title: Combinatorial one-pot chemoenzymatic synthesis of heparin.
abstract: Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal-derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin's characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Ujjwal
authors.initials: U
authors.lastname: Bhaskar
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Guoyun
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Fu
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Onishi
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Mathew
authors.initials: M
authors.lastname: Suflita
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, USA; Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA. Electronic address: linhar@rpi.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology*; D057005:Bioengineering / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011516:Prothrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D011516:Prothrombin; C025337:Factor IIa; D006493:Heparin; D015238:Sulfotransferases; D015951:Factor Xa
keywords: Bioengineered heparin; Liquid chromatography–mass spectrometry; Nuclear magnetic resonance spectroscopy; One-pot synthesis; United States Pharmacopeia
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