Some biological activities of a series of 9a-homo-9,11-epoxy prostanoic acid analogues

A number of stereochemical variants at C-8, C-12 and C-15 of 9a-homo-9,11-epoxy prostaglandins (PGs) have been examined for in vivo activity on blood pressure, bronchial resistance, tracheal segment pressure, heart rate and on intestinal and uterine contractility in artificially respired anaesthetised guinea-pigs; and on blood pressure and blood platelet aggregation in rats (using the extra-corporeal filter-aorta loop technique). In vitro tests for smooth muscle activity were carried out on the isolated rat fundus strip, the guinea-pig tracheal chain and the rat uterus. The following was found: 1. In the guinea-pig, in vivo, all the homo-epoxy PGs were vasopressor and bronchoconstrictor following bolus injections of 250 micrograms i.v. The effects on heart rate, and intestinal and uterine contractility were equivocal. The configurations at the chiral centres, C-8, C-12 and C-15 play an important role in determining potency. The 15-(S)-hydroxy derivatives were the most potent in stimulating vascular and respiratory muscle. The 8-iso configuration appeared to enhance potency amongst the 15-(S)-hydroxy compounds. The 15-(R)-hydroxy configuration markedly reduced constrictor potency. The same pattern of activity was seen on rat blood pressure, in vivo. The 15-(S)-hydroxy configuration combined with the 8-iso configuration had the most potent constrictor activity, while the 15-(R)-hydroxy group negated this and even led, in the case of the natural configuration at C-8 and C-12, to vasodepression. 2. In vitro, the activity on the rat fundus and guinea-pig tracheal chain followed the same pattern. The 15-(S)-hydroxy derivatives were very much more potent than the 15-(R)-hydroxy derivatives at contracting the smooth muscle preparations. Uterine muscle appeared to be relaxed by the PGs with the natural configuration at C-8 and C-12, with the 15-(R)-hydroxy compound exhibiting greater activity. 3. Inhibition of ADP-induced rat blood platelet aggregation after "intra-arterial" administration was shown only by the derivatives with a single change in the natural configuration either at C-8 or at C-15. Additional changes either resulted in inactivity or, in the case of the 8,12-di-iso-15-(S)-hydroxy compound, even reversed the effect to aggregation. The inhibition of aggregation was long lasting with both the 8-iso-15-(S)-hydroxy derivative and the 8,12-nat-15-(R)-hydroxy derivative. In the case of the latter compound, GBR-30731, activity increased during the 30 min after administration. GBR-30731 deserves further investigation as a platelet aggregation inhibitor because of its relatively low smooth muscle stimulant (sometimes even relaxant effects) and its long lasting platelet aggregation inhibiting activity.     

Some biological activities of a series of 9a-homo-9,11-epoxy prostanoic acid analogues

A number of stereochemical variants at C-8, C-12 and C-15 of 9a-homo-9,11-epoxy prostaglandins (PGs) have been examined for in vivo activity on blood pressure, bronchial resistance, tracheal segment pressure, heart rate and on intestinal and uterine contractility in artificially respired anaesthetised guinea-pigs; and on blood pressure and blood platelet aggregation in rats (using the extra-corporeal filter-aorta loop technique). In vitro tests for smooth muscle activity were carried out on the isolated rat fundus strip, the guinea-pig tracheal chain and the rat uterus. The following was found:

1. In the guinea-pig, in vivo, all the homo-epoxy PGs were vasopressor and bronchoconstrictor following bolus injections of 250 μg i.v. The effects on heart rate, and intestinal and uterine contractility were equivocal. The configurations at the chiral centres, C-8, C-12 and C-15 play an important role in determining potency. The 15-(S)-hydroxy derivatives were the most potent in stimulating vascular and respiratory muscle. The 8-iso configuration appeared to enhance potency amongst the 15-(S)-hydroxy compounds. The 15-(R)-hydroxy configuration markedly reduced constrictor potency. The same pattern of activity was seen on rat blood pressure, in vivo. The 15-(S)-hydroxy configuration combined with the 8-iso configuration had the most potent constrictor activity, while the 15-(R)-hydroxy group negated this and even led, in the case of the natural configuration at C-8 and C-12, to vasodepression.

