Analysis of pyruvylated beta-carrageenan by 2D NMR spectroscopy and reductive partial hydrolysis

A polysaccharide rich in 4',6'-O-(1-carboxyethylidene)-substituted (i.e., pyruvylated) beta-carrageenan has been prepared by solvolytic desulfation of a polysaccharide containing predominantly pyruvylated alpha-carrageenan, which was extracted from the red seaweed, Callophycus tridentifer. The 13C and 1H NMR chemical shifts of pyruvylated beta-carrageenan have been fully assigned using 2D NMR spectroscopic techniques. The 4',6'-O-(1-methoxycarbonylethylidene) group, generated during chemical methylation of the polysaccharide, has been shown to survive under the conditions of acidic hydrolysis that cleave the 3,6-anhydro-alpha-D-galactosidic bonds in permethylated samples of both pyruvylated beta- and pyruvylated alpha-carrageenans. As a result, two novel pyruvylated carrabiitol derivatives have been prepared.     

Structural studies on kappa-carrageenan derived oligosaccharides

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum carrageenan. Three oligosaccharides were purified by strong-anion exchange high-performance chromatography. Their structure was elucidated using mass spectral and NMR data. Negative-ion electrospray ionization (ESI) mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the kappa-carrageenan oligosaccharides. 2D NMR techniques, including 1H-(1)H COSY, 1H-(1)H TOCSY and 13C-(1)H HMQC, were performed to determine the structure of a trisulfated pentasaccharide. 1D NMR and ESIMS were used to determine the structures of a kappa-carrageenan-derived pentasaccharide, heptasaccharide, and an undecasaccharide. All the oligosaccharides characterized have a 4-O-sulfo-D-galactopyranose residue at both the reducing and nonreducing ends.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy.

The high-field 1H-NMR spectra of various carrageenan oligosaccharides at room temperature are given. The assignments were faciliated by the use of proton double-quantum coherence (DQCOSY) and 1H-13C chemical shift correlation 2D NMR spectroscopy, and by comparing high-field 1H-NMR spectra of various 4-sulphated oligosaccharides of the neocarrabiose type. The effects of anomeric configuration on the 1H resonances on the same or neighbouring units are discussed. The 13C-NMR shift data are given for the tetrasaccharide of kappa-carrageenan.     

Positional isomers of sulfated oligosaccharides obtained from agarans and carrageenans: preparation and capillary electrophoresis separation

Partial reductive hydrolysis was used to produce oligosaccharide alditols from repetitive sulfated galactans obtained from four Rhodophyta species: kappa-carrageenan (from Kappaphycus alvarezii), theta-carrageenan (Gigartina skottsbergii-alkali-treated lambda-carrageenan), agarose 6-sulfate (Gracilaria domingensis), and pyruvylated agarose 2-sulfate (Acanthophora spicifera-alkali-treated pyruvylated agaran sulfate). Each hydrolyzate was submitted to anion-exchange and gel-filtration chromatography, and the isolated oligosaccharide alditols were identified by 1D and 2D NMR spectroscopy and by ESI mass spectrometry. The positional isomers of the sulfated oligosaccharide alditols were then completely resolved by capillary electrophoresis in a borate buffer. Attempts to correlate the availability of the hydroxyl groups for borate complexation with the relative migration of the oligosaccharides are presented.     

Synthesis and NMR structural analysis of O-succinyl derivative of low-molecular-weight κ-carrageenan

Low-molecular-weight (LMW) κ-carrageenan was achieved through mild hydrochloric acid hydrolysis of κ-carrageenan. The acylation of LMW κ-carrageenan was performed by use of tetrabutylammonium (TBA) salt of the anionic polysaccharide fragments, succinic anhydride, 4-dimethylaminopyridine and tributylamine under homogeneous conditions in N,N-dimethylformamide at 80 °C. Investigation of FT-IR spectrum of the succinylated LMW κ-carrageenan showed that a monoester derivative with succinyl group was formed when LMW κ-carrageenan reacted with succinic anhydride. The ¹H and ¹³C NMR spectroscopy has been used to characterize the fine structure of O-succinyl derivative of the LMW κ-carrageenan. The ¹³C and ¹H NMR chemical shifts of disaccharide unit of O-succinyl LMW κ-carrageenan have been fully assigned using 2D NMR spectroscopic techniques.     

