Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones

Biaryl compounds are ubiquitous metabolites that are often formed by dimerization through oxidative phenol coupling. Hindered rotation around the biaryl bond can cause axial chirality. In nature, dimerizations are catalyzed by oxidative enzymes such as laccases. This class of enzymes is known for non‐specific oxidase reactions while inherent enantioselectivity is hitherto unknown. Here, we describe four related fungal laccases that catalyze γ‐naphthopyrone dimerization in a regio‐ and atropselective manner. In vitro assays revealed that three enzymes were highly P‐selective (ee >95 %), while one enzyme showed remarkable flexibility. Its selectivity for M‐ or P‐configured dimers varied depending on the reaction conditions. For example, a lower enzyme concentration yielded primarily (P)‐ustilaginoidin A, whereas the M atropisomer was favored at higher concentration. These results demonstrate inherent enantioselectivity in an enzyme class that was previously thought to comprise only non‐selective oxidases.     

Enzyme catalytic promiscuity: asymmetric aldol addition reaction catalyzed by a novel thermophilic esterase in organic solvent

The asymmetric aldol addition of 2-butanone and 4-nitrobenzaldehyde catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. APE1547 exhibited a good enzyme activity and enantioselectivity in the reaction. The effects of organic solvent, temperature, water content, and substrate concentration were investigated. The reaction provided optically active secondary alcohol with satisfying enantioselectivity (71.2 %ee) and enzyme activity (38.1 µmol/g/h) under the optimum conditions. A high yield (68.7%) could be obtained when the reaction time was approximately 120 h.     

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

A method for determining lipase enantioselectivity in the transacylation of sec‐alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1‐phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel‐coated 96‐well microtiter plates through a histidine tag (His₆‐tag), screened for transacylation of 1‐phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild‐type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.     

Investigation of the Impact of Water on the Enantioselectivity Displayed by CALB in the Kinetic Resolution of δ‐Functionalized Alkan‐2‐ol Derivatives

It is shown that the low enantioselectivity of Candida antarctica lipase B (CALB)‐catalyzed transesterification of a δ‐functionalized alkan‐2‐ol to its acetate does not correlate at all with the high enantioselectivity of the CALB‐catalyzed hydrolysis of the corresponding acetate in water. This lack of correlation is unusual and for unfunctionalized alkan‐2‐ol derivatives there is a very good correlation between the enantioselectivity of transesterification of the alcohol and hydrolysis of the corresponding acetate (E>200 in both cases). The results confirm previous predictions from molecular modeling. The water effect was mimicked by CALB variant Ala281Ser, which showed an enhanced enantioselectivity in transesterification of δ‐functionalized alkan‐2‐ols compared to wild‐type CALB.     

Water Activity Dependence of Lipases in Non-aqueous Biocatalysis

Eleven lipases are tested and it was found that lipases can be divided into three types according to water activity dependence. The first type is lipase that has low water activity dependence and works in a low water activity, its performance changes little with the change of water activity. The optimum water activity is 0.19 and Newlase F (Rhizopus niveus), lipase FAP-15 (Rhizopus oryzae) belong to this type. The second type is lipase that has medium water activity dependence and its performance changes with the change of water activity. Most lipases belong to this type and the optimum water activity in this type is about 0.60. The third type is lipase that has a high water activity dependence and works only in a high water activity (a _w  > 0.75). WGL (wheat germ) belongs to this type and the optimum water activity is 0.90. The relationship between enantioselectivity and water activity is also discussed and the enantioselectivity seems to be independent of water activity. And we also compared the two control methods of water activity, it was found that the method which add solid salt hydrates to the reaction mixture (method II) is more stable and effective throughout the reaction than the method that pre-equilibrate via the vapor phase (method I). The addition concentration of salt hydrates is also investigated and the optimum concentration is 1 g/l.     

Chirally Functionalized Hollow Nanospheres Containing L‐Prolinamide: Synthesis and Asymmetric Catalysis

Chirally functionalized hollow nanospheres with different surface properties were successfully synthesized by co‐condensation of (2S,1′R,2′R)‐N‐tert‐butyloxycarbonylpyrrolidine‐2‐carboxylic acid [2′‐(4‐trimethoxysilylbenzylamide)cyclohexyl] amide with 1,2‐bis(trimethoxysilyl)ethane or tetramethoxysilane using F127 (EO₁₀₆PO₇₀EO₁₀₆) as surfactant in water. The TEM and N₂ sorption characterizations show that the particle size of the hollow nanosphere is 15–21 nm with a core diameter of 10–16 nm. These L ‐prolinamide‐functionalized hollow nanospheres are highly efficient solid catalysts for the direct asymmetric aldol reaction between cyclohexanone and aromatic aldehydes. It was found that the addition of water in the reaction system not only enhanced the catalytic activity but also increased the enantioselectivity, which is probably due to the enhanced hydrogen bond between the amide oxygen atom and the hydroxyl group of water. Moreover, the catalytic activity increases sharply as the surface hydrophobicity of the hollow nanospheres increases. These hollow nanospheres are quite stable and can be reused with almost the same enantioselectivity and only a slight decrease in catalytic activity.     

