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Isolation and characterization of methylated sugars from the tube of the hydrothermal vent tubiculous annelid worm Alvinella pompejana
1. INTRODUCTION
pllvirtrllu porn&ejuncr [l), the Pompeii W”arm, is a polychaetous annelicl found srrictly srourrd dccg-sea hydrorhermal vents, clt 8 depth of 2600 m. It lives under %ry cxccptional conditions (high temperatures, high pressures, metal-rich watfr), in tubes directly secreted on the white smackers whose temperatures can reach 2WC, while the surrounding water is 2°C. The tube is build Out of organo-mineral material where minerals make up 450/oof the total [2]. The carbohydrate part of the tube is made, largely, of unknown sugars which are probably methylated sugars according to their chromatographic mobilities. This kind of sugar is rare in nature. We report here Chc isolation and the characterization of 3 neutral methylatcd sugars from the tube of Alvinella pompejana, 2, MATERIALS AND METHODS 2. I, Uiologicol
mmrinl Tubes from Alvine/lupo~~r~e~urtrr werecollected at 2600 m depth by the submersible Cyan in April 1984 during the Riocyarisc cruise (12?48’ N, 103*56’ W). Tubes were preserved in formal. They were rinsed and air-dried before experiments.
Motloraccharido deferminntionx weft carried 0111 n&r merhnnolyrir (0.5 N McOHIHCI, 24 h, 80°C) by gaa.liq\tid ohrema. fography of perrr~rnrrtry~~sily~atctl mcthylglyrosidca hydrolysis (4 N VA, 4 Ii, IW’C), reduction borohydridc, pcracrtytrtion (pyridinc/asfticrnhydridc, liquid cRromnro#raphy of alditol acetates,
[3,4) and rfrrr with potassium v/v)andges.
GLC was done with a Girdcl series 300 or SpcctraGhysics model 7100 yns chromntopraph fitted with a fl;~mc.ionisation detector. A capillary colt~mn(0,3 mm inner diameter x 25 m) fused silica OV 101 was used with the following tcmpcraturc propra~m: (a) nlditol acctntcs, I IO-24O“C at 3YZ/min; (b) pcrtrimcthylsilylatcd methyl and butyl-glycosidcs, 120-240°C at 2YYmin: (c) permcthylated sugars, I IO-180°C at 2°C/niin. Nitrogen was the carrier gas at 0.5 atni. 2.4 I Merhylrred t?,otrosucclrari~le,~ isolatiotr Tubes wcrc reduced to powder, treated with chloroform (10 ml), and sonicated (three times). After ccntrifugation (IO 000 x g, 5 min) the dry pellet was trcatcd with 2 N TFA, 2 h at 100°C, After cooling, the hydrolysatc was centrifuged for IS min at IO 000 x g, The hydrolysate was evaporated under pressl.lrc with methanol to remove the acid. The extract was dissolved in distilled water and passed through a column of Dowex 50 x 8 resin (200-400 mesh, H * form, 20 x 2 cm) coupled with a column of Dowex I x 8 resin (200-400 mesh, C&COOform, 20 x 2 cm) and the eluate was freeze-dried. Prcparativc paper chromatography was carried out on Whatman no. 3 paper with +butanol/acetic acid/water (4; 1:5)as solvent [S]. Sugars were visualized with aniline oxalatc [6],
Co~es~onde~e uddrew B. Fournct, Laboratoire de Chimie Biologiquc (Unit6 Mixte de Recherchc du Centre National de la Recherche Scientifique, No1 I l), UniversitC des Sciences et Techniques de Lille Flandres Artois, 59655 Villeneuvc d’Ascq Cedex, France
2.5. Dealkylurion qf merhyla~ed srrgnrs /7,8J The experiments were performed on acetylated sugars in order to increase their solubilities in the reaction medium. The dry sugar (1 mg) is dissolved in 0.5 ml of pyridine and 0.5 ml of acetic anhydride during 24 h at room temperature. After elimination of the solvents under a stream of nitrogen, the acetylated sugar is dissolved in 0.5 ml
ADbrevicrtons: TFA, trifluoroacetic acid; GLC, gas-liquid chromatography; GLC-MS, gas-liquid chromatography-mass spcctrometry; McOH/HCI, methanol chloride
of dichtoromethane, and boron tribromide (50 at) is added. After remaining for 2 min at room temperature, the mixture is evriljoraied to dryness. The product is deacetylated by treatment with sodium tnethoxide in methanol (15 min). After acidification with Dowex
Published
by Elscvier Sciettce Publishers B. V.
