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Structural features of the gum exudates from some Acacia species of the series Phyllodineae and Botryocephalae
C~h’wdrareResearch, 55
STRUCTURAL FEA TURFS OF THE GUM EXUDATES SOME Acncia SPECIES OF THE SERIES- Phyhdimze AND BotryocephaiaP SHZRLEY C. CHURMS,EDWZN H. MERRIFELD, AN;) Ausrm C.S.P.R. Carbohydrate Research Unit, Deprtment University of Cape Town (South Africa)
FRDM
M. STEPHEN
of Organic Chemistry,
(Received July 9th 1976: accepted for publication, October 1Sth. 1976)
Smith degradation of each of the polydisperse, gum polysaecharides from and A. podalyriaefk&a, for which molecular-tieight distributions have been measured by gel-permeation chromatography, gives, in good yield, a methanol-insoluble polysaccharide that shows a single peak on examination by this technique. The molecular weights of the Smith-degraded polysaccharides are close to the values expected if the respective gum polysaccbarides, on losing periodate-vulnerable, peripheral sugar residues, were to be split also at regnlar intervals between the otherwise (1+3)-linked D-galactose chains. The structures of these gums, which are similar iu many respects, conform to the pattern shown reizntly to occur in the gum of A. baileyma. Acacia pycnantha, A. &&rmis, A. j&z~oZi4
INIXODUCITON
Since the technique of periodate oxidation, borohydride reduction, and controhedacid hydrolysis was l%rstapplied by F. Smith and his co-workers to *“urn arabic’, Acacia gum samples from numerous species have been shown, without exception, to contain an internal core of &(1+3)-linked D-galactopyranose residues’: Sequential Smith-degradations3 have given additional information about the nature of peripheral sugar groups, and measurements of the molecular weights of the periodate-resistant portions of the polysaccharides have been of value in the subclassification of gum-forming Acacia species 4. Recently, Smith-degradation procedures have shown that certain low molecular-weight Acacia gums, although polymohzcnlar, contain blocks of galactose residues that are not attacked by periodate but which are separated at regular intervals by sugar residues that are; the consequence is that suitably controlkd, acid hydrolysis of the oxidised, and subsequently reduced gum yields a product that shows ei’cher a single peak on gel chromatography5, or *Dedicated to the memcizy of Professor J. K. N. Jones, F.R.S.
4
S. C. CISUFMS, E. H. hERRWELD, A. M.
tiHEN
two or more peaks that bear a simple molecular-weight x!elationshipto one aqother6. Accordingly, further examples of Acacia gums have been examined in order to test whether the structural Pattern that has emerged is of generai applicability. It was of particular interest to apply Smith degradation to the gum of A. pycnantha (Series Phy&&zeae) because of the detaifed structural model that was available through the extensive researches of other workers’, including Barry degradation’ of the oxidised polysaccharide. The gums of A. dz@mis (Series Phyllodineae)g and A. jilicz~oiiu (Stxies Botryocephuiae) 1’ were also selected for examination because published analytical data for these gums indicate close similarity to the gums from these Series studied earliersV6. EXPERIMENTAL
Sources of gum specimens. - Samples of the gums of Acacia dzJj2ormis and A. j%czjXia, collected in New South Wales, Australia in January, 1970 and February, 1970, respectively, were made available through the courtesy of Dr. D. M. W. Anderson (Edinburgh), who has publishedg*” anal_yticaldata for these specimens. The samples of A. pycnantha gum used in these studies were collected near StelIenbosch, Cape Province, South Africa in November, 1974 and October, 1975, and several different samples of A. podafyriaefolia gum, originating in Johannesburg, Port Elizabeth, and Cape Town (South Africa) were collected in July, 1964, July, 1975 and May, 1976, respectively. Purification of gum samples. - Small samples of the gums, as collected from Acacia stems or branches, were examined by gel-permeation chromatography, but most of the experimental work was performed on bulked material that had been recovered by precipitation with ethanol (4 volumes) from clear, aqueous solutions of these gums. The precipitated polysaccharides were washed with ethanol and etber, redissolved in water, and freeze-dried. General experim,jl?iai condirions. - Opt&I rotations were measured at 20” on a Perk&Elmer 141 polarimeter, by using aqueous soiutions in the concentration range OS-l% unless otherwise specified. The solvent systems used in paper chromatography were (A) 4:1:5 I-butanol-ethanol-water (upper layer), (B) 2:1:1 lbutanol-acetic acid-water, and (C) 8:2: 1 ethyl acetate-pyridine-water. Gel-permeation chromatography was conducted on Bio-Gel P-300 or P-10 as appropriate; with M sodium chloride as eluent 3*11_Proportions of neutral sugars were determined by g.1.c. of the derived alditol acetates I2 , following hydrolysis of the polysaccharides and their degradarion products in sealed tubes, under nitrogen, with 2~ trifIuoroacetic acid for 8 h at 100”; the aqueous acid was normally removed by freeze-drying. Periodate consumption was monitored by the arsenite method 13_ Methylafion analysis. - After methylation of the gums by the Hakomori I4 and Furdie 1 ’ (2-4 times) procedures, the mixtures of methylated sugars produced on hydrolysis (trifluoroacetic acid as already described, for I8 h} were analysed by g.1.c. of the trimethylsilyl ethers of the derived alditolsr6.
