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Studies on uronic acid materials
STUDIES ON URONIC PART XXX*_ MAMINATION FRO&%Acacia
ACID MATERIALS OF THF3Z
drepanolobium
FRACTIONS
OBTAINED
GUM
D. M. W. ANDERSON AND 1. C. M. DEA
Department of Chemistry, The University, Edinburgh 9 (Great Britain) (Received
March 25th, 1968)
ABSTRACT
Dissolution of A. drepanolobizun gum Fraction C (the water-insoluble gel) has been attempted with eight solvents. Sodium borohydride solution (1%) was found to be the best solvent, giving a 69% yield of Soluble-fraction C. Molecularsieve chromatography with Bio-Gel P3QO indicated that Soluble-fraction C has a much higher molecular weight than the cold water-soluble gum (Fraction A), and this has been con&med by light-scattering measurements. Smith-degradation and methylation analyses have indicated that Fractions A, B, and Soluble-fraction C are structuralIy very similar. INTRODUCTION
In a previous paper’, Fraction A (water soluble), Fraction B (soluble in M sodium chloride) and Fraction C (the insoluble gel) of A. drepanolobium gum were compared analytically. Four aldobiouronic acids were also characterised after hydrolysis of Fraction’A with N sulphuric acid. In this Paper, methods for dissolving Fraction C are explored. After dissolution in 1% sodium borohydride solution, Soluble-fraction C is compared with Fractions A and B in terms of (a) Smith degradation and methylation analyses, and (b) molecular weight measurements, in order to ascertain whether the insolubility of Fraction C arises from structural or from molecular weight differences. MATERIALS
AND
MElEHODS
The gum nodules from A. drepanolobizun Harms ex Sjijstedt were collected by Mr. W. M. C. Bagshawe, Frovincial Forest Officer, at Tabora, Western Province, Tanganyika, in July 1961. Powdered A. drepanolobium gum (45 g) was fractionated as described previously’ to give Fractions A (35 g), B (3.5 g), and C (5.0 g). The standard analytical methods have been described previously3. Paper chromatography was carried out on Whatman No. 1 paper with the following solvent *for
Part XXIX,
Carbohyd. Res.,
see Ref. 1. 8 (1968)
440-447
URONIC ACIDS. XXX
441
systems (v/v); (a) benzene-butyl alcoho!-pyridine-water (1:5:3:3, upper layer); (b) ethyl acetate-acetic acid-formic acid-water (l&3:1:4); (c) ethyl acetate-pyridine+ water (104~3); (d) butyl alcohol-ethanol-water (4:1:5, upper layer); (e) butyl alcoholacetic acid-water (4: 1:5, upper layer); cf) butanone-water-cone. ammonia (200: 17: 1). R, values of methylated sugars refer to distances moved relative to 2,3,4,6-tetra-0methyl-D-glucose in solvent (6)_ G.1.c. of mixtures of O-methyl sugars was carried out as described previously4 on columns of (i) 15% by weight of poly@utane-i,4diol succinate) on tX-70 mesh Celite (120 x 0.5 cm) at 175”, and (ii) 15% by weight of poly(ethylene glycol adipate) on 60-70 mesh Celite (75 x 0.5 cm) at 160’. Retention times (7’) are given relative to that of methyl 2,3,4,6-tetra-O-methyl-P-D-glucopyranoside. Polysaccharides were methylated successively with methyl sulphate and sodium hydroxide solution, and with methyl iodide and silver oxide. Methanolyses were carried out under reflux for 7 h with 5% methanolic hydrogen chloride. Light-scattering measurements were made with a SOFICA Model 42000 Photo Gonio Diffusometer, using unpolarised green light (5460 A). Weight-average molecular weights, m,, were calculated as described previously5*6. RESULTS
Attempts to dissolve Fraction C. - It has already been shown’ that Fraction C is soluble in N sodium hydroxide to the extent of 1% (w/v). This method of dissolution is not entirely satisfactory, since alkaline degradation of the polysaccharide will probably occur. Different methods of dissolution were therefore attempted. Samples of Fraction C (120 mg) were extracted with (ii)distilled water; (E) 3M sodium chloride; (iii) 3~ magnesium chloride; (iv) 7~ urea; (v) 2% disodium ethylenediaminetetraacetate solution (w/v); (vi) 2% sodium hexametaphosphate solution (w/v); (uii) 1% sodium borohydride solution (w/v); (viii) 0.1~ phosphate buffer, pH 9.0; (ix) 3% sodium borate soiution (w/v)_ Each extract was filtered, dialysed against tap water for 48 h, and freeze-dried. Table I shows the percentage of Fraction C dissolved by these solvents. All of the freeze-dried products were water-soluble. TABLE I SOLUEILITY
OF FRACTION
c
IN
DIFFERENT
SOLVENTS
Solvents
Percentage
