UC San Diego

The human extracellular endosulfatases Sulf-1 and Sulf-2 remodel heparan sulfate (HS) after its biosynthesis by selectively removing 6-O-sulfate groups from internal glucosamine residues, thereby altering the sulfation patterns that regulate growth-factor signaling. How these enzymes recognize individual sulfate groups within heterogeneous HS remains unresolved. Existing analytical workflows often require depolymerization, which loses the local sequence context on which recognition may depend. A chemical probe that converts the transient Sulf–HS complex into a covalent conjugate could preserve this information for downstream analysis.This thesis establishes the chemical foundation for such a strategy. Type I sulfatases depend on catalytic Cα-formylglycine (FGly), and cyclic aryl sulfamates are reported to cause irreversible, active-site-directed inhibition, although the resulting adduct has not been structurally assigned. A literature cyclic sulfamate inhibitor was reproduced in three steps and used as a reference compound, and a bifunctional aryl azide–cyclic sulfamate probe was synthesized in five steps. The cyclic sulfamate provides the inhibitory scaffold, whereas the aryl azide serves as either a click handle or as a photoaffinity handle under ultraviolet light.Due to limited access to active recombinant Sulf-1 and Sulf-2, both compounds were evaluated against commercial Helix pomatia arylsulfatase as a surrogate FGly-dependent enzyme. Both reduced enzyme activity in a concentration-dependent manner, and neither compound-treated enzyme recovered activity during 96-hour dialysis. These results are consistent with effectively irreversible, mechanism-based inactivation but do not establish the identity or site of covalent modification. Click-mediated fluorescent labeling was inconclusive; however, the experiments defined three operational constraints: sufficient protein loading, avoidance of high-temperature treatment before click labeling, and fluorophore stoichiometry that accounts for residual free probe. Together, the synthetic platform and these operational constraints define the foundation for extending the probe to active Sulfs and pursuing covalent capture of Sulf-bound heparan sulfate.