Chemicals

p-Nitrocatechol sulfate (PNCS) and p-nitrophenyl glycosides were purchased from Sigma (France). The pure sulfated fucose isomers (2-O-, 3-O- and 4-O-sulfated l-fucose) prepared as a mixture were kindly provided by M. McLean (Grampian Enzymes, Orkney, Scotland). Fucoidan (M_r 13 000) used in this study was extracted from the brown algae A. nodosum and purified as previously described [9,10]. It contains ≈ 43% (w/w) fucose units, 34% (w/w) sulfate groups and 5% (w/w) uronic acid functions. Oligosaccharides were prepared by mild hydrolysis of fucoidan in 75 mm H₂SO₄ for 3 h at 60 °C. After neutralization with NaOH, the resulting hydrolysate was filtered on a membrane (molecular weight cut-off = 1000 Da). The oligosaccharides contained in the filtrate were then purified by gel filtration on a Biogel P4 column (1.5 × 100 cm) equilibrated with 0.5 m ammonium hydrogenocarbonate buffer, pH 8.3 (flow rate 7 mL·h⁻¹). Oligosaccharides containing fractions were freeze-dried. Anhydrous sodium sulfate and oxalic acid were from Merck (Darmstadt, Germany) and sodium dichromate was from Sigma (France). Others chemicals and reagents were obtained from current commercial sources at the highest level of purity available. All buffers and solutions were prepared with ultrapure water produced by a laboratory water purification system (Purite).

Preparation of the sulfoesterase

The marine mollusk Pecten maximus was kindly provided by P. Roy (IFREMER, Nantes, France). Digestive glands of P. maximus were homogenized, and proteins were extracted by fractionated ammonium sulfate precipitation according to a previously described procedure [11]. The resulting protein solution was dialyzed against 50 mm sodium acetate buffer, pH 5.2, and the lipids were removed by a washing step with 30% (v/v) ethyl acetate. The aqueous phase, which retained the sulfoesterase activity, was recovered by centrifugation and dialyzed against 50 mm sodium acetate buffer, pH 5.2. After concentration by ultrafiltration with a Millipore Ultrafree 5K device, the protein solution (15 mL) was applied to a Sulfopropyl Fast Flow Sepharose column (5 × 12 cm) equilibrated with the above-mentioned acetate buffer as mobile phase at a flow rate of 1 mL·min⁻¹. After a wash step with 3 col. vol. of the same buffer, the elution was carried out with a 0–0.5 m linear gradient of NaCl. The fractions containing sulfoesterase activity were eluted at a NaCl concentration of 0.15 m. The active fractions were concentrated by ultrafiltration using a Millipore Ultrafree 5K device, and then aliquots (200 µL) were applied to a Superose 12 column (1 × 30 cm) equilibrated with 0.1 m sodium acetate buffer, pH 5.5 (flow rate 0.5 mL·min⁻¹; fraction volume 0.25 mL). Active fractions were pooled and concentrated. The resulting concentrate was stored at −80 °C and made up the enzyme preparation used in this study. Protein concentrations were determined by the Bio-Rad dye binding method with BSA as the standard [12].

Spectrophotometric assays

Sulfoesterase activity was routinely assayed with p-nitrocatechol sulfate as substrate using the spectrophotometric determination of p-nitrocatechol at 515 nm (ε₅₁₅ = 12 200 m⁻¹·cm⁻¹) [13]. The reaction was initiated by the addition of enzyme extract (5 µL) in 0.5 mL of 0.1 m acetate buffer, pH 5.5, containing 3.5 mm substrate. The reaction was carried out at 50 °C for 10 min, then 750 µL of 1 m NaOH were added to stop the reaction and to reveal the absorbance at 515 nm. Enzyme specific activity was defined as the amount of µmoles of p-nitrocatechol released per minute per milligram of protein.

