Development of a new nonpeptidic self-immolative spacer. application to the design of protease sensing fluorogenic probes

Article abstract of DOI:10.1021/ol800198f

The design and synthesis of novel self-immolative spacer systems aiming at the release of phenol-containing compounds are described. The newly designed traceless linkers proved to be conveniently stable under physiological conditions and operate through spontaneous decomposition of an hemithioaminal intermediate under neutral aqueous conditions. Their utility was then illustrated by the preparation of original fluorogenic substrates of penicillin amidase whose strong fluorescence is unveiled through enzyme-initiated domino reactions.

Full text of DOI:10.1021/ol800198f

ORGANIC
LETTERS
2008
Vol. 10, No. 8
1517-1520
Development of a New Nonpeptidic
Self-Immolative Spacer. Application to
the Design of Protease Sensing
Fluorogenic Probes
Yves Meyer,†,‡ Jean-Alexandre Richard,†,‡ Marc Massonneau,‡
Pierre-Yves Renard,*,†,§ and Anthony Romieu*,†,§
Equipe de Chimie Bio-Organique, UniVersite´ de Rouen, Place Emile Blondel,
76821 Mont-Saint-Aignan, France, IRCOF, UMR 6014, rue Lucien Tesnie`re,
76131 Mont-Saint-Aignan, France, and QUIDD, Technopoˆle du Madrillet, 50,
rue Ettore Bugatti, 76800 Saint-Etienne du RouVray, France
pierre-yVes.renard@uniV-rouen.fr
Received January 28, 2008
ABSTRACT
The design and synthesis of novel self-immolative spacer systems aiming at the release of phenol-containing compounds are described. The
newly designed traceless linkers proved to be conveniently stable under physiological conditions and operate through spontaneous decomposition
of an hemithioaminal intermediate under neutral aqueous conditions. Their utility was then illustrated by the preparation of original fluorogenic
substrates of penicillin amidase whose strong fluorescence is unveiled through enzyme-initiated domino reactions.
During the past decade, traceless linkers have emerged as
powerful tools in solid-phase organic synthesis, especially
for the rapid production of highly diverse organic compound
libraries created through combinatorial or parallel chemistry
methods.1 Traceless linkers are so-called because their
cleavage results in an aliphatic or aromatic hydrogen atom,
so that an examination of the final compound reveals no trace
of the point of linkage to the solid phase (unlike classical
cleavable linkers that generally leave functionalities such as
carboxylic acid, carboxamide, ...). Recent innovations in the
chemistry of such linkers and especially the use of enzymes
to release the desired products under very mild neutral and
aqueous conditions,2 have enabled the broadening of the
scope of these chemical tools to the preparation of efficient
drug delivery agents for pharmaceuticals applications,3 as
well as various reversible bioconjugates and sophisticated
spectroscopic bioprobes for applications in the field of
biological analysis.4,5 Regarding these latter multicomponents
(bio)molecular systems, traceless linkers (the term “self-
immolative spacer” is also used in this context) are used as
reversible covalent bonds between two molecular species:
as their self-decomposition, often triggered by enzyme-
initiated domino reactions,6 leaves no further reactive
(2) Reents, R.; Jeyaraj, D. A.; Waldmann, H. DDT 2002, 7, 71-76.
(3) Kratz, F.; Mu¨ller, I. A.; Ryppa, C.; Warnecke, A. ChemMedChem
2008, 3, 20-53.
(4) Chen, X.; Sun, M.; Ma, H. Curr. Org. Chem. 2006, 10, 477-489.
(5) Goddard, J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22, 363-
370.
† COBRA-CNRS UMR 6014.
‡ QUIDD (QUantitative Imaging in Drug Development).
§ Universite´ de Rouen.
(1) Gil, C.; Bra¨se, S. Curr. Opin. Chem. Biol. 2004, 8, 230-237. Wills,
A. J.; Balasubramanian, S. Curr. Opin. Chem. Biol. 2003, 7, 346-352.
Comely, A. C.; Gibson, S. E. Angew. Chem., Int. Ed. 2001, 40, 1012-
1032. Bra¨se, S.; Dahmen, S. Chem. Eur. J. 2000, 6, 1899-1905.
(6) Mayer, S. F.; Kroutil, W.; Faber, K. Chem. Soc. ReV. 2001, 30, 332-
339.
10.1021/ol800198f CCC: $40.75
© 2008 American Chemical Society
Published on Web 03/22/2008

functionality, the structure and properties of each released
molecule are indeed guaranteed to be the same as before
conjugation.
and non activated thioalkyl moieties onto this masked
hemiaminal, yielding the corresponding N-acylhemithioami-
nal 2. In order to have a self-immolative linker, the chosen
thioalkyl moiety was obtained by introducing a thioalky-
loxycarbonyl (namely, 2-thioethyl- or 3-thiopropyloxycar-
bonyl). These latter linkers have been recently used by us
for the design of thiol-labile amine and alcohol protecting
groups10 and by other academic-industry research teams for
the preparation of bioconjugates suitable for bioimaging11
and drug delivery applications.12 The mechanism leading to
their self-decomposition has been intensively studied and
protocols allowing their conjugation with many N- and
O-nucleophiles involving the corresponding activated car-
bonate derivatives are now well established.
In the area of fluorescent bioprobes, recent work has
clearly shown that self-immolative spacers are key compo-
nents in the smart design and synthesis of latent fluorophores,
exhibiting valuable properties for the illumination of numer-
ous biochemical processes.7 Indeed, these three-component
probes (trigger-linker-fluorophore) unmask their intense
fluorescence only by a specific enzymatic cleavage that
produces a labile self-immolative linker-fluorophore deriva-
tive that in turn eliminates spontaneously to release the
original fluorescent marker. These latent fluorophores thus
display a unique selectivity and limited interferences associ-
ated with the probe concentration, excitation intensity, and
emission sensitivity.
Some latent fluorophores of proteolytic enzymes have been
reported4 based on the direct linkage between a fluorophore
moiety and an enzyme recognition unit but the absence of a
spacer between the two compartments happened to cause
two main problems: (1) pro-fluorophores designed for
enzymatic detection have often revealed to be poor substrates
for their respective enzymes; these problems are caused by
steric hindrance brought by the presence of the (bulky)
fluorophore molecule next to the cleavage site which may
prevent access to the active pocket of the enzyme; (2) the
choice of fluorophores is almost exclusively restricted to
aromatic amines such as 7-amino-4-methylcoumarin, cresyl
violet, and rhodamine 110, which do not cover a wide range
of detection wavelengths in the visible and near-infrared
spectra. To try and solve the aforementioned limitation (1),
Jones et al.8 successfully described the use of a reactive linker
for the design of an image contrast agent selectively activated
by the serine protease PSA (i.e., prostate specific antigen),
but the approach turned out to be only applicable to amine-
based fluorophores.
We report here the synthesis and evaluation of three such
water-soluble self-immolative spacers derived from the
original N-acylhemithioaminal core structure 2 and their
application for the preparation of latent fluorescent probes
aimed at the detection of a model protease, penicillin amidase
(also known as penicillin G acylase, PGA).
In the design of the traceless linker, the biocatalyzed
transformation (i.e., the enzymatic hydrolysis of a carboxa-
mide bond) is thus combined with a domino reaction
involving fragmentation of the hemithioaminal 5 and sub-
sequent self-cyclization of the resulting thioalkyl carbonate
6, yielding to the release of the phenol derivatives (Figure
1). To validate the postulated mechanism of this cascade
reaction and to check its efficacy under physiological
conditions, we have chosen to work with PGA, a com-
mercially available and widely used biocatalyst in the
enzymatic synthesis of â-lactam antibiotics, since it allows
for the deprotection of phenacetyl-protected amines13 and
the subsequent release of umbelliferone (i.e., 7-hydroxycou-
marin),5 at the blue fluorescent phenolic fluorophore. Thus,
it was possible to follow the enzymatic hydrolysis and the
following decomposition of the self-immolative spacer by
means of a simple and rapid in vitro fluorescence assay. In
addition to these structural features, the low water solubility
of first generation probes drove us to design fluorogenic
probes 3 and 4, bearing an additional carboxyl group on the
aromatic part of the masked benzyl hemithioaminal.
