EDWINE. BUDZINBKI AND HAROLD C. Box
2564
NzO-
Nz
+
C5H10+ negative ion
Here H’is an atom that reacts while hot and is not scavengable by olefins; H possesses less energy than does H‘ and is scavengable by olefins.
+
H 01 +Hal. (16) The curves drawn through the hydrogen yields in Figure 3 and curve a in Figure 5 were drawn using the value k16/ICl6 = 140 for both propylene and cyclopentene in reaction 16. The fraction of the residual hydrogen yield at a given nitrous oxide concentration that is scavengable by an olefin is about 50% at all nitrous oxide concentrations, the same as that in the absence of nitrous oxide. Kitrous oxide inhibits hydrogen formation by intercepting reaction 9.
NzO
+ e- -+- SzO-
-
+ 0no H or H2
(3)
(18)
The present results lead t o the approximate G values listed after reactions 7-15 for pure cyclopentane, The value G(H2) = 5.3 obtained from the purified cyclopentane in the present work, and the effect of added hydrogen chloride, indicate that a small amount of electron scavenger such as carbon dioxide remained in the cyclopentane. Cyclopentene-Nitrous Oxide Solutions. The fact that the electron scavenging efficiency of nitrous oxide is lower in liquid olefins than in saturated hydrocarbons (Figure 4 and ref 2) is associated with the fact that the ion-electron separation distance in spurs is lower in the former than in the latter.ls The smaller ion-electron separation distance results in a shorter geminate neutralization time and makes it more difficult for a solute molecule to intercept the electron before it reacts with its geminate ion. (18) W. F. Schmidt and A. 0. Allen, J . Chem. Phys., 52, 2345
(17)
(1970).
The Oxidation and Reduction of Organic Compounds by Ionizing Radiation: L-Penicillamine Hydrochloride by Edwin E. Budzinski and Harold C. Box* Biophysics Department, Roswell Park Memorial Institute, Buflalo, New York
I4203
(Received December 29, 1970)
Publication costs assisted by the Public Health Service
Esr measurements on single crystals of L-penicillamine HC1 X-irradiated and observed at 4.2’K reveal one reduced and two oxidized products, The oxidized products are “sulfur” radicals SC(CH3)&H(NW3C1)COOH and chlorine atoms. The reduced product is the anion HSC(CH3),CH(NH,C1)C(OH)O-. On warming, the yield of “sulfur” radicals isaugmented by hole transfer from chlorine atoms to sulfur atoms. Dissociation of the anion also occurs on warming, producing the radical KSC(CHs)&HCOOH. At room temperature the latter radical also disappears, and the final absorption is entirely due to “sulfur” radicals. It appears that processes initiated by both oxidation and reduction contribute to the formation of “sulfur” radicals. Altogether nine paramagnetic absorption spectra were observed in this study including several due to different conformations of the aforementioned radicals.
Introduction I n a previous paper1 it was pointed out that it is often possible to stabilize the primary products of oxidation and reduction formed by ionizing radiation in organic compounds by maintaining the irradiated sample at the (4’20K)’ The damaged temperature Of liquid species can usually be identified using esr SpeCtrOSThe Journal of Physical Chemistry, Val. 76, N o . 1’7, 2971
copy. I n this investigation the technique was used to study radiation damage in single crystals of penidlamine HC1 HS-C(CHg)2-CH(NHs +Cl-)-COOH
I (1) H. C. Box, H. G. Freund, K. T. Lilga, and E. E. Budrinski, J. Phys. Chem., 74,40 (1970).
