Design and synthesis of an affinity probe that targets caspases in proteomic experiments

Article abstract of DOI:10.1016/S0040-4039(02)02724-7

The field of proteomics aims to study all proteins in the human proteome. This huge task may be accelerated by using active-site directed probes which profile proteins in an activity-dependent manner. Herein, we have developed a fluorescently-labeled affinity probe containing chemical reactivity specific towards caspases. Preliminary assays and proof-of-concept experiments demonstrated that this probe exhibits strong chemical reactivity towards caspase-1 over other enzymes, capable of covalently labeling caspapse-1 over other non-caspase enzymes. This thus demonstrates its selectivity and potential in high-throughput screenings of other unknown caspases in a large-scale proteomics experiment.

Full text of DOI:10.1016/S0040-4039(02)02724-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1043–1046
Design and synthesis of an affinity probe that targets caspases in
proteomic experiments
Ming-Lee Liau,a Resmi C. Panickera and Shao Q. Yaoa,b,
*
aDepartment of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
bDepartment of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
Received 12 September 2002; revised 18 November 2002; accepted 29 November 2002
Abstract—The field of proteomics aims to study all proteins in the human proteome. This huge task may be accelerated by using
active-site directed probes which profile proteins in an activity-dependent manner. Herein, we have developed a fluorescently-
labeled affinity probe containing chemical reactivity specific towards caspases. Preliminary assays and proof-of-concept experi-
ments demonstrated that this probe exhibits strong chemical reactivity towards caspase-1 over other enzymes, capable of
covalently labeling caspapse-1 over other non-caspase enzymes. This thus demonstrates its selectivity and potential in high-
throughput screenings of other unknown caspases in a large-scale proteomics experiment. © 2003 Elsevier Science Ltd. All rights
reserved.
The completion of the Human Genome Project has
opened up new and exciting possibilities in the under-
standing of the workings of the cell and diseases. How-
ever, genes alone do not tell the whole story of cellular
functions. Proteins are ultimately responsible for most
processes that take place within the cell. Hence, the
onus is now on scientists, equipped with the sequence
of 30 000–40 000 genes,1 to elucidate the human pro-
teome. Only then can the wealth of information gleaned
from the genome project be translated into tangible
benefits in medicine. Researchers expect the 30 000–
40 000 genes in humans to produce more than 100 000
different proteins in each cell.
To help cope with the enormity of the task, rapid
technological advances in the techniques and methods
involved in proteomic studies would be necessary.
High-throughput screening methods and automation
would enable scientists to analyze proteins on a global
scale quickly and efficiently. Already researchers are
developing new methods to simplify protein analysis
and allow high-throughput screenings.2–6 The activity-
based, affinity tag approach can potentially filter out
proteins that are not of interest and focus on certain
classes of proteins (e.g. based on activity),2 while
protein chips and microarrays offer the attractive
prospect of screening thousands of proteins simulta-
neously.3–6 Unknown proteins could be spotted on
arrays and incubated with potential ligands, which bind
and allow the detection of the target proteins. The
whole process of separating and identifying proteins in
a complex mixture hence may be simplified, by zooming
in only onto specific target proteins.2
Figure 1. Structure of a typical tetrapeptide fmk inhibitor of
caspase and the generalized probe used in target caspases.
Caspases are enzymes which play a key mediating role
in apoptosis, or programmed cell death. All members of
this protease family are characterized by their unique
requirement for an aspartic acid at the P1 position of
the substrate cleavage site. Insufficient caspase activity
promotes cell accumulation implicated in cancer and
Note that amino acids are (
indicated.
L)-configured unless otherwise
* Corresponding author. Tel.: +0065-68741683; fax: +0065-67791691;
e-mail: chmyaosq@nus.edu.sg
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02724-7

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M.-L. Liau et al. / Tetrahedron Letters 44 (2003) 1043–1046
autoimmune diseases, while excessive caspase activity
could lead to accelerated cell death, believed to result in
neurodegenerative disorders such as Huntington’s and
Alzheimer’s diseases.7–9 Given their pivotal role, cas-
pases are attractive therapeutic targets for treatment of
these conditions. It is hence in our interest to discover
new caspases, and to design therapeutic drugs to target
caspases and their regulation.
capable of detecting caspases over other non-caspase
enzymes, a linker unit consisting of a simple alkyl
chain, as shown in Figure 1, was chosen, which is
approximately the length of the P2–P4 tripeptide
sequence in a typical caspase inhibitor and, at the same
time, does not impose any specificity upon a particular
caspase. Other linkers may be chosen when needed to
fine-tune the specificity of the probes against their
targeting enzymes.
Herein, we aim to utilize the affinity tag approach2 to
focus on specific classes of proteins, one class at a time.
The ultimate goal is to develop fluorescently-labeled
affinity probes containing chemical reactivity specific
towards different classes of proteins, such as kinases,
serine proteases and caspases. These probes, combined
with standard gel-based proteomic experiments, or with
newly developed protein array-based strategy,10 may
serve as a high-throughput tool to study the human
proteome.
Initial attempted synthesis of the probe indicated that
the fmk group in the probe is a potential complication
as the presence of fluorine increases the electrophilicity
of the carbonyl carbon.15 Together with its acidic a-
proton, the fmk is rather susceptible to reactions
involving bases and nucleophiles, which can lead to
dimerization, epimerization and Schiff base reactions.16
Therefore, a synthetic route (Scheme 1) was devised, in
which reactions involving the use of potential nucle-
ophiles and strong bases were intentionally avoided.
Strategically, the oxidation of the alcohol to generate
the fmk group was left to the final steps. Briefly, the
t-butyl ester of 3-nitro-propionic acid, 2, was generated
from 1 using the classical isobutene/concentrated acid
approach.17 Fluoroacetaldehyde, generated in situ by
oxidation of fluoroethanol under Swern Conditions,18
was then added to 2 to yield the key intermediate 3 via
a nitro-aldol reaction.19 Hydrogenation19 of 3 over
freshly prepared Raney nickel catalyst20 gave amino-
alcohol 4 in 73% yield. The amino-alcohol was reacted
with succinic anhydride to give the acid 5. The singly
Boc-protected ethylenediamine, prepared as previously
reported,21 was coupled to 5 using standard coupling
reagents. Swern oxidation of the resulting compound 6
yielded 7, which was subsequently treated with 50%
TFA to give 8. Conjugation of the dye molecule, Cy3,
to the probe was carried out by coupling 8 with the
NHS ester of Cy3, which was synthesized from Cy3
using standard coupling methods.22 Overall, the probe 9
was obtained in eight steps with yield of ꢀ10%.23
The general structure of our affinity probes consist of
three units—the reactive unit which may be fine tuned
to target different classes of proteins based on their
enzymatic activity, the linker unit and the fluorescence
unit which facilitates the detection of proteins upon
labeling with the probe. In this report, we have success-
fully developed a fluorescently-labeled affinity probe
capable of selective labeling of caspases. Fluo-
romethylketone (fmk)-containing tetrapeptides (Fig. 1)
are well-documented examples known to covalently
react with the active site residues in many cysteine
proteases.11,12 It was therefore chosen as the elec-
trophilic moiety in the Asp-mimic reactive unit in our
probe structure (Fig. 1). Other classes of cysteine
proteases may also be targeted by simply replacing the
Asp portion with other structures. Cyanine dye, Cy3,
has many outstanding properties, and was thus chosen
as the dye unit.13,14 The linker unit decides the probe’s
specificity towards individual caspases. Since our goal
in this work was to design a general caspase probe
Scheme 1.

