The configuration of 2,6-diamino-3-hydroxypimelic acid in microbial cell walls

beta-Hydroxydiaminopimelic acid, together with some diaminopimelic acid, occurs in the cell-wall mucopeptide of certain Actinomycetales. These components were converted into their di-DNP derivatives and separated by chromatography. Hence the relative proportions present in the cell walls of a number of species were measured. The problem of acid-induced inversion of configuration was studied. Of the diaminohydroxypimelic acids isomer B (see Scheme 2; amino groups meso, hydroxy group threo to its neighbouring amino group) always predominated but a small proportion of isomer D (amino groups l, hydroxy group erythro) also occurred. The configuration of the diaminohydroxypimelic acids was determined by periodate oxidation to glutamic gamma-semialdehyde, which underwent spontaneous ring-closure. Reduction with sodium borohydride produced optically active proline, the configuration of which was determined by direct measurement of the optical rotation of DNP-proline. Un-cross-linked diaminohydroxypimelic acid in the cell wall was oxidized with periodate in the presence of ammonia. Since the remaining amino group was bound in peptide linkage, ring-closure was prevented and borohydride reduction of the aldehyde-ammonia presumed to be present resulted in the formation of ornithine. The quantity of ornithine was used as a measure of the degree of cross-linking.     

Assay for lytic transglycosylases: a family of peptidoglycan lyases

An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel. Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time. Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis. The optimal conditions for this acid hydrolysis were established to be incubation at 96 degrees C for 1 h in 6 M HCl, in vacuo. The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer. To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12. The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment. On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid. This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa.     

2-C-carboxyaldoses and aldonic acids from cellobiose,maltose, and 4-O-methyl-d-glucose with 2-anthraquinone-sulfonic acid

Cellobiose, maltose, and 4-O-methyl-d-glucose were treated with 0.1–20mm 2-anthraquinonesulfonic acid in 0.1m sodium hydroxide at 40°. The hydroxy carboxylic acids formed were separated by ion-exchange, and analyzed by g.l.c.-m.s. as their per(trimethylsilyl) derivatives. The acidic oxidation products of cellobiose were further fractionated into aldonic acids and carboxylaldoses by ion-exchange chromatography. The isolated carboxyaldoses were reduced with sodium borohydride, and then analyzed by g.l.c.-m.s. before and after hydrolysis. The O-d-glucosyl- and O-methyl-substituted products of the sugars consisted of erythronic, arabinonic, ribonic, gluconic, and mannonic acids, in addition to 2-C-carboxypentoses. The nonsubstituted products of the reducing d-glucose unit were formic, glycolic, 2-deoxytetronic, and 3-deoxypentonic acids, and 2-C-carboxy-3-deoxypentoses.     

Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues.

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.     

A glycopeptide resistant to exoglycosidases

The carboxyl group of the terminal N-acetylneuraminic acid residue of the glycopeptide, prepared from α₁-acid glycoprotein by protease digestion, was esterified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then reduced with sodium borohydride. The reduced glycopeptide, thus prepared, containing the reduced N-acetylneuraminic acid, was resistant to hydrolysis by neuraminidase, and consequently to other exoglycosidases. The penultimate β-d-galactosyl residue of the oligosaccharide chain of the reduced glycopeptide was hydrolyzed by β-d-galactosidase only after the removal of the terminal, reduced, sialic acid by mild hydrolysis with acid. The reduced glycopeptide should be a useful substrate for the assay of endoglycosidases in the presence of exoenzymes. It should also find use as a carbon source in the growth of endoglycosidase-elaborating bacteria.     

Characterization of alkaline-degradation products of some 3,4-di- and 3,4,6-tri-methyl ethers of hexoses by G.L.C.-mass spectrometry of O-trimethylsilyl derivatives

G.l.c.-mass spectrometry has been used to provide information on the O-trimethylsilyl derivatives of the products of alkaline degradation of 3,4-di- and 3,4,6-tri-O-methyl-D-glucose, and 3,4,6-tri-O-methyl-D-galactose. During reaction with sodium hydroxide-sodium borohydride mixtures, reduction occurs more rapidly than β-elimination and the only detectable products were the corresponding alditols and the epimeric 3-deoxyalditols. Extended reaction with sodium hydroxide alone, followed by treatment with sodium borohydride, gives mixtures of aldonic acids including the epimeric 3-deoxy-4-O-methylaldonic acids (metasaccharinic acids), 3-deoxyaldonic acids (with loss of the 4-O-methyl substituent), and 3,4-dideoxy-aldonic acids. Possible reaction-pathways are discussed.     

