A Convergent, One-Step Biocatalytic Cascade for the Synthesis of Structurally Diverse Noncanonical Amino Acids

Noncanonical amino acids (ncAAs) are crucial building blocks for the development of peptide drugs. Despite their importance, a general and scalable approach to the synthesis of ncAA remains an unmet need in synthetic chemistry. Here, we report the development of a one-pot 4-enzyme cascade for the production of ncAAs starting from commodity aldehydes and glycine. This cascade, which leverages a phenylserine dehydratase enzyme engineered for stability and tolerance to high-substrate loading, generates up to 37 g/L of the target ncAA and fills an existing gap by providing a single platform technology for producing both l- and d- ncAAs, featuring both aromatic and aliphatic side-chains. Additionally, we provide a demonstration that by incorporating an alcohol dehydrogenase, the reaction can be run as a redox-neutral hydrogen-borrowing cascade to the desired ncAA from readily available alcohol starting materials and expand the scope to a β-methyl ncAA. This work represents a significant expansion of the scope and practical application of biocatalytic ncAA synthesis and will enable protecting-group-free access to a broad range of ncAA with fewer steps and less waste.

非天然氨基酸（Noncanonical amino acids, ncAAs）是多肽药物开发的核心构建模块。尽管其应用价值突出，但合成化学领域至今仍缺乏通用且可规模化的ncAA合成方案。本研究开发了一种一锅法四酶级联反应体系，可从商用醛类与甘氨酸出发合成ncAAs。该级联反应依托经过工程化改造以提升稳定性并耐受高底物负载量的苯丝氨酸脱水酶，目标产物ncAA的产量可达37 g/L；该体系可同时制备带有芳香族与脂肪族侧链的L型及D型ncAAs，通过单一平台技术填补了现有技术的空白。此外，本研究证实，通过引入乙醇脱氢酶，该反应可构建氧化还原中性的借氢级联体系，从易获取的醇类起始原料直接合成目标ncAA，并将底物范围拓展至β-甲基ncAA。本研究大幅拓展了生物催化ncAA合成的应用边界与实用价值，可实现无需保护基的多类ncAA合成，且反应步骤更少、废弃物产生量更低。