Sequence selective dual-emission detection of (i, i + 1) bis-phosphorylated peptide using diazastilbene-type Zn(ii)-Dpa chemosensor

Article abstract of DOI:10.1039/b905814a

This paper describes a new fluorescent chemosensor for phosphorylated peptide, which comprises a rigid trans-4,4′-diazastilbene and two Zn(ii)-Dpa (2,2′-dipicolylamine) units; this chemosensor sequence-selectively binds to a (i, i + 1) bis-phosphorylated peptide and displays a dual-emission fluorescence change.

Full text of DOI:10.1039/b905814a

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COMMUNICATION
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Sequence selective dual-emission detection of (i, i + 1)
bis-phosphorylated peptide using diazastilbene-type
Zn(II)-Dpa chemosensorwz
Yoshiyuki Ishida, Masa-aki Inoue, Tomonori Inoue, Akio Ojida and Itaru Hamachi*
Received (in Cambridge, UK) 23rd March 2009, Accepted 16th April 2009
First published as an Advance Article on the web 24th April 2009
DOI: 10.1039/b905814a
This paper describes
a
new fluorescent chemosensor for
complex 1-2Zn(II) as a fluorescent chemosensor for bis-
phosphorylated peptide. The unique molecular design of
1-2Zn(II), in which the Zn(II)-Dpa sites and the fluorescent
phosphorylated peptide, which comprises a rigid trans-4,40-
diazastilbene and two Zn(II)-Dpa (2,20-dipicolylamine) units;
this chemosensor sequence-selectively binds to a (i, i + 1)
unit are directly conjugated with
a
rigid trans-4,40-
bis-phosphorylated peptide and displays
fluorescence change.
a
dual-emission
diazastilbene unit, provides a high binding selectivity for
(i, i + 1) bis-phosphorylated peptide and a clear dual-emission
signal change in the formation of the binding complex.
The synthesis of 1-2Zn(II) is outlined in Scheme 1. Briefly,
vinylpyridine derivative 4 was obtained by Stille coupling,
followed by an olefin metathesis reaction using Grubb’s
catalyst to yield trans-4,40-diazastilbene 5. Acid catalyzed
deprotection and subsequent reductive amination with
N-methylpicolylamine afforded the ligand 1. Finally,
complexation with 2 equiv. of Zn(NO3)2 yielded the 1-2Zn(II).
Details of the synthetic procedure for 1-2Zn(II) are provided in
the ESI.z
Protein phosphorylation is a prevalent post-translational
modification crucial for controlling many protein functions.
A variety of biological and chemical techniques have been
recently developed to realize precise and sensitive detection of
these phosphorylation events.1,2 Among them, fluorescence
sensing with a small molecular-based chemosensor is a
promising technique to directly detect protein phosphorylation
in a convenient manner without the need for special instru-
ments. Several fluorescent chemosensors have been recently
developed for this purpose, which displayed a fluorescent
signal change upon binding to phosphorylated proteins and
peptides.3–7 Although they might be useful to simply detect
whether protein is in a phosphorylated state or not,8–10
sequence- or site-specific detection of a certain protein phospho-
rylation among various phosphorylation sites still remains a
challenge.
The absorption maximum of 1-2Zn(II) is observed at
294 nm, and when the complex is excited at 294 nm, the
emission maximum is 385 nm (Fig. 2). The fluorescence
quantum yield (F) of 1-2Zn(II) is below 0.01 under aqueous
neutral conditions (50 mM HEPES buffer, pH 7.2). To
evaluate the sensing selectivity of 1-2Zn(II) for bis-phosphorylated
peptides, we performed the fluorescence titration with a series
of phosphorylated peptides possessing two phosphorylated
amino acid residues at (i, i + n) positions (n = 1, 2, 3, 4, 6),
all of which are derived from the natural sequence of the
hyperphosphorylated tau protein reported in the literature
(Table 1).11 Fig. 2 shows the fluorescence spectral change of
1-2Zn(II) upon addition of Tau(400–409)-2P(i + 1) peptide.
Interestingly, a clear dual-emission change took place, that is
the initial emission maximum at 385 nm gradually decreased
We have recently reported that binuclear Zn(II)-Dpa
(2,20-dipicolylamine) complexes such as 2-2Zn(II) strongly
bind to bis-phosphorylated peptides in neutral aqueous
conditions through cross-linking interaction with the two
phosphorylated residues (Fig. 1).5,6 Although 2-2Zn(II)
showed a high binding selectivity for bis-phosphorylated
peptides (maximally 107 Mꢀ1 in binding affinity) against
mono-phosphorylated ones, the binding selectivity among
the doubly phosphorylated peptides at the different (i, i + n)
positions (n = 4, 8, 12) was moderate. This is partly due to its
rather flexible structure, which allows the conformation
change to result in non-selective binding with these peptides.