Distilled water 3~ Sodium chloride 3~ Magnesium chloride 7M Urea 3% Solution (w/v) of sodium borate 0.1~ Phosphate buffer, pH 9.0 2% Solution (w/v) of sodium salt of ethylenediamine tetraacetate 2% Solution (w/v) of sodhim hexametaphosphate 1% Solution (w/v) of sodium borohydride
1 1.5 1.5 2 7 10.5 17.5
of Fraction C dissolved
47 69
Carbohyd. Res., 8
(1968) 440-447
D. M. W. ANDERSON, I. C. M. DEA
442
The best solvent for Fraction C is undoubtedly 1% sodium borohydride solution (w/v). Fraction C (3.75 g) was therefore extracted with this solvent (400 ml), filtered, dialysed against tap water for 48 h, electrodialysed, and freeze-dried, to give Solublefraction C (2.25 g), for which the analytical data are shown in Table II. Hydrolysis (N sulphuric acid for 7 h at 1000) of Soluble-fraction C, followed by paper chromatography, indicated the presence of galactose, arabinose, rhamnose, and the same four aldobiouronic acids which were detected2 in Fractions A, B, and C. TABLE
II
ANALYTICAL
DATA
FOR
SOLUBLE-FRACTION
c
Soluble-fraction Moisture, %
Nitrogen, % Intrinsic viscosity, cm3g-l [ah (c 1.0, water) Equivalent weight Hence, uranic acid anhydride. % Galactose, % Arabinose, % Rhamnose, % Formic acid released on periodate oxidation (moles/g x 103) Periodate reduced (moles/g x 103)
C
10.2 0.85 11.1 + 79.5”
1955
9.0 51 39 1 1.05 3.95
In order to check whether Fraction C had been degraded by treatment with 1% sodium borohydride solution (w/v), Fractions A, B, and Soluble-fraction C were examined by molecular-sieve chromatography with a previously calibrated3*’ “BioGel P300” column (45 x 4.5 c-m). The elution volume of Soluble-fraction C was less than that of “Blue Dextran 2000”; Fractions A and B were eluted at the same elution volume as “3lue Dextran” (Ph armacia, IJppsala, Sweden). This suggested that Soluble-fraction C had a higher molecular weight tnan Fractions A and B. Light-scattering measurements subsequently gave weight-average molecular weights of 9.5 x 105, 10.2 x lo*, and 22.0 x IO’, for Fractions A, B, and Soluble-fraction C, respectively. Methyl&ion analysis of Fractions A, B, and Solubie-fraction C. - The three fractions (300 mg) were methylated exhaustively_ Yields, specific rotations, and methoxyl contents (not raised on further attempted methylation) of the products are shown in Table III. On methanolysis and g.1.c. examination of the mixture of methyl glycoaides, the methylated products of each fraction gave identical traces by using columns (i) and (Z). Results of g.1.c. examination are shown in Table XV; retention times are comparable with those for methyl glycosides from authentic O-methyl sugars. A portion of the methanolysate from each methylated product was hydrolysed with N sulphuric acid for 4 h on a boiling-water bath. The cooled solutions were neutralised with barium carbonate, filtered, treated with Amberlite resin IRCarbohyd.
Res., 8 (1968) 440-447
ACIDS. XXX
URONIC
12O(H+),
443
and concentrated.
Paper-chromatographic
examination
of the hydrolysates
in solvents (d) and cf) showed the presence of 2-U-methylgalactose and CO-methylarabinose, in addition to those O-methyl sugars already identified by g.1.c. of their
methyl glycosides (Table IV). TASLE DATA
III
FOR
MBTAYLATED
A, B,
FRACTIONS
AND
SOLUBLE-FRACTION
c
Fraction B
Fraction A
Solublefraction C
Yield” (mg) gke(c: -0, chloroform) .oo
240 f60” 40.8
200 +59”
250 +61’
41.0
40.7
aFrom 300 mg of each fraction. TABLE
IV
EXAMINATION AND
OF
BIETHYLATED
hiETHANOLYSIS
Relative retention times (T) glycosides on colunmsn: (ii) (i) 0.48
0.55 ; (1.05) 1.80 ; (1.05); 1.98 1.68 (3.05); (3.89) ; 6.42 14.6 ; (2.36);
AND
SOLUBLE-FRACTION
HYDROLYSIS
PRODUCTS
of methyl
3.20 (2.36) (3.89); (4.22) (4.22) 16.4 (3.05)
(0.50); (0.82) 1.26 ; (0.82); 1.43 1.65 2.44 ; (3.00) ; 5.08
0.64 (2.20) 1.76 (3.00); (3.44) (3.44) 11.4
; 2.75
METHYLATED
FRACTIONS
A
AND
B,
RG
in
O-Metlt_vlargars
solvent (d)
(0.50)
0.73
FRCM
c
0.97 0.79 0.82 0.82 0.56 0.88 0.73 0.73 0.73 0.52 0.32 0.35
2,3,4-tri-0-methyl-L-rhamnose 2,3,5-tri-0-methyl-I_-arabinose 2,3,4-tri-0-methyl-r_-arabinose 2,5-di-0-methyl-r_-arabinose 3,5-di-0-methyl-r-arabinose 3,4-di-0-methyl+arabl~ose 2,3,4,6-tetra-0-methyl-D-galactose 2,3,6-tri-O-methyl-D-galactose 2,4,6-tri-0-methyl-D-galactose 2,3.4-tri-O-methyl-o-galactose 2,4-di-0-methyl-D-galactose 2,3,4-tri-0-methyl-D-glucuronic acidb 2-O-methyl-~galactose 4-0-methyk-arabinose
a Figures in parentheses indicate T values of components which are incompletely resolved. bAs methyl ester methyl glycoside.
