Asymmetric Reduction Research Articles

Development of an enzymatic process for the synthesis of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol, the key intermediate of ticagrelor

Asymmetric bioreductions catalyzed by ketoreductases have shown considerable potential in the synthesis of chiral alcohols. However, the synthesis routes of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL), a key chiral intermediate in the synthesis of ticagrelor, still pose significant challenges in term of unsatisfactory substrate loading and production cycle. In this study, a structure-guided rational design was applied to a ketoreductase from Candida glabrata (CgKR1) for the asymmetric reduction of the ketone precursor 2-chloro-1-(3, 4-difluorophenyl) ethanone (CFPO). A number of mutants were obtained with remarkably improved activity and inversed stereoselectivity compared with CgKR1-WT. In particular, F92V/Y208T mutant and F92T/Y208V mutant exhibited strict S-stereoselectivity and extremely high catalytic activity for CFPO. In addition, CFPO with the concentration of 200 g/L can be completely converted into (S)-CFPL on a preparative scale (5.0 g, 92.8% isolated yield) in 4 h through optimizing some parameters in the reduction process, delivering a high space-time yield (STY) of 1200 g L−1 day−1. This study not only provides insight into the relationship between the structure and function of CgKR1, but also establishes an efficient enzymatic process for the biocatalytic synthesis of crucial chiral intermediate (S)-CFPL of ticagrelor.

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and β-halohydrins

Asymmetric reduction of prochiral ketones, particularly, reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to produce enantiopure chiral alcohols is challenging. Herein, an anthrol reductase CbAR with the ability to accommodate diverse bulky substrates, like emodin, for asymmetric reduction is identified. We firstly solve crystal structures of CbAR and CbAR-Emodin complex. It reveals that Tyr210 is critical for emodin recognition and binding, as it forms a hydrogen-bond interaction with His162 and π-π stacking interactions with emodin. This ensures the correct orientation for the stereoselectivity. Then, through structure-guided engineering, variant CbAR-H162F can convert various 2,2-disubstituted 1,3-cyclodiketones and α-haloacetophenones to optically pure (2S, 3S)-ketols and (R)-β-halohydrins, respectively. More importantly, their stereoselectivity mechanisms are also well explained by the respective crystal structures of CbAR-H162F-substrate complex. Therefore, this study demonstrates that an in-depth understanding of catalytic mechanism is valuable for exploiting the promiscuity of anthrol reductases to prepare diverse enantiopure chiral alcohols.

Pricing and Quantity Decisions under Asymmetric Carbon Emission Reduction Information and Cap-and-Trade Mechanism.

With the continuous spread of cap-and-trade mechanisms and consumers' great concerns about environmental issues, manufacturers strive to reduce carbon emissions. Unfortunately, they are not always willing to disclose their accurate emission reductions or may even falsify emission reduction information. By developing a signaling model, we explore the impact of the cap-and-trade mechanism and asymmetric information on the decision-making of supply chain members composed of a manufacturer regulated by the cap-and-trade mechanism, and a retailer. As a result, we show that the low-type manufacturer has the incentive to mimic the pricing behavior of the high-type manufacturer under information asymmetry. Thus, in order to avoid this mimicry, the high-type manufacturer will distort the wholesale price. Moreover, the impact of the cap-and-trade mechanism on the manufacturer depends on the initial quotas. Only when the initial quota is in the middle range does the high-type manufacturer benefit, while the low-type manufacturer suffers. Furthermore, the low-type manufacturer tends to hide emission reduction information, while the high-type manufacturer tends to disclose the information. We also address how information asymmetry weakens the emission reduction advantages of the high-type manufacturer. However, the cap-and-trade mechanism can effectively alleviate this negative effect.

