Two-photon uncaging with fluorescence reporting: Evaluation of the o-hydroxycinnamic platform

Article abstract of DOI:10.1021/ja0722022

This paper evaluates the ohydroxycinnamic platform for designing efficient caging groups with fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by ¹H NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10% range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a series of techniques based on fluorescence emission to measure the action uncaging cross sections with two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish embryos. Reliable structure property relationships were extracted from analysis of the present collected data. In particular, the careful kinetic analysis allows us to discuss the relevance of the ohydroxycinnamic platform for diverse caging applications with one- and two-photon excitation.

Full text of DOI:10.1021/ja0722022

Published on Web 07/21/2007
Two-Photon Uncaging with Fluorescence Reporting:
Evaluation of the o-Hydroxycinnamic Platform
Nathalie Gagey,† Pierre Neveu,†,‡ Chouaha Benbrahim,† Bernard Goetz,#
Isabelle Aujard,† Jean-Bernard Baudin,† and Ludovic Jullien*,†
Contribution from the EÄ cole Normale Supe´rieure, De´partement de Chimie, UMR
CNRS-ENS-UniVersite´ Paris 6 8640 PASTEUR, 24, rue Lhomond,
75231 Paris Cedex 05, France, EÄ cole Normale Supe´rieure, Laboratoire de Physique Statistique,
UMR CNRS-ENS-UniVersite´ Paris 6 and Paris 7 8550, 24, rue Lhomond,
75231 Paris Cedex 05, France, and EÄ cole Normale Supe´rieure, De´partement de Chimie, UMR
CNRS-ENS-UniVersite´ Paris 6 8642; 24, rue Lhomond, 75231 Paris Cedex 05, France
Received March 29, 2007; E-mail: ludovic.jullien@ens.fr
Abstract: This paper evaluates the o-hydroxycinnamic platform for designing efficient caging groups with
fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in
one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption
that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by 1H
NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the
caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly
fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield
of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10%
range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a
series of techniques based on fluorescence emission to measure the action uncaging cross sections with
two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they
satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging
behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish
embryos. Reliable structure property relationships were extracted from analysis of the present collected
data. In particular, the careful kinetic analysis allows us to discuss the relevance of the o-hydroxycinnamic
platform for diverse caging applications with one- and two-photon excitation.
Introduction
biologically nonactive precursor the desired chemical after two-
photon photoactivation (or uncaging).6
The spatio-temporal delivery pattern of precise given amounts
of chemicals is of major importance in chemistry and biology.
Syringes have been used to achieve this goal at the microscopic
level. However such microinjections, by their invasive nature,
are not appropriate if the targeted organism has to remain intact.
Given the excellent intrinsic localization in two-photon excita-
tion,1 photoactivation has been envisioned to provide an
attractive noninvasive alternative: the “optical microsyringe’’.2-5
In the corresponding approach, a focused light pulse is used to
release within an organism perfused with a solution of a
A large enough two-photon action uncaging cross section is
a key factor to satisfactorily implement the preceding two-
photon photoactivation strategy in sensitive samples. In fact,
the cross section should be as large as possible to reduce the
illumination duration and power needed to release a given
amount of effector without generating detrimental effects. For
instance, it was claimed to have to exceed 0.1 or 10 Goeppert-
Mayer (GM; 1 GM ) 10-50 cm4s/photon) for biological
applications.3,7 In fact, the 1 GM range has already been
reached,3,8,9 and the corresponding caging groups have been
successfully used in biological systems. Nevertheless the
improvement of the efficiency of a photolabile protecting group
upon two-photon excitation remains difficult, and it is therefore
useful to gain reliable structure property relationships. Indeed
we still lack general rules to predict the cross section for two-
† UMR CNRS-ENS-Universite´ Paris 6 8640 PASTEUR.
‡ UMR CNRS-ENS-Universite´ Paris 6 and Paris 7 8550.
# UMR CNRS-ENS-Universite´ Paris 6 8642.
(1) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 10763-10768.
(2) Cambridge, S. B.; Davis, R. L.; Minden, J. S. Science 1997, 277, 825-
828.
(3) Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.;
Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Nat. Acad. Sci. U.S.A.,
1999, 96, 1193-1200.
(6) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed., 2006, 45, 4900-4921.
(7) Kiskin, N.; Chillingworth, R.; McCray, J. A.; Piston, D.; Ogden, D. Eur.
Biophys. J. 2002, 30, 588-604.
(4) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001, 28,
317-325.
(8) Lu, M.; Fedoryak, O. D.; Moister, B. R.; Dore, T. M. Org. Lett. 2003, 5,
2119-2122.
(5) Momotake, A.; Lindegger, N.; Niggli, E.; Barsotti, R. J.; Ellis-Davies, G.
C. R. Nat. Methods 2006, 3, 35-40.
(9) Gagey, N.; Neveu, P.; Jullien, L. Angew. Chem., Intl. Ed. 2007, 46, 2467-
2469.
9
9986
J. AM. CHEM. SOC. 2007, 129, 9986-9998
10.1021/ja0722022 CCC: $37.00 © 2007 American Chemical Society

Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
photon absorption of a chromophore from its structure. In
addition, it is not obvious to predict the quantum yield of
uncaging after two-photon excitation, even when the corre-
sponding quantum yield of uncaging after one-photon excitation
is known. Indeed the mechanism leading to uncaging could
depend on the excitation mode.
of the thermal reactions involved in the formation of the
coumarin coproduct accompanying the release of the caged
substrate are already well-documented.22,23 Eventually the
reported cinnamates intrinsically do not fluoresce, in marked
contrast with the photoreleased coumarin coproduct. The latter
behavior is here particularly attractive in relation to the intended
purpose of having a fluorescent reporter upon uncaging.
The present paper examines the o-hydroxycinnamic platform
to tailor efficient photolabile protecting groups that exhibit large
two-photon uncaging action cross sections and that release
strongly fluorescent coproducts upon uncaging. The first section
deals with the design and the syntheses of two series of model
o-hydroxycinnamate that differ by the substitution pattern of
the double bond. The second section evaluates the present series
of photolabile protecting groups with one-photon excitation.
After an initial screen to identify the most promising substitution
patterns, we characterize the one-photon absorption properties
of the compounds, then we determine the uncaging mechanism,
the corresponding kinetics associated to the photochemical step
and the subsequent thermal step, and the uncaging action cross
section with one-photon excitation. In the third section, we
determine the uncaging action cross sections with two-photon
excitation of the most promising caging groups in the investi-
gated series. Then we demonstrate the relevance of the o-
hydroxy platform for biological applications by investigating
two-photon uncaging in a zebrafish embryo. Section 4 is devoted
to the discussion. In particular, we show that the choice of the
“best’’ o-hydroxycinnamic caging moiety is not intrinsic but
depends on the considered application and on the experimental
constraints. Concluding remarks are eventually given in
section 5.
Another important issue is the quantification of delivery upon
uncaging, when one is interested to reach a critical molecule
concentration to induce a specific activity. First, calibration of
the laser power can be made difficult in complex systems (e.g.,
a living organism) because of the uncertainties associated to
many unknown medium characteristics regarding volume,
viscosity, optical homogeneity, etc. Second, the system response
can strongly depend on the amount of uncaged substrate (e.g.,
biological response resulting from the interaction between a
photoreleased ligand and its biological target). An elegant way
to address this quantification task relies on the simultaneous
release of a fluorescent reporter and the desired caged substrate
from the same nonfluorescent caged precursor. Quantitative
analysis could be then simply obtained from analyzing the
increase of fluorescence emission after or better during uncaging
at the targeted site. Whereas numerous caging groups have been
continuously introduced with the goal to improve their photo-
physical and photochemical characteristics,10-12 uncaging quan-
tification using an optical method has attracted little attention.
Indeed, in most cases, the caged substrate and the coproduct
from the photolabile moiety after uncaging exhibit similar
brightnesses. From the latter point of view, the coumaryl
photolabile protecting group was shown to be satisfactory in
several occurrences.13 Nevertheless the absence of fluorescence
emission in the caged substrate was not intrinsic in the reported
systems but was associated to the quenching of the reporter
fluorescence by the substrate moiety: Hence, beyond the
restriction for caging acidic substrates, the coumaryl photolabile
protecting group lacks of generality for the reporting purpose.
Design and syntheses
Our investigation of the o-hydroxycinnamic platform for
uncaging applications starts from the original design of Porter
(Figure 1).24
The preceding considerations led us to consider with high
interest the o-hydroxycinnamic platform that was introduced
to cage alcohols and amines by Porter et al.14-16 In this series,
the primary event leading to uncaging is light absorption by
the cinnamic backbone that belongs to the extensively inves-
tigated series of donor-acceptor ethylenic systems. In the
context of two-photon excitation, the wavelength maximum and
the value of the cross section for two-photon absorption of such
donor-acceptor ethylenic systems can be now reasonably
predicted from the molecular structure.17,18 Their photophysical
behavior is also satisfactorily established, in particular the
competition between fluorescence emission and isomerization
in the excited state.19-21 In addition, the mechanism and kinetics
Retaining a same cinnamate series leading to alcohol release
upon uncaging, we aimed at studying the significance of
different structural parameters to optimize the photorelease
efficiency. As suggested in the introduction, we were especially
interested in establishing structure-property relationships to
improve the uncaging action cross sections with two-photon
excitation as well as the fluorescence reporting features.
Nevertheless, we also considered other aspects such as thermal
(22) Hershfield, R.; Schmir, G. L. J. Am. Chem. Soc. 1973, 95, 7359-7369.
(23) Hershfield, R.; Schmir, G. L. J. Am. Chem. Soc. 1973, 95, 8032-8040.
(24) In the course of the present work, we also considered relying on the
o-aminocinnamic platform for uncaging applications as reported by Porter
in ref 16. Indeed we expected that this series should be particularly favorable
to get large k2 constants because of the lower electronegativity of the
nitrogen atom with regard to the oxygen one. Thus we synthesized the
3-(6-amino-benzo(1,3)dioxo-5-yl)-acrylic acid ethyl ester in two steps by
condensing the 1-carboxymethyliden triphenyl phosphorane on 6-amino-
3,4-methylenedioxybenzaldehyde that was obtained from Fe/HCl reduction
of 6-nitro-3,4-methylenedioxybenzaldehyde in a mixture of acetic acid,
ethanol, and water. 3-(6-Amino-benzo(1,3)dioxo-5-yl)-acrylic acid ethyl
ester exhibits satisfactory absorption properties: in Tris (pH ) 7) 20 mM
(10) Pillai, V. N. R. Synthesis 1980, 1-26.
(11) Bochet, C. G. J. Chem. Soc., Perkin Trans. 2002, 1, 125-142.
(12) Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002, 1, 441-458.
(13) Hagen, V.; Frings, S.; Bendig, J.; Lorenz, D.; Wiesner, B.; Kaupp, U. B.
Angew. Chem., Intl. Ed. 2002, 41, 3625-3628.
(14) Turner, A. D.; Pizzo, S. V.; Rozakis, G.; Porter, N. A. J. Am. Chem. Soc.
1988, 110, 244-250.
NaCl 100 mM buffer/acetonitrile 1/1 (v/v), ꢀE(λ(E1)) ) 13 × 103 M-1 cm-1
(15) Wang, B. H.; Zheng, A. Chem. Pharm. Bull. 1997, 45, 715-718.
