isolation and characterization of …the journal or riolo~ical chemistry vol. 241, no. 8, issue of...

THE JOURNAL or RIOLO~ICAL CHEMISTRY Vol. 241, No. 8, Issue of April 25, 1966

Printed in U.S.A.

Isolation and Characterization of Lipopolysaccharides Containing 6-O-Methyl-D-glucose from

Mycobacterium Species*

(Received for publication, November 17, 1965)

YUAN CHAUN LEE$

From the Department of Biochemistry, University of California, Berkeley 4, California

SUMMARY

A polysaccharide, composed of 6-O-methyl-D-glucose and D-glucose in a molar ratio of 6:4, was isolated from the water- soluble portion of the deacylated crude lipids of Mycobac- ferium phlei and M. tuberculosis. The polysaccharide was purified by gel filtration, ion exchange chromatography, and borate complex formation, and was shown to have a molecular weight of about 3000 and [a]: +160° (c, 0.1; water).

Methylation analysis of the polysaccharide showed that it has a branched structure with an average chain length of 8 to 9 hexose units. The major type of glycosidic linkage is CY-(1 -+ 4), and the branching involves position 3 of one of the 6-O-methyl-D-glucose residues.

Oligosaccharides were prepared by acetolysis of the polysaccharide, and were purified by paper chromatography. Di-, tri-, tetra-, penta-, hexa-, and heptasaccharides of a-(1 + 4)-linked 6-O-methyl-n-glucose, in addition to maltose and maltotriose, were isolated. The only hetero- oligosaccharide found was 0-CY-n-glucopyranosyl-(1 + 3)-6- O-methyl-D-glucose.

&Amylase had no action on the polysaccharide, whereas a-amylase liberated about 20% of the total carbohydrate, mainly as D-glucose and maltose. Limited amylolysis yielded D-glucose, maltose, and unidentified oligosaccharides containing D-glucose.

Intact lipopolysaccharide, containing 2 to 3 moles of ester- linked fatty acid, was prepared from a 70% ethanol extract of M. phlei. The lipopolysaccharide was soluble in water and in a 2: 1 chloroform-methanol mixture, but was only slightly soluble in methanol or ethanol. Proton magnetic resonance and infrared and ultraviolet spectra were consistent with the results obtained by chemical analysis.

Methyl ethers of monosaccharides have been found in a few plant polyssccharides. Examples are 3-O-methyl-n-galactose

* This work was supported in part by Grant AM 00884 from the United States Public Health Service, and Grant GB-2032 from the National Science Foundation.

$ Present address, Department of Biology and McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218.

(l), 6-O-methyl-n-galactose (2), and 4-O-methyl-n-glucuronic acid (3). Mono- and di-O-methyl ethers of deoxyaldoses exist in a number of cardiac glycosides (4), and methyl ethers of 6- deoxygalactose, 6-deoxymannose, and 6-deoxytalose have been isolated from several Mycobucterium species (5).

During our studies of mycobacterial mannophospholipids (6), we isolated from the crude phospholipid fractions a polysaccharide containing 6-O-methyl-n-glucose. An account of the structural proof of this sugar has already appeared (7). Prior to this time, the only methyl ether of n-glucose known to occur in nature was 3-@methyl-o-glucose, a component of the sea cucumber toxin holothurin A (8).

In this report is described the isolation of and some structural studies on the 6-o-methyl-n-glucose polysaccharides of nfyco- bacterium phlei and M. tuberculosis. The polysaccharides appear to be linked covalently to lipid material.

EXPERIMENTAL PROCEDURE

MateriaZsSephadex G-25 (fine), G-50 (fine), and DEAE- Sephadex A-25 (medium), all in irregular (not bead) form, were obtained from Pharmacia. DEAE-Sephadex was converted into its borate form as follows. After treatment of DEAE- Sephadex with HCl and NaOH, the resin was washed on a Buch- ner funnel with saturated sodium tetraborate until the effluent was free of chloride ion, washed with water to remove most of the residual borate, and then packed into a column and washed free of borate.

Swine pancreas cr-amylase and sweet potato fl-amylase were obtained from Worthington, and were used without further purification. The amylolytic activities of the enzymes were assayed by the dinitrosalicylate method (9). Under the conditions described by Bernfeld, a unit of c~- or fl-amylase will produce per mm at 25” 1 pmole of reducing group, calculated as maltose. Glucoamylase I from Aspergillus niger (10) was generously supplied by Drs. John Pazur and D. R. Lineback. A unit of enzyme activity is defined as the quantity of enzyme that produces after 1 hour at 30” 1 mg of n-glucose from 60 mg of starch dissolved in 2 ml of citrate buffer, pH 4.8. Glucostat Special, a glucose oxidase preparation for specific enzymatic assay of n-glucose, was obtained from Worthington.

Genera2 Procedures-Analytical procedures for total carbohydrate (1 I), reducing sugar (12), and phosphorus (13) have been described previously (5). A modification (14) of the method of Somogyi for reducing sugar determination was used in conjunc- tion with the amylolysis studies.

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1900 Lipopolysaccharides Containing 6-O-Methyl-o-glucose Vol. 241, No. 8

Whereas borate is known to suppress the color reaction of sugars with the phenol-H&O1 reagent, it has been shown (15) to enhance color production in the resorcinol-HzS04 reaction (16). Therefore, the latter method was modified as follows for analysis of fractions when borate was used for column chromatography. A borate-HzSO1 mixture (about 0.04 M tetraborate) was prepared by adding 900 ml of 98% H&O4 to a mixture of 1.5 g of Na2B40r. 10 Hz0 and 90 ml of water. The heat of dilution produced during the mixing facilitated complete solution of the borate. The resorcinol reagent was prepared by dissolving 5 g of recrystallized resorcinol in 100 ml of water; this solution could be stored for several weeks at 4”. To a 2-ml sample solution, containing 5 to 40 pg of sugar, was added 0.1 ml of 5% resorcinol solution followed by 4.5 ml of the borate-HzSO1 solution. The latter was added rapidly with the use of a pipette with a large opening (11). After 30 min, the absorbance was determined at 430 mp. A gradual decrease in the absorbance was observed after 4 hours. n-Glucose, 6-O-methyl-n-glucose, and n-mannose all showed a maximum absorption at 430 rnp; the ratio of molar absorptivities for these sugars was 1.00:0.97: 1.29, respectively. In the phenol-H&04 method, with 40 mg of phenol, the ratio of molar absorptivities at 490 rnp was 1.00: 0.98:1.08.

