A highly selective fluorescent ESIPT probe for the dual specificity phosphatase MKP-6

Article abstract of DOI:10.1039/b911145j

A highly selective fluorescent probe for a protein tyrosine phosphatase (PTP) was designed by a simple phosphorylation of the 2-(2′-hydroxyphenyl) benzothiazole (HBT) chromophore: upon selective enzymatic hydrolysis, an excited-state intramolecular proton

Full text of DOI:10.1039/b911145j

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A highly selective fluorescent ESIPT probe for the dual specificity
phosphatase MKP-6w
a
b
Tae-Il Kim,z Hyo Jin Kang,z Garam Han,a Sang J. Chung*b and Youngmi Kim*a
Received (in Cambridge, UK) 8th June 2009, Accepted 24th July 2009
First published as an Advance Article on the web 17th August 2009
DOI: 10.1039/b911145j
A highly selective fluorescent probe for a protein tyrosine
phosphatase (PTP) was designed by a simple phosphorylation
of the 2-(20-hydroxyphenyl)benzothiazole (HBT) chromophore:
upon selective enzymatic hydrolysis, an excited-state intra-
molecular proton transfer (ESIPT) occurs, resulting in a large
Stokes shift.
Protein tyrosine phosphatases (PTPs), together with protein
tyrosine kinases (PTKs), play crucial roles in cellular regulation
and signal transduction pathways.1 Several human diseases,
including cancer, diabetes, and Alzheimer’s are associated
with abnormal phosphatase and/or kinase activities.2
Therefore, monitoring PTP activity is of great interest in
biomedical research.3
Due to unparalleled sensitivity, short detection times, and
Fig. 1 General mechanism of ESIPT process and chemical structures
applicability to high throughput screening (HTS), fluorescence-
based assays are promising tools to detect protein phos-
phorylation states. The most popular fluorescent probes for
monitoring PTP activity have been coumarin or fluorescein
derivatives, in which an increase in fluorescence intensity
occurs upon the enzyme-catalyzed hydrolysis of a phosphate
group.4 Nagano and co-workers reported a ratiometric
fluorescence probe based on fluorescence resonance energy
transfer (FRET) to monitor PTP activity.5a In addition,
FRET was also employed to detect PTP–substrate interactions
in live cells by Bastiaens and collegues.5b
of the enol and keto forms in the ESIPT process of HBT.
of the HBT chromophore converts the enol (E*) to the
excited-state keto species (K*) in the subpicosecond time scale
(Fig. 1). The resulting emission from K* exhibits an unusually
large Stokes shift. Since K* emission occurs in a region of the
spectra that is clear of interferences (self-absorption, etc.), we
sought to explore turn-on fluorescence probes that rely on an
ESIPT process. As a proof-of-principle, we based the design of
probe 3 on the extensively studied 2-(20-hydroxyphenyl)-
benzothiazole model ESIPT fluorophore (HBT, 1).7
Phosphorylation inhibits ESIPT process in probe 3, resulting
exclusively in enol-like emission. However, enzymatic cleavage
of phosphate group in probe 3 generates HBT, resulting in the
dual emission bands associated with keto-enol tautomerization
upon photoexcitation.
Although these probes are adept at detecting generic
phosphatase activity, the detection of a specific PTP still
remains a challenge. The human genome encodes 107 distinct
PTPs.6 Due to their structural similarity and inherent
substrate specificity toward phosphotyrosine, there has been
no report of small molecule probes to selectively detect a specific
PTP. Herein, we report a highly selective fluorescent probe for a
specific PTP that relies on an excited state intramolecular
proton transfer (ESIPT) for signal transduction.
Probe 3 was synthesized in two steps according to Scheme 1.
Commercially available 2-(20-hydroxyphenyl)benzothiazole
(HBT, 1) was phosphorylated with diethyl chlorophosphate
and NaH in THF to give phosphate triester 2. Selective
hydrolysis of 2 was achieved with bromotrimethylsilane to
give the water soluble probe 3 after neutralization with NaOH.
The UV-vis absorption spectrum of the probe 3 in aqueous
solution (0.1 M PBS buffer, pH 7.4) displays a maximum at
Intramolecularly hydrogen-bonded molecules often undergo
an ESIPT process, in which a rapid photoinduced proton
transfer results in tautomerization. For example, irradiation
a Department of Chemistry, Dankook University, 126 Jukjeon-dong,
Yongin-si, Gyeonggi-do, 448-701, Korea.