GUM EXiJDATEs
OF hX.7CiU
5
Oxidation of each polysaccharide with aqueous sodium metaperiodate6 was terminated by addition of the calculated quantity of barium acetate, and the centrifugate was reduced with sodium borohydride. The reduced, oxidised polysaccharides, recovered as previously dcscribed5, were submitted to mild hydrolysis in M trifluoracetic acid for periods ranging from 54 to 120 h at room temperature. The decision to terminate the degradation, by freeze-drying the acidic solution, was governed in each case by the extent of diminution in mm of the polysaccharide and the degree of polymolecularity of the products as shown by gelpermeation chromatography on Bio-Gel P-10. Hydrolysis was stopped when the molecular-weight distribution of the resultant, degraded polysaccharide remained constant over a 24-h period, or longer, in contact with M trikluoroacetic acid. After Smith degradation had been carried to completion, all of the gums *yielded methanolinsoluble, degraded polysaccharides that were monodisperse and did not decrease in molecular weight on further contact with M trifluoroacetic acid at room temperature. The degraded polysaccharides (after paper chromatography of their hydrolysates had demonstrated the absence of uranic acid) were then treated in aqueous solution with mixed ion-exchange resins and freeze-dried. The methanolsoluble fractions of the Smith-degradation products were shown by paper chromatography to consist largely of polyol (glycerol, ma&y), together with low molecular-weight glycosides. Smith degradation. -
RESULaTS AND DISCUSSION
The gums of A. pycnantha, A. d@ormis~ and A. Jiricifolia show a close resemblance, both in the properties listed in Table I and in their structural components as indicated by meth;rlation analysis (Table II), to the gums of A. podaiyriaefoolia, 4. Jata, and A. baiieyana previously examined5p6. Partial-hydrolysis experiments reported elsewhere I7 have confirmed that A. pycnantha gum has the same general structural futures as the last three gums just named’* I** lg, notably acid-labile rhamnose, arabinose and galactose residues in short branches attached to a galactan core that ultimately breaks down to a series of neutral oligosaccharides contaiting /3-(l-+3)-linked D-galactose residues and, in small proportions, the aldobiouronic acid 6-O-(/3-D-glucopyranosyluronic acid)-D-galactose. Paper chromatography (SOL vents B and C) of partial hydrolysates (0.01~ trifiuoroacetic acid, 48 h, 100’) shows that the gums of A. dQ?bmis and A. jilicifolia also yield these products. The approach of using Smith degradation to ascertain the distribution of the B-(1 +3)linked D-galactose residues in the polysaccharide molecule has proved illuminating in previous studies3-6, particularly with A. baileyana gum5 which yields, after a single Smith degradation, a monodisperse, degraded polysaccharide (molecular weight 2500) in which (143)~linked D-galactose residues preponderate, although a small proportion of constituent arabinose remains. There is strong evidence reIat.ing the production of this single, degraded polysaccharide to the occurrence of repeating sub-units having molecular weight 4600 in the molecule of the intact polysaccharide.
E. &66EREIIREiT), 23.c. c X%U&4S;
6 TABLE
I
PROPERTIIZJ
Am
Gum
A. M. STEP-
OF GUM
POLYSACCHARIDES
FROM
Acacia pycnantha,
A. diffarnds,
.
=
A. J?ci/olirr
of
MokcuIar-weight distribution’.*
Equivalent wei8ht
A. pycaantha
A. dioxmia
A. GlicifoIia
-7 12ooO
1s
f7 13cW
31680(2); 24000(6); 17800(18); I2000(36); SOOO(17);6ooO(21)
34000(3); 28200(8); 23500(14); 15000(37); 12cloo(10); UMoo(ls); 7000(13)
3700 (ref. 7)
Hence uranic acid (mol %) Proportions of neutral sugars (mol %) Galactose Arnbinose Rhamnose Periodate consumption (mmoI.g-‘)
28200(2): 21400(S); 16800(16); 14c@O(9); 108G0(40); s400(25) 4300 (re& IO)
3400 (ref. 9)
5
5
4
67 26 2
73 20 2
81 14 1
5.7
6.7
6.2
“Bio-Gel P-300. *Mokcu!ar weights corresponding to peaks in gel chromatograms; relative proportions by weight in parentheses. TABLE
II
hnrrF~UrI0~
ANALYSIS
OF
A. pycnanrha, A. diflomlis,
AND
A. fiiicifotia
GUMSO
Portia&y nwthylated sugar (positions of O-methyl grasps giaen by Iocants)
Moiar propottions X. pyenantha gunP
A. difformis gumC
A. fikifolia
2,:,5-Ara 2.s-Al-a 3,5-Ara 2,3,4-Rha 53-Ara 3,kXla > 2,3,4,6-GaI z4,6-Gal 2,3,6Gal 2,3,4Gal 2?6-Gal 2&Gd 2-G&
18
15
11
5
3
I
3
20 3 I 7 4 30 6
20 3
gumC
; 3 33 3
“iajc for chlorothrm soiutious of the metbylated gums, - 50 (Ref- 22), -45, and -47”, respectively. ‘Compare ref. 22, where the proportions given are simifar except for end-group D-g&CtOpyraOOSe (found to be higher by assay based upon methyl gfycosides). Wakes given were wrr~~box%ed by g.1.c. aria&is of the alditol acetates; compare ref. 23, where methyylgiycosides were employed.
