Anticytokinin activity of 4-furfurylamino-7-(beta-D-ribofuranosyl)pyrrolo(2,3)pyrimidine

4-Furfurylamino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidine, the 7-deaza analog of kinetin riboside, has been synthesized and found to be a potent anticytokinin in the tabacco callus bioassay.     

Chemical structure of the polysaccharide antigen of Eubacterium saburreum, strain O2.

The polysaccharide antigen produced by Eubacterium saburreum, strain O2, is composed of (1 leads to 6)-linked beta-D-glycero-D-galacto-heptopyranosyl residues, all of which are substituted with 6-deoxy-alpha-D-altro-heptofuranosyl groups at O-3.     

Purification of 2,5-anhydro-d-hexitol bis(phosphates) and identification of a major 1,4,6-tris(phosphate) contaminant by 31P-, 13C-, and 1H-N.M.R. spectroscopy

The reaction of two equivalents of diphenylchlorophosphate in cold pyridine with 2,5-anhydrohexitols has been assumed to result in only 1,6-bis(diphenylphosphate) products. However, by thin-layer, silica gel dry-column, and DEAE-Sephadex A-25 column chromatography, the products of this reaction have been shown to contain three major components; monophosphates (32 or 30%, by weight), 1,6-bis(phosphates) (40 or 56%), and 1,4,6-tris(phosphates) (28 or 14%): the former percentages for the product from 2,5-anhydro-d-mannitol (1) and the latter for the product from 2,5-anhydro-d-glucitol (10). The identity of each bis- and tris-(phosphate) of 1 or 10 was established by ³¹P- and ¹³C-n.m.r. spectroscopy. Acetylated bis- and tris-(diphenylphosphates) of 1 were also examined by ¹H-n.m.r. The significance of these findings on the interpretation of studies of the anomeric specificity of enzymes and on the specificity of the reagent diphenylchlorophosphate are discussed. The formation of only a 1,4,6-tris(phosphate) of 10 suggests that the 1,6-bis(diphenylphosphate) of 10 may undergo formation of a 1,3-cyclic phosphate triester by transesterification with elimination of phenol. A method for the determination of the number of cyclohexylammonium groups crystallizing with a sugar phosphate is proposed that simplifies the elemental analysis of this type of salt.     

Preparation of acetylenic sugar derivatives by way of 2-(N-nitroso)acetamido sugars

N-Nitrosation with dinitrogen tetraoxide was used to convert 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranose (1) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-galactopyranose (4) in high yield into the N-nitroso derivatives 2 and 5, respectively. Similarly, 3-acetamido-1,2,4,6-tetra-O-acetyl-3-deoxy-β-D-glucopyranose (12) and methyl 2-acetamido-3,4,5,6-tetra-O-acetyl-2-deoxy-D-gluconate (15) gave their respective, crystalline N-nitroso derivatives 13 and 16. Various other 2-acetamido sugar derivatives were likewise nitrosated. In ethereal solution, compounds 2 and 16 reacted with potassium hydroxide in isopropyl alcohol to give the C₅ acetylene, 1,2-dideoxy-D-erythro-pent-1-ynitol, isolated as the known triacetate 3. By the same procedure, the galacto derivative 5 was converted in high yield into the 3-epimeric C₅ acetylene, 1,2-dideoxy-D-threo-pent-1-ynitol, isolated as its triacetate 6 and characterized by conversion into the known, crystalline 1,2-dideoxy-3-O-(3,5-dinitrobenzoyl)-4,5-O-isopropylidene-D-threo-pent-1-ynitol (7).     

Reversible inactivation of avian lysozymes by dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide

The reaction of hen egg white lysozyme with a 4 molar excess of dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide at pH 6.0 leads to total loss of enzymatic activity within 5 minutes. Upon standing, the inactivated enzyme spontaneously regains activity, leveling off at 60% of the original activity after 72 hours. Under the same conditions, turkey egg white lysozyme is reduced to less than 5% of its original activity within 5 minutes, then spontaneously reactivates to 85% of its original activity after 24 hours. Human lysozyme shows no dramatic loss of activity when treated under these conditions. The presence of the substrate, chitotetraose, prevents the initial inactivation of both hen and turkey enzymes.     

