Studies on uronic acid materials

Curbohydrnre &search Ekevicr Publishing Compctny. Printed in Belgium

STUDIES

ON

PART

INTER-NODULE

XVI1_

Acacia nilofica

URONIC

(Received

of

AND

Chemistry,

March

ACID

MATERIALS

VARIATION

AND

THE

ACIDIC

COMPONENTS

IN

GUM

D. M. W. ANDERSON Deportment

403

Amsterdam

ztst,

K. A.

KARAMALW

The Utticersity,

Edittbur~h

g (Grear

Br~kiitt)

1966)

INTRODUCTION

In a preliminary study of Acacia nilotica (L.) Willd. ex Del. respects from those species studied positive specific rotation (+ 106O), only traces of rhamnose that could graphy.

the gum exudates from several Acacia species, was found2 to differ in a number of interesting prior to 1963. Thus, A. nilotica gum gave a high, a high methoxyl content (I .05x), and contained not be estimated satisfactorily by paper chromato-

In addition, A. nilotica gum gave solutions of low viscosity, and its unusually low nitrogen content (0.08 %) was decreased to 0.02% on electrodialysis (corresponding decreases are not shown by other Acacia gums9). A. nilotica could, therefore, constitute a useful limiting case in investigations into the extent of the dependence of the physico-chemical properties of Acacia gum solutions on their natural nitrogenous content, and it was therefore selected for study in preference to other available species”. In view of the inter-nodule analytical survey of ten nodules

variation observed in A. sepal gum3, a preliminary of A. nilotica gum was undertaken.

EXPERIMENTAL

Origin qf Specimens. Ten large nodules of gum from Acacia nilotica were collected by (the late) M.P. Vidal-Hall, formerly Gum Research Officer to the Republic of the Sudan, at Hawata, Kassala Province, Eastern Sudan, on February 3rd, 1963. All of the nodtiles originated by natural exudation; nodules (I), (2), (3), and (4) were taken

from

individual

small

trees,

nodules

5-10

from

individual

large

trees,

all

growing in close proximity. Analytical Methods. The general methods have been described2, except that paper chromatography was carried out with the following solvent systems (v/v): (A) butanr-ol-ethanol-water (4:1:5, top layer); (B) butan-I-ol-pyridine-water-benzene (5:3:3:1, top layer); (C) ethyl acetate-pyridine-water (10:4:3); (D) ethyl acetateacetic acid-formic acid-water (18:3:1:4); (~7) ethyl acetate-acetic acid-water (g:zz); (F)

butan-I-ol-acetic

acid-water

(4:1:5,

top layer). Carboh_vdmre

Res.,

z (1966)

403-4

Io

ST’JDIES ON URONIC

ACID MATERIALS.

XVI

405

RESULTS

Studies on the crude gum. The results of analyses of nodule Cl)have been pubIished2 under the heading “A. nilorica” in Table II therein. Studies on gum samples purijied by electrodia1y.A Each of the ten nodules of gum was purified separately by electrodialysis4, and the free gum acids were isolated by freeze-drying; analytical data are given in Table I. Particular care was taken with duplicate analyses when it was observed that the results for nodule (5) differed significantly from the others; the results quoted are average values for satisfactory replicates. Partial hydrolysis of the gum acid, and separation of acidic sugars. Electrodialysed specimen (1) (16 g) was hydrolysed with N sulphuric acid (300 ml) for 12 h at 100~. After neutralisation (barium carbonate), de-ionisation [Amberlite IR-120 (Hf form)], and concentration, paper chromatography (solvents B and 0) revealed the presence of galactose, arabinose, glucuronic acid, and 4-O-methylglucuronic acid. Two acidic disaccharides were also indicated (these were, in fact, two mixtures of acidic disaccharides). The neutral and acidic components were separated by passage through Duolite A4 (formate form). Elution with water gave a neutral syrup (10.7 g) which contained galactose and arabinose; this fraction was not examined further. The acidic components were eluted with dilute formic acid to give a syrup (4.4 g) after the necessary isolation stages. Fractionation qf the acidic sugars. The acidic syrup (4.4 g) was added to a cellulose

