Lipase‐catalyzed dynamic kinetic resolution of (R,S)‐fenoprofen thioester in isooctane

A lipase‐catalyzed enantioselective hydrolysis process under in situ racemization of the remaining (R)‐thioetser substrate with trioctylamine as the catalyst was developed for the production of (S)‐fenoprofen from (R,S)‐fenoprofen 2,2,2‐trifluoroethyl thioester in isooctane. Detailed investigations of trioctylamine concentration on the enzyme activation and the kinetic behavior of the thioester in racemization and enzymatic reactions were conducted, in which good agreement between the experimental data and theoretical results was observed. © 2002 Society of Chemical Industry     

Lipase‐catalyzed dynamic hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenyl acetate in water‐saturated isooctane

The hydrolytic resolution of (R,S)‐2,2,2‐trifluoroethyl α‐chlorophenylacetate in water‐saturated isooctane containing Lipase MY(I) at 35 °C is selected as the best reaction condition for producing (R)‐α‐chlorophenyl acetic acid. The kinetic constants, and hence an enantiomeric ratio of 33.6, are estimated and employed for the modeling of time‐course conversions of both substrates by considering product inhibition and enzyme deactivation effects. A successful dynamic kinetic resolution is also achieved, giving the desired (R)‐α‐chlorophenylacetic acid of 93.0% yield and ee_P = 89.5% when 80 mmol dm^?3 trioctylamine acting as the racemization catalyst and enzyme activator is initially added. Copyright © 2006 Society of Chemical Industry     

One‐pot regioselective and stereoselective terpolymerization of rac‐lactide,CO2 and rac‐propylene oxide with TPPMCl (M = Cr,Co, Al)/PPNCl binary catalyst

Tetraphenyl porphyrin metal compound (TPPMCl) (where the TPPMCl was TPPCrCl, TPPCoCl, TPPAlCl), in combination with cocatalyst PPNCl (bis(triphenylphosphine)iminium chloride, the molar ratio of TPPMCl to PPNCl was 1:0.5), was used to catalyze the polymerization of racemic lactide (rac‐LA) in racemic propylene oxide (rac‐PO) medium and the terpolymerization of rac‐LA, CO₂ and rac‐PO. It was found that these TPPMCl/PPNCl binary catalysts could initiate the stereoselective polymerization of rac‐LA in rac‐PO medium to form enriched isotactic polylactide (PLA) (P_i ≥ 68.0%) and terpolymerization of CO₂, rac‐LA, rac‐PO to form PPC‐PLA‐PPO (PPC, poly(propylene carbonate); PPO, poly(propylene oxide)) multiblock copolymer. In particular the PPC‐PLA‐PPO multiblock copolymer thus formed displayed high regioregularity and stereoregularity, and has high head‐to‐tail structure content in the PPC block (H‐T% ≥ 63.6%) and high isotacticity in the PLA block (P_i ≥ 64.0%). The influence of catalyst formula, the monomer feeding ratio, reaction temperature, carbon dioxide pressure and reaction time on the terpolymerization was investigated by ¹H NMR, ¹³C NMR, gel permeation chromatography, DSC and TGA. © 2018 Society of Chemical Industry     

Experimental and Computation Studies on Candida antarctica Lipase B‐Catalyzed Enantioselective Alcoholysis of 4‐Bromomethyl‐β‐lactone Leading to Enantiopure 4‐Bromo‐3‐hydroxybutanoate

