Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis

Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.     

Enantiocomplementary enzymatic resolution of the chiral auxiliary: cis,cis-6-(2,2-dimethylpropanamido)spiro[4.4]nonan-1-ol and the molecular basis for the high enantioselectivity of subtilisin Carlsberg

cis,cis-(+/-)-6-(2,2-Dimethylpropanamido)spiro[4.4]nonan-1-ol, 1, a chiral auxiliary for Diels-Alder additions, was resolved by enzyme-catalyzed hydrolysis of the corresponding butyrate and acrylate esters. Subtilisin Carlsberg protease and bovine cholesterol esterase both showed high enantioselectivity in this process, but favored opposite enantiomers. Subtilisin Carlsberg favored esters of (1S,5S,6S)-1, while bovine cholesterol esterase favored esters of (1R,5R,6R)-1, consistent with the approximately mirror-image arrangement of the active sites of subtilisins and lipases/esterases. A gram-scale resolution of 1-acrylate with subtilisin Carlsberg yielded (1S,5S,6S)-1 (1.1 g, 46 % yield, 99 % ee) and (1R,5R,6R)-1-acrylate (1.3 g, 44 % yield, 99 % ee) although the reaction was slow. The high enantioselectivity combined with the conformational rigidity of the substrate made this an ideal example to identify the molecular basis of the enantioselectivity of subtilisin Carlsberg toward secondary alcohols. When modeled, the favored (1S,5S,6S) enantiomer adopted a catalytically productive conformation with two longer-than-expected hydrogen bonds, consistent with the slow reaction rate. The unfavored (1R,5R,6R) enantiomer encountered severe steric interactions with catalytically essential residues in the model. It either distorted the catalytic histidine position or encountered severe steric strain with Asn155, an oxyanion-stabilizing residue.     

Modeling and prediction of enantioselectivity of lipases by covalent docking method

以SYBYL软件包中的FlexX对接模块为研究工具,模拟预测了4种脂肪酶对66对手性底物的水解及转酯反应的对映体选择性。模拟过程中,将四面体中间态底物类似物共价对接到脂肪酶的活性位点,以3个必需氢键的形成为筛选条件,去除不符合标准的酶-底物对接构象。对接结果表明：当催化反应的E值较小时（E＜100）,酶与R/S两种底物的结合自由能差不足以准确预测酶的优先反应构型;而当底物的E≥100,并且主链碳原子数目较少时,该方法对脂肪酶对映体选择性的预测率明显提高,达到81.5%。由于模拟中,酶与绝大多数底物形成了含有3个氢键的反应型构象,符合理论催化模型,同时由于该方法计算速度快,因而该方法可用于高通量模拟预测脂肪酶的潜在底物。     

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

The Escherichia coli esterase YbfF displays high activity towards 1,2‐O‐isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R‐enantiomer of these substrates, producing the S‐enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R‐enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2‐fold higher enantioselectivity than wild‐type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.     

Simultaneous Use of in Silico Design and a Correlated Mutation Network as a Tool To Efficiently Guide Enzyme Engineering

In order to improve the efficiency of directed evolution experiments, in silico multiple‐substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3‐phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta‐analysis of the docking conformations in wild‐type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15‐fold improved enantioselectivity was found.     

A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.     

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (¹H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.     

Synthesis of some new heterocyclic systems derived from 2-acetylbenzimidazole

The named compound was reacted with thiosemicarbazide and/or semicarbazide to produce the corresponding condensation products II and V respectively. Reaction of II with chloroacetic acid in ethanol containing anhydrous sodium acetate yielded III. Condensation of III with aromatic aldehydes yielded the corresponding arylidene derivatives (IV). Oxidation of the semicarbazone V with selenium dioxide gave 2-(1,2,3-selenadiazole-4-yl)benzimidazole (VIa, b) while with thionyl chloride it gave 2-(1,2,3-thiadiazole-4-yl)benzimidazole (VIIa, b). The chalcones of 2-acetyl and/or 1-methyl-2-acetylbenzimidazole were condensed with hydrazine hydrate, phenylhydrazine and/or hydroxylamine to produce 2-(5-aryl-1(H)-pyrazolin-3-yl)-, 2-(5-aryl-1-phenyl-2-pyrazolin-3-yl)- and 2-(5-aryl-2-isoxazolin-3-yl)benzimidazole (IX, X, XI) respectively.     

