Chemical modification and organelle-specific localization of Orlistat-like natural-product-based probes

Article abstract of DOI:10.1002/asia.201100306

Orlistat, also known as tetrahydrolipstatin (THL), is an FDA-approved anti-obesity drug with potential anti-cancer activity. Previously, we developed a chemical proteomic approach, based on the Orlistat-like probe (1a) for large-scale identification of unknown cellular targets of Orlistat in human hepatocytes. In this article, we report the chemical synthesis and biological evaluation of an expanded set of Orlistat-like compounds, with the intention to further dissect and manipulate potential cellular targets of Orlistat. In doing so, we carried out proteome-wide activity-based profiling and large-scale pull-down/LCMS analysis of these compounds in live HepG2 cells, and successfully identified many putative cellular targets for Orlistat and its structural analogues. By qualitatively assessing the spectra counts of potential protein hits against each of the seventeen Orlistat analogues, we obtained both common and unique targets of these probes. Our results revealed that subtle structural modifications of Orlistat led to noticeable changes in both the cellular potency and target profiles of the drug. In order to further improve the cellular activity of Orlistat, we successfully applied the well-established AGT/SNAP-tag technology to our cell-permeable, benzylguanine (BG)-containing Orlistat variant (4). We showed that the drug could be delivered and effectively retained in different sub-cellular organelles of living cells. This strategy may provide a general and highly effective chemical tool for the potential sub-cellular targeting of small molecule drugs.

Full text of DOI:10.1002/asia.201100306

FULL PAPERS
DOI: 10.1002/asia.201100306
Chemical Modification and Organelle-Specific Localization of Orlistat-Like
Natural-Product-Based Probes
Peng-Yu Yang,[a] Kai Liu,[b] Chongjing Zhang,[a] Grace Y. J. Chen,[a, b] Yuan Shen,[a]
Mun Hong Ngai,[a] Martin J. Lear,[a] and Shao Q. Yao*[a, b]
On the occasion of the 10th anniversary of click chemistry
Abstract: Orlistat, also known as tetra-
hydrolipstatin (THL), is an FDA-ap-
proved anti-obesity drug with potential
anti-cancer activity. Previously, we de-
large-scale pull-down/LCMS analysis of
these compounds in live HepG2 cells,
and successfully identified many puta-
tive cellular targets for Orlistat and its
structural analogues. By qualitatively
assessing the spectra counts of poten-
tial protein hits against each of the sev-
enteen Orlistat analogues, we obtained
both common and unique targets of
these probes. Our results revealed that
subtle structural modifications of Orli-
stat led to noticeable changes in both
the cellular potency and target profiles
of the drug. In order to further im-
prove the cellular activity of Orlistat,
we successfully applied the well-estab-
lished AGT/SNAP-tag technology to
our cell-permeable, benzylguanine
(BG)-containing Orlistat variant (4).
We showed that the drug could be de-
livered and effectively retained in dif-
ferent sub-cellular organelles of living
cells. This strategy may provide a gen-
eral and highly effective chemical tool
for the potential sub-cellular targeting
of small molecule drugs.
veloped
a chemical proteomic ap-
proach, based on the Orlistat-like
probe (1a) for large-scale identification
of unknown cellular targets of Orlistat
in human hepatocytes. In this article,
we report the chemical synthesis and
biological evaluation of an expanded
set of Orlistat-like compounds, with the
intention to further dissect and manip-
ulate potential cellular targets of Orli-
stat. In doing so, we carried out pro-
teome-wide activity-based profiling and
Keywords: chemical proteomics
·
click chemistry · orlistat · SNAP—
tag technology · structure–activity
relationships
Introduction
therapeutic potential and minimize the potential cellular
toxicity of a drug.[1] Recent advances in chemical proteo-
mics, a multidisciplinary research area integrating biochem-
istry and cell biology with organic synthesis and mass spec-
trometry, have enabled a more-direct and unbiased analysis
of a drugꢀs mechanism of action in the context of the pro-
teomes as expressed in the target cell or the tissue of inter-
est.[2] However, at present, few methods are available for
the large-scale profiling of drug–protein interactions in
vivo.[3] We recently reported a chemical proteomic method
that makes use of natural-product-like small-molecule
probes for proteome-wide profiling of putative drug targets
in living cells.[4] That strategy is based on the concept of ac-
tivity-based protein profiling (ABPP), originally coined by
the Cravatt group and further developed by other
groups.[3,5,6] In our studies, terminal-alkyne-containing
probes based on Orlistat or tetrahydrolipstatin (THL,
Scheme 1), an FDA-approved anti-obesity drug, were devel-
oped.[7]
Accurate and thorough determination of drug–target inter-
actions is of great importance in drug discovery. It offers in-
valuable biological insights for a drug candidate, such as its
mode of action both in vitro and in vivo, and also provides
clues for compound optimization in order to maximize the
[a] P.-Y. Yang, C. Zhang, G. Y. J. Chen, Y. Shen, M. H. Ngai,
Prof. Dr. M. J. Lear, Prof. Dr. S. Q. Yao
Department of Chemistry
National University of Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
Fax : (+65)6779-1691
E-mail: chmyaosq@nus.edu.sg
[b] K. Liu, G. Y. J. Chen, Prof. Dr. S. Q. Yao
Department of Biological Sciences
National University of Singapore
14 Science Drive 4, Singapore 117543 (Singapore)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100306.
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listat analogues by performing
various chemical modifications
on the aliphatic side-chains
around the b-lactone core of
the drug.[4b,10] In our prelimina-
ry studies using different Orli-
stat-like compounds, we found
a direct link between the chem-
ical modification of Orlistat and
its cellular activity.[4b] However,
no further studies were carried
out to explain the potential cel-
lular targets of these analogues
and their biological consequen-
ces. Another way to achieve
good in vivo efficacy of bioac-
tive compounds is to deliver
them into the sub-cellular com-
partment of cells where the
target enzyme resides. In this
way, an increase in the effective
concentration of the inhibitor
(i.e. improved potency) with a
better selectivity would be ex-
pected. In fact, such sub-cellu-
lar targeting has recently been
demonstrated successfully for
several inhibitors and small-
Scheme 1. a) Representative structures of the lipstatin family of natural products possessing trans-3,4-disub-
stitued-b-lactones. b) Previously published Orlistat-like probes (1a–1g, 2a, and 3a) and the key intermediate
10a.[4a, c]
Orlistat works primarily on pancreatic and gastric lipases
within the gastrointestinal (GI) tract.[7] Recently, Orlistat
and other lipstatin-based natural products have shown
promising anti-tumor activities by potently inhibiting human
fatty acid synthase (FAS) in tumor cells.[8] FAS is an enzyme
essential for the survival of cancer cells.
molecule probes in cultured cells.[11] Herein, we report fur-
ther chemical and cellular modifications of Orlistat with the
ultimate aim to improve its cellular activities (Figure 1 and
Scheme 3). By using an expanded set of new Orlistat-like
probes, we performed in situ proteome-profiling experi-
ments following treatment of live HepG2 cells with our
probes (Figure 1). Large-scale pull-down/LCMS identifica-
tions of potential cellular targets were subsequently con-
ducted. From these studies, both common and unique pro-
tein targets of Orlistat-like analogues were identified. In
order to further improve Orlistatꢀs cellular activities, we de-
veloped a “cellular modification” strategy to deliver our
cell-permeable Orlistat variant to different sub-cellular or-
ganelles in living cells (Scheme 3). This strategy was success-
fully implemented by taking advantage of the well-estab-
lished AGT/SNAP-tag technology.[12]
In our approach, extremely conservative modifications
(i.e., an alkyne handle; see 1a in Scheme 1) were introduced
in the parental Orlistat structure to provide the necessary
functionality for target identification via copper(I)-catalyzed
1,3-dipolar cycloaddition (click chemistry) or copper cata-
lyzed azide–alkyne cycloadditions (CuAAC),[9] whilst main-
taining the native biological properties of the drug. When
combined with affinity purification and LCMS analysis, we
were able to identify and putatively validate several previ-
ously unknown cellular targets of Orlistat, including
GAPDH, b-tubulin, Annexin A2, Hsp90AB1 and three ri-
bosomal proteins (RPL7a, RPL14, and RPS9).[4a] Although
some of these are highly expressed endogenously, and their
presence might be explained primarily by their relative cel-
lular abundance, such information is important to better un-
derstand the full biological effects of a drug.
