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Regioselective Formation of Enolates

Regioselective Formation of Enolates

As depicted in the figure below, the unsymmetrical ketones can form two possible enolates: less substituted or more substituted enolates. Usually, the thermodynamic enolates are formed from the more substituted α-carbon atom, while the kinetic enolates are formed faster by deprotonation from the less substituted position. The thermodynamic enolates have lower energy, so they are more stable. But the energy required to form kinetic enolates is less.

Keto–Enol Tautomerism: Mechanism

Keto–Enol Tautomerism: Mechanism

The keto and enol forms are known as tautomers and they constantly interconvert (or tautomerize) between the two forms under acid or base catalyzed conditions. Both the reactions involve the same steps—protonation and deprotonation— although in the reverse order.

Feedback Inhibition

Feedback Inhibition

Biochemical reactions are occurring constantly in cells, converting starting substances to different products, usually with the help of enzymes that speed the reactions. Without enzymes, it would take far too long for most reactions to occur to be useful to the cell!

Intramolecular Claisen Condensation of Dicarboxylic Esters: Dieckmann Cyclization

Intramolecular Claisen Condensation of Dicarboxylic Esters: Dieckmann Cyclization

Dieckmann cyclization is an intramolecular Claisen condensation of diesters. The reaction occurs in the presence of a base and generates a cyclic β-ketoester as the final product. Commonly, 1, 6 and 1, 7-diesters are preferred substrates for the reaction since the generated five, and six-membered cyclic β-keto esters are particularly more stable.

Esters to β-Ketoesters: Claisen Condensation Mechanism

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...

Alkylation of β-Diester Enolates: Malonic Ester Synthesis

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.

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Related Experiment Video

Updated: May 28, 2025

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

Published on: January 13, 2017

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Tyler M McCullough^1,2, Vishakha Choudhary^1,2, David L Akey¹

¹Life Sciences Institute, Mary Sue Coleman Hall, 210 Washtenaw Ave., University of Michigan, Ann Arbor, MI 48109-2216, United States.

ACS Catalysis

|February 10, 2025

Summary

Researchers engineered thioesterases (TEs) using 1,3-diaminopropionic acid (DAP) to trap intermediates, revealing how these enzymes form macrolactone antibiotics. This work aids in developing new biocatalysts for drug discovery.

Keywords:

Macrolactonizing enzyme Polyketide synthase Substrate trapping Thioesterase Unnatural amino acid incorporation X-ray crystallography

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Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Published on: February 25, 2011

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Last Updated: May 28, 2025

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028

Published on: January 13, 2017

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Published on: January 16, 2016

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Published on: February 25, 2011

Area of Science:

Biochemistry
Molecular Biology
Synthetic Biology

Background:

Antibiotic resistance necessitates novel antimicrobial drug development, particularly macrolides.
Polyketide synthase (PKS) thioesterases (TEs) are crucial for macrolactone scaffold formation in macrolide biosynthesis.
Limited understanding of TE mechanisms hinders their use as versatile biocatalysts.

Purpose of the Study:

To elucidate the mechanism of TE selectivity in macrolactone formation.
To engineer TEs for accommodating diverse natural and non-natural substrates.
To provide insights for TE engineering and optimization.

Main Methods:

Acyl-enzyme intermediates were trapped as stable amides by substituting the active site serine hydroxyl with 1,3-diaminopropionic acid (DAP).
DAP-modified TEs (TE_DAP) from pikromycin and erythromycin pathways were generated and purified.
TE_DAP variants were tested with various polyketide substrates, and crystal structures were determined.

Main Results:

The erythromycin TE exhibited permissive substrate selectivity, while the pikromycin TE was selective for its native substrates.
Crystal structure of a pikromycin TE_DAP-substrate complex revealed a curled heptaketide with high complementarity to the TE's acyl cavity.
Distinct acyl cavity shapes were observed across different TEs, including juvenimicin, tylosin, and fluvirucin pathways.

Conclusions:

TEs control macrolactone formation through specific substrate interactions within their acyl cavities.
Engineering TEs with unnatural amino acids like DAP provides mechanistic insights and potential for biocatalyst development.
Structural diversity of TE acyl cavities offers a basis for designing TEs with tailored substrate specificities for antibiotic synthesis.