,’
60
288
229
248
‘*p--------131
--*---m-I-“--
t
WCOMfs
233
/’
266 -
,
________ 1--_-_-__---NZOAc
dr
275
ncom
“________ Ii
+
117
----r---“--d
I*
/
OAc
‘i BCOAc I NSOAc
CU20AC
I ,,,__ --______ I
WCOAc ----v__
8'
I
___-
203 HCOMe 189 ,/ __--___--c Ir --,,,,,,,.._J HCOAc
I l-i?OAC
lb8
Fig. 1. Paper chromatography fucose, (4)AlvineNapotnpejuna’s
, , , , , I , ( 1EA ZBB 228 248 268
,
in Partridge solvent [S], of monosaccharides obtained after acid hydrolysis of tubes, (1) glucose, (2) galactose, (3) tube hydrolysate (2 N TFA, 2 h), X1,2,4-di-0-methyl-L-fucosc; X2,2-mono-0-methyl-L-fucosc; 353,3-monoHOmethyl-L-fucose,
Since
tlic
rnbc
of
Atuirrslla
psntpq?rrrc~
conraino
sulfur [2,12), it has been removed with chloroform. af tuber, reduced to powder, were treated
2g
with chloret’arm. After extraction of sulfur, we obtained 1,9 g of tubes which was hydrolysed with 2 N TFA for 2 h at IOVC, After ccntrifugation of the solution the .pupernatant was reduced under vacuum and freezedried. The hyclrolyrate was deionized on Dowex eolumns and rhe unknown sugars were isolated by preparative paper chromatography on Wharman No. 3 paper In Partridge solvent [5] (Fig. I), WC can observe 3 monosaccharides (XI, X2, X3) which move faster than fu~osc.Their Wrurvalues are: RrtlcXI = 2.05; Rr,,eXr = I A; 1.4. The preparative paper hXB = chromatography yielded 3.5 rng of X1,1,2 mg of X2 and 2.7 mg of XZ, from 28 mg of starting material. The 3 compounds were converted into aldicol acetates for GLC and GLC-MS studies since alditol acetates have relatively simple fragmentation patterns [ 13-151. Mass spectra of alditol acetates from each unknown sugar and their fragmentation (electron impact mode) schemes are given in Fig. 2. GLC-MS of the alditol acetate derivative of the XI sugar gave primary fragments at /n/z 117, 131, 233 and 247 allowing to establish the sugar as a 2,4-di-0-methyl-6-deoxyhexose. GLC-MS of the alditol acetate derivative of XL sugar gave major primary fragments at nr/z 117 and 275 allowing to establish this alditol acetate as a 2-O methyl-6.deoxy-hexitol. From the fragmentation scheme of the alditol acetate derivative of X3 sugar, we observe two primary fragments m/z 203 and m/z 189. By elimination of acetic (mol. wt = 60) and a subsequent loss of ketene (mol. wt = 42) secondary fragments tn/z 143 and m/z 101 arise from the primary fragment m/z 203. In analogy, secondary fragments /n/z 129 and m/z 87 arise from the primary fragment m/z 189. This mass spectrometric analysis thus allows the identification of the X3 unknown sugar as a 3.O-methyl-6-deoxy-hexose. The mass spectra in a chemical ionization mode confirm the mono-O-methyl-6-deoxy-hexose nature for X2 and X3 sugars (M-t-I%&” E= 366, in peracetylated form) and the di-0-methyl-6-deoxy-hexose nature for Xl sugar (M -!