Thus, there is a growing need for original self-immolative
spacers that enable the reversible conjugation of a peptide
substrate to a fluorophore bearing either a reactive amino,
hydroxyl, or thiol group. In that context, we want to describe
herein our efforts to design and evaluate a new self-
immolative linker.
The design of the linker was based on the improvement
of the enzyme-labile linker 1 initially developed by Bo¨hm
et al. for the reversible covalent attachment of alcohols to
solid supports.9 As the chemical derivatization of the
activated form of this linker (i.e., benzotriazole derivative)
with aniline, phenol, or thiophenol derivatives is tricky and
leads to unstable conjugates, we chose to introduce stable
(10) Lapeyre, M.; Leprince, J.; Massonneau, M.; Oulyadi, H.; Renard,
P.-Y.; Romieu, A.; Turcatti, G.; Vaudry, H. Chem. Eur. J. 2006, 12, 3655-
3671.
(7) Ho, N.-H.; Weissleder, R.; Tung, C.-H. ChemBioChem 2007, 8, 560-
566. Lavis, L. D.; Chao, T.-Y.; Raines, R. T. ChemBioChem 2006, 7, 1151-
1154. Lavis, L. D.; Chao, T.-Y.; Raines, R. T. ACS Chem. Biol. 2006, 1,
252-260. Chandran, S. S.; Dickson, K. A.; Raines, R. T. J. Am. Chem.
Soc. 2005, 127, 1652-1653.
(8) Jones, G. B.; Crasto, C. F.; Mathews, J. E.; Xie, L.; Mitchell, M. O.;
El-Shafey, A.; D’Amico, A. V.; Bubley, G. J. Bioorg. Med. Chem. 2006,
14, 418-425.
(9) Bo¨hm, G.; Dowden, J.; Rice, D. C.; Burgess, I.; Pilard, J.-F.; Guilbert,
B.; Haxton, A.; Hunter, R. C.; Turner, N. J.; Flitsch, S. L. Tetrahedron
Lett. 1998, 39, 3819-3822.
(11) Jones, L. R.; Goun, E. A.; Shinde, R.; Rothbard, J. B.; Contag, C.
H.; Wender, P. A. J. Am. Chem. Soc. 2006, 128, 6526-6527.
(12) El Alaoui, A.; Schmidt, F.; Amessou, M.; Sarr, M.; Decaudin, D.;
Florent, J.-C.; Johannes, L. Angew. Chem., Int. Ed. 2007, 46, 6469-6472.
Vlahov, I. R.; Santhapuram, H. K. R.; Kleindl, P. J.; Howard, S. J.; Stanford,
K. M.; Leamon, C. P. Bioorg. Med. Chem. Lett. 2006, 16, 5093-5096.
Henne, W. A.; Doorneweerd, D. D.; Hilgenbrink, A. R.; Kularatne, S. A.;
Low, P. S. Bioorg. Med. Chem. Lett. 2006, 16, 5350-5355.
