Nature, type of linkage, quantity, and absolute configuration of (3-hydroxy) fatty acids in lipopolysaccharides from Bacteroides fragilis NCTC 9343 and related strains.

The main fatty acids present in lipopolysaccharides from Bacteroides fragilis NCTC 9343 were identified as 13-methyl-tetradecanoic, D-3-hydroxypentadecanoic, D-3-hydroxyhexadecanoic, D-3-hydroxy-15-methyl-hexadecanoic, and D-3-hydroxyheptadecanoic acids. Of these, 13-methyl-tetradecanoic acid is exclusively ester bound, and 3-hydroxy-15-methyl-hexadecanoic acid is exclusively involved in amide linkage. The other 3-hydroxy fatty acids are both ester and amide bound. All 3-hydroxy fatty acids possess the D configuration, and the 3-hydroxyl group of ester-linked 3-hydroxy fatty acids is not substituted. Lipopolysaccharides of related Bacteroides species (B. thetaiotaomicron, B. ovatus, B. distasonis, and B. vulgatus) showed a fatty acid spectrum with both similar and distinct features compared to that of B. fragilis lipopolysaccharides.     

Chemical structure of the lipid A component of lipopolysaccharides from Fusobacterium nucleatum.

The lipid A component of lipopolysaccharides from Fusobacterium nucleatum Fev 1 consists of beta-1',6-linked D-glucosamine disaccharides, which carry two phosphate groups: one in glycosidic and one in ester linkage. The amino groups of the glucosamine disaccharides are substituted by D-3-hydroxyhexadecanoic acid. The hydroxyl groups of the disaccharide backbone are acylated by tetradecanoic, hexadecanoic, and D-3-hydroxytetradecanoic acids. Part of the ester-bound D-3-hydroxytetradecanoic acid is 3-O-substituted by tetradecanoic acid. Whereas a similar pattern of fatty acids was detected in lipopolysaccharides from two other F. nucleatum strains, the amide-bound fatty acid in F. varium and F. mortiferum was D-3-hydroxytetradecanoic acid. The chemical relationships of lipid A from Fusobacteria and other gram-negative bacteria are discussed.     

Lipopolysaccharides from Pseudomonas maltophilia: composition of the lipopolysaccharide and structure of the side-chain polysaccharide from strain N.C.I.B. 9204

Lipopolysaccharide was extracted from defatted cell-walls of Pseudomonas maltophilia N.C.I.B. 9204. The major fatty acid components were 9-methyldecanoic acid, 2-hydroxy-9-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-11-methyldodecanoic acid. Monosaccharide components of the phosphorylated core-oligosaccharide were D-glucose, D-mannose, D-galacturonic acid, 2-amino-2-deoxyglucose, and a 3-deoxyoctulosonic acid. The putative O-specific polysaccharide was composed mainly of 2-amino-2-deoxy-D-glucose, D-arabinose, and 6-deoxy-L-talose, but also contained an O-acetyl group and small proportions of rhamnose and 6-deoxy-3-O-methyltalose. Degradative and n.m.r. (1H and 13C) studies showed that the polymer had a branched trisaccharide repeating-unit with the following structure; the O-acetyl group was tentatively assigned to C-2 of the 6-deoxytalopyranosyl residue. (Formula: see text).     

L-2-hydroxytetradecanoic acid as a constituent of Salmonella lipopolysaccharides (lipid A)

L-2-Hydroxytetradecanoic acid was recognized as a characteristic, although minor, constituent of the lipid A component of Salmonella lipopolysaccharides. The 2-hydroxy fatty acid was present in lipid A as an ester, probably bound to the hydroxyl group of some D-3-hydroxytetradecanoic acid residues. A survey of enterobacterial lipopolysaccharides showed that L-2-hydroxytetradecanoid acid was also present in Klebsiella and Serratia strains. It was absent, however, from lipopolysaccharides of other genera of the family including Escherichia, Shigella, Proteus, Enterobacter and Yersinia. This restricted distribution of the 2-hydroxy acid may be of significance for taxonomic studies of bacterial genera.     