-NM4 + = 338, in peracetylated form). Demethylation of each sugar with boron tribromide, followed by methylglycssylation and trimethylsilylation allows to identify (by GLC-IvIS) each sugar as a methyl fucoside. These results were confirmed by the
x3 x2
identification, by SK-MS of the pcrmethyl derivatives obtained after permethylation of each sugar. For all components, we identified the 2,3,4-tri-Q-methyl fucoside, Absolute configurations of demethylated sugars have been determined by GLC and GLC-MS using a chiral alcohol. The three sugars, analysed as butyl-pertrimcthyl-silylated glycosides have the same retention time as L-fucose. In conclusion, we have identified in the tube of Alvinelfuponzpejuna, the three unknown sugars as: XI, 2,4-di-O-methyl-L-fucose; X2, 2-mono-O-methyl-Lfucose; X-l, 3-mono-0-methyl-L-fucose. 4. DISCUSSION Naturally occurring methyl ethers of sugars are relatively rare in nature. 3-0-methyl-fucose (digitalose) is known as a constituent of cardiac glycosides [16], as a free sugar from the brown seaweed Desmaresta ucateatu [17], in glycolipids from shellfishes [l&203 and from Mycobacrerium [21]. The configuration of the sugar from the last 3 sources was not determined. The L configuration of 3.O-methyl-fucose was found as constituent ot’ Khizobiurn extracellular polysaccharides [22]. 2.Q-methyl-fucose has been identified in Desmaresta acuteata [ 1719 in glycolipids from Mycobacterium [21,23]. The L isomer of 2.O-methylfucose was found in lipopolysaccharides from photosynthetic prokaryotes [24]. The present work ap57
AcA’novkrf~rntcr~rs: This wark WIIS~;upparrcd by :I grant ((0 F.T.) from rhe lnxrirur Frantgrirdc Kcehrr&e pour I’Espbirariun dcla hlcr (IFREMER) nnd rhe Candcil RCginnnl do Btcteync, nnd by rhrCcmrc Narlanel dc la Rcchcrchc Scicndfiquc (Unirt! Mixcc tlr Rcc’hrrchr WI II, Dirrercur A. Vcrbcr4). Wc wish 16 Ibank Rr, Qimicl DsshruyErcs (IFREMER Brc$r) for furniuhiny lubes.
REFERENCES (I] Drrbruybrca, 267.274.
D. nnd Lnubirr,
L. (1980) Occ?nnul. Acfn 3,
968, 753-759. 131 #omrrliny, J.P., Cirrwig, G.J., Vlirycnfharr, J.P.G. and Clnmp, J.R. (1975) Biochcm. J, ISI. 491-495. (41 ManUcuil,~ J., Bauquclct, S., Dcbrny, H,, Fournct, B., Spik, G, nnd Slrrckcr, 6. (1986) in: Cnrbohydrarc Analysis: A Pracdcal Approach (Chaplin, M,F. nnd Kcnncdy. J.F., eds,) pp, 143-204, IRL press, Oxford.
