Chemoenzymatic synthesis of chiral carboxylic acids via nitriles

BACKGROUND: Enantiomerically pure 1,4‐benzodioxane‐2‐carboxylic acid derivatives are useful building blocks for the synthesis of pharmaceuticals and biologically active compounds whose interaction with their biological target (enzyme, receptor) depends very much on the absolute configuration of the chiral carbon at the 2‐position. The aim of the present work is to investigate the route to racemic nitriles and the subsequent selective enzymatic hydrolysis by nitrilase to optically active 1,4‐benzodioxane‐2‐carboxylic acid and 6‐formyl‐1,4‐benzodioxane‐2‐carboxylic acid. RESULTS: A range of microbial nitrilases from Rhodococcus, Alcaligenes and Pseudomonas strains have been prepared and screened for the desired biotransformations using a chiral high performance liquid chromatography (HPLC) analytical method. The nitrilase from Alcaligenes faecalis ATCC 8750 showed the highest and the nitrilase from Rhodococcus rhodochrous NCIMB 11216 the lowest activity towards 2‐cyano‐6‐formyl‐1,4‐benzodioxane. Lyophilised cells of Rhodococcus R 312 gave the (R)‐1,4‐benzodioxane‐2‐carboxylic acid with high enantioselectivity after 25% conversion. Excellent enantioselectivities for the hydrolysis of both 2‐cyano‐1,4‐benzodioxane as well as 2‐cyano‐6‐formyl‐1,4‐benzodioxane have been achieved and the absolute configuration of 1,4‐benzodioxane‐2‐carboxylic acid was determined to be R by comparison with the specific rotation of commercially available (R)‐1,4‐benzodioxane‐2‐carboxylic acid. CONCLUSIONS: This new nitrilase‐catalysed kinetic resolution of 2‐cyano‐ and 2‐cyano‐6‐formyl‐1,4‐benzodioxane opens a mild route to optically active 1,4‐benzodioxane‐2‐carboxylic acids. As the formyl functional group would be damaged in chemical nitrile hydrolysis, nitrilase‐catalysed hydrolysis solves this synthetic bottleneck and advances nitrilase biocatalytic tools for the preparation of more complex 1,4‐benzodioxane‐2‐carboxylic acids. Copyright © 2007 Society of Chemical Industry     

Enantioselective Biotransformations of Nitriles in Organic Synthesis

Recent advances in enantioselective biotransformations of nitriles utilizing nitrile-hydrolyzing microorganisms and enzymes are summarized in this article. Various examples of biocatalytic synthesis of highly enantiopure carboxylic acids including amino acids, hydroxyl acids, cyclopropanecarboxylic acids and amide derivatives and oxiranecarboxamides are presented. The enantioselectivity of the nitrile hydratases, nitrilases and amideses is discussed.     

Engineering residues on C interface to improve thermostability of nitrilase for biosynthesis of Pregabalin precursor

Nitrilase-mediated bioprocesses exhibited great potential in the production of value-added carboxylic acids. However, poor thermostability of nitrilases usually restricts their industrial applications. Herein, the thermostability of nitrilase BaNIT_M0 was significantly improved by engineering the amino acid residues on the intersection of two dimers (C interface). Except for simultaneous enhancement of enantioselectivity and activity, the best variant V82L/M127I/L159M/F166Q/C237S/Q260H (BaNIT_M4) showed a 10.8-fold increase in half-life at 30°C compared with BaNIT_M0. Structural analysis demonstrated that additional hydrogen bonds were formed between the residues on the C interface, which strengthened the interactions of two symmetrical regions and spirals. Subsequently, the engineered nitrilase was immobilized onto epoxy resins LXTE-603 and the immobilized nitrilase exhibited excellent stability over 12 repeated cycles, which indicated a great industrial potential for biosynthesis of Pregabalin precursor.     

Nitrilase‐Catalyzed Selective Hydrolysis of Dinitriles and Green Access to the Cyanocarboxylic Acids of Pharmaceutical Importance

To further explore its synthetic applications, the nitrilase bll6402 from Bradyrhizobium japonicum strain USDA110 has been examined toward the hydrolysis of various dinitriles. It has been found that nitrilase bll6402 effectively hydrolyzed α,ω‐dinitriles to ω‐cyanocarboxylic acids, and the selectivity was independent of the substrate chain length. This feature is distinct from all the known nitrilases of various sources. Nitrilase bll6402 was thus applied to the synthesis of 1‐cyanocycloalkaneacetic acids, the useful precursors for the synthesis of gabapentin and its analogues.     

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C₄–C₈) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.     

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short‐chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C₂ to C₆. In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole‐cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane‐1,2‐diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)‐1,2‐PDO (1.0 % yield) or 9.6 mm (S)‐1,2‐PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.     