Capillary electrophoresis assays

The sulfoesterase reactions on fucoidan were performed at 30 °C in 20 mL of 0.1 m sodium acetate buffer, pH 5.5. The fucoidan was incubated at a concentration of 5 mg·mL⁻¹ with 0.5% (v/v) of enzyme extract. Fifty-microlitre aliquots taken from the reaction mixture were mixed with an equal volume of a stop solution (prepared with 2 m ethanolamine, 15 mm sodium hydroxide and 0.44 mm oxalic acid, added as internal standard) prior to performing capillary electrophoresis (CE) assay. For a full kinetic study, this protocol was repeated throughout the time course of the enzymatic reaction.

CE assays were carried out with a HP^3DCE capillary electrophoresis system (Hewlett Packard, Waldbronn, Germany) equipped with a diode array detector. The data was handled by a HP Vectra XA computer equipped with chemstation acquisition software. Bare fused silica capillaries, (50 µm inside diameter, 360 µm outside diameter × 38.5 cm in length, 30 cm to detector) were from SGE (Villeneuve-Saint-Georges, France). Samples were introduced in the electrokinetic mode, applying a negative voltage of 10 kV at the capillary inlet for 180 s. Separations were performed under a negative voltage of 15 kV (electric field: 390 V·m⁻¹, current intensity: 45 µA). The temperature in the capillary cartridge was set at 25 °C. The separation electrolyte was prepared by mixing 10 mm sodium dichromate and 40 mm Tris so that its final pH was 8.1, and its final composition was 20 mm chromate, 20 mm Tris/H⁺, 20 mm Tris, 20 mm Na⁺. It was sonicated for 5 min and filtered through 0.2-µm filter units before being loaded into the apparatus. Prior to each sample injection, the capillary was rinsed with the separation electrolyte for 3 min. A blank electrokinetic injection of electrolyte was systematically performed before proceeding with any quantitative measurement. Analytes were detected by indirect UV absorbance at 254 nm using chromate anion in the electrolyte.

The sulfate quantitation was performed as previously described [14] using oxalate as the internal standard. The standard matrix used for calibration purposes was prepared by mixing 5 mm acetic acid, 15 mm NaOH and 20 p.p.m oxalic acid, added as internal standard. The final composition of this matrix was 5 mm acetate, 0.22 mm oxalate, 15 mm Na⁺ and 9.5 mm OH^– (pH 12). The standard sulfate solutions were prepared from a 0.1% anhydrous sodium sulfate stock solution in the standard matrix and subsequent appropriate dilutions in the same matrix. The sulfate-to-oxalate peak area ratio (relative area) was plotted as a function of sulfate concentration.

NMR spectroscopy experiments

NMR spectroscopy was performed on a Bruker DMX 500 spectrometer, operating at the proton Larmor frequency of 500.11 MHz. The experiments were performed with a 5-mm probe, equipped with self-shielded Z-gradients. Spectra were recorded at 298 K or 323 K with suppression of residual solvent (H₂O/D₂O) signal by presaturation. Chemical shifts are reported in p.p.m. using sodium 3-trimethylsilylpropanoate as the internal reference.

One-dimensional spectra were acquired over 32 000 data points using a spectral width of 5000 Hz. Two-dimensional TOCSY, NOESY, ROESY, HSQC, HMQC and HSQC-TOCSY experiments were recorded in the phase-sensitive mode using time proportional phase incrementation [15]. HMBC were recorded in the magnitude mode. TOCSY spectra were run with a spin lock field of about 8 kHz and mixing times of 25 ms and 150 ms. NOESY spectra were obtained with mixing times of 300 and 500 ms, and ROESY spectra with spin-lock duration of 300 or 500 ms ¹H–¹³C long range coupling were investigated with an inverse detected HMBC experiment using delays of 50 and 80 ms. TOCSY and NOESY spectra were collected over a 5000-Hz spectral width using a 2048 × (256–512) data points matrix. Data points were zero-filled in the indirect F1 dimension and apodization by a 90° shifted cosine-squared weighting function was applied in both dimensions prior to Fourier transformation.

The samples of monosulfated fucopyranoses and of fucoidan were exchanged twice with 99.8% D₂O (Sigma) with intermediate lyophilization and dissolved in 0.5 mL of 100% D₂O for NMR analysis. The enzymatic reaction was performed in the NMR tubes containing the substrate, i.e. either the mixture of sulfated fucopyranose isomers (12.5 mg) or fucoidan (10 mg) dissolved in 0.5 mL of deuterated acetate buffer 0.1 m, pH 5.5. The reaction was initiated by addition of enzyme which was obtained as a lyophilized powder after two exchanges in D₂O.

