NMR spectra of oligosaccharides at ultra-high field (900 MHz) have better resolution than expected due to favourable molecular tumbling

Abstract

Nuclear magnetic resonance (NMR) remains the most promising technique for acquiring atomic-resolution information in complex carbohydrates. Significant obstacles to the acquisition of such data are the poor chemical-shift dispersion and artifacts resultant from their degenerate chemical structures. The recent development of ultra-high-field NMR (at 900 MHz and beyond) gives new potential to overcome these problems, as we demonstrate on a hexasaccharide of the highly repetitive glycosaminoglycan hyaluronan. At 900 MHz, the expected increase in spectral dispersion due to higher resonance frequencies and reduction in strong coupling-associated distortions are observed. In addition, the fortuitous molecular tumbling rate of oligosaccharides results in longer T2-values that further significantly enhances resolution, an effect not available to proteins. Combined, the resolution enhancement can be as much as twofold relative to 600 MHz, allowing all 1H-resonances in the hexasaccharide to be unambiguously assigned using standard natural-abundance experiments. The use of ultra-high-field spectrometers is clearly advantageous and promises a new and exciting era in carbohydrate structural biology.

Graphical abstract

Figure 1

(Top) Chemical structure of a hyaluronan (HA) hexasaccharide, which comprises three repeats of a disaccharide of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc). Ring positions are numbered from the reducing terminus. (Bottom) Comparison of free-induction decays of HA hexasaccharides at 600 and 900 MHz (720 ms shown in both cases). Upon Fourier-transformation (FT), the smaller rate of exponential decay (i.e., longer T₂ relaxation time) at 900 MHz results in narrower line-widths and a concomitant increase in resolution (the most crowded region is shown, from 4.0 to 3.7 ppm).

Figure 2

(Top) Comparison in hertz of line-widths at half-maximum (dotted line) for the GlcA H-1^II doublet at 600 MHz (black) and 900 MHz (grey). (Middle) Dependence of T₂ relaxation rate on the overall correlation time (τ_m) at high field (600 MHz) and ultra-high fields (900 MHz and 1.2 GHz). Regions typically associated with oligosaccharides and proteins are marked. (Bottom) Comparison of the resolution enhancement (R_ν/R₆₀₀) gained by increasing the B₀ field-strength for small oligosaccharides (with τ_m = 0.25 ns), a HA hexasaccharide with segmental motion (mass ∼1 kDa, τ_m = 0.63 ns, τ_e = 0.071 ns and S² = 0.57, as measured previously at ring III^1,20) and a protein with a mass of ∼12 kDa (τ_m = 9.2 ns, τ_e = 0.02 ns and S² = 0.94, as measured for Ala33 in the C-terminal SH2 domain of phospholipase Cγ1¹⁶).

Figure 3

Strong coupling between GlcA H-3^VI and GlcA H-4^VI results in virtual coupling wings (∗) to the GlcA H-2^VI quartet. The intensity of these wings relative to the first-order resonances (i.e., those from ³J_1,2 and ³J_2,3) is considerably less at 900 MHz. The resonance indicated by § is part of the GlcA H-2^IV multiplet.

Figure 4

Comparison of DQF-COSY spectra recorded in an identical manner on a sample of HA hexasaccharide at 600 and 900 MHz. Several cross-peaks that are considerably easier to assign at 900 MHz are indicated (A–G), as well as an artifact caused by strong coupling (H). A—GlcNAc, H-4^V, 3^V (δ 3.539, 3.705 ppm); B—GlcNAc, H-4^III, 3^III (3.519, 3.707); C—GlcNAc, H-4^I, H-3^I (3.513, 3.714) (β anomer); D—GlcA, H-4^VI, H-5^VI (3.498, 3.722); E—GlcNAc, H-6b^V, H-6a^V (3.918, 3.776); F—GlcNAc, H-6b^III, H-6a^III (3.910, 3.764); G—GlcNAc, H-6b^I, H-6a^I (3.891, 3.749) (β anomer); H—GlcA, H-2^VI, H-5^VI (3.318, 3.722); I—GlcA, H-2^VI diagonal peak (3.318).