RADIATION DECOMPOSITION
April 20, 1953 [CONTRIBUTION FROM
THE
OF C14-LABELED COMPOUNDS
1867
RADIATION LABORATORY A N D DEPARTMENT OF CHEMISTRY, UNIVERSITY OF CALIFORNIA, ] BERKELEY
Observations on the Radiation Decomposition of Some C I 4 - L a b e l e d Compounds1 BY B. M. TOLBERT,P. T. ADAMS,EDWARD L. BENNETT, ANNM. HUGHES,MARTHAR. KIRK, R . M. LEMMON, R. M. NOLLER,R. OSTWALD AND MELVINCALVIN RECEIVED FEBRUARY 16, 1953 Varying amounts of radiation decomposition during storage in the dry form have been found in the cases of C14-labeled valine, norvaline, norleucine, choline chloride, calcium glycolate and cholesterol. These data indicate that investigators using labeled organic compounds should make frequent checks of the purity of these compounds in order to exclude the possibility of the presence of decomposition products.
TABLE I The work described in this paper was initiated by a recent observation that considerable radiation COMPOUNDS SHOWING MORETHAN ONE PERCENT.RADIAdecomposition had occurred to a sample of cholineTION DECOMPOSITION Non-vol. m e t h ~ 1 - C chloride '~ which had been synthesized in radioac. prods. these laboratories last spring. This observation, obs. on sp. paper together with a similar one made by Dr. Sidney act. R . ~ . P . ~chroDeDate (pc./ X mato- comp., Weinhouse on samples of C14-labeledcalcium glycoCompound prepared mg.j 10-6 gramsb % late which were synthesized here four years ago, has Valine-4.4'-CaId.HCl Nov. 1951 6.0 10 1 1-2 illustrated the need for information on the rate of Norvaline-8-C~~.HCl Jan. 1953 1 7 . 7 46 6 5 decomposition of C14-labeled organic compounds. Norleucine-2-Cl~ March 1952 1 7 . 4 20 4 2 Since this group (Bio-Organic Group of the Radia- Choline-methyl-CI'4 13.0 11 0' 63 chloride May 1952 tion Laboratory) has carried out during the past Calciumglycolale-I-CL' Feb. 1949 5.8 80 1 13d six years the syntheses of several dozen labeled Calcium glycolate-2-C" Feb. 1949 4 0 21 1 zzd organic compounds, we haSe available a number of Cholesterol-4-Cli 7 Oe -40' Aug. 1951 6.5 such compounds which could be examined for evi"-These values were calculated: (R.e.p.) Roentgens dence of radiation decomposition. From these equivalent physical = kNE(R.e.p./e.v.)/wt. in g., where compounds we picked a list of twelve which had k = fraction of radiation absorbed (arbitrarily given the been exposed to large amounts of self-radiation since value of 1 in our calculations); N = total number of events the time of synthesis; E = average energy of the radiatheir syntheses and for which there are un- since tion ( = 50,000 e.v. for C14); R.e.p./e.v. is taken as 1.9 X equivocal data to show their high degree of purity 10-". These observations were made in February, 1953. The main decomposition product of the methyl-labeled a t the time of synthesis. All of these compounds had been stored in dry, solid form in a dark cabinet choline is the volatile trimethylamine. Further details are in the experimental part of this paper. These a t room temperature. The extent of the radiation recorded values were determined (private communication) by Dr. decomposition was established by making paper Sidney Weinhouse who found that radioactive formate was chromatograms of each of the compounds and de- present in these percentages. The formic acid was steam