Biocatalytic reaction racemates resolution

The enantioselectivity of biocatalytic reactions is normally expressed as the enantiomeric ratio or the E value [la], a biochemical constant intrinsic to each enzyme that, contrary to enantiomeric excess, is independent of the extent of conversion. In an enzymatic resolution of a racemic substrate, the E value can be considered equal to the ratio of the rates of reaction for the two enantiomers, when the conversion is close to zero. More precisely, the value is defined as the ratio between the specificity constants (k st/Ku) for tho two enantiomers and can be obtained by determination of the k<-at and Km of a given enzyme for the two individual enantiomers. 

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. 

Biocatalytic Dynamic Kinetic Resolution of (R,S)-1- 2,3-Dihydrobenzo[b]Furan-4-yl -Ethane-1,2-Diol. Most commonly used biocatalytic kinetic resolution of racemates often provide compounds with high e.e., although the maximum theoretical yield of product is only 50%. In many cases, the reaction mixture contains a roughly 50 50 mixture of reactant and product which have only slight differences in physical properties (e.g., a hydrophobic alcohol and its acetate), and thus separation may be very difficult. These issues with kinetic resolutions can be addressed by employing a Dynamic Kinetic Resolution process involving a biocatalyst or biocatalyst with metal-catalyzed in situ racem-ization (26,27). 

Biocatalytic processes have become very important in the chemical industry [1-4], Of particular importance is one property of enz3unes— their stereoselectivity—which enables either of the two enanhomers to be reacted or formed preferentially in chemical reactions with chiral or prochiral compounds. Thus, resolution of racemic mixtures and more importantly the direct synthesis of enantiomerically pure products can be achieved without the need to protect group chemistry. Biocatalytic reactions are usually carried out under mild conditions, thus avoiding xmwanted side reactions [5,6]. Particularly in combination with enz)me immobilization, which enables easy workup of the product and reusability of the catalyst, biocatalysis is a promising approach in green chemistry. Moreover, the availability of protein engineering techniques also makes evolved or tailor-made biocatalysts more suitable for meeting the requirements of industrial applications. 

There are two principles of biocatalytic reactions leading to chiral products the asymmetric synthesis of meso- and prochiral compounds, and the kinetic resolution of racemates [17]. The latter is dominant by far in the number of stereospecific nitrile-converting enzymes, which might be in part due to the easier access of suitable racemates compared to prochiral substrates. In some cases racemic resolutions are convenient because both the (i ) and the (5) enantiomer can be obtained [114]. However, from the commercial aspect, usually racemic resolutions are of disadvantage due to a limited theoretical yield of 50%, the subsequent, often laborious separation of product and remaining substrate, and a time-dependent decrease of the enantioselectivity due to a kinetic rather than an absolute preference for one enantiomer by the enzyme. In contrast, asymmetric syntheses are advantageous due to a theoretical yield of 100%. However, the limited yield of racemic resolutions can be partly counteracted by deracemization techniques [129], some of which have been described here (Figs. 3, 6, 19, 32, and 38). 

For recent extensive reviews on biotransformations with lipases, see Kazlauskas and Bom-scheuer [77], Johnson [78], Rubin and Dennis [79], Itoh et al. [80], and Boland et al. [81]. The most widespread and frequently used biocatalytic reaction involving chiral compounds is kinetic resolution of racemates. Other biocatalytic stereoselective methods, although less frequently used, are asymmetrization of prochiral and meso compounds. These will be briefly discussed in Secs. C and D, respectively. 

Recently, another interesting application of nitrilases has been demonstrated for the synthesis of pregabalin-the API of the neurophatic pain drug Lyrica. In this approach, the key step is the resolution of racemic isobutylsuccinonitrile (Scheme 10.8) [18], the process takes place with total regio- and stereoselectivity, and the (S)-acid is obtained and the (R)-substrate can be recycled under basic conditions. To improve the biocatalytic step, directed evolution was applied using the electronic polymerase chain reaction and in the first round of evolution a single C236S mutation led to a mutant with 3-fold increase in activity [19]. 

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. 

Several synthetic examples that successfully exploit TK for biocatalytic conversions have been reported. For example, the total synthesis of the beetle pheromone (+)-exo-brevicomin utilizes TK from Baker s yeast as the sole chiral reagent. Starting with racemic 2-hydroxybutanol, the ability of TK to effect kinetic resolution of substrates was exploited (Scheme 5.56). The smooth reaction of 2-hydroxybutanol with HPA was catalyzed by TK at pH 7.5 to yield the enantioenriched ketone in 90% yield. This intermediate was chemically converted into (+)-exo-brevicomin.101... 

Biocatalytic resolution has been applied efficiently by BASF for the manufacture of optically active amines, such as phenylethylamine [96]. The process is based on a highly stereoselective resolution of racemic amines by means of an acylation reaction in the presence of a lipase as a catalyst (Scheme 22). The products are obtained in high yields and with excellent enantioselectivities. The unrequited enantiomer can be racemized subsequently. Thus, starting from a racemate, efficient access (with theoretically 100% yield) to the (/ )- and (5)-amine, respectively, is available. This technology, which is said to be carried out on a > 1000 mt scale, has been extended recently by BASF to the production of chiral alcohols. 

