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-galactose-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-lyxose 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 sugar 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 reducing sugar 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 <lI th e e nd ufthis pape r . the preci pil i.lle ll\U st be thoroughly was hed. in ord e r to avoid loss of mat e ria l. 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 optical rotation, 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.