Enantioselective biocatalysts

Basically, there are three ways to tune enzyme enantioselectivity by means of additives (i) the additives are placed in the reaction medium together with the organic solvent, the enzyme, and the reagents (ii) the additives are co-lyophilized with the biocatalyst before use in the organic solvent (iii) the additives are complexed with the substrates before their transformation in the organic medium. 

As shown in this chapter, by focusing on the modulation of enzyme selectivity by medium engineering, quite simple modifications of the solvent composition can really have significant effects on the performances of the biocatalysts. The main drawback remains the lack of reliable predictive models. Despite the significant research efforts (particularly in the last decade), it is likely that a reasonable foresight of the enantioselective outcome of an enzymatic transformation will continue to be based solely on a careful analysis of the increasingly numerous literature reports. 

Biocatalysts usually require mild reaction conditions for an optimal activity (physiologic temperature and pH) and, in general, they show high activity, chemo- and enantioselectivity. Furthermore, when using enzymes, many functional group protections and/or activations can be avoided, allowing shorter synthetic transformations. The use of enzymes is therefore very attractive from an environmental and economic point of view. 

Since stereoselectivities of biocatalytic reductions are not always satisfactory, modification of biocatalysis are necessary for practical use. This section explains how to find, prepare, and modify the suitable biocatalysts, how to recycle the coenzyme, and how to improve productivity and enantioselectivity of the reactions. 

Other biocatalysts were also used to perform the dynamic kinetic resolution through reduction. For example, Thermoanaerobium brockii reduced the aldehyde with a moderate enantioselectivity [30b,c], and Candida humicola was found, as a result of screening from 107 microorganisms, to give the (Jl)-alcohol with 98.2% ee when ester group was methyl [30dj. 

Furthermore, the biocatalysts will be even more important with the shift of the raw materials from oil to biomass. Since biomass is a mixture of various multifunctional compounds, chemo-, regio-, and enantioselective catalysts will be... 

The change of the anion results in alteration of enzymatic activity and also allows improvement in the enantioselectivity from an unacceptable level (1.1) to a synthetically useful value of 24. The cationic component of the IL also affects the activity and selectivity of the biocatalyst. Scheme 5.16 presents the study on the kinetic resolution of adrenaline-type aminoethanol in ILs [64]. 

As each BVMO is limited in substrate specificity, it is crucial to have a large collection of these oxidative biocatalysts available. Except for expanding the scope of possible reactions, a large toolbox of BVMOs would also increase the chance of being able to perform any wanted specific chemo-, regio- and/or enantioselective reaction. This contrasts with the present situation as only a relatively small number of BVMOs can be exploited for biocatalytic purposes. Therefore, it is still crucial to discover or engineer BVMOs with novel biocatalytic properties. 

Catalytic transformations can be divided on the basis of the catalyst-type - homogeneous, heterogeneous or enzymatic - or the type of conversion. We have opted for a compromise a division based partly on type of conversion (reduction, oxidation and C-C bond formation, and partly on catalyst type (solid acids and bases, and biocatalysts). Finally, enantioselective catalysis is a recurring theme in fine chemicals manufacture, e.g. in the production of pharmaceutical intermediates, and a separate section is devoted to this topic. 

The use of rhodium catalysts for the synthesis of a-amino acids by asymmetric hydrogenation of V-acyl dehydro amino acids, frequently in combination with the use of a biocatalyst to upgrade the enantioselectivity and cleave the acyl group which acts as a secondary binding site for the catalyst, has been well-documented. While DuPhos and BPE derived catalysts are suitable for a broad array of dehydroamino acid substrates, a particular challenge posed by a hydrogenation approach to 3,3-diphenylalanine is that the olefin substrate is tetra-substituted and therefore would be expected to have a much lower activity compared to substrates which have been previously examined. 

Over the years of evolution, Nature has developed enzymes which are able to catalyze a multitude of different transformations with amazing enhancements in rate [1]. Moreover, these enzyme proteins show a high specificity in most cases, allowing the enantioselective formation of chiral compounds. Therefore, it is not surprising that they have been used for decades as biocatalysts in the chemical synthesis in a flask. Besides their synthetic advantages, enzymes are also beneficial from an economical - and especially ecological - point of view, as they stand for renewable resources and biocompatible reaction conditions in most cases, which corresponds with the conception of Green Chemistry [2]. 

Vorlop et al. described a novel cross-linked and subsequently poly(vinyl alcohol-entrapped PaHNL for synthesis of (//(-cyanohydrins. These immobilized lens-shaped biocatalysts have a well-defined macroscopic size in the millimeter range, show no catalyst leaching, and can be recycled efficiently. Furthermore, this immobilization method is cheap and the entrapped (/ )-oxynitrilases gave similar good results compared with those of free enzymes. The (//(-cyanohydrin was obtained in good yields and with high enantioselectivity of up to >99% ee [55],... 

Van Langen, L.M., Selassa, R.P, van Rantwijk, F. and Sheldon, R.A. (2005) Cross-linked aggregates of (A )-oxynilrilase a stable, recyclable biocatalyst for enantioselective hydrocyanation. Organic Letters, 7, 327-331. 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

By screening 53 Rhodococcus and Pseudomonas strains, an NHase-amidase biocatalyst system was identified for the production of the 2,2-dimethylcyclopropane carboxylic acid precursor of the dehydropeptidase inhibitor Cilastatin, which is used to prolong the antibacterial effect of Imipenem. A systematic study of the most selective of these strains, Rhodococcus erythropolis ATCC25 544, revealed that maximal product formation occurs at pH 8.0 but that ee decreased above pH 7.0. In addition, significant enantioselectivity decreases were observed above 20 °C. A survey of organic solvent effects identified methanol (10% v/v) as the... 

Rhodococcus sp. AJ270 was applied to the transformation of a number of racemic cis- and traray-3-aryl-2-methyloxiranecarbonitriles (Figure 8.7). In all cases, the NHase activity proceeded very rapidly and with poor enantioselectivity. In contrast, the amidase activity was strongly dependent upon substrate structure. In general, the biocatalyst displays a strong preference for the unsubstituted phenyl side chain or /wa-substituted phenyl side chain compared with ortho- or meta-, and this is manifest both with respect to observed conversion and rate and also observed enantioselectivity. In contrast, the biotransformations of... 

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