2.4.3. Non-Acetylated Isomeric Congeners

Based on the chemical evidence the structure of Lessoniosides F/G were defined as 16,22-diketo-holosta-9(11)-23(24)-25(27)-triene-3β,12α,17α-triol, which is shown in Figure 1. They are of the lanosterol type featuring the characteristic ring-D-fused γ-lactone (C=O) function and a Δ<sup>9</sup> double bond. The aglycone of Lessonioside F/G differs from E by the presence of a double bond at Δ<sup>25</sup> and a loss of the AcO group at C-25 to form an exo double bond, Δ25.

On fragmentation these isomers generate ions at 981.3 corresponding to the loss of aglycone. Subsequent losses of the sugar moieties are described in detail in Figure 3 (k and l) and Supplementary Figure S4 for Lessoniosides F and G. These non-acetylated Lessoniosides possess similar aglycones

but different sugar moieties. It is notable that all three feasible independent fragmentation pathways might occur simultaneously which generated several different fragmentation sequences.

The molecular weights of the deacetylated aglycone moieties in some of these new saponins (Lessoniosides A, B and D) coincided with those reported for Philinopside B, from the sea cucumber *Pentacta quadrangularis* using ESI-MS by Zhang *et al.* [34], however, the structures are very different, because they are from a different family of sea cucumber, with ring closure of the C-20 side chain.

The cleavage of the *O*-acetyl group of the aglycone residue results in the loss of AcOH (60 Da). However, Song and co-workers [27] noted the neutral losses of CH2O (30 Da) and C2H4O2 (60 Da) in cross-ring reactions of the sugar residues. The cross-ring cleavage of the sugar also leads to the loss of C2H4O2 (60 Da).

The losses of H2O and CO2 or their combination results from cleavage at the glycosidic linkages as noted by Waller and Yamasaki [2]. The losses of CO2 (44 Da), H2O (18 Da), AcOH (60 Da), Acetyl group (42 Da) and CH2O (30 Da) were detected from the spectra which affords different product ions and several peaks were assigned to those molecules. For instance, the ion at *m/z* 1373.7 was generated by the loss of CO2 from the deacetylated parent ions (*m/z* 1417.7), or the sequential losses of H2O and acetyl molecule from the ions at *m/z* 511.2 generated ions at *m/z* 493.1 and 451.1, respectively. Further the ion at *m/z* 493.1 can be stemmed from the ion at *m/z* 553.2 (sodiated aglycone) by the loss of the acetoxy group. Additionally, the loss of CH2O from the ion at *m/z* 451.1 yields the ion at *m/z* 421.1. However, Kelecom and coworkers [35], and Kitagawa and associates [36], stated the latter ion as an aglycone fragment. The complete analyses can be seen in the Supplementary Figure S3.

The MS<sup>2</sup> analyses of ions at *m/z* 1477.7 revealed a similar fingerprint profile with those reported for Holothurinosides X, Y and Z, in particular in the area of 100 to 600 Da where the signals were coincident with those of ion at *m/z* 1127.6, which show the intrinsic relationship between these saponin congeners [12].

Lessoniosides A, B, C, D and E are the only examples of glycosides from *H. lessoni* containing an acetoxy side chain in their aglycone moieties. Most of the glycosides isolated from sea cucumber *Holothuria* are Δ9,11- glycosides. In general, 3β-hydroxyholost-9(ll)-ene based aglycones were characterized in Holothurins isolated from animals of the order *Aspidochirota* [17].
