A blue-absorbing photolabile protecting group for in vivo chromatically orthogonal photoactivation

Article abstract of DOI:10.1021/cb400178m

The small and synthetically easily accessible 7-diethylamino-4- thiocoumarinylmethyl photolabile protecting group has been validated for uncaging with blue light. It exhibits a significant action cross-section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features have been implemented in living zebrafish embryos to perform chromatic orthogonal photoactivation of two biologically active species controlling biological development with UV and blue-cyan light sources, respectively.

Full text of DOI:10.1021/cb400178m

Articles
pubs.acs.org/acschemicalbiology
A Blue-Absorbing Photolabile Protecting Group for in Vivo
Chromatically Orthogonal Photoactivation
Ludovic Fournier,† Carole Gauron,‡ Lijun Xu,⊥ Isabelle Aujard,† Thomas Le Saux,†,§
Nathalie Gagey-Eilstein,† Sylvie Maurin,† Sylvie Dubruille,∥ Jean-Bernard Baudin,† David Bensimon,⊥,¶
Michel Volovitch,‡ Sophie Vriz,‡ and Ludovic Jullien*,†,§
†Ecole Normale Super
Cedex 05, France
‡Colleg
e de France, Center for Interdisciplinary Research in Biology (CIRB), CNRS, UMR 7241, INSERM, U1050, 11, Place
Marcelin Berthelot, 75231 Paris Cedex 05, France
⊥Ecole Normale Super
́
ieure, Departement de Physique and Departement de Biologie, Laboratoire de Physique Statistique, UMR
́ ́
ieure, Departement de Chimie, UMR CNRS-ENS-UPMC 8640 PASTEUR, 24, rue Lhomond, 75231 Paris
̀
́
́
CNRS-ENS 8550, 24 rue Lhomond, F-75231 Paris, France
§UPMC, 4, Place Jussieu, 75232 Paris Cedex 05, France,
∥Institut Curie, Centre de Recherche, CNRS, UMR 176, 26, rue d’Ulm, Paris F-75248, France
¶Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California, United States
S
* Supporting Information
ABSTRACT: The small and synthetically easily accessible 7-
diethylamino-4-thiocoumarinylmethyl photolabile protecting
group has been validated for uncaging with blue light. It
exhibits a significant action cross-section for uncaging in the
470−500 nm wavelength range and a low light absorption
between 350 and 400 nm. These attractive features have been
implemented in living zebrafish embryos to perform chromatic
orthogonal photoactivation of two biologically active species
controlling biological development with UV and blue-cyan
light sources, respectively.
protein kinases (MAPK) such as p38 and c-Jun-N-terminal
kinase (JNK).14,15 Carotenoids have been also incriminated
since they are prone to UV A-induced photodegradation and
are involved in the control of gene expression, either directly or
hotoactivation methods have recently proved attractive in
1−10
Pvarious fields of chemistry and biology.
Among
multiple photoactivation strategies, caged molecules have
especially found a widespread use. These molecules rely on
photolabile protecting groups that can be cleaved after light
absorption by the photoactive moiety. In particular, they have
been used to study the dynamics of several biological processes
with high spatiotemporal resolution.1−10 In such a context, one
important constraint is to ensure that the illumination does not
affect the biological system. This is particularly significant to
discriminate the impact of the photoreleased substrate from the
one of illumination (for instance during embryonic develop-
ment11,12). This concern can be hardly fulfilled for living beings
making extensive use of light to control their biological
functions (photosynthetic procaryotes and plants for instance).
However, it may be also relevant in a broader context since
many endogenous biologically active molecules significantly
absorb light even in the ultraviolet A range (UV A; 320−400
nm). Hence illumination of riboflavin or flavin mononucleotide
(FMN) can photosensitize triplet dioxygen,13 and the resulting
16
1
via their O2 quenching action.
Most presently reported caging groups exhibit a maximum of
wavelength absorption lying below the 450 nm range where
riboflavin, FMN, and carotenoids absorb light.1−10,17 To
overcome this limit, a first strategy is to retain the established
photochemistries with extension of the conjugation of the
absorbing moiety. Depending on the series, this approach has
met success18,19 but has also exhibited limitations20,21 when
absorption red-shift was associated to a drop of the quantum
yield for uncaging. The alternative strategy is to tailor caging
groups based on chromophores absorbing in the visible range.
This approach has been implemented by adapting the
rearrangement of an amino-substituted 1,4-benzoquinone to
Received: March 13, 2013
Accepted: April 24, 2013
Published: May 7, 2013
1
singlet state O2 can subsequently induce mitogen-activated
© 2013 American Chemical Society
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a
Scheme 1. Syntheses of Caging Alcohol tcOH and Caged Inducer tcInd
a
Reagents and conditions: (a) acetic acid, DCC, DMAP, CH2Cl2, room temperature, 12 h; (b) Lawesson’s reagent, toluene, reflux, 24 h; (c) 1.25 M
HCl in ethanol, reflux, 15 h; (d) CDI, CH2Cl2, reflux, 4 h; (e) DMAP, CH2Cl2, reflux, 4 h.
photorelease carboxylic acids and phenols using visible
light.22,23 The xanthene chromophore has been used to
photorelease carboxylates and phosphates upon illumination
at 520 nm.24 Ruthenium complexes have also been used to
photorelease complexed amines.25,26 We have chosen here to
investigate the opportunities provided by the small and
synthetically easily accessible thiocoumarin backbone.
Coumarins have led to the development of a successful series
of synthetically easily accessible caging groups.27−29 Consider-
ing that the light absorption of the thiocarbonyl group is
significantly red-shifted with respect to the carbonyl ana-
logue,30,31 we have been interested in examining whether
thiocoumarin analogues would exhibit a similar generic
photochemical behavior32−35 as suggested by the recent
introduction of photocleavable protecting groups for carboxylic
acids absorbing slightly below 400 nm.36 In this work, we
targeted the 7-diethylamino-4-methanol-thiocoumarin putative
caging group, to benefit from its strongly donating substituent
conjugated to the thiocarbonyl group to shift the wavelength of
maximal absorption.
In this paper, we demonstrate that the 7-diethylamino-4-
methanol-thiocoumarin moiety is a relevant blue-absorbing
caging group, which can be used in living zebrafish embryos in
the context of developmental biology. Relying on a caged
cyclofen-OH37,38 analogue, we were able to photocontrol with
blue light the activity of a transcription factor (En2) fused to
the modified estrogen receptor ligand-binding domain
ERT2.39,40 Moreover, taking advantage of the low absorption
of 7-diethylamino-4-methanol-thiocoumarin in the 350−400
nm wavelength range, we used two different wavelengths (365
and 488 nm) to orthogonally photoinduce41−47 two distinct
phenotypes upon independently photogenerating two bio-
logically active substrates: an analogue of the previously
investigated cyclofen-OH37,38 and 13-cis-retinoic acid (13-cis-
RA).
from the commercially available 7-diethylamino-4-methylcou-
marin, we first prepared the 7-diethylamino-4-hydroxymethyl-
coumarin cOH in a one-pot process in 42% yield, upon
adapting reported two-step procedures (SeO2, dioxane/water,
reflux, 14 days; NaBH4, ethanol, RT, 12 h).52,53 The alcohol
cOH was then esterified with acetic acid using the N,N′-
dicyclohexylcarbodiimide (DCC) coupling reagent to yield the
ester cA with 85% yield. This ester was subsequently converted
into the corresponding thiocoumarin tcA (Lawesson’s reagent,
toluene, reflux, 24 h) in 92% yield. The thiocoumarin tcA was
eventually hydrolyzed (1.25 M HCl in ethanol, reflux, 15 h) to
provide the putative caging alcohol tcOH with 75% chemical
yield (see Supporting Information).
To evaluate the relevance of the present caging group, we
adopted a substrate acting as an inducer for controlling the
activity of target proteins fused to the extensively used modified
estrogen receptor ligand-binding domain (ERT2) specific for the
non-endogenous 4-hydroxy-tamoxifen inducer.39,40 Relying on
an extensive study of the relationship between inducer structure
and function,54 we targeted an analogue of the previously
investigated cyclofen-OH,37,38 which is denoted Ind in the
following (Scheme 1). Ind contains a terminal primary amino
moiety,55 to be engaged in the carbamate group, which has
been widely used for caging in the coumarin series for
biological purposes.27 Ind was easily synthesized in three steps
from 4-[cyclohexylidene(4-hydroxyphenyl)methyl]-phenol (see
Supporting Information).37,38 The caged inducer tcInd was
eventually obtained in 30% yield by condensing Ind on the
carbamate intermediate resulting from reaction between N,N′-
carbonyldiimidazole and tcOH.
tcInd is thermally stable at the day time scale in 20 mM pH
7.5 Tris buffer/acetonitrile 1/1 (v/v) at RT. Its solution
strongly absorbs light in the visible range: A maximum of
absorption originating from the thiocoumarin chromophore is
observed at λmax(tcInd) = 469 nm (εtcInd(469 nm) = 2.7 × 104
M−1 cm−1), and its molar absorption coefficient remains larger
than 1.0 × 104 M−1 cm−1 beyond 500 nm (Figure 1a). Its molar
absorption is also interestingly 10 times lower (∼3 × 103 M−1
cm−1) in the 350−400 nm range than at its maximal absorption.
