Chemical screening of Brazilian marine algae producing carrageenans

The yield, IR spectra, 3,6-anhydro-d-galactose and sulfate content are given for several species of putative carrageenophytes from different sites on the Brazilian coast. Kappa carrageenan was found in Hypnea musciformis and Gigartina spp. ; all the other species tested yield an iota type. The lowest carrageenan yield was found in Cryptonemia crenulata (5%), and the highest in Gigartina sp. (72%); 3,6-anhydrogalactose content was highest in Hypnea musciformis and Gigartina spp., whereas sulfate was lowest in H. musciformis. Slight variations in the yield of carrageenans were found during the period of observation (March 1988 to May 1989), but no clear pattern could be recognized. No significant variation was observed in plants from different collection sites.     

Antiherpetic (HSV-1) activity of carrageenans from the red seaweed <Emphasis Type="Italic">Solieria chordalis</Emphasis> (Rhodophyta,Gigartinales) extracted by microwave-assisted extraction (MAE)

Carrageenan yield, physicochemical properties, and antiviral activity of the carrageenan from Solieria chordalis (Rhodophyceae, Solieriaceae) harvested at the Brittany coast (France) were investigated. S. chordalis carrageenan was extracted by conventional and the microwave-assisted extraction (MAE) methods. The effect of different parameters during MAE extraction such as alkali concentration (0, 0.5 and 1% KOH), extraction time (10, 20, and 25 min) and temperature (90 and 105 °C) were evaluated. Native carrageenan extracted by MAE had the highest yield (29.3%) after 10 min at 90 °C. After alkali treatment, carrageenan yield ranged from 10.7 to 18.4%. No significant differences in the carrageenan yield were observed between MAE and conventional method under alkaline conditions. Chemical analysis and FTIR spectra revealed the presence of a predominant iota-carrageenan. Evaluation of the antiviral activity of S. chordalis carrageenan against HSV-1 (Herpes simplex virus type 1) showed a EC₅₀ of the iota-carrageenans fractions in the range of 3.2 to 54.4 μg mL^?1 (MOI 0.01 ID₅₀ mL^?1) without cytotoxicity in that range of concentrations.     

Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red alga Hypnea musciformis

Polysaccharides, galactans, obtained from edible red seaweed Hypnea musciformis were characterized by molecular weight and infrared spectroscopy analysis and were evaluated for antioxidant activity in vitro and for their effects on cell viability. The main components were galactose and sulfate presenting low protein contamination. These sulfated galactans (F1.0) showed a polydisperse profile, and signs in infrared analysis were attributed to a sulfate ester S?=?O bond, the presence of a 3,6-anhydrogalactose C–O bond, nonsulfated β-d-galactose, and a C–O–SO₄ bond in galactose C4. The NMR analysis showed signals at about 95 and 92 attributed to anomeric carbon of 4-linked 3,6-anhydro-α-d-galactopyranose residue of κ-carrageenans and 4-linked 3,6-anhydro-α-d-galactopyranose2-sulfate of ι-carrageenans. Sulfated galactan F1.0 showed strong antioxidant activity under lipid peroxidation assay where F1.0 at 8 mg mL^?1 promoted 57.92% peroxidation inhibition and displayed the scavenging activity on hydroxyl radicals in a dose-dependent manner leading to 32.5% scavenging of these radicals when 5 mg mL^?1 of sulfated galactan F1.0 was used. The sulfated galactan fraction also exhibited strong inhibition on the H₂O₂-induced hemolysis model. Sulfated galactan F1.0 displayed low cytotoxic action in 3 T3 cells and moderate antitumoral action in HeLa cells. These results suggest that sulfated galactan F1.0 from H. musciformis has antioxidant potential, which is a great effect for a compound used as food and in the food industry.     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Influence of tissue source and growth rates on dry weight and carrageenan composition of Chondrus crispus (Gigartinales,Rhodophyta)

Carrageenan analyses were conducted on vegetative female clones of Chondrus crispus that were cultured to provide tissues with differing growth rates. Tissue dry weights increased from apex to base of fronds. Total carrageenan contents were lower in apical 1 to 2 cm segments than elsewhere in the frond, except when the alga was grown at high photon irradiances. Clone 373A contained more carrageenan than clone G8. The proportion of 0.3 M KCl-soluble polymers in the total native carrageenans varied from 44 to 92%, being highest in older tissues of fronds cultured at high photon irradiances. The apical 1 cm segments contained less KCl-soluble carrageenans than other tissues from the corresponding fronds. The KCl-soluble carrageenans, when alkali-modified and refractionated, afforded the expected kappa-iota carrageenan in > 79% yields. The remainder consisted of a polymer containing 23.1% SO₃Na and 8.4% 3,6-anhydrogalactose. Lambda carrageenan was not detected. Variations in carrageenan distribution between the apical region and other parts of the frond may reflect the increasing influence of medullary tissue developed as the immature cells differentiate.     