GUM EXUDATES OF
-&qc&
7
The data- presetit& Sn ‘TaHe m show clearly that the gums of A. pycnantha, A. d#%rmis, and A. jiIic~j&h &so yield, after one Smith degradation, monodisperse
degraded polysaccharides that are essentially galactans, containing arabinose as a minor constituent. The specific rotations of these three degraded gums and the amounts of pcriodate consumed by the whoIe gums vable I) are similar to those found for gums of this type &died earlier. Mtinodispersity of the degraded polysaccharidcs is indicated by both gel-permeation chromatography and ult.racentrifugationzo, and the molecular-weight values obtained by these two independent procedures are in good agreement (Table In). T.1.c. on plates coated with Ki,eselguhr G [solvent systern” 4:4:2-@/v) 2-propanol-acetbne-M lactic acid, to which 34 parts by volume of water had been added] of each of these three Smith-degraded polysaccharides and that from A. baileyarragum gave single spots (RF -0.8). TABLE
III
PROPERTIES
OF hlETHANOL-INSOLUBLE
Smihdegraded
PRODUCTS
gum of:
[aln (de@eW Galactos&ambinose
ratio
Methyiated derivative: [alo in CHCls (degrees) MoIetXhi weight of Smith-degraded polysaccharide Calculated molecular weight of corresponding block in whole gum
%rom gel-permeation chromatography ments=*. The predominance of (1+3) by methyiation analysis.
OF SMITH
DEGRADATION
OF GUMS
A. pycnantha
A. difformis
A. Nicifolia
+28
+32
+29
955
97:3
97:3
-6
-5
35006 410@
36004 38W
21cW 21oob
6600
6800
42ClO
-12
(Bio-Gel P-10). ‘From sedinientation and diffusion measurelinkages in al1 three Smith-degradation products was proved
During the Smith-degradation procedure, the mild acid-hydrolysis s&p that ultimately yielded these moaodisperse, degraded polysaccharides was monitored by gel-permeation chromatography; this method proved more sensitive than others used previously6. Before the degradation was complete, gel-permeation chromatography @G-Gel P-10) of all of these gums showed the presence of components having molecular weights that approximated to multiples (2 and 3 times) of the molecular weights of the Gal products (see, for example, Fig. 1). This observation indicates a periodicity in the galactan skeletons of the -gums, with acetal linkages between the blocks of sugar residues that survive the periodate oxidation; these linkages are separated at regular intervals, and arc broken relatively slow!y during the hydrolysis step. Precise definition of the length of the galactan chains is not yet possible, although the narrowness of the elution band on g&permeation chromatography of smaller samples th%n that illustrated in Fig. 1, and preliminary fractionation experiments with high-pxzssure liquid chromatography (eluting with 2-propanol-water from a Partisil $0 column), support the view that the range caunot he large.
s. C. CHURMS, E. H. ?&RFWIELD,
A. M. sTEE!HEN
n 0
P N
80
110
IO
Eluted volume
Fig. 1. Gel-permeation chromatograms (A)_ Reduced, oxidised gum after perature (B) and for 92 h (C). Further Eluent: M sodium chloride. Columns: (A) gum
60 (ml)
60
70
showing progress of Smith degradation of A. filicifolia hydrolysis in .M trifluoroacetic acid for 54 h at room temexposure to acid for 24 h left the pattern (C) unchanged. Bio-Gel P-300,85 X I .5 cm; (B, C) Bio-de1 P-IO, 55 x 1.5 cm.
Allowing for the proportion of sugar residues removed by the Smith degradation (end-groups, and some arabinose and galactose present as glycosidegn the methanolsoluble material produced) permits calculation, as in the case of A. baileyana gums, of the molecular weights of the blocks of sugar residues in the intact polysac&arides from these gums that give rise to the degraded polysaccharides (see Table In). Scrutiny of the molecular-weight distributions of the gums (TabIe I) shows that most of the components present have molecular weights that are approximately multiples of the calculated values for the sub-units in the gum. The gum of A. podalyriaefoolia was previously reported6 to yield a polymolecular, Smith-degraded polysaccharide, but further contact of this product with 0.01~ triffuoracetic acid for 65 h at room temperature results in the disappearance of (minor) components having molecular weights 8400 and 5000, yielding a monodisperse polysaccharide of molecular weight 2100. This corresponds to a sub-unit, in the gum, of molecular weight approximately 4000. The various samples of the gum examined have been found to vary considerably in aW (as reported4 for some gums from other Acacia species), with values ranging from 9500 to 32090, even among samples taken from different branches of the same young tree. However, this variation was due to the presence, in differing proportions in the different samples, of components having molecular weights of the order of 4000, SOOO,16000, and 32000 in all cases. These are, once again, integral multipies of the molecular weight of the sub-unit indicated by Smith degradation. It has thus been shown that five polymolecular arabinogalactan gums, having relatively low HD, from Acacia species of the series PhyIlodineae (A:podalyrzizefoolia,-
GUM EXUDATE&
OF
Acacin
9
A. pycmn?ha, ahd A. dt~ormis) and Bott-yocephaiae (A. baileyana and A. filicifolia) give a monodisperse, degraded polysaccharide after a single Smith degradation. A notable exception is the otherwise similar gum of A. elata (series Burryocephalae), which yields two Smith-degraded polysaccharides, in nearly equal proportions by weight, havipg molecular weights 2100 and 4400, respectively6. Neither of these products undergoes any diminution in molecular weight when treated with M triIiuoroa+c acid for up to 83 h at room temperature, and therefore neither can be ascribed to the result of incomplete degradation_ In the five examples cited here, a sugar is present, between blocks of (l-+3)-linked galactose residues, so linked as to be vulnerable to attack by periodate and thus to very mild acid hydrolysis in the resulting oxidised, and subsequently reduced polysaccharide. That the polysaccharide itself is resistant to such mild hydrolysis has been demonstrated by gelpermeation chromatography of a sample of A. pycnarzrha gum that had been kept in M tritluoracetic acid for 144 h at room temperature; although paper chromatography showed the release of some arabinose, there was no discernible change in ME and therefore no cleavage of glycosidic bonds in the polysaccharide chains under these conditions. The nature of the inter-sugar linkages between the (1+3)-linked subunits in these Acacia gums remains to be investigated; ionic association between acid groups, for example, is not a major factor here24. ACKNOWLEDGhlENTS
The financial support of the Research and the University of acknowledged. We thank Dr. D. Oliver (Botanical Research Unit, Professor E. A. C. L. E. Schelpe classifying some of the specimens.
