A comparative study of the self-immolation of para-aminobenzylalcohol and hemithioaminal-based linkers in the context of protease-sensitive fluorogenic probes

Article abstract of DOI:10.1039/b926316k

This study focuses on the disassembly-behavior of self-immolative pro-fluorescent linkers under physiological conditions and through an enzyme-initiated domino reaction. The targeted linkers are based on para-aminobenzylalcohol (PABA) or hemithioaminal de

Full text of DOI:10.1039/b926316k

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
A comparative study of the self-immolation of para-aminobenzylalcohol and
hemithioaminal-based linkers in the context of protease-sensitive fluorogenic
probes†
Yves Meyer,a,b Jean-Alexandre Richard,a,b Bruno Delest,b Pauline Noack,b Pierre-Yves Renard*a,c,d and
Anthony Romieu*a,c
Received 14th December 2009, Accepted 24th February 2010
First published as an Advance Article on the web 8th March 2010
DOI: 10.1039/b926316k
This study focuses on the disassembly-behavior of
self-immolative pro-fluorescent linkers under physiologi-
cal conditions and through an enzyme-initiated domino
reaction. The targeted linkers are based on para-
aminobenzylalcohol (PABA) or hemithioaminal derivatives
of para-carboxybenzaldehyde or glyoxilic acid. We found that
a fine tuning of the kinetic properties could be obtained
through the modulation of the linker structure, giving either
a fast signal response or free-adaptable systems suitable for
the design of protease-sensitive fluorogenic probes or prodrug
systems.
The design and optimisation of new enzymatically triggered
self-immolative linkers take an even more critical place in the
development of efficient prodrug systems1 and smart optical
bioprobes suitable for bioanalytical and biological applications
such as high-throughput screening or biomolecular imaging.2
Fig. 1 General principle for the activation of protease-sensitive fluoro-
Indeed, in such molecular constructions, self-immolative linkers
are key components to control the release of the free active
product (i.e., drug or optical marker) upon designed (enzymatic,
chemical or physical) activation under mild neutral and aq.
conditions. For instance, in the emerging and promising field
of pro-fluorescent bioprobes, the targeted tripartate sensor (i.e.,
a molecular probe composed of a trigger recognition unit, a
linker and a fluorophore moiety) unmasks its intense fluorescence
only by a specific enzymatic cleavage that produces a labile self-
immolative linker-fluorophore intermediate that in turn eliminates
spontaneously to release the original fluorescent marker through
a domino reaction3–5 (see Fig. 1 for the application of this concept
to proteases).
genic probes based on a self-immolative linker strategy.
such linkers is potentially beneficial to improve their chemical
stability and enzymatic reactivity by moving away the bulky
hydrophobic fluorophore from the enzyme recognition unit.4,5
Such a self-immolative linker strategy also widens the scope of the
available pro-fluorophores (i.e., aniline-, phenol- or thiophenol-
based fluorophores) for a targeted enzymatic reaction. For ex-
ample, as illustrated by us, it allows the development of efficient
remote protease-coupled fluorescent phenol release systems.5,7
To perform real-time biomolecular imaging, the release of the
latent fluorophore after target recognition should be as fast as
possible in order to accurately track the biological event. However
during self-immolation, the domino (or cascade) reaction is often
slower than the enzyme activation step due to the occurrence
of electronic rearrangement or cyclisation processes which may
induce a significant delay time between the enzyme cleavage
and the observation of a significant emission of the free excited
fluorophore.
