Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'-14C]hexadecyl-sn-glycerol or rac-1-O-[1'-14C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'-14C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.     

Metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol and nutritional effects of dietary rac-1-O-dodecylglycerol in mice

The metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol was investigated in mice. The substrate was rapidly absorbed in the intestine and then incorporated into ether glycerolipids of various organs and tissues in high proportions. In intestine and liver, however, large amounts of rac-1-O-[1'-14C]dodecylglycerol were catabolized by oxidative cleavage of the ether bond followed by degradation of the radioactive cleavage product, i.e., lauric acid, to water-soluble metabolites that were excreted in the urine at a fast rate. The feeding of a rac-1-O-dodecylglycerol-containing diet (1 g rac-1-O-dodecylglycerol/kg body weight X day) given over a period of 4 weeks did not significantly alter body weights or organ weights of mice. Analysis of total lipids revealed that high proportions of the substrate were incorporated into ether lipids of all organs and tissues during the feeding period, generally accompanied by a remarkable increase in saturated acyl moieties and a concomitant decrease of linoleoyl moieties of total lipids. Yet, 4 weeks after removal of the rac-1-O-dodecylglycerol-containing diet, the lipids of murine organs and tissues showed a close resemblance to those of the control group.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Polyoxypregnane glycosides from Caralluma retrospiciens

Six related polyoxypregnane glycosides were isolated and characterised from Caralluma retrospiciens leaves. The compounds were identified as 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-]-beta-D-cymaropyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-beta-D-cymaropyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], 12beta-benzoyloxy-11alpha-isovaleroyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], and 12beta-benzoyloxy-11alpha-isovaleroyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl (1 --> 4)-3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-beta-D-cymaropyranoside]. The structures were determined by detailed analysis of one- and two-dimensional NMR spectra as well as by chemical means. The compounds showed cytotoxic activities towards brine shrimp having IC50 values of 1.19 x 10(-4), 8.83 x 10(-5), 2.64 x 10(-4), 2.26 x 10(-4), 2.39 x 10(-4) and 1.70 x 10(-4) M, respectively. This is the first report of the isolation of these compounds from a natural source.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

Selective synthesis of the hexaenoic molecular species of ether-linked glycerophosphoethanolamine of Ehrlich ascites tumor cells

In a previous study [Waku, K. and Nakazawa, Y. (1978) Eur. J. Biochem. 88, 489-494], we observed the rapid turnover rate of the molecular species of alkylacyl glycerophosphoethanolamine (Gro-P-Etn) containing docosahexaenoic acid and the high selectivity for this molecular species of ethanolamine phosphotransferase was suggested. To clarify this point, the incorporation of [14C]ethanolamine and [14C]CDP-ethanolamine into the individual molecular species of alkenylacyl, alkylacyl and diacyl Gro-P-Etn has been determined in Ehrlich ascites tumor cells. [14C]Ethanolamine was highly incorporated into the pentaenoic + hexaenoic species of alkenylacyl, alkylacyl and diacyl Gro-P-Etn, whereas incorporation of [14C]ethanolamine into molecular species other than the pentaenoic + hexaenoic species was quite low. The selectivity of ethanolamine phosphotransferase to form the molecular species of alkylacyl and diacyl Gro-P-Etn was examined by incubation of [14C]CDP-ethanolamine and microsomes of Ehrlich ascites tumor cells. The incorporation of [14C]CDP-ethanolamine was found to occur most into the pentaenoic + hexaenoic species of both alkylacyl and diacyl Gro-P-Etn. The present results suggest that the pentaenoic + hexaenoic species are preferentially synthesized among the various kinds of molecular species of alkylacyl and diacyl Gro-P-Etn by the ethanolamine phosphotransferase in Ehrlich ascites tumor cells.     