column (80 x4 cm) and eluted with solvent E. Fractions (IO ml) were collected; the contents of every third tube were examined in solvents D and E. Five main fractions were obtained : fraction a (tubes 24--28), 216 mg; fraction b (30-61), 1498 mg; fraction c (62-IOO), 931 mg; fraction d (134-307), 941 mg; fraction e (492-600), 3og mg. The total recovery was therefore 88%. Examination qf the acidic fractions. (a) Fraction

a had RG~Z 3.0 (solvent E) and

crystallised readily. Recrystallisation from water gave D-glucopyranurono-63lactone, m.p. and mixed m.p. 177O, [oL]~ j-19” Cc 1.0, water). (b) Fraction b gave a single chromatographic spot having RRha 0.91 (solvent D) and Raaz 0.39 (solvent B). It had [LY]~t37” Cc 1.5, water) (Found: 0CH3, 14.6. Calc. for a monomethylhexuronic acid: OCHa, t4-9%). The pale syrup (0.4 g) was converted into the methyl ester methyl glycoside (0.34 g) with dry, 2% methanolic hydrogen chloride, reduced with potassium borohydride, and hydrolysed. The product (0.24 g) had R~ha 0.94 in solvent A and gave a single spot, having RG~Z1-94 and 2. I, respectively, in solvents B and B. This behaviour was identical withthat of 4-O-methylglucose. After purification on WhatmanNo. 3MM Curbohydrafe Res.,

2

(x966)

403-410

406

D. M. W. ANDERSON,

K. A. KARAMALLA

paper in solvent A, the product had OCHs, 15.0% (talc. for GH1406: OCH3, 15.6%), and the crystalline phenylosazone had m-p. 156” (lit., 157-160”), after two recrystallisations from hot water. Fraction b was thus 4-O-methyl-D-glucuronic acid; the amide of methyl 4-O-methyl-a-D-glucopyranuronoside was obtained as large, colourless plates, m-p. 231~ (lit., 232-236“), [c&? +140” (c 0.5, water). (c) Fraction c gave a single spot corresponding to D-glucuronic acid in solvent D: examination in solvent B showed the presence of a trace of galactose. In view of the identity of fraction (a), this fraction was not examined further. (d> Fraction d had [cz]~ f55” (c 1.0, water), and partially crystallised. Examination in solvents D &d E gave spots (brown with aniline oxalate spray) having RG=~o-69 and 0.66, respectively. Hydrolysis (2~ sulphuric acid, 6 h) and subsequent examination (solvent D) showed the presence of galactose and 4-O-methylgfucuronic acid in equal proportions (visual examination). A small portion of the fraction (80 mg) was treated with methanolic hydrogen chloride, reduced with potassium borohydride, hydrolysed, and fractionated on 3MM paper in solvent A, to give 4-O-methyl-D-glucose [28 mg; phenylosazone, m-p. 158” (lit., 15g”)], [c&! +58” (c 0.1, water); and D-galactose (31 mg; m-p. and mixed m-p. 156”). Fraction d (500 mg) was methylated with dimethyl sulphate and sodium hydroxide, followed by methyl iodide and silver oxide, to give a product (286 mg; OCHs, 52. I %) which was reduced with lithium aluminium hydride. The methylated, neutral product (213 mg) was hydrolysed (N hydrochloric acid, 4 h); the hydrolysate gave 3 spots (& 0.84, 0.72, o-69) in solvent A. The three components were separated on a cellulose column (2 x 38 cm) using butan-I-o&light petroleum (7:3), saturated with water, as eluant. The first component (68 mg) had & 0.85 (solvent A), was identical with 2,3,4-tri-U-methyl-D-glucose in solvents A, B, and -F, and gave an anilide, m.p. 152“ (lit., 145-150~). The second component (18 mg) had RG 0.71 (solvent A), was identical with 2,3,6-t&O-methyl-D-galactose in solvents A and F, and was oxidised with bromine to 2,3,6-tri-O-methyl-D-galactonolactone, m-p. 96-97” (lit., 98”). The third component (17 mg) was identical with 2,3,+tri-O-methyl-Dgalactose in solvents A, B, and F, and gave an anilide, m.p. and mixed m.p. 166” (Iit., 167-170~). Separation of the aldobiouroltic acids in Fraction d