Enzymatic desulfation of algal fucoidan

Investigation of hydrolase activities contained in a protein extract from digestive glands of P. maximus showed a high sulfoesterase activity on the synthetic substrate p-nitrocatechol sulfate. An average specific activity of 0.6 µmol·min⁻¹·mg⁻¹ at pH 5.5 was usually obtained. As the protein extract was a complex medium containing other hydrolases like glycosidases (not shown), the protein extract was submitted to a two-step chromatographic procedure in order to obtain a semipurified sulfoesterase preparation free of fucosidase activity. Indeed, fucosidase activity could interfere with the action of sulfoesterase on fucoidan. After a first cation-exchange chromatography, fractions with sulfoesterase activity and free of fucosidase were eluted from a Superose 12 gel filtration column at a molecular mass around 47 kDa. Study of the pH effect (not shown) indicated that the sulfoesterase was an acid hydrolase with the maximal activity on synthetic substrate at pH 5.

The sulfoesterase activity on algal fucoidan (M_r 13 000) was analyzed by a capillary electrophoresis method previously described [14]. This method allows the direct measurement of sulfate anions in a complex biological mixture containing high molecular mass polyanions. The analysis of the produced sulfate was achieved in counter-electro-osmotic flow migration mode (negative separation voltage) and with electrokinetic injection of the sample (negative injection voltage) so that fast migrating sulfate anions can be loaded without loading the ionic polymer and other slow migrating anions that are present in the matrix, thus obviating most of interference from the reaction mixture. A fucoidan sample derived from the brown algae A. nodosum was incubated with the sulfoesterase preparation at pH 5.5, slightly higher than the optimal pH in order to prevent the acid hydrolysis of the polysaccharide. The variation of the free sulfate concentration in the fucoidan solutions during the incubation is shown in Fig. 1. A trace of sulfate was detected at time zero in the reaction medium containing fucoidan, mainly resulting from the residual sulfate content in the purified fucoidan and is in keeping with its extraction procedure (see Materials and methods). The sulfoesterase activity on fucoidan was clearly demonstrated by the increase of the sulfate concentration in the fucoidan/enzyme mixture for a period of three days. No desulfation was observed in the control incubation (fucoidan alone) indicating no self-desulfation of fucoidan in the reaction conditions. Sulfur elemental analysis of fucoidan confirmed this desulfation upon enzymatic reaction. A rough calculation based on the initial sulfate content of the fucoidan sample (34% w/w) led to a hydrolyzed sulfate ratio of ≈ 10%. We checked that the slow down of the desulfation reaction was not due to enzyme inactivation as sulfoesterase activity on the synthetic substrate remained almost constant during the incubation. We assume that it is probably related to the enzyme regiospecificity, which is of a major interest for subsequent structure elucidation and property alteration.

Enzymatic desulfation of the sulfated-l-fucose isomers

It was then important to ascertain the regioselectivity of the sulfoesterase. Sulfoesterase was incubated with a mixture of the three isomers of sulfated-l-fucose, i.e. the 2-O-, 3-O- and 4-O-sulfated fucose in D₂O acetate buffer. The action of the sulfoesterase on these isomers was easily followed by ¹H-NMR (500 MHz) as the three sulfated fucose derivatives exhibited distinct chemical shifts, notably for the anomeric protons H-1. We point out that these noncommercial sulfated fucose isomers are the building units of fucoidan, and therefore they have to be considered as standards, a key element lacking in the published structural studies of fucoidan. We took the opportunity to report here the exhaustive list of all proton and carbon chemical shifts for each of the isomers, as useful reference values for this and forthcoming studies.

