Rad. and Environm. Biophys. 13, 63--69 (i976)
Radiation and Environmental Biophysics © Springer-Verlag1976
Selective Radiolysis of Enantiomers O. Merwitz Jfilich l~uelear Research Centre, Institute for Chemistry Jfilich, Fed. Rep. Germany
Summary. The gamma-induced decarboxylation of fl-phenylalanine in the solid state is a chain reaction. Both enantiomers and the raeemate offl-phenylalanine-l-z4C show different exponential dose-effect curves for the radiationinduced cleavage of z4C02. Introduction The main effects in the solid state radiolysis of amino acids are the following: hydrogen cleavage deamination decarboxylation Hydrogen cleavage and deamination give rise to the formation of free radicals, which in the case of being long-lived, can be detected b y ESR-spectroscopy. The decarboxylation causes the formation of a saturated amine the concentration of which in the irradiated substance is identical with the concentration of the produced CO m. In the case of ~-phenylalanine, ~-phenylethylamine is produced: CeHsCH~CH(NH~)COOH-~ CeH~CH2CH~NH~ ~- CO n The analysis of the irradiation products of solid amino acids was previously carried out partly b y mass spectrometry or chromatographically (Gottschall and Tolbert, 1968; Clark et al., 1970), and partly after dissolving the substance irradiated under vacuum in water (Dale et al., 1949; Tolbert et al., 1963; Meshitsuka et al., 1964; Minegishi et al., 1967; Garrison, 1972). The radiation decarboxylation of carboxyl-labeled amino acids in the solid state was also determined using 14C02, which was trapped as Baz4C03, weighed, converted to z4C02, and swept into an ion chamber where the activity was measured. The G (C02)-value was found to be between i and 2 for most of the amino acids studied (Castrillon et al., 1961). l~oreover the variations in the irradiation effect on the isomeric forms of the amino acids were studied. When irradiating DL-tyrosine with ~-particles, the D-isomer is decomposed faster than the L-isomer (Garay, i968). The activation
64
O. Merwitz
energies for the decay of gamma-induced radicals in L- or D-alanine are different (30.3 and 25.9 kcal/1V[, resp.) (Horan et al., t968). The G-values for the radiation damage to DL-phenylalanine and L-phenylalanine at a dose of 4.10 a rad are also different (2.9 and 3.8, resp.) (Korgaonkar and Donde, 1962). However, the accuracies of the applied measuring methods are not sufficient to make an interpretation possible. The irradiation experiments outlined in the literature were carried out either for one dose or in a very narrow dose range so that the question as to whether the decarboxylation follows a linear or an exponential dose-effect curve remained undecided. _As to the selection of phenylalanine for studying the radiation-induced decarboxylation, physico-ehemical and biological reasons have been the decisive factors. Previous investigations of the radiation damage to solid all-labeled nucleic acid bases had proved the fact that the dose-effect curves are measurable for the cleavage of labeled hydrogen in a wide dose range (Merwitz, i972a; Merwitz, 1972 b ; Merwitz, t 974 a; Merwitz and Otto, 1974 b). The measurements were based on the condition of thermal stability of the irradiated substances due to the fact that they had to be sublimed or, at least, heated up for the liberation of the hydrogen trapped in them. Thermal stability is also necessary in the case of carboxyl-labeled amino acids as they also must be sublimed for direct quantitative determination of laC02. Some of the important amino acids are sublimable. This holds true for phenylalanine as well (Gross and Grodsky, 1955). Phenylalanine was chosen because both enantiomers and the racemate are commercially available in a carboxyl-labeled form (Radiochemical Centre Amersham). I t was possible by this fact to detect differences in the radiochemical behaviour of enantiomers. Phenylalanine is of biological interest as an essential amino acid and precursor for the formation of tyrosine and of adrenaline ; its absence causes thyroidal and cortico-adrenal disfunctions. The failure of the catabolisms gives rise to phenylketonuria. Phenylalanine radicals inactivate DNA (de Jong et al., i972). Material and Method
Analytical Quality and Purity of the Substances The serf-decomposition of the labeled compounds was examined before doing the irradiation experiments. I t was found that autoradiolysis at a specific activity of 20 ~Ci/g was negligeable over periods of l0 rain to i2 days necessary for irradiation, and that reasonable counting rates for 14CO~-measurements resulted. In order to reduce the radioactivity of the purchased compounds D-, DL-, and L-/~-phenylalanine-l-laC (total activity 250~Ci each) to the level mentioned above, their aqueous solutions were added to boiling solutions of the corresponding non-labeled compounds at appropriate concentrations. After cooling and filtering, all substances were recrystallized and dried to constant weight in vacuum. Such purification would be sufficient for ordinary chemical operations; but in this case a repeated sublimation is also necessary, since after recrystallizing and drying,
Selective Radiolysis of Enantiomers
[
65
:
t
counting t u b e CH4
glass tu be
I
/
Fig. 1. Schematic diagram of counting apparatus
traces of volatile radioactive impurities are still present. Normally a very low, constant counting rate (t00 to 200 epm/i00 mg of substance) is reached after 2 to 3 sublimations. Immediately before irradiation, the specific activity of the samples was measured by liquid scintillation. The non-labeled isomers and the racemate were of the highest obtainable purity (Fluka: purissimum grade). The radiochemieal purity of DL-fi-phenylalanine-l-14C and L-~-phenylalanine-t-l~C was determined as 99% b y paper chromatography, and t h a t of D-/~-phenylalanine-l-l~C as 97% b y thin-layer chromatography (Radioehemical Centre Amersham). The isotopic abundance of the earboxyl C-atom was 97 % in D-fi-phenylalanine1-14C, 95% in I)L-fi-phenylalanine-l-14C, and 9 t % in L-fi-phenylalanine-l-14C. The remaining radioactivity was distributed among the other carbon atoms, i.e. it appears after decarboxylation in the phenylethylamine which readily condenses.
