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Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide a
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a
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Ryo Takano , Takashi Ueda , Yasuhide Uejima , Kaeko Kamel-Hayashi , Saburo Hara & Susumu Hirase
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Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Published online: 12 Jun 2014.
To cite this article: Ryo Takano, Takashi Ueda, Yasuhide Uejima, Kaeko Kamel-Hayashi, Saburo Hara & Susumu Hirase (1992) Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide, Bioscience, Biotechnology, and Biochemistry, 56:9, 1413-1416, DOI: 10.1271/bbb.56.1413 To link to this article: http://dx.doi.org/10.1271/bbb.56.1413
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Biosci. Biotech. Biochem., 56 (9), 1413-1416, 1992
Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide Ryo TAKANO, Takashi Susumu HIRASE**
VEDA,
Yasuhide VEJIMA, Kaeko KAMEl-HAYASHI, Saburo HARA,* and
Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received March 2, 1992
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Methyl cx- and fJ-o-galactopyranosides and 4-0-fJ-o-galactopyranosyl-3,6-anhydro-L-galactose dimethylacetal were sulfated with sulfuric acid and dicyclohexylcarbodi-imide as a condensation reagent. The sulfated sugars were isolated by ion-exchange chromatography, characterized, and assigned by methylation analyses. On the basis of the yield of each sulfated product that was isolated, sulfation on 0-6 appeared to be predominant.
A variety of carbohydrate sulfates have been found in animals and plants. They are important substances playing physiological roles, and are also potential materials for various uses. 1 ,2) Sulfation of sugars and related compounds is usually carried out by using such reagents as sulfurylchloride, chlorosulfuric acid, sulfur trioxide-pyridine or -dimethylformamide complex, and aryl sulfurylchloride. 3 ,4) Some of these are, however, very hygroscopic for handling, and those reagents with very high reactivity may result in an undesirable degradation and decreased yield. Roiberg and Mumma 5 ) have reported another convenient method using sulfuric acid as the sulfating reagent with dicyc1ohexylcarbodi-imide (DCC) as a condensation reagent in dimethylformamide. This method was applied to carbohydrate derivatives by blocking appropriate hydroxyl groups to obtain the desired sulfate esters. 6 ) As this method employs mild conditions, the reaction can be expected to have high regioselectivity. In the present work, we applied the sulfuric acid-DCC system to methyl cx- and f3-D-galactopyranosides and to 4- 0- f3 -D- galactopyranosy 1- 3, 6-anhydro- L- galactose dimethylacetal, a derivative of the repeating disaccharide of agarose,7) to examine its regiose1ectivity.
Results and Discussion Methyl cx- and f3-D-galactopyranoside and 4-0-f3-Dgalactopyranosyl-3,6-anhydro-L-galactose dimethyl acetal (agarobiose dimethylacetal) were sulfated with sulfuric acid and DCC as described by Mumma et al. 6 ) The sugar sulfates were adsorbed to a column of IR-45 (OR form) and eluted with aqueous ammonia to isolate them from the reaction mixtures. On the basis of quantitative analyses of the unchanged sugar passing through the ion exchanger, ca. 85% of the starting materials were found to have been sulfated in each case. Mixtures of the sugar sulfate isomers were fractionated by the method of Forrester et al.,8) who had isolated fucose sulfates by ion-exchange chromatography, using DEAE-cellulose (borate form) in a borate buffer. The
* **
fractions were purified by rechromatography, and some of them were obtained in crystalline form. Their characterization and assignment are summarized in Table I, the details of the assignments being discussed later in this paper. With the presen't chromatography system, the compounds appear to have been separated by the difference in ionic Table I. Methylation Analyses of the Fractions from Sulfated Methyl and f3-Galactosides, and from Agarobiosc Dimcthylacctal
(X-
Fraction
Methylation product(s)a
Location of sulfatc(s)b
At A2 A3 A4 A5 A6
2,4,6-Me 3 -Gal 2,3,4-Mc 3 -Gal, 2,4-Me 2 -AGal 2,3,4-Me 3 -Gal, 2,3,6-Me 3 -Gal 2,3,6-Me 3 -Gal 2,4-Me 2 -Gal 4,6-Mc 2 -Gal, 2,3-Me 2 -Gal, 3,4-Me 2 -Gal 3,4-Me 2 -Gal,4-Me-AGal
36mix. of 6- and 4-
A7 Bl B2 B3 B4
43,6mix. of 2,3-, 4,6-, and 2,62,6363,6mix. of 3,6-, 4,6-, and 2,6-
B7
2,4,6-Me 3 -Gal 2,3,4-MerGal 2,4-Mc 2 -Gal 2,4-Me 2 -Gal,2,3-Me 2 -Gal, 3,4-Mc 2 -Gal 4-Me-AGal,2,3-Me 2 -Gal, 3,4-Mez-Gal 4-Me-AGal,2,3-Me 2 -Gal, 3,4-Me z-Gal 3,4-Me z-Gal, 4-Me-AGal
Cl C2 C3 C4 C5 C6
2,4,6-Me 3 -Gal, 2,5-Me 2 -AGal 2,3,4-Mc 3 -Gal, 2,5-Me 2 -AGal 2,3,4,6-Me 4 -Gal,2-Me-AGal 2,4-Me 2 -Gal,2,5-Me 2 -AGal 3,4-Mez-Gal,2,5-Mez-AGal 2,3,4-Me 3 -Gal,2-Me-AGal
365'3,62,65'6-
B5 B6
a
h
mix. of 4,6- and 2,6mix. of 4,6- and 2,62,6-
2,4,6-Me 3 -Gal = 1,4,5-tri-0-acetyl-2,4,6-tri-0-methyl-galactitol; 2,4Me 2 -AGal = 1,5-di-0-acetyl-2,4-di-0-methyl-3,6-anhydrogalactitol, etc. Expressed as the sulfated position(s) in the galactose residue. 0-5 of the 3,6-anhydrogalactose dimethylacetal residue is expressed as 5'.
To whom correspondence should be addrcssed. Present address: Research Laboratory, Tallo Co., Ltd., 126 Higashi Shiriike-Shinmachi, Nagata-ku, Kobe 653, Japan.