The chemistry and immunochemistry of carrageenans from Eucheuma and related algal species

Carrageenans from several species of Eucheuma have been fractionated into KCI-soluble and KCI-insoluble fractions and analyzed by the usual chemical procedures. An anti-K-carrageenan, the reactivity of which is directed to K-structures (i.e., 3-linked galactose 4-sulphate, and 4-linked 3,6-anhydrogalactose) was used to analyze these carrageenans immunochemically. The antibody preparation shows only a small amount of cross-reactivity with i-type carrageenans and thus could be used to distinguish K- and i-type carrageenans, the latter having an index of homology of less than 0.2. A comparison of chemical and immunochemical data yielded further information as to the nature of the carrageenan-anti-carrageenan interaction, as well as elucidating the finer structure of carrageenans.     

Methylation analysis of carrageenans from the seaweed Iridaea undulosa

Two carrageenans from Iridaea undulosa, isolated by precipitation of the crude polysaccharide at O.70–1.05 M and 1.55–1.65 M KCl concentrations, were studied by methylation analysis. Acid hydrolysis of the methylated derivative of the less soluble carrageenan (molar ratio galactose: 3,6-anhydrogalactose: sulphate 1.00: 0.50: 1.20) yielded major amounts of 2,6-di-O-methylgalactose (51.3 mol %), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (51.3mol%), 4,6-di-O-methylgalactose (25.6%) and 4-O-methylgalactose (13.4%). Minor quantities of 3-O-methylgalactose (4.6%) and 6-O-methylgalactose (3.2%) were found together with traces of 2,3,6- and/or 2,4,6-tri-O-methylgalactose, 2-O-methylgalactose and galactose. Oxidative acid hydrolysis produced 3,6-anhydro-2-O-methylgalactonic acid and 3,6-anhydrogalactonic acid in a molar ratio 3.5-4.0:1.0. The methylated derivative of the more soluble carrageenan (molar ratio galactose:3,6-anhydrogalactose:sulphate 1.00:0.04:1.43) gave on acid hydrolysis, 2,3,4,6-tetra-O-methylgalactose (4.6%), 2,3,6-tri-O-methylgalactose (4.2%), 2,4,6-tri-O-methylgalactose (10.7%), 4,6-di-O-methylgalactose (24.1%), 3,6-di-0-methylgalactose (8.0%), 2,3-di-O- methylgalactose (3.4%), 2,4-di-O-methylgalactose (4.6%), 2,6-di-O-methylgalactose (4.2%), 3-O-methylgalactose (19.5%),4-O-methylgalactose (9.6%),6-O-methylgalactose(3.1%),galactose (3.4%)and traces of 2-O-methylgalactose.     

Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes of the rhodophyte Mastocarpus stellatus (Gigartinales) were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν-, μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 80 % of the total carrageenans). Mean total carrageenan content in gametophytes was of 37.32?±?1.21 % DW. Spatial and seasonal variations were observed, mainly related to changes on environmental and oceanographic factors and the role of carrageenans in adapting the fronds to these changes. Maximum values in carrageenan content were observed for San Román (Biscay Bay) in May and for Laxe and Mougás (Atlantic coast) in June. The results of this study indicated that spatial differences in carrageenan content were due to interactions of different factors, rather than the effect of a single factor. Fronds from San Román had higher carrageenan content (43.23?±?1.87 % DW) than those collected at two sites of the Atlantic coast, Mougás and Laxe (32.20?±?1.14 % DW). San Román is exposed to the open sea, windy and oriented to the north, and the water temperature is higher in summer than in the Atlantic coast. However, seasonal variations in carrageenan content resulted to be more related to other factors directly correlated with the input of energy in the ecosystems (irradiance, sunshine hours and insolation). Thus, carrageenan content began to increase in early spring when the number of sunlight hours increased. Maximum values were reached in late spring or early summer, just before maximum values of irradiance and air temperature were achieved.     

