How can we make a diverse range of aromatic sulfates to explore sulfatase substrate relationships?

Abstract

Antibody–drug conjugates (ADCs) are an emerging class of targeted therapeutics that combine the high specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. A critical component of ADC design is the linker between mAbs and payload, as its chemical stability and cleavage behaviour directly determine therapeutic efficacy, selectivity, and safety. While peptide-based cleavable linkers dominate current clinical ADC platforms, their susceptibility to premature cleavage and instability in certain biological contexts has motivated the exploration of alternative enzymatically cleavable linkers. Arylsulfatase-cleavable linkers have recently attracted increasing interest as a promising alternative due to their excellent efficiency and reported stability in both human and mouse plasma. The cleavage of sulfate is catalysed by lysosomal sulfatases. Sulfatases were expressed in some tumour environments, which further support the selectivity of ADCs. However, systematic study for the characterization of structure and the analysis of properties is still insufficient, which limited the development of sulfatase-cleavable linker in certain degree. In this study, a diverse library of substituted aryl sulfates was synthesised to probe sulfatase–substrate interactions and to establish a robust analytical framework for their characterisation. The synthetic strategy enabled the preparation of aryl sulfates bearing a range of electron-donating and electron-withdrawing substituents at different positions on the aromatic ring, allowing systematic evaluation of electronic and structural effects. The resulting compounds were fully characterised using nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), mass spectrometry (MS), and UV–visible (UV–Vis) spectroscopy.