Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR

We demonstrate an atom-efficient and easy to use H ₂-driven biocatalytic platform for the enantioselective incorporation of ²H-atoms into amino acids. By combining the biocatalytic deuteration catalyst with amino acid dehydrogenase enzymes capable of reductive amination, we synthesised a library of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as ²H ₂O and ¹⁵NH ₄ ⁺. The chosen approach avoids the use of pre-labeled ²H-reducing agents, and therefore vastly simplifies product cleanup. Notably, this strategy enables ²H, ¹⁵N, and an asymmetric centre to be introduced at a molecular site in a single step, with full selectivity, under benign conditions, and with near 100% atom economy. The method facilitates the preparation of amino acid isotopologues on a half-gram scale. These amino acids have wide applicability in the analytical life sciences, and in particular for NMR spectroscopic analysis of proteins. To demonstrate the benefits of the approach for enabling the workflow of protein NMR chemists, we prepared l-[α- ²H, ¹⁵N, β- ¹³C]-alanine and integrated it into a large (>400 kDa) heat-shock protein oligomer, which was subsequently analysable by methyl-TROSY techniques, revealing new structural information.