Nov., 1958
RECOILTRITIUM LABELING REACTION
has several limitations: (1) in the C14 production the nitrogen source dilutes the target substance and the result is loss of some activity in the main products; (2) 2-methylpyrazine employed as the nitrogen source might conceivably react with some of the reactive fragments in the system and thereby affect the ratio and quantity of benzene-C14 or toluene-C14; (3) during the pile irradiation of samples for a relatively long period (5 to 10 days) the system suffers radiation damage. Factors 1 and 2 affect the carbon-11 production also. The effect of radiation damage on the yields of activity is twofold: partial destruction of active compounds formed and progressive destruction of the target compound with time during irradiation. The fast neutron irradiations of benzene for Cll production were of short duration and do not involve significant radiation damage to the system. In view of these differences in the systems under comparison it is to be noted that the synthesis and re-entry figures for the recoil labeling with carbon-14 and carbon-11 were of the same order of magnitude. The vast difference in the initial recoil energies does not seem to influence significantly the efficiency of labeling. From the results of experiments no. 20 and 21 it can be seen that the presence of %methylpyrazine in the system lowered the activity produced in benzene to a value closer to the benzeneC14yield from the (n,p) reaction. These considerations indicate that the reactions leading to labeled benzene and toluene take place below 45,000 e.v. (recoil energy of C14) and indeed it seems reasonable to assume that they occur at energies considerably lower than this. Conclusion Radical scavengers and phase change from liquid to solid decreased the yield of benzene-cll from the reaction Cl2(n,2n)C" carried out in benzene,
1373
The toluene-Cll yield in the same system was essentially unaffected by the changes in phase and temperature and by the presence of radical scavengers. It seems, therefore, that benzene-C'l was formed by both hot and thermal processes and toluene-Cl1 mainly by hot reactions. The thermal reactions appeared to be diffusion controlled and might not be confined to the reactive site where the carbon-11 was localized. By using recoil carbon-14, it should be possible to adduce evidence for the formation of certain intermediates leading to the observed products. Useful information to verify the mechanisms also can be obtained by extending the present work to isolation of other possible labeled products such as norcaradiene and cycloheptatriene. The formation of the latter is suspected from the presence of active impurities in the tail of the toluene fraction (g.1.c.) where cycloheptatriene was found to elute. It is becoming increasingly clear from the recent l i t e r a t ~ r e ~ ~ , ~ ~ that chemical forces do play an important role in the processes of synthesis and re-entry quite apart from the considerations of physical processes of energy transfer. The present investigation supports this conclusion. Acknowledgment.-We are indebted to Dr. Jerome Hudis for his interest and help with the target design. We wish to thank Dr. Charles P. Baker and the operating staff of the Brookhaven 60-inch cyclotron for their help and cooperation in making the facilities for irradiations. Grateful appreciation is made of help and advice from Dr. David R. Christman on the counting methods used. B.S. acknowledges a travel grant received from the J. N. Tata Endowment for Higher Education of Indians, Bombay, India. (22) G. Harbottle and N. Sutin, THWJOURNAL, 62, 1344 (1958). (23) A. A. Gordus and J. E. Willard, J . A m . Chem. Soc., 79, 4609
(1957).
STUDIES OF THE RECOIL TRITIUM LABELING REACTION. V. FURTHER REACTIONS WITH GLU COSEl BY HOWARD KELLERAND F. S. ROWLAND Contribution from the Chemistry Departments, University of Kansas, Lawrence, Kansas, and Princeton University, Princeton, N. J. Received April 8, 1968
Energetic tritium atoms reacting with glucose molecules in the condensed phase show variations in the percentage of total tritium bound to carbon in the otherwise unchanged glucose molecule, and in the percentage found in each individual position in the molecule. Less than 0.03% of the total tritium is found as labeled galactose in irradiated crystalline glucose. Most of the tritium is present as labile activity in irradiated glucose solution; about 1% is found as labeled glucose.
