Characterization of Novel Glycolipids from the Giant Cockroach (Blaberus colosseus)*

A novel class of glycolipids, assigned the trivial name blaberosides, was isolated from whole head tissues of the giant cockroach (Blaberus colosseus). The class consists of two closely related families, blaberoside I and blaberoside 11, each containing species differing by 26 atomic mass units. The structure of these gentio- biose-based glycoglycerolipids was elucidated by chro-matographic behavior, nuclear magnetic resonance spectroscopy, mass spectrometry, and analysis of chemical degradation products and derivatives. Species in the blaberoside I family have been identified as (blabero-side Ia) and 2-0-[6’-0-(6“-0-3-hydroxy-ll-eicosen-oy~-~-D-g~ucopyranosy~)-~-~-g~ucopyranosy~]-~-(6-

glycolipids (blaberosides) and found them distinctly different from any previously reported glycolipids. This paper reports the first isolation of a glycoglycerolipid from an insect and presents structural characterizations of members of a novel class of glycolipids.

RESULTS
Isolation and Purification of Blaberosides-The average yield of total lipids in six preparative extractions of heads from B. colosseus was 43 mg/g fresh tissue. The yield of acetone fraction obtained by Unisil column chromatography of total lipids was about 56 mg/g total lipid.
TLC of the acetone fraction presented two bands. The band with an R F above mammalian cerebrosides was named blaberosides I. The band with an RF slightly below mammalian hydroxy cerebrosides but above mammalian sulfatides was named blaberosides 11. These two blaberosides were separately isolated by preparative TLC. Various spot tests indicated that both blaberosides contained nonreducing carbohydrate but no primary amino groups, nitrogen, or phosphorus. The compounds appeared to be homogeneous on high performance liquid chromatography analysis when derivatized by benzoylation (2), each producing single peaks with variable shoulder patterns in two separation systems, indicating considerable homogeneity with the possible presence of closely related homologs similar to those encountered due to the varying lengths of the fatty acids in glycosphingolipids (data not presented).
The average yield of blaberosides I was 26 pg from a cockroach head (332 pg/g tissue) or 294 pg/mg acetone fraction. The mean yield of blaberosides I1 was 176 pg/mg acetone fraction.
Structures of Blaberosides I and 11-From the following experimental observations, structures are proposed for blaberosides I and I1 (Fig. 1).
Infrared and Ultraviolet Spectrometry-The infrared spectrum blaberosides I1 was compatible with the known classes of glycoglycerolipids (3). It demonstrated hydroxy 0-H stretching (3700-3100 cm"), methine, methylene, and methyl C-H stretching (2970-2935 cm"), ester C=O stretching (1755 cm"), and a trans C=C stretching (1650 cm"). In the lower wave numbers, the blaberosides showed bands due to methylene deformation (1480 cm"), and C-0 stretch of a long chain ester (1170 cm"). Primary C-0 stretching of a primary The "Materials and Methods" are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. alcohol (1050 cm") and an alkyl ether with C-0 stretching (1080 cm") (4) were also shown. A minor band at 899 cm" suggested the presence of @-glycosidic linkages, and the minor absorbance at 720 cm" was indicative of skeletal vibrations in an unbranched CH2 chain at least four carbons long. The absence of any absorbances in the range of amides or sulfates eliminated glycosphingolipids from consideration (5). No absorbance between 200 and 800 nm was observed in UV spectra of the compounds.
Methanolysis Experiments-When crude acetone fraction lipids were subjected to mild alkaline methanolysis (NaOH) and separated by TLC, the blaberoside spots disappeared, indicating the presence of ester linkages in these lipids. Acid methanolysis of purified blaberosides yielded methyl ester of @-hydroxyl C20-enoic acid and methyl glucoside, identified as described below.
An ether extractable fragment from acid methanolysis had an identical RF value with a @-hydroxyl palmitic acid methyl ester standard on TLC in hexane:ether development. On GC2/ flame ionization detection the @-hydroxyl fatty acid methyl ester from both blaberosides I and I1 yielded identical single peaks confirming a single species of @-hydroxyl fatty acid was present in both lipids. GC/MS spectra indicated that this compound was Cz0 @-hydroxyl fatty acid methyl ester with one double bond (6)(7). The lack of other unbound nonpolar compounds indicated that the @-hydroxyl group of this fatty acid was unbound in the parent molecule.
The water-soluble acid methanolysis products produced a single spot on TLC with an RF identical to that of methylglucoside. Sugar analysis by liquid chromatography after acid hydrolysis of blaberosides I1 as described by Lee (8) also showed a single peak corresponding to glucose.