Temperature‐tuned enantioselectivity in transfer hydrogenation: preparation and catalytic property of amino acid‐modified benzimidazole

A series of l ‐amino acid‐modified benzimidazoles have been synthesized and their application in Ru‐catalysed asymmetric transfer hydrogenation (ATH) of ketones has been evaluated using i‐propanol as hydrogen donor as well as solvent. The enantioselectivity of the product was sensitive to the reaction conditions and showed reversed temperature effect. Better reaction activity and enantioselectivity were obtained at higher reaction temperature and higher molar ratio of substrate to catalyst (S/C). At an S/C of 2000:1 and 5000:1, the turnover frequency (TOF) and the enantiomeric excess (ee) value of the ATH of acetophenone promoted by the combination of l ‐proline benzimidazole 7c and [RuCl₂(p‐cymene)]₂ were up to 6880, 13 000 h^?1 and 67%, 51% respectively. The ee value dropped off after the reaction reached equilibrium. The high activity and enantioselectivity of [RuCl₂(p‐cymene)]₂/ 7c was primarily influenced by the (S,R) configuration, whereas the (S,S) configuration had low activity and enantioselectivity. Copyright © 2012 John Wiley & Sons, Ltd.     

S‐endo‐2‐Norbornyl‐N‐n‐butylcarbamate as a Potential Pseudomonas Lipase Inhibitor to Probe the Enantioselectivity of the Enzyme by Kinetic and Molecular Docking Evaluation

Obesity is a complex health issue and it can cause many health and social problems. Previous studies reported that lipase is a main target for obesity treatment. We synthesized R‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate as potential pseudomonas lipase inhibitors to probe the enantioselectivity of the enzyme and demonstrated that R‐exo‐2‐norbornyl‐N‐n‐butylcarbamate had better enzyme enantioselectivity, ki and the docking model with Pseudomonas species lipase in our previous studies. In this article, we reported the property of the Pseudomonas species lipase inhibitors, R‐and S‐endo‐2‐norbornyl‐N‐n‐butylcarbamate and compared the docking models of these two compounds with R‐ and S‐exo‐2‐norbornyl‐N‐n‐butylcarbamates by AutoDock. We found that S‐endo‐2‐norbornyl‐N‐n‐butylcarbamate has the best enantioselectivity, ki and docking model and this study could provide useful information about enzyme enantioselectivity for the development of Pseudomonas species lipase inhibitors for obesity treatment.     

非水相中微生物脂肪酶催化转酯化拆分(R,S)-α-苯乙醇

研究了非水有机溶剂体系中脂肪酶不对称转酯化拆分(R,S)-α-苯乙醇反应,比较了15种不同微生物来源的脂肪酶,从中优选出催化活性及对映选择性较高的脂肪酶Lipase PS,系统考察了影响该酶催化不对称转酯化反应的关键因素,获得了优化的催化拆分工艺条件.结果表明,脂肪酶Lipase PS在非水反应体系中,以正己烷为反应介...     

Chiral Mesoporous Organosilica Nanospheres: Effect of Pore Structure on the Performance in Asymmetric Catalysis

(R)‐(+)‐1,1′‐Bi‐2‐naphthol ((R)‐(+)‐Binol)‐functionalized (Binol=2,2′‐dihydroxy‐1,1′‐binaphthyl) chiral mesoporous organosilica nanospheres with uniform particle size (100 to 300 nm) have been synthesized by co‐condensation of tetraethoxysilane and (R)‐2,2′‐di(methoxymethyl)oxy‐6,6′‐di(1‐propyl trimethoxysilyl)‐1,1′‐binaphthyl in a basic medium with cetyltrimethylammonium bromide as the template. Nanospheres with a radiative 2D hexagonal channel arrangement exhibit higher enantioselectivity and turnover frequency than those with a penetrating 2D hexagonal channel arrangement (94 versus 88 % and 43 versus 15 h^?1, respectively) in the asymmetric addition of diethylzinc to aldehydes. In addition, under similar conditions, the enantioselectivity of the nanospheres can be greatly improved as the structural order of the framework increases. These results clearly show that the structural order of nanospheres affects enantioselective reactions. The enantioselectivity of the nanospheres synthesized by the co‐condensation method is higher than that of nanospheres prepared by a grafting method and even higher than that of their homogeneous counterpart. These results indicate that the bite angle of (R)‐(+)‐Binol bridging in a more rigid porous network is in a more favorable position for achieving higher enantioselectivity. The efficiency of a co‐condensation method for the synthesis of high‐performance heterogeneous asymmetric catalysts is also reported.     

