Identification and Suppression of β-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis

The formation of 3-(1-piperidinyl)alanyl-containing peptides via phosphoryl beta-elimination was identified from the application of Fmoc-Ser(PO(3)Bzl,H)-OH in peptide synthesis as shown by RP-HPLC, ES-MS and P-31-NMR analysis. An N-alpha-deprotection study using the model substrates, Fmoc-Xxx(PO(3)Bzl,H)-Val-Glu((OBu)-Bu-t)-Resin (Xxx = Ser, Thr or Tyr) demonstrated that piperidine-mediated phosphoryl beta-elimination occurred in the N-terminal Ser(PO(3)Bzl,H) residue at a ratio of 7% to the target phosphopeptide, and that this side reaction did not occur in the corresponding Thr(PO(3)Bzl,H)- or Tyr(PO(3)Bzl,H)- residues. The generation of 3-(1-piperidinyl) alanyl-peptides was also shown to be enhanced by the use of microwave radiation during Fmoc deprotection. An examination of alternative bases for the minimization of byproduct formation showed that cyclohexylamine, morpholine, piperazine and DBU gave complete suppression of beta-elimination, with a 50% cyclohexylamine/DCM (v/v) deprotection protocol providing the crude peptide of highest purity. Piperidine-induced beta-elimination was found only to occur in Ser(PO(3)Bzl,H) residues that were in the N-terminal position, since the addition of the next residue in the sequence rendered the phosphoseryl residue stable to multiple piperidine treatments. The application of the alternative N-alpha-deprotection protocol using 50% cyclohexylamine/DCM (v/v) is therefore recommended for deprotection of the Fmoc group from the Fmoc-Ser(PO(3)Bzl,H) residue, with particular benefit anticipated for the synthesis of multi-phosphoseryl peptides.