Lu Y A, Felix A M
Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey.
Int J Pept Protein Res. 1994 Feb;43(2):127-38.
General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.
介绍了使用固相Fmoc/tBu方法在肽的NH2末端、侧链位置（赖氨酸或天冬氨酸/谷氨酸）或COOH末端进行位点特异性聚乙二醇化的一般程序。本研究选择了白细胞介素-2的一个模型十三肽片段IL-2(44 - 56)-NH2，因为它含有几个三官能氨基酸，可作为聚乙二醇化的潜在位点。聚乙二醇化试剂设计为含有Nle或Orn，用作诊断性氨基酸以确认每摩尔肽存在1个PEG单元。通过将PEG-CH2CO-Nle-OH偶联到肽树脂的游离NH2末端进行NH2末端聚乙二醇化。赖氨酸或天冬氨酸的侧链聚乙二醇化通过两条途径之一实现。直接侧链聚乙二醇化通过与Fmoc-Lys(PEG-CH2CO-Nle)-OH或Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH偶联，随后进行聚乙二醇化肽树脂的固相组装和TFA裂解来完成。或者，通过Fmoc-Lys(Alloc)-OH或Fmoc-Asp(O-烯丙基)-OH引入烯丙基保护基，并在肽树脂组装后通过钯催化脱保护选择性去除。然后在最后阶段分别用PEG-CH2CO-Nle-OH或H-Nle-NH-(CH2)2-PEG对赖氨酸或天冬氨酸的侧链进行固相聚乙二醇化。通过Fmoc-Orn(PEG-CH2CO)-OH最初连接到固相载体上，然后使用Fmoc/tBu策略进行固相肽合成来实现COOH末端聚乙二醇化。聚乙二醇化肽通过透析和制备型HPLC纯化，并通过分析型HPLC、氨基酸分析、1H-NMR光谱和激光解吸质谱进行全面表征。