Extraction of Phenylalanine Enantiomers by Aqueous Two Phase Systems Containing Combinatorial Chiral Selector

In order to obtain a better enantioselectivity of phenylalanine enantiomers and establish the optimal chiral extraction conditions, the distribution behavior was investigated in aqueous two‐phase systems which were composed of polyethylene glycol and ammonium sulfate containing combinatorial chiral selector: β‐cyclodextrin and HP‐β‐cyclodextrin. The influence of the molar concentration ratio of combinatorial chiral selectors, the total molar concentration of combinatorial chiral selectors, pH value, buffer type and its concentration were thoroughly studied, respectively. The results show that the enantioselectivity reaches 1.53 under the optimal chiral extraction conditions. This extraction is a potential economical and effective way for chiral resolution.     

Aqueous micro and nanoreactors based on alternating copolymers of phenylmaleimide and vinylpyrrolidone bearing pendant l‐proline stabilized with PEG grafted chains

Proline may work efficiently in water as catalyst of aldol reactions if it is hydrophobically activated. In this work, we have maximized this hydrophobic activation by the preparation of linear alternating copolymers of hydrophobic phenylmaleimide and a vinylpyrrolidone derivative bearing proline. These copolymers were water soluble above pH 5.0 and, unlike the free proline, exhibited efficient catalysis at pH 7.0. Moreover, they catalyzed and presented enantioselectivity in an aggregated form at pH 4.0 (close to the isoelectric point, IEP, of the polymer). This enantioselectivity has been related to the exclusion of water at this IEP. To control the size and stabilize the aggregates, PEG grafted copolymers were prepared by the incorporation of a PEG‐macromer (2–10 mol%), which rendered stable nano‐aggregates in water at the IEP. At this pH they catalyzed the aldol reaction in a higher rate than the non‐grafted polymer, but the enantioselectivity was decreased. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 55, 1228–1236     

A parametric study on biphasic medium conditions for the enantioselective production of naproxen by <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

A parametric study to increase the enantioselectivity of Candida rugosa lipase (CRL) toward S-Naproxen production by the hydrolysis of racemic Naproxen methyl ester in an aqueous-organic biphasic batch system was carried out. Effects of organic solvent type, aqueous phase/organic solvent volume ratio, agitation rate, concentrations of the substrate and the enzyme, pH of the aqueous phase, and temperature on the en antiomeric excess for the product (ee_p), on the enantiomeric ratio (E) and on the conversion (x) were evaluated. Employing isooctane as the solvent resulted in higher ee_p, E, and x than those obtained in hexane, cyclohexane, and toluene. The higher the volume ratio of aqueous phase/organic solvent employed, the higher the conversion and enantioselectivity achieved. The increase in agitation rate increased the hydrolysis rate. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. The increase in crude CRL concentration resulted in enhancement of x, but the decrease of fee_p and E. Acidic pH led to higher conversion and enantioselectivity than the medium and alkaline pH values. A further increase in temperature to over 45°C decreased the conversion and enantioselectivity. The highest enantiomeric ratio achieved in the S-Naproxen production was E=171.1, with x=49.8% and ee_p=95.7%.     

Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein

Chiral 1,2‐amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O‐pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron‐nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering.     

Rapid Asymmetric Transfer Hydroformylation (ATHF) of Disubstituted Alkenes Using Paraformaldehyde as a Syngas Surrogate

As an alternative to conventional asymmetric hydroformylation (AHF), asymmetric transfer hydroformylation (ATHF) by using formaldehyde as a surrogate for syngas is reported. A catalyst derived from commercially available [Rh(acac)(CO)₂] (acac=acetylacetonate) and 1,2‐bis[(2S,5S)‐2,5‐diphenylphospholano]ethane(1,5‐cyclooctadiene) (Ph‐BPE) stands out in terms of both activity and enantioselectivity. Remarkably, not only are high selectivities achievable, the reactions are very simple to perform, and higher enantioselectivity (up to 96 % ee) and/or turnover frequencies than those achievable by using the same catalyst (or other leading catalysts) can be obtained by using typical conditions for AHF.     