In addition, tcInd is only weakly fluorescent upon excitation at
RESULTS AND DISCUSSION
■
Thiocoumarins can be obtained in good yields in one step from
the corresponding coumarins using the Lawesson’s re-
agent.48−51 Our syntheses are shown in Scheme 1. Starting
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Figure 1. Evolution of the composition of a 5 μM tcInd solution in 20 mM pH 7.5 Tris buffer/acetonitrile (v/v) upon illumination at 470 nm for
various durations t (00(470) = 2.2 × 10−8 einstein s−1 cm−2). (a) Temporal evolution of the solution absorbance normalized to its concentration to
afford its molar absorption coefficient ε(λ) (t = 0, 1, 3, 7, 11, 20 min; solid line). The absorption spectrum of 13-cis-RA (to be used below as a
photoactive precursor) is shown as a dotted line, and the wavelength ranges of the UV and blue light sources used for embryo illumination as violet
and blue zones, respectively. (b) Temporal evolution of the concentrations in tcInd (diamonds) and Ind (circles) extracted from the peak areas in
the HPLC chromatogram. Markers: experimental data. Lines: exponential fits [tcInd]t = [tcInd]0 e−k t (dotted) and [Ind]t = [Ind]∞(1 − e−k t
)
u
u
(solid). The fits provide [tcInd]0 = 4.9 0.1 μM and [Ind]∞ = 4.5 0.2 μM, and 0.5 0.1 and 0.3 0.1 for ku expressed in min−1. T = 293 K.
λexc = 470 nm. We estimated its quantum yield of light emission
to be Φem(tcInd) = 1% with a maximum at λem(tcInd) = 550
nm. Since the tcOH alcohol resulted from tcInd uncaging (vide
infra), we also examined the tcOH photophysical features. As
anticipated from the similarity of the chromophores in both
compounds, the alcohol tcOH exhibits a similar photophysical
behavior. In the same solvent, we observed λmax(tcOH) = 467
nm (εtcOH(467 nm) = 2.4 × 104 M−1 cm−1), λem(tcOH) = 551
nm (Φem(tcOH) = 1%).
We illuminated a 5.0 0.2 μM tcInd solution in 20 mM pH
7.5 Tris buffer/acetonitrile (v/v) with blue light in order to
evidence any photorelease of the inducer. Aliquots of the tcInd
solution illuminated at 470 nm for increasing durations were
analyzed both by HPLC−mass spectrometry and UV−vis
absorption. Analyses of HPLC retention times and of mass
spectra with original samples first demonstrated that illumina-
tion converted tcInd into Ind and the alcohol tcOH. We
analyzed the evolutions of the concentrations of tcInd and Ind
as a function of the illumination duration (Figure 1b). As
expected, the concentration of the photoproduct increased with
time and converged toward the concentration range of the
initial tcInd solution (5 μM); we estimated the final
illuminated tcInd solution asymptotically shifted toward the
maximal absorption of the reference alcohol tcOH. However,
we also noticed that the molar absorption dropped below that
expected for tcOH. Indeed HPLC evidenced that tcOH
photodegraded at the longest times (see Supplementary Figure
1S). From the exponential decay of the normalized absorbance
at the shortest times, we derived another independent estimate
of ku = 0.3
0.1 min−1, in reasonable agreement with the
previous values. After calibration of the photon flux at the
sample, we extracted the quantum yield and the action cross-
section with one-photon excitation associated to tcInd
photoconsumption leading to Ind liberation. We found
Φ
tcInd(470) = 5 1 × 10−3 and εtcInd(470)ΦtcInd(470) = 120
25 M−1 cm−1 at λmax = 470 nm respectively. Such values were
in the range typically observed for the widely used 4,5-
dimethoxy-2-nitrobenzyl caging group (respectively, 6 × 10−3
and 48 M−1 cm−1 at 350 nm in ref 20), but with an absorption
range significantly red-shifted by 120 nm. They also notably
exceeded by more than 1 order of magnitude the uncaging
efficiency at 470 nm of the popular diethylaminocoumarin
photolabile moiety.47
After evidencing tcInd uncaging in vitro, we evaluated the
relevance of this system in vivo. We chose to photocontrol in
developing zebrafish embryos the function of En2, a well-
established cell-autonomous transcription factor,56 which is a
key element in the formation of the diencephalic-mesencephalic
boundary (DMB) in zebrafish. En2 overexpression moves the
DMB rostrally, whereas En2 knockdown moves the DMB
caudally.57 For example, En2 overexpression induces a
reduction of the size of the diencephalon, which results in a
reduction of the size or a total absence of eyes (Figure 2a).58
We injected the mRNA coding for the En2-ERT2 fusion protein
at the one-cell stage in zebrafish embryos. We then attempted
to induce overexpression of En2 activity at 70% epiboly
(epiboly is a cell movement occurring in the early embryo) by
adding Ind or by photoactivating its caged precursor tcInd with
blue light. With respect to control embryos developing in the
concentration in photoreleased inducer Ind to be 4.5
0.2
μM. Meanwhile tcInd concentration exponentially decayed
toward zero. These observations suggest that uncaging was
essentially quantitative. Moreover the exponential decays
allowed us to extract two independent estimates of the rate
constant ku associated to tcInd photoactivation after one-
photon excitation (see Methods). From the data associated
with tcInd and Ind, we derived, respectively, 0.5 0.1 and 0.3
0.1 min−1. These results were further confirmed by analyzing
the temporal evolution of the absorption spectrum of the tcInd
solution. Figure 1a shows that the absorbance in the visible
range blue-shifted from 469 to 465 nm upon increasing the
illumination duration. Since the photoreleased inducer did not
contribute to the absorbance in the corresponding wavelength
range, it was noticeable that the maximal absorption of the
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thiocoumarin chromophore to orthogonally photogenerate two
biologically active substrates with two different wave-
lengths.41−47 Besides tcInd photoactivation by blue light, we
chose to photoactivate 13-cis-retinoic acid (13-cis-RA) by UV
(365 nm) light, a wavelength range that is extensively used for
uncaging in a biological context. Indeed we recently showed
that 13-cis-RA illumination close to its absorption maximum
(see Figure 1a) caused its photoisomerization into all-trans-
retinoic acid (all-trans-RA),12 leading to rescue of hindbrain
formation in embryos whose all-trans RA synthetic pathway had
been impaired by adding diethylaminobenzaldehyde (DEAB)
as an inhibitor (see Supplementary Figure 2S).60 More
specifically, rescue was evidenced upon photoactivating 13-cis-
RA at 90% epiboly in a transgenic line, which expressed green
fluorescent protein (GFP) both in the third (r3) and in the fifth
(r5) rhombomeres. Using the ratio of total fluorescence from
r5 with respect to r3, we showed that r5 development was
restored only after UV illumination.
