Reaction kinetics and mechanism of acid-catalyzed anomerization of 1-O-acetyl-2,3,5-tri-O-benzoyl-l-ribofuranose

The mechanism of the acid-catalyzed anomerization of 1-O-acetyl-2,3,5-O-benzoyl-α- and -β-l-ribofuranoses in different acetic acid-acetic anhydride mixtures was investigated. The progress of the reactions was followed by NMR spectroscopy and the rate constants for the reactions were determined by the use of a kinetic model. The site of anomeric activation was clarified by the use of ¹³C-labeled acetic acid and acetic anhydride, respectively, proving that the anomerization takes place by exocyclic C-O cleavage, thus ruling out anomerization via acyclic intermediates. The role of the acetyl cation as the catalytically active species was further verified.     

Dowex-1-induced reactions of reduced nicotonamide adenine dinucleotide (NADH)

Dowex 1-formate has been found to cause both anomerization and oxidation of NADH, and when NADH is chromatographed on a column of this resin, the major products observed are NAD⁺ and αNAD⁺. Completing with the oxidation reaction is the conversion of NADH and α-NADH to unstable acid-modification products that subsequently break down during chromatography to give APD-ribose and and a variety of neutral and cationic degradation products. The effects of DOWEX 1-formate on NADH differ from those of acid as oxidation is minimal when NADH is incubated in acid solution, although anomerization, acid-modification, and degradation to ADP-ribose and other products readily occur. The neutral and cationic acid-degradation products that are formed from acid-modified NADH have been resolved by chromatography into 12 components, 6 of which react with 3-methyl-2-benzothiazolinone hydrazone and thus are identified as carbonyls. These substances gradually disappear from acid solution over a period of days and are replaced by polymeric pigments.     

Chromatographic methods for the determination of alpha- and beta-5-phospho-D-ribose-alpha-1-pyrophosphate pools in bacteria.

Different methods for the determination of 5-phospho-d-ribose-α-1-pyrophosphate (PRPP) are described. PRPP from ³²P-labeled microorganisms is determined directly either alone or together with the ribo- or deoxyribonucleosidetriphosphates in thin-layer chromatographic systems, using chemicals of high purity. As little as 0.1 pmol of both the α- and β-anomeric forms can be detected. α-PRPP is also determined by a new enzymatical radioactive microassay; the lower limit of detection is 1 pmol. An anomerization from α- to β-PRPP was found to take place during the application of the PRPP onto polyethyleneimine cellulose plates if formic acid or cell extract or both is added to α-PRPP. If the α-PRPP is purified from the cell extract before application, no anomerization will take place. It is also found that PRPP is stable in dilute organic acids. During accumulation of PRPP in purine-starved S. typhimurium, 10% of the PRPP was found as β-PRPP in vivo.     

Synthesis and enzymatic evaluation of phosphoramidon and its β anomer: Anomerization of α-l-rhamnose triacetate upon phosphitylation

A novel and efficient strategy for the synthesis of phosphoramidon and its β anomer has been developed by manipulating the anomerization of α-l-rhamnose triacetate upon phosphitylation. The experimental results suggest that proton transfer, bond rotation, and N atom are the key factors for the anomerization. The determined K_i and K_d values establish that phosphoramidon prepared by this method possesses excellent biological activity, and indicate that the contacts of rhamnose moiety with the enzyme have limited contribution to the binding.     

1,2-acylspiroorthoesters of 3,4,6-tri-O-acetyl-α-d-glucopyranose

The 1,2-O-(2-oxa-3-oxocyclopentylidene) derivative of 3,4,6-tri-O-acetyl-α-d-glucopyranose was prepared in both the exo (4) and endo (5) forms. The compounds were prepared by bromide-ion promoted cyclization of 3,4,6-tri-O-acetyl-2-O-(3-carboxypropanoyl)-α-d-glucopyranosyl bromide. The similar acylorthoester derivatives of phthalic acid were prepared from 3,4,6-tri-O-acetyl-2-O-(2-carboxybenzoyl)-α-d-glucopyranosyl bromide. The cyclizations produced a much higher ratio of the endo forms than would have been expected from their relative thermodynamic stabilities. The configurations were established by nuclear Overhauser enhancement studies and their conformations deduced from ¹H-n.m.r. parameters. The greater stability of the exo isomers appears to have a stereoelectronic origin. Preliminary efforts to engage the acylorthoesters in reactions with isopropyl alcohol to form glycosides are reported. It was discovered that a carboxylic acid provides powerful catalysis for the β to α anomerization of O-acetylated glucopyranosides by stannic chloride.     

Chemical and 13C-n.m.r. studies of an arabinogalactan from Larix sibirica L.

An arabinogalactan isolated from the wood of Larix sibirica L. was investigated by methylation analysis, partial hydrolysis, enzymic oxidation, and ¹³C-n.m.r. spectroscopy. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy were consistent with the data from methylation analysis. The polysaccharide is highly branched and similar in structure to those of arabino-3,6-galactans isolated from other Larix species.     

31P- and 13C-N.M.R.-spectral and chemical characterization of the end-group and repeating-unit components of oligosaccharides derived by acid hydrolysis of haemophilus influenzae type b capsular polysaccharide

Haemophilus influenzae type b capsular polysaccharide [repeating unit, →3)-β- d-Ribf-(1 → 1)- d-Ribol-5-(PO ₂H→] was partially hydrolyzed with HCl to give oligosaccharides that were isolated by size-exclusion chromatography, and then characterized by ³¹P- and ¹³C-n.m.r.-spectral and chemical methods, in order to determine the end-group composition and, hence, the number-average chain-length がL. The ratio (～17:8) of monophosphate end-groups to d-ribofuranose end-groups revealed the relative rates of hydrolysis of the phosphoric diester linkage and the glycosidic linkage in the repeating-unit structure. Cleavage of the phosphoric diester linkage was ～92% regioselective, as indicated by the ～ 12:1 ratio of d-ribofuranose monophosphate end-groups to d-ribitol monophosphate end-groups. The n.m.r. spectra of the oligosaccharide repeating-unit provided evidence for partial stereomutation (～3–8%) that involved rearrangement of the d-ribofuranose phosphoric diester linkage and anomerization at C-1 of d-ribofuranose. Variously sized oligosaccharides (がL = 4, 7, and 12) that had d-ribofuranose end-groups reacted with bovine serum albumin that had an average of ～9 adipyl hydrazide functionalities, to give, within experimental error, quantitative yields of the corresponding, hydrazone-linked, oligosaccharide-protein conjugates.     

Debenzylation of carbohydrate benzyl ethers and benzyl glycosides via free-radical bromination

The dealkylation of benzylated carbohydrates by free-radical bromination and hydrolysis has been further examined. Free-radical bromination of methyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside (1) methyl 2,3-di-O-benzyl-α-D-glucopyranoside (2) 6-O-benzyl-3,5-O-benzylidene-1,2-O-isopropylidene-α-D-glucofuranose (4) and 6-O-benzyl-D-glucose (3) appears to be quantitative. Spectroscopic evidence of a CBr bond indicates that an α-bromobenzyl ether is the product. Alkaline hydrolysis yielded methyl α-D-glucopyranoside from 1 and 2 and D-glucose from 3 and 4. A benzyl group present as an aglycon could be removed in the same way. Reaction of benzyl α-D-glucopyranoside tetraacetate (6) with bromine and chlorine under free-radical conditions and examination of the products by t.l.c. and i.r. spectrophotometry indicated that the first product was an α-halobenzyl glycoside and that the aglycon could be displaced by Br^- or Cl^- to form the tetra-O-acetyl-D-glucopyranosyl halide, undoubtedly with anomerization. Treatment of the mixture of products with moist ether and silver carbonate yielded only 2,3,4,6-tetra-O-acetyl-D-glucopyranose.     

Multiple conformations coexist and interchange in natural B DNA fibers

³¹P nuclear magnetic resonance (n.m.r.) of highly oriented NaDNA and LiDNA fibers was measured as a function of relative humidity over the range from 66% to 98%. The humidity dependence of the spectral patterns of NaDNA fibers shows that the A form has a single conformation while the B form has multiple conformations, and that interconversion between the A and B conformers in the transition region is slow compared to the n.m.r. time-scale (~10^?5 s). Two major conformations of the B form of LiDNA are found to be stable at low relative humidity and they rapidly interchange at high relative humidities. The spectral patterns of immobilized LiDNA are compatible with the single-crystal structure of double-stranded oligo nucleic acids.     

