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Lemon-peel pectin
CarbohydrofeResearch
442
Elsevier Publishing Company. Amsterdam Printed in Belgium
LEMON-PEEL
PECTIN
PART
I. FRACTIONATION
G. 0.
&PINALL*,
Department
J. w. T.
of Chemistry,
AND
PARTIAL
CRAIG,
AND
HYDROLYSIS
OF WATER-SOLUBLE
PECTIN
J. L. WHYn
University of ISiinburgh, Edinburgh (Great Britain)
(Received February 26th, 1968; in revised form, March 29th, 1968)
ABSTRACT
The pectin isolated from dried lemon-peel by extraction with cold water has been subjected to partial depolymerisation by acid hydrolysis, acetolysis, and enzymic degradation. Acidic oligosaccharides whose structures were established, or indicated by chromatographic identification of the products of methylation linkage-analysis, include 2-O-(a-D-galactopyranosyluronic acid)-L-rhamnose, higher oligosaccharides containing residues of gaIacturonic acid and rhamnose, 4-O-(a-D-galactopyranosyluranic acid)-D-galacturonic acid and the polymer-homologous trisaccharide, 6-O(glucopyranosyluronic acid)galactose and 4-O-(glucopyranosyluronic acid)fucose, and 3-Q-xylopyranosylgalacturonic acid. INTRODUCTION
Many pectins in which D-galacturonic acid is the principal constituent contain neutral sugars, especially D-galactose, r._-arabinose, and t.-rhamnose, as integral constituents. Polysaccharides of this type have been examined recently from several sources including sisal fiesh’, Iucerne (alfalfa)2*3, the bark of Amabilis fir4, apple$, white-mustard cotyledon$‘, sycamore cambial and callus tissues7, soybean cotyledons’, and soybean hulls’. Investigations in this laboratory on a commercial sample of citrus pectin from oranges and lemons” provided further evidence for L-rhamnose as a constituent sugar, and also indicated that chains of (1+4)-linked B-D-galactopyranose residues were present in the acidic polysaccharide, since 4-0-p-D-galactopyranosyl-D-galactose and its polymer-homologues were formed as products of partial hydrolysis. However, in view of the mixed origin of the pectin sample and of the uncertain method of extraction, a more detailed examination has been carried out on the pectin isolated from dried lemon-peel by extractions with cold water. We report here the characterisation of acidic oligosaccharides formed on partiai depolymerisation of this pectin. RESULTS AND DISCUSSION
Extraction
of dried lemon-peel with cold water furnished a polysaccharide
*Present address: Department
of Chemistry,
Carbohyd. Res., 7 (1968) 442452
Trent University,
Peterborough,
Ontario,
Canada.
LEMON-PEEL PECTIN. PART I
443
preparation constituting some 10% by weight of the raw material. Subsequent experiments (unpublished results) have shown that considerable further quantities of pectin may be extracted under more drastic conditions, and a detailed examination of this material will be undertaken later. Pectin A of constant composition was isolated from the water-soluble fraction after two reprecipitations from water with acetone. Pectin A was apparently homogeneous when examined by boundary eIectrophoresis. Chromatography on 0-(2-diethylaminoethyl)cellulose, however, gave two main fractions of acidic polysaccharides, which probably represented pectins differing in degree of esterification, together with a small fraction (ca. 5 %) of neutral polysaccharide which gave an arabinose” and a galactose* on hydrolysis. This pectin was, however, considered suitable for studies on the acidic oligosaccharides formed on enzymic hydrolysis. Fractionation of pectin A by precipitation of the insoluble copper salt gave pectin B, but associated neutral polysaccharide was still present. Complete removal of accompanying neutral polysaccharide was achieved by preparative chromatography on O-(Zdiethylaminoethyl)-Sephadex, which gave pectin C and two minor polysaccbaride fractions D and E, to be examined later. Pectin C had a uranic acid content of 76%, and the methoxyl content indicated a degree of esterification of 76%. Hydrolysis gave galacturonic acid, together with arabinose, gaIactose, rhamnose, and a trace of xyli ;e. The latter polysaccharide fractions gave arabinose and galactose as the main products of hydrolysis; one, as the sole product, and the other, together with acidic sugars. Partial hydrolysis by acid of pectin B gave a mixture of acidic oligosaccharides which was separated by chromatography on 0-(2-diethylaminoethyl)-Sephadex, followed where necessary by filter-sheet chromatography, and six oligosaccharides were identified (Scheme I). 2-0-(a-D-Galactopyranosyhtronic acid)-r_-rhamnose (1) was characterised by conversion into the crystalline methyl glycoside pentamethyl ether. The nature of the linkages in oligosaccharides 2 and 3 was indicated by the results of methylation. Although no direct evidence was obtained for anomeric and enantiomorphic configurations, the oligosaccharides were chromatographically indistinguishable from 6-0-(/I-D-glucopyranosyluronic acid)-D-galactose and 4-0(B-D-glucopyranosyluronic acid)-r_-fucose. The structure of the tetrasaccharide 4 was assigned as an 0-(galactopyranosyluronic acid)-( l-+2)-0-rhamnopyranosyl-(1+4)0-(galactopyranosyluronic acid)-(1 +2)-rhamnose on the basis of (a) partial hydrolysis and (6) methylation of the oligosaccharide and the derived alditol. 4-0-(or-D-Galactopyranosyluronic acid)-D-galacturonic acid (5) and the polymer-homologous trisaccharide (6) were each characterised as their crystalline calcium salts and by methylation of the corresponding neutral methyl glycosides obtained by the sequence of reactions involving conversion of the acidic oligosaccharides into the methyl ester gIycosides, Q-imethylsiIylation, reduction with Iithium ahrminium hydride, and removal of protecting groups. *Omission of configurational prefixes indicates that the sugars or their derivatives were identified by chromatographic methods. Carbohyd.
Res., 7 (1968) 442-452
G. 0. ASPINALL,
J. W. T. CRAIG, J. L. WHYTE
Acetolysis of pectin C resulted in the isolation of acidic oligosaccharides only, and each of these contained residues of galacturonic acid and rhamnose. Oligosaccharides 1 and 4 were characterised as described previously, and oligosaccharide 7 was characterised as 0-(galactopyranosyluronic acid)-(1 -+2)-0-rhamnopyranosyl-(1+2)rhamnose by methylation of the trisaccharide and the derived alditol. P a-D-GalpA-(l-+2)-L-Rha 2 GpA-(1+6)-Gal
3 4 5 6
GpA-(1+4)-Fuc GalpA-(l+2)-Rhap-(I+4)-GaIpA-(l+2)-~~
H”voH
&
a-D-GalpA-(l-w%)-D-GalpA M-D-GalpA-(l-+4)-D-GalpA-(14)-D-GalpA 7 GalpA-(l-t2)-Rhap-(1+2)-Rha 8 Xylp-(l-+3)-GalA 10 GalpA-(1+2)-Rhap-(1+4)-GalpA
6H
9
Scheme I
Degradation of pectin A by a commercial enzyme-preparation afforded three further acidic o!igosaccharides. Methylation analysis indicated that oligosaccharide 8 had the structure 3-0-xylopyranosylgalacturonic acid. The tentative structure, 2-0-(4-deoxy-p-L-three-hex4enopyranosyluronic acid)L-rhamnose, was assigned to oligosaccharide 9 on the basis of the folIowing observations: (i) it had a light-absorption maximum at 231 nm characteristic of a& unsaturated carboxylic acids; (ii> it gave a red colour (R,,,547 nm) with thiobarbituric acid; (iii) hydrolysis gave rhamnose as the sole detectable product; and (iz~)cleavage of the methylated disaccharide gave the methyl glycoside of 3,4-di-0-methylrhamnose as the only recognisabie product. The formation of oligosaccharide 9 shows that the enzyme preparation used, like other commercial “pectinases”“, contains some pectin and/or pectic acid hydro-lyase activity in addition to the hydrolytic activity. Oligosaccharide 10 was assigned the structure, 0-(galactopyranosylurqnic acid)-(I +2)-Orhamnopyranosyl-(I +4)-galacturonic acid, since it contained a single rhamnose and two galacturonic acid residues, one at the reducing position. The site of substitution of the rhamnose residue was apparent from methyiation studies, but that of the reducing galacturonic acid residue has been assigned only tentatively. Additional evidence for the overall structure of the pectin was obtained by a series of reactions involving completion of the esterification of the galacturonic acid residues on treatment of pectin C with ethylene oxide, acetylation, and reduction with Iithium borohydride, which resulted in the formation of the carboxyl-reduced polysaccharide. Hydrolysis of this derivative gave galactose as the preponderant product with only traces of other sugars. The presence and nature of other sugar residues were established by gas-chromatographic examination of the methanolysis products from the methylated polysaccharide. The major products were methyl glycosides of 2,3,6-tri-0-methylgalactose, but small proportions of methyl glycosides of 2,3,4,6tetra- and 2,6-di-0-methylgalactose, 2,3,5-t& and 2,3-di-0-methylarabinose, and 3,4-di- and 3-0-methylrhamnose were also present. Cdmhyd.