Measurement o/Radiation-induced 14C02-Cleavage ]rom D-, DL-, and L-fi-Phenylalanine- 1-14C The irradiations of D-, DL-, ancl L-fi-pheny]a]anine-i-14C were carried out with a 6°Co-source in the dose range f r o m 50 to 10 ~ rad. The dose rate amounted to 6 . 1 0 2 rad/h for doses up to 100 rad, and to 3.4.103 rad/h for higher doses. t00 mg of substance, each with a specific activity of 20 ~Ci/g, were irradiated in evacuated glass tubes at room temperature. Each tube was then connected to the counting apparatus (Fig. 1) and the volume, limited b y the stopcocks t, 2 and 4, was pumped out and sealed. Then the break-seal of the glass tube was cracked by the iron core and the phenylalanine was sublimed inside the irradiation tube at a temperature of i60 to t90 ° C under a vacuum of l0 -~ Tort to liberate the trapped
66
14co2
O. Merwitz
I06:
I05
e
o
o
o
104
103
I
I
I
J 11
Illl0
2
I
i
I
I Ill
l 103
I
I
I
I III
Ili04
I
I
I
I II
I Ii105
I
dose
[
I
I II
[,od]
I'r06~
Fig. 9. Dose-effect curves for the 14C02-eleavage from D-, DL- and L-fl-phenylalanine-t-14C
14CO2. Under these conditions phenylalanine is sublimed in an undecomposed state, as was proved by the identity of the uv-speetra and the optical rotation of sublimed and non-sublimed phenalalanine. After cooling, stopcock 3 was closed, methane was filled into the counter via stopcock 2 and the 14C02-counting rate was measured in antieoincidence. Converted to the total volume and referred to one g of substance, the cleavage rates were then obtained for 14C02.
Influence o/Impurities on the 14C02-Counting Rate I t has been shown by many experiments that the G-values for the decarboxylation of amino acids in the solid state are not very different. I f another amino acid labeled on C-1, which had the same thermal properties, were present as impurity, e.g. to the extent of 1%, and the cleavage rate was 0.1% in both cases; then 0.i % of i % and 0.1% of 99% would be cleaved. The cleavage rate of the impurities would have to be i00 times as great as that of phenylalanine to give the same effect. It can thus be deduced that even if solid impurities were present to an extent of several percent, the l~C02-eounting rate would not be altered appreciably. Volatile impurities are removed during sublimation. Results Fig. 2 shows the exponential dose-effect curves determined in this manner. The counting rate of the 1~C02 cleaved from ± g of labeled phenylalanine with the specific activity of 20 ~zCi/g is plotted against the dose on a log-log scale. Below 50 rad the counting rates cannot be measured with sufficient accuracy. In the range from l02 to t04 rad the cleavage rate of the D-isomer is higher b y a maximum factor of 2.7 than that of the L-isomer. The difference in the reaction
Selective l%adiolysis of Enantiomers
67
Table l. Counting rates, percentages and dose-dependent G-values for the C02-eleavage; G-value ratio D/L, D/DL and DL/L Dose rad
50
5. t02
5- 10 a
5.104
5.105
D
cpm/g %C02 G(C03)
1.3.104 0.028 3.2.104
2.4.104 0.054 6.3. t03
3.0.104 0.067 7.7.103
4.6- 104 0.t01 1.2.103
1.5 • t05 0.337 39
DL
cpm/g %C03 G (CO2)
9.0.103 0.020 2.3.104
t.6.104 0.036 4.2.103
2.2. t04 0.050 5.8- 102
3.5.104 0.078 90
1.4.105 0.310 35
L
cpm/g %C02 G(C02)
5.7 • 10 a 0.013 1.5. t04
9.0.103 0.020 2.3- I0 a
t.2.104 0.026 3.0.102
2.5.105 0.056 65
t.3 • 105 0.300 35
D/L
2.1
2.7
2.6
1.8
IA
DL/L
1.5
t.8
1.9
t.4
1.0
D/DL
t.4
1.5
t.3
t.3
1.1
r a t e b e t w e e n t h e D- a n d L-forms is m a n i f e s t e d here v e r y m a r k e d l y . A b o v e 104 r a d t h e differences becomes smaller. The D L - e u r v e in t h e range b e t w e e n t 0 ~ a n d 104 r a d lies a l m o s t e x a c t l y h a l l w a y b e t w e e n t h e D- a n d t h e L-curve. The p e r c e n t a g e s for t h e 14CO~-cleavage d e t e r m i n e d from t h e r a d i o a c t i v i t y in t h e gas phase b y c o m p a r i n g i t w i t h t h a t in t h e solid substance, a n d t h e dosed e p e n d e n t G-values c a l c u l a t e d t h e r e f r o m b y neglecting t h e isotope effect are i n d i c a t e d in T a b l e 1. T h e high G-values give rise to t h e conclusion t h a t t h e r a d i a t i o n - i n d u c e d d e c a r b o x y l a t i o n of p h e n y l a l a n i n e is a chain reaction. There are chain r e a c t i o n s in t h e ease of G-values being higher