Authors
Erna Zukic, M.Sc. (acib GmbH ), Dániel Mokos (Universität Graz), Melanie Weber (Universität Graz), Niklas Stix (Universität Graz), Prof. Dr. Klaus Ditrich (BASF SE), Valerio Ferrario (University of the study of Trieste), Henrik Müller (BASF SE), Dr. Christian Willrodt (BASF SE), Univ.-Prof. Mag. Dr.rer.nat. Karl Gruber (Universität Graz), Dr.techn. Bastian DANIEL, BSc. MSc. (Universität Graz) and Prof. Dr. Wolfgang Kroutil (Universität Graz)
The synthesis of amide bonds is crucial in pharmaceutical chemistry, with many small-molecule drugs featuring this motif. However, conventional methods often require stoichiometric activation reagents and organic solvents, generating significant waste. This study identifies a lipase from Sphingomonas sp. HXN-200 (SpL) capable of efficiently catalyzing the formation of amides from heteroaromatic ethyl esters and various amines directly in aqueous buffer (pH 7.5, 30°C).
A screening of 35 enzymes identified SpL, PestE, and Est2 as promising candidates for amide formation in buffer, with SpL demonstrating superior substrate scope and activity. SpL successfully catalyzed the amidation of various sterically demanding six-membered N-heterocyclic (e.g., pyridine, pyrazine based), five-membered O- or S-heterocyclic (e.g., furan, thiophene based), and bicyclic heteroaromatic ethyl esters with primary amines like benzylamine, aniline, n-hexylamine, and allylamine. Preparative scale reactions (up to 84 mM ester, 3.5 eq. amine) yielded up to 99% isolated product, often within minutes to hours, achieving space-time yields as high as 864 g L⁻¹ d⁻¹ for N-benzylpicolinamide. Product precipitation in the aqueous medium likely drives the reaction equilibrium. Methylamine and the secondary amine piperidine were not accepted substrates.
Structural analysis of SpL via X-ray crystallography (1.6-2.0 Å resolution) revealed a canonical Ser-His-Asp catalytic triad (Ser159, His281, Asp251) within an alpha-beta hydrolase fold. Crucially, an additional aspartate residue (Asp158) adjacent to the catalytic serine was identified. Mutagenesis studies (e.g., D158I variant) confirmed that Asp158 is essential for efficient amide formation, likely by facilitating the deprotonation of the amine substrate (present as aminium ion in buffer) to generate the nucleophilic amine. This suggests a catalytic tetrad (Asp158-Ser159-His281-Asp251) mechanism. Furthermore, the enzyme possesses two distinct access tunnels leading to the active site: an acyl channel and an amine channel. This unique architecture allows the enzyme to accommodate sterically demanding ester and amine substrates simultaneously without interference, facilitating their coupling. Similar structural features (catalytic tetrad precursor, dual tunnels) were observed in PestE and Est2.
This biocatalytic approach offers a sustainable and efficient alternative for synthesizing complex heteroaromatic amides in water, avoiding harsh reagents and organic solvents. The high efficiency and broad substrate scope, enabled by the unique catalytic tetrad and dual-tunnel structure, highlight the potential of SpL for greener industrial synthesis processes, particularly in the pharmaceutical and agrochemical sectors, contributing significantly to sustainable chemistry and advancing biocatalytic methods.