Nitrilase-catalyzed Hydrolysis Research Articles

Development of a Sustainable Chemoenzymatic Process for (S)-Pregabalin Synthesis via Nitrilase-Catalyzed Hydrolysis and Continuous Flow Racemization

Development of a Sustainable Chemoenzymatic Process for (<i>S</i>)-Pregabalin Synthesis via Nitrilase-Catalyzed Hydrolysis and Continuous Flow Racemization

Rational Regulation of Reaction Specificity of Nitrilase for Efficient Biosynthesis of 2-Chloronicotinic Acid through a Single Site Mutation.

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for the efficient synthesis of 2-chloronicotinic acid (2-CA). The development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potential. Herein, a nitrilase from Rhodococcus zopfii (RzNIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN to bind more deeply in the pocket and form a delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. IMPORTANCE 2-CA is an important building block for agrochemicals and pharmaceuticals with a rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, the byproduct 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeded under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN has not been reported because the enzymes showed either extremely low activity or surprisingly high hydration activity toward 2-CN. Herein, the reaction specificity of RzNIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without the formation of byproducts, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

Photoinduced Carboborative Ring Contraction Enables Regio- and Stereoselective Synthesis of Multiply Substituted Five-Membered Carbocycles and Heterocycles.

We report herein a photoinduced carboborative ring contraction of monounsaturated six-membered carbocycles and heterocycles. The reaction produces substituted five-membered ring systems stereoselectively and on preparative scales. The products feature multiple stereocenters, including contiguous quaternary carbons. We show that the reaction can serve as a synthetic platform for ring system alteration of natural products. The reaction can also be used in natural product synthesis. A concise total synthesis of artalbic acid has been enabled by a sequence of photoinduced carboborative ring contraction, Rauhut-Currier reaction, and nitrilase-catalyzed hydrolysis. The synthetic utility of the reaction has been further demonstrated by converting the intermediate organoboranes to alcohols, amines, and alkenes.

Efficient biosynthesis of β-alanine with a tandem reaction strategy to eliminate amide by-product in the nitrilase-catalyzed hydrolysis

An efficient biosynthesis of β-alanine from 3-aminopropionitrile at high concentration has been developed using a one-pot bienzymatic cascade of a nitrilase and an amidase. The nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 catalyzes the hydrolysis of 3-aminopropionitrile to β-alanine at the concentration up to 3.0mol/L with the formation 23% of 3-aminopropanamide. In order to eliminate the by-product 3-aminopropanamide, we cloned and characterized a new amidase from Pseudomonas nitroreducens through gene mining. Under the optimal conditions (50mmol/L Na2HPO4-NaH2PO4 buffer, pH 6.0, 40°C), 2.0mol/L (176g/L) of 3-aminopropanamide was completely hydrolyzed within 12h. A tandem reaction system was then established to eliminate the by-product 3-aminopropanamide and increase the production of β-alanine to 90% isolated yield with 15.02g/(L.h) space-time-yield. These results demonstrated that the tandem reaction strategy was an effective method of eliminating the amide by-products in the nitrilase-catalyzed hydrolysis at high substrate concentration.

A nitrilase-mediated entry to 4-carboxymethyl-β-lactams from chemically prepared 4-(cyanomethyl)azetidin-2-ones

4-(Cyanomethyl)azetidin-2-ones were efficiently prepared from 1,2:5,6-di-O-isopropylidene-d-mannitol, followed by a nitrilase-catalyzed hydrolysis to 4-carboxymethyl β-lactams without affecting the sensitive four-membered ring system.

Nitrilase-catalyzed hydrolysis of 3-aminopropionitrile at high concentration with a tandem reaction strategy for shifting the reaction to β-alanine formation

Given the importance of β-alanine, the nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 was examined toward the hydrolysis of 3-aminopropionitrile. It has been found that nitrilase BjNIT3397 effectively hydrolyzed 3-aminopropionitrile with substrate concentration up to 3M (210g/L) at the pH 7.3 and temperature 30°C. With the increase of substrate concentration from 0.6 to 3M, 3-aminopropanamide was formed and its percentage in the products was increased up to 33%. In order to reduce the formation of 3-aminopropanamide, aspartate ammonia-lyase and fumaric acid were added into the reaction system to consume the byproduct ammonia. As expected, the reaction was shifted toward the formation of β-alanine, resulting in the decrease of 3-aminopropanamide from 33% to 3%. Therefore, a tandem reaction strategy was developed to effectively prevent the formation of 3-aminopropanamide. This might also offer a possibility of producing β-alanine and l-aspartic acid in one process.