South African Council for Scientific and industrial Cape Town (Staff Research Fund) is gratefully M. W. Anderson (Edinburgh) and Dr. E. G. H. Stellenbosch) for providing gum specimens, and (Bolus Herbarium, University of Cape Town) for
REFERENCES F.
SMITH AND D. R. SPRIEZXERSBACH. Abstracts Papers Am. Chem. Sot. Meeting. 128 (1955) 15~; I. J. GOLDSKEIN, G. W. HAY, B. A. LEWIS, AND F. SMITH. Methods Carbohydr. Chem., 5
(196.5) 361-370. G. 0. ASPINALL, Ado. Carbohydr. Chem. Biochem., 24 (1969) 333-379. D. M. W. A--N, SIR EDhmD Hr~sr, AND J. F. STODDART, J_ Chem. Sot. (C), (1966) 1959-
1966. D. M. W. ANDER~N
AND I. C. M. DEA, Phytochem., 8 (1969) 167-176; J. Sot. Cosmetic Chem., 22 (1971) 61-76. S. C. CHURUS AND A. M. STEPHEN, Ckrbohyak. Res., 45 (1975) 291-298. P. I. BEKKER, S. C. CHURMS, A. M. STEPHEN, AND G. R. WOOLARD, J. S. Afr. Chenr. Inst., 25 (1972) 115-130. E. L. Hmm AND k S. PERIXN, J. Chem. Sot_, (1954) 2622-2627:G. 0. A~PINALL, E. L. HIRST, AND A. NICOLSON, ibid., (1959) 1697-1706; D. M. We ANDERSON AND G. M. CWEE, Carbohy&. Res.. 6 (1965) 214-219. V. C. BARRY, Nature, 152 (1943) 537: V. C. BARRY AND P. W_ D. MITCHELL, J. Chem. SOL,
(1954) 40203023. D. M. W. ANDERSON, P. C. Brr,~m
C. G. A. McNks,
Phytochem, ll(l972)
175I-1754.
10
S.-‘it!. CHfmMS,.'E,. H; MBRRlFdD;
$L’.M:--SHBE+
: .10 D. M. W. ANDERSON,P. c. BELL,AND C. G. A..M~~~~&a&jhy&~~Re& ZO (1971)2&3+27P;. 11 S. 6. C&uds~yD~A. M.~~, S:Afii &fed.-+;43 (1%9j iti. - -‘:,.:. ~.:: : .:’ 12 .j. S: SAiVARIjti; J;i!i. Stir&&t,- AND it: JE&s; AMI.-C&5&; j;i it%5)f602+04; 13 P:FUZJEYAND J. L~~ti~.J.:.Fhtwaz. CXm... 17 (1933)~107~113;~ 14 S. .H;\K?.HO&J. Rioc&em. @‘bityu), 55. (1964) 2@-208. ‘.- ._ .--. 15 T_ PUIUNEAE~,3. C. hvme, J. -Che+. So& !33-(f!$3) 102hO37. 16 B. l-I. FREEUN, A. ha: STEkEN, AND P. VANDERBZFL,I.‘C&&o&, 73 (19i2)2P-33. 17 J. ADAM, S. C. ,@iui; A. M. SIZPHEN,D.-G. Smim&~~, ti ,E H. ?Uxf&i~& &zr&hy&: Rex, 54 (1977) 304-307. 18 S. C. ‘CHURMSAND A. M- STEPA; &&&& R&i, i 5 (197&l t-19: 19 S. C. C&ws AND A. M; STEPHEN,. Cmbohy&. Res.; Z1 ‘(1972)s91-98..: 20 S. C. ihJR~, k hf. STEPHEN, AND A. PO-N, S. ,4fi; 1. S&72 (1976) ISl-i52. 21 S. A. HANSEN,J. Chromatagr., 105 (1975) 388-390. : . .. 22 M. KAPLAN AND A. M. STEPHEN, Teirahedrwz,~2~ (1967) 193_1?8; 23 D. M. W. A~ahso~ m.P. C. BELL,Phyrocheti.; 15.(1976).3Ol-303. 24 S. C. CHXJR?@., E. H. MERKI~ELD,AND A. M. Sxsa~~, unpubl~+zd_rtzshs.
