Torbjörn Norin

Molecular Modeling of the Enantioselectivity in Lipase-Catalyzed Transesterification Reactions

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.

Improved Enantioselectivity of a Lipase by Rational Protein Engineering

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1‐Halo‐2‐octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast‐reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivtiy as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

CHIRAL RECOGNITION OF ALCOHOL ENANTIOMERS IN ACYL TRANSFER REACTIONS CATALYSED BY CANDIDA ANTARCTICA LIPASE B

A description of the substrate-enzyme interactions involved in the discrimination of see-alcohol enantiomers in acyl transfer reactions catalysed by the highly enantioselective Candida antarctica lipase B is presented. Experimentally found activities and enantioselectivities from kinetic resolutions of a series of secondary alcohol substrates were used together with molecular modelling for the elucidation of the stereoselective substrate-enzyme interactions. Matching experimental and calculated results allowed conclusions regarding the orientation of the tetra-hedral intermediates in the active site. The finding, valid for substrates of a specific activity above 1 mol min-1 mg-1 protein, describes the origin of enantioselectivity as a combination of a binding site of limited size, the ”stereospecificity pocket“, and principally different productive orientations of the two enantiomers.

Molecular recognition of sec-alcohol enantiomers by Candida antarctica lipase B

Abstract A model to explain the enantioselectivity of Candida antarctica lipase B towards sec -alcohols based on structure activity and molecular modelling is presented. The origin of the enantioselectivity was found to be due to different modes of binding for the enantiomers. The fast enantiomer places its medium substituent in a site of limited size, the stereoselectivity pocket, whereas the slow enantiomer has to position the large substituent in that same pocket. Our model is in agreement with the 24 different substrates tested. Only substituents smaller than n -propyl can be accommodated by the stereoselectivity pocket. Moreover, important unfavourable electrostatic interactions are involved between this region and halogenated substituents. The former requirement entails a high enantiomeric ratio ( E ) for sec -alcohols with a medium group smaller than n -propyl and a large group larger than n -propyl. The latter requirement allows high E only for short chain vic -halogenated alcohols.

Differences in attraction to semiochemicals present in sympatric pine shoot beetles,Tomicus minor andT. piniperda.

The chemical ecology of host- and mate-finding in the pine shoot beetles,Tomicus minor andT. piniperda, was studied in southern Sweden. Beetles were collected in the field from defined attack phases on Scots pine. Using gas chromatography-mass spectroscopy, a number of oxygen-containing monoterpenes, e.g., 3-carene-10-ol, myrtenol,trans-verbenol, and verbenone, were identified from hindgut extracts of both sexes of both species. Compared toT. minor,T. piniperda contained additional compounds and in larger amounts. The amounts were highest in both species at the time when the beetles had bored into contact with the resin-producing xylem-phloem tissue. The synthesis of (1S,6R)-3-carene-10-ol by photooxidatipn of (+)-(1S,6R)-3-carene is described. In comparative electroantennogram (EAG) measurements on males and females of both species, the most active of the tested compounds wastrans-verbenol. Laboratory bioassays of walking beetles showed thatT. piniperda was attracted to uninfestèd pine logs.T. minor was more strongly attracted to pine logs infested with females than to uninfested pine logs, indicating a female-produced aggregation pheromone. Field tests confirmed thatT. piniperda was strongly attracted to pine logs. The attraction ofT. minor to logs was significant only when logs were combined with racemictrans-verbenol and (1S,6R)-3-carene-10-ol.T. minor was also attracted to a combination of these monoterpene alcohols alone. We suggest that host and mate location inT. piniperda is achieved by means of a kairomone composed of host monoterpenes, whileT. minor utilizes a primitive pheromone synergized by host odors. Evolution of host colonization strategies of the two beetles are discussed.

Chiral synthesis of (2s,3s,7s)-3,7-dimethylpentadecan-2-yl acetate and propionate, potential sex pheromone components of the pine saw-fly neodiprion sertifer (geoff.)

Abstract A synthesis of (2 S ,3 S ,7 S )-3,7-dimethylpentadecan-2-yl acetate ( 2 ) and propionate ( 3 ) is described. (2 S )-2-Methyldecan-1-yl lithium ( 5 ) was reacted with (3 S ,4 S )-3,4-dimethyl-γ-butyrolactone ( 6 ) to yield the ketoalcohol 19 which upon Huang-Minlon reduction furnished (2 S ,3 S ,7 S )-3,7-dimethylpentadecan-2-ol ( 1 ). Acylations gave the esters 2 and 3 . The (2 S )-2-methyldecan-1-yl lithium was obtained via asymmetric synthesis. The chiral lactone 6 was obtained from (2 S ,3 S )- trans -2,3-epoxybutane and dimethyl malonate.