(16) Li, H.; Yang, J. H.; Porter, N. A. J. Photochem. Photobiol. A 2005, 169,
289-297.
at 385 nm. The quinolone resulting from uncaging is also reasonably
fluorescent (λ(F1) ) 390 nm; Φ(F1) ) 7%), although less than the coumarins
obtained from the present o-hydroxycinnamic derivatives. Unfortunately,
whereas k2 was indeed so large that we were unable to measure it (subsecond
time scale; see ref 16), k1 was found to be extremely low (1.7 10-5 s-1
(17) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481-491.
(18) Cogne´-Laage, E.; Allemand, J. F.; Ruel, O.; Baudin, J. B.; Croquette, V.;
Blanchard-Desce, M.; Jullien, L. Chem.sEur. J. 2004, 10, 1445-1455.
(19) Dauben, W. G.; Salem, L.; Turro, N. J. Acc. Chem. Res. 1975, 8, 41-54.
(20) Burghardt, I.; Cederbaum, L. S.; Hynes, J. T. Faraday Discuss. 2004, 127,
395-411.
with I0(λ1exc) ) 2.1 10-7 einstein min-1; k1 is here typically two orders of
magnitude smaller than in the o-hydroxycinnamate series; see Table 1S)
forbidding to rely on the o-aminocinnamic platform to develop an efficient
series of photolabile protecting groups for two-photon uncaging applications.
(21) Burghardt, I.; Hynes, J. T. J. Phys. Chem. A 2006, 110, 11411-11423.
9
J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007 9987

A R T I C L E S
Gagey et al.
Chart 2. Structures of the E Series of Photolabile Protecting
Groups Investigated in the Present Study
Scheme 1. Syntheses of the Caged Model Alcohols (X ) H and
X ) Me in the E and E′ Series, Respectively)
Figure 1. The o-hydroxycinnamic platform to illustrate uncaging with
fluorescence reporting. The intrinsically nonfluorescent cinnamate of the
alcohol substrate ROH is illuminated in a wavelength range where it absorbs
light (violet and red arrows with one- and two-photon excitation, respec-
tively). Light absorption initiates a series of photochemical and thermal
processes in the o-hydroxycinnamate backbone leading to release the ROH
substrate together with the fluorescent coumarin F coproduct in a one-to-
one stoichiometric ratio. As the starting cinnamate and the coumarin
coproduct absorb light in the same wavelength range (black solid and dotted
lines, respectively), the F coumarin continuously emits fluorescence (blue
solid line) upon uncaging and its fluorescence intensity reports on the
uncaging extent. The absorption and emission properties of the cinnamate
Ec at 293 K in Tris(pH ) 7) 20 mM NaCl 100 mM buffer/acetonitrile 1/1
(v/v) are used for a purpose of illustration.
related to its neutrality with regards to the photophysical and
photochemical properties of the cinnamate backbone.
In both series, the substitution pattern of the phenyl ring was
designed to locate the maximum of two-photon absorption in
the 700-900 nm wavelength range that is currently available
with the common IR pulsed laser sources. In particular, we
grafted several electron-releasing moieties on the phenyl position
para to the vinyl carboxylate group. The molecular design was
here facilitated by the resulting unsymmetrical donor-acceptor
conjugated structure for which the position of the two-photon
absorption maximum usually satisfactorily compares with twice
the corresponding one of the one-photon absorption.17,18
In the present series, the syntheses are short, easy, and rely
on commercially available or readily accessible synthons. All
the cinnamic esters displayed in Charts 1 and 2 were synthesized
with good yields in one step by condensing the substituted
salicylaldehydes with the appropriate phosphorane (Scheme 1).25
It is essential here to pay attention to protecting the reactive
medium from light and to accurately controlling heat and time
to reduce the formation of the coumarin resulting either from
photo- or thermal isomerizations.
Chart 1. Structures of the E′ Series of Photolabile Protecting
Groups Investigated in the Present Study
Uncaging with One-Photon Excitation. The physicochem-
ical experiments with one-photon excitation were performed
stepwise. First, we screened the photophysical and photochemi-
cal behavior of the E′a-h series in aqueous solutions. The
purpose was here to evaluate the solubility issue, the wavelength
of maximum light absorption with one-photon excitation and
the robustness of the photochemical behavior leading to the
alcohol release. The results led us to identify (i) the members
of the second series Ea-i by keeping the most promising
substituents on the phenyl group of the cinnamate and (ii)
appropriate experimental conditions, in particular the solvent,
to allow comparisons between the different cinnamates.
Preliminary Screening. In view of possible biological
applications, the E′a-h series has been first tentatively evaluated
in aqueous solutions. At that step, its members that did not
exhibit enough water solubility at the submillimolar concentra-
tions required for NMR, and UV-vis absorption experiments
were dissolved after adding a small amount of cosolvent. The
cinnamate solutions were irradiated with one-photon excitation
stability, water solubility, kinetics of substrate release, etc.,
which can prove to be crucial for biological applications.
To examine the role of the substitution pattern of the double
bond, we synthesized two closely related series of cinnamates
sharing the same (E) stereochemistry. Chart 1 and Chart 2
displays the two different E′ and E series that were evaluated
during the present study.
The E′ series relies on the original design of Porter: It
incorporates a methyl substituent on the trisubstituted double
bond. In contrast, the E series contains only disubstituted
derivatives in which the double bond bears the carboxylate group
on one hand and the substituted aromatic moiety on the other
hand. Both series are ethyl cinnamate leading to the release of
ethanol upon uncaging. The choice of the ethyl moiety was here
(25) Porter, N. A.; Thuring, J. W., H. Li J. Am. Chem. Soc. 1999, 121, 7719-
7717.
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Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
Table 1. Photophysical and Photochemical Properties of the Investigated (E)-Cinnamates That Are Relevant to the Uncaging Process with
One- and Two-Photon Excitationa
(1)
exc
(1)
EZ
(1)
EZ
1
(2)
EZ
10-3ꢀE
(
λ(1)
)
λ(F1)
nm[%]
[
Φ(F1)
]
λ
Φ
ꢀE
(
λ(1)
)
Φ
k2, k2
10-3 s-
δ
E(750)
Φ
E
exc
M-1 cm-1 (nm)
nm
QE/QF
%
mM-1 cm-
K1(λ(1)
)
exc
GM
1
Ea
Eb
Ec
Ed
Ei
E′a
E′b
E′d
E′f
E′g
18(331)
16(360)
25(369)
28(378)
20(394)
17(319)
14(344)
31(362)
15(317)
6(339)
452 [40]
423 [30]
454 [70]
475 [60]
482 [50]
461 [50]
421 [40]
473 [40]
391 [40]
408 [40]
350
350
350
360
370
330
350
350
330
350
0.005
c
9
3
5
2
3
1.0
0.4
1.2
0.4
0.4
1.0
1.3
2.7
0.8
1.2
0.2
0.2
0.5
0.1
0.1
0.8
3.5
0.9
0.3
0.1
1.6,1.6
3.2,4.8
2.2,2.2
3.7,6.2
9.5,9.1
10,8
30,35
12,11
13,13
20,30
b
3.8
0.6
2.0
4.7
b
b
b
b
2.5
0.025
0.060
0.015
0.070
0.050
0.050
0.055
0.003
7
10
10
7
25
a Maxima of single-photon absorption (λ(E1)) and molar absorption coefficients for single-photon absorption at λE(1), ꢀE(λ(1)) ( (5%) of the (E)-cinnamates;
E
wavelength maxima (λF(1)) and quantum yields (Φ(F1) (10%) of fluorescence emission of the coumarin coproducts upon uncaging after one-photon
excitation; excitation wavelength used for the series of uncaging experiments with one-photon excitation, λ(e1x)c, relative brightness of the starting
(1)
exc
(E)-cinnamate with regard to brightness of the F coumarin coproduct at λ , QE/QF, quantum yield of (E) to (Z) photoisomerization after one-photon
excitation at λe(1x)c, Φ(1) ( (10%), action cross section for (E) to (Z) photoisomerization with one-photon excitation at λ(1) , ꢀE(λe(1x)c)Φ(1) ( (5%), steady-state
EZ
exc
EZ
constant K1(λ(1) ) ) k1(λ(1) )/k-1(λ(e1x)c) ( (5% ) associated to the E to ZI exchange with one-photon excitation at λ(e1x)c, rate constant for the thermal (Z)-
cinnamate to ecxocumarin leaxcctonization extracted from continuous illumination (k2) and relaxation (k 2) experiments respectively ((10%), and action cross
section for (E) to (Z) photoisomerization with two-photon excitation at λ(2) )750 nm, δE(750) Φ(2) ((20%; GM, 1 GM ) 10-50 cm4 s (photon)-1) for the
exc
EZ
(E)-cinnamate. Except for δE(750) Φ(E2Z) given in acetonitrile, acetonitrile/Tris (pH ) 7) 20 mM NaCl 100 mM buffer 1/1 (v/v) is the solvent. T ) 293 K (see
text and Supporting Information). b Not evaluated. c Too low to be reliably evaluated.
around their wavelength of maximum absorption, and the
absorption and steady-state emission spectra were recorded after
different illumination durations. The results are given in Table
1S in the Supporting Information. The main conclusions of this
preliminary screening are the following: (i) The uncaging
behavior is robust. All the E′a-h cinnamates release alcohol
together with a strongly fluorescent coumarin coproduct upon
one-photon illumination. Noticeably E′h exhibit a limited or
very slow photoisomerization. (ii) Except for E′e in which the
steric hindrance introduced by the 6-methoxy substituent
probably forbids the phenyl conjugation with the double bond,
the members of the E′ series exhibit a significant absorption
with one-photon excitation in the 300-400 nm wavelength
range. Taking into account their donor-acceptor conjugated
structure, the latter feature makes the series potentially ap-
propriate to observe significant absorptions with two-photon
excitation between 700 and 900 nm; (iii) E′c and E′d are not
thermally stable (see Supporting Information). At room tem-
perature, they isomerize in a few hours and thermally release
the ethanol and the coumarin coproduct.
In fact, the latter preliminary observations made with the E′
compounds led us to design the second E series of cinnamates
displayed in Chart 2. We first removed the methyl group on
the double bond to increase the energy barrier for thermal
isomerization (see Supporting Information for a theorical
schematic picture to analyze thermal and photochemical isomer-
ization in the cinnamate series). Then we adopted the phenyl
substituents leading both to significant absorptions beyond 300
nm and to fast enough uncaging.
absorption spectra. In Tris (pH ) 7) 20 mM NaCl 100 mM
buffer/acetonitrile 1/1 (v/v), the investigated E and E′ cinnama-
tes exhibit an absorption spectrum in the 300-400 nm range.
In the case of the less donating phenyl substituents (a and b
derivatives), two bands are visible in the corresponding wave-
length range (Figure 1S; see Supporting Information). In
contrast, one notices the presence of a main band with a shoulder
on its blue edge for the more electron-releasing phenyl substit-
uents (see Figure 1). In all the investigated cases, the band at
the lowest wavelength is the weakest among the two absorption
bands. Noticeably, the position of the band associated to the
more energetic transition is only weakly dependent on the
substituent nature. In contrast, the band at the longest wavelength
is red-shifted upon increasing the electron donating power of
the phenyl substituent. Table 1 summarizes the absorption
features, λ(E1) and ꢀE(λ(1)), associated to the band at the longest
E
wavelength of the investigated (E)-cinnamates.