t h a n t 0 (Henglein et al., 1969). U p to now, G-values higher t h a n i 0 h a v e been o b s e r v e d o n l y in a few eases of r a d i a t i o n - i n d u c e d r e a c t i o n s in organic solids ( L i n d b l o m et al., 1961; D i z d a r o g l u et al., 1973; v o n S o n n t a g et al., 1974). I t is impossible to develop a kinetic m o d e l of t h e m e c h a n i s m of t h e c h a i n r e a c t i o n m e r e l y on t h e basis of t h e s e m e a s u r e m e n t s . F o r this p u r p o s e i t is also necessary to a n a l y s e t h e o t h e r i r r a d i a t i o n p r o d u c t s . However, such analysis p r o b a b l y c a n n o t be carried o u t w i t h t h e same accuracy. I t is t r i e d now to establish a m o d e l i n t e g r a t i n g t h e results o b t a i n e d b y E S R - s p e c t r o s e o p y . T h e counting r a t e s i n d i c a t e d in T a b l e 1 p a r t l y e x h i b i t m e a s u r e d values, a n d p a r t l y values t a k e n from t h e curves. I n t h e m o s t u n f a v o u r a b l e ease, a v a r i a t i o n b y 5 % of t h e G-values for t h e D- a n d L-forms, l y i n g in t h e r a n g e of t h e m e a s u r i n g accuracy, will result in v a r i a t i o n of t h e D / L - r a t i o b y 10 %. Discussion
According to t h e classical conception, e n a n t i o m e r i e forms do n o t f e a t u r e a n y differences of energy, b u t for some t i m e t h e r e h a v e also been certain d o u b t s concerning this opinion (for a review see T h i e m a n n , 1974). G e n e r a l l y it is postul a t e d t h a t such differences of energy, if existing a t all, w o u l d be v e r y small so t h a t t h e y are not, or h a r d l y not, accessible to an e x p e r i m e n t a l d e t e r m i n a t i o n . Therefore t h e a t t e n t i o n was d r a w n to r e a c t i o n s where v e r y s m a l l differences of energy can
68
O. Merwitz
become manifest b y amplification or cascading. P o l y m e r i z a t i o n ( Y a m a g a t a , 1966) or p r e c i p i t a t i o n ( T h i e m a n n a n d W a g e n e r , i970) were considered as such reactions. Nuclear-physicM conceptions too were associated w i t h t h e p r o b l e m of isomerism. Some a u t h o r s d e a l t w i t h t h e a n a l o g y b e t w e e n t h e p a r i t y v i o l a t i o n in w e a k intera c t i o n a n d t h e c h i r a l i t y of biochemical c o m p o u n d s (Sagan, 1957; Vester a n d U l b r i c h t , 1959; U l b r i c h t a n d Vester, J962) a n d e s t i m a t e d t h e m a g n i t u d e of e n e r g y v a r i a t i o n (Rein, 1974). This m a t t e r was d e a l t w i t h in a d e t a i l e d m a n n e r on t h e occasion of a recent s y m p o s i u m on t h e origin a n d amplification of o p t i c a l a s y m m e t r y in chemical s y s t e m s (Thiemann, i973). The possibility of cascading b y r a d i a t i o n - i n d u c e d chain reactions has n o t been so far t a k e n into consideration. B y m e a n s of t h e r a d i o a c t i v e t r a c e r m e t h o d this possibility was realized w i t h t h e amino acid p h e n y l a l a n i n e in t h e p r e s e n t experiments. B y utilizing 14C-labeled e n a n t i o m e r s it was possible to m e a s u r e t h e doseeffect curves in t h e low dose range where t h e differences in t h e r a d i a t i o n effect can be observed. I n this conncction~ it is of special i n t e r e s t to note t h a t chain r e a c t i o n s will also occur in i r r a d i a t i n g c a r b o h y d r a t e s in t h e solid s t a t e (Dizdaroglu et al., 1973; von S o n n t a g et al., i974), b u t t h e r e are n o t y e t a n y e x p e r i m e n t s a v a i l a b l e on e n a n t i o m e r i c forms. T h e y would give i n f o r m a t i o n on w h e t h e r t h e p r e d o m i n a n c e of t h e L - f o r m in t h e amino acids a n d of t h e D - f o r m in t h e sugars can be i n t e r p r e t e d as a result of a n a t u r a l r a d i a t i o n damage. The p r e s e n t results are i n d i c a t i v e of t h e e x t e n t of t h e p r e s u m e d e n e r g y difference b e t w e e n D- a n d L - p h e n y l M a n i n e o n l y if t h e d e p e n d e n c e of t h e chain l e n g t h on t h e dose r a t e a n d on t h e crystal s t a t e is known. A r e p o r t of these e x p e r i m e n t s will follow shortly.
