J. Biochem. 82, 1673-1680 (1977)

GLC Analysis of the Hydroxy and Oxo Compounds Produced by the Smith Degradation of Reducing Di and

Haruki YAMAGUCHI and Takafumi MUKUMOTO Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591

Received for publication, June 18, 1977

Reducing di and trisaccharides were oxidized with periodate under conditions minimizing overoxidation, and the hydroxy and oxo compounds produced by subsequent reduction and were analyzed by GLC after trimethylsilylation. The present study has demon strated that GLC analysis of the hydroxy and oxo compounds produced by Smith degradation is useful for linkage analysis of reducing di and trisaccharides. In addition, it is suggested that the rate of periodate oxidation of the internal hexopyranose residue of trisaccharides depends markedly upon their structures.

Periodate oxidation, followed by borohydride re of the intermediate formyl esters, leading to ad duction and mild hydrolysis with acid (Smith ditional periodate consumption, so-called overoxi degradation), has been generally used for structural dation. It seems likely, however, that the inter- studies of and complex carbohy mediate esters are relatively stable in weak acid drates. The determination of hydroxy and oxo (6). Based on these suggestions, therefore, it is compounds produced by Smith degradation is expected that each of the intermediate products, expected to provide useful information on the upon subsequent reduction followed by hydrolysis structures of . with acid, should give a characteristic set of hy Previously, we have attempted to determine droxy and oxo compounds depending on the link the hydroxy and oxo compounds resulting from ing position of the original , as illus the Smith degradation of non-reducing disac trated for glucobioses with different linking charides by GLC (1, 2), and also applied the positions in Table I. Further, reducing trisac technique to a glycopeptide (3) and polysaccharides charides subjected to Smith degradation might also (4, 5) to elucidate their fine structures. However, give hydroxy and oxo compounds which are GLC analysis of the hydroxy and oxo compounds different in types and/or molar ratios depending on produced by Smith degradation of reducing di and the original linking positions. The study reported trisaccharides has never been attempted. here was undertaken to determine the hydroxy and It is known that aldohexoses and reducing oxo compounds resulting from the Smith degrada end-aldohexose residues linked at positions other tion of reducing di and trisaccharides. It has than C-2 are oxidized by periodate in their pyra now been shown that GLC determination of these nose forms to give formyl esters (6-9). Alkaline compounds is useful for linkage analysis of re or strongly acidic conditions cause rapid hydrolysis ducing di and trisaccharides.

Vol. 82, No. 6, 1977 1673 1674 H. YAMAGUCHI and T. MUKUMOTO

TABLE I. Expected values of hydroxy and oxo compounds produced from D-glucopyranosyl-D-glucopyranoses by normal Malaprade oxidation followed by borohydride reduction and subsequent hydrolysis.

Smith degradation of reducing

EXPERIMENTAL and GLC analysis of the products were carried out substantially as reported previously (2), but

Materials-All chemicals were of reagent with some improvements. A reducing oligosac

grade. and were purchased from charide (ca. 0.15 ƒÊmol) was dissolved in 0.2 ml

Wako Pure Chem. Co. , melibiose, (0.1 ml for (1•¨4)-ƒÀ-n-hexotrioses) of 0.5 M acetate gentiobiose, and were obtained from buffer (pH 3.2) containing 2.14 mg of sodium Sigma Chem. Co. 4-O-ƒÀ-D-Mannopyranosyl-D- metaperiodate and the solution was kept at 30•Ž

mannopyranose and O-ƒÀ-D-mannopyranosyl-(1•¨ for a certain period (up to 12 h). The reaction

4)- ƒÀ-D-mannopyranosyl-(1•¨4)-D-mannopyranose mixture was subsequently neutralized by the ad

were supplied by Dr. T. Mizuno of Shizuoka Uni dition of 0.1 ml of 1 N sodium hydroxide (0.5 N

versity and kojibiose was a gift of Dr. S. Chiba of sodium hydroxide for the reaction mixtures of

Hokkaido University. Isomaltotriose and panose (1•¨4)-ƒÀ-D-hexotrioses) and immediately treated were gifts from Dr. M. Takagi of this University. with 0.1 ml of 0.01 N sodium hydroxide containing