Comparative analysis of sulfated galactans from red algae by reductive hydrolysis and mild methanolysis coupled to two different HPLC techniques

The sugar determination of the sulfated galactans, agars and carrageenans of various red algae was performed using two different techniques of depolymerisation with subsequent HPLC analysis: 1) reductive hydrolysis/ HPAEC-PAD; 2) mild methanolysis/ RPLC-DR. Both techniques were optimized to release quantitatively the composite sugars (galactose, 6-O-methyl-galactose, the labile 3,6-anhydrogalactose and 2-O-methyl-3,6-anhydrogalactose residues) and precise relative response factors of authentic 3,6-anhydrogalactose were determined. The methanolysate neutralisation step, performed subsequently to methanolysis depolymerisation, was demonstrated as a key step for the quantitative recovery of the anhydrogalactose residues. The yield of the main sugars released by the two techniques were in good agreement for the commercial agarose and iota and kappa carrageenans studied. This revised version was published online in June 2006 with corrections to the Cover Date.     

A novel high-performance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3,6-anhydrogalactose

A novel method has been developed to determine the sugar composition of 3,6-anhydrogalactose-containing polysaccharides, such as carrageenan and agar. The method is based on reductive hydrolysis with a methylmorpholine-borane complex in the presence of acid and subsequent high-performance anion-exchange chromatography analysis of the alditols without any derivatization. The method was validated by 13C NMR analysis of six carrageenans and three agars and by a previously used method based on derivatization to alditol acetates and gas-liquid chromatography analysis. The new method was found to be superior to the gas-liquid chromatography method as the analysis time was less than half. Also it was found to be more accurate and reproducible and no derivatization was required. The analysis of the six different carrageenan samples revealed that homogeneous mu- and nu-carrageenan, theoretically without 3,6-anhydrogalactose residues, cannot be isolated from red seaweeds. Consequently, the question arose if mu- and nu-carrageenans at all are present in seaweeds and if the current hypotheses regarding biosynthesis of carrageenans in the seaweeds are correct. The data demonstrated that carrageenans are highly complex natural polysaccharides, which are more irregular than assumed hitherto. The new analytical technique will permit elucidation of the detailed structure of seaweed polysaccharides and determination of their structure-property relationships.     

CO-OCCURRENCE OF CARRAGEENANS AND AGAROIDS IN GYMNOGONGRUS TORULOSUS (GIGARTINALES, PHYLLOPHORACEAE) FROM ARGENTINA

The red seaweeds, Gigartina skottbetgii and Sarcothalia crispata , have commercial value as raw material for the industrial production of phycocolloids (carrageenans) in Argentina. The third alga with potencial possibilities for the carrageenan production is Gymnogongrus torulosus. Herein, we report the study of the polysaccharide system present in Gymnogongrus torulosus , which contribute to the estimation of the importance of this algae in the seaweed industry. Analysis of the hot water-soluble extract (C1), by FT-IR, methylation and ¹³C NMR, showed mainly the presence of iota/kappa carrageenan hybrid (molar ratio ∼ 2:1). The soluble fractions obtained after KCl fractionation (F3, 16.1 % of C1) and soluble after alkaline treatment and KCl fractionation (F3T3, 34.6% of F3T) gave negative optical rotations (−15.5 and −55.4, respectively), considerably lower than those reported for kappa/iota carrageenans (from 56.1 to 66.5). These fractions (F3 and F3T3) have significant quantities of L-galactose (11.1% and 29.8%) and L-3,6-anhydrogalactose (19.2% and 4.3%). The direct relationship between the optical rotation and the percentage of L-galactose indicated that its structural influence is similar in all the fractions. The results suggest that Gymnogongrus torulosus biosynthesizes a polysaccharide system with co-occurrence of carrageenans and agaroids in the same thallus.     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

Growth rate and carrageenan analyses in four strains of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) farmed in the subtropical waters of São Paulo State,Brazil

