Fluorescence probes for tyrosine dephosphorylation based on coumarin-proline conjugates

Article abstract of DOI:10.1246/cl.2011.290

Proline-substituted coumarin compounds were prepared and used as environment-sensitive fluorescence probes. Phosphorylation and dephosphorylation of tyrosine derivatives labeled with the coumarin-proline conjugate induced marked changes in fluorescence in

Full text of DOI:10.1246/cl.2011.290

doi:10.1246/cl.2011.290
290
Published on the web February 11, 2011
Fluorescence Probes for Tyrosine Dephosphorylation Based on Coumarin-Proline Conjugates
Kihang Choi,* Bongjeong Park, So-Yeong Han, and Hee Choon Ahn
Department of Chemistry and Research Institute for Natural Sciences, Korea University, Seoul 136-701, Korea
(Received January 12, 2011; CL-110033; E-mail: kchoi@korea.ac.kr)
R
O
R
O
F3C
O
Proline-substituted coumarin compounds were prepared and
used as environment-sensitive fluorescence probes. Phospho-
rylation and dephosphorylation of tyrosine derivatives labeled
with the coumarin-proline conjugate induced marked changes in
fluorescence intensity allowing phosphatase activity to be
monitored.
O
O
O
N
N
N
CO2H
CO2H
CO2H
D-CP
R = CF3
L-CP
R = CF3
CS
D-Me-CP R = CH3
L-Me-CP R = CH3
Phosphorylation and dephosphorylation are among the most
important and commonly found modifications of hydroxy-
containing amino acids (Ser, Thr, and Tyr).1 These posttransla-
tional modifications are involved in many cell signal trans-
duction pathways and catalyzed by tightly regulated enzyme
reactions. To explore these biologically important reactions,
many chemical probes have been developed that show fluores-
cence changes upon the phosphorylation and dephosphorylation
of amino acids.2,3 One of the key issues in the design of
fluorescence probes is the proper connection of a fluorophore to
the amino acid being modified with optimal sensitivity.
Coumarin derivatives are one of the most widely used
fluorophores in biological studies with large Stokes shifts, and
we have recently reported the synthesis and conformational
properties of a coumarin-proline conjugate (CP in Chart 1).4 In
this coumarin derivative, the proline is used as an electron-
donating group and can also serve as a rigid linker bridging the
coumarin and an amino acid or peptide that can be attached to
the proline carboxyl group. Thus, this coumarin-proline con-
jugate is a potentially useful fluorescence label to monitor the
structural modification of neighboring amino acids. In this paper,
we report that the phosphorylation and dephosphorylation of
CP-labeled tyrosine derivatives induce modest changes in
fluorescence intensity allowing phosphatase-catalyzed reactions
to be monitored.
First, we connected coumarin-proline conjugate CP to L-
tyrosine methyl ester, and this tyrosine derivative was then
converted to the corresponding phosphotyrosine (Scheme 1).
After phosphorylation of the tyrosine residue, the fluorescence
intensity was increased (Figure 1a) presumably because the
aromatic ring of the phosphotyrosine residue is electron-
deficient and, therefore, cannot quench the coumarin fluores-
cence as efficiently as the electron-rich aromatic ring of the
tyrosine residue.5 Phosphorylation of D-CP-Y induced relatively
large enhancement of emission intensity (ca. 400%) whereas
phosphorylation of L-CP-Y produced only a marginal intensity
change (ca. 50%). According to our previous study on N-
arylproline amide conformations,6 the tyrosine residue would be
located more closely to the coumarin ring in D-CP-Y than in L-
CP-Y so that more efficient fluorescence quenching is expected
for D-CP-Y.
Chart 1.
F3C
O
O
a
OR
CP-Y R = H
D- or L-CP
O
b
c
N
R = PO(OBn)2
N
CO2CH3
H
CP-pY R = PO(OH)2
Scheme 1. Reagents and conditions: (a) L-Tyr-OCH3, BOP-Cl,
i-Pr2EtN, DMF, 95-99%; (b) i-Pr2NP(OBn)2, tetrazole, THF; m-
Cl-C6H4CO3H, CH2Cl2, 74-79%; (c) H2, Pd/C, AcOH, MeOH,
76-78%. BOP-Cl: bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
ride.
Figure 1. Fluorescence emission spectra of tyrosine and
phosphotyrosine compounds with (a) CP and (b) Me-CP
conjugates. [compound] = 1 ¯M in 10 mM Tris buffer
(pH 7.0) with 1% MeOH. -ex = 390 and 365 nm for (a) and
(b), respectively.
serves as a conformationally flexible linker. The tyrosine and
phosphotyrosine derivatives of CS showed little difference in
their emission spectra (data not shown) suggesting that the
selection of a rigid linker is important for the connection of the
tyrosine modification to the coumarin fluorescence change.7
Next, we prepared a second version of coumarin-proline
conjugate, Me-CP, and compared the fluorescence changes
induced by tyrosine phosphorylation. For this CH3-substituted
coumarin conjugate, we devised
a short-step synthesis
(Scheme 2) starting from the nucleophilic aromatic substitution
reaction of 4¤-fluoro-2¤-hydroxyacetophenone with proline. The
coumarin fluorophore was constructed efficiently through the
Wittig reaction of the proline-substituted acetophenone followed
by intramolecular transesterification.8 This new coumarin con-
To further examine the relationship between the linker
structure and the phosphorylation-induced fluorescence change,
we prepared coumarin-sarcosine conjugate CS where sarcosine
Chem. Lett. 2011, 40, 290-291
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

