Enzymatic resolution of racemic mixture

In carrying out kinetic resolution, these in the standard approach are limited to 50% yield regarding the racemate. However, different approaches were developed [28] to overcome this limitation. The classical standard solution is to reracemize the unconverted enantiomer. A more advanced solution is the establishment of a dynamic kinetic resolution that has considerably expanded the synthetic scope of chemical processes. Here, the unconverted enantiomer is, in contrast to the latter method, racemized in situ. A great number of novel enzymatic methods have been developed [29]. Within this chapter, process solutions for enzymatic resolutions of racemic mixtures will be highlighted. 

In recent years biotransformations have also shown their potential when applied to nucleoside chemistry [7]. This chapter will give several examples that cover the different possibiUties using biocatalysts, especially lipases, in order to synthesize new nucleoside analogs. The chapter will demonstrate some applications of enzymatic acylations and alkoxycarbonylations for the synthesis of new analogs. The utQity of these biocatalytic reactions for selective transformations in nucleosides is noteworthy. In addition, some of these biocatalytic processes can be used not only for protection or activation of hydroxyl groups, but also for enzymatic resolution of racemic mixtures of nucleosides. Moreover, some possibilities with other biocatalysts that can modify bases, such as deaminases [8] or enzymes that catalyze the synthesis of new nucleoside analogs via transglycosylation [9] are also discussed. 

Paclitaxel analogs bearing a side chain containing heterocyclic or cycloalkyl groups have also been shown to possess anticancer activity, and the enzymatic resolution of racemic mixtures of such intermediates has again been investigated at Bristol-Meyers Squibb [70]. Racemic ds-34 could be stereoselectively hydrolyzed by Pseudomonas cepacia lipase (Amano PS-30) immobilized on Ac-curel and led to high optical purities of the desired (3R,4R)-enantiomer 34 (Scheme 11). Contrary to the use of the free enzyme, the immobilization reduced the required amount of biocatalyst by a factor of 10. 

Before we leave the enzymatic modification of terpenoids, we should point out that enzymes are also employed to resolve racemic mixtures of terpenoids. The principles of Bus are similar to those employed in the resolution of racemic mixtures of amino acids (see Chapter 8). 

Conduritols and inositols are cyclic polyalcohols with significant biological activity. The presence of four stereogenic centers in the stmcture of conduritols allows the existence of 10 stereoisomers. Enzymatic methods have been reported for the resolution of racemic mixtures or the desymmetrization of meso-conduritols. For example, Mucor miehei lipase (MML) showed enantiomeric discrimination between all-(R) and all-(S) stereoisomers ofconduritol E tetraacetate (Figure 6.52). Alcoholysis resulted in the removal of the four acetyl groups ofthe all-(R) enantiomer whereas the all-(S) enantiomer was recovered [141]. 

D-Pantolactone and L-pantolactone are used as chiral intermediates in chemical synthesis, whereas pantoic acid is used as a vitamin B2 complex. All can be obtained from racemic mixtures by consecutive enzymatic hydrolysis and extraction. Subsequently, the desired hydrolysed enantiomer is lactonized, extracted and crystallized (Figure 4.6). The nondesired enantiomer is reracemized and recycled into the plug-flow reactor [33,34]. Herewith, a conversion of 90-95% is reached, meaning that the resolution of racemic mixtures is an alternative to a possible chiral synthesis. The applied y-lactonase from Fusarium oxysporum in the form of resting whole cells immobilized in calcium alginate beads retains more than 90% of its initial activity even after 180 days of continuous use. The biotransformation yielding D-pantolactone in a fixed-bed reactor skips several steps here that are necessary in the chemical resolution. Hence, the illustrated process carried out by Fuji Chemical Industries Co., Ltd is an elegant way for resolution of racemic mixtures. 

Racemic pipecolic acid (6) is obtained by ring closure of TV-alkylglycines by ionic 203 or radical 204 mechanisms. It also may be obtained by conversion of suitable substituents at the C2 of piperidine into the 2-carboxy group, e.g. hydrolysis of a nitrile group 205 or oxidation of a 1,2-dihydroxyethyl group. 206 Resolution of the racemic mixture can be carried out by fractional crystallization. 207-209 Enzymatic resolution of racemic pipecolic acid 210-213 or of synthetic intermediates 214 has been reported. 

The principle of the optical resolution of racemic pantolactone is shown in Fig. 13. If racemic pantolactone is used as a substrate for the hydrolysis reaction by the stereospecific lactonase, only the d- or L-pantolactone might be converted to d- or L-pantoic acid and the l- or D-enantiomer might remain intact, respectively. Consequently, the racemic mixture could be resolved into D-pan-toic acid and L-pantolactone, or D-pantolactone and L-pantoic acid. In the case of L-pantolactone-specific lactonase, the optical purity of the remaining d-pantolactone might be low, except when the hydrolysis of L-pantolactone is complete. On the other hand, using the D-pantolactone-specific lactonase, d-pantoic acid with high optical purity could be constantly obtained independently of the hydrolysis yield. Therefore, the enzymatic resolution of racemic pantolactone with D-pantolactone-specific lactonase was investigated [138 140]. 