OXIDATIOKAND REDUCTION OF IOXIZIXG RADIATION The effect of ionizing radiation on sulfhydryls is of particular interest in radiation biology, Cysteine HC1,
2565
ta
HS-CHZ-CH(NH3 +Cl-)-COOH I1 which is a more biologically significant sulfhydryl compound, has been studied extensively. Wheaton and OrmerodZand Akasaka3 have used esr spectroscopy to examine single crystals of cysteine HC1 irradiated a t liquid nitrogen temperature. However, this temperature is not low enough to stabilize the primary radiation damage products. We have irradiated and examined cysteine HC1 at 4.2'1K and found clear evidence that a more primitive stage of the radiation damage process can be observed at the lower t e m p e r a t ~ r e . ~The esr absorption spectra are not as well defined, however, as those obtained from penicillamine. Consequently, we have concentrated our efforts on the latter compound. As an irradiated penicillamine HC1 crystal is allowed t o warm above 4.2OK various secondary reactions takes place. It is possible to follow the fate of both the oxidized and reduced species created by the ionization process. Ultimately the chain of chemical events initiated by oxidation and reduction leads to the formation of radicals of the type
5-C (CHa)Z-CH (KH3+Cl-)-COOH IIIa Altogether nine paramagnetic absorption spectra, including several associated with various conformations of the aforementioned radicals, were observed in this investigation. Experimental Section Single crystals of L-penicillamine HC1 were obtained by slow cooling of supersaturated aqueous solutions. The crystals are monoclinic belonging to the space group P21Lvith two molecules per unit cell.? The asymmetric unit also contains a molecule of water. A crystal, mounted on a Cu wire, was suspended along the axis of the cylindrical sample cavity of a K band esr spectrometer. The technique used to X-irradiate and measure the absorption in crystals at 4.2'K has been described previous1y.l A thermocouple embedded in the sample holder just above the crystal mount was used t o record temperature during warming experiments. The temperature indicated by the thermocouple was probably somewhat above the actual crystal temperature.
Results Five distinct stages in the radiation damage process could be observed in penicillamine HC1 following irradiation at 4.2"K. The esr spectrum obtained at each stage for a particular crystal orientation is shown in Figure 1. The integrated absorption relative to the integrated absorption at the first stage is indicated. The sequence of events was as follows.
';i
(C)
L t
K t
,
250
GAUSS
?
Figure 1. T h e sequence of esr spectra obtained from a single crystal of X-irradiated penicillamine HC1 at 42°K. ( a ) Following irradiation a t 4.2'K with no warming. ( b ) After warming to a n indicated temperature of 65°K. (c) Warming to an indicated temperature of 200°K. ( d ) After warming to a n indicated temperture of 235'K. (e) After warming t o an indicated temperature of 275°K. These spectra were recorded with t h e magnetic field parallel t o t h e (010) plane in a direction 31.4 and 135' away from the a and c axes, respectively. T h e numbers inclosed in boxes indicate the total integrated absorption relative to t h a t meaured in (a) T h e arrows indicate t h e magnetic field correspding to free spin resonance.
First Stage. The esr absorption obtained at, 4.2'K without warming is shorvn in Figure la. The absorption consists of three distinguishable components. The component labeled ,Bo is due to chlorine atomsa6 The (2) R . F. Wheaton and M. G . Ormerorl, Trans. Faradau Soc., 6 5 , 1638 (1969). (3) K. Akasaka, J . Chem. Phys., 43, 1182 (1965). (4) onpublished data. ( 5 ) D. Crowfoot, C. W. Bunn, R, W. Rogers-Low, and A. TurnerJones, "The Chemistry of Penicillin," Princeton University Press, Princeton, N. J., 1949, pp 310-367. More recently the crystal structure of lrpenicillamine HC1 has been investigated by R. Parthasarathy and F. Cole of the Center for Crystallographic Research, Roswell Park Memorial Institute. (6) Esr spectra labeled with an O( me associated with the reduction process: those labeled with a fi are associated with the oxidation process. This notation is consistent with that used by K. Akasaka, et al., J . Chem. Phys., 40, 3110 (1964), in their study of cystine IlCl irradiated at 77OK. We have used primes, wherever necessary, t o distinguish between spectra attributed t o different conformations of the same paramagnetic species.
