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Radiation damage to nucleosides and nucleotides. II. EPR of uridine single crystals at 295 K
JOURNAL
OF MAGNETIC
RESONANCE
hi,
518-530 (1981)
Radiation Damage to Nucleosidesand Nucleotides. II. EPR of Uridine Single Crystals at 295 K EINAR SAGSTUEN Institute of Physics, Department of Biophysics, University of Oslo, Blindern, Received February
N-Ok0 3, Norway
12, 1981
Single crystals of uridine were irradiated with 4.0-MeV electrons at room temperature and studied by electron paramagnetic resonance techniques. Three radicals were formed and trapped at this temperature, and two of them have been assigned to specific molecular structures. One (I) is the uracil-5-yl radical formed by a net hydrogen atom addition to C6 of the uracil base, and another (II) is a &-centered species exhibiting hyperfme interaction with three nonequivalent /3 protons. The third radical (III) appears to be a product formed by the decay of I, and is probably also formed in the ribose moiety. A reversible, light-induced transformation between I and II was observed. The radical structures and possible mechanisms of formation are discussed. INTRODUCTION
The solid-state radiation chemistry of the nucleic acid constituents has been the subject of vigorous research during the last two decades (I, 2). In particular, derivatives of the pyrimidine bases thymine, cytosine, and uracil have attracted much attention and recently some of the corresponding nucleosides and nucleotides have been investigated as well. The most thoroughly studied of the latter compounds are those derived from cytosine (2-4) and the S-halogen-substituted uracils (2,5,6), whereas less information so far is available about the radiationinduced processes in uracil nucleosides and nucleotides (7-11). In a previous paper (7), radical formation in uridine-5’-phosphate *2Na+ (5’-UMP/Na) was investigated. 0
R =-OH
: uridina
A;-OF-O;: 5’
OH
OH
Evidence was given that the uracil base acts as an electron scavenger, whereas the oxidative processes mainly take place in the sugar-phosphate region of the 518 0022-2364/81/090518-13$02.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
RADIATION
DAMAGE
TO URIDINE
519
molecule. Thus, at 77 K the uracil anion radical was identified, converting into the well-known 5,6-dihydrouracil-5-yl radical at elevated temperatures. At 77 K a fsecondary alkoxy radical was also observed, for which the main part of the unpaired spin density is localized on Ox, in the sugar-phosphate moiety. Upon heatin,g, this radical transformed into a C,,-centered H-abstraction radical. After irradiation at 270 K, a third radical was observed in addition to the 5-yl and C,,centered species. This radical was originally assumed to be the uracil-6-yl radical (7’, 11). Recently it was demonstrated that the EPR parameters for this resonance are also compatible with a radical in the ribose moiety (10). This radical would have the main part of the spin density localized on C4,, which implies an opening of the ribose ring upon radical formation. In the present report, the free radical products trapped in single crystals of uridine after irradiation at room temperature are discussed. As usual for all uracil derivatives, the uracil-5-yl radical (called I) is observed. A second product (II) exhibits a resonance which makes it reasonable to assume that it is a ribose radical formed by a net hydrogen abstraction from C,,. The effects of light illumination and heat treatments are discussed, including a description of a further resonance (III) which appears as a consequence of the heat-induced decay of the 5-yl radical. EXPERIMENTAL
Polycrystalline samples of uridine were obtained from Sigma Chemical Company. Crystals were formed from saturated ethanolic solutions by slow evaporation at 37°C. Partially deuterated crystals were obtained by a similar procedure from solutions of C2H20D (IC GmbH, 95% OD). Care was taken to select single crystals, since twinning was observed for most of the samples obtained. The crystals are generally small and irregularly shaped, but they always exhibit a well-defined axis of elongation (a). Single crystals of uridine are monoclinic with space groupP2, (12). The unit cell contains two asymmetric units, each consisting of two independent uridine molecules (denoted A and B) with slightly different conformations. The dihedral angle between the pyrimidine ring planes of A and B is 12”. The crystal structure is characterized by an extensive network of hydrogen bonds. All crystals used for EPR measurements were examined by X-ray Weissenberg techniques and the crystal axes were localized. The orthogonal a, b, and c* = a :< b reference system was adopted for EPR measurements. The crystals were irradiated at 295 K with 4.0-MeV electrons from a linear accelerator up to doses of 16 Mrad at a dose rate of 0.9 Mrad/min. The EPR measurements were mainly carried out with a Jeol X-band spectrometer. Some spectra were also recorded on a Varian E-9 spectrometer equipped with a E-l 10 Q-band microwave bridge. For calibration purposes, the microwave frequency was monitored using a frequency Imeter, and a reference sample containing DPPH and Mn2+ in MgO could be placed close to the sample in the cavity. First estimates of the g tensor and proton hyperfine coupling tensors were obtained using the methods of Schonland (13 ) and Lund and Vlinngard (14 ), respectively, including the usual linear least-squares fitting procedure (15). These
520
EINAR SAGSTUEN
estimates were then used as input trial tensors in a nonlinear iterative least-squares fitting routine, using an effective spin Hamiltonian which includes the nuclear Zeeman term, as described previously (16). In this analysis one adjustment angle in each plane of rotation was included as additional variational parameters. Error estimates in eigenvalues and eigenvectors were made following the procedure of Fouse and Bernhard (17), which makes use of the variance-covariance matrix for each set of least-squares-adjusted tensor elements. All calculations reported were performed on a CDC CYBER 74 computer with a single precision standard of 60 bits word length. RESULTS
When crystals of uridine are irradiated radical species are formed and trapped.
at room temperature, altogether three Figure 1 shows spectra obtained im-
1450
FIG. 1. Second-derivative
EPR spectra of a uridine crystal after irradiation
at 295 K.
RADIATION
DAMAGE
521
TO URIDINE
mediately after irradiation, recorded at two orientations of the crystal in the applied magnetic field. The resonance lines attributable to two of the radicals, designated by I and II in the following, dominate the spectra of all orientations, whereas lines from a third radical only weakly contribute to the central features of the spectra at this temperature. Radical
Conversions
The analysis of the different resonances was facilitated by several spectral changes induced by light exposure or heat treatment of the crystals. This is demonstrated in Fig. 2. Upon light exposure (A > 300 nm) at temperatures below -3O”C, the conversion II + I was induced (Figs. 2A and B). Upon subsequent storage at room temperature the original spectrum was recovered (Fig. 2C), which indicates that the light-induced conversion is reversible. The backtransformation (I + II) does not, however, continue upon heat treatment above room temperature. Thus, u.p to about 80°C the spectra remained almost unchanged. However, at this temperature a gradual change into the spectrum shown in Fig. 2D occurred. This latter spectrum indicates that radical I had decayed, radical II remained unchanged, and the third resonance in the central region of the spectra had increased in intensity.
u uuuuuu -* I
u
II
FIG. 2. Effect of uv exposure and heat treatment upon the EPR spectra of uridine crystals after irradiation at 295 K. (A) Spectrum recorded immediately afker irradiation at 295 K. (B) Spectrum recorded after 30 min light exposure (A > 300 nm) at -50°C. (C) As (B), but recorded after annealing at room temperature for 2 hr. (D) As (C), but recorded at room temperature after annealing at 85°C for 1 hr. All spectra were obtained with the magnetic field along the crystallographic 6 axis without moving the crystal in the cavity and at identical spectrometer conditions.
