Applying small molecule microarrays and resulting affinity probe cocktails for proteome profiling of mammalian cell lysates

Article abstract of DOI:10.1002/asia.201100523

Small molecule microarrays (SMMs) are proving to be increasingly important tools for assessing protein-ligand interactions, as well as in screening for enzyme substrates and inhibitors, in a high-throughput manner. We previously described an SMM-facilitated screening strategy for the rapid identification of probes against γ-secretase, an aspartic protease. In this article, we extend upon this work with an expanded library of hydroxyethylamine-derived inhibitors which non-exclusively target aspartic proteases. Our library is diversified across P₂, P₁, P₁ ^′, and P₂^′ positions. Accordingly, 86 new inhibitors are synthesized using a combinatorial, solid-phase synthetic approach, bringing the total library size to 284-biotinylated compounds, which were arrayed onto avidin slides. In order to elucidate enzymatic activity and profiles within complex biological samples, screening is performed using fluorescently-labeled mammalian cell lysates. This yielded reproducible profiles or binding fingerprints that correspond with interactions from aspartic proteases or accessory proteins as well as other interacting targets that were present in the sample. The brightest microarray hits were converted to affinity-based probes (AfBPs) using convenient, 1-step "click" chemistry with benzophenone from the relevant building blocks. Pull-down/mass spectrometric analysis with these probes (individuals or cocktail) yielded putative protein targets that include well-known aspartic proteases, such as cathepsinD which is a clear marker for breast cancer cell lines, T47D. Many other hits were also identified, which may be secondary or tertiary interactors of aspartic proteases, or yet unreported off-targets of the hydroxyethylamine pharmacophore. Our work herein thus provides a candidate list of biomarkers for further investigations. Taken together, this SMM-facilitated strategy for the discovery of new AfBPs should provide a useful tool for high-throughput development of novel small molecule probes and the identification of new aspartic proteases as well as related biomarkers in the future.

Full text of DOI:10.1002/asia.201100523

DOI: 10.1002/asia.201100523
Applying Small Molecule Microarrays and Resulting Affinity Probe
Cocktails for Proteome Profiling of Mammalian Cell Lysates
Haibin Shi,[a, c] Mahesh Uttamchandani,[a, d] and Shao Q. Yao*[a, b, c]
On the occasion of the 10th anniversary of click chemistry
Abstract: Small molecule microarrays
(SMMs) are proving to be increasingly
important tools for assessing protein–
ligand interactions, as well as in screen-
ing for enzyme substrates and inhibi-
tors, in a high-throughput manner. We
previously described an SMM-facilitat-
ed screening strategy for the rapid
identification of probes against g-secre-
tase, an aspartic protease. In this arti-
cle, we extend upon this work with an
expanded library of hydroxyethyla-
mine-derived inhibitors which non-ex-
clusively target aspartic proteases. Our
order to elucidate enzymatic activity
and profiles within complex biological
samples, screening is performed using
fluorescently-labeled mammalian cell
lysates. This yielded reproducible pro-
files or binding fingerprints that corre-
spond with interactions from aspartic
proteases or accessory proteins as well
as other interacting targets that were
present in the sample. The brightest
microarray hits were converted to af-
finity-based probes (AfBPs) using con-
venient, 1-step “click” chemistry with
benzophenone from the relevant build-
ing blocks. Pull-down/mass spectromet-
ric analysis with these probes (individu-
als or cocktail) yielded putative protein
targets that include well-known aspart-
ic proteases, such as cathepsin D which
is a clear marker for breast cancer cell
lines, T47D. Many other hits were also
identified, which may be secondary or
tertiary interactors of aspartic proteas-
es, or yet unreported off-targets of the
hydroxyethylamine
pharmacophore.
Our work herein thus provides a candi-
date list of biomarkers for further in-
vestigations. Taken together, this
SMM-facilitated strategy for the dis-
covery of new AfBPs should provide a
useful tool for high-throughput devel-
opment of novel small molecule probes
and the identification of new aspartic
proteases as well as related biomarkers
in the future.
ꢀ
library is diversified across P2, P1, P1 ,
ꢀ
and P2 positions. Accordingly, 86 new
inhibitors are synthesized using a com-
binatorial, solid-phase synthetic ap-
proach, bringing the total library size
to 284-biotinylated compounds, which
were arrayed onto avidin slides. In
Keywords: click chemistry · fluo-
rescence · microarrays · proteins ·
proteomics
[a] H. Shi, M. Uttamchandani, Prof. Dr. S. Q. Yao
Department of Chemistry
Introduction
National University of Singapore
3 Science Drive 3, Singapore 117543
Fax : (+65)6779-1691
Approaches in functional proteomics have classically includ-
ed two-dimensional gel electrophoresis (2DE),[1] liquid chro-
matography-mass spectrometry (LC-MS), and DNA micro-
array technology.[2,3] These methods have been applied to
the large-scale characterization of cellular, subcellular, or
circulating proteins and biomarkers.[4] However, the majori-
ty of such technologies are geared towards detecting
changes in protein abundance rather than actual functional
activity.[5] To address this gap, activity-based protein profil-
ing (ABPP) has emerged as a powerful chemical proteomic
alternative for assessing the differing functional states of en-
zymes and other protein sub-classes within a complex pro-
teome.[6] To date, small molecule probes (the so-called activ-
ity-based probes, or ABPs) needed for ABPP studies have
E-mail: chmyaosq@nus.edu.sg
[b] Prof. Dr. S. Q. Yao
Department of Biological Sciences
National University of Singapore
[c] H. Shi, Prof. Dr. S. Q. Yao
NUS MedChem Program of the Life Sciences Institute
National University of Singapore
[d] M. Uttamchandani
Uttamchandani Mahesh. Defence Medical and Environmental Re-
search Institute
DSO National Laboratories
27 Medical Drive, Singapore 117510
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100523.
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been successfully developed for a wide range of mechanisti-
cally distinct enzymes, such as serine hydrolases, cysteine
proteases, aspartic proteases, metalloproteases, phosphatas-
es, kinases, and others.[7–13] However, gel-based separation of
complex samples labeled with ABPs is highly limited, in
that only a few probes, or a narrow pool of samples, may be
screened at any given time. Small molecule microarrays
(SMMs), on the other hand, offer high scalability in the as-
sessment of ligand-protein interactions.[14] We and others
previously described SMM-facilitated screenings, using com-
binatorial small molecule libraries, for the screening of puri-
fied enzymes and proteins.[15] This included a recent example
for the high-throughput identification of affinity-based
probes (AfBPs) against g-secretase,[9c] an aspartic protease
implicated in the progression of Alzheimerꢀs disease.[16]
More recently, we have successfully demonstrated the first
example of SMMs for large-scale functional profiling of cys-
teine proteases present in native apoptotic biological sam-
ples, including lysates from mammalian cells and parasite-in-
fected red blood cells (RBCs).[17] Herein, we extend this
SMM approach to the screening of a variety of mammalian
cellular lysates from numerous tumor cell lines using small
molecule probes derived from hydroxyethylamine-based
pharmacophores. With the type of throughput provided by
SMMs in the initial screening, we were able to quickly iden-
tify selected AfBPs suitable for the large-scale profiling of
aspartic proteases and other potential biomarkers in mam-
malian proteomes. Additionally, unknown cellular targets
identified from screening with such probes might provide
opportunities for future applications of hydroxyethylamine-
based inhibitors in novel therapeutics (Figure 1).
