Methods and compositions for the enzymatic production of pseudouridine triphosphate

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).