*2.1. Positive and Negative Ion Mode ESI-MS*/*MS Analyses of Oxidized Cerebrosides*

The molecular formulae, C49H93NO12 for allylic hydroperoxides (**1a**, **1b**, **2a**, **2b**, **3c**, **3d**), C49H93NO11 for allylic alcohols (**1a**/ , **1b**/ , **2a**/ , **2b**/ , **3c**/ , **3d**/ ), and C49H91NO11 for enones (**1a**//, **1b**//, **2a**//, **2b**//, **3c**//, **3d**//), were determined by HR-ESI-MS (high resolution ESI-MS) analyses in positive-((+)ESI-MS) and negative-((–)ESI-MS) ion modes. Complementary (+)ESI- and (–)ESI-MS/MS analyses of these glucosylceramides, containing 2-hydroxy acyl chains and phytosphingosine-type backbones, resulted in a series of fragment ions, as shown in Scheme 1.

**Scheme 1.** Designations of the fragment ions observed in the ESI-MS/MS spectra of (**a**) [M + Na]<sup>+</sup> and (**b**) [M − H]<sup>−</sup> ions of the cerebrosides reported here. These designations are consistent with the ones proposed by Ann and Adams [9]. In addition, [*Z*0/*Q* − C3H5N (55 Da)] ions (not shown) were observed in the MS/MS spectra of the [M <sup>−</sup> H]– ions. The fragment *<sup>Z</sup>*0/*<sup>Q</sup>* was also referred to as a *<sup>Z</sup>*0/*<sup>K</sup>* ion [10].

Many of the fragment ions, shown in Scheme 1, have also been detected in our ESI-MS/MS studies of non-oxidized cerebrosides isolated from *Aulosaccus* sp. Namely, (+)ESI-MS/MS spectra of sodium adducts from non-oxidized glycosphingolipids have been characterized by prominent peaks, corresponding to [M + Na]<sup>+</sup> (base peak), *Y*0, *Z*0, *O*, and *C*<sup>1</sup> ions, and by small peaks, representing [M + Na <sup>−</sup> H2O]<sup>+</sup>, *E*, and *B*<sup>1</sup> ions. However, the (+)ESI-MS/MS spectrum of the [M + Na]<sup>+</sup> ion of isomeric hydroperoxy cerebrosides (Figure 2) showed other relative abundances for a variety of these ions. In particular, the *O* ions of *m*/*z* 528.35, representing isomeric monoglucosylated monounsaturated C20 sphingoid base backbones **1** and **2**, constituted the base peak of this spectrum. The spectrum also exhibited a homologous, less abundant *O*/ ion (*m*/*z* 542.36), containing a monoglucosylated cyclopropane C21 sphingoid base backbone **3**. A relatively low intensity pseudo-molecular ion peak (*m*/*z* 910.65, [M + Na]+) and very small peaks, corresponding to *Y*<sup>0</sup> (*m*/*z* 748.60) and *Z*<sup>0</sup> (*m*/*z* 730.58) ions (not shown), were observed. The presence of the [M + Na − Н] <sup>+</sup> peak, comparable with the pseudo-molecular ion peak of the hydroperoxides, was explained by hydrogen atom abstraction, followed by electronic delocalization in the resulting radical, which might yield rearranged products. At the same time, the (+)ESI-MS/MS spectrum revealed more abundant [M <sup>+</sup> Na <sup>−</sup> 102]<sup>+</sup> (*m*/*<sup>z</sup>* 808.55), [M + Na <sup>−</sup> 88]<sup>+</sup> (*m*/*z* 822.56), [*Y*<sup>0</sup> <sup>−</sup> 102] (*m*/*z* 646.505), [*Y*<sup>0</sup> <sup>−</sup> 88] (*m*/*z* 660.52), [*Z*<sup>0</sup> <sup>−</sup> 102] (*m*/*z* 628.49),

and [*Z*<sup>0</sup> −88] (*m*/*z* 642.50) ions. These fragments and fragment [*W* −102] (*m*/*z* 275.20), each with a terminal α,β-unsaturated aldehyde, were thought to arise from specific α-cleavages of lipid hydroperoxides (Figure 2). Related fragment ions, formed by CnH2n+2O losses from [M + Na]<sup>+</sup> ions, were also found in the MS/MS studies of some monoenoic [6] and polyenoic [11–13] FA moieties or free FAs, in which an allylic hydroperoxy group was between double bond(s) and a terminal methyl group. In general, the [M + Na <sup>−</sup> CnH2n+2O]<sup>+</sup> ions were also more abundant in MS/MS spectra of those compounds compared to their [M + Na]<sup>+</sup> ions.