For a same phenyl substituent (entries a, b, and d), the
absorption maximum with one-photon excitation λ(E1) of the E
derivative is systematically red-shifted by 14 kJ mol-1 (12-16
nm) compared to the one of the corresponding E′. This behavior
is most probably related to a partial loss of planarity associated
to a weaker conjugation in the E′ series that would result from
the steric hindrance between the methyl double-bond substituent
and the o-hydroxy phenyl substituent.
As anticipated, λ(E1) shifts to the red when the donating
power of the conjugated phenyl substituent increases in both E
and E′ series. Hence, the longest maximal wavelength of
(1)
Ee
absorption was observed for the julolidine derivative Ei (λ
)
After this preliminary screening, we eventually retained
Tris (pH ) 7) 20 mM NaCl 100 mM buffer/acetonitrile 1/1
(v/v) as a common solvent for one-photon experiments and
Ea-i, E′a-b, E′d, E′f, and E′g as caged substrates for the
following series of thorough investigations with one-photon
excitation.
One-Photon Absorption of the Cinnamates. The absorption
properties with one-photon excitation of the investigated (E)-
cinnamates have been obtained by recording their UV-vis
394 nm) for which the absorption band significantly extends
up to 450 nm. One should here notice the discrepancy
between the nonbrominated Ea and dibrominated Ec 4-phenol
cinnamates. In fact, the electron withdrawing properties of the
bromine atoms in Ec decrease the protonation constant of its
4-hydroxy group by about 5 orders of magnitude (see Supporting
Information). As a consequence, Ec is ionized at pH ) 7 which
gives rise to a phenoxy double bond substituent that is
9
J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007 9989

A R T I C L E S
Gagey et al.
Scheme 2. Mechanism of the Uncaging Process Leading to the
Release of the Caged Alcohol A (Here Ethanol) Together with a
Coumarin F When a Solution of (E) o-Hydroxycinnamate E Is
Illuminated with One-Photon Excitation (Inspired from Schmir et
al.)22
significantly more electron-donating than the corresponding
phenol in Ea.
Eventually, the molar absorption coefficients at the wave-
length of maximal absorption are satisfactorily large. Except
for the naphthalene derivative E′g, ꢀE(λ(E1)) ≈ 2 104 M-1 cm-1
.
Irradiation Experiments with One-Photon Excitation. The
irradiation experiments with one-photon excitation were per-
formed stepwise.
In a first series of experiments, we used 1H NMR and UV-
vis absorption spectroscopy to analyze the species resulting from
illumination of the caged (E)-cinnamates E and E′ (see
Supporting Information). In the case of Ec, we first showed
that the uncaging process obeys the reaction displayed in Figure
1: The caged alcohol A (here ethanol) is released in a one-to-
one stoichiometry together with a coumarin coproduct F upon
illumination with one-photon excitation. This behavior is in line
with Porter’s previous observation.14,26 Noticeably the (E)-
cinnamates E and the corresponding coumarin F exhibit similar
absorption properties making it possible to perform uncaging
and excitation of the reporting fluorescent molecule with a same
excitation source.
Figure 2. (a) Temporal evolution of the fluorescence emission at λem
)
450 nm from a 5.7 µM Ec solution in acetonitrile/Tris (pH ) 7) 20 mM
(1)
NaCl 100 mM buffer 1/1 (v/v) upon one-photon irradiation (λexc ) 350
nm) at several incident light intensities I0(350) (from bottom to top in 10-8
Einstein min-1: 2.7, crosses; 5.1, disks; 8.6, squares; 12.9, diamonds; 17.7,
pluses). Markers indicate the experimental data; lines indicate fits with eq
14. (b) Dependence on the incident light intensities I0(350) of the rate
constants k1 (circles) and k-1 (crosses) extracted from the evolutions
displayed in Figure 2a. T ) 293 K (see Experimental Section).
Scheme 3. Reduction of the Mechanism of the Photochemical
Isomerization of the (E)-Cinnamate E Leading to the Simultaneous
Release of the Caged Alcohol A and of the Coumarin F
During this series of 1H NMR experiments, we also identified
an intermediate containing a double bond exhibiting a (Z)
stereochemistry during the course of the photoconversion. Such
an observation is in line with the previous observations of
a Complete mechanism corresponding to the reactive scheme displayed
in Scheme 2. The rate constants associated to each elementary step are
denoted κi, where the subscript i indicates the corresponding step. bReduced
mechanism after taking into account the relative values of κ2, κ-2, κ3, and
Schmir on a series of closely related coumarinic acids.22,23
Z
would be the primary intermediate that results from (E) to (Z)
photoisomerization of the starting (E)-cinnamates (Scheme 2).
A second intermediate I would be subsequently obtained from
the nucleophilic addition of the o-hydroxy group onto the ethyl
ester moiety.
We then turned to analyze the kinetics of the uncaging process
in the present (E)-cinnamate series. Noticing that only the (E)-
to-(Z) and (Z)-to-(E) cinnamate exchanges should be photo-
chemically activated upon excitation at a large enough wave-
length, we considered retrieving the photochemical properties
of the present caging groups by analyzing their uncaging
kinetics.
κ
- 3. The rate constants associated to each step of the reduced mechanism
are denoted ki, where the subscript i indicates the corresponding step.
is continuously illuminated at different light intensities at a
(1)
exc
wavelength λ closely located to its maximal wavelength of
absorption λ(E1). One can observe a large increase of fluores-
cence emission as a function of time that gets faster when the
illumination intensity is increased.
The analysis of the curves displayed in Figure 2a requires a
modeling of the uncaging process. In principle, Scheme 2
suggests consideration of a three step-six reactions mechanism
that would involve six rate constants (Scheme 3a). However, it
is possible to reduce the mechanism to a two step-three
reactions mechanism (see Scheme 3b).
Figure 2a displays the typical temporal evolutions of the
fluorescence emission from a solution of a (E)-cinnamate that
(26) Porter, N. A.; Bruhnke, J. D. J. Am. Chem. Soc. 1989, 111, 7616-7618.
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Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
Taking advantage of the similar electronic demands of the
hydroxy and ethoxy groups, we first relied on the thorough
investigation of the cyclization of para substituted derivatives
of coumarinic acid yielding the corresponding coumarins.22,23
In the preceding work, the conversion of the I-type tetrahedral
intermediate to the F coumarin with water release was reported
to be the rate-limiting step of the Z-like coumarinic acid to the
coumarin conversion in the case of coumarinic acids para
substituted with electron donating conjugated substituents and
at neutral pH like in the present study. Thus Z and I are
suggested to be in fast exchange at the time scale of the I to F
cyclization. At the same time, the rate constants associated to
the photochemical exchange between the E and Z states and
the rate constant for the I to F cyclization were in a comparable
range under the present illumination conditions suggesting a
consideration of Z and I in a fast exchange regime during the
whole uncaging process. Eventually the final coumarin F is
thermodynamically strongly favored among the different states
displayed in Scheme 2 22,23 so as to neglect the backward
reaction from the coumarin F to the I intermediate in the final
model. After introducing the ZI species that is relevant in
describing Z + I when Z and I are in a fast exchange, we
eventually retained the reduced model that is displayed in
Scheme 3b to analyze the data.
the o-hydroxycinnamic series is especially favorable from this
point of view: The brightness of the (E)-cinnamate caged
compounds at the excitation wavelength required for uncaging
is uniformly very low whereas the one of the coumarin
coproduct is large. In fact, the emission from the starting
cinnamate can be typically neglected in front of the one of the
coumarin photoproduct beyond 1% extent of the uncaging
process.
Quantum Yield of Photoisomerizations Involved in Un-
caging. The quantum yields of (E)- to (Z)-cinnamate photoi-
somerization leading to alcohol uncaging are significant in both
the E and E′ series. They are always larger than 1% and reach
up to 25% for E′g; in fact, they seem to be slightly larger in
the E′ series than in the E one (see Supporting Information for
an interpretation). In particular, our 10% result for E′d
satisfactorily compares with the 13% value reported by Porter
et al. in Tris buffer.27 Such values are in the range of the
quantum yields associated to other relevant uncaging photo-
chemistries. Lower values are generally observed for the most
popular o-nitro benzyl photoisomerization,28 whereas similar
values are reported in the case of the coumaryl photolabile
protecting group.3
Upon one-photon illumination, one does not only observe
the (E)- to (Z)- but also the corresponding (Z)- to (E)-cinnamate
photoisomerization in the present investigated series. In fact,
the apparent thermodynamic constant K1 ) k1/k-1 which
describes the exchange between the starting cinnamate E and
the intermediate ZI is always lower than one except for
E′c (Table 1). Such a result has been already observed by
Porter et al. for the E′d derivative.27 As anticipated from eq
17, K1 depends on the excitation wavelength and peak at
0.6 at 375 nm (see Supporting Information). This behavior
can be accounted for by the difference of the absorption spectra
of the (E)- and (Z)-cinnamate as suggested from studying an
ortho methoxy analogue of Ec, EcMe (see Supporting Informa-
tion).
Rate Constant for the Thermally Driven Events Leading
to Uncaging. Both series of experiments devoted to measure
the rate constant k2 associated to the thermal cyclization of the
(Z)-cinnamates leading to alcohol release together with the
formation of the corresponding coumarin coproduct provide
similar values (Table 1). In the buffer-acetonitrile 1:1 (v:v)
mixture, they lie in the 10-2 s-1 range for all the investigated
compounds and are about 1 order of magnitude larger in the E′
series than in the E one. One may additionally notice a trend
for increasing the rate constant k2 upon increasing the donating
power of the phenyl para substituent in the E series.
The fit of data such as displayed in Figure 2a provides the
values of the rate constants k1, k-1, and k2 (see Experimental
Section). Figure 2b shows that k1 and k-1 depend linearly on
the incident light intensity: this supports the model adopted in
Scheme 3b. To bring further support to the model, we performed
a complementary series of experiments to independently access
the value of the rate constant k2 by observing by UV-vis
absorption spectroscopy the relaxation of solutions of the (E)-
cinnamates after a short illumination with a UV lamp of large
power; under such experimental conditions, only the ZI to F
conversion of Scheme 3b is assayable. As displayed in Table
1, we obtained a satisfactory agreement between the k2 values
extracted from both series of experiments.
After the preceding model validation, we derived the pho-
tochemical properties of the o-hydroxycinnamate photolabile
protecting group with one-photon excitation from k1 and k-1
(see Experimental Section). k1 gives the action (E) to (Z)
photoisomerization cross section with one-photon excitation of
the (E)-cinnamate E. The analysis of k-1 is more difficult
because the equilibrium constant K2 associated to the fast
exchange between Z and I cannot be measured which forbids
us to derive the action (Z) to (E) photoisomerization cross
section with one-photon excitation of the (Z)-cinnamate Z. The
fit also yields the relative brightness of the starting (E)-cinnamate
E with regards to the one of the coumarin coproduct F. Table
1 summarizes the results of the different parameters extracted
from the series of irradiation experiments with one-photon
excitation performed in the whole investigated E and E′ series.
The evidenced trends are examined and discussed in the
following paragraphs.
The preceding observations are in line with the behavior
observed in the coumarinic series for which the I to F reaction
is the rate-limiting step during the thermal Z to F conversion
at neutral pH (Scheme 2).22,23 Indeed, the presence of the methyl
substituent on the double bond is expected to accelerate the I
to F reaction by increasing the associated release of steric
hindrance with regards to the corresponding hydrogen derivative.
