COMBINED NMR/LC-MS APPROACH FOR STRUCTURAL CHARACTERIZATION OF HEPARINS AND NON-ANTICOAGULANT GLYCOL-SPLIT HEPARINS

Heparin is a sulfated polysaccharide extracted from animal organs, which is successfully used since more than fifty years in clinics as anticoagulant and antithrombotic drug. There is also a growing interest in investigating and exploiting “new” activities of heparin, especially for cancer therapy. Nowadays, a number of non-anticoagulant glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction are being considered as potential anticancer due to a potent inhibitory effect on heparanase, overexpressed by tumors. Additionally, the majority of their biological properties are preserved upon glycol-splitting. It is also worth noting that glycol-splitting of glucuronic acid within antithrombin binding region (ATBR) causes a drastic decrease of heparin anticoagulant activity, helping avoiding undesirable effects when being used for clinical therapy. Gs-derivatives of low molecular weight heparins (LMWHs) are of higher interest due to their better bioavailability. In spite of the numerous studies on their biological activity, detailed structural analysis of gs heparins as well as structure–activity relationship have not been reported yet. Their structural characterization remains a very challenging task, firstly, due to the high microheterogeneity of the starting heparin (significantly depending on tissue and animal sources), and to the various side reactions that can occur under sometimes uncontrolled reaction conditions. In the case of more bioavailable and, consequently, more pharmacologically attractive, low molecular weight heparins (LMWHs) used as starting material, the end groups introduced by the depolymerisation processes induce additional heterogeneity of the corresponding gs-derivatives. Evidently, an appropriate analytical approach is required for a pharmaceutical development of these potential drugs. The present study is focused on the development and application of an efficient analytical approach combining advanced techniques, such as NMR and LC-MS methods, for structural characterization of various gs-heparins and gs-LMWHs. The structural information obtained for gs-samples was expected to provide structural details about the sequences of the parent heparin/LMWH material. The long-term aim of this project includes the exploration of structural basis of heparin-related gs-oligosaccharide sequences for their optimal binding motif with heparanase and other target proteins. In Chapter I the state-of-art of the present topic is overviewed. Structural features, biosynthesis and interaction with proteins of heparin and HS as well as structure and bioactivity of LMWHs are discussed. The potential anticancer and anti-inflammation activity of gs-heparins/gs-LMWHs and the main problems in their preparation and structural analysis are also highlighted. In the last part of the Chapter I the main analytical techniques used for analysis of heparin-related samples (NMR, LC-MS, SEC-TDA) are reviewed. The overall goal of the study and the short-term objectives are described in Chapter II. Chapter III is focused on the development of the combined NMR/LC-MS approach using a gs-derivative of a porcine mucosal heparin (PMH) sample as a model. The potentiality of this method for both characterization and differentiation of gs-products originated from heparins of various tissue and animal sources are discussed in terms of average monosaccharide composition determined by NMR as well as LC-MS qualitative and quantitative analysis of the enzymatically digested samples. The advantages of the combination of glycol-splitting and enzymatic depolymerisation for the characterization of gs-products and starting heparin samples are also discussed. A particular attention has been dedicated to the relevance of the diversity of antithrombin binding region (ATBR) in different heparins and to the length of undersulfated domains within their chains. Molecular weight measurements as well as the dependence of the hydrodynamic properties on the introduced glycol-split units (determined by SEC-TDA) are also described. The possible control and monitoring of glycol-splitting reaction (including side-reactions) using the applied techniques is discussed. The applications of the developed approach for the characterization of the more biologically attractive but often more complex gs-LMWHs (gs-tinzaparin, gs-enoxaparin, gs-dalteparin) are reported in Chapter IV. The developed LC-MS method for direct qualitative profiling of gs-LMWHs (possible because of the molecular weight lower than for unfractionated heparins) is discussed as a possible additional analytical tool for their compositional and structural analysis. The studied susceptibility to periodate oxidation/borohydride reduction of particular end-groups (at both reducing and non-reducing ends) of all the three LMWHs is highlighted. Among those, one of the most unexpected result was the oxidation of the enoxaparin 1,6-anhydro-D-mannosamine-N-sulfate, which was studied in detail. The potential application of the LC-MS method for the heparin sequencing is shown for sequences adjacent to the linkage region tetrasaccharide. General conclusions and future perspectives are discussed in Chapter V.