Feruloylated xyloglucan and p-coumaroyl arabinoxylan oligosaccharides from bamboo shoot cell-walls

Phyrochemwy,Vol 29,No 6,pp 1999-2003, 1990

0031-9422/90 %300+000

Pnnted in Great Brrtam

0 1990Pergamon

Press

plc

FERULOYLATED XYLOGLUCAN AND p-COUMAROYL ARABINOXYLAN OLIGOSACCHARIDES FROM BAMBOO SHOOT CELL-WALLS TADASHI ISHII,* TADAKAZU HIROI and JERRY R.

THOMAS~

Forestry & Forest Products Research Institute, P.O. Box 16, Tsukuba Norm Kenkyu Danchmal, Ibarakl, 305, Japan (Recmed Key Word Index-Phyllostachys

1 September 1989)

edulrs, Grammeae; shoot, cell walls, ohgosacchandes,

feruloylated xyloglucan,

p-coumaroylated arabmoxylan

Abstract-A novel feruloylated xyloglucan disaccharide and p-coumaroylated arabinoxylan trlsaccharlde were isolated from cell walls of growing bamboo (Phyllostachys edulls) shoots. On the basis of chemical and spectral data, their structures were determined to be O-(4-O-trans-feruloyl-a-r?_xylopyranosyl)-( l-+6)-D-glucopyranose and 0-[S-O(trans-p-coumaroyl)-cc-L-arabinofuranosyl]-( 1-+ 3)-0-p-D-xylopyranosyl-( l-4)-D-XylOpyranOSe, respectively. This 1s the first reported evidence of a phenohc acid covalently associated with the cell wall hemicellulose, xyloglucan.

INTRODUCTION Phenolic

compounds

such as ferulic acid and p-coumaric acid are covalently bound to the cell walls of higher plants. They are esterlfied to cell-wall polysaccharldes and they are released from cell walls on treatment with cold aqueous alkali. Two feruloylated disaccharides 4-0(6-0-feruloyl-/?-D-galactopyranosyl)-D-galactose and 30-(3-O-feruloyl-u-L-arabtnofuranosyl)-L-arabinose, have been isolated from walls of rapidly growing spinach cells [l]. Ferulic and p-coumanc acid esters of arabmoxylan ohgosaccharldes have been isolated from some grammaceous cell walls including those of wheat [2], sugarcane baggase [3], maize shoots [4], barley aleurone layers [S], barley straw [6], and bamboo shoot [7]. These arabmoxylan ohgosaccharides include O-[S-O-(trans-pcoumaroyl)-cr-L-arabmofuranosyll-( 1+ 3)-O+D-xylopyranosyl-( 1 + 4)-D-xylopyranose C619 O-[S-O-(transferuloyl)-a-L-arabmofuranosyll-( 1+ 3)-0-/?-D-xylopyranosyl-( 1+ 4)-D-xylopyranose [3 - 71, and O-B-Dxylopyranosyl-( 1+4)-O-[S-O-(trans-feruloyl)-cr-~-arabinofuranosyl-( 1 -+ 3)]-O-~-D-XylOpyranOSyl-( 1-+4)-Dxylopyranose [7,8]. Wall-bound feruhc and related hydroxycinnanmic acids are important because they are sites at which covalent cross-links may form between wall polymers by oxldative coupling [9-161. Such cross-linking, catalysed in the cell wall by peroxidase and requumg an oxidant (e.g. H,O,), could restrict wall extensibdity [19-171 and digestibility [18-201. However, cross-linked polysaccharides have yet to be Isolated from cell walls and characterized In a previous paper [7], we isolated two feruloylated arabmoxylan ohgosaccharldes from cell-walls of growing bamboo shoots. On further study of the carbohydrate-

*Author to whom correspondence should be addressed t Present address: Department of Blochemlstry, Umverslty of Dundee, Dundee DDI 4HN, Scotland.

phenolic acid complexes of bamboo shoot, we characterized a feruloylated xyloglucan dlsacchande, and a pcoumaroylated arabinoxylan trisaccharide. This paper describes their structures.

