A self-immobilizing and fluorogenic unnatural amino acid that mimics phosphotyrosine

Article abstract of DOI:10.1039/c1cc14653j

Synthesis of the first self-immobilizing, fluorogenic unnatural amino acid that mimics phosphotyrosine (pTyr) is reported. By using solid-phase peptide synthesis, it was subsequently incorporated into peptide-based probes which found applications in bioim

Full text of DOI:10.1039/c1cc14653j

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A self-immobilizing and fluorogenic unnatural amino acid that mimics
phosphotyrosinew
Jingyan Ge, Lin Li and Shao Q. Yao*
Received 29th July 2011, Accepted 31st August 2011
DOI: 10.1039/c1cc14653j
Synthesis of the first self-immobilizing, fluorogenic unnatural amino
acid that mimics phosphotyrosine (pTyr) is reported. By using
solid-phase peptide synthesis, it was subsequently incorporated into
peptide-based probes which found applications in bioimaging and
fluorescence-activated cell sorting (FACS).
Reversible protein phosphorylation plays a fundamental role
Fig. 1 Representative examples of previously reported fluorogenic
in signal transduction. Protein tyrosine phosphatases (PTPs;
PTP substrates, 2-FMPT and Withers’ sugar probe.
4100 members in humans), together with protein tyrosine
reagents due to their tendency to diffuse from the site of the
reaction.7 Activity-based probes of PTPs have been developed
with varied degrees of success, but they generally inhibit PTPs
irreversibly and do not provide Turn-ON fluorescence read-
outs, and thereby are not suitable for cell-based imaging and
FACS experiments.5a–c An alternative strategy to minimize
product diffusion is to, in the probe design, couple fluores-
cence release to an enzyme–probe covalent linkage.6 Based on
this principle, Withers and co-workers recently reported self-
immobilizing fluorogenic imaging agents based on modified
derivatives of coumarin glycosides (Fig. 1) for histological and
FACS studies of glycosidases.8 Inspired by this work, our
newly developed unnatural amino acid (2 shown in Fig. 2) is a
modified version of the phosphorylated coumaryl amino acid 1
(and the ‘‘caged’’ version 10; Fig. 1), which was recently shown
by Barrios and Mitra to be a close mimic of phosphotyrosine
(pTyr).5d We had previously shown in a separate study of another
novel pTyr mimic, 2-FMPT (Fig. 1), that the introduction of a
2-fluoromethyl group in the aromatic ring of pTyr does not
disrupt PTP recognition, but upon dephosphorylation gener-
ates a highly reactive quinone methide intermediate which
subsequently attaches itself covalently to the nearby proteins.9
2-FMPT, however, was not fluorogenic and cannot report
PTP activities in real time in cells. Thus, our new pTyr mimic 2
not only is self-immobilizing and fluorogenic (i.e. like Wither’s
sugar probe), it also possesses both N- and C-terminus (as in
the case of naturally occurring amino acids), allowing essential
PTP-recognizing peptide elements to be introduced when
needed (Fig. 2).
kinases (PTKs), have an essential role in controlling this
key cellular process and maintaining the intricate balance of
the phosphoproteome network. Defective regulation of these
enzymes has significant implications in human diseases.1
Elevated levels of PTP activities are found in numerous
tumor-associated cells and tissues.2 PTPs dephosphorylate
proteins with a high degree of specificity inside the cell,
although in vitro they have shown only moderate specificity
towards synthetic peptide substrates.3 Consequently, chemical
and biological methods that report not only the global PTP
activities, but more importantly the precise enzymatic activity of
PTPs at single cellular and subcellular levels (and ultimately at
the molecular level), may offer unprecedented views on how
these enzymes work under physiological settings.4 Existing
chemical tools have so far focused on the detection of PTP
activities either in vitro or in situ using recombinant PTPs
or cellular lysates.5 More recently, a small molecule-based
imaging probe capable of detecting PTP activities in live cells
has appeared.6 Notwithstanding, most probes are not able to
detect PTP enzymatic activities on the basis of their substrate,
organelle or cell specificity.6 Herein, we have developed the
first self-immobilizing and fluorogenic unnatural amino acid,
which, upon incorporation into suitable peptide sequences,
gave rise to chemical probes suitable for cell-based analysis
(e.g. imaging and FACS) of endogenous PTP activities, thus
successfully addressing some of the above-mentioned issues.
