[Agr. Biol. Chem., Vol. 33, No. 1, p. 103109, 1969]

Acetolysis of Polysaccharides

Part V. Isolation of Trisaccharides Containing ƒ¿-1,3 Linkage

from a Dextran of Leuconostoc mesenteroides B

By Fumio YAMAUCHI and Kazuo MATSUDA

Department of Agricultural Chemistry, Faculty of Agriculture Tohoku University, Sendai Received July 5, 1968

Fragmentation of a dextran from Leuconostoc mesenteroides B* by controlled acetolysis

gave five trisaccharides; isomaltotriose (0.12%), 3-ƒ¿-isomaltosylglucose (0.80%), 6-ƒ¿-nigero sylglucose (0.43%), 3,6-di-ƒ¿-glucosylglucose (0.18%) and nigerotriose (0.12%). Among these trisaccharides the last three were first isolated from dextran. Isolation of nigerotriose in dicated that some of the 1,3-linkages in the dextran molecule were contiguous.

Previous studies in this series1,2) have shown fragments from the periodate oxidized dex the value of partial acetolysis in obtaining tran also supported the above results. The the non-1,6-linked fragments from dextrans. usefulness of partial acetolysis was reconfirmed In this way nigerose and kojibiose have by Suzuki and Hehre,4) who applied this been isolated from the partial acetolyzates method to sixteen selected dextrans and ob of dextrans of Leuconostoc mesenteroides B and tained a new information on the correlation Leuconostoc mesenteroides NRRL B-1299 (or K), of the yields of kojibiose with the reactivity respectively. Goldstein and Whelan3) also with type-12 pneumococcus antiserum. obtained nigerose and 3-ƒ¿-isomaltosylglucose Weigel and his coworkers,5,6) isolated a following acetolysis of a dextran from Leucono number of branched oligosaccharides with stoc mesenteroides NRRL B-1355. This was degrees of polymerization (DP) greater than the first isolation of a trisaccharide with non- four from the enzymatic hydrolyzates of 1,6-linkage from dextran. The structure of some dextrans. These oligosaccharides con this trisaccharide proposed by the same tained one glucose unit joined through a ƒ¿- authors afforded a direct evidence for the 1,3 or ƒ¿-1,4 linkage to a glucose unit of a coexistence of ƒ¿-1,6 and ƒ¿-1,3 linkages in the main chain of isomaltodextrins. The struc same dextran molecule. However, no nigero ture of these oligosaccharides supported the triose was detected in the fragments of this polysaccharide structure in which a large dextran, suggesting that the majority of the portion of the side chains consisted of a single ƒ¿-1 ,3 linkages does not occur in sequence glucose unit. Again no oligosaccharide merely with each other. Characterization of the consisted of non-1,6 linkage was obtained, suggesting that few of the non-1,6 linkages * In the former report, the name of this strain was erroneously described as NRRL-B-421. 1) K. Matsuda, H. Watanabe and K. Aso, Nippon N 4) H. Suzuki and E.J. Hehre, Arch. Biochem. ogeikagaku Kaishi, 35, 1228 (1961). Biophys., 104, 305 (1964). 2) K. Matsuda, K. Fujimoto and K. Aso, ibid., 5) R. W. Bailey, D. H. Hutson and H. Weigel, 35, 1232 (1961). Biochem. J., 80, 514 (1961). 3) I. J. Goldstein and W.J. Whelan, J. Chem. Soc., 6) D. Abbot and H. Weigel, J. Chem. Soc. (C), 1962, 170. 1966, 821. 104 F. YAMAUCHI and K. MATSUDA