Lipase‐catalysed kinetic resolution of hydroxymethylchromanes

An effective kinetic resolution of hydroxymethylchromanes racemic 2a and 3a has been achieved by means of enantioselective transesterification with vinyl acetate in organic solvents. The alcohols (‐)‐R‐ 2a and (‐)‐S‐ 3a were obtained with high optical purities (94 and 98% ee) in 70% and 38% yields, respectively. The influence of the enzyme source and the character of the solvent on the enantioselectivity were studied.     

Ultrasound-promoted Lipase-catalyzed Enantioselective Transesterification of (R,S)-Glycidol

Enantioselective transesterification of glycidol with vinyl butyrate as an acyl donor was investigated in the presence of Bacillus subtilis lipase(BSL2) as catalyst. Comparison studies demonstrate the advantage of ultrasound over the conventional shaking for the enzymatic reaction in non-aqueous media. The effects of reaction conditions(ultrasound power, temperature, water activity and pH) on the activity and enantioselectivity were also investigated. Under the optimum conditions, the synthetic activity of BSL2 was 2.95 μmol·min?1·mg?1 and the enantioselectivity(E value) was 52.2. Compared with conventional shaking, ultrasound made the synthetic activity and the enantioselectivity increase 9.5-fold and 1.4-fold, respectively. Furthermore, the repeated use of BSL2 for five cycles resulted in no obvious loss of enzyme activity, suggesting that the enzyme is stable under low power ultrasound conditions.     

Asymmetric Sulfonium Ylide Mediated Cyclopropanation: Stereocontrolled Synthesis of (+)‐LY354740

The reaction of ester‐stabilized sulfonium ylides with cyclopentenone to give (+)‐ 5 ((1S,5R,6S)‐ethyl 2‐oxobicyclo[3.1.0]hexane‐6‐carboxylate), an important precursor to the pharmacologically important compound (+)‐LY354740, has been studied using chiral sulfides operating in both catalytic (sulfide, Cu(acac)₂, ethyl diazoacetate, 60 °C) and stoichiometric modes (sulfonium salt, base, room temperature). It was found that the reaction conditions employed had a major influence over both diastereo‐ and enantioselectivity. Under catalytic conditions, good enantioselectivity with low diastereoselectivity was observed, but under stoichiometric conditions low enantioselectivity with high diastereoselectivity was observed. When the stoichiometric reactions were conducted at high dilution, diastereoselectivity was reduced. This indicated that base‐mediated betaine equilibration was occurring (which is slow relative to ring closure at high dilution). Based on this model, conditions for achieving high enantioselectivity were established as follows: use of a preformed ylide, absence of base, hindered ester (to reduce ylide‐mediated betaine equilibration), and low concentration. Under these conditions high enantioselectivity (95 % ee) was achieved, albeit with low diastereocontrol. Our model for selectivity has been applied to other sulfonium ylide mediated cyclopropanation reactions and successfully accounts for the diastereoselectivity observed in all such reported reactions to date.     

Asymmetric oxidation of sulfides catalyzed by chiral (salen)Mn(III) complexes with a pyrrolidine backbone

Catalytic properties of a series of chiral (pyrrolidine salen)Mn(III) complexes for asymmetric oxidation of aryl methyl sulfides were evaluated. Moderate activity, good chemical selectivity and low enantioselectivity were attained with iodosylbenzene as a terminal oxidant. Enantioselectivity of sulfide oxidation was affected slightly by polar solvent and the sulfoxidation carried out in THF for thioanisole and in CH₃CO₂Et for electron‐deficient sulfides gave better enatioselctivities. The addition of the donor ligand PPNO (4‐phenylpyridine N‐oxide) or MNO (trimethylamine N‐oxide) only has a minor positive effect on the enantioselectivity. Also explored was the steric effect of the N_aza‐substituent in the backbone of (pyrrolidine salen)Mn(III) complexes on the enantioselectivity of sulfide oxidation. The sulfides' access pathway is discussed based on the catalytic results. Copyright © 2006 John Wiley & Sons, Ltd.     

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane ( 1 ) to give (S)‐2‐octanol ( 2 ) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C? H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene ( 3 ) to afford (S)‐1‐phenyl‐2‐propanol ( 4 ) with 95 % ee and 98 % subterminal selectivity.