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strength arising from their sulfate content and from their potential to form a borate complex. The mono sulfates were eluted with the lower concentration of ammonium borate, and the disulfates with the higher, in agreement with the sulfate content of each. Among the mono sulfates of galactosides, 3-sulfates were eluted faster than 6-sulfates, which would more easily form the borate complex between 0-2 and 0-3 than 3-sulfates would between 0-4 and 0-6. The separation of the disaccharide sulfates was better than that of the monosaccharide sulfates. With the monosaccharide sulfates, the ionic strength of sulfate groups linked to the sugar residue would presumably mask the contribution of the ionic effect of the sugarborate complex, thus interfering with the chromatographic separation. Methylation analyses of the isolated sugar sulfates were carried out by the method of Hakomori. 9) With this method, the methylation product is usually isolated from the reaction mixture by extraction to avoid, in the subsequent hydrolysis step, undesirable reactions arising from the use of iodide and methylsulfoxide. The extraction of the methylated sugar sulfates was unsuccessful because of their sulfate groups, which solubilized the permethylated compounds into water. The products were successfully isolated from these contaminants by gel filtration on Biogel P-2. The permethylated products obtained were hydrolyzed, converted into alditol acetates, and then analyzed by GLC (Table I). In some compounds, 3,6-anhydrogalactose derivatives were identified besides the normal partially methylated derivatives. The formation of the anhydride, which is a known reaction caused by the elimination of sulfates at 0-6 of the sugar residues by free 0-3 to simultaneously form 3,6-anhydro rings,10) probably occurred in the methylation step carried out under alkaline conditions. The anhydrides that were identified also support the assignment of the sulfate's location. The relative reactivity of the hydroxyl groups in methyl galactosides and agarobiose dimethylacetal by the present sulfation method was calculated from the sum of the yields of the corresponding sugar esters obtained (Table II). In all cases, 0-6 atoms of the galactose residues exhibited the highest reactivity, the next highest being 0-5 of the 3,6anhydrogalactose residue in agarobiose dimethylacetal.
Such relatively high reactivity might have arisen from the lack of steric hindrance in the corresponding hydroxyl groups, which project from the sugar residues. In decreasing degree of reactivity after the hydroxyl groups, the reactivity of 0-2 and 0-3 of a-galactoside was comparable, while 0-2 of p-galactoside was much less reactive than 0-3, possibly due to the steric hindrance by the neighboring methyl group in the p configuration. In the case of agarobiose dimethylacetal, the anhydrogalactose dimethylacetal group as the aglycon, which is much more bulky than a methyl group, is likely to have caused a further reduction in the reactivity at 0-2 of its galactose residue. In all the cases observed, the reactivity of axial 0-4 was lower than that of other equatorial hydroxyl groups. As another reason, the sulfate group introduced to 0-6 might have hindered further sulfation on sterically closed 0-4, like the case of dextran with equatorial 0-4, which can hardly be sulfated. 11) However, disulfates were also formed in the present sulfation process as will be discussed later. The trend in reactivity of the hydroxyl groups with this sulfation method is similar to that described by Turvey and Williams,12) who adopted a sulfur trioxide-pyridine system. In the present case, however, the reactivity of the secondary hydroxyl groups was much lower, indicating higher regioselectivity. Such selectivity might be explained by the bulk of the DCC-sulfuric acid complex, which would reduce the accessibility of the reagent to the sterically hindered hydroxyl groups. Smaller amounts of disulfates were formed during the sulfation of methyl galactosides, although only a 10% excess of reagent was used. This might suggest that the introduced sulfate accelerated the rate of further sulfation at the other positions, while oversulfation of 6-sulfated polysaccharide, in contrast, has been reported to be difficult. 13 ) The excess formation of di- or tri-sulfate has also been reported from sulfation with the sulfur trioxidepyridine system. 14) As predominant sulfation on 0-6 was indicated, the present method would be effective for the selective sulfation of carbohydrates without any protection of the hydroxyl groups.
Experimental General methods. Concentration of the solutions was carried out in vacuo below 40°C with a rotary evaporator. Optical rotation in an
Table II. Relative Reactivitt of the Hydroxyl Groups in Methyl Galactoside and Agarobiose Dimethylacetal after DCC-H 2 S04 Sulfation Hydroxyl grouph
o:-Galactoside
[3-Galactoside
2 3 4 6d 5'
19 20 2 100
7 31
Galactose
C
Agarobiose dimethyl acetal
--------_._-_._.
a
b
C
d
+ 100
45 80 20 100
__.._--_.+ 17
+ 100 50
Calculated from the yield of each fraction obtained by chromatography of the reaction mixtures. Fractions assigned as a mixture of sulfates are excluded from the calculation. Expressed as the sulfated position in the galactose residue. 0-5 of the 3,6-anhydrogalactose dimethyl acetal residue is expressed as 5'. Results from sulfation by the pyridine-sulfur trioxide system in ref. 11. Values for the 6-sulfates are taken as 100.