Having said this, there are still a number of challenges
that prevent Orlistat from being considered as a potential
anti-tumor drug; it has poor solubility and bioavailability,
and, most importantly, it lacks sufficient potency and specif-
icity. To begin to address some of these issues, we and
others previously focused on the development of highly effi-
cient chemistry which has enabled the facile synthesis of Or-
Results and Discussion
Design and Synthesis of Orlistat-Like Probes
The design of Orlistat-like probes was based on the general
structure of a number of naturally occurring congeners of
lipstatin (namely lipstatin, esterastin, and valilactone;
Scheme 1a), and our previously reported compounds, 1a–g,
2a, and 3a (Scheme 1b),[4a,c] in which a terminal C C single
À
bond in one of the aliphatic chains in the parental Orlistat
À
structure was substituted for a C C triple bond. This ex-
tremely conservative modification was previously shown to
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Figure 1. Biological screening of Orlistat-like compounds in live HepG2 cells. a) Overall workflow of the large-scale affinity pull-down/LCMS experi-
ments. The chemically cleavable linker, 6, used in study is shown in red. b) Dose-dependent inhibition of HepG2 cell-proliferation by the 21-member Or-
listat library using an XTT assay. The data represent the average standard deviation for two trials. Wild-type Orlistat (positive control) and DMSO (nega-
tive control) were included in the assay. c) In situ proteome-profiling of Orlistat analogues against HepG2 cells. Probe-labeled proteins were detected by
click-chemistry-mediated coupling to a rhodamine–azide reporter tag, followed by SDS-PAGE and in-gel fluorescence scanning. The arrow indicates the
labeled FAS band. d) Summary of proteins identified in all seventeen Orlistat-like compounds. Top: total number of proteins identified from each probe;
Center: total number of unique proteins identified from each probe; Bottom: relative percentage abundance of FAS (calculated from the emPAI value
for FAS in the experimental sample/the sum of the emPAI values for FAS in all seventeen samples) obtained from each probe.
enable full retention of the native biological activities of Or-
listat, and at the same time allow for subsequent chemical
profiling/target identification by the downstream conjuga-
tion of reporter tags by bio-orthogonal click chemistry.[4a]
We now report the synthesis, anti-proliferation evaluation,
and proteomic-profiling results of an expanded list of Orli-
stat analogues that incorporate various degrees of molecular
complexity (i.e. changes in the position of the terminal-
alkyne handle, the amino ester, and in the chirality;
Scheme 2). This detailed investigation has refined our pres-
ent understanding of the anti-proliferative role played by
Orlistat and has established certain structure–activity rela-
tionships (SARs) within this drugꢀs structural motif from
which the next generation of designed analogues may
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Orlistat-Like Natural-Product-Based Probes
Scheme 2. Synthesis of probes 1h–j, 2b–f, and 3b–c used in this study. THF=tetrahydrofuran, DIAD=diisopropyl azodicarboxylate, binol=1,1’-binaph-
thalene-2,2’-diol, Imi=imidazole, DMAP=4-dimethylaminopyridine.
emerge. To this end, ten new analogues of Orlistat were syn-
thesized (Scheme 2). In the analogue series 1h–j, the left-
hand aliphatic side-chain remained intact, whilst the nature
of the amino ester and/or the chirality at the C3, C4, and C6
positions were varied strategically. In the analogue series
2b–f, the right-hand aliphatic side-chain was kept constant,
whilst the structure of the amino ester (2b–e) and the chiral-
ity at the C3, C4, and C6 positions (2 f, containing an N-
formyl-l-leucine moiety) were altered. For the analogue 3b,
further structural changes were made at the N-formyl group
by the introduction of a long alkyne-containing acyl group.
In the case of 3c, the two terminal alkyne handles were si-
multaneously introduced (top and right).
As shown in Scheme 2, the synthesis of Orlistat analogues
1h–j began with the previously prepared enantiopure alde-
hyde 7b from commercially available dodecan-1-ol.[4a] This
synthetic sequence, which relied on the kinetic resolution of
racemic allylic alcohols, was chosen because of its applicabil-
ity to large-scale synthesis and convenient variation of the
chirality at the C3, C4, and C6 positions. Briefly, aldehyde
7b was treated with thiopyridyl ketene acetal 8 under stan-
dard tandem Mukaiyama-aldol/lactonization (TMAL) reac-
tion conditions,[10] thus providing the silyloxy-b-lactone inter-
mediates as a mixture of diastereomers (ca. 8:1 anti/syn)
with complete selectivity for the trans-b-lactones. Subse-
quently, direct O-desilylation allowed the isolation of the
enantiomerically pure g-hydroxyl b-lactones 9b. Subsequent
C-desilylation with AgNO3/2,6-lutidine gave the terminal
acetylene 10b, which was subjected to Mitsunobu conditions
with various N-formyl amino acids, thus giving rise to the
corresponding configurationally inverted products 1h–j.
Similar to 2a, compound 2b–e were conveniently synthe-
sized from key intermediate 19a with various N-formylated
amino acids under Mitsunobu conditions, thereby giving rise
to the corresponding configurationally inverted products in
a single step. The synthesis of 2 f commenced with the opti-
cally active aldehyde 16b, which was obtained by enantio-
meric enrichment using an asymmetric allylation (allyltribu-
tyltin/binol/InCl3) reaction.[13] Following tert-butyldimethyl-
silyl (TBS) protection and oxidative cleavage, the desired al-
dehyde 18b was obtained, which, following further TMAL
and Mitsunobu reactions, led to the desired Orlistat ana-
logues 2 f. For the synthesis of 3b, the known hydroxyl-b-
lactone 20 was first prepared as previously described.[4a] Sub-
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sequent treatments with 20 and 21 under Mitsunobu condi-
tions furnished the targeted analogue 3b. Compound 3c was
conveniently synthesized from the key intermediate 10a
with 22 under Mitsunobu conditions.
of incubation, the cells were washed (to remove excess
probes), homogenized, incubated with rhodamine–azide (for
the structure, see the Supporting Information, Figure S1)
under click chemistry conditions, separated by SDS-PAGE
gel, and analyzed by in-gel fluorescence scanning. As shown
in Figure 1c, FAS in general was the main cellular target of
these probes in HepG2 cells, as evident from the in-gel fluo-
rescence scanning in which it showed up as the most strong-
ly labeled band amongst all of the labeled bands. This pro-
file is similar to the labeling profiles obtained from another
class of Orlistat compounds reported previously.[4b] The two
key intermediates, 10a and 19a, produced only weakly la-
beling profiles (including weak FAS labeling), again rein-
forcing the importance of an N-formylated amino ester
moiety in the Orlistat structure. However, covalent labeling
of other cellular targets could still be readily detected in the
fluorescence gel, with different probes generating marginally
but noticeably different labeling profiles. This result indi-
cates that our probes targeted both common and unique cel-
lular targets in HepG2 cells, which might have accounted
for the subtle differences in their anti-proliferative activities
as well. To further identify these potential cellular targets,
we performed large-scale affinity pull-down/LCMS analysis
of protein lysates obtained from live HepG2 cells treated
with each of the probes. Previously, we used a standard,
non-cleavable biotin reporter tag to enrich covalently cap-
tured proteins and eluted them by boiling them in the pres-
ence of an SDS-containing buffer.[4a] However, under such
harsh denaturing conditions, both endogenously biotinylated
and highly expressed cellular proteins (nonspecifically
bound to the avidin beads) were still present in the eluent
even after repeated washes, resulting in an unacceptable
number of false positives and greatly interfering with the
identification of low-abundance cellular targets. In this
study, improvement was made by making use of an azoben-
zene-functionalized biotinylated linker (compound 6 in Fig-
ure 1a; for the synthesis, see the Supporting Information,
Scheme S1),[15] which allows selective release of probe-la-
beled proteins under mild elution conditions (25 mm
Na2S2O4) that are compatible with mass spectrometric analy-
sis. Consequently, fewer false positives and more reliable pu-
tative protein hits were obtained in our assay. Briefly, as
summarized in Figure 1a, cellular lysates containing protein
that has been covalently labeled with the Orlistat-like
probe, were treated with the cleavable biotin–diazo-azide
linker (6) under standard click chemistry conditions, precipi-
tated with acetone, washed with methanol, solubilized with
an SDS buffer, followed by affinity purification with Neutra-
vidin beads and SDS-PAGE gel separation. The entire lane
from each pull-down experiment was excised into 10 contig-
uous gel slices, which were subsequently processed individu-
ally for in-gel trypsin digestion as described in the Experi-
mental Section. As negative controls, the entire large-scale
proteomic experiment (from cell treatment to LCMS analy-
sis) was repeated with dimethyl sulfoxide. The peptides ob-
tained from each gel slice were eluted and subjected to
nano-LC-MS/MS analysis. The LC-MS/MS data were
Biological Screening and In Situ Proteome Profiling of
Orlistat Analogues
With these ten new Orlistat analogues in hand (1h–1j, 2b–
2 f, 3b–3c), together with the wild-type Orlistat, the nine
previously reported Orlistat analogues (1a–1g, 2a, 3a) and
two key intermediates (10a and 19a), a total of twenty-two
compounds were tested for their anti-proliferative activities
against the human liver cancer line HepG2 using the XTT
assay described previously,[4a–b] followed by in situ proteome
profiling and target identification using affinity pull-down/
LCMS approaches (Figure 1). The overall workflow of the
strategy is shown in Figure 1a.
As shown in Figure 1b, nine of the compounds examined
(1a–1c, 1d, 1 f, 2a, 2d, 3a and 3c) displayed comparable ac-
tivities to Orlistat in blocking the proliferation of HepG2
cancer cells. A closer examination of the data revealed a
noteworthy structure–activity relationship (SAR) between
the compounds tested and their anti-proliferative activities.