occur in sequence in these dextran molecules. sodium bisulfite solution.11) The reagents used for Recently, Misaki and his coworker7) applied the detection of the compounds were aniline hydrogen the Smith degradation technique to a dextran phthalate12) for reducing sugars except in the case of electrophoresis in bisulfite solution, and silver from a certain Leuconostoc species and obtained nitrate-sodium hydroxide13) for sugar alcohols and a new information on the location of 1,3- reducing sugars in the case of electrophoresis in glucosidic linkage in the dextran. Mild acid bisulfite solution. RG refers to the movement of a hydrolysis of the methylated dextran poly sugar on paper chromatography relative to glucose. alcohol, which had been obtained by periodate MG and MS refer to the mobility of a substance on oxydation and subsequent borohydride reduc paper electrophoresis with respect to glucose and tion, yielded a trace amount of 2,4,6-tri-O- sorbitol, respectively. methyl-D-glucose in addition to the major Complete acid hydrolysis was performed by heating fragments, such as 1-O-methyl-glycerol or 2,4- the sugars (ca. 3 mg) with I N hydrochloric acid in di-O-methyl-D-glucose. This finding is the a boiling water bath for two hours. Partial hydrolysis first conclusive evidence for the presence of was made with 0.1 N hydrochloric acid in a boiling water bath for one hour. Reduction of the sugars 1,3-glucosidic linkage, which occurs in the into the corresponding sugar alcohols was achieved linear part of the dextran. However, no by treating the aqueous sugar solution (0.20, w/v) evidence was given for the presence of the with an equal volume of sodium borohydride solution contiguous 1,3-glucosidic linkages. (0.2%, w/v) at room temperature. After the reaction The present study was designed to isolate mixture was allowed to stand overnight, the solution the trisaccharides from the acetolyzate of the was then deionized with Amberlite IR-120 (H+ form) dextran, and characterize those trisaccharides followed by repeated distillation with anhydrous other than isomaltotriose and 3-ƒ¿-isomaltosyl methanol.

glucose. Acetolysis of the dextran. A scaled-up modification of the method described in the previous paper1) was EXPERIMENTAL used. Finely powdered dextran (300g, dry weight)

Material. The dextran was prepared in a large was suspended in cold acetic anhydride (1440 ml) and scale with a strain of Leuconostoc mesenteroides B treated with a cold mixture of glacial acetic acid essentially by the same method as described in the (960 ml) and conc. sulfuric acid (180 ml). The mix ture was kept at 30°C for several days with occasional previous paper.1) shaking until most of the dextran dissolved. The dark brown solution was poured into ice water (10 General methods. Paper chromatography was per liters) and carefully neutralized first with saturated formed on Toyo No. 2 filter paper by four times aqueous sodium hydroxide and then with sodium ascending method with a mixture of n-butanol-pyri bicarbonate to pH 5.0. The neutralized mixture was dine-water (6 : 4: 3, by vol.).8) Paper electrophoresis extracted in portion with chloroform and the com was carried out on Toyo No. 51 filter paper at about bined extract was washed with water and dried over 15 V/cm for three hours in borate buffer (pH 10.0),9) anhydrous sodium sulfate. Evaporation of the solvent and at about 25 V/cm for one hour in molybdate left a brown sirup, which was distilled with methanol buffer (pH 5.5).10) Paper electrophoresis for the to remove the last traces of acetic anhydride. The determination of the molecular size of sugars was dried sirup (380g) was deacetylated with 0.05N carried out at about 10 V/cm for two hours in 0.4 M sodium methoxide (5 liters) for four hours at room temperature; water was added to dissolve the result- 7) A. Misaki and S. Kanamaru, Agr. Biol. Chem., 32, 432 (1968). 8) A. Jeanes, C. S. Wise and R. J. Dimler, Anal. 11) J. L. Frahn and J. A. Mills, Chem. and Ind., Chem., 23, 415 (1951). 1956, 1137. 9) A. B. Foster, J. Chem. Soc., 1953, 982. 12) S. M. Partridge, Nature, 164, 443 (1949).13)W 10) E.J. Bourne, D. H. Hutson and H. Weigel, . E. Travelyan, D. P. Procter and J. S. ibid., 1961, 35. Harrison, ibid., 166, 444 (1950). Acetolysis of Polysaccharides. Part V. 105

ing precipitate, and the solution was neutralized isomaltose, isomaltotriose (RG 0.33) and a substance with hydrochloric acid to pH 5.8 and finally eva having RG 0.50 and MG 0.59. A small portion of this porated in vacuo. fraction (200 mg) was separated by preparative paper chromatography using Toyo No. 514 thick filter paper.