The NMR data for each O-sulfated l-fucose isomer are presented in Tables 1 and 2. TOCSY spectra lead to connectivity for H-1 through to H-4 and for H-5 to H-6. TOCSY experiment performed with a mixing time of 150 ms, affords H-6, H-5 and H-4 assignments in spite of small coupling constant between H-4 and H-5 (J_4,5 < 1 Hz) in the case of fucopyranosides. Assignments of H-6, H-5 and H-4 were confirmed with NOESY and HMBC experiments. Proton chemical shifts determined here confirmed those previously reported for certain sulfated fucose isomers obtained by mild hydrolysis of fucosylated chondroitin sulfate [16]. The ¹³C chemical shift values for each component were determined by ¹H-¹³C HSQC and HSQC-TOCSY experiments.

All of the O-sulfated l-fucose isomers were mainly present in the fucopyranosyl form based on their coupling constants and chemical shifts of anomeric protons (Table 1). A low proportion of furanosyl form (< 5%) could also be observed (data not shown). l-Fucose is the more abundant enantiomer in nature, and has only been found in the pyranose configuration. The less abundant d-fucose could also occurs as furanoside and pyranoside form [17]. β-l and β-d fucofuranosides have been recently synthesized and exhibit typical coupling constants (α-f, J_1,2 : 3–5 Hz; β-f, J_1,2 : 0.5–1.5 Hz) different from those of fucopyranosides [17–19]. According to the relative signal intensities in the ¹H spectra, the three isomers were present at different proportion in the mixture, and with different anomeric ratio (Fig. 2A): 24.5% of α-l-Fucp-2-O-SO₃^– (ratio α/β = 80/20), 64% of α-l-Fucp-3-O-SO₃^– (ratio α/β = 32/68), 11.5% of α-l-Fucp-4-O-SO₃^– (ratio α/β = 37/63). For the reference sample α-l-fucopyranose in solution, the equilibrium anomeric ratio α/β was 30 : 70. It is well known that anomeric ratio α/β of pyranoses differ considerably between aldoses, and that different substitutions exert varying degrees of α-anomerization in saccharides [20]. In the O-sulfated fucoses mixture, it is interesting to note that 2-O-sulfo-fucose is the only isomer to exhibit major α-anomerization. This effect is probably related to an increase of the anomeric effect from the substitution on the vicinal 2-O position. Downfield shift effects of sulfation observed for ¹H and ¹³C sulfated positions (Tables 1 and 2) are in agreement with data described for sulfated polysaccharides [2,4,21–23].

Enzymatic reactions were carried out in NMR tubes, and spectra were recorded at appropriate time intervals. When the mixture of O-sulfated l-fucopyranosides was incubated at 298 K, the ¹H NMR spectra revealed a continuous decrease in the signals at 5.49 p.p.m. and 4.69 p.p.m. originating, respectively, from the anomeric protons H-1-α and H-1-β of 2-O-sulfo-l-fucopyranoside. Signals at 5.20 p.p.m. and 4.55 p.p.m. appeared and increased concomitantly, resulting from the formation of free unsulfated fucose, respectively, in the α and β anomeric form (Fig. 2B). Anomeric signals of H-1-α- and H-1-β-2-O-sulfo-l-fucopyranose vanished completely within 20 h of incubation with the enzyme. Anomeric signals of α/β-3-O- and 4-O-sulfated l-fucopyranosides remained unaffected. These results demonstrated a high regioselectivity of the sulfoesterase, which hydrolyzes only the sulfate group at the O-2 position of the fucopyranoside. Given its very high regioselectivity, this sulfoesterase is a helpful tool in the structural study of the fucoidan, particularly to address the question of the sulfation pattern and of its role in the biological properties of the polysaccharide.

Enzymatic desulfation of fucoidan oligosaccharides

Given the regioselectivity on synthetic monosulfated fucose, the sulfoesterase activity was then studied on fucoidan oligosaccharide prepared by mild hydrolysis of A. nodosum fucoidan. The oligosaccharides preparation obtained was analyzed by NMR prior to start the enzymatic reaction. The one-dimensional ¹H-NMR spectrum (Fig. 3A) displayed characteristic anomeric proton chemical shifts and coupling constants consistent with the presence of α-l-fucopyranosyl and β-l-fucopyranosyl units. Anomeric H-1 signals spread out from 5.6 to 4.9 p.p.m. correspond to α-l-isomers, whereas β-l-anomers exhibited H-1 signals from 4.9 to 4.5 p.p.m. These latter signals are strongly overlapped by the other ring proton resonances, but could be unambiguously assigned by their ¹³C anomeric chemical shifts through heteronuclear ¹³C–¹H-HMQC spectrum. The integral value of the methyl signals accounted for three protons and was normalized to 3 units. To one methyl group, i.e. three protons, correspond one anomeric proton so that the integral value of all anomeric protons (α + β) is normalized to 1 unit with a ratio of 0.74 unit for α-anomeric protons and 0.26 for β-anomeric protons, indicating that the oligosaccharides were mainly composed of α-l-fucopyranosyl residues.

Signals for the anomeric proton were distributed in several main groups on the spectrum, and were labeled from A to H for H-1-α-l-fucopyranosyl anomers, and I, J and K for H-1-β-l-fucopyranosyl anomers (Fig. 3A). Relative integrations of groups A to H showed that the major groups A and E accounted, respectively, for about 22% and 51% of α-L-H-1 signals. Owing to overlap of β-L-H-1 signals with other proton resonances, the relative ratio of group I, J, K could be only very roughly estimated as 25 : 50 : 25.

Starting from anomeric protons, connectivity from H-1 to H-4 could be established for all group resonances with TOCSY spectra. The TOCSY spectrum with 150 ms mixing time allowed determination of some H-5 and nearly all H-6 chemical shifts, which were confirmed by ROESY experiments.

The analysis of the ¹H chemical shifts of group A revealed that this signal at δ = 5.49 p.p.m. belongs to at least four residues (labeled A1, A2, A3 and A4 in Table 3), all sulfated at the O-2-position (downfield for H-2 of +0.6 to +0.7 p.p.m. with respect to H-2 of fucose). ¹H chemical shifts of A4 coincide exactly with those of the monosaccharide standard 2-O-sulfated fucose above described. The heteronuclear ¹³C–¹H-HMQC spectrum showed that C-1 of the four units A1, A2, A3 and A4 exhibited identical chemical shifts at 93.4 p.p.m. The HMBC spectrum showed only C-1/H-5 intramolecular coupling. It indicates that A1, A2, A3 correspond to terminal reducing fucose units or to monosulfated fucose for A4. For the second major group E, TOCSY and ROESY spectra revealed a strong overlap of at least four residues labeled E1, E2, E3 and E4 in Table 3. The units E1, E2, E3 are 2-O-sulfated according to downfield shift of H-2 (+0.70 p.p.m.), whereas E4 is a 2-O unsulfated unit. The minor unit E3 is also 4-O-sulfated (+0.91 p.p.m. for H-4 with respect to fucose) and is then a 2,4-di-O-sulfo-α-l-fucose unit. ROESY spectrum showed strong cross-peak correlation between H-1 of E1 and both H-4 (3.95 p.p.m.) and H-6 (1.36 p.p.m.) of α-A1 unit. The methyl signal at 1.36 p.p.m. for A1 was characteristic of a fucose branched at O-4 with another fucose [21]; all these data point to a 2-O-di-sulfated difucose α-E1-(1→4)-α-A1 as one of the main oligosaccharides. This α-(1→4) linkage is confirmed by HMBC spectrum where an anomeric carbon at 102.1 p.p.m. (E1) showed interresidue correlation with H-4 of A1 (3.95 p.p.m.).

Residues A2 and A3 that showed a methyl signal at 1.24 p.p.m. were not O-4 branched. Compared to A1 and A4, their stronger downfield shift of H-2 (+ 0.73 and 0.75 p.p.m. with respect to fucose) was in agreement with 2-O-sulfo-3-O branched fucose [2]. The ¹H chemical shifts of group B revealed that the signals belong to two residues B1 and B2 having similar nearest environment (Δδ < 0.02 p.p.m. between each proton). The strong downfield shift of H-2 and H-3 (+ 0.83 and + 0.86 p.p.m., respectively, with respect to fucose) corresponded probably to 2,3-di-O-sulfated residues, assuming that sulfation increments of chemical shifts are additive (Table 1). H-1 resonances of B1 and B2 are upfield shifted of −0.15 and −0.17 p.p.m and indicated an O-1 linkage. Methyl signals for B1 and B2 occurred at 1.23 p.p.m. (no O-4 linkage). The HMQC spectrum showed that H-1 for B1 and B2 were correlated, respectively, with carbons at 96.7 and 97.2 p.p.m. In the ROESY spectrum interresidue cross-peaks can be seen between H-1 of B1 and protons at 3.94 and 4.06 p.p.m. relative to H-4 and H-3 of A2 (A3). These data account for a tri-sulfated disaccharide α-B1-(1→3)-α-A2.