Figure 2. Photoactivation of tcInd by blue illumination in En2-ERT2
mRNA-injected embryos (00(470) = 3.2 × 10−8 einstein s−1 cm−2).
(a) En2 positive phenotype (absent or reduced eye; red stars were
introduced to identify important structures) observed in lateral and
dorsal views of three embryos at 72 hpf after uncaging of tcInd (10
μM) in the presence of (2-hydroxypropyl)-β-cyclodextrin (10 μM).
Untreated wild type embryos are displayed on the left as controls. The
corresponding wild type phenotype was similarly observed at 83% for
non-illuminated En2-ERT2 mRNA-injected embryos. (b) Percentage of
positive En2 phenotype observed in incubating solutions containing
various Ind concentrations (left) or 10 μM tcInd photoactivated with
blue light for 10 min in the presence of 10 μM (2-hydroxypropyl)-β-
cyclodextrin (right).
In a first step, we evaluated in vitro whether we could observe
preferential photoactivation of tcInd (respectively, 13-cis-RA)
upon illuminating at 488 (respectively, 365) nm. Hence we
submitted separate 10 μM solutions of tcInd and 13-cis-RA in
20 mM pH 7.5 Tris buffer/acetonitrile 1/1 (v/v) either to 5
min of 488 nm or/and 1 min of 365 nm illumination at RT.
The resulting concentrations in photoreleased inducer Ind and
all-trans-RA are displayed in Figure 3a and Supplementary
Table 5S. Five minutes of 488 nm illumination liberated 6.5 μM
Ind without simultaneously generating any significant amount
of all-trans-RA. On the other hand, 1 min of 365 nm
illumination yielded about 3 μM all-trans-RA, which amounted
to 30% yield, in quantitative agreement with our previous 25−
30% results.12,60 At the same time we observed the formation of
0.75 μM Ind, which allowed us to extract the quantum yield of
tcInd photoconsumption at 365 nm, ΦtcInd(365) = 6 × 10−3, in
close agreement with the value upon illuminating at 470 nm. In
fact, the observed orthogonal photogeneration of both
biologically active substrates with 365 and 488 nm wavelengths
was in quantitative agreement with the calculated ratio of the
photoactivation cross-sections of tcInd and 13-cis-RA at 488
absence of inducer, we first checked that zebrafish embryos did
not exhibit any significant abnormal development when
incubated in various Ind concentrations (up to 10 μM). We
then examined En2-ERT2-injected zebrafish embryos. In the
absence of inducer, the embryos displayed a normal phenotype
at 72 h post fertilization (hpf): no difference could be observed
between non-injected embryos and embryos injected with En2-
ERT2 mRNA (Figure 2a). In contrast, upon incubation in a
medium containing the inducer, the percentage of embryos
with eye defect (heterogeneous phenotype from complete
absence of eye to reduction of eye size) increased, reaching
50% at an inducer concentration Ind1/2 = 3 1 μM (Figure 2a
and Supplementary Table 1S). Equipped with this information,
we exposed En2-ERT2-injected zebrafish embryos to a 10 μM
tcInd solution in the presence of 10 μM (2-hydroxypropyl)-β-
cyclodextrin added to achieve embryo perfusion with the caged
inducer (see Supporting Information).59 In the absence of blue
illumination, we observed a three times higher incidence of En2
phenotype than in the control without caged inducer (Figure
2b), which probably originated from some partial thermally
driven tcInd hydrolysis after 3 days of incubation. However,
such a leakage was not detrimental to considerably increase the
incidence of En2 phenotype, when tcInd was illuminated by
blue light for a duration similar to its uncaging time (Figure
2b). More precisely, the embryos displayed the characteristic
positive phenotype they showed in the presence of similar
concentrations of the free inducer upon taking into account
that half of the photoreleased Ind molecules remained trapped
within (2-hydroxypropyl)-β-cyclodextrin after uncaging so as to
reduce the concentration in free inducer for embryo perfusion
(see Supporting Information).
