Biocatalytic reaction proteases

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. 

The principal methods for the hydrolase-promoted synthesis of enantiomerically pure alcohols are depicted in Figure 6.44. Biocatalytic acylation and alcoholysis have been reviewed recently [116,117]. Lipases, esterases, and proteases catalyze these reactions, but CAL-B [118-120], CRL [121,122], and diverse lipase preparations from Pseudomonas species are common place. 

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. 

The range of nucleophiles that lipases accept is not confined to water or alcohols. There are numerous examples of amines, hydrazine, phenols," and hydrogen peroxide. Proteases have frequently been used in biocatalytic transformations involving ester hydrolysis and esterification reactions and their different stereoselection often provides a useful complement to the lipases. - ... 

There are various biocatalytic hydrolysis and esterification reactions that result in dynamic kinetic resolution (Fig. 10.43). For example, a protease-catalyzed ester hydrolysis... 

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. 

The first biocatalytic asymmetric Mannich reaction was reported recently by Guan and coUeagues. They found that protease type XIV from Streptomyces griseus was able to catalyze the Mannich reaction between cyclohexanone, j9-nitrobenzaldehyde, and anUine in moderate yields though but in good diastereo- and enantioselectivities (dr=85/15 syn/anti 82% ee) (Scheme 11.41) [126]. Remarkably, control experiments with metal ion or urea-denatured enzyme still afforded the Mannich product in high yields (>51% yield) compared to the blank reaction without enzyme (28% yield), but almost all stereoselectivity was lost. These... 

C. Li, Y.-l. Zhou, N. Wang, X.-W. Feng, K. Li, X.-Q. Yu, Promiscuous protease-catalyzed Aldol reactions a facile biocatalytic protocol for carbon-carbon bond formation in aqueous media, 1. Biotechnol. 150 (2010) 539-545. 

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