The ¹H chemical shifts of residue C are consistent with O-2 sulfation and O-1 linkage (H-1 upfield shifted of −0.14 p.p.m. with respect to H-1 of 2-O-sulfo-fucose). H-1 correlated in the HMQC spectrum with C-1 at 97.1 p.p.m. ROE between H-1 and a proton at 4.08 p.p.m. is obvious and could be assigned to H-3 of A2 or A3, but it remains ambiguous due to strong overlap of H-3 protons for residues A.

Residue D showed a downfield shift for H-3 (+0.73 p.p.m. relative to fucose) and for H-6 (δ = 1.36 p.p.m.). It can be therefore deduced that unit D is 3-O-sulfated and 4-O linked, in agreement with the ¹³C chemical shift of anomeric carbon (95.2 p.p.m.) that is similar to the C-1 chemical shift of 3-O-sulfo-α-l-fucose (Table 2). Despite the difficulty of ascertaining the linkage for E3 and E4, interresidue ROE from cross-peak between H-1 of these minor units at 5.22 p.p.m. and H-4 of D provides evidence for a α-E-(1→4)-α-D linkage.

Signals labeled G in Fig. 3A belong to two units G1 and G2 having a similar nearest environment. The strong downfield shift of H-3 (+0.75 p.p.m.) means that G1 and G2 units are 3-O-sulfated. An upfield shift of H-1 (−0.15 p.p.m.) with respect to 3-O-α-l-sulfo-fucose and a ROESY cross-peak between H-1 of G2 and a proton at 4.08 p.p.m. is in agreement with a linkage α-G2-(1→3)-α-A2 (or α-A3 because overlap of H-3 for A units).

Unit H is 4-O-sulfated (downfield shift of + 0.83 p.p.m. for H-4). The methyl chemical shift at 1.31 p.p.m. and ROESY correlation between H-1 and a methyl at 1.35 p.p.m. as well as with a proton at 4.14 p.p.m. indicated α(1→4) linkage. The proton at 4.14 p.p.m. was not clearly identified and the linked unit remained ambiguous.

The minor unit F (3%) corresponds to free α-l-fucose (same ¹H and ¹³C chemical shifts as for the reference fucose).

For β-l-fucosyl units labeled I, J, K in Fig. 3A, the only sulfated unit is I, which is 3-O-sulfated (downfield shift of +0.71 p.p.m. for H-3). Analysis as described above of α-l-units evidenced a 4-O link for I. A 1→4 linkage between the 2-O-sulfated α-l-fucopyranosyl E2 and I is indicated by HMBC spectrum where anomeric carbon of E2 (101.2 p.p.m.) showed a correlation with H-4 of I at 4.14 p.p.m. Furthermore, the ROESY spectrum showed strong cross-peak correlation between H-1 of E2 and H-6 of the β-I unit. This data is in agreement with the disaccharide α-E2 (1→4)-β-I.

Signal J corresponds to the overlap of two units J1 and J2. Analysis as described above of α-l-units showed a 3-O link of J1. Interresidue cross-peaks with H-1 of B2 was observed at 3.70 and 3.99 p.p.m., which correspond to β-H-3 and β-H-4 of J1 residue. This data agrees with a α-(1→3) linkage between residues B2 and J1.

Signal K displayed the same chemical shifts for all protons as for free β-l-fucose.