and 365 nm. Indeed, retaining ΦtcInd = 5 × 10−3 and Φ13cisRA
=
1.5 × 10−2 (ref 61) for the quantum yields of photoactivation of
tcInd and 13-cis-RA at 488 and 365 nm, respectively, we
derived from absorption spectra [εtcInd(488)ΦtcInd]/
[ε13cisRA(488)Φ13cisRA] > 100 and [ε13cisRA(365)Φ13cisRA]/
[εtcInd(365)ΦtcInd] ≃ 50.
After the latter in vitro validation, we incubated transgenic
embryos injected with the mRNA of En2-ERT2 in a medium
containing 10 μM DEAB, 10 μM tcInd, and 5 nM 13-cis-RA
(see Supporting Information). We subsequently submitted
these embryos either to 5 min of 488 nm62 or/and 1 min of 365
nm illumination (see Figure 1a). The results are displayed in
Figure 3b−f and Supplementary Table 6S. In the absence of
any illumination, we essentially observed neither size reduction
or loss of eye (positive En2 phenotype) nor r5 rescue (positive
all-trans-RA phenotype) (Figure 3b). In contrast, when the
embryos were illuminated by blue light, we observed the
characteristic tcInd photoinduced positive phenotype without
interference with the 13-cis-RA one (Figure 3c); more precisely,
we observed the En2 phenotype level in the range expected
upon photoreleasing 6.5 μM Ind, yielding 3.5 μM free inducer
available for its biological activity upon using 1.2 × 105 for Ind
association constant to the cyclodextrin (see Supporting
Information). Conversely, when the embryos were illuminated
After validation of tcInd photoactivation by blue light in vivo,
we have been interested in exploiting the major red shift of the
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Figure 3. (a) Concentrations in photoreleased inducer Ind and all-trans-RA after either 5 min blue (488 nm) or/and 1 min UV (365 nm)
illumination of separate 10 μM solutions of tcInd and 13-cis-RA in 20 mM pH 7.5 Tris buffer/acetonitrile 1/1 (v/v) at RT. Red bar: all-trans-RA.
Blue bar: Ind. (b−e) En2- and all-trans-RA-induced phenotypes in En2-ERT2 mRNA-injected mp311+/+GFP embryos conditioned with tcInd (10
μM) and 13-cis-RA (5 nM) in a medium containing 10 μM DEAB subsequently submitted to 488 nm (c), 365 nm (d), and 365 and 488 nm
illumination (e) (00(488) = 8.6 × 10−8 einstein s−1 cm−2) and 00(365) = 3.0 × 10−8 einstein s−1 cm−2). A non-illuminated embryo at 48 hpf is
displayed in panel b. One observes the following: in panel b, the eyes (no En2 overexpression) and no r5 (no all-trans-RA); in panel c, no eyes (blue
light photogenerated En2 overexpression) and no r5 (no all-trans-RA); in panel d, the eyes (no En2 overexpression) and r5 (UV light
photogenerated all-trans-RA); in panel e, no eyes (blue light photogenerated En2 overexpression) and r5 (UV light photogenerated all-trans-RA).
Scale bar = 500 μm. (f) Percentages of positive phenotypes resulting from photoactivation of tcInd and 13-cis-RA by 488 and 365 nm illuminations
in En2-ERT2 mRNA-injected mp311+/+GFP zebrafish embryos in a medium containing 10 μM DEAB. Blue bar: positive En2 phenotype. Red bar:
positive all-trans-RA phenotype.
Reagents and Solutions. Tris base (2-amino-2-(hydroxymethyl)-
1,3-propanediol 99.8+% A.C.S. reagent) and acetonitrile (spectropho-
tometric grade) were from Sigma-Aldrich. All solutions were prepared
using water purified through a Direct-Q 5 (Millipore, Billerica, MA).
All in vitro experiments were performed at 293 K in a buffered solution
obtained by mixing in equal volumes acetonitrile and a 20 mM Tris
base aqueous solution acidified to 7.5 (measured with a pH meter
PHM 210; Radiometer Analytical calibrated at pH = 4 and 7) with 1
M chlorhydric acid.
Photophysical Properties. The UV−vis absorption spectra were
recorded at 293 K either on a Kontron Uvikon-940 having cell holders
thermostatted with a circulating bath (Polystat 34-R2, Fisher Bioblock
Scientific, Illkirch, France) or on a Agilent Cary 300 spectropho-
tometer equipped with 1 × 1 cm2 Peltier cell holders. Molar
absorption coefficients were extracted while checking the validity of
the Beer−Lambert law.