(13) Ninkovic, M.; Riester, D.; Wirsching, F.; Dietrich, R.; Schwienhorst,
A. Anal. Biochem. 2001, 292, 228-233.
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Org. Lett., Vol. 10, No. 8, 2008

selected since it was proven to be stable to the acidic and
basic conditions encountered during the synthesis of 3 and
4 and could be selectively removed by treatment with Pd-
(PPh3)4 and dimedone without affecting the acid-sensitive
N-acylhemithioaminal moiety and the fragile carbonate
derived from phenol-containing fluorophores. The benzot-
riazole derivative 10 was prepared in 63% yield by refluxing
a mixture of aldehyde 9, benzotriazole, and phenylacetamide
in dry toluene for 3 days. Reaction of 10 with the sodium
salt of O-(tert-butyldiphenylsilyl)-protected derivative of
â-mercaptoethanol 11 or 3-sulfanylpropanol 12 in dry ethanol
afforded the full-protected hemithioaminals 13 and 14 in 93%
and 30% yields, respectively.15 The modest yield obtained
in the case of the preparation of compound 14 was explained
by a competitive transesterification of the allyl ester. Removal
of the tert-butyldiphenylsilyl (TBDPS) group was achieved
under standard conditions by treatment of 13 and 14 with a
2-fold excess of tetrabutylammonium fluoride (TBAF) in
THF to give the corresponding alcohols 15 and 16. Activation
of alcohols 15 and 16 with N,N′-disuccinimidyl carbonate
(DSC) in dry acetonitrile in the presence of triethylamine
afforded carbonates 17 and 18. Their reaction with the
sodium salt of umbelliferone gave the non water-soluble
coumarin-based fluorogenic probes 19 and 20 in good yields.
Finally, the allyl esters of 19 and 20 were removed under
palladium catalysis in the conditions reported by Zhang et
al.16 The use of a weak nucleophile (i.e., a 3:2 (mol/mol)
mixture of dimedone/benzylamine) as the allyl scavenger was
required to minimize the undesirable nucleophile-mediated
cleavage of the sensitive carbonate moiety. Purification was
achieved by semipreparative RP-HPLC to give the resulting
water-soluble PGA sensitive probes 3 and 4 in moderate
yields (33% for 3 and 10% overall yield for the last three
steps for 4). All spectroscopic data (see the Supporting
Information), especially NMR and mass spectrometry, were
in agreement with the structures assigned.
Figure 1. Principle of the original self-immolative linker studied
in this work and its application to the release of phenol-based
fluorophores after carboxamide bond cleavage.
The synthesis of self-immolative spacers and their chemi-
cal derivatization with the fluorescent reporter group was
achieved as depicted in Scheme 1. First, the enzyme
Scheme 1. Synthesis of Fluorogenic Probes 3 and 4a
Fluorogenic probes 3 and 4 were evaluated under simu-
lated physiological conditions (PBS, pH 7.5, see Figure 2
and the Supporting Information). As expected, profluorescent
probe 3 exhibited no significant fluorescence emission at 458
nm (diminished by 30-fold compared to the fluorescence of
free umbelliferone at the same concentration, see Figure 2A).
Figure 2B showed the time-course for the enzyme catalyzed
hydrolysis of 3. After adding recombinant PGA to the
substrate solution, a strong fluorescence signal generated at
460 nm indicated the catalytic cleavage of the carboxamide
bond and the release of free umbelliferone. The maximum
fluorescence intensity reflecting the quantitative conversion
of 3 into 7-hydroxycoumarine was reached after about 10
min. When substrate 3 was incubated in PBS only, a slow
linear increase in fluorescence intensity was observed,
corresponding to the nonenzymatic hydrolysis of its carbon-
ate moiety. The moderate stability of monoaryl carbonates
derived from â-mercaptoethanol in aqueous buffers has
already been observed by Jones et al. in the context of
a Overall yield for the three last steps because the synthesis of
fluorogenic probe 4 was performed in a small scale without isolation
of intermediates 18 and 20 in a pure form.
recognition unit (i.e., phenylacetamide moiety) was intro-
duced onto the activated form of the linker by using a
methodology developed by Katritzky et al.14 The three-
component reaction was performed with the allyl ester of
4-carboxybenzaldehyde 9: the allyl protecting group was
(15) Katritzky, A. R.; Takahashi, I.; Fan, W. Q.; Pernak, J. Synthesis
1991, 1147-1150.
(16) Zhang, H. X.; Guibe´, F.; Balavoine, G. Tetrahedron Lett. 1988, 29,
623-626.
(14) Katritzky, A. R.; Drewniak, M.; Lue, P. J. Org. Chem. 1988, 53,
5854-5856.
