Structural Analysis of Oxidized Cerebrosides from the Extract of Deep-Sea Sponge Aulosaccus sp.: Occurrence of Amide-Linked Allylically Oxygenated Fatty Acids

The structural elucidation of primary and secondary peroxidation products, formed from complex lipids, is a challenge in lipid analysis. In the present study, rare minor oxidized cerebrosides, isolated from the extract of a far eastern deep-sea glass sponge, Aulosaccus sp., were analyzed as constituents of a multi-component RP-HPLC (high-performance liquid chromatography on reversed-phase column) fraction using NMR (nuclear magnetic resonance) spectroscopy, mass spectrometry, GC (gas chromatography), and chemical transformations (including hydrogenation or derivatization with dimethyl disulfide before hydrolysis). Eighteen previously unknown β-D-glucopyranosyl-(1→1)-ceramides (1a–a//, 1b–b//, 2a–a//, 2b–b//, 3c–c//, 3d–d//) were shown to contain phytosphingosine-type backbones (2S,3S,4R,11Z)-2-aminoeicos-11-ene-1,3,4-triol (in 1), (2S,3S,4R,13Z)-2-aminoeicos-13-ene-1,3,4-triol (in 2), and (13S*,14R*)-2-amino-13,14-methylene-eicosane-1,3,4-triol (in 3). These backbones were N-acylated with straight-chain monoenoic (2R)-2-hydroxy acids that had allylic hydroperoxy/hydroxy/keto groups on C-17/ in the 15/E-23:1 chain (a–a//), C-16/ in the 17/E-23:1 (b–b//) and 14/E-22:1 (c–c//) chains, and C-15/ in the 16/E-22:1 chain (d–d//). Utilizing complementary instrumental and chemical methods allowed for the first detailed structural analysis of a complex mixture of glycosphingolipids, containing allylically oxygenated monoenoic acyl chains.


Introduction
Lipid hydroperoxides are labile compounds derived from lipids containing carbon-carbon double bonds. The formation of these primary peroxidation products occurs in enzymatic and non-enzymatic (autooxidation, photo-oxidation) reactions [1]. Lipid hydroperoxides, formed in biological systems, have not only multiple damaging effects on cellular macromolecules, but are also important regulators of many cellular processes [2]. In some pathological situations, lipid hydroperoxides are generated at higher than normal rates. Such overproduction is implicated in several human diseases and exposures including atherosclerosis, cancer, diabetes, acute lung injury, chronic alcohol exposure, and neurodegenerative disorders. The complex nature of lipid peroxidation and its potential biological significance have attracted the attention of scientists across many disciplinary fields, ranging from chemistry and biochemistry to biology and clinical science (see review [3] and references cited herein).
positions of the oxygenated groups prior to chemical degradation of the oxidized glycosphingolipids. In addition, attempts were made to fix the double bonds of allylic substructures by reacting with dimethyl disulfide (DMDS) because, in our preliminary research, the DMDS adduct of methyl oleate did not lose S-methyl groups during hydrolysis with HCl in MeCN-H2O. To minimize possible allylic rearrangements (1,3-isomerizations) and other alterations, we avoided elevated temperatures and strong acid/base conditions in the derivatization reactions before hydrolysis. Thus, our attention was mainly focused on procedures suitable for an initial detailed structural analysis of a complex mixture of glycosphingolipids, containing an allylic hydroperoxy, hydroxy, or keto group in the monoenoic acyl chain.
In UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) analysis of oxidized cerebrosides, base peak chromatogram, and extracted-ion chromatograms provided limited information because many components eluted simultaneously. In particular, peaks for major isomeric allylic hydroperoxides 1a and 1b were not well-resolved (Supplementary Materials, Figure  S1a, b).
