Cell-based proteome profiling using an affinity-based probe (AfBP) derived from 3-deazaneplanocin A (DzNep)

Article abstract of DOI:10.1002/asia.201300303

3-Deazaneplanocin A (DzNep), a global histone methylation inhibitor, has attracted significant interest in epigenetic research in recent years. The molecular mechanism of action and the cellular off-targets of DzNep, however, are still not well-understood

Full text of DOI:10.1002/asia.201300303

FULL PAPER
DOI: 10.1002/asia.201300303
Cell-Based Proteome Profiling Using an Affinity-Based Probe (AfBP)
Derived from 3-Deazaneplanocin A (DzNep)
Eric K. W. Tam,[a] Zhengqiu Li,[b] Yi Ling Goh,[a] Xiamin Cheng,[b] Sze Yue Wong,[a]
Sridhar Santhanakrishnan,[a] Christina L. L. Chai,*[a, c] and Shao Q. Yao*[b]
Abstract:
3-Deazaneplanocin
A
imal structural modifications. The
probe was found to be highly cell-per-
meable, and possessed similar anti-
apoptotic activities as DzNep in MCF-
7 mammalian cells. Two additional con-
trol probes were made as negative la-
beling/pull-down probes in order to
minimize false identification of back-
ground proteins due to unavoidable, in-
trinsic nonspecific photo-crosslinking
reactions. All three probes were subse-
quently used for in-situ proteome
profiling in live mammalian cells, fol-
lowed by large-scale pull-down/LC-MS/
MS analysis for identification of poten-
tial cellular protein targets that might
interact with DzNep in native cellular
environments. Our LC-MS/MS results
revealed some highly enriched proteins
that had not been reported as potential
DzNep targets. These proteins might
constitute unknown cellular off-targets
of DzNep. Though further validation
experiments are needed in order to un-
equivocally confirm these off-targets,
our findings shed new light on the
future use of DzNep as a validated
chemical probe for epigenetic research
and as a potential drug candidate for
cancer therapy.
(DzNep), a global histone methylation
inhibitor, has attracted significant inter-
est in epigenetic research in recent
years. The molecular mechanism of
action and the cellular off-targets of
DzNep, however, are still not well-un-
derstood. Our aim was to develop
novel DzNep-derived small-molecule
probes suitable to be used in live mam-
malian cells for identification of poten-
tial cellular targets of DzNep under
physiologically relevant settings. In the
current study, we have successfully de-
signed, synthesized, and tested one
such probe, called DZ-1. DZ-1 is
Keywords: affinity-based probes ·
click chemistry · epigenetics · inhib-
itors · proteomics
a
ꢀclickableꢁ affinity-based probe
(AfBP) derived from DzNep with min-
Introduction
tions.[1] In recent years, there has been renewed interest in
the application of DzNep in epigenetic research, primarily
due to the discovery that DzNep could modulate histone
methylation by disrupting the activity of histone-lysine N-
methyltransferase EZH2.[2] The latter is a catalytic subunit
of the polycomb repressive complex 2 (PRC2) that functions
to silence tumor suppressor genes by the addition of three
methyl groups to lysine 27 of histone 3, a modification lead-
ing to chromatin condensation.[3] EZH2 is also known to as-
sociate with numerous cellular proteins, such as the embry-
onic ectoderm development protein, the VAV1 oncoprotein,
and the X-linked nuclear protein (XNP).[4] It may also play
a role in the hematopoietic and central nervous systems.
EZH2 overexpression has been shown to have implications
in tumorigenesis and to correlate with poor prognosis in sev-
eral tumor types.[5] EZH2 inhibition by DzNep has been
documented in cultured cancer cell types, including primary
and acute myeloid leukemia (AML) cells.[6,7] For example,
human AML cells treated with DzNep were found to have
elevated expression levels of several cell-cycle regulators in-
cluding p21, p27, and FBXO32, before undergoing cell cycle
arrest and apoptosis.[6] These findings have led to the pursuit
of using DzNep and similar compounds as potential thera-
peutic agents in the treatment of cancer.[7] Notwithstanding
the intensive efforts on DzNep in epigenetic research, exist-
ing biochemical and cellular data on DzNep and its poten-
Post-translational modification of histones is an essential
event for the regulation of chromatin structure and gene ex-
pression in eukaryotes. 3-Deazaneplanocin
Figure 1) is a carbocyclic analog of adenosine that was origi-
nally developed as a potent inhibitor of S-adenosylhomocys-
teine hydrolase (SAHH) for potential therapeutic applica-
A (DzNep,
[a] Dr. E. K. W. Tam,+ Y. L. Goh, S. Y. Wong, Dr. S. Santhanakrishnan,
Prof. Dr. C. L. L. Chai
Institute of Chemical and Engineering Sciences
8 Biomedical Grove, Neuros #07-01, 138665 (Singapore)
[b] Dr. Z. Li,+ X. Cheng, Prof. Dr. S. Q. Yao
Department of Chemistry
National University of Singapore
3 Science Drive 3, 117543 (Singapore)
Fax : (+65)6779-1691
E-mail: chmyaosq@nus.edu.sg
[c] Prof. Dr. C. L. L. Chai
Department of Pharmacy
National University of Singapore
18 Science Drive 4, 117543 Singapore
E-mail: phacllc@nus.edu.sg
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201300303.
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Figure 1. (A) Overall workflow of the cell-based proteome profiling approach followed by large-scale pull-down/LC-MS/MS for identification of poten-
tial cellular targets of DzNep using affinity-based probes (AfBPs) shown in panel B. (B) Structure of DzNep, the DzNep-derived probe DZ-1, and the
negative control probes NP-1 and NP-2. The two click reporters, rhodamine-N3 and biotin-N3, used in the current study are also shown.
tial biological targets are at best inconclusive.[8] As an ade-
nosyl nucleoside analog, DzNep might be expected to bind
to a variety of other cellular proteins that possess intrinsic
binding affinity toward AMP/ADP/ATP, S-Adenosyl me-
thionine (SAM), NADH/NADPH, and other nucleotide an-
alogs, in addition to its putative targets SAHH and EZH2.
Many such proteins are present in mammalian cells, includ-
ing kinases, methyltransferases, ATPases, oxidoreductases,
and others. It is therefore imperative to decipher the precise
molecular targets of DzNep and to elucidate its mechanism
of action. Strategies capable of large-scale proteome-wide
analysis and identification of potential cellular targets of
DzNep under physiological settings have, however, not been
reported thus far.
Several chemical profiling strategies for large-scale, pro-
teome-wide protein target identification exist, but most of
them use affinity beads immobilized with the compound of
interest (e.g., the probe) to capture its interacting proteins
from cellular lysates.[9,10] In more recent examples, protein–
drug interactions were studied under native cellular settings
by adding the drug of interest directly to cells and initiate
binding to its intended cellular targets. Upon cell lysis, im-
mobilized probes were then added to the cell lysates as
“baits”. By measuring the amount of a protein captured on
beads by quantitative mass spectrometry, with and without
drug treatment, this method, when compared to previous
methods,[9] is a step forward in the right direction and ena-
bles the in-situ profiling of protein–drug interactions indi-
rectly.[10] Nevertheless, the strong reliance on bead-bound
probes and/or cellular lysates to capture protein–drug inter-
actions inevitably leads to many ꢀꢀfalse positivesꢁꢁ and
ꢀꢀmissed targetsꢁꢁ in all these strategies, as genuine protein–
drug interactions (defined hereafter as those that occur in
living cells under physiologically relevant conditions) are
governed by a variety of other factors besides the protein/li-
gands themselves, including protein compartmentalization
and localized protein concentrations. Our long-term re-
search aim has been to develop chemical strategies capable
of interrogating protein–drug interactions in native cellular
environments (i.e., in live cells, not cell lysates).[11,12] This
calls for the design of small-molecule probes that are cell-
permeable, minimally disrupt protein–drug interactions, and
effectively capture interacting cellular targets in situ (Fig-
ure 1A).[12] Previously, we had successfully carried out sever-
al large-scale liquid chromatography–tandem mass spec-
trometry (LC-MS/MS) experiments on small-molecule drugs
(e.g., Orlistat and Dasatinib) by using such design principle-
s.[12a,c] From these studies, we have firmly established that
such a cell-based proteome profiling approach is indeed ef-
fective in the rapid identification of potential cellular targets
of biologicallly relevant small molecules. A key element to
the success of this approach is the design of suitable cell-per-
meable, small-molecule probes that are modified versions of
the target compounds but are capable of recapitulating most
if not all the genuine protein–interacting events under phys-
iological settings (Figure 1B). As part of our on-going inter-
est in DzNep and related compounds, we have undertaken
the challenge of developing DzNep-derived probes that
might be useful for identification of potential cellular targets
(both on- and off-targets) of DzNep. Herein, we report the
successful design, synthesis, and biological testing of DZ-
1 (Figure 1B&Scheme 1), a ꢀclickableꢁ affinity-based probe
(AfBP) derived from DzNep with minimal structural modi-
fications. The probe was found to be highly cell-permeable
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Scheme 1. Synthesis of probes DZ-1, NP-1, and NP-2 used in this study. DIAD=diisopropyl azodicarboxylate, DMAP=4-dimethylaminopyridine,
TBAF=tetra-n-butylammonium fluoride, TBAI=tetrabutylammonium iodide, TMSN3 =trimethylsilyl azide.
