Fig 1.
The Figures were prepared with ChemSketch [27] and edited with Inkscape [28].
Fig 2.
Investigated oligosaccharides.
Investigated disaccharides: trehalose, methyl-1α-2α-mannobiose (M12), methyl-1α-3α-mannobiose (M13), and methyl-1α-6α-mannobiose (M16), together with defined glycosidic dihedral angles ϕ1, ϕ2, and ϕ3 (explicit atom definitions in S1 File). Bottom raffinose trisaccharide.
Table 1.
Summary of MD simulations performed in this work.
Fig 3.
M13 free energy surface and sampled structures.
Left: Calculated free energy surface (FES) of M13 disaccharide in {ϕ1, ϕ2} dihedral angles. Middle: Calculated FES, together with 250 extracted structures from unbiased 500 ns MD simulations (MD). Right: Calculated FES, together with 250 extracted structures for each biased 200 ns MD simulation (biased MD; md1/md2/md3/md4; restrain {ϕ1, ϕ2} values in Table 2). White regions represent area with the free energy >40 kJ/mol.
Fig 4.
Comparison of experimental (exp.) and calculated spectra of M13 disaccharide. Top left—best fit: Best fit of md1/md2/md3/md4 Raman/ROA spectra to experimental data. Top right—MD: Simulated Raman/ROA spectra obtained using structures from unbiased MD simulation (MD). Bottom—local-conformers: Calculated ensemble averaged Raman/ROA spectra of M13 disaccharide prepared in 4 distinct conformations md1/md2/md3/md4 as described in Fig 3.
Table 2.
Restrain values [rad] of ϕ1/ϕ2 glycosidic angles used in biased MD simulations of M13 yielding md1/md2/md3/md4 conformers.
Table 3.
Abundance of conformers of M13 disaccharide as obtained by MD, NMR, and the best fit to Raman/ROA (error bars in brackets).
Fig 5.
M16 free energy surface and sampled structures.
Left: Calculated free energy surface (FES) of M16 disaccharide in terms of the {ϕ1, ϕ2} dihedral angles. Middle: Calculated FES, together with 250 extracted structures from unbiased 500 ns MD simulations (MD). Right: Calculated FES, together with 250 extracted structures per each biased 200 ns MD simulation (biased MD; md1/md2/md3/md4/md5/md6; restrain {ϕ1, ϕ2} values in Table 4). White regions represents area with the free energy >40 kJ/mol.
Fig 6.
Comparison of experimental (exp.) and calculated spectra of M16 disaccharide. Top left—best fit: Best fit of md1/md2/md3/md4/md5/md6 Raman/ROA spectra to experimental data. Top right—MD: Simulated Raman/ROA spectra of disaccharide obtained using structures from unbiased MD simulation (MD). Bottom—local-conformers: Calculated ensemble averaged Raman and ROA spectra of disaccharide prepared in 6 distinct conformations md1/md2/md3/md4/md5/md6 as described in Fig 5.
Table 4.
Restrain values [rad] of ϕ1/ϕ2 glycosidic angles used in biased MD simulations of M16 yielding md1/md2/md3/md4/md5/md6 conformers.
Table 5.
Conformer abundances of M16 disaccharide obtained from MD and the best fit to Raman/ROA experimental data. Estimated error ranges for the latter in parenthesis.
Table 6.
Comparison of conformer populations of M16 disaccharide obtained using MD, NMR, and the best fit to experimental Raman/ROA data.
Fig 7.
MeGlcA free energy surface and sampled structures.
Left: Calculated FES of MeGlcA in ϕ/θ puckering coordinates. Middle: Calculated FES, together with 250 extracted structures from unbiased 500 ns MD simulations (MD). Right: Calculated FES, together with 250 extracted structures per each biased 200 ns MD simulation(biased MD; 1C4/4C1/OS2/1S3; restrain {ϕ, θ} values in Table 7). All plots are shown as equal area Mollweide projection.
Fig 8.
Comparison of experimental (exp.) and calculated spectra of MeGlcA. Top left—best fit: Best fit of 1C4/4C1/OS2/1S3 Raman/ROA spectra to experimental data. Top right—MD: Simulated Raman/ROA spectra of the monosaccharide obtained using structures from unbiased MD simulation (MD). Bottom—local-conformers: Calculated ensemble averaged Raman and ROA spectra of the monosaccharide prepared in 4 distinct conformations 1C4/4C1/OS2/1S3 as described in Fig 7.
Table 7.
ϕ/θ restrain values of MeGlcA.
Restrain values [rad] of ϕ/θ puckering coordinates used in biased MD simulations of methyl-β-d-glucuronic acid yielding 1C4/ 4C1/OS2/1S3 conformers. 1C4 and 4C1 conformer were restrained only in θ puckering variable.
Table 8.
MeGlcA puckering conformer populations.
Abundance of puckering conformers of MeGlcA as obtained by MD, NMR, and the best fit to Raman/ROA experimental data. Estimated error ranges for the NMR and the best fit approaches are in parenthesis.
Fig 9.
Raman/ROA spectra of raffinose trisaccharide.
Comparison of calculated Raman and ROA spectra of raffinose trisaccharide with experiment.
Fig 10.
Raman/ROA spectra of Glc, GlcA, and GlcNAc.
Left: Calculated Raman/ROA spectra for the α/β anomers (Glc, GlcA, and GlcNAc). Right: Best fit to experimental data.
Table 9.
Calculated anomeric ratios of Glc, GlcA, and GlcNAc.
Experimental and calculated anomeric ratios (β fraction) for investigated reducing sugars. Calculated data were obtained as the best fit of the Raman and ROA spectra to experimental data.
Fig 11.
Raman/ROA spectra of mixtures of MeGlc:MeGlcNAc.
In blue the experimental Raman/ROA spectra of MeGlc and MeGlcNAc, and their 3:1, 1:1, and 1:3 mixtures (MeGlc:MeGlcNAc). The best fit simulation spectra to the experiment using the spectra of simulated pure substances to fit them are shown in red (see Fig 12 and Table 10 for the results of the fit).
Fig 12.
Prediction of molar fractions of mixtures of MeGlc:MeGlcNAc.
Summary of calculated molar fractions and estimated errors of mixtures (MeGlc:MeGlcNAc) obtained by the best fitting corresponding experimental Raman/ROA spectra of known composition (black,xMeGlc = 0.00, 0.25, 0.5, 0.75, 1.00) using simulation(red, calculated spectra) or experimental(green, experimental spectra) spectra of pure substances to fit them.
Table 10.
Calculated molar fractions of MeGlc:MeGlcNAc mixtures.
Summary of calculated molar fractions and estimated errors of prepared mixtures (MeGlc:MeGlcNAc; xMeGlc = 0.00, 0.25, 0.5, 0.75, 1.00) obtained by the best fitting corresponding experimental Raman/ROA spectra using simulation() or experimental(
) spectra of pure substances to fit them.
Fig 13.
Raman/ROA spectra of two MeGlc in close proximity.
Calculated Raman/ROA spectra of methyl-β-glucose at infinite dilution, i.e., single molecule (black), of two interacting methyl-β-glucose sugar moieties (red, representative snapshot in the inset). In blue the experimental spectra at 1 M concentration for comparison.