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] + (base peak), Y0, Z0, O, and C1 ions, and by small peaks, representing [M + Na − H2O] + , E, and B1 ions. However, the (+)ESI-MS/MS spectrum of the [M + Na] + 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 С20 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 С21 sphingoid base backbone 3. A relatively low intensity pseudo-molecular ion peak (m/z 910.65, [M + Na] + ) and very small peaks, corresponding to Y0 (m/z 748.60) and Z0 (m/z 730.58) ions (not shown), were observed. The presence of the [M + Na − Н] + 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.  [9]. In addition, [Z 0 /Q − C 3 H 5 N (55 Da)] ions (not shown) were observed in the MS/MS spectra of the [M − H]ions. The fragment Z 0 /Q was also referred to as a Z 0 /K 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] + (base peak), Y 0 , Z 0 , O, and C 1 ions, and by small peaks, representing [M + Na − H 2 O] + , E, and B 1 ions. However, the (+)ESI-MS/MS spectrum of the [M + Na] + 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 C 20 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 C 21 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 0 (m/z 748.60) and Z 0 (m/z 730.58) ions (not shown), were observed. The presence of the [M + Na − Н] + 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.  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 C n H 2n+2 O losses from [M + Na] + ions, were also found in the MS/MS studies of some monoenoic [6] and polyenoic [11][12][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 − C n H 2n+2 O] + ions were also more abundant in MS/MS spectra of those compounds compared to their [M + Na] + 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] + 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 ( 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 [
protons, the coupling constants J = 15.6 Hz and J = 15.4 Hz for the trans-olefinic protons of the allylic hydroperoxides and allylic alcohols, respectively, were detected. The characteristic NMR resonances of two trans-alkenyl CH (δН 6.105, d, J = 15.9 Hz, and 6.91, dt, J = 7.0, 15.9 Hz), conjugated C=O (δС 204.3), and α-CH2 (δН 2.565, t, J = 7.4 Hz) groups were used for determining substructures of enones in acyl chains a // -d // (Figure 5c). The structures of the allylic hydroperoxides, allylic alcohols, and enones were confirmed by HMBC correlations, as depicted in Figure 5. Some very weak signals in the 1 Н-NMR, 1 Н, 1 Н-COSY, and HSQC spectra of oxidized cerebrosides found in the present study were attributed to cis-double bonds of allylic hydroperoxides and allylic alcohols (Appendix A, Figure A1). The complete structures of these compounds could not be elucidated due to their trace amounts.

Analyses of FAs, Sphingoid Bases, and Sugar Obtained from Oxidized Cerebrosides
The RP-HPLC oxidized cerebroside fraction was divided into two parts (parts 1 and 2), which were treated using different chemical procedures before hydrolysis. Then, we applied MeCN/HCl hydrolysis [24] for chemical degradation of cerebrosides. In our experience [8], this procedure causes less disruption of spingoid bases than methanolysis (MeОН/HCl), which is most widely used in studies of complex lipids.
Analysis of FAs from Part 1. Part 1 of the oxidized cerebrosides was subjected to hydrogenation (with Adams' catalyst) to fix the positions of allylic oxygen-containing groups before hydrolysis. Upon hydrolysis, liberated FAs were acetylated and methylated. The 1 Н-NMR spectrum (CDCl3) of FA derivatives showed proton signals of mid-chain substructures, including -H2C-CO-CH2-at δН 2.38 (t, J = 7.4 Hz) and -СН(OАс)-at δH 4.855 (m). GC-MS analysis (electron impact ionization) of these derivatives revealed methyl esters of 2-acetyloxy C23 and C22 acids, containing an isolated keto or acetyloxy group or no additional oxygenated group. Similar products, namely keto, hydroxy, and non-oxygenated acid derivatives, have been previously reported for the hydrogenation (in EtOH over Adams' catalyst) of allylic 9-hydroperoxides and 10-hydroperoxides, obtained from methyl oleate [25].
In the present report, mass spectra  Figures S4-S7). Ions produced by cleavage β to a keto group and ions formed from the methyl end of the molecules by cleavage α to the keto group were prominent in the mass spectra, as described Some very weak signals in the 1 Н-NMR, 1 Н, 1 Н-COSY, and HSQC spectra of oxidized cerebrosides found in the present study were attributed to cis-double bonds of allylic hydroperoxides and allylic alcohols (Appendix A, Figure A1). The complete structures of these compounds could not be elucidated due to their trace amounts.

