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1.
J Org Chem ; 84(11): 6982-6991, 2019 06 07.
Article in English | MEDLINE | ID: mdl-31066559

ABSTRACT

Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes, or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone, or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl- or phenylglyoxal promoted by SmI2, resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl-substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn-products (de = 60-99%), with moderate enantiomeric excesses (ee = 43-56%) but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI2-promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenyl-substituted 2,3-dihydroxybutanones at yields between 40-60%. Finally, the biocatalytic approach resulted in enantiopure syn-(3 R,4 S) 1,3,4-trihydroxypentan-2-ones.


Subject(s)
Butanones/chemical synthesis , Butanones/metabolism , Cinchona/chemistry , Fructose-Bisphosphate Aldolase/metabolism , Pentanones/chemical synthesis , Pentanones/metabolism , Butanones/chemistry , Catalysis , Molecular Structure , Pentanones/chemistry , Stereoisomerism
2.
Biochemistry ; 57(40): 5877-5885, 2018 10 09.
Article in English | MEDLINE | ID: mdl-30204427

ABSTRACT

d-Fructose 6-phosphate aldolase (FSA) catalyzes the asymmetric cross-aldol addition of phenylacetaldehyde and hydroxyacetone. We conducted structure-guided saturation mutagenesis of noncatalytic active-site residues to produce new FSA variants, with the goal of widening the substrate scope of the wild-type enzyme toward a range of para- and meta-substituted arylated aldehydes. After a single generation of mutagenesis and selection, enzymes with diverse substrate selectivity scopes were identified. The kinetic parameters and stereoselectivities for a subset of enzyme/substrate combinations were determined for the reactions in both the aldol addition and cleavage reaction directions. The achieved collection of new aldolase enzymes provides new tools for controlled asymmetric synthesis of substituted aldols.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Fructose-Bisphosphate Aldolase/chemistry , Fructosephosphates/chemistry , Escherichia coli Proteins/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Fructosephosphates/metabolism , Substrate Specificity
4.
FEBS J ; 284(22): 3895-3914, 2017 11.
Article in English | MEDLINE | ID: mdl-28963762

ABSTRACT

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (a) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (b) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate-binding modes. DATABASE: Structural data are available in the Protein Data Bank with accession codes 5o8q for A2, 5o8h for A2C2, 5o9f for A2C3, and 5o9d for A2C2B1.


Subject(s)
Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Coenzymes/metabolism , Phenylethyl Alcohol/metabolism , Rhodococcus/enzymology , Alcohol Dehydrogenase/genetics , Binding Sites , Catalysis , Catalytic Domain , Kinetics , Mutagenesis, Site-Directed , Mutation/genetics , Oxidation-Reduction , Protein Conformation , Stereoisomerism , Substrate Specificity
5.
Chembiochem ; 16(18): 2595-8, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26449620

ABSTRACT

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format.


Subject(s)
Aldehyde-Lyases/metabolism , Acetaldehyde/analogs & derivatives , Acetaldehyde/chemistry , Acetaldehyde/metabolism , Aldehydes/chemistry , Biocatalysis , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , High-Throughput Screening Assays , Kinetics , Substrate Specificity
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