1211 GatlnmbidcX3dicr, M, und Vilh?, C. (1970) IW, Sot. Wm. Biol. 52, 679-693. [22l licnncdy, LB. (1980) Curbahydr. Rer. 87, 156-160. 123) MsLenni\n, A.P. :1n0 RnndiIll, M,M< (1961) tfiarhmr. J, 80, 309-317. 1241 Wc&rxrcr,J., L)rcbvri, C,. and Mayer, H. (1979) Annu. Rev. Microbiul. 33, 215-239. 125) Akira. E., Macdn, P;. and Omcsaun, f-l. (IOQ4) 1. Antibior. II, 37-38. [Xi] McNeil, M., Chartcrjcc, D., Wu Hunter, S. i\nd kcnnnn, P.J. (1989) Methods Enzymol, 173, 715-242. (27) Dcfrelin, R. (1971) in: Comprchrnxivc Biochemistry (Flarkin, M. and SIOIZ, E-H,, cds.) pp. 713-745, Elscvicr, Ams~crdnnr,
pllvirtrllu porn&ejuncr [l), the Pompeii W”arm, is a polychaetous annelicl found srrictly srourrd dccg-sea hydrorhermal vents, clt 8 depth of 2600 m. It lives under %ry cxccptional conditions (high temperatures, high pressures, metal-rich watfr), in tubes directly secreted on the white smackers whose temperatures can reach 2WC, while the surrounding water is 2°C. The tube is build Out of organo-mineral material where minerals make up 450/oof the total [2]. The carbohydrate part of the tube is made, largely, of unknown sugars which are probably methylated sugars according to their chromatographic mobilities. This kind of sugar is rare in nature. We report here Chc isolation and the characterization of 3 neutral methylatcd sugars from the tube of Alvinella pompejana, 2, MATERIALS AND METHODS 2. I, Uiologicol
mmrinl Tubes from Alvine/lupo~~r~e~urtrr werecollected at 2600 m depth by the submersible Cyan in April 1984 during the Riocyarisc cruise (12?48’ N, 103*56’ W). Tubes were preserved in formal. They were rinsed and air-dried before experiments.
Motloraccharido deferminntionx weft carried 0111 n&r merhnnolyrir (0.5 N McOHIHCI, 24 h, 80°C) by gaa.liq\tid ohrema. fography of perrr~rnrrtry~~sily~atctl mcthylglyrosidca hydrolysis (4 N VA, 4 Ii, IW’C), reduction borohydridc, pcracrtytrtion (pyridinc/asfticrnhydridc, liquid cRromnro#raphy of alditol acetates,
[3,4) and rfrrr with potassium v/v)andges.
GLC was done with a Girdcl series 300 or SpcctraGhysics model 7100 yns chromntopraph fitted with a fl;~mc.ionisation detector. A capillary colt~mn(0,3 mm inner diameter x 25 m) fused silica OV 101 was used with the following tcmpcraturc propra~m: (a) nlditol acctntcs, I IO-24O“C at 3YZ/min; (b) pcrtrimcthylsilylatcd methyl and butyl-glycosidcs, 120-240°C at 2YYmin: (c) permcthylated sugars, I IO-180°C at 2°C/niin. Nitrogen was the carrier gas at 0.5 atni. 2.4 I Merhylrred t?,otrosucclrari~le,~ isolatiotr Tubes wcrc reduced to powder, treated with chloroform (10 ml), and sonicated (three times). After ccntrifugation (IO 000 x g, 5 min) the dry pellet was trcatcd with 2 N TFA, 2 h at 100°C, After cooling, the hydrolysatc was centrifuged for IS min at IO 000 x g, The hydrolysate was evaporated under pressl.lrc with methanol to remove the acid. The extract was dissolved in distilled water and passed through a column of Dowex 50 x 8 resin (200-400 mesh, H * form, 20 x 2 cm) coupled with a column of Dowex I x 8 resin (200-400 mesh, C&COOform, 20 x 2 cm) and the eluate was freeze-dried. Prcparativc paper chromatography was carried out on Whatman no. 3 paper with +butanol/acetic acid/water (4; 1:5)as solvent [S]. Sugars were visualized with aniline oxalatc [6],
Co~es~onde~e uddrew B. Fournct, Laboratoire de Chimie Biologiquc (Unit6 Mixte de Recherchc du Centre National de la Recherche Scientifique, No1 I l), UniversitC des Sciences et Techniques de Lille Flandres Artois, 59655 Villeneuvc d’Ascq Cedex, France
2.5. Dealkylurion qf merhyla~ed srrgnrs /7,8J The experiments were performed on acetylated sugars in order to increase their solubilities in the reaction medium. The dry sugar (1 mg) is dissolved in 0.5 ml of pyridine and 0.5 ml of acetic anhydride during 24 h at room temperature. After elimination of the solvents under a stream of nitrogen, the acetylated sugar is dissolved in 0.5 ml
ADbrevicrtons: TFA, trifluoroacetic acid; GLC, gas-liquid chromatography; GLC-MS, gas-liquid chromatography-mass spcctrometry; McOH/HCI, methanol chloride
of dichtoromethane, and boron tribromide (50 at) is added. After remaining for 2 min at room temperature, the mixture is evriljoraied to dryness. The product is deacetylated by treatment with sodium tnethoxide in methanol (15 min). After acidification with Dowex
Published
by Elscvier Sciettce Publishers B. V.
