Affinity-based covalent sialyltransferase probes enabled by ligand-directed chemistry

Area of Science:

• Biochemistry
• Chemical Biology
• Microbiology

Background:

• Sialyltransferases (ST) are crucial enzymes synthesizing sialylated glycans, vital for mammalian and bacterial functions in health and disease.
• Existing chemical tools for ST study are limited to non-covalent inhibitors, hindering enzyme isolation and profiling.

Purpose of the Study:

• To develop a new class of covalent affinity-based probes (AfBP) for sialyltransferases (ST) using ligand-directed chemistry (LDchem).
• To enable specific labeling, isolation, and profiling of STs in both recombinant and native biological systems.

Main Methods:

• Chemoenzymatic synthesis of CMP-Neu5Ac based probes featuring an O-nitrobenzoxadiazole (O-NBD) warhead.
• Application of ligand-directed chemistry (LDchem) for probe design and synthesis.
• Testing probe specificity and sensitivity against recombinant STs and endogenous ST in live bacteria.

Main Results:

• Developed robust covalent ST AfBPs with a lysine-specific O-NBD warhead exhibiting turn-on fluorescence.
• Demonstrated high specificity and submicromolar sensitivity in labeling various recombinant STs.
• Successfully labeled endogenous lipooligosaccharide ST (Lst) in live Neisseria gonorrhoeae.

Conclusions:

• The novel covalent ST AfBPs provide a powerful and robust platform for ST enzyme profiling.
• This technology facilitates the study of STs in their native environments, including pathogens.
• The developed probes advance the chemical biology toolkit for investigating sialylation pathways.