*2.3. 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 <sup>1</sup>Н-NMR spectrum (CDCl3) of FA derivatives showed proton signals of mid-chain substructures, including −**H**2C−CO−C**H**2− at δ<sup>Н</sup> 2.38 (t, *J* = 7.4 Hz) and −CН(OAс)– at δ<sup>H</sup> 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 exhibited base peaks at *m*/*z* 339 and 325 ([M − MeOCO − CH2CO]<sup>+</sup>) for methyl esters of 2-acetyloxy keto C23 (440 [M]<sup>+</sup>) and C22 (426 [M]+) acids, respectively (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 C23 and C22 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, CH2CO, or MeOH (Figures S4–S7).

Hydrogenation of part 1, followed by hydrolysis, acetylation, and methylation, also yielded the methyl esters of 2-acetyloxy C23 and C22 acids, containing an additional isolated acetyloxy group. Expectedly, [M]<sup>+</sup> peaks were absent in the mass spectra of the methyl esters of these diacetylated acids, but [M <sup>−</sup> CH3CO]<sup>+</sup> and [M <sup>−</sup> AcOH]<sup>+</sup> ions were observed in high mass regions (Figures S8–S11). Additionally, these compounds, as derivatives of 2-acetyloxy FAs, fragmented to give abundant [M <sup>−</sup> MeOCO <sup>−</sup> AcOH]<sup>+</sup> ions of *m*/*z* 365 and 351 for methyl esters of C23 and C22 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 CH2CO 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 C23 acids, respectively, and the ions of *m*/*z* 371 and 385 (Figures S10 and S11) for the 2,15- and 2,16-diacetyloxy C22 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 C23 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 C22 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 NaBH4/CeCl3 [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 1H-NMR resonances (CDCl3) of two *trans*-olefinic CH (δН 5.175 dd, *J* = 9.0, 15.2 Hz, and 5.40 dt, *J* = 6.9, 15.2 Hz) and one CH (δН 2.99 m), bearing a –SMe group (δ<sup>H</sup> 1.97 s). The presence of the mid-chain allylic substructure was confirmed by a TOCSY (total correlation spectroscopy) experiment.

**Scheme 3.** Transformations of acyl substructures, containing allylic hydroperoxy/hydroxy/keto groups, into methylthio derivatives.

Cleavage patterns for methyl esters of 2-acetyloxy C23 (470 [M]<sup>+</sup>) and C22 (456 [M]+) acids, containing an allylic methylthio group, are depicted in Scheme 4. The positional isomers of these allylic thioethers were only partially GC-separated. The mass spectra of isomeric allylic thioethers (Figures S21–S28) were characterized by diagnostic peaks, corresponding to ions produced by α-cleavage. The fragments, formed by cleavage α to the carbon carrying an allylic methylthio group on the side remote from the carboxyl group included *m*/*z* 385 and 399 ions for the methyl esters of 2-acetyloxy C23 acids with –SMe group in the (*n*–7) and (*n*–6) positions, respectively (Figures S22 and S24). The peaks at *m*/*z* 371 and 385, observed in the mass spectra of methyl esters of homologous 2-acetyloxy 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 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.

**Scheme 4.** GC-MS cleavage patterns for the methyl esters of 2-acetyloxy C23 and C22 acids, containing an allylic methylthio group or three methylthio groups.

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.

We clarified the acyl structures (Figure 1) using a logical approach. The GC-MS analyses of the methyl esters of 2-acetyloxy methylthio FAs (Scheme 4) revealed that the three-carbon allylically functionalized substructures of oxidized cerebrosides included C-15/ –C-16/ –C-17/ and C-16/ –C-17/ –C-18/ fragments for 2-hydroxy C23 acyl chains and C-14/ –C-15/ –C-16/ and C-15/ –C-16/ –C-17/ fragments for 2-hydroxy C22 acyl chains. According to the GC-MS analyses of the hydrogenated FA derivatives, the amide-linked FAs of oxidized cerebrosides contained hydroperoxy, hydroxy, or keto groups in the (*n*–8) or (*n*–7) positions, more specifically in the 16/ and 17/ positions of 2-hydroxy C23 acyl chains and the 15/ and 16/ positions of 2-hydroxy C22 acyl chains. A priori, an allylic hydroperoxy, hydroxy, or keto group should be located in the terminal points of the previously mentioned three-carbon substructures. Consequently, the C-15/ –C-16/ –C-17/ fragments of the C23 acyl chains (**a**–**a**//) contained such groups in position C-17/ and the double bond between C-15/ and C-16/ , while the C-16/ –C-17/ –C-18/ fragments of the other C23 acyl chains (**b**–**b**//) had 16/ -hydroperoxy/hydroxy/oxo groups and 17/ ,18/ -double bonds. Similarly, the C-14/ –C-15/ –C-16/ fragments of the C22 acyl chains (**c**–**c**//) contained 16/ -hydroperoxy/hydroxy/oxo groups and 14/ ,15/ -double bonds, while the C-15/ –C-16/ –C-17/ fragments of the other C22 acyl chains (**d**–**d**//) had 15/ -hydroperoxy/hydroxy/oxo groups and double bonds between C-16/ and C-17/ .

