Enantiomer separation enzymatic resolution

Unless asymmetric induction is complete, it is necessary to remove the undesired enantiomer from the product mixture. Whereas in conventional diastereoselective asymmetric syntheses this removal can typically be readily accomplished by crystallization or chromatography, the separation of enantiomeric products can be problematic. Often, though, with enantio-enriched samples it is possible to recrystallize either the racemate from the pure enantiomer or, preferably, one enantiomer from the other [I2a,16,17], Another very effective method to produce enan-tiopure compounds is by enzymatic resolution of the enantio-enriched product from chiral PTC [16,18]. These methods are illustrated by examples in the alkylation section of this chapter (Chart 10.6). 

The separation of a racemic compound into its enantiomers is called resolution. Various methodologies have been used for the resolution of the enantiomers on both analytical and preparative scales. The different techniques may be categorized into two classes the classical approach, using enzymatic degradation of one of the enantiomers, and preferential crystallization. Modem technologies include spectroscopic, electrophoretic, and chromatographic methods. 

Reetz, M.T., W. Wiesenhofer, G. Francio and W. Leitner, Continuous Flow Enzymatic Kinetic Resolution and Enantiomer Separation Using Ionic Liquid/Supercritical Carbon Dioxide Media, Advanced Synthesis Catalysis, 345, 1221-1228 (2003). 

Enzymatic resolution is also used to separate the enantiomers of amino acids. 

Several multi-ton industrial processes still use enzymatic resolution, often with lipases that tolerate different substrates. BASF, for example, makes a range of chiral amines by acylating racemic amines with proprietary esters. Only one enantiomer is acylated to an amide, which can be readily separated from the unreacted amine. Many fine chemicals producers also employ acylases and amidases to resolve chiral amino acids on a large scale. l-Acylases, for example, can resolve acyl d,l-amino acids by producing the I-amino acids and leaving the N-acyl-l-amino acid untouched after separation, the latter can be racemized and returned to the reaction. d-Acylase forms the alternative product. Likewise, DSM and others have an amidase process that works on the same principle d,l-amino acid amides are selectively hydrolyzed, and the remaining d-amino acid amide can be either racemized or chemically hydrolyzed. 

Reetz MT, Wiesenhofer W, Francio G et al (2003) Continuous flow enzymatic kinetic resolution and enantiomer separation using ionic Uquid/supercritical carbon dioxide media. Adv Synth Catal 345 1221-1228... 

Table IV contains selected examples of the enzymatic resolution of esters with various structures. As was discussed above, enhancement of the optical purity was possible by changing the acyl group or the enzyme (Run 1,2 or 6,7). Noteworthy is the fact that acetate 5 with a trifluoromethyl group was converted by lipase MY into the alcohol with (R) absolute configuration without exception in every case, when the stereochemistry has been determined. However, substrates with the other fluorine-containing substituents furnished alcohols whose asymmetric configuration depended on their structures. Particularly interesting is the relationship between Runs 4 and 5 (18), when resolution was accompanied with simultaneous separation of diastereomers. To the best of our knowledge, this is the first example of the type of resolution of both diastereomers and enantiomers by enzymatic hydrolysis in a single transformation.

Resolution of enantiomers, separation of diastereomers, asymmetric synthesis, microbial and enzymatic reactions and synthesis from chiral starting materials are all methods for producing optically active compounds. 

Enzymatic resolution is also used to separate the enantiomers of amino acids. Enzymes are chiral molecules with specific catalytic activities. For example, when an acylated amino acid is treated with an enzyme like hog kidney acylase or carboxypeptidase, the enzyme cleaves the acyl group from just the molecules having the natural (l) configuration. The enzyme does not recognize D-amino acids, so they are unaffected. The resulting mixture of acylated D-amino acid and deacylated L-amino acid is easily separated. Figure 24-5 shows how this selective enzymatic deacylation is accomplished. 

One of the initial routes considered for the preparation of isomers was the enzymatic resolution using 5 -nucleotidase and alkaline phosphatase that would allow access to both enantiomers, racemic compound. The chemically synthesized monophosphate derivative ( )-121 was resolved using 5 -nucleotidase from Crotalus atrox venom, and the resulting mixture was separated by chromatography and purified on silica gel to give (+)-123 with a 90% ee. Hydrolysis of the remaining monophosphate (-)-122 with alkaline phosphatase from E. coli afforded Epivir [155]. 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

Despite its widespread application [31,32], the kinetic resolution has two major drawbacks (i) the maximum theoretical yield is 50% owing to the consumption of only one enantiomer, (ii) the separation of the product and the remaining starting material may be laborious. The separation is usually carried out by chromatography, which is inefficient on a large scale, and several alternative methods have been developed (Figure 6.2). For example, when a cyclic anhydride is the acyl donor in an esterification reaction, the water-soluble monoester monoacid is separable by extraction with an aqueous alkaline solution [33,34]. Also, fiuorous phase separation techniques have been combined with enzymatic kinetic resolutions [35]. To overcome the 50% yield limitation, one of the enantiomers may, in some cases, be racemized and resubmitted to the resolution procedure. 

Before an asymmetric synthesis appeared of levofloxacin (1, (—)-ofloxacin), (—)- ofloxacin was isolated via optical, enzymatic, and crystallization resolution of the racemic ofloxacin (17) Drugs Future, 1992 Hayakawa et al., 1986, 1991). For instance, tricyclic core 52 was converted to ( + )-3,5-dinitrobenzoyl derivative 54 in 75% yield (Scheme 4.5). The enantiomers were then separated via high-performance liquid chromatography (HPLC) with a SUMIPAX OA-4200 column to deliver optically pure benzoyl esters 55a and 55b (Drugs Future, 1992 Hayakawa et al., 1986, 1991). 

Contrary to the optical resolutions described in Sections 2.1.1.-2.1.3., which depend on the solubility or chromatographic properties ( Thermodynamic resolution ), the kinetic resolution rests on rate differences shown by the enantiomers when reacted with an optically active reagent. In the ideal case, only one enantiomer is converted into the envisaged product and the other enantiomer is unchanged. In this way, optical resolution is reduced to the more simple separation of two different reaction products. In practice, only two methods of kinetic resolution are reasonably general and reliable the Sharpless epoxidation of allylic alcohols and the enzymatic transesterification of racemic alcohols or carboxylic acids. 

The application of enzymatic acylation for the resolution of racemic alcohols in organic solvent has shown to be an effective method to rapidly synthesize chiral alcohols. The racemic alcohols are treated with the lipase and acylating agent one enantiomer remains unconverted whereas the second enantiomer is esterified and easily separated by distillation (Scheme 7.2). Vinyl acetate or isopropenyl acetate are typical acylating agents, as the generated vinyl alcohol tautomerizes rapidly... 

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