Ions, possibly formed by Hock cleavage [14,15], were insignificant in our (+)ESI-MS/MS analysis of allylic hydroperoxides. These fragments included *m*/*z* 796.55 (for **1b**, **2b**, and **3d**) and 782.54 (for the allylic isomers of **1a**, **2a**, and **3c**) ions, as illustrated in Scheme S1 (Supplementary Materials) and Figure 2 (allylic rearrangements for acyl chains **a** and **c** are not shown). In contrast, the relatively abundant ions, presumably formed by analogous cleavage from hemiacetal derivatives, were reported for MS/MS fragmentations of [M + Na]<sup>+</sup> precursors of some free polyenoic FAs in which an allylic hydroperoxy group was between the double bond system and C-1 [12,13].

Favorable cleavage of the isomers, formed by allylic rearrangements of acyl chains **b** and **d** (Figure 2), occurred, losing an 88 Da fragment. At the same time, compounds with parent acyl chains **b** and **d** underwent other favorable fragmentations, yielding a distinct group of homologous ions (from *m*/*z* 668.44 to 738.50), containing the most significant fragment of *m*/*z* 724.49. We suggest that the occurrence of these ions may be connected with homolysis of a weak RO-/-OH bond, formation of an alkoxyl radical, and subsequent formation of a radical centered on a remote and non-activated carbon atom of a saturated hydrocarbon chain. This may lead to fast cyclization that, in turn, leads to MS fragmentations of the resulting cyclic ethers, shown in Scheme 2. The proposed cyclization reaction is reminiscent of the formation of cyclic (mainly, five-membered) ethers from acyclic saturated monohydroxy alcohols, occurring via radical (primarily alkoxyl) intermediates under appropriate chemical and thermal conditions (for reviews, see References [16,17]). In this process, secondary aliphatic alcohols yielded 2,5-dialkyltetrahydrofurans [18]. A possible mechanism for the formation of such products includes 1,5-transposition of the radical center from the oxygen atom of the alkoxyl radical to δ-carbon atom, involving a 1,5-hydrogen transfer through a chair-like six-membered transition state. For a linear hydrocarbon chain with an initial alkoxyl radical, the 1,5-hydrogen atom transfer may be considered the most common reaction, even though intramolecular abstractions of the hydrogen atom from other positions (1,4-migrations, 1,6-migrations, and 1,7-migrations, etc.) may be observed [17]. Similar processes may have occurred in our MS/MS experiment because alkyl hydroperoxides are known to form alkoxyl radicals by thermal or photolytic decomposition [17,19]. Apparently, the ability to undergo favorable cyclic transition states affected the fragmentation process, leading to the formation of major homologous ions, as illustrated in Scheme 2.

Ion intensity profiles, obtained for cerebrosides with allylic hydroxy or keto groups, were, in general, similar to those of non-oxidized cerebrosides found in *Aulosaccus* sp. In particular, the MS/MS spectra of sodiated molecular ions of allylic alcohols (Figure 3a: *m*/*z* 894.66 [M + Na]+) and enones (Figure 3b: *m*/*z* 892.64 [M + Na]+) showed predominant peaks of pseudo-molecular ions, along with peaks of lower intensities, which represented [M <sup>+</sup> Na <sup>−</sup> H2O]<sup>+</sup>, *<sup>Y</sup>*0, *<sup>Z</sup>*0, and *<sup>O</sup>* ions. However, unlike [M <sup>+</sup> Na]<sup>+</sup> ions of non-oxidized cerebrosides, the sodium adducts of allylic alcohols and enones fragmented to give discernible ions of *W*-type and minor *U* and *T* ions. Additionally, the acyl-containing ions of allylic alcohols had a tendency to lose one, or even two, hydrogen atoms. In this case, a trend was observed toward the increased loss of hydrogen atoms with decreasing ion masses. For example, a significant difference was noted between the relative intensities of [M + Na]<sup>+</sup> and [M <sup>+</sup> Na <sup>−</sup> H] + peaks ([M <sup>+</sup> Na <sup>−</sup> 2H]<sup>+</sup> ions were not even detected), but the intensities of *<sup>W</sup>*, [*<sup>W</sup>* <sup>−</sup> H], and [*<sup>W</sup>* <sup>−</sup> 2H] peaks were comparable (Figure 3a).

