Glycans or carbohydrates covalently linked to proteins or lipids play important roles in the interactions of bacteria and their hosts. One of the main sugars that are used to form these glycan structures is Uridine 5’-diposho-N-acetylglucosamine (UDP-GlcNAc) which is primarily used as a donor for N-acetylglucosamine (GlcNAc). GlcNAc is important since it is a key component of bacterial cell wall peptidoglycan, as well as other bacterial surface structures. In addition, UDP-GlcNAc can be modified to form other sugar moieties to be used in other bacterial glycan biosynthetic pathways with the use of sugar-modifying enzymes. This study aims to optimize the production of UDP-GlcNAc in a ‘one-pot synthesis’ format to collect on a larger scale to then exploit the versatility to form other UDP-linked sugars that are not commercially available. These enzymatic reactions are tracked by high performance liquid chromatography (HPLC) to visualize the formation of UDP-GlcNAc. This system can then be used to analyze the shift in retention times when modifying UDP-GlcNAc to other UDP-linked sugars. This study also aims to compare the structures of different sugar-modifying enzymes that can impact their substrate promiscuity and selectivity