[Agr. Biol. Chem. Vol. 31, No. 3, p. 261266, 1967]

Glycosides and Oligosaccharides in the L-Rhamnose Series

Part II. Syntheses of Certain ƒ¿-L-Rhamnosyl Disaccharides

By Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

Laboratory of Food Chemistry, Shizuoka Women's Junior College, Shizuoka

Received July 9, 1966

An attempt was made to synthesize certain ƒ¿-L-rhamnosyl disaccharides. In this

publication the following sugars were prepared by employing Helferich or Bredereck reaction; 6-O-ƒ¿-L-rhamnosyl-D-glucose (rutinose), 6-O-ƒ¿-L-rhamnosyl-D-galactose (robinobiose),

2-O-ƒ¿-L-rhamnosyl-D-glucose (neohesperidose), all of which are known as sugar portions of

plant glycosides, 6-O-ƒ¿-L-rhamnosyl-D-mannose and 1-O-ƒ¿-L-rhamnosyl-D-fructose, both of which have not yet been found in nature.

INTRODUCTION configuration, but it is now known to be the

In order to study the substrate-specificity of a-form." Robinobiose is the sugar portion of ƒ¿-L-rhamnosidase induced in Aspergillus species, the glycoside robinin which occurs in Robinia certain ƒ¿-L-rhamnosyl disaccharides were in- pseudoacacia flowers," Puerarea thunbergiana6) and tended to synthesize. In this publication new several phaseolus species. The sugar was also synthetic methods of the following sugars, synthesized chemically by G. Zemplen7) and namely, 6-O-ƒ¿-L-rhamnopyranosyl-D-glucopyra was considered to contain P-link, but it is now nose (rutinose), 6-O-ƒ¿-L-rhamnopyranosyl-D- assigned as a-link." Neohesperidose forms the galactopyranose (robinobiose), 2-O-ƒ¿-L-rhamno glycosidic portion of neohesperidin, naringin and poncirin which are present in the peel of pyranosyl-D-glucopyranose, which are naturally occurring disaccharides, 6-O-ƒ¿-L-rhamnopy citrus. ranosyl-D-mannopyranose and 1-O-ƒ¿-L-rhamno In order to know the suitable synthetic method for ƒ¿-L-rhamnosyl disaccharides, an pyranosyl-D-fructopyranose,which have not been discovered in nature, are reported. attempt was made to synthesize them by the

Rutinose occurs as the sugar portion of a following procedures, namely, Konigs-Knorr number of plant glycosides, e.g., rutin, linarin, reactions' by the use of silver salt as a con diosmin, hesperidin, etc. This disaccharide densing agent in chloroform solution, Zemplen may be readily liberated by hydrolysis of reaction" by the use of mercuric acetate in rutin with dilute acids' or with enzyme which benzene solution, Helferich reactions' by the is formed in Rhamnus utilis2) and other plant species. Rutinose was chemically synthesized 4) P. A. J. Gorin and A. S. Perlin, Can. J. Chem., by G. Zemplen.3) 37, 1930 (1959). 5) G. Zemplen and A. Gerecs, Ber., 68, 2054 On the basis of this synthesis L-rhamnose (1934). in rutinose was originally assigned as the ƒÀ- 6) S. Hattori, M. Shimokoriya and M. Nakajima, Chem. Abstracts, 47, 705 (1953). 7) G. Zemplen, A. Gerecs and H. Flesch, Ber., 1) G. Zemplen and A. Gerecs, Ber., 71, 2520 71, 2511 (1938). (1938). 8) W. Konigs and E. Knorr, Sitzungsber. Bayr. 2) C. Charaux, Comp. Rend., 178, 1312 (1924). Akad. Wiss., 30, 103 (1900); Ber., 34, 957 (1901). 3) G. Zemplen and A. Gerecs, Ber., 67, 2049 9) B. Helferich and J. Zirner, Chem. Ber., 95, (1934). 2604 (1962). 262 Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