522
EINAR SAGSTUEN
This conversion is irreversible upon cooling to room temperature. Identical spectra are also obtained if a crystal, not previously light exposed, is heated to 80°C. Radical I-The
UraciE Syl Radical
Figures 3A and B show spectra attributable to radical I, obtained after light exposure at -5O”C, at crystal orientations corresponding to those in Fig. 1. The resonance from radical I is characterized by three large proton interactions. One of these seems to be of the CYtype, whereas the two others may be ascribed to p protons. The results from a full analysis of the EPR data are given in Table 1. The g value is only slightly anisotropic, indicating a carbon-centered radical. The angle between the eigenvectors for the minimum principal g value and the intermediate (Ytensor principal value is 2.7”, suggesting a planar n-electron radical. The assignment of radical I as the 5,6-dihydrouracil-5-yl radical unequivocally emerges from the excellent agreement between the different eigenvectors and corresponding molecular reference vectors calculated from the crystal structure, as shown in Table 1. The coupling tensors are all very similar to those observed for 5-yl radicals formed in other uracil derivatives (2,7,8,16,18,29). It appears that the formation of radical I specifically takes place in molecule B of the asymmetric unit (12). A pronounced line broadening was observed at crystal orientations where the magnetic field is close to the perpendicular of the pyrimidine ring. This suggests a small positive spin density on a nitrogen atom. At other orientations a small DPPH
I
FIG. 3. Second-derivative EPR spectra of an uridine crystal after irradiation at room temperature. (A), (B) Spectra recorded after 30 min light exposure (A > 300 nm) at -50°C. (C), (D) Spectra recorded at room temperature after annealing at 80°C for 2 hr.
2X045(2) 2.0035(l) 2.0019(2)
-0.5683 0.7044 0.1406 0.7734
0.0077 0.6595 0.3587 0.9480
0.43(7) -0.58(S) 0.69(3)
0.67(2) 0.39(26) -O&4(14)
0.420(10) 0.654(17) -0.629(14) 0.18(8) -0.70(5) 0.70(4)
a
0.5358
0.9810
-0.0442 -0.5254
0.3952 -0.6989 0.9182 0.2940
0.82(2) -O%(5) -0.57(3)
0.47(4) -0.45(32) 0.76(17)
0.98(2) 0.03(8) -0.22(8)
-0.053(21)
0.816(08) -0.576(11)
E
0.8216 0.4773 -0.1340 0.3387
-0.1679 0.1218
0.2767
0.9186
0.37(7) 0.81(6) 0.45(6)
0.58(2) 0.81(6) 0.12(35)
0.13(2) 0.72(5) 0.68(4)
0.397(10) 0.492(20) 0.775(09)
c*
12.5
32.8
+1.3 -1.4
f 0.4
c 0.4
14.2 r 0.2 k 0.1
46.7 20.4
Molecule A
3.0
f1.4 -3.0
15.7 k 0.8
4.2 " 0.5 4.4 2 0.4 15.3 f 0.8
Molecule B
AV
2
4
2 1 3
Ref. vector
” The number in parentheses is the standard deviation in the corresponding Iast digit(s) of the quoted value. b This is the angle of deviation between the eigenvector and a molecular reference vector, identified by a number. The interval of uncertainty is given at a 95% confidence level. c The negative sign of this splitting is assumed, in accordance with theoretical considerations upon a-proton interaction. d These vectors are calculated from the crystal structure (12). The positions of the protons H,, and H,, are calculated by assuming a symmetrical arrangement with respect to the ring plane, an angle H,,-C,-H,, = 1 IO” and C-H bond lengths of 1.09 A.
G G G
G G
37.2(2) 3X6(3) 54.9(3) 50-O(3) 49.4(4)
G G G G
-30.1(2) - 17.2(4) -8.9(5) 39.9(l)
Principal values
Ref. vectors,d Molecule A 1 C,- H bond direction 2 Pyrimidine ring normal C,-H,, direction 3 4 C,- H,, direction Ref. vectors, Molecule B 1 Q-H bond direction 2 Pyrimidine ring normal 3 C,-%, direction 4 C,- Kt;, direction
2.0033(l)
51.4(2)
H,&,
R
37.6(l) G
H&S)
G
- 18.7(2) G
H,(G)
)
Isotropic value
Tensor
Principal directions
TABLE 1 EPR PARAMETERS FOR RADICAL I IN IRRADIATED SINGLE CRYSTALS OF URIDINE AI 2% K'
VI z
$ m
g $
g
'3
w
EINAR SAGSTUEN
524 DPPH
,25G,
DPPH 1 c
FIG. 4. Effect of partial deuteration upon the EPR spectra of uridine crystals after irradiation at 295 K. Spectra (A) and (C) are of crystals grown from C,H,OH, and spectra (B) and (D) from partially deuterated crystals. (A), (B) Spectra recorded atler light exposure (A > 300 nm) at -50°C. (C), (D) Spectra recorded at room temperature after annealing at 80°C for 2 hr.
doublet splitting was resolved, exhibiting a maximum value of about 3.5 G with the magnetic field nearly perpendicular to the N3-H bond, in the pyrimidine plane. This is illustrated in Fig. 4A. In Fig. 4B the corresponding spectrum from a partially deuterated crystal is shown, clearly demonstrating that the responsible proton is exchangeable. Furthermore, the minimum linewidth for radical I (about 2 G(pp)) was observed with the magnetic field directed nearly parallel to the N,-H bond direction. Thus, the angular behavior of this additional proton interaction together with the observed linewidth variation suggests that some spin density is localized to N3 and that the proton bonded to N3 is responsible for the doublet splitting. Quantitatively, the 5-yl radical accounts for approximately 50% of the total EPR absorption prior to light exposure. Radical
II-A
Cd,-Centered Radical
Upon the decay of radical I at temperatures above 8O”C, the outer resonance lines attributable to radical II become more amenable for study. Figures 3C and D show the spectra obtained after heat treatment at the same crystal orientations as for the spectra in Fig. 1. As indicated by the stick spectra, the resonance from radical II is characterized by three large proton interactions. Two of these are almost equivalent at most orientations. The total width of the resonance varied between 82 and 93 G, and the rotation data show that all three couplings are relatively isotropic, which suggests P-proton interactions only. A mutual overlap of
RADIATION
DAMAGE TABLE
EPR
PARAMETERS
525
TO URIDINE 2
FOR RADICAL II IN IRRADIATED OF URIDINE AT 295 K”
SINGLE
CRYSTALS
Principal directions Tensor
Isotropic value
Principal values
a
b
c
g
2.0032(2)
2.0041(2) 2.0031(3) 2.0024(2)
-0.03(7) -0.36(13) 0.93(7)
-0.13(5) 0.93(8) 0.35(15)
0.99(4) O.ll(17) 0.08(4)
HP1
36 G
22 G
HP2
25 G
-c3 G
Hi33
24 G
?3 G
u The number in parentheses is the standard deviation digit(s) of the quoted value.
