Synthesis of phosphopeptides by the Multipin method:: Evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH

The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiester-protected derivatives, Fmoc-Tyr(PO(3)Bzl,H)-OH, Fmoc-Ser(PO(3)Bzl,H)-OH and Fmoc-Thr(PO(3)Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu-Glu-Asp-Pro-Ala-NH2 (Xxx = Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9) DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIG-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx(PO(3)Bzl,H)OH derivatives. In the subsequent synthesis of the alpha-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr-His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Sr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO(3)Bzl,H)-OH.