Indian Journal of ChemistryVol. 37B, April 1998, pp.323 - 324

Rapid Communication

A simplified and practical methodfor methylation analysis of

polysaccharides

Chen Lia, Chenggang Huang", Zhixiao Ua* &

YaozuChen" b

'State Key Laboratory of Applied Organic Chemistry,Lanzhou University, Lanzhou 730000, PR.China'Department of Chemistry, Zhejiang University,

Hangzhou 310027, PR.ChinaReceived 27 January 1998; accepted 9 March 1998

Butyllithium is directly added to the solution ofpolysaccharides in dimethyl sulfoxide. The methylsulfinylcarbanion, as sugar-alkoxide-fonning reagent, is generated inthe solution. All the operations are performed as one-potreaction. Five polysaccharides from different traditionalChinese medicines and four dextrans have been methylatatedand satisfactory results achieved.

Although IH NMR, J3C NMR and 20 NMR havebeen widely used in structural elucidation ofpolysaccharides, methylation analysis is still anindispensable method to determine the types oflinkage between different glycosidic residues'".Characterization of partially methylated alditolacetate (PMAA) derivatives produced fromhydrolysis, reduction and acetylation, reveals thepositions of the unmethylated hydroxyl groups.Carbon atoms carrying these free hydroxyl groupsare involved in linking the sugar units inpolysaccharides. Moreover, the number of residuesin the average repeating unit, the nature of terminalunits, and the units at which branching occurs mayalso be deduced form the analysis of methylatedproducts.

There exists no perfect methylation method.Hakomori's procedure" is now almost universallyused among various methylation methods. In thisprocedure, CH3SOCH2-, generated from thereaction of dimethyl sulphoxide (DMSO) andsodium hydride (NaH), is considered to be theeffective basic agent which plays a critical role insugar alkoxide formation. However, it has to beprepared before use, and generally, the methylation

manipulation has to be repeated several times forthe completion of the reaction. Cincanu et al? havereported a method for sugar alkoxide formationusing a solid base (NaOH or KOH) in DMSOsolution. Their method is simple and fast incomparison with Hakomori's, for it is unnecessaryto first prepare methylsulfinyl methanide. Never-theless, York et al.6,7 indicated that as NaOH andCH3I are added simultaneously, oxidative side-reactions can occur which make the polysaccharidesdegraded and then, give incorrect results. Therefore,this method is more suitable for analysis ofoligosaccharides than for polysaccharides. In recentyears, the lithium salt of methylsulfinyl carbanion,obtained by treatment of DMSO with butyllithium,has been shown to have superior performance incontrast to sodium and potassium salts':". Thesemethods are characterized by short reaction periodand clean gas chromatograms, but the Li-methysulfiny carbanion still has to be preparedseparately before use.

We report herein a simplified procedure in whichbutyllithium is directly added to a solution of apolysaccharide in DMSO. The CH3SOCH2-,generated in the solution, participates in the reactionof sugar alkoxide formation inunediately. Alloperations are performed in one flask with only onetreatment. The procedure is both rapid andconvenient. Five polysaccharides from traditionalChinese medicines (Astragalus mongholiousBunges; Angelic sinensis oliv Diels; Codonopsispilosula Franch. Nannf; Asparagus cochinChinensis Lour; Sophora subprostrate et TChen)and four dextrans were fully methylated by thesimplified procedure. The methylated derivatives ofthe nine polysaccharides were analyzed by IR.Complete disappearance of the hydroxyl band in theregion of 3300-3600 em" from the spectra showedthe completion of perm ethylat ion. By analysis of thepartially methylated alditol acetates (PMAA) usingGC-MS, the structures of the four dextransaccorded with the data of known 1,6-linked glucans,and the results of the five plant polysaccharideswere in good agreement with the results obtained by

324 INDIAN J CHEM, SEC. B, APRIL 1998

Hakomori's procedure (repeated three times)for the same samples. (Details of theirstructures will be published elsewhere.) Thus,the viability of this method has been initiallyproved.

