Activity-based proteome profiling of potential cellular targets of orlistat - An FDA-approved drug with anti-tumor activities

Article abstract of DOI:10.1021/ja907716f

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Full text of DOI:10.1021/ja907716f

Published on Web 12/22/2009
Activity-Based Proteome Profiling of Potential Cellular
Targets of Orlistat - An FDA-Approved Drug with Anti-Tumor
Activities
Peng-Yu Yang,†,‡,§ Kai Liu, Mun Hong Ngai,†,§ Martin J. Lear,†,§ Markus R. Wenk,‡
and Shao Q. Yao†,‡,§,
*
Department of Chemistry, Department of Biological Sciences and, NUS MedChem Program of the
Life Sciences Institute, 3 Science DriVe 3, National UniVersity of Singapore, Singapore 117543
Received September 10, 2009; E-mail: chmyaosq@nus.edu.sg
Abstract: Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor
activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not
been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive
probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental
THL structure to maintain the native biological properties of Orlistat, while providing the necessary
functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike,
cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach
is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected
fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated
by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis.
Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at
its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target
identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers
of their biological targets.
Introduction
cellular FAS activity, Orlistat induces endoplasmic reticulum
stress in tumor cells, inhibits endothelial cell proliferation and
Drug discovery is a long and costly process, yet most drugs
have side effects, ranging from simple nuisances to life-
threatening complications.1 Unanticipated effects of a drug,
often revealed either during clinical trials or sometimes after
the drug enters the market, could lead to termination of a
drug development program/recall of the drug, or, in some
rare cases where the effects are beneficial, new drug
applications.1b,c Therefore, one of the most critical steps in
the drug discovery process is the effective identification of
the so-called off-targets and anticipation of their potential
side effects a priori.
angiogenesis, and consequently delays tumor progression on a
variety of cancer cells, including prostate, breast, ovary, and
melanoma cancer cells.3 As a result, this compound (as well as
other Orlistat-like analogues with improved potency and bio-
availability5) has been proposed as a promising anticancer drug.
Cellular off-targets and potential side effects of Orlistat in cancer
therapies, however, have not been extensively explored thus far.6
Our long-term research goals focus on developing novel
chemical proteomic strategies that enable large-scale studies of
Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved
antiobesity drug, which works primarily on pancreatic and
gastric lipases within the gastrointestinal (GI) tract.2 Recently,
Orlistat was found to inhibit the thioesterase domain of fatty
acid synthase (FAS), an enzyme essential for the growth of
cancer cells but not normal cells.3,4 By effectively blocking the
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Res. 2004, 64, 2070–2075. (b) Pemble, C. W.; Johnson, L. C.; Kridel,
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K. G.; Seguin, F.; Bastos, D. C.; Agostini, M.; Rangel, A. L. C. A.;
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R. D.; Graner, E. Int. J. Cancer 2008, 123, 2557–2565. (k) Menendez,
J. A.; Vellon, L.; Lupu, R. Int. J. Gynecol Cancer 2006, 16, 219–
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2006, 20, 2027–2035. (m) Cheng, F.; Wang, Q.; Chen, M.; Quiocho,
F. A.; Ma, J. Proteins 2008, 70, 1228–1234.
* To whom correspondence should be addressed. Tel: +65 65162925, Fax:
+65 67791691.
† Department of Chemistry.
‡ Department of Biological Sciences.
§ NUS MedChem Program of the Life Sciences Institute.
(1) (a) Giacomini, K. M.; Krauss, R. M.; Roden, D. M.; Eichelbaum, M.;
Hayden, M. R.; Nakamura, Y. Nature 2007, 446, 975–977. (b)
Campillos, M.; Kuhn, M.; Gavin, A.-C.; Jensen, L. J.; Bork, P. Science
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10.1021/ja907716f  2010 American Chemical Society

Cell-Based Proteome Profiling of Orlistat
A R T I C L E S
Figure 1. Overall strategy for activity-based proteome profiling of potential cellular targets of Orlistat using alkyne-containing, cell-permeable THL analogues
(1, 2, and 3). The alkyne handles in the probes are shaded (purple circle). The “Click”-based reporters used in the manuscript are shown (in red rectangle).
therapeutically relevant enzymes, as well as small molecules
(i.e., potential drug candidates) that can modulate these enzymes’
cellular activities.7 In the current study, we set out to look for
new cellular targets, including off-targets, of Orlistat at its early
stages of development for cancer therapy. Herein, we report,
for the first time, the identification and putative validation of
several previously unknown cellular targets of Orlistat by using
a natural product-based, chemical proteomic approach.
Results and Discussion
Design and Synthesis of Orlistat-like Probes. Our strategy is
based on the well-established activity-based protein profiling
(ABPP) approach,8 by making use of THL-like protein-reactive
probes 1, 2, and 3 (i.e., THL-R, THL-L, and THL-T,
respectively, in Figure 1). We took advantage of several key
properties known to THL in the design of our activity-based
probes:3,5 (1) THL (being derived from a natural product) is
(5) (a) Ma, G.; Zancanella, M.; Oyola, Y.; Richardson, R. D.; Smith, J. W.;
Romo, D. Org. Lett. 2006, 8, 4497–4500, and references cited therein.
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5285–5296, and references cited therein.
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Mikhailidis, D. P.; Elisaf, M. S. Drug Saf. 2008, 31, 53–65.
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Yang et al.
Scheme 1. Total Synthesis of the Three Probes
cell-permeable, making our probes applicable for direct in situ
cell-based screening; (2) THL reacts with its known cellular
targets via a covalent reaction through its reactive ꢀ-lactone
moiety and the nucleophilic active-site residue (typically Ser/
Cys residues, e.g. Ser2308 in FAS3b) of the target protein,
resulting in the formation of an isolatable protein/THL complex;
(3) previous minor structural modifications at either the 16-
carbon or 6-carbon aliphatic chains of THL did not significantly
alter its native biological activities.5b Accordingly, probes 1 and
2, in which a C-C triple bond was introduced into THL to
replace the terminal C-C single bond of the aliphatic chains,
were synthesized. We also synthesized probe 3 by substituting
the CHO group in the N-formyl-L-leucine moiety of THL with
a propiolic acyl group (Figure 1, shaded in purple). These
extremely conservative modifications of introducing an alkyne
handle in the parental THL structure were aimed at maintaining
the native biological properties of Orlistat, while providing the
necessary functionality for identification and characterization
(i.e., imaging) of previously unknown cellular targets by
downstream conjugation of the protein/probe complex to reporter
tags via the bio-orthogonal click chemistry.8,9
Numerous strategies for the total synthesis of THL have been
reported.5 Our own efforts for the preparation of these probes
followed similar tracks (Scheme 1). The synthesis of 1 (THL-
R) commenced with the tandem Mukaiyama aldol-lactonization
(TMAL) reaction between the optically pure aldehyde, 8a
(obtained in five steps involving separation of a diastereomeric
mixture of racemic homoallylic acohols; Scheme S1 in the
Supporting Information), and TMS-protected thiopyridyl ketene
acetal 12 (prepared in four steps from 6-bromohexanoic acid;
Scheme S3 in the Supporting Information), giving the desired
ꢀ-lactone as a mixture of diastereomers (∼9:1 anti/syn) with
complete selectivity for the trans-ꢀ-lactone. Following O-
desilylation and silica gel separation, the enantiomerically pure
γ-hydroxy ꢀ-lactone 13 was isolated. The trans configuration
of the ꢀ-lactone core was unambiguously confirmed by coupling
constant analysis (JH2, H3 ) ∼4.4 Hz).5a Subsequent C-desily-
lation with AgNO3/2,6-lutidine gave terminal acetylene 14,
which was subjected to Mitsunobu conditions with N-formyl-
L-leucine to give the configurationally inverted product 1. For
the synthesis of 2 (THL-L), an alternative route to obtain the
(9) Kalesh, K. A.; Yang, P.-Y.; Srinivasan, R.; Yao, S. Q. QSAR Comb.
Sci. 2007, 26, 1135–1144.
