Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland.
J Am Chem Soc. 2021 Feb 24;143(7):2736-2740. doi: 10.1021/jacs.1c00925. Epub 2021 Feb 11.
Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.
非核糖体肽（NRPs）是一类具有治疗意义的次级代谢产物，由模块化合成酶以装配线的方式合成。我们之前曾报道，在短杆菌肽合成酶的初始苯丙氨酸加载氨酰化结构域中，单个色氨酸到丝氨酸的突变完全改变了其对点击反应类似物的特异性。在这里，我们报告了这种微创策略能够有效地在生物活性 NRP 途径水平上进行功能化。在重建的短杆菌肽合成酶中，W227S 点突变允许起始模块选择性地掺入带有炔基、卤素和苯甲酰取代基的苯丙氨酸类似物。类似地，在模块 4 中的 W2742S 突变同样允许这些功能化的底物类似物在位置 4 处有效地掺入，从而将该策略扩展到延伸模块。在短杆菌肽 A 的位置 1 或 4 上高效地掺入炔烃手柄，允许通过 Cu(I)-催化的炔烃-叠氮化物环加成反应对相应的短杆菌肽类似物进行一步选择性荧光标记。通过将合成生物学与生物正交化学相结合，该方法在 NRPs 分离和分子靶标阐明以及 NRPs 治疗药物的组合优化方面具有很大的潜力。