Both enantiomers of optically pure 4‐bromo‐3‐hydroxybutanoate, which is an important chiral building block in the syntheses of various biologically active compounds including statins, were synthesized from rac‐4‐bromomethyl‐β‐lactone through kinetic resolution. Candida antarctica lipase B (CAL‐B) enantioselectively catalyzes the ring opening of the β‐lactone with ethanol to yield ethyl (R)‐4‐bromo‐3‐hydroxybutanoate with high enantioselectivity (E>200). The unreacted (S)‐4‐bromomethyl‐β‐lactone was converted to ethyl (S)‐4‐bromo‐3‐hydroxybutanoate (>99% ee), which can be further transformed to ethyl (R)‐4‐cyano‐3‐hydroxybutanoate, through an acid‐catalyzed ring opening in ethanol. Molecular modeling revealed that the stereocenter of the fast‐reacting enantiomer, (R)‐bromomethyl‐β‐lactone, is ∼2 Å from the reacting carbonyl carbon. In addition, the slow‐reacting enantiomer, (S)‐4‐bromomethyl‐β‐lactone, encounters steric hindrance between the bromo substituent and the side chain of the Leu278 residue, while the fast‐reacting enantiomer does not have any steric clash.     

Inclusive separation of acetophenone from petrochemical by‐product with 1‐phenylethanol via noncovalent interactions

Separation of the barely studied mixture of acetophenone and 1‐phenylethanol, a typical by‐product obtained by oil refinery plant, based on the preferential affinity of β‐cyclodextrin (abbreviated as β‐CD) for acetophenone is focused. To demonstrate the potential application of β‐CD for separation of acetophenone from 1‐phenylethanol, the noncovalent interactions of β‐CD with acetophenone and 1‐phenylethanol were compared from thermodynamic and conformational points of view. For the purpose of separation, a multicomponent coprecipitation technique has been established based on the selective noncovalent binding property of β‐CD, which has been proved rigorously. Under the optimized conditions, the acetophenone/1‐phenylethanol equimolar binary mixture can be separated with a separation factor >37. For the petrochemical by‐product, which contains 74.93 wt % of acetophenone, 17.79 wt % of 1‐phenylethanol, and other minor compounds, expanding the separation scale, content of acetophenone in complex can get 99.2%, and the separation efficiency of β‐CD kept stable after recycling twice. © 2014 American Institute of Chemical Engineers AIChE J, 60: 2962–2975, 2014     

Access to Enantiopure α‐Alkyl‐β‐hydroxy Esters through Dynamic Kinetic Resolutions Employing Purified/Overexpressed Alcohol Dehydrogenases

α‐Alkyl‐β‐hydroxy esters were obtained via dynamic kinetic resolution (DKR) employing purified or crude E. coli overexpressed alcohol dehydrogenases (ADHs). ADH‐A from R. ruber, CPADH from C. parapsilosis and TesADH from T. ethanolicus afforded syn‐(2R,3S) derivatives with very high selectivities for sterically not impeded ketones (‘small‐bulky’ substrates), while ADHs from S. yanoikuyae (SyADH) and Ralstonia sp. (RasADH) could also accept bulkier keto esters (‘bulky‐bulky’ substrates). SyADH also provided preferentially syn‐(2R,3S) isomers and RasADH showed in some cases good selectivity towards the formation of anti‐(2S,3S) derivatives. With anti‐Prelog ADHs such as LBADH from L. brevis or LKADH from L. kefir, syn‐(2S,3R) alcohols were obtained with high conversions and diastereomeric excess in some cases, especially with LBADH. Furthermore, due to the thermodynamically favoured reduction of these substrates, it was possible to employ just a minimal excess of 2‐propanol to obtain the final products with quantitative conversions.     

酰基供体对动态动力学拆分1-四氢萘胺的影响

采用新型消旋催化剂耦合Novozym 435成功构建1-四氢萘胺的动态动力学拆分体系用于制备光学纯(R)-1-四氢萘胺。该反应存在着自催化酰胺化反应,会降低反应的对映体选择性。从改变酰基供体结构的角度出发来抑制这种自催化酰胺化反应,考察了不同酸部以及不同醇部的酰基供体对1-四氢萘胺动态动力学拆分反应的影响,发现随着酰基供体结构变得复杂,1-四氢萘胺动态动力学拆分反应结果也相应变得越好,当采用戊酸对氯苯酯作为酰基供体时,动态动力学拆分反应结果就可达到最佳,即转化率>99%,光学纯度eeP>99%。     

