Highly sensitive peptide-based probes for protein tyrosine phosphatase activity utilizing a fluorogenic mimic of phosphotyrosine

Article abstract of DOI:10.1016/j.bmcl.2005.08.054

Fluorescent probes that can be incorporated into peptides and proteins are in high demand, with applications ranging from cellular imaging to binding and activity assays. Here, we report the high yielding synthesis of an enantiomerically pure phosphocoumarin-based amino acid and its incorporation into peptides via standard solid-phase peptide synthesis methodologies. Peptides containing this new amino acid serve as highly sensitive fluorogenic probes for protein tyrosine phosphatase activity.

Full text of DOI:10.1016/j.bmcl.2005.08.054

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5142–5145
Highly sensitive peptide-based probes for protein
tyrosine phosphatase activity utilizing a fluorogenic
mimic of phosphotyrosine
Sayantan Mitra and Amy M. Barrios*
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA
Received 30 July 2005; revised 18 August 2005; accepted 22 August 2005
Available online 3 October 2005
Abstract—Fluorescent probes that can be incorporated into peptides and proteins are in high demand, with applications ranging
from cellular imaging to binding and activity assays. Here, we report the high yielding synthesis of an enantiomerically pure phos-
phocoumarin-based amino acid and its incorporation into peptides via standard solid-phase peptide synthesis methodologies. Pep-
tides containing this new amino acid serve as highly sensitive fluorogenic probes for protein tyrosine phosphatase activity.
Ó 2005 Elsevier Ltd. All rights reserved.
There is a great need for versatile fluorescent probes that
can be incorporated into proteins. Such tags are useful
not only for visualizing a modified protein or enzyme
activity in vivo and in vitro, but also for assaying en-
zyme activity and determining the substrate specificity
of enzymes. For example, fluorogenic and colorimetric
substrates are commonly used to assay protease activity
and substrate specificity1 and have been used to a lesser
extent to assay the activity of many other enzymes
including glycosidases, phosphatases, sulfatases, and
esterases.2,3 With an ever-expanding catalog of post-
translational modifications and an analogous library
of enzymes catalyzing the corresponding transforma-
tions, the need for adaptable fluorogenic substrates is
growing.4
ethylumbelliferone phosphate (DiFMUP).7 Although
these substrates are readily hydrolyzed by many PTPs,
they do not work with all members of this enzyme fam-
ily. To measure PTP activity against peptide substrates,
the small increase in absorbance or fluorescence upon
hydrolysis of a phosphotyrosine residue in a peptide
substrate can be followed.7–9 Alternatively, MS10 and
protease-coupled assays11 have been reported. Unfortu-
nately, these methods either lack sensitivity or require
specialized equipment and expertise, hampering their
widespread adoption.
Recent work indicates that many PTPs have distinct
substrate specificities mediated both by interactions with
residues on either side of the target phosphotyrosine res-
idue and also by interactions of other PTP domains with
the substrate.12 A variety of approaches to defining the
substrate sequence specificities of PTPs have been uti-
lized, including substrate-trapping mutants,13 affinity
selection of inhibitory peptides,14 phosphotyrosine-con-
taining phage libraries,15 and inverse alanine scanning.16
A detailed understanding has been slow to emerge be-
cause the chemical tools necessary for studying PTP
activity against peptide substrates are not widely avail-
able.6,5 To date, there is no method for assaying PTP
activity that is highly sensitive, continuous, widely avail-
able, and applicable to high-throughput screening of
peptide substrate libraries. A well-designed probe based
on these criteria would provide crucial information
about the similarities and differences in peptide sub-
strates recognized by members of the PTP family of
One example of a family of enzymes whose study would
benefit from improved chemical probes is the protein
tyrosine phosphatase (PTP) enzyme family. The PTPs
are a diverse family of enzymes involved in key signaling
pathways and implicated in a number of disease states
including cancer and diabetes.5,6 Currently, PTP activity
is followed through the use of small, non-peptidic sub-
strates such as para-nitrophenylphosphate (p-NPP), 4-
methylumbelliferone phosphate (MUP), and difluorom-
Keywords: Protein tyrosine phosphatases; Fluorogenic probes; Fluo-
rescent amino acid.
*
Corresponding author. Tel.: +1 213 821 2554; fax: +1 213 740
0930; e-mail: amyb@usc.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.054

