Pyruvic Acid Ketal Research Articles

Structural Diversity in Galactans From Red Seaweeds and Its Influence on Rheological Properties.

Galactans are important components of many plant cell walls. Besides, they are the major polysaccharides in extracellular matrixes from different seaweeds, and other marine organisms, which have an acidic character due to the presence of sulfate groups in their structures. In particular, most of the red seaweeds biosynthesize sulfated galactans with very special linear backbones, constituted by alternating (1→3)-β-d-galactopyranose units (A-unit) and (1→4)-α-galactopyranose residues (B-unit). In the industrially significant seaweeds as source of hydrocolloids, B-units belong either to the d-series and they produce carrageenans (as in the order Gigartinales), or to the l-series, and they are sources of agarose and/or structurally related polymers (i.e., Gelidiales, Gracilariales). In both cases, the latter units appear as cyclized 3,6-anhydro-α-galactose in certain amounts, which can be increased by alkaline cyclization of α-galactose 6-sulfate units. Besides, it has been clearly shown that some red algae produce different amounts of both galactan structures, known as d/l-hybrids. It is not yet clear if they comprise both diasteromeric types of units in the same molecule, or if they are mixtures of carrageenans and agarans that are very difficult to separate. It has been reported that the biosynthesis of these galactans, showing that the nucleotide transport for d-galactopyranose units is UDP-d-Gal, while for l-galactose, it is GDP-l-Gal, so, there is a different pathway in the biosynthesis of agarans. However, at least in those seaweeds that produce carrageenans as major galactans, but also agarans, both synthetic pathways should coexist. Another interesting characteristic of these galactans is the important variation in the sulfation patterns, which modulate their physical behavior in aqueous solutions. Although the most common carrageenans are of the κ/ι- and λ-types (with A-units sulfated at the 4- and 2-positions, respectively) and usually in agarans, when sulfated, is at the 6-position, many other sulfate arrangements have been reported, greatly influencing the functional properties of the corresponding galactans. Other substituents can modify their structures, as methyl ethers, pyruvic acid ketals, acetates, and single stubs of xylose or other monosaccharides. It has been shown that structural heterogeneity at some extent is essential for the proper functional performance of red algal galactans.

Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green alga Codium divaricatum

A sulfated polysaccharide, designated CP2-1, was isolated from the green alga Codium divaricatum by water extraction and purified by anion-exchange and size-exclusion chromatography. CP2-1 is a galactan which is highly sulfated and substituted with pyruvic acid ketals. On the basis of chemical and spectroscopic analyses, the backbone of CP2-1 was mainly composed of (1→3)-β-d-galactopyranose residues, branched by single (1→)-β-d-galactopyranose units attached to the main chain at C-4 positions. The degree of branching was estimated to be about 12.2%. Sulfate groups were at C-4 of (1→3)-β-d-galactopyranose and C-6 of non-reducing terminal galactose residues. In addition, the ketals of pyruvic acid were found at 3,4- of non-reducing terminal galactose residues forming a five-membered ring. CP2-1 possessed a high anticoagulant activity as assessed by the activated partial thromboplastin time and thrombin time assays. The investigation demonstrated that CP2-1 was an anticoagulant-active sulfated polysaccharide distinguishing from other sulfated polysaccharides from marine green algae.

Mutation in the pssM gene encoding ketal pyruvate transferase leads to disruption of Rhizobium leguminosarum bv. viciae-Pisum sativum symbiosis.