aqueous solution was measured by Laurent's saccarimeter with a cell of 1 dm in length. Melting points were measured by a micro-melting point apparatus (Model MP-S2, Yanagimoto Co.). GLC was carried out by a gas chromatograph (Model 163, Hitachi Co.) equipped with a hydrogen flame ionization detector under the following conditions: injection temperature, 210°C; carrier gas (nitrogen) flow rate, 20 ml/min; hydrogen pressure, 0.6kg/cm 2 ; air pressure, 2.0kg/cm 2 . Stainless steel columns (3 mm x I m) were packed with Chromo sorb W NAW coated with following stationary phases: column a, 3% (w/w) Silar 'lOC;15) column b, 0.5% PEG 20 M. GLC-MS was carried out by a combined gas chromatograph-mass spectrometer (QP-1000, Shimadzu Corp.), using a fused silica capillary column (OJ mm x 25 m) coated with SP-IOOO and operating at a temperature of 210°C. Helium was used as the carrier gas at a 2 ml/min flow and 20: 1 split ratio. Mass spectra were recorded at 70eV ionization voltage, and infrared spectroscopy was carried out with a spectrometer (Model 215, Hitachi Co.) by the KBr disk method. Methyl O:-D-galactopyranoside and [3-D-galactopyranoside were obtained by treating commercial D-galactose with N methanolic hydrogen chloride, chromatographically separating as described by Matsushima and Miyazaki,16) and purifying by recrystallization. Agarobiose dimethyl-
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acetal was obtained by partial methanolysis of commercial agar, with subsequent charcoal chromatography and recrystallization. Sulfation of methyl a-galactoside. A 5.15 g portion (25 mmol) of DCC was dissolved in 60 ml of dried DMF, and 1.00 g (5 mmol) of methyl a-galactopyranoside in 40 ml of DMF was added. To the solution was next added 0.55 g (5.5 mmol) of sulfuric acid in 22 ml of DMF at O°C under a nitrogen stream, and the solution was stirred for 15 min. The reaction mixture was poured into 200 g of crushed ice and immediately neutralized with barium carbonate. The resulting barium sulfate and the precipitate of N,N'-dicyc1ohexylurea were removed by filtration. The filtrate was applied to an Amberlite IR-120 cation exchanger (H form, 1.26 x 23.5 cm), and the eluate was further treated by an Amberlite IR-45 anion exchanger (OH form, 1.26 x 23.5 cm). The column was eluted with deionized water to remove the nonsulfated sugar until the eluate showed negative in the anthrone test. The amount of unchanged galactoside passing through in the eluate was estimated as 133 mg on the basis of a colorimetric determination of the eluate. The anion exchanger was further eluted with N/2 ammonium hydroxide and then with N ammonium hydroxide. Each eluate was evaporated and afforded a syrupy sulfate mixture (1.36 g from N/2 eluate, and 0.008 g from N eluate). The former eluate was further investigated. Sulfation of methyl fJ-galactoside and agarobiose dimethylacetal. Methyl fJ-galactoside (1.00 g) was sulfated under exactly the same conditions as those used for methyl a-galactoside, the reaction mixture being applied to IR-120 and IR-45 columns. The amount of unchanged galactoside which passed through the columns was estimated as 160 mg, and that of the recovered sulfate mixture obtained from elution of the anion exchanger by N/2 ammonium hydroxide was 1.52 g. Essentially no sugar sulfate was eluted by N ammonium hydroxide. Agarobiose dimethyl acetal (1.11 g, 3 mmol) was sulfated with 3.16 g (15 mmol) ofDCC and 0.44 g (4.5 mmol) of sulfuric acid in 81 ml ofDMF, and then treated similarly to the galactosides. The amount of unchanged disaccharide was 0.14 g (13% of the starting disaccharide). The reaction mixture was successively applied to the IR-120 and IR-45 columns. The anion exchanger was eluted with NII0 and N ammonium hydroxide, before evaporating to afford 1.21 g and 0.095 g of a syrupy sulfate mixture, respectively. The former eluate was further investigated. Anion exchange chromatography of the sulfate mixture from methyl a-galactoside. A 288 mg portion of the sulfate mixture from the N/2
ammonium hydroxide eluate of IR-45 was dissolved in a borate buffer containing 200mM boric acid and 10mM ammonium hydroxide, and then applied to a Whatman DE 52 DEAE-cellulose column (borate form, 2.2 x 50 cm) with elution by the same borate buffer. The eluate in each tube (containing 10 ml) was checked for sugar content by the orcinol test and recombined into 3 fractions, AI, A2 and A3. From tube number 200, the column was eluted with a buffer containing 300 mM boric acid and 60 mM ammonium hydroxide to afford 4 fractions, A4 to A 7. This procedure was repeated twice, and finally 1.07 g of the sulfate mixture was fractionated. Each recombined fraction was concentrated to dryness, before adding and evaporating methanol in order to remove the remaining ammonium borate. The recombined fractions were rechromatographed under the same conditions, characterized and identified by methylation analyses as already described. Fraction AI. The amorphous solid (41 mg), eluted with the 200mM borate buffer (tube numbers 100 to 140), was identified as methyl a-Dgalactoside 3-sulfate, and yielded white crystals (22.8 mg) from ethanol. [aJt/ + 148.6° (c 1.2); mp 192-193°C (dec.); IR spectrum: 1250--60, 840,800cm- 1 . Fraction A2. The amorphous solid (446 mg), eluted with the 200 mM borate buffer (tube numbers 141 to 177), was identified as methyl a-Dgalactoside 6-sulfate, and yielded white crystals (323.4 mg) from ethanol. [aJb5 + 124.6° (c 1.2); mp 127-129°C (dec.); IR spectrum: 1240--60, 800cm- 1 . Fraction A3. The syrup (12 mg), eluted with the 200 mM borate buffer (tube numbers 178 to 199), was identified as a mixture of the 6-sulfate and 4-sulfate of methyl galactoside. Fraction A4. The syrup (13 mg), eluted with the 300 mM borate buffer (tube numbers 200 to 212), was identified as methyl a-D-galactoside 4sulfate. [aJb5 + l09S (c 1.1); IR spectrum: 1240--60,830, 8l0cm- I • Fraction A5. The syrup (98 mg), eluted with the 300 mM borate buffer (tube numbers 271 to 304), was identified as methyl galactoside 3,6disulfate. IR spectrum: 1240-60, 820-40 cm - 1.