S. Mitra, A. M. Barrios / Bioorg. Med. Chem. Lett. 15 (2005) 5142–5145
5143
enzymes. This detailed knowledge would be invaluable
in the design of potent, selective PTP inhibitors for bio-
chemical and therapeutic applications. Furthermore, an
appropriate fluorogenic probe for PTP activity would
find use in visualizing PTP activity in whole cells.
be found in the Supplementary information available
online. This synthesis is superior to other reported syn-
theses of coumaryl amino acids in its simplicity, yield,
and versatility. We anticipate that this new phosphoty-
rosine mimic will be useful for imaging PTP activity
both in vitro and also in vivo.
To facilitate the study of PTP activity, substrate specific-
ity, and inhibition, a fluorogenic, peptide-based PTP
substrate based on a phosphorylated coumaryl amino
acid moiety has been developed. Fluorescent amino
acids are highly desirable synthons for a variety of bio-
chemical applications, and a handful of enantioselective
syntheses of coumaryl amino acids have been reported
in the literature recently.17–22 In this work, we have syn-
thesized and characterized an appropriately protected,
phosphorylated coumaryl amino acid that can be incor-
porated directly into peptide substrates using standard
Fmoc-based solid-phase peptide synthesis (SPPS)
methodologies.
To demonstrate the utility of pCAP in in vitro PTP as-
says, we synthesized a series of peptide substrates with
the general formula DADE-X-GPAA-NH2, where X is
phosphotyrosine (9a), tyrosine (9b), pCAP (10a) or
CAP (10b). The coumarin-based amino acids are com-
patible with standard, Fmoc-based SPPS methodolo-
gies.23 Any standard coupling procedure can be used to
extend the growing peptide chain, with the exception of
pCAP and CAP addition, which couples more efficiently
when PyBOP and HOBt are used as the coupling re-
agents. Deprotection of the phosphate moiety was
accomplished by the addition of trimethylsilyliodide to
the peptide prior to the cleavage from the resin. The pep-
tides were then fully deprotected and cleaved from the
resin, purified by reverse-phase HPLC, and characterized
by mass spectrometry. The peptide sequence DADE-X-
GPAA-NH2 is expected to be a reasonable, though not
optimal, substrate for a variety of PTPs.
Enantiomerically pure N-a-Fmoc-L-aspartic acid b-tert-
butyl ester (1) was chosen as the starting point in this
synthesis, outlined in Scheme 1. The a-carboxylic acid
of (1) was first protected as a trichloroethyl (Tce) ester
(2). The b-carboxylic acid was then deprotected and
acylated using isopropenyl chloroformate and Mel-
drumÕs acid to obtain the b-ketoester (4) in an overall
yield of 90% starting from (1). Methanesulfonic acid
was recently reported to catalyze the condensation be-
tween an amino acid derived b-ketoester and resorcinol
at room temperature, preserving the chirality of the ste-
reocenter.14 In our hands, the reaction gave higher yields
of the N- and C-protected coumaryl amino acid (6)
when carried out at 0–4 °C. Phosphorylation of (6) with
diethylchlorophosphate and diisopropylethylamine gen-
erated the phosphorylated coumarin (7). Subsequent
removal of the Tce group using 50% acetic acid in tetra-
hydrofuran in the presence of activated zinc dust gener-
ated the phosphorylated coumaryl amino acid (pCAP)
(8) quantitatively from (7). As illustrated in Scheme 1,
this approach generates an enantiomerically pure,
appropriately protected phosphocoumaryl-amino-pro-
pionic acid (8, pCAP) in high yield via six facile steps
from readily available starting materials. Product purifi-
cation through column chromatography was only neces-
sary for compounds 6 and 7. Full synthetic details can
If the pCAP-containing peptides are to be useful in en-
zyme activity assays, they must exhibit a large change
in fluorescence upon hydrolysis. As anticipated based
on the success of MUP in PTP activity assays,7 we found
that a 22 lM solution of peptide 10b exhibited fluores-
cence (kex = 334 nm, kem = 460 nm) that is 104-fold more
intense than a 22 lM solution of peptide 10a (see inset,
Fig. 1). As shown in Figure 1, a large increase in fluores-
cence is observed when 10a is hydrolyzed by a PTP. Fur-
thermore, the fluorescence intensity of the peptide 10b
increases linearly in proportion to concentration over
a wide concentration range, indicating that the PTP-cat-
alyzed hydrolysis of 10a to form 10b should provide a
facile, sensitive assay for PTP activity. In addition, no
hydrolysis of the fluorogenic peptide substrate 10a was
observed under the conditions of the reaction in the ab-
sence of enzymes.
Because the pCAP moiety is slightly larger than phos-
photyrosine, we set out to establish the efficiency of
O
NHFmoc
OH
O
O
NHFmoc
OTce
O
NHFmoc
OTce
O
NHFmoc
OTce
(i)
(iiia)
(iiib)
(ii)
ButO
ButO
O
HO
O
O
1
O
O
2
4
3
NHFmoc
OTce
NHFmoc
OTce
NHFmoc
OH
5
OH
O
O
(v)
O
O
(vi)
EtO
HO
(iv)
O
P
O
O
HO
O
O
O
P
O
EtO
EtO
O
6
O
7
EtO
8 (pCAP)
Scheme 1. Reagents and conditions: synthesis of 8 (pCAP) (i) CCl3CH2OH, dicyclohexylcarbodiimide, DMAP, 0 °C, 15 h, 97%. (ii) 50% TFA in
CH2Cl2, rt, 15 min, >99%. (iiia) Isopropenyl chloroformate, MeldrumÕs acid, DMAP, CH2Cl2, 0 °C, 2 h. (iiib) CH3OH/benzene (4:1), reflux, 12 h,
94%. (iv) CH3SO3H, 0 °C, 6 h, 60%. (v) (EtO)2POCl, diisopropylethylamine, CHCl3, rt, 14 h, 85%. (vi) Zn dust (6 equiv) 50% acetic acid in THF, rt,
8 h, 99%.

