Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Biocatalytic enantioselective synthesis of cenobamate, an antiepileptic drug.

A green and efficient process for the synthesis of cenobamate has been accomplished in 70% yield and >99% ee through the bio-reduction of ß-ketotetrazole using Daucus carota whole plant cells. The corresponding ß-hydroxytetrazole was isolated in 60% yield and >98% ee. This is the first report on the biocatalytic reduction of ß-ketotetrazole using plant enzymes derived from D. carota root cells with excellent enantioselectivity.

Chromium Catalyzed Asymmetric Reformatsky Reaction.

This study describes an unprecedented chromium-catalyzed asymmetric Reformatsky reaction, enabling the synthesis of chiral ß-hydroxy carbonyl compounds from α-chlorinated or α-brominated esters and amides. By employing a chiral chromium/diarylamine bis(oxazoline) catalyst, we achieved relatively broad functional group tolerance. Distinct from known reports, the protocol operates under both classical and photoredox conditions, facilitated by the in situ formation of a nucleophilic chiral chromium intermediate through a radical-polar crossover mechanism. Preliminary mechanistic insights, supported by DFT calculations, identify the nucleophilic aldehyde addition as the key stereo-determining step. This approach not only overcomes the limitations of existing Reformatsky reactions but also provides a versatile strategy for accessing complex chiral molecules.

Mining of a novel reductase and its application for asymmetric reduction of p-methoxyacetophenone.

(R)-1-(4-methoxyphenyl) ethanol [(R)-1b] is an essential precursor for the synthesis of aryl propanoic acids' anti-inflammatatory drugs. Biocatalysts for (R)-1b preparation are limited and reductase has problems of low substrate concentration and low conversion rate. As a result, there is a constant need for discovering novel biocatalysts with excellent catalytic performances. In this study, a novel reductase LpSDR from Lacisediminihabitans profunda for the biocatalytic reduction of p-methoxyacetophenone (1a) to (R)-1b was obtained based on gene-mining technology, and some key reaction parameters were also investigated to improve the conversion rate of 1a using whole cells of recombinant Escherichia coli expressing reductase LpSDR as biocatalysts. It was found that the optimal concentration of isopropanol, ZnSO4·7H2O solution, 1a, and recombinant E. coli resting cells, the optimal reaction temperature, buffer pH, and reaction time were 1.95 mol l-1, 0.75 mmol l-1, 75 mmol l-1, 250 g (wet weight) l-1, 28°C, 7.0, and 21 h, respectively. Under the above conditions, a conversion rate of 99.5% and an enantiomeric excess of 99.6% were obtained, which were superior to the corresponding values previously reported. This study provides a novel reductase LpSDR, which is helpful in reducing 1a to (R)-1b.

Enhancing cofactor regeneration of cyanobacteria for the light-powered synthesis of chiral alcohols.

Cyanobacteria Synechocystis sp. PCC 6803 was exploited as green cell factory for light-powered asymmetric synthesis of aromatic chiral alcohols. The effect of temperature, light, substrate and cell concentration on substrate conversions were investigated. Under the optimal condition, a series of chiral alcohols were synthesized with conversions up to 95% and enantiomer excess (ee) > 99%. We found that the addition of Na2S2O3 and Angeli's Salt increased the NADPH content by 20% and 25%, respectively. As a result, the time to reach 95% substrate conversion was shortened by 12 h, which demonstrated that the NADPH regeneration and hence the reaction rates can be regulated in cyanobacteria. This blue-green algae based biocatalysis showed its potential for chiral compounds production in future.

Construction and characterization of novel bifunctional fusion proteins composed of alcohol dehydrogenase and NADH oxidase with efficient oxidized cofactor regeneration.

To tune the efficiency of oxidized cofactor recycling between alcohol dehydrogenase (ADH) and NADH oxidase (NOX) for the production of aromatic chiral alcohols, we designed and constructed four novel bifunctional fusion proteins composed of thermostable ADH and NOX from Thermococcus kodakarensis KOD1. ADH was linked to the N- or C-terminus of NOX with a typical rigid linker (EAAAK)3 and a flexible linker (GGGGS)3 , respectively. Compared with the parental enzymes, the NOX moieties in the four fusion proteins exhibited higher specific activities (141%-282%), while the ADH moieties exhibited varying levels of specific activity (69%-167%). All fusion proteins showed decreased affinities toward the cofactors, with increased Km values toward NADH (159%-406%) and NAD+ (202%-372%). In the enantioselective oxidation of (RS)-1-phenylethanol coupled with cofactor regeneration, the four fusion proteins displayed different positive and negative effects on the recycling efficiency of the oxidized cofactor. The two fusion proteins composed of NOX at the N-terminus exhibited higher total turnover numbers than the corresponding mixtures of individual enzymes with equal activities, particularly at low cofactor concentrations. These findings suggest high cofactor recycling efficiencies of the fusion proteins with appropriate design and their potential application in the biosynthesis of chiral alcohols.