Metabolism of platelet-activating factor in primary cultured adult rat hepatocytes by a new pathway involving phospholipase C and alkyl monooxygenase

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) upon interaction with primary cultured adult rat hepatocytes was investigated. [3H]PAF-acether was transformed time-dependently into [3H]lyso-PAF-acether, 1-O-[3H]alkylglycerol and finally converted to 3H-labeled fatty aldehyde. 1-O-[3H]Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) was formed after a long incubation time and with a smaller amount compared with that formed in platelets and neutrophils. When lipids from cells, cell surfaces and incubation medium were analyzed separately, most of the transformed products of [3H]PAF-acether remained in the cells. When 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine was incubated with hepatocytes, it was mainly converted into 1-O-[3H]alkylglycerol. 3H-labeled fatty aldehyde and [3H]alkylacyl-GPC were also found. Hepatocytes metabolized slowly from 1-O-[1-14C]hexadecylglycerol to 3H-labeled fatty aldehyde and 3H-labeled phospholipid. These findings suggest that cultured hepatocytes mainly catabolize exogeneous PAF-acether by removing the acetyl residue and the polar head group and, finally, by cleaving an ether bond. The deacetylation-reacylation step, which is important in platelets and neutrophils, was not shown to be a main metabolic pathway of PAF-acether in cultured hepatocytes.     

Inhibition by derivatives of diguanidines of cell proliferation in Ehrlich ascites cells grown in cultures.

The anti-proliferative effects of 1,1'-[(methylethanediylidene)dinitrilo]diguanidine [methylglyoxal bis(guanylhydrazone)] and 1,1'-[(metHYLETHANEDIYLIDENE)dinitrilo]bis-(3-aminoguaNIDINE) HAVE BEEN STUDIED IN Ehrlich ascites carcinoma cells grown in suspension cultures. Both compounds are potent inhibitors of S-adenosyl-L-methionine decarboxylase from the tumour cells. In the presence of putrescine (but not in its absence), the inhibition produced by 1,1'-[methylethanediylidene)dinitrilo]bis-(3-aminoguanadine) was apparently irreversible, as judged by persistent depression of the enzyme activity even after extensive dialysis. The two compounds produced similar increases in adenosylmethionine decarboxylase activity, which resulted from a striking stabilization of the enzyme in cells grown in the presence of the drugs. The inhibitory effect of the two diguanidine derivatives on the synthesis of DNA and protein became evident after an exposure of 4--8 h. At that time, the only change seen in tumour polyamines in cells grown in the presence of the inhibitors was an increase in cellular putrescine. To find out whether the compounds initially interfered with the energy production of the tumour cells, the cultures were grown in the presence of uniformly labelled glucose, and the formation of lactate, as well as the oxidation of the sugar into CO2, were measured. The activation of glycolysis upon dilution of the tumour cells with fresh medium and the subsequent formation of labelled CO2 were siliar in control cells and in cells exposed to methylglyoxal bis(buanylhydrazone), 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) or diaminopropanol. Only a marginal decrease in the cellular content of ATP was found in cells exposed to the inhibitors for 24 h. The diguanidine-induced growth inhibition was fully reversed by low concentrations of exogenous polyamines. However, the possibility remained that the reversal by polyamines was due to a decrease of intracellular diguanidine concentration. Our results indicate that the mode of action of 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) is fully comparable to that of methylglyoxal bis(guanylhydrazone), as regards stabilization of adenosylmethionine decarboxylase and the appearance of growth inhibition in Ehrlich ascites cells. The data tend to support the view that both compounds apparently have an early anti-proliferative effect unrelated to polyamine metabolism.     

Synthesis and antineoplastic properties of an ether glycerophosphonocholine, and analog of ET-18-OCH3-GPC.

A glycerophosphonocholine analog of the ether-linked lipid, rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3-GPC), was synthesized in which the head group is nonhydrolyzable by phospholipase C. The phosphonate analog used in this study is rac-3-octadecyloxy-2-methoxy-propyl-phosphonocholine, C18H37OCH2CH(OCH3)CH2P(O)(O)OCH2CH2N+(CH3)3. The activity of the synthetic phosphonate was tested in the human leukemic cell line, HL-60, and the human undifferentiated cervical carcinoma, C-41. The glycerophosphonocholine inhibited [3H]thymidine uptake by HL-60 cells with an EC50 value of 5-7 microM. The glycerophosphate ET-18-OCH3-GPC had an EC50 value of approximately 2 microM against HL-60 cells. The EC50 values estimated from cell viability experiments were similar to that for [3H]thymidine uptake. The EC50 value for C-41 cells was about 10-15 microM. The data demonstrate that the glycerophosphonocholine is a promising anti-cancer drug for the treatment of both leukemia and solid tumors. Furthermore, the data demonstrate that phospholipase C-catalyzed hydrolysis of ET-18-OCH3-GPC does not play an important role in the cytotoxic action of the ether-linked glycerolipids.     

