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Related Concept Videos

Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis01:07

Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis

Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the SN2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an alkylated β-keto acid.
Esters to Carboxylic Acids: Acid-Catalyzed Hydrolysis01:13

Esters to Carboxylic Acids: Acid-Catalyzed Hydrolysis

Hydrolysis of esters under acidic conditions proceeds through a nucleophilic acyl substitution. In the presence of excess water, the reaction proceeds in a reversible manner, forming carboxylic acids and alcohols.
During hydrolysis, the ester is first activated towards nucleophilic attack through the protonation of the carboxyl oxygen atom by the acid catalyst. The protonation makes the ester carbonyl carbon more electrophilic. In the next step, water acts as a nucleophile and adds to the...
Alkylation of β-Diester Enolates: Malonic Ester Synthesis01:14

Alkylation of β-Diester Enolates: Malonic Ester Synthesis

Malonic ester synthesis is a method to obtain α substituted carboxylic acids from ꞵ-diesters such as diethyl malonate and alkyl halides.
Carboxylic Acids to Esters: Acid-Catalyzed (Fischer) Esterification Mechanism01:13

Carboxylic Acids to Esters: Acid-Catalyzed (Fischer) Esterification Mechanism

Carboxylic acids react with alcohols to yield esters via an acid-catalyzed condensation reaction called Fischer esterification. This is a nucleophilic acyl substitution reaction that proceeds via a tetrahedral intermediate, where a water molecule is eliminated as the leaving group.
Carboxylic Acids to Esters: Acid-Catalyzed (Fischer) Esterification Overview01:20

Carboxylic Acids to Esters: Acid-Catalyzed (Fischer) Esterification Overview

The Fischer esterification reaction was developed by the German chemist Emil Fischer in 1895. It is a condensation reaction between carboxylic acids and alcohols in an acidic medium to give esters and water.
Esters to β-Ketoesters: Claisen Condensation Mechanism01:08

Esters to β-Ketoesters: Claisen Condensation Mechanism

Regular Claisen condensation involves the synthesis of β-ketoesters by combining identical ester molecules bearing two α hydrogens in the presence of an alkoxide base. The reaction commences with the deprotonation of the acidic α hydrogen by the base to form a resonance stabilized ester enolate. This nucleophilic ion then attacks the carbonyl center of another ester molecule to generate a tetrahedral alkoxide intermediate. Next, the expulsion of the alkoxide group from the intermediate restores...

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Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile
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Engineering a Carboxylesterase for Enantioselective Acyl Transfer Reaction.

Wencai Fan1, Sanyang Li1, Xinxin Liu1

  • 1Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210023, China.

The Journal of Organic Chemistry
|July 8, 2026
PubMed
Summary

We developed hyper-steric engineering to enhance enzyme catalytic activity. This method increases amino acid steric size, boosting enzyme efficiency and enantioselectivity for acyl transfer reactions.

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Area of Science:

  • Biocatalysis and enzyme engineering
  • Protein engineering and directed evolution

Background:

  • Enzyme catalytic cavity optimization is crucial for maximizing catalytic efficiency.
  • Conventional hypo-steric engineering decreases amino acid size to enlarge cavities.
  • A novel hyper-steric engineering approach is needed to enhance enzyme performance.

Purpose of the Study:

  • To introduce a hyper-steric engineering strategy for enzyme improvement.
  • To enhance the catalytic activity and enantioselectivity of carboxylesterase EstFF1.
  • To achieve efficient acyl transfer to a wide range of alcohols.

Main Methods:

  • Developed a hyper-steric engineering method by increasing amino acid steric size.
  • Engineered the carboxylesterase EstFF1, creating variants R386V, R390V (VIII) and G359L (IX).
  • Utilized specific amino acid substitutions to modulate the enzyme's catalytic cavity size and shape.

Main Results:

  • The R386V, R390V variant (VIII) enabled efficient acyl transfer to primary alcohols under mild conditions.
  • Hyper-steric engineering of G359 to L359 (IX) on variant VIII enlarged the catalytic cavity.
  • This modification allowed acyl transfer to diverse secondary alcohols with high S-enantioselectivity.

Conclusions:

  • Hyper-steric engineering is a powerful strategy for enhancing enzyme catalytic activity and selectivity.
  • This approach successfully improved the performance of carboxylesterase EstFF1 for acyl transfer reactions.
  • The engineered enzymes demonstrate broad substrate scope and high enantioselectivity, valuable for biocatalysis.