E-mail: youngmi@dankook.ac.kr; Fax: +82 31-8005-3148;
Tel: +82 31-8005-3156
b BioNanotechnology Research Center, KRIBB, and Nanobio Division,
UST, 52 Eoeun-dongYuseong, Daejeon 305-333, Korea.
E-mail: sjchung@kribb.re.kr; Fax: +82 42-879-8594;
Tel: +82 42-879-8433
w Electronic supplementary information (ESI) available: Detailed
experimental procedures, additional UV-vis excitation, and emission
spectra and enzyme assay studies. See DOI: 10.1039/b911145j
z Contributed equally to this work.
Scheme 1 Reagents and conditions: a. (EtO)2POCl, NaH, THF,
51%; b. (i) TMSBr, CH2Cl2 (ii) aq. NaOH (pH B 8), 80%.
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Fig. 3 Absorption (dashed lines) and emission spectra (solid lines) of
HBT in 2% DMSO–water (v/v) at different concentrations at 25 1C.
[HBT] = 0.2 (black), 2 (blue), and 20 mM (red). Excitation at 330 nm.
Fig. 2 Absorption (dashed lines) and emission spectra (solid lines) of
probe 3 (blue) and HBT (red) in aqueous solution at 25 1C. Probe 3 in
PBS buffer solution (pH 7.4) and HBT in 2% DMSO–H2O (v/v).
Excitation at 318 nm for probe 3 and at 350 nm for HBT. [probe 3] =
10 mM and [HBT] = 20 mM.
HBT. The strong emssion observed from the keto tautomer
within aggregates is attributed to the increased planarity and
rigidity by aggregation as well as the inhibition of free rotation
about the biaryl C–C bond such that the radiationless
deactivation may be reduced. The emission spectra of HBT
also depend on the excitation wavelengths (see ESIw).
To apply our sensing scheme in aqueous media, a relatively
high concentration of probe 3 was tested for catalytic
hydrolysis by PTP. Twenty six human PTPs were randomly
selected, overexpressed in Escherichia coli, purified by metal
affinity chromatography, and their catalytic activities were
confirmed with DiFMUP, a commercially available PTP
substrate (see ESIw). The selected PTPs consisted of classical
PTPs that specifically recognize phosphotyrosine and dual
specific PTPs that recognize phosphoserine and phosphothreonine
as well as phosphotyrosine as substrates (see ESIw).6
264 nm with shoulder peak at 318 nm and the emission
spectrum shows a peak maximum at 384 nm, which is
characteristic of an enol-like emission (Fig. 2).
To validate the proposed sensing mechanism, the photo-
physical properties of HBT, the expected product after
reaction with PTP, were examined. As well-documented in
the literature,8 the fluorescence spectra of HBT show a strong
keto tautomer emission (B535 nm) in nonpolar solvents with
a large Stokes shift resulting from ESIPT. With increasing
solvent polarity, the emission spectra of HBT exhibit a gradual
increase in the intensity of enol emission peak (B368 nm),
which arises from the excited state of intermolecularly hydrogen-
bonded enol (see SI).
Since the enzyme assay is performed in aqueous media
where the ESIPT process could be inhibited by intermolecular
H-bonding to solvent, the photophysical properties of
HBT, were further examined in aqueous media at different
concentrations. Due to its low solubility in aqueous media, the
absorption and fluorescence spectra of HBT were acquired in
2% DMSO–water (v/v). In relatively dilute solution (0.2 and
2 mM), the emission spectra of HBT display a broad peak
at ca. 470 nm and a weak intensity peak at 375 nm, but
the characteristic emission peak of the keto tautomer at
longer wavelengths is absent (Fig. 3). However, at a higher
concentration (20 mM), the fluorescence spectrum shows an
intense, broad emission peak at 520 nm together with a weak
shoulder at 456 nm (Fig. 3). Based on previous reports of
similar photophysical behavior in compounds that undergo
ESIPT,9 the emission peak at 520 nm can be assigned to the
keto tautomer within aggregates and emission at 470 nm might
be due to the formation of species that are intermolecularly
hydrogen-bonded to solvent molecules. In addition, the
solution of 20 mM HBT in 2% DMSO–water was visibly
turbid, indicating the formation of aggregates, while less
concentrated solutions (0.2 and 2 mM) remained clear. These
results suggest that HBT, which would be generated upon
enzymatic cleavage of phosphate group, exists as aggregates
or as the solvated enol form depending on concentration of
When probe 3 (50 mM) was incubated with PTPs, unexpectedly,
a strong fluorescence signal increase at 535 nm occurred only
in the presence of MKP-6, also known as DUSP14 (Fig. 4).10
Fig. 4 Fluorescence responses of probe 3 to various PTPs. Probe
3 was incubated for 60 min in the presence of 100 nM (blue bar) and
1 mM (red bar) PTPs, respectively. Data were obtained at 25 1C in
20 mM Tris buffer (pH 8.0) containing NaCl (150 mM), 0.01% Triton
X-100, DTT (5 mM), and BSA (5 mM). Excitation at 355 nm, emission
collected at 535 nm. [probe 3] = 50 mM.