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Cell-Based Proteome Profiling of Orlistat
A R T I C L E S
Figure 2. (A) Dose-dependent inhibition of HepG2 cell proliferation by Orlistat and three analogues (1, 2, and 3) using XTT assay. Data represent the
average (s.d. for two trials. (B) Western blot analysis of eIF2R phosphorylation in PC-3 cells upon treatment with the four compounds. GAPDH was used
as a loading control. (C) Inhibition of protein synthesis in HepG2 cells treated with the indicated concentrations of Orlistat, THL-R, or CHX (cycloheximide,
an inhibitor of protein biosynthesis) for 12 h and then pulsed with AHA (L-azidohomoalanine) for 4 h. Cell lysates were prepared and subjected to click
chemistry with rhodamine-alkyne (provided with the AHA kit) following vendor’s protocols, SDS-PAGE analysis, and in-gel fluorescence scanning (fluorescent
gel is shown in grayscale). (D) Activation of caspase-8 in MCF-7 cells treated with the indicated concentrations of Orlistat/analogues.
optically active aldehyde 23 was explored. First, 10-bromo-1-
decanol was converted to the chiral homoallylic alcohol 21 in
five steps (Scheme S5 in the Supporting Information) involving
asymmetric allylation with allyltributyl stannane in the presence
of chiral (S)-BINOL-InCl3 complex.10 The absolute configura-
tion of 21 was unequivocally determined using Mosher ester
analysis (Scheme S6 in the Supporting Information).11 Subse-
quently, a two-step reaction gave 23, which reacted with 16
under TMAL conditions to give 24 and eventually 2. For the
synthesis of 3 (THL-T), the known hydroxyl-ꢀ-lactone 30 was
first prepared based on published procedures,5a then reacted with
29 (obtained in four steps from Fmoc-Leu-OH; Scheme S7 in
the Supporting Information) under Mitsunobu conditions, giving
3 in 52% yield.
Effects on Cell Proliferation, Phosphorylation of eIf2r and
Activation of Caspase-8. Next, the three probes were evaluated
against Orlistat (as a positive control) for potential biological
activities. Three different types of cellular assays based on
previously established Orlistat biology were used.3a,c-e First,
the antiproliferation activity of the four compounds (1, 2, 3,
and Orlistat) against HepG2 cells (a human hepatocellular liver
carcinoma cell line) was evaluated using the XTT assay (part
A of Figure 2);3a all four compounds showed a dose-dependent
inhibition of tumor cell proliferation over a 72 h time period
with comparable potency. Second, we carried out comparative
analysis of the compounds in their ability to induce phospho-
rylation of eIf2R in the prostate cancer PC-3 cells.3c Previous
studies had shown that inhibition of FAS by Orlistat induces
endoplasmic reticulum (ER) stress and results in the phospho-
rylation of the translation initiation factor eIF2R. As shown in
part B of Figure 2, PC-3 cells treated with different amounts of
each compound showed similarly elevated eIF2R phosphory-
lation. Because the phosphorylation of eIF2R is known to inhibit
cellular protein synthesis,3c we carried out metabolic labeling
experiments with azidohomoalanine (AHA, pioneered by Di-
eterich et al.12) to measure the level of newly synthesized
proteins in cells treated with Orlistat, THL-R or cycloheximide
(a well-known protein synthesis inhibitor). As shown in part C
of Figure 2, greatly reduced levels of protein synthesis were
observed in cells treated with each of the three compounds. The
inhibition of protein synthesis was dose-dependent, which is
consistent with previous findings carried out using radiolabeled
35S-methionine.3c Lastly, inhibition of FAS by Orlistat was
previously shown to induce tumor cell apoptosis by activating
caspase-8.3e We therefore tested the compounds against the
invasive human breast cancer MCF-7 cells (part D of Figure
2); similar degrees of caspase-8 activation (as evidenced by the
appearance of the p41/43 bands corresponding to cleaved
caspase-8) were observed against all four compounds (at either
5 or 25 µM). Collectively, these data show the introduction of
a terminal alkyne handle at various designated locations in
Orlistat scaffold did not noticeably affect its biological activities,
and 1, 2, and 3 were indeed suitable chemical probes for cell-
based proteome profiling and identification of previously
unknown cellular targets of Orlistat.
In Situ and in Vitro Proteome Profiling. We next compared
the in situ proteome reactivity profiles of the three probes to
identify proteins which were covalently labeled by the probes
in live HepG2 cells.13 Probes (1-20 µM) were directly added
to the cell culture medium, either alone or in the presence of
(12) Dieterich, D. C.; Link, A. J.; Graumann, J.; Tirrell, D. A.; Schuman,
E. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9482–9487.
(13) Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Nat.
Biotechnol. 2005, 23, 1303–1307.
(10) Teo, Y.-C.; Tan, K.-T.; Loh, T.-P. Chem. Commun. 2005, 1318–1320.
(11) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451–
2458.
(14) Martin Moche, M.; Schneider, G.; Edwards, P.; Dehesh, K.; Lindqvist,
Y. J. Biol. Chem. 1999, 274, 6031–6034.
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Figure 3. Proteome profiling of HepG2 cells using THL analogues 1, 2, and 3 (over a concentration range of 1-20 µM). Both in situ (live cell) and in vitro
(whole-cell lysates) labelings were carried out. (A and B) In situ proteome labeling of HepG2 cells, with or without Orlistat (100 µM), followed by click
chemistry with rhodamine-azide (33), SDS-PAGE analysis, and in-gel fluorescence scanning. The labeled FAS (marked with red arrow) and multiple Orlistat-
sensitive targets (marked with blue arrows) were isolated and identified subsequently using biotin-azide (34), and summarized in Table 1. In (B), asterisks
show the expected locations of FAS and other Orlistat-sensitive bands. (C) showed the fluorescent profile of the gel from part A of Figure 3 upon treatment
with hydroxylamine. The fluorescence labeling of most proteins (but not FAS) were reversed by hydroxylamine, indicating a thioester/ester linkage. (D) In
vitro labeling of HepG2 whole-cell proteome lysates (for in vitro competitive ABPP profile, part D of Figure S2 in the Supporting Information).
Figure 4. Target validation of the identified “hits”. (A) Western blotting analysis of pulled-down fractions of HepG2 live cells treated with THL-R (or
DMSO as negative controls; right lanes) with their respective antibodies. Biotin-azide 34 was used in the click chemistry with avidin-agarose beads for
pull-down experiments. (B) In situ labeling of recombinantly expressed ꢀ-tubulin, GAPDH, RPL7a, and RPL14 by THL-R (fluorescent gel shown in grayscale).
(C) Comparative labeling analysis of wild-type GAPDH and Cys151Ala mutant (upper panel, fluorescent gel shown in grayscale); (lower panel, silver-stained
gel) comparable amounts of protein loading were demonstrated in all three lanes.
