# US 9,434,968 B2 · Transaminase reactions

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,434,968 B2
- Document ID: US9434968B2
- Status: Granted
- Kind: B2
- Application number: 14/547,339
- Filed: 2014-11-19
- Published: 2015-03-19
- Granted: 2016-09-06
- Earliest claimed: 2009-06-22
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Gregory Hughes; Paul N. Devine; Fred J. Fleitz; Brendan T. Grau; John Limanto; Christopher Savile; Emily Mundorff
- Web page: https://patentgrep.com/patent/US9434968B2

## CPC

- C12P 17/10: Nitrogen as only ring hetero atom
- C12N 9/1096: {transferring nitrogenous groups (2.6)}
- C12P 13/001: {Amines; Imines}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 17/12: containing a six-membered hetero ring
- C12P 17/182: {Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00)}
- C12Y 206/01: Transaminases (2.6.1)
- Y02P 20/52: using catalysts, e.g. selective catalysts
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

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

## Claims

### Claim 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 R1 is a substituted or unsubstituted aryl or heteroaryl; R2 is a substituted or unsubstituted C1-C6 alkyl, —R3C(O)R4, or —R3OC(O)R5; R3 is an substituted or unsubstituted C1-C4 alkyl; and R4 is H, a substituted or unsubstituted C1-C4 alkyl, NR6R7, or OR8, wherein R5, R6, R7, and R8 are independently H or C1-C4 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.

### Claim 2 (depends on 1)

The process of claim 1, wherein: i) R1 is a substituted or unsubstituted phenyl a substituted or unsubstituted pyridinyl, or a substituted aryl or heteroaryl; ii) the substitution on the C1-C6 alkyl and R3 are selected from halogen, NR5R6, or OR8, where R5 and R6 are defined above and R8 is H or C1-C4 alkyl; or iii) R2 is methyl or halo substituted methyl, wherein the halo substituted methyl optionally is CF2H or CF3.

### Claim 3 (depends on 1)

The process of claim 1, wherein: i) the amine product of formula (I) is:
wherein R9 is H, Cl, Br, F, CH3, CF3, CN, SO2, —OCH3, —C(O)CH3, or NO2, and the ketone substrate of formula (II) is:
wherein R9 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 R7 is substituted or unsubstituted C1-C4 alkyl, and R10 is R9 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 R11 is halogen, OH, —C(O)R4, —OC(O)R5, or NR6R7, wherein R4, R5, R6, R7, R7 and R10 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:

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 1)

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 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;
iii) 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; and optionally 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; X284 is G; X297 is S; X321 is P and X329 is H; or
iii) the transaminase polypeptide has an amino acid sequence that corresponds to the sequence of SEQ ID NO: 58, 72, 80, 86, 96, 98, 100, or 102.

### Claim 9

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, R10 is Cl, Br, F, CH3, CF3, CN, SO2, —OCH3, or NO2,
the process comprising:
(a) contacting a ketone substrate of formula:
wherein R11 is halogen, OH, —C(O)R4, —OC(O)R5, or NR6R7, wherein R4, R5, R6, and R7 is H or C1-C4 alkyl, and R10 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 95% sequence identity to SEQ ID NO:74; and
(b) cyclizing the amine product under suitable conditions to form the compound of formula (III).

### Claim 10 (depends on 9)

The process of claim 9, 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.