Oxynitrilase enzymes

Solvent effects upon the formation and stereoselectivity of 4-methoxycyclohexylanhydrin using the (R)-oxynitrilase enzyme derived from P. amygdalus was also determined by the author and summarized below ... 

Oxynitrilase enzymes from various plants catalyse the addition of cyanide ion to aldehydes to give the cyanohydrins present in many plants. Amygdalin, the cyanohydrin of a trisaccharide, is perhaps the most notorious of these as it releases HCN from oil of bitter almonds and has been responsible for the death of people drinking old samples of the liqueur noyeau.37... 

The most popular and selective catalysts for the asymmetric transformation of aldehydes into cyanohydrins are oxynitrilase enzymes. However, as the emphasis of this book is on chemically catalysed transformations, a discussion ofbiocatalysed... 

The enantiomerically pure amino acids also can be produced through a similar synthetic pathway catalyzed by enzymes. For example, reaction of hydrogen cyanide with benzaldehyde catalyzed by either (R)- or (S)-oxynitrilase enzyme yields the enantiomeric cyanohydrins (R)- or (S)-mandelonitrile. Alternatively, by adding carbon dioxide and hydrogen cyanide and ammonia as feedstocks, aldehydes can be converted to hydantoins (4-alkylimidazolidine-2,5-diones), which can be then hydrolyzed with either D- or L- hydan-toinases to produce D- or L-a-amino acids, respectively. 

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). 

The apphcation of (5)-oxynitrilase has been reported only recendy (159). The enzyme isolated from shoots of Sorghum catalyzes the condensation between various 3- and 4-substituted benzaldehydes and hydrogen cyanide resulting in (5)-cyanohydrins in 80—90% yield and up to 99% ee. 

As early as 1908, Rosenthaler found in the ferment mixture of emulsin a u-oxynitrilase , which directed the addition of hydrocyanic acid (hydrogen cyanide) to benzaldehyde asymmetrically to give x-hydroxybenzeneacetonitrilc (mandelonitrile)9. This result was confirmed1 °, however, it was not until 1963 that Pfeil ct al. first isolated and characterized the enzyme (R)-oxyni-trilase [EC 4.1.2.101 from bitter almonds (Prunus amygdalus)1 12. The yellow-colored enzyme contains a flavin-adenine dinucleotide (FAD)11 and loses its activity by splitting off this prosthet-... 

Chapters 5-8 are directed to emerging enzymes, which include oxynitrilases, aldolases, ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications especially in synthesis of chiral drugs and intermediates. 

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],... 

The best results for the asymmetric cyanohydrination reactions are obtained through biocatalysis, using the readily available enzyme oxynitrilase. This provides cyanohydrins from a number of substances with over 98% ee.146... 

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. 

This enzyme [EC 4.1.2.10], also known as hydroxynitrile lyase and (i )-oxynitrilase, catalyzes the conversion of mandelonitrile to cyanide and benzaldehyde. 

Nature often uses two pathways to produce oxygenated molecules. One is oxidative fictionalization of substrates through enzymatic hydration, monohydroxylation, dihydroxylation or epoxidation. Alternatively, oxygenated compounds can be generated by C—C formation catalyzed by aldolase, transketolase, oxynitrilase and related enzymes, where chiral centers are created simultaneously without an overall change in oxidation states [78]. 

Oxynitrilases are enzymes that catalyze the formation and cleavage of cyanohydrins through the stereoselective addition of hydrogen cyanide to aldehydes or methyl ketones giving enantiopure a-hydroxynitriles. The use of (R)-oxynitrilases for the preparation of chiral cyanohydrins has dramatically grown in the last decade because of their possibihties as precursors for the synthesis of many compounds with physiological properties [50]. 

Other interesting applications of the ophcally active cyanohydrins obtained by (R)-oxynitrilase-catalyzed processes are the produchon of the blockbuster clopido-grel (Scheme 10.23) [52], and the angiotensin-converhng enzyme inhibitors enala-pril and lisinopril (Scheme 10.24) [53, 54]. 

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. 

The lyases comprise enzyme class 4. They are enzymes cleaving C-C, C-0, C-N and other bonds by elimination, not by hydrolysis or oxidation. Lyases also catalyse addition to donble bonds. The types of reactions catalysed by lyases are decarboxylation (decarboxylase), hydration/dehydration (hydratase/dehydratase), ammonia addition/deamination (ammonia-lyase), cyanohydrin formation/cleavage (oxynitrilase),... 

