Quantitative methods for determining the points of O-(carboxymethyl) substitution in O-(carboxymethyl)-guar

Two methods are described for locating the O-(carboxymethyl) groups in O-(carboxymethyl)guar. In Method I, O-(carboxymethyl)guar was depolymerized by methanolysis, the O-(carboxymethyl) groups were reduced, and the mixture of methyl glycosides and O-(2-hydroxyethyl)-substituted methyl glycosides was converted into a mixture of per-O-acetylated alditols and partially O-(2-acetoxyethyl)ated, partially O-acetylated alditols. Analysis of these alditols by gas-liquid chromatography-mass spectrometry allowed the positions of substitution of the O-(carboxymethyl) groups on the galactosyl groups and mannosyl residues to be determined. However, this method did not distinguish between O-(carboxymethyl) substitution on 4-linked and 4,6-linked mannosyl residues. This limitation was overcome by the more-detailed analysis provided by Method II, in which O-(carboxymethyl)guar was carboxyl-reduced, the product methylated, the glycosyl residues hydrolyzed, the sugars reduced, and the alditols acetylated to yield a mixture of partially O-acetylated, partially O-methylated alditols and partially O-acetylated, partially O-(2-methoxyethyl)ated, partially O-methylated alditols. These derivatives, when separated and quantitated by g.l.c., and identified by g.l.c.-m.s., gave a quantitative measure of every type of carboxymethyl substitution in guar.     

Polymer-mediated cyclodehydration of alditols and ketohexoses

The polymer PEDOT⁺ (1 or 2) mediates a cyclodehydration reaction with alditols 3, 5, 7, 9, in hydrocarbon solvents, to give cyclic ethers 4, 6, 8, or 10, respectively, in high yield with a trivial isolation protocol. Polymers 1 or 2 also mediate the cyclodehydration of ketohexoses such as d-fructose, but not aldohexoses, to the important industrial intermediate 5-hydroxymethylfurfural (17), under milder conditions when compared to reactions mediated by mineral acids. A cascade reaction with ketohexoses is observed in toluene via cyclodehydration followed by Friedel–Crafts alkylation of the initially formed benzylic alcohol to give 16.     

Analysis of derivatives of carbohydrates by high-pressure liquid chromatography

The behaviour of benzoylated derivatives of alditols, monosaccharides, disaccharides, amino sugars, and methyl glycosides in high-pressure liquid chromatography (h.p.l.c.) has been investigated. A system was devised, using the most basic equipment of a single pump and fixed-wavelength u.v. detector, which gave good separations of the components of mixtures of derivatised methyl glycosides. Fractionation of complex mixtures of many of the other benzoylated carbohydrates was achieved in less than 30 min. The 4-nitrobenzoates were less useful for routine analyses.     

Determination of carbohydrates, sugar alcohols, and glycols in cell cultures and fermentation broths using high-performance anion-exchange chromatography with pulsed amperometric detection

Cell cultures and fermentation broths are complex mixtures of organic and inorganic compounds. Many of these compounds are synthesized or metabolized by microorganisms, and their concentrations can impact the yields of desired products. Carbohydrates serve as carbon sources for many microorganisms, while sugar alcohols (alditols), glycols (glycerol), and alcohols (methanol and ethanol) are metabolic products. We used high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) to simultaneously analyze for carbohydrates, alditols, and glycerol in growing yeast (Saccharomyces cerevisiae) cultures and their final fermentation broths. Both cultures were grown on complex undefined media, aliquots centrifuged to remove particulates, and the supernatants diluted and directly injected for analysis. Pulsed amperometry allowed a direct detection of the carbohydrates, alditols, and glycols present in the cultures and fermentation broths with very little interference from other matrix components. The broad linear range of three to four orders of magnitude allowed samples to be analyzed without multiple dilutions. Peak area RSDs were 2-7% for 2, 3-butanediol, ethanol, glycerol, erythritol, rhamnose, arabitol, sorbitol, galactitol, mannitol, arabinose, glucose, galactose, lactose, ribose, raffinose, and maltose spiked into a heat-inactivated yeast culture broth supernatant that was analyzed repetitively for 48 h. This method is useful for directly monitoring culture changes during fermentation. The carbohydrates in yeast cultures were monitored over 1 day. A yeast culture with medium consisting primarily of glucose and trace levels of trehalose and arabinose showed a drop in sugar concentration over time and an increase in glycerol. Yeast growing on a modified culture medium consisting of multiple carbohydrates and alditols showed preference for specific carbon sources and showed the ability to regulate pathways leading to catalysis of alternative carbon sources.     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