Fractionation of the acetolyzate on charcoal:Celite Chromatographically pure laminaribiose and isomalto triose were obtained. The former spontaneously column. Deacetylated acetolyzate of the dextran crystallized on being evaporated (yield 35 mg), showed prepared as above was diluted to 4.5 liters and frac tionated on a series of two charcoal: Celite (1:1, by [ƒ¿]_??_=+22.5•‹ at equilibrium (c=2.1, water). This value and the infrared spectrum were identical with wt.) columns (350 x 200 mm diam., each containing those of authentic laminaribiose.15) The latter (yield 1.5 kg of charcoal) using the following solvents as 16 mg) showed [ƒ¿]_??_=+145° (c=1.6, water) which successive elution developer; water (140 liters), 5% was identical with the reported value of isomalto- (60 liters), 7% (60 liters) and 20% (100 liters) aqueous triose.16) ethanol. Each 10 liters of the effluent was tested for carbohydrate component with the Molisch reagent, and Fractions 11-18 eluted with 7% ethanol (total a small portion of the Molisch positive fraction was yield 10.0g) mainly consisted of a substance having submitted to paper chromatography after being Re 0.50 and MG 0.59. Electrophoresis of the corre evaporated in vacuo. sponding sugar alcohol of this substance in molybdate The water effluent contained only glucose, 50 and buffer revealed the presence of two components. One

7% ethanol effluents contained nigerose as a sole was immobile in molybdate buffer (trisaccharide A), disaccharide, and the 200, ethanol effluent contained and the other had MS 0.60 (trisaccharide B). trisaccharides and higher oligosaccharides accompanied Among these fractions fraction 11 contained a with a small amount of disaccharide. large part of trisaccharide A and only a trace of The 5 and 7% ethanol effluents were combined trisaccharide B. A portion of this fraction (200 mg) and evaporated to a sirup (32.0g), from which was acetylated with acetic anhydride and anhydrous nigerose was crystallized as reported previously;14) sodium acetate in usual manner. The crude acetate mp 156°C, [ƒ¿]_??_=+125•¨130•‹ (c=2.0, water). This was purified on Magnesol: Celite column (300 x 30 mm sugar was further characterized as its crystalline ƒÀ- diam.) with 700 ml of benezene to give 190 mg of acetate; mp 150°C, [ƒ¿]_??_= +84•‹ (c=1.4, chloroform). purified material. This was crystallized from ethanol, yield 105 mg. It had mp 120•Ž, [ƒ¿]_??_=+111•‹, (c=2.3, chloroform). The free sugar obtained by deacetyla Refractionation of the oligosaccharides. The 20% tion showed [ƒ¿]D=+152•‹ (c=2.0, water). These ethanol effluent was evaporated to give 16.5 g of sirup. values were identical with the reported values of The similar fractions obtained from three preliminary 3-ƒ¿-isomaltosylglucose.3,17) A mixed melting point fractionations were combined (total amount 64 g), of the above acetate with the known specimen of redeacetylated with sodium methoxide, and refrac 3-ƒ¿-isomaltosylglucose undecaacetate, which was kindly tionated on a charcol: Cetite column (500 x 160 mm diam., containing 1 kg of charcoal) by stepwise provided by Dr. Sato, showed no depression. elution with 0-25% aqueous ethanol. Each 5 liters Fraction 19 eluted with 7.5% ethanol gave on of the effluents was collected, a small portion of paper electrophoresis in molybdate buffer almost which was applied for paper chromatography and single component migrating with trisaccharide B. electrophoresis as before. Acetylation of a portion of this fraction (600 mg)

The effluent with water (fractions 1-5) gave no gave 980 mg of crude acetate, which was crystallized carbohydrate. Elution of the column with 5%, from ethanol, yield 320 mg. It had mp 120•Ž, aqueous ethanol gave fractions 6•`7, from which a [ƒ¿]_??_=+101•‹ (c= 1.9, chloroform). These values were sirup containing glucose, nigerose (RG 0.87) and very close to those of the acetate of trisaccharide A, isomaltose (RG 0.64) was obtained. Fractions 8•`10 eluted with 5% ethanol yielded a 15) P. Bachli and E. G. V. Percival, J. Chem. Soc., sirup (4.3 g) containing laminaribiose (RG 0.93), 1952, 1243. 16) J. R. Turvey and W.J. Whelan, Biochem. J., 67, 49 (1957). 14) F. Yamauchi and K. Matsuda, Nature, 204, 17) A. Sato and H. Ono, Rep. Ferm. Res. Inst. 1088 (1964). Japan, 24, 147 (1963). 106 F. YAMAUCHI and K. MATSUDA