This reaction is known to be catalyzed by the enzyme oxynitrilase to produce the optically pure cyanohydrin 76). Since this reaction proceeds with a base catalyst, Jnoue et al. 75) used cyclic and linear dipeptides containing (S)-histidine. The catalysts employed are as follows benzyloxycarbonyl-R-(S)-histidine methyl ester with R = (S)-alanyl, (R)-alanyl, (S)-phenylalanyl,[Z-(S)-Ala-(S)-His-OCH3, Z-(R)-Ala-(S)-His-OCH3, and Z-(S)-Phe-(S)-His-OCH3] as linear dipeptides, and cyclic (S)-histidine containing dipeptides Gly—(S)—His,... 

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],... 

The Sorghum (S)-oxynitrilase exclusively catalyzes the addition of hydrocyanic acid to aromatic aldehydes with high enantioselectivity, but not to aliphatic aldehydes or ketones [519, 526], In contrast, the Hevea (S)-oxynitrilase was also found to convert aliphatic and a,/ -unsaturated substrates with medium to high selectivity [509, 527]. The stereocomplementary almond (R)-oxynitrilase likewise has a very broad substrate tolerance and accepts both aromatic, aliphatic, and a,/ -unsaturated aldehydes [520, 521, 523, 528, 529] as well as methyl ketones [530] with high enantiomeric excess (Table 9). It is interesting to note that this enzyme will also tolerate sterically hindered substrates such as pivalaldehyde and suitable derivatives 164 which are effective precursors for (R)-pantolactone 165 [531],... 

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. 

For the synthesis of cyanohydrins nature provides the chemist with R- and S-selective enzymes, the hydroxynitrile lyases (HNL) [4-7]. These HNLs are also known as oxynitrilases and their natural function is to catalyze the release of HCN from natural cyanohydrins like mandelonitrile and acetone cyanohydrin. This is a defense reaction of many plants. It occurs if a predator injures the plant cell. The reaction also takes place when we eat almonds. Ironically the benzaldhyde released together with the HCN from the almonds is actually the flavor that attracts us to eat them. 

Analogously, Van Pelt et al. (2009) have realized a one-pot bienzymatic cascade combining the (5 )-selective oxynitrilase from M. esculenta and a purified nitrile hydratase from Nitriliruptor alkaliphilus for the synthesis of aliphatic S)-a-hydroxycarboxylic amides (Table 17.6) both enzymes were immobilized as CLEAs to enhance their stability (Van Pelt et al., 2009). 

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>. 

The enzyme mandelorutrile lyase (oxynitrilase) isolated from almond flour has been shown to catalyze the stereospedfic cyanohydrination of... 

In catalytic processes with enzymes such as D-oxynitrilase and (R) xynitrilase (mandelonitrilase) or synthetic peptides such as cyclo[(5)-phenylalanyl-(5)-histidyl], or in reaction with TMS-CN pro-mot by chiral titanium(IV) reagents or with lanthanide trichlorides, hydrogen cyanide adds to numerous aldehydes to form optically active cyanohydrins. The optically active Lewis acids (8) can also be used as a catalyst. Cyanation of chiral cyclic acetals with TMS-CN in the presence of titanium(IV) chloride gives cyanohydrin ethers, which on hydrolysis lead to optically active cyanohydrins. An optically active cyanohyrMn can also be prepared from racemic RR C(OH)CN by complexation with bru-... 

For the production of (5)-m-phenoxybenzaldehyde cyanohydrin (47) DSM established an enzymatic hydrocyanation process on an industrial scale (Scheme 31). An efficient (S)-oxynitrilase biocatalyst has been developed. This enzyme is derived from the plant Hevea brasiliensis, and has been cloned and overexpressed in a microbial host organism [117]. In the presence of this biocatalyst the desired product 47 has been obtained with high enantioselec-tivity. 

Several industrial processes using lyases as catalysts have been reported. Perhaps the most prominent lyase-catalyzed process is the production of acrylamide from acrylnitrile. This process is carried out by the Nitto Chemical Company of Japan at a scale of more than 40,000 tons per year. Another example is the use of a fumarase for the production of (5 )-malic acid from fumaric acid. As shown in Fig. 7, a water molecule is added to the double bond in fumarate by means of an addition reaction. The result is a cleavage of the carbon-carbon double bond, and a formation of a new carbon-oxygen bond. A third example is bio-catalytic production of a cyanohydrin from a ketone. This reaction is catalyzed by a lyase called oxynitrilase. It consists of the cleavage of one carbon-oxygen bond, and the addition of a HCN molecule. The chirality of the product is based on the form of the enzyme used (/ -oxynitrilase or 5-oxynitrilase). ... 

Oxynitrilases or hydroxynitrile lyases (HNL) constitute a group of enzymes that catalyze the reversible addition of HCN to ketones and aldehydes. The natural role of these enzymes is a defence mechanism of higher plants against herbivores, whereby HCN is liberated from cyanoglucosides such as prunasin (almond, cherry, apple) by the action of a glycosidase and a hydroxynitrile lyase. 

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