Angewandte
Chemie
Having quantitatively characterized the behavior of the
caged substrate 3 in vitro, we checked that the protecting
group was suitable for biological applications in a living
organism. We chose the zebrafish, which seems particularly
promising for the use of light to control biological activity: its
embryo is transparent, unpigmented strains are available, and
it has recently emerged as an attractive model animal for
Keywords: donor–acceptor systems · fluorescence ·
photochemistry · photolysis · two-photon excitation
.
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numerous studies. We incubated a zebrafish embryo at the
0–15-somite stage in a 10 mm solution of 3 for 60 min and
1
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ure 2b displays the fluorescence emission spectra with two-
photon excitation recorded within the targeted embryo cell
and the corresponding spectrum of 4 in water. The good
agreement between the spectra suggests that the emitting
fluorophore is indeed coumarin 4 in an aqueous environment.
Then the emission intensity from the fluorescent cell can be
converted to 4 concentration by using the 4 emission rate per
molecule measured by fluorescence correlation spectroscopy
after two-photon excitation. We found values in the 10 mm
range which suggest that the initial intracellular concentration
of caged precursor 3 was essentially similar to the concen-
tration of the incubating solution. As shown in Figure 1b, the
targeted single cell can be easily identified by its final strong
fluorescence emission.
Thus, the 3,5-dibromo-2,4-dihydroxycinnamic caging plat-
form seems promising for implementing the optical-syringe
approach in vivo. The solubility of the 3,5-dibromo-2,4-
dihydroxycinnamic moiety favors cell permeation. Its robust
photophysical and photochemical properties after two-
photon excitation put this caging group among the best
available for substrate uncaging in biological samples. Quan-
titative control of delivery can be achieved by recording
fluorescence emission from the water-soluble coumarin
coproduct 4 that reports on the concentration of photo-
released substrate.
Davies, Nat. Methods 2006, 3, 35 – 40.
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with the release of the desired substrate was already reported
[
(
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3
absence of fluorescence emission in the caged precursor was
associated with the quenching of coumarin fluorescence by the
substrate moiety. This behavior is markedly different from the
present approach, in which the difference in brightness is
expected to be systematically large between the caged substrate
and the released coumarin coproduct whatever the substrate
moiety.
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Received: November 10, 2006
Revised: December 15, 2006
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[17] L. I. Zon, R. T. Peterson, Nat. Rev. Drug Discovery 2005, 4, 35 –
Published online: February 20, 2007
44.
Angew. Chem. Int. Ed. 2007, 46, 2467 –2469
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim