Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Routes to higher-carbon sugar derivatives having keto-acetylenic and -alkenic,and α,β-unsaturated aldehyde functionality

Oxidative dimerization of 7,8-dideoxy-1,2:3,4-di-O-isopropylidene-d-glycero-α-d-galacto-oct-7-ynopyranoside (1) gave a high yield of the diyne 2, readily reduced by lithium aluminum hydride to the trans,trans-diene (4). The structures of 2 and 4 were established spectroscopically and by degradation of 4 to d-glycero-d-galacto-heptitol (perscitol). A mixture of the alkyne 1 and its 7-epimer 10 was readily oxidized by dimethyl sulfoxide-acetic anhydride to the 6-ketone 11, and the 8-alkene analog was similarly prepared from the alkenes derived from 1 and 10. Likewise, oxidation of 6,7-dideoxy-1,2-O-isopropylidene-α-d-gluco(and β-L-ido)-hept-6-enopyranose gave the corresponding 5-ketone. The acetylenic ketone 11 gave a crystalline oxime and (2,4-dinitrophenyl)hydrazone, the latter being accompanied by the product of attack of the reagent at the acetylene terminus (C-8). Previous work had shown that formyl-methylenetriphenylphosphorane did not convert 1,2:3,4-di-O-isopropylidene-6-aldehydo-α-d-galacto-hexodialdo-1,5-pyranose into the corresponding C₈ unsaturated aldehyde, although the latter was obtainable via1 and 10 by an ethynylation-hydroboration sequence. The Wittig route with formylmethylenetriphenylphosphorane is shown to be satisfactory for obtaining C₇ unsaturated aldehydes from 3-O-benzyl-1,2-O-isopropylidene-5-aldehydo-α-d-xylo-pentodialdo-1,4-furanose (22) and the 3-epimer of 22, respectively. These reactions provide convenient access to higher-carbon sugars and chiral dienes for synthesis of optically pure products of cyclo-addition reactions.     

An interaction between lysozyme and mucus glycoproteins. Implications for density-gradient separations.

1. Some mucus glycoproteins form soluble complexes with lysozyme at neutral pH values. 2. The extent of complex-formation was determined, by an ultracentrifugal difference method, for a range of glycoproteins covering the common blood-group specificities. 3. Interaction was strongest with those glycoproteins of blood-group Lea specificity; these were also richest in sialic acid. 4. Interaction diminished with increase of ionic strength, and was not detectable at I 0.50; however, an asialoglycoprotein was found to retain some activity. The interaction is accordingly primarily, but probably not exclusively, coulombic in origin. 5. The buoyant density of lysozyme in CsCl, CsBr, CsI and Cs2SO4 was determined; the values in the last three salts are anomalously high. This finding accounts for the previously noted difficulty of separating free protein from glycoproteins by single-stage centrifugation in CsBr. 6. Conditions for effective separation of glycoproteins from secretions containing lysozyme by density-gradient centrifugation are reported.     

Synthesis of C-substituted triazoles,isoxazoles, and pyrazoles by 1,3-dipolar cycloaddition to acetylenic sugars

Addition of phenyl azide to 3,5-di-O-acetyl-6,7-dideoxy-1,2-O-isopropylidene-β-l-idio-hept-6-ynofuranose (1) and subsequent saponification gave a 4-substituted 1-phenyl-1,2,3-triazole derivative (3) whose optical rotatory dispersion (o.r.d.) curve was positive. The α-d-gluco analog (5) of 1 similarly gave the 5-epimer (7) of 3; its o.r.d. curve was negative. Both 3 and 7 were degraded to the known 1-phenyl-1,2,3-triazole-4-carboxaldehyde. Similarly, addition of 2,4,6-trimethylbenzonitrile N-oxide to 1 or 5 gave the corresponding, crystalline 3-mesitylisoxazoles as single products; ¹³C-n.m.r. spectroscopy was used to establish the orientation of addition. Related 3-mesitylisoxazoles (11 and 13) were obtained from 1,2:3,4-di-O-isopropylidene-d-glycero-α-d-galacto-oct-7-ynopyranose (10) and its l-glycero 6-epimer (12), respectively; 11 showed the expected, large levorotation, and the 6-epimer 13 was also levorotatory. Benzonitrile (N-phenyl)imine, prepared in situ from 1-(α-chlorobenzylidene)-2-phenylhydrazine and base, did not react with 10 (or its 6-epimer 12), but did react with the 6-keto analog to give a 5-substituted 1,3-diphenyl-1,2-diazole.     

Reaction of derivatives of methyl 2,3-O-benzylidene-6-deoxy-α-L-mannopyranoside with butyllithium: Synthesis of methyl 2, 6-dideoxy-4-O-methyl-α-L-erytho-hexopyranosid-3-ulose

Methyl 2,3-O-benzylidene-6-deoxy-α-L-mannopyranoside (2) reacted with butyllithium to give a mixture of 1,5-anhydro-3-C-butyl-1,2,6-trideoxy-L-ribo-hex-1-enitol (3) and its L-arabino analogue (4), together with methyl 2,3,6-trideoxy-α-L-erythro-hex-2-enopyranoside (5). In contrast, the 4-O-methyl analogue (8) of 2 was converted by butyllithium into methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexo-pyranosid-3-ulose (9), which was further characterized as its oxime 10. The 4-O-benzyl analogue of 8, obtained as two separate diastereoisomers (6 and 7) differing in configuration at C-2 of the dioxolane ring, gave a complex mixture of products on treatment with butyllithium.