Screening enoxaparin tetrasaccharide SEC fractions for 3-O-sulfo-N-sulfoglucosamine residues using [1H,15N] HSQC NMR

Abstract

Heparin and heparan sulfate (HS) are important in mediating a variety of biological processes through binding to myriad different proteins. Specific structural elements along the polysaccharide chains are essential for high affinity protein binding, such as the 3-O-sulfated N-sulfoglucosamine (GlcNS3S) residue, a relatively rare modification essential for heparin's anticoagulant activity. The isolation of 3-O-sulfated oligosaccharides from complex mixtures is challenging because of their low abundance. Although methods such as affinity chromatography are useful in isolating oligosaccharides that bind specific proteins with high affinity, other important 3-O-sulfated oligosaccharides may easily be overlooked. Screening preparative-scale size-exclusion chromatography (SEC) fractions of heparin or HS digests using [(1)H,(15)N] HSQC NMR allows the identification of fractions containing 3-O-sulfated oligosaccharides through the unique (1)H and (15)N chemical shifts of the GlcNS3S residue. Those SEC fractions containing 3-O-sulfated oligosaccharides can then be isolated using strong anion-exchange (SAX)-HPLC. Compared with the results obtained by pooling the fractions comprising a given SEC peak, SAX-HPLC analysis of individual SEC fractions produces a less complicated chromatogram in which the 3-O-sulfated oligosaccharides are enriched relative to more abundant components. The utility of this approach is demonstrated for tetrasaccharide SEC fractions of the low molecular weight heparin drug enoxaparin facilitating the isolation and characterization of an unsaturated 3-O-sulfated tetrasaccharide containing a portion of the antithrombin-III binding sequence.

1H and 15N NMR Analyses on Heparin, Heparan Sulfates and Related Monosaccharides Concerning the Chemical Exchange Regime of the N-Sulfo-Glucosamine Sulfamate Proton

“…Despite the significant concentrations of GlcNS units in both GAG types, 1 H- 15 N cross-peaks derived from this unit have not been detected through the series of 1 H- 15 N HSQC spectra depicted at Figure 2 . Many previous works have, however, observed and identified the 1 H- 15 N cross-peak of the GlcNS unit [ 7 , 16 , 18 , 19 , 20 , 23 , 24 ]. The 1 H- 15 N pair of GlcNS resonates to a far upfield region than GlcNAc in both 1 H and 15 N dimensions, more exactly close to δ H /δ N at 5.5/93.5 ppm [ 16 ].…”

“…Most of these analyses are based on the 1 H and 13 C nuclei. However, a great content of works using the less employed isotope 15 N have been appearing in the literature lately [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. This nucleus can be found at the composing amino sugars of GAGs such as GlcN, GlcNAc, GlcNS and N -acetyl galactosamine.…”

Obtaining the Enoxaparin Sodium Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the “Greenness” of the Purification Stage

“…The analysis of samples for comparison was carried out according to the internal specification developed based on the pharmacopoeial monograph, as well as according to specific methods. Since the structure requires accurate, painstaking analysis of saccharide units and their sequence, additional methods of analysis of similar structures were introduced [13,14].…”