Biocatalysts and methods for the synthesis of armodafinil
Inventors
Ang, Ee Lui • Alvizo, Oscar • Behrouzian, Behnaz • Clay, Michael D. • Collier, Steven J. • Eberhard, Ellen D. • Fan, Fu • Song, Shiwei • Smith, Derek J. • Widegren, Magnus • Wilson, Robert • XU, Junye • Zhu, Jun
Assignees
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Publication Number
US-12344866-B2
Publication Date
2025-07-01
Expiration Date
Abstract
The present invention relates to non-naturally occurring polypeptides useful for preparing armodafinil, polynucleotides encoding the polypeptides, and methods of using the polypeptides. The non-naturally occurring polypeptides of the present invention are effective in carrying out biocatalytic conversion of the (i) 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil), or (ii) benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid, which is a pivotal intermediate in the synthesis of armodafinil, in enantiomeric excess.
Core Innovation
The patent relates to engineered cyclohexanone monooxygenase (CHMO) polypeptides and non-naturally occurring polynucleotides encoding such polypeptides. The engineered CHMO polypeptides have CHMO activity and an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2, and comprise an amino acid substitution at a position corresponding to position 143 in the polypeptide of SEQ ID NO: 2.
The engineered CHMO polypeptides are described as designed to convert CHMO substrates identified as compound (1a)/(1b) to compound (2a)/(2b) with enhanced enantioselectivity. The document states that the engineered CHMO variants outperform wild-type CHMO (SEQ ID NO: 2) with improved enantiomeric excess, improved activity, enhanced thermostability, and enhanced substrate/product tolerance.
The invention further describes engineered polynucleotides encoding the engineered CHMO polypeptides, along with recombinant vectors and host cells for expression. The document also describes biocatalytic process elements conceptually, including cofactor recycling using KRED in combination with oxygenation and conversion to (R)-BHSO and armodafinil as described in the schemes and examples.
Claims Coverage
The claims cover one independent inventive feature set directed to a non-naturally occurring CHMO polynucleotide/polypeptide combination defined by sequence identity to SEQ ID NO: 2 and a specified substitution at the residue position corresponding to position 143. Dependent claims add further substitution options, additional sequence-identity constraints, and performance-related conversion, activity, and enantiomeric excess features.
Non-naturally occurring CHMO polypeptide sequence identity to SEQ ID NO: 2
A non-naturally occurring polynucleotide encoding a non-naturally occurring polypeptide having cyclohexanone monooxygenase (CHMO) activity, wherein the amino acid sequence of the polypeptide has at least 90% sequence identity to SEQ ID NO: 2.
Position 143 amino acid substitution for engineered CHMO activity
The non-naturally occurring polynucleotide wherein the CHMO polypeptide comprises an amino acid substitution at a position corresponding to position 143 in the polypeptide of SEQ ID NO: 2, and wherein the amino acid at the position corresponding to position 143 is selected from cysteine, glutamate, glycine, histidine, lysine, methionine, proline, glutamine, serine, threonine, and tryptophan.
Additional substitution positions corresponding to SEQ ID NO: 2
The polynucleotide defines a corresponding polypeptide containing at least one amino acid substitution at one or more of the listed numbered positions corresponding to SEQ ID NO: 2.
Sequence identity relative to SEQ ID NO: 1
The polynucleotide further includes a sequence having at least 90% identity to SEQ ID NO: 1.
At least 2-fold increased activity for converting compound (1b) to compound (2b)
The polynucleotide encodes a non-naturally occurring polypeptide that converts compound (1b) to compound (2b) with at least a 2-fold increased activity relative to the polypeptide of SEQ ID NO: 2.
At least 90% conversion in 24 h with about 50 g/L substrate loading for converting compound (1b) to compound (2b)
The polynucleotide encodes a non-naturally occurring polypeptide capable of converting compound (1b) to compound (2b) with at least 90% conversion in 24 h with a substrate loading of about 50 g/L.
R-enantiomer production with at least 50% enantiomeric excess for converting compound (1b)
The polypeptide encoded by the polynucleotide can convert the acid substrate of compound (1b) to the R-enantiomer compound (2b) in at least 50% enantiomeric excess.
The claims center on engineered CHMO polypeptides encoded by non-naturally occurring polynucleotides, with at least 90% sequence identity to SEQ ID NO: 2 and a specific engineered substitution at the residue position corresponding to position 143. The dependent claims further refine the engineered substitution set and define additional sequence identity constraints and performance thresholds for conversion, activity, and enantiomeric excess for compound (1b) to compound (2b).
Stated Advantages
Improved enantiomeric excess compared to wild-type CHMO (SEQ ID NO: 2).
Improved activity compared to wild-type CHMO (SEQ ID NO: 2).
Enhanced enantioselectivity in converting CHMO substrates (compound (1a)/(1b)) to (2a)/(2b).
Enhanced thermostability versus SEQ ID NO: 2.
Enhanced substrate/product tolerance versus SEQ ID NO: 2.
High conversion with stereoselectivity in a 24 h period under claimed performance constraints.
Documented Applications
Biocatalytic asymmetric oxidation steps in armodafinil synthesis by converting 2-(benzhydrylsulfinyl)acetamide to (−)-2-[(R)-(diphenylmethyl)sulfinyl]acetamide (armodafinil).
Conversion of benzhydryl-thioacetic acid to (R)-2-(benzhydrylsulfinyl)acetic acid (R-BHSO/modafinic acid) using engineered CHMOs.
Conversion of CHMO substrates compound (1a)/(1b) to compound (2a)/(2b) using engineered CHMO polypeptides, including production of (R)-2-(benzhydrylsulfinyl)acetic acid (R-BHSO/BHSO) and conversion to armodafinil (compound (2a)) as described in the schemes and examples.
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