Introduction

The release of hydrocyanic acid (HCN) as a defense against herbivores is widely distributed in higher plants (cyanogenesis). The highly toxic HCN is chemically masked in the plants in the form of cyanohydrins, which are stabilized by an O-^-glycosidic linkage to saccharides (mainly D-glucose).1 During cyanogenesis, a specific ^-glycosidase cleaves the cyanoglycoside first into the corresponding cyanohydrin and a carbohydrate. In a second step, the cyanohydrin is decomposed enzymatically into HCN and the corresponding carbonyl compound by hydroxynitrile lyases (HNLs) (Scheme 1).

It is interesting to note that in the case of R1 = R2, an optically active cyanohydrin results in all known cases of cyanogenesis. For the enzymatic

Scheme 1: Cyanogenesis in higher plants.

Scheme 1: Cyanogenesis in higher plants.

CN NC

Scheme 2: HNL catalyzed enantioselective addition of HCN to aldehydes.

cleavage of the resulting cyanohydrins, enantiospecific HNLs (R- or S-HNLs) are required. As any other catalyst, HNLs catalyze the reaction in both directions. An enantioselective addition of HCN to carbonyl compounds (R1 = R2) forming optically active cyanohydrins (Scheme 2) could therefore be deduced from cyanogenesis.

The first attempts to prepare optically active cyanohydrins using the HNL from bitter almonds (Prunus amygdalus), (R)-PaHNL [EC.4.1.2.10], as catalyst gave only moderate optical yields.2 Especially with slower-reacting aldehydes, enantioselectivity was very poor because the non-enzymatic catalyzed chemical addition yielding racemates becomes dominant. The decisive breakthrough for synthetic applications of HNLs in the enantioselective synthesis of cyanohydrins was the discovery that the chemical addition can be suppressed considerably by working in organic solvents immiscible with water.3 Table 1 shows a comparison of the optical yields of the (R)-PaHNL-catalyzed cyanohydrin formation in water/ethanol and in an organic solvent, respectively.3

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 (S)-cyanohydrins. (S)-SbHNL [EC 4.1.2.11], isolated from Sorghum bicolor, was the first HNL used for the preparation of (S)-cyanohydrins.4 Since the substrate range of SbHNL is limited to aromatic and heteroaromatic aldehydes as substrates, other enzymes with (S)-cyanoglycosides have been investigated as catalysts for the synthesis of (S)-cyanohydrins. The (S)-HNLs from cassava (Manihot esculenta, MeHNL)5 and from Hevea brasiliensis (HbHNL)6 proved to be highly promising candidates for the preparation of (S)-cyanohydrins. Both MeHNL5 and HbHNL7 have been overexpressed successfully in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris.

In Table 2, the properties and characteristics of the five HNLs presently applied in the enzyme-catalyzed large-scale preparation of chiral cyanohydrins are summarized.78

Although considerable progress has been made in metal-catalyzed syntheses of non-racemic cyanohydrins in recent years, the HNL-catalyzed preparation of chiral cyanohydrins is by far the most important method especially for large-scale processes, due to the easy access of (R)- and (S)-HNLs and the high optical and chemical yields of the cyanohydrins obtained.9-12

Table 1

(R)-PaHNL catalyzed cyanohydrin formation in H2O/EtOH and in organic solvents, respectively

O OH

Aldehyde R = H2O/EtOH Organic solvent

(%)

ee (%)

Solv.

(%)

ee (%)

Ph

1

99

86

EtOAc

2.S

9S

99

•-Pr2O

3

96

>99

3-PhO-C6H4

S

99

1G.S

EtOAc

192

99

98

2-furyl

2

86

69

EtOAc

4

88

99

MeCH=CH

1.S

68

76

EtOAc

3

68

97

Me3C

2.S

S6

4S

•-Pr2O

4.S

84

83

MeS(CH2)2

3

87

6G

•-Pr2O

16

98

HNLs currently applied as catalysts in the enantioselective preparation of chiral cyanohydrins7'8

Table 2

HNLs currently applied as catalysts in the enantioselective preparation of chiral cyanohydrins7'8

MeHNL/HbHNL SbHNL

PaHNL

LuHNL

Family Euphorbiaceae

FAD No

Carbohydrate No

Structure Homotetramer

MW of subunits 30 000

R/S specificity S

Homologies pir7 protein

Gramineae No Yes

Heterotetramer 33 GGG, 18 GGG S

Serine carboxypeptidases

Rosaceae

Monomer 6G GGG R

Linaceae

Homodimer 42 GGG R

Flavoproteins Alcohol dehydrogenases

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