and to possess similar anti-apoptotic activities as DzNep in
MCF-7 mammalian cells. We have successfully carried out
in-situ proteome profiling of DZ-1 in MCF-7 cells, followed
by large-scale pull-down LC-MS/MS analysis for the iden-
tification of potential cellular protein targets of DzNep. Our
LC-MS/MS results revealed some highly enriched proteins
that were previously unknown DzNep targets. These might
constitute potential cellular off-targets of DzNep.
Results and Discussion
Overall Workflow of Cell-Based Proteome Profiling
As shown in Figure 1A, in our cell-based proteome profiling
approach, a cell-permeable DzNep-derived probe (e.g., DZ-
1) bearing a photoreactive group and an alkyne handle was
used to convert non-covalent protein–drug interactions into
covalent linkage in situ. Upon cell lysis, these protein–probe
cross-linked complexes would be subsequently conjugated
to an azide-containing reporter tag (e.g., rhodamine-N3 or
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biotin-N3) by the well-established bioorthogonal click
chemistry.[13] Further in-gel fluorescence scanning and/or
pull-down (PD)/LC-MS/MS analysis would enable large-
scale identification of potential DzNep-binding cellular pro-
teins. This approach could alleviate many of the aforemen-
tioned problems encountered in the ꢀtraditionalꢁ PD ap-
proaches,[9,10] in that it would enable genuine noncovalent
protein–probe intereactions to be faithfully captured in situ
prior to downstream in-vitro cell lysis, protein enrichment,
and LC-MS/MS analysis for target identification.
tionality under copper-catalyzed microwave conditions,
thereby giving rise to 5a (61% yield). The corresponding
amine side-product 5b was also obtained in 11% yield. The
exo-amino functionality on the purine ring of 5a was then
protected as the N,N-diBoc derivative 6. Subsequent depro-
tection of the trityl group in 6 was achieved by treatment
with p-toluene sulfonic acid to yield the cyclopentendiol 7 in
74% yield. Dess–Martin periodinane oxidation of 7 gave
the corresponding aldehyde 8 (quantitative yield), which
was used in a subsequent Grignard reaction without further
purifications. The Grignard reaction between the aldehyde 8
and 4-[(trimethylsilyl)ethynyl]phenyl magnesium bromide
delivered compound 9 as an inseparable 10:1 diastereomeric
mixture (56% yield). Finally, deprotection of the TMS
group on the alkyne with TBAF and treatment with HCl
gave the desired target probe DZ-1 as a mixture of diaste-
reomers (58% yield over two steps).
The synthesis of the two negative probes, NP-1 and NP-2,
is summarized in Scheme 1. Probe NP-1 was prepared in
three steps from (4-aminophenyl)methanol. The first step in-
volved the formation of azide 11 by diazotization–azidation
of the aromatic amine starting material with sodium nitrite
and sodium azide in acetic acid at 08C (83% yield). The hy-
droxy group in 11 was then converted into a bromo group to
give 12 (77% yield). The subsequent SN2 reaction between
the alkoxide form of 12 and pent-4-yn-1-ol furnished NP-
1 in 37% yield. The other negative probe, NP-2, was synthe-
sized in a three-step route. First, a direct coupling reaction
between the chloropurine and pent-4-yn-1-ol was successful-
ly carried out under Mitsunobu reaction conditions (with tri-
phenylphosphine and diethyl azodicarboxylate as coupling
reagents) to afford 13 in 24% yield. Next, direct amine-to-
azide conversion from 13 into 14 in a single step was suc-
cessful by using n-butyl nitrite and trimethylsilyl azide
(TMS-N3) in acetonitrile at ꢀ158C (46% yield). Finally,
amination of the chloro moiety on 14 gave the expected
product NP-2 in 39% yield. All probes including their inter-
mediates were fully characterized by NMR spectroscopy
and mass spectrometry before being used in subsequent bio-
logical and proteomic experiments.
Design and Synthesis of DZ-1
The probe DZ-1 contained several critical elements of
design needed for a successful cell-based proteome profiling
experiment. First, the probe preserved most of the core
structure in DzNep, namely the adenine and the cyclopen-
tendiol moieties, thus retaining most if not all of the pro-
tein-binding ability and apoptotic activities when compared
with wild-type DzNep (see below). Second, a photo-reactive
group that allows conversion of non-covalent protein–probe
interaction in situ to covalent linkage upon UV irradiation
was introduced into the probe, and this was accomplished
through an aryl azide installed at the C2 position of the ade-
nine group, which retained the essential structural features
in adenine and, at the same time, delivered an aryl azide
that can be converted into a highly reactive nitrene upon
UV irradiation (254 nm). Finally, an aryl alkyne moiety was
installed at a position remote from the core pharmacophore.
Based on known structure–activity relationships of DzNep
analogs and their apoptotic activities (data not shown),[1b]
this position was not expected to interfere with protein–
probe binding. The alkyne would serve as a convenient click
reporter moiety for subsequent detection, purification, and
isolation of probe-labeled proteins. For the purpose of re-
ducing false-positive hits, which are intrinsic and in most
cases unavoidable in protein photo-crosslinking experi-
ments,[14] two negative probes, NP-1 and NP-2 (Figure 1B),
were designed and used in all subsequent cell-based pro-
teome profiling and PD/LC-MS/MS experiments.
The synthesis of DZ-1 is summarized in Scheme 1. It com-
menced from the optically pure O-protected cyclopentendiol
1 which was obtained from d-ribose in 8 steps.[15] Mitsunobu
coupling between compound 1 and 2-amino-6-chloropurine
gave the single adduct 2 in excellent yield (95%). Unfortu-
nately, subsequent direct amine-to-azide conversion on the
purine ring of 2 to deliver the corresponding azide 5a in
a single step was unsuccessful when n-butyl nitrite in the
presence of trimethylsilylazide or sodium azide was used.
Thus, a longer sequence was subsequently employed to ach-
ieve the desired chemical transformation through the forma-
tion of intermediates 3 and 4. In this synthetic route, the ar-
omatic amine 2 was initially converted into the correspond-
ing iodide 3 by a diazotization–iodination protocol. This was
followed by amination at the 4-chloro position of the purine
ring to furnish 4-amino-2-iodoadenine (i.e., compound 4).
