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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