Chemoenzymatic Route to Oxyfunctionalized Cembranoids Facilitated by Substrate and Protein Engineering

Area of Science:

• Organic Chemistry
• Biocatalysis
• Medicinal Chemistry

Background:

• Cembranoids are 14-membered macrocyclic diterpenoids with therapeutic potential.
• Selective late-stage oxidation of cembranoids is challenging for chemical catalysts.

Purpose of the Study:

• To develop a novel chemoenzymatic route for oxyfunctionalized macrocycles, including cembranoids.
• To investigate the regioselectivity and diastereoselectivity of engineered P450 enzymes in cembranoid synthesis.

Main Methods:

• Metal-catalyzed ring-closing metathesis combined with P450 BM3-catalyzed hydroxylation.
• Systematic substrate probing to determine enzyme regioselectivity factors.
• Site-directed mutagenesis of P450 BM3 (V78A/F87A variant).
• NMR analysis and X-ray crystallography for stereochemical determination.

Main Results:

• A chemoenzymatic route yielding cembranoid-like analogues was established.
• P450 BM3 regioselectivity was influenced by macrocycle rigidity and substituent properties.
• The V78A/F87A P450 BM3 mutant showed 90% regioselectivity for C5 hydroxylation.
• The mutant exhibited high diastereoselectivity, producing syn-cembranoid-diols exclusively.

Conclusions:

• The developed chemoenzymatic strategy enables efficient synthesis of functionalized macrocycles.
• Enzyme engineering significantly enhances regioselectivity and diastereoselectivity in cembranoid oxidation.
• This approach provides access to novel cembranoid analogues for potential therapeutic applications.