Synthesis and diazomethane-catalysed 1→2 acyl migration of the l-arabinosyl esters of N-acylamino acids

The fully benzylated α- and β-l-arabino-pyranosyl (1 and 2) and -furanosyl esters (3 and 4) of N-acetyl-d-alanine and N-tert-butoxycarbonyl-l-phenylalanine have been synthesised. Catalytic hydrogenation of 3 and 4 gave both anomers of 1-O-(N-tert-butoxycarbonyl-l-phenylalanyl)-l-arabino-pyranose (5) and -furanose (6), which were characterised as the triacetates 7 and 8, respectively. Treatment of the cis-oriented β-anomers of 5 and 6 with 0.5 equiv. of diazomethane at 0° for 1 h led to the 1→2 acyl rearrangement, with pyranose—furanose interconversion and anomerisation, to give, upon acetylation, a mixture of 1,3,4- and 1,3,5-tri-O-acetyl-2-O-(N-tert-butoxycarbonyl-l-phenylalanyl)-α,β-l-arabino-pyranose and -furanose, the structures of which were determined by ¹H- and ¹³C-n.m.r. spectroscopy. The 1→2 acyl-migration step in the l-arabino series is immediately followed by isomerisation into the four possible forms.     

Occurrence of diaminopimelic epimerase in maize

Extracts of maize leaves catalyzed the interconversion of meso-diaminopimelic acid its L-isomer. Three observations support the existence of this epimerase activity: (i) detection of the reversible interconversion of L-diaminopimelic acid and meso-diaminopimelic acid by paper chromatography after incubation of either isomer with extract; (ii) formation of [¹⁴C]CO₂ from L-[¹⁴C]diaminopimelic acid in an incubation mix containing meso-diaminopimelic acid decarboxylase; and (iii) inhibition of [¹⁴C]CO₂ evolution from L-diaminopimelic acid by unlabeled meso-diaminopimelic acid. The demonstration of the diaminopimelic acid epimerase lends support to the occurrence in plants of the complete diaminopimelic acid pathway for biosynthesis of lysine as it occurs in Escherichia coli and most bacteria.     

Iron-sulfur cluster dynamics in biotin synthase: A new [2Fe-2S] cluster

Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S]²⁺ cluster per monomer. However, active BioB contains in addition a [4Fe-4S]²⁺ cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S]¹⁺ cluster on reduction with dithionite and report the observation of a new [2Fe-2S]¹⁺ cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.     

Alternate g−g−. conformation for dinucleoside phosphates in solution

An alternative g^?g^? conformation (conformer I') for dinucleosides in solution has been deduced, based on potential energy calculations and nuclear magnetic resonance spectroscopy. This conformation is characterized by larger glycosidic torsional angles (X=94–111°) than those of conformer 1 (X=8–35°), although the other torsional angles are similar. There are thus four stable confonners (I, I', II and III) for dinucleosides in equilibrium with the open forms. The structure of conformer I' supports that of the ‘vertical’ double helix constructed by Olson (W.K. Olson. Proc. Natl. Acad. Sci. U.S.A. 74, (1977) 1775). Our data may suggest the possibility of interconversion between the vertical double helix and the regular double helix of A-form DNA, RNA or A'-form RNA.     

Treatment of peptidoglycan monomer with aqueous ammonia: Formation of lactoylpeptide and a saturated disaccharide

Treatment of peptidoglycan monomer (1) from Brevibacterium divaricatum with aqueous ammonia led to cleavage of the C-3 ether linkage in the N-acetylmuramoyl residue to give the d-lactoylpentapeptide and a saturated disaccharide. By using ¹³C-n.m.r. spectroscopy, the disaccharide was identified as chitobiosamine. Alkaline treatment of model compounds under similar conditions showed that N-acetylmuramoyl derivatives with C-1 unsubstituted undergo cleavage at C-3 to give the corresponding 2-acetamido-2-deoxy-d-glucopyranose derivative. The reaction of 1 with ammonia was monitored by ¹H-n.m.r. spectroscopy and, from the data obtained, rate constants and the activation energy were calculated.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