Creation of an Engineered Amide Synthetase Biocatalyst by the Rational Separation of a Two-Step Nitrile Synthetase

The synthesis of amides through acid and amine coupling is one of the most commonly used reactions in medicinal chemistry, yet still requires atom-inefficient coupling reagents. There is a current demand to develop greener, biocatalytic approaches to amide bond formation. The nitrile synthetase (NS) enzymes are a small family of ATP-dependent enzymes which catalyse the transformation of a carboxylic acid into the corresponding nitrile via an amide intermediate. The Bacillus subtilis QueC (BsQueC) is an NS involved in the synthesis of 7-cyano-7-deazaguanine (CDG) natural products. Through sequence homology and structural analysis of BsQueC we identified three highly conserved residues, which could potentially play important roles in NS substrate binding and catalysis. Rational engineering led to the creation of a NS K163A/R204A biocatalyst that converts the CDG acid into the primary amide, but does not proceed to the nitrile. This study suggests that NSs could be further developed for coupling agent-free, amide-forming biocatalysts.     

Influence of different carboxy-terminal mutations on the substrate-, reaction- and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191

Different members of the nitrilase superfamily (D-carbamoylases, Nit-Fhit proteins, amidases, cyanide dihydratases and nitrilases) were compared by multiple sequence alignments and a long carboxy-terminal extension (about 50 amino acids) identified in all nitrilases and cyanide dihydratases which was not present in other members of the nitrilase superfamily. The function of this C-terminal part was experimentally analysed in the arylacetonitrilase of Pseudomonas fluorescens EBC191 by the construction of various deletion mutants, chimeric enzymes with other bacterial nitrilases and site-specific mutagenesis. The enzyme variants were tested with the substrates 2-phenylpropionitrile and mandelonitrile and compared regarding specific activities, degree of amide formation and enantioselectivity. The enzyme variants containing deletions up to 32 amino acids did not show significant differences in comparison with the wild-type enzyme. Deletion mutants with 47-67 amino acids missing generally demonstrated reduced enzyme activities, increased amounts of amide formation and increased proportions of the (R)-enantiomers of the amides and acids formed. Also certain exchanges of H296 in the C-terminal motif DpvGHY led to enzyme variants with a similar phenotype. Chimeric enzymes which contained up to 59 amino acids deriving from the nitrilases of Rhodococcus rhodochrous NCIMB11216 or Alcaligenes faecalis ATCC8750 were active and resembled, with respect to the enantioselectivity and degree of amide formation, the wild-type enzyme of P.fluorescens.     

A Tandem Enzymatic sp2‐C‐Methylation Process: Coupling in Situ S‐Adenosyl‐l‐Methionine Formation with Methyl Transfer

A one‐pot, two‐step biocatalytic platform for the regiospecfic C‐methylation and C‐ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S‐adenosyl‐l ‐methionine (SAM), followed by alkylation of aromatic substrates by the C‐methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled ¹³CH_3, ¹³CD₃ and CD₃ groups from their corresponding SAM analogues formed in situ.     

From Bugs to Bioplastics: Total (+)-Dihydrocarvide Biosynthesis by Engineered Escherichia coli

The monoterpenoid lactone derivative (+)-dihydrocarvide ((+)-DHCD) can be polymerised to form shape-memory polymers. Synthetic biology routes from simple, inexpensive carbon sources are an attractive, alternative route over chemical synthesis from (R)-carvone. We have demonstrated a proof-of-principle in vivo approach for the complete biosynthesis of (+)-DHCD from glucose in Escherichia coli (6.6 mg L⁻¹). The pathway is based on the Mentha spicata route to (R)-carvone, with the addition of an ′ene′-reductase and Baeyer–Villiger cyclohexanone monooxygenase. Co-expression with a limonene synthesis pathway enzyme enables complete biocatalytic production within one microbial chassis. (+)-DHCD was successfully produced by screening multiple homologues of the pathway genes, combined with expression optimisation by selective promoter and/or ribosomal binding-site screening. This study demonstrates the potential application of synthetic biology approaches in the development of truly sustainable and renewable bioplastic monomers.     

Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

Biocatalytic dealkylation of aryl methyl ethers is an attractive reaction for valorization of lignin components, as well as for deprotection of hydroxy functionalities in synthetic chemistry. We explored the demethylation of various aryl methyl ethers by using an oxidative demethylase from Pseudomonas sp. HR199. The Rieske monooxygenase VanA and its partner electron transfer protein VanB were recombinantly coexpressed in Escherichia coli and they constituted at least 25 % of the total protein content. Enzymatic transformations showed that VanB accepts NADH and NADPH as electron donors. The VanA–VanB system demethylates a number of aromatic substrates, the presence of a carboxylic acid moiety is essential, and the catalysis occurs selectively at the meta position to this carboxylic acid in the aromatic ring. The reaction is inhibited by the by-product formaldehyde. Therefore, we tested three different cascade/tandem reactions for cofactor regeneration and formaldehyde elimination; in particular, conversion was improved by addition of formaldehyde dehydrogenase and formate dehydrogenase. Finally, the biocatalyst was applied for the preparation of protocatechuic acid from vanillic acid, giving a 77 % yield of the desired product. The described reaction may find application in the conversion of lignin components into diverse hydroxyaromatic building blocks and generally offers potential for new, mild methods for efficient unmasking of phenols.     