Asymmetric Guerbet Reaction to Access Chiral Alcohols.

The first example of an asymmetric Guerbet reaction has been developed. Using commercially available, classic Noyori RuII -diamine-diphosphine catalysts, well-known in asymmetric hydrogenation, racemic secondary alcohols are shown to couple with primary alcohols in the presence of a base, affording new chiral alcohols with enantiomeric ratios of up to 99:1. Requiring no reducing agents, the protocol provides an easy, alternative route for the synthesis of chiral alcohols. Mechanistic studies reveal that the reaction proceeds via a Ru-catalyzed asymmetric hydrogen autotransfer process in concert with a base-promoted allylic alcohol isomerization.

A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%. CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

Production of (S)-1,2-propanediol from l-rhamnose using the moderately thermophilic Clostridium strain AK1.

Clostridium strain AK1 was investigated for its capacity of producing 1,2-propanediol from l-rhamnose but not l-fucose. The maximum yields of 1,2-propanediol from rhamnose was 0.81 mol 1,2-PD/mol l-rhamnose. The influence of different initial substrate concentrations as well as the effect of temperature and pH on 1,2-PD production was investigated.

Assessing the stereoselectivity of carbonyl reductases toward the reduction of OPBE and docking analysis.

The asymmetric reduction of prochiral carbonyl compounds by NAD(P)H-dependent carbonyl reductases represents a powerful method for the production of optically active alcohols. The stereoselectivity of a series of carbonyl reductases were evaluated toward the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). A majority of reductases produced the ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE) with low to excellent enantiomeric excess (e.e.), whereas about 30% reductases catalyzed OPBE to form (S)-HPBE. Among them, the carbonyl reductase from Saccharomyces cerevisiae (SeCR) and short-chain dehydrogenase from Gluconobacter oxydans (GoKR) exhibited 100% e.e., yielding the corresponding (R) and (S)-HPBE, respectively. However, the SeCR showed relative higher activity (29.0 U/mg) and affinity (Km of 0.22 mM) than those of GoKR. Docking analysis found that the interaction of OPBE with enzyme-NADPH complex determined the NADPH-provided hydrogen transfer and the configuration of reductive product. These results indicated that the three-dimensional (3D) structure of enzymes controlled the stereoselectivity of the reductive product based on the geometry of the substrate and cofactor.

Highly enantioselective production of a chiral intermediate of sitagliptin by a novel isolate of Pseudomonas pseudoalcaligenes.

OBJECTIVE: To produce (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one (S)-1 from 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-1-(2,4,5-trifluorophenyl)butan-2-one (2) by microbial bioreduction. RESULTS: A new isolate of Pseudomonas pseudoalcaligenes reduced enantioselectively prochiral ketone 2 to chiral alcohol (S)-1. Whole cells of the bacterium were tolerant towards 20 % (v/v) DMSO and 10 g 2/l. Under the optimal conditions, the preparative-scale bioreduction yielded (S)-1 at 90 % yield and >99 % ee. Cells could be re-used with the yield and ee of product being 45 % and >99 %, respectively, after five cycles. CONCLUSION: Bioreduction using whole cells of P. pseudoalcaligenes is an attractive approach to produce (S)-1, as a chiral intermediate of the anti-diabetic drug, sitagliptin.

Optimized expression of (S)-carbonyl reductase in Pichia pastoris for efficient production of (S)-1-phenyl-1, 2-ethanediol.

The recombinant (S)-carbonyl reductase (SCR) in Escherichia coli catalyzed the reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) with low efficiency. In this work, its 6× histidine fusion gene his6 -scr was cloned in Pichia pastoris under the control of the AOX1 methanol inducible promoter. The heterologous protein SCR was expressed through a Mut(s) phenotype. Under the optimal conditions: pH 7.0, initial OD600 2.5, methanol daily addition concentration 1.0% and induction duration 4-5 days, the recombinant protein SCR was produced at the highest level. The enzyme activity in the cell-free exacts of P. pastoris was 0.38, which was over twofold than that of the recombinant E. coli-SCR. The enzyme was purified to homogeneity with a specific activity of 3.41 U mg(-1) , and it catalyzed the biotransformation of (S)-PED with a high optical purity of 96.9% in a high yield of 89.7% at optimum pH of 7.0. The developed effective system of P. pastoris-SCR will facilitate the preparation of pure chiral alcohol in industry.