In addition, the presence of an electron donating conjugated
substituent in para of the phenyl group is expected to facilitate
the release of the leaving-alcohol moiety.
Brightness of the (E)- and (Z)-Cinnamates and of the
Coumarin Uncaging Coproducts. The release of a strongly
fluorescent molecule compared to the initial caged molecule
would be of great interest in quantifying the uncaging reaction
in environments where reaction calibrations can prove to be
daunting, such as in biological applications. Table 1 shows that
(27) Koenigs, P. M.; Faust, B. C.; Porter, N. A. J. Am. Chem. Soc. 1993, 115,
9371-9379.
(28) Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.;
Jullien, L. Chem.sEur. J. 2006, 12, 6865-6879.
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A R T I C L E S
Gagey et al.
In fact, the k2 value is expected to strongly depend on the
nature of the caged substrate as well as on the solvent. Indeed,
the rate limiting I f F step probably obeys here an E1
mechanism associated to the loss of the alcohol moiety.22 Then
one anticipates k2 to increase either with an increase of the
withdrawal electronic properties of the alcohol or with an
increase of the solvent polarity. During the present work, we
did not attempt to modify the nature of the caged alcohol.
Nevertheless it is here interesting to notice that Porter mentioned
that the thermal cyclization of a (Z)-cinnamate to the corre-
sponding coumarin occurred in less than 1 ms in the case of a
phenol protected with an o-hydroxycinnamate moiety.14 In
contrast, we benefited from the favorable solubility properties
of the Ec cinnamate to reproduce uncaging experiments in
different solvents. We found the same k2 ) 11 10-3 s-1 value
in pure acetonitrile and in the acetonitrile/Tris (pH ) 7) 20 mM
NaCl 100 mM buffer 1/1 (v/v) mixture. In contrast, we observed
a spectacular 300-fold increase of the corresponding rate
constant in Tris (pH ) 7) 20 mM NaCl 100 mM buffer: we
found k2 ) 0.3 s-1. The latter results are in line with Porter’s
previous k2 measurements for E′d in mixtures of ethanol and
pH 7.4 Tris buffer in different proportions.26 In particular, Porter
found that k2 varied by more than 3 orders of magnitude when
going from ethanol/buffer 98:2 (v/v) (k2 ) 7 10-4 s-1) to
ethanol/buffer 2:98 (v/v) (k2 ) 2 s-1).
Irradiation Experiments with Two-Photon Excitation. In
Vitro Uncaging. We took advantage of the release upon
uncaging of the fluorescent coumarin in a one-to-one stoichi-
ometry with the caged alcohol to measure the uncaging action
cross section.
Figure 3. (a) Temporal evolution of the fluorescence emission from a 1.8
(2)
µM Ec solution in acetonitrile upon irradiation at λ ) 750 nm at two
exc
We first characterized the uncaging process by evidencing
the photoinduced formation of the coumarin coproduct by means
of its photophysical properties that can be investigated even at
low concentrations. Figure 3a displays the temporal evolution
of the fluorescence emission from a 1.8 µM Ec solution in
acetonitrile upon irradiation at 750 nm at two laser powers.29
One observes a continuous increase. Such a behavior is in line
with the formation of a photoproduct that is more fluorescent
than the starting (E)-cinnamate ester Ec. In a preceding
communication,9 we relied on a spectral analysis to conclude
that this photoproduct was the coumarin Fc: both species exhibit
the same emission spectrum with two-photon excitation. Fluo-
rescence correlation spectroscopy (FCS) measurements also lead
to the same conclusion (Figure 3b). Indeed the fluorophore that
is formed upon illumination with two-photon excitation exhibits
the same diffusion coefficient and flux of emitted photons per
molecule and per second as the reference coumarin Fc.
laser powers P (mW), 1.5 (dots) and 1.7 (pluses). The initial value of the
two traces were made identical to favor the comparison of the slopes (see
Experimental Section). (b) FCS autocorrelation curves G(τ) with two-photon
excitation from the illuminated volume Vexc resulting from focusing the
Ti-Sapphire laser beam at P ) 1.7 mW after different illumination times:
0-1500 (circles) and 4500-6000 (pluses) s. Fitting (solid line) of the
experimental points (markers) with eq 2, respectively provided 7 ( 3 µs
and 7 ( 3 µs for the diffusion time τD and 15 and 25 for the number of
fluorescent molecules contained within the excitation volume Vexc. After
taking into account the intensity of fluorescence emission from Vexc that
was measured at the APD, we evaluate at 300 Hz the flux of emitted photons
per fluorescent molecule and per second that satisfactorily compared with
the value measured for the reference Fc coumarin. The data shown in Figure
3a and 3b are used to extract the action (E) to (Z) photoisomerization cross
(2)
exc
section with two-photon excitation at λ
solvent, acetonitrile; T ) 293 K.
) 750 nm, δE(750) Φ(2)
:
EZ
(E)- to (Z)-cinnamate photoisomerization with two-photon
excitation can be extracted from the slope of the linear temporal
evolution of the fluorescence intensity displayed in Figure 3a.
In particular, we verified in the course of this series of
experiments that the dependence of the rate constant k1 with
the average laser power was quadratic so as to conclude that
the observed behavior did result from two-photon absorption.
Table 1 displays the (E) to (Z) photoisomerization action cross
Then we analyzed the kinetics of the uncaging process. In
reference to the kinetic model displayed in Scheme 3b, the rate
constant k2 is now much larger than the rate constants k1 and
k-1 in the present conditions of illumination with two-photon
excitation. Indeed the illuminated volume is lower with two-
than with one-photon excitation during in vitro experiments (see
eqs 15, 16, 18). Then only k1 is here accessible: the Experi-
mental Section explains that k1 and the action cross section for
sections with two-photon excitation of several (E)-cinnamates
(2)
exc
at λ ) 750 nm in acetonitrile. They are all 1 order of
magnitude larger than the 0.1 GM limit which was claimed to
be the lowest value relevant for biological applications.3 In fact,
the Ee value equal to 4.7 GM at 750 nm is the largest reported
to date.3
In Vivo Uncaging. Having quantitatively characterized in
vitro the behavior of a caged alcohol, we were willing to
investigate whether the o-hydroxycinnamic moiety would be
(29) To study uncaging with two-photon excitation for the whole series of
cinnamates in a same solvent, we had to switch from the acetonitrile/buffer
1:1 (v/v) mixture to pure acetonitrile. Indeed, we observed in the former
solvent that the fluorescence traces such as in Figure 3a periodically
exhibited plateaus for the most hydrophobic derivatives (noticeably except
for Ec which is fairly water-soluble).
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9992 J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007

Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
to the exponential fit of the fluorescence emission from the focal
spot was in line with the signal intensity expected from the
coumarin Fc in aqueous solution at a typical 10 µM concentra-
tion. We further checked the agreement between the plateau
value and the incubating solution concentration by conducting
the same experiment with a more dilute solution (500 nM) and
recorded the FCS autocorrelation curve upon uncaging comple-
tion. We measured a 500 nM Fc concentration inside cells (see
eq 2 in Experimental Section). Such observations suggest that
the initial intracellular concentration in caged precursor Ec was
essentially similar to the concentration of the incubating solution.
The rate given by the exponential fit is the one of the limiting
step in the uncaging reaction. We measured 6 × 10-3 s-1. The
expected rate from a 1.6 GM uncaging action cross section with
two-photon excitation of Ec at 750 nm9 would be 20 s-1 at 4.5
mW power in this closed environment (see eq 18 in Experi-
mental Section and data not shown). This value is 3 orders of
magnitude greater than the measured rate. We thus can rule out
the (E)-to-(Z) photoisomerization of Ec as limiting step. A first
possible rate-limiting step could be here the ZIc to Fc
conversion. Indeed, we do not know what is actually the location
of the (E)-cinnamate Ec within an embryo cell, and we observed
that k2 is considerably decreased if Ec is not located in water
but in an organic-like medium. Another possible explanation
could be that the uncaging process is limited by the off-rate
constant of some complex involving Ec that would be located
out from the laser focal point. In both cases, it is here important
to notice that the observed uncaging rate constant would
considerably depend on the structure of the caged alcohol.
Figure 4. Evaluation of two-photon uncaging with fluorescence reporting
of the o-hydroxycinnamic platform in vivo (a zebrafish embryo is used for
illustration). The embryo was incubated in a 10 µM aqueous solution of
nonfluorescent caged alcohol Ec that penetrates in the whole organism.
The embryo was subsequently submitted to a continuous two-photon
excitation by focusing a laser beam (red arrows) in the caudal fin area (a)
and the fluorescence signal emitted from the illuminated tiny zone was
recorded as a function of time. The uncaging process is specifically revealed
by the exponential dependence on time of singular fluorescence rises
observed along the fluorescence trace (b) (see text and Experimental
Section).
Discussion
Evaluating the efficiency of a caging group is a complex issue.
Indeed the demands on the caging group can vary considerably
depending on the application. In the following discussion, we
are essentially concerned with discussing two aspects of the
o-hydroxycinnamic platform for uncaging applications in biol-
ogy. We first examine processing issues that are relevant to
condition the biological sample. We subsequently discuss the
uncaging opportunities provided by the o-hydroxycinnamic
photolabile moiety for two-photon uncaging in a biological
context.
appropriate as a photolabile protecting group for biological
applications in a living organism. We adopted the zebrafish
animal model that 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 numerous studies.30
We first incubated a zebrafish embryo at the 10-15 somite
stage in a 10 µM solution of Ec for 60 min. We subsequently
illuminated at 750 nm the caudal fin area (Figure 4a) and we
continuously recorded the fluorescence emission arising from
the focal point. We observed a smooth increase of the
fluorescence signal (see Figure 4b). Ethanol uncaging occurred
in the illuminated cell releasing the fluorescent coumarin
coproduct Fc characterized from its emission spectrum:9 Thus
we concluded that two-photon uncaging with fluorescence
reporting is active at the single-cell level in vivo in the
o-hydroxycinnamic series.
It can be shown that the temporal dependence of the
fluorescence emission during uncaging in a closed volume is
exponential if the starting cinnamate Ec and the coumarin
photoproduct Fc do not permeate at the uncaging time scale
(see Supporting Information). Such an exponential behavior was
indeed observed by analyzing individual fluorescence rises such
as shown in Figure 4b. The final asymptotic value associated
During this study, we have been particularly concerned with
the solubility of the caged substrate. This point is especially
significant for biological applications. For instance, in the
zebrafish embryo considered here, the caged substrate has first
to be dissolved in an aqueous solution, which requires an
exhibition of some hydrophilicity, and then reach the targeted
cell, which involves crossing hydrophobic barriers. From the
latter point of view, the o-hydroxycinnamic platform is par-
ticularly favorable: this photolabile moiety is synthetically easily
accessible, and the different substituents on the benzene ring
can be easily changed to finely tune the hydrophilic/hydrophobic
balance in the caged derivatives as well as in the coumarin
coproduct. In particular, we consider as satisfactory the results
obtained with Ec (and Fc) in which we introduced in the
o-hydroxycinnamic caging moiety a conjugated acidic phenol
with a protonation constant in the range of intracellular pH (4.5-
7.5). First, the native acidic state absorbs only rather far in the
UV: the strong UV-vis absorption yielding uncaging only
occurs after dissolution in water, and the pure caged substrate
(30) Zon, L. I.; Peterson, R. T. Nat. ReV. Drug DiscoVery 2005, 4, 35-44.
