# US 9,040,262 B2 · Biocatalysts for ezetimibe synthesis

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- Number: US 9,040,262 B2
- Document ID: US9040262B2
- Status: Granted
- Kind: B2
- Application number: 13/695,856
- Filed: 2011-05-04
- Published: 2013-02-28
- Granted: 2015-05-26
- Earliest claimed: 2010-05-04
- Assignees: Codexis, Inc.
- Applicants: Michael Crowe; Oscar Alvizo; Behnaz Behrouzian; Yong Koy Bong; Steven J. Collier; Anupam Gohel; Jagadeesh Mavinahalli; Naga Modukuru; Emily Mundorff; Derek Smith; Shiwei Song; Wan Lin Yeo
- Inventors: Michael Crowe; Oscar Alvizo; Behnaz Behrouzian; Yong Koy Bong; Steven J. Collier; Anupam Gohel; Jagadeesh Mavinahalli; Naga Modukuru; Emily Mundorff; Derek Smith; Shiwei Song; Wan Lin Yeo
- Web page: https://patentgrep.com/patent/US9040262B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C07K 14/47: from mammals
- C12P 17/10: Nitrogen as only ring hetero atom
- C12Y 101/00: Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y 101/01184: Carbonyl reductase (NADPH) (1.1.1.184)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12P 17/14: Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)}
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present disclosure relates to non-naturally occurring polypeptides useful for preparing Ezetimibe, polynucleotides encoding the polypeptides, and methods of using the polypeptides.

## Claims

### Claim 1

A method for preparing compound of Formula (Ia) in diastereomeric excess
wherein,
X is C or S;
R1 is selected from —H, —F, —Cl, —Br, or —I;
R2 is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protected with a hydroxyl protecting group), —CH2NH2 (optionally protected with a nitrogen protecting group), and any one of the following optionally substituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, cycloalkyl, aryl, heteroaryl, or heterocycle;
R3 is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protected with a hydroxyl protecting group), —CH2NH2 (optionally protected with a nitrogen protecting group), and any one of the following optionally substituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, cycloalkyl, aryl, heteroaryl, or heterocycle;
R4 is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protected with a hydroxyl protecting group); and
R5 is selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protected with a hydroxyl protecting group), —CH2NH2 (optionally protected with a nitrogen protecting group), and any one of the following optionally substituted groups: alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, cycloalkyl, aryl, heteroaryl, or heterocycle;
said method comprising:
contacting a compound of Formula (IIa), wherein, X, R1, R2, R3, R4, and R5 are as defined above,
with a non-naturally occurring ketoreductase polypeptide in the presence of cofactor NADPH or NADH under suitable reaction conditions, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and the following amino acid residues: R at position X40; I or L at position X153; A, or P at position X190; A, C, N, S, or T at position X196; F or W at position X199; and I at position X206.

### Claim 2 (depends on 1)

The method claim 1 in which R2 and R3 are each independently selected from —H, —F, —Cl, —Br, —I, —CN, —OH (optionally protected with a hydroxyl protecting group), —CH2NH2 (optionally protected with a nitrogen protecting group), and any one of the following optionally substituted groups: —(C1-C6)alkyl, —(C1-C6)alkoxy, —(C1-C6)alkenyl, —(C1-C6)alkenoxy, —(C1-C6)alkynyl, —(C1-C6)alkynoxy, —(C1-C6)cycloalkyl, or a heterocycle, having from 1 to 4 carbon atoms and 1 to 2 hetero atoms.

### Claim 3 (depends on 1)

The method claim 1 in which any one or more of R2, R3, or R5 is an —OH or an —OH group protected with a hydroxyl protecting group.

### Claim 4 (depends on 1)

The method of claim 1 in which R1 is —F, R4 is —H, and R5 is —H.

### Claim 5 (depends on 1)

The method of claim 1, wherein the compound of Formula (Ia) is compound (1)
and the compound of Formula (IIa) is compound (2)

### Claim 6 (depends on 5)

The method of claim 5 in which the suitable reaction conditions comprise a compound (2) loading of at least 100 g/L and an engineered polypeptide concentration of about 0.1-3.0 g/L.

### Claim 7 (depends on 5)

The method of claim 5 in which the suitable reaction conditions comprise a co-solvent system comprising an aqueous buffer solution and IPA, wherein the IPA concentration is about 55-75% (v/v).

### Claim 8 (depends on 5)

The method of claim 5 in which the method further comprises converting NADP+ to NADPH with a cofactor recycling system, wherein the cofactor recycling system comprises an NADPH or NADH cofactor loading of about 0.03-0.5 g/L, IPA at a concentration of about 55-75% (v/v), and the non-naturally occurring ketoreductase polypeptide.

### Claim 9 (depends on 5)

The method of claim 5 in which the suitable reaction conditions comprise:
(1) substrate loading of about 50-200 g/L compound (2);
(2) polypeptide concentration of about 1.5-2.5 g/L;
(3) NADPH cofactor concentration of about 0.1-0.2 g/L;
(4) a co-solvent solution of an aqueous buffer and about 60-70% (v/v) IPA;
(5) about pH 6.6-7.0; and
(6) temperature of about 28-30° C.

### Claim 10 (depends on 5)

The method of claim 5 in which the suitable reaction conditions comprise a substrate loading of about 100 g/L compound (2) and wherein the method results in at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater conversion of compound (2) to compound (1) in 24 h.