Ketoreductase Engineering for Stereoselective 1,3-Indane Diol Synthesis
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Solution Overview
Problem
There is a need for additional ketoreductase enzymes that can efficiently convert ketones to chiral alcohols, particularly for the synthesis of specific compounds like 1,3-indane diols, which are useful in the production of pharmaceuticals such as belzutifan, a hypoxia-inducible factor inhibitor.
Innovation Solution
Development of ketoreductase enzymes capable of stereoselectively reducing fluoro hydroxyindanone to fluorodiol, utilizing cofactors like NADH or NADPH, and processes for preparing and using these enzymes to synthesize diastereomerically pure 1,3-indane diols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type ketoreductase enzymes are used, then the basic function of converting ketones to chiral alcohols is achieved, but the enzymatic activity is insufficient for efficient synthesis of specific compounds like 1,3-indane diols
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the ketoreductase enzyme sequence (e.g., residues at positions 2, 11, 94, 135, 136, 138, 139, 145, 163, 170, 171, 173, 183, 186, 187, 203, 207, 226, 227, 229, 238, 242, 243, 276, 277, 282, 286, 291, 309, 313, 325, 342, 343) to optimize the enzyme's catalytic activity and substrate specificity for converting fluoro hydroxyindanone to fluorodiol, achieving 150 to 7000 times greater enzymatic activity than wild-type enzymes
2Productivity
If ketoreductase enzymes are engineered for high activity, then productivity increases, but the complexity of enzyme development and characterization increases
Solution Approach 1:
The patent segments the enzyme development process into systematic steps: identifying key amino acid positions through structural analysis, creating focused mutation sets at these positions, and testing individual variants. This segmentation of the complex engineering process into manageable steps reduces overall development complexity while achieving high productivity
Solution Approach 2:
The patent applies partial action by focusing mutations on specific critical amino acid positions rather than random comprehensive mutagenesis. This targeted approach achieves high enzymatic activity with reduced complexity compared to exhaustive screening of all possible mutations
3Manufacturing precision
If wild-type ketoreductase enzymes are used, then the general conversion of ketones to chiral alcohols occurs, but the stereoselectivity and diastereomeric purity of the product is insufficient
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at critical positions in the enzyme active site that locally enhance stereoselectivity for forming specific diastereomers (e.g., (1S,2S,3R)-1,3-indane diols). These localized changes at specific residue positions improve manufacturing precision without requiring changes to the entire enzyme structure, thereby preserving overall productivity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The enzymes achieve high enzymatic activity, with improvements ranging from 150 to 7000 times greater than wild-type enzymes, enabling efficient synthesis of chiral alcohols for pharmaceutical applications.
Implementation Method 1
Ketoreductases, also known as alcohol dehydrogenases, are enzymatic reducing agents, a specific class of enzymes that catalyze the selective reduction of ketones to chiral alcohols
Implementation Method 2
catalyze the selective reduction of ketones to chiral alcohols
Implementation Method 3
Enzymatic reduction of ketones and aldehydes requires participation of a co-factor that can act as an electron donor
Data Source
AI summary
The present disclosure provides ketoreductase enzymes having improved enzymatic properties including the capability of reducing hydroxy indanones to provide diastereomerically pure 1,3-indane diols useful for the synthesis of belzutifan. Also provided are polynucleotides encoding the ketoreductase enzymes, and host cells capable of expressing the ketoreductase enzymes. A purification procedure for isolating the ketoreductase enzymes is also provided.