Analyses of FAs, Sphingoid Bases, and Sugar Obtained from Oxidized Cerebrosides
The RP-HPLC oxidized cerebroside fraction was divided into two parts (parts 1 and 2), which were treated using different chemical procedures before hydrolysis. Then, we applied MeCN/HCl hydrolysis [24] for chemical degradation of cerebrosides. In our experience [8], this procedure causes less disruption of spingoid bases than methanolysis (MeOН/HCl), which is most widely used in studies of complex lipids.
Analysis of FAs from Part 1. Part 1 of the oxidized cerebrosides was subjected to hydrogenation (with Adams' catalyst) to fix the positions of allylic oxygen-containing groups before hydrolysis. Upon hydrolysis, liberated FAs were acetylated and methylated. The 1 Н-NMR spectrum (CDCl 3 ) of FA derivatives showed proton signals of mid-chain substructures, including −H 2 C−CO−CH 2 − at δ Н 2.38 (t, J = 7.4 Hz) and −CН(OAс)at δ H 4.855 (m). GC-MS analysis (electron impact ionization) of these derivatives revealed methyl esters of 2-acetyloxy C 23 and C 22 acids, containing an isolated keto or acetyloxy group or no additional oxygenated group. Similar products, namely keto, hydroxy, and non-oxygenated acid derivatives, have been previously reported for the hydrogenation (in EtOH over Adams' catalyst) of allylic 9-hydroperoxides and 10-hydroperoxides, obtained from methyl oleate [25].
In the present report, mass spectra exhibited base peaks at m/  Figures S4-S7). Ions produced by cleavage β to a keto group and ions formed from the methyl end of the molecules by cleavage α to the keto group were prominent in the mass spectra, as described for MS fragmentations of several methyl esters of oxo (keto) FAs [26]. In particular, mass spectra of methyl esters of C 23 and C 22 acids displayed homologous pairs of m/z 127/142 and 113/128 fragments, containing methyl ends of the molecules with keto groups on the (n-8) or (n-7) carbon, respectively. These keto group positions were confirmed by a number of ions containing the polar end of the FA esters that also produced daughter ions due to loss of AcOH, CH 2 CO, or MeOH ( Figures S4-S7).
Hydrogenation of part 1, followed by hydrolysis, acetylation, and methylation, also yielded the methyl esters of 2-acetyloxy C 23 and C 22 acids, containing an additional isolated acetyloxy group. Expectedly, [M] + peaks were absent in the mass spectra of the methyl esters of these diacetylated acids, but [M − CH 3 CO] + and [M − AcOH] + ions were observed in high mass regions ( Figures S8-S11). Additionally, these compounds, as derivatives of 2-acetyloxy FAs, fragmented to give abundant [M − MeOCO − AcOH] + ions of m/z 365 and 351 for methyl esters of C 23 and C 22 acids, respectively. Isomers, containing an isolated acetyloxy group in different positions, were discerned based on the presence of diagnostic α-cleavage ions and more abundant product ions, formed by elimination of CH 2 CO or AcOH from α-fragments, as described for acetates of secondary alcohols [27]. In particular, the α-fragments included the ions of m/z 385 and 399 ( Figures S8 and S9) for the derivatives of 2,16-, and 2,17-diacetyloxy C 23 acids, respectively, and the ions of m/z 371 and 385 (Figures S10 and S11) for the 2,15-and 2,16-diacetyloxy C 22 acid derivatives, respectively. Moreover, some homologous ions, which arose from C-1-⁄-C-2 bond fission and cleavage α to an isolated acetyloxy group, were specific for the different positions of this group in acyl chains. For example, the abundant ions of m/z 283 and 297 ( Figures S8 and S9), which were detected in the mass spectra of the methyl esters of isomeric 2-acetyloxy C 23 acids, confirmed the presence of second acetyloxy groups on C-16 and C-17, respectively. Similarly, m/z 269 and 283 ions in the mass spectra of the methyl esters of 2-acetyloxy C 22 acids (Figures S10 and S11) indicated a second acetoxy group on C-15 and C-16, respectively. Additionally, α-fragmentation of isomers with an acetyloxy group in the (n-8) or (n-7) positions and subsequent loss of AcOH gave rise to m/z 111 and 97 ions, respectively, containing the methyl end of acyl chains.
