Combined biocatalytic reductive amination and deuteration to prepare isotopically labelled amino acids for NMR analysis of large proteins

02 September 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Biocatalysis offers many advantages for selective isotopic labelling of valuable small molecules, such as the deuterated amino acids utilised in protein NMR. Until recently, applications of biocatalytic deuteration systems have been restricted by their requirement for a supply of super-stoichiometric quantities of a specifically labelled 2H-pre-cursor, which can be both costly to purchase and complex to prepare. Overcoming this hurdle, we have demonstrated a novel and easy to use H2-driven biocatalytic platform for the incorporation of 2H-atoms across a number of molecular functional groups. By combining the biocatalytic deuteration catalyst with enzymes capable of reductive amination, we synthesised a suite of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. Notably, this strategy enables the introduction of a 15N-label, 2H-label, and chiral centre all in a single-step, and gives rise to amino acid isotopologues on a half gram scale for use directly in the preparation of isotopically labelled proteins. To demonstrate the applicability of the approach in 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, which was subsequently analysable by Methyl-TROSY techniques, revealing new structural information.


Isotopic labelling
Asymmetric catalysis
Deuterium oxide
Structural biology
Large proteins


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