Geahlen R L, Loudon G M, Paige L A, Lloyd D
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Anal Biochem. 1992 Apr;202(1):68-70. doi: 10.1016/0003-2697(92)90207-n.
A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.
描述了一种制备生物素化树脂的方法，该树脂可通过固相肽合成的标准方法进行延伸，以得到在羧基末端生物素化的肽。这种方法对于制备需要游离氨基末端的生物素化肽尤为重要。将Nε-9-芴甲氧羰基-(Fmoc)-Nα-叔丁氧羰基(Boc)-L-赖氨酸偶联到对甲基苯二甲胺树脂上，然后除去Fmoc保护基团并与(+)-生物素-4-硝基苯酯反应，得到Nα-Boc-生物胞素-对甲基苯二甲胺树脂。通过合成生物素化肽Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-生物胞素-NH2来测试该树脂的效用，该肽用作肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶(NMT)的体外底物，NMT是催化蛋白质N-肉豆蔻酰化的酶。通过HPLC和质谱对肽衍生物进行分析，结果显示出预期质量的单一主要产物，表明生物素基团在HF裂解和脱保护过程中未被破坏。生物素化肽用作NMT的底物，所得的肉豆蔻酰化肽可通过吸附到固定化抗生物素蛋白上进行定量回收。