in the corresponding
last
resonance lines in the central portion of the spectra made a thorough analysis of the hyperfine coupling tensors difficult. The g value varied slightly around the freespin g value suggesting a carbon-centered free radical. The available EPR data are collected in Table 2. The spectra in Figs. 3C and 4C show two additional small doublet splittings which are resolved only at a few orientations. Experiments with partially deuterated crystals demonstrated that the large P-proton interactions are attributable to nonexchangeable protons, whereas both the smaller doublet splittings collapse upon deuteration and consequently are attributable to exchangeable protons. This is illustrated by the spectrum in Fig. 4D. A free radical in uridine, which exhibits splittings from three p protons is probably formed in the ribose part of the molecule. Furthermore, the magnitude of the couplings shows that the main part of the spin density is localized to one position only (i.e., there is no substantial spin delocalization through aromatic conjugation). The observation of three large nonexchangeable couplings suggest that C+ is essential with regard to spin localization. Assuming that no large rearrangements take place, a possible structure for the radical is
The radical is formed by a net hydrogen abstraction from C4,. Here the hydrogen atoms bonded to CS, and Cg, are responsible for the large couplings, whereas the
526
EINAR
SAGSTUEN
y protons at 03, and OS, may be responsible for the two exchangeable doublet splittings. The g-value variation for radical II is relatively small, indicating a limited amount of spin density localized on the oxygen atoms near C4,. Like radical I, radical II also seems to be selectively formed in one of the two types of uridine molecules. Thus, the geometry of the two undamaged ribose residues is sufficiently different so as to reveal the coexistence of two conformations of the radical. The available data are not sufficient to determine in which of the two molecules the radical is formed. Nor is it possible to arrive at the detailed molecular structure of the radical. The minimum g values occur in a direction 23” away from the C,-H bond direction in the undamaged molecule (and 18” from the perpendicular to the Ct,-C5,-01, fragment). Furthermore, from the crystal structure the dihedral angles between the C4,-H bond and the C3,-H and the two C,,-H bonds in both molecules are about 13, 73, and 46”, respectively. From the Heller-McConnell relation (20) these angles correspond to p splittings very different from those observed. Thus, geometrical rearrangements must have taken place during radical formation. Only a full knowledge of the interaction tensors combined with the detection of precursor radicals will permit the evaluation of a detailed structure for radical II. The resonance from radical II accounts for approximately 35% of the total EPR absorption prior to heat treatment. Radical
III-A
Second Ribose Radical
As described above, heat treatments at 80°C induce the decay of radical I and the intensity of the central portion of the spectra increases. The central part of the resonance, mainly attributable to radical III, consists of a narrow multiplet which never exceeds a total width of 25 G. Upon heating a crystal above 80°C radical II disappears at about 100°C and new resonance lines appear in the center, superimposed on those from radical III. The combination of severe overlap with other resonances (either radical II or its decay product) and a relatively isotropic hyperfine splitting pattern again makes the determination of coupling tensors impossible from EPR measurements alone. Some general comments to the observations may, however, be made. Figure 5 presents some spectra obtained after annealing of a crystal at 105°C. In this figure, spectra from protiated as well as partially deuterated crystals are shown. The stick spectra indicates the resonance lines from radical III. Resonance lines from the decay product(s) of radical II partly obliterate the spectra. Together with the spectra in Figs. 2 to 4 these data show that the resonance attributable to radical III may be interpreted as arising from interactions between the unpaired electron and three protons. Two of these interactions are relatively isotropic (both of the order of 10 G), and are attributable to nonexchangeable hydrogens. The third coupling is smaller (maximum observed splitting is approximately 4.5 G), is somewhat more anisotropic, and disappears upon deuteration. Thus, this splitting is attributable to interaction with an exchangeable proton. The g-value anisotropy is small, varying between 2.0038 and 2.0024. Also, the linewidth variation is small, indicating no interactions with nitrogen atoms.
RADIATION
DAMAGE
TO URIDINE
527
FIG. 5. Second-derivative EPR spectra of uridine crystals after irradiation at room temperature alnd subsequent annealing at 110°C. The spectra were recorded at room temperature. (A) and (C) are of crystals grown from C,H,OH and (B) and (D) from partially deuterated crystals. The stick spectra indicate the resonance lines from radical III. The fully drawn sticks represent interaction with nonexchangeable protons, and the broken sticks represent an interaction with an exchangeable proton. The vertical arrows indicate the position of the DPPH resonance.
The magnitude of the g-value variation seems to exclude the possibility that a large fraction of the unpaired spin density is localized to a keto oxygen. Furthermore, the failure to detect anisotropic coupling from an (Y proton makes a conjugated radical structure less likely. Then, with the main part of the unpaired spin density residing on one carbon atom only, the magnitude of the P-proton couplings suggests that the two /3 hydrogens are bonded to two different atoms. These considerations suggest the following structure for the radical:
Although the g-value variation does not exclude a radical in the uracil base, the above discussion renders a ribose radical more likely. As to the position in which t.he radical is centered in the ribose moiety, these data do not give any conclusive evidence. The I e II transition described above suggests, however, that it may be localized in the C3,-C4,-C5, region of the molecule. DISCUSSION
AND CONCLUSIONS
Irradiation of uridine crystals at room temperature produces three free radical species altogether. Radical I is the well-known uracil 5-yl radical. The spectral
528
EINAR
SAGSTUEN
