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)-L -Δ4,5GlcA-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,}.