Laminaribiose and O-ƒÀ-D-galactopyranosyl-(1•¨6)- 2.5 mg of sodium borohydride. The additions of

ƒ¿ -D- galactopyranosyl-(1•¨6)-D- galactopyranose the aqueous sodium hydroxide and the sodium

were prepared by partial acid hydrolysis of a (1 borohydride solution were carried out with external

•¨ 3)-ƒÀ-D- (curdran, (10)) and a heterogalactan cooling in an ice-water bath. The slightly alka

(5), respectively, and purified by repeated gel-filtra line solution was allowed to stand overnight at tions through a column of Bio-gel P-2 (100-200 4-6•Ž. The excess sodium borohydride was de-

mesh). and cellotriose were also ob composed by addition of 0.1 ml of 1 N acetic acid tained by partial acid hydrolysis of and and the resulting acidic solution was evaporated

purified as described above. 3-O-ƒ¿-D-Mannopyra to a syrup. The residue was dissolved in 0.3 ml nosyl-D-mannopyranose was prepared by partial of methanol and allowed to stand at 50•Ž for

acetolysis of a glycopeptide obtained from Taka 15 min. The alcoholic solution was evaporated to

A (11). A strongly acidic cation-exchange dryness under reduced pressure, and the residue

resin (Dowex 50•~8, 200-400 mesh) was reactivated was subjected to two further evaporations from

with 2 N hydrochloric acid immediately before use. methanol. The residue was dissolved in 0.2 ml

A weakly basic anion-exchange resin (Amberlite of water and immediately passed through ion

IR-4B) which had been stored in the chloride form exchange resin. To desalt the reaction mixture, a

after activation, was treated successively with 2 N glass column (5.0•~1.0 cm) was packed with ammonium hydroxide and water just before use. Amberlite IR-4B (OH, 0.3 ml), then Dowex

General Methods-All evaporations were con 50x8 (H+, 0.3 ml) was layered over the basic

ducted under reduced pressure at a bath temper resin. After applying the sample solution to the

ature not exceeding 30•Ž. The consumption of column, the resin was quickly washed twice with

periodate was estimated by measuring the ab 0.1 ml volumes of water and then with 1.6 ml of sorbance at 290 nm (12). Oligosaccharides were water. The effluent was collected in a glass tube

determined by the orcinol-sulfuric acid reaction (5.0•~1.4 cm) with a Teflon-lined screw cap. The

(13). desalting procedure was completed within about

J. Biochem. SMITH DEGRADATION PRODUCTS OF REDUCING OLIGOSACCHARIDES 1675

5 min. To the neutral effluent was added 0.2 ml

of 0.175 N hydrochloric acid containing 5 mg of

hydroxylamine hydrochloride. The glass tube was

tightly stoppered and maintained at 80•Ž for

90 min. Then the reaction mixture was evaporated

to dryness under reduced pressure and the residue

was subjected to repeated evaporations from

methanol (3 times). The residue was dissolved in

50ƒÊ1 of anhydrous pyridine containing 25 ƒÊg of

2-ethyl-2-hydroxymethyl-1, 3-propanediol as an in

ternal standard and treated with 25 pl of hexa methyldisilazane and 15 pl of trimethylchlorosilane

for trimethylsilylation. The reaction mixture was Fig. 1. Production of hydroxy and oxo compounds centrifuged and an aliquot (ca. 1 ƒÊ1) of the super- and consumption of periodate in the Smith degradation natant was injected into a gas chromatograph. of maltose. Analyses of the degradation products were

GLC was performed with a Shimadzu 4APF gas performed at intervals during periodate oxidation. chromatograph equipped with a hydrogen-flame Reaction conditions were as described in "EXPERI-

ionization detector. A steel column (300•~0.3 cm MENTAL." •ü, Glycerol; •œ, meso-erythritol; ?,

i.d.) was used, packed with 5% SE-30 on Chro ; ?, ;•~, periodate.

mosorb W (60-80 mesh), and the column temper

ature was programmed from 100•Ž to 190•Ž at a C-1, C-2, and C-3 of the non-reducing end

rate of 4•Ž increment per min. unit. The course of this oxidation reaction may be interpreted by conformational analysis. Figure I also shows that the intermediate formyl ester RESULTS AND DISCUSSION derived from maltose was essentially stable at From previous findings (6-9), it is clear that the pH 3.2, and little overoxidation occurred over a pH at which periodate oxidation is performed period of 7 h. significantly affects both the rate of oxidation and 4-0- ƒÀ-D-Glucopyranosyl-D-glucopyranose the kind of oxidation products formed. We there- (cellobiose), 4-0- ƒÀ-D-galactopyranosyl-D-gluco

fore checked carefully the effect of pH on the peri (lactose), and 4-0-ƒÀ-D-mannopyranosyl- odate oxidation of some reducing glucobioses and D-mannopyranose were subjected to periodate glucotrioses in acetate buffer at various pH's. oxidation, and the hydroxy and oxo compounds

Our experiments clearly indicated that overoxi produced by subsequent reduction followed by dation has a minimum in the pH 3 to 3.4 region, hydrolysis were analyzed as performed on maltose. in agreement with the results previously reported Cellobiose gave glycolaldehyde more rapidly, and for maltose (6) and cellobiose (8). Therefore, the therefore a smaller amount of glyceraldehyde, than acetate buffer was adjusted to pH 3.2 for periodate maltose, whereas erythritol production was a little

oxidation throughout the present study. slower. Clear-cut Malaprade oxidation was ob- GLC determination of the hydroxy and oxo served for lactose. Only the initial 1 h of peri compounds resulting from the Smith degradation odate oxidation led to the production of gly of maltose was carried out with simultaneous ceraldehyde from lactose. The progress of estimation of the periodate consumption (Fig. 1). oxidation of 4-0-ƒÀ-D-mannopyranosyl-D-manno

The production of glycolaldehyde increased gradu pyranose was similar to that of cellobiose, though ally and approached I mol/mol, accompanied by a the mannopyranose residue has a cis-glycol group decrease of glyceraldehyde. On the other hand, at C-2 and C-3. The analytical values obtained hydroxy compounds were rapidly produced. for these are summarized in Table II These results show that the oxidation of the non- together with those for maltose. reducing end glucose unit proceeded by primary The intermediate formyl ester from lami attack between the hydroxyl groups at C-3 and naribiose was not as stable as that from maltose. C-4, because glyceraldehyde should be derived from As shown in Fig. 2, slow overoxidation was ob-

Vol. 82, No. 6, 1977 1676 H. YAMAGUCHI and T. MUKUMOTO

TABLE II. Hydroxy and oxo compounds obtained by the Smith degradation of reducing di and trisaccharides.

Disaccharides were oxidized with 0.05 M Na104 at 30•Ž for 5 h, (1•¨f)-ƒÀ-D-hexotrioses with 0.1 M Na104 at 35•Ž for 10 h, and other trisaccharides with 0.05 M NaIO4 at 30•Ž for 7 h. The subsequent analytical procedures were as described in "EXPERIMENTAL." The values are expressed as molar ratios of the products to the nearest integral values of glycerol, calculated on a molar basis.

a 4-O-ƒ¿-D-Glucopyranosyl-D-glucopyranose, etc.

served, resulting in a relatively low yield of ara binitol. However, the yield was still in approxi

mate agreement with expectation, based on the

normal Malaprade oxidation of laminaribiose. The

periodate oxidation of 3-O-ƒ¿-D-mannopyranosyl- D-mannopyranose progressed rapidly at the re

ducing-end mannopyranose residue with the pro duction of , whereas the non-reducing

end mannopyranose was more slowly oxidized

than in the case of laminaribiose.

