Chemiluminescent probe for the in vitro detection of protease activity

Article abstract of DOI:10.1021/ol702190y

(Chemical Equation Presented) A strategy involving the use of a self-immolative linker has been investigated for the chemiluminescent sensing of proteases. The reactive linker enabled the release of a 1,2-dioxetane light precursor. As a proof of principle

Full text of DOI:10.1021/ol702190y

ORGANIC
LETTERS
2
007
Vol. 9, No. 23
853-4855
Chemiluminescent Probe for the in Vitro
Detection of Protease Activity
4
Jean-Alexandre Richard,†,‡ Ludovic Jean, Anthony Romieu,*
‡
,†
‡
‡
,†
Marc Massonneau, Pauline Noack-Fraissignes, and Pierre-Yves Renard*
Equipe de Chimie Bio-Organique, UniVersit e´ de Rouen, INSA de Rouen, CNRS UMR
014, COBRA, IRCOF, 1 rue Lucien Tesni e` re, 76131 Mont-Saint-Aignan Cedex,
6
France, and QUIDD, 50 rue Ettore Bugatti, Technop oˆ le du Madrillet 76800
Saint-Etienne du RouVray Cedex, France
pierre-yVes.renard@uniV-rouen.fr
Received September 6, 2007
ABSTRACT
A strategy involving the use of a self-immolative linker has been investigated for the chemiluminescent sensing of proteases. The reactive
linker enabled the release of a 1,2-dioxetane light precursor. As a proof of principle, caspase-3, a key peptidase involved in apoptosis has
been targeted. An in vitro assay has been carried out and proved the decomposition of the linker and the selectivity for caspase-3.
Chemiluminescence is defined as the electromagnetic radia-
tion (ultraviolet, visible, or infrared) produced when a
chemical reaction yields an electronically excited inter-
mediate or product, which either luminesces (direct chemi-
luminescence) or donates its energy to another molecule
responsible for the emission (indirect or sensitized chemi-
luminescence). Oxalate esters, luminol, acridinium esters, and
fication of target analytes in tissue selections offered by
chemiluminescence as compared to those by fluorescence,
chemiluminescent probes have been also used for the
3
development of immuno- and immunohistoassays. In order
to widen the scope of chemiluminescent probes, our interest
is focused on the development of chemiluminescent assays
for new classes of enzymes. We have thus previously
reported the design and synthesis of a chemiluminescent
probe for the detection of acetylcholinesterase activity using
1
,2-dioxetanes are well-known chemiluminescent species
1
emitting light following a chemical reaction. The advantage
of using chemiluminescence in bioanalytical assays is
associated with a rapid and sensitive detection of the target
through reduction of the background noise thanks to the
absence of a photonic excitation. Thus, chemiluminescent
probes, especially those derived from 1,2-dioxetanes 1, have
been designed for the highly sensitive detection of various
enzymes such as alkaline phosphatase, â-galactosidase,
4
an unprecedented thiophenol trigger. We report here our
first investigations regarding the tricky detection of proteases.
Despite recent advances in the development of chemi-
5
luminescent probes, the detection of proteases still remains
challenging. The issue mainly lies in the difficulty to obtain
an efficient light signal following the cleavage of the peptide
2
(2) Matsumoto, M. J. Photochem. Photobiol., C 2004, 5, 27-53 and
references therein.
neuramidinase. Taking into account the versatility, the
sensitivity, and the higher specific localization and quanti-
(
3) Kricka, L. J. Anal. Chim. Acta 2003, 500, 279-286.
(4) Sabelle, S.; Renard, P.-Y.; Pecorella, K.; de Suzzoni-Dezard, S.;
†
Equipe de chimie Bio-Organique.
QUIDD (QUantitative Imaging in Drug Development).
Creminon, C.; Grassi, J.; Mioskowski, C. J. Am. Chem. Soc. 2002, 124,
4874-4880.
(5) For a recent example, see Hewage, H. S.; Wallace, K. J.; Anslyn, E.
V. Chem. Commun. 2007, 3909-3911.
‡
(
1) Beck, S.; Koster, H. Anal. Chem. 1990, 62, 2258-2270; Erratum
1
991, 63, 848.
1
0.1021/ol702190y CCC: $37.00
© 2007 American Chemical Society
Published on Web 10/16/2007