use of a mixture of mercuric cyanide and other hand the configuration of synthetic mercuric bromide in acetonitrile solution and rutinose has been confirmed as a-type because Bredereck reaction"' by the use of silver specific rotation value of its oxidation product perchlorate in nitromethane solution. with periodic acid was in accordance with Essentially these reactions involve the elimi that of the oxidation product of natural nation of a hydrogen halide (usually hydrogen rutinose. bromide) between a substituted glycosyl halide Rutinose could also be synthesized by the and unsubstituted hydroxyl groups of a second similar manner in which only 1,2,3,4-tetra-O- monosaccharide in the presence of appropriate acetyl-ƒÀ-D-glucose" was replaced by 1,2,3,5- catalyst. The experiment was carried out on di-O-isopropy lidene-ƒ¿-D-glucofuranose.13) a microscale and the products were examined Furthermore another synthetic route is by paper chromatography. described. In the case of Konigs-Knorr reaction the Condensation of 2, 3, 4-tri-O-acetyl-ƒ¿-L- main products were two orthoesters and only rhamnosyl bromide"' with 6-O-trityl-1,2,3,4- a small amount of rutinose was detected, and tetra-O-actyl-D-glucosell) by the Bredereck it was considered that this method was not reaction yielded the condensed products, which suitable for ƒ¿-L-rhamnosyl disaccharide syn- upon deacetylation and subsequent fractiona thesis. In contrast, in the case of the other tion by carbon-column chromatography gave reaction the main product was rutinose, giving pure rutinose. fortunately no orthoesters. It was also led to crystalline heptaacetate. Especially the latter two procedures gave Robinobiose was chemically synthesized by the best results and these methods were chiefly G. Zemplen in 1934. used in the following experiments. Rutinose3) In this paper it was synthesized by the two was already synthesized chemically in 1934 by different methods. 2,3,4-Tri-O-benzoyl-ƒ¿-L- G. Zemplen by condensing 2,3,4-tri-O-acetyl-ƒ¿- rhamnosyl bromide"' was condensed with L-rhamnosyl bromide with 2,3,4,6-tetra-O- 1,2: 3,4-di-O-isopropylidene-D-galactose by the acetyl-ƒ¿-D-glucosyl chloride, followed by treat Helferich reaction to give a new crystalline ment with mercuric acetate. But the present 6-ƒ¿-(2, 3, 4-tri-O-benzoyl-L-rhamnosyl)-1, 2 : 3, 4- paper describes other new synthetic methods di-O-isopropylidene-D-galactose. The latter of rutinose. 2,3,4-Tri-O-acetyl-ƒ¿-L-rhamnosyl compound lost its acetone and benzoyl groups bromide"' was condensed with 13-1,2,3,4-tetra- upon mild acid and alkaline hydrolyses, re O-acetyl-D-glucose11) by the Helferich reaction spectively, and gave pure robinobiose. The to form the mixture of sugar acetates, which configuration of the sugar was established as upon deacetylation yielded a mixture of free a-type by its oxidation with periodic acid, sugars. On account of isolation and purifica which led through customary reactions to the tion of rutinose carbon-column chromatography same compound that was obtained similarly was applied to the sugar mixtures. from rutinose. After adsorption of them on the column Upon acetylation of robinobiose it is con rutinose fractions were collected by stepwise verted to the amorphous heptaacetate, which elution with water and ethanol of various on treatment with titanium tetrachloride concentrations. Rutinose was obtained as an yielded ƒ¿-acetochlororobinobiose in crystalline amorphous hygroscopic powder, which upon form. An other synthetic route to robinobiose acetylation afforded a heptaacetate. On the was also established. The condensation of 2,3,4-tri-O-acetyl-ƒ¿-L-rhamnosyl bromide with 10) H. Bredereck, A. Wagner, G. Faber, H. Ott 1,2,3,4-di-O-isopropylidene-D-galactose by the and J. Rauther, Chem. Ber., 92, 1135 (1959). Helferich reaction led to the condensation Glycosides and Oligosaccharides in the L-Rhamnose Series . Part II 263

