Enantioselective synthesis using enzymes

Several approaches to enantioselective synthesis have been taken, but the most efficient are those that use chiral catalysts to temporarily hold a substrate molecule in an unsymmetrical environment—exactly the same strategy that nature uses when catalyzing reactions with chiral enzymes. While in that unsymmetrical environment, the substrate may be more open to reaction on one side than on another, leading to an excess of one enantiomeric product over another. As an analog)7, think about picking up a coffee mug in your... 

The same reasoning applies to the synthesis of pure enantiomers as to organic synthesis in general processes should be atom efficient and have low E factors, i.e. involve catalytic methodologies. This is reflected in the increasing attention being focused on enantioselective catalysis, using either enzymes or chiral metal complexes. 

Some developments have been carried out for the enantioselective synthesis of biologically active compounds. One such example is the synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate, an important intermediate for the angiotensinconverting enzyme (ACE) inhibitor benazepril, or for coenzyme A, using the NORPHOS ligand (Scheme 33.9) [21]. 

In contrast to Mori s synthesis, Pawar and Chattapadhyay used enzymatically controlled enantiomeric separation as the final step [300]. Butanone H was converted into 3-methylpent-l-en-3-ol I. Reaction with trimethyl orthoacetate and subsequent Claisen-orthoester rearrangement yielded ethyl (E)-5-methyl-hept-4-enoate K. Transformation of K into the aldehyde L, followed by reaction with ethylmagnesium bromide furnished racemic ( )-7-methylnon-6-ene-3-ol M. Its enzyme-catalysed enantioselective transesterification using vinylacetate and lipase from Penicillium or Pseudomonas directly afforded 157, while its enantiomer was obtained from the separated alcohol by standard acetylation. 

Another class of enzymes that can be used for the enantioselective synthesis of amines and amino acids is the aminotransferases or transaminases (TAs) [29]. As shown in Scheme 6.15, they can be employed in a kinetic resolution or an asymmetric synthesis mode. 

Enzyme-based processes for the resolution of chiral amines have been widely reported [2, 3] and are used in the manufacture of pharmaceuticals, for example, BASF s process for chiral benzylic amine intermediates. Scheme 13.1 [4]. The methods used are enantioselective hydrolysis of an amide and enantioselective synthesis of an amide, both of which are kinetic resolutions. For high optical purity products the processes depend upon a large difference in the catalyzed reaction rates of each enantiomer. 

Only a little research on the separation and synthesis of chiral compounds has been published so far. Because enzymes have extremely high selectivity, and owing to the great importance of enantioselective synthesis or enantiomeric resolution in the pharmaceutical industry, the most intense research in this area can be expected, along with minimizing the use of substances and maximizing their effect. 

Initial preparative work with oxynitrilases in neutral aqueous solution [517, 518] was hampered by the fact that under these reaction conditions the enzymatic addition has to compete with a spontaneous chemical reaction which limits enantioselectivity. Major improvements in optical purity of cyanohydrins were achieved by conducting the addition under acidic conditions to suppress the uncatalyzed side reaction [519], or by switching to a water immiscible organic solvent as the reaction medium [520], preferably diisopropyl ether. For the latter case, the enzymes are readily immobilized by physical adsorption onto cellulose. A continuous process has been developed for chiral cyanohydrin synthesis using an enzyme membrane reactor [61]. Acetone cyanhydrin can replace the highly toxic hydrocyanic acid as the cyanide source [521], Inexpensive defatted almond meal has been found to be a convenient substitute for the purified (R)-oxynitrilase without sacrificing enantioselectivity [522-524], Similarly, lyophilized and powered Sorghum bicolor shoots have been successfully tested as an alternative source for the purified (S)-oxynitrilase [525],... 

A similiar approach was performed by van de Velde (1999), using incorporation of vanadate into an acid phosphatase (phytase) to create a semi-synthetic peroxidase similar to the heme-dependent chloroperoxidase. The latter is a useful enzyme for the asymmetric epoxidation of olefins, but less stable due to oxidation of the porphyrin ring and difficult to express outside the native fungal host. The authors exploited the structural similarity of active sites from vanadate-dependent halo-peroxidases and acid phosphatases and have shown the useful application as an enantioselective catalyst for the synthesis of chiral sulfoxides (van de Velde, 1999). 

The development of accurate methods for the determination of enantiomeric purity, which began in the late 1960 s, has been critical for the assessment of enantioselective synthesis. Thus a prerequisite in the enzyme-catalyzed kinetic resolution of racemates is a precise and reliable assessment of the degree of enantioselectivity (E), enantiomeric excess (ee) and conversion (c). Among these methods are 1) polarimetric methods, 2) gas chromatographic methods, 3) liquid chromatographic methods and 4) NMR spectroscopy. The most convenient and sensitive methods used are chiral GC and HPLC. 

