JOU RNAL OF RESEAR CH of t he Na tional Bureau of Standa rds - A. Ph ysi cs a nd Chem istry Vol. 7 1A, No.2, March- April 1967

Synthesis of ex -L-Fucose-I-1 4 C (6-Deoxy-L--I-14 C)

H. S. Isbell, H. L. Frush, and N. a. Holt

Institute for Materials Research, National Bureau of Standards, Washington, ·D.C. 20234

(October 31. 1966)

ll'-L-Fucose- / -14C was synthesized in a radiochem ical yie ld of 30 perce nl. The synthesis in volved degradati on of nonradioac ti ve L' fu coni c acid to 5-deoxy-L- a nd synthesis from this of a·L-fu cose· J.14C by use of 14C- labeled cya ni d e in the cyanohydrin reacti on. The resulting epi me ric, 14C· labeled aldoni c acids were separated as eithe r the bari u m o r the sodi u m salt s. Bo th salt s of L·fuconi c acid crystalli ze more readil y tha n the correspond ing salt s of the e pi me ri c 6·deoxY· L·talo ni c acid. T he preparati on of barium L-fu conate b y the electro lyti c ox idati on of L·fucose in the prese nce of bHium car bona te a nd barium bro mi de is descri bed.

Key Words: Carbon·14·labeled (.fucose, C· fuconic acid·I . I'C, L-fucose· / · 14C, radi oactive carbo· h ydrates, s ynthesis of radioactive s ugars.

1. Introduction tio n is favored by the presence of a general acid­ catal ys t. P olysaccharides containing L-fu cose are found in A porti on of the crude 5-deoxy-L-Iyxose was then 14 the cell wall s of marine algae, in a few gums, and in treated with spdium cyanide- C in th e presence of certain substances of animal ori gin, s uch as red blood­ sodium bicarbonate. The cyanohydrins were carried cells, blood serum, gastric mucin, ova ri an cystofluid , through th e reaction steps shown in fi gure 1. The 4 and many other materi als of biological importance. resulting a -L -fu cose-l -1 C crys talli zed directl y from Because of the widespread occurrence of L-fu cose, the solution (without th e intermedi ate pre parati on of the 14C -l abeled is a valuable research tooL We phenylh ydrazone, a ste p ordinaril y fo und necessary have carried out the synthesis of a -L-fu cose-l-1 4C, and in the preparati on of L-f ucose from seaweed). The 14 have suppli ed the radi oacti ve sugar to nume rous sodium 6-d eoxY- L-talonate- l - C obtained as a by­ work e rs engaged in bi ological research. The method produ ct was retain ed for future use. developed for the pre parati on is outlined below. The a-L-fucose used as the starting material for th e separati on of the labeled product was obtained 2. Experimental Details from the seaweed Ascophyllum nodosum by a pro­ 2.1. Barium L-Fuconate [2] cedure developed earlier, as described in [1].1 The sugar was oxidized electrolyti cally by the method of A mixture of 20.5 g (0.125 mole) of a-L-fucose, 4 g of Isbell and Frush [2], and the product was separated barium bromide, and 12.5 g of barium carbonate in as crys talline barium L-fu conate. The barium salt 500 ml of water was placed in a 2-liter, 3-n ecked fla sk was converted into the calcium salt, and this was fitted with two graphite electrodes (1 cm in diameter) degraded to 5-deoxy-L-l yxose by the method of Ruff [3] and an effi cient stirrer. The flask was cooled in ice as modifi ed by Hockett and Hudson [4], the proportion water, and a dir~c t current of 0.2 A was passed through of redu cin g s ugar formed from calciu m L-fu conate the stirred solution. After 34 hr (6.8 A-hr), a test for being determined by iodine ti trati on in alk a lin e solu­ with Benedi ct reagent [8] was faint, ti on [5]. In ord er to ascertain conditio ns fav orable and the electrolys is was stopped. The residual bro­ for formation of the fuco (gaLacto) confi guration in mine and the barium bromide in the solution were th e cyanohydrin reacti on, test syntheses, beginning res pectively re moved by addin g 5 g of a decolori zin g with 5-deoxy-L-l yxose, were conducted at the tracer carbon and 4.2 g of sil ver s ulfate. T he sus pension level, and the produ cts were analyzed by the isotope­ was filt ered , and the residue on the filt er was washed dilution technique [6 , 7]. The results s howed that with hot water. 2 The fi ltrate was treated with hy­ forma ti on of a hi gher proportion of the fuco confi gura- drogen s ulfide to remove residual silver salts, refil-

2 Barium L-fuconal e lIlay occas iu na ll y c rystallize before filtrat iun is conduc ted . H ence. r I F'ij.!;urcs in brad,cls indicate the lite rature refer Clltes

  • 133 L-Fucose 1electrolytic oxidation Barium L-fuconate t Calcium L-fuconate

    1degradation (H 20 2 + Fe2 +) 5-Deoxy-L-lyxose

    1cyanohydrin reaction (Na14CN)

    Epimeric cyanohydrins-1-14C

    hydrolysis, and separation of sodium salts

    Sodium L-fuconate-1- 14 C Sodium 6-deoxY-L-talonate-1-14 C ! L-Fucono-1,4-lactone-I-14C 1reduction (N aHg,r) L-Fucose-I- 14C .

    FIGURE 1. Synthesis of a-L-Fucose-l-'4C. tered, and concentrated under reduced pressure to a passed through a column containing 25 ml of mixed thin syrup, which was diluted with ethyl alcohol to the (1 : 1) cation· and anion-exchange resins. The column point of incipient turbidity and nucleated with crystal­ was well washed with water, and the effluent was line barium L-fuconate. concentrated to about 25 m!. Titration of an aliquot After several hours, a crop of crystals was separated with iodine in alkaline solution [5] showed the presence by filtration; by reconcentrating the filtrate and dilut· of 3.25 mmoles of reducing sugar (54% of the theoretical ing it with ethanol, a ser.ond crop was obtained; com­ yield). bined yield, 26 g (84%). The salt, Ba(C6Hl106h, was recrystallized from hot water. It was only slightly 2.3. Reaction of S-DeoxY-L-lyxose With Sodium Cyanide soluble in water, and showed no , in In order to ascertain favorable conditions for forma­ agreement with the reported properties of barium tion of the L-fuconic cyanohydrin from 5-deoxy-L­ L-fuconate [9).3 lyxose, the following aqueous reaction mixtures were sealed in glass ampoules and kept for 4 days at about 2.2. S-Deoxy-L-Iyxose 5°C. Under vigorous stirring, 1.5 g (3 mmoles) of barium 1. 0.079 mmole of 5-deoxy-L-Iyxose in 0.65 ml of L-fuconate in 50 ml of hot water was successively solution, treated with 6.2 ml of 1 N sulfuric acid and 350 mg of 0.07 mmole of sodium cyanide-14C (0.73 mCi/ calcium carbonate. Without removal of the precipi­ mmole), and tate, the mixture, now containing a solution of calcium 0.30 mmole of sodium hydroxide in 1 ml of L-fuconate, was placed in a water bath at 50°C; 62 solution. mg of barium acetate, 63 mg of ferrous sulfate, and, finally, 0.75 ml of 30-percent hydrogen peroxide were 2. 0.079 mmole of 5-deoxy-L-lyxose in 0.65 ml of added, whereupon the mixture turned' brown. After solution, 1 hr, a second quantity (0.75 ml) of hydrogen peroxide 0.05 g (approximately) of solid carbon dioxide, was added. After an additional hour, the suspension 0.07 mmole of sodium cyanide·14C (0.73 mCi/ was diluted with 25 ml of methyl alcohol and 50 ml of mmole), and ethyl alcohol, and filtered. The filtrate was concen­ 0.30 mmole of sodium hydroxide in 1 ml of trated under reduced pressure to about 25 ml, and solution. In the preparation of the second reaction mixture, 3 For IHc paring s mall qu a ntities of L-fuconic acid. oxidation of L-fuc ose by bromine [101 the solid carbon dioxide was shaken with the sugar or by iodine ill alka line solutio n 15 1 is morc conve nient than the electrol yti c method de­ "cribcd here. The result ant brumide or i(lclide is removed as the silv er salt . solution and allowed to vaporize before the alkaline

