Chemical synthesis of o-thiophosphotyrosyl peptides

The synthon for O-thiophosphotyrosine, Fmoc-TyrPS(OBzl)₂]-OH ( 1c ), was prepared in 63%;, yield from Fmoc-Tyr-OH by first transient protection as the ^tBuMe₂Si-ester and phosphinylation with (BzlO)₂PNⁱPr₂/ tetrazole followed by oxidation of P(III) to P(V) vith S₈x in CS₂. Building block 1c was incorporated in the Fmoc solid-phase synthesis of two O-thiophosphotyrosine-containing peptides H-Thr-Glu-Pro-Gln-Tyr(PS)-Gln-Pro-Gly-Glu-OH ( 2 ) and H-Thr-Arg-Asp-Ile-Tyr(PS)-Glu-Thr-Asp-Phe-Phe-Arg-Lys-OH ( 3 ), corresponding to sequences of the p60^src (523–531) protein and an insulin receptor ( IR ) (1142–1153) analogue, respectively. An alternative approach of synthesis, the global phosphorylation of a resin-bound peptide, also proved useful. Thus, the free tyrosyl side-chain containing-peptide IR (1142–1153) on support was phosphinylated with the above phosphoramidite reagent followed by oxidation with either S₈/CS₂ or tetraethylthiuram disulfide/CH₃CN solutions. Deprotection and peptide-resin cleavage was performed with a TFA/thiophenol (H₂O) mixture. Crude peptides 2 and 3 were stable to the acidolytic deprotection. Preparative RP(C₁₈)HPLC was initially performed using 0.1% TFA(aq) EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosphopeptide in solution. Stability measurements both as a function of time and pH. further confirmed this initial finding. Purifications performed at intermediate pH using a triethylammonium acetatc (pH 7.5) CH₃CN solvent system overcame this problem.