2. In vitro, the activity on the rat fundus and guinea-pig tracheal chain followed the same pattern. The 15-(S)-hydroxy derivatives were very much more potent than the 15-(R)-hydroxy derivatives at contracting the smooth muscle preparations. Uterine muscle appeared to be relaxed by the PGs with the natural configuration at C-8 and C-12, with the 15-(R)-hydroxy compound exhibiting greater activity.

3. Inhibition of ADP-induced rat blood platelet aggregation after “intra-arterial” administration was shown only by the derivatives with a single change in the natural configuration either at C-8 or at C-15. Additional changes either resulted in inactivity or, in the case of the 8,12-di-iso-15-(S)-hydroxy compound, even reversed the effect to aggregation.

The inhibition of aggregation was long lasting with both the 8-iso-15-(S)-hydroxy derivative and the 8,12-nat-15-(R)-hydroxy derivative. In the case of the latter compound, GBR-30731, activity increased during the 30 min after administration. GBR-30731 deserves further investigation as a platelet aggregation inhibitor because of its relatively low smooth muscle stimulant (sometimes even relaxant effects) and its long lasting platelet aggregation inhibiting activity./lt     

Synthesis of eicosa-2-trans-8,11,14-all cis-tetraenoic acid-3-14C and DL-3-hydroxy eicosa-8,11,14-all cis-trienoic acid-3-14C

A general procedure for the synthesis of 2-trans polyenoic fatty acids and of dl-3-hydroxypolyenoic acids is described. The 2-trans acids are prepared by LiAlH(4) reduction of a suitable polyenoic fatty acid ester to the alcohol, formation of the tosylate, oxidation to the aldehyde, and Doebner condensation of the latter with malonic acid. The 3-hydroxy acids are obtained by reaction of the acyl chloride of a suitable polyenoic acid with the sodium enolate of methyl acetoacetate and sodium methoxide to give the 3-keto ester, the keto group of which is reduced with sodium borohydride to the alcohol. These procedures were applied to the synthesis of eicosa-2-trans-8, 11, 14-all cis-tetraenoic acid-3-(14)C and DL-3-hydroxy eicosa-8, 11, 14-trienoic acid-3-(14)C.     

Purification and characterization of a novel keto ester reductase from the green alga, <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>: comparison of enzymological properties with other microbial keto ester reductases

A novel keto ester reductase (Chlorella sorokiniana keto ester reductase, CSKER) from Chlorella sorokiniana SAG 211-8k cells was purified. The CSKER had a monomeric structure based on gel filtration chromatography (37 kDa) and SDS–polyacrylamide gel electrophoresis (34 kDa). The purified CSKER showed a high reducing activity with β-keto esters, in particular, ethyl 4-chloro-3-oxobutanoate and ethyl 2-chloro-3-oxobutanoate. However, the purified enzyme did not show any reducing activity with α-keto esters and 2-chlorobenzoylformamide (aromatic α-keto amide). The CSKER catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate, ethyl 3-oxobutanoate, and methyl 3-oxobutanoate to the corresponding (R)-, (S)-, and (S)-hydroxy ester, respectively, with high enantioselectivity (>99% e.e.), respectively. Furthermore, the reduction of ethyl 2-methyl-3-oxobutanoate by CSKER exclusively yielded the corresponding syn-(2R, 3S)-hydroxy ester. The purified CSKER was inactive with NADH, used instead of NADPH. None of the keto ester-reducing enzymes already isolated from other microorganisms was identical to the CSKER. These results suggested that CSKER is a novel keto ester reductase that has not yet been reported.     

Tetrahydroisoquinolines as subtype selective estrogen agonists/antagonists

Two series of 6-hydroxy and 7-hydroxy tetrahydroisoquinolines were prepared. Evaluating a range of C-1, C-4, and N-substituents led to the discovery of ER alpha and ER beta selective analogs.     

Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor-oxaloacetate (OA) or alpha-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Regiospecific and enantioselective metabolism of 8,9-epoxyeicosatrienoic acid by cyclooxygenase.

8(S),9(R)-epoxyeicosatrienoic acid, a major product of the renal cortex, was found to be a substrate for cyclooxygenase from human platelets and ram seminal vesicles. 11(R)-hydroxy-8(S),9(R)-epoxyeicosatrienoic acid was the sole metabolic product. The 8(R),9(S)-enantiomer formed both C-11 and C-15 hydroxylated metabolites. These novel findings suggest that the cyclooxygenase-dependent renal vasoconstrictor activity of 8(S),9(R)-epoxyeicosatrienoic acid may be due to the 11(R)-hydroxy metabolite.     