Results and Discussion
Rationale of Our Strategy
Aspartic proteases are the smallest of all protease classes in
the human genome, with only 15 members documented thus
far.[18] However, these enzymes have received considerable
attention as potential therapeutic targets since many have
been shown to play important roles in the physiological and
pathological processes.[19] For example, HIV-1 protease is an
important target in the fight against AIDS.[19a,b] Renin is a
target in the treatment of hypertension.[19c] Cathepsin D is
implicated in breast cancer metastasis.[19d] Plasmepsins are a
key mediator in malaria pathogenesis.[19e] Aspartic proteases
are characterized by two catalytic aspartic acid residues lo-
cated in their active sites and, in most cases, by the con-
served Asp-Thr-Gly (DTG) sequence in the primary struc-
ture. Although the catalytic mechanism of aspartic proteases
is poorly understood, it has been generally accepted that the
aspartic residues in the enzyme active site bind a molecule
of water through extensive H-bonding. Of the numerous
known aspartic protease inhibitors, hydroxyethylamine-con-
taining compounds are amongst the most potent and suc-
cessful, some of which are already FDA-approved drugs.[20]
For example, Amprenavir and Nelfinavir are just two exam-
ples of hydroxyethylamine-based drugs currently used in the
treatment of AIDS (Figure 2a). By mimicking the transition
state formed during the proteolytic reaction of aspartic pro-
teases, the hydroxyl group on the hydroxyethylamine iso-
stere binds tightly to the enzyme active site through H-
bonding with the catalytic aspartic residues, thereby replac-
ing the active site-bound water molecule. We previously
showed that this pharmacophore could be strategically and
conveniently diversified to generate inhibitor libraries that
ꢀ
ꢀ
vary structurally at the corresponding P2, P1, P1 , and P2 po-
sitions (Figure 2b).[9c]
In the current study, with the aim to develop new AfBPs,
which may be used to gain further insight into potential hy-
Figure 1. Overall strategy for small molecule microarray (SMM)-facilitated development of AfBPs for proteome profiling of aspartic proteases in mam-
malian cell lysates.
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Proteome Profiling of Mammalian Cell Lysates
Design and Synthesis of
Hydroxyethylamine-Containing
Inhibitor Library
Hydroxyethylamine
isosteres
are transition-state mimics that
were introduced as the back-
bone replacement of the amide
ꢀ
bond located between the P1/P1
position. We previously devel-
oped
a solid-phase synthetic
strategy capable of diversifying
all four positions around the
hydroxyethylamine core, that is,
ꢀ
ꢀ
P1/P1 and P2/P2 residues (Fig-
ure 2b).[9c] From that study, two
sublibraries (N-terminal and C-
terminal as shown in Figure 2b)
with a total of 198 different
compounds were already avail-
able. To further expand the cov-
erage of our library of com-
pounds to be spotted on the
SMM, an additional 86 new
compounds were prepared in
the current study (Scheme 1).
In the new N-terminal subli-
brary, the R1 position, corre-
sponding to the P1 residue upon
binding to aspartic proteases,
Figure 2. a) Representative structures of two commercially available drugs against HIV protease, an aspartic
protease. b) The 184-member library against aspartic proteases based on the hydroxyethylamine pharmaco-
phore.[9c] In both N- and C-terminal sublibraries, the P1/P1ꢀ positions were diversified by building blocks derived
from three amino acid residues (Phe, Leu, and Ala; in red). The P2/P2 positions (highlighted in gray boxes),
were diversified by various carboxylic acids and sulfonyl chloride building blocks.
ꢀ
droxyethylamine-interacting proteins in various mammalian
proteomes and to discovery new cancer biomarkers, we con-
ceived a multi-step approach, as depicted in Figure 1; in the
first stage, an SMM platform was established, on which a
structurally diverse library of hydroxyethylamine inhibitors,
each containing a biotin tag (and a connecting linker) for
SMM immobilization, was used to probe the binding profiles
of endogenous aspartic proteases and other proteins from
complex proteome samples. We had recently shown this is
feasible with fluorescently-labeled crude proteome lysates.[17]
Subsequently, positive hits (e.g., bright spots) obtained from
the SMM screening were identified, and the corresponding
compounds were conveniently converted to affinity-based
probes (AfBPs) by the CuI-catalyzed 1,3-dipolar cycloaddi-
tion (click chemistry or CuAAC).[21] This step was made
possible owing to the modular design of our compound li-
braries, and the introduction of a benzophenone (Bp) as
well as a reporter tag to the vicinity of these hydroxyethyla-
mine pharmacophores enabled the covalent capture of any
potential interacting proteins. In the next step, the resulting
AfBPs, either as individual probes or a mixture (that is,
“cocktail”) of probes, are used for gel-based proteome
profiling against mammalian cell lysates. Finally, large-scale
pull-down experiments followed by mass spectrometric anal-
ysis with selected probes allows positive identification of hy-
droxyethylamine-binding protein targets that may be further
developed into potential cancer biomarkers.
was varied with three amino acids (Tyr, Lys, and Val). The
R2 position, corresponding to the P2 residue, was diversified
with 24 aromatic/aliphatic moieties (Figure S3 in the Sup-
porting Information). In the new C-terminal sublibrary, the
ꢀ
ꢀ
P1 residue (denoted as R1 in the inhibitor library) was fixed
with Tyr for its general importance at this position amongst
ꢀ
many known aspartic protease substrates/inhibitors. The P2
ꢀ
residue (denoted as R2 ) was similarly varied with a variety
of commercially available sulfonyl chlorides (see Supporting
Information). As shown in Scheme 1, the synthesis of these
compounds was carried out by following our previously re-
ported solid-phase approaches on different pre-loaded 3,4-
dihydro-2H-pyran (DHP) resins.[9c] Starting from scaffolds
4d–f and 8d, which were synthesized from the correspond-
ing commercially available l-amino acids following previ-
ously reported procedures with some modifications,[22] com-
À
pounds 4d–f and 8d were immobilized at their OH groups
onto the DHP resin under acidic conditions, giving preload-
ed resins for N- and C-terminal sublibraries, respectively.
Next, for the N-terminal sublibrary, Fmoc deprotection of
the resin was carried out, followed by standard acylation
with 24 different acid building blocks (1–24 in Figure S3 in
the Supporting Information). Subsequent azide reduction,
which yielded the corresponding amines, attachment of a
biotin linker, and TFA cleavage yielded the 61-member N-
terminal compounds. For the synthesis of C-terminal com-
pounds, the corresponding preloaded DHP resin was first al-
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Scheme 1. Solid-phase synthesis of the N- and C-terminal sublibraries using preloaded DHP resins. A total of 86 new hydroxyethylamine-derived com-
pounds were synthesized. Fmoc=fluorenylmethyloxycarbonyl, DCE=1,2-dichloroethane, DMF=N,N-dimethylformamide, PyBrop=bromo-tris-pyrroli-
dino phosphoniumhexafluorophosphate, HOAt=1-hydroxy-7-azabenzotriazole, DIEA=ethyldiisopropylamine, NMP=1-methyl-2-pyrrolidone,
PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, THF=tetrahydrofuran.
kylated with iso-butylamine, then acylated with commercial-
ly available sulfonyl chloride building blocks (1–25 in Fig-
ure S4 in the Supporting Information). Subsequent azide re-
duction with SnCl2/PhSH provided the corresponding
amines, which, upon coupling with the biotin linker and
TFA cleavage, afforded the 25-member C-terminal com-
pounds. The resulting 86 new compounds were analyzed by
LC-MS and shown to be of sufficient purity (>80%) for
subsequent microarray immobilization and screening
(Table S1 in the Supporting Information).