Efficient Kilogram-Scale Synthesis of a Novel Oxazolidinone Antibacterial Candidate YG-056SP

The development of an improved kilogram-scale synthesis of a novel oxazolidinone antibacterial candidate YG-056SP for the treatment of multidrug-resistant Gram-positive bacterial infection is described. The new process is highlighted by a step economical construction of the chiral N-methyloxazolidone, employing an asymmetric reduction of the carbonyl and methylamine substitution, followed by the cyclization with N,N′-carbonyldiimidazole. Another highlight is the safe construction of the fused ring with high optical purity. Epoxy intermediates were obtained by intramolecular ring closure, and reacted with phenol fragments, followed by reduction, protection, and an intramolecular tandem reaction to afford the fused ring. The endgame process features the completion of the Miyaura reaction and the Suzuki coupling reaction in one pot and tert-butyldimethylsilyl deprotection to make dibenzyl phosphate. Finally, cheap hydrochloric acid was used to deprotect the benzyl group, which delivered YG-056SP with a defined particle-size distribution suitable for preclinical studies after crystal transformation. Compared with the initial synthetic route, the overall yield of this optimized process increased significantly from 2.3 to 29.6%.

Enzymatic (2R,4R)‐Pentanediol Synthesis – “Putting a Bottle on the Table”

Abstract(2R,4R)‐Pentanediol is an interesting precursor for the synthesis of chiral ligands. A ketoreductase (KRED) was employed for the asymmetric reduction of acetylacetone to this diol. Biocatalysis often suffers from low concentrations of hydrophobic substrates and low stability of the enzyme in unconventional media. Here, we present an engineered KRED variant applicable in a neat substrate system, including upscaling to the multi‐liter scale and downstream processing (DSP). Our engineered KRED applied in a neat substrate system is a powerful technique for the synthesis of chiral diols yielding product concentrations of 208 g L−1.

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.

Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.

An Inconvenient Truth: Welfare Losses From Asymmetric Reductions in Transportation Costs

Recent empirical studies find that the magnitudes of reductions in bilateral shipping costs are asymmetric within country pairs. Motivated by these findings, I study the welfare effects of asymmetric reductions in transportation costs in Helpman and Itskhoki's [(2010). Labour market rigidities, trade and unemployment. Review of Economic Studies, 77(3), 1100–1137] model of international trade with heterogeneous firms and frictional labor markets. I show that sufficiently asymmetric reductions in bilateral transportation costs result in welfare losses in the importing country, while a uniform reduction in the bilateral transportation costs of both imports and exports always benefits both countries. This paper raises a cautionary note that the phrase ‘welfare gains from trade liberalization’ implicitly assumes an ‘approximately equal’ reduction in transportation costs.

Chiral phosphoric acid catalyzed redox deracemization of triarylmethanes

Described here is the first deracemization of triaryl-substituted carbon stereocenters, which is in contrast to the well-established processes to deracemize monoaryl- and diaryl-substituted ones. This one-pot redox process involves in situ generation of a para-quinone methide intermediate followed by asymmetric reduction by chiral phosphoric acid catalysis. A wide range of highly enantioenriched triarylmethanes could be generated with high efficiency under mild conditions.

Biocatalytic asymmetric reduction of fluoroalkyl ketones to access enantiopure fluoroalkyl secondary alcohols.

An efficient biocatalytic reduction of difluoroalkyl ketones for accessing chiral fluoroalkyl secondary alcohols is reported using commercial NADPH-dependent ketoreductase K234 with 2-propanol as a co-substrate for NADPH regeneration. This bioreduction reaction could be applied to a broad range of prochiral fluoroketones including aryl difluoroketones, aliphatic difluoroketones, and trifluoroacetophenones with excellent conversion and favorable enantioselectivity at high substrate concentrations (100 g L-1). These results indicate the potential of K234 for the industrial production of valuable chiral fluorohydrins. Moreover, this biocatalytic protocol could also be effective without the addition of an external nicotinamide cofactor, which provides useful guidance for further application of ketoreductase K234 in the asymmetric synthesis of chiral secondary alcohols.

