Activity and Enantioselectivity of Lipase Immobilized in CTAB Microemulsion-based Gels

Introduction The formation of gelatin-containing mieroemulsionbased gels（MBGs） was first described in 1986 and the physical/structural characterization was carried out by a number of groups with a variety of techniques including tracer diffusion, electrical conductivity, NMR, X-ray and small angle neutron scattering. The MBGs were proposed to comprise an extensive, rigid, interconnected network of gelatin/water rods stabilized by a monolayer of surfactant, in coexistence with a po- pulation of conventional W/O microemulsion droplets.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

Preparative Resolution of Gatifloxacin Enantiomers with Pre-Column Esterification Strategy and Comparing Their Enantioselectivity to Bacteria and Antibody

Gatifloxacin （GFX） is a kind of chiral fluoroquinolones compound due to the methyl group at the C-3 position of the piperazine ring[1]. Although the enantiomers of GFX show similar levels of antimicrobial activity and pharmacokinetics[2], the other biological activities （i.e., toxicity or enantioselective recognition to various receptors in vivo） of GFX enantiomers have not yet been studied. With this in mind, we developed a rapid and cost-effective high performance liquid chromatographic （HPLC） separation procedure for GFX enantiomers with a pre-column esterification strategy.

Rapid Estimation of Enantioselectivity in Lipase-catalyzed Resolution of Glycidyl Butyrate Using pH Indicator

A simple method for rapid estimation of the enantioselectivity of lipase in resolution of chiral esters is described. The enantioselectivity of lipase can be estimated rapidly through comparing the difference of hydrolysis rates for the racemic ester and its slow reacting enantiomer under the same condition because the difference mainly depends on the enantioselective ratio（E values）. The higher the enantioselectivity of enzyme, the larger the difference of hydrolysis rate. The bromothymol blue（BTB） can be used as pH indicator for microplate reader to monitor the formation of acid in lipase-catalyzed hydrolysis of esters. This method has been successfully used to rapidly estimate the enantioselectivity of several lipases in the resolution of glycidyl butyrate.

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.

How can aprotic ionic liquids affect enzymatic enantioselectivity?

The enantioselectivity of α-chymotrypsin in the esterification of N-acetyl-tryptophan with ethanol was examined in aprotic ionic liquids. The enantioselectivity was found to be strongly affected by a kind of solvent, water content, and reaction temperature. The (kcatKM)L/(kcat/KM)D ratio in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([C2mim][FSI]) containing 1.0% (v/v) water at 25°C exhibits 32,000, while that in 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim][BF4]) containing 1.0% (v/v) water at 25°C shows 190. The (kcat/KM)L/(kcat/KM)D ratio in [C2mim] [BF4] at 25°C varies from 190 at 1.0% (v/v) water to 65000 at 5.0% (v/v) water. Moreover, the (kcat/KM)L/ (kcat/KM)

A Machine Learning Model for Predicting Enantioselectivity in Hypervalent Iodine(III)Catalyzed Asymmetric Phenolic Dearomatizations

Catalytic asymmetric dearomatization(CADA)of phenols has emerged as a powerful strategy for constructing stereochemically complicated architectures from planar aromatic feedstocks.However,the development of novel catalysts for highly enantioselective phenolic oxidative dearomatization continues to be a time-and resource-intensive endeavor,attributable mainly to the paucity of a reliable predictive catalyst design strategy.In this study,we systematically compiled a dataset of 847 literaturereported asymmetric phenolic dearomatization by hypervalent iodine(III)catalysts(HVI-CADA dataset),a unique type of catalyst that is gaining increasing attention owing to their ecofriendly features.Leveraging this reaction dataset,we established a machine learning predictive model to predict enantioselectivity.The XGBoost algorithm exhibited the optimal performance,with a root-mean-square error of 0.26(kcal/mol)and an R^(2)of 0.84.This established model can effectively guide the selection of the optimal catalyst and additives in out-of-sample tests.Subsequent independent experiments were conducted to validate the results obtained from the model predictions.We anticipate that our current work will facilitate further design,optimization,and development of novel chiral hypervalent iodine catalysts for new asymmetric phenolic dearomatization reactions.

