Fig. 3. Synthesis of sulfo-4-thio-HS analogs.

The starting hemi-A thio-heparosan analog (where m = ~45 and n = 0 or 1; R’ = H or GlcA-C₂H₄-thioamido-fluorescein) contains a hemi-A S-linked structure (boxed). Chemical O-sulfation (top pathway) results in 6-O-sulfo GlcNAc or 2-O-sulfo GlcA residues. Enzymatic N- and 6-O-sulfation, catalyzed by NDST and 6-OST using the sulfo-donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) (bottom pathway), results primarily in 6-O-sulfo and NS, 6-O-sulfo GlcNAc residues. (Highlighted atoms of the major structures: S, yellow; O, red; Y = acetyl or sulfo, aqua; X = sulfo or hydroxyl, blue. Experimental details and sulfation isomer levels are provided in the Supplementary Information).