Light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers

Article abstract of DOI:10.1002/chem.201301298

Three procedures that rely on photoactivation are introduced to accurately analyze the disassembly kinetics of a collection of self-immolative spacer groups within the window 10^-2-10³ s. Our results are relevant for deriving quantitative structure-property relationships. In particular, we have been able to access 20 ms temporal resolution, which made possible the measurement of the shortest ever reported disassembly time for an activated self-immolative spacer. Quick as lightning! Accurate kinetic analysis of the disassembly of various self-immolative spacers in a 10 ^-2-10³ s window was achieved. An epifluorescence setup allowed the measurement of the shortest time (20 ms) for a self-immolation event. Copyright

Full text of DOI:10.1002/chem.201301298

FULL PAPER
DOI: 10.1002/chem.201301298
Light Activation for the Versatile and Accurate Kinetic Analysis of
Disassembly of Self-Immolative Spacers
Ahmed Alouane,[a, b, c] Raphaꢀl Labruꢁre,*[a, b, c] Thomas Le Saux,[c, d] Isabelle Aujard,[c]
Sylvie Dubruille,[a, b] Frꢂdꢂric Schmidt,*[a, b] and Ludovic Jullien*[c, d]
Abstract: Three procedures that rely on photoactivation are introduced to accu-
rately analyze the disassembly kinetics of a collection of self-immolative spacer
Keywords: disassembly
cence · kinetics · photochemistry ·
self-immolation
· fluores-
groups within the window 10ꢀ2–103 s. Our results are relevant for deriving quantita-
tive structure–property relationships. In particular, we have been able to access
20 ms temporal resolution, which made possible the measurement of the shortest
ever reported disassembly time for an activated self-immolative spacer.
Introduction
Self-immolative spacers are covalent assemblies that are
tailored to correlate the cleavage of two chemical bonds.[1]
Such spacer groups were first introduced to overcome chem-
ical limitations in the design of enzymatic substrates,[2]
which typically contain two moieties that react with the
enzyme (A) and report on its activity (B). When the two-
ꢀ
part compound (A B) cannot be obtained or cleaved by
using the planned enzyme, moieties A and B can be linked
together through a stable self-immolative spacer group,
which decouples the enzyme-triggered bond cleavage (in-
volving A) from the spontaneous bond cleavage that leads
Scheme 1. In a self-immolative spacer, cleavage of bond 1 spontaneously
causes the cleavage of bond 2. Thus, a primary event cleaves the initial
precursor into three moieties: A, the spacer, and B.
to the release of B (Scheme 1).
Self-immolative spacer groups have been extensively used
in prodrug strategies in which B is a drug and A is a recog-
nition motif that is responsible for the selective reaction
with an enzyme.[3] Beyond preventing steric hindrance at the
enzyme cleavage site, such a design brings further degrees
of freedom to tune the solubility, physiological stability, and
metabolization of the substrate. Moreover, it also provides
the ability to control the kinetics of drug release after enzy-
matic activation, in which the self-immolative spacer group
acts as an autonomous “molecular clock”.
Self-immolative spacers are also increasingly used in the
context of bioanalysis.[4] Enzymatic activation causes the re-
lease of a probe, which shows strongly altered reporting
properties. In this context, accelerating the disassembly of
an activated spacer is essential. Indeed, the decomposition
rate of a self-immolative spacer controls the spatial correla-
tion between the enzyme position and the labeling location.
For instance, in the emergent context of assisting surgeons
by selectively labeling cells that contain specific enzymes
(biomarkers),[5] too slow a self-immolation would generate
the labeling of surrounding healthy cells.
Hence, versatile and accurate kinetic analysis of the disas-
sembly of self-immolative spacers is essential for determin-
ing their scope of application. Previous kinetic studies have
already provided useful structure–property relationships,
thus allowing the prediction of significant features, such as
[a] Dipl.-Chem. A. Alouane, Dr. R. Labruꢀre, S. Dubruille,
Dr. F. Schmidt
Institut Curie, Centre de Recherche
26 rue d’Ulm, 75248 Paris (France)
E-mail: raphael.labruere@curie.fr
frederic.schmidt@curie.fr
[b] Dipl.-Chem. A. Alouane, Dr. R. Labruꢀre, S. Dubruille,
Dr. F. Schmidt
CNRS, UMR 176, 26 rue d’Ulm, 75248 Paris (France)
[c] Dipl.-Chem. A. Alouane, Dr. R. Labruꢀre, Dr. T. Le Saux,
Dr. I. Aujard, Prof. Dr. L. Jullien
Ecole Normale Supꢁrieure, Dꢁpartement de Chimie
UMR CNRS-ENS-UPMC 8640 PASTEUR
24 rue Lhomond, 75231 Paris (France)
E-mail: ludovic.jullien@ens.fr
[d] Dr. T. Le Saux, Prof. Dr. L. Jullien
UPMC Univ. Paris 6, 4 Place Jussieu, 75232 Paris (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301298.
ꢀ
ꢀ
the stability of the A spacer B precursor and the rate of B
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release.[1a,6] However, these studies used enzymatic/chemical
reactions to trigger the disassembly of the spacer, which has
limited the temporal resolution of the kinetic analysis to the
minute range. In particular, no precise information is pres-
ently available for the fastest and highly significant (see
above) self-immolative spacers.
Following our recent development of a self-immolative
spacer-based caging group that showed fluorescence report-
ing, we have estimated that photochemical activation could
yield significant improvements for investigating the kinetics
of self-immolation reactions. Indeed, light gives access to ex-
clusive temporal and spatial resolution, so as to permit the
analysis of the kinetics of the fastest processes. Herein, we
implemented three procedures that relied on light activation
to analyze the kinetics of the disassembly of self-immolative
spacer groups at a high temporal resolution of 20 ms. In
fact, such a limit is precisely relevant for investigating the
behavior of the fastest reported self-immolation processes.
Beyond this achievement, we show that those procedures
are relevant for analyzing the kinetics of self-immolation
processes over a kinetic window that is five orders of magni-
tude wide (10ꢀ2–103 s), which should be favorable for most
current applications of self-immolative spacers.
Scheme 2. Caged self-immolative spacers cSR1 LG.
marin, C2) and DDAO (1,3-dichloro-9,9-dimethyl-9H-acri-
din-2(7)-one, D1), which respectively exhibited strong emis-
sion in the green- and red-light regions, only after liberation
of their phenol/aniline groups.[9]
Results and Discussion
Experimental design: Most self-immolative spacer groups
are based on phenyl cores that contain a heteroatom that is
involved in bond 1, the scission of which causes the cleavage
of bond 2 through elimination or cyclization reactions
(Scheme 1). We chose to employ a phenol core, along with a
quinone-methide-elimination process, because this latter
process has been shown to be versatile for releasing two or
more leaving groups.[7] We eventually targeted a series of
caged-substituted phenol-containing spacers (cSR1 LG) with
different appended leaving groups (Scheme 2), which were
expected to exhibit various disassembly rates after their
photoactivation.
Synthesis: The caged self-immolative spacers, cSR1 LG, were
synthesized by coupling caged benzyl alcohol modules,
cSR1 OH (see the Supporting Information), with reporting
fluorophores after appropriate activation processes
(Scheme 3).
Carbonates cSH1 C and cS1RD were synthesized in high over-
all yields according to literature procedures[10] in a two-step
To deactivate the precursor, as well as to permit its photo-
activation, the phenol oxygen atom was protected with the
widely used 4,5-dimethoxy-2-nitrobenzyl caging group. In
particular, such 2-nitrobenzyl caging groups have been re-
ported to liberate phenol groups on the millisecond time-
scale, which herein makes it possible to analyze self-immola-
tion processes that occur more slowly than within 10 ms.[8]
Various electron-donating (methyl or methoxy) and elec-
tron-withdrawing substituents (nitro or bromo) were intro-
duced onto the phenyl ring. Eventually, the benzylic posi-
tions of the spacers were bonded to fluorescent reporting
moieties through carbonate/carbamate or ether groups. To
finely analyze the stoichiometry and the kinetics of the self-
immolation of the photoactivated spacers, we chose latent
fluorophores for which the emission was quenched and
blue-shifted in the caged precursor with respect to in the
free state. We retained two coumarins (7-hydroxy-4-(trifluor-
omethyl)coumarin, C1, or 7-amino-4-(trifluoromethyl)cou-
Scheme 3. Synthetic pathway for the synthesis of cSR1 LG.
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Self-Immolative Spacers in Disassembly
FULL PAPER
sequence by using triethylamine in dry THF to couple either
coumarin C1 or DDAO D1 with the chloroformates that
were obtained from the cS1ROH alcohols in the presence of
phosgene. Carbamate cSH1 C’ was obtained in 52% yield, ac-
cording to a closely related procedure that involved carba-
moylation of anilino-coumarin C2 with triphosgene, fol-
lowed by reaction with cSH1 OH. Eventually, the synthesis of
cSH1 C’’ was conducted in two steps with an overall yield of
69% by alkylating coumarin C1 in the presence of K2CO3
with the iodo derivative that was obtained after the iodina-
tion of cSH1 OH.
Photoactivated self-immolation: Stability and photoactiva-
tion experiments of cSR1 LG were performed in a mixture of
MeCN and 0.1m Britton–Robinson buffer (1:1, v/v). From
the absence of any temporal evolution of the absorption
spectrum over the typical 24 h timescale, we concluded that
the cSR1 LG molecules were stable in the dark under all of
our experimental conditions.
To analyze the self-immolation kinetics of the photoacti-
vated cSR1 LG intermediates, we recorded the temporal evo-
lution of the fluorescence emission from the reporting fluo-
rophore upon submitting solutions of cSR1 LG to continuous
illumination at 365 nm to both photoactivate and excite the
reporting fluorophores. In a first series of experiments, we il-
luminated the stirred solutions in a cuvette in a standard
spectrofluorimeter, which allowed us to obtain the typical
1 s temporal resolution that is associated with homogeneous
mixing. For the fastest systems, we subsequently implement-
ed a stopped-flow mixing technique and performed epifluo-
rescence microscopy, which made it possible to access 40
and 20 ms temporal resolutions, respectively. Our kinetic ex-
periments were performed at three different pH values (to
study the incidence of the ionization state of the phenol
spacer on the rate of disassembly) within the range 293–
318 K (in view of the relevance of self-immolative spacers
for biological applications at various temperatures).