Efficient enzymatic kinetic resolution of 2-heptylamine with a highly active acyl donor

Novozyme435 facilitated kinetic resolution of 2-heptylamine was here presented. Methyl methoxyacetate was used as acyl donor. A survey of influencing factors including hydrogen bonding effect, solvent effect, steric effect, temperature and the amount of acyl donor were investigated in detail. At the optimum conditions, the enantiomeric separation was successfully obtained within 8 h at 20 °C, and gave high conversion and optical purity of (R)-2-heptylamine, 48.9% and over 99% respectively. The immobilized lipase B was found to be suitable for the enantiomeric separation of aliphatic amines with good recyclability.     

Kinetic resolution of 1‐phenylethanol integrated with separation of substrates and products by a supported ionic liquid membrane

BACKGROUND: In the present study, the kinetic resolution of rac‐1‐phenylethanol by transesterification with several vinyl esters catalysed by a commercial immobilized Candida antarctica lipase B (Novozym 435) was carried out in n‐hexane at different water contents. The subtrates and products involved in the kinetic resolution were separated using a membrane bioreactor containing a supported liquid membrane based on the ionic liquid 1‐butyl‐3‐methylimidazolium tetrafluoroborate [bmim⁺][BF₄^?]. RESULTS: Variables affecting the kinetic resolution performance of the enzyme were studied. First, the influence of water content of the medium on the synthetic activity, selectivity and enantioselectivity of the enzyme was analysed in order to establish the optimal amount of water. The use of vinyl esters of different alkyl chain length (vinyl propionate, vinyl butyrate and vinyl laurate) as acyl donors to kinetic resolution was studied. Finally, the integrated reaction/separation process for the resolution of rac‐1‐phenylethanol was carried out in the optimal conditions found. CONCLUSION: These investigations demonstrate that the coupling of lipase enantioselectivity with the selective separation of supported liquid membranes based on ionic liquids provides a promising basis for practical production of enantiomerically pure or enriched compounds. Copyright © 2008 Society of Chemical Industry     

Enantioselective Hydrolysis of d,l‐Menthyl Benzoate to L‐(–)‐Menthol by Recombinant Candida rugosa Lipase LIP1

The stereoselective synthesis of L ‐menthol is an attractive process in the flavor and fragrance industry. One promising way to obtain optically pure menthol is the enantioselective hydrolysis of menthol esters under enzymatic catalysis. We developed an effective and highly enantioselective method for the synthesis of L ‐(−)‐menthol (>99% EE) by hydrolyzing the key industrial starting compound, d, l ‐menthyl benzoate. The enzyme of choice was the lipase from Candida rugosa (CRL). While commercially available preparations of this lipase showed only minor selectivity (E=15), excellent enantiomeric purity (E>100) was achieved using the heterologously expressed isoenzyme LIP1.     

Enantiopure N‐Benzyloxycarbonyl‐β2‐amino Acid Allyl Esters from Racemic β‐Lactams by Dynamic Kinetic Resolution using Candida antarctica Lipase B

The dynamic kinetic resolution of α‐substituted racemic β‐lactams by alcoholytic ring‐opening, catalyzed by immobilized lipase B from Candida antarctica is described. With this process, a variety of racemic α‐substituted N‐Cbz‐azetidinones (Cbz=benzyloxycarbonyl) was transformed to the corresponding N‐Cbz‐protected β²‐amino acid allyl esters with high enantioselectivity (up to 99%) and high yields (up to quantitative) at room temperature.

