*3.4. Analyses of FAs, Sphingoid Bases, and Sugar from Oxidized Cerebrosides (Parts 1 and 2)*

The constituents of part 1 (3.0 mg) were hydrogenated in EtOH over Adams' catalyst at room temperature (10 h). The resulting hydrogenation product was hydrolyzed in MeCN (0.45 mL) and 5 N HCl (0.05 mL) for 4 h at 75 ◦C in a capped vial. Hydrolysate was concentrated in vacuo, partitioned between CHCl3 (1.0 mL) and H2O (3 × 1.0 mL). The H2O extract was evaporated to dryness to give sugar-containing subfraction. The CHCl3 layer was concentrated in vacuo, and a dry residue was acetylated with Ac2O (0.2 mL) in pyridine (0.2 mL), overnight. The acetylated material was subjected to chromatography over silica gel column (3.0 cm × 1.2 cm), using hexane-ethylacetate (5:1→2:1→1:1, *v*/*v*) system, and then CHCl3-EtOH (1:1, *v*/*v*) system. Elution with 60 mL of hexane-ethylacetate (2:1, *v*/*v*) gave a subfraction of per-acetylated sphingoid bases (0.8 mg). 2-Acetyloxy FA (0.7 mg) was eluted with 20 mL of CHCl3-EtOH (1:1, *v*/*v*) system. The subfraction of the acetates of sphingoid bases was re-purified by column chromatography (SiO2, 3.5 cm × 1.2 cm) eluting with 60 mL of hexane-ethyl acetate (2:1, *v*/*v*). The eluate was evaporated in vacuo yielding a mixture (0.5 mg) of (2*S*,3*S*,4*R*)-2-aminoeicosane-1,3,4-triol (*n*-t20:0, using shorthand nomenclature [42]) and (2*S*,3*S*,4*R*,13*S*\*,14*R*\*)-2-amino-13,14-methylene-eicosane-1,3,4-triol (*cis*-13,14-methylene-t21:0): [α] 25D = +27.9 (*c* = 0.03, CHCl3), 1H-NMR (500 MHz, CDCl3): 5.94 (d, *J* = 9.4 Hz, NH), 5.10 (dd, *J* = 3.0, 8.1 Hz, H-3), 4.94 (dt, *J* = 3.0, 9.9 Hz, H-4), 4.475 (m, H-2), 4.29 (dd, *J* = 4.8, 11.6 Hz, H-1b), 4.01 (dd, *J* = 3.0, 11.6 Hz, H-1a), 2.08 (s, −OCOCH3 at C-3), 2.05 (two s, −OCOCH3 at C-4 and at C-1), 2.025 (s, −NHCOC**H3**), 1.64 (m, H2C-5), 1.45−1.05 (m, –(CH2)n–), 0.88 (t, *J* = 6.9 Hz, H3C-20 of *n*-t20:0), 0.89 (t, *J* = 6.9 Hz, H3C-20 of *cis*-13,14-methylene-t21:0), 0.65 (m, H-13, H-14 of *cis*-13,14-methylene-t21:0), 0.565 (ddd, *J* = 4.1, 8.3, 8.3 Hz, H-21b of *cis*-13,14-methylene-t21:0), −0.33 (dt, *J* = 4.1, 5.5 Hz, H-21a of *cis*-13,14-methylene-t21:0); (2*S*,3*S*,4*R*)-2-aminoeicosane-1,3,4-triol MS, *m*/*z* (relative intensity, %): 452/453/454 [M − AcOH − H/M − AcOH/M − AcNH2] <sup>+</sup> (1.0/0.3/0.9), 380 [M <sup>−</sup> AcOCH2 <sup>−</sup> AcOH]<sup>+</sup> (15), 338 [M <sup>−</sup> AcOCH2 <sup>−</sup> AcOH <sup>−</sup> CH2CO]<sup>+</sup> (14), 333 [M <sup>−</sup> 3AcOH]<sup>+</sup> (19), 320 [M <sup>−</sup> AcOCH2 <sup>−</sup> 2AcOH]<sup>+</sup> (23),144 [AcOCH2CH(NHAc)]<sup>+</sup> (90), 102 [AcOCH2CH(NHAc) <sup>−</sup> CН2CO]<sup>+</sup> (45), 84 [AcOCH2CH(NHAc) − AсOН] <sup>+</sup> (100); (13*S*\*,14*R*\*)-2-amino-13,14-methylene-eicosane-1,3,4-triol MS, *<sup>m</sup>*/*<sup>z</sup>* (relative intensity, %): 525 [M]<sup>+</sup> (2), 465 [M <sup>−</sup> AcOH]<sup>+</sup> (21), 406 [M <sup>−</sup> AcOH <sup>−</sup> AcNH2] <sup>+</sup> (9), 394 (6), 380 (5), 332 [M <sup>−</sup> AcOCH2 <sup>−</sup> 2AcOH]<sup>+</sup> (12), 144 [AcO-CH2CH(NHAc)]<sup>+</sup> (52), 102 [AcOCH2CH(NHAc) <sup>−</sup> CН2CO]<sup>+</sup> (52), 84 [AcOCH2CH(NHAc) <sup>−</sup> AсOН] <sup>+</sup> (100). The components of the subfraction of 2-acetyloxy FAs were methylated by a standard method with *N*-nitroso-*N*-methylurea. The resulting compounds were purified by column chromatography (SiO2, 3.5 cm × 1.2 cm) eluting with 44 mL of hexane-ethyl acetate (10:1, *v*/*v*). The elution gave a mixture of the methyl esters of 2-acetyloxy FAs (0.5 mg): 1H-NMR (CDCl3, 700 MHz): 4.99 (dd, *J* = 6.6, 6.9 Hz, H-2), 4.855 (m, mid-chain –CН(OAс)– of diacetyloxy acids), 3.74 (s, H3CO– at C-1), 2.38 (t, *J* = 7.4 Hz, mid-chain *–***H**2C–CO–C**H**2– of keto acids), 2.135 (s, −OCOCH3 at C-2), 1.82 (m, H2C-3), 1.45–1.15 (m, –(CH2)n–), 0.88 (a series of triplets about this point, *J* ≈ 7.0 Hz), the methyl esters of keto C23 and C22 acids MS: Figures S4–S7, S12–S16; the methyl esters of diacetyloxy C23 and C22 acids MS: Figures S8–S11, S17–S20, the methyl ester of saturated 2-acetyloxy C23 acid MS: 426 [M]<sup>+</sup> (0.04%), 384 [M <sup>−</sup> CH2CO]<sup>+</sup> (100%), 352 [M <sup>−</sup> CH2CO <sup>−</sup> MeOH]<sup>+</sup> (34%), and the methyl ester of saturated 2-acetyloxy C22 acid MS: 412 [M]<sup>+</sup> (0.04%), 370 [M <sup>−</sup> CH2CO]<sup>+</sup> (100%), 338 [M <sup>−</sup> CH2CO <sup>−</sup> MeOH]<sup>+</sup> (35%).