Methanolysis of purified blaberosides in methanolic triethylamine provided a single water soluble fraction on TLC The abbreviations used are: GC, gas chromatography; TMS, tetramethylsilane; MS; mass spectrometry; LD, laser desorption; PDMS, plasma desorption; FAB, fast atom bombardment; FID, flame ionization detection; GLC, gas liquid chromatography. which was identified as gentiobiose attached to glycerol 3alkyl ether from the following observations. The RF value consistently ran above methylglucoside and methyldiglucoside standards (gentiobiose, cellobiose, trehalose). The watersoluble product contained a nonreducing sugar. When examined by GC/MS as a TMS derivative, a single peak at 25.2 min represented the intact sugar with a glycerol ether still attached. Prominent mle 452 and 361 fragments indicated the presence of one sugar which was completely unsubstituted prior to TMS derivatization with the exception of the anomeric carbon. The lack of the mle 203 fragment, the presence of a prominent mle 295 fragment, and the abundance of mle 204 compared to mle 217, ruled out a trehalose 1-1 and suggested a 1-4 (cellobiose), 1-6 (gentiobiose), or 1-2 (sophorose) linkage (9)(10)(11)(12)(13). The assignment of a 1-6 linkage was supported by the presence of mle 583 and the very high abundance of mle 451 in the experimental spectrum matching a gentiobiose standard (Table I). Further methanolysis of the water-soluble material yielded a nonpolar fragment. GC/MS of TMS derivatives of the nonpolar fraction matched spectra of TMS batyl alcohol and TMS chimyl alcohol standards and indicated that homolog heterogeneity was due to this component of blaberosides. Chromotropic analysis of blaberosides for glycerol was negative. This observation suggested that the glycerol must be substituted at its @ carbon, preventing the formation of formaldehyde by this reaction.
Methylation Experiments-The position of attachment of the @-hydroxyl fatty acid in blaberoside I1 was determined by permethylation experiments. Two prominent spots of permethylation products appeared near the solvent front. The materials from these spots were eluted separately and subjected to acid methanolysis. The methylated sugars obtained from each spot were examined by GC/MS. The total ion chromatographs of both samples were indistinguishable, presenting single major peaks at 6.3 min with fragmentation patterns corresponding to methyl 2,3,4-tri-O-methylglucoside. Very minor anomer peaks at 5.03 min were present in each chromatograph, representing less than 10% of the total ion abundance. The overwhelming abundance of methyl 2,3,4-tri-0-methylglucoside established that only the six positions of glucose were protected from methylation. This observation, in combination with a disaccharide backbone, as discussed above, suggested a 1-6 sugar linkage and acylation of the nonreducing sugar's 6 position by the @-hydroxyl fatty acid. The nonpolar products of the acid methanolysis of the blaberoside permethylation product were fractionated by TLC and the products examined by GC/MS. The TLC indicated two nonpolar products, one with an R,v matching a (3-methoxypalmitate methyl ester standard and the other with an RF matching batyl alcohol methyl ether standard. The GC/MS analysis of the former material yielded a major peak with a retention time of 12.3 min, whose mass spectrum revealed a fragmentation pattern similar to that seen in @-methoxypalmitate standard and consistent with @-methoxyeicosenoic acid methyl ester. This pattern was interpreted to confirm the lack of participation of the @-hydroxyl group in structural covalent bonding. A smaller peak eluted at 11.3 min and produced a mass spectrum with a fragmentation pattern consistent with methyl @-methoxyeicosaenoate based on methyl @-methoxyhexadecaenoate standard. Retention times and fragmentation patterns of these components did not change following attempted TMS derivatization. The derivatives of monoalkyl ethers of glycerols were also examined by GC/MS. Authentic standards of hexadecyl glycerol ether and batyl alcohol were used for comparison. The monoalkylglycerol products of the acid methanolysis of permethylated blaberoside I1 had molecular ions and fragmentation patterns compatible with monomethylated selachyl and chimyl alcohol, indicating the presence of a free alcohol group on the glycerol backbone before methylation. The fragmentation patterns had prominent m/e 45 fragment abundance due to CH20CH3 ions and low abundance of m/e 31 fragments due to CHzOH ions (4-8 or greater m/e 54/31 ratio) indicating the glycerol ethers were linked to the sugar at their 2 position.
These studies indicated the presence of the diglucoside backbone with 1'-6 linkage and the esterification of the fatty acid to the 6' position of the nonreducing end sugar. They also suggested the unusual attachment of the carbohydrate to the second carbon of the glycerol ether (14).