Existing fluorogenic substrates, such as difluoromethyl-
umbelliferone phosphate (DiFMUP; Fig. 1), are widely used
to detect PTP activities in vitro but are not suitable cell-based
Compound 2, the Fmoc-protected form of the unnatural
amino acid, was synthesized in several steps from commercially
available N-a-Fmoc–L-aspartic acid as shown in Fig. 2b.
A 2-nitrobenzyl photolabile protecting group was introduced
in 2 to facilitate chemical synthesis and at the same time
provide temporal control over PTP-probe recognition.6,9
Department of Chemistry, National University of Singapore, 3 Science
Drive 3, Singapore 117543, Singapore. E-mail: chmyaosq@nus.edu.sg;
Fax: +65 67791691; Tel: +65 65162925
w Electronic supplementary information (ESI) available: Detailed
experimental procedures and results. See DOI: 10.1039/c1cc14653j
c
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Fig. 2 (a) Overall working principle of the pTyr mimic 2 and its peptide probes. With SPPS, PTP-recognizing elements or CPPs may be
introduced into the probe. Upon UV-irradiation to uncage the phosphate, the peptide binds to its intended PTP, and gets dephosphorylated. The
ensuring charge delocalization followed by spontaneous elimination of –F generates the highly reactive quinone methide intermediate which either
gets quenched by H2O or diffuses away from the enzyme active site before forming covalent protein–probe complexes (with nearby PTP or other
proteins). (b) Chemical synthesis of 2 and Fmoc-based SPPS of the three CPP-containing probes.
Briefly, the first four steps in the synthesis were based on
previously reported procedures,5d with some modifications, giving
the allyl-protected coumaryl amino acid 6. Subsequently,
formylation of 6 by Duff’s reaction gave 7 in moderate yield
(25%).10 Next, phosphorylation with di(2-nitrobenzyl) chloro-
phosphate under basic conditions provided 8 (46% yield),
which was reduced with NaBH4 (giving 9; 85% yield), followed
by fluorination with DAST to give 10 (74% yield). Finally, the
allyl group in 10 was removed by treatment with Pd(PPh3)4,
giving 11 (90% yield). To obtain the new unnatural amino acid
2, compound 11 was treated with TEA/thiophenol to remove
one of the 2-nitrobenzyl groups. To incorporate the unnatural
amino acid into peptide-based probes, the precursor 11 was
directly used in standard solid-phase peptide synthesis (SPPS)
using Fmoc chemistry (Fig. 2b). We chose to incorporate the
self-immobilizing fluorogenic pTyr mimic into three different
cell-penetrating peptides (CPPs), giving the resulting peptide-
based probes (pMem, pER, and pMito; Fig. 2b). These locali-
zation peptides had previously been used to successfully
deliver enzyme inhibitors into subcellular organelles of live
cells.11 A flexible (Gly)4 linker was inserted between the CPPs
and the unnatural amino acid to minimize potential interferences
of PTP recognition by the CPPs. In the present work, we chose
to focus on the prospect of these peptide-based probes as
agents to study endogenous PTP activities through subcellular
imaging and FACS experiments. In future, additional PTP-
binding peptide fragments may be similarly introduced, by
using the chemistry developed herein, to generate agents that
address the substrate specificity of PTPs (Fig. 2).9
We first examined the photo- and bio-chemical properties of
compound 2 and pER peptide (as a representative example). It
was established that nearly complete uncaging (495%) of the
2-nitrobenzyl group, giving the corresponding PTP-responsive
adducts, could be achieved under 500 mJ cmꢀ2 UV exposure
(inset in Fig. 3a for pER; see ESIw for 2). Subsequent addition
of recombinant PTP1B (a human PTP involved in diabetes
and obesity1) or lysates of HeLa cells (a cancer cell line with
elevated PTP activities6) led to a time-dependent increase in
fluorescence (Fig. 3a and ESIw), with a concomitant release of
the phosphate group (observed by LC-MS; see ESIw). In a
recent study, it was found that the quinone methide generated in
live cells underwent both self-quenching (490%; with aqueous
media) and self-immobilizing (o10%; with nearby proteins)
pathways,6 where the former produced a highly fluorescent
diffusible dye molecule and the latter generated localized
fluorescence. We wondered whether the UV-irradiated 2, when
treated with PTP1B, behaves similarly and produces covalent
adducts with proteins nearby (Fig. 2a).9 As shown in Fig. 3b,
the labelling reaction between PTP1B (over-expressed in bacterial
lysates) and uncaged 2, upon SDS separation and in-gel
fluorescence scanning, confirmed that, while the majority of
2 was self-quenching (see free probe at the gel front; lane 1), a
small amount of PTP1B and other cellular proteins were also
labeled (caused by self-immobilization of 2). Bariros’ coumaryl
amino acid 1, which is a non-self-immobilizing analog of 2,
was used as a negative control; no labeled protein was detected
(lane 2). Lastly, PTP1B treated with uncaged 2 was further
analyzed by mass spectrometry and enzymatic assay; results
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10940 Chem. Commun., 2011, 47, 10939–10941
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Fig. 4 Fluorescence images of HeLa cells treated with different
peptide probes (20 mM, 30–90 min incubation), UV-irradiated, further
incubated (1.5 h) then imaged (in coumarin channel). ‘‘Overlay’’:
images from the coumarin channel overlaid with the tracker channel.
