title: Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates.
abstract: A general synthetic method for the efficient preparation of Tyr(P)-containing peptides is described by the use of Fmoc-Tyr(PO3tBu2)-OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P)-Tyr(P)-OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8-13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using 32P-radiolabeled [Tyr(P)]4-angiotensin II, both unlabeled synthetic [Tyr(P)]4-angiotensin II and Ser-Ser-Ser-Tyr(P)-Tyr(P) reduced the release of 32P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]4-neurotensin 8-13 was ineffective as a competitive substrate and indicated that this particular Tyr(P)-containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P)-Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P)-Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.
authors.affiliation: Biochemistry and Molecular Biology Unit, School of Dental Science, University of Melbourne, Victoria, Australia.
authors.forename: J W
authors.initials: JW
authors.lastname: Perich
authors.forename: M
authors.initials: M
authors.lastname: Ruzzene
authors.forename: L A
authors.initials: LA
authors.lastname: Pinna
authors.forename: E C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives*
chemical_list: D000596:Amino Acids; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D010744:Phosphoric Monoester Hydrolases