Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate...

Area of Science:

• Biocatalysis and enzyme engineering
• Organic chemistry
• Medicinal chemistry

Background:

• Enzymatic C-H activation offers selective oxidation but faces limitations in substrate scope and enzyme stability.
• Non-heme iron and α-ketoglutarate-dependent dioxygenases are promising for hydroxylation reactions.
• Current methods for synthesizing belzutifan involve a lengthy five-step chemical process.

Purpose of the Study:

• To engineer a robust biocatalyst for direct, stereo- and regio-selective hydroxylation of a fluoroindanone precursor.
• To replace a multi-step chemical synthesis with a single enzymatic step for belzutifan production.
• To improve enzyme stability and reduce product overoxidation through directed evolution.

Main Methods:

• Engineering of a non-heme iron and α-ketoglutarate-dependent dioxygenase via directed evolution.
• Mechanistic studies to identify limitations in enzyme activity and stability.
• Scale-up of the biocatalytic oxidation to kilogram scale for drug synthesis.

Main Results:

• Developed an engineered dioxygenase capable of direct enantioselective hydroxylation of a non-native substrate.
• Improved enzyme stability and reduced product overoxidation, achieving over 15,000 total turnovers.
• Successfully synthesized belzutifan precursor via a high-yielding, green, and efficient one-step biocatalytic oxidation at kilogram scale.

Conclusions:

• Engineered biocatalysts can overcome limitations in enzyme stability and substrate scope for industrial applications.
• Biocatalytic hydroxylation provides a significantly more efficient and sustainable alternative to traditional chemical synthesis for complex molecules like belzutifan.
• This work demonstrates the practical utility of enzyme engineering in advancing green chemistry for pharmaceutical manufacturing.