Mass Spectrometry on Intact Bluberosides-Laser desorption, plasma desorption (PDMS), and fast atom bombardment (FAB) mass spectrometry were used to confirm the molecular weights of the structures shown in Fig. 1. Even electron molecular ions were observed, formed by the addition of protons or other cations to the neutral molecules. The results are summarized in Table 11. It should be noted that the FAB technique produces measurements of the monoisotopic mass, whereas both LD and PDMS, which are obtained on low resolution instruments, produce measurements of the average mass of the isotopically unresolved molecular ion cluster. Calculated values for the molecular weights are shown in Table I11 and are in good agreement with the measured values.
In general, laser desorption mass spectra (Fig. 2) were the most informative, since fragment ions were also observed. Peaks which differ by 26 mass units can be assigned to the fragments containing hexadecyloxy and octadeceloxy homologs, whereas other peaks represent the loss of this heterogeneous portion of the molecule. The most prominent peaks in    Fig. 3 shows 'H NMR of blaberosides 11. The peak at 5.25 ppm was due to ethylene protons. Numerous peaks between 5.2 and 2.8 ppm represented protons from the sugar, the fatty acid @-hydroxyl group and the free alcohol of the glycerol ether. From the chemical analysis described above, as many as three primary alcohols could be in the structure of blaberoside 11, one on the glycerol ether and one at each 6 position of the two glucoses. Only one triplet, which disappeared with deuterium exchange (see below), was found downfield, indicating that two of the three possible primary alcohols were substituted and only one was free.
Deuterium exchange greatly simplified the proton spectrum. The two most downfield doublets above the vinyl proton peak, retained after deuterium exchange, represented the anomeric protons of two hexose rings (data not shown). The spin-spin (J1,*) coupling constant of these peaks was 7.7 and 7.5 Hz indicating that both anomeric carbons were in p configuration.
'H NMR of the polar triethanolamine methanolysis products, blaberosides IIa and IIb without their P-hydroxylated fatty acids, showed a loss of the doublet centered on 4.6 ppm corresponding to the @hydroxyl group, the observation which confirmed that the hydroxyl group was free in the parent compound. The 5.25-ppm vinyl proton peak was also reduced about in half, confirming the presence of an unsaturation on the fatty acid.
13C Nuclear Magnetic Resonance Spectroscopy-13C NMR was useful in establishing the position of substitutions in blaberosides (Fig. 4). The shift of the sixth carbon peak of the reducing end sugar from 60.6 to 69.8 ppm confirmed a 1-6 linkage and the presence of a gentiobiose (Glul-Glu') sugar backbone, accounting for one of the substituted primary alcohols in the structure. It also further supported the glycosidic bond to the glycerol by eliminating any possible binding to sugar secondary alcohols, leaving only primary alcohol binding sites as alternatives to the anomeric position. The possi-   Too close to dimethyl sulfoxide-de signal to measure accurately.
' Too close to HDO signal to measure accurately.
bility of a glucose-glucose 1-4 linkage was eliminated by 13C NMR by the lack of a four-position carbon shifted to around 80 ppm. This shift was present in cellobiose (G1u'-Glu4) standard run under identical conditions. The possibility of 1-2 and 1-3 linkages were eliminated from the observation that C p and C3 were not shifted downfield. All other possible linkages were ruled out similarly by the lack of predicted shifts of the carbon supposedly involved in the linkage from the reducing end sugar.

Two-dimensional ' H Nuclear Magnetic
Resonance Spectroscopy-By establishing homonuclear shift correlated coupling relationships between protons on adjacent carbons, two-dimensional COSY provided adjunctive evidence for the sugar- NMR studies, therefore, supported the proposed structure of blaberoside I1 with the fatty acid esterified to the 6' position of gentiobiose and a glycosidic bond to the secondary alcohol dimethyl sulfoxide-d6, was adequate to show coupling between 1 and 5' and 6 and 6' sugar hydrogens establishing their relatively near through space proximity. Although more quantitative nuclear Overhauser effect data are required to assign specific through space distances, the coupling of these nuclei in this experiment indicates proximities of approximately 5 A. This would require the two sugar molecules to be folded over each other at the 1'-6 linkage.
