Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Automated summary

Enoxaparin, a widely used antithrombotic drug with a highly microheterogeneous structure, was studied in this research to identify 16 saturated and 23 unsaturated tetramers, revealing a unique enoxaparin signature and the structural relationship between non-reducing ends and biosynthesis of parent heparin. Additionally, glucosamine tetrasaccharides at the NRE may be associated with heparanase hydrolytic action, with high sulfation degree and 3-O-sulfation characteristic for both types of NRE.