John Boutelje

Enzyme catalysed hydrolysis of dialkylated propanedioic acid diesters, chain length dependent reversal of enantioselectivity

Abstract Enzyme catalysed hydrolyses of dialkylated propanedioic acid diesters have been studied. A novel change of enantioselectivity from pro- S to pro- R in the hydrolysis of ester groups was observed, depending on the chain length of the alkyl substituents of the substrate. Optically pure ( S )-(-)-α-methylphenylalanine was prepared by α-chymotrypsin catalysed hydrolysis of benzylmethylpropanedioic acid dimethyl ester.

Lipase immobilized by adsorption

SummaryCandida cylindraceae lipase was immobilized by adsorption to various hydrophilic and hydrophobic supports and studied with respect to the esterification rates of a primary and a secondary alcohol, respectively, in organic media. The reaction rates were compared with the rate of esterification with “free lipase”. When the secondary alcohol, (R, S)-1-phenylethanol, was used the highest reaction rates were measured for lipase adsorbed to the hydrophobic supports. When the primary alcohol, heptanol, was used “free lipase” exhibited the highest reaction rate. A kinetic explantation of these results is proposed.

The effect of dimethyl sulfoxide on the enantioselectivity in the pig liver esterase catalyzed hydrolysis of dialkylated propanedioic acid dimethyl esters

Abstract The effect of dimethyl sulfoxide (DMSO) on the enantioselectivity in the pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of dialkylated propanedioic acid diesters was studied. Increasing concentrations of DMSO increased the ratio of the (R)-enantiomers of the produced monoesters. This effect was not due to the change in pHapp caused by the solvent. The reaction rate was lowered to one-tenth of the rate without DMSO when the DMSO concentration was increased to 50%.

Control of the stereoselectivity of pig liver esterase by different reaction conditions in the hydrolysis of cis-N-benzyl-2,5-bismethoxycarbonylpyrrolidine and structurally related diesters

Abstract The enantiotopic selectivity of pig liver esterase in the enzyme-catalyzed hydrolysis of cis-N-benzyl-2,5-bismethoxycarbonylpyrrolidine (1) was investigated under different reaction conditions. Optically pure monoester 2 (≥98% enantiomeric excess) was obtained in Tris-buffered incubations at pH 7.5 when the reaction medium contained 25% dimethyl sulfoxide or 10% methanol. Reactions run in Tris buffer were faster and gave higher stereoselectivity than reactions performed in a pH-stat. Similar observations were made with structurally related diesters (3,5,7,9,11–13). Tris acts as an alternative nucleophile in the pig liver esterase-catalyzed hydrolysis of diester 1 and the Tris-amide formed lowers the chemical yield of monoester 2.

Highly enantioselective route to (R)-proline derivatives via enzyme catalysed hydrolysis of cis-N-benzyl-2,5-bismethoxycarbonylpyrrolidine in an aqueous dimethyl sulphoxide medium

Pig liver esterase catalysed hydrolysis of diester (1) with dimethyl sulphoxide as cosolvent in buffered solutions gave optically pure monoester (2) which was further transformed to the (R)-proline related ester (4) by radical decarboxylation.

Computer Graphics As A Tool For The Prediction Of The Stereoselectivity Of Enzyme Catalyzed Reactions. α-Cxhymotrypsin Catalyzed Hydrolysis Of Substituted Propanedioic Acid Diesters.

Computer graphics can be used for the prediction of substrate specificity and stereoselectivity in the α-chymotrypsin catalyzed hydrolysis of Disubstituted propanedioic acid diesters. This enzyme catalyzed reaction is useful for the preparation of chiral starting materials, e.g. for the synthesis of optically pure α-methylaminoacids.

Enhanced stereoselectivity in pig liver esterase catalysed diester hydrolysis. The role of a competitive nucleophile

The enantioselectivity of pig liver esterase catalysed hydrolysis of cis-N-benzyl-2,5-bis(methoxy-carbonyl)pyrrolidine (1) has previously been shown to be very dependent on the reaction conditions. Hydrolysis performed in media buffered with tris(hydroxymethyl)aminomethane (Tris) afforded a monoester with much higher optical purity than hydrolysis in media without Tris. Detailed product studies in a Tris-buffered medium have been performed using NMR-techniques and a 13C-labelled ester. The NMR-studies revealed the presence of (2S,5R)-N-benzyl-2-methoxycarbonyl-5-[[[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]amino]carbonyl]pyrrolidine (4) as an intermediate, which together with the isolated product (2S,5R)-N-benzyl-2-carboxy-5-[[[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino]carbonyl]pyrrolidine (3) suggested Tris as a competitive nucleophile to water. The increased enantioselectivity seen in the produced (2R,5S)-N-benzyl-2-methoxy-carbonyl-5-carboxypyrrolidine (2) was explained by the preference of Tris to react faster with one of the diastereomeric acyl enzymes over the other.

Mannitol dehydrogenase from industrial waste mycelium of Penicillium chrysogenum. Purification, properties and possible use in cofactor regenerating systems in vitro

SummaryMannitol dehydrogenase, NADP specific (EC 1.1.1.138), was purified 823 fold from industrial waste mycelium of Penicillium chrysogenum. The enzyme had a molecular weight of 160,000 and was composed of four subunits of equal size. The pH optimum for activity was 7.3 and for stability 6.0.The substrate specificity was limited to d-mannitol for the purified enzyme. Zinc and cadmium ions as well as sulphhydryl compounds were inhibitors of the enzyme. The enzyme had a Km for mannitol of 760 mM and a Km for NADP of 14μM.Mannitol dehydrogenase in crude extracts made from mycelium harvested before 92 h of fermentation had double the half life compared to extracts made from mycelium harvested after this time.The use of mannitol dehydrogenase in an in vitro NADPH regenerating system is discussed.

Enzyme catalyzed hydrolysis of the diesters of cis- and trans-cyclohexanedicarboxylic acids

SummaryPig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of the dimetrhy ester of meso-cis-1,2-cyclohexanedicarboxylic acid yielded the optically pure (1S,2R)-monoester. The corresponding diethyl ester yielded racemic monoester.The diethyl ester of racemic trans-1,2-cyclohexanedicarboxylic acid was kinetically resolved by partial hydrolysis with subtilisin (EC 3.4.21.14) or pig liver esterase. The (1R,2R)-monoester had an enantiomeric excess of 45% and was obtained in an enantiomerically pure form through recrystallisation. The remaining (1S,2S)-diester exhibited an enantiomeric excess of 83%. The nature of the ester function (methyl, ethyl, and propyl esters) had a great influence on the enantiomeric excess obtained and on the kinetic parameters.