Components of part 2 (1.4 mg) were acetylated with Ac2O (0.2 mL) in pyridine (0.2 mL), overnight. After vacuum drying, CHCl3 (two drops) and CeCl3·7H2O (18.6 mg) were added to the acetylated material, and the mixture was dissolved in MeOH (0.125 mL). NaBH4 (5 mg, excess) was slowly added with stirring. After 5 min, the reaction was quenched by the addition of NH4Cl water solution (5.4 mg in 0.2 mL H2O). A dry residue was obtained after complete evaporation of the reaction mixture. It was extracted with CHCl3 to remove most inorganic admixtures. After vacuum drying of the CHCl3 extract, the resulting dry material was dissolved in DMDS (0.1 mL), and a solution (0.025 mL) of iodine in diethyl ether (60 mg/mL) was added. The mixture was kept for 4 days at room temperature. Then the reaction was stopped by adding aqueous solution of Na2S2O3 (5%, 0.2 mL), and the mixture was extracted with hexane (5 × 0.5 mL). The hexane extract was evaporated to dryness, and the resulting compounds were hydrolyzed in MeCN/HCl. Then, subfractions, containing sugar, peracetylated sphingoid bases (0.4 mg), and 2-acetyloxy FAs (0.3 mg), were obtained, as described above. The subfraction of peracetylated sphingoid bases consisted of peracetylated (13*S*\*,14*R*\*)-2-amino-13,14-methylene-eicosane-1,3,4-triol and DMDS derivatives of peracetylated (11*Z*)-2-aminoeicos-11-ene-1,3,4-triol and (13*Z*)-2-aminoeicos-13-ene-1,3,4-triol: 1H-NMR (500 MHz, CDCl3): 5.93 (d, *J* = 9.5 Hz, NH), 5.095 (dd, *J* = 3.1, 8.3 Hz, H-3), 4.94 (dt, *J* = 3.1, 9.9 Hz, H-4), 4.47 (m, H-2), 4.29 (dd, *J* = 4.8, 11.8 Hz, H-1b), 4.00 (dd, *J* = 3.1, 11.8 Hz, H-1a), 2.685 (m, –C**H**(SCH3)–C**H**(SCH3)–), 2.10 (s, –CH(SC**H3**)–CH(SC**H3**)–), 2.08 (s, −OCOCH3 at C-3), 2.05 (two s, −OCOCH3 at C-4 and at C-1), 2.025 (s, −NHCOC**H3**), 1.84 (m; H-b, –**H**2CCH(SCH3)–CH(SCH3)C**H**2–), 1.64 (m, H2C-5), 1.42−1.18 (m, –(CH2)n–), 1.32 (m, H-a, –**H**2CCH(SCH3)–CH(SCH3)C**H**2–), 0.88 (t, *J* = 6.9 Hz, H3C-20 of *n*-t20:0), 0.89 (t, *J* = 6.9 Hz, H3C-20 of *cis*-13,14-methylene-t21:0), 0.645 (m, H-13, H-14 of *cis*-13,14-methylene-t21:0), 0.56 (ddd, *J* = 4.2, 8.2, 8.2 Hz, H-21b of *cis*-13,14-methylene-t21:0), −0.33 (dt, *J* = 4.2, 5.4 Hz, H-21a of *cis*-13,14-methylene-t21:0). The DMDS derivative of per-acetylated (11*Z*)-2-aminoeicos-11-ene-1,3,4-triol MS, *m*/*z* (relative intensity, %): 557 [M <sup>−</sup> HSMe]<sup>+</sup> (1.4), 432 [M − H3CSC9H18] <sup>+</sup> (24), 372 [M <sup>−</sup> H3CSC9H18 <sup>−</sup> CH3COOH]<sup>+</sup> (100), 330 (11), 312 (6), 173 [H3CSC9H18] <sup>+</sup> (30), 102 [AcOCH2CH(NHAc) <sup>−</sup> CН2CO]<sup>+</sup> (25), 84 [AcOCH2CH(NHAc) <sup>−</sup> AсOН] <sup>+</sup> (45). The DMDS derivative of per-acetylated (13*Z*)-2-aminoeicos-13-ene-1,3,4-triol MS, *m*/*z* (relative intensity, %): 557 [M <sup>−</sup> HSMe]<sup>+</sup> (2), 460 [M <sup>−</sup> H3CSC7H14] <sup>+</sup> (24), 