Biocatalytic process for preparing eslicarbazepine and analogs thereof

Patent metadata

Field	Value
Publication number	US-2016244792-A1
Application number	US-201615150686-A
Country	US
Kind code	A1
Filing date	May 10, 2016
Priority date	Apr 13, 2011
Publication date	Aug 25, 2016
Grant date	—

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 80% sequence identity to SEQ ID NO:10 and has at least the following features: X80 is T; X96 is V or R; X145 is L; X153 is T; X190 is P; X196 is L or M; and X226 is V.

2 . The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence has at least 93% sequence identity to SEQ ID NO: 36.

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 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 X199 is M.

4 . 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.

5 . 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.

6 . 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; X196 is M or L; X199 is M; and X226 is V.

7 . 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.

8 . 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.

9 . The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence 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; X196 is M or L; X199 is M; X200 is P; and X226 is V.

10 . The non-naturally occurring polynucleotide of claim 9 , 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.

11 . The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence comprises a sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

12 . A non-naturally occurring polynucleotide that encodes a polypeptide sequence comprising an amino acid sequence having at least 80% 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 X71, X87, and X131, wherein the polypeptide has ketoreductase activity.

13 . The non-naturally occurring polynucleotide of claim 12 , wherein encoded polypeptide sequence comprises one or more of the following features: X71 is P or G; X87 is L, and X131 is C.

14 . The non-naturally occurring polynucleotide of claim 1 , wherein the encoded polypeptide sequence is immobilized on a solid support.

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

16 . An isolated polynucleotide encoding the polypeptide of claim 12 .

17 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.

18 . An expression vector comprising the polynucleotide of claim 1 .

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

20 . An expression vector comprising the polynucleotide of claim 17 .

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P17/10 Primary

  Nitrogen as only ring hetero atom · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N11/00

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

• C12Y101/00

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

• C12N9/004

  Cytochrome-b5 reductase (1.6.2.2) · CPC title