Marc Massonneau

7-hydroxycoumarin-hemicyanine hybrids: a new class of far-red emitting fluorogenic dyes.

The design and synthesis of novel water-soluble far-red emitting phenol-based fluorophores derived from 7-hydroxycoumarin are described. These hemicyanine-coumarin hybrids display promising spectroscopic features such as large apparent Stokes shift (ranging from 60 to 140 nm) and fluorescence emission maxima between 620 and 720 nm in physiological conditions. Their utility was then illustrated by the preparation of an original fluorogenic probe of penicillin G acylase (PGA) whose fluorescence is unveiled through an enzyme-initiated domino reaction.

Development of a New Nonpeptidic Self-Immolative Spacer. Application to the Design of Protease Sensing Fluorogenic Probes

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.

Latent Fluorophores Based on a Self-Immolative Linker Strategy and Suitable for Protease Sensing†

The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9 H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.

Distribution and functional characterization of pituitary adenylate cyclase–activating polypeptide receptors in the brain of non-human primates

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

A novel and unusually long-lived chemiluminophore based on the 7-hydroxycoumarin scaffold.

Synthesis and chemiluminescent properties of a new 1,2-dioxetane chemiluminophore bearing a 7-hydroxycoumarin moiety are presented. The 1,2-dioxetane decomposition ended up with strong and long-lived emission of light. This new structure opens way to the development of a new generation of bright chemiluminescent bio-probes.

In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis

Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo.

Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.