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Fraction A6. The syrup (17 mg), eluted with the 300 mM borate buffer (tube numbers 307 to 333), was identitied as a mixture of the 2,3-disulfate, 4,6-disulfate, and 2,6-disulfate of methyl galactosides. Fraction A7. The syrup (129 mg), eluted with the 300 mM borate buffer (tube numbers 334 to 376), was identified as methyl a-D-galactoside 2,6-disulfate, and yielded white crystals (71.3 mg) from ethanol. [aJb5 + 102.2° (c 1.0); mp 182-183°C (dec.); IR specrum: 1240--60, 820cm- 1 • Anion exchange chromatography of the sulfate mixture from methyl fJ-galactoside. A 1.00 g portion of the sulfate mixture from the N/2 am-
monium hydroxide eluate of IR-45 was chroma to graphed under the same conditions as those for methyl a-galactoside, except that the elution buffer was changed to the 300 mM borate buffer at tube 270. The seven fractions obtained, B1 to B7, were rechromatographed, characterized, and identified (Table I). Fraction BI. The syrup (19 mg), eluted with the 200 mM borate buffer (tube numbers III to 142), was identified as methyl fJ-D-galactoside 3-sulfate. [aJb5 + 15.4° (c 1.1); IR spectrum: 1240--50, 805cm- 1 • Fraction B2. The syrup (408 mg), eluted with the 200 mM borate buffer (tube numbers 146 to 179), was identified as methyl fJ-D-galactoside 6-sulfate, and yielded white crystals (139.2mg) from ethanol. [aJb5 -5.1° (c 1.0); mp 142-l43°C; IR spectrum: 1240--60, 820cm- 1 . Fraction B3. The amorphous solid (177 mg), eluted with the 300 mM borate buffer (tube numbers 270 to 295), was identified as methyl fJ-Dgalactoside 3,6-disulfate, and yielded white crystals (119.1 mg) from ethanol. [aJb5 +22.0° (c 1.0); mp 188-l89°C (dec.); IR spectrum: 1240--60, 800--820 cm -1. Fraction B4. The syrup (7 mg), eluted with the 300 mM borate buffer (tube numbers 296 to 306), was identified as a mixture of the 3,6-disulfate, 4,6-disulfate, and 2,6-disulfate of methyl gaJactosides. Fraction B5. The syrup (14mg), eluted with the 300mM borate buffer (tube numbers 307 to 317), was identified as a mixture of the 4,6-disulfate and 2,6-disulfate of methyl galactosides. Fraction B6. The syrup (15 mg), eluted with the 300 mM borate buffer (tube numbers 318 to 327), was identified as a mixture of the 4,6-disulfate and 2,6-disulfate of methyl galactosides. Fraction B7. The amorphous solid (48mg), eluted with the 300mM borate buffer (tube numbers 328 to 349), was identified as methyl fJ-Dgalactoside 2,6-disulfate. [aJt/ - 3.66° (c 1.0); IR spectrum: 1240--60, 820cm- 1 • Anion exchange chromatography of the sulfate mixture from agarobiose dime thylace tal. A 401 mg portion of the sulfate mixture from IR-45 was
applied to the same column as that used for methyl galactosides. The column was eluted with the buffer containing 200 mM boric acid and 10 mM ammonium hydroxide from tubes 1 to 167, with subsequent elution with the buffer containing 200 mM boric acid and 40 mM ammonium hydroxide, to afford 6 fractions, C1 to C6. These fractions were rechromatographed, characterized and identified (Table I). Fraction Cl. The syrup (12 mg), eluted with the first buffer (tube numbers 74 to 112), was identified as agawbiose dimethylacetaI3'-sulfate. [aJ&5 19.3° (c 0.8); IR spectrum: 1240--50, 81Ocm- l . Fraction C2. The amorphous solid (134mg), eluted with the first buffer (tube numbers 113 to 143), was identified as agarobiose dimethylacetal 6'-sulfate. [aJb5 -30.7° (c 0.8); IR spectrum: 1240--60, 810cm- 1 • Fraction C3. The syrup (32mg), eluted with the first buffer (tube numbers 144 to 166), was identified as agarobiose dimethylacetal 5sulfate. [aJb5 -28S (c 0.7); IR spectrum: 1240--60, 790--81Ocm- 1 • Fraction C4. The syrup (28 mg), eluted with the second buffer (tube numbers 256 to 289), was identified as agarobiose dimethyl acetal 3',6'disulfate. [aJb5 -4S (c 1.2); IR spectrum: 1240, 780--800 em -1. Fraction CS. The syrup (23 mg), eluted with the second buffer (tube numbers 290 to 322), was identified as agarobiose dimethylacetal 2',6'disulfate. [aJb5 -15.7° (c 0.8); IR spectrum: 1240--60, 810cm- 1 . Fraction C6. The syrup (72 mg), eluted with the second buffer (tube numbers 323 to 414), was identified as agarobiose dimethylacetal 5,6'disulfate. [aJb5 -32.0° (c 0.8); IR spectrum: 1240--60, 81Ocm- 1 • Methylation analysis of the mono- and disaccharide sulfates. Each 5 mg portion of the sulfated methyl galactoside and agarobiose fractions from anion-exchange chromatography was methylated in methyl sulfoxide, using dimethylsulfinyl carbanion as described by Hakomori. 9 ) The reaction mixtures were chromatographed on Biogel P-2 (2.2 x 200 cm) with deionized water as the eluant. On the basis of the anthrone test, carbohydrates were eluted faster than methylsulfoxide and iodide. The methylated
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products isolated were hydrolyzed with N sulfuric acid at 100°C for 18 hr, and then neutralized with barium carbonate. For those compounds from agarobiose dimethylacetal and some of the galactosides liable to be converted into 3,6-anhydride by methylation (see the result and discussion section), hydrolysis by N/50 sulfuric acid at 100°C for 2 hr and subsequent borohydride reduction were carried out before hydrolysis by N sulfuric acid to protect the acid-labile 3,6-anhydrogalactose. The hydrolyzates were identified by GLC with columns a and b and by GLC-MS as alditol acetates. 15 ,17)
References 1) 2) 3)
6) 7) 8) 9) 10)
11) 12) 13) 14) 15) 16) 17)
218. C. P. Hoiberg and R. O. Mumma, J. Amer. Chem., Soc., 91, 4273-4278 (1969). R. O. Mumma, C. P. Hoiberg, and R. Simpson, Carbohydr. Res., 14, 119-122 (1970). C. Araki, Bull. Chem. Soc. Jpn., 29, 543-544 (1956). P. F. Forrester, P. F. Lloyd, and C. H. Stuart, Carbohydr. Res., 49, 175-184 (1976). S. Hakomori, J. Biochem., 55, 205-208 (1964). D. A. Rees, J. Chem. Soc., 1961, 5168-5171. J. R. Turvey and T. P. Williams, J. Chem. Soc., 1963, 2242-2246. J. R. Turvey and T. P. Williams, J. Chem. Soc., 1962, 2119-2122. H. Miyaji and A. Misaki, J. Biochem., 74, 1131-1139 (1973). K. Nagasawa, H. Uchiyama, and N. Wajima, Carbohydr. Res., 158, 183-190 (1986). S. Hirase, K. Watanabe, and R. Takano, Agric. BioI. Chem., 42, 1065-1068 (1978). Y. Matsushima and T. Miyazaki. J. Biochem., 55, 464-465 (1964). P.-E. Jansson, L. Kenne, H. Liedgren, B. Lindberg, and J. Longrenn, Chem. Commun., Univ. Stockholm, 8, 1-28 (1976).
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T. J. Painter, "The Polysaccharides, "Vol. 2, ed. by G. O. Aspinall, Academic Press, New York, 1983, pp. 195-285. L.-A. Fransson, "The Polysaccharides," Vol. 3, ed. by G. O. Aspinall, Academic Press, New York, 1985, pp. 337-415. R. L. Whistler, W. W. Spencer, and J. N. BeMiller, "Methods in Carbohydrate Chemistry," Vol. II, ed. by R. L. Whistler and M. L. Wolfrom, Academic Press, New York, 1963, pp. 298-303. J. R. Turvey, "Advances in Carbohydrate Chemistry," Vol. 20, ed. by M. L. Wolfrom, Academic Press, New York, 1965, pp. 183-
5)
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Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide a
a
a
a
a
Ryo Takano , Takashi Ueda , Yasuhide Uejima , Kaeko Kamel-Hayashi , Saburo Hara & Susumu Hirase
a
a
Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Published online: 12 Jun 2014.
