Application of paramagnetic NMR-validated molecular dynamics simulation to the analysis of a conformational ensemble of a branched oligosaccharide

Abstract

Oligosaccharides of biological importance often exhibit branched covalent structures and dynamic conformational multiplicities. Here we report the application of a method that we developed, which combined molecular dynamics (MD) simulations and lanthanide-assisted paramagnetic NMR spectroscopy, to evaluate the dynamic conformational ensemble of a branched oligosaccharide. A lanthanide-chelating tag was attached to the reducing end of the branched tetrasaccharide of GM2 ganglioside to observe pseudocontact shifts as the source of long distance information for validating the conformational ensemble derived from MD simulations. By inspecting the results, the conformational space of the GM2 tetrasaccharide was compared with that of its nonbranched derivative, the GM3 trisaccharide.

Figure 1

Torsion angle density maps of the combined MD trajectory of (a, b, and c) GM2 tetrasaccahride and (d and e) GM3 trisaccahride [13]. (a) The GalNAc-Gal, (b and d) the Neu5Ac-Gal, and (c and e) the Gal–Glc linkages. The d and e parts of this figure were reproduced by permission of The Royal Society of Chemistry. The definitions of Φ and ψ were used for the GalNAc–Gal and the Gal–Glc linkages, Φ = H1–C1–O'4–C'4 and ψ = C1–O'4–C'4–H'4, and the Neu5Ac–Gal linkage, Φ = C1–C2–O'3–C'3 and ψ = C2–O'3–C'3–H'3.

Scheme 1

Paramagnetic tagging of the GM2 tetrasaccharide.

Figure 2

¹H-¹³C HSQC spectra of the GM2 tetrasaccharide tagged with Tm³⁺ (magenta) and La³⁺ (blue).

Figure 3

Correlations between the experimentally observed and back-calculated PCS values of the GM2 tetrasaccharide with Tm³⁺.

Figure 4

The differences of proton PCS values shown as subtraction of Δδ¹Hof the GM2 tetrasaccharide from Δδ¹Hof the GM3 trisaccharide with Tm³⁺ (blue) and Tb³⁺ (red). n.d., not detected due to extensive line broadening.

Figure 5

Three-dimensional structural model of one of the major conformers of the GM2 tetrasaccharide. Cylinder (left) and space-filling (right) models are shown in the same orientation.

Scheme 2

Preparation of azide 2.

Scheme 3

Preparation of amine 3.

Scheme 4

Preparation of compound 4.

Scheme 5

Preparation of the GM2 tetrasaccharide with the tag.