Topspin version 3

Deuterium oxide (D₂O, 99.9% D) and 3-trimethylsilyl-²H₄-propionic acid sodium salt (TSP) were purchased from Cambridge Isotope Laboratories, Inc. (Miami, U.S.A.) and from Sigma-Aldrich (St. Louis, MO). Spectra were obtained from solutions in D₂O (580 µL plus 20 µL of TSP 1 mg/mL at 30 °C, using TSP as reference (δ = 0.00 ppm), using a Bruker AVANCE III NMR spectrometer operating at 14.1 Tesla (600.13 MHz for ¹H) equipped with a QXI probe with gradient on the z-axis (Bruker Biospin, Germany). 2D ¹³C/¹H multiplicity-edited HSQC NMR experiments were carried out using heteronuclear correlation via double inept transfer with decoupling during acquisition, using trim pulses in inept transfer with multiplicity editing during the selection step (hsqcedetgpsisp2.2). The 2D HSQC experiments were recorded for quadrature detection in the indirect dimension, using 8 scans per series of 2 K × 256 W data points with zero filling in F1 (4 K) prior to Fourier transformation^{42 (link)}. Data processing and integration were performed using the software Topspin version 3.1 (Bruker Biospin, Rheinstetten, Germany). ¹³C/¹H correlations were assigned according to Nascimento et al.^{11 (link)}.

Vacuum dried polysaccharide samples were solubilized in 0.65 mL of deuterium oxide (99.9 % atom D, Sigma Aldrich) to reach a final concentration ranging from 8 to 15 mg/mL and transferred to 5-mm NMR tubes (Wilmad). ¹H and ¹³C NMR spectra were acquired using a 5-mm broadband probe on Bruker Avance III 400 or 500 MHz spectrometers, equipped with a high precision temperature controller. Data acquisition and processing were performed using TopSpin version 3.1 (Bruker).
¹H NMR experiments were acquired at 25 +/- 0.1 °C using a mono-dimensional standard pulse-program with 32k data points over a 10 ppm spectral width, accumulating 128 scans. The spectra were weighted with 0.2 Hz line broadening and Fourier-transformed. The transmitter was set at the water frequency, which was used as the reference signal (4.79 ppm).
To obtain ¹³C spectra with adequate signal-to-noise ratio, all data were acquired using distortionless enhancement by polarization transfer (DEPT). DEPT experiments were collected at pulse angle of 3π/4 at 25 ± 0.1 °C, accumulating a number of scans > 16,384. The transmitter was set at the ethanol frequency which was used as the reference signal (17.47 ppm). All mono-dimensional proton NMR spectra were obtained in quantitative manner using a total recycle time to ensure full recovery of each signal (5 x Longitudinal Relaxation Time T₁).

Mono-dimensional ¹H-NMR measurements were performed at 353 °K on a Bruker AVANCE I instrument equipped with a 5-mm probe operating at 400.2 MHz (Bruker, Billerica, MA, USA). A total of 3 mg of polymer was dissolved in 0.5 mL of D₂O, and 32 scans were recorded. Chemical shifts of the protons were expressed as δ (ppm) using H₂O at 4.25 ppm at 80 °C as reference. The data were collected and processed using the Software TOPSPIN, version 3.1 (Bruker Biospin, Rheinstetten, Germany).
The degree of methyl-esterification (DM) values was determined by ¹H-NMR spectroscopy, integrating the hydrogen areas corresponding to -CH₃ at 3.9 ppm, H-1 (5.1–5.3 ppm), H-5 of unesterified α-D-GalAp units at 4.7 ppm, and H-5′ of esterified α-D-GalAp units at 5 ppm, as discussed by Grasdalen, Bakøy, and Larsen [21 (link)]. The hydrogen from -CH₃ around 3.9 ppm gives DM, using H-1 integral as reference [22 (link)]. Nevertheless, the H-1 signal of Gal-A units is very near the H-1 of arabinogalactan. The DM % is also determined by the ratio of -CH₃ using (H-5 + H-5′) integrals or from the ratio H-5′/(H-5 + H-5′) with a relatively good accordance.