100 µM of the competing Orlistat. After two hours, the cells
were washed (to remove excessive probes), homogenized,
incubated with rhodamine-azide (33) under click-chemistry
conditions, separated by SDS-PAGE gel, and analyzed by in-
gel fluorescence scanning (Figure 3). In addition to the expected
FAS band (265 kDa, marked with red arrow; part A of Figure
3), confirmed by treatment with an anti-FAS antibody (part A
of Figure 4), we also observed a number of Orlistat-sensitive
targets, that is, those labeled bands that were competed away
by treatments with excessive Orlistat (marked with blue arrows
and asterisks in parts A and B of Figure 3). Most of these labeled
bands were clearly visible even at a low probe concentration
(e.g., 1 µM of THL-R; lane 1 in part A of Figure 3). Both
probe 1 and 2 gave similar proteome labeling profiles, whereas
probe 3 (THL-T) consistently produced weaker labeled bands
(possibly caused by inefficient click-chemistry conjugation due
to inaccessibility of the alkyne handle located at the N-formyl-
L-leucine end, which, based on X-ray structure of FAS/Orlistat
complex, was buried deep into the protein3b). To assess which
nucleophilic residue of the labeled proteins might have been
covalently modified by our probes, SDS-PAGE gels from the
in situ labeling were subjected to in-gel treatment with hy-
droxylamine (NH2OH), which preferentially cleaves thioesters,
and esters to a lesser extent, under neutral pH conditions.15 As
shown in part C of Figure 3, the labeled FAS and most other
Orlistat-sensitive bands showed a much reduced fluorescence
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A R T I C L E S
Table 1. Proteins Identified by Pull Down and Mass Spectrometry
#
protein name
calcd/obsd mass Da/kDa
localization
protein function
2
3
4
5
6
7
8
9
Hsp90AB1
unnamed
ꢀ-tubulin
ANXA2
GAPDH
RPL7a
75 088/72
56 156/56
49 671/52
38 808/39
36 201/36
30 148/30
23 902/23
22 635/22
cytoplasm, nucleus
cytoplasm, nucleus
nucleus
cytoplasm, nucleus
cytoplasm, nucleus
cytoplasm, ribosome
cytoplasm, ribosome
cytoplasm, ribosome
molecule chaperone with ATPase acitivity, stress response
protein biosynthesis, translational elongation
GTPase acitivity and cell division cell division
calcium binding, cell division
glycolysis, energy production
biogenesis, protein synthesis
RPL14
RPS9
biogenesis, protein synthesis
biogenesis, protein synthesis
signal upon treatment with NH2OH, suggesting likely involve-
ment of a cysteine/serine residue in the labeling between these
proteins and THL (vide infra). It is interesting to note that the
labeled FAS band was to some degree resistant to NH2OH
treatment, clearly indicating the formation of a more chemically
stable ester linkage between Ser2308 in FAS3b and THL-R. In a
related experiment, we also performed competitive ABPP with
Cerulenin, a known FAS inhibitor which irreversibly inactivates
the ꢀ-ketoacyl-ACP synthase domain but not the thioesterase
domain of FAS;14 as expected (Figure S2 of the Supporting
Information), Cerulenin did not abolish the labeling of the THL
probes toward FAS as well as most other Orlistat-sensitive
proteins. This indicates that the labeling of our probes is both
target- and domain-specific, and they may in the future be used
to distinguish different domains of FAS. As shown in part D
of Figure 3, when compared to in situ (live cell) labeling, similar
proteome labeling profiles, albeit with significantly higher
background labeling, were obtained when whole-cell lysates
were used instead. This shows the importance of the in situ
labeling (made possible by the cell-permeable property of the
THL probes) as a prerequisite for accurate and specific target
identification.
Target Identification and Validation. Subsequently, the
labeled protein extract was enriched (following click-chemistry
conjugation with biotin-azide 34) by avidin-agarose beads,
separated by SDS-PAGE gel, confirmed by strepavidin blotting,
subjected to in-gel trypsin digestion, and identified by MS/MS
analysis. In addition to FAS, eight new proteins were identified
(numbered 2-9, in part A of Figure 3 and Table 1), of which
one is an unnamed protein. Two of the proteins, GAPDH and
ꢀ-Tubulin, are house-keeping genes constitutively expressed in
most cell lines but are known to be expressed more highly in
cancer cells.16 Both GAPDH (a dehydrogenase) and ꢀ-tubulin
(a hydrolase) possess nucleophilic active-site cysteine residues.
It is therefore not surprising they were targets of Orlistat and
their labeling was reversed by NH2OH treatment. It should be
noted that tubulins are well-known targets of anticancer drugs
(e.g., taxol17). Three other proteins identified, RPL7a, RPL14,
and RPS9, are ribosomal proteins. They are known to be
implicated in protein synthesis, control of cellular transforma-
tion, tumor growth, aggressiveness and metastasis. Overexpres-
sion of these proteins had previously been reported in colon,
brain liver, breast and prostate carcinoma.18a-d In retrospect,
our earlier findings that protein synthesis was greatly inhibited
in HepG2 cells treated with Orlistat, as shown in part C of Figure
2, may also imply that these ribosomal proteins are probable
cellular targets of the drug. The remaining two proteins, Annexin
A2 and Hsp90AB1, are involved in cell proliferation/division
and protein degradation, respectively.
In an effort to further validate the MS results, we carried out
pull-down and Western blotting experiments with the respective
antibodies (part A of Figure 4);19 results confirmed all proteins
tested (including FAS) were indeed positively labeled by THL-
R, and thus likely are true cellular targets of Orlistat. Four of
the proteins (ꢀ-tubulin, GAPDH, RPL7a, and RPL14) were
taken for additional validation experiments. First, the c-Myc
fusions of these proteins were transiently expressed in HEK-
293T cells (Figure S3 in the Supporting Information), labeled
(by THL-R in situ), immune-purified (with c-Myc agarose
beads), subjected to click chemistry (with rhodamine-azide 33),
and analyzed by SDS-PAGE (part B of Figure 4); results
unambiguously confirmed that all four proteins were fluores-
cently labeled by the probe. To confirm whether labeling of
the probe is active site-directed, GAPDH, whose active site
residue Cys151 had previously been characterized,16b was taken
as an example for further site-directed mutagenesis experiments.
GAPDH is a multifunction protein and well known for its
primary role as a glycolytic enzyme. Recently, increasing
evidence has suggested that this enzyme is also involved in a
variety of activities which are unrelated to energy production,
including membrane fusion, microtubule bundling, DNA repair,
and apoptosis.16b An active site (Cys151 to Ala) mutant of
GAPDH was therefore generated, transiently expressed and
purified from HEK-293T cells, labeled with THL-R, reacted
with rhodamine-azide 33, then finally analyzed by SDS-PAGE
followed by in-gel fluorescence scanning (part C of Figure 4);
as expected, probe THL-R only labeled the wild-type GAPDH
and not the Cys151Ala mutant, thus confirming Cys151 as the
residue in GAPDH being covalently labeled by THL-R. This
also confirms the active site-directed nature of the probe. It
should be noted that, in a series of recent reports,20 Sieber et
al. had developed activity-based probes based on small molecule
libraries containing a reactive ꢀ-lactone moiety. The authors
concluded that ꢀ-lactones are promising privileged structures
and could be used to identify a variety of mechanistically distinct
enzymes. Our Orlistat-based probes, though structurally much
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Shamir, E.; Hang, H. C. J. Am. Chem. Soc. 2009, 131, 4967–4975,
and references cited therein.
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P. V. Prostate 2008, 68, 1555–1560. (b) Phadke, M. S.; Krynetskaia,
N. F.; Mishra, A. K.; Krynetskiy, E. J. Pharmacol. Exp. Therapeut.
2009, 331, 77–86, and references cited therein.
(17) Kingston, D. G.; Newman, D. J. Curr. Opin. Drug DiscoV. DeV. 2007,
10, 130–144.
(18) (a) Zhu, Y.; Lin, H.; Li, Z.; Wang, M.; Luo, J. Breast Cancer Res.
Treat. 2001, 69, 29–38. (b) Liu, Y.; Zhu, X.; Liao, S.; Tang, Q.; Liu,
K.; Guan, X.; Zhang, J.; Feng, Z. Oncol. Rep. 2007, 18, 943–951. (c)
Wang, Y.; Cheong, D.; Chan, S.; Hooi, S. C. Int. J. Oncol. 2000, 16,
757–762. (d) Vaarala, M. H.; Porvari, K. S.; Kyllonen, A. P.;
Mustonen, M. V. J.; Lukkarinen, O.; Vihko, P. T. Int. J. Cancer 1998,
78, 27–32.
(19) Commerically available antibody for RPL14 failed to detect even
endogenous RPL14 and was thus not pursued further in our experiments.
(20) (a) Bo¨ttcher, T.; Sieber, S. A. Angew. Chem., Int. Ed. 2008, 47, 4600–
4603. (b) Bo¨ttcher, T.; Sieber, S. A. J. Am. Chem. Soc. 2008, 130,
14400–14401. (c) Bo¨ttcher, T.; Sieber, S. A. ChemBioChem 2009,
10, 663–666.
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Yang et al.
Figure 5. Cellular imaging of HepG2 cells with probe 1 (for 2 and 3, see Figure S5 in the Supporting Information). Live HepG2 cells were treated with
1 (20 µM) for 2 h, fixed, permeabilized, reacted with rhodamine-azide (10 µM, false-colored in red) under click-chemistry conditions, stained with Hoechst
(blue), anti-FASN primary antibodies, followed by FITC-conjugated antimouse IgG antibody (green), or ER-Tracker Green (glibenclamide BODIPY FL),
mounted, and imaged. Samples were imaged with an Olympus IX71 inverted microscope, equipped with a 60X oil objective (NA 1.4, WD 0.13 mm) and
CoolSNAP HQ CCD camera (Roper Scientific, Tucson, AZ, USA). Images were processed with MetaMorph software (version 7.1.2.; Molecular Devices,
PA, USA). Scale bar ) 8 µm. All images were acquired similarly. Cells treated with DMSO (negative controls) are also shown.
more complex, appear to behave similarly and are capable of
targeting a number of other cellular proteins in addition to FAS.