Iridium-Catalyzed Site-and Enantioselective C(sp^(2))-H Borylation of Benzhydryl Ethers:Enantioselectivity Amplification by Kinetic Resolution Relay

Comprehensive Summary,We herein report a simple ether-directed iridium-catalyzed site-and enantioselective C(sp^(2))-H borylation of benzhydryl ethers for the first time.Various chiral benzhydryl ethers were obtained with high enantioselectivities in the presence of a tailor-made chiral bidentate boryl ligand.We found that the kinetic resolution relay significantly amplified the enantioselectivity.The synthetic utility of the current method was demonstrated by gram-scale C—H borylation and C—B bond transformations.

Understanding the endocrine disruption of chiral pesticides:The enantioselectivity in estrogenic activity of synthetic pyrethroids

Synthetic pyrethroids(SPs) ,a family of chiral insecticides consisting of multiple stereoismers,have been regarded as estrogenic endocrine-disrupting chemicals(EDCs) .In this study,we applied the yeast two-hybrid and molecular docking(MD) assay to assess the enantioselective estrogenic activities of three commonly used SPs,bifenthrin(cis-BF) ,permethrin(PM) and fenvalerate(Fen) .The β-galactosidase analyses indicated that all of the testing pyrethroids displayed significant(p【0.05) enantioselectivity.The results showed that the estrogenic potential of cis-BF was mainly attributed to 1S-cis-BF.Neither PM nor Fen showed estrogenic effects.However,two stereoisomers of PM possessed estrogenic potential activities.αR-2R-Fen and αS-2S-Fen also induced the β-galactosidase activity.The inability to initiate the reporter gene expression by the racemic chemicals may be due to the low ratios of these isomers or the antagonism among them.The strong hydrophobic interaction and the hydrogen bond between positive estrogenic isomers and ERα support our biological testing results.This research demonstrated that the enantioselective estrogenic activity of chiral SPs was due to selective binding between their isomers and the ERαreceptor.The data suggests that enantioselectivity of these chiral pesticides is significant to their estrogenic activities.

Determination of the enantioselectivity of six chiral aryloxy aminopropanol drugs transport across Caco-2 cell monolayers

This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers,a model of human intestinal epithelial membrane.Six chiral aryloxy enantiomers(atenolol,sotalol,celiprolol,carvedilol,metoprolol and propafenone)were tested in bi-directional transport studies.The separation and quantitation of these enantiomers were performed by reversed-phase high-performance liquid chromatography(RP-HPLC)using 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate(GITC)as a pre-column derivatizing agent.Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested.The efflux ratio(ER)for(R)-(t)-celiprolol was 8.96,but it was much lower for(S)-(-)-celiprolol which is 3.42 at the concentration of 96.0 mM;carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0 mM.They are 2.41 for(R)-(t)-carvedilol and 1.98 for(S)-(-)-carvedilol.But in the transport studies of racemic atenolol,sotalol,metoprolol and propafenone,no enantioselective transport were observed over the concentration range tested.Because P-glycoprotein(P-gp)is highly expressed in Caco-2 cells,we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.

DFT Investigation on the Enantioselectivity of Olefin Carboacylation Catalyzed by a Rh(Ⅰ) Complex

The C-C bond activation and recyclization of benzocyclobutenone to poly-fused rings catalyzed by the[Rh(R,S-L)]+complex producing the R,S-,S,R-,R,R-and S,S-product were investigated systematically at the BP86/6-31G(d,p)level in gas phase and THF,and the R,S-and S,R-reaction pathways were revisited at the M062X/6-31G(d,p)level in THF.The computational results reveal that THF only marginally alters the free-energy barriers,but elevates the relative energies of all species.The BP86 functional testifies that in both gas phase and THF,the activation of strained C-C bonds bears relatively low free-energy barriers,and the rate-determining steps of S,R-and R,R-channels are different from those of R,S-and S,S-channels.The BP86 functional also predicts that the R,S-channel is energetically most favorable in gas phase,but the S,R-product is dominant in THF.The change of NPA charges can mirror the variation of molecular structures to elucidate reaction mechanisms.