Figure 1. Temporal evolution of the fluorescence emission at lem =500 nm
upon illumination at lexc =(365ꢁ25) nm of: a) (1.0ꢁ0.3) mm solution of
cSH1 C’’, or b) (5ꢁ1) mm solution of cS1HC’ at 24.5ꢃ10ꢀ9 Einsꢀ1 light inten-
sity at various temperatures. Circles denote the experimental data (293,
303, 310, and 318 K); solid lines show the fits to Equation (13) (a and b
at 310 and 318 K) and Equation (45) (b at 293 and 303 K), respectively
(see the Supporting Information for all Equations). Solvent: MeCN with
0.1m Britton–Robinson pH 8 buffer (1:1 v/v).
Figure 1a shows the temporal evolution of the fluores-
cence emission, ICF1(t), on the release of coumarin C1 upon
continuous illumination of a (1.0ꢁ0.3) mm solution of caged
precursor cSH1 C’’ on the fluorimeter setup. Starting from a
nonfluorescent solution of cSH1 C’’, the fluorescence emission
increased, as anticipated from the photoreleasing of coumar-
in C1. The final signal demonstrated the quantitative photo-
release of coumarin C1 from cSH1 C’’ at all of the investigated
temperatures: By using standard solutions of coumarin C1
for the fluorescence calibration, we derived a value of (1.5ꢁ
0.5) mm for the final concentration, C11. Similar behaviors
were observed for all of the investigated compounds,
cSR1 LG, thus demonstrating the quantitative release of the
reporting fluorophore (C1, C2, or D1) after photoactivation.
A cascade of reactions leads from the photoactivated
cSR1 LG precursors to the final reporting fluorophores, F. In
line with our previous work, for the kinetic analysis, we
adopted the shortened scheme as shown in Scheme 4, which
retained steps that typically occurred more slowly than on
the millisecond timescale (see the Supporting Information).
Illumination of caged precursors cSR1 LG first yielded
phenol intermediates SR1 LG, which subsequently disassem-
bled into a benzenic core (thereby ultimately yielding struc-
ture SR1 ), together with a leaving group, LG. The reporting
coumarin C1 directly resulted from the disassembly of
ether-bounded spacer SH1 C’’. In the other intermediates,
SR1 LG, LG was a carbonic/carbamic ester, which further de-
composed to eventually afford carbon dioxide and the re-
porting fluorophore, F, in a third step. In Scheme 4, rate
constant k1 is associated with photoactivation, whereas rate
constants k2 and k3 refer to the self-immolation and decar-
boxylation steps, respectively.
First, we performed the kinetic analysis of the disassembly
of compound SH1 C’’, which only involved two steps. Fig-
ure 1a shows that our kinetic model was appropriate for ob-
taining satisfactory fits at all of the investigated tempera-
tures. Hence, we could determine several parameters:
1) The photoactivation quantum yield, f
G
0.3)ꢃ10ꢀ4, which was in accordance with reported values for
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R. Labruꢀre, F. Schmidt, L. Jullien et al.
HOCOC2 with respect to that
of coumarin C2, 0.4; 3) k2
values, which ranged from
(0.27ꢁ0.01) to (0.8ꢁ0.1) sꢀ1
between 293 and 318 K; 4) k3
values, (2.4ꢁ0.5)ꢃ10ꢀ3 and
(8ꢁ1)ꢃ10ꢀ3 sꢀ1 at 293 and
303 K respectively,[12] in agree-
ment with reported values for
the decarboxylation of car-
bamic acid.[13]
Then, the corresponding ex-
periments and analyses were
performed for the caged self-
immolative spacers that con-
Scheme 4. Disassembly processes after the photoactivation of caged self-immolative spacer cSR1 LG.
uncaging with the ortho-nitrobenzyl group;[11] 2) the relative
brightness of phenol intermediate SH1 C’’ with respect to that
of coumarin C1, 0.4, which was in line with our previous re-
sults;[10] 3) k2 values, which ranged from (6.2ꢁ0.3)ꢃ10ꢀ4 to
(7ꢁ1)ꢃ10ꢀ3 sꢀ1 between 293 and 318 K. Then, these latter
values were used to extract the disassembly times of SH1 C’’,
according to td =1/k2, within the range 293–318 K. The re-
sults are summarized in Table 1.
tained a carbonate group (for an exhaustive report of the
experimental data, see the Supporting Information). In
those cases, we could notably use a condensed kinetic
model, which focused on the two first steps shown in
Scheme 4, because the final decarboxylation step was faster
(below the millisecond timescale[14]) than the temporal reso-
lution of our fluorescence-acquisition setup. Again, the ex-
tracted values of the photoactivation quantum yields, f-
AHCTUNGTRENNUNG
(365 nm), were in line with literature expectations[11] and we
could retrieve the disassembly times according to td =1/k2
(Table 1).
Table 1. Disassembly times (td, in s) of the activated self-immolative
spacers at various temperatures and pH 8 (unless otherwise indicated).