Even though the exact physiological roles of these proteins in
connection with Orlistat and its pharmacological effects have
not been established from this study, we believe these putative
cellular targets of Orlistat should be carefully evaluated when
one considers using Orlistat in cancer therapy.
of Orlistat. It is also possible that the antitumor activities of
Orlistat might have originated from the drug’s ability to inhibit
both FAS (as previously reported3) and some of these newly
identified targets. In either case, our findings have important
implications in consideration of Orlistat as a potential anticancer
drug. Finally, our strategy should be broadly useful for off-
target identification against quite a number of existing drugs
and/or candidates, which are also covalent modifiers of their
biological targets.21
Cellular Imaging Using THL-R. To demonstrate the utility
of our cell-permeable probes for potential cellular imaging of
Orlistat targets, we performed fluorescence microscopy to
visualize probe-treated cells (Figure 5 and Figure S4 in the
Supporting Information). Live HepG2 cells were first treated
with THL-R (1), fixed with PFA, permeabilized with Triton
X-100, conjugated to rhodamine-azide (33) by click chemistry,
and imaged (colored in red). Immunofluorescence was also
carried out on the same cells to visualize the localization of
endogenous FAS (colored in green in top panels). Minimal
fluorescence was observed in samples treated with only DMSO,
whereas, in THL-R treated cells, fluorescence was mostly
distributed in endoplasmic reticulum (ER, bottom panels in
Figure 5). Note that endogenous FAS was mostly cytosolic
(which includes ER, top panels). Thus, our imaging results are
consistent with previous findings that inhibition of FAS with
Orlistat induces ER stress specifically in tumor cells.3c We take
note, however, that, at its current state, THL-R might not be
the most suitable chemical probe for bioimaging of FAS, as it
also labels a number of other cellular proteins (as evidenced
from our studies herein). Work is in progress to develop other
Orlistat analogues, which may confer much greater specificities,
and results will be reported in due course.
Experimental Section
Chemicals and Antibodies. Orlistat (98%), Tris(2-carboxyethyl)
phosphine (TCEP), and the click chemistry ligand, tris[(1-benzyl-
1H-1,2,3-triazol-4-yl)methyl]amine (“ligand”), were purchased from
Sigma-Aldrich. Antibody against FAS (Cat No. 610963) was from
BD Transduction Laboratories (San Diego, CA). Antibodies against
eIF2R (#9722), phospho-eIF2R (#9721), and cleaved caspase-8
(#9746) were from Cell Signaling Technologies (Beverly, MA).
Antibodies against HSP90b (sc-1057), Annexin A2 (sc-1492),
RPL14 (KQ-16, sc-100826), ꢀ-tubulin (sc-58882), and c-Myc
(9E10) were from Santa Cruz Biotechnology Inc. Antibodies against
GAPDH (ab9484), RPL7a (ab70753), and RPS9 (ab74711) were
from Abcam. (S)-(+)-N-formyl leucine 9, thioketene acetal 16,
hydroxyl-ꢀ-lactone 30, amino azide linker 31, and rhodamine B
acid 32 were prepared as described.22-26 The AHA metabolic
labeling kit was from Invitrogen.
Cell Culture. Cell lines were obtained from the National Cancer
Institute Developmental Therapeutics Program (NCI60 cell line
panel). HepG2 and HEK-293T were grown in DMEM (Invitrogen,
Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum
(21) (a) Potashman, M. H.; Duggan, M. E. J. Med. Chem. 2009, 52, 1231–
1246. (b) Drahl, C.; Cravatt, B. F.; Sorensen, E. J. Angew. Chem.,
Int. Ed. 2005, 44, 5788–5809. (c) Mukherji, D.; Spicer, J. Exp. Opin.
InVest. Drugs 2009, 18, 293–301.
Conclusions
We have developed a novel chemical proteomic approach
that enabled, for the first time, identification and putative
validation of several previously unknown cellular targets of
Orlistat. The potential of these cell-permeable probes to be used
as future imaging probes has also been explored. Whereas
further studies are needed to better understand the exact
relevance of Orlistat and its pharmacological effects in relation
to these newly identified cellular targets, our findings point to
a likely scenario that these proteins might be potential off-targets
(22) Boyle, G. A.; Govender, T.; Kruger, H. G.; Maguire, G. E. M.
Tetrahedron: Asymmetry 2004, 15, 2661–2666.
(23) Yin, J.; Yang, X. B.; Chen, Z. X.; Zhang, Y. H. Chin. Chem. Lett.
2005, 16, 1448–1450.
(24) Schwabacher, A. W.; Lane, J. W.; Schiesher, M. W.; Leigh, K. M.;
Johnson, C. W. J. Org. Chem. 1999, 63, 1727–1729.
(25) Nguyen, T.; Francis, M. B. Org. Lett. 2003, 5, 3245–3248.
(26) Liu, K.; Shi, H.; Xiao, H.; Chong, A. G. L.; Bi, X.; Chang, Y. T.;
Tan, K.; Yada, R. Y.; Yao, S. Q. Angew. Chem., Int. Ed. 2009, 48,
8293–8297. (b) Shi, H.; Liu, K.; Xu, A.; Yao, S. Q. Chem. Commun.
2009, 5030–5032.
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(FBS, Gibco Invitrogen), 100 U/mL penicillin and 100 µg/mL
streptomycin (Thermo Scientific, Rockford, IL) and maintained in
a humidified 37 °C incubator with 5% CO2. MCF-7 and PC-3 were
maintained in RPMI 1640 medium supplemented with 10% FBS
and 100 U/mL penicillin and 100 µg/mL streptomycin. To generate
protein lysates, cells were washed twice with cold phosphate-
buffered saline (PBS), and harvested with a cell scraper, and
collected by centrifugation. Cell pellets were resuspended in PBS
and lysed by sonication. Protein concentration was determined by
the Bradford assay. Cell lysates were diluted with PBS to achieve
final concentration of ∼1 mg/mL for labeling reactions.
hydrochloride (TCEP) (1 mM, 50 mM freshly prepared stock
solution in deionized water), tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl] amine (TBTA) (100 µM, 10 mM stock solution in
DMSO) and CuSO4 (1 mM, 50 mM freshly prepared stock solution
in deionized water)] was added and vortexed, then incubated for
2 h at room temperature with gentle mixing. The reactions were
terminated by the addition of prechilled acetone (0.5 mL), placed
at -20 °C for 30 min and centrifuged at 13000 rpm for 10 min at
4 °C to precipitate proteins. The supernatant was discarded and
the pellet washed two times with 200 µL of prechilled methanol.
The protein pellets were allowed to air-dry for 10 min, resuspended
in 25 µL 1 × standard reducing SDS-loading buffer and heated
for 10 min at 95 °C; ∼20 µg of protein was loaded per gel lane for
separation by SDS-PAGE (12% or 8-16% gradient precast gel),
then visualized by in-gel fluorescence scanning using a Typhoon
9410 Variable Mode Imager scanner.
For in situ labeling, cells were grown to 80-90% confluence in
24-well plates under the conditions described above. The medium
was removed, and then cells were washed twice with cold PBS,
and treated with 0.5 mL of DMEM-containing probe (1-20 µM),
with or without Orlistat or Cerulenin (100 µM). Probes were applied
from DMSO stocks whereby DMSO never exceeded 1% in the
final solution. The same volume of DMSO was used as a negative
control. After 2 h of incubation at 37 °C/5% CO2, the growth
medium was aspirated, and cells were washed twice with PBS to
remove the excessive probe, trypsined, and pelleted by centrifuga-
tion. The cell pellet was resuspended in PBS (50 µL), homogenized
by sonication, and diluted to ∼1 mg/mL with PBS. Probe targets
were detected by click chemistry with rhodamine-azide 33, SDS-
PAGE analysis, and in-gel fluorescence scanning.