**Scheme 2.** Possible formation of the homologous ions of *m*/*z* 738.50, 724.49, and 710.48.

**Figure 3.** (+)ESI-MS/MS spectra of [M + Na]<sup>+</sup> ions of isomeric (**a**) allylic alcohols **1a**/ , **1b**/ , **2a**/ , **2b**/ , **3c**/ , **3d**/ and (**b**) enones **1a**//, **1b**//, **2a**//, **2b**//, **3c**//, **3d**//.

In (−)ESI-MS/MS experiments with [M − H]<sup>−</sup> and [M + Cl]<sup>−</sup> ions (Figure 4a–c), allylic hydroperoxides again produced more fragments than allylic alcohols and enones. The main feature of (−)ESI-MS/MS fragmentation of [M − H]−, *Y*0, *Z*0, *Z*0/*Q*, *T*, *W*, and other precursor ions, containing an allylic hydroperoxy group, was the loss of water to produce fragments with enone functionality in acyl chains, as described for [M − H]<sup>−</sup> ions of hydroperoxy-eicosatetraenoic acids [20]. In particular, the homologous ions of *m*/*z* 436.4 ([*Z*0/*Q* − H2O]) and 422.4 ([*Z*<sup>0</sup> / /*Q* − H2O]), containing acyl C23 and C22 chains, respectively (Figure 4a), were also observed in (−)ESI-MS/MS spectrum of enones (Figure 4c). Then, relatively low-intensity pseudo-molecular ion peaks ([M + Cl]<sup>−</sup> and [M − H]−) and very small peaks, corresponding to *Y*<sup>0</sup> and *Z*<sup>0</sup> ions (not shown), were seen in the (−)ESI-MS/MS spectrum of allylic hydroperoxides. A discernible [M − 2H] peak was comparable with a pseudo-molecular [M − H] peak. This spectrum also exhibited [M + Cl − 102]−, [M + Cl − 88]−, [M − H − 102]−, [M − H − 88] −, and [*Z*0/Q − 102] ions, interpreted as α-cleavage ions with a terminal α,β-unsaturated aldehyde. Additionally, the two minor ions of *m*/*z* 743.6 and 729.55 could be formed by α-cleavages of compounds in which an allylic hydroperoxy group was between a double bond and C-1/ . In particular, the *m*/*z* 743.6 ions could be fragments of isomeric compounds **1b**, **2b** (C-15/ <sup>−</sup>C-16/ bond fission), and **3d** (C-14/ <sup>−</sup>C-15/ bond fission), while the less abundant *m*/*z* 729.55 ions could be fragments of the allylic isomers of compounds **1a**, **2a** (C-14/ <sup>−</sup>C-15/ bond fission), and **3c** (C-13/ <sup>−</sup>C-14/ bond fission).

**Figure 4.** *Cont*.

**Figure 4.** (−)ESI-MS/MS spectra of [M + Cl]<sup>−</sup> and [M − H]<sup>−</sup> ions of isomeric (**a**) allylic hydroperoxides **1a**, **1b**, **2a**, **2b**, **3c**, **3d**, (**b**) allylic alcohols **1a**/ , **1b**/ , **2a**/ , **2b**/ , **3c**/ , **3d**/ , and (**c**) enones **1a**//, **1b**//, **2a**//, **2b**//, **3c**//, **3d**//.

Like the (−)ESI-MS/MS spectra of the non-oxidized cerebrosides of *Aulosaccus* sp., those of allylic alcohols (Figure 4b) and enones (Figure 4c) exhibited significant peaks corresponding to [M − H]<sup>−</sup> and *Z*0/*Q* ions, with lower intensity peaks representing [M + Cl]−, *Y*0, *Z*0, [*Z*0/*Q* − C3H5N], and *W* ions. The negatively charged acyl-containing ions of allylic alcohols (Figure 4b), like their positively charged acyl-containing counterparts (Figure 3a), tended to lose hydrogen atoms in the MS/MS experiment.