Fraction d was suspected to be a mixture of monomethylaldobiouronic acids when the rotation for the corresponding fraction from the other specimens of A. nilotica was observed to vary signifkantly from +48 to f71”. Separation of fraction d into two aldobiouronic acids was achieved on strips (4” wide) of 3MM paper, in solvent D for g6 h. The chromatograms were freed from acidic solvent by air-drying for 48 h, followed by heating for 5 min at 150”. The located zones were eluted with cold water to give aldobiouronic acids A (IOI mg) and B (95 mg). Excmination of aldobiouronic acids A and B Acid A (IOI mg) had &oZ 0.79 (solvent 0) and [cz]~ fg3” Carbohydrate Res., 2 (1966)403-410

[c 1.0, water).

STUDlEs ON URONIC ACID MATERIALS.XVI

407

A sample (40 mg) was converted into the methyl ester methyl glycoside; one halfportion of the product was reduced with potassium borohydride and hydrolysed. The products were chromatographically identified (solvent A) as galactose and 4-O-methylglucose. The second half-portion reduced 1.8 mol. of sodium periodate. In duplicate experiments, the aldobiouronic acid gave, on periodate oxidation, 1.01 and 1.04 mol. of formaldehyde, indicating that C-6 of the D-galactose residue was not involved in a linkage. These experiments, considered in conjunction with the methylation evidence reported above for acidic fraction (d), led to identification of acid A as 4-0-(4-0-methyl-a-D-glucopyranosyluronic acid)-D-galactose. Acid B (yield 95 mg) had RG=Z0.68 (solvent 0) and [z]g +6” (c 0.95, water). Methanolysis, followed by potassium borohydride reduction and subsequent hydrolysis, gave only galactose and 4-O-methylglucose(solvent 0). Periodate oxidation of the aldobiouronic acid at pH 8 gave no formaldehyde. The methyl ester methyl glycoside, on oxidation with sodium periodate in darkness for 2 days at room temperature consumed 3-1 mol. of periodate. These experiments, considered in conjunction with the methylation evidence reported above for acidic fraction (d), led to the identification of acid B as 6-O-(4-0-methyl-fl-D-glucopyranosyluronic acid)-D-galactose. (e) Fraction e (309 mg) had [a]g +31” (c 1.2, water) and gave, after hydrolysis, only galactose and glucuronic acid (solvents B and 0) Methanolysis, followed by potassium borohydride reduction and hydrolysis, gave only galactose and glucose. Chromatographic separation in solvent D for 120 h was required to reveal the presence of two components having Rcaz 0.21 (major component) and 0.28. Fraction e was then fractionated on 3MM paper (4” wide) in solvent D for 160 h to give aldobiouronic acids C and D. 2Zxanzinatiorz of aldobiozcronicacids C and D