In general, the chirality of the Orlistat analogues is impor-
tant for activity. For example, in a direct head-to-head com-
parison, compounds with an inverted stereochemistry at the
C3, C4 and C6 positions (S to R), i.e., 1b–d versus 1h–j, and
2a versus 2 f, were shown to have significantly lower anti-
proliferative activities. The stereochemistry of the a-carbon
position in the N-formylated amino ester moiety of Orlistat
also had a notable effect on its activity: both 1a and 2a
(which are structurally identical to Orlistat, except with a
ꢀ
switch in the position of the C C group between aliphatic
chains) appeared to be more active than their corresponding
epimers, 1g, and 2e, respectively. The N-formylated amino
ester moiety as a whole appeared to be essential for anti-
proliferative activity, as demonstrated by the lack of activity
in the two hydroxyl lactone intermediates, 10a and 19a (in
which the N-formylated amino ester was completely delet-
ed). It should be noted that, in a previous study, the N-
formyl group of Orlistat had also been shown to be highly
sensitive to the inhibition of FAS.[10b] The different anti-pro-
liferative profiles displayed by Orlistat-like compounds have
likely been accumulated through their inhibition against a
multitude of cellular targets, including FAS, in HepG2 cells.
Indeed, many drugs appear to work by synergistically target-
ing multiple proteins.[14]
In situ Proteome Profiling and Target Identification
We then carried out in situ proteome-reactivity profiles of
all probes to identify potential cellular targets which were
covalently labeled by the probes in live HepG2 cells. Fol-
lowing previously published procedures with some modifica-
tions (summarized in Figure 1a),[4a] each probe (20 mm) was
added directly to the cell culture medium. After two hours
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Orlistat-Like Natural-Product-Based Probes
Table 1. Potentially unique proteins identified by mass spectrometry.[a]
Gene
Protein name
Probe Localization
Function
symbol
HLA-A
HLA class I histocompatibility antigen, A-68
alpha chain
1a
membrane
host-virus interaction, immune response
PFN1
RPL22
MIF
RPL12
PC
PGM1
BCAP31
CLTCL1
PFKM
TPP2
Profilin-1
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1c
cytoplasm
cytoplasm, nucleus
cytoplasm
nucleus
mitochondrion
cytoplasm
ER, nucleus
cytoplasm
n/a
cytoplasm, nucleus
membrane, cyto-
plasm, nucleus
membrane
ER, nucleus, cyto-
plasm
cytoskeletal protein binding
protein metabolism
cell proliferation, signal transduction
protein metabolism
gluconeogenesis, lipid synthesis
glucose metabolism
transport, apoptosis
receptor-mediated endocytosis
glycolysis
proteolysis
60S ribosomal protein L22
Macrophage migration inhibitory factor
60S ribosomal protein L12
Pyruvate carboxylase, mitochondrial
Phosphoglucomutase 1
B-cell receptor-associated protein 31
Isoform 1 of Clathrin heavy chain 2
Isoform 2 of 6-phosphofructokinase, muscle type
Tripeptidyl-peptidase 2
HLA class I histocompatibility antigen, B-59
alpha chain
Prostaglandin E synthase 2
Acidic leucine-rich nuclear phosphoprotein 32
family member A
HLA-B
immune response
PTGES2
ANP32A
1c
1c
fatty acid biosynthesis, lipid synthesis
tumor suppressor, transcriptional repression
ATP1B3
P4HB
Sodium/potassium-transporting ATPase subunit
beta-3
cDNA FLJ59430, highly similar to Protein disul-
fide-isomerase
HLA class I histocompatibility antigen, Cw02
alpha chain
Eukaryotic translation initiation factor 3 subunit
E
1c
1c
1d
1d
membrane
transport
ER
cell redox homeostasis
immune response
protein biosynthesis
HLA-C
EIF3E
membrane
cytoplasm, nucleus
NOP56
MYOF
GNAI2
Nucleolar protein 56
Isoform 5 of Myoferlin
Guanine nucleotide-binding protein G(i), alpha-2
subunit
1d
1d
1e
nucleus
membrane
membrane
ribosome biogenesis
phospholipid binding
signal transduction
SCARB1
Scavenger receptor class B member 1
1e
membrane
receptor for phospholipids, cholesterol ester, lipopro-
teins, phosphatidylserine
transcription regulation
transport
transport
cytokinesis
transport
muscle organ development
transcription regulation
STAT3
SCAMP3
Signal transducer and activator of transcription 3
Secretory carrier-associated membrane protein 3
1 f
1 f
1 f
1 f
1 f
1g
1h
cytoplasm, nucleus
membrane
nucleus
cytoplasm, nucleus
ER
nucleus
TMEM48 Isoform 3 of Nucleoporin NDC1
MYH9 Isoform 2 of Myosin-9
SEC61A2 Sec61 alpha form 2 isoform b
LMNA
BZW1
Isoform C of Lamin-A/C
Basic leucine zipper and W2 domain-containing
protein 1
cytoplasm, nucleus
DNAJC11 Isoform 3 of DnaJ homolog subfamily C member
11
1h
n/a
unknown
RAB4B
NIT2
KIAA0368 KIAA0368 protein
GTF2I
ARF1
TOP2B
LY6K
Ras-related protein Rab-4B
Omega-amidase NIT2
1h
1h
1h
1h
1h
1h
1j
membrane
cytoplasm
ER, centrosome
cytoplasm, nucleus
Golgi
nucleus
membrane, nucleus
cytoplasm
vesicular trafficking
hydrolase, metabolism
protein metabolism
transcription regulation
transport, signal transduction
transcription regulation
useful as a tumor biomarker
fatty acid biosynthesis, lipid synthesis
signal transduction
Isoform 1 of General transcription factor II-I
ADP-ribosylation factor 1
DNA topoisomerase 2-beta
Lymphocyte antigen 6 K
Acyl-protein thioesterase 2
Guanine nucleotide-binding protein G(o) subunit
alpha
LYPLA2
GNAO1
1j
1j
membrane
ATP2A1
SERCA1B of Sarcoplasmic/endoplasmic reticu-
lum calcium ATPase 1
2a
ER
apoptosis in response to ER stress
SH3KBP1 SH3 domain-containing kinase-binding protein 1
2a
2b
3b
cytoplasm
cytoplasm
membrane
signal transduction
actin binding
unknown
PLEC1
ESYT2
Isoform 1 of Plectin
Isoform 4 of Extended synaptotagmin-2
[a] 44 potentially unique proteins were listed. References are given in the Supporting Information. n/a=not available.
searched against the IPI (International Protein Index)
human protein database using an in-house MASCOT server
for protein identification. All proteins were identified by a
minimum score of 40 and at least two unique peptides.
Based on these criteria, around 200 proteins on average
were identified for each of the seventeen Orlistat probes
used (Figure 1d, top). Details of the identified protein hits,
including their accession numbers, emPAI values, and pro-
tein masses, are listed in the Supporting Information
(Table S2, Table 1 lists the unique proteins identified). A
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total of sixty proteins were identified in all of our probes,
which may constitute the common cellular targets for these
probes (for a detailed list and relevant references, please see
the Supporting Information). These include well-known
serine-type proteins, such as enolases (ENO1, ENO3), elon-
gation factors (EEF2, EEF1A1, and EEF1G) and cysteine-
type proteins, such as transferrin receptor protein (TFRC),
ubiquitin-like modifier-activating enzyme (UBA1), and pro-
tein disulfide isomerase (PDIA6), and several lipid-metabo-
lism-related proteins, such as 3-hydroxyacyl-CoA dehydro-
genase type-2 (HSD17B1), triosephosphate isomerase, and
trans-2,3-enoyl-CoA reductase. Five of our previously iden-
tified targets against 1a (FASN, GAPDH, b-tubulin, Annex-
in A2, and HSP90AB1),[4a] also emerged as common hits for
our new probes. Not surprisingly, some other lipid- and/or
fatty-acid-metabolism-related proteins, such as palmitoyl-
protein thioesterase (PPT1), carnitine O-palmitoyltransfer-
ase (CPT1A), ATP-citrate synthase (ACLY), dihydrolipoyl
false positives/non-specific proteins binders could be mini-
mized but not eliminated entirely from our results. Conse-
quently, proper follow-up studies and validation experiments
will be needed before any biological conclusions can be
made for some of these protein hits.
