Preparation Of D-Arabinose-1-C 14 And D-Ribose-1-C 14

2.4. Prep a r a tion of Potassium D-Arabona te-l -C I4 fromD-Erythrose by the Cyanohydrin Synthesis in thePresence of Ammonium CarbonateIn another preparation of D-arabinose-1-CI4, theammonium cal'bonaLe meLh od was employed. Fivemilliliters of a solution containing 0.7 millimole ofsodium hydroxide and about 0.7 millimole of sodiumcyanide with 5.4 mc of carbon 14 was frozen on thewalls of a small glass-stopper ed t ub e. Five milliliters of approxim ately 1 A1 ammonitUn carbonatewas then frozen in the tube, and finally 1 ml of asolution containing 1 millimole of D-erythrose 8 wasadded. The mix ture was allowed to thaw in an icebath and was then kept at room temperature for3 days. It was treated essentially in the mannerdescribed in section 2.3, except that a crop of potassium D-arabonate-1-C 14 (52 mg, containing 2,030MC) 9 was separated without the addition of carrier.The total radiochemical yield (2,446 }lC, or 45 percent) was slightly less than that of the formerpreparation . However, as greater mechanical losswas incm-red in this prepara tion because of th e higherlevel of activity, it is believed that the two methodsare equally satisfac tory in the syn th esis of potassium D-arabonate-l-C I4.2 .5 . Preparation of D-Arabono-'Y -Lacton e-l-C 14 a n dReduction to D-Ar abin ose-l -C14An aqueous solution con taining 446 mg (2.19millimoles) of potassium D-arabonate wiLh 1,190 MCof activity was passed through a column of cationexchange r esin (see foo tnote 7). The column wasthoroughly washed, and the effluent was evaporatedat 60 C substantially to dryness. The residue,dissolved in a few milliliters of methanol, was transferred to a r eduction tube such as that describedpreviously [3] . Briefly, it consists of a test tube 2.8by 20 cm fitted with a stirrer reaching to the bottomof the tube and with a sidearm for the introductionof the amalgam. The solution in the tube was concentrated in an air stream and seeded with D-arabono'Y-lactone. 1O The tube was finally stored in a desiceator, and the contents was moistened from time totime with methanol. When lactonization was complete, as indicated b y the disappearance of anysirupy phase (1 or 2 weeks), the material was reduced with sodium amalgam by Lhe proceduredescribed later.The lactonization described above required considerable time. In ord er to avoid this delay, thefollowing rapid procedm-e was developed: Twomillimoles of potassium D-arabonate-l-CI4 was passedB Because of uncertainty in the concentration of the cyanide solution, theerythrose was in larger excess than would ordinarily bc added.'Except for t he analyses in table 1, all assays of carbon 14 were made in formamide solution [l J . Details of tbe analysis of tbis crop of potassium D-arabon ate1-0" are illustrative: T he material was dissolved in water and t he volume wasadjusted to 10 1111. A 5O-XaJiquot was dil uted to 1 ml with water in amicromixingpipette. A 2O-X aliq uot of t his was dil uted to 1 ml With form amide, and thesolu tion gave 74.9 counts per second in a 2-. proportional counter in which 1count per second is eq ui valent to O,002711'c.10 Tbe free acid may crystallize from the freshly prepared sirup at t his point.Conversion of t be acid to the lactone at room temperature requires at least a week.through a column of cation-exchange r e in, and theeffluent was concentrated tUlder reduced pressure toa sirup, which was tran ferred wi Lb a few drops ofwater to a r eduction tube. Five milliliters of glacialacetic acid was added, and tbe tube was placed in aboiling-water batb and heated in a stream of airfor 5 hr. After 1 hr of this period, 1 ml of water and5 ml of acetic acid were added. At tln'cc I-hI' intervals thereafter, tbe material was treated with 5 mlof acetic acid only. At the end of the heating period,the pale-yellow residue was dissolved in 3 or 4 dropsof methanol, seeded with D-arabono-'Y-lactone, andplaced in a desiccator over calcium chloride. Itcrystallized well within 24 hr.When samples of nonradioactive D-arabonic acidwere subj ected to this procedm e, the optical rotations, [altO, 24 and 72 In' after the sirups were seededwith the crystalline lactone were, respectively, 66 .0 and 67 .6 (based on th e weight of thelactone). These values correspond approximatelyto conversion of 91 and 94 percent, respectively, ofthe D-arabonic acid to the lactone.Prior to the preparation of th e labeled sugar, aseries of sodium amalgam reductions was carriedou t on the nonradioactive lactone in order to determine optimum conditions. Sodium acid oxalatewas employed as a neutralizing agent [6], and afterr emoval of the salts in the m anner describ ed below,the reduction product was analyzed for reducingsugars by the modified Scales method [13]. It wasfound that a maximum yield of sugar (85 percent)is obtained with 4.6 g of 5-percent sodium amalgamper millimole of lactone.In a typical reduction of the labeled lactone to thesugar, the reduction tube, containing 6.4 g of crystalline sodium acid oxalate and the lactone preparedfrom 446 mg of potassium D-arabonate-l -C 14, having1,190 MC of activity, was placed in an ice bath. Afterthe addition of 20 ml of ice water, the stirrer was adjusted to produce vigorous stirring at th e bottom of thetube, and 9.2 g of 5-percent sodium amalgam pellets 11was added tlu'ough the side arm. Stirring wa continued for 3 In', at the end of which tim e the mercmywas removed, and the aqueous mixture was dilutedwith 5 volumes of methanol. The crystalline saltswere separated , washed with methanol, and discarded, after it was ascertained tha t they were inactive. Phenolphthalein indica tor was added to themother liquor, previously cooled in ice water , andthen aqueous sodium hydroxide until a faint butpermanent pink color was obtained. The solutionwas concentrated under r educed pressure to about 10ml, and treated with 5 volumes of methanol ; the precipitated salts were separated, washed with methanol,tested for radioactivity, and discarded . The solution was concentrated to r emove the alcohol, andpassed over a column containing 25 ml of mixedanion and cation exchange resins (see footnotes 5 and6); the resins were washed until the activity of thewashings, "was negligible. The effluent was con-'11 T he amalgam was prepared in small pellets by pouring it in molten conditionthrough a heated alund um tbimble, having a small bole in tbe bottom, into a2-ft "shot tower" of mineral oil. T he pellets were stored under mineral oil, ant!just before use, were blotted dry, weighed. and rinsed with benzene.309,

centl'uted under reduced pressure and at a temperature less than 40 0 to about 10 ml ; at this stage thesolution was filtered into a round-bottomed flasktlll'ough a microfilter containing a small bed of decolorizing carbon,12 and the combined solution andwashings were freeze-dried in a 50-ml flask. Theresidue in the flask was dissolved in a few drops ofmethanol, and 2-propanol was added to incipientturbidity. Crystallization began immediately afternucleation. At the end of 2 days, the mother liquorwas separated, and the crystals were washed in placewith a few drops of a m ethanol-2-propanol mixture(2: 1). For recrystallization, the crude material, dissolved in a few drops of water, was transferred to astandard-tap er test tube, and the water was r emovedunder reduced pressure . The thick sirup was dissolved in a few drops of methanol, and 2-pl'opanolwas cautiously added, until saturation was approached. The crystals that formed in 24 hI'weighed 185 mg and contained 672 MC of activity.With the aid of 100 mg of nonradioactive D-arabinoseas carrier, an additional 39 MC of activity was obtained from the mother liq uor. The yield of D-arabinose-1-014 (711 MC) from potassium D-a.rabonate-l0 14 was thus 60 percent .132 .6 . Separation of Cadmium D-Ribonate-l-C14The mother liquor from the preparation of potassium D-arabonate-1-0 14 , described in section 2.3, wasevaporated to remove the alcohol present. Theresidue was dissolved in water and passed over acolumn of cation exchange resin (see footnote 7).The effluent, containing 6,900 MC of activity, wasthen neutralized with cadmium h ydroxide to the endpoint of phenolphthalein. After the addition of 1 gof nonradioactive cadmium D-ribonate, the solutionwas filtered with the aid of decolorizing carbon, andth e filtrate was evaporated under r educed pressureto a thin sirup. Nucleation of the sirup with crystalline cadmium D-ribonate and careful addition ofaqueous m ethanol resulted in a voluminous crystallization. The crystals were separated by filtration ,and recrystallized by dissolving in 4 ml of hot waterand adding successively 5 ml of m ethanol and water(1: 1) and 2 ml of m ethanol and wa tel' (2: 1). The ho tsolution was th en saturated with m eth anol and allowed to cool slowly. The fil's·t crop of recrystallizedcad mium D-ribonate-l-014 weighed 678 .6 mg and hadan activity of 1,796 MC. After successive additionsof two I-g quantities of nonradioactive cadmiumD-ribonate as carrier , and r ecrystallization of theresulting crops of salt, an addi tional quantity ofcadmium D-ribonate-l-014 containing 1,416 MC wasrecovered. 'The total yield of cadmium D-ribonate-1 0 14 was thus 3,212 MC, corresponding to 23 percentof the 014-labeled cyanide used ."This step removes a traoe of oil tbat may be introduccd with the sodiumamalgam." Dl other preparations, rad iochemical yields up to 70 pOI'coat have beenobtainod.2.7. Praparation of D-Ribono-y-lactone-l-C14 andReduction to D-Ribose-l-C I 4A solution containing 679 mg of cadmiumD-ribonate-1-C I 4, with an activity of 1,800 MC, waspassed over a column of cation exchange resin (seefootnote 7), and the effluent was concentrated underreduced pressure to a sirup. The sirup was transferred by use of water to tID'ee reduction tubes andevaporated to dryness by heating on a steam bathunder a stream of dry air. The residue in each