Partitioning of 14C-labeled photosynthate to allelochemicals and primary metabolites in source and sink leaves of aspen: evidence for secondary metabolite turnover

Theories on allelochemical concentrations in plants are often based upon the relative carbon costs and benefits of multiple metabolic fractions. Tests of these theories often rely on measuring metabolite concentrations, but frequently overlook priorities in carbon partitioning. We conducted a pulse-labeling experiment to follow the partitioning of ¹⁴CO₂-labeled photosynthate into ten metabolic pools representing growth and maintenance (amino acids, organic acids, lipids plus pigments, protein, residue), defense (phenolic glycosides, methanol:water and acetone-soluble tannins/phenolics), and transport and storage (sugars and starch) in source and importing sink leaves of quaking aspen (Populus tremuloides). The peak period of ¹⁴C incorporation into sink leaves occurred at 24 h. Within 48 h of labeling, the specific radioactivity (dpm/mg dry leaf weight) of phenolic glycosides declined by over one-third in source and sink leaves. In addition, the specific radioactivity in the tannin/phenolic fraction decreased by 53% and 28% in source and sink leaves, respectively. On a percent recovery basis, sink leaves partitioned 1.7 times as much labeled photosynthate into phenolic glycosides as source leaves at peak ¹⁴C incorporation. In contrast, source leaves partitioned 1.8 times as much ¹⁴C-labeled photosynthate into tannins/phenolics as importing sink leaves. At the end of the 7-day chase period, sink leaves retained 18%, 52%, and 30% of imported ¹⁴C photosynthate, and labeled source leaves retained 15%, 66%, and 19% of in situ photosynthate in metabolic fractions representing transport and storage, growth and maintenance, and defense, respectively. Analyses of the phenolic fractions showed that total phenolics were twice as great and condensed tannins were 1.7 times greater in sink than in source leaves. The concentration of total phenolics and condensed tannins did not change in source and sink leaves during the 7-day chase period. Received: 31 July 1998 / Accepted: 8 February 1999     

The Fate of l-Phenylalanine Fed to Germinating Pea Seeds, Pisum sativum (L.) var. Alaska, during Imbibition

Radioactive l-phenylalanine-l-¹⁴C or -U-¹⁴C was fed to pea seeds during imbibition. More than 95% was imbibed. Less than 1% of the radioactivity was respired as CO₂. Of the radioactivity taken into the embryos, 80% was still in the cotyledons by 3 days. About half of this was unchanged phenylalanine: 5% free, 10 to 20% in soluble proteins, 1 to 6% in cell wall proteins, and 14% released by mild acid hydrolysis. No other radioactive amino acid was found. About 0.3% of the radioactivity was identified as free caffeic, ferulic, and coumaric acids or their glycosides, and a further 5% was released by mild acid hydrolysis into a phenolic acid fraction. About half of the radioactivity in the cotyledons was lost in the fractionation procedures.     

Biosynthesis of heterocyst glycolipids of Anabaena cylindrica

The incorporation of sodium acetate-[1-¹⁴C] into the heterocyst glycolipids of Anabaena cylindrica cultures from 60–234 hr old is reported. Incorporation of radioactivity was maximal in 88 hr old cultures. In 60 hr and 88 hr cultures about 90 % of the radioactivity of the heterocyst glycolipids was found in the non-saponifiable glycolipid fraction, whereas in older cultures this fraction contained only 75 % of the radioactivity. Acid hydrolysis of non-saponifiable heterocyst glycolipid fractions showed that in 60 hr cultures, 81 % of the radioactivity occurs in the lipid moiety, whereas in older cultures a greater proportion (40–53 %) of the radioactivity was found in the sugar residue. The lipid fraction obtained by acid hydrolysis contained a mixture of labelled long chain mono-, di- and trihydric alcohols. In young (60 hr) cultures the primary alcohol fraction was most heavily labelled (57.3 % of the radioactivity in the non-saponifiable glycosides) with much smaller amounts in the diol and triol (8.4 and 15.1 % respectively), whereas in older cultures (234 hr) the primary alcohol (23.6 %) diol (22.5 %) and triol (18.9 %) fractions contained ca equal amounts of radioactivity.     

