Studies on uronic acid materials

STUDIES PART

ON URONIC METHYLATION

XIV*.

DIMETHYL

SULPHOXlDE

ACID WITH

MATERIALS

THE SODIUM HYDRIDE-METHYL

IODIDE-

SYSTEM

D. M. W. ANDERSON AND

Departmenr (Received

G. M. CREE of Clrembfry, The Uniuersity, Edinburglt 9 f Great Britain)

December

znd, I 965)

1NTRODUCTION

Recently, there has been renewed interest in methods for methylating polysaccharides. Attempts have been made to improve yields and also to reduce the number of repetitive treatments required with the classical Haworth and Purdie3 techniques. Kuhn” and his co-workers used NJV-dimethylformamide as solvent with silver oxide and methyl iodide for permethylations and have since proposed3 the use of fV,N-dimethylformamide and dimethyl sulphoxide, separately or in admixture, as solvents for methylations with barium hydroxide and methyl iodide or dimethyl sulphnte. Our attempts to methylate Acacia gum polysaccharides, using barium salts. gave reasonable methoxyi contents, but emulsions, which were extremely difficult to brcak.tended to form.The use of dimethyl sulphoxide, with powdered sodium hydroxide and dimethyl sulphate, has been reported6 to give high yields of almost full) methylated. neutral polysaccharides, but, in our experience, this technique is less successful with acidic materials. We have investigated the use of sodium hydride and methyl iodide for the mtthylation of acidic Acacia polysaccharides dissolved in dimerhyl sulphoxidc. (A similar technique has been used to methylate gfycoproteins and neutral polysnccharides’, but its use with acidic materiaIs has not. to our knowledge. been reported previously). We have also used this reaction for the rapid methylation of mono- and di-saccharides: sodium hydride has been used in the methylation of monosaccharide dcrivutives in ether-type solvent&s or in N&V-dimethylformamide9. In the presence of acidic groups, the sodium hydride reaction might lead to the following side-reactions. with the formation of artifacts: (u) condensation of ester groups with the methylsulphinyl carbanion to give a sulphoxide; (b) in the presence of cstcr groups, /Sclimination could occur to give 4,gunsaturated acids*0 (esters of hot11 glucuronic and galacturonic acids can undergo” such eliminations): (~1 the product from (h) could react further to give a variety of products. The methylated products were therefore examined to ascertain whether such artlfocts had arisen.

STUDIFS

ON URONIC

ACID

MATERIALS.

XIV

163

EXPERIMENTAL

Paper chromatography was Izarried out on Whatman No. I paper with the following solvent systems (v/v): (a) butan-x-al-ethanol-water (4:r:5, upper layer): (b) butan-r-o&-acetic acid-water (4: 1~5, upper layer); (c) ethyl acetate-acetic acidformic acid-water (r&3: r:4). Gas-liquid chromatography (g,.l.c.) was carried out on columns (3 ft x 0.25 in) of polyethyleneglycol adipate (I 5% by weight. on acid-washed Celite, 80-100 mesh) at I 50”; the carrier gas was nitroger! at a flow rate of too ml/min. The chromatograph (Model S3A, Gas Chromatography Ltd, Maidenhead) was fitted with flame-ionisation detectors, Retention times are given reIative to that of methyl 2,3,4,6-tctra-U-methylj?-D-glucopyranoside. Weights recorded are those co\-rected for moisture content (by drying I o constant weight at 105”). Hydrolyses and rrlethanolyses were effected with N sulphuric acid for 8 h at 100’ and with 5% meth..lnolic hydrogen chloride for 6 h at 100~ (sealed tube), respectively. Reagent-grade dimethyl sulphoxidc and methyl iodide were redistilled before use. Methoxyl cements were determined by a specific,vapour-phase, infrared methodlz. Absorption spectra were taken with a Pcrkin-Elmer 137 UV Spectrophotometer. E.~periri?enta/pre~aufions. Powdcrecl sodium hydride was used without dificulty throughout our experiments; due :?recautions were obscrvcd when handling this reagent. Where preferred, the commercial dispersion of sodium hydride in oil mny PC used. A convenient method is to ;ldd the dispersion to 2::~ dimcthyl slllphoxidr: the layer containing sodium hydride dissolved in dimcthyl SLJphoxidc tn:ly then 1~. added to a solution. in dirncthyl suMoxide. of the material t:~ hc mcthylul~d. RE.sCIL’rs (a) Acidic polysaccharides Merlylation of IJWgum jront Acacia nubica (Bent/d.) The gum (4.64 g) was dissolved in dimethyl sulphoxide (250 ml), and powdcrcd sodium hydride (2 g) was added in small portions, with gentle stirring, during I Il. The solution turned yellow and, ultimately, became semi-solid. Methyl iodide (5 ml) was added dropwise with stirring during z h. The solution was stirred overnight. and one drop then gave a neutral reactiol.1 when added to water. A further three addition3 of sodium hydride and methyl iodide were made to the reaction mixture on iucccssivc days, as described for the first add tion. The mixture was then poured into water (1.5 I) to precipitate the methylated polysaccharide, and any excess or methyl iodide was removed by aspiration. The prec pitate was collected (centrifuge) and dissolved in chloroform. The supernatant solutic n (aqueous dimethyl sutphoxide) was cxtriictcti with chloroform, and the extract was combined with the solotion of prccipit,ltcd gtltn. The chloroform solution was wash:d with water to remove dimcthyI sL~IphoxicIc.