containing 1.05 g of ~-1,2,3,4-tetraacetyl D-glucose, products which, followed by deacetylation and deacetonation, afforded a mixture of free 0.75 g of silver oxide, 0.1 g of iodine and 0.3 g of Drierite in dry chloroform was added, with stirring, sugars. After treatment of them with carbon- a solution of 1.2 g of 2,3,4-tri-O-acetyl-ƒ¿-L-rhamnosyl column chromatography robinobiose was ob bromide in 5 ml of chloroform. The mixture was tained. shaken for 18 hours. It was characterized as crystalline ƒ¿-aceto The sirup was treated with 5 ml of 0.05 N sodium chloroderivative. Similarly, 6-O-ƒ¿-L-rhamnosyl- methoxide solution at 0°C overnight. After neutrali D-mannose was prepared by Bredereck reaction zation with a slight excess of Amberlite IR-120(H+),

condensing 2,3,4-tri-O-acetyl-ƒ¿-L-rhamnosyl the filtrate was concentrated and was taken up in

bromide with 6-trityl-ƒÀ-1,2,3,4-tetra-O-acetyl- water. Examination of paper chromatogram showed D-mannose.'" Condensing products, after losing the presence of glucose (RF 0.30), rhamnose (RF 0.52),

its protecting groups followed by fractionation two orthoesters (RF 0.60, 0.65) and rutinose (RF 0.20). ii) Synthesis of rutinose by the Zemplen's by carbon column, gave amorphous hygroscopic method. A mixture of 2.08 g of ƒÀ-1,2,3,4-tetraacetyl 6-O-ƒ¿-L-rhamnosyl-D-mannose. D-glucose, 2.1 g of 2,3,4-triacetyl ƒ¿-L-rhamnosyl bro The configuration of the sugar was deter- mide and 0.9 g of mercuric acetate in 30 ml of absolute mined as a-type by the similar procedure as benzene was kept for four days at room temperature. described in the case of rutinose. In the case After washing with water several times and dried of 1-O-ƒ¿-L-rhamnosyl-D-fructose synthesis, 2,3, over calcium chloride, the benzene phase was evapo 4-tri-O-benzoyl-ƒ¿-rhamnosyl bromide was con rated in vacuo to a sirup and was taken up in

densed with 2,3: 4, 5-di-O-isopropylidene-D- methanol. Chromatographic examination of the sirup

fructopyranose by Helferich reaction. As after deacetylation with sodium methoxide showed

condensation product, 1-ƒ¿-(2,3,4-tri-O-benzoyl- the presence of glucose (RF 0.30), rhamnose (RF 0.52) and two lesser components, rutinose (RF 0.20) and L-rhamnosyl)-2, 3 : 4, 5-di-O- isopropylidene-D- unknown sugar (RF 0.15). No orthoester products fructopyranose was obtained in crystalline were detected. form. By losing protecting groups followed 2. Synthesis of 6-ƒ¿-L-rhamnosyl-D-glucose (ru by purification by carbon column, 1-O-ƒ¿-L- tinose) by Helferich reaction rhamnosyl-D-fructose was given. It was i) Condensation of 2,3,4-tri-O-acetyl-ƒ¿-L-rham characterized as a crystalline phenylhydrazone. nosyl bromide12) with ƒÀ-1,2,3,4-tetra-O-acetyl-ƒÀ-D- Neohesperidose was synthesized by Helferich glucose.11) To ~ solution of 3.59g of mercuric reaction. Condensation of 2,3,4-tri-O-acetyl- cyanide and 5.08 g of mercuric bromide in 99 ml of ƒ¿-L-rhamnosyl bromide with ƒ¿-1,3,4,6-tetra-0- absolute acetonitrile were added 9.57 g of ƒÀ-l,2,3,4- acetyl glucose yielded condensed products, tetraacetyl D-glucose and 9.9g of 2,3,4-triacetyl-ƒ¿-L-