A number of major pharmaceutical companies have used biocatalytic approaches based on esterases and lipases lo prepare target drugs or intermediates [70,122-126]. Most of these approaches involve resolutions that start with a racemic ester or amide, and as such, yields of < 50% can only be realized. Recent examples of resolutions applied to pharmaceutical intermediates such as the paclitaxel (Taxol) side chain and A-(+)-BMY-14802, an antipsychotic agent, have been described by the Bristol-Myers group [70]. The following sections discuss selected examples of the use of esterases and lipases to hydrolyze prochiral or we o-substrates, where theoretical yields of 100% can be realized, followed by a brief discussion of dynamic kinetic resolution where reaction yields of 100% can also be achieved. 

The reaction concept with this new hydantoinase-based biocatalyst is economically highly attractive since it represents a dynamic kinetic resolution process converting a racemic hydantoin (theoretically) quantitatively into the enantiomerically pure L-enantiomer [19]. The L-hydantoinase and subsequently the L-carbamoylase hydrolyze the L-hydantoin, l-11, enantioselectively forming the desired L-amino acid, l-2. In addition, the presence of a racemase guarantees a sufficient racemiza-tion of the remaining D-hydantoin, d-11. Thus, a quantitative one-pot conversion of a racemic hydantoin into the desired optically active a-amino acid is achieved. The basic principles of this biocatalytic process in which three enzymes (hydan-toinase, carbamoylase, and racemase) are integrated is shown schematically in Fig. 9. 

The reduction of ketones into alcohols can be achieved using biocatalytic methods. Amongst the most popular of the available methods is the use of Baker s yeast, BY (Saccharomyces cerevisiae). The use of P-ketoesters as substrates leads to the corresponding p-hydroxy esters, often with high enantioselectivity. In the particular case of a-substituted P-ketoesters, the substrates spontaneously racemize, and this provides the basis for many reports of dynamic resolution reactions, some of which are described in the following discussion. In 1976, Deol and co-workers showed that cycloalkyl p-ketoesters could be reduced under dynamic resolution conditions (Fig. 9-26) >58l... 

Biocatalytic kinetic resolution of racemic hydroxymethylphosphinates 271 via their lipase-promoted acetylation in supercritical carbon dioxide as the reaction medium was investigated. The reaction was fastest when pressure was closer to the critical pressure at 11 MPa the reaction rate reached its maximum when the pressure was increased to 15 MPa. The optimal conditions were obtained at 13 MPa (yields 50%, 30% ee). The stereoselectivity of the reaction depended on solvent, substituents at phosphorus, and solubility of substrates in SCCO2. The best results were obtained with the Candida antarctica lipase (Novozym 435) (Scheme 89) [183, 184]. 

Sequential Biocatalytic Resolutions. For a racemic substrate bearing tv o chemically and stereochemically identical reactive groups, an enzymatic resolution proceeds through two consecutive steps via an intermediate monoester stage. During the course of such a reaction the substrate is forced to enter the active site... 

In practice, however, deracemization via repeated resolution is often plagued by low overall yields due to the harsh reaction conditions required for (chemical) racemization [71]. In view of the mild reaction conditions displayed by enzymes, there is a great potential for biocatalytic racemization based on the use of racemases of EC-class 5 [72, 73]. 

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. 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

A selection of biocatalytic deoxygenation reactions is shovm in Figure 1.8. The reducing power of baker s yeast in an ethanol-water mixture and sodium hydroxide at 60° C has been found effective for the rapid and selective reduction of a series of N-oxides like aromatic and heteroaromatic N-oxide compounds [118]. DMSO reductase from Rhodobacter sphaeroides f sp. denitrificans catalyzed the (S)-enantioselective reduction of various sulfoxides and enabled the resolution of racemic sulfoxides for the synthesis of (R)-sulfoxides with >97% ee [119,120]. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus resolved a racemic mixture of methyl p-tolyl sulfoxide by catalyzing the reduction of (S)-methyl p-tolyl sulfoxide and gave enantio-merically pure (J )-methyl p-tolyl sulfoxide in 88% yield, while whole cells of E. coli,... 

The BBE from Eschscholzia califomica (California poppy) is the best-characterized member of this enzyme class due to the availability of an efficient heterologous expression system using Pichia pastoris [158,159]. Moreover, its biochemical properties, structure, and reaction mechanism were thoroughly investigated [160-162]. In addition, the potential of BBE was investigated for biocatalytic applications such as the kinetic resolution of different racemic nonnatural benzylisoquinolines [16,163,164]. 

The combination of a resin and covalently supported IL with SCCO2 was also used in the KR and dynamic kinetic resolution (DKR) of 1-phenylethanol with vinyl propionate catalyzed by Candida antarctica lipase B (CALB) [125]. The IL molecule covalently supported on Merrifield resin was realized through the reaction of 1-butyl imidazole with chloromethylated resin. Subsequently, NTf2 was introduced via ion exchange. Under improved conditions, the conversion of 1-phenylethanol was 50% with 99.9% ee to the product. In order to develop a more efficient process, the KR of 1-phenylethanol was tested on a flow system, and it remained stable for 6 days with 99% ee Moreover, by combing two fixed-bed reactors loaded with the supported enzyme (biocatalytic reactor, CALB-SILLP (SILLP, supported ionic liquid-like phase) 11, 150 mg) and an additional one with an acid zeolite (chemical racemization catalyst, 100 mg). Figure 2.40, the DKR of 1-phenylethanol... 

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