Engineered Phenylalanine Ammonia‐Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives

Abstract Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia‐lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron‐deficient substrates is often poor. Here, we report the structure‐based engineering of PAL from Planctomyces brasiliensis ( Pb PAL), enabling preparative‐scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide‐ and ester‐containing fumaric acid derivatives. Through the elucidation of cryo‐electron microscopy (cryo‐EM) Pb PAL structure and screening of the structure‐based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron‐deficient substrates. Our engineered PALs demonstrated exclusive α‐regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative‐scale hydroamination yielding tert ‐butyl protected l ‐aspartic acid, widely used as intermediate in peptide solid‐phase synthesis.