One of the questions we were interested in addressing
was whether subtle chemical modifications of the Orlistat
structure led to changes in its cellular targets, which was al-
ready suggested by the earlier in situ proteome-profiling ex-
periments (Figure 1c). If so, one would expect that unique
proteins targeted by each of the seventeen Orlistat probes
could be identified from our affinity pull-down/LCMS ex-
periments. Upon closer examination of our MS results, we
found a total of forty-four unique proteins from twelve of
the seventeen Orlistat probe used (Figure 1d, bottom, and
Table 1). Among those proteins are several HLA (human
leukocyte antigen) class I antigens that are part of the genet-
ic region known as the major histocompatibility complex
(MHC) class I proteins,[25] and G proteins (guanine-nucleo-
tide-binding proteins) which belong to the larger group of
enzymes called GTPases.[26] Specifically, HLA-A, HLA-B,
and HLA-C were targeted uniquely by 1a, 1c, 1d, and
GNAI2, GNAO1 were detected only with 1e, 1j, respective-
ly. Interestingly, serine exopeptidase TPP2 (tripeptidyl-pep-
tidase 2), which is essential for some MHC class I antigen
presentation,[27] was selectively pulled-down by 1b. STAT3
(signal transducer and activator of transcription 3), which
has recently been identified as a tumor suppressor,[28] was
uniquely pulled down by 1 f. NIT2, an omega-amidodicar-
boxylate amidohydrolase and a known tumor-suppressor
protein,[29] was pulled down only by 1h. Again, we caution
that these MS-derived results are only preliminary findings,
and need to be further confirmed with suitable validation
experiments. Nevertheless, our data concludes that subtle
chemical modifications could change the selectivity profiles/
cellular targets of Orlistat, and some of its analogues may
be further developed into drugs targeting some of the
unique proteins mentioned above.
dehydrogenase
(DLD),
acyl-CoA
dehydrogenases
(ACADM, ACADVL), fatty aldehyde dehydrogenase
(ALDH3A2), sterol O-acyltransferase (SOAT1), long-chain
acyl-CoA synthetase (SLC27A2), acetyl-CoA acetyltransfer-
ases
(ACAT1),
lysophospholipid
acyltransferase
(MBOAT7), were also pulled-down by most (but not all) of
our Orlistat probes.[16–22]
Recent advances in quantitative mass spectrometry have
now allowed the direct evaluation of drug–protein interac-
tions based on the relative peak intensity (or the spectra
count) of proteins/peptides obtained from a mass spectrom-
eter.[23] Although semi-quantitative at best, this evaluation
provides a very simple and quick method to evaluate the
relative binding efficiency of a drug against its protein
target.[24] Specifically, the so-called emPAI (Exponentially
Modified Protein Abundance Index) MS quantitation ap-
proach provides an estimate of protein concentration from
the number of peptide sequences of a protein identified by
tandem mass spectrometry.[24c,d] The abundance of an identi-
fied protein in a given pull-down sample relative to the
abundance of the same protein in all pull-down samples was
then calculated as the ratio of its emPAI value over the sum
of the emPAI values for that protein in all seventeen sam-
ples. These ratios where then plotted in a heat map for all
protein/probe combinations (see the Supporting Informa-
tion, Figure S2). As shown in Figure 1d, bottom, FAS which
was successfully pulled down by all seventeen Orlistat
probes showed varied relative percentage abundance values
ranging from as low as 3.0% (with probe 3a) to as high as
12.8% (with probe 1e), thereby indicating small but rela-
tively significant differences in the relative binding affinity
between this protein and the different Orlistat probes. It
should be noted that all MS-based results obtained herein
(including all the putative protein hits identified and their
emPAI values) should only be used as preliminary data. In
our current study, improvement on our previous affinity
pull-down procedures was made.[4a] Nevertheless, owing to
the highly complex cellular environment, the intrinsic limita-
tion of affinity pull-down assay and mass spectrometry,[23,24]
Design and Synthesis of AGT/SNAP-Orlistat Bioconjugates
as Organelle-Targetable Probes
Sub-cellular targeting of a drug, whereby the drug is deliv-
ered to a specific organelle inside living cells and subse-
quently retained there (thus greatly increasing the effective
concentration of the drug), is expected to not only improve
its in vivo efficacy but also minimize its off-target effects
and cytotoxicity.[30] A number of drug-delivery vehicles, in-
cluding the mannose cluster for mannose-receptor interac-
tion targeting the endolysosomal pathway,[11a] cell-penetrat-
ing and cell-localization peptides,[11b,c] have been successfully
used for sub-cellular delivery. We were particularly intrigued
by the AGT/SNAP-tag method, which is widely used in the
bioimaging field for highly efficient in vivo protein labeling
using benzylguanine (BG)-containing small-molecule
probes.[12,31] The Johnsson group and other groups recently
adopted this method for the sub-cellular delivery of small-
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Orlistat-Like Natural-Product-Based Probes
molecule-based fluorescence sensors for the sensitive detec-
Next, we confirmed that BG-containing Orlistat 4 could
indeed serve as a substrate of AGT/SNAP and be success-
fully conjugated (i.e. Scheme 3b, Step I). As shown in
Figure 2, recombinantly purified hexahistidine-tagged AGT
protein (His-AGT), pre-incubated with 4 in PBS for 4 hours
at room temperature, was shown to completely stop the
fluorescence labeling between His-AGT and its natural sub-
[11d]
tion of zinc(II),[31b] calcium(II),[31c,d] and H2O2
in cultured
cells. We were interested to know whether the same concept
could also be applied for sub-cellular drug delivery. Because
FAS expresses mostly in the endoplasmic reticulum (ER),
modifying Orlistat to be delivered and retained in the ER
should in principle greatly improve the drugꢀs anti-tumor ac-
tivity (both in potency and selectivity). As shown in
Scheme 3, in a proof-of-principle study, we envisaged a
drug-delivery system consisting of two critical components:
a benzylguanine (BG)-derivatized Orlistat (4; Scheme 3c)
and a model mammalian cell line over-expressing a SNAP
protein fused to a desired protein-localization sequence
(PLS). Organelle-targetable probe 4 was designed on the
basis of our previous findings that, having a terminal alkyne
handle on the right-hand aliphatic chain and structural var-
iations on the left-hand aliphatic chain of Orlistat do not sig-
nificantly interfere with the drugꢀs inhibition of FAS.[4a,b]
Therefore, we were hopeful that once 4 enters the cells and
becomes conjugated by the SNAP protein (Scheme 3b), the
resulting AGT/SNAP-Orlistat conjugate would be locally
“concentrated” and retained in the desired sub-cellular or-
ganelle (Scheme 3a). Subsequently, sub-cellular protein tar-
geting may take place. Compound 4 was conveniently pre-
pared using click chemistry between benzylguanine azide
(24), synthesized in two steps from 6-chloroguanine (see the
Supporting Information, Scheme S2), and the previously re-
ported compound 5 (Scheme 3c).[4b]
Figure 2. Competition assay for AGT labeling with 4. Aliquots taken
from the reaction mixture at the indicated time and incubated with
BGFL (100 mm) for 15 minutes, followed by SDS-PAGE and in-gel fluo-
rescence scanning.
Scheme 3. a) Principle of AGT/SNAP-tag fusion strategy for sub-celllular protein targeting using a cell-permeable, benzylguanine (BG)-containing Orli-
stat analogue (4). b) Bioconjugation reaction of AGT/SNAP+BG-Orlistat that occurs inside living cells, followed by sub-cellular protein targeting/bio-
imaging. c) Schematic showing of click chemistry-facilitated synthesis of the organelle-targeting, BG-containing Orlistat probe (4). DMSO=dimethyl
sulfoxide.
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S. Q. Yao et al.
FULL PAPERS
strate, BGFL, thereby indicating the successful formation of
the desired AGT/SNAP-Orlistat (Scheme 3b). Further con-
firmation was obtained from ESI-Q-TOF mass analysis
(Table 2); a mass shift of 535 between the untreated His-
AGT and (His-AGT+4) labeling reaction was observed,
which is consistent with the calculated mass of the AGT/
SNAP-Orlistat conjugate. The reactive b-lactone ring in Or-
listat also remained intact after conjugation.
elles (Figure 3; green channels), compound 4 was directly
applied to the media and the (SNAP+4) conjugation reac-
tion was left to proceed for another four hours followed by
extensive washing of the cells (to remove excessive probe).
Subsequently, in situ click chemistry was carried out in these
live cells, using a previously reported procedure,[4a] to visual-
ize the localization/retention of 4 (Scheme 3b, Step III). As
shown in Figure 2 (red channels), the successful accumula-
tion of 4 in the desired organelles (mitochondria, ER, and
nucleus), as guided by the localization of AGT-SNAP, was
observed (see the merged images of the green and red chan-
nels). Cells transfected with a control plasmid containing
AGT/SNAP gene without a protein localization sequence
displayed uniform fluorescence labeling throughout the
entire cell (data not shown). Taken together, these data
show that the Orlistat probe 4 could be successfully deliv-
ered/retained in sub-cellular organelles by using the AGT/
SNAP-tag strategy.[12d] At present, owing to the lack of a
suitable AGT-deficient HepG2 cell line, we were unable to
carry out subsequent in situ proteome profiling/LCMS ex-
periments for target identification. However, as a proof-of-
concept experiment, we believe the AGT/SNAP-tag strategy
represents an interesting and viable tool for sub-cellular
drug delivery and organelle targeting. It might even be used
in the future for in vivo organelle proteomics applications.[33]
Table 2. Observed mass peaks for ESI-Q-TOF mass analysis of His-AGT
with and without 4.[a]
Sample
MW [Da]
Mass difference
His-AGT
His-AGT+4
23071
23606
535
[a] For original mass spectra, see the Supporting Information.