For analysis of the constituent sugars of oligo- and polysaccharides, 0.2 to 1.0 mg of carbohydrate was hydrolyzed at 100” in 1 ml of N HzS04 for 5 to 6 hours, or in 0.25 N HzS04 at 100’ for 18 hours. The hydrolysate was treated with Amberlite IR-45 (acetate form) to remove the acid, concentrated, and subjected to paper chromatography.

The hydroxamic acid method for acyl esters (17) was adapted as follows. To a l-ml aqueous solution containing 0.1 to 1.0 pmole of ester were added 0.2 ml of 2 M NHpOH.HCl solution and 0.2 ml of 3.5 N sodium hydroxide. After 20 min at room temperature, 0.2 ml of 4 N HCl and 0.2 ml of 0.37 M Fe& in 0.1 N HCI were added to the solution. Absorbance was measured at 520 rnp in a 2-ml cuvette with a l-cm light path. Under these conditions, 1 Fmole of ethyl acetate in aqueous solution yielded 0.63 absorbance unit.

Descending paper chromatography was carried out on What- man No. 1 filter paper with the following solvent systems (composition in volume ratios) ; Solvent A, l-butanol-pyridine- water, 10:3:3; Solvent B, ethyl acetate-pyridine-water, 10:4:3;

TABLE I

Gas chromatography of methyl glucoeides

Methyl sugars

Methyl glucosides 2,3,4,6-. 155” 2.8 3.9

2,3,4-. 155 5.5 7.6

2,3,6-. 155 7.8 10.4

2,4,6-. 155 7.3 10.8 2,3-. 202 5.8

2,6-. 202 4.8 6.2

Methylated maltose. 235 9.6 12.1

Methylated cellobiose 235 8.2 9.9

5 The assignment of anomers is based on the generalization made by Bishop (21).

Retention time”

Temperature

@ I a

min

0.8 I I I B DEAE-Sephadex !

0.6 -

0.2 - A

EFFLUENT VOLUME (ml)

FIG. 1. Elution patterns obtained by fractionation of the deacylated phospholipids on DEAE-Sephadex. The solid line represents carbohydrate, while the dashed line represents phosphate. Peak 1-A contained primarily trehalose. Peak 1-B is the polysaccharide containing 6-O-methyl-n-glucose. The remaining peaks are the deacylated mannophospholipids described in Reference 6.

Solvent C, 1-butanol saturated with water (18); Solvent D, l- butanol-ethanol-water, 4 : 1:5, upper phase (19) ; Solvent E, ethyl acetate-pyridine-2.5 y. aqueous boric acid, 60 : 25 : 20. For detection of sugars on chromatograms, an aniline-trichloracetate reagent (20) was used in addition to the AgN03-NaOH and periodate-benzidine reagents (6).

The conditions for gas chromatographic analysis of sugar derivatives have been described (6, 7). Gas chromatographic data of reference methylated sugars are listed in Table I.

Sugars were reduced in 0.5 to 1.0% solutions of NaBH4 at 5” for 16 to 24 hours. The method of Park and Johnson (12) for reducing sugar was used to determine the extent of the reaction. After reduction, excess NaBHe was destroyed by adding Dowex 50 (H+ form), and boric acid was removed as methyl borate by the repeated addition and evaporation of methanol. The conditions of Hanahan and Olley (22) were used for periodation and formaldehyde determination of sugar alcohols and of glycerol.

Proton magnetic resonance spectra were determined at 60 mc with a Varian model A-60 spectrometer, and optical rotation was measured wit,h a Rudolph photoelectric polarimeter.

RESULTS

6-O-Methyl-o-glucose Polysaccharide from M. phlei-The methods for extraction and deacylation of phospholipids from M. phki have been described (6). About 250 g of wet cells yielded 2.5 g of crude phospholipids. The water-soluble mixture (1 g) of the deacylated phospholipids was fractionated on a column, 75 x 1.6 cm, of DEAE-Sephadex (carbonate form). Elution of the column with 0.07 M ammonium carbonate, pH 8.5, gave the pattern shown in Fig. 1. Peak 1-A contained primarily trehalose, while Peak 1-B contained polysaccharide material consisting mainly of n-glucose and 6-O-methyl-n-glucose. The other peaks contained deacylated mannophospholipids (6). In some experiments, Peaks 1-A and 1-B overlapped considera- bly.

The phosphate-free material containing 6-@methyl-u-glucose (Peaks 1-A and 1-B combined), obtained from several runs of DEAE-Sephadex chromatography, was combined and passed


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Issue of April 25, 1966 Y. C. Lee 1901

through a Sephadex G-25 column (150 x 3 cm) equilibrated with 0.1 M pyridine acetate, pH 4. B-O-Methyl-n-glucose was found only in the peak emerging near the void column (Fig. 2~). In another experiment, the water-soluble portion of the deacylated phospholipid mixture was applied directly to a Sephadex G-25 column (150 X 3 cm) equilibrated with 0.1 M lithium acetate, pH 6.0. The material containing 6-@methyl-n-glucose was found in the first peak (Fig. 2b). Further purification by gel filtration was achieved with a column of Sephadex G-50 (150 X 2 cm), with wat.er as the eluent. The major fraction was collected as indicated in Fig. 3; it contained material giving upon hydrolysis primarily 6-O-methyl-n-glucose and n-glucose.

The product (about 0.5 g) obtained from the Sephsdex G-50 column was applied to a DEAE-Sephadex column (carbonate


FIG. 2. a, gel filtration pattern in 0.1 M pyridine acetate, pH 4, of combined Peaks 1-A and 1-B from Fia. 1. b, gel filtration nat- tern in 0.1 M lithium acetate, pH 6.0, OF the deaiylated phospholipids. The solid lines represent carbohydrate, and the dashed Zinc represents ph0sphat.e. The first peak in both figures contains the 6-O-methyl-n-glucose polysaccharide.

100 200 300 400


FIG. 3. Gel filtration pattern in water of the polysaccharide fractions containing 6-O-methyl-n-glucose obtained from Fig. 2, a and b. The portion above the solid bar was combined for further purification (Fig. 4).