The mono(methylthio) and tris(methylthio) derivatives of allylically oxygenated FAs were also used to explain other structural peculiarities of the starting material. Upon hydrogenation, all the methylthio derivatives lost *S*-methyl groups, giving saturated hydrocarbon chains. In particular, the transformations, shown in Scheme 3, with subsequent hydrodesulfurization allowed us to convert –CH=CH–CH(OOH/OH)– and –CH=CH–CO– substructures to –CН2–CН2–CН2– chains without reducing other oxygen-containing groups in the molecules. As a result, the unbranched structures of the parent amide-linked FAs were clarified using retention times of the 2-acetyloxy tricosanoic and docosanoic acid methyl esters, obtained from the methylthio derivatives. Then, these methyl esters were converted into (2*S*)-oct-2-yl esters of 2-hydroxy acids. The resulting (2*S*)-oct-2-yl esters of 2-hydroxy tricosanoic and docosanoic acids coeluted in GC analyses with the reference (2*S*)-oct-2-yl ester of (2*R*)-2-hydroxy tricosanoic or docosanoic acids, respectively, indicating (2*R*)-configurations of 2-hydroxy acids liberated from oxidized cerebrosides.

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 (2*R*)-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.

*Analyses of Sphingoid Bases and Sugar from Parts 1 and 2*. The sphingoid bases, liberated by hydrolysis of oxidized cerebrosides, were obtained as acetylated derivatives. The <sup>1</sup>Н-NМR data (δ<sup>Н</sup> values of CH2-1–CH-4; CDCl3) and optical rotation value ([α] 25D = + 27.9, CHCl3) of the hydrogenated sphingoid base acetates, isolated from the hydrolysate of part 1, indicated their (2*S*,3*S*,4*R*)-configuration [31]. The <sup>1</sup>Н-NМR spectrum of these compounds also showed signals of the terminal methyl groups of dominant normal-chain (δН 0.88, t, *J* = 6.9 Hz) and minor cyclopropane-containing (δН 0.89, t, *J* = 6.9 Hz) constituents. Under our conditions of hydrogenation, ring opening was not the dominant process for the minor constituent, so the signals of *cis*-cyclopropane protons at δ<sup>Н</sup> −0.33 (dt, *J* = 4.2, 5.5 Hz), 0.56 (ddd, *J* = 4.2, 8.3, 8.3 Hz), and 0.645 (m) [32] were observed. The hydrolysis of derivatized oxidized cerebrosides from part 2 with subsequent acetylation of products gave three sphingoid base derivatives. Two spingoid base derivatives were the DMDS adducts of acetylated isomeric monoenoic

C20 compounds. The mass spectrum of the DMDS adduct of major acetylated C20 monoene exhibited significant peaks at *m*/*z* 432 [M − H3CSC9H18] <sup>+</sup>, 372 [M <sup>−</sup> H3CSC9H18 <sup>−</sup> AcOH]<sup>+</sup> (base peak), and 173 [H3CSC9H18] <sup>+</sup>, indicating 11,12-double bond in backbone **1**. Key peaks in the mass spectrum of the DMDS adduct of isomeric minor acetylated C20 monoene, which have a longer retention time in GC-MS, were observed at *m*/*z* 460 [M − H3CSC7H14] <sup>+</sup>, 400 [M <sup>−</sup> H3CSC7H14 <sup>−</sup> AcOH]<sup>+</sup> (base peak), and 145 [H3CSC7H14] <sup>+</sup>, indicating Δ13 unsaturation in backbone **2**. A third acetylated sphingoid base, derived from cyclopropane-containing backbone **3**, did not give a DMDS adduct. In the mass spectrum of this compound, an [M <sup>−</sup> AcOH]<sup>+</sup> ion fragmented to give discernible peaks at *<sup>m</sup>*/*<sup>z</sup>* 380 [M <sup>−</sup> AcOH <sup>−</sup> (CH2)6CH3] <sup>+</sup> (<sup>α</sup> cleavage to a cyclopropane ring, at the C-14–C-15 position) and 394 [M <sup>−</sup> AcOH <sup>−</sup> (CH2)5CH3] <sup>+</sup> (β-cleavage, at the C-15–C-16 position) that was characteristic of the peracetate of the C21 sphingoid base containing a ring between C-13 and C-14.

