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Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide02:44

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Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double bond with two different stereochemical approaches, namely anti and syn. Dihydroxylation using osmium tetroxide progresses with syn stereochemistry.
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Oxidative Cleavage of Alkenes: Ozonolysis01:46

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In ozonolysis, ozone is used to cleave a carbon–carbon double bond to form aldehydes and ketones, or carboxylic acids, depending on the work-up.
Ozone is a symmetrical bent molecule stabilized by a resonance structure.
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Oxidation of Alkenes: Anti Dihydroxylation with Peroxy Acids02:04

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Diols are compounds with two hydroxyl groups. In addition to syn dihydroxylation, diols can also be synthesized through the process of anti dihydroxylation. The process involves treating an alkene with a peroxycarboxylic acid to form an epoxide. Epoxides are highly strained three-membered rings with oxygen and two carbons occupying the corners of an equilateral triangle. This step is followed by ring-opening of the epoxide in the presence of an aqueous acid to give a trans diol.
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Preparation of Epoxides03:00

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Overview
Epoxides result from alkene oxidation, which can be achieved by a) air, b) peroxy acids, c) hypochlorous acids, and d) halohydrin cyclization.
Epoxidation with Peroxy Acids
Epoxidation of alkenes via oxidation with peroxy acids involves the conversion of a carbon–carbon double bond to an epoxide using the oxidizing agent meta-chloroperoxybenzoic acid, commonly known as MCPBA. Since the O–O bond of peroxy acids is very weak, the addition of electrophilic oxygen of...
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Oxymercuration-Reduction of Alkenes02:36

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Oxymercuration–reduction of alkenes is one of the major reactions converting alkenes to alcohols. It involves the hydration of alkenes with mercuric acetate in a mixture of tetrahydrofuran and water, forming an organomercury adduct. This is followed by a demercuration step in which the adduct is reduced to an alcohol using sodium borohydride.
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Hydroboration-Oxidation of Alkenes03:08

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In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.
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Computationally Designed Peroxygenases That Exhibit Diverse and Selective Terpene Oxyfunctionalization.

Judith Münch1, Jordi Soler2, Ofir Gildor-Cristal3

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Summary

Computational enzyme design rapidly created 50 variants of unspecific peroxygenase (MthUPO) with enhanced terpene oxyfunctionalization. These designed enzymes show significantly improved activity, selectivity, and novel product synthesis, outperforming the wild type in various applications.

Keywords:
FuncLibSaccharomyces cerevisiaecomputational chemistryenzymesoxyfunctionalizationterpenesunspecific peroxygenaseyeast

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Computational Chemistry and Molecular Modeling
  • Organic Synthesis and Chemical Transformations

Background:

  • Selective oxyfunctionalization of terpenes is crucial for industrial synthesis but remains challenging.
  • Unspecific peroxygenases (UPOs) show potential but often lack desired selectivity and activity.
  • Computational enzyme design offers a pathway to engineer biocatalysts with tailored functions.

Purpose of the Study:

  • To computationally design variants of MthUPO with improved terpene oxyfunctionalization capabilities.
  • To enhance enzyme activity, regioselectivity, chemoselectivity, and stereoselectivity for specific terpene substrates.
  • To demonstrate the efficacy of in silico-guided enzyme engineering for biocatalyst development.

Main Methods:

  • Utilized an AlphaFold2 model of MthUPO as a basis for computational design.
  • Employed the FuncLib algorithm to generate a library of 50 enzyme variants.
  • Performed extensive substrate testing to evaluate activity and selectivity profiles of designed variants.

Main Results:

  • All 50 designed variants retained measurable activity, with significant improvements observed across terpene substrates (2.2-fold to 7.1-fold increase).
  • Demonstrated dramatic shifts in selectivity, including increased regioselectivity for 3-hydroxy-β-damascone (3% to 46%) and enhanced chemoselectivity for citral A (>99%) and citral B (89%).
  • Achieved novel product synthesis (e.g., isopiperitenol, epoxides) and inverted enantioselectivity for β-ionone hydroxylation (R:S ratio 1:99).

Conclusions:

  • FuncLib-enabled active-site remodeling successfully generated a small, diverse enzyme panel outperforming wild-type MthUPO.
  • Computational design, particularly leveraging epistatic effects in variants like design 4 and 11, enables rapid development of highly selective biocatalysts.
  • In silico approaches are powerful tools for creating tailored enzymes for challenging synthetic applications.