"Click" synthesis of small molecule probes for activity-based fingerprinting of matrix metalloproteases

Article abstract of DOI:10.1039/b609446e

By using "Click Chemistry", we achieved the facile synthesis of various affinity-based hydroxamate probes that enable generation of activity-based fingerprints of a variety of metalloproteases, including matrix metalloproteases (MMPs), in proteomics experiments. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b609446e

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COMMUNICATION
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‘‘Click’’ synthesis of small molecule probes for activity-based
fingerprinting of matrix metalloproteases{
Jun Wang,a Mahesh Uttamchandani,b Junqi Li,a Mingyu Hua and Shao Q. Yao*abc
Received (in Cambridge, UK) 4th July 2006, Accepted 2nd August 2006
First published as an Advance Article on the web 15th August 2006
DOI: 10.1039/b609446e
affinity, but more importantly specificity, of the enzyme/probe
By using ‘‘Click Chemistry’’, we achieved the facile synthesis of
various affinity-based hydroxamate probes that enable genera-
tion of activity-based fingerprints of a variety of metallopro-
teases, including matrix metalloproteases (MMPs), in
proteomics experiments.
complex.5 Most activity-based profiling experiments have been
routinely carrried out in gel-based experiments. Recent extension
of ABP into a protein microarray has promised even higher
throughput, as well as miniaturization, in future enzyme assays.5,6
However, no report has thus far documented the application of
affinity-based probes (such as ones that target MMPs4) on a
protein microarray.
Enzymes play a key role in virtually every biological process. They
have long been considered valuable drug targets for potential
treatments of major human diseases. Matrix metalloproteases
(MMPs), for example, are a family of zinc-containing proteases
which represent a family of at least 23 members in humans alone,
and have become attractive targets for drug discovery because of
their implication in diseases such as arthritis, Alzheimer’s disease,
cancer, and heart disease.1 Despite their well-documented pro-
tumorigenic actions, only three MMPs, namely MMP-1, -2 and -7,
have thus far been experimentally validated as potential cancer
targets. Another three (i.e. MMP-3, -8 and -9) have recently been
classified as antitargets due to the key role they play in normal
tissue homeostasis. With the precise biological functions of other
human MMPs remaining largely unknown, the development of
novel chemical and biological methods capable of high-throughput
identification and characterization of MMPs has become increas-
ingly urgent. Activity-based profiling (ABP), originally developed
by Cravatt et al.,2 is one such technique that has recently been
adapted for the study of metalloproteases including MMPs.3 ABP
works by selectively targeting enzymes of choice from a crude
proteome, in an activity-based manner, using the so-called activity-
and affinity-based small molecule probes. In the case of MMPs,
small molecule probes possessing a) a hydroxamic acid recognition
moiety known to chelate to the active-site zinc of MMPs (as well
as other zinc-containing proteins such as thermolysin and
collagenase), b) a photolabile group (usually benzophenone or
diazirine) capable of covalent crosslinking to the target enzyme,
and c) a fluorescent/affinity tag for easy visualization/isolation of
the cross-linked enzyme, have been successfully documented.4
Furthermore, in cases where a repertoire of probes having different
recognition elements are available, one can obtain the ‘‘activity-
based fingerprint’’ of the target enzyme, which reveals not only
As shown in Scheme 1, our previously reported first-generation
MMP probes were peptides containing a C-terminal hydroxamic
acid.4b Despite simplicity in their chemical synthesis, these so-called
‘‘left-handed’’ probes suffer a major drawback in that they bind to
MMPs with relatively low affinity, making them less suitable for
sensitive detection of MMPs from a complex proteome.1,2 In this
Communication, we report 1) the synthesis of second-generation
‘‘right-handed’’ probes aided by ‘‘click chemistry’’, 2) their
application in gel-based activity-based fingerprinting of numerous
metalloproteases (including MMPs), and 3) the related preliminary
microarray-based experiments which, for the first time, demon-
strate that affinity-based probes are indeed compatible with
protein microarrays for potential enzyme profiling experiments.