Smith degradation’ of Fractions, A, B, and Soluble-fraction C. - The purified fractions (1.5 g of polysaccharide) were dissolved in water (37.5 ml), and 0.25~ sodium metaperiodate (37.5 ml) was added. The oxidation mixtures were left in darkness for 48 h at room temperature, when the reactions were stopped by addition of ethylene glycol (2 ml). The solutions were dialysed against tap water for 48 h. Sodium borohydride (OS g) was added to each solution, and the mixtures were kept for 30 h at room temperature and then dialysed for a further 48 h. The three polyCarbohyd. Res., 8 (1968) 440-447
D. M. W. ANDERSON, I. C. M. DEA
alcohols were hydrolysed in N sulphuric acid for 48 h at room temperature, and the hydrolysates were neutralised with barium carbonate, atered, and deionised with Amberlite resin IR-120 @If). The solutions were each dialysed against distihed water (1 litre) for 24 h, and then against tap water for 48 h. The three Smith-degraded polysaccharides were isolated as freeze-dried products, and the dialysates were concentrated to syrups. Yields and analytical data for the three Smith-degraded polysaccharides are shown in Table V. Hydrolysis of the Smith-degraded poIysaccharides (N sulphuric acid for 7 h at lOO”), followed by paper chromatography, indicated the presence of only galnctose and arabinose. The Smith-degraded polysaccharides were hydrolysed with 0.5~ sulphuric acid for 1 h on a boiling-water bath. Paper-chromatographic examination indicated the presence of galactose, arabinose, and two neutral disaccharides having the mobilities of 3-O+-D-galactopyranosyl-D-galactose [RGrrl0.49 in solvent (a>, 0.54 in solvent (c)l and 6-0-B-D-galactopyranosyl-D-galactose [RGaI0.31 in solvent (a), 0.38 in solvent (c), major component]. TABLE ANALYTICAL
V DATA
FOP. THE
ShfITH-DEGRADED
POLYSACCHARIDES
Moisture, % Nitrogen, % Yield of polysaccharide (g) Yield, % Galac:ose, % Arabinose, % EC& (;-_1.0, water) Formic acid released on periodate oxidation [mole/g] X 10s Periodate consumed [mole/g] x 1Oa periodate consumption Ratio of formic acid released
A
Fraction B
Soluble-fraction
1.01 1.11 55.5 43 57 +4s” 0.94
10.5 0.98 1.14 57 44 56 -l-45” 0.96
10.8 0.32 1.12 56 44 56 +45” 0.97
3.38
3.47
3.45
3.59
3.62
3.56
Fracrion 11.9
C
Chromatographic examination of the three dialysates in solvents (a) and (c) showed the presence of glycolaldehyde and glycerol, and traces of ethylene glycol and threitol. The traces of ethyIene gIycol wouId be expected from non-reducing, arabinopyranosyl end-groups. The traces of threitol would be expected from D-galactopyranosyl residues having a substituent on C-4, as in the a-D-(1+4)-linked aldobiouronic acids previously detected in the hydrolysates of Fractions A, 3, and C. Methylation analysis of Smith-degradedproducts of Fractions A, B, and Solublefraction C. - The three degraded products (200 mg) were methylated exhaustively by using the methods of Haworth and Purdie. Yields, specific rotations, and methoxyl contents (not raised on further attempted methylation) of the products are shown in Table VI. On methanolysis and g.1.c. examination of the mixtures of methyl Carbohyd.
Res., 8 (1968)
440447
URONIC
ACIDS.
445
XXX
glycosides with columns (i) and (ii), the thee methyIated products gave identical results. The results of the g.1.c. examination are shown in Table VII. A portion of each methanolysate was hydrolysed (N sulphuric acid for 4 h at 100”). The cooled solutions were neutralised with barium carbonate, filtered, treated with Amber&e resin IR-120(W), and concentrated. Paper-chromatographic examination of the hydrolysates in solvents (d) and u) showed the presence of 2,6-d& and 2-O-methylgalactose, in addition to those O-methyl sugars already identified by g.1.c. of their methyl glycosides (Table VII). TABLE DATA
VI FOR
hfETHYLATED,
SMITH-DEGRADED
FRACIXONS
A, B,
AND
SOLUBLE-FRACTION
Fraction A
Fraction B
Fraction C
156 -t-39” 40. I
150 +3g”
15s +38”
YieIdc (mg) [ah (c 1.0, chloroform) OMe,%
40.0
c
40.1
QFrom 200 mg of each fraction. TABLE
VII
EXAMINATION
OF
METHANOLYSIS
AND
HYDROLYSIS
PRODUCTS
FROM
METHYLATED,
SMITH-DEGRADED
POLYSACCKARIDES
Relativeretention rimes (JY)of methyl glycosides on columns: (ii) (i)
Ro in solvent (d)
Hence, O-methyl sugars identified
0.56; 0.74 1.06; 2.36 1.80; 3.22 1.68 3.90; 4.22 6.45 14.6; 16.4
0.97 0.82 0.82 0.88 0.73 0.73 0.52 0.55 0.32
2,3,5-tri-U-methyl-L-arabinose 3,5-di-0-methyl-r-arabinose 2,5-di-U-methyl+arabiiose 2,3,4,6-tetra-0-methyl-n-galactose 2,4,6-tri-0-methyl-o-galactose 2,3,4&i-O-methyl-n-galactose 2,4-di-0-methyl-D-galactose 2,6-di-0-methyl-n-galactose 2-O-methyl-D-galactose unknown sugars
1.53
0.50; 0.63 0.81; 1.76 1.26; 2.20 1.65 3.00; 3.45 5.08 9.9; 11.4 1.05
DISCUSSION
A. drepanolobium gum Fraction C is sparingly soluble ($ 1% w/v) in M sodium hydroxide. This method of dissolution is not satisfactory, since degradation may occur under such alkaline conditions. Eight different aqueous solvents for Fraction C were therefore investigated (Table I)_ 3~ Sodium chloride, 3~ magnesium chloride, and 7~ urea were unsuccessful solvents; hydrogen bonding between polysaccharide molecules is therefore unlikely to be the major reason for the insolubility of Fraction C. 3% Sodium borate solution and 0.1~ phosphate buffer dissolved Fraction C to the Carbohyd. Res., 8 (1968) 44CM47
D. M. W. ANDERSON,
I. C. M. DEA
extent of 7 and 10.5%, respectively; these solvents have pH 9, and this may account for the slight solubilisation effected. Fraction C was dissolved to a greater extent by 2% disodium ethylenediaminetetra-acetate solution (17.5%) and by 2% sodium hexametaphosphate solution (47%). The action of these solvents is probably dependent on the chelation of calcium and magnesiumg~‘o, and cross-linking by polyvalent metal ions might contribute to the insolubility of Fraction C in aqueous solutions. The higher solubility in sodium hexametaphosphate solution is expected, since its optimum condition? are at neutral pH and room temperature (i.e., the conditions of extraction); the optimum conditions for disodium ethylenediaminetetra-acetate solution’ are at pH 10 and 60-70”.