Acid C (103 mg) had Rear 0.21 (solvent 0) and gave [cc]~ -5” (c 0.4, water). It was chromatographically homogeneous and identical in solvents D, E, and F with 6-0-(p-D-glucopyranosyluronic acid)-D-galactose. Reduction of the methyl ester methyl glycosides with potassium borohydride, followed by hydrolysis, gave only galactose and glucose. Methylation of a portion (80 mg) by the Haworth and the Purdie methods, followed by reduction (lithium aluminium hydride), gave a product (38 mg) which, after hydrolysis, was fractionated on 3MM paper in solvent A to give 2,3,4-tri-O-methyl-D-glucose (14 mg) [Rcrsr 0.85 (solvent A); anilide, m.p. f148” (lit., 145~1500)] and 2,3,4-tri-O-methyl-D-galactose (I I mg) [Roar 0.67 (solvent A), chromatographically identical with an authentic specimen in solvents A, 0, and F; attempted preparation of the an&de did not yield a crystalline product]. Acid D (18 mg) had Rccrr 0.28 and 0.32 on Whatman No. I and 3MM papers, respectively, in solvent D, and had [cL]~ -I- 107’ (c o. I, water). Hydrolysis gave only gaIactose and glucuronic acid; periodate oxidation at pH 8 for 24 h gave 0.93 moI. of formaldehyde. This acid has subsequently been obtained from other Acacia species, and has been more rigorously characteriseds as 4-O-(a-D-glucopyranosyluronk

acid)-D-galactose. CarbohycGare Rex, z (1966)

403-410

D. M. W. ANDERSON,

408

K. A. KARAMALLA

DISCUSSION

The analytical results in Table I supplement the limited data of this type extant3~6; the additional work involved in such an approach is justified for the following reasons. For such complex polysaccharides, it is reasonable to expect that some variation in composition and properties may exist between gum specimens exuded by different trees of -a particular species (it is becoming apparent that the inter-nodule variation for one species may be greater than that for another). This investigation has shown that a knowledge of the inter-nodule variation is useful whenever an aspect of heterogeneity, or the possibility of fractionation, is involved. In this study of A. niictica, the natural exudates, collected on the same day, from ten different trees growing in close proximity, have been examined. This approach can be extended to examine (i) the seasonal variation for a species, (ii) the variation between specimens from geographical locations differing in climate and type of soil, and (iii> the variation between exudations resulting from different stimuli, e.g., from tapping, from natural processes, from diseased trees, and from trees attacked by ants or borer beetles. Such studies could provide useful evidence regarding the mode of biosynthesis of gum and the nature of the carbohydrate systems serving as gum precursors in the tree. The available analytical data substantiate the view3 that a single, gum nodule is itself complex and offers the simplest system available for structural investigation_ Recent developments in analysis make such an approach possible if the collection of reasonably large nodules can be arranged. A preliminary analytical survey of several nodules is, nevertheless, required, to select the most representative nodule of the species for structural study, and to establish the extent to which it varies from other specimens. Thevalue of this approach is seen in Table I,fromwhich specimen (5) must be regarded as atypical (in some respects) of the A. niloticu species. On the basis of present knowledge, there is, however, no evidence that specimen (5) did not originate from A. nifotica. The ten specimens studied were collected by an accepted authority’ on the Sudanese Acacias, whose undertaking was, in the research coliaboration between the Sudanese Department of Forests and this laboratory, to collect, personally, only specimens which could be authenticated beyond doubt. Acacia nilotica is, moreover, distinctly characteristic to a trained fieldsman; the species resembling it most closeIy is A. arabica. Inspection of the available analytical data for Acacia species” indicates that although A. pycnantha, A. arabica, and A. fistuia each have SOltlefeature in common with specimen (5) in Table I, its analytical characteristics, taken as a whole, do not suggest that it would be more correctly assigned to any other species for which information exists. Although this view may require alteration in the future, the present reserve associated with this specimen further justifies a limited, preliminary, analytical survey of the nodules of any gum species under structural investigation. Determinations of the traces of rhamnose in A. nilotica could only be achieved Carbohydrare

Res.,

2


STUDIES ON URONIC

ACID MATERIALS.

by a spectroscopic

method

XVI

developed7

409 for the purpose.