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A R T I C L E S
Gagey et al.
can be easily stored as an inert compound. Furthermore, at
neutral pH such as in a cell, the caged substrate constantly
switches between its acidic uncharged phenol state (making it
soluble in a lipophilic environment such as a bilayer) and its
basic negatively charged phenate form (conferring water
solubility). Water solubility facilitates organism exposition to
the caged substrate and the dual hydrophilic/hydrophobic
character probably explains why we found Ec at the concentra-
tion of the incubating Ec solution even in cells deeply embedded
in the zebrafish embryo. At the same time, the coumarin
coproduct Fc is significantly water-soluble which permits a
reliable interpretation of its fluorescence emission in vivo.9
Two-photon uncaging has been especially considered for
biological applications in view of its promising spatial and
temporal resolution. However most photochemically driven
substrate releases involve a multistep mechanism with both
photochemical and thermal reactions.11 The primary photo-
chemical event initiated at the focal spot of the laser beam and
the subsequent substrate release are consequently most often
decoupled in time and, correspondingly, in space since the
intermediates generated after illumination would be generally
free to diffuse. Thus a detailed kinetic analysis not restricted to
the photochemical events is required to appreciate the efficiency
of a photolabile protecting group for given two-photon uncaging
applications.7,31 In the two following paragraphs, we examine
two generic situations according to the position of the focal spot
with regard to the cellular environment.
In extracellular photolysis, uncaging occurs in a volume which
can be considered as infinite and two-photon uncaging is used
to generate in the focal spot a concentration in photoreleased
substrate that significantly departs from its concentration in the
environment. Then an “efficient’’ caging group has to exhibit
rate constants associated to the primary photochemical event
as well as to the subsequent thermal reactions larger than the
rate of diffusion through the laser beam waist (typically 104
s-1).31,32 Moreover increasing the latter rate constants beyond
the diffusion rate still improves the uncaging efficiency. To have
a tunable delivery at nondetrimental laser powers, the rate-
limiting step should be the primary photochemical event in
which a two-photon uncaging action cross section should be at
least be equal to 0.1-1 GM at 750 nm.
intracellular distribution in photoreleased substrate. In fact, the
constraint on the rate constant in the case of intracellular
photolysis is rather permissive in terms of action cross section
with two-photon excitation of the primary photochemical
event: 1 mGM range is now sufficient to be “efficient’’ at
nondetrimental laser powers.
In the present o-hydroxycinnamate system, the primary
photochemical event is the (E)- to (Z)-cinnamate photoisomer-
ization which is associated to action cross sections with two-
photon excitation exceeding the 1 GM range. From the latter
point of view, the o-hydroxycinnamate is an excellent photo-
chemical platform belonging to the most efficient available to
date.3,5,8 Nevertheless, one has also to take into account the
thermal reactions involved in the substrate release. Although
Porter reported rate constants larger than 103 s-1 for phenol
uncaging, we here observed at most the 1 s-1 range for alkyl
alcohol uncaging with Ec in water. To increase the latter value,
the present study suggests some possible improvement associ-
ated with modifying the substitution pattern of the cinnamate
double bond: For instance, we observed that the addition of a
methyl substituent increased k2 by a factor of ten. At the present
time, we estimate that the o-hydroxycinnamate system should
be essentially restricted for alcohol uncaging in intracellular
photolysis where its excellent fluorescence reporting features
are particularly appropriate to yield information on the amount
of released substrate. As an example, it should prove to be useful
to control gene expression through the release of ligands for
transcription factors. Indeed, tetracyclin and ecdysone are the
inducers of two widely used gene expression inducible sys-
tems;34,35 they contain alcohol groups which can be used to graft
o-hydroxycinnamate groups to inactivate their biological func-
tions. Moreover, the relevant biological timescales associated
with gene expression are fully compatible with release rates in
the 1 s-1 or even the 10-2 s-1 range.
Conclusion
The present investigation underlines the significance of the
o-hydroxycinnamic platform to design versatile photolabile
protecting groups with fluorescence reporting for alcohols upon
one- and two-photon excitation. The o-hydroxycinnamate series
is synthetically easily accessible. An appropriate choice of the
different substituents on the benzene ring is expected to allow
for fine-tuning of the hydrophilic/hydrophobic balance in the
caged derivatives as well as in the coumarin coproduct upon
alcohol uncaging. In addition, the robust photophysical and
photochemical properties are particularly attractive. The maxi-
mal wavelength for one-photon absorption of the o-hydroxy-
cinnamates can be easily tuned at the UV-vis limit where the
cinnamates exhibit a strong absorption. The caged alcohol is
essentially nonfluorescent and quantitatively releases upon one-
photon excitation the alcohol substrate together with a strongly
fluorescent coumarin coproduct in a one-to-one molar ratio.
Uncaging and excitation of the reporting fluorescent molecule
can be additionally done using the same excitation source. We
eventually observed maximal wavelength and action cross
sections associated to uncaging upon two-photon excitation that
favorably compare with the best reported values.
The boundary conditions are very different in intracellular
photolysis: Two-photon uncaging now occurs in a closed
volume and one is concerned with generating a homogeneous
intracellular concentration in released substrate from a caged
precursor faster than the biological phenomena to be triggered.
The lower temporal resolution is now fixed by an homogeniza-
tion time which is at least in the 10-100 ms range for a typical
“small’’ substrate in a 1000 µm3 cell.33 Then an “efficient’’
caging group only requires the rate constants of the primary
photochemical event and of the subsequent thermal reactions
to be larger than the rate of diffusion within the closed territory
(typically 101-102 s-1). Noticeably increasing the preceding
rate constant does not improve here the uncaging efficiency but
only forces diffusion to be taken into account to yield the
(31) Brown, E. B.; Shear, J. B.; Adams, S. R.; Tsien, R. Y.; Webb, W. W.
Biophys. J. 1999, 76, 489-499.
(32) Kiskin, N. I.; Ogden, D. Eur. Biophys. J. 2002, 30, 571-587.
(33) In fact, one should also consider here that a caged compound might form
a complex with some intracellular components (bilayer, proteins) not located
at the laser focus. Then the apparent homogeneization time could be
associated to the off-rate of the caged substrate from the complex.
(34) Cambridge, S. B.; Geissler, D.; Keller, S.; Curten, B. Angew. Chem., Int.
Ed. 2006, 45, 2229-2231.
(35) Lin, W.; Albanese, C.; Pestell, R. G.; Lawrence, D. S. Chem. Biol. 2002,
12, 1347-1353.
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Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
(E)-3-(3,5-Dibromo-2,4-dihydroxy-phenyl)-2-methyl Acrylic Acid
Ethyl Ester (E′c). Yield, 35%; mp 112-114 °C. H NMR (ppm, 250
MHz, CDCl3, 298 K) δ: 7.67 (s, 1 H), 7.38 (s, 1 H), 5.99 (s, 1 H),
5.88 (s, 1 H), 4.27 (q, 2 H, J ) 7.1 Hz), 2.03 (d, 3 H, J ) 1.4 Hz),
1.34 (t, 3 H, J ) 7.2 Hz). C NMR (ppm, 62.8 MHz, CDCl3, 298 K) δ:
168.2, 151.1, 149.7, 132.0, 131.3, 129.7, 117.4, 100.1, 99.0, 61.0, 14.2
(2C). Anal. Calcd (%) for C12H12O4Br2 (380.03): C, 37.93; H, 3.18.
The o-hydroxycinnamic caging group appears particularly
attractive in biological applications that do require precise but
not necessarily “fast’’ two-photon excitation-induced substrate
release. In particular, our results suggest that quantitative control
of substrate delivery could be achieved in vivo at the second
time scale by recording the fluorescence emission from the
coumarin F coproduct that reports on the concentration in
photoreleased substrate.
Found: C, 37.70; H, 3.10. Mass spectrometry, MS (CI, CH4): m/z 381
79
[M + 1]. MS (CI, CH4, HR): m/z 380.9161 (calcd mass for C12H13O4
-
Br81Br, 380.9153).
Experimental Section
(E)-3-(4-Diethylamino-2-hydroxy-phenyl)-2-methyl Acrylic Acid
Ethyl Ester (E′d).25 Yield, 70%; mp 96-97 °C. H NMR (ppm, 250
MHz, CDCl3, 298 K) δ: 7.71 (s, 1 H), 7.12 (d, 1 H, J ) 8.2 Hz), 6.21
(dd, 1 H, J1 )8.7 Hz, J2 )2.4 Hz), 6.1 (d, 1 H, J ) 2.4 Hz), 5.3 (s, 1
H), 4.18 (q, 2 H, J ) 7.1 Hz), 3.27 (q, 4 H, J ) 7.1 Hz), 2.01 (s, 3 H),
1.26 (t, 3 H, J ) 7.17 Hz), 1.1 (t, 6 H, J ) 7.1 Hz). 13C NMR (ppm,
62.8 MHz, CDCl3, 298 K) δ 169.3, 155.6, 149.6, 133.6, 131.2, 125.1,
110.2, 104.3, 98.0, 60.7, 44.4 (2C), 14.4 (2C), 12.7 (2C). Anal. Calcd
(%) for C16H23O3N (277.36): C, 69.28; N, 5.05; H, 3.18. Found: C,
69.14; N, 5.07; H, 8.65. Mass spectrometry, MS (CI, CH4): m/z 278
[M + 1]. MS (CI, CH4, HR): m/z 278.1761 (calcd mass for C16H24O3N,
278.1756).
Syntheses. 2,4-Dihydroxybenzaldehyde, 2-hydroxy-4-methoxybenz-
aldehyde, 4-diethylamino-2-hydroxybenzaldehyde, 2-hydroxy-5-ni-
trobenzaldehyde, 2-hydroxy-4,6-dimethoxybenzaldehyde, carboethoxy-
ethylidenetriphenylphosphorane and carboethoxymethylidenetriphenyl-
phosphorane were commercially available. The syntheses of the other
starting substituted benzaldehydes and of the 1-carboxymethylidene
triphenylphosphorane are reported in Supporting Information.
General Procedures. The commercially available chemicals were
used without further purification. Anhydrous solvents were freshly
distilled before use. Column chromatography (CC) used silica gel 60
(0.040-0.063 mm) from Merck; analytical and thin layer chromatog-
raphy (TLC) was performed with Merck silica gel 60 F254 precoated
plates, detection by UV (254 nm); melting point used a Bu¨chi 510. 1H
NMR and 13C NMR spectra were obtained by AM 250 SY Bruker.
Chemical shifts (δ) in ppm related to protonated solvent as internal
reference: (1H) CHCl3 in CDCl3, 7.26 ppm; CHD2COCD3 in CD3-
(E)-3-(2-Hydroxy-4,6-dimethoxy-phenyl)-2-methyl Acrylic Acid
1
Ethyl Ester (E′e). Yield, 30%; mp 99 °C. H NMR (ppm, 250 MHz,
CDCl3, 298 K) δ: 7.49 (d, 1 H, J ) 1.1 Hz), 6.12 (dd, 2 H, J )2.1
Hz, J )1.1 Hz), 4.93 (s, 1 H), 4.27 (q, 2 H, J ) 7.1 Hz), 3.79 (s, 3 H),
3.78 (s, 3 H), 1.57 (s, 3 H), 1.34 (t, 3 H, J ) 7.1 Hz). 13C NMR (ppm,
62.8 MHz, CDCl3, 298 K) δ: 168.1, 161.8, 158.8, 154.5, 131.5, 131.3,
104.6, 93.2, 91.3, 60.9, 55.6, 55.4, 15.0, 14.3. Anal. Calcd (%) for
C14H18O5 (266.12): C, 63.15; H, 6.81. Found : C, 63.14; H, 6.72. Mass
spectrometry, MS (CI, NH3): m/z 367 [M + 1].