Next, the iodo group in 4 was converted into an azido func-
Biological Evaluation of the Probes
We first assessed the cell permeability of the probes. DzNep
was used as the positive reference compound. Many small-
molecule probes developed thus far have bulky reporter
groups such as a fluorophore or biotin directly attached to
the compound of interest.[16] This makes downstream protein
analysis and isolation straightforward, but the main problem
is that the chemical structure of the resulting probe becomes
significantly different from that of the original unmodified
compound. Consequently, the biological properties, includ-
ing cell permeability, cellular localization, and protein-bind-
ing ability, of such probes might be significantly altered. Our
design of DZ-1 took a “minimalist” approach to minimize
structural changes to DzNep, and we thus expected DZ-
1 and DzNep to possess similar cellular properties including
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Figure 2. (A) Results of the cell-permeability assay with the three probes and DzNep. (B) Dose-dependent inhibition of cell proliferation in MCF-7 and
DLD1 cancer cells with DzNep and the three probes (DZ-1, NP-1, and NP-2) by using the XTT anti-proliferation assay. The data represent the average
and standard deviation for two trials. (C) In-vitro and in-situ proteome labeling profiles of MCF-7 and DLD1 by the three probes at different probe con-
centrations (5 and 20 mm). Only the in-gel fluorescence scanning profiles are shown. (D) Bioimaging of MCF-7 and DLD1 cells treated with DZ-1 (5 and
20 mm). Panels 1, 3, 5, and 7: Cells treated with the probe followed by click chemistry with rhodamine-N3; panels 2, 4, 6, and 8: Merged images of the
images shown in panels 1, 3, 5, and 7 with Hoechst-stained nuclei (pseudocolored in blue). Scale bar, 10 mm.
cell permeability. To confirm this expectation, we subjected
all three probes (DZ-1, NP-1, and NP-2) and DzNep to a bi-
directional permeability assay in MDCK cells and the re-
sults are summarized in Figure 2A. With apparent permea-
bility (Papp) values of 18.57, 10.16, and 32.35 mmsꢀ1 for DZ-
1, NP-1, and NP-2, respectively, all three probes had excel-
lent cell permeability that is comparable to that of DzNep,
which gave a Papp value of 12.29 mmsꢀ1 under identical assay
conditions. These results indicate that our DzNep-derived
probes are suitable for experiments in live cells.
DzNep is known to induce apoptotic cell death in
a number of cancer cells, such as MCF-7 (breast cancer),
but not in normal cells.[2a,5] In addition, a combination treat-
ment of DzNep and suberoylanilide hydroxamic acid
(SAHA), which is a well-known histone deacetylase inhibi-
tor on DLD-1 cells (a colorectal cancer cell line) was found
to effectively induce cell death (unpublished results).[1b,6] In
order to determine whether DZ-1 and DzNep possess simi-
lar cellular activities, we performed anti-proliferative assays
against MCF-7 and DLD1 cell lines to compare their cell-
killing activities (Figure 2B). Following drug treatment for
72 h, both DzNep and DZ-1 showed good anti-proliferative
activities against MCF-7 cells. The effect was dose-depen-
dent and noticeable even at concentrations as low as 1 mm.
Side-by-side comparison of the two compounds showed that
DzNep consistently displayed a slightly higher cell-killing
effect than DZ-1 at all tested concentrations, thus indicating
that the structural modifications introduced into DZ-1 did
cause marginal but tolerable changes in the probeꢁs cellular
activities. The fact that neither DZ-1 nor DzNep showed
any effect against DLD1 cells indicates that these com-
pounds alone were insufficient to induce apoptotic cell
death in this cell line. The two negative control probes, NP-
1 and NP-2, showed no apparent inhibitory activities against
either MCF-7 or DLD1 cell growth. This is expected as nei-
ther probe possesses the two obligatory structural features
of DzNep. Our results thus indicate that DZ-1 likely pre-
served most if not all of DzNepꢁs original biological and cel-
lular activities, and therefore may be used for subsequent
cell-based proteome profiling experiments for large-scale
identification of potential cellular targets (on- and off-tar-
gets) of DzNep.
In-Vitro and In-Situ Proteome Profiling
Next, we carried out endogenous proteome labeling, under
both in-vitro (cell lysates) and in-situ (live cells) settings,
using both MCF-7 and DLD1 cell lines. All three probes
(DZ-1, NP-1, and NP-2) were run side-by-side to compare
their proteome labeling profiles under two different probe
concentrations (5 and 20 mm). Upon probe labeling, followed
by rhodamine-N3 click chemistry, the resulting samples were
separated by SDS-PAGE and visualized by in-gel fluores-
cence scanning (Figure 2C). Relatively weak fluorescence
labeling profiles were observed for both negative probes
(NP-1 and NP-2) at both low and high concentrations (e.g.,
5 and 20 mm, respectively), thus indicating that neither probe
had any significant number of specific cellular targets. On
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the other hand, DZ-1 showed weak labeling at 5 mm probe
concentration, but at 20 mm probe concentration strongly la-
beled bands, which likely corresponded to specific cellular
targets of the probe, started to appear. This indicates that
the effective cellular concentraction of DZ-1 was relatively
high. The overall number and intensity of the fluorescent
bands produced by DZ-1 labeling were consistently higher
than those produced by the two negative probes (e.g., NP-
1 and NP-2) under the same probe concentrations in both
MCF-7 and DLD1 cell lines. This result also indicates that
more cellular proteins were targeted by DZ-1 in identical
proteome environments. There were obvious differences in
the in-vitro and in-situ proteome profiles of both MCF-7
and DLD1 produced by DZ-1, suggesting that the cellular
targets of this probe might be different under different cel-
lular settings. Similar results were observed in our previous
cell-based proteome profiling studies.[12] This underscores
the significance of our approach in its ability to label endog-
enous protein targets in live cells in their native cellular en-
vironments. There were clear differences in the labeling be-
tween the two cell lines, under both in-vitro and in-situ set-
tings, indicating that DzNep likely has different cellular tar-
gets in MLC-7 and DLD1 cells. Both in-vitro and in-situ
proteome profiling experiments were repeated on cells pre-
treated with an excessive amount of DzNep (Figure S1 in
the Supporting Information). The results showed that most
DZ-1 labeled bands were effectively competed away, indi-
cating that these labeled proteins were likely true cellular
targets of DzNep.
croscopy (Figure 2D). In MCF-7 cells, strong fluorescence
signals were observed throughout the whole cell excluding
the nucleus at 20 mm probe concentration. When the probe
concentration was decreased to 5 mm, there was a corre-
sponding decrease in the overall fluorescence signals of the
cells, with most of the fluorescence localized mainly on the
edge of cell nuclei, indicating that, upon uptake into cells,
DzNep likely was localized to these regions under physio-
logical settings. In DLD1 cells, we only observed significant
fluorescence signals at 20 mm probe concentration, which
was in accordance with both the anti-proliferative results
and the in-situ proteome labeling experiments (Figure 2B
and 2C) that suggested that DzNep had fewer cellular tar-
gets in this cell line. Of the two putative cellular targets of
DzNep, SAHH is a cytosolic protein and EZH2 is localized
to the nucleus of mammalian cells. The fact that our imaging
results showed mostly cytosolic but not nuclear localization
of the DZ-1 probe provides yet another line of evidence
that DzNep likely has many other unknown cellular targets
that reside outside of the cell nucleus. The endogenous ex-
pression of EZH2 was either too low to be detected by the
probe, or EZH2 may not be a true cellular target of DzNep
(see below).
Large-Scale Pull-Down/LC-MS/MS Analysis
Finally, we performed large-scale pull-down/LC-MS/MS ex-
periments on live MCF-7 cells in situ labeled with DZ-1,
NP-1, and NP-2 to identify potential cellular targets of
DzNep using previously published procedures, with some
modifications.[12c] NP-1 and NP-2 were used as the negative
control probes in order to subtract background protein la-
beling originating from intrinsic non-specific labeling caused
by photo-crosslinking experiments.[14] Protein extracts from
the labeled cells were enriched (following click-chemistry
conjugation with biotin-N3) by avidin-agarose beads, sepa-
rated by SDS-PAGE (see Figure S2 in the Supporting Infor-
mation), subjected to in-gel trypsin digestion, and identified
by LC-MS/MS analysis. The complete LCMS results of all
three probes are shown in the Supporting Information, with
selected results summarized in Figure 3. As in the case with
most large-scale LC-MS/MS experiments, a large number of
proteins hits were identified in our results (>1000 different
proteins). Among them, many were inevitably highly abun-
dant, nonspecific proteins that were labeled indiscriminately
in live cells due to their “sticky” nature and high levels. As
a result, they were labeled by all three probes. These pro-
teins were automatically eliminated as false positives. Of the
remaining proteins, we focused on those that met the follow-
ing two criteria: 1) proteins with an exponentially modified
protein abundance index (emPAI) value of greater than 0.1,
and 2) proteins with a protein score of greater than 30. The
emPAI offers an approximate, label-free, relative quantita-
tion of proteins in our pull-down samples based on the pro-
tein concentration. In total, there were 41 such proteins
present and they were deemed likely true binders of DZ-1.