References Castrillon, J. A., Despopoulos, A., Tolbert, :B. M. : t39th Meeting of the American Chemical Society, St. Louis, Miss., March 21--30, 1961 Clark, J., Kushelewsky, A. P., Slifkin, M. A. : :Determination of radioIysis products of amino acids by mass spectrometry. Radiat. Effects 2, 303--304 (1970) ])ale, W. M., Davies, J. V., Gilbert, W. C. : The kinetics and specificities of deamination of nitrogeneous compounds by X-irradiation. Biochem. J. 45, 93 99 (i949) ])izdarogln, M., yon Sonntag, C., Schulte-Frohlinde, D., Dahlhoff, W. V.: l~otiz fiber die Darstellung von 5-Desoxylactobionsiiure auf strahlenchemischem Wege. Liebigs Ann. Chem. 1973, 1592--1594 Garay, A. : Origin and role of optical isomery in life. Nature (Lond.) 219, 338--340 (t968) Garrison, W. M. : Radiation-induced reactions of amino acids and peptides. Radiat. Rcs. Revs. 3, 305--326 (1972) GottschM1, W. C., Tolbert, B. M. : The solid-state radiation chemistry of selected transition metal chelates of glycine and alaninc. J. Phys. Chem. 72, 922--925 (1968) Gross, I)., Grodsky, G. : On the sublimation of amino acids and peptides. J. Amcr. chem. Soc. 77, 4678--t680 (4955) Henglein, A., Schnabel, W., Wcndcnberg, J. : Einffihrung in die Strahlenchemie, 4. Anti., S. 20. Weinheim: Vcrlag Chemic 4969 Horan, P. K., Taylor, W. D., Strother, G. :K., Snipes, W.: Stability of radiation-induced organic free radicals. Biophys. J. 8, t64--474 (4968) de Jong, J., Loman, H., Blok, J. : Inactivation of biologically active DNA by radiationinduced phenylManine radicals. Int. J. P~adiat. Biol. 22, 44--24 (4972) Korgaonkar, K. S., Donde, R. B. : The effects of ionizing radiation on L-, DL-phenylalanine and L-, DL-tryptophan studied by ultra-violet and infra-red spectrophotometry. Int. J. Radiat. Biol. 5, 67--77 (t962)
Selective l~adiolysis of Enantiomers
69
Lindblom, R. O., Lemmon, t~. M., Calvin, M. : Kinetic and electron spin resonance studies of the radiation decomposition of crystalline choline chldride. J. Amer. chem. Soc. 83, 2484 2489 (196t) Merwitz, O. : Die Tritiumabspaltung aus festem Adenin-2-T und Adenin-8-T unter der Einwirkung yon 6°Co-y-Strahlung. ]3iophysik 8, 245--253 (1972a) Merwitz, O. : Die Tritiumabspaltung aus festem, gammabestrahltem Uracil-5-T und Uracil-6-T. ]3iophysik 8, 310--315 (1972b) Merwitz, O. : Die Tritiumabspaltung aus festem, gammabestrahltem Cytosin-5-T. Rad. and Environm. ]3iophysics 11, 63--68 (t 974a) Merwitz, 0., Otto, R. : Linear and sigmoid dose-effects relations: investigations with gammairradiated thymine-(methyl-T) in aqueous solution and in the solid state, l~ad. and Environm. Biophysics 11, 69--77 (1974b) Meshitsuka, G., Shindo, K., Minegishi, A., Suguro, H., Shinozaki, Y.: Radiolysis of solid glycine. Bull. chem. Soc. Jap. 37, 928--930 (1964) Minegishi, A., Shinozaki, Y., Meshitsuka, G. : Radiolysis of solid L-a-alanine. Bull. Chem. Soc. Jap. 40, 1271--1272 (t967) Rein, D. : Some remarks on parity-violating effects of intramolecular interaction. J. mol. Evol. 4, 15--22 (t974) Sagan, C. : l~adiation and the origin of the gene. Evolution 11, 40--55 (t957) yon Sonntag, C., Neuwald, K., Dizdaroglu, M. : Radiation chemistry of DNA model compounds. III. y-radiolysis of 2-deoxy-D-ribose in the crystalline state. Conversion of 2-deoxy-D-ribose into 2,5-dideoxy-D-erythro-pentonic acid via a chain reaction, l~adiat. Res. 58, I - - 8 (1974) Thiemann, W. : International Symposium on Generation and Amplification of Asymmetry in Chemical Systems, 540 p. Jfllich, 24--26 September t973 Thiemann, W. : The origin of optical activity. Naturwissenschaften 61, 476--483 (1974) Thiemann, W., Wagener, K. : Is there an energy difference between enantiomorphic structures ? Angew. Chem. Intern. Ed. 9, 740--74t (1970) Tolbert, ]3. M., Stansfield, R., Krinks, M. It. : l~adiation decomposition of solid amino acids. EU1%-1625 e, 575--581 (1963) Ulbricht, T. L. V., Vester, F. : Attempts to induce optical activity with polarized fi-irradiation. Tetrahedron 18, 629--637 (1962) Vester, F., Ulbricht. T, L. V. : Optische Aktivitiit und Pariti~tsverletzung im fi-Zerfall. Naturwissenschaften 46, 68 (1959) ¥amagata, Y. : A hypothesis for the asymmetric appearance of biomolecules on earth. J. theor. Biol. 11, 495--498 (1966)
Received July 23, 1975, in revised form December 8, i975
Dr. O. Merwitz Institut ffir Chemic der Kernforschungsanlage D-5170 Jiilich Postfach 365 Fed. Rep. Germany
Radiation and Environmental Biophysics © Springer-Verlag1976
Selective Radiolysis of Enantiomers O. Merwitz Jfilich l~uelear Research Centre, Institute for Chemistry Jfilich, Fed. Rep. Germany
