IP Library › Granted Patent US 12,188,069
Granted Patent B2
US 12,188,069 · App. 16/966,359 · Granted Jan 7, 2025

Biocatalyst and methods for synthesizing mixed disulfide conjugates of thienopyridine compounds

Inventor: Haoming Zhang (Ann Arbor, MI)
Assignee: The Regents of the University of Michigan
C12P17/167A61K31/4535A61K45/06C12Y106/02004
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Quick Facts
Patent No.
US 12,188,069
App. No.
16/966,359
Granted
Jan 7, 2025
Kind
B2
Abstract

The present invention relates to methods for synthesizing mixed disulfide conjugates of thienopyridine compounds with a genetically engineered variant of cytochrome P450 BM3 or CYP102A1 as a catalyst, and belongs to the field of chemical synthesis.

Claims (21)

1. A mutant CYP102A1 enzyme capable of catalyzing conjugation between 2-oxo thienopyridine and heterocyclic thiols in the presence of a reducing reagent, wherein the reducing agent is NADPH or NADH,

wherein the mutant CYP102A1 enzyme comprises SEQ ID NO: 2 and having substitutions selected from: A82F/L188Q, A82F/L188Q/R47L, A82F/F87V/R47L, A82F/F87V/L188Q, A82F/R47L, A82F/T365N, A82F/F87V/L188Q/H116Q, A82F/F87V/L188Q/K31T, A82F/F87V/L188Q/S56R/A135S, A82F/F87V/L188Q/V299D/1458F/P481H, A82F/W1046A, A82F/L188Q/R47L/F87V/W1046A, A82F/F87V/L188Q/W1046A, A82F/F87V/L188Q/W1046L, and A82F/F87V/L188Q/K31T/W1046A.

2. The mutant CYP102A1 enzyme of claim 1 , wherein the catalyzing of conjugation between 2-oxo thienopyridine and heterocyclic thiols in the presence of a reducing reagent results in the generation of mixed disulfide conjugates of thienopyridine compounds.

3. The mutant CYP102A1 enzyme of claim 1 , wherein the catalyzing of conjugation between 2-oxo thienopyridine and heterocyclic thiols in the presence of a reducing reagent selectively generates cis stereoisomers of the mixed disulfide conjugates of thienopyridine compounds.

4. The mutant CYP102A1 enzyme of claim 1 , wherein the enzyme is encoded by a nucleic acid sequence having at least 99% homology with SEQ ID NO: 1.

5. The mutant CYP102A1 enzyme of claim 1 , wherein the enzyme is encoded by a nucleic acid sequence having at least 100% homology with SEQ ID NO: 3.

6. A method for synthesizing cis stereoisomers of mixed disulfide conjugates of thienopyridine compounds, comprising mixing a 2-oxo thienopyridine moiety, a heterocyclic thiol moeity, and the mutant CYP102A1 enzyme of claim 1 in the presence of a reducing reagent, wherein the reducing agent is NADPH or NADH.

7. The method of claim 6 , wherein the 2-oxo thienopyridine moiety is represented by

wherein R1 is either Chlorine or Fluorine; wherein R2 is H, COOCH3, or COCHCH2CH2.

8. The method of claim 6 , wherein the heterocyclic thiol moiety is represented by R3-SH; wherein R3 is selected from 3-nitropyridine-2-thiol, 2-mercaptopyridine, 2-mercapto-6-methylpyridine, 5-chloropyridine-2-thiol, 2-mercapto-5-trifluoromethyl-pyridine, 3-(trifluoromethyl) pyridine-2-thiol, 2-mercaptopyridine-3-carbonitrile, 4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile, 2-quinolinethiol, 1-amino-3-mercaptoisoquinoline, 6-chloropyridazine-3-thiol, and 2,5-dimethylfuran-3-thiol.

9. The method of claim 6 , wherein the mixing occurs at ambient temperature, wherein the mixing occurs for a time period between twenty and sixty minutes.

10. The method of claim 6 , wherein the mutant CYP102A1 enzyme is comprised within a bacterial cytosolic fraction.

11. The method of claim 6 , wherein the amount of mutant CYP102A1 enzyme is between approximately 0.1 and 1 μM.

12. The method of claim 6 , wherein the mixing results in the generation of approximately 100 mg of cis stereoisomers of mixed disulfide conjugates of thienopyridine compounds per liter of the 2-oxo thienopyridine moiety, the heterocyclic thiol moeity, the mutant CYP102A1 enzyme, and the reducing agent.

13. A method of treating, ameliorating, or preventing a cardiovascular disease in a patient comprising administering to said patient a therapeutically effective amount of a compound generated with the method of claim 6 .

14. The method of claim 13 , wherein said administration is selected from the group consisting of oral administration and intravenous administration.

15. The method of claim 13 , wherein said cardiovascular disease is selected from the group consisting of coronary artery disease, peripheral vascular disease, atherothrombosis, and cerebrovascular disease.

16. The method of claim 13 , wherein said compound reduces aggregation of platelets.

17. The method of claim 16 , wherein said reduces aggregation of said platelets occurs through

irreversible binding to P2Y 12 receptors and/or blocking ADP receptors.

18. The method of claim 13 , further comprising co-administration of at least one agent selected from the group consisting of a HMG-CoA reductase inhibitor, an ACE Inhibitor, a Calcium Channel Blocker, a Platelet Aggregation Inhibitor, a Polyunsaturated Fatty Acid, Fibric Acid Derivative, a Bile Acid Sequestrant, an Antioxidant, a Thrombolytic Agent, and an Antianginal Agent.

Assignments (2)
CONFIRMATORY LICENSE Recorded Jan 23, 2024
From: UNIVERSITY OF MICHIGAN
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 066363/0541 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 18, 2021
From: ZHANG, HAOMING
To: THE REGENTS OF THE UNIVERSITY OF MICHIGAN
Reel/Frame 055313/0603 →
Continuity (2)
Provisional Application 62624494 · Jan 31, 2018
Related Publication 20200370081A1 · Nov 26, 2020
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