parameters as given in Table 1 are very similar to those for the same radical formed in crystals of uracil (18), and some derivatives (7, 8, 12, 16). In all these compounds, a weak interaction with N3 and its attached proton has been observed. Flossman et al. (22) suggested, based on INDO-UHF calculations, that this observation together with the magnitude of the P-methylene couplings indicate that the radical is protonated at the C,=O, group. Direct experimental evidence supporting this suggestion has not been obtained, either by EPR (7, 26) or by ENDOR (2.?,24) measurements. In a very recent ENDOR analysis of the 5-thymyl radical in thymidine, 0, was found nor to be protonated (J. N. Herak, personal communication). RHF/CI calculations at the INDO level of approximation also indicate that O4 is unprotonated in the uracil 5-yl radical itself as well as in its 5-halogen substituted derivatives (25). Protonation of the 5-yl radical may have some relevance to the observed radical transformations in uridine. As described above, light exposure at temperatures below -30°C results in a transformation of radical II into radical I, or to be more precise, into a radical with a resonance indistinguishable from that of radical I. The reverse transformation is readily completed by storage at room temperature (see Fig. 2). Clearly, there is no equilibrium between I and II at room temperature, since the spectrum does not change further by heating above room temperature (below 80°C). Two explanations may be offered. In the first place, uv illumination may produce (from radical II) a radical I’ chemically different from I. The difference may, for example, be that I’ is protonated at 0, and I is not. Bernhard (26) recently considered this possibility in an attempt to rationalize the lightinduced transformation between 5-yl and 6-yl radicals in uracil and cytosine derivatives. In the present case this possibility is considered less likely, since the spectral parameters from a protonated and an unprotonated version of I are expected to differ significantly. A second possibility is that I and I’ are chemically identical but trapped in different environments. I and I’ must, however, both be formed in molecule B of the asymmetric unit, since the spectra from I’ in A and B should (by virtue of their crystallographic inequivalent orientations) be distinguishable. It is of interest to note that no D adduct corresponding to I was observed in partially deuterated crystals. Neglecting the possibility for a fast H-D backexchange, this indicates that the added H atom originates from a nonexchangeable position. The mechanism of formation of radical I in uridine will be discussed elsewhere, when the low-temperature experiments have been analyzed. The analysis for radical II makes is reasonable to consider a structure featuring Cd, as the radical locus. Similar resonances have not previously been detected in irradiated nucleosides or nucleotides. A radical involving three /3 protons was recently observed, however, in crystals of methylglucopyranoside after irradiation at 77 K (27). A detailed ENDOR analysis revealed that this radical retained its tetrahedral bonding (by sp3 hybrids) at the carbon on which the main unpaired spin densities become localized. The available data indicate that this is not the case for II. Two modes of formation for radical II may be considered. One consists in a 4’,5’-H atom transfer in a precursor hydroxyalkyl radical (27). A second possibility may be deprotonation at Cd9of a parent cation radical. As discussed previ-
RADIATION
DAMAGE
TO URIDINE
529
ously (10,28), if an electron vacancy is localized on 01,, the resulting positively charged radical may be stabilized by a deprotonation. Likely positions for deprotonation in the ribose moiety are those next to O,,, i.e., C1, and C4,. It has been speculated that the conformation of the ribose moiety in part determines which site is selected (28). In 5’-UMP a radical was detected which may be attributable to a primary deprotonation at C,, (10). The conformation of the ribose sugar in uridine is different from that in 5’-UMP. Thus, in uridine, H4, is situated in a conformation similar to that for H,, in 5’-UMP. Hence, rather than Cl,, C4, should be the site for deprotonation in uridine. Again, low-temperature studies are needed for further experimental evidences. Radical III, which exhibits a very weak resonance after irradiation at room temperature, is most probably a decay product from radical I. Several recent observations indicate that the uracil-S-y1 radical is not a stable end product in irradiated nucleosides and nucleotides. Thus, in 5’-UMP this radical decays upon storage at room temperature (I I ). Eventual decay products have not been identified. In uridine (this work) and in uracil-p-D-arabinofuranoside (unpublished results) the 5-yl radicals decay by heat treatment. The species formed exhibit similar resonance patterns. In uridine the experimental data indicate that the radical is formed in the ribose part of the molecule. The H-adduct radicals are among the most important products in the radiolysis of nucleic acids. It is of biological i-mportance if these species are unstable and if the decay leads to alterations in the ribose-phosphate region of the nucleotides. ACKNOWLEDGMENTS Professor T. Henriksen is acknowledged for the thorough reading of this manuscript and many suggestions for its improvement. Thanks are due to the Studsvik Science Research Laboratory, and in particular to Dr. Anders Lund, for making their Varian Q-band spectrometer available and for valuable discussions. Discussions with Drs. D. M. Close, H. Oloff, and J. N. Herak are also acknowledged. Thanks are due to the Norsk Hydros Institute for Cancer Research for use of their linear electron accelerator. REFERENCES I. H. C. Box. “Radiation 2. J. HUTTERMANN, 3.
4. 5. 6.
7. 8. 9. 10. II. 12. i3. 1’4.
Effects. ESR and ENDOR Analysis,”
W. KGHNLEIN,
R. TEOLE,
Academic Press, New York, 1977. (Eds.), “Effects of Ionizing
AND A. J. BERTINCHAMPS
Radiation on DNA,” Springer-Verlag, Berlin, 1978. W. A. BERNHARD, J. H~TTERMANN, A. MOLLER, D. M. CLOSE, AND G. W. FOUSE, Radiat. Res. 68, 3% (1976). D. M. CLOSE AND W. A. BERNHARD, J. Chem. Phys. 68, 210 (1979). J. HOTTERMANN, W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT,J. Phys. Chem. 81,228(1977). H. OLOFF, E. HAINDL, J. HUTTERMANN, AND J. KRAUSS, Radiat. Res. 80, 447 (1979). E. SAGSTUEN, Radiat. Res. 81, 188 (1980). R. BER~ENE AND R. A. VAUGHAN, Znt. J. Radiat. Biol. 29, 145 (1976). H. C. Box, W. R. POTTER, AND E. E. BUDZINSKI, J. Chem. Phys. 62, 3476 (1975). E. SAGSTUEN, Radiat. Res. 84, 164 (1980). B. RAKVIN AND J. N. HERAK, Znt. J. Radiat. Biol. 38, 129 (1980). E. A. GREEN, R. D. ROSENSTEIN, R. SHIONE, D. J. ABRAHAM, B. L. TRUS, AND R. E. MARSH, Acta Crystallogr. Ser. B 31, 102 (1975) [erratum: Acta Crystallogr. Ser. B 31, 1221 (1975)]. D. S. SCHONLAND, Proc. Phys. Sot. London 73, 788 (1958). A. LUND AND T. VANNGARD, J. Chem. Phys. 42, 2979 (1965).
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15. E. SACSTUEN AND C. ALEXANDER, JR., Mol. Phys. 32, 743 (1976). 16. 0. CLAESSON, A. LUND, J.-P. JORGENSEN, AND E. SAGSTUEN, J. Map. Reson. 41, 229 (1980). 17. G. W. FOUSE AND W. A. BERNHARD, J. Magn. Reson. 31, 191 (1978). 18. H. ZEHNER, W. FLOSSMANN, E. WESTHOF, AND A. MOLLER, Mol. Phys. 32, 869 (1976). 19. 20. 21.
I. EGTVEDT, E. SAGSTUEN, R. BERGENE, AND T. HENRIKSEN, Radiat. Res. 75, 252 (1978). C. HELLER AND H. M. MCCONNELL, J. Chem. Phys. 32, 1.535 (1960). J. A. POPLE AND D. L. BEVERIDGE, “Approximate Molecular Orbital Theory,” McGraw-Hill, New York, 1970. 22. W. FLOSSMANN, H. ZEHNER, AND E. WESTHOF, Znt. J. Radiat. Biol. 36, 577 (1979). 23. J. HOTTERMANN, W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT, Mol. Phys. 32, 1111 (1976). 24. E. HAINDL AND J. HOTTERMANN, J. Magn. Reson. 30, 13 (1978). 25’. H. OLOFF AND J. H~TTERMANN, J. Magn. Reson. 40, 415 (1980). 26. W. A. BERNHARD, Adv. Radiat. Biol. 10, in press. 27. K. P. MADDEN AND W. A. BERNHARD, J. Chem. Phys. 70, 2431 (1979). 28. K. P. MADDEN AND W. A. BERNHARD, J. Phys. Chem. 83,2643 (1979).
