nu structures

Room temperature, low-field lZC-n.m.r. spectra of degraded carrageenans.

Part II. O.n the sp.ecifi.'city of the. autohydrolysm reaction tn kap,pa/ ota and mu/nu structures Marina Ciancia, Maria C. Matulewicz~', Carlos A. Stortz~ and Alberto S. Cerezo~,:~ Departmento de Quimica Orgdnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabell6n 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina (Received 11 April 1991; revised 26 June 1991) A kappa/iota carrageenan from Gigartina skottsbergii and a partially cyclized mu/nu carrageenan from Iridaea undulosa were submitted to autohydrolysis. The 13C-n.m.r. spectra of the degraded products give structural information on the polysaccharides and show clearly that besides the known splitting of 3,6-anhydrogalactosidic linkaoes, the linkage between ot-o-galactose2,6-disulphate and fl-o-galactose 4-sulphate is cleaved with similar specific reaction rate. Keywords: laC-n.m.r.;spectroscopy;carrageenan;autohydrolysis;specificity

Introduction Autohydrolysis of kappa/iota carrageenans splits the glycosidic linkages between 3,6-anhydrogalactose (or its 2-sulphate) and fl-D-galactose 4-sulphate residues 1'2. Therefore, a controlled degradation is produced yielding representative fragments which give non-viscous, concentrated solutions necessary for ~3C-n.m.r. spectroscopy determinations with low-field equipment and at room temperature 1. These spectra render not only structural information but also a key to the specificity of the autohydrolysis reaction. Herein are reported the ~3Cn.m.r, spectra of degraded kappa/iota-type and partially cyclized m u / n u carrageenans; these spectra give structural information on the polysaccharides and show that, besides the above mentioned splitting of the 3,6anhydrogalactosidic linkage, the linkage between ~-Dgalactose 2,6-disulphate and fl-D-galactose 4-sulphate is cleaved with similar specific reaction rate.

Experimental The retrieval of kappa/iota 3 and m u / n u 4 carrageenan samples, autohydrolysis 1.2 and borohydride reduction 1 procedures are described elsewhere. Molecular weight determinations were carried out by measurement of reducing power 5'6. 13C-n.m.r. was obtained at 25.2MHz on a Varian XL-100 apparatus equipped with a 620 L-100 computer. Spectra were run at room temperature using solutions * For Part I, see ref. 1. ~"Research Member of the National Research Council of Argentina (CONICET). :~To whom correspondenceshould be addressed. 0141-8130/91/060337-04 © 1991 Butterworth-HeinemannLimited

of the oligosaccharide mixtures ( ~ 3 5 rag) in 0.4 ml of 1:1 H 2 0 / D 2 0 in a 5-mm tube. A pulse angle of 90 °, maximum acquisition time (0.8 s) and no pulse delay were classic parameters. Chemical shifts were measured relative to dioxan as external standard, and indicated referred to TMS according to the equation 6TMS 6diogan"~ 67.4 ppm. Proton-decoupling and deuterium-lock were used in all cases. The number of acquisitions varied between 100 000 and 200 000. =

Results The kappa/iota-like (for idealized repeating units of carrageenans see Table 1) carrageenan (molar ratio Gal: 3,6-AnGal: sulphate 1.00:0.64:1.32) was isolated from the cystocarpic stage of Gioartina skottsberoii (carrageenan 1C 1, Ref. 3). This carrageenan was autohydrolysed giving the results shown in Table 2. The rate constants of the reaction were calculated from molecular weight (0.077 h - ~) and internal 3,6-anhydrogalactose (0.071 h-1) determinations. After 62.5 h autohydrolysis, the loss of sulphate groups was ~ 2 0 % and the remaining glycosidicallylinked 3,6-anhydrogalactose was 11% of the original. Table 1 Idealized repeating units of carrageenans

Kappa Iota Mu Nu Lambda

4-1inked ~-galactose

3-1inked fl-galactose

3,6-anhydro 3,6-anhydro 2-sulphate 6-sulphate 2,6-disulphate 2,6-disulphate

4-sulphate 4-sulphate 4-sulphate 4-sulphate 2-sulphate (70%)

Int. J. Biol. Macromol., 1991, Vol. 13, December

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~3C-n.m.r. of degraded carrageenans. II: M. Ciancia et al. The partially cyclized m u / n u carrageenan (molar ratio Gal: 3,6-AnGal: sulphate 1.00:0.21:1.21) was isolated from Iridaea undulosa and subjected to structural analysis as previously reported (carrageenan As, Ref. 4); methylation analysis indicated ~ 2 5 % contamination

with lambda structures 4. Results of its autohydrolysis are reported elsewhere2. The reaction constants were 0.081 h-1 (from molecular weight determinations) or 0.076h -1 (from linked 3,6-anhydrogalactose determinations). After 36 h autohydrolysis the sulphate loss was ~ 30% and the remaining glycosidically-linked 3,6-anhydrogalactose was also ~ 3 0 % of the original. The room temperature, 25 MHz 13C-n.m.r. spectra of the degraded carrageenans are shown in Figure 1. The chemical shifts and relative areas of the anomeric signals are given in Table 3 with the corresponding assignments 7. As the relative area of the peak corresponding to C-1 of an open chain, reducing 3,6-AnGal is considerably smaller and not comparable to that of the cyclic, linked galactose units, areas in Table 3 were calculated normalized to the latter. The assignments were determined with the aid of methylation analysis of the parent carrageenans 4'8. The chemical shifts of the non-anomeric carbons (not shown) agree with those assignments. Borohydride-reduced products gave spectra in which the peaks corresponding to the reducing ends were replaced by those corresponding to primary hydroxyl groups (around 64 ppm).