Structural analysis of Herbaspirillum seropedicae lipid-A and of two mutants defective to colonize maize roots

Lipid-A was isolated by mild acid hydrolysis from lipopolysaccharides extracted from cells of Herbaspirillum seropedicae, strain SMR1, and from two mutants deficient in the biosynthesis of rhamnose (rmlB(-) and rmlC(-)). Structural analyzes were carried out using MALDI-TOF and derivatization by per-O-trimethylsilylation followed by GC-MS in order to determine monosaccharide and fatty acid composition. De-O-acylation was also performed to determine the presence of N-linked fatty acids. Lipid-A from H. seropedicae SMR1 showed a major structure comprising 2-amino-2-deoxy-glucopyranose-(1→6)-2-amino-2-deoxy-glucopyranose phosphorylated at C4' and C1 positions, each carrying a unit of 4-amino-4-deoxy-arabinose. C2 and C2' positions were substituted by amide-linked 3-hydroxy-dodecanoic acids. Both rhamnose-defective mutants showed similar structure for their lipid-A moieties, except for the lack of 4-amino-4-deoxy-arabinose units attached to phosphoryl groups.     

Lipopolysaccharides from Mesorhizobium huakuii and Mesorhizobium ciceri: chemical and immunological comparative data

Lipopolysaccharides of two Mesorhizobium species of different host specificity were compared: M. huakuii and M. ciceri. M. huakuii sp. was represented by five strains with special consideration of M. huakuii IFO 15243(T). SDS/PAGE profiles revealed that all M. huakuii LPS preparations contained low molecular mass fractions (LPS-II) of the same molecular size. All of lipopolysaccharides contained high molecular mass fractions (LPS-I). However, the high molecular mass fraction from each strain possessed an individual molecular size distribution pattern. The crossreactivity of blotted lipopolysaccharides with rabbit polyclonal antibodies against Mesorhizobium huakuii IFO 15243(T) whole bacteria indicated the presence of common epitope(s) within the investigated Mesorhizobium huakuii strains. Moreover, LPS from M. huakuii S52 also reacted with anti M. ciceri HAMBI 1750 serum showing that there are epitopes common for different mesorhizobial species. LPS isolated from Mesorhizobium huakuii strain IFO 15243(T) contained neutral sugars: L-6-deoxytalose, L-rhamnose, D-galactose and D-glucose, aminosugars:D-quinovosamine, D-glucosamine, D-2,3-diamino-2,3-dideoxyglucose and D-galacturonic and D-glucuronic acids. In the LPS preparation, fatty acids typical for Mesorhizobium strains were detected. 3-Hydroxydodecanoic, 3-hydroxy-iso-tridecanoic, 3-hydroxyeicosanoic, 3-hydroxyheneicosanoic and 3-hydroxydocosenoic acids were the major amide linked fatty acids, while iso -heptadecanoic, eicosanoic, docosenoic, as well as 27-hydroxyoctacosanoic and 27-oxooctacosanoic acids were the dominant ester linked fatty residues.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxy octadecadienoic acids and monohydroxy eicosatetraenoic acids

The metabolism of arachidonic and linoleic acids by VX₂ carcinoma tissue was determined. Prostaglandin E₂ was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3– 8% of arachidonic acid metabolism were 11-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (11-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40–80% 9-HODD and 20–60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX₂ carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E₂ biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E₂ biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX₂ carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E₂ via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.     

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species.

Two strains of Bacteroides asaccharolyticus and two strains of Bacteroides fragilis were analyzed for total fatty acid, total lipid fatty acid, and total bound fatty acid profiles. Extracted lipids and defatted cell residues were subjected to sequential alkaline and acid methanolyses to distinguish ester- and amide-linked fatty acids in each fraction. In the lipid fractions, all the ester-linked fatty acids were nonhydroxylated, whereas all of the amide-linked fatty acids were hydroxylated. In the nonextractable fractions, both hydroxy and nonhydroxy fatty acids were found in both ester and amide linkage, although hydroxy acids predominated. The fatty acid profiles of the bound fractions differed widely from those of the lipid fractions. Bound fatty acid represented approximately 10% of the total cellular fatty acids.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absolute configuration of 3-hydroxy fatty acids present in lipopolysaccharides from various bacterial groups.

