Cassava, cyanohydrins

The hydroxynitrile lyase (HNL)-catalyzed addition of HCN to aldehydes is the most important synthesis of non-racemic cyanohydrins. Since now not only (f )-PaHNL from almonds is available in unlimited amounts, but the recombinant (S)-HNLs from cassava (MeHNL) and rubber tree (HbHNL) are also available in giga units, the large-scale productions of non-racemic cyanohydrins have become possible. The synthetic potential of chiral cyanohydrins for the stereoselective preparation of biologically active compounds has been developed during the last 15 years. 

Until 1987, the (R)-PaHNL from almonds was the only HNL used as catalyst in the enantioselective preparation of cyanohydrins. Therefore, it was of great interest to get access to HNLs which catalyze the formation of (5 )-cyanohydrins. (5 )-SbHNL [EC 4.1.2.11], isolated from Sorghum bicolor, was the first HNL used for the preparation of (5 )-cyanohydrins. Since the substrate range of SbHNL is limited to aromatic and heteroaromatic aldehydes as substrates, other enzymes with (5 )-cyanoglycosides have been investigated as catalysts for the synthesis of (5 )-cyanohydrins. The (5 )-HNLs from cassava (Manihot esculenta, MeHNL) and from Hevea brasiliensis (HbHNL) proved to be highly promising candidates for the preparation of (5 )-cyanohydrins. Both MeHNL and HbHNL have been overexpressed successfully in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. 

Citrate buffer (0.1 m, pH 2.5, 20 /iL), 3-pyridinecarboxaldehyde (300 mg) and CLEA -immobilized HNL (40 mg) were added to 9.7 mL of the dichloromethane-HCN solution and the reaction was aged for 2-20 h at 5 °C. Commercially available CLEAS of almond HNL (PaHNL-CLEA ) and cassava HNL (MeHNL-CLEA ) were used to catalyze the syntheses of R)- and (5)-cyanohydrin respectively. 

Bitter almonds contain amygdalin, which is the P-D-glucoside of prunasin, so it hydrolyses sequentially to the same products. Cassava, which is used in many parts of the world as a food plant, contains linamarin, which is the P-D-glucoside of acetone cyanohydrin. Preparation of the starchy tuberous roots of cassava for food involves prolonged hydrolysis and boiling to release and drive off the HCN before they are suitable for consumption. 

The commercially available CLEA particles of hydroxynitrile lyases (HNLs) from cassava and almond were used to produce both enantiomers. The use of dichloromethane, free hydrogen cyanide, and low temperatures was important for improving the cyanohydrin stereoselectivity and suppressing the background reaction. The aggregate particles were easily separated from the reaction by filtration followed by washing, and up to ten times reuse was demonstrated. 

Native HNLs from bitter almonds (Prunus amygdalus), cassava (Manihot escu-lenta), millet (Sorghum bicolor), and flax (Linum usitatissimum) were repeatedly used in the synthesis of chiral cyanohydrins [39, 41, 197]. Cyanohydrins are versatile building blocks in natural product synthesis, giving organic chemists the possibility of introducing all kinds of functional groups (Fig. 38) [198]. 

The levels of HCN equivalent (HCN, linamarin and cyanohydrins combined) must not exceed 50mg/kg in the complete feed and the maximum level of cassava permitted in poultry in Canada is 200g/kg. (From CFIA, 2005.)... 

The reversibility of cyanohydrin formation is of more than theoretical interest. In parts of Africa the staple food is cassava. This food contains substantial quantities of the glucoside of acetone cyanohydrin (a glucoside is an acetal derived from glucose). We shall discuss the structure of glucose later in this chapter, but for now, just accept that it stabilizes the cyanohydrin. 

We saw an example In Chapter 6 where acetone cyanohydrin is found in the cassava plant as a glucoside and suitable precautions must be taken when eating cassava to avoid poisoning byHCN. 

Both linamarin and amygdalin are toxic compounds because they are metabolized to cyanohydrins, which are hydrolyzed to carbonyl compounds and toxic HCN gas. This second step is merely the reconversion of a cyanohydrin to a carbonyl compound, a process that occurs with base in reactions run in the laboratoiy (Section 21.9A). If cassava root is processed with care, linamarin is enzymatically metabolized by this reaction sequence and the toxic HCN is released before the root is ingested, making it safe to eat. 

Naturally occurring cyanohydrin derivatives—linamarin, from cassava root and amygdahn, commonly called laetrile, from apricot, peach and wild cherry pits (Section 21.9B)... 

Progress has also been made in the overexpression of the oxynitrilase from Manihot esculenta (cassava) [67,70] in Escherichia coli. As mentioned previously, this oxynitrilase is very similar to the Hevea enzyme [16,32], because both plants belong to the same plant family, the Euphorbiaceae. A fermentation on the 40 1 scale gave, after simple purification, a total amount of about 40,000 lU of oxynitrilase activity and allowed the exploration of its ability to catalyse the formation of (S)-cyanohydrins [67]. 

HNL from Manihot esculenta Crantz (termed E.C. 4.1.2.37 at this time because E. C. 4.1.2.39 was not created earlier than 19991981) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava in 1994 471. First experiments demonstrated a broad substrate range, but only unsatisfactory optical purities were obtained1"1. The overexpression of the cloned M. esculenta HNL gene in E. coli increased the accessibility and specific activity of the biocatalyst as well as the ee of produced cyanohydrins 871. 

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