To cite this article: Ryo Takano, Takashi Ueda, Yasuhide Uejima, Kaeko Kamel-Hayashi, Saburo Hara & Susumu Hirase (1992) Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide, Bioscience, Biotechnology, and Biochemistry, 56:9, 1413-1416, DOI: 10.1271/bbb.56.1413 To link to this article: http://dx.doi.org/10.1271/bbb.56.1413
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (9), 1413-1416, 1992
Regioselectivity in Sulfation of Galactosides by Sulfuric Acid and Dicyclobexylcarbodi-imide Ryo TAKANO, Takashi Susumu HIRASE**
VEDA,
Yasuhide VEJIMA, Kaeko KAMEl-HAYASHI, Saburo HARA,* and
Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received March 2, 1992
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Methyl cx- and fJ-o-galactopyranosides and 4-0-fJ-o-galactopyranosyl-3,6-anhydro-L-galactose dimethylacetal were sulfated with sulfuric acid and dicyclohexylcarbodi-imide as a condensation reagent. The sulfated sugars were isolated by ion-exchange chromatography, characterized, and assigned by methylation analyses. On the basis of the yield of each sulfated product that was isolated, sulfation on 0-6 appeared to be predominant.
A variety of carbohydrate sulfates have been found in animals and plants. They are important substances playing physiological roles, and are also potential materials for various uses. 1 ,2) Sulfation of sugars and related compounds is usually carried out by using such reagents as sulfurylchloride, chlorosulfuric acid, sulfur trioxide-pyridine or -dimethylformamide complex, and aryl sulfurylchloride. 3 ,4) Some of these are, however, very hygroscopic for handling, and those reagents with very high reactivity may result in an undesirable degradation and decreased yield. Roiberg and Mumma 5 ) have reported another convenient method using sulfuric acid as the sulfating reagent with dicyc1ohexylcarbodi-imide (DCC) as a condensation reagent in dimethylformamide. This method was applied to carbohydrate derivatives by blocking appropriate hydroxyl groups to obtain the desired sulfate esters. 6 ) As this method employs mild conditions, the reaction can be expected to have high regioselectivity. In the present work, we applied the sulfuric acid-DCC system to methyl cx- and f3-D-galactopyranosides and to 4- 0- f3 -D- galactopyranosy 1- 3, 6-anhydro- L- galactose dimethylacetal, a derivative of the repeating disaccharide of agarose,7) to examine its regiose1ectivity.
Results and Discussion Methyl cx- and f3-D-galactopyranoside and 4-0-f3-Dgalactopyranosyl-3,6-anhydro-L-galactose dimethyl acetal (agarobiose dimethylacetal) were sulfated with sulfuric acid and DCC as described by Mumma et al. 6 ) The sugar sulfates were adsorbed to a column of IR-45 (OR form) and eluted with aqueous ammonia to isolate them from the reaction mixtures. On the basis of quantitative analyses of the unchanged sugar passing through the ion exchanger, ca. 85% of the starting materials were found to have been sulfated in each case. Mixtures of the sugar sulfate isomers were fractionated by the method of Forrester et al.,8) who had isolated fucose sulfates by ion-exchange chromatography, using DEAE-cellulose (borate form) in a borate buffer. The
* **
fractions were purified by rechromatography, and some of them were obtained in crystalline form. Their characterization and assignment are summarized in Table I, the details of the assignments being discussed later in this paper. With the presen't chromatography system, the compounds appear to have been separated by the difference in ionic Table I. Methylation Analyses of the Fractions from Sulfated Methyl and f3-Galactosides, and from Agarobiosc Dimcthylacctal
(X-
Fraction
Methylation product(s)a
Location of sulfatc(s)b
At A2 A3 A4 A5 A6
2,4,6-Me 3 -Gal 2,3,4-Mc 3 -Gal, 2,4-Me 2 -AGal 2,3,4-Me 3 -Gal, 2,3,6-Me 3 -Gal 2,3,6-Me 3 -Gal 2,4-Me 2 -Gal 4,6-Mc 2 -Gal, 2,3-Me 2 -Gal, 3,4-Me 2 -Gal 3,4-Me 2 -Gal,4-Me-AGal
36mix. of 6- and 4-
A7 Bl B2 B3 B4
43,6mix. of 2,3-, 4,6-, and 2,62,6363,6mix. of 3,6-, 4,6-, and 2,6-
B7
2,4,6-Me 3 -Gal 2,3,4-MerGal 2,4-Mc 2 -Gal 2,4-Me 2 -Gal,2,3-Me 2 -Gal, 3,4-Mc 2 -Gal 4-Me-AGal,2,3-Me 2 -Gal, 3,4-Mez-Gal 4-Me-AGal,2,3-Me 2 -Gal, 3,4-Me z-Gal 3,4-Me z-Gal, 4-Me-AGal
Cl C2 C3 C4 C5 C6
2,4,6-Me 3 -Gal, 2,5-Me 2 -AGal 2,3,4-Mc 3 -Gal, 2,5-Me 2 -AGal 2,3,4,6-Me 4 -Gal,2-Me-AGal 2,4-Me 2 -Gal,2,5-Me 2 -AGal 3,4-Mez-Gal,2,5-Mez-AGal 2,3,4-Me 3 -Gal,2-Me-AGal
365'3,62,65'6-
B5 B6
a
h
mix. of 4,6- and 2,6mix. of 4,6- and 2,62,6-
2,4,6-Me 3 -Gal = 1,4,5-tri-0-acetyl-2,4,6-tri-0-methyl-galactitol; 2,4Me 2 -AGal = 1,5-di-0-acetyl-2,4-di-0-methyl-3,6-anhydrogalactitol, etc. Expressed as the sulfated position(s) in the galactose residue. 0-5 of the 3,6-anhydrogalactose dimethylacetal residue is expressed as 5'.
To whom correspondence should be addrcssed. Present address: Research Laboratory, Tallo Co., Ltd., 126 Higashi Shiriike-Shinmachi, Nagata-ku, Kobe 653, Japan.