Growth rate, semi-refined and refined carrageenan yields, 3,6-anhydrogalactose and sulphate contents, and gel strength were investigated in four strains of Kappaphycus alvarezii (brown, green and red tetrasporophytic strains, and one strain derived from tetraspores progeny, called G11) farmed in subtropical waters of São Paulo State, Brazil. All studied strains showed higher growth rates from February to May, decreasing from July to December. The G11 strain exhibited lower growth rates, but had semi-refined and refined carrageenan yields significantly higher than the others and similar to a commercial sample. A negative correlation between growth rate and semi-refined carrageenan yield was verified for all strains. The brown strain had the lowest content of 3,6-anhydrogalactose, while G11 and green strains showed higher values. No clear pattern of variation and no relationship were observed for sulphate and gel strength. However, all strains presented gel strength values near the one obtained from commercial sample. These results show that the carrageenan extracted from brown, green, red, and G11 strains of K. alvarezii cultured in subtropical waters of São Paulo State, Brazil with commercial potentials.     

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.     

Postharvest culture in the dark: An eco-friendly alternative to alkali treatment for enhancing the gel quality of κ/ι-hybrid carrageenan from Chondrus crispus (Gigartinales,Rhodophyta)

After harvesting from natural stocks, female gametophytes of the red seaweed Chondrus crispus were cultured for several days under a light-deprived regime in order to determine if dark treatment can improve the gel quality of carrageenan extracted from the seaweed. An increase in the gel strength of native carrageenan was observed after dark treatment for 10 d, but not after 5 d, as compared to that of freshly harvested (baseline) seaweed. Corresponding decrease in sulfate and increase in 3,6-anhydrogalactose (3,6-AG) contents of the carrageenan extract were also observed. We posit that during dark treatment, the production of sulfated carrageenan precursor units was prevented while the enzymatic conversion of the prevailing precursor units to the gelation-promoting 3,6-AG was allowed to proceed. The observed increase in carrageenan gel strength with a 10-d dark treatment was comparable to the effect of the widely-used industrial procedure of alkali treatment in improving the extract’s gel quality. Hence, postharvest culture in the dark for 10 d can be an eco-friendly alternative to alkali treatment.     

Carrageenans from Cystocarpic and Sterile Plants of Chondrus Pinnulatus (Gigartinaceae, Rhodophyta) Collected from the Russian Pacific Coast

The chemical structure, gel properties and biological activity of the carrageenans isolated from cystocarpic and sterile plants of Chondrus pinnulatus were investigated. The total carrageenan content of the sterile plant was observed to be twice that of the cystocarpic plants. According to data obtained by ¹³C-NMR and FT IR, the gelling polysaccharides from cystocarpic and sterile plants of C. pinulatus have similar structures and were identified as κ/ι-carrageenans. The difference between these polysaccharides was in the ratio of the κ- and ι-segments, with a predominant content of κ-segments in cystocarpic plants (80%). Moreover, KCl-insoluble fractions possibly contain hetero-disperse μ/ν precursor: amounts of this in the polysaccharide from sterile plants were more than that extracted from the cystocarpic plants. The KCl-soluble fractions (non gelling) were λ-carrageenans with another carrageenan type that had a low amount of 3,6-anhydrogalactose. Carrageenans from cystocarpic stages showed good gelling properties, whereas those from sterile plants formed a very weak gel. Structural differences and molecular weight of carrageenans obviously determine the biological activity of the polysaccharides. Non gelling-carrageenans from both types of ιt C. pinnulatus plants showed high macrophage-phosphatase activity and κ/ι-carrageenan from cystocarpic plant possessed a potent anti-coagulant activity, which was extremely strong in a low concentration of 100 μg ml⁻¹.     

Cell wall composition of the carrageenophyte Kappaphycus alvarezii (Gigartinales, Rhodophyta) partitioned by wet sieving

