Journal of Research of the Nationa l Bureau of Standards Vol. 50, No.3, March 1953 Research Paper 2400 Synthesis of Lactose-I-C14 and Lactobionic-I-C 14 Delta Lactone From 3.f3-D-Galactopyranosyl-a-D-Arabinose I Harriet L. Frush and Horace S. Isbell Lact ose-I-CH, which has been prepared for t he first time, was obtained in 38-percent radiochemical yield by the cyanohydrin synthesis with 3- (JS-D-galactopyranosyl)-n-arabinose. The latter substance was prepared in crystalline form and its reaction with N a CH]'\ was studied under a variety of conditions. Acid catalysts favor production of the epimer having the mannose configuration. An improved procedure is given for the lactonization of lactobionic acid; by slow crystallizat ion from methyl cellosolve, large crystals of high-purity lact obionic-I-C14 delta lactone were obtained . The lact one was reduced to lactose by sodium amalgam in the presence of sodium acid oxalate with a yield (by analysis) of 84 percent. A modification of the process in which the separation of the epimeric acids or lact ones is omitted, was also found t o be practicable for t he production of lactose-I-CIl. 1. Introduction To provide a basis for directing the cyanohydrin reaction to lactobionic nitrile rather than to 4-galac­ There are many unsolved problems in human and tosyl-mannonic nitrile, the condensation of 3-galac­ animal nutrition that can be attacked by means of tosyl-arabinose with cyanide was studied by means of certain position-labeled carbohydrates. Lactose, one a tracer technique. Tlli involved conden ation of of the most important of these carbohydrates, has no t the sugar with labeled cyanide in buffered solution , heretofore been available in radioactive form. This hydrolysis of the cyanohydrins, and separation of the paper repOTts the preparation of high-activity labeled lactobionic acid as the calcium lactobionate I-Ou-Iabeled lactose in good yield by means of a calcium bromide double alt [4] . It was found that cyanohydrin synthesis on 3-galactosyl-arabinose. sodium bicarbonate and presumably other general acid catalysts in the cyanohydrin mixture favor 2 . Discussion of the Experimental Method formation of the 4-galacto yl-mannonic epimer; cal­ cium chloride or sodium carbonate, however , favor The over-all plan for the production of labeled formation of the 4-galacto yl-gluconic (lactobionic) lactose consisted of the following steps: epimer. These results obtained by tracer methods, Lactose correspond qualitatively to those obtained by optical rotation and isolation methods in the tudy of the 1E lectrolytic oxidation [1] 2 cyanohydrin synthe i for the production of D-glu­ cose-I-Ol4 and D-mannose-l-0 14 from D-arabinosc [5]­ Calcium lactobionate In the preparation of labeled lactose, both the cal­ H 20 2+ Fe+++ [2] cium chloride method [6], and the sodium carbonate 1 method for conducting the cyanide condensation 3-(JS-D-Galactopyrano yl)-D-arabino e 3 were used, but the lat ter is preferable because it Cyanohydrin reaction avoids the introduction and subsequent troublesome 1 removal of the chloride ion. In early preparations Epimeric nitriles by the calcium chloride method, lactobionic acid wa isolated from the cyanohydrin hydrolyzate as the 1H ydrolysis crystalline calcium lactooionate calcium chloride Epimeric aldobionic acids double salt [7] . The procedure involved several steps later eliminated by condensation in the presence of Lactonization and sodium carbonate, and crystallization of lactobionic separation of epimers lactone from the hydrolyzate. 'rhe latter step wa r 1 made practicable by the development of a procedure Lactobionic-a-Iactone 4-(JS-D-Galactopyranosyl) for the complete conversion of lactobionic acid to -D-mannonic acid the lactone. 1N aHg", reduction To ascertain favorable conditions for reduction of the lactone to lactose, numerous samples of nonradio­ 1Cry tallization active lactobiomc delta lactone were reduced with Lactose-I-CH various quantities of sodium amalgam in the presence 1 Part of a project on the development of methods for the synthesis of radIo­ of sodium acid oxalate. The reduction wa con­ active carbohydrates, spon80red by the Atomic Energy Co=ission . ducted in the apparatus described previously [5], but • Figures in brackets indicate t.he literature references at the end of this paper. • The alpha modification of this sugar, 3·(p·D·galactopyranosyl)·a·D·arahinose modified by a sidearm that permitted addition of had been crystallized several years ago by Zempl ~ u [31 , but no seed crystals were available in this laboratory. However, after abo ut 2 months, crystals were ob­ amalgam while the solution was being tin'ed. It tained from a sirup containing methyl cellosolve aud isopropyl ether. There­ was found by analysis that, under suitable conditions, after, crystallization of other preparations was readily effected by use of seed crystals. 