**Appendix C**

Methyl esters of 11-hydroxy and 8-hydroxy elaidic acids were prepared from methyl oleate according to the method described by Li et al. [46]. Methyl 11-hydroxy elaidate was treated with DMDS to give a mixture, consisting of 59.2% of the expected DMDS adduct, 26.8% of isomeric allylic thioethers (9-(methylthio)octadec-10-enoate and 11-(methylthio)octadec-9-enoate), and 8.0% of 9,10,11-tris(methylthio)octadecenoate (Scheme A1). Two GC peaks that showed identical major fragments in GC-MS represented stereoisomeric *bis*(methylthio) adducts with a nearly 1:1 ratio. Assuming a specific anti-addition of DMDS [33], these stereoisomeric adducts could be racemic erythro-9,10-bis(methylthio)octadecanoates, differing in configuration only at the carbon atom linked to the –OH group. Based on the NOESY experiment, two pairs of singlets at δН 2.14, 2.22 and δН 2.06, 2.185 in the 1H-NMR spectrum (CDCl3) of the mixture were assigned to vicinal –SMe groups of different racemates. Two GC peaks (major and minor) for mono(methylthio)octadecenoates also had identical mass spectra. For major *trans*-allylic thioethers, the 1H,1H-COSY diagram (CDCl3) showed a spin system, which consisted of the protons of two *trans*-olefinic CH (δН 5.17, dd, *J* = 9.2, 15.2 Hz,

and 5.40, dt, *J* = 6.8, 15.2 Hz) and CH (δ<sup>Н</sup> 2.985, m), bearing an –SMe group (1H-NMR spectrum: δ<sup>H</sup> 1.965, s). Accordingly, the major GC peak for overlapping 9-(methylthio)octadec-10-enoates and 11-(methylthio)octadec-9-enoates represented isomers with a *trans*-double bond (23.0%), and the minor GC peak, observed before the major peak, likely represented their *cis*-isomers (3.8%). Similarly, two GC peaks (major and minor) for stereoisomeric 9,10,11-tris(methylthio)octadecanoates were observed. Upon MeCN/HCl hydrolysis of this methylthio derivative mixture, the stereoisomeric bis(methylthio) adducts were destroyed, so major allylic thioethers (9/11-(methylthio)octadec-10/9-enoic acids, 59.3%) and minor tris(methylthio) derivatives (stereoisomeric 9,10,11-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 11-hydroxy elaidate. This was confirmed by the <sup>1</sup>Н-NMR spectra, recorded before and after hydrolysis. In particular, the <sup>1</sup>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 (δ<sup>H</sup> 2.10, s), and α-CH2 (δ<sup>Н</sup> 1.845, m; 1.32, m) groups of *bis*(methylthio) oleic acid. Analogously, the *bis*(methylthio) derivatives of monoenoic sphingoid bases were obtained after MeCN/HCl hydrolysis of derivatized cerebrosides (part 2).

As for the allylic alcohol acetate, methyl 8-acetyloxy elaidate did not react appreciably with DMDS under the conditions used for methyl 11-hydroxy elaidate (room temperature, 24 h). However, with longer reaction times (4 days), methyl 8-acetyloxy elaidate reacted with DMDS to give major allylic ethers, 8-(methylthio)octadec-9-enoate and 10-(methylthio)octadec-8-enoate (61.5%, mainly *trans*-forms), and minor 8,9,10-tris(methylthio)octadecanoates (25.4%), formed after deacetylation of the starting compound. Surprisingly, the expected bis(methylthio)derivative of methyl 8-acetyloxy elaidate was not detected (Scheme A1). For experimental details and mass spectra (Figures S33–S37), see the supplementary materials.
