*2.2. NMR Analysis*

NMR spectroscopy analysis is the most frequently used analytical technique for the structural characterization of sulfated glycans [25]. The analysis of 1H-NMR, as seen in Figures S2 and S3, COSY, as seen in Figures S4 and S5, and HSQCed, as seen in Figure 2 and Figure S6, spectra resulted in the identification of four main structural units of SP1 and SP2, labeled A to D, as seen in Figure 2A,C. The signals of the anomeric hydrogens of units A, B, C, and D were 4.69, 4.52, 4.82, and 4.69 ppm, respectively. These anomeric values indicated that all identified units are in the β configuration [26,27]. With the chemical shifts of the anomeric hydrogens, it was possible to determine the 1H/13C correlation in an HSQCed experiment, as seen in Figure 2A.

**Figure 2.** 2D-NMR spectra of SP1. (**A**) 1H/13C correlation 2D-NMR spectrum (HSQCed). (**B**) 1H/13C correlation 2D-NMR spectrum (HSQCed) of pyruvate. (**C**) 1H/1H correlation 2D-NMR spectrum (COSY). (**D**) 1H/13C correlation 2D-NMR spectrum (HMBC) of pyruvate.

Table 2 presents the results of the HSQCed analysis. As can be observed, SP1 and SP2 are very similar because these polysaccharides have many common structural characteristics, which can be confirmed by analyzing the spectra present in Supplementary Figures S2–S6. Therefore, only the spectra of SP1 are presented in Figure 2. The main difference between the two SPs was the signal of position 4 of unit B: in SP1, it was at 4.27/69.8 ppm, whereas in SP2, it was at 5.00/78.3 ppm. This result indicated the presence of a sulfate group at position 4 of a galactose in SP2 [28]. Furthermore, anomeric signals of 4.90/103.6 and 4.87/104.5 ppm were identified in SP2, but it was not possible to identify the other positions of the unit. These values have been described as characteristic of pyruvate galactan structural units in positions 4 and 6 [29] and in positions 3 and 4 [30], respectively.


**Table 2.** Signal assignments (ppm) of NMR spectra of SP1 and SP2 from *C. cupressoides*.

n.d. = not detected. Signals reported in literature: a Fernández et al. [30]; b Bilan et al. [28]; c Arata et al. [29].

Through the analysis of HSQCed spectra, it was also possible to determine each of the 4 structural unit constituents of the SPs. Unit A presents anomeric correlation between hydrogen and carbon at 4.69/105.5 ppm. In addition, it was possible to observe the presence of a signal at 1.48/26.4 ppm corresponding to pyruvate, as seen in Figure 2B, as one of the substituents of units A and C, as seen in Table 2, linked at O-4 and O-6. The presence of pyruvate was also confirmed in the HMBC spectrum, as seen in Figure 2D. Thus, both the A and C units are in the form of 4,6-*O*-(1-carboxyethylidene)-β-<sup>d</sup>-galactose or <sup>→</sup>3)4,6Pyr-β-<sup>d</sup>-Gal*p*-(1<sup>→</sup>. Unit B has 4.52/104.4 ppm as H1/C1 correlation. In addition, there are chemical shifts at 3.81/84.4 and 4.00/71.0 ppm at positions 3 and 6, respectively, which are characteristics of β(1→3,6)-linked units [28]. Unit C presents an anomeric signal at 4.82/103.9 ppm, but also shows signals of pyruvylation at positions 4 and 6. However, when compared to unit A, it is possible to observe that positions C1 and C2 are deshielded, probably due the presence of a sulfate group at C2 [29]; this unit was identified as <sup>→</sup>3)4,6Pyr-β-D-Gal*p*2S-(1<sup>→</sup>. Unit D has 4.69/105.5 ppm as H1/C1 correlation and a chemical shift of position 3 (3.81/84.4 ppm) corresponding to →3)-β-D-Gal*p*-(1→ [28].

We detected some signals that are not part of any of the four systems we have marked, as seen in Table 2. Costa et al. [23] showed CBB-F1.0 made of galactose:xylose:mannose (1.0:0.6:0.1) and traces of fucose and rhamnose. Thus, these signals may be indicative of the presence of these monosaccharides in SP1 and SP2. The signal at 4.41/68.5–69.5 ppm corresponds to H5/C5 correlation of fucose units [31]. The presence of O-methyl-mannose was confirmed by the signal at 3.49/57.8–58.8 ppm [32]. We also found two signals that correspond to O-methyl-rhamnose (3.57/80.6 ppm) and (3.42/81.8 ppm) [33,34]. However, we were not able to identify spin systems that confirm the presence of these monosaccharides. Most likely, these sugars can be randomly distributed throughout the galactan structures.

Overall, the studies of NMR spectra ensure that the <sup>→</sup>3,6)-β-<sup>d</sup>-Gal*p*-(1<sup>→</sup> is the predominant unit of these polysaccharides, as seen in Figure 3B. However, galactopyranosyl units linked by β1→3

linkages are also found, as seen in Figure 3D, as there are units containing 3,4-*O*-(1' carboxy)-ethylidene, as seen in Figure 3A, and 2-*O*-sulfate, as seen in Figure 3C.

**Figure 3.** Proposed structures of the components found in the SP1 and SP2. The radical R corresponds to hydrogen atoms (H) or sulfate groups (SO4); however, for SP1, R = H. (**A**): <sup>→</sup>3)4,6Pyr-β-<sup>d</sup>-Gal*p*-(1<sup>→</sup>; (**B**): <sup>→</sup>3,6)-β-<sup>d</sup>-Gal*p*-(1<sup>→</sup>, for SP1 and <sup>→</sup>3,6)-β-<sup>d</sup>-Gal*p*4S-(1<sup>→</sup>, for SP2; (**C**): <sup>→</sup>3)4,6Pyr-β-<sup>d</sup>-Gal*p*2S-(1<sup>→</sup>; (**D**): <sup>→</sup>3)-β-<sup>d</sup>-Gal*p*-(1<sup>→</sup>.

The presence of β-<sup>d</sup>-galactans in green algae has also been reported in other works. Ciancia et al. [35] identified a 3-linked β-<sup>d</sup>-galactan, partially sulfated at C6 and pyruvylated at positions 4 and 6, from *Bryopsis plumosa*. Species of the genus *Codium* synthesize galactans consisting of partially sulfated C4 and/or C6 β-<sup>d</sup>-galactopyranose residues with significant pyruvate content [26–28,30]. Sulfated and pyruvylated galactans were also isolated from the tropical green algae species *Penicillus capitatus*, *Udotea flabellum*, and *Halimeda opuntia* [29]. However, studies on sulfated and pyruvylated galactans obtained from green algae with immunostimulating properties are scarce. Because of that, a question arose. Would these galactans be responsible for the immunomodulatory activity that was observed in CCB-1.0 and was it shown by Barbosa et al. [24]? Therefore, the effect of these galactans on the levels of some immunomodulation mediators (NO, ROS, and cytokines) was evaluated.
