Characterizing the microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1H and 15N NMR chemical shift analysis.

Heparin and heparan sulfate (HS) are members of a biologically important group of highly anionic linear polysaccharides called glycosaminoglycans (GAGs). Because of their structural complexity, the molecular-level characterization of heparin and HS continues to be a challenge. The work presented herein describes an emerging approach for the analysis of unfractionated and low molecular weight heparins, as well as porcine and human-derived HS. This approach utilizes the untapped potential of (15)N NMR to characterize these preparations through detection of the NH resonances of N-sulfo-glucosamine residues. The sulfamate group (1)H and (15)N chemical shifts of six GAG microenvironments were assigned based on the critical comparison of selectively modified heparin derivatives, NMR measurements for a library of heparin-derived oligosaccharide standards, and an in-depth NMR analysis of the low molecular weight heparin enoxaparin through systematic investigation of the chemical exchange properties of NH resonances and residue-specific assignments using the [(1)H,(15)N] HSQC-TOCSY experiment. The sulfamate microenvironments characterized in this study include GlcNS(6S)-UA(2S), ΔUA(2S)-GlcNS(6S), GlcNS(3S)(6S)-UA(2S), GlcNS-UA, GlcNS(6S)-red(α), and 1,6-anhydro GlcNS demonstrating the utility of [(1)H,(15)N] HSQC NMR spectra to provide a spectroscopic fingerprint reflecting the composition of intact GAGs and low molecular weight heparin preparations.

Sulfamate proton solvent exchange in heparin oligosaccharides: evidence for a persistent hydrogen bond in the antithrombin-binding pentasaccharide Arixtra.

Sulfamate groups (NHSO(3)(-)) are important structural elements in the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). In this work, proton nuclear magnetic resonance (NMR) line-shape analysis is used to explore the solvent exchange properties of the sulfamate NH groups within heparin-related mono-, di-, tetra- and pentasaccharides as a function of pH and temperature. The results of these experiments identified a persistent hydrogen bond within the Arixtra (fondaparinux sodium) pentasaccharide between the internal glucosamine sulfamate NH and the adjacent 3-O-sulfo group. This discovery provides new insights into the solution structure of the Arixtra pentasaccharide and suggests that 3-O-sulfation of the heparin N-sulfoglucosamine (GlcNS) residues pre-organize the secondary structure in a way that facilitates binding to antithrombin-III. NMR studies of the GlcNS NH groups can provide important information about heparin structure complementary to that available from NMR spectral analysis of the carbon-bound protons.

Detection of the 1H and 15N NMR resonances of sulfamate groups in aqueous solution: a new tool for heparin and heparan sulfate characterization.

Sulfamate (NHSO(3)(-)) groups are critically important structural elements of the glycosaminoglycans heparin and heparan sulfate (HS). Experimental conditions are presented for detection of the sulfamate (1)H NMR resonances in aqueous solution. NMR spectra reported for N-sulfoglucosamine (GlcNS) and the synthetic pentasaccharide drug fondaparinux demonstrate the broad utility of the sulfamate group (1)H chemical shifts to reflect differences in molecular structure. The sulfamate protons also provide an efficient route for detection of (15)N chemical shifts through proton-nitrogen correlations measured with the heteronuclear single quantum coherence (HSQC) experiment. The HSQC spectra of GlcNS, fondaparinux, and the low-molecular weight heparin enoxaparin illustrate the power of the (1)H and (15)N chemical shifts of the sulfamate NH groups for the structural characterization of heparin and HS.

NMR methods to monitor the enzymatic depolymerization of heparin.

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used (1)H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the (1)H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion.

Hydroxyl-proton hydrogen bonding in the heparin oligosaccharide Arixtra in aqueous solution.

Heparin is best known for its anticoagulant activity, which is mediated by the binding of a specific pentasaccharide sequence to the protease inhibitor antithrombin-III (AT-III). Although heparin oligosaccharides are thought to be flexible in aqueous solution, the recent discovery of a hydrogen bond between the sulfamate (NHSO3(-)) proton and the adjacent 3-O-sulfo group of the 3,6-O-sulfated N-sulfoglucosamine residue of the Arixtra (fondaparinux sodium) pentasaccharide demonstrates that definable elements of local structure are accessed. Molecular dynamics simulations of Arixtra suggest the presence of additional hydrogen bonds involving the C3-OH groups of the glucuronic acid and 2-O-sulfo-iduronic acid residues. NMR measurements of temperature coefficients, chemical shift differences, and solvent exchange rate constants provide experimental confirmation of these hydrogen bonds. We note that the extraction of rate constants from cross-peak buildup curves in 2D exchange spectroscopy is complicated by the presence of radiation damping in aqueous solution. A straightforward model is presented that explicitly takes into account the effects of radiation damping on the water proton relaxation and is sufficiently robust to provide an accurate measure of the proton exchange rate between the analyte hydroxyl protons and water.