400 [M <sup>−</sup> H3CSC7H14 <sup>−</sup> CH3COOH]<sup>+</sup> (100), 358 (10), 340 (11), 145 [H3CSC7H14] <sup>+</sup> (36), 102 [AcOCH2CH(NHAc) <sup>−</sup> CН2CO]<sup>+</sup> (36), 84 [AcOCH2CH(NHAc) <sup>−</sup> AсOН] <sup>+</sup> (55). The 2-acetyloxy acids from FA subfraction were methylated using *N*-nitroso-*N*-methylurea. The resulting methyl esters were purified by column chromatography (SiO2, 3.5 cm × 1.2 cm), eluting with 44 mL of hexane-ethyl acetate (10:1, *v*/*v*). The elution gave a mixture, containing the methyl esters of major mono(methylthio) and minor tris(methylthio) derivatives of 2-acetyloxy FAs (0.2 mg): 1H-NMR (CDCl3, 700 MHz): 5.40 (dt, *J* = 6.9, 15.2 Hz, –**H**C=CH–CH(SCH3)–), 5.175 (dd, *J* = 9.0, 15.2 Hz, –HC=C**H**–CH(SCH3)–), 4.99 (dd, *J* = 6.2, 6.9 Hz, H-2), 3.74 (s, H3CO– at C-1), 2.99 (m, –HC=CH–C**H**(SCH3)–), 2.135 (s, −OCOCH3 at C-2), 2.04 (m, allylic CH2), 1.965 (s, –HC=CH–CH(SC**H3**)–), 1.82 (m, H2C-3), 1.45–1.15 (m, –(CH2)n–), 0.88–0.89 (a series of triplets, *J* ≈ 7.0 Hz), the methyl esters of the mono(methylthio) derivatives of 2-acetyloxy C23 acid MS: Figures S21–S24, the methyl esters of the mono(methylthio) derivatives of 2-acetyloxy C22 acid MS: Figures S25–S28, the methyl esters of the tris(methylthio) derivatives of 2-acetyloxy C23 acid MS: Figures S29 and S30, the methyl esters of the tris(methylthio) derivatives of 2-acetyloxy C22 acid MS: Figures S31 and S32. For the methyl esters of 2-acetyloxy C23 acids containing an allylic –SMe group, the order of elution was 15-methylthio-16-ene→17-methylthio-15-ene→16-methylthio-17-ene→18-methylthio-16-ene. The elution order for the methyl esters of 2-acetyloxy C22 acids containing an allylic –SMe group was 14-methylthio-15-ene→16-methylthio-14-ene→15-methylthio-16-ene→17-methylthio-15-ene. The mixture of these methylthio derivatives was hydrogenated over Adams' catalyst in AcOH at room temperature (12 h). The hydrogenation gave methyl 2-(acetyloxy)tricosanoate and methyl 2-(acetyloxy)docosanoate. The straight-chain structures of these compounds were clarified, using the GC-MS data on the methyl esters of straight-chain 2-acetyloxy tricosanoic and docosanoic acids, released from the non-oxidized cerebrosides of *Aulosaccus* sp. Then, methyl 2-(acetyloxy)tricosanoate and methyl 2-(acetyloxy)docosanoate, obtained by hydrodesulfurization, were de-esterified in MeCN (0.45 mL) and 5N HCl (0.05 mL) for 4 h at 73−75 ◦C to give free FAs. These FAs were converted into (2*S*)-oct-2-yl esters of 2-hydroxy FAs by treatment with 2% H2SO4 in (2*S*)-octan-2-ol (0.2 mL) for 4 h at 75 ◦C in a capped vial [8]. In GC analyses, the retention times of the resulting (2*S*)-oct-2-yl ester of 2-hydroxy tricosanoic and docosanoic acids were identical with those of the standard (2*S*)-oct-2-yl esters of (2*R*)-2-hydroxy tricosanoic and docosanoic acids, respectively.

