Racemic Resolution Using Hydrolytic Enzymes

Due to the reasons mentioned above (costs, substrate availability, development time, patent issues, etc.) many chiral products are produced first as a racemate. At a later stage, usually, the enantiomers (or stereoisomers) have to be separated in 

More than 60% of all biotransformations using isolated enzymes are hydrolase-dependent processes. Several reviews have summarized the achievements in this field [33-37]. From the numerous examples published, two will be discussed in order to highlight the potential of enzyme-catalyzed kinetic resolution for the production of chiral pharmaceuticals. 

Subtilisin Carlsberg, a laundry enzyme, is extraordinarily cheap and stable even at extreme concentrations of substrate and salt at elevated temperature. A major disadvantage of this chemoenzymatic route was the fact that no racemization procedure for the unwanted (R)-enantiomer [(R)-3] could be established [40]. 

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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. 

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... 

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. 

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. 

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. 

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. 

Hydrolytic enzymes are often able to catalyze the esterification of one enantiomer of a racemic alcohol, thus providing one enantiomer of the alcohol and the ester of the other enantiomer, readily separable by extraction or other means. Candida rugosa lipase, immobilized on DEAE-Sephadex-A-25, has been used in such a resolution of ( )-menthol (Eq. 3.2) [20]. 

Lipase-Catalyzed Hydrolysis of Cyanohydrin Acetates. Hydrolytic enzymes, especially lipases, are widely used for enantioselective transformations, and have been used to prepare optically active cyanohydrins. For example, the lipase-catalyzed kinetic resolution of racemic w-phenoxybenzaldehyde cyanohydrin acetate was an essential step in the synthesis of (li ,ci5,aiS)-cypermethrine 19). Another recent report described the lipase-catalyzed kinetic resolution of pentafluorobenzaldehyde cyanohydrin acetate 20). To examine this approach, 2- and 6-fluoro-3,4-dibenzyloxybenzaldehyde cyanohydrin acetates (12b,d) were prepared from the aldehydes 10b,d. Preliminary attempts to carry out lipase-catalyzed kinetic resolutions of Aese cyanohydrin acetates were unsuccessfiil (unpublished results). 

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... 

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... 

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. 

BMY 14802 88 has also been prepared by lipase-catalyzed resolution of racemic BMY 14802 acetate ester 90 [148]. Lipase from Geotrichum candidum (GC-20 from Amano Enzyme Co.) catalyzed the hydrolysis of acetate 90 to / -(+)-BMY 14802 (Fig. 28) in a biphasic solvent system in 48% reaction yield (theoretical maximum yield is 50%) and 98% e.e. The rate and enantioselectivity of the hydrolytic reaction was dependent on the organic solvent used. The enantioselectivity E values) ranged from 1 in the absence of solvent to more than 100 in dichloromethane and toluene. S-(—)-BMY 14802 was also prepared by the chemical hydrolysis of undesired BMY 14802 acetate obtained during enzymatic resolution process. 

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