Cell Proliferation Assay. Cell viability was determined using
the XTT colorimetric cell proliferation kit (Roche) following
manufacturer’s guidelines. Briefly, cells were grown to 20-30%
confluence (since they will reach ∼90% confluence within 48 to
72 h in the absence of drugs) in 96-well plates under the conditions
described above. The medium was aspirated, and then washed with
PBS, and then treated, in duplicate, with 0.1 mL of the medium
containing different concentrations of THL analogues (1-50 µM)
or Orlistat (1-50 µM, as a positive control). Probes were applied
from DMSO stocks whereby DMSO never exceeded 1% in the
final solution. The same volume of DMSO was used as a negative
control. Fresh medium, along with THL analogues or Orlistat, were
added every 24 h. After a total treatment time of 72 h, proliferation
was assayed using the XTT colorimetric cell proliferation kit
(Roche) following manufacturer’s guidelines (read at 450 nm). Data
represent the average (s.d. for two trials.
Western Blotting. To monitor the effects of THL analogues on
inducing phosphorylation of eIF2R, PC-3 cells were treated with
indicated concentrations of Orlistat and THL analogues for 16 h.
Samples from treated cells were then separated on 12% SDS-PAGE
gel and further transferred to PVDF membranes. Membranes were
blocked with 5% BSA in TBS. After blocking, membranes were
incubated with anti-eIF2R (#9722 from Cell Signaling, 1/5000) or
antiphospho-eIF2R (#9721 from Cell Signaling, 1/2000). After
incubation, membranes were washed with TBST for three times
and then incubated with an appropriate secondary antibody [anti-
mouse conjugated HRP (1/5000) or antirabbit conjugated HRP
(1/5000)]. After secondary incubation, blots were washed again with
TBST before the development with SuperSignal West Pico kit
(Pierce).
Hydroxylamine Treatment of Gels. After the proteins were
separated by SDS-PAGE gel, the gel was soaked in 40% MeOH,
10% acetic acid, shaking overnight at room temperature, washed
with deionized water (2 × 5 min), and scanned for the prehydroxy-
lamine treatment fluorescence. The gel was then soaked in PBS,
shaking 1 h at room temperature, followed by boiling in neutralized
hydroxylamine (Alfa Aesar) (2.5% final concentration) for 5 min,
washing with deionized water (2 × 5 min), and soaking in 40%
MeOH, 10% acetic acid, shaking overnight at room temperature.
The gel was washed with deionized water (2 × 5 min) and scanned
for the posthydroxylamine fluorescence.
Pull-Down and Mass Spectrometry Identification. To identify
the in situ targets of THL-R in HepG2 cell line, pull-down followed
by immunoblot or MS/MS identification experiments were carried
out as described below. In situ labeling sample of HepG2 by
THL-R were prepared as described previously. After in situ
labeling, cells were washed with PBS, detached from T75 culturing
flask and pelleted by centrifuge for 1000 rpm for 15 min. Five
milligrams of the lysates were reacted by click chemistry with
biotin-azide 34 under the conditions described above, acetone
precipitated, and resolubilized in 0.1% SDS in PBS with brief
sonication. This resuspended sample was then incubated with
avidin-agarose beads (100 µL/mg protein) at room temperature for
30 min. After centrifugation, supernatant were removed and the
beads were washed with 1% SDS in PBS for 4 times. After washing,
the beads were boiled in elution buffer (200 mM Tris pH 6.8, 400
mM DTT, 8% SDS).
For immunoblotting analysis, this pulled-down sample was then
separated on 8-16% gradient precast gel (Biorad), transferred to
PVDF membrane and probed with purified mouse anti-FAS (Cat
No. 610963, BD Transduction Laboratories).
For MS/MS identification, proteins from pulled-down fractions
were separated on 12% SDS-PAGE gel, followed by silver staining.
Trypsin digestion was performed with In-Gel Trypsin Digestion
Kit from Pierce for respective visible protein bands. After digestion,
digested peptides were then extracted from the gel with 50%
acetonitrile and 1% formic acid. Tryptic peptide extracts were
evaporated by speedvac and reconstituted with 10 µL of 0.1% TFA,
a volume of 2 µL of the peptide extracts were manually spotted
To monitor the effects of THL analogues on inducing activation
of the caspase-8 pathway, MCF-7 cells were treated with indicated
concentrations of Orlistat and THL analogues for 36 h. Samples
from treated cells were then separated on a 12% SDS-PAGE gel
and further transferred to PVDF membranes. Membranes were
blocked with 5% BSA in TBS. After blocking, membranes were
incubated with anticaspase 8 (#9746 from Cell Signaling, 1/2000).
After incubation, membranes were washed with TBST for three
times, and then incubated with antimouse conjugated HRP (1/5000).
After secondary incubation, blots were washed again with TBST
before the development with SuperSignal West Pico kit (Pierce).
Measurement of Protein Synthesis. Live HepG2 cells were
treated with the indicated concentrations of Orlistat/THL-R or CHX
(cycloheximide, an inhibitor of protein biosynthesis) for 12 h,
washed twice with PBS, and then pulsed with AHA (L-azi-
dohomoalanine, 20 µM) for 4 h. Cells were collected, washed, and
cell lysates were prepared and subjected to click chemistry with
rhodamine-alkyne (provided with the AHA kit), SDS-PAGE
analysis, and in-gel fluorescence scanning.
In Vitro and In Situ Proteome Labeling and Analysis. For in
vitro proteome labeling, probes were added to cell lysates (50 µg)
in 50 µL of PBS at a final concentration of 1-20 µM in the presence
or absence of excess Orlistat or Cerulenin competitor (a final
concentration of 100 µM). Unless indicated otherwise, samples were
incubated for 2 h with varying concentrations of probe at room
temperature. After incubation, 10 µL of the freshly premixed click
chemistry reaction cocktail in PBS [rhodamine-azide 33 (100 µM,
10 mM stock solution in DMSO), tris(2-carboxyethyl)phosphine
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A R T I C L E S
Yang et al.
onto a Prespotted AnchorChip MALDI target plate for MALDI-
TOF Mass step with 10 µL of 10 mM ammonium phosphate in
0.1% TFA, and allowed to dry at ambient temperature. MALDI
TOF mass spectra were recorded using Ultraflex III TOF/TOF mass
spectrometer (Bruker Daltonics) with the Compass 1.2 software
package including flexControl 3.0 and flexAnalysis 3.0, calibrated
with PAC peptide calibration standards. MS/MS analysis for the
major peaks in PMF spectra were carried out by autoLIFT on the
MALDI-TOF/TOF instrument. MS and MS/MS Peak lists with
intensity value were submitted to Matrix Science Mascot server
(http://www.matrixscience.com/) through BioTools 3.0 (Bruker
Daltonics) using database NCBInr 090202 version, variable modi-
fications of carbamidometyl on cysteine (C) and oxidation on
methione (M), allowing maxium of trypsin missed cleavage, peptide
mass tolerance at 200 ppm; MS/MS mass tolerance of 0.7 Da.
Target Validation by Western Blotting. Pull-down sample from
labeled lysates was separated on 12% SDS-PAGE gel together with
pull-down sample from DMSO-treated, unlabeled lysates (negative
controls). After SDS-PAGE gel separation, proteins were then
transferred to a PVDF membrane and subsequently blocked with
2.5% (w/v) BSA/PBST. Membranes were incubated for 1 h at room
temperature with the respective antibodies (i.e., anti-RPL7a, anti-
Annexin A2, anti-HSP90b, anti-RPS9, anti-GAPDH, and anti-ꢀ-
tubulin). After three times of washes with PBST, blots were further
incubated with the appropriate secondary antibody for 1 h at room
temperature. After incubation, the blot was washed again with PBST
for 3 times and the SuperSignal West Pico kit (Pierce) was used to
develop the blot.
Target Validation by Recombinant Protein Expression in
HEK-293T Cells. Mammalian expression vectors overexpressing
each of the four targets were purchased from Origene (USA).