The best solvent found for Fraction C is 1% sodium borohydride solution. This has pH 9, and contains borate ions, but these facts cannot account for the dissolution action of this solvent in view of the resu1t.s reported above for sodium borate solution and for phosphate buffer. Dilute sodium borohydride solution has also been found” to be an effective solvent for water-insoluble gums from the genera Combrelum, Lannea, and Teclea.
Fraction C was extracted with 1% sodium borohydride solution in a largescale experiment to yield Soluble-fraction C as the freeze-dried, electrodialysed product. The yield of solubilised gum was 69%, based on the estimated dry weight of the insoluble gel. This represents virtually complete recovery of the polysaccharide present; all of the bark and other foreign matter in the gum sample had inevitably become concentrated in the gel (Fraction C). Analysis showed Soluble-fraction C to have the same sugar percentages as Fraction C; on periodate oxidation, the two fractions released the same amount of formic acid and reduced the same amount of periodate. Fractions A, B, and Soluble-fraction C were compared by using methylation analysis. The methylated products were obtained in similar yields, and had the same specific rotations. On methanolysis and g.1.c. examination, the three methylated products gave identical results. On Smith degradation, Fractions A, B, and Solublefraction C gave similar yields of Smith-degraded products. The analytical data
(Table V) for the Smith-degraded polysaccharides are almost identical, and methylation analyses of the three degraded polysaccharides gave identical results. There is therefore a close structural similarity between Fractions A, B, and Soluble-fraction C. Soluble-fraction C has a lower intrinsic viscosity than that obtained’ in M sodium hydroxide solution for Fraction C. Molecular-sieve chromatography on a previously calibrated Bio-Gel P300 column was therefore carried out to determine if the decrease in viscosity was the result of degradation of the polysaccharide during the extraction with sodium borohydride solution. Soluble-fraction C was eluted
before the elution volume of “blue dextran”; in contrast, Fractions A and B were eluted at the same elution volume as for ‘-blue dextran”. SolubIe-fraction C could therefore differ from Fractions A and B in having either a much higher molecular weight, or a different molecular shape l3 . Differences in shape imply differences in molecular structure, such as the frequency or pattern of branching. Since the Smith degradation Carbohyd. Res., 8 (1968) 440447
.
URONIC
ACIDS.
XXX
447
and methylation analyses showed that the three fractions are closely similar structurally, it seems unlikely that the differences between Fractions A, B, and Solublefraction C are due to differences in molecular shape. Light-scattering measurements confirmed that Soluble-fraction C has a much higher weight-average molecular weight (22.0 x 10’) than Fraction A (9.5 x 10’) and Fraction B (10.2 x 10’). The precise reasons why sodium borohydride solution dissolves Fraction Care not known; the interactions which cause the polysaccharide molecules to aggregate seem to be counteracted irrevocably, since, after freeze-drying, the product (Soluble-fraction C) remains water-soluble. Although it appears that gross degradation does not occur during the solubilising process, the possibility of some degradation’s occurring cannot be excluded. The true molecular weight of the gel (Fraction C) may beconsiderably higher than that indicated by the experiments involving Soluble-fraction C. ACKNOWLEDGMENTS
We thank Professor Sir Edmund Hirst, C. B. E., F. R. S., for his interest, the Science Research Council for a maintenance award (to I. C. M. D.), and Rowntree and Co. Ltd. (York) and Laing-National Ltd. (Manchester) for financial support. REFERENCES 1 Part XXIX: D. M. W. ANDERSON, I.C. M. DEA, AND R. N. SMITH, Carbolryd. Rcs., 7 (1968)320. D. M. W. ANDERSON AND I. C. M. DEA, Carbo/zyd_ Res., 5 (1967) 461. 3 D. IM. W. ANDERSON AND J. F. STODDART, C’arbohyd. Res., 2 (1966) 104. 4 D. M. W. ANDERSON, SIR EDM~D HIR.?.T,AND J. F. STODDART, J. Clrem. Sm. (CT), (1966) 19.59. 5 D. M. W. ANDERSON. SIR EDMUND HIR~T. S. RArihiAN, AND G. STAINSBY, Carbohyd, Res., 3 (1967) 308. 6 D. M. W. ANDERSON AND S. RAHMAN, Curbohyd. Res., 4 (1967) 298. 7 D. M. W. ANDERSON, I. C. M. DEA, S. IIAHt.fAN, AND J. F. STODDART, Cllem. Conzmun., (1965) 145. 8 F. SMITH AND R. MONTGOhlERY, The Chemistry of Plant Gums and Mucilages, ReinhoId, New York, 1959. 9 D. S. LETHAM, Australian J. Agri. Res., 12 (1961) 60. 10 E. C. COCKING, Biochem. J., 76 (1960) 51 P. 11 D. S. LETHAM, JZxptL CeN Research, 27 (196:!) 352. 12 I. C. M. DEA, unpublished work. 13 D. M. W. ANDERSON AND J. F. STODDART, Lab. Pratt., 16 (1967) 841.