,’
60
288
229
248
‘*p--------131
--*---m-I-“--
t
WCOMfs
233
/’
266 -
,
________ 1--_-_-__---NZOAc
dr
275
ncom
“________ Ii
+
117
----r---“--d
I*
/
OAc
‘i BCOAc I NSOAc
CU20AC
I ,,,__ --______ I
WCOAc ----v__
8'
I
___-
203 HCOMe 189 ,/ __--___--c Ir --,,,,,,,.._J HCOAc
I l-i?OAC
lb8
Fig. 1. Paper chromatography fucose, (4)AlvineNapotnpejuna’s
, , , , , I , ( 1EA ZBB 228 248 268
,
in Partridge solvent [S], of monosaccharides obtained after acid hydrolysis of tubes, (1) glucose, (2) galactose, (3) tube hydrolysate (2 N TFA, 2 h), X1,2,4-di-0-methyl-L-fucosc; X2,2-mono-0-methyl-L-fucosc; 353,3-monoHOmethyl-L-fucose,
Since
tlic
rnbc
of
Atuirrslla
psntpq?rrrc~
conraino
sulfur [2,12), it has been removed with chloroform. af tuber, reduced to powder, were treated
2g
with chloret’arm. After extraction of sulfur, we obtained 1,9 g of tubes which was hydrolysed with 2 N TFA for 2 h at IOVC, After ccntrifugation of the solution the .pupernatant was reduced under vacuum and freezedried. The hyclrolyrate was deionized on Dowex eolumns and rhe unknown sugars were isolated by preparative paper chromatography on Wharman No. 3 paper In Partridge solvent [5] (Fig. I), WC can observe 3 monosaccharides (XI, X2, X3) which move faster than fu~osc.Their Wrurvalues are: RrtlcXI = 2.05; Rr,,eXr = I A; 1.4. The preparative paper hXB = chromatography yielded 3.5 rng of X1,1,2 mg of X2 and 2.7 mg of XZ, from 28 mg of starting material. The 3 compounds were converted into aldicol acetates for GLC and GLC-MS studies since alditol acetates have relatively simple fragmentation patterns [ 13-151. Mass spectra of alditol acetates from each unknown sugar and their fragmentation (electron impact mode) schemes are given in Fig. 2. GLC-MS of the alditol acetate derivative of the XI sugar gave primary fragments at /n/z 117, 131, 233 and 247 allowing to establish the sugar as a 2,4-di-0-methyl-6-deoxyhexose. GLC-MS of the alditol acetate derivative of XL sugar gave major primary fragments at nr/z 117 and 275 allowing to establish this alditol acetate as a 2-O methyl-6.deoxy-hexitol. From the fragmentation scheme of the alditol acetate derivative of X3 sugar, we observe two primary fragments m/z 203 and m/z 189. By elimination of acetic (mol. wt = 60) and a subsequent loss of ketene (mol. wt = 42) secondary fragments tn/z 143 and m/z 101 arise from the primary fragment m/z 203. In analogy, secondary fragments /n/z 129 and m/z 87 arise from the primary fragment m/z 189. This mass spectrometric analysis thus allows the identification of the X3 unknown sugar as a 3.O-methyl-6-deoxy-hexose. The mass spectra in a chemical ionization mode confirm the mono-O-methyl-6-deoxy-hexose nature for X2 and X3 sugars (M-t-I%&” E= 366, in peracetylated form) and the di-0-methyl-6-deoxy-hexose nature for Xl sugar (M -!-NM4 + = 338, in peracetylated form). Demethylation of each sugar with boron tribromide, followed by methylglycssylation and trimethylsilylation allows to identify (by GLC-IvIS) each sugar as a methyl fucoside. These results were confirmed by the