STRUCTURAL FEA TURFS OF THE GUM EXUDATES SOME Acncia SPECIES OF THE SERIES- Phyhdimze AND BotryocephaiaP SHZRLEY C. CHURMS,EDWZN H. MERRIFELD, AN;) Ausrm C.S.P.R. Carbohydrate Research Unit, Deprtment University of Cape Town (South Africa)
FRDM
M. STEPHEN
of Organic Chemistry,
(Received July 9th 1976: accepted for publication, October 1Sth. 1976)
Smith degradation of each of the polydisperse, gum polysaecharides from and A. podalyriaefk&a, for which molecular-tieight distributions have been measured by gel-permeation chromatography, gives, in good yield, a methanol-insoluble polysaccharide that shows a single peak on examination by this technique. The molecular weights of the Smith-degraded polysaccharides are close to the values expected if the respective gum polysaccbarides, on losing periodate-vulnerable, peripheral sugar residues, were to be split also at regnlar intervals between the otherwise (1+3)-linked D-galactose chains. The structures of these gums, which are similar iu many respects, conform to the pattern shown reizntly to occur in the gum of A. baileyma. Acacia pycnantha, A. &&rmis, A. j&z~oZi4
INIXODUCITON
Since the technique of periodate oxidation, borohydride reduction, and controhedacid hydrolysis was l%rstapplied by F. Smith and his co-workers to *“urn arabic’, Acacia gum samples from numerous species have been shown, without exception, to contain an internal core of &(1+3)-linked D-galactopyranose residues’: Sequential Smith-degradations3 have given additional information about the nature of peripheral sugar groups, and measurements of the molecular weights of the periodate-resistant portions of the polysaccharides have been of value in the subclassification of gum-forming Acacia species 4. Recently, Smith-degradation procedures have shown that certain low molecular-weight Acacia gums, although polymohzcnlar, contain blocks of galactose residues that are not attacked by periodate but which are separated at regular intervals by sugar residues that are; the consequence is that suitably controlkd, acid hydrolysis of the oxidised, and subsequently reduced gum yields a product that shows ei’cher a single peak on gel chromatography5, or *Dedicated to the memcizy of Professor J. K. N. Jones, F.R.S.
4
S. C. CISUFMS, E. H. hERRWELD, A. M.
tiHEN
two or more peaks that bear a simple molecular-weight x!elationshipto one aqother6. Accordingly, further examples of Acacia gums have been examined in order to test whether the structural Pattern that has emerged is of generai applicability. It was of particular interest to apply Smith degradation to the gum of A. pycnantha (Series Phy&&zeae) because of the detaifed structural model that was available through the extensive researches of other workers’, including Barry degradation’ of the oxidised polysaccharide. The gums of A. dz@mis (Series Phyllodineae)g and A. jilicz~oiiu (Stxies Botryocephuiae) 1’ were also selected for examination because published analytical data for these gums indicate close similarity to the gums from these Series studied earliersV6. EXPERIMENTAL
Sources of gum specimens. - Samples of the gums of Acacia dzJj2ormis and A. j%czjXia, collected in New South Wales, Australia in January, 1970 and February, 1970, respectively, were made available through the courtesy of Dr. D. M. W. Anderson (Edinburgh), who has publishedg*” anal_yticaldata for these specimens. The samples of A. pycnantha gum used in these studies were collected near StelIenbosch, Cape Province, South Africa in November, 1974 and October, 1975, and several different samples of A. podafyriaefolia gum, originating in Johannesburg, Port Elizabeth, and Cape Town (South Africa) were collected in July, 1964, July, 1975 and May, 1976, respectively. Purification of gum samples. - Small samples of the gums, as collected from Acacia stems or branches, were examined by gel-permeation chromatography, but most of the experimental work was performed on bulked material that had been recovered by precipitation with ethanol (4 volumes) from clear, aqueous solutions of these gums. The precipitated polysaccharides were washed with ethanol and etber, redissolved in water, and freeze-dried. General experim,jl?iai condirions. - Opt&I rotations were measured at 20” on a Perk&Elmer 141 polarimeter, by using aqueous soiutions in the concentration range OS-l% unless otherwise specified. The solvent systems used in paper chromatography were (A) 4:1:5 I-butanol-ethanol-water (upper layer), (B) 2:1:1 lbutanol-acetic acid-water, and (C) 8:2: 1 ethyl acetate-pyridine-water. Gel-permeation chromatography was conducted on Bio-Gel P-300 or P-10 as appropriate; with M sodium chloride as eluent 3*11_Proportions of neutral sugars were determined by g.1.c. of the derived alditol acetates I2 , following hydrolysis of the polysaccharides and their degradarion products in sealed tubes, under nitrogen, with 2~ trifIuoroacetic acid for 8 h at 100”; the aqueous acid was normally removed by freeze-drying. Periodate consumption was monitored by the arsenite method 13_ Methylafion analysis. - After methylation of the gums by the Hakomori I4 and Furdie 1 ’ (2-4 times) procedures, the mixtures of methylated sugars produced on hydrolysis (trifluoroacetic acid as already described, for I8 h} were analysed by g.1.c. of the trimethylsilyl ethers of the derived alditolsr6.