FIG. 4. Purification of the 6-@methyl-n-glucose polysaccharide on DEAE-Sephadex. The second peak, eluted with ammonium carbonate, yielded 6-O-methyl-n-glucose and D-ghCOSe on acid hydrolysis.

form) which was then washed with water. About 10% of the carbohydrate was not retained by the column. The adsorbed material was eluted with 0.04 M ammonium carbonate, pH 8.6 (Fig. 4). The fraction eluted with ammonium carbonate gave 6-o-methyl-n-glucose and n-glucose on acid hydrolysis, but traces of mannose and of an unidentified compound which had an RF value slightly greater than that of 6-O-methyl-n-glucose were also found by paper chromatography with the use of Solvent A. Phosphorus was not detected in this fraction, when an amount that would have been sufficient to demonstrate 1 mole of phosphate per 50 moles of hexose was used. Acid hydrolysis of the material not retained by the column yielded, as sugar constituents, glucose, mannose, 6-@methyl-n-glucose, 6-deoxymannose, and 6-deoxytalose.

The substance eluted by ammonium carbonate was hetero- geneous when subjected to paper electrophoresis in 0.02 M sodium tetraborate, pH 9.2, at 25 volts per cm for 90 min. Final purification was effected with a column (65 X 0.9 cm) of DEAE- Sephadex (borate form) with a linear gradient (0 to 0.075 M;

total volume, 2 liters) of sodium tetraborate, pH 9.2. The fractions shown in Fig. 5a were analyzed by the modified resorcinol-H&SC4 method, and the material in the major peak was combined as indicated by the bar on the figure. Sodium tetraborate was removed as described above. The final product, designated as MGP, appeared to be homogeneous by paper electrophoreeis in the borate buffer, and contained 6-O-methyl- n-glucose and n-glucose as the only detectable sugar constituents. The molar ratio of the two sugars was 6:4, as determined by paper and gas chromatography. The over-all yield of MGPr was estimated to be about 5% (w/w) of the crude phospholipid fraction.

6-O-Methyl-o-glucose Polysmcharides from h4. tuberculosis- 6-O-Methyl-n-glucose was also found in a polysaccharide-like material in the phospholipid fraction of M. tuberculosis (6, 7). The scheme for isolation of polysaccharides containing 6-O

1 Abbreviations: MGP, defined earlier in this paragraph; ND, NDR, CMA, CMR, CMR’, and CMRW, see text and Chart 1.


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1902 Lipopolysaccharides Gntaining B-O-Methyl-D-glucose Vol. 241, No. 8

0.6 c M.phlei

I\

-2 E 0.2- a d 8 5 0 ' I

E I I 1 I I I 1 M.tuberculosis

0 600 800 1000 1200


FIG. 5. a, ion exchange chromatography of the &O-methyl-n- glucose polysaccharide from M. ph.Zei on DEAE-Sephadex in borate solution. b, chromatography of the polysaccharide from M. tuberculosis under the same conditions as a. The portions above the solid bars were collected for further study.

methyl-n-glucose from M. tuberculosis was essentially the same as that applied to M. phlei. The behavior of the polysaccharides from d4. tuberculosis did not differ significantly from the corresponding polysaccharide from M. phlei (MGP) during fractionation, except for the borate column chromatography. Here, in contrast to MGP, the polysaccharides from M. tuberculosis separated into two distinct fractions (Fig. 5b). The same molar ratio of 6-O-methyl-n-glucose to n-glucose, 6:4, was observed in each of the two fractions from M. tuberculosis.

Properties of MGP-Molecular weight determination2 of MGP in 0.1 M NaCl by the sedimentation equilibrium method (23) gave a value of 3060 f 3%. Titration of MGP, which had been decationieed previously with Dowex 50 (H+ form), gave an equivalent weight of 3080. The acidic group showed a single inflection point with pK, 3.5.

The infrared spectrum of MGP showed a strong carbonyl group absorption band at 1640 cm-r and a band at 860 cm-r probably associated with a! anomeric C-H deformational vibration. No significant absorption was seen near 890 cm-r. An aqueous solution of MGP showed some ultraviolet absorption (E:pm = 0.35 at 220 mp).

The proton magnetic resonance spectrum of MGP showed anomeric hydrogen signals concentrated at 5.2 f 0.1 ppm, indicating that the majority of the anomeric carbons are in a! configuration. The spectra of the 6-O-methyl-n-glucose polysaccharides from M. tuberculosis showed the same features. The specific optical rotation was [a]:’ -1-160” (c, 0.1; water).

The following color reactions were negative: “direct Ehrlich” reaction for sialic acid (24); Elson-Morgan reaction for amino sugars and muramic acid (25) ; the periodate-thiobarbituric acid reaction for 2-keto-3-deoxyoctonate (26). The carbazole reaction for uranic acids (27) gave some color, but the same color

1 The author is indebted to Dr. H. K. Schachman for a molecular weight determination of MGP.

intensity was obtained when equivalent concentrations of the two hexoses were tested. MGP showed only negligible reducing power by the method of Park and Johnson (12).

Interaction of iodine with MGP was studied in the following way (28). The polysaccharide, 1.44 mg, was dissolved in 2 ml of 0.006 N I? solution and the absorption spectrum was com- pared with a reagent blank. In contrast to glycogen or amylo- pectin (28), no maximum was observed between 400 and 700 mCc.

Methylation AnaZysLs-MGP was methylated repeatedly by the methyl iodide-silver oxide method (5, 29), until completion of methylation was indicated by infrared spectroscopy (6). After methanolysis of the methylated polysaccharide, gas chromatography at 155” showed the presence of the methyl glycosides of 2,3,4,6-tetra-O-methyl-n-glucose and 2,3,6- tri-@methyl-n-glucose in the ratio of 1:7 to 8. At 202”, in addition to the peaks of the tetra- and trimethylglucoses, two peaks were observed with retention times of 4.8 and 6.2 min, and were concluded to be the anomeric pair of a methyl di-O- methylglucoside. Standard methyl 2,3-di-O-methyl-cr-n-gluco- side, under the same conditions, had a retention time of 5.8 min. When the methanolysate of the methylated polysaccharide was treated with 0.044 M NaI04, some periodate consumption was observed. After deionization, the product was analyzed by gas chromatography. The peaks believed to be the anomers of a methyl di-O-methylglucoside were no longer observed.

A portion of the methylated MGP was hydrolyzed with acid, and the hydrolysate was examined by paper chromatography in Solvent D. A di-O-methylglucose (RF 0.57)) different from 2,3- di-O-methyl-n-glucose (RF 0.53), was observed in the hydrolysate.

