Enzyme-Catalyzed Efficient Synthesis of Enantiomerically Pure (S)- and (R)-n-Butylphthalide.

Area of Science:

• Biocatalysis
• Organic Synthesis
• Chemical Engineering

Background:

• 3-n-Butylphthalide (NBP) is a key drug for acute ischemic stroke.
• (S)-NBP shows superior clinical efficacy compared to (R)-NBP.
• Stereoselective synthesis of NBP enantiomers is challenging.

Purpose of the Study:

• To develop an efficient and highly stereoselective enzymatic synthesis for (S)- and (R)-NBP.
• To engineer carbonyl reductases for asymmetric reduction.
• To implement a continuous-flow enzymatic process for NBP production.

Main Methods:

• Engineering of two carbonyl reductases, SmCR_K6 and SsCR_K1.
• Asymmetric reduction of 2-pentanoyl benzonitrile using engineered enzymes.
• Continuous-flow synthesis in a 3D microfluidic reactor.

Main Results:

• Engineered SmCR_K6 achieved a 23-fold increase in catalytic activity and 94% ee for (S)-NBP.
• Engineered SsCR_K1 showed a fourfold increase in activity and 99% ee for (R)-NBP.
• Continuous-flow process yielded 9-fold and 30-fold higher space-time yields for (S)-NBP and (R)-NBP, respectively.

Conclusions:

• A novel enzymatic strategy enables efficient and highly stereoselective synthesis of NBP enantiomers.
• Engineered reductases and microfluidic technology offer a scalable approach for industrial NBP production.
• This method provides a promising route for manufacturing chiral drugs like NBP.