Summary. The gamma-induced decarboxylation of fl-phenylalanine in the solid state is a chain reaction. Both enantiomers and the raeemate offl-phenylalanine-l-z4C show different exponential dose-effect curves for the radiationinduced cleavage of z4C02. Introduction The main effects in the solid state radiolysis of amino acids are the following: hydrogen cleavage deamination decarboxylation Hydrogen cleavage and deamination give rise to the formation of free radicals, which in the case of being long-lived, can be detected b y ESR-spectroscopy. The decarboxylation causes the formation of a saturated amine the concentration of which in the irradiated substance is identical with the concentration of the produced CO m. In the case of ~-phenylalanine, ~-phenylethylamine is produced: CeHsCH~CH(NH~)COOH-~ CeH~CH2CH~NH~ ~- CO n The analysis of the irradiation products of solid amino acids was previously carried out partly b y mass spectrometry or chromatographically (Gottschall and Tolbert, 1968; Clark et al., 1970), and partly after dissolving the substance irradiated under vacuum in water (Dale et al., 1949; Tolbert et al., 1963; Meshitsuka et al., 1964; Minegishi et al., 1967; Garrison, 1972). The radiation decarboxylation of carboxyl-labeled amino acids in the solid state was also determined using 14C02, which was trapped as Baz4C03, weighed, converted to z4C02, and swept into an ion chamber where the activity was measured. The G (C02)-value was found to be between i and 2 for most of the amino acids studied (Castrillon et al., 1961). l~oreover the variations in the irradiation effect on the isomeric forms of the amino acids were studied. When irradiating DL-tyrosine with ~-particles, the D-isomer is decomposed faster than the L-isomer (Garay, i968). The activation
64
O. Merwitz
energies for the decay of gamma-induced radicals in L- or D-alanine are different (30.3 and 25.9 kcal/1V[, resp.) (Horan et al., t968). The G-values for the radiation damage to DL-phenylalanine and L-phenylalanine at a dose of 4.10 a rad are also different (2.9 and 3.8, resp.) (Korgaonkar and Donde, 1962). However, the accuracies of the applied measuring methods are not sufficient to make an interpretation possible. The irradiation experiments outlined in the literature were carried out either for one dose or in a very narrow dose range so that the question as to whether the decarboxylation follows a linear or an exponential dose-effect curve remained undecided. _As to the selection of phenylalanine for studying the radiation-induced decarboxylation, physico-ehemical and biological reasons have been the decisive factors. Previous investigations of the radiation damage to solid all-labeled nucleic acid bases had proved the fact that the dose-effect curves are measurable for the cleavage of labeled hydrogen in a wide dose range (Merwitz, i972a; Merwitz, 1972 b ; Merwitz, t 974 a; Merwitz and Otto, 1974 b). The measurements were based on the condition of thermal stability of the irradiated substances due to the fact that they had to be sublimed or, at least, heated up for the liberation of the hydrogen trapped in them. Thermal stability is also necessary in the case of carboxyl-labeled amino acids as they also must be sublimed for direct quantitative determination of laC02. Some of the important amino acids are sublimable. This holds true for phenylalanine as well (Gross and Grodsky, 1955). Phenylalanine was chosen because both enantiomers and the racemate are commercially available in a carboxyl-labeled form (Radiochemical Centre Amersham). I t was possible by this fact to detect differences in the radiochemical behaviour of enantiomers. Phenylalanine is of biological interest as an essential amino acid and precursor for the formation of tyrosine and of adrenaline ; its absence causes thyroidal and cortico-adrenal disfunctions. The failure of the catabolisms gives rise to phenylketonuria. Phenylalanine radicals inactivate DNA (de Jong et al., i972). Material and Method
Analytical Quality and Purity of the Substances The serf-decomposition of the labeled compounds was examined before doing the irradiation experiments. I t was found that autoradiolysis at a specific activity of 20 ~Ci/g was negligeable over periods of l0 rain to i2 days necessary for irradiation, and that reasonable counting rates for 14CO~-measurements resulted. In order to reduce the radioactivity of the purchased compounds D-, DL-, and L-/~-phenylalanine-l-laC (total activity 250~Ci each) to the level mentioned above, their aqueous solutions were added to boiling solutions of the corresponding non-labeled compounds at appropriate concentrations. After cooling and filtering, all substances were recrystallized and dried to constant weight in vacuum. Such purification would be sufficient for ordinary chemical operations; but in this case a repeated sublimation is also necessary, since after recrystallizing and drying,
Selective Radiolysis of Enantiomers
[
65
:
t
counting t u b e CH4
glass tu be
I
/
Fig. 1. Schematic diagram of counting apparatus
traces of volatile radioactive impurities are still present. Normally a very low, constant counting rate (t00 to 200 epm/i00 mg of substance) is reached after 2 to 3 sublimations. Immediately before irradiation, the specific activity of the samples was measured by liquid scintillation. The non-labeled isomers and the racemate were of the highest obtainable purity (Fluka: purissimum grade). The radiochemieal purity of DL-fi-phenylalanine-l-14C and L-~-phenylalanine-t-l~C was determined as 99% b y paper chromatography, and t h a t of D-/~-phenylalanine-l-l~C as 97% b y thin-layer chromatography (Radioehemical Centre Amersham). The isotopic abundance of the earboxyl C-atom was 97 % in D-fi-phenylalanine1-14C, 95% in I)L-fi-phenylalanine-l-14C, and 9 t % in L-fi-phenylalanine-l-14C. The remaining radioactivity was distributed among the other carbon atoms, i.e. it appears after decarboxylation in the phenylethylamine which readily condenses.