When we further grouped these proteins based on their
Bioimaging
There has been enormous interest in the development of
small-molecule-based imaging probes capable of reporting
in-vivo protein–drug interactions as well as enzymatic activi-
ties.[17–19] In some recent examples, several groups have suc-
cessfully reported small-molecule probes that could be used
to image different endogenous kinases in live cells.[12c,19]
Given the excellent cell permeability of DZ-1, as well as its
ability to closely imitate endogenous cellular activities of
DzNep, we wondered if this probe could also serve as
a useful imaging reagent to study the cellular uptake and lo-
calization of DzNep in live mammalian cells. It should be
noted that, since DzNep could likely bind to a variety of dif-
ferent cellular proteins, DZ-1 would therefore not be very
useful in bioimaging of specific protein targets. We carried
out live-cell imaging experiments in MCF-7 and DLD1 cells
using DZ-1. First, the cells were treated with DZ-1 (5 and
20 mm) for 2 h. No cell death was observed at the end of
probe treatment. Subsequently, the cells were irradiated by
UV light to initiate covalent protein–probe linkage, thereby
“fixing” the probe to its cellular targets/locations. Cells were
then fixed with formaldehyde, permeabilized by using Triton
X-100, and treated with rhodamine-N3 following our previ-
ously optimized click chemistry protocols.[12c] The same cells
were further stained with Hoechst to visualize their nuclei.
Finally, the cells were imaged by confocal fluorescence mi-
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Figure 3. (A) The number and percentage of each protein subgroup enriched from the MCF-7 cell line. (B) Representative proteins hits identified from
MCF-7 cells. “1” signals detection only in positive PD/LC-MS/MS (see the Supporting Information for details).
functions (Figure 3A), we found 6 methyltransferases, 7 kin-
ases, 8 ATPases, and 5 phosphatases, most of which possess
a nucleotide-binding pocket in their active sites and there-
fore likely have an intrinsic binding affinity toward DzNep
as well. Some of the most interesting proteins hits are also
summarized in the table shown in Figure 3B. Both MAPK
and PKA, which are well-known protein kinases, as well as
histone arginine methyltransferase and phosphatase methyl-
transferase, which like EZH2 are methyltransferases that
utilize S-adenosylhomocysteine as a cofactor, are well repre-
sented in this list. While our list of potential cellular protein
targets of DzNep shown in Figure 3 should be further vali-
dated by other independent experiments and more detailed
biological studies, the results themselves clearly support the
assumption we set out to confirm at the start of the current
study, that is, compounds such as DzNep, given their struc-
tural homology to adenosine, are likely binders to a variety
of cellular proteins. Interestingly, neither SAHH nor EZH2
was positively identified from our LCMS results (see the
Supporting Information). This again indicates that either
both proteins were not endogenously expressed to a signifi-
cant extent to be effectively labeled by DZ-1 in our experi-
ments or that they are not true cellular targets of DzNep.
methyltransferases. Although further studies will be needed
in order to validate some of these newly identified, potential
DzNep targets, our current findings show that DzNep likely
has many unknown cellular targets and that both existing
putative targets, EZH2 and SAHH, might not be true tar-
gets of DzNep under native cellular settings. Our findings
should have important implications in the consideration of
using DzNep as a validated chemical probe for epigenetic
research (especially against EZH2) and as a potential drug
candidate for cancer therapy. Finally, our strategy should be
generally useful for the off-target identification of other
suitable drugs and/or candidates.
Experimental Section
General Information
All chemicals were purchased from Aldrich or Alfa Aesar and used as
received, unless specified otherwise. Dried solvents (CH2Cl2, DMF, THF,
acetonitrile, Et2O, and toluene) were drawn from a Glass Contour sol-
vent dispensing system. All reactions requiring anhydrous conditions
were carried out under argon atmosphere using oven-dried glassware.
The reaction progress was monitored by thin-layer chromatography
(TLC) on pre-coated silica plates (Merck 60 F254, 0.25 mm), and spots
were visualized by UV light or phosphomolybdic acid stain. Flash
column chromatography was carried out using Merck 60 F254, 0.040–
0.063 mm silica gel. 1H and 13C NMR spectra were recorded on a Bruker
400 MHz instrument equipped with a CryoProbe. Chemical shifts for pro-
tons are reported in parts per million (ppm) that were referenced to the
NMR solvent (CDCl3: 7.26 ppm; [D6]DMSO: 2.50 ppm; CD3OD:
3.31 ppm). Chemical shifts for carbon are given in ppm that were refer-
enced to the solvent (CDCl3: 77.0 ppm; [D6]DMSO: 39.5 ppm; CD3OD:
49.0 ppm). Data are presented as follows: chemical shift, multiplicity
(br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet),
integration and coupling constants (J) in Hertz (Hz). Electron impact
mass spectra (EIMS) (low and high resolution) were measured using
a Finnigan MAT95XP double-focusing mass spectrometer. Electrospray
ionization (ESI) mass spectra were recorded using a Waters Quattro Mi-
croTM API instrument for low-resolution and an Agilent 6210 Time-of-
Flight LC/MS instrument for high-resolution mass spectrometry. In-gel
fluorescence scanning of SDS-PAGE gels was carried out with a Typhoon
9410 fluorescence gel scanner (Amersham Biosciences), and where appli-
Conclusions
We have successfully developed a “clickable”, cell-permea-
ble probe, DZ-1, suitable for cell-based proteome profiling
and target identification of DzNep in live MCF-7 cells. The
probe possessed similar cell permeability and cellular apop-
totic activities as the wild-type DzNep. Through a cell bio-
imaging experiment we confirmed that DZ-1 might be
useful for the study of cellular uptake and cellular localiza-
tion of DzNep. From our in-situ large-scale pull-down/LC-
MS/MS results, we have successfully obtained a list of poten-
tial cellular targets (on- and off-targets) of DzNep. Among
the list were several kinases, phosphatases, ATPases, and
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Christina L. L. Chai, Shao Q. Yao et al.
cable, bands were quantified using ImageQuant 3.3 (Molecular Dynam-
ics). Tris(2-carboxyethyl) phosphine (TCEP) and tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (“ligand”) were purchased from Sigma–Al-
drich. Hoechst 33342 was purchased from Invitrogen. DzNep (SML0305)
was purchased from Sigma–Aldrich. MCF-7 mammalian cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen,
Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum
(FBS; Invitrogen), 100 UmLꢀ1 penicillin, and 100 mgmLꢀ1 streptomycin
(Thermo Scientific, Rockford, IL), and maintained in a humidified 378C
incubator with 5% CO2. Rhodamine-N3 and biotin-N3 were synthesized
as previously reported.[12]
2-Azido-9-((3aS,4R,6aR)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-
3aH-cyclopenta[d][1,3]dioxol-4-yl)-9H-purin-6-amine (5a)
AHCTUNGTRENNUNG
To a solution of CuI (31.2 mg, 0.164 mmol) and N,N-dimethylethylene di-
amine (53 mL, 0.49 mmol) in water (22 mL) was added NaN3 (213 mg,
3.276 mmol) to form the catalyst mixture. Subsequently, this mixture was
added to a microwave vial containing 4 (1.1 g, 1.63 mmol) in EtOH
(52 mL). The resulting suspension was heated in a microwave reactor at
908C for 90 min. Next, EtOH was removed under reduced pressure and
the mixture was dissolved in CH2Cl2, washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. Further purification by flash
chromatography (EtOAc/petroleum 40:60!70:30, followed by MeOH/
CH2Cl2 3:97!6:94) afforded the azido product 5a (610 mg; 61%) and
the amine side-product 5b (100 mg, 11%). 5a: 1H NMR (400 MHz,
CDCl3): d=7.53–7.39 (m, 6H), 7.37–7.21 (m, 9H), 5.97 (brs, 2H), 5.49
(brs, 1H), 5.25 (s, 1H), 5.10 (s, 1H), 4.65 (brs, 1H), 4.10 (d, J=15.5 Hz,
1H), 3.85 (d, J=15.5 Hz, 1H), 1.42 (s, 3H), 1.32 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=156.9, 150.2, 146.9, 143.7, 128.5, 127.9, 127.9,
127.2, 121.5, 112.6, 87.3, 84.5, 84.2, 65.2, 61.5, 27.5, 26.1 ppm; HR-MS
(ESI+) m/z calcd for C33H30N8O3: 287.2589 [M+H]+, found 587.2498. 5b:
1H NMR (400 MHz, CDCl3): d=7.46 (dd, J=3.30, 5.31 Hz, 6H), 7.33–
7.19 (m, 9H), 5.98 (s, 1H), 4.88 (d, J=5.46 Hz, 1H), 4.74 (t, J=5.44 Hz,
1H), 4.58 (s, 1H), 3.88 (d, J=14.26 Hz, 1H), 3.67 (d, J=14.28 Hz, 1H),
2.71 (d, J=9.18 Hz, 1H), 1.37 ppm (s, 6H); HR-MS (ESI+) m/z calcd for
C33H33N6O3: 561.2614 [M+H]+, found 561.2593.