Photochemical Studies on 5-Methylbicyclo[1.1.1]pentane Derivatives： p-Orbital Overlap Controlled Enantioselectivity

The first example of the p-orbital overlap controlled enantioselectivity of Norrish type II photocyclization reaction was described. Irradiation of 5-methyl bicyclo[l. 1.1 ]pentanyl ketone with UV in the solid state as well as in the acetonitrile solution afforded the Norrish/Yang photocyclization compound as the sole product. Solid-state asymmetric photochemical studies using ionic chiral auxiliary technique led to the enantioselectivity as high as 60%. The results were rationalized by Xray single crystal structure.

Operationally Simple Enantioselective Silane Reduction of Ketones by the [Ir(OMe)(cod)]2/Azolium Catalytic System

An operationally simple protocol was designed for the enantioselective silane reduction (ESR) of ketones using air- and moisture-stable [Ir(OMe)(cod)]2 (cod = 1,5-cyclooctadiene) (3) as a metal catalyst precursor. This reaction was driven by chiral hydroxyamide-functionalized azolium salt 2. The catalytic ESR reaction could be performed under benchtop conditions at room temperature. Treatment of 2 with 3 in THF yielded the monodentate IrCl(NHC)(cod) (NHC = N-heterocyclic carbene) complex 4 in 93% yield, herein the anionic methoxy ligand of 3 serves as an internal base that deprotonates the azolium ring of 2. The well-defined Ir complex 4 catalyzed the ESR reaction of propiophenone (6) with (EtO)2MeSiH using the pre-mixing reaction procedure. Based on this success, the catalytic ESR reaction was designed and implemented using an in situ-generated NHC/Ir catalyst derived from 2 and 3. Thus, a wide variety of aryl ketones could be reduced to the corresponding optically active alcohols in moderate to excellent stereoselectivities at room temperature without temperature control. Since the high catalytic activity of 3 was observed, we next evaluated several other transition metal catalyst precursors for the catalytic ESR reaction under the influence of 2. This evaluation revealed that Ir(acac)(cod) (acac = acetylacetonate) (28) and [IrCl(cod)]2 (5) can be successfully used as metal catalyst precursors in the ESR reaction.

An efficient chiral synthesis of (R)-N-[3-acetyl-4-(2-hydroxy-3-isopropylamino-propoxy)phenyl]-butanamide with high enantioselectivity

R-Enantiomer of the β-receptor antagonist N-[3-acetyl-4-(2-hydroxy-3-isopropylamino-propoxy)phenyl] butanamide with high enantioselectivity was synthesized from cheap starting materials and enantiopure chiral reagent through an efficient,convenient and practical synthetic strategy.Title product was detected by 1H NMR,13C NMR,and MS,and the enatiomeric excess was determined by chiral HPLC analysis using a chiracel AD-H column.

Enantioselectivity of Photochemical Reactions within Polymer Microcapsules

Polymer microcapsule was employed as a reaction medium to achieve enantioselectivity in photochemical reduction of phenyl cyclohexyl ketone and photoelectrocyclization of tropolone methyl ether under the influence of various chiral inductors. In all cases, low but evident enantioselectivity was observed. The poor enantioselectivity is probably due to the facts that not all the capsules include simultaneously both the chiral inductor and the reactant molecules, and the wall of the microcapsule is not rigid enough to hold the reactant and the chiral inductor molecules in close contact.

DFT Study on the Origin of the Enantioselectivity of （S）-4-Hydroxylproline-Catalyzed Direct Aldol Reaction between Acetone and 4-Nitrobenzaldehyde

DFT-B3LYP calculations were carried out to study the enantioselectivity of the （S）-4-hydroxylproline-catalyzed direct aldol reaction between acetone and 4-nitrobenzaldehyde. Four transition structures associated with the stereo-controlling step of the reaction have been determined. They are corresponding to the anti and syn arrangements of the methylene moiety related to the carboxylic acid group in enamine intermediate and the si and re attacks to the aldehyde carbonyl carbon. The effect of DMSO solvent on the stereo-controlling step was investigated with polarized continuum model （PCM）. The computed energies of the transition states reveal the moderate enantioselectivity of the reaction.