The cortical and medullary cells of Kappaphycus alvarezii fractions were screened by wet sieving after aqueous extraction of carrageenans. The cell populations obtained showed a clear partition between these two cell types. The main monosaccharide in hydro-insoluble cell walls was cellulosic glucose (70% dry weight), the crystallinity of which was shown torange from 20% in the cortical cells to 45% in the large medullary cells (over 250 μm diameter). Minor monosaccharides in the insoluble fraction were galactose, 3,6-anhydrogalactose (indicating presence of residual carrageenans), mannose and xylose. However, the major part of the remaining galactose probably originated from another galactoglycan strongly linked to insoluble polymers in the large medullarycell walls. The mannose concentration was maximum in the cortical cells and decreased with increasing size of the medullary cells. Thus, besides cellulose, two other types of polysaccharides were detected in insolublecell walls, mannoglycans and galactoglycans in cortical and medullary cellwalls, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes and tetrasporophytes of Chondrus crispus were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν- and μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 50% of the total carrageenans). In tetrasporophytes, the presence of λ-carrageenan was confirmed. Carrageenan content in gametophytes (37.4?±?1.68% DW) was higher than in tetrasporophytes (29.13?±?0.76% DW). Spatial and temporal variation in carrageenan content in both life cycle phases appears to be related mainly to seawater and air temperatures, insolation, water movement and desiccation. The highest values of carrageenan content were recorded in those localities where higher values of precipitation, wind speed or water movement occurred. A bimodal temporal pattern on carrageenan content was observed. Fronds showed a high carrageenan content in spring and autumn. During these seasons, the content was over 40% in gametophytes and 30% in the tetrasporophytes. In summer and winter, these values down in both life cycle phases below 30%. In general the highest carrageenan contents were related to highest seawater temperatures. On the contrary, high air temperature and high insolation appeared to be unfavourable for carrageenan production. GLM models were obtained to predict carrageenan production from natural C. crispus populations, along Galician coast.     

DIFFERENTIAL LOCALIZATION OF CARRAGEENAN GELLING SEQUENCES IN KAPPAPHYCUS ALVAREZII VAR. TAMBALANG (RHODOPHYTA) WITH FITC-CONJUGATED CARRAGEENAN OLIGOSACCHARIDES1

The ability of kappa (κ) and iota (ι) carrageenans to form gels is dependent upon the regular repeat of disaccharide units along the carbohydrate chain. Short, chemically- and enzymatically-purified fragments of κ and ι carrageenan were conjugated to fluorescein and used as specific hybridization probes for localization of κ and ι carrageenan gelling sequences within the cells walls and intercellular matrices of Kappaphycus alvarezii (Doty) Doty. The probes label cell walls and intercellular matrices under ionic conditions appropriate for gelation of κ and ι carrageenans. The distribution of κ and ι carrageenans in the matrix and cell walls of K. alvarezii was determined with respect to cell type (epidermis, cortex, medulla, and central axis) and age. The κ-probe labels the cell walls of all cell types except epidermal in both young and old tissues. In contrast, the ι-probe labels the cell walls of the epidermis in both young and old tissue and the cell walls of the thylles only in old tissue. Both probes label intercellular matrix material; however, ι-probe labelling is very much weaker than κ-probe labelling. The results indicate that FITC-conjugated carrageenan oligosaccharides are useful tools that provide information on gelling subunit distribution.     

Potential energy surfaces of carrageenan models: carrabiose,beta-(1 --> 4)-linked D-galactobiose,and their sulfated derivatives

The adiabatic conformational surfaces of several beta-linked disaccharides, which correspond to the repeating structures of carrageenans, were calculated using the MM3 force-field. The studies were carried out on the disaccharide beta-D-Galp-(1 --> 4)-alpha-D-Galp and eight sulfated derivatives, as well as on carrabiose (beta-D-Galp-(1 --> 4)-3,6-An-alpha-D-Galp) and five sulfated derivatives. The presence of 3,6-anhydrogalactose does not change the main features of the maps, although it increases the flexibility of the glycosidic linkage. Sulfation neither produces a striking effect on the map shape, nor a shift on the global minimum, which always remains with psi (theta(C-1'-O-4-C-4C-5)) in trans orientation, and phi (theta(O-5'-C-1'-O-4-C-4)) with a value close to -80 degrees. This effect differs from that occurring on the alpha linkage of equivalent disaccharides, for which the sulfation pattern on the beta-galactose unit shifts the global minima to different positions. A reduction in the flexibility (originated in a deepening of the global minimum well) is observed by sulfation on position 2 of the beta-D-galactose unit, and by sulfation of position 6 of the alpha-D-galactose unit (when the beta-D-galactose unit is 4-sulfated). Within the compounds containing 3,6-anhydrogalactose, the effect of sulfation is even less noticeable. The calculated low-energy regions on carrabiose derivatives agree with X-ray diffraction data on carrageenan fibers and on peracetylated carrabiose dimethyl acetal, and with NOE calculations carried out on kappa-carrabiose.