     

Optical resolution of (R,S)‐2‐phenyl‐1‐propanol through enantioselective ethycellulose membranes

Enantioselective membrane was prepared using ethyl cellulose (EC) as membrane material. The flux and permselective properties of membrane using aqueous solution of (R,S)‐2‐phenyl‐1‐propanol as feed solution was studied. The employed membrane process was a pressure driven process. All kinds of important conditions including preparation and operation of membranes were investigated in this experimentation. When the membrane was prepared with 18 wt % EC, 20 wt % N,N‐dimethylformamide in casting solution, 13 min evaporation time and 0°C temperature of water bath for the gelation of the membrane, and the operating pressure and feed solution of (R,S)‐2‐phenyl‐1‐propanol were 0.2 MPa and 1.5 mg/mL, respectively, over 90% of enantiomeric excess (e.e.) and 44.2 (mg/m² h) of flux were obtained. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol—4‐(1‐hydroxyethyl)‐2‐methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent‐inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p‐substituents while tolerating o‐methoxy substituents.     

Lipase‐catalyzed enantioselective esterification of S(+)‐naproxen ester prodrugs in cyclohexane

A lipase‐catalyzed enantioselective esterification process in cyclohexane was developed for the synthesis of S(+)‐naproxen ester prodrugs containing the moiety of N,N‐dialkylamino, ethylene glycol or alkyl ether of ethylene glycol. A high enantiomeric ratio of 44 was obtained when di(ethylene glycol) was selected as the best acyl acceptor. A reversible ping‐pong Bi Bi mechanism has been employed to elucidate the enzymatic behavior of the initial conversion rate for S(+)‐naproxen and the time‐course conversions for both enantiomers. Improvement of the enzyme activity was demonstrated when alcohol in excess of its cyclohexane solubility limit was used. The application of excess racemic naproxen in the presence of solid substrate suspensions showed enhanced productivity and enantioselectivity for the desired S(+)‐ester. Studies of the recovery and racemization of the remaining R(−)‐naproxen are also reported. © 1999 Society of Chemical Industry     

Efficient Lipase‐Catalyzed Kinetic Resolution and Dynamic Kinetic Resolution of β‐Hydroxy Nitriles. Correction of Absolute Configuration and Transformation to Chiral β‐Hydroxy Acids and γ‐Amino Alcohols

Chemoenzymatic dynamic kinetic resolution of β‐hydroxy nitriles 1 has been carried out using Candida antarctica lipase B and a ruthenium catalyst. The use of a hydrogen source to depress ketone formation in the dynamic kinetic resolution yields the corresponding acetates 2 in good yield and high enantioselectivity. It is shown that the ruthenium catalyst and the enzyme can be recycled when used in separate reactions. We also report on the preparation of various enantiomerically pure β‐hydroxy acid derivatives and γ‐amino alcohols from 1 and 2. The latter compounds were also used to establish the correct absolute configuration of 1 and 2.     

Screening,Molecular Cloning,and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β‐Hydroxy‐β‐trifluoromethyl Ketone

The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP⁺‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.     

Development of a Novel Biocatalyst for the Resolution of rac‐Pantolactone

A novel L ‐pantolactone hydrolase, Lph, from Agrobacterium tumefaciens Lu681 was characterized, which stereospecifically hydrolyses L ‐pantolactone to L ‐pantoic acid yielding D ‐pantolactone with > 95% enantiomeric excess. The enzyme was found to be a 30 kDa‐Zn²⁺‐hydrolase with a K_m for L ‐pantolactone of 7 mM and a V_max of 30 U/mg. The corresponding lph gene was identified as an 807 bp ORF and cloned into E. coli. It was overexpressed under control of P_tac and P_rha yielding enzyme activities of up to 600 U/g dry weight. Resolution of d,l ‐pantolactone in repeated batches with isolated Lph and enzyme recovery by membrane filtration gave D ‐pantolactone with 50% yield and 90–95% ee over 6 days. Covalent immobilization to EupergitC led to a stable biocatalyst easy to handle in a repeated batch production of D ‐pantolactone. Further improvements in the activity of Lph were achieved by directed evolution of the enzyme. Activities of mutants F62S, K197D and F100L were increased 2.3, 1.7, and 1.5 fold, respectively.