Synthesis of Optically Active β-Alkyl-α-methylene-γ-butyro- lactones from Enantioselective Biotransformation of Nitriles, an Unusual Inversion of Enantioselectivity

A new approach to optically active β-alkyl-α-methylene-γ-butyrolactone derivatives was reported from the Rhodococcus sp. AJ270-catalyzed hydrolysis of appropriate nitriles. The inversion of enantioselectivity of the amidase has been observed when a methyl protection was introduced into the hydroxy group of the parent substrate.

Plant-Derived Enzymes Producing Chiral Aroma Compounds and Potential Application

Aroma(volatile)compounds play important ecological functions in plants,and also contribute to the quality of plant-derived foods.Moreover,chiral aroma compounds affect their functions in plants and lead to different flavor quality properties.Formations of chiral aroma compounds are due to the presence of enzymes producing these compounds in plants,which are generally involved in the final biosynthetic step of the aroma compounds.Here,we review recent progress in research on the plant-derived enzymes producing chiral aroma compounds,and their changes in response to environmental factors.The chiral aroma enzymes that have been reported produce(R)-linalool,(S)-linalool,(R)-limonene,and(S)-limonene,etc.,and these enzymes are found in various plant species.We also discuss the origins of enantioselectivity in the plant-derived enzymes producing chiral aroma compounds and summarize the potential use of plants containing enzymes producing chiral aroma compounds for producing chiral flavors/fragrances.

Biomimetic asymmetric Michael addition reactions in water catalyzed by amino-containing β-cyclodextrin derivatives

Nineβ‐cyclodextrin derivatives containing an amino group were synthesized via nucleophilic sub‐stitution from mono（6‐O‐p‐tolylsulfonyl）‐β‐cyclodextrin and used in asymmetric biomimetic Mi‐chael addition reactions in water at room temperature. The mechanism responsible for the moder‐ate activity and enantioselectivity of the β‐cyclodextrin derivatives was explored using nuclear magnetic resonance spectroscopy, namely 2D 1H rotating‐frame overhauser effect spectroscopy （ROESY）, ultraviolet absorption spectroscopy, and quantum chemical calculations, which provide a useful technique for investigating the formation of inclusion complexes. The effects of the pH of the reaction medium, theβ‐cyclodextrin derivative dosage, the structure of the modifying amino group, and various substrates on the yield and enantioselectivity were investigated. The results indicated that these factors had an important effect on the enantiomeric excess （ee） in the reaction system. Experiments using a competitor for inclusion complex formation showed that a hydrophobic cavity is necessary for enantioselective Michael addition. A comparison of the reactions using 4‐nitro‐β‐nitrostyrene and 2‐nitro‐β‐nitrostyrene showed that steric hindrance improved the enan‐tioselectivity. This was verified by the optimized geometries obtained from quantum chemical cal‐culations. An ee of 71%was obtained in the asymmetric Michael addition of cyclohexanone and 2‐nitro‐β‐nitrostyrene, using （S）‐2‐aminomethylpyrrolidine‐modified β‐CD as the catalyst, in an aqueous buffer solution, i.e., CH3COONa‐HCl （pH 7.5）.

Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin

Enantioselectivity of chiral pollutants is receiving growing concern due to the difference in toxicology and environment fate between enantiomers.In this study,enantiomers of insecticide beta-cypermethrin （beta-CP） were separated on selected chiral column by HPLC,and the toxicity of enantiomers was evaluated using the zebrafish embryo-larval assays.The enantiomers of beta-CP were baseline separated on Chiralcel OD and Chiralpak AD columns and detected by circular dichroism （CD） at 236 nm.Better separation could be achieved at lower temperature （e.g.,20°C） and with lower levels of polar modifiers.Pure enantiomers were obtained on Chiralcel OD.The CD spectra of enantiomers were recorded.By comparing the elution order with a previous similar study,the absolute configuration of beta-CP enantiomers was determined.The individual enantiomers were used in zebrafish embryo test,and the results showed that beta-CP enantioselectively induced yolk sac edema,pericardial edema and crooked body.The 1R-cis-αS and 1R-trans-αS enantiomers showed strong developmental toxicities at concentration of 0.1 mg/L,while the 1S-cis-αR and 1S-trans-αR induced no malformations at higher concentration （e.g.,0.3 mg/L）.The results suggest that the enantioselective toxicological effects of beta-CP should be considered when evaluating its ecotoxicological effects.