which by deacetylation and subsequent frac rhamnosyl bromide with stirring until the solution

tionation afforded a sirup which showed a was complete. After being kept overnight at room temperature single spot on paper chromatogram. It was the solvent was removed in vacuum below 30°C and reacetylated giving ƒÀ-heptaacetyl derivative in the residual sirup was extracted with absolute chloro crystalline form. form and filtered.

The filtrate was washed three times with 1 N potas EXPERIMENTAL sium bromide solution and dried over anhydrous 1. Comparison of the synthetic methods of the sodium sulphate. ƒ¿-L-rhamnosyl disaccharide After filtering, the filtrate was concentrated in Paper chromatography was carried out with paper vacuo to an amorphous powder, which presumably using butanol : acetic acid : water (4 : 1 : 2) as solvent and aniline hydrogen phthalate as spraying reagent. 11) B. Helferich and W. Klein, Ann., 455, 173 i) Synthesis of rutinose by the Konigs-Knorr (1927). reaction. Active silver oxide was prepared accord- 12) E. Fischer, M. Bergmann and A. Rabe, Ber., ing to the procedure of B. Helferich. To a mixture 53, 2362 (1920). 264 Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

contained rutinose heptaacetate as a main product. zobromorhamnose with stirring and the mixture was As it was failed to crystallize it from any solvent, allowed to stand for three hours. After the reaction it was deacetylated by dissolving in 80 ml of 0.05 N was over, the solution was treated according to the sodium methylate in methanol on cooling, and allowed procedure described above and 2.2g of sirup was to stand overnight in an ice box. obtained.

Then the sodium was removed by stirring with a As the sirup was failed to crystallize from any slightly excess of Amberlite resin IR-120(H+) until solvent, it was deacetylated and deacetonated as usual the solution became neutral, and the resin was filtered manner, and fractionated by carbon column as de off and the filtrate was evaporated in vacuo to a scribed above. Rutinose (0.31 g) was obtained as an sirup. amorphous hygroscopic powder, which was acetylated

The sirup (7.55g) was dissolved in 63 ml of water again as a usual manner to yield ƒÀ-heptaacetate. It to make a 10% solution, and chromatographed on was recrystallized from ethanol. Its m.p. of 168° column of carbonceltite (1 : 1). Fractionation of the showed no depression with authentic specimen. materials adsorbed on the column by gradient elution 3. Synthesis of rutinose by Bredereck reaction10) with water and aqueous ethanol was carried out. To the solution of 12.42g of silver perchlorate10) The fractions which contained only rutinose were in 108 ml of anhydrous nitromethane was added 2g collected and evaporated to dryness giving amorphous, of dried calcium sulphate. After while 35.4g of 6- hygroscopic rutinose (2 g). [a]D -10° (c 1, EtOH). O-trityl-ƒÀ-l, 2, 3, 4-tetra-O-acetyl-D-glucose11) and 30g In order to determine the configuration of the disac of ƒ¿-acetobromorhamnose were dissolved in the solu charide, rutinose (60 mg) was dissolved in 2 ml of tion with cooling and agitated vigorously for ten water and the solution was mixed with 2 ml of a minutes at room temperature. solution containing 300 mg of sodium periodate. After the reaction was over, the mixture was Within 24 hr., the rotational value was constant, filtrated in order to remove silver bromide and trityl giving the specific rotation of the oxidized product of -107° (c 0.4, H2O). In order to make acetyl perchlorate, and the filtrate was washed twice with an aqueous saturated sodium bicarbonate solution rutinose, 1.6g of well dried rutinose was dissolved in a mixture of 10 ml of acetic anhydride and 2.5 g (0°C) and several times with cold water and filtered. Chloroform was added to the filtrate and it was dried of anhydrous sodium acetate, and heated for 2 hours over anhydrous sodium sulphate. Then the filtrate at 100°C. was distilled off in vacuo, yielding 13.5g of sirup After cooling the solution was poured into ice water which did not crystallized from any kind of solvent. with stirring and left overnight in the refrigerator. On account of purification, it was chromatographed The amorphous precipitates were collected and on carbon-celite column after deacetylation as usual washed with water and dried. It was crystallized manner. from absolute ethanol in prisms, and yielded 0.9 g; Rutinose was obtained as an amorphous hygroscopic m.p. 167-169° (from EtOH). Its m.p. showed no depression on admixture with powder, yield (2.6g). Upon acetylation it afforded a heptaacetate of m.p. 166-,-167°C, which gave no authentic specimen. [a]D -31.2° Lit. [a]D -28.7° depression on admixture with authentic specimen. Anal. Found: C, 50.31; H, 5.70. Calcd. for C26H36O17: 4. Synthesis of 6-O-ƒ¿-L-rhamnosyl-D-galactose C, 50.32; H, 5.80%a.