In summary, the fucoidan sample chosen for incubation with the sulfoesterase enzyme is a mixture of monosaccharides (α- and β-l-fucose and 2-O-sulfo-α-l-fucose) and disaccharides (mono, di and trisulfated), α-(1→4) and α-(1→3) linked. The NMR spectroscopy analysis of the mixture is in agreement with data reported for fractions of A. nodosum fucoidan of higher molecular mass (M_r≈ 3900–10 000) [4].

After incubation at 25 °C for 24 h, the ¹H-NMR spectrum (Fig. 3B) exhibited a significant decrease in the signals originating from the 2-O sulfated residues of the main groups of α-l-anomers A and E. With regards to all α-H-1-anomeric protons, a decrease of 41% and 66% was observed, respectively, for H-1-A units and for H-1-E units. Simultaneously, free α-l-fucose F and β-l-fucose K exhibited an increase in signal (10% instead of 3% initially for F).

It is noteworthy that protons of the initial major groups A and E, of which all but E4 were 2-O-sulfo-α-l-fucopyranosyl units, strongly decrease, whereas 3-O or 4-O-sulfo-α-l-fucosyl units (G and H) remained unaffected. In the same manner chemical shifts observed initially for β-H-1 protons of the 3-O-sulfo-β-l-fucosyl unit I remained unchanged (Δδ≈ 0.02 p.p.m.). New additional signals labeled M, N and P simultaneously appeared that accounted, respectively, for 7, 17 and 15% of all α-H-1 anomer signals and were 2-O-unsulfated units.

The remaining signals A (labeled A′1, A′2, A′3, Table 4, Fig. 4) exhibited the same chemical shifts for all protons as initially (Δδ ≈ 0.02 p.p.m.) and remained 2-O-sulfated, but anomeric signals were more spread out. For example, the methyl signal of unit A′1 appeared at 1.30 p.p.m. instead of 1.36 p.p.m. before the enzymatic reaction. This indicated that the initial 4-O linked unit E1 is modified, probably due to the enzymatic hydrolysis of its O-2 sulfate group. The remaining signals arising from the group E corresponded to at least three units (labeled E′1, E′2, E′3, Table 4, Fig. 4), and showed two kinds of anomeric carbon shifts at 95 and 101.5 p.p.m. (Fig. 5). Anomeric carbon at 101.5 p.p.m. correlated in HSQC-TOCSY spectrum with H-1 to H-4 protons of unit E′1; these protons showed downfield shifts (downfield ≈ +0.7 and +0.9 p.p.m. of H-2 and H-4 resonances) characteristic of a 2,4-di-O-sulfation. Both E′2 and E′3 are 2-O-unsulfated, E′3 being in addition 4-O-sulfated. As it did not exist previously, it is likely that E′3 results from the 2-O-desulfation of the initial E3 unit (2,4-di-O-sulfo-fucose). In NOESY spectra, cross-peak correlations between H-1 of E′1 (E′2), E′3 and methyl signals at 1.36 and 1.33 p.p.m. displayed 1→4 linkages with J and I-β-units.

A new anomeric α-H-1 labeled M corresponds to a 3-O-sulfated unit (downfield of H-3 proton), 4-O linked given the ¹H methyl resonance at 1.31 p.p.m. and significant downfield of + 7 p.p.m. for C-4 carbon. New emergent units labeled N and P with H-1 at 5.00 p.p.m. and 4.96 p.p.m. are 2-O-unsulfated and showed anomeric carbons at 102.8 and 103.5 p.p.m. In the NOESY spectrum, interresidue NOEs can be seen between H-1 at 5.00 p.p.m. and a methyl signal at 1.33 p.p.m. (large envelope) as well as with signals at 4.12 and 4.17 p.p.m. Cross-peak correlation is observed between H-1 at 4.96 p.p.m. and a methyl signal at 1.30 p.p.m. as well as with a H-4 signal at 3.92 p.p.m. HMBC spectrum confirmed those H-1/H-4 correlations and established 1→4 linkages. The pattern of NOEs observed for the anomeric proton of N could be compatible with both 1→4 linked disaccharides: α-N-(1→4)-β-I and α-N-(1→4)-α-M. Correlations seen for H-1 of P could be in agreement with α-P-(1→4)-α-A′1.