Backbone protection and its application to the synthesis of a difficult phosphopeptide sequence

Abstract

The N-(2-hydroxy-4-methoxybenzyl)(Hmb) backbone amide-protecting group has been applied to the synthesis of phosphopeptides via post-assembly global phosphorylation. Reversible protection of the Hmb 2-hydroxy moiety was mandatory in order to prevent its phosphorylation and resultant irreversible stabilisation to acidolysis. This was achieved through the use of either the acetyl (Ac) or allyloxycarbonyl (Allot) groups, introduced through their respective anhydrides in the presence of tertiary base. Following global phosphorylation, Ac or Alloc could be removed from resin-bound Hmb backbone-substituted fully protected phosphopeptide by either hydrazinolysis or palladium-catalysed cleavage to re-establish Hmb acid-lability. Similarly, hydrazinolysis in solution of otherwise deprotected, backbone-substituted phosphopeptide was found to be efficient and free from phosphoroamino acid β-elimination side-reactions. The optimised protocols were applied in the preparation of peptides from human Tau [390–406; Ser(PO₃H)³⁹⁶] and the MAP kinase ERK 2 [178–188; Thr(PO₃H)¹⁸³; Tyr(PO₃H)¹⁸⁵].