During the past decade, significant research efforts have been
devoted to the development of sophisticated prodrug technolo-
gies involving drug delivery vehicles with self-eliminating and
multiple release properties.1,8 Since the most commonly used
self-eliminating structures are based on para-aminobenzylalcohol
(PABA) linker (or its hydroxylated analogue),9 some academic
groups have recently reported comprehensive kinetic studies for
the disassembly of such self-immolative systems through 1,4-
and/or 1,6-benzyl elimination reactions. Thus, Shabat et al. have
These latent fluorescent probes thus display a unique selectivity
and limited interferences associated with the probe concentration,
excitation intensity, and emission sensitivity.6 Moreover, the use of
aEquipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038,
rue Lucien Tesnie`re, 76131, Mont-Saint-Aignan, France. E-mail: pierre-
yves.renard@univ-rouen.fr, anthony.romieu@univ-rouen.fr; Fax: +33 2-35-
52-29-71; Tel: +33 2-35-52-24-14 (or 24-15)
bQUIDD, Technopoˆle du Madrillet, 50, rue Ettore Bugatti, 76800, Saint-
Etienne du Rouvray, France
cUniversite´ de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan,
France
dInstitut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris,
France
† Electronic supplementary information (ESI) available: Detailed synthetic
procedures and characterisation data for compounds 3, 5 and 6. See DOI:
10.1039/b926316k
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1777–1780 | 1777
©

View Article Online
published an elegant and sophisticated molecular system that
can disassemble through para- and ortho-azaquinone-methide
elimination reactions in which the 1,6-elimination process was
found to be faster than the 1,4-elimination one.10 Similarly, Shabat
et al. have shown that the pyridinone-methide elimination was
even faster than the parent azaquinone-methide process under
physiological conditions.11 Interestingly, Lee et al. have also
reported a kinetic study on self-immolation reactions concerning
the 1,4- and 1,6-elimination reactions of quinone-methide systems
showing an efficient “shortcut” that outperforms conventional
pathways involving repetitive quinone-methide rearrangements
and elimination.12 In the context of protease-sensitive fluorogenic
probes, we have explored three different self-immolative spacer
strategies based either: (1) on the use of an intramolecular lactam-
cyclisation process with the enzyme-labile safety catch linker
reported by Waldmann et al.13 (Fig. 2), (2) on the use of the
self-cleavable PABA linker or (3) on the design of an original
hemithioaminal core.14 The resulting pro-fluorophores are able
to release fluorescent umbelliferone (or its 4-acetic acid derivative)
within few minutes in the presence of the model protease penicillin
G acylase (PGA) under physiological conditions. However, in
the first case, the limited stability of the aryl ester moiety
prevented its generalised use for the latent fluorescent probes
design.15 In the third case, a slow linear increase in fluorescence
intensity was observed without the enzyme, corresponding to
the non-enzymatic hydrolysis of its carbonate moiety (compound
1, Fig. 3a). To improve its stability, the carbonate linkage was
replaced by two carbamate moieties separated each other by a
N,N¢-dimethylethylenediamine linker (compound 2, Fig. 3b). This
modification significantly improved the overall stability of the
probe but has a detrimental effect on the kinetics for fluorophore
release. These observations led us to study the kinetic behavior
of the PGA-catalysed disassembly process of several amidase-
sensitive fluorogenic probes bearing various self-immolative spac-
ers including PABA or hemithioaminal derivatives, in order to
find the best compromise between stability, self-reactivity upon
enzyme triggering, and substrate scopes and limitations. Here, we
present the results of this comparative study whose ultimate goal
is to select an optimised linker for the construction of protease-
sensitive fluorogenic probes fulfilling all requirements for in cellulo
or in vivo imaging applications.
Fig. 3 a) Fluorescence emission time-course of pro-fluorescent com-
pound 1 (concentration: 4.5 mM) with recombinant PGA (0.12 U, 37 ◦C) in
PBS buffer at 460 nm (lex = 360 nm). b) Fluorescence emission time-course
of pro-fluorescent compound 2 (concentration: 3.0 mM) with recombinant
PGA (0.12 U, 37 ◦C) in PBS buffer at 460 nm (lex = 360 nm).
In this context, five model compounds 2–6 (Table 1) were
synthesised (see ref. 14 for 2 and ref. 5 for 4, see ESI† for synthesis
details of 3, 5 and 6). These molecules were designed specifically
to integrate: (1) a phenylacetamide moiety to be cleaved by PGA
functioning as the triggering agent, and(2) a maskedumbelliferone
(i.e., 7-hydroxycoumarin) unit to be released as the final product
and to elicit turn-on fluorescence. Concerning the hemithioaminal
derivative 3, the starting material para-carboxybenzaldehyde used
for the preparation of 1 and 2, was replaced by glyoxilic acid in
order to get an amino acid-like spacer (i.e., a-substituted glycine)
which may be further inserted within peptide architectures accord-
ing to standard peptide synthesis protocols.16 In addition to these
PABA and hemithioaminal-based linkers which were elongated
with the N,N¢-dimethylethylenediamine moiety for compounds
2, 3 and 5, this latter bis-amine cyclisation spacer alone was
equipped with phenylacetamide trigger and 7-hydroxycoumarin
as reporter group to give 6. Indeed, to our knowledge, there is no
information in the literature about the ability of PGA to cleave
such N-disubstituted peptide bond, and such a fluorogenic probe
enables us to address this issue.
All these fluorogenic probes were incubated at the same concen-
tration at 37 ◦C with PGA (0.12 U) in phosphate buffered saline
(PBS, pH 7.5) and the corresponding fluorescence time-courses
were recorded at l = 460 nm (Fig. 4). Comparison of the resulting
curves clearly indicates that the decomposition of PABA-based
Fig. 2 General structure of phenol-based fluorogenic probes derived from
the Waldmann traceless linker.
1778 | Org. Biomol. Chem., 2010, 8, 1777–1780
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 1 Disassembly mechanisms of self-immolative spacers PABA and hemithioaminal derivatives for the PGA-induced release of 7-hydroxycoumarin
Compounda
Self-immolative spacer
Reaction type
Release mechanismb
2
Fragmentation + dual cyclisation
3
4
5
1,6-Benzyl elimination
1,6-Benzyl elimination + Cyclisation
6
Cyclisation
a R = H for compounds 2–5 and Me for compound 6 b OF = 7-hydroxycoumarin
self-immolative linkers was faster than with the hemithioaminal
counterparts. Thus, the 1,6-benzyl elimination clearly occurs much
more rapidly than the fragmentation-cyclisation process involving
a thioalkyl carbamate intermediate. Yet, it is interesting to note
that the elongated pro-fluorescent probe 5 was able to release
the phenol-based fluorophore even faster than the parent PABA-
based fluorogenic probe 4. As already observed by Lee et al.,12
the carbamate decarboxylation occurring during the domino
reaction provides an additional thermodynamic driving force that
is responsible for the experimentally observed faster fluorescence
response of 5 compared to 4. Similarly, pro-fluorescent probe 2 un-
derwent a slower fragmentation when compared with compound 5
because the first cyclisation reaction involves a nucleophilic attack
of the free thiol onto a less electrophilic carbamate which leads to
the slow release of ethylene monothiocarbonate instead of CO2
(see Table 1 for the different PGA-initiated domino reactions
explored in this study). An alternative disassembly mechanism
involving the internal nucleophilic attack of the thiol group on the
a carbon of the 2-thioethyl moiety and subsequent elimination of
ethylene episulfide and CO2, can be also considered.17 However,
we and others have clearly demonstrated that the decomposition
of 2-thioethyl carbamate intermediates (through one of these
two disassembly mechanisms) is favoured by the formation of
a thiolate anion, a better nucleophile than the corresponding
thiol, and so readily occurred at pH over 8.0 and not under
physiological conditions.18 Pro-fluorescent probes 2 and 3 exhibit
similar kinetics for the self-immolation process, revealing that
some structural variations close to the active site of the enzyme did
not interfere with the substrate recognition. However, enzymatic
cleavage did not occur with fluorogenic probe 6, suggesting that the
N-methylation of the cleavable phenylacetamide bond prevents its
hydrolysis by PGA. Furthermore, no nonspecific cleavage of these
pro-fluorescent probes was detected in control reactions where
2–6 were incubated only in PBS (see ESI† for the corresponding
fluorescence emission time-course curves). These results clearly
demonstrate that the use of elongated PABA or hemithioaminal
based linkers enables to get enzyme-reactive pro-fluorophores with
high chemical stability.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1777–1780 | 1779
©

View Article Online
Notes and references
1 For selected reviews, see: (a) F. Kratz, I. A. Muller, C. Ryppa and A.
Warnecke, ChemMedChem, 2008, 3, 20; (b) I. Tranoy-Opalinski, A.
