biostudies-literatureStancanelli EEshelman Institute for Innovation, University of North CarolinaNHLBI NIH HHSNational Institutes of HealthNational Science Foundation1207-1214https://www.ebi.ac.uk/biostudies/studies/S-EPMC9294993biostudies-literatureUnknown17(5)Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of <b>H/C 12-mer 5</b> is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosacchaACS chemical biologyChemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.PMC9294993R01 HL158932R01 HL094463HL144970GM134738R01 HL144970HL094463HL158932GM123792DMR-1933525Liu JLi JArnold KPadmanabhan APham TQSu GStancanelli ELiu WWander RPagadala VWang ZKanack AXu YfalseChemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of <b>H/C 12-mer 5</b> is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccha2022-01-01T00:00:00Z2022 May2025-04-25T23:27:05.638Z2025-04-06T09:21:14.082ZS-EPMC92949933542077710.1021/acschembio.2c00146