Cyanohydrin enzymatic hydrolysis

Reisinger, C., Osprian, I., Glieder, A. et al. (2004) Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from Rhodococcus erythropolis. Biotechnology Letters, 26, 1675-1680. 

Pyrethroids are commercially important insecticides that usually contain a cyclopropyl unit that is m-substituted and a cyanohydrin derivative. They are usually sold as a mixture of isomers. However, asymmetric routes have been developed, especially because these compounds are related to chrysanthemic esters (Chapter 12).251 The pyrethroids can be resolved through salt formation or by enzymatic hydrolysis.252... 

The first total synthesis of amiclenomycin, an inhibitor of biotin biosynthesis, was completed by A. Marquet and co-workers. In order to prove its structure unambiguously, both the cis and trans isomers were prepared. The L-amino acid functionality was installed by a Strecker reaction using TMSCN in the presence of catalytic amounts of Znla. The resulting O-TMS protected cyanohydrin was exposed to saturated methanolic ammonia solution, which gave rise to the corresponding a-amino nitrile. Enzymatic hydrolysis with immobilized pronase afforded the desired L-amino acid. 

Chemical or enzymatic hydrolysis of the chiral cyanohydrin gives access to 2-hy-droxycarboxylic acids. The most prominent examples are (S)- and (R)-mandclic acids, which are mainly used for racemate resolution. Other chiral acids, such as (R)-2-chlorom an del i c acid, are used as precursors for pharmaceuticals (see Fig. 11). Scale-up and production of (R)-2-chloromandelic acid was successfully performed with a space-time yield of 250 g/L/d (ee = 95%). 

Esterase or lipase as catalyst. Application of hydrolytic enzymes is realized in three different systems enzymatic hydrolysis or transesterification of racemic cyanohydrin esters (see Figure 14.7-4.) as well as enzymatic acylation of racemic cyanohy-... 

A series of cyanohydrin acetates with an e.e. up to 98% has been prepared by enzymatic hydrolysis of their racemic acetates in the presence of an esterase from Pseudomonas spJ137]. Lipoprotein lipase from Pseudomonas sp. catalysed irreversible transesterification using enol esters was applied to the resolution of different aromatic cyanohydrins[138> 139). 

The vindoline synthesis required prior preparation of the amine coupling partner, the 2,4-dinitrobenzenesulfonamide 713, which was prepared from the pent-anal 710, as shown in Scheme 43. A notable feature of this route was the enzyme-mediated resolution of the cyanohydrin acetate 711, via enzymatic hydrolysis to selectively afford a diastereomeric mixture of only the (5)-cyanohydrins 712. 

Table 3. (R)-Cyanohydrins by Enzymatic Formation from Ketones and Hydrocvamic Acid as well as (7 )-a-Hydroxy-a-methyl Carboxylic Acids by Hydrolysis...

Osprian, 1., Fechter, M.H. and Griengl, H., Biocatal3ftic hydrolysis of cyanohydrins an efficient approach to enantiopure a-hydroxy carboxylic acids. J. Mol. Catal. B Enzymatic, 2003,24-25, 89-98. 

Figure 7.8 a) Enzymatic (oxynitrilase-catalyzed) formation of cyanohydrins from aldehydes, b) Chemical hydrolysis of cyanohydrins to hydroxy acids. 

Interest in the synthesis of enantiopure 2-hydroxycarboxylic acids via asymmetric enzymatic transformations is still increasing and two pathways have risen into prominence recently. The first is based on enantioselective hydrocyanation of the appropriate aldehyde in the presence of an oxynitrilase (hydroxynitrile lyase, EC 4.1.2.10), which gives rise to the corresponding enantiomerically pure cyanohydrin, followed by chemical hydrolysis in the presence of strong acid (Figure 16.1, route a). This latter step generates copious quantities of salt and is not compatible with sensitive functional groups, which is a serious limitation. 

The nitrilase mediated DKR route to enantiomerically pure 2-hydroxycarboxylic acids is restricted to the (R)-enantiomers because, to our knowledge, no (S)-selec-tive nitrilases for cyanohydrin substrates are commonly available [11]. We reasoned that a fully enzymatic route to the (S)-acids should be possible by combining an (S)-selective oxynitrilase (hydroxynitrile lyase, EC 4.1.2.10, (S)-hydroxynitrile lyase) and a non-selective nitrilase in a bienzymatic cascade (see Figure 16.3). Besides being more environmentally acceptable than chemical hydrolysis, the mild reaction conditions of the combined enzymatic reaction would be compatible with a wide range of hydrolysable groups. 

The reports mentioned above provide a systematic coverage of the nonimmobi-lized enzymatic reactors used in biocatalytic reactions under continuous flow operation. Results from microreactor experiments were comparatively higher than conventionally mixed batch reactors in terms of conversion rate and improvement of product yield as demonstrated for hydrolysis [140], dehalogenation [141], oxidation [142], esteriflcation [143], synthesis of isoamyl acetate [144,145], synthesis of cyanohydrins [147,148], synthesis of chiral metabolites [153], reduction [151], and bioluminescent reaction [149]. The small volumes involved and the favorable mass transfer inherent to these devices make them particularly useful for the screening of biocatalysts and rapid characterization of bioconversion systems. The remarkable results of such studies revealed that the product yield could be enhanced significantly in comparison with the conventional batch runs. 

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