(E)-3-(2-Hydroxy-4-methoxyphenyl)-2-methyl Acrylic Acid Ethyl
Ester (E′f). Yield, 60%; mp 99 °C. 1H NMR (ppm, 250 MHz, CDCl3,
298 K) δ: 7.73 (s, 1 H), 7.11 (d, 1 H, J ) 9.2 Hz), 6.8 (bs, 1 H), 6.42
(s, 1 H), 6.39 (d, 1 H, J ) 9.2 Hz), 4.17 (q, 2 H, J ) 7.2 Hz), 3.68 (s,
3 H), 1.96 (s, 3 H), 1.24 (t, 3 H, J ) 7.1 Hz). 13C NMR (ppm, 62.8
MHz, CDCl3, 298 K) δ: 169.4, 161.1, 155.7, 134.2, 130.8, 127.4, 115.7,
106.1, 101.5, 61.0, 55.2, 14.2, 14.1. Anal. Calcd (%) for C13H16O4
(236.10): C, 66.09; H, 6.83. Found: C, 66.03; H, 6.78. Mass
spectrometry, MS (CI, NH3): m/z 237 [M + 1].
13
COCD3, 2.20 ppm; CHD2SOCD3 in CD3SOCD3, 2.54 ppm; (13C)
-
CDCl3 in CDCl3, 77.0 ppm; 13CD3COCD3 in CD3COCD3, 29.8 ppm;
13CD3SOCD3 in CD3SOCD3, 39.6 ppm. Coupling constants J are given
in Hz. Mass spectrometry (chemical ionization and high resolution with
NH3 or CH4) was performed at the Service de Spectrome´trie de masse
de l’ENS. Microanalyses were obtained from the Service de Mi-
croanalyses de l’Universite´ Pierre et Marie Curie, Paris.
General Procedure for Wittig Reactions.25 A mixture of aldehyde
and carboethoxyethylidenetriphenylphosphorane or carboethoxy-
methylidenetriphenylphosphorane (1.5 equiv) in toluene (10 mL for 1
mmol of aldehyde) was heated at 60 °C under argon upon protecting
from light. The course of the reaction was followed by TLC (cyclo-
hexane/AcOEt). After 2 to 4 h, the reaction was completed. After
cooling to room temperature, toluene was removed in a vacuum. The
crude residue was purified by flash chromatography on silica gel
(mixtures of ethyl acetate and cyclohexane as eluent) to yield the desired
cinnamate in high yield (from 60 to 90%; about 10% of the coumarin
resulting from thermal trans-cis isomerization followed by lactonization
was formed during the reaction).
(E)-3-(2-Hydroxy-naphtalen-1-yl)-2-methyl Acrylic Acid Ethyl
1
Ester (E′g). Yield, 80%; mp 94-96 °C. H NMR (ppm, 250 MHz,
CDCl3, 298 K) δ: 7.9 (s, 1 H), 7.7 (m, 3 H), 7.47-7.21 (m, 3 H), 5.6
(bs, H), 4.35 (q, 2 H, J ) 7.0 Hz), 1.87 (d, 3 H, J ) 0.9 Hz), 1.40 (t,
3 H, J ) 7.3 Hz). 13C NMR (ppm, 62.8 MHz, CDCl3, 298 K) δ: 167.6,
150.0, 134.5, 132.8, 132.1, 130.2, 128.7, 128.3, 126.8, 123.9, 123.7,
117.6, 114.6, 61.2, 14.6, 14.3. Anal. Calcd (%) for C16H16O3 (256):
C, 75.00; H, 6.25. Found: C, 74.95; H, 6.28. Mass spectrometry, MS
(CI, CH4): m/z 257 [M + 1]. MS (CI, CH4, HR): m/z 257.1176 (calcd
mass for C16H17O3, 257.1178).
(E)-3-(2,4-Dihydroxyphenyl)-2-methyl Acrylic Acid Ethyl Ester
1
(E′a). Yield. 60%; mp 121-122 °C. H NMR (ppm, 250 MHz, CD3-
COCD3, 298 K) δ: 8.63 (bs, 1 H), 7.9 (s, 1 H), 7.27 (d, 1 H, J ) 8.4
Hz), 6.5 (d, 1 H, J ) 2.3 Hz), 6.45 (dd, 1 H, J ) 8.4 Hz, J ) 2.3 Hz),
4.22 (q, 2 H, J ) 7 Hz), 2.07 (s, 3 H), 1.31 (t, 3 H, J ) 7 Hz). 13C
NMR (ppm, 62.8 MHz, CD3COCD3, 298 K) δ: 168.6, 159.7, 157.7,
134.5, 131.5, 125.1, 115.2, 107.3, 102.9, 60.3, 14.1, 14.0. Anal. Calcd
(%) for C12H14O4 (222.09): C, 64.85; H, 6.35. Found: C, 64.63; H,
6.31. Mass spectrometry, MS (CI, NH3): m/z 223 [M + 1].
(E)-3-(6-Hydroxy-benzo(1,3)dioxo-5-yl)-2-methyl Acrylic Acid
Ethyl Ester (E′b). Yield, 70%; mp 113-114 °C. 1H NMR (ppm, 250
MHz, CDCl3, 298 K) δ: 7.65 (s, 1 H), 6.70 (s, 1 H), 6.47 (s, 1 H),
5.94 (s, 1 H), 5.02 (s, 2 H) 4.27 (q, 2 H, J ) 7.1 Hz), 2.02 (s, 3 H),
1.34 (t, 3 H, J ) 7.1 Hz). 13C NMR (ppm, 62.8 MHz, CD3COCD3,
298 K) δ: 168.9, 125.5, 149.7, 141.6, 135.0, 126.5, 115.7, 109.3, 102.3,
98.6, 61.0, 14.7, 14.5. Anal. Calcd (%) for C13H14O5 (250): C, 62.39;
H, 5.64. Found: C, 62.27; H, 5.59. Mass spectrometry, MS (CI, CH4):
m/z 251 [M + 1]. MS (CI, CH4, HR): m/z 251.0924 (calcd mass for
C13H15O5, 251.0919).
(E)-3-(2-Hydroxy-5-nitrophenyl)-2-methyl Acrylic Acid Ethyl
Ester (E′h). Yield, 50%; mp 115-115.5 °C. 1H NMR (ppm, 250 MHz,
CDCl3, 298 K) δ: 8.15 (m, 2 H), 7.63 (s, 1 H), 6.99 (d, 1 H, J ) 9.2
Hz), 6.10 (s, 1 H), 4.31 (q, 2 H, J ) 7.1 Hz), 2.03 (d, 3 H, J ) 1.2
Hz), 1.37 (t, 3 H, J ) 7.2 Hz). 13C NMR (ppm, 62.8 MHz, CDCl3, 298
K) δ: 169.1, 160.1, 140.9, 132.4, 131.9, 126.1, 125.9, 123.3, 116.1,
61.8, 14.2 (2C). Anal. Calcd (%) for C12H13NO5 (251): C, 57.37; N,
5.58; H, 5.22. Found: C, 57.45; N, 6.58; H, 5.30. Mass spectrometry,
MS (CI, CH4): m/z 252 [M + 1].
(E)-3-(2,4-Dihydroxyphenyl) Acrylic Acid Ethyl Ester (Ea). Yield,
70%; mp 193-195 °C. 1H NMR (ppm, 250 MHz, CD3SOCD3, 298 K)
δ: 10.12 (bs, 1 H), 9.9 (bs, 1 H), 7.78 (d, 1 H, J ) 16 Hz), 7.42 (d,
1 H, J ) 8.6 Hz), 6.4-6.35 (m, 2 H), 6.28 (dd, 1 H, J ) 8.4 Hz,
J ) 2.1 Hz), 4.15 (q, 2 H, J ) 7.2 Hz), 1.24 (t, 3 H, J ) 7.2 Hz). 13
C
NMR (ppm, 62.8 MHz, CD3SOCD3, 298 K) δ: 167.1, 160.9, 158.5,
9
J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007 9995

A R T I C L E S
Gagey et al.
140.3, 130.3, 113.0, 112.6, 107.8, 102.4, 59.4, 14.3. Anal. Calcd
(%) for C11H12O4 (208.07): C, 63.45; H, 5.81. Found: C, 63.37; H,
5.80.
Windows systems) to extract the pKa of the ground state (see Supporting
Information).37
Steady-State Fluorescence Emission. Corrected fluorescence spec-
tra upon one-photon excitation were obtained from a Photon Technology
International LPS 220 spectrofluorimeter. Solutions for fluorescence
measurements were adjusted to concentrations such that the absorption
maximum was around 0.15 at the excitation wavelength. The overall
fluorescence quantum yields after one-photon excitation Φ(F1) were
calculated from the relation (1):
(E)-3-(6-Hydroxy-benzo(1,3)dioxo-5-yl) Acrylic Acid Ethyl Ester
(Eb). Yield, 60%; mp 149-150 °C. 1H NMR (ppm, 250 MHz, CDCl3,
298 K) δ: 8.02 (d, 1 H, J ) 16.1 Hz), 6.92 (s, 1 H), 6.41 (s, 1 H), 6.31
(d, 1 H, J ) 15.9 Hz), 6.02 (s, 1 H) 5.95 (s, 2 H), 4.26 (q, 2 H, J )
7.1 Hz), 1.29 (t, 3 H, J ) 7.1 Hz). 13C NMR (ppm, 62.8 MHz, CD3-
COCD3, 298 K) δ: 167.8, 153.7, 151.5, 142.6, 140.1, 115.5, 114.7,
106.5, 102.5, 98.6, 60.4, 14.7. Anal. Calcd (%) for C12H12O5 (236):
C, 61.01; H, 5.12. Found: C, 60.86; H, 5.21. Mass spectroscopy, MS
(CI, CH4): m/z 237 [M+1]. MS (CI, CH4, HR): m/z 237.0762 (calcd
mass for C12H13O5, 237.0763).
ref(λexc
)
(1)1 - 10-A
D
Dref
n
2
Φ(F1) ) Φ
(1)
ref
)
(n )
1 - 10-A(λ
exc
ref
where the subscript ref stands for standard samples. A(λexc) is the
absorbance at the excitation wavelength, D is the integrated emission
spectrum, and n is the refractive index for the solvent. The uncertainty
in the experimental value of Φ(F1) was estimated to be (10% . The
standard fluorophore for the quantum yields measurements was quinine
(E)-3-(3,5-Dibromo-2,4-dihydroxyphenyl) Acrylic Acid Ethyl
Ester (Ec). Yield, 40%; mp 118-118.5 °C. 1H NMR (ppm, 250 MHz,
CDCl3, 298 K) δ: 7.81 (d, 1 H, J ) 16.1 Hz), 7.60 (s, 1 H), 6.47 (d,
1 H, J ) 16.1 Hz), 6.07 (bs, 2 H), 4.22 (q, 2 H, J ) 7.0 Hz), 1.33 (t,
3 H, J ) 7.0 Hz). 13C NMR (ppm, 62.8 MHz, CD3COCD3, 298 K) δ:
167.3, 154.2, 153.7, 138.7, 131.6, 118.8, 117.8, 102.1, 101.8, 60.7,
14.6. Anal. Calcd (%) for C11H10O4Br2 (365.9): C, 36.10; H, 2.75.
Found: C, 36.06; H, 2.63. Mass spectrometry, MS (CI, CH4): m/z 367
[M + 1]. MS (CI, CH4, HR): m/z 364.9024, 366.9006, and 368.8992
(calcd mass for C11H10O4Br2: 364.9024, 366.9004, and 368.8985).
sulfate in 0.1 M H2SO4 with Φ(1) ) 0.55.38
ref
Irradiation Experiments. Three different protocols have been used
to perform the irradiations relying on one-photon excitation.
