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.
Richard
Jean-Alexandre
JA
UMR CNRS 6014, COBRA, IRCOF, rue Lucien Tesnière, 76130 Mont-Saint-Aignan, France.
Meyer
Yves
Y
Jolivel
Valérie
V
Massonneau
Marc
M
Dumeunier
Raphaël
R
Vaudry
David
D
Vaudry
Hubert
H
Renard
Pierre-Yves
PY
Romieu
Anthony
A
D010129:4-Aminobenzoic Acid / Q000737:chemistry*; D053148:Caspase 3 / Q000378:metabolism; D003116:Color; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010399:Penicillin Amidase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010447:Peptide Hydrolases; D053148:Caspase 3; D010399:Penicillin Amidase; D010129:4-Aminobenzoic Acid