Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC

详细信息    查看全文

• 作者：Natalie W. Nairn ; Pauline A. Bariola ; Thomas J. Graddis ; Michael Pete VanBrunt ; Aijun Wang ; Gary Li ; Kenneth Grabstein
• 刊名：Bioconjugate Chemistry
• 出版年：2015
• 出版时间：October 21, 2015
• 年：2015
• 卷：26
• 期：10
• 页码：2070-2075
• 全文大小：267K
• ISSN：1520-4812

文摘

Bioconjugation by copper-catalyzed azide鈥揳lkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28鈥?3 (LEYCLK) and 137鈥?47 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2鈥? h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems.