Chemical synthesis of o‐thiophosphotyrosyl peptides

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Abstract
The synthon for O-thiophosphotyrosine, Fmoc-Tyr[PS(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 1cwas 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 p60src (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.

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