Native FG, dHG-5 and Oligosaccharides Contained in dHG-5.
Preparation of native FG, dHG-5 and its oligosaccharides. The native FG was extracted and purified fromH. fuscopunctata with yield of ~ 0.8 %. The1H NMR spectrum (Figure S1) showed native FG had a backbone consisted of repeated {4)-D -glucuronic acid (GlcA)-β(1,3)-N-acetyl-D -galactosamine (GalNAc)-β(1,} disaccharide units, and abundant α-L -fucose sulfate (FucS) branches linked to C3 of each GlcA residue in backbone. The main type of FucS branches in the native FG wasL -fucose-3,4-disulfates (Fuc3S4S), and the minor wereL -fucose-2,4-disulfates (Fuc2S4S) andL -fucose-4-sulfates (Fuc4S). The depolymerized fraction, dHG-5, was prepared from native FG by β-eliminative depolymerization as previously described (Zhou, Gao et al. , 2020) and the high performance gel permeation chromatography (HPGPC) profiles of dHG-5 (Figure 1A and Figure S2) showed that dHG-5 was the mixture of oligosaccharides with different degree of polymerization (dp). The molar percentages of the main oligosaccharides (oHG-5, oHG-8, oHG-11, oHG-14, oHG-17, oHG-20, oHG-23, oHG-26, oHG-29 and those with dp > 29) in dHG-5 were estimated as 4.86, 17.39, 17.55, 15.93, 13.62, 11.22, 6.62, 4.74, 2.95 and 5.12%, respectively, according to the proportion of peak area in HPGPC. dHG-5 was further size-fractionated using GPC with Bio-Gel P6 and P10 columns (Bio-Rad) under monitor of HPGPC using a TSK G2000SWXL column, and nine homogeneous oligosaccharides were obtained (Figure 1A). The Mw of dHG-5 was calculated as 5236 Da, according to the calibration curve from the data of purified oligosaccharides using GPC software. Structures of dHG-5 and its purified oligosaccharide fractions were analysed by 1D/2D NMR and ESI-MS.
Structure of dHG-5 . According to the1H/13C NMR spectra, the non-reducing ends of dHG-5 were Δ4,5-unsaturated glucuronic acids (ΔU) and the reducing terminals were the alditol of glucuronic acids (D -GlcA-ol, L -gulonic acid). The chemical structure of dHG-5 was further confirmed by analyzing its 1D/2D NMR spectra (Figure S3-S6). The position 4 and 6 of GalNAc were both sulfated (GalNAc4S6S) according to signals of H4 and H6 that shifted downfield by approximately 0.5−0.7 ppm. Based on the cross-signals in 1H-1H ROESY and1H-13C HMBC spectra and the anomeric proton-proton coupling constant (3JH-H ) values of GlcA and GalNAc4S6S, GlcA and GalNAc4S6S residues were linked with alternating β1,3 and β1,4 glycosidic linkage. Likewise, the Fuc3S4S branches were linked to the position 3 of GlcA residues through an α1,3 glycosidic linkage.
Structure of oligosaccharides from dHG-5. The complete structural analysis of compound oHG-5, oHG-8, oHG-11, oHG-14 and oHG-17 was also conducted with full assignments of 1D/2D NMR spectra (Figure S7-S26). The1H and 13C NMR spectra of oHG-8 and oHG-11 were shown as representatives in Figure 1B. For signals in1H-, 13C- and 2D-NMR spectra of oHG-11, oHG-14, oHG-17 were similar except differences in integral area of corresponding signals (Figure S27), the 2D-NMR spectra of oHG-20, oHG-23, oHG-26 and oHG-29 weren’t recorded.
The 1H NMR spectrum of oHG-8 recorded in D2O showed four well resolved single proton resonances in the 5.0-6.0 ppm region. The low-field signal at 5.68 ppm indicated a typical proton at position 4 of the unsaturated uronic acid residue (ΔU) resulting from β-eliminative cleavage (Vitor Hugo Pomin, 2013). The signals at 5.28 ppm and 5.20 ppm were anomeric proton signals of Fuc3S4S residue (F) linked to GlcA (U) and Fuc3S4S residue (dF) linked to ΔU, respectively. The signal at 5.02 ppm was anomeric proton signal of Fuc3S4Sresidue (rF) linked to reducing termini (rU). The1H-1H COSY spectrum provided eight complete spin connectivity information for each sugar residue (Figure S12). Signals of all four protons of ΔU residue were identified by starting from the signal at 5.68 ppm assigned to the H-4, the H-1, H-2 and H-3 signals were observed with chemical shifts of 4.846, 3.830, 4.426 ppm, respectively. All protons of other residues were also clearly identified in a similar manner by starting from their respective anomeric proton signals.
Total assignment of the carbon spectrum of oHG-8 (Figure S12) was achieved by analysis its 1H-13C HSQC (Figure S14). The C-1 chemical shifts of ΔU, dF, dA, F, U, A, rU and rF residues were at 105.81, 100.94, 102.39, 101.97, 106.44, 104.17, 65.21 and 104.17 ppm, respectively. The C4 and C5 of ΔU were at 109.44 ppm and 149.61 ppm, respectively, in accordance with the presence of double bond. The carbonyl of ΔU at 171.65 ppm shifted highfield by about 6 ppm. In addition, negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) analysis revealed several multiply charged ions in the spectrum (Figure 1C). The four charged ions [M-4Na]4− with m/z at 591.9546 were observed as the most abundant ions. The molecular mass was identical to the calculated value of 2459.8184, confirming that the molecular formula of oHG-8 is C52H69N2Na13O70S10(octasaccharide). Therefore, oHG-8 was determined to be an octasaccharide with the sequenceL -Fuc3S4S-α(1,3)-L4,5GlcA-α(1,3)-D -GalNAc4S6S-β(1,4)-[L -Fuc3S4S-α(1,]3)-D -GlcA-β(1,3)-D -GalNAc4S6S-β(1,4)-[L -Fuc3S4S-(α1,] 3)-D -GlcA-ol.
The structures of compound oHG-5, oHG-11, oHG-14 and oHG-17 were determined using the similar approach. They were identified as penta-, hendeca-, tetradeca- and heptadeca-saccharide by 1H NMR spectra, respectively, based on the integral area ratio of the anomeric proton signals of Fuc3S4Sresidues linked to different positions of their backbones (Figure S27). The main components of oHG-20, oHG-23, oHG-26, oHG-29 were eicosa-, tricosa-, hexacosa- and nonacosasaccharide, respectively, according to their HPGPC (Figure 1) and their 1H NMR spectra (Figure S27). In short, oHG-5, oHG-8, oHG-11, oHG-14 and oHG-17 were highly regular oligosaccharides, and the non-reducing terminal of these compounds was ΔU produced by the β-eliminative cleavage. The internal sequence of these oligosaccharides was constituted by the repeating trisaccharide unit {4)-[L -Fuc-α(1,]3)-D -GlcA-β(1,3)-D -GalNAc-(β1,}, where Fuc side chains and GalNAc residues were primarily Fuc3S4S and GalNAc4S6S, respectively. It is worth noting that the reducing end was a hexuronic acid alditol residue instead of sulfated GalNAc, which were resulted from the peeling reaction (Shang, Gao et al. , 2018). The structural difference among these depolymerized oligo-saccharides was the number of the repeating trisaccharide unit {3)-D -GalNAc4S6S-β(1,4)-[L -FucS-α(1,]3)-D -GlcA-β(1,}.