Hydroxynitrile lyase substrate selectivity

Chiral cyanohydrins are versatile intermediates in the synthesis of a-hydroxy acids, /3-amino alcohols, amino nitriles, a-hydroxy ketones and aziridines. For the synthesis of enantiopure cyanohydrins, the use of hydroxynitrile lyases is currently the most effective approach.Application of an organic-solvent-free system allows thermodynamically hindered substrates to be converted with moderate to excellent yields. With the use of the highly selective hydroxynitrile lyase from Manihot esculenta, the syntheses of several acetophenone cyanohydrins with excellent enantioselectivities were developed (Figure 8.2). (5)-Acetophenone cyanohydrin was synthesized on a preparative scale. ... 

The hydroxynitrile lyase (HNL) class of enzymes, also referred to as oxynitrilases, consists of enzymes that catalyze the formation of chiral cyanohydrins by the stereospecific addition of hydrogen cyanide (HCN) to aldehydes and ketones (Scheme 19.36).275 279 These chiral cyanohydrins are versatile synthons, which can be further modified to prepare chiral a-hydroxy acids, a-hydroxy aldehydes and ketones, acyloins, vicinal diols, ethanolamines, and a- and P-amino acids, to name a few.280 Both (R)- and (.S )-selective HNLs have been isolated, usually from plant sources, where their natural substrates play a role in defense mechanisms of the plant through the release of HCN. In addition to there being HNLs with different stereo-preferences, two different classifications have been defined, based on whether the HNL contains a flavin adenine dinucleotide (FAD) co-factor. 

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 reason why these enzymes have received considerable attention over the years is that they display a high degree of enantiotopic selectivity on the prochiral aldehyde and ketone substrates. The selectivity of these enzymes is in many instances masked by the rate of spontaneous racemization of the cyanohydrins, which are prone to racemization under non-acidic conditions. This balance of selectivity of the enzymes versus the competition with the spontaneous racemization reaction as a function of the pH was described as early as 1921 using the hydroxynitrile lyase enzyme from peach leaves [22], These early experiments describe one of the challenges of applying hydroxynitrile lyases on an industrial scale. 

F. Substrate Selectivities of Hydroxynitrile Lyases from Different Sources... 

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