Solid phase synthesis of pp60src-related phosphopeptides via ‘global’ phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2 Original Research Article

The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60src protein kinase family, Asn-Glu-Tyr(P)-Ser-Ala, Ala-Glu-Tyr(P)-Ser-Ala, Ala-Ser-Tyr(P)-Ser-Ala, Ala-Ser(P)-Tyr-Ser-Ala, Ala-Thr-Tyr(P)-Ser-Ala, Ala-Thr(P)-Tyr-Ser-Ala and Ala-Ser(P)-Tyr(P)-Ser-Ala, were prepared in good yield using the ‘global’ ‘phosphite-triester’ phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP® coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final ‘global’ phosphorylation of the peptide resins was accomplished using di-tert-butyl N,N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di-t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr(P) residue in the −1 position was significantly more important than the Ser(P)/Thr(P) residue in the −2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the −2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr(P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser(P), Thr(P), Glu}. In addition to the Tyr(P) residue directing phosphorylation to the + 1 position, the good phosphorylation of both ASY(P)SA and ATY(P)SA by casein kinase-2 indicated that the Tyr(P) residue was also able to direct phosphorylation to a Ser/Thr in the −1 position.