Introduction Energetic tritium atoms from the nuclear reaction Lis(n,a)Ha have been shown to undergo reactions with a variety of organic molecules.2 (1) Supported in part by Atomic Energy Commission Contract No. AT-(ll-1)-407. (2) (a) R. Wolfgang, F. S. Rowland and C . N. Turton, Science, 121, 715 (1955); (b) F.,S. Rowland, C. N. Turton and R. Wolfgang, J . A m . Chem. SOL, 78, 2354 (1956); (c) F. S. Rowland and R. Wolfgang, Nucleonics 14, No. 8 , 58 (1956); (d) R. Wolfgang, J. Eigner and F. S. Rowland, THISJOURNAL, 6 0 , 1137 (1956); (e) M. El-Bayed and R. Wolfgang, J . A m . Chem. Soc., 79, 3286 (1957) : (f) A. Gordus, M. Sauer
The previous experimental data on the reactions of recoiling tritium with crystalline glucose and galactose have shown that an important fraction (-10%) of the tritium is bound in non-labile form in the otherwise unchanged hexose.2b Degradation of the hexoses into suitable derivatives demonstrated further that the tritium activity was disand J. Willard, ibid., 79, 3284 (1957); (9) W. J. Hoff, Jr., and F. 6. Rowland, ibid., 79,4867 (1957); (h) J. Evans, J. C. Quinlan, M. Sauer, Jr., and J. Willard, THISJOURNAL, 62, 1351 (1958); (i) M. El-Sayed, P. Estrup and R. Wolfgang, ibid., 62, 1356 (1958).
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HOWARD KELLER AND F. S. ROWLAND
tributed in a very non-uniform manner throughout the molecules. No investigation was made of the chemical location of the remaining tritium activity. The number of potential labeled products resulting from tritium reactions with the glucose molecule makes smaller, simpler molecules preferable for much of the experimental work on recoil tritium reactions. Similarly, study of the gas phase reactions permits isolation of the reactions involving individual molecules from the specific environmental effects of condensed phases. However, further investigations of hexose molecules seemed worthwhile along certain specific lines. We have investigated the observed distribution of tritium activity among non-labile positions under a variety of experimental conditions. Glucose samples have been irradiated in aqueousxolution and in crystalline form under different pile irradiation conditions. From each irradiation, a pure glucose fraction has been isolated. Additional degradations of the glucose molecules have been performed in order to determine the intramolecular distribution of tritium activity. I n addition, aliquots of one irradiated crystalline glucose sample have been dissolved in several solvents at different temperatures. Partial degradations have been performed on these samples to determine the effects of the solution processes on the intramolecular tritium distribution. In both the glucose and galactose of the previous experiments, the intramolecular degradation showed substantial activity in C-H bonds at asymmetric carbon atoms, indicating that the substitution had taken place with the retention of optical configuration. We have therefore investigated the possibility that substitution reactions at these carbon atoms might also be occurring with inversion of optical configuration. Carrier galactose, differing from glucose only in reversed configuration at the no, 4 carbon position, has been added to two of these irradiated glucose samples. The galactose carrier has then been purified both chemically and radiochemically.
Experimental Irradiations .-The irradiations are summarized below. All irradiations were carried out at the Brookhaven National Laboratory in a flux of 1.8 X n./cm.2/sec. Irradiations I and I1 lasted 24 hours, and I11 for 5 days. ( I ) 7 ml. of aqueous solution, containing 3.0 g. of glucose and 0.21 g. of LiC1. The solution was pale yellow after irradiation. (11) A mixture of 10.0 g. of crystalline glucose and 0.30 g. of LizCOa. This sample was light tan in color after irradiation. (111) A mixture of 30 g. of crystalline glucose and 1.5 g. of LtCOa. After irradiation. the samnle was light tan to brown an"d slightly gummy. . Purification and Isolation of Hexoses.-(I) Three aliquots were taken from the irradiated solution. (a) 2 ml. of ( I ) was added to 10.0 g. of inactive glucose in 6 ml. of 2-1 methanol-water. This solution was concentrated in vacuo, diluted with methanol, cooled, and the resultant glucose recrystallized twice. An aliquot from the second recrystallization was converted to glucose pentaacetate. (b) 2 ml. of (I) was added to 4.9 g. of galactose and 3 ml. of ethanol. This solution was concentrated in vacua, diluted with methanol, cooled and the resultant galactose recrystallized three times. Aliquots were converted to galactose pentaacetate and to mucic acid. (c) 2.2 ml. of (I) was evaporated to near dryness and then I
Vol. G2