Another drawback of 2-2Zn(II) is its small fluorescence signal
change at a relatively shorter wavelength upon binding to a
bis-phosphorylated peptide, which makes it difficult to
precisely analyze a certain protein phosphorylation event. In
this communication, we report a new binuclear Zn(II)-Dpa
Department of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto University, Katsura campus,
Kyoto, 615-8510, Japan. E-mail: ihamachi@sbchem.kyoto-u.ac.jp
w Dedicated to Seiji Shinkai on the occasion of his 65th birthday.
z Electronic supplementary information (ESI) available: Syntheses and
compound characterizations of 1-2Zn(II) and the phosphorylated peptides
listed in Table 1. The CD data of 1-2Zn(II). See DOI: 10.1039/b905814a
Fig. 1 Structures of fluorescence probes for bis-phosphorylated
peptides (above), and illustration of their cross-linking binding with
bis-phosphorylated peptide (below).
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Scheme 1 Synthesis of 1-2Zn(II).
Fig. 3 Isothermal calorimetric titration of 1-2Zn(II) (0.1 mM) with 25
aliquots (10 mL each) of Tau(400–409)-2P(i + 1) (2.0 mM) in 50 mM
HEPES buffer at pH 7.2 and 25 1C. The solid line represents the best fit
of the experimental data.
the bis-phosphorylated peptide. None of the fluorescence
change was induced by the corresponding mono-phosphorylated
peptide Tau(400–409)-1P (Table 1), implying that 1-2Zn(II)
interacts with the bis-phosphorylated Tau(400–409)-2P(i + 1)
peptide through the cross-linking binding mode so as to
Fig. 2 Fluorescence spectral changes of 1-2Zn(II) (10 mM) in 50 mM
HEPES buffer at pH 7.2 upon addition of Tau(400–409)-2P(i + 1) at
25 1C, lex = 323 nm. Inset: Fluorescence titration profile of 1-2Zn(II)
(lem = 378 nm and 427 nm) against the concentration of
Tau(400–409)-2P(i + 1). The solid line is the best fit of the data to
show
a dual-emission change with a strong binding
affinity. CD measurement of the mixture of 1-2Zn(II) and
Tau(400–409)-2P(i + 1) showed a large Cotton peak in the
absorbance region of 1-2Zn(II) from 230 to 380 nm (Fig. S1z),
further supporting the formation of a conformationally
restricted cross-linking binding complex. The dual-emission
change was also observed in the titration with the (i, i + 2) bis-
phosphorylated peptides such as Tau(204–217)-2P(i + 2) and
Tau(231–238)-2P(i + 2), however the emission changes
(Fig. 4) and the binding affinities (Table 1) were apparently
smaller than in the case of Tau(400–409)-2P(i + 1) peptide.
The fluorescence changes and the binding affinities were
also small for other peptides that have the two phosphorylated
residues at more distant positions, such as Tau(210–220)-
2P(i + 3), Tau(204–217)-2P(i + 4), and Tau(204–217)-
2P(i + 6) (Fig. 4 and Table 1). Overall these results indicate
that 1-2Zn(II) is a selective chemosensor for (i, i + 1) bis-
phosphorylated peptide with a dual-emission signal change.
This high sequence selectivity would be reasonably ascribed to
the equation for 1 : 1 complex formation.
and a new emission at a longer wavelength (lmax = 425 nm)
concomitantly emerged. This signal change allowed us to
detect 10ꢀ6 M of the peptide in the fluorescence titration.
The ratio plot of the fluorescence intensity at two wavelengths
(378 nm and 427 nm) shows that the ratio value R increases up
to nearly 4-fold (Fig. 4). The binding constant of 1-2Zn(II)
with Tau(400–409)-2P(i + 1) peptide was evaluated to be
6.1 ꢂ 105 Mꢀ1 by curve fitting analysis. This binding behavior
was further evaluated by ITC (isothermal titration calorime-
try, Fig. 3), showing a binding constant (Ka) of 6.3 ꢂ 105 Mꢀ1
with a 1 : 1 stoichiometry (N = 1.02), almost identical with
data from the fluorescence titration. It is clear that the
emission response of 1-2Zn(II) directly reflects the binding to
Table 1 Binding constants for the complexation of 1-2Zn(II) with phosphorylated peptide determined by a fluorescence titration experiment
Peptide sequencea
P-sitesb
105 Kapp/Mꢀ1c,d
e
—
Tau(400–409)-1P :
YSGDTpSPRHLS
i
Tau(400–409)-2P(i + 1) :
Tau(204–217)-2P(i + 2) :
Tau(231–238)-2P(i + 2) :
Tau(210–220)-2P(i + 3) :
Tau(204–217)-2P(i + 4) :
Tau(204–217)-2P(i + 6) :
YSGDpTpSPRHLS
YGTPGSRSRpTPpSLPT
YTPPKpSPpSS
YSRTPpSLPpTPPT
YGTPGpSRSRpTPSLPT
YGTPGpSRSRTPpSLPT
i, i + 1
i, i + 2
i, i + 2
i, i + 3
i, i + 4
i, i + 6
6.1 (6.3)
0.2 (0.8)
0.6
0.2
0.08
e
—
a
b
N-terminal tyrosine (Y) was introduced for determination of the peptide concentration from UV absorbance. P-sites designates the position of
c
phosphorylated amino acid residues on the peptide. Binding constants (Kapp) were determined by curve fitting with a theoretical equation for 1 : 1
d
complexation. The values in parentheses were determined by an ITC experiment. Binding constant could not be evaluated due to the small
e
increase of the fluorescence intensity (lem = 427 nm).
