Nitrilases reaction

Not surprisingly, some nitrilase reactions were accompanied by the formation of the corresponding amides, such as pipecolic amide 12b (up to 10%) and pyrrolidine-3-carbo3amide 10b (for a discussion of nitrile hydratase activity of nitrilases see Section 15.3.3). 

The formation of amides during nitrilase reactions was observed as early as 1964... 

Mahadevan and Thimann [43] postulated the first nitrilase reaction mechanism, suggesting that the nitrile carbon present in the substrate displays a partial positive charge that is subjed to nudeophihc attack by one of the two SH groups in the nitrilase active site. The resulting thioimidate is then hydrolyzed to a thioester, with the release of ammonia as a by-product Hydrolysis of the acyl-enzyme then results in the release of the final acid product. 

The discovery and exploitation of enzymes in aldoxime-nitrile pathway nitrile hydratase, amidase, nitrilase, aldoxime dehydratase, etc., are shown along with the use of methodologies, such as organic chemistry, microbial screening by enrichment and acclimation culture techniques, enzyme purification, gene cloning, molecular screening by polymerase chain reaction (PCR). 

Hydrolases lipase, protease, esterase nitrilase, nitrile hydratase glycosidase, phosphatase hydrolysis reactions in H20... 

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

The preparation of malonic acid monoesters has been demonstrated using the microbial nitrilase activity of Corynebacterium nitrilophilus ATCC 21 419, Gordona terrae MA-1, or Rhodococcus rhodochrous ATCC 33 025 to hydrolyze methyl cyanoacetate, ethyl cyanoace-tate, M-propyl cyanoacetate, isopropyl cyanoacetate, M-butyl cyanoacetate, tertbutyl cyanoacetate, 2-ethylhexyl cyanoacetate, allyl cyanoacetate, and benzyl cyanoacetate [96]. By maintaining the concentration of nitrile in a reaction mixture at <5 wt%, significant inactivation of the nitrilase activity was avoided for example, a total of 25 g of M-propyl cyanoacetate was added in sequential 5g portions to a lOOmL suspension of Rhodococcus rhodochrous ATCC 33 025 cells (OD630 = 5.6) in 50 mM phosphate buffer (pH 7.0) over 30h at 25 °C to produce mono-M-propyl malonate in 100% yield (Figure 8.17). 

In summary, the formation of optically active compounds through hydrolysis reactions is dominated by biocatalysis mainly due to the availability and ease of use of a wide variety of esterases, lipases and (to a lesser extent) acylases. Epoxide ring-opening (and related reactions) is likely to be dominated by salen-metal catalysts while enzyme-catalysed nitrile hydrolysis seems destined to remain under-exploited until nitrilases or nitrile hydratases become commercially available. 

DeSantis et have reported the discovery of new nitrilases through the screening of genomic libraries created by the extraction of DNA from various environments (metagenomics). In preliminary experiments, using 25 mM mandelonitrile in pH 8 buffer containing 10% methanol and 0.12 g mL of one of these nitrilases, the acid was produced quantitatively with 98 % ee within 10 min. The product was subsequently shown to be (7 )-mandelic acid after isolation in 86 % yield. In a parallel reaction, (/ )-2-chloromande-lic acid was produced at a seventeenth of the rate (Scheme 1.44). 

Nitrilase-mediated conversion of 3-cyanopyridine into nicotinic acid is an attractive alternative to chemical methods of nicotinic acid synthesis.It has been synthesized using whole cells (containing nitrilase) of some microorganisms and involves the following reaction ... 

Nitrilases, Nitrile Hydratases, and Amidases 5.03.8.1 The Reactions and the Enzymes... 

Recently, another interesting application of nitrilases has been demonstrated for the synthesis of pregabalin-the API of the neurophatic pain drug Lyrica. In this approach, the key step is the resolution of racemic isobutylsuccinonitrile (Scheme 10.8) [18], the process takes place with total regio- and stereoselectivity, and the (S)-acid is obtained and the (R)-substrate can be recycled under basic conditions. To improve the biocatalytic step, directed evolution was applied using the electronic polymerase chain reaction and in the first round of evolution a single C236S mutation led to a mutant with 3-fold increase in activity [19]. 

Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

Chemical reactions enhanced by catalysts or enzymes are an integral part of the manufacturing processes for the majority of chemical products. The total market for catalysts and enzymes amounts to 11.5 billion (2005), of which catalysts account for about 80%. It consists of four main applications environment (e.g., automotive catalysts), 31% polymers (e.g., polyethylene and polypropylene), 24% petroleum processing (e.g., cracking and reforming), 23% and chemicals, 22%. Within the latter, particularly the catalysts and enzymes for chiral synthesis are noteworthy. Within catalysts, BINAPs [i.e., derivatives of 2,2 -bis(diphenylphosphino) -1, l -bis-l,l -binaphthyl) have made a great foray into chiral synthesis. Within enzymes, apart from bread-and-butter products, like lipases, nitrilases, acylases, lactamases, and esterases, there are products tailored for specific processes. These specialty enzymes improve the volumetric productivity 100-fold and more. Fine-chemical companies, which have an important captive use of enzymes, are offering them to third parties. Two examples are described here ... 

Figure 5.5 Degradation routes for nitriles. The first route is a two-step reaction involving a nitrile hydratase, which converts the nitrile to the amide, and an amidase, which converts the amide to the corresponding acid. The second pathway involves direct hydrolysis of the nitrile to the carboxylic acid and ammonia by a nitrilase.

FIGURE 19.1 General reactions of hydrolase enzymes. In the same group as nitrilase enzymes are the amidases. This includes amino acid amidase ... 

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