Methylthiolation of *cis*-monoenes and *trans*-monoenes with DMDS, as an anti-addition, leads to the threo-adducts and erythro-adducts, respectively [33,34]. The threo-diastereomers and erythro-diastereomers can be easily distinguished by NMR shifts of protons and carbons in and close to the 1,2-bis(alkylthio) moiety [35]. In addition to the data presented in the NMR study of Knothe and Steidley [35], we used δН values of the DMDS adducts of two standards, methyl palmitoleate (Figure 6a) and its *trans*-isomer (Figure 6b), to confirm the configurations of double bonds in the monoenoic sphingoid base moieties of the starting oxidized cerebrosides. The δ<sup>H</sup> values for CH2, α to the –CH(SMe)–CH(SMe)– moiety, were obtained through correlations in <sup>1</sup>Н, <sup>1</sup>Н-COSY diagrams. The <sup>1</sup>Н-NМR and <sup>1</sup>Н, <sup>1</sup>Н-COSY spectra (CDCl3) of the DMDS derivatives of acetylated sphingoid bases, derived from backbones **1** and **2**, showed superimposed resonances of two vicinal CH groups (δ<sup>Н</sup> 2.685, m), linked to –SMe (δ<sup>H</sup> 2.10, s), and two α-CH2 groups (δ<sup>Н</sup> 1.84, m; 1.32, m), characteristic of threo-diastereomers. Consequently, *cis*-monoenes (**1** and **2**) were precursors of these compounds.

**Figure 6.** Some δ<sup>H</sup> values (700 MHz, CDCl3) for the *S*-methylated substructures of (**a**) methyl threo-9,10-bis(methylthio)hexadecanoate and (**b**) methyl erythro-9,10-bis(methylthio)hexadecanoate. Unlike the signals of the methylthio groups of the threo-isomer, the signals of –SMe groups of the erythro-isomer were two partially resolved singlets at δ<sup>H</sup> 2.121 and 2.119.

GC analyses of peracetylated (2*R*)- and (2*S*)-oct-2-yl glucosides showed a D-configuration of glucose, released from parts 1 and 2 [36].

## *2.4. 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. These <sup>β</sup>-D-glucopyranosyl-(1→1)-ceramides (**1a**–**a**//, **1b**–**b**//, **2a**–**a**//, **2b**–**b**//, **3c**–**c**//, **3d**–**d**//) were shown to contain phytosphingosine-type backbones, (2*S*,3*S*,4*R*,11*Z*)-2-aminoeicos-11-ene-1,3,4-triol (in **1**), (2*S*,3*S*,4*R*,13*Z*)-2-aminoeicos-13-ene-1,3,4-triol (in **2**), and (13*S*\*,14*R*\*)-2-amino-13,14-methyleneeicosane-1,3,4-triol (in **3**). These backbones were *N*-acylated with straight-chain monoenoic (2*R*)-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**//). 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 1H-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**, Δ11:Δ<sup>13</sup>

≈ 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.

Previously, the possible precursors of the oxidized cerebrosides were found in the major RP-HPLC fraction of glycosphingolipids, isolated from the extract of *Aulosaccus* sp. These potential precursors were <sup>β</sup>-d-glucopyranosyl-(1→1)-ceramides that contained backbones **<sup>1</sup>** (60% in the fraction) and **<sup>2</sup>** (20%), *N*-acylated with (2*R*,16*Z*)-2-hydroxytricos-16-enoic acid, and backbone **3** (20%), *N*-acylated with (2*R*,15*Z*)-2-hydroxydocos-15-enoic acid [8]. Perhaps, peroxidation of the amide-linked FAs occurred symmetrically about the *cis*-(*n*–7) double bond, so a hydroperoxy, hydroxy, or keto group, found in the major*trans*-monoenoic peroxidation products, was located at each of the carbon atoms, which originally formed the double bond (namely, in the (*n*–8) and (*n*–7) positions, <sup>≈</sup>1:1). In particular, structures **<sup>a</sup>**–**a**// and **b**–**b**// could be formed from C23Δ16*<sup>Z</sup>* acyl chain while structures **c**–**c**// and **d**–**d**// could be formed from the C22Δ15*<sup>Z</sup>* acyl chain.

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, Δ5-, Δ7-, and Δ8(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 H2O2. We suggest that H2O2 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.
