S -specific amidase

An example of a very efficient asymmetric transformation is the preparation of (W)-phcnylgly-cine amide (Scheme 7.8) (see also Chapter 25).40 This offers a good alternative to the enzymatic resolution of (fCS )-phcnylglycinc amide with the (S)-specific amidase from Pseudomoms putida.41 This amide is used in a coupling process for semi-synthetic antibiotics.42... 

Figure 16 Conversion of (i ,S)-2-aiylpropionitriles by an (i )-selective nitrile hydratase and an (S)-specific amidase from Rhodococcus rhodochrous ATCC 21197.

Pseudomonas sp. B21C9 was isolated from soil by an enrichment culture using (jR,S) 2-isopropyl-4-chlorophenylacetonitrile as the sole source of nitrogen [62]. The strain converted this nitrile to both enantiomerically pure (5)-acid and (/ )-amide (Fig. 19). In order to increase the yield of the (S )-acid, which is part of a commercial pyrethroid insecticide, the (.R)-amide was racemized by heating. Besides a strictly (S)-specific amidase, a poorly... 

The hydrolysis with a (R)-specific amidase from Klebsiella oxytoca resolved 2-hydroxy- and 2-amino-3,3,3-trifluoro-2-methylpropionamide, giving the (R)-acid and the remaining (S)-amide (Figure 6.39) [104,105]. 

Shaw, N.M., Naughton, A., Robins, K., et al. (2002) Selection, purification, characterisation, and cloning of a novel heat-stable stereo-specific amidase from Klebsiella oxytoca, and its application in the synthesis of enantiomerically pure (R)- and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide. Organic Process Research Development, 6, 497-504. 

Pantothenic acid is largely excreted unchemged by mammds. Some phos-phopantetheine may also be excreted in the urine tifter administration of [ C]pantothenic acid, some of the label may be recovered in exhaled CO2. This is probably the result of intestinal bacterid metabolism, because many bacteria have ptmtothenase, a specific amidase that cleaves ptmtothenic acid to /S-dtmine and pantoic acid. Pseudomonas species are capable of using pantothenic acid as their sole carbon source. 

Immobilized Rhodococcus sp. SP 361 is an example that shows how complex stereoselective nitrile conversions might sometimes appear [57]. In the bioconversion of R,S)-2-alkylarylacetonitriles an unusual enantioselective diversity was observed. Whereas most racemic 2-alkylarylacetonitriles were converted to (S)-acids and (/ )-amides (Fig. 17), indicating the presence of an (>S)-selective amidase, ibuprofen nitrile was only converted to the ( )-acid (e.e. 32%) without any intermediate amide formation. A slow and strictly R) specific nitrile hydratase and a fast and less strict (5)-amidase were accounted for the (/ )-specific ibuprofen synthesis. The aryl-bound isobutyl moiety of ibuprofen nitrile seemed to invert the molecule orientation at the catalytic center of the nitrile hydratase. Furthermore, in the conversion of (.R,5)-2-(4-methylphenyl)propionitrile, not only the chiral S) acid (e.e. more than 95%, yield 41%) and (/ )-amide (e.e. more than 95%, yield 18%), but also enantiomerically almost pure (fi )-nitrile (e.e. more than 95%, yield 25%) was obtained. In this instance, the nitrile was hydrated with a partial (5)-selectivity. Overall, the absolute configuration of the products was rationalized according to a model assuming the presence of an (5)-selective amidase and a nitrile hydratase with (5)-, (JR)-, or nonspecificity depending on the type of substrate. 

Furthermore, Agrobacterium tumefaciens d3, which was enriched on racemic 2-phen-ylpropionitrile as the sole source of nitrogen, was used for the conversion of 2-arylpro-pionitriles to the corresponding (5)-acids [63]. The conversion of both racemic 2-phenyl-propionitrile and -amide by whole cells led to the formation of the (S)-acid with an e.e. above 96% after 47.5% conversion and was caused by the combined action of an (5)-specific nitrile hydratase and a highly (5)-specific amidase (Fig, 17). The stereoselectivity of the nitrile hydratase was used for the preparation of (5)-amides by whole cells in the presence of an amidase inhibitor or with the purified nitrile hydratase [64]. The highest... 

