Organic hydrolytic kinetic resolution

Several reports regarding the directed evolution of enantioselective epoxide hydrolases (EHs) have appeared [23,57-59]. These enzymes constitute important catalysts in synthetic organic chemistry [4,60]. The first two reported studies concern the Aspergillus niger epoxide hydrolase (ANEH) [57,58]. Initial attempts were made to enhance the enantioselectivity of the AN E H -catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-19). The WT leads to an Evalue of only 4.6 in favor of (S)-20 (see Scheme 2.4) [58]. 

Lipases are the most frequently used enzymes in organic chemistry, catalyzing the hydrolysis of carboxylic acid esters or the reverse reaction in organic solvents [3,5,34,70]. The first example of directed evolution of an enantioselective enzyme according to the principle outlined in Fig. 11.2 concerns the hydrolytic kinetic resolution of the chiral ester 9 catalyzed by the bacterial lipase from Pseudomonas aeruginosa [8], This enzyme is composed of 285 amino acids [32]. It is an active catalyst for the model reaction, but enantioselectivity is poor (ee 5 % in favor of the (S)-acid 10 at about 50 % conversion) (Fig. 11.10) [71]. The selectivity factor E, which reflects the relative rate of the reactions of the (S)- and (R)-substrates, is only 1.1. 

In the hydrolytic kinetic resolution of lactones, the separation of the formed (water-soluble) hydroxycarboxylic acid from unreacted (lipophilic) lactone is particularly easy via extraction using an aqueous-organic system. 

Jacobsen s hydrolytic kinetic resolution of epoxides catalyzed by a Co(salen) catalyst analogous to the one used for asymmetric epoxidation has brought a considerable advance to the use of epoxides. Indeed, these substrates are among the most useful reagents in organic synthesis. One of the two epoxide enantiomers is selectively opened by a nucleophile (including water), which leads to both the terminal epoxide and the functional alcohol in quantitative yields (i.e. 50% of each) and more than 98 e.e. for both products. This system has been applied industrially by Rhodia on ton-scales for hydrolysis of propylene oxide and epichlorhydrin. - ... 

Karboune, S., Archelas, A. and Baratti, J.C. (2010) Free and immobilized Aspergillus niger epoxide hydrolase-catalyzed hydrolytic kinetic resolution of racemic p-chlorostyrene oxide in a neat organic solvent medium. Process Biochem., 45,210-216. 

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

Biocatalysts, mainly hydrolytic enzymes and oxidoreductases, have been used for organic reactions due to their excellent enantioselectivities and environmentally friendliness.1 Typical enzymatic reactions used for the organic synthesis are shown in Figure 1. Especially, hydrolytic enzymes for kinetic resolutions of racemates have been utilized widely because of their high stabilities, wide substrate specificities, lack of cofactor requirements and high availabilities. 

Lipases have been extensively used for the kinetic resolution of racemic alcohols or carboxylic acids in organic solvents. Chiral alcohols are usually reacted with achiral activated esters (such as vinyl, isopropenyh and trichloroethyl esters) for shifting the equilibrium to the desired products and avoiding problems of reversibility. For the same reasons, chiral acids are often resolved by using acidolysis of esters. In both cases, the overall stereoselectivity is affected by the thermodynamic activity of water of water favors hydrolytic reactions leading to a decrease in the optical purity of the desired ester. Direct esterifications are therefore difficult to apply since water formed during the reaction may increase the o of the system, favors reversibiUty, and diminishes the overall stereoselectivity. 

Scheme 2.15 gives some examples of the use of epoxide hydrolases in organic synthesis. Entries 1 to 3 are kinetic resolutions. Note that in Entry 1 the hydrolytic product is obtained in high e.e., whereas in Entry 2 it is the epoxide that has the highest e.e. In the first case, the reaction was stopped at 18% conversion, whereas in the second case hydrolysis was carried to 70% completion. The example in Entry 3 has a very high E (> 100) and both the unreacted epoxide and diol are obtained with high e.e. at 50% conversion. Entry 4 shows successive use of two separate EH reactions having complementary enantioselectivity to achieve nearly complete... 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

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