GUM EXiJDATEs
OF hX.7CiU
5
Oxidation of each polysaccharide with aqueous sodium metaperiodate6 was terminated by addition of the calculated quantity of barium acetate, and the centrifugate was reduced with sodium borohydride. The reduced, oxidised polysaccharides, recovered as previously dcscribed5, were submitted to mild hydrolysis in M trifluoracetic acid for periods ranging from 54 to 120 h at room temperature. The decision to terminate the degradation, by freeze-drying the acidic solution, was governed in each case by the extent of diminution in mm of the polysaccharide and the degree of polymolecularity of the products as shown by gelpermeation chromatography on Bio-Gel P-10. Hydrolysis was stopped when the molecular-weight distribution of the resultant, degraded polysaccharide remained constant over a 24-h period, or longer, in contact with M trikluoroacetic acid. After Smith degradation had been carried to completion, all of the gums *yielded methanolinsoluble, degraded polysaccharides that were monodisperse and did not decrease in molecular weight on further contact with M trifluoroacetic acid at room temperature. The degraded polysaccharides (after paper chromatography of their hydrolysates had demonstrated the absence of uranic acid) were then treated in aqueous solution with mixed ion-exchange resins and freeze-dried. The methanolsoluble fractions of the Smith-degradation products were shown by paper chromatography to consist largely of polyol (glycerol, ma&y), together with low molecular-weight glycosides. Smith degradation. -
RESULaTS AND DISCUSSION
The gums of A. pycnantha, A. d@ormis~ and A. Jiricifolia show a close resemblance, both in the properties listed in Table I and in their structural components as indicated by meth;rlation analysis (Table II), to the gums of A. podaiyriaefoolia, 4. Jata, and A. baiieyana previously examined5p6. Partial-hydrolysis experiments reported elsewhere I7 have confirmed that A. pycnantha gum has the same general structural futures as the last three gums just named’* I** lg, notably acid-labile rhamnose, arabinose and galactose residues in short branches attached to a galactan core that ultimately breaks down to a series of neutral oligosaccharides contaiting /3-(l-+3)-linked D-galactose residues and, in small proportions, the aldobiouronic acid 6-O-(/3-D-glucopyranosyluronic acid)-D-galactose. Paper chromatography (SOL vents B and C) of partial hydrolysates (0.01~ trifiuoroacetic acid, 48 h, 100’) shows that the gums of A. dQ?bmis and A. jilicifolia also yield these products. The approach of using Smith degradation to ascertain the distribution of the B-(1 +3)linked D-galactose residues in the polysaccharide molecule has proved illuminating in previous studies3-6, particularly with A. baileyana gum5 which yields, after a single Smith degradation, a monodisperse, degraded polysaccharide (molecular weight 2500) in which (143)~linked D-galactose residues preponderate, although a small proportion of constituent arabinose remains. There is strong evidence reIat.ing the production of this single, degraded polysaccharide to the occurrence of repeating sub-units having molecular weight 4600 in the molecule of the intact polysaccharide.
E. &66EREIIREiT), 23.c. c X%U&4S;
6 TABLE
I
PROPERTIIZJ
Am
Gum
A. M. STEP-
OF GUM
POLYSACCHARIDES
FROM
Acacia pycnantha,
A. diffarnds,
.
=
A. J?ci/olirr
of
MokcuIar-weight distribution’.*
Equivalent wei8ht
A. pycaantha
A. dioxmia
A. GlicifoIia
-7 12ooO
1s
f7 13cW
31680(2); 24000(6); 17800(18); I2000(36); SOOO(17);6ooO(21)
34000(3); 28200(8); 23500(14); 15000(37); 12cloo(10); UMoo(ls); 7000(13)
3700 (ref. 7)
Hence uranic acid (mol %) Proportions of neutral sugars (mol %) Galactose Arnbinose Rhamnose Periodate consumption (mmoI.g-‘)
28200(2): 21400(S); 16800(16); 14c@O(9); 108G0(40); s400(25) 4300 (re& IO)
3400 (ref. 9)
5
5
4
67 26 2
73 20 2
81 14 1
5.7
6.7
6.2
“Bio-Gel P-300. *Mokcu!ar weights corresponding to peaks in gel chromatograms; relative proportions by weight in parentheses. TABLE
II
hnrrF~UrI0~
ANALYSIS
OF
A. pycnanrha, A. diflomlis,
AND
A. fiiicifotia
GUMSO
Portia&y nwthylated sugar (positions of O-methyl grasps giaen by Iocants)
Moiar propottions X. pyenantha gunP
A. difformis gumC
A. fikifolia
2,:,5-Ara 2.s-Al-a 3,5-Ara 2,3,4-Rha 53-Ara 3,kXla > 2,3,4,6-GaI z4,6-Gal 2,3,6Gal 2,3,4Gal 2?6-Gal 2&Gd 2-G&
18
15
11
5
3
I
3
20 3 I 7 4 30 6
20 3
gumC
; 3 33 3
“iajc for chlorothrm soiutious of the metbylated gums, - 50 (Ref- 22), -45, and -47”, respectively. ‘Compare ref. 22, where the proportions given are simifar except for end-group D-g&CtOpyraOOSe (found to be higher by assay based upon methyl gfycosides). Wakes given were wrr~~box%ed by g.1.c. aria&is of the alditol acetates; compare ref. 23, where methyylgiycosides were employed.