To study the question whether acidic exopolysaccharide (EPS) modification, e.g. pyruvylation, plays any role in the development of Rhizobium leguminosarum/Pisum sativum symbiosis. The amino acid sequence deduced from the pssM gene, localized within the pss (polysaccharide synthesis) gene locus, was shown to be homologous to several known and putative ketal pyruvate transferases, including ExoV from Sinorhizobium meliloti and GumL from Xanthomonas campestris. Rh. l. bv. viciae strain VF39 carrying a Km-cassette insertion into the pssM gene was obtained by the gene replacement technique. Knock-out of pssM led to the absence of the pyruvic acid ketal group at the subterminal glucose in the repeating unit of EPS as it was shown by (13)C and (1)H nuclear magnetic resonance (NMR) analysis. Complementation in trans restored the EPS modification in the pssM mutant. Disruption of the pssM gene resulted also in the formation of aberrant non-nitrogen-fixing nodules on peas. Ultrastructural studies of mutant nodules revealed normal nodule invasion and release of bacteria into the plant cell cytoplasm, but further differentiation of bacteroids was impaired, and the existing symbiosomes underwent lysis. PssM encodes ketal pyruvate transferase involved in the modification of the Rh. l. bv. viciae EPS. The absence of subterminal glucose pyruvylation in the EPS repeating units negatively influences (directly or indirectly) the formation of the nitrogen-fixing symbiosis with peas. Our finding that the absence of modification even at the single position of EPS is likely to be crucial for establishment of nitrogen-fixing symbiosis argues in favour of the idea concerning their specific signalling role in this process.

Galactans from Cryptonemia species . Part II: Studies on the system of galactans of Cryptonemia seminervis (Halymeniales) and on the structure of major fractions

Cryptonemia seminervis biosynthesizes a family of d, l-hybrid galactans based on the classical 3-linked β- d-galactopyranosyl→4-linked α- d- and α- l-galactopyranosyl alternating sequence (A-units→B-units) with major amounts of α- d- and α- l-galactose and 3,6-anhydro- d- and l-galactose and lesser percentages of 3,6-anhydro-2- O-methyl- l-galactose, 2- O-methyl-, 4- O-methyl- and 6- O-methylgalactoses. The dispersion of structures in this family is based on five structural factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and glycosyl side-chain (4- O-methyl galactopyranosyl and/or xylosyl); (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d, l-galactoses and d, l-3,6-anhydrogalactoses also in the B-units, (d) the formation of diads and (e) the sequence of the diads in the linear backbone. Considering these variables it is not unexpected to find in the fractions studied at least 18 structural units producing highly complex structures. Structural studies carried out in two major fractions (S2S-3 and S2S-4) showed that these galactans were formed mainly by β- d-galactopyranosyl 2-sulfate (20 and 11.9 mol %), β- d-galactopyranosyl 2-sulfate 4,6- O-(1′-carboxyethylidene) (8.9 and 6.0 mol %) and β- d-galactopyranosyl 2,6-sulfate (5.4 and 18.6 mol %), together with 3,6-anhydro-α- l-galactopyranosyl (11.4 and 7.3 mol %) and 3,6-anhydro-α- l-galactopyranosyl 2-sulfate (4.9 and 15.4 mol %) and minor quantities of 12–15 other structural units. Preparative alkaline treatment carried out on fraction (S2S-3) produced a quantitative formation of 3,6-anhydro α- l-galactopyranosyl units from precursor units (α- l-galactose 6-sulfate and α- l-galactose 2,6-sulfate). Kinetic studies on this 3,6-anhydro cyclization show a rate constant of 5.2 × 10 4 s −1 indicating diads of the type G→L6S/2,6S. Data from chemical, spectroscopic and kinetic studies suggest that, in S2S-3, the agaran block in the d, l-hybrid galactan is composed of the following diads: G(6R)→L6S/2,6S and G2S(P)(2,6S)→LA(2S)(2R)(2M) and the carrageenan block of G2S(P)→D(2S)(2,3S)(3S)(3,6S) in a molar ratio of agaran to carrageenan structures of ∼2:1.

