Jürgen Roos

Substrate Specificity of Mutants of the Hydroxynitrile Lyase from Manihot esculenta

Several tryptophan128‐substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL‐catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL‐W128A) are in most cases superior to the wild‐type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3‐phenoxybenzaldehyde. These experimental results are in accordance with the X‐ray crystal structure of MeHNL‐W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3‐phenoxybenzaldehyde with wtMeHNL and MeHNL‐W128A in both aqueous citrate buffer and a two‐phase system of water/methyl tert‐butyl ether again reveal the superiority of the mutant enzyme: 3‐phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space‐time yield of 57 g L−1 h−1.

Enzyme catalyzed addition of hydrocyanic acid to substituted pivalaldehydes — A novel synthesis of (R)-pantolactone

Abstract (R)-Cyanohydrins (R)- 2b-h are obtained in good optical yields by (R)-oxynitrilase catalyzed enantioselective addition of HCN to β-substituted pivalaldehydes 1b-h. Under optimized reaction conditions with highly purified (R)-oxynitrilase, hydroxypivalaldehyde (1a) is converted to (R)- 2a in satisfactory chemical and optical yields. By acid-catalyzed hydrolysis the cyanohydrins (R)- 2a-h cyclize directly to give crude (R)-pantolactone (R)- 3 with ee-values of 56–95% which, after recrystallization, go up to ≥98 %ee in all cases.

Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris - utilization of the recombinant enzyme for enzymatic analysis and site-directed mutagenesis

Abstract Hydroxynitrile lyases (HNL) are involved in the catabolism of cyanogenic glycosides in cyanogenic plants and are powerful tools in the stereoselective synthesis of cyanohydrins. The recent cloning of the hydroxynitrile lyase from flax ( Linum usitatissimum; LuHNL) reveals that this enzyme defines a novel class of HNL. Thorough biochemical and mutational analysis of LuHNL have been hampered by low expression levels of the recombinant enzyme in Escherichia coli . To overcome this impediment, we have cloned a myc-His-tagged LuHNL-cDNA under control of the methanol-inducible AOX1 (alcohol oxidase) promotor of Pichia pastoris and introduced it in the SMD1168 strain. Recombinant LuHNL was kinetically indistinguishable from the authentic flax enzyme. Immobilized LuHNL was used for synthesis of several aliphatic ( R )-cyanohydrins in a preparative scale to analyze the products according to enantiomeric excess and yield of reaction. LuHNL has significant homologies to members of the Zn 2+ -containing alcohol dehydrogenases (Zn 2+ -ADHs). In particular, residues responsible for coordination of Zn 2+ ions or fulfilling structural or functional tasks in Zn 2+ -ADHs are conserved. We found about 2–4 mol zinc per mol of recombinant LuHNL using atom absorption spectroscopy in a non His-tagged version of LuHNL. Using site-directed mutagenesis, we substituted several of the conserved residues against alanine in LuHNL and found that in most cases, HNL-activity was impaired. Hence, it seems that LuHNL and Zn 2+ -ADHs have similar structural requirements with respect to maintaining a catalytically active structure. Residues essentially involved in catalysis of Zn 2+ -ADHs are also of functional importance in LuHNL, suggesting that the removal of the proton from alcohol and cleavage of cyanohydrins can be fulfilled by similar active site structures.

Hydroxynitrile lyase catalyzed enantioselective HCN addition to O-protected α-hydroxyaldehydes

Abstract Various O -protected glycol- and racemic lactaldehydes 3 and 6 as well as O -allyl protected racemic α-hydroxyaldehydes 7 (R 1 =Et, Pr, Bu) have been prepared to investigate and perform a stereoselective Kiliani–Fischer synthesis by hydroxynitrile lyase (HNL) catalyzed addition of HCN. From all protecting groups investigated the allyl moiety was most suitable. ( R )-PaHNL from bitter almonds ( Prunus amygdalus ), yielding the (2 S )-cyanohydrins 8 – 10 , was found to be a more stereoselective catalyst than ( S )-MeHNL from maniok ( Manihot esculenta ). While ( R )-PaHNL led to enantiomeric excesses ≥93%, with ( S )-MeHNL the (2 R )-cyanohydrins 8 – 10 were obtained with enantiomeric excesses ≤78%.

Synthesis of (1R,cis,αS)-cypermethrine via lipase catalyzed kinetic resolution of racemic m-phenoxybenzaldehyde cyanohydrin acetate

Abstract A technical scale preparation of optically active (1 R , cis ,α S )-cypermethrine 4 from racemic m -phenoxybenzaldehyde cyanohydrin acetate ( RS )- 1 and (1 R , cis )-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid chloride (1 R , cis )- 3 is described. Key steps of the new procedure are a lipase catalyzed enantioselective transesterification of ( RS )- 1 with n -butanol and direct acylation of the mixture of ( R )- 1 and ( S )-cyanohydrin ( S )- 2 with (1 R , cis )- 3 to give enantiomerically pure (1 R , cis ,α S )- 4 . The unchanged ( R )- 1 is removed from (1 R , cis ,α S )- 4 by distillation, and is racemized with triethylamine to give ( RS )- 1 which is returned to the process. The total yield of (1 R , cis ,α S )- 4 referred to ( RS )- 1 is 80%.

Stereoselective synthesis of 3-amino-4,5-dihydroxyaldehydes—a novel preparation of N-acetyl-l-daunosamine

Abstract A general route for the stereoselective synthesis of 3-amino-4,5-dihydroxyaldehydes, with almost any desired configuration at the three stereogenic centers, is described by applying a combination of enzymatic and chemical steps. l -Daunosamine 1 , for example, the glycosidic fragment of many important anthracycline antibiotics has been prepared by this route starting from O -allyl- l -lactaldehyde ( S )- 6a . ( R )-Hydroxynitrile lyase (HNL) catalyzed addition of HCN to ( S )- 6a yields the 2,3-dihydroxynitrile (2 S ,3 S )- 7a with high stereoselectivity (91% de ) in 75% yield. The addition of allyl Grignard to the O -protected 2,3-dihydroxynitrile (2 S ,3 S )- 9a and subsequent hydrogenation of the imino intermediate leads to 4-amino-2,3-dihydroxy-1-heptene (4 S ,5 S ,6 S )- 12a , which after ozonization and deprotection gives N -acetylated l -daunosamine 14a in a total yield of 15% referring to ( S )- 6a . The general applicability of this chemoenzymatic multistep procedure is demonstrated in the stereoselective synthesis of the unnatural aminodeoxy sugar (2 S ,3 S ,4 S )- 14b , starting from isovaleraldehyde 3 .

Stereoselective synthesis of β-amino-γ-butyrolactones

Abstract A novel synthesis of optically active β-amino-γ-butyrolactones is described. O -Silylated ( R )-cyanohydrins ( R )- 3 (derived from aldehydes 1 by ( R )-hydroxynitrile lyase (( R )-PaHNL)-catalyzed addition of HCN) were reacted with allyl Grignard to give amino alcohols (4 R ,5 S )- 5 after reduction. In the addition of crotyl Grignard reagent, workup conditions are decisive for the formation of amino alcohol 8 , which was isolated as a diastereoisomeric mixture; the acetylated main diastereoisomer (3 S ,4 R ,5 R )- 10 was separated. Ozonolysis of the acetylated amino alcohols (4 S ,5 R )- 7a , b and (3 S ,4 S ,5 R )- 10 affords the aldehydes 12a – c , which were directly oxidized with CrO 3 in dilute H 2 SO 4 to yield the β-acetamido-γ-acetoxycarboxylic acids (3 S ,4 R )- 13a , b and (2 R ,3 S ,4 R )- 13c . Compounds 13 cyclized spontaneously under acidic conditions to afford β-acetamido-γ-butyrolactones (4 S ,5 R )- 14a , b and (3 R ,4 S ,5 R )- 14c .