As peptides gained interest as new drugs in the past years, their synthetic routes had been the subject of review and improvement. Fmoc/tBu-based solid-phase peptide synthesis (SPPS) is the most convenient technique, and the implementation in continuous flow has allowed for single-pass coupling and deprotection reactions. The focus of this research is to evaluate the relationship between undesired solvent-promoted reactions and final crude purity, by studying volume changes of a variable bed flow reactor through the synthesis. Based on the temperature, typical solvents for SPPS such as dimethylformamide (DMF) or N-methyl-2-pyrrolidone (NMP) can cause unwanted Fmoc removal during wash steps. It was found that for every millilitre of DMF used at 80°C, up to 1 μmol of Fmoc-protected peptide is deprotected, leading to additional impurities. This effect can, however, be minimised by adding additives such as HOBt, which reduces such unwanted deprotection to just 0.1 μmol/ml.

中文翻译：

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固相肽合成中意外保护基团去除的评估：使用连续流动方法进行量化

随着肽在过去几年中作为新药引起人们的兴趣，它们的合成路线一直是审查和改进的主题。基于 Fmoc/tBu 的固相肽合成 (SPPS) 是最方便的技术，连续流动的实施允许单程偶联和脱保护反应。本研究的重点是通过研究可变床流反应器在合成过程中的体积变化来评估不希望的溶剂促进反应与最终粗品纯度之间的关系。根据温度，SPPS 的典型溶剂如二甲基甲酰胺 (DMF) 或N-methyl-2-pyrrolidone (NMP) 会在洗涤步骤中导致不必要的 Fmoc 去除。发现在 80°C 下使用的每毫升 DMF，多达 1 μmol 的 Fmoc 保护的肽被脱保护，导致额外的杂质。然而，这种影响可以通过添加诸如 HOBt 之类的添加剂来最小化，从而将这种不需要的脱保护降低到仅 0.1 μmol/ml。