US 8,273,554 B2 · Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

Claims31

Dependent claims

A recombinant ketoreductase polypeptide capable of converting the substrate 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to the product (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one, which comprises an amino acid sequence that is at least 90% identical to a reference sequence of SEQ ID NO:

130, with the proviso that the amino acid sequence has at the residue corresponding to residue X145 a serine and at the residue corresponding to residue X190 a cysteine.

A recombinant ketoreductase polypeptide comprising a region with an amino acid sequence having at least 90% identity to residues 90 to 211 of a reference sequence of SEQ ID NO:

130, with the proviso that the ketoreductase polypeptide has at the residue corresponding to X145 a serine and at the residue corresponding to residue X190 a cysteine.

Dependencies are inferred from the claim text.

Facts

Application

12/545,034Filed 20 Aug 2009 · us provisional application

Priority

29 Aug 2008

Published

US 2010/0062499 A111 Mar 2010

Granted

25 Sep 2012

Term ends

Not estimatedThe source records don’t support an estimate.

Classification13 codes

C12N 9/0006
Oxidoreductases: acting on CH-OH groups as donors
C12P 7/02
Preparation of oxygen-containing organic compounds: containing a hydroxy group
C12P 17/10
Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms: nitrogen as only ring hetero atom
C12P 17/14
Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms: nitrogen or oxygen as hetero atom and at least one…

Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

Life of this patent

Ketoreductase polypeptides for the stereoselective production of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one

Life of this patent

Abstract

Claims31