termining, by means of radioautographs of the distilled from the glycolic acid and its radioactivity measas COa after oxidation by mercuric ion. e Approxichromatograms, if other radioactive constituents ured mately 40% destruction (based on the amount no longer had appeared during storage. Similar paper precipitable by digitonin) of the labeled cholesterol has chromatograms had been prepared a t the times of been reported to us (private communication) by Professors synthesis and in all cases there had been no ob- It'. G. Dauben and I. L. Chaikoff. These observations will servable (or, a t most, less than one per cent.) be reported in detail in a separate publication. radioactive impurity. By comparing the old TABLE I1 radioautographs with the ones recently prepared, COMPOUNDS SHOWING LESS THAN ONE PER CENT.RADIAwe have measured the percentage of radioactive imTION DECOMPOSITION purities which have appeared since the times of Date Sp. act. R.e.p. synthesis. In the case of choline, which had been COmpound prepared (pc./mg.j x 10-9 stored in an evacuated sealed tube, we were able to Gly~ine-2-C'~.HCI 6 Dec. 1951 4.9 determine the amount of a volatile radioactive prod- Guanine hydrochloride-4-C14 March 1950 2 . 6 9 uct, namely, trimethylamine, which had formed dur- 8-Azaguanine-4-C14 13 March 1950 3.8 ing storage. None of the other compounds were Adenine sulfate-4,6-C214 10 2 9 March 1950 stored in sealed tubes and any volatile decomposi- 8-Azaadenine-1,6-Cz1? 4.1 March 1950 15 tion products would not be detected unless thev Stilbamidine diisethionatecomprised a considerable fraction (at least loc&) 31 amidine-C2I4 Feb. 1951 13.7 of the radioactivity of the freshly prepared com- Thyroxine-ol-C14 3 May 1950 1.0 pound. The data which were obtained are sum- Succinic a ~ i d - 2 - C ' ~ 14 Dec. 1949 3.9 marized in Tables I and 11. The data of Tables I and I1 show that there is a Our data are too scanty to permit predictions as to general relationship between the amounts of radia- the susceptibility of various organic groups to radiation (R.e.p.) and the likelihood of finding some tion damage-this we hope to do later with the acradiation decomposition ; however, the choline, cho- cumulation of further information. For the preslesterol and stilbamidine provide major exceptions. ent, however, it should be emphasized that users of C14-labeled compounds would be well advised to (1) The work Aei~ribed t h i s p ~ p e rW A S i~iiinsoredti> t h e II 5 check the purity of their compounds frequently, h t o m l r htierg! C'ommi\iiori 111
1868
TOLBERT, ADAMS, BENNETT,
HUGHES,KIRK,LEMMON, NOLLER,OSTWALD AND
CALVIN
vel. 75
particularly if the compound is of high specific activity and/or was prepared several years ago. For checking such purity, paper chromatography provides a very convenient tool, especially for finding non-volatile radioactive impurities. However, it may also be useful for volatile impurities if these compounds have either a basic or acidic function and can be kept in a non-volatile form on a paper chromatogram by using, respectively, an acidic or a basic solvent (for example, the chromatography of choline which is reported in this paper). There are several references in the to the use of paper chromatography for determining the purity of radioactive preparations.