As a result of the FA analyses, hydrogenated minor derivatives of allylically oxygenated FAs were also detected. While the major derivatives formed from fatty acyl groups containing hydroperoxy/hydroxy/keto groups in the (n-8) or (n-7) positions, the minor derivatives were rearranged products of these FA moieties (Appendix B, Figures S12-S20).
Analysis of FAs from Part 2. In this investigation, we tried to use S-methyl groups as markers for oxidized cerebroside double bonds. However, enones with polarized double bonds that did not react with DMDS under mild conditions, and labile allylic hydroperoxides were not suitable for this purpose. Therefore, enones and allylic hydroperoxides were converted into allylic alcohols that were expected to add to DMDS.
The oxidized cerebrosides from part 2 were acetylated to increase solubility of these relatively polar compounds in DMDS and other low-polarity or non-polar organic solvents. In this process, allylic hydroperoxides were transformed into enones (Scheme 3), as reported by Porter and Wujek [23]. The mixture obtained after acetylation was treated with NaBH 4 /CeCl 3 [28] to convert enones into allylic alcohols. The resulting derivatives, containing an allylic hydroxy or acetyloxy group, were treated with DMDS, and the products of this reaction were hydrolyzed in MeCN/HCl. Liberated 2-hydroxy FAs were acetylated, methylated, and analyzed by a GC-MS method that revealed major mono(methylthio) and minor tris(methylthio) derivatives (Schemes 3 and 4). The mono(methylthio) compounds, containing an allylic methylthio group, were characterized by 1 S24). The peaks at m/z 371 and 385, observed in the mass spectra of methyl esters of homologous 2acetyloxy C22 acids, represented fragments of the same origin ( Figures S26 and S28). α-fragmentation of other mono(methylthio) derivatives gave rise to the ions at m/z 171 and 157, containing the methyl end of isomers with an allylic -SMe group in the (n-9) and (n-8) positions, respectively ( Figures S21,  S23, S25, and S27). Relative abundances of α-fragments in average mass spectrum were used to quantify isomer distribution, and approximately equal amounts of the four isomeric allylic thioethers were found. This finding may reflect the fact that these S-methyl derivatives were possibly products of allylic rearrangements, occurring prior to GC-MS analysis.
The minor tris(methylthio) derivatives of the methyl esters of 2-acetyloxy C23 (564 [M] + ) or C22 (550 [M] + ) acids produced more diagnostic fragments than the previously mentioned major mono(methylthio) derivatives. Expectedly, the cleavages of minor S-methylated compounds occurred between methylthio-carrying carbons to yield substantial fragment ions, as illustrated in Scheme 4 and Figures S29-S32. A cluster of four GC peaks for isomeric tris(methylthio) derivatives was observed. According to fragmentation patterns, there were two peaks representing positional isomers and two peaks that represented stereoisomers of these regioisomers on the chromatogram.
The results of the transformations of allylic alcohols and their acetates into methylthio derivatives (Scheme 3) were confirmed by experiments with model compounds, methyl esters of 11hydroxy and 8-acetyloxy elaidic acids (prepared from methyl oleate, Appendix C, Scheme A1, Figures S33-S37). Under the conditions used here, the allylic alcohol acetates reacted with DMDS to give major allylic thioethers and minor tris(methylthio) derivatives. Allylic alcohols reacted with DMDS to give major DMDS adducts and minor allylic thioethers and tris(methylthio) derivatives. However, in contrast to the DMDS adducts of monoenes with an isolated double bond, the bis(methilthio) derivatives of allylic alcohols were destroyed during MeCN/HCl hydrolysis.  S23, S25, and S27). Relative abundances of α-fragments in average mass spectrum were used to quantify isomer distribution, and approximately equal amounts of the four isomeric allylic thioethers were found. This finding may reflect the fact that these S-methyl derivatives were possibly products of allylic rearrangements, occurring prior to GC-MS analysis.