Selective acylation of alkyllysophospholipids by docosahexaenoic acid in Ehrlich ascites cells

Ehrlich ascites cells were cultured with 1-O-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) or 1-O-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC) to reveal the selective retention of polyunsaturated fatty acids at second position of ether-containing phospholipids. Although small percentages of the lysophospholipids were degraded into long-chain alcohol, both alkyllyso-GPE and -GPC were acylated at the rate of approximately 2 nmol/30 min per 10(7) cells. Alkylacylacetylglycerols were prepared from the acylated products by phospholipase C treatment, acetylation and TLC, and fractionated according to the degree of unsaturation by AgNO3-TLC. The distribution of the radioactivity among the subfractions indicated that both alkyllysophospholipids were mainly esterified by docosahexaenoic acid and to a somewhat lesser extent by arachidonic acid. The selectivity for docosahexaenoic acid in the esterification of 1-alkyl-GPE was much stronger than in that of 1-alkyl-GPC. Although acyl-CoA: 1-alkyl-glycerophosphoethanolamine acyltransferase activity of Ehrlich cell microsomes with arachidonoyl-CoA and docosahexaenoyl-CoA as acyl donors was negligible compared with the acyl-CoA:1-alkyl-glycerophosphocholine acyltransferase activity, a significant amount of 1-alkyl-GPE was acylated in the microsomes without exogenously added acyl-CoA. HPLC analysis revealed that docosahexaenoic acid and arachidonic acid were mainly esterified by the microsomal transferase. Acylation of 1-alkyl-GPC with docosahexaenoic acid and arachidonic acid was also observed in the absence of added acyl-CoA, but the activity was lower than that for 1-alkyl-GPE. Although the source of the acyl donor in the acylation has not been determined, the acylation is probably due to the direct transfer of acyl groups between intact phospholipids. The above results provided the first evidence that the lysophospholipid acyltransferase system including the transacylase activity participates in the selective retention of docosahexaenoic acid in intact cells and a cell free system.     

Biologically active ether lipids. Biotransformation of rac-1(3)-O-alkylglycerols in cell suspension cultures of rape and semisynthesis of 1-O-alkyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamines, potent antitumor agents

Biotransformation of rac-1(3)-O-hexadecylglycerol by photomixotrophic rape (Brassica napus) cells in suspension culture leads to 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines and small proportions of other ether lipids, e.g. 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamines. Reaction of the hexadecylacyl-glycerophosphocholines with ethanolamine in the presence of phospholipase D from Streptomyces chromofuscus yields additional hexadecylacylglycerophosphoethanolamines. Partial hydrolysis of the combined hexadecylacylglycerophosphoethanolamines followed by reacylation of the resulting lyso compound with palmitic anhydride gives 1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl) ethanolamine, a nontoxic ether glycerophospholipid with antitumor activity. The corresponding 1-O-tetradecyl,1-O-octadecyl, and 1-O-[(Z)-9'-octadecnyl] derivatives are prepared similarly.     

An alpha-D-galactosyltransferase activity in Ehrlich ascites tumor cells. Biosynthesis and characterization of a trisaccharide (alpha-D-galactose-(1 goes to 3)-N-acetyllactosamine)

An alpha-D-galactosyltransferase activity has been detected in membranous fractions (42,000 x g) of Ehrlich ascites cells which transfers galactosyl groups from UDP-galactose to endogenous and exogenous acceptors. The products of the reaction contain alpha-D-galactopyranosyl groups at the nonreducing termini. A solid state assay was developed to follow alpha-D-galactosyltransferase activity in the presence of beta-D-galactosyltransferase. Examination of a variety of insolubilized exogenous acceptors indicated that the most active acceptors for the alpha-D-galactosyltransferase had the structure beta-D-Gal-(1 goes to 4)-beta-D-GlcNAc(1 goes to at their nonreducing termini. Incubation of UDP-[14C]galactose and beta-D-gal-(1 goes to 4)-D-GlcNAc (N-acetyllactosamine) or of UDP-galactose and beta-D-[14C]Gal-(1 goes to 4)-D-GlcNAc in the presence of the alpha-D-galactosyltransferase resulted in the enzymic synthesis of a 14C-labeled trisaccharide. Chemical and enzymic methods of analysis revealed the structure of the trisaccharide to be alpha-D-Gal-(1 goes to 3)-beta-D-Gal-(1 goes to 4)-D-GlcNAc. These data indicate that the alpha-D-galactosyltransferase in Ehrlich ascites cells transfers galactosyl groups to suitable acceptors to form an alpha-(1 goes to 3)-D-galactosidic linkage.     

