Enzymes, hydrolytic, racemic resolution

The KR of secondary alcohols by some hydrolytic enzymes has been well known. The combinations of these hydrolytic enzymes with racemization catalysts have been explored as the catalysts for the efficient DKR of the secondary alcohols. Up to now, lipase and subtilisin have been employed, respectively, as the R- and 5-selective resolution enzymes in combination with metal catalysts (Scheme 2). 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

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. 

Typical commercial enzymes reported for resolution of amino acids were tested. Whole cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids" the acylase-catalyzed resolution of Xacyl amino acids had extremely low rates toward a-dialkylated amino acids and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-ethylhexanoic amide. Finally, a large-scale screening of hydrolytic enzymes for enantioselective hydrolysis of racemic amino esters was carried out. Of all the enzymes and microorganisms screened, pig hver esterase (PLE) and Humicola langinosa lipase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

Biotransformations are now firmly established in the synthetic chemist s armoury, especially reactions employing inexpensive hydrolytic enzymes for the resolution of racemates and for the desymmetrization of prochiral substrates. From a practical viewpoint, biocatalytic resolution is arguably the simplest method available to obtain synthetically useful quantities of chiral synthons. As an illustration of this point, many racemic secondary alcohols ROH can be resolved without prior derivatization by combining with a lipase and a volatile acyl donor (usually vinyl acetate) in an organic solvent, to effect irreversible transesterification once the desired degree of conversion has been reached, routine filtration to remove the enzyme and concentration of the filtrate affords the optically enriched products ROAcyl and ROH directly. 

It is generally beheved that selectivity of hydrolytic enzymes strongly depends on the proximity of the chiral center to the reacting carbonyl group, and only a few examples of successful resolutions exist for compounds that have the chiral center removed by more than three bonds. A noticeable exception to this rule is the enantioselective hydrolysis by Pseudomonasfluorescens Hpase (PEL) of racemic dithioacetal (5) that has a prochiral center four bonds away from the reactive carboxylate (24). The monoester (6) is obtained in 89% yield and 98% ee. 

However, the most common and important method of synthesis of chiral non-racemic hydroxy phosphoryl compounds has been the resolution of racemic substrates via a hydrolytic enzyme-promoted acylation of the hydroxy group or hydrolysis of the 0-acyl derivatives, both carried out under kinetic resolution conditions. The first attempts date from the early 1990s and have since been followed by a number of papers describing the use of a variety of enzymes and various types of organophosphorus substrates, differing both by the substituents at phosphorus and by the kind of hydroxy (acetoxy)-containing side chain. 

Figure 2.2 Production of enantiopure compounds using hydrolytic enzymes. In (a) a prochiral diester is hydrolysed to yield predominance (in theory 100%) of one enantiomer. In the next example (b) a raeso-diester is hydrolysed to yield predominance (in theory 100%) of one enantiomer of the monoester. If kj>k2 the (IS, 2i )-enantiomer is formed to the greatest extent. Due to the preference of the enzyme k4>kj and the lower monoester (IR, 2S) will be removed fastest. Hence both steps will lead to an increase of the upper enantiomer at the monoester stage. If the reaction proceeds to completion, however, the result will be another raeio-compound, a diol. In example (c) a racemic secondary ester is resolved by hydrolysis. One monoester is hydrolysed faster than the other and this leads to kinetic resolution.

Optical resolution of racemic compounds by biocatalysts has been a useful method as shown in this review. For this purpose, two types of biocatalysts are mainly used hydrolytic enzymes and oxidoreductases. 

A second example of the use of directed molecular evolution for natural product synthesis is the use of lipases by Reetz and colleagues. This work is based on the kinetic hydrolytic resolution of racemic mixtures, in which one enantiomer is preferentially hydrolyzed and the chiral product is thus enriched. Utilizing both random mutagenesis and directed techniques such as CAST,64 they have improved the stereoselectivity of a lipase from Pseudomonas aeruginosa (PAL) on a number of occasions with different substrates. One of the first examples utilized the model substrate 2-methyldecanoic acid /xnitrophenyl ester, for which the wild-type enzyme has an enantioselectivity of E= 1.1. As a consequence of five mutations accumulated through random mutagenesis, followed by saturation mutagenesis, the enantioselectivity was increased to 25.8.123 More... 

Hydrolytic enzymes have long been used for the kinetic resolution of racemic alcohols and carboxylic acids. In recent years, the corresponding transformation on Table 14,1 List of suppliers of enzymes referred in this chapter. 

Alternative hydrolytic enzymes, such as amidases and acylases, have also been employed for the resolution of racemic amines. Unlike lipases, these enzymes are typically not commercially available but they can be isolated from microorganisms. 

Lipase- or esterase-catalyzed hydrolytic resolution of ketoprofen esters was usually employed for the preparation of (isolated microorganisms or commercial lipases have been applied for the enantioselective hydrolysis of racemic ketoprofen esters. A commercial enzyme. Lipase OF from Candida rugosa (Meito Sangyo, Japan), was found to be a good biocatalyst for kinetically resolving the 2-chloroethyl ester of ketoprofen (2-(3-benzoylphenyl)propionic acid). ... 

Among the principal methods for the enzymatic synthesis of enantiomerically pure amino acids depicted in Scheme 2.10, the most widely applied strategy is the resolution of racemic starting material (synthetically prepared from inexpensive bulk chemicals) employing easy-to-use hydrolytic enzymes such as proteases, esterases, and lipases. In contrast, more complex procedures requiring special expertise are the (1) reductive amination of a-keto acids using a-amino acid... 

Lipases have also been widely applied for the resolution of racemic chiral amines. In principle, these reactions can be carried out in both the hydrolytic mode as well as under conditions favouring acylation. As amines are more nucleophilic than alcohols, it is necessary to use less reactive acyl donors in order to minimize the background reaction of non-enzyme catalysed acylation, and in this respect it appears that simple esters such as ethyl acetate are optimal. 

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