‘‘Click Chemistry’’ is a concept originally introduced by
Sharpless et al. which refers to several classes of chemical
transformations that share a number of important properties
including very high reaction efficiency (in both conversion and
selectivity) under mild conditions, and a simple workup.7 The
Cu(I)-catalyzed 1,3-cycloaddition between an azide and an alkyne
is one such reaction that has recently been explored in the
discovery of drugs and materials. Herein, we take advantage of the
‘‘click chemistry’’ between an alkyne-derivatized succinyl hydro-
xamate warhead and an azide-containing trifunctional tag for the
highly modular and facile synthesis of a total of 12 different
activity-based probes against MMPs, which are otherwise
challenging to make synthetically (Scheme 2).
aDepartment of Chemistry, National University of Singapore, 3 Science
Drive 3, Singapore 117543. E-mail: chmyaosq@nus.edu.sg;
Fax: (+65) 67791691; Tel: (+65) 68741683
bDepartment of Biological Sciences, National University of Singapore,
Singapore 117543
cNUS MedChem Program of the Office of Life Sciences, National
University of Singapore, Singapore 117543
{ Electronic supplementary information (ESI) available: Procedures for
the synthesis and characterization of inhibitors, microarray and microplate
experiments, as well as kinetic experiments and computer modeling. See
DOI: 10.1039/b609446e
Scheme 1 General structures of the 1st and 2nd generation MMP
probes.
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Scheme 2 General structures of the 12 MMP probes used in this work. Val (A); Leu (B); Ile (C); Phe (D); Long-Phe (E); Cyclohexyl (F); Cyclopentyl (G);
O–Ph (H); Long-OH (I); Lys (J); Asp (K); Sulfone (L).
It is noted that, while our manuscript was in preparation,
Cravatt et al. reported a similar MMP-targeting, succinyl
hydroxamate library, in which ‘‘click chemistry’’ was used to
facilitate cell permeability, rather than chemical synthesis, of the
probes per se.8
oxazolidinone auxiliary, with different side chains being introduced
from their corresponding alkyl bromides. The hydroxamate was
protected with a trityl group, ensuring its compatibility with
standard Fmoc peptide chemistry/TFA cleavage procedures. The
four warheads were synthesized in 5–8 steps from readily available
starting materials, with good to excellent yields in almost all steps.
Of the twelve succinyl hydroxamates, eight contain various alkyl
and aromatic hydrophobic side chains (A to H in Scheme 2). They
were synthesized based on previously reported procedures,9 details
of which will be published elsewhere. The remaining four
hydroxamates (I to L) introduce side chains possessing acidic
and basic groups, as well as hydrophilic groups with the property
of hydrogen-bonding, at the P19 position, thus are able to form
favorable electrostatic interactions with different S19 binding
pockets of MMPs. The four corresponding precursors, in suitably
protected form as shown in Fig. 1, were accordingly prepared. The
acid-labile protecting groups in 1–4 were chosen such that the
warheads are compatible with standard solid-phase Fmoc
chemistry, allowing them to be used in future for large-scale
synthesis of probe libraries.
With the sulfone-containing warhead, i.e. 4,
a base-labile
protection group was avoided in order to minimize the risk of
b-elimination side reaction caused by the release of the sulfone.
With all twelve warheads in hand, propargylamine was subse-
quently used to install the alkyne handle. Prior to or upon TFA
treatments, the resulting warheads (in either protected or free form,
respectively) were assembled to the azide-containing trifunctional
molecule (which contains benzophenone as the photolabile group
and rhodamine as the fluorescent tag; see ESI) using ‘‘click
chemistry’’, furnishing the twelve desired MMP probes as shown
in Scheme 2. The robustness of ‘‘click chemistry’’ was once again
proven to be the key to successful synthesis of our final probes, in
that all twelve probes were quantitatively assembled with no trace
of side products.
Detailed synthesis of the above four warheads is described in the
ESI. We took into consideration that the synthetic strategy needs
to be general, enabling the facile incorporation of a wide variety of
P19 side chains and, when necessary, with precise stereospecific
control over the chiral center located in the warhead. This was
accomplished by standard enolate chemistry coupled with Evans’
We next sought to fingerprint different metalloproteases
including MMPs with our probes. Protein fingerprinting, as its
name suggests, is a distinctive pattern generated against a panel of
focused small molecule probes, which reflects the protein’s catalytic
activity or binding property. Since our probes were designed based
on known inhibitors of metalloproteases, the resulting inhibitor-
based fingerprint not only offers invaluable information for
decoding the enzymes’ physiological roles, but also facilitates the
discovery of potent and selective inhibitors as potential drugs. A
diverse group of different classes of metalloproteases were chosen
in our experiment, such that they highlight the potential of the
strategy not only in distinguishing between both close and distant
members, but also in potential identification and characterization
of various disease-related enzymes, i.e. MMPs and anthrax lethal
factor. In addition, carbonic anhydrase, a well-known zinc-binding
protein (but not a metalloprotease) was also tested. Fig. 2 displays
the results obtained using gel-based fingerprinting with the probe
library. Notably, we were able to produce distinct and reprodu-
cible fingerprints for each of these proteins, thus providing a
unique capability of identifying and classifying these proteins
Fig. 1 Structures of four new hydrophilic succinyl warheads.