During the course of the preceding experiments, we no-
ticed that the two-step kinetic model was poorly reliable in
the case of the cSO1 MeD caged spacer. Indeed, we successfully
accounted for the experimental behavior by using a one-
step kinetic model (see the Supporting Information, Fig-
ure S11a), which suggested that the self-immolation step oc-
curred faster than the temporal resolution of our experimen-
tal design.[10,15] To gain further insight into the self-immola-
tion rate constant of cSO1 MeD, we first equipped our fluorime-
ter with a stopped-flow accessory, thus allowing us to ach-
ieve a temporal resolution of 40 ms. Indeed, we have now
reliably analyzed the results of the illumination experiments
by using the two-step kinetic model (see the Supporting In-
formation, Figure S11b), which yielded td(SO1 MeD) values in
the range 70–300 ms (Table 1).
cSR1 LG
293 K
303 K
310 K
318 K
cSH1 C
cSH1 C’
cSH1 C’’
cSH1 D
(2.3ꢁ0.3)
(3.7ꢁ0.1)
(1600ꢁ100)
(6.2ꢁ0.9)
(1.3ꢁ0.1)
(2.9ꢁ0.6)
(770ꢁ60)
(4.9ꢁ0.1)
–
–
[a]
[a]
(2.5ꢁ0.1)
(290ꢁ60)
(4.8ꢁ0.6)
(1.2ꢁ0.1)
(140ꢁ15)
(4.0ꢁ0.3)
cSB1 r
D
pH 4
pH 8
–
(140ꢁ15)
–
(100ꢁ5)
(29ꢁ1)
–
(14ꢁ4)
–
–
(2.6ꢁ0.2)
–
[a]
pH 10
–
cSN1 O
D
2
pH 4
pH 8
pH 10
–
(133ꢁ2)
(2.6ꢁ0.4)
–
(2.5ꢁ0.1)
–
–
(2.8ꢁ0.2)
(2.1ꢁ0.1)
–
(1.2ꢁ0.1)[b]
(0.12ꢁ0.02)[b]
–
–
cSO1 Me
D
(0.3ꢁ0.1)[b]
(0.7ꢁ0.2)[c]
(0.067ꢁ0.008)[b]
[a] Below the accessible temporal resolution under the measurement con-
ditions. [b] Values extracted from the stopped-flow experiments.
[c] Values extracted from the microscopy experiments.
To confirm the reliability of our estimates, we devised an
alternative experiment that provided us with a temporal res-
olution of down to 20 ms (limited by the acquisition fre-
quency of our camera). An 80 mm solution of cSO1 MeD was in-
troduced into multiple micrometric cavities of a PDMS mi-
crofluidic device, which were submitted to homogeneous il-
lumination at 360 nm and optical recording on an epifluores-
cence setup (Figure 2a and b and the file Movie.avi[23] in the
Supporting Information).[10,16] The temporal evolution of the
fluorescence signal from several neighboring cavities is
shown in Figure 2c, together with the fit that was obtained
by using the two-step kinetic model. Thus, we extracted
td(SO1 MeD)=700 ms at 293 K, which was in reasonable agree-
ment with the result that was obtained by stopped-flow anal-
ysis.
Equipped with these satisfactory results, we performed
the more demanding kinetic analysis of the self-immolation
of the spacers that contained either carbonate or carbamate
groups. Figure 1b shows the temporal evolution of the fluo-
rescence emission, ICF2(t), on the release of coumarin C2
upon the continuous illumination of a (5ꢁ1) mm solution of
caged precursor cSH1 C’ on the fluorimeter setup. Now, rely-
ing on a three-step kinetic model (see the Supporting Infor-
mation), we satisfactorily fitted the experimental data and
derived: 1) The photoactivation quantum yield, fACTHNUGRTNEUNG(365 nm)=
(23.3ꢁ0.2ꢃ10ꢀ4, which was within the expected range for
the ortho-nitrobenzyl caging group;[11] 2) the relative bright-
nesses of phenol SH1 C’ and of carbamic intermediate
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Self-Immolative Spacers in Disassembly
FULL PAPER
Discussion
This investigation shows that this series of self-immolative
spacer groups can be tailored to release a substrate from the
10ꢀ1 to the 103 s timescales. These results are in good agree-
ment with previously reported structure–property relation-
ships,[1a] but the precise determination of the associated ki-
netic information is now possible. The elimination of qui-
none methide is typically ꢃ103-times slower when the re-
porting phenol fluorophore is directly grafted on through an
ether bond rather than through a carbonate or carbamate
linkage (cf. cS1HC’’ with cS1HC, cS1HD, and cSH1 C’).[17] Notably,
the carbonyl moiety acts as an efficient uncoupling frag-
ment; despite major differences between the nucleofugacity
of structures C1, D1, and C2, the elimination of quinone
methide occurs at similar timescales in cSH1 C, cS1HD, and
cSH1 C’. However, the slow decarboxylation of the carbamic-
acid intermediate postpones the release of the final sub-
strate in carbamate cSH1 C’. Phenol ionization typically accel-
erates the disassembly of the spacer by a factor of ꢃ102 (cf.
Br
cSN1 O D and cS1 D in the range pH 4–10; the associated phe-
2
nols exhibit pK 8.1 and 9.5, respectively; see the Supporting
Information). Retaining the nonionized phenol state to ana-
lyze the influence of the substitution pattern, we relied on
data that were acquired at pH 8 for cSH1 D and cS1NO D (with
2
associated phenols exhibiting pK 11.6 and 11.7) and pH 4
for cSB1 rD and cSN1 O D. Hence, in line with the development
2
of a positive charge on the exocyclic methylene during the
elimination of quinone methide,[6] we observed that append-
ing electron-donating groups onto the phenyl core accelerat-
ed the disassembly of the spacer. The disassembly time typi-
cally decreased by a factor of 20 on replacing an appending
inductive electron-withdrawing group (m-NO2 or o,p-Br in
Br
cSN1 O D and cS1 D) by an inductive electron-donating group
2
(m-Me in cSH1 D). Another drop by a factor of 50 was ob-
served upon replacing an inductive electron-donating group
(m-Me in cSH1 D) by a conjugated strongly mesomeric elec-
tron-donating group (o-OMe in cSO1 MeD). Eventually, the
disassembly time is typically expected to drop by a factor of
10 upon increasing the temperature by 25 K from room tem-
perature.
