Methanolysis Products Research Articles

Biosynthetic studies of the polar lipids of Halobacterium cutirubrum. Formation of isoprenyl ether intermediates

Biosynthetic studies were carried out on the complex polar lipids of the extreme halophile, Halobacterium cutirubrum. Cells were pulse-labeled with [ 32P]phosphate, [ 14C]mevalonate and [ 14Clglycerol for short terms (1, 15, 30, 60 min); incorporation of radiolabels into total lipids and their distribution among individual lipid components were determined as a function of incubation time. Glycerol was incorporated 9-fold more rapidly into total lipids than mevalonate, but at the same rate as phosphate. Several intermediate components were detected by short-term (1–30 min) pulse labeling, some of which were shown to be allyl ether-bound isoprenyl intermediates of the major saturated diphytanylglycerol ether lipids, phosphatidylglycerophosphate (PGP), phosphatidylglycerol (PG), phosphatidylglycerosulfate (PGS) and sulfated triglycosyldiphytanyl-glycerol (S-TGD). These intermediate components were detected by their products of acid methanolysis, alkaline hydrolysis and Vitride reduction, before and after catalytic hydrogenation. Another intermediate detected was the isoprenyl ether analogue of phosphatidic acid, which showed a product-precursor relationship with PGP and PG for all three radioisotope precursors. Other intermediates remain unidentified, but their product-precursor relationship was established by their labeling patterns with the three radioisotope precursors. These studies suggest that the biosynthetic pathways for the major diphytanylglycerol ether phospho- and glycolipids involve formation of the respective isoprenyl intermediates that are reduced in a stepwise fashion to the fully saturated diphytanylglycerol ether phospho- and glycolipids. Chase studies, following a 24 h labeling period, showed that the major phospholipid (PGP) has a very low turnover rate for phosphate groups, but a 3-fold higher turnover of the phytanyl groups; the minor phospholipids PG and PGS have much higher phosphate turnover rates.

Oxidation of 1-O-(alk-1-enyl)-2,3-di-O-acylglycerols: models for plasmalogen oxidation.

Alk-1-enyl diacyl glycerides, model compounds for plasmalogen lipids, were synthesized for use as substrates in oxidation studies. The neutral plasmalogen glycerides prepared included 1-O-(hexadec-1-enyl)-2,3-di-O-stearoylglycerol (4a) and 1-O-(hexadec-1-enyl)-2-O-linoleoyl-3-O-stearoylglycerol (4b). Oxidative disappearance of these glycerides was followed directly by high performance liquid chromatography (HPLC). The extent of oxidation of the side chains of these glycerides, i.e. the alkenyl ether and fatty acyl functions, was monitored by conversion of the side chains to dimethylacetal and methyl ester by methanolysis, followed by subsequent gas chromatography (GC) of the methanolysis products. Both analytical approaches show that the alkenyl ether function of neat 4a oxidizes more slowly than neat ethyl linoleate. However, the rate of alkenyl ether loss from 4a is accelerated in the presence of added ethyl linoleate. Moreover, when the linoleoyl group is incorporated into the 2-position of the alkenyl glyceride, as in 4b, the rate of loss of the alkenyl group was shown to be similar to the rate of loss of the linoleoyl group. These results suggest that oxidation of plasmalogen glycerides should not be ignored as a factor that contributes to the oxidative instability of animal tissue.

Isolation of 1-monomethylphosphoinositol 4,5-bisphosphate [a product of methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate] from Swiss mouse 3T3 cells.

We have noted two previously undescribed inositol polyphosphates in neutral methanol extracts from Swiss mouse 3T3 cells that were grown in [3H]inositol and stimulated with platelet-derived growth factor. They have been identified as 1-monomethylphosphoinositol 4,5-bisphosphate and 1-monomethylphosphoinositol 4-phosphate by comparison to a synthesized standard using HPLC chromatography, paper electrophoresis, and enzymatic dephosphorylation with inositol polyphosphate 5-phosphomonoesterase and intestinal alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1]. We propose that these compounds are formed by methanolysis of inositol 1,2-(cyclic)-4,5-trisphosphate and inositol 1,2-(cyclic)-4-bisphosphate present in the cells. Inositol cyclic phosphates did not react with neutral methanol in the absence of the cells, which are required for the methanolysis reaction. These findings suggest a role for inositol cyclic phosphates as reactive compounds that are added to as yet unidentified cellular acceptors.

Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid.

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125I-labeled proteins with a series of monoclonal antibodies (MAbs). Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selective partitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 was concomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [35S]methionine, 14C-amino acids, or [3H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquid chromatography identification of [3H]methyl palmitate after acid methanolysis of delipidated proteins. An additional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid also attached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. These studies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins are major surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within this species.

