Biocatalytic process for preparing eslicarbazepine and analogs thereof

Patent metadata

Field	Value
Publication number	US-9963683-B2
Application number	US-201715433852-A
Country	US
Kind code	B2
Filing date	Feb 15, 2017
Priority date	Apr 13, 2011
Publication date	May 8, 2018
Grant date	May 8, 2018

Abstract

The present disclosure relates to biocatalysts and its uses for the efficient preparation of eslicarbazepine, eslicarbazepine acetate, and analogs thereof.

First claim

What is claimed is:

1. A non-naturally occurring polynucleotide encoding a polypeptide capable of converting compound (2c)

to compound (1c)

in enantiomeric excess in presence of NADPH, the ketoreductase polypeptide comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2 and has at least the following features and the amino acid at position 196 is lysine or methionine.

2. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises one or more of the following features: X80 is T; X96 is V or R; X145 is L; X153 is T; X190 is P; X226 is V X17 is H; X29 is T; X43 is R; X71 is P or G; X87 is L; X95 is Y; X131 is C; X173 is L; and/or X199 is M.

3. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises one or more of the following features: X17 is M or H; X29 is T; X40 is R; X43 is R or V; X64 is V; X94 is G or A; X95 is Y or M; X147 is Q or M; X152 is L or A; X157 is C or S; X173 is L; X199 is M; and X200 is P.

4. The non-naturally occurring polynucleotide of claim 1 , where the encoded polypeptide sequence further comprises one or more of the following features: X25 is T; X76 is A; X144 is V; X150 is L; X194 is R; X233 is G; and X249 is W or F.

5. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence comprises at least the following features: X64 is V; X71 is P or G; X80 is T; X87 is L; X94 is G or A; X96 is V or R; X145 is L; X147 is Q or M; X153 is T; X173 is L; X190 is P; X199 is M; and X226 is V.

6. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises one or more of the following features: X17 is M or H; X29 is T; X40 is R; X43 is R or V; X95 is M or Y; X131 is C; X152 is L or A; and X200 is P.

7. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises one or more of the following features: X25 is T; X76 is A; X144 is V; X150 is L; X157 is C or S; X194 is R; X233 is G; and X249 is W or F.

8. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises at least the following features: X17 is H or M; X25 is T; X29 is T; X40 is R; X43 is R or V; X64 is V; X71 is G or P; X80 is T; X87 is L; X94 is G; X95 is Y or M; X96 is R or V; X131 is C; X145 is L; X147 is Q or M; X152 is A or L; X153 is T; X157 is S or C; X173 is L; X190 is P; X199 is M; X200 is P; and X226 is V.

9. The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence further comprises one or more of the following features: X76 is A; X144 is V; X150 is L; X194 is R; X233 is G; and X249 is W or F.

10. A non-naturally occurring polynucleotide that encodes a polypeptide sequence comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 and having one or more residue differences as compared to the reference sequence of SEQ ID NO:2 at residue positions corresponding to X196, wherein the polypeptide has ketoreductase activity.

11. The non-naturally occurring polynucleotide of claim 10 , wherein encoded polypeptide sequence comprises one or more of the following features: X196 is L or M; X71 is P or G; X87 is L, and X131 is C.

12. The non-naturally occurring polynucleotide of claim 10 , wherein the encoded polypeptide sequence is immobilized on a solid support.

13. The non-naturally occurring polypeptide of claim 1 , wherein said non-naturally occurring polypeptide is immobilized on a solid support.

14. An isolated polynucleotide encoding the polypeptide of claim 1 .

15. An expression vector comprising the polynucleotide of claim 10 .

16. An expression vector comprising the polynucleotide of claim 11 .

17. A host cell comprising the expression vector of claim 15 .

18. A method for producing a non-naturally occurring polypeptide, comprising culturing the host cell of claim 17 , under conditions such that said non-naturally occurring polypeptide is produced.

19. The method of claim 17 , further comprising purifying the non-naturally occurring polypeptide.

20. The method of claim 18 , wherein said host cell is E. coli.

Assignees

• Codexis Inc

Inventors

• Gohel Anupam
• Smith Derek J
• Wong Brian
• Sukumaran Joly
• Yeo Wan Lin
• Collier Steven J
• Novick Scott

Classifications

• C12Y101/00

  Oxidoreductases acting on the CH-OH group of donors (1.1) · CPC title

• C07D223/22

  Dibenz [b, f] azepines; Hydrogenated dibenz [b, f] azepines · CPC title

• C12N11/00

  Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title

• C12N9/004

  Cytochrome-b5 reductase (1.6.2.2) · CPC title