Enhancing the asymmetric reduction activity of ene-reductases for the synthesis of a brivaracetam precursor

Brivaracetam is an effective third-generation antiepileptic drug, and its two chiral centers are a major challenge for designing an inexpensive large-scale synthesis process. Enzymatic asymmetric reduction offers an alternative biotransformation method for reducing 2-(5H)-furanone-4-propyl (3a) to (R)-4-propyldihydrofuran-2(3 H)-one (3b), which is a key intermediate in synthesizing brivaracetam. However, no enzymes have been reported to catalyze this reaction with both high activity and stereoselectivity. In this study, fourteen ene-reductases were compared, and the old yellow enzyme OYE2 was identified as having strict stereoselectivity but unsatisfactory activity. Through evaluations with structurally similar substrates and alanine-screening methods, two key residues were identified, T37 and Y82, playing critical roles in catalytic activity. The hydrogenation activity of mutants Y82W and T37C increased by 2.97 and 7.13 times, respectively. Molecular dynamics simulation indicates that mutant Y82W might shorten the distance between the electron acceptor and donor, while T37A might decrease the redox potential of FMN and then accelerate hydrogen transfer. These results indicate that tuning the redox potential properties of FMN is an important consideration for engineering FMN-dependent oxidoreductases. During the gram-scale synthesis of 3b, T37C exhibited excellent stereoselectivity (>99 % ee) and conversion (>99 %), laying a foundation for a simple one-step route in producing the chiral precursor brivaracetam.

An efficient, concise, and scalable synthesis of Izenamide A and B via asymmetric reduction of γ-amino β-keto ester using 2-methyl-CBS-oxazaborolidine catalysts

A efficient and concise synthesis of izenamides A and B is reported. We have followed a more convergent approach while avoiding costly and harmful chemicals. Major attraction of this synthesis is the first ever report of highly asymmetric reduction of N-Boc γ-amino β-ketoester using 2-Methyl-CBS-oxazaborolidine catalysts to generate the key intermediate. Further, this reaction can be of immense significance as γ-amino β-ketoesters constitutes an important motif in drug discovery.

Efficient Biosynthesis of (S)-1-chloro-2-heptanol Catalyzed by a Newly Isolated Fungi Curvularia hominis B-36

(S)-1-chloro-2-heptanol is an enantiopure chemical of great value that can synthesize Treprostinil for treating primary pulmonary hypertension. In this work, a new strain B-36, capable of asymmetric reduction of 1-chloro-2-heptanone to (S)-1-chloro-2-heptanol, was screened and identified as Curvularia hominis B-36 (CCTCC M 2017654) based on the morphological and internally transcribed spacer (ITS) sequence. The reductive capacity of Curvularia hominis B-36 was investigated as a whole-cell biocatalyst in the bioreduction, and the excellent yield (97.2%) and enantiomeric excess (ee) value (99.9%) were achieved under the optimal conditions as follows: 75 mM 1-chloro-2-heptanone, K2HPO4-KH2PO4 (100 mM, pH 6.0), 50 g L−1 resting cells (dry cell weight; DCW), 15% (v/v) isopropanol as co-substrate, 200 rpm, 30 °C, 20 h. The scaled-up biocatalytic process was accomplished at a bioreactor in a 1.5 L working volume, showing superb yield (~97%) and selectivity (99.9%). The product (S)-1-chloro-2-heptanol was purified and characterized by NMR. Curvularia hominis B-36 is a novel catalyst and the asymmetric synthesis route is benign and eco-friendly.

Recycling of Cofactors in Crude Enzyme Hydrogels as Co-immobilized Heterogeneous Biocatalysts for Continuous-Flow Asymmetric Reduction of Ketones.

