Transaminase reactions

Patent metadata

Field	Value
Publication number	US-10138503-B2
Application number	US-201615226397-A
Country	US
Kind code	B2
Filing date	Aug 2, 2016
Priority date	Jun 22, 2009
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

The present disclosure relates to methods of using transaminase polypeptides in the synthesis of chiral amines from prochiral ketones.

First claim

What is claimed is:

1. A process for preparing the compound of formula (III):

having the indicated stereochemical configuration at the stereogenic center marked with an * in an enantiomeric excess over the opposite enantiomer,
wherein, R 10 is Cl, Br, F, CH 3 , CF 3 , CN, SO 2 , —OCH 3 , or NO 2 ,
the process comprising:
(a) contacting a ketone substrate of formula:

wherein R 11 is halogen, OH, C(O)R 4 , OC(O)R 5 , or NR 6 R 7 , wherein R 4 , R 5 , R 6 , and R 7 is H or C 1 -C 4 alkyl, and R 10 is as defined above,

with a transaminase polypeptide in the presence of an amino donor, wherein said amino donor comprises isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine, under reaction conditions suitable for converting the ketone substrate to an amine product of formula:

wherein the amino acid sequence of the transaminase polypeptide has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue of the transaminase polypeptide which corresponds to the residue at position 223 of SEQ ID NO: 2 is proline, and wherein the transaminase polypeptide is capable of converting the ketone substrate to the amine product at a rate that is greater than that of a transaminase polypeptide comprising the amino acid sequence of SEQ ID NO:2; and
(b) cyclizing the amine product under suitable conditions to form the compound of formula (III).

2. The process of claim 1 , wherein the ketone substrate is 4-chloro-1-(2-fluorophenyl)butan-1-one:

and the amine product is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

thereby forming (R)-2-(2-fluorophenyl)pyrrolidine:

in enantiomeric excess.

3. The process of claim 1 , wherein a reaction condition comprises a temperature of 20° C. to 65° C.

4. The process of claim 3 , wherein a reaction condition comprises a temperature of 40° C. to 65° C.

5. The process of claim 1 , wherein the amine product is produced in at least 90% enantiomeric excess.

6. The process of claim 1 , wherein the amine product is produced in at least 99% enantiomeric excess.

7. The process of claim 1 , further comprising the step of removing a carbonyl by-product of the reaction.

8. The process of claim 7 , wherein the amino donor is alanine or 3-aminobutyric acid and the carbonyl by-product is a keto acid.

9. The process of claim 7 , wherein the carbonyl by-product has a vapor pressure higher than water, and removal of the carbonyl byproduct is by sparging with a nonreactive gas or by applying a vacuum.

10. The process of claim 9 , wherein the non-reactive gas is nitrogen gas.

11. The process of claim 9 , wherein the amino group donor is isopropylamine and wherein the carbonyl by-product is acetone.

12. The process of claim 1 , wherein a reaction condition is a pH of 7.0 to a pH of 11.0.

13. The process of claim 12 , wherein the pH is maintained by adding isopropylamine.

14. The process of claim 1 , wherein a reaction condition comprises dimethylsulfoxide (DMSO) as a solvent.

15. The process of claim 14 , wherein the concentration of DMSO is between 10% to 40% (v/v).

16. The process of claim 1 , wherein the ketone substrate is present at 5 to 25 g/L.

17. The process of claim 1 , wherein one or more residue positions corresponding to the residue positions of SEQ ID NO: 2 selected from: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X69; X81; X94; X96; X102; X117; X120; XI22; X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211; X215; X217; X225; X230; X252; X269; X273; X282; X284; X292; X297; X306; X321; and X329 is/are substituted in the transaminase polypeptide.

18. The process of claim 17 , wherein the one or more residue positions corresponding to the residue positions of SEQ ID NO:2 selected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X225, X282, and X284 is/are substituted in the transaminase polypeptide.

19. The process of claim 17 , wherein the type of amino acid substitution is selected from:
X4 is an aromatic residue,
X8 is a constrained residue;
X26 is an aromatic or constrained residue;
X48 is a polar, acidic, aliphatic or non-polar residue;
X60 is an aromatic residue;
X61 is an aromatic residue;
X62 is an aromatic or polar residue;
X65 is an aliphatic residue;
X69 is a cysteine (C) or non-polar, polar, or aliphatic residue;
X81 is a non-polar residue;
X94 is an aliphatic residue;
X96 is an aliphatic residue;
X102 is an aliphatic or basic residue;
X122 is a constrained, non-polar or aliphatic residue;
X124 is a polar or constrained residue;
X136 is an aromatic residue;
X137 is a polar or aliphatic residue;
X150 is aromatic, constrained or polar residue;
X152 is cysteine (C), non-polar, aliphatic, or polar residue;
XI60 is an aliphatic residue;
X163 is an aliphatic or constrained residue;
X169 is an aliphatic residue;
X174 is an aliphatic residue;
X178 is a polar residue;
X195 is an aromatic or polar residue;
X199 is an aliphatic or aromatic residue;
X208 is cysteine (C) or constrained, non-polar, aromatic, polar, or basic residue;
X209 is an aliphatic residue;
X211 is an aliphatic residue;
X215 is a cysteine (C);
X217 is a polar residue;
X225 is an aromatic residue;
X230 is an aliphatic residue;
X252 is an aromatic or aliphatic residue;
X269 is a constrained residue;
X273 is an aromatic residue;
X282 is a polar residue;
X284 is a non-polar residue;
X292 is a polar residue;
X297 is a polar residue;
X306 is an aliphatic residue;
X321 is a constrained residue, and
X329 is a constrained or aromatic residue.

20. The process of claim 17 , wherein the amino acid substitution is selected from: X4 is Y; X8 is P; X26 is H; X48 is Q, D, V, G, or A; X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S; X81 is G; X94 is I or L; X96 is L; X102 is L or K; X122 is M, I, L, V, or H; X124 T, H or N; X136 is Y or F; X137 is T or I; X150 is F, H, or S; X152 is I, L, S or C; X160 is L; X163 is H or V; X169 is L; X174 is A; X178 is S; X195 is F or Q; X199 is W or I; X208 is H, C, G, K, N, Y, D or S; X209 is L; X211 is I; X215 is C; X217 is N; X225 is Y; X230 is V; X252 is F; X269 is P; X273 is Y; X282 is S; X284 is G; X292 is T; X297 is S; X306 is L; X321 is P; and X329 is H.

21. The process of claim 20 , wherein the amino acid substitution is selected from: X8 is P; X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S; X81 is G; X94 is I or L; X96 is L; X122 is M, I, L, V, or H; X124 T, H or N; X136 is Y or F; X169 is L; X178 is S; X199 is W or I; X209 is L; X215 is C; X217 is N; X269 is P; X273 is Y; X282 is S; X284 is G; X297 is S; X321 is P and X329 is H.

22. The proces

Assignees

• Codexis Inc

Inventors

• Hughes Gregory
• Devine Paul N
• Fleitz Fred J
• Grau Brendan T
• Limanto John
• Savile Christopher
• Mundorff Emily

Classifications

• C12P17/10 Primary

  Nitrogen as only ring hetero atom · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P17/182

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• Y02P20/52

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