Transaminase reactions

Patent metadata

Field	Value
Publication number	US-9434968-B2
Application number	US-201414547339-A
Country	US
Kind code	B2
Filing date	Nov 19, 2014
Priority date	Jun 22, 2009
Publication date	Sep 6, 2016
Grant date	Sep 6, 2016

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 a substituted or unsubstituted aryl or heteroaryl; R 2 is a substituted or unsubstituted C 1 -C 6 alkyl, —R 3 C(O)R 4 , or —R 3 OC(O)R 5 ; R 3 is an substituted or unsubstituted C 1 -C 4 alkyl; and R 4 is H, a substituted or unsubstituted C 1 -C 4 alkyl, NR 6 R 7 , or OR 8 , wherein 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 presence of an amino donor under reaction conditions suitable for converting the ketone substrate to the amine product, wherein the transaminase polypeptide has at least 95% sequence identity to SEQ ID NO:74.

2. The process of claim 1 , wherein: i) R 1 is a substituted or unsubstituted phenyl a substituted or unsubstituted pyridinyl, or a substituted aryl or heteroaryl; ii) 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 iii) 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 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 formula (II) is:

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

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

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

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

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

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

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

and the ketone substrate of formula (II) is:

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

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

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

and the ketone substrate of formula (II) is:

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 , R 7 , R 7 and R 10 are defined above; or VIII) wherein the amine product of formula (I) is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

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

4. The process of claim 1 , wherein:
i) the reaction condition comprises a temperature of 20° C. to 65° C.;
ii) the reaction condition comprises a temperature of 40° C. to 65° C.;
iii) the amine product is produced in at least 90% enantiomeric excess;
iv) the amine product 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 condition is from a pH of about 7.0 to a pH of about 11.0, wherein the pH optionally is maintained by adding isopropylamine;
vii) the reaction condition comprises a solvent of dimethylsulfoxide (DMSO), wherein the DMSO is between about 10% to about 40% (v/v); and/or
viii) wherein the substrate 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 is nitrogen gas; or
ii) the amino group donor 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 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;
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 constr

Assignees

• Codexis Inc

Inventors

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

Classifications

• C12P13/001 Primary

  Amines; Imines · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · 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

• C12P17/10 Primary

  Nitrogen as only ring hetero atom · CPC title