High-performance liquid chromatographic method to resolve and determine lipopolysaccharide sub-groups of Escherichia coli endotoxin in isolated perfused rat liver perfusate.

A high-performance liquid chromatographic method was developed for resolving heterogeneous preparations of fluorescently labelled endotoxin derived from Escherichia coli (Serotype 0111:B4) into separate lipopolysaccharide sub-groups. The endotoxin was chromatographed on an analytical gel permeation column using a mobile phase of acetonitrile (20%, v/v) and 100 mM phosphate buffer (pH 7.75). Four fluorescent peaks were resolved, representing sub-groups of markedly different molecular sizes. Three of the four sub-groups contained the core polysaccharide 2-keto-3-deoxyoctonate, confirming that they contained lipopolysaccharide. Fluorescein isothiocyanate (FITC)-labelled endotoxins derived from Vibrio cholerae and Salmonella minnesota chromatographed using the same system eluted with distinctly different patterns of peaks from each other and from E. coli. Extraction of E. coli FITC-endotoxin from a buffer solution using a phenol-diethyl ether method and subsequent chromatography allowed the determination of three of the four fluorescent sub-groups over the concentration range 1-15 micrograms/ml.     

Comparative structural study of the lipopolysaccharides of Y. enterocolitica serovars 0:7.8 and 0:19.8

The lipopolysaccharides ofYersinia enterocolitica, serovars 0:7.8 (strain 106) and 0:19.8 (strain 842), isolated from the microbial mass by phenol-water extraction, contained residues of L-fucose, 6-deoxy-D-gulose, D-mannose, D-galactose, D-glucose, D- and L-glycero-D-mannoheptoses, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and 2-keto-3-deoxyoctonic acid (KDO). The polysaccharides obtained by mild acid hydrolysis of the lipopolysaccharides followed by gel filtration on Sephadex G-50 were a mixture of the O-specific polysaccharide and the core, which could not be separated even by repeated rechromatography because of the comparability of their molecular masses. On the basis of the results of monosaccharide analysis, methylation, Smith degradation, and partial hydrolysis, a structure has been suggested for the repeating unit of the O-specific polysaccharides of the lipopolysaccharides ofY. enterocolitica of the serovars studied. Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Branch, USSR Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 763–770, November–December, 1989.     

Comparative structural study of the lipopolysaccharides of Y. enterocolitica serovars 0:7.8 and 0:19.8

The lipopolysaccharides ofYersinia enterocolitica, serovars 0:7.8 (strain 106) and 0:19.8 (strain 842), isolated from the microbial mass by phenol-water extraction, contained residues of L-fucose, 6-deoxy-D-gulose, D-mannose, D-galactose, D-glucose, D- and L-glycero-D-mannoheptoses, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and 2-keto-3-deoxyoctonic acid (KDO). The polysaccharides obtained by mild acid hydrolysis of the lipopolysaccharides followed by gel filtration on Sephadex G-50 were a mixture of the O-specific polysaccharide and the core, which could not be separated even by repeated rechromatography because of the comparability of their molecular masses. On the basis of the results of monosaccharide analysis, methylation, Smith degradation, and partial hydrolysis, a structure has been suggested for the repeating unit of the O-specific polysaccharides of the lipopolysaccharides ofY. enterocolitica of the serovars studied.Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Branch, USSR Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 763–770, November–December, 1989.     

The use of thermal lensing for the determination of pyrogens

Based on the optimized spectrophotometric determination of pyrogens (of various classes ( p-aminophenol and endotoxins), thermal lensing was applied to the determination of these substances at the submicrogram level. The limit of detection of p-aminophenol, a pyrogenic impurity in pharmaceutical formulations of paracetamol, by reaction with resorcinol in alkaline solutions is 100 ng mL(-1). Phloroglucinol was considered as an analog of resorcinol as a reagent in this reaction. The conditions of spectrophotometric determination of pyrogenic lipopolysaccharides (endotoxins) by ion-pair formation with methylene blue (the limit of detection is 100 ng mL(-1)), by ion-pair formation with Stains-All (1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide) (the limit of detection is 500 ng mL(-1)), and by reaction of 2-keto-3-deoxyoctonic acid with thiobarbituric acid (the limit of detection is 800 ng mL(-1)) were proposed. The optimized procedure for 2-keto-3-deoxyoctonic acid was applied for thermal lensing that provided a decrease in the limit of detection to 70 ng mL(-1) and was also used for lipopolysaccharide determination in the endotoxin standard from E. coli.     