GUM EXUDATES OF
-&qc&
7
The data- presetit& Sn ‘TaHe m show clearly that the gums of A. pycnantha, A. d#%rmis, and A. jiIic~j&h &so yield, after one Smith degradation, monodisperse
degraded polysaccharides that are essentially galactans, containing arabinose as a minor constituent. The specific rotations of these three degraded gums and the amounts of pcriodate consumed by the whoIe gums vable I) are similar to those found for gums of this type &died earlier. Mtinodispersity of the degraded polysaccharidcs is indicated by both gel-permeation chromatography and ult.racentrifugationzo, and the molecular-weight values obtained by these two independent procedures are in good agreement (Table In). T.1.c. on plates coated with Ki,eselguhr G [solvent systern” 4:4:2-@/v) 2-propanol-acetbne-M lactic acid, to which 34 parts by volume of water had been added] of each of these three Smith-degraded polysaccharides and that from A. baileyarragum gave single spots (RF -0.8). TABLE
III
PROPERTIES
OF hlETHANOL-INSOLUBLE
Smihdegraded
PRODUCTS
gum of:
[aln (de@eW Galactos&ambinose
ratio
Methyiated derivative: [alo in CHCls (degrees) MoIetXhi weight of Smith-degraded polysaccharide Calculated molecular weight of corresponding block in whole gum
%rom gel-permeation chromatography ments=*. The predominance of (1+3) by methyiation analysis.
OF SMITH
DEGRADATION
OF GUMS
A. pycnantha
A. difformis
A. Nicifolia
+28
+32
+29
955
97:3
97:3
-6
-5
35006 410@
36004 38W
21cW 21oob
6600
6800
42ClO
-12
(Bio-Gel P-10). ‘From sedinientation and diffusion measurelinkages in al1 three Smith-degradation products was proved
During the Smith-degradation procedure, the mild acid-hydrolysis s&p that ultimately yielded these moaodisperse, degraded polysaccharides was monitored by gel-permeation chromatography; this method proved more sensitive than others used previously6. Before the degradation was complete, gel-permeation chromatography @G-Gel P-10) of all of these gums showed the presence of components having molecular weights that approximated to multiples (2 and 3 times) of the molecular weights of the Gal products (see, for example, Fig. 1). This observation indicates a periodicity in the galactan skeletons of the -gums, with acetal linkages between the blocks of sugar residues that survive the periodate oxidation; these linkages are separated at regular intervals, and arc broken relatively slow!y during the hydrolysis step. Precise definition of the length of the galactan chains is not yet possible, although the narrowness of the elution band on g&permeation chromatography of smaller samples th%n that illustrated in Fig. 1, and preliminary fractionation experiments with high-pxzssure liquid chromatography (eluting with 2-propanol-water from a Partisil $0 column), support the view that the range caunot he large.
s. C. CHURMS, E. H. ?&RFWIELD,
A. M. sTEE!HEN
n 0
P N
80
110
IO
Eluted volume
Fig. 1. Gel-permeation chromatograms (A)_ Reduced, oxidised gum after perature (B) and for 92 h (C). Further Eluent: M sodium chloride. Columns: (A) gum
60 (ml)
60
70
showing progress of Smith degradation of A. filicifolia hydrolysis in .M trifluoroacetic acid for 54 h at room temexposure to acid for 24 h left the pattern (C) unchanged. Bio-Gel P-300,85 X I .5 cm; (B, C) Bio-de1 P-IO, 55 x 1.5 cm.
Allowing for the proportion of sugar residues removed by the Smith degradation (end-groups, and some arabinose and galactose present as glycosidegn the methanolsoluble material produced) permits calculation, as in the case of A. baileyana gums, of the molecular weights of the blocks of sugar residues in the intact polysac&arides from these gums that give rise to the degraded polysaccharides (see Table In). Scrutiny of the molecular-weight distributions of the gums (TabIe I) shows that most of the components present have molecular weights that are approximately multiples of the calculated values for the sub-units in the gum. The gum of A. podalyriaefoolia was previously reported6 to yield a polymolecular, Smith-degraded polysaccharide, but further contact of this product with 0.01~ triffuoracetic acid for 65 h at room temperature results in the disappearance of (minor) components having molecular weights 8400 and 5000, yielding a monodisperse polysaccharide of molecular weight 2100. This corresponds to a sub-unit, in the gum, of molecular weight approximately 4000. The various samples of the gum examined have been found to vary considerably in aW (as reported4 for some gums from other Acacia species), with values ranging from 9500 to 32090, even among samples taken from different branches of the same young tree. However, this variation was due to the presence, in differing proportions in the different samples, of components having molecular weights of the order of 4000, SOOO,16000, and 32000 in all cases. These are, once again, integral multipies of the molecular weight of the sub-unit indicated by Smith degradation. It has thus been shown that five polymolecular arabinogalactan gums, having relatively low HD, from Acacia species of the series PhyIlodineae (A:podalyrzizefoolia,-
GUM EXUDATE&
OF
Acacin
9
A. pycmn?ha, ahd A. dt~ormis) and Bott-yocephaiae (A. baileyana and A. filicifolia) give a monodisperse, degraded polysaccharide after a single Smith degradation. A notable exception is the otherwise similar gum of A. elata (series Burryocephalae), which yields two Smith-degraded polysaccharides, in nearly equal proportions by weight, havipg molecular weights 2100 and 4400, respectively6. Neither of these products undergoes any diminution in molecular weight when treated with M triIiuoroa+c acid for up to 83 h at room temperature, and therefore neither can be ascribed to the result of incomplete degradation_ In the five examples cited here, a sugar is present, between blocks of (l-+3)-linked galactose residues, so linked as to be vulnerable to attack by periodate and thus to very mild acid hydrolysis in the resulting oxidised, and subsequently reduced polysaccharide. That the polysaccharide itself is resistant to such mild hydrolysis has been demonstrated by gelpermeation chromatography of a sample of A. pycnarzrha gum that had been kept in M tritluoracetic acid for 144 h at room temperature; although paper chromatography showed the release of some arabinose, there was no discernible change in ME and therefore no cleavage of glycosidic bonds in the polysaccharide chains under these conditions. The nature of the inter-sugar linkages between the (1+3)-linked subunits in these Acacia gums remains to be investigated; ionic association between acid groups, for example, is not a major factor here24. ACKNOWLEDGhlENTS
The financial support of the Research and the University of acknowledged. We thank Dr. D. Oliver (Botanical Research Unit, Professor E. A. C. L. E. Schelpe classifying some of the specimens.
