Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation

Abstract

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS-R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.

Keywords: FRET; fluorescent P-hydroxyphospholes; fluorescent labelled GHSR ligands; peptide coupling; phospholamide.