The interconversion of glycine and serine by plant tissue extracts

1. Extracts prepared from a variety of higher-plant tissues by ammonium sulphate fractionation were shown to catalyse the interconversion of glycine and serine. This interconversion had an absolute requirement for tetrahydrofolate and appeared to favour serine formation. 2. The biosynthesis of serine from glycine was studied in more detail with protein fractionated from 15-day-old wheat leaves. Synthesis of [¹⁴C]serine from [¹⁴C]glycine was not accompanied by labelling of glyoxylate, glycollate or formate. 3. The synthesis of serine from glycine was stimulated by additions of formaldehyde, and [¹⁴C]formaldehyde was readily incorporated into C-3 of serine in the presence of tetrahydrofolate. 4. The results are interpreted as indicating that serine biosynthesis involves a direct cleavage of glycine whereby the α-carbon is transferred via N⁵N¹⁰-methylenetetrahydrofolate to become the β-carbon of serine.     

The biosynthesis of C55 polyprenols by a cell-free preparation of Lactobacillus plantarum

A cell-free supernatant of lysates of Lactobacillus plantarum catalyses the synthesis of lipids from [2-¹⁴C]mevalonate. Of the added mevalonate, 7.5% is incorporated into lipids, which were fractionated into five components. About 4% of the radioactivity in these lipids co-chromatographs with compounds shown by mass spectrometry, n.m.r. and i.r. spectroscopy to be C₅₅ polyprenols, and about 2% co-chromatographs with a hexamer. The rest of the radioactivity is in more complex fractions. Analysis by mass spectrometry, n.m.r. and i.r. spectroscopy shows that the major C₅₅ polyprenol is undecaprenol, accompanied by an isomer containing one reduced isoprene unit. A Kuhn–Roth degradation of [¹⁴C]polyprenols indicates that the supernatant catalyses synthesis of these compounds de novo.     

Zn2+, Mg2+, and H+ binding to d-fructose 1,6-bisphosphate studied by 31P and 1H NMR

The anomeric composition and mutarotation rates of fructose 1,6-bisphosphate were determined in the presence of 100 mm KCl at pH 7.0 by ³¹P NMR. At 23 and 37 °C the solution contains (15 ± 1)% of the α anomer. The anomeric rate constants at 37 °C are (4.2 ± 0.4) s^?1 for the β → α anomerization and (14.9 ± 0.5) s^?1 for the reverse reaction. A D₂O effect between 2.1 and 2.6 was found. From acid base titration curves it appeared that the pK values of the phosphate groups range from 5.8 to 6.0. Mg²⁺ and Zn²⁺ bind preferentially to the 1-phosphate in the α-anomeric position. Zn²⁺ has a higher affinity for this phosphate group than Mg²⁺ has. At increasing pH the fraction α anomer decreases slightly. At increasing Mg²⁺/fructose 1,6-bisphosphate ratios the fraction α anomer increases till 19% at a ratio of 20. Proton and probably Mg²⁺ binding decreases the anomerization rate. The time-averaged preferred orientation of the 1-phosphate along the C₁O₁ bond of the α conformer is strongly pH dependent, gauche rotamers being predominant at pH 9.4. In the presence of divalent cations the orientation is biased toward trans. A mechanistic model is proposed to explain the Zn²⁺, Mg²⁺, and pH-dependent behavior of the gluconeogenic enzyme fructose 1,6-bisphosphatase.     

Monomer sequence and acetylation pattern in some bacterial alginates

The sequential structures and acetylation patterns of alginates from several strains of Azotobacter vinelandii and Pseudomonas species, including P. aeruginosa, P. putida, P. fluorescens, and P. mendocina, have been studied by ¹H-n.m.r. spectroscopy. O-Acetyl groups were exclusively associated with the d-mannuronic acid residues and the degree of acetylation varied in the range 4–5%, depending upon the proportion of this acid in the polymer. ¹H-N.m.r. spectroscopy of a naturally occurring and an artifically acetylated d-mannuronan made it possible to determine the degrees of acetylation at O-2, O-3, and O-2,3. The most conspicuous difference between alginates from A. vinelandii and the four Pseudomonas species was the complete absence of consecutive l-guluronic acid residues in the latter.