Challenges and Recent Advances in O ‐Sulfation

Comprehensive Summary O ‐Sulfation is a critical biochemical modification that involves the covalent attachment of a sulfuryl group to hydroxyl groups, functioning as a key regulator in protein activity, glycan function, and metabolic detoxification. In biological systems, this modification is catalyzed by sulfotransferases using 3'‐phosphoadenosine‐5'‐phosphosulfate (PAPS) as the sulfuryl donor, thereby profoundly influencing the structure, function, and biological activity of diverse biomacromolecules. However, the chemical synthesis of organic sulfates faces considerable challenges, including poor stability, limited solubility in organic solvents, and difficulties in regioselective sulfation. Over the past decades, significant progress has been made in the development of sulfating reagents and synthetic strategies. In this review, we comprehensively summarize the development of O ‐sulfation methodologies focusing on three main aspects: (1) chemical sulfation using various reagents, including strong sulfur‐containing acids, sulfur trioxide amine/amide complexes, and sulfate diesters; (2) regioselective sulfation for selective modification of specific hydroxyl groups; and (3) chemoenzymatic strategies that combine chemical synthesis with enzymatic catalysis using PAPS as sulfuryl donor for the efficient and precise construction of functional sulfated molecules. The current challenges and future opportunities are also discussed. Key Scientists Efforts toward the development of sulfating reagents date back to 1926, when Baumgarten reported the first synthesis of SO₃·Py and used it for the O ‐sulfation of ethanol. In the following decades, Newman pioneered O‐ sulfation with concentrated H₂SO₄ in 1950, and Mumma reported DCC‐mediated O ‐sulfation of alcohols with H₂SO₄ in 1966. Turning to sulfation strategies, in 1981, Penny introduced the first protection/deprotection strategy using phenyl chlorosulfate to access masked sulfate diesters. In 2016, Yu demonstrated microwave‐assisted O , N ‐sulfation of HP and HS‐like saccharides, achieving rapid and complete sulfation. This strategy was extended by Cao (2019) to complex O ‐mannose glycans bearing the HNK‐1 epitope. In 2020, Niu established O ‐sulfation via sulfur(VI) fluoride exchange (SuFEx), offering a scalable and mild protection/deprotection platform. In 2025, Li developed late‐stage O ‐sulfation using PNPS as a bioinspired sulfuryl donor, achieving tunable chemoselectivity. In the realm of chemical biology, significant progress has also been made. In 2000, Wong achieved tyrosine sulfation on P‐selectin glycoprotein ligand‐1, revealing the modulatory role of sulfation in glycosyltransferase activity. In 2004, Hung reported the synthesis of heparin oligosaccharides. In 2014, Liu and Linhardt pioneered the chemoenzymatic synthesis of low‐molecular‐weight heparins, enabling cost‐effective production of homogeneous anticoagulant oligosaccharides. In 2018, Li and Wu synthesized a fucosylated chondroitin sulfate nonasaccharide as a potent anticoagulant. The year 2024 witnessed several breakthroughs: Li completed the chemoenzymatic synthesis of a comprehensive sulfated ganglioside glycan library, enabling high‐throughput glycan microarray studies; Huang developed chemoenzymatic synthesis of chondroitin sulfate proteoglycan glycopeptides on solid phase; and Lei, Li, and Yu performed structure‐guided discovery of bile acid derivatives, identifying 3‐sulfated deoxycholic acid as a key activator of MRGPRX4 in cholestatic itch. Finally, in 2025, Ye achieved comprehensive synthesis and anticoagulant evaluation of a diverse fucoidan library.