Evidence that the substance was 2,6-di-O-methyl-n-glucose was obtained as follows. Authentic 2,3,6-tri-O-methyl-n-glu- case, 37 mg, was partially demethylated by heating at 100” for 5 min with 1 ml of 48% hydrobromic acid in a sealed tube. The hydrolysate was deionized with Amberlite MB-3, and the product was chromatographed on paper in Solvent D to isolate the di-O-methylglucose fraction produced by demethylation. The mixture of di-O-methylglucose was treated with methanolic HCl, and the glycosides were examined by gas chromatography before and after periodate oxidation. Two of the peaks repre- senting periodate-susceptible di-O-methylglucosides had the same retention times as the di-O-methylglucosides derived from the methylated MGP. By partial demethylation 2,3,6-trimethylglucose could yield only one dimethylglucose the methyl glycoside of which would be destroyed by periodate, that being the 2,6 isomer.

Acetolysis-MGP, 80 mg, was shaken with a mixture of acetic acid (0.5 ml), acetic anhydride (0.5 ml), and 98% sulfuric acid (0.05 ml) at room temperature. After 15 min, a clear solution was obtained, which was allowed to stand at room temperature for 40 hours. The acetolysate, slightly brown in color, was poured into 30 g of ice water. The mixture was slowly warmed to room temperature and was adjusted to pH 7.5 with dilute NaOH. The suspension of sugar acetates was extracted repeatedly with chloroform until carbohydrate was no longer ex- tractable; the carbohydrate remaining in the aqueous phase represented 4.7% of the starting material. The chloroform extracts were combined, dried over anhydrous sodium sulfate, and evaporated to yield 135 mg of sugar acetates.

The mixture of sugar acetates was deacetylated in methanol with barium methoxide (30). The deacylation mixture was


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poured into a suspension of Dowex 50 (H+ form) in water and was stirred vigorously. The filtrate from the resin was concentrated to a small volume for gel filtration as described below.

In order to increase the yield of higher oligosaccharides, 78 mg of MGP were acetolyzed for 8 hours at room temperature, then for 10 hours at 5”. Isolation and deacetylation of the products were carried out in the same way.

Gel Filtration and Paper Chromatography of Oligosaccharides- The oligosaccharide mixture obtained from the acetolysis-deacetylation reactions was applied to a column (220 x 1.6 cm) of Sephadex G-25 (fine), and was eluted with water at the rate of 10 ml per hour. The elut,ion pattern, as determined by the phenol-

0.6

180 210 240 270 300 330

EFFLUENT (ml)

FIG. 6. Fractionation on Sephadex G-25 of the oligosaccharides obtained from MGP by acetolysis. The solid line indicates the products from the 40-hour acetolysis, and the dashed line the products from the 8-hour acetolysis. For explanation of Peaks A, B, C, D, E, F, G, and H see the text.

@Cl

@El 0 Bz

@F 0c3 c) B3

CBG OH

8 E, OD2 QC4

I I I / E3, , , ,

HGFEDCBA

c

TABLE II

Oligosaccharides obtained from acetolvsate

Oligosac- charide

Rp in Solvent B

A

Bl

BP

B3

Cl CZ

C3

C4

Dl

D*

El

E2

El F

G H

0.189 0.280 0.147 0.116 0.253 0.215 0.081 0.068 0.230

0.068 0.201 0.064 0.053 0.180 0.157 0.142

T

Yielda structure

%

1.4 5.0 1.1 1.1 7.8 1.2 0.6 0.6 4.4

0.5d l.Bd l.ld l.ld

n-Glucose 6.O-Methyl-n-glucose Glucoseb Maltose cu.MGlc-(1 -+ 4)-MGlcc a-Glc-(1 -+ 3)-MGlc Glucoseb Maltotriose a-MGlc-(1+4)+MGlc-(1+4)-

MGlc Glucoseb (a-1,4-MGlc) 4 Glucose, MGlcb Glucose, MGlcb (ol-1,4-MGIc)b (a-1,4-MGIc)G (a-1,4-MGlc),

-- o From the 40.hour acetolysis. b Constituent sugars. c MGlc, 6-O-methyl-n-glucose. d From the &hour acetolysis.

”

HzSO1 method, is shown in Fig. 6. Fractions A, B, C, and D were isolated as indicated, and the oligosaccharides were examined by paper chromatography in Solvents A and B. The products from the 8-hour acetolysis were fractionated similarly and examined by paper chromatography. Fractions E, F, G, and H, in addition to Fractions A, B, C, and D were obtained.

The relative mobility, in Solvent A, of the major oligosaccharides in each fraction is represented schematically in Fig. 7. The individual oligosaccharides were isolated by preparative paper chromatography on Whatman No. 1 paper with Solvent A and elution for 2 to 3 days. The purity of each oligosaccharide was ascertained by chromatography in Solvent B. The yields of the isolated oligosaccharides are listed in Table II.

Maltose series

I I 1 I I I I HGFEDCB

2.5

2.0

Y- IL cr” 0 8- I

1.5 ”

5

1.0

FRACTIONS FROM SEPHADEX COLUMN -INCREASING MOLECULAR SIZE

FIG. 7. a, the paper chromatographic properties of the oligosaccharides from MGP. The migration distance was measured in Solvent A. 0, uncharacterized oligosaccharides; @,a-(1 --t 4)-6-O-methyl-n-glucose oligosaccharides; @, ~(1 ---f 4)-n-glucose oligosaccharides ;

l , hetero-oligosaccharide. b, the plot of log [1006 RF/(1 - RF)] against molecular size. The RF values were measured in Solvent B.


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1904 Lipopolysaccharides Containing 6-O-Methyl-o-glucose Vol. 241, No. 8

Characterization of Oligosaccharides (Table II)-Each of the isolated sugars gave a reducing value when treated by the method of Park and Johnson (12). Sugar A was chromatographically identical with n-glucose in Solvents A and B, and was oxidized by glucose oxidase at a rate similar to that of n-glucose. Sugar Bi could not be distinguished from authentic 6O-methyl-n- glucose in Solvents A, B, and C. Glucose oxidase showed only negligible action on this component.

When Cl, [aID +116” (c, 0.06; water), was hydrolyzed with 1 N H2S04 for 6 hours at loo“, only 6-O-methyl-n-glucose was detected by paper chromatography in Solvents A and B. Reduc- tion of Cl with sodium borohydride resulted in a 53 y0 decrease in the color value as determined by the modified resorcinol-H2S04 method. Periodate oxidation of the reduced C1 produced 0.89 mole of formaldehyde per mole of the original compound. Acid hydrolysis of the reduced Ci yielded 6-O-methyl-n-glucitol and 6-O-methyl-n-glucose (Solvent E). About 1 mg of C1 was methylated and the product was methanolyzed and analyzed by gas chromatography. Methyl glycosides of 2,3,4,6-tetra- O-methyl-n-glucose and 2,3,6-tri-O-methyl-n-glucose were found.