TABLE I. YIELDS AND SOME PROPERTIES OF TRISACCHARIDES ISOLATED FROM DEXTRAN OF Leuconostoc mesenteroides B

* Relative to glucose . ** Relative to sorbitol .

but the melting point was depressed to 110•`111•Ž showed the same MG value (0.55) corresponding to on admixture with trisaccharide A acetate. The that of trisaccharide (maltotriose was used as a free sugar of trisaccharide B, prepared by deacetyla reference compound). tion of the acetate, showed [ƒ¿]_??_=+153•‹ (c=2.5, High mobility of these trisaccharides in borate water). The corresponding sugar alcohol prepared buffers) indicated the presence of 1,3- or 1,6-linkage by reduction with sodium borohydride showed MS at the reducing end-group. 0.60 on paper electrophoresis in molybdate buffer. Mobility of the corresponding sugar alcohols of However, a trace of another sugar alcohol which these trisaccharides during electrophoresis in molyb was immobile in molybdate buffer was still present. date buffer gave further structural information.10) Recrystallization or purification on Magnesol: Celite Trisaccharide B alcohol as well as isomaltotriitol had column of the acetate could not remove a trace of

this substance.

Fractions 20-28 eluted with 7.5 and 10% ethanol

yielded 5.90g of sirup which contained two sub- stances having RG 0.50 (trisaccharide C) and RG 0.69

(trisaccharide D). The former had the same RG value as trisaccharide A or B and the corresponding sugar alcohol was immobile during electrophoresis in molybdate buffer. These characteristic were quite

similar to those of trisaccharide A, but the MG value

(0.51) in borate buffer was slightly different from that of trisaccharide A (0.59). A portion of this

fractions (450 mg) was separated on cellulose column

(400•~30mm diam.) with a mixture of isopropanol: butanol: water (7:1:3 by vol.) to give chromato

graphically pure trisaccharide C (141 mg) having

[ƒ¿]_??_=+133•‹ (c=0.7, water) and trisaccharide D (138mg) having [ƒ¿]_??_=+159•‹ (c=1.2, water). At tempts to crystallize either the free sugars or the

acetates of these trisaccharides were not successful. The yields and properties of these trisaccharides

(A-D) are shown in Table I.

Characterization of the trisaccharides by paper chro matography and electrophoresis. The molecular size of the trisaccharides was ascertained by paper electro FIG. 1. Paper Chromatographic Mobilities of Oligo- phoresis in sodium bisulfite.11) These trisaccharides saccharides a'=Rf/l-Rf. Acetolysis of Polysaccharides. Part V. 107

Ms 0.60, indicating the presence of 1,6-linkage at dimethyl sulfoxide (3ml) was mixed with methyl the reducing end-group. Trisaccharide A, C and D sulfinyl carbanion solution, which was prepared by alcohols were immobile, indicating the presence of dissolving sodium hydride (200mg) in dimethyl

