Biocatalytic reaction lipases

Thanks to their special properties and potential advantages, ionic liquids may be interesting solvents for biocatalytic reactions to solve some of the problems discussed above. After initial trials more than 15 years ago, in which ethylammonium nitrate was used in salt/water mixtures [29], results from the use of ionic liquids as pure solvent, as co-solvent, or for biphasic systems have recently been reported. The reaction systems are summarized in Tables 8.3-1 and 8.3-2, below. Table 8.3-1 compiles all biocatalytic systems except lipases, which are shown separately in 8.3-2. Some of the entries are discussed in more detail below. 

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. 

Significant research efforts have been directed towards the performance of biocatalytic reactions in RTIL media and this field has recently been reviewed. A wide range of reactions have been studied (Table 6.6), but it should be noted that most of the enzymes that have worked particularly well in RTILs are lipases. 

Hydrolases were in the first catalogue after the company was founded in 1950 but, not surprisingly, the chiral molecules originated mainly from the chiral pool. The first biocatalytic reactions were developed with kidney acylases and later with esterases and lipases, in the beginning mainly animal-derived biocatalysts [10], The set-up of in-house biocatalyst production from microbial and plant sources as well as the construction of a new biotechnology laboratory with ten fermenters of up to 300 L total volume, allow the development and production of improved biocatalysts and for them to be applied in the asymmetric synthesis of laboratory chemicals. There are today more than 100 biocatalytic processes in routine production and a project management team is handling custom biotransformations. 

Clearly, most biocatalytic reactions for the production of fine chemicals are used to obtain enantiopure or enantioemiched compoimds, and only a minor nimiber of syntheses lead to products without chiral centers. More than 65 ap-pHcations of immobilized enzymes or whole cells for industrial research and production have been treated in this review, and it can be stated that approximately 80% utilize the class of hydrolytic enzymes. This number reflects the ease of handling and the broad utility of these enzymes. The reported hydrolytic enzyme applications mainly involve lipases, whereas other hydrolases can only be found in fewer but nevertheless just as attractive cases. The broad field of asymmetric synthesis (e.g., asymmetric reduction/oxidation) is defi-... 

Examples of Biocatalytic Reactions in Ionic Liquids 645 Table 8-2 Enzymes other than lipases in ionic liquids... 

In a further example, a biocatalytic route for the production of optically pure 3-substituted cyclohexylamine derivatives from prochiral bicychc P-diketones was established by employing three biocatalytic reaction steps (Scheme 4.16) [53]. The sequence combined the stereoselective hydrolysis of a C-C bond catalyzed by a P-diketone hydrolase [54] (6-oxocamphor hydrolase (OCH) from Rhodococcus sp. [55]), followed by an Upase-catalyzed esterification [Candida antarctica lipase B (CAL-B), Novozyme 435], and a subsequent asymmetric amination by either an (S)-or (1 )-selective m-TA [V.fluvialis [27] or a variant of the Arthrobacter sp. TA [16a] (ArRmutll)]. 

It is interesting to note that a combination of surfactants such as lecithin and AOT appears to be more advantageous for some biocatalytic reactions. An increased activity of Chromo. viscosum lipase in AOT or CTAB reverse micelles observed in the presence of nonionic surfactants (such as tetraethylene glycol dodecyl ether, C12E4) was attributed to the possible action of nonionic surfactant presumably as a protecting agent which prevents unfavorable ionic and hydrophobic interactions between anionic AOT molecules and the enzyme [99,104,133]. 

The use of organic solvents as reaction media for biocatalytic reactions can not only overcome the substrate solubility issue, but also facilitate the recovery of products and biocatalysts as well. This technique has been widely employed in the case of lipases, but scarcely applied for biocatalytic reduction processes, due to the rapid inactivation and poor stability of redox enzymes in organic solvents. Furthermore, all the advantages for nonaqueous biocatalysis can take effect only if the problem of cofactor dependence is also solved. Thus, bioreductions in micro- or nonaqueous organic media are generally restricted to those with substrate-coupled cofactor regeneration. 

Deep eutectic solvents based on choline acetate (ChOAc), which have lower viscosities as compare to the ChCl/Urea eutectic mixture, have been also used as reaction media in several biocatalyzed transesterification reactions. In this sense, Zhao et al. reported the transesterification of ethyl sorbitate with 1-propanol by the lipase Novozym 435 Candida Antarctica lipase B immobilized on acrylic resins), achieving high initial rates (1 pmolmin g ) and selectivity (99%). Furthermore, in a model biodiesel synthesis system, the authors examined the transeterification of the lipid Miglyol oil 812 (a mixture of triglycerides of caprylic acid (C8) and capric acid (CIO)) with methanol, catalyzed by Novozym 435 in ChOAc/Gly (1 1.5 molar ratio). The biocatalytic reaction was very rapid in this eutectic mixture, with 97% conversion achieved after only 3 hours. 

Z. Guan, J.-P. Fu, Y.-H. He, Biocatalytic promiscuity lipase-catalyzed asymmetric Aldol reaction of heterocyclic ketones with aldehydes, TeUahedron Lett. 53 (2012) 4959-4961. 

A fimctional, easily assembled, operated and cleaned microbioreactor packed with immobilized Candida antarctica lipase B (Novozyme 435) was recently developed by Pohar et al. (2010). So far, microbioreactor was used for studying continuous mode ester synthesis within bis(trifluoromethylsulfonyl)imide - based ionic liquid media. Ionic liquid containing substrates was pumped into the microbioreactor at various flow rates, and at the outlet of the reactor the product was collected and analyzed. With fuUy adjustable length, width and depth, the developed packed bed microbioreactor was proven to be a very successful and versatile tooling for biocatalytic reactions such as isoamyl acetate or butyl butyrate synthesis (Cvjetko et al, 2010 Pohar et al., 2010). 

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. 

The principal methods for the hydrolase-promoted synthesis of enantiomerically pure alcohols are depicted in Figure 6.44. Biocatalytic acylation and alcoholysis have been reviewed recently [116,117]. Lipases, esterases, and proteases catalyze these reactions, but CAL-B [118-120], CRL [121,122], and diverse lipase preparations from Pseudomonas species are common place. 

The rate of the reaction in [BDMIM]BF4 was superior to that in [BMIM]BF4. It is significant that no drop in the reaction rate was observed in the [BDMIM]BF4 system after 10 repeated uses of the enzyme, whereas the reaction rate was significantly reduced when the reaction was conducted in [BMIM]BF4 or in [BMIM]PF6 (282). [BDMIM]BF4 was found to be the best solvent for the recycled use of the enzyme under normal pressure conditions when vinyl acetate was used as the acyl donor. No reaction took place when [BDMIM]PF6 was used as the solvent in the lipase-catalyzed reaction. (The [BDMIM]PF6 was purified with particular care to rule out the possibility of contamination.) The replacement of the C2 proton of [BMIM]PF6 by a methyl group was therefore concluded to have a large influence on its biocatalytic compatibility. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

The range of nucleophiles that lipases accept is not confined to water or alcohols. There are numerous examples of amines, hydrazine, phenols," and hydrogen peroxide. Proteases have frequently been used in biocatalytic transformations involving ester hydrolysis and esterification reactions and their different stereoselection often provides a useful complement to the lipases. - ... 

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. 

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