Res., 7 (1968) 442-452
LEMON-PEEL PECTIN. PART I
445
The present results show that many of the structural features which have been established for lucernezS3 and soybean’s9 pectins are also encountered in this lemonpeel pectin. The hexuronic acid content (76 Oh) of the pectin indicates that ca. 79 % of the sugar residues are D-galacturonic acid. Although glucuronic acid is a trace constituent of this, as of other, pectins, it probably accounts for less than 1% of the sugar residues. Of the neutral sugar constituents, the clearest evidence for a structural role has been obtained in the ease of L-rhamnose. It is evident from the characterisation of the acidic oligosaccharides 1, 4, 7, and 10, which were formed in different ways on partial fragmentation, that residues of this sugar are present in the main chains of the pectin, where they interrupt blocks of 4-O-substituted a-D-galacturonic acid residues. The rhamnose residues, however, are unevenly distributed in the galacturonorhamnan chains and are probably concentrated in certain regions, where they may alternate with galacturonic acid residues, as in oligosaccharide’4, or occur as adjacent units, as in oligosaccharide 7. The other neutral-sugar constituents of lemon-peel pectin probably occur in outer chains. Methylation of the carboxyl-reduced pectin showed that arabinofuranose residues were present together with some non-terminal units. Apart from the partial characterisation of the aldobiouronic acid, namely, 6- O-(glucopyranosyluronic acid)galactose (2), as a minor product of partial hydrolysis, nothing is yet known of the location of the galactose residues. However, the failure to detect galactose-containing oligosaccharides as acetolysis products under conditions in which they were formed from soybean pectins**’ suggests that chains of contiguous galactose residues are largely absent in the lemon pectin. Although xylose is only a trace constituent of the pectin, the isolation of the oligosaccharide 3-O-xylopyranosylgalacturonic acid (8) on enzymic hydrolysis showed that residues of this sugar are attached to the galacturonan chain, as in soybean-cotyledon pectin’ and tragacanthic acidr3. As indicated by the isolation of the two aldobiouronic acids 2 and 3, this pectin, like those from soybeans’*’ and lucerne2v3, contains glucuronic acid and fucose as trace constituents, but there is no evidence for their structural location. EXPERIMENTAL
General. - Unless otherwise stated, sugars and oligosaccharides were identified by paper chromatography, which was performed on Whatman Nos. 1 and 3 MM papers, with the following solvent systems (v/v): (A) 10:4:3 ethyl acetate-pyridinewater; (B) 18:3: 1:4 ethyl acetate-acetic acid-formic acid-water; (C) l&8:3:9 ethyl acetate-acetic acid-formic acid-water; (D) 4: I :5 butyl alcohol-ethanol-water (upper layer); Q 200:17: 1 butanone-water-ammonia; (F) 9: 1: 1 butanone-acetic acid-water (saturated with boric acid)_ The R, values of methylated sugars refer to rates of movement reiative tc hat of 2,3,4,6-tetra-O-methyl-D-glucose (as unity) in solvent D. Paper ionophoresis was conducted in borate buffer at pH 10. Unless otherwise stated, optical rotations were observed for aqueous solutions at ca. 18”. Gas chromatography was performed on columns of acid-washed Celite coated Carbohyd. Res., 7 (1968) 442-452
G. 0.
ASPINALL,
J. W. T. CRAIG,
J. L. WHYTE
with (G) 10% by weight of butane l&diol succinate polyester (operating temperature, l?Y), (6) 10% by weight of m-bis(m-phenoxyphenoxy)benzene (200”), and (c) 3% or 5 % by weight of neopentylglycol adipate polyester (125 or 150”). All compounds whose identification was by gas chromatography, alone, had the same retention times as those of authentic reference compounds. Isolation and fractionation of lemon-peel pectin. Powdered lemon-peel, which was kindly supplied by Mr. W. C. Platt of the Ventura Coastal Lemon Corporation, Ventura, California, had been pre-extracted with isopropyl alcohol to remove lemon oil. Powdered peel (I kg) was stirred in water (30 liters) at pH 4.8 for 24 h at room temperature. Residual peel was removed in a centrifuge, and the extraction was repeated. The combined, aqueous extracts were poured into acetone (1.5 vol), and the resulting precipitate was washed with acetone-water, dissolved in water, and freeze-dried to give crude pectin (80 g), [aID+ 193” (c 0.32). The crude pectin was reprecipitated three times from aqueous solution with acetone, to give
pectin A (68 g), [a], +218” (c 0.31), of constant composition [Found: uranic acid (by decarboxylation), 75; OMe, 10.7%]. A sample of pectin A was chromatographed on O-(2-diethylaminoethyl)cellulose (phosphate form)14 with 0.025~, 0.05~, 0. IM, 0.25~, and 0.5~ sodium dihydrogen phosphate buffer at pH 6 as eluant. The fractions were monitored with the phenol-sulphuric acid rea’gent15, appropriate fractions were dialysed and concentrated, and polysaccharides were precipitated with acetone, redissolved in water, and isolated by freeze-drying. Hydrolysis of the polysaccharides indicated that a neutral fraction (ca. 5%) was eluted with 0.025~ buffer, but that all later fractions (largely eluted with 0.1~ and 0.25~ buffer) were of essentially similar composition. Aqueous, 7% cupric acetate (200 ml) was added dropwise, with stirring, to pectin A (20 g) in water (4 I). The insoluble copper salt that resulted was removed in a centrifuge, and decomposed by washing with acetone containing 1% of hydrogen chloride, and the precipitate remaining was washed free of acid with ethanol, dissolved in water, and freeze-dried to give pectin B (18.2 g), [cz],, f221 o (c 0.28) [Found: uranic acid residues, 76; OMe, 10.2%]. Chromatography of a sample of pectin B on U-(2-diethylaminoethyl)cellulose as described above showed that the neutral polysaccharide fraction was still present, although in slightly diminished amount. Pectin A (12 g) in water (600 ml) was adsorbed on a column of O-(2-diethylaminoethyl)Sephadex A-50 (100 g, formate form), and polysaccharide fractions were eluted with water (12 liters), 0.2~ formic acid (12 liters), and M formic acid (20 liters). The formic acid solutions were dialysed for 36 h, the pH of the solutions was adjusted to 4.5 by the addition of potassium acetate, and the solutions were concentrated, the pH being kept at 4.5 by the addition of acetic acid. The polysaccharide contents were precipitated with ethanol containing 4% of acetic acid, washed with ethanol, redissolved in water, and freeze-dried_ In a typical separation, polysaccharide D (196 mg, eluted with water) had [a& + 38” (c 0.2), and gave, on hydrolysis, arabinose, galactose, and smaller amounts of galacturonic acid, xylose, fucose, and rhamnose [Found: uranic acid residues, lo’/,]; polysaccharide E (381 mg, eluted with Carbohyd. Res., 7 (1968) 442-452
LEMON-PEEL
PECTIN.
PART
447
I
0.2M formic acid had [a], + 78” (c 0.2), and gave, on hydrolysis, galactose, arabinose, galacturonic acid, and a trace of rhamnose [Found: uranic acid resldues, 27%]; and pectin C (7.29 g, eluted with M formic acid) had [a], +218” (c OS), and on hydrolysis gave galacturonic acid, together with arabinose, galactose, and traces of xylose [Found: uranic acid residues, 76; OMe, 10.2 %I. Chromatography of pectin C on 0-(2-diethylaminoethyl)cellulose showed that neutral polysaccharide was absent. Partial, acidhydrolysis ofpectin. - Pectin B (17 g) was heated in 0.5~ sulphuric acid (150 ml) for 4 h on a boiling-water bath, during which time degraded polysaccharide separated. Degraded polysaccharide was removed at the centrifuge, and further polysaccharide was precipitated by the addition of acetone (1 vol) and removed by centrifugation. The supernatant liquid was concentrated to remove acetone, rendered neutral with barium hydroxide and barium carbonate, filtered, passed through a column of Amberlite resin IR-120 (H+) to remove barium ions, and evaporated to a syrup. The barium sulphate precipitate was washed with very dilute sulphuric acid (pH 4), and the washings were rendered neutral by shaking with Amberlite resin LA-l (5% in chloroform), passed through a column of Amberlite resin IR-120 (Hf), and evaporated to a syrup. The combined, degraded polysaccharides were rehydrolysed, and the products were isolated in the same way. Residual, degraded polysaccharide (5.94 g) gave, on hydrolysis, galacturonic acid and only traces of neutral sugars. The combined syrups (8.25 g) were adsorbed batchwise on columns of U-(2-diethylaminoethyl)-Sephadex A-25 (formate form)_ Elution of the columns with water removed neutral sugars, and elution with a gradient of water containing Od0.4~ formic acid, and with o.Jhf, M, and 3M formic acid gave fractions containing galacturonic acid and acidic oligosaccharides. Further separations by filter-sheet chromatography in solvents B and C gave chromatographically pure samples of oligosaccharides’-6. Oligosaccharide 1. The sugar (47 mg), R,,,0.83 in solvent B, IW~ 0.56, had [01]nt96” (c 0.47), gave galacturonic acid and rhamnose on hydrolysis, and was chromatographically indistinguishable from 2-0-(a-D-galactopyranosyluronic acid)L-rhamnose. The aldobiouronic acid was characterised by conversion into its methyl glycoside pentamethyl ether dihydrate, m.p. and mixed m.p. 69” (hot-stage) and 119” (capillary tube), [a]u + 91 o (c 0.63, chloroform), and by X-ray powder photography’. Oligosaccharide 2. The sugar (8 mg). R,,,0.26 in solvent B, MG 1.04, had [I&, +6” (c 0.4) gave glucuronic acid, glucurono-6,3-lactone, and galactose on hydrolysis, and was chromatographically and ionophoretically indistinguishable from 6-0-(B-D-glucopyranosyluronic acid)lD-galactose. Gas chromatography of the methanolysis products from the methyfated derivative on column a showed the presence of methyl glycosides of 2.3,4-tri-0-methylglucuronic acid, and 2,3,4- and 2,3,5-tri-0-methylgalactose. Oligosaccharide 3. The sugar (15 mg). Rc,,0.60 in solvent B, Mo 0.63, -70” (c 0.8), gave glucuronic acid, glucurono-6,3-lactone, and fucose on had blD hydrolysis, and was chromatographically and ionophoretically indistinguishable from 4-0-(/?-D-glucopyranosyluronic acid)-L-fucose’. Gas chromatography of the
Carbohyd.