Together with R. rhodochrous ATCC 21197 [43] and Pseudomonas putida NRRL 18668 [51], also Rhodococcus sp. C3II md Rhodococcus erythropolis MP 50 were used for the enantiospecific preparation of (S)-naproxen [65]. Rhodococcus sp. C3II lacks a nitrilase but exhibits nitrile hydratase and amidase activities, both of which are constitutive and prefer the (5 )-enantiomers of naproxen derivatives. On the other hand, the enzymes from R. erythropolis MP 50 were induced by nitriles and its nitrile hydratase was (R)-specific [44]. Due to the presence of a strictly (5)-specific amidase, both strains finally formed (5)-naproxen with high enantioselectivity (Fig. 18). Evidence for the enantioselectivity of the nitrile hydratases of both strains was obtained by the formation of optically active amides in the presence of the amidase inhibitor diethyl phosphoramidate [63,65]. The nitrile hydratase of Rhodococcus sp. C3II whole cells was used for the sjmthesis of (>S)-naproxen amide with 94% e.e. after 30% conversion in the presence of the amidase inhibitor [63]. In addition, the highly stereoselective amidases of these two strains were used to prepare (5)-ketoprofen (Fig. 28), and the amidase from R. erythropolis MP 50 was used to prepare (5)-2-phenylpropionic acid with more than 99% e.e. and more than 49% conversion [66,67]. 

In contrast, the nitrile hydratase of R. rhodochrous IFO 15564, which was isolated by a screening using 3-hydroxypropionitrile and benzonitrile as the sole sources of nitrogen, was found to be (5)-selective toward substituted 3-hydroxyglutaronitriles [70]. Together with an (5)-specific amidase, it converted 3-benzoyloxyglutarodinitrile to the sole product (S)-cyanocarboxylic acid in an optically pure form without leaving any intermediate ( )-cyano amide (Fig. 21). 

Figure 32 Synthesis of (S)-dimethylcyclopropane carboxamide by an (/ )-specific amidase from Comamonas acidovorans A18.

Sonke T, S Ernste, RF Tandler, B Kaptein, WPH Peeters, FBI van Assema, MG Wubbolts, HE Schoemaker (2005) L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321. Appl Environ Microbiol 71 7971-7963. 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

S. -H. Hung, L. Hedstrom, Converting trypsin to elastase substitution of the SI site and adjacent loops reconstitutes esterase specificity but not amidase activity, Prot. Eng. 1998, 11, 669-673. 

Although carboxylesterases and amidases were thought to be different, no purified carboxylesterase has been found that does not have amidase activity toward the corresponding amide. Similarly enzymes purified on the basis of their amidase activity have been found to have esterase activity. Thus these two activities are now regarded as different manifestations of the same activity, specificity depending on the nature of R, R and R groups and, to a lesser extent, on the atom (O, S, or N) adjacent to the carboxyl group. 

Despite the presence of water throughout the body, hydrolysis reactions of esters and amides require enzymes to proceed at an appreciable rate. Numerous enzymes throughout the body carry out hydrolysis reactions. The enzymes, both esterases and amidases, are found in the digestive system, individual cells, and plasma. The exact site of hydrolysis of a specific drug depends on the drug s structure and functionality. The ester in aspirin... 

Novo, C., Farnaud, S., Tata, R., et al. 2002. Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. Biochemistry Journal, 365 731-8. 

The first generation process started with the chemical synthesis of the (R,S)-amide. There were several possible synthetic routes (Fig. 8) via the (R,S)-nitrile or (R,S)-acid [19, 20], A microbial screening program resulted in the isolation of several bacterial strains containing amidases that could specifically hydrolyze the (R)-amide. One of these strains, Comomonas acidivorans A 18 was particularly effective [21]. After the hydrolysis of the unwanted isomer the product (S)-2,2-dimethylcy-clopropane carboxamide was isolated from the bio-solution using a combination of salting-out and solvent extraction. This process had some intrinsic problems ... 

In the first step the (R,S)-nitrile is rapidly and quantitatively hydrolyzed to the (R,S)-amide. The amidase containing biomass is then added so that the (R)-amide is specifically hydrolyzed to the (R)-acid, and the product, the (S)-amide, remains. A completely new isolation process was developed ultra-filtration, electrodialysis, ion-exchange chromatography, reverse osmosis, crystallization, centrifugation, and drying. The product has been produced at a 15 m3 scale and has an ee value of >98%. The isolated yield calculated from the nitrile was >35%. The (R)-acid can be recycled by reacting it with thionyl chloride and ammonia to produce the (R,S)-amide. This results in minimal waste and higher yields. 

In addition to the more common (S)-amidase activities, (RJ-specific enzymes have also been identified. Thus (S)-ketoprofenamide has been derived from the racemic mixture using a biocatalyst from Comamonas acidovorans KPO 2771-4 to hydrolyze the (R)-enantiomer with high selectivity1141 (see Scheme 12.2-6). 

Although the liver is the body s principal centre for metabolic degradation, this process also goes on elsewhere. For example the bloodstream carries non-specific esterases which rapidly hydrolyse almost every kind of ester, natural or fabricated, and they are accompanied by a rather less active nonspecific amidase. Many degradative enzymes operate in the bowel-wall and the kidney. An inactivator of catecholamines, catechol 0-methyltransferase, is found in many tissues, and Section 12.5 describes the enzyme that hydrolyses acetylcholine in the neuromuscular junction. 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

Pollmann, S. et al. (2006) Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana. Planta 224,1241-1253... 

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