Accurate assignments of NMR spin systems of monosaccharide residues for homopolysaccharide structural characterization

Abstract

2D COSY and TOCSY experiments are commonly employed for chemical shift assignment of monosaccharide residues in glycans. However, for homopolysaccharides composed of a single type of monosaccharide residues, extensive signal overlap in these ¹H homonuclear correlation spectra often hinders unambiguous chemical shift assignments. DREAMTIME experiment can highly selectively excite specific J-coupled protons in each monosaccharide residue.

Here, we present an integrated NMR approach combining 2D DQF-COSY, TOCSY, and 1D DREAMTIME TOCSY to achieve precise spin system assignments in APS-N, a glucan from Astragalus membranaceus. J-coupled H-1/H-2 protons were initially assigned from well-resolved cross-peaks in the 2D DQF-COSY spectrum. The distinct chemical shifts of the anomeric protons allowed clear differentiation between α- and β- d-glucose residues, which exhibit typical ³J_H-1,H-2-coupling constants of 4.5 and 7.3 Hz, respectively. These essential informations were exploited to guide selective excitation of H-1 and H-2 in DREAMTIME. An extended TOCSY mixing concatenated to the DREAMTIME element enabled efficient magnetization transfer from the initially excited protons (H-1, H-2) across the full spin system. The unique selectivity of 1D DREAMTIME TOCSY allows for the validation of spin system identification from the 2D TOCSY spectrum, while enabling more precise and unambiguous chemical shift assignments for each monosaccharide residue.