Advances in aldo-keto reductases immobilization for biocatalytic synthesis of chiral alcohols.

Chiral alcohols are essential building blocks of numerous pharmaceuticals and fine chemicals. Aldo-keto reductases (AKRs) constitute a superfamily of oxidoreductases that catalyze the reduction of aldehydes and ketones to their corresponding alcohols using NAD(P)H as a coenzyme. Knowledge about the crucial roles of AKRs immobilization in the biocatalytic synthesis of chiral alcohols is expanding. Herein, we reviewed the characteristics of various AKRs immobilization approaches, the applications of different immobilization materials, and the prospects of continuous flow bioreactor construction by employing these immobilized biocatalysts for synthesizing chiral alcohols. Finally, the opportunities and ongoing challenges for AKR immobilization are discussed and the outlook for this emerging area is analyzed.

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 150.3 µmol/L to 681.8 µmol/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.

Coupling metal and whole-cell catalysis to synthesize chiral alcohols.

BACKGROUND: The combination of metal-catalyzed reactions and enzyme catalysis has been an essential tool for synthesizing chiral pharmaceutical intermediates in the field of drug synthesis. Metal catalysis commonly enables the highly efficient synthesis of molecular scaffolds under harsh organic conditions, whereas enzymes usually catalyze reactions in mild aqueous medium to obtain high selectivity. Since the incompatibility between metal and enzyme catalysis, there are limitations on the compatibility of reaction conditions that must be overcome. FINDINGS: We report a chemoenzymatic cascade reaction involved Palladium (Pd) catalyzed Suzuki-Miyaura coupling and whole-cell catalyzed C = O asymmetric reduction for enantioselective synthesis of value-added chiral alcohol. The cell membrane serves as a natural barrier can protect intracellular enzymes from organic solvents. CONCLUSIONS: With dual advantages of cascade catalysis and biocompatibility, our work provides a rational strategy to harvest chiral alcohols in high yield and excellent enantioselectivity, as a channel to establish chemoenzymatic catalysis.

Effective asymmetric preparation of (R)-1-[3-(trifluoromethyl)phenyl]ethanol with recombinant E. coli whole cells in an aqueous Tween-20/natural deep eutectic solvent solution.

(R)-1-[3-(Trifluoromethyl)phenyl]ethanol ((R)-MTF-PEL) is an important chiral building block for the synthesis of a neuroprotective compound, (R)-3-(1-(3-(trifluoromethyl)phenyl)ethoxy)azetidine-1-carboxamide. In this work, an effective whole-cell-catalyzed biotransformation was developed to produce (R)-MTF-PEL, and its productivity was increased by medium engineering strategy. The recombinant E. coli BL21(DE3)-pET28a(+)-LXCAR-S154Y variant affording carbonyl reductase was adopted for the reduction of 3'-(trifluoromethyl)acetophenone to (R)-MTF-PEL with enantiomeric excess (ee) > 99.9%. The addition of 0.6% Tween-20 (w/v) boosted the bioreduction, because the substrate concentration was increased by 4.0-fold than that in the neat buffer solution. The biocatalytic efficiency was further enhanced by introducing choline chloride: lysine (ChCl:Lys, molar ratio of 1:1) in the reaction medium, because the product yield reached 91.5% under 200 mM substrate concentration in the established Tween-20/ChCl:Lys-containing system, which is the highest ever reported for (R)-MTF-PEL production. The optimal reduction conditions were as follows: 4% (w/v) ChCl:Lys, 12.6 g (DCW)/L recombinant E. coli cells, pH 7.0, 30 ℃ and 200 rpm, reaction for 18 h. The combined strategy of surfactant and NADES has great potential in the biocatalytic process and the synthesis of chiral alcohols.

Efficiently Enantioselective Hydrogenation Photosynthesis of (R)-1-[3,5-Bis(trifluoromethyl)phenyl] ethanol over a CLEs-TiO2 Bioinorganic Hybrid Materials.