Res., 7 (1968) 442452
448
G. 0.
ASPINALL.
J. W. T. CRAIG,
J. L. WHY-l-E
methanolysis products from the methylated derivative on column a showed the presence of methyl glycosides of 2,3,4-tri-U-methylglucuronic acid and 2,3-di-Umethylfucose. ’ Oligosaccharide 4. -The sugar (8 mg). RGol 0.12 and 0.56 in solvents B and C, McO.67, had [a]n +92” (c 0.4), and gave, on partial hydrolysis, galacturonic acid, rhamnose, and oligosaccharide 1. CoIorimetric determinations of galacturonic acid (carbazole method)16 and rhamnose (cysteine method)” residues indicated their presence in the approximate molar ratio of I:1 in the oligosaccharide and of 2: 1 in the derived alditol (borohydride reduction). Controlled, partial hydrolysis of the derived aldito1 gave 2-0-(galactopyranosyluronic acid)rhamnose and 2-O-(galactopyranosyluronic acid)rhamnitoI (separated in solvent F). Gas chromatography of the methanolysis products from the methylated oligosaccharide alditol showed the presence of 1,3,4,5-“cetra-O-methylrhamnitol, and methyl glycosides of 2,3,4-triand 2,3-di-0-methylgalacturonic acid, and 3,4-di-O-methyl-rhamnose. Oligosaccharide 5. - The sugar (508 mg), Rcol 0.21 and 0.50 in solvents B and C, MG 0.95, was chromatographicahy and ionophoretically indistinguishable from 4-0-(a-D-galactopyranosyluronic acid)-D-galacturonic acid, gave galacturonic acid, only, on hydrolysis, and furnished a calcium salt, [a], + 115” (c 0.68, 0.5M hydrochloric acid)‘. The sugar (30 mg) was treated with methanolic 1% hydrogen chIoride for 18 h at room temperature, the acid was neutralised with silver carbonate, and the mixture was filtered, and the filtrate evaporated. The resulting methyl ester methyl glycosides (31 mg) were treated with hexamethyldisilazane (0.6 ml) and chlorotrimethylsilane (0.3 ml) in pyridine (3 ml) for 1 h at 25”. The resulting solution was evaporated to dryness. The residue was extracted with ether (2 x 10 ml), and the extract was evaporated to a syrup (52 mg)). The trimethyIsiIy1 ethers were reduced by refiuxing with lithium ahrminium hydride (100 mg) in ether (5 ml) for 2 h, and ethyl acetate was added to decompose the excess of hydride. The precipitate that separated was removed by centrifugation, and shaken with 0.25~ sulphuric acid (3 ml) for 1 h. The resulting solution was rendered neutral with barium carbonate, the suspension was filtered, and the filtrate was treated with Amberlite IR-120 (H+) resin to remove barium ions, and evaporated to a syrup (21 mg). Hydrolysis of a sample of the syrup gave gaIactose only. The major portion of the neutral disaccharide glycoside was methylated with methyl iodide and silver oxide in NJ%dimethylformamide, and gas chromatography of the methanolysis products of the methylated derivative on column c showed the presence of methyl glycosides of 2,3,4,6-tetraand 2,3,6-tri-0-methylgalactose. Oligosaccharide 6. - The sugar (923 mg), RGllI0.24 in solvent C, Mc 0.95, was chromatographicahy and ionophoretically indistinguishable from the “galacturonotriose” isolated from other pectins, gave galacturonic acid, only, on hydrolysis, and furnished a calcium salt, [OL],,+ 135” (c 0.72, 0.5M hydrochloric acid)‘. The oligosaccharide (110 mg) was converted into the methyl ester methyl glycosides as described for oligosaccharide 5,.and thence into the trimethylsilyl ethers, which were reduced with lithium aluminium hydride. The resuhing methyl galactotricsides Carbohyd. Res., 7 (1968) 442452
LEMON-PEEL
PECTIN.
PART I
449
were methylated with methyl sulphate and sodium hydroxide, and methyl iodide and silver oxide, to give the methylated trisaccharide (66 mg). A sample of this derivative was heated with methanolic hydrogen chloride, and gas chromatography of the products on column c showed the presence of methyl glycosides of 2,3,4,6-tetra-, 2,3,6-t&, and (in traces) 2,6-di-0-methylgalaclose. The major portion (60 mg) of the methylated trisaccharide was hydrolysed in hi hydrochloric acid (3 ml) for 8 h on a boiling-water bath, and the cooled solution was rendered neutral with silver carbonate, centrifuged, treated with Amberlite IR-120 (H+) resin to remove silver ions, and concentrated to a syrup (57 mg). The syrup was adsorbed on a column of 1:1 charcoal-Celite (20 g), and elution with water containing increasing proportions of ethanol gave fractions i (4 mg), ii (29 mg), and iii (17 mg). Fraction i, R, 0.56 and 0.19 in solvents D and E, gave methyl glycosides having the retention times of those of 2,6-di-0-methylgalactose. Fraction ii, R, 0.76 and 0.51 in solvents D and E, [c& +82” (c 0.58, chloroform), gave methyl glycosides having the retention times of those of 2,3,6-tri-0-methyl-D-galactose. The sugar was characterised by conversion into 2,3,6-tri-0-methyl-D-galactono-l&lactone, m-p. and mixed m-p. 97-98”. Fraction iii, R, 0.89 and 0.87 in solvents D and E, [a], f97” (c 0.34, chloroform), gave methyl glycosides having the retention times of those of 2,3,4,6-tetra-O-methyl-Dgalactose, and was characterised by conversion into the aniline derivative, mp. and mixed m.p. 196-197”. Acetolysis ofpectin. - Pectin C (12 g) was acetylated by the method of Carson and Maclay’s, and the acetylated polysaccharide (12.3 g) was dispersed in acetic acid (320 ml). Acetic anhydride (320 ml) was added dropwise to the solution, and concentrated sulphuric acid (32 ml) was added dropwise, with vigorous stirring, during 2 h. The resulting solution was kept for 75 h at room temperature_ A precipitate that separated gave galacturonic acid, only, on hydrolysis. The supernatant liquid was poured into ice-water, and brought to pH 3 by the addition of sodium hydrogen carbonate. The precipitated acetates were separated by centrifugation, and dissolved in chloroform, and the aqueous solution was extracted with chloroform (4 x 150 ml). The combined chloroform extracts were evaporated to a syrup (6.5 g), which was deacetylated by treatment of a solution in chloroform (15 ml) and methanol (30 ml) with 0.25&rbarium methoxide in methanol (30 ml) for 24 h at 0”. The reaction mixture was poured into water, rendered neutral with dilute sulphuric acid, filtered, treated with .~,mberlite IR-120 (H’) resin to remove barium ions, and evaporated to a syrup (2.0 g). The syrup was adsorbed onto a column of 0-(2-diethylaminoethyl)Sephadex A-25 (formate form) (25 g). Elution with water gave neutral sugars (181 mg), including arabinose, galactose. xylose, and rhamnose, but no oligosaccharides could be detected, either by direct examination or after fslrther separation by chromatography on charcoal-Celite. Elution of the ion-exchange column sequentially with 0.05~~ a gradient of 0.05 --, 0.4M, 0.4Mr, and hl formic acid gave a series of fractions, from which chromatographically pure samples of oligosaccharides 1 (20 mg), 4 (8 mg), and 7 (11 mg) were isolated after further separation by filter-sheet chromatography. Oligosaccharides 1 and 4 were characterised as described previously, and later fractions, Carboh_vd. Res., 7
(1968)442-452
450
G. 0. ASPINALL,
J. W. T. CRAIG,
J. L. WHYTE