. now known to contain (1% of rhamnose, major constituent in alI Acacia gums (c$

Several

Acacia

species

are

and this cannot now be considered a ref. 8) Furthermore, polysaccharides

containing both D-glucuronic acid and its 4-O-methyl ether are no longer unusual (CA ref. 8); these acids occur conjointly in species of the AZbizid>s, Khqd” and, now, of the Acacia genera. Methoxyl groups were recently reported9 to occur in Acacia species; the relatively high methoxyl content in A. nilotica has facilitated the present confirmation that the methoxyl groups are structurally significantg. Prior to this investigation, only A. karroo” and A. Senegal1 had been reported to contain two aldobiouronic acids; on re-examination, those species previously reported to contain only 6-0(b-D-glucopyranosyluronic acid)-D-galactose may be found to be more complex. The optical rotation data presented for acid fraction (6) and its components give an indication of the extent to which heteropolymolecularityl is displayed by

A. nilotica gum. Calculation shows that the proportions of the monomethylaldobiouronic acids A and B vary, iu the nodules examined, from approximately I:I to 3:1_ Furthermore, with the recovery of these monomethyl acids accounting for the observed methoxyl content, and with no evidence of alternative locations for the variations in methoxyl groups in this or other Acacia species 135, the inter-nodule methoxyl content suggest that the heteropolymolecularity extends to differences in acids the proportions of the monomethyl acids (A + B) and the non-methylated (C -I- 0). ACKNOWLEDGMENTS

We thank Professor Sir Edmund Hirst, C.B.E., F.R.S., for his interest in these studies, the Sudanese Ministry of Education for a scholarship (to K.A.K.), and Samuel Jones Ltd. (London), Laing-National Ltd. (Manchester), and Rowntree Ltd. (York) for financial support_ SU;MMARY Inter-nodule variations in the composition and properties of Acacia nilotica gum have been investigated. The results confirm the value of this type of analytical survey of a species, prior to structural studies on a single, representative nodule. The acidic components of the gum have been examined in detail. For the first time in the genus Acacia, four aldobiouronic acids, two of which contain 4-O-methylD-glucuronic acid, were present and were identified as 4-O-(&O-methyl-a-Dglucopyranosyluronic acid)-D-galactose (A), 6-O-(4-O-methylq%D-glucopyranosyluranic acid)-D-galactose (B), 6-0-(B-D-glucopyranosyluronic acid)-D-galactose (C), and 4-O-(a-D-glucopyranosyluronic acid)-D-galactose (0) The analytical data indicate that there is an inter-nodule variation in the proportions of acids A and B, and: further, in the proportions and the unsubstituted acids (C + 0).

of the monomethyl

Carbohydrate

Res.,

acids (A + B)

2 (1966)

403-410

-410

D. M. W. ANDERSON,

K. A. -LA

REFERENCES I Part XVz’Dl kf. W. AN~RSON AND J. F..STODDART, Carbohydrate Rti., i (1966) -4, J. Chem. Sot. (C), 8 (1966) 764 D.M.W_ANDERSONANDK.& 3 D. M. W. ANDERSONAND M. A..~~ERBIcH, J. Chem. Sot., (1963) I. Carbohydrate 4 D. M. W. &‘lDERSON, G. M. CW, J. J_ MARSHALL, AND S. ELUIMAN,

104.

2

63.

Res., 2 (1966)

5 0. M. W. AND&SON AND G. M. CREE, unpublished results. 6 D. &f-W. ANDERSON, E. L. Hm, AND N. J_.KING, Talanfn, 3 (rgsg) 118. 7 D. M. W. ANDERSON AND J. F. STODDART, in P. W. SHALLIS (Ed.), Proceedings o_f the SAC Conference, Nottingham 1965, Heffer, Cambridge, p_ 232. 8 D. W. DRUMMONDANDE.PERCIVAL,L Chem.Soc.,(rg61)3go8. g D. M. W. ANDERSON, G. M. CREE, M. A. HERBICH, K. A. KAR&MALLA, AND I_ F_ STODDART, Talanta, II (1964) 1559. IO G. 0. ASPMALL, M. J. JOHNSTON,AND R. YOUNG, J. Chem. Sot., (1965) 2701. AND A. M. STEPHEN,J. Chem. Sot., (1955) 1428. II A.J.C HARISON, J. R. NT, CarbohyrGate Res., 2 (1966) 403-410