Reversed-phase ion-pair ultra-high-performance-liquid chromatography-mass spectrometry for fingerprinting low-molecular-weight heparins.

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC-MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC-MS fingerprints are presented and compared for tinzaparinand enoxaparin.

A closer look at the nitrogen next door: 1H-15N NMR methods for glycosaminoglycan structural characterization.

Recently, experimental conditions were presented for the detection of the N-sulfoglucosamine (GlcNS) NHSO(3)(-) or sulfamate (1)H and (15)N NMR resonances of the pharmaceutically and biologically important glycosaminoglycan (GAG) heparin in aqueous solution. In the present work, we explore further the applicability of nitrogen-bound proton detection to provide structural information for GAGs. Compared to the detection of (15)N chemical shifts of aminosugars through long-range couplings using the IMPACT-HNMBC pulse sequence, the more sensitive two-dimensional (1)H-(15)N HSQC-TOCSY experiments provided additional structural data. The IMPACT-HNMBC experiment remains a powerful tool as demonstrated by the spectrum measured for the unsubstituted amine of 3-O-sulfoglucosamine (GlcN(3S)), which cannot be observed with the (1)H-(15)N HSQC-TOCSY experiment due to the fast exchange of the amino group protons with solvent. The (1)H-(15)N HSQC-TOCSY NMR spectrum reported for the mixture of model compounds GlcNS and N-acetylglucosamine (GlcNAc) demonstrate the broad utility of this approach. Measurements for the synthetic pentasaccharide drug Arixtra® (Fondaparinux sodium) in aqueous solution illustrate the power of this NMR pulse sequence for structural characterization of highly similar N-sulfoglucosamine residues in GAG-derived oligosaccharides.

Glycosaminoglycans: oligosaccharide analysis by liquid chromatography, capillary electrophoresis, and specific labeling.

Glycosaminoglycans (GAGs) are a class of biopolymers that include chondrotin sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid, heparin, and heparan sulfate. The GAGs are linear polysaccharides that are microheterogeneous in composition and polydisperse in size. Because they have the most complex structures, this article is aimed at describing a step-by-step procedure for processing and analyzing heparin and heparan sulfate-derived oligosaccharides, although the basic protocols and procedures apply equally well to other members of the GAG family. The methods described in this manuscript include the preparation of oligosaccharides through enzymatic depolymerization, size fractionation by preparative scale size-exclusion chromatography (SEC), and disaccharide isomer analysis by reverse-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) and capillary electrophoresis (CE).

Getting to know the nitrogen next door: HNMBC measurements of amino sugars.

Long-range ¹H-¹5N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the ¹J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. ¹5N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6-O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the ¹5N chemical shifts of the α anomer are downfield of the ß form. For the N-acetylated sugars, the ß anomer has a unique long-range ¹5N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the ¹5N chemical shift of the glucosamine analogs, 6-O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D2O solution, peaks in the ¹5N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to ²H-¹5N coupling.

The efficient structure elucidation of minor components in heparin digests using microcoil NMR.

The structural complexity and microheterogeneity of the glycosaminoglycans heparin and heparan sulfate make their characterization a daunting task. The methodology described herein utilizes a combination of enzymatic digestion, size-exclusion chromatography, strong anion-exchange HPLC, reverse-phase ion-pair ultrahigh performance liquid chromatography-mass spectrometry, and microcoil NMR for the efficient sequencing of heparin-derived tetrasaccharides. The high mass sensitivity of microcoil NMR makes this technique well suited for the characterization of mass-limited samples removing a bottleneck in the analysis workflow and permitting structural characterization of minor components isolated from a heparin enzymatic digestion. Complete characterization of one tetrasulfonated, five pentasulfonated isomers and two hexasulfonated tetrasaccharide sequences is described. To our knowledge, two of the identified minor tetrasaccharides are unique, and have not been previously reported: IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S) and ΔUA(2S)-GlcNS(6S)-IdoA-GlcNS(6S).

Heparin characterization: challenges and solutions.

Although heparin is an important and widely prescribed pharmaceutical anticoagulant, its high degree of sequence microheterogeneity and size polydispersity make molecular-level characterization challenging. Unlike nucleic acids and proteins that are biosynthesized through template-driven assembly processes, heparin and the related glycosaminoglycan heparan sulfate are actively remodeled during biosynthesis through a series of enzymatic reactions that lead to variable levels of O- and N-sulfonation and uronic acid epimers. As summarized in this review, heparin sequence information is determined through a bottom-up approach that relies on depolymerization reactions, size- and charge-based separations, and sensitive mass spectrometric and nuclear magnetic resonance experiments to determine the structural identity of component oligosaccharides. The structure-elucidation process, along with its challenges and opportunities for future analytical improvements, is reviewed and illustrated for a heparin-derived hexasaccharide.