Lastly, we assessed whether the direct application of 4 to
live mammalian cells leads to successful localization and re-
tention of the Orlistat probe in the desired sub-cellular or-
ganelle. However, we were unable to carry out experiments
directly in HepG2 cancer cells, because an AGT-deficient
cell-line is currently not available,[32] and the intrinsically
low transfection efficiency of HepG2 cells also precluded
the accumulation of a sufficient amount of transiently ex-
pressed PLS-SNAP in the cells. Therefore, we used an AGT-
deficient CHO-9 cell-line instead. Sub-cellular expression of
AGT-SNAP protein was performed by transient transfection
of the CHO-9 cells with mammalian DNA constructs con-
taining the AGT-SNAP gene fused to the different protein
localization sequences (PLS; including membrane, ER, and
nucleus). Forty eight hours later, after the proteins were suc-
cessfully expressed and detected in the corresponding organ-
Conclusions
We have synthesized and evaluated a set of Orlistat-like
compounds for their anti-proliferative activity in HepG2
cancer cells. Our results showed some of the analogues (1a–
1d, 1 f, 2a, 2d, 3a, and 3c) displayed comparable activities
Figure 3. Images of CHO-9 cells expressing AGT-SNAP-tag in mitochondria (top), ER (center), or nuclei (bottom), then treated with 4, fixed, permeabi-
lized, clicked with rhodamine-azide, and imaged (red channels). Cell nuclei were stained with Hoechst (blue channels), anti-FLAG M2 primary antibody,
followed by FITC-conjugated antimouse IgG antibody to detect AGT/SNAP protein (green channels). Red and Green channels were overlaid, giving
merged images shown. Scale bar=10 mm. PLS: Cox8A (mitochondria); KDEL (ER); H2B (nucleus).
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Orlistat-Like Natural-Product-Based Probes
In Situ Proteomic Profiling
when compared to the parent drug Orlistat, whilst others
showed marginally weaker activities. Subsequent in situ pro-
teome-activity profiling, followed by large-scale affinity pull-
down/LCMS identifications of putative protein hits enabled
us to identify potential common and unique targets and to
assess their relative binding to our probes, thus providing
some preliminary structure–activity-relationship (SAR) in-
formation. This chemical-proteomics approach may be used
for the discovery of new cellular targets of Orlistat and for
the design of better Orlistat analogues. It may also provide
a general method for both on- and off-target identification
of other suitable drugs. Finally, in order to further improve
the cellular activity of Orlistat, we have successfully applied
the well-established AGT/SNAP-tag technology to a benzyl-
guanine (BG)-containing Orlistat variant (4), and in a
proof-of-concept bioimaging experiment, showed the drug
could be delivered and effectively retained in different sub-
cellular organelles of living cells. Owing to the lack of an
AGT-deficient HepG2 cell line, subsequent in situ proteome
profiling and pull-down/LCMS experiments were not possi-
ble at this stage.
Cells were grown to 80–90% confluence in 24-well plates under the con-
ditions described above. The medium was removed, and then cells were
washed twice with cold PBS, and treated with 0.5 mL of DMEM-contain-
ing probe (20 mm). All compounds were solubilized in DMSO. To avoid
adverse effects on cell growth, the final DMSO concentration in the
assay never exceeded 1% in cultivation medium. Medium containing 1%
DMSO was used as a negative control. After incubation, the growth
medium was aspirated, and cells were washed twice with PBS to remove
the excess probe, trypsinized, and pelleted at 1,000 rpm for 10 min,
washed twice with PBS, and re-suspended in PBS (100 mL). Cells were
homogenized by sonication, and diluted to ~1 mg/mL with PBS. To ini-
tiate the click chemistry reaction, 20 mL of freshly premixed solution con-
taining rhodamine–azide (100 mm final concentration), TCEP (1 mm final
concentration), ligand (100 mm final concentration), and CuSO4 (1 mm
final concentration) were added. The reaction was incubated at 108C for
2 h with gentle mixing. Termination of the reaction was done by addition
of pre-chilled acetone (0.5 mL). The resulting solution was then placed at
À208C for 30 min, followed by centrifugation (13000 rpm ꢂ 10 min) at
48C. The supernatant was discarded and the precipitated protein pellets
were washed with pre-chilled methanol (2ꢂ200 mL), air-dried for 10 min,
resuspended in 1 ꢂ standard reducing SDS-loading buffer (25 mL) then
heated for 10 min at 958C. Finally, the protein sample (ca. 50 mg/lane)
was loaded onto 4% to 15% gradient precast SDS-PAGE (16ꢂ20 cm),
separated, followed by in-gel fluorescence scanning with a Typhoon 9410
Variable Mode Imager scanner (GE Amersham).
Pull-Down and Mass Spectrometry Identification
Experimental Section
For proteomic experiments, living HepG2 cells (T75 culturing flask) la-
beled in growth media with probes or DMSO (negative control) were
harvested, washed, and homogenized in PBS. CuAAC reagents were
added at the same concentrations as described above, except that biotin–
diazo-azide 6 was substituted for rhodamine–azide. Acetone-precipitated
and methanol-washed protein pellets were solubilized in PBS containing
0.1% (w/v) SDS by brief sonication. Insoluble materials were precipitat-
ed by centrifugation (13,000 g ꢂ 10 min) at 48C. The supernatants were
then incubated with gentle shaking at 48C overnight with Neutravidin
agarose beads (50 mLmgÀ1 protein, Prod # 29204, Thermo Scientific,
USA) which have been pre-washed twice with PBS. After centrifugation,
the bead/complexes were washed extensively 4 times with 1% (w/v) SDS
in PBS, three times with PBS, and twice with 250 mm ammonium bicar-
bonate (ABC). Beads were re-suspended in 500 mL of 8m urea, and then
25 mL of 200 mm TCEP and 25 mL of 400 mm iodoacetamide were added
for capping of the reactive cysteine residues. After 30 min, the beads
were washed twice with 250 mm of ABC. Elution of bound proteins from
the beads was then performed with a total of 400 mL freshly prepared
elution buffer (0.5% SDS, 25 mm Na2S2O4 in 50 mm ABC) by rocking for
1 h at room temperature. Protein samples were concentrated using an
YM-10 Centricon spin column (Millipore, USA). Following SDS-PAGE
separation, protein bands were visualized by Coomassie blue/silver stain-
ing. Gel lanes corresponding to both DMSO-treataed and probe-treated
samples were then each cut into 10 slices. Subsequent trypsin digestion
(using In-Gel Trypsin Digestion Kit, Pierce Co., USA) and peptide ex-
traction (with 50% acetonitrile and 1% formic acid) generated a total of
10 LCMS samples for each pull-down experiment. All samples were
dried in vacuo and stored at À208C until LCMS analysis. Each LCMS
sample was re-suspended in 0.1% formic acid for mass spectrometry
analysis as previously described.[34a] Briefly, peptides were separated and
analyzed on a Shimadzu UFLC system (Shimadzu, Kyoto, Japan) coupled
to an LTQ-FT Ultra (Thermo Electron, Germany). Mobile phase A
(0.1% formic acid in H2O) and mobile phase B (0.1% formic acid in ace-
tonitrile) were used to establish the 60 min gradient comprised of 45 min
of 5–35% B, 8 min of 35–50% B, and 2 min of 80% B, followed by re-
equilibrating at 5% B for 5 min. Peptides were then analyzed on LTQ-
FT with an ADVANCEꢃ CaptiveSprayꢃ Source (Michrom BioResour-
Chemicals and Antibodies
Orlistat (98%), Tris(2-carboxyethyl) phosphine (TCEP), the click
chemistry ligand, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(“ligand”) were purchased from Sigma–Aldrich. Hoechst33342 was pur-
chased from Invitrogen. Monoclonal ANTI-FLAG M2 antibody was pur-
chased from Sigma. The bacterial His-AGT plasmid, mammalian plas-
mids FLAG-Cox8A-SNAP, FLAG-H2B-SNAP, FLAG-KDEL-SNAP,
FLAG-NK1R-SNAP, mCherry-Cox8A, and mCherry-KDEL were gener-
ous gifts from Christopher J. Chang (University of California, Berkeley).
AGT-deficient CHO-9 cell line was a generous gift from the Institute of
Toxicology, University of Mainz (Germany). Recombinant expression
and purification of His-AGT protein was as previously reported.[11d]
Cell-Culture Conditions
HepG2 cells were grown in DMEM (Invitrogen, Carlsbad, CA) contain-
ing 10% heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen),
100 UmLÀ1 penicillin, and 100 mgmLÀ1 streptomycin (Thermo Scientific,
Rockford, IL) and maintained in a humidified 378C incubator with 5%
CO2. CHO-9 cells were maintained in F12-Nutrient Mixture (Invitrogen)
supplemented with 5% fetal bovine serum and 100 U/mL penicillin and
100 mgmLÀ1 streptomycin.