2
Carbohyd. Res., 8 (1968) 440-447
ACID MATERIALS OF THF3Z
drepanolobium
FRACTIONS
OBTAINED
GUM
D. M. W. ANDERSON AND 1. C. M. DEA
Department of Chemistry, The University, Edinburgh 9 (Great Britain) (Received
March 25th, 1968)
ABSTRACT
Dissolution of A. drepanolobizun gum Fraction C (the water-insoluble gel) has been attempted with eight solvents. Sodium borohydride solution (1%) was found to be the best solvent, giving a 69% yield of Soluble-fraction C. Molecularsieve chromatography with Bio-Gel P3QO indicated that Soluble-fraction C has a much higher molecular weight than the cold water-soluble gum (Fraction A), and this has been con&med by light-scattering measurements. Smith-degradation and methylation analyses have indicated that Fractions A, B, and Soluble-fraction C are structuralIy very similar. INTRODUCTION
In a previous paper’, Fraction A (water soluble), Fraction B (soluble in M sodium chloride) and Fraction C (the insoluble gel) of A. drepanolobium gum were compared analytically. Four aldobiouronic acids were also characterised after hydrolysis of Fraction’A with N sulphuric acid. In this Paper, methods for dissolving Fraction C are explored. After dissolution in 1% sodium borohydride solution, Soluble-fraction C is compared with Fractions A and B in terms of (a) Smith degradation and methylation analyses, and (b) molecular weight measurements, in order to ascertain whether the insolubility of Fraction C arises from structural or from molecular weight differences. MATERIALS
AND
MElEHODS
The gum nodules from A. drepanolobizun Harms ex Sjijstedt were collected by Mr. W. M. C. Bagshawe, Frovincial Forest Officer, at Tabora, Western Province, Tanganyika, in July 1961. Powdered A. drepanolobium gum (45 g) was fractionated as described previously’ to give Fractions A (35 g), B (3.5 g), and C (5.0 g). The standard analytical methods have been described previously3. Paper chromatography was carried out on Whatman No. 1 paper with the following solvent *for
Part XXIX,
Carbohyd. Res.,
see Ref. 1. 8 (1968)
440-447
URONIC ACIDS. XXX
441
systems (v/v); (a) benzene-butyl alcoho!-pyridine-water (1:5:3:3, upper layer); (b) ethyl acetate-acetic acid-formic acid-water (l&3:1:4); (c) ethyl acetate-pyridine+ water (104~3); (d) butyl alcohol-ethanol-water (4:1:5, upper layer); (e) butyl alcoholacetic acid-water (4: 1:5, upper layer); cf) butanone-water-cone. ammonia (200: 17: 1). R, values of methylated sugars refer to distances moved relative to 2,3,4,6-tetra-0methyl-D-glucose in solvent (6)_ G.1.c. of mixtures of O-methyl sugars was carried out as described previously4 on columns of (i) 15% by weight of poly@utane-i,4diol succinate) on tX-70 mesh Celite (120 x 0.5 cm) at 175”, and (ii) 15% by weight of poly(ethylene glycol adipate) on 60-70 mesh Celite (75 x 0.5 cm) at 160’. Retention times (7’) are given relative to that of methyl 2,3,4,6-tetra-O-methyl-P-D-glucopyranoside. Polysaccharides were methylated successively with methyl sulphate and sodium hydroxide solution, and with methyl iodide and silver oxide. Methanolyses were carried out under reflux for 7 h with 5% methanolic hydrogen chloride. Light-scattering measurements were made with a SOFICA Model 42000 Photo Gonio Diffusometer, using unpolarised green light (5460 A). Weight-average molecular weights, m,, were calculated as described previously5*6. RESULTS
Attempts to dissolve Fraction C. - It has already been shown’ that Fraction C is soluble in N sodium hydroxide to the extent of 1% (w/v). This method of dissolution is not entirely satisfactory, since alkaline degradation of the polysaccharide will probably occur. Different methods of dissolution were therefore attempted. Samples of Fraction C (120 mg) were extracted with (ii)distilled water; (E) 3M sodium chloride; (iii) 3~ magnesium chloride; (iv) 7~ urea; (v) 2% disodium ethylenediaminetetraacetate solution (w/v); (vi) 2% sodium hexametaphosphate solution (w/v); (uii) 1% sodium borohydride solution (w/v); (viii) 0.1~ phosphate buffer, pH 9.0; (ix) 3% sodium borate soiution (w/v)_ Each extract was filtered, dialysed against tap water for 48 h, and freeze-dried. Table I shows the percentage of Fraction C dissolved by these solvents. All of the freeze-dried products were water-soluble. TABLE I SOLUEILITY
OF FRACTION
c
IN
DIFFERENT
SOLVENTS
Solvents
Percentage
