*2.2. NMR Characterization of Oxidized Cerebrosides*

The <sup>1</sup>Н-NMR and 13C-NMR spectra (CD3OD) of oxidized cerebrosides (Table 1, Figures S2 and S3), as well as the corresponding spectra of the non-oxidized cerebrosides of *Aulosaccus* sp. [8], showed signals of β-glucopyranosyl-(1→1)-ceramides that had monoenoic or cyclopropane-containing phytosphingosine-type backbones, *N*-acylated with 2-hydroxy FAs. In particular, H-2 (δН 4.25, m) of the sphingoid base moieties displayed a characteristic cross signal with *N*-acyl C-1/ (δ<sup>C</sup> 177.7), as demonstrated by an НMBC experiment with sphingolipids **1**–**3**. <sup>1</sup>Н, <sup>1</sup>Н-COSY diagram indicated that several protons, starting from −O−CH2− (δ<sup>Н</sup> 3.80 and 4.045, dd, CH2-1) and ending with alkyl −CH2− (δН 1.31 and 1.55, m, CH2-6), formed a linear spin system of phytosphingosine-type moieties of **1**–**3**. Another spin system consisted of CH-2/ (δ<sup>Н</sup> 4.01, dd), CH2-3/ (δ<sup>Н</sup> 1.60 and 1.74, m), and CH2-4/ (δ<sup>Н</sup> 1.42, m) protons of 2-hydroxy acyl chains. The signals of a β-glucopyranoside moiety were sequentially assigned by <sup>1</sup>Н, <sup>1</sup>Н-COSY, HSQC (heteronuclear single-quantum correlation spectroscopy), and НМBC experiments, starting from the signal of anomeric CH-1// (δ<sup>Н</sup> 4.28, d, *J* = 7.8 Hz; δ<sup>C</sup> 105.3). Accordingly, cross signals CН**2**-1/**C**-1// and CН-1///**C**-1 were observed in the НМBC diagram of glycosides **1**–**3**. Then, the <sup>1</sup>Н-and 13C-NMR spectra of these sphingolipids showed signals of long hydrocarbon chains (−(CH2)n−, δ<sup>Н</sup> 1.22–1.42, m, δ<sup>C</sup> 30.6–32.0) and terminal methyl groups (δ<sup>Н</sup> 0.89–0.90, several overlapping triplets, δ<sup>C</sup> 15.0–15.05, broad signal). The δ values of allylic CH2 (δ<sup>C</sup> 28.7–28.85, δН 2.02, m) and olefinic CH (δН 5.34, m) were used to characterize *cis*-double bonds of backbones **1** and **2**. These data were in good agreement with <sup>1</sup>Н-NMR and 13C-NMR (CD3OD) data on some cerebrosides [21] and FA standards [22] with isolated *cis*-double bonds (*cis*-isomers: δ<sup>C</sup> 28.7–28.9, δН 2.02–2.03 m, allylic CH2, and δ<sup>H</sup> 5.335–5.34 m, olefinic CH, *trans*-isomers: δ<sup>C</sup> 34.15–34.2, δН 1.97–1.975 m, allylic CH2, and δН 5.37–5.38 m, olefinic CH). A *cis*-cyclopropane ring in backbone **3** caused three upfield shifted signals at δ<sup>Н</sup> −0.33 (dt, *J* = 4.1, 5.3 Hz, H-21a), 0.58 (ddd, *J* = 4.1, 8.3, 8.3 Hz, H-21b), and 0.67 (m, H-13, H-14).


**Table 1.** <sup>1</sup>Н-(500 MHz) and 13C-(125 MHz) NMR data (CD3OD) <sup>1</sup> for the RP-HPLC fraction, containing compounds **1a**–**a**//, **1b**–**b**//, **2a**–**a**//, **2b**–**b**//, **3c**–**c**//, and **3d**–**d**//.

<sup>1</sup> All signals were assigned based on data of <sup>1</sup>Н−1НCOSY, HSQC, and НМBC experiments. <sup>2</sup> The two resolved triplets were observed in 700-MHz spectra. <sup>3</sup> δ<sup>H</sup> and δ<sup>C</sup> values for acyl fragments with allylic –OOН/–OН/=O are given separately (Figure 5).

Apart from the previously mentioned NMR resonances, the signals of *trans*-monoenoic acyl moieties with an allylic hydroperoxy, hydroxy, or keto group (Figure 5a–c) were observed in the NMR spectra of the RP-HPLC fraction, containing oxidized cerebrosides. The δ<sup>H</sup> values (CD3OD) of the signals for –**H**C(OOH)– (4.16, dt), –**H**C(OH)– (3.94, q), and −CH=C**H**–CO– (6.105, d) were close to δ<sup>H</sup> values (CDCl3) of signals of corresponding protons in the *trans*-monoenoic allylic hydroperoxides (4.2, q), allylic alcohols (4.0, q), and enones (6.1, d), respectively, prepared from oleic acid [23]. Deviations of δ<sup>H</sup> values (this study) from those in other experiments [23] arose from solvent effects. According to <sup>1</sup>Н, <sup>1</sup>Н-COSY correlations, a spin system, assigned to acyl chains **a**–**d** (Figure 5a), included protons of allylic hydroperoxide, especially two olefinic CH (δН 5.35 and 5.665, m) and allylic CH (δ<sup>Н</sup> 4.16, dt, *J* = 6.6, 7.0 Hz), bearing −OOH. Allylic CH (δ<sup>Н</sup> 3.94, q, *J* = 6.8 Hz), bearing –OH, and two olefinic CH (δН 5.395 and 5.59, m) groups, belonging to allylic alcohol, were part of a spin system within acyl chains **a**/ –**d**/ (Figure 5b). In experiments, involving selective irradiation of allylic 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 (δ<sup>C</sup> 204.3), and α-CH2 (δ<sup>Н</sup> 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.

**Figure 5.** The δ<sup>H</sup> (500 MHz) and δ<sup>C</sup> (125 MHz) values and key HMBC correlations for acyl fragments with allylic (**a**) hydroperoxy (acyl chains **a**–**d**), (**b**) hydroxy (acyl chains **a**/ –**d**/ ), and (**c**) keto (acyl chains **a**//–**d**//) groups (solvent: CD3OD).

Some very weak signals in the <sup>1</sup>Н-NMR, <sup>1</sup>Н, <sup>1</sup>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.