Measurement o/Radiation-induced 14C02-Cleavage ]rom D-, DL-, and L-fi-Phenylalanine- 1-14C The irradiations of D-, DL-, ancl L-fi-pheny]a]anine-i-14C were carried out with a 6°Co-source in the dose range f r o m 50 to 10 ~ rad. The dose rate amounted to 6 . 1 0 2 rad/h for doses up to 100 rad, and to 3.4.103 rad/h for higher doses. t00 mg of substance, each with a specific activity of 20 ~Ci/g, were irradiated in evacuated glass tubes at room temperature. Each tube was then connected to the counting apparatus (Fig. 1) and the volume, limited b y the stopcocks t, 2 and 4, was pumped out and sealed. Then the break-seal of the glass tube was cracked by the iron core and the phenylalanine was sublimed inside the irradiation tube at a temperature of i60 to t90 ° C under a vacuum of l0 -~ Tort to liberate the trapped
66
14co2
O. Merwitz
I06:
I05
e
o
o
o
104
103
I
I
I
J 11
Illl0
2
I
i
I
I Ill
l 103
I
I
I
I III
Ili04
I
I
I
I II
I Ii105
I
dose
[
I
I II
[,od]
I'r06~
Fig. 9. Dose-effect curves for the 14C02-eleavage from D-, DL- and L-fl-phenylalanine-t-14C
14CO2. Under these conditions phenylalanine is sublimed in an undecomposed state, as was proved by the identity of the uv-speetra and the optical rotation of sublimed and non-sublimed phenalalanine. After cooling, stopcock 3 was closed, methane was filled into the counter via stopcock 2 and the 14C02-counting rate was measured in antieoincidence. Converted to the total volume and referred to one g of substance, the cleavage rates were then obtained for 14C02.
Influence o/Impurities on the 14C02-Counting Rate I t has been shown by many experiments that the G-values for the decarboxylation of amino acids in the solid state are not very different. I f another amino acid labeled on C-1, which had the same thermal properties, were present as impurity, e.g. to the extent of 1%, and the cleavage rate was 0.1% in both cases; then 0.i % of i % and 0.1% of 99% would be cleaved. The cleavage rate of the impurities would have to be i00 times as great as that of phenylalanine to give the same effect. It can thus be deduced that even if solid impurities were present to an extent of several percent, the l~C02-eounting rate would not be altered appreciably. Volatile impurities are removed during sublimation. Results Fig. 2 shows the exponential dose-effect curves determined in this manner. The counting rate of the 1~C02 cleaved from ± g of labeled phenylalanine with the specific activity of 20 ~zCi/g is plotted against the dose on a log-log scale. Below 50 rad the counting rates cannot be measured with sufficient accuracy. In the range from l02 to t04 rad the cleavage rate of the D-isomer is higher b y a maximum factor of 2.7 than that of the L-isomer. The difference in the reaction
Selective l%adiolysis of Enantiomers
67
Table l. Counting rates, percentages and dose-dependent G-values for the C02-eleavage; G-value ratio D/L, D/DL and DL/L Dose rad
50
5. t02
5- 10 a
5.104
5.105
D
cpm/g %C02 G(C03)
1.3.104 0.028 3.2.104
2.4.104 0.054 6.3. t03
3.0.104 0.067 7.7.103
4.6- 104 0.t01 1.2.103
1.5 • t05 0.337 39
DL
cpm/g %C03 G (CO2)
9.0.103 0.020 2.3.104
t.6.104 0.036 4.2.103
2.2. t04 0.050 5.8- 102
3.5.104 0.078 90
1.4.105 0.310 35
L
cpm/g %C02 G(C02)
5.7 • 10 a 0.013 1.5. t04
9.0.103 0.020 2.3- I0 a
t.2.104 0.026 3.0.102
2.5.105 0.056 65
t.3 • 105 0.300 35
D/L
2.1
2.7
2.6
1.8
IA
DL/L
1.5
t.8
1.9
t.4
1.0
D/DL
t.4
1.5
t.3
t.3
1.1
r a t e b e t w e e n t h e D- a n d L-forms is m a n i f e s t e d here v e r y m a r k e d l y . A b o v e 104 r a d t h e differences becomes smaller. The D L - e u r v e in t h e range b e t w e e n t 0 ~ a n d 104 r a d lies a l m o s t e x a c t l y h a l l w a y b e t w e e n t h e D- a n d t h e L-curve. The p e r c e n t a g e s for t h e 14CO~-cleavage d e t e r m i n e d from t h e r a d i o a c t i v i t y in t h e gas phase b y c o m p a r i n g i t w i t h t h a t in t h e solid substance, a n d t h e dosed e p e n d e n t G-values c a l c u l a t e d t h e r e f r o m b y neglecting t h e isotope effect are i n d i c a t e d in T a b l e 1. T h e high G-values give rise to t h e conclusion t h a t t h e r a d i a t i o n - i n d u c e d d e c a r b o x y l a t i o n of p h e n y l a l a n i n e is a chain reaction. There are chain r e a c t i o n s in t h e ease of G-values being higher