The experiments devoted to the identification of the products from
the photochemical isomerization of the cinnamate Ec by 1H NMR were
performed for various durations at 20 °C by putting a bench top UV
lamp (6 W 365 nm UV lamp; Fisher Bioblock) above a cuvette
containing V ) 2.5 mL of illuminated solution that was subsequently
transferred into a NMR tube.
The experiments devoted to evidencing the formation of the coumarin
Fc from the photochemical isomerization of the cinnamate Ec by UV-
vis absorption as well as to analyze the kinetics of E photoisomerization
were performed on 2.5 mL samples in 1 × 1 cm2 quartz cuvettes under
constant stirring. Excitations were performed with the 75 W xenon lamp
of a Photon Technology International LPS 220 spectrofluorometer. In
the case of the kinetics experiments, the continuous illumination was
performed at several slit widths to cover a significant range of incident
light intensities. The corresponding incident light intensities were
systematically calibrated by determining the kinetics of photoconversion
of the R-(4-dimethylaminophenyl)-N-phenylnitrone into 3-(4-dimethy-
laminophenyl)-2-phenyloxaziridine in fresh dioxane as described in ref
39. During the present series of experiments, typical incident light
intensities were in the 10-7 einstein min-1 range.
The experiments devoted to analyze the kinetics of the thermal
processes following the (E) to (Z) cinnamate photoisomerization were
performed on 2.5 mL samples in 1 × 1 cm2 quartz cuvettes. The cuvette
was continuously submitted to the irradiation from a bench top UV
lamp (6 W 365 nm UV lamp; Fisher Bioblock) for 1 min. The
absorbance was subsequently recorded as a function of time on the red
edge of the absorption band.
Two-Photon Excitation. The two-photon experiments were per-
formed with a home-built setup9,40 using fluorescein for calibration.
Illumination comes from a mode-locked titanium-sapphire laser (Mira
pumped by Verdi, Coherent). Fluorescence photons were collected with
an Olympus UPlanApo 60 × 1.2 NA water immersion objective through
filters (AHF Analysentechnik) and optical fibers (FG200LCR multi-
mode fiber, Thorlabs) connected to two avalanche photodiodes
(SPCM-AQR-14, Perkin-Elmer, Vaudreuil, Canada) coupled to an
ALV-6000 correlator (ALV GmbH). The excitation input power was
measured with a Lasermate powermeter (Coherent) or a Nova II
(E)-3-(4-Diethylamino-2-hydroxy-phenyl) Acrylic Acid Ethyl
1
Ester (Ed). Yield, 65%; mp 134-137 °C. H NMR (ppm, 250 MHz,
CDCl3, 298 K) δ: 7.88 (d, 1 H, J ) 15.9 Hz), 7.31 (d, 1 H, J ) 8.8
Hz), 6.33 (d, 1 H, J ) 15.9 Hz), 6.25 (d, 1 H, J ) 8.8 Hz), 6.03 (s, 1
H), 5.66 (s, 1 H), 4.24 (q, 2 H, J ) 7.1 Hz), 3.34 (q, 4 H, J ) 7.0 Hz),
1.32 (t, 3 H, J ) 7.0 Hz), 1.16 (t, 6 H, J ) 7.0 Hz). 13C NMR (ppm,
62.8 MHz, CDCl3, 298 K) δ: 168.6, 156.8, 150.7, 140.3, 130.7, 112.2,
110.0, 105.1, 98.0, 60.0, 44.5 (2C), 14.4, 12.6 (2C). Anal. Calcd (%)
for C15H21O3N (263.3): C, 68.40; H, 8.03; N, 5.31. Found: C, 67.87;
H, 7.98; N, 5.65. Mass spectroscopy, MS (CI, CH4): m/z 264 [M +
1]. MS (CI, CH4, HR): m/z 264.1596 (calcd mass for C15H22O3N:
264.1600).
(E)-3-(8-Hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido(3,2,1-ij)quinolin-
9-yl) Acrylic Acid Ethyl Ester (Ei). Yield, 75%; mp 167-169 °C.
1H NMR (ppm, 250 MHz, CDCl3, 298 K) δ: 7.86 (d, 1 H, J ) 15.8
Hz), 6.97 (s, 1 H), 6.24 (d, 1 H, J ) 15.8 Hz), 4.95 (s, 1 H), 4.22 (q,
2 H, J ) 7.1 Hz), 3.38 (q, 4 H, J ) 5.2 Hz), 2.67 (t, 4 H, J ) 6.4 Hz),
1.94 (m, 4 H), 1.31 (t, 3 H, J ) 7.0 Hz). 13C NMR (ppm, 62.8 MHz,
CD3 OCD3, 298 K) δ: 168.4, 153.8, 146.7, 141.5, 126.5, 115.2, 111.5,
111.0, 108.0, 59.9, 50.6, 49.8, 28.0, 22.8, 22.1, 22.0, 14.8. Anal. Calcd
(%) for C17H21O3N (287): C, 71.06; N, 4.87; H, 7.37. Found: C, 70.83;
N, 4.70; H, 7.5. Mass spectroscopy, MS (CI, CH4): m/z 288 [M + 1].
MS (CI, CH4, HR): m/z 288.1603 (calcd mass for C17H22O3N:
288.1600).
Solutions. Except for pKa measurements performed in the Britton-
Robinson buffer36 (acetic acid, boric acid, phosphoric acid) at 0.1 M,
in vitro experiments were made in CH3CN/20 mM Tris (pH ) 7) 100
mM NaCl buffer 1/1 v/v or in acetonitrile. No thermal (E) to (Z)
isomerization or thermal ester hydrolysis were ever observed at the
time scale of the present experiments (24 h).
pH Measurements. pH measurements were performed on a Standard
pH meter PHM210 Radiometer Analytical that was calibrated with
buffers at pH 4 and 7.
Spectroscopic Measurements and Irradiation Experiments. One-
Photon Excitation. UV-Visible Absorption. UV/vis absorption
spectra were recorded at 293 K on a Kontron Uvikon-940 spectropho-
tometer. Molar absorption coefficients were extracted while checking
the validity of the Beer-Lambert law.
(37) Gampp, H.; Maeder, M.; Meyer, C. J.; Zuberbu¨hler, A. D. Talanta 1985,
32, 95-101. Gampp, H.; Maeder, M.; Meyer, C. J.; Zuberbu¨hler, A. D.
Talanta 1985, 32, 257-264. Gampp, H.; Maeder, M.; Meyer, C. J.;
Zuberbu¨hler, A. D. Talanta 1985, 32, 1133-1139. Gampp, H.; Maeder,
M.; Meyer, C. J.; Zuberbu¨hler, A. D. Talanta 1986, 33, 943-951.
(38) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991-1024.
(39) Wang, P. F.; Jullien, L.; Valeur, B.; Filhol, J. S.; Canceill, J.; Lehn, J. M.
New J. Chem. 1996, 20, 895-907.
The evolutions of the absorbance as a function of pH were analyzed
with the SPECFIT/32 Global Analysis System (version 3.0 for 32-bit
(40) Charier, S.; Meglio, A.; Alcor, D.; Cogne´-Laage, E.; Allemand, J. F.; Jullien,
L.; Lemarchand, A. J. Am. Chem. Soc. 2005, 127, 15491-15505.
(36) Frugoni, C. Gazz. Chim. Ital. 1957, 87, 403-407.
9
9996 J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007

Evaluation of the o-Hydroxycinnamic Platform
A R T I C L E S
powermeter (Ophir Optronics Ltd.). Powers were kept under 5 mW at
the sample to stay in the quadratic range for absorption and to be not
detrimental to cells in the in vivo application.41 The intensity and the
temporal correlation function of the collected fluorescence emission
are recorded.
µL and τV ) 4 min, whereas τu ) 1/(k1 + k-1) = 4700 min in the
fastest situations encountered during that work.
Thus the following equations can be considered as valid beyond a
characteristic homogeneization time scale by considering concentrations
averaged over the whole volume V.
Then we rely on the mechanism displayed in Scheme 3a to write
eqs (3-6) describing the concentration evolutions:
FCS Experiments. FCS autocorrelation curve G(τ) were recorded
during the irradiation experiments devoted to extract the action
photoisomerization cross sections with two-photon excitation (vide
infra). At the beginning of the irradiation experiment, the fluorescence
emission from the solution of the cinnamate E is too weak to record
any tractable FCS curve. Therefore we first irradiate the E solution to
produce enough coumarin coproduct F to get enough signal and
appropriate FCS curves: We typically excite the solution at 750 nm
with a laser power in the 1-2 mW range for ∆t0 ) 2 h which typically
corresponds to 1% of E f F photoconversion. Beyond ∆t0, any FCS
autocorrelation curve G(τ) can be reliably fitted by assuming that the
solution contains one freely diffusing species only: the coumarin F.
Indeed, the contribution of the residual caged alcohol E in the G(τ)
curve can be neglected owing to the weak E brightness. We conse-
quently used eq 2 to fit the experimental FCS autocorrelation curves
G(τ) displayed in Figure 3b:42
dE
dt
) - κ1E + κ-1
Z
(3)
(4)
(5)
(6)
dZ
dt
) κ1E - (κ-1 + κ2)Z + κ-2
I
dI
dt
) κ2Z - (κ-2 + κ3)I + κ-3F × A
dF
) κ3I - κ-3F × A
dt
where A designates the concentration in photoreleased alcohol.
Then we first take into account that the exchange between Z and I
is fast at the time scale of the in vitro irradiation experiments; beyond
the relaxation time associated to the Z a I exchange, it is meaningful
to introduce the average species ZI (concentration ZI ) Z + I; then
the concentrations in Z and I are (1/(1+K2)) ZI and (K2/(1+K2)) ZI,
respectively, where K2 designates the equilibrium constant associated
to the conversion of Z into I). We additionally consider the large
coumarin stability to neglect the terms associated to the back reaction
(-3) in eqs 5-6. Thus eqs 3-6 transform into eqs 7-9:
1
1
G(τ) )
(2)
NF 1 + τ/τD
In eq 2, NF and τD designate the average number of F molecules in the
illuminated spot described as 2D-Gaussian and the diffusion time of F
through the beam waist.
Irradiation Experiments. The irradiations were performed on 2.5
µL samples contained in a cylindrical well made of parrafin (radius,
1.25 mm; height, 2 mm) sealed with Parafilm (no evaporation occurs
on a time scale of several days).
