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Formation of Halohydrin from Alkenes02:41

Formation of Halohydrin from Alkenes

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An alkene, such as propene, reacts with bromine in the presence of water to yield a halohydrin. Halohydrins contain a halogen and a hydroxyl group attached to adjacent carbons. When the halogen is bromine, it is called a bromohydrin, while a chlorohydrin has chlorine as the halogen.
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Alcohols from Carbonyl Compounds: Reduction02:23

Alcohols from Carbonyl Compounds: Reduction

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Reduction is a simple strategy to convert a carbonyl group to a hydroxyl group. The three major pathways to reduce carbonyls to alcohols are catalytic hydrogenation, hydride reduction, and borane reduction.
Catalytic hydrogenation is similar to the reduction of an alkene or alkyne by adding H2 across the pi bond in the presence of transition metal catalysts like Raney Ni, Pd–C, Pt, or Ru. Aldehydes and ketones can be reduced by this method, often under mild to moderate heat (25–100°C) and...
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[4+2] Cycloaddition of Conjugated Dienes: Diels–Alder Reaction01:16

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The Diels–Alder reaction is an example of a thermal pericyclic reaction between a conjugated diene and an alkene or alkyne, commonly referred to as a dienophile. The reaction involves a concerted movement of six π electrons, four from the diene and two from the dienophile, forming an unsaturated six-membered ring. As a result, these reactions are classified as [4+2] cycloadditions.
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Aldehydes and Ketones with Water: Hydrate Formation01:20

Aldehydes and Ketones with Water: Hydrate Formation

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An oxygen-based nucleophile, like water, can undergo addition reactions with aldehydes and ketones. The reaction leads to the formation of hydrates, also referred to as 1,1-diols or geminal diols.
The formation of hydrates is a reversible reaction. Hydrate formation is influenced by steric and electronic factors accompanying the alkyl substituents on the carbonyl group: The rate of hydrate formation increases with a decrease in the number of alkyl groups attached to the carbonyl carbon. Hence,...
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Catalytic hydrogenation of alkenes is a transition-metal catalyzed reduction of the double bond using molecular hydrogen to give alkanes. The mode of hydrogen addition follows syn stereochemistry.
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Halogenation of Alkenes02:46

Halogenation of Alkenes

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Halogenation is the addition of chlorine or bromine across the double bond in an alkene to yield a vicinal dihalide. The reaction occurs in the presence of inert and non-nucleophilic solvents, such as methylene chloride, chloroform, or carbon tetrachloride.
Consider the bromination of cyclopentene. Molecular bromine is polarized in the proximity of the π electrons of cyclopentene. An electrophilic bromine atom adds across the double bond, forming a cyclic bromonium ion intermediate.
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A robust cosolvent-compatible halohydrin dehalogenase by computational library design.

Hesam Arabnejad1, Marco Dal Lago2, Peter A Jekel1

  • 1Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Protein Engineering, Design & Selection : PEDS
|December 22, 2016
PubMed
Summary
This summary is machine-generated.

Researchers enhanced halohydrin dehalogenase (HheC) enzyme stability for organic cosolvent reactions using computational design. A 12-fold mutant (HheC-H12) showed increased melting temperature and cosolvent resistance, preserving catalytic activity.

Keywords:
biocatalysiscosolvent stabilityhaloalcohol dehalogenaseprotein engineeringthermostability

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Area of Science:

  • Enzyme engineering
  • Computational biochemistry
  • Biocatalysis

Background:

  • Halohydrin dehalogenase (HheC) is a valuable biocatalyst.
  • Organic cosolvents often limit enzyme applicability.
  • Enzyme stabilization is crucial for industrial applications.

Purpose of the Study:

  • To computationally design and experimentally validate stabilizing mutations for HheC.
  • To improve HheC's performance in organic cosolvents.
  • To create a more robust enzyme for industrial biocatalysis.

Main Methods:

  • Computational library design (Framework for Rapid Enzyme Stabilization by Computational libraries).
  • In silico evaluation: energy calculations, disulfide bond predictions, molecular dynamics simulations.
  • Experimental validation: site-directed mutagenesis, melting temperature assays, cosolvent resistance tests, enzyme kinetics, crystal structure analysis.

Main Results:

  • Identified 218 potential stabilizing point mutations and 35 disulfide bonds computationally.
  • Experimentally confirmed 29 stabilizing point mutations, primarily in two regions.
  • Developed a 12-fold mutant (HheC-H12) with a 28°C higher melting temperature and enhanced cosolvent resistance.
  • HheC-H12 exhibited a higher optimal catalytic temperature while retaining low-temperature activity.
  • Crystal structures revealed mutations improved surface charge distribution and inter-subunit interactions.

Conclusions:

  • Computational enzyme design is effective for improving HheC stability.
  • The HheC-H12 mutant demonstrates significantly enhanced stability and broader applicability in organic cosolvents.
  • This engineered enzyme serves as a platform for further optimization of enantioselectivity and activity.