bond. 1,2-Dioxetanes indeed usually emit light through
spontaneous decomposition of a compound such as 2, bearing
a negatively charged atom onto an aromatic ring bearing the
far,10 none of them is based on chemiluminescence. More-
over, other caspases have a close substrate profile as
compared to casapse-3; thus, providing that the probe shows
a high sequence selectivity, this strategy should allow the
construction of a flexible set of caspase chemiluminescent
substrates. This enzyme thus appeared to be an interesting
case study before extending our device to other proteases.
Hence, the linker 8 was introduced onto the DEVD (for
Asp-Glu-Val-Asp) peptide sequence 7 which is specifically
recognized by caspase-3 and cleaved after the Asp residue
1,2-dioxetane, via a mechanism chemically initiated electron
exchange luminescence (CIEEL). 2 can be, in the case of
enzymes probes, formed specifically through the enzymatic
cleavage of 1 (Figure 1). One possible approach for the
1
1
at the C-terminal side. 7 was prepared in a highly efficient
3-step procedure by solution peptide synthesis using a Boc
1
strategy (93% mean yield for each step, see Supporting
Information [SI]). In order to have an orthogonal set of
protective groups, the three carboxylic acids of peptide 7
were protected as 2-trimethylsilylethyl (TMSE) esters,12
easily removed by treatment with fluoride ion sources under
mild conditions, shown to be compatible with the 1,2-
dioxetane structures.
Figure 1. Enzymatic activation of 1 leading to 2 whose decom-
position triggers light emission.
detection of proteases would thus be to connect the C-
terminus of a peptide sequence directly to the aromatic
moiety (i.e., X ) NH). However, the amount of light emitted
by the release of aniline is by far much weaker than that by
Then, benzylic alcohol 9 was converted to its mesylate
derivative, and enol ether 10, prepared from 3-hydroxy-
benzoic acid methyl ester in three steps according to liter-
1
3
ature procedures, was incorporated on the C-terminus of
6
the phenol or thiophenol equivalent (i.e., X ) O or S).
peptide-benzyl alcohol 9 (Scheme 1).
Thus, inspired by the chemistry of prodrugs widely used
7
in medicinal chemistry for drug delivery applications, we
designed probe 3 which incorporates a reactive linker able
to transfer the amide bond-breaking event to a phenolate
release (Figure 2). p-Aminobenzyl alcohol 8 (PABA) was
Scheme 1. Synthesis of Chemiluminescent Probe 12
Figure 2. Self-immolative linker strategy for the release of the
phenolate moiety 5 after peptide bond cleavage.
Finally, compound 11 underwent the key step of the
synthesis: a [2+2] cycloaddition between enol ether 11 and
singlet oxygen ( O ). The formation of the 1,2-dioxetane
2
derivative was checked by RP-HPLC, and the compound was
subsequently isolated in a pure form by semipreparative RP-
HPLC. Final deprotection with tetrabutylammonium fluoride
1
4
8
chosen as a self-immolative linker. Thus, aniline 4, formed
by the amide bond cleavage, should spontaneously decom-
pose to release phenolate 5, leading to the excited compound
6, which returns to the ground state through light emission.
As a proof of concept, we applied our device to the sensing
(
7) (a) Damen, E. W. P.; Nevalainen, T. J.; van den Bergh, T. J. M.; de
of caspase-3, an effector peptidase which plays a crucial role
Groot, F. M. H.; Scheeren, H. W. Bioorg. Med. Chem. 2001, 10, 71-77.
(b) de Groot, F. M. H.; Loos, W. J.; Koekkoek, R.; van Berkom, L. W. A.;
Busscher, G. F.; Seelen, A. E.; Albrecht, C.; de Bruijn, P.; Scheeren, H.
W. J. Org. Chem. 2001, 66, 8815-8830. (c) Amir, R. J.; Pessah, N.; Shamis,
M.; Shabat, D. Angew. Chem., Int. Ed. 2003, 42, 4494-4499. (d) Haba,
K.; Popkov, M.; Shamis, M.; Lerner, R. A.; Barbas, C. F., III; Shabat, D.
Angew. Chem., Int. Ed. 2005, 44, 716-720. (e) Amir, R. J.; Danieli, E.;
Shabat, D. Chem. Eur. J. 2007, 13, 812-821. (f) Shamis, M.; Shabat, D.
Chem. Eur. J. 2007, 13, 4523-4528.
9
in the last steps of the apoptotic process. Although many
fluorescent caspase-3-activity probes have been reported so
(
6) (a) Bronstein, I.; Edwards, B.; Martin, C.; Sparks, A.; Voyta, J. C.
Tropix, Inc., U.S.A.). WO 9624849, 1996. (b) Watanabe, N.; Ichikawa,
M.; Ono, A.; Murakami, H.; Matsumoto, M. Chem. Lett. 2005, 34, 718-
19.
(
7
4854
Org. Lett., Vol. 9, No. 23, 2007