Flow biocatalysis involving oxidoreductase is limited by the difficulty in recycling expensive cofactors. In this study, an enzyme-rich hydrogel monolithic microreactor was developed via in situ microfluidic assembly of inexpensive crude enzymes. This porous gel biocatalyst exhibited good tethering functions to nicotinamide cofactors; thus, they were retained by the hydrogel to controllably form a novel heterogeneous biocatalyst with self-sufficient cofactors. The flow asymmetric production of a chiral alcohol in this cofactor-entrapped gel microreactor achieved >99 % enantioselectivity and a high space-time yield of 46.3 g L-1 h-1 at 94.8 % conversion. Moreover, the turnover number of cofactors reached as high as 4800 after continuous operation of 160 reactor volumes, realizing significantly higher utilization of the cofactors compared with many reported strategies. Furthermore, this engineered heterogeneous biocatalyst exhibited improved performance in terms of product tolerance and storage stability, paving the way for a green, cost-effective, and sustainable continuous-flow production of enantiopure alcohols.

Green Asymmetric Reduction of Methylacetophenone

Green Asymmetric Reduction of Methylacetophenone

Crowd Evacuation through Crossing Configurations: Effect of Crossing Angles and Walking Speeds on Speed Variation and Evacuation Time

The design of safe and efficient pedestrian facilities necessitates the knowledge of complex human movements, such as intersecting pedestrian streams, under different conditions. This study aims to experimentally investigate the impact of intersecting angles on collective crowd dynamics under two different urgency levels. Data were collected from a controlled laboratory experiment with scenarios consisting of three intersection angles (30°, 90°, and 150°) and two desired speed levels (normal walking and slow running). Trajectory data of individual experiment participants were extracted from the recorded video footage. The results indicate that the 30° intersection has the lowest bottleneck effect compared to the other angles. Moreover, the time-to-target analysis shows that the 150° intersection has a higher waiting time at the intersection compared to the other angles for the jogging scenarios. The speed distribution and space utilization maps implied an asymmetrical reduction in speed in the two corridors of the intersection, even though the physical and geometrical configurations are symmetric. The lane-based analysis of collective speeds revealed that the inner lane (the lane that initially encounters the intersecting flow) has the maximum reduction in speed. The outcomes of this study may be useful to evaluate the congestion effects associated with crossing configurations and in calibrating and validating simulation tools to reproduce such effects accurately.

Understanding the effectiveness of enzyme pre-reaction state by a quantum-based machine learning model

Prediction of enzymatic stereochemistry is a significant challenge in computational chemistry because of targeting very small energy gaps in highly complicated macromolecular systems. Here, we report a scenario of four substrates (2-pentanone, 2-hexanone, 2-heptanone, and 2-octanone) within four enzyme variants (wild type, W116A, F285A, and W286A) of a medium-chain dehydrogenase from Candida parapsilopsi s. The relative stabilities of pro-R and pro-S pre-reaction states are calculated by umbrella sampling, approximately consistent with the observed stereoselectivity in experiment. Besides, a LASSO-SVM machine-learning model is constructed with structural information of 704 pairs of quantum-mechanistic/molecular-mechanistic transition states (TSs) and pre-reaction states (PRSs), achieving the explanatory power of 99.6% for the calculated barriers. Intriguingly, the explanatory power with the PRS-alone structural information reaches 90.7%, but it decreases to 55.4% with the TS-alone structural information. Thus, the outcomes support that the enzymatic stereoselectivity is substantially determined by the frontier-molecular-orbital-related pre-organization of the enzyme-substrate reacting complexes. • Machine-learning models with transition states and PRSs • Machine-learning, umbrella sampling, and frontier molecular orbital validate PRS theory • Rationalization of the overthrow of Prelog’s rule in CpRCR-catalyzed ketone reductions Luo et al. present a quantum-based machine learning model to validate the effectiveness of pre-reaction state theory for a zinc-dependent medium-chain dehydrogenase CpRCR. Specifically, the anti-Prelog rule of asymmetric reductions is rationalized by the pre-reaction state and frontier molecular orbital analysis.