1,3-linkage at the reducing end-group. sulfoxide (5ml). The mixture was stirred under Further structural evidence was obtained from the nitrogen stream for one hour at room temperature, relation between log al and molecular size, where ƒ¿' then 1ml of methyl iodide was added and further was defined as Rf/1-Rf.18) As shown in Fig. 1, stirred for one and half hours. The reaction mixture plot of ƒ¿' of glucose, isomaltose and isomaltotriose was diluted with water and extracted with chloroform. against DP is linear (line I). This indicates that The extract was washed with water, dried over an the line I represents ƒ¿-1,6 homologous series. Simi hydrous sodium sulfate, and evaporated to dryness larly, plot of ƒ¿' of glucose, nigerose and trisaccharide (yield 20•`30mg). D against DP is also linear (line II). This suggests Methanolysis of the methylated sugars was per that trisaccharide D is composed merely of ƒ¿-1,3- formed by heating, in a sealed tube, with 2.5% glucosidic linkage. Trisaccharides A, B and C have methanolic hydrogen chloride in a boiling water bath the same Rf value and ƒ¿' of these trisaccharides for one hour. The resulting solution was evaporated are plotted, against DP, on the intersecting point of to dryness after removal of hydrogen chloride with line III and line IV which are parallel to line I and anion exchanger IR-4B (carbonate form). line II, respectively. This suggests that these three trisaccharides are composed of ƒ¿-1,6 and aƒ¿-1,3- Gas liquid chromatography. Gas liquid chromato glucosidic linkages. graphy was performed on a Hitachi-Perkin Elmer Model F 6 gas chromatography apparatus fitted with Characterization by acid hydrolysis. Each trisac a flame ionization detector under isothermal condition charide was completely hydrolyzed as described (205•‹). Two columns of stainless steel tube (2 m x 3 before. Paper chromatography of the deionized mm diam.) were used: column A was packed with hydrolyzate showed, in each case, a single component 5% Ucon LB 550X on Gaschrome CLH and was identical with glucose. operated at gas flow rate of 15.7 ml nitrogen/min.; Partial hydrolyzates of trisaccharide A, B and C and column B was packed with 5% diethylene glycol were shown by paper chromatography to contain the succinate polyester on Celite 545 (50•`100 mesh), and components corresponding to glucose, isomaltose and was operated at gas flow rate of 26 ml nitrogen/min. nigerose in addition to the mother compounds. Paper The trisaccharides were permethylated as described chromatography of the partial hydrolyzate of trisac before and, after subsequent methanolysis, submitted charide D showed glucose and nigerose in addition to to gas chromatography. As the reference compounds, the mother compound. methyl tri-O-methyl-D-glucopyranosides were prepared Partial hydrolysis of the corresponding sugar alcohols by methylating the glucobioses of known structures of the trisaccharides gave rise to glucose and iso followed by subsequent methanolysis. The retention maltose from trisaccharide A, glucose and nigerose volumes of the tri-O-methyl-glucosides relative to from trisaccharides B and D and only glucose from methyl 2,3,4,6-tetra-O-methyl-a-D-glucopyranoside are trisaccharide C as the components which were detected shown in Table 11. by paper chromatography using aniline hydrogen Methanolyzates of the methylated trisaccharides A phthalate as a spray reagent. and B gave peaks representing almost equal amount

of methyl 2,3,4- and 2,4,6-tri-O-methyl glucopyrano Gas liquid chromatography of the methanolyzates of the sides in addition to methyl 2,3,4,6-tetra-O-methyl-D- methylated trisaccharides glucopyranosides. Methanolyzate of the methylated trisaccharide D gave peaks representing only methyl Permethylation and methanolysis. Permethylation of 2,4,6-tri-O-methyl-D-glucopyranosides in addition to the trisaccharides essentially followed the method of methyl 2, 3, 4, 6-tetra-O-methyl-D-glucopyranosides. Hakomori.19) A solution of sugars (30•`40mg) in Methanolysis of the methylated trisaccharide C gave no peak representing tri-O-methyl-D-glucosides, but 18) D. French and G. M. Wild, J. Am. Chem. Soc., 75,, 2612 (1953). two peaks with retention volumes 2.38 and 3.14 on 19) S. Hakomori, J. Biochem. Japan, 55, 205 (1964). column A and 5.70 and 8.20 on column B in addition 108 F. YAMAUCHI and K. MATSUDA

TABLE II. RELATIVE RETENTION VOLUMES OF METHYLSUGARS ON GAS LIQUID CHROMATOGRAPHY

* Retention time , 7.5 min. + Retention time , 3.0 min.