which contained “galacturonobiose” and “galacturonotriose” and gave galacturonic acid, only, on hydrolysis, were not examined further. Oligosaccharide 7. - The sugar, RGIII0.49 in solvent B, had [a],, + 84” (c 0.2), and gave galacturonic acid and rhamnose on hydrolysis. Partial, acid hydrolysis gave 2-0-(galactopyranosyluronic acjd)rhamnose (oligosaccharide 1) and rhamnose, and similar treatment of the derived alditol gave the same aldobiouronic acid and rhamnitol. Gas chromatography of the methanolysis products from the methylated oligosaccharide alditol on column c showed the presence of 1,3,4,5-tetra-O-methylrhamnitol and methyl glycosides of 2,3,4-tri-0-methylgalacturonic acid and 3,4-di0-methylrhamnose. Enzymic degradation of pectin. - Pectin A (20 g) was digested with “Pectinase” (Koch-Light Laboratories) (2 g) in water (10 liters) for 24 h at room temperature, and the enzyme and degraded polysaccharide were precipitated by the addition of acetone (1 vol). The supernatant liquid was concentrated, poured into acetone (4 vol), and kept for 2 days at 4”. The resulting precipitate appeared to contain crystalline galacturonic acid, and was removed by centrifugation. It was dissolved h water, the pH of the solution was adjusted to 7 by the addition of ammonia, and evaporation gave syrup S, (9 g). The supernatant liquid was also adjusted to pH 7 by the addition of ammonia, and evaporated to a syrup S, (ll g). Although galacturanic acid was the main component of both syrups, the syrups were separately chromatographed on 0-(2-diethylaminoethyl)-Sephadex A-25 (formate form) (50 g), using water to elute neutral monosaccharides, and 0.05~ and a gradient of 0.05 + 0.4~ formic acid to elute galacturonic acid and acidic oligosaccharides. Fractions containing three previously unrecognised oligosaccharides were obtained, and chromatographically pure samples of oligosaccharides 8 (12 mg), 9 (19 mg), and 10 (29mg) were isolated by filter-sheet chromatography. Ohgosaccharide 8. - The sugar, RGolA 0.53 in solvent B, [&, t28” (c 0.24), was chromatographically similar to 3-O-&D-xylopyranosyl-D-galacturonic acid, and on hydrolysis gave galacturonic acid and xylose”. Hydrolysis of the derived alditol gave xylose as the sole reducing sugar. Gas chromatography of the methanolysis products from the methylated alditol on column c showed the presence of 2,3,5,6tetra-O-methylgalactono-l,4-lactone and methyl glycosides of 2,3,4-tri-O-methylxylose’. Oligosaccharide 9. - The sugar, RGIIIA 2.02 in solvent B, had [01]o+ 87” (~0.30) and 1,,, 231 nm. Rhamnose was the only detectable hydrolysis product of the sugar, and rhamnitol, of the derived alditol, but quantitative estimation by the cysteine method” indicated the presence of 46% of rhamnose residues in the sugar. The sugar reacted with thiobarbituric acid in a manner characteristic of glycosides of 4-deoxy-L-tflreo-hex-4-enosuronic acid” to give a red colour (A,,, 547 nm). Gas chromatography of the methanolysis products from the methylated alditol on column c showed the presence of the methyl glycoside of 3,4-di-0-methylrhamnose and unidentified components. Oligosaccharide 10. - The sugar, RClrlA0.12 and 0.50 in solvents B and C, had &i+&,&. Rex., 7 1I968) 44%&Z
LEMON-PEEL
451
PECTIN., PART I
[a]n + 85” (c 0.4), and gave galacturonic acid and rhamnose on hydrolysis. Colorimetric estimations’6*‘7 of galacturonic acid and rhamnose indicated that the sugars were present in the ratio of 1.9: 1, and, after reduction to the alditol, in the ratio of 1: 1. Treatment of the oligosaccharide for 16 h at 100” in 1:l methanol-water containing 2.5 % of triethylamine gave 2-O-(galactopyranosyluronic acid)rhamnose as the sole reducing sugar. Gas chromatography of the methanolysis products from the methylated trisaccharide alditol on columns b and c showed the presence of methyl glycosides of 2,3,4-tri-O-methylgalacturonic acid and 3,4-di-O-methylrhamnose, and an unidentified component (T3.70 and 9.95 on columns b and c). This component may have been a derivative of 2,4,5,6-tetra-O-methylgalactonic acid, since compounds having the same retention time were observed in the methanolysis products from methylated 3-O-(a-D-galactopyranosyluronic acid)-L-galactonic acid (“galacturonobiitol”) and methylated 3-O-(a-D-galactopyranosyl)-D-galactonic acid (from oxidation of the corresponding galactobiose). Methylation of carboxyl-reduced pectin. Pectin C (1 g) in water (100 ml) was passed through Amberlite IR-120 (HC) resin to remove metal ions, ethylene oxide (25 ml) was added, and the solution was kept for 5 weeks at room temperature, whereupon esterification was complete (pH 6.8). The polysaccharide ester (1 g) was isolated by precipitation with ethanol (3 vol), dissolved in water, and freeze-dried, and was then acetylated by the method of Carson and Maclay” to give the acetate (1.1 g). Lithium borohydride (I. 1 g) in tetrahydrofuran (25 ml) was added to a suspension of the polysaccharide acetate (1.1 g) in tetrahydrofuran (25 m!), and the mixture was refluxed, with stirring, for 20 hzo. Excess of hydride was decomposed by’ the dropwise addition of water, more water (50 ml) was added, and tetrahydrofuran was removed under diminished pressure. Addition of dilute sulphuric acid to the solution (to pH 7) gave a precipitate P, and further polysaccharide precipitates were isolated after diaiysis and after addition of ethanol. All precipitates gave galactose as the main sugar on hydrolysis, with only traces of other sugars. Precipitate P was dissolved in 0.5hi sodium hydroxide, and precipitation with ethanol gave carboxylreduced pectin (322 mg), [c& + 175” (c 0.5, 0.5M sodium hydroxide)_ Carboxyl-reduced pectin (220 mg) was treated successively with methyl sulphate and sodium hydroxide, and methyl iodide and silver oxide in N,N-dimethylformamide, to give methylated, carboxyl-reduced pectin (148 mg), [oc]o + 177’ (c 0.3, chloroform) [Found: OMe, 42.8%]. A sample of the methylated polysaccharide was heated with methanolic hydrogen chloride, and examination of the cleavage products by gas chromatography on columns a, 6, and c indicated the presence of methyl glycosides of the following sugars (approximate proportions in parentheses): 2,3,4,6-tetra(+ +), 2,3,6-tri- (+ + -t -I-), and 2,6-di-O-methylgalactose (+ +), 2,3,5-tri- (+ +) and 2,3-di-O-methylarabinose (+), and 3,4-di- (+), and 3-O-methylrhamnose (+)_ ACKNOWLEDGMENTS
The authors thank Professor Sir Edmund Hirst, C. B. E., F. R. S., for his interest and advice, and Mr. W. C. Platt for the supply of powdered lemon-peel. Cnrbulyd.
Res., 7 ( ! 968) 442-452
452
G. 0. ASPINALL, J. W. T. CRAIG, J. L. WHYTE
This work was conducted under the auspices of the Brewing Industry Research Foundation, to whom the authors are indebted for the award of scholarships (to J. W. T. C. and J. L. W.). REFERENCES 1 G. 0. A~PINALL AND A. CANAS-RODRIGUEZ, J. Chem. Sot., (1958) 4020. 2 G. 0. ASPMALL AND R. S. FANSHAWE, J. Chem. Sot., (1961) 4215. 3 G. 0. ASPINALL, B. GE~TETNER, J. A. MOLLOY, AND M. UDDIN, J. Chem. Sot., submitted for publication. 4 S. S. BHA~ACHARJEE AND T. E. TIMELL, Calf. J. Cbem., 43 (1965) 617. 5 A. J. BAR= AND D. H. NORTHCOT& Biochem. J.94 (196.5) 617. 6 S. E. B. GOULD, D. A. REINS,N. G. RICHARDSON, AND I. W. STEELE, Narure, 208 (1965) 876. 7 R. W. STODDART, A. J. BARRETT, AND D. H. NORTHCOTE, Biochem. L, 102 (1967) 194. 8 G. 0. ASPINALL, I. W. CO~ELL, S. V. EGAN, I. M. MORRISON, AND J. N. C. WHYTE, J. Chem. Sot. (C), (1967) 1071. 9 G. 0. A~PINALL, K. HUNT, AND I. M. MORRISON, J. Chem. Sot. (C), (1967) 1080. 10 J. L. WHITE, Ph. D. Thesis, Edinburgh (1964). 11 P. ALBERSHEIM, H. NEuKohl, AND H. DEUEL, Arch. Biochem. Biophys., 60 (1960) 46. 12 I?. ALBERSHEIM, H. NEUKOM, AND H. DEUEL, Helu. Chim. Acta, 43 (1960) 1422; J. H. MCCLENDON AND J. H. KREISHER, Anal. Biochem., S (1963) 295; J. H. MCCLENDON AND J. L. HESS, J. Food Sci., 28 (1963) 289. 13 G. 0. A~PINALL AND J. BAILLIE, J. Chem. Sot., (1963) 1702. 14 H. NEUKOM, H. DEIJEL, W. J. HERI, AND W. K~~NDIG, Helv. Chim. Actu, 43 (1960) 64. IS M. DUBOIS, K. A. GILLES, J. ic. HAMILTON, P. A. REBERS, AND F. Shfmi, Anal. Chem., 28 (1956) 350. 16 E. A. MCCOK.B AND R. MCCREADY, Anal. Chem., 24 (1952) 1630. 17 Z. DISCHE, Methods Carbobyd. Chem., 5 (1962) 501. 18 G. 0. ASPINALL AND J. BAILLIE, J. Chem. Sot., (1963) 1702. 19 J. F. CARSON AND W. D. MACLAY, J. Amrr. Chem. Sot., 68 (1946) 1015. 20 D. A. REES AND J. W. B. SAhwEL, Chem: Ind (London), (1965) 2008. Carbohyd. Res., 7 (1968) 442452
