Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols

2019 ◽  
Vol 400 (3) ◽  
pp. 313-321 ◽  
Author(s):  
Yijie Dong ◽  
Peiyuan Yao ◽  
Yunfeng Cui ◽  
Qiaqing Wu ◽  
Dunming Zhu ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Directed Evolution ◽  
Kinetic Resolution ◽  
Building Blocks ◽  
Binding Pocket ◽  
Model Reaction ◽  
Transition Metal Catalysts ◽  
Saturation Mutagenesis ◽  
Enantiomerically Pure ◽  
Amino Acid Alphabet

Abstract Chiral arylpropanols are valuable components in important pharmaceuticals and fragrances, which is the motivation for previous attempts to prepare these building blocks enantioselectively in asymmetric processes using either enzymes or transition metal catalysts. Thus far, enzymes used in kinetic resolution proved to be best, but several problems prevented ecologically and economically viable processes from being developed. In the present study, directed evolution was applied to the thermostable alcohol dehydrogenase TbSADH in the successful quest to obtain mutants that are effective in the dynamic reductive kinetic resolution (DYRKR) of racemic arylpropanals. Using rac-2-phenyl-1-propanal in a model reaction, (S)- and (R)-selective mutants were evolved which catalyzed DYRKR of this racemic substrate with formation of the respective (S)- and (R)-alcohols in essentially enantiomerically pure form. This was achieved on the basis of an unconventional form of iterative saturation mutagenesis (ISM) at randomization sites lining the binding pocket using a reduced amino acid alphabet. The best mutants were also effective in the DYRKR of several other structurally related racemic aldehydes.

scholarly journals Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

2019 ◽  
Vol 20 (13) ◽  
pp. 3353 ◽  
Author(s):  
Gustavo de Almeida Santos ◽  
Gaurao V. Dhoke ◽  
Mehdi D. Davari ◽  
Anna Joëlle Ruff ◽  
Ulrich Schwaneberg
Keyword(s):  
Directed Evolution ◽  
Bacillus Megaterium ◽  
Coupling Efficiency ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Building Blocks ◽  
Saturation Mutagenesis ◽  
P450 Monooxygenase ◽  
Initial Oxidation ◽  
P450 Bm3 ◽  
Total Turnover

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.

scholarly journals Lipase Catalyzed Synthesis of Enantiopure Precursors and Derivatives for β-Blockers Practolol, Pindolol and Carteolol

Catalysts ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 503
Author(s):  
Morten Gundersen ◽  
Guro Austli ◽  
Sigrid Løvland ◽  
Mari Hansen ◽  
Mari Rødseth ◽  
...  
Keyword(s):  
Building Block ◽  
Kinetic Resolution ◽  
Enantiomeric Excess ◽  
Catalytic Amount ◽  
Building Blocks ◽  
Β Blocker ◽  
Β Blockers ◽  
Optical Rotations ◽  
Reported Data ◽  
By Products

Sustainable methods for producing enantiopure drugs have been developed. Chlorohydrins as building blocks for several β-blockers have been synthesized in high enantiomeric purity by chemo-enzymatic methods. The yield of the chlorohydrins increased by the use of catalytic amount of base. The reason for this was found to be the reduced formation of the dimeric by-products compared to the use of higher concentration of the base. An overall reduction of reagents and reaction time was also obtained compared to our previously reported data of similar compounds. The enantiomers of the chlorohydrin building blocks were obtained by kinetic resolution of the racemate in transesterification reactions catalyzed by Candida antarctica Lipase B (CALB). Optical rotations confirmed the absolute configuration of the enantiopure drugs. The β-blocker (S)-practolol ((S)-N-(4-(2-hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide) was synthesized with 96% enantiomeric excess (ee) from the chlorohydrin (R)-N-(4-(3-chloro-2 hydroxypropoxy)phenyl)acetamide, which was produced in 97% ee and with 27% yield. Racemic building block 1-((1H-indol-4-yl)oxy)-3-chloropropan-2-ol for the β-blocker pindolol was produced in 53% yield and (R)-1-((1H-indol-4-yl)oxy)-3-chloropropan-2-ol was produced in 92% ee. The chlorohydrin 7-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one, a building block for a derivative of carteolol was produced in 77% yield. (R)-7-(3-Chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one was obtained in 96% ee. The S-enantiomer of this carteolol derivative was produced in 97% ee in 87% yield. Racemic building block 5-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one, building block for the drug carteolol, was also produced in 53% yield, with 96% ee of the R-chlorohydrin (R)-5-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one. (S)-Carteolol was produced in 96% ee with low yield, which easily can be improved.

Stereochemistry of epoxide carbonylation using bimetallic Lewis acid/metal carbonyl complexes

2004 ◽  
Vol 76 (3) ◽  
pp. 557-564 ◽  
Author(s):  
Y. D. Y. L. Getzler ◽  
Viswanath Mahadevan ◽  
E. B. Lobkovsky ◽  
G. W. Coates
Keyword(s):  
Lewis Acid ◽  
Kinetic Resolution ◽  
Metal Carbonyl ◽  
Carbonyl Complexes ◽  
Enantiomerically Pure ◽  
Metal Carbonyl Complexes ◽  
Pure Complex

The stereochemistry of epoxide carbonylation using bimetallic [Lewis acid]+[Co(CO)4]- complexes is reported. The achiral complex [(salph)Al(THF)2][Co(CO)4] stereospecifically carbonylates cis- and trans-2-butene oxide to the trans- and cis-β-lactones, respectively. Preliminary experiments regarding the carbonylative kinetic resolution of racemic trans-2-butene oxide using the enantiomerically pure complex [(R,R-salcy)Al(THF)2][Co(CO)4] are also reported.

Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis

2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Enoate Reductase ◽  
Iterative Saturation Mutagenesis

Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution

2015 ◽  
Vol 127 (42) ◽  
pp. 12587-12592 ◽  
Author(s):  
Zhoutong Sun ◽  
Richard Lonsdale ◽  
Xu-Dong Kong ◽  
Jian-He Xu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Binding Pocket ◽  
Enzyme Binding

scholarly journals Refactoring the Genetic Code for Increased Evolvability

2017 ◽  
Author(s):  
Gur Pines ◽  
James D. Winkler ◽  
Assaf Pines ◽  
Ryan T. Gill
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Single Gene ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Mutagenic Potential ◽  
Single Nucleotide ◽  
Common Error ◽  
Mutational Landscape ◽  
Genetic Codes

AbstractThe standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.Significance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

Enzymes in organic synthesis. 32. Stereospecific horse liver alcohol dehydrogenase-catalyzed oxidations of exo- and endo-oxabicyclic meso diols

1984 ◽  
Vol 62 (11) ◽  
pp. 2578-2582 ◽  
Author(s):  
J. Bryan Jones ◽  
Christopher J. Francis
Keyword(s):  
Alcohol Dehydrogenase ◽  
Organic Synthesis ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Preparative Scale ◽  
Enantiomerically Pure ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
One Step ◽  
Catalyzed Oxidation ◽  
Enzymes In Organic Synthesis

Preparative-scale horse liver alcohol dehydrogenase-catalyzed oxidation of mesoexo- and endo-7-oxabicyclo[2.2.1]heptane diols provides a direct one-step route to enantiomerically pure chiral γ-lactones of the oxabicyclic series.

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