The minor tris(methylthio) derivatives of the methyl esters of 2-acetyloxy C 23 (564 [M] + ) or C 22 (550 [M] + ) acids produced more diagnostic fragments than the previously mentioned major mono(methylthio) derivatives. Expectedly, the cleavages of minor S-methylated compounds occurred between methylthio-carrying carbons to yield substantial fragment ions, as illustrated in Scheme 4 and Figures S29-S32. A cluster of four GC peaks for isomeric tris(methylthio) derivatives was observed. According to fragmentation patterns, there were two peaks representing positional isomers and two peaks that represented stereoisomers of these regioisomers on the chromatogram.
The results of the transformations of allylic alcohols and their acetates into methylthio derivatives (Scheme 3) were confirmed by experiments with model compounds, methyl esters of 11-hydroxy and 8-acetyloxy elaidic acids (prepared from methyl oleate, Appendix C, Scheme A1, Figures S33-S37). Under the conditions used here, the allylic alcohol acetates reacted with DMDS to give major allylic thioethers and minor tris(methylthio) derivatives. Allylic alcohols reacted with DMDS to give major DMDS adducts and minor allylic thioethers and tris(methylthio) derivatives. However, in contrast to the DMDS adducts of monoenes with an isolated double bond, the bis(methilthio) derivatives of allylic alcohols were destroyed during MeCN/HCl hydrolysis. Previously, the synthesis of allylic thioethers from allylic alcohols and thiols was reported by Zhang et al. ( [29]: iodine-catalyzed process) and Tabarelli et al. ( [30]: catalyst-free approach). Regioisomeric mixtures of allylic thioethers were produced when the allylic alcohol contained two different substituents. To explain the presence of regio-isomer products of 1,3-isomerization, the allylic cation, formed by water loss from the allylic alcohol, was proposed to be an intermediate in the reaction pathway [30]. The existence of a similar mechanism explains the formation of regio-isomeric allylic thioethers and their tris(methilthio) derivatives in the iodine-catalyzed reaction of allylic alcohols and their acetates with DMDS reported here. Additionally, allylic thioethers, which could undergo 1,3isomerization under acidic conditions, were possibly formed from the bis-DMDS adducts of allylic alcohols during MeCN/HCl hydrolysis. As a result of these rearrangements, the FA methylthio derivatives, obtained from oxidized cerebrosides, gave characteristic mass spectra, permitting locations of three-carbon allylically oxygenated substructures, rather than double bonds in the starting acyl chains. Previously, the synthesis of allylic thioethers from allylic alcohols and thiols was reported by Zhang et al. ( [29]: iodine-catalyzed process) and Tabarelli et al. ( [30]: catalyst-free approach). Regio-isomeric mixtures of allylic thioethers were produced when the allylic alcohol contained two different substituents. To explain the presence of regio-isomer products of 1,3-isomerization, the allylic cation, formed by water loss from the allylic alcohol, was proposed to be an intermediate in the reaction pathway [30]. The existence of a similar mechanism explains the formation of regio-isomeric allylic thioethers and their tris(methilthio) derivatives in the iodine-catalyzed reaction of allylic alcohols and their acetates with DMDS reported here. Additionally, allylic thioethers, which could undergo 1,3-isomerization under acidic conditions, were possibly formed from the bis-DMDS adducts of allylic alcohols during MeCN/HCl hydrolysis. As a result of these rearrangements, the FA methylthio derivatives, obtained from oxidized cerebrosides, gave characteristic mass spectra, permitting locations of three-carbon allylically oxygenated substructures, rather than double bonds in the starting acyl chains.
Thus, we used two complementary approaches for determining oxidized acyl chain structures in glycosphingolipids. Analysis of the FA derivatives from part 1 indicated the allylic oxygen-containing group positions, but hydrogenation resulted in the loss of information regarding the positions of double bonds in the starting material (approach 1). In the analysis of part 2 (approach 2), the data on the locations of three-carbon allylically oxygenated substructures in the FA derivatives and the information on their straight-chain structures and (2R)-configurations were obtained. Although there was no direct information about the position of the double bond in the FA esters, the combined data of approaches 1 and 2 allowed for determination of the double bond and allylic hydroperoxy, hydroxy, or keto group locations in each acyl chain.