Synthesis
6-Chloro-9-((3aS,4R,6aR)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-
3aH-cyclopenta[d]ACHTUNGTRENNUNG[1,3]dioxol-4-yl)-9H-purin-2-amine (2)
To a solution of 2-amino-6-chloropurine (751 mg, 4.43 mmol) in anhy-
drous THF (170 mL) was added 1 (1.46 g, 3.41 mmol, prepared based on
previously published procedures[15]) and PPh3 (1.162 g, 4.43 mmol). Sub-
sequently, diisopropyl azodicarboxylate (DIAD, 872 mL, 4.43 mmol) was
added dropwise and the reaction was stirred at 508C for 18 h under
reflux conditions. Following removal of the solvent under reduced pres-
sure, the resulting residue was purified by flash chromatography (Et2O/
petroleum ether, 10:90!60:40) to afford the desired product 2 (1.56 g;
95%) as a white solid. 1H NMR (400 MHz, CDCl3): d=7.62 (s, 1H),
7.52–7.38 (m, 6H), 7.36–7.20 (m, 9H), 5.97 (brs, 1H), 5.49 (brs, 1H), 5.26
(d, J=5.5 Hz, 1H), 4.66 (d, J=5.5 Hz, 1H), 4.03 (d, J=16.0 Hz, 1H),
3.87 (d, J=16.0 Hz, 1H), 1.43 (s, 3H), 1.33 ppm (s, 3H); 13C NMR
(101 MHz, CDCl3): d=159.0, 153.4, 151.4, 150.4, 143.6, 140.4, 128.5,
128.8, 127.2, 125.7, 121.4, 112.6, 87.3, 84.1, 84.1, 77.3, 64.3, 61.3, 27.5,
26.1 ppm; HR-MS (ESI+) m/z calcd for C33H31ClN5O3: 580.2115 [M+H]+
, found 580.2118.
2-Azido-9-((3aS,4R,6aR)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-
3aH-cyclopenta[d]ACTHUNRGTNEUNG[1,3]dioxol-4-yl)-N,N-diboc-9H-purin-6-amine (6)
To a solution of 5a (600 mg, 1.02 mmol) in CH2Cl2 (51 mL) was added
Boc anhydride (893 mg, 4.09 mmol) and DMAP (25 mg, 0.205 mmol).
The reaction was allowed to stir at room temperature for 4 h. Following
the removal of CH2Cl2 under reduced pressure, the resulting residue was
purified by flash chromatography (EtOAc/petroleum ether, 10:90!
35:65) to afford the desired product 6 (780 mg, 89%) as a white foam.
1H NMR (400 MHz, CDCl3): d=7.85 (s, 1H), 7.50–7.38 (m, 6H), 7.35–
7.18 (m, 9H), 5.97 (s, 1H), 5.59 (s, 1H), 5.36–5.27 (m, 1H), 4.74 (d, J=
5.5 Hz, 1H), 4.02 (d, J=15.5 Hz, 1H), 3.87 (d, J=15.5 Hz, 1H), 1.48 (s,
18H), 1.42 (s, 3H), 1.32 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=
155.9, 154.3, 151.3, 150.5, 150.3, 143.7, 142.9, 128.5, 127.9, 127.2, 126.2,
121.4, 112.7, 87.4, 84.4, 84.2, 84.1, 65.0, 61.4, 27.8, 27.5, 26.1 ppm.
6-Chloro-9-((3aS,4R,6aR)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-
3aH-cyclopenta[d]ACHTUNGTRENNUNG[1,3]dioxol-4-yl)-2-iodo-9H-purine (3)
To a solution of 2 (1.56 g, 2.69 mmol) in anhydrous acetonitrile (54 mL)
was added CuI (51.2 mg, 0.27 mmol) and CH2I2 (10.8 mL, 134.5 mmol).
The reaction was then cooled to 08C while n-butyl nitrite (1.57 mL,
13.5 mmol)[20] was added. Subsequently, the reaction was heated to 508C
and further stirred for 3 h. Acetonitrile was removed under reduced pres-
sure and the resulting mixture was dissolved in CH2Cl2, washed with di-
luted bicarbonate, dried over Na2SO4, filtered, and concentrated in
vacuo. Purification by flash chromatography (Et2O/petroleum ether,
20:80!50:50) afforded the desired product 3 as a pale yellow foam
(1.86 g; 80%). 1H NMR (400 MHz, CDCl3): d=7.87 (s, 1H), 7.48–7.40
(m, 6H), 7.36–7.20 (m, 9H), 5.94 (brs, 1H), 5.64 (brs, 1H), 5.33 (d, J=
5.5 Hz, 1H), 4.72 (d, J=5.5 Hz, 1H), 4.05 (d, J=16.0 Hz, 1H), 3.89 (d,
J=16.0 Hz, 1H), 1.43 (s, 3H), 1.33 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=152.1, 151.2, 150.6, 143.7, 143.6, 132.1, 128.5, 128., 127.9,
127.3, 121.0, 112.9, 87.4, 84.3, 84.1, 65.2, 61.3, 27.5, 26.2 ppm; HR-MS
(ESI+) m/z calcd for C33H30IN5O3: 671.1393 [M+H]+, found: 671.1399.