Quantitative determination of lipids and their constituents by the Chromarod TLC‐FID system

AbstractThe methanolysis products of neutral sphingolipid and archaebacterial neutral glycolipid were separated on Chromarods S‐II (silica gel) with a double developing system. The lipid constituents separated on the rods were scanned automatically with a hydrogen flame ionization detector (Iatroscan). The molar ratios of the constituents determined by this system were very close to the theoretical values of the lipid.

Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine

rac-1-[1- 14C]Lauroyl-2-oleylglycero-3-phosphol[methyl- 3H]clioline and rac-1-lauroyl-2-[1- 14C]oleoylglycero-3-phospllo [ methyl- 3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmito- yl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative intial rates of hydrolysis of sn-1,2,- and sn-2,3-rmenantiomers were 100–200:1 for phospholipase C and 40–50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8–13:1) and total for phospholipase D. There was a parallel dramatic decrease (500–1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylglycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.

Resolution and absolute configuration of 7,12-dimethylbenz[a]anthracene 5,6-epoxide enantiomers

The enantiomers of 7,12-dimethylbenz[a]anthracene (DMBA) 5,6-epoxide were directly resolved by normal-phase high-performance liquid chromatography with an ionically bonded chiral stationary phase. The absolute configurations of the resolved enantiomers were determined by comparison of circular dichroism spectra of the methanolysis products formed from the epoxide enantiomers with that of a DMBA trans -5,6-dihydrodiol enantiomer of known absolute stereochemistry. DMBA 5R,6S-epoxide is hydrated by rat liver microsomal epoxide hydrolase predominantly (95%) to a 5S,6S-dihydrodiol. The results indicate that the 5S,6S-dihydrodiol formed from the metabolism of DMBA by microsomes prepared from the livers of 3-methylcholanthrene-treated rats is predominantly derived from a 5R,6S-epoxide intermediate.

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z-ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.

Effect of para-substituents on the photochemical ring-opening reactivity of 1-(p-substituted-benzoyl)aziridines in methanol

Photolysis of 1-(p-substituted-benzoyl)aziridines (1a–e) in Ar-purged methanol led to N-(2-methoxyethyl)benzamides (3b–e) as the methanolysis products and/or N-ethylbenzamides (2a and b). Penta-1,3-diene quenched the formation of (2a and b) but not (3b–e).

Application of a thin-layer chromatography—flame-ionization detection system for the determination of complex lipid constituents

A simple and rapid method for the determination of relative amounts of complex lipid constituents was developed using a thin-layer chromatography—flame ionization detection (TLC—FID) system. The methanolysis products from a sphingoglycolipid (ceramide monohexoside) were separated on Chromarod SII (silica gel) with doouble development to distances of 6 and 10 cm from the start using chloroform—methanol—15 N ammonia solution (60:10:1) and n-hexane—diethyl ether (50:2). The components separated on the rod were automatically scanned with a hydrogen flame-ionization detector (Iatroscan). The relative responses of methyl glucoside and methyl galactoside to sphingosine and a fatty acid methyl ester were in the same proportions as the molecular ratio. The methanolysis products of sphingomyelin were also separated by the same system. Methanolytes of glyceroglycolipids and glycerophospholipids were separated into individual components with triple development using ethyl acetate—formic acid (70:2) twice and n-hexane—diethyl ether (50:20) to distances of 5 and 10 cm, respectively, from the start.

Methyl-2,6-Anhydro-α-D-Mannofurahosid Eine Verbesserte Darstellungsmethode Sowie Die Bestimmung Der Kristall- Und Molekolstruktur

Abstract The title compound 3, which is the first member of a new class of anhydroglycosides, was initially isolated from the methanolysis mixture of the pyranoid isomer 1 in about 1% yield. Improved yields of greater than 30 % of 3 are obtained by treating the main product of the aforementioned methanolysis, the dimethylacetal 2, with a catalytic amount of p-toluenesulfonic acid in boiling xylene. 3 (C7H12O5) crystallizes from diisopropyl ether in the monoclinic space group P21 (Z=2) with a = 552.4(1), b = 685.2(1), c = 1052.9(3) pm, α = 90.0, β = 98.95(6) and Y = 90.0°. The structure was solved by X-ray crystal structure analysis using direct methods to R indices of 0.036 and 0.045, respectively, for 1180 independent reflections. The furanoid ring in 3 adopts a conformation intermediate between 2T3(D) and E3(D) (puckering parameters: Q = 48.9 pm; Φ = 102.4°). The oxane ring adopts an a2Ca5(D) conformation, which is heavily distorted by flattening at C-6 (puckering parameters: Q = 64.2 pm; Φ = 58.1° and ...