164

II. Y.

W.

ANDtRSON.

Ci. bl. C-ftft

dried (MgSOa), concentrated (rotary evaporator at less than 30 9. and then added lo light petroleum (b.p. 60-80”) to precipitate the methylated polysacsharide, which was removed by centrifugation and dried at room temperature under diminished pressure yield, 3.98 g (85%) (Found: OMe, 4o.8%). Single-step methylation of a degraded gum from Acacia nubica Degraded gum (1.86 g), prepared from A. nubicu gum by autohydrolysis. was dissolved in dimethyl sulphoxide (50 ml). Sodium hydride (z g) was added in small portions, followed by methyl iodide (5 ml), as for the whole gum. The mixture was stirred overnight, water (zoo ml) was added, and the mixture was extracted with chloroform. The isolation of the methylated, degraded gum (1.62 g. 87%) then foilowed the sequence already described for the whole gum (Found: QMc, 37.1 Y$)_ Tws for the formation of artifacts The following tests were made on the methylated whole-gum and methylated. degraded gum. The methylated gum, dissoIved in spectroscopic grade ethanol, showed no absorption at 235 mEA,indicating the absence of &elimination products. The methylated gum was tested for the presence of unsaturation by the thiobsrbituric acid method’*. Methylated gum (IO mg) was shaken with water (1 ml). and hydrochloric acid (5 ml, 0.5~) and thiobarbituric acid (IO ml. 0.01~) were then udded. The solution was immersed in a boiling water-bath for 30 min and cooled. and the absorption spectrum was examined. There was no absorption at 547 tlilr. indicating the absence of 4,5-unsaturated derivatives of DgJucuronic acid. Strwfl-scale mrtllytation of degraded A. nubica gum To degraded gum (30 mg) in dimethyl sulphoxide ( IO ml) was added. as drxrlhcd ahovc, sodium hydride (500 mg) followed by methyl iodide (1.3 ml). and the mixture was stirred ger!Jy overnight. A second addition of reagents was then made. the milrturc W;IS stirred overnight, and the methylated product (20 mp) was isolated as dcscribrd for the methylation of whole gum (Found: OMe, 4o.o%)_ A half-portion of the product was dissolved in chloroform and examined by g.1.c. The remainder of the product was hydrolysed and then examined by paper chromatography in solvents (a) and (6). In all of these examinations. the chromatograms were identical with those obtained from the product of the large-scale methylalion of degraded A. nubica gum. (A) Acidic ~nom- and di-saccharides Methyiatiun of D-glucuronic acid. b-Glucuronic acid [200 mg, chromatographically homogeneous in solvent (c) rind containing no b-glucurone] was dissolved in dimethyi sulphoxide (IO ml), and sodium hydride (500 mg) was added in small portions with gentle stirring during I h. Methyl iodide (1.4 ml) was added dropwise, and the mixture :a.s stored o*ieroight.

STUDtESON URONICACIDMATERIALS.XIV

165

The methylated product wa: then isolated by pouring into water, followed by extraction with chloroform at roo0J.ntemperature. The solution of the product in chloroform was divided into four parts. Portion I was examined direc?!y by g.!.c. and gave only three peaks, having relative retention times of 0.17, 2.14, and 2.8-1. The &St component had the same retention time as dimethyl sulphoxide. The other two peaks had the same retention times as were given by an authentic specimen of methyl (methyl 2,3,4-tri-U-methyl-a&D-glucopyranosid)uronate. Portion z was concentWed to dryness and subjected to methanolysis, and the neutralized (silver carbonate) solution was concentrated to dryness. A solution of the residue in chloroform was el amined by g.1.c. as for portion I. The same peaks were obtained, but with a decreastd proportion of dimethyl suIphoxide. Portion 3 was concef trated to dryness and hydrolysed. The product was neutralised (barium carbona:.e) and examined by paper chromatography in solvents (rr) and {b). Only one comr,onent, chromatographically identical with 2,3,4-tri-Omethyl-D-ghzuronic acid, was detected. Portion 4 was concentr-ited to dryness and warmed with water (L ml), and then hydrochloric acid (5 ml, 0.5’4) and thiobarbituric acid (IO ml, 0.01~) were added. The solution was kept for 30 nin at 100~ and then cooled; there was no absorption at 547 mfl. Methylation