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Addition of sodium vanadate—a known PTP inhibitor11—
entirely blocked the fluorescence increase, providing
This work was supported by a Korea Research Foundation
(KRF) grant funded by the Korea government (MEST)
(No.2009-0030100) and by the GRRC program of Gyeonggi
province (GRRG 2008-A01).
a
confirmation that the enzymatic activity of MKP-6 is required
to hydrolyze probe 3 and result in fluorescent HBT (1).
The fluorescence increase was dependent upon enzyme
concentration and reaction time, with 1 mM and 100 nM of
MKP-6, respectively resulting in 90% and 34% hydrolysis of
probe 3 in 60 min. Kinetic parameters for the hydrolysis of
probe 3 by MKP-6 were determined by Lineweaver-Burk
analysis (see ESIw), yielding values of Km = 535 mM and
kcat = 0.15 sꢁ1. Enzymatic efficiency, as estimated by a
Notes and references
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k
cat/Km = 0.28 mMꢁ1 sꢁ1 for probe 3, is lower than that of
DiFMUP (kcat/Km = 8.1 mMꢁ1 ꢁ1).
s
Considering the structural similarity between PTPs,
the selective hydrolysis of 3 with MKP-6 is remarkable.
To our knowledge, probe 3 is the first small molecule
fluorescent probe that selectively detects a specific PTP
among the large PTP family. Dual specific PTPs have been
reported to possess a wider and shallower active site than
classical PTPs, which might contribute to the selectivity of our
assay.12 Therefore, we infer that classical PTPs do not bind
probe 3 due to the presence of a bulky substituent ortho to the
phenyl phosphate group. The differentiation of MKP-6 from
other dual specific PTPs by probe 3 is however difficult to
explain in this stage because the three dimensional structure
of MKP-6 remains to be elucidated. MKP-6 has been
reported to inhibit the apoptosis of gastric cancer cells
via suppression of the catalytic activity of MAP kinase
subfamilies such as JNK1/2, Erk1/2 and p38 Map kinases.13
Thus, aberrant MKP-6 activity in gastric cells can imply
cancer.
9 J. Huang, A. Peng, H. Fu, Y. Ma, T. Zhai and J. Yao, J. Phys.
Chem. A, 2006, 110, 9079.
In summary, phosphorylation of HBT, a well known
ESIPT compound, was successful in the design of a new
turn-on fluorescence probe. The enzymatic cleavage of probe
3 in the aqueous media showed a strong red-shifted emission
from the keto tautomer within aggregates. In addition,
probe 3 is highly selective to a dual specific PTP, MKP-6,
which is implicated in gastric cancer, but not to other 25 PTPs
tested in this study. Based on this successful proof-of-concept
demonstration, a membrane-permeable derivative of probe 3
for the detection of intracellular MKP-6 activity is now
under investigation.
10 In the PTP assay condition, the characteristic emission peak of the
keto tautomer from HBT was further red-shifted to 535 nm and
even at low concentrations (see ESIw). This might be due that the
assay condition including BSA (5 mM) and NaCl (150 mM),
accentuated red-shifted emission signal from keto tautomer via
interaction or adsorption of HBT on marcromolecules.
11 (a) D. Heffetz, I. Bushkin, R. Dror and Y. Zick, J. Biol. Chem.,
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FEBS J., 2008, 275, 867.
13 L. Bai, S. O. Yoon, P. D. King and J. L. Merchant, Cell Death
Differ., 2004, 11, 663.
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