Turnover rates of the molecular species of alkenyl ether phospholipids of Ehrlich ascites tumor cells.

[1-14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1-O-Alkenyl-2-acyl-3-acetylglycerols were prepared from the ethanolamine phosphoglycerides and fractionated by AgNO3-impregnated thin-layer chromatography into five molecular species. Bands 1--5 obtained in this way can be designated as saturated, monoene, diene, tetraene and hexaene species, respectively. Band 5, composed mainly of hexaene species was predominant among those derived from alkenyl ether phospholipids. The specific radioactivities of the fractionated species were determined. Hexaene molecular species turned over most rapidly and disaturated and tetraene species turned over at a high rate, while monoene and diene molecular species turned over very slowly.     

Augmentation of DNA synthesis in guinea pig bone marrow cells by platelet-activating factor (PAF)

When guinea pig bone marrow cells were incubated in the presence of 10(-7) to 10(-5) M platelet-activating factor (PAF) for 24 to 72 hours, [3H]thymidine incorporation of cells was time-dependently augmented. The enantiomer of PAF and lysoPAF, a major metabolite of PAF, did not show significant enhancement. A non-metabolizable potent PAF agonist, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-sn-glycero-3-phospholine, enhanced the [3H]thymidine incorporation at 10(-10) to 10(-8) M. This augmentation of DNA synthesis in bone marrow cells was abolished by specific PAF antagonists, CV-6209 or FR-900452. When the conditioned medium of PAF-stimulated bone marrow cells was added to another culture of bone marrow cells, the augmentation of DNA synthesis was also observed. These results suggest that PAF may affect the proliferation of one or some classes of guinea pig bone marrow cells through release of soluble factor(s).     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Concentrated hydriodic acid in simultaneous deprotections of multifunctional inositols

l-1-Deoxy-1-fluoro-6-O-methyl-myo-inositol was epimerized by chloral/DCC in boiling 1,2-dichloroethane yielding D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol. The latter and l-4-O-benzyl-3-O-cyclohexylcarbamoyl-5-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-muco-inositol, l-4-O-benzyl-3-O-cyclohexylcarbamoyl-1,2-O-ethylidene-5-O-methyl-muco-inositol, d-1-O-cyclohexylcarbamoyl-2-deoxy-5,6-O-ethylidene-2-fluoro-3-O-methyl-chiro-inositol, as well as D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol were deprotected with boiling 57% aq hydrogen iodide. Ether, urethane and ethylidene acetal functions were simultaneously cleaved by the reagent, whereas the trichloroethylidene groups were still intact or were only removed in small quantities. Especially, the urea function of D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol was decomposed to a cyclohexylamino group. The hydrodechlorination of D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol using Raney-Nickel yielded a mixture of the corresponding 5,6-O-ethylidene- and 5,6-O-chloroethylidene derivatives. The three synthetic steps-hydrodehalogenation, HI-deprotection and peracylation- were combined without purification of the intermediates.     

Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

Pregnane steroidal glycosides and their cytostatic activities

Four new steroidal glycosides such as 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-12-β-tigloyl-14-β-hydroxy-17-β-pregnane (1), 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-12-β-(2'-amino)-benzoyl-14-β-hydroxy-17-β-pregnane (2), 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-12-β-14-β-dihydroxy-17-α-pregnane (3) and 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-12-β-14-β-dihydroxy-17-β-pregnane (4) were isolated from the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae), a crassulacean acid metabolism plant, an endemic species to the Canary Islands that has been used in traditional medicine as a cicatrizant, vulnerary and disinfectant. The dichloromethane extract exhibited significant cytostatic activity against HL-60, A-431 and SK-MEL-1 cells, human leukemic, epidermoid carcinoma and melanoma cells, respectively. As shown in Table I, compounds 1 and 2 showed very similar IC(50) values. The acetylation of 1 to give the diacetate 5 increases 5-fold the cytotoxicity against HL-60 cells. Compounds 3 and 4 did not show cytotoxicity at the assayed concentrations. With respect to the compounds containing only the steroid ring (6-8), the presence of a charged O-amino-benzoyl but not a tigloyl group improved the cytotoxicity.