442
Elsevier Publishing Company. Amsterdam Printed in Belgium
LEMON-PEEL
PECTIN
PART
I. FRACTIONATION
G. 0.
&PINALL*,
Department
J. w. T.
of Chemistry,
AND
PARTIAL
CRAIG,
AND
HYDROLYSIS
OF WATER-SOLUBLE
PECTIN
J. L. WHYn
University of ISiinburgh, Edinburgh (Great Britain)
(Received February 26th, 1968; in revised form, March 29th, 1968)
ABSTRACT
The pectin isolated from dried lemon-peel by extraction with cold water has been subjected to partial depolymerisation by acid hydrolysis, acetolysis, and enzymic degradation. Acidic oligosaccharides whose structures were established, or indicated by chromatographic identification of the products of methylation linkage-analysis, include 2-O-(a-D-galactopyranosyluronic acid)-L-rhamnose, higher oligosaccharides containing residues of gaIacturonic acid and rhamnose, 4-O-(a-D-galactopyranosyluranic acid)-D-galacturonic acid and the polymer-homologous trisaccharide, 6-O(glucopyranosyluronic acid)galactose and 4-O-(glucopyranosyluronic acid)fucose, and 3-Q-xylopyranosylgalacturonic acid. INTRODUCTION
Many pectins in which D-galacturonic acid is the principal constituent contain neutral sugars, especially D-galactose, r._-arabinose, and t.-rhamnose, as integral constituents. Polysaccharides of this type have been examined recently from several sources including sisal fiesh’, Iucerne (alfalfa)2*3, the bark of Amabilis fir4, apple$, white-mustard cotyledon$‘, sycamore cambial and callus tissues7, soybean cotyledons’, and soybean hulls’. Investigations in this laboratory on a commercial sample of citrus pectin from oranges and lemons” provided further evidence for L-rhamnose as a constituent sugar, and also indicated that chains of (1+4)-linked B-D-galactopyranose residues were present in the acidic polysaccharide, since 4-0-p-D-galactopyranosyl-D-galactose and its polymer-homologues were formed as products of partial hydrolysis. However, in view of the mixed origin of the pectin sample and of the uncertain method of extraction, a more detailed examination has been carried out on the pectin isolated from dried lemon-peel by extractions with cold water. We report here the characterisation of acidic oligosaccharides formed on partiai depolymerisation of this pectin. RESULTS AND DISCUSSION
Extraction
of dried lemon-peel with cold water furnished a polysaccharide
*Present address: Department
of Chemistry,
Carbohyd. Res., 7 (1968) 442452
Trent University,
Peterborough,
Ontario,
Canada.
LEMON-PEEL PECTIN. PART I
443
preparation constituting some 10% by weight of the raw material. Subsequent experiments (unpublished results) have shown that considerable further quantities of pectin may be extracted under more drastic conditions, and a detailed examination of this material will be undertaken later. Pectin A of constant composition was isolated from the water-soluble fraction after two reprecipitations from water with acetone. Pectin A was apparently homogeneous when examined by boundary eIectrophoresis. Chromatography on 0-(2-diethylaminoethyl)cellulose, however, gave two main fractions of acidic polysaccharides, which probably represented pectins differing in degree of esterification, together with a small fraction (ca. 5 %) of neutral polysaccharide which gave an arabinose” and a galactose* on hydrolysis. This pectin was, however, considered suitable for studies on the acidic oligosaccharides formed on enzymic hydrolysis. Fractionation of pectin A by precipitation of the insoluble copper salt gave pectin B, but associated neutral polysaccharide was still present. Complete removal of accompanying neutral polysaccharide was achieved by preparative chromatography on O-(Zdiethylaminoethyl)-Sephadex, which gave pectin C and two minor polysaccbaride fractions D and E, to be examined later. Pectin C had a uranic acid content of 76%, and the methoxyl content indicated a degree of esterification of 76%. Hydrolysis gave galacturonic acid, together with arabinose, gaIactose, rhamnose, and a trace of xyli ;e. The latter polysaccharide fractions gave arabinose and galactose as the main products of hydrolysis; one, as the sole product, and the other, together with acidic sugars. Partial hydrolysis by acid of pectin B gave a mixture of acidic oligosaccharides which was separated by chromatography on 0-(2-diethylaminoethyl)-Sephadex, followed where necessary by filter-sheet chromatography, and six oligosaccharides were identified (Scheme I). 2-0-(a-D-Galactopyranosyhtronic acid)-r_-rhamnose (1) was characterised by conversion into the crystalline methyl glycoside pentamethyl ether. The nature of the linkages in oligosaccharides 2 and 3 was indicated by the results of methylation. Although no direct evidence was obtained for anomeric and enantiomorphic configurations, the oligosaccharides were chromatographically indistinguishable from 6-0-(/I-D-glucopyranosyluronic acid)-D-galactose and 4-0(B-D-glucopyranosyluronic acid)-r_-fucose. The structure of the tetrasaccharide 4 was assigned as an 0-(galactopyranosyluronic acid)-( l-+2)-0-rhamnopyranosyl-(1+4)0-(galactopyranosyluronic acid)-(1 +2)-rhamnose on the basis of (a) partial hydrolysis and (6) methylation of the oligosaccharide and the derived alditol. 4-0-(or-D-Galactopyranosyluronic acid)-D-galacturonic acid (5) and the polymer-homologous trisaccharide (6) were each characterised as their crystalline calcium salts and by methylation of the corresponding neutral methyl glycosides obtained by the sequence of reactions involving conversion of the acidic oligosaccharides into the methyl ester gIycosides, Q-imethylsiIylation, reduction with Iithium ahrminium hydride, and removal of protecting groups. *Omission of configurational prefixes indicates that the sugars or their derivatives were identified by chromatographic methods. Carbohyd.
Res., 7 (1968) 442-452
G. 0. ASPINALL,
J. W. T. CRAIG, J. L. WHYTE
Acetolysis of pectin C resulted in the isolation of acidic oligosaccharides only, and each of these contained residues of galacturonic acid and rhamnose. Oligosaccharides 1 and 4 were characterised as described previously, and oligosaccharide 7 was characterised as 0-(galactopyranosyluronic acid)-(1 -+2)-0-rhamnopyranosyl-(1+2)rhamnose by methylation of the trisaccharide and the derived alditol. P a-D-GalpA-(l-+2)-L-Rha 2 GpA-(1+6)-Gal
3 4 5 6
GpA-(1+4)-Fuc GalpA-(l+2)-Rhap-(I+4)-GaIpA-(l+2)-~~
H”voH
&
a-D-GalpA-(l-w%)-D-GalpA M-D-GalpA-(l-+4)-D-GalpA-(14)-D-GalpA 7 GalpA-(l-t2)-Rhap-(1+2)-Rha 8 Xylp-(l-+3)-GalA 10 GalpA-(1+2)-Rhap-(1+4)-GalpA
6H
9
Scheme I
Degradation of pectin A by a commercial enzyme-preparation afforded three further acidic o!igosaccharides. Methylation analysis indicated that oligosaccharide 8 had the structure 3-0-xylopyranosylgalacturonic acid. The tentative structure, 2-0-(4-deoxy-p-L-three-hex4enopyranosyluronic acid)L-rhamnose, was assigned to oligosaccharide 9 on the basis of the folIowing observations: (i) it had a light-absorption maximum at 231 nm characteristic of a& unsaturated carboxylic acids; (ii> it gave a red colour (R,,,547 nm) with thiobarbituric acid; (iii) hydrolysis gave rhamnose as the sole detectable product; and (iz~)cleavage of the methylated disaccharide gave the methyl glycoside of 3,4-di-0-methylrhamnose as the only recognisabie product. The formation of oligosaccharide 9 shows that the enzyme preparation used, like other commercial “pectinases”“, contains some pectin and/or pectic acid hydro-lyase activity in addition to the hydrolytic activity. Oligosaccharide 10 was assigned the structure, 0-(galactopyranosylurqnic acid)-(I +2)-Orhamnopyranosyl-(I +4)-galacturonic acid, since it contained a single rhamnose and two galacturonic acid residues, one at the reducing position. The site of substitution of the rhamnose residue was apparent from methyiation studies, but that of the reducing galacturonic acid residue has been assigned only tentatively. Additional evidence for the overall structure of the pectin was obtained by a series of reactions involving completion of the esterification of the galacturonic acid residues on treatment of pectin C with ethylene oxide, acetylation, and reduction with Iithium borohydride, which resulted in the formation of the carboxyl-reduced polysaccharide. Hydrolysis of this derivative gave galactose as the preponderant product with only traces of other sugars. The presence and nature of other sugar residues were established by gas-chromatographic examination of the methanolysis products from the methylated polysaccharide. The major products were methyl glycosides of 2,3,6-tri-0-methylgalactose, but small proportions of methyl glycosides of 2,3,4,6tetra- and 2,6-di-0-methylgalactose, 2,3,5-t& and 2,3-di-0-methylarabinose, and 3,4-di- and 3-0-methylrhamnose were also present. Cdmhyd.