Kinetic Analysis of the Photoisomerization of the Cinnamates E
to the Coumarins F. The Model. We are here interested in the
derivation of the theoretical expressions of the concentrations in E, Z,
I, and F (E, Z, I, and F, respectively) during the in vitro uncaging
process.
dE
dt
) - k1E + k-1ZI
(7)
(8)
(9)
dZI
dt
) k1E - (k-1 + k2)ZI
dF
) k2ZI
dt
We first assume that the system (volume V) is homogeneous at any
time of its evolution. In fact, the whole system is not homogeneously
illuminated during the irradiation experiments; only part of the system
(volume Vexc) is submitted to light. Thus matter transport has to be
considered to support the homogeneity assumption:
(1) In the case of the one-photon illumination, the cuvette content
is continuously stirred, and the homogeneity assumption is fulfilled as
soon as uncaging does not occur much below the second time scale
which is here the case.
with k1 ) κ1, k-1 ) (1/(1+K2))κ-1, and k2 ) (K2/(1+K2))κ3. Equations
7-9 make clear the interpretation of the reduced mechanism displayed
in Scheme 3b, that it is only valid beyond the time scale for exchange
between Z and I.
During the continuous illumination experiments, the differential
equation governing the temporal evolution of F is
d3F
dt3
d2F
dt2
dF
+ (k1 + k-1 + k2)
+ k1k
) 0
(10)
2 dt
(2) In contrast, there is no such stirring upon two-photon illumination;
homogeneization is controlled by diffusion. In fact, we take here again
advantage from the slow uncaging rates with regards to the homoge-
neization process: the uncaging relaxation time is much larger than
the characteristic time τV required for E, Z, I, and F to diffuse in the
whole system. Indeed, for in vitro two-photon experiments, V ) 2.5
Considering that the system initially only contains E (initial
concentration E0), we get after integration:
x
E0
∆ + k1
2
+k +x∆)t/2)
1
-1
2
E )
e(-(k +k
+
(
x
∆
x
∆ - k1
2
x∆)t/2)
-1+k2-
e(-(k +k
(11)
(12)
1
(41) During the present series of experiments, we observed the formation of
holes from focusing the laser beam in the zebrafish embryo at laser powers
beyond 15 mW at 750 nm at the sample. We do not think that this
detrimental effect results from heat associated with light absorption with
residual one-photon excitation at 750 nm followed by nonradiative
deexcitation. First, below 5 mW at 750 nm at the sample, water does not
lead to any significant heating (see: Scho¨nle, A.; Hell, S. W.; Opt. Lett.
1998, 23, 325-327). In addition, the zebrafish embryo does not contain
any significant amount of interfering endogeneous chromophores at that
step of development. In particular, we were never able to express GFP
under control of a heat-shock promoter by simply focusing the IR laser
beam in any targeted cell of an appropriate transgenic embryo. In fact, we
estimate that our observation is more probably related to plasma formation
that occurs at 10 mW at 750 nm even in pure solvents (see: Sacchi, C. A.
J. Opt. Soc. Am. B 1991, 8, 337-345. Vogel, A.; Noack, J.; Hu¨ttman, G.;
Paltauf, G.; App. Phys. B 2005, 85, 1015-1047).
)
k1E0
x
-1+k2-
x∆)t/2)
)
-1+k2+
(e(-(k +k
∆)t/2) - e(-(k +k
1
1
ZI )
x
∆
2k1k2E0
1
x
-1+k2+
F )
(- 1 + e(-(k +k
∆)t/2)) +
1
[
x
∆
x
k1 + k-1 + k2 +
∆
1
x∆)t/2)
-1+k2-
(1 - e(-(k +k
)
(13)
1
]
x
k1 + k-1 + k2 -
∆
(42) Krichevsky, O.; Bonnet, G. Rep. Prog. Phys. 2002, 65, 251-297.
where ∆ ) (k1 + k-1 + k2)2 - 4k1k2.
9
J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007 9997

A R T I C L E S
Gagey et al.
Introducing QE, QZI and QF the brightnesses of E, ZI,43 and F,
respectively, we eventually derive the temporal evolution of the
fluorescence intensity IF as
1/k1 which was analyzed here. Then the corresponding slope of the
linear IF variation is equal to I˙F ) RQFE0k1.
RQF can be independently measured by recording and analyzing the
APD counts and the autocorrelation curve from a solution of the
coumarin F. The APD counts provide RQFNFP2 where NF and P
designate the average number of F molecules in the illuminated spot
and the laser power at the sample. The FCS autocorrelation curve gives
NF from the ordinate at the origin, and P is measured with the
powermeter.
x
E0
∆ - k1
2
x∆)t/2)
(-(k1+k-1+k2-
x
IF ) R
∆QF + e
QZIk1 + QE
-
(
[
x
∆
2k1k2
x∆)t/2)
-1+k2+
1
QF
+ e(-(k +k
)
x
k1 + k-1 + k2 -
∆
E0 being known, k1 can be thus derived from I˙F.44 The action
(2)
EZ
photoisomerization cross section with two-photon excitation δE(2)
Φ
x
∆ + k
2
2k1k2
- QZIk1 + QE
1 + QF
(14)
is eventually derived from the expressions of k1 following Kiskin et
al.:7
(
)
]
x
k1 + k-1 + k2 +
∆
where R is a collecting factor of fluorescence emission.
2V
(2)u
λ
k1 ) 0.737δEΦ
excP2
V
(18)
In the case of relaxation experiments devoted to independently
measuring the rate constant associated to the thermal conversion of ZI
to F, the evolution of the sample absorption with one-photon excitation
A is followed as a function of time after an initial strong illumination
pulse. Adopting the second step displayed in Scheme 3b to model the
system relaxation, the absorbance A obeys an exponential law: A(t)
) A0 exp(-k2t) + A∞[1 - exp(-k2t)].
Extraction of the Action Photoisomerization Cross Sections with
One- and Two-Photon Excitation. One-Photon Illumination. The
orders of magnitude of k1, k-1, and k2 are similar under the present
experimental conditions with one-photon excitation. The fluorescence
evolution is thus directly fitted with eq 14 using ø1 ) (k1 + k-1 + k2
EZτ
2
(
)
P πhcwxy
In eq 18, δE, Φ(2), u, τP, and wxy designate the cross sections for two-
EZ
photon absorption leading to (E)- to (Z)-photoisomerization at the
(2)
exc
excitation wavelength λ of E, the quantum yields associated to the
(E)- to (Z)-photoconversions after two-photon excitation, the period
of the laser pulses (13.6 ns in our case), the duration of the laser pulses
(200 fs), and the waist of the focused laser beam (0.3 µm).
Acknowledgment. Prof. Sophie Vriz is gratefully acknowl-
edged for generous gifts of zebrafish embryos. Dr. Jean-Franc¸ois
Allemand is gratefully acknowledged for his help in designing
FCS experiments. This research is financially supported by the
“Association pour la Recherche sur le Cancer’’ (ARC) (2005,
Project n3787).
x
x
-
∆)/2, ø2 ) (k1 + k-1 + k2 + ∆)/2, A1 ) ((QZI/QF)k1 + QE/QF
x
(
x
∆ - k1)/2 - k1k2/ø1) and A2 ) (- (QZI/QF)k1 + QE/QF ( ∆ + k1)/
2) + k1k2/ø2) as floating parameters. k1, k-1, and k2 are then extracted
from analyzing the dependence of ø1 and ø2 on light intensity. In
addition, A1 and A2 provide QZI/QF and QE/QF.
The action photoisomerization cross sections with one-photon
excitation are eventually derived from the expressions of k1 and k-1
given by the relations (15-16):39
Supporting Information Available: Syntheses of the non-
commercially available starting substituted benzaldehydes and
of the 1-carboxymethyliden triphenyl phosphorane; preliminary
screening of the thermal stability and the photophysical and the
photochemical properties of the caged model compounds E′a-h
in aqueous solutions (Table 1S); a schematic picture to analyze
the photochemical and thermal isomerizations in the cinnamate
series (Schemes 1S and 2S); UV-vis absorption spectra of Ea
and Eb (Figure 1S); measurement of the Ec and Fc protonation
constants (Figures 2Sa-c); identification of the products from
the photochemical isomerization of the cinnamate Ec with one-
photon excitation (Figures 3Sa-b, 4Sa-d, and 5S); dependence
of the apparent thermodynamic constant for the Ec to ZIc
exchange on the one-photon excitation wavelength (Figures 6S
and 7S, Scheme 3S); model for the kinetics of two-photon
uncaging in vivo. This material is available free of charge via
the Internet at http://pubs.acs.org.
2.3ꢀE(λe(1x)c)φE(1Z)l
(1)
exc
k1 )
I0(λ
)
(15)
(16)
V
2.3ꢀZ(λ(e1x)c)φZ(1E)l
1
(1)
k-1
)
I0(λ )
exc
1 + K2
V
In eqs (15,16), ꢀE(λ(1) ) and ꢀZ(λe(1x)c), φ(E1Z), and φ(Z1E), l, V, and I0(λ(1)
)
exc
exc
designate the molar absorption coefficients of the (E) and (Z) cinnamate
stereoisomers, the quantum yields for the (E)- to (Z)- and (Z)- to (E)-
photoconversions, the path length of the light beam, the irradiated
volume, and the light intensity at the excitation wavelength, respectively.
In Table 1, we introduce the steady-state constant K1(λ(e1x)c) associ-
(1)
exc
ated to the E to ZI exchange with one-photon excitation at λ
:
ꢀE(λ(e1x)c)φ(1)
EZ
K1 ) (1 + K2)
(17)
JA0722022
(1)
ꢀZ(λ(e1x)c)φ
ZE
(44) Although here less precise, a satisfactory order of magnitude of k1 can
also be extracted from the FCS data. In the considered kinetic regime, E
can be considered to directly afford F with k1 rate constant and the number
of F molecules in the focal point, NF, increases linearly with time with a
Two-Photon Illumination. To derive the expressions of k1 and k-1
when two-photon excitation is used, we consider that the reactions (1)
and (-1) displayed in Scheme 3b take place only in the focal volume
Vexc resulting from laser focusing. The value k2 is now much larger
than k1 and k-1 under the present experimental conditions with two-
photon excitation. In particular, ø1 ) k1 and ø2 ) k2 at first order. Upon
assuming that the reservoir of starting cinnamate is infinite (E ) E0)
and by neglecting the brightness of E in front of the one of F, one
easily demonstrates that the temporal fluorescence evolution given in
eq 14 becomes linear in the kinetic regime between the times 1/k2 and
slope E0k1. Then the average number of molecules 1/G(0) ) < NF
>
[t1;t2]
extracted from the FCS experiment between time t1 and t2 (0 and 1500 s,
and then 4500 and 6000 s in the experiment shown in Figure 3b) is given
by the expression:
E0k1t2+N0F
t2
1
1
dt
1
)
)
ln
∫
0
[t1;t2]
E0k1t+N0F
E0k1t1+NF
(
)
<NF>
t2-t1
t1
E0k1(t2-t1)
where N0F is the number of F molecules in the focal point a t ) 0 s. Then
the results from at least two FCS experiments provide NF and k1. In the
0
case of the curves displayed in Figure 3b, we can extract in particular k1
) 1.2 × 10-6 s-1 which is in good agreement with the value obtained
(43) QZI ) (1/(1 + K2))QZ + (K2/(1 + K2))QI where QZ and QI designate the
brightnesses of Z and I, respectively.
from the slope extracted from Figure 3a, k1 ) 1.0 × 10-6 s-1
.
9
9998 J. AM. CHEM. SOC. VOL. 129, NO. 32, 2007