Distilled water 3~ Sodium chloride 3~ Magnesium chloride 7M Urea 3% Solution (w/v) of sodium borate 0.1~ Phosphate buffer, pH 9.0 2% Solution (w/v) of sodium salt of ethylenediamine tetraacetate 2% Solution (w/v) of sodhim hexametaphosphate 1% Solution (w/v) of sodium borohydride
1 1.5 1.5 2 7 10.5 17.5
of Fraction C dissolved
47 69
Carbohyd. Res., 8
(1968) 440-447
D. M. W. ANDERSON, I. C. M. DEA
442
The best solvent for Fraction C is undoubtedly 1% sodium borohydride solution (w/v). Fraction C (3.75 g) was therefore extracted with this solvent (400 ml), filtered, dialysed against tap water for 48 h, electrodialysed, and freeze-dried, to give Solublefraction C (2.25 g), for which the analytical data are shown in Table II. Hydrolysis (N sulphuric acid for 7 h at 1000) of Soluble-fraction C, followed by paper chromatography, indicated the presence of galactose, arabinose, rhamnose, and the same four aldobiouronic acids which were detected2 in Fractions A, B, and C. TABLE
II
ANALYTICAL
DATA
FOR
SOLUBLE-FRACTION
c
Soluble-fraction Moisture, %
Nitrogen, % Intrinsic viscosity, cm3g-l [ah (c 1.0, water) Equivalent weight Hence, uranic acid anhydride. % Galactose, % Arabinose, % Rhamnose, % Formic acid released on periodate oxidation (moles/g x 103) Periodate reduced (moles/g x 103)
C
10.2 0.85 11.1 + 79.5”
1955
9.0 51 39 1 1.05 3.95
In order to check whether Fraction C had been degraded by treatment with 1% sodium borohydride solution (w/v), Fractions A, B, and Soluble-fraction C were examined by molecular-sieve chromatography with a previously calibrated3*’ “BioGel P300” column (45 x 4.5 c-m). The elution volume of Soluble-fraction C was less than that of “Blue Dextran 2000”; Fractions A and B were eluted at the same elution volume as “3lue Dextran” (Ph armacia, IJppsala, Sweden). This suggested that Soluble-fraction C had a higher molecular weight tnan Fractions A and B. Light-scattering measurements subsequently gave weight-average molecular weights of 9.5 x 105, 10.2 x lo*, and 22.0 x IO’, for Fractions A, B, and Soluble-fraction C, respectively. Methyl&ion analysis of Fractions A, B, and Solubie-fraction C. - The three fractions (300 mg) were methylated exhaustively_ Yields, specific rotations, and methoxyl contents (not raised on further attempted methylation) of the products are shown in Table III. On methanolysis and g.1.c. examination of the mixture of methyl glycoaides, the methylated products of each fraction gave identical traces by using columns (i) and (Z). Results of g.1.c. examination are shown in Table XV; retention times are comparable with those for methyl glycosides from authentic O-methyl sugars. A portion of the methanolysate from each methylated product was hydrolysed with N sulphuric acid for 4 h on a boiling-water bath. The cooled solutions were neutralised with barium carbonate, filtered, treated with Amberlite resin IRCarbohyd.
Res., 8 (1968) 440-447
ACIDS. XXX
URONIC
12O(H+),
443
and concentrated.
Paper-chromatographic
examination
of the hydrolysates
in solvents (d) and cf) showed the presence of 2-U-methylgalactose and CO-methylarabinose, in addition to those O-methyl sugars already identified by g.1.c. of their
methyl glycosides (Table IV). TASLE DATA
III
FOR
MBTAYLATED
A, B,
FRACTIONS
AND
SOLUBLE-FRACTION
c
Fraction B
Fraction A
Solublefraction C
Yield” (mg) gke(c: -0, chloroform) .oo
240 f60” 40.8
200 +59”
250 +61’
41.0
40.7
aFrom 300 mg of each fraction. TABLE
IV
EXAMINATION AND
OF
BIETHYLATED
hiETHANOLYSIS
Relative retention times (T) glycosides on colunmsn: (ii) (i) 0.48
0.55 ; (1.05) 1.80 ; (1.05); 1.98 1.68 (3.05); (3.89) ; 6.42 14.6 ; (2.36);
AND
SOLUBLE-FRACTION
HYDROLYSIS
PRODUCTS
of methyl
3.20 (2.36) (3.89); (4.22) (4.22) 16.4 (3.05)
(0.50); (0.82) 1.26 ; (0.82); 1.43 1.65 2.44 ; (3.00) ; 5.08
0.64 (2.20) 1.76 (3.00); (3.44) (3.44) 11.4
; 2.75
METHYLATED
FRACTIONS
A
AND
B,
RG
in
O-Metlt_vlargars
solvent (d)
(0.50)
0.73
FRCM
c
0.97 0.79 0.82 0.82 0.56 0.88 0.73 0.73 0.73 0.52 0.32 0.35
2,3,4-tri-0-methyl-L-rhamnose 2,3,5-tri-0-methyl-I_-arabinose 2,3,4-tri-0-methyl-r_-arabinose 2,5-di-0-methyl-r_-arabinose 3,5-di-0-methyl-r-arabinose 3,4-di-0-methyl+arabl~ose 2,3,4,6-tetra-0-methyl-D-galactose 2,3,6-tri-O-methyl-D-galactose 2,4,6-tri-0-methyl-D-galactose 2,3.4-tri-O-methyl-o-galactose 2,4-di-0-methyl-D-galactose 2,3,4-tri-0-methyl-D-glucuronic acidb 2-O-methyl-~galactose 4-0-methyk-arabinose
a Figures in parentheses indicate T values of components which are incompletely resolved. bAs methyl ester methyl glycoside.