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strength arising from their sulfate content and from their potential to form a borate complex. The mono sulfates were eluted with the lower concentration of ammonium borate, and the disulfates with the higher, in agreement with the sulfate content of each. Among the mono sulfates of galactosides, 3-sulfates were eluted faster than 6-sulfates, which would more easily form the borate complex between 0-2 and 0-3 than 3-sulfates would between 0-4 and 0-6. The separation of the disaccharide sulfates was better than that of the monosaccharide sulfates. With the monosaccharide sulfates, the ionic strength of sulfate groups linked to the sugar residue would presumably mask the contribution of the ionic effect of the sugarborate complex, thus interfering with the chromatographic separation. Methylation analyses of the isolated sugar sulfates were carried out by the method of Hakomori. 9) With this method, the methylation product is usually isolated from the reaction mixture by extraction to avoid, in the subsequent hydrolysis step, undesirable reactions arising from the use of iodide and methylsulfoxide. The extraction of the methylated sugar sulfates was unsuccessful because of their sulfate groups, which solubilized the permethylated compounds into water. The products were successfully isolated from these contaminants by gel filtration on Biogel P-2. The permethylated products obtained were hydrolyzed, converted into alditol acetates, and then analyzed by GLC (Table I). In some compounds, 3,6-anhydrogalactose derivatives were identified besides the normal partially methylated derivatives. The formation of the anhydride, which is a known reaction caused by the elimination of sulfates at 0-6 of the sugar residues by free 0-3 to simultaneously form 3,6-anhydro rings,10) probably occurred in the methylation step carried out under alkaline conditions. The anhydrides that were identified also support the assignment of the sulfate's location. The relative reactivity of the hydroxyl groups in methyl galactosides and agarobiose dimethylacetal by the present sulfation method was calculated from the sum of the yields of the corresponding sugar esters obtained (Table II). In all cases, 0-6 atoms of the galactose residues exhibited the highest reactivity, the next highest being 0-5 of the 3,6anhydrogalactose residue in agarobiose dimethylacetal.
Such relatively high reactivity might have arisen from the lack of steric hindrance in the corresponding hydroxyl groups, which project from the sugar residues. In decreasing degree of reactivity after the hydroxyl groups, the reactivity of 0-2 and 0-3 of a-galactoside was comparable, while 0-2 of p-galactoside was much less reactive than 0-3, possibly due to the steric hindrance by the neighboring methyl group in the p configuration. In the case of agarobiose dimethylacetal, the anhydrogalactose dimethylacetal group as the aglycon, which is much more bulky than a methyl group, is likely to have caused a further reduction in the reactivity at 0-2 of its galactose residue. In all the cases observed, the reactivity of axial 0-4 was lower than that of other equatorial hydroxyl groups. As another reason, the sulfate group introduced to 0-6 might have hindered further sulfation on sterically closed 0-4, like the case of dextran with equatorial 0-4, which can hardly be sulfated. 11) However, disulfates were also formed in the present sulfation process as will be discussed later. The trend in reactivity of the hydroxyl groups with this sulfation method is similar to that described by Turvey and Williams,12) who adopted a sulfur trioxide-pyridine system. In the present case, however, the reactivity of the secondary hydroxyl groups was much lower, indicating higher regioselectivity. Such selectivity might be explained by the bulk of the DCC-sulfuric acid complex, which would reduce the accessibility of the reagent to the sterically hindered hydroxyl groups. Smaller amounts of disulfates were formed during the sulfation of methyl galactosides, although only a 10% excess of reagent was used. This might suggest that the introduced sulfate accelerated the rate of further sulfation at the other positions, while oversulfation of 6-sulfated polysaccharide, in contrast, has been reported to be difficult. 13 ) The excess formation of di- or tri-sulfate has also been reported from sulfation with the sulfur trioxidepyridine system. 14) As predominant sulfation on 0-6 was indicated, the present method would be effective for the selective sulfation of carbohydrates without any protection of the hydroxyl groups.
Experimental General methods. Concentration of the solutions was carried out in vacuo below 40°C with a rotary evaporator. Optical rotation in an
Table II. Relative Reactivitt of the Hydroxyl Groups in Methyl Galactoside and Agarobiose Dimethylacetal after DCC-H 2 S04 Sulfation Hydroxyl grouph
o:-Galactoside
[3-Galactoside
2 3 4 6d 5'
19 20 2 100
7 31
Galactose
C
Agarobiose dimethyl acetal
--------_._-_._.
a
b
C
d
+ 100
45 80 20 100
__.._--_.+ 17
+ 100 50
Calculated from the yield of each fraction obtained by chromatography of the reaction mixtures. Fractions assigned as a mixture of sulfates are excluded from the calculation. Expressed as the sulfated position in the galactose residue. 0-5 of the 3,6-anhydrogalactose dimethyl acetal residue is expressed as 5'. Results from sulfation by the pyridine-sulfur trioxide system in ref. 11. Values for the 6-sulfates are taken as 100.
