CHEMICAL SYNTHESIS OF O-THIOPHOSPHOTYROSYL PEPTIDES

The synthon for O-thiophosphotyrosine, Fmoc-Tyr[PS(OBzl)(2)]-OH (1c), was prepared in 63% yield from Fmoc-Tyr-OH by first transient protecti on as the (t)BuMe(2)Si-ester and phosphinylation with (BzlO)(2)(PNPr2) -Pr-i/ tetrazole followed by oxidation of P(III) to P(V) with Ss in CS 2. Building block Ic was incorporated in the Fmoc solid-phase synthesi s of two O-thiophosphotyrosine-containing peptides H-Thr-Glu-Pro-Gln-T yr(PS)Gln-Pro-Gly-Glu-OH (2) and Arg-Asp-Ile-Tyr(PS)-Glu-Thr-Asp-Phe-P he-Arg-Lys-OH (3), corresponding to sequences of the p60(src) (523-531 ) protein and an insulin receptor (IR) (1142-1153) analogue, respectiv ely. An alternative approach of synthesis, the global phosphorylation of a resin-bound peptide, also proved useful. Thus, the free tyrosyl s ide-chain containing-peptide IR (1142-1153) on support was phosphinyla ted with the above phosphoramidite reagent followed by oxidation with either S-8/CS2 or tetraethylthiuram disulfide/CH3CN solutions. Deprote ction and peptide-resin cleavage was performed with a TFA/thiophenol ( H2O) mixture. Crude peptides 2 and 3 were stable to the acidolytic dep rotection. Preparative RP(C-18)HPLC was initially performed using 0.1% TFA(aq)/EtOH solvents. However, analyses of fractions resulting from the purification step indicated significant decomposition of thiophosp hopeptide in solution. Stability measurements both as a function of li me and pH, further confirmed this initial finding. Purifications perfo rmed at intermediate pH using a triethylammonium acetate (pH 7.5)/CH3C N solvent system overcame this problem.(C) Munksgaard 1994.