Corrected emission spectra upon one-photon excitation were
obtained from a Photon Technology International QuantaMaster
spectrofluorimeter having a Peltier thermostatted cell holder (TLC50,
Quantum Northwest, USA). Solutions for emission measurements
were adjusted to concentrations such that the absorption maximum
was around 0.15 at the excitation wavelength. The overall emission
quantum yields after one-photon excitation Φem were calculated from
the relation
by UV light, they exhibited the 13-cis-RA photoinduced positive
phenotype without any interference with the tcInd one (Figure
3d); more precisely, the phenotype levels were in line with the
photogeneration of 30% of all-trans-RA from 5 nM 13-cis-RA
(1.5 nM generates 90% of positive phenotype; see ref 60) and
0.75 μM Ind (e.g., 0.35 μM uncomplexed Ind) from 10 μM
tcInd. The achievement of the orthogonally chromatic
photoactivation was further emphasized by analyzing the
phenotype observed upon applying both blue and UV
illuminations (Figure 3e), which distinctly evidenced positive
phenotypes induced by photogeneration of Ind and all-trans-
RA.
Concluding Remarks. The 7-diethylamino-4-thiocoumar-
inylmethyl moiety exhibits attractive features as a caging group.
It is synthetically easily accessible, exhibits a significant action
cross-section for uncaging in the blue wavelength range
(εtcInd(470)ΦtcInd(470) = 120 25 M−1 cm−1), and has been
validated in a biological context. Moreover its low action cross-
section for uncaging in the 350−400 nm wavelength range
(εtcInd(365)ΦtcInd(365) = 12 2 M−1 cm−1) makes it attractive
to perform chromatic orthogonal uncaging in combination with
many presently available UV-absorbing caging groups, which do
not exhibit any significant absorption beyond 450 nm. Indeed
such a value remains significantly lower than the action cross-
section for uncaging of even modestly efficient UV-absorbing
caging groups,1−10 so as to avoid any major 7-diethylamino-4-
thiocoumarinylmethyl photoactivation when UV illumination is
applied. In particular, the 7-diethylamino-4-thiocoumarinyl-
methyl caging group could be used to orthogonally photo-
release one or two biologically active species with a variable
delay in different territories, thus opening a way to analyze self-
and cross-feedbacks during embryo development, both in time
and space.
2
ref (λexc
)
1 − 10−A
ref
D
n
n
ref
⎛
⎜
⎝
⎞
⎟
⎠
Φ
= Φ
em
1 − 10−A(λ
)
exc
D
(1)
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 Φem was estimated to be 10%. The
standard fluorophore for the quantum yields measurements was
Fluoresceine in sodium hydroxide 0.1 M with Φref = 0.92.63
The quartz cuvettes (Hellma) were 1 cm × 1 cm (3 mL samples).
The temperature was directly measured in the cuvettes using a type K
thermocouple connected to a ST-610B digital pyrometer (Stafford
Instruments, Stafford, U.K).
HPLC Coupled to Mass Spectrometry. High pressure liquid
chromatography was carried out with an Accela System liquid
chromatograph (Thermo Finnigan, Les Ulis, France) equipped with
a Hypersil Gold column (1.9 μm, 2.1 mm × 50 mm) connected to a
METHODS
■
Syntheses. The experimental subsection reporting on the
syntheses is in the Supporting Information.
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Thermo-Finnigan TSQ Quantum Discovery Max triple quadrupole
mass spectrometer. Five microliters of illuminated sample solution was
injected in the chromatographic column. The analytes eluted from the
column with a mobile phase composed of two solvents A (water
containing formic acid at 0.05% v/v) and B (acetonitrile). A gradient
was used to optimize the separation of the analytes. Initially, the
column was equilibrated at 0.20 mL min−1 with a mobile phase
consisting of 90% A and 10% B. One minute after injection, the
proportion of B was linearly increased to 80% within 4 min and
remained at that proportion for an additional 2 min. After this step,
composition of the mobile phase was set to initial conditions, and the
column was equilibrated for 3 min prior to the next injection.
Detection was performed by mass spectrometry. After separation, the
analytes were introduced in the mass spectrometer through a heated
electrospray ionization source (50 °C, 4000 V) operating in the
positive mode. The temperature of the capillary transfer was set at 270
°C. Nitrogen was employed as nebulizing (35 psi) and auxiliary gas
(30 arbitrary units). Argon was used as collision gas (1.0 mTorr in
Q2). Peak areas were converted into concentrations after preliminary
calibrations. Instrument control and data collection were handled by a
computer equipped with Xcalibur software (version 2.0).
where V is the irradiated volume and Iu(λexc) is expressed in einstein
per unit of time. Provided that the total absorbance at the excitation
wavelength is much lower than 1, one obtains at first order64
[SPG] = [SPG]0 exp(−kut)
(4)
with
2.3ϕI0(λexc)εu(λexc)l
u
ku =
(5)
V
where [SPG]0 is the initial concentration in caged species, and εu(λexc
)
and l are the effective molar absorption coefficient for photo-
consumption of SPG at the excitation wavelength and the path length
of the light beam, respectively. By assuming that the system initially
contains only SPG, one then easily derives
A
A
tot(λexc, t)
tot(λexc, 0)
εSH(λexc) + εPG(λexc)
=
εSPG(λexc
)
⎛
⎞
⎟
⎠
ε
SH(λexc) + εPG(λexc
)
+ 1 −
exp(−k t)
⎜
u
εSPG(λexc)
⎝
(6)
Irradiation Experiments. Two different protocols have been used
to perform the irradiations.
where εSH, εPG, and εSPG designate the molar absorption coefficients of
SH, PG, and SPG respectively.