RESULTS AND DISCUSSION

The results of FAB mass spectra indicated that the material obtained from bamboo shoot cell walls after Drtselase treatment, LH-20 chromatography, and reversed-phase HPLC contained two compounds. Compound 1 was M, 488 and compound 2 was M, 560. The positive-ion spectrum of the native material contained mtenseions at m/z 511 [M, +Na]+ and 583 [M,+Na]+, and weaker tons at m/z 489 [M1+l]+, 537[M1+K]‘, 561 [M, + l]‘, and 599 [M, + K]‘. After addmg potassium iodide, peak intensities at m/z 527 [M, +K]+ and m/z 599 CM, + K] + increased, and after adding cesmm iodide, intense peaks appeared at m/z 621 [M, +Cs]’ and m/z 693 [M, +Cs]+. In addition to the molecular tons, fragments ions at m/z 309 and 177, generated from 1 by loss of one hexose residue and one pentose residue, respectively, were observed. Fragments ions at m/z 411, 279 and 147 were derived from 2 by loss of one, two and three pentose residue, respectively. These FABMS results showed that 1 consisted of one ferulic acid, one pentose residue, and one hexose residue and that the ferulic acid was linked to the pentose residue, and that 2 consisted of one p-coumartc acid and three pentose residues. Composittonal analysis by GC of alditol acetates showed that the material containing 1 and 2 consisted of arabinose, xylose and glucose in a molar ratio of ca 1: 3 : 1. The absolute configurations of the arabmose, xylose and glucose were L, D, and D, respectively. Alkaline hydrolysis yielded feruhc acid and p-coumanc acid in a molar ratio of ca 1: 1. Methylation analysts (Table 1) gave five deriva1,4-di-O-acetyl-2,3,5-tn_O-methyl-aranamely, tives, binitol (derived from terminal arabinofuranosyl residues) 1,5-dl-O-acetyl-2,3,4-tri-0-methylxylitol (derived from

1999

2ooo

T

kHIl

1

2 termmal xylopyranosyl residues), 1,3,5-tn-O-acetyl-2,4dl-0-methylxyhtol (derived from 3-linked xylopyranosyl residues), 1,4,5-tn-O-acetyl-2,3-dl-0-methylxyhtol (presumably derived from 4-hnked xylopyranosyl residues), and 1,5,6-tn-O-acetyl-2,3,4-tri-0-methylglucltol (derived from 6-linked glucopyranosyl residues) Compounds 1 and 2 were reduced with sodium borodeutride to give ferulic acid and p-coumaric acid and ohgosaccharyl alditols 1 and 2 Methylatlon analysis of the ohgosaccharyl alditols (Table 1) revealed termmal arabmofuranosyl, terminal xylopyranosyl, 3-lmked xylopyranosyl, 4-hnked xyhtol, and 6-hnked glucltol residues Comparison of the results of methylatlon analysis before and after reduction Indicates that 6-lmked glucose and 4hnked xylose are present at the reducmg termmus of 1 and 2, respectively By comparison of the retention times and mass spectra of per-0-methylated oligosaccharyl aldltols 1 and 2 with those of authentic compounds, oligosaccharyl aldltols I and 2 were determined to be a-D-xylopyranosyl-( 1 + 6)-

Table

1 Methylatlon

analysts

et al

D-glucltol and cc-L-arabmofuranosyl-( 1+ 3)-[j-D-xylopyranosyl-( 1 + 4)-D-xyhtol, respectively Olrgosaccharyl aldltol 1 gave ions at m/z 143 (bA,, lOO%), 175 (bA,, 85 O%), 236 (aJ,, 66 3%), 296 (aJ,, 26 7%), 38l~(ald,2.0%), 395 (M-32, 5 3%) Ohgosaccharyl aidltol 2 gave Ions at m/z 143 (CA,, 42%), 175 (CA,, 30%), 192 (aJ,, lOO%), 238 (aJ,, 4 O%, 303 (cbA,, 2 4%), 335 (cbA,, 3 O%), 412 (abJ,, 4 So/,), 453 (ald, 1 4%) and 497 (ald. 0 9%) The presence m the EI mass spectrum of an aJ, fragment ton. and absence of an aJ, fragment Ion at nljz 252, mdlcated the penultlmate glycosyl restdue of ohgosaccharyl aldltol 2 was 3-lmked [2l] Analysis of the mixture of 1 and 2 by ‘“CNMR spectroscopy was used to elucidate the posItIon to which p-coumarlc acid and feruhc acid are linked to the ohgosaccharides Assignments of the signals m the 125 MHz 13C NMR spectrum m D,O are given m Table 2 The signals of carbon atoms of compounds 1 and 2 were asslgned by using published data [22-261 The presence of slgnal at 6 1079 m the region foI anomerlc carbon atoms suggests that arabmofuranose in compound 2 IS Xhnked After mltlal ‘jC NMR spectrum of the mixture had been recorded, the sample was recovered and then treated with 0 5 M NaOH The sample obtained from the alkali hydrolysate was then subjected to ‘“CNMR A comparison of the spectrum of compounds I and 2 with that of the saponified sample revealed an appreciable shift of C-5 (+ 3 85 ppm) and C-4 (-2 87ppm) of the Larabmofuranosyl residue of compound 2, mdlcatmg that C-5 1s esterlfied [3-71 The chemical shifts of the xylose residue m 1, and the changes Induced by treatment with sodmm hydroxide are summarized m Table 3 It has been shown [22. 231 m studies of acetylated methyl xylopyranostdes that the carbon chemical shift of C-5 IS changed only upon acetylatlon of C-4, and not upon acetylatlon of C-2 or C3, while acetylatlon of C-2 and C-3, but not of C-4, changes the chemical shift of C-2 The values of the carbon chemical shrfts of the esterlfied xylopyranose m 1 obtained m the present study comclde closely with the values obtained m the two previous studies, except for the value for C-3 obtamed by Petrakova and Kovac 1221 (Table 3). The reason for this discrepancy IS not known Nevertheless, the results indicate that C-4 of compound 1 IS estenfied. Assignments of the signals m ‘HNMR spectrum of compounds 1 and 2 showed that feruhc and pcoumarlc acids both have a trans-configuration from the wide coupling constants of H-7 and H-8 (ca I6 Hz) From

