Automated hypoiodite oxidation of carbohydrates

An automated system is described for the hypoiodite oxidation of aldoses and substituted aldoses to the corresponding aldonic acids. Automated determination of the glyoxylic acid and formaldehyde obtained on oxidation with periodate enables the 3-O-, 4-O-, and 6-O-substituted aldonic acids to be distinguished. The method is applied to the analysis of oligosaccharides in column eluates.     

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]⁻ together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]⁺), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.     

Gas chromatographic determination of polyols and aldoses in human urine as polyacetates and aldononitrile polyacetates

Thirty urinary collections from female and male neonates, juveniles, and adults have been analyzed by a new gas chromatographic (GC) method. Sixty meter high resolution open tubular glass capillary columns with a coating containing a dispersion of small particles of silanized silicic acid (Silanox) in SE-30 liquid phase were employed. Seventeen urinary polyols and aldoses were quantified through use of an internal reference compound and detector response factors. The components were analyzed as polyacetyl esters (from the polyols) and poly-O-acetylaldonic nitriles (from the aldoses).     

Resolution of the enantiomers of aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates

Acyclic diastereoisomers, namely, 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates are readily obtained by reductive amination of aldoses with chiral α-methylbenzylamine (MBA) in the presence of sodium cyanoborohydride, and may be separated by reversed-phase 1.c. and, more effectively, by adsorption 1.c. According to this procedure, enantiomers of the common, neutral aldoses are resolved. In adsorption 1.c., l-l_* [defined as an adduct of an l-aldose and l-(-)-MBA] is eluted before d-l_* for erythrose, xylose, ribose, glucose, 4-O-methylglucose, galactose, and fucose, and the elution order is the reverse for arabinose, lyxose, mannose, rhamnose, and glyceraldehyde. This behavior is probably related to the configuration of C-2 of the aldoses.     

Carbon-13-enriched carbohydrates. Preparation of erythrose,threose, glyceraldehyde,and glycolaldehyde with 13C-enrichment in various carbon atoms

Two-, three-, and four-carbon aldononitriles were prepared, and catalytically reduced with palladium-barium sulfate (5%) to the coresponding aldoses in high yields at pH 1.7 ± 0.1 and atmospheric pressure. Carbon-13-enriched glycolaldehyde, glyceraldehyde, erythrose, and threose were prepared with enrichment in various carbon atoms, permitting unequivocal assignment of chemical shifts for all carbons and determination of the proportions of cyclic hemiacetals and linear gem-diol forms in solution. Carbon-carbon and carbon-hydrogen coupling-constants for the furanose ring and linear hydrates of these short-chain aldoses are reported and discussed.     

The mass spectra of some per-O-acetylaldononitriles

The mass spectra of some per-O-acetylaldono- and per-O-acetyldeoxyaldononitriles have been recorded. The major fragmentation-pathways are discussed in terms of the application of electron-impact mass-spectrometry to structural studies of aldoses. Both acetyl and deuterium-labelled acetyl derivatives are included. The spectra are useful in verifying the position of the deoxy group(s) of deoxyaldoses.     

Identification of ketoses by use of their peracetylated oxime derivatives: A G.L.C.-M.S. approach

A method based on peracetylated oxime (PAKO) derivatives has been developed for rapid g.l.c.-m.s. survey of ketoses. This derivatization procedure (and the chromatographic analysis of these derivatives) is identical to one previously employed to identify aldoses by means of peracetylated aldononitrile (PAAN) derivatives. The production of chemically different derivatives from the aldoses and ketoses by the same derivatization procedure greatly simplifies the chromatographic separation of the derivatives of the ketoses from those of the aldoses, and also results in distinctively different, mass-spectral fragmentation-pathways for the two sets of derivatives. Both the electron-impact (e.i.) and ammonia chemical-ionization (c.i.) mass spectra of PAKO derivatives have been examined. Extensive differences between the fragmentation-pathways of the PAAN and the PAKO derivatives have been observed both by e.i.m.s. and ammonia c.i.m.s. The g.l.c.-m.s. of these PAKO derivatives, in conjunction with various, isotopic variants of the derivatization process, can yield extensive structural information with regard to the starting saccharides associated with the known, or unknown, g.l.c. peaks. The g.l.c. and mass-spectral properties of highly O-methylated PAKO derivatives of d-fructose are compared, and contrasted, to those of the PAKO derivatives of non-O-methylated saccharides. The chromatographic properties of derivatives of oligosaccharides that result from the PAAN-PAKO derivatization procedure have also been studied.     

In silico studies on the substrate specificity of an l-arabinose isomerase from Bacillus licheniformis

l-Arabinose isomerase (BLAI) from Bacillus licheniformis was found to be active only with l-arabinose, unlike other l-arabinose isomerases (l-AIs) active with a variety of aldoses. Therefore, the differences in molecular interactions and substrate orientation in the active site of l-AIs have been examined and the residue at position 346 is proposed to be responsible for the unique substrate specificity of BLAI.     