Biosynthetic origin of hydrogen atoms in the lipase inhibitor lipstatin

The lipase inhibitor lipstatin is biosynthesized in Streptomyces toxytricini via condensation of a C(14) precursor and a C(8) precursor, which are both obtained from fatty acid catabolism. To study the mechanism of this reaction in more detail, S. toxytricini was grown in medium containing a mixture of U-(13)C,U-(2)H-lipids and unlabeled sunflower oil or in a medium containing 70% D(2)O. Lipstatin was isolated and analyzed by (1)H,(2)H, and (13)C NMR spectroscopy. Hydrogen atoms at C-2, C-3, and C-4 of lipstatin were found to be derived from solvent protons. The formation of the lipstatin precursor 3-hydroxy-Delta(5,8)-tetradecadienoyl-CoA by beta oxidation of linoleic acid explains the incorporation of solvent hydrogen into the 4 position of lipstatin. The hydrogen in position 3 of lipstatin is most probably introduced from solvent by proton/deuterium exchange of a redox cofactor involved in the reduction of the keto group in the branched chain beta keto acid arising by a decarboxylative condensation. The incorporation of solvent hydrogen at position 2 can be explained by epimerization of a chiral intermediate at C-2 and C-3. Epimerization may involve a dehydration-rehydration mechanism.     

Gibberellins in shoots and developing capsules of Populus species

Extracts of stems of growing shoots of Populus deltoides and P. trichocarpa, and developing capsules of P. deltoides were analysed for gibberellins (GAs) by gas chromatography-mass spectrometry. The following known GAs were identified by comparison of their Kovats retention indices (KRIs) and mass spectra with those of standards: GA1, GA8, GA9, GA19, GA20, 16 beta,17-dihydro-17-hydroxy GA20, GA23, GA28, GA29, GA34, GA44, and GA97. Several of these have not been previously reported from Populus. In addition, two new GAs were identified as 12 beta-hydroxy GA53 (GA127) and 16 beta,17-dihydro-17-hydroxy GA53 and their structures were confirmed by partial synthesis. Evidence was found of 16,17-dihydro-16,17-dihydroxy GA9, 16,17-dihydro-16,17-dihydroxy GA12, 12-hydroxy GA14, and GA34-catabolite by comparison of mass spectra and KRIs with published data. Several putative GAs (hydroxy- and dihydroxy-GA12-like) were also found. The catabolites of active GAs or of key precursors, hydroxylated at C-2 in stems and either C-2, C-12, C-17, or C-16,17 in capsules, were the major proportion of the GAs.     

Functional analysis of γ-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans

Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans . Analysis of the carotenoids in the resultant mutant R1ΔcrtO demonstrated that dr0093 encodes γ-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of γ-carotene derivatives to form ketolated carotenoids. The mutant R1ΔcrtO became more sensitive to H₂O₂ treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans . Carotenoid extracts from mutant R1ΔcrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.     

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.     

Glucosylation of phenolic compounds by Pharbitis nil hairy roots: I. Glucosylation of coumarin and flavone derivatives

Hairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation. Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.     

The metabolism of 1-phospho-5-methylthioribose

In partially fractionated rat liver homogenate, 1-phospho-5-methylthioribose is oxidatively converted to 2-keto, 4-methylthiobutyric acid, 2-hydroxy, 4-methylthiobutyric acid and formate. The amount of formate formed is equal to the amount of 2-keto, 4-methylthiobutyric acid plus 2-hydroxy, 4-methylthiobutyric acid formed. The data suggest that the keto acid is the precursor of the hydroxy acid. No readily dissociable cofactors are involved in the reaction. A mechanism is proposed for the conversion of 1-phospho-4-methylthiobutyric acid to formate and 2-keto, 4-methylthiobutyric acid.     

Bromine oxidation of 1,2-O-isopropylidene-α-d-Glucofuranose and sucrose

Sucrose and $\text{1,2-O-}\text{isopropylidene-α-}\text{d}\text{-glucofuranose}$ (1) were oxidised with bromine in aqueous solution at pH 7 and room temperature. The resulting keto derivatives were converted into their more-stable O-methyloximes, which were characterised by spectroscopic and chromatographic methods. Oxidation of 1 occurred at C-3 and C-5, with a preference for C-5. In the sucrose derivatives isolated after oxidation, those having a keto group in the glucopyranosyl moiety preponderated. The axial fructofuranosyl aglycon protects position 3 in the glucopyranosyl group and oxidation occurs only at C-2 and C-4. Small amounts of sucrose oxidised at C-3 in the fructofuranosyl moiety were also found.     

