Dimethylcyclopropane Carboxamide

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  

The second-generation process introduced the hydrolysis of the racemic nitrile to the racemic amide (Fig. 9) by nitrile hydratase containing whole cells. The nitrile hydratase step led to several improvements  

This resulted in a two-step, one pot biotransformation. In this process the gene for the amidase was also cloned into Escherichia coli, resulting in a number of additional advantages  

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. 

An alternative synthesis starting from L-arginine, and involving the biotransformation of (S)-5-[(amino-iminomethyl) amino]-2-chloropentanoic acid (L-Cl-argi-nine) to (S)-5-amino-2-chloropentanoic acid (L-Cl-omithine), which then sponta- 

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A key step in the synthesis of the /3-lactamase inhibitor cilastatin (Bayer, Leverkusen, Germany) is the preparation of (S)-2,2-dimethylcyclopropane carboxamide. The chemically synthesized corresponding nitrile, l-cyano-2,2-dimethylcyclopropane, is hydrolyzed by a highly active but enanhounspecific nitrile hydratase to the racemic carboxamides. An amidase from Comomonas acidovorans overexpressed in E. coli selectively hydrolyzes the undesired (R)-isomer to the acid. The remaining (S) enantiomer is obtained with > 99% e.e. and 48% conversion (the resulting E value thus exceeds 100). The (R)-acid is recycled by chemical amidation with thionyl chloride and ammonia. The process has been developed by Lonza (Visp, Switzerland) and runs on a 15 m3 scale (Rasor, 2001). 

Fig. 8 Possible synthetic routes for the production of (R,S)-2,2-dimethylcyclopropane carboxamide.

A stereospecific amidase from Comamonas acidovorans A18 was used for the biotechnological synthesis of (S)-dimethylcyclopropane carboxamide, which is employed as a precursor for the synthesis of the dihydropeptidase inhibitor Cilastin [89]. The (/ )-acid was chemically recycled to the racemic amide (Fig. 32). The amidase gene was cloned into a faster growing E. coli strain leading to a constitutive enzyme expression and a shortened fermentation process. 

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

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