Aldose-ketose interconversion in pyridine in the presence of aluminium oxide

The reaction rate of the Lobry de Bruyn-Alberda van Ekenstein transformation of aldoses to ketoses in boiling pyridine was strongly increased by the addition of aluminium oxide. In addition to aldose-ketose transformation, 2-epimers of the starting aldoses and 3-epimers of the primarily produced ketoses were formed to some extent, as reported also when these reactions are carried out without aluminium oxide. The relative amounts of the primary ketose and the starting aldose in the reaction mixtures may be explained on the basis of their stability, predicted from reported free energy calculations. Isomerisation of ketoses to aldoses was much slower than the reverse reaction. The relative free energies are also in these cases important, the very stable xylo-2-hexulose gave only 7% and 6% of the aldoses gulose and idose, respectively, after boiling for 7h in pyridine in the presence of aluminium oxide.     

Carbon-13-enriched carbohydrates. Preparation of aldononitriles and their reduction with a palladium catalyst

Cyanide condenses with aldoses at 25° in aqueous solution between pH 7.0–9.0 to produce aldononitriles in high yield. These nitriles may be reduced catalytically over palladium-barium sulfate (5%) at pH 4.2 ± 0.1 and 25° to yield the corresponding aldoses in 60–90% yield, depending on the structure of the nitrile. 1-Amino-1-deoxyalditols are produced in approximately 10% yield, and their formation is favored when hemiacetal formation is hindered in the parent aldose. Generally, the product epimeric aldoses can be separated from contaminating by-products and from each other by ion-exchange and adsorption chromatography. This procedure has been applied to the preparation of [1-¹³C]-enriched pentoses and hexoses.     

Kinetics and mechanism of oxidation of some aldoses by vanadium(v) in perchloric acid medium

The kinetics of oxidation of some aldoses by vanadium(V) in perchloric acid media have been investigated. Each reaction is first order with respect to both [Vanadium(V)] and [Aldose]. The reactions are catalysed by acid. The addition of sodium perchlorate accelerates the rate of reaction. Kinetic evidence for the formation of an intermediate compound between vanadium(V) and aldoses is insignificant, and a mechanism is suggested in which vanadium(V) reacts with the aldoses by a fast step to form a transition state, followed by the decomposition of the latter to give the products of reaction in a slow step. The formation of free-radical intermediates has been demonstrated, and one-electron reduction of vanadium(V) by aldoses seems to be the most plausible mechanism. The oxidation rates follow the order: xyloses arabinose galactose mannose. The activation parameters are reported.     

A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.     

Purification,characterization and structure analysis of NADPH-dependent d-xylose reductases from Candida tropicalis

NADPH-dependent d-xylose reductases (XRs) from Candida tropicalis IFO 0618 were purified and characterized. Mono Q HPLC revealed three XR isomers. The K_m values of XR1, XR2 and XR3 for d-xylose were 37, 30 and 34 mM, and for NADPH 14, 18 and 9 μM, respectively. NADH did not act as a cofactor. The specificities of the three XRs for several aldoses were essentially the same. Gel filtration and cross-linking analysis showed that both XR1 and XR2 were dimers composed of identical subunits. The pI values of XR1 and XR2 were estimated to be 4.15 and 4.10, respectively. Comparison of the peptide maps of XR1 and XR2 showed that the molecular weights of 8 fragments of lysylendopeptidase-digested XR1 and XR2 were essentially the same as each other. The amino acid composition of each XR was also very similar. The molecular weights of XR1 and XR2 by mass spectra analysis were 36,497.91 and 36,539.68, respectively. The amino acid sequences of two XR1 peptide fragments (Nos. 4 and 5) were highly homologous with those of Pichia stipitis XR and mammalian aldose reductases.     

Interaction of vanadate with monosaccharides and nucleosides: a multinuclear NMR study

Proton, 13C and 51V nuclear magnetic resonance spectroscopy has been used to study the interaction of vanadate with several molecules containing more than one hydroxyl group, including various aldoses and nucleosides. The aldoses D-mannose and D-ribose mainly form tridentate complexes, of trigonal bipyramidal geometry, with vanadate at pH 7. These sugars use three consecutive hydroxyl groups, cis to each other, of their pyranose forms to bind vanadate in those cyclic triesters. Other aldoses, like D-glucose, which do not have this unique structural characteristic, do not form tridentate complexes, but can form weaker bidentate cyclic diesters using two consecutive pyranose cis hydroxyl groups. Of course, the pyranose forms of D-mannose and D-ribose, as well as the furanose form of D-ribose, also yield cyclic diesters of vanadate. All these aldoses form weak monodentate noncyclic monoesters of tetrahedral geometry using a single hydroxyl group. The nucleosides uridine, cytidine and adenosine form two complexes of trigonal bipyramidal geometry with vanadate. In these complexes, having 1:1 and 2:1 ligand-to-metal stoichiometries, the nucleosides form cyclic diesters with vanadate using their C2, and C3, hydroxyl groups.     

