"Self-immolative" spacer for uncaging with fluorescence reporting

Article abstract of DOI:10.1002/anie.201204032

Dual photoliberation: A caged, branched, self-immolative spacer (see scheme, gray box) was designed to rapidly and simultaneously release a desired compound (green) and a fluorophore (red) upon photoactivation. Careful kinetic analysis of the disassembly of the spacer shows that it occurs on the shortest time scale reported to date.

Full text of DOI:10.1002/anie.201204032

Angewandte
Chemie
DOI: 10.1002/anie.201204032
Chemical Biology
“Self-Immolative” Spacer for Uncaging with Fluorescence
Reporting**
Raphaꢀl Labruꢁre, Ahmed Alouane, Thomas Le Saux, Isabelle Aujard, Philippe Pelupessy,
Arnaud Gautier, Sylvie Dubruille, Frꢂdꢂric Schmidt,* and Ludovic Jullien*
The spatio-temporal delivery of known amounts of chemicals
is of major importance in chemistry, biology, and medicine.
Pipettes and syringes are used in various macro- and micro-
formats to fulfill this goal. However, they are intrusive and
rely on convective motion, which may be detrimental to
delivery to embedded and fragile samples, such as targeted
cells in a living organism. Activating the release of a desired
compound from an inactive precursor by introducing a non-
invasive trigger (e.g., ultrasound,[1] UV-IR light,[2] or X-rays[3])
provides an attractive alternative. However, quantification of
the amount delivered remains a significant problem. First,
calibration of the triggering beam can be difficult in complex
systems (e.g., a living organism) because of the unknown and
heterogenous nature of these systems. Second, the local
concentration of inactive precursor may be unknown, which
makes it difficult to deliver a specific concentration of
substrate. One possible way to address this quantification
problem relies on the simultaneous release of a reporter (in
a one-to-one molar ratio) and the desired substrate from
a common precursor. Quantitative analysis then can be done
simply from analyzing the increase in the reporter signal
after—or even better, during—activation at the targeted site.
Numerous caging groups have been introduced that allow
release of the substrate following light activation of the caged
precursors. Improvement of their photophysical and photo-
chemical features has been extensively addressed.[2] In con-
trast, optical reporting of uncaging has attracted much less
attention. Strategies relying on the concomitant release of
a fluorophore were developed.[4] These strategies used caging
groups that were designed to release a fluorophore as a side
product. However, efficient optical reporting of their release
has remained limited to specific substrates. One way to
overcome this limitation is to decouple the fluorescent
reporter from the caging group. Herein, we adopt a modular
approach in which photoactivation of a caged precursor leads
to the release of not one, but two molecules: the desired
substrate and the reporting fluorophore.
Our approach is based on a so-called “self-immolative”
spacer.[5] In these covalent chemical assemblies, commonly
used for therapeutic or bioanalytical aims, a primary (e.g.
enzymatic) reaction initiates internal molecular rearrange-
ments driving a second reaction causing the release of
a substrate (e.g. a drug or a probe). Self-immolative spacers
uncouple the primary and the secondary reactions, which has
made it possible to increase the diversity of enzyme substrates
in prodrug strategies for targeted drug delivery.[6] In addition,
branched self-immolative spacers were recently shown to
release two (or more) molecules after a single triggering
cleavage.[7] Thus we synthesized a caged, branched, self-
immolative spacer to liberate the desired compound and
fluorophore upon photoactivation (Scheme 1).
[*] Dr. R. Labruꢀre, Dipl.-Chem. A. Alouane, S. Dubruille, Dr. F. Schmidt
Institut Curie, Centre de Recherche
26 rue d’Ulm, 75248 Paris (France)
E-mail: frederic.schmidt@curie.fr
Dr. R. Labruꢀre, Dipl.-Chem. A. Alouane, Dr. T. Le Saux, Dr. I. Aujard,
Dr. A. Gautier, Prof. Dr. L. Jullien
Ecole Normale Supꢁrieure, Dꢁpartement de Chimie, UMR CNRS-
ENS-UPMC 8640 PASTEUR
Scheme 1. A branched self-immolative spacer for uncaging with fluo-
rescence reporting. Upon removal of a photolabile protecting group
(circle), illumination initiates self-immolation leading to release of
both the substrate (green ellipse) and the fluorophore (red square).