As shown in Fig. 3, 1 mol of isomaltose

reduced about 6 mol of periodate and gave I mol

each of glycerol and glyceraldehyde. These results Fig. 2. Production of hydroxy and oxo compounds suggest that the formic acid ester produced from and consumption of periodate in the Smith degradation isomaltose is unstable and hydrolyzed rapidly. of laminaribiose. Analyses of the degradation products were performed at intervals during periodate oxidation. After the hydrolytic cleavage of the ester linkage, Reaction conditions were as described in "EXPERI the sixth mole of oxidant appears to attack the MENTAL." •ü, Glycerol; •œ, D-arabinitol; ?, glycol- newly appearing ƒ¿-hydroxylcarbonyl groups, con

aldehyde; ?, glyceraldehyde; •~, periodate. verting the glyceraldehyde group into a glycol-

aldehyde residue. This suggestion was supported

by the observation that 1,2-ethanediol, though it

J. Biochem. SMITH DEGRADATION PRODUCTS OF REDUCING OLIGOSACCHARIDES 1677

Fig. 3. Production of hydroxy and oxo compounds and consumption of periodate in the Smith degradation Fig. 4. Production of hydroxy and oxo compounds of isomaltose. Analyses of the degradation products and consumption of periodate in the Smith degradation were performed at intervals during periodate oxidation. of kojibiose. Analyses of the degradation products Reaction conditions were as described in "EXPERI were performed at intervals during periodate oxidation. MENTAL." •ü, Glycerol; ?, glycolaldehyde; ?, Reaction conditions were as described in "EXPERI glyceraldehyde; x, periodate. MENTAL." •ü, Glycerol; ?, glycolaldehyde; ?,

glyceraldehyde; x, periodate. was not estimated quantitatively, was detected with glycerol and glycolaldehyde on the gas

chromatograms. These results were in accord mained almost unchanged during the progress of

with those previously reported by Neumuller and overoxidation, suggesting that the hydrolysis of

Vasseur (6) and by Charlson and Perlin (14), but the intermediate compound produced by normal

not with those recently reported by Honda and Malaprade oxidation is the rate-controlling process,

co-workers (15). The intermediate compound thus and that the subsequent oxidation processes are

formed appeared to be relatively stable, because much faster. Similar rapid overoxidation was also

the periodate consumption remained unchanged observed for 3-O-ƒ¿-D- glucopyranosyl-D-fructo

during prolonged treatment with periodate. pyranose (), which has a structure similar 6-O-ƒÀ-D-Glucopyranosyl -D- glucopyranose to that of kojibiose.

(gentiobiose) was more rapidly oxidized by peri Figure 5 shows the production of hydroxy odate than was isomaltose, and gave equimolar and oxo compounds and consumption of peri

quantities of glycerol and glycolaldehyde as final odate in the Smith degradation of O-ƒ¿-D-gluco

products. 6-O-ƒ¿-D- Galactopyranosyl -D- gluco pyranosyl (1•¨4)-ƒ¿-D-glucopyranosyl-(1•¨4)- D-

pyranose (melibiose) resembled isomaltose closely glucopyranose (maltotriose). One mole of glycerol in the time course of production of hydroxy and was produced rapidly, whereas the amounts of

oxo compounds. The analytical values found for glycolaldehyde and erythritol gradually approached these disaccharides coincided well with those for the values expected from the results for maltose.

isomaltose (Table II). On inspection of this figure, it is clear that the 2-O-ƒ¿-D- Glucopyranosyl-D-glucopyranose intermediate formyl ester of maltotriose is rather

(kojibiose) was much more prone to overoxidation stable, as observed for maltose. by periodate than hexobioses with other linking There was a marked difference in the rate of

positions, and gave considerably lower yields of periodate oxidation between maltotriose and its glycolaldehyde and glycerol than expected (Fig. 4). p-isomer, cellotriose. The rate of periodate oxi This observation was consistent with the results dation of the non-reducing end glucose residue of

obtained by Chanson and Perlin (14). However, cellotriose, which was observed as glycerol pro

kojibiose gave a glycolaldehyde: glycerol molar duction after reduction, was rapid, like that of

ratio of approximately 1 : 3 after the completion of maltotriose, under comparable conditions, whereas

normal Malaprade oxidation, and the ratio re- the internal glucose residue of cellotriose was only

Vol. 82, No. 6, 1977 1678 H. YAMAGUCHI and T. MUKUMOTO

Fig. 5. Production of hydroxy and oxo compounds Fig. 6. Production of hydroxy and oxo compounds and consumption of periodate in the Smith degradation and consumption of periodate in the Smith degradation of maltotriose. Analyses of the degradation products of cellotriose. Analyses of the degradation products were performed at intervals during periodate oxidation. were performed at intervals during periodate oxidation. Reaction conditions were as described in "EXPERI Reaction conditions were as described in "EXPERI MENTAL." •ü, Glycerol; •œ, meso-erythritol; ?, MENTAL." •ü, Glycerol; •œ, mesoerythritol; ?, glycolaldehyde; ?, glyceraldehyde; x, periodate. glycolaldehyde; x, periodate.

gradually oxidized, resulting in a slow production of erythritol and glycolaldehyde. From this

observation, it may be concluded that both end

residues in cellotriose are sufficiently close

to the hydroxyl groups on C-2 and C-3 of the

internal residue to hinder the attack of periodate.

The features of degradation of cellotriose are

shown in Fig. 6, in which periodate oxidation

was carried out with higher concentrations of both

periodate and the substrate at a higher temperature. The yield of erythritol was not very high, suggesting that overoxidation gradually occurred at the

reducing-end residue under such conditions. Similar results were found for O-ƒÀ-D-manno Fig. 7. Production of hydroxy and oxo compounds pyranosyl-(1•¨4)- P -D-mannopyranosyl-(1•¨4)-D- and consumption of periodate in the Smith degradation mannopyranose. The rate of periodate oxidation of isomaltotriose. Analyses of the degradation products of the internal mannopyranose residue of (1•¨4)- were performed at intervals during periodate oxidation.

ƒÀ-D-mannotriose appeared to be almost equal to Reaction conditions were as described in "EXPERI

that of cellotriose, though the internal manno MENTAL." •ü, Glycerol; •œ, meso-erythritol; ?, glycolaldehyde; ?, glyceraldehyde; x, periodate. pyranose residue has a cis-glycol group as a single site for periodate oxidation. These observations

suggest that the rate of periodate oxidation is much The production of a considerable amount of more significantly affected by steric inhibition glyceraldehyde was observed at oxidation times between sugar residues than by the stability of the shorter than about I h. The degradation features

cyclic periodate complexes of cis and trans-glycols. of isomaltotriose suggest that the reducing-end The analytical values obtained from these trisac glucopyranose residue of this was charides are summarized in Table II. oxidized to a glycolaldehyde group, which is in

The features of degradation of O-ƒ¿-D-gluco accord with the result found for isomaltose. This pyranosyl-(1•¨6)-ƒ¿-D-glucopyranosyl-(1•¨6)-D- suggestion was supported by the detection of glucopyranose (isomaltotriose) are shown in Fig. 7. 1,2-ethanediol on the gas chromatograms, as in

J. Biochem. SMITH DEGRADATION PRODUCTS OF REDUCING OLIGOSACCHARIDES 1679 the case of isomaltose, though this is not indicated dation of trisaccharides may be significantly in Fig. 7. Similar features of degradation closely affected by steric hinderance between sugar resi

resembling those of isomaltotriose were obtained dues, depending upon their structures, suggests that with O-ƒ¿-D-galactopyranosyl-(1•¨6)-ƒ¿-D-galacto- periodate oxidation should be carried out with pyranosyl-(1•¨6)-D-galactopyranose. care to complete the normal Malaprade oxidation The present method was also applied to of higher oligosaccharides and polysaccharides

O-ƒ¿-D-glucopyranosyl-(1•¨6) -ƒ¿-D-gl ucopyranosyl- which are structurally more complicated.