t h a n t 0 (Henglein et al., 1969). U p to now, G-values higher t h a n i 0 h a v e been o b s e r v e d o n l y in a few eases of r a d i a t i o n - i n d u c e d r e a c t i o n s in organic solids ( L i n d b l o m et al., 1961; D i z d a r o g l u et al., 1973; v o n S o n n t a g et al., 1974). I t is impossible to develop a kinetic m o d e l of t h e m e c h a n i s m of t h e c h a i n r e a c t i o n m e r e l y on t h e basis of t h e s e m e a s u r e m e n t s . F o r this p u r p o s e i t is also necessary to a n a l y s e t h e o t h e r i r r a d i a t i o n p r o d u c t s . However, such analysis p r o b a b l y c a n n o t be carried o u t w i t h t h e same accuracy. I t is t r i e d now to establish a m o d e l i n t e g r a t i n g t h e results o b t a i n e d b y E S R - s p e c t r o s e o p y . T h e counting r a t e s i n d i c a t e d in T a b l e 1 p a r t l y e x h i b i t m e a s u r e d values, a n d p a r t l y values t a k e n from t h e curves. I n t h e m o s t u n f a v o u r a b l e ease, a v a r i a t i o n b y 5 % of t h e G-values for t h e D- a n d L-forms, l y i n g in t h e r a n g e of t h e m e a s u r i n g accuracy, will result in v a r i a t i o n of t h e D / L - r a t i o b y 10 %. Discussion
According to t h e classical conception, e n a n t i o m e r i e forms do n o t f e a t u r e a n y differences of energy, b u t for some t i m e t h e r e h a v e also been certain d o u b t s concerning this opinion (for a review see T h i e m a n n , 1974). G e n e r a l l y it is postul a t e d t h a t such differences of energy, if existing a t all, w o u l d be v e r y small so t h a t t h e y are not, or h a r d l y not, accessible to an e x p e r i m e n t a l d e t e r m i n a t i o n . Therefore t h e a t t e n t i o n was d r a w n to r e a c t i o n s where v e r y s m a l l differences of energy can
68
O. Merwitz
become manifest b y amplification or cascading. P o l y m e r i z a t i o n ( Y a m a g a t a , 1966) or p r e c i p i t a t i o n ( T h i e m a n n a n d W a g e n e r , i970) were considered as such reactions. Nuclear-physicM conceptions too were associated w i t h t h e p r o b l e m of isomerism. Some a u t h o r s d e a l t w i t h t h e a n a l o g y b e t w e e n t h e p a r i t y v i o l a t i o n in w e a k intera c t i o n a n d t h e c h i r a l i t y of biochemical c o m p o u n d s (Sagan, 1957; Vester a n d U l b r i c h t , 1959; U l b r i c h t a n d Vester, J962) a n d e s t i m a t e d t h e m a g n i t u d e of e n e r g y v a r i a t i o n (Rein, 1974). This m a t t e r was d e a l t w i t h in a d e t a i l e d m a n n e r on t h e occasion of a recent s y m p o s i u m on t h e origin a n d amplification of o p t i c a l a s y m m e t r y in chemical s y s t e m s (Thiemann, i973). The possibility of cascading b y r a d i a t i o n - i n d u c e d chain reactions has n o t been so far t a k e n into consideration. B y m e a n s of t h e r a d i o a c t i v e t r a c e r m e t h o d this possibility was realized w i t h t h e amino acid p h e n y l a l a n i n e in t h e p r e s e n t experiments. B y utilizing 14C-labeled e n a n t i o m e r s it was possible to m e a s u r e t h e doseeffect curves in t h e low dose range where t h e differences in t h e r a d i a t i o n effect can be observed. I n this conncction~ it is of special i n t e r e s t to note t h a t chain r e a c t i o n s will also occur in i r r a d i a t i n g c a r b o h y d r a t e s in t h e solid s t a t e (Dizdaroglu et al., 1973; von S o n n t a g et al., i974), b u t t h e r e are n o t y e t a n y e x p e r i m e n t s a v a i l a b l e on e n a n t i o m e r i c forms. T h e y would give i n f o r m a t i o n on w h e t h e r t h e p r e d o m i n a n c e of t h e L - f o r m in t h e amino acids a n d of t h e D - f o r m in t h e sugars can be i n t e r p r e t e d as a result of a n a t u r a l r a d i a t i o n damage. The p r e s e n t results are i n d i c a t i v e of t h e e x t e n t of t h e p r e s u m e d e n e r g y difference b e t w e e n D- a n d L - p h e n y l M a n i n e o n l y if t h e d e p e n d e n c e of t h e chain l e n g t h on t h e dose r a t e a n d on t h e crystal s t a t e is known. A r e p o r t of these e x p e r i m e n t s will follow shortly.
