Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO₃R₂)-OH derivatives

Summary

The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a ‘large-scale’ three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, ‘phosphite-triester’ phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.