**Elena A. Santalova \*, Vladimir A. Denisenko and Pavel S. Dmitrenok**

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, Pr. 100 let Vladivostoku 159, 690022 Vladivostok, Russia; vladenis@piboc.dvo.ru (V.A.D.); paveldmt@piboc.dvo.ru (P.S.D.)

**\*** Correspondence: santalova@piboc.dvo.ru or santalova.e.a@yandex.ru; Tel.: +7-(4232)-31-14-30; Fax: +7-(4232)-31-40-50

Academic Editors: Daniela Rigano and RuAngelie Edrada-Ebel Received: 1 November 2020; Accepted: 19 December 2020; Published: 21 December 2020

**Abstract:** 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 <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**//). 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.

**Keywords:** glycosphingolipids; cerebrosides; peroxidation products; structure elucidation; allylic thioether; NMR; ESI-MS; GC-MS; mass spectra; glass sponge