Decomposition process of organic matter derived from freshwater phytoplankton

We investigated the biodegradation process of freshwater phytoplanktonic organic matter using incubation experiments, with special reference to changes in three major biomolecules: neutral aldoses, amino acids and fatty acids. The concentration of neutral aldoses decreased drastically relative to amino acids and fatty acids during the early decomposition phase (days 0–7), owing largely to the rapid decomposition of storage carbohydrate. This resulted in a temporary decrease in the C/N ratio of organic matter. During the late phase (days 7–60), however, the rate of decrease in neutral aldoses slowed, while the considerable decrease in amino acids and fatty acids continued. The inconspicuous change in amino acid composition was probably due to the fact that no protein and/or peptide is composed of a limited species of amino acid. Although the compositional variety of organic matter among the phytoplankton was clearly observed at the start of decomposition, it became obscure in the course of 60 days. This indicates that while the organic composition of the labile fraction of phytoplanktonic organic matter varies depending on the phytoplankton groups, the refractory fraction has similar composition. The addition of bacterial organic matter is likely another reason for the similar composition of the remaining organic matter at day 60.     

Active transport into the human erythrocyte; evidence from comparative kinetics and competition among monosaccharides

1. Applicability of the previously postulated active transport system for conveying glucose into the human red cell was tested in connection with a number of related substances, comprising 6-carbon aldoses and ketoses, 5-carbon aldoses, and 5- or 6-carbon polyhydric alcohols. 2. The alcohols did not perceptibly penetrate the cells; all the sugars penetrated, the rates differing, but all of the same order of magnitude. 3. All the aldoses penetrated according to the pattern previously reported for glucose, in that the rate of penetration was not directly related to the gradient, but subject to some limiting factor. 4. The ketoses penetrated approximately according to the pattern of passive diffusion. 5. When present in equal concentrations, any aldose prevented or greatly delayed the entrance of a ketose, while the ketose did not perceptibly alter the rate of aldose uptake. Within each class, similar inhibitory relations were observed. 6. Penetration of all the sugars showed a high temperature coefficient and was inhibited by the mercuric ion or p-chloromercuribenzoate; certain of the sugars showed a residual degree of penetration not thus inhibitable. 7. Penetration of the ketoses was selectively inhibited by barbital. 8. These observations are interpreted in terms of simple equilibria between the various sugars and a hypothetical carrier molecule in the membrane, with which the sugars form a complex during their passage through the membrane. Comparisons between the sugars in relation to their reactions with the carrier system are indicated.     

General methods for enriching aldoses with oxygen isotopes

Synthetic methods are described for enriching 4-, 5-, and 6-carbon aldoses with oxygen isotopes. The general approach includes exchange between H2(1)8O and the aldehyde group of an aldose, exchange of O-1 onto C-2 of both of the 2-epimeric aldoses formed by molybdate-resin epimerization, and chain extension using cyanide addition. These methods make possible the production of all 16 aldohexoses enriched at 5 of the 6 oxygen atoms, all 8 aldopentoses enriched at 4 of the 5 oxygen atoms, and the four aldotetroses enriched at 2 of the 4 oxygen atoms. The general applicability of these methods is illustrated by the synthesis of a group of 22 different, 18O-enriched, biologically important D-aldoses having 4, 5, and 6 carbon atoms. The group includes D-[1-, 2-, 3-, 4-, O]glucose, D-[1-, 2-, 3-, 4-, and 6-18O]mannose, D-[1-, 2-, 3-, and 5-18O]arabinose, D-[1- abd 2- 18O] erythorose, and D-[1- and 2-18O]threose. The g.l.c.-m.s. characterization of these sugars with respect to the position and degree of 18O-enrichment is reported. The potential of the methods for producing aldoses having oxygen labels at multiple positions, or aldoses labeled simultaneously with oxygen, hydrogen, and carbon isotopes is discussed.     

An in vivo analysis of the energetics of aldose oxidation by Acinetobacter calcoaceticus

Summary A continuous culture study was made of the energetics of oxidation of various aldose sugars by Acinetobacter calcoaceticus LMD 79.41. The consumption of aldoses during carbon- and energy-limited growth of the organism on mixtures of acetate and an aldose was independent of the pH of the culture. Acid production, however, was strongly dependent on this parameter. It is shown that aldose consumption without concurrent acid production is due to formation of the corresponding lactone, the hydrolysis of which is pH-dependent. The cell yield of A. calcoaceticus on mixtures of acetate and glucose or xylose was much higher than during growth on acetate alone. This increase in cell yield was, however, dependent on the pH of the culture. Only at pH values which permitted a high rate of lactone hydrolysis an enhancement of the cell yield was observed. These results suggest that lactone hydrolysis has an important bearing on the efficiency of periplasmic oxidation of aldoses in bacteria.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.