microarray, based on previously developed procedures.[17] To
ensure that all fluorescence signals were indeed attributable
to activity-dependent interactions between the hydroxye-
thylamine inhibitors and their targeted proteins, the heat-de-
natured form of representative cellular lysates (that is,
T47D) was routinely applied to the same array as a negative
control experiment. As shown in Figure 3a, all eight mam-
malian cell lysates produced highly distinctive and similar
SMM binding profiles, with most of the brightest spots origi-
nating from selected members of the C-terminal sublibrary
(Figure 3b). We speculated that this arises from the pres-
ence of some commonly targeted proteins in both normal
and cancer cells. Colored heat maps were generated from
the corresponding SMM images in order to better visualize
and compare different binding fingerprints obtained from
the eight different cell lines (Figure 3b); each mammalian
cell line displayed characteristic patterns, and closer exami-
nation further revealed that spots containing hydroxyethyla-
Proteome Profiling of Mammalian Cell Lysates on SMMs
The 86 new compounds were combined with the 198 previ-
ously reported compounds, giving a total of 284 hydroxye-
ꢀ
ꢀ
thylamines with diversities at all P2/P1/P1 /P2 positions. They
were robotically spotted onto avidin-functionalized slides in
duplicate to generate the corresponding small molecule mi-
croarray (see Supporting Information for spotting pattern).
Eight different human cell lines, seven of which are derived
from tumor cells (e.g., human normal kidney cell line
HEK293T, Ovary cancer cells OVCAR-3, erythromyeloblas-
toid leukemia cells K562, colon cancer cells HCT116, hepa-
tocellular liver carcinoma cells HepG2, and three breast
cancer cell lines MDA-MB-435, MCF7, and T47D), were se-
lected for SMM screening (Figure 3). Briefly, cellular pro-
teome lysates from these cell lines were minimally labeled
by an amino-reactive Cy3 dye and directly applied onto the
ꢀ
mine compounds having Tyr and Ala at the P1 position pro-
vided the strongest binders. However, the effect of different
ꢀ
substituent groups at the P2 position did not have much of
an effect on the binding signals observed. Taken together,
the SMM screening provided a rapid tool to quickly delin-
eate the protein–ligand interaction and identify potential
“hit” compounds for subsequent follow-up studies (that is,
conversion to AfBPs and subsequent proteome profiling ex-
periments). Additionally, the unique binding fingerprints ob-
tained from these SMM experiments may also provide
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Proteome Profiling of Mammalian Cell Lysates
azide-containing WH, TACTHNUTRGENNU(G 1–5), and AACHUTNGTRENN(GUN 3–8), which were ac-
tually key intermediates in the solid-phase synthesis of our
hydroxyethylamine libraries (Scheme 1), were “click” as-
sembled with one of the two alkyne-containing photo-cross-
linkers, TER-Bp-ꢀ and Biotin-Bp-ꢀ, giving 11 fluorescent-
ly labeled AfBPs (for subsequent in-gel fluorescence-based
activity-based profiling) and 11 biotin-labeled AfBPs (for
subsequent pull-down/target identification). Introduction of
the photoreactive moiety benzophenone (Bp) enabled us to
covalently capture potential protein binders from within the
proteome and perform MS analysis for de novo target iden-
tification. A variety of “click” conditions were investigated;
eventually, conditions (0.1 eq CuSO4/0.4 eq NaAsc with
DMSO/H2O as co-solvents) were found that afforded the
desired 22 AfBPs with excellent yields and purities, within
12 h.
Activity-Based Profiling of the AfBP Library with
Recombinant Cathepsin D
To demonstrate the ability of our SMM-derived AfBPs for
UV-initiated, activity-based profiling of aspartic proteases,
we first carried out in-gel fluorescence scanning of recombi-
nant cathepsin D labeled with each of the eleven tetraethylr-
hodamine (TER) AfBPs. Cathepsin D is a well-known lyso-
somal aspartic protease endogenously expressed in mamma-
lian cells and has been reported to play an important role in
protein degradation and tumor progression. Following previ-
ously published protocols with some modifications,[9c] indi-
vidual probes were incubated with cathepsin D for 30 min
before being exposed to UV irradiation (at ~350 nm) for
25 min. This was followed by SDS-PAGE separation and in-
gel fluorescence scanning. As shown in Figure 4a, one dis-
tinct labeled band, which corresponded to cathepsin D, was
highly visible across all eleven probes, with varying labeling
intensities. It thus indicated that all selected probes could
positively label recombinant cathepsin D, albeit with differ-
ent degrees of reactivity. The labeling intensity of each
probe also indicated the relative binding preference of the
enzyme to the corresponding hydroxyethylamine pharmaco-
phore. In order to investigate the detection limit of the la-
beling reaction, varied amounts of recombinant cathepsin D
were labeled by a cocktail of the eleven probes (that is, a
mixture of an equal amount of each probe; Figure S7 in the
Supporting Information); results showed proportional in-
creases in fluorescence intensity of the labeled cathepsin D
with increased amounts of the enzyme. We estimated that
by using this method, as little as 1.5 pmol of cathepsin D
could be readily detected.
Figure 3. SMM profiles obtained by screening with eight different mam-
malian cell lysates. a) The binding profiles of Cy3-labeled mammalian
cell lysates obtained from screening against the 284-member hydroxye-
thylamine SMM. All 284 compounds were spotted in duplicate (see Sup-
porting Information for spotting patterns/IDs). Positions of 11 brightest
inhibitor spots are highlighted on the SMM. b) Color heat maps display-
ing binding of the 284-member compound library against eight Cy3-lal-
eled mammalian cell lysates. The zoomed heat map shows inhibitor po-
ꢀ
tencies of the C-terminal sublibrary presenting Ala and Tyr at the P1 po-
sition. The scale bar represents the relative potency ratio.
useful clues for future design and diversification of hydrox-
yethylamine pharmacophores as potential drug candi-
dates.[23]
Click Assembly of Select AfBPs
To further examine the positive cellular protein targets that
exhibited strong binding to members of our hydroxyethyla-
mine library, the eleven brightest spots (boxed in Figure 3a)
were chosen, and converted into the corresponding affinity-
based probes (AfBPs). As was mentioned earlier, this step
was made possible owing to the modular design of our com-
pound libraries, and the implementation of click chemistry
into our design principle. The highly efficient and modular
CuI-catalyzed cycloaddition reaction between alkynes and
azides (that is, click chemistry) had previously been success-
fully exploited to facilitate the synthesis of other classes of
activity-based probes.[21] As shown in Scheme 2, selected
In vitro Proteome Profiling
To further investigate potential cellular binding targets of
the probes within complex mammalian proteomes, we next
carried out in vitro proteome profiling experiments. Four
TER probes (T1, T3, A3, and A6) were used to label the
eight aforementioned mammalian cell lysates. Briefly, each
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bands were observed, indicating
that significantly more positive-
ly interacting proteins were
present in the total cellular pro-
teome of these cell lines. On
the contrary, far fewer fluores-
cently labeled bands were ob-
served for MDA-MB-435 and
HCT116 cell lines (which was
consistent with their earlier
comparatively weaker fluores-
cence binding signals on the
SMM; Figure 3). It is also inter-
esting to note that, while the
four TER probes did generate
mostly similar proteome pro-
files within each mammalian
cell line as expected, relative
fluorescence intensities amongst
the labeled bands varied from
one probe to another.
