Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-β-d-glucosamine or N-sulfated 1,6-anhydro-β-d-mannosamine residues

Mascellani, Giuseppe; Guerrini, Marco; Torri, Giangiacomo; Liverani, Lino; Spelta, Franco; Bianchini, P

doi:10.1016/j.carres.2006.12.010

Cited by 30 publications

(30 citation statements)

References 16 publications

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“…This process affords a polydisperse mixture of LMWH chains having unnatural, unsaturated uronate residues at their non-reducing ends and unnatural 1,6-anhydro amino sugar residues at the reducing ends of some of their polysaccharide chains. 10,11 …”

Section: Introductionmentioning

confidence: 99%

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Zhang²,

et al. 2012

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Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography and by chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycerSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

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Section: Introductionmentioning

confidence: 99%

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Zhang²,

et al. 2012

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“…5 This chemical method used in the manufacturing of enoxaparin can modify its structure, not only changing the polydispersity and sequence heterogeneity of LMWH but also introducing unnatural saccharide residues, such as unsaturated uronic acid residues at the nonreducing ends and unnatural 1,6-anhydro ring structures at the reducing ends of some of the enoxaparin’s chains. 7,8 …”

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confidence: 99%

Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Steppich

Wang³

et al. 2014

Anal. Chem.

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Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-O-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production.

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“…In fact, the observed values are similar to those of some disaccharides with 1,6-anhydro-terminal units (1,6aM NS 5.57 ppm and 1,6aA NS 5.61 ppm). 15 The anomeric 1,6aM NS signal showed a notable intensity decrease after 5 h of reaction, and after 20 h it was present only in trace amount (Fig. 2).…”

Section: Resultsmentioning

confidence: 90%

“…The absence of A NS -G disaccharide unit within the isolated tetrasaccharides permitted to monitor the anomeric signal of 1,6aM NS by monodimensional 1 H NMR spectroscopy. In the case of more complex fractions or unfractionated enoxaparin, the 1,6aM NS signal (at 5.57–5.59 ppm) 13,15 overlaps with the anomeric signal of A NS followed by G (at 5.58 ppm) 18 in the 1 H NMR spectrum. Even if their differentiation is possible by 2D HSQC NMR spectroscopy, it could not permit monitoring reaction kinetics because of much longer acquisition times.…”

Section: Resultsmentioning

confidence: 96%

“…Enoxaparin oligosaccharides bear various amino sugars at their reducing end (RE): glucosamine N -sulfate (A NS(6S) , ~9% of total amino sugar content 13 ) and unnatural residues formed during depolymerisation of heparin under alkaline conditions 14 , namely mannosamine N -sulfate (M NS(6S) ), 1,6-anhydro-glucosamine N -sulfate (1,6aA NS ) and 1,6-anhydro-mannosamine N -sulfate (1,6aM NS ), about 3, 2 and 2.5% of total amino sugar content 13 ). It was shown that unlike for 1,6aA NS residues, the NMR cross peaks of its epimer 1,6aM NS 13,15 dissappeared in the HSQC spectrum of enoxaparin upon glycol-splitting. 12 Interestingly, LC/MS analysis of both intact gs-enoxaparin and its heparinase-digest indicated the generation of unknown structures by glycol-splitting.…”

Section: Introductionmentioning

confidence: 99%

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Susceptibility of enoxaparin reducing end amino sugars to periodate oxidation

Алексеева

Elli

Cosentino

et al. 2014

Carbohydrate Research

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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 characterisation 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 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.

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Characterization of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-β-d-glucosamine or N-sulfated 1,6-anhydro-β-d-mannosamine residues

Cited by 30 publications

References 16 publications

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Susceptibility of enoxaparin reducing end amino sugars to periodate oxidation

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