291
O
O
system was successfully used to monitor the tyrosine dephos-
phorylation catalyzed by phosphatases. We are currently work-
ing to apply the coumarin-proline conjugates to study enzyme
reactions responsible for the modification of tyrosine and other
amino acid residues.
O
OH
O
OH
a
c
Me-CP-Y and
Me-CP-pY
N
N
CO2R
CO2R
F
R = H
R = CH3
R = CH3
R = H
This work was supported by a grant (No. 2010-0000176)
and Priority Research Centers Program (No. 2010-0020209) of
MEST/NRF.
b
d
Scheme 2. Reagents and conditions: (a) L- or D-proline,
K2CO3, DMSO, ¦, 99%; (b) CH3I, K2CO3, DMF, 93%; (c)
Ph3P=CHCO2CH3, Ph2O, ¦, 78%; (d) LiOH, H2O, THF, 78%.
References and Notes
1
2
3
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Figure 2. Fluorescence changes during phosphatase-catalyzed
reactions (solid, no enzyme; dashed, phosphatase from potato;
dotted, phosphatase from wheat germ). [D-CP-pYG]0 = 1 ¯M
¹1
and [enzyme] = 6 U L in 50 mM citric acid buffer (pH 5.0)
with 1% MeOH, -ex = 390 nm.
4
5
E.-K. Park, B. Park, J.-H. Choi, K. Choi, M. Cho, J. Phys.
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jugate was then converted to its tyrosine and phosphotyrosine
derivatives following the same procedure described in
Scheme 1. We observed relatively large increase in emission
intensity for the coumarin conjugate with D-proline linker
(Figure 1b), and this is consistent with the previous result with
CP conjugates. However, the intensity change is much smaller
for the tyrosine-phosphotyrosine pair with Me-CP than for the
one with CP analog, probably due to the difference in the
reduction potentials,9 suggesting that the CF3-substituted cou-
marin conjugate is better for the monitoring of tyrosine
phosphorylation.
With the effect of the tyrosine modification on the fluores-
cence at hand, we tried to apply the coumarin-proline conjugate
system to enzyme-catalyzed reactions. To avoid possible
interference caused by the hydrolysis of carboxylic acid
esters, we prepared D-CP-pYG as a phosphotyrosine substrate
(Figure 2). Dephosphorylation by acid phosphatases proceeded
smoothly showing a modest decrease in fluorescence intensity.
At the completion of the reaction, the intensity was reduced
by 75%, and this change is consistent with the fluorescence
difference observed with the tyrosine-phosphotyrosine pair with
D-CP (Figure 1a).
For the examples of fluorescence quenching by a tyrosine
residue, see: a) Q. Wang, S. M. Cahill, M. Blumenstein, D. S.
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Although detailed photophysical studies are required to fully
understand this phenomenon, it has been shown that a rigid
linker can facilitate fluorescence quenching by limiting the
conformational degree of freedom and, as a result, stabilizing
the conformations responsible for donor-acceptor interaction.
See: a) F. D. Lewis, J. M. Wagner-Brennan, J. M. Denari,
J. Phys. Chem. A 1998, 102, 519. b) C. Toniolo, F.
Formaggio, M. Crisma, J.-P. Mazaleyrat, M. Wakselman, C.
George, J. R. Deschamps, J. L. Flippen-Anderson, B. Pispisa,
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Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.
html.
6
7
8
9
In summary, we have prepared coumarin-proline conjugates
and studied the fluorescence changes induced by the phospho-
rylation and dephosphorylation of a neighboring tyrosine
residue. Two structurally similar coumarin fluorophores showed
quite different fluorescence behaviors, and the CF3-substituted
compound with D-proline linker afforded modest changes in
emission intensity upon the tyrosine modification. This probe
For the comparison of reduction potentials and quenching
efficiencies of CH3- and CF3-substituted coumarin deriva-
tives, see: a) C. A. M. Seidel, A. Schulz, M. H. M. Sauer,
J. Phys. Chem. 1996, 100, 5541. b) R. J. Cave, E. W. Castner,
Jr., J. Phys. Chem. A 2002, 106, 12117.
Chem. Lett. 2011, 40, 290-291
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/