Smith degradation’ of Fractions, A, B, and Soluble-fraction C. - The purified fractions (1.5 g of polysaccharide) were dissolved in water (37.5 ml), and 0.25~ sodium metaperiodate (37.5 ml) was added. The oxidation mixtures were left in darkness for 48 h at room temperature, when the reactions were stopped by addition of ethylene glycol (2 ml). The solutions were dialysed against tap water for 48 h. Sodium borohydride (OS g) was added to each solution, and the mixtures were kept for 30 h at room temperature and then dialysed for a further 48 h. The three polyCarbohyd. Res., 8 (1968) 440-447
D. M. W. ANDERSON, I. C. M. DEA
alcohols were hydrolysed in N sulphuric acid for 48 h at room temperature, and the hydrolysates were neutralised with barium carbonate, atered, and deionised with Amberlite resin IR-120 @If). The solutions were each dialysed against distihed water (1 litre) for 24 h, and then against tap water for 48 h. The three Smith-degraded polysaccharides were isolated as freeze-dried products, and the dialysates were concentrated to syrups. Yields and analytical data for the three Smith-degraded polysaccharides are shown in Table V. Hydrolysis of the Smith-degraded poIysaccharides (N sulphuric acid for 7 h at lOO”), followed by paper chromatography, indicated the presence of only galnctose and arabinose. The Smith-degraded polysaccharides were hydrolysed with 0.5~ sulphuric acid for 1 h on a boiling-water bath. Paper-chromatographic examination indicated the presence of galactose, arabinose, and two neutral disaccharides having the mobilities of 3-O+-D-galactopyranosyl-D-galactose [RGrrl0.49 in solvent (a>, 0.54 in solvent (c)l and 6-0-B-D-galactopyranosyl-D-galactose [RGaI0.31 in solvent (a), 0.38 in solvent (c), major component]. TABLE ANALYTICAL
V DATA
FOP. THE
ShfITH-DEGRADED
POLYSACCHARIDES
Moisture, % Nitrogen, % Yield of polysaccharide (g) Yield, % Galac:ose, % Arabinose, % EC& (;-_1.0, water) Formic acid released on periodate oxidation [mole/g] X 10s Periodate consumed [mole/g] x 1Oa periodate consumption Ratio of formic acid released
A
Fraction B
Soluble-fraction
1.01 1.11 55.5 43 57 +4s” 0.94
10.5 0.98 1.14 57 44 56 -l-45” 0.96
10.8 0.32 1.12 56 44 56 +45” 0.97
3.38
3.47
3.45
3.59
3.62
3.56
Fracrion 11.9
C
Chromatographic examination of the three dialysates in solvents (a) and (c) showed the presence of glycolaldehyde and glycerol, and traces of ethylene glycol and threitol. The traces of ethyIene gIycol wouId be expected from non-reducing, arabinopyranosyl end-groups. The traces of threitol would be expected from D-galactopyranosyl residues having a substituent on C-4, as in the a-D-(1+4)-linked aldobiouronic acids previously detected in the hydrolysates of Fractions A, 3, and C. Methylation analysis of Smith-degradedproducts of Fractions A, B, and Solublefraction C. - The three degraded products (200 mg) were methylated exhaustively by using the methods of Haworth and Purdie. Yields, specific rotations, and methoxyl contents (not raised on further attempted methylation) of the products are shown in Table VI. On methanolysis and g.1.c. examination of the mixtures of methyl Carbohyd.
Res., 8 (1968)
440447
URONIC
ACIDS.
445
XXX
glycosides with columns (i) and (ii), the thee methyIated products gave identical results. The results of the g.1.c. examination are shown in Table VII. A portion of each methanolysate was hydrolysed (N sulphuric acid for 4 h at 100”). The cooled solutions were neutralised with barium carbonate, filtered, treated with Amber&e resin IR-120(W), and concentrated. Paper-chromatographic examination of the hydrolysates in solvents (d) and u) showed the presence of 2,6-d& and 2-O-methylgalactose, in addition to those O-methyl sugars already identified by g.1.c. of their methyl glycosides (Table VII). TABLE DATA
VI FOR
hfETHYLATED,
SMITH-DEGRADED
FRACIXONS
A, B,
AND
SOLUBLE-FRACTION
Fraction A
Fraction B
Fraction C
156 -t-39” 40. I
150 +3g”
15s +38”
YieIdc (mg) [ah (c 1.0, chloroform) OMe,%
40.0
c
40.1
QFrom 200 mg of each fraction. TABLE
VII
EXAMINATION
OF
METHANOLYSIS
AND
HYDROLYSIS
PRODUCTS
FROM
METHYLATED,
SMITH-DEGRADED
POLYSACCKARIDES
Relativeretention rimes (JY)of methyl glycosides on columns: (ii) (i)
Ro in solvent (d)
Hence, O-methyl sugars identified
0.56; 0.74 1.06; 2.36 1.80; 3.22 1.68 3.90; 4.22 6.45 14.6; 16.4
0.97 0.82 0.82 0.88 0.73 0.73 0.52 0.55 0.32
2,3,5-tri-U-methyl-L-arabinose 3,5-di-0-methyl-r-arabinose 2,5-di-U-methyl+arabiiose 2,3,4,6-tetra-0-methyl-n-galactose 2,4,6-tri-0-methyl-o-galactose 2,3,4&i-O-methyl-n-galactose 2,4-di-0-methyl-D-galactose 2,6-di-0-methyl-n-galactose 2-O-methyl-D-galactose unknown sugars
1.53
0.50; 0.63 0.81; 1.76 1.26; 2.20 1.65 3.00; 3.45 5.08 9.9; 11.4 1.05
DISCUSSION
A. drepanolobium gum Fraction C is sparingly soluble ($ 1% w/v) in M sodium hydroxide. This method of dissolution is not satisfactory, since degradation may occur under such alkaline conditions. Eight different aqueous solvents for Fraction C were therefore investigated (Table I)_ 3~ Sodium chloride, 3~ magnesium chloride, and 7~ urea were unsuccessful solvents; hydrogen bonding between polysaccharide molecules is therefore unlikely to be the major reason for the insolubility of Fraction C. 3% Sodium borate solution and 0.1~ phosphate buffer dissolved Fraction C to the Carbohyd. Res., 8 (1968) 44CM47
D. M. W. ANDERSON,
I. C. M. DEA
extent of 7 and 10.5%, respectively; these solvents have pH 9, and this may account for the slight solubilisation effected. Fraction C was dissolved to a greater extent by 2% disodium ethylenediaminetetra-acetate solution (17.5%) and by 2% sodium hexametaphosphate solution (47%). The action of these solvents is probably dependent on the chelation of calcium and magnesiumg~‘o, and cross-linking by polyvalent metal ions might contribute to the insolubility of Fraction C in aqueous solutions. The higher solubility in sodium hexametaphosphate solution is expected, since its optimum condition? are at neutral pH and room temperature (i.e., the conditions of extraction); the optimum conditions for disodium ethylenediaminetetra-acetate solution’ are at pH 10 and 60-70”.