Fernandes, M. Thomas, J. P. Gesson and S. Papot, Anticancer Agents
Med. Chem., 2008, 8, 618.
2 For selected reviews, see: (a) N. Johnsson and K. Johnsson, ACS Chem.
Biol., 2007, 2, 31; (b) J.-L. Reymond, V. S. Fluxa and N. Maillard, Chem.
Commun., 2009, 34.
3 For selected recent examples, see: (a) S. S. Chandran, K. A. Dickson
and R. T. Raines, J. Am. Chem. Soc., 2005, 127, 1652; (b) G. B. Jones,
C. F. Crasto, J. E. Mathews, L. Xie, M. O. Mitchell, A. El-Shafey,
A. V. D’Amico and G. J. Bubley, Bioorg. Med. Chem., 2006, 14, 418;
(c) L. D. Lavis, T.-Y. Chao and R. T. Raines, ChemBioChem, 2006, 7,
1151; (d) L. D. Lavis, T.-Y. Chao and R. T. Raines, ACS Chem. Biol.,
2006, 1, 252; (e) E. Danieli and D. Shabat, Bioorg. Med. Chem., 2007,
15, 7318; (f) M. Waibel, X.-B. Zhang and J. Hasserodt, Synthesis, 2009,
318; (g) X.-B. Zhang, M. Waibel and J. Hasserodt, Chem.–Eur. J., 2010,
16, 792.
4 N.-H. Ho, R. Weissleder and C.-H. Tung, ChemBioChem, 2007, 8, 560.
5 J.-A. Richard, Y. Meyer, V. Jolivel, M. Massonneau, R. Dumeunier, D.
Vaudry, H. Vaudry, P.-Y. Renard and A. Romieu, Bioconjugate Chem.,
2008, 19, 1707.
Fig. 4 Fluorescence emission time-course of pro-fluorescent probes 2–6
(concentration: 3.0 mM) with recombinant PGA (0.12 U, 37 ◦C) in PBS
buffer at 460 nm (lex = 360 nm).
6 X. Chen, M. Sun and H. Ma, Curr. Org. Chem., 2006, 10, 477.
7 J.-A. Richard, M. Massonneau, P.-Y. Renard and A. Romieu, Org.
Lett., 2008, 10, 4175.
8 For selected reviews, see: (a) D. V. McGrath, Mol. Pharmaceutics, 2005,
2, 253; (b) P. Gomes, N. Vale and R. Moreira, Molecules, 2007, 12,
2484.
9 For selected examples about the use of PABA (or its hydroxylated
analogue), see: (a) C. Fossey, A.-H. Vu, A. Vidu, I. Zarafu, D. Laduree,
S. Schmidt, G. Laumond and A.-M. Aubertin, J. Enzyme Inhib. Med.
Chem., 2007, 22, 591; (b) C. Fossey, N.-T. Huynh, A.-H. Vu, A. Vidu,
I. Zarafu, D. Laduree, S. Schmidt, G. Laumond and A.-M. Aubertin,
J. Enzyme Inhib. Med. Chem., 2007, 22, 608; (c) K. A. Ajaj, M. L.
Biniossek and F. Kratz, Bioconjugate Chem., 2009, 20, 390; (d) A.
Fernandes, A. Viterisi, F. Coutrot, S. Potok, D. A. Leigh, V. Aucagne
and S. Papot, Angew. Chem., Int. Ed., 2009, 48, 6443.
