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| 2. |
- Al-Smadi, Derar, 1983-, et al.
(författare)
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Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di-, and Tri-Hydroxypentanones
- 2019
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Ingår i: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 84:11, s. 6982-6991
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Tidskriftsartikel (refereegranskat)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 phenyl glyoxal 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-phenylsubstituted 2,3-dihydroxybutanones, at yields between 40−60%. Finally, the biocatalytic approach resulted in enantiopure syn (3R,4S) 1,3,4-trihydroxypentan-2-ones.
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| 3. |
- Al-Smadi, Derar, 1983-, et al.
(författare)
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Synthesis of substrates for aldolase-catalyzed reactions : A comparison of methods for the synthesis of substituted phenylacetaldehydes
- 2018
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Ingår i: Synlett. - : Georg Thieme Verlag KG. - 0936-5214 .- 1437-2096. ; 29:9, s. 1187-1190
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Tidskriftsartikel (refereegranskat)abstract
- Methods for the synthesis of phenylacetaldehydes (oxidation, one-carbon chain extension) were compared by using the synthesis of 4-methoxyphenylacetaldehyde as a model example. Oxidations of 4-methoxyphenylethanol with activated DMSO (Swern oxidation) or manganese dioxide gave unsatisfactory results; whereas oxidation with 2-iodoxybenzoic add (IBX) produced 4-methoxyphenylacetaldehyde in reasonable (75%) yield. However, Wittig-type one-carbon chain extension with methoxymethylene-triphenylphosphine followed by hydrolysis gave an excellent (81% overall) yield of 4-methoxyphenylacetaldehyde from 4-methoxybenzaldehyde (a cheap starting material). This approach was subsequently used to synthesise a set of 10 substituted phenylacetaldehydes in good to excellent yields.
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- Enugala, Thilak Reddy, 1984-
(författare)
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Engineered Alcohol Dehydrogenases for Stereoselective Chemical Transformations
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes are biomolecules built from amino acids and catalyze the chemical transformations in a cell. Enzymes are by nature stereoselective, biodegradable, environmentally friendly, and can perform catalysis in aqueous solutions and at ambient temperatures. Due to these advantages the use of enzymes as biocatalysts for chemical transformations has emerged as an attractive “greener” alternative to conventional chemical synthesis strategies. And, if naturally occurring enzymes cannot carry out the desired chemical transformations, the functional properties of enzymes can be modified by directed evolution or protein engineering techniques. Since enzymes are genetically encoded they can be optimized for desired traits such as substrate selectivity or improved catalytic efficiency. Considering these advantages and also keeping the synthetic and industrial application in mind, we have employed alcohol dehydrogenase-A (ADH-A) from Rhodococcus ruber DSM 44541 as a study object in engineering for new catalytic properties. ADH-A tolerates water miscible organic solvents, accepts a relatively wide range of aromatic sec-alcohols/ketones as substrates and is therefore a potentially useful biocatalyst for asymmetric synthesis of organic compounds. Presented research work in this thesis has been primarily focused on engineering of ADH-A and characterization of resulting enzyme variants. The engineering efforts have aimed for altered substrate scope, as well as stereo- and regioselectivities. Furthermore, possible substrate promiscuity in engineered enzyme variants has also been addressed. In short, i). Paper I: three sub sites, each consisting of two-three amino acid residues within the active-site cavity were exposed to saturation mutagenesis in step-wise manner, coupled to an in vitro selection for improved catalytic activity with the unfavored (R)-1-phenylethanol. The observed stereoselectivity could be explained partly by a shift in nonproductive substrate binding. ii). Paper II is aimed specifically towards the improving the catalytic activity with aryl-substituted vicinal diols, such as (R)-1-phenylethane-1,2-diol, and the possibility to link the ADH-A reaction with a preceding epoxide hydrolysis to produce the acyloin 2-hydroxyacetophenone from rac-styrene oxide. iii). Paper III is mainly focused towards studies of regioselectivity. Here, ADH-A and engineered variants were challenged with a substrate containing two sec-alcohol functions and the cognate di-ketone. The regioselectivity in wild type as well as in engineered variants could in part be explained by a combination of experimental and computer simulations. iv). Paper IV is focused on elucidating possible effects on substrate promiscuities in engineered variants as compared to the wild type parent enzyme, when challenged with a spectrum of potential previously untested substrates.
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| 6. |
- Enugala, Thilak Reddy, et al.
(författare)
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The Role of Substrate-Coenzyme Crosstalk in Determining Turnover Rates in Rhodococcus ruber Alcohol Dehydrogenase
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435 .- 2155-5435. ; 10:16, s. 9115-9128
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Tidskriftsartikel (refereegranskat)abstract
- Eight related alcohol dehydrogenases that had been originally isolated by laboratory evolution of ADH-A from Rhodococcus ruber DSM44541 for modified substrate scopes, were together with their parent wild-type, subjected to biochemical characterization of possible activities with a panel of chiral alcohols and pro-chiral ketones. Determinations of rates of catalyzed alcohol oxidations and ketone reductions, and of cofactor release, pointed out to the role of a W295A substitution as being decisive in steering enantioselectivity in the oxidation of arylated 1-methyl substituted alcohols. Molecular dynamics simulations of enzyme-substrate interactions in the Michaelis complexes of wild-type and a Y294F/W295A double mutant could rationalize the experimentally observed shift in enantioselectivity and differences in catalytic activity with 4-phenyl-2-butanol. Finally, we present herein evidence for apparent inter-dependency between substrate/product and the cofactor in the ternary complex, that directly affects the NADH dissociation rates, and thus that this substrate-coenzyme crosstalk plays a direct role in determining the turnover rates.
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| 8. |
- Hamnevik, Emil, et al.
(författare)
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Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
- 2017
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 284:22, s. 3895-3914
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Tidskriftsartikel (refereegranskat)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: (1) 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. (2) 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.
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| 9. |
- Ma, Huan, et al.
(författare)
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A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates
- 2015
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Ingår i: ChemBioChem (Print). - : Wiley. - 1439-4227 .- 1439-7633. ; 16:18, s. 2595-2598
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Tidskriftsartikel (refereegranskat)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 presented as 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 these type of bulky acceptor substrates 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 in vitro evolved aldehyde reductase that reduces the aldehyde product formed after FSA-afforded cleavage of the aldol. The assay is suitable both for steady state enzyme kinetics and real-time activity screening in a 96-well format.
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