Oxidized Cerebrosides from the Extract of Aulosaccus sp.: Structures and Possible Origins
As a result of our study, structures of 18 previously unknown compounds, found in the complex mixture of the oxidized cerebrosides from the extract of Aulosaccus sp., were elucidated.  Cerebrosides, having backbones 1, 2, and 3, comprised, respectively, 60%, 20%, and 20% of the mixture. The percentages were calculated from the integration of signals of a cyclopropane ring in the 1 H-NMR spectra of this mixture (backbone 3) and from relative intensities of GC peaks, represented by DMDS derivatives of two acetylated isomeric monoenoic sphingoid bases (backbones 1 and 2, ∆ 11 :∆ 13 ≈ 3:1). The GC-MS analysis of the hydrogenation products of amide-linked FAs indicated that the a:b, c:d, a / :b / , c / :d / , a // :b // , and c // :d // isomer ratios were approximately 1:1.Therefore, the employed complementary instrumental and chemical methods clarified structures of oxidized cerebrosides in a complex mixture, without requiring isolation or complete separation.
According to the product composition, photo-oxidation and autooxidation [1] are possible mechanisms involved in the formation of the oxidized cerebrosides from the extract of Aulosaccus sp. However, we would like to point to another possible origin of the oxidized cerebrosides in Aulosaccus sp., taking into account the relationship between these oxidation products and other compounds isolated from the same sponge sample. In particular, some bacterial branched-chain, cyclopropane-containing FAs, and their monoenoic precursors were present in significant amounts in Aulosaccus sp. [32], and an overwhelming number of the sterols (stanols, ∆ 5 -, ∆ 7 -, and ∆ 8(14) -sterols) of this sponge were oxidized to the corresponding 3-ketosteroids [37]. The occurrence of these FAs and steroids in Aulosaccus sp. suggested this sponge was associated with actinobacteria, known as sponge-specific microorganisms [38] and sterol degraders [39]. Cholesterol oxidase, produced by a variety of actinobacteria [40], could catalyze the transformations of the previously mentioned sterols into 3-ketosteroids [41] with the generation of H 2 O 2 . We suggest that H 2 O 2 production in the enzymatic oxidation of Aulosaccus sp. sterols led to oxidative transformations of a certain part of cerebrosides, located in the membranes of eukaryotic cells together with sterols.

Animal Material
The sample of the genus Aulosaccus (phylum Porifera, class Hexactinellida, order Lyssacinosida, family Rossellidae), 930 g (wet weight), was collected in July 2011 by dredging (with small Sigsbi trail) from a 505-m depth near Iturup Island (Kuril Islands, 45 • 01.05 / N, 147 • 00.03 / E) during a cruise onboard the r/v "Academik Oparin". The species was identified by Dr A.L. Drozdov (A.V. Zhirmunskii Institute of Marine Biology, Far Eastern Branch of RAS, Vladivostok, Russia). The collected sponge was stored at −15 • C.

Extraction and Isolation
The isolation of total cerebroside sum (238 mg) from the EtOH extract of Aulosaccus sp. was described in our previous article [8].  Table 1 and Figures S2 and S3. This fraction was divided into two parts. The constituents of parts 1 and 2 were analyzed using different chemical procedures.
The absolute configuration of glucose, released from oxidized cerebrosides, was determined by the GC analyses of per-acetylated (2R)-oct-2-yl glycosides according to the method of Leontein et al. [41]. The sugar (1.2 mg), (2R)-octan-2-ol (0.4 mL), and one drop of trifluoroacetic acid in a capped vial were kept for 7 h at 120 • C with stirring. Then, the mixture was concentrated in vacuo and acetylated with Ac 2 O (0.4 mL) in pyridine (0.4 mL), overnight. The acetylated material was purified by column chromatography (SiO 2 , 3.0 cm × 1.2 cm), eluting with a mixture of hexane/ethylacetate (5:1, v/v). The eluate was evaporated, yielding per-acetylated (2R)-oct-2-yl glucoside. Standards, D-Glc (1.0 mg) and L-Glc (1.0 mg), were treated and derivatized under the same conditions that were applied to the sugar subfractions, liberated from parts 1 and 2. The GC profiles (the retention times and intensities of GC peaks) of the derivatives of D-Glc and sugar, obtained from cerebrosides, were proven to be identical.