aqueous solution was measured by Laurent's saccarimeter with a cell of 1 dm in length. Melting points were measured by a micro-melting point apparatus (Model MP-S2, Yanagimoto Co.). GLC was carried out by a gas chromatograph (Model 163, Hitachi Co.) equipped with a hydrogen flame ionization detector under the following conditions: injection temperature, 210°C; carrier gas (nitrogen) flow rate, 20 ml/min; hydrogen pressure, 0.6kg/cm 2 ; air pressure, 2.0kg/cm 2 . Stainless steel columns (3 mm x I m) were packed with Chromo sorb W NAW coated with following stationary phases: column a, 3% (w/w) Silar 'lOC;15) column b, 0.5% PEG 20 M. GLC-MS was carried out by a combined gas chromatograph-mass spectrometer (QP-1000, Shimadzu Corp.), using a fused silica capillary column (OJ mm x 25 m) coated with SP-IOOO and operating at a temperature of 210°C. Helium was used as the carrier gas at a 2 ml/min flow and 20: 1 split ratio. Mass spectra were recorded at 70eV ionization voltage, and infrared spectroscopy was carried out with a spectrometer (Model 215, Hitachi Co.) by the KBr disk method. Methyl O:-D-galactopyranoside and [3-D-galactopyranoside were obtained by treating commercial D-galactose with N methanolic hydrogen chloride, chromatographically separating as described by Matsushima and Miyazaki,16) and purifying by recrystallization. Agarobiose dimethyl-
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acetal was obtained by partial methanolysis of commercial agar, with subsequent charcoal chromatography and recrystallization. Sulfation of methyl a-galactoside. A 5.15 g portion (25 mmol) of DCC was dissolved in 60 ml of dried DMF, and 1.00 g (5 mmol) of methyl a-galactopyranoside in 40 ml of DMF was added. To the solution was next added 0.55 g (5.5 mmol) of sulfuric acid in 22 ml of DMF at O°C under a nitrogen stream, and the solution was stirred for 15 min. The reaction mixture was poured into 200 g of crushed ice and immediately neutralized with barium carbonate. The resulting barium sulfate and the precipitate of N,N'-dicyc1ohexylurea were removed by filtration. The filtrate was applied to an Amberlite IR-120 cation exchanger (H form, 1.26 x 23.5 cm), and the eluate was further treated by an Amberlite IR-45 anion exchanger (OH form, 1.26 x 23.5 cm). The column was eluted with deionized water to remove the nonsulfated sugar until the eluate showed negative in the anthrone test. The amount of unchanged galactoside passing through in the eluate was estimated as 133 mg on the basis of a colorimetric determination of the eluate. The anion exchanger was further eluted with N/2 ammonium hydroxide and then with N ammonium hydroxide. Each eluate was evaporated and afforded a syrupy sulfate mixture (1.36 g from N/2 eluate, and 0.008 g from N eluate). The former eluate was further investigated. Sulfation of methyl fJ-galactoside and agarobiose dimethylacetal. Methyl fJ-galactoside (1.00 g) was sulfated under exactly the same conditions as those used for methyl a-galactoside, the reaction mixture being applied to IR-120 and IR-45 columns. The amount of unchanged galactoside which passed through the columns was estimated as 160 mg, and that of the recovered sulfate mixture obtained from elution of the anion exchanger by N/2 ammonium hydroxide was 1.52 g. Essentially no sugar sulfate was eluted by N ammonium hydroxide. Agarobiose dimethyl acetal (1.11 g, 3 mmol) was sulfated with 3.16 g (15 mmol) ofDCC and 0.44 g (4.5 mmol) of sulfuric acid in 81 ml ofDMF, and then treated similarly to the galactosides. The amount of unchanged disaccharide was 0.14 g (13% of the starting disaccharide). The reaction mixture was successively applied to the IR-120 and IR-45 columns. The anion exchanger was eluted with NII0 and N ammonium hydroxide, before evaporating to afford 1.21 g and 0.095 g of a syrupy sulfate mixture, respectively. The former eluate was further investigated. Anion exchange chromatography of the sulfate mixture from methyl a-galactoside. A 288 mg portion of the sulfate mixture from the N/2
ammonium hydroxide eluate of IR-45 was dissolved in a borate buffer containing 200mM boric acid and 10mM ammonium hydroxide, and then applied to a Whatman DE 52 DEAE-cellulose column (borate form, 2.2 x 50 cm) with elution by the same borate buffer. The eluate in each tube (containing 10 ml) was checked for sugar content by the orcinol test and recombined into 3 fractions, AI, A2 and A3. From tube number 200, the column was eluted with a buffer containing 300 mM boric acid and 60 mM ammonium hydroxide to afford 4 fractions, A4 to A 7. This procedure was repeated twice, and finally 1.07 g of the sulfate mixture was fractionated. Each recombined fraction was concentrated to dryness, before adding and evaporating methanol in order to remove the remaining ammonium borate. The recombined fractions were rechromatographed under the same conditions, characterized and identified by methylation analyses as already described. Fraction AI. The amorphous solid (41 mg), eluted with the 200mM borate buffer (tube numbers 100 to 140), was identified as methyl a-Dgalactoside 3-sulfate, and yielded white crystals (22.8 mg) from ethanol. [aJt/ + 148.6° (c 1.2); mp 192-193°C (dec.); IR spectrum: 1250--60, 840,800cm- 1 . Fraction A2. The amorphous solid (446 mg), eluted with the 200 mM borate buffer (tube numbers 141 to 177), was identified as methyl a-Dgalactoside 6-sulfate, and yielded white crystals (323.4 mg) from ethanol. [aJb5 + 124.6° (c 1.2); mp 127-129°C (dec.); IR spectrum: 1240--60, 800cm- 1 . Fraction A3. The syrup (12 mg), eluted with the 200 mM borate buffer (tube numbers 178 to 199), was identified as a mixture of the 6-sulfate and 4-sulfate of methyl galactoside. Fraction A4. The syrup (13 mg), eluted with the 300 mM borate buffer (tube numbers 200 to 212), was identified as methyl a-D-galactoside 4sulfate. [aJb5 + l09S (c 1.1); IR spectrum: 1240--60,830, 8l0cm- I • Fraction A5. The syrup (98 mg), eluted with the 300 mM borate buffer (tube numbers 271 to 304), was identified as methyl galactoside 3,6disulfate. IR spectrum: 1240-60, 820-40 cm - 1.
