Lactobacillus brevis ADH (LBADH) is an alcohol dehydrogenase that is commonly employed to reduce alkyl or aryl ketones usually bearing a methyl, an ethyl or a chloromethyl as a small ketone ...substituent to the corresponding (R)-alcohols. Herein we have tested a series of 24 acetophenone derivatives differing in their size and electronic properties for their reduction employing LBADH. After plotting the relative activity against the measured substrate volumes we observed that apart from the substrate size other effects must be responsible for the activity obtained. Compared to acetophenone (100% relative activity), other small substrates such as propiophenone, α,α,α-trifluoroacetophenone, α-hydroxyacetophenone, and benzoylacetonitrile had relative activities lower than 30%, while medium-sized ketones such as α-bromo-, α,α-dichloro-, and α,α-dibromoacetophenone presented relative activities between 70% and 550%. Moreover, the comparison between the enzymatic activity and the obtained final conversions using an excess or just 2.5 equiv. of the hydrogen donor 2-propanol, denoted again deviations between them. These data supported that these hydrogen transfer (HT) transformations are mainly thermodynamically controlled. For instance, bulky α-halogenated derivatives could be quantitatively reduced by LBADH even employing 2.5 equiv. of 2-propanol independently of their kinetic values. Finally, we found good correlations between the IR absorption band of the carbonyl groups and the degrees of conversion obtained in these HT processes, making this simple method a convenient tool to predict the success of these transformations.
A biocatalytic redox‐neutral reaction cascade was designed for the deracemisation of racemic mandelic acid to yield optically pure L‐phenylglycine employing three enzymes. The cascade consisted of ...three steps: a racemisation, an enantioselective oxidation and a stereoselective reductive amination. The enantioselective oxidation of D‐mandelic acid to the corresponding oxo acid was coupled with the stereoselective reductive amination of the latter; thus the oxidation as well as the reduction reactions were performed simultaneously. The formal hydrogen ed in the first step – the oxidation – was consumed in the reductive amination allowing a redox‐neutral cascade due to a cascade‐internal cofactor recycling. The enantiomers of the starting material were interconverted by a racemase (mandelate racemase) ensuring that in theory 100% of the starting material can be transformed. Using this set‐up racemic mandelic acid was transformed to optically pure L‐phenylglycine (ee >97%) at 94% conversion without the requirement of any additional redox reagents in stoichiometric amounts.
The enzymatic kinetic resolution (EKR) of racemic alcohols or esters is a broadly recognized methodology for the preparation of these compounds in optically active form. Although EKR approaches have ...been developed for the enantioselective transesterification of a vast number of secondary alcohols or hydrolysis of their respective esters, to date, there is no report of bio- or chemo-catalytic asymmetric synthesis of non-racemic alcohols possessing 1,2,3,4-tetrahydroquinoline moiety, which are valuable building blocks for the pharmaceutical industry. In this work, the kinetic resolution of a set of racemic 1,2,3,4-tetrahydroquinoline-propan-2-ols was successfully carried out in neat organic solvents (in the case of CAL-B and BCL) or in water (in the case of MsAcT single variants) using immobilized lipases from Candida antarctica type B (CAL-B) and Burkholderia cepacia (BCL) or engineered acyltransferase variants from Mycobacterium smegmatis (MsAcT) as the biocatalysts and vinyl acetate as irreversible acyl donor, yielding enantiomerically enriched (S)-alcohols and the corresponding (R)-acetates with E-values up to 328 and excellent optical purities (>99% ee). In general, higher ee-values were observed in the reactions catalyzed by lipases; however, the rates of the reactions were significantly better in the case of MsAcT-catalyzed enantioselective transesterifications. Interestingly, we have experimentally proved that enantiomerically enriched 1-(7-nitro-3,4-dihydroquinolin-1(2H)-yl)propan-2-ol undergoes spontaneous amplification of optical purity under achiral chromatographic conditions.