Asymmetric synthesis with lipases and esterases can basically be performed by two different approaches - the desymmetrization of prochiral or meso compounds and the enzymatic kinetic resolution of racemic mixtures. The main bottleneck of kinetic resolutions, product yields of maximum 50%, can be overcome if an in situ racemization of the starting material is possible. In this case all starting material can theoretically be converted to the desired product [34],... 

Recent studies in the pharmaceutical field using MBR technology are related to optical resolution of racemic mixtures or esters synthesis. The kinetic resolution of (R,S)-naproxen methyl esters to produce (S)-naproxen in emulsion enzyme membrane reactors (E-EMRs) where emulsion is produced by crossflow membrane emulsification [38, 39], and of racemic ibuprofen ester [40] were developed. The esters synthesis, like for example butyl laurate, by a covalent attachment of Candida antarctica lipase B (CALB) onto a ceramic support previously coated by polymers was recently described [41]. An enzymatic membrane reactor based on the immobilization of lipase on a ceramic support was used to perform interesterification between castor oil triglycerides and methyl oleate, reducing the viscosity of the substrate by injecting supercritical CO2 [42],... 

The number of enzymes for industrial synthetic applications is growing fast. Enzymatic synthesis can be performed under mild reaction conditions so that many problems of chemical synthesis like isomerization orracemization can be prevented. Furthermore, enzymes are highly specific and selective, especially for enantio- or regio-selective introduction of functional groups. For the preparation of chiral enantiopure compounds, the resolution of racemic mixtures by hydrolases is a well-established route, which has the advantage to be able to use enzymes free of coenzymes. Otherwise, only a maximum yield of 50% can be reached by the primary reaction and further steps of reracemization must follow to avoid loss of the undesired enantiomer. 

Roche D, Prasad K, Repic O (1999) Enantioselective acylation of 3-aminoesters using penicillin G acylase in organic solvents. Tetrahed Lett 40 3665-3668 Rolinson GN, Batchelor ER, Butterworth D et al. (1960) Formation of 6-aminopenicillanic acid from penicillin by enzymatic hydrolysis. Nat Lond 187 236-237 Rolinson GN, Geddes AM (2007) The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA). Internatl J Antimicrob Agents 29 3-8 Resell CM, Ferndndez-Lafuente R, Guisdn JM (1993) Resolution of racemic mixtures by synthesis reactions catalyzed by immobilized derivatives of the enzyme peniciUin G acylase. J Mol Catal 84 365-371... 

Most of the enzymatic peptide forming reactions are strictly stereo-specific for L-amino acids so that racemization that often accompanies chemical coupUng of optically active amino acids does not occur. The formation of only the L-amino acid derivative out of a D,L-mixture, in an enzymatic formation e.g. of an anilide from a D,L-Z-amino acid ester and aniUne, makes proteolytic enzymes useful reagents for the resolution of racemic mixtures. This is supplementary to the enzymatic stereospecific deacylation of D,L-iV-acylamino acid mixtures where exclusively the L-derivative will be deacylated. 

Enzymatic conversions have also found application in the resolution of racemic mixtures. When an organic synthesis involves a chiral center, one typically obtains a racemic mixture of the end products. Very commonly, chiral isomers differ in sensory properties, and one desires one enantiomer as opposed to the racemic mixture, e.g., L-menthol is the desired form of menthol. It is very diflicult to separate enantiomers by chemical means on a commercial basis (cyclodextrin-based colnnms have some utility in this application). However, a characteristic of enzymatic reactions is their stereospeciflcity, i.e., they will act on one enantiomer but not another. Thus, enzymatic processes may be incorporated into chemical synthesis to obtain a pure optical isomer. This may be done in either of two ways. 

Optical isomers in solution can be separated by a number of techniques, e.g. enzymatic methods, mechanical resolution, etc. In mechanical resolution of racemic mixtures of optical isomers, a supersaturated solution of a racemic mixture is seeded with the pure crystal of one of the isomers. This crystal grows and one of the isomers is separated from the solution. However, the solution remains supersaturated in the other isomer, which tends to precipitate, resulting in poor separation of the isomers. 

In Chapter 7, we briefly mentioned that there were a number of biological methods for resolution of racemic mixtures of chiral compounds. Because enzymes, biological catalysts, are composed of chiral amino acids, they will generally accept only the natural enantiomer of a compound as their substrate. Some enzymes are very specific as to their substrate, but others can be used with whole classes of substrate, even where these are not very close in overall structure to their natural substrates. The simplest form of resolution involves a racemate, where one enantiomer is transformed by the enzyme and the other is not. This is called kinetic resolution, since it depends on the relative rates of enzymatic catalysis of the reactions of one enantiomer over the other. Our first example is shown in Figure 15.22. When the epoxide is opened by the Grignard reagent, only the tra s-product is formed, but this is a racemic mixture. The alcohol is converted to an ester, which is then hydrolyzed enzymatically using a lipase enzyme from Pseudomonas fluorescens. Notice the very mild conditions for... 