Inhibitory effect of methyl jasmonate and its related compounds on kinetin-induced retardation of oat leaf senescence

The senescence-promoting activities of methyl jasmonate and its related compounds were compared with respect to structure-activity relationships. The activities were assayed by using oat ( A vena saliva L. cv. Victor) leaf segments in the presence of 2 μg/ml kinetin. Dextrorotatory methyl jasmonate prepared from an authentic sample of the racemate mixture was less active than the naturally occurring levorotatory form especially at its low concentrations (0.1 to 2.5 μg/ml). The activity of jasmonic acid, the free acid form of methyl jasmonate, was much less than the methyl ester, and this relationship was true for the other compounds tested. The reduction of the unsaturated bond in the substituent at the C-2 position and the keto group at the C-3 position greatly reduced the activity. The length of the n -alkyl substituents at the C-2 position had also a significant effect on the activity. From these results, it is concluded that the important functional groups for the high senescence-promoting activity of the methyl jasmonate related compounds are the methyl acetate substituent at the C-l position, the 2' cis -pentenyl or n -pentyl group at the C-2, position and the keto group at the C-3 position in methyl jasmonate.     

Levels of 13,14-dihydro-15-keto-PGF2α in biological fluids as measured by radioimmunoassay

Antibodies directed toward 13,14-dihydro-15-keto-PGF_2α were prepared in rabbits. The C-15 keto group of the metabolite is immunodominant. The antibodies recognize, although to a lesser extent, the reduced 13,14-double bond, the C-9 hydroxyl group and the C-11 hydroxyl group of the metabolite. With previously described antibodies to 15-keto-PGF_2α the C-15 keto group and the 13,14-double bond of the homologous metabolite were equally immunodominant. Therefore, assay of biological samples with both antisera permits identification and quantitation of either metabolite. The levels of 13,14-dihydro-15-keto-PGF_2α in human peripheral venous sera and the levels of cross reacting metabolites in urine have been determined.     

Identification of intramembrane hydrogen bonding between 13 keto group of bacteriochlorophyll and serine residue α27 in the LH2 light-harvesting complex

Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 α-subunit, and the C-13¹ keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the β-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13¹ keto carbonyl and a methyl hydrogen of the alanine replacing serine(−4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13¹ not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 Å in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.     

Structure-activity relationships in a series of 11-deoxy prostaglandins

A series of 11-deoxy prostaglandin derivatives and some naturally occurring prostaglandins have been investigated in the anaesthetized artificially respired guinea-pig for their effect on blood pressure, bronchial resistance (overflow pressure at constant volume), tracheal segment pressure, and on intestinal and uterine smooth muscle. The compounds were administered intravenously. Prostaglandins E₁, E₂ and F_2α produced responses that were qualitatively similar to those in the literature. Prostaglandin A₂ (100 μg) was a bronchoconstrictor, although it decreased tracheal segment pressure and blood pressure. Prostaglandin B₂ (100 μg) caused double elevations in blood pressure, tracheal segment pressure and bronchial resistance. The intensity of bronchoconstriction produced by PGB₂ was of the same order as with PGF_2α. A number of structure-activity relationships were found. 11-Deoxygenation lowered the biological activity of the natural prostaglandins PGE₁ and PGF_1α. The vasodepressor and bronchodilator responses of 11-deoxy PGE₁ were converted to vasopressor and bronchoconstrictor by epimerisation at C-15. Introduction of a methyl group at C-15 of 11-deoxy PGF_1α both increased and prolonged vasopressor and bronchoconstrictor activity. At C-9 both the keto and β-hydroxy group imparted vasodepressor and bronchodilator activity, while the α-hydroxy led to vasopressor and bronchoconstrictor activity. Extension of the omega sidechain by two methylene groups radically reduced the activity of 11-deoxy PGF_1α and its derivatives.These experiments indicate that steric differences in the prostaglandin structures studied can result in diametrically opposed profiles of biological activity. Further, small variations in the prostaglandin molecule can lead to differences in potency and/or profile of activity in the guinea-pig.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide