Deoxy-D-Glucose, by Transglucosylation of Schizosaccharomyces Pombe A-Glucosidase

Agric. Biol. Chem., 44 (3) , 549•`554, 1980 549

Enzymatic Synthesis of a New Disaccharide , 6-O-a-Glucopyranosyl-2- deoxy-D-glucose, by Transglucosylation of Schizosaccharomyces pombe a-Glucosidase

Seiya CHIBAand Osamu YAMANA*

Departmentof AgriculturalChemistry , Faculty of Agriculture, HokkaidoUniversity, Sapporo, Japan ReceivedJuly 27, 1979

A new a-D-glucosyl-2-deoxy-D-glucose was enzymatically synthesized by the transglucosylation of Schizosaccharornyces pombe a-glucosidase under the co-existence of maltose and 2-

deoxy-D-glucose. The disaccharide was chromatographically isolated in a semicrystalline

powder (hemihydrate). It was concluded that the sugar was 6-O-a-D-glucopyranosyl-2-deoxy-

D-glucose ([a•nD22+108•‹, in water), which was the main product formed in this transglucosylation .

In the previous paper,1) we reported on two deoxy-D-glucose. kinds of new disaccharides, 3- and 4-a-D- glucosyl-2-deoxy-D-glucose, enzymatically synthesized by the transglucosylation of buck- MATERIALS AND METHODS wheat a-glucosidase. However, 6-a-D- Materials. Maltose (SP grade) and 2-deoxy-D- glucosyl-2-deoxy-D-glucose was not formed in glucose were purchased from Nakarai Chemical Co., the reaction system with buckwheat a-gluco- Ltd. and Sigma Chemical Co., Ltd., respectively. a- sidase which was an enzyme synthesizing D-Glucosyl-(1-.3)-or (1•¨4)-2-deoxy-D-glucosel) were mainly glucodisaccharides having a-1, 2-, a- enzymatically synthesized by the transglucosylation of buckwheat a-glucosidase. 1, 3- or a-1, 4-glucosidic linkages.2) From Schizosaccharomyces pombe a-glucosidase was pre- the action pattern of transglucosylation, the pared according to the method described in the enzyme such as Schizosaccharormyces pombe previous paper.3) One unit of enzyme was expressed a-glucosidase3,4) capable of producing pre- as the amount of enzyme which hydrolyzed 1 ƒÊmol of ferentially a-1, 6-glucosidic linkage seems to maltose per min at 0.4% maltose, 37•Ž and pH 5.0 be more suitable for the synthesis of 6-0- (M/25 sodium acetate buffer). substituted 2-deoxy-D-glucose. Color reaction of 2-deoxy sugars.5) A mixture of When 2-deoxy-D-glucose acts as an acceptor 3.5 ml of a solution containing 5 ƒÊg of 2-deoxy-D- in the transglucosylation of a-glucosidase, it is glucose or each 10 ƒÊg of disaccharides and 0.5 ml of a assumed that the formation of glucosidic periodic acid solution (4.8 mg of HIO4 per ml of 0.125N linkage may be performed through C3, C4 or H2S04) was allowed to stand for 40 min at room temperature, and then 1 ml of 2 % sodium arsenite solution C6 hydroxyl group of 2-deoxy-D-glucose, be- in 0.5 N HCI was added to terminate the oxidation. cause this deoxysugar in free state is con- To 1 ml of the reaction mixture was added 2 ml of a sidered to exist in a pyranose form. Thus, 2-thiobarbituric acid solution (7.1 mg of 2-thiobarbi- the formation of 5-0-substituted 2-deoxy-D- turic acid per ml of 0.007 N NaOH). The mixture was heated for 20 min in boiling water, cooled and optical glucose is unlikely. density was measured at 532 nm. The present paper is concerned with the structure of a new a-D-glucosyl-2-deoxy-D- Periodate oxidation. Four hundred ƒÊl of 20mm glucose synthesized by the transglucosylation sodium metaperiodate, 100 ƒÊ1 of carbohydrate solu- of an a-glucosidase. This disaccharide was tion, in which 0.3•`0.7mg of each carbohydrate was contained, and 500 ƒÊl of 0.1 M sodium acetate buffer concluded to be 6-O-a-D-glucopyranosyl-2- (pH 5.3) were allowed to react in an amber tube at * Present address: Meiji Milk Products Co., Ltd. 0•`2•Ž or 15•Ž. At time intervals chosen a 50 ƒÊl 550 S. CHIBA and 0. YAMANA

aliquot of the reaction mixture was pipetted out, and used as the carrier gas at a flow rate of 30 ml/min.

the course of the consumption of periodate was Column temperature was 250•Ž. estimated up to 24 hr according to the colorimetric method described by Avigade6) using 2, 4, 6-tripyridyl- RESULTS AND DISCUSSION s-triazine reagent.

Isolation of the transglucosylation product Trimethylsilvlation. The preparation of trimethyl- silyl (TMS) derivatives was performed by the method Five hundred ml of a reaction mixture con-

of Sweeley et al.7) taining 25g of maltose, 25g of 2-deoxy-D-

Enzyme hydrolysis. Two ml of the mixture con- glucose, 100ml of M/10 sodium acetate buffer taining 5 mg of the isolated disaccharide, 30 units of (pH 5.0) and 120ml of the enzyme solution

buckwheat a-glucosidase and 0.5 ml of M/10 sodium (100 units) was incubated at 37•‹C for 8 hr. acetate buffer (pH 5.0) was incubated at 37•‹C. Then, the mixture was heated in boiling water

to terminate the reaction and concentrated to Thin layer and paper chromatography. Paper chro-

matography (PPC) was carried out by multiple ascend- 120 ml in vacuo.

ing technique (three times) on Toyo Roshi No. 50 The concentrated solution was subjected to filter paper. The solvent system was n-butanol, chromatography of a carbon-Celite (2: 1, by pyridine and water (6:4:3 by volume). The sugars weight) column (5.5•~44cm). The elution were detected with anisidine-phthalate reagent.8) The sugar extracted from filter paper was determined by pattern is shown in Fig. 1. After monosac- charides were washed out from the column phenol-sulfuric acid method.9) Thin layer chromatography (TLC) was carried out with water (fraction no. 1•`25), and then by multiple ascending technique (double) on Kieselgel elution was carried out stepwise with 2.5%, G chromatoplate. The solvent system was n-butanol, 5%, 7.5%, 10% and 25•“ ethanol, succes- isopropanol and water (10: 5: 4, by volume). Anisal- sively.. dehyde reagent10) was used for detection of sugars. The effluent with 2.5 ethanol (fraction

Gas liquid chromatography. Gas liquid chroma- no. 40•`70) was concentrated to a syrup under toraphy (GLC) was performed on a Hitachi 063 gas reduced pressure. The syrup contained malt- liquid chromatograph equipped with a flame-ioniza- ose, a small amount of isomaltose and an tion detector. A stainless steel column (0.3•~200 cm) a-D-glucosyl-2-deoxy-D-glucose which was ab- packed with 3 % Silicone SE-30 on Chromosorb WAW- DMCS (60•`80 mesh) was employed. Nitrogen was breviated as •gGDG". As Fig. I shows, a

Fraction No. (200ml/fraction)

FIG. 1. Elution Pattern of Reaction Products from a Carbon-Celite Column.

Sample, 120 ml; carbon-Celite (2: 1) column, 5.5•~44 cm; flow rate, 3.5 ml/min. The sugar

content in each fraction was determined by the phenol-sulfuric acid method.0) Enzymatic Synthesis of 6-a-D-Glucosyl-2-deoxy-D-glucose 551

small amount of sugar was also eluted in other

ethanol fraction: which included 3- and 4-a-D-

glucosyl-2-deoxy-D-glucose, maltose, isomaltose, panose and an unidentified trisaccharide

consisting of D-glucose and 2-deoxy-D-glucose . Subsequently, the syrup of 2 .5% ethanol

fraction was developed on Toyo Roshi No . 50 filter paper (40 x 40cm) by multiple ascending

technique (three times). The zone correspond-

ing to the spot of GDG was cut out from the

chromatogram. From the zone strips , GDG was extracted with hot water and then the

aqeous extract was evaporated to a syrup . The isolated GDG gave single spot on paper

and thin layer chromatogram. GDG was separated in semicrystalline powder from the

syrup with ethanol. The yield was ca . 1g. FIG. 2. Paper Chromatogram of GDG and its Hy- drolyzate by an a-Glucosidase.

Characteristics of GDG S, standard; H, hydrolyzate. GDG was a hygroscopic reducing sugar

which reduced Fehling's reagent. However, was chromatographed, the sugars extracted

GDG was negative to triphenyltetrazolium from filter paper were determined by phenol- chloride (TTC) reagent11) reacting specifically sulfuric acid method.9 ) with a-keto alcohol group (-CO-CHOH-) of The sugar alcohol 12) derived from GDG sugar to give a red triphenylformazan com- gave only D-glucose as reducing sugar on hy- pound. This suggested that the sugar was drolysis by acid (a few mg of sugar alcohol, 2-O-substituted-D-glucose, or the reducing with 1 N H2SO4 for 2 hr at 100•‹C). Accord- moiety was 2-deoxy-D-glucose . The color ingly, the reducing moiety of GDG should be with the detecting reagents and Rg values on 2-deoxy-D-glucose. paper and thin layer chromatogram are pre- GDG showed a specific rotation of [a]22D+ sented in Table I. 108•‹ (c=0.5, in water). Anal. Found: C, GDG was found to possess an a-glucosyl 43.25;H,7.12. Calcd. for C,_H22010.1/2H20: configuration, because of being completely C, 42.99; H, 6.87%.. This result indicates hydrolyzed to D-glucose and 2-deoxy-D-glucose that the semicrystalline preparation is hemi-

(1: 1) by buckwheat a-glucosidase13) (5 mg of hydrate, like the crystals of 3- and 4-a-D-

GDG, 30 units of enzyme, for 5 hr at 37•‹C) glucosyl-2-deoxy-D-glucose. Acetylation of as shown in Fig. 2. After the hydrolyzate GDG was tried, but the product was not

TABLE I Rg VALUES AND COLOR DEVELOPMENTS OF GDG AND RELATED CARBOHYDRATES 552 S. CHIBA and O. YAMANA

separated in a crystalline form. ment of GDG was nearly the same as that of free 2-deoxy-D-glucose, and 3- or 4-a-D- Color test of malonaldehyde-thiobarbituric acid glucosyl-2-deoxy-D-glucose showed no colora- reaction tion. It has been generally known that the method of Waravdekar and Saslaw is highly sensitive Gas liquid chromatography of TMS-derivatives for the estimation of 2-deoxy sugar, as shown from GDG and other related sugars in Scheme 1, which is based on the results of TMS-derivatives from glucitol, GDG and periodate oxidation and color reaction of the 3- or 4-a-D-glucosyl-2-deoxy-D-glucose were ensuing malonaldehyde with 2-thiobarbituric applied to GLC. As Fig. 3 shows, TMS- acid. The method is utilizable for the ex- derivatives from GDG gave the pattern amination of the position of glucosidic linkage distinct from the patterns for TMS-derivatives in 0-substituted 2-deoxy-D-glucose. As can be seen from Scheme 1, if the C3 or C4 hydroxyl group of 2-deoxy-D-glucose takes part in bond formation, malonaldehyde will not be derived, in contrast with the case of 6-0-substituted 2-deoxy-D-glucose. Therefore, the 0-substituted 2-deoxy-D-glucose having a-1, 3- or a-1, 4-glucosidic linkage can be distinguished from that having a-1, 5- or a-1, 6-glucosidic linkage.

SCHEME 1. A Colorimetric Method of Estimation of 2-Deoxy-sugars.

TABLE 11. OPTICAL DENSITY OF MALONALDEHYDE- THIOBARBITURIC ACID REACTION WITH GDG AND RELATED CARBOHYDRATES

FIG. 3. Gas-liquid Chromatogram of TMS-derivatives from GDG and Related Carbohydrates. G-H2, D-glucitol: 3GDG, 3-a-D-glucosyl-2-deoxy-D- The data of colorimetric measurements are glucose; 4GDG, 4-a-D-glucosyl-2-deoxy-D-glucose; presented in Table II. The color develop- Mix, mixture of GDG and other sugars. Enzymatic Synthesis of 6-a-D-Guucosyl-2-deoxy-D-glucose 553 from the authentic 3- and 4-0-substituted 2- deoxy-D-glucose, although 5-0-substituted 2- deoxy-D-glucose was not tested because there was no sample. Since it seems unlikely that

