2.4.1. ESI- MS<sup>2</sup> Analyses of Ion at *m/z* 1477.7

Tandem MS analyses revealed the presence of two different peaks with *m/z* value of 947.5 and 981.3, corresponding to the losses of different aglycone moieties with *m/z* values of 530 and 496, respectively, confirming the presence of chemical structural isomers. Further this MS<sup>2</sup> analysis also distinguished the presence of an acetoxy group in both isomer types.

Similar to sulfated compounds, after collisional activation, the parent ions are subjected to three independent dissociation pathways shown using full and dotted arrows (Figure 4). First, the consecutive losses of the deacetylated aglycone, acetoxy group, MeGlc, Qui, Xyl, Xyl and MeGlc residues (blue arrows) followed by Glc afford product ions as shown in Figure 4 confirmed the structure of Lessonioside A. Therefore, in this case, the ions at *m/z* 493.1 correspond to the sodiated key diagnostic sugar residue; [MeGlc-Glc-Xyl + Na]+.

Secondly, the decomposition of the parent ion could also be triggered by the loss of sugar moieties followed by the deacetylated aglycone residue which generated daughter ions as shown in Figure 3e confirming once more the structure of Lessonioside A. It is clear that the ions at *m/z* 493.1 correspond to the sodiated deacetylated aglycone moiety (*m/z* value of 470). Alternatively, ions corresponding to the sequential losses of Glc, Qui, Glc, AcOH, MeGlc, Qui, and Xyl (red dotted arrows) were detected in Figure 3g indicating the presence of another isomer, Lessonioside B, which possesses two Qui units. The presence of two Qui in the carbohydrate chain of sea cucumber glycoside is a very rare characteristic.

Finally, the fragmentation of the parent ions can also be initiated with the loss of the acetoxy group. The consecutive losses of the acetic acid (AcOH) and the deacetylated aglycone unit followed by the sequential losses of the sugar moiety (Figure 3b) further confirmed the structure of Lessonioside A. Alternatively, the decomposition of the deacetylated saponins can be accomplished by the sequential

losses of monosaccharides in the sugar chain, namely ion detected at 1417.7 [M − AcOH + Na]<sup>+</sup> (black dotted arrows, Figure 3h). In this case, the ions at 493.1 corresponds to the DeAc Agl moiety [M − sugar residue − AcOH + Na]+. Alternatively, the sequential losses of AcOH and sugars from the parent ions (Figure 3i), afforded daughter ions that assisted in postulating the structure of another new isomer, Lessonioside D. The above evidence suggested that Lessonioside A possesses the same aglycone as Lessoniosides B and D, but differs in the hexasaccharide chain. The complete analyses can be seen in Supplementary Figure S3.

The MALDI-MS2 and ESI-MS2 analyses for all possible isomers were carried out in a similar manner as described above for Lessoniosides A, B, C and D. A comprehensive list of possible fragmentation patterns based on the MS<sup>2</sup> ions generated from the ion at *m/z* 1477.7 is shown in Figure 3.

The sugar moiety of Lessonioside A was found to be identical to those of Cladolosides C1 and C2 isolated from the sea cucumber *Cladolabes schmeltzii* [16], confirming the constituents of the hexasaccharide chain (Figures 1 and 3). The sugar component also had some similarity to those of Violaceuside B isolated from the sea cucumber *Pseudocolochirus violaceus* [29]. This group also stated the ions at *m/z* 625.2 and 493.1 corresponded to [MeGlc + Xyl + Glc + Xyl + Na]+ and [MeGlc + Xyl + Glc + Na]+, respectively, which confirmed our results. Yayli and associates [30], however, stated the ions at *m/z* 493 and 325, corresponding to [MeGlc-*O*-Xyl-*O*-Qui(*O*)-*O*] <sup>+</sup> and [MeGlc-*O*-Xyl]+, respectively, which are under question. The structure of the aglycone moiety was also very similar to that of Holothurinoside Y [12], the difference being the addition of an acetoxy group at C-16. The assignments of the MS2 signals associated with the aglycone moiety Lessoniosides A, B and D showed a close similarity to those reported for 16β-acetoxy-holosta-9-ene-3β,12α,17α-triol, the aglycone of Nobiliside C [*m/z* 715], *m/z* 656 [M − OAc + Na]+, isolated from the sea cucumber *Holothuria nobilis* [31].

These saponin congeners identified from Fraction 18 are more conjugated with glycosides compared with the new saponins previously reported in this species [3,12]. Lessoniosides C, D and E possess the same terminal saccharide moiety (MeXyl), which is a rare structural feature among naturally occurring sea cucumber glycoside and has been infrequently reported.