Vectors were transfected with Lipofectamine reagent (Qiagen) at
80% confluence. After 24 h of transfection, either nontransfected
and transfected cell were incubated with THL-R. After THL-R
incubation, small fractions of the samples were analyzed by Western
blotting with 1/2000 anti-c-Myc antibody (Santa Cruz). The rest
of the cells were lysed and immunopurified with c-Myc agarose
beads (Santa Cruz). Eluted fractions were clicked with rhodamine-
azide 33 as previously described. After click chemistry, samples
were concentrated, separated on a SDS-PAGE gel and scanned by
in-gel fluorescence.
of rhodamine-azide 33 in PBS for 2 h at room temperature with
vigorous shaking. Cells were washed with PBS three times, and
then with 20 mM of HEPES, pH 7.5, 500 mM of NaCl, 2% triton
for overnight at room temperature, then washed again by PBS for
three times. For colocalizing in situ targets of THL analogues with
FAS, cells were further incubated with anti-FASN primary antibod-
ies (1:200) for 1 h at room temperature (or overnight at 4 °C), and
washed twice with PBS. The cells were incubated with FITC-
conjugated antimouse IgG (1:500) for 1 h, washed again. Cells were
stained with 1 µg/mL Hoechst for 10 min at room temperature,
washed again before mounting. For colocalizing in situ targets of
THL-R with ER, cells were further incubated with ER-Tracker
Green (glibenclamide BODIPY FL) for 1 h at room temperature
(or overnight at 4 °C), and washed twice with PBS. Cells were
stained with 1 µg/mL Hoechst for 10 min at room temperature,
washed again before mounting.
Chemical Synthesis. All chemicals were purchased from com-
mercial vendors and used without further purification, unless
otherwise noted. Tetrahydrofuran (THF) was distilled over sodium
benzophenone and used immediately. Dichloromethane (CH2Cl2)
was distilled over CaH2. All nonaqueous reactions were carried out
under nitrogen atmosphere in oven-dried glassware. Reaction
progress was monitored by TLC on precoated silica plates (Merck
60 F254, 250 µm thickness) and spots were visualized by ceric
ammonium molybdate, basic KMnO4, UV light, or iodine. 1H NMR
and 13C NMR spectra were recorded on a Bruker model Avance
300 MHz or DPX-300 MHz or DPX-500 MHz NMR spectrometer.
Chemical shifts are reported in parts per million relative to internal
standard tetramethylsilane (Si(CH3)4 ) 0.00 ppm) or residual
1
solvent peaks (CHCl3 ) 7.26 ppm). H NMR coupling constants
(J) are reported in Hertz (Hz) and multiplicity is indicated as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
br s (broad singlet), dd (doublet of doublet), dt (doublet of triplet),
dq (doublet of quartet), tq (triplet of quartet). Mass spectra were
obtained on Shimadzu IT-TOF-MS or Shimadzu ESI-MS system.
(E)-2-(1-(tert-butyldimethylsilyloxy)-8-(trimethylsilyl)-oct-1-
en-7-ynylthio)-pyridine (12). Thiopyridyl ester 11 (prepared in
three steps from 6-bromohexanoic acid, Scheme S3 in the Sup-
porting Information) (5.42 g, 17.9 mmol) was dried azeotropically
with xylenes. After purging with N2, anhydrous DMF (1.57 g, 21.5
mmol) and anhydrous Et3N (3.02 g, 21.5 mmol) in dry CH2Cl2
(120 mL) was added and the mixture was cooled to -78 °C. After
10 min, LiHMDS (1.0 M, 41.3 mL, 41.3 mmol) was added
dropwise. The reaction mixture was stirred for 30 min and TBSCl
(5.4 g, 35.8 mmol) was added dropwise to the reaction mixture at
-78 °C. The reaction mixture was stirred for 2 h at this temperature
and then quenched with pH 7 phosphate buffer (20 mL × 2). The
organic layer was separated, dried over Na2SO4 and evaporated.
The resulting yellowish oil was purified by flash chromatography
(10% EtOAc/hexane) to afford ketene acetal 12 (5.60 g, 74%) as
Identification of GAPDH Labeling Site. The mammalian
expression vector overexpressing GAPDH was used as template
for generating the GAPDH active-site mutant, using the Site-
Direceted Mutagenesis System provided by Invitrogen (USA).
Cys151 was mutated to Ala. Primers used for site-directed mutagen-
esis were designed following the vendor’s protocols. They are
shown below:
5′ -ATCAGCAATGCCTCCGCCACCACCAACTGC-3′
5′-GCAGTTGGTGGTGGCGGAGGCATTGCTGAT-3′
1
a yellow oil. H NMR (500 MHz, CDCl3) δ 8.40-8.50 (m, 1H),
Vectors overexpressing both wild-type and mutant GAPDH were
transfected to HEK-293T cell lines. Recombinant protein was
purified, labeled and clicked as previously described. Further,
samples were separated on SDS-PAGE gel and fluorescently
scanned with Typhoon Scanner. After scanning, gel was fixed and
silver stained to visualize the total protein bands.
Cellular Imaging Using THL-R. HepG2 cells were seeded onto
24-well plates containing sterile glass coverslips and grown until
70-80% confluence. Further, cells were treated with 0.5 mL of
DMEM with 20 µM THL-R or DMSO. After 24 h, the growth
medium was removed, and cells were washed twice with PBS. After
fixation in 3.7% paraformaldehyde in PBS for 15 min at room
temperature, cells were washed twice with cold PBS again. Cells
were permeabilized with 0.1% Triton X-100 in PBS 10 min at room
temperature, and blocked with 2% BSA in PBS for 30 min at room
temperature, and then washed twice with PBS. Cells were then
treated with a freshly premixed click chemistry reaction solution
in a 250 µL volume at final concentrations of the following reagents:
1 mM of CuSO4, 1 mM of TCEP, 100 µM of TBTA, and 10 µM
7.54 (dt, J ) 1.9, 7.6, Hz, 1H), 7.31-7.33 (d, J ) 8.2 Hz, 1H),
6.98-7.01 (m, 1H), 5.39 (t, J ) 7.0 Hz, 1H), 2.34 (dd, J ) 6.9,
13.8 Hz, 2H), 2.20 (dd, J ) 5.7, 12.6 Hz, 2H), 1.50-1.56 (m, 4H),
0.88 (s, 9H), 0.14 (s, 9H), 0.08(s, 6H); 13C NMR (125 MHz, CDCl3)
δ 174.1, 160.4, 149.4, 139.7, 136.5, 123.4, 121.5, 119.6, 107.3,
84.5, 35.9, 28.4, 28.3, 26.3, 25.7, 25.6, 25.0, 19.7, 18.1, 17.6, 0.2.
-4.8; ESI-MS: [M+1]+ calcd: 419.213, found: 420.244.
(3S,4S)-4-((R)-2-hydroxytridecyl)-3-(6-(trimethylsilyl)hex-5-
ynyl)oxetan-2-one (13). TMAL Reaction. ZnCl2 (796 mg, 5.84
mmol) was fused under vacuum and then allowed to cool to room
temperature under a flow of nitrogen. Fifteen milliliters of dry
CH2Cl2 was then added via syringe followed by a solution of 8a
(547 mg, 2.92 mmol) in 5 mL of CH2Cl2 and ketene acetal 12 (1.72
g, 4.08 mmol). The reaction mixture was stirred for 72 h at room
temperature. Ten milliliters of pH 7 phosphate buffer were then
added and the mixture was stirred vigorously for 45 min, filtered
through a pad of Celite, and washed with CH2Cl2. The organic
filtrate was dried over Na2SO4, filtered, and concentrated. The
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Cell-Based Proteome Profiling of Orlistat
A R T I C L E S
residue was purified by flash chromatography on SiO2 (10% EtOAc/
mixture was cooled to -78 °C and stirred for 10 min. Aldehyde
20 (1.19 g, 6.6 mmol) in DCM (10 mL) was added dropwise and
stirred at -78 °C for 4 h then warmed to room temperature
overnight. Saturated NaHCO3 solution (25 mL) was added to the
reaction mixture and stirred for 30 min. The aqueous phase was
extracted with DCM. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The crude product was purified by column
chromatography (hexane/EtOAc, 95:5) to give homoallylic alcohol
21 (1.06 g, 71%, 62.4% ee) as a colorless oil. 1H NMR (500 MHz,
CDCl3) δ 5.79-5.87 (m, 1H), 5.15 (br d, J ) 3.2 Hz, 1H), 5.12 (s,
1H), 3.62-3.67 (m, 1H), 2.28-2.33 (m, 1H), 2.11-2.19 (m, 3H),
1.93 (t, J ) 2.5 Hz, 1H), 1.29-1.57 (m, 16H); 13C NMR (125
MHz, CDCl3) δ 134.9, 118.1, 84.8, 70.7, 68.0, 42.0, 36.8, 29.6,
29.5, 29.4, 29.1, 28.7, 28.5, 25.6, 18.4.
(R)-tert-Butyldimethyl(pentadec-1-en-14-yn-4-yloxy)silane (22).