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine‐tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross‐linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross‐linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross‐linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)‐mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nit‐CLEAs). The nitrilase activity in the CLEAs was at 30 °C and 60 °C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)‐mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 °C to 50 °C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.     

Asymmetric Synthesis of 3‐Substituted Cyclohexylamine Derivatives from Prochiral Diketones via Three Biocatalytic Steps

Prochiral bicyclic diketones were transformed to a single diastereomer of 3‐substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a C C hydrolase (6‐oxocamphor hydrolase) in the first step and by an ω‐transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the C C hydrolytic step as well as the esterification could be run simultaneously in a one‐pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio‐amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω‐transaminase employed and the substrate the cis‐ as well as the trans‐diastereomers could be obtained in optically pure forms.     

Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.     

Biocatalytic synthesis of some chiral pharmaceutical intermediates by lipases

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of three pharmaceutical drug candidates. These include (i) the synthesis of [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone2 for the semi-synthesis of paclitaxel (taxol)5, an anticancer compound; (ii) synthesis of chiral (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimenthanol monoacetate ester9 for the chemoenzymatic preparation of a thromboxane A₂ antagonist; (iii) the enzymatic synthesis ofS-(−) 3-benzylthio-2-methylpropanoic acid, a key chiral intermediate for the synthesis of antihypertensive drugs captopril10 or zofenopril13.     

Phenylalanine meta-Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation

The rare nonproteinogenic amino acid, meta-l -tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorubidus. Insights from kinetic analyses of the wild-type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta-hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3-position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme-catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2-hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta-l -tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.     

一种腈水解酶的高通量筛选方法

水解腈的酶类(腈水解酶或腈水合酶/酰胺酶)在制药行业中生产有机羧酸及其衍生物方面有着广泛的应用,为了获得较多的酶源,建立高通量筛选方法是非常必要的.今利用改进的羟肟酸铁分光光度比色法建立了一种简单、快速、高通量的筛选方法,同时利用产酶菌株Rhodococcus sp. CCZU10-1静息细胞催化反应来进行方法的准确性验证.并且通过与液相色谱法检测结果进行对比,结果表明羟肟酸铁比色法对有机羧酸的检测具有较高的准确性.因此,建立的高通量筛选方法在产腈水解酶微生物筛选及应用方面有很大应用前景.     

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate‐2‐Kinase

The efficient synthesis of pure d ‐glycerate‐2‐phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one‐step biocatalytic phosphorylation of glyceric acid. Glycerate‐2‐kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate‐2‐kinase‐catalyzed phosphorylation was followed by ³¹P NMR and showed excellent enantioselectivity towards phosphorylation of the d ‐enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d ‐glycerate 2‐phosphate. This phosphorylation reaction, using recombinant glycerate‐2‐kinase, yielded d ‐glycerate 2‐phosphate in fewer reaction steps and with higher purity than chemical routes.     

Selective Extraction and Spectrophotometric Determination of Vanadium(V) with N-Hydroxy-N-p-chlorophenyl-N′-(2-methyl-5-chloro)phenyl-p-toluamidine Hydrochloride and Various Monobasic Carboxylic Acids

Abstract

N-Hydroxy-N-p-chlorophenyl-N′-(2-methyl-5-chloro)phenyl-p-toluamidine hydrochloride (HCPMCPTH), a newly synthesized bidentate chelating agent, reacts with vanadium(V) in the presence of monobasic carboxylic acids to give blue-violet water-insoluble adducts. The adducts have been extracted into various organic solvents such as chloroform, benzene, and carbon tetrachloride, and employed for the spectrophotometric determination of vanadium(V) and its separation from diverse ions. Investigations of the vanadium-HCPMCPTH-carboxylic acid complexes by spectrophotometric and solvent extraction techniques showed that the complex had the composition VO(OAm)₂OH.HOOCR (where HOAm = hydroxyamidine and HOOCR = carboxylic acid). The molar absorptivity and wavelength of maximum absorption of the mixed complex depend on the nature of the carboxylic acid used.     

β-Mercaptoalkanoic carboxylic esters: versatile synthons in heterocyclic chemistry

This review presents approaches to the synthesis of heterocyclic annelated thiophenes of the last 35 years. Annelated thiophenes have been reported by the cyclocondensation reaction of β -mercaptoalkanoic carboxylic esters with 1,3-bifunctional substrates such as α -haloenones, β -haloenones, α -halogenoacrylic acid derivatives. The reaction takes place by nucleophilic addititon, followed by Dieckmann cyclization with the elimination of hydrogen halide. In case of other substrates such as β -chlorocinnamonitriles, β -haloacrylonitriles, arylsulfonylacrylonitriles, α,β -unsaturated nitriles, Dieckmann–Thorpe–Ziegler cyclization occurs resulting in aminothiophene derivatives. Functionalized annulated thiophene derivatives have potent industrial applications because of significant biological properties.