Smith Kendrick L, Arbuckle Madeline A, Kim Alexander T, Tsai Chun-Yi, McIntosh John A, Verma Deeptak, Shim Eunjae, Narayan Alison R H
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
J Am Chem Soc. 2025 Jul 23;147(29):25191-25200. doi: 10.1021/jacs.5c04098. Epub 2025 Jul 9.
The elaboration of amine substrates through C-C bond-forming reactions is important in the synthesis of bioactive small molecules. Pyridoxal-5'-phosphate (PLP)-dependent enzymes have emerged as valuable biocatalysts for this class of reactions, due to their high stereoselectivity and ability to forge new C-C bonds on unprotected α-amino acid substrates. However, the use of abiological primary amines as pronucleophiles with enzymes such as threonine aldolase has been unexplored, moderating the utility of a biocatalytic approach in the synthesis of diverse 1,2-amino alcohols. In this report, we disclose the discovery and engineering of a PLP-dependent aldolase that accepts (2-azaaryl)methanamines in an aldol-type transformation. The 1,2-amino alcohol products generated, which contain representative heteroaromatic pharmacophores, are delivered with control over both the diastereoselectivity and enantioselectivity in the C-C bond-forming event. Protein engineering provided variants with improved binding affinity for the abiological substrate and decreased affinity for the native α-amino acid, overcoming inhibition of the abiotic reaction by components of lysate, a major challenge in reaction discovery with PLP-dependent enzymes such as threonine aldolases. This work represents the first known example of C-C bond formation on nonamino acid substrates with threonine aldolase and provides a platform for further development of complexity-building biocatalytic reactions with abiotic amine substrates.
通过碳 - 碳键形成反应来构建胺类底物在生物活性小分子的合成中具有重要意义。5'-磷酸吡哆醛（PLP）依赖性酶已成为这类反应中很有价值的生物催化剂，这是由于它们具有高立体选择性，并且能够在未受保护的α-氨基酸底物上形成新的碳 - 碳键。然而，将非生物伯胺作为亲核试剂与苏氨酸醛缩酶等酶一起使用的情况尚未得到探索，这限制了生物催化方法在合成多种1,2 - 氨基醇中的应用。在本报告中，我们揭示了一种PLP依赖性醛缩酶的发现与工程改造，该酶能在醛醇型转化中接受（2 - 氮杂芳基）甲胺。所生成的1,2 - 氨基醇产物含有代表性的杂芳基药效基团，在碳 - 碳键形成过程中对非对映选择性和对映选择性均有控制。蛋白质工程提供了对非生物底物具有更高结合亲和力且对天然α-氨基酸亲和力降低的变体，克服了裂解液成分对非生物反应的抑制作用，而裂解液成分的抑制是利用PLP依赖性酶（如苏氨酸醛缩酶）进行反应发现时的一个主要挑战。这项工作代表了用苏氨酸醛缩酶在非氨基酸底物上形成碳 - 碳键的首个已知实例，并为进一步开发使用非生物胺底物构建复杂生物催化反应提供了一个平台。