of methyl

a-D-gkopyranoside

and methyl

a-n-galactopyranoside

Quantities and procedu .e were as for D-glucuronic acid, except that the mixtures were stirred for only I h aftrr addition of the methyl iodide. Examination by g.1.c. of the products from each glyc-osidc showed that only the fully methylated sugars were present. Methylation

of an aldobiourori ic arid

6-O-V-D-Glucopyranos qluronic acid)-D-galactose (60 mg) was dissolved in dimethyt sulphoxide (IO ml). Reaction with sodium hydride (500 mg) and methyl iodide (1.4 ml) was then carried out as described above for the smali-scale methylation of the degraded gum from A. nubica. A half-portion of the pr+)duct was hydrolysed and examined by paper chromatography in solvents (a) and (6) Only two components, chromatographically identical with reference samples of z,3,4.-tri-O-methyl-D-glucuronic acid and 2,3,4-tri-O-methylD-galactose, were detected. Th : remainder of the product was hydrolysed in mcthanoiic hydrogen chloride and neutra ised (silver carbonate), and the methanol was removed by evaporation. The product ‘vas dissolved in dry chloroform and examined by g.1.c. Components having retention times identical with those of the methyl glycosidcs of and of the methyl ester of z,3~4-tri-O-mcthyl-l,2,3,4-tri-O-methyl-D-galactose glucuronic acid were found*. *hIore added may also

irr prwfi In some rea:tions. be formed, presumabty tt rough

it has been observed that reaction of the disaccharide

z.3,5-tri-U-methyl-r~-~;ll~ct~se in the furanose form.

166

D. M. W. ANDERSON, G. M. CRt;E

DISCUSSION

The methylation

technique described gives complete methylation of monosaahain one stage, and methoxyl contents exceeding 36”/, have consistently been achieved for acidic polysaccharides by making 2-4 additions of the reagents. Careful tests have failed to indicate the formation of artifacts. The products are given in high yield, and satisfactory results have been obtained with 3o-mg samples. Yields of only zo-30% were obtained when classical methods were used, in mu&stage processes, to methylate acidic polysaccharides from A. se~@s and A. niloticu~4. In the light of this direct comparison, the sodium hydride method clearly offers distinct advantages. It is hoped that this communication will lead other investigators to assess the performance of the method with a wider range of polysaccharides.

rides and acidic disaccharides

ACKNOWLEDGEMENT

We are grateful to Sir Edmund Hirst, C.B.E., F. R-S., for his interest in these studies, and we thank the Science Research Council for the award of a maintenance grant (to G.M.C.). We acknowledge the generous gift by Messrs. I.C.I. Ltd_ of the gas chromatograph and its ancillary equipment.

Reaction of acidic poly-, mono-, and di-saccharides with sodium hydride and methyl iodide, in dimethyl sulphoxide as solvent, requires very few repetitive treatments to give highly methylated products in very gocid yield. Artifacts could not be dctectcd in the products.

RF FERFNCES t D. M. W. ANIXRSON, G. M. CR~E, J. J. MARSHALL, AND S. RAHMAN, CurbdqdrmeRe~.. z ( r96hr6~ N. HAWOHTJI. 1. Ckm. Sot., 107 (1915) 8. P 7 ?%RIm AND J. C. IWVINE,J. Ckm SW.. 83 (1903) 1021. 4 R KI~IIN. Ii. -rHISCWMANN, AND I. Lliw, Atrgew. C/rem., 67 (1955) 32. $ K. WAU~NFLXS, G. BECIITLER, R. KUHN, H. TRISCHMANN,AND H. EGGE, Angmr. Chem. /nrrm. W.. a U

Z, $ I 9

80 D8 14

I1 84

2 (1963) 515. II. C. SIIRIVASTAVA,P. P. SINGH, S. N. HARSHE, AND K. VIRK, Tetmhedmn fetrcrs, (1964) 493. S. IhKOMORI, 1. Rlochenr. (Tokyo). 55 (1964) 205. D. M. LBMAL. P, D. PACHT, AND R. B. WOODWARD, Tetrahedron, 18 (1961) 1275. 1. 8. ~WlMACOMOOAND D. PolcrshfouTn, Carbohydrate Res.. I (1965) ~28: J. S. BRIMACOWI~~ It. D. JONIIS, M. STACEY AND J. J. WILLARD, Carbohydrate Res.. z (1966) 167. P. A~J!MSHIIIM,M. NBUKOM, AND H. DEUEL. Arch. Biochem. Biuphys., go (rg6ot 46. D. A. Rms AND J. W. B. SAMUEL. personal communication. n. M. W. ANDIG~SON.S. GARBUTT, AND S. S. H. ZAIDI, Anal. Chim. Acta, 29 (1963) 39. M, A. HFHBII‘M,Ph. D. Thesis, Edinburgh. 1963. K. A. KMIAMALLA, Ph. D. Thesis, Edinburgh, 1965.