a new chemical tool for characterization and partial depolymerization

8/7/2019 A new chemical tool for characterization and partial depolymerization

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Hydrobiologia 260/261: 641-645, 1993.A. R. O. Chapman, M. T. Brown & M. Lahaye (eds), Fourteenth International Seaweed Symposium. 1993 Kluwer Academic Publishers.Printed in Belgium.

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A new chemical tool for characterization and partial depolymerizationof red algal galactans

A. I. UsovN.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47,Moscow 117913, Russia

Key words: agar, carrageenan, 3,6-anhydrogalactose, reductivehydrolysis, sulfated galactans, redalgae

Abstract

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine com-plex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by theirreduction to the corresponding 3,6-anhydro-galactitols, whereasall the other monosaccharides are lib-erated essentially in the non-reduced form; the reductive hydrolysis products may be determined quan-titatively using gas-liquid chromatography (GLC). The method is recommended for preliminary char-acterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactansin the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based onthis reaction, the attribution of unknown galactans to the agar orcarrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass withsubsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantiallyretained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elu-cidation of structures of reduced sulfated oligosaccharides may be of great value for the structuralanalysis of complex red algal galactans.

Introduction

Acid hydrolysis is a common procedure for de-termination of monosaccharide composition ofoligo- and polymeric carbohydrates. Mostmonosaccharides are sufficiently stable to acidsunder the conditions of glycosidic bond cleavage.These monosaccharides are released by hydroly-sis in a free state and may be easily identified andquantified by different chromatographic methods.But several types of monosaccharides, e.g. keto-ses (including 3-deoxy-2-keto-octonic and sialicacids), 3,6-dideoxyhexoses, and 3,6-anhydrohex-

oses, are acid-labile and require protection from

the acid degradation during hydrolysis. Recentlyit was shown that reduction at the moment ofhydrolysis may be a method of choice for suchprotection, and borane - 4-methylmorpholinecomplex was recommended as a good reducingagent for this approach (Garegg et al., 1988). Ourwork is devoted to the application of reductivehydrolysis to the structural analysis of red algalgalactans. A similar study was independently un-dertaken by Stevenson & Furneaux (1991).

It is well known that red algae, a phylogeneti-cally very old division of marine macrophytes,differ from all the other plants in polysaccharide

composition (Painter, 1983; Usov, 1992). They


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usually contain sulfated galactans as the mainpolysaccharide components, but, in some species,sulfated xylomannans or neutral xylans may pre-dominate. Several representatives of galactansknown as agars or carrageenans have valuablegelling and stabilizing properties and are preparedfrom algae in large scale as products of great in-dustrial importance. Red algal galactans usuallyhave a 'masked repeating' structure. Their carbo-hydrate chains are e ssentially linear and based ona common structural backbone of alternatingalpha- and beta-galactose residues substituted atpositions 4 and 3, respectively. The beta-galactoseresidues always belong to the D-series, but thealpha-galactose residues are D in carrageenans

and L in agars. Various hydroxyls may be sub-stituted by ester sulfate or methyl groups, pyruvicacid acetals and sometimes by additionalmonosaccharides, as, for example, D-xylose or4-0-methyl-L-galactose residues. A substantialpart of the alpha-galactose residues may exist inthe form of 3,6-anhydro derivatives. The proper-ties of polysaccharides are influenced very muchby all these modifications, and high 3,6-anhydro-galactose content is especially important for goodgelling ability.

Various chemical methods were developed toanalyze the composition and other structural fea-tures of sulfated galactans, but they are, as a rule,rather tedious and time- and substance-consum-ing. Many of them are now substituted by 13C-NMR spectroscopy. The anomeric carbon chem-ical shifts were shown to be sensitive to theabsolute configuration of 4-0-linked galactosederivatives, so the attribution of the polysaccha-ride to the agar or carrageenan group may be

done at the very beginning of the structural in-vestigation (Usov, 1984). The position of otherresonances are sensitive to the presence ofO-methyl and sulfate groups and 3,6-anhydroga-lactose residues. In fact, the 13 C-NMR spectra ofmany disaccharide repeating units usually presentin red algal galactans are now completely inter-preted and may be used to identify the corre-sponding structural elements in new polysaccha-rides (Lahaye et al., 1989).

In spite of these achievements, it is desirable to

have some methods which may accelerate thescreening procedure and give the possibility tocharacterize and to quantify polysaccharidesprior to their isolation from the algal biomass.Improvement of methods of partial depolymer-ization to obtain oligosaccharides, which are ofgreat value for determination of polysaccharidestructures, represents another very important fieldin the study of red algal galactans. We have shownthat reductive hydrolysis may be effectively usedin both directions mentioned above.