((3aR,6R,6aS)-6-(2-Azido-6-(dibocamino)-9H-purin-9-yl)-2,2-dimethyl-
6,6a-dihydro-3aH-cyclopenta[d]ACTHNUGRTNEUNG[1,3]dioxol-4-yl)methanol (7)
To a solution of 6 (780 mg, 0.99 mmol) in acetone (9.9 mL) was added
2,2-dimethoxypropane (10.32 mL, 9.91 mmol) and p-toluenesulfonic acid
dihydrate (95.26 mg, 0.50 mmol). The reaction was allowed to stir at
room temperature for 18 h, and stopped by quenching with saturated bi-
carbonate until the pH of the solution was basic. Following removal of
acetone under reduced pressure, the resulting mixture was dissolved in
CH2Cl2, washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. Further purification by flash chromatography (EtOAc/petrole-
um ether, 10:90!100:0) afforded the desired product 7 (400 mg, 74%)
as a white solid. 1H NMR (400 MHz, CDCl3): d=7.90 (s, 1H), 5.73 (s,
1H), 5.54 (s, 1H), 5.47 (d, J=5.5 Hz, 1H), 4.78 (d, J=5.5 Hz, 1H), 4.52–
4.39 (m, 2H), 1.52–1.44 (m, 21H), 1.37 ppm (s, 3H); 13C NMR (101 MHz,
CDCl3): d=156.3, 154.0, 152.3, 150.9, 150.0, 143.0, 124.0, 121.5, 113.0,
84.6, 84.3, 84.2, 65.3, 59.9, 27.9, 27.8, 27.4, 25.9 ppm; HR-MS (ESI+) m/z
9-((3aS,4R,6aR)-2,2-Dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-
cyclopenta[d]ACHTUNGTRENNUNG[1,3]dioxol-4-yl)-2-iodo-9H-purin-6-amine (4)
To a solution of 3 (1.134 g, 1.64 mol) in THF (16.4 mL) was added 33%
NH3 (8.2 mL) in a sealed tube. The reaction was heated to 508C and al-
lowed to stir for 6 h. Next, THF was removed under reduced pressure
and the mixture was dissolved in CH2Cl2, washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo to afford pure 4 (1.1 g,
100%) as a white solid. 1H NMR (400 MHz, CDCl3): d=7.71 (s, 1H),
7.49–7.37 (m, 6H), 7.36–7.20 (m, 9H), 6.30 (s, 2H), 5.93 (d, J=0.9 Hz,
1H), 5.60 (s, 1H), 5.33–5.27 (m, 1H), 4.71 (d, J=5.5 Hz, 1H), 4.03 (d, J=
15.5 Hz, 1H), 3.88 (d, J=15.5 Hz, 1H), 1.41 (s, 3H), 1.33 ppm (s, 3H);
13C NMR (101 MHz, CDCl3): d=154.2, 151.0, 149.6, 146.9, 143.7, 138.4,
128.5, 128.0, 127.9, 127.2, 121.3, 112.8, 87.4, 84.3, 84.2, 65.0, 61.3, 27.5,
26.2 ppm; HR-MS (ESI+) m/z calcd for C33H31IN5O3: 672.1472 [M+H]+,
found 672.1462.
calcd for C24H33N8O7: 545.2472ACHTUNGTRNEUNG
[M+H]+, found 545.2469.
(3aR,6R,6aS)-6-(2-Azido-6-(dibocamino)-9H-purin-9-yl)-2,2-dimethyl-
6,6a-dihydro-3aH-cyclopenta[d][1,3]dioxole-4-carbaldehyde (8)
AHCTUNGTRENNUNG
To a solution of 7 (186 mg, 0.34 mmol) in CH2Cl2 (3.4 mL) was added
Dess–Martin periodinane (DMP, 174 mg, 0.41 mmol), and the reaction
was allowed to stir at room temperature for 2 h. Next, the reaction mix-
ture was filtered through a pack of Celite (2 times). Upon removal of
CH2Cl2 in vacuo, the desired product 8 was obtained, which was used di-
rectly in the next step without further purification. 1H NMR (400 MHz,
CDCl3): d=9.96 (s, 1H), 7.90 (s, 1H), 6.70 (s, 1H), 5.77 (d, J=5.5 Hz,
Chem. Asian J. 2013, 8, 1818 – 1828
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Christina L. L. Chai, Shao Q. Yao et al.
1H), 5.71 (s, 1H), 4.94 (d, J=5.5 Hz, 1H), 1.49–1.48 (bs, 21H), 1.39 ppm
found 405.1345; calcd for C13H16N8NaO3: 427.1243 [M+Na]+, found
(s, 3H).
427.1249.
4-Azidophenyl)methanol (11)[21]
((3aR,6R,6aS)-6-(2-azido-6-(dibocamino)-9H-purin-9-yl)-2,2-dimethyl-
6,6a-dihydro-3aH-cyclopenta[d]ACTHNUTRGNEUNG[1,3]dioxol-4-yl)(4-
((trimethylsilyl)ethynyl)phenyl)methanol (9)
To a stirred solution of (4-aminophenyl)methanol (600 mg, 4.87 mmol) in
acetic acid (36 mL) and water (4 mL) was added NaNO2 (672 mg,
9.74 mmol) and then NaN3 (633 mg, 9.74 mmol) at 08C. The reaction
mixture was stirred for 30 min and subsequently poured into water and
neutralized to pH 7 by slow addition of solid NaHCO3. The resulting
mixture was extracted with EtOAc. The combined layer was washed with
water and brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography (EtOAc/petroleum
ether, 0:100!15:85) to afford 11 (600 mg, 83%) as a colorless oil.
1H NMR (400 MHz, CDCl3): d=7.35 (d, J=8.6 Hz, 2H), 7.02 (d, J=
8.5 Hz, 2H), 4.66 ppm (s, 2H).
To a 2-neck round-bottom flask under argon was added magnesium
(48.7 mg, 2.0 mmol) in anhydrous THF (1 mL), and the reaction was al-
lowed to stir at 508C. Subsequently, a solution of [(4-bromophenyl)ethy-
nyl]trimethylsilane (507 mg, 2.0 mmol) in anhydrous THF (3 mL) was
added dropwise to the above mixture, and the reaction was stirred at
508C for 30 min under reflux conditions. Next, a small amount of iodine
was added to magnesium (9.7 mg, 0.4 mmol) in a separate vial and this
was then added to the reaction mixture. Stirring was continued at 508C
for another 3 h to finally afford 4-[(trimethylsilyl)ethynyl]phenyl magne-
sium bromide (“the Grignard reagent”) as a 0.5m solution in THF. Subse-
quently, the above Grignard reagent in THF (2.05 mL, 1.02 mmol) was
added to a solution of 8 (185 mg; 0.34 mmol) in anhydrous THF (3.4 mL)
under argon, and the reaction was allowed to stir at room temperature
for 18 h. The reaction was then quenched with saturated NH4Cl. Upon
removal of THF under reduced pressure, the resulting mixture was dis-
solved in CH2Cl2, washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. Further purification by flash chromatography
(EtOAc/petroleum ether, 0:100!50:50) afforded the desired product 9
(139 mg; 56%) as a yellow solid (mixture of isomers 10:1). 1H NMR
(400 MHz, CDCl3): d=7.84 (s, 1H), 7.50 (d, J=8.3 Hz, 2H), 7.38 (d, J=
8.3 Hz, 2H), 6.23 (s, 1H), 5.90 (s, 1H), 5.53 (s, 1H), 5.06 (d, J=5.5 Hz,
1H), 4.73 (d, J=5.5 Hz, 1H), 1.57 (s, 9H), 1.49 (s, 3H), 1.44 (s, 9H), 1.31
(s, 3H), 0.26 ppm (s, 9H); 13C NMR (101 MHz, CDCl3): d=152.3, 152.1,
150.5, 150.3, 149.5, 140.2, 136.4, 132.5, 127.8, 124.1, 121.9, 118.2, 113.2,
104.5, 95.4, 84.2, 83.8, 83.0, 82.9, 75.6, 65.3, 28.2, 27.8, 27.8, 27.4, 26.1,
0.0 ppm; HR-MS (ESI+) m/z calcd for C35H45N8O7Si: 717.3180 [M+H]+,
found 717.3198.
Azido-4-(bromomethyl)benzene (12)[21]
To a solution of 11 (200 mg, 1.34 mmol) in dry CH2Cl2 (5 mL) under
argon was added CBr4 (889 mg, 2.68 mmol) and PPh3 (703 mg,
2.68 mmol) at 08C, and the reaction was stirred for 30 min. Following sol-
vent evaporation in vacuo, the resulting residue was purified by flash
chromatography (EtOAc/petroleum ether, 0:100!2:98) to afford 12
(218 mg, 77%) as a colorless oil. 1H NMR (400 MHz, CDCl3): d=7.38
(d, J=8.5 Hz, 2H), 7.00 (d, J=8.5 Hz, 2H), 4.48 ppm (s, 2H).
1-Azido-4-((pent-4-yn-1-yloxy)methyl)benzene (NP-1)
To a solution of pent-4-yn-1-ol (63 mg, 0.75 mmol) in dry THF (2 mL)
was added sodium hydride (23 mg, 0.56 mmol, 60%) under argon at 08C.
Following stirring for 30 min, a solution of 12 (80 mg, 0.38 mmol) in THF
was cannulated, followed by addition of Bu4NI (70 mg, 0.19 mmol). The
reaction mixture was stirred further for 5 h, before being quenched by
slow addition of a saturated aqueous NH4Cl solution. The resulting mix-
ture was then extracted with EtOAc. The combined layer was washed
with water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography (EtOAc/petro-
leum ether, 0:100!5:95) to afford NP-1 (30 mg, 37%) as a colorless oil.