The best solvent found for Fraction C is 1% sodium borohydride solution. This has pH 9, and contains borate ions, but these facts cannot account for the dissolution action of this solvent in view of the resu1t.s reported above for sodium borate solution and for phosphate buffer. Dilute sodium borohydride solution has also been found” to be an effective solvent for water-insoluble gums from the genera Combrelum, Lannea, and Teclea.
Fraction C was extracted with 1% sodium borohydride solution in a largescale experiment to yield Soluble-fraction C as the freeze-dried, electrodialysed product. The yield of solubilised gum was 69%, based on the estimated dry weight of the insoluble gel. This represents virtually complete recovery of the polysaccharide present; all of the bark and other foreign matter in the gum sample had inevitably become concentrated in the gel (Fraction C). Analysis showed Soluble-fraction C to have the same sugar percentages as Fraction C; on periodate oxidation, the two fractions released the same amount of formic acid and reduced the same amount of periodate. Fractions A, B, and Soluble-fraction C were compared by using methylation analysis. The methylated products were obtained in similar yields, and had the same specific rotations. On methanolysis and g.1.c. examination, the three methylated products gave identical results. On Smith degradation, Fractions A, B, and Solublefraction C gave similar yields of Smith-degraded products. The analytical data
(Table V) for the Smith-degraded polysaccharides are almost identical, and methylation analyses of the three degraded polysaccharides gave identical results. There is therefore a close structural similarity between Fractions A, B, and Soluble-fraction C. Soluble-fraction C has a lower intrinsic viscosity than that obtained’ in M sodium hydroxide solution for Fraction C. Molecular-sieve chromatography on a previously calibrated Bio-Gel P300 column was therefore carried out to determine if the decrease in viscosity was the result of degradation of the polysaccharide during the extraction with sodium borohydride solution. Soluble-fraction C was eluted
before the elution volume of “blue dextran”; in contrast, Fractions A and B were eluted at the same elution volume as for ‘-blue dextran”. SolubIe-fraction C could therefore differ from Fractions A and B in having either a much higher molecular weight, or a different molecular shape l3 . Differences in shape imply differences in molecular structure, such as the frequency or pattern of branching. Since the Smith degradation Carbohyd. Res., 8 (1968) 440447
.
URONIC
ACIDS.
XXX
447
and methylation analyses showed that the three fractions are closely similar structurally, it seems unlikely that the differences between Fractions A, B, and Solublefraction C are due to differences in molecular shape. Light-scattering measurements confirmed that Soluble-fraction C has a much higher weight-average molecular weight (22.0 x 10’) than Fraction A (9.5 x 10’) and Fraction B (10.2 x 10’). The precise reasons why sodium borohydride solution dissolves Fraction Care not known; the interactions which cause the polysaccharide molecules to aggregate seem to be counteracted irrevocably, since, after freeze-drying, the product (Soluble-fraction C) remains water-soluble. Although it appears that gross degradation does not occur during the solubilising process, the possibility of some degradation’s occurring cannot be excluded. The true molecular weight of the gel (Fraction C) may beconsiderably higher than that indicated by the experiments involving Soluble-fraction C. ACKNOWLEDGMENTS
We thank Professor Sir Edmund Hirst, C. B. E., F. R. S., for his interest, the Science Research Council for a maintenance award (to I. C. M. D.), and Rowntree and Co. Ltd. (York) and Laing-National Ltd. (Manchester) for financial support. REFERENCES 1 Part XXIX: D. M. W. ANDERSON, I.C. M. DEA, AND R. N. SMITH, Carbolryd. Rcs., 7 (1968)320. D. M. W. ANDERSON AND I. C. M. DEA, Carbo/zyd_ Res., 5 (1967) 461. 3 D. IM. W. ANDERSON AND J. F. STODDART, C’arbohyd. Res., 2 (1966) 104. 4 D. M. W. ANDERSON, SIR EDM~D HIR.?.T,AND J. F. STODDART, J. Clrem. Sm. (CT), (1966) 19.59. 5 D. M. W. ANDERSON. SIR EDMUND HIR~T. S. RArihiAN, AND G. STAINSBY, Carbohyd, Res., 3 (1967) 308. 6 D. M. W. ANDERSON AND S. RAHMAN, Curbohyd. Res., 4 (1967) 298. 7 D. M. W. ANDERSON, I. C. M. DEA, S. IIAHt.fAN, AND J. F. STODDART, Cllem. Conzmun., (1965) 145. 8 F. SMITH AND R. MONTGOhlERY, The Chemistry of Plant Gums and Mucilages, ReinhoId, New York, 1959. 9 D. S. LETHAM, Australian J. Agri. Res., 12 (1961) 60. 10 E. C. COCKING, Biochem. J., 76 (1960) 51 P. 11 D. S. LETHAM, JZxptL CeN Research, 27 (196:!) 352. 12 I. C. M. DEA, unpublished work. 13 D. M. W. ANDERSON AND J. F. STODDART, Lab. Pratt., 16 (1967) 841.
2
Carbohyd. Res., 8 (1968) 440-447