One should emphasize the relevance of this photoactiva-
tion approach for reliably extracting the kinetic parameters
that are associated with the self-immolation process over a
broad kinetic window. In particular, whereas the fastest self-
immolations have been reported to occur within a few sec-
onds,[17a,18] this investigation is the first to show that those
self-immolation events occur precisely on the 100 ms time-
scale. For instance, this information will be significant for
choosing the relevant concentration of a prodrug to circum-
vent drug activation to the close perimeter of an activating
enzyme.[19] Furthermore, this approach will permit access to
even much faster timescales. Indeed, whereas temporal reso-
lution of the cuvette experiments are ultimately limited by
the homogenization timescales (at most 40 ms with our stop-
ped-flow equipment), this procedure, which relies on homo-
genous illumination with an epifluorescence microscope
Figure 2. Disassembly processes after the photoactivation of caged self-
immolative spacer cSR1 LG. a) Schematic representation of the epifluores-
cence microscope. b) Micrographs (lem =(675ꢁ45) nm) of a polydime-
thylsiloxane (PDMS) microfluidic device that initially contained (80ꢁ
5) mm cSO1 MeD before (t=0 s) and after (t=20 s) photoactivation at lexc
=
(360ꢁ20) nm with 2.5ꢃ10ꢀ3 Einsꢀ1 mꢀ2 light intensity. c) Temporal evolu-
tion of the fluorescence emission, ICF1(t), from six neighboring cS1OMeD-
containing wells (a vertical offset has been added for comparison). Cir-
cles denote the experimental data; solid lines show the fits to Equa-
tion (46), thus yielding <k1 > =(0.116ꢁ0.003) sꢀ1 and <k2 > =(1.11ꢁ
0.07) sꢀ1. Solvent: MeCN with 0.1m Britton–Robinson pH 8 buffer (1:1,
v/v), T=293 K.
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R. Labruꢀre, F. Schmidt, L. Jullien et al.
cSH1 D: Yellow solid (61% yield); 1H NMR (300 MHz, CDCl3, 258C): d=
7.78 (s, 1H), 7.65 (d, J=9 Hz, 1H), 7.63 (s, 1H), 7.48 (s, 1H), 7.29 (d, J=
2 Hz, 1H), 7.28 (d, J=2 Hz, 1H), 7.18 (dd, J=9, 2 Hz, 1H), 7.17 (dd, J=
8, 2 Hz, 1H), 6.91 (d, J=8 Hz, 1H), 5.55 (s, 2H), 5.45 (s, 2H), 3.97 (s,
6H), 2.33 (s, 3H), 1.86 ppm (s, 6H); 13C NMR (75 MHz, CDCl3, 258C):
d=173.1, 154.3, 154.1, 153.4, 153.0, 150.0, 147.8, 140.2, 139.5, 139.4, 138.9,
138.6, 137.5, 135.7, 133.1, 131.8, 131.2, 130.8, 129.3, 122.5, 120.8, 119.1,
112.1, 109.3, 107.9, 67.1, 66.9, 56.5, 56.4, 39.1, 26.6, 20.4 ppm; LRMS
(ES+): m/z: 689 [M+Na]+; HRMS (ES+): m/z calcd for C33H29Cl2N2O9:
667.1244 [M+H]+; found: 667.1238.
setup, will be only limited by the acquisition frequency of
the camera. Hence, it should easily yield information on the
ms timescale with a fast camera.
Conclusion
We have introduced and implemented three procedures that
rely on light activation to accurately analyze the disassembly
kinetics of a collection of self-immolative spacer groups
over a wide kinetic window (10ꢀ2–103 s). Our results are rel-
evant for deriving quantitative structure–property relation-
ships, which should be especially significant in the context of
prodrugs. In particular, we have been able to access a tem-
poral resolution of 20 ms, which made it possible to measure
the shortest disassembly time ever reported for an activated
self-immolative spacer.
1
cSB1 rD: Yellow solid (55% yield); H NMR (300 MHz, CDCl3, 258C): d=
7.79 (s, 1H), 7.65 (d, J=9 Hz, 1H), 7.63 (s, 1H), 7.35 (s, 1H), 7.29 (d, J=
2 Hz, 1H), 7.24 (s, 1H), 7.17 (dd, J=9, 2 Hz, 1H), 5.67 (s, 2H), 5.54 (s,
2H), 3.97 (s, 3H), 3.95 (s, 3H), 2.60 (s, 3H), 1.85 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3, 258C): d=173.1, 155.5, 154.1, 153.3, 152.8, 150.0, 148.2,
140.1, 139.4, 139.1, 138.6, 137.5, 135.7, 133.1, 131.4, 129.9, 127.7, 126.9,
122.6, 120.6, 119.1, 116.2, 109.3, 108.2, 68.1, 66.1, 56.6, 56.4, 39.1, 26.6,
23.9 ppm; LRMS (ES+): m/z: 825 [M+H]+; HRMS (ES+): m/z calcd for
C33H27Br2Cl2N2O9: 824.9440 [M+H]+; found: 824.9435.