In cuvettes. Illumination Experiments. The experiments devoted
to the identification of the photoproducts and to the analysis of
photoconsumption kinetics were performed at 5 μM concentration on
3 mL samples in 1 × 1 cm2 quartz cuvettes under constant stirring.
Excitation at 470 nm was performed with the 75 W xenon lamp of the
spectrofluorometer. After each illumination delay, 200 μL were
removed for subsequent analysis with HPLC/Mass spectrometry.
The 470 nm overall incident light intensity, I0(470), was calibrated
using a procedure relying on analyzing the intensity IDCM of
fluorescence emission of a 4-dicyanomethylene-2-methyl-6-p-dimethy-
lamino-styryl-4H-pyrane (DCM) solution in absolute ethanol upon
exciting at two different wavelengths. The fluorescence emission
IDCM(470) associated with I0(470) was first recorded upon exciting at
470 nm where DCM absorbs light with molar absorption coefficient
εDCM(470). Then the corresponding fluorescence emission IDCM(365)
was recorded upon exciting at 365 nm with light intensity I0(365)
(molar absorption coefficient εDCM(365)). I0(365) was subsequently
extracted from determining the kinetics of photoconversion at 365 nm
of the α-(4-dimethylaminophenyl)-N-phenylnitrone into 3-(4-dime-
thylaminophenyl)-2-phenyloxaziridine in absolute ethanol as described
in reference 64. Eventually, the blue light intensity I0(470) was
computed from [εDCM(365)IDCM(470)]/[εDCM(470)IDCM(365)]. The
surfacic photon flux 00(470) was then derived from dividing the 470
nm overall incident light intensity I0(470) by the cuvette surface (3
cm2). During the present series of experiments, typical overall and
surfacic photon fluxes at the sample were in the 10−7 einstein s−1 and 2
× 10−8 einstein s−1 cm−2 ranges, respectively.
Exponential fit of experimental data yielded ku, from which we
extracted the action cross-section with one-photon excitation
associated with tcInd photoconsumption:
kuV
2.3lI0
εu(λexc)ϕ(λexc) =
u
(7)
The associated quantum yield ϕu(λexc) was eventually obtained using
the value of the molar absorption of the caged inducer from its
absorption spectrum.
In Dishes Containing Zebrafish Embryos. One-photon illumina-
tion experiments were also performed at 20 °C. Three light sources
have been used:
• a blue light source (470 nm LED, essentially a 40 nm wide, at half
height, Gaussian distribution centered at 470 nm; LXML-PB01, Philips
Lumileds, San Jose, CA) delivering typical 3 × 10−8 einstein s−1 cm−2
surfacic photon fluxes 00 at the illuminated sample. It was used for the
blue illumination experiments involving tcInd only
• a blue laser light source (163-C12, Spectra Physics, Santa Clara
CA) delivering typical 9 × 10−8 einstein s−1 cm−2 surfacic photon
fluxes 00 at the illuminated sample. The laser typically delivers 15 mW
at 488 nm. The laser beam was filtered through a LL01-488-25 laser
cleanup filter (Semrock, Rochester, NY) and introduced with two
mirrors (BB1-E02, Thorlabs, Newton, NJ) into a liquid light guide
(LLG0338-4, Thorlabs). The exiting beam was collimated with a 50
mm focal lens. This blue laser light source was used for the blue
illumination experiments involving both tcInd and 13-cis-RA
• a benchtop UV lamp (365 nm; essentially a 40 nm wide, at half
height, Gaussian distribution centered at 350 nm; 6 W; Fisher
Bioblock) delivering typical 3 × 10−8 einstein s−1 cm−2 surfacic photon
fluxes 00 at the illuminated sample.
Extraction of the Photochemical Parameters. Assuming that
photoactivation is rate-limiting among the processes leading to SH
liberation, we considered that the illuminated system was submitted to
the reaction
We checked that, when illuminated with these light sources at the
time scale of our experiments, zebrafish embryos developed normally.
Experiments in Zebrafish Embryos. General Information. The
transgenic line mp311+/+GFP has been reported in our previous
work.60 Fish were raised and bred according to standard methods.65
The embryos were incubated at 28 °C. Developmental stages were
determined as hours post fertilization (hpf).