Next, all eleven probes were
used to label T47D cell lysates
in order to obtain comprehen-
sive proteome “fingerprinting”
profiles as a result of hydroxye-
thylamine/protein binding inter-
action (Figure 4c); each probe
again showed highly similar yet
distinctive proteome labeling
profiles, indicating that the both
ꢀ
R1/R2 residues exerted a con-
siderable influence over probe
interactions with cellular pro-
tein targets. The experiments
also revealed that each probe
had different reactivity profiles.
For example, a 90 kDa protein
showed extremely strong label-
ing with probes T4, A3, A4,
and A6 (labeled with an arrow
in Figure 4c), but not with
Scheme 2. Hit-to-probe conversion by click chemistry and structures of the twenty-two AfBPs. DMSO=di-
methyl sulfate.
other probes. However,
a
55 kDa-protein was positively
labeled by most of the probes
except probe A4 and A5 (la-
probe (1 mm final probe concentration; an equal amount of
lysates was used in each labeling reaction) was treated with
each of the eight mammalian cellular lysates for 30 min fol-
lowed by 25 min UV irradiation. Subsequent SDS-PAGE
separation and in-gel fluorescence scanning revealed the
sub-proteome binding profiles of the mammalian lysates as
a result of positive interactions between endogenous pro-
teins and the hydroxyethylamine-derived probe. As shown
in Figure 4b, different human cell lines were each distinctly
labeled despite obvious similarities. For example, in T47D
and HepG2 cancer cell lines, many more positively labeled
beled with an asterisk in Figure 4c). To validate whether
this 55 kDa labeled band corresponded to endogenous cath-
epsin D, which is widely over-expressed in human breast
cancer cells, we performed pull-down experiments using the
biotinylated probe of T2 which had the best labeling perfor-
mance for T47D cell lysates based on the labeling profile.
Upon labeling with probe, T47D cell lysates were subjected
to affinity enrichment and subsequently analyzed by immu-
noblotting with cathepsin D antibody (right gel in Fig-
ure 4c); results clearly showed that cathepsin D was success-
fully labeled and isolated by the biotinylated probe of T2.
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Proteome Profiling of Mammalian Cell Lysates
Figure 4. In-gel fluorescence images of recombinantly purified cathepsin D and mammalian cell lysates labeled with different AfBPs (individual probe
or/and mixture cocktails). a) Fluorescence labeling profiles of recombinant cathepsin D with 11 individual fluorescently labeled TER probes. b) Fluores-
cence labeling profiles of eight mammalian cell lysates with four representative TER probes (T3, T1, A3, and A6; structures are shown in Scheme 2).
The final concentration of each probe used in the labeling reaction was 1 mm. c) In vitro proteome profiling of T47D cell lysates with 11 individual TER
probes. The asterisk indicates the protein band corresponding to cathepsin D, which was confirmed by pull-down/immunoblotting with anti-cathepsin D
antibody (right gel). The arrow indicates the distinct 90 kDa protein band that appeared only in selected cell lines. d) In vitro proteome profiles of eight
different mammalian cell lysates with a mixture cocktail of 11 TER probes. The final concentration of each probe within the cocktail was 0.5 mm. Abbre-
viations: PD=positive pull-down assay; Ctrl=negative pull-down assay (avidin beads without probe).
This experiment thus demonstrates the feasibility of our af-
finity-based probe for profiling of endogenous cathepsin D
in cellular lysates. At this stage, attempts were not made to
determine whether some other fluorescently labeled bands
in our proteome profiling experiments corresponded to
other endogenous aspartic proteases (vide infra).
lar lysates (Figure 4d); in general, cancer cell line K562 and
three human breast cancer cell lines, namely MCF-7, MDA-
MB-435, and T47D, showed similar proteome labeling pro-
files, with the exception of a 100 kDa band which appeared
only in the T47D cell line. Similar proteome labeling pro-
files were also observed for ovary cancer cell line OVCAR-
3 and colon cancer cell line HCT116. Comparatively weak
proteome labeling profiles were seen for HEK293T and
HepG2 cell lines. Collectively, the success of this cocktail
approach indicates it may be more widely adopted in future
for large-scale proteome profiling experiments, and, in some
cases, to differentiate different types of mammalian cell ly-
sates.
While the detailed proteome profiling analysis of T47D
cell lysates with the 11 individual probes provided useful in-
formation about the interaction between cellular proteins
and the hydroxyethylamine pharmacophore, the experiment
itself was laborious, severely limiting these probesꢀ potential
application in large-scale proteome profiling against multi-
ple mammalian cell proteomes. Previously, a cocktail-based
approach, in which a mixture of several activity-based
probes instead of individual probes, had been successfully
applied in the profiling of metalloprotease activities in com-
plex proteomes.[24] We speculated that a similar approach
may be adopted herein to increase the throughput of our
profiling experiments, therefore enabling much more com-
prehensive proteome profiles of multiple mammalian cell ly-
sates to be readily obtained within a reasonable time frame.
We therefore applied an equal amount of all eleven AfBPs
as a single mixture cocktail in our profiling experiments. By
applying the same labeling protocol, the cocktail (compris-
ing 0.5 mm of each probe, giving a final probe concentration
of ~5 mm) was applied to each of the eight mammalian cellu-
Pull-Down Experiments and Target Identification
Next, in order to identify potential cellular targets of the hy-
droxyethylamine pharmacophore, we performed affinity
pull-down experiments with the biotinylated version of the
probe cocktail. The mixture of 11 biotinylated probes
(0.5 mm of each probe) was incubated with T47D cell lysates
for 30 min, followed by 25 min UV irradiation to initiate co-
valent cross-linking of interacting protein-probe pairs. Upon
precipitation with acetone and washing with methanol, the
resulting biotinylated proteins were reconstituted with SDS
buffer, then affinity-enriched with Neutravidin beads. Fol-
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lowing SDS-PAGE gel separation and silver staining
(Figure 5), the entire gel lane from the pull-down sample
was then cut into slices and each slice was independently
processed for in-gel trypsin digestion as described in the Ex-
perimental Section. As a negative pull-down control, the
entire experiment was repeated with the same cell lysates
treated with 5% DMSO instead of the actual probe cock-
tail.
mass spectrometry,[25,26] false positives/non-specific protein
binders could be minimized but not eliminated entirely from
our results. Consequently, proper follow-up studies and vali-
dation experiments will be needed before any biological
conclusion can be made to some of these protein hits. Of
the 39 identified proteins, cathepsin D was one of them.
This result is well expected as our previous immunoblotting
results had independently verified this (Figure 4c). It is in-
teresting to note that missing from the list are other known
mammalian aspartic proteases, which might have been
caused by their relatively lower expression level in the
T47D cell line. Among other proteins identified, both
HSP5A and b-tubulin are two of the well-known interactors
of cathepsin D.[27,28] Their positive interaction with our
AfBPs were further confirmed by pull-down/western blot-
ting with their respective antibodies (Figure 5b). Another
two proteins known to be regulated by cathepsin D, namely
superoxide dismutase (SOD) and eukaryotic translation
elongation factor 1 delta isoform 4 (eEF1),[27] were also
identified. Notably, a known pepstatin A-binding protein, di-
sulfide-isomerase (PDI),[29] was also identified with a high
MOWSE score. Additionally, a number of other targeted
proteins were pulled down including one well-known serine
protease (lon protease homolog or LONP1), several riboso-
mal proteins (60S ribosomal protein L5 or RPL5 and iso-
form 1 of plectin-1 or PLEC1), RNA recognition proteins
(isoform 1 of heterogeneous nuclear ribonucleoprotein M or
HNRNPM and Isoform 1 of heterogeneous nuclear ribonu-
cleoprotein Q SYNCRIP), leucyl-tRNA synthetase, cyto-
plasmic (or LARS), and DNA-directed RNA polymerase II
subunit RPB2 (or POLR2B). At this stage, we do not have
further evidence to establish whether these hits directly in-
teracted with our probes as well as hydroxyethylamine phar-
macophores in general, or were captured indirectly through
interactions with other binding proteins. Another plausible
explanation is that these hydroxyethylamine moieties may
also potentially interact/target other protein classes besides
aspartic proteases, and some of the hits identified herein
may be considered potential off-targets of hydroxyethyla-
mine-derived drugs in cellular environments.[13c] Collectively,
it demonstrates that our SMM-facilitated AfBP strategy
should potentially be useful in the application of large inhib-
itor libraries for comparative proteomic profiling, with the
potential for novel and disease-related biomarker identifica-
tion.