Synthesis of tri-, hexa-, and nonasaccharide subunits of the atypical O-antigen polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains

Synthesis of trisaccharide repeating unit, -->3)-alpha-D-Rhap-(1-->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rha p-(1-->, and its dimeric hexa- and trimeric nonasaccharide subunits of the atypical O-antigen polysaccharide of the lipopolysaccharide from Danish H. pylori strains D1, D3, and D6 has been accomplished. Successful synthesis of the hexasaccharide and the nonasaccharide was possible by dimerization and trimerization of the suitably protected trisaccharide repeating unit, in which three monosaccharide moieties were arranged in a proper order by placing the sterically demanding 3-C-methyl-D-mannose moiety in between D- and L-rhamnoses. Key steps include the coupling of three monosaccharide moieties and dimerization and trimerization of the trisaccharide unit by glycosylations employing the 2'-carboxybenzyl glycoside method. Also presented is a method for the synthesis of the novel branched sugar, 3-C-methyl-D-mannose moiety by elaboration of its equatorial hydroxyl and axial methyl groups at C-3' in the disaccharide stage.     

Structural investigation of the lipopolysaccharide ofYersinia enterocolitica serovar 0:8

The lipopolysaccharide ofYersinia enterocolitica serovar 0:8 (strain 161) isolated from the microbial mass by aqueous-phenol extraction contains residues of L-fucose-6-deoxy-D-gulose, D-mannose, D-galactose, D-glucose, D- and L-glycero-D-mannoheptoses, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and 2-keto-3-deoxyoctonic acid (KDO). The polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide followed by gel filtration on Sephadex G-50. On the basis of the results of monosaccharide analysis, methylation, Smith degradation, and partial hydrolysis the following structure is suggested for the repeating unit of the O-specific polysaccharide of the LPS ofYersinia enterocolitica, serovar 0:8:Pacific Ocean Institute of Bioorganic Chemistry of the Far-Eastern Scientific Center of the USSR Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh Soedenenii, No. 5, pp. 657–664, September–October, 1987.     

Structural investigation of the lipopolysaccharide ofYersinia enterocolitica serovar 0:8

The lipopolysaccharide ofYersinia enterocolitica serovar 0:8 (strain 161) isolated from the microbial mass by aqueous-phenol extraction contains residues of L-fucose-6-deoxy-D-gulose, D-mannose, D-galactose, D-glucose, D- and L-glycero-D-mannoheptoses, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and 2-keto-3-deoxyoctonic acid (KDO). The polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide followed by gel filtration on Sephadex G-50. On the basis of the results of monosaccharide analysis, methylation, Smith degradation, and partial hydrolysis the following structure is suggested for the repeating unit of the O-specific polysaccharide of the LPS ofYersinia enterocolitica, serovar 0:8:     

Full structure of the carbohydrate chain of the lipopolysaccharide of Providencia rustigianii O34

A lipopolysaccharide isolated from an opportunistic pathogen of the Enterobacteriaceae family Providencia rustigianii O34 was found to be a mixture of R-, SR-, and S-forms consisting of a lipid moiety (lipid A) that bears a core oligosaccharide, a core with one O-polysaccharide repeating unit attached, and a long-chain O-polysaccharide, respectively. The corresponding carbohydrate moieties were released from the lipopolysaccharide by mild acid hydrolysis and studied by sugar and methylation analyses along with one- and two-dimensional NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. As a result, the structures of the core and the O-polysaccharide were established, including the structure of the biological repeating unit (an oligosaccharide that is preassembled and polymerized in biosynthesis of the O-polysaccharide), as well as the mode of the linkage between the O-polysaccharide and the core. Combining the structure of the carbohydrate moiety thus determined and the known structure of lipid A enabled determination of the full lipopolysaccharide structure of P. rustigianii O34.     