    134 cyanide solution was added. After 4 days, each exchange resin. The effluent was concentrated under a mpoule was opened, and the contents were hydrolyzed reduced pressure, and then divided equally between and treated with cation·exchange resin in a manner two reduction tubes described previously [6]. The . similar to that described below for the large pre pa· aqueous solution in each tube was concentrated to a . ration. Barium L·fuconate 4 (l00 mg) was added to syrup by means of a stream of air introduced through each solution; after several recrystallizations of tbe a capillary tube. The acid was lactonized by heating salt, the activities and the corresponding yield s of the syrup at 70 °C with glacial acetic acid. After 1 hr barium (,·fuconate·I-14C in the cyanohyClrln reaction of heating, the completeness of lactonization was were as follows: (1) 0.22 /LCi/mg, 46.5 percent; (2) tested by paper chromatography and by scanning of 0.28 /Lei/mg, 60.5 percent. He nce, tbe presence of the radioactivity in a paper-chromatogram scanner. bi carbonate (a general acid·catalyst) favors the for­ (U nlactonized acid remains near the origin in the usual mation of the L-fuconi c epimer. de veloping solvents.) Heating was repeated for an hour, and the tubes were allowed to stand over soda­ lime in a desiccator for several days. Lactonization 2.4. Sodium L-Fuconate-l-14 C appeared to be nearly complete, as shown by test chroma tograms. In a 100-ml, round-bottomed flask in an ice-bath, The lactone (not crystalline) was reduced by the 6 ml of 0.13 M 5-deoxY-L-lyxose (0.78 mmole) and 202 pre viously described method [6 , 11], by use of 20 ml mg of sodium bi carbonate were shake n witb a small of water in each tube, and, per mmole of lactone, 4.6 g lump (0.1 g) of solid carbon di ox ide. To tbis mixture of 5-percent sodium amalgam in the form of pellets was added 5 ml of a cold solution containing 0.71 [12] and 3.2 g of sodium hydrogen oxalate. The reduc­ mmole (9.36 mC i) of sodium cyanide- 14C and 1.72 ti on produc ts in the tubes were combined, and the mmoles of sodium hydroxide. The mixture was ke pt residual acids were ne utralized with sodiu m hydroxide. in a refri ge rator for one day, '1nd at room te mperature Five volumes of methyl alcohol were added, and the for four days. Tbe n, 530 mg of sodium carbonate in me rc ury and precipitated salts were separated on a 10 ml of water was added , and the fla s k, equipped with filt er, wa shed with methyl alcohol and di scarded. The an air condenser, was heated in a boiling-water bath filtrate was concentrated under reduced pressure to until evolution of am monia had ceased. The solution about 10 ml and diluted with fiv e volumes of me thyl was stirred with 5 ml of a cati on-excha nge resin 5 and alcohol. The salts that precipitated were removed passed through a column co nt aining 20 ml of the resin. by filtration , a nd the filtrate was concentrated to The efflu ent (co ntaining 7.74 mCi of activity) was re move the alcohol. The syrup was di ssolved in partially concentrated unde r reduced pressure (to water, a nd the solution was passed through a column remove carbon dioxide), and ne utrali zed with sodium containing 25 ml of a 1:1 mixture of cation- and anion­ hydroxide. exchange res in s. Nonradioactive sodium L-fu conate (100 mg) was The e fflu e nt (w hi c h was shown to be salt-free by added, and the solution was concentrated to a syrup testing with a co mme rcial co nductivity·me ter) was which was satura ted with meth yl alcohol a nd nucleated. freeze-dried. The residue was di ssolved in me thyl The crystals of sodium L-fu co nate-/-14C that formed alcohol, isopropyl alcohol was added to the point of were separated , and washed with 50 percent aqueous incipie nt turbidity, and the