ii) Condensation of 2, 3, 4-tri-O-benzoyl-ƒ¿-L- (robinobiose) by Helferich reaction. i) Condensation of ƒ¿-benzobromorhamnose with rhamnosyl bromide (ƒ¿-benzobromorhamnose)14) diaeetonegalactose.16) A solution containing 8.34g with 1,2:3,5-di-O-isopropylidene-ƒ¿D-glucofuranose of mercuric cyanide, 11.83g of mercuric bromide, (isodiacetone glucose).13) To a solution of 0.356g 17.1 g of diacetonegalactose and 35.48g of ƒ¿-ben of mercuric cyanide and 0.504 g of mercuric bromide zobromorhamnose was. allowed to stand three hours in 14 ml of absolute acetonitrile were added 0.73g and was treated as described above. The residual of 1, 2 : 3, 5-diisopropylidene D-glucose and ƒ¿-ben sirup was crystallized easily from absolute ethanol in needles, yield 32.3 g (64.8%). 13) K. A. Petrov and E. E. Nifantiv, Zh. Obshch. Khim., 34(5), 145962 (1964). 14) R. K. Ness, H. G. Fletchner, Jr., and C . S. 15) K. Freudenberg and R. M. Hixon, Ber., 56, Hvdson, J. Am. Chem. Soc., 73, 296 (1952). 2119 (1923). Glycosides and Oligosaccharides in the L-Rhamnose Series . Part II 265

The crystals were 6-ƒ¿-(2', 31, 4'-tribenzoyl-L- acetonegalactose and 4.11 g of ƒ¿-acetobromorhamnose rhamnosyl)-1, 2 : 3,4- diisopropylidene-D-galactose (I): in 42 ml of absolute acetonitrile was treated similarly m.p. 162---164°C, [a]D +76°. Anal. Found: C, 65.05; as in the case of rutinose synthesis. The robinobiose

H, 5.87. Calcd. for C39H42013: C, 65.18; H, 5.85%. fractions collected by carbon-column chromatography In order to remove benzoyl groups of (I), 10 g of (1) were evaporated to a sirup, yield 0.756g (37.8%). was dissolved in 250 ml of methanol and 25 ml of Acetylation and chlorination of 0.63 g of robinobiose