Asymmetric reduction of conjugated C[dbnd]C bonds by immobilized fusion of old yellow enzyme and glucose dehydrogenase

Asymmetric reduction of the conjugated CC bonds by the old yellow enzymes (OYEs) presents a promising field in the synthesis of chiral chemicals. Nevertheless, few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability. Herein, a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase (GDH) from Bacillus megaterium was constructed to stereoselectively reduce the conjugated CC bonds in a self-sufficient continuous process. The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation. The best fusion YqjM_G_GDH gave 98% conversion of 100 ​mmol/L 2-methylcyclopentenone with an excellent ee value (>99%) in 3 ​h, while the mixture of individual enzymes only obtained 68% conversion after more than 8 ​h. The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated. A one-pot process was designed to purify and immobilize the fusion on the Ni2+-nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers. The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β-unsaturated ketones (20 ​mmol/L) continuously with only 50 ​μmol/L NADP+ to initiate the whole process, but also retained more than 82% of the initial activity after seven cycles, serving as a good candidate for the industrial applications.

Imine Reductases: Multifunctional Biocatalysts with Varying Active Sites and Catalytic Mechanisms

AbstractThe synthesis of chiral amines is significant in the pharmaceutical industry. Imine reductase (IRED), a promising biocatalyst that was previously known to only catalyze asymmetric imine reduction (IR), was revealed to achieve direct asymmetric reductive amination (RA) of ketones with excess amines, producing secondary and tertiary amines. Moreover, conjugate reduction (CR) and RA activity by a single IRED has been reported for the preparation of valuable amine diastereomers. IREDs catalyzing these different reactions share the same standard quaternary structure, but possess varying active sites, indicating correlations and differences between their catalytic mechanisms. In this review, we trace the catalytic mechanisms reported for IRED‐catalyzed IR, RA, and CR‐RA. Insights obtained from structural and protein engineering in understanding the IRED‐catalyzed asymmetric synthesis are also included. This review will help readers acquire comprehensive insights into the catalytic mechanism of IREDs and ultimately inspire the engineering of IREDs for industrial applications.

Transborylation-Enabled Boron Catalysis

AbstractThis review highlights transborylation (controlled boron-boron exchange) and its applications as a turnover strategy in boron-catalysed methodologies. Catalytic applications of B–C, B–O, B–N, B–F, B–S, and B–Se transborylations are discussed in the context of transborylation-enabled catalysis, across a wide range of organic transformations including hydroboration, C–C bond formation, C–H borylation, chemoselective reduction, and asymmetric reduction.1 Introduction2 B–C Transborylation3 B–O Transborylation4 B–N Transborylation5 B–F Transborylation6 B–S Transborylation7 Conclusion

Enhancing intracellular NADPH bioavailability through improving pentose phosphate pathway flux and its application in biocatalysis asymmetric reduction reaction

The intracellular NAD(P)H insufficiency is the key factor which limits the reduced product (such as chiral alcohols) synthesis by whole cell biocatalysis or microbial cell factory. In this paper, we reported a novel solution to increase NADPH supply through strengthening the pentose phosphate pathway (PPP) flux with overexpression of extra zwf (gene for glucose 6-phosphatedehydrogenase) and glk (gene for glucokinase) by recombinant Escherichia coli BL21(DE3)/pETDuet-1-glk-zwf and pET28a-bccr containing a carbonyl reductase gene bccr. The amount of intracellular NADPH was significantly increased from 150.3μmol/L to 681.8μmol/L after strengthening the PPP flux, which was 4.5-fold to that of the control. It was applied to improve the asymmetric reduction of 4-chloroacetoacetate to ethyl S-4-chloro-3-hydroxybutylate catalyzed by the BcCR, which increased the reaction yield 2.8-fold to the control. This strategy provides a new way to increase NADPH supply in E.coli cell factories.