x3 x2
identification, by SK-MS of the pcrmethyl derivatives obtained after permethylation of each sugar. For all components, we identified the 2,3,4-tri-Q-methyl fucoside, Absolute configurations of demethylated sugars have been determined by GLC and GLC-MS using a chiral alcohol. The three sugars, analysed as butyl-pertrimcthyl-silylated glycosides have the same retention time as L-fucose. In conclusion, we have identified in the tube of Alvinelfuponzpejuna, the three unknown sugars as: XI, 2,4-di-O-methyl-L-fucose; X2, 2-mono-O-methyl-Lfucose; X-l, 3-mono-0-methyl-L-fucose. 4. DISCUSSION Naturally occurring methyl ethers of sugars are relatively rare in nature. 3-0-methyl-fucose (digitalose) is known as a constituent of cardiac glycosides [16], as a free sugar from the brown seaweed Desmaresta ucateatu [17], in glycolipids from shellfishes [l&203 and from Mycobacrerium [21]. The configuration of the sugar from the last 3 sources was not determined. The L configuration of 3.O-methyl-fucose was found as constituent ot’ Khizobiurn extracellular polysaccharides [22]. 2.Q-methyl-fucose has been identified in Desmaresta acuteata [ 1719 in glycolipids from Mycobacterium [21,23]. The L isomer of 2.O-methylfucose was found in lipopolysaccharides from photosynthetic prokaryotes [24]. The present work ap57
AcA’novkrf~rntcr~rs: This wark WIIS~;upparrcd by :I grant ((0 F.T.) from rhe lnxrirur Frantgrirdc Kcehrr&e pour I’Espbirariun dcla hlcr (IFREMER) nnd rhe Candcil RCginnnl do Btcteync, nnd by rhrCcmrc Narlanel dc la Rcchcrchc Scicndfiquc (Unirt! Mixcc tlr Rcc’hrrchr WI II, Dirrercur A. Vcrbcr4). Wc wish 16 Ibank Rr, Qimicl DsshruyErcs (IFREMER Brc$r) for furniuhiny lubes.
REFERENCES (I] Drrbruybrca, 267.274.
D. nnd Lnubirr,
L. (1980) Occ?nnul. Acfn 3,
968, 753-759. 131 #omrrliny, J.P., Cirrwig, G.J., Vlirycnfharr, J.P.G. and Clnmp, J.R. (1975) Biochcm. J, ISI. 491-495. (41 ManUcuil,~ J., Bauquclct, S., Dcbrny, H,, Fournct, B., Spik, G, nnd Slrrckcr, 6. (1986) in: Cnrbohydrarc Analysis: A Pracdcal Approach (Chaplin, M,F. nnd Kcnncdy. J.F., eds,) pp, 143-204, IRL press, Oxford.
1211 GatlnmbidcX3dicr, M, und Vilh?, C. (1970) IW, Sot. Wm. Biol. 52, 679-693. [22l licnncdy, LB. (1980) Curbahydr. Rer. 87, 156-160. 123) MsLenni\n, A.P. :1n0 RnndiIll, M,M< (1961) tfiarhmr. J, 80, 309-317. 1241 Wc&rxrcr,J., L)rcbvri, C,. and Mayer, H. (1979) Annu. Rev. Microbiul. 33, 215-239. 125) Akira. E., Macdn, P;. and Omcsaun, f-l. (IOQ4) 1. Antibior. II, 37-38. [Xi] McNeil, M., Chartcrjcc, D., Wu Hunter, S. i\nd kcnnnn, P.J. (1989) Methods Enzymol, 173, 715-242. (27) Dcfrelin, R. (1971) in: Comprchrnxivc Biochemistry (Flarkin, M. and SIOIZ, E-H,, cds.) pp. 713-745, Elscvicr, Ams~crdnnr,