1H NMR (400 MHz, CDCl3): d=7.32 (d, J=8.6 Hz, 2H), 7.01 (d, J=
8.5 Hz, 2H), 4.48 (s, 2H), 3.57 (t, J=6.2 Hz, 2H), 2.32 (td, J=7.1, 2.7 Hz,
2H), 1.94 (t, J=2.7 Hz, 1H), 1.88–1.77 ppm (m, 2H); 13C NMR
(101 MHz, CDCl3): d=139.3, 135.3, 129.1 (2C), 118.9 (2C), 83.8, 72.4,
68.7, 68.5, 28.6, 15.3 ppm; FTIR (neat): n˜max =3302, 2256, 2110,
((3aR,6R,6aS)-6-(2-azido-6-(dibocamino)-9H-purin-9-yl)-2,2-dimethyl-
6,6a-dihydro-3aH-cyclopenta[d]ACTHUNGTREN[NUNG 1,3]dioxol-4-yl)(4-
ethynylphenyl)methanol (10)
To a solution of 9 (139 mg; 0.19 mmol) in anhydrous THF (9.7 mL) was
added 1m tetra-n-butylammonium fluoride (TBAF) in THF (233 mL,
0.23 mmol), and the reaction was allowed to stir at room temperature for
2 h, before being quenched with saturated NH4Cl. Following removal of
THF under reduced pressure, the resulting mixture was dissolved in
CH2Cl2, washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. Further purification by flash chromatography (EtOAc/petrole-
um ether, 10:90!40:60) afforded 10 (86 mg; 69%) as a yellow solid.
1H NMR (400 MHz, CDCl3): d=7.72 (s, 1H), 7.54 (d, J=8.1 Hz, 2H),
7.42 (d, J=8.1 Hz, 2H), 6.25 (s, 1H), 5.90 (s, 1H), 5.50 (s, 1H), 5.07 (d,
J=5.4 Hz, 1H), 4.74 (d, J=5.4 Hz, 1H), 3.11 (s, 1H), 1.56 (s, 9H), 1.50
(s, 3H), 1.44 (s, 9H), 1.31 ppm (s, 3H); 13C NMR (101 MHz, CDCl3): d=
156.8, 152.2, 150.6, 149.8, 149.5, 140.6, 136.8, 132.7, 127.9, 123.0, 122.2,
119.2, 113.1, 84.1, 83.8, 83.1, 83.0, 82.7, 78.0, 77.2, 75.5, 65.1, 28.1, 27.7,
27.7, 27.4, 26.1 ppm; HR-MS (ESI+) m/z calcd for C32H37N8O7: 645.2785
[M+H]+, found 645.2772.
1507 cmꢀ1
;
HR-MS (ESI+) m/z calcd for C12H13N3NaO: 238.0951
[M+Na]+, found 238.0961.
6-Chloro-9-(pent-4-yn-1-yl)-9H-purin-2-amine (13)
To a suspension of 2-amino-6-chloropurine (1.5 g, 8.85 mmol) in anhy-
drous THF (30 mL) under argon was sequentially added pent-4-yn-1-ol
(1.63 mL, 17.69 mmol), PPh3 (5.8 g, 22.12 mmol), and DIAD (4.36 mL,
22.12 mmol). The reaction mixture was stirred at 508C for 18 h under
reflux conditions. Following evaporation of THF under reduced pressure,
the residue was purified by flash chromatography (MeOH/CHCl3, 1:99!
5:95) to afford 13 (500 mg, 24%) as a white solid. 1H NMR (400 MHz,
CDCl3): d=7.79 (s, 1H), 5.10 (s, 2H), 4.24 (t, J=6.8 Hz, 2H), 2.22 (dd,
J=6.8, 2.6 Hz, 1H), 2.13–2.01 ppm (m, 4H); 13C NMR (101 MHz,
CDCl3): d=159.0, 153.8, 151.3, 142.5, 125.3, 81.9, 70.3, 42.5, 27.6,
15.6 ppm; HR-MS (ESI+) m/z calcd for C10H11ClN5: 236.0697 [M+H]+,
found 236.0700.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
(DZ-1)
Compound 10 (30 mg, 0.047 mmol) in MeOH (1 mL) was stirred in 2n
HCl (116.7 mL, 0.233 mmol) at room temperature for 18 h. Upon solvent
removal under reduced pressure, a yellowish solid was obtained. Purifica-
tion using reverse-phase preparative HPLC was performed to afford DZ-
2-Azido-6-chloro-9-(pent-4-yn-1-yl)-9H-purine (14)
To a stirred solution of 17 (156 mg, 0.67 mmol) in dry acetonitrile (4 mL)
under argon was added butyl nitrate (780 mL, 6.68 mmol) and TMS-N3
(877 mL, 6.88 mol) at ꢀ158C. After stirring at the same temperature for
12 h, the solvent was removed under reduced pressure. The resulting resi-
due was purified by flash chromatography (EtOAc/petroleum ether,
0:100!30:70) to afford 14 (80 mg, 46%) as a white solid. 1H NMR
(400 MHz, CDCl3): d=8.05 (s, 1H), 4.38 (t, J=6.8 Hz, 2H), 2.25 (m,
2H), 2.18–2.08 (m, 2H), 2.06 ppm (t, J=2.6 Hz, 1H); 13C NMR
(101 MHz, CDCl3): d=156.1, 153.3, 152.0, 145.1, 128.9, 81.5, 70.5, 43.0,
1
1 (17.2 mg, 84%) as a pure white solid. H NMR (400 MHz, MeOD): d=
7.94 (s, 1H), 7.54–7.45 (m, 4H), 6.06 (s, 1H), 5.42 (brs, 1H), 5.39 (brs,
1H), 4.36 (apparrent, J=5.5 Hz, 1H), 4.26 (d, J=5.5 Hz, 1H), 3.47 ppm
(s, 1H); 13C NMR (101 MHz, MeOD): d=155.9, 154.0, 153.9, 151.2,
143.3, 142.0, 133.3, 128.8, 124.4, 123.5, 114.1, 84.2, 78.9, 78.0, 74.3, 72.6,
68.2 ppm; HR-MS (ESI+) m/z calcd for C19H17N8O3: 405.1424 [M+H]+,
Chem. Asian J. 2013, 8, 1818 – 1828
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Christina L. L. Chai, Shao Q. Yao et al.
27.6, 15.5 ppm; HR-MS (ESI+) m/z calcd for C10H9ClN7: 262.0602
Cellular Imaging in MCF-7 Cells
[M+H]+, found 262.0600.
MCF-7 cells were seeded in glass bottom dishes (Mattek) and grown
until 70–80% confluence.[12c] Cells were then treated with 0.5 mL of
DMEM containing the probe at different concentrations. After 2 hat
378C, the medium was removed, and cells were gently washed twice with
PBS, followed by UV irradiation (254 nm) for 5 min. The cells were sub-
sequently fixed for 15 min at room temperature with 3.7% formaldehyde
in PBS, washed twice with cold PBS, and permeabilized with 0.1% Triton
X-100 in PBS for 10 min. Cells were then blocked with 2% BSA in PBS
for 30 min, washed twice with PBS, and subsequently treated with a fresh-
ly prepared premixed click chemistry reaction solution in a volume of
100 mL (final concentrations of reagents: 1 mm CuSO4, 1 mm TCEP,
100 mm TBTA, and 10 mm rhodamine-N3 in PBS) for 2 h at room tempera-
ture under vigorous shaking. Cells were washed with PBS at least three
times. The same cells were then incubated in PBS containing
0.25 mgmLꢀ1 of Hoechst for 15 min at room temperature to stain nuclear
DNA, and washed with PBS for 5 min with gentle agitation and a final
wash with deionized water (1–2 min with gentle agitation) before mount-
ing. Cellular imaging was done using a Leica TCS SP5X confocal micro-
scope system equipped with Leica HCX PL APO 63ꢃ/1.20 W CORR
CS, 405 nm diode laser, white laser (470–670 nm, with 1 nm increments,
with eight channels AOTF for simultaneous control of eight laser lines,
each excitation wavelength provides 1.5 mV), and a photomultiplier tube
(PMT) detector ranging from 410 to 700 nm for steady-state fluorescence.