Table 2 Autohydrolysis of the kappa/iota carrageenan Time (h)

Linked 3,6-AnGal (%)

Mol. wt.

0 1 2 3 4 5 6 7 8 11.5 15.5 19 23 25.5 28.5 32.5 36.5 42.5 46.5 51 58 62.5

37.5 34.5 31.7 28.8 25.6 18.6 18.3 17.8 16.9 16.2 15.6 14.5 12.1 11.5 10.6 8.9 7.5 6.5 6.2 5.4 4.3 4.2

22200 6680 3730 2830 2370 2070 1880 1340 1230 1030 911 911 878 787 770 761 751 716 668 752 752 668

Discussion Previous knowledge 1'2 showed that autohydrolysis of kappa/iota carrageenans proceeds through splitting of the glycosidic linkages between 3,6-anhydrogalactose (or its 2-sulphate) and fl-I~-galactose 4-sulphate residues. The autohydrolysis of the kappa/iota and the partially cyclized m u / n u carrageenans produced major amounts of two disaccharides, namely: (a) kappa carrabiose, in

i

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,

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I 80

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Figure 1 The 25 MHz ~3C-n.m.r.s~ctra ofcarrageenan samples of the kappa/iota (a) and mu/nu (b) type in the range 110-50 ppm determined at room temperature

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Int. J. Biol. Macromol., 1991, Vol. 13, December

13C-n.m.r. of de,Traded carra~Teenans. II: M. Ciancia et al Table

3 Relative areas and assignments of the anomeric signals in the spectra of the degraded carrageenans Assignmentsc

C- 1

A r e a s a'b

(ppm)

(%)

C-1 of

linked to

Kappa/iota carraoeenan 105.3 104.0 103.2 101.6 98.8 98.4 97.4 96.5 93.2 92.4 92.0 91.0

12 22 5 5 3 5 11 3 4 6 4 22

fl-Gal (4S) fl-Gal 4S /~-Gal (4S) fl-Gal 2, 4S ~t-Gai 2, 6S n-Gal 6S Red.fl-Gal (6S)e ct-Gal 6S Red.~t-Gal (6S) ~t-3,6-AnGal 2S ~,-3,6-AnGal 2S Red.3,6-AnGal

(Red.) ~-Gal 6(2, 6)dS Red.3,6-AnGal ~t-3,6-AnGal 2S ~t-3,6-AnGal 2S fl-Gal 4S fl-Gal 4S -~-Gal -fl-Gal 4(2, 4)S fl-Gal --

105.2

39

103.9

7

101.4 98.1 97.2 95.0 94.6 93.1 n.f.f

4 11 15 4 4 8 7

/~-Gal (4S) fl-Gal (6S) //-Gal 4S /~-Gal 2(2, 4) (2, 6)S fl-Gal 2S ~t-Gal 6S Red.fl-Gal 6S" ~t-3,6-AnGal fl-Gal 2(2, 6)S Red.~t-Gal (6S) Red.3,6-AnGal

(Red.) ~t-Gal 6S ~t-Gal 2(2, 6)S Red.3,6-AnGal (Red.) ~t-Gal (6S) ~t-3,6-AnGal fl-Gal 4(2, 4)S -fl-Gal 2S ~t-Gal 2(2, 6) (6)S ---

Mu/nu carraoeenan

• Areas were calculated as average of three different expansions of spectra. bAreas for fl-Gal 4S units (103.9 - 104.0ppm) and Red.3,6-AnGalunits (91.0ppm) are consideredequal, as the absorption values for C-1 of the hydrated aldehyde are not comparable with those of anomericcarbons. "Assignmentswere determinedwith the aid of methylationanalysisof the parent carrageenans. d Data in parenthesis indicate optional assignments. ' Mutarrotated from a linked u-Gal unit. f n.f. = not found.