Chloroplasts Isolated from Kidney Bean Leaves Are Capable of Phenolic Compound Biosynthesis

The homogenate and chloroplast fractions isolated from the leaves of 10–14-day-old kidney-bean (Phaseolus vulgaris L.) seedlings were incubated with ¹⁴C-L-phenylalanine for 30 min in the light, and the incorporation of radioactivity into phenolic compounds was determined. Label incorporation into phenolic compounds of the homogenate and chloroplast fractions amounted to 15–17 and 4–5% of the introduced radioactivity, respectively. The chloroplasts were about an order of magnitude higher than the homogenate in the specific radioactivity of phenolic compounds. Chloroplasts contained four flavonol glycosides (kaempferol and quercetin aglycones), which were the major components of soluble phenolic compounds of leaves. It was concluded that kidney-bean leaf chloroplasts were capable of performing phenolic compound biosynthesis.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Izumoring: a strategy for bioproduction of all hexoses

Izumoring is a new concept for the bioproduction of all hexose sugars - 16 aldohexoses, eight ketohexoses and 10 hexitols -- using enzymatic and microbiological reactions. The biocatalysts necessary for the strategy were (i) d-tagatose 3-epimerase [which epimerizes all ketohexoses at C-3 of the sugar], (ii) polyol dehydrogenases [which catalyze oxidation-reduction reactions between ketohexoses and the corresponding hexitols], and (iii) aldose isomerases [which catalyze isomerization reactions between aldohexoses and corresponding aldoketoses]. All ketohexoses, aldohexoses and hexitols may be arranged in a symmetric ring connected by the biochemical reactions, allowing the design for the bioproduction of all 34 hexose sugars. The ring shows there are four entrances to the l-hexose world from the natural d-hexoses. The Izumoring ring shows clearly the relationship and the position of all 34 six carbon sugars.     

Effect of hexachlorobiphenyl on vitamin A homeostasis in the rat

Vitamin A status and turnover were examined in rats that had been exposed to chronic dietary treatment of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 1 mg/kg diet. HCB caused hepatic depletion and renal accumulation of vitamin A, and a 1.7-fold increase in the serum retinol concentration. Intravenously administered [3H]retinol bound to retinol binding protein-transthyretin complex (RBP-TTR complex) was used to study the dynamics of circulatory retinol in these rats. In HCB-treated rats, the plasma turnover rate of retinol was increased compared to vitamin A-adequate untreated controls. HCB caused a 50% reduction of total radioactivity in liver, and, except for 0.5 h after the [3H]retinol-RBP-TTR dose, the specific activity of the hepatic retinyl ester pool was greater compared to control rats. The kidneys of HCB-treated rats accumulated radioactivity in the retinyl ester fraction. HCB also caused a 50% reduction in adrenal radioactivity compared with control rats. Urinary and fecal excretion of radioactivity was 3-fold higher in HCB-treated rats as compared to controls. Our findings demonstrate that chronic HCB feeding results in expansion of plasma vitamin A mass, in changes of liver and kidney retinol and retinyl ester pool dynamics and in an increased metabolism of vitamin A.     

Prebiotic sugar synthesis: Hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide

Summary Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 MM dl-glyceraldehyde to ketohexoses at 25°C (pH 5–6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.     

Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber discs

The accumulation of glycoalkaloids that normally takes place in aerobically incubated potato ( Solanum tuberosum L.) tuber discs has been found to be inhibited by the ethylene-releasing substance ethephon. Using ethephon and the ethylene action inhibitor norborna-2,5-diene, the effect of ethylene on the synthesis of sterols and glycoalkaloids, which partly share their biosynthetic pathway, was investigated.
Control discs showed incorporation of (2-¹⁴C)mevalonic acid into free sterols, steryl esters, steryl glycosides and acylated steryl glycosides at 24 h, thereafter the radioactivity decreased in free sterols and steryl esters concomitant with the appearance of radioactivity in glycoalkaloids. Discs with ethephon additions contained more radioactivity in all sterol classes at all time-points, but no glycoalkaloids were formed.
The enzyme S-adenosyl- l -methionine:sterol C24 methyltransferase (SMT, EC 2. 1. 1. 41), located at one presumed branching point in the sterol and glycoalkaloid pathway, was characterized and found to exhibit similar characteristics as in other plants, but a lower specific activity. The activity of SMT increased in ageing tuber discs and this increase was further stimulated by ethephon, but inhibited by norborna-2,5-diene. The activity of the glycoalkaloid-specific enzyme UDP-glucose:solanidine glucosyltransferase (EC 2. 4. 1) also increased after slicing, but here ethephon additions counteracted the induction. The activity of the sterol-specific UDP-glucose:sterol glucosyltransferase (EC 2. 4. 1) was unaffected by either tuber slicing or ethephon additions.
The results indicate that ethylene stimulates sterol synthesis in wounded potato discs, and that the wound-induction of SMT is regulated by ethylene.     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