(1•¨4)-D-glucopyranose (panose). The analytical In the present study, the Smith degradation values found for this trisaccharide were consistent and the subsequent procedures for GLC analysis with expectation, based on the results from malto of the products were carried out substantially and isomaltotriose (Table II). according to the previously proposed method (2), Glycolaldehyde was the only oxo compound but with some significant improvements. The found for the oligosaccharides tested in the present pretreatment of the polyalcohol derivative with study. However, a reducing trisaccharide in which methanol markedly improved its recovery from the the internal hexopyranose residue is linked at C-2 resin used for desalting. This suggests that the should give another oxo compound, glyceraldehyde, polyalcohol derivative readily forms a negatively as found previously from a kojibiosyl group (2). charged borate complex, which is decomposed by It is evident from Table II that, for each linking methanolysis. Further, repeated evaporations position of reducing disaccharides composed of with methanol appeared to be useful as a pre hexopyranoses, a characteristic set of degradation treatment for pertrimethylsilylation of the hydroxy products is obtained. This suggests that GLC compounds and oxime derivatives of oxo com- analysis of the hydroxy and oxo compounds may pounds. These observations are consistent with be useful for linkage analysis of reducing disac the fact that small amounts of borate tend to charides. Further, linear reducing trisaccharides interfere with the acetylation of alditols to be composed of hexopyranose residues might all be assayed by GLC (16). Such treatments with distinguishable by GLC analysis of the degradation methanol might also be useful in the analytical products, since each of them should be charac method (1, 2) previously proposed for the non- terized by types and/or molar ratios of hydroxy reducing derivatives of disaccharides. and oxo compounds produced from it. This The authors are indebted to Professor T. Mizuno for expectation was confirmed in part by the results gifts of 4-O-ƒÀ-D-mannopyranosyl-D-mannopyranose and obtained with some trisaccharides in the present O-ƒÀ-D-mannopyranosyl -(1•¨4)-ƒÀ -D-mannopyranosyl-

study. Therefore, the proposed method may be (1•¨4)-D-mannopyranose, and also to Dr. S. Chiba for useful for linkage analysis of reducing di and a gift of kojibiose. Thanks are also due to Dr. M. trisaccharides. Takagi for supplying isomaltotriose and panose. The In biochemical investigations of polysac technical assistance of Mr. K. Okamoto is gratefully charides and complex carbohydrates, it is fre acknowledged. quently necessary to elucidate the structures of REFERENCES reducing oligosaccharides which may be produced by partial hydrolysis. The structures of reducing 1. Yamaguchi, H., Ikenaka, T., & Matsushima, Y. oligosaccharides have usually been elucidated by (1968) J. Biochem. 63, 553 the methylation procedure followed by combined 2. Yamaguchi, H., Ikenaka, T., & Matsushima, Y. GLC-mass spectrometry. These procedures, how (1970) J. Biochem. 68, 253 3. Yamaguchi, H., Ikenaka, T., & Matsushima, Y. ever, are not easy, especially on a small scale, (1970) J. Biochem. 68, 843 though clear-cut data can be expected. Compared 4. Fukagawa, K., Yamaguchi, H., Uotani, 0., Tsuji with methylation analysis, the present method moto, T., & Yonezawa, D. (1975) Agric. Biol. Chem. possesses the advantages of rapidity and simplicity. 39, 1703 The features of the formation of degradation 5. Mukumoto, T. & Yamaguchi, H. (1977) Carbohyd. products from these oligosaccharides provide a Res. in press basis for the periodate oxidation of carbohydrates. 6. Neumuller, G. & Vasseur, E. (1952) Arkiv Kemi 5, The finding that the progress of periodate oxi- 235

Vol. 82, No. 6, 1977 1680 H. YAMAGUCHI and T. MUKUMOTO

7. Fleury, P.F., Courtois, J.E., & Bieder, A. (1952) 12. Ikenaka, T. (1963) J. Biochem. 54, 328 Bull. Soc. Chim. France 118 13. Anai, M., Ikenaka, T., & Matsushima, Y. (1966) 8. Head, F.S. & Hughes, G. (1954) J. Chem. Soc. 603 J. Biochem. 59, 57 9. Hough, L., Taylor, T.J., Thomas, G.H.S., & Woods, 14. Charlson, A.J. & Perlin, A.S. (1956) Can. J. Chem. B.M. (1958) J. Chem. Soc. 1212 34,1804 10. Harada, T., Misaki, A., & Saito, H. (1968) Arch. 15. Honda, S., Kakehi, K., & Takiura, K. (1976) Biochem. Biophys. 124, 292 Carbohyd. Res. 47, 213 11. Yamaguchi, H., Ikenaka, T., & Matsushima, Y. 16. Yamaguchi, H. & Makino, K. (1977) J. Biochem. (1971) J. Biochem. 70, 587 81,563

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