agreement with the before mentioned specificity of the reaction, and (b) 4-O-(4-sulpho-fl-o-galactopyranosyl)D-galactose, indicating that also some of the ~t-ogalactosidic linkages are hydrolysed. The fact that the rate constants, calculated using molecular weight and linked 3,6-anhydrogalactose were similar, suggests that the energies of both 0t-linkages, 3,6-anhydrogalactosidic and galactosidic, are levelled off, possibly by the influence of the sulphate groups present in certain positions. It has been found L2 that during autohydrolysis part of the sulphate ester groups is cleaved. The identification of the products of the reaction (Table 3) and methylation analysis of the parent carrageenans '~'s showed that most of these groups were on C-2 of the 0t-o-galactose units whose linkages were broken. Besides, when the kappa carrageenan from Eucheuma cottonii 2 and a kappa fraction from Gioartina skottsberoii 8 were subjected to autohydrolysis the glycosidic linkage between 3,6anhydrogalactose and fl-o-galactose 4-sulphate was also split but with a lower specific rate constant (ca. 0.04 h - ~ against ca. 0.08 h-1 for a iota-like carrageenan). These facts indicate that sulphation on C-2 of either ~-galactose or 3,6-anhydrogalactose units increases the rate of the autohydrolysis reaction. This has also been shown for regular acid hydrolysis9. On the basis that the 2-sulphate of the 0t-galactose unit is necessary for the autohydrolysis reaction, but that the reducing galactose units produced do not carry sulphate

on C-2, it becomes evident that this sulphate is cleaved after glycosidic hydrolysis, as occurred with the 3,6anhydrogalactose units a. In the case of the production of kappa carrabiose, the hydrolysis of the sulphate group from the 3,6-anhydro-0t-o-galactose 2-sulphate residue was explained 1 considering that after hydrolysis of the 3,6-anhydrogalactosidic linkage, the pyranose ring opens, being C-1 and C-2 in an open chain; therefore, the acid lability of the 2-sulphate groups may increase considerably. Obviously, this explanation cannot be applied to the preferential hydrolysis of the C-2-sulphate of the ~t-ogalactose 2-sulphate units. Clancy and Turvey 1° found an increase in the rate of hydrolysis of the sulphate group of o-glucose 3-sulphate when compared with the methyl glycosides (208% faster than the/% and 35% than the ~-). It is noteworthy that re-investigation of the 13C-n.m.r. spectrum of a kappa/iota hybrid carrageenan from Iridaea undulosa (mainly hydrolysed to kappa carrabiose) a led to the conclusion that the 3,6-anhydrogalactosidic linkages not broken during autohydrolysis probably corresponded to the diads 3,6-AnGal-,fl-Gal and 3,6-AnGal 2-sulphate --, B-Gal. The same fact was observed with the carrageenans studied in this paper (Table 3); therefore, sulphation on C-4 of the fl-o-galactose unit plays a significant role in this reaction. Autohydrolysis of the kappa/iota carrageenan produced ~ 5 0 % of kappa carrabiose; this fact and the above mentioned cleavages indicate the presence of major

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13C-n.m.r. o f degraded carrageenans. II: M. Ciancia et al.

quantities of the triad fl-Gal 4-sulphate-~ 3,6-AnGal (or its 2-sulphate) ~ fl-Gal 4-sulphate. In the same way, a triad fl-Gal 4-sulphate-~ct-Gal 2-sulphate (or 2,6disulphate) ~ fl-Gal 4-sulphate can be suggested too, confirming thus the presence of ,~20% of m u / n u carrageenans. The spectrum also shows as minor units (Table 3): fl-galactose units non-sulphated, sulphated on C-2 or on C-2 and C-4, linked to ~-galactose 6-sulphate and to 3,6-anhydrogalactose 2-sulphate. The autohydrolysis of the degraded m u / n u carrageenan yielded a small amount of kappa carrabiose, indicating again the present of the triad fl-Gal 4-sulphate-~3,6-AnGal (or its 2-sulphate) ~ f l - G a l 4-sulphate in the undegraded product. On the other hand, major amounts of the disaccharide fl-Gal 4-sulphate ~ Gal (or its 6-sulphate) confirm the above mentioned triad, and thus the m u / n u structure. Although 25% oflambda-like carrageenan was present in this fraction 4, only small amounts of its diads can be seen in the spectrum. This is possibly due to the meagre (or no) fragmentation suffered by this molecule,

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thus leading to short spin-spin relaxation times usually associated with these large molecules.

Acknowledgements The authors are indebted to the UMYMFOR (FCEyNCONICET) for technical assistance. This work was supported by grants from CONICET, IFS (Sweden), TWAS (BC 89-22) and UNESCO (871.716.9(2) & (3)).

References 1 Stortz,C. A. and Cerezo,A. S. Int. J. Biol.Macromol. 1991,13, 101 2 Stortz,C. A. and Cerezo,A. S. Carbohydr. Res. 1987, 166, 317 3 Matulewicz,M. C., Ciancia, M., Noseda, M. D. and Cerezo, A. S. Phytochemistry 1989,28, 2937 4 Stortz,C. A. and Cerezo,A. S. Carbohydr. Res. 1986, 145, 219 5 Somogyi,M. J. Biol. Chem. 1952, 195, 19 6 Park,J. T. and Johnson, M. J. J. Biol. Chem. 1949, 181, 149 7 Stortz,C. A. and Cerezo,A. S. Carbohydr. Polym. (in press) 8 Ciancia, M., Matulewicz, M. C. and Cerezo, A. S. unpublished results 9 Lloyd,P. F. and Forrester, P. F. Carbohydr. Res. 1971, 19, 430 10 Clancy,M. J. and Turvey,J. R. J. Chem. Soc. 1961,2935