Figure 5. Pull-down experiments using the biotinylated probe cocktail
and subsequent target validation of selected “hits” by Western blotting
analysis. a) Silver-stained gel of the samples from pull-down experiment.
Asterisks show the expected locations of cathepsin D, HSP5A, and b-tu-
bulin. b) Western blotting analysis of pulled-down fractions of T47D cell
lysates treated with the biotinylated probe cocktail (or negative controls
without the cocktail; right lanes) and target validation of selected hits
with their respective antibodies. Abbreviations: SS, Silver stain; PD, posi-
tive pull-down assay; Ctrl, Neutravidin beads without probes (5%
DMSO instead).
Following LC-MS/MS analysis of the trypsin-digested
samples, all proteins were identified by setting a minimum
MOWSE (molecular weight search) score of 40 as the cut-
off threshold. In addition, proteins that appeared in the neg-
ative control pull-down experiment (beads only) were ex-
cluded as potential false positives. In order to further mini-
mize false positives in our protein list and obtain more relia-
ble putative hits, we also performed competitive pull-down
experiments in the presence of pepstatin A which is a well-
known general aspartic protease inhibitor. Briefly, the entire
positive pull-down/LC-MS experiment was repeated, with
the introduction of a pre-incubation step of T47D lysates
with pepstatin A (100 mm) for 30 min, prior to the addition
of the probe cocktail. By further subtracting the protein list
obtained from the pepstatin A-competitive pull-down ex-
periment from that of the positive pull-down experiment, a
total of 39 proteins were finally identified as highly confi-
dent putative proteins hits (Table 1). It should be noted that
all MS-based results obtained herein (including all the puta-
tive protein hits listed in Table 1) should only be used as
preliminary data. In our current study, deliberate efforts
were made to improve our pull-down/LC-MS results. Never-
theless, owing to the highly complex cellular environment
and the intrinsic limitation of affinity pull-down assay and
Conclusions
We have synthesized a new 86-member library of hydroxye-
thylamine-derived pharmacophores in this study. By com-
bining with a previously published compound library, we
have, for the first time, developed a small molecule microar-
ray (SMM) platform for facilitated screening and identifica-
tion of potential affinity-based probes (AfBPs) suitable to
be used in large-scale profiling of multiple mammalian cell
lysates. Aided by our SMM screening results, a panel of
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Proteome Profiling of Mammalian Cell Lysates
Table 1. Proteins identified by pull-down and mass spectrometry.
#
ID
Protein Description
MS
Score Peptides
matched
Family
1
2
3
4
5
6
7
8
9
IPI00011229 Cathepsin D
IPI00023598 Tubulin beta-4 chain
45.0 163
4
Aspartic protease
Tubulin family
Protein kinase
Biotin binding
Tubulin family
50
47
1497 74
801
IPI00786995 protein kinase, DNA-activated, catalytic polypeptide
IPI00299402 Pyruvate carboxylase, mitochondrial
IPI00646779 TUBB6 protein
IPI00017454 Putative tubulin-like protein alpha-4B
IPI00024993 Enoyl-CoA hydratase, mitochondrial
IPI00171903 Isoform 1 of Heterogeneous nuclear ribonucleoprotein M
IPI00020984 highly similar to Calnexin
38
21
23
18
14
22
13
22
10
19
10
6
130.3 531
50.5 531
27.8 510
31.8 412
77.7 376
71.9 289
46.3 259
34.6 244
53.3 188
53.7 187
57.5 181
24.6 180
Tubulin family
Enoyl-CoA hydratase
RNA recognition domain
Calreticulin family
Tubulin family
Ribosomal protein
Ribosomal protein L18P
Plakin or cytolinker
Heat shock protein
Helicase family.
10 IPI00641706 TUBB6 46 kDa protein
11 IPI00000494 60S ribosomal protein L5
12 IPI00014898 Isoform 1 of Plectin-1
13 IPI00037070 HSPA8 54 kDa protein
14 IPI00010796 Protein disulfide-isomerase
15 IPI00420014 Isoform 1 of U5 small nuclear ribonucleoprotein 200 kDa
helicase
6
16 IPI00292496 Tubulin beta-8 chain
17 IPI00003362 HSPA5 protein
18 IPI00003925 Isoform 1 of Pyruvate dehydrogenase E1 component subunit 39.6 172
beta,
50.3 177
72.5 175
17
10
5
Tubulin
Heat shock protein
–
19 IPI00022891 ADP/ATP translocase 1
20 IPI00029737 Isoform Long of Long-chain-fatty-acid–CoA ligase 4
33.3 167
80.2 156
9
10
Mitochondrial carrier
ATP-dependent AMP-binding enzyme
family.
21 IPI00005040 Medium-chain specific acyl-CoA dehydrogenase, mitochon-
drial
47
128
4
Acyl-CoA dehydrogenase
22 IPI00219682 Erythrocyte band 7 integral membrane protein
23 IPI00943207 Ribonucleoside-diphosphate reductase
24 IPI00103994 Leucyl-tRNA synthetase, cytoplasmic
31.9 125
81.7 125
135.6 123
5
9
5
Bad 7/mec-2 family
–
Class-I aminoacyl-tRNA synthetase
family.
25 IPI00003348 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit 38.05 107
beta-2
5
WD repeat G protein
26 IPI00027808 DNA-directed RNA polymerase II subunit RPB2
27 IPI00022314 Superoxide dismutase [Mn], mitochondrial
28 IPI00009634 Sulfide:quinone oxidoreductase, mitochondrial
29 IPI00031517 DNA replication licensing factor MCM6
30 IPI00413641 Aldose reductase
135.2 107
24.9 99
50.2 94
93.8 92
36.2 89
4
6
3
3
10
2
RNA polymerase beta chain family
iron/manganese superoxide dismutase
SQRD family.
MCM family
Aldo/keto reductase
–
31 IPI00465233 Eukaryotic translation initiation factor 3, subunit E interact- 71.1 86
ing protein
32 IPI00005158 Lon protease homolog, mitochondrial
33 IPI00021048 Isoform 1 of Myoferlin
34 IPI00009253 Alpha-soluble NSF attachment protein
35 IPI00018140 Isoform 1 of Heterogeneous nuclear ribonucleoprotein Q
36 IPI00290462 Carbonyl reductase [NADPH] 3
37 IPI00064086 Eukaryotic translation elongation factor 1 delta isoform 4
38 IPI00169383 Phosphoglycerate kinase 1
39 IPI00008986 Large neutral amino acids transporter small subunit 1
106.9 81
23.6 81
33.7 80
69.8 80
31.2 80
28.7 78
44.9 73
55.7 72
4
2
4
3
4
2
4
1
Serine protease
Ferlin family
SNAP family
RNA recognition
Dehydrogenases
EF-1-beta family
Kinase
l-type amino acid transporter
AfBPs were rapidly synthesized by click chemistry. Subse-
quent in vitro proteome profiling experiments confirmed
these probes are useful tools for interrogation of endoge-
nous aspartic proteases present in a variety of mammalian
cell lines. A ꢂcocktailꢀ screening approach was further devel-
oped to facilitate rapid and simultaneous profiling of multi-
ple cellular lysates. Large-scale affinity pull-down/LC-MS
identifications of putative protein hits enabled us to identify
both direct and indirect binding targets of the hydroxyethyl-
amine pharmacophore. Our SMM-facilitated AfBP discov-
ery strategy thus might provide a useful tool for future dis-
covery of new aspartic proteases and other potential cancer
biomarkers.