Appendix B
Although a single peak in the GC chromatogram represented overlapping esters of four isomeric keto acids, the mass spectra, obtained from different points in the GC peak, exhibited fragmentations of samples, enriched in the isomers with keto groups in the (n-9)→(n-8)→(n-7)→(n-6) positions (in this order of elution). Mass spectra of methyl esters of minor keto C23 and C22 acids displayed homologous pairs of m/z 141/156 and 99/114 fragments, containing methyl ends of the molecules with keto groups on the (n-9) or (n-6) carbon, respectively (Figures S12, S13, S15, and S16). The total mass spectra, obtained by averaging the spectra over the selected GC peaks, showed significant differences between relative abundances of several ions, characteristic of different isomers. For the overlapping esters of 15-keto, 16-keto, 17-keto, and 18-keto C23 acids, the abundance ratio of the characteristic ions of m/z 342, 356, 370, and 384 were about 1:3:3:1 ( Figure S14). Accordingly, the mixture of C23 acid derivatives contained significant amounts of isomers with a keto group in the (n-8) and (n-7) positions and minor amounts of isomers with a keto group located at the (n-9) and (n-6) positions. The overlapping methyl esters of homologous keto C22 acids exhibited similar fragmentation and elution behavior.
GC-MS analysis also revealed the presence of clusters of closely overlapping chromatographic peaks for derivatives of regio-isomeric diacetyloxy C23 and C22 acids, including minor components. In particular, the α-fragments included the ions of m/z 371 and 413 (Figures S17 and S18) for the derivatives of 2,15-diacetyloxy and 2,18-diacetyloxy C23 acids, respectively, and the ions of m/z 357

Appendix B
Although a single peak in the GC chromatogram represented overlapping esters of four isomeric keto acids, the mass spectra, obtained from different points in the GC peak, exhibited fragmentations of samples, enriched in the isomers with keto groups in the (n-9)→(n-8)→(n-7)→(n-6) positions (in this order of elution). Mass spectra of methyl esters of minor keto C 23 and C 22 acids displayed homologous pairs of m/z 141/156 and 99/114 fragments, containing methyl ends of the molecules with keto groups on the (n-9) or (n-6) carbon, respectively (Figures S12, S13, S15, and S16). The total mass spectra, obtained by averaging the spectra over the selected GC peaks, showed significant differences between relative abundances of several ions, characteristic of different isomers. For the overlapping esters of 15-keto, 16-keto, 17-keto, and 18-keto C 23 acids, the abundance ratio of the characteristic ions of m/z 342, 356, 370, and 384 were about 1:3:3:1 ( Figure S14). Accordingly, the mixture of C 23 acid derivatives contained significant amounts of isomers with a keto group in the (n-8) and (n-7) positions and minor amounts of isomers with a keto group located at the (n-9) and (n-6) positions. The overlapping methyl esters of homologous keto C 22 acids exhibited similar fragmentation and elution behavior.
