By using the solution NMR technique and the “cleave and analyze” approach, we have modeled the kinetics of coupling reactions between activated Fmoc-Arg(Pbf)-OH and NH₂-Arg(Pbf)-Arg(Pbf)-Ala-Arg(Pbf)-Rink amide-AM resin, determined the reaction rates, and correlated the extent of conversion to the Kaiser and TNBS color test results. Since NMR spectroscopy is a highly specific and quantitative tool with structural elucidation capability, the NMR assay for determination of the reaction conversion in solid-phase peptide synthesis (SPPS) can be developed by comparing the peaks from the reactant peptide containing the free amino groups with the product peptide containing the newly coupled Fmoc-amino acid moiety in the one-dimensional ¹H NMR spectrum. We utilized the NMR assay to measure the conversion for each cycle in SPPS to assemble Ac-c(C)arrrar-NH₂ and provided an in-process testing (IPT) time limit for the full-scale production. The data showed that after 18 h of reaction, the conversion for all cycles achieved >99.9%, demonstrating the robustness of the SPPS process and ensuring the control of certain impurities, such as deletion sequences, below 0.1% in the active pharmaceutical ingredient.

中文翻译：

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NMR固相肽合成AMG 416的动力学和颜色转化反应的耦合动力学研究

通过使用溶液NMR技术和“裂解和分析”方法，我们对活化的Fmoc-Arg（Pbf）-OH与NH ₂ -Arg（Pbf）-Arg（Pbf）-Ala-Arg之间的偶联反应动力学进行了建模。（Pbf）-Rink酰胺-AM树脂，确定反应速率，并将转化程度与Kaiser和TNBS颜色测试结果相关联。由于NMR光谱学是具有结构阐明能力的高度特异性和定量工具，因此可以通过比较来自包含游离氨基的反应物肽的峰与NMR来开发用于测定固相肽合成（SPPS）中反应转化率的NMR分析方法。一维包含¹个新偶联的Fmoc-氨基酸部分的产物肽1 H NMR谱。我们利用NMR分析来测量SPPS中每个循环的转化率，以组装Ac-c（C）arrrar-NH _2，并为全面生产提供了过程中测试（IPT）时限。数据显示，反应18小时后，所有循环的转化率均> 99.9％，这证明了SPPS工艺的稳健性，并确保了对活性药物成分中某些杂质（如缺失序列）的控制在0.1％以下。