Alcohol uncaging with fluorescence reporting: Evaluation of o-acetoxyphenyl methyloxazolone precursors

Article abstract of DOI:10.1021/ol800284g

(Chemical Equation Presented) This paper evaluates a series of photolabile protecting groups with built-in fluorescence reporting. They rely on readily available o-acetoxyphenyl methyloxazolones as activated precursors. Alcohol substrates are easily caged. The resulting photoactivable esters exhibit large one- and two-photon uncaging cross sections. The alcohol substrates are quantitatively released in a 1:1 molar ratio with a strongly fluorescent coumarin coproduct that serves as a reporter to quantify substrate delivery.

Full text of DOI:10.1021/ol800284g

ORGANIC
LETTERS
2008
Vol. 10, No. 12
2341-2344
Alcohol Uncaging with Fluorescence
Reporting: Evaluation of
o-Acetoxyphenyl Methyloxazolone
Precursors
Nathalie Gagey,† Matthieu Emond,† Pierre Neveu,†,‡ Chouaha Benbrahim,†
Bernard Goetz,† Isabelle Aujard,† Jean-Bernard Baudin,† and Ludovic Jullien*,†
´
De´partement de Chimie and Laboratoire de Physique Statistique, Ecole Normale
Supe´rieure, 24, rue Lhomond, 75231 Paris Cedex 05, France
ludoVic.jullien@ens.fr
Received March 4, 2008
ABSTRACT
This paper evaluates a series of photolabile protecting groups with built-in fluorescence reporting. They rely on readily available o-acetoxyphenyl
methyloxazolones as activated precursors. Alcohol substrates are easily caged. The resulting photoactivable esters exhibit large one- and
two-photon uncaging cross sections. The alcohol substrates are quantitatively released in a 1:1 molar ratio with a strongly fluorescent coumarin
coproduct that serves as a reporter to quantify substrate delivery.
Uncaging1–3 with one- and two-photon excitation4 is a
method of choice for a precise delivery of chemicals both
spatially and temporally in a noninvasive way. Its use in
biological applications is now well-established.5–10 However,
because of the possible damaging of biological samples by
light, illumination duration and power should ideally be kept
low. There is thus a lower limit on the two-photon uncaging
action cross section needed to deliver a given amount of
molecules: values of 0.1 or 10 Goeppert-Mayer (GM; 1 GM
) 10-50 cm4 s/photon) are usually cited for a biological
application.6,11 In fact the 1 GM range has already been
reached, leading to a successful use in biological sys-
tems.6,12–14
As biological samples often respond in a dose-dependent
manner, a tool making it possible to vary, in a well-
established way, the amount of delivered molecules would
be of great interest. In theory, uncaging fulfills that aim, but
it is often hampered by the need to characterize the uncaging
rate in the biological system. Therefore an easy way to
quantify the uncaging reaction would be extremely useful.
One possibility is to use a fluorescent reporter.
† De´partement de Chimie UMR CNRS-ENS-UPMC Paris 06 8640 and
8642.
‡ Laboratoire de Physique Statistique UMR CNRS-ENS-UPMC Paris
06-Paris 07 8550.
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.
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Using the o-hydroxy cinnamate series that had been
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introduced by Porter et al.,15 we showed that such photolabile
458
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10.1021/ol800284g CCC: $40.75
Published on Web 05/27/2008
2008 American Chemical Society

protecting groups were good candidates to fulfill those goals.
Indeed they exhibit a large two-photon uncaging action cross-
section up to 4.7 GM at 750 nm in acetonitrile.14In addition,
whereas they are intrinsically nonfluorescent, uncaging leads
to the release of a fluorescent reporter and a chosen alcohol
in a 1:1 molar ratio. The increase of fluorescence intensity
at the uncaging site can be used as a probe for a quantitative
analysis of the uncaging reaction (Figure 1).14 This property
is independent of the protected substrate.16
that this platform could be used to develop caging groups.
We were especially interested in the easy and reliable
coupling of the desired substrate. Indeed, the preparation of
the carboxylic acid unit to cage a given alcohol in Porter’s
original series proved to be delicate from the synthetically
accessible ethyl ester precursor. This feature could hamper
further developments of the cinnamic platform to cage
alcohols. In the series presented hereinafter, the direct
reaction between the chosen alcohol and an appropriate
oxazolone cycle (playing the role of an activated carboxylic
acid) leads simply, in one step only, to the envisioned
o-hydroxy cinnamate ester.
The relevance of the o-hydroxy oxazolone platform for
uncaging applications was assayed using the original design
of Cornforth et al. with a phenyl group as the oxazolone
substituent. Results proved to be promising: (i) the syntheses
of the o-hydroxyphenyl phenyloxazolone were easy, (ii) the
oxazolone ring could be opened with stoichiometric amounts
of a model alcohol, and (iii) the uncaging properties of the
resulting cinnamates were attractive (see Supporting Infor-
mation). However, the 3-benzoylamido coumarin coproduct
formed upon uncaging was poorly fluorescent. We therefore
prepared a series of 3-acyloylamido coumarins to identify a
more appropriate fluorescent reporter for uncaging. Among
these, we found that 3-acetamido coumarins were strongly
fluorescent for all of the investigated electron-donating
substituents (see Supporting Information). Hence we decided
to focus on the o-hydroxyphenyl methyloxazolone platform.
The syntheses of the corresponding o-hydroxyphenyl
methyloxazolone derivatives with the Plo¨chl-Erlenmeyer
synthesis19 were less straightforward than in the o-hydrox-
yphenyl phenyloxazolone series; this might be due to a poorer
solubility, which would forbid product precipitation after
oxazolone formation.20–23 We therefore considered an al-
ternative synthetic pathway (Scheme 1). We first activated
Figure 1. Uncaging with fluorescence reporting. Light absorption
by the nonfluorescent o-hydroxyphenyl cinnamate cA resulting from
oxazolone opening with the alcohol substrate ROH (violet and red
arrows with one- and two-photon excitation, respectively) initiates
a series of photochemical and thermal processes leading to release
the ROH substrate together with a fluorescent coumarin F coproduct
in a 1:1 stoichiometric ratio. cA and F absorb light in the same
wavelength range (black solid and dotted lines, respectively), and
the uncaging extent can be correspondingly extracted from the
intensity of F fluorescence emission (blue solid line). The absorption
and emission properties of the cinnamate 4c 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.
Scheme 1. Syntheses of Caged Model Alcohols cA (4a-c)
In parallel to the development of the o-hydroxy cinnamate
series, we considered another cinnamate series relying on
the o-hydroxyphenyl oxazolone precursor. During the syn-
thesis of those cinnamates, Cornforth et al. noticed that, upon
heating, isomerization occurs around their trisubstituted
double bond, leading to the formation of a coumarin.17 Such
a reaction could also be triggered by light, as later noted.18
Their resemblance with Porter’s series led us to hypothesize
(12) Lu, M.; Fedoryak, O. D.; Moister, B. R.; Dore, T. M. Org. Lett.
2003, 5, 2119–2122.
(13) Gagey, N.; Neveu, P.; Jullien, L. Angew. Chem. Int. Ed. 2007, 46,
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J.- B.; Jullien, L. J. Am. Chem. Soc. 2007, 129, 9986–9998.
(15) Turner, A. D.; Pizzo, S. V.; Rozakis, G.; Porter, N. A. J. Am. Chem.
Soc. 1988, 110, 244–250.
the aldehyde electrophilic center by alkyl imine formation
which was shown to be especially efficient for reactions
related to the nucleophilic condensation of N-acetyl glycine.24
The butylimines 2a-c were prepared quantitatively from
butylamine and the parent easily accessible benzaldehydes
1a-c (the benzaldehyde 1c was obtained according to the
literature).25 We subsequently coupled N-acetyl glycine to
the 2-hydroxyphenylbutylimines 2a-c in acetic anhydride.
