Biocatalysts and methods for the synthesis of armodafinil

Patent metadata

Field	Value
Publication number	US-12344866-B2
Application number	US-202217833788-A
Country	US
Kind code	B2
Filing date	Jun 6, 2022
Priority date	Dec 8, 2010
Publication date	Jul 1, 2025
Grant date	Jul 1, 2025

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.

First claim

What is claimed is:

1. 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, wherein said polypeptide comprises an amino acid substitution at a position corresponding to position 143 in the polypeptide of SEQ ID NO: 2, wherein the amino acid at the position corresponding to position 143 of the polypeptide of SEQ ID NO: 2 is selected from cysteine, glutamate, glycine, histidine, lysine, methionine, proline, glutamine, serine, threonine, and tryptophan.

2. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting the acid substrate compound (1b) to compound (2b) (R-enantiomer) or its opposite enantiomer compound (S-enantiomer)

with at least 2-fold improved activity relative to the wild-type polypeptide of SEQ ID NO: 2.

3. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 2, wherein the polypeptide comprises an alanine, glutamic acid, glycine, isoleucine, lysine, proline, serine, threonine, or valine at a position corresponding to position 246 of SEQ ID NO: 2.

4. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting the acid substrate of compound (1b) to the R-enantiomer compound (2b)

in at least 50% enantiomeric excess.

5. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 2, wherein said polypeptide further comprises one or more substitutions corresponding to substitutions in SEQ ID NO: 2 selected from the group consisting of a glycine at position 278, and lysine at position 490.

6. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide further comprises an isoleucine at a position corresponding to position 277 of SEQ ID NO: 2, alanine or glycine at a position corresponding to position 278 of SEQ ID NO: 2, threonine or tyrosine at a position corresponding to position 280 of SEQ ID NO: 2, isoleucine at a position corresponding to position 281 of SEQ ID NO: 2, arginine at a position corresponding to position 326 of SEQ ID NO: 2, and lysine or glutamine at a position corresponding to position 490 of SEQ ID NO: 2.

7. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1a) to compound (2a)

in at least 75% enantiomeric excess under suitable reaction conditions.

8. The non-naturally occurring polynucleotide of claim 1 , wherein said non-naturally occurring polypeptide is further capable of converting compound (1a) to compound (2a)

with an activity increased at least 2-fold relative to the activity of the polypeptide of SEQ ID NO: 2, under suitable reaction conditions.

9. The non-naturally occurring polynucleotide of claim 6 , wherein said non-naturally occurring polypeptide further comprises a combination of amino acid substitutions at positions corresponding to positions in the polypeptide of SEQ ID NO: 2, wherein said combination is selected from the group consisting of:
(a) the amino acid at position 3 is threonine, the amino acid at position 43 is glycine, the amino acid at position 75 is methionine, the amino acid at position 143 is glycine, the amino acid at position 166 is alanine, the amino acid at position 280 is tyrosine, the amino acid at position 395 is arginine, the amino acid at position 412 is leucine, the amino acid at position 426 is serine, the amino acid at position 432 is serine, the amino acid at position 433 is glycine, the amino acid at position 435 is alanine, the amino acid at position 491 is valine, the amino acid at position 503 is alanine, the amino acid at position 504 is isoleucine, the amino acid at position 512 is asparagine, and the amino acid at position 532 is proline;
(b) the amino acid at position 3 is threonine, the amino acid at position 43 is glycine, the amino acid at position 75 is methionine, the amino acid at position 99 is valine, the amino acid at position 143 is glycine, the amino acid at position 161 is aspartic acid, the amino acid at position 166 is alanine, the amino acid at position 174 is isoleucine, the amino acid at position 273 is serine, the amino acid at position 280 is tyrosine, the amino acid at position 324 is lysine, the amino acid at position 395 is arginine, the amino acid at position 412 is leucine, the amino acid at position 426 is serine, the amino acid at position 432 is serine, the amino acid at position 433 is glycine, the amino acid at position 435 is alanine, the amino acid at position 491 is valine, the amino acid at position 503 is alanine, the amino acid at position 504 is isoleucine, the amino acid at position 512 is asparagine, and the amino acid at position 532 is proline;
(c) the amino acid at position 3 is threonine, the amino acid at position 43 is glycine, the amino acid at position 75 is methionine, the amino acid at position 79 is threonine, the amino acid at position 82 is alanine, the amino acid at position 99 is valine, the amino acid at position 110 is methionine, the amino acid at position 143 is glycine, the amino acid at position 161 is aspartic acid, the amino acid at position 166 is alanine, the amino acid at position 174 is isoleucine, the amino acid at position 208 is threonine, the amino acid at position 273 is serine, the amino acid at position 280 is tyrosine, the amino acid at position 324 is lysine, the amino acid at position 395 is arginine, the amino acid at position 412 is leucine, the amino acid at position 426 is serine, the amino acid at position 432 is serine, the amino acid at position 433 is glycine, the amino acid at position 435 is alanine, the amino acid at position 491 is valine, the amino acid at position 503 is alanine, the amino acid at position 504 is isoleucine, the amino acid at position 505 is lysine, the amino acid at position 512 is asparagine, and the amino acid at position 532 is proline;
(d) the amino acid at position 3 is threonine, the amino acid at position 43 is glycine, the amino acid at position 75 is methionine, the amino acid at position 79 is threonine, the amino acid at position 82 is alanine, the amino acid at position 99 is valine, the amino acid at position 110 is methionine, the amino acid at position 143 is glycine, the amino acid at position 161 is aspartic acid, the amino acid at position 166 is alanine, the amino acid at position 174 is isoleucine, the amino acid at position 208 is threonine, the amino acid at position 273 is serine, the amino acid at position 280 is tyrosine, the amino acid at position 324 is lysine, the amino acid at position 395 is arginine, the amino acid at position 412 is leucine, the amino acid at position 426 is serine, the amino acid at position 432 is serine, the amino acid at position 433 is glycine, the amino acid at position 435 is alanine, the amino acid at position 472 is isoleucine, the amino acid at position 48

Assignees

• Codexis Inc

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

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P13/02

  Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)} · CPC title

• C12Y114/13022

  Cyclohexanone monooxygenase (1.14.13.22) · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12P11/00

  Preparation of sulfur-containing organic compounds · CPC title