Cell-Proliferation Assay
Cell viability was determined using the XTT colorimetric cell-prolifera-
tion kit (Roche) following the manufacturerꢀs guidelines. Briefly, cells
were grown to 20–30% confluence (because they will reach approximate-
ly 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,
washed with PBS, and then treated, in duplicate, with 0.1 mL of the
medium containing different concentrations of the probe (1–50 mm) or
Orlistat (1–50 mm; 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 the probes and 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 manufacturerꢀs
guidelines (read at 450 nm). Data represent the average (Æs.d.) of two
trials.
ces, USA) at an electrospray potential of 1.5 kV. A gas-flow of 2 LminÀ1
,
ion-transfer tube temperature of 1808C and collision-gas pressure of 0.85
mTorr were used. The LTQ-FT was set to perform data acquisition in the
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S. Q. Yao et al.
FULL PAPERS
positive ion mode as previously described except that the m/z range of
350–1600 was used in the full MS scan.[34a] The raw data were converted
into the mgf format as described previously.[34b] The database (76708 se-
quences, 33362815 residues) used for the Mascot search was a concaten-
ated IPI protein database. The database search was performed using an
in-house Mascot server (version 2.2.07, Matrix Science, UK) with an MS
tolerance of 10 ppm and MS/MS tolerance of 0.8 Da. Two missed cleav-
age sites of trypsin were allowed. Carbamidomethylation (C) was set as a
fixed modification, and oxidation (M) and phosphorylation (S, T, and Y)
were set as variable modifications.
13C NMR spectra were recorded on a Bruker model DPX-500 MHz
NMR spectrometer. Chemical shifts are reported in parts per million rel-
ative to internal standard tetramethylsilane (SiACHTNUGTRNEG(UN CH3)4 =0.00 ppm) or re-
sidual solvent peaks (CHCl3 =7.26 ppm). Mass spectra were obtained at
the center for Chemical Characterization and Analysis at NUS. b-lactone
1a-g, 2a, 3a, 5, 7b, 10a, 19a, and 22 were synthesized as previously de-
scribed.[4]
(S)-(R)-1-((2R,3R)-3-(hex-5-yn-1-yl)-4-oxooxetan-2-yl)tridecan-2-yl 2-
formamidopropanoate (1h)
b-lactone 10b (14 mg, 0.04 mmol), triphenylphosphine (21 mg,
0.08 mmol), N-formyl-l-alanine (10 mg, 0.08 mmol) were placed in a
round-bottom flask and azeotroped under vacuum with 0.5 mL of xylene
for 30 min. Addition of 1 mL dry THF was followed by cooling to 08C;
DIAD (12 mL, 0.08 mmol) was then added via syringe. The mixture was
stirred at 08C for 10 min, and allowed to warm to ambient temperature.
The reaction was monitored by TLC. After the reaction was complete,
the mixture was then concentrated in vacuo and purified by flash chro-
matography on silica gel (20% EtOAc/hexanes) to afford 1h (7 mg,
36%) as a colorless oil. 1H NMR (500 MHz, CDCl3): d=8.18 (s, 1H),
6.17 (br s, 1H), 5.04—5.09 (m, 1H), 4.62 (quin, J=7.6 Hz, 1H), 4.34—
4.37 (m, 1H), 3.24 (dt, J=7.6, 3.8 Hz, 1H), 2.21 (dt, J=6.3, 2.5 Hz, 2H),
2.19–2.14 (m, 1H), 2.03 (dt, J=13.9, 5.0 Hz, 1H) 1.96 (t, J=2.5 Hz, 1H),
1.84–1.54 (m, 10H), 1.45 (d, J=6.9 Hz, 3H), 1.27 (br s, 17H), 0.88 ppm
(t, J=7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=172.2, 170.5, 160.4,
83.9, 74.6, 72.7, 68.7, 56.8, 47.1, 38.7, 33.9, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2,
27.9, 27.1, 25.7, 25.2, 22.7, 18.2, 18.1, 14.1 ppm; ESI-MS: (m/z) calcd for
C26H43NO5 [M+Na]+ 472.3, found 472.2.
In Vitro His-AGT Labeling with (4) and Analysis by ESI-Q-TOF MS
His-AGT (2 mm) was incubated in PBS with 1 mm of 4 for different
lengths of time (15 min, 2 h, and 4 h) at room temperature, then incubat-
ed with BGFL (100 mm) for 15 min followed by SDS-PAGE separation,
and in-gel fluorescence scanning. The deconvoluted MS data were col-
lected by ESI-Q-TOF mass analysis.
Fluorescence Microscopy
AGT-deficient CHO-9 cells were seeded on sterile 12-mm glass cover-
slips contained in 24-well plates and left to adhere for 24 h prior to trans-
fection. Transient transfection was carried out according to protocol in-
structions by using EndoFectin (GeneCopoeia). The medium was re-
placed 12 h post-transfection. After 36 h, the cells were incubated with 4
(30 mm) for 4 h in growth medium at culture temperature and 5% CO2.
Medium containing 1% DMSO was used as a negative control. The cells
were then washed twice with PBS, and fixed with 4% paraformaldehyde
in PBS for 15 min at room temperature, washed with PBS (2ꢂ5 min with
gentle agitation), and permeabilized with 0.1% Triton-X 100 in PBS for
15 min at room temperature, and washed with PBS (2ꢂ5 min with gentle
agitation). The cells were blocked with 2% bovine serum albumin (BSA)
in PBS for 30 min at room temperature, and washed with PBS (2ꢂ5 min
with gentle agitation). The cells were then treated with a freshly pre-
mixed click chemistry reaction solution of rhodamine–azide (10 mm final
concentration from a 10 mm stock solution in DMSO), TCEP (1 mm final
concentration from a 50 mm freshly prepared stock solution in deionized
water), TBTA (100 mm final concentration from a 10 mm stock solution in
DMSO), and CuSO4 (1 mm final concentration from a 100 mm freshly
prepared stock solution in deionized water), in PBS for 1 h at room tem-
perature. The cells were washed with PBS (1ꢂ5 min with gentle agita-
tion), and cold methanol (1ꢂ5 min with gentle agitation), followed by
1% Tween-20 and 0.5 mm of EDTA in PBS (3ꢂ2 min with gentle agita-
tion), and with PBS (2ꢂ5 min with gentle agitation). The cells were then
incubated in PBS containing 0.25 mgmLÀ1 of Hoechst for 15 min at room
temperature to stain the nuclear DNA, and washed with PBS (2ꢂ5 min
with gentle agitation) and a final wash with deionized water (1ꢂ5 min
with gentle agitation) before mounting. For co-localization of SNAP pro-
tein expressed, indirect immunofluorescent cytochemical staining was
carried out according the manufacturerꢀs instructions. Cells were blocked
with 10% BSA in PBS for 30 min, and washed with PBS (2ꢂ5 min with
gentle agitation).The cells were incubated with monoclonal Anti-FLAG
M2 antibody diluted in 1:2000 in 3% BSA in PBS for 2 h at 378C, and
washed with PBS (3ꢂ5 min with gentle agitation). Then secondary anti-
body goat-anti-mouse IgG-FITC (Santa Cruz) diluted in 1:100 in 3%
BSA in PBS for 45 min at 378C, and washed PBS (2ꢂ5 min with gentle
agitation). Images were acquired using Observer Z1 (Zeiss, Germany)
equipped with a 63X NA1.4 objective and a CoolSNAP HQ2 CCD
camera (Photometrics, USA), or LSM 510 META (Zeiss, Germany)
equipped with an EC Plan-Neofluar 100 ꢂ NA1.3 objective.
(R)-1-[(2R,3R)-3-(hex-5-yn-1-yl)-4-oxooxetan-2-yl]tridecan-2-yl 2-
formamidoacetate (1i)
Prepared according to the representative Mitsunobu reaction procedure
using b-lactone 10b (14 mg, 0.04 mmol), triphenylphosphine (21 mg,
0.08 mmol), N-formyl-glycine (10 mg, 0.1 mmol) and DIAD (12 mL,
0.08 mmol) in 1.0 mL of THF. Purification by flash chromatography on
silica gel (15% EtOAc/hexanes) gave 1i (8 mg, 46%) as a colorless oil.
1H NMR (500 MHz, CDCl3): d=8.26 (s, 1H), 6.11 (br s, 1H), 5.10—5.15
(m, 1H), 4.33–4.36ACTHNUTRGNEU(GN m, 1H), 4.12 (dd, J=19.0, 5.7 Hz, 1H), 4.04 (dd, J=
19.0, 5.0 Hz, 1H), 3.23 (dt, J=7.6, 3.8 Hz, 1H), 2.22 (dt, J=7.0, 2.5 Hz,
2H), 2.1 (dt, J=15.1, 7.6 Hz, 1H), 2.03 (dt, J=14.5, 4.4, 1H), 1.96 (t, J=
2.5 Hz, 1H), 1.86–1.73 (m, 9H), 1.26 (br s, 14H), 0.88 ppm (t, J=7.6 Hz,
3H); 13C NMR (125 MHz, CDCl3): d=170.5, 169.2, 160.9, 83.8, 74.9,
73.0, 68.8, 56.9, 40.1, 38.9, 34.1, 31.9, 29.6, 29.5, 29.4, 29.32, 29.26, 27.9,
25.7, 25.2 22.7, 18.1, 14.1 ppm; ESI-MS: (m/z) calcd for C25H41NO5 [M+
H]+ 436.3, found 436.2.
