Efficient Fmoc/solid‐phase peptide synthesis of O‐phosphotyrosyl‐containing peptides and their use as phosphatase substrates

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Abstract
A general synthetic method for the efficient preparation of Tyr(P) ‐containing peptides is described by the use of Fmoc‐Tyr(PO31Bu2) ‐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 un‐labeled 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.

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