Kinetics and modeling of 1-phenyl-1,2-propanedione hydrogenation

Kinetics and modeling of 1-phenyl-1,2-propanedione hydrogenation over cinchonidine-modified Pt/Al₂O₃ catalyst is reported. Hydrogenation experiments carried out in a pressurized autoclave (288 K, 1.2–6.5 bar hydrogen) revealed interesting kinetic effects which inspired the model development. The enantioselectivity towards the (R)-configuration, as well as the reaction rate and regioselectivity, depended on the modifier concentration having a maximum. The enantio- and regioselective effects were explained by the kinetic model, which assumes different number of sites for adsorption of the carbonyl groups of the 1-phenyl-1,2-propanedione as well as for the cinchonidine adsorbed in flat and tilted modes. The number of adsorption sites needed for the different species were obtained from molecular considerations and the hydrogenation rate constants were determined along with the adsorption parameters by non-linear regression analysis. A comparison of model predictions with experimental data revealed that the model accounts for the kinetic regularities.     

Evaluation of Pseudoenantiomeric Mixed Carbonates as Efficient Fluorogenic Probes for Enantioselectivity Screening

We report mixed carbonates as enzyme substrates and demonstrate their application as fluorogenic probes for lipase and esterase enantiopreference screening. By the application of pseudoenantiomers with distinct fluorescence behaviors, it is possible to evaluate the activity and enantiopreference of hydrolytic enzymes. In order to validate our method, enantioselectivities calculated from fluorometric measurements were compared with the results obtained from larger‐scale kinetic resolution.     

Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active‐site residues of potato epoxide hydrolase StEH1 combined with an enzyme‐activity screen allowed the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3‐epoxypropyl)benzene. The wild‐type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots: W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.     

High pressure enhances activity and selectivity of Candida rugosa lipase immobilized onto silica nanoparticles in organic solvent

High hydrostatic pressure has been increasingly utilized to improve functions of enzymes, and most of such studies are currently focused on free enzymes in aqueous solution or organic solvent. In this work, Candida rugosa lipase (CRL) was immobilized onto silica nanoparticles and its activity and enantioselectivity in organic solvent were evaluated at high pressures under different water activities. The application of high hydrostatic pressures (50–200 MPa) led to improved activities of immobilized CRL for transesterification of (R)-1-phenylpropan-2-ol with vinyl acetate by 4–6 folds. Additionally the immobilization of CRL resulted in a significant change of selectivities, shifting the enantiomeric excess from the (R)- towards (S)-1-phenylpropan-2-yl acetate product at atmospheric pressure. The application of high pressures led to either enantiomeric excess towards (R)-1-phenylpropan-2-yl or no enantiomeric selectivity, depending on the water activities in the organic solvent and the level of pressures. The interesting behaviour of immobilized CRL under high pressures offers new opportunities to modulate enzyme functions through combination of high pressures and enzyme immobilization.     

Increased Enantioselectivity by Engineering Bottleneck Mutants in an Esterase from Pseudomonas fluorescens

Four hydrophobic and bulky amino acid residues (F126, F144, F159, and I225) were identified to form a bottleneck guarding the entrance to the active site of an esterase from Pseudomonas fluorescens (PFE I). Hence, a range of nonpolar amino acids were introduced into PFE I to broaden the substrate range and to increase enantioselectivity while preserving the hydrophobicity of the tunnel. First, single variants were created and then the most enantioselective ones were combined to find cooperative effects. This resulted in several mutants, which showed substantially enhanced enantioselectivity; for instance, in the kinetic resolution of 1‐phenyl‐1‐propyl acetate, with which the wild type only showed E=1.2, two mutants gave E>46. For 1‐phenyl‐1‐ethyl acetate enantioselectivity increased from ～50 to >100 for all mutants studied. Furthermore, higher conversions could be found at shorter reaction times; this indicates that the mutations not only enhanced selectivity, but that also the entrance into the active site was indeed facilitated by these mutations. The experimental results could be explained by computer modeling.     

微波辐射下1,2,4-三唑硫酮席夫碱的合成及表征

微波辐射条件下,以氨基均三唑硫醇与查尔酮为原料,通过亲核取代反应,制备了3-(4-氨基-5-硫基-3-苯基均三唑-1-氮代)-1,3-二苯-1-丙酮(Ⅳ),Ⅳ与系列芳香醛经缩合反应,合成了7种三唑硫酮席夫碱Ⅴa～Ⅴg。探讨了原料摩尔比、催化剂用量、反应时间、溶剂、微波辐射功率对收率的影响,得到了优化的工艺条件:n(芳香醛)∶n(氨基三唑硫酮)=1∶1.1,微波功率500 W,催化剂冰醋酸2 mL,反应时间5～7 min,溶剂为N,N-二甲基甲酰胺(DMF),收率为71%～87%。用IR、MS、1HNMR、元素分析对合成中间体和目标产物进行了结构表征。     

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high‐volume, low‐price sectors, such as polymers, to low‐volume, high‐price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas‐phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a “synthetic platform technology”. Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2‐arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.     

Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).     

Engineering a Carbonyl Reductase for Scalable Preparation of (S)-3-Cyclopentyl-3-hydroxypropanenitrile,the Key Building Block of Ruxolitinib

(S)-3-Cyclopentyl-3-hydroxypropanenitrile is the key precursor for the synthesis of ruxolitinib. The bioreduction of 3-cyclopentyl-3-ketopropanenitrile ( 1 a ) offers an attractive method to access this important compound. A carbonyl reductase (PhADH) from Paraburkholderia hospita catalyzed the reduction of 1 a giving the (S)-alcohol ( 1 b ) with 85 % ee. Rational engineering of PhADH resulted in a double mutant H93C/A139L, which enhanced the enantioselectivity from 85 % to >98 %, as well as a 6.3-fold improvement in the specific activity. The bioreduction of 1 a was performed at 200 g/L (1.5 M) substrate concentration, leading to isolation of (S)- 1 b in 91 % yield. Similarly, using this mutant enzyme, 3-cyclohexyl-3-ketopropanenitrile ( 2 a ) and 3-phenyl-3-ketopropanenitrile ( 3 a ) were reduced at high concentration affording the corresponding alcohols in >99 % ee, and 90 % and 92 % yield, respectively. The results showed that the variant H93C/A139L was a powerful biocatalyst for reduction of β-substituted-β-ketonitriles.     

Design,Synthesis, and Structure-Activity Relationship Study of Pyrazolones as Potent Inhibitors of Pancreatic Lipase

Pancreatic lipase (PL), a key target for the prevention and treatment of obesity, plays crucial roles in the hydrolysis and absorption of in dietary fat. In this study, a series of pyrazolones was synthesized, and their inhibitory effects against PL were assayed by using 4-methylumbelliferyl oleate (4-MUO) as optical substrate for PL. Comprehensive structure–activity relationship analysis of these pyrazolones led us to design and synthesize a novel compound P32 (5-(naphthalen-2-yl)-2-phenyl-4-(thiophen-2-ylmethyl)-2,4-dihydro-3H-pyrazol-3-one) as a potent mixed-competitive inhibitor of PL (IC₅₀=0.30 μM). In addition, P32 displayed some selectivity over other known serine hydrolases. A molecular docking study for P32 demonstrated that the inhibitory activity of P32 towards PL could be attributed to the π-π interactions of 2-naphthyl unit (R¹) and hydrophobic interactions of phenyl moiety (R³) with the active site of PL. Thus, P32 could serve as promising lead compound for the development of more efficacious and selective pyrazolones-type PL inhibitors for biomedical applications.     

乙酸芳樟酯的合成研究

以芳樟醇和醋酐为原料 ,无水K2 CO3为催化剂合成了乙酸芳樟酯。反应最佳工艺条件为 :醇酐质量比为 1∶1 2 ,催化剂用量为芳樟醇投料量的 4 % ,反应时间为 2 4h ,反应温度为 116± 2℃。芳樟醇转化率大于 95 2 % ,产品中乙酸芳樟酯含量大于 94 1%。     

Chemoselective hydrolysis of methyl 2‐acetoxybenzoate using free and entrapped esterase in K‐carrageenan beads

Porcine liver esterase was entrapped in natural polysaccharides K‐carrageenan and retention of its activity was determined using p‐nitrophenyl acetate as the substrate. The optimum pH for esterase activity of entrapped enzyme showed a little shift towards acidic side. Immobilized enzyme showed improved thermal and storage stability. The entrapped esterase retained 50% of its activity after eight repetitive cycles. Michaelis constant K_m for the free and entrapped enzymes was almost same indicting no conformational change during immobilization. Maximum velocity V_max was observed to decrease on immobilization. The free and entrapped esterase was used for selective hydrolysis of methyl 2‐acetoxybenzoate to methyl 2‐hydroxybenzoate in batch process as well as in a fixed bed reactor. The hydrolysis was observed to be 99% within 2 h for free as well as immobilized enzyme in batch process. The rate of hydrolysis was found to depend on pH. The turn over number of selective hydrolysis in batch and fixed bed reactor was 3.08 × 10⁶ and 1.19 × 10⁷, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008