Tegge W
Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig, Germany.
Int J Pept Protein Res. 1994 May;43(5):448-53. doi: 10.1111/j.1399-3011.1994.tb00543.x.
The 13 amino acid EGFR-sequence AENAEYLRVAPQS-NH2 containing the in vivo autophosphorylated Tyr 1171, was synthesized by Fmoc continuous-flow SPPS with and without N-terminal Boc protection. In addition to the native sequence, peptides in which tyrosine was exchanged by serine and threonine were prepared. Global phosphorylation of the unprotected hydroxyl amino acids on the resin with di-tert-butyl-N,N-diethylphosphoramidite and 1H-tetrazole followed by in situ oxidation of the resulting phosphites with tert-butyl hydroperoxide or with dibenzoyl tetrasulfide resulted in the tyrosine-, serine- and threonine-phosphorylated and -thiophosphorylated sequences, respectively. The quality of the products after phosphorylation with N-terminal protection was better than without. Whereas the serine- and threonine-thiophosphate group was stable, tyrosine-thiophosphate turned out to be hydrolytically labile under acidic conditions. The rate of hydrolysis was determined with the tyrosine-thiophosphorylated model dipeptide Ac-Tyr-Gly-OH between pH 0.1 and 8. Hydrolysis was fastest at pH 3, with a half-time of 12.5 h at room temperature. The tyrosine-thiophosphate group was completely stable at pH 8.
包含体内自磷酸化酪氨酸1171的13个氨基酸的表皮生长因子受体（EGFR）序列AENAEYLRVAPQS-NH2，通过Fmoc连续流动固相肽合成法（SPPS）合成，分别有无N端Boc保护。除天然序列外，还制备了酪氨酸被丝氨酸和苏氨酸取代的肽段。用二叔丁基-N,N-二乙基磷酰胺和1H-四氮唑对树脂上未保护的羟基氨基酸进行全局磷酸化，然后用叔丁基过氧化氢或二苯甲酰四硫化物对生成的亚磷酸酯进行原位氧化，分别得到酪氨酸、丝氨酸和苏氨酸磷酸化及硫代磷酸化序列。N端保护下磷酸化后的产物质量优于未保护的情况。虽然丝氨酸和苏氨酸硫代磷酸基团稳定，但酪氨酸硫代磷酸在酸性条件下被证明在水解方面不稳定。在pH 0.1至8之间用酪氨酸硫代磷酸化的模型二肽Ac-Tyr-Gly-OH测定水解速率。在pH 3时水解最快，室温下半衰期为12.5小时。酪氨酸硫代磷酸基团在pH 8时完全稳定。