Images were processed with Leica Application Suite Advanced Fluores-
cence (LAS AF).
2-Azido-9-(pent-4-yn-1-yl)-9H-purin-6-amine (NP-2)
Compound 14 (25 mg, 0.09 mmol) was heated with methanolic ammonia
(3 mL) in a sealed tube at 508C for 12 h. The reaction mixture was
poured into water and the resulting solid was filtered, washed with water,
and dried to afford NP-2 (9 mg, 39%) as
a
white solid. 1H NMR
(400 MHz, CDCl3): d=7.73 (s, 1H), 4.24 (t, J=6.7 Hz, 2H), 2.71 (s, 2H),
2.17 (td, J=6.7, 2.5 Hz, 2H), 2.10–1.98 ppm (m, 3H); 13C NMR
(101 MHz, CDCl3): d=156.8, 155.9, 151.1, 140.1, 116.3, 81.9, 70.1, 42.5,
27.8, 15.4 ppm; HR-MS (ESI+) m/z calcd for C10H10N8Na: 265.0921
[M+Na]+, found 265.0928.
Cell-Permeability and Proliferation Assays
The cell-permeability assay was carried out, as previously described,[22]
with MDCK (Madin–Darby canine kidney) cells and with caffeine and
Lucifer yellow as controls. Cell viability was determined using the XTT
colorimetric cell-proliferation kit (Roche). Briefly, cells were grown to
20–30% confluence (as they will reach approximately 90% confluence
within 48 to 72 h in the absence of drugs) in 96-well plates under the con-
ditions described above. The medium was aspirated, washed with PBS,
and then treated, in duplicate, with 0.1 mL of the medium containing dif-
ferent concentrations of the probe (1–20 mm) or DzNep (1–20 mm; as
a positive control). Probes were applied from DMSO stocks whereby
DMSO does not exceed 1% in the final solution. The same volume of
DMSO was used as a negative control. Fresh medium, along with the
probes and DzNep, were added every 24 h. After a total treatment time
of 72 h, proliferation was assayed using the XTT kit by following the
guidelines provided by the manufacturer (read at 450 nm). Data repre-
sent the average (ꢁs.d.) of two trials.
Pull-Down (PD) and LC-MS/MS Analysis
The general in-situ proteome labeling and pull-down procedures were
based on previously reported procedures,[12] with the following optimiza-
tions. Briefly, the probe (10 mm) was directly added to live MCF-7 cells,
followed by incubation for 5 h at 378C under 5% CO2. DMSO never ex-
ceeded 1% in the final solution. The medium was aspirated, and cells
were washed twice gently with PBS to remove excess probe, followed by
UV irradiation (254 nm) for 5 min on ice. The cells were then trypsinized
and pelleted by centrifugation. Subsequently, the cell pellets were resus-
pended in PBS (50 mL), homogenized by sonication, and diluted to
1 mgmLꢀ1 with PBS. The labeled lysates were then subjected to click re-
action with biotin-N3, and all subsequent experiments were carried out as
previously described.[12c] Control labeling/PD experiments using the nega-
tive probe (NP-1 or NP-2 at 10 mm final concentration) was carried out
concurrently. After PD, all protein samples were separated on 10% SDS-
PAGE gels, followed by coomassie or silver staining. Trypsin digestion
was performed as previously described.[12] Digested peptides were ex-
tracted from the gel with 50% acetonitrile and 1% formic acid. The pep-
tides were separated and analyzed on a Shimadzu UFLC system (Shi-
madzu, Japan): A mobile phase A (0.1% formic acid in H2O) and mobile
In-Vitro and In-Situ Proteome Profiling
For in-vitro labeling, the probe was added to 50 mg of freshly prepared
cell lysates (prepared as previously described[12]) in 50 mL of Hepes
buffer at a desired concentration. Unless indicated otherwise, samples
were incubated for 30 min at room temperature and then irradiated with
UV light (254 nm) for 5 min. Four microliters of a freshly premixed click
chemistry reaction cocktail in PBS containing 100 mm rhodamine-N3
(from a 1 mm stock solution in DMSO), 0.1 mm TBTA (from a 2.4 mm
freshly prepared stock solution in deionized water), 1 mm TCEP (from
a 24 mm freshly prepared stock solution in deionized water), and 1 mm
CuSO4 (from a 24 mm freshly prepared stock solution in deionized
water) were added. The reaction was further incubated for 2 h with
gentle mixing, before it was terminated by the addition of prechilled ace-
tone (0.5 mL; 30 min incubation at ꢀ208C). Precipitated proteins were
subsequently collected by centrifugation (13000 g for 10 min at 48C).
The supernatant was discarded and the pellet was washed with 200 mL of
prechilled MeOH. Subsequently, the sample was heated at 958C in 2ꢃ
SDS loading buffer for 10 min. Around 20 mg (per gel lane) of proteins
were separated by SDS-PAGE (10% gel) and then visualized by in-gel
fluorescence scanning. For in-situ labeling, cells were grown to 80–90%
confluence in 6-well plates under conditions described above. The
medium was removed, and cells were washed twice with cold PBS and
then treated with 0.5 mL of probe-containing DMEM (the probe was di-
luted from DMSO stocks whereby DMSO never exceeded 1% in the
final solution). After 5 h of incubation at 378C under 5% CO2, the
medium was aspirated, and cells were washed gently with PBS (2ꢃ) to
remove excess probe, followed by UV irradiation for 5 min on ice. The
cells were then trypsinized and pelleted by centrifugation. Subsequently,
the cell pellets were resuspended in PBS (50 mL), homogenized by soni-
cation, and diluted to 1 mgmLꢀ1 with PBS. All subsequent procedures
were similar to those from in-vitro experiments. The protein pellets were
then resuspended in 20 mL of 1ꢃ SDS loading buffer and heated for
10 min at 958C. Around 20 mg (per gel lane) of proteins were separated
by SDS-PAGE (10% gel) and then visualized by in-gel fluorescence
scanning.
phase
B (0.1% formic acid in acetonitrile) were used to establish
a 60 min gradient comprising 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 mass spectrometer with an Ad-
vance CaptiveSpray Source (Michrom Bio Resources) at an electrospray
potential of 1.5 kV. A gas flow of 2 Lminꢀ1, ion transfer tube tempera-
ture of 1808C, and collision gas pressure of 0.85 mTorr were used. The
LTQ-FT was set to perform data acquisition in the positive-ion mode as
previously described,[23] except that the m/z range of 350–1600 was used
in the full MS scan. The raw data were converted to mgf format. The da-
tabase search was performed with an in-house Mascot server (version
2.2.07, Matrix Science) with MS tolerance of 10 ppm and MS/MS toler-
ance of 0.8 Da. Two missed cleavage sites of trypsin were allowed. Carba-
midomethylation (C) was set as a fixed modification, and oxidation (M)
and phosphorylation (S, T, and Y) were set as variable modifications.
LC-MS/MS results obtained from the above experiments (in situ, and for
negative probes) were processed as shown below, and results are sum-
marized in the Supporting Information. As in the case of most large-scale
LCMS experiments,[12] a large number of proteins were identified from
each LCMS run, many of which were “sticky” and/or highly abundant
proteins. These were automatically removed. “False” hits that appeared
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Christina L. L. Chai, Shao Q. Yao et al.
in negative control pull-down/LCMS experiments were further eliminat-
ed. Detailed LC-MS/MS results are presented in the Supporting Informa-
tion.
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Acknowledgements
Funding was provided by the National Medical Research Council
(NMRC/1260/2010) and the Agency for Science Technology and Re-
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the scale-up of some compounds and Dr. Yu Qiang for the supply of
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Received: March 7, 2013
Revised: April 11, 2013
Published online: June 7, 2013
Chem. Asian J. 2013, 8, 1818 – 1828
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