to the peaks representing methyl 2,3,4,6-tetra-O-methyl- lation of this sugar from dextran suggested D-glucopyranosides. These peaks were assigned, that ƒ¿-1,3-glucosidic linkages occur not only though not conclusively, to methyl 2,4-di-O-methyl-D- at the branch points but at least some of them glucopyranosides. are contiguous in the dextran molecule. Trisaccharides A, B and C had the same RESULTS AND DISCUSSION RG (0.50) on paper chromatography and seemed to be made up of ƒ¿-1,6 and ƒ¿-1,3 linkages On repeated fractionation by charcoal: from their ƒ¿' value. Partial hydrolyzates of Celite column chromatography the acetolyzate these trisaccharides were also shown to con of a dextran from Leuconostoc mesenteroides B tain, by paper chromatography, both iso yielded five trisaccharides. Among these maltose and nigerose in addition to glucose trisaccharides, isomaltotriose was characterized and mother compounds. by its mobility on paper chromatography and During electrophoresis in molybdate buffer, electrophoresis and [ƒ¿]D value. trisaccharide B alcohol migrated with the Trisaccharide D seemed to contain only ƒ¿- same MG as isomaltotriitol (0.60) indicating 1,3-glucosidic linkage, since mobility of this the presence of 1,6-linkage at the reducing trisaccharide on paper chromatography (RG end-group. Partial hydrolysis of the reduction 0.69) was higher than those of other sugars and the plot of log ƒ¿' of glucose, nigerose product of trisaccharide B yielded nigerose as a reducing disaccharide. These results indicated and this sugar against molecular size (DP) that trisaccharide B has the structure O-ƒ¿- was linear. This was further supported by D-glucopyranosyl-(1•¨ 3)-O-ƒ¿-D-glucopyranosyl- the results that nigerose was a sole disac charide detected in the partial hydrolyzate of (1•¨6)-O-ƒ¿-D-glucose (or 6-ƒ¿-nigerosylglucose). The reduction products of trisaccharides A trisaccharide D and its corresponding sugar and C were both immobile during electro alcohol. This was the first isolation and characterization of nigerotriose (O-ƒ¿-D-gluco phoresis in molybdate buffer. However, on partial hydrolysis the former gave glucose pyranosyl-(1•¨3)-O-ƒ¿-D-glucopyranosyl-(1•¨ and isomaltose as the reducing products detec 3)-D-glucose, or 3-ƒ¿-glucosylnigerose), although the table by paper chromatography presence of this sugar had been suggested in , whereas the only reducing product so identified in the the partial hydrolyzate of isolichenan.20) Iso partial hydrolyzate of the latter was glucose. 20) S. Peat, W.J. Whelan, J. R. Turvey and K . On this evidence, the trisaccharides A and,C Morgan, J. Chem. Soc., 1961, 623. were assigned to the structures of O-ƒ¿-D-gluco- Acetolysis of Polysaccharides. Part V. 109

pyranosyl-(1•¨6)-O-ƒ¿-n-glucopyranosyl-(1-A)- consist of single glucose unit. O-ƒ¿-D-glucose (or 3-ƒ¿-isomaltosylglucose) and O- The structures of these trisaccharides were ƒ¿ -D-glucopyranosyl-(1•¨3)-[O-ƒ¿-D-glucopyrano- also confirmed by the gas chromatographic syl-(1•¨6)]-O-ƒ¿-D-glucose (or 3,6-di-ƒ¿-glucosyl- analysis of the methanolysis products of the glucose), respectively. The former (trisac methylated sugars. charide A) was conclusively characterized by It is interesting that a small amount of

mixed mp of its acetyl derivative with the laminaribiose was obtained from the aceto

known specimen of 3-ƒ¿-isomaltosylglucose lyzate of this dextran, although occurrence

undecaacetate. of this disaccharide in the acetolyzate of

Isolation of the branched trisaccharide another dextran had been already mentioned

(trisaccharide C) gave a direct evidence for by Goldstein et al.3) It is not still certain the branched structure of the original dextran. whether this anomalous linkage was derived

Isolation of trisaccharide A, which has been from the original structure of the dextran or,

already obtained from another dextran,31 in as speculated by Goldstein et al., produced

dicated that at least more than one glucose by the conversion of ƒ¿-1,3-glucosidic linkage

unit were joined through a 1,3-linkage to a during acetolysis.

glucose unit of 1,6-linked main chain or some of the 1,3-linkages occurred in the linear Acknowledgement. The authors wish to thank Dr. part of the dextran molecule. This structure Akio Sato for his kind gift of 3-ƒ¿-isomaltosylglucose represents marked contrast with that of undecaacetate.

another dextran5,6,21) in which the branches

21) R W. Jones, R.J. Dimler, A. Jeanes, C. A. Wilham and C. E. Rist, Abstracts Papers Am. Chem. Soc., 126, 13D (1954).