Synthesis and peptide incorporation of an unnatural amino acid containing activity-based probe for protein tyrosine phosphatases

Article abstract of DOI:10.1016/j.bmcl.2009.04.081

An unnatural amino acid was synthesized to incorporate a quinone methide-generating activity-based probe for protein tyrosine phosphatases (PTPs) and then integrated into a PTP1B-specific substrate. The resulting probe led to preferential labeling of PTP1

Full text of DOI:10.1016/j.bmcl.2009.04.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3264–3267
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and peptide incorporation of an unnatural amino acid
containing activity-based probe for protein tyrosine phosphatases
*
Kui Shen , Lixin Qi, Mohini Ravula, Krzysztof Klimaszewski
Northern Illinois University, Department of Chemistry and Biochemistry, DeKalb, IL 60115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An unnatural amino acid was synthesized to incorporate a quinone methide-generating activity-based
probe for protein tyrosine phosphatases (PTPs) and then integrated into a PTP1B-specific substrate.
The resulting probe led to preferential labeling of PTP1B in cell lysates in the presence of PTP4A3.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 7 April 2009
Accepted 20 April 2009
Available online 23 April 2009
Keywords:
Protein tyrosine phosphatase
Activity-based probe
Unnatural amino acid
Quinone methide
Affinity labeling
PTP1B
PTP4A3
Protein tyrosine phosphatases (PTPs), just like their counteract-
ing enzymes protein tyrosine kinases (PTKs), mediate protein tyro-
sine phosphorylation, a central regulatory mechanism for cellular
signal transduction.1 For example, PTP1B functions as a positive
regulator of signaling events associated with breast and ovarian
tumorigenesis, in addition to playing a role in downregulating
insulin and leptin signaling.1 Another PTP, PTP4A3, is implicated
in cell proliferation, migration and cancer metastasis.1 Since there
are more than 100 PTPs in humans, it is desirable to clearly define
the partnerships between individual PTPs and phosphoproteins.1
While substrate-trapping mutants of PTPs can be used to identify
their physiologically relevant substrates,2 no methods are available
to allow direct identification of PTPs for phosphopeptide sub-
strates. In this regard, recent progress in the proteomic applica-
tions of mechanism-based enzyme inactivators is encouraging.3
In these applications, activity-based probes have been used to label
enzymes including phosphatases.
probes are reactive towards thiols even in the absence of PTPs,
especially in neutral or basic aqueous solutions, and thiols are
commonly present in the reducing cellular environments. Further-
more,
a-bromobenzylphosphonate undergoes solvolysis under
similar conditions.6 It has been hypothesized that the integration
of a turnover-based probe into a PTP-specific phosphotyrosyl sub-
strate will avoid the disadvantages of other types of probes as well
as improve the specificity in labeling the PTP. To test this hypoth-
esis, we began our investigation by synthesis and peptide incorpo-
ration of a quinone methide-based probe-containing amino acid,
thus converting a ‘miniature’ PTP1B-specific substrate into a turn-
over-based probe (Fig. 1). We report herein our preliminary results
of this specific investigation.
First of all, a novel unnatural amino acid 5, the 3-difluoromethyl
analog of phosphotyrosine, was synthesized. While both conver-
gent and divergent routes are possible,7 we started the synthesis
with orthoformylation of the tyrosine phenol moiety,8 followed
by routine functional group transformations4,9 (Scheme 1).
The ‘miniature’ substrate 6, or, when converted into a PTP-resis-
tant form, inhibitor 7, is highly efficient and selective for PTP1B,
and exhibits a PTP1B-interacting pattern that is typical of a physi-
ologically relevant or optimal PTP1B phosphopeptide substrate.9
Then the presumably PTP1B-specific probe 8 was assembled via
solid phase synthesis, that consisted of a PTP-probing component,
derived from the ‘miniature’ PTP1B substrate, and a biotin for Wes-
tern blot detection by streptavidin-horse radish peroxidase (HRP),
with the two separated from each other by a polyamide-polyether
linkage. The flexible PEG-like linkage was used to minimize the po-
Among activity-based PTP-targeting probes (Fig. 1), turnover-
based suicidal substrates containing fluoromethylphenyl phos-
phate (e.g., 1 and 2) have been intensively studied.4 Hydrolysis of
such substrates by PTPs or other phosphatases generates a highly
reactive quinone methide, which then reacts with a nucleophile
near the phosphatase active site. More recently, the derivatives
of
a
-bromobenzylphosphonate (3)5 and phenyl vinylsulfonate
(4)6 have been used as class-specific PTP probes. However, these
* Corresponding author. Tel.: +1 815 753 1463; fax: +1 815 753 4802.
E-mail address: kshen@niu.edu (K. Shen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.081

K. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3264–3267
3265
O
O
OH
OH
O
O
OH
OH
CHF2
O
OH
OH
O
O
O
X
PO3H2
CF2PO3H2
P
P
Br
P
S
6
7
X = O
X = CF2
CO2H
NH2
H
N
N
H
F
CO2H
NH2
2
NH2
3
N3
4
O
O
O
1
O
CHF2
OPO3H2
NH2
PTP(1B)-probing
HN
H
NH
H
5
HO2C
OPO3H2
CHF2
CF2PO3H2
O
S
8
Linker
O
OH
OH
HN
CO2H
O
Br
P
H
N
O
O
NH
HN
O
NH2
H
N
9
N
H
O
O O
O
O
Figure 1. Design of an activity-based probe 8 that is presumably specific for PTP1B: PTP(1B) probing component is linked to biotin via a flexible linker. 1–4, structures of
precursors or components of previously studied activity-based probes for PTPs; 5, the 3-difluoromethyl analog of phosphotyrosine; 6 and 7, highly efficient and selective
PTP1B substrate and inhibitor, respectively; and 9, a PTP inactivator.
Z
NH
Z
NH
Z NH
O
O
b
a
MeO2C
MeO2C
MeO2C
OH
OH
OPO3Et2
c
Z
NH
CHF2
OPO3Et2
Fmoc NH
HO2C
CHF2
OPO3H2
d,e,f
MeO2C
5
Scheme 1. Synthesis of Fmoc-protected unnatural amino acid 5. Reagents and conditions: (a) anhydrous MgCl2, Et3N, (CHO)n, acetonitrile, reflux, 18 h, 43%; (b) Et3N,
ClPO3Et2, CH2Cl2, ice bath to room temperature, overnight, 85%; (c) Et2NSF3 (DAST), CH2Cl2, ice bath to room temperature, overnight, 64%; (d) TMSBr, CH2Cl2, ice bath to room
temperature, 16 h; (e) Fmoc-OSu, 1,4-dioxane/aqeous NaHCO3, 3 h; (f) LiOH, THF/water, ice bath, 1.5 h; 61%.
tential interference between biotin and the PTP-probing
component.
is PTP activity-dependent, since incubation of the probe with
dPTP1B, the catalytically deficient C215S mutant of PTP1B, did
not show any detectable signal. As expected, the probe-PTP cova-
lent complexes run at slightly higher molecular weights than the
unmodified PTPs. Multiple bands, that indicated multiple labeling
of a single protein molecule, were detected at the higher concen-
tration of 8 in both PTP1B and PTP4A3 labeling experiments. This
result is consistent with the previous observations in that the dif-
fusable quinone methide-based labeling is not highly active site-
When 8 interacts with a PTP, it will be dephosphorylated and
activated, producing a highly reactive quinone methide, which
then reacts with a nearby nucleophile, such as a thiolate. If this
nucleophile originates from the PTP, that is indeed in the proximity
of the newly-generated quinone methide product, then such a
reaction leads to crosslinking of the probe with the PTP (Scheme
2). Since the probe 8 carries specificity for PTP1B, that is, selec-
tively recognized by PTP1B’s catalytic machinery, such labeling is
expected to recapitulate comparable selectivity for PTP1B over
other PTPs. To our knowledge, this is the first example of using
an activity-based probe to selectively label an individual PTP in
the presence of other PTPs.
specific and may attack the N-terminal
a-amino group as well as
non-active-site cysteines.4 This kind of single-site labeling may
not always completely inactivate the PTP although the probe 8
showed time- and dose-dependent inactivation for PTP1B (data
not shown).
To evaluate 8 as an activity-based PTP probe, two purified re-
combinant PTPs—that are over 100-fold different in kcat/Km for
6—PTP1B and PTP4A3, were respectively incubated at neutral to
basic pH with the probe (Fig. 2A). As shown in Figure 2B, incuba-
tion with the probe at a low concentration led to detectable biotin
labeling of PTP1B but not of PTP4A3. However, at a higher concen-
tration of 8, both PTP1B and PTP4A3 could be labeled. Such labeling
To assess the selectivity of 8 for PTP detection in a complex pro-
teome, Escherichia coli lysates were spiked with purified recombi-
nant PTPs (Fig. 3A). While both PTP4A3 and PTP1B are readily
detectable (Fig. 3A, the left lane), diffusable quinone methides
did not result in significant nonspecific labeling of other proteins.
When PTP4A3 and dPTP1B were spiked into the lysates, only
PTP4A3 was detected (Fig. 3A, the right lane). This demonstrates
O
OH
CHF
O
Substrate
OPO3
PTP
F
F
CHF
CHF
CHF2
PTP-Nu
PTP-Nu
8
Scheme 2. Proposed mechanism of PTP labeling by the probe 8 (adapted from Ref. 4).