OF MAGNETIC
RESONANCE
hi,
518-530 (1981)
Radiation Damage to Nucleosidesand Nucleotides. II. EPR of Uridine Single Crystals at 295 K EINAR SAGSTUEN Institute of Physics, Department of Biophysics, University of Oslo, Blindern, Received February
N-Ok0 3, Norway
12, 1981
Single crystals of uridine were irradiated with 4.0-MeV electrons at room temperature and studied by electron paramagnetic resonance techniques. Three radicals were formed and trapped at this temperature, and two of them have been assigned to specific molecular structures. One (I) is the uracil-5-yl radical formed by a net hydrogen atom addition to C6 of the uracil base, and another (II) is a &-centered species exhibiting hyperfme interaction with three nonequivalent /3 protons. The third radical (III) appears to be a product formed by the decay of I, and is probably also formed in the ribose moiety. A reversible, light-induced transformation between I and II was observed. The radical structures and possible mechanisms of formation are discussed. INTRODUCTION
The solid-state radiation chemistry of the nucleic acid constituents has been the subject of vigorous research during the last two decades (I, 2). In particular, derivatives of the pyrimidine bases thymine, cytosine, and uracil have attracted much attention and recently some of the corresponding nucleosides and nucleotides have been investigated as well. The most thoroughly studied of the latter compounds are those derived from cytosine (2-4) and the S-halogen-substituted uracils (2,5,6), whereas less information so far is available about the radiationinduced processes in uracil nucleosides and nucleotides (7-11). In a previous paper (7), radical formation in uridine-5’-phosphate *2Na+ (5’-UMP/Na) was investigated. 0
R =-OH
: uridina
A;-OF-O;: 5’
OH
OH
Evidence was given that the uracil base acts as an electron scavenger, whereas the oxidative processes mainly take place in the sugar-phosphate region of the 518 0022-2364/81/090518-13$02.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
RADIATION
DAMAGE
TO URIDINE
519
molecule. Thus, at 77 K the uracil anion radical was identified, converting into the well-known 5,6-dihydrouracil-5-yl radical at elevated temperatures. At 77 K a fsecondary alkoxy radical was also observed, for which the main part of the unpaired spin density is localized on Ox, in the sugar-phosphate moiety. Upon heatin,g, this radical transformed into a C,,-centered H-abstraction radical. After irradiation at 270 K, a third radical was observed in addition to the 5-yl and C,,centered species. This radical was originally assumed to be the uracil-6-yl radical (7’, 11). Recently it was demonstrated that the EPR parameters for this resonance are also compatible with a radical in the ribose moiety (10). This radical would have the main part of the spin density localized on C4,, which implies an opening of the ribose ring upon radical formation. In the present report, the free radical products trapped in single crystals of uridine after irradiation at room temperature are discussed. As usual for all uracil derivatives, the uracil-5-yl radical (called I) is observed. A second product (II) exhibits a resonance which makes it reasonable to assume that it is a ribose radical formed by a net hydrogen abstraction from C,,. The effects of light illumination and heat treatments are discussed, including a description of a further resonance (III) which appears as a consequence of the heat-induced decay of the 5-yl radical. EXPERIMENTAL
Polycrystalline samples of uridine were obtained from Sigma Chemical Company. Crystals were formed from saturated ethanolic solutions by slow evaporation at 37°C. Partially deuterated crystals were obtained by a similar procedure from solutions of C2H20D (IC GmbH, 95% OD). Care was taken to select single crystals, since twinning was observed for most of the samples obtained. The crystals are generally small and irregularly shaped, but they always exhibit a well-defined axis of elongation (a). Single crystals of uridine are monoclinic with space groupP2, (12). The unit cell contains two asymmetric units, each consisting of two independent uridine molecules (denoted A and B) with slightly different conformations. The dihedral angle between the pyrimidine ring planes of A and B is 12”. The crystal structure is characterized by an extensive network of hydrogen bonds. All crystals used for EPR measurements were examined by X-ray Weissenberg techniques and the crystal axes were localized. The orthogonal a, b, and c* = a :< b reference system was adopted for EPR measurements. The crystals were irradiated at 295 K with 4.0-MeV electrons from a linear accelerator up to doses of 16 Mrad at a dose rate of 0.9 Mrad/min. The EPR measurements were mainly carried out with a Jeol X-band spectrometer. Some spectra were also recorded on a Varian E-9 spectrometer equipped with a E-l 10 Q-band microwave bridge. For calibration purposes, the microwave frequency was monitored using a frequency Imeter, and a reference sample containing DPPH and Mn2+ in MgO could be placed close to the sample in the cavity. First estimates of the g tensor and proton hyperfine coupling tensors were obtained using the methods of Schonland (13 ) and Lund and Vlinngard (14 ), respectively, including the usual linear least-squares fitting procedure (15). These
520
EINAR SAGSTUEN
estimates were then used as input trial tensors in a nonlinear iterative least-squares fitting routine, using an effective spin Hamiltonian which includes the nuclear Zeeman term, as described previously (16). In this analysis one adjustment angle in each plane of rotation was included as additional variational parameters. Error estimates in eigenvalues and eigenvectors were made following the procedure of Fouse and Bernhard (17), which makes use of the variance-covariance matrix for each set of least-squares-adjusted tensor elements. All calculations reported were performed on a CDC CYBER 74 computer with a single precision standard of 60 bits word length. RESULTS
When crystals of uridine are irradiated radical species are formed and trapped.
at room temperature, altogether three Figure 1 shows spectra obtained im-
1450
FIG. 1. Second-derivative
EPR spectra of a uridine crystal after irradiation
at 295 K.
RADIATION
DAMAGE
521
TO URIDINE
mediately after irradiation, recorded at two orientations of the crystal in the applied magnetic field. The resonance lines attributable to two of the radicals, designated by I and II in the following, dominate the spectra of all orientations, whereas lines from a third radical only weakly contribute to the central features of the spectra at this temperature. Radical
Conversions
The analysis of the different resonances was facilitated by several spectral changes induced by light exposure or heat treatment of the crystals. This is demonstrated in Fig. 2. Upon light exposure (A > 300 nm) at temperatures below -3O”C, the conversion II + I was induced (Figs. 2A and B). Upon subsequent storage at room temperature the original spectrum was recovered (Fig. 2C), which indicates that the light-induced conversion is reversible. The backtransformation (I + II) does not, however, continue upon heat treatment above room temperature. Thus, u.p to about 80°C the spectra remained almost unchanged. However, at this temperature a gradual change into the spectrum shown in Fig. 2D occurred. This latter spectrum indicates that radical I had decayed, radical II remained unchanged, and the third resonance in the central region of the spectra had increased in intensity.
u uuuuuu -* I
u
II
FIG. 2. Effect of uv exposure and heat treatment upon the EPR spectra of uridine crystals after irradiation at 295 K. (A) Spectrum recorded immediately afker irradiation at 295 K. (B) Spectrum recorded after 30 min light exposure (A > 300 nm) at -50°C. (C) As (B), but recorded after annealing at room temperature for 2 hr. (D) As (C), but recorded at room temperature after annealing at 85°C for 1 hr. All spectra were obtained with the magnetic field along the crystallographic 6 axis without moving the crystal in the cavity and at identical spectrometer conditions.