Synergistic Interaction between Kappa-Carrageenan and Locust-bean Gum in Aqueous Media

Although neither kappa-carrageenan nor locust-bean gum gelled alone, a mixed aqueous solution of the above gums gave a gel at the concentration of 0.6% total gums in a range of low temperatures. The solution also gelled even at the concentration of 0.4% total gums in the presence of 0.1% KC1. The maximum dynamic modulus was obtained with a series of the samples composed of kappa-carrageenan and locust-bean gum in the mixing ratios of 1:1 and 3:1 at the concentration of 0.6 and 0.8% total gums at 0°C. The dynamic modulus of a mixed solution of kappa-carrageenan and locust-bean gum was not influenced by pH between pH 7.0 to 11.5, but decreased in the acidic range.

We concluded that intermolecular interactions, at low temperature, between kappa-carrageenan and locust-bean gum may take place on the K⁺-bridge of the former and the backbone of the latter molecule at low concentrations, but at high concentration of the gums, self-association of kappa-carrageenan molecules might also occurred.     

Rheology of kappa-carrageenan in mixtures of sodium and cesium iodide: two types of gels

Recent studies on dilute solutions (Borgström et al. (1996), Int. J. Biol. Macromol. 18, 223) have shown that kappa-carrageenan helices associate into superhelical rigid rods in mixed 0.1 M aqueous solutions of NaI and CsI above a critical mole fraction (x_Cs = 0.4) of Cs. This work concerns the temperature-dependent rheology of more concentrated systems in mixed and pure solutions of the same salts. Gels with low moduli were even found in NaI alone, although this salt is known to impede the gelation of kappa-carrageenan, but only above 0.9% (w/w) of carrageenan. These gels were reminiscent of iota-carrageenan gels in two respects: the (low) magnitude of the shear storage modulus (G′), and the absence of hysteresis in the sol-gel transition. On the other hand, both the threshold concentration for gelation and the ratio between the loss and storage moduli were substantially higher for the kappa-carrageenan gels in NaI. In mixed solutions of CsI and NaI, two types of kappa-carrageenan gels could be distinguished, depending on the cesium content. The transition occurred at x_Cs = 0.4, as in the previous studies on dilute solutions. Below x_Cs = 0.4, the gels were similar to those in NaI alone. Above x_Cs = 0.4, the gels were similar to ‘conventional’ kappa-carrageenan gels, formed in salts such as KC1: a pronounced thermal hysteresis appeared in the sol-gel transition, the gels showed tendencies for syneresis, and G′ increased dramatically with increasing cesium content.     

Characterization of a fucoidan from Lessonia vadosa (Phaeophyta) and its anticoagulant and elicitor properties

Blades of Lessonia vadosa (Phaeophyta) were extracted with 2% CaCl(2) solution, affording in 4.4% yield a polysaccharide which contained fucose and sulfate groups in the molar ratio 1.0:1.12. The high negative optical activity value ([alpha](D)(22)=-134.0 degrees ), FT-IR and NMR analysis suggest the presence of a fucoidan. (13)C NMR spectrum of the polysaccharide obtained by solvolytic desulfation of native fucoidan indicated the major presence of 1-->3 linked alpha-l-fucan. Depolymerization of the native fucoidan with H(2)O(2) in the presence of copper(II) acetate gave in 54.8% yield a fraction with 33.7% of sulfate content. The native fucoidan (MW 320,000) showed good anticoagulant activity whereas the radical depolymerized fraction (MW 32,000) presented a weak anticoagulant activity. These polysaccharides showed significant activation of phenylalanine-ammonia lyase (PAL), lipooxygenase (LOX) and glutathione-S-transferase (GST) defence enzyme activities in tobacco plants.     