The absolute configuration of 3-hydroxy fatty acids has been studied, which are present in the lipopolysaccharides of the following bacteria: Phodopseudomonas gelatinosa, Rh. viridis, Rhodospirillum tenue, Chromobacterium violaceum, Pseudomonas aeruginosa, Bordetella pertussis, Vibrio metchnikovii, Vibrio cholerae, Salmonella spp., Escherichia coli, Shigella flexneri, Proteus mirabilis, Yersinia enterocolitica and Fusobacterium nucleatum. The 3-hydroxy acids were liberated by strong alkaline hydrolysis, converted to 3-methoxy acid L-phenylethylamides and analyzed by gas-liquid chromatography. With the aid of authentic D-3-hydroxy fatty acids it was shown for all lipopolysaccharides that the 3-hydroxy acids, regardless of chain lengths, branching, 3-O-substitution or type of linkage, possess the D-configuration. 2-Hydroxydodecanoic acid, which is present in some lipopolysaccharides, was analyzed in an analogous way and shown to possess the L-configuration.     

Occurrence of Branched-Chain Hydroxy Fatty Acids in Pseudomonas maltophilia

Three branched-chain hydroxy acids not previously reported in other bacteria were found in extracts from saponified whole cells of Pseudomonas maltophilia. On the basis of evidence from mass spectrometry, infrared spectroscopy, and gas chromatographic procedures, they were identified as 2-hydroxy-9-methyldecanoic acid, 3-hydroxy-9-methyldecanoic acid, and 3-hydroxy-11-methyldodecanoic acid. These acids appeared to be tightly bound to other cellular components since they were not extracted from lyophilized cells with a chloroform-methanol (3:1) mixture.     

Hydroxy fatty acids in Bacteroides species: D-(--)-3-hydroxy-15-methylhexadecanoate and its homologs.

Acid hydrolysates of 140 strains, representing 11 species of the genus Bacteroides, were analyzed by capillary gas-liquid chromatography for total cellular fatty acid. All samples contained components which appeared to be hydroxy fatty acid. The relative amount and chain length distribution of the hydroxy fatty acids, as well as the nonhydroxy fatty acids, varied according to species. To characterize the presumed hydroxy acids, a composite of some 40 of these samples was analyzed by thin-layer and capillary gas-liquid chromatography, mass spectrometry, infrared spectrophotometry, and polarimetry. The hydroxy acids were shown to be of the D-(--)-3-hydroxy acid family. The predominant component was the iso-branched D-(--)-3-hydroxy-15-methylhexadecanoic acid. Lesser amounts of the iso-branched 15-carbon, straight-chain 16-carbon, and anteiso-branched 17-carbon acids were also found.     

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.     

Myristyl and palmityl acylation of the insulin receptor

The presence of covalently bound fatty acids in the insulin receptor has been explored in cultured human (IM-9) lymphocytes. Both alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor incorporate [3H]myristic and [3H]palmitic acids in a covalent form. The effects of alkali and hydroxylamine on the labeled subunits indicate the existence of two different kinds of fatty acid linkage to the protein with chemical stabilities compatible with amide and ester bonds. The alpha subunit contains only amide-linked fatty acid while the beta subunit has both amide- and ester-linked fatty acids. Analysis by high performance liquid chromatography after acid hydrolysis of the [3H]myristate- and [3H]palmitate-labeled subunits demonstrates the fatty acid nature of the label. Furthermore, both [3H]myristic and [3H]palmitic acids are found attached to the receptor subunits regardless of which fatty acid was used for labeling. The incorporation of fatty acids into the insulin receptor is dependent on protein synthesis and is also detectable in the Mr = 190,000 proreceptor form. Fatty acylation is a newly identified post-translational modification of the insulin receptor which may have an important role in its interaction with the membrane and/or its biological function.     

Lipopeptide structure determines TLR2 dependent cell activation level

Bacterial lipoproteins/peptides are composed of di-O-acylated-S-(2,3-dihydroxypropyl)-cysteinyl residues N-terminally coupled to distinct polypeptides, which can be N-acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester-bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide-bound and another ester-bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno-stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.     

Characterization ofCampylobacter jejuni lipopolysaccharide

Lipopolysaccharides were isolated from dehydratedCampylobacter jejuni by combination of the phenol-chloroform-petroleum ether and phenol-water extraction techniques. Biochemical characterizations of lipopolysaccharide were performed on the two fractions of highest purity. Neutral sugar analyses detected galactose, glucose, trace amounts of mannose, and an unidentified deoxy-hexose. The primary amino sugars were galactosamine, glucosamine, and glucosamine-phosphate. Chemical analyses of other lipopolysaccharide components included phosphate, 3-deoxy-d-manno-octulosonic acid (KDO), and fatty acids. The predominant fatty acids were 3-hydroxytetradecanoic and hexadecanoic acids with lesser amounts of tetradecanoic acid. 3-Hydroxytetradecanoic acids were bound to lipid A by both amide and ester linkages.     