tubewas dissolved in a few drops of water and the solution, after the addition of 5 ml of m ethyl cellosolve,was evaporated again. This process was repeated4 times over a period of 2 days. Each residue wasthen dissolved in 2 ml of m ethyl cellosolve, and thesolution was seeded with crystalline D-ribono-y-lactone and stored in a desiccator over calcium chloride.From time to time the residues were moistened withm ethyl cellosolve. Orystallization occurred slowly,but ultimately all sirup disappear ed, and the residu esappeared to be entirely crystalline.A series of reductions on nonradioactive D-ribonoy-lactone showed that a maximum yield of sugar(70 percent) was obtained when 9.2 g of 5-percentamalgam and excess sodium acid oxalate were employed for the reduction of 1 millimole of lactone.Each of the three samples of lactone mentioned abovewas dissolved in 20 ml of ice water and reduced b yuse of the above quantities of amalgam and oxalate.The product from the three reductions was combined ,the m er cury was separated, and the mixture wasdiluted with 5 volumes of m ethanol. The resultingcrystalline salts were separated by filtration , washedwith m ethanol, and discarded after a test showedn egligible radioactivity. The filtrate was concentrated under reduced pressure to a volume of about15 ml and diluted with 75 ml of m ethanol ; the sodiumsalts that separated were removed b y filtration andwashed with methanol. As a test showed that thissecond crop of salts likewise contained no appreciableradioactivity, it was discarded. The alcoholic filtratewas concentrated to about 10 ml, neutralized withaqueous sodium h ydroxide in the presence of phenolphthalein until a permanent pink color was obtained ,and immediately deionized by passage over a columnof mixed cation and anion exch ange r esins (see footno tes 5 and 6). The effluent and wash liquor werecombined and con centra ted under reduced pressureto a volume of 6 ml ; 750 mg of carrier D-ribose wasdissolved in the sirup , and the solut ion was filteredthrough 1 ml of a mixture of d ecolorizing carbon anddiatomaceous earth . The filtered solution was fl'eezedried, and the residue was dissolved in 0.5 ml ofethanol. A mixture of ethanol and isoamyl alcoholwas added to the point of incipient turbidity, and thesolution was seeded with D-ribose and stored in arefrigerator. \Vhen crystal growth seem ed complete(5 days), the mother liquor was removed with acapillary pipette, and the crystals were carefullywashed , in place, with ethanol and dried over calcium3101

chlorid e. The crude prod L1ct (768 mg) had an activityof 667 }Joe, or 37 percen t of the activity of the cadmiumD-ribonl1te- l- 0 4 used.For recrystallization, the product was dissolved ina few drops of water, a drop of acetic acid was added,and the solu tion was filtered through decolorizingcarbon. Th e filtrate was con centrated under reduced pressure to a thick sirup. The residue wasdissolved in ethanol, and the solution was concentmted again under reduced pressure. The residuewas dissolved in 0.5 ml of ethanol, and after additionof isoamyl alcohol to incipient turbidity, the solutionwas seeded and stored, first at room temperature andfinally in a refrigerator. After 5 days, the productwas sepamted, washed in place with a few drops ofethanol, and dried. The recrystallized D-ribose-1-CI4(540 mg) contained 465 }JoC of carbon 14. By use ofcarri ers on the mother liquor from the recrystallization, nearly all of the radioactivity of th e crud eD-ribose- I-C I4 was reclaimed.3 . References[1] H. S. Isbell , J. V. I ar abi nos, H . L. Fru h , X . B. Hol t,A. Schwebel, and T. T . Galkows ki, J . Resea rch :\BS48,163 (1952) RP2301.[2] H . S. I sbell, a nd J. V. Karabinos, J . R e ea reh :--JBS lB,2334 (1952) RP2335.[3] H. L. Frush and H . S. Is bell, J . Research NBS 50, 133(1953) RP2400 .[4] H. L . Frush and H . S. I sbell, U. S. Ato.mic Ene rgyComm ission Report No . NBS- 2308 (1953); J . ResearchN BS 51, 167 (1953) RP2446.[5] H . S. Is bell , U . S. Patent 2606918, August 1952.[6] H. S. Isbell , U. S. Patent 2632005, March 1953.[7] D. A. Rappopor t and VV. Z. Hassid, J. Am. Chem . Soc.73, 5524 (1951) .[8] J . C. Sowden and H. O. L. Fischer, J. Am. Chell1. Soc.69, 1963 (1947) .[9] J . C. Sowde n, J . Am Chell1. Soc. 72, 808 (1950) .[10] R. C. Hockett, D. V. Collins, and A. Scattergood, J .Am . Chem. Soc. 73, 599 (1951) .[11] iVI. Calvin, C. Heide lberger, J . C. Reid, B . M. TolberL,and P . F. Yankwich, I sotopic carbon, p. 92 (J. Wiley& Sons, Inc., New York, N. Y., 1949) .[12] A. Schwebel, H. S. I sbell, and J . V. Karabino , Science113, 465 (1951) .[13] H. S. Isbell, 'liT . W. Pigman , and H . L. Frush, J . R esearchNBS 24, 241 (1940) RP1282 .VVAS HING1' ON,II311Sep tembcr 1, 1953 .

Preparation of D-Arabinose-I-C'4 and D-Ribose-l-C14 I Harriet L. Frush and Horace S. Isbell 1 t By application of the cyanohydrin synthesis to D-erythroso, D-arabinose-I-C14 and D ribose-I-C" have been prepared in overall