Intramolecular pyrophosphate formation during N^α-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of peptides containing adjacent phosphotyrosine residues

The derivative N^α-9-fluorenyimethyloxycarbonyl-O-phospho-L-tyrosine [FmocTyr(PO₃H₂)-OH] has been used successfully for the solid-phase synthesis of a wide varietv of phosphorylated peptides. However, when it is used to incorporate consecutive phosphotymsine residues, apyrophosphate linkage can form between the Two adjacent tyrosines. Incorporation of unprotected phosphotyrosine during the synthesis of peptides with multiple phosphotymsine residues has been studied as a function of coupling conditions and the absence or presence of intervening amino acid residues. The pyrophosphate-forming side reaction is more severe with increased coupling times and/or repetitions of coupling and occurs only when the phosphotyrosine residues are directh adjacent to one another.