References Castrillon, J. A., Despopoulos, A., Tolbert, :B. M. : t39th Meeting of the American Chemical Society, St. Louis, Miss., March 21--30, 1961 Clark, J., Kushelewsky, A. P., Slifkin, M. A. : :Determination of radioIysis products of amino acids by mass spectrometry. Radiat. Effects 2, 303--304 (1970) ])ale, W. M., Davies, J. V., Gilbert, W. C. : The kinetics and specificities of deamination of nitrogeneous compounds by X-irradiation. Biochem. J. 45, 93 99 (i949) ])izdarogln, M., yon Sonntag, C., Schulte-Frohlinde, D., Dahlhoff, W. V.: l~otiz fiber die Darstellung von 5-Desoxylactobionsiiure auf strahlenchemischem Wege. Liebigs Ann. Chem. 1973, 1592--1594 Garay, A. : Origin and role of optical isomery in life. Nature (Lond.) 219, 338--340 (t968) Garrison, W. M. : Radiation-induced reactions of amino acids and peptides. Radiat. Rcs. Revs. 3, 305--326 (1972) GottschM1, W. C., Tolbert, B. M. : The solid-state radiation chemistry of selected transition metal chelates of glycine and alaninc. J. Phys. Chem. 72, 922--925 (1968) Gross, I)., Grodsky, G. : On the sublimation of amino acids and peptides. J. Amcr. chem. Soc. 77, 4678--t680 (4955) Henglein, A., Schnabel, W., Wcndcnberg, J. : Einffihrung in die Strahlenchemie, 4. Anti., S. 20. Weinheim: Vcrlag Chemic 4969 Horan, P. K., Taylor, W. D., Strother, G. :K., Snipes, W.: Stability of radiation-induced organic free radicals. Biophys. J. 8, t64--474 (4968) de Jong, J., Loman, H., Blok, J. : Inactivation of biologically active DNA by radiationinduced phenylManine radicals. Int. J. P~adiat. Biol. 22, 44--24 (4972) Korgaonkar, K. S., Donde, R. B. : The effects of ionizing radiation on L-, DL-phenylalanine and L-, DL-tryptophan studied by ultra-violet and infra-red spectrophotometry. Int. J. Radiat. Biol. 5, 67--77 (t962)
Selective l~adiolysis of Enantiomers
69
Lindblom, R. O., Lemmon, t~. M., Calvin, M. : Kinetic and electron spin resonance studies of the radiation decomposition of crystalline choline chldride. J. Amer. chem. Soc. 83, 2484 2489 (196t) Merwitz, O. : Die Tritiumabspaltung aus festem Adenin-2-T und Adenin-8-T unter der Einwirkung yon 6°Co-y-Strahlung. ]3iophysik 8, 245--253 (1972a) Merwitz, O. : Die Tritiumabspaltung aus festem, gammabestrahltem Uracil-5-T und Uracil-6-T. ]3iophysik 8, 310--315 (1972b) Merwitz, O. : Die Tritiumabspaltung aus festem, gammabestrahltem Cytosin-5-T. Rad. and Environm. ]3iophysics 11, 63--68 (t 974a) Merwitz, 0., Otto, R. : Linear and sigmoid dose-effects relations: investigations with gammairradiated thymine-(methyl-T) in aqueous solution and in the solid state, l~ad. and Environm. Biophysics 11, 69--77 (1974b) Meshitsuka, G., Shindo, K., Minegishi, A., Suguro, H., Shinozaki, Y.: Radiolysis of solid glycine. Bull. chem. Soc. Jap. 37, 928--930 (1964) Minegishi, A., Shinozaki, Y., Meshitsuka, G. : Radiolysis of solid L-a-alanine. Bull. Chem. Soc. Jap. 40, 1271--1272 (t967) Rein, D. : Some remarks on parity-violating effects of intramolecular interaction. J. mol. Evol. 4, 15--22 (t974) Sagan, C. : l~adiation and the origin of the gene. Evolution 11, 40--55 (t957) yon Sonntag, C., Neuwald, K., Dizdaroglu, M. : Radiation chemistry of DNA model compounds. III. y-radiolysis of 2-deoxy-D-ribose in the crystalline state. Conversion of 2-deoxy-D-ribose into 2,5-dideoxy-D-erythro-pentonic acid via a chain reaction, l~adiat. Res. 58, I - - 8 (1974) Thiemann, W. : International Symposium on Generation and Amplification of Asymmetry in Chemical Systems, 540 p. Jfllich, 24--26 September t973 Thiemann, W. : The origin of optical activity. Naturwissenschaften 61, 476--483 (1974) Thiemann, W., Wagener, K. : Is there an energy difference between enantiomorphic structures ? Angew. Chem. Intern. Ed. 9, 740--74t (1970) Tolbert, ]3. M., Stansfield, R., Krinks, M. It. : l~adiation decomposition of solid amino acids. EU1%-1625 e, 575--581 (1963) Ulbricht, T. L. V., Vester, F. : Attempts to induce optical activity with polarized fi-irradiation. Tetrahedron 18, 629--637 (1962) Vester, F., Ulbricht. T, L. V. : Optische Aktivitiit und Pariti~tsverletzung im fi-Zerfall. Naturwissenschaften 46, 68 (1959) ¥amagata, Y. : A hypothesis for the asymmetric appearance of biomolecules on earth. J. theor. Biol. 11, 495--498 (1966)
Received July 23, 1975, in revised form December 8, i975
Dr. O. Merwitz Institut ffir Chemic der Kernforschungsanlage D-5170 Jiilich Postfach 365 Fed. Rep. Germany
