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

Abstract

We demonstrate an atom-efficient and easy to use H₂-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 ²H₂O and ¹⁵NH₄⁺. 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.

Fig. 1. Routes to α-deuterated chiral amino acids (l-alanine in this case). (A) Stereoretentive hydrogen isotope exchange methodologies (HIE) utilise chemo- or bio-catalysts to install a ²H atom at a pre-formed asymmetric centre. (B) The stereoselective reductive amination approach used in this work installs the N-atom and ²H-atom with simultaneous formation of the chiral centre, enabling many labelling patterns from a generic starting compound.

Fig. 2. A H₂-driven route to selectively isotopically labelled forms of l-alanine bearing an asymmetric deuterium centre. The scheme utilises H₂ gas to drive the formation and recycling of [4-²H]-NADH from NAD⁺ and ²H₂O by the action of a soluble hydrogenase.

Fig. 3. ¹H NMR spectra of variously isotopically labelled l-alanine molecules prepared biocatalytically (500 MHz, 293 K, ²H₂O, p²H 8.0).

Fig. 4. Preparation of ²H and ¹⁵N-labelled l-leucine and l-phenylalanine by H₂ driven biocatalytic reductive amination and deuteration. *For 5, β-²H atoms were incorporated by pre-incubation of the substrate in ²H₂O overnight before the enzymes were added.

Fig. 5. In order to simulate the rotational environment of the labelled amino acid inside a protein, l-[α-²H,¹⁵N, β-¹³C]-alanine was dissolved in a viscous glycerol medium and studied by NMR. The figure shows the ¹³C–¹H decoupled HSQC experiment of the sample in ²H₂O at 298 K (red) and 90 vol% glycerol at 288 K (blue trace). Under 0 vol% glycerol, the ratio of intensities is 3 : 1 : 1 : 3, as expected for a small molecule. At 90 vol% glycerol and low temperature, the resonances remained intense. The intensity ratio changed to approximately 2 : 1 : 1 : 2. Due to the outer resonances having 9× faster relaxation than the inner lines, in the macromolecular limit when the molecule tumbles slowly. Although the effects of slow tumbling are clear, the resonances remain sharp indicating the utility of this reagent inside high molecular weight proteins.

Fig. 6. (a) The X-ray crystallographic structure of the small heat shock protein (HSP) from M. jannaschii, (PDB id 1SHS). The structure is built from dimeric sub-units, and the expected alanine ¹³CH₃ groups are shown. The homo 24-mer has a molecular weight of 396 kDa. (b) Alanine region of methyl spectrum revealing 12 clear resonances with a range of linewidths. This contrasts with the X-ray structure that anticipates 8 unique environments. In solution, the complex adopts multiple conformations, and a range of environments with different motional regimes. This spectrum demonstrates that l-alanine produced using our synthesis scheme can be readily incorporated into high molecular weight biomolecules for mechanistic and structural studies.