The 2-acetoxyphenyl methyloxazolones 3a-c were obtained
(16) The photorelease of favorable substrates can be reported by a
fluorescence increase in another caging system. See: Hagen, V.; Frings, S.;
Bendig, J.; Lorenz, D.; Wiesner, B.; Benjamin Kaupp, U. Angew. Chem.,
Int. Ed. 2002, 41, 3625–3628.
(17) Cornforth, Sir J.; Ming-Hui, D J. Chem. Soc., Perkin Trans. 1 1991,
2183–2187.
(18) Walter, R.; Purcell, T. C.; Zimmer, H. J. Heterocycl. Chem. 1966,
235–236.
2342
Org. Lett., Vol. 10, No. 12, 2008

Table 1. Photophysical and Photochemical Properties of Investigated (Z)-Cinnamates Relevant to the Uncaging Process with One- and
Two-Photon Excitationa
εZ (λ(Z1)),
-1
-1
(1)
(1)
(1)
ZE
mM
cm
(nm)
λ(F1)[Φ(F1)],
nm[%]
λ
,
Φ
,
εZ(λ(e1xc))Φ
mM
,
103 k2,
s-1
δZ(750)
δF(750)
Φ(F2), GM
exc
ZE
-1
-1
(2)
nm
103QZ/QF
%
cm
Φ
, GM
ZE
4a
4b
4c
17(330)
28(380)
17(356)
404[100]
483[70]
426[100]
330
360
350
1.8
1.6
4.4
4
5
7
0.7
1.1
1.1
35
30
130
b
0.3
2.0
b
6.2
4.6
a Maxima of single-photon absorption (λ(Z1)) and molar absorption coefficients for single-photon absorption at λ(Z1), εZ(λ(Z1)) ((5%) of the (Z)-cinnamates;
wavelength maxima (λ(F1)) and quantum yields (Φ(F1) ( 10%) of fluorescence emission of the coumarin coproducts upon uncaging after one-photon excitation;
(1)
excitation wavelength used for the series of uncaging experiments with one-photon excitation, λ ; relative brightness of the starting (Z)-cinnamate with
exc
(1)
regard to brightness of the F coumarin coproduct at λ(ex1c), QZ/QF; quantum yield of (Z) to (E) photoisomerization after one-photon excitation at λ(1), ΦZE
((10%); action cross section for (Z) to (E) photoisomerization with one-photon excitation at λ(ex1c), εZ(λ(1))ΦZE ((5%); rate constant for the theerxmc al (E)-
(1)
exc
cinnamate to coumarin lactonization extracted from continuous illumination experiments, k2 ((10%); action cross section for (Z) to (E) photoisomerization
(2)
(2)
with two-photon excitation at λ ) 750 nm, δZ(750) ΦZE ((20%; GM, 1 GM ) 10-50 cm4 s/photon) for the (Z)-cinnamate, and action cross section for
exc
(2)
(2)
fluorescence emission of the F coumarin with two-photon excitation at λ ) 750 nm, δF(750) Φ(F2) ((20%; GM). Except for δZ(750) ΦZE given in
exc
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.
with reasonably good yields. These methyloxazolone precur-
sors 3a-c were eventually condensed with a model alcohol
to characterize the photophysical and photochemical proper-
ties of the resulting cinnamates. n-Butanol was retained here
because of the neutrality of the butyl chain with regards to
the photophysical and photochemical properties of the
photoactive backbone. The final model (Z)-cinnamates 4a-c
were easily obtained with moderate to good yields from
condensing 2 equiv of sodium butylate in THF on 3a-c.
The UV-vis absorption spectra of the investigated (Z)-
cinnamates 4a-c were used to determine their absorption
properties upon one-photon excitation. In Tris pH 7, 20 mM
NaCl, 100 mM buffer/acetonitrile 1/1 (v/v), the absorption
spectrum of 4a-c lies in the 300-400 nm range (Figure 1).
Table 1 summarizes the absorption features of 4a-c, maximum
λ(Z1) and molar absorption coefficient εZ(λ(Z1)) for single-photon
absorption, associated with the band at the longest wavelength.
The absorption maximum with one-photon excitation λ(Z1) in
4a-c is systematically red-shifted by 20 kJ mol-1 (12-18 nm)
compared to that of the corresponding methyl-trisubstituted
cinnamate in the Porter series.14 This behavior is most probably
related to the additional length of the conjugation pathway due
to the N-acyl substituent on the double bond compared to the
methyl group. As anticipated, λ(Z1) shifts to the red when the
donating power of the conjugated phenyl substituent increases.
Hence, the longest maximal wavelength of absorption was
it is thus possible to perform uncaging and excitation of the
reporting fluorescent molecule with the same excitation
source.
We then analyzed the uncaging kinetics, which is critical
in view of possible biological applications.13,27 Figure 2
displays the typical temporal evolution of the fluorescence
emission from a solution of a (Z)-cinnamate that is continu-
ously illuminated at a wavelength λ(e1xc) close to λZ(1) . A large
increase of fluorescence emission as a function of time can
be observed.
The curves displayed in Figure 2 were analyzed using the
model introduced in our previous work.14 We considered a
three-step-six-reaction mechanism (Scheme 2a), which is
reduced to a two-step-three-reaction mechanism involving
only three rate constants (see Scheme 2b).
We derived the photochemical properties of the present
caging groups with one-photon excitation from k1 and k-1
(see Supporting Information). The fit also yields the relative
brightness of the starting (Z)-cinnamate Z with regards to
the one of the coumarin coproduct F. Table 1 displays the
results of the different parameters extracted from these series
of irradiation experiments. The coumarin coproduct emits a
very strong fluorescence emission in the 400-500 nm range
with quantum yields exceeding 60%. In contrast, the bright-
ness of the starting o-hydroxycinnamates is uniformly very
low at the excitation wavelength required for uncaging.
Hence uncaging can be easily evidenced and quantified using
the fluorescence increase with the present photolabile
protecting groups.
(1)
observed for the diethylamino derivative 4b (λ ) 380 nm)
4b
for which the absorption band significantly extends up to 420
nm. Eventually, the molar absorption coefficients at the
wavelength of maximal absorption are satisfactorily large:
εZ(λZ(1)) ≈ 2 × 104 M -1 cm -1
.
The photochemical properties of the present cinnamate
series were first analyzed with one-photon excitation. We
used H NMR and UV-vis absorption spectroscopy to
analyze the species formed upon illumination of the caged
(Z)-cinnamates. We showed using 4c that the uncaging
process obeys the reaction displayed in Figure 1: upon one-
photon excitation, the caged alcohol A (here butanol) is
released with the coumarin coproduct F in a 1:1 molar ratio
(see Supporting Information). This behavior is similar to
previous observations in the related Porter series.14,15,26
Noticeably, the present (Z)-cinnamates and the corresponding
coumarin coproduct F exhibit similar absorption properties;
(19) Erlenmeyer, E. Ann. 1893, 275, 1–8
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Org. Lett., Vol. 10, No. 12, 2008
2343

Scheme 2. (a) Mechanism of uncaging yielding the caged
alcohol A (here n-butanol) together with a coumarin F from
illumination of the (Z) o-hydroxy cinnamate cA precursor. (b)
Reduced mechanism for kinetic analysis. The rate constants
associated to each elementary step (a) or to the reduced
mechanism (b) are denoted, respectively, κi and ki where the
subscript i indicates the corresponding step.
