Enzyme catalyzed reaction cyanohydrination

The optical purities were determined solely from the optical rotations of the (/ -cyanohydrins thus obtained. Only for (/ )-a-hydroxybcnzeneacetonitrile, available from benzaldehyde, was an optical purity determined by comparison with the natural product. Variation of the reaction conditions (pH, temperature, concentration) in water/ethanol led to no appreciable improvementsl4. The use of organic solvents that are not miscible with water, but in which the enzyme-catalyzed reaction can still take place, resulted in suppression of the spontaneous addition to a significant extent, whereas the enzyme-catalyzed formation of cyanohydrins was only slightly slower (Figure l)13. 

Biihler, H., Bayer, A. and Effenberger, F. (2000) Enzyme-catalyzed reactions, part 39. A convenient synthesis of optically active 5,5-disubstituted 4-amino- and 4-hydroxy-2(5f/)-furanones from (5)-ketone cyanohydrins. Chemistry - A European Journal, 6, 2564—2571. 

Since the reaction has been reviewed recently (12) only a few additional facts will be mentioned. Many optically active cyanohydrins can be prepared (33) with e.e. s of 84 to 100% by the use of the flavopnotein D-oxynitrilase adsorbed on special (34) cellulose ion-exchange resins. Although the enzyme is stable, permitting the use of a continuously operating column, naturally only one enantiomer, usually the R isomer, is produced in excess. This (reversible) enzyme-catalyzed reaction is very rapid (34). Nonenzymic catalysts, such as the cinchona alkaloids, permit either enantiomer to be prepared in excess. 

F. Effenberger, B. Hoersch, S. Foerster, andT. Ziegler, Enzyme-catalyzed reactions. 5. Enzyme-catalyzed synthesis of (S)-cyanohydrins and subsequent hydrolysis to (S)-a-hydroxy-carboxylic adds, Tetrahedron Lett. 1990, 31, 1249-1252. 

T. Ziegler, and S. Kuehner, Enzyme catalyzed reactions. 9. Enzyme-catalyzed synthesis of (R)-ketone cyanohydrins and their hydrolysis to (R)-a-hydroxy-a-methyl carboxylic adds, Tetrahedron Letters 1991,... 

Effenberger, R, Ziegler, T. and Forster, S. (1987) Enzyme-catalyzed reaction. 15. Preparation of (R)-2-(sulfonyloxy)-nitriles and their reactions with acetates inversion of the configuration of optically active cyanohydrins. Chem. Ber., 126, 779-86. 

Enzyme-catalyzed reactions in this area include reaction with glycine catalyzed by L-threonine aldolase to afford 164 <2000SL1046> and the use of almond oxynitrilase to catalyze the formation of cyanohydrin 165 by reaction of 161 with acetone cyanohydrin <2001T2213>. 

In most enzyme-catalyzed reactions two or more substrates are involved, such as in the enzyme-catalyzed aldol reaction, the cyanohydrin reaction or enzyme-catalyzed peptide synthesis (examples used before). For many reaction schemes kinetic models have been derived using the steady-state assumption. Some important reaction mechanisms and the corresponding rate equations are summarized in Table 7-1. An approach to the steady-state method and a detailed discussion of the resulting kinetic models is difficult and is not the aim of this chapter. 

Effenberger F, Gutterer B, Ziegler T, Eckhardt E, Aichholz R. Enzyme-catalyzed reactions. 7. Enantioselective esterification of racemic cyanohydrins and enantioselective hydrolysis or transesterification of cyanohydrin esters by lipases. Liebigs Ann. Chem. 1991 47-54. 

S-Cyanohydrins by Selective Cleavage of the R Form in a Racemate. In principle it should be possible to obtain S-cyanohydrins by selective enzymatic decomposition of the R enantiomer in a racemic mixture. This is difficult to achieve on a practical scale because the HCN that is liberated can engage in both enzyme-catalyzed and non-enzyme-catalyzed reactions by which the R enantiomer is again formed. This problem has been elegantly solved by Gotor and co-workers by combining this approach with that of the transcyanation [102]. A mixture of aldehyde and racemic cyanohydrin from a methyl ketone was treated with almond meal. The / -cyanohydrin of the methyl ketone was selectively cleaved and the / -cyanohydrin of the aldehyde was selectively formed by trans-... 

