	US 12,247,236 B2
	Methods and compositions for the enzymatic production of pseudouridine triphosphate
Alexander Koglin, Santa Fe, NM (US); Michael Humbert, Santa Fe, NM (US); Matthias Strieker, Santa Fe, NM (US); and Grant Eliason, Rio Rancho, NM (US)
Assigned to NATURE'S TOOLBOX, INC., Rio Rancho, NM (US)
Filed by Natures's Toolbox, Inc., Rio Rancho, NM (US)
Filed on Mar. 21, 2024, as Appl. No. 18/612,615.
Application 18/612,615 is a continuation of application No. 18/206,319, filed on Jun. 6, 2023, granted, now 11,959,117.
Application 18/206,319 is a continuation in part of application No. PCT/US2023/064934, filed on Mar. 24, 2023.
Claims priority of provisional application 63/323,145, filed on Mar. 24, 2022.
Prior Publication US 2024/0247294 A1, Jul. 25, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/32 (2006.01); C12N 9/12 (2006.01); C12N 9/88 (2006.01)

CPC C12P 19/32 (2013.01) [C12N 9/1205 (2013.01); C12N 9/1229 (2013.01); C12N 9/88 (2013.01); C12Y 207/01015 (2013.01); C12Y 207/04001 (2013.01); C12Y 402/0107 (2013.01)]

20 Claims

1. A method producing pseudouridine, the method comprising:

protecting a pseudouridine-5′-monophosphate (ΨMP) with a protecting agent that reacts with hydroxyl groups of said ΨMP forming a protected ΨMP;

methylating the protected ΨMP with a methylating agent forming a protected N1-methyl-pseudouridine-5′-monophosphate (protected m1ΨMP);

deprotecting the protected m1ΨMP with a deprotection agent forming a N1-methyl-pseudouridine-5′-monophosphate (m1ΨMP); and

sequentially phosphorylating the m1 ΨMP with a phosphorylating agent forming N1-methyl-pseudouridine-5′-diphosphate (m1ΨDP) and N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).