5-0-substituted 2-deoxy-D-glucose is formed where the oxidative degradation probably through the transglucosylation as stated above, occur in the same process as in the case of the results on the color test in the preceding malonic acid,14) consuming 3 mol of the oxi- section and GLC suggest that the C6 hydroxyl dant per 1 mol of malonaldehyde. Conse- group of 2-deoxy-D-glucose would be involved quently, 1 mol of 6-a-D-glucosyl-2-deoxy-D- in a-glucosidic linkage. glucose consumes 7 mol of periodate. The same overoxidation is expected to occur in the

Oxidative degradation with periodate oxidation of 2-deoxy-D-glucose, resulting in

The application of this method is considered the consumption of 6 mol of oxidant per to be useful for the discrimination between TABLE ITI. SODIUMMETAPERIODATE 3-, 4- or 5-0-substituted 2-deoxy-D-glucose CONSUMPTIONBY GDG AND from 6-0-substituted 2-deoxy-D-glucose. When RELATEDCARBOHYDRATES 1 mol of 3-, 4- or 5-a-D-glucosyl-2-deoxy-D- glucose is oxidized by periodate under the conditions mentioned above, 2 mol of the oxidant may be consumed, because the bonds between C2, C3 and C4 in nonreducing end, glucosyl moiety only, are selectively cleaved. On the other hand, 6-a-D-glucosyl-2-deoxy-D- glucose is oxidatively degraded according to the procedures in Scheme 2, consuming 4 mol of the oxidant per 1 mol of sugar. However, malonaldehyde formed from reducing end may be also oxidized at 15•‹C by accompaning with a Overoxidation, accompanied by the oxidation of the oxidation of active methylene group in malonaldehyde. the way: b As6-a-D-glucosyl-2-deoxy-D-glucose.

SCHEME 2. Oxidative Degradation of 6-a-D-Glucopyranosyl-2-deoxy-D-glucose with Sodium Metaperiodate. 554 S. CHIBA and 0. YAMANA

1 mol of sugar. Such a reaction of malonal- 2) M. Takahashi, T. Shimomura and S. Chiba, ibid., dehyde, overoxidation, would be retarded under 33, 1399 (1969); S. Chiba and T. Shimomura, Denpun Kagaku, 26, 59 (1979). appropriate condition, e. g., at retatively low 3) S. Chiba and T. Shimomura, Agric. Biol. Chem., temperature. 29, 540 (1965).

As represented in Table III, 1 mol of GDG 4) S. Chiba and T. Shimomura, ibid., 30, 536 (1966). consumed about 4 mol of periodate at 0•`2•Ž 5) V. S. Waravdekar and L. D. Saslaw, J. Biol. and 7 mol at 15•‹C. These experimental Chem., 234, 1945 (1959). 6) G. Avigad, Carbohyd. Res., 11, 119 (1969). values agree with the theoretical values, and 7) C. C. Sweeley, R. Bentley, M. Makita and W. W. GDG will be distinguished from 3-, 4- or 5-a- Wells, J. Am. Chem. Soc., 85, 2497 (1963).

D-glucosyl-2-deoxy-D-glucose. 8) A. Schweiger, J. Chromatog., 9, 374 (1962).

On the basis of the experimental results 9) M. Dubois, R. Gills, J. K. Hamilton, P. A. described above, it seemed most reasonable Rebers and F. Smith, Anal. Chem., 28, 350 (1959). to conclude that the structure of GDG was 10) E. Stab, •gDunnschicht-Chromatographie," Springer-Verlag, 1962, p. 514. 6-O-a-D-glucospyrano syl-2-deosy-D-glucose. 11) K. Wallenfels, Naturwissenschaften, 37, 491

Acknowledgment. We wish to thank Emeritus Pro- (1950). fessor T. Shimomura, Hokkaido University, for his 12) M. Abdel-Akher, J. K. Hamilton and F. Smith, encouragement and discussion. J. Am. Chem. Soc., 73, 4691 (1951).

13) K. Kanaya, S. Chiba, T. Shimomura and K.

REFERENCES Nishi, Agric. Biol. Chem., 40, 1929 (1976). 14) J. R. Dyer,•gMethods of Biochemical Analysis,"

1) S. Chiba T. Shimomura and K. Hatakeyama, Vol. III, ed. by D. Glick, Interscience Publishers Agric. Biol. Chem., 39, 591 (1975). Inc., New York and London, 1956, p. 111.