Cyanohydrin Synthesis. Another synthetically useful enzyme that catalyzes carbon—carbon bond formation is oxynitnlase (EC 4.1.2.10). This enzyme catalyzes the addition of cyanides to various aldehydes that may come either in the form of hydrogen cyanide or acetone cyanohydrin (152—158) (Fig. 7). The reaction constitutes a convenient route for the preparation of a-hydroxy acids and P-amino alcohols. Acetone cyanohydrin [75-86-5] can also be used as the cyanide carrier, and is considered to be superior since it does not involve hazardous gaseous HCN and also virtually eliminates the spontaneous nonenzymatic reaction. (R)-oxynitrilase accepts aromatic (97a,b), straight- (97c,e), and branched-chain aUphatic aldehydes, converting them to (R)-cyanohydrins in very good yields and high enantiomeric purity (Table 10). 

Today, the most promising synthesis of optically active cyanohydrins, especially with respect to the enantioselectivity of the reaction, is the enzyme-catalyzed addition of hydrogen cyanide to aldehydes and ketones, respectively. 

Enantiomeric or specific synthesis of cyanohydrin is influenced by the reaction medium, cyanide source, water content, buffer pH, enzyme, and temperature during the HNL-catalyzed reaction. To maximize the enantiomeric excess of the cyanohydrin product, care must be taken to minimize the parallel chemical (nonenzymatic) condensation and racemi-zation of products. 

The enzyme-catalyzed cyanohydrin reaction offers new and interesting perspectives for the synthesis of different kinds of chiral cyanohydrins, because over the next few years the continuous development of new genetically modified oxynitrilases will be without any doubt of great utility for the preparation of pharmaceuticals. 

Willeman et al. [26] modeled the enzyme-catalyzed cyanohydrin synthesis in a stirred batch tank reactor. Assumption of a mass transfer limitation (Figure 9.3b) is made, which results in a low concentration of substrate in the aqueous phase, thus suppressing the non-enzymatic reaction. In a well-stirred biphasic system the enzyme concentration was varied, keeping the phase ratio constant A maximum rate of conversion is reached at the concentration where mass transfer of the substrate becomes limiting. Further increase of enzyme concentration does not enhance the reaction rate [27]. The different results achieved by the two groups are explained by the different process strategies. No mass transfer limitation could be detected by Hickel et al. because the stirring rate in the aqueous phase was not varied [26]. 

Reactions catalyzed by solid bases were obvious candidates for testing hypotheses on the nature and the mode of action of enzymes. Bredig [40] used aminated cellulose (B2) as a model because an enzyme was thought to consist of "a specific active function and a colloidal carrier". Indeed, cyanohydrin 40 was formed with an enantiomeric excess of 22% Fig. 3 and Table 3 contain a summary of the reported results for base-catalyzed reactions. It is not clear whether the ZnO/ffuctose catalyst (Bl) described by Erlenmeyer [39] is really heterogeneous but it is the first report on using sugars as modifiers. Some reactions are probably just curiosities (39, 41), but two... 

The bifunctional nature and the presence of a stereocenter make a-hydroxyketones (acyloins) amenable to further synthetic transformations. There are two classical chemical syntheses for these a-hydroxyketones the acyloin condensation and the benzoin condensation. In the acyloin condensation a new carbon-carbon bond is formed by a reduction, for instance with sodium. In the benzoin condensation the new carbon-carbon bond is formed with the help of an umpolung, induced by the formation of a cyanohydrin. A number of enzymes catalyze this type of reaction, and as might be expected, the reaction conditions are considerably milder [2-4, 26, 27]. In addition the enzymes such as benzaldehyde lyase (BAL) catalyze the formation of a new carbon-carbon bond enantioselectively. Transketolases (TK)... 