of compounds 1 and 2, and ohgosaccharoyl I and 2

Glycosyl residue

Methylated derwatlve

Lmkage posItIons

1 and 2

Arabmofuranosyl Xylopyranosyl

2, 3, 5 2, 3, 4 2, 3

Termmal Termmal 4

177 17 8 186

199 35 5 *

2, 2, 1, 1,

3 6 4 6

300 159

22 5

Ohgosaccharoyl aldltols 1 and 2

mol%

Glucopyranosyl Xyhtol Gltwtol *Not detected

4 3, 4 2, 3, 5 2, 3, 4

aldltols

62 155

Feruloylated Table

2 13C NMR spectral

C-l Compound 1 Ix-D-Xylose a-D-Glucose B-D-Glucose Furuhc acid Compound 2 a+Arabmose /I-D-Xylose Ct-D-XylOSe

P-D-Xylose p-Coumanc

acid

c-2

c-3

xyloglucan

from bamboo

data of compounds c-4

c-5

2001

1 and 2 (125 76 MHz m D,O)

C-6

c-7

C-8

c-9

OMe

56.20

98 35 92.40 96 28 126 62

71 58 7171 74 30 11175

70 87 73 20 76 15 148.03

7171 69 73” 69 91” 148 62

58 70 70 31” 74 55 11540

66.40” 66 50b 123 71

146 97

11400

168 81

108 36 101 93 92.22 9671 12646

8168 72 98 70 87 74 20 130 72

76 72 82 00 71 17 74 14 11623

8133 67.93 76 65 77 03 158 78

65.22 64.00 59 09 63.20 11623

130.72

146 58

114 10

169.43

residue

m compound

“.“May be interchanged Table

3. Comparison

of the carbon

Methyl G(-DXylopyranoslde

chemical shifts of the xylopyranose previously published values

Compound

1

Methyl

1 with

4-0-acetyl-a-D-xylopyranoside

C

1231

post-NaOH

Native

c221

1231

1 2 3 4 5

10009 7188 73 37 69 90 6161

9849(+014) 71 58(O) 73 28(+241) 6965(-206) 61.63( + 2 93)

98 35 7158 70 87 7171 58 70

996(+049) 71 5(+0 38) 723(+107) 71 5(-1.60) 586(+301)

9996(+013) 71 81(+007) 7ooq+3 31) 7175(-l 85) 57.88( + 3.73)

Values for methyl a-D-xylopyranoside and methyl 4-O-acetyi-a-D-xylopyranoslde (nght-hand column) were taken from ref 1231. Values for methyl 4-O-acetyl-a-D-xylopyranoside m the left-hand column were taken from ref [22] Values m parentheses are changes m carbon chemical shifts induced by deestenficatlon; the chemical shifts of methyl 4-O-acetyl-cc-D-xylopyranoside were subtracted from those of methyl a-Dxylopyranoslde, while those of native compound 1 were subtracted from the correspondmg values obtained after-ireatment with 0 5 M sodmm hydroilde. these results, the proposed structures of 1 and 2 are O(4-0-trans-feruloyl-a-D-xylopyranosyl)-(1 -+ 6)-D-glucopyranose and 0-[S-0-(trans-p-coumaroyl)+t-arabinofuranosyl]-( 1 + 3)-0-b-D-xylopyranosyl-( l-4)-D-xylopyranose, respectively We have characterized a feruloylated xyloglucan oligosaccharide and a p-coumaroyl arabmoxylan oligosaccharide from bamboo shoot cell walls. This is the first report that feruhc acid is linked to xyloglucan in plant cell walls. We have already isolated two feruloylated arabinoxylan oligosaccharides from the same plant material [7]. Compound 2 has recently been isolated from the cell walls of barley straw [6]. It has been suggested that cinnamic acid derivatives esterified with polysaccharides are responstble for the formation of cross-linked polysaccharides [9-171. Our present results suggest that feruloylated xyloglucan also participate m the cross-linkmg of polysaccharides in primary cell walls, m addttton to feruloylated and pcoumaroylated arabinoxylan. Further studies are in progress to characterize the fraction containmg dlferulic acid residues. EXPERIMENTAL General ‘H and 13CNMR spectra. 503 04 MHz, 125 76 MHz respectively in D,O Chemical shifts were referenced to an ext.