24 rue Lhomond, 75231 Paris (France)
E-mail: ludovic.jullien@ens.fr
Dr. R. Labruꢀre, Dipl.-Chem. A. Alouane, S. Dubruille, Dr. F. Schmidt
UMR 176
26 rue d’Ulm, 75248 Paris (France)
The usefulness of this strategy is governed by two main
aspects: 1) the photochemical efficiency of the photolabile
protecting group; 2) the stoichiometry and the kinetics for
releasing the substrate and its fluorescent reporter. Herein,
we did not attempt to optimize the photochemical step; thus,
as a caging group, we used the 4,5-dimethoxy-2-nitrobenzyl
moiety, which is widely used and acts also as a quencher (see
below). In contrast, we designed the spacer to accelerate its
self-immolation after photoactivation, as well as decrease the
delay between the release of the substrate and its reporter
(Scheme 2). We chose a 4,6-dibromo-2-hydroxy-5-methyl-1,3-
Dr. P. Pelupessy
Ecole Normale Supꢁrieure, Dꢁpartement de Chimie, UMR CNRS-
UPMC-ENS 7203 Laboratoire des Biomolꢁcules
24 rue Lhomond, 75231 Paris (France)
[**] This work has been supported by the ANR PCV 2008 (Proteophane)
and ANR Blanc 2010 (Kituse).
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201204032.
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uncaging, by recording fluorescence intensities at two emis-
sion wavelengths.[4c–e]
The spacer cS2CD was synthesized in five steps from 3,5-
dibromo-4-methylphenol (see Scheme 2 and Supporting
Information). Briefly, 4,6-dibromo-2-hydroxy-5-methyl-1,3-
phenylene)dimethanol was obtained by bishydroxymethyla-
tion in 98% yield[10] and its hydroxymethyl groups were
protected with tert-butyldimethylsilyl groups (81% yield).
The remaining phenol was subsequently alkylated with 4,5-
dimethoxy-2-nitrobenzyl bromide (68% yield). After
removal of the silyl protecting groups (60% yield), the
bis(hydroxyethyl) intermediate was treated with phosgene
followed by the fluorophores C and D, to afford cS2CD in
33% yield. For kinetic analysis, we also synthesized 1) the
symmetrical spacer cS2D2 incorporating two identical fluo-
rophores D and 2) a model of our caged spacer in which one
branch involved in self-immolation was removed (cS1C). We
anticipated that cS1C would release C alone upon photo-
activation (see Supporting Information, Chart S1).
First, stability and uncaging of cS2CD were investigated in
1:1 mixture of acetonitrile/0.1m Britton–Robinson buffer
pH 8 (v/v) at room temperature. We first showed that
cS2CD is stable in solution in the dark for 48 h, owing to the
absence of a change in the absorption spectrum. Then we
performed two series of UV-illumination experiments. To
determine the molar ratio of the photo-induced release of C
and D, we analyzed their concentrations by LC/MS after
various durations of illumination at 365 nm. To analyze the
self-immolation kinetics of cS2CD, we then recorded the
temporal change in the fluorescence intensity from a cS2CD
solution under continuous illumination at 365 nm.
Figure 1a displays the concentrations of C and D after
illumination of duration t, as determined by LC/MS analysis.
As expected, their concentration increases with time and
shows an exponential evolution (see Supporting Information,
Section 2.2.3). Upon illuminating a 5.1 ꢀ 0.4 mm cS2CD solu-
tion, we calculated C1 = 5.3 ꢀ 0.5 mm and D1 = 4.9 ꢀ 0.5 mm
for the final concentrations of C and D, in agreement with
a quantitative photoinduced release of both substrates.
Furthermore, we also calculated from the global fit of
cS2CD uncaging a quantum yield f(365 nm) = 8.6 ꢀ 0.9 ꢀ
10ꢁ4, in line with reported values for the ortho-nitrobenzyl
caging group.[11]
Figures 1b,c show the temporal evolutions of the fluores-
cence emissions IFCðtÞ and IFDðtÞ of C and D, respectively, upon
continuous illumination of a 5.1 ꢀ 0.4 mm solution of cS2CD at
298 K. Starting from a nonfluorescent cS2CD solution,
fluorescence emission increases, as anticipated, from photo-
release of C and D. Again, the final signals demonstrated
quantitative, equimolar photorelease of C and D from cS2CD.