GC-MS analysis also revealed the presence of clusters of closely overlapping chromatographic peaks for derivatives of regio-isomeric diacetyloxy C 23 and C 22 acids, including minor components. In particular, the α-fragments included the ions of m/z 371 and 413 (Figures S17 and S18) for the derivatives of 2,15-diacetyloxy and 2,18-diacetyloxy C 23 acids, respectively, and the ions of m/z 357 and 399 (Figures S19 and S20) for the 2,14-diacetyloxy and 2,17-diacetyloxy C 22 acid derivatives, respectively. The abundant ions of m/z 269 and 311 ( Figures S17 and S18), which were detected in the mass spectra of the methyl esters of isomeric minor 2-acetyloxy C 23 acids, confirmed the presence of second acetyloxy groups on C-15 and C-18, respectively. Similarly, m/z 255 and 297 ions in the mass spectra of the methyl esters of minor 2-acetyloxy C 22 acids (Figures S19 and S20) indicated a second acetoxy group on C-14 and C-17, respectively. α-Fragmentation of isomers with an acetyloxy group in the (n-9), (n-8), (n-7), or (n-6) positions (in this order of elution) and subsequent loss of AcOH gave rise to m/z 125, 111, 97, and 83 ions, respectively, containing the methyl end of acyl chains. Among these α-fragments, the largest fragment of m/z 125 was much less abundant than the related ions. Unfortunately, the percentages of all the methyl esters of isomeric diacetylated FAs, which were only partially separated by GC, could not be accurately evaluated on the basis of mass spectral data because the presence of diagnostic α-fragments, formed from isomers with an acetyloxy group in the (n-9) positions, were masked by other peaks in averaged mass spectrum. However, for diacetyloxy C 23 acid derivatives with an isolated acetyloxy group in the (n-8), (n-7), or (n-6) positions, the abundance ratio of the diagnostic α-fragments of m/z 385, 399, and 413, respectively, were about 3:3:1. The abundance ratio of homologous α-fragments (m/z 371, 385, and 399) were nearly the same for the derivatives of diacetyloxy C 22 acids. Accordingly, the spectra, recorded at the top of the most intense overlapping GC peaks, exhibited fragmentation patterns of major isomers with an acetyloxy group in the (n-8) or (n-7) positions, while the mass spectra, recorded at lower points, belonged to minor isomers with an acetyloxy group on the (n-9) or (n-6) carbons.
We assumed that the abundance ratio of components with oxygen-containing groups on the (n-9), (n-8), (n-7), or (n-6) carbons (about 1:3:3:1, respectively) were nearly the same for isomeric hydrogenated products and initial hydroperoxy FA moieties. This suggestion was supported by (-)ESI-MS/MS study of isomeric allylic hydroperoxides (Figure 4a), showing two pairs of characteristic peaks with a 3:1 intensity ratio, including peaks at m/z 743.6/729.55 (fragments, formed from acyl chains with -OOH in the (n-8)/(n-9) positions) and m/z 784.6/798.6 (fragments, formed from acyl chains with -OOH in the (n-7)/(n-6) positions). Thus, the previously mentioned minor hydrogenated products were possibly derived from minor amide-linked FA with allylic hydroperoxy/hydroxy/keto groups in the (n-9) or (n-6) positions. However, complete structures of the corresponding oxidized cerebrosides could not be comprehensively elucidated due to their minor amounts. Therefore, only major cerebrosides with oxygen-containing groups in the (n-8) or (n-7) positions of acyl chains were the focus of our research.
Molecules 2020, 25, x FOR PEER REVIEW 23 of 26 tris(methylthio)octadecanoic acids, 12.4%), analogous to that shown in Scheme 3, along with octadecadienoic acid (11.7%), were found in the hydrolysate. Apparently, the bis(methilthio) adduct of allylic alcohol could lose an -OH group and one -SMe group during MeCN/HCl hydrolysis, giving rise to an additional amount of allylic thioethers.
Scheme A1. Allylic mono-hydroxylation of methyl oleate, followed by transformations of allylic alcohols into S-methyl derivatives. GC-MS cleavage patterns for the derivatives, obtained from methyl oleate, are depicted.
The DMDS adduct of methyl oleate can be de-esterified in MeCN/HCl without detectable degradation of the -CH(SMe)-CH(SMe)-fragment, in contrast to the DMDS adduct of methyl 11hydroxy elaidate. This was confirmed by the 1 Н-NMR spectra, recorded before and after hydrolysis. In particular, the 1 Н-NМR spectrum (CDCl3) of the product, obtained after hydrolysis of the DMDS adduct of methyl oleate, showed the superimposed signals of two vicinal CH (δН 2.685, m), linked to -SMe groups (δH 2.10, s), and α-CH2 (δН 1.845, m; 1.32, m) groups of bis(methylthio) oleic acid. Scheme A1. Allylic mono-hydroxylation of methyl oleate, followed by transformations of allylic alcohols into S-methyl derivatives. GC-MS cleavage patterns for the derivatives, obtained from methyl oleate, are depicted.