Experimental Section
Chemicals and Antibodies
Pepstatin A and plain glass slides were purchased from Sigma Aldrich
(USA), and modified to generate the corresponding avidin-coated sur-
face as previously described.[30] Recombinant cathepsin D was purchased
from Invitrogen, and monoclonal cathepsin D antibody was purchased
from Santa Cruz.
Cell Culture Conditions
HEK293T, HepG2, OVCAR-3, T47D, MDA-MB-435, HCT116, and
MCF7 cells were grown in DMEM (Invitrogen, Carlsbad, CA) containing
10% heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen),
100 UmLÀ1 penicillin, and 100 gmLÀ1 streptomycin (Thermo Scientific,
Rockford, IL) and maintained in a humidified 378C incubator with 5%
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CO2. K562 cells were grown in RPMI 1640 medium (Invitrogen Corpora-
tion) supplemented with 10% heat-inactivated fetal bovine serum,
100 UmLÀ1 penicillin, and 100 gmLÀ1 streptomycin and maintained in a
humidified 378C incubator with 5% CO2.
PBS. After washing, the beads were boiled in the elution buffer (200 mm
Tris at pH 6.8 containing 400 mm DTT and 4% SDS) for 15 min. Eluted
proteins were separated on 12% SDS-PAGE gel (together with negative
pull-down control), then visualized by coomassie blue/silver staining.
In order to identify potential cellular targets of the hydroxyethylamine
pharmacophore, pull-down experiments with a cocktail of 11 biotinylated
probes were also carried out. Fresh T47D cell lysates (~5 mg) were incu-
bated with the cocktail of 11 biotinylated probes (0.5 mm for each probe)
for 0.5 h at 48C in an acidic buffer (50 mm HEPES, pH 5.4, 1 mm EDTA,
100 mm NaCl). Similarly, the same volume of DMSO was incubated with
T47D lysates as a negative control. Subsequently the reactions were irra-
diated on ice for 25 min under UV lamp and quenched by pre-chilled
acetone (10 mL) before being placed at À208C for 30 min. The mixtures
were then centrifuged at 13,000 rpm for 10 min at 48C to precipitate the
proteins. The supernatant was discarded and the pellet was washed two
times with 10 mL of pre-chilled methanol. The protein pellets were reso-
lubilized with 0.1% SDS-PBS buffer. Next, the solutions were incubated
with Neutravidin agarose beads for 2 h at 48C. The supernatant was re-
moved by centrifugation, and the beads were washed with 0.1% SDS in
PBS once, and washed four times with PBS. After washing, the beads
were boiled in the elution buffer (200 mm Tris at pH 6.8 containing
400 mm DTT and 4% SDS) for 15 min. Eluted proteins were separated
on 12% SDS-PAGE gel (together with negative pull-down control), then
visualized by coomassie blue/silver staining.
Preparation of Cellular Lysates
Mammalian lysates were prepared as follows: fresh cell pellets were
thawed on ice by suspension in 100 mL of lysis buffer (50 mm HEPES,
pH 5.4, 1 mm EDTA, 100 mm NaCl, 10% Glycerol, 0.1% NP-40), incu-
bated for 15 min on ice, and then spun for 20 min at 13000 rpm at 48C.
The cleared supernatant was decanted to an Eppendorf tube and kept at
48C. Protein concentration was quantified by Bradford assay (Bio-Rad,
USA), aliquoted and stored in À808C, and used for subsequent screening
on array.
Small Molecule Microarray Preparation and Screening
Procedures for the construction of the SMM were based on previously
published protocols,[30] and are described in detail in the Supporting In-
formation. Cellular lysates were minimally labeled with the Cy3 dye for
1 h on ice following the manufacturerꢀs protocols. The excessive dye was
quenched with a 10-fold molar excess of hydroxylamine for a further 1 h,
then removed by buffer exchange with a Microcon centrifugal filter (Mil-
lipore, USA) or extensive dialysis at 48C overnight (Amersham, GE
Healthcare, USA). The labeled lysate was reconstituted in a final buffer
volume of 30 mL of HEPES (pH 5.4) containing 1% bovine serum albu-
min. In a standard microarray experiment, the labeled lysate (4 mg in
30 mL of HEPES) was applied under a cover slip to the array. The sam-
ples were then incubated with the array in a humidified chamber for 1 h
at RT before repeated rinses with PBST (PBS containing 0.05% of
Tween 20) with gentle shaking. Slides were scanned using an ArrayWoRx
microarray scanner installed with the relevant filters (Cy3, lex/em=548/
595 nm).
Each gel lane was cut into multiple slices. Subsequently, trypsin digestion
(using In-Gel Trypsin Digestion Kit, Pierce) and peptide extraction (with
50% acetonitrile and 1% formic acid) were carried out. The samples
were then dried in vacuo and stored at À208C. LC-MS/MS analysis was
performed using an LTQ-FT ultra mass spectrometer (Thermo Electron,
Germany) coupled with an online Shimadzu UFLC system utilizing
nanospray ionization. Peptides were first enriched with a Zorbax 300SB
C18 column (5 mmꢃ0.3 mm, Agilent Technologies), followed by elution
into an integrated nanopore column (75 mmꢃ100 mm) packed with C18
material (5 mm particle size, 300 ꢄ pore size). Mobile phase A (0.1%
formic acid in H2O) and mobile phase B (0.1% formic acid in acetoni-
trile) were used to establish the 90 min gradient, comprising 3 min of 0–
5% B, then 52 min of 5–30% B, followed by 12 min of 30–60% B; main-
tained at 80% B for 8 min before re-equilibrating at 5% B for 15 min.
Sample was injected into the MS with an electrospray potential of 1.8 kV
without sheath and auxiliary gas flow, ion transfer tube temperature of
1808C, and collision gas pressure of 0.85 mTorr. A full-survey MS scan
(350–2000 m/z range) was acquired in the 7-T FT-ICR cell at a resolution
of 100000 and a maximum ion accumulation time of 1000 ms. Precursor
ion charge-state screening was activated. The linear ion trap was used to
collect peptides where the 10 most intense ions were selected for colli-
sion-induced dissociation (CID) in MS2, performed concurrently with a
maximum ion accumulation time of 200 ms. Dynamic exclusion was acti-
vated for this process, with a repeat count of one and exclusion duration
of 30 s. For CID, the activation Q was set at 0.25, isolation width (m/z)
2.0, activation time 30 ms, and normalized collision energy 35%. The Ex-
tract-Msn (version 4.0) program found in Bioworks Browser 3.3 (Thermo
Electron, Germany) was used to extract tandem MS spectra in the data
format from the raw data of the LTQ-FT ultra. These data files were
then converted into the MASCOT generic file format using an in-house
program. Intensity values and fragment ion m/z ratios were not manipu-
lated. These data were used to obtain protein identities by searching
against the corresponding database by means of an in-house MASCOT
server (version 2.2.03, Matrix Science, Boston, MA). The search was lim-
ited to a maximum of two missed trypsin cleavages, #13C of 2, mass toler-
ances of 10 ppm for peptide precursors and 0.8 Da for fragment ions.