Regioselective Acylation of Ginsenosides by Novozyme 435 to Generate Molecular Diversity

Ginsenosides are major bioactive constituents of ginseng (Panax spp.; Araliaceae), a traditional Chinese medicinal herb. In order to increase the molecular diversity and broaden the potential usage of ginsenosides, ginsenosides Rd ( 1 ), Rg3 ( 2 ), (20R)‐Rg3 ( 3 ), Rh2 ( 4 ), Re ( 5 ), Rh1 ( 8 ), Rg2 ( 9 ), gypenoside XVII ( 6 ), and pseudoginsenoside F11 ( 7 ) were regioselectively acylated with vinyl acetate, catalyzed by Novozyme 435 (lipase B from Candida antarctica), in organic solvents to afford different mono‐acetyl ginsenosides. Ginsenoside Rd ( 1 ) was also acylated with vinyl decanoate or vinyl cinnamate to generate 1b and 1c , respectively. Acylation of glucosylated ginsenosides ( 1 – 4, 6, 8 ) occurred at the primary 6‐OH function of the terminal glucose (Glc) moiety of the sugar at C(3) or C(20) of the dammarane‐type aglycone. In contrast, ginsenosides 5, 7 , and 9 , containing mixed sugar moieties, resulted in acylation of both the rhamnose (Rha) and the glucose (Glc) moieties. In the case of ginsenoside Re ( 5 ) and pseudoginsenoside F11 ( 7 ), acylation at the secondary 4‐OH function of the terminal Rha moiety, attached at C(3) of the aglycone, is preferred. The structures of all acylated products were determined by extensive MALDI‐TOF‐MS and NMR analyses.     

Three New Resin Glycosides and a New Tetrahydropyran Derivative from the Seeds of <i>Quamoclit pennata</i>

Three new resin glycosides, quamoclins V (1), VI (2), and VII (3) and a new tetrahydropyran derivative, quamopyran (4), were isolated from the seeds of Quamoclit pennata BOJER (Convolvulaceae). The chemical structures of these compounds were determined primarily on the basis of spectroscopic data. The carboxyl group of the aglycone, 11S-convolvulinolic acid, of 1 and 2 was linked intermoleculary with a hydroxy group of the sugar moiety to form a macrocyclic ester structure, as in already known jalapins, and 3 was an acylated glycosidic acid methyl ester. All of the sugar moieties of 1-3 were acylated by one 2S-methylbutyric acid. Compound 4 was a diketone having a tetrahydropyran ring.     

Recent results on the biochemistry of the cell wall lipopolysaccharides of Salmonella bacteria

Lipopolysaccharides, which are located in the cell wall of gram-negative bacteria, are characterized by their biological versatility. They represent the O antigens of the bacteria, they are potent endotoxins, and they often function as the receptor sites for bacteriophages. The study of the mode of action of lipopolysaccharides and the search for structures in the macromolecules that are responsible for biological activity became promising when principles of the chemical fine structure of lipopolysaccharides were identified. The following review summarizes the results of recent investigations regarding the structure of lipopolysaccharides, their biosynthesis and its genetic determination.     

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.     

Synthesis of the trisaccharide repeating unit of the atypical O-antigen polysaccharide from Danish Helicobacter pylori strains employing the 2'-carboxybenzyl glycoside

[reaction: see text] Synthesis of the unique trisaccharide repeating unit of the O-polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains has been accomplished. Key steps include the coupling of three monosaccharide moieties by glycosylations employing the 2'-carboxybenzyl glycoside method. Also presented is a method for the synthesis of the novel branched sugar, 3-C-methyl-D-mannose, which is one of three monosaccharide components.     

The biosynthesis of acarbose and validamycin

The studies reported here have established the biosynthetic origin of the mC7N units of acarbose and validamycin from sedo-heptulose 7-phosphate, and have identified 2-epi-5-epi-valiolone as the initial cyclization product. The deoxyhexose moiety of acarbose arises from glucose with deoxythymidyl-diphospho-4-keto-6-deoxy-D-glucose (dTDP-4-keto-6-deoxy-D-glucose) as a proximate intermediate. However, despite the identical origin of the aminocyclitol moieties in acarbose and validamycin A, the pathways of their formation seem to be substantially different. Validamycin A formation involves a number of discrete ketocyclitol intermediates, 5-epi-valiolone, valienone, and validone, whereas no free intermediates have been identified on the pathway from 2-epi-5-epi-valiolone to the pseudodisaccharide moiety of acarbose. The stage is now set for unraveling the mechanism or mechanisms by which the two components of the pseudodisaccharide moieties of acarbose and validamycin are uniquely coupled to each other via a nitrogen bridge.     

