Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments

Abstract

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.

Fig. 1

Simplified formula of a representative ATBR-containing chain of porcine mucosal heparin (a) and the corresponding glycol-split derivative (b) obtained by periodate oxidation and borohydride reduction of nonsulfated glucuronic (G/G′) and iduronic (I) residues, to generate gsG/gsG′, and gsI [13] LR = linkage region [23] at the “reducing” end (RE). The biochemical formula of ATBR is indicated in structure (a), starting with a residue I that is not part of the pentasaccharidic active site, but most often precedes it. Structure (a) is adapted from [22]; a major modification involves the A_NS,6S residue at the non-reducing end (NRE), assumed to be the consequence of cleavage (by an endo-β-D-glucosidase) of a G-A_NS6S glycosidic bond in the chains of the heparin proteoglycan precursor (“macromolecular heparin”). The structure of the “full” linkage region LR is reported in (c) and (d) for heparin and RO-heparin, respectively, where G_LR is the G residue in the LR, Gal1 and Gal2 are two galactose residues, Xyl is xylose, Ser is serine [23]. The two glycol-split residues (gsG_LR and gsXyl) in the LR of gs-heparin were not previously described (see text). Circled residues in the unmodified heparin structure (a) and (c) highlight unsubstituted diol groups susceptible of glycol-splitting

Fig. 2

Simplified structures of tinzaparin, enoxaparin, and dalteparin Internal sequences are indicated as substantially the same as in non-depolymerized heparin. The end groups susceptible of being modified by periodate oxidation or borohydride reduction are circled

Fig. 3

Anomeric (a) and “ring” (b) regions of the ¹H/¹³C HSQC NMR spectra of RO-tinzaparin (red spots) superimposed on the spectrum of the corresponding unmodified LMWH (blue spots) Symbols for typical end-group signals are framed with rectangles A – glucosamine, A* – N-sulfate-glucosamine-3-O-sulfate, ΔU2S – 4,5-unsaturated 2-O-sulfated uronic acid, I/G – iduronic and glucuronic acids, G_LR – glucuronic acid of the linkage region sequence, gsI/gsG – glycol-split iduronic and glucuronic acids, gsU – glycol-split uronic acids, Gal – galactose, Xyl – xylose, Ser - serine

Fig. 4

LC-MS profiles of enoxaparin (a), RO-enoxaparin (c) and the corresponding expanded chromatograms (b, d) LC conditions: column C18 100 x 2.1 mm,.2.6 μm; eluents A and B – 10 mM DBA and 10 mM CH₃COOH in H₂O-CH₃CN = 9:1 (v/v) and CH₃CN, respectively; gradient: 0 min – 0%B, 5 min – 0%B, 130 min – 35%B, 140 min – 70%B, 145 min – 70%B, 148 min – 0%B, 170 min – 0%B; flow rate – 0.25 ml/min

Fig. 5

Terminal residues at the non-reducing end (NRE) and at the reducing end (RE) confirmed or found for unmodified and RO-LMWHs NOTE: Since structures have been confirmed or elucidated by MS spectroscopy, the anionic groups (COOH, SO₃H) are represented in their undissociated form

Fig. 6

LC-MS chromatogram of the heparinase-digest of RO-enoxaparin LC conditions: column C18 100 x 2.1 mm, 2.6 μm; eluents A and B – 10 mM DBA and 10 mM CH₃COOH in H₂O-CH₃OH = 9:1 (v/v) and CH₃OH, respectively; 0 min – 17% B, 10 min – 17%B, 30 – 42%, 50 min – 50% B, 65 min – 90%, 75 min – 90%B, 76 min – 17% B, 95 min – 17 %B; flow rate – 0.1 ml/min; injection volume – 5 μl. Sodium borohydride reduction was used to distinguish between internal gs-unit and alditol end residue. For example, the double-charged ion with m/z 497.02 could correspond to both ΔU4,4,0,1gs and ΔU4,4,0-ol. After sodium borohydride reduction two peaks with m/z values were observed: a) m/z 498.02, indicating that this oligosaccharide containing one internal gs-unit (gsU), corresponds to the structure ΔU_S-A_NS6(S)-gsU-A_NS6(S), where only one of the two sulfate groups within parenthesis is present; b) unmodified m/z 497.02, which should have an internal 2-O-sulfated uronic acid (U_S) and corresponds to ΔU_S-A_NS-U_S-A.ol_NS structure. The additional NaBH₄ reduction of the RO-enoxaparin digest proved also the structure assigned to the minor tetrasaccharides terminating with an uronic acid remnant (ΔU4,4,1,1gs-R, m/z 577.03 for [M-2H]²⁻ ion form, and ΔU4,5,0,1gs-R, m/z 596.01 for [M-2H]²⁻ ion form), for which the m/z values remained constant after the additional reduction