standard of MeOH (49.30 ppm from TMS) FABMS: m glycerin at 3 kV GC-MS was operated m the El mode with an electron energy of 70 eV Plant materml Shoots (ca 2Ocm long) of Mouso-chlku bamboo (Phyllostachys edulu) were collected m Tsukuba, Ibarakl Prefecture, m May 1988 The youngest growing portions (2 kg) were cut mto shces, and homogemzed m 50 mM Tns-HCI (pH 7 6) at 4” with a Waring blender The homogenate was filtered through 3 layers of cheese cloth The residue was washed successively with 50mM Tns-HCI (pH 7 6), H,O, EtOH, C,H,-EtOH (2 1) for 5 hr to remove hplds, and finally Me&O, then air-dried Phloroglucmol reaction of the crude cell walls showed no existence of hgnm. The crude cell walls contamed 4 6 mg phenohc acids per g dry wt The ratio of p-coumanc acid, feruhc acid, and dlferuhc acid, determmed by HPLC, was 100 300 3 Isolatron of compounds 1 and 2. The cell wails (20 g) were suspended m 10 I 10 mM NaOAc buffer (pH 5.0) and Incubated for 16 hr at 30” after addltlon of 3 ml of a 60 mg -’ soln of Drlselase (purchased from Kyowa Hakko, Tokyo, and punfied as described [l]) The suspension was heated m a boding-water bath for 10 mm to stop the reaction, then centnfuged. The supernatant soln was passed through Dowex-50 (H+ form) to were removed from remove Na+, and coned Polysaccharldes the dIgestIon products by addltlon of 5 vols of EtOH; the ppt. was removed by centrlfugatlon and the supernatant evapd to dryness The EtOH-soluble Drlselase dIgestIon products were

