CHEMICAL SYNTHESIS OF PHOSPHORYLATED PEPTIDES OF THE CARBOXY-TERMINALDOMAIN OF HUMAN P53 BY A SEGMENT CONDENSATION METHOD

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce pho sphorylated and non-phosphorylated derivatives of the C-terminal tetra merization and regulatory domains of human p53 (residues 303-393). Eff icient condensation synthesis of the 91 residue p53 domain was achieve d in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the nonphosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc c hemistry. The C-terminal segment p53(361-393) (3) and its derivative p hosphorylated at serine 392 (3P392) were synthesized as partially prot ected peptides in the solid phase using Fmoc chemistry. Phosphoamino a cid was incorporated into the N-terminal segment (1P315) at the residu e corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)(2))-OH during sy nthesis. Serine 392 in the C-terminal segment was selectively phosphor ylated after synthesis by phosphitylation followed by oxidation. A der ivative phosphorylated at serine 378 was synthesized in a one-step con densation of the unphosphorylated N-terminal segment (1) and the phosp horylated long C-terminal segment p53(335-393) (2-3P378). Yields of th e ligated peptides after removal of the protecting groups and HPLC pur ification averaged 60% for the first condensation and 35% for the seco nd condensation. All five p53 peptides exhibited monomer-tetramer asso ciation as determined by analytical ultracentrifugation. Circular dich roism spectroscopy revealed that phosphorylation at Ser315 increased t he alpha-helical content, which was abolished when Ser392 also was pho sphorylated, suggesting an interaction between N-terminal and C-termin al residues of the C-terminal domain of p53. (C) Munksgaard 1996.