Res., 7 (1968) 442-452
LEMON-PEEL PECTIN. PART I
445
The present results show that many of the structural features which have been established for lucernezS3 and soybean’s9 pectins are also encountered in this lemonpeel pectin. The hexuronic acid content (76 Oh) of the pectin indicates that ca. 79 % of the sugar residues are D-galacturonic acid. Although glucuronic acid is a trace constituent of this, as of other, pectins, it probably accounts for less than 1% of the sugar residues. Of the neutral sugar constituents, the clearest evidence for a structural role has been obtained in the ease of L-rhamnose. It is evident from the characterisation of the acidic oligosaccharides 1, 4, 7, and 10, which were formed in different ways on partial fragmentation, that residues of this sugar are present in the main chains of the pectin, where they interrupt blocks of 4-O-substituted a-D-galacturonic acid residues. The rhamnose residues, however, are unevenly distributed in the galacturonorhamnan chains and are probably concentrated in certain regions, where they may alternate with galacturonic acid residues, as in oligosaccharide’4, or occur as adjacent units, as in oligosaccharide 7. The other neutral-sugar constituents of lemon-peel pectin probably occur in outer chains. Methylation of the carboxyl-reduced pectin showed that arabinofuranose residues were present together with some non-terminal units. Apart from the partial characterisation of the aldobiouronic acid, namely, 6- O-(glucopyranosyluronic acid)galactose (2), as a minor product of partial hydrolysis, nothing is yet known of the location of the galactose residues. However, the failure to detect galactose-containing oligosaccharides as acetolysis products under conditions in which they were formed from soybean pectins**’ suggests that chains of contiguous galactose residues are largely absent in the lemon pectin. Although xylose is only a trace constituent of the pectin, the isolation of the oligosaccharide 3-O-xylopyranosylgalacturonic acid (8) on enzymic hydrolysis showed that residues of this sugar are attached to the galacturonan chain, as in soybean-cotyledon pectin’ and tragacanthic acidr3. As indicated by the isolation of the two aldobiouronic acids 2 and 3, this pectin, like those from soybeans’*’ and lucerne2v3, contains glucuronic acid and fucose as trace constituents, but there is no evidence for their structural location. EXPERIMENTAL
General. - Unless otherwise stated, sugars and oligosaccharides were identified by paper chromatography, which was performed on Whatman Nos. 1 and 3 MM papers, with the following solvent systems (v/v): (A) 10:4:3 ethyl acetate-pyridinewater; (B) 18:3: 1:4 ethyl acetate-acetic acid-formic acid-water; (C) l&8:3:9 ethyl acetate-acetic acid-formic acid-water; (D) 4: I :5 butyl alcohol-ethanol-water (upper layer); Q 200:17: 1 butanone-water-ammonia; (F) 9: 1: 1 butanone-acetic acid-water (saturated with boric acid)_ The R, values of methylated sugars refer to rates of movement reiative tc hat of 2,3,4,6-tetra-O-methyl-D-glucose (as unity) in solvent D. Paper ionophoresis was conducted in borate buffer at pH 10. Unless otherwise stated, optical rotations were observed for aqueous solutions at ca. 18”. Gas chromatography was performed on columns of acid-washed Celite coated Carbohyd. Res., 7 (1968) 442-452
G. 0.
ASPINALL,
J. W. T. CRAIG,
J. L. WHYTE
with (G) 10% by weight of butane l&diol succinate polyester (operating temperature, l?Y), (6) 10% by weight of m-bis(m-phenoxyphenoxy)benzene (200”), and (c) 3% or 5 % by weight of neopentylglycol adipate polyester (125 or 150”). All compounds whose identification was by gas chromatography, alone, had the same retention times as those of authentic reference compounds. Isolation and fractionation of lemon-peel pectin. Powdered lemon-peel, which was kindly supplied by Mr. W. C. Platt of the Ventura Coastal Lemon Corporation, Ventura, California, had been pre-extracted with isopropyl alcohol to remove lemon oil. Powdered peel (I kg) was stirred in water (30 liters) at pH 4.8 for 24 h at room temperature. Residual peel was removed in a centrifuge, and the extraction was repeated. The combined, aqueous extracts were poured into acetone (1.5 vol), and the resulting precipitate was washed with acetone-water, dissolved in water, and freeze-dried to give crude pectin (80 g), [aID+ 193” (c 0.32). The crude pectin was reprecipitated three times from aqueous solution with acetone, to give
pectin A (68 g), [a], +218” (c 0.31), of constant composition [Found: uranic acid (by decarboxylation), 75; OMe, 10.7%]. A sample of pectin A was chromatographed on O-(2-diethylaminoethyl)cellulose (phosphate form)14 with 0.025~, 0.05~, 0. IM, 0.25~, and 0.5~ sodium dihydrogen phosphate buffer at pH 6 as eluant. The fractions were monitored with the phenol-sulphuric acid rea’gent15, appropriate fractions were dialysed and concentrated, and polysaccharides were precipitated with acetone, redissolved in water, and isolated by freeze-drying. Hydrolysis of the polysaccharides indicated that a neutral fraction (ca. 5%) was eluted with 0.025~ buffer, but that all later fractions (largely eluted with 0.1~ and 0.25~ buffer) were of essentially similar composition. Aqueous, 7% cupric acetate (200 ml) was added dropwise, with stirring, to pectin A (20 g) in water (4 I). The insoluble copper salt that resulted was removed in a centrifuge, and decomposed by washing with acetone containing 1% of hydrogen chloride, and the precipitate remaining was washed free of acid with ethanol, dissolved in water, and freeze-dried to give pectin B (18.2 g), [cz],, f221 o (c 0.28) [Found: uranic acid residues, 76; OMe, 10.2%]. Chromatography of a sample of pectin B on U-(2-diethylaminoethyl)cellulose as described above showed that the neutral polysaccharide fraction was still present, although in slightly diminished amount. Pectin A (12 g) in water (600 ml) was adsorbed on a column of O-(2-diethylaminoethyl)Sephadex A-50 (100 g, formate form), and polysaccharide fractions were eluted with water (12 liters), 0.2~ formic acid (12 liters), and M formic acid (20 liters). The formic acid solutions were dialysed for 36 h, the pH of the solutions was adjusted to 4.5 by the addition of potassium acetate, and the solutions were concentrated, the pH being kept at 4.5 by the addition of acetic acid. The polysaccharide contents were precipitated with ethanol containing 4% of acetic acid, washed with ethanol, redissolved in water, and freeze-dried_ In a typical separation, polysaccharide D (196 mg, eluted with water) had [a& + 38” (c 0.2), and gave, on hydrolysis, arabinose, galactose, and smaller amounts of galacturonic acid, xylose, fucose, and rhamnose [Found: uranic acid residues, lo’/,]; polysaccharide E (381 mg, eluted with Carbohyd. Res., 7 (1968) 442-452
LEMON-PEEL
PECTIN.
PART
447
I
0.2M formic acid had [a], + 78” (c 0.2), and gave, on hydrolysis, galactose, arabinose, galacturonic acid, and a trace of rhamnose [Found: uranic acid resldues, 27%]; and pectin C (7.29 g, eluted with M formic acid) had [a], +218” (c OS), and on hydrolysis gave galacturonic acid, together with arabinose, galactose, and traces of xylose [Found: uranic acid residues, 76; OMe, 10.2 %I. Chromatography of pectin C on 0-(2-diethylaminoethyl)cellulose showed that neutral polysaccharide was absent. Partial, acidhydrolysis ofpectin. - Pectin B (17 g) was heated in 0.5~ sulphuric acid (150 ml) for 4 h on a boiling-water bath, during which time degraded polysaccharide separated. Degraded polysaccharide was removed at the centrifuge, and further polysaccharide was precipitated by the addition of acetone (1 vol) and removed by centrifugation. The supernatant liquid was concentrated to remove acetone, rendered neutral with barium hydroxide and barium carbonate, filtered, passed through a column of Amberlite resin IR-120 (H+) to remove barium ions, and evaporated to a syrup. The barium sulphate precipitate was washed with very dilute sulphuric acid (pH 4), and the washings were rendered neutral by shaking with Amberlite resin LA-l (5% in chloroform), passed through a column of Amberlite resin IR-120 (Hf), and evaporated to a syrup. The combined, degraded polysaccharides were rehydrolysed, and the products were isolated in the same way. Residual, degraded polysaccharide (5.94 g) gave, on hydrolysis, galacturonic acid and only traces of neutral sugars. The combined syrups (8.25 g) were adsorbed batchwise on columns of U-(2-diethylaminoethyl)-Sephadex A-25 (formate form)_ Elution of the columns with water removed neutral sugars, and elution with a gradient of water containing Od0.4~ formic acid, and with o.Jhf, M, and 3M formic acid gave fractions containing galacturonic acid and acidic oligosaccharides. Further separations by filter-sheet chromatography in solvents B and C gave chromatographically pure samples of oligosaccharides’-6. Oligosaccharide 1. The sugar (47 mg), R,,,0.83 in solvent B, IW~ 0.56, had [01]nt96” (c 0.47), gave galacturonic acid and rhamnose on hydrolysis, and was chromatographically indistinguishable from 2-0-(a-D-galactopyranosyluronic acid)L-rhamnose. The aldobiouronic acid was characterised by conversion into its methyl glycoside pentamethyl ether dihydrate, m.p. and mixed m.p. 69” (hot-stage) and 119” (capillary tube), [a]u + 91 o (c 0.63, chloroform), and by X-ray powder photography’. Oligosaccharide 2. The sugar (8 mg). R,,,0.26 in solvent B, MG 1.04, had [I&, +6” (c 0.4) gave glucuronic acid, glucurono-6,3-lactone, and galactose on hydrolysis, and was chromatographically and ionophoretically indistinguishable from 6-0-(B-D-glucopyranosyluronic acid)lD-galactose. Gas chromatography of the methanolysis products from the methyfated derivative on column a showed the presence of methyl glycosides of 2.3,4-tri-0-methylglucuronic acid, and 2,3,4- and 2,3,5-tri-0-methylgalactose. Oligosaccharide 3. The sugar (15 mg). Rc,,0.60 in solvent B, Mo 0.63, -70” (c 0.8), gave glucuronic acid, glucurono-6,3-lactone, and fucose on had blD hydrolysis, and was chromatographically and ionophoretically indistinguishable from 4-0-(/?-D-glucopyranosyluronic acid)-L-fucose’. Gas chromatography of the
Carbohyd.
Res., 7 (1968) 442452
448
G. 0.
ASPINALL.
J. W. T. CRAIG,
J. L. WHY-l-E
methanolysis products from the methylated derivative on column a showed the presence of methyl glycosides of 2,3,4-tri-U-methylglucuronic acid and 2,3-di-Umethylfucose. ’ Oligosaccharide 4. -The sugar (8 mg). RGol 0.12 and 0.56 in solvents B and C, McO.67, had [a]n +92” (c 0.4), and gave, on partial hydrolysis, galacturonic acid, rhamnose, and oligosaccharide 1. CoIorimetric determinations of galacturonic acid (carbazole method)16 and rhamnose (cysteine method)” residues indicated their presence in the approximate molar ratio of I:1 in the oligosaccharide and of 2: 1 in the derived alditol (borohydride reduction). Controlled, partial hydrolysis of the derived aldito1 gave 2-0-(galactopyranosyluronic acid)rhamnose and 2-O-(galactopyranosyluronic acid)rhamnitoI (separated in solvent F). Gas chromatography of the methanolysis products from the methylated oligosaccharide alditol showed the presence of 1,3,4,5-“cetra-O-methylrhamnitol, and methyl glycosides of 2,3,4-triand 2,3-di-0-methylgalacturonic acid, and 3,4-di-O-methyl-rhamnose. Oligosaccharide 5. - The sugar (508 mg), Rcol 0.21 and 0.50 in solvents B and C, MG 0.95, was chromatographicahy and ionophoretically indistinguishable from 4-0-(a-D-galactopyranosyluronic acid)-D-galacturonic acid, gave galacturonic acid, only, on hydrolysis, and furnished a calcium salt, [a], + 115” (c 0.68, 0.5M hydrochloric acid)‘. The sugar (30 mg) was treated with methanolic 1% hydrogen chIoride for 18 h at room temperature, the acid was neutralised with silver carbonate, and the mixture was filtered, and the filtrate evaporated. The resulting methyl ester methyl glycosides (31 mg) were treated with hexamethyldisilazane (0.6 ml) and chlorotrimethylsilane (0.3 ml) in pyridine (3 ml) for 1 h at 25”. The resulting solution was evaporated to dryness. The residue was extracted with ether (2 x 10 ml), and the extract was evaporated to a syrup (52 mg)). The trimethyIsiIy1 ethers were reduced by refiuxing with lithium ahrminium hydride (100 mg) in ether (5 ml) for 2 h, and ethyl acetate was added to decompose the excess of hydride. The precipitate that separated was removed by centrifugation, and shaken with 0.25~ sulphuric acid (3 ml) for 1 h. The resulting solution was rendered neutral with barium carbonate, the suspension was filtered, and the filtrate was treated with Amberlite IR-120 (H+) resin to remove barium ions, and evaporated to a syrup (21 mg). Hydrolysis of a sample of the syrup gave gaIactose only. The major portion of the neutral disaccharide glycoside was methylated with methyl iodide and silver oxide in NJ%dimethylformamide, and gas chromatography of the methanolysis products of the methylated derivative on column c showed the presence of methyl glycosides of 2,3,4,6-tetraand 2,3,6-tri-0-methylgalactose. Oligosaccharide 6. - The sugar (923 mg), RGllI0.24 in solvent C, Mc 0.95, was chromatographicahy and ionophoretically indistinguishable from the “galacturonotriose” isolated from other pectins, gave galacturonic acid, only, on hydrolysis, and furnished a calcium salt, [OL],,+ 135” (c 0.72, 0.5M hydrochloric acid)‘. The oligosaccharide (110 mg) was converted into the methyl ester methyl glycosides as described for oligosaccharide 5,.and thence into the trimethylsilyl ethers, which were reduced with lithium aluminium hydride. The resuhing methyl galactotricsides Carbohyd. Res., 7 (1968) 442452
LEMON-PEEL
PECTIN.
PART I
449
were methylated with methyl sulphate and sodium hydroxide, and methyl iodide and silver oxide, to give the methylated trisaccharide (66 mg). A sample of this derivative was heated with methanolic hydrogen chloride, and gas chromatography of the products on column c showed the presence of methyl glycosides of 2,3,4,6-tetra-, 2,3,6-t&, and (in traces) 2,6-di-0-methylgalaclose. The major portion (60 mg) of the methylated trisaccharide was hydrolysed in hi hydrochloric acid (3 ml) for 8 h on a boiling-water bath, and the cooled solution was rendered neutral with silver carbonate, centrifuged, treated with Amberlite IR-120 (H+) resin to remove silver ions, and concentrated to a syrup (57 mg). The syrup was adsorbed on a column of 1:1 charcoal-Celite (20 g), and elution with water containing increasing proportions of ethanol gave fractions i (4 mg), ii (29 mg), and iii (17 mg). Fraction i, R, 0.56 and 0.19 in solvents D and E, gave methyl glycosides having the retention times of those of 2,6-di-0-methylgalactose. Fraction ii, R, 0.76 and 0.51 in solvents D and E, [c& +82” (c 0.58, chloroform), gave methyl glycosides having the retention times of those of 2,3,6-tri-0-methyl-D-galactose. The sugar was characterised by conversion into 2,3,6-tri-0-methyl-D-galactono-l&lactone, m-p. and mixed m-p. 97-98”. Fraction iii, R, 0.89 and 0.87 in solvents D and E, [a], f97” (c 0.34, chloroform), gave methyl glycosides having the retention times of those of 2,3,4,6-tetra-O-methyl-Dgalactose, and was characterised by conversion into the aniline derivative, mp. and mixed m.p. 196-197”. Acetolysis ofpectin. - Pectin C (12 g) was acetylated by the method of Carson and Maclay’s, and the acetylated polysaccharide (12.3 g) was dispersed in acetic acid (320 ml). Acetic anhydride (320 ml) was added dropwise to the solution, and concentrated sulphuric acid (32 ml) was added dropwise, with vigorous stirring, during 2 h. The resulting solution was kept for 75 h at room temperature_ A precipitate that separated gave galacturonic acid, only, on hydrolysis. The supernatant liquid was poured into ice-water, and brought to pH 3 by the addition of sodium hydrogen carbonate. The precipitated acetates were separated by centrifugation, and dissolved in chloroform, and the aqueous solution was extracted with chloroform (4 x 150 ml). The combined chloroform extracts were evaporated to a syrup (6.5 g), which was deacetylated by treatment of a solution in chloroform (15 ml) and methanol (30 ml) with 0.25&rbarium methoxide in methanol (30 ml) for 24 h at 0”. The reaction mixture was poured into water, rendered neutral with dilute sulphuric acid, filtered, treated with .~,mberlite IR-120 (H’) resin to remove barium ions, and evaporated to a syrup (2.0 g). The syrup was adsorbed onto a column of 0-(2-diethylaminoethyl)Sephadex A-25 (formate form) (25 g). Elution with water gave neutral sugars (181 mg), including arabinose, galactose. xylose, and rhamnose, but no oligosaccharides could be detected, either by direct examination or after fslrther separation by chromatography on charcoal-Celite. Elution of the ion-exchange column sequentially with 0.05~~ a gradient of 0.05 --, 0.4M, 0.4Mr, and hl formic acid gave a series of fractions, from which chromatographically pure samples of oligosaccharides 1 (20 mg), 4 (8 mg), and 7 (11 mg) were isolated after further separation by filter-sheet chromatography. Oligosaccharides 1 and 4 were characterised as described previously, and later fractions, Carboh_vd. Res., 7
(1968)442-452
450
G. 0. ASPINALL,
J. W. T. CRAIG,
J. L. WHYTE
which contained “galacturonobiose” and “galacturonotriose” and gave galacturonic acid, only, on hydrolysis, were not examined further. Oligosaccharide 7. - The sugar, RGIII0.49 in solvent B, had [a],, + 84” (c 0.2), and gave galacturonic acid and rhamnose on hydrolysis. Partial, acid hydrolysis gave 2-0-(galactopyranosyluronic acjd)rhamnose (oligosaccharide 1) and rhamnose, and similar treatment of the derived alditol gave the same aldobiouronic acid and rhamnitol. Gas chromatography of the methanolysis products from the methylated oligosaccharide alditol on column c showed the presence of 1,3,4,5-tetra-O-methylrhamnitol and methyl glycosides of 2,3,4-tri-0-methylgalacturonic acid and 3,4-di0-methylrhamnose. Enzymic degradation of pectin. - Pectin A (20 g) was digested with “Pectinase” (Koch-Light Laboratories) (2 g) in water (10 liters) for 24 h at room temperature, and the enzyme and degraded polysaccharide were precipitated by the addition of acetone (1 vol). The supernatant liquid was concentrated, poured into acetone (4 vol), and kept for 2 days at 4”. The resulting precipitate appeared to contain crystalline galacturonic acid, and was removed by centrifugation. It was dissolved h water, the pH of the solution was adjusted to 7 by the addition of ammonia, and evaporation gave syrup S, (9 g). The supernatant liquid was also adjusted to pH 7 by the addition of ammonia, and evaporated to a syrup S, (ll g). Although galacturanic acid was the main component of both syrups, the syrups were separately chromatographed on 0-(2-diethylaminoethyl)-Sephadex A-25 (formate form) (50 g), using water to elute neutral monosaccharides, and 0.05~ and a gradient of 0.05 + 0.4~ formic acid to elute galacturonic acid and acidic oligosaccharides. Fractions containing three previously unrecognised oligosaccharides were obtained, and chromatographically pure samples of oligosaccharides 8 (12 mg), 9 (19 mg), and 10 (29mg) were isolated by filter-sheet chromatography. Ohgosaccharide 8. - The sugar, RGolA 0.53 in solvent B, [&, t28” (c 0.24), was chromatographically similar to 3-O-&D-xylopyranosyl-D-galacturonic acid, and on hydrolysis gave galacturonic acid and xylose”. Hydrolysis of the derived alditol gave xylose as the sole reducing sugar. Gas chromatography of the methanolysis products from the methylated alditol on column c showed the presence of 2,3,5,6tetra-O-methylgalactono-l,4-lactone and methyl glycosides of 2,3,4-tri-O-methylxylose’. Oligosaccharide 9. - The sugar, RGIIIA 2.02 in solvent B, had [01]o+ 87” (~0.30) and 1,,, 231 nm. Rhamnose was the only detectable hydrolysis product of the sugar, and rhamnitol, of the derived alditol, but quantitative estimation by the cysteine method” indicated the presence of 46% of rhamnose residues in the sugar. The sugar reacted with thiobarbituric acid in a manner characteristic of glycosides of 4-deoxy-L-tflreo-hex-4-enosuronic acid” to give a red colour (A,,, 547 nm). Gas chromatography of the methanolysis products from the methylated alditol on column c showed the presence of the methyl glycoside of 3,4-di-0-methylrhamnose and unidentified components. Oligosaccharide 10. - The sugar, RClrlA0.12 and 0.50 in solvents B and C, had &i+&,&. Rex., 7 1I968) 44%&Z
LEMON-PEEL
451
PECTIN., PART I
[a]n + 85” (c 0.4), and gave galacturonic acid and rhamnose on hydrolysis. Colorimetric estimations’6*‘7 of galacturonic acid and rhamnose indicated that the sugars were present in the ratio of 1.9: 1, and, after reduction to the alditol, in the ratio of 1: 1. Treatment of the oligosaccharide for 16 h at 100” in 1:l methanol-water containing 2.5 % of triethylamine gave 2-O-(galactopyranosyluronic acid)rhamnose as the sole reducing sugar. Gas chromatography of the methanolysis products from the methylated trisaccharide alditol on columns b and c showed the presence of methyl glycosides of 2,3,4-tri-O-methylgalacturonic acid and 3,4-di-O-methylrhamnose, and an unidentified component (T3.70 and 9.95 on columns b and c). This component may have been a derivative of 2,4,5,6-tetra-O-methylgalactonic acid, since compounds having the same retention time were observed in the methanolysis products from methylated 3-O-(a-D-galactopyranosyluronic acid)-L-galactonic acid (“galacturonobiitol”) and methylated 3-O-(a-D-galactopyranosyl)-D-galactonic acid (from oxidation of the corresponding galactobiose). Methylation of carboxyl-reduced pectin. Pectin C (1 g) in water (100 ml) was passed through Amberlite IR-120 (HC) resin to remove metal ions, ethylene oxide (25 ml) was added, and the solution was kept for 5 weeks at room temperature, whereupon esterification was complete (pH 6.8). The polysaccharide ester (1 g) was isolated by precipitation with ethanol (3 vol), dissolved in water, and freeze-dried, and was then acetylated by the method of Carson and Maclay” to give the acetate (1.1 g). Lithium borohydride (I. 1 g) in tetrahydrofuran (25 ml) was added to a suspension of the polysaccharide acetate (1.1 g) in tetrahydrofuran (25 m!), and the mixture was refluxed, with stirring, for 20 hzo. Excess of hydride was decomposed by’ the dropwise addition of water, more water (50 ml) was added, and tetrahydrofuran was removed under diminished pressure. Addition of dilute sulphuric acid to the solution (to pH 7) gave a precipitate P, and further polysaccharide precipitates were isolated after diaiysis and after addition of ethanol. All precipitates gave galactose as the main sugar on hydrolysis, with only traces of other sugars. Precipitate P was dissolved in 0.5hi sodium hydroxide, and precipitation with ethanol gave carboxylreduced pectin (322 mg), [c& + 175” (c 0.5, 0.5M sodium hydroxide)_ Carboxyl-reduced pectin (220 mg) was treated successively with methyl sulphate and sodium hydroxide, and methyl iodide and silver oxide in N,N-dimethylformamide, to give methylated, carboxyl-reduced pectin (148 mg), [oc]o + 177’ (c 0.3, chloroform) [Found: OMe, 42.8%]. A sample of the methylated polysaccharide was heated with methanolic hydrogen chloride, and examination of the cleavage products by gas chromatography on columns a, 6, and c indicated the presence of methyl glycosides of the following sugars (approximate proportions in parentheses): 2,3,4,6-tetra(+ +), 2,3,6-tri- (+ + -t -I-), and 2,6-di-O-methylgalactose (+ +), 2,3,5-tri- (+ +) and 2,3-di-O-methylarabinose (+), and 3,4-di- (+), and 3-O-methylrhamnose (+)_ ACKNOWLEDGMENTS
The authors thank Professor Sir Edmund Hirst, C. B. E., F. R. S., for his interest and advice, and Mr. W. C. Platt for the supply of powdered lemon-peel. Cnrbulyd.
Res., 7 ( ! 968) 442-452
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G. 0. ASPINALL, J. W. T. CRAIG, J. L. WHYTE
This work was conducted under the auspices of the Brewing Industry Research Foundation, to whom the authors are indebted for the award of scholarships (to J. W. T. C. and J. L. W.). REFERENCES 1 G. 0. A~PINALL AND A. CANAS-RODRIGUEZ, J. Chem. Sot., (1958) 4020. 2 G. 0. ASPMALL AND R. S. FANSHAWE, J. Chem. Sot., (1961) 4215. 3 G. 0. ASPINALL, B. GE~TETNER, J. A. MOLLOY, AND M. UDDIN, J. Chem. Sot., submitted for publication. 4 S. S. BHA~ACHARJEE AND T. E. TIMELL, Calf. J. Cbem., 43 (1965) 617. 5 A. J. BAR= AND D. H. NORTHCOT& Biochem. J.94 (196.5) 617. 6 S. E. B. GOULD, D. A. REINS,N. G. RICHARDSON, AND I. W. STEELE, Narure, 208 (1965) 876. 7 R. W. STODDART, A. J. BARRETT, AND D. H. NORTHCOTE, Biochem. L, 102 (1967) 194. 8 G. 0. ASPINALL, I. W. CO~ELL, S. V. EGAN, I. M. MORRISON, AND J. N. C. WHYTE, J. Chem. Sot. (C), (1967) 1071. 9 G. 0. A~PINALL, K. HUNT, AND I. M. MORRISON, J. Chem. Sot. (C), (1967) 1080. 10 J. L. WHITE, Ph. D. Thesis, Edinburgh (1964). 11 P. ALBERSHEIM, H. NEuKohl, AND H. DEUEL, Arch. Biochem. Biophys., 60 (1960) 46. 12 I?. ALBERSHEIM, H. NEUKOM, AND H. DEUEL, Helu. Chim. Acta, 43 (1960) 1422; J. H. MCCLENDON AND J. H. KREISHER, Anal. Biochem., S (1963) 295; J. H. MCCLENDON AND J. L. HESS, J. Food Sci., 28 (1963) 289. 13 G. 0. A~PINALL AND J. BAILLIE, J. Chem. Sot., (1963) 1702. 14 H. NEUKOM, H. DEIJEL, W. J. HERI, AND W. K~~NDIG, Helv. Chim. Actu, 43 (1960) 64. IS M. DUBOIS, K. A. GILLES, J. ic. HAMILTON, P. A. REBERS, AND F. Shfmi, Anal. Chem., 28 (1956) 350. 16 E. A. MCCOK.B AND R. MCCREADY, Anal. Chem., 24 (1952) 1630. 17 Z. DISCHE, Methods Carbobyd. Chem., 5 (1962) 501. 18 G. 0. ASPINALL AND J. BAILLIE, J. Chem. Sot., (1963) 1702. 19 J. F. CARSON AND W. D. MACLAY, J. Amrr. Chem. Sot., 68 (1946) 1015. 20 D. A. REES AND J. W. B. SAhwEL, Chem: Ind (London), (1965) 2008. Carbohyd. Res., 7 (1968) 442452