Dynamic kinetic resolution enables the limit of 50 % theoretical yield of kinetic resolution to be overcome. The application of lipase-catalyzed enzymatic resolution with in situ thiyl radical-mediated racemization enables the dynamic kinetic resolution of non-benzylic amines to be obtained. This protocol leads to (/f)-amides with high enantioselectivities. It can be applied either to the conversion of racemic mixtures or to the inversion of (5)-enantiomers. 

The second-resolution approach relied on enzymatic resolution of acetate esters 62 (Scheme 4.7) (Hayakawa et ah, 1991). The sequence opened with the alkylation of 2,3-difluoro-6-nitrophenol (59) with l-acetoxychloro-2-propane (60) to deliver ether 61. Reduction of the nitro group of 61 gave an intermediate anihne that cyclized to give racemic benzoxazine 62 in 62% yield. A variety of lipases were then examined for the resolution. The best results arose from use of LPL Amano 3, derived from P. aeruginosa, which gave a ratio of 73 23 in favor of the desired (—)-enantiomer. Benzoylation of the enantiomerically-enriched mixture followed by chromatography of the aryl amides delivered enantiomerically pure 63. 

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). 

Resolution of racemic 1,3/4/6,7,llb-hexahydro-2H-pyrazino[l,2-a]iso-quinolin-4-one into enantiomers was unsuccessful either through the crystallization of diastereomeric chiral salts prepared from enantiopure acids in different solvent mixtures, or with kinetic resolution by an enzymatic acylation using different enzymes (08EJO895). 

In order to increase the efficiency of biocatalytic transformations conducted under continuous flow conditions, Honda et al. (2006, 2007) reported an integrated microfluidic system, consisting of an immobilized enzymatic microreactor and an in-line liquid-liquid extraction device, capable of achieving the optical resolution of racemic amino acids under continuous flow whilst enabling efficient recycle of the enzyme. As Scheme 42 illustrates, the first step of the optical resolution was an enzyme-catalyzed enantioselective hydrolysis of a racemic mixture of acetyl-D,L-phenylalanine to afford L-phenylalanine 157 (99.2-99.9% ee) and unreacted acetyl-D-phenylalanine 158. Acidification of the reaction products, prior to the addition of EtOAc, enabled efficient continuous extraction of L-phenylalanine 157 into the aqueous stream, whilst acetyl-D-phenylalanine 158 remained in the organic fraction (84—92% efficiency). Employing the optimal reaction conditions of 0.5 gl min 1 for the enzymatic reaction and 2.0 gl min-1 for the liquid-liquid extraction, the authors were able to resolve 240 nmol h-1 of the racemate. 

Enzymatic methods offer in principle the possibility of a direct enantioselective synthesis of amino acids. Enzymes are often used for separation of racemic mixtures, as examplified in the case of methionine. Although racemic methionine is adequate for the animal feed sector, other applications require the enan-tiomerically pure (L)-form. For the resolution, (L)-acylases from Aspergillus sp. are often used, since they can accept a broad spectrum of substrates, are highly active, and very stable under the production conditions. [62]... 

As can be concluded on the basis of Figure 9, only one of the enantiomers reacts in the best case of kinetic resolution. The reaction in the initially racemic mixture [S + S ] then stops at 50% conversion giving 50% of the enantiopure product [P ] and 50% of the enantiopure substrate Enzyme- or chemocat-alyzed racemization of the less reactive enantiomer in situ allows kinetic resolution to be changed to dynamic kinetic resolution when the substrate racemizes fast compared with the product formed and when the enzyme and the product p ) stable under the racemization conditions (Fig. 10). In dynamic kinetic resolution, a racemic mixture is transformed into the product enantiomer with 100% theoretical yield. Since 2003, a kind of revolution has been occurring in the development of dynamic kinetic resolution methods through enzymatic transesterification reactions (15). 

Unlike enzymatic reactions, microorganisms have the ability to perform multiple reactions, and they do not require cofacors for regeneration (albeit they require nutrients). They may be used to generate a flavor compound from a nonvolatile precursor (e.g., produce a lactone from castor oil), to effect the bioconversion of one volatile to another (e.g., valencene to nootkatone), or effect a chiral resolution (a racemic mixture of menthol). The primary limitation of using microoganisms for... 

This chapter illustrates the application of lipases and esterases as user-friendly biocatalysts in (i) desymmetrization of prochiral or meso-diols and diacetates, (ii) kinetic resolution of racemic alcohols, and (iii) preparation of enantiopure intermediate(s) from a mixture of stereoisomers by enzymatic differentiation. All the examples were taken from our own works in natural products synthesis. 

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