Conversion of a 2-(N-nitroso)acetamido hexose into a five-carbon, acetylenic sugar derivative

Dinitrogen tetraoxide was used to convert 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-glucopyranose (1) in high yield into the syrupy N-nitroso derivative 2, and benzyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranose (6) into the crystalline N-nitroso analog 7. The N-nitroso derivative 2 in acetonitrile underwent photolysis by pyrex-filtered, u.v. light to regenerate the starting acetamide 1 in high yield; spontaneous decomposition of 2 afforded β-D-glucopyranose pentaacetate (3) and other products. In ethereal solution, compound 2 reacted with potassium hydroxide in isopropyl alcohol with loss of the 2-substituent and C-1, to give a C₅ acetylene, 1,2-dideoxy-D-erythro-pent-1-ynitol, isolated in high yield as its triacetate 4 and characterized by conversion into the known, crystalline 1,2-dideoxy-3-O-(3,5- dinitrobenzoyl)-4,5-O-isopropylidene-D-erythro-pent-1-ynitol (5).     

Zinc ion-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime: bimolecular participation of hydroxozinc(II) ion

The Zn²⁺-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime (A) proceeds through three paths. The first two paths, the water path and the hydroxide path, have been observed in related systems. The third path, whose rate is bimolecular with respect to hydroxozinc(II) ion, is newly observed. The methyl group of A raises reaction rates through steric compression. The rate data obtained with A provide further evidences for the intramolecular nucleophilic attack of the metal-bound water molecule or hydroxide ion at the complexed ester. The third path is attributed either to the general base participation of free hydroxozinc(II) ion in the nucleophilic reaction of the Zn(II)-bound hydroxide ion or to the intramolecular reaction of dimeric hydroxozinc(II) ions. Implications of the present results to the actions of metalloenzymes, especially carbonic anhydrase and carboxypeptidase A, are also discussed.     

Stepwise synthesis of methyl 4-O-[3-O-(β-d-xylopyranosyl)-β-d-xylopyranosyl]-β-d-xylopyranoside

The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α-d-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K_m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.     

Overestimates of the size of poly(A) segments.

Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.     

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Multifunctional catalysis by metal complexes: 1. Ester hydrolysis catalyzed by oximinatozinc(II) ions

Hydrolysis of p-nitrophenyl acetate catalyzed by a Zn(II) complex of 2-acetylpyridineketoxime or 2-pyridinecarboxaldoxime was studied as a model of multifunctional catalysis by metalloproteases. The reaction proceeded exclusively through the formation of an acylcatalyst intermediate under the experimental conditions, and both the formation and the breakdown of the acyl intermediate were much faster than the spontaneous reaction. The metal ion, the metal-bound water molecule or hydroxide ion, the oximate ion, and general bases contributed to the multifunctional catalysis in ester hydrolysis by the oximinatozinc(II) ions.     

1,5-anhydro-2-deoxy-3-O-(2-deoxy-beta-D-arabino-hexopyranosyl)-D-arabino-hex-1-enitol, the by-product in the enzymic hydration of D-glucal by beta-D-glucosidase from emulsin.

The by-product (3) in the hydration of D-glucal (1) catalyzed by emulsin beta-D-glucosidase has been identified as 1,5-anhydro-2-deoxy-3-O-(2-deoxy-beta-D-arabino-hexopyranosyl)-D-arabino-hex-1-enitol. Two models for the formation of 3 are discussed, involving transfer of a 2-deoxy-D-arabino-hexopyranosyl cation to HO-3 of D-glucal (glycon transfer) and transfer of an allylic D-pseudoglucal cation to HO-1 of 2-deoxy-D-arabino-hexopyranose (aglycan transfer). The enzymic production of 3 is highly regiospecific, which lends support to the second model and implies the presence of a specific binding-site for the aglycon moiety.     

Synthesis of N-(1-deoxyhexitol-1-yl)amino acids, reference compounds for the nonenzymic glycosylation of proteins

The N-(1-deoxy-D-mannitol-1-yl) and N-(1-deoxy-D-glucitol-1-yl) derivatives of L-valine, L-alanine, L-threonine, and L-leucine were prepared by reductive amination of D-mannose and D-glucose with the appropriate amino acids, in the presence of sodium cyanoborohydride. N epsilon-(1-Deoxy-D-mannitol-1-yl)- and N epsilon-(1-deoxy-D-glucitol-1-yl)-L-lysine were prepared by similar reactions of hexoses with N alpha-tert-butoxycarbonyl and N alpha-benzyloxycarbonyl-L-lysine, followed by removal of the protecting groups. The structures were confirmed by 1H-n.m.r. spectroscopy, which showed that each compound was completely free of its C-2 epimer. The synthetic compounds may be used as reference compounds for the identification of N-(1-deoxyhexitol-1-yl)amino acids formed when N-(1-deoxy-D-fructose-1-yl) groups of nonenzymically glycosylated proteins, of the hemoglobin A1c type, are reduced with sodium borohydride, and the protein is subjected to acid-catalyzed hydrolysis.