Lipase enzymes, stereoselectivity

In an alternative approach to prepare the chiral side chain of captopril (14) and zofenopril (18), the lipase-catalyzed stereoselective esterification of racemic 3-benzoylthio-2-methylpropionic acid (19) (Fig. 8B) in an organic solvent system was demonstrated to yield i -(+)-methyl ester (20) and unreacted acid enriched in the desired S-( )-enantiomer (19) [45], Using lipase PS-30 with toluene as solvent and methanol as nucleophile, the desired S-(-)-(19) was obtained in 37% reaction yield (theoretical max. 50%) and 97% e.e. Substrate was used at 22-g/ liter concentration. The amount of water and the concentration of methanol supplied in the reaction mixture was very critical. Water was used at 0.1 % concentration in the reaction mixture. More than 1% water led to the aggregation of enzyme in the organic solvent, with a decrease in the rate of reaction which was due to... 

Using a resolution process, chiral alcohol i -(+)-57 was also prepared by the lipase-catalyzed stereoselective acetylation of racemic 57 in organic solvent [85]. Various lipases were evaluated among which lipase PS-30 (Amano International Enzyme Co.) and BMS lipase efficiently catalyzed the acetylation of the undesired enantiomer of racemic 57 to yield S-(—)-acetylated product 60 and unreacted desired R-(+)-57 (Fig. 17). A reaction yield of 49 M% (theoretical maximum yield is 50 M%) and an e.e. of 98.5% were obtained for J -(+)-57 when the reaction was conducted in toluene as solvent in the presence of isopropenyl acetate as acyl donor. In methyl ethyl ketone at 50 g/L substrate concentration, a reaction yield of 46 M% (theoretical maximum yield is 50 M%) and optical purity of 96.4% were obtained for R-(+)-57. 

In all cases, the immobilized and the native enzyme stereoselectively esterify the 5-(+) enantiomer. The enantiomeric excess obtained with the native enzyme increases with the amount of water as described by Hoegberg et al. [125], whereas the immobilized derivatives are not affected by the additional water, except in the case of the derivatives on SiOa. Finally, we can observe in Table 10 that at the same reaction time (192 h) and at the same amount of water (1 ml buffer), only the immobilized lipase on Si02 displayed the same enzymatic activity and enantioselectivity as the native form. 

Esterases, proteases, and some lipases are used in stereoselective hydrolysis of esters bearing a chiral or a prochiral acyl moiety. The substrates are racemic esters and prochiral or meso-diesters. Pig liver esterase (PLE) is the most useful enzyme for this type of reaction, especially for the desymmetrization of prochiral or meso substrates. 

Enzyme-catalyzed stereoselective hydrolysis allows the preparation of enantio-merically enriched lactones. For instance. Pseudomonas sp. lipase (PSL) was found to be a suitable catalyst for the resolution of 5-undecalactone and 5-dodecalactone (Figure 6.20). Relactonization of the hydroxy acid represents an efficient method for the preparation of both enantiomers of a lactone [67]. 

The lipase-catalyzed DKRs provide only (/ )-products to obtain (5 )-products, we needed a complementary (5 )-stereoselective enzyme. A survey of (5 )-selective enzymes compatible to use in DKR at room temperature revealed that subtilisin is a worthy candidate, but its commercial form was not applicable to DKR due to its low enzyme activity and instability. However, we succeeded in enhancing its activity by treating it with a surfactant before use. At room temperature DKR with subtilisin and ruthenium catalyst 5, trifluoroethyl butanoate was employed as an acylating agent and the (5 )-products were obtained in good yields and high optical purities (Table 3)P... 

Another approach to the synthesis of chiral non-racemic hydroxyalkyl sulfones used enzyme-catalysed kinetic resolution of racemic substrates. In the first attempt. Porcine pancreas lipase was applied to acylate racemic (3, y and 8-hydroxyalkyl sulfones using trichloroethyl butyrate. Although both enantiomers of the products could be obtained, their enantiomeric excesses were only low to moderate. Recently, we have found that a stereoselective acetylation of racemic p-hydroxyalkyl sulfones can be successfully carried out using several lipases, among which CAL-B and lipase PS (AMANO) proved most efficient. Moreover, application of a dynamic kinetic resolution procedure, in which lipase-promoted kinetic resolution was combined with a concomitant ruthenium-catalysed racem-ization of the substrates, gave the corresponding p-acetoxyalkyl sulfones 8 in yields... 

The authors then used a modification of their Lipase-AK route to produce the natural enantiomer, as described in detail in the chapter by Kenji Mori in this volume. Instead of using the enzyme to execute a stereoselective monohydrolysis of meso diacetate 140, the enzyme was used to esterify selectively one of the hydroxy groups of meso diol 128, resulting in the antipodal hydroxyester. After oxidation of the free hydroxyl to the acid, and recrystallization of its salt with (JR)-l-naphthylethylamine, the purified acid was then carried through the remaining steps to furnish the chiral pheromone compound (see the chapter by Kenji Mori in this volume). 