tablished by the preparation directly, from the choline sample dissolved in water, of the 2,4-dinitrophenylhydrazone. This derivative, after recrystallization once from glacial acetic acid and twice from ethanol, was found t o have a melting point of 146' (literature value? 147"). I t was also found to b: complttely noi-radioactive. About two-thirds of the radioactivity of the original choline preparation was volatile-it was lost in the operation of making a plate for counting the choline chloride. That this volatile product is trimethylamine was demonstrated as follows : ( 1)Aqueous alkali was added to a freshly opened tube of the choline and the volatile products were collected in a connected trap which contained a saturated solution of picric acid in ethanol and which was cooled in a liquid nitrogen bath. The trap was then removed, closed off from the atmosphere, and warmed, with shaking, to about 60". After the trap was cooled back to 0", crystalline picrate appeared. The solvent was removed by filtration and the precipitate was dried. Its melting point after two recrystallizations Experimental from ethanol was 211" (literature value8 216') and its acThe valine-4,4'-Cz14,norvaline-3-C1*,and norleucine-2-CI4 tivity was 1.67 mc./mmole (the activity of the original which were found to show radiation decomposition were all choline chloride was 1.82 mc./mmole; both values deterpreparedvia an acetamidomalonate synthesis using as starting mined by direct plating). (2) When the labeled choline materials, respectively, isopropyl-methyl-C214iodide, propyl- chloride from a freshly opened tube was placed in acid solu1-C14iodide, and b ~ t y 1 - l - Ciodide '~ All of these preparations tion and an aliquot portion paper chromatographed (onewill be the subject of a forthcoming publication. The final dimensionally) using a n-butanol-concd. HC1-water (4 : 1:1 purification of the labeled compounds was accomplished by by vol.) solvent, two radioactive spots were obtained. The elution from an ion-exchange column. Paper chromatog- faster moving spot (higher RF value) contained 6370 of the raphy of the preparations (phenol-water in one direction, total activity and the slower moving spot (choline) conbutanol-propionic acid-water in the other) showed that tained 37y0 of the activity on the paper. The faster moving there was less than one per cent. of radioactive impurities spot was cut out, eluted, and the material co-chromatopresent. graphed with labeled trimethylamine hydrochloride. These The preparation of the labeled choline has been reported materials proved to be chromatographically identical (oneearlier.e The material described in the present paper was dimensional paper chromatogram with the same acidic prepared a t a later date (May, 1952) using the same pro- butanol solvent). cedure. The later preparation included, in addition, .the The syntheses of the labeled glycolic acids, and the criteria same paper chromatographic search for radiochemical im- for their purity a t the time of preparation, have been depurities as was carried out on all the labeled compounds scribed in an earlier p ~ b l i c a t i o n . ~The same applies to the mentioned in this report. Again, these impurities were less guanine, 8-azaguanine, adenine and 8-azaadenine: to the than one per cent. of the preparation. glycine,l0 to the succinic acid" and to the stilbamidine.I2 Since the radiation decomposition of the choline is so ex- The thyroxine synthesis (R. M. Lemmon, unpublished tensive (63%) we made some efforts to determine what the work) was carried out using g l y ~ i n e - 2 - C and ~ ~following the principal decomposition products were. Upon opening a original procedure of Haringt0t1.l~ sealed tube of the choline a pronounced odor of acetaldehyde was observed. The presence of acetaldehyde was es- BERKELEY, CALIF. ( 2 ) R Ostwald, P. T. Adams and B. &I.Tolbert, THISJ O U R N A74, I, 2425 (1952). (3) D. M.Hughes, R. Ostwald and B. M . Tolbert, ibid., 74, 2434 (1952). (4) A. A. Benson, J. A. Bassham, M . Calvin, T. C. Goodale, V . A. Haas and W. Stepka, ibid , 73, 1710 11950). (5) W. Tarpey, H. Hauptmann, B. Tolbert and H. Rapoport, ibid., T I , 5126 (1950). (6) F. Christenson and B. M . Tolbert, Univ. of Calif. Radiation Lab. Report ?To. 962, unclassified, July 1.5, 1049.
(7) Shriner and Fuson, "Identification of Organic Compounds," 3rd Edition, John Wiley and Sons,Inc., New York, N. Y., p. 229. (8) Beilstein, "Handbuch der Organischen Chemie," Val. VI, p. 280. (9) Edward L. Bennett, THISJOURNAL, 74, 2420 (1952). (IO) B. hl. Tolbert and D. M. Hughes, Univ. of Calif. Radiation Lab. Report No. 705. unclassified, May 16, 1950. (11) E. C. Jorgensen, J. A. Bassham, M. Calvin and B. M. Tolbert. THISJ O U R N A L , 74, 2418 (1952). (12) Edward L. Bennett, Concer Research, in press. (13) C. R. Harington and G. Barger, Biochcm. J.,21, 169 (1927).