10 R. Erez and D. Shabat, Org. Biomol. Chem., 2008, 6, 2669; a similar
study was published by Warnecke et al. but the self-eliminating structure
was activated with a chemical reagent and solubility problems did not
allow evaluation of the disassembly under physiological conditions.
See: A. Warnecke and F. Kratz, J. Org. Chem., 2008, 73, 1546.
11 R. Perry-Feigenbaum, P. S. Baran and D. Shabat, Org. Biomol. Chem.,
2009, 7, 4825.
12 H. Y. Lee, X. Jiang and D. Lee, Org. Lett., 2009, 11, 2065.
13 Unpublished results.
14 Y. Meyer, J. A. Richard, M. Massonneau, P.-Y. Renard and A. Romieu,
Org. Lett., 2008, 10, 1517.
15 A model pro-fluorescent compound composed of phenylacetamide,
Waldmann linker and 7-hydroxycoumarin-4-acetic acid moieties was
firstly synthesised and subjected to PGA hydrolysis. However, a
significant non-specific hydrolysis was observed and evaluated to
30% by RP-HPLC quantification of the released 2-(aminomethyl)-4,5-
dimethoxy-benzeneacetic acid.
In conclusion, self-immolative linkers based on the hemithioam-
inal core disassemble slower than PABA analogues when us-
ing N,N¢-dimethylethylendiamine as a stabilising spacer. Thus,
PABA derivatives will be used for getting fast-response probes
whereas the hemithioaminal spacers will be preferred for the
construction of tunable (bio)functionalised probes. Indeed, these
latter compounds exhibit a conveniently positioned carboxylic
acid group which may be used as an anchoring point for
grafting either: (1) a water-solubilising group such as poly-
sulfonated peptide-based linkers19 especially to counterbalance
the hydrophobicity of the grafted fluorophore, (2) an additional
peptide as part of the substrate for endoproteases, to increase the
enzyme specificity/selectivity or binding or (3) a targeting ligand
suitable for driving the designed pro-fluorescent probe or prodrug
to the targeted tissue and/or proteolytic enzyme. The present
study thus both widens the scope of accessible self-immolative
linkers, and provides additional informations to those recently
published by the groups of Lee, Shabat and Warnecke. This
thus should aid the choice and design of suitable self-immolative
systems useful for the development of optical bioprobes or drug
delivery vehicles suitable, for instance, for cancer imaging and
chemotherapy.
Acknowledgements
16 M. P. Samant, R. White, D. J. Hong, G. Croston, P. M. Conn, J. A.
Janovick and J. Rivier, J. Med. Chem., 2007, 50, 2067.
This work was supported by La Re´gion Haute-Normandie via
the CRUNCh program (CPER 2007-2013), QUIDD and Institut
Universitaire de France (IUF). We thank CNRS and QUIDD
for a PhD grant to Yves Meyer. Recombinant A. faecalis PGA
was gratefully furnished by Pr. L. Fischer, Universita¨t Hohenheim
(Institut fu¨r Lebensmitteltechnologie, Fachgebiet Biotechnologie,
Stuttgart).
17 A. Satyam, Bioorg. Med. Chem. Lett., 2008, 18, 3196.
18 (a) S. Far and O. Melnyk, Tetrahedron Lett., 2004, 45, 7163; (b) M.
Lapeyre, J. Leprince, M. Massonneau, H. Oulyadi, P.-Y. Renard, A.
Romieu, G. Turcatti and H. Vaudry, Chem.–Eur. J., 2006, 12, 3655;
(c) O. P. Varghese, W. Sun, J. Hilborn and D. A. Ossipov, J. Am. Chem.
Soc., 2009, 131, 8781.
19 A. Romieu, D. Brossard, M. Hamon, H. Outaabout, C. Portal and P.-Y.
Renard, Bioconjugate Chem., 2008, 19, 279.
1780 | Org. Biomol. Chem., 2010, 8, 1777–1780
This journal is
The Royal Society of Chemistry 2010
©