RESUMEN
Two phosphoserine tetradecapeptides corresponding to sequences 987-1000 (peptide pSer994) and 1017-1030 (peptide pSer1023/1025) from the human insulin receptor involved in the regulation of its activity were successfully synthesized using Fmoc-based chemistry. Phosphorylation was performed by post-assembly phosphitylation followed by oxidation. The selective phosphorylation of Ser residues was achieved incorporating into the peptide chain the Ser (Trt) derivative and t-Bu blocking groups at sites other than those intended to be phosphorylated. The Trt group was selectively removed with dichloroacetic acid while under this condition t-Bu protecting groups remained unaltered. Following conjugation to keyhole limpet hemocyanin phosphopeptides were used as immunogens to generate sequence-specific phosphoserine antibodies. Peptide pSer994 induced antibodies in New Zealand white rabbits which discriminated between the phosphorylated and nonphosphorylated forms of the peptide, thus representing promising candidates to recognize signaling pathways associated to the regulation of the human insulin receptor.
Asunto(s)
Fosfopéptidos/síntesis química , Fosfopéptidos/inmunología , Fosfoserina/inmunología , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fosfopéptidos/genética , Fosforilación , Fosfoserina/química , Conejos , Receptor de Insulina/genética , Receptor de Insulina/inmunología , Serina/química , Serina/metabolismoRESUMEN
We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.