Elsevier

Carbohydrate Research

Volume 339, Issue 8, 1 June 2004, Pages 1459-1466
Carbohydrate Research

A nearly idealized 6-O-methylated η-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

https://doi.org/10.1016/j.carres.2004.03.019Get rights and content

Abstract

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and 13C, 1H, and 13C/1H heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6-O-methylcarrabiose 2,4-disulfate (the repeating unit of 6-O-methylated η-carrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported.

Introduction

The commercially valuable extracellular polysaccharides extracted from red algae are primarily sulfated galactans comprised of repeating disaccharide units.[1], [2] The fundamental repeating disaccharide consists of 3-linked β-d-galactopyranose (Galp) and 4-linked α-Galp residues. The configuration of the 4-linked residues determines whether the galactan is classified as an agar (l-) or a carrageenan (d-). The 4-linked residues often occur in the form of 3,6-anhydrogalactopyranose (AnGalp), and the repeating units containing l- and d-AnGalp as the nominal reducing end sugar are referred to as agarobiose and carrabiose, respectively.2 The repeating disaccharide units may be substituted to varying degrees by sulfate ester, methyl ether, pyruvate acetal, and/or glycosyl residues. During the last decade, increasingly sophisticated techniques have been applied to the analysis of red algal galactans and have enabled the discovery and characterization of unusual or novel substitution patterns.[3], [4], [5], [6], [7] These techniques include chemical methods, such as reductive hydrolysis,[8], [9] which permits the quantitative recovery and analysis of the acid-labile AnGal; reductive partial hydrolysis[9], [10] and reductive amination[11], [12] to derive configurational information about constituent sugars in combination with their linkage patterns; and two-dimensional nuclear magnetic resonance (NMR) spectroscopy.[3], [4], [5] A recent report4 used a combination of essentially all these techniques to characterize pyruvated β-carrageenan and highlighted the benefit of such studies by providing empirically derived reference data for specific substitution patterns.

One notable substitution pattern occurring in red algal galactans is the 6-O-methylation of 3-linked d-Galp residues (6-MeGal). The gel setting temperatures of agars have been correlated with their 6-MeGal content,[13], [14] and the effect of this substitution on gel setting temperatures has implications for the commercial end use of agars with high 6-MeGal content, which are regarded as suitable for applications in processed foods but not for microbiological or most biotechnological applications.[15], [16] Although 6-MeGal is relatively common and abundant in agars, it is relatively rare in carrageenans. The most conspicuous O-methylated repeating disaccharide documented in carrageenans is probably 6-O-methylcarrabiose 2,4-disulfate (the repeating unit for 6-O-methylated η-carrageenan). First identified 20 years ago through extensive fractionation as a minor and somewhat anomalous component of the commercially important κ-carrageenan from Kappaphycus alvarezii (Doty) Doty (as Eucheuma cottonii),17 this repeating disaccharide unit has more recently been found to be a relatively significant component of carrageenans with comparatively complex substitution patterns from species of Rhabdonia, Erythroclonium, Austroclonium, and Areschougia,[6], [18], [19], [20] as well as a relatively minor component of carrageenans from Meristotheca and other Kappaphycus species,[21], [22], [23] and it has attracted interest for its potential chemotaxonomic and commercial implications.[19], [23], [24] We have analyzed the sulfated galactans from an Australian endemic red alga, Claviclonium ovatum (Lamouroux) Kraft et Min-Thein. The structure of these galactans approaches that of an idealized 6-O-methylated η-carrageenan, providing the opportunity to characterize in detail the 6-O-methylcarrabiose 2,4-disulfate disaccharide unit.

Section snippets

Algal material

The sample of C. ovatum used for this study (MELU #A40751) was a sterile specimen collected by Dr. J. Huisman from the drift at Mangles Bay, Point Peron, Western Australia on 10 June 1993.

Extraction and treatment of the polysaccharides

The dried algal fronds were cleaned of surface debris and epiphytes and milled to a powder. The polysaccharides were extracted from the milled algal material in distilled water at 95 °C, clarified, and precipitated with 2-propanol as described previously.25 A portion of this polysaccharide preparation was

Compositional analyses

The native polysaccharide preparation was obtained in 32% yield (w/w of the dried algal material, Table 1) and contained a relatively high level of sulfate (35.8% w/w of the polysaccharide preparation, Table 1). Constituent monosaccharide analysis of the native preparation (Table 1) confirmed that the polysaccharides were mainly galactans, with 93 mol % of the total monosaccharides comprising 6-MeGal, AnGal, and Gal. The remainder consisted of Glc (6 mol %) and Xyl (1 mol %).

Following alkali

Conclusion

The chemical and spectroscopic data showed that the alkali-modified C. ovatum galactans were composed of a nearly idealized repeating unit of 6-O-methylcarrabiose 2,4-disulfate, with some minor substitution patterns also present. The C. ovatum carrageenans therefore are the most highly methylated carrageenans reported. The 13C, 1H, and HMQC NMR spectra for the repeating unit were fully assigned.

Acknowledgments

A.C. acknowledges the support of a Postdoctoral Research Fellowship (# F00103165) and a Discovery Project Grant (# DP0210153) provided by the Australian Research Council.

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