Org. Lett., Vol. 10, No. 8, 2008
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moieties and the N-methyl-N-[2-(methylamino)ethyl] car-
bamate linker is one of the most popular self-immolative
spacer for the release of alcohols and phenols especially in
the context of prodrugs.17 In vitro fluorescence assay and
LC-MS analyses (see the Supporting Information) showed
that 21 is not prone to nonspecific hydrolysis and was
suitable for PGA sensing, although the kinetics of the enzyme
triggered domino reaction was significantly slowed down.
In summary, we have designed and synthesized a novel
self-immolative spacer system that allowed the release of
phenol-containing compounds through an original two-step
cascade reaction triggered by the enzymatic formation of a
free amine. The potential utility of this traceless linker
strategy in bioconjugate chemistry was illustrated by the
preparation of three fluorogenic probes aimed at the detection
of the activity of a model enzyme penicillin amidase.
Interestingly, the water-solubilizing carboxyl group of these
acylhemithioaminal derivatives could be alternatively used
for anchoring a targeting ligand suitable for guiding the
profluorophore or the prodrug to the tumor-expressed pro-
teolytic enzyme, aimed at designing optical bioprobes or drug
delivery systems suitable for cancer imaging and chemo-
therapy.18
Figure 2. (A) Emission spectra of (ex λ ) 323 nm) of umbellifer-
one (-) and fluorogenic probe 3 (- - -) at 25 °C in PBS (concentra-
tion 2.4 µM). (B) Fluorescence emission time-course of probe 3
(concentration 3.0 µM) with recombinant PGA (0.12 U, incubation
time 30 min, 37 °C) in PBS buffer at 460 nm (ex λ ) 360 nm).
releasable luciferin-transporter conjugates11 and could be
partially circumvented by the lengthening of the alkyl chain
between the sulfur atom and the carbonate group. Indeed,
when the fluorogenic probe 4 derived from 3-sulfanylpro-
panol was incubated in PBS, a slow linear increase in
fluorescence intensity at 460 nm was also observed but the
nonspecific hydrolysis rate decreased significantly compared
to 3 (ratio between slopes 4/3: 0.7), whereas the specific
enzymatic cleavage showed equivalent kinetics (see the
Supporting Information).
In addition to these spectral measurements, LC-MS
analyses of the enzymatic hydrolysis mixture of 3 were
performed to confirm the enzyme-initiated self-decomposi-
tion mechanism of the N-acylhemithioaminal linker and
proved both the identity of the released compounds 4-car-
boxybenzaldehyde (tR ) 6.4 min, [M - H]- ) 149.11) and
7-hydroxycoumarin (tR ) 14.3 min, [M - H]- ) 161.09)
and the efficacy of this release process (see the Supporting
Information).
Acknowledgment. This work was supported by La
Re´gion Haute-Normandie and QUIDD. We thank Christophe
Portal (QUIDD) and Freddy Rivault (QUIDD) for careful
reading and English corrections of the manuscript, POLY-
INTELL company for the open access to their LC-MS
apparatus and Dr. Ce´line Pe´rollier (Project Leader, POLY-
INTELL) for her technical assistance during LC-MS
analyses.
Supporting Information Available: Procedures and
additional data for syntheses and analyses reported herein.
This material is available free of charge via the Internet at
http://pubs.acs.org.
To further improve the stability of enzyme-activable
conjugates bearing the self-immolative N-acylhemithioaminal
2 toward nonspecific hydrolysis, we also explored the
synthesis of an elongated PGA fluorogenic probe 21 that
contained a well-known bis-amine cyclization spacer (see
the Supporting Information). Indeed, carbamate linkages are
known to be more stable than the corresponding carbonate
OL800198F
(17) Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.; Engelhardt,
E. L. J. Med. Chem. 1990, 33, 97-101.
(18) For an example of such drug delivery system, see: Gopin, A.;
Pessah, N.; Shamis, M.; Rader, C.; Shabat, D. Angew. Chem., Int. Ed. 2003,
42, 327-332.
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Org. Lett., Vol. 10, No. 8, 2008