(2)
excitation for 4b and 4c at λ ) 750 nm in acetonitrile (see
exc
Figure 2. Temporal evolution of the fluorescence emission at λem
Table 1). Both are larger than the 0.1 GM limit that was
claimed to be the lowest value relevant for biological
applications.6 In fact, the 4c value, which is equal to 2.0
GM at 750 nm, is among the largest reported to date.
) 450 nm from a 5 µM 4c solution in acetonitrile/Tris pH 7, 20
mM NaCl, 100 mM buffer 1/1 (v/v) upon one-photon irradiation
(1)
-8
(λ ) 350 nm) at light intensity I0(350) ) 2.4 × 10
einstein
exc
-1
min
. Markers: experimental data. Line: fit (see Supporting
Information). T ) 293 K.
Thus the present o-acetoxyphenyl methyloxazolones are
favorable precursors yielding robust and attractive photolabile
protecting groups with fluorescence reporting for alcohols
upon one- and two-photon excitation. The corresponding
o-hydroxy cinnamate incorporating the targeted alcohol can
be easily synthesized. Their maximal wavelength for one-
photon absorption can be 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 and a strongly
fluorescent coumarin coproduct in a 1:1 molar ratio. In
addition, uncaging and excitation of the reporting fluorescent
molecule can be achieved using the same excitation source.
We eventually measured two-photon uncaging action cross
sections that compare favorably with the best reported values.
In conclusion, the present o-acetoxy methyloxazolone pre-
cursors appear attractive to implement the “optical microsy-
ringe′′ strategy in biological applications that 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
by recording the fluorescence emission from the coumarin
F coproduct that acts as a reporter of the concentration of
the photoreleased substrate.
The quantum yields of (Z)- to (E)-cinnamate photoisomer-
ization leading to alcohol uncaging are in the 5% range; this
is significant compared to other uncaging photochemistries.
Various values ranging from more than 10%28–30 to less than
1%31 have been reported for the most popular o-nitrobenzyl
photoisomerization whereas similar values are reported in
the case of the coumaryl photolabile protecting group.6
The values for the rate constant k2 associated to the thermal
cyclization of the (E)-cinnamates leading to alcohol release
and the formation of the coumarin coproduct are in the 10
-1 s -1 range in the buffer-acetonitrile 1/1 (v/v) mixture.32
This range is 10- to 100-fold larger than for Porter’s
cinnamates bearing a doubly substituted double bond.14 From
the latter point of view, the present observation supports the
trend already observed in ref 14: the triple substitution pattern
of the cinnamate double bond appears as the most favorable
mean to accelerate substrate release.
We take advantage of the release upon uncaging of a
strongly fluorescent coumarin coproduct in a 1:1 stoichiom-
etry with the caged alcohol to measure the two-photon
uncaging action cross section.14 The kinetic analysis of the
uncaging process of 4b and 4c leading to the formation of
the coumarins Fb and Fc yields the action cross section for
(Z)- to (E)-cinnamate photoisomerization with two-photon
Acknowledgment. This research was financially supported
by the “Association pour la Recherche sur le Cancer′′ (ARC)
(2005, project n3787).
Supporting Information Available: Screening of the
o-hydroxyphenyl phenyloxazolone platform and of the
3-acyloylamido coumarins; experimental section; identifica-
tion of the products from the photochemical isomerization
of the cinnamate 4c with one-photon excitation. This material
is available free of charge via the Internet at http://pubs.acs.org.
(28) Corrie, J. E. T. J. Chem. Soc., Perkin Trans. 1 1993, 2161–2166.
(29) Specht, A.; Goeldner, M. Angew. Chem., Int. Ed. 2004, 43, 2008–
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Neveu, P.; Jullien, L. Chem. Eur. J. 2006, 12, 6865–6879.
(32) We already underlined in ref 14 that the k2 value is expected to
strongly depend on the solvent. In particular, it should be considerably larger
in a more polar solvent such as in aqueous solutions.
OL800284G
2344
Org. Lett., Vol. 10, No. 12, 2008