Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus

Abstract

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO₃^-)-α(1-3)-l-Fucp2,4(SO₃^-)-α(1-3)-l-Fucp2(SO₃^-)-α(1-3)-l-Fucp2(SO₃^-)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H₂O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space ¹H-¹H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.

Keywords: Conformation; Fucose; NMR spectroscopy; Structural glycobiology; Sulfated fucan.

Fig. 1.

(A) 1D ¹H, δ_H expansions 5.5–3.0 ppm and 1.4–1.1 ppm, (B) and 2D ¹H–¹³C, δ_H/δ_C expansions 5.4–3.0/100–65 ppm spectra of l-Fucp in D₂O at 25 °C. The signals were assigned to α and β anomeric configurations of l-Fucp in solution, respectively. The relative abundance of the anomeric ¹H’s is shown below the respective peaks in the 1D ¹H NMR spectrum. Blue signals correspond to the cross-peaks of α-l-Fucp and red signals correspond to the cross-peaks of β-l-Fucp.

Fig. 2.

1D ¹H, δ_H expansion 10.0–0.0 ppm, NMR spectra of l-Fucp in 85:15% H₂O:acetone-d₆ at 5–25 °C. The colors and temperatures are yellow for 25 °C, navy blue for 20 °C, light blue for 15 °C, green for 10 °C, and red for 5 °C. The inset on the left intensifies the magnitude of the resultant downfield peaks generated from the exchangeable ¹H’s. Signal assignments are indicated accordingly in the spectrum.

Fig. 3.

Build-up curves of the NOE resonances (inset panels show the connections) seen in the series of NOESY spectra of l-Fucp in D₂O at 25 °C (A), and in 85:15% H₂O:acetone-d₆ at 5 °C (B and C). While panels A and B show the pair of connections of the non-exchangeable carbon-bound ¹H’s, panel C shows the exchangeable ¹H’s from hydroxyl groups. Note that the horizontal and vertical scales, designating mixing times and peak intensities, respectively, are different in the panels.

Fig. 4.

2D ¹H–¹H, δ_H/δ_H expansions 8.0–5.3/8.0–3.0 ppm from (A) COSY and (B) NOESY spectra (5 ms mixing time) of l-Fucp in 85:15% H₂O:acetone-d₆ at 5 °C. Blue signals correspond to the cross-peaks of α-l-Fucp, red signals correspond to the cross-peaks of β-l-Fucp, and gray signals to the auto-peaks of the diagonal.

Fig. 5.

(A) 1D ¹H, δ_H expansions 5.6–3.4 ppm and 1.5–1.0 ppm, and (B) 2D ¹H–¹³C HSQC, δ_H/δ_C expansions 5.7–3.0/105–50 ppm, spectra of the L. variegatus SF pentasulfated tetrasaccharide in D₂O at 25 °C. The signals were assigned respectively to the four chemically different α-l-Fucp units of the tetrasaccharide in solution.

Fig. 6.

1D ¹H, δ_H expansion 10.0–0.0 ppm, NMR spectra of L. variegatus SF pentasulfated tetrasaccharide in 85:15% H₂O:acetone-d₆ at 5–25 °C. The colors and temperatures are yellow for 25 °C, navy blue for 20 °C, light blue for 15 °C, green for 10 °C, red for 5 °C, and black for 0 °C. The inset on the left intensifies the magnitude of the resultant peaks generated from the exchangeable ¹H’s. Signal assignments of the exchangeable ¹H’s are indicated accordingly in the inset.

Fig. 7.

(A and B) Build-up curves of the NOE resonances (inset panels show the assignments) seen in the series of NOESY spectra, and (C) NOESY spectrum (12 ms mixing time), δ_H/δ_H expansions 8.0–6.5/8.0–3.0 ppm, of the exchangeable ¹H’s obtained for the L. variegatus SF pentasulfated tetrasaccharide in 85:15% H₂O:acetone‑d₆ at 0 °C. While panel A shows the resonances of the hydroxyl ¹H’s, panel B shows those from the sulfamate groups. Note that the horizontal and vertical scales designating, respectively, mixing times and peak intensities are different in the panels.

Fig. 8.

NMR NOE-restrained 3D structural representations of the L. variegatus SF-derived pentasulfated tetrasaccharide [l-Fucp4(SO₃⁻)-α(1–3)-l-Fucp2,4(SO₃⁻)-α(1–3)-l-Fucp2(SO₃⁻)-α(1–3)-l-Fucp2(SO₃⁻)] in both (A) stick molecular models, and (B) surface molecular models. In both molecular models, atoms of hydrogen, oxygen, carbon, and sulfur are shown in white, red, grey, and yellow, respectively. The molecular representations with 180° turn have also been shown for both models. Hydrogen bonds are represented by dashed blue lines.