522
EINAR SAGSTUEN
This conversion is irreversible upon cooling to room temperature. Identical spectra are also obtained if a crystal, not previously light exposed, is heated to 80°C. Radical I-The
UraciE Syl Radical
Figures 3A and B show spectra attributable to radical I, obtained after light exposure at -5O”C, at crystal orientations corresponding to those in Fig. 1. The resonance from radical I is characterized by three large proton interactions. One of these seems to be of the CYtype, whereas the two others may be ascribed to p protons. The results from a full analysis of the EPR data are given in Table 1. The g value is only slightly anisotropic, indicating a carbon-centered radical. The angle between the eigenvectors for the minimum principal g value and the intermediate (Ytensor principal value is 2.7”, suggesting a planar n-electron radical. The assignment of radical I as the 5,6-dihydrouracil-5-yl radical unequivocally emerges from the excellent agreement between the different eigenvectors and corresponding molecular reference vectors calculated from the crystal structure, as shown in Table 1. The coupling tensors are all very similar to those observed for 5-yl radicals formed in other uracil derivatives (2,7,8,16,18,29). It appears that the formation of radical I specifically takes place in molecule B of the asymmetric unit (12). A pronounced line broadening was observed at crystal orientations where the magnetic field is close to the perpendicular of the pyrimidine ring. This suggests a small positive spin density on a nitrogen atom. At other orientations a small DPPH
I
FIG. 3. Second-derivative EPR spectra of an uridine crystal after irradiation at room temperature. (A), (B) Spectra recorded after 30 min light exposure (A > 300 nm) at -50°C. (C), (D) Spectra recorded at room temperature after annealing at 80°C for 2 hr.
2X045(2) 2.0035(l) 2.0019(2)
-0.5683 0.7044 0.1406 0.7734
0.0077 0.6595 0.3587 0.9480
0.43(7) -0.58(S) 0.69(3)
0.67(2) 0.39(26) -O&4(14)
0.420(10) 0.654(17) -0.629(14) 0.18(8) -0.70(5) 0.70(4)
a
0.5358
0.9810
-0.0442 -0.5254
0.3952 -0.6989 0.9182 0.2940
0.82(2) -O%(5) -0.57(3)
0.47(4) -0.45(32) 0.76(17)
0.98(2) 0.03(8) -0.22(8)
-0.053(21)
0.816(08) -0.576(11)
E
0.8216 0.4773 -0.1340 0.3387
-0.1679 0.1218
0.2767
0.9186
0.37(7) 0.81(6) 0.45(6)
0.58(2) 0.81(6) 0.12(35)
0.13(2) 0.72(5) 0.68(4)
0.397(10) 0.492(20) 0.775(09)
c*
12.5
32.8
+1.3 -1.4
f 0.4
c 0.4
14.2 r 0.2 k 0.1
46.7 20.4
Molecule A
3.0
f1.4 -3.0
15.7 k 0.8
4.2 " 0.5 4.4 2 0.4 15.3 f 0.8
Molecule B
AV
2
4
2 1 3
Ref. vector
” The number in parentheses is the standard deviation in the corresponding Iast digit(s) of the quoted value. b This is the angle of deviation between the eigenvector and a molecular reference vector, identified by a number. The interval of uncertainty is given at a 95% confidence level. c The negative sign of this splitting is assumed, in accordance with theoretical considerations upon a-proton interaction. d These vectors are calculated from the crystal structure (12). The positions of the protons H,, and H,, are calculated by assuming a symmetrical arrangement with respect to the ring plane, an angle H,,-C,-H,, = 1 IO” and C-H bond lengths of 1.09 A.
G G G
G G
37.2(2) 3X6(3) 54.9(3) 50-O(3) 49.4(4)
G G G G
-30.1(2) - 17.2(4) -8.9(5) 39.9(l)
Principal values
Ref. vectors,d Molecule A 1 C,- H bond direction 2 Pyrimidine ring normal C,-H,, direction 3 4 C,- H,, direction Ref. vectors, Molecule B 1 Q-H bond direction 2 Pyrimidine ring normal 3 C,-%, direction 4 C,- Kt;, direction
2.0033(l)
51.4(2)
H,&,
R
37.6(l) G
H&S)
G
- 18.7(2) G
H,(G)
)
Isotropic value
Tensor
Principal directions
TABLE 1 EPR PARAMETERS FOR RADICAL I IN IRRADIATED SINGLE CRYSTALS OF URIDINE AI 2% K'
VI z
$ m
g $
g
'3
w
EINAR SAGSTUEN
524 DPPH
,25G,
DPPH 1 c
FIG. 4. Effect of partial deuteration upon the EPR spectra of uridine crystals after irradiation at 295 K. Spectra (A) and (C) are of crystals grown from C,H,OH, and spectra (B) and (D) from partially deuterated crystals. (A), (B) Spectra recorded atler light exposure (A > 300 nm) at -50°C. (C), (D) Spectra recorded at room temperature after annealing at 80°C for 2 hr.
doublet splitting was resolved, exhibiting a maximum value of about 3.5 G with the magnetic field nearly perpendicular to the N3-H bond, in the pyrimidine plane. This is illustrated in Fig. 4A. In Fig. 4B the corresponding spectrum from a partially deuterated crystal is shown, clearly demonstrating that the responsible proton is exchangeable. Furthermore, the minimum linewidth for radical I (about 2 G(pp)) was observed with the magnetic field directed nearly parallel to the N,-H bond direction. Thus, the angular behavior of this additional proton interaction together with the observed linewidth variation suggests that some spin density is localized to N3 and that the proton bonded to N3 is responsible for the doublet splitting. Quantitatively, the 5-yl radical accounts for approximately 50% of the total EPR absorption prior to light exposure. Radical
II-A
Cd,-Centered Radical
Upon the decay of radical I at temperatures above 8O”C, the outer resonance lines attributable to radical II become more amenable for study. Figures 3C and D show the spectra obtained after heat treatment at the same crystal orientations as for the spectra in Fig. 1. As indicated by the stick spectra, the resonance from radical II is characterized by three large proton interactions. Two of these are almost equivalent at most orientations. The total width of the resonance varied between 82 and 93 G, and the rotation data show that all three couplings are relatively isotropic, which suggests P-proton interactions only. A mutual overlap of
RADIATION
DAMAGE TABLE
EPR
PARAMETERS
525
TO URIDINE 2
FOR RADICAL II IN IRRADIATED OF URIDINE AT 295 K”
SINGLE
CRYSTALS
Principal directions Tensor
Isotropic value
Principal values
a
b
c
g
2.0032(2)
2.0041(2) 2.0031(3) 2.0024(2)
-0.03(7) -0.36(13) 0.93(7)
-0.13(5) 0.93(8) 0.35(15)
0.99(4) O.ll(17) 0.08(4)
HP1
36 G
22 G
HP2
25 G
-c3 G
Hi33
24 G
?3 G
u The number in parentheses is the standard deviation digit(s) of the quoted value.