Fraction C2, [cy], +84.5” (c, 0.02; water), consisted of n-glucose and 6-O-methyl-n-glucose. Lu-Glucoamylase showed no action on this oligosaccharide over 12 hours at 40”, with the use of a ratio of 450 units of enzyme per pmole of hexose equivalent. Upon reduction with sodium borohydride, the original color value by the resorcinol-H2S04 method decreased by 48%. Periodate oxidation produced 0.85 mole of formaldehyde per mole of the original oligosaccharide. The reduced CB was hydrolyzed and examined by paper chromatography. Only glucose could be detected with the aniline-trichloracetate reagent. About 0.5 mg of CZ was methylated and the methylated disaccharide was analyzed by gas chromatography without methanolysis. At 235”,

0.8

0.2

1

I

: \ i

180 240 300

(4 T

1 .: I i

/, 270 300 330

1 ii I, I’ i i I i i i I B

II ’ ’ 04 ! Sephadex I i

G-25

i i i i I

23 hr

-1

-0

-C


FIG. 8. Gel filtration pattern of the amylolytic products of MGP. a, exhaustive amylolysis: dashed line represents intact MGP; solid line, the products after 23 hours of enzyme action. Carbohydrate was determined by the phenol-H%SOd method. b, oligosaccharides from limited amylolysis. Reducing power was determined by the method of Park and Johnson.

retention time of the methylated CZ was 9.0 min (major peak), whereas methylated maltose showed a major peak at 9.6 min and a minor peak at 12.1 min. After methanolysis, the methylated CZ produced 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of glucose.

Fraction Di, [a], +130” (c, 0.06; water), produced mainly 6-0- methyl-D-glucose and a small amount of glucose. However, after purification of D1 by rechromatography on the Sephadex G-25 column (220~cm), only a trace of glucose could be found in the acid hydrolysate. Reduction of D1 followed by periodate oxidation produced 0.90 mole of formaldehyde per 2 moles of remaining reducing sugar. Methylation analysis of D1 showed the presence of 2,3,4,6-tetra-o-methyl- and 2,3,6-tri-o-methylglucose in a ratio of approximately 1: 2.

Fractions El, F, G, and H all produced mainly 6-O-methyl-n- glucose, but also a trace of glucose, after acid hydrolysis. When log[RF/(l - RF)] values for the oligosaccharides, B1, Cl, El, D1, F, G, and H, were plotted against the degree of polymerization a straight line was obtained (Fig. 7). This suggests that these oligosaccharides belong to a homologous series consisted of a-(1 -+ 4)-linked 6-O-methyl-n-glucose.

Acid hydrolysis of Bz and B3 produced glucose as the only detectable sugar. B3 could not be distinguished from maltose in Solvents A and B. The rate of digestion of BB by a-glucoamylase was comparable to that of maltose, whereas Bz was not hydrolyzed by the same enzyme. Similarly, acid hydrolysis of Cs, Cd, and Dz yielded only glucose. Cq and DP behaved like maltotriose on paper chromatography in Solvents A and B. Oligo- saccharides E2 and Et both contained B-O-methyl-n-glucose in addition to glucose.

Digestion of MGP by Amylases-MGP, 40 mg, was dissolved in 4 ml of 0.02 M phosphate buffer, pH 6.8, containing 0.006 M NaCl, and digested at 40” with 448 units of pancreatic a-amylase. Increase in the reducing power of the digestion mixture was measured by the modification by Paleg of the Somogyi method (14). Either the substrate or the enzyme was omitted from con- trol incubation mixtures. After 103 hours of incubation, the reducing power, expressed as maltose, reached 14.6 pmoles/lO@ pmoles of hexose unit, and the value did not increase thereafter. Bfter 23 hours, the incubation mixture was heated for 10 min at loo”, filtered to remove denatured protein, concentrated in a vacuum to about 2 ml, and applied to a column (220 cm) of Sephadex G-25.

As shown in Fig. 8a, the first peak, emerging slightly later than the starting material, contained 82% of the applied carbohydrate. Two additional peaks, corresponding to maltose and glucose, represented 12 and S%, respectively, of the total carbohydrate. However, paper chromatography revealed that there was another component in the maltose peak with an RF value slightly greater than that of glucose. When the high molecular weight fragment of the first a-amylase digestion was subjected to a second incubation with cr-amylase under the same conditions, no increase in the reducing power was observed.

The cr-amylase-resistant, high molecular weight material, 18.7 mg in 1.5 mu of water, was treated at room temperature with 18 units of a-glucoamylase of A. niger. The increase in reducing power, measured as n-glucose by the method of Paleg, reached a maximum value equivalent to 3.2% of the total hexose units after 32 hours. After passage of the digestion mixture through the Sephadex G-25 column (220 cm), monosaccharide and the resistant fragment were separated. The monosaccharide liberated


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by cr-glucoamylase M-as characterized by paper chromatography and the glucose oxidase reaction as D-ghlcose. A second incubation of the high molecular weight fragment with the cr-glucoamylase did not result in further liberation of glucose.

In an attempt to obtain larger oligosaccharides by limited (y- amylolysis, 30 mg of MGP were digested at room temperature with 224 units of pancreatic cr-amylase in 4 ml of 0.01 M phosphate buffer, pH 6.8, containing 0.003 M NaCl. After 9 hours, the reducing power measured by the method of Park and John- son reached a value of 4.1 ~moIes/lOO pmoles of hexose unit. The reaction was stopped by heating, and gel filtration of the limited amylolysis products gave four oligosaccharide peaks, corresponding to mono-, di-, tri-, and tetrasaccharides, as shown in Fig. 86.

Paper chromatography in Solvents A and B showed that Peak IV was glucose and Peak III was mainly maltose. The RF value (Solvent A) of the oligosaccharide in Peak II was about 25 y0 greater than that of malt,ose, while the oligosaccharide in Peak I migrated faster than maltotriose to about the same extent. Similar relationships were observed in Solvent B. Upon acid hydrolysis, the oligosaccharide of Peak II produced glucose and another unidentified product, which was detectable by the AgN03-NaOH reagents and had an RF value 15% greater than that of 6-O-methyl-n-glucose in Solvent A.