cSN1 O D: Yellow solid (85% yield); 1H NMR (300 MHz, CDCl3, 258C):
2
d=8.43 (d, J=9 Hz, 1H), 8.33 (dd, J=9, 3 Hz, 1H), 7.81 (s, 1H), 7.67 (d,
J=9 Hz, 1H), 7.64 (s, 1H), 7.36 (s, 1H), 7.31 (d, J=2 Hz, 1H), 7.19 (dd,
J=9, 2 Hz, 1H), 7.14 (d, J=9 Hz, 1H), 5.69 (s, 2H), 5.50 (s, 2H), 3.99 (s,
6H), 1.87 ppm (s, 6H); 13C NMR (75 MHz, CDCl3, 258C): d=173.1,
160.7, 154.2, 153.1, 152.8, 150.1, 148.4, 141.8, 140.1, 139.6, 139.4, 139.2,
138.7, 137.5, 135.7, 133.2, 126.9, 126.8, 126.1, 124.1, 120.6, 119.1, 111.8,
109.2, 108.2, 67.9, 65.3, 56.6, 56.5, 39.1, 26.6 ppm; LRMS (ES+): m/z: 720
[M+Na]+; HRMS (ES+): m/z calcd for C32H26Cl2N3O11: 698.0938
[M+H]+; found: 698.0953.
cSO1 MeD: Yellow solid (82% yield); 1H NMR (300 MHz, CDCl3, 258C):
d=7.70 (s, 1H), 7.63 (s, 1H), 7.62 (s, 1H), 7.60 (d, J=9 Hz, 1H), 7.23 (d,
J=2 Hz, 1H), 7.06 (dd, J=9, 2 Hz, 1H), 6.67 (s, 1H), 5.44 (s, 2H), 5.43
(s, 2H), 4.00 (s, 3H), 3.93 (s, 3H), 3.91 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H),
1.86 ppm (s, 6H); 13C NMR (75 MHz, CDCl3, 258C): d=173.5, 154.3,
153.8, 153.1, 150.3, 149.9, 149.4, 148.0, 142.3, 140.6, 139.8, 139.1, 138.9,
136.0, 133.4, 130.8, 122.7, 121.0, 119.5, 109.9, 108.0, 100.5, 72.6, 62.2, 61.7,
56.9, 56.7, 39.5, 26.9 ppm; LRMS (ES+): m/z: 743 [M+H]+; HRMS
(ES+): m/z calcd for C35H33Cl2N2O12: 743.1405 [M+H]+; found: 743.1400.
cSH1 C’: To a solution of 7-amino-4-(trifluoromethyl)-2H-chromen-2-one
(10 mg, 0.04 mmol) and Et3N (24 mL, 0.08 mmol) in anhydrous CH2Cl2
(1 mL) at 08C under an Ar atmosphere was added a solution of triphos-
gene (15 mg, 0.05 mmol) in anhydrous CH2Cl2 (1 mL). The solution was
stirred at 08C for 2 h, a solution of alcohol cSH1 OH (14 mg, 0.04 mmol) in
anhydrous CH2Cl2 (2 mL) was slowly added at 08C, and the reaction mix-
ture was stirred for a further 2 h at RT. The resulting suspension was fil-
tered and the crude precipitate was purified by preparative HPLC (A/B,
90:10) to afford compound cSH1 C’ as a white solid (12 mg, 52% yield).
1H NMR (300 MHz, [D7]DMF, 258C): d=10.61 (s, 1H), 7.94 (s, 1H), 7.93
(d, J=2 Hz, 1H), 7.87 (d, J=9 Hz, 1H), 7.71 (dd, J=8, 2 Hz, 1H), 7.70
(s, 1H), 7.49 (d, J=2 Hz, 1H), 7.41 (dd, J=9, 2 Hz, 1H), 7.31 (d, J=
8 Hz, 1H), 7.04 (s, 1H), 5.68 (s, 2H), 5.52 (s, 2H), 4.16 (s, 3H), 4.11 (s,
3H), 2.48 ppm (s, 3H); LRMS (ES+): m/z: 606 [M+NH4]+; HRMS
(ES+): m/z calcd for C28H27F3N3O9: 606.1693 [M+NH4]+; found:
606.1688.
cSH1 C’’: To a stirring solution of PPh3 (60 mg, 0.23 mmol), imidazole
(16 mg, 0.23 mmol), and iodine (58 mg, 0.23 mmol) in anhydrous CH2Cl2
(10 mL) was added alcohol cSH1 OH (87 mg, 0.15 mmol). The resulting
mixture was stirred at RT for 4 h and filtered to remove the precipitate.
A saturated aqueous solution of Na2S2O3 (20 mL) and CH2Cl2 (30 mL)
were successively added to the filtrate. The organic phase was separated,
dried over MgSO4, and evaporated under reduced pressure. The crude
residue was purified by column chromatography on silica gel (cyclohex-
ane/EtOAc, 8:2 v/v) to afford 1-((2-(iodomethyl)-4-methylphenoxy)meth-
yl)-4,5-dimethoxy-2-nitrobenzene as a white solid (50 mg, 75% yield).
1H NMR (300 MHz, CDCl3, 258C): d=7.79 (s, 1H), 7.60 (s, 1H), 7.15 (s,
1H), 7.07 (d, J=8 Hz, 1H), 6.80 (d, J=8 Hz, 1H), 5.54 (s, 2H), 4.56 (s,
2H), 4.07 (s, 3H), 3.98 (s, 3H), 2.28 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3, 258C): d=154.6, 153.9, 148.2, 139.1, 131.1, 130.8, 129.9, 127.4,
Experimental Section
Synthesis: The commercially available chemicals were used without fur-
ther purification. Anhydrous solvents were freshly distilled before use.