in the corresponding
last
resonance lines in the central portion of the spectra made a thorough analysis of the hyperfine coupling tensors difficult. The g value varied slightly around the freespin g value suggesting a carbon-centered free radical. The available EPR data are collected in Table 2. The spectra in Figs. 3C and 4C show two additional small doublet splittings which are resolved only at a few orientations. Experiments with partially deuterated crystals demonstrated that the large P-proton interactions are attributable to nonexchangeable protons, whereas both the smaller doublet splittings collapse upon deuteration and consequently are attributable to exchangeable protons. This is illustrated by the spectrum in Fig. 4D. A free radical in uridine, which exhibits splittings from three p protons is probably formed in the ribose part of the molecule. Furthermore, the magnitude of the couplings shows that the main part of the spin density is localized to one position only (i.e., there is no substantial spin delocalization through aromatic conjugation). The observation of three large nonexchangeable couplings suggest that C+ is essential with regard to spin localization. Assuming that no large rearrangements take place, a possible structure for the radical is
The radical is formed by a net hydrogen abstraction from C4,. Here the hydrogen atoms bonded to CS, and Cg, are responsible for the large couplings, whereas the
526
EINAR
SAGSTUEN
y protons at 03, and OS, may be responsible for the two exchangeable doublet splittings. The g-value variation for radical II is relatively small, indicating a limited amount of spin density localized on the oxygen atoms near C4,. Like radical I, radical II also seems to be selectively formed in one of the two types of uridine molecules. Thus, the geometry of the two undamaged ribose residues is sufficiently different so as to reveal the coexistence of two conformations of the radical. The available data are not sufficient to determine in which of the two molecules the radical is formed. Nor is it possible to arrive at the detailed molecular structure of the radical. The minimum g values occur in a direction 23” away from the C,-H bond direction in the undamaged molecule (and 18” from the perpendicular to the Ct,-C5,-01, fragment). Furthermore, from the crystal structure the dihedral angles between the C4,-H bond and the C3,-H and the two C,,-H bonds in both molecules are about 13, 73, and 46”, respectively. From the Heller-McConnell relation (20) these angles correspond to p splittings very different from those observed. Thus, geometrical rearrangements must have taken place during radical formation. Only a full knowledge of the interaction tensors combined with the detection of precursor radicals will permit the evaluation of a detailed structure for radical II. The resonance from radical II accounts for approximately 35% of the total EPR absorption prior to heat treatment. Radical
III-A
Second Ribose Radical
As described above, heat treatments at 80°C induce the decay of radical I and the intensity of the central portion of the spectra increases. The central part of the resonance, mainly attributable to radical III, consists of a narrow multiplet which never exceeds a total width of 25 G. Upon heating a crystal above 80°C radical II disappears at about 100°C and new resonance lines appear in the center, superimposed on those from radical III. The combination of severe overlap with other resonances (either radical II or its decay product) and a relatively isotropic hyperfine splitting pattern again makes the determination of coupling tensors impossible from EPR measurements alone. Some general comments to the observations may, however, be made. Figure 5 presents some spectra obtained after annealing of a crystal at 105°C. In this figure, spectra from protiated as well as partially deuterated crystals are shown. The stick spectra indicates the resonance lines from radical III. Resonance lines from the decay product(s) of radical II partly obliterate the spectra. Together with the spectra in Figs. 2 to 4 these data show that the resonance attributable to radical III may be interpreted as arising from interactions between the unpaired electron and three protons. Two of these interactions are relatively isotropic (both of the order of 10 G), and are attributable to nonexchangeable hydrogens. The third coupling is smaller (maximum observed splitting is approximately 4.5 G), is somewhat more anisotropic, and disappears upon deuteration. Thus, this splitting is attributable to interaction with an exchangeable proton. The g-value anisotropy is small, varying between 2.0038 and 2.0024. Also, the linewidth variation is small, indicating no interactions with nitrogen atoms.
RADIATION
DAMAGE
TO URIDINE
527
FIG. 5. Second-derivative EPR spectra of uridine crystals after irradiation at room temperature alnd subsequent annealing at 110°C. The spectra were recorded at room temperature. (A) and (C) are of crystals grown from C,H,OH and (B) and (D) from partially deuterated crystals. The stick spectra indicate the resonance lines from radical III. The fully drawn sticks represent interaction with nonexchangeable protons, and the broken sticks represent an interaction with an exchangeable proton. The vertical arrows indicate the position of the DPPH resonance.
The magnitude of the g-value variation seems to exclude the possibility that a large fraction of the unpaired spin density is localized to a keto oxygen. Furthermore, the failure to detect anisotropic coupling from an (Y proton makes a conjugated radical structure less likely. Then, with the main part of the unpaired spin density residing on one carbon atom only, the magnitude of the P-proton couplings suggests that the two /3 hydrogens are bonded to two different atoms. These considerations suggest the following structure for the radical:
Although the g-value variation does not exclude a radical in the uracil base, the above discussion renders a ribose radical more likely. As to the position in which t.he radical is centered in the ribose moiety, these data do not give any conclusive evidence. The I e II transition described above suggests, however, that it may be localized in the C3,-C4,-C5, region of the molecule. DISCUSSION
AND CONCLUSIONS
Irradiation of uridine crystals at room temperature produces three free radical species altogether. Radical I is the well-known uracil 5-yl radical. The spectral
528
EINAR
SAGSTUEN
parameters as given in Table 1 are very similar to those for the same radical formed in crystals of uracil (18), and some derivatives (7, 8, 12, 16). In all these compounds, a weak interaction with N3 and its attached proton has been observed. Flossman et al. (22) suggested, based on INDO-UHF calculations, that this observation together with the magnitude of the P-methylene couplings indicate that the radical is protonated at the C,=O, group. Direct experimental evidence supporting this suggestion has not been obtained, either by EPR (7, 26) or by ENDOR (2.?,24) measurements. In a very recent ENDOR analysis of the 5-thymyl radical in thymidine, 0, was found nor to be protonated (J. N. Herak, personal communication). RHF/CI calculations at the INDO level of approximation also indicate that O4 is unprotonated in the uracil 5-yl radical itself as well as in its 5-halogen substituted derivatives (25). Protonation of the 5-yl radical may have some relevance to the observed radical transformations in uridine. As described above, light exposure at temperatures below -30°C results in a transformation of radical II into radical I, or to be more precise, into a radical with a resonance indistinguishable from that of radical I. The reverse transformation is readily completed by storage at room temperature (see Fig. 2). Clearly, there is no equilibrium between I and II at room temperature, since the spectrum does not change further by heating above room temperature (below 80°C). Two explanations may be offered. In the first place, uv illumination may produce (from radical II) a radical I’ chemically different from I. The difference may, for example, be that I’ is protonated at 0, and I is not. Bernhard (26) recently considered this possibility in an attempt to rationalize the lightinduced transformation between 5-yl and 6-yl radicals in uracil and cytosine derivatives. In the present case this possibility is considered less likely, since the spectral parameters from a protonated and an unprotonated version of I are expected to differ significantly. A second possibility is that I and I’ are chemically identical but trapped in different environments. I and I’ must, however, both be formed in molecule B of the asymmetric unit, since the spectra from I’ in A and B should (by virtue of their crystallographic inequivalent orientations) be distinguishable. It is of interest to note that no D adduct corresponding to I was observed in partially deuterated crystals. Neglecting the possibility for a fast H-D backexchange, this indicates that the added H atom originates from a nonexchangeable position. The mechanism of formation of radical I in uridine will be discussed elsewhere, when the low-temperature experiments have been analyzed. The analysis for radical II makes is reasonable to consider a structure featuring Cd, as the radical locus. Similar resonances have not previously been detected in irradiated nucleosides or nucleotides. A radical involving three /3 protons was recently observed, however, in crystals of methylglucopyranoside after irradiation at 77 K (27). A detailed ENDOR analysis revealed that this radical retained its tetrahedral bonding (by sp3 hybrids) at the carbon on which the main unpaired spin densities become localized. The available data indicate that this is not the case for II. Two modes of formation for radical II may be considered. One consists in a 4’,5’-H atom transfer in a precursor hydroxyalkyl radical (27). A second possibility may be deprotonation at Cd9of a parent cation radical. As discussed previ-
RADIATION
DAMAGE
TO URIDINE
529
ously (10,28), if an electron vacancy is localized on 01,, the resulting positively charged radical may be stabilized by a deprotonation. Likely positions for deprotonation in the ribose moiety are those next to O,,, i.e., C1, and C4,. It has been speculated that the conformation of the ribose moiety in part determines which site is selected (28). In 5’-UMP a radical was detected which may be attributable to a primary deprotonation at C,, (10). The conformation of the ribose sugar in uridine is different from that in 5’-UMP. Thus, in uridine, H4, is situated in a conformation similar to that for H,, in 5’-UMP. Hence, rather than Cl,, C4, should be the site for deprotonation in uridine. Again, low-temperature studies are needed for further experimental evidences. Radical III, which exhibits a very weak resonance after irradiation at room temperature, is most probably a decay product from radical I. Several recent observations indicate that the uracil-S-y1 radical is not a stable end product in irradiated nucleosides and nucleotides. Thus, in 5’-UMP this radical decays upon storage at room temperature (I I ). Eventual decay products have not been identified. In uridine (this work) and in uracil-p-D-arabinofuranoside (unpublished results) the 5-yl radicals decay by heat treatment. The species formed exhibit similar resonance patterns. In uridine the experimental data indicate that the radical is formed in the ribose part of the molecule. The H-adduct radicals are among the most important products in the radiolysis of nucleic acids. It is of biological i-mportance if these species are unstable and if the decay leads to alterations in the ribose-phosphate region of the nucleotides. ACKNOWLEDGMENTS Professor T. Henriksen is acknowledged for the thorough reading of this manuscript and many suggestions for its improvement. Thanks are due to the Studsvik Science Research Laboratory, and in particular to Dr. Anders Lund, for making their Varian Q-band spectrometer available and for valuable discussions. Discussions with Drs. D. M. Close, H. Oloff, and J. N. Herak are also acknowledged. Thanks are due to the Norsk Hydros Institute for Cancer Research for use of their linear electron accelerator. REFERENCES I. H. C. Box. “Radiation 2. J. HUTTERMANN, 3.
4. 5. 6.
7. 8. 9. 10. II. 12. i3. 1’4.
Effects. ESR and ENDOR Analysis,”
W. KGHNLEIN,
R. TEOLE,
Academic Press, New York, 1977. (Eds.), “Effects of Ionizing
AND A. J. BERTINCHAMPS
Radiation on DNA,” Springer-Verlag, Berlin, 1978. W. A. BERNHARD, J. H~TTERMANN, A. MOLLER, D. M. CLOSE, AND G. W. FOUSE, Radiat. Res. 68, 3% (1976). D. M. CLOSE AND W. A. BERNHARD, J. Chem. Phys. 68, 210 (1979). J. HOTTERMANN, W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT,J. Phys. Chem. 81,228(1977). H. OLOFF, E. HAINDL, J. HUTTERMANN, AND J. KRAUSS, Radiat. Res. 80, 447 (1979). E. SAGSTUEN, Radiat. Res. 81, 188 (1980). R. BER~ENE AND R. A. VAUGHAN, Znt. J. Radiat. Biol. 29, 145 (1976). H. C. Box, W. R. POTTER, AND E. E. BUDZINSKI, J. Chem. Phys. 62, 3476 (1975). E. SAGSTUEN, Radiat. Res. 84, 164 (1980). B. RAKVIN AND J. N. HERAK, Znt. J. Radiat. Biol. 38, 129 (1980). E. A. GREEN, R. D. ROSENSTEIN, R. SHIONE, D. J. ABRAHAM, B. L. TRUS, AND R. E. MARSH, Acta Crystallogr. Ser. B 31, 102 (1975) [erratum: Acta Crystallogr. Ser. B 31, 1221 (1975)]. D. S. SCHONLAND, Proc. Phys. Sot. London 73, 788 (1958). A. LUND AND T. VANNGARD, J. Chem. Phys. 42, 2979 (1965).
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EINAR SAGSTUEN
15. E. SACSTUEN AND C. ALEXANDER, JR., Mol. Phys. 32, 743 (1976). 16. 0. CLAESSON, A. LUND, J.-P. JORGENSEN, AND E. SAGSTUEN, J. Map. Reson. 41, 229 (1980). 17. G. W. FOUSE AND W. A. BERNHARD, J. Magn. Reson. 31, 191 (1978). 18. H. ZEHNER, W. FLOSSMANN, E. WESTHOF, AND A. MOLLER, Mol. Phys. 32, 869 (1976). 19. 20. 21.
I. EGTVEDT, E. SAGSTUEN, R. BERGENE, AND T. HENRIKSEN, Radiat. Res. 75, 252 (1978). C. HELLER AND H. M. MCCONNELL, J. Chem. Phys. 32, 1.535 (1960). J. A. POPLE AND D. L. BEVERIDGE, “Approximate Molecular Orbital Theory,” McGraw-Hill, New York, 1970. 22. W. FLOSSMANN, H. ZEHNER, AND E. WESTHOF, Znt. J. Radiat. Biol. 36, 577 (1979). 23. J. HOTTERMANN, W. A. BERNHARD, E. HAINDL, AND G. SCHMIDT, Mol. Phys. 32, 1111 (1976). 24. E. HAINDL AND J. HOTTERMANN, J. Magn. Reson. 30, 13 (1978). 25’. H. OLOFF AND J. H~TTERMANN, J. Magn. Reson. 40, 415 (1980). 26. W. A. BERNHARD, Adv. Radiat. Biol. 10, in press. 27. K. P. MADDEN AND W. A. BERNHARD, J. Chem. Phys. 70, 2431 (1979). 28. K. P. MADDEN AND W. A. BERNHARD, J. Phys. Chem. 83,2643 (1979).