0.1 N sodium methylate solution was added to the gave ƒ¿-acetochloroderivative, m.p. 178•`180•Ž. solution, which was heated under reflux for five 5. Synthesis of 1-O-ƒ¿-L-rhamnosyl-D-fructose by minutes and allowed to stand overnight at room Helferich reaction temperature. i) Condensation of 2, 3, 4-tri-O-benzoyl-ƒ¿-L- The solution was neutralized by the addition of the rhamnosyl bromide with 2, 3 : 4, 5-di-O-isopro Amberite IR-120(H+) to remove sodium ion and was pylidene-D-fructopyranose.16) A mixture of 4.445g extracted with chloroform in order to remove methyl mercuric cyanide, 6.3 g of mercuric bromide, 9.2 g benzoate. of ƒÀ-diacetonefructose and 18.865 g of ƒ¿-benzobromo- The aqueous phase was made 0.1 N with sulphuric rhamnose in 98 ml of acetonitrile was allowed to acid and was heated for hours at 70°C to release stand overnight at room temperature. A part of acetone group. needle crystals were deposited during the reaction. After cooling and adding barium carbonate in The reaction mixture was treated similarly as de order to remove sulphate ion, the filtrate was con scribed in the case of robinobiose synthesis. centrated in vacuo to a sirup which weighed 4.32 g The sirup obtained was crystallized from absolute (90.18%). [a]D 0° [ƒ¿]_??_Oxid -113°. alcohol in needles, which were 1-ƒ¿-(2,3,4-tribenzoyl- Periodate oxidation of robinobiose should yield L-rhamnosyl)-2, 3 : 4, 5-diisopropylidene D-fructopy theoretically the same trialdehyde as in the case of ranose (II) and yielded 15g (69%): [a]n +100° (c 5, rutinose oxidation. CHCl3); m.p. 190191.5°C (from EtOH). Anal. From [ƒ¿]_??_Oxid value of robinobiose it was thought Found: C, 65.31; H, 5.80. Calcd. for C39H43013: C, to have a-link. The well dried robinobiose (0.63g) 65.09; H, 5.847%. The protecting groups of (II) were was acetylated as usual manner, giving an amorphous lost by the similar manner as described in the case heptaacetate, 0.42 g: m.p. 84.5-85°C. Lit. m.p. 83°C. of (I), and was obtained 1-O-ƒ¿-L-rhamnosyl-D-fructose Anal. Found: C, 50.39: H, 5.78. Calcd. for C26H36017: as a sirup which was contaminated with a small C, 50.32; H, 5.80%. To a solution of robinobiose amount of rhamnose and fructose. It was transferred heptaacetate (500 mg) in 3.5 ml of anhydrous chloro to a mixture of carbon and celite (1 : 1), which was form 150 mg of titanium tetra chloride was added. eluted with water and 2•`5% ethanol. A yellow precipitates were formed, which did not The eluate containing only the disaccharide fraction dissolve during the reaction. After being kept for was recovered as an amorphous solid: yield, 4.67 g three hours at room temperature the reaction mixture (61.340); [a]o -4.85° (c 0.58, CHCl3), [a]_??_Osid -107°. was poured into ice wate, whereupon the precipitates The configuration of the dissccharide was assigned as dissolved and the chloroform phase became almost a-type. Upon acetylation as usual manner it gave colourless. only a sirup acetate. In order to characterize the

The latter was separated, washed with water, dried disaccharide, phenylhydrazone of it was prepared. over calcium chloride and concentrated in vacuo. To 100 mg of sample in 10 ml of water were added 250 mg of phenylhydrazine hydrochloride and 350 mg The residual sirup was dissolved in small quantity of crystalline sodium acetate, and the mixture was of ether and kept at 0°C overnight. The colourless heated at 100°C for 30 minutes. After cooling, needle crystals of ƒ¿-acetochlororobinobiose were deposited, crystals of slightly yellow colouration were separated, which were collected by filtration: yield, 200 mg; m.p. 178.180°C; [a]D -6.6° (c 3.02, HCl3). Lit. which were collected and dried to yield 50 mg of m.p. 178.-180°C. Anal. Found: C, 48.59; H, 5.65. the product, 193°C(decomp.) (EtOH). [a]D 20.4° (c 1, EtOH). Anal. Found: C, 57.20;H, 6.18;N, 11.21. Calcd. for C24H32015Cl: C, 48.32; H, 5.53%.

