Thomas Döring, Kevin Weiland, Carolin Plattner, Tobias Huber, Dirk Bächle, Daniel Samson
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引用次数: 0
Abstract
The loading (i.e., substitution) of solid supports for oligonucleotide synthesis is an important parameter in large-scale manufacturing of oligonucleotides. Several key process parameters are dependent on the substitution of the solid support, including the number of phosphoramidite nucleoside equivalents used in the coupling step. For dimethoxytrityl (DMTr)–loaded solid supports, the substitution of the resin is determined by quantitatively cleaving the DMTr protecting group from the resin under acidic conditions and then analyzing the DMTr cation extinction by UV/vis spectroscopy. The spectrometric measurement can be performed at 409 nm or the global extinction maximum of 510 nm. The substitution is then calculated based on the Lambert-Beer law analogously to the substitution determination of Fmoc-substituted resins. Below, the determination of the molar extinction coefficient at 510 nm in a solution of 10% dichloroacetic acid in toluene and subsequent determination of the DMTr loading of DMTr-substituted resins is reported. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1: Determination of the molar extinction coefficient at 510 nm in DCA Deblock solution
Basic Protocol 2: Substitution determination of DMTr-substituted resins by cleavage of the DMTr cation
用于大规模寡核苷酸合成的 DMTr 取代树脂的负载测定
用于寡核苷酸合成的固体支持物的负载(即替代)是大规模生产寡核苷酸的一个重要参数。几个关键的工艺参数取决于固体支持物的替代情况,包括偶联步骤中使用的磷酰胺核苷当量的数量。对于负载二甲氧基三苯甲基(DMTr)的固体支持物,在酸性条件下定量裂解树脂上的 DMTr 保护基,然后通过 UV/vis 光谱分析 DMTr 阳离子的消光来确定树脂的取代度。光谱测量可在 409 纳米或 510 纳米的全消光最大值处进行。然后根据朗伯-比尔定律计算取代度,类似于 Fmoc 取代树脂的取代度测定。下面报告了在 10%二氯乙酸的甲苯溶液中测定 510 纳米波长处摩尔消光系数以及随后测定 DMTr 取代树脂的 DMTr 负载的情况。© 2024 作者。基本方案 1:在 510 纳米波长处测定 DCA Deblock 溶液中的摩尔消光系数基本方案 2:通过裂解 DMTr 阳离子测定 DMTr 取代树脂的取代度
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