Detection of Virulence Genes in Vibrio cholerae Isolated from Aquatic Environment in Kerala, Southern India

Vibrio cholerae is the etiologic agent of cholera. It is an autochthonous inhabitant of all aquatic environments. The virulence of V. cholerae is maintained by the CTX genetic element and tcpA gene. In the present investigation, environmental strains of V. cholerae isolated from different aquatic biotopes in Kerala were identified and serotyped. The antibiotic resistance pattern and presence of virulence and regulatory genes were examined. We found the presence of toxigenic non-O1/non-O139 strains harboring the CTX genetic element, heat-stable enterotoxin, rtxA gene, El Tor hemolysin, and Vibrio pathogenicity island (VPI). The strains also produced the cholera toxin (CT) as determined by monosialoganglioside enzyme-linked immunosorbent assay. A few strains belonging to the O1 serogroup but lacking the CTX genetic element were also observed. The majority of the environmental strains belonged to non-O1/non-O139 serogroup with many possessing toxR, ompU, heat-stable enterotoxin, and rtxA gene. The toxigenic non-O1/non-O139 strains exhibited resistance to trimethoprim, ampicillin, and polymixin B and intermediate resistance to co-trimoxazole. However, all other environmental strains were found resistant to ampicillin and polymixin B. Our findings demonstrate that the virulence genes are dispersed among the environmental strains of V. cholerae and a complex aquatic environment can give rise to pathogenic V. cholerae.     

Practical synthesis of 2-keto-3-deoxy-D-glycero-D-galactononulosonic acid (KDN)

Reaction of propargylmagnesium bromide with 2,3;5,6-di-O-isopropylidene-D-mannonolactone followed by highly stereoselective reduction of the so-formed lactol with sodium borohydride gives a syn-diol from which practical syntheses of 2-keto-3-deoxy-D-glycero-D-galactononulosonic acid (KDN) and various partially protected derivatives have been achieved all of which feature the oxidative unmasking of the α-keto acid moiety from the alkyne.     

Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 15 degrees C and 25 degrees C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Lipooligosaccharides (LOSs) are macromolecules present on the external cellular membrane of Gram-negative bacteria, structurally made of two distinct regions, lipid A and Core. By varying their growth temperature, bacteria such as psychrophiles change the phosphorylation distribution of the LOSs produced. The level of phosphorylation and the phosphate group positions in LOSs produced by the extremophile psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125, grown at 15 degrees C and 25 degrees C, were investigated by nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) and tandem mass spectrometry (MS/MS). The samples, obtained by phenol/chloroform/petroleum ether (PCP) extraction of dried cells, were treated with hydrazine at 37 degrees C in order to reduce the heterogeneity by removal of the ester-linked fatty acid moieties. The molecular ion distributions in these LOS fractions were investigated in negative ion mode. Based on these data it was postulated that the sample grown at 25 degrees C contained four phosphate groups while that at 15 degrees C contained three. In order to determine phosphorylation sites in sugar chains, the samples were submitted to low collision energy MS/MS for sequencing. In the sample with three phosphates, one was found to be linked to the tetrasaccharide Core region, more precisely to position C-4 of the Kdo unit. The two remaining phosphate groups were both linked to the 2-acylamide-2-deoxy-D-glucopyranose of the lipid A moiety, and two possible distributions could be postulated on the basis of the fragmentation pattern obtained; in the first case both phosphate groups are linked as a pyrophosphate moiety to position C-1 of the proximal glucosamine (reducing residue), while in the second case one phosphate is linked to position C-1 of the proximal glucosamine and the other to position C-4' of the distal glucosamine (non-reducing residue). This distribution was also found in the lipid A moiety of the tetraphosphorylated sample grown at 25 degrees C, which bears two phosphate groups on the Core region, one on position C-4 of the Kdo and the other on position C-7 or C-8 of the same residue. The phosphate locations were derived from the intra-ring cleavage ions of sugar moieties in the LOSs obtained by an optimized CID procedure using negative ion QTOF-MS/MS.     

以氨基酸为配体的血液灌流用内毒素吸附剂的制备及性能研究

以琼脂凝胶为载体,氨基酸为配体,合成了内毒素吸附剂,并对其吸附效果进行了比较.对吸附剂的各种性质进行了表征,选取琼脂-赖氨酸吸附剂对内毒素血症的兔子进行血液灌流,可将血浆中的内毒素降至接近正常值,最高清除率达73.6%,且吸附剂具有良好的血液相容性.     

Structure of the fully modified left-handed cyclohexene nucleic acid sequence GTGTACAC

CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 A, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3'-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking.     

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.