Only proteins with a MOWSE score higher than 40, corresponding to p<
0.05, were considered significant. The peptide/protein lists obtained were
exported as an html file.
Probe Labeling and In-gel Fluorescence Scanning
For labeling of individual probes, lysates from mammalian cells (~50 mg
of total proteins as determined by Bradford assay) were incubated with
the TER probe (1 mm final concentration; 5% DMSO) in HEPES buffer
(50 mm HEPES, pH 5.4, 1 mm EDTA, 100 mm NaCl) for 30 min at RT,
then irradiated on ice for 25 min using a B100 A lamp (UVP) at a dis-
tance of 5 cm. The reactions were then resuspended in 50 mL 1ꢃSDS-
loading buffer followed by heating for 10 min at 958C; around 20 mg of
protein was loaded per gel lane for separation by SDS-PAGE (12% SDS
gel), then visualized by in-gel fluorescence scanning using a Typhoon
9410 Variable Mode Imager scanner. For cocktail labeling, lysates from
mammalian cells (~50 mg of total proteins) were incubated with a cock-
tail of 11 TER probes (0.5 mm of each probe, 5% DMSO) in HEPES
buffer (50 mm HEPES, pH 5.4, 1 mm EDTA, 100 mm NaCl) for 30 min at
RT, then irradiated on ice for 25 min using a B100 A lamp (UVP) at a
distance of 5 cm. The reaction was quenched by addition of pre-chilled
acetone (0.5 mL), then placed at À208C for 30 min. Upon centrifugation
at 13,000 rpm for 10 min at 48C to precipitate proteins, the supernatant
was discarded and the resulting pellet was washed two times with 200 mL
of pre-chilled methanol, then resuspended in 50 mL 1ꢃSDS-loading
buffer followed by heating for 10 min at 958C; around 20 mg of protein
was loaded per gel lane for separation by SDS-PAGE (12% SDS gel),
then visualized by in-gel fluorescence.
Affinity Pull Down and Mass Spectrometric Target Identification
Two sets of pull-down assays were carried out in this study. For pull
down with an individual probe, biotinylated probe T2 (10 mm final con-
centration) was incubated with fresh T47D cell lysates (~5 mg) for 0.5 h
at 48C in an acidic buffer (50 mm HEPES, pH 5.4, 1 mm EDTA, 100 mm
NaCl). Meanwhile, the same volume of DMSO was incubated with T47D
cell lysates as the negative control experiment. Subsequently, the samples
were irradiated on ice for 25 min under UV lamp. The resulting mixtures
were incubated with Neutravidin agarose beads for further 2 h at 48C.
After that, the supernatant was removed by centrifugation, and the beads
were washed with 0.1% SDS in PBS once, and washed four times with
For immunoblotting experiments, samples from pull-down experiments
were resolved by SDS-PAGE gel and transferred to polyvinylidene fluo-
ride membranes. Membranes were then blocked with 3% BSA in TBST
(0.05% Tween in TBS) for 1 h at RT. After blocking, membranes were
incubated with cathepsin D primary antibody (1:500), HSP5A primary
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Proteome Profiling of Mammalian Cell Lysates
antibody (1:500), and b-tubulin (1:500) for 1 h at RT, respectively. The
membranes were then washed four times with TBST (10 min/time), and
incubated with the respective secondary antibody. After 1 h incubation at
RT, blots were washed again with TBST before the development with Su-
perSignal West Dura Kit (Thermo Scientific).
trated under reduced pressure. Purification by silica-gel chromatography
with 80:20 hexanes/EtOAc afforded compound 8d as white solid. Yield=
1
77%. H NMR (300 MHz, CDCl3): d=1.33 (s, 9H), 2.51 (dd, J=5.07 Hz,
1H), 2.79 (d, J=5.66 Hz, 1H), 2.90–3.14 (m, 2H), 3.52–3.59 (m, 1H),
3.63–3.75 (m, 2H), 6.96 (d, J=8.40 Hz, 2H), 7.17 ppm (d, J=6.60 Hz,
1H); 13C NMR (75 MHz, CDCl3): d=154.3, 146.9, 131.8–131.5(m), 129.8,
Chemical Synthesis
124.5, 72.2, 71.9, 69.2, 65.0, 37.3, 36.4, 28.8 ppm. LC-MS: m/z [M+H]+
=
327.350.
All chemicals were purchased at the highest grade available from com-
mercial vendors and used without further purification, unless otherwise
noted. All reactions were carried out under N2 atmosphere with HPLC-
grade solvents, unless otherwise stated. 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 13C NMR spectra were recorded on a Bruker
Avance 300 MHz, or Bruker model DPX-500 MHz NMR spectrometer.
Chemical shifts are reported in parts per million relative to internal stan-
Immobilization of 4ACTHNUTRGNEUGN(d–f) onto DHP Resin
According to previously published procedures,[9c] HM DHP resin (40 mg)
was first swelled in DCE (5 mL) for 2 h. The solvent was then removed
and 4ACHTNUTRGNEUNG(d–f) (4.0 eq) and a catalytic amount of PPTS (pyridinium p-tolue-
nesulfonate) (1.5 eq) in DCE (10 mL) were added at RT. The reaction
was then stirred for 12 h at 608C. The resulting resin was washed with
NMP (3ꢃ), THF (3ꢃ), CH2Cl2 (3ꢃ), and Et2O (3ꢃ) and dried in vacuo,
then stored at À208C.
dard tetramethylsilane (SiACHTNUTRGNEUNG(CH3)4 =0.00 ppm) or residual solvent peaks
(CHCl3 =7.26 ppm). Mass spectra were obtained at the Center for Chem-
ical Characterization and Analysis at NUS. Analytical HPLC was carried
out on Shimadzu LC-IT-TOF or Shimadzu LC-ESI system equipped with
an autosampler, using reverse-phase Phenomenex Luna 5 mm C18 100 ꢄ
50ꢃ3.0 mm columns. TFA/H2O (0.1%) and TFA/acetonitrile (0.1%)
were used as eluents for all HPLC experiments.
Deprotection of Fmoc Group
The resulting resin was suspended in a solution of 20% piperidine in
NMP and shaken for 2 h at RT. Upon solvent removal by filtration, the
resin was washed with NMP (3ꢃ), THF (3ꢃ), CH2Cl2 (3ꢃ), and Et2O
(3ꢃ), then dried under reduced pressure. The progress of the reaction
was monitored by the ninhydrin test. Blue beads indicate the presence of
primary amine.
Synthesis of a-Azido alcohols 4ACTHNUTRGENUG(N d–f)
a-Azido ketones 3 (5 mmol) were dissolved in 20 mL of THF followed
by addition of sodium borohydride (6 mmol) in 2 mL H2O at 08C. The
reaction mixture was then stirred for 1 h and neutralized with aqueous
1 n HCl. After extraction, the organic layers were washed with brine and
dried over anhydrous Na2SO4, and concentrated under reduced pressure.