(TBAF) led to chemiluminescent probe 12 after purification
by semipreparative RP-HPLC. The structure of this chemi-
luminescent caspase-3 substrate was confirmed by ESI mass
spectrometry (see SI).
With this tool in hand, we investigated the efficiency of
our device against recombinant human caspase-3. As the
chemiluminescence of a 1,2-dioxetane happens to be less
14
efficient in an aqueous media compared to organic solvents,
specific enhancers (namely 5-(stearoylamino)fluorescein
emission wavelength ) 530 nm) as well as a surfactant
(
cetyltrimethylammonium bromide [CTAB]) were added to
the medium. Thus, caspase-3-mediated cleavage of probe 12
was carried out following a classical final time detection
procedure, and a significant light emission at 530 nm could
be observed (see SI). The light was persistent for more than
10 min, corresponding to a “glow” chemiluminescence which
is known to allow a more sensitive detection than a “flash”
1
emission. The long-lived time course of this light emission
is thus an interesting feature for further biological assays
(Figure 3A). The maximum of light emission was also
represented as a function of time, thus enabling us to follow
the kinetics of the enzymatic reaction (Figure 3B).
Finally, the caspase-3 detection limit in this nonoptimized
assay format was estimated by decreasing the amount of
enzyme until no more light could be significantly detected
(see SI). Hence, an encouraging detection limit of 1.31 pmol
of enzyme was determined (Figure 3C). Furthermore, control
reactions in which probe 12 was incubated with caspase-3
buffer alone, with penicillin amidase (penicillin G acylase),
or with other initiator caspases were undertaken. In these
conditions, no emission of light was detected, showing that
neither spontaneous hydrolysis nor nonspecific enzymatic
cleavage of 12 occurred (see SI).
Figure 3. (A) Luminescence intensity (area under the emission
curve in the range 490-570 nm) recorded every 9 s with probe 12
-
3
after incubation with recombinant human caspase-3 (1.6 10 U,
incubation time 105 min). (B) Maximum light emission of probe
12 as a function of the incubation time with recombinant human
In conclusion, we have designed and synthesized an
efficient chemiluminescent probe suitable for the in vitro
detection of protease activity. A strategy involving the use
of a self-immolative spacer 8 (PABA) was developed to
release the light precursor phenolate 5. An application of
this device was undertaken, and the first chemiluminescent
caspase-3 probe 12 was synthesized. An in vitro assay
enabled the detection of a “glow” luminescence suitable for
caspase-3. (C) Determination of the detection limit of caspase-3.
further biological applications. Moreover, preliminary assays
proved that the probe was selective and exhibited a detection
limit of around 1 pmol. This new tool constitutes a great
innovation in the field of protease-sensing assays and optical
bioprobes. Currently, work is in progress in our laboratory
to extend this device to other proteases of biological interest.
(
8) Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. J. Med. Chem.
1
981, 24, 479-480.
Acknowledgment. This work was supported by La
R e´ gion Haute-Normandie and QUIDD. The contribution of
Alicia Foucourt (CNRS UMR 6014) and Yves Meyer
(
9) Denault, J.-B.; Salvesen, G. S. Chem. ReV. 2002, 102, 4489-4499.
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Bernardin, A.; Chipon, B.; Massonneau, M.; Renard, P.-Y.; Romieu, A.
Bioconjugate Chem. 2007, 18, 1303-1317 and references 55-58 therein.
(QUIDD) to the total synthesis of tetrapeptide 7 is greatly
(11) (a) Philchenkov, A. A. Biochemistry (Moscow) 2003, 68, 365-376.
acknowledged. We thank Dr. J e´ r oˆ me Leprince (U413 IN-
SERM/IFRMP 23) for MALDI-TOF mass measurements and
Annick Leboisselier (IRCOF) for the determination of
elemental analyses.
(
b) Fuentes-Prior, P.; Salvesen, G. S. Biochem. J. 2004, 384, 201-232.
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L. F. M. L.; Weiss, D.; Beckert, R.; Baader, W. J. Synthesis 2006, 1781-
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.
1
786. (c) Renard, P.-Y.; Romieu, A.; Massonneau, M. (Quidd, Fr.;
Universite De Rouen). WO 2886292, 2006.
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