The absolute configuration of glucose, released from oxidized cerebrosides, was determined by the GC analyses of per-acetylated (2*R*)-oct-2-yl glycosides according to the method of Leontein et al. [41]. The sugar (1.2 mg), (2*R*)-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 Ac2O (0.4 mL) in pyridine (0.4 mL), overnight. The acetylated material was purified by column chromatography (SiO2, 3.0 cm × 1.2 cm), eluting with a mixture of hexane/ethylacetate (5:1, *v*/*v*). The eluate was evaporated, yielding per-acetylated (2*R*)-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.

**Supplementary Materials:** The following are available online. Figure S1a: Base peak chromatogram from UPLC-MS analysis of oxidized cerebrosides (positive ion mode). Figure S1b: Extracted-ion chromatograms from UPLC-MS analysis of oxidized cerebrosides. Scheme S1: Hock fragmentation of some isomeric allylic hydroperoxides found in the present study. Figures S2, S2a–S2c: <sup>1</sup>Н-NMR spectra (CD3OD, 500 MHz) of the RP-HPLC fraction, containing oxidized cerebrosides. Figure S3 13C-NMR spectrum (CD3OD, 125 MHz) of the RP-HPLC fraction containing oxidized cerebrosides. Figures S4–S7: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C23 and C22 acids with a keto group in the (*n*–8) or (*n*–7) positions. Figures S8–S11: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C23 and C22 acids with an additional acetyloxy group in the (*n*–8) or (*n*–7) positions. Figures S12, S13: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C23 acids with a keto group in the (*n*–9) or (*n*–6) positions. Figure S14: Averaged mass spectrum for the four methyl ester of isomeric 2-acetyloxy keto C23 acids. Figures S15, S16: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C22 acids with a keto group in the (*n*–9) or (*n*–6) positions. Figures S17–S20: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C23 and C22 acids with an additional acetyloxy group in the (*n*–9) or (*n*–6) positions. Figures S21–S28: Mass spectra of samples, enriched in the methyl esters of 2-acetyloxy C23 and C22 acids with an allylic methylthio group. Figures S29–S32: Mass spectra of the methyl esters of 2-acetyloxy tris(methylthio) C23 and C22 acids. Figure S33: Mass spectrum of the bis(methylthio) adduct of methyl 11-hydroxy elaidate. Figure S34: Superimposed mass spectra of overlapping methyl 9-(methylthio)octadec-10-enoate and 11-(methylthio)octadec-9-enoate. Figure S35: Mass spectrum of

methyl 9,10,11-tris(methylthio)octadecanoate. Figure S36: Superimposed mass spectra of overlapping methyl 10-(methylthio)octadec-8-enoate and 8-(methylthio)octadec-9-enoate. Figure S37: Mass spectrum of methyl 8,9,10-tris(methylthio)octadecanoate. A section before Figures S33–S37 provide experimental details, concerning allylic monohydroxylation of methyl oleate and methylthiolation of allylic hydroxy/acetyloxy elaidates.

**Author Contributions:** Investigation, data interpretation, methodology, writing, E.A.S. NMR analyses, V.A.D. ESI-MS analyses, validation, P.S.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partly funded by the RFBR (Russian Foundation for Basic Research), grant NO. 20-03-00014, and by the Ministry of Science and Education (Russian Federation), grant NO. 13.1902.21.0012 (number of agreement: 075-15-2020-796).

**Acknowledgments:** The study was carried out on the equipment of the Collective Facilities Center "The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS". The authors express their gratitude to O.P. Moiseenko and L.P. Ponomarenko for their kind help in the GC-MS and GC analyses and V.A. Stonik for reading the manuscript and discussing its content. We thank D.N. Pelageev and O.S. Radchenko for the provision of some reagents.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.