Asymmetric Synthesis of Both Antipodes of β-Hydroxy Nitriles and β-Hydroxy Carboxylic Acids via Enzymatic Reduction or Sequential Reduction/Hydrolysis

Use of isolated carbonyl reductases in the reduction of aromatic beta-ketonitriles have completely eliminated the competing alpha-ethylation, which is often observed with whole cell biocatalysts. By choosing suitable recombinant carbonyl reductase, the reduction of beta-ketonitriles afforded (R)- or (S)-beta-hydroxy nitriles with excellent optical purity and yield. Subsequently, nitrilase-catalyzed hydrolysis of the obtained optically pure beta-hydroxy nitriles led to the corresponding beta-hydroxy carboxylic acids in high yields. More importantly, the sequential enzymatic reduction and hydrolysis could be carried out in "two-step-one-pot" fashion without the isolation of intermediates beta-hydroxy nitriles, lowering the cost and minimizing the environmental impact. This allows ready access to both antipodes of chiral beta-hydroxy nitriles and beta-hydroxy carboxylic acids of pharmaceutical importance with excellent optical purity.

Asymmetric Reduction of β-Ketonitriles with a Recombinant Carbonyl Reductase and Enzymatic Transformation to Optically Pure β-Hydroxy Carboxylic Acids

Alpha-ethylation is concomitant with the reduction of aromatic beta-ketonitriles catalyzed by whole-cell biocatalysts. Use of isolated carbonyl reductase has completely eliminated this competing reaction. (R)-beta-Hydroxy nitriles were obtained via a reduction catalyzed by a recombinant carbonyl reductase with excellent optical purity and were further converted to (R)-beta-hydroxy carboxylic acids via a nitrilase-catalyzed hydrolysis. The present study allows ready access to both chiral beta-hydroxy nitriles and beta-hydroxy carboxylic acids of pharmaceutical importance.

Unexpected Stereorecognition in Nitrilase‐Catalyzed Hydrolysis of β‐Hydroxy Nitriles.

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Unexpected Stereorecognition in Nitrilase-Catalyzed Hydrolysis of β-Hydroxy Nitriles

Biocatalytic enantioselective hydrolysis of beta-hydroxy nitriles to corresponding (S)-enriched beta-hydroxy carboxylic acids has been achieved for the first time by an isolated nitrilase bll6402 from Bradyrhizobium japonicum USDA110. This offers a new "green" approach to optically pure beta-hydroxy nitriles and beta-hydroxy carboxylic acids. The observed remote stereorecognition is surprising because this nitrilase shows no enantioselectivity for the hydrolysis of alpha-hydroxy nitriles such as mandelonitrile.

A metal ion-based method for the screening of nitrilases

In this paper we describe a colorimetric method for the screening of nitrilases. When a buffered solution of CoCl 2 is added to a nitrilase-catalyzed hydrolysis reaction, the ammonia product forms a complex with the cobalt ion resulting in a color change from light pink to yellow, which can readily be quantified using a spectrophotometer at 375 nm. This method has been demonstrated for both wild-type and evolved nitrilases. ▪

Optimization of an Immobilized-Cell Biocatalyst for Production of 4-Cyanopentanoic Acid

Optimization of microbial cell immobilization, catalyst specific activity, and volumetric productivity were required for scale-up of the nitrilase-catalyzed hydrolysis of 2-methylglutaronitrile to 4-cyanopentanoic acid, an intermediate in the preparation of 1,5-dimethyl-2-piperidone. As an alternative to the immobilization of Acidovorax facilis 72W cells in carrageenan, immobilization in alginate, followed by cross-linking with glutaraldehyde and polyethylenimine, produced a catalyst which was stable in reaction mixtures containing high concentrations of 4-cyanopentanoic acid ammonium salt. Immobilization in alginate produced catalysts with a higher nitrilase specific activity than was achieved in carrageenan, and volumetric productivity of 4-cyanopentanoic acid was increased from 19 to 49 g/L/h. Substituting alginate for carrageenan also eliminated one process step in the immobilization. A further increase in volumetric productivity to 79 g/L/h was achieved by using an immobilized Escherichia coli transformant which expresses A. facilis 72W nitrilase.

Enzymatic Routes to Enantiomerically Pure Aromatic α-Hydroxy Carboxylic Acids: A Further Example for the Diversity of Biocatalysis