(2S)-(R)-1-[(2R,3R)-3-(hex-5-yn-1-yl)-4-oxooxetan-2-yl]tridecan-2-yl 2-
formamido-3-methylpentanoate (1j)
Prepared according to the representative Mitsunobu reaction procedure
using b-lactone 10b (14 mg, 0.04 mmol), triphenylphosphine (21 mg,
0.08 mmol), N-formyl-l-isoleucine (14 mg, 0.08 mmol) and DIAD (12 mL,
0.08 mmol) in 1.0 mL THF. Purification by flash chromatography on
silica gel (15% EtOAc/hexanes) gave 1j (8 mg, 41%) as a colorless oil.
1H NMR (500 MHz, CDCl3): d=8.23 (s, 1H), 6.16 (br s, 1H), 5.02—5.03
(m, 1H), 4.61 (dd, J=8.2, 4.4 Hz, 1H), 4.34—4.37 (m, 1H), 3.23 (dt, J=
7.6, 3.8 Hz, 1H), 2.20–2.15 (m, 3H), 1.93 (t, J=2.5 Hz, 1H), 1.60–1.25
(m, 30H), 0.96 (d, J=7.0 Hz, 2H), 0.93 (d, J=7.6 Hz, 3H), 0.87 ppm (t,
J=6.9 Hz, 3H); IT-TOF-MS: (m/z) calcd for C29H49NO5 [M<M+H]+
calcd: 491.361, found: 492.288.
Chemical Synthesis
(S)-(S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridec-12-yn-2-yl 2-
formamidopropanoate (2b)
All chemicals were purchased as reagent grade and used without further
purification, unless otherwise noted. Tetrahydrofuran (THF) was distilled
over sodium benzophenone and used immediately. All non-aqueous reac-
tions were carried out under nitrogen atmosphere in oven-dried glass-
ware. Reaction progress was monitored by TLC on pre-coated silica
plates (Merck 60 F254, 250 mm thickness) and spots were visualized by
ceric ammonium molybdate, basic KMnO4, or UV light. 1H NMR and
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 19a (13 mg, 0.04 mmol), PPh3 (47 mg, 0.18 mmol), N-formyl-l-
alanine (21 mg, 0.18 mmol) and DEAD (27 mL, 0.17 mmol) in THF
(1 mL). Purification by flash chromatography on silica gel (hexanes/
EtOAc, 100:0 to 4:1) to give 2b (13 mg, 73%) as a white solid. 1H NMR
(500 MHz, CDCl3): d=8.18 (s, 1H), 6.21 (br d, J=6.3 Hz, 1H), 5.05—
2772
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Orlistat-Like Natural-Product-Based Probes
5.07 (m, 1H), 4.61—4.67 (m, 1H), 4.30 (m, 1H), 3.21 (dt, J=7.6, 3.8 Hz,
1H), 2.15 (dt, J=7.0, 2.5 Hz, 3H), 2.00 (dt, J=15.1, 4.5 Hz, 1H), 1.93 (t,
J=2.5 Hz, 1H), 1.80–1.50 (m, 8H), 1.45 (d, J=6.9 Hz, 3H), 1.39–1.27 (m,
18H), 0.88 ppm (t, J=7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=
172.1, 170.7, 160.4, 84.7, 74.9, 73.0, 68.1, 57.1, 47.1, 38.9, 34.2, 31.4, 29.3,
29.2, 29.16, 29.0, 28.9, 28.7, 28.4, 27.6, 26.7, 25.1, 22.5, 18.3, 14.4,
14.0 ppm; TOF-MS: (m/z) calcd for C26H43NO5 [M+Na]+ 472.304, found
472.267.
(500 MHz, CDCl3): d=8.20 (s, 1H), 5.95 (br s, 1H), 5.02—5.04 (m, 1H),
4.66—4.69 (m, 1H), 4.26–4.31 (m, 1H), 3,20—3.24 (m, 1H), 2.17 (dt, J=
6.9, 2.5 Hz, 2H), 2.02–1.98 (m, 1H), 1.93 (t, J=2.5 Hz, 1H) 1.82–1.65 (m,
6H), 1.58–1.48 (m, 4H), 1.28 (br s, 18H), 0.96 (d, J=6.3 Hz, 6H),
0.88 ppm (t, J=6.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=172.2,
170.9, 160.6, 84.7, 74.5, 72.5, 68.0, 57.0, 47.6, 41.5, 38.7, 33.8, 31.4, 29.3,
29.2, 29.0, 28.9, 28.7, 28.4, 27.6, 26.7, 25.2, 24.9, 22.8, 22.5, 21.8, 18.3,
14.0 ppm; ESI-MS: (m/z) calcd for C29H49NO5 [M+Na]+ 514.4, found
514.3 [M+Na]+.
(S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridec-12-yn-2-yl 2-
formamidoacetate (2c)
(S)-(S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridecan-2-yl 4-methyl-2-(oct-
7-ynamido)pentanoate (3b)
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 19a (12 mg, 0.03 mmol), PPh3 (45 mg, 0.17 mmol), N-formylgly-
cine (18 mg, 0.17 mmol) and DEAD (25 mL, 0.16 mmol) in THF (1 mL).
Purification by flash chromatography on silica gel (hexanes/EtOAc, 100:0
to 4:1) to give 2c (15 mg, 70%) as a colorless oil: 1H NMR (500 MHz,
CDCl3): d=8.04 (s, 1H), 6.21 (br s, 1H), 5.09—5.14 (m, 1H), 4.31—4.34
(m, 1H), 4.11 (dd, J=18.3, 5.7 Hz, 1H), 4.02 (dd, J=18.3, 5.1 Hz, 1H),
3.18–3.22 (m, 1H), 2.17 (dt, J=6.9, 2.5 Hz, 2H), 2.10—2.15 (m, 1H), 2.01
(dt, J=15.2, 4.2 Hz, 1H), 1.93 (t, J=2.5 Hz, 1H), 1.81–1.71 (m, 5H), 1.51
(quin, J=7.0 Hz, 2H), 1.41–1.27 (m, 16H), 0.88 ppm (t, J=6.3 Hz, 3H);
13C NMR (125 MHz, CDCl3): d=170.8, 169.2, 161.0, 84.7, 75.0, 73.0, 68.1,
57.0, 40.1, 38.9, 34.1, 31.4, 29.3, 29.2, 29.0, 28.9, 28.7, 28.4, 27.6, 26.7, 25.1,
22.5, 18.4, 14.0 ppm; TOF-MS: (m/z) calcd for C25H41NO5 [M+Na]+
458.288, found 458.301.
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 21 (100 mg, 0.28 mmol), PPh3 (110 mg, 0.42 mmol), acid 22
(106 mg, 0.42 mmol) and DIAD (77 mL, 0.39 mmol) in THF (5 mL). Pu-
rification by flash chromatography on silica gel (hexanes/EtOAc, 100:0 to
80:20) to give 3b (49 mg, 30%) as a white solid. 1H NMR (500 MHz,
CDCl3): d=5.77 (d, J=8.2 Hz, 1H), 5.00 (m, 1H), 4.59 (dt, J=8.8,
5.1 Hz, 1H), 4.27–4.30 (m, 1H), 3.21 (dt, J=7.6, 3.8 Hz, 1H), 2.23 (t, J=
7.6 Hz, 2H), 2.19 (dt, J=8.0, 2.5 Hz, 2H), 2.14 (t, J=7.6 Hz, 2H), 1.98
(dt, J=15.2, 4.4 Hz, 1H), 1.93 (t, J=2.5 Hz, 1H), 1.71–1.82 (m, 2H),
1.42–1.67 (m, 14H), 1.26 (br s, 22H), 0.96 (d, J=5.1 Hz, 3H), 0.95 (d, J=
5.1 Hz, 3H), 0.88 ppm (t, J=7.0 Hz, 6H). 13C NMR (125 MHz, CDCl3):
d=172.6, 172.5, 170.7, 84.4, 74.8, 72.4, 68.3, 57.1, 51.0, 41.7, 38.7, 36.3,
34.1, 31.9, 31.5, 29.61, 29.60, 29.5, 29.4, 29.3, 29.0, 28.3, 28.1, 27.7, 26.7,
25.1, 25.01, 25.0, 22.9, 22.7, 22.5, 21.9, 18.2, 14.1, 14.0 ppm; ESI-MS: calcd
for C36H63NO5 [M+H]+ 590.5; found 590.4.