Nature of nonenzymatically bound hexose in hemoglobin, albumin, and crystallin

Glucose incorporated in vitro during nonenzymatic glucosylation into albumin and hemoglobin was fully reducible by sodium borohydride unlike native albumin. Further, a prior hydrolysis under mild conditions (1 M oxalic acid:2 M HCl, 4 hr) was not required for in vitro incorporated glucose to yield maximal color intensity in the phenol-sulfuric acid reaction. Glucosyl-albumin, glucosyl-crystallin, and hemoglobin A1 behaved similarly in this respect. Hexose bound to HbA0 which alone showed an enhanced color intensity on prior acid hydrolysis was also not easily reduced by sodium borohydride. L-Cysteine (0.023 M) enhanced the color yield of glucosyl-hemoglobin, glucosyl-albumin, and glucosyl-crystallin to a lesser extent compared to fructose in the phenol-sulfuric acid reaction. Urea (6 M) also marginally increased the color intensity of glucosyl proteins and fructose.     

1,6-diamino-2,5-anhydro-1,6-dideoxy-l-iditol and some derivatives thereof

1,6-Diamino-2,5-anhydro-1,6-dideoxy-l-iditol (31) and its derivatives were synthesized, starting from 2,4-O-benzylidene-1,6-di-O-tosyl-d-glucitol. The 1,6-bis-(acetamido)-l-talo epoxide was readily hydrolyzed to the corresponding l-iditol derivative under anchimeric assistance of the 1-acetamido group. On treatment with formaldehyde-formic acid, diamine 31 gave a tricyclic, 1,4:3,6-bis(N,O-methylene) derivative which was stable under acidic conditions but, according to ¹³C-n.m.r. spectroscopy, was readily hydrolyzed to an equilibrium mixture in neutral, aqueous solution. The corresponding 1,6-bis(dimethylamino) derivative could be obtained by reducing this equilibrium mixture with borohydride. The different, quaternary salts obtained on methylation of the corresponding 1,6-bis(dimethylamino) derivatives with methyl iodide (aiming at the structure of epi-allo-muscarine) showed no muscarine-like, biological activity.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

Alginic acids in Lessonia vadosa: Partial hydrolysis and elicitor properties of the polymannuronic acid fraction

Extraction of Lessonia vadosa(Laminariales, Phaeophyta) collectedin three different localities near PuntaArenas in the south of Chile, with 3%aqueous sodium carbonate solutions gave asodium alginate yield in the range3.0–17.7% dry weight. There were markeddifferences in the mannuronic acid toguluronic acid ratio (M/G) (0.21–1.69) inthe alginic acids, samples collected inPuerto del Hambre in winter were composedmainly of mannuronic acid residues. Thealginate samples were characterized bypartial hydrolysis and FT-IR spectroscopy.No relationship was found between tissuetype and polyguluronic acid content. Theelicitor activity of the polymannuronicacid enriched fraction from alginic acid ofblades from Puerto del Hambre was assayedin wheat plants. The polymannuronic acidenriched fraction induced substancialelicitation of phenylalanine ammonia-lyase(PAL) and peroxidase (POD) activities.     

Release of fatty acids from virus glycoproteins by hydroxylamine

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.     

