iduronate and metaperiodate

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

Topics: Borohydrides; Chromatography, Reverse-Phase; Dalteparin; Enoxaparin; Glucuronic Acid; Heparin Lyase; Heparin, Low-Molecular-Weight; Hydrolysis; Iduronic Acid; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Periodic Acid; Tinzaparin

There is a growing interest on glycol-split low-molecular weight heparins (gs-LMWHs), obtained by periodate oxidation of LMWHs, optionally followed by borohydride reduction, as potential anticancer and anti-inflammatory drugs. However, their structural characterization is still a challenging task, mainly because of the high microheterogeneity of the starting material. In addition, susceptibility to oxidation of some end-groups of LMWHs induces additional heterogeneity, making analysis of gs-LMWHs more complex. In our previous study we showed that 1,6-anhydro-d-mannosamine N-sulfate was affected by periodate, while its epimer 1,6-anhydro-d-glucosamine N-sulfate was resistant. In order to understand the apparently anomalous behavior of terminal 1,6-anhydro-d-mannosamine N-sulfate residues, in the present work we have studied by NMR spectroscopy and LC/MS the behavior of the reducing end amino sugar residues of the tetrasaccharides, isolated from the LMWH enoxaparin, in the presence of periodate. Their molecular mechanics conformational characterization has been also performed. We have shown that the C(2)-C(3) bond of the 1,6-anhydro-d-mannosamine residue can be split by periodate despite the N-substitution. Moreover, we have found that both terminal d-mannosamine N-sulfate and d-glucosamine N-sulfate, lacking the 1,6-anhydro-bridge, can be also oxidized by periodate but with a significantly lower rate. The present results suggest that the cis-e-/a-position of OH and NHSO3(-) groups of N-sulfated 1,6-anhydro-d-mannosamine is not the only factor that makes these end residues susceptible to the oxidation. The 1,6-anhydro-bridge that 'blocks' the ring conformation appears another crucial factor for oxidation to occur. Moreover, we have shown that controlling the reaction time could permit to selectively split non-sulfated iduronic acids of enoxaparin chains without oxidizing terminal amino sugar residues, a finding that may be useful to obtain more structurally homogeneous gs-LMHWs.

Topics: Amino Sugars; Enoxaparin; Heparin, Low-Molecular-Weight; Hexosamines; Iduronic Acid; Magnetic Resonance Spectroscopy; Molecular Conformation; Oligosaccharides; Oxidation-Reduction; Periodic Acid; Sulfates