Combinatorial one-pot chemoenzymatic synthesis of heparin

Carbohydr Polym. 2015 May 20;122:399-407. doi: 10.1016/j.carbpol.2014.10.054. Epub 2014 Nov 7.

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.

Keywords: Bioengineered heparin; Liquid chromatography–mass spectrometry; Nuclear magnetic resonance spectroscopy; One-pot synthesis; United States Pharmacopeia.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticoagulants / chemistry
  • Anticoagulants / pharmacology*
  • Bioengineering / methods
  • Chromatography, High Pressure Liquid
  • Chromatography, Liquid
  • Factor Xa / chemistry
  • Factor Xa / metabolism
  • Heparin / biosynthesis*
  • Heparin / chemistry
  • Heparin / pharmacology
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Prothrombin / antagonists & inhibitors
  • Prothrombin / metabolism
  • Sulfotransferases / metabolism*

Substances

  • Anticoagulants
  • Prothrombin
  • Factor IIa
  • Heparin
  • Sulfotransferases
  • Factor Xa