Gel--sol transition in kappa-carrageenan systems: microviscosity of hydrophobic microdomains, dynamic rheology and molecular conformation.

The effect of gel-sol transition in kappa-carrageenan systems on the microviscosity of hydrophobic microdomains, as well as its relation to macroscopic rheology and molecular conformation, was studied in kappa-carrageenan systems. The microdomains were probed by 1,3-di(-1-pyrenyl)propane (P3P) for which the excimer intensity (Ie) provides relative measures of the microviscosity in the immediate probe surroundings. In particular the applicability of P3P to monitor the gel--sol transition was proved, the results showing a dramatic decrease in microviscosity in the vicinity of the transition point. The corresponding changes in rheological properties and carrageenan conformation were investigated by dynamic viscometry (DV) and optical rotation (OR), respectively. The temperature of onset of the transition as indicated by the microviscosity data (T0) was found to correlate well with the OR and DV-results. The application of microviscosity and OR-measurements allowed an estimation of the helical content at T0 to be determined. P3P-data indicate a microenvironment viscosity for the probe sites in the kappa-carrageenan system comparable to that found in SDS micelles.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

Testing the validity of comparisons between the rheological and the calorimetric glass transition temperatures

Dynamic mechanical techniques are used increasingly in the investigation of vitrification phenomena in biological materials, thus posing the question of whether the rheological T(g) should be compared with the established practice of obtaining T(g) values from differential scanning calorimetry. The nature of the rheological T(g) is discussed and its frequency dependence is established with a view to facilitating comparisons with calorimetric data. Despite claims made in the literature, results on high sugar-kappa-carrageenan mixtures, hydrated gelatin films, and thermoset epoxy resins demonstrate that there is no clear reference point for comparison of the glass transition temperatures derived with the two techniques. Furthermore, the structure-forming ability of kappa-carrageenan and other biopolymers impacts primarily upon the mechanical manifestation of vitrification and contributes to the state of complexity of comparisons between thermal and mechanical data.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

A sulfated glucuronofucan containing both fucofuranose and fucopyranose residues from the brown alga Chordaria flagelliformis

A fucoidan fraction composed of l-fucose, sulfate, and d-glucuronic acid in a molar proportion of about 1:1:0.25 and small amount of acetyl groups was isolated from the brown alga Chordaria flagelliformis. Several modified polysaccharides were prepared from the native fucoidan using solvolytic desulfation, carboxyl reduction, and partial acid hydrolysis. Polysaccharide structures were elucidated by methylation analysis and 1D and 2D NMR spectroscopy. The fucoidan was shown to contain a backbone of 3-linked α-l-fucopyranose residues, about one-third of which are glycosylated at C-2 by α-d-glucopyranosyluronic acid residues. About half of the latter residues are glycosylated at C-4 by single α-l-fucofuranose residues or by disaccharides α-l-Fucf-(1→2)-α-l-Fucf-(1→. Fucofuranose residues are mono- and disulfated at different positions, whereas some additional sulfate groups occupy C-2 and C-4 of the backbone, the latter position being also partially acetylated.     

The disordered conformation of kappa-carrageenan in solution as determined by NMR experiments and molecular modeling

