Chemoenzymatic Synthesis and Purification of Bioorthogonally Tagged UDP‐GlcNAc and UDP‐GalNAc Analogues

Nucleotide-sugar donors containing bioorthogonal moieties are important tools to study cellular glycosylation. Typically, the acetamide moiety in N-acetylhexosamines such as GlcNAc and GalNAc is replaced by an acylamide with a clickable tag and converted to the corresponding uridine diphosphate analogue. These probes can then be tested for acceptance by glycosyltransferase enzymes in vitro. Lengthy procedures in synthetic chemistry currently limit the availability of bioorthogonal uridine diphosphate (UDP)-sugar analogues. Chemoenzymatic synthesis has proven to be a powerful and effective alternative, and multiple approaches have been published to date. In this protocol, we describe a streamlined method for the generation of bioorthogonal UDP-GlcNAc and UDP-GalNAc analogues. We describe the chemical modification of D-glucosamine and D-galactosamine to incorporate bioorthogonal acylamides, the subsequent one-pot multienzyme conversion to the corresponding UDP-sugar analogues, and reproducible purification. Our approach features the bacterial kinase NahK and human pyrophosphorylase AGX1 as well as a recombinantly expressed AGX1 variant with an expanded substrate profile. The approach further features an inorganic pyrophosphatase and an alkaline phosphatase to improve enzymatic turnover and aid the purification process, respectively. The use of biosynthetic enzymes with substrate promiscuity extends the scope of bioorthogonal nucleotide-sugar analogue structures to aid efforts in chemical glycobiology. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc Alternate Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc from a protected GlcNH₂ or GalNH₂ precursor Basic Protocol 2: Conversion of D-GlcNAc or D-GalNAc analogues to UDP-sugars using analytical- (reaction scouting) and preparative-scale enzymatic synthesis and purification.