Troglitazone as an Active Affinity Probe
Figure 1 Structures of TGZ and its biotinylated analogue designed.
Scheme 1 Synthesis of TGZ-biotin
Reagents and conditions: (a) LiAlH4, THF, 70 ℃, 3 h, 93.6%; (b) tert-butyl 2-[2-(2-bromoethoxy)ethoxy]ethylcarbamate, K2CO3, n-Bu4NI, 80 ℃,
butanone, 24 h, 59.0%; (c) (i) KOBu-t, DMF, room temperature, 1 h; (ii) 4-fluorobenzaldehyde, room temperature, 15 h, 29.2%; (d) thia-
zolidine-2,4-dione, piperidine, benzoic acid, reflux to separate water, 4 h, 59.6%; (e) Mg, CH3OH, 45 ℃, 8 h, 70.8%; (f) (i) 2 mol•L－ HCl in EtOAc,
1
room temperature, 1 h; (ii) (D)-biotin-N-succinimidyl ester, i-Pr2NEt, CH2Cl2, room temperature, 12 h, 58.4%.
Chin. J. Chem. 2010, 28, 2240—2244
© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
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Li et al.
FULL PAPER
moval of the tert-butyloxycarbonyl protecting group of
5 and subsequent incorporation of the biotin tag fur-
nished TGZ-biotin at last.
purification. Dichloromethane (DCM) was distilled
from CaH2. Toluene and tetrahydrofuran (THF) were
distilled from sodium. DMF was distilled in vacuo. Re-
actions were monitored by thin-layer chromatography
1
(TLC). H NMR (300 MHz) and 13C NMR (75 MHz)
Affinity chromatography employing TGZ-biotin
Previous work has shown that TGZ triggered activa-
tion of Erk1/2 dependent on epidermal growth factor
receptor (EGFR) instead of PPARγ.16 Besides,
pre-incubation of porcine aorta endothelial (PAE) cells
stably expressing human EGFR with TGZ caused a dose
and time-dependent inhibition of rhodamine tagged
EGF binding to cell surface at 4 ℃.16 These results im-
plied that TGZ might direct bind EGFR for signaling.
To validate this assumption, 293T cells overexpressing
spectra were recorded on a Varian Mercury VX300
Fourier transform spectrometer. The chemical shifts δ
were reported using the δ＝7.26 signal of CDCl3 as in-
ternal standard. Low-resolution mass data were obtained
on an Agilent 6100 Series Quadrupole LC/MS systems.
High-resolution mass data were obtained on a Micro-
mass Q-Tof UltimaTM spectrometer.
Synthesis
－1
EGFR were simulated with TGZ-biotin (50 µmol•L )
(2R/S)-(6-Hydroxy-2,5,7,8-tetramethylchroman-
2-yl)-carbinol (1) To a stirred solution of trolox (215
mg, 0.86 mmol) in dry THF (15 mL) was added LiAlH4
(33 mg, 0.86 mmol) and the mixture was heated to re-
flux and stirred for 3 h while maintaining refluxing.
Na2SO4•nH2O (200 mg) was then added at 0 ℃ to
quench the reaction and the mixture was stirred at 0 ℃
till becoming clear. The solid was removed by filtration
and the filtrate concentrated under reduced pressure to
give a brown gum (190 mg, 93.6%), which was used in
the next step without further purification.19 1H NMR
(CDCl3) δ: 1.22 (s, 3H), 1.64—1.75 (m, 1H), 1.95—
2.06 (m, 1H), 2.12 (s, 6H), 2.21 (s, 3H), 2.67 (brs, 2H),
3.58—3.68 (m, 2H); 13C NMR (CDCl3) δ: 11.50, 12.00,
12.40, 20.37, 20.47, 27.89, 69.41, 75.14, 117.38, 119.15,
121.73, 126.61, 144.95, 145.20; ESIMS m/z: 259.1
(M＋Na)＋.
for 1 h at room temperature. The cells were then ren-
dered to trypsin digestion and cell lysates were then in-
cubated with streptavidin magnetic beads at room tem-
perature for 1h. The bound proteins were resolved in
SDS-PAGE and immunoblotted with anti-EGFR anti-
body. As shown in Figure 2, the affinity chromatogra-
phy could extract EGFR from the cells. While in con-
trast, EGFR could not be extracted if the cells were not
treated with the probe first. These results further vali-
date the notion that TGZ functionally binds EGFR and
shed a new light on its anti-tumor mechanisms.
(2R/S)-(6-(2-(2-(2-(tert-Butoxycarbonylamino)eth-
oxy)ethoxy)ethoxy)-2,5,7,8-tetramethylchroman-2-
yl)-carbinol (2) Anhydrous K2CO3 (439 mg, 3.17
mmol) and n-Bu4NI (78 mg, 0.22 mmol) were added to
a solution of 1 (500 mg, 2.12 mmol) and tert-butyl
2-(2-(2-bromoethoxy)ethoxy)ethylcarbamate (990 mg,
3.17 mmol) in butanone (35 mL). After being stirred at
80 ℃ for 24 h, H2O was added at room temperature to
quench the reaction and the product was extracted into
EtOAc and purified by chromatography on a silica gel
eluted with PE∶EtOAc (V∶V＝1.5∶1) to get a yel-
Figure 2 TGZ-biotin direct binds EGFR. Streptavidin-biotin
affinity chromatography could extract EGFR from lysates of
293T cells which overexpressed EGFR and were first stimulated
with the probe (50 µmol•L－ 1). The bound protein was detected to
be EGFR by western blotting with anti-EGFR.