Low-actinic glassware was used for all experiments with compounds that
contained nitroveratryl moieties. Column chromatography was performed
on Merck silica gel 60 (0.040–0.063 mm). Analytical and thin layer chro-
matography (TLC) was performed on Merck silica gel 60 F-254 precoat-
ed plates; detection was performed by using UV light (l=254 nm).
NMR spectra were recorded on an AC Bruker spectrometer at 300 MHz
(for H nuclei) and 75 MHz (for 13C nuclei). Coupling constants (J) are in
1
Hz. HPLC analyses and purifications of the final caged species were per-
formed on a Waters system with a Wdelta 600 pump and a PDA 996 UV
detector at l=245 nm (analytical HPLC: X-Terra Waters MS C18
column, 150 mmꢃ4.6 mm, 5 mm, flow rate: 1 mLminꢀ1
; preparative
HPLC: X-Terra Waters Prep MS C18 column, 150 mmꢃ19 mm, 5 mm,
flow rate: 10 mLminꢀ1; elution with MeCN/water mixtures). For informa-
tion on the syntheses of the intermediate benzylic alcohols, cSR1 OH, see
the Supporting Information.
General procedure for the preparation of caged self-immolative spacers
that contain carbonic leaving groups: To a solution of the desired benzyl-
alcohol intermediate (cSR1 OH, 0.01 mmol) in anhydrous THF (10 mL) at
08C under an Ar atmosphere was quickly added a 20% solution of phos-
gene in toluene (25 mL, 0.05 mmol) by using a syringe. The mixture was
stirred at RT for 2 h and Ar gas was then bubbled through the solution
for 15 min to remove any unreacted phosgene. The purged solution was
added dropwise to a solution of Et3N (8 mL, 0.06 mmol) and the desired
reporter, 7-hydroxy-4-(trifluoromethyl)-2H-chromen-2-one or DDAO
(0.02 mmol), in anhydrous THF (10 mL) and the mixture was cooled to
08C and stirred for 15 min. The resulting suspension was diluted with
CH2Cl2 (10 mL) and a 1m aqueous solution of HCl (20 mL) was added.
The organic phase was separated, dried over MgSO4, and evaporated
under reduced pressure. The crude product was purified by preparative
HPLC (MeCN/water 90:10 v/v).
cSH1 C: White solid (40% yield); 1H NMR (300 MHz, CDCl3, 258C): d=
7.79 (s, 1H), 7.73 (d, J=9 Hz, 1H), 7.45 (s, 1H), 7.27 (d, J=2 Hz, 1H),
7.26 (d, J=2 Hz, 1H), 7.19 (dd, J=9, 2 Hz, 1H), 7.18 (dd, J=8, 2 Hz,
1H), 6.92 (d, J=8 Hz, 1H), 6.78 (s, 1H), 5.54 (s, 2H), 5.46 (s, 2H), 3.98
(s, 3H), 3.96 (s, 3H), 2.33 ppm (s, 3H); 13C NMR (75 MHz, CDCl3,
258C): d=158.2, 154.9, 154.0, 153.9, 152.4, 147.8, 138.9, 131.7, 131.1,
130.6, 129.3, 129.0, 126.3, 122.2, 118.1, 115.4, 112.0, 111.3, 110.3, 109.3,
107.8, 67.0, 56.3, 20.3 ppm; LRMS (ES+): m/z: 612 [M+Na]+; HRMS
(ES+): m/z calcd for C28H22F3NNaO10
:
612.1088 [M+Na]+; found:
612.1081.
11722
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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 11717 – 11724

Self-Immolative Spacers in Disassembly
FULL PAPER
112.6, 109.9, 108.3, 67.3, 57.8, 56.8, 20.8, 2.4 ppm; LRMS (ES+): m/z: 466
[M+Na]+; HRMS (ES+): m/z calcd for C17H22IN2O5: 461.0567
[M+NH4]+; found: 461.0570.
densities were recorded on a Nova II power meter (Laser Measurement
Instruments). Typical light fluxes were within the range 2.5ꢃ
10ꢀ3 Einsꢀ1 mꢀ2
.
To a stirring solution of 1-((2-(iodomethyl)-4-methylphenoxy)methyl)-
4,5-dimethoxy-2-nitrobenzene (30 mg, 0.07 mmol) in anhydrous DMF
(10 mL) was added K2CO3 (14 mg, 0.1 mmol) and 7-hydroxy-4-(trifluoro-
methyl)-2H-chromen-2-one (16 mg, 0.07 mmol). The mixture was stirred
at RT for 2 h and then CH2Cl2 (20 mL) and water (20 mL) were added.