See ESIw for details.
Fig. 3 (a) Time-dependent fluorescence measurements of UV-irradiated
pER (4 mM) in HeLa cell lysates (lex = 360 ꢁ 10 nm; lem = 460 ꢁ 10 nm).
Control (’): non-irradiated pER + lysates. Inset: HPLC profiles of
50 mM pER before (0 s) and after UV-irradiation (120 s). (b) Fluorescence
gel (imaged under a UV transilluminator) of PTP1B-overexpressed
bacterial lysates treated with uncaged 2 (1 mM; lane 1) and 1 (0.5 mM;
lane 2). The corresponding coomassie gel is shown on the left. A small
amount of covalent labelling between 2 and PTP1B and other cellular
proteins was observed. Most of 2 was released as the highly fluorescent free
probe (arrowed). (c) FACS histogram of HeLa cells treated with 80 mM
of uncaged 1 or 2 (‘‘ꢀUV’’ represents cells treated with non-irradiated 2,
a negative control). (d) Relative numbers of stained cells (by uncaged 2;
80 mM) from the FACS results of different mammalian cells (Fig. S9 in
ESIw).
cells were subsequently imaged to detect subcellularly localized,
endogenous PTP activities (Fig. 4); all three peptides gave rise to
strong fluorescence signals only in their intended organelles,
indicating successful delivery and subcellular imaging of PTPs.
In conclusion, we have shown the utility of this newly
developed, self-immobilizing and fluorogenic pTyr mimic in
studying endogenous PTP activities using FACS and bioimaging
experiments. Future work will focus on incorporation of this
unnatural amino acid into peptides and proteins, to engineer
the corresponding enzyme sensors capable of addressing the
substrate specificity of individual PTPs.3,9
Funding support was provided by Ministry of Education
(R-143-000-394-112) and Agency for Science, Technology and
Research (A*Star) of Singapore (R-143-000-391-305).
indicated that most of the PTP1B (490%) in the reaction was
not covalently labeled and still retained its original enzymatic
activity (Fig. S6 and S4, ESIw, respectively).
The self-immobilizing Turn-ON fluorescence properties of
these new PTP probes make them suitable for FACS experi-
ments.8 HeLa cells were first incubated with 2 for 30 min,
allowing sufficient time for the compound to enter the cells.
Subsequently the cells were UV-irradiated, further incubated
for 2 h, then sorted. Compound 1 was used as a negative
control. As shown in Fig. 3c, we observed a noticeable increase
in the number of highly fluorescently labeled cells, over both
non-irradiated cells and cells treated with 1. Repeated washes
of 2-treated cells did not cause significant fluorescence leakage,
indicative of a covalent linkage. We next used 2 to detect PTP
activities in a cell type-specific manner, in anticipation that
most cancers have elevated PTP expression.2 While UV irra-
diation itself did not result in any apparent effect on the cells
(Fig. S8, ESIw), FACS analysis of 5 common mammalian cells,
shown in Fig. 3d, indicated significantly higher fluorescence
counts in cancer cells treated with 2 (MCF-7, HeLa &
HepG2), possibly caused by their elevated endogenous PTP
activities.2
Notes and references
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Finally, we showed that 2, upon incorporation into suitable
peptide sequences, could be used for subcellular imaging of PTP
activities in live cells. pMem, pER, and pMito, each containing a
CPP, served to deliver our pTyr mimic to different subcellular
organelles (membrane, endoplasmic reticulum and mitochondria,
respectively11). Upon UV irradiation, the probe-treated HeLa
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10939–10941 10941