Location of Double Bonds-The unsaturated 3-hydroxyeicosenoic acid methyl ester obtained from alkaline methanolysis of either blaberosides I or I1 was acetylated and then ozonized. The acetylation was necessary to avoid further degradation of the ozonolysis product. GC/MS analysis of the ozonolysis product, after purification by TLC, revealed only methyl esters of an 11 carbon 3-hydroxyl dicarboxylic acid and a nine-carbon fatty acid from samples of blaberoside I or 11. These results clearly indicated that the double bond in this fatty acid was located between the 11th and 12th carbons.

DISCUSSION
This report presents two major findings. First, the four blaberoside homologs characterized in this study represent the first identification and characterization of glycoglycerolipids from an insect. Second, these novel glycolipids are structurally unique from other known glycolipids of animal or plant origin.
Glycolipids are generally classified into two broad major divisions, the glycosphingolipids, containing long chain amino alcohols, and the glycoglycerolipids, which contain glycerol (15). The glycoglycerolipids, which would include the class blaberosides, are most abundant in plants and bacteria (3,16), but also occur in a variety of animal tissues including the mammalian brain as minor components (17). Among various glycoglycerolipids, blaberosides represent a previously unreported class of glycolipids distinct from the glycosylmonoacylglycerides, glycosyldiacylglycerols, and glycomonoalkylmonoacylglycerides, which occur commonly in nature and have their sugar attached to the tertiary alcohol position of a glyceroether and an ester-linked fatty acid at the 2 position.
The unique structure of blaberosides suggests several characteristics of the molecules. These in turn may prove important in the eventual determination of their role in the biology of the cockroach. The strict conservation of the unusual 201 structure of the 8-hydroxyl fatty acid implies very specific function of that moiety. The similarity in chain length to the alkyl chain of selachyl alcohol may also indicate similar functions, possibly anchoring the molecule in specific membrane environments. If blaberosides are integral membrane glycolipids, they could be anchored by any or all of their aliphatic chains.
The 1-6 linkage of the gentiobiose backbone is the most flexible of all of the disaccharide linkages (18). If blaberosides are anchored at both ends of the sugar backbone, the flexibility of the gentiobiose backbone would allow for the dynamic restructuring of the sugar by lateral adjustment of the positions of the lipophilic anchors providing the potential for "tuned reception" by a blaberoside receptor.
Membrane anchoring by the glycerol ether alone would leave the very flexible gentiobiose 1-6 linkage of blaberosides to serve as a bearing capable of considerable positional change for a P-hydroxyl fatty acid which could have important biological function. For example, the P-hydroxyl group and the first 20 carbons of the primary chain of the mycolic acids in lipid A from mycobacteria are thought to be responsible for lipid A's ability to initiate the complement cascade (19). The composition of the glycerol ethers is, like the P-hydroxyl fatty acids, highly conserved with only two homologs present. These two glycerol ethers, chimyl and selachyl alcohols, are the predominant monoalkylglycerols found in other animal tissues, including those of humans (20). The alkyl chains of the glycerol ethers could provide preferential stabilization in specific membrane loci with different membrane environments.
The diglucosyl double sugar backbone of the blaberosides is analogous to the digalactosyl backbone in the digalactosyldiacyl and monoalkylmonoacyl glycerolipids of vertebrates and may represent an evolutionary divergence of hexose metabolism similar to that postulated in the cerebrosides (1). This may have further implications for the presence of sulfoglycoglycerolipids in insects if the lack of insect sulfatide sphingolipids is indeed due to the inability of glucose to be sulfated in a manner similar to galactose (1). In addition, the P-1-6-diglucoside linkage (gentiobiose) is unique in the characterized glycoglycerolipids, although commonly present in a variety of other important nonlipid plant compounds including Crocetin mono-and digentiobiosyl esters from crocus plants and saffron spice (21) and the drug amygdalin or laetrile (0-€3-D-gentiobiosyl-D-(-)-mandelonitrile) from the seeds of rosaceae plants (22).
The p-glycosidic linkage of a glycerol ether to a sugar backbone is characteristic of glycoglycerolipids; however, assignment of this linkage to the second position of the glycerol rather than the first position is unusual. The characterized glycoglycerolipids, with the exception of blaberoside 11, have complete substitution of their glycerol moiety which may affect the ability to achieve sugar attachment to the second glycerol position.
Only 12 insect glycolipids have been previously described (1, 23-26), the majority from whole body homogenates and all glycosphingolipids containing glucose, galactose, mannose, N-acetylglucosamine, or N-acetylgalactosamine, analogous to the vertebrate glycosphingolipids. Strong evidence in vertebrate species suggests insect glycolipids, including blaberosides, should serve detection, reception, and selection roles at the tissue, cellular, membrane, and molecular level.