Enzyme and Nonenzyme Catalysts By nature, enzymes themselves are chiral and they catalyze a variety of chemical reactions with stereoselectivity. These reactions include oxidation, reduction, and hydration. Examples of enzymes are oxidases, dehydrogenases, lipases, and proteases. Metoprolol, an adrenoceptor-blocking drug, is produced using an enzyme-catalyzed method. 

Another method for assaying the activity and stereoselectivity of enzymes at in vitro concentrations is based on surface-enhanced resonance Raman scattering (SERRS) using silver nanoparticles (116). Turnover of a substrate leads to the release of a surface targeting dye, which is detected by SERRS. In a model study, lipase-catalyzed kinetic resolution of a dye-labeled chiral ester was investigated. It is currently unclear how precise the method is when identifying mutants which lead to E values higher than 10. The assay appears to be well suited as a pre-test for activity. 

The beneficial effect of the hydrophobicity of [BMIM]PFg was shown to extend to other enzymes a remarkably enhanced enantioselectivity was observed for lipases AK and Pseudomonas fluorescens for the kinetic resolution of racemic P-chiral hydroxymethanephosphinates (Scheme 31) (278). The ee values of the recovered alcohols and the acetates were about 80% when the enzymatic reactions were conducted in the hydrophobic [BMIMJPFg. In contrast, there was little enantioselectivity (<5%) observed with the enzymes in hydrophilic [BMIM]BF4. The lack of stereoselectivity in [BMIM]BF4 was attributed to the high miscibility of [BMIM]BF4 with water. The relatively hydrophilic ionic liquid is capable of stripping off the essential water from the enzyme surface, leading to insufficient hydration of the enzyme and a consequently strong influence on its performance (279). 

As mentioned in part 2.1.3 hydrolytic enzymes are the most frequently used enzymes in organic chemistry. There are several reasons for this. Firstly, they are easy to ttse because they do not need cofactors like the oxidoreductases. Secondly, there are a large amormt of hydrolytic enzymes available because of their industrial interest. For instance detergent enzymes comprise proteases, celltrlases, amylases and lipases. Even if hydrolytic enzymes catalyse a chemically simple reaction, many important featirres of catalysis are still contained such as chemo-, regio- and stereoselectivity and specificity. 

Esters are widespread in fruits and especially those with a relatively low molecular weight usually impart a characteristic fruity note to many foods, e.g. fermented beverages [49]. From the industrial viewpoint, esterases and lipases play an important role in synthetic chemistry, especially for stereoselective ester formations and kinetic resolutions of racemic alcohols [78]. These enzymes are very often easily available as cheap bulk reagents and usually remain active in organic reaction media. Therefore they are the preferred biocatalysts for the production of natural flavour esters, e.g. from short-chain aliphatic and terpenyl alcohols [7, 8], but also to provide enantiopure novel flavour and fragrance compounds for analytical and sensory evaluation purposes [12]. Enantioselectivity is an impor-... 

Enzymatic Hydrolysis. Enzymatic hydrolysis has received enormous attention. The enzymes generally employed are lipases from microorgan isms, plants, or mammalian liver. The great advantage of the enzymatic process is its high chemo- and stereoselectivity. 

In an enzymatic resolution approach, chiral (+)-tra .s-diol (60) was prepared by the stereoselective acetylation of racemic diol with lipases from Candida cylindraceae and P. cepacia. Both enzymes catalyzed the acetylation of the undesired enantiomer of racemic diol to yield monoacetylated product and unreacted desired (+)-trans-diol (60). A reaction yield of 40% and an e.e. of >90% were obtained using each lipase [104],... 

Until the last decade or so, the only synthetically useful catalytic asymmetric acyl transfer processes were biotransformations using hydrolase enzymes particularly lipases and esterases [24]. Various lipases and esterases provide high levels of stereoselectivity (s) for the acylative KR and ASD of a wide variety of sec-alcohols and some amines, although the latter transformations have been less thoroughly explored [25-28]. However, the preparative use of enzymes is associated with a number of well-documented limitations, including their generally... 

Candida rugosa lipase (CRL) hydrolysed the other enantiomer selectively (Scheme 6.11). This proves once again that in most cases enzymes with the desired stereoselectivity are available. 

Chiral diols have also been prepared starting from meso-compounds [68-71]. Since meso-compounds are, in essence, symmetric molecules, the same applies as for the other symmetric starting materials. Indeed, this is exactly what was found Even though the stereocenters of the protected heptane tetrol are far away from the ester groups that are to be hydrolysed stereoselectively, this is what happens [69, 70]. The high selectivity is partly due to the fact that the secondary alcohol groups are protected as a cyclic acetal, giving additional structural information to the enzyme (Scheme 6.20 A). A cyclic acetal also provides additional structural information in the enantioselective hydrolysis of a pentane tetrol derivative (Scheme 6.20 B) [71]. In both cases Pseudomonas fluorescens lipase (PFL) proved to be the enzyme of choice. 

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