P-Amylase from sweet potato showed no action toward MGP during an incubation period of 2 days at 37”, at a level of 250 units of enzyme per 40 mg of the polysaccharide. Under the same conditions, the enzyme digested more than 50% of soluble starch within 4 hours.

Extraction of Intact Lipopolysaccharide-Moist M. phlei cells (974 g) were extracted with 70% ethanol.3 The extract was di- aIyzed against water for a week at 4”, and the diaIysis residue gave 7.4 g of solids after lyophilization; this product is designated as Fraction ND (nondialyzable). Acid hydrolysis of ND, followed by paper chromatography, revealed the presence of glucose, [email protected], mannose, galactose, 6-deoxymannose, 6-deoxytalose, and myoinositol.

Fraction ND, 4.0 g, was extracted twice with a mixture of chloroform (400 ml) and methanol (100 ml) then with a mixture of chloroform (ZOO ml) and methanol (200 ml). The insoluble material was designated as NDR (nondialyzable residue). The material extracted with the first chloroform-methanol mixture (2.0 g) was combined with that of the second mixture (0.2 g), evaporated to dryness, and extracted three times with acetone (100 ml, 40 ml, and 40 ml). The acetone extracts were combined and evaporated to dryness to yield 1.06 g of solid material, which was designated as CMA (chloroform-methanol, acetone-soluble). The residue, 1.07 g, was designated as CMR (chloroform-methanol residue). A flow diagram of the extraction scheme is shown in Chart 1.

Acid hydrolysis of NDR gave primarily glucose and mannose, but galactose, 6-deoxymannose, and 6-deoxytalose (Fig. 9) were also detected. On the other hand, CMR produced glucose and 6-O-methyl-n-glucose as the major components, although mannose, 6-deoxytalose, myoinositol, and a small amount of glycerol were also detected. While CMA reacted strongly with the phenol-HzSOI reagents, acid hydrolysis followed by paper chromatography and detection with the AgNO$-NaOH reagents showed insignificant quantities of monosaccharides. Further

J J. A. Ferguson and C. E. Ballou, unpublished results.

Dialysis residue (4.0 g)

Extrac‘t.(20 g) $ . ..L-.3OH (4~1)

Residue

1 CH~X-CXI,~ (1~1) *

Extract (0.2 g)

4

Residue (1.90 g) NDR

Acetone

Extract’(l.OG g) Residue (1.07 g; 218 rni of sugar, 15 mg of P) CMA CMR

)(o.6gg 65mgof Upper layer (0.31 g; 164 mg of sugar, 1 mg of P) sugar, 14 mg of P)’

CMRW CMR’ CHART 1. Diagrammatic representation of the procedure for

extraction of the intact lipopolysaccharide.

experiments showed that hydrolysis with 1 N H&04 at 100” destroyed over 90% of the phenol-HpSOr-positive material in CMA.

The CMR fraction was dissolved in a mixture of chloroform (80 ml) and methanol (40 ml), and washed with water (30 ml). The lower layer was washed repeatedly with fresh upper phase of a chloroform-methanol-water mixture (8:4:3, v/v). The combined washings (CMRW), weighing 0.3 g after evaporation, contained about 75% of the total sugar in the CMR fraction but only 7% of the total phosphorus. In contrast, the washed residue (CMR’) contained more than 90% of the total phosphate, but only about 25% of the total sugar.

Column Chromatography-The CMRW fraction was passed through a column (150 x 2 cm) of Sephadex G-50, equilibrated in 0.1 M acetic acid, and the carbohydrate concentration and ultraviolet absorbance (280 mp) were measured. As shown in Fig. 10, most of the carbohydrate material (280 mg) appeared in Peak 10-B, whereas Peak 10-A and Peak IO-C, both of which showed strong ultraviolet absorption, contained little carbohydrate. The material in Peak 10-B (about 250 mg) was further fractionated on a column of DEAE-Sephadex (carbonate), pH 7.8 (Fig. 11). About 50 mg of the applied material emerged in the water wash (150 ml). A linear gradient of 0 to 0.1 M sodium bicarbonate (total volume, 500 ml) eluted two more peaks which were decationized with Dowex 50 (H+ form), neutralized with dilute ammonium bicarbonate, and evaporated to dryness. Each fraction was desalted by means of the column (220 cm) of Sephadex G-25 and elution with water to give 45 mg from Peak 11-B and 67 mg from Peak 11-C. Constituent sugars in Fractions 11-A, 11-B, and 11-C were analyzed by paper chromatography following acid hydrolysis (Fig. 9). Mannose was the major monosaccharide in Fraction 11-A, but glucose and 6-0- methyl-n-glucose were also present. In addition, there was a dif- fuse spot, RGlc 1.60, in Solvent A. In Fractions 11-B and 11-C,


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1906 Lipopolysaccharides Containing 6-O-Methyl-~-glucose Vol. 241, No. 8

glucose and B-O-methyl-n-glucose were the main monosaccharide units.

Characteristics of LipopolysaccharidesBoth 11-B and 11-C were readily soluble in water or in a mixture of chloroform- methanol (2: 1, v/v), but only slightly soluble in ethanol or methanol. Proton magnetic resonance spectra of Compounds 11-B and 11-C indicated, by the signals in the region of 0.9 to 1.6 ppm, the presence of aliphatic carbon chains. There was a signal at about 2.2 ppm, which could be assigned to hydrogens adjacent to a moderately electronegative substituent, such as a carboxyl group. There was also a broad signal at 2.6 to 3.0 ppm in the spectrum of Compound 11-C suggesting the presence of a stronger electronegative group.

Acyl ester bond determination by the hydroxamic acid method showed the presence of 1 mole of ester per 7.2 moles of hexose unit in II-B, and 1 mole of ester bond per 8.3 moles of hexose unit in 11-C. About 20 mgeach of 11-B and 11-C were dissolved in 5 ml of chloroform-methanol (2:1, v/v), and 0.2 ml of 4 N

FIG. 9. Paper chromatography in Solvent A of the sugar components in the acid hydrolysates of the fractions from Fig. 11. Samples 1, 2, and 3 are from Peaks 11-A, 11-B, and 11-C, respectively, of Fig. 11. Sample 4 is from the NDR fraction (see text).

I I I I I

B i ii

i

i I I

i : \ , : : \

I '.

'. I :'

I 1 , ‘: I I ' : I : i

\ 1 '8. /',

90 180 270 360 450

Sephadex G-50

EFFLUENT(ml)

FIG. 10. Gel filtration of CMRW (see text) in 0.1 M acetic acid. The solid line indicates carbohydrate, and the dash.ed line, absorbance at 280 rnp. The portion above the solid bar contained the lipopolysaccharide.

