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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.

Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis

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...

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.

Transcription Attenuation in Prokaryotes

Transcription Attenuation in Prokaryotes

Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure. Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated...

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.

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Updated: Jun 22, 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, Vishakha Choudhary, David L Akey

Biorxiv : the Preprint Server for Biology

|July 1, 2024

Summary

Researchers engineered thioesterase enzymes (TEs) using 1,3-diaminopropionic acid (DAP) to trap intermediates, revealing how macrolide antibiotic scaffolds form and enabling biocatalyst optimization.

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The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

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Last Updated: Jun 22, 2025

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

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The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

Published on: January 13, 2013

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

Area of Science:

Biochemistry
Synthetic Biology
Drug Discovery

Background:

Antibiotic resistance necessitates new antimicrobial drugs, particularly macrolides.
Polyketide synthase (PKS) thioesterases (TEs) catalyze macrolactone formation, crucial for macrolide antibiotics.
Limited understanding of TE mechanisms hinders biocatalyst development for diverse substrates.

Purpose of the Study:

To elucidate the mechanism of TE substrate selectivity in macrolactone formation.
To engineer TEs as biocatalysts for a wider range of natural and non-natural substrates.
To understand how TEs direct nucleophilic attack for macrolactone ring closure.

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 purified.
TE_DAP variants were tested with various polyketide intermediates, and crystal structures were determined.

Main Results:

Erythromycin TE exhibited permissive substrate selectivity, while pikromycin TE was selective for its native substrates.
Crystal structure of pikromycin TE_DAP revealed a curled heptaketide substrate with high shape complementarity to the active site.
Distinct acyl cavity shapes were observed across different TEs, including those from juvenimicin, tylosin, and fluvirucin biosynthesis.

Conclusions:

TEs exhibit varied substrate selectivity based on active site architecture, particularly the acyl cavity.
Engineering TEs with DAP provides insights into substrate binding and catalytic mechanisms.
Understanding TE structural diversity facilitates the engineering of novel biocatalysts for macrolide synthesis.