Conclusion
We have developed a biotinylated TGZ analogue for
its target validation via affinity chromatography. The
biotin-tagged probe turned out to be useful for the pull-
down of TGZ’s direct binding target by affinity chro-
matography, which helps to gain a new insight con-
cerning the mechanisms for TGZ’s antiproliferative and
pro-apoptotic effects. Considering that TGZ has shown
anticancer efficacy in humans and might exert this ac-
tivity through several different pathways, this probe
represents an appropriate tool for further biological
studies to clarify the molecular mechanisms of the
promising antitumor reagent.
1
low oil (584 mg, 59.0%). H NMR (CDCl3) δ: 1.22 (s,
3H), 1.42 (s, 9H), 1.70—1.78 (m, 1H), 1.92—2.02 (m,
1H), 2.07 (s, 3H), 2.15 (s, 3H), 2.18 (s, 3H), 2.63 (brs,
2H), 3.25—3.38 (m, 2H), 3.50—3.60 (m, 2H), 3.60—
3.65 (m, 2H), 3.65—3.75 (m, 2H), 3.75—3.80 (m, 2H),
3.82 (s, 4H), 5.00—5.10 (m, 1H); ESIMS m/z: 490.3
(M＋Na)＋.
4-(6-(2-(2-(2-(tert-Butoxycarbonylamino)ethoxy)-
ethoxy)ethoxy)-2,5,7,8-tetramethylchroman-2-ylme-
thoxy)benzaldehyde (3) To a stirred solution of 2
(111 mg, 0.24 mmol) in dry DMF (2.5 mL) at room
temperature was added t-BuOK (81 mg, 0.72 mmol).
The mixture was stirred for 1 h first and then
4-fluorobenzaldehyde (51 µL, 0.48 mmol) in dry DMF
(20 mL) was added. The mixture was continued to be
stirred for 15 h at room temperature and was then
Experimental
General methods
Unless otherwise noted, all reagents were purchased
from commercial suppliers and used without further
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Chin. J. Chem. 2010, 28, 2240—2244

Design and Synthesis of Biotinylated Troglitazone as an Active Affinity Probe
quenched with water (5 mL) and extracted with EtOAc
thrice. The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4, and concentrated.
The residue was chromatographed over silica gel using
PE∶EtOAc (V∶V＝2∶1) as eluent to give 40 mg
lowed by the N-hydroxylsuccinimide ester of Biotin (6
mg, 0.018 mmol). The mixture was stirred at room
temperature for 12 h and concentrated under reduced
pressure. Purification by chromatography on a silica gel
column eluted with CHCl3∶MeOH (V∶V＝20∶1)
yielded the product Biotin-TGZ as a white solid (7 mg,
1
(29.2%) of 3 as a viscous liquid. H NMR (CDCl3) δ:
1
1.41 (s, 12H), 1.85—2.00 (m, 1H), 2.05 (s, 3H), 2.10—
2.20 (m, 1H), 2.14 (s, 3H), 2.18 (s, 3H), 2.61 (brs, 2H),
3.25—3.40 (m, 2H), 3.50—3.62 (m, 2H), 3.62—3.70
(m, 2H), 3.70—3.80 (m, 2H), 3.82 (s, 4H), 3.97—4.08
(m, 2H), 5.05—5.15 (m, 1H), 7.04 (d, J＝8.4 Hz, 2H),
7.82 (d, J＝8.4 Hz, 2H), 9.89 (s, 1H).
58.4%). H NMR (CDCl3) δ: 1.20—1.40 (m, 2H), 1.42
(s, 3H), 1.50—1.70 (m, 4H), 1.80—1.95 (m, 2H), 2.00
—2.20 (m, 11H), 2.55—2.65 (m, 2H), 2.67—2.73 (m,
1H), 2.85—2.95 (m, 1H), 3.10—3.25 (m, 2H), 3.30—
3.40 (m, 1H), 3.40—3.55 (m, 2H), 3.60—3.65 (m, 2H),
3.65—3.80 (m, 4H), 3.80—4.00 (m, 6H), 4.25—4.35
(m, 1H), 4.45—4.55 (m, 2H), 5.80 (brs, 1H), 6.28 (brs,
1H), 6.69 (brs, 1H), 6.85 (d, J＝7.8 Hz, 2H), 7.11 (d,
J＝7.8 Hz, 2H); ESIMS m/z: 821.4 (M＋Na)＋and 797.4
(M—H)－; HRMS calcd for C40H54N4O9S2 821.3230,
found 821.3208.
5-(4-(6-(2-(2-(2-(tert-Butoxycarbonylamino)eth-
oxy)ethoxy)ethoxy)-2,5,7,8-tetramethylchroman-2-yl-
methoxy)phenylmethylene)thiazolidine-2,4-dione (4)
A mixture of 3 (40 mg, 0.07 mmol), thiazolidine-
2,4-dione (10 mg, 0.084 mmol), benzoic acid (2 mg,
0.016 mmol) and piperidine (1 µL, 0.01 mmol) in
toluene (4 mL) was refluxed for 4 h with continuous
removal of water using a Dean-Stark water separator.
The reaction mixture was cooled to room temperature,
and the solvent evaporated. The resulting residue was
chromatographed on a silica gel to give the product as a
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Chin. J. Chem. 2010, 28, 2240—2244
© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPER
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Chin. J. Chem. 2010, 28, 2240—2244