Biocatalytic reaction ester synthesis

Preparation of optically active P-aminoesters, P-aminonitriles, and P-aminocarbox-amides are of special relevance for the synthesis of enantiomerically pure P-aminoacids compounds of special relevance in several areas of medicinal chemistry. The resolution of P-aminoesters can be carried out by acylation of the amino groups or by other biocatalytic reactions of the ester groups, such as hydrolysis, transesterification, or aminolysis. The resolution of ethyl ( )-3-aminobutyrate... 

Since the beginning of the 20th century, organic solvents have been used in enzymatic reaction media [30]. Biocatalytic reactions in water-organic biphasic media were first carried out by Cremonesi et al. [31] and by Buckland et al. [32] less than 30 years ago. Their work aimed at the conversion of high concentrations of poorly water soluble components, particularly steroids. Later, biphasic systems were used for enzyme-catalyzed synthesis reactions that were unfavored in water, changing the reaction equilibrium towards the higher yield of the product, such as esters or peptides. 

A cost effective and easily scaled-up process has been developed for the synthesis of (S)-3-[2- (methylsulfonyl)oxy ethoxy]-4-(triphenylmethoxy)-1 -butanol methanesulfonate, a key intermediate used in the synthesis of a protein kinase C inhibitor drug through a combination of hetero-Diels-Alder and biocatalytic reactions. The Diels-Alder reaction between ethyl glyoxylate and butadiene was used to make racemic 2-ethoxycarbonyl-3,6-dihydro-2H-pyran. Treatment of the racemic ester with Bacillus lentus protease resulted in the selective hydrolysis of the (R)-enantiomer and yielded (S)-2-ethoxycarbonyl-3,6-dihydro-2H-pyran in excellent optical purity, which was reduced to (S)-3,6-dihydro-2H-pyran-2-yl methanol. Tritylation of this alcohol, followed by reductive ozonolysis and mesylation afforded the product in 10-15% overall yield with excellent optical and chemical purity. Details of the process development work done on each step are given. 

A fimctional, easily assembled, operated and cleaned microbioreactor packed with immobilized Candida antarctica lipase B (Novozyme 435) was recently developed by Pohar et al. (2010). So far, microbioreactor was used for studying continuous mode ester synthesis within bis(trifluoromethylsulfonyl)imide - based ionic liquid media. Ionic liquid containing substrates was pumped into the microbioreactor at various flow rates, and at the outlet of the reactor the product was collected and analyzed. With fuUy adjustable length, width and depth, the developed packed bed microbioreactor was proven to be a very successful and versatile tooling for biocatalytic reactions such as isoamyl acetate or butyl butyrate synthesis (Cvjetko et al, 2010 Pohar et al., 2010). 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

More recently, a biocatalytic manufacturing route was developed in which deacylation was accomplished by penicillin G acylase in water at room temperature, requiring no protection and deprotection (Scheme 8.9) [58]. Moreover, through reaction engineering, penicillin G acylase also catalyzes the acylation of 6-APA with either amino esters or aminoamides to produce a wide range of semi-synthetic P-lactam antibiotics such as amoxicillin and ampicillin. A similar approach could be applied to the synthesis of the 7-ADCA derivatives cefaclor, cephalexin, and cefadroxil. 

The synthesis of DPP-IV inhibitor Saxagliptin 5 also required (55)-5-amino-carbonyl-4,5-dihydro-lH-pyrrole-l-carboxylic acid, l-(l,l-dimethylethyl)ester 10 (Figure 16.3C). Direct chemical ammonolyses were hindered by the requirement for aggressive reaction conditions, which resulted in unacceptable levels of amide race-mization and side-product formation, while milder two-step hydrolysis-condensation protocols using coupling agents such as 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) [41] were compromised by reduced overall yields. To address this issue, a biocatalytic procedure was developed based on the Candida antartica lipase B (CALB)-mediated ammonolysis of (55)-4,5-dihydro-lH-pyrrole-l,5-dicarboxylic acid, l-(l,l-dimethylethyl)-5-ethyl ester 9 with ammonium carbamate to furnish 10 without racemization and with low levels of side-product formation. 

At this moment, fractionating reactors are mostly studied and applied outside the fine-chemical field. Examples are the large-scale production of the fuel ethers MTBE and TAME via reactive distillation. Also, biocatalytic studies have been performed. Malcata and co-workers investigated the integration of ester formation by Upases and distillative separation of the final products ester and water [44]. A number of synthesis reactions have been studied such as the esterification of ethanol and acetic acid to form ethyl acetate and water [45] in an SMB reactor with chemocatalysts (acidic ion exchange resins). Another, fairly similar appUcation was presented by Kawase et al. [46] to manufacture an ester from 2-phenylethanol. Mensah and Carta [47] used a chromatography column with lipases immobilised on resin to produce esters as well. 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

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