7-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose. A constituent of the endotoxin of Bordetella pertussis.

Hydrolysis of the Bordetella pertussis endotoxin, extracted from both "phase I" and "phase IV" bacteria, with 4 M HCl for 1 h at 100 degrees C, released the disaccharide named in the title; it was isolated by paper electrophoresis or by ion-exchange chromatography in about 1% yield (w/w). The structure of the heptose could be rigorously established by chemical degradation; the facts that the glucosaminidic linkage was hydrolysed by an enzyme preparation containing both, alpha and beta-N-acetylglucosaminidase activities, whereas it was resistant to cleavage by pure beta-N-acetylglucosaminidase strongly support the assumption that the disaccharide contains an alpha-D-glucosaminide linkage.     

Identification of 2-amino-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucose as a major constituent of the hydrophobic region of the Bordetella pertussis endotoxin

2-Amino-6-O-(2-amino-2-deoxy-β- d-glucopyranosyl)-2-deoxy- d-glucose substituted on the amino group of the reducing 2-amino-2-deoxy- d-glucose unit by a 3-hydroxytetradecanoyl group was shown to be a major constituent of the “Lipid A” fragment obtained by acid hydrolysis of the Bordetella pertussis endotoxin.     

Direct regioselective 2-O-(p-toluenesulfonylation) of sucrose

2-O-(p-Toluenesulfonyl)sucrose was regioselectively synthesized by direct p-toluenesulfonylation of sucrose using N-(p-toluenesulfonyl)imidazole in the presence of molecular sieves at 40 degrees C. The reactivities of the sucrose hydroxy groups toward this sulfonylation increased in the order as follows: OH-2>OH-1'>OH-3'>OH-6>OH-6'. These results were diametrically opposite to the expected sulfonylation with p-toluenesulfonyl chloride in pyridine, for which the reactivity increased in the order as follows: OH-6', OH-6>OH-1'>OH-2. The desired 2-O-(p-toluenesulfonyl)sucrose was readily isolated by simple open reversed-phase column chromatography, followed by recrystallization, thus overcoming the main difficulties associated with regioselectivity, efficiency, and isolation techniques for the practical preparation.     

Synthesis and Properties of Modified Oligodeoxyribonucleotides Containing 2"-O-(2,3-Dihydroxypropyl)uridine and 2"-O-(2-Oxoethyl)uridine

A new uridine derivative, 2"-O-(2,3-dihydroxypropyl)uridine, and its 3"-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2"-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2"-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2" position of the carbohydrate moiety was confirmed by the interaction withp-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units does not practically differ from that of the natural analogues.     

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl-beta-D- 3-O-(2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl- beta-D-glucopyranosyl)-beta-D-galactoyranoside. pyranosyl)-beta-D-galactopyranoside

Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.     

2-amino-2-desoxy-6-O-(D-glykofuranosylurono-6,3-lacton)-D-galaktopyranosen

Condensation of 2-amino-2-deoxy-D-galactopyranose with D-glucuronic acid or D-mannurono-6,3-lactone in the presence of hydrochloric acid gave the corresponding 2-amino-2-deoxy-6-O-(D-glycofuranosylurono-6,3-lactone)-D-galactopyranoses. The α-D configuration of the disaccharide derived from D-glucuronic acid was determined by its resistance towards β-D-glucuronidase.     

Relationship between endotoxin and prostaglandin (PGE2 and PGFM) concentrations and ovarian function in dairy cows with puerperal endometritis