The organic phase was separated, dried over MgSO4, and evaporated
under reduced pressure. The crude residue was purified by column chro-
matography on silica gel (cyclohexane/EtOAc, 8:2 v/v) to afford com-
pound cSH1 C’’ as a white solid (35 mg, 92% yield). 1H NMR (300 MHz,
CDCl3, 258C): d=7.75 (s, 1H), 7.62 (d, J=9 Hz, 1H), 7.31 (s, 1H), 7.25
(d, J=2 Hz, 1H), 7.15 (dd, J=8, 2 Hz, 1H), 6.99 (dd, J=9, 2 Hz, 1H),
6.98 (d, J=2 Hz, 1H), 6.91 (d, J=8 Hz, 1H), 6.62 (s, 1H), 5.51 (s, 2H),
5.25 (s, 2H), 3.96 (s, 3H), 3.76 (s, 3H), 2.32 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3, 258C): d=162.8, 159.2, 156.3, 153.9, 147.9, 139.2, 131.1,
130.6, 128.9, 126.4, 123.4, 113.9, 112.5, 109.3, 108.1, 107.1, 102.2, 67.6,
66.5, 56.4, 56.1, 20.5 ppm; LRMS (ES+): m/z: 568 [M+Na]+; HRMS
(ES+): m/z calcd for C27H22F3NNaO8: 568.1189 [M+Na]+; found:
568.1183.
Acknowledgements
This work was supported by the ANR PCV 2008 (Proteophane) and the
ANR Blanc 2010 (Kituse). We thank Katherine J. Silvestre and Gꢁral-
dine Hallais for providing technical assistance.
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Analytical solutions: All of the experiments were performed in MeCN
with 0.1m Britton–Robinson buffer (1:1, v/v).[20] All of the solutions were
prepared by using water that was purified on a Direct-Q 5 system (Milli-
pore, Billerica, MA).
pH measurements: pH measurements were performed on a Standard pH
meter PHM210 Radiometer Analytical (calibrated with aqueous buffers
at pH 4 and 7 or 10) with a Crison 5208 Electrode (Barcelona, Spain),
which was accurate over the pH range 0–14.
UV/Vis absorption spectroscopy: UV/Vis absorption spectra were re-
corded in 1 cmꢃ1 cm quartz cuvettes (Hellma) on a diode-array UV/Vis
spectrophotometer (Evolution Array, Thermo Scientific) at 298 K. Molar
absorption coefficients were extracted whilst checking the validity of the
Beer–Lambert law.
The evolution of the absorbance as a function of pH value were analyzed
by using the SPECFIT/32 Global Analysis System (version 3.0 for 32-bit
Windows systems) to extract the pK values of the core phenol groups of
the self-immolative spacers (see the Supporting Information).[21]
Steady-state fluorescence emission spectroscopy: Corrected fluorescence
spectra upon one-photon excitation were recorded on a Photon Technol-
ogy International QuantaMaster QM-1 spectrofluorimeter (PTI, Mon-
mouth Junction, NJ) that was equipped with
a Peltier cell-holder
(TLC50, Quantum Northwest, Shoreline, WA). Solutions for the fluores-
cence measurements were adjusted to concentrations such that the ab-
sorption maximum was about 0.15 at the excitation wavelength.
Irradiation experiments: Three different procedures were used to per-
form the one-photon irradiation, that is, on a spectrofluorimeter (with or
without stopped-flow) or on an epifluorescence microscope.
On the spectrofluorimeter, irradiations were performed on 400 mL sam-
ples in 0.2ꢃ1 cm2 quartz fluorescence cuvettes (Hellma) by using a fil-
tered 75 W xenon lamp at several slit widths under constant stirring. The
incident-light intensities were calibrated by using a-(4-dimethylamino-
phenyl)-N-phenylnitrone actinometer, as reported previously.[22] Typical
integral incident-light intensities were within the range 1ꢃ10ꢀ9 Einsꢀ1
.
On the epifluorescence microscope, solutions were introduced in 40ꢃ
10 mm2 (Øꢃh) cylindrical microcuvettes that were generated in a PDMS
stamp after standard photolithography. The PDMS microdevice was fixed
onto the microscope stage and submitted to irradiations with a 100 W
xenon lamp (LOT-Oriel, Palaiseau, France) that was equipped with an
excitation filter (360ꢁ20) nm and
a dichroic mirror (T425 LPXR,
Chroma Technology, Bellows Falls, VT, USA); the fluorescence emission
was recorded by using a ꢃ10 objective (Fluor NA 0.5; Zeiss, Le Pecq,
France), which was mounted onto a home-built microscope that was
equipped with a Luca-R CCD camera (Andor Technology, Belfast, UK),
at (675ꢁ45) nm (Chroma Technology, Bellows Falls, VT, USA).
3000 Images were recorded at 25 Hz (exposure time: 39 ms). The signal
was averaged within each well over 20ꢃ20 mm2 square areas. Photon-flux
Chem. Eur. J. 2013, 19, 11717 – 11724
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11723

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[23] Left: movie displaying the temporal evolution of the epifluorescence
image of the PDMS microfluidic device initially containing (80ꢁ
5) mm cSO1 MeD upon photoactivation at lexc =(360ꢁ20) nm with 2.5ꢃ
10ꢀ3 Einsꢀ1 mꢀ2 light intensity and imaging at lem =(675ꢁ45) nm.
Right: temporal evolution of the fluorescence emission from six
neighboring cSO1 MeD-containing wells (a vertical offset was intro-
duced to facilitate reading). Markers: experimental data; red solid
lines: fits with Equation (46) yielding <k1 > =(0.116ꢁ0.003) sꢀ1 and
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<k2 > =(1.11ꢁ0.07) sꢀ1
. Solvent: CH3CN/0.1m Britton–Robinson
pH 8 buffer 1:1 (v/v); T=293 K.
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Received: April 6, 2013
Published online: July 16, 2013
11724
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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 11717 – 11724