The Candida antarctica lipase B catalysed kinetic resolution of seudenol in non-aqueous media of controlled water activity

Abstract For further investigation of the kinetic resolutions in transesterification reactions with the highly enantioselective Candida antarctica lipase B, an easy to study model reaction with one typical substrate, the Douglas Fir Beetle pheromone 3-methyl-2-cyclohexen-1-ol (Seudenol), was developed. The influence of the nature of the solvent and thermodynamic water activity was studied. Initial rates showed constant or progressively increasing values with increasing water activity. Enantioselectivity depended on the choice of solvent and descended in most cases with increasing water activity. No general correlations of enantioselectivity or activity with physicochemical constants of the solvent were found. However, at a water activity of 0.11 a tendency toward optimum hydrophobicity (i.elog P ≈ for enantioselectivity was observed. Enantiomeric ratios were in the range 8–32.

Relative amounts and enantiomeric compositions of monoterpene hydrocarbons in xylem and needles of Picea abies

Abstract The relative amounts of 23 monoterpene hydrocarbons, including enantiomers, present in extracts of branch xylem and needles from 41 Picea abies plus-trees of widely different origins, were determined. A two-dimensional gas chromatographic system was used for the determination of the enantiomeric compositions of the seven major chiral monoterpenes. Data were evaluated by multivariate data analysis. Large variations were found in the enantiomeric compositions, as well as in the relative amounts of the monoterpenes, both within and among the trees. Trees from the same geographic area did not show a greater similarity in their composition of volatiles than trees from different geographic areas. In the xylem, (−)-β-pinene and (−)-β-phellandrene, and in the needle samples, (−)-α-pinene, (−)-limonene and (−)-camphene, dominated over their (+)-enantiomers. Among-tree variation in enantiomeric composition was higher in xylem samples than in needle samples, except for β-pinene and β-phellandrene.

S-ethyl thiooctanoate as acyl donor in lipase catalysed resolution of secondary alcohols

Abstract The use of S-ethyl thiooctanoate as acyl donor in resolution of secondary alcohols by lipase catalysed transesterification, resulted in an efficient displacement of the equilibrium towards esterification of the alcohol. A lipase (component B) from Candida antarctica was used as the catalyst. On a semi-preparative scale, 0.5 g of 2-octanol was resolved in less than one hour, affording an enantiomeric excess of >98% of the remaining alcohol and >97% of the produced ester. Three other alcohols, 1-phenyl ethanol, 1-cyclohexyl and trans -2-methyl cyclohexanol were also resolved with good optical yields. After 50% conversion on a preparative scale, 15 g of alcohol, the ( S )-enantiomer of 2-octanol could be obtained with an enantiomeric excess of 96%. The ( R )-enantiomer was isolated with an enantiomeric excess of 97% after hydrolysis of the produced ester.

Kinetic resolutions of amine and thiol analogues of secondary alcohols catalyzed by the Candida antarctica lipase B

Abstract Chiral amine and thiol analogues of secondary alcohols were tested as substrates for the Candida antarctica lipase B with emphasis on lipase enantioselectivity and substrate reactivity. Although the apparent enantiomeric ratios were lower for the amines than for the corresponding alcohols, the amines 2-octylamine and 1-phenyl-ethylamine were produced with high optical purity (ee-93–99%) in a lipase-catalyzed acyl transfer reaction with ethyl octanoate as the acyl donor. The initial rates were lower and the reaction times longer for the amines than for the corresponding alcohols. The thiols 2-octanethiol and 1-phenylethanethiol did not yield any thiol ester product in the analogous lipase-catalyzed acyl transfer reaction. By preparing the thiooctanoate of 1-phenyl-ethanethiol and employing it as acyl donor in a lipase-catalyzed reaction with 1-phenylethanol as acyl acceptor, however, 1-phenylethanethiol could be resolved with high enantiomeric excess of the ( R )-enantiomer (ee-95%) and moderate enantiomeric excess of the ( S )-enantiomer (ee-75%).