Blood concentrations of progesterone, 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM) and endotoxin, and uterine fluid concentrations of prostaglandin E(2) (PGE(2)), PGFM and endotoxin were evaluated in 14 dairy cows with puerperal endometritis (mild (n=6) and heavy (n=8)). Endotoxin was measured using a quantitative kinetic assay. Cows with heavy endometritis had significantly higher concentrations of plasma PGFM (P<0.01) and uterine fluid PGE(2) and endotoxin (P<0.05) than cows with mild endometritis. Concentrations of PGFM in plasma and uterine fluid, of PGFM and PGE(2), and PGE(2) and endotoxin in uterine fluid were positively and significantly (P<0.05) correlated. The presence of endotoxin in plasma was detected in one out of six mild and in eight out of eight heavy endometritis cows. Peak plasma endotoxin concentrations (0.08-9.14 endotoxin units/ml (EU/ml) were observed between 1 and 12 days postpartum (pp) and thereafter amounts generally remained below 0.1 EU/ml (last day of detection: Day 27 pp). Abnormal ovarian function was observed in six cows (four with prolonged anoestrus and two with long luteal phase after the first postpartum ovulation). Plasma endotoxin concentrations were detected in the anoestric cows. The results suggest that: (i) concentrations of uterine fluid endotoxin and PGE(2) and of plasma PGFM are related to the degree of endometritis; (ii) absorption of endotoxin from the uterus to the bloodstream occurs, mainly in heavy endometritis cows; and (iii) there is a relationship between uterine infection, endotoxin production and resumption of pp ovarian activity.     

Synthesis of allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyranoside, a unique plant N-glycan motif containing arabinose

The synthesis of the trisaccharide allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyra-noside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl beta-mannoside with the acetylated alpha-D-mannosyl bromide gave the alpha-(1 --> 6)-disaccharide as the main product and the crystalline 3,6-branched trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1 --> 6) disaccharide with L-arabinofuranosyl bromide furnished a mixture of 3-O- and 2-O-alpha-L-Ara-trisaccharides from which the title compound was isolated.     

KILO-SCALE SYNTHESIS PROCESS FOR 2′-O-(2-METHOXYETHYL)-PYRIMIDINE DERIVATIVES

We describe an improved process to produce 2′-O-(2-methoxyethyl)-pyrimidines. Starting with commercially available O-2,2′-anhydro-5-methyluridine and tris-(2-methoxyethyl)borate, we modified the ring-opening reaction conditions and changed to a continuous extraction purification method to give 2′-O-(2-methoxyethyl)-5-methyluridine. The dimethoxytritylation 5′/3′ ratios and yield were improved by the use of 2,6-lutidine as the base. Conditions to convert to the 5-methylcytidine analog and its isolation by crystallization were optimized. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities.     

Water-soluble 3-O-(2-methoxyethyl)cellulose: synthesis and characterization

The synthesis of novel 3-O-(2-methoxyethyl)cellulose via 2,6-di-O-thexyldimethylsilyl ethers was successfully carried out. Treatments of 3-O-(2-methoxyethyl)-2,6-di-O-thexyldimethylsilylcellulose with tetrabutylammonium fluoride trihydrate led to a complete removal of the protecting groups. Structure characterization carried out by means of 1D and 2D NMR spectroscopy proves a high regioselectivity. The novel cellulose ether is soluble in dimethyl sulfoxide, N,N-dimethylacetamide, N-methylpyrrolidone, and water. Size-exclusion chromatography revealed a distinct aggregation behavior in water.     

A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl Diglyceride, from Arabidopsis thaliana

The cyclic derivative of 13(S)-hydroperoxolinolenic acid, 12-oxophytodienoic acid, serves as a signal transducer in higher plants, mediating mechanotransductory processes and plant defenses against a variety of pathogens, and also serves as a precursor for the biosynthesis of jasmonic acid, a mediator of plant herbivore defense. Biosynthesis of 12-oxophytodienoic acid from alpha-linolenic acid occurs in plastids, mainly in chloroplasts, and is thought to start with free linolenic acid liberated from membrane lipids by lipase action. In Arabidopsis thaliana, the glycerolipid fraction contains esterified 12-oxophytodienoic acid, which can be released enzymatically by sn1-specific, but not by sn2-specific, lipases. The 12-oxophytodienoyl glycerolipid fraction was isolated, purified, and characterized. Enzymatic, mass spectrometric, and NMR spectroscopic data allowed us to establish the structure of the novel oxylipin as sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride. The novel class of lipids is localized in plastids. Purified monogalactosyl diglyceride was not converted to the sn1-(12-oxophytodienoyl) derivative by the combined action of (soybean) lipoxygenase and (A. thaliana) allene oxide synthase, an enzyme ensemble that converts free alpha-linolenic acid to free 12-oxophytodienoic acid. When leaves were wounded, a significant and transient increase in the level of (12-oxophytodienoyl)-monogalactosyl diglyceride was observed. In A. thaliana, the major fraction of 12-oxophytodienoic acid occurs esterified at the sn1 position of the plastid-specific glycerolipid, monogalactosyl diglyceride.     

Synthesis of 1-O-(2′,4′-Dichlorophenoxyacetyl)-d-glucopyranose

1-O-Glucosyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) were easily prepared from 4,6-O-benzylideneglucose. The configuration of 1-O-ester linkage was affected by pH at the end of the reaction, that is, β-type was a major product at a neutral or acidic condition and α-type at an alkaline condition. Both of the anomers showed the same biological activities as sodium salt of 2,4-D.     

Synthesis of 2-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-5a-carba-alpha-D- mannopyranose, and 5a-carba-2-O- and 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoses.

Protected derivatives of the title carba-trisaccharide 1, and carba-disaccharides 4 and 6 have been synthesised by conventional glycosylation of the precursors 8, 9, or 22 of 5a-carba-alpha-D-mannopyranose with sugar halides. These are carba-sugar analogues of the oligosaccharide chains frequently occurring as structural units in biologically important glycoconjugates, and could be utilised as the building blocks for construction of higher carba-oligosaccharides of biological interest.     

Involvement of tachykinin NK(1) and NK(2) receptors in changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia in Guinea pigs

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK(1) receptor antagonist), SR-48,968 (a tachykinin NK(2) receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R(L)) and dynamic lung compliance (C(dyn)) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R(L) and decreased C(dyn) 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK(2) receptors is responsible for the former response, while activation of both tachykinin NK(1) and NK(2) receptors is involved in the latter response.     

Synthesis and antibacterial activities of a novel alkylide: 3-O-(3-aryl-2-propargyl) and 3-O-(3-aryl-2-propenyl)clarithromycin derivatives

A series of novel 3-O-(3-aryl-E-2-propenyl)clarithromycin derivatives 8 and 3-O-(3-aryl-2-propargyl)clarithromycin derivatives 11 were designed, synthesized, and evaluated for their in vitro antibacterial activities. Compared with 8c and 11c (Ar was 5-pyrimidyl), 3-O-(3-(5′-pyrimidyl)-Z-1-propenyl) counterpart 6c displayed 4- to 64-fold more potent activities against erythromycin-susceptible Staphylococcus aureus and Streptococcus pneumoniae. Moreover, the activities of 6c, 8c, and 11c against erythromycin-resistant S. aureus and S. pneumoniae were in general 4-fold higher than those of the reference compound, clarithromycin and azithromycin.     

Removal of endotoxin during purification of poly(3-hydroxybutyrate) from gram-negative bacteria.

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

静脉注射用人免疫球蛋白细菌内毒素检测

探索静脉注射用人免疫球蛋白(IVIG)细菌内毒素含量的鲎试验检测方法。根据《中国药典》(2000版)细菌内毒素含量的检测方法进行,将待检品用NaOH调pH至中性,稀释至2.4倍可用标示灵敏度为0.25EU/m l的特异性鲎试剂进行细菌内毒素检测,结果与家兔法进行比较,并且在样品中加入定量内毒素0.6 EU/m l用两种方法进行对比试验。结果表明用细菌内毒素检测法(鲎试验法)检测静脉注射用人免疫球蛋白是可行的。     

Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s)

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.     

Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-negative bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. PHB was recovered from these bacteria by two different methods, and the endotoxin levels were determined. When PHB was recovered by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB content in the cell. The NaOH digestion method, which was particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 10⁴ EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.