Imidazole (848 mg, 12.5 mmol), DMAP (51 mg, 0.42 mmol) and
TBSCl (1.13 g, 1.47 mmol) was added to a solution of alcohol 21
(980 mg, 4.15 mmol) in DCM (20 mL) at 0 °C. The reaction
mixture was warmed to room temperature and stirred for 7.5 h.
The organic phase was washed with water, brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude product
was then purified by flash column chromatography (hexane/EtOAc,
96:4) to provide 22 (1.35 g, 97%) as a colorless oil.1H NMR (500
MHz, CDCl3) δ 5.77-5.85 (m, 1H), 5.03 (br d, J ) 8.8 Hz, 1H),
5.01 (s, 1H), 3.67 (quint., J ) 5.7 Hz, 1H), 2.16-2.22 (m, 4H),
1.94 (t, J ) 2.5 Hz, 1H), 1.54 (q, J ) 7.6 Hz, 1H), 1.26-1.41 (m,
15H), 0.89 (s. 9H), 0.04 (s, 6H); 13C NMR (125 MHz, CDCl3) δ
135.5, 116.5, 84.8, 72.0, 68.0, 41.9, 36.8, 29.7, 29.6, 29.4, 29.1,
28.8, 28.5, 25.9, 25.7(2), 25.3, 18.4, 18.2, 18.1, -2.9.
(R)-3-(tert-Butyldimethylsilyloxy)tetradec-13-ynal (23). Ozone
was bubbled through a solution of 22 (900 mg, 2.67 mmol) and
Et3N (540 mg, 5.34 mmol) in DCM (25 mL) cooled at -78 °C.
After reaction was complete (monitored by TLC), Me2S (332 mg,
5.34 mmol) was added to the reaction mixture and warmed to room
temperature. After stirring for 2 h, the solvent was removed under
reduced pressure. The crude aldehyde was then purified by column
chromatography (hexane/EtOAc, 100:0 to 9:1) to furnish aldehyde
23 (720 mg, 80%) as a colorless oil.1H NMR (500 MHz, CDCl3)
δ 9.81 (t, J ) 2.6 Hz, 1H), 4.17 (quint., J ) 5.7 Hz, 1H), 2.51 (dd,
J ) 2.6, 5.0 Hz, 2H), 2.18 (dt, J ) 2.5, 6.9 Hz,, 2H), 1.93 (t, J )
2.5 Hz, 1H), 1.29-1.55 (m, 16H), 0.88 (s, 9H), 0.07 (s, 3H), 0.06
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 202.4, 84.8, 68.3, 68.0,
50.8, 37.8, 29.6, 29.5, 29.4, 29.0, 28.7, 28.5, 25.8, 25.1, 18.4, 18.0,
-4.4, -4.7.
hexane) to provide the mixture of silyloxy-ꢀ-lactone diastereomers
1
as a white solid. H NMR (500 MHz) analysis of the mixture of
diastereomers indicated a diastereomeric ratio of 8.5:1. Without
further purification, the mixture was used directly in the next step.
O-Desilylation. To a stirred solution of the mixture of silyloxy-
ꢀ-lactones in 48 mL of CH3CN cooled to 0 °C, 4.8 mL of HF
(48%) was added dropwise. The mixture was stirred at 0 °C for
2 h, then allowed to warm to room temperature and stirred for an
additional 8 h. The reaction mixture was diluted with 150 mL of
Et2O, quenched carefully with cold saturated NaHCO3 solution, and
washed with brine. The organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography (10% EtOAc/hexane) to provide the hydroxy-ꢀ-
lactone 13 (360 mg, 46%) and a mixture of the two diastereomers
(90 mg, 12%) as white solids (58% overall two steps). Spectroscopic
1
data are reported for the major diastereomeric-ꢀ-lactone 13. H
NMR (500 MHz, CDCl3) δ 4.50 (app quint., J ) 4.4 Hz, 1H),
3.78-3.83 (m, 1H), 3.27 (dt, J ) 3.8, 7.6 Hz,1H), 2.24 (t, J ) 7.0
Hz, 2H), 1.77-1.96 (m, 4H), 1.49-1.57 (m, 8H), 1.26 (br s, 18H),
0.88 (t, J ) 7.0 Hz, 3H), 0.15 (s, 9H); 13C NMR (125 MHz, CDCl3)
δ 171.3, 106.7, 85.0, 75.6, 68.5, 56.5, 41.8, 38.2, 31.9, 29.6, 29.5,
29.3, 28.1, 27.3, 26.9, 25.9, 25.4, 22.7, 19.6, 14.1.; ESI-MS:
[M+1]+ calcd: 422.32, found: 423.25.
C-Desilylation. To a solution of (trimethylsily1)-acetylene 13
(134 mg, 0.5 mmol) in 20 mL of a mixture of acetone/H2O/2,6-
lutidine (1:1:0.l) was added solid AgNO3 (850 mg, 5 mmol). The
white suspension was stirred vigorously for 4 h, and then 5 mL of
a 1.0 M aqueous KH2PO4 solution was added. The resulting yellow
slurry was stirred for an additional 30 min. Filtration of the reaction
mixture through Celite removed most of the yellow precipitate. The
filtrate was extracted with Et2O, and the combined organic extracts
were washed once with brine and dried over Na2SO4. Evaporation
of the solvent under reduced pressure afforded a pale-yellow oil.
Purification by chromatography on silica gel (15:l hexane/EtOAc)
1
gave 81 mg of 14 (83%) as a sticky, colorless oil. H NMR (500
MHz, CDCl3) δ 4.50 (app quint., J ) 4.4 Hz, 1H), 3.78-3.83 (m,
1H), 3.28 (dt, J ) 3.8, 8.2 Hz, 1H), 2.19-2.23 (m, 2H), 1.91-1.97
(m, 2H), 1.77-1.87 (m, 2H), 1.47-1.62 (m, 8H), 1.26 (br s, 18H)
0.88 (t, J ) 6.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.4,
84.0, 75.6, 68.7, 68.5, 56.4, 41.8, 38.2, 31.9, 29.6(2), 29.5(2), 29.3,
28.0, 27.2, 25.8, 25.4, 22.7, 18.1, 14.1.; IT-TOF-MS: m/z [M+1]+
calcd: 350.282, found: 351.288.
Mitsunobu Reaction. ꢀ-lactone 14 (70 mg, 0.2 mmol), PPh3
(79 mg, 0.3 mmol), and N-formyl-L-leucine 9 (128 mg, 0.8 mmol)
were placed in a RB-flask and azeotroped under vacuum with 0.5
mL of xylene for 30 min. Addition of 5.0 mL of dry THF was
followed by cooling to 0 °C, DIAD (60 µL, 0.3 mmol) was then
added via syringe. The mixture was stirred at 0 °C for 10 min,
allowed to warm to room temperature. The reaction was monitored
by TLC. After the reaction was complete, the mixture was then
concentrated in vacuo and polar impurities were separated by flash
chromatography (60:40, hexane/EtOAc). Further purification of the
residue by flash chromatography (20% EtOAc/hexane) yielded
ꢀ-lactone 1. (74 mg, 75%). 1H NMR (500 MHz, CDCl3) δ 8.22 (s,
1H), 5.92 (d, J ) 8.2 Hz, 1H), 5.01-5.06 (m, 1H), 4.68 (dt, J )
4.4, 8.8 Hz, 1H), 4.30 (q, J ) 4.4 Hz, 1H), 3.23 (dt, J ) 3.8, 7.6
Hz, 1H), 2.14-2.23 (m, 3H), 1.96 (t, J ) 2.5 Hz, 1 H), 1.95-2.04
(m, 1H), 1.55-1.82 (m, 11H), 1.25-1.27 (m, 18 H), 0.96-0.98
(m, 6H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
δ 171.9, 170.5, 160.6, 83.9, 74.7, 72.7, 68.8, 56.9, 49.7, 41.5, 38.7,
34.1, 31.9, 29.6, 29.5, 29.4, 29.3(2), 28.0, 27.1, 25.7, 25.1, 24.9,
22.9, 22.7, 21.9, 21.7, 18.1, 14.1.; ESI-MS: m/z [M+1]+ calcd:
491.4, found: 492.3.
