# US 10,767,202 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 10,767,202 B2
- Document ID: US10767202B2
- Status: Granted
- Kind: B2
- Application number: 16/164,365
- Filed: 2018-10-18
- Published: 2019-02-07
- Granted: 2020-09-08
- 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/US10767202B2

## 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 the compound of formula (I):
wherein, R7 is optionally substituted C1-C4 alkyl, and R10 is H, Cl, Br, F, CH3, CF3, CN, SO2, —OCH3, —C(O)CH3, or NO2,
the process comprising:
contacting a ketone substrate of formula:
wherein R7 and R10 are as defined above,
with a transaminase polypeptide in the presence of an amino donor, wherein said amino donor comprises isopropylamine, under reaction conditions suitable for converting the ketone substrate to the compound of formula (I),
wherein the transaminase polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 74, wherein said transaminase polypeptide comprises a substitution at the position corresponding to residue 223 of SEQ ID NO: 2, and is capable of converting the ketone substrate to the compound of formula (I) at a rate that is increased as compared to SEQ ID NO:2.

### Claim 2 (depends on 1)

The process of claim 1, wherein the ketone substrate is ethyl 3-oxo-3-(pyrindin-2-yl)propanoate:
and the compound of formula (I) is (R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate:

### Claim 3 (depends on 1)

The process of claim 1, wherein the reaction conditions comprise a temperature of 20° C. to 65° C.

### Claim 4 (depends on 3)

The process of claim 3, wherein the reaction conditions comprise a temperature of 40° C. to 65° C.

### Claim 5 (depends on 1)

The process of claim 1, wherein the compound of formula (I) is produced in at least 90% enantiomeric excess.

### Claim 6 (depends on 1)

The process of claim 1, wherein the compound of formula (I) is produced in at least 99% enantiomeric excess.

### Claim 7 (depends on 1)

The process of claim 1, wherein the amino donor is isopropylamine.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

The process of claim 8, 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.

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 9)

The process of claim 9, wherein the carbonyl by-product is acetone.

### Claim 12 (depends on 1)

The process of claim 1, wherein the reaction conditions comprise a pH of 7.0 to a pH of 11.0.

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 1)

The process of claim 1, wherein the reaction conditions comprise dimethylsulfoxide (DMSO) as a solvent.

### Claim 15 (depends on 14)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 1)

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; X122; 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.

### Claim 18 (depends on 17)

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.

### Claim 19 (depends on 17)

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;
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;
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.

### Claim 20 (depends on 17)

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.

### Claim 21 (depends on 20)

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.

### Claim 22 (depends on 17)

The process of claim 17, wherein the transaminase polypeptide has the amino acid sequence of SEQ ID NO: 74.