Application of the Drugs. Ind and its caged precursor cInd, DEAB
(diethylaminobenzaldehyde; Sigma-Aldrich), and 13-cis-retinoic acid
(13-cis-RA; Sigma-Aldrich) were solubilized at 10 mM in DMSO
(those stock solutions were stored at −20 °C where they proved stable
for several months). Aliquots of those solutions and (2-hydrox-
ypropyl)-β-cyclodextrin 45% (w/v) in H2O (Sigma-Aldrich) were
added to Volvic water66 containing the zebrafish embryos to reach the
concentrations indicated in the text. Incubations were carried out in
hν
(2)
SPG → SH + PG
to derive the cross-section for tcInd photoconsumption after one-
photon excitation. In eq 2, SPG, SH, and PG designate the caged
species, the species that was initially caged, and the product from the
protecting moiety after SH release. The reaction rate for the
photoconsumption of the caged compound SPG is proportional to
the intensity of the monochromatic excitation light absorbed by SPG
at the excitation wavelength, Iu(λexc) and to the photoconsumption
cross-section with one-photon excitation, ϕu:
Iu(λexc)ϕ
d[SPG]
dt
u
−
=
(3)
V
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ACS Chemical Biology
Articles
the dark in plastic arrays of eight square 1.0 cm2 fields (μ-Slides 8-well;
Ibidi) containing 500 μL of solution and 10 zebrafish embryos.
en2-ERT2 Coding Plasmids. myc-cEn2-ERT2 coding plasmid: An
intermediate vector (pC5fiERT) was first constructed by inserting
Cre-ERT2 coding sequence (fragment XhoI-Ecl136I from pCre-ERT2, a
kind gift of D. Metzger and P. Chambon, Strasbourg, France 67) into
pcDNA5/FRT (Invitrogen, digested with XhoI and EcoO109I blunted
with Klenow). We then prepared pC5fEn2ERT, with the coding
sequence for chicken En2 (cEn2), fused in N-ter to myc tag and in C-
ter to ERT2 coding sequence, downstream CMV and T7 promoters
and upstream the bovine growth hormone polyadenylation signal, via a
trimolecular ligation between the NheI-HindIII fragment from
pCL9mEn2,68 pC5fiERT cleaved by NheI and XhoI, and a HindIII-
XhoI adaptor. The final construct, pCSmEn2ERT, was built by
transferring the NheI-XhoI fragment (NheI end blunted with Klenow
enzyme) of pC5fEn2ERT into pCSgfpERT,37,38 cleaved by BstBI
(blunted with Klenow enzyme) and XhoI. pCSmEn2ERT contains
the myc-cEn2-ERT2 coding sequence downstream simian CMV and
SP6 promoters and upstream the late SV40 polyadenylation signal and
was used to prepare synthetic RNA in vitro for injection in Zebrafish
embryos. All conditions for restriction digests, gel purification, ligation
and bacteria transformation are standard.69 Plasmid complete
sequences are available upon request.
Zebrafish Embryo Experiments. Zebrafish embryos were injected at
one-cell stage with the synthesized mRNA (30 ng/μL) with an in vitro
transcription kit (mMessage mMachine, Ambion). They were
subsequently incubated in aqueous solutions of the various substrates
according to the conditioning sequences reported in the main text. In
the series of experiments devoted to orthogonal photoactivation of
tcInd and 13-cis-RA, we first conditioned En2-ERT2 mRNA-injected
mp311+/+GFP zebrafish embryos by incubating them with 10 μM
DEAB (solution 1) from the sphere stage.60 The embryos were
subsequently transferred at 50% epiboly into a medium containing 10
μM DEAB and 5 nM 13-cis-RA (solution 2) for 5 min. They were then
transferred into a medium containing 10 μM DEAB, 10 μM tcInd, and
10 μM (2-hydroxypropyl)-β-cyclodextrin (solution 3) in which they
were incubated up to the photoactivation step: at 70% epiboly with
488 nm illumination for 5 min or/and at 90% epiboly with 365 nm
illumination for 1 min, both durations being similar to photoactivation
times. The zebrafish embryos were scored for their positive phenotype
at 24 hpf (Figure 2a and Supplementary Figure 2S).60 The results are
shown in Supplementary Table 5S. Illuminated embryos were
observed at 24, 48, and 72 hpf.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Ludovic.Jullien@ens.fr.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work has been supported by the ANR (PCV 2008,
■
Proteophane), the Minister
̀
e de la Recherche (a fellowship to
L.F.), and the LEA-Nabi and the LIA CNRS-CNSI. The
authors thank V. Jullien and G. Pons at Service de
Pharmacologie Clinique, Saint Vincent de Paul Hospital Paris,
for access to their HPLC−MS instrument, and Nicole
Quenech’Du for technical help with the imaging platform.
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missing eye(s) could be observed (see Figure 2). Zebrafish embryos
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̈
ASSOCIATED CONTENT
■
S
* Supporting Information
Syntheses (including Scheme 1S), the validation of the Ind
inducer (including Table 1S), zebrafish embryo conditioning
with the caged inducer tcInd (including Table 2S), the
preliminary in vivo uncaging experiments (including Tables 3S
and 4S), and the supplementary figures and tables. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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