Reference Standards Laboratory, United States Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD, 20852, United States.
Reference Standards Laboratory, United States Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, MD, 20852, United States.
J Pharm Biomed Anal. 2021 Sep 5;203:114136. doi: 10.1016/j.jpba.2021.114136. Epub 2021 May 21.
Exenatide is a peptide based anti-diabetic prescription medication. Until now, the literature has lacked a comprehensive atom-specific molecular characterization for this complex large peptide by NMR spectroscopy that can be effortlessly and rapidly utilized for biopharmaceutical structural veracity. Peptide structure verification by NMR is challenging and cumbersome when reliant on traditional proton-based methodology (through-bond and through-space proton connectivity) alone due to increasing complexity, low signal dispersion, and overlap. These challenges are overcome by using 2D heteronuclear (H-C and H-N) maps that not only allow unambiguous signal assignment, but also condense the structural verification information within simplified peptide amide and carbon fingerprint maps. Here we report such simplified amide and carbon fingerprint maps for exenatide; made possible by the first ever comprehensive heteronuclear (H,C, and N) atom specific assignment of exenatide. These heteronuclear assignments were obtained without any isotopic enrichments i.e. at natural abundance, and hence are easily deployable as routine procedures. Furthermore, we compare the 2D heteronuclear maps of exenatide to a chemically identical peptide differing only in the isomerism of the Cα position of the first amino acid, [dHis1]-exenatide, to demonstrate the uniqueness of these maps. We show that despite deliberate changes in pH, temperature, and concentrations, the differences between the amide maps of exenatide and [dHis1]-exenatide are retained. The work presented here not only provides a facilitated structure verification of exenatide but also a framework for heteronuclear NMR data acquisition and signal assignment of large peptides, at natural abundance, in creating their respective unique 2D fingerprint maps.
艾塞那肽是一种基于肽的抗糖尿病处方药。到目前为止，文献中缺乏通过 NMR 光谱对这种复杂的大型肽进行全面的原子特异性分子表征的方法，而这种方法可以轻松快速地用于生物制药结构的真实性。由于复杂性增加、信号分散和重叠，仅依赖于传统的基于质子的方法（通过键和通过空间质子连接）时，通过 NMR 验证肽结构具有挑战性和繁琐性。这些挑战通过使用二维异核（H-C 和 H-N）图谱得到克服，这些图谱不仅允许明确的信号分配，而且还将结构验证信息浓缩在简化的肽酰胺和碳指纹图谱中。在这里，我们报告了艾塞那肽的这种简化酰胺和碳指纹图谱；这是通过首次对艾塞那肽进行全面的异核（H、C 和 N）原子特异性分配实现的。这些异核分配是在没有任何同位素丰度增强的情况下获得的，即在自然丰度下，因此可以轻松地作为常规程序部署。此外，我们将艾塞那肽的二维异核图谱与化学上相同但仅在第一个氨基酸的 Cα 位置的异构体不同的肽（[dHis1]-艾塞那肽）进行比较，以证明这些图谱的独特性。我们表明，尽管故意改变 pH、温度和浓度，艾塞那肽和[dHis1]-艾塞那肽的酰胺图谱之间的差异仍然保留。这里介绍的工作不仅提供了艾塞那肽结构验证的便利方法，而且还提供了在自然丰度下为大型肽获取和分配异核 NMR 数据以及创建各自独特的二维指纹图谱提供了框架。
