JAMES W. SINCLAIR AND MELVINW. HANNA
84
Electron Paramagnetic Resonance Study of L-Alanine Irradiated at Low Temperaturesla
by James W.Sinclair and Melvin W. Hannalb Department of Chemistry, Univeraity of Colorado, Boulder, Colorado (Received September 67, 1966)
Single crystals of L-alanine have been X-irradiated a t 80°K and the electron paramagnetic resonance (epr) of the fragments formed have been studied as a function of temperature. The initial fragment formed a t 80°K has the unpaired electron localized mainly on the carboxyl group. Upon warming to 140-150°K, this initial fragment is quantitatively converted to CH&HR (R = COOH), but at a different orientation in the crystal than CH2CHR formed by irradiation at room temperature. Upon warming to room temperature, a reorientation of the fragment occurs giving the usual room-temperature epr spectrum. An interesting feature about the intermediate fragment is that the methyl protons remain equivalent even when recooled to 80'K. This is in contrast to the roomtemperature fragment where the rapid interconversion of methyl protons is frozen out by cooling to 77OK. A mechanism for radiation damage in amino acids is proposed.
Introduction Electron paramagnetic resonance has proved to be a powerful tool for elucidating the structure of paramagnetic fragments formed by X-irradiation of single crystals.2& Most studies of this type are carried out by irradiating a single crystal of the material to be studied at room temperature and studying the paramagnetic resonance spectrum as a function of the orientation of the crystal with respect to the applied magnetic field. There were several early indications that the paramagnetic fragments formed at room temperature were the final products of a chain of events involving several intermediate ~ p e c i e s , ~ and ~ -recent ~ work has shown this to be the case for succinic for aaminoisobutpric acid,6and for g l y ~ i n e . ~ , ~ Single crystals of L-alanine were first studied by Miyagawa and Gordys and later by hliyagawa and Itohlo and by Morton and co-workers." The roomtemperature fragment has been identified as CH&HR (R = COz- or COOH). An interesting feature of this fragment is that the methyl protons are interchanging rapidly by rotation or tunnelling at room temperature and are magnetically equivalent. At 77"K, however, this interconversion has been slowed so that the three methyl protons are no longer equivalent. Temperature dependence studies of the interchange rate indicate The Journal of Physical Chemistry
an activation energy for the process of approximately 4 kcal/mole. l o , l l In this work we have found that if single crystals of L-alanine are irradiated at 77"K, an initial fragment is formed which is very different from the room-temperature fragment discussed above. At 14O-15OoK, this initial fragment changes to the room-temperature (1) (a) Supported in part by Grant Ghf-09187-05 from the National Institutes of Health, U. S. Public Health Service. (b) Alfred P. Sloan Fellow. (2) (a) See J. R. Morton, Chem. Rev., 64, 453 (1964), for a recent .review of this work; (b) I. Miyagawa and W. Gordy, J . Am. Chem. Soc., 83, 1036 (1961). (3) (a) Yu. N. Molin, I. I. Chkheidze, N. Ya. Buben, and V . V. Voevodskii, Zh. Struct. K h i m . , 2, 293 (1961); (b) Yu. N. h'lolin, A. T. Koritskii, N. Ya. Buben, and V. V. Voevodskii, Dokl. Akud. N a u k SSSR, 124, 127 (1959). (4) T . Henrikson, "Free Radicals in Biological Systems," Academic Press, New York, N. Y., 1961, p 279. (5) H. C. Box, H.G. Freund, and K. T . Lilga, J . Chem. Phys., 42, 1471 (1965). (6) H. C. Box and H. G. Freund, ibid., 44, 2345 (1966). (7) H. C. Box, H. G. Freund, and E. E. Budzinski, J. Am. Chem. SOC., 88, 658 (1966). (8) M. A. Collins and D. H. Whiffen, Mol. Phys., 10, 317 (1966). (9) I. Miyagawa and W. Gordy, J . Chem. P h y s . , 32, 255 (1960). (10) I. Miyagawa and K. Itoh, ibid., 36, 2157 (1962). (11) J. R. Morton and A. Horsfield, ibid., 39, 427 (1963); A. Horsfield, J. R. Morton, and D. H. Whiffen, M o l . Phys., 4, 425 (1961); 5, 115 (1962).
EPRSTUDYOF L-ALANINE IRRADIATED AT Low TEMPERATURES
85
*ALANINE- LOW TEMPERATURE FRAGMENT A. PROTONATED SPECIES
8. DEUTERATI
(0011
(010)
c
_
c _ _
20 GAUSS
Figure 1. The esr spectra of the radical produced by X-irradiation of Lalanine a t temperatures below 140% measured along the three crystallographic axes: (A) normal calanine; (B) Galanine in which the polar protons have been replaced by deuterons.
fragment, CH&HR, but this fragment does not have the same orientation as the fragment obtained by Xirradiation a t room temperature. A second unique feature of this species is that the methyl protons maintain their rapid interconversion upon recooling to 80°K. Finally, at room temperature, the C H d H R becomes reoriented to give the usual room-temperature spectrum.
Experimental Section Single crystals of L-alanine were grown from aqueous solution. The crystals were mounted on the wall of a metal microwave cavity operating in the TE 101 mode. The cavity was attached to the cold finger of a special cryostat constructed by Andonian Associates. The cryostat has a beryllium window in the outer heat
shield and holes in the inner heat shields to allow in situ X-irradiation to be carried out. The temperature of the cavity can be controlled by adjusting a small heater on the cold finger and the pressure of heat exchange gas which couples the cold finger to the liquid refrigerant bath. The epr spectra were taken using a Varian Associates spectrometer with superheterodyne detection. A Varian 9-in. rotating magnet was used to study the angular dependence of the spectra. Crystals in which the three polar protons were replaced by deuterons were prepared by four recrystallizations of normal L-alanine from 99.6% DzO.
Results Irradiation of a single crystal of L-alanine at 7 7 4 5 ° K produces a paramagnetic fragment with either a threeVolume 71,Number 1
Januarv 1967
JAMES
86
A. INTERMEDIATE
(0101
W. SINCLAIR AND MELVIN W. HANNA
FRAGMENT
8. ROOM .TEMPERATURE FRAGMENT
(0011 ~~~
M
25 GAUSS
Figure 2. ( A ) The esr spectra of the stable intermediate fragment of irradiated Galanine measured along the three crystallographic axes. (B) The esr spectra of calanine irradiated a t room temperature.
or four-line epr spectrum depending on orientation. Spectra for HOalong the 100, 010, and 001 crystal axes are shown in Figure 1A. The average line width is about 6 gauss. If the same experiment is performed on a sample in which the polar protons have been replaced by deuterons, a two-line spectrum is obtained at all orientations. The spectra of the fragment in the deuterated crystal along the three crystallographic axes are shown in Figure 1B. The doublet spacing in this deuterated fragment shows only small anisotropy. These results are consistent with a paramagnetic fragment in which the unpaired electron interacts with two nonequivalent protons. One proton (A) has an appreciable anisotropic coupling and is easily exchanged with deuterium. The second proton (B) has an almost isotropic coupling and is not easily replaced by deuterium. The hyperfine coupling tensor for proton A is almost diagonal in the crystal axis system as is shown by the data given in Table I. Because of the large The JOUTW~ of Physical Chemistry
line widths and consequent errors in identifying the hyperfine couplings, the data only serve &s rough inTable I : Hyperfine Parameters for the Low-Temperature Fragment in X-Irradiated I,-Alanine
Proton
A
Hyperfine splittings, gauss
-Direction 1
-10.2" -18.7 -16.6
Isotropic value
B
0.98 0.20 0.0
cosines-m
n
0.17 0.98 0.0
0.0 0.0 1.0
- 1 5 . 1 gauss
18.4" 18.4
19.6 Isotropic value 18.8 gauss
(100) (010) (001)
Negative sign for proton A and positive sign for proton B assumed.
EPRSTUDYOF L-ALANINEIRRADIATED AT Low TEMPERATURES
Table 11: Magnetic Resonance Parameters for the Intermediate Fragment in >Alanine Hyp-rfine splittings,
Nucleus
CH
CH3
gauss
-Direction 1
-2i3.0" 10.283 -23.5 10.999 -13.6 10.045 Isotropic value -21.4 25, sa 23.5 28 5 Isotropic value 23.8
a Sign assumed. direction cosines.
Table 111: Magnetic Parameters for the Room-Temperature Fragment in L-Alanine"J Hyperfine splittings,
cosine-
m
zk0.056 zk0.028 10,975
87
n
rt0.957 i.0.033 10.218
Proton
CH
(010)
Direction cosines m
I
1
-16.6 0.277 -0.727 -32.0 -0.486 0,460 -8.4 0.822 0.523 Isotropic value 19.0
-
CHs (001)
gauss
27.5 0.000 0.000 25.0 25.0 1.000 Isotropic value 25.8
0.642 0.765 0.000
.--. n
0.628 0.743 0.225 0.765 -0.642 0.000
* Anisotropy too small to calculate accurate
dicators of the principal axis system for this proton. Since the anisotropy in the splittings for proton B is much less than the line width, only the values of the hyperfine splittings along the three principal axes are given. The spectrum of the low-temperature fragment discussed above remains unchanged until the temperature is increased to 145-15OoK. Above this temperature, the spectra ch:tnge irreversibly into those shown in Figure 2A. The average line width in these spectra is 2.5 gauss. In Figure 2B are shown the spectra along the three crystallographic axes of the usual roomtemperature fragment. Comparison of these two sets of spectra shows that this second fragment is similar to the room-temperature fragment but that the orientation dependence of the hyperfine couplings are different. The magnetic parameters for this fragment are given in Table 11. Appropriate values for the roomtemperature fragment from the literature are given in Table I11 for comparison. The spectra of this intermediate fragment are unchanged when the polar protons are replaced by deuterons. More important, in view of past work, is the fact that the spectra also remain unchanged when the sample was recooled to 80°K. If the crystal was now allowed to stand at room temperature for several hours, the spectra which have been observed by other workers in crystals irradiated a t room temperature mere observed. 9-11 When the crystal was now recooled to 77-80°K, the typical freezing out of the methyl group rotation was observed.
Discussion The epr spectra of the low-temperature fragment can be explained if one assumes that in the fragment responsible the unpaired electron interacts with one a proton (a a-type proton) and one ,t3 proton (a n-type
proton). The a proton must be easily exchangeable with deuterium, which means that it must be attached to an amino nitrogen or a carboxyl oxygen. A possible fragment having these properties is I1
k I
I1
which could be formed from fragment I by simply transferring a proton or deuteron across a hydrogen bond from a neighboring molecule. The very large line widths in the spectra of this low-temperature fragment appear to be due to second-order transitions or satellite lines from nearby protons. At one orientation (loo), these satellite lines spaced at approximately the proton resonance frequency from the main line are almost resolved. The large &proton hyperfine coupling is consistent with the odd electron being in an antibonding orbital of a carboxyl group since this orbital has a large density on the carbon atom. Similar ionic fragments have been postulated in succinic acid5 and 2aminoisobutyric acid,6 and would also explain the lowtemperature spectra recently obtained with glycine.8 The epr spectra of the intermediate fragment formed at 150-160° indicates that this paramagnetic species is similar to, but not identical with, the room-temperature fragment. The intermediate fragment differs from the room-temperature fragment in the following ways. (1) The orientation of the principal axes of the hyperfine tensor is different. (2) The a proton in the intermediate fragment has a larger isotropic but smaller anisotropic hyperfine coupling than the roomtemperature fragment. (3) The methyl group in the intermediate fragment is not subject to restricted rotation above 77°K as is the methyl group in the roomtemperature fragment. The crystal structure of L-alanine has recently been Volume 71, Number I
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JAMESW. SINCLAIR AND MELVIN W. HANNA
88
determined by Simpson and Marsh,I2 and the direction cosines which the a-carbon-hydrogen bond makes with the three crystallographic axes are 0.059,0.96, and 0.21, re~pective1y.l~If, as is usually the case, the direction of greatest positive anisotropic coupling lies along the C-H bond, then a consideration of the data in Table I1 shows that in the intermediate fragment, the C-H bond has the same orientation as it does in the undamaged molecule. The differences in the a-proton hyperfine couplings between the intermediate and roomtemperature fragments also suggest that the hybridization around the a-carbon atom is different in the intermediate fragment than in the room-temperature fragment. The smaller anisotropic couplings would be consistent with the odd electron orbital having some sp3 hybrid character since that would decrease the value of ( l / r 3 ) between the odd electron and the proton. Thus, in making the transition between the intermediate and room temperature fragments, the alanine radical both changes its orientation in the crystal and its hybridization about the a-carbon atom. Many of the orientation and hybridization questions will be resolved by a CY3 study which is currently in progress. There is a convenient mechanism which can be written to rationalize the formation of the intermediate fragment (111) from the low-temperature fragment (11) that does not involve the formation of an additional paramagnetic species. This mechanism involves loss of free ammonia as shown in (B).
Thus, the general mechanism for radiation damage in amino acids that is suggested by this work is the following.
(1) Ionization of an electron from one alanine molecule leaves a positive hole and the negative ion I. (2) The negative ion decays according to reactions A and B. (3) The positive ion decays even at 77°K according to the well-known reaction +NE3
I
a"+
CHaCHCOz.
I +CHaCH.
+ COz
IV
The ethyl ammonium radical IV is not observed because of the fact that the epr intensity is spread among so many lines compared with radicals I and 11. It is significant that this mechanism predicts the formation of both of the principle fragments in glycine.'.S
Discussion C. THOMSON (University of St. Andrews, Scotland). Have you any data on Cla splittings in these species, both in the -COOH group and the CHaCH- group?
J. W. SINCLAIR.We are currently preparing C13-labeled materials and will have these data in the near future. M. C. R. SYMONS (Leicester University). For the radical
H
RC022- or RC
