Transaminase reactions

Patent metadata

Field	Value
Publication number	US-12247233-B2
Application number	US-202217751200-A
Country	US
Kind code	B2
Filing date	May 23, 2022
Priority date	Jun 22, 2009
Publication date	Mar 11, 2025
Grant date	Mar 11, 2025

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 an amine product of structural formula (I):

having the indicated stereochemical configuration at the stereogenic center marked with an *; in an enantiomeric excess over the opposite enantiomer, wherein
R 1 is optionally substituted aryl or heteroaryl;
R 2 is an optionally substituted C 1 -C 6 alkyl, —R 3 C(O)R 4 , or —R 3 OC(O)R 5
R 3 is an optionally substituted C 1 -C 4 alkyl, and R 4 is H, an optionally substituted C 1 -C 4 alkyl, NR 6 R 7 , or OR 8 , where R 5 , R 6 , R 7 , and R 8 are independently H or C 1 -C 4 alkyl;

the process comprising contacting a ketone substrate of structural formula (II):

with a transaminase polypeptide in the presence of an amino donor under reaction conditions suitable for converting the ketone substrate to the amine product, wherein the transaminase polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 102 and is capable of converting the ketone substrate to the amine product of structural formula (I) at a rate that is increased as compared to SEQ ID NO:2.

2. The process of claim 1 , wherein:
i) R 1 is an optionally substituted phenyl;
ii) R 1 is an optionally substituted pyridinyl;
iii) R 1 is a substituted aryl or heteroaryl;
iv) the substitution on the C 1 -C 6 alkyl and R 3 are selected from halogen, NR 5 R 6 , or OR 8 , where R 5 and R 6 are defined above and R 8 is H or C 1 -C 4 alkyl; or
v) R 2 is methyl or halo substituted methyl, wherein the halo substituted methyl optionally is CF 2 H or CF 3 .

3. The process of claim 1 , wherein:
i) the amine product of structural formula (I) is:

wherein R 9 is H, Cl, Br, F, CH 3 , CF 3 , CN, SO 2 , —OCH 3 , —C(O)CH 3 , or NO 2 , and the ketone substrate of structural formula (II) is:

wherein R 9 optionally is in the para position on the phenyl ring;
ii) the amine product of structural formula (I) is (S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine:

and the ketone substrate of structural formula (II) is 1-(4-bromophenyl)-2,2,2-trifluoroethanone:

iii) the amine product of structural formula (I) is (S)-2,2,2-trifluoro-1-p-tolylethanamine:

and the ketone substrate of structural formula (II) is 2,2,2-trifluoro-1-p-tolylethanone:

iv) the amine product of structural formula (I) is (S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine:

and the ketone substrate of structural formula (II) is 2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanone:

v) the amine product of structural formula (I) is:

and the ketone substrate of structural formula (II) is:

wherein R 7 is optionally substituted C 1 -C 4 alkyl, and R 10 is R 9 defined above;
vi) the amine product of structural formula (I) is (R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate:

and the ketone substrate of structural formula (II) is ethyl 3-oxo-3-(pyrindin-2-yl)propanoate:

vii) the amine product of structural formula (I) is:

and the ketone substrate of structural formula (II) is:

wherein R 11 is halogen, OH, —C(O)R 4 , —OC(O)R, or NR 6 R 7 , wherein R 4 , R 5 , R 6 , R 7 , R 7 and R 10 are defined above; or
viii) wherein the amine product of structural formula (I) is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

and the ketone substrate of structural formula (II) is 4-chloro-1-(2-fluorophenyl)butan-1-one:

4. The process of claim 1 , wherein:
i) the reaction conditions comprise a temperature of 20° C. to 65° C.;
ii) the reaction conditions comprise a temperature of 40° C. to 65° C.;
iii) the amine product of structural formula (I) is produced in at least 90% enantiomeric excess;
iv) the amine product of structural formula (I) is produced in at least 99% enantiomeric excess;
v) the amino donor is selected from isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine;
vi) the reaction conditions comprise a pH of 7.0 to a pH of 11.0, wherein the pH optionally is maintained by adding isopropylamine;
vii) the reaction conditions comprise a solvent of dimethylsulfoxide (DMSO), wherein the DMSO is between 10% to 40% (v/v); and/or
viii) wherein the amino donor is present at 5 to 25 g/L.

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

6. The process of claim 5 , wherein the amino donor is an amino acid and the carbonyl by-product is a keto acid.

7. The process of claim 6 , 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, wherein:
i) the non-reactive gas optionally is nitrogen gas; or
ii) the amino donor optionally is isopropylamine and the carbonyl by-product is acetone.

8. 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, wherein:
i) the residue difference optionally occurs at one or more residue positions selected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X223, X225, X282, and X284;
ii) 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;
X

Assignees

• Codexis Inc

Inventors

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

Classifications

• Y02P20/52

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

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12P13/04

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

• C12P13/001

  Amines; Imines · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title