Enzymatic Synthesis of Cyanohydrins

In the following discussion the method of reactor optimization will be demonstrated using two enzyme systems introduced earlier, namely the enzymatic synthesis of N-acetylneuraminic acid and the enzymatic synthesis of cyanohydrins using oxynitrilase. 

In the case of the enzymatic synthesis of cyanohydrins, enantioselectivity is the most important criterion. Investigation of the kinetics of the whole system (enzymatic and non-enzymatic reaction) offers the possibility to optimize reaction conditions to... 

Enzymatic Synthesis of Cyanohydrins 974 Martin H. Fechter and Herfried Cringl... 

The enzymatic synthesis of cyanohydrin 6 reported by the group of Ruges, demonstrates several advantages of flow over batch conditions. Toxic HCN, necessary for the enzyme catalyzed addition to aldehyde 5, was... 

The enzymatic synthesis of chiral cyanohydrins has reached a high stage of development. The different reaction systems give the possibility to convert a great... 

Recently, it has been demonstrated that the enzymatic synthesis of (S)-cyanohydrins was possible using an oxynitrilase isolated from Sorghum biocolor (95,96), These optically active cyanohydrins can be subsequently converted chemically to chiral ot-hydroxyadds, aminoalcohols, and acyloins. 

The pH value has an influence not only on the activity of enzymes but also on chemical reactions. The chemical cyanohydrin reaction is base-catalysed, as, compared to HCN, the cyanide ion more easily attacks the carbonyl group. As a result, a distinct decrease of the reaction rate for the non-enzymatic synthesis of mandeloni-trile occurs at lower pH values. Also, the enzyme activity decreases but not to the same extent therefore, the enantioselective enzymatic reaction becomes dominant at lower pH (see Fig. 7-11). 

Figure 7-32. Enzymatic synthesis of benzaldehyde cyanohydrin in an EMR calculated and experimental enantioselectivity as a function of conversion 155- 60l...

The enzymatic synthesis of enantiopure cyanohydrins has been brought to a high stage of development. Both (R)- and (S)-cyanohydrins are accessible for a broad variety of substrates in as a rule excellent yield and enantiopurity. Following recent progress in overexpression, HNLs are also available in quantities needed for industrial production. The procedures for safe handling of cyanides are well established so that they do not restrict the exploitation of HNLs. 

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. 

One of the major issues that need to be controlled is the nonspecific chemical addition of HCN to the substrate, which negatively impacts the enantiopurity of the product, as enantiopurity is the result of the ratio of enzymatic to nonenzymatic cyanohydrin formation. This nonenz3unatic background reaction is pH and temperature dependent performing the conversions at low pH values (<4-5) and low temperature (5-8 °C) allows the suppression of the xmdesired racemic product formation, thereby enabling the synthesis of cyanohydrins with higher enantiopurity. However, as an aqueous... 

Costes, D., Wehtje, E. and Adlercreutz, P. (2001) Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins. Journal of Molecular Catalysis B-Enzymatic, 11, 607-612. 

Figure 8.3 Enzymatic enantiocompiementary synthesis of the cyanohydrins from pyridine-3-carboxaidehyde.

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],... 

Relatively few of the enzymatic methods applicable to the preparation of secondary cyanohydrins have been adapted successfully to the synthesis of optically pure tertiary cyanohydrins [1,3,4]. Similarly, progress in asymmetric hydro-cyanation of ketones with synthetic catalysts lagged far behind advances in aldehyde cyanation. This situation has changed fairly dramatically over the past... 

For recent reviews/highlights on the asymmetric synthesis (by enzymatic, organocatalytic and organometallic methods) of cyanohydrins and their applications, see references 2-8. 

Lu W, Chen P et al (2008) New stereoselective synthesis of thiamphenicol and florfenicol from enantiomerically pure cyanohydrin a chemo-enzymatic approach. Tetrahedron 64 7822-7827... 

Cyanohydrins are versatile building blocks that are used in both the pharmaceutical and agrochemical industries [2-9]. Consequently their enantioselective synthesis has attracted considerable attention (Scheme 5.1). Their preparation by the addition of HCN to an aldehyde or a ketone is 100% atom efficient. It is, however, an equilibrium reaction. The racemic addition of HCN is base-catalyzed, thus the enantioselective, enzymatic cyanide addition should be performed under mildly acidic conditions to suppress the undesired background reaction. While the formation of cyanohydrins from aldehydes proceeds readily, the equilibrium for ketones lies on the side of the starting materials. The latter reaction can therefore only be performed successfully by either bio- or chemo-cat-... 

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 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. 

A suitable catalyst for the synthesis of (R)-cyanohydrins is the enzyme (R)-oxynitrilase from bitter almonds. It catalyzes exclusively si-face addition of hydrogen cyanide to benzaldehyde or other aldehydes. A competing non-enzymatic parallel reaction lowers the enantiomeric excess of the product155, 56]. 

The addition reaction of carbon-11 labelled cyanide ion to the bisulphite addition adduct of an aldehyde has been extended to prepare carbon-11 labelled amines. Maeda and coworkers prepared both p- and m-octopamine [2-(p-and m-hydroxyphenyl)-2-hydroxyethyl-amine] from the corresponding benzaldehyde by reducing the cyanohydrin formed in the reaction between the appropriate benzaldehyde and cyanide ion both under enzymatic conditions and by the basic modification of the Bucherer-Strecker synthesis, with borane-THF. The synthesis of / -octopamine is presented in equation 64. 

The chemoenzymatic synthesis of (13S)-hydroxy-18 2(9Z,ll ) was achieved in nine steps starting from (2 )-octenal. Of importance was the enzymatic conversion of (2 )-octenal to the (5)-cyanohydrin [5] with (S)-hydroxynitrile lyase cloned from Hevea brasiliensis (41). 13- Hydroxy-lO-oxo-18 1(11 ) was synthesized via a Kno-evenagel-type reaction of Isopropyl 11 - phenylsulfinyl-10-oxoundecanoate with hep-tanal to form y-hydroxyenone functionality together with carbon chain elongation (42). The regiospecific oxidation of a number of substituted unsaturated fatty esters with /7-benzoquinone in the presence of palladium(II) chloride under concomitant ultrasonic irradiation was reported. For example, methyl 9-hydroxy-18 1 (12Z) furnished methyl 9-hydroxy-12-keto-18 0 exclusively (43). 

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