Purification by silica-gel chromatography with 85:15 hexane/EtOAc af-
Coupling Reactions for Acids and Sulfonyl Chlorides
The resulting resin firstly was swelled in dry THF for 2 h, then the sol-
vent was removed. To the resin was added a preactived solution contain-
ing the corresponding acid (4.0 eq), PyBOP (4.0 eq), HOAt (4.0 eq), and
DIPEA (8.0 eq) in THF or a solution of the corresponding sulfonyl chlo-
ride (4.0 eq) and DIEA (8.0 eq) in dry THF. The reaction mixtures were
shaken overnight at RT and the resin was finally washed with NMP (3ꢃ),
THF (3ꢃ), CH2Cl2 (3ꢃ), and Et2O (3ꢃ) before being dried under re-
duced pressure. The completeness of the reaction was monitored by the
ninhydrin test. A negative test indicated the absence of primary amine
and the completeness of the reaction.
forded a-azido alcohol 4ACHTUNGTRENNUNG(d–f) in 50–70%.
N-a-Fmoc-isopropylalanylazidoalcohol (4d)
Yield=79%. 1H NMR (500 MHz, CDCl3): d=0.72 (d, J=9.55 Hz, 6H),
2.58 (d, J=5.65 Hz, 1H), 3.19–3.41 (m, 2H), 3.53–3.58 (m, 1H), 3.67 (s,
1H), 4.49 (t, J=5.98 Hz, 1H), 4.59–4.64 (m, 2H), 7.33 (t, J=7.57 Hz,
2H), 7.40 (t, J=3.78 Hz, 2H), 7.59 (t, J=5.98 Hz, 2H), 7.77 ppm (d, J=
7.55 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=157.1, 143.7, 141.5, 127.7,
127.1, 124.9, 120.0, 71.2, 66.3, 58.6, 54.4, 47.5, 28.0, 20.0 ppm; LC-MS: m/
z [M+Na]+ =403.154.
Immobilization of 8d onto DHP Resin
HM DHP resin (40 mg) was swelled in DCE (5 mL) for 2 h. The solvent
was then removed and 8d (4.0 eq) and a catalytic amount of PPTS
(1.5 eq) in DCE (10 mL) were added at RT. The reaction was then stirred
for 12 h at 608C. The resulting resin was washed with NMP (3ꢃ), THF
(3ꢃ), CH2Cl2 (3ꢃ), and Et2O (3ꢃ) and dried in vacuo, then stored at
À208C.
N-a-Fmoc-4-tert-butoxy-phenylalanylazidoalcohol (4e)
1
Yield=90%. H NMR (500 MHz, CDCl3): d=1.30 (s, 9H), 2.78–2.84 (m,
2H), 3.25–3.32 (m, 2H), 3.72–3.78 (m, 2H), 4.11–4.17 (m, 1H), 4.39–4.45
(m, 2H), 4.79 (d, J=6.95 Hz, 1H), 5.08 (d, J=8.85 Hz, 1H), 6.88 (d, J=
7.55 Hz, 2H), 7.02–7.07 (m, 2H), 7.30 (t, J=7.23 Hz, 2H), 7.39 (t, J=
7.25 Hz, 2H), 7.51 ppm (t, J=7.55 Hz, 2H), 13C NMR (125 MHz,
CDCl3): d=156.4, 154.2, 143.6, 141.4, 132.2–124.8(m), 124.2, 119.9, 78.4,
Amine Alkylation and Coupling Reactions for C-Terminal Library
Support-bound bromomethyl alcohols were added to a vial, followed by
addition of a solution of iso-butylamine (10 eq) in NMP (10 mL). The
vial was then sealed and the reaction mixture was heated at 808C for
36 h. The resin was subsequently washed with NMP (3ꢃ), THF (3ꢃ),
CH2Cl2 (3ꢃ), and ether (3ꢃ), then dried in vacuo. Acylation with the sul-
fonyl chloride (4.0 eq) and DIEA (8.0 eq) in dry THF was carried out
overnight. The resin was then washed with NMP (3ꢃ), THF (3ꢃ),
CH2Cl2 (3ꢃ), and ether (3ꢃ), then dried under reduced pressure.
72.1, 66.5, 54.9, 54.2, 47.3, 35.2, 28.8 ppm; LC-MS: m/z [M+Na]+
=
523.207.
N-a-Fmoc-4-Boc-butylalanylazidoalcohol (4 f)
1
Yield=88%. H NMR (500 MHz, CDCl3): d=1.23–1.28 (m, 4H), 1.45 (s,
9H), 3.00–3.09 (m, 2H), 3.24–3.28 (m, 2H), 3.48–3.75 (m, 2H), 4.19 (t,
J=6.3 Hz, 1H), 4.42–4.61 (m, 2H), 5.01 (s, 1H), 5.13 (d, J=8.85 Hz,
1H), 7.31 (t, J=7.55 Hz, 2H), 7.40 (t, J=7.55 Hz, 2H), 7.58 (d, J=
6.95 Hz, 2H), 7.76 ppm (d, J=7.55 Hz, 2H); 13C NMR (125 MHz,
CDCl3): d=156.5, 143.7, 141.3, 127.7, 127.0, 124.9, 119.9, 79.3, 73.1, 66.4,
Reduction of Azido Group to Primary Amine
Reduction of the azide was accomplished using 0.2m SnCl2, 0.8m PhSH,
and 1.0m Et3N in THF (1 mL) for 4 h. The resin was then washed with
50% (vol) of aqueous THF solution (3ꢃ), THF (3ꢃ), CH2Cl2 (3ꢃ), and
ether (3ꢃ), then dried in vacuo. The reaction was monitored by using the
ninhydrin test. If primary amine is present, the beads will become blue
after treatment with ninhydrin reagents.
60.4, 53.9, 47.4, 39.5, 31.3, 29.5, 22.4, 20.9 ppm; LC-MS: m/z [M+H]+
=
410.205.
a-Azido bromomethyl-TyrACTHNUGTRNEUNG(tBu)-alcohol (8d)
To the solution of a-azido bromomethyl ketones 7 (8 mmol, 1 eq) in
20 mL of 95:5 THF/H2O, sodium borohydride (12 mmol. 1.5 eq) was
added gradually at 08C. The reaction mixture was stirred for 1 h and
then neutralized with aqueous 1 n HCl. After extraction with EtOAc, the
organic extracts were washed with brine, dried with Na2SO4, and concen-
Acylation of Primary Amine with Long-Chain Biotin Acids
The above resulting support-bound amine was then acylated in a preac-
tived solution of biotin acid (4.0 eq), PyBOP (4.0 eq), HOAt (4.0 eq),
and DIPEA (8.0 eq) in THF. The reaction mixture was shaken overnight
Chem. Asian J. 2011, 6, 2803 – 2815
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2813

S. Q. Yao et al.
FULL PAPERS
at RT, and the resins were washed with NMP (3ꢃ), THF (3ꢃ), CH2Cl2
(3ꢃ), and Et2O (3ꢃ) and dried under vacuum. The completeness of the
reaction was monitored by the ninhydrin test. If the coupling reaction is
completed, the beads will exhibit no color change after treatment with
ninhydrin reagents.
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1H NMR (300 MHz, [D6]DMSO): d=0.54 (d, J=7.02 Hz, 6H), 0.66 (d,
J=6.15 Hz, 2H), 1.04–1.19 (m, 4H), 1.23–1.41 (m, 10H), 1.86–2.03 (m,
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Acknowledgements
This work was supported by the Competitive Research Program (R143-
000-218-281; NRF-G-CRP 2007-04) from the National Research Founda-
tion (NRF) of Singapore, the Agency for Science, Technology and Re-
search (R-143-000-391-305) and the Ministry of Education (R-143-000-
394-112). We thank Jigang Wang for technical assistance and Dr. Siu
Kwan Sze (Nanyang Technological University, Singapore) for advice and
support for the LC-MS experiments.
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Received: June 8, 2011
Published online: September 2, 2011
Chem. Asian J. 2011, 6, 2803 – 2815
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2815