Experimental SectionGeneral. IR spectra were recorded in KBr

pellets on a FT-170SX spectrometer. Gas-liquidchromatography was performed on a Shimazu GC-9A apparatus equipped with a fused-silica capillarycolumn of OV-225 (0.29mrnx25m) and pro-grammed from 180-250 °C at 3 °C/min. GLC-MSanalysis was carried out on QP-IOOOA with a fusedsilica and with a programmed temperature-increaseof 4 °C Imin from 180-250 °C at a He flow of ImLimin.

Materials. Butyllithium was prepared in ourlaboratory according to the reported procedure'P".Other reagents were obtained from AldrichChemical Company. CH31 and DMSO were driedand distilled before use. Polysaccharides: DextranTIO (MW 10,000), T40 (MW 40,000), T70 (MW70,000), TllO (MW 110,000), Pharmacia. Fiveplant polysaccharides were extracted, isolated andpurified in our laboratory from the above-mentionedtraditional Chinese medicines separately.

Methylation procedure. Polysaccharide sample(5 mg) was dried in a conical flask (10 mL)containing a magnetic stirrer at 110°C for 6 hr in agun-drier under vacuum over P20S. Sealing theflask with a serum cap inserted with two syringeneedles, one needle for introducing argon, and theother for evacuation. Anhydrous DMSO (1.5 mL)was added by syringe after displacing gas in theflask twice. The solution was stirred at roomtemperature until the sample dissolved completely(about 30min.). 1 mL of 1.2 M butyllithium inhexane was then added dropwise, and the solutionstirred for 1 hr at room temperature. The gas in theflask should be displaced 1-2 times during thereaction. 1 mL of anhydrous methyl iodide wasadded through a syringe. After pulling out the twoneedles (stopping argon) and sealing the needle-cellwith laboratory film at once, and then stirring forlhr at room temperature, the flask was opened upand 2mL of water added to it. The derivatives wereextracted twice with 1 mL chloroform. The organiclayer was washed twice with water and dried over

anhydrous Na2S04 overnight. After evaporatingthe solvent under reduced pressure and drying theresidue at 50°C in vacuum overnight, thepermethylated product of polysaccharide wasobtained.

Hydrolysis. The product was hydrolyzed firstwith formic acid (2 mL, 90%) at 100°C for 6 hrand then with trifluoroacetic acid (2mL, 2M) at 120°C for 6 hr. The acids were removed afterhydrolysis by evaporation in a stream of argon in awater-bath at 50°C. CH30H was added three timesduring this step.

Reduction. The sample was dissolved in H20 (2mL) and. NaB~ (20 mg) added to it. The solutionwas kept for 2 hr at room temperature and thenneutralized with acetic acid. Three additionalevaporations were performed with methanol (I mLeach), followed by evaporation to dryness. Theresidue was dried in vacuum at 50°C over P20S for4 hr.

Acetylation. The residue was acetylated byheating in Ac20 (l mL) and pyridine (l mL) for 2hr at 121°C. The mixture was allowed to evaporateat room temperature, and then PMAA wasperformed.Acknowledgement

This project was supported by National NaturalScience Foundation of China. (Grant No.39730480.)References1 Fournet B, Montreuil J, Strecker G, Dorland L,

Haverkamp J, Yliegenthart F G, Binette J P & Schmid K,Biochemistry, 17, 1978 , 5206.

2 Montreuil J, Adv Carbohydr Chem Biochem, 37, 1980 ,157.

3 Yliegenthart J F G, Dorland L & Halbeek H Y, AdvCarbohydr Chem Biochem , 41,1983,209.

4 Hakomori S, J Biochem (Tokyo), 55, 1964 ,205.5 Ciucanu I & Kerek F, Carbohydr Res, 131, 1984,209.6 York W S, Kiefer L L, Albersheim P & Darvill A G,

Carbohydr Res, 208, 1990 , 175.7 Kiefer L L, York W S, Albersheim P s: Darvil A,

Carbohydr Res, 197, 1990, 1398 Blakeny A B & Stone B A, Carbohydr Res, 140, 1985 ,

319.9 Parente J P, Cardon P, Leroy Y, Montreuil J & Fournet

B, Carbohydr Res, 14J, 1985 , 4 I.10 Gilman H & Haubein A H, J Am Chern Soc, 66, 1944 ,

1515.II Gilman H, Beel J A, Brannen C G, Bullock M W, Dunn

G E & Miller L S, J .-Im Chem Soc, 71, 1949, 1499.