Lyases are the enzymes responsible for catalyzing addition and elimination reactions. Lyase-catalyzed reactions involve the breaking of a bond between a carbon atom and another atom such as oxygen, sulfur, or another carbon atom. They are found in cellular processes, such as the citric acid cycle, and in organic synthesis, such as in the production of cyanohydrins. 

A more recent example of enzyme-catalyzed synthesis performed in micro reactors was reported by Rutjes and co-workers [81], who demonstrated the use of crude enzyme lysates, containing hydroxynitrile lyase, for the enantioselective synthesis of cyanohydrins. Employing a wet-etched borosilicate glass micro reaction channel, containing pillars to promote biphasic laminar flow, the authors evaluated the... 

Hydroxynitrile lyase enzymes catalyze the asymmetric addition of hydrogen cyanide onto a carbonyl group of an aldehyde or a ketone thus forming a chiral cyanohydrin [1520-1524], a reaction which was used for the first time as long ago as 1908 [1525]. Cyanohydrins are rarely used as products per se, but they represent versatile starting materials for the synthesis of several types of compounds [1526] ... 

Along the same lines, the remarkable synthetic potential of enzyme-catalyzed irreversible acyl transfer in nearly anhydrous organic solvents can be demonstrated particularly weU by the transformation of alcoholic substrates (such as organo-metallics or cyanohydrins) which are prone to decomposition reactions in an aqueous medium and thus cannot be transformed via enzyme-catalyzed hydrolysis reactions. 

Enzymes of the hydroxynitrilase dass catalyze the addition of HCN to aldehydes, produdng cyanohydrins. Recendy, the reaction has been extended to a few ketones with modified hydroxynitrilase enzymes. In many cases, these are formed with good optical purities and such reactions are the simplest type of enzyme catalyzed carbon-carbon bond formation. By pairing hydroxynitrile lyases with nitrilases or nitrile hydratases, one-pot, multistep conversions become possible, and this also shifts the equilibrium to favor the addition products. Such concerns are particularly important when applying these catalysts to ketones where the equilibrium generally favors the starting carbonyl compound (Figure 1.17). 

The NHase and amidase were largely nonselective for cyanohydrins and the corresponding 2-hydroxy amides, respectively, but they were suitable for the hydrolysis of enantiopure cyanohydrins prepared from aldehydes and HCN by oxynitrilases [89, 90]. The cascade of NHase and amidase, in which the latter enzyme catalyzed an acyl transfer reaction, was suitable for the preparation of... 

As any other catalyst, enzymes also catalyze reactions in both directions. An enan-tioselective addition of HCN to aldehydes in the presence of a HNL, forming an optically active cyanohydrin, could therefore be deduced from the enantioselectivity of cyanogenesis. 

In Table 9 the results of the preparation of (5)-cyanohydrins starting from both aldehydes and ketones with HCN and recombinant HbHNL as catalyst are summarized [9,29]. The reactions with HbHNL were performed normally in a biphasic system consisting of a concentrated aqueous enzyme solution and an organic solvent not miscible with water, e.g., ferf-butyl methyl ether. It must be noted that the reactions and optical yields in the HbHNL-catalyzed cyanohydrin formation (Table 9) were achieved by using the 10-fold higher amount of enzyme, in comparison to MeHNL-catalyzed reactions (Tables 7 and 8) (see notes to Tables 7 and 9). 

Racemic hydantoins result from the reaction of carbonyl compounds with potassium cyanide and ammonium carbonate or the reaction of the corresponding cyanohydrins with ammonium carbonate (Bucherer-Bergs reaction). Hydantoins racemize readily under basic conditions or in the presence of hydantoin racemase, thus allowing DKR (Figure 6.43). Hydantoinases (EC 3.5.2.2), either isolated enzymes or whole microorganisms, catalyze the hydrolysis of five-substituted... 

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