I I I I DEAE-Sephadex

-H20.- NaHC03

A *

B 4

C

I ,_/

.’

100 200 300 400

0.10 m

H 0.05 g

z

1

FIG. 11. Gradient elution on DEAE-Sephadex, carbonate form, of the lipopolysaccharide from Fig. 10. Peaks 11-B and 11-C contain related MGP lipopolysaccharides.

NaOH was added. The solution, clear at first, gradually be- came turbid, and finally some precipitate was formed. After 10 min at room temperature, the suspension was neutralized by adding Dowex 50 (H+ form) and water, and the products were separated into lipophilic (lower layer) and hydrophilic (upper layer) portions as described previously (6). The lower layer, containing fatty acids, was treated with diazomethane, and the resulting methyl esters were analyzed by gas chromatography with a 10% diethylene glycol succinate column (31). Both 11-B and 11-C gave a peak corresponding to palmitic acid and an unidentified peak with a retention time of 0.89 relative to that of palmitic acid. Several minor fatty acid peaks were also observed, but were not identified.


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DISCUSSION

Isolation of 6-O-n~eth,yl-D-gl~ose PolysaccharidesChemical analysis of the cellular components of many Mycobucterium species has received considerable attention during the last three decades. However, the presence of 6-O-methyl-n-glucose in some of these fractions was noted only recently (7). The 6-O- methyl-n-glucose polysaccharide, which we have isolated from the water-soluble fraction of the deacylated lipid mixture, was fractionated on the basis of molecular size (Figs. 2 and 3), acidity (Fig. 4), and borate complex formation (Fig. 5). The final product appeared to be homogeneous by these three criteria.

Although both Al. phlei and M. tuberculosis produce the same type of compound, a slight difference in their characteristics of borate complex formation was observed (Fig. 5). In spite of this difference, the ratio of 6-O-methyl-n-glucose to o-glucose was the same for MGP (from JV. phlei) and the two corresponding substances from ill. tuberculosis. Since the procedures for ob- taining the crude lipids from these species were somewhat different, the subtle difference observed between the compounds from AT. phlei and M. tuberculosis during borate column chromatography should not be interpreted at this time as a species difference.

General Properties of JfGP-The signal position of anomeric hydrogens in the proton magnetic resonance spectrum indicates that most, if not all, of t,he glycosidic linkages are of the cy configuration. The optical rotation is consistent with this assignment, although the value is somewhat lower than those of most LY- (1 + 4)linked glucans.

Despite its molecular size and its average chain length, MGP failed to react with iodine in a fashion expected of an a-(1 -f 4)- linked glucan. This may result from the 6-O-methyl groups and from branching which would prevent formation of the helical structure needed for polarization of the iodine molecules (32).

The molecular weight determined in the ultracentrifuge (3060) was in good agreement with the equivalent weight (3080) and indicated the presence of only one acidic group per mole of MGP. The acidic group showed a pK, value of 3.5 and is probably a carboxylic acid, as is suggested also by the infrared and ultraviolet spectra. The ratio of 6-O-methyl-n-glucose to n-glucose was 6:4. Since the average anhydrohexosyl unit may be considered to have a molecular weight of 170, it can be calculated from this value that there are about 18 hexose units in the molecule.

Methylation of XGP---The average chain length of MGP was 8 to 9 hexose units, judged from the ratio of tetramethylglucose to trimethylglucose. Since MGP contains about 18 hexose units, it is obvious that there is at least one, but probably no more than one, branching point. The trimethylglucose component was the 2,3,6 isomer, indicating that the majority of the glycosidic linkages are 1 --t 4.

A di-O-methylglucose also was obtained from the methylated MGP. It was distinguished from 2,3-di-O-methyl derivative by paper chromatography, and its methyl glycosides were oxidiza- ble by periodate. The latter indicates that the substance was either 2,6- or 4,6-di-O-methyl-n-glucose. The dimethylglucose from the methylated polysaccharide behaved on paper and gas chromatography like one of the dimethylglucoses obtained by the partial demethylation of 2,3,6-tri-O-methyl-n-glucose. The methyl glycoside of this synthetic dimethyl ether was also susceptible to periodate oxidation. Since only 2,3-, 2,6-, or 3,6-di-O-methyl-n-glucose can be oxidized by periodate, it is

reasonable to conclude that the substance from methylated MGP is 2,6-di-O-methyl-n-glucose. The presence of this dimethylglucose is consistent with the branched structure suggested for MGP. If the main glucosidic linkages involve C-4 positions, then branching would be at a C-3 position.

Oligosaccharides from MGP-Since partial acetolysis is very effective for controlled fragmentation of polysaccharides, the same technique was used in this study to obtain oligosaccharides from MGP. However, the 6-O-methyl groups present in the oligosaccharides resulted in unexpected complication in separa- tion of the fragments. Although Peaks A and B (Fig. 6) appear to contain mono- and disaccharides, respectively, paper chromatography revealed that Peak A contained mainly D-ghlCOSe,

and Peak B, 6-O-methyl-n-glucose. This was confirmed by a successful resolution of a synthetic mixture of these sugars on the same column. Also, as indicated in the discussion below, di-, tri-, tetra-, and higher cligosaccharides consisting only of 6-O- methyl-n-glucose were found in Peaks C, D, E, F, G, and H, while maltose and maltotriose were found in Peaks B and C. The results suggest that an O-methyl group at position 6 con- tributed nearly as much molecular volume as a hexopyranosyl unit, as far as gel filtration properties are concerned.

Since each of the oligosaccharide peaks contained more than one component, preparative paper chromatography was used for further fractionation (Table II). The disaccharide Cr was obtained in the highest yield (7.8%). The result of borohydride reduction, periodate oxidation, and methylation indicated that this sugar was a (1 + 4)-linked disaccharide of 6-O-methyl-n- glucose. From its highly positive optical rotation, the glycoside linkage was concluded to be in cx configuration. Hence, it can be described as 0-(6-0-methyl-cY-n-glucopyranosyl)-(1 --f 4)-6- O-methyl-n-glucose. The trisaccharide Dr (4.4% yield) was analyzed by the same techniques, and was found to be 0-(6-0- methyl-a-n-glucopyranosyl)-(1 --) 4)-0-(6-O-methyl-ol-o-glucopyranosyl)-(1 + 4).6-O-methyl-o-glucose.

Since the disaccharide Cr and the trisaccharide Dr were obtained in comparatively high yield, they are considered to represent the predominant type of linkage occurring in MGP. This speculation was substantiated by the isolation of a series of cu-(1 + 4)-linked B-O-methyl-n-glucose oligosaccharides: El (tetrasaccharide), F (pentasaccharide), G (hexasaccharide), and H (heptasaccharide). Although complete structural analysis was performed only for Ci and Dr, the paper chromatographic properties of the oligosaccharides suggest that they belong to a homologous series (33).

The only hetero-oligosaccharide isolated (C,) was shown to be 0.a-o-glucopyranosyl-(1 --t 3)-6-O-methyl-u-glucose. The isolation of this oligosaccharide is compatible with the presence of 2,6-di-O-methyl-n-glucose in the methylated MGP, and indicates that C? represents a partial structure of the branching point. The failure of the A. niger cu-glucosamylase to act on Cz emphasizes the importance of the free hydroxyl group at C-6 position for the enzymic action.

Maltose (B3) and maltotriose (CL) were the only oligosaccharides composed entirely of n-glucose that were characterized. Fraction Bz, although it appeared to contain n-glucose as the only sugar constituent, was not digested by cr-glucoamylase from A. niger, and was not chromatographically identical with maltose. Fraction C3 also gave only n-glucose upon hydrolysis, but it was chromatographically different from maltotriose. Further struc-


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1908 Lipopolysaccharides Conta .ining 6-o-Methyl-o-glucose

tural characterization of Bz, as well as CB, must await preparation linkage, and the nature of the lipid portion require further in- of these oligosaccharides in larger quantity. vestigation.

Fraction Dz consisted only of n-glucose and behaved like Cq on paper chromatography. Therefore it was concluded that D2 was also maltotriose. On the other hand, Ez and Ea yielded glucose and some 6-O-methyl-n-glucose upon acid hydrolysis. Their peak positions during gel filtration and their RF values on paper chromatography suggested that they are probably penta- or hexasaccharides of n-glucose in which one or two of the monosaccharide units are 6-O-methyl-n-glucose.

As shown in Fig. 7, the presence of the B-O-methyl groups drastically changed the RF value of a given oligosaccharide. The effect is so pronounced that a pentasaccharide of 6-0- methyl-n-glucose (F) migrated even faster than maltose (BJ, and a trisaccharide (DJ migrated as fast as glucose (A).

In summary, the lipopolysaccharide may be visualized as a polysaccharide which contains about 18 moles of hexose and 1 mole of an unidentified carboxylic acid. The lipopolysaccharide is esterified by approximately 2 moles of fatty acid. The backbone of the polysaccharide portion is a chain of at least 7 residues of cr-(1 --f 4)-linked 6-O-methyl-n-glucose, to which is attached, by an ar-(1 + 3)-linkage, an oligosaccharide chain consisting of 3 to 4 residues of cr-(1 -+ 4)-linked n-glucose. The backbone structure may also contain several n-glucosyl residues.

Acknowledgment-1 wish to express my appreciation to Dr. Clinton E. Ballou for his valuable advice and generous support of this work.

Action of Amylases on MGP-/SAmylase from sweet potato showed no action on MGP, indicating that the polysaccharide has no nonreducing terminal with a sequence of three or more a-(1 + 4)n-glucopyranosyl units. On the other hand, exhaustive digestion with cr-amylase from pig pancreas liberated about 20’% of the total carbohydrate as mono- and disaccharides. The remainder of the substrate was not drastically smaller in molecular size than the original polysaccharide. These results indicate that the amylase acted on MGP as an exoglycosidase. No oligosaccharide larger than a tetrasaccharide could be found in the limited a-amylolysis products, suggesting that the seg- ment of the polysaccharide susceptible to the cu-amylase is no larger than a tetrasaccharide.

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2. NUNN, J. R., AND VON HOLDT, M. M., J. Chem. Sot., 1094 (1957).

3. WHITE, E. W., J. Am. Chem. Sot., 70, 367 (1948). 4. REICHSTEIN, T., Angew. Chem., 74, 887 (1962). 5. LEDERER, E.., Advak Carbohydrate Chem., 16, 207 (1961). 6. LEE. Y. C.. AND BALLOU. C. E.. J. Biol. Chem.. 239.1316 (1964). 7. LEE: Y. C:, AND BALLOT, C. E., J. Bid. Chem.,.239, PC3602

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8. CHANLEY, J. D., LEDERER, R., WAX, J., NIGRELLI, R. F., AND SOBOTKA, H., J. Am. Chem. SW., 81, 5180 (1959).

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Isolation of Intact Lipopolysaecharide-Isolation of the polysaccharide, MGP, from the chloroform-methanol extract of mycobacterial cells suggests that the substance existed originally as a lipopolysaccharide. However, the initial isolation of MGP

13. BARTLETT, G. R., J. Biol. Chem., 234, 466 (1959). 14. PALEG, L. G., Anal. Chem., 31, 1902 (1959). 15. MONTGOMERY, R., LEE, Y. C., AND WV, Y. C., Biochemistry, 4,

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Vol. 241, No. 8

__ _ involved a deacylation step which ‘would have altered the lipid portion of the compound. Therefore, an extraction scheme in- volving milder conditions was developed to isolate the polysaccharide in its intact form.

The aqueous fraction, CMRW (Chart l), containing most of the 6-O-methyl-n-glucose in the CMR fraction, was purified in aqueous systems by gel filtration and ion exchange chromatography. A column of DEAE-Sephadex (carbonate form) separated two compounds (11-B and 11-C, Fig. 11) containing 6-O- methyl-n-glucose and n-glucose. The signals at 0.9 to 1.6 ppm and at about 2.2 ppm in the proton magnetic resonance spectra of these compounds indicated that they contained fatty acyl esters, and the hydroxamic acid method showed the presence of 2.5 and 2.2 moles of esters per 18 moles of hexose unit in 11-B and 11-C, respectively. The solubility of 11-B and 11-C in the chloroform-methanol mixture and the appearance of turbidity during the deacylation reaction also suggest the presence of fatty acyl esters. Gas chromatographic analyses confirmed that methyl esters of fatty acids could be derived from 11-B and 11-C. These results, therefore, suggest that 11-B and 11-C are lipopolysaccharides, although the exact mode of lipid-polysaccharide

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Yuan Chaun Lee SpeciesMycobacterium-Methyl-d-glucose from

OIsolation and Characterization of Lipopolysaccharides Containing 6-

1966, 241:1899-1908.J. Biol. Chem.

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