Complete reductive hydrolysis

Hydrolysis of a mixture of model compounds,namely, methyl glycosides of xylose, 3,6-anhydro-galactose, and galactose (2 M CF 3 COOH,100 C, 8 h) in the presence of inositol as an in-ternal standard with subsequent conversion intoacetylated aldononitriles and GLC was shown toafford a three-component mixture in the absenceof reducing agent (3,6-anhydrogalactose wascompletely degraded), but four components werefound, when the reaction was carried ou t in thepresence of borane -4-methylmorpholine com-plex. This additional compound was proved to be3,6-anhydrogalactitol acetate, and good correla-tion was observed between its peak area and thequantity of methyl 3,6-anhydrogalactoside in thestarting mixture. It should be noted that practi-cally no xylose or galactose was reduced underconditions of reductive hydrolysis, since decom-position of the reducing agent preceded the lib-eration of these monosaccharides in appreciableamounts. Hence, these sugars may also be deter-

mined quantitativelyby the usual GLC procedureas acetylated aldononitriles together with 3,6-anhydrogalactitol acetate. The average molar re-sponse factors were calculated from numerousexperiments, and the method was proved to besatisfactorily accurate and reproducible (Usov &Elashvili, 1991a).

When applied to several galactans of knownmonosaccharide composition, the method gavereasonable values for the main components, suchas 3,6-anhydrogalactose and galactose, as well as


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for some minor mono-O-methyl-galactoses inusual agar or carrageenan samples. The proce-dure of reductive hydrolysis permits 3,6-anhydro-galactose and 3,6-anhydro-2-0-methylgalactoseto be determined separately, since these monosac-charides are converted into different alditols. Infact, the corresponding derivatives of all themonosaccharides usually found in the hydrolyz-ates of red algal biomass (besides components ofgalactans, xylose, mannose, and glucose are oftenformed as the hydrolysis products of xylans, man-nans, and floridean starches, respectively) are wellresolved by GLC. The procedure is rather insen-sitive to many non-carbohydrate componentspresent in the algal biomass, and therefore it may

be used to characterize the polysaccharide com-position of algae without any pretreatment. Theanalysis may be carried out rapidly and econom-ically, using small amounts of starting material.The advantage of this approach was demon-strated by determination of monosaccharidecomposition in hydrolyzates of 40 algal speciesfrom Kamchatka coastal waters (Table 1). Thiswork was accomplished shortly after the collec-

tion of seaweeds in summer 1990 (Usov & Kloch-kova, 1992).

Partial reductive hydrolysis

Hydrolysis of galactans with high 3,6-anhydroga-lactose content in the presence of borane -4-methylmorpholine complex at lower tempera-ture and acid concentration (0.5 M CF 3 COOH,65 C, 8 h) gave rise to reduced disaccharideshaving terminal 3,6-anhydrogalactitol residue.For example, agarobiitol (3,6-anhydro-4-0-,B-D-galactopyranosyl-L-galactitol) wasobtained afterpartial reductive hydrolysis of agar, whereas car-

rabiitol (3,6-anhydro-4-0-fl-D-galactopyranosyl-D-galactitol), carrabiitol 4'-sulfate, and carrabii-tol 2,4'-disulfate were isolated as the mainhydrolysis products of undersulfated carrageenanfrom Tichocarpus crinitus (Gmel.) Rupr., kappa-carrageenan, and iota-carrageenan, respectively(Usov & Elashvili, 1989). The latter result indi-cates that partial reductive hydrolysis of galac-tans may be carried out with substantial retention

Table 1. Polysaccharide composition of Northwestern Pacific red seaweeds as revealed by complete and partial reductive hy-drolysis of biomass.

Order Number of Main poly- Group 3,6-Anhydrogalactose(family) species saccharide attribution contentb)

(number of (number of of galactanssamples a)) species) C) d)

Bangiales 4 (5) Galactan Agars M HCryptonemiales 9 (9) Galactan (6) Agars or carrageenans L M

Glucan (3)Gigartinales 6 (10) Galactan Carrageenans He)Rhodymeniales 1(3) Galactan Agars L LPalmariales 7 (14) Xylan (6) Agars L M

Glucan (1)Ceramiales 2 (2) Galactan Agars L L(Delesseriaceae)Ceramiales 11(11) Galactan (10) Agars H -(Ceramiaceae Glucan (1)and Rhodomelaceae)

a) Samples of the same species differ in habitat or belong to different generations.b Abbreviations: L: - low, M - moderate, H - high.c) Before alkaline modification.d) After alkaline modification.') Low 3,6-anhydrogalactose content was found only in tetra-sporophyte of Iridaea cornucopiae P. et R.


