A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation

• Published in: Organic & biomolecular chemistry 2008-09, Vol.6 (17), p.3065-3078
• Main Authors: Clavé, Guillaume, Boutal, Hervé, Hoang, Antoine, Perraut, François, Volland, Hervé, Renard, Pierre-Yves, Romieu, Anthony
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid; Cross-Linking Reagents - chemical synthesis; Cross-Linking Reagents - chemistry; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemistry; Fluoroimmunoassay - methods; Substance P - chemistry; Substance P - immunology; Substance P - metabolism; Sulfhydryl Compounds - chemistry
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/b807263a

A convenient, versatile and straightforward synthesis of a novel heterotrifunctional peptide-based linker molecule is described. This generic bio-labelling reagent contains an amine-reactive N-hydroxysuccinimidyl carbamate moiety, an aldehyde/ketone-reactive aminooxy group and a thiol group with a propensity to form urea, oxime and thioether linkages respectively. The full chemical orthogonality between the free aminooxy and thiol functionalities was demonstrated through the preparation of a fluorescent reagent suitable for the selective staining of a carboxaldehyde-modified surface by means of oxime ligation. The absence of reactivity of these two functions toward the nucleophile-sensitive active carbamate was obtained by using temporary aminooxy- and thiol-protecting groups removable under mild conditions. The utility of the linker molecule to cross-link three different molecular partners has been illustrated by the preparation of fluorescent tripod-functionalised surfaces which may be useful in developing new peptide microarrays and related immunosensors.