Engineering of biological pathways with man-made materials provides inspiring blueprints for sustainable drug production. (R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethanol [(R)-3,5-BTPE], as an important artificial chiral intermediate for complicated pharmaceutical drugs and biologically active molecules, is often synthesized through a hydrogenation reaction of 3,5-bis(trifluoromethyl)acetophenone (3,5-BTAP), in which enantioselectivity and sufficient active hydrogen are the key to restricting the reaction. In this work, a biohybrid photocatalytic hydrogenation system based on an artificial cross-linked enzymes (CLEs)-TiO2-Cp*Rh(bpy) photoenzyme is developed through a bottom-up engineering strategy. Here, TiO2 nanotubes in the presence of Cp*Rh(bpy) are used to transform NADP+ to NADPH during the formation of chiral alcohol intermediates from the catalytic reduction of a ketone substrate by alcohol dehydrogenase CLEs. Hydrogen and electrons, provided by water and photocatalytic systems, respectively, are transferred to reduce NADP+ to NADPH via [Cp*Rh(bpy)(H2O)]2+. With the resulting NADPH, [(R)-3,5-BTPE] is synthesized using our efficient CLEs obtained from the cell lysate by nonstandard amino acid modification. Through this biohybrid photocatalytic system, the photoenzyme-catalyzed combined reductive synthesis of [(R)-3,5-BTPE] has a yield of 41.2% after reaction for 24 h and a very high enantiomeric excess value (>99.99%). In the case of reuse, this biohybrid system retained nearly 95% of its initial catalytic activity for synthesizing the above chiral alcohol. The excellent reusability of the CLEs and TiO2 nanotubes hybrid catalytic materials highlights the environmental friendliness of (R)-3,5-BTPE production.

The production of enantiomerically pure 1-phenylethanol by enzymatic kinetic resolution method using response surface methodology.

As the enantiomers of 1-phenylethanol are valuable intermediates in several industries, the lipase catalyzed kinetic resolution of (R,S) -1-phenylethanol is a relevant research topic. In this study, the goal was to determine the optimum reaction parameters to produce enantiomerically pure 1-phenylethanol by lipase (Novozyme 435) catalyzed kinetic resolution using response surface methodology (RSM). Reactions were performed with 40-400 mM (R,S)-1-phenylethanol, 120-1200 mM vinyl acetate and 2-22 mg/mL biocatalyst concentrations (BC L ), at 20-60 °C and with a stirring rate of 50-400 rpm for 5-120 min. The samples were analyzed using high performance liquid chromatography (HPLC) with a Chiralcel OB column. Optimum reaction parameters to reach 100% enantiomeric excess for the substrate ( ee s ) were determined as follows: substrate concentration (C s ): 240 mM, BC L : 11 mg/mL, at 42 °C with a reaction time of 75 min. Model validation was performed using these conditions and ee s was calculated as 100%, which indicates the predicted model was efficient and accurate. When compared to the literature, it was observed that the reaction time decreased significantly. This is an important result considering the industrial scale perspective.

Dataset describing ethanol and 1,2-propanediol production by a stenothermal moderately thermophilic anaerobe, Clostridium strain AK1.

The dataset details the fermentation of D-glucose, L-rhamnose, and L-fucose and their end-product formation by the moderate thermophile Clostridium strain AK1 (DSM 18778) as related to the work described in "Propanediol from L-rhamnose using the moderately thermophilic Clostridium strain AK1" [1]. The influence of culture conditions on end product formation from D-glucose and L-rhamnose by AK1 was investigated in batch culture. Strain AK1 was cultivated at initial substrate concentrations varying from 0 to 60 mM and initial pH values varying from 4.5 to 8.5. Additionally different cultivation temperatures (30-65 °C), the influence of liquid-gas phase ratio as well as different phosphate concentrations on growth were investigated.

Magnetic combined cross-linked enzyme aggregates (Combi-CLEAs) for cofactor regeneration in the synthesis of chiral alcohol.

Magnetic Fe3O4 nanoparticles were prepared and embedded into the Combi-CLEAs to produce the magnetic Combi-CLEAs in this work. The process for magnetic Combi-CLEAs preparation was optimized, and its properties were investigated. The optimum temperature, thermal stability and optimum pH of magnetic Combi-CLEAs were similar to those of Combi-CLEAs. The catalytic performance of magnetic Combi-CLEAs was tested with the biosynthesis of (S)-ethyl 4-chloro-3-hydroxybutyrate ((S)-CHBE). Magnetic Combi-CLEAs could tolerate higher substrate concentration in the biphasic system. The catalytic efficiency and long-term operational stability of magnetic Combi-CLEAs were obviously superior to those of Combi-CLEAs in both aqueous and biphasic systems. Embedding of magnetic Fe3O4 nanoparticles endowing rigidity contributed to these improvements. Furthermore, the preparation of magnetic Combi-CLEAs was easy, and its recovery during multiple batches of reactions could be fulfilled by magnetic field. Aforementioned advantages make the magnetic Combi-CLEAs hold obvious potential for industrial application.