diluted with methanol five times. Evaporation in vacuo to total dryness yielded a hygroscopic fluffy tan precipitate. (11) (a) The irradiated glucose mixture was dissolved in 50 ml. of water to produce a clear, dark brown solution. Ten mg. of galactose was added as hold-back carrier. The solution was treated with Darco 600 charcoal, diluted with methanol, passed through Dowex X-8 and Duolite OHform (A-4) resins and concentrated in vacuo. More than 2.5 g. of glucose crystallized out. Two recrystallizations were performed followed by dilution with 1OX weight of inactive glucose and another crystallization. (b) 5.0 g. of inactive galactose was added to the filtrate from the original glucose crystallization. The galactose solution was concentrated, diluted with methanol and crystallized. (111) 0.60 g. of irradiated glucose was thoroughly mixed and ground four times with 59.4 g. of inactive glucose to produce a fine powder, with a light tan tinge. Aliquots of this glucose were dissolved a8 (1) 1.0 g. in 2 ml. of water a t 100' (2) 0.5 g. in 10 ml. of ethyl alcohol af 78" (3) 0.5 g. in 10 ml. of dioxane at 100 (4) 0.5 g. in 10 ml. of pyridine at 100' (5) 1.0 g. in 2 ml. of HzO after 48 hr. at 50' After solution, the glucose was crystallized, and the formaldehyde dimedone derivative formed from each sample. Chemical Procedure. Derivatives.-Distributions of tritium activity among the various non-labile positions of the hexoses have been determined for several samples containing appreciable amounts of radioactivity. I n those cases for which the total activity was limited, some of the derivatives were formed. The derivatives usually used were those listed in a previous paper.2b These derivatives were formed as follows: The crystalline hexoses were acetylated with acetic anhydride and sodium acetate by the usual procedure and the resultant penta-0acetyl-D-hexoses recrystallized from an ethanol-water mixture. The radiochemically pure sugars were used to prepare the phenylosazone, phenylosotriazole, 2-phenyl-4formylosotriazole, 2-phenyl-4-carboxylic acid osotriaaole, benzoyl glycolaldehyde semicarbazone and hexonic acid derivatives by previously described methods .a The formaldehyde dimedone derivatives were prepared by oxidation of sugar samples with sodium metaperiodate and precipitation of the resulting formaldehyde with d i m e d ~ n e . ~Mucic acid was made from D-galactose by oxidation with nitric acid. As additional verification of this method for determining the distribution of tritium activity in the hexose molecule, additional derivatives were prepared from the glucose Sam le used by Rowland, Turton and Wolfgang,zb and labefed RTW in Table 111. This sample also had been exposed in crystalline form in a mixture with LizCOa,although at a flux of loQn./cm.*/sec. These derivatives included: 1-phenyl-4-phenyl-hydrazonopyrazolone-5, 2- -nitrophenyl4-formylosotriazole, 2-p-nitrophenyl-osotriazo~-4-carboxylic acid, 1,2-bisphenylhydrazone of mesoxaldehyde and amethyl glucoside. Tritium Assay.-Samples were converted to gas by the method of KaDlan, Wilzbach and Brown.6 A standard aliquot of the resulting methanehydrogen gas was introduced into a silver-walled gas proportional counter with an active volume of 85 cc. The counter filling m s completed with ethane, propane or 90% argon-10% methane mixture, all of which give good plateaus. These measurements are reproducible to approximately 1yofor identical samples, and to about 2T0 for different samples containing the same labeled roups. The over-all accuracy of the assays is about 5h.
Results Sample (I).-Most of the tritium activity produced in the aqueous glucose solution was found in the solution in labile form. N o assay was made of the solution itself or of H20 separated from the (3) J. C. Bevington, E. J. Bourne and C. N. Turton, Chemistry & Industry, 1390 (1953). (4) R. E. Reeves, J . Am. Clem. SOC.,63, 1470 (1941). (6) K. Wilzbach, L. Kaplan and W. G. Brawn, Science, 118, 522 (1953).
RECOILTRITIUM LABELING REACTION
Nov., 1958
solution. However, calculations from the exchangeable activity present in the precipitates gave an estimate of 80f2070 of the tritium in labile form. Not more than 1% of the tritium reached chemical stability as non-labile hydrogen in glucose, as shown in Table I. Less than 0.1% of the tritium was present as labeled galactose. The residue from (I) (e) contained more activity than could be expected from labile hydrogens alone, leading to an estimate of 5 f 2% bound as non-labile tritium in degradation or polymeric products of glucose. TABLE I CHEMICAL LOCATION OF RECOILTRITIUM IN AQUEOUSGLUCOSE SOLUTION (42y0 GLUCOSE BY WEIQHT) CPM/ mg.
(a) Glucose aliquot Glucose crystals 1st recryst. 2nd recryst. Glucose pentaacetate 1st recryst.
DPM/mmole original"
% of total
3860 15.7 X 106 886 3.60 X 106 658 2.68 X 106 246 247
2.16 X 106 2.18 X 106
51
(b) Galactose aliquot Galactose crystals 4990 4.5 X 108 1st recryst. 550 1.01 X 106 2nd recryst. 509 '0.93 X 106 3rd recryst. 517 .95 X 106 Galactose pentaacetate 60 .24 X 108 Mucic acid (1st recryst.) 39 .08 X 108