The Journal of Physical Chemistry, Vol. 76, .Vo. 17, 1971
EDWIN E. BUDZINSKI AXD HAROLD C. Box
2566 component labeled
is attributed to the radical cation
HS *-C(CH3)2-CH(YHa+Cl-)COOH IIIb or its neutralized form S-C(CHa)&H(T\THs +Cl-)-COOH IIIa The component labeled a0 is attributed to the anion 0-
HS-C (CH~)?-CH(SHB +Cl-)-C
/
\
OH
IV The hyperfine structure associated with the Po absorption in Figure l a clearly indicates that the absorption is due to atomic chlorine. There are four clusters of lines arising from the interaction of the unpaired electron with the chlorine nucleus. The two isotopes of chlorine, 35Cl(75% abundant) and 37Cl(25% abundant), have a spin of but slightly different magnetic moments. There is an additional interaction between the unpaired electron and two protons. Although the structure analysis5 of penicillamine HC1 has not determined the position of the hydrogen atoms, there is evidence of hydrogen bonding between the chlorine ion and oxygen atoms of a carboxyl group and two water molecules and between the chlorine ion and the nitrogen of an amino group. Two of these hydrogen bonds undoubtedly account for the proton hyperfine structure associated with the chlorine atom absorption. The protons are equivalently coupled for the crystal orientation used for Figure 1 and the interaction gives rise to a triplet hyperfine splitting in which the intensities of the lines are in the ratio of 1:2: 1. Thus the hyperfine lines in the Po absorption in Figure l a can be reconstructed by assigning two parameters : a hyperfine coupling of 87.0 G for the 35Cl nucleus (and consequently 73.2 G for the 37Clnucleus) and a coupling of 12.1 G for the protons. A stick diagram of the computed hyperfine pattern is compared with the observed pattern in Figure 2 . Since the hydrogen atoms involved in hydrogen bonding Tvith the chlorine ion are easily exchangeable, the proton hyperfine pattern is absent in crystals grown from heavy water solution. The chlorine hyperfine pattern and the g value of the PO absorption are extremely anisotropic as would be expected for a C1 atom with the unpaired electron in a 3p state. For most crystal orientations the Po spectrum is much less well resolved than that shown in Figure la. Consequently, the tensors required to characterize this absorption fully were not determined. The PI absorption has a large anisotropy associated with its g value. The principal values and direction T h e Journal of Physical Chemistry, Vol. 76, -Yo. 1'7, 1071
m m
4
Figure 2. T h e spectra expected from chlorine atoms interacting with two protons indicated by stick diagrams for the two isotopes of chlorine. These two absorptions partially overlap to produce the hyperfine structure shown in t h e 00 components of the esr absorption.
cosines for the principal axes of the g tensor are given in Table I. The g value characteristics suggest a radical in which the unpaired electron is localized on an atom of high atomic number (and consequently has a large spin-orbit coupling). Thus the absorption is attributed to the radical I11 in which the unpaired electron is localized mainly on the sulfur atom. Since there is no evidence of hyperfine coupling due to the sulfhydryl proton, it is more plausible to ascribe the absorption to the neutral form of the radical IIIa. The principal values of the g tensor for the absorption are very similar to the values (2.29, 1.99, 1.99) found by Akasakaa in cysteine HC1 irradiated at 77°K for a radical attributed to the analog of IIIa. As can be seen from Table I, the g value associated with the a. absorption is only slightly anisotropic. The hyperfine doublet is attributed to the exchangeable proton of the carboxyl group.7 An experiment, which is described later, using a partially deuterated penicillamine HC1 crystal confirms this interpretation. Anion (7) J . W.Sinclair and M. 7.V. Hanna, J. Phys. Chem., 71, 84 (1967); P. B. Ayscough and A. K . Roy, Trans. Faraday Soc., 64, 582 (1968).