South African Council for Scientific and industrial Cape Town (Staff Research Fund) is gratefully M. W. Anderson (Edinburgh) and Dr. E. G. H. Stellenbosch) for providing gum specimens, and (Bolus Herbarium, University of Cape Town) for
REFERENCES F.
SMITH AND D. R. SPRIEZXERSBACH. Abstracts Papers Am. Chem. Sot. Meeting. 128 (1955) 15~; I. J. GOLDSKEIN, G. W. HAY, B. A. LEWIS, AND F. SMITH. Methods Carbohydr. Chem., 5
(196.5) 361-370. G. 0. ASPINALL, Ado. Carbohydr. Chem. Biochem., 24 (1969) 333-379. D. M. W. A--N, SIR EDhmD Hr~sr, AND J. F. STODDART, J_ Chem. Sot. (C), (1966) 1959-
1966. D. M. W. ANDER~N
AND I. C. M. DEA, Phytochem., 8 (1969) 167-176; J. Sot. Cosmetic Chem., 22 (1971) 61-76. S. C. CHURUS AND A. M. STEPHEN, Ckrbohyak. Res., 45 (1975) 291-298. P. I. BEKKER, S. C. CHURMS, A. M. STEPHEN, AND G. R. WOOLARD, J. S. Afr. Chenr. Inst., 25 (1972) 115-130. E. L. Hmm AND k S. PERIXN, J. Chem. Sot_, (1954) 2622-2627:G. 0. A~PINALL, E. L. HIRST, AND A. NICOLSON, ibid., (1959) 1697-1706; D. M. We ANDERSON AND G. M. CWEE, Carbohy&. Res.. 6 (1965) 214-219. V. C. BARRY, Nature, 152 (1943) 537: V. C. BARRY AND P. W_ D. MITCHELL, J. Chem. SOL,
(1954) 40203023. D. M. W. ANDERSON, P. C. Brr,~m
C. G. A. McNks,
Phytochem, ll(l972)
175I-1754.
10
S.-‘it!. CHfmMS,.'E,. H; MBRRlFdD;
$L’.M:--SHBE+
: .10 D. M. W. ANDERSON,P. c. BELL,AND C. G. A..M~~~~&a&jhy&~~Re& ZO (1971)2&3+27P;. 11 S. 6. C&uds~yD~A. M.~~, S:Afii &fed.-+;43 (1%9j iti. - -‘:,.:. ~.:: : .:’ 12 .j. S: SAiVARIjti; J;i!i. Stir&&t,- AND it: JE&s; AMI.-C&5&; j;i it%5)f602+04; 13 P:FUZJEYAND J. L~~ti~.J.:.Fhtwaz. CXm... 17 (1933)~107~113;~ 14 S. .H;\K?.HO&J. Rioc&em. @‘bityu), 55. (1964) 2@-208. ‘.- ._ .--. 15 T_ PUIUNEAE~,3. C. hvme, J. -Che+. So& !33-(f!$3) 102hO37. 16 B. l-I. FREEUN, A. ha: STEkEN, AND P. VANDERBZFL,I.‘C&&o&, 73 (19i2)2P-33. 17 J. ADAM, S. C. ,@iui; A. M. SIZPHEN,D.-G. Smim&~~, ti ,E H. ?Uxf&i~& &zr&hy&: Rex, 54 (1977) 304-307. 18 S. C. ‘CHURMSAND A. M- STEPA; &&&& R&i, i 5 (197&l t-19: 19 S. C. C&ws AND A. M; STEPHEN,. Cmbohy&. Res.; Z1 ‘(1972)s91-98..: 20 S. C. ihJR~, k hf. STEPHEN, AND A. PO-N, S. ,4fi; 1. S&72 (1976) ISl-i52. 21 S. A. HANSEN,J. Chromatagr., 105 (1975) 388-390. : . .. 22 M. KAPLAN AND A. M. STEPHEN, Teirahedrwz,~2~ (1967) 193_1?8; 23 D. M. W. A~ahso~ m.P. C. BELL,Phyrocheti.; 15.(1976).3Ol-303. 24 S. C. CHXJR?@., E. H. MERKI~ELD,AND A. M. Sxsa~~, unpubl~+zd_rtzshs.