84 percent of the lactone i converted to the sugar. 133 After removal of sodimTI salts, the lactose was sep­ anion exchange resins.' The combined efRuent and al'ated by crystallization from aqueous methanol wash liquor was evaporated under reduced pressure with the addition of isopropanol. In radioactive to a thick sirup, which was dissolved in about 50 ml preparations, additional labeled sugar was recovered of methvl cellosolve. From this solution several from the mother liquor by the carrier technique. In frac tions" of siruP? 3-galactosyl-arabinose were pre­ a preparation starting with 3.5 mc of C14-labeled cipitated by the addition of isopropyl ether. Each sodium cyanide the yield of labeled lactobionic delta of the fraction s was dissolved in methyl cellosolve lactone was 54 percent. In the reduction step 68 and th e solutions, brought to incipient turbidity by percent of th e lactone was isolated as crys talline means of isoprop:d ether, wer e stored in a desiccator lactose. The over-all radiochemical yield of lactose- over calcium chloride. After 2 months, the sirupy 1-C14 was thus 37 percen t. residue of one fraction crystallized. The r emaining It was also found practicable to prepare lactose fractions crystallized when seeded, and a total of without the separation and purification of any inter­ 50 g of crude crystalline 3-(j3 -D-galactopy-ranosyl)-a-D­ mediate, by reducing the crude lactone mixture pre­ arabinose was obtained. To r ecrystalliz e, the mate­ pared from the cyanohydrin reaction. The resulting rial was dissolved in a minimum of hot water, and solution gave one crop of crystalline lactose without the solution was filt ered with the aid of deeolorizin6 carrier ; additional crops, obtained by use of carriers, carbon and treated 'with isopropanol almost to the raised the radioch emical yield to 38 percent. point of turbidity. It was also recrystalliz ed from a thick aqueous sirup (80° Brix) by the addition of 3. 'Experimental Details m ethyl cellosolve. [albD of the purified product was - 62.5 0 at equilibrium (water , c= 2) in approximate 3.1. Preparation of 3-(j3 -D-Galactopyranosyl)- a-D­ agreement with the value previously reported by Arabinose Zemplen [3] ([al1D= - 50 .3°"""'-63. 1°). A mixture of 375 g of calcium lactobionate penta­ 3.2. Epimeric Proportions of the Products Formed by hydrate, 20 g of barium acetate monohydrate, and Condensation of 3- (j3 -D-Galactopyronosyl)-D-aro­ 10 g of ferric sulfate (with abou t 6H20 ), was added to 3 liters of boiling wa ter [2]. The mixture was binose with Cyanide cooled to 350 C, and 120 ml of 30-percent hydrogen The experiments outlined in table 1 were conducted peroxide was added . During the ensuing reaction to determine optimum conditions for the formation period, the solution was cooled to maintain a temp­ of the lactobionic epimer. In each experimen t 2 ml erature of less than 50 ° C. After about 1 hI' the of a solution containing 0.0636 millimole of 0 14_ solution, which had turned darI-.: bn~)\vn , was cooled labeled cyanide with 5.12 J1.c of activity was placed to 40° C, and a second 120-ml portlOn of hydro!$en in a glass-stoppered tube. The solution was frozen peroxide was added. ,Vhen the seco.n~ reactlOn in a carbon dioxid e-ace tone freezing mixture to pre­ was complete, about 25 g of a decolol1zmg carbon ven t loss of cyanidc, after which 2 ml of a solution, and 10 g of eal ci~m carbonate were added. The containing 0.0705 millimole of 3-galactosyl-arabinose mixture was filtered , and the fil trate was evaporated and the v arious substances listed in table 1, was under reduced pressure t o a sirup of about 80-percen t introduced . The mixture was shaken gently until total solids. The sirup was mixed first with 300 ml the ice dissolved and was then allowed to stand at of methanol and then 600 ml of a 1: 1 mixture of room temperature. After 48 hI', th e mixture was methanol a~d ethanol was added. The resulting heatcd at 80° C for 5 hI' to eff ect hydrolysis, and precipitate was separated by fil ~ra t ion , and was then was evaporated to dryn ess in an air stream. wash ed, fu'st on the filter, then III a beaker, and T he residue in each tube was dissolved in a few drops finally on the 6.1te l' with a total of 500 ml of ethanol. of water, and the solution was neu tralized to the The r esidue 'was discarded. The filtrate and wash end point of phenolphthalein by the addition of liquor deposited some gummy material fr~m which dilu te hydrobromic acid. N omadioactive calcium the clear liquid was decanted . The r esIdue was lactobionate calcium bromid e (531.3 mg of Ca triturated with 100 ml of ethanol, and the ethanol (CI2H 21 0 12 )2 .CaBr2 .6H20 ) [4] was dissolvcd in each hy­ extract was fil ter ed .