solution was nucleated. me thyl alcohol. The mother liquor was diluted with The crystals of a-L-fu cose-l -14C that form ed in 24 a solution of 500 mg of nonradioactive sodium L-fu con­ hr were separated and recrystallized; yield, 113 mg ate and co ncentrated to a syrup, and the crystal s that having an activity of 1,168 /L Ci. A second crop, weigh­ formed were co mbined with the first crop and rec rystal­ ing 214 mg after recrystallization, and having an 1 lized; yield, 522 mg, 4,330 /LCi. A third c rop, obtained activity of 655 /LCi, was obtained from the mother by use of nonradioactive carrier, contained 40 /LCi of liquors by use of 200 mg of nonradioactive L-fucose activity_ The total yield, 4,370 /LCi , is 46.7 percent, as the carrier. 14 based on the sodium cyanide- C used. When the ion-exchange column was eluted with 10- The mother liquors, containing approximately 3 percent aqueous acetic acid, it yielded salts having mCi of activity, chiefly in the form of sodium 6-deoxy­ an activity of 2,057 /LCi . The effluent was freeze­ L-talonate, were held for future use. dried, and the residue was dissolved in water and passed through a column containing 15 ml of a cation­ 2.5. L- Fucono-l,4-lactone-l-14C and Reduction to exchange resin. The acid solution was concentrated L-Fucose-I-14C under reduced pressure, transferred to a reduction tube, lactonized , and reduced with sodium amalgam A solution containing 2.58 mmoles of sodium L­ as described a bove. After removal of the salts and fuconate·l-14C having an activity of 4,330 /LCi was unreacted L-f'uconic acid-1- 14C, the sugar solution was passed through a column containing 10 ml of cation- combined with th e mother liquor of the first prepara­ r tion , yielding crystalline a·L-fucose-l-14C which, when j "Although barium L-fuconate was used in the tracer experiments, sodium L-fuconate recrystallized , weighed 288 mg and had an activity was la ler f(lund tu ha ve more sati s fa ctury crysta lli zi ng: properties. Accordingly_ the laller of 777 /LCi. 1 sa lt was used fU I" separatiun uf the c pimc rs in the hi gh-act ivit y pre paratiun re purted in the fu lluw in g sect io ll s. The elution, lactonization, and reduction procedures ~ This caused the evolution of carbon dioxide. and obviated. in part. the formation of gas pockets ill til e resin culumn. The culumn was a lso back· washed several times during; use. were again repeated, and yielded 222 mg of a-L­ Fina ll y. all I>f th e resin was washe d until it was s ubsta ntially free frum radiuacti vit y. fucose-l-14C having an activity of 222 /LCi. Thus, the 135 total radiochemical yield of a-L-fucose-1- 14C from [5] C. M. Kline and S. F. Acree, BS J. Res. 5,1063 (1930) RP2.47 ; sodium L-fuconate-1- 14C was 2.8 mCi or 65 percent; Ind. Eng .. Chem. , An al. Ed. 2, 413 (1930). 14 [6] H. L. Frush and H. S. Isbell , J. Res. NBS 50, 133 (T953 ) RP2.400. the yield of the labeled sugar from sodium cyanide- C [7] H. L. Frush and H. S. Isbell , J. Res. NBS 51,307 (1953) RP2.458. was 30 percent. [8] S. R. Benedict, J. BioI. Chern. 5, 485 (1909). [9] A. Miither and B. Toll ens, Ber. deut. chem. Ces. 37,306 (1904). [10] C. S. Hudson and H. S. Isbell . J. Am . Chem. Soc. 51,2225 3. References (1929). .; [11] H. S. Isbell, H. L. Frush, and N. B. Holt , J. Res. NBS 53, 217 [1] F. 1. Bates, ed., Polarimetry, saccharimetry and the , (1954) RP2536. NBS Circ. C440 (1942) p. 460. [1 2] H. S. Isbell , H. L. Frush, and N. B. Holt, J. Res. NBS 64A [2] H. S. Isbell and H. L. Frush, BS J. Res. 6, 1145 (1931) RP328. (phys. and Chern.) No.1, 135 (1963). [3] O. Ruff, Ber. deut. chern. Ces. 32,550 (1899) ; 35,2360 (1902). [4] R. C. Hockett and C. S. Hudson, J. Am . Chem. Soc. 56, 1632 (1934). (paper 71A2-444)

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