Scheme 2.

Chemoenzymatic synthesis of HS mimetics having two NS domains separated by an NA domain of different length. A) Compound 1 was transformed to 23–27 by repeated enzymatic extension using PmHS2 in combination with UDP‐GlcNAc and UDP‐GlcA step by step, resulting compounds 23–27 and unmodified 1 were capped with GlcNAc‐N₃ to obtain 3–8. B) Compound 1 was functionalized with an alkyne moiety by treatment of N‐hydroxysuccinamide (NHS)‐activated 4‐pentynoic acid in a mixture of 0.3 M NaHCO₃/acetonitrile/MeOH (5/5/1) to give 2. C) Compounds 9–14 with two NS domains separated by an NA domain of defined length were obtained by CuAAC click reaction of 2 with 3–8, respectively.