Jack S.
Rowbotham‡
*a,
Jake H.
Nicholson
a,
Miguel A.
Ramirez
a,
Kouji
Urata
a,
Peter M. T.
Todd
a,
Gogulan
Karunanithy§
b,
Lars
Lauterbach¶
c,
Holly A.
Reeve||
a,
Andrew J.
Baldwin
*bd and
Kylie A.
Vincent
*a
aDepartment of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford, UK. E-mail: jack.rowbotham@manchester.ac.uk; kylie.vincent@chem.ox.ac.uk
bDepartment of Chemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, Oxford, UK
cTechnische Universität Berlin, Institute for Chemistry, Straße des 17. Juni 135, 10437 Berlin, Germany
dKavli Institute for Nanoscience Discovery, University of Oxford, Oxford OX1 3QU, UK. E-mail: andrew.baldwin@chem.ox.ac.uk
First published on 26th October 2023
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.
From a synthetic standpoint, chiral-deuteration of target precursors is challenging. Chemical strategies to prepare α-deuterated amino acids often rely on precious metals and expensive chiral ligands or auxillaries.11–16 Such approaches can suffer from imperfect selectivity, diminished isotopic purities, and complex work-up procedures, which can make the product cost prohibitively high for structural biology research. As an alternative to chemo-routes, several biocatalytic strategies have also been demonstrated for preparing α-deuterated amino acids.17–21 These methods benefit from the mild reaction conditions and inherent selectivity associated with enzyme reactivity, but the complex reaction mixtures hinder product isolation.
A number of key advances have been made recently in hydrogen isotope exchange (HIE) strategies for the synthesis of isotopically labelled amino acids.22–24 Both Chun and Narayan25 and Doyon and Buller26 have published elegant biocatalytic HIE methods for stereoretentive α-deuteration of amino acids, both of which operate through pyridoxal phosphate (PLP)-dependent enzymes. The Doyon and Buller approach is particularly notable for the ability to control both α- and β-deuteration. Similarly, in the chemo-catalytic space, the groups of Roche27 and Pieters14 have shown ruthenium (supported on carbon or as nanoparticles on polyvinylpyrrolidone) to be particularly effective for the stereoretentive HIE of amino acids, and Valero et al. have demonstrated Kerr-type homogeneous iridium catalysts for similar reactions.28 These diverse state-of-the-art stereoretentive HIE strategies are summarised in Fig. 1A.
Whilst HIE strategies for the preparation of [2H]-amino acids have advanced significantly, such methods still require a pre-formed amine chiral centre prior to the installation of the deuterium. In the case of amino acids for protein NMR, multiple isotope labels {2H, 15N, 13C} are usually required,29 which are ideally installed in as few steps as possible from low-cost isotope pre-cursors. Motivated by this problem, we sought to develop a direct and inexpensive route to α-deuterated amino acids with high stereoselectively, minimal work-up requirements, and in-built versatility regarding the pattern of isotopic labels.
In contrast to the HIE strategies employed elsewhere, we utilised a reductive amination approach to deuteration, whereby the N-atom, 2H-label, and chiral centre are all introduced into the target molecule in a single-step (Fig. 1B). This transformation is achievable by the use of an amino acid dehydrogenase (AADH); these are robust, widely available enzymes with naturally high activity and selectivity.30–35 The challenge in employing amino acid dehydrogenases for α-deuteration is the requirement to supply a suitably [2H]-labelled cofactor: [4-2H]-NADH. We have developed an atom-efficient strategy for generating [4-2H]-NADH from NAD+in situ using only H2 as a clean reductant and 2H2O as a cheap source of deuterium.36 The approach to obtain the deuterated cofactor uses an electronically coupled H2-oxidising site and NAD+ reducing site, in this case as part of the same enzyme, the soluble hydrogenase from Ralstonia eutropha, (now known as Cupriavidus necator), ReSH.37,38 The [4-2H]-NADH can then be supplied to an amino acid dehydrogenase (such as L-alanine dehydrogenase, L-AlaDH) in the presence of 14NH4+/15NH4+ and substrate to give a chiral [2H]-amino acid (Fig. 2).
This method effectively uses two enzymes in one solution to produce α-deuterated amino acids in gram quantities, with H2 in the local atmosphere providing the driving force. The approach is customisable, to allow for different 2H/13C/15N labelling patterns with little variation of the method. We demonstrate the effectiveness of this procedure by incorporating labelled L-alanine into a 400 kDa molecular chaperone, Hsp16.5, and acquiring a 1H–13C heteronuclear multiple quantum coherence (HMQC) spectrum suitable for high resolution structural and dynamical study. We anticipate that access to larger quantities of specifically labelled amino acids at substantially lower costs using our method will increase their use in biochemical studies.