Polysaccharides from the green seaweeds Codium fragile and C. vermilara with controversial effects on hemostasis

Codium fragile and Codium vermilara biosynthesize water-soluble sulfated arabinans and galactans (and/or sulfated arabinogalactans), α(1 → 4)- d-glucans and β(1 → 4)- d-mannans. The former polysaccharides are composed by 3-linked β- d-galactopyranose and β- l-arabinopyranose residues, they are highly sulfated and substituted with pyruvic acid ketals. For both seaweeds, they have the same main structural units, but in different percentages. All the room-temperature water extracts from both seaweeds showed a dual haemostatic effect: they prevented coagulation, but they induced platelet aggregation. Anticoagulant activity and platelet aggregation were higher in the samples with polysaccharides richer in sulfate, mainly in those from C. vermilara, which have a higher degree of sulfation and arabinose content.

The system of galactans from Cryptonemia crenulata (Halymeniaceae, Halymeniales) and the structure of two major fractions. Kinetic studies on the alkaline cyclization of the unusual diad G2S→D(L)6S

Cryptonemia crenulata biosynthesizes a family of dl-hybrid galactans that are based on the classical 3-linked β- d-galactopyranosyl→4-linked α-galactopyranosyl alternating sequence (A-units→B-units). The dispersion of structures in these galactans is based on four factors, namely: (a) the amount and position of substituent groups as sulfate (major), pyruvic acid ketals, methoxyl and side substituents of β- d-xylose and/or β- d-galactose; (b) the ratio galactose/3,6-anhydrogalactose in the B-units; (c) the ratio d-/ l-galactoses and 3,6-anhydrogalactoses also in the B-units and (d) the sequence of the diads in the linear backbone. Alkali treatment carried out on the major fraction produced a nearly quantitative formation of 3,6-anhydrogalactose units from precursor units (α-galactose 6-sulfate (major) and α-galactose 2,6-sulfate, minor). Kinetic studies show a rate constant, for the diad G2S-D(L) 6-S, of 1.7 × 10 4 s −1 indicating a reaction faster than in λ-carrageenans but slower than in porphyrans.

Behaviour of Polysaccharides Under Harsh Conditions

Abstract The implementation of EOR polymers for high salinity and high temperature reservoirs requires a detailed knowledge of their solution behaviour. This paper describes the results of comparativestudies of the most commonly used polysaccharides, i.e., xanthan and scleroglucan. Improved laboratory techniques, such as equipment for stability tests under anaerobic conditions and high-temperature low-shear viscometer, have recently been designed to characterize their behavior under field conditions. It has been shown that:The stability and good filterability of improved quality polymers arc maintained for several months at 90 ºC.The viscosity of polysaccharides is decreased only slightly by an increase in temperature to 90 ºC,From flow experiments and batch tests, adsorption levels on reservoir rocks decrease with temperature. Introduction The use of polymers in harsh reservoir conditions for EOR applications requires understanding their behaviour in high salinity brine at high temperature. The favourable candidates already known are polysaccharides such as xanthan and scleroglucan. Due to their. Structure in multistranded chains, which gives them molecular rigidity, their viscosities in concentrated brine are higher and less sensitive to multivalent ions than other EOR polymers such as polyacrylamide Moreover, their most interesting characteristic is their good performance at high temperature. The laboratory techniques described in this paper have been improved to analyze polymer solution behaviour under high temperature conditions (≥90 ºC). The stability and the filterability of polysaccharide solutions were studied in a controlled oxygen atmosphere. The rheological characteristics were determined by using a newly devised capillary viscometer. Adsorption on rocks was derived from both batch tests and core flow experiments. The overall results provide criteria for evaluating the respective advantages of both polysaccharides under given field conditions. Experimental Polymers The polymers used, i.e., xanthan and scleroglucan, are polysaccharides both obtained by fermentation process and provided as broths. Their characteristics are given in Table 1. Xanthan (XCPS) is a strong polyelectrolyte containing D-glucuronic acid units and pyruvic acid ketals(1). Scleroglucan (SGPS) is a nonionic polymer made up of linear glucose units branched at every' third residue(2). Polymer solutions were prepared by simple dilution of the broths in salt water. Then, they were clarified by filtration to remove any cells. Low-molecular-weight impurities were eliminated by ultrafiltration. Some solutions, called "high quality" in this paper, were extensively purified by a thermal treatment to remove impurities, especially proteins bound to the polymer molecules(3). This thermal treatment consisted in heating the ultrafiltered solution during 48 hours at 90 ºC and then if necessary, reultrafiltering the solution to eliminate the last impurities. The brine used are a sodium chloride solution or a reconstituted sea water containing 27.8g.l−1 NaCl, 9.54.g.l−1 MgCl22,6H2O, 1.62g.l−1 CaCl2,2H2O, 0.03g.l−1 SrCl22,6H2O, 7.73gl−1 Na2SO4, 10H2O. Procedure Polymer concentrations were determined with a Dohrmann DC80 carbon analyzer, which gives the total carbon content of solutions, and/or with the Technicon Auto Analyser System, which gives the glucose content

