Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.
Departments of Pharmacology and Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520, United States.
Acc Chem Res. 2023 Jun 20;56(12):1656-1668. doi: 10.1021/acs.accounts.3c00183. Epub 2023 May 23.
Analytical methods allow for the structure determination of submilligram quantities of complex secondary metabolites. This has been driven in large part by advances in NMR spectroscopic capabilities, including access to high-field magnets equipped with cryogenic probes. Experimental NMR spectroscopy may now be complemented by remarkably accurate carbon-13 NMR calculations using state-of-the-art DFT software packages. Additionally, microED analysis stands to have a profound effect on structure elucidation by providing X-ray-like images of microcrystalline samples of analytes. Nonetheless, lingering pitfalls in structure elucidation remain, particularly for isolates that are unstable or highly oxidized. In this Account, we discuss three projects from our laboratory that highlight nonoverlapping challenges to the field, with implications for chemical, synthetic, and mechanism of action studies. We first discuss the lomaiviticins, complex unsaturated polyketide natural products disclosed in 2001. The original structures were derived from NMR, HRMS, UV-vis, and IR analysis. Owing to the synthetic challenges presented by their structures and the absence of X-ray crystallographic data, the structure assignments remained untested for nearly two decades. In 2021, the Nelson group at Caltech carried out microED analysis of (-)-lomaiviticin C, leading to the startling discovery that the original structure assignment of the lomaiviticins was incorrect. Acquisition of higher-field (800 MHz H, cold probe) NMR data as well as DFT calculations provided insights into the basis for the original misassignment and lent further support to the new structure identified by microED. Reanalysis of the 2001 data set reveals that the two structure assignments are nearly indistinguishable, underscoring the limitations of NMR-based characterization. We then discuss the structure elucidation of colibactin, a complex, nonisolable microbiome metabolite implicated in colorectal cancer. The colibactin biosynthetic gene cluster was detected in 2006, but owing to colibactin's instability and low levels of production, it could not be isolated or characterized. We used a combination of chemical synthesis, mechanism of action studies, and biosynthetic analysis to identify the substructures in colibactin. These studies, coupled with isotope labeling and tandem MS analysis of colibactin-derived DNA interstrand cross-links, ultimately led to a structure assignment for the metabolite. We then discuss the ocimicides, plant secondary metabolites that were studied as agents against drug-resistant . We synthesized the core structure of the ocimicides and found significant discrepancies between our experimental NMR spectroscopic data and that reported for the natural products. We determined the theoretical carbon-13 NMR shifts for 32 diastereomers of the ocimicides. These studies indicated that a revision of the connectivity of the metabolites is likely needed. We end with some thoughts on the frontiers of secondary metabolite structure determination. As modern NMR computational methods are straightforward to execute, we advocate for their systematic use in validating the assignments of novel secondary metabolites.
分析方法可用于确定亚毫克量复杂次生代谢物的结构。这在很大程度上得益于 NMR 光谱能力的进步，包括获得配备低温探针的高场磁铁。现在，使用最先进的 DFT 软件包，实验 NMR 光谱可以通过非常准确的碳-13 NMR 计算来补充。此外，微 ED 分析有望通过提供分析物微晶的类似于 X 射线的图像对结构阐明产生深远影响。尽管如此，结构阐明仍然存在挥之不去的陷阱，特别是对于那些不稳定或高度氧化的分离物。在本报告中，我们讨论了来自我们实验室的三个项目，这些项目突出了该领域相互独立的挑战，对化学、合成和作用机制研究具有启示意义。我们首先讨论了 lomaiviticins，这是 2001 年披露的复杂不饱和聚酮天然产物。最初的结构是通过 NMR、高分辨质谱、紫外-可见和红外分析得出的。由于其结构带来的合成挑战以及缺乏 X 射线晶体学数据，近二十年来，这些结构分配一直未经测试。2021 年，Caltech 的 Nelson 小组对（-）-lomaiviticin C 进行了微 ED 分析，结果令人震惊地发现，lomaiviticins 的原始结构分配是不正确的。更高场（800 MHz H，冷探头）NMR 数据的获取以及 DFT 计算提供了对原始错误分配的基础的深入了解，并进一步支持了微 ED 确定的新结构。对 2001 年数据集的重新分析表明，这两种结构分配几乎无法区分，这突显了基于 NMR 的特征的局限性。然后，我们讨论了 colibactin 的结构阐明，colibactin 是一种复杂的、不可分离的微生物组代谢物，与结直肠癌有关。colibactin 生物合成基因簇于 2006 年被检测到，但由于 colibactin 的不稳定性和低产量，它无法被分离或表征。我们使用化学合成、作用机制研究和生物合成分析的组合来鉴定 colibactin 中的亚结构。这些研究，加上 colibactin 衍生的 DNA 链间交联的同位素标记和串联 MS 分析，最终确定了代谢物的结构分配。然后，我们讨论了 ocimicides，这是一种作为抗耐药性药物的植物次生代谢物。我们合成了 ocimicides 的核心结构，并发现我们的实验 NMR 光谱数据与天然产物报告的数据存在显著差异。我们确定了 32 种 ocimicides 的理论碳-13 NMR 位移。这些研究表明，很可能需要修订代谢物的连接。最后，我们对次生代谢物结构确定的前沿进行了一些思考。由于现代 NMR 计算方法易于执行，我们提倡系统地使用它们来验证新次生代谢物的分配。