2002

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chromatographed usmg a column (2 5 x 90 cm) of Sephadex LH-20 eluted with H,O. Fracttons of 4 8 ml were collected and assayed for total carbohydrate by PhOH-H,SO, [27] and phenohc actds A fractton, havmg K,, value of 3 14, contamed predommantly feruloylated ohgosacchartdes These ohgosacchartdes were fractionated usmg prep. reversed-phase HPLC with a 25 x 2.0cm td column [Sham-Pak Prep ODS(H)-kit from Shlmadzu] at 40” eluted with 14% (v/v) aq MeCN at 4 ml mu- ‘, the eluate was monitored at 320 nm and a peak of UV-postttve maternal elutmg at 57-69 mm was collected and lyophthzed Usmg an analytical HPLC column at 40” eluted with 1’4% MeCm‘ at tJ6mimin-‘; this traction yteided. an asymmetric broad peak Further attempts at purrficatton were unsuccessful, and included reversed-phased HPLC using a couple of ODS columns (Shim-Pack ODS(H), 0 46 x 25 cm from Shtmadzu), normal-phase HPLC using an NH, column (Kasetsorb LCNH,, 0 46 x 25 cm from Tokyo Kaset) eluted with MeCN-H,O (4 1 v/v) and GPC using a column of Tosoh TSKgel G 2500 PWXL (0 78 x 30 cm) eluted with H,O Alkalnte hydrolyszs and reductwn A sample (ca 300 ng of each) was dissolved m 250 ~1 of a 10 mgml-’ soln of NaBD, m 1 5 M NH,OH After 3 hr at room temp, the soln was actdtfied wtth glacial HOAc, and extracted wtth Et,0 The Et,0 phase was washed wtth Ha0 and evapd The aq phase was passed through Dowex 50 w (H+ form), to remove Na+, and dried. Evapns with HOAc-MeOH (1 9, 250~1) and MeOH (250~1) were performed 3 and 4 x , respectively, to remove borate The Et,0 extract was fracttonated by reversed-phase HPLC using an ODS column (Shim-Pack prep-ODS(H), 0 46 x 25 cm, from Shtmadzu) Samples were chromatographed m 40% aq MeOH WlJfilJJJJJJ~~ t% ktoh ti. a fhw. rtie d 0.4. mJ.mra- 1 !?t!mok acids were monitored at 320 nm Feruhc, and p-coumartc actds in UV-postttve HPLC liactions were 1dentlfFed. using directmsertton, EIMS The ohgosaccharyl aldttols m the q. phase and 1 and 2 were per-O-methylated accordmg to a modtticatton [28] of the mejhnd. d r& [x9]. Pe~-r2-rn&&a&. d~gm2dx3mk~. and olrgosaccharyl aldttols were purtfied as described [30] and analysed by GC-MS Per-@methylated ohgosacchary!~ ahhtoJs were dtssolved m 30 nl Me&O m prepratton for analysts by GC Andy GC-MS Ca f part m 30 ni of the sampfe was inJected, and anatysed by G~C-MS~ using spiitfess nqection, a ffow rate of 30 ml mm- ’ He, the inJector at 250”, and 30 m x 0 25 mm fusedsthca DB-1 column (J&W Sctenttfic), with a temp program starting at 50” for 3 mm, then mcreasmg to 150” at 3O”mn-‘, and finally to 340” at 6” mn-‘, and holdmg at 340” for 10 mm Each peak was identified by comparmg the R,s and MS with those of authenttc a-D-xylopyranosyl-( 1 + 6)-o-glucitol and aL-arabmofuranosyl-(1~3)-~-D-xylopyranosyl-(l~4)-o-xylitol, which were prepared from the correspondmg oligosacchartde as described above Analysts of glycosyl residues Samples (.. 50 ng) were hydrolyzed in 250 nl 2 M TFA at 121” for 20 mm The hydrolysates were converted to aldrtoL acetates as dessrrbed [31]_ SampJes. were dtssolved in 10 ~1 Me&O, and ca 1 nl was lqected GC was performed using a 15 m x 0 25 mm SP-2330 fused-slhca-column (Supelco) operated isothermally at 235” in the split mode (1 40), at flow rate of 40 ml mm _ ’ He, and nqector and detector at 250 Glycosyl hnkage analysis Per-O-methylated ohgosacchartdes and ohgosccharyl aldrtols were hydrolysed m 2 M TFA for 121” for 3 hr. The hydrolysates were converted to partially methylated aldttol acetates as described 1321 Samples were dissolved m 10 ~1 Me&O, and ca 1~1 was qected Partrally methylated aldnol acetates were separated using GC and GC-MS m the split mode (1 40) a flow rate of40 ml mm 1He, InJector and detector at 250”, and a 30m x 0 25 mm SP-2330 (Supelco), with a temp

program starting at 170” for 2 mm, then mcreasmg to 235” at @/mm, and holding at 235” for 10 mm. Determmatlon of absolute conjiguratlon Analysts of absolute configuratton of the glycosyl residues m the compounds was performed using a previously described method 1331 A sample (100 pg) was reacted with (S)-( +)-2-butanohc 1 M HCI (200 11) 16 hr at 80” to form the S-(+)-2-butyl glycostdes The butyl glycostdes were converted to the TMSl ethers and analysed by GC [32]. Standards were prepared by reactton of monomeric D(+)-xylose, L-(-)-arabmose and D-(+)-glucose, wtth (S)-( +)-2butanohc 1 M HCI GC was performed usmg a 30 m x 0 25 mm tused&ica DB-1 coiumn (MW~Sctentitfcj, split inJection (1 4Uj, 250” detector, and a flow rate of 40 ml mm ’ He, wtth a temp program starting at 140‘ for 2 mm, mcreasing to 220” at 2” mm I, then to 275‘ at 30’ mm I. and holdmg at 275” for 10 mm Acknowledgements-We thank Dr Y Kato (Htrosakt Umverstty, Aomort) for the gift of a-D-xylopyranosyl-(l-6)-o-glucose and xyloglucan from barley, Dr T Tobtta (Japan Tabacco, Inc .) for the NMR spectroscopy, and Mrs M Ntshlda for typmg the manuscript The work was supported by research grant (Btomedia ProJects BMP-89-I-l-6) from the Mnnstry of Agriculture, Forestry and Fisheries J R T acknowledges the Sctence and Technology Agency for a vtsttmg sctenttst fellowshtp

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2003

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