M.-L. Liau et al. / Tetrahedron Letters 44 (2003) 1043–1046
1045
Figure 2. List of enzymes (Sigma Catalog c). (a): (1) a-chymotrypsin (C-4129), (2) b-chymotrypsin (C-4629), (3) g-chymotrypsin
(C-4754), (4) chymotrypsinogen (C-4879), (5) pepsin (P-6887), (6) proteinase K (P-6556), (7) subtilisin (P-5380), (8) trypsin
inhibitor (T-9378), (9) trypsinogen (T-1143), (10) trypsin (T-1426), (11) thrombin (T-4648), (12) BSA (A-2512), (13) lysozyme
(L-6876), (14) phosphatase, acid (P-3627), (15) phosphatase, alkaline (P-7640), (16) phosphatase, alkaline (P-2265), (17)
phosphatase, alkaline (P-4002), (18) lipase (L-1754), (19) lipase (L-3001), (20) lipase (L-9031), (21) protease (P-6911), (22)
phosphatase, acid (P-3752). (b): (23) Caspase-1, (24) papain (P-4762), (25) chymopapain (C-8526), (26) bromelain (B-4882).
pase-1 is a prerequisite for the labeling reaction to
occur.
Next, the probe was tested for selective labeling of
proteins based on their enzymatic activity. A panel of
22 commercially available proteins was used, none of
which were caspases. Upon incubation with the probe
for 30 min under appropriate conditions,24 the proteins
were first separated using SDS-PAGE (Fig. 2a), fol-
lowed by detection of fluorescence labeling using a
fluorescence-based gel scanner. None of these proteins
gave any significant fluorescent bands, except for BSA,
which is known to bind to many molecules non-
specifically.
In conclusion, we have successfully designed, synthe-
sized, and tested a fluorescent, small molecule probe
capable of labeling caspases in a highly specific, activ-
ity-based fashion. Our studies clearly demonstrated the
potential of this probe in the selective detection of
caspases. The selectivity of this probe towards caspase-
1, over non-cysteine protease enzymes, as well as other
cysteine proteases, might enable the selective detection
of caspase-1 in a complex protein sample. Subsequent
experiments with this probe would involve other cas-
pase members as well as in vivo samples to further
verify the versatility of the probe. Furthermore, our
probe may be readily modified to target other classes of
cysteine protease, or a specific cysteine protease.
We next investigated the selective labeling of the probe
against its targeting enzyme, caspase. Caspase-1 was
incubated with probe for differing lengths of time and
the resulting labeled reaction was analyzed by SDS-
PAGE and fluorescence detection. In order to unam-
biguously confirm the selectivity of the probe against
ONLY caspases, three other non-caspase cysteine
proteases were used as controls and analyzed simulta-
neously. As shown in Figure 2b, only caspase-1 was
selectively labeled by the probe following 0.5 h incuba-
tion. No labeling was observed for the other three
non-caspase cysteine proteases, indicating the high spe-
cificity of this probe for the detection of caspase activ-
ity. As the incubation time of the labeling reaction
increases (3.5 h), other non-caspase cysteine proteases
started to react with the probe, as evidenced by appear-
ance of some fluorescence bands on the gel. However, a
comparatively much stronger band was still observed
for the caspase reaction. In order to further confirm
that the fluorescence labeling of caspase-1 by the probe
is due to its enzymatic activity, the enzyme was first
inactivated by heat, then treated with the probe fol-
lowed by SDS-PAGE analysis. No labeling of caspase-1
was observed, indicating the enzymatic activity of cas-
Acknowledgements
Funding support from the National University of Sin-
gapore (NUS) and the Agency for Science, Technology
and Research (A*STAR) of Singapore are acknowl-
edged. S.Q.Y. is the recipient of the 2002 Young Inves-
tigator Award (YIA) from the Biomedical Research
Council (BMRC).
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