Transaminase reactions

Patent metadata

Field	Value
Publication number	US-2016333381-A1
Application number	US-201615226397-A
Country	US
Kind code	A1
Filing date	Aug 2, 2016
Priority date	Jun 22, 2009
Publication date	Nov 17, 2016
Grant date	—

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 a 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 presence of an amino donor under reaction conditions suitable for converting the ketone substrate to an amine product of formula:

wherein the transaminase polypeptide has at least 80% sequence identity to SEQ ID NO:74 and is capable of converting the ketone substrate to the amine product at a rate that is improved as compared to 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 the reaction condition comprises a temperature of 20° C. to 65° C.

4 . The process of claim 3 , wherein the 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 , wherein the amino donor is selected from isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine.

8 . The process of claim 7 , wherein the amino donor is isopropylamine.

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

10 . The process of claim 9 , wherein the amino donor is an amino acid and the carbonyl by-product is a keto acid.

11 . The process of claim 10 , wherein the carbonyl by-product has a vapor pressure higher than water, and removal of the carbonyl byproduct is by sparging with a non-reactive gas or by applying a vacuum.

12 . The process of claim 11 , wherein the non-reactive gas is nitrogen gas.

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

14 . The process of claim 1 , wherein the reaction condition is from a pH of about 7.0 to a pH of about 11.0.

15 . The process of claim 14 , wherein the pH is maintained by adding isopropylamine.

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

17 . The process of claim 16 , wherein the DMSO is between about 10% to about 40% (v/v).

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

19 . The process of claim 1 , wherein the transaminase comprises an amino acid sequence having a residue difference as compared to SEQ ID NO:2 at one or more residue positions selected from: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X81; X94; X96; X102; X117; X120; 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, X292; X297; X306; X321; and X329.

20 . The process of claim 19 , wherein the residue difference with a transaminase polypeptide of SEQ ID NO:2 occurs at one or more residue positions selected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X223, X225, X282, and X284.

21 . The process of claim 19 , wherein the type of amino acid residue at the position of the residue difference 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; X160 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; X223 is a constrained 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.

22 . The process of claim 19 , wherein the amino acid residue at the position of the residue difference 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; X223 is P; 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.

23 . The process of claim 22 , wherein the amino acid residue at the position of the residue difference 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; X223 is P; X269 is P; X273 is Y; X282 is S;

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12P17/10 Primary

  Nitrogen as only ring hetero atom · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P17/12

  containing a six-membered hetero ring · CPC title