Kinetic analysis of the experimental data must take into
account the cascade of reactions involved in the self-
immolation process (see Supporting Information, Sec-
tion 2.2). We adopted the simplified kinetic Scheme shown
in Scheme 2, which includes the steps typically occurring
beyond the millisecond time scale.[12] The caged precursor
yields the phenol intermediate S2CD, which subsequently
disassembles along one of two pathways to give the same final
products, a benzenic core S2 together with C and D. In
Scheme 2. Simplified kinetic scheme of self-immolation of the spacer
cS2CD. After cleavage of the photolabile protecting group, a cascade of
internal reactions leads to the release of coumarin (C) and DDAO (D)
fluorophores, which represent the desired substrate and reporter,
together with carbon dioxide and the benzenic core S2.
phenylenedimethanol motif, which is predicted to self-immo-
late upon removal of the phenol group. Besides synthetic
considerations, its symmetrical pattern of conjugated benzylic
substituents should reduce the delay between the release of
the desired product and the reporter fluorophore. Further-
more, to accelerate the reaction[8] and make the release as
substrate-independent as possible, we attached the benzylic
positions of the spacer to the substrate/reporter moieties
through carbonate linkages. To analyze the stoichiometry and
the kinetics of self-immolation of the photoactivated spacer,
we chose as a model substrate/reporter pair two fluorophores,
whose emission is quenched and blue-shifted in the caged
precursor with respect to the free state.
Hence we used coumarin (7-hydroxy-4-(trifluoromethyl)-
coumarin, C) and DDAO (1,3-dichloro-9,9-dimethyl-9H-
acridin-2(7)-one, D), which respectively exhibit strong emis-
sion in the blue and in the red regions only after ionization of
their phenol group.[9] These fluorophores absorb and emit in
different wavelength ranges, which enabled us to separately
investigate the temporal dependence of their release upon
2
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ultimate release of C in the self-immolation processes.
Further evidence supporting our kinetic interpretation was
found from analysis of the brightness of the intermediates
involved in S2CD self-immolation (see Supporting Informa-
tion).
The preceding kinetic analysis makes it possible to
evaluate the present strategy for uncaging with fluorescent
reporting. The substrate and its reporter are released in a one-
to-one molar ratio about ten seconds after removal of the
phenol group. In fact, this time is among the shortest times
ever reported for disassembly of a self-immolative linker.[2h,13]
Furthermore, using the values of the uncaging cross-section
ef(365 nm), where e is the molar absorption coefficient, and
the rate constants k21, k22, and k31, we simulated the change in
the concentrations of C and D upon illumination of a cS2CD
solution (see Figure 1d). C is released about ten seconds after
D. Although this delay would be detrimental to quantitative
reporting of uncaging in an open system, it is sufficiently short
to analyze most biological processes occurring in a closed
system (e.g., a cell). Indeed the delay between the release of
the substrate and its reporter remains moderate with respect
to characteristic times of cellular events, many of which
exceed the minute range (e.g., phosphorylation, protein
translation, protein degradation).
Next, we reproduced the preceding uncaging experiments
in human embryonic kidney (HEK) 293 cells. We incubated
cells for 30 minutes in a solution of 5.3 mm cS2CD in 100 mm
PBS buffer pH 7.4. We observed identical cellular fluores-
cence following uncaging with 365 nm light when cells were
incubated with 5 mm cS2CD for 30 minutes or 2 hours (data
not shown), suggesting that an incubation time of 30 min is
sufficient to equilibrate the distribution of cS2CD between the
extracellular solution and the cytoplasm. After rinsing with
PBS, cells were continuously illuminated at 365 nm and
imaged in real time with epifluorescence microscopy using
two independent channels, collecting the emission of C and D
(see the Movie in the Supporting Information).[14] We
analyzed the kinetics of photoinduced release of C and D
within HEK 293 cells (Supporting Information, Figure S8).
We did not notice any difference in the uncaging behavior
among cells that were similarly illuminated. Proceeding as
above, we calculated f(365 nm) = 3.5 ꢀ 0.2 ꢀ 10ꢁ4, k21 ꢂ
0.2 sꢁ1, and k31 ꢂ 0.1 sꢁ1 in good agreement with the values
found in vitro, showing no significant alteration of the rate
constants associated with cS2CD photoactivation in living
cells.