Studies on the constituents of aceraceae plants. V. Two diarylheptanoid glycosides and an arylbutanol apiosylglucoside from Acer nikoense.

From the stem bark of Acer nikoense MAXIM. (Aceraceae), three glycosides were isolated, namely aceroside III (1), C30H40O12, mp 138-141°C, [α]D-98.4°, aceroside VI (2), C25H32O8·1/2H2O, mp 124-125°C, [α]D-69.3°, and apiosylepirhododendrin (3), amorphous film (C21H32O11), [α]D-59.5°. On acid hydrolysis, 1 yielded acerogenin A (4), apiose, glucose, and a partially hydrolyzed product 2, while 3 afforded (+)-rhododendrol (11), apiose, and glucose. Inspection of the carbon-13 nuclear magnetic resonance (13C NMR) and the PRFT-NMR spectra of 1 and 3 disclosed that they are apiosyl-(1→6)-glucosides. The proton nuclear magnetic resonance (1H NMR) spectral data of their permethylates (6 and 12) and the analytical data of methanolysis products of 6 and 12 established the structures of 1, 2 and 3 as acerogenin A 11-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, acerogenin A 11-O-β-D-glucopyranoside and (+)-rhododendrol 2-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, respectively.

Chromato-mass spectrometric study of 3,6-dideoxyhexoses

The main features of the fragmentation of the acetates of methyl 3,6-dideoxyhexopyranosides and -furanosides have been established with the use of deutero and partially methylated analogs. A considerable proportion of the ion current of the pyranosides is due to the fragmentation of the monosaccharide ring: K, C, H. In the case of the furanosides, on the other hand, the main portion of the ion current is due to the ions of the furanose ring, A, E. It is proposed to use mass spectra and the gas-chromatographic properties of the 3,6-dideoxhexoses studied for their identification in the products of the methanolysis of lipopolysaccharides.

Zur Kenntnis des Faktors F430 aus methanogenen Bakterien: Struktur des porphinoiden Ligandsystems

Factor F430 from Methanogenic Bacteria: Structure of the Porphninoid Ligand SystemA structure is proposed for F430M, a non‐cristalline methanolysis product of isolates of the nickel‐containing, porphinoid factor F430 from Methanobacterium thermoautotrophicum.Crucial to the structure determination are five incorporation experiments with M. thermoautotrophicum (strain Marburg) in which the specifically mono‐13C‐labeled biosynthetic precursors (2‐13C), (3‐13C), (4‐13C)‐, (5‐13C) ALA (ALA = δ‐amino‐levulinic acid) and L‐(methyl‐13C)methionine were incorporated into F430 with high efficiency. The 13C‐NMR,‐spectra of the specifically labeled F430M samples derived therefrom, together with the UV./VIS. spectral data of F430M, contain all the information necessary for the deduction of the constitution of the F430M chromophore, assuming the established pattern of porphinoid biosynthesis to be operative in F430 biosynthesis. 1H‐NMR. spectroscopy and, in particular, 1H‐NMR.‐NOE‐difference spectroscopy corroborates and completes the constitutional assignments and, furthermore, makes possible an almost complete derivation of the molecule's relative configuration. Schemes 3 and 4 summarize the results of 1H‐NMR. spectroscopy, presenting them within the context of the proposed structure for F430M. The assignment of absolute configuration implied in the formula is given preference because of F430M's very close structural and (assumed) biosynthetic relationship to sirohydrochlorin and vitamin B12 (with respect to ring C, the assignment is based on degradative evidence).According to the proposed structure, the nickel complex F430M possesses an uroporphinoid (Type III) ligand skeleton with an additional carbocyclic ring and a chromophore system not previously encountered among natural porphinoids. It can be considered to be a (tetrahydro) derivative of the corphin system, combining structural elements of both porphyrins and corrins.

Products and Kinetic Substituent Effects in the Reactions of Diaryldiazomethanes with 2,5-Dichloro-p-benzoquinone

Abstract Substituted diphenyldiazomethanes[(x-C6H4)2CN2; x=p-OCH3, p-CH3, H, p-Cl] were found to react with 2,5-dichloro-p-benzoquinone at its conjugated C=C and C=O bonds to give bicyclic diones and poly(2,5-dichlorohydroquinone benzhydryl ether)s respectively, obeying the second-order kinetic law. The product ratios of the polyethers to the bicyclic diones increased from 1.1 to 8.1 with the electron-donating ability of the substituents. The individual rate constants were well correlated with the Hammett equation: logk3=−2.15–1.55 σ (r=0.996) and logk4=−2.03–1.85 (σ0+0.515Δ\barσR+) (r=1.00) for the bicyclic diones and polyethers processes respectively. The presence of methanol as a trapping agent stopped the polymerization completely and afforded the methanolysis product, though no essential change was found in the formation of the bicyclic dione.