Using the combination of ReSH and L-AlaDH it was possible to generate a library of ten L-alanine isotopologues (compounds 1a–d, 2a–c, 3a–c in Fig. 3) simply by varying the solvent (H2O/2H2O), ammonium salt (14NH4+/15NH4+), and the pyruvate substrate (12C/13C). In each case, the 1H signals from the methyl peak of the alanine (–C3, δ = 1.46 ppm) in the corresponding NMR spectra could be used to verify the product (full characterisation given in SI Section S.3.1†). Within the limits of detection, only a single isotopologue was formed in each case, verifying the high selectivity of the biocatalytic system, even in the presence of unlabelled H2 gas. It was found that the commercial L-AlaDH used to generate this library led to an enantiomeric excess (ee) of 90%, owing to the presence of a contaminating racemase. As such, a purified L-AlaDH (produced in house, see ESI Section S.2.2†) was used for all subsequent experiments, and delivered the expected >99% ee.
Fig. 3 1H NMR spectra of variously isotopically labelled L-alanine molecules prepared biocatalytically (500 MHz, 293 K, 2H2O, p2H 8.0). |
In addition to the experiments with L-AlaDH, the H2-driven system was also coupled to commercial L-leucine dehydrogenase (L-LeuDH) and L-phenylalanine dehydrogenase (L-PheDH) enzymes. Using this system, it was straightforward to prepare L-[α-2H,15N]-leucine (4) and L-[α-2H,15N, β-2H2]-phenylalanine (5) with similarly high levels of conversion and isotopic and enantioselectivity (see Fig. 4 and ESI Section S.3.1†).
This initial scaled-up reaction was set up anaerobically in a pressure-vessel, which may not be available in all laboratories. Therefore, in order to probe the wider applicability of the ReSH as a deuteration catalyst, a similar reaction to synthesise 3c was set up on the bench in standard laboratory glassware. Here, [3-13C]-pyruvate (35 mM) was reacted in a stirred solution of 2H2O (65 mL, p2H 8.0) in a round-bottom flask with a balloon of H2, under otherwise identical conditions. Again, this reaction proceeded to full conversion, and enabled the isolation of 198 mg of product.
Finally, the reaction conditions were further intensified to increase the amount of isolated product. Here, the synthesis of 1b was performed using 100 mM pyruvate in 60 mL 2H2O with 150 mM NH4HCO3, again in a round-bottom flask. Whilst full conversion was observed after 19 hours, giving rise to 520 mg of product (95% isolated yield), a slight decrease in ee was observed (97%) under the higher loading conditions. In all cases however, the isotopic purity of the isolated products (1d, 1b, 3c) closely followed the isotopic purity of the starting reaction mixture and simple precautions (such as washing the enzymes in 2H2O) were sufficient to keep the %2H high (>95%).
Initially, a decoupled 13C–1H HSQC spectra of the methyl group of the sample were recorded at 298 K in 2H2O (Fig. 5, red trace), yielding a pair of quartets with signal intensity 3:1:1:3 in the 13C dimension, as expected for a small molecule where we detect an anti-phase coherence (which would be in the ratio 1:3:3:1 in a 13C 1D spectrum detecting in-phase coherence). In a spectrum acquired at 288 K in 90 vol% glycerol (Fig. 5, blue trace), the spectra were highly similar. Under these conditions, the viscosity was increased by a factor of approximately 450, effectively simulating the local tumbling behaviour of the molecule as if it were part of a 50 kDa protein. This demonstrates that we expect sharp signals from this amino acid, even when incorporated in high molecular weight complexes. We therefore sought to incorporate this in a suitably chosen protein. We incorporated L-[α-2H,15N, β-13C]-alanine (3c) into the small heat shock protein Hsp16.5. This protein is a molecular chaperone from Methanococcus jannaschii that assembles into a 24-mer of molecular weight 396 kDa (Fig. 6a).44In vitro, this protein is a potent inhibitor of protein aggregation and amyloid formation. L-[α-2H,15N, β-13C]-Alanine (3c, 100 mg) was added 1 hour prior to induction, and the incorporation was >99%. A 13C–1H HMQC NMR spectrum was acquired. 12 resonances were clearly observed, with a range of linewidths, indicating differential mobility within different parts of the complex. As 8 unique resonances were expected from the symmetric structure characterised by X-ray crystallography, our precursor reveals multiple conformations of the protein (Fig. 6b). This spectrum validates the utility of this precursor in future biochemical studies. This is among the largest proteins for which an alanine methyl-TROSY spectrum has been recorded.4,42
Fig. 6 (a) The X-ray crystallographic structure of the small heat shock protein (HSP) from M. jannaschii, (PDB id 1SHS).34 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. |
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3sc01718d |
‡ Current address: Department of Chemistry, University of Manchester, Manchester Institute of Biotechnology, Manchester, UK. |
§ Current address: Department of Structural and Molecular Biology, Division of Biosciences, University College London, Darwin Building, Gower Street, WC1E 6BT, London, UK. |
¶ Current address: RWTH Aachen University iAMB – Institute of Applied Microbiology Worringer Weg 1, 52074 Aachen, Germany. |
|| Current address: HydRegen Limited, Centre for Innovation and Enterprise, Begbroke Science Park, Oxford, OX5 1PF, UK. |
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