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of sulfate groups. It is interesting to note the char-acteristic difference between pairs of G1 (ano-meric carbon of /i-D-galactopyranose residue,103.7 and 104.8 p.p.m.) and A4 (carbon 4 of 3,6-anhydrogalactitol residue, 86.9 and 88.8 p.p.m.)resonances in carbon-13 NMR spectra of agar-obiitol and carrabiitol caused by different abso-lute configuration of 3,6-anhydrogalactitol resi-dues, whereas the positions of signals of othercarbon atoms are practically the same. The site ofsulfate may be easily determined from the largelow-field shift of the corresponding carbon reso-nance in the 13 C-NMR spectra of oligosaccha-rides.

Agarobiitol or carrabiitol, as the products of

partial reductive hydrolysis, may be unambigu-ously identified as acetates by GLC. This factmakes it possible to determine the absolute con-figuration of 3,6-anhydrogalactose residues, usingthis chromatographic technique alone, and to at-tribute unknown galactans to the agar or carrag-eenan group without isolation of polysaccharides,according to the results of partial reductive hy-drolysis of small amounts of algal biomass (Usov& Elashvili 1991a).

It should be noted that the yields of reduceddisaccharides depend not only on the proportionof 3,6-anhydrogalactose and galactose, but alsoon the degree of regularity of polysaccharide mol-ecules and on degree of sulfation, since agarobi-itol or carrabiitol sulfates as well as higher oli-gosaccharides are not registered by GLC. If theratio of 3,6-anhydrogalactose to galactose isless than 0.1, the detection of disaccharides afterpartial reductive hydrolysis of biomass may bedifficult. The situation may be improved by pre-

liminary alkaline treatment of the biomass, whereupon the 4-linked galactose 6-sulfate residuesin galactans are transformed into 3,6-anhydroga-lactose residues (Rees, 1961). Based on this ap-proach, it was possible to attribute galactans of40 species of Kamchatka red algae to the agar orcarrageenan groups according to the partial re-ductive hydrolysis data (Usov & Klochkova,1992). Some interesting observations were madeconcerning the relationship between the polysac-charide content and taxonomic position of the

algae (Table 1). For example, representatives ofPalmariales differ from all the other orders, sincethey contain xylans as the main polysaccharidecomponents. Cryptonemiales is the only orderwhere both agars and carrageenans were found indifferent species. In Ceramiales representatives ofthe family Delesseriaceae differ from those of twoother families in 3,6-anhydrogalactose content,and so on. Some of these observations may besignificant for chemical taxonomy of red sea-weeds.

Methylated derivatives of agarobiitol may beformed besides agarobiitol itself after partial re-ductive hydrolysis of highly methylated agarspresent in several algae. For example, 2-0-

methyl-, 6'-O-methyl-, and 2.6'-di-O-methyl-agarobiitol were identified in the partial hydrolyz-ate of Gelidiella acerosa (Forssk.) Feld. et Hamelbiomass. Structures of these compounds wereconfirmed using GLC - mass spectrometry (Usov& Ivanova, 1992). Identification and quantifica-tion of these methyl ethers together with fraction-ation may be useful in the study of distribution ofmethylated sugars in agar molecules.

The application of partial reductive hydrolysiswas especially effective in the structural investi-gation of a complex sulfated galactan isolatedfrom Laurencia nipponica Yamada. Its composi-tion and 13 C-NMR spectrum were characteristicfor non-regular structure. Among the partial re-ductive hydrolysis products agarobiitol and its2'-sulfate were shown to be the main compo-nents. Also isolated were 2-O-methyl-agarobiitol,two reduced disulfated tetrasaccharides of theporphyran type, one reduced branched pentasac-charide disulfate containing a xylose residue, and

a high-molecular fraction devoid of 3,6-anhydro-galactose residues. Structures of higher oligosac-charides were established using compositionanalysis, secondary ion mass spectrometry, and13 C-NMR spectroscopy. The tetrasaccharideswere shown to have identical sugar chains anddiffer only in the position of one sulfate group (atC-2 of internal or terminal /B-D-galactopyranoseresidue, respectively). The pentasaccharide hadthe same backbone and sulfation pattern with anadditional P-D-xylopyranose unit located at po-


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sition 3 of a 4-linked a-L-galactopyranose 6-sul-fate residue. It was concluded from these datathat the parent polysaccharide is really a xyloga-lactan with hybrid backbone containing both aga-rose and porphyran type segments, where sulfategroups occupy mainly position 2 of 3-linked E-D-galactopyranose residues (Usov & Elashvili,1991b).

