*2.1. Structure Determination of Saponins by ESI-MS*

ESI-MS*<sup>n</sup>* is a very effective and powerful technique to distinguish isomeric saponins as they generate different MS*<sup>n</sup>* fragmentation profiles [27,28]. All saponin ions perceived in the ESI-MS spectrum of the HPCPC fractions were also analyzed by ESI-MS2 in the positive ion mode. Previous MS<sup>2</sup> studies on HPCPC fractions 12, 14, 15 [3], 17, 18, 20 and 22 [12] obtained from the butanolic extract of viscera of sea cucumber *H. lessoni* yielded a number of new saponins. This analysis of fraction 18 gave complex spectra representing several saponin classes, also confirmed the presence of saponins reported in the literature and identified new saponin congeners (Supplementary Figure S1, and Figure 1 of [12]). Fifteen major peaks were detected which corresponded to several known triterpene compounds (as summarized in Table 1 of [12]), including Holothurinosides C/C1, Desholothurin A1 and Desholothurin A (synonymous with Nobiliside 2a), Holothurinoside J1, Fuscocinerosides B/C or Scabraside A or 24-dehydroechinoside A and Holothurin E, Holothurin A, Holothurinosides E/E1/O/P, Holothurinoside M, Holothurinosides A/A1/R/R1/S/Q, Holothurinoside N, Holothurinoside I and Holothurinoside K1 in addition to several new saponins [3,12]. The spectrum displays one dominant peak at *m/z* 1477.7, which corresponds to unidentified (new) saponins, with elemental compositions of C68H110O33, C66H102O35 and C66H118O34.

**Figure 1.** The structures of the new acetylated saponins in the viscera of *H. lessoni*, Lessoniosides (**A**–**E**) along with the non-acetylated Lessoniosides (**F**–**G**) compounds are described in this figure.

This analysis revealed that HPCPC Fraction 18 contains several saponin congeners showing that the absolute purification of the saponins was not possible within a single HPCPC run with these closely related compounds.

#### *2.2. Structure Identification of Saponins by MALDI-MS*

Similar to the ESI-MS, the MALDI MS of the isobutanol-enriched saponin extract obtained from the viscera of the *H. lessoni* revealed the presence of at least 75 saponin congeners, including 39 new sulfated, non-sulfated and acetylated triterpene glycosides, and 36 congeners which were previously reported in other holothurians [3].

To elucidate the chemical structure of saponins based on the MS<sup>2</sup> spectra, as described previously [3,12], precursor ions were selected, fragmented and fragmentation profiles built. The molecular structures of the saponins were determined by the identification of the mass transitions between the successive collision-induced fragmentation peaks on the basis of the accurate mass of the individual sugar components.

Based on the literature, MeGlc and MeXyl are always terminal sugars and Xyl is always the first sugar, which is bound to C-3 of the aglycone. Further, the exact mass of each sugar, such as MeGlc = 176 Da, Glc = 162 Da, Xyl = 132 Da, Qui = 146 Da, and the determination of the mass transitions between the peaks on the basis of the accurate mass of the individual sugar moieties, and mass and sequence of the key diagnostic peaks helped us build the sequence of these sugar moieties. Using this strategy the structure of seven new triterpene glycosides from *H. lessoni* with an *m/z* value of 1477.7 from HPCPC fraction 18 were characterized.

The chemical structures of the new acetylated saponins from the viscera of *H. lessoni* are illustrated in Figure 1. Lessoniosides A, B, C, D and E are the only published examples of glycosides from *H. lessoni* containing the side chain of the acetoxy group in their aglycone moieties. We now provide an account of the structure elucidation of these saponins using this approach.