3784 | Chem. Commun., 2006, 3783–3785
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Fig. 3 Protein microarray of various metalloenzymes screened by the
Leu probe. Five different proteins were spotted in triplicate: 1. carbonic
anhydrase (300 mg/ml); 2. collagenase (300 mg/ml); 3. thermolysin
(300 mg/ml); 4. anthrax LF (6 mg/ml); 5. b-chymotrypsin (300 mg/ml).
zinc-binding enzyme), but not with b-chymotrypsin. Despite
several attempts, we were unable to detect the fluorescence
labeling of anthrax LF, as well as several MMPs (data not shown),
on the microarray. As these proteins were only available from
commercial sources in very low stock concentrations, we attributed
our failure to the less-than-optimal immobilization of the proteins.
Work is in progress to confirm this by recombinantly expressing
these proteins in sufficient quantity/concentration for protein
microarray fabrication, and results will be reported in due course.
In conclusion, we have used ‘‘click chemistry’’ to successfully
synthesize a second-generation library of metalloprotease probes
containing succinyl warheads with a variety of P19 functionalities.
With these probes, we have been able to generate unique activity-
based fingerprints against various metalloproteases including
MMPs and other therapeutically important enzymes such as
anthrax LF. Such fingerprinting strategies may lead to future
identification and characterization of new MMPs, and the
development of potential potent and selective inhibitors. We have
also for the first time shown that affinity-based probes may be
equally amenable for high-throughput screening of metallopro-
tease activities in a protein microarray.
Fig. 2 Fingerprints of 12 probes against 7 metalloenzymes. Strongest
relative labeling is visualized in red according to the scale shown. The
fingerprints were further hierarchically clustered according to their labeling
profiles (see ESI).
according to their labeling profiles. Generally the Lys and Ile
probes showed the greatest degree of labeling and seemed to
strongly label nearly all the enzymes tested. The other probes were
however more discerning in their labeling patterns. It was observed
that the strongest labeling for MMP-3 was that of Long-OH and
Lys probes. This agrees well with the known long hydrophobic
pocket of MMP-3 that has been previously reported to bind
designed inhibitors with such long hydrophilic scaffolds.1 MT-1
MMP also shares a similar long pocket to MMP-3 and is observed
to possess greater affinity to the Long-Phe as well as the Lys probe
relative to the other probe scaffolds. The short S19 pocket of
MMP-7 was shown to accommodate both the Val and Phe probes.
The Asp and Sulfone probes showed the weakest labeling with
most of the enzymes, indicating these moieties are generally
unfavored for most of the metalloproteases tested. Overall the
fingerprints enabled different enzymes to be classified according to
their similarity. MMP-3 gave a distinct profile compared to the
other enzymes screened. The labeling pattern of carbonic
anhydrase was similar to that of MMP-7. Both anthrax lethal
factor and MT-1 MMP show strong selective labeling with one of
the probe library set, namely Ile and Lys respectively. Importantly
the panel of probes we have designed enables sufficient coverage
for one enzyme to be distinguished from the next. We have further
shown the fingerprints obtained are activity-based (that is,
dependent upon the enzyme catalytic activity), and may be carried
out with enzymes present in a crude proteome mixture (that is, in
the presence of other unrelated proteins) (see ESI).
Funding support was provided by the National University of
Singapore (NUS) and the Agency for Science, Technology and
Research (A*STAR) of Singapore.
Notes and references
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3 For a recent review, see: A. Saghatelian and B. F. Cravatt, Nat. Chem.
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4 (a) A. Saghatelian, N. Jessani, A. Joseph, M. Humphrey and
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(b) E. W. S. Chan, S. Chattopadhaya, R. C. Panicker, X. Huang and
S. Q. Yao, J. Am. Chem. Soc., 2004, 126, 14435–14446.
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7 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8,
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We next tested the feasibility of these probes to be used in a
protein microarray for potential high-throughput discovery of
metalloproteases. Previously, only activity-based, and not affinity-
based, probes have been shown to detect enzymes immobilized in a
protein microarray.5a,6 Five different enzymes, comprising three
metalloproteases (i.e. collagenase, thermolysin and anthrax LF),
one serine protease (i.e. b-chymotrypsin) and carbonic anhydrase,
were spotted in triplicate on a glass slide, and subsequently
screened with the Leu probe (Fig. 3). Results indicate that the
probe was in general able to distinguish metalloenzyme activity
over other non-metalloenzyme activities, in most cases generating
positive fluorescence signals only with metalloproteases (e.g.
collagenase and thermolysin), as well as carbonic anhydrase (a
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Chem. Commun., 2006, 3783–3785 | 3785