5144
S. Mitra, A. M. Barrios / Bioorg. Med. Chem. Lett. 15 (2005) 5142–5145
7 106
6 106
5 106
4 106
3 106
2 106
1 106
Table 1. Kinetic data for the hydrolysis of substrates by YOP and
TCPTP
Enzyme
Substrate
kcat (sÀ1
100 20
41
)
Km (mM)
9a
2.7 0.8
0.65 0.07
0.8 0.2
YOP
10a
MUP
10a
2
5.7 0.5
35 10
TCPTP
0.8 0.3
4 104
3 104
2 104
1 104
0 100
In conclusion, we have developed a facile, high-yielding
synthesis of a highly fluorescent, phosphorylated amino
acid that can be incorporated into peptides using stan-
dard SPPS techniques. The resultant substrates are effi-
ciently hydrolyzed by PTPs and exhibit a large increase
in fluorescence upon hydrolysis. This work represents a
critical step forward in the characterization of PTPs,
both because it demonstrates that the pCAP residue
can be incorporated into peptides and hydrolyzed by
PTPs, and more importantly because it verifies that
pCAP is accepted as a phosphotyrosine mimic by both
bacterial and human PTPs. The pCAP-containing pep-
tides provide a highly sensitive, continuous assay for
PTP activity that is widely accessible and readily incor-
porated into peptide substrates for high-throughput
screening or activity-based assays. Much less enzyme is
required for this assay than for others that have been
reported previously.12,13 In principle, CAP could also
be derivatized with a variety of other electrophiles to
create fluorogenic substrates for aryl sulfatases, glycosi-
dases, etc. Work is currently underway in our laboratory
to incorporate CAP and pCAP into combinatorial pep-
tide libraries for assaying enzyme substrate specificities
and to synthesize fluorinated derivatives of pCAP
(pCAPF and pCAPF2), which may have greater sensi-
tivity, especially at low pH.7 Furthermore, this series
of fluorogenic peptides may aid in the visualization of
enzyme activity in vivo.
300 400 500 600
Wavelength
0
5
10
15
Time (min)
20
25
30
Figure 1. Raw data showing the initial increase in fluorescence over
time when a 250 lM solution of 10a is allowed to react with YOP.
Inset: emission spectra of 22 lM solutions of peptides 10a (solid line)
and 10b (dashed line) with kex = 334 nm.
enzymatic turnover of 10a. As shown in Figure 2A,
10a is readily dephosphorylated by the PTP from Yer-
sinia enterocolitica (YOP). Peptide 10a (kcat/Km =
63,000 MÀ1 sÀ1) is hydrolyzed significantly more effi-
ciently by YOP than MUP (kcat/Km = 6200 MÀ1 sÀ1
,
Supplementary Figure 1) and approximately as effi-
ciently as 9a (kcat/Km = 37,000 MÀ1 sÀ1, Supplementary
Figure 2). The turnover rate (kcat) of 10a by YOP is
approxmately 9-fold faster than MUP and about 2-
fold slower than 9a, while the Km is about the same
as MUP and 4-fold lower than 9a. The Yersinia en-
zyme is one of the most active PTPs known and read-
ily accepts the pCAP moiety as a phosphotyrosine
mimic. To determine if these substrates are likely to
be broadly applicable, we also tested the activity of
human T-cell PTP (TCPTP) against 10a. Notably,
with TCPTP we were unable to obtain satisfactory
kinetic data with 9a, while hydrolysis of 10a was read-
ily detected, as shown in Figure 2B. The kinetic data are
summarized in Table 1.
Acknowledgments
The authors thank Ms. Caitlin Hubbard for helpful dis-
cussions. A.M.B. acknowledges the College of Letters
Arts and Sciences, and the WiSE program at USC for
research support.
7 10-8
6 10-8
5 10-8
-8
A
B
1 10
8 10-9
6 10-9
4 10-9
2 10-9
0 100
4 10-8
3 10-8
2 10-8
1 10-8
0 100
0
0.0001 0.0001 0.0002 0.0002 0.0003
0
0.0005
0.001 0.0015
0.002 0.0025
[S] (M)
[S] (M)
Figure 2. Hydrolysis of 10a by (A) YOP and (B) TCPTP. The curve represents the best fit of the data to the Michaelis–Menten equation.

S. Mitra, A. M. Barrios / Bioorg. Med. Chem. Lett. 15 (2005) 5142–5145
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