The availability of 38C2 as a broad scope, enantioselective, efficient aldolase enzyme has had a significant impact on organic synthesis. Some of the molecules we have synthesized with 38C2 include the natural products ( + ) —frontalin [( + )— 27] (List et al., 1999), some brevicomins [( —) —28 and (—) —29] (List etal., 1998a), epothilones A (30) and C (31) (Sinha et al., 1998), and the Wieland-Miescher ketone [( ) — ( + )—32] (Hoffmann et al., 1998 Zhong et al., 1997). The brevicomin examples represent the first use of a catalytic antibody to decrease the total number of synthetic steps and increase the enantioselectivity of natural product syntheses. 

Because of the specificity and the enantioselectivity of some enzyme-catalyzed reactions, the application of enzymes is increasingly important in asymmetric induction and kinetic resolution in organic synthesis. A large number of publications were recently reviewed, focusing on utilization of enzymes and microorganisms to stereospecific hydrolysis and other reactions to produce pure stereoisomers (2,3). However, the use of an enzyme as a catalyst has usually been limited to small-scale experiments in the laboratory. 

The enantioselective synthesis of a-hydroxyketones via a carbon-carbon bond forming reaction has received a significant impulse during recent years. Four different enzymes are commonly used for this reaction BAL, BFD, pyruvate decarboxylase (PDC) and TK. Many different compounds can be prepared with... 

Epoxide formation using biocatalysis is a useful process for the formation of chiral oxiranes (Scheme 29). The synthesis of enantioenriched epoxides using enzymes has been reviewed <1995BCSF769>. Chloroperoxidase has been examined for the oxidation of 2-methyl-l-alkenes, among other alkenes. The yields in some cases can be low, but the enantioselectivities can be high <1995JA6412, 1997JA443>. This enzyme has been used in a synthesis of... 

Note should also be made that in some cases recrystallization reduces the enantiomeric excess, which can lead to crystallization of the racemate (94). In these cases the mother liquors contain moderately to highly enriched material. It is therefore important to plan the strategy at which point the enantiomer is recrystallized to optical purity. This may be from an enzymic resolution, or in the event that an asymmetric synthesis has failed, to deliver enantiopure product. As discussed in Section 3, the liquors from the diastereomeric resolution with DTTA of 88%de can be cleaved to the free base, and crystallization of the hydrochloride salt gives >98% ee. This is because of the fact that methylphenidate hydrochloride has a eutectic point of 30% ee. Davieset al. (95) and Winkler et al. (96) have prepared single enantiomer methylphenidate (29), Their approaches use an enantioselective synthesis the enantiomeric excesses are 86% and 69%, respectively, thus requiring recrystallization... 

Several hydrolytic enzymes other than esterases have been applied for synthetic purposes. One important subject is the chemoenzymatic preparation of amino acids. An industrial method for the synthesis of unnatural d- or L-amino acids employs the enzymatic hydrolysis of hydantoins, prepared by Bucherer-Bergs condensation using either D- or L-hydantoinase (cf Section 3.2.1.4) [33]. Another efficient method of preparing natural and unnatural amino acids is the two-step synthesis which features a Pd-catalyzed amidocarbonylation (eq. (2) cf Section 2.1.2.4) to afford racemic A-acyl amino acids followed by enantioselective hydrolysis using various acylases [34]. 

Abstract Various approaches to the preparation of enantiomerically pure (2/i,27i)-(+)-Z/irco-mclhyl-phenidate hydrochloride (1) are reviewed. These approaches include synthesis using enantiomerically pure precursors obtained hy resolution, classical and enzyme-hased resolution approaches, enan-tioselective synthesis approaches, and approaches based on enantioselective synthesis of (2S,2 R)-ery-Zftro-methylphenidate followed by epimerization at the 2-position. 

After the first preparation of enantiomerically pure (27 ,2 7 )-t/zreo-methylphenidate hydrochloride (1) in 1958, it is only recently that a great deal of interest has been demonstrated in the synthesis of this molecule. Various approaches to the preparation of enantiomerically pure (2R,2>R)-(-i-)-t/zreo-methylpheni-date hydrochloride (1) are reviewed. These approaches include synthesis using enantiomerically pure precursors obtained by resolution, classical and enzyme-based resolution approaches, enantioselective synthesis approaches, and approaches based on enantioselective synthesis of 2S, 2 Ryerythro-methylphenidate followed by epimerization at the 2-position. Classical resolution approaches have been successfully upscaled to produce 1 on a multi-kilo-gram scale due to the ready availability of racemic ( )-t/zreo-methylphenidate hydrochloride (10). VVfiiIt some enantioselective approaches are short, they do not provide 1 of the desired enantiomeric purity necessary for drug development. Enantioselective synthesis approaches to produce 1, however, will be-... 

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