Effects of Thallus Age and Alkali Treatment on Pyruvic Acid Contents of Agars from the Red Alga Gracilaria chorda.

The pyruvic acid content of agars from Gracilaria chorda Holmes were analyzed in relation to thallus (agarophyte) age and alkali treatment. Its distribution in agar fractions obtained by sequential solvent extraction [cold water (22°C), boiling 99.5, 80, 60, 40, and 20% EtOH and water (100°C)] before and after alkali treatment of young, mature, and senescent thalli were compared. Pyruvic acid content increased with thallus age (young, 0.11%; mature, 0.17%; and senescent, 0.33%) indicating increasing substitution of the agar repeating unit with pyruvic acid ketal groups as the agarophyte grows. This substitution increased the solubility of agar molecules particularly in cold water. Alkali treatment did not affect pyruvic acid concentration; however, its distribution among the fractions changed after alkali treatment. Pyruvic acid concentration in the agar frac-tions ranged from 0.05 to 0.76% and from 0.08 to 1.25% before and after alkali treatment, respec-tively. The agars from G. chorda had a relatively lower pyruvic acid content than most of the commercial agars examined.

Affinity interactions between agarose and β-1,4-glycans: a model for polysaccharide associations in algal cell walls

This paper reviews the extensive and previously unpublished work on the interactions between agarose and 1,4-linked β- d-glycans carried out at Unilever Research, Colworth Laboratory, UK. The effect of the following variables is discussed: (i) galactose content of galactomannans; (ii) substitution patterns in the agarose molecule; (iii) structural variations in the 1,4-β- d-glycan main chain; and (iv) molecular size of the 1,4-β- d-glycans. Double helices of agarose, a non-substituted regular polysaccharide comprising 1,3-linked β- d-galactose and 1,4-linked 3,6-anhydro-α- l-galactose, bind in an ordered cooperative fashion to an extended ribbon ordered conformation of sequences of 1,4-linked β- d-mannopyranose residues in plant galactomannans to give mixed gelling systems. This interaction survives, in a modified form, substitution along the agarose molecule by O-methyl ether and O-sulphate esters at O6 of the d-galactose and O2 of the 3,6-anhydro- l-galactose, and 4,6-linked pyruvic acid ketal groups on the d-galactose. The higher the level of substitution on the agarose, the weaker the interaction with galactomannan. In general, the higher the level of galactose substitution in the galactomannan the lower the extent of interaction with agarose. Evidence is presented, however, which indicates that the fine structural distribution of galactose along the galactomannan molecule is also an important determinant for the co-gelling interaction. Substituted 1,4-linked β- d-glucomannans, β- d-glucans and β- d-xylans which can form closely similar extended ribbon order conformations to the galactomannans also participate in co-gelling interactions with agarose. These β- d-glycans are similar in structure to important skeletal polysaccharides such as hemicelluloses and cellulose. This suggests that the binding between agars and β- d-glycans might mimic biological cohesion between skeleton and gel phases in natural red seaweed cell walls. The sensitivity of the interactions studied to fine details of agar and β- d-glycan structure is what might be expected on biological grounds, since the wide and subtle variations of natural polysaccharide structure are presumed to represent a mechanism for control of their intermolecular interactions.

