The present invention relates inter alia to methods of biosynthetic production of QS-21, precursors and variants thereof, and to related aspects.

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 disclosure relates to the identification of a novel nucleoside modification, referred to herein as anhydride U, present in tRNA molecules within the cell. The present disclosure further relates to the enzymatic and chemical production of anhydride U as well as methods for detecting and regulating the presence of anhydride U within the cell.