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 protection as the tBuMe2Si-ester and phosphinylation with (BzlO)2PNiPr2/tetrazole followed by oxidation of P(III) to P(V) with S8 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 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 acetate (pH 7.5)/CH3CN solvent system overcame this problem.
Kitas
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Pharma Research New Technologies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Küng
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Bannwarth
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W
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D005449:Fluorenes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000138:chemical synthesis*; D010766:Phosphorylation; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000596:Amino Acids; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds