Detection of the ¹H and ¹⁵N NMR Resonances of Sulfamate Groups in Aqueous Solution: A New Tool for Heparin and Heparan Sulfate Characterization

Abstract

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.

Unravelling Glycobiology by NMR Spectroscopy

“…The three isotopes with magnetically active nuclei spin-½ mostly studied in glycobiology NMR are 1 H, 13 C, and 15 N. Each one has its own magnetic relative susceptibility (Table 1), its own precessional frequency (Larmor frequencies at 800 MHz for 1 H, for example, in Table 1), relaxation properties, and principally differential atomic positions within a molecule. These differenced localizations ultimately provide useful information concerning structural features, in the atomic perspective, and dynamic properties, of specific regions or sites within a molecule.…”

“…The side-chain amino group of this amino acid is responsible to donate the NH2 group to the amino sugar during hexosamine biosynthesis in the cytosol. Through this in vivo labeling method, 15 N-HSQC spectra became recordable for a couple of few hundred micrograms of GAG isolated from cellular sources [10]. On the other hand, as standard GAGs can be readily available from suppliers, the HSQC spectra using 15 N-isotope at natural abundance proved to be successfully recordable at 15 mg/ml concentration-samples within just a couple of hours of acquisition.…”

“…Through this in vivo labeling method, 15 N-HSQC spectra became recordable for a couple of few hundred micrograms of GAG isolated from cellular sources [10]. On the other hand, as standard GAGs can be readily available from suppliers, the HSQC spectra using 15 N-isotope at natural abundance proved to be successfully recordable at 15 mg/ml concentration-samples within just a couple of hours of acquisition. 15 N-HSQC spectrum has been proved to be very straightforward in structural characterization of GAGs in solution ( Figure 4).…”

“…On the other hand, as standard GAGs can be readily available from suppliers, the HSQC spectra using 15 N-isotope at natural abundance proved to be successfully recordable at 15 mg/ml concentration-samples within just a couple of hours of acquisition. 15 N-HSQC spectrum has been proved to be very straightforward in structural characterization of GAGs in solution ( Figure 4). Figure 4C, and further more downfield 15 N-chemical shift of 6-sulfated units, Figure 4A, and B), and adjacent uronic acid type (glucuronic acid at upfield 1 H-chemical shifts, Figures 4A-C, as opposed to iduronic acid at the more downfield 1 H-chemical shifts, Figure 4D) can be easily determined through this method [10].…”

“…Although the 1 H-15 N cross-peaks from 15 N-HSQC spectra of residual N-acetylated glucosamines (GlcNAc) can be fairly used to quantify the amounts and types of uronic acid units, to which GlcNAc are linked ( Figure 5), the remaining amounts of uronic acids linked to N-sulfated glucosamines (GlcNS) can be missed under normal conditions. Alternative ways to force the protonated states in sulfamate groups is the use of controlled samples at narrow pH range (7.0-8.0) [15], or to slow down the fast 1 H-amide exchange rates recording experiments at very low-temperatures, as detailed next with preliminary results using commercially available sodiated GlcNS as a molecular model to mimic the composing N-sulfated amino sugars in heparins and heparan sulfates.…”