Ketoreductase enzymes for the synthesis of 1,3-diol substituted indanes

Patent metadata

Field	Value
Publication number	US-2026078354-A1
Application number	US-202318880970-A
Country	US
Kind code	A1
Filing date	Jul 5, 2023
Priority date	Jul 8, 2022
Publication date	Mar 19, 2026
Grant date	—

Abstract

The present disclosure provides ketoreductase enzymes having improved enzymatic properties including the capability of reducing hydroxy indanones to provide diastereomerically pure 1,3-indane diols useful for the synthesis of belzutifan. Also provided are polynucleotides encoding the ketoreductase enzymes, and host cells capable of expressing the ketoreductase enzymes. A purification procedure for isolating the ketoreductase enzymes is also provided.

First claim

1 . A polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2.

2 . The polypeptide according to claim 1 , wherein the amino acid sequence has at least a 95% sequence identity to SEQ ID NO: 2.

3 . The polypeptide according to claim 1 , wherein the amino acid sequence has at least a 98% sequence identity to SEQ ID NO: 2.

4 . The polypeptide according to claim 1 , wherein the amino acid sequence consists of SEQ ID NO: 2.

5 . The polypeptide according to claim 1 , consisting of SEQ ID NO: 2.

6 . The polypeptide according to claim 1 , wherein at least at least one of the following conditions are satisfied:
(a) amino acid (aa) residue 2 of SEQ ID NO:2 is other than alanine, or (b) aa residue 11 of SEQ ID NO:2 is other than glutamic acid.

7 . A polynucleotide encoding the polypeptide according to claim 1 .

8 . The polynucleotide of claim 7 , wherein the polynucleotide comprises SEQ ID NO:3.

9 . The polynucleotide of claim 7 , wherein at least one of the following conditions are satisfied:
(a) the triplet codon encoding the amino acid residue at position 2 of the polypeptide is other than GCT; (b) the triplet codon encoding the amino acid residue at position 3 of the polypeptide is other than AAA; (c) the triplet codon encoding the amino acid residue at position 4 of the polypeptide is other than ATC; or (d) the triplet codon encoding the amino acid residue at position 11 of the polypeptide is other than GAA.

10 . An expression vector comprising the polynucleotide according to claim 7 , operably linked to one or more control sequences suitable for directing expression of the encoded polypeptide in a host cell.

11 . The expression vector of claim 10 , wherein the control sequence comprises a promoter.

12 . The expression vector of claim 11 , wherein the promoter comprises an E. coli promoter.

13 . A host cell comprising the expression vector of claim 11 .

14 . The host cell of claim 12 , wherein said host cell is E. coli.

15 . A process for precipitating proteins from a cell lysate containing a polypeptide comprising an amino acid sequence having at least a 90% sequence identity to SEQ ID NO: 2, comprising treating the cell lysate containing the polypeptide to result in a cell lysate composition comprising >20% isopropanol.

16 . The process of claim 15 , wherein the volume percent of isopropanol in the cell lysate composition is about 25%.

Assignees

• Merck Sharp & Dohme Llc

Inventors

• Cahn Jackson Kenai Blender
• Cheung-Lee Wai Ling
• Chun Stephanie W
• Hiraga Kaori
• Kosjek Birgit
• Knight John
• Makarewicz Amanda M
• Mccann Scott
• Moore Jeffrey C
• Verma Deeptak

Classifications

• C12Y101/01

  with NAD+ or NADP+ as acceptor (1.1.1) · CPC title

• C12P11/00

  Preparation of sulfur-containing organic compounds · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title