ii) Condensation of ƒ¿-acetobromorhamnose with diacetonegalactose. A mixture of 2.16 g of mercuric 16) E. Pacsu, E.J. Wilson,Jr., and L. Graf, J. bromide, 1,52g of mercuric cyanide, 2.8g of di Am. Chem.Soc., 61, 2675 (1939). 266 Shintaro KAMIYA, Sachiko ESAKI and Misao HAMA

Calcd. for C24H31O8N: C, 57.25; H, 6.16; N, 11.13%. anhydride and 12 ml of pyridine was allowed to stand for three days in the refrigerator. 6. Synthesis of 6-0-ƒ¿-L-rhamnosyl-D-mannose by Then the solution was poured into ice water and the Bredereck reaction the amorphous precipitates was collected and crystal The disaccharide was synthesized similarly as in lized from ethanol in needles: yield, 240 mg; m.p. the case of rutinose synthesis by the Bredereck reac 160°C (from ethanol); [a]D -5.2° (c 3.8, CHCl3). tion. 8.5 g of silver perchlorate 73.6 ml of nitro Anal. Found: C, 50.34; H, 5.80. Calcd. for C26H36O17: methane, 4.1 g of calcium sulphate, 13.5 g of ƒ¿- C, 50.32; H, 5.80%. benzobromorhamnose and 24g of 6-O-trityl-ƒÀ-1,2,3,4- tetra-O-acetyl-D-mannose were used to obtain 26.6g 7. Synthesis of 2-0-ƒ¿-L-rhamnosyl D-glucose

of the sirup which could not be crystallized from (neohesperidose) by the Helferich reaction any kind of solvent. It was deacetylated as usual A mixture of 3.59g of mercuric cyanide, 5.08g of manner and 8.5 g of free sugar was obtaind. The mercuric bromide, 9.75 g of ƒ¿-1,3,4,6-tetra-O-acetyl-

sirup was found chromatographically to contain four D-glucose and 9.89 g of ƒ¿-acetobromorhamnose in compounds. The main component had RF 0.25, and 100 ml of acetonitrile was allowed to stand overnight the minors were RF 0.31 (glucose), RF 0.51 (rhamnose) at room temperature and was treated as usual manner.

and RF 0.12 (unknown), respectively. The sirup was As the sirup obtained could not be crystallized, it dissolved in water to make 10% solution and frac was deacetylated as usual manner and chromatographed

tionated on carbon-celite (1: 1) column as usual on the carboncelite column. The disaccharide frac manner. tions were collected and, on removal of the solvent, The disaccharide fractions were collected and amorphous power was obtained: yield, 7 g; [a]1 -5.4°

evaporated to an amorphous powder: yield, 1.73g; (c 1, H2O). In order to prepare acetyl derivative of [a]D -8.2° (c 1, H2O). Sodium metaperiodate oxida the sugar, 1 g of the sample was acetylated as usual tion of sample should give the same trialdehyde as manner affording neohesperidose heptaacetate in was obtained in the case of rutinose oxidation. needles: yield, 0.4g; m.p. 148•`148•Ž (EtOH), lit. 151°C; [a]D 0°- Anal. Found: C, 50.07; H, 5.71. [ƒ¿]_??_Oxid -110.3° Thus the configuration of the disaccharide was assigned as a-type. In order to Calcd. for C26H32O17: C, 50.32; H, 5.80%. characterize the disaccharide, 6-O-ƒ¿-L-rhamnosyl-D-

mannose heptaacetate was prepared. Acknowledgement. The authors wish to ex- A mixture of 500 mg of sugar, 14 ml of acetic press their thanks to Mr. Kusuo Narita of 17) D. D. Reynolds and W. L. Evans, J. Am. Chem. Shizuoka Pharmaceutical College for elemental Soc., 62, 66 (1940). analyses.