^
Figure 1. a) Photoreleased concentrations in C ( ) and D (*) after
illumination for a duration t of a 5.1 mm cS2CD solution at
84ꢂ10ꢁ9 einsteinsꢁ1 light intensity. Solid line: fits of the data from
Equations (34),(37) in the Supporting Information. b) Fluorescence
emission of C ICF ðtÞ (lem =500 nm) over time during illumination at
lexc =365 nm of a 5.1 mm cS2CD solution at various light intensities
(40, 80, 120, and 160ꢂ10ꢁ9 einsteinsꢁ1). Diamonds: experimental
data; solid lines: fits from Equation (49) in the Supporting Informa-
tion. c) Fluorescence emission of D IDF ðtÞ (lem =660 nm) over time
during illumination at lexc,1 =365 nm and lexc,2 =645 nm of a 5.1 mm
cS2CD solution at varying light intensities of lexc,1 (40, 80, 120, and
160ꢂ10ꢁ9 einsteinsꢁ1). Circles: experimental data; solid lines: fits
from Equation (50) in Supporting Information. d) Simulation of the
change in concentration of C (solid line) and D (dashed line) over time
upon continuous illumination of a 5 mm cS2CD solution at
10ꢁ7 einsteinsꢁ1 light intensity, as obtained from Equations (46)–
(48),(51),(52) using the values of ef(365 nm), k21, k22, and k31
extracted from the fits. Solvent: 1:1 (v/v) CH3CN/0.1m Britton–
Robinson buffer pH 8. T=298 K.
Scheme 2, the rate constant k1 is associated with uncaging,
whereas the rate constants k21, k22, k31, and k32 all refer to the
steps of self-immolation (see Supporting Information).
We obtained satisfactory fits of the kinetic data using
f(365 nm) = 8.8 ꢀ 0.9 ꢀ 10ꢁ4 for the cS2CD uncaging quantum
yield, k21 = 0.10 ꢀ 0.02 sꢁ1, k22 < 0.02 sꢁ1, and k31 = 0.11 ꢀ
0.06 sꢁ1. To support our kinetic analysis, we first noted that
the uncaging quantum yield f(365 nm) was in line with the
result calculated from the LC/MS analysis. The values of k21
and k31 were then compared with the values calculated from
the analysis of self-immolation of the model cS1C and cS2D2
spacers (see Supporting Information, Section 2.2.1 and 2.2.2).
A value for k21 of approximately 0.1 sꢁ1 compares fairly well
with the rate constant associated with the first S2D2 self-
immolation step (also approximately 0.1 sꢁ1). In fact, this
result was as expected, because the same moiety D was first
released from both S2CD and S2D2 phenols. We also saw an
agreement between k31 (approximately 0.1 sꢁ1) and the rate
constant for cS1C self-immolation (approximately 0.3 sꢁ1).
Indeed, these rate constants both corresponded to the
Finally, we used confocal microscopy to analyze the final
concentrations of C and D within the cells after uncaging with
365 nm light. Excitation at 405 nm and 633 nm light were
subsequently used to image C and D under optimized
conditions. Figures 2a–c show confocal micrographs of non-
illuminated cS2CD-incubated control cells. Under these
imaging conditions, some auto-fluorescence was observed in
the green emission channel upon exciting at 405 nm (Fig-
ure 2b). In contrast, no significant auto-fluorescence was
observed in the far-red emission channel with 633 nm light
excitation (Figure 2c). Figures 2d,e show confocal micro-
graphs of cS2CD-incubated cells that were exposed to 365 nm
light until a steady state of fluorescence was achieved. We
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Figure 2. Confocal micrographs of HEK 293 cells incubated with
5.3 mm cS2CD before (a–c) and after irradiation at 365 nm for 80 s (d–
f). a,d) Transmission, b,e) green fluorescence emission with excitation
at lexc =405 nm (emission of C), and c,f) far-red fluorescence emission
with excitation at lexc =633 nm (emission of D). Buffer: 100 mm PBS
pH 7.4 with 1% DMSO. T=298 K. Scale bar=8 mm.
observed strong green and far-red fluorescence emission from
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living cells, as was also shown in vitro.
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a desired substrate and a fluorescent reporter, in a one-to-
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and simultaneously enough to consider this caged platform
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Received: May 24, 2012
Published online: && &&, &&&&
Keywords: caging groups · fluorescence · imaging agents ·
.
kinetics · self-immolative spacers
4
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Communications
Chemical Biology
R. Labruꢁre, A. Alouane, T. Le Saux,
I. Aujard, P. Pelupessy, A. Gautier,
S. Dubruille, F. Schmidt,*
L. Jullien*
&&&& — &&&&
“Self-Immolative” Spacer for Uncaging
with Fluorescence Reporting
Dual photoliberation: A caged, branched,
self-immolative spacer (see scheme, gray
box) was designed to rapidly and simul-
taneously release a desired compound
(green) and a fluorophore (red) upon
photoactivation. Careful kinetic analysis
of the disassembly of the spacer shows
that it occurs on the shortest time scale
yet reported.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!