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Fraction A6. The syrup (17 mg), eluted with the 300 mM borate buffer (tube numbers 307 to 333), was identitied as a mixture of the 2,3-disulfate, 4,6-disulfate, and 2,6-disulfate of methyl galactosides. Fraction A7. The syrup (129 mg), eluted with the 300 mM borate buffer (tube numbers 334 to 376), was identified as methyl a-D-galactoside 2,6-disulfate, and yielded white crystals (71.3 mg) from ethanol. [aJb5 + 102.2° (c 1.0); mp 182-183°C (dec.); IR specrum: 1240--60, 820cm- 1 • Anion exchange chromatography of the sulfate mixture from methyl fJ-galactoside. A 1.00 g portion of the sulfate mixture from the N/2 am-
monium hydroxide eluate of IR-45 was chroma to graphed under the same conditions as those for methyl a-galactoside, except that the elution buffer was changed to the 300 mM borate buffer at tube 270. The seven fractions obtained, B1 to B7, were rechromatographed, characterized, and identified (Table I). Fraction BI. The syrup (19 mg), eluted with the 200 mM borate buffer (tube numbers III to 142), was identified as methyl fJ-D-galactoside 3-sulfate. [aJb5 + 15.4° (c 1.1); IR spectrum: 1240--50, 805cm- 1 • Fraction B2. The syrup (408 mg), eluted with the 200 mM borate buffer (tube numbers 146 to 179), was identified as methyl fJ-D-galactoside 6-sulfate, and yielded white crystals (139.2mg) from ethanol. [aJb5 -5.1° (c 1.0); mp 142-l43°C; IR spectrum: 1240--60, 820cm- 1 . Fraction B3. The amorphous solid (177 mg), eluted with the 300 mM borate buffer (tube numbers 270 to 295), was identified as methyl fJ-Dgalactoside 3,6-disulfate, and yielded white crystals (119.1 mg) from ethanol. [aJb5 +22.0° (c 1.0); mp 188-l89°C (dec.); IR spectrum: 1240--60, 800--820 cm -1. Fraction B4. The syrup (7 mg), eluted with the 300 mM borate buffer (tube numbers 296 to 306), was identified as a mixture of the 3,6-disulfate, 4,6-disulfate, and 2,6-disulfate of methyl gaJactosides. Fraction B5. The syrup (14mg), eluted with the 300mM borate buffer (tube numbers 307 to 317), was identified as a mixture of the 4,6-disulfate and 2,6-disulfate of methyl galactosides. Fraction B6. The syrup (15 mg), eluted with the 300 mM borate buffer (tube numbers 318 to 327), was identified as a mixture of the 4,6-disulfate and 2,6-disulfate of methyl galactosides. Fraction B7. The amorphous solid (48mg), eluted with the 300mM borate buffer (tube numbers 328 to 349), was identified as methyl fJ-Dgalactoside 2,6-disulfate. [aJt/ - 3.66° (c 1.0); IR spectrum: 1240--60, 820cm- 1 • Anion exchange chromatography of the sulfate mixture from agarobiose dime thylace tal. A 401 mg portion of the sulfate mixture from IR-45 was
applied to the same column as that used for methyl galactosides. The column was eluted with the buffer containing 200 mM boric acid and 10 mM ammonium hydroxide from tubes 1 to 167, with subsequent elution with the buffer containing 200 mM boric acid and 40 mM ammonium hydroxide, to afford 6 fractions, C1 to C6. These fractions were rechromatographed, characterized and identified (Table I). Fraction Cl. The syrup (12 mg), eluted with the first buffer (tube numbers 74 to 112), was identified as agawbiose dimethylacetaI3'-sulfate. [aJ&5 19.3° (c 0.8); IR spectrum: 1240--50, 81Ocm- l . Fraction C2. The amorphous solid (134mg), eluted with the first buffer (tube numbers 113 to 143), was identified as agarobiose dimethylacetal 6'-sulfate. [aJb5 -30.7° (c 0.8); IR spectrum: 1240--60, 810cm- 1 • Fraction C3. The syrup (32mg), eluted with the first buffer (tube numbers 144 to 166), was identified as agarobiose dimethylacetal 5sulfate. [aJb5 -28S (c 0.7); IR spectrum: 1240--60, 790--81Ocm- 1 • Fraction C4. The syrup (28 mg), eluted with the second buffer (tube numbers 256 to 289), was identified as agarobiose dimethyl acetal 3',6'disulfate. [aJb5 -4S (c 1.2); IR spectrum: 1240, 780--800 em -1. Fraction CS. The syrup (23 mg), eluted with the second buffer (tube numbers 290 to 322), was identified as agarobiose dimethylacetal 2',6'disulfate. [aJb5 -15.7° (c 0.8); IR spectrum: 1240--60, 810cm- 1 . Fraction C6. The syrup (72 mg), eluted with the second buffer (tube numbers 323 to 414), was identified as agarobiose dimethylacetal 5,6'disulfate. [aJb5 -32.0° (c 0.8); IR spectrum: 1240--60, 81Ocm- 1 • Methylation analysis of the mono- and disaccharide sulfates. Each 5 mg portion of the sulfated methyl galactoside and agarobiose fractions from anion-exchange chromatography was methylated in methyl sulfoxide, using dimethylsulfinyl carbanion as described by Hakomori. 9 ) The reaction mixtures were chromatographed on Biogel P-2 (2.2 x 200 cm) with deionized water as the eluant. On the basis of the anthrone test, carbohydrates were eluted faster than methylsulfoxide and iodide. The methylated
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products isolated were hydrolyzed with N sulfuric acid at 100°C for 18 hr, and then neutralized with barium carbonate. For those compounds from agarobiose dimethylacetal and some of the galactosides liable to be converted into 3,6-anhydride by methylation (see the result and discussion section), hydrolysis by N/50 sulfuric acid at 100°C for 2 hr and subsequent borohydride reduction were carried out before hydrolysis by N sulfuric acid to protect the acid-labile 3,6-anhydrogalactose. The hydrolyzates were identified by GLC with columns a and b and by GLC-MS as alditol acetates. 15 ,17)
References 1) 2) 3)
6) 7) 8) 9) 10)
11) 12) 13) 14) 15) 16) 17)
218. C. P. Hoiberg and R. O. Mumma, J. Amer. Chem., Soc., 91, 4273-4278 (1969). R. O. Mumma, C. P. Hoiberg, and R. Simpson, Carbohydr. Res., 14, 119-122 (1970). C. Araki, Bull. Chem. Soc. Jpn., 29, 543-544 (1956). P. F. Forrester, P. F. Lloyd, and C. H. Stuart, Carbohydr. Res., 49, 175-184 (1976). S. Hakomori, J. Biochem., 55, 205-208 (1964). D. A. Rees, J. Chem. Soc., 1961, 5168-5171. J. R. Turvey and T. P. Williams, J. Chem. Soc., 1963, 2242-2246. J. R. Turvey and T. P. Williams, J. Chem. Soc., 1962, 2119-2122. H. Miyaji and A. Misaki, J. Biochem., 74, 1131-1139 (1973). K. Nagasawa, H. Uchiyama, and N. Wajima, Carbohydr. Res., 158, 183-190 (1986). S. Hirase, K. Watanabe, and R. Takano, Agric. BioI. Chem., 42, 1065-1068 (1978). Y. Matsushima and T. Miyazaki. J. Biochem., 55, 464-465 (1964). P.-E. Jansson, L. Kenne, H. Liedgren, B. Lindberg, and J. Longrenn, Chem. Commun., Univ. Stockholm, 8, 1-28 (1976).
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T. J. Painter, "The Polysaccharides, "Vol. 2, ed. by G. O. Aspinall, Academic Press, New York, 1983, pp. 195-285. L.-A. Fransson, "The Polysaccharides," Vol. 3, ed. by G. O. Aspinall, Academic Press, New York, 1985, pp. 337-415. R. L. Whistler, W. W. Spencer, and J. N. BeMiller, "Methods in Carbohydrate Chemistry," Vol. II, ed. by R. L. Whistler and M. L. Wolfrom, Academic Press, New York, 1963, pp. 298-303. J. R. Turvey, "Advances in Carbohydrate Chemistry," Vol. 20, ed. by M. L. Wolfrom, Academic Press, New York, 1965, pp. 183-
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