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Almost the same excitation spectra were obtained from the
solution of 1-2Zn(II) (lem = 385 nm) and its mixture with
Tau(400–409)-2P(i + 1) (lem = 427 nm), indicating that the
emission shift is caused by the electronic configuration change
in the excitation state. Also, we noticed that an almost
identical spectral change occurred on addition of inorganic
phosphate although the affinity is much smaller than for
the (i, i + 1) peptide (See Fig. 4).y This implies that the
cross-linking binding which may affect the planarity of the
diazastilbene fluorophore is not essential for the dual-emission
changes.12z Instead, it may be proposed that the coordination
of phosphate anions to Zn(II)-Dpa sites modulates an excited-
state through several factors such as a solvent reorientation
process, resulting in the emission shift. Further photophysical
study is required to fully understand the detailed sensing
mechanism.
Fig. 4 Changes of the ratio value R (F427/F378) of 1-2Zn(II) (10 mM in
50 mM HEPES buffer at pH 7.2) in the titration with Tau(400–409)-
2P(i + 1) (K), Tau(204–217)-2P(i + 2) (.), Tau(210–220)-2P(i + 3)
(E), Tau(204–217)-2P(i + 4) (’) and inorganic phosphate (J).
In summary, we have revealed that 1-2Zn(II) is a selective
fluorescent chemosensor for (i, i + 1) bis-phosphorylated
peptide with
a dual-emission change among various
phosphorylated peptides. This sensing selectivity and signal
change are achieved on the basis of the simple but sophisticated
molecular design of 1-2Zn(II), i.e., direct conjugation of the
Zn(II)-Dpa sites to the rigid fluorophore with a minimized
conformational flexibility. As a general design strategy, exten-
sion of the conjugated olefin unit of the chemosensor may
allow us to create fluorescent chemosensors selective for other
types of peptides doubly phosphorylated at more distant
positions. Our research is ongoing along this line.
Notes and references
y 1-2Zn(II) also showed a similar dual-emission change towards
inorganic pyrophosphate (PPi) and inositol 1,4,5-triphosphate (IP3)
with a strong binding affinity (Kapp 4106 Mꢀ1), suggesting that these
poly-phosphate species competitively inhibit the sensing of the
phosphorylated peptides.
z Little emission shift was observed in the temperature-dependent
fluorescence measurement from 25 to ꢀ78 1C, also suggesting that the
emission shift is not simply caused by the conformational rigidification
of the diazastilbene unit in the cross-linking binding.
Fig. 5 Photograph of the aqueous solutions of 1-2Zn(II) (20 mM)
in the absence (a) and presence of the phosphorylated peptide
(40 mM): Tau(400–409)-2P(i + 1) (b), Tau(204–217)-2P(i + 2) (c),
Tau(210–220)-2P(i + 3) (d), Tau(204–217)-2P(i + 4) (e) irradiated
with 365 nm light.
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the rigid and compact structure of 1-2Zn(II), which is suitable
to bind to the two phosphate groups positioned adjacent to
each other.
Fig. 5 displays a photograph of the aqueous solution of
1-2Zn(II) in the absence and presence of various bis-
phosphorylated peptides. Distinct blue–green fluorescence
was selectively observed in the solution containing
Tau(400–409)-2P(i + 1) peptide, whereas the blue emission
was negligible in the case of Tau(204–217)-2P(i + 2),
Tau(210–220)-2P(i + 3) and Tau(204–217)-2P(i + 4). Thus,
we can visually discriminate the (i, i + 1) peptide from other
bis-phosphorylated peptides by using 1-2Zn(II) as a fluorescent
indicator.
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To gain insight into the mechanism of the dual-emission
change, we performed several spectroscopic experiments.
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This journal is The Royal Society of Chemistry 2009
2850 | Chem. Commun., 2009, 2848–2850