Synthetic media for production of quality xanthan gum in 20 liter fermentors

AbstractXanthan gum is a heteropolysaccharide synthesized by Xanthomonas campestris NRRL B‐1459 and is composed of D‐glucose, D‐mannose, and D‐glucuronic acid, in addition to acetic and pyruvic acids. Different amounts of pyruvic acid ketal are found in various preparations which can influence the viscosities of dilute xanthan solutions. Polysaccharide production on synthetic media was studied in small‐scale fermentors. Fermentation conditions were established for production of both high and low pyruvic acid gums (about 4 and 2% pyruvic acid, respectively). Low nitrogen [0.1% (NH4)2HPO4] and air (0.25 vol/liter/min) levels favor production of low pyruvate gum; increasing (NH4)2HPO4 to 0.15%, adding K2HPO4, and increasing the air flow to 1.5 vol/liter/min favored production of normal gum. Both processes gave xanthan yields of 50 to 60%, based on 2.5% initial D‐glucose substrate, in two to three days. Differences in pyruvic acid content and in the quantity of gum produced under a given set of conditions were attributed to strain variability. Substrains were isolated that have desirable characteristics for production of xanthan gum; i.e., the ability to give good yields of high‐pyruvate gum when grown on both complex and synthetic media.

The Structure of the Capsular Polysaccharide from Klebsiella K-Type 21

Methylation, periodate oxidation, and partial hydrolysis studies on the capsular polysaccharide, and on the carboxyl reduced polymer, of Klebsiella K21 show the structure to consist of a repeating unit.[Formula: see text]The anomeric linkages were determined by p.m.r. spectroscopy of isolated oligosaccharides and, in part, by specific enzymes. P.m.r. spectroscopy of the original polysaccharide in methyl sulfoxide-d6 showed clearly a ratio of one pyruvic acid ketal (CH3, τ 8.5) to five anomeric protons (τ 4.65–5.40).

The occurrence of pyruvic acid ketal in a glucan from a fungus, Microsporum quinckeanum.

A glucan containing 0.6% of pyruvic acid has been isolated from the mycelium of Microsporum quinckeanum. Methylation studies on the native glucan and on a depyruvated sample, proved that the pyruvic acid was present as a ketal and probably as a 4,6-ketal (4,6-O-1-carboxyethylidene) attached to nonreducing terminal units in the polysaccharide. The native glucan was capable of eliciting immediate-type cutaneous-hypersensitivity reactions in guinea pigs sensitized by cutaneous infection with M. quinckeanum and reacted with homologous antiserum but gave no reaction with type XXVII pneumococcal antiserum that contained antibodies directed towards the pyruvic ketal.

P. M. R. Spectroscopic Analysis of Bacterial Polysaccharides Containing Pyruvic Acid

Abstract The characteristic p.m.r. signal at τ 8.4–8.6 given by the CH3-C group of a pyruvic acid ketal (1-carboxyethylidene derivative)may be used to demonstrate the presence of such a substituent in polysaccharides. Similar signals are given when the pyruvic acid ketal is methylated or reduced. The method is convenient for monitoring purification procedures and chemical transformations of polysaccharides such as are involved in structural studies. In certain cases the molar ratio of pyruvic acid may be estimated.

Degradation of a pneumococcal type-specific polysaccharide with exposure of group-specificity.

Pyruvic acid is an immunological determinant of the quite rigorously type-specific capsular polysaccharide of pneumococcal type IV (S IV). Removal of pyruvic acid by mild hydrolysis converts the capsular polysaccharide of type IV into an analog of the pneumococcal group-specific C-substance. Depyruvylated S IV resembles C-substance so closely immunologically that it not only precipitates a high proportion of the anti-C in antipneumococcal sera, regardless of their immunological types, but also, like C, precipitates human C-reactive protein in the presence of calcium ions. Apparently, the removal of pyruvic acid ketal rings from adjacent sugars unmasks N-acetylgalactosamine residues which must be linked and spaced much as are those in C-substance. Groupings reactive with suitably linked N-acetylgalactosamine, therefore, appear to be located on the surfaces of molecules of human C-reactive protein.