The conformation of kappa-carrageenan in solution was studied combining 1H and 13C NMR with molecular mechanics. The experimental conditions were chosen to characterize the disordered conformation of the polymer. Particular attention has been given to explore a wide range of experimental conditions as to the dependence on solvent (water and Me2SO), polymer concentration, temperature, pH, presence of a denaturing agent (guanidinium chloride), and of ions otherwise able to induce conformational order of the carrageenan chains, either in solution (I-) or in the gel state (Rb+). Two-dimensional NOE experiments were analyzed to obtain information on internuclear distances, and molecular mechanics provided the range of energetically accessible conformations. Two inter-residue topological constraints were clearly identified: their combination is rather restricting for the chain and suggests that the disordered conformation of kappa-carrageenan is characterized by an intrinsic stiffness with high values of persistent length and characteristic ratio. They also rule out any postulated interchain hydrogen bonds. In contrast, experiments on the temperature dependence of the chemical shift in Me2SO reveal the existence of two inter-residue intramolecular H-bonds which might contribute positively to the rigidity of the polymer chain. The overall picture emerging from the present results is that of a locally elongated 'loose single helix'.     

Effect of partial desulfation and oversulfation of sodium spirulan on the potency of anti-herpetic activities

The aim of the present study is to evaluate the effect of partial desulfation and oversulfation of sodium spirulan (Na-SP) isolated from Spirulina platensis on the exhibition of anti-herpes simplex viruses type 1 and 2 (HSV-1 and -2) activities. Partially desulfated (PDS-SPs) and oversulfated derivatives (OS-SPs) were obtained by solvolytic desulfation and sulfation with dicyclocarbodiimide-sulfuric acid, respectively. When PDS-SPs were subjected to anti-HSV-1 assay, antiviral potency was dependent on their sulfate content, and PDS-SPs with lower sulfate content than 8.6% were found to be inactive. Some derivatives showing anti-HSV-1 effect also showed anti-HSV-2 activity. Anti-HSV-1 effect of OS-SPs was equivalent to that of Na-SP when they were added to the medium during viral infection and throughout the incubation thereafter, while they were enhanced as compared with Na-SP when they were added to the medium immediately after viral infection. The results of time-of-addition experiments suggested that the most sensitive phase of OS-SP-2 and -5 might be the early steps of viral adsorption and penetration.     

Cation effects on swelling of kappa-carrageenan: a photon transmission study

Swelling behavior of kappa-carrageenan gels in water and KCl solutions was investigated by photon transmission experiments following the preparation of gels in the presence and absence of externally added K+ ion as a gel promoting agent. Transmitted photon intensity, Itr, increased continuously during swelling depending on the carrageenan and ion content in the gel. This increase in Itr was modeled using the Li-Tanaka equation. Both the experimental work and the model showed that the swelling of low carrageenan and ion content gels took less time than that of high ion content gels. It is confirmed that double helices in a swollen gel move much faster in pure water than in KCl solution during swelling processes. Swelling time constants, tau1, and collective diffusion coefficients, Do, were measured for the gels swollen in water and KCl solutions.     

Heterogeneity of carrageenans from Chondrus crispus.

Carrageenans extracted from gametophytic and sporophytic Chondrus crispus were analysed by hydrolysis, KCl fractionation and 1H NMR spectroscopy. The carrageenan from gametophytic plants is composed predominantly of two KCl insoluble fractions which contain kappa-carrageenan as the major component with 1-carrageenan and sulphated galactans as minor components. The precursor mu- and v-carrageenans were not found in the soluble fraction. The extract from sporophytic plants is composed mainly of a KCl soluble fraction which could be separated into 10 fractions by ion-exchange chromatography. The major component did not show a lambda-type structure but one of a xi-carrageenan.     

Interpenetrating network formation in agarose--kappa-carrageenan gel composites

Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the seaweed polysaccharides agarose and kappa-carrageenan. Over a range of ionic strength, and potassium content, there is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. At low ionic strength, where the agarose gels first, differential scanning calorimetry evidence shows some influence of the carrageenan on the agarose ordering enthalpy, particularly at higher polymer concentrations. As the potassium level is increased, however, and the order of gelling is reversed, this effect disappears. Cure behavior for the systems at high ionic strength can be described as a simple summation of the pure component contributions. At low ionic strength, on the other hand, the modulus behavior is more complex, suggesting either a modification, in the mixture, of the kappa-carrageenan gelling parameters or a more complex modulus additivity rule.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.