Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis

Abstract

A series of N-acetyl-chitooligosaccharides (GlcNAc)(1-6) have been studied by a nuclear magnetic resonance (NMR) method, diffusion ordered spectroscopy (DOSY). DOSY has also been applied to two additional synthetic related oligosaccharides [GlcNH(2)-(GlcNAc)(4) and GlcNH(2)-(GlcNAc)(2)-GlcNAcSO(3)Na]. A plot of the log of the determined diffusion coefficients (logD) of (GlcNAc)(n) versus the log of molecular weight was linear (6 points, R(2) = 0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within 10% error. The processed NMR data of all the chitooligosaccharides was also plotted in a polyacrylamide gel-like format to aid visual interpretation. Moreover, the logD value of the NMR signal resonances of a chitin-binding protein (hevein) changed as a function of a given titrated ligand, (GlcNAc)(6). Evidence for a 2:1 hevein:(GlcNAc)(6) complex is detected by DOSY at high hevein:(GlcNAc)(6) ratios. This data is consistent with published analytical ultracentrifugation and isothermal titration calorimetry data. A 1:1 complex is preferred at higher ligand concentrations. DOSY can complement size exclusion chromatography in carbohydrate research with the advantage that oligosaccharides are more readily detected by NMR.

Diffusion-ordered nuclear magnetic resonance spectroscopy for analysis of DNA secondary structural elements

“…to provide MW information for samples being analyzed [32,[34][35][36][37][38][39]. Different forms of diffusion NMR experiments have been applied for diverse varieties of systems including inorganic materials [37,[40][41][42], organic molecules [17,23,24,28,[43][44][45][46][47][48][49] and polymers of different kinds (inorganic and organic polymers, polysaccharides, proteins) [21,37,[50][51][52][53][54][55][56][57][58][59][60][61][62]. Application for nucleic acids has been rather limited because the well-defined double helical structures have been investigated for most cases.…”

Mimicking Chitin: Chemical Synthesis, Conformational Analysis, and Molecular Recognition of the β(1→3) N‐Acetylchitopentaose Analogue

“…1-2 mm), the molecule behaved as a monomer, since at these concentrations the diffusion coefficient was basically identical to that previously reported for the chitin pentasaccharide. [11] Thus, the experimental data can be unambiguously correlated with a single species.…”

“…To properly evaluate the NOEs, and to discard the possibility of aggregates at the working concentration, NMR diffusion order spectroscopy (DOSY) experiments [11] were used to confirm the aggregation state of the molecules for the employed experimental conditions. It was confirmed that, at the working concentration (ca.…”

Assessing Carbohydrate–Carbohydrate Interactions by NMR Spectroscopy: The Trisaccharide Epitope from the Marine Sponge Microciona prolifera

“…[11] Among the NMR methods suitable for analyzing ligand binding, transferred NOE [12] and DOSY [13,14] are particularly useful for detection of binding based on changes in the rotational (TR-NOESY) and translational (DOSY) properties of the ligands upon binding to large receptors. These techniques are particularly useful in the medium-to-low affinity range, and, therefore it is speculated on whether they could be adopted to detect weak-affinity carbohydrate-carbohydrate interactions such as those described above.…”

“…Each measured log D value is a weighted average of the diffusion coefficients of receptorbound and free ligand. Therefore, ligands with NMR signals that show increases in apparent molecular weight signify a receptor-bound population, whereas ligands that do not bind to the receptor exhibit no change in log D. This approach has previously been used to characterize the binding of a small protein receptor (hevein) to different oligosaccharides, [13] to detect the binding of plant extracts to the lectin domain of cholera toxin, [14] and to characterize the interactions of organic ligands in suspensions of titanium dioxide nanoparticles. [15] In particular, pulsed field gradient NMR spectroscopy is an in situ methodology that enables the resolution of mixtures of different species in solution solely on the basis of their differentiated translational mobility.…”