Ketones and imines as substrates of ThDP-dependent enzymes
Final Report Abstract
The formation of tertiary alcohols through C–C bond formation by ThDP-catalyzed addition to ketones, and the planned formation of 2-amino ketones through addition to imines, were in the focus of P6. For this, structural elucidation of YerE or a homologue (in collaboration with the groups of Gunter Schneider and Doreen Doritzsch) was planned. For the use of imines as a starting point TK was proposed. In the long term, we envisaged broadening the substrate range of other ThDP-dependent enzymes towards ketones and possibly imines. We set out to i) elucidate structure–activity relationship of YpYerE and PpYerE (formerly named ‘IlvB’-Pf5), ii) characterize Ao:DCPIP (formerly ‘acetylacetoin synthase’) from B. licheniformis, iii) develop CDH as a multipurpose catalyst, and iv) test TK as starting point for the use of imines as substrates. All projects were performed as proposed. Detailed analysis and biochemical, biogentic, structural, as well as mutational characterization of YerE from two different strains resulted in a broad understanding of ketone-accepting ThDP-dependent enzymes. Based on these results we were able to identify several enzymes accepting hydroxylated C4, C5, C6, and C7 α-keto acids as donor substrates and ketones and many different aldehydes as acceptor substrates. Such enzymatic C–C bond-formations represent a valuable and sustainable entry into chiral tertiary alcohols, as this group of compounds is notoriously difficult to be synthesized by other means. Bacterial Ao:DCPIP OR is the first wild-type enzyme to be characterized to give access to (S)-PAC and derivatives with high yield and good enantioselectivity. Before that, the synthesis of (S)-PAC was achieved by rationally designed variants of (R)-selective enzymes. Additionally, the hitherto unreported use of the Ao:DCPIP OR–methylacetoin pair permits the suppression of the homo-coupling side reaction associated with the utilization of other acyl anion precursors. Moreover, 1,2-diketones are alternative donor substrates. This new donor might enable access to new acyloins, such as the cross coupling with aliphatic aldehydes, an as yet unsolved problem in enzymatic as well as non-enzymatic catalysis. ThDP-dependent CDH can be used for a broad range of different applications ranging from desymmetrizations, formation of (S)-acetoin, and synthesis of many different secondary 2-hydroxy ketones. While wild-type CDH catalyzes the cleavage of cyclohexane-1,2-dione, the rationally designed variant CDH-H28A/N484A uses the same substrate for C–C bond-ligation. The variant was applied in cross-benzoin reactions with ketones as acceptor substrates, resulting in the formation of tertiary 2-hydroxy ketones. Despite many different attempts, we were not able to show ThDP-dependent enzyme-catalyzed formation of 2-amino ketones. This, and the difficulties regarding the unsolved biosynthesis of the ephedrine alkaloids, prompted us to look for alternative biocatalytic access to 2-amino ketones by C–C bond-ligation. Indeed, PLP-dependent ligases might provide a suitable alternative for this longstanding problem. This unexpected outcome of this project might represent a valuable alternative for the enzymatic formation of 2-amino ketones and hence, after reduction, β-amino alcohols, another motif often found in bioactive natural products. Moreover, the reductive amination performed by an IRED and methylamine with (R)-PAC opens another, supposedly biomimetic synthetic pathway. In summary, the interdisciplinary and internationally supported work on enzymes accepting ketones as substrates resulted in a broad understanding of the structural and biochemical characteristics. Moreover, alternative enzyme-catalyzed formation of 2-amino ketones by C–C bond-ligation has been proposed.
Publications
- Enantioselective Intermolecular Aldehyde-Ketone Cross-Coupling through an Enzymatic Carboligation Reaction, Angew. Chem. Int. Ed. 2010, 49: 2389–2392
Lehwald P, Richter M, Röhr C, Liu Hw, Müller M
(See online at https://doi.org/10.1002/anie.200906181) - Elucidation of the Enantioselective Cyclohexane-1,2- dione Hydrolase (CDH) Catalyzed Formation of (S)-Acetoin. ChemCatChem 2014, 6: 969–972
Loschonsky S, Waltzer S, Müller M
(See online at https://doi.org/10.1002/cctc.201300904) - Extended Reaction Scope of Thiamine Diphosphate Dependent Cyclohexane-1,2- dione Hydrolase: From C–C Bond Cleavage to C–C Bond Ligation. Angew. Chem. Int. Ed. 2014, 53: 14402–14406
Loschonsky S, Wacker T, Waltzer S, Giovannini PP, McLeish MJ, Andrade SLA, Müller M
(See online at https://doi.org/10.1002/anie.201408287) - Regio- and Stereoselective Aliphatic-Aromatic Cross-Benzoin Reaction: Enzymatic Divergent Catalysis. Chem. Eur. J. 2016, 22: 13999–14005
Beigi M, Gauchenova E, Walter L, Waltzer S, Bonina F, Stillger T, Rother D, Pohl M, Müller M
(See online at https://doi.org/10.1002/chem.201602084) - Substrate-determined diastereoselectivity in an enzymatic carboligation. ChemBioChem 2016, 17: 1207–1210
Lehwald P, Fuchs O, Nafie LA, Müller M, Lüdeke S
(See online at https://doi.org/10.1002/cbic.201600202) - Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens, ChemBioChem 2018, 19: 2282–2293
Hampel S, Steitz J-P, Baierl A, Lehwald P, Wiesli L, Richter M, Fries A, Pohl M, Schneider G, Dobritzsch D, Müller M
(See online at https://doi.org/10.1002/cbic.201800325)