Conclusion

The reductive hydrolysis procedures were used inthe present work for two purposes. The first onewas the rapid preliminary characterization of the

polysaccharide composition fora large series ofred seaweeds. In this case small samples of algalbiomass were subjected to complete and partialreductive hydrolysis. Monosaccharide composi-tion of the biomass reflected the content of dif-ferent types ofpolysaccharides. Chromatographicevidence on the absolute configuration of 3,6-anhydrogalactose residues permittedalgal galac-tans to be attributed to the agar or carrageenangroups, and determination of 3,6-anhydrogalac-tose to galactose ratio made it possible to predictsome practically valuable properties of thesepolysaccharides without their isolation from thealgal biomass. The method provided a real pos-sibility to collect data on the polysaccharide com-position for use in the chemical taxonomy of redseaweeds.

The second purpose was the partial fragmen-tation of a complex agar-like galactan. Practicallycomplete splitting of 3,6-anhydrogalactosidicbonds took place regardless of the position of

other substituents, giving rise to reduced oligosac-charides where sulfate groups and even xyloseresidues were retained. This splitting was muchmore extensive than enzymatic hydrolysis withbeta-agarase. The resulting reduced oligosaccha-rides were more stable and more convenient forchromatographic separation than the products ofpartial acid hydrolysis or methanolysis. Their

structures were elucidated using mass spectrom-etry and 13C-NMR spectroscopy, affording valu-able information on the structural features of theparent polysaccharide.

References

Garegg, P. J., B. Lindberg, P. Konradsson & I. Kvarnstram,1988. Hydrolysis of glycosides under reducing conditions.Carbohydr. Res. 176: 145-148.

Lahaye, M., W. Yaphe, M. T. Phan Viet & C. Rochas, 1989.'3 C-N.M.R. Spectroscopic investigation of methylated andcharged agarose oligosaccharides and polysaccharides.Carbohydr. Res. 190: 249-265.

Painter, T. J. 1983. Algal polysaccharides. In G. O. Aspinall(ed.), The Polysaccharides. Acad. Press, New York2: 195-285.

Rees, D. A., 1961. Estimation of the relative amounts of iso-meric sulphate esters in some sulphated polysaccharides.J.Chem. Soc. 5168-5171.

Stevenson, T. T. & R. H. Furneaux, 1991. Chemical methodsfor the analysis of sulphated galactans from red algae. Car-bohydr. Res. 210: 277-298.

Usov, A. I., 1984. NMR spectroscopy of red seaweedpolysaccharides: agars, carrageenans, and xylans. Bot. mar.27: 189-202.

Usov, A. I., 1992. Sulfated polysaccharides of the red sea-

weeds. Food Hydrocolloids 6: 9-23.Usov, A. I. & M. Ya. Elashvili, 1989. Specific fragmentation

of red algal galactans under reducing conditions. InF. E. C. S. Fifth Int. Conf. Chem. Biotechnol. Biol. ActiveNat. Prod., Conf. Proc. Bulgarian Acad. Sci., Sofia, 2:346-350.

Usov, A. I. & M. Ya. Elashvili, 1991a. Quantitative determi-nation of 3,6-anhydrogalactose derivatives and partial frag-mentation of the red algal galactans under reductive hy-drolysis conditions. Bioorg. Khim. 17: 839-848 (inRussian).

Usov, A. I. & M. Ya. Elashvili, 1991b. Polysaccharides ofalgae. 44. Investigation of sulfated galactan from Laurencianipponica Yamada (Rhodophyta, Rhodomelaceae) usingpartial reductive hydrolysis. Bot. mar. 34: 553-560.

Usov, A. I. & E. G. Ivanova, 1992. Polysaccharides of algae.46. Studies on agar from the red seaweed Gelidiella ace-rosa. Bioorg. Khim. 18: 1108-1116 (in Russian).

Usov, A. I. & N. G. Klochkova, 1992. Polysaccharides ofalgae. 45. Polysaccharide compositionof red seaweeds fromKamchatka coastal waters (Northwestern Pacific) studiedby reductive hydrolysis of biomass. Bot. mar. 35: 371-378.

a new chemical tool for characterization and partial depolymerization

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