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Preparation of Nitriles01:12

Preparation of Nitriles

2.8K
One of the common methods to prepare nitriles is the dehydration of amides. This method requires strong dehydrating agents like phosphorous pentoxide or boiling acetic anhydride for converting amides to nitriles. Another reagent namely, thionyl chloride also accomplishes the dehydration of amides, where amide acts as a nucleophile. The first step of the mechanism involves the nucleophilic attack by the amide on the thionyl chloride to form an intermediate. In the next step, the electron pairs...
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Aldehydes and Ketones with HCN: Cyanohydrin Formation Overview01:32

Aldehydes and Ketones with HCN: Cyanohydrin Formation Overview

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Cyanohydrins are compounds that contain –CN and –OH groups on the same carbon atom. They are formed by the nucleophilic addition of the cyanide ions to the carbonyl group. Cyanide ions are highly basic and nucleophilic and can be generated from HCN under aqueous conditions. However, since HCN is a weak acid, the number of cyanide ions generated is very small. Hence, a small amount of base or KCN/NaCN is added to HCN to increase the concentration of the cyanide ions in the reaction...
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Aldehydes and Ketones with HCN: Cyanohydrin Formation Mechanism01:10

Aldehydes and Ketones with HCN: Cyanohydrin Formation Mechanism

4.8K
Cyanohydrins are formed when cyanide nucleophiles and carbonyl compounds like aldehydes and ketones react. A strong base, the cyanide ion, catalyzes cyanohydrin formation. The ions are generated from HCN under aqueous conditions. Once the cyanide ions are generated, the first step involves the nucleophilic attack of the cyanide ions on the electrophilic carbonyl carbon. This attack shifts the π electrons from the C=O to the oxygen atom forming the alkoxide ion intermediate. The alkoxide...
4.8K
Preparation of Alkynes: Dehydrohalogenation02:34

Preparation of Alkynes: Dehydrohalogenation

18.9K
Introduction
Alkynes can be prepared by dehydrohalogenation of vicinal or geminal dihalides in the presence of a strong base like sodium amide in liquid ammonia. The reaction proceeds with the loss of two equivalents of hydrogen halide (HX) via two successive E2 elimination reactions.
18.9K
Preparation of Carboxylic Acids: Hydrolysis of Nitriles01:19

Preparation of Carboxylic Acids: Hydrolysis of Nitriles

6.7K
Nitriles (R–CN) can be converted into carboxylic acids (R–COOH) upon treatment with aqueous acids, i.e., upon hydrolysis of nitriles. Under base-catalyzed conditions, carboxylate anions (R–COO−) are formed.
6.7K
Formation of Halohydrin from Alkenes02:41

Formation of Halohydrin from Alkenes

15.3K
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.
15.3K

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A single point mutation enhances hydroxynitrile synthesis by halohydrin dehalogenase.

Marcus Schallmey1, Peter Jekel1, Lixia Tang2

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

Enzyme and Microbial Technology
|February 10, 2015
PubMed
Summary

Researchers enhanced epoxide ring-opening reactions using halohydrin dehalogenase (HheC) by introducing a T134A mutation. This modification significantly boosted enzyme activity, creating valuable β-hydroxynitriles for synthetic chemistry.

Keywords:
CyanideEpoxidesHalohydrin dehalogenaseHydroxynitrileLigand bindingProtein engineering

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Synthetic Chemistry

Background:

  • Halohydrin dehalogenases (HheC) catalyze epoxide ring opening with cyanide, producing valuable β-hydroxynitriles.
  • The HheC enzyme from Agrobacterium radiobacter shows limited cyanolysis activity.
  • Sequence analysis suggested specific active site residue substitutions could enhance cyanolytic activity.

Purpose of the Study:

  • To engineer a more active halohydrin dehalogenase variant for improved β-hydroxynitrile synthesis.
  • To investigate the structural and mechanistic basis for enhanced cyanolytic activity.

Main Methods:

  • Site-directed mutagenesis was used to create the T134A variant of HheC.
  • Enzyme kinetics were measured to quantify cyanolysis activity.
  • Structural analysis was performed on the mutant enzyme.

Main Results:

  • The T134A HheC variant exhibited up to an 11-fold increase in cyanide-mediated epoxide ring-opening activity.
  • The mutation disrupted a hydrogen bond between residue 134 and active site serine 132.
  • Enhanced activity was attributed to altered substrate oxygen interactions, not changes in the nucleophile binding site.

Conclusions:

  • The T134A mutation effectively enhances the cyanolytic activity of HheC.
  • Modifying active site hydrogen bonding networks is a viable strategy for enzyme engineering.
  • This engineered enzyme offers improved biocatalytic routes to β-hydroxynitriles.