Total synthesis and conformational studies of hapalosin, N-desmethylhapalosin and 8-deoxyhapalosin.

Hapalosin (2), a 12-membered cyclic depsipeptide possessing MDR-reversing activity, and analogues (3) and (4) have been synthesized using macrolactamization as an important ring-forming step. Three building blocks: (2S,3R)-3-(tert-butyldimethylsilyloxy)-2-methyl-decanoic acid (13), benzyl (S)-2-hydroxy-3-methylbutanate (14), and (4S,3R)-4-(benzyloxycarbonyl-methylamino)-3-methoxymethoxy-5-pheny l-pentanoic acid (28) were prepared from Evans's chiral imide (9), L-valine, and L-N-Boc phenylalanine (17), respectively, and were assembled together by applying twice Yamaguchi's coupling methodology. A new and efficient selective N-methylation of gamma-hydroxy-beta-amino ester taking advantage of the vicinal amino alcohol function was uncovered in the course of this study. Thus, treatment of compound 19 with HCHO in the presence of catalytic amount of pTsOH followed by reduction (NaBH3CN, TFA, CH2Cl2) of the so-formed oxazolidine 24 gave the N-methylated product 25. Furthermore, a dual role of oxazolidine as protecting group of vicinal amino alcohol and latent N-methyl function was established which allowed synthesizing both hapalosin (2) and N-desmethylhapalosin (3) from the same linear precursor 32 in a step-efficient and atom economic way. In contrast to hapalosin (2) and N-desmethyl analogue (3), the amide bond of 8-deoxy hapalosin (4) exists at room temperature (CDCl3) exclusively in s-cis conformation as evidenced by NOE studies. This observation has been explained on the basis of computational studies. No significant MDR reversing activity of 8-deoxy hapalosin (4) was observed in K562 R and S/Adriblastine against human erythroleucemic cell lines indicating thus the important contribution of hydroxy group to the bioactivity of hapalosin.     

Characterization of a structural series of lipid A obtained from the lipopolysaccharides of Neisseria gonorrhoeae. Combined laser desorption and fast atom bombardment mass spectral analysis of high performance liquid chromatography-purified dimethyl derivatives

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of serum-sensitive strains of Neisseria gonorrhoeae was fractionated on a silicic acid column to yield the hexaacyl and pentaacyl MLAs. The dimethyl derivative of the hexaacyl MLA was analyzed by proton nuclear magnetic resonance spectroscopy. The dimethyl esters of hexaacyl and pentaacyl MLAs were further purified by reverse-phase high performance liquid chromatography, and all of the peaks were analyzed by laser desorption mass spectrometry. Considerable structural information was obtained by laser desorption mass spectrometry due to three kinds of specific fragmentations of the sugar at the reducing end. Two major fractions were also analyzed by positive ion fast atom bombardment mass spectrometry. High performance liquid chromatography was able to separate the dimethyl MLA according to number, nature, and position of the fatty acyl groups. Since almost no structural information is available, the mass spectra of the samples were interpreted on the basis of the established structure of a model lipid A (hexaacyl MLA derived from Salmonella minnesota). Thirteen different structures of dimethyl MLA were identified. The four prominent dimethyl MLAs found in the fractionated samples were M1 (Mr = 1463), M2 (Mr = 1479), M3 (Mr = 1661), and M4 (Mr = 1677). These MLAs appear to have a 1'----6 linked glucosamine disaccharide backbone. The most prominent hexaacyl MLA was M3. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage and lauroxymyristate at the 2- and 2'-positions in amide linkage of the glucosamine disaccharide. The most abundant pentacyl MLA was M2. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage, lauroxymyristate at the 2'-position in amide linkage, and hydroxymyristate at the 2-position in amide linkage of the disaccharide. The lipid A of N. gonorrhoeae appeared to differ from that of the Salmonella strains by the presence of shorter-chain fatty acids and by the normal fatty acid distribution in the reducing and distal subunits.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.