Novel Hydrolase Protein for Hepatitis C Intermediate Synthesis
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Solution Overview
Problem
Current methods for producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, an intermediate for hepatitis C therapeutic agents, suffer from insufficient optical selectivity and purity, making them unsuitable for industrial-scale production.
Innovation Solution
A novel hydrolase protein derived from Bacillus subtilis, with specific amino acid substitutions, is used to hydrolyze dialkyl 2-vinylcyclopropane-1,1-dicarboxylate, achieving higher selectivity and optical purity of (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, suitable for industrial production of hepatitis C therapeutic agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional enzyme hydrolysis is used to produce (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, then the production process can be established, but the optical selectivity and purity are insufficient (90% e.e.), making it unsuitable for industrial application
Solution Approach 1:
The invention applies parameter changes by modifying the amino acid sequence of the enzyme (changing biological parameters) to improve optical selectivity. Specifically, the patent identifies and modifies key amino acid residues in the enzyme structure to enhance its stereoselectivity toward producing (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, thereby achieving over 99% e.e. and making the process industrially viable
Solution Approach 2:
The invention uses copying by creating a modified version of the existing enzyme based on the known para-nitrobenzyl esterase structure from Bacillus subtilis. The patent copies the fundamental enzyme structure and active site architecture but introduces specific amino acid substitutions to improve optical selectivity while maintaining catalytic function
2Reliability
If enzyme hydrolysis is performed to obtain the intermediate, then the method can be established, but the optical selectivity of the enzyme is insufficient for high-purity production
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid parameters in the enzyme sequence. Through site-directed mutagenesis and structure-activity relationship analysis, the invention optimizes key residues to enhance enantioselectivity, achieving over 99% e.e. and making the process suitable for industrial production of optically pure intermediates
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 novel hydrolase protein significantly enhances the selectivity and purity of (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid production, enabling an industrially applicable method for producing therapeutic agents for hepatitis C.
Implementation Method 1
a para-nitrobenzyl hydrolase protein derived from Bacillus subtilis hydrolyzes diethyl 2-vinylcyclopropane-1,1-dicarboxylate
Implementation Method 2
the inventors have found that a para-nitrobenzyl hydrolase protein derived from Bacillus subtilis hydrolyzes diethyl 2-vinylcyclopropane-1,1-dicarboxylate at good selectivity
Data Source
AI summary
It is an object of the present invention to provide a novel hydrolase, which is used when dialkyl 2-vinylcyclopropane-1,1-dicarboxylate is hydrolyzed with an enzyme, so as to efficiently obtain (1S,2S)-1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid that is useful as an intermediate for synthesizing therapeutic agents for hepatitis C. According to the present invention, there is provided a hydrolase protein, which consists of the amino acid sequence shown in any one of SEQ ID NOS. 2 to 5 and which has activity of catalyzing, at higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO. 1, a reaction of producing (1S,2S)-1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid from diethyl 2-vinylcyclopropane-1,1-dicarboxylate.