3266
K. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3264–3267
A
B
MW
(KD)
97 -
66 -
MW
(KD)
66 -
45 -
PTP1B/dPTP1B
PTP4A3
45 -
PTP1B
30 -
20 -
30 -
20 -
PTP4A3
Loaded protein
(Coomassie)
14 -
Figure 2. Labeling of purified recombinant PTPs—PTP1B, PTP4A3 and dPTP1B (4 lg each). (A) SDS–PAGE and coomassie staining of purified recombinant PTPs. (B) Western
blot using streptavidin-HRP after 1 h incubation with 20
l
M (dilute) or 200
lM (conc.) of the probe 8.
A
B
MW
(KD)
MW
(KD)
97 -
66 -
45 -
97 -
66 -
45 -
PTP1B
PTP1B
30 -
30 -
20 -
PTP4A3
20 -
Loaded protein
(Coomassie)
PTP1B/dPTP1B
PTP4A3
Streptavidin mAb IP+Streptavidin
Figure 3. Labeling of PTPs by 0.8 mM of the probe 8 in cell lysates in the absence or presence of 6 mM of the general PTP inactivator 9. (A) E. coli lysates (70
lg total proteins)
spiked with 4 g each of PTP4A3 and PTP1B/dPTP1B at 1 h incubation, as detected by Western blot using streptavidin-HRP. (B) Jurkat cell lysates (25 g total proteins) at 5 h
l
l
incubation, as detected by Western blot using streptavidin-HRP (left lane), Western blot using monoclonal antibody to PTP1B (mAb) + secondary antibody-HRP (middle lane),
and PTP1B immunoprecipitation (IP) + Western blot using streptavidin-HRP (right lane), respectively.
that the labeling by the probe 8 is selective for active PTPs in the
presence of the inactive PTP and other endogenous bacterial
proteins.
It was of interest to examine whether 8 could distinguish PTP1B
from PTP4A3 or other PTPs. Since high doses of the probe 8 would
otherwise eventually label any coexisting PTPs, we introduced a
lane). At this point, it is not clear whether the other band repre-
sents another phosphatase having substrate specificity comparably
to PTP1B or indeed a partner protein that is in complex with PTP1B
(and hence presumably subject to diffusion-dependent labeling).
Nonetheless, further identification is possible with affinity-enrich-
ment, limited proteolysis and LC–MS analysis.
nonspecific, competing but non-detecting PTP inactivator
9
In conclusion, we present here a novel phosphotyrosine analog
that incorporates a PTP activity-based probe, which in turn can be
incorporated into an individual PTP-specific substrate, leading to
the observed specificity in labeling the PTP in the presence of other
PTPs. Whereas peptide substrates coupled with small molecule
crosslinkers have been used for mechanism-based labeling of spe-
cific protein kinases,12 our results imply that phosphotyrosyl pep-
tide substrates combined with activity-based probes and general
suppressors can be used to label specific PTPs responsible for their
dephosphorylation. More detailed work on the application of this
and other related probes are ongoing and will be reported in due
course.
(Fig. 1) into our labeling experiments, reasoning that signals result-
ing from less specific PTPs would be more suppressed.10 We chose
a
-bromobenzylphosphonate 9 because of its ready synthetic avail-
ability.11 It was not attached to biotin and hence it is non-detecting
in our system. Indeed, with the addition of 9, PTP1B was readily
and preferentially detected in the presence of PTP4A3 in E. coli ly-
sates (Fig. 3A, the middle lane). This implies that selective labeling
of a PTP in the presence of other PTPs can be facilitated by nonspe-
cific suppression of general PTP activities.
To confirm the above perception, we decided to test 8 against a
mammalian proteome that is known to contain PTP1B and multi-
ple other PTPs. To this end, Jurkat cell lysates were studied
(Fig. 3B) by the combination of 8 and 9. Two major bands were ob-
served (Fig. 3B, the left lane), with the stronger signal at the lower
molecular weight corresponding to PTP1B, as detected by a mono-
clonal antibody (Fig. 3B, the middle lane). The identity of PTP1B
was further confirmed by PTP1B enrichment from immunoprecip-
itation using the PTP1B-specific monoclonal antibody and subse-
quent Western blot using strepavidin-HRP (Fig. 3B, the right
Acknowledgments
We thank Professor Narayan Hosmane for helpful discussions,
Professor Zhenghe Wang for help with Jurkat cells, and Northern
Illinois University (Department of Chemistry and Biochemistry
and Center for Biochemical and Biophysical Studies) for financial
support.

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