(3S,4S)-3-Hexyl-4-((R)-2-hydroxytridec-12-ynyl)oxetan-2-
one (24). This was prepared according to the general TMAL
reaction procedure using aldehyde 23 (544 mg, 1.61 mmol), ZnCl2
(438 mg, 3.22 mmol), and ketene acetal 16 (740 mg, 2.42 mmol)
in DCM (10 mL). The crude mixture was purified by column
chromatography (hexane/EtOAc: 100:0 to 97:3) to provide a mixture
of silyloxy-ꢀ-lactone diastereomers as a colorless oil. 1H NMR (500
MHz) analysis of the mixture of diastereomers indicated that a
diastereomeric ratio of 11:1. Without further purification, the
1
mixture was used directly in the next step. H NMR (500 MHz,
CDCl3) δ 4.41-4.44 (m, 1H), 3.83-3.85 (m, 1H), 3.16-3.24 (m,
1H), 2.18 (dt, J ) 2.5, 6.9 Hz, 3H), 1.94 (t, J ) 2.5 Hz, 1H),
1.28-1.85 (m, 27H), 0.90 (s, 9H), 0.89 (t, J ) 7.0 Hz, 3H), 0.07
(s, 6H).
Prepared according to the general O-desilylation procedure using
silyloxy-ꢀ-lactone 23 (409 mg, 0.9 mmol), and 40% HF (782 µL)
in CH3CN (12 mL). The crude mixture was purified by flash
chromatography (hexane/EtOAc, 100:0 to 9:1) to provide the
hydroxy-ꢀ-lactone 24 (173 mg, 32%) and a mixture of the two
diastereomers (42 mg, 8%) as a colorless oil (40% overall two
steps). Spectroscopic data are reported for the major diastereomeric
ꢀ-lactone 24: 1H NMR (500 MHz, CDCl3) δ 4.49 (dt, J ) 4.4, 8.8
Hz, 1H), 3.78-3.83 (m, 1H), 3.24-3.27 (m, 1H), 2.18 (dt, J )
2.5, 7.0 Hz, 2H), 1.93 (t, J ) 2.5 Hz, 1H), 1.72-1.92 (m, 4H),
(R)-Pentadec-1-en-14-yn-4-ol (21). (S)-BINOL (415 mg, 1.45
mmol) was added to a suspension of InCl3 (292 mg, 1.32 mmol,
azeotropically dried over THF (2 × 3 mL)) in CH2Cl2 (15 mL)
and stirred for 2 h at room temperature. Allyltributyltin (4.37 g,
13.2 mmol) was added and stirred for 5 min, then the reaction
9
J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010 665

A R T I C L E S
Yang et al.
1.29-1.53 (m, 25H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 171.6, 84.7, 75.6, 68.5, 68.0, 56.6, 41.8, 38.1, 31.5,
29.4, 29.0, 28.9, 28.7, 28.4, 27.7, 26.7, 25.4, 22.5, 18.4, 14.0; IT-
TOF-MS: m/z [M+1]+ calcd: 350.281, found: 351.301.
29.5, 29.3, 29.0, 27.7, 26.8, 25.4, 22.7, 14.1, 14.0; ESI-MS: m/z
[M+Na]+ calcd: 354.3, found: 377.2.
(R)-((S)-1-((2S,3S)-3-Hexyl-4-oxooxetan-2-yl)tridecan-2-yl)-4-
methyl-2-propiolamidopentanoate (3). Prepared according to the
general Mitsunobu reaction procedure using ꢀ-lactone 30 (71 mg,
0.20 mmol), PPh3 (68 mg, 0.26 mmol), acid 29 (46 mg, 0.25 mmol)
and DIAD (52 µL, 0.26 mmol) in THF (3 mL). Purification by
flash chromatrography (hexane/EtOAc, 100:0 to 85:15) afforded 3
(S)-((S)-1-((2S,3S)-3-Hexyl-4-oxooxetan-2-yl)tridec-12-yn-2-
yl)-2-formamido-4-methylpentanoate (2). Prepared according to
the general Mitsunobu reaction procedure using ꢀ-lactone 24 (11
mg, 0.03 mmol), DIAD (23 µL, 0.12 mmol), N-formyl-L-leucine
(20 mg, 0.13 mmol), and triphenylphosphine (33 mg, 0.13 mmol)
and in THF (1 mL). Purified by flash chromatography (hexane/
EtOAc, 9:1 to 4:1) to give ꢀ-lactone 2 as a colorless oil (44%). 1H
NMR (500 MHz, CDCl3) δ 8.22 (s, 1H), 5.91 (br d, J ) 8.2 Hz,
1H), 5.00-5.03 (m, 1H), 4.66-4.71 (m, 1H), 4.27-4.34 (m, 1H),
3.22 (dt, J ) 4.4, 7.6 Hz, 1H), 2.18 (dt, J ) 2.5, 6.9 Hz, 2H), 2.00
(dt, J ) 4.4, 14.5 Hz, 1H), 1.93 (t, J ) 2.5 Hz, 1H), 1.28-1.85
(m, 30H), 0.96-0.99 (m, 6H), 0.89 (t, J ) 7.6 Hz, 3H); IT-TOF-
MS: m/z [M+1]+ calcd: 491.361, found: 492.320.
1
(54 mg, 52%) as a colorless oil: H NMR (500 MHz, CDCl3) δ
6.31 (br d, J ) 7.6 Hz, 1H), 5.01-5.04 (m, 1H), 4.60 (dt, J ) 4.4,
8.8 Hz, 1H), 4.26-4.30 (m, 1H), 3.21 (dt, J ) 3.8, 7.6 Hz, 1H),
2.84 (s, 1H), 2.15 (dt, J ) 7.6, 15.1 Hz, 1H), 1.99 (dt, J ) 4.5,
15.7 Hz, 1H), 1.54-1.80 (m, 8H), 1.25 (br s, 25H), 0.96 (d, J )
6.3 Hz, 3H), 0.95 (d, J ) 6.3 Hz, 3H), 0.874 (t, J ) 7.0 Hz, 3H),
0.867 (t, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 171.4,
170.7, 151.7, 83.3, 74.7, 74.1, 72.9, 57.0, 51.4, 41.2, 38.6, 34.0,
31.9, 31.4, 29.6, 29.5, 29.4, 29.3, 29.2, 28.9, 27.6, 26.7, 25.1, 24.9,
22.8, 22.6, 22.5, 21.7 14.1, 14.0; IT-TOF-MS: m/z [M+1]+ calcd:
519.392, found: 520.391.
(3S,4S)-3-Hexyl-4-((R)-2-hydroxytridecyl)oxetan-2-one (30).5a
Prepared according to the general TMAL reaction procedure using
aldehyde 8a (432 mg, 1.27 mmol), ZnCl2 (346 mg, 2.54 mmol)
and ketene acetal 16 (652 mg, 1.85 mmol). Purification by flash
chromatrography (hexane/EtOAc, 100:0 to 95:5) gave a mixture
of silyloxy-ꢀ-lactone diastereomers as colorless oils, which was
used directly in the next O-desilylation step.
Acknowledgment. Funding support was provided by the
National Research Foundation (Competitive Research Programme
R143-000-218-281) of Singapore.
Supporting Information Available: Details concerning the
synthesis of 8a, 9, 11, 16, 29, and additional figures containing
in vitro competition studies with Orlistat, cellular imaging with
probe 2 and 3, Western blotting analysis of recombinant
enzymes, mass spectrometry data, complete Table 1, and spectral
characterization of new compoounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Prepared according to the general O-desilylation reaction pro-
cedure using silyloxy-ꢀ-lactone (500 mg, 1.07 mmol) and 40% HF
(2.5 mL). Purification by flash chromatrography (hexanes/EtOAc,
100:0 to 96:4) gave the hydroxy-ꢀ-lactone 30 (174 mg, 38%) and
a mixture of the two diastereomers (47 mg, 10%) as white solids
1
(48% overall, 2 steps): H NMR (500 MHz, CDCl3) δ 4.50 (m,
1H), 3.81 (m, 1H), 3.26 (dt, J ) 7.6, 3.8 Hz, 1H), 1.95-1.26 (m,
35H), 0.88 (t, J ) 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ
171.6, 75.6, 68.5, 56.6, 41.8, 38.1, 31.9, 31.5, 29.63, 29.61, 29.55,
JA907716F
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666 J. AM. CHEM. SOC. VOL. 132, NO. 2, 2010