An important class of compounds which can be produced by means of enzymatic routes are enantiomerically pure aromatic α-hydroxy carboxylic acids, in particular mandelic acid and derivatives thereof. Numerous different types of enantioselective biocatalytic approaches to these target molecules have been developed. Among them are chiral enzymatic resolution processes using racemic precursors as well as asymmetric catalytic methods starting from prochiral compounds. Regarding the resolution processes, nitrilase-catalyzed hydrolysis and dehydrocyanation of racemic cyanohydrins as well as stereocontrolled ester cleavage of O-acetylcyanohydrins and α-hydroxy carboxylic acid esters, respectively, have been developed. Main contributions in the field of asymmetric catalytic concepts are the asymmetric reduction of α-keto acids as well as the asymmetric hydrocyanation of aldehydes. In the latter case, a subsequent chemical hydrolysis gives the desired products. In the following, these different concepts for the biocatalytic preparation of aromatic α-hydroxy carboxylic acid are discussed showing that biocatalysis can provide the organic chemist with many versatile synthetic pathways to obtain chiral aromatic α-hydroxy carboxylic acids. 1 Introduction 2 Overview: Biocatalytic Routes to Aromatic α-Hydroxy Carboxylic Acids 3 Route A: Enzymatic Hydrolysis of an Ester Group 4 Route B: Biocatalytic Transesterification − Enzymatic Hydrolysis of the O-Acetyl Group 5 Route C: Enzymatic Hydrolysis of the Cyano Group 6 Route D: Enzymatic Cleavage of rac-Cyanohydrins 7 Route E: Enantioselective Biocatalytic Hydrocyanation of Aromatic Aldehydes 7.1 Enzymatic Enantioselective Hydrocyanation in Organic Media 7.2 Enzymatic Enantioselective Hydrocyanation in Aqueous Media 7.3 Enzymatic Enantioselective Hydrocyanation in Biphasic Solvent Systems 7.4 Chemical Hydrolysis of Enantiomerically Enriched Cyanohydrins 8 Route F: Enantioselective Reduction of α-Keto Acids and Esters Thereof 9 Summary and Outlook

Enzymatic Routes to Enantiomerically Pure Aromatic α-Hydroxy Carboxylic Acids: A Further Example for the Diversity of Biocatalysis

An important class of compounds which can be produced by means of enzymatic routes are enantiomerically pure aromatic α-hydroxy carboxylic acids, in particular mandelic acid and derivatives thereof. Numerous different types of enantioselective biocatalytic approaches to these target molecules have been developed. Among them are chiral enzymatic resolution processes using racemic precursors as well as asymmetric catalytic methods starting from prochiral compounds. Regarding the resolution processes, nitrilase-catalyzed hydrolysis and dehydrocyanation of racemic cyanohydrins as well as stereocontrolled ester cleavage of O-acetylcyanohydrins and α-hydroxy carboxylic acid esters, respectively, have been developed. Main contributions in the field of asymmetric catalytic concepts are the asymmetric reduction of α-keto acids as well as the asymmetric hydrocyanation of aldehydes. In the latter case, a subsequent chemical hydrolysis gives the desired products. In the following, these different concepts for the biocatalytic preparation of aromatic α-hydroxy carboxylic acid are discussed showing that biocatalysis can provide the organic chemist with many versatile synthetic pathways to obtain chiral aromatic α-hydroxy carboxylic acids. 1 Introduction 2 Overview: Biocatalytic Routes to Aromatic α-Hydroxy Carboxylic Acids 3 Route A: Enzymatic Hydrolysis of an Ester Group 4 Route B: Biocatalytic Transesterification − Enzymatic Hydrolysis of the O-Acetyl Group 5 Route C: Enzymatic Hydrolysis of the Cyano Group 6 Route D: Enzymatic Cleavage of rac-Cyanohydrins 7 Route E: Enantioselective Biocatalytic Hydrocyanation of Aromatic Aldehydes 7.1 Enzymatic Enantioselective Hydrocyanation in Organic Media 7.2 Enzymatic Enantioselective Hydrocyanation in Aqueous Media 7.3 Enzymatic Enantioselective Hydrocyanation in Biphasic Solvent Systems 7.4 Chemical Hydrolysis of Enantiomerically Enriched Cyanohydrins 8 Route F: Enantioselective Reduction of α-Keto Acids and Esters Thereof 9 Summary and Outlook

Electronic substituent effects in the nitrilase-catalyzed hydrolysis of para-substituted benzyl cyanides

The initial rates of the nitrilase (Novo)-catalyzed hydrolysis of a series of para-substituted benzyl cyanides (R = NO 2, Cl, OCH 3, OH, NH 2) were found to be susceptible to the nature of the para-substituent of the substrate and a Hammett-type linear free energy correlation was observed with ϱ = 0.96. In a separate study, effective solubilization of substituted benzyl cyanide substrates having electron-donating groups (OH, NH 2, OCH 3) was achieved upon mixing with β-cyclodextrin to form 1:1 mol ratio inclusion complexes, but para-substituted benzyl cyanides with electron-withdrawing groups (C l, NO 2) were not fully solubilized under the same conditions. In addition, it was shown that the presence of β-cyclodextrin not only had no inhibitory effect on the enzyme activity, but it actually increased the initial rate of hydrolysis of the unsubstituted benzyl cyanide:β-cyclodextrin inclusion complex. However, the initial rates of hydrolysis were observed to be smaller when β-cyclodextrin was added to the para-substituted benzyl cyanides.