The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5′-triphosphate (m1ΨTP).

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine (?) and its variants, such as N1-methyl-pseudouridine-5-triphosphate (m1?TP).

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine (?) and its variants, such as N1-methyl-pseudouridine-5-triphosphate (m1?TP).

Provided herein is a one-pot method of synthesising a C-nucleoside-5-monophosphate such as pseudouridine-5-monophosphate, from an N-nucleoside using a multi-step enzymatic pathway. The method comprises reacting the N-nucleoside with a phosphate source and nucleoside phosphorylase to form a pentose-1-phosphate intermediate, reacting the pentose-1-phosphate intermediate with a phosphomutase to form pentose-5-phosphate intermediate, and reacting the pentose-5-phosphate intermediate with a nucleoside-5-phosphate C-glycosidase to form the C-nucleoside-5-monophosphate.

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine () and its variants, such as N1-methyl-pseudouridine-5-triphosphate (m1TP).

The present invention includes novel systems, methods, and compositions for the enzymatic/chemical production of pseudouridine () and its variants, such as N1-methyl-pseudouridine-5-triphosphate (m1TP).