Brown seaweed species from Strangford Lough: compositional analyses of seaweed species and biostimulant formulations by rapid instrumental methods

The aims of this study were to characterise the composition of five seaweed species (Ascophyllum nodosum, Fucus serratus, Fucus vesiculosus, Laminaria hyperborea and Sargassum muticum), their extracts and commercial formulations, using thermogravimetry (TGA), energy dispersive X-ray microanalysis (EDX), Fourier-transform infrared spectroscopy (FTIR) and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). Analyses of the samples by TGA and EDX provided information on the proportions of algal cell wall, inorganic fractions and minerals. The main carbohydrate constituents of the five species and extracts were identified by their pyrolysis products, e.g. 1-(2-furanyl) ethanone, 5-methyl-2-furcarboxaldehyde, 2-hydroxy-3-methyl-2-cyclopenten-1-one, diannhydromannitol, 1,6-anhydromannopyranose and 1,6-anhydromannofuranose, using Py-GC/MS. The differences in relative intensities of the infrared bands of the five species were enhanced, especially after acid extraction compared with alkaline or neutral treatments, resulting in improved understanding of the compositional changes. In addition four commercial formulations and two acidic extracts of A. nodosum were evaluated for composition using the techniques. The dry matter, pH, electrical conductivity, ash, carbon and nitrogen content of the six preparations showed significant differences in composition. Variations in fatty acid, alginic acid, mannitol, laminarin and fucoidan content of the six formulations were reported. The results have shown that TGA, EDX, Py-GC/MS and FTIR are complementary techniques for rapid evaluation of seaweed materials and products.     

Evidence for a beta-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO₄ at pH 6.5 and then treated with sodium [³H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110°C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [³H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the β-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.     

Action of porcine-pancreatic amylase on oxidized-reduced amylose of low degree of modification

Amylose was oxidized with 0.1–0.2 mol of periodate per glucose residue (G), and then reduced with sodium borohydride or borotritide to give an oxidized-reduced amylose of low degree of modification. Mild acid hydrolysis gave erythritol, 2-O-α-d-glucosyl-l-erythritol, higher homologs, and other products. Extensive action of porcine-pancreatic amylase on the polymer gave, besides d-glucose and maltose, oligosaccharides containing one or more oxidized-reduced (modified, M), acyclic residues. The enzymic products containing only one oxidized-reduced residue were identified as a modified tetrasaccharide (MG₃) and a modified pentasaccharide (MG₄). Structures of MG₃ and MG₄ were identified by a combination of enzymic and chemical approaches. With glucoamylase, MG₄ was converted into MG plus d-glucose, whereas MG₃ was totally resistant. On mild acidic hydrolysis, MG₃ was converted into 2-O-α-d-glucosyl-d-erythritol plus maltose. These results indicate that MG₃ is G-M-G-G and that MG₄ is G-G-M-G-G. In principle, MG₄ could occupy the five d-glucose residue, substrate-binding site of porcine-pancreatic amylase in such a way that M, the acyclic structure replacing a d-glucose residue, is placed just to the “left” of the catalytic site. The modified structure, being very vulnerable to acidic hydrolysis, might then be expected to be very readily attacked by the amylase, but in fact, it is not.     

Synthesis of eicosa-2-trans-8,11,14-all cis-tetraenoic acid-3-14C and DL-3-hydroxy eicosa-8,11,14-all cis-trienoic acid-3-14C

A general procedure for the synthesis of 2-trans polyenoic fatty acids and of dl-3-hydroxypolyenoic acids is described. The 2-trans acids are prepared by LiAlH(4) reduction of a suitable polyenoic fatty acid ester to the alcohol, formation of the tosylate, oxidation to the aldehyde, and Doebner condensation of the latter with malonic acid. The 3-hydroxy acids are obtained by reaction of the acyl chloride of a suitable polyenoic acid with the sodium enolate of methyl acetoacetate and sodium methoxide to give the 3-keto ester, the keto group of which is reduced with sodium borohydride to the alcohol. These procedures were applied to the synthesis of eicosa-2-trans-8, 11, 14-all cis-tetraenoic acid-3-(14)C and DL-3-hydroxy eicosa-8, 11, 14-trienoic acid-3-(14)C.     

Synthesis of 1,4:5,8-dianhydro-octitol derivatives

The carbon chain of 1,6-dibromo-1,6-dideoxy-3,4-O-isopropylidene-d-mannitol was elongated via reaction with sodium cyanide to give the 1,6-dicyanide. Hydrolysis and esterification then gave dimethyl 2,7-dideoxy-4,5-O-isopropylidene-d-manno-octarate, treatment of which with 2,2-dimethoxypropane and an acid catalyst gave dimethyl 2,7-dideoxy-3,6:4,5-di-O-isopropylidene-d-manno-octarate. Reduction of the terminal methoxycarbonyl groups then gave 2,7-dideoxy-3,6:4,5- di-O-isopropylidene-d-manno-octitol, which was converted into 1,4:5,8-dianhydro- 3,6-diazido-2,3,6,7-tetradeoxy-l-manno- and -l-ido-octitol.     

Synthesis of 4-cyano- and 4-nitrophenyl 2,5-anhydro- 1,6-dithio-alpha-D-gluco- and -alpha-L-guloseptanosides carrying different substituents at C-3 and C-4

Treatment of 1,6:2,5-dianhydro-3,4-di-O-methanesulfonyl-1-thio-D-glucitol in methanol with sodium hydroxide afforded 1,6:2,5:3,4-trianhydro-1-thio-allitol, 1,4:2,5-dianhydro-6-methoxy-1-thio-D-galactitol, 1,6:2,5-dianhydro-4-O-methyl-1 -thio-D-glucitol, 1 ,6:2,5-dianhydro-3-O-methanesulfonyl-1 -thio-D-glucitol and 1 ,6:2,5-dianhydro-4-deoxy-1-thio-D-erythro-hex-3-ulose (14) in 5, 4, 28, 5.5 and 41% yield, respectively. Formation of these derivatives can be explained via a common sulfonium intermediate. Reduction of 14 with sodium borohydride and subsequent acetylation afforded 3-O-acetyl-1,6:2,5-dianhydro-4-deoxy-1-thio-D-xylo-hexitol, the absolute configuration of which was proved by X-ray crystallography. The 1,6:2,5-dianhydro-1-thio-D-hexitol derivatives in which the free OH groups were protected by acetylation, methylation or mesylation were converted by a Pummerer reaction of their sulfoxides into the corresponding 1-O-acetyl hexoseptanose derivatives which were used as donors for the glycosidation of 4-cyano- and 4-nitrobenzenethiol, respectively. The Pummerer reaction of 1,6:2,5-dianhydro-4-deoxy-3-O-methyl-1-thio-D-xylo-hexitol S-oxide gave, besides 1-O-acetyl-2,5-anhydro-3-deoxy-4-O-methyl-6-thio-alpha-L- (23) and 1-O-acetyl-2,5-anhydro-4-deoxy-3-O-methyl-6-thio-alpha-D-xylo-hexoseptanose (25), 1-O-acetyl-4-deoxy-2,6-thioanhydro-D-lyxo-hexopyranose, formed in a rearrangement reaction. The same rearrangement took place, when a mixture of 23 and 25 was used as donor in the glycosidation reaction with 4-cyanobenzenethiol, applying trimethylsilyl triflate as promoter. The oral antithrombotic activity of the obtained alpha-thioglycosides was determined in rats, using Pescador's model.     

Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin

Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.     

Analysis of glycated amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives

A method has been developed for the analysis of hexitolamino acids formed by acid-catalyzed hydrolysis of nonenzymatically glycated proteins that have been treated with sodium borohydride. The hexitolamino acids are converted into phenylthiocarbamyl (PTC) derivatives which are analyzed by reverse-phase HPLC. The PTC derivatives of N alpha-hexitolamino acids behave like lactones, migrating on the column more slowly than the corresponding PTC-amino acids. The PTC derivatives of N epsilon-glucitol- and N epsilon-mannitol-lysine are probably free acids, since they migrate faster than PTC-lysine. The method, which can be used to determine the degree of glycation of N-terminal and lysyl residues, has been applied successfully to human hemoglobin, serum albumin, and ocular lens proteins.     

4-O-α-d-galactopyranosyl-d-galactose: Efficient synthetic routes from “polygalacturonic acid”

Enzymic hydrolysis of “polygalacturonic acid” gave a mixture of oligomers which was fractionated by ion-exchange chromatography. The resulting di- and tri-saccharides were treated, respectively, with methanol and ethylene oxide, and the resulting esters were reduced with sodium borohydride. Treatment of the products with acetic anhydride and sulfuric acid, followed by deacetylation, produced the title compound.