The stereoinversion of one enantiomer into its mirror‐image counterpart within a racemate furnishes a single stereoisomeric product in 100 % theoretical yield. This extremely efficient type of ...deracemization, whereby substrate and product possess an identical chemical structure, can be achieved by using bio‐ or chemo‐catalysts or combinations thereof and is applicable to secondary alcohols, amines and α‐substituted carboxylic acids. Special emphasis is devoted to the theoretical background of the one‐pot, single‐step deracemization of sec‐alcohols.
A biocatalytic system is presented for the stereoselective amination of ketones at the expense of NH3 and molecular hydrogen. By using a NAD+-reducing hydrogenase, an alanine dehydrogenase, and a ...suitable ω-transaminase, the R- as well as the S-enantiomer of various amines could be prepared with up to >99% ee and 98% conversion.
Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently ...scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: ( S )-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), ( R )-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R 2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.
Ketones with two bulky substituents, named bulky-bulky ketones, as well as less sterically demanding ketones were successfully reduced to the corresponding optically highly enriched alcohols using a ...novel identified recombinant short-chain alcohol dehydrogenase RasADH from Ralstonia sp. DSM 6428 overexpressed in E. coli.
Three cloned enoate reductases from the “old yellow enzyme” family of flavoproteins were investigated in the asymmetric bioreduction of activated alkenes. 12‐Oxophytodienoate reductase isoenzymes ...OPR1 and OPR3 from Lycopersicon esculentum (tomato), and YqjM from Bacillus subtilis displayed a remarkably broad substrate spectrum by reducing α,β‐unsaturated aldehydes, ketones, maleimides and nitroalkenes. The reaction proceeded with absolute chemoselectivity – only the conjugated CC bond was reduced, while isolated olefins and carbonyl groups remained intact – with excellent stereoselectivities (ees up to >99%). Upon reduction of a nitroalkene, the stereochemical outcome could be determined via choice of the appropriate enzyme (OPR1 versus OPR3 or YqjM), which furnished the corresponding enantiomeric nitroalkanes in excellent ee. Molecular modelling suggests that this “enzyme‐based stereocontrol” is caused by subtle differences within the active site geometries.
Functionalisation of polycyclic aromatic hydrocarbons (PAHs) and their
-heteroarene analogues (NPAHs) is a tedious synthetic endeavour that requires diverse bottom-up approaches. Cytochrome P450 ...enzymes of white-rot fungi were shown to participate in the fungal detoxification of xenobiotics and environmental hazards via hydroxylation of PAH compounds. In this paper, the recently discovered activity of the monooxygenase CYP5035S7 towards (N)PAHs was investigated in detail, and products formed from the substrates azulene, acenaphthene, fluorene, anthracene, and phenanthrene by whole-cell biocatalysis were isolated and characterised. The observed regioselectivity of CYP5035S7 could be explained by a combination of the substrate's electron density and steric factors influencing the substrate orientation giving insight into the active-site geometry of the enzyme.
The (+)‐ as well as the (−)‐enantiomer of the pyrrolizidine alkaloid xenovenine were prepared within five steps with 17 and 30% overall yields, respectively, in optically pure form, >99% ee as well ...as >99% de. In the asymmetric key step a transaminase performed a regio‐ and stereoselective monoamination of a triketone. By employing two enantiocomplementary transaminases from Arthrobacter sp. both enantiomers were accessible. The triketone was readily prepared via two steps starting from commercially available, achiral 2‐(n‐heptyl)furan. In the final catalytic hydrogenation step, the newly introduced chiral centre directed hydrogen addition to form preferentially the desired (5Z,8E)‐diastereomer. The regio‐ and stereoselective amination of a single ketone moiety out of three allowed the performance of the shortest and highest yielding total synthesis of the bicyclic showcase pyrrolizidine alkaloid without the need for protecting strategies.
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