Hedda K. Weber

Enzymatic hydroxylation reactions

During the past 18 months, considerable progress has been made in the understanding of the key enzyme-substrate interactions that control the regioselectivity and stereoselectivity of the hydroxylation reaction performed by cytochrome-P450-dependent enzymes of mammalian origin. The manipulation of microbial hydroxylating enzymes, in both whole-cell and cell-free environments, has also been examined in the context of controlling the regioselectivity and stereoselectivity of the hydroxylation reaction. Several new applications for hydroxylating enzymes have been reported, and the construction of chimeric hydroxylating enzymes has been used both for mechanistic studies and for the production of enzymes with high hydroxylating activity for a defined substrate.

`Watching' lipase-catalyzed acylations using 1H NMR: competing hydrolysis of vinyl acetate in dry organic solvents

Abstract Lipase-catalyzed acetylations of 1-phenylethanol with vinyl acetate were monitored in situ by 1 H NMR spectroscopy. Surprisingly, even under dry conditions (no added water) the major reaction was hydrolysis of the vinyl acetate, not acetylation of the substrate. Because this competing hydrolysis consumes water and releases acetic acid, the reaction conditions in lipase-catalyzed acylations are not constant, but vary with the reaction time. Addition of a chiral shift reagent reveals the enantiomeric purity of the starting alcohol and allows calculation of the enantiomeric ratio, E , for the reaction.

Molecular reasons for lipase-sensitivity against acetaldehyde

Abstract The molecular reasons for the sensitivity of microbial lipases towards acetaldehyde, emerging as unavoidable by-product from acyl transfer reactions employing vinyl esters as acyl donors, were shown to be associated with specific properties of lysine residues. Since the mechanism of deactivation involves the formation of Schiff bases at the lysine e-amino groups, the relative reactivity (i.e. nucleophilicity) of each residue was estimated by using an electronic (p K a value) and a steric parameter (accessible surface area of the side chain). Sensitive lipases, as from Candida rugosa and Geotrichum candidum , possess several lysine residues that have high p K a values (> 12) and are highly exposed to the solvent (surface areas of 210–220 A 2 ). In contrast, the lysine groups of stable lipases like from Rhizomucor miehei, Candida antarctica B and Pseudomonas glumae have moderate p K a values (up to 11.6) and are rather buried (surface areas of 130–150 A 2 ). A close investigation of Candida rugosa lipase revealed that the most exposed lysine residues are located in the lid region (Lys75 and Lys85). The data suggest that Lys75, which is involved in fixing the lid in its open conformation, is presumably the prime target for deactivation by acetaldehyde.

Improving hydrolases for organic synthesis

Improving hydrolases by site-directed mutagenesis continues to be important, but an alternative method - directed evolution - also gains favor. Directed evolution combines random mutagenesis with screening or selection for the desired property. Directed evolution is especially useful for cases like solvent tolerance or thermostability where current theories are inadequate to predict which structural changes will give improvement. Researchers have also recently made significant progress on several practical problems: how to maintain the high activity of proteases and lipases in nonpolar organic solvents, how to resolve amines, and how to efficiently recycle the unwanted enantiomer in kinetic resolutions. Besides the lipases and proteases, researchers are also developing new hydrolases, notably dehalogenases and epoxide hydrolases.

Microbial hydroxylation of 2-cycloalkylbenzoxazoles. Part III. Determination of product enantiomeric excess and cleavage of benzoxazoles

Abstract An HPLC-system to measure the e.e. of trans -3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol 6 , cis -3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol 38 , 3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one 8 and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol 7 simultaneously in the fermentation mixture of the substrate 2-cyclopentyl-1,3-benzoxazole 5 with Cunninghamella blakesleeana DSM 1906 is presented. The scope and limitations of the benzoxazole cleavage reactions is discussed.

Microbial hydroxylation of 2-cycloalkylbenoxazoles. Part II. Determination of product structures and enhancement of enantiomeric excess

Abstract The determinations of product structures obtained in the microbial hydroxylations of various 2-cycloalkyl-1,3-benzoxazoles using Cunninghamella blakesleeana DSM 1906 and Bacillus megaterium DSM 32 are described. The initially low e.e. of 3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol 6 , 2-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol 14 and 4-(2-benz-1,3-oxazol-2-yl)cycloheptan-1-ol 21 can be enhanced to 98 % using lipase catalyzed resolution.

Online NMR for monitoring biocatalysed reactions—the use of lipases in organic solvents

The lipase-catalysed acetylation of 2-hydroxymethylpiperidine was carried out in the NMR tube and monitored by 1 H NMR spectroscopy for defined periods of time. Two different acylating agents were used: ethyl acetate in C 6 D 6 and vinyl acetate in C 6 D 6 . The lipase employed was porcine pancreatic lipase (PPL). Ethyl acetate in C 6 D 6 was hydrolysed by the enzyme but no formation of acylated product could be detected in these dilute solutions. In case of vinyl acetate as the acyl donor, the reaction did not give the desired N-acylated compound but the corresponding oxazolidine-derivative, which was formed by the reaction of the aminoalcohol and the acetaldehyde. This compound was assigned unambiguously without isolation from the reaction medium by total correlation spectroscopy (TOCSY) and gHSQC experiments.

Rapid determination of γ-value and xanthate group distribution on viscose by liquid-state (1)H NMR spectroscopy.

A method for the determination of the γ-value and more importantly the distribution of xanthate groups on cellulose xanthate produced during the viscose process is presented. The method is based upon stabilization of xanthate groups attached to the cellulose chain by reaction with 4-methylbenzyl bromide and analysis of the resulting product by liquid-state (1)H NMR. Careful analysis of the proton-spectrum using deconvolution gave a very fast method for the measurement of the γ-value which compared well with the data obtained by IR spectroscopy. In addition it could be shown that the distribution of the xanthate groups on the anhydroglucose monomeric unit (xanthation at position 2, 3 or 6) changes significantly during ripening. The method gave useful results even for viscose with low γ-values of about 25.