Evidence that the acidic polysaccharide secreted by Agrobacterium radiobacter (ATCC 53271) has a seventeen glycosyl-residue repeating unit.

The extracellular anionic polysaccharide produced by the bacterium Agrobacterium radiobacter (ATCC 53271) contains D-galactose, D-glucose, and pyruvic acid in the molar ratio 2:15:2. Analysis of the methylated polysaccharide indicated the presence of terminal, non-reducing glucosyl, 3-, 4-, 6-, 2,4-, and 4,6-linked glucosyl residues, 3-linked 4,6-O-[(S)-1-carboxyethylidene]glucosyl residues, and 3-linked galactosyl residues. Partial acid hydrolysis of the methylated polysaccharide, followed by reduction with NaB2H4 and then O-ethylation, gave a mixture of alkylated oligoglycosyl alditols that were separated by reversed-phase h.p.l.c. and analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. Smith degradation of the polysaccharide gave three diglycosyl alditols that were separated by semi-preparative, high-pH anion-exchange chromatography, and were analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The polymer obtained by NaBH4 reduction of the periodate-oxidized polysaccharide was methylated, and the noncyclic acetals were hydrolyzed with aq. 90% formic acid to generate a mixture of partially O-methylated mono- and di-glycosyl alditols. The partially O-methylated oligoglycosyl alditols were O-ethylated. The resulting alkylated oligoglycosyl alditols were separated by reverse-phase h.p.l.c. and then characterized by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The results from the studies described here provide strong evidence that the acidic polysaccharide secreted by A. radiobacter (ATCC 53271) has a heptadecasaccharide repeating unit.     

Sulfur metabolism in Thiorhodaceae. IV. Assimilatory reduction of sulfate byThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711, andChromatium strains 1611 and 6412 can grow with molecular hydrogen replacing sulfide as the electron donor. Sulfate suffices as the sulfur source. The incorporation of radioactive sulfur from³⁵S-sulfate was measured in growing cells in which molecular hydrogen or acetate was the electron donor. In cells pre-grown in sulfide, the incorporation of radioactivity began slowly after a lag period; in contrast, cells grown in sulfate took up the marker at a faster rate and without a lag. The radioactivity appeared in protein as cysteine and methionine. No elimination of sulfide was detected during growth. Thus, the reduction of sulfate was purely assimilatory.     

Regioselective synthesis of alditol vicinal bis-cyclic thionocarbonates via alditol stannylene acetal complexes as a short and efficient route to alpha,omega-diiodoalditol derivatives.

The bis-cyclic thionocarbonates of alditols (pentitols and hexitols) were quickly and easily obtained from alditol stannylene complexes and phenyl chlorothionoformate (PhOC(S)Cl) in good yields. Acetylation of isolated free alditol bis-thionocarbonates and subsequent iodination using methyl iodide under pressure led to alpha,omega-diiodo derivatives of alditols in good to excellent isolated yields (67-93%).     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.     

Studies on a microchemical method for the determination of the degree of polymerization of neutral oligo- and polysaccharides. I. Quantitative separation of trace amounts of alditols from mixtures with a large excess of monosaccharides.

A method has been devised for the quantitative separation of trace amounts of alditols from mixtures with large amounts of monosaccharides, using a strongly basic ion-exchange resin. Ten kinds of common reducing monosaccharides used were strongly retained by a very basic anion-exchange resin in the hydroxyl form, whereas the corresponding alditols showed no significant affinity for the basic resin. Model studies showed excellent recoveries of alditol from known mixtures of alditol and the corresponding aldose at ratios in the range from 1 : 1X10(3) TO 1 : 1X10(4) (by weight). Application of this procedure to dextrans after reduction and hydrolysis resulted in quantitative separation of the terminal alditol.