NMR experiments were recorded at 308 K either on an 800 MHz Avance NEO with an 18.8 Tesla magnetic field or on a 600 MHz Avance III HD spectrometer with a 14.1 Tesla magnetic field, both from Bruker (Billerica, MA, USA). Spectrometers were equipped with cryogenically cooled triple resonance 1H[13C/15N] probes. Spectra were recorded using TopSpin 4.0.7 on the 800 MHz spectrometer or Topspin 3.6.1 for the 600 MHz spectrometer (Bruker Biospin). 1H and 13C chemical shifts were referenced to external DSS (2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt).
Polysaccharide samples (5 to 20 mg) were dissolved either in 200 µL or 300 µL of D2O (99.97% 2H atoms, Eurisotop, Saclay, France) and placed in 3 mm tubes (Norell HT, Sigma-Aldrich) or 4 mm Shigemi tubes, depending on the amount of the sample. Resonance assignment, glycosidic bonds identification, and J coupling measurements were achieved from homonuclear 1H-1H COSY [22 (link)] and natural abundance heteronuclear 1H-13C experiments: HSQC spectra recorded with or without decoupling [23 (link)], H2BC [24 (link)], HMBC [25 (link)], and HSQC-TOCSY [26 (link)] with a 200 ms mixing time. Monosaccharide residues were identified from the anomeric region; their anomeric configuration was established from the corresponding chemical shifts and the 1JH1,C1 coupling constant. 3JH1,H2 coupling constants were measured from 1H-1H COSY experiments obtained with a resolution of 1 Hz. Glycosidic bonds were identified using HMBC experiments. The proportion of the different monosaccharide residues was estimated from the integrals of the anomeric peaks on the 1H-13C-HSQC spectrum.
Stimulated echo diffusion experiments of the yet undescribed mannan (G3Man, see Results) were performed at 298°K with convection compensation and bipolar gradients [27 (link)]. These diffusion-ordered experiments (DOSY) were recorded with a diffusion delay of 140 ms; gradients were applied during 3.4 ms with 16 varying intensities and a total recycle delay of 3 s. Experiments were performed in triplicate. The diffusion coefficients were calculated with Topspin DOSY standard routines.
The apparent diffusion coefficient (d) of mannan for selected individual signals was obtained by fitting the integral (I) of selected signals to gaussian decays as a function of the gradient intensity (G):
where Io represents the signal that is integral in the absence of gradients. Integral errors were estimated with five times the noise standard deviation. Fits were performed with Kaleidagraph™ 4.5 (Synergy software).
Polysaccharide samples (5 to 20 mg) were dissolved either in 200 µL or 300 µL of D2O (99.97% 2H atoms, Eurisotop, Saclay, France) and placed in 3 mm tubes (Norell HT, Sigma-Aldrich) or 4 mm Shigemi tubes, depending on the amount of the sample. Resonance assignment, glycosidic bonds identification, and J coupling measurements were achieved from homonuclear 1H-1H COSY [22 (link)] and natural abundance heteronuclear 1H-13C experiments: HSQC spectra recorded with or without decoupling [23 (link)], H2BC [24 (link)], HMBC [25 (link)], and HSQC-TOCSY [26 (link)] with a 200 ms mixing time. Monosaccharide residues were identified from the anomeric region; their anomeric configuration was established from the corresponding chemical shifts and the 1JH1,C1 coupling constant. 3JH1,H2 coupling constants were measured from 1H-1H COSY experiments obtained with a resolution of 1 Hz. Glycosidic bonds were identified using HMBC experiments. The proportion of the different monosaccharide residues was estimated from the integrals of the anomeric peaks on the 1H-13C-HSQC spectrum.
Stimulated echo diffusion experiments of the yet undescribed mannan (G3Man, see Results) were performed at 298°K with convection compensation and bipolar gradients [27 (link)]. These diffusion-ordered experiments (DOSY) were recorded with a diffusion delay of 140 ms; gradients were applied during 3.4 ms with 16 varying intensities and a total recycle delay of 3 s. Experiments were performed in triplicate. The diffusion coefficients were calculated with Topspin DOSY standard routines.
The apparent diffusion coefficient (d) of mannan for selected individual signals was obtained by fitting the integral (I) of selected signals to gaussian decays as a function of the gradient intensity (G):
where Io represents the signal that is integral in the absence of gradients. Integral errors were estimated with five times the noise standard deviation. Fits were performed with Kaleidagraph™ 4.5 (Synergy software).
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