OXIDATION ASD REDUCTION OF IONIZING RADIATION
Table I : Principal Values of g Tensors and Hyperfine Coupliiig Tensor6 (it1 Gauaij) and Direct'ioii Coaines of Principal Axes Associated wit8hVarious i2bsorptions Showii in Figilre 1" a' 0 0
g value
010
hyperfine
CYIO
g value
01'0
hypehie
PI
g value
P '1 g value
P"1
g value
Y g value
2,0033 2.0024 2.0016 10 10 6
2.00:31 2.0023 2.0012 20 16 13
011
0 1
hyperfine
Pa g value
Ca-S bond
0.0 -0.13 0.99
1.00 0.00 0.00 1.0 0.0 0.0
c
0.00 -0.60 0.80 0.0 0.99 0.13
0.00 0.13 0.99
1.OO 0.00 0.00
0.00 -0.99 0.13
0.83 0.00 0.52
0.00 1.00 0.00
-0.52 0.00
0.85
2,297 2,037 1.921
0.2.i -0.74 0.62
0,9$5 0.03 -0.32
0.20 0.67 0.72
2,217 2.000 1.983
0.33 0.72 -0.60
0.91 -0.41 0.02
0.23 0.56 0.80
2.058 2,023 2.002
0.96 -0.19 -0.20
0.12 -0.36 0.93
0.25 0.91 0.32
2.248 1,996
0.07 -0.70 0.71
0.44 0.66 0.61
0.89 -0.27 -0.36
0.49 O.H6 0.14
0.84 -0.43 -0.33
0.23 -0.28 0.93
0.54 0.83 0.14
0.81 -0.46 -0.37
0.24 -0.31 0.92
-0.44 0.79 -0.43
0.35 0.59 0.73
0.83 0.17 -0.54
1.978 q value
0.00 0.80 0.60
b
2,0037 2,0022 2.000x 32 18 7 2.054 2.024 2.000
0.22x
0.937
0.238
direction Direction cosines for the C3-S bond direction are also given. T h e direction a' is orthogonal t o b and c. There is another set of tensors related t o this set by a twofold rotation about b.
I V is analogous t o the anions observed in succinic acid8 and amino acid^^,^ irradiated a t low temperature. Second Stage. By raising the crystal above the level of the liquid helium the crystal could be warmed to a temperature indicated approximately by a thermocouple embedded in the sample holder. Warming t o 65°K and subsequent reimmersion in liquid helium produced the spectrum shown in Figure lb. The only significant change is that the a,, absorption has been replaced by another which is labeled ato. The g tensor and the hyperfine coupling tensor for the atoabsorption
2567
are given in Table I, The L Y 'absorption ~ is attributed to a different conformation of the anion IV. Changes in the conformation of free radicals produced at low temperature are often observed as the temperature increases.1o The hyperfine coupling is larger for the a'o absorption, indicating a reorientation of the anion such that the projection of 0-H bond perpendicular to the carboxyl plane is larger."" Third Stage. The Po absorption disappears when the crystal is warmed to 200°1< and the absorption labeled pfl becomes apparent. The latter absorption is probably present even in the initial stage (Figure l a ) but is less intense and partially masked by the Po absorption. The principal values of the g tensors for the and 0'1 absorptions (Table I) are very similar. The absorption is attributed to a different conformation of the radical IIIa. Experience teaches us to expect that free radicals produced by irradiation at low temperatures are likely to reorient or "relax" at higher temperat~res.2,','~'~~'~3 For example, several conformations of the radical analogous to I I I a have been observed in cysteine HC1 crystals irradiated x i t h X-rays2 or ultraviolet light12 at low temperature and subsequently allowed to warm. A new absorption labeled y appears in Figure IC. The large anisotropy associated with the g value for this absorption suggests a free radical with the unpaired electron localized on the sulfur atom. However, the principal axes of the g tensor for the y absorption (Table I) are such that we are uncertain whether this absorption should be ascribed to an oxidation or a reduction product. This point is discussed more fully in the last section of this paper. Fourth Stage. Warming to 235OK yields an absorption pattern, Figure Id, consisting of two components. The y and PI absorptions have disappeared and the P'I absorption is enhanced. The a'o absorption has also disappeared and a new absorption, labeled a1 in Figure Id, has appeared. The al absorption has a well resolved and markedly anisotropic hyperfine doublet associated with it. Additional hyperfine lines appear at some crystal orientations which are due to forbidden transition^.'^ The anisotropic coupling and the existence of forbidden transitions (at I< band) are characteristic of radicals in which the unpaired electron on a carbon atom interacts with the proton of a neighboring bonded hydrogen atom. The absorption is attributed (8) H. C. Box, H. G . Freund, and I