Methanolysis products of asparagine-linked N-acetylglucosamine and a new method for determination of N- and O-glycosidic N-acetylglucosamine in glycoproteins that contain asparagine-linked carbohydrates

Amino acid analyzer and gas chromatographic analyses of methanolysates of model compounds of N-glycosidic and O-glycosidic N-acetylglucosamine shows that the main product of N-glycosidic N-acetylglucosamine is glucosamine and that of O-glycosidic N-acetylglucosamine is methyl α-glucosaminide. The amounts of N-acetylglucosamine in glycoproteins determined by methanolysis followed by gas-liquid chromatography represent the amount of O-glycosidic N-acetylglucosamine. A new method was developed to determine O-glycosidic and N-glycosidic N-acetylglucosamine in glycoproteins. Glucosamine and methyl α-glucosaminide in the methanolysates of glycoproteins are determined in an amino acid analyzer using the buffer system that is usually used for analysis of basic amino acids in the two-column system. The contents of O- and N-glycosidic N-acetylglucosamine of Taka-amylase A, ovalbumin, and egg white and yolk flavoproteins determined by the method agree reasonably well with the values calculated from the carbohydrate structures or compositions of the compounds. Therefore, this method can be used to determine the number of N-glycosidically linked carbohydrate chains together with the amount of O-glycosidic N-acetylglucosamine in glycoproteins.

Glycosides from Muscari comosum. 1. Eucosterol glycoside and structure of its methanolysis products

A mixture of at least four glycosides has been isolated from bulbs of Muscari comosum. Acidic methanolysis of this mixture afforded a crystalline fraction consisting of four diols 1a differing in the configuration at the epimerizable C-20 and/or C-24 centers. Benzoylation of this diol fraction allowed the isolation of one pure dibenzoate, mp 175–176 °C, to which structure 1b was assigned on the basis of chemical and spectral evidence. This allowed the deduction that one of the aglycone moieties was 27-norlanostane triterpene eucosterol 4a. This conclusion has been confirmed by identification of 4a as the major product of the enzymatic hydrolysis of the glycoside mixture.

Naphthalene tetrachlorides and related compounds. Part 7. Products of methanolysis of a 1-p-tolylnaphthalene tetrachloride; evidence for a rapid bimolecular elimination of methanol from the 2,3-dichloro-1-methoxy-1-p-tolyl-1,2-dihydronaphthalenes

Unimolecular methanolysis of r-1,c-2,t-3,t-4-tetrachloro-1-p-tolyltetralin gives two isomeric 1-methoxy-1-p-tolyl-2,3,4-trichlorotetralins, each of which undergoes bimolecular elimination in two defined stages having similar rates, and giving 2,3-dichloro-1-p-tolylnaphthalene as the nearly exclusive final product. The intermediate 2,3-dichloro-1-methoxy-1-p-tolyl-1,2-dihydronaphthalenes, which can be characterised through signals in their 1H n.m.r. spectra, must, therefore, undergo bimolecular elimination of methanol at rates which can be estimated from the rates of build-up of their precursors. The theoretical implications of these results are discussed; it is suggested that this elimination must be far on the E1cb side of the spectrum of bimolecular eliminations.

Quantitative analysis of carbohydrate residues of glycoproteins and glycolipids by gas—liquid chromatography : An appraisal of experimental details

Many investigators have reported problems with the use of d-mannitol and other alditos as internal standards for gas chromatographic quantification of trimethylsilyl ether derivatives of methyl glycosides. Quantification of carbohydrate residues of glycoproteins and glycolipids requires neutralization of the products of dry acidic methanolysis. Silver carbonate and Amberlite IR-45 ion exchange resin (OH −) were compared as neutralization agents for 0.75 N HCL methanolysates (80°, 20 h). Only 4/2-17% of the d-mannitol internal standard was recovered when a 50 nmole equimolar mixture of d-mannitol and α-methyl glycosides was neutralized with silver carbonate. Addition of 0.1 ml of acetic anhydride or glacial acetic acid after silver carbonate neutralization did not prevent significant loss of d-mannitol, myo- inositol and perseitol at the 50 nmole level. There was no appreciable loss of d-mannitol internal standard, when compared to α-methyl glycosides, after IR-45 resin (OH −) neutralization. Resin neutralization allowed 10/2-200 nmoles of d-mannitol internal standard to be added to acidic methanolysates immediately after methanolysis and carried directly through the analytical procedure.

Methanolysis products of sulfomycin I

Methanolysis products of sulfomycin I