Structural analysis of colanic acid from Escherichia coli by using methylation and base-catalysed fragmentation. Comparison with polysaccharides from other bacterial sources.

Essentially the same methanolysis products were obtained after methylation of the slime and capsular polysaccharides from Escherichia coli K12 (S53 and S53C sub-strains) and the slime polysaccharides from E. coli K12 (S61), Aerobacter cloacae N.C.T.C. 5290 and Salmonella typhimurium SL1543. These were the methyl glycosides of 2-O-methyl-l-fucose, 2,3-di-O-methyl-l-fucose, 2,3-di-O-methyl-d-glucuronic acid methyl ester, 2,4,6-tri-O-methyl-d-glucose, 2,4,6-tri-O-methyl-d-galactose and the pyruvic acid ketal, 4,6-O-(1'-methoxycarbonylethylidene)-2,3-O-methyl-d-galactose. All were identified as crystalline derivatives from an E. coli polysaccharide. The structure of the ketal was proved by proton-magnetic-resonance and mass spectrometry, and by cleavage to pyruvic acid and 2,3-di-O-methyl-d-galactose. All these polysaccharides are therefore regarded as variants on the same fundamental structure for which the name colanic acid is adopted. Although containing the same sugar residues, quite different methanolysis products were obtained after methylation of the extracellular polysaccharide from Klebsiella aerogenes (1.2 strain). The hydroxypropyl ester of E. coli polysaccharide, when treated with base under anhydrous conditions, underwent beta-elimination at the uronate residues with release of a 4,6-O-(1'-alkoxycarbonylethylidene)-d-galactose. Together with the identification of 3-O-(d-glucopyranosyluronic acid)-d-galactose as a partial hydrolysis product, this establishes the nature of most, if not all, of the side chains as O-[4,6-O-(1'-carboxyethylidene)-d-galactopyranosyl]-(1-->4)-O-(d-glucopyranosyluronic acid)-(1-->3)-d-galactopyranosyl...

Configuration of pyruvic acid ketals, 4,6-O-linked to d-galactose units, in bacterial and algal polysaccharides

Methyl 4,6-O-(hydroxyisopropylidene)-α-d-glucopyranosides (X and XI) were synthesized and the ketal configuration of X was established by a Smith degradation to 1,3-O-(hydroxyiso-propylidene)-l-erythritol (XIII), which was prepared from 2,8-anhydro-1-deoxy-d-glycero-β-d-gulo-octulopyranose (XVI). Methyl 4,6-O-(hydroxyisopropylidene)-α-d-galactopyranosides (II and V) were prepared and their ketal configurations determined by comparison of molecular rotations. Methyl 4,6-O-(1-carbomethoxyethylidene)-α-d-galactopyranosides (IX and XII) from II and V and methyl 4,6-O-(1-carbomethoxyethylidene)-α-d-glucopyranosides (XIV and XV) from X and XI were utilized for this purpose. Also, the hydrogen-bonding characteristics of methyl 4,6-O-(hydroxyisopropylidene)-2,3-di-O-methyl-α-d-galactopyranosides (III and VI) from II and V were compared with those of methyl 4,6-O-(hydroxyisopropylidene)-2,3-di-O-methyl-α-d-glucopyranosides (VII and VIII) from X and XI. Based on the finding that X contains an axial C-methyl group, the results from the two approaches show that methyl 4,6-O-(hydroxyisopropylidene)-α-d-galactopyranoside (II) contains an axial C-methyl group in the conformations depicted. The stereoisomer of this compound was prepared from 4,6-O-(1-carboxyethylidene)-d-galactose (IV), a partial hydrolysis product of Corynebacterium insidiosum polysaccharide and Difco agar and establishes that the natural products contain equatorial C-methyl ketal groups.