(2S)-(S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridec-12-yn-2-yl 2-
formamido-3-methylpentanoate (2d)
(S)-(S)-1-[(2S,3S)-3-(hex-5-yn-1-yl)-4-oxooxetan-2-yl]tridecan-2-yl 4-
methyl-2-propiolamidopentanoate (3c)
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 19a (12 mg, 0.03 mmol), PPh3 (45 mg, 0.17 mmol), N-formyl-l-
isoleucine (27 mg, 0.17 mmol) and DEAD (25 mL, 0.16 mmol) in THF
(1 mL). Purification by flash chromatography on silica gel (hexanes/
EtOAc, 100:0 to 4:1) to give 2d (11 mg, 63%) as a white solid. 1H NMR
(500 MHz, CDCl3): d=8.25 (s, 1H), 6.05 (br d, J=8.8 Hz, 1H), 5.00—
5.05 (m, 1H), 4.66 (dd, J=8.8, 5.1 Hz, 1H), 4.28 (dt, J=7.6, 4.4 Hz, 1H),
3.23 (dt, J=8.2, 4.4 Hz, 1H), 2.15 (dt, J=14.5, 5.1 Hz, 3H), 2.01 (dt, J=
14.5, 5.1 Hz, 1H), 1.93 (t, J=2.5 Hz, 1H), 1.85–1.25 (m, 26H), 0.96 (d,
J=7.0 Hz, 3H), 0.95 (d, J=7.0 Hz, 3H), 0.88 ppm (t, J=7.6H, 3H);
13C NMR (125 MHz, CDCl3): d=170.9, 170.7, 160.7, 84.8, 74.6, 72.9, 68.1,
57.1, 55.4, 38.6, 37.6, 33.9, 31.5, 29.3, 29.2, 29.0, 28.9, 28.7, 28.4, 27.7, 26.7,
25.1, 24.9, 22.5, 18.4, 15.6, 14.0, 11.5 ppm; TOF-MS: (m/z) calcd for
C29H49NO5 [M+H]+ 492.361, found 492.379.
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 10a (30 mg, 0.086 mmol), PPh3 (34 mg, 0.13 mmol), acid 20
(24 mg, 0.13 mmol) and DIAD (26 mL, 0.13 mmol) in THF (1 mL). Purifi-
cation by flash chromatography on silica gel (10% EtOAc/hexanes) to
give 3c (19 mg, 66%) as a white solid. 1H NMR (500 MHz, CDCl3): d=
6.25 (d, J=8.2 Hz, 1H), 5.00–5.05 (m, 1H), 4.58–4.62 (m, 1H), 4.28–4.31
(m, 1H), 3.23 (dt, J=3.8, 7.6 Hz, 1H), 2.85 (s, 1H), 2.22 (dt, J=2.5,
6.3 Hz, 2H), 2.15 (t, J=8.2 Hz, 1H), 2.01 (dt, J=4.4, 15.2 Hz, 1H), 1.95
(t, J=2.5 Hz, 1H), 1.73–1.85 (m, 2H), 1.63–1.68 (m, 3H), 1.54–1.68 (m,
8H), 1.25 (br s, 16H), 0.97 (d, J=6.3 Hz, 3H), 0.96 (d, J=6.3 Hz, 3H),
0.88 ppm (t, J=7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): d=171.4,
170.4, 151.7, 83.9, 76.7, 74.6, 74.1, 72.8, 68.7, 56.9, 51.4, 41.2, 38.7, 34.0,
31.9, 29.6, 29.5, 29.4, 29.3(2), 28.0, 27.1, 25.7, 25.1, 24.9, 22.8, 22.7, 21.8,
18.1, 14.1 ppm. ESI-MS: calcd for C31H49NO5 [M+H]+ 516.4; found
516.1.
(R)-(S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridec-12-yn-2-yl 2-
formamido-4-methylpentanoate (2e)
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 19a (15 mg, 0.04 mmol), PPh3 (56 mg, 0.22 mmol), N-formyl-d-
leucine (34 mg, 0.22 mmol) and DEAD (41 mL, 0.21 mmol) in THF
(1 mL). Purification by flash chromatography on silica gel (hexanes/
EtOAc, 100:0 to 9:1) followed by a second purification by flash chroma-
tography on silica gel (hexanes/EtOAc, 9:1 to 4:1) to give 2e (8 mg,
(S)-(S)-4-(1-(4-((2-amino-9H-purin-6-yl)methoxy)benzyl)-1H-1,2,3-
triazol-4-yl)-1-((2S,3S)-3-(hex-5-yn-1-yl)-4-oxooxetan-2-yl)butan-2-yl 2-
formamido-4-methylpentanoate (4)
To a mixture of di-alkyne b-lactone 5 (10.2 mg, 0.022 mmol) and BG-
azide 24 (6.5 mg, 0.022 mmol) in a mixture of DMSO/H2O (3:1, 400 mL)
was added CuI (4.2 mg, 0.022 mmol) and DIPEA (8 mL, 0.044 mmol).
The reaction mixture was stirred at room temperature for 72 h. The reac-
tion mixture was then diluted with water (1 mL) and extracted with
EtOAc (3ꢂ1 mL). The combined organic phase was extracted with 1m
EDTA solution (3ꢂ1 mL), brine (2 mL) and dried over anhydrous
Na2SO4. The product was used in the next step without purification. LC-
ESI-MS (m/z) calcd for C38H52N9O6Si [M+H]+ 758.4, found 758.4. To a
solution of the above mixture in DMF (200 mL) was added a solution of
tris(dimethylamino)sulfonium difluorotrimethylsilicate (TAS-F) (8.0 mg,
0.029 mmol) in DMF (200 mL). The reaction mixture was stirred at room
temperature for 12 h. The reaction mixture was diluted with water
(500 mL) and the aqueous phase was extracted with EtOAc (3ꢂ1 mL).
The combined organic phase was washed with brine (1 mL) and dried
over anhydrous NaSO4. The residue was purified by preparative HPLC
(Phenomenex Luna C18, 5 mm, 50ꢂ3 mm; 0–10 min, 20–95% CH3CN;
10–12 min, 100% CH3CN; flow rate: 0.5 mLminÀ1) to give 4 (2.6 mg,
1
36%) as a white solid. H NMR (500 MHz, CDCl3): d=8.20 (s, 1H), 5.91
(br d, J=5.3 Hz, 1H), 5.00—5.05 (m, 1H), 4.64—4.69 (m, 1H), 4.34—
4.37 (m, 1H), 3.22 (dt, J=6.9, 4.4 Hz, 1H), 2.18 (dt, J=6.9, 2.5 Hz, 1H),
2.02 (dt, J=14.5, 4.5 Hz, 1H), 1.93 (t, J=2.5 Hz, 1H), 1.86–1.27 (m,
29H), 0.97 (d, J=5.0 Hz, 6H), 0.88 ppm (t, J=7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d=172.2, 170.9, 160.6, 84.7, 74.6, 72.6, 68.1, 57.0,
49.6, 41.5, 38.5, 33.8, 31.5, 29.34, 29.32, 29.2, 29.0, 28.9, 28.7, 27.6, 26.7,
25.6, 25.2, 24.9, 22.8, 22.5, 21.9, 18.4, 14.0 ppm; TOF-MS: (m/z) calcd for
C29H49NO5 [M+H]+ 492.361, found 492.344.
(S)-(R)-1-[(2R,3R)-3-hexyl-4-oxooxetan-2-yl]tridec-12-yn-2-yl 2-
formamido-4-methylpentanoate (2 f)
Prepared according to the general Mitsunobu reaction procedure using
b-lactone 19b (15 mg, 0.04 mmol), PPh3 (43 mg, 0.16 mmol), N-formyl-l-
leucine (27 mg, 0.17 mmol) and DEAD (25 mL, 0.16 mmol) in THF
(1 mL). Purification by flash chromatography on silica gel (hexanes/
1
l
EtOAc, 100:0 to 4:1) to give 2 f (13 mg, 81%) as a colorless oil: H NMR
17% yield over two steps) as a colorless oil. H NMR (500 MHz, CDCl3):
Chem. Asian J. 2011, 6, 2762 – 2775
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2773

S. Q. Yao et al.
FULL PAPERS
d=7.99 (s, 1H), 7.63 (s, 1H), 7.50 (d, J=7.6 Hz, 2H), 7.41 (s, 1H), 7.24
(d, J=7.6 Hz, 2H), 5.62 (d, J=14.5 Hz, 1H), 5.52—5.55 (m, 1H), 5.43 (d,
J=12 Hz, 1H), 5.38 (d, J=14.5 Hz, 1H), 4.94 (br s, 1H), 4.85 (br s, 1H),
4.49 (br s, 1H), 4.27 (dt, J=8.2, 4.4 Hz, 1H), 3.20 (dt, J=8.3, 3.8 Hz,
1H), 2.79 (t, J=6.3 Hz, 1H), 2.17–2.20 (m, 2H), 2.09–2.15 (m, 2H), 2.00–
2.07 (m, 2H), 1.95 (t, J=2.5 Hz, 1H), 1.51–1.77 (m, 5H), 0.97 (d, J=
6.3 Hz, 3H), 0.94 ppm (d, J=6.3 Hz, 3H). LC-TOF-MS (m/z) calcd for
C35H43N9O6 [M+H]+ 686.3336, found 686.3423.
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Acknowledgements
This work was supported by the National Research Foundation (NRF),
Competitive Research Program (R143-000-218-281; NRF-G-CRP 2007-
04). We gratefully acknowledge a generous gift of plasmids from Prof.
Christopher J. Chang (University of California, Berkeley). We thank
Jigang Wang for technical assistance and Dr. Siu Kwan Sze (Nanyang
Technological University, Singapore) for advice and support for LCMS
experiments.
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